Pyrano[3,2-c]quinoline - 6-chlorotacrine hybrids as a novel family of acetylcholinesterase-and β-amyloid-directed anti-Alzheimer compounds

Article abstract of DOI:10.1021/jm900859q

Two isomeric series of dual binding site acetylcholinesterase (AChE) inhibitors have been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase, AChE-induced and self-induced β-amyloid (Aβ) aggregation, and β-secretase

Full text of DOI:10.1021/jm900859q

J. Med. Chem. 2009, 52, 5365–5379 5365
DOI: 10.1021/jm900859q
Pyrano[3,2-c]quinoline-6-Chlorotacrine Hybrids as a Novel Family of Acetylcholinesterase-
and β-Amyloid-Directed Anti-Alzheimer Compounds
†
†
†
†
‡
Pelayo Camps, Xavier Formosa, Carles Galdeano, Diego Munoz-Torrero,*,† Lorena Ramı
rez, Elena Gomez,
ꢀ
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‡
‡,^
§
§
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#
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Nicolas Isambert, Rodolfo Lavilla, Albert Badia, M. Victoria Clos, Manuela Bartolini, Francesca Mancini,
´
Vincenza Andrisano,# Mariana P. Arce,¥ M. Isabel Rodrı
¥
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guez-Franco, Oscar Huertas, Thomai Dafni, and F. Javier Luque
†
´
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Laboratori de Quımica Farmaceutica (Unitat Associada al CSIC), Facultat de Farmacia, and Institut de Biomedicina (IBUB), Universitat de
ꢁ
Barcelona, Av. Diagonal 643, E-08028, Barcelona, Spain, ‡Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10-12,
E-08028, Barcelona, Spain, ^Laboratori de Quımica Organica, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028,
´
Barcelona, Spain, Departament de Farmacologia, Terapeutica i Toxicologia, Institut de Neurociencies, Universitat Autonoma de Barcelona,
ꢁ
ꢁ
§
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E-08193, Bellaterra, Barcelona, Spain, #Department of Pharmaceutical Sciences, Alma Mater Studiorum, Bologna University, Via Belmeloro 6,
¥
´
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´
I-40126, Bologna, Italy, Instituto de Quımica Medica (CSIC), Juan de la Cierva, 3, E-28006, Madrid, Spain, and Departament de Fisicoquımica,
ꢁ
Facultat de Farmacia, and Institut de Biomedicina (IBUB), Universitat de Barcelona, Av. Diagonal 643, E-08028, Barcelona, Spain
Received March 16, 2009
Two isomeric series of dual binding site acetylcholinesterase (AChE) inhibitors have been designed,
synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase, AChE-induced and self-
induced β-amyloid (Aβ) aggregation, and β-secretase (BACE-1) and to cross blood-brain barrier. The
new hybrids consist of a unit of 6-chlorotacrine and a multicomponent reaction-derived pyrano[3,2-c]-
quinoline scaffold as the active-site and peripheral-site interacting moieties, respectively, connected
through an oligomethylene linker containing an amido group at variable position. Indeed, molecular
modeling and kinetic studies have confirmed the dual site binding of these compounds. The new hybrids,
and particularly 27, retain the potent and selective human AChE inhibitory activity of the parent
6-chlorotacrine while exhibiting a significant in vitro inhibitory activity toward the AChE-induced and
self-induced Aβ aggregation and toward BACE-1, as well as ability to enter the central nervous system,
which makes them promising anti-Alzheimer lead compounds.
Introduction
emerged as promising disease-modifying anti-Alzheimer drug
candidates.6 Of particular interest are those AChEIs able to
simultaneously bind to both peripheral and catalytic sites,
which are separated by about 14 A, as they are located at the
mouth and at the bottom of the gorge leading to the active
site.7 Apart from the Aβ antiaggregating effects arising from
blockade of the peripheral site, dual binding site AChEIs are
usually endowed with a potent AChE inhibitory activity
because of the increased number of drug-target interactions,
thus overcoming the low activity of selective peripheral site
AChEIs.8-18 Indeed, in vitro inhibitory activities of AChE
and AChE-induced Aβ aggregation have been reported for
different families of dual binding site AChEIs,19-34 which in
cases such as memoquin26,27 and NP-6135 have been shown to
reduce brain amyloid burden and increase cognition in animal
models of AD. Some dual binding site AChEIs such as
memoquin26,27 or bis(7)-tacrine9,13,36,37 are undergoing pre-
clinical evaluation, while NP-61 entered phase I clinical trials
for AD in the U.K. in April 2007.38 Prompted by these results
and the tremendous potential of dual binding site AChEIs to
impact both the course of AD and its symptomatology, the
design and synthesis of novel families of dual binding site
AChEIs have been actively pursued in the past years.39-52
The design of dual binding site AChEIs is carried out by
linking through a tether of suitable length an active-site
interacting unit, usually derived from a known active site
AChEI, with a peripheral-site interacting unit suited to inter-
act with Trp286 (human AChE (hAChE) numbering), the
In the past decade, the design of novel classes of inhibitors
of the enzyme acetylcholinesterase (AChEa) as therapeutic
interventions for Alzheimer’s disease (AD) has been mostly
driven by the pivotal finding that AChE can bind the
β-amyloid peptide (Aβ), thereby promoting Aβ aggregation
as an early event in the neurodegenerative cascade of AD.1,2
The Aβ proaggregating effect of AChE results in cognitive
impairment in doubly transgenic mice expressing human
amyloidprecursorprotein(APP) andhuman AChE.3,4 Block-
ade of the peripheral site of AChE, the Aβ recognition zone
within the enzyme,5 was therefore expected to affect the
AChE-induced Aβ aggregation and could be a potential
strategy to modulate the progression of AD.
On the basis of these premises, novel classes of AChE
inhibitors (AChEIs) targeting the peripheral site have
*To whom correspondence should be addressed. Phone: þ34
þ 934024533. Fax: þ34 þ 934035941. E-mail: dmunoztorrero@ub.edu.
a Abbreviations: Aβ, β-amyloid peptide; AChE, acetylcholinesterase;
AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease; APP,
amyloid precursor protein; bAChE, bovine acetylcholinesterase;
BACE-1, β-secretase; BBB, blood-brain barrier; BChE, butyrylcholi-
nesterase; CNS, central nervous system; DDQ, 2,3-dichloro-5,6-dicya-
no-1,4-benzoquinone; DTNB, 5,50-dithiobis(2-nitrobenzoic) acid;
hAChE, human acetylcholinesterase; hBChE, human butyrylcholines-
terase; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; PAMPA, parallel ar-
tificial membrane permeation assay; PBS, phosphate buffered saline;
PDB, Protein Data Bank; PTFE, polytetrafluoroethylene; TcAChE,
Torpedo californica acetylcholinesterase.
r
2009 American Chemical Society
Published on Web 08/07/2009
pubs.acs.org/jmc

5366 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Camps et al.
characteristic residue of the peripheral site. With a few excep-
tions in which a peripheral-site interacting unit containing an
aliphatic amine, protonated at physiological pH, or a qua-
ternary ammonium group establishes cation-π interactions
with Trp286, in most cases the peripheral-site interacting unit
contains aromatic moieties able to establish π-π stacking
interactions with Trp286, in some cases reinforced by con-
comitant cation-π interactions due to the presence of proto-
natable or quaternary nitrogen atoms in the aromatic system.
The prototype of peripheral site AChEI is propidium
(1, Chart 1), which binds the AChE peripheral site in two
orientations related by a flip of 180ꢀ around the phenanthri-
dinium pseudosymmetry axis.53,54 The driving force for the
binding of 1 to the peripheral site is the π-π stacking,
reinforced by cation-π interactions, between the phenanthri-
dinium moiety and Trp286, which is supplemented by a
hydrogen bond between one of the aromatic amino groups
and His287.
Chart 1. Structures of Propidium, Scaffold 2, and 6-Chlorota-
crine
Scheme 1. Synthesis of Tricyclic Esters 11 and 12 and Car-
boxylic Acids 13 and 14
Some of us recently reported the synthesis of the multi-
component reaction-derived pyrano[3,2-c]quinoline scaffold
2 (Chart 1).55 We thought that a 5-phenyl-substituted deriva-
tive thereof would resemble the 6-phenylphenanthridinium
moiety of propidium and could serve as the peripheral-site
interacting unit of a novel family of dual binding site AChEIs.
Because the nitrogen atom of this tricyclic moiety is not
expected to be protonated at physiological pH (Table S1,
Supporting Information), this aromatic system should estab-
lish π-π stacking with Trp286. Noteworthy, the neutral
character of this moiety could result in a better penetration
into the central nervous system (CNS).
Herein, we describe the synthesis, pharmacological eva-
luation, and molecular modeling of a novel family of
potent dual binding site AChEIs that combine a 5-phen-
ylpyrano[3,2-c]quinoline moiety with 6-chlorotacrine, 3
(Chart 1), a potent AChEI already used in other dual binding
site AChEIs,22,23,31,32,39,44,52,56,57 through an amido-contain-
ing oligomethylene linker. The pharmacological evaluation of
these novel compounds includes AChE and butyrylcholines-
terase (BChE) inhibition, as well as inhibition of the AChE-
and self-induced Aβ aggregation and inhibition of β-secretase
(BACE-1), which altogether comprises an interesting set of
effects shared by some dual binding site AChEIs. To prove the
starting hypothesis on a good blood-brain barrier (BBB)
permeability, the brain penetration of the novel hybrids has
been assessed using an artificial membrane assay.
Recently, Martınez and co-workers developed a novel
´
series of indole-tacrine hybrids as dual binding site AChEIs,
which contained an amido group within the linker either
directly bound to the indole system (i.e., the peripheral-site
interacting unit) or separated by one to three atoms
(methylene groups in most cases), and reported important
differences in the AChE inhibitory activity upon shift of
the amido group within the tether chain while keeping the
same total length of the linker.23 Specifically, separation of the
amide from the indole ring by two methylene groups increased
up to 2300-fold the AChE inhibitory potency relative to the
indole directly bound counterpart. In view of these results, we
designed the tricyclic ester 12 (Scheme 1) as the precursor of
the parallel series of hybrids 23-27 (Scheme 2), containing
linkers of the same total length as 18-22 but with the amido
group shifted two positions within the tether. Regarding the
length of the linker, oligomethylene chains of 6-10 and 4-8
members for hybrids 18-22 and 23-27, respectively, were
considered suitable to provide the dual site binding.
