Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a new family of anti-Alzheimer agents targeting β-amyloid, tau, and cholinesterase pathologies

Article abstract of DOI:10.1016/j.ejmech.2014.07.021

Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c] quinoline carboxylic ester derivative as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O → NH bioisosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS) inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and octamethylene-linked homologues 5b-d have been also synthesized for comparison purposes, and found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological profiling of hybrids 5a-d has shown that they are also potent inhibitors of human butyrylcholinesterase and moderately potent Aβ42 and tau anti-aggregating agents, with IC ₅₀ values in the submicromolar and low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have predicted a good brain permeability for hybrids 5a-d, and hence, their ability to reach their targets in the central nervous system. The multitarget profile of the novel hybrids makes them promising leads for developing anti-Alzheimer drug candidates with more balanced biological activities.

Full text of DOI:10.1016/j.ejmech.2014.07.021

European Journal of Medicinal Chemistry 84 (2014) 107e117
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids as a
new family of anti-Alzheimer agents targeting
cholinesterase pathologies
b-amyloid, tau, and
a
a
b
c
ꢀ
ꢀ
ꢀ
ꢀ
Ornella Di Pietro , F. Javier Perez-Areales , Jordi Juarez-Jimenez , Alba Espargaro ,
d
d
e
b, *
ꢁ
ꢀ
ꢀ
ꢀ c
M. Victoria Clos , Belen Perez , Rodolfo Lavilla , Raimon Sabate , F. Javier Luque
,
a, **
~
Diego Munoz-Torrero
a
ꢁ
ꢁ
Laboratori de Química Farmaceutica (Unitat Associada al CSIC), Facultat de Farmacia, and Institut de Biomedicina (IBUB), Universitat de Barcelona,
Av. Joan XXIII, 27-31, E-08028 Barcelona, Spain
b
ꢁ
Departament de Fisicoquímica, Facultat de Farmacia (Campus Torribera), and IBUB, Universitat de Barcelona, Prat de la Riba 171,
E-08921 Santa Coloma de Gramenet, Spain
Departament de Fisicoquímica, Facultat de Farmacia, and Institut de Nanociencia i Nanotecnologia (IN UB), Universitat de Barcelona,
c
2
ꢁ
ꢁ
Av. Joan XXIII, 27-31, E-08028 Barcelona, Spain
d
ꢁ
ꢁ
ꢁ
Departament de Farmacologia, de Terapeutica i de Toxicologia, Institut de Neurociencies, Universitat Autonoma de Barcelona,
E-08193 Bellaterra, Barcelona, Spain
e
ꢁ
ꢁ
Laboratori de Química Organica, Facultat de Farmacia, Universitat de Barcelona, and Barcelona Science Park, Baldiri Reixac 10-12,
E-08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of an essentially inactive 3,4-dihydro-2H-pyrano[3,2-c]quinoline carboxylic ester derivative
as acetylcholinesterase (AChE) peripheral anionic site (PAS)-binding motif by double O / NH bio-
isosteric replacement, combined with molecular hybridization with the AChE catalytic anionic site (CAS)
inhibitor 6-chlorotacrine and molecular dynamics-driven optimization of the length of the linker has
resulted in the development of the trimethylene-linked 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine
e6-chlorotacrine hybrid 5a as a picomolar inhibitor of human AChE (hAChE). The tetra-, penta-, and
octamethylene-linked homologues 5bed have been also synthesized for comparison purposes, and
found to retain the nanomolar hAChE inhibitory potency of the parent 6-chlorotacrine. Further biological
profiling of hybrids 5aed has shown that they are also potent inhibitors of human butyrylcholinesterase
Received 4 March 2014
Received in revised form
18 June 2014
Accepted 6 July 2014
Available online 7 July 2014
Keywords:
Tetrahydrobenzo[h][1,6]naphthyridines
Multitarget compounds
and moderately potent Ab42 and tau anti-aggregating agents, with IC50 values in the submicromolar and
Dual binding site AChE inhibitors
low micromolar range, respectively. Also, in vitro studies using an artificial membrane model have
predicted a good brain permeability for hybrids 5aed, and hence, their ability to reach their targets in the
central nervous system. The multitarget profile of the novel hybrids makes them promising leads for
developing anti-Alzheimer drug candidates with more balanced biological activities.
© 2014 Elsevier Masson SAS. All rights reserved.
Ab aggregation inhibitors
Tau aggregation inhibitors
1. Introduction
upcoming decades, unless efficient disease-modifying drugs
become available [1].
Alzheimer's disease (AD) currently represents one of the most
important unmet medical needs worldwide. Worryingly, because
prevalence and mortality figures associated with AD will keep
increasing, this condition will be even more pronounced in the
Current treatments for AD involve the use of acetylcholines-
terase (AChE) inhibitors (donepezil, rivastigmine and galantamine)
or NMDA receptor antagonists (memantine), which restore
neurotransmitter deficits that are responsible for the symptomatic
phase of the disease (cognitive, functional, and neuropsychiatric
deficits) that appears a decade or more after the onset of the
neurodegenerative process.
* Corresponding author.
** Corresponding author.
It is becoming increasingly apparent that the simultaneous
modulation of several crucial targets that play early roles in the
E-mail addresses: fjluque@ub.edu (F.J. Luque), dmunoztorrero@ub.edu
neuropathogenic process is
a promising approach to derive
~
(D. Munoz-Torrero).
http://dx.doi.org/10.1016/j.ejmech.2014.07.021
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

108
O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
Cl
N
HN
N
n
O
X
O
H
Cl
9
1
X
X
N
N
N
5
H2N
6-chlorotacrine, 2
Cl
Cl
1, X = O
3, X = O; n = 8
molecular hybridization
IC50 (hAChE) > 10 μM
IC50 (hBChE) > 10 μM
IC50 (hAChE) = 19 nM
IC50 (hBChE) = 223 nM
optimization of PAS-binding moiety
optimization of PAS-binding moiety
optimization of tether length
molecular hybridization
optimization of tether length
4, X = NH
5a, X = NH; n = 3
IC50 (hAChE) = 65 nM
IC50 (hBChE) = 920 nM
Fig. 1. Rational design of the tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrid 5a.
effective drugs that can modify the course of AD [3,4]. Obviously,
administration of these multitarget drugs depends on a precise
knowledge of the timing of the critical neuropathologies to be hit
and on the development of suitable biomarkers that enable timely
therapeutic interventions and the assessment of their impact on AD
progression. Whereas these important issues are addressed [2,5,6],
the parallel development of multitarget drugs hitting different
combinations of key biological targets should be actively pursued.
chlorotacrine unit at the CAS, the 5-(4-chlorophenyl)-3,4-
dihydro-2H-pyrano[3,2-c]quinoline ester 1, used as synthetic pre-
cursor of 3, was found to be essentially inactive as AChE inhibitor.
With the aim of optimizing this tricyclic scaffold as a PAS-binding
moiety, we recently designed a family of tetrahydrobenzo[h][1,6]
naphthyridines, which mainly resulted from the substitution of the
oxygen atom at position 1 of the 3,4-dihydro-2H-pyrano[3,2-c]
quinoline system of ester 1 by an amine nitrogen atom [10]. The
rationale behind this structural modification was that the
increased basicity of the pyridine nitrogen atom would enable i) its
protonation at physiological pH, and hence, ii) the establishment
Neuropathologies related to the b-amyloid peptide (Ab) and tau
protein are thought to be at the root of the neurodegenerative
process [2]. Also, AChE seems to play a role in the early phases of
the disease, inasmuch as it can bind to A
aggregation into oligomers and fibrils and increasing the neuro-
toxicity of A aggregates [7]. The A proaggregating action of AChE
has been reported to reside in its peripheral anionic site (PAS) [7c],
which is located at the mouth of a 20 Å deep gorge that leads to the
catalytic anionic site (CAS) of the enzyme [8].
We have recently developed a new family of potent AChE in-
hibitors able to simultaneously bind at the CAS and PAS of the
enzyme, i.e. dual binding site inhibitors, which were designed by
molecular hybridization of the novel 3,4-dihydro-2H-pyrano[3,2-c]
quinoline scaffold of ester 1 and the potent AChE CAS inhibitor 6-
chlorotacrine, 2 (Fig. 1) [9]. The most potent hybrid of the series,
compound 3 (Fig. 1) retained the high human AChE inhibitory ac-
tivity of the parent 6-chlorotacrine (3: IC50 ¼ 7 nM in human
erythrocyte AChE; IC50 ¼ 19 nM in human recombinant AChE
(hAChE)), and additionally exhibited a potent inhibitory activity of
b
, thereby accelerating its
of cationep interactions, apart from pep stacking, with the aro-
matic PAS residues. Indeed, the most potent compound of the
series, 4 (Fig. 1), resulting from a double O / NH bioisosteric
replacement from ester 1 at position 1 and at the side chain in
b
b
position 9, exhibited
(IC50 65 nM) [10]. Molecular dynamics (MD) simulations
confirmed the expected binding of the tricyclic moiety of 4 at the
AChE PAS (catione interactions with Trp286) and hydrogen
a nanomolar hAChE inhibitory activity
¼
p/pep
bond between the protonated pyridine nitrogen atom and the
hydroxyl group of the PAS residue Tyr72) as well as additional
interactions of the amide function at position 9 with midgorge
residues (hydrogen bond between the amide NH group and the
hydroxyl group of Tyr124) [10].
