Design, synthesis and biological evaluation of ambenonium derivatives as AChE inhibitors

Article abstract of DOI:10.1016/S0014-827X(03)00150-2

Ambenonium (1), an old AChE inhibitor, is endowed with an outstanding affinity and a peculiar mechanism of action that, taken together, make it a very promising pharmacological tool for the treatment of Alzheimer's disease (AD). Unfortunately, the bisquaternary structure of 1 prevents its passage through the blood brain barrier. In a search of centrally active ambenonium derivatives, we planned to synthesize tertiary amines of 1, such as 2 and 3. In addition, to add new insights into the binding mechanism of the inhibitor, we designed constrained analogues of ambenonium by incorporating the diamine functions into cyclic moieties (4-12). The biological evaluation of the new compounds has been assessed in vitro against human AChE and BChE. All tertiary amine derivatives resulted more than 1000-fold less potent than 1 and, unlike prototype, did not show any selectivity between the two enzymes. This result, because of recent findings concerning the role of BChE in AD, makes our compounds, endowed with a well-balanced profile of AChE/BChE inhibition, valuable candidates for further development. To better clarify the interactions that account for the high affinity of 1, docking simulations and molecular dynamics studies on the AChE-1 complex were also carried out.

Full text of DOI:10.1016/S0014-827X(03)00150-2

Il Farmaco 58 (2003) 917ꢀ928
/
www.elsevier.com/locate/farmac
Design, synthesis and biological evaluation of ambenonium
derivatives as AChE inhibitors
Maria Laura Bolognesi, Andrea Cavalli, Vincenza Andrisano, Manuela Bartolini,
Rita Banzi, Alessandra Antonello, Michela Rosini, Carlo Melchiorre *
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
Received 11 December 2002; accepted 28 January 2003
Abstract
Ambenonium (1), an old AChE inhibitor, is endowed with an outstanding affinity and a peculiar mechanism of action that, taken
together, make it a very promising pharmacological tool for the treatment of Alzheimer’s disease (AD). Unfortunately, the
bisquaternary structure of 1 prevents its passage through the blood brain barrier. In a search of centrally active ambenonium
derivatives, we planned to synthesize tertiary amines of 1, such as 2 and 3. In addition, to add new insights into the binding
mechanism of the inhibitor, we designed constrained analogues of ambenonium by incorporating the diamine functions into cyclic
moieties (4ꢀ12). The biological evaluation of the new compounds has been assessed in vitro against human AChE and BChE. All
/
tertiary amine derivatives resulted more than 1000-fold less potent than 1 and, unlike prototype, did not show any selectivity
between the two enzymes. This result, because of recent findings concerning the role of BChE in AD, makes our compounds,
endowed with a well-balanced profile of AChE/BChE inhibition, valuable candidates for further development. To better clarify the
interactions that account for the high affinity of 1, docking simulations and molecular dynamics studies on the AChE-1 complex
were also carried out.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Alzheimer’s disease; Acetylcholinesterase inhibitor; Acetylcholinesterase; Butyrylcholinesterase; Ambenonium analogues
1. Introduction
accumulation of intracellular neurofibrillary tangles
that remain the definitive diagnostic markers for the
disease. In the absence of a definitive cure for AD, the
treatment has focused on therapy to restore the choli-
nergic tone in the brain, providing symptomatic benefits
and slowing progression of the disease [2].
Among the possible therapeutic approaches toward
improving cholinergic transmission in AD, acetylcholi-
nesterase inhibitors (AChEIs) are the only FDA-ap-
proved pharmacological treatment and the drugs
tacrine, donepezil, rivastigmine, and galantamine have
been shown to be really effective in the treatment of the
cognitive, behavioral, and functional deficits of AD.
Ambenonium (1) is an old AChEI, which is structu-
rally dissimilar from those above mentioned and en-
dowed with one of the highest known affinity (sub
nanomolar range) [3,4]. This outstanding potency is
unique for a compound that does not form any covalent
bond at the active site of the enzyme, although its
bisquaternary oxamide structure prevents the passage
Alzheimer’s disease (AD) is the most common age-
related neurodegenerative disorder and, with the con-
tinuing increase of the elderly population, it has become
an urgent public health problem in western countries.
Paradoxically, this devastating disease is still of un-
known etiology, with the exception of the rare familial
case of autosomal dominant inheritance of gene defects
[1]. A key biochemical hallmark of AD is a selective loss
of cholinergic neurons in the forebrain with a marked
decrease in cholineacetyltransferase (ChAT) in the
hippocampus and cerebral cortex, which correlates
with brain pathology and cognitive dysfunction. Asso-
ciated with this neurochemical changes are the extra-
cellular deposition of b-amyloid peptide and the
* Corresponding author.
E-mail address: camelch@kaiser.alma.unibo.it (C. Melchiorre).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00150-2

