Structure-activity relationships of acetylcholinesterase noncovalent inhibitors based on a polyamine backbone. 4. Further investigation on the inner spacer

Article abstract of DOI:10.1021/jm8009684

Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties that might be relevant to Alzheimer's disease. 15 was the most interesting, inhibiting AChE in the nanomolar range and inhibiting AChE-induced and self-promoted β-amyloid aggregation in the micromolar range.

Full text of DOI:10.1021/jm8009684

7308
J. Med. Chem. 2008, 51, 7308–7312
Structure-Activity Relationships of Acetylcholinesterase Noncovalent Inhibitors Based on a
Polyamine Backbone. 4. Further Investigation on the Inner Spacer
Vincenzo Tumiatti,*,†,§ Andrea Milelli,† Anna Minarini,† Michela Rosini,† Maria Laura Bolognesi,† Marialuisa Micco,†
Vincenza Andrisano,† Manuela Bartolini,† Francesca Mancini,† Maurizio Recanatini,† Andrea Cavalli,† and Carlo Melchiorre†
Department of Pharmaceutical Sciences, Alma Mater Studiorum, UniVersity of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and Polo
Scientifico-Didattico di Rimini, UniVersity of Bologna, 40100 Bologna, Italy
ReceiVed July 31, 2008
Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with
less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties
that might be relevant to Alzheimer’s disease. 15 was the most interesting, inhibiting AChE in the nanomolar
range and inhibiting AChE-induced and self-promoted ꢀ-amyloid aggregation in the micromolar range.
Introduction
Alzheimer’s disease (ADa) is a multifactorial disease and age-
related neurodegenerative disorder clinically characterized by
loss of memory and progressive deficits in different cognitive
domains. Most currently prescribed AD drugs aim to increase
the level of acetylcholine (ACh) in the brain by inhibiting AChE.
But clinical experience shows that cholinesterases inhibition is
a palliative treatment, which does not address AD’s etiology.1
Research into newer and more potent AChE inhibitors (AChEIs)
is thus focused on compounds whose properties go beyond
AChE inhibition.2-4
The “one-molecule-multiple-targets” paradigm suggests that
a single molecule could hit several targets responsible for the
onset and/or progression of AD.5,6 To this end, the design and
synthesis of multi-target-directed ligands (MTDLs) useful for
combating neurodegenerative diseases6 have been published.7
This seems more appropriate for addressing the complexity of
AD and may provide new drugs for tackling the multifactorial
nature of AD, stopping its progression.
In 1998, we reported on derivatives displaying affinity for
(i) AChE active and peripheral binding sites and (ii) muscarinic
M2 receptors.8 The prototypes caproctamine (1) and its ethyl
analogue (2)9 antagonized muscarinic M2 receptors and inhibited
active and peripheral sites of AChE. Because AChE may act
as a chaperone in inducing ꢀ-amyloid (Aꢀ) aggregation through
the interaction of its peripheral anionic site (PAS) with the
peptide, the inhibition of PAS might be relevant to the search
for AChEIs endowed with Aꢀ antiaggregating properties.10
Although 1 and 2 contacted PAS and active AChE binding sites,
they did not inhibit the AChE-induced Aꢀ aggregation. To verify
whether this failure was due to their high structural flexibility,
their inner octamethylene spacer was partially or totally
incorporated into a more constrained moiety.11 This led to the
discovery of the dipiperidino derivative 3, which displayed
improved AChE inhibitory potency and the ability to partially
Figure 1. Design strategy for 4-16.
inhibit AChE-induced Aꢀ aggregation, suggesting that inhibition
of Aꢀ aggregation is achieved by polyamines incorporating a
constrained spacer between the two inner nitrogen atoms rather
than a flexible polymethylene chain.11 It is known that aromatic
residues may give additional interactions with the aromatic rings
lined in AChE gorge12 and may confer ꢀ-sheet-breaking
properties that might lead to the inhibition of self-mediated Aꢀ
aggregation.13
Thus, to obtain new AChE-inhibiting MTDLs endowed with
additional properties such as inhibition of AChE-induced Aꢀ
aggregation and ꢀ-sheet-breaking ability,14 we replaced the
dipiperidino moiety of 3 with less flexible cyclic systems,
leading to 4-16 (Figure 1). Monoamine 16 was included in
this study to verify the importance, if any, of the two aminoalkyl
side chains in the interaction with AChE and Aꢀ.
