Design, synthesis, and evaluation of benzophenone derivatives as novel acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2008.02.035

Starting from a structure-based drug design, new acetylcholinesterase inhibitors were designed and synthesized as analogues of donepezil. The compounds were composed by an aromatic function and a tertiary amino moiety connected by a suitable spacer. In pa

Full text of DOI:10.1016/j.ejmech.2008.02.035

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1341e1348
http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis, and evaluation of benzophenone derivatives
as novel acetylcholinesterase inhibitors
*
Federica Belluti , Lorna Piazzi, Alessandra Bisi, Silvia Gobbi, Manuela Bartolini,
Andrea Cavalli, Piero Valenti, Angela Rampa
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received 17 January 2008; received in revised form 14 February 2008; accepted 15 February 2008
Available online 8 March 2008
Abstract
Starting from a structure-based drug design, new acetylcholinesterase inhibitors were designed and synthesized as analogues of donepezil.
The compounds were composed by an aromatic function and a tertiary amino moiety connected by a suitable spacer. In particular, the benzo-
phenone nucleus and the N,N-benzylmethylamine function were selected. The easily accessible three-step synthesis of these compounds resulted
to be significantly less difficult and expensive than that of donepezil. Several compounds possess anti-cholinesterase activity in the order of
micro and sub-micromolar. Particularly, compounds 1 and 10 were the most potent inhibitors of the series.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Alzheimer’s disease; Benzophenone; AChE inhibitors
1. Introduction
AD. The crystallographic structure of the Torpedo californica
AChE gorge complex with donepezil was described by Suss-
man [8] and clearly showed that the molecule has a distinctive
orientation along the active-site gorge, extending from the ac-
tive anionic site to the PAS, placed at the bottom and at the
top, respectively, where it establishes specific interactions (ar-
omatic pep or cationep stacking) with conserved aromatic
residues. The study allowed characterization of the binding
site and identification of the moieties that play a fundamental
role in the biological activity of the inhibitor, such as the dime-
thoxyindanone function, the charged piperidine nitrogen atom
and the benzyl group [9]. The synthetic route employed to
obtain donepezil [10] was elaborate, quite difficult and also
expensive, due to the involvement of many steps and to the
employment of unstable or expensive synthetic intermediates
or of hazardous reagents.
Alzheimer’s disease (AD) is a progressive neurodegenera-
tive disorder characterized by some major pathological signs
such as synaptic loss, reduced levels of the neurotransmitter
acetylcholine (ACh), b-amyloid senile plaques (SP) and neu-
rofibrillary tangles (NFT). The most dramatic abnormalities
are those of the cholinergic system [1]. Acetylcholinesterase
(AChE), besides its hydrolyzing activity, also plays a proaggre-
gating (non-catalytic) role, accelerating b-amyloid peptide
(Ab) aggregation and deposition into the fibrils that compose
the SP [2]. This action is associated with a specific motif
located near the entrance of the active-site gorge termed pe-
ripheral anionic site (PAS) [3]. Several efforts have been
done for the development of potent and selective AChE inhib-
itors (AChEI) and to date these drugs (such as tacrine [4],
rivastigmine [5], donepezil [6] and galantamine [7]) represent
the main therapeutic approach for symptomatic treatment of
As a part of our ongoing project aimed at identifying re-
versible AChEIs [11,12], we developed a new class of com-
pounds endowed with an interesting biological profile and
characterized by an easily affordable preparation methodol-
ogy. The presence of lipophilic moieties and a tertiary amino
group represents the key requirement for a good anti-AChE
* Corresponding author. Tel.: þ39 051 2099732; fax: þ39 051 2099734.
E-mail address: federica.belluti@unibo.it (F. Belluti).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.035

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F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
Table 1
Structures of the synthesized compounds 1e16
activity because of their interaction with the aromatic residues
that line the wall of the AChE gorge and with the anionic site
of the enzyme. Taking these considerations into account, the
examination of the schematic drawing of the molecule of
donepezil furnished the rationale for a structure-based drug
design as depicted in Fig. 1. Briefly, two main molecular mod-
ifications were carried out: (a) ring formation, accomplished
by the inclusion of the methylene unit in a six-membered ar-
omatic ring, joining the indanone and the piperidine functions;
and (b) opening of the piperidine ring and of the five-mem-
bered ring of the indanone cycle. Our efforts culminated in
the design of a molecule in which the 3,4-dimethoxybenzo-
phenone nucleus and the N,N-benzylmethylamine moiety
were connected by a methylene spacer (1). The positional
effect of the substitution of the [(benzylmethylamino)methyl]
moiety on the 3,4-dimethoxybenzophenone was then examined,
to confirm our working hypothesis, and only the para-
substituted derivative (1) showed high potency, while the
corresponding meta-substituted analogue proved to be inactive.
Therefore, with the aim to identify some essential pharmaco-
phore elements, various portions of the lead molecule (1) were
modified, while the carbonyl group and the phenyl rings of the
benzophenone scaffold, together with the tertiary nitrogen
atom, were maintained throughout the present investigation.
Several compounds were synthesized and reported in Table 1.