Once the PAS-binding moiety had been optimized, we inferred
that molecular hybridization with the CAS binder 6-chlorotacrine
should provide further improvement of the AChE inhibitory activ-
ity. The disposition of the amide chain at position 9 of the tetra-
hydrobenzo[h][1,6]naphthyridine 4 along the midgorge, with its
nitrogen atom at a distance of ~6 Å from the position occupied by
the exocyclic amino group of tacrine in its complex with Torpedo
californica AChE (Fig. 2) [11], suggested that a linker of 3 methylenes
should be optimal to connect both moieties retaining all their in-
teractions with AChE residues all along the enzyme gorge. Thus, the
tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrid 5a
human butyrylcholinesterase (hBChE) and
aggregating activity (29% inhibition of AChE-induced A
gation at 100 and 21% inhibition of self-induced
aggregation at 50 M) [9].
Even though the 5-(4-chlorophenyl)-3,4-dihydro-2H-pyrano
[3,2-c]quinoline moiety of 3 might establish stacking in-
a
weak
A
b
anti-
40 aggre-
42
b
mM
Ab
m
pep
teractions with the aromatic PAS residues Trp286 and Tyr72
(hAChE numbering), concomitant to the interactions of the 6-

O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
109
(Fig. 1) was rationally designed as a novel multiple-site AChE in-
hibitor, bearing optimized PAS-binding moiety and tether length.
Herein, we describe the synthesis of the tetrahydrobenzo[h][1,6]
naphthyridinee6-chlorotacrine hybrid 5a and its longer tetra-,
penta-, and octa-methylene-linked homologues 5bed, the evalu-
ation of their inhibitory activities against hAChE and hBChE, and
the study of their binding mode to hAChE by MD simulations. To
further expand the potential multitarget profile of hybrids 5aed,
Saponification of ester 6, followed by treatment of the resulting
carboxylate with an Et2O solution of HCl afforded the corre-
sponding carboxylic acid, which was isolated as the naphthyridine
hydrochloride. Coupling of the carboxylic acid with the known
aminoalkyltacrine derivatives 7aed [14], using HOBt and EDC in
the presence of Et3N in a 10:1 mixture EtOAc/DMF, followed by
silica gel column chromatography purification afforded the ex-
pected N-Boc-protected amides, in some cases together with
directly deprotected amides (5a and 5c). Treatment of the N-Boc-
protected amides with 4 M HCl in dioxane at room temperature
afforded the target amides 5aed in 34e80% total overall yields
from ester 6 (Scheme 1).
The novel tetrahydrobenzonaphthyridine-6-chlorotacrine hy-
brids 5aed were fully characterized in the form of dihydrochloride
salts through their spectroscopic data (IR, 1H and 13C NMR) and
HRMS and their purity was assessed by elemental analysis and
HPLC measurements. The biological characterization was also per-
formed with the dihydrochloride salts.
their inhibitory activities against the aggregation of Ab42 and tau
protein in intact Escherichia coli cells, as a simplified in vivo model of
aggregation of amyloidogenic proteins, were also evaluated.
Moreover, the brain penetration of these hybrids, and therefore the
ability to reach their targets at the central nervous system (CNS),
was assessed by a parallel artificial membrane permeation assay
(PAMPA-BBB).
2. Results and discussion
2.1. Synthesis of the tetrahydrobenzo[h][1,6]naphthyridine-6-
chlorotacrine hybrids
2.2. Acetylcholinesterase inhibition
2.2.1. Evaluation of AChE inhibitory activity
Apart from the rationally designed trimethylene-linked hybrid
5a, we planned the synthesis of the longer tetra- and penta-
methylene homologues 5b and 5c, still bearing relatively short
linkers. We also envisioned the synthesis of the octamethylene-
linked analogue 5d, mainly for comparison with the
The inhibitory activity of the novel hybrids 5aed against hAChE
was evaluated by the method of Ellman et al. [15]. The 3,4-dihydro-
2H-pyrano[3,2-c]quinoline derivatives 1 and 3, as well as 6-
chlorotacrine, 2, were also evaluated under the same assay condi-
tions as reference compounds. Also, the reported activity of com-
pound 4 [10] was considered for comparison purposes.
octamethylene-linked
3,4-dihydro-2H-pyrano[3,2-c]quinoline-
based hybrid 3, to gain further insight into the effect of the O / NH
bioisosteric replacement at position 1 of the tricyclic system, while
keeping the same tether length.
For the synthesis of hybrids 5aed, we used as starting material
the N-Boc-protected tetrahydrobenzo[h][1,6]naphthyridine 6,
readily available by a multicomponent Povarov reaction between 4-
chlorobenzaldehyde, ethyl 4-aminobenzoate and N-Boc-3,4-
dihydro-2H-pyridine under the catalysis of Sc(OTf)3 in acetonitrile
[12], followed by DDQ oxidation [13] of the resulting diastereo-
meric mixture of octahydrobenzo[h][1,6]naphthyridines [10].
All the tetrahydrobenzonaphthyridine-6-chlorotacrine hybrids
turned out to be very potent inhibitors of hAChE (Table 1). Indeed,
in agreement with the rational design strategy, the trimethylene-
linked hybrid 5a was the most potent hAChE inhibitor of the se-
ries, exhibiting a surprising picomolar IC50 value (IC50 ¼ 6.27 pM).
The highly potent inhibitory activity of 5a is indicative of the suc-
cess of the rational design strategy, in terms of both the molecular
hybridization and the optimization of the tether length. On the one
hand, molecular hybridization has been successful because 5a is
roughly 1000-fold and >10000-fold more potent than the parent
compounds 6-chlorotacrine, 2, and 4, respectively (Table 1). On the
other hand, the MD-driven optimization of the tether length has
been also successful, inasmuch as the trimethylene-linked hybrid
5a is much more potent than the tetra-, penta-, and octa-
methylene-linked counterparts (>2000-fold more potent than 5b
and 5c and >300-fold more potent than 5d).
The O / NH bioisosteric replacement, which had proven to be
successful in the optimization from the 3,4-dihydro-2H-pyrano
[3,2-c]quinoline ester 1 to the tetrahydrobenzo[h][1,6]naphthyr-
idine amide 4 [10], has been also successful in the corresponding
hybrids with 6-chlorotacrine, the tetrahydrobenzo[h][1,6]naph-
thyridine-based hybrid 5d being roughly 10-fold more potent than
the 3,4-dihydro-2H-pyrano[3,2-c]quinoline-based hybrid 3, with
the same tether length (Table 1).
Overall, hybrid 5a constitutes one of the most potent non-
covalent inhibitors of hAChE so far reported, even though a few
examples of other picomolar or even femtomolar inhibitors of AChE
have also been described [14a,16].
2.2.2. Binding mode within AChE: molecular modelling studies
To shed light on the structural basis of the surprisingly high
AChE inhibitory activity determined for compound 5a, the binding
mode to hAChE was explored by means of MD simulations, taking
advantage of the X-ray crystallographic structure of the hAChE
complex with huprine W (PDB entry 4BDT [17]). The initial pose of
the ligand was guided by the structural information available for
the binding mode of huprine X to T. californica AChE (PDB entry
Fig. 2. Superimposition of the MD-predicted binding mode of tetrahydrobenzo[h][1,6]
naphthyridine 4 in AChE with the X-ray structure of the complex Torpedo californica
AChEetacrine.

110
O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
Cl
6"
7"
8"
5"
1) a) KOH, MeOH; b) HCl, Et2O
2) a) HOBt, EDC, Et3N, EtOAc/DMF
1'
10"
N
n'
9"
Cl
4"
3"
HN
N
n
b)
O
H
CO2Et
1"
9
2"
10
8
7
Boc
N
N
H
N
1
2
3
H2N
N
n
H
N
N
6
7a-d
5
4
6
5a-d
3) 4 M HCl / dioxane
a, n = 3; b, n = 4; c, n = 5; d, n = 8
Cl
Cl
Scheme 1. Synthesis of the target tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids.