Chemistry
The structures of the novel 5-phenylpyrano[3,2-c]-
quinoline-6-chlorotacrine hybrids 18-27 are shown in
Scheme 2. Alignment of the 5-phenylpyrano[3,2-c]quinoline
system with the phenanthridinium moiety of propidium in its
complex with mouse AChE53 (Figure S1, Supporting In-
formation) revealed that if the novel hybrids were to interact
with AChE by placing the tricyclic system in a way similar to
that of the tricyclic system of propidium, position 9 of the
pyrano[3,2-c]quinoline system would be suitable for attach-
mentofthelinker, whichfrom thispointcould snakedownthe
active site gorge. An ester group at position 9 was chosen as a
suitable functionalization to allow attachment of the linker by
reaction with an appropriate aminoalkyltacrine. Thus, tricyc-
lic ester 11 (Scheme 1) was designed as the precursor of the
peripheral-site interacting unit of the novel hybrids 18-22
(Scheme 2).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5367
Scheme 2. Synthesis of Hybrids 18-27
1 equiv of aminoalkyltacrines 17c-g and 17a-e, respectively,
afforded hybrids 18-22 and 23-27 in low to moderate yields
(19-37% and 25-48% yield, respectively). The novel hybrids
18-27 were fully characterized as dihydrochlorides through
their spectroscopic data, HRMS, and elemental analyses. Not
unexpectedly, in a study to assess the potential chemical
instability at physiological pH of the new hybrids due to the
presence of a hydrolyzable amido group, hybrid 23 did not
undergo any noticeable decomposition in 1:1 acetonitrile/
Sorensen phosphate buffer at pH 7.4 up to 4 days at 37 ꢀC
(see Supporting Information).
Pharmacology and Molecular Modeling
Cholinesterase Inhibition. AChE Inhibition. The AChE
inhibitory activity of hybrids 18-27 was assayed by the
method of Ellman et al.64 on AChE from bovine (bAChE)
and human (hAChE) erythrocytes (Table 1). The hybrids of
the first series (18-22) are potent inhibitors of both bAChE
and hAChE, with IC50 values in the low nanomolar range in
most cases. The most potent hAChE inhibitor was 20, and
shortening or lengthening of the linker led to a 3- to 7-fold
decrease of inhibitory activity. The hybrids of the second
series (23-27) are also potent inhibitors of both enzymes. In
contrast with the first series, hybrids 23-27 turned out to be
2- to 3-fold more potent toward the human enzyme. Also,
unlike the first series, no significant dependency on the length
of the linker was found for the hAChE inhibitory activity of
23-27. Hybrids 24 and 25 were the most potent hAChE
inhibitors of the second series, they being roughly equipotent
to compound 20, in which the total length of the linker is
equivalent to that of 25. Although dual site binding to AChE
is expected to increase the inhibitory potency relative to the
monomeric parent compounds from which they were de-
signed, this assumption is not always fulfilled.65,66 Indeed,
the hAChE inhibitory activity of hybrids 20, 24, and 25 is
comparable with that of the parent 6-chlorotacrine and is
clearly higher than the activity measured for propidium and
the tricyclic ester precursors 11 and 12.
The synthesis of hybrids 18-27 was envisaged through the
coupling of tricyclic esters 11 and 12 with readily available
aminoalkyltacrines 17.23 A straightforward access to 11 and
12 was carried out through a Povarov multicomponent reac-
tion.58 Thus, reaction of 3,4-dihydro-2H-pyran, 4, with ethyl
p-aminobenzoate, 5, and p-chlorobenzaldehyde, 7, under
Y(OTf)3 catalysis in CH3CN afforded in 76% yield a 1.3:1
diastereomeric mixture of pyranotetrahydroquinolines 9,
whose 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
oxidation55 yielded the desired tricyclic ester 11 in 75% yield.
The Povarov reaction of methyl 3-(4-aminophenyl)propano-
ate 659 with 4 and benzaldehyde 8 under Sc(OTf)3 catalysis,
followed by DDQ oxidation of the diastereomeric mixture of
pyranotetrahydroquinolines 10 and catalytic hydrogenation
of the unseparable mixture of the desired ester 12 and its
cinnamate derivative, formed by the competitive oxidation of
the ethylene bridge, afforded in 82% overall yield the tricyclic
ester 12 (Scheme 1). This latter compound lacked the chlorine
atom at the phenyl substituent, present in 11. However, this
chlorine atom, easily removable in the catalytic hydrogena-
tion conditions, was not expected to play a major role in the
interaction of this moiety with the AChE peripheral site.
Aminoalkyltacrines 17a-g were synthesized in 35-78%
yield following a procedure23,60,61 that involves amination of
dichloroacridine 1562 with commercially available R,ω-dia-
mines 16a-g in refluxing 1-pentanol (Scheme 2). In this
reaction, significant amounts of dimers of 6-chlorotacrine,
28a-g,56,62,63 were formed despite the fact that an excess of
diamines (4 equiv) was used.
Molecular Modeling Studies. To gain insight into the
molecular determinants that modulate the hAChE inhibi-
tory activity of the novel hybrids, the binding mode of
compounds 20, 25, and 27 was investigated by means of
docking computations.
The conformation of the active site gorge appears to be
highly conserved in different X-ray crystallographic struc-
tures. In particular, the observed structural changes at the
catalytic binding site are small except for those of Tyr337 (in
hAChE; Phe330 in Torpedo californica AChE, TcAChE).67,68
Accordingly, the binding mode of the 6-chlorotacrine unit
of these hybrids can be inferred from the X-ray crystal-
lographic structures of TcAChE complexes with tacrine7
and its structurally related analogue huprine X,69 where the
9-aminotetrahydroacridine unit is stacked against Trp86
and Tyr337 (Trp84 and Phe330 in TcAChE), the protonated
pyridine nitrogen atom is hydrogen-bonded to His447
(His440 in TcAChE), and the chlorine atom fits a hydro-
phobic pocket formed by Trp439, Met443, and Pro446
(Trp432, Met436, and Ile439 in TcAChE). In fact, a common
pose is found for the tacrine moiety in the X-ray structures of
a variety of dual binding site inhibitors, including bis(5)-
tacrine and bis(7)-tacrine,70 tacrine(8)-4-aminoquinoline,70
NF595,71 tacrine(10)-hupyridone,72 and TZ2PA6.73 The
structural features of the binding of the pyrano[3,2-c]-
quinoline moiety of the hybrids at the peripheral site are
Hydrolysis of esters 11 and 12 (Scheme 1), followed by
treatment of the corresponding carboxylic acids 13 and 14
with 1 equiv of ethyl chloroformate and 2.2 equiv of Et3N in
CH2Cl2, and reaction of the resulting mixed anhydrides with

5368 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Camps et al.
Table 1. AChE and BChE Inhibitory Activities of the Hydrochlorides of 6-Chlorotacrine and Tricyclic Esters 11 and 12, Propidium Iodide, and the
Dihydrochlorides of the Pyrano[3,2-c]quinoline-6-Chlorotacrine Hybridsa
IC50 (nM)
compd
18 2HCl
19 2HCl
bAChE
hAChE
hBChE
AChE selectivityb
20.4 ( 0.9
10.3 ( 0.4
10.4 ( 0.7
24.0 ( 1.6
93.7 ( 3.2
48.1 ( 2.0
23.9 ( 1.3
30.8 ( 1.9
29.9 ( 0.6
34.1 ( 1.0
>10000
19.2 ( 1.5
18.3 ( 2.6
7.03 ( 0.3
24.9 ( 1.5
50.0 ( 3.0
16.6 ( 1.0
9.64 ( 1.4
11.1 ( 0.1
14.4 ( 1.4
14.0 ( 1.2
>10000
1074 ( 178
1931 ( 47
331 ( 42
1391 ( 31
1622 ( 117
586 ( 16
290 ( 9.8
218 ( 3.2
234 ( 7.8
1076 ( 78
>10000
56
3
3
106
47
20 2HCl
3
3
3
3
3
3
3
3
21 2HCl
56
32
22 2HCl
23 2HCl
24 2HCl
35
30
25 2HCl
20
16
26 2HCl
27 2HCl
77
11 HCl
ndc
ndc
0.4
110
3
3
12 HCl
propidium iodide
>10000
6289 ( 377
5.73 ( 0.4
ndc
ndc
13200 ( 400d
916 ( 19
32300 ( 2200d
8.32 ( 0.7
6-chlorotacrine HCl
3
a Values are expressed as the mean ( standard error of the mean of at least four experiments. IC50 inhibitory concentration (nM) of AChE (from
bovine or human erythrocytes) or BChE (from human serum) activity. b IC50(hBChE)/IC50(hAChE). c Not determined. d Data from ref 21.
angles close to -120ꢀ (χ1) and þ50ꢀ (χ2) are found in com-
plexes with bis(7)-tacrine,70 tacrine(8)-4-aminoquinoline,70
and NF595.71 At this point, it is worth stressing how the
different lengths of the tether in bis(5)-tacrine and bis(7)-
tacrine lead to a distinct arrangement of the indole ring of
Trp286.70 Finally, an alternative orientation defined by dihe-
dral angles close to -160ꢀ (χ1) and -120ꢀ (χ2) is found in the
complex with syn-TZ2PA6.73 Overall, this analysis stresses the
conformational plasticity of Trp286 and its capability to adopt
different orientations depending on the chemical features of
the ligand.
On the basis of the preceding discussion, the binding
modes of 20, 25, and 27 were investigated using three models
of hAChE, in which Trp286 was imposed to adopt each one
of the three above-mentioned conformational orientations
(denoted as A, B, and C; Figure 1). Moreover, suitable
restraints were introduced to fix the orientation of the
6-chlorotacrine moiety, thus enhancing the conformational
sampling of the ligand at the peripheral binding site and
along the gorge. Noteworthy, this restrained docking proto-
col was able to predict the X-ray binding mode of bis(5)-
tacrine, bis(7)-tacrine, tacrine(8)-4-aminoquinoline, (R)- and
(S)-tacrine(10)-hupyridone, and syn- and anti-TZ2PA6
within the first 10 poses and with root-mean square devia-
tions less than 1.8 A (Table S3, Supporting Information).
Following previous studies in the literature,79-81 the re-
lative stabilities of the first 50 poses obtained in the docking
of every ligand on each one of the targets were reranked using
MM-PBSA calculations (Table S4, Supporting In-
formation). In all cases the most favorable binding is found
for target C, as this binding mode is favored by 4.3-7.6 and
2.4-9.9 kcal/mol when dielectric permittivities of 2 and 4 are
considered for the interior of the ligand-hAChE complex,
respectively, relative to the second most stable complex.
Moreover, the results also show that there are small differ-
ences in the affinities estimated for the binding of hybrids 20,
25, and 27 to target C. Keeping in mind the range of
uncertainty expected for MM-PBSA calculations,80,82 this
finding is in agreement with the similar inhibitory potencies
measured for these compounds (IC50 values ranging from 7
to 14 nM, Table 1).