O
O
R3
R
R
R2
N
R1
R4
e
R4
R1
R
R1
R2
R3
H
1
2
OCH3 OCH3 CH3
O
OCH3 OCH3
OCH3 OCH3
e
e
e
e
N
CH3
3
N
CH3
CH3
N
CH3
4
5
6
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
e
e
e
e
e
e
N
CH3
CH3
N
N
7
8
9
OCH3 OCH3 CH3
OCH3 OCH3 CH3
OCH3 OCH3 CH3
m-NO2
o-OCH3
m-CH3
H
e
e
e
e
e
e
2. Chemistry
10 OCH3 OCH3 C2H5
11 OCH3 OCH3 C2H4OH
12 OCH3 OCH3 C2H4N(CH3)2
Scheme 1 outlines the synthetic route followed for the prep-
aration of the benzophenone derivatives. FriedeleCrafts acyl-
ation of the appropriate anisole derivative with p-toluoyl
chloride in the presence of SnCl4 as Lewis acid yielded the
4-methylbenzophenones 17e19 in good yields. Lithiation of
2-bromonaphtalene with n-BuLi and subsequent treatment
with p-tolunitrile gave, upon acid hydrolytic workup, the
naphthalen-2-yl-p-tolylmethanone derivative (20). Bromina-
tion with N-bromosuccinimide (NBS) of compounds 17e20
afforded the bromomethyl derivatives 21e24, that were then
subjected to nucleophilic substitution reaction by the selected
amines affording the desired final compounds 1e12, 14e16.
Another part of the synthetic plan led to the insertion of the
H
H
N
CH3
13 OCH3 OCH3
e
e
14
15
H
F
OCH3 CH3
OCH3 CH3
H
H
H
e
e
e
16 CH]CHe
CH]CH
CH3
E-propenyl side chain in the 4-position of the benzophenone
nucleus: the aldehyde 25, obtained by treatment of 21 with
hexamethylenetetramine, was coupled with the ylide, achieved
from triphenylethylphosphonium bromide, according to the
classic Wittig procedure, to afford the derivative 26, this was
then brominated with NBS to give 27, which was treated
with N,N-benzylmethylamine, as reported above, to give the
final compound 13.
O
O
H3C
H3C
N
O
donepezil
O
3. Results and discussion
O
O
H3C
H3C
CH3
N
The biological profiles of the novel benzophenone deriva-
tives listed in Table 1 toward the enzymes human AChE and
butyrylcholinesterase (BChE) were determined and expressed
as IC50, values, and the inhibitory activities were reported in
Table 2. The ability of compounds 1e16 to inhibit AChE
1
Fig. 1. Schematic drawing of donepezil and structure-based design of com-
pound 1.

F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
1343
O
X
R
R
a or b
c
R1
R1
CH3
17-20
O
O
e
R
R
O
Br
R1
R1
25
21-24
f
d
O
O
d
R
R
R1
R4
R1
CH3
2-6
26
c
O
O
R3
R
R
R2
N
Br
R1
R1
1, 7-12, 14-16
27
d
O
R
CH3
N
R1
13
Scheme 1. Reagents and conditions: (a) X ¼ H; R ¼ H, F, OCH3; R1 ¼ OCH3: p-toluoyl chloride, SnCl4; (b) X ¼ Br; R, R1 ¼ CH]CHeCH]CH: n-BuLi, THF,
ꢀ78 ꢁC, 4-methoxybenzonitrile, r.t., then conc. H2SO4, H2O, dioxane, reflux; (c) NBS, (PhCO)2O2, CCl4, reflux; (d) selected amine, TEA, toluene, reflux; (e)
hexamethylenetetramine, formic acid, EtOH 60%, reflux; (f) triphenylethylphosphonium bromide, NaH, THF, r.t.
and BChE activities was assessed employing the Ellman’s
method [13] and using donepezil as reference compound.
The compound 1 showed an interesting inhibitory profile
with IC50 ¼ 0.46 mM. Concerning modifications focused on
the amino function, some small aliphatic groups were inserted
to assess the importance of the N,N-benzylmethylamino por-
tion: compounds 2e5 showed a remarkable decrease in activ-
ity, underlying the importance of the benzyl group. We then
examined the rigid analogue 6 and found that its potency
was in the same way decreased by one order of magnitude.
Placement of substituents on the benzyl ring (compounds 7e
9) again decreased the inhibitory potency by one order of mag-
nitude in comparison with 1. A further modification involved
the replacement of the methyl group with different alkyl func-
tions (compounds 10e12): only the ethyl moiety maintained
the same potency as 1, while the hydroxyethyl and the dime-
thylaminoethyl functions decreased the potency by one order
of magnitude. Moreover, we explored the effect of elongating
the lead: the potency of the vinylog 13 was lower than 1.
Finally, the importance of the 3,4-dimethoxy substitution pat-
tern was investigated: the 3-methoxy group was removed (14)
or substituted by a fluorine atom (15) leading to less potent
compounds, and the introduction of a naphthalene moiety
(16), instead of the 3,4-dimethoxyphenyl function, led to a sig-
nificant decrease in activity.
Most of the derivatives proved to be selective for AChE
with respect to BChE. Particularly, compounds 1 and 10 pre-
sented a noteworthy anti-AChE activity, even if lower than
that of donepezil, and a remarkable selective behaviour. The
structural modifications carried out on compound 1 allowed
us to elucidate the nature of the interactions with AChE bind-
ing sites. The unmodified benzyl group on the nitrogen atom
proved to be an essential requirement for the inhibitory activ-
ity. Since both the increase of steric hindrance, due to the pres-
ence of substituents, and the elongation of the molecule were
poorly tolerated, we could assume that this moiety was prop-
erly arranged in such a way as to completely fill the bottom of
the AChE gorge. The previous considerations could be

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F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
Table 2
Inhibition of human recombinant AChE and BChE from human serum by the
studied compounds
Compounds
IC50 (mM) ꢂ SEM
AChE
IC50 ratio BChE/AChE
BChE
1
0.46 ꢂ 0.04
345 ꢂ 74
60.7 ꢂ 1.3
n.t.
n.t.
