Structure-activity relationships of acetylcholinesterase noncovalent inhibitors based on a polyamine backbone. 2. Role of the substituents on the phenyl ring and nitrogen atoms of caproctamine

Article abstract of DOI:10.1021/jm021055+

Continuing our studies on polyamine-based compounds of potential interest in the field of Alzheimer's disease therapeutics, we investigated the structure - activity relationships (SAR) of a lead compound (caproctamine, 3) identified in a previous work. In particular, we varied the substituents on the phenyl ring and on the nitrogen functions (both the amine and the amide), and studied the effects of such modifications on the inhibitory potency against isolated acetyl- and butyryl-cholinesterase (AChE and BChE). Moreover, the ability of selected compounds to reverse the D-tubocurarine-induced neuromuscular blockade and their antagonism toward muscarinic M₂ receptors in guinea pig left atrium were assayed. The most interesting SAR result was the identification of a relationship between the electronic characteristics of 2-substituents (measured by pK_a) and the AChE inhibitory potency (pIC₅₀) of tertiary amine compounds 6-12, which was confirmed by the invariance of the pIC₅₀ values of the corresponding methiodide derivatives 14-20. With regard to the biological profile, the most interesting compound was the N-ethyl-analogue of caproctamine (9), that showed pIC₅₀ values of 7.73 (±0.02) and 5.65 (±0.03) against AChE and BChE, respectively. The ability to increase the acetylcholine level was maintained in the functional assay (pAI₅₀ for reversing the neuromuscular blockade was 6.45 (±0.07)), as well as the ability to antagonize the M₂ receptors (pK_b = 5.65 (±0.06)). Moreover, 9 showed a long duration of action as AChE inhibitor, an useful property in view of a possible development of this compound as a therapeutic agent.

Full text of DOI:10.1021/jm021055+

954
J . Med. Chem. 2003, 46, 954-966
Str u ctu r e-Activity Rela tion sh ip s of Acetylch olin ester a se Non cova len t
In h ibitor s Ba sed on a P olya m in e Ba ck bon e. 2. Role of th e Su bstitu en ts on th e
P h en yl Rin g a n d Nitr ogen Atom s of Ca p r octa m in e
Vincenzo Tumiatti,† Michela Rosini,† Manuela Bartolini,† Andrea Cavalli,† Gabriella Marucci,‡
Vincenza Andrisano,† Piero Angeli,‡ Rita Banzi,† Anna Minarini,† Maurizio Recanatini,† and Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and Department of
Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
Received October 2, 2002
Continuing our studies on polyamine-based compounds of potential interest in the field of
Alzheimer’s disease therapeutics, we investigated the structure-activity relationships (SAR)
of a lead compound (caproctamine, 3) identified in a previous work. In particular, we varied
the substituents on the phenyl ring and on the nitrogen functions (both the amine and the
amide), and studied the effects of such modifications on the inhibitory potency against isolated
acetyl- and butyryl-cholinesterase (AChE and BChE). Moreover, the ability of selected
compounds to reverse the D-tubocurarine-induced neuromuscular blockade and their antagonism
toward muscarinic M2 receptors in guinea pig left atrium were assayed. The most interesting
SAR result was the identification of a relationship between the electronic characteristics of
2-substituents (measured by pKa) and the AChE inhibitory potency (pIC50) of tertiary amine
compounds 6-12, which was confirmed by the invariance of the pIC50 values of the
corresponding methiodide derivatives 14-20. With regard to the biological profile, the most
interesting compound was the N-ethyl-analogue of caproctamine (9), that showed pIC50 values
of 7.73 ((0.02) and 5.65 ((0.03) against AChE and BChE, respectively. The ability to increase
the acetylcholine level was maintained in the functional assay (pAI50 for reversing the
neuromuscular blockade was 6.45 ((0.07)), as well as the ability to antagonize the M2 receptors
(pKb ) 5.65 ((0.06)). Moreover, 9 showed a long duration of action as AChE inhibitor, an useful
property in view of a possible development of this compound as a therapeutic agent.
In tr od u ction
mission through the activation of the synaptic nicotinic
and muscarinic receptors. It has been demonstrated that
AChE could play a key role during an early step in the
development of the senile plaques, as revealed by the
finding that AChE accelerates â-amyloid peptide (Aâ)
deposition.9 Active site inhibitors, such as edrophonium,
did not affect this peculiar feature of AChE, which was
affected by peripheral binding site ligands, such as
decamethonium and propidium. Interestingly enough,
butyrylcholinesterase (BChE), an enzyme that lacks the
peripheral binding site, did not affect amyloid formation.
This finding suggests clearly that the catalytic site of
AChE does not participate in the interaction of the
enzyme with Aâ, whereas it is possible that the periph-
eral binding site of AChE may be involved in amyloid
formation.9
Consequently, the design of ligands able to interact
simultaneously with catalytic and peripheral sites
should implicate advantages over the known AChE
inhibitors. Clearly, inhibition of the peripheral binding
site would prevent the aggregation of Aâ induced by
AChE. Following this reasoning, our group has started
a research project aimed at producing novel ligands
based on a polyamine backbone, having affinity for both
AChE active and peripheral binding sites and for
muscarinic M2 receptors as well. It was advanced that
drugs able to antagonize selectively presynaptic mus-
carinic M2 autoreceptors may also be useful in AD as
they would facilitate ACh release in the synapse.10
Inhibitors of acetylcholinesterase (AChE) are impor-
tant in controlling diseases that involve impaired ace-
tylcholine-mediated neurotransmission, including Alz-
heimer’s disease (AD), the most common cause of
dementia in elderly patients, which involves selective
loss of cholinergic neurons in the brain.1-3 To date, the
cholinergic hypothesis is still the most successful ap-
proach in alleviating the symptoms of AD. However,
despite an enormous amount of work, only few inhibi-
tors of AChE, such as tacrine, donepezil, rivastigmine,
and galantamine (Figure 1), have been approved for the
symptomatic treatment of AD and they do not even
address the etiology of the disease for which are used.4-8
Recent efforts have led to the development of additional
AChE inhibitors whose efficacy is under clinical evalu-
ation.7
AChE is the enzyme involved in the hydrolysis of the
neurotransmitter acetylcholine (ACh) at cholinergic
synapses in the central and peripheral nervous system.
Inhibitors of AChE activity promote an increase in the
concentration and the duration of action of synaptic ACh
thus causing an enhancement of the cholinergic trans-
* Corresponding author: Carlo Melchiorre, Department of
Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6,
40126 Bologna, Italy. Tel. +39-051-2099706. Fax: +39-051-2099734.
E-mail: camelch@kaiser.alma.unibo.it.
† University of Bologna.
‡ University of Camerino.
10.1021/jm021055+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/14/2003

Structure-Activity of Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 955
active as the prototype. Furthermore, we have estab-
lished that optimum inhibition of AChE activity is
dependent on the carbon chain separating the inner
nitrogen atoms of carbon analogues of 1. Although
optimum inhibition of AChE was observed for the
tetraamine bearing a heptamethylene spacer between
the inner nitrogen atoms, we have chosen as lead
compound the higher homologue tetraamine methoc-
tramine (2, Figure 2), carrying eight methylenes, be-
cause it displayed optimum affinity for muscarinic M2
receptors.10 Taking into account the observation that a
diamine diamide backbone retained significant affinity
for muscarinic M2 receptors, the structure of 2 was
modified by replacing the two inner amine functions
with amide groups such as to improve the overall
lipophilicity, which might be important for penetrating
the brain. This study led to the discovery of caprocta-
mine (3, Figure 2), which became our new lead. It
emerged as a powerful tool for investigating the neu-
rological disorders due to a loss in the cholinergic
system. In comparison with 1, 3 was 42-fold more potent
at AChE, while being 2-fold less potent at BChE with a
AChE/BChE selectivity ratio of 68. Furthermore, it was
a weak antagonist at both muscarinic M1 and M3
receptors, while displaying an affinity toward muscar-
inic M2 receptors similar to the affinity for AChE as
revealed by a comparison of pA2 (M2) and pIC50 (AChE)
values which were 6.39 and 6.77, respectively. It was
also established that 3 caused a mixed type of inhibition,
that is, inhibition of both the active site and a second
distal site of the enzyme.10
F igu r e 1. Chemical structure of AChE inhibitors in current
use for the treatment of Alzheimer’s disease.
The starting point of our study was the observation
that benextramine (1, Figure 2), the prototype of tetra-
amine disulfides for irreversible inhibition of R-adreno-
receptors,11 displayed also a significant affinity for
cardiac muscarinic M2 receptors and potentiated the
effect of ACh on the frog rectus.12,13 Subsequently, the
finding that 1 was a reversible inhibitor of AChE has
provided the opportunity to apply the universal tem-
plate approach to the design of polyamines displaying
affinity for AChE and muscarinic M2 receptors.10
In a preliminary study, we have shown that the
disulfide bridge of 1 is not required for AChE inhibition
because the corresponding carbon analogue was as
The present article expands on the study of another
aspect of structure-activity relationships of prototype
3, namely, the effect of substituents on the phenyl ring
and of N-alkyl substituents on the four nitrogen atoms.
The presence of a 2-methoxybenzyl group seems to play
a crucial role for the affinity toward AChE as revealed
by the unsubstituted analogue of 1, which inhibited
AChE only at millimolar concentration. This finding
offers the opportunity to examine the effect of different
aromatic substituent parameters such as Hammett σ
and Hansch π values on both affinity and selectivity for
F igu r e 2. Chemical structure of benextramine (1), methoctramine (2), and caproctamine (3). The design strategy for the
development of 3 is also shown.