Figure 1. Representation of the three orientations adopted by
Trp286 in targets A (blue), B (red), and C (orange) of hAChE
(χ1 and χ2 amount to -60ꢀ and -80ꢀ for A, to -120ꢀ and þ50ꢀ for B,
and to -160ꢀ and -120ꢀ for C; see text). The backbone of hAChE is
shown in ribbon (light-blue). Trp86 at the catalytic site is colored by
atom, and the gorge leading from the peripheral site to the catalytic
pocket is shown in gray.
more delicate because of the conformational flexibility of
Trp286 (Trp279 in TcAChE), as noted in the X-ray struc-
tures available for several peripheral site ligands,53 including
propidium, and dual binding site inhibitors70-73 as well as
from molecular dynamics simulations.23,31,74 In fact, three
main arrangements of the indole ring of Trp286 can be
identified upon inspection of the X-ray structures (Table
S2, Supporting Information). The first orientation is char-
acterized by dihedral angles χ1 (N-CR-Cβ-Cγ) and χ2 (CR-
Cβ-Cγ-Cδ2) close to -60ꢀand -80ꢀ, respectively, as found
in the apo form of the enzyme,53,75 and in complexes with
catalytic (tacrine,7 huprine X,69 and (-)-huperzine A76) and
peripheral (propidium, decidium, and gallamine)53 binding
site inhibitors. Moreover, this arrangement is found in com-
plexes with dual binding site inhibitors (decamethonium,7
donepezil,77 bis(5)-tacrine,70 tacrine(10)-hupyridone,72 and
anti-TZ2PA673) and in the complex with fasciculin.78 Dihedral
Inspection of the best poses found for hybrid 20 bound
to target C shows that the 6-chlorotacrine moiety
roughly matches the tacrine unit of syn-TZ2PA6. The slight

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5369
Figure 2. (Top) Representation of the most favorable binding mode of hybrid 20 (colored by atom) determined from MM-PBSA
computations performed with internal permittivities of 2 (left) and 4 (right). Relevant residues at the catalytic (Trp86 and Tyr337)
and peripheral (Trp286 and Tyr72) binding sites are colored by atom. (Bottom) Representation of the poses predicted for hybrid 20
from MM-PBSA computations and the orientation of syn-TZ2PA6 (blue) in the X-ray crystallographic structure of mouse AChE (PDB entry
1Q83).
displacement observed for the tetrahydroacridine systems
can be ascribed to the positioning of the chlorine atom in 20
in the hydrophobic pocket formed by Trp439, Met443, and
Pro446 (see above) and particularly to the active role played
by the triazole ring in the tether of syn-TZ2PA6 in mediating
interactions with specific residues along the gorge (see
Figure 2),73 which also explain the different arrangement
of the linker observed for syn-TZ2PA6 and for the two poses
of 20. At the peripheral binding site the pyrano[3,2-c]-
quinoline moiety adopts two main orientations (Figure 2),
which can be roughly interconverted by a 180ꢀ rotation
through the C5-C9 axis. At first sight, this finding might
be surprising, keeping in mind the well-defined arrangement
observed for the phenanthridinium moiety of syn-TZ2PA6
in the X-ray structure,73 but this can be ascribed to addi-
tional interactions involving the buried phenanthridinium
amino group that are absent in the case of the pyrano[3,2-c]-
quinoline moiety. Accordingly, the stacking of the pyrano-
[3,2-c]quinoline moiety might involve distinct arrangements
provided that there exists a significant overlap with the
aromatic rings of Trp286 and Tyr72 and that steric clashes
with neighboring residues are avoided. Similar overall ar-
rangements are found for hybrids 25 and 27 (see Figures S2
and S3, Supporting Information).
binding modes shown in Figure 2 for compound 20 (and
Figures S2 and S3 for 25). Only the trajectories run for the
ligand with the pyran ring oriented toward the bulk solvent
yielded stable trajectories, as noted by inspection of the time
dependence of both the potential energy and the root-mean-
square deviation of selected atoms in the protein backbone,
the binding site, and the ligand in the ligand-receptor
complexes (Figure S4, Supporting Information). There is a
large resemblance between the snapshots collected at the end
of the trajectories and those used as starting structures
(Figure 3). The main difference lies in the orientation of
the tether, which adopts a more extended conformation at
the end of the trajectories. Nevertheless, the tacrine unit
remains stacked between the aromatic rings of Trp86 and
Tyr337, and the pyrano[3,2-c]quinoline unit retains the π-π
stacking with Trp286 and Tyr72. Finally, a well-defined
interaction pattern between the amido functionality and
residues lining the entrance of the gorge is not observed.
This finding is in contrast with the binding mode reported
from modeling studies for a series of structurally related
indole-tacrine hybrids containing an amido group within
the linker,23 where this group was proposed to participate in
hydrogen-bond interactions with several residues in the
gorge. These distinct features could presumably stem from
the different arrangement of the pyrano[3,2-c]quinoline and
indole units at the peripheral binding site.
To further explore the proposed binding mode, a series of
10 ns molecular dynamics simulations were run for com-
pounds 20 and 25, which were chosen as representative
members of the two series of dual binding site inhibitors.
The simulations were run for each of the two potential
Kinetic Analysis of AChE Inhibition. The mechanism of
AChE inhibition was investigated in vitro using compound
20, the most potent inhibitor of the two series. Graphical

5370 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Camps et al.
Figure 3. Representation of the structures (blue) collected at the end of the 10 ns molecular dynamics simulations of compounds 20 (left) and
25 (right) bound to AChE showing the stacking of the tacrine moiety with Trp86 and Tyr337 and of the phenylpyrano[3,2-c]quinoline unit with
Trp286 and Tyr72 (yellow). The starting structure used in molecular dynamics simulations is shown in green.
analysis of the overlaid reciprocal Lineweaver-Burk plots
(Figure 4) showed both increasing slopes (decreased Vmax
)
and increasing intercepts (higher Km) at increasing inhibitor
concentration. This pattern indicates mixed-type inhibition.
Therefore, in agreement with molecular modeling studies,
the pattern in Figure 4 shows that 20 is able to bind the
peripheral site as well as the active site of hAChE. Replots of
the slope versus concentration of 20 give an estimate of the
competitive inhibition constant, Ki, of 1.91 nM.
BChE Inhibition. Recent evidence has shown that inhibi-
tion of BChE might be valuable in the search for anti-
Alzheimer agents.83,84 Consequently, the inhibitory activity
on human serum BChE (hBChE) was also assayed by the
method of Ellman et al. (Table 1).64 6-Chlorotacrine inhibits
hAChE 110-fold more potently than hBChE.31 The presence
of the chlorine atom at the tacrine unit, which leads to an
increased AChE inhibitory activity relative to unsubstituted
tacrine,85-87 becomes detrimental for hBChE inhibition. A
steric hindrance due to the proximity of the chlorine atom to
the terminal methyl group of Met437 in the hBChE active
site seems to account for the detrimental influence of this
substituent on the hBChE inhibitory activity relative to
tacrine,23,65 which is 21-fold more potent toward hBChE
than 6-chlorotacrine.31 As expected, hybrids 18-27 are more
potent inhibitors of hAChE than hBChE (32- to 106-fold and
16- to 77-fold in the first and second series, respectively).
Hybrids 23-27 exhibit IC50 values in the nanomolar range,
they being 1.5- to 7-fold more potent than their counterparts
of the first series and up to 3- to 4-fold more potent than
6-chlorotacrine.
Inhibition of Aβ Aggregation and Formation. Inhibition of
AChE-Induced Aβ Aggregation. With the design of this novel
family of dual binding site AChEIs, an interference with the
AChE-induced Aβ aggregation was also pursued. Thus, the
new hybrids were tested for their ability to inhibit the AChE-
induced aggregation of Aβ1-40, by using a thioflavin T
fluorescence method.88 Also, the Aβ-antiaggregating effect
of one of the tricyclic ester precursors, 12, was determined,
while those of propidium88 and 6-chlorotacrine31 were al-
ready described. The new hybrids significantly inhibit, at
100 μM, the hAChE-induced Aβ aggregation, with per-
centages of inhibition ranging from 23% to 46% (Table 2),
while 6-chlorotacrine and 12 were the almost inactive. Most
of the new hybrids could be classified as weak inhibitors
of the AChE-induced Aβ aggregation, with expected
IC50 values in the high micromolar range, while the
Figure 4. Kinetic study on the mechanism of AChE inhibition by
20. Overlaid Lineweaver-Burk reciprocal plots of AChE initial
velocity at increasing substrate concentration (ATCh, 0.56-
0.11 mM) in the absence of inhibitor and in the presence of 20
(1.25-3.75 nM) are shown. Lines were derived from a weighted
least-squares analysis of the data points.
most potent described inhibitors display potencies in the
low micromolar range.21,23,26,27,30,33 Nevertheless, the in-
hibitory activity of the most potent hybrids, 26 and 27, is
in the same range as that of other known dual binding site
AChEIs.20,22,24,28,29,31,32
The Aβ antiaggregating effect of the novel hybrids seems
to depend on the position of the amido group within the
linker, the hybrids of the second series being more potent
than their counterparts of the first series. Moreover, the Aβ
antiaggregating effect seems to be independent of the length
of the linker within the first series, while a slightly increased
effect is observed upon lengthening of the linker in the
second series. Overall, the most potent AChE-induced Aβ
aggregation inhibitors within both series were hybrids 26 and
27. The higher Aβ antiaggregating activity of the novel
hybrids relative to 6-chlorotacrine and 12 seems to be
indicative of their dual binding site character. In turn, their
lower Aβ antiaggregating activity relative to propidium
could be ascribed to the different binding mode of the
pyrano[3,2-c]quinoline moiety of these hybrids at the AChE
peripheral site and/or to a less efficient interaction with
Trp286 due to the lack of cation-π interaction in our
hybrids, which are not protonated at the quinoline nitrogen
atom of this moiety at physiological pH.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5371
Table 2. Aβ Aggregation and BACE-1 Inhibitory Activities of the Hydrochlorides of 6-Chlorotacrine and Tricyclic Ester 12, Propidium Iodide,
and the Dihydrochlorides of the Pyrano[3,2-c]quinoline-6-Chlorotacrine Hybridsa
compd
AChE-induced Aβ1-40 aggregationb (%)
Aβ1-42 self-induced aggregationc (%)
BACE-1 activityd (%)
18 2HCl
19 2HCl
20 2HCl
27.2 ( 7.1
22.9 ( 3.2
28.6 ( 0.5
ndf
16.4 ( 2.6
28.6 ( 3.3
21.5 ( 1.8
ndf
nae
3
3
3
14.5 ( 5.7
18.5 ( 7.6
ndf
21 2HCl
3
3
3
3
3
3
3
22 2HCl
27.9 ( 5.2
34.4 ( 1.0
37.9 ( 3.4
38.9 ( 2.6
45.9 ( 2.8
45.7 ( 0.3
10.5 ( 4.7
82.0 ( 2.5g
8.5 ( 1.6h
20.5 ( 1.1
45.3 ( 0.9
12.2 ( 1.1
30.8 ( 8.6
49.1 ( 15.1
47.3 ( 8.8
nae
nae
23 2HCl
nae
24 2HCl
nae
25 2HCl
19.6 ( 5.1
34.4 ( 3.6
77.8 ( 6.4
nae
ndf
nae
26 2HCl
27 2HCl
12 HCl
3
propidium iodide
6-chlorotacrine HCl
89.8 ( 0.9
7.1 ( 1.2
3
a Values are expressed as mean ( standard error of the mean from two independent measurements, each performed in duplicate. b A 100 μM
concentration of the inhibitor was used. c A 50 μM concentration of the inhibitor was used ([Aβ]/[I] = 1/1). d A 2.5 μM concentration of the inhibitor was
used. e Not active. f Not determined. g Data from ref 88. h Data from ref 31.