132
2
3
36.8 ꢂ 0.5
2.37 ꢂ 0.09
27.2 ꢂ 0.1
54.0 ꢂ 9.0
4.27 ꢂ 0.52
3.71 ꢂ 0.22
1.65 ꢂ 0.26
0.57 ꢂ 0.04
1.41 ꢂ 0.14
8.64 ꢂ 0.66
2.10 ꢂ 0.09
1.82 ꢂ 0.08
1.57 ꢂ 0.08
19.6 ꢂ 0.11
0.02 ꢂ 0.01a
4
88.8 ꢂ 3.1
n.t.
n.t.
33
5
6
7
>100
8
16.2 ꢂ 0.9
125 ꢂ 21
71.5 ꢂ 3.7
172 ꢂ 36
33.2 ꢂ 1.3
44.0 ꢂ 1.9
>100
4.4
76
9
10
127
122
4
11
12
13
14
21
15
16
>100
n.t.
Donepezil
7.42 ꢂ 0.39
371
n.t.: not tested.
a
Data obtained from Ref. [14].
extended to the N-alkyl function: placement of a small alkyl
group (methyl or ethyl) resulted favorable, suggesting the im-
portance of the hydrophobic character of the middle region of
the enzyme, while compounds bearing an hydrophilic moiety,
such as hydroxyethyl or dimethylaminoethyl, could badly in-
teract with the biological counterpart. Moreover, the 3,4-dime-
thoxyphenyl group proved to be important for the biological
activity since structural complication, achieved by inserting
an additional phenyl moiety, or simplification, obtained by
removing the 3-methoxy group, resulted detrimental for the
inhibitory potency.
Molecular modeling studies were carried out to provide
a better interpretation of the biological profile of 1 toward
hAChE. In particular, docking simulations were carried out
with the software GOLD [15] and outcomes were rationalized
by means of the clustering algorithm AClAP [16,17]. Com-
pound 1 was studied according to the computational strategy re-
ported in the supplementary data. In Fig. 2, the binding mode of
1 (Fig. 2A) at the hAChE gorge is reported. It can be seen that 1
is properly positioned into the enzyme gorge interacting with
the internal residue Trp86 by means of a pep stacking. More-
over, some aromatic and hydrophobic interactions were also
identified between 1 and aromatic residues lining the hAChE
gorge. A possible interaction could likely be identified for the
carbonyl oxygen of the benzophenone moiety, that in some
docking poses was positioned at the right distance for establish-
ing an H-bond with the Phe295 backbone. However, 1 was un-
able to properly contact Trp286, the key residue of the hAChE
PAS. This could be a possible explanation for its lower activity
when compared to donepezil, which seems to contact such res-
idue slightly better than our compound (Fig. 2B). Furthermore,
such a docking outcome prevented us to embark toward the high
costly biological assays to assess the capability of the present
series of compounds to interfere with the AChE-induced Ab
Fig. 2. (A) Docking model of 1 (carbon atoms are green) at the human AChE
gorge. It clearly arises that the benzophenone moiety does not properly contact
the key PAS residue Trp286. (B) X-ray complex between Torpedo californica
AChE and donepezil (carbon atoms are magenta). Donepezil seems to contact
Trp279 slightly better than 1 (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.).

F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
1345
aggregation. It is well-established that only inhibitors able to
properly interact with AChE PAS and to protrude out of the
enzyme gorge have the potential to be inhibitors of the
AChE-induced Ab aggregation.
saturated solution (10 mL), water (10 mL), dried over anhy-
drous Na2SO4 and evaporated to dryness. The crude solid
was further purified by crystallization from toluene.
5.1.1.1. (3,4-Dimethoxyphenyl)-p-tolylmethanone (17). Start-
ing from 1,2-dimethoxybenzene (6.9 g, 50 mmol), compound
17 was obtained, 90% yield, white needles: mp 105e107 ꢁC
4. Conclusion
1
A structure-based drug design allowed us to synthesize
a new series of AChEIs as analogues of donepezil. Particu-
larly, the key features for the high inhibitory potency of done-
pezil, the 3,4-dimethoxy group and the N-benzylamino moiety,
were inserted on a benzophenone backbone. Compounds 1 and
10 were the most potent inhibitors of the series, while the dif-
ferent structural modifications carried out on compound 1
were not able to improve the AChE inhibitory potency of
the new derivatives. Still compound 1, characterized by a fairly
good inhibitory activity and by a simple and affordable synthe-
sis, could be considered as a new lead for further optimization.
(lit. [18] 127e128 ꢁC). H NMR (CDCl3) d 2.40 (s, 3H), 3.95
(s, 3H), 3.98 (s, 3H), 6.81 (d, J ¼ 8.4 Hz, 1H), 7.38 (dd, J ¼ 8.4
and 1.8 Hz, 1H), 7.45e7.55 (m, 3H), 7.77 (d, J ¼ 8.4 Hz, 2H).
5.1.1.2. (4-Methoxyphenyl)-p-tolylmethanone (18). Starting
from anisole (5.4 g, 50 mmol), compound 18 was obtained,
1
71% yield, white needles: mp 94e95 ꢁC (lit. [19] 92 ꢁC). H
NMR (CDCl3) d 2.40 (s, 3H), 3.95 (s, 3H), 7.10 (d,
J ¼ 8.8 Hz, 2H), 7.35 (d, J ¼ 8.4 Hz, 2H), 7.60 (d,
J ¼ 8.4 Hz, 2H), 7.70 (d, J ¼ 8.8 Hz, 2H).