956 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Tumiatti et al.
Sch em e 1a
a
CBZ, C6H5CH2OCO-; i, NaBH4, MeCOMe; ii, CBZ-NH(CH2)5COOH, Et3N, EtOCOCl, dioxane; iii, HBr, CH3COOH; iv, R2-C6H4CHO,
toluene; v, NaBH4, EtOH; vi, (EtO)2SO2; vii, HCOOH, HCHO; viii, MeI, MeCOMe.
AChE over BChE. It is known that for relevant quan-
titative structure-activity relationships at least 12
carefully selected compounds are necessary to obtain a
significant two-parameter equation. In the present
study, our aim was to determine only whether electronic
and/or lipophilic properties of aromatic substituents of
3-related compounds could exert any favorable effect on
AChE selectivity and affinity rather than assess a
quantitative relationship. It seemed this could be de-
termined with a few properly chosen substituents. These
were selected to have σ and π values in a positive or
negative direction, in all combinations. Comparison of
the AChE inhibition of these substituted derivatives
with the unsubstituted analogue 6 should reveal the
importance, if any, of one or both these parameters. The
compounds used were the chloro and trifluoromethyl
(+π, +σ) (7 and 8), methoxy (-π, -σ) (9), n-propoxy and
methyl (+π, -σ) (10 and 11), and nitro (-π, +σ) (12)
derivatives. To verify whether the aromatic substituent
at position 2 might have any influence on the basicity
and, as a consequence, on the extent of protonation of
the outer nitrogen atom, we synthesized also the cor-
responding methiodides 14-21 of 6-13, which bear a
permanent positive charge.
case, the choice of 3 as lead compound was dictated by
the fact that the synthesis of N-methyl derivatives was
easier than that of the corresponding N-ethyl analogues.
Next, to verify the effect of the N-alkylation of
nitrogen atoms of the tetraamine backbone we replaced
the hydrogen atom of 33 with a methyl, ethyl, and
i-propyl group, affording 3, 9, and 22, respectively.
Similarly, we replaced the N-methyl group of 9 with an
N-i-propyl group, affording 13. Notwithstanding several
efforts, we were not able to synthesize the N-ethyl
homologue of 9.
Finally, to investigate on the role of amide functions
of 9 in the interaction with AChE, we included in the
present study compound 23 and its methiodide analogue
24.
Ch em istr y
All the compounds were synthesized by standard
procedures (Schemes 1-3) and were characterized by
1
IR, H NMR, mass spectra, and elemental analysis.
Compound 2914 was the starting material for the
synthesis of compounds 6-12, 14-20, and 22. Alkyla-
tion of the commercially available diamine 25 with
acetone via Schiff base in the presence of NaBH4
afforded 26, which was reacted with N-[(benzyloxy)-
carbonyl]-6-aminocaproic acid to give 27. Removal of the
N-(benzyloxy)carbonyl group of 27 by hydrolysis with
HBr gave diamine diamide 28.
Since the presence of a 2-methoxy function, as in 9,
conferred optimum affinity for AChE, we investigated
also analogues 4 and 5 in which the 2-methoxy group
was moved at positions 3 and 4, respectively. In this

Structure-Activity of Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 957
Sch em e 2a
the amide C-N bond, a unique singlet should be
observed. The presence of rotamers (E/E, E/Z, and Z/Z)
complicated also 13C NMR spectra. In compound 9, the
two amide N-CH3 groups were split into three signals
at δ 33.88, 33.60, and 33.27 ppm, respectively.
Biology
To determine the potential interest of compounds
4-24 for the treatment of AD, their AChE inhibitory
activity was determined by the method of Ellman et al.16
on AChE from human erythrocytes. Furthermore, to
establish the selectivity of 4-24, their BChE inhibitory
activity was also calculated by the same method on
BChE from human serum. The inhibitory potency was
expressed as pIC50 values, which represent the minus
logarithm of the concentration of inhibitor required to
decrease enzyme activity by 50%. To allow comparison
of the results, 1-3, tacrine, and physostigmine were
used as the reference compounds.
Selected compounds were further analyzed in a
peripheral cholinergic synapse, such as skeletal neuro-
muscular junction, by determining the ability to reverse
the D-tubocurarine-induced neuromuscular blockade,
which is a well-known effect of AChE inhibitors. The
results were expressed as pAI50 values, which represent
the minus logarithm of the concentration of inhibitor
that reaches 50% of antagonism index (AI).17,18 Tacrine,
physostigmine, and 3 were used as the reference com-
pounds.
a
i, H2/Pd, MeCOMe; ii, 2-MeO-C6H4-CH2Cl, DMF.
Sch em e 3
The mechanism of the AChE inhibition was deeply
studied for one representative compound (9) of the
series, using prototype 3 as the reference compound.
Functional activity at muscarinic receptors was de-
termined by the use of the muscarinic M2 receptor-
mediated negative inotropism in driven guinea pig left
atria (1 Hz). This method has been described in detail
earlier.19 The biological results were expressed as pKb
values determined according to van Rossum.20
Diamine diamides 28 and 2914 were treated with the
appropriate substituted benzaldehyde followed by re-
duction with NaBH4 of the formed Schiff base to the
corresponding dibenzyl derivatives 30-39 (Scheme 1).
Diethylation of 30-39 to afford 6-13 was performed
by using diethylsulfate in toluene at refluxing temper-
ature (Scheme 1). The state of the reaction was continu-
ously monitored to avoid possible quaternarization of
amine functions. Similarly, 4115 was treated with di-
ethylsulfate to give 23 (Scheme 3). Methiodides 14-21
and 24 were prepared by methylation of the correspond-
ing tertiary diamine with an excess of methyl iodide
(Schemes 1 and 3).
3-Methoxy and 4-methoxy 4 and 5 derivatives were
synthesized by Eschweiler-Clarke methylation of com-
pounds 37 and 38, respectively (Scheme 1).
Compound 22 was synthesized using a different
synthetic pathway as shown in Scheme 2. Thus, dialky-
lation of 29 was performed by alkylation with acetone
in reducing conditions, using catalytic hydrogenation
over 10% palladium on charcoal, to give 40. Finally,
compound 40 was treated with 2-methoxybenzyl chlo-
ride to afford derivative 22.
Resu lts a n d Discu ssion
The inhibitory potency, expressed as pIC50 values, of
compounds 4-24 of AChE and BChE from human
erythrocytes and human serum, respectively, is reported
in Table 1 in comparison with that of prototype
polyamines benextramine (1), methoctramine (2), and
caproctamine (3), and well-known AChE inibitors such
as tacrine and physostigmine. Furthermore, 6-12 and
17 were evaluated also for their ability to reverse the
skeletal neuromuscular junction blockade in rat phrenic
nerve-hemidiaphragm in comparison with 3, tacrine,
and physostigmine. In addition, the antagonism at
muscarinic M2 receptors was determined for compounds
7-9.
Recently, we reported that 3 was endowed with a well-
balanced affinity profile as an AChE inhibitor and a
competitive muscarinic M2 receptor antagonist.10 Fur-
thermore, it was shown that 3 was able to bind at both
the catalytic and the peripheral sites of AChE. Conse-
quently, 3 emerged as a valuable tool in investigating
AD because, besides its effects on the cholinergic system,
it might also prevent AChE-mediated Aâ aggregation
by interacting with the peripheral anionic binding site
of AChE. These promising results prompted us to
perform structural modifications on the structure of 3
An inspection of 1H NMR data reveals that different
rotamers around the amide C-N bond are present in
solution. In fact, all the signals relative to N-CH3
protons are split into two singlets. For instance, in
compound 9 (and many others, see Experimental Sec-
tion), the signals at δ 2.90 ppm (s, 3H) and 2.96 ppm (s,
3H) correspond to different rotamers of the amide
N-CH3 groups. Under freely rotating conditions around

