Cholinesterase inhibitors: Xanthostigmine derivatives blocking the acetylcholinesterase-induced β-amyloid aggregation

Article abstract of DOI:10.1021/jm049515h

In continuing research that led us to identify a new class of carbamate derivatives acting as potent (Rampa et al. J. Med. Chem. 1998, 41, 3976) and long-lasting (Rampa et al. J. Med. Chem. 2001, 44, 3810) acetylcholinesterase (AChE) inhibitors, we obtained some analogues able to simultaneously block both the catalytic and the β-amyloid (Aβ) proaggregatory activities of AChE. The key feature of these derivatives is a 2-arylidenebenzocycloalkanone moiety that provides the ability to bind at the AChE peripheral site responsible for promoting the Aβ aggregation. The new carbamates were tested in vitro for the inhibition of both cholinesterases and also for the ability to prevent the AChE-induced Aβ aggregation. All of the compounds had AChE IC ₅₀ values in the nanomolar range and showed the ability to block the AChE-induced Aβ aggregation, thus supporting the feasibility of this new strategy in the search of compounds for the treatment of Alzheimer's disease.

Full text of DOI:10.1021/jm049515h

4444
J. Med. Chem. 2005, 48, 4444-4456
Cholinesterase Inhibitors: Xanthostigmine Derivatives Blocking the
Acetylcholinesterase-Induced â-Amyloid Aggregation
Federica Belluti, Angela Rampa, Lorna Piazzi, Alessandra Bisi, Silvia Gobbi, Manuela Bartolini,
Vincenza Andrisano, Andrea Cavalli, Maurizio Recanatini,* and Piero Valenti
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received June 21, 2004
In continuing research that led us to identify a new class of carbamate derivatives acting as
potent (Rampa et al. J. Med. Chem. 1998, 41, 3976) and long-lasting (Rampa et al. J. Med.
Chem. 2001, 44, 3810) acetylcholinesterase (AChE) inhibitors, we obtained some analogues
able to simultaneously block both the catalytic and the â-amyloid (Aâ) proaggregatory activities
of AChE. The key feature of these derivatives is a 2-arylidenebenzocycloalkanone moiety that
provides the ability to bind at the AChE peripheral site responsible for promoting the Aâ
aggregation. The new carbamates were tested in vitro for the inhibition of both cholinesterases
and also for the ability to prevent the AChE-induced Aâ aggregation. All of the compounds
had AChE IC50 values in the nanomolar range and showed the ability to block the AChE-
induced Aâ aggregation, thus supporting the feasibility of this new strategy in the search of
compounds for the treatment of Alzheimer’s disease.
Introduction
several evidences point out a connection between these
phenomena and the cholinergic system. Actually, some
years ago, Nitsch showed that muscarinic receptors are
involved in the regulation of the metabolism of the Aâ
precursor protein (APP),4 while, recently, evidence of the
decrease of the total level of Aâ in the cerebrospinal fluid
of AD patients treated with a M1 muscarinic agonist was
reported.5 Another link between the amyloid and the
cholinergic hypotheses was provided by the discovery
of Inestrosa and co-workers that AChE is able to
promote the aggregation of Aâ.6 Several studies on this
topic have been carried out following the first report,
whose most interesting outcomes are that (a) the
peripheral anionic site (PAS) of AChE is involved in this
action and AChE inhibitors (AChEIs) targeted at the
PAS can prevent it7-9 and (b) the Aâ proaggregating
effect of AChE is operating also in vivo, as recently
shown by experiments carried out on transgenic mice.10
Alzheimer’s disease (AD), the most common cause of
dementia in the elderly, is a chronic, slowly progressive
disorder characterized by an impairment of intellectual
capacity. This syndrome is associated with widespread
neuronal loss and consequent altered neurotransmission
regarding several central neurotransmitter systems.
The most dramatic abnormalities are those involving
the central cholinergic system, thus supporting the
foundation of the so-called “cholinergic hypothesis” of
AD, which postulates that the cognitive decline ex-
perienced by AD patients is due to an extensive loss of
cholinergic neurons, especially in certain regions of the
brain as the neocortex, the hippocampus, and the
amygdala. The cholinergic hypothesis asserted that
drugs able to restore the CNS cholinergic tone would
slow the cognitive decline and formed the rationale for
the current therapeutic treatment of AD aimed at
controlling the symptoms of the disease.1,2
Presently, only a few AChEIs (i.e., tacrine, donepezil,
rivastigmine, and galantamine) are available for a
symptomatic treatment of AD patients, while several
different approaches (such as the use of neurotrophic
agents, glutamate antagonists, benzodiazepine receptor
ligands, calcium antagonists, antiinflammatory agents,
antioxidants, and compounds interfering with APP
processing) are being followed in the search for a
disease-modifying therapeutic strategy that still re-
mains elusive. In this situation, and considering also
the positive results obtained with the current attempts
of associating AChEIs and other drugs acting at a
different level in the AD pathological scenario,11,12 the
idea of multipotent compounds that combine the func-
tions needed to hit more than one pharmacological
target into one molecule can be considered a feasible
one. Actually, we and others have already shown that
it is possible to obtain compounds that show anti-
cholinesterase activity plus some other action relevant
in the neurodegenerative context of AD.13-16
On the other side, noticeable features of AD pathology
include intracellular neurofibrillary tangles and extra-
cellular neuritic plaques (or senile plaques, SP). Espe-
cially SP, dense deposits containing the Aâ as a primary
component, are localized in the midbrain region associ-
ated with cognition and memory. There is strong
evidence that an abnormal overproduction of Aâ, along
with its extracellular accumulation, is an early event
in the development of AD. All of this pathological,
biochemical, and genetic evidence has promoted the
“amyloid hypothesis” of AD pathogenesis, which states
that the production and the deposition of Aâ in the form
of fibrils lead to neuronal cell death and to the clinical
symptoms.3
However, even though the central role played by the
aggregation of Aâ as the major and early event in the
pathogenesis of the disease is generally recognized,
* Corresponding author: Prof. Maurizio Recanatini. Tel: +39 051
2099720. FAX: +39 051 2099734. E-mail: maurizio.recanatini@unibo.it.
10.1021/jm049515h CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/27/2005

Cholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4445
Chart 1
The three-dimensional structure of AChE has been
known for a long time, and also, on its basis, much
evidence has been provided about the existence of two
distinct binding sites for substrate and inhibitors: a
catalytic and an allosteric site, or peripheral binding site
(the PAS), localized at the bottom and the entrance of
the active-site gorge, respectively. As already men-
tioned, the PAS was proposed as a key player in the
process of accelerating Aâ fibril formation.8 Given these
assumptions, in previous papers, we reported the dis-
covery of both covalent and noncovalent AChEIs tar-
geted at both the catalytic and the peripheral sites of
AChE.17 In some of these studies, we carried out
structure-activity relationship (SAR) investigations
about a subset of compounds that allowed us to identify
some of the key structural features responsible for good
anti-AChE activity in terms of inhibitory potency and
selectivity (vs butyrylcholinesterase, BuChE). The most
suitable structural framework was composed by an
heteroaryl moiety, an N-methyl, N-alkoxy spacer chain,
and a methylcarbamoyloxybenzyl group defining the
class of [N-methyl-N-(3-methylcarbamoyloxyphenyl)-
methyl]aminoalkoxyeteroaryl derivatives. Among the
derivatives investigated, xanthostigmine (1) and its
seven-methylene analogue 2 (Chart 1) were found to be
potent AChEIs, providing the best results in terms of
inhibitory activity against isolated AChE (IC50 values
equal to 0.30 and 0.32 nM, respectively).18,19
aimed at characterizing the structural requirements
necessary to realize a good interaction with the hydro-
phobic environment of the PAS in addition to the AChE
inhibiting ability. The attention was then focused on the
heteroaryl moiety that should be able to establish an
interaction mainly with the indole ring of Trp279 (the
numbering of residues from the Torpedo californica
sequence will be used throughout this paper) and
consequently to prevent the Aâ proaggregating action
of AChE. For this purpose, our approach entailed the
replacement of the dibenzopyran-4-one moiety of com-
pounds 1 and 2 with groups characterized by greater
flexibility and space extension.
Given these premises, this study describes the design,
synthesis, in vitro evaluation, and SAR of a series of
2-arylidenebenzocycloalkanone analogues prepared with
the aim to target simultaneously both the catalytic and
the peripheral AChE binding sites. To allow the mol-
ecules to span all of the AChE gorge from the entrance,
where the PAS is located, to the catalytic cavity, the
following structural variations have been explored: (i)
insertion of an alkoxy spacer chain of suitable length
(three or seven methylene units); (ii) introduction of a
sterically encumbering π-electron-rich arylidene moiety,
also different substituents were inserted into the
arylidene aryl ring to increase the contact area with the
PAS (part A of Chart 1); (iii) variation in the size and
kind of cycloaliphatic ring to vary the relative positions
of different functional groups (part B of Chart 1); the
selected benzocycloalkanone moieties were benzofuran-
3-one, 3,4-dihydro-2H-naphthalen-1-one, chroman-4-
one, and indan-1-one.
As part of our ongoing project, 1 and 2 were chosen
for a more detailed investigation and were subjected to
structural modifications to further evaluate the SAR of
this class of compounds (Chart 1). When the drug design
strategy outlined above was taken into account, we

4446 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Belluti et al.
Scheme 1a
a Reagents and conditions: (i) X-(CH2)n-Br, K2CO3, acetone, reflux, 8 h; (ii) KI, methyl ethyl ketone, 8 h; (iii) Ar-CHO, (a) KOH,
EtOH, rt, 3 h and (b) Al2O3, CH2Cl2, rt, 3 h; (iv) 3-methylaminomethylphenol, toluene, reflux, 10 h; (v) methylisocyanate, NaH, toluene,
rt, 24 h.
Scheme 2a
Chemistry. The studied compounds were prepared
by means of an aldol condensation of the appropriate
benzocycloalkanone with the selected commercially
available aryl aldehydes. Different catalysts were em-
ployed according to reported methods: a basic medium
(KOH in ethanol),20 an acid one (gaseous HCl in etha-
nol),21 or, alternatively, an inorganic solid support
(neutral Al2O3 in dichloromethane).22 The adopted
synthetic approach was subordinated to the stability of
the intermediates. These condensations are known to
proceed almost stereoselectively providing a mixture of
geometric isomers, in which the trans isomer is present
in the greater amount.23,24 Further purification by
crystallization in a suitable solvent afforded the desired
pure geometrical isomer. In the case of the 2-arylidene-
benzofuran-3-one (aurone) set (9a-c and 10a-g), the
δ value of the exocyclic olefinic carbon determined by
13C NMR spectroscopy was around 113 ppm revealing
the Z configuration.25 For the derivatives 20-22, the E
geometry was verified through a nuclear Overhauser
effect experiment proving the absence of the coupling
relationship between the signals of the exocyclic olefinic
hydrogen and the adjacent methylene group.
a Reagents and conditions: (i) Br-(CH2)7-Br, K2CO3, acetone,
reflux, 8 h; (ii) 3,4,5-trimethoxybenzaldehyde, (a) KOH, EtOH, rt,
3 h and (b) HCl gas, EtOH, 0 °C, 1 h, rt, 18 h; (iii) 3-methyl-
aminomethylphenol, toluene, reflux, 10 h; (iv) methylisocyanate,
NaH, toluene, rt, 24 h.
The 2-arylidenebenzocycloalkanone derivatives were
synthesized following the general reactions depicted in
Schemes 1 and 2 using ω-haloalkoxybenzocycloalkanone
derivatives (3a, 4a, and 11-13) as key intermediates.
These were obtained by alkylation of phenolic benzo-
cycloalkanones, some of which are commercially avail-
able (5-hydroxyindan-1-one and 3,4-dihydro-2H-naph-
thalen-1-one), whereas others were previously syn-
thesized (6-hydroxybenzofuran-3-one26 and 7-hydroxy-
chroman-4-one27). 1-Chloro-3-bromopropane and 1,7-

Cholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4447
Scheme 3a
3-methylaminomethylphenol in refluxing toluene18 to
afford 7a-c, 8a-g, and 17-19; these derivatives were
subsequently treated with methylisocyanate to obtain
the desired final compounds 9a-c, 10a-g, and 20-22.
The synthesis of compound 27 was accomplished as
illustrated in Scheme 3. For reasons of chemical stability
of the intermediates, the following route was adopted:
1-(2,4-dihydroxyphenyl)ethanone was condensed with
1,7-dibromoheptane in the presence of K2CO3 to afford
the 7-bromoheptyloxy derivative 23 that was then
reacted with 3-methylaminomethylphenol in refluxing
toluene to afford 24. The phenolic intermediate was
treated with CuBr2 to afford the corresponding 2-bromo-
phenylethanone derivative 25 that was cyclized by
means of sodium acetate in hot water to obtain 26 that,
in turn, gave the desired compound 27 after treatment
with methylisocyanate.
a Reagents and conditions: (i) Br-(CH2)7-Br, K2CO3, acetone,
reflux, 8 h; (ii) 3-methylaminomethylphenol, toluene, reflux, 10
h; (iii) CuBr2, CHCl3, AcOEt, reflux, 10 h; (iv) sodium acetate, H2O,
reflux, 1 h; (v) methylisocyanate, NaH, toluene, rt, 24 h.
Biology. The biological profiles of the novel carbam-
ates listed in Table 1 against AChE (catalytic and Aâ
proaggregatory actions) and BuChE were determined.
The inhibitory potencies against recombinant human
AChE and isolated human BuChE were evaluated by
studying the hydrolysis of acetylthiocholine (ATCh) and
butyrylthiocholine (BuTCh), respectively, following the
method of Ellman.28 The IC50 values for inhibition of
both cholinesterases were determined to allow an
estimation of the eventual selectivity of the compounds.
The ability of all of the compounds to inhibit the
proaggregatory action of AChE toward Aâ was assessed
dibromoheptane were employed in the alkylation that
was achieved in hot acetone and in the presence of K2-
CO3. The ω-chloro- and ω-bromo-alkoxybenzofuran-3-
one intermediates (3 and 4) were converted into the
corresponding more chemically stable ω-iodoalkoxy
derivatives (3a and 4a) by treatment with NaI in
refluxing methyl ethyl ketone.
Key compounds 3a, 4a, and 11-13 reacted with the
selected arylaldehydes by the methods stated above to
afford the corresponding 2-arylidenebenzocycloalkanones
5a-c, 6a-g, and 14-16, which were then coupled with
Table 1. Inhibition of Human AChE and BuChE and Inhibition of Human AChE-Induced Aâ Aggregation by the Studied Compounds
IC50 (nM) (
SE AChE
IC50 (nM) (
SE BuChE
IC50 ratio
BuChE/AChE
% inhibition of Aâ(1-40)
aggregationa ( SE
compd
Ar
Z
n
C log Pb
1
3
7
3
3
3
7
7
7
7
7
7
7
7
7
7
7
7
0.30 ( 0.01c
0.32 ( 0.09d
2.99 ( 0.14
6.86 ( 0.56
3.41 ( 0.42
0.52 ( 0.02
2.76 ( 0.11
2.09 ( 0.30
79.1( 4.3
48 ( 4c
16.5 ( 1.4d
155 ( 10
44.3 ( 3.8
70.3 ( 10.5
136 ( 13
40.2 ( 1.0
74.8 ( 2.9
761 ( 4.3
57.9 ( 6.3
94.6 ( 5.8
130 ( 5
81.3 ( 8.3
153 ( 14
84.4 ( 7.7
4650 ( 540
11400 ( 600
23.1 ( 1.0c
160c
51.6d
51.8
6.45
20.6
261
14.5
35.5
9.6
29.7
11.8
2.91
45.2
15.6
17.1
0.09
7.08
1.6c
< 10
2
< 10
9a
a
b
c
a
b
c
d
e
f
g
a
a
a
b
O
O
O
O
O
O
O
O
O
O
29.2 ( 1.9
37.2 ( 1.2
35.0 ( 2.6
41.0 ( 6.3
66.7 ( 7.3
53.3 ( 4.8
65.3 ( 3.2
43.3 ( 5.7
49.1 ( 3.4
47.6 ( 7.2
32.8 ( 1.7
45.3 ( 0.8
33.3 ( 2.5
49.5 ( 2.9
17.5 ( 4.2
30 ( 3f
9b
9c
10a
10b
10c
10d
10e
10f
10g
20
3.38
5.25
5.25
6.43
4.08
5.38
5.50
3.12
3.08
3.68
1.95 ( 0.12
8.00 ( 0.57
44.6 ( 2.1
CH2
CH2O
CH2CH2
O
1.80 ( 0.57
9.81 ( 0.12
4.94 ( 0.25
49600 ( 400
1610 ( 70
21
22
8be
27
O
Phys
14.1 ( 0.2c
a Determined at [inhibitor] ) 100 µM, [Aâ(1-40)] ) 230 µM, and [AChE] ) 2.3 µM. b Calculated31 for the 2-arylidenebenzocycloalkanone
fragments combining the Ar and Z features shown in the general structure. c From ref 18. d From ref 19. e No carbamate, see Scheme 1
for the structure. f From ref 9.

4448 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Belluti et al.
by means of a thioflavin T-based fluorometric assay
early reported by Inestrosa7 and recently validated by
us through circular dichroism.9 By this method, the
aggregation of Aâ can be evaluated in vitro based on
the determination of the fluorescence emission of thio-
flavin T. This yellow dye binds to amyloid fibrils giving
rise to an intense specific emission band (λmax ) 490
nm) in its fluorescent spectrum. Under the appropriate
conditions of pH, buffer type, and concentration, the
AChE-induced Aâ fibril formation can be monitored,
while minimizing the spontaneous Aâ aggregation. After
incubation of an Aâ solution with AChE and the
inhibitor, the fluorescence intensities were compared to
a blank lacking the test compound and the percent
inhibition due to the presence of the inhibitor was
calculated.
Figure 1. Overlaid time courses of fluorescence intensity (IF,
λem ) 490 nm with λexc ) 446 nm) related to (a) Aâ, (b) Aâ
plus compound 10d, (c) Aâ and AChE, and (d) Aâ and AChE
plus compound 10d samples incubated for 24 h in phosphate
buffer 0.215 M, pH 8.0, at room temperature. Samples were
diluted with 2.0 mL of 1.5 µM thioflavin T in 50 mM glycine-
NaOH buffer, pH 8.5, for the evaluation of the fluorescence
intensities.
Results
The inhibitory activities against both AChE and
BuChE of the ω-[N-methyl-N-(3-methylcarbamoyloxy-
phenyl)methyl]-aminoalkoxyaryl derivatives together
with those of the reference compounds 1, 2, and phys-
ostigmine are reported in Table 1, expressed as IC50
values. As we already reported in previous papers,18,19
the carbamate analogues show a time-dependent pat-
tern of inhibition, strongly dependent on the bulkiness
of the N-carbamic substituent. Here, we did not deter-
mine the value of the carbamoylation constants for all
of the compounds but verified that even in this series
the maximum inhibition could be reached within the
incubation time with the two enzymes. Under these
conditions, the IC50 values are indicative of the potency
of inhibition and the risk of potency underestimation is
overcome.
The data regarding the subset of compounds 9a-c
and 10a-c allowed us to examine the consequence of
changing the N-alkoxy spacer chain and, particularly,
the introduction of a three-methylene and a seven-
methylene alkyl spacer: lengthening the chain from
three to seven methylene units slightly affected the
AChE inhibitory activity. As a matter of fact, only for
10a, an improvement of potency of 1 order of magnitude
over the average value for the subset was noticed.
With regards to the 2-arylidenebenzocycloalkanone
moiety, it emerged that the presence of bulky arylidene
groups was of pivotal importance for good AChE inhibi-
tory potency, as compound 27, lacking this portion, was
a very weak AChEI (IC50 ) 1.61 ( 0.07 µM). All of the
compounds bearing a seven-unit methylene chain (10a-
g, and 20-22) showed a nanomolar potency for inhibit-
ing AChE, except for 10d and 10g. On the other hand,
the inhibitory action against BuChE was more variable
within the series, always remaining 1 or 2 orders of
magnitude lower than that against AChE. The IC50
values for the AChE and BuChE inhibition by 10a (0.52
( 0.02 and 136 ( 13 nM, respectively, IC50 ratio of
BuChE/AChE ) 261) show that this compound is a
potent, and rather selective, AChEI.
AChE inhibitory potency was further diminished and
the same detrimental effect was noticed upon the
insertion of the anthracene ring as in 10d.
With reference to the cycloalkanone ring of 10a, we
also explored the replacement of the oxygen atom of the
furanone nucleus by synthesizing other cyclic fragments
as the cyclopentanone, pyranone, and cyclohexanone
analogues 20-22. These structural changes negatively
affected the AChE inhibiting potency of the compounds
that was lowered by 1 order of magnitude with respect
to compound 10a.
All of the compounds of Table 1 were also tested for
their action against Aâ aggregation by determining their
ability to inhibit the AChE-induced Aâ fibril formation.
The percentage of inhibition of the AChE-induced
fibrillogenesis for each compound at 100 µM is reported.
In Figure 1, the fluorometric thioflavin T test results
for compound 10d are illustrated; four fluorometric plots
are overlaid, related to the monitoring of fluorescence
emission of thioflavin T at λem ) 490 nm with λexc
)
446 nm, which reveals Aâ fibril formation in the
presence (c) and in the absence (a) of AChE and in the
presence of AChE and inhibitor (d). The time-dependent
loss of fluorescent signal shown in Figure 1 was already
reported by LeVine29 and was explained in terms of a
decrease in the number of dye molecules bound or a
change in the fluorescent yield of the bound dye
molecules. Thioflavin T binds also to AChE,30 and
therefore the related fluorescence emission at the same
wavelength was subtracted in each sample to obtain
only the fluorescence contribution due to the formation
of Aâ fibrils.
It may be noticed that the inhibitor concentration in
the aggregation assay (100 µM) is much higher than the
IC50 values of the same compounds (in the range of nM,
see Table 1). As we pointed out before,9 this is related
to the higher AChE and Aâ concentrations that are
necessary to speed the aggregation process up to an
extent suitable for analytical purposes. Nevertheless,
these high concentration values should be “normalized”
in order to compare the inhibition data shown in Table
1. Specifically, it should be considered that the amount
of enzyme used in the antiaggregation assay is 1.2 ×
Considering the arylidene moiety of the compounds,
replacing the 3,4,5-trimethoxyphenyl group of 10a with
R- or â-naphthalenyl, anthracenyl, phenyl, or dichloro-
phenyl fragments (10b-g) weakened the AChE inhibi-
tory activity of the resulting derivatives. Moreover, in
the case of the 3,5-dichlorophenyl analogue (10g), the

Cholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4449
105 times higher than the amount used in Ellman’s
assay, and the amount of inhibitor is proportionally
higher. As a consequence, taking the inhibitor/AChE
concentration ratio, it results in the same magnitude
for both the Ellman’s and the antiaggregation assays
([inhibitor]/[AChE] ) ∼50, if an IC50 value in the
nanomolar range is considered). In this respect, it seems
plausible that similar amounts of inhibitor can carry
out simultaneously both the anticholinesterasic and the
antiaggregating actions.
By observation of the data reported in Table 1, it
emerges that all of the carbamates except for 1 and 2
show a significant reduction of the Aâ aggregation
induced by AChE. When the subsets 9a-c and 10a-c
are compared, it appears that the seven-methylene
chain favors the antiaggregating action, likely because
it allows a better positioning of the large aromatic
portion onto the Aâ-binding site. The most potent agents
are 10b and 10d, which bear a â-naphthalene and an
anthracene nucleus, respectively, in the arylidene por-
tion. The compounds bearing a benzylidene fragment
(9a, 10a, and 10e-g), as well as those replacing the
furanone ring with different cyclic moieties (20-22),
were somewhat weaker inhibitors of AChE-induced Aâ
fibril formation. Interestingly, two compounds (27 and
8b) each lacking one of the important structural fea-
tures of this series (the arylidene moiety is missing in
27, and 8b bears no carbamate function) showed op-
posite behaviors, the first one being much less potent
than the second one in blocking the AChE-induced Aâ
aggregation. Finally, all of the tested compounds (except
for 9a and 27) showed a greater potency in blocking the
Aâ aggregation promoted by AChE than the natural
lead physostigmine, while our lead 2 showed only a
marginal reduction of Aâ fibril formation.
Figure 2. Hypothetical binding mode of the covalent adduct
between 10b (green) and the AChE active site gorge (only a
few amino acid residues are shown). The model was obtained
by following the procedure described in ref 18 for inhibitor
docking and complex optimization.
Considering the molecular features responsible for the
dual activity, in a previous work, we had already shown
that the co-presence in 2 of an N-methyl carbamic group,
a dibenzopyran-4-one nucleus, and a seven-methylene
spacer was an optimal characteristic for blocking the
AChE catalytic activity.19 However, surprisingly, this
compound showed only marginal activity against the
AChE-induced Aâ aggregation (Table 1). Here, we
demonstrate that the latter action can be obtained if
the heteroaromatic fragment is replaced with a struc-
turally more complex group (2-arylidenebenzocyclo-
alkanone) like in the novel derivatives. The most evident
feature of these compounds is the increased extension
of the conjugated π-system constituted by the benzo-
cycloalkanone nucleus connected through a double bond
to an aromatic (poly)cycle. Although the planar conju-
gated structure might play a specific role in interfering
with the Aâ/AChE interaction at the PAS, a merely
lipophilic effect of the whole fragment cannot be ruled
out. In fact, the calculated31 log P values (Table 1) of
the 2-arylidenebenzocycloalkanone parts of the mol-
ecules (combining the Ar and Z features of the general
structure reported in Chart 1) are roughly correlated
(r2 ) 0.64) with the percent values of inhibition of Aâ
aggregation for the seven-methylene derivatives 10a-
g, 20-22, and 8b. In this regard, it is quite remarkable
that compound 8b, lacking the carbamate function and
being a very weak AChE inhibitor, fits well this cor-
relation, confirming that the block of the AChE-induced
effect on Aâ is independent from the action of the
inhibitor at the catalytic site of the enzyme. This can
be an interesting result considering the rather poor
druglike properties of this class of compounds. Actually,
carbamic AChEIs (like physostigmine) show rather
rapid metabolic degradation, and the elimination of the
carbamate function with retention of the blocking action
toward AChE-induced Aâ aggregation as in 8b might
provide a way to circumvent the likely nonoptimal
pharmacokinetic characteristics of the xanthostigmine
derivatives.
Discussion
The new carbamates presented in this paper (9a-c,
10a-g, 20-22, and 27) were prepared to further explore
our previously proposed drug design strategy16,17,19
aimed at obtaining potent AChEIs that were able also
to prevent the AChE-induced Aâ aggregation. A suitable
starting point was identified in xanthostigmine (1),18
a
compound that includes in its structure two functions
of known ability to bind the catalytic site of AChE (the
carbamic group and the N-methyl, N-benzylaminic
moiety), as well as an heteroaromatic nucleus capable,
if properly spaced, of binding to the PAS at the mouth
of the AChE gorge. The data reported in Table 1 indicate
that indeed we succeeded in obtaining new carbamates
displaying a good potency in blocking both the catalytic
and the Aâ proaggregating actions of AChE: compound
10b represents the best combination of the two actions,
even though 10a is more potent against the AChE
catalytic activity, and 10d, while being a less potent
AChEI, has a comparable potency against the induction
of Aâ aggregation. Of course, the relevance of these
results has to be referred to the in vitro system, where
the AChE-induced Aâ aggregation is tested: the inhibi-
tor concentration used in our assay system (100 µM) is
rather high, but it derives from the need to use a high
concentration of AChE to induce the Aâ aggregation in
an experimentally suitable time.9 Experiments are
planned to assess the in vivo relevance of this effect.
A possible AChE binding mode for 10b is shown in
Figure 2, where the numerous possible interactions
between the enzyme and the inhibitor are evident. The
covalent adduct is displayed with the carbamic C of 10b
bound to the Ser200 Oγ; other favorable interactions of
the benzylaminic part of this type of carbamic deriva-
tives with the AChE catalytic site are described in detail

