Structure-activity relationships of acetylcholinesterase noncovalent inhibitors based on a polyamine backbone. 3. Effect of replacing the inner polymethylene chain with cyclic moieties

Article abstract of DOI:10.1021/jm0494366

In the present paper we expanded SAR studies of 3, the ethyl analogue of the AChE inhibitor caproctamine (2), by investigating the role of its octamethylene spacer separating the two amide functions through the replacement with dipiperidine and dianiline

Full text of DOI:10.1021/jm0494366

6490
J. Med. Chem. 2004, 47, 6490-6498
Structure-Activity Relationships of Acetylcholinesterase Noncovalent
Inhibitors Based on a Polyamine Backbone. 3. Effect of Replacing the Inner
Polymethylene Chain with Cyclic Moieties
Vincenzo Tumiatti,* Vincenza Andrisano, Rita Banzi, Manuela Bartolini, Anna Minarini, Michela Rosini, and
Carlo Melchiorre*
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received July 16, 2004
In the present paper we expanded SAR studies of 3, the ethyl analogue of the AChE inhibitor
caproctamine (2), by investigating the role of its octamethylene spacer separating the two amide
functions through the replacement with dipiperidine and dianiline moieties. Compounds 4 and
8 were the most interesting of the two series of compounds. Compound 4 was the most potent
AChE inhibitor with a pIC50 value of 8.48 ( 0.02, while displaying also significant muscarinic
M2 antagonistic activity (pKb value of 6.18 ( 0.20). The availability of a suitable assay allowed
us to verify whether 2, 3, 4, and 8 inhibit AChE-induced Aâ aggregation. Although all four
derivatives caused a mixed type of AChE inhibition (active site and PAS), only 4 and 8, which
bear an inner constrained spacer, were able to inhibit AChE-induced Aâ aggregation to a greater
extent than donepezil. Clearly, the ability of an AChE inhibitor, based on a linear polyamine
backbone, to bind both AChE sites may not be a sufficient condition to inhibit also AChE-
induced Aâ aggregation. Dipiperidine derivative 4 emerged as a valuable pharmacological tool
and a promising lead compound for new ligands to investigate and, hopefully, treat Alzheimer’s
disease.
Introduction
Alzheimer’s disease (AD), the most common form of
dementia in adults, is a neurodegenerative disorder
characterized by loss of cognitive ability and severe
behavioral abnormalities, and it ultimately results in
total degradation of intellectual and mental activities.1
Much effort is devoted to elucidate the relationships
among the hallmarks of the disease, that is, amyloid
plaques, neurofibrillary tangles, and loss of neurons in
the hippocampus and nucleus basalis of Maynart. AD
is characterized by a pronounced degradation of the
cholinergic system and by alteration in other neu-
rotransmitter systems as glutamatergic and serotonin-
ergic ones.2
The actual therapeutic approaches are based on
different morphological and biochemical characteristics
of AD and focused on the following directions: (i)
restoring of the native levels of the cholinergic trans-
mission in CNS; (ii) decreasing the production or ag-
Figure 1. Chemical structure of drugs in current use for the
gregation of â-amyloid peptide (Aâ), the major compo-
treatment of Alzheimer’s disease.
nent of the senile plaques, or increasing its removal; (iii)
protection of nerve cells from toxic metabolites formed
(Figure 1).3,4 These drugs have been approved for the
in neurodegenerative processes; (iv) activation of other
symptomatic treatment of AD as they do not even
neurotransmitter systems to compensate indirectly the
address the etiology of the disease for which are used.5-7
cholinergic function deficit.
Some experimental evidence suggests that AChE plays
Despite an enormous amount of work, many aspects
also a noncholinergic function8,9 in the development of
of both the etiology and the physiological pathway of
AD. In particular, its consistent presence in senile
AD remain still unclear. To date, only acetylcholinest-
plaques could represent the cause of Aâ aggregation and
erase (AChE) inhibitors, such as tacrine, donepezil,
deposition.10,11 It accelerates the assembly of Aâ into
rivastigmine, and galantamine, and an NMDA receptor
insoluble fibrils containing both Aâ and AChE, which
antagonist, memantine, are available for AD treatment
are more toxic to cells than Aâ alone.12 Several studies,
carried out in the presence of either competitive or
noncompetitive inhibitors of AChE to determine the
molecular domain of AChE involved in the interaction
* Corresponding authors. C.M.: tel, +39-051-2099706; fax, +39-051-
2099734; e-mail, carlo.melchiorre@unibo.it. V.T.: tel, +39-051-2099709;
fax, +39-051-2099734; e-mail, vincenzo.tumiatti@unibo.it.
10.1021/jm0494366 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/16/2004

SARs of AChE Noncovalent Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6491
with Aâ, suggest that the catalytic site of AChE does
not participate in the interaction with Aâ. A potential
locus of interaction between Aâ and AChE has been
identified by crystal structure data on the external
surface in proximity of the catalytic gorge of AChE and
called the “AChE peripheral site”.13 The specific role of
AChE in amyloid formation is confirmed by observations
on butyrylcholinesterase (BChE). BChE shares many
structural and physicochemical properties with AChE14
and has been detected in senile plaques and in neu-
rofibrillary tangles, where it is colocalized with the Aâ.15
However, BChE does not bear a peripheral site and does
not enhance the assembly of Aâ into amyloid fibrils.16,17
It derives that compounds able to bind simultaneously
to both the catalytic and peripheral sites of AChE should
implicate advantages over inhibitors that act only on
the catalytic site because the inhibition of the peripheral
binding site might prevent the aggregation of Aâ
induced by AChE. Our group has been involved for a
long time in the design and synthesis of ligands having
affinity for (i) both AChE active and peripheral binding
sites and (ii) muscarinic M2 receptors18 by applying the
“cholinergic hypothesis”.6 Such derivatives would facili-
tate ACh release in the synaptic cleft by antagonizing
muscarinic M2 autoreceptors and would potentiate as
well the remaining cholinergic transmission in affected
brain regions by inhibition of AChE activity.
Application of the universal template approach has
allowed us to design polyamines, displaying affinity for
AChE and muscarinic M2 receptors, structurally related
to methoctramine (1), a well-known selective muscarinic
M2 receptor antagonist.18,19 It turned out that capro-
ctamine (2) antagonized muscarinic M2 receptors with
a pA2 value of 6.39 and inhibited AChE with a pIC50
value of 6.77. Owing to its interesting biological profile,
2 was investigated further by inserting different sub-
stituents on the terminal phenyl rings and on the four
nitrogen atoms, leading to the N-ethyl analogue 3 with
Figure 2. Chemical structure of methoctramine (1) and
caproctamine (2) and of its ethyl analogue 3. The design
strategy for the development of 4-16 is also shown.