958 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Ta ble 1. Inhibition of AChE and BChE Activities by Polyamines
Tumiatti et al.
b
pIC50
d
e
no.a
R1
X
R2
AChE
BChE
AChE/BChEc
pAI50
pKa
pKb (M2)f
1g
2g
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5.14 ( 0.02 5.21 ( 0.03
5.27 ( 0.03 6.01 ( 0.02
0.9
0.2
68
12
1
21
37
36
121
5
132
69
118
69
133
640
118
5
388
196
204
12
ndh
nd
5.79 ( 0.07
nd
nd
nd
nd
nd
nd
nd
nd
7.92 ( 0.08i
2-OMe
3-OMe
4-OMe
H
2-Cl
2-CF3
2-OMe
NMe
Me 6.77 ( 0.01 4.93 ( 0.04
Me 6.17 ( 0.04 5.08 ( 0.03
Me 5.24 ( 0.02 5.26 ( 0.03
Me 7.31 ( 0.03 5.97 ( 0.02
Me 6.95 ( 0.15 5.39 ( 0.04
Me 5.34 ( 0.04 3.78 ( 0.08
Me 7.73 ( 0.02 5.65 ( 0.03
Me 7.47 ( 0.03 6.78 ( 0.02
Me 7.25 ( 0.05 5.13 ( 0.03
Me 6.10 ( 0.03 4.26 ( 0.08
iPr 7.74 ( 0.04 5.67 ( 0.04
6.39 ( 0.23i
nd
nd
nd
5.61 ( 0.31
<5
NMe
NMe
NEt
NEt
NEt
NEt
6.32( ( 0.13 7.42 ( 0.04
7.19 ( 0.04
6.84 ( 0.02
6.45 ( 0.07
6.92 ( 0.04
6.15 ( 0.04
7.80( ( 0.06 5.65 ( 0.6
2-OnPr NEt
6.19( ( 0.02 7.75 ( 0.05
6.35 ( 0.01
5.93 ( 0.02
nd
nd
nd
nd
7.21 ( 0.02
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
2-Me
2-NO2
2-OMe
H
2-Cl
2-CF3
2-OMe
NEt
NEt
NEt
7.46 ( 0.02
6.26 ( 0.03
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
N+(Me)Et Me 7.43 ( 0.02 5.60 ( 0.03
N+(Me)Et Me 7.71 ( 0.02 5.59 ( 0.02
N+(Me)Et Me 7.93 ( 0.13 5.12 ( 0.03
N+(Me)Et Me 7.92 ( 0.02 5.85 ( 0.01
2-OnPr N+(Me)Et Me 7.76 ( 0.03 7.08 ( 0.04
2-Me
N+(Me)Et Me 7.78 ( 0.05 5.19 ( 0.05
N+(Me)Et Me 7.91 ( 0.06 5.62 ( 0.02
N+(Me)Et iPr 8.09 ( 0.02 5.78 ( 0.04
2-NO2
2-OMe
2-OMe
NiPr
Me 7.14 ( 0.13 6.06 ( 0.03
6.69 ( 0.06 5.58 ( 0.02
7.11 ( 0.02 6.12 ( 0.03
6.60 ( 0.02 7.27 ( 0.02
7.75 ( 0.18 7.64 ( 0.15
NEt
13
10
0.2
1
N+(Me)Et
tacrine
physostigmine
6.33 ( 0.03
6.50 ( 0.04
a
b
1, 2, tetrahydrochlorides; 3-13, 23, dioxalates; 14-21, 24, diiodides. AChE and BChE were from human erythrocytes and human
serum, respectively. pIC50 values represent the negative logarithm of the concentration of inhibitor required to decrease enzyme activity
by 50% and are the mean of two independent measurements, each performed in triplicate. c The AChE/BChE selectivity ratio is the
d
antilog of the difference between pIC50 values at AChE and BChE, respectively. pAI50 values represent the negative logarithm of inhibitor
concentration that reaches 50% of antagonism index (AI), i.e., the ability to reverse the D-tubocurarine-induced neuromuscular blockade.17
e Apparent dissociation constants taken from ref 19. f pKb values (( SE, n ) 4) were calculated according to Van Rossum20 at 10 µM
g
h
i
concentration at muscarinic M2 receptors in guinea pig left atrium. See Figure 2 for structure. nd, not determined. Data from ref 10.
to improve its biological profile. An examination of the
results in Table 1 shows how AChE inhibitory activity
can be affected markedly by an appropriate substituent
on the nitrogen atoms and on the phenyl ring.
-σ) has a role in the interaction with AChE and BChE,
it was replaced by selected groups, affording analogues
6-8 and 10-12, which have π and σ values in a positive
or negative direction, in all combinations. An analysis
of the results in Table 1 reveals that the 2-substituent
has a role in the interaction with AChE. The 2-methoxy
derivative 9 was the most potent of the series being 3-
and 245-fold more potent than the unsubstituted ana-
logue 6 and the 2-trifuoromethyl derivative 8, respec-
tively, which was the least potent of the series. The
overall results obtained with these derivatives clearly
indicate that lipophilic (π) or steric (expressed by molar
refractivity, MR) characteristics of 2-substituents have
little, if any, effect on AChE inhibitory activity in
comparison with electronic properties (σ). It suffices to
say that AChE inhibitory activity of 9 and 12, albeit
their π (-0.02 vs - 0.28) and MR (7.87 vs 7.38) values
are comparable, differed by a factor of 43. A possible
interpretation of these results is that the substituents
in 6-12 affect N-protonation through mixed inductive
and mesomeric effects. This view was confirmed by the
good correlation found for pIC50 values and dissociation
constants (pKa)21 of basic AChE inhibitors 6-12 (Figure
3). If a most relevant effect of the 2-substituent of 6-12
on AChE inhibitory activity were to increase the basic-
ity, then the pIC50 value of an inhibitor having a
permanent positive charge should not be dependent on
Our study began with an evaluation of AChE and
BChE inhibitory activity due to the substituent on the
outer and inner nitrogen atoms of the polyamine
backbone of 3. We had already shown that N-methyla-
tion of the corresponding analogue of 3, bearing second-
ary amine and amide functions, resulted in a significant
increase (∼10-fold) in AChE inhibitory activity, while
not affecting BChE inhibitory activity.10 In the present
investigation, we replaced the methyl group of amine
functions of 3 with ethyl and i-propyl groups, affording
9 and 22, respectively. The N-ethyl analogue 9 was more
potent than both N-methyl (3) and N-i-propyl (22)
derivatives as revealed by their pIC50 values at AChE
of 7.73 ( 0.02, 6.77 ( 0.01, and 7.14 ( 0.13, respectively
(Table 1). Then, the inner nitrogen atoms of 9 were
modified by exchanging the methyl groups with i-propyl
ones, affording 13, which resulted as active as 9 at both
AChE and BChE. It would be of interest to evaluate the
corresponding N-ethyl analogue because it might help
in identifying the substituent that would confer opti-
mum AChE inhibitory activity. Unfortunately, we did
not succeed in synthesizing the N-ethyl analogue of 9.
To verify whether the 2-methoxy function of 9 (-π,

Structure-Activity of Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 959
in AChE inhibitory activity. Similarly, the correspond-
ing methiodide 24 of 23 was less potent than the
corresponding methiodide 17 of 9. Altogether, these
results suggest clearly that the amide function of 9
cannot be exchanged for an ether moiety.
All of the compounds were significantly less potent
in inhibiting BChE than AChE activity. The most potent
compounds of the series in inhibiting BChE activity
were the 2-n-propoxy derivatives 10 and 18, whereas
the least active were the 2-trifluoromethyl (8) and
2-nitro (12) derivatives. Most of the tertiary amino
compounds were very selective for AChE, especially
those having negative σ values such as 9, 11, and 13 by
factors between 118 and 132. Since the AChE inhibitory
activity of methiodides appeared to be not affected by
the 2-substituent, the selectivity of methiodides bearing
electron-withdrawing groups (positive σ values) for
AChE was significantly increased in comparison with
the corresponding tertiary amino derivatives.
F igu r e 3. Relationship between AChE inhibiting activity
(pIC50) and the apparent dissociation constant (pKa) of com-
pounds 6-12. The substituent at position 2 of the phenyl ring
is indicated in the graph. The inset gives the squared correla-
tion coefficient (r2). Data from Table 1.
Ta ble 2. Effect of pH on AChE Inhibitory Activity, Expressed
as pIC50 Values, of 9 and 12 and of Their Corresponding
Methiodides 17 and 20
pIC50
Tertiary amino derivatives, which might have greater
potential in AD over the corresponding methiodides,
because of their lipophilicity, were assayed in rat nerve-
hemidiaphragm preparation to determine their ability
to reverse the neuromuscular blockade induced by
D-tubocurarine. All of the tested compounds were more
active than prototype 3, and most of them were also
more potent than tacrine (Table 1). The results in this
assay did not parallel perfectly those obtained in
determining AChE inhibitory activity. The most potent
in reversing neuromuscular blockade were the 2-chloro
(7) and 2-trifluoromethyl (8) derivatives with pIC50
values of 7.19 ( 0.04 and 6.84 ( 0.02, respectively. The
2-methoxy derivative 9 was 19-fold less potent in
reversing the neuromuscular blockade than in inhibiting
AChE activity. The discrepancy between the results
obtained in the two assays were observed also for AChE
inhibitors with a different structure.22 It was argued
that the determination of the AChE inhibition by
biochemical studies determine the ability of a compound
to inhibit enzyme activity, analyzing only the effect of
a drug in a single mechanism. The pharmacological
testing through the determination of neuromuscular
blockade reversion analyzes the magnitude of an effect
regardless of the implicated mechanisms. In other
words, the pharmacological assay may give additional
information relative to biochemical studies because it
evaluates how the inhibitor enhances the activity of a
cholinergic synapse and not merely the action on an
isolated enzyme. Thus, it might be that a compound
increases ACh release through mechanisms other than
AChE inhibitory action.
no.
pH ) 7.0
pH ) 7.5
pH ) 8.0
9
12
17
20
7.79 ( 0.04
7.49 ( 0.06
7.96 ( 0.04
7.90 ( 0.05
7.76 ( 0.05
7.10 ( 0.05
7.96 ( 0.06
7.89 ( 0.07
7.73 ( 0.02
6.10 ( 0.03
7.92 ( 0.02
7.91 ( 0.06
the electronic properties of the substituent. To this end,
we evaluated the corresponding methiodides 14-20 of
6-12. It turned out that these compounds inhibited
AChE activity with comparable pIC50 values comprised
within a range of 0.5 log units, whereas pIC50 values
for 6-12 differed by 2.4 log units. Clearly, the ortho-
substituents of 14-20, unlike those of the corresponding
tertiary amino derivatives 6-12, did not appear to play
a role in the interaction with AChE.
To further support the hypothesis that the electronic
properties affect the protonation of tertiary amino
functions, we evaluated AChE inhibitory activity of
compounds 9 and 12 in comparison with their corre-
sponding methiodides 17 and 20 at different pH values,
that is, 7.0, 7.5, and 8.0. The results reported in Table
2 show clearly that pIC50 values for the 2-methoxy
derivative 9 and methiodides 17 and 20 were not
affected by pH, whereas the pIC50 value of the 2-nitro
derivative (σ ) 0.78) was affected markedly by pH,
confirming that the extent of protonation of the inhibitor
has a significant effect on AChE inhibitory activity.
Since the 2-methoxy function was endowed with
optimal electronic properties, we investigated also the
effect on AChE inhibitory activity of moving the meth-
oxy function from position 2 to positions 3 and 4,
affording compounds 4 and 5. It turned out that the
affinity for AChE is dependent on the position of the
methoxy function and follows the order 2-MeO > 3-MeO
> 4-MeO, which is not in line with the observation that
the pKa was the most important parameter for AChE
inhibitory activity, as one would expect the 4-MeO
derivative 5 to be even more potent than 9. However,
this may be only an apparent contradiction because
different effects (for instance, steric hindrance) other
than the extent of protonation of the amine function
might determine the lower potency of 5 relative to 9.
Replacement of the amide function of 9 with a
methylenoxy unit, affording 23, resulted in a decrease
To verify whether diamine diamides related to 3
possess affinity also for muscarinic M2 receptors besides
their AChE inhibition properties, compounds 7-9 were
tested in the guinea pig left atrium. It turned out that
7 and 9 are weaker muscarinic M2 receptor antagonists
than prototype 3, whereas the 2-trifluoromethyl deriva-
tive was devoid of affinity at 10 µM concentration (Table
1).
Clearly, the 2-methoxy derivative 9 was the most
potent among tertiary amino derivatives in inhibiting
AChE activity, being 9-fold more potent than prototype
3 with an AChE/BChE selectivity ratio of 121. Further-
more, 9 was more potent than tacrine and as active as