5.1.1.3.
(3-Fluoro-4-methoxyphenyl)-p-tolylmethanone
5. Experimental protocols
(19). Starting from 1-fluoro-2-methoxybenzene (1.0 g, 8.0
mmol), compound 19 was obtained, 87% yield, white solid:
mp 79e81 ꢁC. 1H NMR (CDCl3) d 2.45 (s, 3H), 3.98 (s,
3H), 7.02 (t, J ¼ 8.4 Hz, 1H), 7.29 (d, J ¼ 8.4 Hz, 2H), 7.55e
7.72 (m, 4H).
All melting points were determined in open glass capillaries
using a Buchi apparatus and are uncorrected. Reaction courses
¨
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on Merk precoated silica gel F254
plates. Nuclear magnetic resonance (1H NMR) spectra were re-
corded with a Varian 300 MHz spectrometers, peaks positions
are given in parts per million downfield from tetramethylsilane
(TMS) as internal standard and spin multiplicities are given as
s (singlet), d (doublet), t (triplet), q (quartet), dd (double dou-
blet), dt (double triplet), m (multiplet) or br (broad). Mass
spectra were obtained with Waters Micromass ZQ 4000 (ES-
MS spectra) or V. G. 7070 E (EI-MS spectra) apparatuses.
Wherever analyses are only indicated with elements symbols,
analytical results obtained for those elements are within
0.4% of the theoretical values. Column chromatography was
carried out with silica gel (Kieselgel 40, 0.040e0.063 mm;
Merck) using the flash technique. Yields were reported after
chromatographic purification. Pure samples were obtained as
hydrochloride salts by treating the product with HCl saturated
methanol and were crystallized from MeOH/Et2O. Compounds
were named following IUPAC rules as applied by Beilstein-
Institut AutoNom (version 2.1), a PC integrated software
package for systematic names in organic chemistry.
5.1.2. Naphthalen-2-yl-p-tolylmethanone (20)
To a stirred solution of 2-bromonaphthalene (1.0 g, 4.8
mmol) in anhydrous THF (50 mL) under N2 at ꢀ78 ꢁC,
n-BuLi (1.6 M hexane solution, 9.6 mL) was added dropwise.
The mixture was stirred for 1 h at the same temperature, and
then 4-methylbenzonitrile (1.20 g, 10.25 mmol) in THF (10
mL) was added dropwise. The solution was stirred for further
1 h at ꢀ78 ꢁC and then was allowed to warm to room temper-
ature. On cooling an acidic solution (conc. H2SO4 0.75 mL,
H2O 10 mL, dioxane 9 mL) was added. The reaction was
then heated at reflux for 2 h, cooled, basified with 2 N
NaOH solution, and extracted with CH2Cl2 (3 ꢃ 50 mL).
The organic extracts were washed with brine (50 mL), dried
over Na2SO4 and evaporated under reduced pressure, giving
a crude solid which was chromatographed on silica gel with
petroleum ether/ethyl acetate (95:5) as eluant, affording 20
(65%), mp 90e92 ꢁC. 1H NMR (CDCl3) d 2.48 (s, 3H),
7.32 (d, J ¼ 8.8 Hz, 2H), 7.50e7.63 (m, 2H), 7.80 (d, J ¼
8.0 Hz, 2H), 7.75e7.98 (m, 4H), 8.26 (s, 1H).
All elemental analyses and mass spectra are referred to the
1
free base. H NMR spectra are referred to the hydrochloride
salt, unless otherwise indicated.
5.1.3. General procedure for the synthesis of
bromomethylbenzophenones (21e24, 27)
A mixture of aryl-p-tolylmethanone derivative (1 equiv), N-
bromosuccinimide (1.1 equiv), a catalytic amount of benzoyl
peroxide in CCl4 (10 mL) was heated at 60 ꢁC for 6 h under
bright lamp. The reaction mixture was hot filtered and the
filtrate was concentrated under reduced pressure. The crude
solid was purified by crystallization from ligroin to afford
the desired bromomethyl derivative.
5.1. Chemistry
5.1.1. General procedure for the synthesis
of benzophenones 17e19
To an ice-cold mixture of the anisole derivative (1 equiv),
p-toluoyl chloride (1.2 equiv) and anhydrous SnCl4 (1.2 equiv)
were added portionwise. After stirring at 60 ꢁC for 4 h, the re-
action mixture was quenched with ice/water. Et2O was added
and the organic layer was separated, washed with NaHCO3
5.1.3.1. (4-Bromomethylphenyl)-(3,4-dimethoxyphenyl)metha-
none (21). Starting from 17 (2.56 g, 10.0 mmol), compound

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F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
1
21 was obtained, 80% yield, white needles: mp 113e115 ꢁC.
1H NMR (CDCl3) d 3.95 (s, 3H), 3.98 (s, 3H), 4.53 (s, 2H),
6.80 (d, J ¼ 8.4 Hz, 1H), 7.35 (dd, J ¼ 8.4 and 1.8 Hz, 1H),
7.45e7.55 (m, 3H), 7.74 (d, J ¼ 8.4 Hz, 2H).
110e112 ꢁC. H NMR (CDCl3) d 1.95 (d, J ¼ 7.5 Hz, 3H),
3.95 (s, 3H), 3.97 (s, 3H), 5.91 (dd, J ¼ 11.7 and 7.5 Hz,
1H), 6.85 (d, J ¼ 11.7 Hz, 1H), 6.91 (d, J ¼ 8.4 Hz, 1H),
6.91 (d, J ¼ 8.7 Hz, 1H), 7.40 (d, J ¼ 8.4 Hz, 2H), 7.49 (d,
J ¼ 2.1 Hz, 1H), 7.75 (d, J ¼ 8.4 Hz, 2H).
