Acetylcholinesterase inhibitors: SAR and kinetic studies on ω-[N-methyl-N-(3-alkylcarbamoyloxyphenyl]methyl] aminoalkoxyaryl derivatives

Article abstract of DOI:10.1021/jm010914b

In this work, we further investigated a class of carbamic cholinesterase inhibitors introduced in a previous paper (Rampa et al. J. Med. Chem. 1998, 41, 3976). Some new ω-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl analogues were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The structure of the lead compound (xanthostigmine) was systematically varied with the aim to optimize the different parts of the molecule. Moreover, such a structure-activity relationships (SAR) study was integrated with a kinetic analysis of the mechanism of AChE inhibition for two representative compounds. The structural modifications lead to a compound (12b) showing an IC₅₀ value for the AChE inhibition of 0.32 ± 0.09 nM and to a group of BuChE inhibitors also active at the nanomolar level, the most potent of which (15d) was characterized by an IC₅₀ value of 3.3 ± 0.4 nM. The kinetic analysis allowed for clarification of the role played by different molecular moieties with regard to the rate of AChE carbamoylation and the duration of inhibition. On the basis of the results presented here, it was concluded that the cholinesterase inhibitors of this class possess promising characteristics in view of a potential development as drugs for the treatment of Alzheimer's disease.

Full text of DOI:10.1021/jm010914b

3810
J . Med. Chem. 2001, 44, 3810-3820
Acetylch olin ester a se In h ibitor s: SAR a n d Kin etic Stu d ies on
ω-[N-Meth yl-N-(3-a lk ylca r ba m oyloxyp h en yl)m eth yl]a m in oa lk oxya r yl
Der iva tives
Angela Rampa, Lorna Piazzi, Federica Belluti, Silvia Gobbi, Alessandra Bisi, Manuela Bartolini,
Vincenza Andrisano, Vanni Cavrini, Andrea Cavalli, Maurizio Recanatini,* and Piero Valenti
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received May 2, 2001
In this work, we further investigated a class of carbamic cholinesterase inhibitors introduced
in a previous paper (Rampa et al. J . Med. Chem. 1998, 41, 3976). Some new ω-[N-methyl-N-
(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl analogues were designed, synthesized,
and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE). The structure of the lead compound (xanthostigmine) was
systematically varied with the aim to optimize the different parts of the molecule. Moreover,
such a structure-activity relationships (SAR) study was integrated with a kinetic analysis of
the mechanism of AChE inhibition for two representative compounds. The structural modifica-
tions lead to a compound (12b) showing an IC50 value for the AChE inhibition of 0.32 ( 0.09
nM and to a group of BuChE inhibitors also active at the nanomolar level, the most potent of
which (15d ) was characterized by an IC50 value of 3.3 ( 0.4 nM. The kinetic analysis allowed
for clarification of the role played by different molecular moieties with regard to the rate of
AChE carbamoylation and the duration of inhibition. On the basis of the results presented
here, it was concluded that the cholinesterase inhibitors of this class possess promising
characteristics in view of a potential development as drugs for the treatment of Alzheimer’s
disease.
Ch a r t 1. Drugs Presently Marketed for the Treatment
of AD
In tr od u ction
The study of new agents useful to treat Alzheimer’s
disease (AD) is one of the most active research fields in
both pharmaceutical industry and academia. AD is a
progressive neurodegenerative syndrome associated
with aging leading to the most common form of senile
dementia. The disease is characterized by the presence
of some neuropathological markers detected in the brain
of AD patients, which are the â-amyloid (âA) plaques
and the neurofibrillary tangles. A pathogenic role is
ascribed to these lesions, and many research programs
focused on drugs able to modify the course of the disease
are targeting both their formation and neurotoxicity.1
On the other hand, a number of pharmacological
agents are already available to treat AD, and although
they are not able to block the progression of the
neurodegeneration, still they are of help in managing
the symptoms of the disease. All these drugs (Chart 1)
can be considered as outcomes of the so-called cholin-
ergic hypothesis,2 that, at least in its earliest formula-
tion,3 predicted a favorable effect for compounds able
to sustain the central cholinergic tone. Tacrine, done-
pezil, and rivastigmine are acetylcholinesterase (AChE)
inhibitors that increase the levels of acetylcholine (ACh)
at the synapse by blocking the breakdown of the
neurotransmitter. Tacrine and donepezil act through a
competitive mechanism, while rivastigmine is an acy-
lating inhibitor able to covalently modify the enzyme
by transferring its carbamoyl group to the serine hy-
droxyl present in the active site.4
Ch a r t 2. Structures of ω-[N-Methyl-N-(3-alkylcarbam-
oyloxyphenyl)methyl]aminoalkoxyaryl Derivatives
In previous papers,5 we introduced and studied a class
of N-methyl-N-(3-carbamoyloxyphenyl)methylamino de-
rivatives characterized by the presence of an heteroaryl
moiety linked to the tertiary amino nitrogen through
an alkoxy chain (Chart 2). The structure-activity
relationships (SAR) of this series of AChE inhibitors
* To whom correspondence should be addressed. Tel: +39 051
2099720. Fax: +39 051 2099734. E-mail: mreca@alma.unibo.it.
10.1021/jm010914b CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/13/2001

Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3811
Ta ble 1. Physicochemical and Analytical Data of the Compounds Studied
compd
Ara
n
m
R
mp (°C)
yield (%)
formula
analysis
b
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
13
Z
Z
Z
Z
6
7
8
9
10
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
104-106
118-120
152-154
129-131
138-140
155-158
135-137
136-139
121-124
100-101
90-93
50
50
50
50
50
20
30
30
20
20
30
40
C29H32N2O5
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
c
c
c
c
c
c
c
c
C30H35ClN2O5
C31H37ClN2O5
C32H39ClN2O5
C33H41ClN2O5
C26H29ClN2O4
C26H31ClN2O3
C23H27ClN2O3
C19H25ClN2O3
Z
Z1
Z2
Z3
Z4
Z5
Z
b
C25H28N2O3
c
c
-
-
C27H25ClN2O5
C27H27ClN2O5
14
Z
158-160
b
15a
15b
15c
15d
Z
Z
Z
Z
3
3
3
3
6
7
8
9
94-97
66-68
32-33
35-36
15
20
15
20
C35H43N3O6
C, H, N
C, H, N
C, H, N
C, H, N
b
C36H45N3O6
b
C37H47N3O6
b
C38H49N3O6
a
b
See Chart 2 (General Structure) for the definitions of Ar, n, m, and R. Free base (crystallizing from ligroin). c Hydrochlorides
(crystallizing from methanol-ether).
were analyzed, and some conclusions were drawn
regarding the features of different parts of the molecular
skeleton.5b In particular, the dibenzopyran-4-one ring,
a three-carbon-atom chain, and a methyl group on both
the amino and the carbamic nitrogen atoms provided
the best results in term of inhibitory activity against
isolated AChE (IC50 ) 0.30 nM for the most potent
compound,5b xanthostigmine in Chart 2). The relevant
level of AChE inhibitory potency reached by some
compounds of the series prompted us to further explore
the SAR of the class, also paying attention to some
aspect of AChE inhibition that recently emerged in
connection with the use of anticholinesterase agents in
the treatment of AD. We thus decided to introduce
modifications in the linker chain, the carbamoyl sub-
stituent, and the aryl moiety of xanthostigmine and to
study the effects of such changes on the AChE inhibitory
activity. The ability to inhibit butyrylcholinesterase
(BuChE) was tested as well, to assess the selectivity of
the compounds. Finally, a kinetic study was carried out
on two representative members of the series, aimed at
characterizing the mechanisms of the AChE inhibitory
action for the xanthone-derived class of anticholinest-
erases.
properly extending the alkoxy chain of aminoalkoxyaryl
derivatives, it is possible to reach the external site, in
such a way as the heteroaryl moiety has the possibility
to establish π-stacking interactions with the indole ring
of Trp279. Accordingly, we designed the subset of
compounds 12a -e characterized by n ) 6-10 (Table
1).