1E66 [18]), which matches well the structure of huprine W bound
to hAChE, and by the recently reported binding mode of compound
4 [10].
formed by the amide group in the tether, as the amide NH group is
involved in a hydrogen bond with Asp74 (average distance of 3.6 Å),
which is in turn hydrogen-bonded to the hydroxyl group of Tyr341
(average distance of 2.8 Å), while the amide carbonyl oxygen forms
either direct or water-mediated interactions with the hydroxyl
group of Tyr337 (average distance of 4.9 Å). As a further test, the
binding free energy of compound 5a was determined with the
Solvated Interaction Energy (SIE) method [19], which relies on MM/
PBSA calculations in conjunction with weighting scaling factors for
the free energy components suitably parameterized to reproduce
the experimental binding affinities for a diverse set of pro-
teineligand complexes. The SIE binding affinity obtained for com-
pound 5a is ꢀ12.5 kcal/mol (Table S1, Supplementary Material),
which is 4 kcal/mol lower than that determined for compound 4
(ꢀ8.5 kcal/mol [10]), which is in agreement with the ~104 ratio
between the IC50 values reported in Table 1.
The analysis of the 100 ns MD trajectory supported the struc-
tural integrity and stability of the ligand bound to the hAChE gorge.
Thus, with the sole exception of a slight reorientation of the tet-
rahydrobenzo[h][1,6]naphthyridine moiety in the PAS, which was
originated from a conformational change in the loop defined by
residues 289e292, the RMSD of the ligand remained fully stable
during the last 60 ns of the trajectory (Fig. S1, Supplementary
Material). The hybrid 5a establishes a complex network of in-
teractions with the residues of the binding site (Fig. 3). As expected,
the 6-chlorotacrine moiety was tightly bound in the CAS due to the
cationep interactions with the aromatic rings of Trp86 and Tyr337
(average distances of 3.9 Å between the indole or phenol rings and
the aminoacridine unit) and the hydrogen bond of the protonated
acridine nitrogen atom with the carbonyl oxygen of His447
(average distance of 2.9 Å). On the other hand, the tetrahydrobenzo
[h][1,6]naphthyridine moiety was firmly stacked against Trp286,
These findings provide a basis to explain the abrupt change in
inhibitory activity between compounds 5a and 5b, as the enlarge-
ment of the oligomethylene chain between tacrine and the amide
group would disrupt the interactions with the midgorge residues.
On the other hand, the amide group that is present in the tether of
thus enabling the formation of a cationep interaction between the
protonated quinoline nitrogen atom and the indole ring of Trp286.
The binding of this moiety was also assisted by transient hydrogen
bond interactions between the NH group and the hydroxyl oxygen
of Tyr72. The most remarkable finding concerns the interactions
~
some tacrineeindole heterodimers reported by Munoz-Ruiz et al.
as picomolar AChE inhibitors was also suggested to participate in a
complex network of interactions with midgorge residues [14a]. In
Table 1
Inhibitory activities of 1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine-6-chlorotacrine hybrids and reference compounds against AChE, BChE, A
b42 and tau aggregation, and
BBB predicted permeabilities.
c
Compd
hAChE IC50 (nM)a
hBChE IC50 (nM)a
A
b
42 aggregation in
Tau protein aggregation in
Pe (10ꢀ6 cm sꢀ1
)
(prediction)
E. coli (% inhib. at 10
m
M)b
E. coli (% inhib. at 10
m
M)b
5a
5b
5c
5d
1
2
3
4
0.006 0.002
14.2 1.7
14.2 1.9
2.06 0.3
nad
5.9 0.6
19.3 1.4
65.0 3.0f
120 13
205 16
337 30
286 35
nad
52.5 0.4
55.8 0.7
54.3 0.7
77.5 0.9
41.8 0.4
11.0 0.6
54.6 0.5
ndg
40.7 0.5
58.9 0.4
57.7 0.5
68.7 0.5
27.9 1.2
1.4 0.7
37.8 0.5
ndg
10.5 0.7 (CNSþ)
13.8 0.4 (CNSþ)
14.3 1.3 (CNSþ)
19.2 1.3 (CNSþ)
9.9 1.4 (CNSþ)
19.8 0.4 (CNSþ)e
13.9 0.8 (CNSþ)
22.9 0.8 (CNSþ)f
114
4
223 35
920 30f
a
IC50 inhibitory concentration (nM) of human recombinant AChE and human serum BChE. IC50 values are expressed as mean standard error of the mean (SEM) of at least
four experiments (n ¼ 4), each performed in duplicate.
b
% Inhibition of A
b42 and tau protein aggregation at 10
m
M in intact E. coli cells. Values are expressed as mean SEM of four independent experiments (n ¼ 4).
c
d
e
f
Permeability values from the PAMPA-BBB assay. Values are expressed as the mean SD of three independent experiments (n ¼ 3).
Not active.
Data from Ref. [9].
Data from Ref. [10].
Not determined.
g

O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
111
[3,2-c]quinoline- and tetrahydrobenzo[h][1,6]naphthyridine-based
hybrids 3 and 5d roughly displaying the same potency (Table 1).
2.4. Ab42 and tau aggregation inhibition
The aggregation of the amyloidogenic proteins A
most aggregation-prone and neurotoxic 42 amino acid form
thereof (A 42), and tau are widely thought to constitute early
b, especially the
b
pathogenic events in AD, and hence, they are the target of many
drug candidates purported to modify the natural course of the
disease [21].
We have recently developed a new methodology for the eval-
uation of the effects of putative inhibitors on the aggregation and
subsequent formation of insoluble inclusion bodies of any amyloi-
dogenic protein that can be overexpressed in E. coli cells [22]. The
method relies on monitoring the changes in the fluorescence of
Thioflavin S (TheS) that are produced upon binding to amyloid
aggregates rich in b-sheet structures. Compounds that are able to
cross the membranes of E. coli cells and inhibit the aggregation of
overexpressed amyloidogenic proteins will lead to a decrease in the
fluorescence of TheS. This method is fast, simple and inexpensive,
as it avoids the use of synthetic peptides. We have successfully used
this method for the evaluation of inhibitors of A
gregation. Interestingly, the results obtained in the screening of
42 aggregation inhibitors correlated very well with the results
previously reported from in vitro assays using synthetic peptides,
thereby validating this methodology [22].
b42 and tau ag-
Fig. 3. Representation of the binding mode of the tetrahydrobenzo[h][1,6]naphthyr-
idine-6-chlorotacrine hybrid 5a (magenta) in the average structure obtained from the
snapshots sampled in the last 5 ns of the trajectory. The residues involved in in-
teractions are shown as green-coloured sticks. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
A
b
The inhibitory activity of hybrids 5aed against Ab42 and tau
aggregation was assessed using this methodology. In general, very
similar results and SAR trends were found for both activities. At
the most potent tacrineeindole heterodimer the amide group,
located at six methylene groups from the tacrine unit, was sug-
gested to interact with Tyr124 and Tyr337. In hybrid 5a the short-
ening of the chain to three methylenes enables the formation of a
distinct interaction pattern with Asp74 and Tyr337. Overall, these
findings reinforce the significant contribution played by the
midgorge in complementing both PAS and CAS and modulating the
affinity of AChE inhibitors.
10
mM, hybrids 5aed exhibited percentages of inhibition in the
ranges 52e77% and 41e69% against Ab42 and tau aggregation,
respectively (Table 1).
Molecular hybridization led to increased A
aggregating activities, hybrids 5aed being more potent than 6-
chlorotacrine (5e7-fold more potent against A 42 aggregation
b42 and tau anti-
b
and 30e50-fold more potent against tau aggregation). The parent
compound 4 could not be tested in these assays, but in in vitro tests
it had been found to be a weak inhibitor of A
b42 aggregation (15%
2.3. Butyrylcholinesterase inhibition
inhibition at 10 M) [10], so presumably hybrids 5aed are also
m
more potent than 4.
BChE is partly responsible for acetylcholine hydrolysis and
hence, for the cholinergic deficit of AD patients, especially in
advanced stages of the disease, when the levels of AChE in CNS
markedly decrease. Thus, BChE also represents a biological target of
interest for AD treatment [20]. The BChE inhibitory activity of hy-
brids 5aed against hBChE was evaluated by the method of Ellman
et al. [15].
The parent compounds 6-chlorotacrine, 2, and 4 are selective
inhibitors of hAChE, albeit still exhibiting a moderately potent
hBChE inhibitory activity, with IC50 values in the submicromolar
range. Not unexpectedly, hybrids 5aed were also more potent
against hAChE than hBChE, displaying submicromolar IC50 values
for hBChE inhibition (Table 1). In this case, molecular hybridization
resulted in an increased hBChE inhibitory activity relative to the
parent compound 4 (3e8-fold) but in a slightly decreased potency
relative to 6-chlorotacrine, 2, with the exception of hybrid 5a,
which was equipotent to 6-chlorotacrine.