Since in a previous study we verified that AChE
inhibition by polyamines related to 3 was dependent on
the extent of protonation of the two terminal amine
functions, we included in this study iodomethylated
derivatives 11-13, which bear two permanent positive
charges to verify further the role of basicity on activity.
Finally, to achieve information on the role of the two
diamide functions on activity, they were reduced to the
corresponding amine functions as in 14-16 (Scheme 1).
The two inner amine functions of these compounds will
be hardly protonated at physiological pH and, conse-
quently, should not alter the overall lipophilicity of the
molecule.
Chemistry
All the compounds were synthesized by standard
procedures (Scheme 1) and were characterized by IR,
1H NMR, mass spectra, and elemental analysis. Di-
amine diamides 17-20 and 21-23 were obtained by
reacting N-[(benzyloxy)carbonyl]-6-aminocaproic acid
with the appropriate dipiperidine or dianiline deriva-
tive, respectively. Removal of the N-(benzyloxy)carbonyl
group by hydrolysis with HBr gave diamine diamides
24-30, which were treated with 2-methoxybenzalde-
hyde followed by reduction with NaBH4 of the formed
Schiff base to the corresponding dibenzyl derivatives
31-37. Diethylation of 31-37 to afford 4-10 was
performed by using diethyl sulfate in toluene at reflux-
ing temperature. The state of the reaction was continu-
ously monitored to avoid possible quaternization of the
basic functions. Tetraamines 14-16 were prepared by
reduction of diamine diamides 8-10 with BACH-EI in
dry diglyme.
a significantly improved inhibitory activity (pIC50
7.73) toward AChE.
)
The present article expands on the study of another
aspect of structure-activity relationships of 3, namely
the investigation on the role of the octamethylene spacer
separating the two amide functions. A previous docking
study carried out on the diprotonated form of 2 revealed
that 2 is able to interact simultaneously with both active
and peripheral sites of AChE and to establish favorable
interactions with a number of residues in the gorge
thanks to the flexibility of the molecule, which allows
it to assume many conformations.20 To reduce the
conformational freedom of the polymethylene chain of
polyamines we designed a series of compounds in which
the inner octamethylene chain of 3 is incorporated
partially or totally into a more constrained moiety as
shown in Figure 2.21 These structural modifications
would afford compounds in which the inner diamide
moiety is forced to assume a more definite arrangement
that would allow the two basic terminal chains to orient
themselves in different spatial regions relative to each
other. Therefore, the highly flexible polymethylene
chain connecting the two amide functions of 3 was
replaced by the less flexible dipiperidine or dianiline
moieties, affording 4-7 or 8-10 (Scheme 1), respec-
tively.
Methiodides 11-13 were synthesized by methylation
of the corresponding tertiary diamine with an excess of
methyl iodide.
Biology
To determine the potential interest of compounds
4-16 for the treatment of AD, their inhibitory potency
against recombinant human AChE and isolated serum
BChE was evaluated by studying the hydrolysis of
acetylthiocholine (ATCh) following the method of Ell-
man et al.22 The inhibitory potency was expressed as
pIC50 values, which represent the negative logarithm
of the concentration of inhibitor required to decrease

6492 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Tumiatti et al.
Scheme 1a
a
Cbz ) C6H5CH2OCO-; (i) Et3N, EtOCOCl, dioxane; (ii) HBr, CH3COOH; (iii) 2-MeOC6H4CHO, toluene, NaBH4, EtOH; (iv) (EtO)2SO2,
toluene; (v) CH3I, acetone; (vi) BACH-EI, diglyme.
enzyme activity by 50%. To allow comparison of the
results, 1-3, tacrine, physostigmine, and donepezil were
used as the reference compounds. Functional activity
of 4 and reference compounds 1-3 at muscarinic recep-
tors was determined by the use of the muscarinic M2
receptor mediated negative inotropism in driven guinea
pig left atria (1 Hz).23 The biological results were
expressed as pKb values according to van Rossum.24
The nature of AChE inhibition caused by these
compounds was investigated by the comparison of the
graphical analysis of steady-state human AChE inhibi-
tion data of the most potent compound of this series (4)
with those of donepezil, a well-known inhibitor of
catalytic and peripheral sites of AChE (Figure 3).25,26
The ability of compounds 4, 6, and 8 to inhibit the
proaggregating action of AChE toward Aâ, in compari-
son with donepezil and reference compounds 2 and 3,
was assessed through a thioflavin T-based fluorometric
assay early reported by Inestrosa27 and recently vali-
dated through circular dichroism.28 The results are
reported in Table 1.
well-known AChE inhibitors tacrine, physostigmine,
and donepezil. Furthermore, compounds 4, 6, and 8
were evaluated also for their ability to inhibit the
proaggregating action of AChE toward Aâ. Compounds
2, 3, and donepezil were used as reference compounds.
In addition, the antagonism at muscarinic M2 receptors
was determined for compound 4.
Recently, we reported that 3 is a potent AChE
inhibitor and a muscarinic M2 receptor antagonist.21 We
demonstrated that the AChE inhibitory potency of
polyamines related to 3 is linearly correlated with the
basicity of the two basic nitrogen atoms, which is
influenced by the nature and the position of substituents
on the two aromatic rings. A graphical analysis of
steady-state inhibition data indicated that 3 was able
to bind at both catalytic and peripheral sites of AChE.
Thus, 3 could represent a valuable lead for the design
of compounds useful to investigate AD because of (a)
their improvement of cholinergic transmission by in-
hibiting the catalytic site of AChE and (b) their interac-
tion with the AChE peripheral anionic site which might
prevent AChE-mediated Aâ aggregation.21 This reason-
ing formed the basis for present study. All the synthe-
sized compounds 4-16 showed AChE inhibition activity
with pIC50 values ranging from 4.51 to 8.48. All of the
compounds were less potent in inhibiting BChE than
AChE activity. The most potent compound against
Results and Discussion
The inhibitory potency, expressed as pIC50 values, of
compounds 4-16 on human recombinant AChE and
BChE from human serum is reported in Table 1 in
comparison with the prototype polyamines 1-3 and the

SARs of AChE Noncovalent Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6493
The reduction of the two amide functions of 8-10,
leading to compounds 14-16, caused a marked loss in
activity, confirming a previous finding that polymeth-
ylene tetraamines are less active on AChE than the
corresponding diamine diamides (for example, compare
the inhibitory potency of 1 vs 2). The finding that 4 and
8, which are endowed of the shortest distance (n ) 0,
Table 1) between the two amide nitrogen atoms in the
dipiperidine and dianiline series, respectively, showed
the highest potency within the two series may indicate
that the octamethylene spacer of 3 interacts with AChE
in a not extended conformation.