Inhibition of Spontaneous Aβ Aggregation. A number of
dual binding site AChE inhibitors exhibit a significant
inhibitory activity on Aβ self-aggregation.23,26,27,29,32-34
The new hybrids significantly inhibit the self-induced Aβ
aggregation when tested at equimolar ratio with Aβ, with
percentages of inhibition ranging from 12% to 49%
(Table 2). In the same assay conditions, 6-chlorotacrine
and the tricyclic ester 12 turned out to be, respectively,
almost and completely inactive, while propidium was more
potent than the new hybrids. Hybrids of the second series
were generally more potent than their counterparts of the
first series, with the sole exception of 24 which was 2-fold less
potent than 19. A clear dependency of the spontaneous Aβ
antiaggregating activity on the length of the linker was not
observed. Compounds 23, 26, and 27 were the most potent
hybrids. It might be hypothesized that a two-methylene
linker between the amido group and the phenylpirano[3,2-
c]quinoline moiety facilitates the formation of hydrogen
bonds, with Aβ leading to an increased affinity. The expected
IC50 values for the most potent hybrids must be around
50 μM, while the strongest inhibitors of spontaneous Aβ
aggregation among known dual binding site AChEIs show
potencies in the low micromolar range.26,27,33,34 Thus, 23, 26,
and 27 can be considered moderate inhibitors of Aβ1-42 self-
aggregation.
Inhibition of BACE-1. BACE-1 has been largely investi-
gated as a therapeutic target for disease-modifying agents in
AD, since BACE-1 is involved in the proteolytic cleavage of
APP to Aβ and BACE-1 knockout mice did not show any
adverse phenotype. Although highly active in vitro peptide
inhibitors with poor pharmacokinetics are known, potent
brain permeable inhibitors have not been developed yet.
Therefore, there is an increasing need for small organic
BACE-1 inhibitors able to cross the BBB.89 The ability of
the novel hybrids to inhibit in vitro human recombinant
BACE-1 was also investigated. Some dual binding site
AChEIs such as donepezil,90 bis(7)-tacrine,91 lipocrine,92
memoquin,26 and AP224393 exhibit BACE-1 inhi-
bitory activity, which increases their potential as disease-
modifying anti-Alzheimer drug candidates. In particular,
since the new hybrids might structurally resemble to some
extent bis(7)-tacrine, which showed in vitro and in vivo
activity,91 their BACE-1 inhibitory activity was first
screened at a single concentration (2.5 μM) by a fluoro-
metric assay.94
Hybrid 27 was the most potent BACE-1 inhibitor, exhibit-
ing a 78% inhibition. Compounds 19, 20, 25, and 26 were less
active than 27 (15-34% inhibition), and the rest of the
hybrids, the tricyclic ester 12, and 6-chlorotacrine were
inactive (Table 2). In general the BACE-1 inhibitory activity
of the hybrids of the second series was higher than that of
their counterparts of the first series. For the second series, the
inhibitory activity increased along with the tether length. The
IC50 value of the most active hybrid (27) was 1.81 ( 0.48 μM,
it being more active than bis(7)-tacrine in vitro (IC50
=
7.5 μM).91 Thus, 27 emerged as a promising anti-Alzheimer
drug candidate endowed with a potent hAChE inhibitory
activity (nanomolar range) and a significant in vitro inhibi-
tory effect on both Aβ formation and AChE-induced and
self-induced Aβ aggregation.
In Vitro Blood-Brain Barrier Permeation Assay. Brain
penetration is a major issue for successful CNS drugs. In the
past years, several in silico/in vitro methods have been used
to predict the BBB permeation potential of test compounds.
Among them, the parallel artificial membrane permeation
assay (PAMPA-BBB) described by Di et al.95 predicts pas-
sive BBB permeation with high success, high throughput,
and reproducibility. To evaluate the brain penetration of the
pyrano[3,2-c]quinoline-6-chlorotacrine hybrids herein de-
scribed, we used the PAMPA-BBB assay, which was success-
fully applied by some of us to different compounds.39,52,96-98
The in vitro permeability (Pe) of selected hybrids (18, 19, and
23-27), the tricyclic ester 12, and 6-chlorotacrine through a
lipid extract of porcine brain was determined by using
phosphate buffered saline (PBS)/EtOH (80:20 or 70:30,
depending on the solubility of compounds). At each solvent
mixture, assay validation was made by comparing the ex-
perimental permeability with the reported values of 15
commercial drugs that gave a good lineal correlation: Pe(exp)
=1.48Pe(bibl) þ 1.91 (R2 = 0.95) for PBS/EtOH (80:20) and
Pe(exp)=1.99 Pe(bibl) þ 1.07 (R2 = 0.92) for PBS/EtOH
(70:30). From these equations and taking into account the
limits established by Di et al. for BBB permeation,95 we
established that compounds with permeability values over
7.8 ꢀ 10-6 cm s-1 (PBS/EtOH, 80:20) or 9.0 ꢀ 10-6 cm s-1
(PBS/EtOH, 70:30) should cross the BBB. All tested hybrids
showed permeability values over the above limits (Table 3;
see also Table S5, Supporting Information), pointing out
that they could cross the BBB and reach their pharmacolo-
gical targets located in the CNS.

5372 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Camps et al.
Table 3. Permeability Results from the PAMPA-BBB Assay for
Selected Pyrano[3,2-c]quinoline-6-Chlorotacrine Hybrids (Pe, 10-6
cm s-1) with Their Predictive Penetration into the CNS
Varian Gemini 300, Varian Mercury 400, and Varian Inova
500 spectrometers, respectively. The chemical shifts are reported
in ppm (δ scale) relative to internal tetramethylsilane, and
coupling 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 18, 27, and
6-chlorotacrine, as model compounds, which in turn were
compd
Pe (10-6 cm s-1 a
)
prediction
18 2HClb
14.9 ( 1.1
17.7 ( 0.2
15.1 ( 0.2
10.6 ( 0.4
13.2 ( 0.4
10.2 ( 0.3
10.1 ( 0.4
21.4 ( 0.6
19.8 ( 0.4
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
3
19 2HClb
3
3
3
3
23 2HClc
1
assigned on the basis of DEPT, COSY H/1H (standard pro-
24 2HClb
cedures), and COSY 1H/13C (gHSQC and gHMBC sequences)
experiments. IR spectra were run on Perkin-Elmer Spectrum
RX I or Thermo Nicolet Nexus spectrophotometers. Absorp-
tion values are expressed as wavenumbers (cm-1); only signifi-
cant absorption bands are given. Column chromatography was
performed on silica gel 60 AC.C (35-70 mesh, SDS, ref
2000027). Thin-layer chromatography was performed with
aluminum-backed sheets with silica gel 60 F254 (Merck, ref
1.05554), and spots were visualized with UV light and 1%
aqueous solution of KMnO4. The analytical samples of all of
the new hybrids that were subjected to pharmacological evalua-
tion possess a purity of g95%, as evidenced by results of their
elemental analyses.
(4aRS,5RS,10bRS)- and (4aRS,5SR,10bRS)-Ethyl 5-(4-Chl-
orophenyl)-3,4,4a,5,6,10b-hexahydro-2H-pyrano[3,2-c]quinoline-9-car-
boxylate (9). Y(OTf)3 (295 mg, 0.55 mmol, 0.2 equiv) was added to a
solution of p-chlorobenzaldehyde, 7 (385 mg, 2.74 mmol, 1 equiv),
and ethyl p-aminobenzoate, 5 (453 mg, 2.74 mmol, 1 equiv), in dry
CH3CN (20 mL). A solution of 3,4-dihydro-2H-pyran, 4 (250 μL,
231 mg, 2.74 mmol, 1 equiv), in dry CH3CN (3 mL) was then added,
and the reaction mixture was stirred at room temperature under
inert atmosphere for 12 h. Saturated aqueous NaHCO3 (20 mL) was
added, and the resulting mixture was extracted with AcOEt (3 ꢀ
10 mL). The combined organic extracts were dried with anhydrous
Na2SO4, filtered, and concentrated under reduced pressure to give a
residue (1.1 g), which was subjected to column chromatography [35-
70 μm silica gel (40 g), hexane/AcOEt mixtures]. On elution with
hexane/AcOEt, 20:80, the desired product 9 (722 mg, 71% yield) was
isolated as a 1.3:1 mixture of diastereoisomers as a waxy solid. A single
diastereoisomer was obtained as a waxy solid in pure form from
fractions isolated from the flash chromatography (51 mg, 5% yield)
and characterized as the all-cis isomer (4aRS,5RS,10bRS):IR(NaCl)
ν 3346 (N-H st), 1685(CdOst) cm-1;1H NMR (400 MHz, CDCl3)
δ 1.27-1.62 (m, 4H, 3-H2 and 4-H2), 1.37 (t, J=7.1 Hz, 3H, CH2-
CH3), 2.16 (m, 1H, 4a-H), 3.43 (dt, J=12.0, J0=1.9 Hz, 1H, 2-Hax),
3.63 (broad d, J≈12.0 Hz, 1H, 2-Heq),4.26(s, 1H,NH), 4.33(m,2H,
CH2-CH3),4.73(d,J=1.7 Hz, 1H, 10b-H), 5.28 (d, J= 5.4 Hz, 1H, 5-
H), 6.58 (d, J=8.4 Hz, 1H, 7-H), 7.30-7.40 (complex signal, 4H, 5-
ArH), 7.79 (d, J=8.4 Hz, 1H, 8-H), 8.11 (s, 1H, 10-H); 13C NMR
(100.6 MHz, CDCl3) δ 14.5 (CH3, CH2-CH3), 18.1 (CH2, C3), 25.3
(CH2, C4), 38.4 (CH, C4a), 58.6 (CH, C5), 60.3 (CH2, CH2-CH3),
60.7 (CH2, C2), 72.1 (CH, C10b), 113.6 (CH, C7), 118.7 (C, C9),
120.3 (C, C10a), 128.0 (CH, C8), 128.7 (CH, 5-Ar-Cmeta), 129.8 (CH,
5-Ar-Cortho), 130.1 (CH, C10), 133.5 (C, 5-Ar-Cpara), 138.7 (C, 5-Ar-
25 2HClc
26 2HClb
3
27 2HClb
3
12c
6-chlorotacrine HClc
3
a Values are expressed as the mean ( standard deviation of three
independent experiments. b Compound dissolved in PBS/EtOH (70:30).
c Compound dissolved in PBS/EtOH (80:20).
Conclusion
We have synthesized a new series of pyrano[3,2-c]-
quinoline-6-chlorotacrine hybrids as a novel class of dual
binding site AChEIs. Variation of the position of an amido
group within the oligomethylene linker gives rise to two
parallel isomeric series. In general, the new hybrids are potent
AChEIs, with IC50 values in the low nanomolar range and
withslight differencesbetweenbothseries. The resultsindicate
that linkage of a 5-phenylpyrano[3,2-c]quinoline moiety to a
unit of the highly potent AChE active site inhibitor 6-chlor-
otacrine through an amido-containing tether does not result
in an increased potency, as the most potent hybrids are
equipotent to 6-chlorotacrine. Moreover, the AChE inhibi-
tory potency of the hybridsshows a modestdependence on the
length of the linker and the position of the amido group.