960 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Tumiatti et al.
F igu r e 4. Effect of pH on the AChE inhibitory activity,
expressed as pIC50 values, of 9 and 12 in comparison with their
corresponding methiodides analogues 17 and 20.
physostigmine, which, contrary to 9, inhibits AChE
activity by way of covalent bond formation with the
enzyme. In addition, 9 was more potent in reversing
neuromuscular blockade at the skeletal junction than
both 3 and tacrine, and was as active as physostigmine.
Interestingly, 9 retained significant affinity also for
muscarinic M2 receptors, a property that may help in
increasing ACh release in the synapse as a result of
presynaptic muscarinic M2 receptor blockade. Owing to
this biological profile, 9 was studied further to charac-
terize the nature of the AChE inhibitory activity.
Inhibition of AChE activity by 9 was very fast and
not time-dependent, as 50% of enzyme inactivation
produced by 18.6 nM concentration following 1-min
incubation was not significantly different (p > 0.01) from
the inhibition observed up to a 40-min incubation. The
graphical analysis of steady-state inhibition data for 9
is shown in Figure 5, whereas the estimates of competi-
tive inhibition constants Ki are reported in Table 3 in
comparison with 3 and tacrine. It was found that 9, like
3, caused a mixed type of inhibition, that is, inhibition
of both the catalytic site and a second distal site of the
enzyme in agreement with the inhibitory behavior
displayed by some recently reported23 bis-tetrahydro-
aminoacridine inhibitors of AChE.
The reversibility of action of 9 was assessed by
dialysis in comparison to prototype 3 and tacrine. It was
found that AChE activity blocked by tacrine was fully
regenerated after 5 h, whereas following blockade by 9
only 50% of AChE activity could be reversed after 15 h
and AChE inhibition could be detected also after 24 h
(Figure 6). The longer duration of action displayed by 9
could tentatively be ascribed to a limited number and
access of water molecules in the gorge, owing to the
binding with a peripheral site besides the catalytic site
of AChE.
F igu r e 5. Steady-state inhibition by 9 of AChE hydrolysis of
acetylthiocholine. Reciprocal plot of initial velocity and sub-
strate concentration (a) and replot of the reciprocal plot versus
inhibitor concentration (b) are reported. Reciprocal plot of
initial velocity in the absence of inhibitor gave an estimate of
kapp for acetylthiocholine of 170 ( 15 µM (four experiments).
Lines were derived from a weighted least-squares analysis of
the data points.
Ta ble 3. Inhibition Constants of AChE Obtained with 9 in
Comparison with 3 and Tacrinea
no.
AChE Ki (nM)
9
3
12.2 ( 0.6
104 ( 16
151 ( 16
tacrine
a
Inhibition constants, expressed as Ki values, were calculated
from kinetic data in Figure 5.
to move from the AChE gorge as many as 16 water
molecules (Figure 7). These numbers can be considered
only as an indication of the number of water molecules
involved in the formation of the AChE/ligand complex,
and their absolute value can vary depending on such
factors as the conformational state of the side chains of
the residues lining the gorge or the computational
scheme used to calculate them. However, the difference
between 9 and tacrine is significant and reflects the
different level of occupancy of the enzyme cavity by the
two ligands.
Considering the much slower regeneration of the
enzyme after inhibition by 9 compared to that after
inhibition by tacrine (Figure 6), one might relate this
phenomenon to the difficulty of resolvating the gorge,
that seems greater when a molecule such as 9 is present
than when tacrine occupies the cavity. Actually, it is
reasonable that, in the case of 9, a larger number of
water molecules have to find their way to penetrate into
the gorge, than in the case of tacrine. Interestingly,
recent crystallographic and theoretical studies on AChE
reached the conclusion that the number of water
molecules able to fill the enzyme gorge is about 20.26,27
Despite the approximation in the determination, the
number of 16 referred to the water molecules displaced
by 9 is consistent with a wide occupation of the AChE
In an attempt to rationalize the long duration of
action of 9 (Figure 6), we computationally investigated
the capability of both 9 and a reference AChE inhibitor
(tacrine) to displace water molecules from the enzyme
gorge. We took advantage of a purposely built AChE/9
docking model and of the crystallographic structure of
the AChE/tacrine complex.24 Then, by means of the
Insight II software25 (default parameters were used),
we obtained a rough estimate of the number of water
molecules present in the AChE gorge both in the
presence and in the absence of ligands.
The simulations with the inhibitors showed that while
tacrine displaced only six water molecules, 9 was able