The graphical analysis of steady-state inhibition data
for 4 in comparison to donepezil is shown in Figure 3,
whereas the estimates of competitive inhibition con-
stants Ki of derivatives 2, 3, 4, and donepezil are
reported in Table 2. It was found that 4, like 2, 3, and
donepezil, caused a mixed type inhibition, that is,
inhibition of both the catalytic site and a second distal
site of the enzyme, according to the reported results
indicating that donepezil is a double site inhibitor.25 In
addition, an analysis of Ki values shown in Table 2
reveals that 4 is the most potent among the investigated
diamine diamides.
Furthermore, the availability of a suitable test for the
determination of the Aâ aggregation induced by AChE
allowed us to verify if an inhibitor of peripheral and
active sites of AChE is able to decrease or block the Aâ
aggregation induced by AChE. To this end, we selected
compounds 4/8 and 6 because they have a shorter or a
longer spacer, respectively, in comparison to prototype
2, 3, and donepezil. It turned out that reference com-
pounds 2 and 3 were not able to inhibit the Aâ
aggregation induced by AChE, although the steady-state
diagrams of AChE inhibition confirmed the binding of
the two ligands to both active and peripheral AChE
sites, whereas 4 showed a 41% inhibition that was
significantly higher than the percent inhibition (22%)
observed for donepezil. Interestingly, 4 was more potent
than 6 (15%) in inhibiting AChE-induced Aâ aggrega-
tion, which may suggest that optimal inhibition is
achieved when the two rings of the inner spacer are
linked to each other, giving raise to a more rigid
structure. This view was in agreement with the inhibi-
tion value of 35% observed for 8, which bears a dianiline
spacer. The finding that 2 and 3, although binding to
both AChE sites, did not affect Aâ aggregation may
indicate that the ability to bind to active and peripheral
sites of AChE is not a sufficient condition to inhibit the
Aâ aggregation induced by AChE. It appears that to
inhibit Aâ aggregation a 2-related polyamine must
incorporate in its structure a constrained spacer be-
tween the two inner nitrogen atoms, rather than a
flexible polymethylene chain.
Figure 3. Steady-state inhibition of AChE hydrolysis of
acetylthiocholine by 4 (a) and donepezil (b). Reciprocal plot of
initial velocity and substrate concentration is reported. Re-
ciprocal plot of initial velocity in the absence of inhibitor gave
an estimate of kapp for ATCh of 170 ( 15µM (four experiments).
Lines were derived from a weighted least-squares analysis of
the data points.
BChE was 12 whereas the least active was 16. Besides
AChE inhibition, the observed BChE inhibition may
have relevance considering the new emerging role of
BChE in AD.29 An analysis of results reported in Table
1 reveals that AChE inhibition can be markedly affected
by the introduction of more constrained spacers, such
as dipiperidine and dianiline moieties, in the structure
of 3. The two most potent AChE inhibitors were 4 and
5, which belong to the dipiperidine series. They dis-
played also the highest AChE/BChE ratios, which were
comparable to each other. Compounds 6 and 7, which
bear a longer spacer in their structure, were only twice
less potent than 4 at AChE, whereas they resulted
slightly more potent in inhibiting BChE activity. The
quaternization of 4 and 5, leading to 11 and 12, did not
improve AChE and BChE inhibitory activity, suggesting
that the 2-methoxy functions on the two aromatic rings
are endowed with optimal electronic properties, in
agreement with previous results obtained with poly-
amines related to 3.
Interestingly diamine diamide 4 besides its AChE and
Aâ-aggregation inhibition properties was also a mus-
carinic M2 receptor antagonist in the guinea pig left
atrium (pKb ) 6.18 ( 0.20). This additional property
may help in increasing ACh release in the synapse as a
result of muscarinic M2 autoreceptors blockade.
Replacing the 1,8-diaminooctane spacer of 3 with a
dianiline moiety, leading to 8-10, caused a decrease in
inhibitory activity on AChE as well as in the selectivity
AChE/BChE ratio. The most potent inhibitor of this
series was 8, which is endowed of the shortest distance
between the two amide functions, confirming the trend
observed for the dipiperidine series. Again, the quater-
nization of the two basic nitrogen atoms of 10, leading
to 13, did not affect the inhibitory activity on AChE as
observed for the dipiperidine series.
Conclusions
The present investigation has demonstrated that the
replacement of the inner linear polymethylene chain of

6494 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Tumiatti et al.
Table 1. Inhibition of AChE and BChE Activities and AChE-Induced Aâ Aggregation by Polyamines
b
pIC50
AChE/
% inhibition of
no.a
X
n
AChE
BChE
BChEc
pKb(M2)d
Aâ aggregatione
1f
2f
3f
4
5.27 ( 0.03
6.77 ( 0.01
7.73 ( 0.02
8.48 ( 0.02
8.48 ( 0.01
8.13 ( 0.02
8.18 ( 0.03
7.44 ( 0.03
7.20 ( 0.03
6.77 ( 0.02
8.24 ( 0.02
8.24 ( 0.02
6.92 ( 0.03
5.58 ( 0.01
5.97 ( 0.04
4.51 ( 0.05
6.37 ( 0.02
7.87 ( 0.18
7.64 ( 0.09
6.01 ( 0.02
4.93 ( 0.04
5.65 ( 0.03
5.07 ( 0.03
5.19 ( 0.03
5.44 ( 0.02
5.47 ( 0.02
5.73 ( 0.02
5.74 ( 0.03
5.77 ( 0.01
5.73 ( 0.01
6.18 ( 0.02
5.96 ( 0.06
4.81 ( 0.02
5.79 ( 0.06
4.02 ( 0.04
7.34 ( 0.02
7.58 ( 0.15
5.12 ( 0.01
0.2
68
7.92 ( 0.08g
ndh
<5i
<5i
41 ( 2
nd
6.39 ( 0.23g
121
2570
1949
490
513
51
5.65 ( 0.6g
6.18 ( 0.20
0
1
2
3
0
1
2
2
3
5
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
6
15 ( 3
nd
7
8
O
O
O
35 ( 5
nd
9
29
10
10
11
12
13
14
15
16
nd
323
115
9
nd
nd
nd
H2
H2
H2
0
1
2
6
nd
2
nd
3
nd
tacrine
physostigmine
donepezil
0.1
2
nd
nd
331
22 ( 3
a 1, tetrahydrochloride; 2-10, dioxalate; 11-13, methiodides. b Human recombinant AChE and BChE from human serum were used.
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 antilog of the difference
between pIC50 values at AChE and BChE, respectively. d pKb values ((SE, n ) 4) were calculated according to Van Rossum24 at 10 µM
concentration at muscarinic M2 receptors in guinea pig left atrium. e Inhibition of AChE-induced Aâ aggregation produced by the tested
compound at 100 µM concentration. f See Figure 2 for structure. g Data from ref 21. h Not determined. i Not significant.
