Design, synthesis, and biological evaluation of conformationally restricted rivastigmine analogues

Article abstract of DOI:10.1021/jm049782n

Rivastigmine (1), an acetylcholinesterase (AChE) inhibitor approved in 2000 for the treatment of Alzheimer disease, bears a carbamate moiety in its structure, which is able to react covalently with the active site of the enzyme. Kinetic and structural stu

Full text of DOI:10.1021/jm049782n

J. Med. Chem. 2004, 47, 5945-5952
5945
Design, Synthesis, and Biological Evaluation of Conformationally Restricted
Rivastigmine Analogues
Maria Laura Bolognesi, Manuela Bartolini, Andrea Cavalli, Vincenza Andrisano, Michela Rosini,
Anna Minarini, and Carlo Melchiorre*
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received March 19, 2004
Rivastigmine (1), an acetylcholinesterase (AChE) inhibitor approved in 2000 for the treatment
of Alzheimer disease, bears a carbamate moiety in its structure, which is able to react covalently
with the active site of the enzyme. Kinetic and structural studies on the interaction of 1 with
different cholinesterases have been published, giving deeper, but not definitive, insights on
the catalysis mechanism. On the basis of these findings and in connection with our previous
studies on a series of benzopyrano[4,3-b]pyrrole carbamates as AChE inhibitors, we designed
a series of conformationally restricted analogues of 1 by including the dimethylamino-R-
methylbenzyl moiety in different tricyclic systems. A superimposition between the conformation
of 1 and the carbon derivative 4, as obtained from Monte Carlo simulations, supported the
idea that the tricyclic derivatives might act as rigid analogues of 1. The biological profile of
4-9, assessed in vitro against human AChE and BChE, validated our rational design.
Compound 5, bearing a sulfur-containing system, showed the highest inhibitory activity, being
192-fold more potent than 1. In the present study, the most potent inhibitors were always
methyl derivatives 3-5, endowed with a nanomolar range potency, whereas the ethyl ones
were 40 times less potent. A reasonable explanation for this finding might be a steric hindrance
effect between the ethyl group of 1 and His440 in the active site, as already suggested by the
crystal structure of the complex AChE/1. The unfavorable influence of the carbamic N-alkyl
chain on AChE inhibition is less striking when considering BChE inhibition, since BChE is
characterized by a bigger acyl binding pocket than AChE. In fact, methyl carbamates 3-5 did
not show AChE/BChE selectivity, whereas compounds 6-9 were significantly more potent in
inhibiting BChE than AChE activity. At 100 µM, 5 was found to inhibit the AChE-induced
aggregation only by 19% likely because it is not able to strongly interact with the peripheral
anionic site of AChE, which plays an essential role in the Aâ aggregation mediated by the
enzyme but is lacking in BChE structure.
Introduction
cholinergic agonist cevimeline,8 prescribed in the U.S.
and Japan for Sjogren’s syndrome and currently in
clinical trial for AD (Chart 1).
Alzheimer’s disease (AD), the most common cause of
dementia, is a complex neurological affection that is
clinically characterized by loss of memory and progres-
sive deficits in different cognitive domains. The consis-
tent neuropathologic hallmark of the disorder, generally
noted on postmortem brain examination, is a massive
deposit of aggregated protein breakdown products,
amyloid plaques, and neurofibrillary tangles. Even if the
primary cause of AD is still speculative, the amyloid
plaques are thought to be mainly responsible for the
devastating clinical effects of the disease.1 In recent
years, significant research attention has also been
devoted to the role of free radical formation, oxidative
cell damage, and inflammation in the pathogenesis of
AD, providing new promising targets and validated
animal models.2 To date, however, the enhancement of
the central cholinergic function is the only clinical
effective approach.3,4 The intensive research of drugs
able to improve the cholinergic transmission in AD has
produced so far five launched products, the acetylcho-
linesterase (AChE) inhibitors tacrine,5 donepezil,6 ri-
vastigmine, and galantamine,7 and the muscarinic
Rivastigmine (1, Figure 1), in particular, represents
a newer-generation inhibitor,9 approved in 2000 under
the name of Exelon, endowed with a carbamate struc-
ture that makes it a slow substrate able to react
covalently with the active site of the enzyme. It belongs
to a series of miotine (2, Figure 1) derivatives,10 all of
which display inhibitory action toward AChE, but 1 was
selected for clinical trials because of its 10-fold greater
affinity for brain monomeric G1 over the peripheral form
of the enzyme, its chemical stability, and good toler-
ability. In addition, 1 has an “in vivo” peculiar phar-
macological profile;11 it shows interesting selectivity for
the brain regions more affected by the neuronal degen-
eration,12 has a dual inhibitory action on both AChE
and butyrylcholinesterase (BChE), and has demon-
strated broad benefits across the severity of AD and also
in patients with the Lewy body variant of AD.13 Recent
evidence suggests that in AD brain, BChE activity rises,
while AChE activity remains unchanged or declines.