* To whom correspondence should be addressed. Address: Department
of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126
Bologna, Italy. Phone: +39-051-2099709. Fax: +39-051-2099734. E-mail:
vincenzo.tumiatti@unibo.it.
Chemistry
Diamine diamides 17-21 were obtained by reacting N-[(ben-
zyloxy)carbonyl]-6-aminocaproic acid with piperazine, cis-
cyclohexane-1,2-diamine, (()-trans-cyclohexane-1,2-diamine,
trans-cyclohexane-1,4-diamine, and ethane-1,2-diamine, respec-
tively. Removal of the N-(benzyloxy)carbonyl group by acidic
hydrolysis gave diamine diamides 22-26, which were treated
†
Department of Pharmaceutical Sciences, University of Bologna.
Polo Scientifico-Didattico di Rimini, University of Bologna.
§
a Abbreviations: Aꢀ, ꢀ-amyloid; ACh, acetylcholine; AChE, acetylcho-
linesterase; AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease;
BChE, butyrylcholinesterase; MTDL, multi-target-directed ligand; NTD,
1,4,5,8-naphthalenetetracarboxylic diimide; PAS, peripheral anionic site.
10.1021/jm8009684 CCC: $40.75
2008 American Chemical Society
Published on Web 10/28/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7309
Scheme 3a
Scheme 1a
a Conditions: Cbz ) C6H5CH2OCO-; (i) Et3N, EtOCOCl, dioxane, room
temp, 72 h, 49-93%; (ii) 30% HBr in CH3COOH, CH3COOH, room temp,
4 h, quantitative yield; (iii) (a) 2-MeOC6H4CHO, toluene, reflux, 6 h; (b)
NaBH4, EtOH, room temp, 6 h, 65-84%; (iv) (EtO)2SO2, toluene, reflux,
48 h, 26-54%; (v) (a) borane-N-ethyl-N-isopropylaniline complex, diglyme,
reflux, 4 h; (b) 6 N HCl, reflux, 1 h, 23%; (vi) diethyloxalate, EtOH, reflux,
12 h, 42%.
a Conditions: (i) amine and anhydride molar ratio, 2:1 for 13-15 and
1:1 for 16, EtOH, reflux, 60 h, 35-51%.
Scheme 2a
toluenesulfonates for 11-14) were prepared to obtain derivatives
easier to handle.
Results and Discussion
To determine the potential interest of 4-16 for AD treatment,
their inhibitory potency was evaluated on recombinant human
AChE and isolated BChE from human serum in comparison
with polyamines 1-3 and the marketed drugs donepezil and
galantamine.
An analysis of the results (Table 1) reveals that replacement
of the dipiperidino moiety of 3 with constrained moieties
strongly influenced the ability to inhibit AChE and BChE. The
new derivatives inhibited AChE activity in the nanomolar range
with the exception of 5-7. The low affinity of 5-7 for AChE
might be due to the difficulty of the two amide functions to
assume a planar arrangement to each other, as is probably
possible for 4, 9, 10, and 12, which were only slightly less potent
than 3. The most potent compounds of the present series (8,
11, 13-15) were characterized by an aromatic residue in the
middle of their structure. This suggests the possibility of
establishing more π-π interactions with several aromatic
residues located in the AChE gorge. In particular, 15, endowed
with a 1,4,5,8-naphthalenetetracarboxylic diimide (NTD) moiety,
showed a very high AChE inhibitory activity and a 9-fold
improvement in potency in comparison with 3. All the synthe-
sized compounds showed a selective inhibitory activity for
AChE relative to BChE, and 15 was the most selective and
potent of the series with an AChE/BChE selectivity ratio greater
than 5000, perhaps relevant when considering the emerging role
of BChE in AD17 and the importance of selectivity toward
AChE in AD treatment.18 Finally, 16, characterized by only one
side chain, was 144-fold less potent than 15, highlighting the
importance of the presence of two side chains for an optimal
interaction with both sites of AChE.