The length of the linker seemed to have a subtle effect on Ab42
and tau anti-aggregating activities. These activities slightly
increased with the tether length so that the longer homologue 5d
was 1.5-fold more potent than the shorter counterpart 5a for both
activities. On the other hand, the O / NH bioisosteric replacement
had a similar effect on both activities, the tetrahydrobenzo[h][1,6]
naphthyridine-based hybrid 5d being roughly 1.5-fold more potent
than the 3,4-dihydro-2H-pyrano[3,2-c]quinoline-based hybrid 3
for A
b42 and tau aggregation inhibition.
The parallel results obtained for these hybrids against both A
b
42
and tau aggregation further support the notion that diseases based
on the pathological aggregation of one or several amyloidogenic
proteins might share common mechanisms and might be con-
fronted with common therapeutic interventions [23].
Overall, hybrids 5aed can be considered as moderately potent
dual A
b
42 and tau anti-aggregating compounds, with IC50 values
M. Because these anti-
that must lie around or below 10
m
The length of the linker has little influence on the hBChE
inhibitory activity. Only a slight trend toward decreased potency
with increased tether length was observed, the shorter homologue
5a being 1.7-, 2.8-, and 2.4-fold more potent than the longer
counterparts 5b, 5c, and 5d, respectively. Finally, the O / NH
bioisosteric replacement had little influence on the hBChE inhibi-
tory activity, the octamethylene-linked 3,4-dihydro-2H-pyrano
aggregating activities have been determined without involving
the presence of AChE, the high AChE inhibitory activity of hybrids
5aed cannot be responsible for their A
activities, which might be ascribed, instead, to a direct interaction
with A 42 and tau. The precise mechanisms through which these
hybrids bind A 42 and tau and/or exert their anti-aggregating ac-
tivities are unknown, even though it has been reported that the
b42 and tau anti-aggregating
b
b

112
O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
presence of several aromatic moieties with extended
systems (including biphenyls and phenyl-substituted benzoheter-
oaromatic systems, similar to the phenylquinoline moiety present
p
-conjugated
method was also used to assess the intestinal absorption of the
novel compounds, which was predicted to be positive in all cases.
Finally, the predicted rat acute toxicity of the hybrids was clearly
lower than that predicted for the anti-Alzheimer AChE inhibitor
tacrine, thereby supporting the safety of these compounds
(Table 2).
in hybrids 5aed) may enable binding to A
b42 [24].
The dual A 42 and tau anti-aggregating profile is of much in-
b
terest for disease-modifying anti-Alzheimer agents. However, it
must be recognized that the anti-aggregating activities of hybrids
5aed are not well balanced relative to their cholinesterase inhibi-
tory activities, especially AChE and particularly in the case of its
picomolar inhibitor 5a, which represents an important issue in
multitarget compounds.
3. Conclusion
In this work we have further advanced in the hit-to-lead opti-
mization process that, starting from the 3,4-dihydro-2H-pyrano
[3,2-c]quinoline carboxylic ester 1, had led to the potent hAChE
inhibitors 3 [9] and 4 [10] by molecular hybridization with 6-
chlorotacrine and by double O / NH bioisosteric replacement,
respectively. Herein, combination of the optimized AChE PAS-
binding moiety present in 4, molecular hybridization with 6-
chlorotacrine, and an MD-driven optimization of the tether
length has led to the discovery of the 1,2,3,4-tetrahydrobenzo[h]
[1,6]naphthyridine-6-chlorotacrine hybrid 5a as a picomolar in-
hibitor of hAChE.
Apart from 5a, other three longer homologues, i.e. 5bed, have
been also synthesized and found to be potent inhibitors of hAChE,
exhibiting IC50 values in the low nanomolar range. Like the parent
compounds 6-chlorotacrine, 2, and the tetrahydrobenzo[h][1,6]
naphthyridine 4, hybrids 5aed have been found to be selective for
hAChE vs. hBChE inhibition, albeit still keeping a potent hBChE
inhibitory activity. Very interestingly, these hybrids turned out to
2.5. Bloodebrain barrier permeation assay
Anti-Alzheimer drug candidates, like any other CNS drug, must
be able to efficiently enter into the brain, which requires a good
ability to cross the bloodebrain barrier (BBB) and a low P-glyco-
protein efflux liability [25]. The large molecular weight of hybrids
5aed (>500) might compromise their ability to cross biological
membranes, including BBB [26]. However, a number of distinct
anti-Alzheimer hybrid compounds with molecular weights over
500 have shown good oral availability and/or brain permeability in
ex vivo and in vivo studies in mice [27]. Indeed, the positive results
obtained for the hybrids 5aed in the aggregation studies in E. coli
cells were already indicative of their ability to cross biological
membranes, but a more accurate determination of their ability to
cross the BBB was necessary. In this light, the brain permeability of
the hybrids 5aed was predicted using an in vitro model of passive
transcellular permeation, namely the widely known PAMPA-BBB
assay [28]. Thus, the in vitro permeability (Pe) through a lipid
extract of porcine brain was determined using a mixture of
phosphate-buffered saline (PBS)/EtOH (70:30). Assay validation
was made by comparing the experimental and reported perme-
ability values of 14 commercial drugs (Table S2, Supplementary
be moderately potent dual inhibitors of Ab42 and tau aggregation
in intact E. coli cells, with IC50 values in the low micromolar range.
Taking into account all the tested activities, hybrid 5d, with the
most potent Ab42 and tau anti-aggregating activities, is likely the
hybrid of the series with the most interesting multitarget profile.
Notwithstanding that better balanced potencies at their different
targets would have been desirable, the multitarget profile of the
novel hybrids, together with their predicted ability to cross the BBB,
make them interesting anti-Alzheimer lead compounds.
Material), which provided
a
good linear correlation: Pe
(exp) ¼ 1.5010 Pe (lit) ꢀ 0.8618 (R2 ¼ 0.9199). Using this equation
and the limits established by Di et al. for BBB permeation [28], the
following ranges of permeability were established: Pe
(10ꢀ6 cm sꢀ1) > 5.1 for compounds with high BBB permeation
(CNSþ); Pe (10ꢀ6 cm sꢀ1) < 2.1 for compounds with low BBB
permeation (CNSꢀ); and 5.1 > Pe (10ꢀ6 cm sꢀ1) > 2.1 for compounds
with uncertain BBB permeation (CNS ). All the tetrahydrobenzo[h]
[1,6]naphthyridine-6-chlorotacrine hybrids, 5aed, were predicted
to be able to cross the BBB. The measured Pe values for 5aed were
found to slightly increase with the tether length, and hence, with
lipophilicity, and were clearly above the threshold for high BBB
permeation (Table 1), thereby anticipating their ability to enter the
brain and reach their different CNS targets.
4. Experimental part
4.1. Chemistry
4.1.1. General methods
Melting points were determined in open capillary tubes with an
MFB 595010M Gallenkamp melting point apparatus. Recrystalli-
zation solvents from which the analytical samples have been ob-
tained are indicated after the melting points. 400 MHz 1H NMR and
500 MHz 1H/125.8 MHz 13C NMR spectra were recorded on Varian
Gemini 400 and Varian Mercury 500 spectrometers, respectively.
The chemical shifts are reported in ppm (d scale) and coupling
Of note, the predicted in vitro ability of the novel hybrids to cross
the BBB was also confirmed through the BBB permeation index
obtained using a recently reported in silico multiclassification
method (Table 2), which was developed utilizing a comprehensive
data set containing around 12,000 diverse compounds [29]. This
constants are reported in Hertz (Hz). Assignments given for the
NMR spectra of the new compounds have been carried out by
comparison with the NMR data of the precursor ester 6 and the
hybrid compound 5d which in turn, were assigned on the basis of
DEPT, COSY 1H/1H (standard procedures), and COSY 1H/13C (gHSQC
or gHMBC sequences) experiments. IR spectra were run on a Per-
kineElmer Spectrum RX I spectrophotometer, using the Attenuated
Total Reflectance (ATR) technique. Absorption values are expressed
as wave-numbers (cmꢀ1); only significant absorption bands are
given. Column chromatography was performed on silica gel 60 AC.C
Table 2
In silico prediction of ADMET properties for hybrids 5aed and the reference com-
pound tacrine.
Compd
Bloodebrain barrier
permeation
Intestinal
absorption
Rat acute toxicity
LD50 (mg/kg)
(35e70
mM, SDS, ref 2000027). Thin-layer chromatography was
5a
5b
5c
5d
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
566
449
803
performed with aluminium-backed sheets with silica gel 60 F254
(Merck, ref 1.05554), and spots were visualized with UV light and
1% aqueous solution of KMnO4. NMR spectra of all of the new
compounds were performed at the Centres Científics i Tecnologics
of the University of Barcelona (CCiTUB), while elemental analyses
493
Tacrine
75 (70)a
ꢁ
a
Taken from ref. [30].