In our previous work on the same class of carbamic
AChE inhibitors,5b we studied the SAR of the heteroaryl
moiety by replacing the xanthone ring with several
different nuclei based on the benzopyran skeleton. In
the series presented here, we aimed at probing the
relevance of the benzopyran oxygenated functions by
introducing a benzophenone (compound 12f) or other
aromatic moieties (compounds 12g-j) as aryl groups.
To explore whether the AChE gorge could tolerate less
flexible analogues, an ethyne fragment was introduced
within the linker chain (compound 13). Moreover, the
tertiary amine function was included in a tetrahydroiso-
quinoline nucleus, in such a way as to mimic the
N-methyl-N-benzyl fragment of xanthostigmine through
a partially saturated ring (compound 14).
In h ibitor Design . In the title compounds, the alkoxy
chain linking the aryl moiety to the tertiary amino
group was lengthened to allow the aryl ring to contact
the Trp279 side chain at the entrance of the AChE
gorge. This aromatic residue together with other ones
(Tyr70, Tyr121) is considered to constitute an essential
part of the AChE peripheral binding site,6 and recent
studies have shown that inhibitors binding to this site
are able to block the âA aggregation induced by AChE.7
A three-dimensional model of the binary complex be-
tween AChE and xanthostigmine5b showed that by
Substituents on the carbamic nitrogen of covalent
AChE inhibitors are known to affect the regeneration
time of the enzyme, in the sense that the presence of

3812 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Rampa et al.
F igu r e 1. Stereoview of the binary complex between 15b (magenta) and AChE. The carbamic N-heptyl chain can position the
protonated morpholine ring at a suitable distance from the indole ring of Trp279 to give a π-cation interaction. The model was
obtained by following the procedure described in ref 5b for inhibitor docking and complex minimization.
longer carbamic N-alkyl chains increases the recovery
time.8 Actually, in the series of heteroarylcarbamic
derivatives previously published,5b we detected such an
effect. Moreover, recently, Bartolucci et al.9 reported the
crystal structure of a carbamoylated AChE bearing a
dimethylmorpholinooctylcarbamic moiety covalently
bound to the catalytic serine of the enzyme. From this
study, it clearly appeared that the protonated morpho-
line interacted with the indole side chain of Trp279 via
a π-cation interaction. Thus, we attempted to combine
an increased duration of action with the possibility to
contact the AChE peripheral site by designing com-
pounds 15a -d , in which the carbamic N-alkyl chain
varies from 6 to 9 carbon units. Additionally, the
terminal morpholino group might counterbalance the
increase of lipophilicity due to the rather long alkyl
chains and improve the pharmacokinetic properties of
the compounds.10
To theoretically verify the binding hypothesis of the
above long chain morpholinoalkyl carbamates, we mod-
eled the tetrahedral intermediate formed by compound
15b in the AChE gorge. From the docking model shown
in Figure 1, it clearly appears that the morpholino ring
has the possibility to contact the peripheral site, pro-
vided that the N-alkyl chain is of appropriate length.
Ch em istr y. The synthesis of the studied compounds
was accomplished as illustrated in Scheme 1.
The selected hydroxy-derivatives were treated with
1-bromo-ω-chloroalkanes or ω-dibromoalkanes (accord-
ing to commercial availability) in the presence of K2-
CO3 to afford ω-haloalkoxy-derivatives 1a -j, which
were treated with NaI in refluxing methyl ethyl ketone
to give the corresponding ω-iodoalkoxy-intermediates
2a , 2f-j, 7. These compounds were then condensed with
N-(3-hydroxybenzyl)methylamine5b to afford 3a -j, 8 or
with 7-nitro-1,2,3,4-tetrahydroisoquinoline11 to give 9.
The nitro-derivative 9 was hydrogenated with Pd/C to
10, which was then diazotated to afford 11. The hy-
droxy-derivatives 3a -j, 11 were treated with methyl
isocyanate to give the desired compounds 12a -j, 14.
3-Hydroxyxanthone was treated with BrCH2CtCH in
the presence of K2CO3 to afford 4, which was condensed
with N-(3-benzyloxybenzyl)methylamine to afford 5,
debenzylated to 6, and then treated with methyl isocy-
anate to give 13.
presence of the inhibitor. The selectivity of the com-
pounds was also tested by determining their inhibitory
activity against BuChE.
The mechanism of the AChE inhibition was studied
in depth for two representative compounds of the series
(12a and 15b). As previously reported,13 the inhibition
of AChE by carbamates involves a reversible complex
(EC) formation, followed by carbamoylation of the
enzyme and production of a covalent adduct (F). The
carbamoylated enzyme is then hydrolyzed to regenerate
the free enzyme. The whole mechanism can be repre-
sented as follows:
k3
\ 98 F 98 E + Q
k5
E + C {kk
1} EC
2
After the reversible complex formation (EC; equilib-
rium constant: KC ≡ k2/k1), the carbamoylation phase
of the reaction is considerably more rapid than the
decarbamoylation phase (i.e., k3 > k5), and the two
phases can be characterized separately.13b,c Therefore,
we investigated on both the first kinetic phase (carbam-
oylation) of the inhibition process, by determining k3,
and the decarbamoylation stage, by determining k5, with
the final aim to compare the mode of action of the new
inhibitors with that of the reference compound physos-
tigmine.
To characterize the carbamoylation step, a traditional
stopped time assay13b was performed, in which AChE
and inhibitor were mixed in the assay buffer, and
aliquots were transferred to the spectrophotometer cell
at various times for the determination of the residual
AChE activity. The data were fitted to the following
equation
R ) R0 exp(-kobst) + R
(1)
where R, R0, and R are ratios of the inhibited enzyme
activity (vi) to the control activity (v0) at times t, 0, and
, respectively. In this way, the values of the observed
pseudo-first-order inhibition rate constant (kobs) for each
concentration of inhibitor were obtained. Double recip-
rocal plots of kobs versus inhibitor concentration ([I])
were then used to compute k3 and KC from the intercept
and from the ratio of the slope to the intercept, respec-
tively, according to the equation:
Compound 8 was treated with the appropriate ω-mor-
pholinoalkylisocyanates10 to afford compounds 15a -d .
En zym e In h ibition . The inhibitory activity against
AChE of the newly synthesized compounds was studied
using the method of Ellmann et al.12 to determine the
rate of hydrolysis of acetylthiocholine (ATCh) in the
1/kobs ) KC/k3 1/[I] + 1/k3
(2)
Concerning the characterization of the last stage of
inhibition (decarbamoylation), the carbamoylated en-
zyme prepared by incubating AChE with the inhibitor

Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3813
Sch em e 1a
a
Reagents: (i) Br(CH2)nX, K2CO3, reflux, 24 h; (ii) NaI, MeCOEt, reflux, 4 h; (iii) N-(3-hydroxybenzyl)methylamine (or 7-nitro-1,2,3,4-
tetrahydroisoquinoline for 9), reflux, 15 h; (iv) BrCH2CtCH, K2CO3, reflux, 24 h; (v) N-(3-benzyloxybenzyl)methylamine, CH2O, CuSO4,
EtOH/H2O, reflux, 24 h; (vi) AlCl3, C6H5NMe2, 0-5 °C, 4 h. (vii) CH3NCO, NaH, rt, 24 h; (viii) ω-morpholine-alkylisocyanate, NaH, rt, 24
h; (ix) H2, Pd/C, rt; (x) NaNO2, 0-5 °C, H+.
was dialyzed to monitor the regeneration of the inacti-
vated enzyme.5b The recovery of the carbamoylated
enzyme activity was determined by interrupting the
dialysis and testing the enzyme activity at fixed time
intervals up to 96 h. For each of the two inhibitors
studied, the enzyme activity (expressed as percentage
related to the noninhibited enzyme) was plotted against
the time of dialysis. The obtained data were fitted to
one-phase or two-phase exponential growth curves, and
the experimental rate constant (k5) was derived.
tions of the chain length (n in the general formula of
Chart 2) influence AChE inhibition more strongly than
BuChE inhibition. This feature is evident also from the
plots of Figure 2a,b, where the variations of activity
(expressed as pIC50) are reported as a function of the
number of methylene units of compounds 12a -e for the
inhibition of AChE and BuChE, respectively. The most
potent AChE inhibitor of the whole series is 12b (n )
7; IC50 ) 0.32 nM), that shows the same subnanomolar
potency as xanthostigmine (n ) 3; IC50 ) 0.30 nM5b),
while retaining a lower level of selectivity with respect
to BuChE inhibition (ratio BuChE/AChE 51.6, instead
of 1605b).