5.1.3.2. (4-Bromomethylphenyl)-(4-methoxyphenyl)methanone
(22). Starting from 18 (2.25 g, 10.0 mmol), compound 22 was
obtained, 85% yield, white needles: mp 108e110 ꢁC (lit. [19]
62 ꢁC). 1H NMR (CDCl3) d 3.96 (s, 3H), 4.55 (s, 2H), 7.10 (d,
J ¼ 8.8 Hz, 2H), 7.35 (d, J ¼ 8.4 Hz, 2H), 7.60 (d, J ¼ 8.4 Hz,
2H), 7.70 (d, J ¼ 8.8 Hz, 2H).
5.1.6. General procedure for the synthesis
of compounds 1e16
To a solution of the bromomethyl intermediate 21e24, 27
(1 equiv) in toluene (10 mL), the selected amine (1.5 equiv)
and triethylamine (1.5 equiv) were added. The resulting mix-
ture was stirred under reflux for 18 h. HCl (3 N, 10 mL) was
added dropwise and the aqueous phase was basified with
K2CO3 and then extracted with dichloromethane (3 ꢃ 30
mL). The combined organic extracts were dried over
Na2SO4 and the solvent was removed under reduced pressure.
Unless otherwise indicated HCl saturated methanol was added
and the crude solid was crystallized from MeOH/Et2O.
Starting from 21 the following compounds were obtained.
5.1.3.3. (4-Bromomethylphenyl)-(3-fluoro-4-methoxyphenyl)metha-
none (23). Starting from 19 (2.0 g, 8.2 mmol), compound 23
1
was obtained, 75% yield, white needles: mp 73e75 ꢁC. H
NMR (DMSO) d 3.99 (s, 3H), 4.58 (s, 2H), 7.03 (t,
J ¼ 8.4 Hz, 1H), 7.51 (d, J ¼ 8.4 Hz, 2H), 7.48e7.77 (m, 4H).
5.1.3.4. (4-Bromomethylphenyl)naphthalen-2-yl-methanone
(24). Starting from 20 (0.28 g, 1.14 mmol), compound 24
was obtained, 37% yield, reddish oil. 1H NMR (CDCl3)
d 4.56 (s, 2H), 7.33 (d, J ¼ 8.4 Hz, 2H), 7.52e7.62 (m, 2H),
7.78 (d, J ¼ 8.0 Hz, 2H), 7.75e8.00 (m, 4H), 8.25 (s, 1H).
5.1.6.1.
{4-[(Benzylmethylamino)methyl]phenyl}-(3,4-dime-
thoxyphenyl)methanone (1). Yield: 75%, hydrochloride salt:
mp 158e160 ꢁC. 1H NMR (CDCl3) d 2.64 (d, J ¼ 4.4 Hz,
3H), 3.96 (s, 3H), 3.98 (s, 3H), 4.04e4.13 (m, 2H), 4.15e
4.23 (m, 2H), 6.86 (d, J ¼ 8.4 Hz, 1H), 7.30e7.42 (m, 6H),
7.52 (d, J ¼ 1.8 Hz, 1H), 7.30e7.40 (m, 4H). ES-MS m/z:
376 (M þ 1). Anal. C24H25NO3 (C, H, N).
5.1.3.5. [4-(3-Bromopropenyl)phenyl]-(3,4-dimethoxyphenyl)me-
thanone (27). Starting from 26 (0.56 g, 2 mmol), compound
27 was obtained, 45% yield, white needles: mp 132e133 ꢁC.
1H NMR (CDCl3) d 3.95 (s, 3H), 3.96 (s, 3H), 4.18 (d,
J ¼ 7.5 Hz, 2H), 6.48e6.58 (m, 1H), 6.72 (d, J ¼ 16.2 Hz,
1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 7.37 (dd, J ¼ 2.1 and 8.4 Hz,
1H), 7.47e7.56 (m, 3H), 7.75 (d, J ¼ 8.4 Hz, 2H).
5.1.6.2. (3,4-Dimethoxyphenyl)-(4-morpholin-4-ylmethylphe-
nyl)methanone (2). Yield: 70%, hydrochloride salt: mp
1
196e199 ꢁC. H NMR (CDCl3) d 2.80e3.05 (m, 2H), 3.35e
3.45 (m, 2H), 3.95e4.05 (m, 8H), 4.20e4.44 (m, 4H), 6.88
(d, J ¼ 8.4 Hz, 1H), 7.35 (dd, J ¼ 8.4 and 1.8 Hz, 1H), 7.53
(d, J ¼ 8.4 Hz, 2H), 7.80e7.85 (m, 4H). ES-MS m/z:
(M þ 1). Anal. C20H23NO4 (C, H, N).
5.1.4. 4-(3,4-Dimethoxybenzoyl)benzaldehyde (25)
A mixture of 21 (2.0 g, 6.0 mmol), hexamethylentetramine
(0.93 g, 6.60 mmol), formic acid (0.6 mL), 60% aqueous
EtOH (300 mL) was heated under reflux for 12 h. The solvent
was removed and the aqueous phase was extracted with
CH2Cl2 (3 ꢃ 50 mL), the organic layers were dried and evap-
orated to give a crude solid that was purified by flash chroma-
tography using petroleum ether/ethyl acetate (80:20) as eluant,
67% yield, mp 113e115 ꢁC. 1H NMR (CDCl3) d 3.96 (s, 3H),
3.98 (s, 3H), 6.90 (d, J ¼ 8.4 Hz, 1H), 7.34 (dd, J ¼ 8.4 and
1.8 Hz, 1H), 7.52 (d, J ¼ 1.8 Hz, 1H), 7.88 (d, J ¼ 8.4 Hz,
2H), 8.01 (d, J ¼ 8.4 Hz, 2H).