Structure-Activity of Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 961
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), medium pressure (Biotage, KP-SIL 60, 0.032-0.063
nm), or gravity column (Kieselgel 60, 0.063-0.200 mm; Merck)
chromatography. Reactions were followed by thin-layer chro-
matography (TLC) on Merck (0.25 mm) glass-packed precoated
silica gel plates (60 F254) that were visualized in an iodine
chamber. The term “dried” refers to the use of anhydrous
sodium sulfate. 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.
N,N′-Diisop r op ylocta n e-1,8-d ia m in e (26). A mixture of
octane-1,8-diamine (0.72 g, 5 mmol), molecular sieves (3 Å),
and acetone (1.1 mL, 15 mmol) in EtOH (20 mL) was stirred
for 30 min at room temperature, and then an excess of NaBH4
(0.9 g, 23.79 mmol) was added, and the stirring was continued
for 6 h. Following removal of molecular sieves, the solution
was made acidic with 6 N HCl. Removal of the solvent gave a
residue that was dissolved in water (40 mL). The solution was
washed with ether (3 × 20 mL) to remove nonbasic materials,
then was made basic with 2 N NaOH, and finally was
extracted with CH2Cl2 (3 × 30 mL). Evaporation of the dried
solvent afforded a residue that was purified by flash chroma-
tography. Elution with CH2Cl2/EtOAc/MeOH/aqueous 28%
ammonia (5:4:1:0.3) gave 26 as a pale yellow oil: 0.85 g (75%
yield); 1H NMR (CDCl3) δ 0.90 (d, 12H), 1.12-1.38 (m, 12H +
2H exchangeable with D2O), 2.43 (t, 4H), 2.63 (m, 2H); EI MS
m/z 228 (M+).
[5-({8-[(6-Ben zyloxyca r b on yla m in oh exa n oyl)isop r o-
p yla m in o]oct yl}isop r op ylca r b a m oyl)p en t yl]ca r b a m ic
Acid Ben zyl E st er (27). Ethyl chloroformate (0.7 mL, 7.3
mmol) in dry dioxane (10 mL) was added dropwise to a stirred
and cooled (5 °C) solution of N-[(benzyloxy)carbonyl]-6-ami-
nocaproic acid (1.7 g, 6.4 mmol) and triethylamine (0.9 mL,
6.4 mmol) in dioxane (30 mL), followed after standing for 30
min by the addition of 26 (0.7 g, 3 mmol) in dioxane (20 mL).
After the mixture was stirred at room temperature for 72 h,
it was evaporated, affording a residue that was suspended in
water (100 mL). The aqueous mixture was extracted with
CHCl3 (4 × 20 mL). The organic phase was washed with 2 N
NaOH, 2 N HCl, and brine. Removal of the dried solvent gave
a yellow oil that was purified by flash chromatography. Elution
with CH2Cl2/EtOAc/EtOH (8.7:1:0.3) afforded 27 as a pale
yellow oil: 0.8 g (37% yield); 1H NMR (CDCl3) δ 1.05-1.66
(m, 36H), 2.20-2.32 (m, 4H), 2.99-3.21 (m, 8H), 3.92-4.05
(m, 1H), 4.56-4.68 (m, 1H), 4.78-4.86 (br s, 2H exchangeable
with D2O), 5.04 (s, 4H), 7.24-7.35 (m, 10H); EI MS m/z 723
(M+).
6-Am in oh exa n oic Acid {8-[(6-Am in oh exa n oyl)isop r o-
p yla m in o]octyl}isop r op yla m id e (28). A solution of 30%
HBr in acetic acid (25 mL) was added to a solution of 27 (2.28
g, 3.1 mmol) in acetic acid and the resulting mixture was
stirred for 4 h at room temperature. Ether (100 mL) was then
added, yielding an oil, which was washed with ether (3 × 20
mL) and dissolved in water (50 mL). The solution was made
basic with KOH pellets and extracted with CHCl3 (3 × 30 mL).
Removal of the dried solvent gave 28 in quantitative yield:
1H NMR (CDCl3) δ 1.05-1.61 (m, 36H), 2.11-2.29 (m, 4H),
2.38-2.51 (br s, 4H exchangeable with D2O), 2.62 (t, 4H),
2.92-3.08 (m, 4H), 3.90-4.06 (m, 1H), 4.51-4.63 (m, 1H).
6-Isop r op yla m in oh exa n oic Acid {8-[(6-Isop r op yla m i-
n oh exa n oyl)m eth yla m in o]octyl}m eth yla m id e (40). A so-
lution of 2914 (1.12 g, 2.8 mmol) in acetone (50 mL) was
hydrogenated over 10% Pd on charcoal for 4 h. Following
catalyst removal, the solvent was evaporated, obtaining a
residue that was purified by flash chromatography. Elution
with MeOH/aqueous 28% ammonia (9:1) afforded 40 as an
oil: 30% yield; 1H NMR (CDCl3) δ 1.02 (d, 12H), 1.20-1.69
(m, 24H), 2.15-2.38 (m, 4H + 2H exchangeable with D2O),
F igu r e 6. Time course of AChE regeneration after inhibition
by 8.5, 0.85, and 13 µM concentration of 3, 9, and tacrine,
respectively (a), and time course of elimination of 9 at 0.85
µM concentration from dialysis bag in the absence of AChE
determined by HPLC (b).
gorge: as a consequence, it might be hypothesized that
the process of enzyme regeneration is slowed because
of the limited possibility of water molecules to diffuse
in and of the inhibitor molecule to diffuse out the
binding cavity.
Con clu sion s
We carried out an extensive SAR study of the poten-
tial anti-Alzheimer lead caproctamine (3), mainly with
regard to the anti-cholinesterase activity. Variation of
the alkyl group on the amine nitrogen of 3 led us to
identify the ethyl group as the best substituent on that
function, while the modification of the alkyl on the inner
nitrogen atoms did not seem to affect significantly the
activity. The substitutions introduced at the ortho
position of the phenyl ring showed a clear effect on the
inhibition of AChE related to the electronic influence
of the substituents on the ionization of the nearby
tertiary amine. After the evaluation of both AChE and
BChE inhibitory potency, of the ability of reversing the
D-tubocurarine-induced neuromuscular blockade, and of
the antagonistic activity on muscarinic M2 receptors, we
individuated compound 9 as a promising derivative
endowed of interesting biological characteristics making
it worthy of further study in the field of Alzheimer
therapeutics.
Exp er im en ta l Section
Ch em istr y. Melting points were taken in glass capillary
tubes on a Buechi SMP-20 apparatus and are uncorrected. IR,
electron impact (EI) mass, and NMR spectra were recorded
on Perkin-Elmer 297, VG 7070E, and Varian VXR 300 instru-
ments, respectively. Chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane (TMS), and spin
multiplicities are given as s (singlet), br s (broad singlet), d
(doublet), t (triplet), q (quartet), or m (multiplet). Although
IR spectra data are not included (because of the lack of unusual
features), they were obtained for all compounds reported and

962 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Tumiatti et al.
F igu r e 7. Docking model of the solvated 9/AChE complex: three water molecules are present in the gorge (a). The solvated
AChE gorge after the removal of 9. The enzyme active site hosts 19 water molecules: 16 water molecules can be displaced by the
interaction between 9 and AChE (b). Solvated tacrine/AChE docking complex (crystal structure): 13 water molecules are present
in the gorge (c). The solvated AChE gorge after the removal of tacrine. The enzyme active site hosts 19 water molecules: six
water molecules can be displaced by the interaction between tacrine and AChE (d). Water molecules are in ball-and-stick
representation. The carbon atoms of 9 and tacrine are green and magenta, respectively.
2.57 (t, 4H), 2.66-2.86 (m, 2H) 2.90 (s, 3H), 2.98 (s, 3H), 3.16-
3.38 (m, 4H); EI MS m/z 482 (M+).
[6-(2-t r iflu or om et h ylb en zyla m in o)h exa n oyl]a m in o}oc-
tyl)a m id e (32), 6-(2-P r op oxyben zyla m in o)h exa n oic Acid
Meth yl-(8-{m eth yl-[6-(2-p r op oxyben zyla m in o)h exa n oyl]-
a m in o}octyl)a m id e (34), 6-(2-Meth ylben zyla m in o)h ex-
a n oic Acid Meth yl-(8-{m eth yl-[6-(2-m eth ylben zyla m in o)-
h exa n oyl]a m in o}octyl)a m id e (35), 6-(2-Nitr oben zyla m i-
n o)h exa n oic Acid Meth yl-(8-{m eth yl-[6-(2-n itr oben zyl-
a m in o)h exa n oyl]a m in o}octyl)a m id e (36), 6-(3-Meth oxy-
b en zyla m in o)h exa n oic Acid (8-{[6-(3-Met h oxyb en zyl-
a m in o)h exa n oyl]m eth yla m in o}octyl)m eth yla m id e (37),
6-(4-Meth oxyben zyla m in o)h exa n oic Acid (8-{[6-(4-Meth -
oxyben zyla m in o)h exa n oyl]m eth yla m in o}octyl)m eth yl-
a m id e (38), 6-(2-Meth oxyben zyla m in o)h exa n oic Acid
Isop r op yl-(8-{isop r op yl-[6-(2-m eth oxyben zyla m in o)h ex-
a n oyl]a m in o}octyl)a m id e (39). A mixture of 2914 for com-
pounds 30-38, or 28 for compound 39 and the suitable
substituted benzaldehyde (in a 1:2 molar ratio) in toluene (50
mL) was stirred at the refluxing temperature in a Dean Stark
apparatus for 6 h. Following solvent removal, the residue was
taken up in EtOH (30 mL) and NaBH4 was added and the
stirring was continued at room temperature for 6 h. The
mixture was then made acidic with 3 N HCl, filtered, and
evaporated. The residue was dissolved in water, and the
6-[Isop r op yl-(2-m eth oxyben zyl)a m in o]h exa n oic Acid
[8-({6-[Isop r op yl-(2-m et h oxyb en zyl)a m in o]h exa n oyl}-
m eth yla m in o)octyl]m eth yla m id e Dioxa la te (22). A mix-
ture of 40 (0.18 g, 0.38 mmol) and 2-methoxybenzyl chloride
(0.12 g, 0.76 mmol) in dry DMF (4 mL) was stirred for 36 h at
room temperature. The removal of the solvent gave a residue
that was purified by flash chromatography. Elution with CH2-
Cl2/MeOH/aqueous 28% ammonia (10:0.5:0.05) afforded 22 as
a pale yellow oil, which was transformed into the dioxalate
1
salt: 25% yield; H NMR (free base, CDCl3) δ 1.05 (d, 12H),
1.40-1.80 (m, 24H), 1.98-2.10 (m, 2H), 2.20-2.38 (m, 4H),
2.41-2.46 (m, 4H), 2.93 (d, 6H), 3.19-3.40 (m, 4H), 3.48-3.70
(m, 4H), 3.82 (s, 6H), 6.80-7.02 (m, 4H), 7.19-7.22 (m, 2H),
7.50-7.60 (m, 2H); EI MS m/z 723 (M+). Anal. (C48H78N4O12
C, H, N.
)
Gen er a l P r oced u r e for th e Syn th esis of 6-Ben zyl-
a m in oh exa n oic Acid {8-[(6-Ben zyla m in oh exa n oyl)m eth -
yla m in o]octyl}m eth yla m id e (30), 6-(2-Ch lor oben zyla m i-
n o)h exa n oic Acid (8-{[6-(2-Ch lor ob en zyla m in o)h exa n -
oyl]m eth yla m in o}octyl)m eth yla m id e (31), 6-(2-Tr iflu o-
r om eth ylben zyla m in o)h exa n oic Acid Meth yl-(8-{m eth yl-