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M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ928
/
through the blood brain barrier, after conventional oral
or intravenous routes of administration. In addition, as
recently suggested for other AChEIs, it has been
reported that 1 promotes the non-amyloidogenic route
of amyloid precursor protein (APP) processing, due to a
stimulatory effect on protein kinase C [5,6]. Taken
together, these two possible mechanisms of action
should make ambenonium a very promising pharmaco-
logical tool in the treatment of AD, and a suitable lead
in the development of a new family of AChEIs.
To investigate the binding mode of 1 with AChE, we
have first carried out docking simulations, providing the
molecular basis of its inhibitory activity. Furthermore,
in a search of ambenonium derivatives able to penetrate
the blood brain barrier, we planned to synthesize
correspondent tertiary amines of 1, by deleting one
ethyl group from each quaternary ammonium center (2
and 3). In particular in 3, the chloro substituent on the
phenyl ring of ambenonium has been substituted with
an O-methoxy, to verify whether the substituent at
position 2 might have any influence on the basicity and,
as a consequence, on the extent of protonation of the
benzylic nitrogen atom, as already demonstrated for
caproctamine derivatives [7]. In addition, to add new
insights into the binding mechanism of ambenonium
and its tertiary derivatives with AChE, we designed
constrained analogues by incorporating the diamine
moiety of 17 with HBr afforded the desired diamine 18,
which upon reaction with oxalyl chloride gave the
corresponding tertiary amine (2) of 1.
For the synthesis of diamine 21, carrying a 2-
methoxybenzyl group on one nitrogen, a more straight-
forward synthetic approach was followed (Scheme 2).
Reductive amination of N-CBZ-1,2-diamino ethane
with 2-methoxybenzaldehyde furnished compound 19,
which, in turn, was alkylated with diethylsulfate and
deprotected in acidic medium to give 21. Again, acyla-
tion of 21 with oxalyl chloride provided final compound
3.
Alkylation of 1-BOC-piperazine with 2-bromobenzyl
bromide and subsequent deprotection of the carbamate
moiety of 22 with CF3COOH gave 23 in two steps,
which, in turn, was acylated with the appropriate acidic
chloride to give 4ꢀ
/
6 (Scheme 3).
Amides 7ꢀ9 were synthesized by reaction of the
/
proper 1,2-diaminocyclohexane with chloroacetyl chlo-
ride followed by nucleophilic substitution with amine 16
(Scheme 4).
Similarly, starting from piperazine and amines 16 and
28 [8], derivatives 10 and 11 were obtained (Scheme 5).
The commercial availability of an appropriate (S)-
leucine derivative dictated a slightly different approach
for the synthesis of analogue 12, as shown in Scheme 6.
N-BOC-leucine-N-hydroxysuccinimide was amidated
with piperazine to give 29, which, upon treatment with
CF3COOH to remove the protecting groups and re-
ductive amination with 2-bromobenzaldehyde, provided
12.
The synthesis of the trifluoroacetyl derivative 13 was
accomplished as illustrated in Scheme 7. Derivative 32,
obtained through reductive amination and subsequent
ethylation of glycine sodium salt, was amidated with
1,2-diamino ethane to 33, which, upon reduction of the
carbonyl functions with borane and acetylation with
ethyl trifluoroacetate, afforded 13.
functions into cyclic moieties. In compounds 4ꢀ6 the
/
diamine functions of the lead have been included in a
piperazine cycle and the distance between the nitrogens
has been varied by introducing methylene units between
the carbonyls. Furthermore, we focused on rigid analogs
7ꢀ12, in which the oxamide function has been reversed
/
and the amide nitrogen included in or linked to a cycle.
In particular, to investigate if additional interaction with
the enzyme might take place, a derivative of amino acid
leucine with a lipophilic isobutyl tail was synthesized
(12). Finally, with the aim of improving the access of the
molecule to the CNS modulating the lipophilicity, the
amide moieties of 3 were transformed into trifluoroace-
tamides, as in derivative 13. The design strategy of our
compounds is shown in Fig. 1.
3. Results and discussion
Before starting the design of this series of ambeno-
mium (1) analogues, we thought it of interest to carry
out docking simulations and molecular dynamics (MD)
studies on the AChE-1 complex, with the aim to
rationalize at molecular level the interactions between
1 and AChE that account for its high potency. 1 was
firstly docked into the enzyme gorge and then the
complex was geometrically relaxed by means of MD
simulations. In Fig. 2 is reported a minimized average
conformation taken from the last 60 ps of MD simula-
tions. This picture indeed shows that 1 is able to contact
both the catalytic and the peripheral AChE sites.
Actually, in our docking model, 1 interacted with
2. Chemistry
Compounds 2ꢀ6 were synthesized through acylation
reaction of the oxalyl, malonyl or succinyl chlorides and
the appropriate diamine.
The diamine bearing a 2-chlorobenzyl substituent (18)
was prepared as outlined in Scheme 1, via the key
intermediate 16 [8] that represented a useful synthon
/
also in the synthesis of 7ꢀ11. Thus, protection of the
/
amino function of 2-aminoethyl bromide (14) with CBZ
group yielded the derivative 15 that alkylated amine 16
to provide intermediate 17. Cleavage of the protecting
Trp84 of the catalytic site by means of a pꢀp stacking,
/

M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
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919
Fig. 1. Design strategy for the synthesis of ambenonium (1) derivatives: one ethyl group has been deleted from each ammonium quaternary center
(a); the diamine functions have been incorporated into a piperazine cycle and the distance between the nitrogens has been varied (b); the oxamide
function has been reversed and the amide nitrogen included in or linked to a cycle (c and d).
and with Trp279 of the peripheral site by means of a p-
cation interaction. Furthermore, other relevant interac-
tions between 1 and AChE were detected, providing the
molecular basis of 1 inhibitory activity. In particular, we
pointed out further p-cation interactions with Tyr330,
and Tyr70, whereas H-bonds were identified with
Tyr121, Tyr70, and Asp72. In the latter, the interaction
was also reinforced by the formal charge of ꢁ1 of
/
Scheme 1.

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M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
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Scheme 2.
Asp72, providing a tighter H-bond. Eventually, an
electrostatic interaction was detected between a qua-
ternary ammonium group of 1 and Asp276.
Fig. 2 clearly points out that 1 is able establishing
highly favorable contacts with several AChE amino
acids of both the central and the peripheral enzyme sites,
thus providing a possible explanation for the striking
potency of such inhibitor.
were investigated by determining the inhibitory poten-
cies on AChE and BChE from human erythrocytes and
serum and are reported in Table 1 in comparison with
that of prototype 1.
An analysis of the results reveals that unfortunately
the replacement of the quaternary ammonium functions
of 1 with tertiary amines, affording 2 (tertiary ambeno-
nium) and 3, caused a dramatic loss of affinity, being the
new derivative more than four orders of magnitude less
potent than the lead. This finding clearly indicates that
the presence of two ammonium centers is an essential
requirement for the binding with the enzyme and that
These theoretical studies strengthened our basic idea
to design a new series of lipophilic ambenonium
derivatives, having access to the central nervous system.
The biological properties of the new compounds 2ꢀ13
/
Scheme 3.