Table 2. Inhibition Constants of AChE Obtained with 4 in
Comparison with 2, 3, and Donepezila
synaptic cleft. Owing to its biological profile, 4 may
represent a new promising lead in the field of AD.
compd
AChE Ki (nM)
Experimental Section
2
3
4
104 ( 0.6b
12.2 ( 16c
5.67 ( 0.15
20.5 ( 3.3d
Chemistry. Melting points were taken in glass capillary
tubes on a Buechi SMP-20 apparatus and are uncorrected.
Electron impact (EI) mass and direct infusion ESI-MS spectra
were recorded on VG 7070E, and Waters ZQ 4000 apparatus,
respectively. 1H NMR spectra were recorded on a Varian VXR
300 instrument. 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), q (quartet), t (triplet), or m (multiplet). The elemen-
tal 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 per-
formed on silica gel columns by flash (Kieselgel 40, 0.040-
0.063 mm; Merck) or gravity column (Kieselgel 60, 0.063-
0.200 mm; Merck) chromatography. 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 sodium sulfate. Compounds were named
following IUPAC rules as applied by Beilstein-Institut Au-
toNom (version 2.1), a PC integrated software package for
systematic names in organic chemistry.
donepezil
a Inhibition constants, expressed as pKi values, were calculated
from kinetic data in Figure 3. b Data from ref 20. c Data from ref
21. d Data from ref 26.
2 and 3 with a bicyclic moiety led to an increase of AChE
inhibitory activity. The most active compounds of this
new series of polyamines were dipiperidines 4 and 5,
and the dianiline 8, which are endowed of the shortest
distance between the two amide functions. These data
may indicate that 2 and 3 interact in a not extended
conformation with AChE.
The analysis of steady-state diagrams of 2, 3, 4, and
8 showed that all derivatives caused inhibition of AChE
catalytic and peripheral sites (dual sites), but only 4 and
8, which incorporate in their polyamine backbone an
inner constrained spacer, were able to inhibit AChE-
induced Aâ aggregation to a greater extent than the
reference compound donepezil. Thus, it is our hypothesis
that inhibition of AChE peripheral site is not a sufficient
condition to inhibit AChE-induced Aâ aggregation,
which may necessitate particular structural require-
ments to prevent the interaction of AChE with Aâ.
Derivative 4 was also a muscarinic M2 antagonist, a
property that can enhance the release of AChE in the
General Procedures for the Synthesis of {6-[1′-(6-
Benzyloxycarbonylamino-hexanoyl)-[4,4′]bipiperidinyl-
1-yl]-6-oxo-hexyl}-carbamic Acid Benzyl Ester (17), (6-
{4-[1-(6-Benzyloxycarbonylamino-hexanoyl)-piperidin-
4-ylmethyl]-piperidin-1-yl}-6-oxo-hexyl)-carbamic Acid
Benzyl Ester (18), [6-(4-{2-[1-(6-Benzyloxycarbonylamino-
hexanoyl)-piperidin-4-yl]-ethyl}-piperidin-1-yl)-6-oxo-
hexyl] Carbamic Acid Benzyl Ester (19), [6-(4-{3-[1-(6-

SARs of AChE Noncovalent Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6495
Benzyloxycarbonylamino-hexanoyl)-piperidin-4-yl]-
propyl}-piperidin-1-yl)-6-oxo-hexyl]-carbamic Acid Benzyl
Ester (20), {5-[4′-(6-Benzyloxycarbonylamino-hexanoy-
lamino)-biphenyl-4-ylcarbamoyl]-pentyl}-carbamic Acid
Benzyl Ester (21), (5-{4-[4-(6-Benzyloxycarbonylamino-
hexanoylamino)-benzyl]-phenylcarbamoyl}-pentyl)-car-
bamic Acid Benzyl Ester (22), and [5-(4-{2-[4-(6-Benzyl-
oxycarbonylamino-hexanoylamino)-phenyl]-ethyl}-
phenylcarbamoyl)-pentyl]-carbamic Acid Benzyl Ester
(23). Ethyl chloroformate (0.95 mL, 10 mmol) in dry dioxane
(20 mL) was added dropwise to a stirred and cooled (5 °C)
solution of N-[(benzyloxy)carbonyl]-6-aminocaproic acid (2.65
g, 10 mmol) and triethylamine (1.4 mL, 10 mmol) in dioxane
(30 mL), followed after standing for 30 min by the addition of
the suitable dipiperidine or dianiline (5 mmol) in dioxane (20
mL). After the mixture was stirred at room temperature for
72 h, the solvent was evaporated, affording a residue that was
suspended in water (100 mL). The aqueous mixture was
extracted with CHCl3 (4 × 20 mL) or the solid residue filtered
off for compounds 21-23. The organic phase or the solid
residue for 21-23 was washed with 2 N NaOH, 2 N HCl, and
brine. Removal of the dried solvent gave the desired crude
products 17-23.
amino-hexanoyl)-piperidin-4-yl]-propyl}-piperidin-1-yl)-
hexan-1-one (27), 6-Amino-hexanoic Acid [4′-(6-amino-
hexanoylamino)-biphenyl-4-yl]-amide (28), 6-Amino-
hexanoic Acid {4-[4-(6-amino-hexanoylamino)-benzyl]-
phenyl}-amide (29), and 6-Amino-hexanoic Acid (4-{2-
[4-(6-amino-hexanoylamino)-phenyl]-ethyl}-phenyl)-
amide (30). A solution of 30% HBr in acetic acid (25 mL) was
added to a solution of 17-23 (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 compounds 24-30 in quantitative yields.
24: foam solid; 1H NMR (CDCl3, free base) δ 0.98-1.83 (m,
22H + 4H exchangeable with D2O), 2.23 (t, 4H), 2.38 (t, 2H),
2.64 (t, 4H), 2.88 (t, 2H), 3.81 (d, 2H), 4.58 (d, 2H).
25: foam solid; 1H NMR (CDCl3, free base) δ 0.98-1.78 (m,
24H + 4H exchangeable with D2O), 2.31 (t, 4H), 2.52 (t, 2H),
2.68 (t, 4H), 2.98 (t, 2H), 3.82 (d, 2H), 4.58 (d, 2H).
26: foam solid; 1H NMR (CDCl3, free base) δ 0.73-1.42 (m,
26H), 1.91-2.08 (m, 4H), 2.18 (br s, 4H exchangeable with
D2O), 2.38 (t, 4H), 2.68 (t, 4H), 3.51 (d, 2H), 4.23 (d, 2H).
27: foam solid; 1H NMR (CDCl3, free base) δ 0.95-1.72 (m,
28H), 2.28 (t, 4H), 2.42-2.54 (m, 2H + 4H exchangeable with
D2O), 2.68 (t, 4H), 2.93 (t, 2H), 3.80 (d, 2H), 4.55 (d, 2H).