Resu lts
The inhibitory activities against both human eryth-
rocyte AChE and human serum BuChE of ω-[N-meth-
yl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxy-
aryl derivatives together with that of the reference
physostigmine are reported in Table 2, expressed as IC50
values. In Table 3, the equilibrium constant KC and the
rate constants k3 and k5 determined for the AChE
inhibition by 12a , 15b, and physostigmine are shown.
ACh E a n d Bu Ch E In h ibitor y Activity. From the
IC50 values of compounds 12a -e, it appears that varia-
With regards to the replacement of the xanthone
nucleus with different aryl groups (Ar in the general
formula of Chart 2), this causes a drop of AChE
inhibitory activity, as shown by the subset 12f-j.
However, compound 12f retains some activity, which
might suggest that the presence of a polar group on the
aromatic moiety is needed, to ensure a favorable inter-
action with the binding pocket into the gorge. Compound
12g carries another bridged aryl group and displays an

3814 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Rampa et al.
Ta ble 2. Inhibitory Activity on Isolated AChE and BuChE and IC50 Ratio of the Compounds Studied
compd
Ar
n
m
R
IC50 (nM) AChE
IC50 (nM) BuChE
IC50 ratio BuChE/AChE
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
13
Z
Z
Z
Z
6
7
8
9
10
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
1.4 ( 0.3
0.32 ( 0.09
6.8 ( 0.7
11.7 ( 0.5
21.8 ( 2.1
5.4 ( 0.8
13.9 ( 1.1
16.5 ( 1.4
25.2 ( 3.0
48.3 ( 3.4
31.6 ( 1.7
24.5 ( 2.6
22.1 ( 0.9
205 ( 27
9.9
51.6
3.7
4.1
1.4
4.5
0.9
2.5
0.3
0.8
2.5
2.7
Z
Z1
Z2
Z3
Z4
Z5
Z
24.7 ( 2.9
82.6 ( 4.1
79.6 ( 3.7
54.6 ( 1.8
210 ( 15
24.8 ( 2.2
42.6 ( 3.0
529 ( 91
-
-
14
Z
12.4 ( 1.3
34.1 ( 3.0
15a
15b
15c
Z
Z
Z
Z
3
3
3
3
6
7
8
9
21.1 ( 2.6
26.2 ( 2.2
27.4 ( 2.9
7.5 ( 0.8
4.9 ( 0.3
3.9 ( 0.4
0.35
0.2
0.1
15d
29.3 ( 4.8
14.1 ( 0.2
3.3 ( 0.4
0.1
1.6
p h ys.
23.1 ( 1.0
Ta ble 3. Kinetic and Thermodynamic Constants for the AChE
Inhibition
compd
KC (nM)
6.4 ( 0.5
k3 (min-1
2.3 ( 0.3
)
k5 (h-1
)
12a
15b
0.77 ( 0.05
0.06 ( 0.01
27.2 ( 3.2
33.5 ( 2.7
0.11 ( 0.01
0.41 ( 0.05
p h ysostigm in e
0.43 ( 0.02
intermediate level of potency between 12f and 12h -j.
The selectivity with respect to the BuChE inhibition is
almost negligible in this subset.
Compounds 13 and 14 containing some constraints
that hinder the mutual flexibility of the extreme aryl
and phenylcarbamic groups show different potencies on
both the cholinesterases but the same (almost null)
selectivity. The introduction of a rigid linear ethyne
moiety in the linking chain is detrimental for both the
AChE and BuChE inhibitory activities, indicating that
a flexible chain is necessary. On the other hand,
incorporation of the tertiary amino group into a satu-
rated ring is not likewise deleterious, even if the activity
decreases with respect to the lead compound, and is also
lower than that of other analogues (the subset 12a -d ).
Finally, the variation of the length of the carbamic
N-substituent has a remarkable effect on the selectivity
of the inhibitors, as compounds 15a -d show a higher
inhibitory activity on BuChE than on AChE. Another
interesting aspect of the IC50 data of this subset is that
the number of methylene units (m ) 6-9) of the
ω-morpholinoalkyl-substituent influences only slightly
the inhibitory potency, which ranges from 21.1 to 29.3
nM against AChE, and from 3.3 to 7.5 nM against
BuChE. Compound 15d is the most potent BuChE
inhibitor of this class of carbamic derivatives, showing
an IC50 value of 3.3 nM.
F igu r e 2. Plots of pIC50 values for AChE (a) and BuChE (b)
inhibition against the number of methylene units of com-
pounds 12a -e. IC50 values for the analogues with 3-5
methylene units were taken from ref 5b.
Mech a n ism of ACh E In h ibition . The equilibrium
and rate constants (KC and k3, respectively) of physos-
tigmine, 12a , and 15b were determined from eq 2
following the method reported by Feaster and Quinn13b
(Table 3). From the plots of Figure 3, it appears that
the two heteroaryloxyalkylamino compounds show a
time-dependent pattern of inhibition, which is similar
to that of physostigmine. The time course of the inhibi-
tion is characterized by an increase until to a steady
state, which is reached after 20 min, almost 2 h, and
40 min, in the case of 12a , 15b, and physostigmine,
respectively. The stability constants of the inhibitor-

Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3815
F igu r e 4. Time course of AChE regeneration after inhibition
by 12a and 15b.
bamoyloxyphenyl)methyl]aminoalkoxyaryl AChE in-
hibitors by appropriately varying some selected struc-
tural features of the molecules. The results presented
in Table 2, integrated with those reported in the
previous article on the same class of compounds,5b allow
us to definitely assess the relative importance of the
different parts of the molecules.
The length of the chain linking the (hetero)aryloxy
moiety to the basic nitrogen influences substantially the
AChE inhibitory potency, even if a simple correlation
between number of carbon units and activity does not
exist (Figure 2a). The compounds characterized by four
and seven methylene units, and showing IC50 values
equal to 37 nM5b and 0.32 nM (12b), respectively, fall
clearly out of the otherwise linear plot of Figure 2a,
indicating that some specific (unfavorable or favorable)
interaction strongly affects their binding to the AChE
gorge. These data suggest that the length and the
flexibility of the spacer chain affect in some rather
unpredictable way the interactions of the compounds
with the residues lining the gorge and do not allow the
establishment of a correlation with physicochemical
properties (like lipophilicity), due also to the simulta-
neous variation of size and lipophilicity in the subset.
The essential role of the intermediate chain flexibility
is pointed out by the weak anti-AChE activity of
compound 13 carrying the ethyne fragment (IC50 ) 210
nM). It is interesting to note that 13 has almost the
same value of calculated15 log P (4.93) as xanthostig-
mine (4.48). The inclusion of the tertiary amine nitrogen
in a cycle, like in compound 14, affects the binding
capability less than the stiffening of the chain, indicat-
ing that the tetrahydroisoquinoline ring mimics a
conformation of the N-methyl-N-benzylamine fragment
compatible with a correct orientation of the molecule
in the enzyme active site.