M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
/928
921
Scheme 4.
tertiary amine functions, although protonated at phy-
siological pH [7], might establish a different binding
mode with the target.
The insertion of the ethylenediamine functions into a
piperazine cycle was detrimental for the activity: com-
exhibited by tertiary ambenonium (2), whereas the other
stereoisomers were devoid of affinity. To explain these
experimental data, a superimposition between the con-
formation of 1, as obtained from MDs simulations, and
the cis (9) and the trans (8) derivatives was carried out.
Low energy conformations of each molecule were
selected and fitted onto 1, as shown in Fig. 3. It is
evident that the overlap is not perfect for the trans
stereoisomer, whereas a good overall superimposition is
shown for 1 and 9, confirming that the spatial arrange-
ment of the diamide moiety of 9 better resembles that of
1.
pounds 4ꢀ6, with different distances between the nitro-
/
gens, were not able to inhibit the enzyme up to 1 mM
concentration, suggesting that a certain degree of
flexibility in the diamine moiety is an important
determinant for the recognition of AChE.
Next, the transformation of the oxamide in cyclic
amides gave raise to interesting results: in the series of
the cyclohexane diamides 7ꢀ
9 retained good inhibitory potency, comparable to that
/
9, only the cis stereoisomer
Cyclization of the oxamide moiety in a piperazine ring
afforded compounds 10 and 11, differing in the sub-
Scheme 5.

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M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
/928
Scheme 6.
stituent at ortho position of the phenyl. Again, these
compounds displayed similar antagonist potencies for
AChE and comparable to that of 2.
Interestingly enough, transformation of the oxamide
function of 3 in trifuoroacetamides, affording 13, did
not affect potency negatively: 13 was slightly more
active than 3 at both AChE and BChE. Regarding the
inhibitory potency toward BChE, all of the tested
compounds, unlike the prototype, were not able to
discriminate between the two enzymes. The most potent
compound of the series was the cis-derivative 9, with an
Derivative 12, obtained through amidation of the
amino acid Leu with piperazine, showed an intriguing
affinity profile: in fact, notwithstanding the presence of
a secondary amine, which is known not conferring
optimum interaction with the enzyme, it is endowed of
inhibitory activity in the micromolar range. This finding
suggests that the isobutyl tail may interact with apolar
residues of the enzyme.
IC50 value of 3.7390.24 mM. This result may be
/
interesting because of the recent findings [9,10] on the
role of BChE in AD, which is viewed as a ‘pathological
Scheme 7.

M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
/928
923
4.1.1. (2-Bromo-ethyl)-carbamic acid benzyl ester (15)
A solution of benzyl chloroformate (1.56 ml, 10
mmol) was added dropwise to a solution of 2-bromo-
ethylamine hydrobromide (14) (2 g, 10 mmol) in 1 M
NaOH (20 ml) and the reaction mixture was vigorously
stirred for 30 min. The formed solid was collected and
dissolved in CHCl3 (40 ml). The obtained solution was
then washed with 1 N HCl (2ꢂ
/
30 ml) and water (2ꢂ30
/
ml). The organic phase was dried and evaporated to give
15 as a solid, which was triturated with ether: 66% yield;
1
m.p. 42 8C; H NMR (CDCl3): d 3.48 (t, 2H), 3.62 (q,
2H), 5.13 (s, 2H), 5.17 (br s, 1H exchangeable with
D2O), 7.39ꢀ7.50 (m, 5H).
/
Fig. 2. The binding mode of ambenonium (1) within the AChE gorge.
A minimized average conformation over the last 60 ps of MD
simulations is shown. Hydrogen atoms explicitly involved in H-bonds
(dotted lines) are shown.
4.1.2. {2-[(2-Chloro-benzyl)-ethyl-amino]-ethyl}-
carbamic acid benzyl ester (17)
A mixture of 15 (550 mg, 2.13 mmol), 16 (399 mg,
1.94 mmol), NEt3 (0.28 ml, 2.13 mmol) and KI (161 mg,
0.97 mmol) in absolute EtOH was refluxed for 16 h.
Removal of the solvent gave a residue that was purified
by flash chromatography. Eluting with EtOAc/cyclo-
cholinesterase’, acting under conditions of decreased
AChE activity. To this end, our compounds, that are
endowed with a well-balanced profile of AChE/BChE
inhibition, may represent valuable candidates for further
development.
1
hexane (3:7) gave 17 as transparent oil: 26% yield; H
NMR (CDCl3): d 0.94 (t, 3H), 2.40ꢀ
(q, 2H), 3.58 (s, 2H), 5.00 (s, 2H), 5.21 (br s, 1H
exchangeable with D2O), 7.02ꢀ7.37 (m, 9H).
/2.57 (m, 4H), 3.16
/
4. Experimental
4.1.3. N1-(2-Chloro-benzyl)-N1-ethyl-ethane-1,2-
diamine (18)
4.1. Chemistry
A solution of 17 (175 mg, 0.51 mmol) and 30% HBr in
CH3COOH (2 ml) in CH3COOH (6 ml) was stirred for 3
h. Ether (10 ml) was added yielding a solid that was
dissolved in water. The aqueous solution was washed
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
with CH2Cl2 (1ꢂ
/
10 ml), made basic with K2CO3 and
recorded on Perkinꢀ
/
Elmer 297, VG 7070E, and Varian
extracted with CH2Cl2 (3ꢂ
/
15 ml). The combined
organic extracts were dried and evaporated to give 18
VXR 300 instruments, respectively. Chemical shifts are
reported in parts per million (ppm) relative to 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 were consis-
tent with the assigned structures. The elemental compo-
1
as yellow oil: 95% yield; H NMR (CDCl3): d 1.03 (t,
3H), 2.45ꢀ
/
2.60 (m, 4H), 2.69 (t, 2H), 3.63 (s, 2H), 7.08ꢀ
/
7.32 (m, 3H), 7.46 (d, 1H).
4.1.4. N,N?-Bis-{2-[(2-chloro-benzyl)-ethyl-amino]-
ethyl}-oxalamide dihydrochloride (2)
sitions of the compounds agreed to within 9
/
0.4% of the
A solution of oxalyl chloride (0.022 ml, 0.25 mmol) in
dioxane (2 ml) was added dropwise at 10 8C to a
solution of 18 (95 mg, 0.45 mmol) in dioxane (3 ml).
The mixture was stirred for 1 h and the formed solid was
collected by filtration and purified by flash chromato-
graphy. Eluting with CHCl3/MeOH/aq. 28% NH3
(9:1:0.1) gave 2 as the free base that was converted
into the dihydrochloride salt: 59% yield; m.p. 197 8C
calculated value. When the elemental analysis is not
included, crude compounds were used in the next step
without further purification. Chromatographic separa-
tions were performed on silica gel columns by flash
(Kieselgel 40, 0.040ꢀ/0.063 mm; Merck) or gravity
column (Kieselgel 60, 0.063ꢀ/0.200 mm; Merck) chro-
matography. Reactions were followed by thin-layer
chromatography (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 Na2SO4.
1
(EtOH/Et2O); H NMR (CDCl3; free base): d 1.04 (t,
6H), 2.53 (q, 8H), 2.62ꢀ
/
2.70 (m, 4H), 3.66 (s, 4H), 7.12ꢀ
/
7.34 (m, 6H), 7.51 (d, 2H). EI MS m/e (relative intensity)
478 (Mꢃ, 1), 182 (100).