4450 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Belluti et al.
in a previous paper.18 The seven-methylene chain of 10b
allows the 2-arylidenebenzocycloalkanone moiety to
interact with the PAS, particularly with the key Trp279
side chain. Moreover, the ethereal O of the spacer chain
of 10b and the OH group of Tyr121 are in the right
position to form an H-bond.
From the hypothetical binding mode here outlined,
the only specific interaction between 10b and the PAS
seems to be that involving the 2-naphthylidenebenzo-
furanone moiety and the indolyl side chain of Trp279,
which show a favorable geometry for a “parallel stacked
and displaced”32 aromatic-aromatic interaction; the
ring center-ring center distance is 4.4 Å (theoretical
4.5 Å32). The great ability (much greater than that of 2,
for example) of this extended π-electron-rich terminal
of 10b to mask the Aâ-binding site at the PAS might
be one reason for the efficacy of this inhibitor in
preventing the AChE-promoted Aâ aggregation.
Certainly, the binding mode depicted in Figure 2
might not be the only one for 10b, as the complexity of
the interaction between carbamates and the AChE
gorge can suggest other possibilities. In attempting an
interpretation of the SAR for the AChE-mediated Aâ
antiaggregating action of the reported compounds, it
should be observed that the percent inhibition values
were determined in a test system requiring high con-
centrations of AChE, Aâ, and inhibitor. However, since
the inhibitor/AChE concentration ratio was comparable
to that reached in Ellman’s assay, the binding mecha-
nism might not have been affected by low affinity and
nonspecific interactions other than those also occurring
in the IC50 assay. Actually, the lack of correlation
between the inhibition of catalytic and aggregating
actions might be indicative of the involvement of non-
catalytic interactions in the AChE binding mechanism
at a higher concentration. Moreover, the prototypic
carbamic AChEI, physostigmine, while not presenting
any extended π-electron-rich terminals, still showed a
30% inhibition of the Aâ proaggregatory action of AChE;
this might be eventually due to the formation of a
transient enzyme-inhibitor intermediate involving the
PAS.33 Differences in the dual activity profile among the
various inhibitors tested might thus arise from contri-
butions of different interaction modes to the overall
binding processes.
In a recent paper on an AChE dual function inhibitor,
we advanced the hypothesis that AChE/BuChE selectiv-
ity might be correlated with the ability to block the
AChE-induced Aâ aggregation, suggesting this property
is an indicator for the activity targeted at Aâ.16 How-
ever, this hypothesis can be valid only for noncovalent
inhibitors, which show a noncompetitive or a mixed
mode of inhibition when they are more strongly bound
to the PAS (first affinity binding site) than to the
catalytic site. In fact, one of the peculiar structural
differences between the two cholinesterase enzymes is
that the PAS is missing in BuChE.34 Conversely, for
covalent inhibitors such as the carbamates described
here, the highest affinity binding site is the catalytic
site where the carbamate group interacts with the
nucleophilic serine leading to the covalent adduct. In
this case, the PAS constitutes a secondary site where
the inhibitor terminal moiety can interact leading to the
inhibition of AChE-promoted Aâ aggregation, while the
driving force for the formation of the covalent bond is
the carbamate affinity for the catalytic site. The stability
of the carbamate/cholinesterase covalent complex can
therefore affect the potency of inhibition and give rise
to an AChE/BuChE selectivity ratio, which is not
influenced by the interaction of the inhibitor with the
AChE PAS. Actually, we have previously found that
compounds of greater dimensions or lipophilicity can be
better accommodated in the spatially wider BuChE
active site gorge showing a reversed selectivity.19 In-
deed, though, this issue requires further study as
conflicting evidence was reported by Guillozet et al.,35
who found that BuChE associated with Aâ in the early
phases of plaque formation. This should also remind us
that BuChE still deserves a lot of consideration as a
viable AD target as recent studies clearly pointed out.36
As a final remark, a comment is deserved on the
perspective AD therapeutic relevance of AChEIs, par-
ticularly those able to block the AChE-induced Aâ
aggregation. This noncatalytic action of AChE was
discovered by Inestrosa some years ago from in vitro
experiments,6 and reports on the pathological relevance
of this effect in vivo (animal models) are starting to
appear.10,37 Certainly, the biological data available today
(included those presented in this paper) may give rise
to some concerns about the potential therapeutic useful-
ness of the inhibition of AChE-induced Aâ proaggregat-
ing effect. For instance, the concentrations of AChE, Aâ,
and inhibitor used in the test system to assess the
inhibitors’ action against the Aâ aggregation are high
and not comparable with the corresponding ones in the
brain. Particularly, the inhibitor concentration used in
the antiaggregation assays is quite different from the
AChE IC50 values, which might lead one to conclude
that the dual effect cannot occur in the physiological
system. As discussed above however, the inhibitor/AChE
concentration ratio is comparable to that occurring in
Ellman’s inhibition assay, and this observation might
bring back a more correct perspective of the data on the
block of the AChE-induced Aâ aggregation. Indeed, it
might be of some concern that the AChE concentration
in the AChE-induced Aâ aggregation assay is nearly 100
times higher than that in vivo (AChE is present in the
different areas of the CNS at concentrations ranging
from 4 to 45 units per gram of tissue38); however it was
found to be necessary to maximize a phenomenon which
requires quite a longer time to fully develop.9 This is
an issue that definitely requires in-depth investigation
to obtain an accurate in vitro model system more closely
resembling the AD brain conditions.
Probably it is too early to retain the Aâ proaggregat-
ing action of AChE as a validated AD target and
extreme caution is required in assessing the potential
therapeutic relevance of blocking this effect, but efforts
in this field seem to be justified by the perspective to
obtain lead candidates covering a spectrum of thera-
peutically useful actions. Moreover, as biological studies
on AChE (and BuChE) proceed, the role of cholinest-
erases inhibitors in AD pharmacology seems to (re)gain
interest, despite the nonoptimal results in the clinic. The
in vitro action of AChEIs toward the AChE-induced Aâ
aggregation together with the evidences of the increase
of nonamyloidogenic APP processing by AChEIs,39,40 and
of the benefits derived by the concomitant inhibition of

Cholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4451
both AChE and BuChE,41 may be indicating new
avenues for those involved in the design and experi-
mental study of cholinesterases inhibitors.
NaI (0.7 g, 4.6 mmol), and the resulting mixture was stirred
under reflux for 8 h. After the mixture was cooled, the solvent
was removed under reduced pressure and the crude residue
was dissolved in dichlorometane (25 mL) and washed with
water (25 mL). The organic layer was dried over sodium sulfate
anhydrous, filtered, and concentrated under reduced pressure.
The semisolid residue was crystallized from toluene to give a
yellow, crystalline compound (1.20 g, 90% yield), mp 115-118
Experimental Section
Chemistry. Starting materials unless otherwise specified
in the Experimental Section were used as high-grade com-
mercial products. All of the melting points were determined
in open glass capillaries, using a Bu¨chi apparatus, and are
uncorrected. Proton NMR spectra were recorded on Varian
Gemini 200 and 300 spectrometers, and chemical shifts are
reported as δ value relative to tetramethylsilane as an internal
standard. Mass spectra were recorded on a Waters ZQ 4000
apparatus operating in electrospray (ES) mode. Wherever
analyses are only indicated with element symbols, analytical
results obtained for those elements are within 0.4% of the
theoretical values.
6-(3-Chloropropoxy)benzofuran-3-one (3). A stirred sus-
pension of 6-hydroxybenzofuran-3-one (5.0 g, 33 mmol), 1-bromo-
3-chloropropane (5.49 g, 33 mmol), and potassium carbonate
(4.6 g, 33 mmol) in dry acetone (150 mL) was refluxed for 8 h.
The hot reaction mixture was filtered, and the solvent was
evaporated under reduced pressure. Crystallization of the
residue from toluene afforded 3 as a white solid (5.5 g, 95%
yield), mp 75-77 °C. 1H NMR (CDCl3, δ): 2.22-2.39 (m, 2H),
3.78 (t, 2H, J ) 6.6 Hz), 4.22 (t, 2H, J ) 6.6 Hz), 4.66 (s, 2H),
6.55 (d, 1H, J ) 1.8 Hz), 6.64 (dd, 1H, J ) 1.8 and 8.4 Hz),
7.59 (d, 1H, J ) 8.4 Hz). Anal. (C11H11ClO3) C, H.
6-(7-Bromoheptyloxy)benzofuran-3-one (4). Following
the previous procedure and starting from 6-hydroxybenzo-
furan-3-one (5.0 g, 33 mmol) and 1,7-dibromoheptane (8.5 g,
33 mmol), 4 was prepared (95% yield), mp 53-54 °C (toluene).
1H NMR (CDCl3, δ): 1.31-1.68 (m, 6H), 1.75-1.95 (m, 4H),
3.41 (t, 2H, J ) 6.6 Hz), 4.01 (t, 2H, J ) 6.6 Hz), 4.61 (s, 2H),
6.51 (d, 1H, J ) 1.8 Hz), 6.64 (dd, 1H, J ) 1.8 and 8.4 Hz),
7.54 (d, 1H, J ) 8.4 Hz). Anal. (C15H19BrO3) C, H.
5-(7-Bromoheptyloxy)indan-1-one (11). Following the
previous procedure and starting from 5-hydroxyindan-1-one
(4.9 g, 33 mmol), 11 was prepared (70% yield), mp 38-40 °C
(toluene). 1H NMR (CDCl3, δ): 1.23-1.90 (m, 10H), 2.43 (t,
2H, J ) 6.6 Hz), 2.98 (t, 2H, J ) 6.6 Hz), 3.30 (t, 2H, J ) 6.6
Hz), 4.00 (t, 2H, J ) 6.6 Hz), 6.56-6.68 (m, 2H), 7.85-7.98
(m, 1H). Anal. (C16H21BrO2) C, H.
1
°C. H NMR (CDCl3, δ): 2.22-2.36 (m, 2H), 3.36 (t, 2H, J )
6.6 Hz), 4.11 (t, 2H, J ) 6.6 Hz), 4.62 (s, 2H), 6.55 (d, 1H, J )
1.8 Hz), 6.64 (dd, 1H, J ) 1.8 and 8.4 Hz), 7.60 (d, 1H, J ) 8.4
Hz). Anal. (C11H11IO3) C, H.
6-(7-Iodoheptyloxy)benzofuran-3-one (4a). Following
the previous procedure and starting from 4, 4a was prepared
(94% yield), mp 67-69 °C (toluene). 1H NMR (CDCl3, δ): 1.30-
1.53 (m, 6H), 1.73-1.95 (m, 4H), 3.21 (t, 2H, J ) 6.6 Hz), 4.03
(t, 2H, J ) 6.6 Hz), 4.63 (s, 2H), 6.53 (d, 1H, J ) 1.8 Hz), 6.68
(dd, 1H, J ) 1.8 and 8.4 Hz,), 7.57 (d, 1H, J ) 8.4 Hz). Anal.
(C15H19IO3) C, H.
(Z)-6-(3-Iodopropoxy)-2-(3,4,5-trimethoxybenzylidene)-
benzofuran-3-one (5a). Sodium hydroxide (0.1 g, 4.2 mmol)
was added to a solution of 3a (1.00 g, 4.2 mmol) and 3,4,5-
trimethoxybenzaldehyde (0.82 g, 4.2 mmol) in ethanol (10 mL).
The reaction mixture was stirred for 3 h at room temperature.
The precipitated was collected by filtration and crystallized
from ethanol to afford the pure 5a as a yellow, crystalline
compound (0.94 g, 59% yield), mp 142-144 °C. 1H NMR
(CDCl3, δ): 2.24-2.29 (m, 2H), 3.36 (t, 2H, J ) 6.6 Hz), 3.88
(s, 3H), 3.92 (s, 6H), 4.11 (t, 2H, J ) 6.6 Hz), 6.71-6.81 (m,
3H), 7.17 (s, 2H,), 7.70 (d, 1H, J ) 8.4 Hz). 13C NMR (CDCl3)
δ 7.27, 34.89, 53.35, 56.3, 56.6, 108.85, 113.92, 116.90, 126.06,
128.74, 129.21, 131.00, 132.42, 148.55, 149.36, 158.08, 165.32,
187.98. Anal. (C21H21IO6) C, H.
(Z)-6-(3-Iodopropoxy)-2-naphthalen-1-ylmethyleneben-
zofuran-3-one (5b). Following the previous procedure and
starting from 3a (1.00 g, 3.16 mmol) and 1-naphthaldehyde
(0.49 g, 3.16 mmol), 5b was prepared (59% yield), mp 142-
144 °C (toluene). 1H NMR (CDCl3, δ): 2.30-2.36 (m, 2H), 3.87
(t, 2H, J ) 6.6 Hz), 4.18 (t, 2H, J ) 6.6 Hz), 6.77-6.8 (m, 2H),
7.48-7.66 (m, 4H), 7.76 (d, 1H, J ) 8.4 Hz), 7.87-7.94 (m,
2H), 8.32 (d, 1H, J ) 8.4 Hz), 8.41 (d, 1H, J ) 8.4 Hz). 13C
NMR (CDCl3) δ 7.1, 34.33, 60.15, 97.99, 112.5, 114.44, 116.89,
123.44, 123.77, 125.54, 125.67, 125.81, 125.99, 128.11, 128.55,
130.76, 133.45, 135.55, 146.76, 165.55, 168.45, 182.33. Anal.
(C22H17IO3) C, H.
(Z)-6-(3-Iodopropoxy)-2-naphthalen-2-ylmethyleneben-
zofuran-3-one (5c). Following the previous procedure and
starting from 3a (1.00 g, 3.16 mmol) and 2-naphthaldehyde
(0.49 g, 3.16 mmol), 5c was prepared (84% yield), mp 115-
118 °C (toluene). 1H NMR (CDCl3, δ): 2.23-2.35 (m, 2H), 3.39
(t, 2H, J ) 6.6 Hz), 4.18 (t, 2H, J ) 6.6 Hz), 6.77 (dd, 1H, J )
1.8 and 8.4 Hz), 6.84 (d, 1H, J ) 1.8 Hz), 6.98 (s, 1H), 7.51-
7.54 (m, 2H), 7.73 (d, 1H, J ) 8.4 Hz), 7.83-7.92 (m, 3H,),
8.06 (dd, 1H, J ) 1.8 and 8.4 Hz,), 8.30 (s, 1H). 13C NMR
(CDCl3) δ 7.1, 34.33, 66.15, 95.59, 108.5, 114.44, 115.89, 123.45,
125.77, 125.80, 126.54, 126.67, 127.99, 128.0, 128.15, 130.76,
134.45, 146.55, 168.55, 165.45, 182.03. Anal. (C22H17IO3) C,
H.
7-(7-Bromoheptyloxy)chroman-4-one (12). Following
the previous procedure and starting from 7-hydroxychroman-
4-one (5.4 g, 33 mmol), 12 was prepared (63% yield) as a
colorless oil purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate 9:1). 1H NMR (CDCl3, δ): 1.23-
1.90 (m, 10H), 2.68-2.90 (m, 2H), 3.30 (t, 2H, J ) 6.6 Hz),
3.99 (t, 2H, J ) 6.6 Hz), 4.40 (t, 2H, J ) 6.6 Hz), 6.50 (s, 1H),
6.70 (dd, 1H, J ) 2.2 and 8.4 Hz), 7.76 (d, 1H, J ) 8.4 Hz).
Anal. (C16H21BrO3) C, H.
6-(7-Bromoheptyloxy)-3,4-dihydro-2H-naphthalen-1-
one (13). Following the previous procedure and starting from
6-hydroxy-3,4-dihydro-2H-naphthalen-1-one (5.35 g, 33 mmol),
13 was prepared (90% yield) as a colorless oil purified by flash
chromatography on silica gel (petroleum ether/ethyl acetate
1
9:1). H NMR (CDCl3, δ): 1.38-1.52 (m, 6H), 1.76-1.88 (m,
(Z)-6-(7-Iodoheptyloxy)-2-(3,4,5-trimethoxybenzylidene)-
benzofuran-3-one (6a). Following the previous procedure and
starting from 4a (0.8 g, 2.1 mmol) and 3,4,5-trimethoxybenz-
aldehyde (0.41 g, 2.1 mmol), 6a was prepared (95% yield), mp
4H), 2.08-2.19 (m, 2H), 2.61 (t, 2H, J ) 6.8 Hz), 2.92 (t, 2H,
J ) 6.8 Hz), 3.42 (t, 2H, J ) 7.0 Hz), 4.01 (t, 2H, J ) 7.0 Hz),
6.69 (d, 1H, J ) 2.2 Hz), 6.81 (dd, 1H, J ) 2.2 and 8.8 Hz),
7.99 (d, 1H, J ) 8.8 Hz). Anal. (C17H23BrO2) C, H.
1
94-96 °C (ethanol). H NMR (CDCl3, δ): 1.38-1.49 (m, 6H),
1-[4-(7-Bromoheptyloxy)-2-hydroxyphenyl]ethanone
(23). Following the previous procedure and starting from
1-(2,4-dihydroxyphenyl)-ethanone (2.5 g, 16.5 mmol), 23 was
prepared (90% yield) as a colorless oil purified by flash
chromatography on silica gel (petroleum ether/ethyl acetate
1.80-1.88 (m, 4H), 3.31 (t, 2H, J ) 6.6 Hz), 3.92 (s, 3H), 3.95
(s, 6H), 4.09 (t, 2H, J ) 6.6 Hz), 6.71 (d, 1H, J ) 1.8 Hz), 6.76
(d, 1H, J ) 8.4 Hz), 7.17 (m, 3H), 7.70 (d, 1H, J ) 8.4 Hz). 13
C
NMR (CDCl3) δ 7.27, 25.77, 28.28, 28.85, 30.33, 34.89, 52.35,
56.35, 57.67, 108.88, 113.82, 116.99, 126.16, 128.84, 129.27,
131.32, 132.42, 148.55, 149.36, 158.08, 165.82, 187.62. Anal.
(C25H29IO6) C, H.
1
9:1). H NMR (CDCl3, δ): 1.31-1.68 (m, 6H), 1.75-1.95 (m,
4H), 2.50 (s, 3H), 3.44 (t, 2H, J ) 6.6 Hz), 4.06 (t, 2H, J ) 6.6
Hz), 4.66 (s, 2H), 6.56 (d, 1H, J ) 1.8 Hz), 6.69 (dd, 1H, J )
1.8 and 8.4 Hz), 7.54 (d, 1H, J ) 8.4 Hz). Anal.(C15H21BrO3)
C, H.
(Z)-6-(7-Iodoheptyloxy)-2-naphthalen-1-ylmethylene-
benzofuran-3-one (6b). Following the previous procedure
and starting from 4a (0.5 g, 1.3 mmol) and 1-naphthaldehyde
(0.2 g, 1.3 mmol), 6b was prepared (50% yield), mp 84-85 °C
6-(3-Iodopropoxy)benzofuran-3-one (3a). A solution of
3 (1,0 g, 4.2 mmol) in butan-2-one (20 mL) was treated with