With regards to the aryl moiety, it appears from both
the data of Table 2 and the consideration of the previous
results5b that the presence of polar groups in this part
of the molecules is essential for a strong binding to
AChE. In fact, all the compounds lacking any polar
(generally oxygen) atom in the aryl ring lose activity
for at least 1 order of magnitude. In an attempt to
explain in terms of molecular properties the effects of
the variation of the aryl moieties on the AChE inhibitory
activity, we calculated the frontier orbitals for model
compounds representing the derivatives having the
same values of n (3), m (0), and R (CH3), and variable
aromatic nucleus. The HOMO and LUMO energies for
the different aryloxymethyl fragments (including those
of the corresponding compounds studied in ref 5b) are
F igu r e 3. Time course of AChE inhibition by 12a (a), 15b
(b), and physostigmine (c).
AChE complex (KC) and the rate constant of the car-
bamoyl-enzyme formation (k3) were different for 12a ,
15b, and physostigmine, compound 12a showing the
highest rate of carbamoyl-AChE formation, nearly 20
times higher than 15b. To further explore some aspects
of the carbamoylation of cholinesterases, the rate of
formation of the carbamoyl adduct (kobs) with BuChE
for the 7-morpholinoheptyl-derivative 15b (at 13 nM
inhibitor concentration) was also measured. The kobs
resulted equal to 1.009 min-1 for BuChE carbamoyla-
tion, while the same kinetic parameter for AChE was
0.0243 min-1
.
To study the last step of the enzymatic reaction, the
carbamoyl-AChE adducts formed by 12a and by 15b
were hydrolyzed to regenerate the free enzyme. The
percentage of recovered cholinesterase activity was
plotted against time (Figure 4). According to previous
studies,14,5b the velocity of this step was related to the
length of the carbamate N-alkyl chain. Compound 12a
(bearing the N-methylcarbamoyl substituent) showed a
two-phase activity recovery, with t1/2 values of 0.9 and
11.2 h, respectively, which seems to account for a two-
site binding mechanism. Instead, the 7-morpholinohep-
tyl-derivative 15b was still inhibited after 96 h (Figure
4). This last behavior was in agreement with the data
reported for long chain analogues of physostigmine.8
Discu ssion
One of the aims of the present work was to extend
the study of the SAR of the ω-[N-methyl-N-(3-alkylcar-

3816 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Rampa et al.
Ta ble 4. Frontier Orbital Energies of the Different Aryl
Moieties
dency to increase selectivity with the chain length.
However, these compounds are quite effective BuChE
inhibitors active at the nanomolar level (IC50 ) 3.3-
7.5 nM). Eptastigmine, the N-heptyl analogue of phy-
sostigmine, has an IC50 against BuChE of 5 nM,16 and,
recently, Yu et al. reported a phenserine-based selective
BuChE inhibitor showing an IC50 value of 1 nM.17
Compound 15d (IC50 ) 3.3 nM) can thus be considered
one of the most potent BuChE inhibitors currently
known, and it is worth further study aimed at improving
its selectivity, at the light of the recent hypothesis
regarding a role for BuChE inhibitors in the treatment
of AD.17,18 In particular, Giacobini proposed that severe
cases of AD might be treated by blocking the glial
BuChE with selective inhibitors, as such agents would
produce an increase of the brain levels of ACh without
eliciting cholinergic side effects.18
E(HOMO)a
E(LUMO)a
model compd
compd
(kcal/mol)
(kcal/mol)
(ref 5b)
-9.2237
-9.4540
-0.6828
-0.9246
(ref 5b)
(ref 5b)
(ref 5b)
-9.2011
-9.4383
-0.9110
-0.5254
The results of the kinetic experiments performed with
physostigmine, 12a , and 15b can be used to tentatively
explain some aspects of the AChE inhibition mecha-
nisms by the different carbamates, which might then
help to understand the different inhibitory potencies.
Indeed, the structure of the three molecules plays a role
in differentiating their inhibitory potency (IC50 values)
and also their kinetic mechanism of action. Physostig-
mine and 15b are widely different in structure and size;
still their IC50 values differ by less than two times. On
the contrary, 12a , not much different from 15b, shows
a 10-fold potency with respect to physostigmine. How-
ever, the inhibitor forming the most stable complex
(12a ) is the fastest one to give the carbamoyl adduct.
(ref 5b)
-9.3537
-9.3568
-0.6864
-0.3810
12f
12g
12h
12i
12j
-9.0769
-8.5933
-9.1140
-8.8296
-0.0804
-0.4155
0.3578
The different biological properties of 12a and 15b
considered thus far clearly indicate that a long inter-
mediate alkyl chain is favorable, while a long carbamic
N-alkyl chain is less optimal for AChE inhibition.
Looking at the KC and k3 values of 12a and 15b (Table
3), it might be hypothesized that these two structural
features influence the carbamoylation reaction more
than the stability of the enzyme-inhibitor noncovalent
complex. In any way, the N-alkyl carbamic side chain
could increase the bulk of the molecule to an extent
where the interaction with the active site is disturbed,
despite the relatively wide dimensions of the gorge. The
nucleophilic attack of the catalytic serine might be
hampered, because of the steric hindrance around the
carbamoyl group.
-0.0582
Energy values calculated by means of the PM3 program.20
a
reported in Table 4. There is no quantitative correlation
between pIC50 and HOMO or LUMO energies, but a
trend clearly emerges: it appears that the most potent
AChE inhibitors have the most negative values of both
orbital energies, while the least active derivatives show
the least negative values of both E(HOMO) and E(LU-
MO). This behavior might be interpreted in the sense
that compounds interact better with the active site
gorge, if they are able to favorably accept electrons in
their LUMO. Thus, the involvement of the LUMO
(which, in the whole molecules, is localized on the aryl
moiety) in the variation of activity might confirm the
hypothesis advanced in the previous paper5b that this
part of the molecule can establish π-stacking interac-
tions with aromatic residues lining the AChE gorge. On
the other hand, no relationships with lipophilicity or size
can be found.
The unfavorable effects of the carbamic N-alkyl chain
on AChE inhibition are reversed when considering
BuChE inhibition. This is evident by comparing the IC50
values against the two enzymes in the series 15a -d . A
clue to interpret such a behavior might be provided by
the consideration of the kobs values determined for the
inhibition of both enzymes by 15b: 1.009 min-1 for
BuChE carbamoylation and 0.0243 min-1 for AChE
carbamoylation. BuChE is characterized by a wider
gorge than AChE,19 and this might be the reason 15b
reacts faster with BuChE leading to a higher potency.
Compounds 12a -j, 13, and 14 considered thus far do
not seem to discriminate between AChE and BuChE,
as their BuChE/AChE IC50 ratios range between 10 and
0.3, with the only exception of 12b (51.6). Within these
small figures, three compounds (12g, 12i, 12j) show a
preference for the BuChE active site. Instead, all the
compounds characterized by long variable ω-morpholi-
noalkyl chains on the carbamic nitrogen (15a -d ) are
slightly selective for BuChE, displaying also the ten-
The results of the experiments of AChE activity
regeneration (illustrated in Figure 4) carried out with
12a and 15b, two xanthone derivatives displaying
different structural characteristics, suggest some ob-
servation about the terminal phase of the AChE-
inhibitors interaction. The recovery of enzyme activity
after inhibition by 12a shows a biphasic time course,13b

Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3817
element symbols, analytical results obtained for those elements
are within 0.4% of the theoretical values.
characterized by t1/2 of 0.9 and 11.2 h, respectively. This
behavior can be compared to that of physostigmine that
bears the same methylcarbamic fragment as 12a , even
if on a different molecular skeleton. As previously
reported,5b in our system, the physostigmine-inhibited
AChE recovers its activity with t1/2 ) 1.6 h, which
implies some difference in the regeneration mecha-
nisms, despite the identity of the hydrolyzed carbamoyl
fragments. The hypothesis might be advanced that the
released alcoholic moieties of the two inhibitors (esero-
line from physostigmine, 3-[N-methyl-N-(3-hydroxyben-
zyl)amino]hexyloxyxanthen-9-one (3a ) from 12a ) have
some different residual binding affinity for a secondary
site in the AChE gorge, that contributes to a longer
lasting inhibition.