924
M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ928
/
Table 1
Inhibition of AChE and BChE activities by ambenonium-related compounds
no.
Stereoisomer
X
n
IC50 (mM) a
AChE
BchE
1
0.0006989
/
0.00004
8.209
/
0.44
2
Cl
OMe
Br
28.09
/
3.1
nd b
3
11.09
/
1.2
21.59
/
0.80
4
0
1
2
ꢀ
/
1000
1000
1000
1000
1000
Nd
Nd
Nd
Nd
Nd
5
Br
Br
ꢀ
/
6
ꢀ
/
7
R,R
S,S
cis
ꢀ
/
8
ꢀ/
9
64.89
4.819
4.729
8.029
4.49
/
7.0
3.739
4.969
4.099
Nd
6.109
/
0.24
0.31
0.25
10
11
12
13
Cl
Br
/
0.35
0.55
0.37
/
/
/
/
/
0.40
/0.25
a
AChE and BChE were from human erythrocytes. 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.
b
nd, not determined.
4.1.5. [2-(2-Methoxy-benzylamino)-ethyl]-carbamic
acid benzyl ester (19)
A mixture of N-CBZ-1,2-diaminoethane (720 mg, 3.7
D2O), 2.71 (t, 2H), 3.29 (q, 2H), 3.75 (s, 2H), 3.80 (s,
3H), 5.09 (s, 2H), 5.38 (br s, 1H exchangeable with
D2O), 6.84ꢀ6.92 (m, 2H), 7.13ꢀ7.36 (m, 7H).
/
/
˚
mmol), molecular sieves (3 A) and 2-methoxybenzalde-
hyde (760 mg, 5.6 mmol) was stirred for 30 min at room
temperature (r.t.), and then NaBH4 (0.21 g, 5.6 mmol)
was added and the stirring was continued for further 14
h. Following removal of molecular sieves, the solution
was made acidic with 3 N HCl. Removal of the solvent
gave a residue which was dissolved in water (50 ml). The
4.1.6. {2-[Ethyl-(2-methoxy-benzyl)-amino]-ethyl}-
carbamic acid benzyl ester (20)
Diethyl sulfate (0.75 ml, 5.7 mmol) was added
dropwise to a stirred solution of 19 (890 mg, 6.5
mmol) in toluene (50 ml) and the resulting mixture
was refluxed for 24 h. After cooling at r.t., the mixture
was shaken with concd. NaOH (50 ml). The organic
layer was separated, dried and evaporated to give a
residue, which was purified by flash chromatography.
Eluting with CH2Cl2/petroleum ether/MeOH/aq. 28%
NH3 (4.6:5.0:0.35:0.035) afforded 20 as an oil: 60%
aqueous solution was washed with ether (3ꢂ
made basic with 35% NaOH and extracted with CH2Cl2
(3ꢂ30 ml). The combined organic extracts were dried
/30 ml),
/
1
and evaporated to give 19 as yellow oil: 80% yield; H
NMR (CDCl3): d 1.59 (br s, 1H exchangeable with