Structure-Activity of Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 963
resulting solution was washed with ether, made basic with 2
N NaOH, and extracted with CHCl3. Removal of the dried
solvent afforded the desired compound as a yellow oil in 34-
98% yields.
30: 56% yield; 1H NMR (CDCl3) δ 1.18-1.72 (m, 24H + 2H
exchangeable with D2O), 2.20-2.46 (m, 4H), 2.62 (t, 4H), 2.88
(s, 3H), 2.98 (s, 3H), 3.17-3.36 (m, 4H), 3.77 (s, 4H), 7.21-
7.31 (m, 10H).
ben zyl)a m in o]h exa n oic Acid [8-({6-[Eth yl-(2-p r op oxy-
ben zyl)am in o]h exan oyl}m eth ylam in o)octyl]m eth ylam ide
(10), 6-[Eth yl-(2-m eth ylben zyl)am in o]h exan oic Acid [8-({6-
[Eth yl-(2-m eth ylben zyl)a m in o]h exa n oyl}m eth yla m in o)-
octyl]m eth yla m id e (11), 6-[Eth yl-(2-n itr oben zyl)a m in o]-
h exa n oic Acid [8-({6-[E t h yl-(2-n it r ob en zyl)a m in o]h ex-
a n oyl}m eth yla m in o)octyl]m eth yla m id e (12), 6-[Eth yl-(2-
m eth oxyben zyl)a m in o]h exa n oic Acid [8-({6-[Eth yl-(2-
m eth oxyben zyl)a m in o]h exa n oyl}isop r op yla m in o)octyl]-
isop r op yla m id e (13), Eth yl-[6-(8-{6-[6-[eth yl-(2-m eth oxy-
ben zyl)a m in o]h exyloxy}octyloxy)h exyl]-(2-m eth oxyben -
zyl)a m in e (23). A mixture of 30, 31, 32, 33,10 34, 35, 36, 39,
or 4115 and diethylsulfate (1:2.5 ratio) was refluxed for 48 h
in toluene. Following removal of the solvent, the residue was
taken up in water and made basic with KOH pellets and
immediately extracted with CHCl3 (3 × 20 mL) or directly
purified by column chromatography to avoid the quaterna-
rization of amine functions. Removal of the dried solvent gave
a residue that was purified by flash chromatography. Elution
with CH2Cl2/EtOAc/MeOH/aqueous 28% ammonia (5:4:1:0.1)
gave the desired compound (6-13, or 23) as a pale yellow oil
in 20-82% yields, that was converted into the dioxalate salt
(foam solid).
6: 20% yield; 1H NMR (free base, CDCl3) δ 1.00 (t, 6H),
1.24-1.68 (m, 24H), 2.24 (t, 4H), 2.38-2.52 (m, 8H), 2.88 (s,
3H), 2.94 (s, 3H), 3.18 (t, 2H), 3.28 (t, 2H), 3.53 (s, 4H), 7.16-
7.31 (m, 10H); EI MS m/z 634 (M+). Anal. (C44H70N4O10) C, H,
N.
7: 58% yield; 1H NMR (free base, CDCl3) δ 0.97 (t, 6H),
1.12-1.68 (m, 24H), 2.20 (t, 4H), 2.38-2.48 (m, 8H), 2.81 (s,
3H), 2.86 (s, 3H), 3.14 (t, 2H), 3.24 (t, 2H), 3.56 (s, 4H), 7.05-
7.19 (m, 4H), 7.21-7.25 (m, 2H), 7.43-7.46 (d, 2H); EI MS
m/z 703 (M+). Anal. (C44H68Cl2N4O10) C, H, N.
31: 62% yield; 1H NMR (CDCl3) δ 1.22-1.79 (m, 24H + 2H
exchangeable with D2O), 2.22-2.38 (m, 4H), 2.64 (t, 4H), 2.80
(s, 3H), 2.84 (s, 3H), 3.20-3.28 (m, 2H), 3.30-3.40 (m, 2H),
3.89 (s, 4H), 7.18-7.28 (m, 4H), 7.31-7.42 (m, 4H).
32: 58% yield; 1H NMR (CDCl3) δ 1.17-1.64 (m, 24H + 2H
exchangeable with D2O), 2.22 (t, 4H), 2.55-2.62 (m, 4H), 2.80-
2.85 (m, 6H), 3.13-3.31 (m, 4H), 3.85 (s, 4H), 7.21-7.30 (m,
2H), 7.40-7.59 (m, 6H).
1
34: 56% yield; H NMR (CDCl3) δ 0.99 (t, 6H), 1.10-1.82
(m, 28H + 2H exchangeable with D2O), 2.11-2.28 (m, 4H),
2.53 (t, 4H), 2.83 (s, 3H), 2.87 (s, 3H), 3.10-3.32 (m, 4H), 3.73
(s, 4H), 3.87 (t, 4H), 6.72-6.88 (m, 4H), 7.09-7.25 (m, 4H).
35: 93% yield; 1H NMR (CDCl3) δ 1.11-1.60 (m, 24H + 2H
exchangeable with D2O), 2.15-2.25 (m, 10H), 2.57 (t, 4H), 2.79
(s, 3H), 2.82 (s, 3H), 3.16 (t, 2H), 3.21 (t, 2H), 3.64 (s, 4H),
7.00-7.27 (m, 8H).
36: 74% yield; 1H NMR (CDCl3) δ 1.18-1.79 (m, 24H + 2H
exchangeable with D2O), 2.26-2.32 (m, 4H), 2.50-2.62 (m,
4H), 2.85-2.98 (m, 6H), 3.18-3.38 (m, 4H), 3.98-4.01 (m, 4H),
7.24-7.92 (m, 8H).
37: 39% yield; 1H NMR (CDCl3) δ 1.15-1.74 (m, 24H + 2H
exchangeable with D2O), 2.15-2.40 (m, 4H), 2.51-2.74 (m,
4H), 2.79-3.02 (m, 6H), 3.07-3.43 (m, 4H), 3.59-3.97 (m,
10H), 6.67-7.00 (m, 6H), 7.07-7.33 (m, 2H); EI MS m/z 638
(M+).
8: 40% yield; 1H NMR (free base, CDCl3) δ 1.03 (t, 6H),
1.10-1.71 (m, 24H), 2.27 (t, 4H), 2.38-2.55 (m, 8H), 2.88 (s,
3H), 2.93 (s, 3H), 3.21 (t, 2H), 3.32 (t, 2H), 3.69 (s, 4H), 7.25
(t, 2H), 7.42-7.59 (m, 4H), 7.88 (d, 2H); EI MS m/z 770 (M+).
Anal. (C46H68F6N4O10) C, H, N.
38: 38% yield; 1H NMR (CDCl3) δ 1.26-1.81 (m, 24H + 2H
exchangeable with D2O), 2.20-2.33 (m, 4H), 2.63 (t, 4H), 2.92
(d, 6H), 3.20-3.28 (m, 2H), 3.30-3.36 (m, 2H), 3.73 (s, 4H),
3.80 (s, 6H), 6.84-6.87 (d, 4H), 7.22-7.26 (d, 4H).
39: 34% yield; 1H NMR (CDCl3) δ 1.10-1.85 (m, 36H + 2H
exchangeable with D2O), 2.21-2.37 (m, 4H), 2.61 (t, 4H), 2.98-
3.19 (m, 4H), 3.79 (s, 4H), 3.85 (s, 6H), 3.86-4.05 (m, 1H),
4.58-4.65 (m, 1H), 6.80-6.95 (m, 4H), 7.18-7.30 (m, 4H); EI
MS m/z 695 (M+).
1
9: 58% yield; H NMR (free base, CDCl3) δ 1.12-1.72 (m,
30H), 2.28 (t, 4H), 2.59-2.79 (m, 8H), 2.90 (s, 3H), 2.96 (s,
3H), 3.19-3.38 (m, 4H), 3.82 (s, 10H), 6.82-6.98 (m, 4H),
7.21-7.32 (m, 2H), 7.48 (d, 2H); 13C NMR (free base, CDCl3)
δ 173.11, 172.99, 157.93, 130.43, 128.48, 127.88, 120.54,
110.45, 55.61, 53.71, 51.53, 50.26, 50.20, 47.91, 35.58, 33.88
(N-CH3), 33.60 (N-CH3), 33.27 (N-CH3), 29.96 and 29.67 and
29.59, 28.81, 27.77 and 27.68 and 27.51, 27.02, 25.74, 25.35,
11.96; EI MS m/z 695 (M+). Anal. (C46H74N4O12) C, H, N.
6-[(3-Meth oxyben zyl)m eth yla m in o]h exa n oic Acid [8-
({6-[(3-Met h oxyben zyl)m et h yla m in o]h exa n oyl}m et h yl-
a m in o)octyl]m eth yla m id e Dioxa la te (4) a n d 6-[(4-Meth -
oxyben zyl)m eth yla m in o]h exa n oic Acid [8-({6-[(4-Meth -
oxyben zyl)m eth yla m in o]h exa n oyl}m eth yla m in o)octyl]-
m eth yla m id e Dioxa la te (5). A solution of 37 or 38 (0.21
mmol) in 95% HCOOH (0.5 mL) and 37% HCHO (0.5 mL) was
refluxed for 12 h. The solution was made basic with 40% NaOH
and extracted with CHCl3 (3 × 10 mL). Removal of the dried
solvent gave a residue that was purified by flash chromatog-
raphy. Elution with CHCl3/MeOH/aqueous 28% ammonia (9:
1:0.1) gave 4 or 5 that was converted into the dioxalate salt.
1
10: 23% yield; H NMR (free base, CDCl3) δ 1.04 (t, 12H),
1.10-1.70 (m, 24H), 1.82 (q, 4H), 2.25 (t, 4H), 2.42-2.59 (m,
8H), 2.89 (s, 3H), 2.94 (s, 3H), 3.21 (t, 2H), 3.32 (t, 2H), 3.61
(s, 4H), 3.92 (t, 4H), 6.80-6.95 (m, 4H), 7.18 (t, 2H), 7.42 (d,
2H); EI MS m/z 751 (M+). Anal. (C50H82N4O12) C, H, N.
1
11: 82% yield; H NMR (free base, CDCl3) δ 1.01 (t, 6H),
1.12-1.72 (m, 24H), 2.20-2.55 (m, 18H), 2.89 (s, 3H), 2.93 (s,
3H), 3.21 (t, 2H), 3.32 (t, 2H), 3.48 (s, 4H), 7.10-7.38 (m, 8H);
EI MS m/z 663 (M+). Anal. (C46H74N4O10) C, H, N.
1
4: 95% yield; H NMR (free base, CDCl3) δ 1.19-1.67 (m,
24H), 2.16 (s, 6H), 2.20-2.38 (m, 8H), 2.88 (s, 3H), 2.93 (s,
3H), 3.18-3.36 (m, 4H), 3.42 (s, 4H), 3.78 (s, 6H), 6.70-6.89
(m, 6H), 7.11-7.25 (m, 2H); EI MS m/z 666 (M+). Anal.
(C44H70N4O12) C, H, N.
12: 45% yield; 1H NMR (free base, CDCl3) δ 0.92 (t, 6H),
1.22-1.62 (m, 24H), 2.20-2.45 (m, 12H), 2.83 (s, 3H), 2.92 (s,
3H), 3.15-3.32 (m, 4H), 3.79 (s, 4H), 7.22-7.78 (m, 8H); EI
MS m/z 725 (M+). Anal. (C44H68N6O14) C, H, N.
1
5: 81% yield; H NMR (free base, CDCl3) δ 1.25-1.73 (m,
13: 34% yield; 1H NMR (free base, CDCl3) δ 1.02-1.70 (m,
42H), 2.21-2.38 (q, 4H), 2.42-2.62 (m, 8H), 3.05-3.19 (q, 4H),
3.61 (s, 4H), 3.83 (s, 6H), 3.96-4.11 (m, 1H), 4.61-4.75 (m,
1H), 6.81-6.99 (m, 4H), 7.18-7.33 (m, 2H), 7.45 (d, 2H); EI
MS m/z 751 (M+). Anal. (C50H82N4O12) C, H, N.
24H), 2.15 (s, 6H), 2.20-2.37 (m, 8H), 2.90-2.94 (m, 6H),
3.16-3.40 (m, 8H), 3.79 (s, 6H), 6.81-6.86 (m, 4H), 7.18-7.21
(m, 4H); EI MS m/z 666 (M+). Anal. (C44H70N4O12) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Dioxa la te
Sa lts of 6-(Ben zyleth yla m in o)h exa n oic Acid (8-{[6-(Ben -
zyle t h yla m in o)h e xa n oyl]m e t h yla m in o}oct yl)m e t h yl-
a m id e (6), 6-[(2-Ch lor oben zyl)eth yla m in o]h exa n oic Acid
[8-({6-[(2-ch lor oben zyl)eth ylam in o]h exan oyl}m eth ylam i-
n o)octyl]m eth yla m id e (7), 6-[Eth yl-(2-tr iflu or om eth yl-
ben zyl)a m in o]h exa n oic Acid [8-({6-[Eth yl-(2-tr iflu or o-
m eth ylben zyl)am in o]h exan oyl}m eth ylam in o)octyl]m eth -
yla m id e (8), 6-[Eth yl-(2-m eth oxyben zyl)a m in o]h exa n oic
Acid [8-({6-[E t h yl-(2-m et h oxyb en zyl)a m in o]h exa n oyl}-
m eth ylam in o)octyl]m eth ylam ide (9), 6-[Eth yl-(2-pr opoxy-
1
23: 20% yield; H NMR (free base, CDCl3) δ 1.04 (t, 6H),
1.25-1.62 (m, 28H), 2.44 (t, 4H), 2.51 (q, 4H), 3.37 (t, 8H),
3.58 (s, 4H), 3.81 (s, 6H), 6.83 (d, 2H), 6.88 (t, 2H), 7.19-7.42
(t, 2H), 7.40 (d, 2H); EI MS m/z 640 (M+). Anal. (C44H72N2O12
)
C, H, N.
Gen er a l P r oced u r es for th e Syn th esis of Meth iod id es
14-21 a n d 24. A solution of the appropriate compound (6-
13 or 23) as the free base in acetone (15 mL) was treated with
methyl iodide (1:10 molar ratio). After standing overnight at