3-(6-Ch lor oh exyloxy)xa n th en -9-on e (1a ). A stirred sus-
pension of 2.12 g (0.01 mol) of 3-hydroxyxanthen-9-one, 3.9 g
(0.02 mol) of 1-bromo-6-chlorohexane, and 1.4 g (0.01 mol) of
K2CO3 in dry acetone was refluxed for 24 h. The reaction was
monitored by TLC. The hot reaction mixture was filtered and
evaporated to dryness. The residue was crystallized from
toluene to give 2.3 g (70%) of 1a : mp 96-97 °C. 1H NMR δ
1.5 (m, 4H), 1.85 (m, 4H), 3.55 (t, 2H), 4.05 (t, 2H), 6.85-8.35
(m, 7H, Ar). Anal. (C19H19ClO3): C, H.
3-(7-Br om oh ep tyloxy)xa n th en -9-on e (1b). Using the
previous procedure and starting from 2.12 g (0.01 mol) of
3-hydroxyxanthen-9-one and 5.18 g (0.02 mol) of 1,7-dibromo-
heptane, 2.1 g (70%) of 1b was obtained: mp 90-93 °C
(toluene). 1H NMR δ 1.5 (m, 6H), 1.85 (m, 4H), 3.45 (t, 2H),
4.1 (t, 2H), 6.85-8.35 (m, 7H, Ar). Anal. (C20H21BrO3): C, H.
With regards to the dramatically longer duration of
action of the 7-morpholinoheptyl-derivative 15b (no
enzyme regeneration after 96 h), it is not a surprise at
the light of a similar result obtained with an N-heptyl
carbamate belonging to the series previously studied.5b
Also, the recently solved crystal structure of a carbam-
oylated AChE9 showed that the substantial irrevers-
ibility of the inhibitory action of 8-(2,6-dimethylmor-
pholino)octylcarbamoyleseroline (MF268) might be
ascribed to limited access of hydrolytic water molecules
to the carbamoylated active site, in consequence of the
filling of the AChE gorge by the long carbamic residue.
Such steric hindrance is further reinforced by the
π-cation interaction of the dimethylmorpholino group
with the Trp279 residue that anchors the hydrolyzed
fragment within the enzyme cavity. It is reasonable to
assume that the 7-morpholinoheptylcarbamoyl residue
of 15b exerts similar effects, thus leading to the
extremely slow reactivation of the inhibited enzyme.
3-(8-Br om ooctyloxy)xa n th en -9-on e (1c). Using the pre-
vious procedure and starting from 2.12 g (0.01 mol) of
3-hydroxyxanthen-9-one and 5.44 g (0.02 mol) of 1,8-dibro-
mooctane, 2.8 g (70%) of 1c was obtained: mp 138-141 °C
(toluene). 1H NMR δ 1.5 (m, 8H), 1.85 (m, 4H), 3.45 (t, 2H),
4.1 (t, 2H), 6.85-8.35 (m, 7H, Ar). Anal. (C21H23BrO3): C, H.
3-(9-Br om on on yloxy)xa n th en -9-on e (1d ). Using the pre-
vious procedure and starting from 2.12 g (0.01 mol) of
3-hydroxyxanthen-9-one and 5.72 g (0.02 mol) of 1,9-dibro-
mononane, 3.3 g (80%) of 1d was obtained: mp 100-102 °C
1
(toluene). H NMR δ 1.4 (m, 10H), 1.85 (m, 4H), 3.45 (t, 2H),
4.1 (t, 2H), 6.85-8.35 (m, 7H, Ar). Anal. (C22H25BrO3): C, H.
3-(10-Br om od ecyloxy)xa n th en -9-on e (1e). Using the
previous procedure and starting from 2.12 g (0.01 mol) of
3-hydroxyxanthen-9-one and 6.0 g (0.02 mol) of 1,10-dibromo-
decane, 3.02 g (70%) of 1e was obtained: mp 124-126 °C
1
(toluene). H NMR δ 1.4 (m, 12H), 1.85 (m, 4H), 3.45 (t, 2H),
4.1 (t, 2H), 6.85-8.35 (m, 7H, Ar). Anal. (C23H27BrO3): C, H.
[4-(3-Ch lor op r op oxy)p h en yl](p h en yl)m eth a n on e (1f).
Using the previous procedure and starting from 1.98 g (0.01
mol) of 4-hydroxyphenyl(phenyl)methanone and 3.14 g (0.02
mol) of 1-bromo-3-chloropropane, 1.85 g (70%) of 1f was
obtained: mp 53-56 °C. (ligroin). 1H NMR δ 2.25 (m, 2H),
Con clu sion s
3.75 (t, 2H), 4.2 (t, 2H), 6.9-7.85 (m, 9H, Ar). Anal. (C16H15
ClO2): C, H.
-
In this paper, the SAR of ω-[N-methyl-N-(3-alkylcar-
bamoyloxyphenyl)methyl]aminoalkoxyaryl AChE and
BuChE inhibitors were studied by the synthesis and
biological characterization of a series of purposely
designed analogues. The effects of different molecular
fragments on the AChE and BuChE inhibitory potency
were determined and were also discussed on the basis
of some structural and physicochemical properties. The
SAR considerations were integrated with the results of
a previous article5b dealing with the same class of
carbamic cholinesterase inhibitors.
The steps of the pseudoirreversible mechanism of
AChE inhibition by two representative compounds were
studied through the determination of the kinetic and
thermodynamic constants. The kinetic data allowed for
enlightenment of the role played by the variation of the
alkyl chain in two different positions of the molecular
skeleton in directing selectivity, affinity, and duration
of action of this kind of AChE inhibitors. The prolonged
duration of the anticholinesterase effect of the mor-
pholino and long alkyl chain derivatives should provide
the basis for their higher therapeutic potential.
1-Ben zyl-3-(3-ch lor op r op oxy)b en zen e (1g). Using the
previous procedure and starting from 1.74 g (0.01 mol) of
3-benzylphenol, 1.82 g (70%) of 1g was obtained: mp 37-39
°C (ligroin). 1H NMR δ 2.3 (m, 2H), 3.8 (t, 2H), 4.0 (s, 2H),
4.15 (t, 2H), 6.9-7.4 (m, 9H, Ar). Anal. (C16H17ClO): C, H.
2-(3-Ch lor op r op oxy)n a p h th a len e (1h ). Using the previ-
ous procedure and starting from 1.44 g (0.01 mol) of 2-naph-
thol, 1.5 g (70%) of 1h was obtained: mp 48-50 °C (toluene).
1H NMR δ 2.35 (m, 2H), 3.8 (t, 2H), 4.25 (t, 2H), 7.15-7.85
(m, 7H, Ar). Anal. (C13H13ClO): C, H.
1-(3-Ch lor op r op oxy)ben zen e (1i). Using the previous
procedure and starting from 0.94 g (0.01 mol) of phenol, 1.2 g
(90%) of 1i was obtained: oil. 1H NMR δ 2.25 (m, 2H), 3.75 (t,
2H), 4.2 (t, 2H), 6.9-7.4 (m, 5H, Ar). Anal. (C9H11ClO): C, H.
4-(3-Ch lor op r op oxy)bip h en yl (1j). Using the previous
procedure and starting from 1.46 g (0.01 mol) of 4-hydroxyl-
biphenyl, 1.7 g (70%) of 1j was obtained: mp 59-61 °C
1
(ligroin). H NMR δ 2.3 (m, 2H), 3.8 (t, 2H), 4.17 (t, 2H), 7.0-
7.6 (m, 9H, Ar). Anal. (C15H15ClO): C, H.
3-(6-Iod oh exyloxy)xa n th en -9-on e (2a ). A mixture of 3.3
g (0.01 mol) of 1a and 1.5 g (0.01 mol) of NaI in 30 mL of
methyl ethyl ketone was refluxed for 4 h. After cooling, the
separated solid was collected by filtration: 3.8 g (90%) of 2a
1
was obtained: mp 75-78 °C (ligroin). H NMR δ 1.5 (m, 4H),
1.85 (m, 4H), 3.2 (t, 2H), 4.05 (t, 2H), 6.8-8.35 (m, 7H, Ar).