Therefore, both enzymes are likely involved in regulat-
ing acetylcholine (ACh) levels and, consequently, may
represent legitimate therapeutic targets for the develop-
* To whom correspondence should be addressed. Phone: +39-051-
2099706. Fax: +39-051-2099734. E-mail: carlo.melchiorre@unibo.it.
10.1021/jm049782n CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/14/2004

5946 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Bolognesi et al.
Chart 1. Chemical Structures of Cholinergic Ligands for the Treatment of AD
Figure 2. Superimposition of a PM3 minimized low-energy
conformation of 4 (carbon atoms are green) onto a PM3
minimized low-energy conformation of 1. The pharmacophoric
functions of 4 fit very well those of 1. The rigid analogue 4
might indicate the bioactive conformation of rivastigmine.
Figure 1. Design strategy for the synthesis of 4-9 by
inserting the dimethylaminoethylphenyl moiety of 1 in differ-
ent tricyclic systems related to 3.
binding site, which are still in debate. In addition, the
role of the carbamoyl nitrogen substituent of 3 was
investigated through the synthesis of different aryl and
alkyl carbamates (6-9).
ment of agents such as 1, which, with the ability to
inhibit BChE in addition to AChE, should lead to
improved clinical outcomes.14
Chemistry
Recent kinetic and structural studies on the interac-
tion of 1 with different cholinesterases gave deeper, but
not definitive, insights into the mechanism of catalysis.15
On the basis of these findings and in connection with
our previous studies16 on a series of benzopyrano[4,3-
b]pyrrole carbamates as AChE inhibitors, we designed
a series of conformationally restricted analogues of 1
and 2 by including the dimethylamino-R-methylbenzyl
moiety in different tricyclic systems related to 3 (Figure
1). To support this basic idea, a superimposition between
the conformation of 1 and the carbon derivative 4, as
obtained from Monte Carlo simulations, was carried out.
Low-energy conformations of each molecule were se-
lected and fitted, as shown in Figure 2. Clearly, the
overlap was satisfactory (rmsd ) 0.28 Å), confirming
that the tricyclic derivatives might act as rigid ana-
logues of 1. Thus, the isosteric replacement of the
endocyclic oxygen of 3 with a sulfur or a carbon atom,
leading to 4 or 5 respectively, might provide information
about the physicochemical requirements of the enzyme
Aryl-functionalized tricyclic pyrrolidines have very
few synthetic precedents in the literature,17 so the
development of an efficient synthesis for target com-
pounds 4 and 5 appealed to us. The previous pathway
to 3, which involved a [3 + 2] dipolar cycloaddition of a
2-allyloxybenzaldeyde, was not deemed practical for the
synthesis of the carbon analogue 4, since the prepara-
tion of the corresponding 2-butenylbenzaldehyde was
not as feasible. Thus, a different synthetic strategy
starting from the commercially accessible tetralone 10
was followed (Scheme 1). Alkylation of the lithium salt
of 5-methoxytetralone with bromoacetonitrile in rigor-
ously anhydrous conditions furnished nitrile 11, which
in turn was cyclized to 12 through catalytic hydrogena-
tion over Raney Ni.18 The coupling constant observed
for the ring-fusion protons (J ) 6.5 Hz) was consistent
with the reported value for cis isomer 3,16 and the cis
stereochemistry was further secured from detailed
nuclear Overhauser effect (NOE) studies. Methylation
of the amine function of 12 and subsequent dealkylation

Rivastigmine Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5947
Scheme 1
Scheme 2
Scheme 3
of the methoxy group of 13 gave phenol 14, which upon
reaction with the appropriate isocyanate afforded final
compounds 4 and 9.
In contrast, the synthesis of 5 (Scheme 2) could be
performed using the strategy previously described for
3,16 through reaction of the key intermediate 2-allyl-
sulfanyl-3-methoxybenzaldehyde (16) and sarcosine tri-
methylsilyl ester. The preparation of 16 was readily
accomplished in one pot by sequential treatment of
1519,20 with sodium hydroxide and allyl bromide. After
formation of the tricyclic derivative 17, the synthesis
proceeded as depicted for 4 in Scheme 1 and, in
agreement with the parent system, the cis fusion ring
was assigned through the coupling constant of the
benzylic methine proton.
Finally, carbamoylation of 19 to give the different
carbamates 6-8 was accomplished as reported for the
preparation of 4 (Scheme 3).
It should be mentioned that the enantiomer separa-
tion of the different racemic mixtures was not per-
formed, since we16 and others21 have noticed that the
chirality does not seem to strongly affect the inhibitory
activity of this class of 1-related compounds.
Biology
To determine the potential interest of compounds 4-9
for the treatment of AD, their AChE inhibitory activity

5948 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Bolognesi et al.