17: white powder; 82% yield from [4,4′]dipiperidine as
starting diamine and purified by gravity chromatography
eluting with CH2Cl2/EtOAc/EtOH (6.5:3:0.5); mp 95-98 °C; 1H
NMR (CDCl3) δ 0.91-1.75 (m, 22H), 2.12-2.42 (m, 6H), 2.82
(t, 2H), 3.08 (d, 4H), 3.75 (d, 2H), 4.48 (d, 2H), 5.01 (s, 4H),
5.45 (br s, 2H exchangeable with D2O), 7.05-7.23 (m, 10H).
18: white powder; 48% yield from 4-(piperidin-4-ylmethyl)-
piperidine as starting diamine and purified by flash chroma-
tography eluting with CHCl3/EtOAc/CH3OH (7.5:2:0.5); mp
1
28: mp 280 °C dec; H NMR (DMSO-d6, free base) δ 1.22-
1.75 (m, 12H), 2.25 (t, 4H), 2.35-2.48 (m, 4H), 3.10 (br s, 4H
exchangeable with D2O), 7.51-7.89 (m, 8H), 9.93 (s, 2H
exchangeable with D2O).
29: mp 130-132 °C; 1H NMR (DMSO-d6, free base) δ 1.18-
1.60 (m, 12H), 2.26 (t, 4H), 2.42-2,56 (m, 4H), 3.21 (br s, 4H
exchangeable with D2O), 3.80 (s, 2H), 7.01-7.56 (m, 8H), 9.78
(s, 2H exchangeable with D2O).
1
99-101 °C; H NMR (CD3OD) δ 0.98-1.91 (m, 24H), 2.38 (t,
4H), 2.61 (t, 2H), 2.98-3.19 (m, 6H), 3.85-4.02 (m, 2H), 4.42-
4.58 (m, 2H), 5.08 (s, 4H), 7.28-7.40 (m, 10H), 7.95 (s, 2H
exchangeable with D2O).
30: mp 174-175 °C; 1H NMR (DMSO-d6, free base) δ 1.20-
1.61 (m, 12H), 2.27 (t, 4H), 2.44-2.57 (m, 4H), 2.77 (s, 4H),
3.25 (br s, 4H exchangeable with D2O), 7.09 (d, 4H), 7.48 (d,
4H), 9.78 (s, 2H exchangeable with D2O).
19: white powder; 59% yield from 4-(2-piperidin-4-ylethyl)-
piperidine as starting diamine and purified by flash chroma-
tography eluting with CH2Cl2/EtOAc/CH3OH (6:3.7:0.3); mp
General Procedures for the Synthesis of 6-(2-Methoxy-
benzylamino)-1-{1′-[6-(2-methoxy-benzylamino)-hex-
anoyl]-[4,4′]bipiperidinyl-1-yl}-hexan-1-one (31), 6-(2-
Methoxy-benzylamino)-1-(4-{1-[6-(2-methoxy-ben-
zylamino)-hexanoyl]-piperidin-4-ylmethyl}-piperidin-1-
yl)-hexan-1-one (32), 6-(2-Methoxy-benzylamino)-1-[4-(2-
{1-[6-(2-methoxy-benzylamino)-hexanoyl]-piperidin-4-
yl}-ethyl)-piperidin-1-yl]-hexan-1-one (33), 6-(2-Methoxy-
benzylamino)-1-[4-(3-{1-[6-(2-methoxy-benzylamino)-
hexanoyl]-piperidin-4-yl}-propyl)-piperidin-1-yl]-hexan-
1-one (34), 6-(2-Methoxy-benzylamino)-hexanoic Acid {4′-
[6-(2-Methoxy-benzylamino)-hexanoylamino]-biphenyl-
4-yl}-amide (35), 6-(2-Methoxy-benzylamino)-hexanoic
Acid (4-{4-[6-(2-Methoxy-Benzylamino)-hexanoylamino]-
benzyl}-phenyl)-amide (36), and 6-(2-Methoxy-benzyl-
amino)-hexanoic Acid [4-(2-{4-[6-(2-Methoxy-benzylami-
no)-hexanoylamino]-phenyl}-ethyl)-phenyl]-amide (37).
A mixture of 24-30 and 2-methoxybenzaldehyde (in a 1:2.2
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),
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 resulting solution was washed with ether,
made basic with 2 N NaOH, and extracted with CHCl3.
Removal of the dried solvent gave the desired crude products
31-37, which, when necessary, were purified by flash chro-
matography.
1
118-120 °C; H NMR (CDCl3) δ 1.02-1.78 (m, 26H), 2.33 (t,
4H), 2.51 (t, 2H), 2.98 (t, 2H), 3.24 (q, 4H), 3.81 (d, 2H), 4.62
(d, 2H), 4.85 (br s, 2H exchangeable with D2O), 5.11 (s, 4H),
7.28-7.42 (m, 10H).
20: white powder; 23% yield from 4-(3-piperidin-4-ylpropyl)-
piperidine as starting diamine and purified by flash chroma-
tography eluting with CH2Cl2/EtOAc/CH3OH (8.5:0.5:1); mp
90-91 °C; 1H NMR (CDCl3) δ 0.95-1.72 (m, 28H), 2.29 (t, 4H),
2.48 (t, 2H), 2.94 (t, 2H), 3.12-3.21 (m, 4H), 3.79 (d, 2H), 4.58
(d, 2H), 4.82 (br s, 2H exchangeable with D2O), 5.10 (s, 4H),
7.25-7.40 (m, 10H).
21: white powder; nearly quantitative yield from biphenyl-
4,4′-diamine as starting diamine; mp 279-281 °C dec; 1H NMR
(DMSO-d6) δ 1.15-1.61 (m, 12H), 2.28 (t, 4H), 2.96 (t, 4H),
4.96 (s, 4H), 7.20-7.35 (m, 10H + 2H exchangeable with D2O),
7.52-7.64 (m, 8H), 10.01 (s, 2H exchangeable with D2O).
22: white powder; 83% yield from 4-(4-aminobenzyl)aniline
1
as starting diamine; mp 178-179 °C; H NMR (DMSO-d6) δ
1.17-1.60 (m, 12H), 2.18 (t, 4H), 2.94 (q, 4H), 3.76 (s, 2H),
4.95 (s, 4H), 7.05 (d, 4H), 7.17-7.37 (m, 10H + 2H exchange-
able with D2O), 7.43 (d, 4H), 9.94 (s, 2H exchangeable with
D2O).