5.1.6.3.
(3,4-Dimethoxyphenyl)-{4-[(ethylpropylamino)me-
thyl]phenyl}methanone (3). Yield: 68%, hydrochloride salt:
mp 154e156 ꢁC. H NMR (CDCl3) d 3.05e3.25 (m, 4H),
3.96 (s, 3H), 3.98 (s, 3H), 4.24 (d, J ¼ 4.8 Hz, 1H), 6.90 (d,
J ¼ 8.4 Hz, 1H), 7.37 (dd, J ¼ 8.4 and 1.8 Hz, 1H), 7.52 (d,
J ¼ 8.4 Hz, 2H), 7.80e7.92 (m, 4H). ES-MS m/z: 328
(M þ 1). Anal. C20H25NO3 (C, H, N).
1
5.1.6.4. (3,4-Dimethoxyphenyl)-(4-{[(2-dimethylaminoethyl)-
methylamino]methyl}phenyl)-methanone (4). Yield: 66%, hy-
1
drochloride salt: mp 216e218 ꢁC. Base: H NMR (CDCl3)
5.1.5. (3,4-Dimethoxyphenyl)-(4-propenylphenyl)
methanone (26)
d 2.28 (s, 9H), 2.45e2.65 (m, 4H), 3.61 (s, 2H), 3.95 (s,
3H), 3.96 (s, 3H), 6.89 (d, J ¼ 8.4 Hz, 1H), 7.35 (dd, J ¼ 8.4
and 1.8 Hz, 1H), 7.42 (d, J ¼ 8.4 Hz, 2H), 7.48 (d,
J ¼ 1.8 Hz, 1H), 7.72 (d, J ¼ 8.4 Hz, 2H). ES-MS m/z: 357
(M þ 1). Anal. C21H28N2O3 (C, H, N).
To a stirred suspension of NaH (30 mg, 1.2 mmol) in anhy-
drous benzene (25 mL) at 0 ꢁC triphenylethylphosphonium
bromide (0.45 g, 1.2 mmol) was added; the mixture was stirred
under the same conditions for 30 min, then compound 25
(0.5 g, 1.0 mmol) dissolved in benzene (10 mL) was added.
The reaction mixture was stirred at r.t. overnight. Ice was
added and the organic phase was separated, and dried to afford
a crude solid that was purified by flash chromatography using
petroleum ether/ethyl acetate (80:20) as eluant, 55% yield, mp
5.1.6.5. (3,4-Dimethoxyphenyl)-{4-[(methylprop-2-ynylamino)-
methyl]phenyl}methanone (5). Yield: 55%, hydrochloride salt:
mp 95e97 ꢁC. 1H NMR (CDCl3) d 2.80 (s, 1H), 2.92
(d, J ¼ 4.4 Hz, 3H), 3.78e3.94 (m, 2H), 3.97 (s, 3H), 3.98

F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
1347
(s, 3H), 4.22e4.29 (m, 2H), 6.90 (d, J ¼ 8.4 Hz, 2H), 7.35 (dd,
J ¼ 8.4 and 1.8 Hz, 1H), 7.53 (d, J ¼ 1.8 Hz, 1H), 7.70e7.78
(m, 4H). ES-MS m/z: 324 (M þ 1). Anal. C20H21NO3 (C, H, N).
7.65e7.78 (m, 3H), 7.78e7.85 (m, 4H). ES-MS m/z: 406
(M þ 1). Anal. C25H27NO4 (C, H, N).
5.1.6.12. (4-{[Benzyl-(2-dimethylaminoethyl)amino]methyl}-
phenyl)-(3,4-dimethoxyphenyl)methanone (12). Yield: 69%,
hydrochloride salt: mp 162e164 ꢁC. Base: 1H NMR
(CDCl3) d 2.22 (s, 6H), 2.45e2.75 (m, 4H), 3.65 (s, 2H),
3.70 (s, 2H), 3.95 (s, 3H), 3.96 (s, 3H), 6.90 (d, J ¼ 8.4 Hz,
1H), 7.20e7.55 (m, 9H), 7.72 (d, J ¼ 7.6 Hz, 1H). ES-MS
m/z: 433 (M þ 1). Anal. C27H32N2O3 (C, H, N).
5.1.6.6. [4-(3,4-Dihydro-1H-isoquinolin-2-ylmethyl)phenyl]-
(3,4-dimethoxyphenyl)methanone (6). Yield: 77%, hydro-
chloride salt: mp 122e124 ꢁC. Base: 1H NMR (CDCl3)
d 2.65e2.85 (m, 2H), 2.87e3.00 (m, 2H), 3.68 (s, 2H), 3.78
(s, 2H), 3.96 (s, 3H), 3.98 (s, 3H), 6.90 (d, J ¼ 8.4 Hz, 2H),
7.15 (d, 1.8 Hz, 1H), 7.39 (dd, J ¼ 1.8 and 8.4 Hz, 1H),
7.50e7.55 (m, 3H), 7.75 (d, J ¼ 8.4 Hz, 2H). ES-MS m/z:
388 (M þ 1). Anal. C25H25NO3 (C, H, N).
5.1.6.13.