M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
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925
4.1.10. 1-(2-Bromo-benzyl)-piperazine (23)
A solution of 22 (780 mg, 2.2 mmol) and TFA (5.5 ml)
in CHCl3 was stirred at r.t. for 3 h. The solvent was
evaporated yielding a solid that was dissolved in water
(30 ml). The resulting solution was then washed with
ether (3ꢂ/20 ml), made basic with K2CO3, and extracted
with CHCl3 (3ꢂ
/
20 ml). The organic extracts were dried
and evaporated to give 23 as an oil: 77% yield; 1H NMR
(CDCl3): d 1.50 (s, 1H exchangeable with D2O), 2.38 (t,
4H), 2.57 (t, 4H), 3.46 (s, 2H), 7.00 (t, 1H), 7.18 (t, 1H),
7.37 (t, 2H).
4.1.11. 1,2-Bis-[4-(2-bromo-benzyl)-piperazin-1-yl]-
ethane-1,2-dione dihydrochloride (4)
It was obtained by reaction of 23 and oxalyl chloride
following the procedure described for 2. The formed
solid was purified by crystallization from i-PrOH/
MeOH: 50% yield; m.p. 185 8C; 1H NMR (CDCl3;
free base): d 2.53 (t, 8H), 3.43 (t, 4H), 3.65 (t, 8H), 7.07ꢀ
/
Fig. 3. Superimposition of 9 and 8 (trans) onto 1 (black).
7.19 (t, 2H), 7.24ꢀ
/
7.35 (t, 2H), 7.39ꢀ
/
7.47 (d, 2H), 7.52ꢀ
/
7.90 (d, 2H). EI MS m/e (relative intensity) 564 (Mꢃ, 4),
169 (100).
yield; 1H NMR (CDCl3, free base): d 1.04 (t, 3H), 2.50ꢀ
2.66 (m, 4H), 3.34 (q, 2H), 3.61 (s, 2H), 3.81 (s, 3H), 5.14
/
(s, 2H), 5.70 (br s, 1H exchangeable with D2O), 6.86ꢀ
/
4.1.12. 1,3-Bis-[4-(2-bromo-benzyl)-piperazin-1-yl]-
propane-1,3-dione dihydrochloride (5)
6.98 (m, 2H), 7.23ꢀ7.43 (m, 7H).
/
It was obtained by reaction of 23 and malonyl
chloride following the procedure described for 2. Eluting
with EtOAc/petroleum ether/MeOH/aq. 28% NH3
(5:4:1:0.05) gave 5 as the free base that was converted
into the dihydrochloride salt: 53% yield; m.p. 205 8C
4.1.7. N1-Ethyl-N1-(2-methoxy-benzyl)-ethane-1,2-
diamine (21)
It was obtained as an oil by deprotection of 20
following the procedure described for 18: 75% yield;
1H NMR (CDCl3): d 1.05 (t, 3H), 2.09 (br s, 2H
1
(EtOH/Et2O); H NMR (CDCl3; free base): d 2.50 (q,
8H), 3.50 (s, 2H), 3.53ꢀ3.68 (m, 12H), 7.10 (t, 2H), 7.21
/
exchangeable with D2O), 2.50ꢀ
(m, 2H), 3.60 (s, 2H), 3.82 (s, 3H), 6.83ꢀ
7.18ꢀ7.43 (m, 2H).
/
2.61 (m, 4H), 2.72ꢀ
/
2.82
6.98 (m, 2H),
(t, 2H), 7.42 (d, 2H), 7.53 (d, 2H). EI MS m/e (relative
intensity) 578 (Mꢃ, 1), 169 (100).
/
/
4.1.13. 1,4-Bis-[4-(2-bromo-benzyl)-piperazin-1-yl]-
butane-1,4-dione dihydrochloride (6)
It was obtained by reaction of 23 and succinyl
chloride following the procedure described for 2. The
formed solid was purified by crystallization from i-
PrOH/MeOH; 69% yield; m.p. 270 8C; 1H NMR (D2O):
4.1.8. N,N?-Bis-{2-[ethyl-(2-methoxy-benzyl)-amino]-
ethyl}-oxalamide dioxalate (3)
It was obtained by reaction of 21 and oxalyl chloride
following the procedure described for 2. Eluting with
CH2Cl2/EtOH/aq. 28% NH3 (9.5:0.5:0.05) gave 3 as the
free base that was converted into the dioxalate salt: 30%
yield; m.p. 197 8C (EtOH/Et2O); 1H NMR (CDCl3; free
d 2.71 (s, 4H), 3.34ꢀ
/
3.57 (m, 8H), 3.61ꢀ
/
3.98 (m, 8H),
7.81 (d, 4H). EI
4.55 (s, 4H), 7.33ꢀ
/
7.52 (d, 4H), 7.73ꢀ
/
MS m/e (relative intensity) 592 (Mꢃ, 10), 169 (100).
base): d 1.04 (t, 6H), 2.45ꢀ
3.63 (s, 4H), 3.84 (s, 6H), 6.84ꢀ
(m, 4H), 7.98 (br s, 2H exchangeable with D2O).
/
2.65 (m, 8H), 3.41 (q, 4H),
/
6.99 (m, 4H), 7.20ꢀ7.42
/
4.1.14. 2-Chloro-N-[2-(2-chloro-acetylamino)-
cyclohexyl]-acetamide (R,R) (24)
A solution of chloroacetyl chloride (0.66 ml, 8.3
mmol) in dioxane (5 ml) was added at 5 8C to a solution
of (1R,2R)-1,2-diaminocyclohexane (500 mg, 4.4 mmol)
and NEt3 (1.16 ml, 8.3 mmol) in dioxane (15 ml) and the
reaction mixture was stirred at r.t. for 2 h. The formed
NEt3 hydrochloride was filtered off and the solution was
evaporated in vacuo yielding a residue that was dis-
solved in CH2Cl2 (30 ml). The obtained solution was
4.1.9. 4-(2-Bromo-benzyl)-piperazine-1-carboxylic acid
tert-butyl ester (22)
It was obtained as an oil by reaction of 1-BOC-
piperazine and 2-bromo benzyl bromide following the
procedure described for 17: 85% yield; 1H NMR
(CDCl3): d 1.39 (s, 9H), 2.82 (s, 4H), 3.63 (s, 4H),
4.10 (s, 2H), 7.13 (t, 1H), 7.31 (q, 1H), 7.51 (d, 1H), 7.82
(d, 1H).
then washed with water (2ꢂ20 ml), with 20% aq.
/