O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
113
and high resolution mass spectra were carried out at the Micro-
analysis Service of the IIQAB (CSIC, Barcelona, Spain) with a Carlo
Erba model 1106 analyzer, and at the CCiTUB with an LC/MSD-TOF
Agilent Technologies spectrometer, respectively. The HPLC mea-
surements were performed using an HPLC Waters Alliance HT
apparatus comprising a pump (Edwards RV12) with degasser, an
autosampler, a diode array detector and a column as specified
below. The reverse phase HPLC determinations were carried out on
organic extracts were dried over anhydrous Na2SO4, and evapo-
rated at reduced pressure to give amide 5a (115 mg, 41% overall
yield from 6, 80% total overall yield of 5a) as a yellowish solid; Rf
0.65 (CH2Cl2/MeOH/50% aq. NH4OH 9:1:0.05).
A solution of amide 5a (95 mg, 0.16 mmol) in MeOH (5 mL) was
filtered through a 0.45 mm PTFE filter and treated with a methanolic
solution ofHCl (0.53 N, 2.6 mL,1.38mmol). The resultingsolutionwas
evaporated at reduced pressure and the solid was washed with
pentane to give, after drying under standard conditions, 5a$2HCl
a YMC-Pack ODS-AQ column (50 ꢁ 4.6 mm, DS. 3
mm, 12 nm).
Solvent A: water with 0.1% formic acid; Solvent B: acetonitrile with
0.1% formic acid, or solvent A: water with NH4HCO3, 10 mM pH 9.0;
Solvent B: acetonitrile. Gradient: 5% of B to 100% of B within
3.5 min. Flux: 1.6 mL/min at 50 ꢂC. The analytical samples of all of
the compounds that were subjected to pharmacological evaluation
were dried at 65 ꢂC/2 Torr (standard conditions) at least for 2 days
and possess a purity ꢃ95% as evidenced by their elemental analyses
and HPLC measurements. Of note, as previously reported for some
tacrine-related dimeric compounds [31], the new hybrids herein
described have the ability to retain molecules of water, which
cannot be removed after drying the analytical samples under the
aforementioned standard conditions. Thus, the elemental analyses
of these compounds showed the presence of variable amounts of
water, which have been indicated in the corresponding compound
formulae.
(95 mg) as a yellowish solid: mp 295e297 ꢂC (MeOH); IR (ATR)
n
3500e2500 (max at 3315, 3231, 3084, 3034, 2941, 2865, 2772, NeH,
þNeH, CeH st),1643,1632,1578,1553,1514 (C]O, AreCeC, AreCeN
st) cmꢀ1 1H NMR (500 MHz, CD3OD)
; d 1.94e2.02 (complex signal,
4H, 200-H2, 300-H2), superimposed in part 2.00 (tt, J¼J0 ¼ 6.0 Hz, 2H, 3-
H2), 2,20 (tt, J¼J0 ¼ 6.5 Hz, 2H, 2’-H2), 2.76 (t, J ¼ 6.0 Hz, 2H, 4-H2),
superimposed in part 2.78 (m, 2H, 100-H2), 2.99 (t, J ¼ 6.5 Hz, 2H, 400-
H2), 3.61 (t, J ¼ 6.5 Hz, 2H,10-H2), 3.73 (t, J ¼ 6.0 Hz, 2H, 2-H2), 4.13 (t,
J ¼ 6.5 Hz, 2H, 30-H2), 4.85 (s, NH, þNH), 7.47 (dd, J ¼ 9.0 Hz, J0 ¼ 2.0 Hz,
1H, 700-H), 7.64 (d, J ¼ 2.0 Hz,1H, 500-H), 7.65 (dm, J ¼ 9.0 Hz, 2H) and
7.68 (dm, J ¼ 9.0 Hz, 2H) [2(6)-H and 3(5)-H 4-chlorophenyl], 7.85 (d,
J ¼ 8.5 Hz, 1H, 7-H), 8.20 (dd, J ¼ 8.5 Hz, J0 ¼ 1.5 Hz,1H, 8-H), 8.41 (d,
J ¼ 9.0 Hz, 1H, 800-H), 8.96 (d, J ¼ 1.5 Hz, 1H, 10-H); 13C NMR
(125.8 MHz, CD3OD) d
20.0 (CH2, C3), 21.7 (CH2, C300), 22.9 (CH2, C200),
24.9 (CH2, C100), 25.0 (CH2, C4), 29.4 (CH2, C400), 31.1 (CH2, C20), 37.9
(CH2, C10), 43.0 (CH2, C2), 46.3 (CH2, C30), 110.0 (C, C4a), 113.7 (C,
C9a00), 115.5 (C, C8a00), 116.1 (C, C10a) 119.0 (CH, C500), 121.0 (CH, C7),
123.8 (CH, C10), 126.7 (CH, C700), 128.7 (CH, C800), 130.5 (2CH) and
131.8 (2CH) [C2(6) and C3(5) 4-chlorophenyl], 132.3 (C, C1 4-
chlorophenyl), 132.5 (CH, C8), 132.8 (C, C9), 138.3 (C, C4 4-
chlorophenyl), 139.9 (C, C600), 140.3 (C, C6a), 140.4 (C, C10a00), 151.6
4.1.2. N-{3-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]
propyl}-5-(4-chlorophenyl)-1,2,3,4-tetrahydrobenzo[h][1,6]
naphthyridine-9-carboxamide 5a
A solution of ester 6 (4.79 g, 10.2 mmol) and KOH pellets (85%
purity, 2.04 g, 30.9 mmol) in MeOH (260 mL) was stirred under
reflux for 24 h. The resulting mixture was cooled to room tem-
perature and evaporated to dryness. The solid residue was treated
with an Et2O solution of HCl (1.2 N, 171 mL, 205 mmol) for 30 min
and the resulting suspension was evaporated at reduced pressure,
to give the corresponding tetrahydrobenzo[h][1,6]naphthyr-
idinecarboxylic acid, in the form of its hydrochloride salt (6.54 g),
which was directly used in the next step without further purifica-
(C, C5),152.3 (C, C4a00),155.8 (C, C10b),158.2 (C, C900),168.2 (C, CONH);
35
HRMS (ESI), calcd for [C
H
Cl2N5O þ Hþ] 610.2135, found 610.2129;
35 33
Anal. C35H33Cl2N5O$2HCl$1.5H2O (C, H, N).
4.1.3. N-{4-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]butyl}-
5-(4-chlorophenyl)-1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine-
9-carboxamide 5b
It was prepared as described for 5a. Starting from crude tetra-
hydrobenzo[h][1,6]naphthyridinecarboxylic acid (570 mg) and
amine 7b (401 mg, 1.32 mmol), a semisolid residue (2.54 g) was
tion: IR (ATR)
st), 1679, 1584, 1574 (C]O, AreCeC, AreCeN st) cmꢀ1
(400 MHz, CD3SO) 1.28 [s, 9H, NCOOeC(CH3)3], 1.76 (m, 2H, 3-H2),
n
3500e2500 (max at 3316, 2966, OeH, þNeH, CeH
;
1H NMR
d
2.66 (t, J ¼ 5.6 Hz, 2H, 4-H2), 3.41 (m, 2H, 2-H2), 7.49 (d, J z 8.4 Hz,
obtained and purified by column chromatography (35e70 mm silica
2H), 7.64 (d, J ¼ 8.4 Hz, 2H) [2(6)-H and 3(5)-H 4-chlorophenyl],
gel, CH2Cl2/MeOH/50% aq. NH4OH mixtures, gradient elution). On
elution with CH2Cl2/MeOH/50% aq. NH4OH 99:1:0.2, N1-Boc-pro-
tected amide (307 mg, 48% overall yield from 6) and a 1:1 mixture
of this amide with starting amine 7b (1H NMR) (234 mg, 18% overall
yield of the protected amide from 6; 66% total overall yield of
protected amide from 6) were successively isolated.
7.78 (d, J ¼ 8.8 Hz, 1H, 7-H), 8.17 (br d, J z 8.8 Hz, 1H, 8-H), 8.34 (br
35
s, 1H, 10-H); HRMS (ESI), calcd for [C
found 439.1410.
H
ClN2O4þHþ] 439.1419,
24 23
To
a solution of crude tetrahydrobenzo[h][1,6]naphthyr-
idinecarboxylic acid (298 mg) in EtOAc/DMF 10:1 (21 mL), Et3N
(0.19 mL, 138 mg, 1.36 mmol), EDC (146 mg, 0.94 mmol), and HOBt
(128 mg, 0.94 mmol) were added, and the mixture was stirred at
room temperature for 5 min. To the resulting mixture, amine 7a
(200 mg, 0.69 mmol) was added and the reaction mixture was
stirred at room temperature for 18 h and concentrated at reduced
pressure, to give a semisolid residue (1.18 g), which was purified by
Treatment of the N1-Boc-protected amide (272 mg, 0.38 mmol)
with 4 M HCl/dioxane (2.51 mL, 10.0 mmol) as described for 5a
afforded amide 5b (237 mg, 66% overall yield from 6) as a yellowish
solid; Rf 0.67 (CH2Cl2/MeOH/50% aq. NH4OH 9:1:0.05).