23: white powder; nearly quantitative yield using 4-(4-
aminophenethyl)aniline as starting diamine; mp 184-185 °C;
1H NMR (DMSO-d6) δ 1.20-1.61 (m, 12H), 2.26 (t, 4H), 2.77
(s, 4H), 2.98 (q, 4H), 5.00 (s, 4H), 6.62 (d, 1H exchangeable
with D2O), 6.85 (d, 1H exchangeable with D2O), 7.09 (d, 4H),
7.30-7.40 (m, 10H), 7.47 (d, 4H), 9.78 (s, 2H exchangeable with
D2O).
General Procedures for the Synthesis of 6-Amino-1-
[1′-(6-amino-hexanoyl)-[4,4′]bipiperidinyl-1-yl]-hexan-1-
one (24), 6-Amino-1-{4-[1-(6-amino-hexanoyl)-piperidin-
4-ylmethyl]-piperidin-1-yl}-hexan-1-one (25), 6-Amino-1-
(4-{2-[1-(6-amino-hexanoyl)-piperidin-4-yl]-ethyl}-
piperidin-1-yl)-hexan-1-one (26), 6-Amino-1-(4-{3-[1-(6-
31: pale yellow oil; 78% yield; 1H NMR (CDCl3) δ 0.98-1.80
(m, 22H), 2.18 (br s, 2H exchangeable with D2O), 2.28-2.38
(m, 4H), 2.45 (t, 2H), 2.64 (t, 4H), 2.88 (t, 2H), 3.78 (s, 4H),
3.82-3.91 (m, 8H), 4.64 (d, 2H), 6.82-6.95 (m, 4H), 7.21-7.31
(m, 4H).
32: pale yellow oil; 85% yield; 1H NMR (CDCl3) δ 0.98-1.83
(m, 24H), 2.22-2.38 (m, 4H + 2H exchangeable with D2O),

6496 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Tumiatti et al.
2.43-2.71 (m, 6H), 2.96 (t, 2H), 3.75-3.89 (m, 12H), 4.62 (d,
2H), 6.81-7.31 (m, 8H); EI MS m/z 648 (M+).
(d, 2H), 6.81-7.00 (m, 4H), 7.22 (t, 2H), 7.42 (d, 2H); EI MS
m/z 718 (M+). Anal. (C48H74N4O12) C, H, N.
33: pale yellow oil; 85% yield; eluting solvent CH2Cl2/EtOAc/
CH3OH/aqueous 30% ammonia (6:3:1:0.2); 1H NMR (CDCl3) δ
0.95-1.72 (m, 26H), 1.98 (br s, 2H exchangeable with D2O),
2.27 (t, 4H), 2.48 (t, 2H), 2.56 (t, 4H), 2.94 (t, 2H), 3.74-3.82
(m, 12H), 4.56 (d, 2H), 6.80-6.90 (m, 4H), 7.16-7.24 (m, 4H);
EI MS m/z 662 (M+).
34: pale yellow oil; 88% yield; eluting solvent CH2Cl2/EtOAc/
CH3OH/aqueous 30% ammonia (5:4:1:0.1); 1H NMR (CDCl3) δ
0.80-1.65 (m, 28H + 2H exchangeable with D2O), 2.20 (t, 4H),
2.25-2.53 (m, 6H), 2.81 (t, 2H), 3.68-3.71 (m, 12H), 4.49 (d,
2H), 6.71-6.82 (m, 4H), 7.09-7.17 (m, 4H).
7: 10% yield; eluting solvent CH2Cl2/CH3OH (7:3); 1H NMR
(free base, CDCl3) δ 1.07 (t, 6H), 1.12-1.65 (m, 28H), 2.28 (t,
4H), 2.43-2.58 (m, 10H), 2.92 (t, 2H), 3.60 (s, 4H), 3.77-3.83
(m, 8H), 4.59 (d, 2H), 6.80-6.95 (m, 4H), 7.21 (t, 2H), 7.40 (d,
2H); EI MS m/z 732 (M+). Anal. (C49H76N4O12) C, H, N.
8: 44% yield; eluting solvent CH2Cl2/EtOAc/CH3OH/aqueous
1
30% ammonia (5:4:1:0.05); H NMR (free base, CDCl3) δ 1.07
(t, 6H), 1.25-1.80 (m, 12H), 2.31-2.63 (m, 12H), 3.62 (s, 4H),
3.82 (s, 6H), 6.83-6.98 (m, 4H), 7.18-7.59 (m, 12H), 7.88 (s,
2H exchangeable with D2O); EI MS m/z 707 (M + 1). Anal.
(C48H62N4O12) C, H, N.
35: colorless oil; 56% yield; purified by gravity column
eluting with CH3OH/toluene/aqueous 30% ammonia (7:3:0.1);
1H NMR (CDCl3) δ 1.35-1.81 (m, 12H + 2H exchangeable with
D2O), 2.39 (t, 4H), 2.64 (t, 4H), 3.81 (s, 4H), 3.84 (s, 6H), 6.84-
6.98 (m, 4H), 7.18-7.38 (m, 4H), 7.48-7.68 (m, 8H + 2H
exchangeable with D2O).
36: colorless oil; 43% yield; purified by gravity column
eluting with CH3OH/toluene/aqueous 30% ammonia (6:4:0.1);
1H NMR (CDCl3) δ 1.31-1.78 (m, 12H), 2.01 (br s, 2H
exchangeable with D2O), 2.29 (t, 4H), 2.61 (t, 4H), 3.79 (s, 4H),
3.82 (s, 6H), 3.87 (s, 2H), 6.82-6.93 (m, 4H), 7.03 (d, 4H), 7.18-
7.25 (m, 4H), 7.38 (d, 4H), 8.10 (s, 2H exchangeable with D2O).
37: colorless oil; 27% yield; eluting solvent CHCl3/CH3OH/
petroleum ether/toluene/aqueous 30% ammonia (5:2:2:1:0.15);
1H NMR (CDCl3) δ 1.18-1.76 (m, 12H), 1.95 (br s, 2H
exchangeable with D2O), 2.26 (t, 4H), 2.57 (t, 4H), 2.81 (s, 4H),
3.73 (s, 4H), 3.82 (s, 6H), 6.82-6.98 (m, 4H), 7.08 (d, 4H), 7.14-
7.25 (m, 4H), 7.34 (d, 4H), 7.94 (br s, 2H exchangeable with
D2O).
9: 16% yield; eluting solvent CH2Cl2/EtAc/MeOH/aqueous
1
30% ammonia (5:4:1:0.06); H NMR (free base, CDCl3) δ 1.04
(t, 6H), 1.20-1.75 (m, 12H), 2.29 (t, 4H), 2.44-2.58 (m, 8H),
3.59 (s, 4H), 3.79 (s, 6H), 3.87 (s, 2H), 6.81-6.95 (m, 4H), 7.08
(d, 4H), 7.18-7.22 (m, 4H), 7.36-7.41 (m, 4H + 2H exchange-
able with D2O); EI MS m/z 721 (M + 1). Anal. (C49H64N4O12)
C, H, N.