Table 1. Inhibition of AChE and BChE Activities by 1-Related
7 turned out to be significantly more potent than both
rivastigmine (1) and the parent derivative miotine (2),
confirming the rational design carried out. Among the
restricted analogues, 5 showed the most high inhibitory
activity, being 192- and 12-fold more potent than 1 and
2, respectively. A reasonable explanation for this phe-
nomenon is that the tricyclic system of the rigid series
acts as a conformationally restricted mimic of the 3-[1-
(dimethylamino)ethy]phenyl fragment of 1, as revealed
by Monte Carlo conformational analyses. Actually, 1
diplayed as many as 29 low-energy conformations
(within an energy window of 3 kcal/mol from the global
minimum), whereas the rigid carbon analogue 4 adopted
only two possible low-energy conformations. In this
respect, the conformation of 4, shown in Figure 2, might
indicate the bioactive conformation of 1. The restricted
compounds have higher affinity because they do not
incur the entropic penalty experienced when 1 and 2,
having a freely rotating skeleton, bind.
Compounds
IC50 ( SEM (nM)a
compd
X
R
AChE
BChE
1
2
3
4
5
6
7
8
9
rivastigmine
1535 ( 64
100 ( 33
301 ( 14
406 ( 13
59.4 ( 2.3
24.5 ( 1.6
miotine
O
Me
Me
Me
30.0 ( 1.7
17.3 ( 1.2
CH2
S
8.11 ( 0.28 10.5 ( 0.5
CH(CH3)Ph 1870 ( 230 209 ( 3
O
O
(CH2)6CH3
Et
Et
20.3 ( 3.1
420 ( 5
393 ( 33
1.26 ( 0.04
44.41 ( 1.8
30.3 ( 2.5
O
CH2
a Human recombinant AChE and BChE from human serum
were used. IC50 values represent the concentration of inhibitor
required to decrease enzyme activity by 50% and are the mean of
two independent measurements, each performed in triplicate. The
incubation time was between 20 and 60 min, according to the
kinetic type.
However, it is well established that several structural
elements determine AChE inhibitory activity of car-
bamate derivatives. For instance, the alkyl substituent
on the carbamoyl nitrogen strongly affects the affinity
profile. In our series of rigid compounds, the most potent
inhibitors resulted in methyl derivatives 3-5, all en-
dowed with potency in the nanomolar range. Increasing
the length of the alkyl chain to ethyl resulted in a 40-
fold reduction in both the oxygen and carbon series (3
vs 8 and 4 vs 9). A similar behavior was shown by 6,
carrying a bulky R-1-phenylethyl carbamate on the [1]-
benzopyrano[4,3-b]pyrrole scaffold. The potency was
restored to a value similar to that of the methyl
derivative 3 by lengthening the chain to n-heptyl,
affording 7, which displayed an IC50 value of 20.3 nM.
These results are in good agreement with the higher
potency reported for monosubstituted carbamate de-
rivatives of 1 carrying a methyl group instead of an
ethyl one.21,24 The anomalous ethyl effect was also
observed by Lieske and co-workers,25 who found, in a
series of indolinyl carbamates, the diethyl derivative to
be about 7400-fold less potent than the dimethylcar-
bamoyl analogue. Actually, to support these experimen-
tal data, the crystal structure of AChE/rivastigmine
complex has disclosed a steric hindrance effect between
the ethyl group of 1 and His440 in the active site,
causing a significant movement of this amino acid away
from its normal partner, Glu327, and resulting in the
disruption of the catalytic triad.15 However, the high
affinity displayed by 7 is not in line with a negative
steric effect between the N-alkyl substituent of the
inhibitor and His440 of the enzyme. Clearly, the n-
heptyl group of 7 interacts in such a way that the
catalytic triad of the active site of AChE is not nega-
tively affected as one would expect simply on the basis
of steric hindrance.
Figure 3. Time course of AChE inhibition at IC50 concentra-
tions for compounds 1-8.
was determined by the method of Ellman et al.22 on
human recombinant AChE. Furthermore, to establish
the selectivity of 4-9, their BChE inhibitory activity
was also calculated by the same method on BChE from
human serum. The inhibitory potency was expressed as
IC50, which represents the concentration of inhibitor
required to decrease the enzyme activity by 50%. To
allow comparison of the results, 1-3 were used as the
reference compounds.
Finally, the ability of 5 to inhibit the proaggregating
action of AChE toward â-amyloid (Aâ) was assessed
through a thioflavin T-based fluorometric assay,23 in
comparison with 1.
Results and Discussion
The inhibitory potency, expressed as IC50 values, of
compounds 4-9 of AChE and BChE is reported in Table
1 in comparison with that of analogue 3 and the two
reference compounds 1 and 2. The overlaid time course
of inhibition experiments at the IC50 concentrations of
inhibitors 1-8 is shown in Figure 3.