a Conditions: (i) Et3N, CH2Cl2, room temp, 96 h, 9%; (ii) 60% NaH,
anhydrous DMF, piperazine-2,5-dione or 1,4-dihydroquinoxaline-2,3-dione
or imidazolidin-2-one, room temp for 35 and 36 and reflux for 37, 24 h,
18-46%; (iii) for 10 and 11, KI, K2CO3, 1-pentanol, reflux, 40 h, 11-57%;
for 8 and 12, CH3CN and Et3N, reflux, 64 h, 27-30%.
with 2-methoxybenzaldehyde followed by reduction with NaBH4
of the formed Schiff base to the corresponding dibenzyl
derivatives 27-31. Diethylation of 27-31 with diethyl sulfate
gave 4-7 (Figure 1) and 32. Reduction of 32 afforded 33, which
was condensed with diethyloxalate to give 9 (Scheme 1).
Acylation of 1,2,3,6,7,8-hexahydrobenzo[lmn][3,8]phenanthro-
line15 with 6-bromohexanoyl chloride gave 34, whereas N-
alkylation of piperazine-2,5-dione, 1,4-dihydroquinoxaline-2,3-
dione, and imidazolidin-2-one with the appropriate dibromod-
erivative afforded intemediates 35-37, which were diaminated
with ethyl-(2-methoxybenzyl)amine to give 8, 10-12 (Figure
1), respectively (Scheme 2).
Finally, 13-16 (Figure 1) were obtained through the con-
densation of the commercially available anhydrides [5,5′]bi-
isobenzofuranyl-1,3,1′,3′-tetraone, benzo[1,2-c;4,5-c′]difuran-
1,3,5,7-tetraone, isochromeno[6,5,4-def]isochromene-1,3,6,8-
tetraone, and benzo[de]isochromene-1,3-dione, respectively,
with N-ethyl-N-(2-methoxybenzyl)hexane-1,6-diamine16 (Scheme
3). Different salts (dioxalates for 4-10, 12, and 15 and di-p-
PAS is well-established as important for Aꢀ-fibrils formation
mediated by AChE;10,19 thus, compounds able to interact with
amino acids located in the PAS area may reduce the formation
of neurotoxic Aꢀ fibrils. PAS may thus be an attractive target
when developing potential AD-modifying drugs. A Line-
weaver-Burk plot obtained at increasing concentrations of
substrate and inhibitor showed that 15 interacted with the
catalytic site and PAS (see Supporting Information). Therefore,
the ability of 4 and 7-16 to inhibit AChE-induced Aꢀ(1-40)

7310 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Brief Articles
codes: 1B41 for AChE,25 1P0M for BChE26). Because of 15’s
flexibility, several docking runs were carried out with GOLD.27
The outcomes were clusterized by AClAP.28 In Figure 2SI of
Supporting Information, a low energy pose representative of a
statistically populated cluster is reported for binary complexes
of AChE and 15 (Figure 2A of Supporting Information) and of
BChE and 15 (Figure 2B of Supporting Information). The
binding mode of 15 at the AChE gorge shows that the ligand
can interact with Trp86 of the internal anionic site and with
Trp286 of PAS (Figure 2A of Supporting Information). The
latter could explain 15’s ability to inhibit AChE-induced Aꢀ
aggregation. 15 might also interact with several aromatic
residues of the enzyme mid-gorge.29 In particular, the NTD
moiety could establish favorable π-π stacking or simple
hydrophobic interactions with Tyr341, Phe338, and Phe295.