926
M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ928
/
Na2CO3 (2ꢂ
/
20 ml) and 0.5 N HCl (2ꢂ
/
20 ml). The
4.1.20. 2-Chloro-1-(4-chloroacetyl-piperazin-1-yl)-
ethanone (27)
organic phase was dried and evaporated to give 24 as a
solid: 47% yield; m.p. 230 8C; 1H NMR (CDCl3): d 1.33
(d, 4H), 1.73ꢀ
It was obtained by reaction of piperazine and
chloroacetyl chloride following the procedure described
/
1.84 (m, 2H), 1.98ꢀ
/
2.11 (m, 2H), 3.66ꢀ
/
1
3.71 (m, 2H), 4.01 (s, 4H), 6.80 (br s, 2H).
for 24: 30% yield; m.p. 118 8C; H NMR (CDCl3): d
3.43ꢀ3.73 (m, 8H), 4.09 (s, 4H).
/
4.1.15. 2-Chloro-N-[2-(2-chloro-acetylamino)-
cyclohexyl]-acetamide (S,S) (25)
It was obtained by reaction of (1S,2S)-1,2-diamino-
cyclohexane and chloroacetyl chloride following the
procedure described for 24: 70% yield.
4.1.21. 2-[(2-Chloro-benzyl)-ethyl-amino]-1-(4-{[(2-
chloro-benzyl)-ethyl-amino]-acetyl}-piperazin-1-yl)-
ethanone dihydrochloride (10)
It was obtained by reaction of 16 and 27 following the
1
procedure described for 7: 41% yield; m.p. 200 8C; H
NMR (DMSO-d6): d 1.29ꢀ
12H), 4.40ꢀ4.55 (m, 8H), 7.49ꢀ
(m, 2H), 9.95 (br s, 2H exchangeable with D2O).
/
1.35 (m, 6H), 3.20ꢀ
/
3.36 (m,
4.1.16. 2-Chloro-N-[2-(2-chloro-acetylamino)-
cyclohexyl]-acetamide (cis) (26)
It was obtained by reaction of cis-1,2-diaminocyclo-
hexane and chloroacetyl chloride following the proce-
dure described for 24: 25% yield; m.p. 137 8C; 1H NMR
/
/
7.58 (m, 6H), 7.78ꢀ
/7.89
4.1.22. 2-[(2-Bromo-benzyl)-ethyl-amino]-1-(4-{[(2-
chloro-benzyl)-ethyl-amino]-acetyl}-piperazin-1-yl)-
ethanone dihydrochloride (11)
It was obtained by reaction of 28 and 27 following the
1
procedure described for 7: 45% yield; m.p. 290 8C; H
(CDCl3): d 1.49ꢀ
/
1.72 (m, 6H), 1.84 (br s, 2H), 4.00ꢀ
/
4.26 (m, 6H), 7.00 (br s, 2H).
4.1.17. 2-[(2-Chloro-benzyl)-ethyl-amino]-N-(2-{2-
[(2-chloro-benzyl)-ethyl-amino]-acetylamino}-
cyclohexyl)-acetamide (R,R) dihydrochloride (7)
A mixture of 16 (300 mg, 1.46 mmol), 24 (194 mg,
0.73 mmol), i-Pr2NEt (0.51 ml, 2.92 mmol) and KI (61
mg, 0.37 mmol) in absolute EtOH (15 ml) was refluxed
for 72 h. Removal of the solvent gave a residue that was
dissolved in CH2Cl2 (30 ml). The obtained solution was
NMR (DMSO-d6): d 1.32 (t, 6H), 3.22ꢀ
/
3.57 (m, 12H),
4.45 (s, 4H), 4.57 (s, 4H), 7.42ꢀ7.94 (m, 8H), 9.96 (br s,
/
2H exchangeable with D2O). EI MS m/e (relative
intensity) 594 (Mꢃ, 2), 226 (100).
4.1.23. {1-[4-(2-tert-Butoxycarbonylamino-4-methyl-
pentanoyl)-piperazine-1-carbonyl]-3-methyl-butyl}-
carbamic acid tert-butyl ester (29)
A solution of N-terbutyloxycarbonyleucine-N-hy-
droxy-succinimmide (500 mg, 1.5 mmol) in CHCl3 (10
ml) was added dropwise to a solution of piperazine (72
mg, 0.84 mmol) in 15 ml of CHCl3. The resulting
mixture was stirred at r.t. for 18 h, then washed with 1
then washed with water (2ꢂ
N HCl (2ꢂ20 ml). The aqueous phase was made basic
with K2CO3 and extracted with CH2Cl2 (3ꢂ30 ml).
/20 ml) and extracted with 1
/
/
Evaporation of the dried solvent gave 7 as the free base
that was converted into the dihydrochloride salt (hygro-
scopic): 40% yield; 1H NMR (DMSO-d6/D2O): d 1.16 (t,
M KHSO4 (2ꢂ
evaporated to give 29 as an oil: 72% yield; H NMR
(CDCl3): d 0.87 (q, 12H), 1.52ꢀ1.75 (m, 20H), 2.75 (s,
2H), 3.12ꢀ3.90 (m, 8H), 4.48ꢀ4.67 (m, 2H), 5.31 (d, 2H
exchangeable with D2O).
/
10 ml) and water (2ꢂ10 ml), dried and
/
10H), 1.54ꢀ1.66 (m, 4H), 3.12 (d, 4H), 3.41 (br s, 2H),
/
1
3.64 (q, 4H), 4.53 (s, 4H), 7.32ꢀ7.48 (m, 6H), 7.57 (d,
/
/
2H). EI MS m/e (relative intensity) 532 (Mꢃ, 3), 182
(100).
/
/
4.1.18. 2-[(2-Chloro-benzyl)-ethyl-amino]-N-(2-{2-
[(2-chloro-benzyl)-ethyl-amino]-acetylamino}-
cyclohexyl)-acetamide dihydrochloride (S,S) (8)
It was obtained by reaction of 16 and 25 following the
procedure described for 7: 70% yield.
4.1.24. 2-Amino-1-[4-(2-amino-4-methyl-pentanoyl)-
piperazin-1-yl]-4-methyl-pentan-1-one (30)
It was obtained as an oil by deprotection of 29
following the procedure described for 23: 95% yield;
1H NMR (CDCl3): d 0.87 (d, 12H), 1.12ꢀ
/
1.36 (m, 6H),
1.70 (s, 4H exchangeable with D2O), 3.32ꢀ3.70 (m,
10H).
/
4.1.19. 2-[(2-Chloro-benzyl)-ethyl-amino]-N-(2-{2-
[(2-chloro-benzyl)-ethyl-amino]-acetylamino}-
cyclohexyl)-acetamide dihydrochloride (cis) (9)
It was obtained by reaction of 16 and 26 following the
procedure described for 7: 57% yield; 1H NMR (DMSO-
4.1.25. 2-(2-Bromo-benzylamino)-1-{4-[2-(2-bromo-
benzylamino)-4-methyl-pentanoyl]-piperazin-1-yl}-4-
methyl-pentan-1-one dihydrochloride (12)
d6/D2O): d 1.13ꢀ
s, 2H), 4.00 (br s, 6H), 4.59 (br s, 4H), 7.39ꢀ
6H), 7.81 (br s, 2H). EI MS m/e (relative intensity) 532
(Mꢃ, 2), 182 (100).
/
1.42 (m, 10H), 1.56 (br s, 6H), 3.20 (br
7.61 (m,
It was obtained by reaction of 30 and 2-bromoben-
zaldehyde following the procedure described for 19: 45%
/
yield; m.p. ꢀ
/
290 8C (MeOH/i-PrOH); 1H NMR (D2O):
d 0.89 (q, 12H), 1.67 (m, 6H), 3.20ꢀ
/
3.53 (m, 8H), 4.20ꢀ
/