The most interesting finding is that the potency
toward AChE is generally increased in the rigidified [1]-
benzopyrano[4,3-b]pyrrole derivatives compared to the
flexible prototype 1. In particular, derivatives 4, 5, and
The results at BChE had a potency trend similar to
that observed at AChE: rigid carbamates 3-5 were 1
order of magnitude more potent than flexible compounds
1 and 2 but, unlike 1 which displayed a preferential
BChE selectivity, showed similar IC50 values for AChE
and BChE inhibition. Recent evidence suggests that
beyond the regulation of synaptic ACh levels BChE may
also have a role in the etiology and progression of AD.
Consequently, the new compounds, which have a dual

Rivastigmine Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5949
methoxyphenyl) ester (15)19,20 were prepared according to
inhibitory action on both AChE and BChE, might show
a better therapeutic profile in AD and related demen-
tia.14 The unfavorable effect of the carbamic N-alkyl
chain on AChE inhibition was reversed when consider-
ing BChE inhibition, as revealed by comparing the IC50
values of the bulky derivatives 6-9 for the two enzymes.
A reasonable explanation might be that BChE is
characterized by a bigger acyl binding pocket than
AChE,26 thus accounting for the lower clash effect of a
wider substituent toward BChE.
Exchanging the oxygen or the carbon atom with a
sulfur (5 vs 3 and 4) resulted in a slight increase of both
AChE and BChE inhibition, suggesting a favorable
short-range interaction between the aromatic moiety of
Trp86 (AChE) and Trp82 (BChE) of the active sites and
the sulfur atom of the inhibitor.27
The lack of AChE/BChE selectivity is consistent with
the finding that our inhibitors weakly prevented the
proaggregating effect of AChE toward Aâ in a thioflavin
T-based fluorometric assay previously reported.23,28 At
100 µM, 5 was found to inhibit the AChE-induced
aggregation only by 19% likely because it is not able to
strongly interact with the peripheral anionic site (PAS)
of AChE, which plays an essential role in Aâ aggrega-
tion mediated by the enzyme, whereas it is lacking in
BChE. In this respect, docking simulations, purposely
addressed to identify a possible binding mode of 5
toward PAS of AChE, turned out to be unable to
establish a univocal binding mode, as a possible conse-
quence of the quite low affinity of present inhibitors
toward this site. In contrast, 1, which showed a slightly
higher affinity for BChE, was completely unable to act
as a proaggregating inhibitor (<5% inhibition at 100
µM), whereas 5 showed a slightly higher affinity for
AChE and a weak effect against the proaggregating
activity of AChE. Inhibitors able to bind to both sites of
AChE are usually weaker inhibitors of BChE and more
potent inhibitors for the proaggregating effect induced
by AChE, suggesting that AChE/BChE selectivity might
be relevant to establish the capability of a new molecule
to contrast Aâ aggregation.29
literature methods.
(5-Methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)-
acetonitrile (11). A solution of 1.6 N n-butyllithium (6.87 mL,
0.011 mol) in hexane was slowly added to a cold (-60 °C)
solution of diisopropylamine (1.68 mL, 0.011 mol) in dry THF
(5 mL). After 0.5 h, a solution of 5-methoxytetralone (1.76 g,
0.010 mol) in THF (2 mL) was added dropwise, followed by
the addition of bromoacetonitrile (0.77 mL, 0.011 mol). The
temperature was allowed to warm from -60 °C to room
temperature, and the reaction mixture was stirred for 16 h.
It was diluted with water and extracted with EtOAc. The
organic layer was dried and concentrated to give a brown oily
residue, which was purified by flash chromatography. Eluting
with petroleum ether/EtOAc (8.5:1.5), the second fraction
corresponded to 11 (230 mg, 11% yield) as clear solid: mp 104-
105 °C; EI-MS m/z ) 215 (M+); IR νmax 2238 cm-1 (CN); 1H
NMR (CDCl3, 200 MHz) δ 7.65 (d, 1H), 7.31 (t, 1H), 7.07 (d,
1H), 3.89 (s, 3H), 3.35-3.20 (dq, 1H), 3.08-2.98 (dd, 1H), 2.95-
2.75 (m complex, 2H), 2.70-2.45 (m complex, 2H), 2.10-1.85
(m, 1H); 13C (CDCl3, 200 MHz) δ 195.99, 156.56, 132.58, 132.34,
127.10, 118.80, 118.45, 114.57, 77.63, 77.00, 76.37, 55.63,
43.87, 27.89, 22.41, 18.25.
cis-6-Methoxy-2,3,3a,4,5,9b-hexahydro-1H-benzo[g]in-
dole (12). A solution of 11 (230 mg, 1.07 mmol) in absolute
EtOH (15 mL) was hydrogenated in the presence of Raney
nickel (0.2 g) at atmospheric pressure and room temperature
for 2 days. The mixture was filtered through Celite, and the
filtrate was evaporated to give a residue, which was purified
by flash chromatography. Eluting with CH2Cl2/MeOH/NH3
(9.5:0.5:0.05) afforded 12 (100 mg, 46% yield) as brown oil: 1H
NMR (CDCl3, 400 MHz) δ 7.15 (t, 1H), 7.07 (d, 1H), 6.72 (d,
1H), 4.07 (d, 1H), 3.84 (br s, 1H exch), 3.80 (s, 3H), 3.20-3.08
(m, 1H), 2.85-3.00 (m + td, 1H + 1H), 2.45-2.30 (m complex,
2H), 2.20-2.10 (m, 1H), 1.78-1.73 (m, 1H), 1.70-1.60 (m, 1H),
1.45-1.35 (dq,1H).