A solution of amide 5b (237 mg, 0.38 mmol) in CH2Cl2 (10 mL)
was filtered through a 0.45
mm PTFE filter and treated with a
column chromatography (35e70
m
m silica gel, CH2Cl2/MeOH/50%
methanolic solution of HCl (0.53 N, 6.3 mL, 3.34 mmol). The
resulting solution was evaporated at reduced pressure and the solid
was washed with pentane to give, after drying under standard
conditions, 5b$2HCl (285 mg) as a yellowish solid: mp 276e279 ꢂC
aq. NH4OH mixtures, gradient elution). On elution with CH2Cl2/
MeOH/50% aq. NH4OH 99.5:0.5:0.2 to 99.2:0.8:0.2, N1-Boc-pro-
tected amide (141 mg, 43% overall yield from 6) and the final amide
5a (111 mg, 39% overall yield from 6) were successively isolated as
yellowish solids.
(CH2Cl2/MeOH 5:3); IR (ATR)
n 3500e2500 (max. at 3226, 3062,
3028, 2937, 2871, 2803, NeH, þNeH, CeH st), 1651, 1632, 1586,
A solution of the N1-Boc-protected amide (141 mg, 0.20 mmol)
in 4 M HCl/dioxane (2.41 mL, 9.64 mmol) was stirred thoroughly at
room temperature for 18 h, and was evaporated at reduced pres-
sure. The resulting solid residue was diluted with H2O (3 mL) and
alkalinized with 10% aqueous Na2CO3 (2 mL). The alkaline solution
was extracted with CHCl3/MeOH 9:1 (4 ꢁ 25 mL) and the combined
1573, 1538 (C]O, AreCeC, AreCeN st) cmꢀ1 1H NMR (500 MHz,
;
CD3OD)
d
1.84 (tt, J¼J0 ¼ 7.0 Hz, 2H, 20-H2), 1.92e2.03 (complex
signal, 8H, 3-H2, 30-H2, 200-H2 and 300-H2), 2.71 (t, J ¼ 6.0 Hz, 2H, 100-
H2), 2.76 (t, J ¼ 6.0 Hz, 2H, 4-H2), 2.98 (t, J ¼ 6.0 Hz, 2H, 400-H2), 3.52
(t, J ¼ 7.0 Hz, 2H, 10-H2), 3.72 (t, J ¼ 6.0 Hz, 2H, 2-H2), 4.05 (t,
J ¼ 7.0 Hz, 2H, 40-H2), 4.85 (s, NH, þNH), 7.50 (dd, J ¼ 9.5 Hz,

114
O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
J0 ¼ 2.5 Hz, 1H, 700-H), 7.66e7.70 [complex signal, 5H, 2(6)-H and
3(5)-H 4-chlorophenyl, and 500-H], 7.84 (d, J ¼ 9.0 Hz, 1H, 7-H), 8.22
(dd, J ¼ 9.0 Hz, J0 ¼ 2.0 Hz, 1H, 8-H), 8.40 (d, J ¼ 9.5 Hz, 1H, 800-H),
amine 7d (400 mg, 1.11 mmol), a semisolid residue (2.45 g) was
obtained and purified by column chromatography (35e70 m silica
m
gel, CH2Cl2/MeOH/50% aq. NH4OH mixtures, gradient elution). On
elution with CH2Cl2/MeOH/50% aq. NH4OH 100:0:1, N1-Boc-pro-
tected amide (370 mg, 63% overall yield from 6) was isolated as a
yellowish solid.
8.98 (d, J ¼ 2.0 Hz, 1H, 10-H); 13C NMR (125.8 MHz, CD3OD)
d 20.0
(CH2, C3), 21.7 (CH2, C300), 22.9 (CH2, C200), 24.7 (CH2, C100), 25.0 (CH2,
C4), 27.3 (CH2) and 28.7 (CH2) (C2’, C3’), 29.3 (CH2, C400), 40.7 (CH2,
C1’), 43.0 (CH2, C2), 48.8 (CH2, C4’), 109.9 (C, C4a), 113.6 (C, C9a00),
115.5 (C, C8a00), 116.2 (C, C10a), 119.0 (CH, C5”), 120.9 (CH, C7), 123.7
(CH, C10), 126.8 (CH, C700), 128.9 (CH, C800), 130.4 (2CH) and 131.8
(2CH) [C2(6) and C3(5) 4-chlorophenyl], 132.3 (C, C1 4-
chlorophenyl), 132.5 (CH, C8), 133.2 (C, C9), 138.3 (C, C4 4-
chlorophenyl), 140.0 (C, C6”), 140.3 (C, C6a), 140.5 (C, C10a”), 151.6
Treatment of the N1-Boc-protected amide (370 mg, 0.47 mmol)
with 4 M HCl/dioxane (3.10 mL, 12.4 mmol) as described for 5a
afforded amide 5d (359 mg, 63% overall yield from 6) as a yellowish
solid; Rf 0.78 (CH2Cl2/MeOH/50% aq. NH4OH 9:1:0.05).
A solution of amide 5d (359 mg, 0.53 mmol) in CH2Cl2 (10 mL)
was filtered through a 0.45
mm PTFE filter and treated with a
(C, C5), 152.1 (C, C4a”), 155.7 (C, C10b), 157.9 (C, C9”), 167.9 (C,
methanolic solution of HCl (0.53 N, 8.73 mL, 4.63 mmol). The
resulting solution was evaporated at reduced pressure and the solid
was washed with pentane to give, after drying under standard
conditions, 5d$2HCl (283 mg) as a yellowish solid: mp 203e204 ꢂC
35
CONH); HRMS (ESI), calcd for [C
H
36 35
Cl2N5OþHþ] 624.2291, found
624.2274; Anal. C36H35Cl2N5O$2HCl$1.5H2O (C, H, N).
4.1.4. N-{5-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]
pentyl}-5-(4-chlorophenyl)-1,2,3,4-tetrahydrobenzo[h][1,6]
naphthyridine-9-carboxamide 5c
(CH2Cl2/MeOH 5:4); IR (ATR)
2929, 2860, NeH, þNeH, CeH st), 1731, 1631, 1573 (C]O, AreCeC,
AreCeN st) cmꢀ1 1H NMR (500 MHz, CD3OD)
1.38e1.50 (com-
n 3500e2500 (max. at 3228, 3039,
;
d
It was prepared as described for 5a. Starting from crude tetra-
hydrobenzo[h][1,6]naphthyridinecarboxylic acid (190 mg) and
amine 7c (141 mg, 0.44 mmol), a semisolid residue (1.34 g) was
plex signal, 8H, 3’-H2, 4’-H2, 5’-H2, 6’-H2), 1.69 (tt, J¼J’ ¼ 7.5 Hz, 2H,
2’-H2), 1.85 (tt, J z J’z7.5 Hz, 2H, 7’-H2), 1.92e2.00 (complex signal,
6H, 3-H2, 2”-H2, 3”-H2), 2.68 (t, J ¼ 6.0 Hz, 2H, 1”-H2), 2.75 (t,
J ¼ 6.5 Hz, 2H, 4-H2), 3.01 (t, J ¼ 6.0 Hz, 2H, 4”-H2), 3.43 (t, J ¼ 7.5 Hz,
2H,1’-H2), 3.71 (t, J ¼ 6.0 Hz, 2H, 2-H2), 3.95 (t, J ¼ 7.5 Hz, 2H, 8’-H2),
4.85 (s, NH, þNH), 7.55 (dd, J ¼ 9.5 Hz, J’ ¼ 2.0 Hz, 1H, 7”-H), 7.67
[complex signal, 4H, 2(6)-H and 3(5)-H 4-chlorophenyl], 7.78 (d,
J ¼ 2.0 Hz,1H, 5”-H), 7.87 (d, J ¼ 9.0 Hz,1H, 7-H), 8.25 (dd, J ¼ 9.0 Hz,
J’ ¼ 2.0 Hz,1H, 8-H), 8.38 (d, J ¼ 9.5 Hz,1H, 8”-H), 8.99 (d, J z 2.0 Hz,
obtained and purified by column chromatography (35e70 mm silica
gel, CH2Cl2/MeOH/50% aq. NH4OH mixtures, gradient elution). On
elution with CH2Cl2/MeOH/50% aq. NH4OH 99.2:0.8:0.2 to 99:1:0.2,
impure N1-Boc-protected amide (51 mg) and deprotected amide 5c
(65 mg, 34% overall yield from 6) were successively isolated as
yellowish solids; Rf 0.71 (CH2Cl2/MeOH/50% aq. NH4OH 9:1:0.05).