4452 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Belluti et al.
(ethanol). 1H NMR (CDCl3) δ 1.37-1.54 (m, 6H), 1.83-1.88
(m, 4H), 3.55 (t, 2H, J ) 6.6 Hz), 4.15 (t, 2H, J ) 6.6 Hz),
6.77-6.8 (m, 2H), 7.48-7.66 (m, 4H), 7.76 (d, 1H, J ) 8.4 Hz),
7.87-7.94 (m, 2H), 8.32 (d, 1H, J ) 8.4 Hz), 8.41 (d, 1H, J )
8.4 Hz). 13C NMR (CDCl3) δ 7.84, 26.67, 28.28, 29.15, 30.33,
31.89, 34.0, 72.3, 96.6, 112.21, 114.52, 115.09, 123.16, 123.27,
125.15, 125.42, 128.32, 128.80, 130.55, 133.08, 135.82, 138.54,
146.11, 149.36, 152.98, 182.62. Anal. (C26H25IO3) C, H.
(Z)-6-(7-Iodoheptyloxy)-2-naphthalen-2-ylmethylene-
benzofuran-3-one (6c). Following the previous procedure and
starting from 4a (0.5 g, 1.3 mmol) and 2-naphthaldehyde (0.2.
g, 1.3 mmol), 6c was prepared (50% yield), mp 84-85 °C
(ethanol). 1H NMR (CDCl3) δ 1.39-1.53 (m, 6H), 1.83-1.87
(m, 4H), 3.22 (t, 2H, J ) 6.6 Hz), 4.11 (t, 2H, J ) 6.6 Hz),
6.75-6.84 (m, 2H), 6.99 (s, 1H), 7.52-7.56 (m, 2H), 7.76 (d,
1H, J ) 8.4 Hz), 7.82-7.92 (m, 3H), 8.06 (dd, 1H, J ) 1.8 Hz,
and 8.4 Hz), 8.30 (s, 1H). 13C NMR (CDCl3) δ 1.04, 7.10, 25.77,
28.23, 28.85, 30.33, 32.89, 68.3, 97.6, 111.93, 112.52, 114.09,
123.75, 125.16, 125.75, 126.22, 126.25, 126.85, 128.12, 128.70,
133.36, 133.55, 134.08, 146.11, 153.54, 158.52, 182.62. Anal.
(C26H25IO3) C, H.
(Z)-2-Anthracen-9-ylmethylene-6-(7-iodoheptyloxy)-
benzofuran-3-one (6d). Following the previous procedure
and starting from 4a (0.5 g, 1.3 mmol) and anthracen-9-
carbaldehyde (0.26 g, 1.3 mmol), 6d was prepared (50% yield),
mp 156-158 °C. 1H NMR (CDCl3) δ 1.25-1.50 (m, 6H), 1.70-
1.90 (m, 4H), 3.18 (t, 2H, J ) 7.0 Hz), 3.93 (t, 2H, J ) 7.0 Hz),
6.45 (d, 1H, J ) 2.2 Hz), 6.86 (dd, 1H, J ) 2.2 and 8.4 Hz),
7.49-7.55 (m, 4H), 7.77 (d, 1H, J ) 8.4 Hz), 7.79 (s, 1H), 8.08
(m, 4H), 8.53 (s, 1H). 13C NMR (CDCl3) δ 7.27, 25.89, 28.35,
28.90, 30.49, 33.51, 68.90, 97.06, 108.85, 112.92, 114.99,
125.51, 126.06, 126.34, 128.74, 128.96, 129.97, 131.36, 150.08,
167.32, 168.98, 186.51. Anal. (C30H27IO3) C, H.
(Z)-2-Benzylidene-6-(7-iodoheptyloxy)benzofuran-3-
one (6e). Neutral Al2O3 was added to a solution of 4a (1.00 g,
2.67 mmol) and benzaldehyde (0.42 g, 4.0 mmol) in CH2Cl2 (6
mL). The reaction mixture was stirred at room temperature
for 3 h. The solid was removed by filtration and washed with
CH2Cl2 the solvent was evaporated under reduced pressure,
and the residue was crystallized from ethanol and afforded
the pure 6e as a yellow, crystalline compound (24% yield), mp
114-116 °C. 1H NMR (CDCl3) δ 1.28-1.39 (m, 6H), 1.79-1.96
(m, 4H), 3.20 (t, 2H, J ) 6.8 Hz), 4.04 (t, 2H, J ) 6.6 Hz),
6.68-6.75 (m, 2H), 6.80 (s, 1H), 7.38-7.48 (m, 3H), 7.68 (d,
1H, J ) 8.4 Hz), 7.88 (dd, 2H, J ) 1.4 and 7.8 Hz). 13C NMR
(CDCl3) δ 25.73, 28.18, 28.78, 30.31, 33.31, 68.74, 96.87, 111.95,
112.45, 114.44, 125.60, 128.67, 129.39, 131.11, 132.28, 147.69,
166.77, 168.37, 182.71. Anal. (C22H23IO3) C, H.
yield), mp 121-123 °C (ethanol). 1H NMR (CDCl3) δ 1.42-
1.48 (m, 6H), 1.79-1.88 (m, 4H), 3.40 (t, 2H, J ) 6.6 Hz), 3.89
(s, 2H), 3.91 (s, 9H), 4.02 (t, 2H, J ) 6.2 Hz), 6.83 (s, 2H),
6.90-6.96 (m, 2H), 7.46 (s, 1H), 7.78 (d, 1H, J ) 8.4 Hz). Anal.
(C26H31BrO5) C, H.
(E)-7-(7-Bromoheptyloxy)-3-(3,4,5-trimethoxybenzyl-
idene)chroman-4-one (15). HCl gas was bubbled through
an ice-cooled solution of 12 (5.61 g, 16.5 mmol) in ethanol for
1.5 h. The reaction mixture was left overnight at room
temperature; the solvent was removed under reduced pressure
to afford a brown solid that was purified by flash chromatog-
raphy on silica gel (petroleum ether/ethyl acetate 4:1). The
solid was crystallized from ethanol to give 15 as a yellow,
crystalline compound (32% yield), mp 70-72 °C. 1H NMR
(CDCl3) δ 1.43-1.56 (m, 6H), 1.78-1.88 (m, 4H), 3.21 (t, 2H,
J ) 6.6 Hz), 3.88-3.93 (m, 9H), 4.00 (t, 2H, J ) 6.6 Hz), 5.37
(d, 2H, J ) 1.8 Hz), 6.39 (d, 1H, J ) 2.2 Hz), 6.52 (s, 2H), 6.61
(dd, 1H, J ) 2.2 and 8.8 Hz), 7.78 (s, 1H), 7.96 (d, 1H, J ) 8.8
Hz). Anal.(C26H31BrO6) C, H.
(E)-6-(7-Bromoheptyloxy)-2-(3,4,5-trimethoxybenzyl-
idene)-3,4-dihydro-2H-naphthalen-1-one (16). Following
the previous procedure and starting from 13 (1.06 g, 3.13
mmol) and 3,4,5-trimethoxybenzaldehyde (0.67 g, 3.44 mmol),
16 was prepared (28% yield), mp 74-76 °C (petroleum ether).
1H NMR (CDCl3) δ 1.20-1.46 (m, 6H), 1.79-1.93 (m, 4H), 2.93
(t, 2H, J ) 6.6 Hz), 3.15 (t, 2H, J ) 6.6 Hz), 3.43 (t, 2H, J )
6.6 Hz), 3.82-3.96 (m, 9H), 4.04 (t, 2H, J ) 6.2 Hz), 6.67 (s,
2H), 6.71 (d, 1H, J ) 2.2 Hz), 6.87 (dd, 1H, J ) 2.2 and 8.8
Hz), 7.77 (s, 1H), 8.11 (d, 1H, J ) 8.8 Hz). Anal. (C27H33BrO5)
C, H.
(Z)-6-{3-[(3-Hydroxybenzyl)methylamino]propoxy}-2-
(3,4,5-trimethoxybenzylidene) benzofuran-3-one (7a). A
solution of 5a (0.59 g, 1.2 mmol) and 3-methylaminomethyl-
phenol (0.33 g, 2.4 mmol) in toluene was refluxed for 12 h.
The reaction mixture was acidified with 2 N HCl and extracted
with ether. The aqueous solution was neutralized with NaH-
CO3 and extracted with ethyl acetate (3 times for 50 mL). The
combined organic layers were dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue
was crystallized from toluene to give 0.3 g of 7a (50% yield),
1
mp 152-154 °C. H NMR (CDCl3) δ 1.98-2.05 (m, 2H), 2.27
(s, 3H), 2.57 (t, 2H, J ) 6.6 Hz), 3.48 (s, 2H), 3.91 (s, 3H), 3.93
(s, 6H), 4.11 (t, 2H, J ) 6.6 Hz), 6.65-6.81 (m, 3H), 7.08-7.20
(m, 6H), 7.66 (d, 1H, J ) 8.4 Hz). 13C NMR (CDCl3) δ 32.7,
42.3, 53.1, 56.3, 62.11, 66.9, 97.32, 108.97, 113.56, 114.36,
114.53, 120.06, 125.57, 125.8, 128.6, 129.2, 132.44, 136.76,
148.55, 149.22, 168.23, 156.84, 187.73. Anal.(C29H31NO7) C,
H, N.
(Z)-2-(3,4-Dichlorobenzylidene)-6-(7-iodoheptyloxy)-
benzofuran-3-one (6f). Following the previous procedure and
starting from 4a (1.0 g, 2.67 mmol) and 3,4-dichlorobenzalde-
hyde (0.56 g, 3.2 mmol), 6f was prepared (66% yield), mp 101-
103 °C (ethanol). 1H NMR (CDCl3) δ 1.43-1.54 (m, 6H), 1.82-
190 (m, 4H), 3.21 (t, 2H, J ) 6.9 Hz), 4.10 (t, 2H, J ) 6.9 Hz),
6.63 (s, 1H), 6.75-6.81 (m, 2H), 7.37 (d, 1H, J ) 1.5 Hz), 7.69
(d, 1H, J ) 8.4 Hz), 7.74 (d, 2H, J ) 2.1 Hz). 13C NMR (CDCl3)
δ 7.06, 25.79, 28.24, 28.84, 29.72, 30.39, 33.39, 68.98, 97.13,
108.18, 113.04, 114.08, 125.93, 128.89, 129.00, 135.25, 148.79,
167.28, 168.62, 182.37. Anal. (C22H21Cl2IO3) C, H.
(Z)-2-(3,5-Dichlorobenzylidene)-6-(7-iodoheptyloxy)-
benzofuran-3-one (6g). Following the previous procedure and
starting from 4a (1.0 g, 2.67 mmol) and 3,5-dichlorobenzalde-
hyde (0.56 g, 3.2 mmol), 6g was prepared (52% yield), mp 119-
121 °C (ethanol). 1H NMR (CDCl3) δ 1.43-1.54 (m, 6H), 1.82-
1.90 (m, 4H), 3.21 (t, 2H, J ) 6.9 Hz), 4.10 (t, 2H, J ) 6.9 Hz),
6.63 (s, 1H), 6.75-6.81 (m, 2H), 7.37 (d, 1H, J ) 1.5 Hz), 7.69
(d, 1H, J ) 8.4 Hz), 7.74 (d, 2H, J ) 2.1 Hz). 13C NMR (CDCl3)
δ 7.06, 25.79, 28.24, 28.84, 29.72, 30.39, 33.39, 68.98, 97.13,
108.18, 113.04, 114.08, 125.93, 128.89, 129.00, 135.25, 148.79,
167.28, 168.62, 182.37. Anal. (C22H21Cl2IO3) C, H.
(Z)-6-{3-[(3-Hydroxybenzyl)methylamino]propoxy}-2-
naphthalen-1-ylmethylenebenzo-furan-3-one (7b). Fol-
lowing the previous procedure and starting from 5b (1.15 g,
2.53 mmol), 7b was prepared (60% yield), mp 139-141 °C
(toluene). 1H NMR (CDCl3), δ 2.00-2.10 (m, 2H), 2.19 (s, 3H),
2.57 (t, 2H, J ) 6.6 Hz), 3.44 (s, 2H), 4.28 (t, 2H, J ) 6.2 Hz),
6.60-6.75 (m, 2H), 6.81-6.87 (m, 2H), 7.02-7.17 (m, 2H), 7.52
(s, 1H), 7.66-7.72 (m, 4H), 7.79-8.04 (m, 2H), 8.32 (d, 1H, J
) 8.4 Hz), 8.41 (d, 1H, J ) 8.4 Hz). 13C NMR (CDCl3) δ 30.7,
42.06, 52.07, 62.03, 66.88, 96.82, 112.54, 113.54, 114.28,
115.73, 117.91, 120.17, 123.06, 125.28, 125.48, 125.84, 126.69,
128.00, 128.60, 128.78, 129.75, 129.9, 131.45, 133.76, 140.17,
146.39, 156.51, 166.84, 168.39, 182.58. Anal. (C30H27NO4) C,
H, N.
(Z)-6-{3-[(3-Hydroxybenzyl)methylamino]propoxy}-2-
naphthalen-2-ylmethylenebenzo-furan-3-one (7c). Fol-
lowing the previous procedure and starting from 5c (1.15 g,
2.53 mmol), 7c was prepared (60% yield), mp 173-175 °C
(toluene). 1H NMR (CDCl3) δ 1.78-1.82 (m, 2H), 2.02 (s, 3H),
2.32-2.37 (m, 2H), 3.24 (s, 2H), 3.99 (t, 2H, J ) 6.6 Hz), 6.48-
6.57 (m, 3H), 6.61 (s, 1H), 6.66 (d, 1H, J ) 1.8 Hz), 6.74 (s,
1H), 6.85-6.88 (m, 1H), 7.29-7.35 (m, 2H), 7.47 (d, 1H, J )
8.4 Hz), 7.62-7.78 (m, 3H), 7.82-7.88 (m, 1H), 8.12 (s, 1H),
8.51 (s, 1H). 13C NMR (CDCl3) δ 30.0, 38.9, 52.1, 62.31, 70.0,
96.14, 112.76, 114.45, 114.58, 114.59, 120.06, 121.57, 123.85,
125.4, 125.7, 125.8, 126.32, 127.55, 128.07, 128.09, 129.11,
(E)-5-(7-Bromoheptyloxy)-2-(3,4,5-trimethoxybenzyl-
idene)indan-1-one (14). Following the previous procedure
and starting from 11 (1.0 g, 3.08 mmol) and 3,4,5-trimethoxy-
benzaldehyde (0.66. g, 3.39 mmol), 14 was prepared (89%

Cholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4453
133.09, 134.12, 137.32, 146.49, 152.73, 157.84, 167.88, 168.73,
182.17. Anal. (C30H27NO4) C, H, N.
1.62-1.78 (m, 4H), 2.71 (s, 3H), 2.98-3.08 (m, 2H), 4.00 (t,
2H, J ) 7.0 Hz), 4.07 (s, 2H), 6.51 (s, 1H), 6.60-6.68 (m, 2H),
6.70-6.87 (m, 3H), 7.15 (t, 1H, J ) 7.2 Hz), 7.41 (d, 1H, J )
8.4 Hz), 7.57-7.62 (m, 2H), 7.91 (d, 1H, J ) 1.8 Hz), 9.13 (s,
1H). 13C NMR (CDCl3) δ 23.69, 25.32, 26.22, 28.29, 54.82,
59.19, 68.51, 90.64, 96.83, 100.80, 108.19, 112.58, 113.75,
117.06, 117.23, 121.58, 125.40, 129.91, 130.34, 131.84, 132.08,
132.82, 136.20, 148.00, 157.62, 166.79, 168.11, 181.96. Anal.
(C30H31Cl2NO4) C, H, N.
(Z)-2-(3,5-Dichlorobenzylidene)-6-{7-[(3-hydroxybenzyl)-
methylamino]heptyloxy}benzofuran-3-one (8 g). Follow-
ing the previous procedure and starting from 6g (0.50 g, 0.94
mmol), 8g was prepared (40% yield), mp 125-127 °C (ethyl
acetate/n-hexane). 1H NMR (CDCl3) δ 1.26-1.58 (m, 8H),
1.68-1.85 (m, 2H), 2.20 (s, 3H), 2.33-2.42 (m, 2H), 3.46 (s,
2H), 4.00 (t, 2H, J ) 6.6 Hz), 6.54 (s, 1H), 6.66-6.80 (m, 5H),
7.12 (t, 1H, J ) 8.0 Hz), 7.27 (s, 1H), 7.58-7.66 (m, 3H). 13C
NMR (CDCl3) δ 25.81, 26.61, 27.32, 28.79, 29.11, 41.83, 57.16,
61.88, 69.03, 96.94, 108.30, 113.05, 113.76, 114.68, 116.73,
120.93, 125.84, 128.78, 128.89, 129.12, 135.11, 139.15, 148.62,
156.57, 167.32, 168.48, 182.34. Anal. (C30H31Cl2NO4) C, H, N.
(E)-5-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}-
2-(3,4,5-trimethoxybenzylidene)-indan-1-one (17). Fol-
lowing the previous procedure and starting from 14 (0.50 g,
0.99 mmol), 17 was prepared (25% yield), mp 122-125 °C
(ethyl acetate/n-hexane). 1H NMR (CDCl3) δ 1.32-1.43 (m,
6H), 1.66-1.82 (m, 4H), 2.44 (s, 3H), 1.61-1.68 (m, 2H), 3.76
(s, 2H), 3.91 (s, 3H), 3.92 (s, 6H), 3.95 (s, 2H), 4.02 (t, 2H, J )
6.3 Hz), 4.86 (s, 1H), 6.74-6.98 (m, 6H), 7.14-7.19 (m, 2H),
7.50 (s, 1H), 7.81 (d, 1H, J ) 8.4 Hz). Anal. (C34H41NO6) C, H,
N.
(E)-7-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}-
3-(3,4,5-trimethoxybenzylidene)chroman-4-one (18). Fol-
lowing the previous procedure and starting from 15 (0.32 g,
0.56 mmol), 18 was prepared (49% yield), mp 91-92 °C (ethyl
acetate/n-hexane). 1H NMR (CDCl3) δ 1.27-1.49 (m, 8H),
1.75-1.79 (m, 2H), 2.22 (s, 3H), 2.37 (t, 2H, J ) 7.6 Hz), 3.43
(s, 2H), 3.88-3.93 (m, 9H), 3.99 (t, 2H, J ) 6.6 Hz), 5.36 (d,
2H, J ) 1.8 Hz), 6.39 (d, 1H, J ) 2.2 Hz), 6.52 (s, 2H), 6.63
(dd, 1H, J ) 1.8 and 8.4 Hz), 6.70-6.90 (m, 3H), 7.17 (t, 1H,
J ) 7.6 Hz), 7.79 (s, 1H), 7.96 (d, 1H, J ) 8.8 Hz). Anal.
(C34H41NO7) C, H, N.
(Z)-6-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}-
2-(3,4,5-trimethoxybenzylidene) benzofuran-3-one (8a).
Following the previous procedure and starting from 6a (0.5 g,
0.9 mmol), 8a was prepared (60% yield), mp 124-126 °C
(toluene). 1H NMR (CDCl3) δ 1.30-1.55 (m, 8H), 1.78-1.86
(m, 2H), 2.21 (s, 3H), 2.39 (t, 2H, J ) 6.6 Hz), 3.46 (s, 2H),
3.91 (s, 3H), 3.93 (s, 6H), 4.07 (t, 2H, J ) 6.6 Hz), 6.71-6.85
(m, 5H), 7.12-7.20 (m, 4H), 7.70 (d, 1H, J ) 8.4 Hz). 13C NMR
(CDCl3) δ 26.7, 28.0, 29.6, 30.64, 42.36, 53.1, 56.01, 56.27,
62.14, 66.9, 97.14, 108.41, 108.60, 112.56, 112.35, 114.06,
114.57, 120.08, 121.84, 125.57, 125.63, 127.76, 128.87, 140.22,
147.31, 151.01, 168.21, 166.84, 182.73. Anal. (C33H39NO7) C,
H, N.
(Z)-6-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}-
2-naphthalen-1ylmethylenebenzofuran-3-one (8b). Fol-
lowing the previous procedure and starting from 6b (0.5 g, 1.0
mmol), 8b was prepared (60% yield), mp 134-135 °C (toluene).
1H NMR (CDCl3) δ 1.21-1.43 (m, 8H), 1.69-1.85 (m, 2H), 2.09
(s, 3H), 2.24-2.31(m, 2H), 3.33 (s, 2H), 3.98 (t, 2H, J ) 6.6
Hz), 6.61-6.76 (m, 5H), 6.99-7.05 (m, 1H), 7.43-7.52 (m, 4H),
7.63 (d, 1H, J ) 8.4 Hz), 7.80-7.83 (m, 2H), 8.21 (d, 1H, J )
8.4 Hz), 8.32 (d, 1H, J ) 8.4 Hz). 13C NMR (CDCl3) δ 7.1, 26.6,
29.7, 34.33, 42.33, 53.07, 62.20, 72.34, 96.59, 112.56, 113.89,
114.34, 114.87, 116.44, 121.77, 123.06, 125.28, 125.49, 125.84,
126.69, 128.00, 128.61, 128.64, 129.76, 129.97, 131.95, 133.45,
140.55, 148.76, 156.55, 168.45, 182.33. Anal. (C34H35NO4) C,
H, N.
(Z)-6-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}-
2-naphthalen-2-ylmethyleneben-zofuran-3-one (8c). Fol-
lowing the previous procedure and starting from 6c (0.5 g, 1.0
mmol), 8c was prepared (60% yield), mp 96-98 °C (toluene).
1H NMR (CDCl3) δ 1.36-1.50 (m, 8H), 1.87-1.94 (m, 2H), 2.24
(s, 3H), 2.42 (t, 2H, J ) 6.6 Hz), 3.46 (s, 2H), 4.09 (t, 2H, J )
6.6 Hz), 6.72-6.86 (m, 4H), 6.99 (s, 1H), 7.12-7.19 (m, 1H),
7.50-7.56 (m, 2H), 7.72 (d, 1H, J ) 8,4 Hz), 7.83-7.96 (m,
3H), 8.04-8.10 (m, 2H), 8.31 (s, 1H). 13C NMR (CDCl3) δ 14.1,
28.6, 29.7, 30.33, 30.53, 42.07, 62.20, 66.88, 97.09, 112.53,
114.04, 114.17, 114.40, 114.64, 120.06, 120.76, 125.68, 126.49,
126.64, 127.22, 127.62, 128.61, 128.38, 128.55, 129.21, 129.44,
131.83, 133.76, 147.55, 150.11, 166.67, 168.45, 182.77. Anal.
(C34H35NO4) C, H, N.
(Z)-2-Anthracen-9-ylmethylene-6-{7-[(3-hydroxybenzyl)-
methylamino]heptyloxy}ben-zofuran-3-one (8d). Follow-
ing the previous procedure and starting from 6d (0.4 g, 0.71
mmol), 8d was prepared (50% yield), mp 95-97 °C (ethanol).
1H NMR (CDCl3) δ 1.20-1.80 (m, 10H), 2.18 (d, 3H, J ) 3.2
Hz), 2.33 (t, 2H, J ) 8.2 Hz), 3.41 (s, 2H), 3.94 (t, 2H, J ) 8.0
Hz), 6.46 (d, 1H, J ) 2.2 Hz), 6.62-6.84 (m, 4H), 7.15 (t, 1H,
J ) 8.0 Hz), 7.46-7.58 (m, 5H), 7.77 (d, 1H, J ) 8.4 Hz), 7.79
(s, 1H), 8.02-8.18 (m, 3H), 8.54 (s, 1H). 13C NMR (CDCl3) δ
7.27, 25.84, 27.25, 28.78, 29.12, 42.30, 57.41, 62.10, 68.89,
96.64, 108.76, 112.82, 113.87, 115.71, 121.29, 125.14, 125.35,
125.92, 126.20, 128.81, 129.23, 131.21, 149.68, 167.26, 168.30,
186.63. Anal. (C38H37NO4) C, H, N.
(E)-6-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}-
2-(3,4,5-trimethoxybenzylidene)3,4-dihydro-2H-naphtha-
len-1-one (19). Following the previous procedure and starting
from 16 (0.46 g, 0.89 mmol), 19 was prepared (27% yield), mp
1
81-83 °C (ethyl acetate/n-hexane). H NMR (CDCl3) δ 1.20-
1.46 (m, 6H), 1.79-1.93 (m, 4H), 2.18 (s, 3H), 2.34-2.40 (m,
2H), 2.90 (t, 2H, J ) 6.2 Hz), 3.08-3.16 (m, 2H), 3.44 (s, 2H),
3.87-3.90 (m, 9H), 3.99 (t, 2H, J ) 6.4 Hz), 6.45 (s, 2H), 6.65-
6.90 (m, 6H), 7.12 (t, 1H, J ) 8.0 Hz), 7.76 (s, 1H), 8.08 (d,
1H, J ) 8.8 Hz). Anal. (C35H46NO6) C, H, N.
1-(2-Hydroxy-4-{7-[(3-hydroxybenzyl)methylamino]-
heptyloxy}phenyl)ethanone (24). Following the previous
procedure and starting from 23 (0.5 g, 1.55 mmol), 24 was
1
prepared (90% yield) as an oil. H NMR (CDCl3) δ 1.30-1.60
(Z)-2-Benzylidene-6-{7-[(3-hydroxybenzyl)methylamino]-
heptyloxy}benzofuran-3-one (8e). Following the previous
procedure and starting from 6e (0.29 g, 0.63 mmol), 8e was
prepared (50% yield), mp 95-97 °C (acetone). 1H NMR (CDCl3)
δ 1.26-1.44 (m, 6H), 1.76-1.83 (m, 4H), 2.37 (s, 3H), 2.60 (t,
2H, J ) 6.6 Hz), 3.68 (s, 2H), 4.05 (t, 2H, J ) 6.6 Hz), 6.38-
6.51 (m, 1H), 6.72-6.76 (m, 2H), 6.84 (s, 1H), 6.86-6.88 (m,
2H), 6.99 (s, 1H), 7.18 (t, 1H, J ) 7.6 Hz), 7.41-7.50 (m, 3H),
(m, 8H), 1.78-1.86 (m, 2H), 2.21 (s, 3H), 2.35 (t, 2H, J ) 6.6
Hz), 2.50 (s, 3H), 3.66 (s, 2H), 4.05 (t, 2H, J ) 6.6 Hz), 6.71-
6.85 (m, 4H), 7.12-7.20 (m, 2H), 7.70 (d, 1H, J ) 8.4 Hz). Anal.
(C23H31NO4) C, H, N.
2-Bromo-1-(2-hydroxy-4-{7-[(3-hydroxybenzyl)methyl-
amino]heptyloxy}phenyl)ethanone (25). A suspension of
CuBr2 in AcOEt (15 mL) was added to a hot solution of 24
(0.45 g, 1.4 mmol) in CHCl3 (15 mL), and the mixture was
refluxed for 12 h. The mixture was filtered, and the organic
phase was evaporated under reduced pressure. The organic
residue was purified by flash chromatography on silica gel
(methylene chloride/methanol 9:1) to obtain 25 as an oil (45%
7.70 (d, 1H, J ) 9.2 Hz), 7.90 (dd, 1H, J ) 1.8 and 8.0 Hz). 13
C
NMR (CDCl3) δ 25.79, 27.09, 28.79, 28.95, 41.31, 56.68, 61.25,
68.87, 96.98, 111.99, 112.66, 114.46, 115.58, 116.95, 121.59,
125.78, 128.76, 129.60, 131.24, 132.30, 136.43, 147.78, 156.56,
167.01, 168.52, 183.03. Anal. (C30H33NO4) C, H, N.
1
yield). H NMR (CDCl3) δ 1.20-1.60 (m, 8H), 1.75-1.85 (m,
(Z)-2-(3,4-Dichlorobenzylidene)-6-{7-[(3-hydroxybenzyl)-
methylamino]heptyloxy}benzofuran-3-one (8f). Following
the previous procedure and starting from 6f (0.29 g, 0.63
mmol), 8f was prepared (48% yield), mp 147-149 °C (ethyl
acetate/n-hexane). 1H NMR (CDCl3) δ 1.20-1.42 (m, 6H),
2H), 2.25 (s, 3H), 2.45 (t, 2H, J ) 6.6 Hz), 3.66 (s, 2H), 3.95 (s,
2H), 4.05 (t, 2H, J ) 6.6 Hz), 6.75-6.89 (m, 4H), 7.12-7.20
(m, 2H), 7.70 (d, 1H, J ) 8.4 Hz). Anal. (C23H29BrNO4) C, H,
N.