cis-6-Methoxy-1-methyl-2,3,3a,4,5,9b-hexahydro-1H-
benzo[g]indole (13). Formic acid (96%, 2.01 mL, 17.4 mmol)
was added dropwise to 12 (100 mg, 0.49 mmol), and then
formaldehyde (37%, 1.8 mL) was added to the resulting
mixture, which was heated at 90 °C for 10 h, cooled (0 °C),
diluted with water, and washed with CH2Cl2. The aqueous
layer was then made basic with aqueous 40% NaOH and
extracted with CH2Cl2 (4×). Removal of the dried solvents gave
a residue, which was purified by flash chromatography.
Eluting with CH2Cl2/MeOH/NH3 (9.3:0.7:0.07) afforded 13 (70
mg, 65% yield) as a clear oil: 1H NMR (CDCl3, 200 MHz) δ
7.28-7.12 (m, 1H), 6.84-6.78 (m, 2H), 3.83 (s, 3H), 3.15-3.05
(m + d, 1H + 1H), 2.86-2.61 (m complex, 2H), 2.59-2.43 (m,
1H), 2.36 (s, 3H), 2.31-2.21 (m, 1H), 2.15-2.00 (m, 1H), 1.86-
1.52 (m, 3H).
Experimental Section
Chemistry. Melting points were taken in glass capillary
tubes on a Bu¨chi SMP-20 apparatus and are uncorrected. IR,
electron impact (EI) mass spectra, and direct infusion ESI
mass spectra were recorded on Perkin-Elmer 297, VG 7070E,
and Waters ZQ 4000 apparatus, respectively. 1H NMR and
COSY experiments were recorded on Mercury 400, Varian
VXR 200, and Varian VXR 300 instruments. Chemical shifts
are reported in parts per million (ppm) relative to tetrameth-
ylsilane (TMS), and spin multiplicities are given as s (singlet),
br s (broad singlet), d (doublet), t (triplet), or m (multiplet).
Although the IR spectral data are not always included (because
of the lack of unusual features), they were obtained for all
compounds reported and were consistent with the assigned
structures. The elemental compositions of the compounds
agreed to within (0.4% of the calculated value. When the
elemental analysis is not included, crude compounds were used
in the next step without further purification. Chromatographic
separations were performed on silica gel columns by flash
(Kieselgel 40, 0.040-0.063 mm; Merck) or gravity column
(Kieselgel 60, 0.063-0.200 mm; Merck) chromatography.
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.
Miotine30 (2) and dimethylthiocarbamic acid S-(2-formyl-6-
cis-1-Methyl-2,3,3a,4,5,9b-hexahydro-1H-benzo[g]indol-
6-ol (14). A sample of 1 M BBr3 in CH2Cl2 (2 mL) was added
to a stirred and cooled (0 °C) solution of 13 (70 mg, 0.32 mmol)
in anhydrous CHCl3 (4 mL) under a stream of dry nitrogen.
When the addition was completed, the reaction mixture was
stirred at room temperature for 4 h. After the mixture was
cooled at 0 °C, excess BBr3 was destroyed by cautious dropwise
addition of anhydrous MeOH (10 mL). The resulting mixture
was heated at reflux for 2 h. Removal of the solvent gave a
viscous solid, which was purified by flash chromatography.
Eluting with CH2Cl2/MeOH/NH3 (9.6:0.4:0.04) afforded 13 (50
mg, 77% yield) as a clear oil: 1H NMR (CDCl3, 200 MHz) δ
6.83 (t, 1H), 6.65 (d, 1H), 6.33 (d, 1H), 5.32 (br s, 1H exch),
3.20 (m + d, 1H + 1H), 2.78-2.53 (m complex, 3H), 2.49 (s,
3H), 2.43-2.29 (m, 1H), 2.13-1.96 (m, 1H), 1.92-1.70 (m
complex, 2H), 1.61-1.43 (m, 1H).
cis-Methylcarbamic Acid 1-Methyl-2,3,3a,4,5,9b-hexahy-
dro-1H-benzo[g]indol-6-yl Ester Hydrochloride (4). MeN-
CO (4 µL, 0.068 mol) was added to a stirred mixture of 14 (10
mg, 0.050 mmol) and NaH (1 mg, 0.044 mmol) in anhydrous
THF (2 mL). Stirring was continued for 20 h at room temper-
ature, and then removal of the solvent gave a residue that was
transformed into the hydrochloride salt: 80% yield; mp 180-

5950 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Bolognesi et al.