964 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Tumiatti et al.
room temperature, the solvent was removed and the residue
was triturated with ether to give a foam pale yellow solid in
quantitative yield.
14: 1H NMR (DMSO-d6) δ 1.01-1.77 (m, 30H), 2.18-2.31
(m, 4H), 2.71 (s, 3H), 2.78 (s, 6H), 2.86 (s, 3H), 3.00-3.25 (m,
12H), 4.40 (s, 4H), 7.47 (s, 10H). Anal. (C42H72I2N4O2) C, H,
N.
15: 1H NMR (DMSO-d6) δ 1.04 (t, 6H), 1.21-1.77 (m, 24H),
2.28 (m, 4H), 2.76 (s, 3H), 2.90 (s, 9H), 3.01-3.35 (m, 12H),
4.61 (s, 4H), 7.49-7.68 (m, 8H). Anal. (C42H70Cl2I2N4O2) C, H,
N.
16: 1H NMR (DMSO-d6) δ 1.15-1.78 (m, 30H), 2.23-2.31
(m, 4H), 2.74-2.92 (m, 12H), 3.15-3.51 (m, 12H), 4.69 (s, 4H),
7.76-7.98 (m, 8H). Anal. (C44H70F6I2N4O2) C, H, N.
17: 1H NMR (DMSO-d6) δ 1.26-1.75 (m, 30H), 2.12-2.19
(m, 4H), 2.77 (s, 3H), 2.82 (s, 6H), 2.91 (s, 3H), 3.06-3.56 (m,
12H), 3.84 (s, 6H), 4.43 (s, 4H), 7.03-7.21 (m, 4H), 7.41-7.59
(m, 4H). Anal. (C44H76I2N4O4) C, H, N.
1.2; NaHCO3, 25; glucose, 11.1. Effects of AChE inhibitors on
neuromuscular junction were assessed as the ability of revers-
ing the partial blockade induced by D-tubocurarine in indirectly
elicited twitch responses. Twitches were obtained by stimulat-
ing the phrenic nerve with square pulses of 0.5-ms duration
at 0.2 Hz and supramaximal voltage. Neuromuscular blockade
was obtained with the addition of D-tubocurarine (1-5 µM).
Drugs were added when a reduction of the twitch response of
70-80% of control values was obtained. The effect of each drug
was evaluated after 15 min of exposure. To avoid the possible
carry-over effects, only one concentration of inhibitor was
tested on each preparation. Several drug concentrations were
evaluated for each AChE inhibitor. To quantify the reversal
effect of each drug, the antagonism index (AI or percent of
antagonism)17 was determined for each concentration and the
AI50 (drug concentration that gives a 50% value of AI) was
calculated.
In h ibition of ACh E a n d BCh E. The method of Ellman
et al. was followed.16 Five different concentrations of each
compound were used to obtain inhibition of AChE or BChE
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 5,5′-dithio-bis(2-nitrobenzoic acid), 0.035 unit/mL
AChE or BChE derived from human erythrocytes (0.39 and
5.9 UI/mg, respectively; Sigma Chemical), and 550 µM acetyl-
thiocholine iodide. Test compounds were added to the assay
solution and preincubated at 37 °C with the enzyme for 20
min followed by the addition of substrate. Assays were done
with a blank containing all components except AChE or BChE
to account for 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, and IC50 values were determined
graphically from log concentration-inhibition curves.
To ascertain the effect of pH on the inhibition of AChE
activity, experiments were also carried out at pH values of 7.0
and 7.5 for few selected inhibitors.
Deter m in a tion of Stea d y-Sta te In h ibition Con sta n t.
To obtain estimates of the competitive inhibition constant Ki,
reciprocal plots of 1/V versus 1/[S] were constructed at
relatively low concentration of substrate (below 0.5 mM). The
plots were assessed by a weighted least-squares analysis that
assumed the variance of V to be a constant percentage of V
for the entire data set. Slopes of these reciprocal plots were
then plotted against 9 concentration (range 7-37 nM) in a
similar weighted analysis and Ki was determined as the ratio
of the replot intercept to the replot slope.
Reciprocal plots involving 9 inhibition show both increasing
slopes (decreased Vmax at increasing inhibitor’s concentrations)
and increasing intercepts (higher Km) with higher inhibitor
concentration. This pattern indicates mixed inhibition, arising
from significant inhibitor interaction with both the free enzyme
and the acetylated enzyme. Replots of the slope versus the
concentration of 9 gives an estimate of competitive inhibition
constant, Ki ) 12.2 ( 0.6 nM.
18: 1H NMR (DMSO-d6) δ 0.98 (t, 6H), 1.05-1.80 (m, 34H),
2.22-2.30 (m, 4H), 2.72 (s, 3H), 2.82 (s, 6H), 2.91 (s, 3H), 3.05-
3.19 (m, 12H), 4.00 (t, 4H), 4.42 (s, 4H), 7.00-7.15 (m, 4H),
7.44-7.51 (m, 4H). Anal. (C48H84I2N4O4) C, H, N.
19: 1H NMR (DMSO-d6) δ 1.10-1.92 (m, 30H), 2.25-2.34
(m, 4H), 2.48 (s, 6H), 2.84 (s, 3H), 2.94 (s, 3H), 3.07 (s, 6H),
3.18-3.76 (m, 12H), 4.77 (s, 4H), 7.21-7.34 (m, 6H), 7.51-
7.59 (m, 2H). Anal. (C44H76I2N4O2) C, H, N.
20: 1H NMR (DMSO-d6) δ 1.12-1.75 (m, 30H), 2.19-2.23
(m, 4H), 2.64-2.84 (m, 12H), 3.02-3.38 (m, 12H), 4.79 (s, 4H),
7.71-7.81 (m, 6H), 8.02-8.11 (m, 2H). Anal. (C42H70I2N6O6)
C, H, N.
21: 1H NMR (DMSO-d6) δ 1.03 (d, 6H), 1.08 (d, 6H), 1.23-
1.79 (m, 30H), 2.19-2.31 (m, 4H), 2.79 (s, 6H), 2.92-3.31 (m,
12H), 3.82 (s, 6H), 3.96-4.06 (m, 2H), 4.40 (s, 4H), 7.03-7.16
(m, 4H), 7.40-7.50 (m, 4H). Anal. (C48H84I2N4O4) C, H, N.
24: 1H NMR (DMSO-d6) δ 1.19-1.81 (m, 34H), 2.81 (s, 6H),
3.02-3.37 (m, 16H), 3.85 (s, 6H), 4.43 (s, 4H), 7.00-7.21 (m,
4H), 7.40-7.62 (m, 4H). Anal. (C42H74I2N2O4) C, H, N.
Biology. F u n ction a l An ta gon ism . Male guinea pigs
(200-300 g) and male Wistar rats (280-300 g) were killed by
cervical dislocation. The organs required were set up rapidly
under 1 g of tension in 20 mL organ baths containing
physiological salt solution (PSS) maintained at an appropriate
temperature and aerated with 5% CO2-95% O2.
Gu in ea P ig Left Atr ia . The heart was rapidly removed,
and right and left atria were separated out. The left atria were
mounted at 30 °C in PSS of the following composition (mM):
NaCl, 118; KCl, 4.7; CaCl2, 2.52; MgSO4‚7H2O, 1.18; KH2PO4,
1.18; NaHCO3, 23.8; glucose, 11.7. Tissues were stimulated
through platinum electrodes by square-wave pulses (1 ms, 1
Hz, 5-10 V) (Tetra Stimulus, N. Zagnoni). Inotropic activity
was recorded isometrically. Tissues were equilibrated for 2 h
and a cumulative concentration-response curve to APE was
constructed. Concentration-response curves were constructed
by cumulative addition of the reference agonist. The concen-
tration of agonist in the organ bath was increased ap-
proximately 3-fold at each step, with each addition being made
only after the response to the previous addition had attained
a maximal level and remained steady. Following 30 min of
washing, tissues were incubated with the antagonist for 1 h,
and a new dose-response curve to the agonist was obtained.
Contractions were recorded by means of a force displacement
transducer connected to the MacLab system PowerLab/800.
In addition parallel experiments in which tissues did not
receive any antagonist were run ito check any variation in
sensitivity.
So the pattern in the graphical representation shows 9 able
to bind to the peripheral anionic site as well as the active site.
Dia lysis. Stock solutions in replicates were prepared as
following: the free enzyme (0.39 units in 3 mL of 0.1 M
phosphate buffer pH 8.0), the same amount of enzyme plus
tacrine 13.0 µM, 8.5 µM 3, and 0.85 µM 9. The enzyme
concentration was 10-fold higher than that used in the Ellman
test, while the inhibitors’ concentration was 50-fold higher
than that of their IC50 values.
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 22 °C against 2 L of 0.1 M
phosphate buffer pH 8.0. Dialysis was interrupted at fixed
times up to 30 h. The solutions were five times diluted 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 spectrophotometrically tested after addition
of 0.034 mL of DTNB and 0.016 mL of acetylthiocoline as
described above. Plots of enzyme activities for each compound,
To quantify antagonist potency, pKb values were calculated
from the equation pKb ) log(DR - 1) - log [B], where DR is
the ratio of EC50 values of agonist after and before treatment
with 10 µM concentration of the antagonist [B].20
Ra t Ner ve-Hem id ia p h r a gm s. Right and left phrenic
nerve-hemidiaphragms were used following a described pro-
cedure.17,18 Briefly, each phrenic nerve-hemidiaphragm was
suspended at 25 °C in PSS of the following composition (mM):
NaCl, 118; KCl, 4.8; CaCl2, 2.5; KH2PO4, 1.2; MgSO4‚7H2O,