{4-[3-(Benzylmethylamino)propenyl]phenyl}-(3,4-
dimethoxyphenyl)methanone (13). Starting from 27 (0.65 g,
0.9 mmol) and N,N-benzylmethylamine (0.17 mL, 1.35 mmol)
compound 13 was obtained, 45% yield, hydrochloride salt:
5.1.6.7. (3,4-Dimethoxyphenyl)-(4-{[methyl-(3-nitrobenzyl)
amino]methyl}phenyl)methanone (7). Yield: 65%, hydrochlo-
ride salt: mp 185e186 ꢁC. 1H NMR (CDCl3) d 2.72 (d,
J ¼ 4.4 Hz, 3H), 3.96 (s, 3H), 3.98 (s, 3H), 4.05e4.18 (m,
2H), 4.20e4.38 (m, 2H), 6.90 (d, J ¼ 8.4 Hz, 1H), 7.34 (dd,
J ¼ 1.8 and 8.4 Hz, 1H), 7.52 (d, J ¼ 1.8 Hz, 1H), 7.43 (t,
J ¼ 8.2 Hz, 1H), 7.80e7.85 (m, 4H), 8.34 (d, J ¼ 8.2 Hz,
1H), 8.40 (d, J ¼ 1.2 Hz, 1H), 8.50 (d, J ¼ 8.2 Hz, 1H). ES-
MS m/z: 421 (M þ 1). Anal. C24H24N2O5 (C, H, N).
1
mp 172e174 ꢁC. H NMR (CDCl3) d 3.15e3.24 (m, 2H),
3.65 (s, 2H), 3.95 (s, 3H), 3.97 (s, 3H), 6.40e6.77 (m, 2H),
6.80 (d, J ¼ 8.4 Hz, 1H), 7.25e7.41 (m, 5H), 7.43e
7.53 (m, 3H). ES-MS m/z: 402 (M þ 1). Anal. C26H27NO3
(C, H, N).
5.1.6.14. {4-[(Benzylmethylamino)methyl]phenyl}-(4-methoxy-
phenyl)methanone (14). Starting from 22 (0.3 g, 1.0 mmol)
and N,N-benzylmethylamine (0.2 mL, 1.5 mmol) compound
14 was obtained as brown oil, 56% yield, hydrochloride salt:
mp 170e172 ꢁC. Base: 1H NMR (CDCl3) d 2.20 (s, 3H),
3.55 (s, 2H), 3.60 (s, 2H), 3.80 (s, 3H), 7.10 (d, J ¼ 8.8 Hz,
2H), 7.20e7.40 (m, 5H), 7.35 (d, J ¼ 8.0 Hz, 2H), 7.60 (d,
J ¼ 8.4 Hz, 2H), 7.70 (d, J ¼ 9.2 Hz, 2H). ES-MS m/z: 346
(M þ 1). Anal. C23H23NO2 (C, H, N).
5.1.6.8. (3,4-Dimethoxyphenyl)-(4-{[(2-methoxybenzyl)methy-
lamino]methyl}phenyl)-methanone (8). Yield: 75%, hydro-
1
chloride salt: mp 168e170 ꢁC. H NMR (CDCl3) d 2.65 (d,
J ¼ 4.4 Hz, 3H), 3.90 (s, 3H), 3.97 (s, 3H), 3.99 (s, 3H),
4.10e4.25 (m, 2H), 4.30e4.43 (m, 2H), 6.89 (d, J ¼ 7.8 Hz,
1H), 6.98 (d, J ¼ 8.4 Hz, 1H), 7.06 (t, J ¼ 8.1 Hz, 1H),
7.35 (d, J ¼ 8.4 Hz, 1H), 7.46 (t, J ¼ 7.8 Hz, 1H), 7.51
(d, J ¼ 1.8 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 1H), 7.80e7.85
(m, 4H). ES-MS m/z: 406 (M þ 1). Anal. C25H27NO4 (C,
H, N).
5.1.6.15. {4-[(Benzylmethylamino)methyl]phenyl}-(3-fluoro-4-
methoxyphenyl)methanone (15). Starting from 23 (0.5 g,
1.3 mmol) and N,N-benzylmethylamine (0.25 mL, 1.95 mmol)
compound 15 was obtained, 68% yield, mp 38e40 ꢁC, hydro-
5.1.6.9. (3,4-Dimethoxyphenyl)-(4-{[methyl-(3-methylbenzyl)a-
mino]methyl}phenyl)methanone (9). Yield: 72%, hydrochlo-
ride salt: mp 219e221 ꢁC. 1H NMR (CDCl3) d 2.63 (d,
J ¼ 4.4 Hz, 3H), 3.96 (s, 3H), 3.98 (s, 3H), 4.00e4.20 (m,
2H), 4.22e4.32 (m, 2H), 6.90 (d, J ¼ 8.4 Hz, 1H), 7.34e
7.43 (m, 5H), 7.52 (d, J ¼ 1.8 Hz, 1H), 7.78e7.88 (m, 4H).
ES-MS m/z: 390 (M þ 1). Anal. C25H27NO3 (C, H, N).
1
chloride salt: mp 201e204 ꢁC. H NMR (CDCl3) d 2.63 (d,
J ¼ 4 Hz, 3H), 3.99 (s, 3H), 4.02e4.20 (m, 2H), 4.22e4.31
(m, 2H), 7.03 (t, J ¼ 8.4 Hz, 1H), 7.45e7.53 (m, 3H), 7.44e
7.68 (m, 4H), 7.78e7.84 (m, 4H). ES-MS m/z: 364 (M þ 1).