Moreover, Tyr72 may interact by H-bonding with the methoxy
substituent of one of the benzylammonium ends. This could
account for the high inhibitory potency of 15 against human
AChE. Conversely, although 15 could favorably interact with
several BChE amino acids (Figure 2B of Supporting Informa-
tion), the lack of the same pool of aromatic residues at the BChE
mid-gorge may explain 15’s AChE/BChE selectivity. Indeed,
besides a cation-π interaction between 15 and the Trp82 indole
ring of BChE, the only other striking interaction was between
15 and Tyr332. Moreover, 15 seemed unable to interact with
Phe278, a fundamental residue for inhibiting BChE activity.29
The presence of BChE Asp70 close to one of the two imide
moieties of the NTD scaffold may further decrease the
molecule’s affinity toward this target.
Table 1. Inhibition of AChE and BChE Activities and of
AChE-Mediated and Self-Induced Aꢀ Aggregation by 4-16 and
Reference Polyamines 1-3
inhibition of Aꢀ
aggregation (%)
IC50 (nM)b
AChE-
inducedc
self-
compda
AChE
BChE
inducedd
1e
2
3
4
5
6
7
8
9
10
11
12
13
170 ( 2e
11600 ( 300e
2250 ( 60e
8490 ( 610g
10700 ( 1900
30800 ( 2300
36800 ( 3300
25300 ( 4200
1040 ( 30
10300 ( 400
2090 ( 130
95.8 ( 1.5
876 ( 24
<5f
20.3 ( 1.9
16.1 ( 0.5e
3.32 ( 0.12g
25.7 ( 1.9
4160 ( 200
5470 ( 240
8550 ( 40
4.83 ( 0.16
68.9 ( 2.4
15.9 ( 0.6
1.41 ( 0.03
72.4 ( 3.2
14.0 ( 0.6
7.70 ( 0.27
0.37 ( 0.02
53.5 ( 5.7
23.1 ( 4.8
2010 ( 150l
32300l
<5
ndh
41.2 ( 2.0
38.9 ( 4.0
ndh
13.7 ( 6
<5f
ndh
ndh
ndh
25.6 ( 4.6
42.1 ( 2.2
47.5 ( 1.6
61.4 ( 4.4
70.8 ( 3.2
53.8 ( 3.7
70.2 ( 4.83
51.1 ( 0.3
>90i
ndh
12.7 ( 2.9
ndh
4.8 ( 1.3
8.8 ( 2.9
<5
293 ( 23
16.5 ( 1.3
<5
14
15
16
3000 ( 200
1910 ( 120
416 ( 32
54.5 ( 5.4j
<5
19.5 ( 2.8
donepezil
galantamine
propidium
Congo red
7420 ( 390
20700 ( 1500l
13200l
22k
<5
17.9 ( 0.1l
82.0 ( 2.5k
ndh
<5
61.1 ( 4.6l
ndh
ndh
78.8 ( 0.9l
a 1, dihydrochloride; 2-10, 12, and 15, dioxalate; 11, 13, 14, di-p-
toluenesulfonate; 16, p-toluenesulfonate. See Figure 1 for structures.
b Human recombinant AChE and BChE from human serum were used. IC50
values represent the concentration of inhibitor required to decrease enzyme
activity by 50% and are the mean of two independent measurements, each
performed in triplicate. c Inhibition of AChE-induced Aꢀ(1-40) aggregation.
The concentrations of the tested inhibitor and Aꢀ(1-40) were 100 and 230
µM, respectively, whereas the Aꢀ(1-40)/AChE ratio was equal to 100:1.
d Inhibition of self-induced Aꢀ(1-42) aggregation (50 µM) produced by
the tested compound at 10 µM concentration. e Data from ref 8. f Not
significant. g Data from ref 30. h nd, not determined. i IC50 ) 8.13 ( 0.97
µM. j IC50 ) 9.69 ( 1.09 µM. k Data from ref 10. l Data from ref 24.