Exp er im en ta l Section
Anal. (C19H19IO3): C, H.
Ch em istr y. Gen er a l Meth od s. All melting points were
determined in open glass capillaries, using a Bu¨chi apparatus,
and are uncorrected. 1H NMR spectra were recorded in CDCl3
solution on a Varian Gemini 300 spectrometer, with Me4Si as
internal standard. Wherever analyses are only indicated with
[4-(3-Iod op r op oxy)p h en yl](p h en yl)m eth a n on e (2f). Us-
ing the previous procedure and starting from 2.74 g (0.01 mol)
of 1f, 2.9 g (80%) of 2f was obtained: mp 199-203 °C. 1H NMR
δ 2.2 (m, 2H), 3.25 (t, 2H), 4.0 (t, 2H), 6.8-7.7 (m, 9H, Ar).
Anal. (C16H15IO2): C, H.

3818 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Rampa et al.
1-Ben zyl-3-(3-iod op r op oxy)ben zen e (2g). Using the pre-
vious procedure and starting from 2.6 g (0.01 mol) of 1g, 2.8 g
(80%) of 2g was obtained: mp 56-59 °C (toluene). H NMR δ
2.25 (m, 2H), 3.3 (t, 2H), 3.95 (s, 2H), 4.05 (t, 2H), 6.8-7.4 (m,
9H, Ar). Anal. (C16H17IO): C, H.
2-(3-Iod op r op oxy)n a p h th a len e (2h ). Using the previous
procedure and starting from 2.2 g (0.01 mol) of 1h , 2.5 g (80%)
of 2h was obtained: mp 128-130 °C. 1H NMR δ 2.25 (m, 2H),
3.35 (t, 2H), 4.1 (t, 2H), 7.0-7.7 (m, 7H, Ar). Anal. (C13H13IO):
C, H.
°C. 1H NMR δ 2.15 (m, 2H), 2.35 (s, 3H), 2.7 (t, 2H), 3.6 (s,
2H), 4.0 (t, 2H), 6.0 (broad, 1H, OH), 6.7-7.7 (m, 9H, Ar). Anal.
(C17H21NO2): C, H, N.
4-[3-[N-Met h yl-N-(3-h yd r oxyb en zyl)a m in o]p r op oxy]-
bip h en yl (3j). Using the previous procedure and starting from
1.23 g (0.005 mol) of 2j, 0.81 g (45%) of 3j was obtained: mp
78 °C. 1H NMR δ 2.1 (m, 2H), 2.27 (s, 3H), 2.6 (t, 2H), 2.7
(broad, 1H, OH), 3.5 (s, 2H), 4.1 (t, 2H), 6.7-7.6 (m, 9H, Ar).
Anal. (C24H27NO2): C, H, N.
1
3-(P r op a r gyloxy)xa n th en -9-on e (4). A stirred suspension
of 2.12 g (0.01 mol) of 3-hydroxyxanthen-9-one, 1.2 g (0.01 mol)
of propargyl bromide, 1.4 g (0.01 mol) of K2CO3 in dry acetone
was refluxed for 24 h. The reaction was monitored by TLC.
The hot reaction mixture was filtered and evaporated to
dryness. The residue was crystallized from toluene to give 2.25
1-(3-Iod op r op oxy)ben zen e (2i). Using the previous pro-
cedure and starting from 1.7 g (0.01 mol) of 1i, 2.3 g (90%) of
2i was obtained: oil. 1H NMR δ 2.25 (m, 2H), 3.35 (t, 2H), 4.0
(t, 2H), 6.9-7.4 (m, 5H, Ar). Anal. (C9H11IO): C, H.
4-(3-Iod op r op oxy)bip h en yl (2j). Using the previous pro-
cedure and starting from 2.46 g (0.01 mol) of 1j, 2.4 g (70%) of
2j was obtained: mp 60-61 °C (ligroin). 1H NMR δ 2.3 (m,
1
g (90%) of 4, mp 142-144 °C. H NMR δ 2.6 (m, 1H), 4.84 (m,
2H), 6.98-8.35 (m, 7H, Ar). Anal. (C16H10O3): C, H.
2H), 3.4 (t, 2H), 4.1 (t, 2H), 7.0-7.6 (m, 9H, Ar). Anal. (C15H15
IO): C, H.
-
3-[[4-[N-Meth yl-N-(3-ben zyloxyben zyl)a m in o]-2-bu ty-
n yl]oxy]xa n th en -9-on e (5). A solution of 1.15 g (4.6 mmol)
of 4 in EtOH/H2O 1:1 (15 mL) was added to a suspension of
CuSO4 (0.1 g), HCHO (0.5 mL), and 1.36 g (6.0 mmol) of N-(3-
benzyloxybenzyl)methylamine. H2SO4 was added until pH 8,
and the mixture was refluxed for 24 h. After cooling, 30 mL
NH3 was added, and the reaction mixture was extracted with
ether, dried (Na2SO4), and evaporated to dryness. The residue
purified by flash chromatography afforded 1.7 g (60%) of 5,
3-[[[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]h exyl]oxy]x-
a n th en -9-on e (3a ). A solution of 2.1 g (0.005 mol) of 2a and
1.37 g (0.01 mol) of N-(3-hydroxybenzyl)methylamine in 100
mL of toluene was refluxed for 15 h. After cooling, the reaction
mixture was washed (H2O), dried (Na2SO4), and evaporated
to dryness. The residue, on crystallizing from toluene, afforded
1
0.86 g (40%) of 3a : mp 92-95 °C. H NMR δ 1.4 (m, 6H), 1.8
1
(m, 2H), 2.2 (s, 3H), 2.4 (t, 2H), 3.45 (s, 2H), 4.05 (t, 2H), 6.7-
8.35 (m, 11H, Ar). Anal. (C27H29NO4): C, H, N.
oil. H NMR δ 2.3 (s, 3H), 3.4 (s, 2H), 3.55 (s, 2H), 4.9 (s, 2H),
5.15 (s, 2H), 6.8-8.4 (m, 16H, Ar). Anal. (C32H27NO4): C, H,
N.
3-[[[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]h ep tyl]oxy]-
xa n th en -9-on e (3b). Using the previous procedure and start-
ing from 1.94 g (0.005 mol) of 1b, 0.89 g (40%) of 3b was
obtained: mp 107-110 °C. 1H NMR δ 1.4 (m, 8H), 1.8 (m, 2H),
2.2 (s, 3H), 2.4 (t, 2H), 3.45 (s, 2H), 4.05 (t, 2H), 6.7-8.35 (m,
11H, Ar). Anal. (C28H31NO4): C, H, N.
3-[[4-[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]-2-bu tyn yl]-
oxy]xa n th en -9-on e (6). A solution of 1.7 g (3.48 mmol) of 5
in dry CH2Cl2 (35 mL) was stirred under N2 at 0 °C; N,N-
dimethylaniline (8.3 mL) was added, and the mixture was
stirred for 5 min. Then 1.8 g (14 mmol) of anhydrous AlCl3
was added, and the reaction was stirred at room temperature
for 6 h. The reaction mixture was quenched with H2O, and
the organic layer was washed with H2O, dried (Na2SO4), and
evaporated to dryness. The residue was purified by flash
chromatography, affording 0.8 g (60%) of 6: mp 195-197 °C.
1H NMR δ 2.25 (s, 3H), 3.3 (s, 2H), 3.45 (s, 2H), 4.85 (s, 2H),
6.7-8.4 (m, 11H, Ar). Anal. (C25H21NO4): C, H, N.