Molecular modeling and kinetic studies have confirmed the
dual site binding of these hybrids to hAChE. Apart from the
characteristic interactions of the 6-chlorotacrine unit of these
hybrids within the active site of AChE, the pyranoquinoline
moiety is proposed to interact at the peripheral site forming a
double near-parallel stacking with Trp286 and Tyr72, respec-
tively, which are positioned in a way similar to the arrange-
ment found in the complex of the enzyme with syn-TZ2PA6.
The presence of a chlorine atom at position 6 of the tacrine
unit, known to be detrimental for BChE inhibition, not
unexpectedly accounts for the hAChE/hBChE selectivity of
these hybrids.
Because oftheir dualbindingsitecharacter, the new hybrids
are able to inhibit the AChE-induced Aβ1-40 aggregation.
Also, these compounds exhibit a significant ability to inhibit
the self-induced Aβ1-42 aggregation, and some of them can
also inhibit BACE-1. In general, these effects seem to be
sensitive to the position of the amido group of the linker,
the hybrids of the second series being more potent than their
counterparts of the first series. Moreover, in the second series
the activities increase along with the tether length. Finally,
these hybrids seem to be able to cross BBB. Overall, 27
emerges as a promising anti-Alzheimer drug candidate able
to hit both AChE (catalytic and noncatalytic activities) and
Aβ (aggregation and production) and, therefore, with poten-
tial symptomatic and disease-modifying effects.
C
ipso), 148.7 (C, C7a), 166.8 (C, CO). HRMS calcd for (C21-
H2235ClNO3 þ Hþ) 372.1361, found 372.1370.
Ethyl 5-(4-Chlorophenyl)-3,4-dihydro-2H-pyrano[3,2-c]quino-
line-9-carboxylate (11). To a solution of the diastereomeric
mixture of tetrahydroquinolines 9 (408 mg, 1.11 mmol) in
CHCl3 (15 mL), DDQ (498 mg, 2.19 mmol, 2 equiv) was added,
and the reaction mixture was stirred at room temperature for
24 h. Saturated aqueous Na2CO3 (50 mL) was added, the
organic phase was separated, and the aqueous one was extracted
with CH2Cl2 (3 ꢀ 10 mL). The combined organic phase and
extracts were dried with anhydrous Na2SO4 and evaporated
under reduced pressure to give a crude residue (870 mg), which
was subjected to column chromatography [35-70 μm silica gel
(20 g), hexane/AcOEt mixtures]. On elution with hexane/
AcOEt, 50:50, pyranoquinoline 11 (305 mg, 75% yield) was ob-
tained as an off-white solid: mp 197-199 ꢀC (hexane/AcOEt, 1:1);
Experimental Section
Chemistry. General Methods. Melting points were deter-
mined in open capillary tubes with a MFB 595010M Gallen-
1
kamp or a Buchi B-540 melting point apparatus. 300 MHz H/
€
75.4 MHz 13C NMR spectra, 400 MHz 1H/100.6 MHz 13C
NMR spectra, and 500 MHz 1H NMR spectra were recorded on
IR (NaCl) ν1715 (CdO st), 1623 (ar-C-Candar-C-Nst) cm-1
1H NMR (400MHz, CD3OD) δ1.36 (t, J=7.1 Hz, 3H, CH2-CH3),
;

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5373
2.09 (m, 2H, 3-H2), 2.76 (t, J = 6.3 Hz, 2H, 4-H2), 4.39 (q, J = 7.1
Hz, 2H, CH2-CH3), 4.60 (m, 2H, 2-H2), 7.60-7.70 (complex signal,
4H, 5-ArH), 8.04 (dd, J = 8.9 Hz, J0 = 0.5 Hz, 1H, 7-H), 8.46 (dd,
J=8.9Hz, J0=1.8 Hz, 1H, 8-H), 8.93 (dd, J=1.8Hz, J0 =0.5Hz,
1H, 10-H); 13C NMR (100.6 MHz, CD3OD) δ 13.4 (CH3, CH2-
CH3), 20.3 (CH2, C3), 22.3 (CH2, C4), 62.0 (CH2, CH2-CH3), 70.3
(CH2, C2), 114.5 (C, C4a), 119.3 (C, C10a), 120.8 (CH, C10), 125.1
(C, C9), 129.4 (CH, C7), 130.1 (CH, C8), 130.3 (CH, 5-Ar-Cortho),
130.8 (CH, 5-Ar-Cmeta), 133.3 (C, 5-Ar-Cpara), 138.0 (C, 5-Ar-
Cipso), 140.6 (C, C6a), 157.2 (C, C10b), 165.0 (C, C5), 166.3 (C,
CO). HRMS calcd for (C21H1835ClNO3 þ Hþ) 368.1048, found
368.1056.
Methyl 3-(3,4-Dihydro-5-phenyl-2H-pyrano[3,2-c]quinolin-
9-yl)propanoate (12). A. Synthesis of the Diastereomeric Mix-
ture of Tetrahydroquinolines 10. To a solution of 3,4-dihydro-
2H-pyran (508 μL, 468 mg, 5.58 mmol, 1 equiv), benzaldehyde
(566 μL, 591 mg, 5.58 mmol, 1 equiv), and the aniline 6 (1.00 g,
5.58 mmol, 1 equiv) in CH3CN (40 mL) was added Sc(OTf)3
(550 mg, 1.12 mmol, 0.2 equiv), and the reaction mixture was
stirred overnight under inert atmosphere at room temperature.
Saturated aqueous NaHCO3 (40 mL) was added, and the
resulting mixture was extracted with AcOEt (3 ꢀ 30 mL). The
combined organic extracts were dried with anhydrous MgSO4
and concentrated under reduced pressure to give a residue
(1.9 g), which was subjected to column chromatography [35-
70 μm silica gel (40 g), hexane/AcOEt/Et3N mixtures]. On
elution with hexane/AcOEt/Et3N, 74:25:1, the expected adduct
10 (1.68 g, 86% yield) was obtained as a diastereomeric mixture.
B. DDQ Oxidation of 10 to 12: Overoxidation of 12 to Its
Cinnamic Derivative. To a solution of the mixture of diastereoi-
somers 10 (4.75 g, 13.5 mmol, 1 equiv) in CHCl3 (50 mL) were
added Et3N (5.70 mL, 4.14 g, 40.8 mmol, 3 equiv) and DDQ
(3.10 g, 13.6 mmol, 1 equiv). The reaction mixture was heated to
reflux for 3 h and concentrated in vacuo. The resulting solid
residue (7.90 g) was subjected to column chromatography [35-
70 μm silica gel (200 g), hexane/AcOEt mixtures]. On elution
with hexane/AcOEt, 20:80, the desired pyranoquinoline 12 (1.60g)
was obtained together with its cinnamic derivative (1.60 g, 35%
combined yield).
successively with aqueous 2 N NaOH (3 ꢀ 3 mL) and water (2 ꢀ
3 mL). The organic phase was dried with anhydrous Na2SO4
and concentrated in vacuo to give a brown oily residue, from
which 1-pentanol and the excess of diamine were removed by
distillation at 100 ꢀC/1 Torr and 140 ꢀC/1 Torr, respectively. The
distillation residue was taken in CH2Cl2 (1.3 mL), filtered, and
concentrated in vacuo to give a brown oily residue which was
subjected to column chromatography (35-70 μm silica gel,
CH2Cl2/MeOH/25% or 50% aqueous NH4OH or AcOEt/
MeOH/Et3N mixtures as eluent) to afford, separately, bis-6-
chlorotacrine 28 and the desired amine 17 as a brown and
yellowish oil, respectively.
For characterization purposes, analytical samples of the
dihydrochlorides of 17 and 28 were prepared as follows: the
amine 17 or the dimer 28 (1 mmol) were dissolved in MeOH (2-
10 mL), the solution was filtered through a polytetrafluoroethy-
lene (PTFE) 0.45 μm filter and treated with an excess of a
methanolic solution of HCl (6-9 mmol), and the resulting
solution was concentrated in vacuo to dryness. The solid was
recrystallized from MeOH/AcOEt mixtures, triturated with
Et2O, and dried at 65 ꢀC/15 Torr for 4 days to give 17 2HCl
3
or 28 2HCl as yellowish solids.
9-[(6-Aminohexyl)amino]-6-chloro-1,2,3,4-tetrahydroacridine
Dihydrochloride (17c 2HCl). From 15 (4.00 g, 15.9 mmol) and
3
3
1,6-diaminohexane, 16c (7.52 g, 64.8 mmol), a brown oily
residue (7.30 g) was obtained and subjected to column chroma-
tography [35-70 μm silica gel (88 g), CH2Cl2/MeOH/25%
aqueous NH4OH mixtures]. On elution with CH2Cl2/MeOH/
25% aqueous NH4OH, 99:1:0.05, bis(6)-6-chlorotacrine, 28c
(246 mg), a mixture 28c/17c in the approximate ratio of 20:80
(1H NMR) (614 mg, 8% total yield of 28c), and amine 17c (3.61
g, 78% total yield) were consecutively isolated as brown-yellow
oils: Rf(17c)=0.39; Rf(28c)=0.80 (CH2Cl2/MeOH/25% aqueous
NH4OH, 9:1:0.01).
17c 2HCl: mp 157-158 ꢀC (MeOH); IR (KBr) ν 3500-2500
3
(max at 3413, 2935, 2864, N-H, þN-H, and C-H st), 1630,
1573, and 1513 (ar-C-C and ar-C-N st) cm-1; 1H NMR (300
MHz, CD3OD, presat/Watergate) δ 1.39-1.52 (complex signal,
4H, 30-H2 and 40-H2), 1.66 (tt, J ≈ J0 ≈ 7.5 Hz, 2H, 50-H2), 1.80
(tt, J ≈ J0 ≈ 7.5 Hz, 2H, 20-H2), 1.88-1.98 (complex signal, 4H,
2-H2 and 3-H2), 2.69 (m, 2H, 1-H2), 2.90 (t, J=7.5 Hz, 2H, 60-
H2), 2.98 (m, 2H, 4-H2), 3.83 (t, J = 7.5 Hz, 2H, 10-H2), 7.48 (dd,
J=9.3 Hz, J0=2.1 Hz, 1H, 7-H), 7.75 (d, J=2.1 Hz, 2H, 5-H),
8.30 (d, J = 9.3 Hz, 1H, 8-H); 13C NMR (75.4 MHz, CD3OD) δ
22.3 (CH2, C3), 23.2 (CH2, C2), 25.2 (CH2, C1), 27.1 (CH2) and
27.3 (CH2) (C30 and C40), 28.5 (CH2), 30.9 (CH2) and 31.4 (CH2)
(C4, C20, and C50), 40.6 (CH2, C60), 49.1 (CH2, C10), 114.3 (C,
C9a), 116.5 (C, C8a), 121.3 (CH, C5), 126.1 (CH, C7), 128.0
(CH, C8), 138.5 (C, C6), 142.8 (C, C10a), 154.5 (C, C4a), 156.2
(C, C9).