Anal. C23H22FNO2 (C, H, N).
5.1.6.16. {4-[(Benzylmethylamino)methyl]phenyl}naphthalen-
2-yl-methanone (16). Starting from 24 (0.15 g, 0.46 mmol)
and N,N-benzylmethylamine (0.08 mL, 0.69 mmol) compound
16 was obtained, 45% yield, hydrochloride salt: mp 173e175 ꢁC.
1H NMR (CDCl3) d 2.65 (d, J ¼ 4.4 Hz, 3H), 4.10e423 (m,
2H), 4.28e4.42 (m, 2H), 7.45e7.73 (m, 6H), 7.80e8.00 (m,
8H), 8.28 (s, 1H). ES-MS m/z: 366 (M þ 1). Anal.
C26H23NO (C, H, N).
5.1.6.10.
{4-[(Benzylethylamino)methyl]phenyl}-(3,4-dime-
thoxyphenyl)methanone (10). Yield: 77%, hydrochloride salt:
mp 178e180 ꢁC. 1H NMR (CDCl3) d 1.52 (t, J ¼ 6.9 Hz,
3H), 3.04 (q, J ¼ 7.0 Hz, 2H), 3.96 (s, 3H), 3.98 (s, 3H),
4.08e4.22 (m, 2H), 4.25e4.38 (m, 2H), 6.90 (d, J ¼ 8.4 Hz,
1H), 7.34 (dd, J ¼ 1.8 and 8.4 Hz, 1H), 7.45e7.50 (m, 3H),
7.51 (d, J ¼ 1.8 Hz, 1H), 7.65e7.78 (m, 3H), 7.80e7.95 (m,
4H). ES-MS m/z: 390 (M þ 1). Anal. C25H27NO3 (C, H, N).
5.2. Determination of inhibitory effect on AChE and
BChE activity
5.1.6.11. (4-{[Benzyl-(2-hydroxyethyl)amino]methyl}phenyl)-
(3,4-dimethoxyphenyl)methanone (11). Yield: 76%, hydrochloride
1
salt: mp 183e185 ꢁC. H NMR (CDCl3) d 3.10e3.20 (m, 2H),
The capacity of compounds 1e16 to inhibit AChE activity
was assessed using Ellman’s method [13]. Initial rate assays
were performed at 37 ꢁC with a Jasco V-530 double beam
spectrophotometer by following the rate of increase in the
3.90e4.10 (m, 8H), 4.20e4.38 (m, 2H), 4.42e4.58 (m, 2H),
4.78e4.85 (br, 1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 7.33 (dd, J ¼ 1.8
and 8.4 Hz, 1H), 7.42e7.50 (m, 4H), 7.55e7.65 (m, 2H),

1348
F. Belluti et al. / European Journal of Medicinal Chemistry 44 (2009) 1341e1348
absorbance at 412 nm for 5 min. AChE stock solution was pre-
pared by dissolving human recombinant AChE (E.C. 3.1.1.7)
lyophilized powder (Sigma, Italy) in 0.1 M phosphate buffer
(pH ¼ 8.0) containing Triton X-100 0.1%. Stock solution of
BChE (E.C. 3.1.1.8) from human serum (Sigma, Italy) was
prepared by dissolving the lyophilized powder in an aqueous
solution of gelatine 0.1%. The final assay solution consisted
of 0.1 M phosphate buffer pH 8.0, with the addition of
340 mM 5,5’-dithio-bis(2-nitrobenzoic acid), 0.02 unit/mL of
human recombinant AChE, or BChE from human serum and
550 mM of substrate (acetylthiocholine iodide, ATCh or butyr-
ylthiocholine iodide, BTCh, respectively). Stock solutions of
tested compounds were prepared in methanol and diluted in
bidistilled water. Five different concentrations of inhibitors
were selected in order to obtain inhibition of the enzymatic ac-
tivity comprised between 20% and 80%. Aliquots (50 mL) of
increasing concentration of inhibitor were added to the assay
solution and preincubated for 20 min at 37 ꢁC with the enzyme
followed by the addition of substrate. Assays were carried out
with a blank containing all components except AChE or BChE
in order to account for the non-enzymatic reaction. The reac-
tion rates were compared and the percent inhibition due to the
presence of increasing concentrations of inhibitor was calcu-
lated. Each concentration was analyzed in duplicate, and
IC50 values were determined graphically from log concentra-
tionepercent inhibition curves (GraphPad Prism 4.03 soft-
ware, GraphPad Software Inc.).
by Sutcliffe and co-workers [21] turned out to be an approach
that meets all of the criteria required for a robust clustering
protocol [16]. The poses reported in Fig. 2 for compound 1
is the one most representative of the most populated cluster
(28 poses) that, according to the Chauvenet criterion, was sta-
tistically populated. The 3D model of 1 was built using the
SYBYL (SYBYL 7.1, Tripos Inc., St. Louis, MO) standard
fragment library and then optimized at density functional level
of theory (B3LYP/6-31G*) by means of the Gaussian03 soft-
ware (Gaussian, Inc., Pittsburgh, PA, 2003).
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˚
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ber of islands was 5, the niche size was 2, migrate was 10, mu-
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generated by GOLD and then clusterized by means of AClAP
(v. 1.0) [16,17]. Briefly, AClAP is a newly developed cluster-
ing protocol implemented in a MATLAB metalanguage pro-
gram, which combines a hierarchical agglomerative cluster
analysis with a clusterability assessment method and a user in-
dependent cutting rule [17]. In particular, when applied to
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the average linkage rule with the cutting function developed
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