183 °C (from EtOH/ether); MS (ESI+) m/z ) 261.2 (M + H)+;
1H NMR (CD3OD, 300 MHz) δ 7.45-7.39 (m, 2H), 7.21 (t, 1H),
4.55 (d, 1H), 3.84-3.71 (m, 1H), 3.40-3.25 (m, 1H), 3.04 (s,
3H), 3.00-2.84 (m, 1H), 2.80 (s, 3H), 2.75 (t, 2H), 2.61-2.41
(m, 1H), 2.10-1.90 (m, 1H), 1.89-1.70 (m, 2H). Anal. (C15H21-
ClN2O2) C, H, N.
2-Allylsulfanyl-3-methoxybenzaldehyde (16). A solution
of 15 (2.80 g, 0.012 mol) in MeOH (11 mL) was stirred under
reflux under nitrogen for 1 h with aqueous 10% NaOH (5.6
mL, 0.014 mol). The cooled alkaline mixture was then treated
with a solution of allyl bromide (1.12 mL, 0.013 mol) in MeOH
(25 mL) and stirred at room temperature overnight. The
formed white solid was filtered off, and the filtrate evaporated
to give a residue, which was purified by flash chromatography.
Eluting with petroleum ether/EtOAc (9:1) afforded 16 as brown
oil: 65% yield; 1H NMR (CDCl3, 300 MHz) δ 10.72 (s, 1H),
7.51 (d, 1H), 7.40 (t, 1H), 7.11 (d, 1H), 5.80-5.60 (m, 1H), 4.88-
4.80 (m, 2H), 3.95 (s, 3H), 3.47 (d, 2H).
cis-6-Methoxy-1-methyl-1,2,3,3a,4,9b-hexahydro-5-thia-
1-azacyclopenta[a]naphthalene (17). A mixture of N-
methylglycine (2.13 g, 0.024 mol) and chlorotrimethylsilane
(30.3 mL, 2.39 mmol) was gently refluxed under nitrogen for
3 h. After the mixture was cooled, the formed solid was
collected by filtration and washed with dry ether. To a
suspension of this solid in dry toluene (70 mL) were added
diisopropylethylamine (6.56 mL, 37.7 mmol) and 16 (1.35 g,
6.5 mmol), the reaction mixture was heated under reflux, and
the water that was formed continuously was removed for 18
h. Removal of the solvent gave an oil that was partitioned
between aqueous NaHCO3 and CH2Cl2. The organic phase was
dried and evaporated to give a residue that was purified by
flash chromatography. Eluting with petroleum ether/EtOAc/
MeOH/NH3 (8:1.5:0.5:0.05) afforded 17 as a clear oil: 60%
yield; 1H NMR (CDCl3, 300 MHz) δ 7.11 (t, 1H), 6.84-6.78
(m, 2H), 3.89 (s, 3H), 3.18-3.10 (d + m, 2H), 3.00-2.80 (m,
1H), 2.74 (d, 2H), 2.30-2.20 (s + m, 3H + 1H), 2.12-2.02 (m,
2H).
cis-1-Methyl-1,2,3,3a,4,9b-hexahydro-5-thia-1-azacyclo-
penta[a]naphthalen-6-ol (18). It was synthesized from 17
(400 mg, 0.017 mol) following the procedure described for 14:
60% yield; EI-MS m/z ) 221 (M+); 1H NMR (CDCl3, 200 MHz)
δ 7.10 (t, 1H), 6.90-6.77 (m, 2H), 4.85 (br s, 1H exch), 3.21-
3.14 (d + m, 2H), 3.14-2.90 (m, 1H), 2.79-2.59 (m, 2H), 2.34-
2.20 (s + m, 4H), 2.19-2.05 (m, 2H).
cis-Methylcarbamic Acid 1-Methyl-1,2,3,3a,4,9b-hexahy-
dro-5-thia-1-azacyclopenta[a]naphthalen-6-yl Ester Hy-
drochloride (5). It was synthesized from 18 (95 mg, 0.43
mmol) following the procedure described for 4: 40% yield; mp
202-206 °C; 1H NMR (free base, CDCl3, 200 MHz) δ 7.20-
7.00 (m, 3H), 5.20 (br s, 1H exch), 3.20-3.10 (d + m, 2H), 3.00-
2.80 (d + m, 4H), 2.80-2.60 (m, 2H), 2.40-2.20 (s + m, 3H +
1H), 2.15-1.15 (m, 2H). Anal. (C14H19ClN2O2S) C, H, N.