M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ
/928
927
4.50 (m, 6H), 7.29ꢀ
/
7.52 (m, 6H), 7.63ꢀ
/
7.89 (t, 2H). EI
to give a residue that was purified by flash chromato-
graphy. Eluting with CH2Cl2/EtOH/aq. 28% NH3
(9:1:0.1) gave 34 as an oil: 30% yield; 1H NMR
(CDCl3): d 1.02 (t, 6H), 2.18 (br s, 2H exchangeable
MS m/e (relative intensity) 397 (Mꢃ, 4), 254 (100).
4.1.26. (2-Methoxy-benzylamino)-acetic acid (31)
It was obtained by reaction of glycine sodium salt and
with D2O), 2.48ꢀ
/
2.68 (m, 16H), 3.58 (s, 4H), 3.80 (s,
7.36 (m, 4H).
2-methoxybenzaldehyde following the procedure de-
1
scribed for 19: 35% yield; m.p. 141ꢀ
6H), 6.82ꢀ6.93 (m, 4H), 7.19ꢀ
/
/
/
143 8C; H NMR
(D2O): d 3.38 (s, 2H), 3.71 (s, 3H), 4.06 (s, 2H), 6.80ꢀ
/
4.1.30. 2,2,2-Trifluoro-N-[2-(2-methoxy-benzylamino)-
ethyl]-N-(2-{[2-(2-methoxy-benzylamino)-ethyl]-
trifluoroacetyl-amino}-ethyl)-acetamide dioxalate (13)
Ethyl trifluoroacetate (0.24 ml, 2.04 mmol) was added
to a solution of 34 (90 mg, 0.2 mmol) in dry MeOH (10
ml) and the reaction mixture was stirred at r.t. for 3
days. Evaporation of the solvent gave a residue that was
purified by flash chromatography. Eluting with CH2Cl2/
EtOH/aq. 28% NH3 (9.5:0.5:0.05) afforded 13 as a free
base which was transformed into the dioxalate salt: 30%
6.97 (m, 2H), 7.11ꢀ7.18 (m, 1H), 7.21ꢀ7.33 (1H).
/
/
4.1.27. [Ethyl-(2-methoxy-benzyl)-amino]-acetic acid
(32)
It was obtained by reaction of 31 and diethyl sulfate
1
following the procedure described for 20: 20% yield; H
NMR (CDCl3): d 1.09 (t, 3H), 2.78 (q, 2H), 3.22 (s, 2H),
3.80 (s, 3H), 3.92 (s, 2H), 6.92ꢀ
/
7.04 (m, 4H), 7.27ꢀ7.42
/
(m, 1H).
yield; 1H NMR (CDCl3, free base): d 1.03 (t, 6H), 2.56ꢀ
2.68 (m, 12H), 3.40ꢀ3.60 (m, 8H), 3.80 (s, 6H), 6.87ꢀ
7.00 (m, 4H), 7.19ꢀ7.38 (m, 4H).
/
4.1.28. 2-(2-Methoxy-benzylamino)-N-{2-[2-(2-
/
/
methoxy-benzylamino)-acetylamino]-ethyl}-acetamide
(33)
/
Ethyl chlorocarbonate (0.30 ml, 2.9 mmol) in dry
dioxane (10 ml) was added dropwise to a stirred and
cooled (5 8C) solution of 32 (0.63 g, 2.9 mmol) and NEt3
(0.81 ml, 5.8 mmol) in dioxane (50 ml), followed after
standing for 15 min by the addition of ethylenediamine
(0.097 ml, 1.5 mmol) in dioxane (20 ml). After stirring at
r.t. for 24 h, the mixture was evaporated, affording a
residue that was suspended in water (100 ml). The
4.2. Biology
4.2.1. Inhibition of acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE)
The method of Ellmann et al. was followed [11]. Five
different concentrations of each compound were used in
order 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 mM 5,5?-dithio-bis(2-nitrobenzoic acid),
0.035 U/ml AChE or BChE derived from human
erythrocytes or human serum (0.5 and 3.4 U.I./mg,
respectively; Sigma Chemical), and 550 mM acetylthio-
coline iodide. Test compounds were added to the assay
solution and preincubated 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 in order to account for non-enzymatic reaction.
The reaction rates were compared and the percent
inhibition due to the presence of test compounds was
calculated. Each concentration was analysed in tripli-
cate, and IC50 values were determined graphically from
aqueous mixture was extracted with CHCl3 (4ꢂ20 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.
Eluting with CH2Cl2/EtOH/aq. 28% NH3 (9.5:0.5:0.04)
1
afforded 33 as a low melting solid: 65% yield; H NMR
(CDCl3): d 1.03 (t, 6H), 2.56 (q, 4H), 3.08 (s, 4H), 3.13ꢀ
3.19 (m, 4H), 3.61 (s, 4H), 3.89 (s, 6H), 6.87ꢀ7.00 (m,
4H), 7.19ꢀ7.38 (m, 4H), 7.92 (br s, 2H exchangeable
with D2O).
/
/
/
4.1.29. N-(2-Methoxy-benzyl)-N?-{2-[2-(2-methoxy-
benzylamino)-ethylamino]-ethyl}-ethane-1,2-diamine
(34)
A solution of 10 M BH3×
/
CH3SCH3 (0.55 ml, 5.5
log concentrationꢀ
4.3. Molecular modeling
The modeling procedure of the AChEꢀ
complex was performed by means of the MacroModel
Ver 5.5 software package [12]. The energy calculations
were carried out with MacroModel implementation of
AMBER force field, i.e. AMBER*, that uses by default
the united atom model [13], providing a special scheme
to incorporate specific structural and energetic data
directly into the force field. This feature allows to treat
explicitly (all-atom model [14]) hydrogen atoms bound
/
inhibition curves.
mmol) in dry diglyme (5 ml) was added dropwise at 5 8C
to a solution of 33 (460 mg, 0.98 mmol) in dry diglyme
(35 ml) with stirring under a stream of dry nitrogen.
When the addition was completed, the reaction mixture
was heated at 120 8C for 10 h. After cooling at 0 8C,
excess borane was destroyed by cautious addition of
MeOH (5 ml). The resulting mixture was left to stand
overnight at r.t., cooled at 0 8C, treated with HCl gas for
10 min, and then heated at 120 8C for 3 h. After cooling,
the formed solid was filtered, dissolved in water. The
aqueous solution was made basic with NaOH pellets,
/ambenonium
extracted with CH2Cl2 (4ꢂ20 ml), dried and evaporated
/