A solution of amide 5c (65 mg, 0.10 mmol) in CH2Cl2 (10 mL) was
1H, 10-H); 13C NMR (125.8 MHz, CD3OD)
d 20.0 (CH2, C3), 21.8 (CH2,
filtered through a 0.45
m
m PTFE filter and treated with a methanolic
C3”), 22.9 (CH2, C2”), 24.8 (CH2, C1”), 25.0 (CH2, C4), 27.6 (CH2, C6’),
28.0 (CH2, C3’), 29.3 (CH2, C4”), 30.1 (CH2) and 30.2 (CH2) (C4’, C5’),
30.4 (CH2, C2’), 31.3 (CH2, C7’), 41.3 (CH2, C1’), 43.0 (CH2, C2), 49.2
(CH2, C8’), 109.8 (C, C4a), 113.3 (C, C9a”), 115.4 (C, C8a”), 116.1 (C,
C10a), 119.1 (CH, C5”), 120.9 (CH, C7), 123.6 (CH, C10), 126.7 (CH,
C7”), 128.8 (CH, C8”), 130.4 (2CH) and 131.9 (2CH) [C2(6) and C3(5)
4-chlorophenyl], 132.2 (C, C1 4-chlorophenyl), 132.6 (CH, C8), 133.5
(C, C9), 138.3 (C, C4 4-chlorophenyl), 140.1 (C, C6”), 140.2 (C, C6a),
140.5 (C, C10a”), 151.4 (C, C5), 152.1 (C, C4a”), 155.7 (C, C10b), 157.8
solution of HCl (0.53 N, 1.6 mL, 0.85 mmol). The resulting solution
was evaporated at reduced pressure and the solid was washed with
pentane to give, after drying under standard conditions, 5c$2HCl
(32 mg) as a yellowish solid: mp 207e208 ꢂC (CH2Cl2/MeOH 20:3);
IR (ATR)
CeH st), 1625, 1614, 1587, 1541, 1519 (C]O, AreCeC, AreCeN st)
cmꢀ1 1H NMR (500 MHz, CD3OD)
1.30 (m, 2H, 3’-H2), 1.58 (tt,
n
3500e2500 (max. at 3376, 3062, 2927, 2860, NeH, þNeH,
;
d
J z J’z6.5 Hz, 2H, 2’-H2), 1.77 (m, 2H, 4’-H2), 1.90e2.02 (complex
signal, 4H, 2”-H2, 3”-H2), superimposed in part 1.99 (tt,
J z J’z5.0 Hz, 2H, 3-H2), 2.69 (m, 2H, 1”-H2), 2.76 (t, J ¼ 6.0 Hz, 2H,
4-H2), 2.98 (m, 2H, 4”-H2), 3.49 (td, J ¼ 7.0 Hz, J’ ¼ 6.5 Hz, 2H, 1’-H2),
3.72 (t, J ¼ 5.0 Hz, 2H, 2-H2), 3.98 (t, J ¼ 7.0 Hz, 2H, 5’-H2), 4.84 (s,
NH, þNH), 7.50 (br d, J z 9.0 Hz, 1H, 7”-H), 7.66e7.72 [complex
signal, 4H, 2(6)-H and 3(5)-H 4-chlorophenyl], 7.73 (d, J ¼ 1.5 Hz,
1H, 5”-H), 7.86 (d, J ¼ 9.0 Hz, 1H, 7-H), 8.24 (d, J ¼ 9.0 Hz, 1H, 8-H),
8.39 (d, J ¼ 9.0 Hz, 1H, 8”-H), 8.66 (m, 1H, CONH), 9.02 (br s, 1H, 10-
(C, C9”), 167.9 (C, CONH); HRMS (ESI), calcd for
35
[C
C
H
Cl2N5O
þ
Hþ] 680.2917, found 680.2900; Anal.
40 43
40H43Cl2N5O$2HCl$2.5H2O (C, H, N).
4.2. Biological assays
4.2.1. Determination of AChE and BChE inhibitory activities
The inhibitory activities against human recombinant AChE
(SigmaeAldrich) and human serum BChE (SigmaeAldrich) were
evaluated spectrophotometrically by the method of Ellman et al.
H); 13C NMR (125.8 MHz, CD3OD)
d 20.0 (CH2, C3), 21.7 (CH2, C3”),
22.9 (CH2, C2”), 24.9 (CH2, C1”), 25.0 (CH2, C4), 25.6 (CH2, C3’), 29.4
(CH2, C4”), 29.9 (CH2) and 30.9 (CH2) (C2’, C4’), 40.8 (CH2, C1’), 43.0
(CH2, C2), 49.0 (CH2, C5’), 109.8 (C, C4a), 113.4 (C, C9a”), 115.5 (C,
C8a”), 116.1 (C, C10a), 119.0 (CH, C5”), 120.9 (CH, C7), 123.6 (CH,
C10), 126.8 (CH, C7”), 128.7 (CH, C8”), 130.4 (2CH) and 131.9 (2CH)
[C2(6) and C3(5) 4-chlorophenyl], 132.3 (C, C1 4-chlorophenyl),
132.7 (CH, C8), 133.3 (C, C9), 138.3 (C, C4 4-chlorophenyl), 140.0 (C,
C6”), 140.2 (C, C6a), 140.4 (C, C10a”), 151.5 (C, C5), 152.2 (C, C4a”),
155.7 (C, C10b), 157.8 (C, C9”), 167.9 (C, CONH), an extra peak at
40.1 ppm was observed; HRMS (ESI), calcd for [C
638.2448, found 638.2435; Anal. C37H37Cl2N5O$2HCl$3H2O (C, H,
N).
[15]. The reactions took place in a final volume of 300
phosphate-buffered solution pH 8.0, containing hAChE (0.02 u/mL)
or hBChE (0.02 u/mL) and 333 M 5,5'-dithiobis(2-nitrobenzoic)
mL of 0.1 M
m
acid (DTNB; SigmaeAldrich) solution used to produce the yellow
anion of 5-thio-2-nitrobenzoic acid. Inhibition curves were per-
formed in duplicates using at least 10 increasing concentrations of
inhibitors and preincubated for 20 min at 37 ꢂC before adding the
substrate [32]. One duplicate sample without inhibitor was always
present to yield 100% of AChE or BChE activities. Then substrates,
35
H
Cl2N5O þ Hþ]
37 37
acetylthiocholine iodide (450
iocholine iodide (300 M; SigmaeAldrich), were added and the
reaction was developed for 5 min at 37 ꢂC. The colour production
mM; SigmaeAldrich) or butyrylth-
m
4.1.5. N-{8-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-
5-(4-chlorophenyl)-1,2,3,4-tetrahydrobenzo[h][1,6]naphthyridine-
9-carboxamide 5d
It was prepared as described for 5a. Starting from crude tetra-
hydrobenzo[h][1,6]naphthyridinecarboxylic acid (480 mg) and
was measured at 414 nm using
spectrophotometer.
Data from concentrationeinhibition experiments of the in-
hibitors were calculated by non-linear regression analysis, using
the GraphPad Prism program package (GraphPad Software; San
a labsystems Multiskan

O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
115
Diego, USA), which gave estimates of the IC50 (concentration of
drug producing 50% of enzyme activity inhibition). Results are
expressed as mean S.E.M. of at least 4 experiments performed in
duplicate.
following the method described by Di et al. [28]. The in vitro
permeability (Pe) of fourteen commercial drugs through lipid
extract of porcine brain membrane together with the test com-
pounds was determined. Commercial drugs and the synthesized
compounds were tested using a mixture PBS:EtOH 70:30. Assay
validation was made by comparing the experimental permeability
of the different compounds with the reported bibliography values
of the commercial drugs, which showed a good correlation: Pe
(exp) ¼ 1.5010 Pe (lit) ꢀ 0.8618 (R2 ¼ 0.9199). From this equation
and taking into account the limits established by Di et al. for BBB
permeation, we established the ranges of permeability as com-
pounds of high BBB permeation (CNSþ): Pe (10ꢀ6 cm sꢀ1) > 5.1;
compounds of low BBB permeation (CNSꢀ): Pe (10ꢀ6 cm sꢀ1) < 2.1,
and compounds of uncertain BBB permeation (CNS ): 5.1 > Pe
(10ꢀ6 cm sꢀ1) > 2.1.