10: 49% yield; eluting solvent CH2Cl2/EtAc/MeOH/aqueous
1
30% ammonia (5:4:1:0.15); H NMR (free base, CDCl3) δ 1.03
(t, 6H), 1.25-1.79 (m, 12H), 2.30 (t, 4H), 2.44-2.56 (m, 8H),
2.83 (s, 4H), 3.58 (s, 4H), 3.80 (s, 6H), 6.84-6.99 (m, 4H), 7.08
(d, 4H), 7.10-7.31 (m, 4H), 7.38-7.46 (m, 4H + 2H exchange-
able with D2O); EI MS m/z 735 (M + 1). Anal. (C50H66N4O12)
C, H, N.
General Procedures for the Synthesis of N4,N4′-Bis-
{6-[ethyl-(2-methoxy-benzyl)-amino]-hexyl}-biphenyl-
4,4′-diamine (14), N-Ethyl-N′-[4-(4-{6-[ethyl-(2-methoxy-
benzyl)-amino]-hexylamino}-benzyl)-phenyl]-N-(2-
methoxy-benzyl)-hexane-1,6-diamine (15), and N-Ethyl-
N′-{4-[2-(4-{6-[ethyl-(2-methoxy-benzyl)-amino]-
hexylamino}-phenyl)-ethyl]-phenyl}-N-(2-methoxy-
benzyl)-hexane-1,6-diamine (16). A solution of 2 M BACH-
EI in tetrahydrofuran (0.9 mL) was added dropwise at room
temperature to a solution of 8-10 (0.4 mmol) in dry diglyme
(10 mL) under a stream of dry nitrogen. When the addition
was completed, the reaction mixture was heated at 200 °C for
2 h. After cooling at room temperature, excess borane was
destroyed by cautious dropwise addition of 2 N HCl (3 mL)
and water (2 mL). The resulting mixture was then heated at
200 °C for 1 h. After cooling at 0 °C, the mixture was made
basic with KOH powder and extracted with CHCl3 (3 × 50
mL). Removal of the dried solvent gave a residue, which was
purified by gravity column chromatography. All the purified
compounds were converted in dioxalate salts (foam solid).
14: colorless oil; 47% yield; eluting solvent CH2Cl2/EtOAc/
petroleum ether/CH3OH/aqueous 30% ammonia (4.5:2:3:0.5:
General Procedure for the Synthesis of 6-[Ethyl-(2-
methoxy-benzyl)-amino]-1-(1′-{6-[ethyl-(2-methoxy-ben-
zyl)-amino]-hexanoyl}-[4,4′]bipiperidinyl-1-yl)-hexan-1-
one (4), 6-[Ethyl-(2-methoxy-benzyl)-amino]-1-[4-(1-{6-
[ethyl-(2-methoxy-benzyl)-amino]-hexanoyl}-piperidin-
4-ylmethyl)-piperidin-1-yl]-hexan-1-one (5), 6-[Ethyl-(2-
methoxy-benzyl)-amino]-1-{4-[2-(1-{6-[ethyl-(2-methoxy-
benzyl)-amino]-hexanoyl}-piperidin-4-yl)-ethyl]-piperidin-
1-yl}-hexan-1-one (6), 6-[Ethyl-(2-methoxy-benzyl)-amino]-
1-{4-[3-(1-{6-[ethyl-(2-methoxy-benzyl)-amino]-hexanoyl}-
piperidin-4-yl)-propyl]-piperidin-1-yl}-hexan-1-one (7),
6-[Ethyl-(2-methoxy-benzyl)-amino]-hexanoic Acid (4′-
{6-[Ethyl-(2-methoxy-benzyl)-amino]-hexanoylamino}-
biphenyl-4-yl)-amide (8), 6-[Ethyl-(2-methoxy-benzyl)-
amino]-hexanoic Acid [4-(4-{6-[Ethyl-(2-methoxy-benzyl)-
amino]-hexanoylamino}-benzyl)-phenyl]-amide (9), and
6-[Ethyl-(2-methoxy-benzyl)-amino]-hexanoic Acid {4-[2-
(4-{6-[Ethyl-(2-methoxy-benzyl)-amino]-hexanoylamino}-
phenyl)-ethyl]-phenyl}-amide (10). A mixture of 31-37 and
diethyl sulfate (1:2.5 ratio) was refluxed for 48 h in toluene.
Following removal of the solvent, the residue was taken up in
water, made basic with KOH pellets, and immediately ex-
tracted with CHCl3 (3 × 20 mL) or directly purified by column
chromatography to avoid the quaternization of the amine
functions. Removal of the dried solvent gave a residue, which
was purified by flash chromatography. All the purified com-
pounds were converted in dioxalate salt (foam solid).
4: 35% yield; eluting solvent CH2Cl2/EtOAc/CH3OH/aqueous
1
0.09); H NMR (CDCl3) δ 1.08 (t, 6H), 1.21-1.78 (m, 16H +
2H exchangeable with D2O), 2.38-2.41 (m, 8H), 3.11 (t, 4H),
3.62 (s, 4H), 3.82 (s, 6H), 6.61 (d, 4H), 6.72-7.01 (m, 4H), 7.08-
7.25 (m, 8H); EI MS m/z 679 (M + 1). Anal. (C48H66N4O10) C,
H, N.
15: colorless oil; 38% yield; eluting solvent CHCl3/EtOAc/
petroleum ether/CH3OH/aqueous 30% ammonia (4:3:2.5:0.5:
1
0.05); H NMR (CDCl3) δ 1.12 (t, 6H), 1.22-1.71 (m, 16H +
2H exchangeable with D2O), 2.41-2.62 (m, 8H), 3.11 (t, 4H),
3.61 (s, 4H), 3.78 (s, 2H), 3.83 (s, 6H), 6.58 (d, 4H), 6.82-7.12
(m, 8H), 7.17-7.42 (m, 4H); EI MS m/z 693 (M + 1). Anal.
(C49H68N4O10) C, H, N.
1
30% ammonia (6:3:1:0.05); H NMR (free base, CDCl3) δ 1.15
(t, 6H), 1.12-1.82 (m, 22H), 2.31 (t, 4H), 2.42-2.71 (m, 10H),
2.93 (t, 2H), 3.78 (s, 4H), 3.81-3.96 (m, 8H), 4.65 (d, 2H), 6.83-
7.02 (m, 4H), 7.22-7.35 (m, 2H), 7.45 (d, 2H); EI MS m/z 691
(M+). Anal. (C46H70N4O12) C, H, N.