C. Hydrogenation of the Mixture of 12 and Its Cinnamic
Derivative. The previous mixture (100 mg) was subjected to
hydrogenation at atmospheric pressure in EtOH (10 mL) in the
presence of 10% w/w Pd/C (20 mg) for 12 h. The resulting
mixture was filtered, and the filtrate was concentrated in vacuo.
The resulting residue was taken up in AcOEt (15 mL) and
washed with saturated aqueous NaHCO3 (20 mL), and the
organic phase was dried with anhydrous MgSO4 and evaporated
at reduced pressure to yield the desired pyranoquinoline 12
(98mg, 95% overallyieldfrom 10), which was obtained as a glassy
white solid: mp 93-94 ꢀC (AcOEt); IR (NaCl) ν 1736 (CdO st)
28c 2HCl [N,N0-Bis(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)-
1
cm-1; H NMR (400 MHz, CDCl3) δ 1.99 (m, 2H, 3-H2), 2.73
3
1,6-hexanediamine]: mp 126-127 ꢀC (MeOH); IR (KBr) ν
3500-2500 (max at 3254, 3049, 2933, 2859, 2792, N-H, þN-
H, and C-H st), 1631, 1603, 1574, 1557, and 1514 (ar-C-C and
ar-C-N st) cm-1; 1H NMR (300 MHz, CD3OD) δ 1.50 (m, 4H,
30-H2), 1.80-2.00 (complex signal, 12H, 2-H2, 3-H2, and 20-H2),
2.66 (m, 4H, 1-H2), 2.98 (m, 4H, 4-H2), 3.91 (t, J ≈ 7.0 Hz, 4H,
10-H2), 4.87 (s, NH and þNH), 7.51 (dd, J = 9.6 Hz, J0 = 2.2 Hz,
2H, 7-H), 7.75 (d, J = 2.2 Hz, 2H, 5-H), 8.35 (d, J ≈ 9.6 Hz, 2H,
8-H); 13C NMR (75.4 MHz, CD3OD) δ 22.5 (CH2, C3), 23.3
(CH2, C2), 25.3 (CH2, C1), 27.4 (CH2), 31.4 (CH2), and 31.6
(CH2) (C4, C20, and C30), 49.2 (CH2, C10), 114.6 (C, C9a), 116.9
(C, C8a), 122.1 (CH, C5), 125.9 (CH, C7), 127.7 (CH, C8), 138.0
(C, C6), 143.6 (C, C10a), 155.3 (C), and 155.6 (C) (C4a and C9).
9-[(8-Aminooctyl)amino]-6-chloro-1,2,3,4-tetrahydroacridine
(complex signal, 4H, 4-H2 and 9-CH2-CH2-COO), 3.13 (t, J=
7.8 Hz, 2H, 9-CH2-CH2-COO), 3.67 (s, 3H, COOCH3), 4.42 (t,
J=5.2 Hz, 2H, 2-H2), 7.43 (complex signal, 4H, 8-H, 5-ArHmeta
and 5-ArHpara), 7.55 (dd, J=8.4 Hz, J’=1.5 Hz, 2H, 5-ArHortho),
7.94 (d, J = 1.5 Hz, 1H, 10-H), 7.97 (d, J=8.4 Hz, 1H, 7-H); 13
,
C
NMR (100.6 MHz, CDCl3) δ 21.7 (CH2, C3), 23.6 (CH2, C4),
31.0 (CH2, 9-CH2-CH2-COO), 35.5 (CH2, 9-CH2-CH2-COO),
51.5 (CH3, COOCH3), 66.9 (CH2, C2), 110.6 (C, C4a), 119.6 (C,
C10a), 119.7 (CH, C10), 128.0 (CH, C7), 128.1 (CH, 5-Ar-Cpara),
128.6 (CH, 5-Ar-Cortho), 129.0 (CH, 5-Ar-Cmeta), 130.1 (CH, C8),
137.5 (C, C9), 140.3 (C, 5-Ar-Cipso), 146.0 (C, C6a), 156.9 (C,
C10b), 160.2 (C, C5), 173.1 (C, COO). HRMS calcd for
(C22H21NO3 þ Hþ) 348.1594, found 348.1588.
General Procedure for the Reaction of 6,9-Dichloro-1,2,3,4-
tetrahydroacridine, 15, with R,ω-Alkanediamines. A mixture of
15 (1 mmol) and an excess of the diamine 16 (4 mmol) in
1-pentanol (1.3 mL) was heated under reflux with magnetic
stirring for 18 h. The resulting mixture was cooled to
room temperature, diluted with CH2Cl2 (3 mL), and washed
Dihydrochloride (17e 2HCl). From 15 (4.00 g, 15.9 mmol) and
3
1,8-diaminooctane, 16e (8.24 g, 57.2 mmol), a brown oily
residue (9.56 g) was obtained and subjected to column chroma-
tography [35-70 μm silica gel (115 g), CH2Cl2/MeOH/25%
aqueous NH4OH mixtures]. On elution with CH2Cl2/MeOH/

5374 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Camps et al.
25% aqueous NH4OH, 96:4:0.1 to 85:15:0.2, bis(8)-6-chlorota-
crine, 28e (2.11 g, 46% yield) and amine 17e (2.00 g, 35% yield)
were consecutively isolated as brown-yellow oils: Rf(17e) = 0.33;
and evaporated at reduced pressure to give a yellow oily residue,
which was subjected to column chromatography (35-70 μm
silica gel, hexane/AcOEt/Et3N or heptane/AcOEt/Et3N mix-
tures as eluent) to afford the amides 18-27 as yellowish oils.
The isolated hybrids 18-27 were transformed into the corre-
sponding dihydrochlorides as follows: A solution of the free
base (1 mmol) in CH2Cl2 (10-60 mL) was filtered through a
0.45 μm PTFE filter and treated with excess of a methanolic
solution of HCl (6 mmol). The solution was concentrated in
vacuo to dryness, and the solid residue was, in general, recrys-
tallized from MeOH/AcOEt mixtures and dried at 65 ꢀC/15
Torr for 4 days.
R
f(28e)=0.88 (CH2Cl2/MeOH/25% aqueous NH4OH, 9:1:0.01).
17e 2HCl: mp 109-110 ꢀC (MeOH/AcOEt, 1:1); IR (KBr) ν
3
3500-2500 (max at 3344, 2929, 2855, N-H, þN-H, and C-H
st), 1630, 1605, 1574, 1558, and 1512 (ar-C-C and ar-C-N st)
cm-1 1H NMR (300 MHz, CD3OD) δ 1.24-1.44 (complex
;
signal, 8H, 30-H2, 40-H2, 50-H2, and 60-H2), 1.63 (tt, J ≈ J0 ≈ 7.5
Hz, 2H, 70-H2), 1.71 (tt, J ≈ J0 ≈ 7.5 Hz, 2H, 20-H2), 1.84-1.94
(complex signal, 4H, 2-H2 and 3-H2), 2.66 (m, 2H, 1-H2), 2.89 (t,
J = 7.5 Hz, 2H, 80-H2), 2.94 (m, 2H, 4-H2), 3.69 (t, J = 7.5 Hz,
2H, 10-H2), 4.89 (s, NH, þNH and þNH3), 7.36 (dd, J = 9.0 Hz,
J0 = 2.1 Hz, 1H, 7-H), 7.71 (d, J = 2.1 Hz, 1H, 5-H), 8.16 (d, J=
9.0 Hz, 1H, 8-H); 13C NMR (75.4 MHz, CD3OD) δ 22.8 (CH2,
C3), 23.5 (CH2, C2), 25.5 (CH2, C1), 27.4 (CH2) and 27.7 (CH2)
(C30 and C60), 28.6 (CH2), 30.1 (2 CH2), 31.8 (CH2), and 32.1
(CH2) (C4, C20, C40, C50, and C70), 40.8 (CH2, C80), 49.4 (CH2,
C10), 115.1 (C, C9a), 117.6 (C, C8a), 123.3 (CH, C5), 125.6 (CH,
C7), 127.4 (CH, C8), 137.3 (C, C6), 144.9 (C, C10a), 155.0 (C,
C4a), 156.6 (C, C9).
6-Chloro-9-{{6-[5-(4-chlorophenyl)-3,4-dihydro-2H-pyrano[3,2-
c]quinoline-9-carboxamido]hexyl}amino}-1,2,3,4-tetrahydroa-
cridine Dihydrochloride (18 2HCl). From crude 13 [173 mg of
3
the crude product obtained from 136 mg (0.37 mmol) of ester
11] and amine 17c (126 mg, 0.37 mmol), a brown oily residue
(212 mg) was obtained and subjected to column chromatog-
raphy [35-70 μm silica gel (20 g), hexane/AcOEt/Et3N
mixtures]. On elution with hexane/AcOEt/Et3N, 20:80:0.05
to 0:100:0.05, hybrid 18 (56 mg, 23% yield) was isolated as a
28e 2HCl [N,N0-Bis(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)-
yellowish oil: Rf = 0.52 (AcOEt/Et3N, 10:0.05). 18 2HCl: mp
3
3
1,8-octanediamine]: mp 134-135 ꢀC (MeOH/AcOEt, 1:1); IR
(KBr) ν 3500-2500 (max at 3229, 3046, 2926, 2853, 2768, N-H,
þN-H, and C-H st), 1630, 1570, and 1515 (ar-C-C and ar-
194-195 ꢀC (CH2Cl2). IR (KBr) ν 3500-2500 (max at 3393,
3253, 3056, 2928, 2856, N-H, þN-H, O-H, and C-H st),
1
1633 and 1575 (CdO, ar-C-C and ar-C-N st) cm-1; H
1
C-N st) cm-1; H NMR (300 MHz, CD3OD, presat/Water-
NMR (500 MHz, CD3OD) δ 1.46-1.56 (complex signal, 4H,
30-H2 and 40-H2), 1.70 (tt, J ≈ J0 ≈ 7.0 Hz, 2H, 50-H2), 1.88 (tt,
J = J0 = 7.0 Hz, 2H, 20-H2), 1.92-1.99 (complex signal, 4H,
2-H2 and 3-H2), 2.16 (tt, J = J0=6.0 Hz, 2H, 300-H2), 2.68 (t,
J = 6.0 Hz, 2H, 1-H2), 2.85 (t, J=6.0 Hz, 2H, 400-H2), 2.98 (t,
J=6.0 Hz, 2H, 4-H2), 3.45 (t, J=7.0 Hz, 2H, 60-H2), 3.96 (t,
J = 7.0 Hz, 2H, 10-H2), 4.76 (t, J ≈ 6.0 Hz, 2H, 200-H2), 4.85 (s,
NH and þNH), 7.53 (dd, J=9.5 Hz, J0=2.0 Hz, 1H, 7-H), 7.69
[dm, J = 8.5 Hz, 2H, 3(5)-H p-chlorophenyl], superimposed in
part 7.73 [dm, J= 8.5 Hz, 2H, 2(6)-H p-chlorophenyl], 7.74 (d,
J = 2.0 Hz, 1H, 5-H), 8.09 (d, J ≈ 9.0 Hz, 1H, 700-H), 8.34 (dd,
J = 9.0 Hz, J0=2.0 Hz, 1H, 800-H), 8.38 (d, J = 9.5 Hz, 1H, 8-
H), 8.79 (d, J = 2.0 Hz, 1H, 1000-H); 13C NMR (100.6 MHz,
CD3OD) δ 21.72 (CH2, C300), 21.74 (CH2, C3), 22.9 (CH2, C2),
23.7 (CH2, C400), 24.7 (CH2, C1), 27.2 (CH2) and 27.5 (CH2)
(C30and C40), 29.3 (CH2, C4), 30.2 (CH2, C50), 31.2 (CH2, C20),
40.9 (CH2, C60), 49.1 (CH2, C10), 70.9 (CH2, C200), 113.4
(C, C9a), 115.2 (C, C4a00), 115.4 (C, C8a), 119.1 (CH, C5),
120.5 (C, C10a00), 122.9 (CH, C700), 123.3 (CH, C1000), 126.7
(CH, C7), 128.8 (CH, C8), 130.4 [CH, C3(5) p-chlorophenyl],
132.0 [CH, C2(6) p-chlorophenyl], 132.7 (CH þ C, C800 and
C1 p-chlorophenyl), 135.0 (C, C900), 138.7 (C, C4 p-chloro-
phenyl), 140.0 (C, C6), 140.5 (C, C10a), 142.2 (C, C6a00),
152.1 (C, C4a), 157.8 (C, C9), 158.6 (C, C500), 166.2 (C,
C10b00), 168.0 (C, CONH). HRMS calcd for (C38H3835Cl2-
N4O2þHþ) 653.2450, found 653.2432. Anal. (C38H38Cl2N4-
O2 2HCl 2.4H2O) C, H, N.