Conclusions
We have demonstrated that constraining the dipiperidino
moiety of 3 led to derivatives with a better biological profile.
The most potent was 15, endowed with an NTD moiety. It
inhibited AChE in the subnanomolar range and inhibited AChE-
induced and self-promoted Aꢀ aggregation in micromolar
concentration. Thus, 15 emerges as an MTDL able to hit several
targets of the AD pathogenesis cascade.
However, the rational design of compounds that simulta-
neously modulate different protein targets at a comparable
concentration for each target remains a challenging task.6 In
the present case, proof of the concept of the biological profile
of 15 in vivo is needed to confirm the relevance of the NTD
moiety in the design of new derivatives for AD treatment.
aggregation was assessed through a thioflavin T-based fluoro-
metric assay.10 5 and 6 were not evaluated because of their poor
AChE inhibitory activity. 4, 8, and 9 were as active as 3 (Table
1), while 10-15 were more potent than 3. It appears that an
inner spacer bearing aromatic residues may be optimal for
inhibiting AChE-induced Aꢀ aggregation. 15 was the most
potent inhibitor with an IC50 lower than that of the reference
compound propidium.10 Notably, neither 3 nor any of the
marketed AChE inhibitors, when tested in the same conditions,
showed comparable antiaggregating activity.10,20 The most
potent published compounds acting as AChE-induced Aꢀ
aggregation inhibitors display a potency in the same range as
15.21-23 The low inhibitory activity shown by 16 emphasizes
the importance of the two aminoalkyl side chains for an optimal
interaction with the two AChE binding sites.
Since it was previously reported that compounds endowed
with a polyamine scaffold may interfere with Aꢀ polymerization
and aggregation,24 the ability of 4, 8, 10-16 to inhibit self-
promoted Aꢀ(1-42) aggregation was also determined (Table
1).10 Again, 15, although less potent that the reference compound
Congo red, displayed an IC50 comparable with that of propidium.
This activity might be due to the ability of 15 to behave as a
ꢀ-sheet breaker because of its planar and constrained aromatic
system.19 Unfortunately, because of its low solubility, the
contribution,ifany,oftheNTDmoietybenzo[lmn][3,8]phenanthroline-
1,3,6,8-tetraone15 and, consequently, its possible contribution
to the different biological properties of 15 could not be
determined.
Experimental Section
General Procedure for the Synthesis of 4-7. A mixture of
27, 28, 29, or 30 and diethyl sulfate (1:2.5 ratio) was heated at the
refluxing temperature for 48 h in toluene. After removal of the
solvent, the residue was taken up in water, made basic with KOH
pellets, and immediately extracted with CHCl3 (3 × 20 mL) or
directly purified by column chromatography to avoid quaternariza-
tion of amine functions. Removal of the dried solvent gave a residue
that was purified by gravity chromatography, providing the desired
4-7, which were converted into the corresponding dioxalate salts
by adding 2 equiv of oxalic acid in ether to an ether solution of the
free base. See Supporting Information for further experimental
details, elemental analysis (EA) data, and spectral data (ESI-MS
1
and H and 13C NMR).
General Procedure for the Synthesis of 8 and 12. A mixture
of ethyl(2-methoxybenzyl)amine (0.125 g, 0.76 mmol) and triethy-
lamine (0.077 g, 0.76 mmol) was added to a solution of the dibromo
derivative 34 or 37 (0.38 mmol) in CH3CN (20 mL). The reaction
mixture was stirred at the refluxing temperature for 64 h. After
solvent evaporation, the crude material was poured in water, made
basic, and extracted with CH2Cl2 (3 × 50 mL). After evaporation
To disclose a possible binding mode of 15 at AChE and BChE
binding pockets, docking simulations were performed using the
available crystallographic structures of the two enzymes (PDB

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7311
of the solvent, the crude material was purified by flash chroma-
tography. The two purified compounds were converted into the
dioxalate salts as described for 4-7.