3-[3-(7-Nitr o-1,2,3,4-tetr a h yd r o-2-isoqu in olyl)p r op oxy]-
xa n th en -9-on e (9). To a solution of 3.8 g (0.01 mol) of 75b in
toluene was added 3.56 g (0.02 mol) of 7-nitro-1,2,3,4-tetrahy-
droisoquinoline,11 and the solution was refluxed for 12 h. The
reaction mixture was washed with H2O, dried (Na2SO4), and
evaporated to dryness. The residue purified by flash chroma-
tography (dichloromethane/ethyl acetate 3:2) afforded 1.3 g
(30%) of 9: mp 144-145 °C (toluene). 1H NMR δ 2.08-2.23
(m, 2H), 2.71-2.87 (m, 4H), 3.04 (t, 2H), 3.78 (s, 2H), 4.22 (t,
2H), 6.9-8.3 (m, 10H, Ar). Anal. (C25H22N2O5): C, H, N.
3-[3-(7-Am in o-1,2,3,4-tetr ah ydr o-2-isoqu in olyl)pr opoxy]-
xa n th en -9-on e (10). A solution of 2.15 g (0.005 mol) of 9 in
tetrahydrofuran (150 mL) was hydrogenated at room temper-
ature and atmospheric pressure over palladium on carbon. The
catalyst was filtered off, and the mixture was evaporated to
dryness. The residue was crystallized from toluene to yield 1.8
g (90%) of 10: mp 126-127 °C. 1H NMR δ 2.05-2.21 (m, 2H),
2.63-2.85 (m, 6H), 3.58 (s, 2H), 4.19 (t, 2H), 6.4-8.3 (m, 10H,
Ar). Anal. (C25H24N2O3): C, H, N.
3-[[[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]octyl]oxy]x-
a n th en -9-on e (3c). Using the previous procedure and starting
from 2.01 g (0.005 mol) of 1c, 0.92 g (40%) of 3c was obtained:
1
mp 110-112 °C. H NMR δ 1.4 (m, 10H), 1.8 (m, 2H), 2.2 (s,
3H), 2.4 (t, 2H), 3.45 (s, 2H), 4.05 (t, 2H), 6.7-8.4 (m, 11H,
Ar). Anal. (C29H33NO4): C, H, N.
3-[[[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]n on yl]oxy]x-
a n th en -9-on e (3d ). Using the previous procedure and starting
from 2.08 g (0.005 mol) of 1d , 0.94 g (40%) of 3d was
obtained: mp 82-85 °C. 1H NMR δ 1.4 (m, 12H), 1.8 (m, 2H),
2.2 (s, 3H), 2.4 (t, 2H), 3.45 (s, 2H), 4.05 (t, 2H), 6.7-8.35 (m,
11H, Ar). Anal. (C30H35NO4): C, H, N.
3-[[[N-Meth yl-N-(3-h yd r oxyben zyl)a m in o]d ecyl]oxy]x-
a n ten -9-on e (3e). Using the previous procedure and starting
from 2.15 g (0.005 mol) of 1e, 0.97 g (40%) of 3e was obtained,
mp 89-91 °C. 1H NMR δ 1.4 (m, 14H), 1.8 (m, 2H), 2.2 (s,
3H), 2.4 (t, 2H), 3.45 (s, 2H), 4.05 (t, 2H), 6.7-8.35 (m, 11H,
Ar). Anal. (C31H37NO4): C, H, N.
4-[[N-Meth yl-N-(3-h ydr oxyben zyl)am in o]pr opoxy]ph e-
n yl(p h en yl)m eth a n on e (3f). Using the previous procedure
and starting from 1.37 g (0.005 mol) of 2f, 0.75 g (40%) of 3f
was obtained as hydrochloride salt: mp 151-153 °C (MeOH/
ether). 1H NMR δ 2.0 (m, 2H), 2.2 (s, 3H), 2.55 (t, 2H), 3.45 (s,
2H), 4.05 (t, 2H), 6.7-7.9 (m, 13H, Ar). Anal. (C24H26ClNO3):
C, H, N.
3-[[[3-(3-Ben zylph en oxy)pr opyl](m eth yl)am in o]m eth yl]-
p h en ol (3g). Using the previous procedure and starting from
1.3 g (0.005 mol) of 2g, 0.72 g (40%) of 3g was obtained: mp
3-[3-(7-Hyd r oxy-1,2,3,4-t et r a h yd r o-2-isoq u in olyl)p r o-
p oxy]xa n th en -9-on e (11). A solution of 2.0 g (0.005 mol) of
10 in 20 mL of H2SO4 and 40 mL of H2O was cooled to -5 °C,
and a solution of 0.34 g (0.005 mol) of NaNO2 in 5 mL of H2O
was added dropwise. The solution was stirred for 30 min, then
poured in H2SO4 1:2 (150 mL), and stirred in a steam bath for
30 min. The mixture was allowed to stand overnight, and the
resulting precipitate was collected by filtration, washed with
H2O, and crystallized from ethanol to give 0.8 g (40%) of 11:
mp 193-194 °C. 1H NMR δ 2.06-2.18 (m, 2H), 2.61-2.88 (m,
6H), 3.62 (s, 2H), 4.20 (t, 2H), 6.8-8.3 (m, 10H, Ar). Anal.
(C25H23NO4): C, H, N.
1
72-75 °C. H NMR δ 2.0 (m, 2H), 2.2 (s, 3H), 2.6 (t, 2H), 3.45
(s, 2H), 3.95 (m, 4H), 6.7-7.3 (m, 13H, Ar). Anal. (C24H27
NO2): C, H, N.
-
3-[[(Meth yl)[3-(2-n a p h th yloxy)p r op yl]a m in o]m eth yl]-
p h en ol (3h ). Using the previous procedure and starting from
1.1 g (0.005 mol) of 2h , 0.64 g (40%) of 3h was obtained: mp
80-84 °C. 1H NMR δ 2.15 (m, 2H), 2.35 (s, 3H), 2.8 (t, 2H),
3.7 (s, 2H), 4.05 (t, 2H), 6.7-7.8 (m, 11H, Ar). Anal. (C21H23
NO2): C, H, N.
-
3-[[(Met h yl)(3-p h en oxyp r op yl)a m in o]m et h yl]p h en ol
(3i). Using the previous procedure and starting from 0.85 g
(0.005 mol) of 2i, 0.81 g (60%) of 3i was obtained: mp 78-80
Gen er a l Meth od for th e P r ep a r a tion of Ca r ba m a tes
(12a -12j, 13, 14, 15a -d ). A mixture of the selected ω-[N-

Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3819
alkyl-N-(3-hydroxy)benzyl]aminoalkoxy derivative (0.001 mol),
the selected alkyl isocyanate (0.001 mol), and 10 mg of NaH
in dry toluene was stirred at room temperature for 24 h,
quenched with water, and then extracted with dichloromethane.
The organic layer was washed with water, dried, and evapo-
rated to dryness. The residue was purified by crystallization
or by flash chromatography.
was analyzed in triplicate. The percent inhibition of the
enzyme activity due to the presence of increasing test com-
pound concentration was calculated by the following expres-
sion: 100 - (vi/v0 × 100), where vi is the rate calculated in
the presence of inhibitor and v0 is the enzyme activity.
Inhibition curves were obtained for each compound by plotting
the percent inhibition versus the logarithm of inhibitor
concentration in the assay solution. The linear regression
parameters were determined for each curve and the IC50
extrapolated.