gate) δ 1.37-1.48 (complex signal, 8H, 30-H2 and 40-H2), 1.83 (tt,
J ≈ J0 ≈ 7.2 Hz, 4H, 20-H2), 1.90-1.98 (complex signal, 8H, 2-H2
and 3- H2), 2.66 (m, 4H, 1-H2), 2.99 (m, 4H, 4-H2), 3.92 (t, J =
7.2 Hz, 4H, 10-H2), 7.54 (dd, J=9.3 Hz, J0 =2.1 Hz, 2H, 7-H),
7.77 (d, J ≈ 2.1 Hz, 2H, 5-H), 8.37 (d, J ≈ 9.3 Hz, 2H, 8-H); 13
C
NMR (75.4 MHz, CD3OD) δ 21.8 (CH2, C3), 22.9 (CH2, C2),
24.8 (CH2, C1), 27.6 (CH2, C30), 29.4 (CH2, C4), 30.2 (CH2) and
31.3 (CH2) (C20 and C40), 49.2 (CH2, C10), 113.2 (C, C9a), 115.3
(C, C8a), 119.0 (CH, C5), 126.6 (CH, C7), 128.7 (CH, C8), 139.9
(C, C6), 140.3 (C, C10a), 152.0 (C, C4a), 157.5 (C, C9).
General Procedure for the Preparation of Amides 18-27 from
Esters 11 or 12 and Amines 17. A.1. Hydrolysis of Ester 11. A
solution of ester 11 (1 mmol) and aqueous 5 N NaOH (1.2 mL, 6
equiv) in MeOH (56 mL) was heated under reflux with magnetic
stirring for 16 h. The resulting mixture was cooled to 0 ꢀC (ice-
water bath), treated with aqueous 2 N HCl (5.5 mL, 11 equiv),
and concentrated in vacuo. The obtained solid residue was
extracted with MeOH (18 mL), and the organic extract was
evaporated at reduced pressure to give the hydrochloride of the
corresponding carboxylic acid, 13, as a white solid, which was
used in the next step without further purification.
A.2. Hydrolysis of Ester 12. A solution of ester 12 (1 mmol)
and KOH pellets (240 mg of 85% purity reagent, 3.6 equiv) in
MeOH (25 mL) was heated under reflux with magnetic stirring
for 24 h. The resulting mixture was cooled to room temperature
and evaporated in vacuo. The obtained solid residue was treated
with a solution of HCl in Et2O (20 equiv) with stirring for
30 min, and the suspension was evaporated in vacuo to give the
hydrochloride of the corresponding carboxylic acid, 14, as a
white solid, which was used in the next step without further
purification.
B. Reaction of Carboxylic Acids 13 or 14 with Amines 17. To a
cold solution (0 ꢀC, ice-water bath) of 13 or 14 (crude product
arising from 1 mmol of the starting ester 11 or 12) and anhy-
drous Et3N (2.2 mmol) in anhydrous CH2Cl2 (50 mL), ethyl
chloroformate (1 mmol) was added and the mixture was thor-
oughly stirred at 0 ꢀC for 30 min. To the resulting solution, a
cold solution (0 ꢀC, ice-water bath) of amine 17 (1 mmol) in
anhydrous CH2Cl2 (50 mL) was added, and the reaction mixture
was stirred at room temperature for 64-72 h and treated with
10% aqueous Na2CO3 until pH 10 (95 mL). The organic phase
was separated, and the aqueous one was extracted with CH2Cl2
(3 ꢀ 40 mL). The organic phase and combined extracts were
washed with water (2 ꢀ 80 mL), dried with anhydrous Na2SO4,
3
3
6-Chloro-9-{{8-[3-(3,4-dihydro-5-phenyl-2H-pyrano[3,2-c]qui-
nolin-9-yl)propanamido]octyl}amino}-1,2,3,4-tetrahydroacridine Di-
hydrochloride (27 2HCl). From crude 14 [126 mg of the crude
3
product obtained from 122 mg (0.35 mmol) of ester 12] and
amine 17e (126 mg, 0.35 mmol), a yellow oily residue (198 mg)
was obtained and subjected to column chromatography [35-70
μm silica gel (19 g), heptane/AcOEt/Et3N mixtures]. On elution
with heptane/AcOEt/Et3N, 20:80:0.1, hybrid 27 (91 mg, 39%
yield) was isolated as a yellowish oil: Rf = 0.68 (AcOEt/MeOH/
Et3N, 90:10:0.1). 27 2HCl: mp 190-191 ꢀC (MeOH). IR (KBr)
3
ν 3500-2500 (max at 3389, 3245, 3056, 2925, 2853, N-H, þN-
H, O-H, and C-H st), 1633 and 1576 (CdO, ar-C-C, and
ar-C-N st) cm-1; 1H NMR (500 MHz, CD3OD) δ 1.20-1.36
(complex signal, 6H, 40-H2, 50-H2, and 60-H2), 1.37-1.45
(complex signal, 4H, 30-H2, and 70-H2), 1.81 (quint, J=7.5 Hz,
2H, 20-H2), 1.91-1.99 (complex signal, 4H, 2-H2, and 3-H2),
2.15 (quint, J ≈ 6.0 Hz, 2H, 300-H2), 2.62 (t, J = 7.5 Hz, 2H,
900-CH2-CH2-CONH), 2.66 (t, J = 6.0 Hz, 2H, 1-H2), 2.83

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5375
(t, J=6.5 Hz, 2H, 400-H2), 2.99 (t, J=6.0 Hz, 2H, 4-H2), 3.10 (t,
J = 7.5 Hz, 2H, 80-H2), 3.18 (t, J=7.5 Hz, 2H, 900-CH2-CH2-
CONH), 3.92 (t, J=7.5 Hz, 2H, 10-H2), 4.76 (t, J = 5.0 Hz, 2H,
200-H2), 4.84 (s, NH and þNH), 7.55 (dd, J=9.5 Hz, J0 = 2.0
Hz, 1H, 7-H), 7.65-7.74 [complex signal, 5H, Ar-H phenyl],
7.77 (d, J = 2.0 Hz, 1H, 5-H), 7.94 (dd, J=9.0 Hz, J0 = 1.5 Hz,
1H, 800-H), 8.00 (d, J = 9.0 Hz, 1H, 700-H), 8.21 (d, J = 1.5 Hz,
1H, 1000-H), 8.37 (d, J ≈ 9.5 Hz, 1H, 8-H); 13C NMR (100.6
MHz, CD3OD) δ 21.8 (CH2, C300), 21.9 (CH2, C3), 22.9 (CH2,
C2), 23.8 (CH2, C400), 24.7 (CH2, C1), 27.6 (CH2, C30), 27.7
(CH2, C60), 29.3 (CH2, C4), 30.1 (CH2) and 30.2 (CH2) (C40
and C50), 30.3 (CH2, C70), 31.3 (CH2, C20), 32.8 (CH2, 900-CH2-
CH2-CONH), 38.1 (CH2, 900-CH2-CH2-CONH), 40.3 (CH2,
C80), 49.2 (CH2, C10), 70.6 (CH2, C200), 113.3 (C, C9a), 114.2
(C, C4a00), 115.4 (C, C8a), 119.1 (CH, C5), 121.0 (C, C10a00),
122.3 (2 CH, C700 and C1000), 126.8 (CH, C7), 128.8 (CH, C8),
130.2 (CH) and 132.2 (CH þ C) [C1, C2(6), C3(5), and C4
phenyl], 135.9 (CH, C800), 139.7 (C, C6a00), 140.1 (C, C6), 140.5
(C, C10a), 142.9 (C, C900), 152.1 (C, C4a), 157.8 (C, C500), 157.9
(C, C9), 165.1 (C, C10b00), 174.3 (C, CONH). HRMS calcd for
(C42H4735ClN4O2 þ Hþ) 675.3460, found 675.3457. Anal.
purchased from Bachem AG (Bubendorf, Switzerland). Aβ1-40
(2 mg mL-1) was dissolved in HFIP and lyophilized. The 1 mM
solutions of tested inhibitors were prepared by dissolution in
MeOH.
Aliquots of 2 μL Aβ1-40 peptide, lyophilized from 2 mg mL-1
HFIP 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 co-incubation
experiments aliquots (16 μL) of hAChE (final concentration
2.30 μM, Aβ/AChE molar ratio 100:1) and AChE in the
presence of 2 μL of the tested inhibitor (final inhibitor concen-
tration 100 μM) in 0.215 M sodium phosphate buffer, pH 8.0,
solution were added. Blanks containing Aβ1-40 alone, human
recombinant AChE alone, and Aβ1-40 plus tested 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.88 The fluorescence in-
tensities due to β-sheet conformation were monitored for 300 s
at λem = 490 nm (λexc=446 nm). The percent inhibition of the
AChE-induced aggregation due to the presence of the tested
compound was calculated by the following expression: 100 -
[(IFi/IFo) ꢀ 100)] where IFi and IFo are the fluorescence
intensities obtained for Aβ plus AChE in the presence and in
the absence of inhibitor, respectively, minus the fluorescence
intensities due to the respective blanks.
(C42H47ClN4O2 2HCl 2.25H2O) C, H, N.