intermediate compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
8. It was synthesized from 34: yellow oil; 27% yield; eluting
solvent, toluene/EtOAc/MeOH/CH2Cl2/aqueous 33% ammonia (4:
4:1:1:0.05); 1H NMR (free base, 200 MHz, CDCl3) δ 1.01 (t, J )
5.4, 6H), 1.17-1.68 (m, 12H), 1.68-2.51 (m, 12H), 3.38 (s, 6H),
3.58 (s, 4H), 4.88 (s, 4H), 5.06 (s, 4H), 6.68-6.96 (m, 4H),
7.17-7.42 (m, 8H); 13C NMR (200 MHz, CDCl3) δ 11.9, 25.3,
27.0, 27.5, 44.7, 47.8, 48.8, 51.4, 53.5, 55.4, 110.3, 120.3, 127.6,
127.9, 128.2, 130.2, 157.7, 198.1; MS (ESI+) m/z ) 733 (M +
H)+. Anal. (C50H64N4O12), C, H, N.
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1
EA, and spectral data (ESI-MS and H and 13C NMR).
General Procedure for the Synthesis of 10 and 11. KI (1.6 g,
10 mmol), K2CO3 (4.7 g, 17 mmol), and ethyl(2-methoxybenzy-
l)amine (0.313 g, 1.9 mmol) were added to a solution of the
dibromoderivative 35 or 36 (0.38 mmol) in n-pentanol (20 mL).
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the desired compound. 10 was converted into the dioxalate salt as
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sulfonate salt by adding 2 equiv of p-toluenesulfonic acid in ether
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10. See Supporting Information for further experimental details,
1
EA, and spectral data (ESI-MS and H and 13C NMR).
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solvent, toluene/EtOAc/MeOH/ aqueous 33% ammonia (4:5:1:0.03);
1H NMR (free base, 200 MHz, CDCl3) δ 1.07 (t, J ) 6.0, 6H),
1.38-1.77 (m, 16H), 2.50 (t, J ) 15, 4H), 2.55 (q, J ) 7.5, 4H),
3.61 (s, 4H), 3.83 (s, 6H), 4.19 (t, J ) 7.8, 4H), 6.85-7.44 (m,
12H); 13C NMR (200 MHz, CDCl3) δ 11.7, 26.8, 26.9, 43.2, 47.6,
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1
details, EA, and spectral data (ESI-MS and H and 13C NMR).
15. It was synthesized from isochromeno[6,5,4-def]isochromene-
1,3,6,8-tetraone: colorless oil; 43% yield; eluting solvent, toluene/
1
EtOAc/CH2Cl2/MeOH/aqueous 33% ammonia (4:4:1:1:0.05); H
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NMR (free base, CDCl3) δ 1.05 (t, J ) 7.4, 6H), 1.43-1.76 (m,
16H), 2.43-2.59 (m, 8H), 3.58 (s, 4H), 3.82 (s, 6H), 4.19 (t, J )
7.6, 4H), 6.18-6.96 (m, 4H), 7.15-7.28 (m, 2H), 7.89-7.43 (m,
2H), 8.77 (s, 4H); 13C NMR (free base, CDCl3) δ 1.12, 11.96, 27.07
27.16, 27.37, 28.22, 41.02, 47.74, 51.44, 53.58, 55.41, 110.25,
120.35, 125.38, 126.69, 127.52, 128.29, 128.40, 129.10, 130.13,
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Acknowledgment. The authors thank Valeria Battelli,
Massimo Gambuti, Marianna Leardini, and Elisa Piersanti for
technical assistance. This research was supported by a grant
from MIUR, Rome (PRIN), and Polo Scientifico-Didattico di
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Supporting Information Available: Experimental details for
chemistry, biology, and modeling; elemental analysis data of target
compounds; and experimental details and characterization of

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