cis-R-(1-Phenylethyl)carbamic Acid 1-Methyl-1,2,3,-
3a,4,9b-hexahydro-5-oxa-1-azacyclopenta[a]naphthalen-
6-yl Ester Hydrochloride (6). It was synthesized from 1916
(70 mg, 0.34 mmol) and R-(+)-1-phenylethyl isocyanate fol-
lowing the procedure described for 4: 80% yield; mp 196-200
°C; EI-MS m/z ) 352 (M+);1H NMR (free base, CDCl3, 300
MHz) δ 7.40-7.20 (m, 5H), 7.10-6.95 (d, 2H), 7.85 (t, 1H),
5.35 (br s, 1H exch), 5.00-4.90 (m, 1H), 4.10-4.00 (m, 1H),
4.95-4.80 (t, 1H), 3.10-3.00 (t, 1H), 3.00-2.90 (d, 1H), 2.40-
2.25 (s + q, 5H), 2.10-2.00 (m, 1H), 1.60-1.40 (d + m, 4H);
1H NMR (CD3OD, 300 MHz) δ 8.15 (br s, 1H exch), 7.37-7.26
(m, 5H), 7.25-7.18 (m, 2H), 7.10-7.05 (t, 1H), 4.90-4.70 (m,
1H), 4.70-4.50 (m, 1H), 4.15-4.00 (m, 1H), 3.95-3.80 (m, 1H),
3.75-3.60 (m, 1H), 3.06 (s + m, 3H + 2H), 2.60-2.40 (m, 1H),
2.10-1.90 (m, 1H), 1.49 (d, 3H). Anal. (C21H25ClN2O2) C, H,
N.
exch), 4.13-4.06 (m, 1H), 3.92 (t, 1H), 3.24 (q, 2H), 3.14-3.05
(m, 1H), 2.97 (d, 1H), 2.50-2.35 (s + m, 3H + 1H), 2.34 (q,
1H), 2.18-2.00 (m, 1H), 1.60-1.20 (m complex, 11H), 0.91 (t,
3H). Anal. (C20H31ClN2O3) C, H, N.
cis-Ethylcarbamic Acid 1-Methyl-1,2,3,3a,4,9b-hexahy-
dro-5-oxa-1-azacyclopenta[a]naphthalen-6-yl Ester Hy-
drochloride (8). It was synthesized from 1916 (100 mg, 0.49
mmol) and ethyl isocyanate following the procedure described
1
for 4: 52% yield; mp 176 °C dec; H NMR (free base, CDCl3,
200 MHz) δ 7.09-7.02 (m, 2H), 6.91 (t, 1H), 5.12 (br s, 1H
exch), 4.13-4.05 (m, 1H), 3.91 (t, 1H), 3.39-3.25 (m, 2H),
3.14-3.04 (m, 1H), 2.97 (d, 1H), 2.55-2.38 (m + s, 3H + 1H),
2.32 (q, 1H), 2.14-1.98 (m, 1H), 1.55-1.39 (m, 1H), 1.27-1.15
(m, 3H). Anal. (C15H21ClN2O3) C, H, N.
cis-Ethylcarbamic Acid 1-Methyl-2,3,3a,4,5,9b-hexahy-
dro-1H-benzo[g]indol-6-yl Ester Hydrochloride (9). It
was synthesized from 14 (50 mg, 0.25 mmol) and ethyl
isocyanate following the procedure described for 4: 30% yield;
EI-MS m/z ) 274 (M+); 1H NMR (free base, CDCl3, 200 MHz)
δ 7.18 (t, 1H), 7.06-7.01 (m, 2H), 5.04 (br s, 1H exch), 3.39-
3.25 (m, 2H), 3.13-3.06 (m, 2H), 2.64-2.51 (m complex, 3H),
2.33-2.21 (s + m, 3H + 1H), 2.15-2.01 (m, 1H), 2.00-1.53
(m complex, 3H), 1.22 (t, 3H). Anal. (C16H23ClN2O2) C, H, N.
NMR Analysis. To assign the protons at positions 3a and
9b of the benzoindole system, the structure of 12 was deter-
mined by 2D 1H and 13C NMR spectroscopy. Irradiation of the
doublet signal produced by the hydrogen atom 9b at 4.07 ppm
resulted in a positive nuclear Overhauser effect (NOE) in the
multiplet corresponding to the 3a proton (2.30-2.34), confirm-
ing the cis ring fusion. 1H, 13C, and 2D NMR spectra were
measured using a 20 mg sample of 12 dissolved in 0.7 mL of
CDCl3 and measured on a Mercury 400 MHz spectrometer at
30 °C.
Biology. Inhibition of AChE and BChE. The method of
Ellman et al.22 was followed. 1 was obtained by extraction from
Exelon tablets, and the purity was confirmed by HPLC. AChE
stock solution was prepared by dissolving 1000 units of
lyophilized powder (Sigma Chemical) in 0.1 M phosphate
buffer (pH 8.0) containing Triton X-100, 0.1%. BChE stock
solution was prepared by dissolving 100 units of lyophilized
powder (Sigma Chemical) in aqueous gelatin solution (0.1%
w/v). Enzymes stock solutions were diluted before use to reach
an activity ranging between 0.13 and 0.100 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 to obtain inhibition of AChE or
BChE activity between 20% and 80%. The assay solution
consisted of 0.1 M phosphate buffer (pH 8.0), with the addition
of 340 µM DTNB (Ellman’s reagent), AChE or BChE, and 550
µM acetylthiocholine iodide (ATCh). The final assay volume
was 1 mL. Initial rate assays were performed at 37 °C with a
Jasco V-530 double beam spectrophotometer: the rate of
increase in the absorbance at 412 nm was followed for 5 min.