16: pale yellow oil; 58% yield; eluting solvent CHCl3/EtOAc/
petroleum ether/CH3OH/aqueous 30% ammonia (4:3:2.5:0.5:
0.05); 1H NMR (DMSO-d6) δ 1.18-1.81 (m, 22H), 2.22 (q, 2H),
2.65 (s, 2H), 2.86-3.18 (m, 8H), 3.85 (s, 4H), 4.15-4.35 (m,
10H), 6.48 (d, 4H), 6.88-7.21 (m, 8H), 7.41-7.58 (m, 4H); EI
MS m/z 707 (M + 1). Anal. (C50H70N4O10) C, H, N.
General Procedures for the Synthesis of Methiodides
11-13. A solution of the appropriate compound (6, 7, or 10)
as free base in acetone (15 mL) was treated with methyl iodide
(1:10 molar ratio). After standing overnight at room temper-
ature, the solvent was removed and the residue was triturated
with ether to give a foam pale sticky solid in quantitative
yield.
5: 17% yield; eluting solvent CH2Cl2/EtOAc/CH3OH/aqueous
1
30% ammonia (5:4:1:0.10); H NMR (free base, CDCl3) δ 1.08
(t, 6H), 1.18-1.73 (m, 24H), 2.25 (t, 4H), 2.32-2.73 (m, 10H),
2.93 (t, 2H), 3.73 (s, 4H), 3.78-3.84 (m, 8H), 4.58 (d, 2H), 6.78-
6.94 (m, 4H), 7.22 (t, 2H), 7.43 (d, 2H); EI MS m/z 705 (M +
1). Anal. (C47H72N4O12) C, H, N.
6: 21% yield; eluting solvent CH2Cl2/CH3OH (7:3); 1H NMR
(free base CDCl3) δ 1.06 (t, 6H), 1.12-1.78 (m, 26H), 2.31 (t,
4H), 2.45-2.59 (m, 10H), 2.97 (t, 2H), 3.58-3.89 (m, 12H), 4.60

SARs of AChE Noncovalent Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6497
11: 1H NMR (DMSO-d6) δ 1.05-1.78 (m, 32H), 2.24 (t, 4H),
2.42 (t, 2H), 2.79 (s, 6H), 2.92 (t, 2H), 3.05-3.35 (m, 8H), 3.73-
3.82 (m, 8H), 4.29 (d, 2H), 4.41 (s, 4H), 7.03 (t, 2H), 7.14 (d,
2H), 7.39-7.54 (m, 4H). Anal. (C46H76I2N4O4) C, H, N.
Reciprocal plots involving 4 or donepezil inhibition show
both increasing slopes (decreased Vmax at increasing inhibitor
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 4 or donepezil gives
estimate of competitive inhibition constant, Ki ) 5.67 ( 0.15
nM or Ki ) 20.5 ( 3.3 nM, respectively.
12: 1H NMR (CDCl3) δ 0.98-1.95 (m, 34H), 2.35 (t, 4H), 2.49
(t, 2H), 2.95 (t, 2H), 3.08 (s, 6H), 3.35-3.75 (m, 8H), 3.73-
3.91 (m, 8H), 4.49 (d, 2H), 4.71 (s, 4H), 6.98 (q, 4H), 7.38-
7.52 (m, 2H), 7.68 (d, 2H). Anal. (C47H78I2N4O4) C, H, N.
1
13: H NMR (DMSO-d6) δ 1.21-1.38 (m, 10H), 1.61-1.83
Thus, the pattern in the graphical representation shows 4
and donepezil able to bind to the peripheral anionic site as
well as the active site of AChE.
(m, 8H), 2.31 (t, 4H), 2.74 (s, 4H), 2.77 (s, 6H), 3.02-3.38 (m,
8H), 3.80 (s, 6H), 4.38 (s, 4H), 6.88-7.18 (m, 8H), 7.36-7.44
(m,8H),9.78(s,2HexchangeablewithD2O).Anal.(C48H68I2N4O4)
C, H, N.
Inhibition of AChE-Induced Aâ Aggregation. Aliquots
of 2 µL of Aâ peptide, lyophilized from 2 mg mL-1 1,1,1,3,3,3-
hexafluoro-2-propanol solution and dissolved in DMSO, were
incubated for 24 h at room temperature in 0.215 M sodium
phosphate buffer (pH 8.0) at a final concentration of 230 µM.
For coincubation experiments aliquots (16 µL) of AChE (final
concentration 2.30 µM, Aâ/AChE molar ratio 100:1) and AChE
in the presence of 2 µL of the tested inhibitor in 0.215 M
sodium phosphate buffer pH 8.0 solution (final inhibitor
concentration 100 µM) were added.
Blanks containing Aâ, AChE, and Aâ plus inhibitors in 0.215
M sodium phosphate buffer (pH 8.0) were prepared. The final
volume of each vial was 20 µL. Each assay was run in
duplicate. To quantify amyloid fibril formation, the thioflavin
T fluorescence method was then applied.28 The fluorescence
intensities due to â-sheet conformation were monitored for 300
s at λem ) 490 nm (λex ) 446 nm). The percent inhibition of
the AChE-induced aggregation due to the presence of the test
compound was calculated by the following expression: 100 -
(IFi/IFo × 100) where IFi and IFo are the fluorescence intensi-
ties obtained for Aâ plus AChE in the presence and in the
absence of inhibitor, respectively, minus the fluorescence
intensities due to the respective blanks.
Biology. Functional Antagonism at Guinea Pig Left
Atria. Male guinea pigs (200-300 g) were killed by cervical
dislocation. The heart was rapidly removed, and right and left
atria were separated out and set up rapidly under 1 g of
tension in 20 mL organ baths containing physiological salt
solution (PSS) maintained at 30 °C and aerated with 5% CO2-
95% O2.
The left atria were mounted 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 concentration of agonist in the organ
bath was increased approximately 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 experi-
ments in which tissues did not receive any antagonist were
run in order to check any variation in sensitivity.
Statistical Analysis. Data were analyzed using a phar-
macological computer program.30 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.
Acknowledgment. This research was supported by
Grants from the University of Bologna (Funds for
Selected Research Topics) and MIUR.
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].24
Supporting Information Available: Elemental analysis
data of reported compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
Inhibition of AChE and BChE. The method of Ellman
et al. was followed.22 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 µM 5,5′-dithio-bis(2-nitrobenzoic acid),
0.035 unit/mL of human recombinant AChE or BChE derived
from human serum (Sigma Chemical), and 550 µM of substrate
(acetylthiocholine iodide or butyrylthiocholine 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 in order 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 concen-
tration-inhibition curves.
Determination of Steady-State Inhibition Constant.
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 4 (range 0-1.93 nM) or donepezil
concentration (range 0-65.1 nM) in a similar weighted
analysis, and Ki was determined as the ratio of the replot
intercept to the replot slope.
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