Structure-Activity of Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 965
(7) Barril, X.; Orozco, M.; Luque, F. J . Towards improved acetyl-
cholinesterase inhibitors: a structural and computational ap-
proach. Mini Rev. Med. Chem. 2001, 1, 255-266.
expressed as a percentage of the dialyzed free enzyme activity
against time of dialysis, were calculated for each inhibitor. A
HPLC method was used to monitor over the time the concen-
tration of 9 incubated alone in the dialysis bag. The stationary
phase was a 5 µm Phenyl-hexyl Luna Phenomenex (150 × 3.0
mm i.d.) column, the mobile phase consisted of a mixture of
acetonitrile/0.02 M triethylammonium phosphate (pH 4.0) (37:
63 v/v), the flow rate was 0.4 mL min-1 and the UV detection
was fixed at 220 nm. Compound 9 at 0.85 µM concentration
in the dialysis bag, without enzyme, decreased to zero in 8 h.
Molecu la r Dyn a m ics (MD) Sim u la tion s. An AChE/9
docking model was built by taking advantage of the energeti-
cally relaxed complex between AChE and 3.10 9 was simply
built by properly modifying 3. With the aim of obtaining an
energetically relaxed AChE/9 docking model, MD simulations
were carried out by using the united atom AMBER* force field
as implemented in MacroModel Ver. 5.5.28 Polar and aromatic
hydrogen atoms were added to the protein, while the aliphatic
portions were treated using the united atom model of AM-
BER.29 It should be remarked that the aromatic ring modeling
by means of the all atom model allowed properly accounting
for the π-cation interaction,30 fundamental when studying
AChE/inhibitor complexes.31
Around the inhibitor (10 Å), a set of atoms was defined,
which was allowed to freely move during the simulations. A
shell was defined 3 Å around such a core of atoms. The atoms
belonging to this shell were constrained by applying an energy
penalty force constant of 100 kJ /mol Å-2. Atoms beyond this
shell were maintained at their crystallographic positions. This
protocol has already been applied to relax AChE/inhibitor
complexes.10,32,33
On this system, a total of 300 ps of MD simulations were
carried out and the last snapshot was energy minimized by
both steepest descent and conjugate gradient until a conver-
gence of 0.05 kJ /mol Å-1 on the gradient was reached. This
energy-minimized structure was then used for the subsequent
solvation study.
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acetylcholinesterase inhibitors: implications in treatment of
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cholinesterase accelerates assembly of amyloid-beta-peptides
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R.; Cavalli, A.; Cavrini, V.; Rosini, M.; Tumiatti, V.; Recanatini,
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a
polyamine backbone for potential use against Alzheimer’s
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properties of the adrenergic R-receptor. 2. Optimum covalent
inhibition by two different prototypes of polyamine disulfides.
J . Med. Chem. 1978, 21, 1126-1132.
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muscarinic blocking and atropinic blocking effects of a tetramine
disulfide and R-adrenoceptor blocking activity. Can. J . Physiol.
Pharmacol. 1979, 57, 41-47.
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substituent effects on M2 muscarinic receptor blocking activity.
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A.; Tumiatti, V. Structure-activity relationships among meth-
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four nitrogens on M2 muscarinic blocking activity as revealed
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Solva tion Sim u la tion s. To study the water molecule
displacement caused by both 9 and tacrine, some simulations
were carried out by means of the software package Insight
II.25 First, the crystal structure of AChE/tacrine complex (PDB
code: 1ACJ ) and the energy minimized docking model of
AChE/9 were solvated. Then, the two inhibitors were removed
from the gorge, and the protein was solvated again. This
allowed us to detect the water molecules displaced by either
tacrine or 9. The procedure was repeated several times, and
average numbers of displaced water molecules were obtained
for 9 and tacrine, respectively.
(20) Van Rossum, J . M. Cumulative dose-response curves. II.
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Sta tistica l An a lysis. Data were analyzed using a phar-
macological computer program.34 Values are given as mean (
standard error of n independent observations. Student’s t-test
was used to assess the statistical significance of the difference
between two means.
Ack n ow led gm en t. This research was supported by
Grants from the University of Bologna (Funds for
Selected Research Topics) and MIUR.
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