Test compounds were added to the assay solution and prein-
cubated with the enzyme for times between 20 and 60 min,
according to the kinetic type, followed by the addition of
substrate. Assays were done with a blank containing all
components except AChE or BChE to account for nonenzy-
matic reaction. The reaction rates were compared, and the
percent inhibition due to the presence of the test compound
was calculated. Each concentration was analyzed in triplicate,
and IC50 values were determined graphically from the log
concentration-inhibition curves.
Inhibition of AChE Induced Aâ Aggregation. Thioflavin
T (Basic Yellow 1), human recombinant AChE lyophilized
powder, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and Triton
X-100 were purchased from Sigma Chemicals. Buffers and
other chemicals were of analytical grade. Absolute DMSO over
molecular sieves was from Fluka. Water was deionized and
doubly distilled. Aâ (1-40), supplied as 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.
cis-Heptylcarbamic Acid 1-Methyl-1,2,3,3a,4,9b-hexahy-
dro-5-oxa-1-azacyclopenta[a]naphthalen-6-yl Ester Hy-
drochloride (7). It was synthesized from 1916 (90 mg, 0.44
mmol) and heptyl isocyanate following the procedure described
for 4: 79% yield; mp 160-161 °C; 1H NMR (free base, CDCl3,
200 MHz) δ 7.10-7.03 (m, 2H), 6.89 (t, 1H), 5.09 (br s, 1H

Rivastigmine Analogues
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 5951
Aliquots of 2 µL of Aâ peptide, lyophilized from 2 mg mL-1
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 of
2.30 µM, Aâ/AChE molar ratio of 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 concentra-
tion ranging between 10 and 250 µM) were added. Blanks
containing Aâ, AChE, and Aâ, plus inhibitors at 100 µM, 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 method was then applied.23,31,32 The fluorescence
intensities were compared and the percent inhibition due to
the presence of test compounds was calculated. The percent
inhibition of the AChE induced aggregation due to the pres-
ence of the inhibitor was calculated by the following expres-
sion: 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.33
Molecular Modeling. (1) Construction of the Models.
1 and 4 were built by properly modifying the 1-(1-(dimethyl-
amino)ethyl)-7-methyl-8,9-dihydro-7H-5-oxa-7-azabenzocyclohepten-6-one
skeleton retrieved from the Cambridge Structural Database
(CSD code KEXXUG). The models were minimized first by
using steepest descent and then conjugate gradient until a
convergence of 0.05 kcal mol-1 Å-1 on the gradient was
reached. Then conformational analyses were carried out, and
the minimum energy conformers were further optimized at
semiempirical Hamiltonian level PM3,34 as implemented in the
SYBYL 6.8 (Tripos Inc., St. Louis, MO) graphic interface to
MOPAC (keywords PRECISE and MMOK).
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(2) Conformational Analyses. Monte Carlo conforma-
tional analyses35 were carried out by means of the MacroModel
package software,36 using the MMFF force field37 and the
generalized Born/surface area (GB/SA) continuum solvation
model to simulate the aqueous environment.38 In a Monte
Carlo study, the phase space of a molecule is sampled by
randomly changing dihedral angle rotations or atom positions.
Then the trial conformation is accepted if its energy has
decreased from the previous one. If the energy is higher,
various criteria can be applied to accept or reject the Monte
Carlo trial. In the present simulations, the number of Monte
Carlo steps was set equal to 5000 and the trial conformation
was accepted if the energy was lower than that of the previous
conformation or if its energy was within an energy window of
100 kJ/mol. Then the conformations were grouped by means
of a cluster analysis39 using geometrical parameters as filtering
screens. The combined use of Monte Carlo and cluster analyses
has turned out to be a potent means for sampling the
conformational space of highly flexible molecules.40
(3) Docking Simulations. To identify the possible binding
mode of the compounds at the AChE PAS, 100 docking
simulations were performed by means of the DOCK 4.0.1
package software41 and using the cocrystal between the AChE
and Fasciculin (PDB code 1B41).42 Fasciculin was removed
from the complex, and the truncated residues Glu268, Gln291,
Glu369, and Arg522 were properly completed by means of the
Biopolymer module of SYBYL 6.8. Hydrogen atoms were added
to the protein amino acids, and the atomic partial charges from
the all-atom Amber force field were loaded.43
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Acknowledgment. This work was supported by
grants from the University of Bologna (Funds for
Selected Research Topics) and MIUR.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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