4.2.2. Determination of Ab42 and tau aggregation inhibitory
activities in intact E. coli cells
Cloning and overexpression of Ab42 peptide: E. coli competent
cells BL21 (DE3) were transformed with the pET28a vector (Nova-
gen, Inc., Madison, WI, USA) carrying the DNA sequence of A 42.
b
Because of the addition of the initiation codon ATG in front of both
genes, the overexpressed peptide contains an additional methio-
nine residue at its N terminus. For overnight culture preparation,
10 mL of lysogeny broth (LB) medium containing 50 m
g mLꢀ1 of
kanamycin were inoculated with a colony of BL21 (DE3) bearing the
plasmid to be expressed at 37 ꢂC. After overnight growth, the
OD600 was usually 2e2.5. For expression of A
overnight culture were transferred into eppendorf tubes of 1.5 mL
containing 960 L of LB medium with 50
g mLꢀ1 of kanamycin,
1 mM of isopropyl 1-thio- -D-galactopyranoside (IPTG), 10 L of a
10 M solution of each hybrid 5 or reference compound in DMSO,
and 10 L of a 25 M solution of TheS in water. The samples were
b
42 peptide, 20
m
L of
4.3. Molecular modelling
m
m
4.3.1. Setup of the system
b
m
Molecular modelling was performed using the X-ray crystallo-
graphic structure of hAChE in complex with huprine W (PDB ID:
4BDT [15]). The structure was refined by removal of N-acetyl-D-
glucosamine and sulphate anions and addition of missing hydrogen
atoms. The enzyme was modelled in its physiological active form
with neutral His447 and deprotonated Glu334, which together
with Ser203 form the catalytic triad. The ionization state for the rest
of ionizable residues was assessed from PROPKA3 [33] calculations.
Accordingly, the standard ionization state at neutral pH was
considered but for residues Glu285, Glu450 and Glu452, which
were protonated. Finally, three disulfide bridges were defined be-
tween Cys residues 257e272, 529e409, and 69e96, respectively.
m
m
m
grown for 24 h at 37 ꢂC and 1400 rpm using a Thermomixer
(Eppendorf, Hamburg, Germany). In the negative control (without
drug) the same amount of DMSO was added in the sample.
Cloning and overexpression of tau protein: E. coli BL21 (DE3)
competent cells were transformed with pTARA containing the RNA-
polymerase gen of T7 phage (T7RP) under the control of the pro-
moter pBAD. E. coli BL21 (DE3) with pTARA competent cells were
transformed with pRKT42 vector encoding four repeats of tau
protein in two inserts. For overnight culture preparation, 10 mL of
M9 medium containing 0.5% of glucose, 50 m
g mLꢀ1 of ampicillin
and 12.5
m
g mLꢀ1 of chloramphenicol were inoculated with a col-
4.3.2. Molecular dynamics simulations
ony of BL21 (DE3) bearing the plasmids to be expressed at 37 ꢂC.
The binding mode of hybrid 5a to hAChE was explored by means
of MD simulations. Starting from that proposed initial pose, a
100 ns MD simulation was performed using the PMEMD module of
AMBER12 [34] software package, the parm99SB [35] force field for
the protein and the GAFF-derived parameters [36] for the ligand,
whose geometrical parameters were optimized at the B3LYP/6-
31G(d) level [37] and its charge distribution was described by us-
ing electrostatic potential charges with the RESP procedure [38].
Naþ cations were added to neutralize the negative charge of the
system with the XLEAP module of AMBER12. The system was
immersed in an octahedral box of TIP3P [39] water molecules,
preserving the crystallographic waters inside the binding cavity.
The final system contained around 52,000 atoms.
The geometry of the system was minimized in four steps. First,
water molecules and counterions were refined through 7000 steps
of conjugate gradient and 3000 steps of steepest descent algorithm.
Then, the position of hydrogen atoms was optimized using 4500
steps of conjugate gradient and 500 steps of steepest descent al-
gorithm. At the third stage, hydrogen atoms, water molecules and
counterions were further optimized using 11500 steps of conjugate
gradient and 2500 steps of steepest descent algorithm. Thermali-
zation of the system was performed in five steps of 25 ps, increasing
the temperature from 50 to 298 K. Concomitantly, the residues that
define the binding site were restrained during thermalization using
After overnight growth, the OD600 was usually 2e2.5. For
expression of tau protein, 20
ferred into eppendorf tubes of 1.5 mL containing 970
medium containing 0.25% of arabinose, 0.5% of glucose, 50
of ampicillin and 12.5 L of a 10
g mLꢀ1 of chloramphenicol, 10
solution of each hybrid 5 or reference compound in DMSO, and
m
L of overnight culture were trans-
L of M9
g mLꢀ1
m
m
m
m
mM
10 mL of a 25 mM solution of TheS in water. The samples were grown
for 24 h at 37 ꢂC and 1400 rpm using a Thermomixer (Eppendorf,
Hamburg, Germany). In the negative control (without drug) the
same amount of DMSO was added in the sample.
TheS steady-state fluorescence: TheS (T1892) and other chemi-
cal reagents were purchased from Sigma (St. Louis, MO). TheS stock
solution (2.5 mM) was prepared in double-distilled water purified
through a Milli-Q system (Millipore, USA). Fluorescent spectral
scans of TheS were analyzed using an Aminco Bowman Series 2
luminescence spectrophotometer (Aminco-Bowman AB2, SLM
Aminco, Rochester, NY, USA). Excitation and emission slit widths of
4 nm were used. Finally, the fluorescence emission at 455 nm, when
exciting at 375 nm, was recorded. In order to normalize the TheS
fluorescence as a function of the bacterial concentration, OD600 was
obtained using a Shimadzu UV-2401 PC UVeVis spectrophotometer
(Shimadzu, Japan). The final fluorescence data were obtained
considering as 100% the TheS fluorescence of the bacterial cells
expressing the peptide or protein in the absence of drug and 0% the
TheS fluorescence of the bacterial cells non-expressing the peptide
or protein. Final data are the average of ten independent
experiments.
a
variable restraining force. Thus,
a
force constant of
25 kcal mole1 ꢀ2 was used in the first stage of the thermalization
Å
and was subsequently decreased by increments of 5 kcal mole1 Åꢀ2
in the next stages. Then, an additional step of 250 ps was performed
in order to equilibrate the system density at constant pressure
(1 bar) and temperature (298 K). Finally, a 100 ns trajectory was run
using a time step of 2 fs. SHAKE was used for those bonds con-
taining hydrogen atoms in conjunction with periodic boundary
4.2.3. PAMPA-BBB assay
To evaluate the brain penetration of hybrids 5, a parallel artificial
membrane permeation assay for bloodebrain barrier was used,

116
O. Di Pietro et al. / European Journal of Medicinal Chemistry 84 (2014) 107e117
(c) G.V. De Ferrari, M.A. Canales, I. Shin, L.M. Weiner, I. Silman, N.C. Inestrosa,
A structural motif of acetylcholinesterase that promotes amyloid beta-peptide
fibril formation, Biochemistry 40 (2001) 10447e10457.
conditions at constant volume and temperature, particle mesh
Ewald for the treatment of long electrostatic interactions, and a
cutoff of 10 Å for nonbonded interactions. Moreover, in the initial
20 ns of the simulation the distance between the protonated ni-
trogen in the 6-chlorotacrine moiety of the inhibitor and the
carbonyl oxygen of His447 was constrained to avoid artefactual
rearrangements in the CAS of the enzyme.
The structural analysis was performed using in-house software
and standard codes of AMBER12. The solvent interaction energies
(SIE) technique developed by Purisima and co-workers [19] was
used to estimate the interaction free energies for the AChE inhibi-
tor. Calculations were performed for a set of 200 snapshots taken
along the last 40 ns of the MD trajectory.
[8] J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman,
Atomic structure of acetylcholinesterase from Torpedo californica e a proto-
typic acetylcholine-binding protein, Science 253 (1991) 872e879.
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V. Andrisano, M.P. Arce, M.I. Rodríguez-Franco, O. Huertas, T. Dafni, F.J. Luque,
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Acknowledgements
ꢀ
This work was supported by Ministerio de Ciencia e Innovacion
(MICINN) (CTQ2011-22433, CTQ2012-30930, SAF2011-27642,
SAF2009-10553, start-up grant of the Ramon y Cajal program for
R.S.), Generalitat de Catalunya (GC) (2009SGR1396, 2009SGR1024,
2009SGR249), and Xarxa de Recerca en Química Teorica i Compu-
tacional (XRQTC). Fellowships from GC to O.D.P. and F.J.P.A., a
contract from the Ramon y Cajal program of MICINN to R.S., a
contract from the Juan de la Cierva program of Ministerio de
Economía y Competitividad to A.E., and the ICREA Academia sup-
port to F.J.L. are gratefully acknowledged. The Center for Scientific
and Academic Services of Catalonia (CESCA) is acknowledged for
providing access to computational facilities. We are grateful to Dr.
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Marc Reves and Dr. F. Spyrakis for their technical assistance in the
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dx.doi.org/10.1016/j.ejmech.2014.07.021.
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X is a novel high-affinity inhibitor of
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