4454 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Belluti et al.
(Z)-6-{7-[(3-Hydroxy-benzyl)methylamino]heptyloxy}-
benzofuran-3-one (26). A solution of 25 (0.20 g, 0.63 mmol)
in water was treated with sodium acetate (0.065 g, 0.75 mmol),
the mixture was refluxed for 1 h, allowed to cool to room
temperature, and extracted with methylene chloride. The
combined organic layers were dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The residue
was crystallized from methanol to give 26 as a white solid,
mp 109-110 °C (67% yield). 1H NMR (CDCl3) δ 1.30-1.60 (m,
8H), 1.74-1.88 (m, 2H), 2.26 (s, 3H), 2.33 (t, 2H, J ) 6.6 Hz),
3.86 (s, 2H), 4.05 (t, 2H, J ) 6.6 Hz), 4.21 (s, 2H), 6.71-6.85
(m, 4H), 7.12-7.20 (m, 2H), 7.70 (d, 1H, J ) 8.4 Hz). Anal.
(C23H29NO4) C, H, N.
(Z)-Methylcarbamic Acid 3-[(Methyl{3-[3-oxo-2-(3,4,5-
trimethoxybenzylidene)-2,3-dihydrobenzofuran-6-yloxy]-
propyl}amino)methyl]phenyl Ester (9a). A solution of 7a
(0.2 g, 0.36 mmol) in dry toluene (70 mL) was treated with
NaH (60% dispersion in mineral oil, 0.015 g, 0.36 mmol) at 0
°C. After the solution had been stirred for 0.5 h, methyl-
isocianate (0.02 g, 0.36 mmol) was added dropwise at room
temperature. The mixture was stirred overnight at room
temperature, decomposed with ice water, and extracted twice
with dichloromethane. The combined organic layers were dried
over sodium sulfate anhydrous, filtered, and concentrated
under reduced pressure to give a semisolid residue that was
further purified by flash chromatography on silica gel (di-
chloromethane/methanol 9.5:0.5) to obtain 9a as a pale yellow
powder (24% yield), mp 52-54 °C (ethyl acetate/n-hexane). 1H
NMR (CDCl3) δ 1.98-2.05 (m, 2H), 2.27 (s, 3H), 2.55 (t, 2H, J
) 6.6 Hz), 2.89 (d, 3H, J ) 4.8 Hz), 3.51 (s, 2H), 3.93-3.98
(m, 9H), 4.16 (t, 2H, J ) 6.6 Hz), 4.91-4.98 (br, 1H), 6.71-
6.75 (m, 2H), 6.76 (s, 2H), 6.93-6.97 (m, 2H), 7.15 (s, 1H), 7.16
(s, 1H), 7.21-7.26 (m, 2H). 13C NMR (CDCl3) δ 26.91, 29.7,
42.3, 53.1, 56.3, 62.11, 66.9, 97.14, 108.66, 112.36, 112.56,
114.53, 120.06, 125.57, 125.8, 128.6, 129.2, 132.44, 136.76,
147.32, 148.55, 152.89, 166.84, 168.23, 182.73. MS (ES) m/z
) 563 (M + H+). Anal. (C31H34N2O8) C, H, N.
hexane). 1H NMR (CDCl3) δ 1.38-1.58 (m, 8H), 1.70-1.90 (m,
2H), 2.21 (s, 3H), 2.19 (s, 3H), 2.39 (t, 2H, J ) 6.6 Hz), 2.89 (d,
3H, J ) 4.8 Hz), 3.48 (s, 2H), 3.91 (s, 3H), 3.95 (s, 3H), 3.98 (s,
3H), 4.07 (t, 2H, J ) 6.6 Hz), 4.98-5.08 (br, 1H), 6.60-6.85
(m, 5H), 6.98-7.05 (m, 2H), 7.26-7.29 (m, 2H), 7.69 (d, 1H, J
) 8.4 Hz). 13C NMR (CDCl3) δ 14.20, 26.91, 28.90, 29.34, 29.7,
31.63, 42.3, 53.1, 57.3, 61.11, 68.9, 97.14, 108.66, 112.36,
112.56, 114.53, 120.06, 125.57, 125.8, 128.6, 129.2, 132.44,
136.76, 147.32, 148.55, 152.89, 168.23, 166.84, 182.73. MS (ES)
m/z ) 619 (M + H+). Anal. (C35H42N2O8) C, H, N.
(Z)-Methylcarbamic Acid 3-({Methyl[7-(2-naphthalen-
1-ylmethylene-3-oxo-2,3-dihydrobenzofuran-6-yloxy)hep-
tyl]-amino}methyl)phenyl Ester (10b). Following the pre-
vious procedure and starting from 8b (0.2 g, 0.39 mmol), 10b
was prepared (27% yield), mp 95-97 °C (ethyl acetate/n-
hexane). 1H NMR (CDCl3) δ 1.35-1.51 (m, 8H), 1.78-1.87 (m,
2H), 2.19 (s, 3H), 2.37 (t, 2H, J ) 6.6 Hz), 2.89 (d, 3H, J ) 4.8
Hz), 3.48 (s, 2H), 4.08 (t, 2H, J ) 6.6 Hz), 4.91-5.01 (br, 1H),
6.65-6.76 (m, 2H), 6.97-7.05 (m, 1H), 7.07-7.17 (m, 2H),
7.52-7.66 (m, 5H), 7.75 (d, 1H, J ) 8.4 Hz), 7.88-7.95 (m,
2H), 8.33 (d, 1H, J ) 8.4 Hz), 8.43 (d, 1H, J ) 8.4 Hz). 13C
NMR (CDCl3) δ 14.20, 25.93, 27.31, 27.76, 28.9, 29.32, 39.9,
31.66, 42.3, 57.45, 61.91, 68.96, 97.14, 107.86, 112.67, 120.96,
121.59, 123.06, 125.35, 125.47, 126.09, 126.92, 128.38, 128.88,
129.33, 130.11, 132.11, 133.62, 140.55, 148.32, 150.93, 166.43,
168.84, 182.88. MS (ES) m/z ) 579 (M + H+). Anal. (C36H38N2O5)
C, H, N.
(Z)-Methylcarbamic Acid 3-({Methyl[7-(2-naphthalen-
2-ylmethylene-3-oxo-2,3-dihydrobenzofuran-6-yloxy)-
heptyl]amino}methyl)phenyl Ester (10c). Following the
previous procedure and starting from 8c (0.2 g, 0.39 mmol),
10c was prepared (20% yield), mp 83-85 °C (ethyl acetate/n-
hexane). 1H NMR (CDCl3) δ 1.20-1.58 (m, 8H), 1.78-1.89 (m,
2H), 2.20 (s, 3H), 2.39 (t, 2H, J ) 6.6 Hz), 2.89 (d, 3H, J ) 4.8
Hz), 3.49 (s, 2H), 4.09 (t, 2H, J ) 6.6 Hz), 4.91-5.01 (br, 1H),
6.98 (d, 1H, J ) 6.4 Hz), 6.83 (m, 1H), 6.90 (s, 1H), 7.08-7.18
(m, 2H), 7.50-7.56 (m, 2H), 7.70 (d, 1H, J ) 8,4 Hz), 7.83-
7.96 (m, 3H), 8.04-8.10 (m, 2H), 8.04 (s, 1H). 13C NMR (CDCl3)
δ 14.20, 25.93, 27.66, 28.91, 29.31, 30.0, 31.66, 40.9, 52.1, 61.31,
70.11, 96.14, 112.76, 114.58, 114.96, 119.59, 120.06, 121.57,
123.85, 125.0, 125.2, 126.14, 126.76, 127.77, 128.00, 128.11,
133.09, 134.55, 136.32, 146.49, 152.73, 157.84, 167.88, 168.73,
182.17. MS (ES) m/z ) 579 (M + H+). Anal. (C36H38N2O5) C,
H, N.
(Z)-Methylcarbamic Acid 3-({[7-(2-Anthracen-9-yl-
methylene-3-oxo-2,3-dihydrobenzo-furan-6-yloxy)heptyl]-
methylamino}methyl)-phenyl Ester (10d). Following the
previous procedure and starting from 8d (0.2 g, 0.35 mmol),
10d was prepared (27% yield), mp 95-97 °C (ethyl acetate/
n-hexane). 1H NMR (CDCl3) δ 1.21-1.75 (m, 10H), 2.16 (s, 3H),
2.34 (t, 2H, J ) 7.2 Hz), 2.87 (d, 3H, J ) 4.8 Hz), 3.48 (s, 2H),
3.93 (t, 2H, J ) 6.2 Hz), 4.92-4.94 (br, 1H), 6.46 (d, 1H, J )
2.2 Hz), 6.75 (dd, 1H, J ) 2.2 and 8.4 Hz), 6.97-7.23 (m, 3H),
7.31 (t, 1H, J ) 7.2 Hz), 7.45-7.55 (m, 4H), 7.77 (d, 1H, J )
8.4 Hz), 7.79 (s, 1H), 8.02-8.14 (m, 4H), 8.52 (s, 1H). 13C NMR
(CDCl3) δ 25.86, 26.98, 27.31, 27.75, 28.81, 29.16, 42.29, 57.43,
61.94, 68.92, 96.94, 108.67, 112.18, 112.82, 119.99, 121.94,
125.15, 125.35, 125.73, 126.20, 128.57, 128.81, 129.85, 131.22,
149.97, 150.97, 167.28, 168.87, 186.48. MS (ES) m/z ) 629 (M
+ H+). Anal. (C40H40N2O5) C, H, N.
(Z)-Methylcarbamic Acid 3-({Methyl[3-(1-naphthalen-
2-ylmethylene-3-oxo-2,3-dihydro-benzofuran-6-yloxy)-
propyl]amino}methyl)phenyl Ester (9b). Following the
previous procedure and starting from 7b (0.2 g, 0.43 mmol),
9b was prepared (27% yield), mp 95-97 °C (ethyl acetate/n-
1
hexane). H NMR (CDCl3) δ 1.96-2.20 (m, 2H), 2.27 (s, 3H),
2.52 (t, 2H, J ) 6.6 Hz), 2.89 (d, 3H, J ) 4.8 Hz), 3.51 (s, 2H),
4.15 (t, 2H, J ) 6.6 Hz), 4.90-4.98 (br, 1H), 6.71-6.75 (m,
2H), 6.78-6.95 (m, 1H), 7.07-7.30 (m, 3H), 7.52-7.66 (m, 4H),
7.74 (d, 1H, J ) 8.4 Hz), 7.90-7.95 (m, 2H), 8.33 (d, 1H, J )
8.4 Hz), 8.43 (d, 1H, J ) 8.4 Hz). 13C NMR (CDCl3) δ 28.31,
30.0, 39.9, 52.3, 61.91, 70.19, 96.14, 112.86, 114.48, 116.96,
119.59, 121.06, 123.35, 123.47, 125.0, 125.4, 126.04, 126.76,
128.77, 128.84, 129.11, 130.09, 133.55, 135.32, 136.49, 149.43,
152.84, 158.73, 166.36, 168.58, 182.18. MS (ES) m/z ) 523 (M
+ H+). Anal. (C32H30N2O5) C, H, N.
(Z)-Methylcarbamic Acid 3-({Methyl[3-(2-naphthalen-
2-ylmethylene-3-oxo-2,3-dihydro-benzofuran-6-yloxy)-
propyl]amino}methyl)phenyl Ester (9c). Following the
previous procedure and starting from 7c (0.2 g, 0.43 mmol),
9c was prepared (25% yield), mp 99-101 °C (ethyl acetate/n-
1
hexane). H NMR (CDCl3) δ 1.98-2.22 (m, 2H), 2.20 (s, 3H),
2.52 (t, 2H, J ) 6.6 Hz), 2.88 (d, 3H, J ) 4.8 Hz), 3.49 (s, 2H),
4.14 (t, 2H, J ) 6.6 Hz), 4.91-5.01 (br, 1H), 6.69 (d, 1H, J )
6.4 Hz), 6.79-6.81 (m, 2H), 6.90 (s, 1H), 7.01-7.09 (m, 1H),
7.08-7.18 (m, 2H), 7.50-7.59 (m, 2H), 7.68 (d, 1H, J ) 8.4
Hz), 7.83-7.97 (m, 3H), 8.06 (s, 1H), 8.30 (s, 1H). 13C NMR
(CDCl3) δ 28.91, 30.0, 40.9, 52.1, 61.31, 70.11, 96.14, 112.76,
114.58, 114.96, 119.59, 120.06, 121.57, 123.85, 125.0, 125.2,
126.14, 126.76, 127.77, 128.00, 128.11, 133.09, 134.55, 136.32,
146.49, 152.73, 157.84, 168.73, 167.88, 182.17. MS (ES) m/z
) 523 (M + H+). Anal. (C32H30N2O5) C, H, N.
(Z)-Methylcarbamic Acid 3-({[7-(2-Benzylidene-3-oxo-
2,3-dihydrobenzofuran-6-yloxy)heptyl]methylamino}-
methyl)phenyl Ester (10e). Following the previous proce-
dure and starting from 8e (0.2 g, 0.42 mmol), 10e was prepared
1
(29% yield), mp 82-84 °C (ethyl acetate/n-hexane). H NMR
(CDCl3) δ 1.28-1.56 (m, 8H), 1.80-1.90 (m, 2H), 2.19 (s, 3H),
2.37 (t, 2H, J ) 6.6 Hz), 2.90 (d, 3H, J ) 4.8 Hz), 3.48 (s, 2H),
4.08 (t, 2H, J ) 6.6 Hz), 4.98-5.01 (br, 1H), 6.73-6.77 (m,
2H), 6.83 (s, 1H), 6.98-7.16 (m, 3H), 7.26-7.29 (m, 1H), 7.39-
7.48 (m, 3H), 7.68-7.72 (d, 1H, J ) 8.4 Hz), 7.89-7.92 (m,
2H). 13C NMR (CDCl3) δ 25.89, 27.26, 27.70, 28.87, 29.19,
29.69, 42.24, 57.41, 61.89, 68.93, 96.95, 111.62, 112.55, 114.49,
128.82, 129.44, 130.77, 131.18, 132.36, 140.77, 147.78, 150.95,
(Z)-Methylcarbamic Acid 3-[(Methyl{7-[3-oxo-2-(3,4,5-
trimethoxybenzylidene)-2,3-dihydrobenzofuran-6-yloxy]-
heptyl}amino)methyl]phenyl Ester (10a). Following the
previous procedure and starting from 8a (0.2 g, 0.36 mmol),
10a was prepared (30% yield), mp 79-81 °C (ethyl acetate/n-

Cholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4455
155.15, 166.95, 168.48, 182.84. MS (ES) m/z ) 529 (M + H+).
4.04 (t, 2H, J ) 6.6 Hz), 4.21 (s, 2H), 4.98-5.08 (br, 1H), 6.60-
6.90 (m, 5H), 7.26-7.29 (m, 1H), 7.72 (d, 1H, J ) 8.4 Hz). MS
(ES) m/z ) 441 (M + H+). Anal. (C25H32N2O5) C, H, N.
Biology. Thioflavin T (Basic Yellow 1), human recombinant
AChE lyophilized powder, BuChE from human serum, ATCh
iodide, BuTCh iodide, 5,5′-dithiobis(2-nitro-benzoic acid) (DTNB),
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), Triton X-100, and
physostigmine were purchased from Sigma-Aldrich. Buffers
and other chemicals were of analytical grade. Absolute DMSO
over a molecular sieves was from Fluka. Water was deionized
and doubly distilled. Aâ(1-40), supplied as a trifluoroacetate
salt, was purchased from Bachem AG. Aâ (2 mg ml-1) was
dissolved in HFIP, lyophilized, and used in this form after
dilution for aggregation experiments.
Inhibition of AChE and BuChE. The method of Ellman
et al. was followed.28 A 0.037 M ATCh iodide solution was
prepared in water. A 0.01 M DTNB solution was prepared in
potassium phosphate buffer (pH 7.0) containing 0.15% (w/v)
sodium bicarbonate. AChE stock solution was prepared by
dissolving 1000 units in 0.1 M phosphate buffer (pH 8.0)
containing Triton X-100 0.1%. The enzyme was diluted before
use, to reach an activity ranging between 0.13 and 0.10 AU/
min in the final assay conditions. Stock solutions of the tested
compounds (1 mM) were prepared in methanol. Five different
concentrations of each compound were used in order to obtain
inhibition of AChE activity ranging between 20 and 80%.
The assay solution consisted of a 0.1 M phosphate buffer
(pH 8.0), with the addition of 340 µM DTNB and AChE and
550 µM ATCh iodide. The final assay volume was 1 mL. Test
compounds were added to the assay solution and preincubated
with the enzyme for 20 min before the addition of the
substrate.
Initial rate assays were performed at 37 °C with a Jasco
V-530 double beam spectrophotometer, and the rate of increase
in the absorbance at 412 nm was followed for 5 min. Assays
were run with a blank containing all of the components except
AChE in order to account for a nonenzymatic reaction. The
reaction rates were compared and the percent inhibition due
to the presence of test compounds was calculated. Each
concentration was analyzed in triplicate. The percent inhibi-
tion of the enzyme activity due to the presence of increasing
test compound concentration was calculated by the following
expression: 100 - (vi/vo × 100), where vi is the initial rate
calculated in the presence of inhibitor and vo is the enzyme
activity. Inhibition curves were obtained for each compound
by plotting the % inhibition vs the logarithm of inhibitor
concentration in the assay solution. The linear regression
parameters were determined for each curve and the IC50 values
were extrapolated. The computer program used to analyze
these data was GraphPad Prism 3.0 (GraphPad Software Inc.).
Inhibition of BuChE was measured as described above,
substituting 0.035 unit/mL of BuChE from human serum and
550 µM BuTCh for the enzyme and substrate, respectively.
Inhibition of AChE-Induced Aâ Aggregation. For the
thioflavin T-based fluorometric assay, analyses were performed
with a Jasco spectrofluorimeter FP-750, using a 3 mL quartz
cell. Aliquots of 2 µL of Aâ lyophilized from 2 mg ml-1 of HFIP
solution and dissolved in DMSO, were incubated for 24 h at
room temperature in 0.215 M sodium phosphate buffer (pH
8.0) at a final concentration of 230 µM. For co-incubation
experiments, aliquots (16 µL) of AChE (final concentration 2.30
µM, Aâ/AChE molar ratio 100:1) and AChE in the presence of
2 µL of the tested inhibitors in 0.215 M sodium phosphate
buffer (pH 8.0) solution (final inhibitor concentration 100 µM)
were added.
Anal. (C32H36N2O5) C, H, N.
(Z)-Methylcarbamic Acid 3-[({7-[2-(3,4-Dichlorobenzyl-
idene)-3-oxo-2,3-dihydrobenzofuran-6-yloxy]heptyl}methyl-
amino)methyl]phenyl Ester (10f). Following the previous
procedure and starting from 8f (0.2 g, 0.37 mmol), 10f was
prepared (25% yield), mp 90-92 °C (ethyl acetate/n-hexane).
1H NMR (CDCl3) δ 1.24-1.60 (m, 8H), 1.80-1.92 (m, 2H), 2.22
(s, 3H), 2.32-2.48 (m, 2H), 2.89 (d, 3H, J ) 4.8 Hz), 3.52 (s,
2H), 4.08 (t, 2H, J ) 6.6 Hz), 4.98-5.01 (br, 1H), 6.67 (s, 1H),
6.73-6.78 (m, 2H), 7.02-7.19 (m, 3H), 7.26-7.30 (m, 1H), 7.49
(d, 1H, J ) 8.4 Hz), 7.63-7.71 (m, 2H), 8.02 (d, 1H, J ) 2.2
Hz). 13C NMR (CDCl3) δ 25.92, 27.26, 27.77, 28.89, 29.20,
42.12, 57.33, 61.77, 69.08, 97.11, 108.68, 112.96, 114.19,
120.25, 122.15, 125.90, 129.00, 130.17, 130.70, 132.28, 132.49,
133.00, 133.35, 148.44, 151.02, 167.28, 168.56, 182.43. MS (ES)
m/z ) 598 (M + H+). Anal. (C32H34Cl2N2O5) C, H, N.
(Z)-Methylcarbamic Acid 3-[({7-[2-(3,5-Dichlorobenzyl-
idene)-3-oxo-2,3-dihydro-benzofuran-6-yloxy]heptyl}methyl-
amino)methyl]phenyl Ester (10g). Following the previous
procedure and starting from 8g (0.2 g, 0.37 mmol), 10g was
prepared (24% yield), mp 109-111 °C (ethyl acetate/n-hexane).
1H NMR (CDCl3) δ 1.35-1.57 (m, 8H), 1.76-1.89 (m, 2H), 2.23
(s, 3H), 2.36-2.46 (m, 2H), 2.89 (d, 3H, J ) 4.8 Hz), 3.53 (s,
2H), 4.09 (t, 2H, J ) 6.6 Hz), 4.98 (s, 1H), 6.63 (s, 1H), 6.74-
6.81 (m, 2H), 6.98-7.30 (m, 4H), 7.33-7.37 (m, 1H), 7.69 (d,
1H, J ) 8.4 Hz), 7.75 (d, 2H, J ) 2.0 Hz). 13C NMR (CDCl3) δ
25.90, 27.23, 27.80, 28.87, 29.17, 39.79, 57.25, 61.96, 69.09,
90.31, 97.13, 108.18, 113.06, 114.02, 122.15, 125.92, 128.89,
129.00, 135.23, 148.79, 151.00, 167.36, 173.24, 182.40. MS (ES)
m/z ) 598 (M + H+). Anal. (C32H34Cl2N2O5) C, H, N.
(E)-Methylcarbamic Acid 3-[(Methyl{7-[1-oxo-2-(3,4,5-
trimethoxybenzylidene)indan-5-yloxy]heptyl}amino)-
methyl]phenyl Ester (20). Following the previous procedure
and starting from 17 (0.2 g, 0.38 mmol), 20 was prepared (27%
yield), mp 116-118 °C (ethyl acetate/n-hexane). 1H NMR
(CDCl3) δ 1.38-1.51 (m, 8H), 1.79-1.86 (m, 2H), 2.19 (s, 3H),
2.37 (t, 2H, J ) 7.6 Hz), 2.89 (d, 3H, J ) 4.8 Hz), 3.48 (s, 2H),
3.91 (s, 3H), 3.92 (s, 6H), 3.95 (s, 2H), 4.07 (t, 2H, J ) 6.2 Hz),
5.00-5.02 (br, 1H), 6.90-6.94 (m, 3H), 6.98-7.03 (m, 2H),
7.05-7.18 (m, 2H), 7.23-7.30 (m, 1H), 7.54 (s, 1H), 7.85 (d,
1H, J ) 8.4 Hz). MS (ES) m/z ) 617 (M + H+). Anal.
(C36H44N2O7) C, H, N.
(E)-Methylcarbamic Acid 3-[(Methyl{7-[4-oxo-3-(3,4,5-
trimethoxybenzylidene)chroman-7-yloxy]heptyl}amino)-
methyl]phenyl Ester (21). Following the previous procedure
and starting from 18 (0.2 g, 0.37 mmol), 21 was prepared (20%
yield) as a hydrochloride salt, mp 99-101 °C (methanol/ethyl
1
ether). H NMR (CDCl3) δ 1.22-1.54 (m, 8H), 1.78-1.82 (m,
2H), 2.21 (s, 3H), 2.40 (t, 2H, J ) 7.2 Hz), 2.88 (d, 3H, J ) 4.8
Hz), 3.52 (s, 2H), 3.86-3.94 (m, 9H), 3.97 (t, 2H, J ) 1.8 Hz),
4.97 (s, 1H), 5.36 (d, 1H, J ) 1.8 Hz), 6.38-6.40 (m, 1H), 6.52
(s, 2H), 6.58-6.68 (m, 1H), 6.98-7.32 (m, 4H), 7.78 (s, 1H),
7.98 (d, 1H, J ) 8.4 Hz). MS (ES) m/z ) 633 (M + H+). Anal.
(C36H45N2O8) C, H, N.
(E)-Methylcarbamic Acid 3-[(Methyl{7-[5-oxo-6-(3,4,5-
trimethoxybenzylidene)-5,6,7,8-tetrahydronaphthalen-
2-yloxy]heptyl}amino)methyl]phenyl Ester (22). Follow-
ing the previous procedure and starting from 19 (0.2 g, 0.35
mmol), 22 was prepared (20% yield) as a hydrochloride salt
mp 102-105 °C (methanol/ethyl ether). 1H NMR (CDCl3) δ
1.23-1.48 (m, 8H), 1.75-1.84 (m, 2H), 2.21 (s, 3H), 2.39 (t,
2H, J ) 7.0 Hz), 2.84-2.92 (m, 5H), 3.10 (t, 2H, J ) 6.6 Hz),
3.51 (s, 2H), 3.82-3.91 (m, 9H), 4.01 (t, 2H, J ) 6.6 Hz), 5.00
(s, 1H), 6.64-6.72 (m, 2H), 6.92 (s, 2H), 6.98-7.29 (m, 4H),
7.88 (s, 1H), 8.02 (d, 1H, J ) 8.8 Hz). MS (ES) m/z ) 631 (M
+ H+). Anal. (C37H46ClN2O7) C, H, N.
Methylcarbamic Acid 3-({Methyl-[7-(3-oxo-2,3-dihydro-
benzofuran-6-yloxy)-heptyl]-amino}-methyl)-phenyl Es-
ter (27). Following the previous procedure and starting from
26 (0.1 g, 0.42 mmol), 27 was prepared (20% yield) as a
hydrochloride salt, mp 106-108 °C (methanol/ethyl ether). 1H
NMR δ 1.35-1.60 (m, 8H), 1.75-1.96 (m, 2H), 2.25 (s, 3H),
2.39 (t, 2H, J ) 6.6 Hz), 2.87 (d, 3H, J ) 4.8 Hz), 3.45 (s, 2H),
Blanks containing Aâ, AChE, and Aâ plus inhibitors, in
0.215 M sodium phosphate buffer (pH 8.0), were prepared. The
final volume of each vial was 20 µL. Each assay was run in
duplicate.
To quantify amyloid fibril formation, the thioflavin T
fluorescence method9 was then applied. The fluorescence
intensities were compared, and the percent inhibition due to
the presence of the test compounds was calculated. The percent
inhibition of the AChE-induced aggregation due to the pres-

4456 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Belluti et al.
(18) Rampa, A.; Bisi, A.; Valenti, P.; Recanatini, M.; Cavalli, A.; et
al. Acetylcholinesterase inhibitors: synthesis and structure-
activity relationships of omega-[N-methyl-N-(3-alkylcarbamoyl-
oxyphenyl)-methyl]aminoalkoxyheteroaryl derivatives. J. Med.
Chem. 1998, 41, 3976-3986.
(19) Rampa, A.; Piazzi, L.; Belluti, F.; Gobbi, S.; Bisi, A.; et al.
Acetylcholinesterase inhibitors: SAR and kinetic studies on
omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]amino-
alkoxyaryl derivatives. J. Med. Chem. 2001, 44, 3810-3820.
(20) Pelter, A.; Ward, R. S.; Heller, H. G. Carbon-13 nuclear magnetic
resonance spectra of (Z)- and (E)-aurones. J. Chem. Soc., Perkin
Trans. 1 1979, 328-329.
ence of the tested compound was calculated by the following
expression: 100 - (IFi/IFo × 100), where IFi and IFo are the
fluorescence intensities obtained for Aâ plus AChE in the
presence and in the absence of inhibitor, respectively, minus
the fluorescent intensities due to the respective blanks.
Acknowledgment. Investigation supported by the
University of Bologna (funds for selected research
topics).
(21) Bayer, H.; Hartmann, R. W. [Pyridyl-substituted tetralone
derivatives: a new class of nonsteroidal aromatase inhibitors].
Arch. Pharm. (Weinheim, Ger.) 1991, 324, 815-820.
(22) Varma, R. S.; Varma, M. Alumina-mediated condensation. A
simple synthesis of aurones. Tetrahedron Lett. 1992, 33, 5937-
5940.
(23) Hastings, J. S.; Heller, H. G. The stereochemistry of aurones
[2-(substituted benzylidene)-3(2H)-benzofuranones]. J. Chem.
Soc., Perkin Trans. 1 1972, 2128-2132.
(24) Brady, B. A.; Kennedy, J. L.; I., O. S. W. The configuration of
aurones. Tetrahedron 1973, 29, 359-362.
(25) Sharma, A.; Chibber, S. S. Carbon-13 NMR spectroscopy of
methoxy- and acetoxyaurones. J. Heterocycl. Chem. 1981, 18,
275-277.
(26) Teo, C. C.; Kon, O. L.; Sim, K. Y.; Ng, S. C. Synthesis of 2-(p-
chlorobenzyl)-3-aryl-6-methoxybenzofurans as selective ligands
for antiestrogen-binding sites. Effects on cell proliferation and
cholesterol synthesis. J. Med. Chem. 1992, 35, 1330-1339.
(27) Koch, K.; Biggers, M. S. General preparation of 7-substituted
4-chromanones: synthesis of a potent aldose reductase inhibitor.
J. Org. Chem. 1994, 59, 1216-1218.
Supporting Information Available: Elemental analysis
for selected compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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