1H NMR for Com p ou n d s 12a -12j, 13, 14, 15a -d . 12a :
δ (CDCl3) 1.45 (m, 6H), 1.75 (m, 2H), 2.1 (s, 3H), 2.3 (t, 2H),
2.8 (d, 3H), 3.4 (s, 2H), 4.0 (t, 2H), 4.85 (broad, 1H, NH), 6.8-
8.3 (m, 11H, Ar). 12b: δ (CDCl3) 1.5 (m, 8H), 1.85 (m, 2H), 2.2
(s, 3H), 2.35 (t, 2H), 2.8 d, 3H), 3.5 (s, 2H), 4.1 (t, 2H), 4.9
(broad, 1H, NH), 6.8-8.35 (m, 11H, Ar). 12c: δ (CDCl3) 1.45
(m, 10H), 1.75 (m, 2H), 2.1 (s, 3H), 2.3 (t, 2H), 2.8 (d, 3H), 3.4
(s, 2H), 4.0 (t, 2H), 4.85 (broad, 1H, NH), 6.8-8.3 (m, 11H,
Ar). 12d : δ (CDCl3) 1.45 (m, 12H), 1.75 (m, 2H), 2.1 (s, 3H),
2.3 (t, 2H), 2.8 (d, 3H), 3.4 (s, 2H), 4.0 (t, 2H), 4.85 (broad, 1H,
NH), 6.8-8.3 (m, 11H, Ar). 12e: δ (CDCl3) 1.45 (m, 14H), 1.8
(m, 2H), 2.15 (s, 3H), 2.35 (t, 2H), 2.8 (d, 3H), 3.4 (s, 2H), 4.1
(t, 2H), 4.95 (broad, 1H, NH), 6.8-8.4 (m, 11H, Ar). 12f: δ
(DMSO-d6) 2.25 (t, 2H), 2.65 (d, 3H), 2.7 (s, 3H), 3.2 (t, 2H),
4.1-4.5 (m, 4H), 7.0-7.8 (m, 13H, Ar). 12g: δ (DMSO-d6) 2.15
(t, 2H), 2.65 (d, 3H), 2.7 (s, 3H), 3.2 (t, 2H), 3.85 (s, 2H), 4.0 (t,
2H), 4.3 (m, 2H), 6.8-7.8 (m, 13H, Ar). 12h : δ (DMSO-d6) 2.25
(t, 2H), 2.65 (d, 3H), 2.75 (s, 3H), 3.2 (t, 2H), 4.1-4.5 (m, 4H),
7.1-7.9 (m, 11H, Ar). 12i: δ (DMSO-d6) 2.25 (t, 2H), 2.65 (d,
3H), 2.7 (s, 3H), 3.2 (t, 2H), 4.0 (t, 2H), 4.3 (m, 2H), 6.9-7.8
(m, 9H, Ar). 12j: δ (CDCl3) 2.0 (m, 2H), 2.25 (s, 3H), 2.6 (t,
2H), 2.87 (d, 3H), 3.55 (s, 2H), 4.08 (t, 2H), 6.95-7.58 (m, 13H,
Ar). 13: δ (DMSO-d6) 2.65 (s, 3H), 2.75 (d, 3H), 4.1 (s, 2H),
4.3 (s, 2H), 5.2 (s, 2H), 7.1-8.2 (m, 11H, Ar). 14: δ (CDCl3)
2.24-2.43 (m, 2H), 2.66 (d, 3H), 3.29-3.48 (m, 4H), 3.71-3.83
(m, 2H), 4.25-4.68 (m, 4H), 7.04-8.12 (m, 10H, Ar), 10.80
(broad, 1H, NH).15a : δ (CDCl3) 1.2-1.6 (m, 8H), 2.05 (t, 2H),
2.3 (m, 5H), 2.45 (m, 4H), 2.6 (t, 2H), 3.25 (m, 2H), 3.55 (s,
2H), 3.75 (m, 4H), 4.2 (t, 2H), 5.05 (broad, 1H, NH), 6.8-8.4
(m, 11H, Ar). 15b: δ (CDCl3) 1.3-1.6 (m, 10H), 2.05 (t, 2H),
2.3 (m, 5H), 2.45 (m, 4H), 2.6 (t, 2H), 3.25 (m, 2H), 3.55 (s,
2H), 3.75 (m, 4H), 4.2 (t, 2H), 5.0 (broad, 1H, NH), 6.9-8.4
(m, 11H, Ar). 15c: δ (CDCl3) 1.2-1.6 (m, 12H), 2.05 (t, 2H),
2.3 (m, 5H), 2.45 (m, 4H), 2.55 (t, 2H), 3.25 (m, 2H), 3.5 (s,
2H), 3.7 (m, 4H), 4.2 (t, 2H), 5.0 (broad, 1H, NH), 6.8-8.4 (m,
11H, Ar). 15d : δ (CDCl3) 1.2-1.6 (m, 14H), 2.05 (t, 2H), 2.3
(m, 5H), 2.45 (m, 4H), 2.55 (t, 2H), 3.25 (m, 2H), 3.5 (s, 2H),
3.7 (m, 4H), 4.2 (t, 2H), 5.0 (broad, 1H, NH), 6.8-8.4 (m, 11H,
Ar).
In h ibition of ACh E a n d Bu Ch E. The method of Ellman12
was followed. ATCh iodide solution (0.037 M) was prepared
in water. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s
reagent) (0.01 M) was dissolved in pH 7.0 phosphate buffer,
and 0.15% (w/v) NaHCO3 was added. AChE solution was
prepared dissolving 20 units in 5 mL of 0.2% aqueous gelatin
by sonication at 35 °C. A dilution 1:1 with water was performed
before use, to obtain the enzyme activity comprised between
0.13 and 0.100 AU/min. Stock solutions of the test compounds
(0.5-1 mM) were prepared in ethanol as well as the physos-
tigmine reference stock solution. The assay solutions were
prepared by diluting the stock solutions in water. Five different
concentrations of each compound were used in order to obtain
inhibition of AChE activity comprised 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, 0.035 unit/mL
AChE derived from human erythrocytes (Sigma Chemical),
and 550 µM ATCh iodide. The final assay volume was 1 mL.
Test compounds were added to the assay solution and prein-
cubated with the enzyme for 20 min, the addition of substrate
following.
Inhibition of BuChE was measured as described above,
substituting 0.035 unit/mL of BuChE from human serum and
550 µM butyrylthiocholine (BuTCh) for enzyme and substrate,
respectively.
Kinetic characterization of carbamoylation events. The
stopped time assay was performed, in which AChE and
inhibitors at five concentrations comprised in the range 0.34-
1.35 nM (12a ), 0.81-57 nM (15b), and 10-200 nM (physos-
tigmine as reference compound) were mixed in the assay buffer
at pH 8.0. After few minutes incubation at 37 °C, the
determination of residual activity of the AChE-catalyzed
hydrolysis of ATCh was followed spectrophotometrically at 412
nm. A parallel control (i.e., no inhibitors in the mixture)
allowed to adjust activities measured at various time.
The data were fitted to eq 1, and kobs values were calculated
accordingly. KC and k3 values were calculated from the plots
of kobs against [I] (eq 2).
Deca r ba m oyla tion Kin etics. Stock solutions in replicates
were prepared as following: the free enzyme (1.0 units in 2
mL of 0.1 M phosphate buffer pH 8.0), the same amount of
enzyme plus 12a (1.39 × 10-8 M) and 15b (2.6 × 10-7 M).
The solutions were gently stirred and incubated at 37 °C
for 20 min. They were then loaded into 12.000 MW cutoff
dialysis bags and dialyzed at 4 °C against 2 L of 0.1 M
phosphate buffer pH 8.0. Dialysis was interrupted after 6-12-
18-24-48-96 h. The solutions were diluted five times with
the same buffer and kept at 20-22 °C under magnetic stirring.
Aliquots of 0.95 mL of each solution were sampled, and the
enzyme activity was spectrophotometrically tested after ad-
dition of 0.034 mL of DTNB and 0.016 mL of ATCh as above-
described. Plots of enzyme activities for each compound
expressed as a percentage of the dialyzed free enzyme activity
against the time of dialysis were calculated for each inhibitors.
The obtained curves were fitted to one phase exponential
growth or two phase exponential growth with the Prism 3.0
program and the experimental rate constants k5 derived. Half
time is 0.69/k5.
Ack n ow led gm en t. Investigation supported by Uni-
versity of Bologna (funds for selected research topics)
and by MURST. M.R. thanks Corwin Hansch for the
access to the C-QSAR program.
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