928
M.L. Bolognesi et al. / Il Farmaco 58 (2003) 917ꢀ928
/
precursor protein processing and protein kinase C level in vitro,
Neurochem. Int. 38 (2001) 219ꢀ226.
[6] M. Pakaski, H. Papp, Z. Rakonczay, I. Fakla, P. Kasa, Effects of
to heteroatoms and aromatic carbons. In this way, we
have been able taking into account also the multiple-ion
contributions of the p-cation interaction. Actually, these
/
acetylcholinesterase inhibitors on the metabolism of amyloid
are fundamental when modeling AChEꢀinhibitor com-
/
precursor protein in vitro, Neurobiology 9 (2001) 55ꢀ57.
/
plexes [15], and their major contribution is indeed
accounted for by the interaction between quadrupole
of aromatic rings and charge of positive groups [16].
[7] V. Tumiatti, M. Rosini, M. Bartolini, A. Cavalli, G. Marucci, V.
Andrisano, P. Angeli, R. Banzi, A. Minarini, M. Recanatini, C.
Melchiorre, Structureꢀactivity relationships of acetylcholinester-
/
ase noncovalent inhibitors based on a polyamine backbone. 2.
The coordinates of the AChEꢀdecamethonium com-
/
Role of the substituents on the phenyl ring and nitrogen atoms of
plex were retrieved from the Protein Data Bank (PDB
code 1ACL [17]). Ambenonium was properly built and
docked into the enzyme active site, taking advantage of
caproctamine, J. Med. Chem. 46 (2003) 954ꢀ966.
/
[8] W.R. Meindl, E. von Angerer, H. Schonenberger, G. Ruck-
deschel, Benzylamines: synthesis and evaluation of antimycobac-
terial properties, J. Med. Chem. 27 (1984) 1111ꢀ1118.
/
AChEꢀ
/
decamethonium interaction model. The AChEꢀ
/
[9] E. Giacobini, Cholinesterase inhibitors: from the Calabar bean to
Alzheimer therapy, in: E. Giacobini (Ed.), Cholinesterase and
Cholinesterase inhibitors, Martin Dunitz, London, 2000, pp.
ambenonium complex was then submitted to a mole-
cular modeling protocol aimed at refining the position
of the inhibitor in the active site pocket. Minimization
and MD simulations were carried out on a core of
˚
unconstrained atoms around the active site (8 A) and on
a shell of constrained atoms (energy penalty force
1
constant of 100 kJ/A /mol ) surrounding the core (6
181ꢀ226.
/
[10] Q. Yu, H.W. Holloway, T. Utsuki, A. Brossi, N.H. Greig,
Synthesis of novel phenserine-based-selective inhibitors of butyr-
ylcholinesterase for Alzheimer’s disease, J. Med. Chem. 42 (1999)
2
˚
1855ꢀ1861.
/
[11] G.L. Ellmann, D. Courtney, V. Andres, Jr., R.M. Featherstone,
˚
A). An initial minimization (2000 steps, steepest descent)
A new and rapid colorimetric determination of acetylcholinester-
and a subsequent temperature constant MD simulation
(100 ps, 298 K, 1.0 fs time step) were carried out. An
equilibration time of 40 ps was allowed before starting
the data collection. The MD average structure of the last
60 ps was energy minimized first by steepest descent and
then by conjugate gradient with a derivative conver-
ase activity, Biochem. Pharmacol. 7 (1961) 88ꢀ95.
[12] F. Mohamadi, N.G.J. Richards, W.C. Guida, R.M.J Liskamp,
/
M.A. Lipton, C.E. Caulfield, G. Chang, T.F. Hendrickson, W.C.
Still, MacroModel*
organic and bioorganic molecules using molecular mechanics, J.
Comput. Chem. 1 (1990) 440ꢀ467.
/an integrated software system for modeling
/
[13] J.S. Weiner, P.A. Kollman, D.A. Case, U.C. Singh, C. Ghio, G.
Alagona, S. Profeta, P.A Weiner, Jr., New force field for
molecular mechanical simulation of nucleic acids and proteins,
2
1
˚
gence criterion of 0.05 kJ/A /mol . This molecular
modeling protocol has already provided reliable results,
when studying AChEꢀinhibitor complexes [18,19].
/
J. Am. Chem. Soc. 106 (1984) 765ꢀ784.
/
[14] W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz, Jr.,
D.M. Ferguson, D.C. Spellmeyer, T. Fox, J.W. Caldwell, P.A.
Kollman, A Second generation force field for the simulation of
proteins, nucleic acids, and organic molecules, J. Am. Chem. Soc.
Acknowledgements
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5197.
Supported by grants from University of Bologna
(Funds for selected research topics) and MIUR.
/
/
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