3
3
Biochemical Studies. AChE and BChE Inhibition Assay.
AChE inhibitory activity was evaluated spectrophotometrically
at 25 ꢀC by the method of Ellman,64 using AChE from bovine or
human erythrocytes and acetylthiocholine iodide (0.53 or 0.13
mM for bAChE and hAChE, respectively) as substrate. The
reaction took place in a final volume of 3 mL of 0.1 M
phosphate-buffered solution, pH 8.0, containing 0.025 or 0.04
units of bAChE or hAChE, respectively, and 333 μM 5,50-
dithiobis(2-nitrobenzoic) acid (DTNB) solution used to pro-
duce the yellow anion of 5-thio-2-nitrobenzoic acid. Inhibition
curves were performed in triplicate by incubating at least 12
concentrations of inhibitor for 15 min. One triplicate sample
without inhibitor was always present to yield 100% of AChE
activity. The reaction was stopped with 100 μL of 1 mM eserine,
and the color production was measured at 414 nm. BChE
inhibitory activity determinations were carried out similarly,
using 0.035 unit of human serum BChE and 0.56 mM butyr-
ylthiocholine, instead of AChE and acetylthiocholine, in a final
volume of 1 mL.
Data from concentration-inhibition experiments of the in-
hibitors were calculated by nonlinear regression analysis, using
the GraphPad Prism program package (GraphPad Software;
San Diego, CA), which gave estimates of the IC50 (concentration
of drug producing 50% of enzyme activity inhibition). Results
are expressed as the mean ( SEM of at least four experiments
performed in triplicate. DTNB, acetylthiocholine, butyrylthio-
choline, and enzymes were purchased from Sigma, and eserine
was purchased from Fluka.
Kinetic Analysis of AChE Inhibition. To obtain estimates of
the mechanism of action of 20, reciprocal plots of 1/velocity
versus 1/[substrate] were constructed at relatively low concen-
tration of substrate (0.56-0.11 mM) by using Ellman’s meth-
od64 and human recombinant AChE (Sigma, Milan, Italy).
Three concentrations of 20 were selected for this study: 1.25,
1.88, and 3.75 nM. The plots were assessed by a weighted least-
squares analysis that assumed the variance of the velocity (v) to
be a constant percentage of v for the entire data set. Data
analysis was performed with GraphPad Prism 4.03 software
(GraphPad Software Inc.).
Slopes of the obtained reciprocal plots were then plotted
against 20 concentration in a similar weighted analysis, and Ki
was determined as the intercept on the negative x-axis.
AChE-Induced Aβ1-40 Aggregation Inhibition Assay. Thio-
flavin T (Basic Yellow 1), human recombinant AChE lyophi-
lized powder, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), were
purchased from Sigma Chemicals. Absolute DMSO over mo-
lecular sieves was from Fluka. Water was deionized and do-
ubly distilled. Αβ1-40, supplied as trifluoroacetate salt, was
Aβ1-42 Self-Aggregation Inhibition Assay. As reported in a
previously published protocol,99 HFIP pretreated Aβ1-42 sam-
ples (Bachem AG, Switzerland) were solubilized with a CH3CN/
Na2CO3/NaOH (48.4:48.4:3.2) mixture. Experiments were per-
formed by incubating the peptide in 10 mM phosphate buffer
(pH 8.0) containing 10 mM NaCl at 30 ꢀC for 24 h (final Aβ
concentration 50 μM) with and without inhibitor (50 μM, Aβ/
inhibitor = 1/1). Blanks containing the tested inhibitors were
also prepared and tested. To quantify amyloid fibrils formation,
the thioflavin T fluorescence method was used.88 After incuba-
tion, samples were diluted to a final volume of 2.0 mL with 50
mM glycine-NaOH buffer (pH 8.5) containing 1.5 μM thio-
flavin T. A 300 s time scan of fluorescence intensity was carried
out (λexc=446 nm; λem=490 nm, FP-6200 fluorometer, Jasco
Europe), and values at plateau were averaged after subtracting
the background fluorescence of 1.5 μM thioflavin T solution.
The fluorescence intensities were compared, and the percent
inhibition due to the presence of the inhibitor was calculated by
the following formula 100 - [(IFi/IFo) ꢀ 100] where IFi and IFo
are the fluorescence intensities obtained for Aβ1-42 in the
presence and in the absence of inhibitor, respectively.
β-Secretase (BACE-1) Inhibition Assay. β-Secretase (BACE-
1, Sigma) inhibition studies were performed by employing a
peptide mimicking APP sequence as substrate (M-2420,
Bachem). The following procedure was employed: an amount
of 5 μL of test compound (or DMSO) was preincubated with
175 μL of the enzyme (c=17.2 nM) for 1 h at room temperature.
The substrate (3 μM) was then added and left to react for 15 min.
The fluorescence signal was read at λem=405 nm (λexc=320 nm).
The fluorescence intensities with and without inhibitor were
compared, and the percent inhibition due to the presence of test
compounds was calculated. The % inhibition due to the pre-
sence of increasing test compound concentration was calculated
by the following expression: 100 - [(IFi/IFo) ꢀ 100], where IFi
and IFo are the fluorescence intensities obtained for BACE-1 in
the presence and in the absence of inhibitor, respectively.
Inhibition curve was obtained for 27 by plotting the % inhibi-
tion versus the logarithm of inhibitor concentration in the assay
sample. The linear regression parameters were determined and
the IC50 was extrapolated, when possible (GraphPad Prism 4.0,
GraphPad Software Inc.).
To demonstrate inhibition of BACE-1 activity, a peptido-
mimetic inhibitor (β-secretase inhibitor IV, Calbiochem) was
serially diluted into the reactions’ wells (IC50=0.013 μM).

5376 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Camps et al.
Molecular Modeling. Docking was performed with the pro-
gram rDock, which is an extension of the program Ribo-
Dock,100 using an empirical scoring function calibrated on the
basis of protein-ligand complexes.101 The reliability of rDock
was assessed by docking a set of known dual binding site
AChEIs taking advantage of the X-ray crystallographic struc-
tures of their complexes with AChE (PDB entries 1Q83, 1Q84,
1ODC, 1ZGB, 1ZGC, 2CKM, and 2CMF), which was also
chosen to identify three different orientations of the indole ring
of Trp286 in the peripheral binding site in hAChE (see Molec-
ular Modeling Studies and also Tables S2 and S3 in Supporting
Information).
predict the binding mode of huprines to AChE.110 Briefly, the
enzyme was immersed in a pre-equilibrated box of TIP3P111
water molecules. The final systems contained the protein-
ligand complex and around 16 000 water molecules (about
57 000 atoms). After thermatilization at 298 K, a series of
10 ns trajectories were sampled for the different compounds in
the receptor-ligand complex. The system was simulated in the
NPT ensemble using periodic boundary conditions and Ewald
sums for treating long-range electrostatic interactions (with the
default Amber-9 parameters). All simulations were performed
with the parmm99 force field of the Amber-9 package. The
structural analysis was performed using in-house software and
standard codes (PTRAJ module) of Amber-9.
The docking of compounds 20, 25, and 27 in hAChE was then
explored using the three structural models of the target hAChE
(named A, B, and C). Structural water molecules that mediate
relevant interactions between the tacrine moiety and the enzyme
were retained in the target models. The docking volume was
defined as the space within 10 A of the ligands spanning both
catalytic and peripheral binding sites. The ligands were consid-
ered in their monocationic form on the basis of the pKa
estimated using ACD software.102 The structure of the ligands
was initially energy minimized at the AM1103 level using Gauss-
ian 03.104 Suitable restraints were introduced to position the
tacrine moiety of the hybrids in the catalytic site, as inspection of
the X-ray crystallographic structures of several tacrine-based
dual binding site AChEIs reveals that the tacrine unit shares a
common binding mode, which in turn mimics the pose found for
AChE complexes with tacrine7 and huprine X69 (see text). This
allowed us to focus the sampling effort on the orientation of the
pyrano[3,2-c]quinoline unit of the hybrids at the peripheral
binding site and the linker along the gorge. Each compound
was subjected to 100 docking runs, and the output docking
modes were analyzed by visual inspection in conjunction with
the docking scores.
In Vitro BBB Permeation Assay. Prediction of the brain
penetration was evaluated using a parallel artificial membrane
permeation assay (PAMPA) in a similar manner as described
previously.39,52,95-98 Commercial drugs, phosphate buffered
saline solution at pH 7.4 (PBS), and dodecane were purchased
from Sigma, Aldrich, Acros, and Fluka. Millex filter units
(PVDF membrane, diameter 25 mm, pore size 0.45 μm) were
acquired from Millipore. The porcine brain lipid (PBL) was
obtained from Avanti Polar Lipids. The donor microplate was a
96-well filter plate (PVDF membrane, pore size 0.45 μm), and
the acceptor microplate was an indented 96-well plate, both
from Millipore. The acceptor 96-well microplate was filled with
180 μL of PBS/EtOH (80:20 or 70:30), and the filter surface of
the donor microplate was impregnated with 4 μL of PBL in
dodecane (20 mg mL-1). Compounds were dissolved in PBS/
EtOH (80:20 or 70:30) at 1 mg mL-1, filtered through a Millex
filter, and then added to the donor wells (180 μL). The donor
filter plate was carefully put on the acceptor plate to form a
sandwich, which was left undisturbed for 120 min at 25 ꢀC. After
incubation, the donor plate was carefully removed and the
concentration of compounds in the acceptor wells was deter-
mined by UV spectroscopy. Every sample was analyzed at five
wavelengths, in four wells, and at least in three independent
runs, and the results are given as the mean ( standard deviation.
In each experiment, 15 quality control standards of known BBB
permeability were included to validate the analysis set.
The ligand-protein poses were reranked using the MM-
PBSA approach (Table S4, Supporting Information). An energy
minimization of the complexes was first carried out in order to
reduce steric clashes that might arise from docking calculations.
All minimizations and MM-PBSA calculations were performed
with the parmm99 force field of the Amber-9 package.105 The
relative binding affinities of the best poses of the ligands were
determined by using eq 1.
Acknowledgment. Financial support from the Ministerio
de Ciencia y Tecnologıa (MCT) and FEDER (Grants
´
CTQ2008-03768/PPQ, BQU2006-03794, SAF2008-05595,
SAF2006-04339, SAF2006-01249), Generalitat de Catalunya
(Grants 2005-SGR00180, 2005-SGR00662), MIUR (Grant
PRIN 2007) (Rome, Italy), and EU’s 7FP funding (BISNES,
Grants NMP-2007-1.1-1 and GA n. 214538) and fellowships
sol
nonpolar
ΔGbinding ¼ ΔGMM þ ΔGseolel þ ΔG
-TΔS
ð1Þ
The partial atomic charges for the compounds were derived using
the RESP protocol106 by fitting to the molecular electrostatic
potential calculated at the HF/6-31G* level with Gaussian 03.
The internal conformational energy (ΔGMM) was determined
using the standard formalism and parameters implemented in
AMBER. The electrostatic contribution (ΔGseolel) was computed
using a dielectric constant of 78.4 for the aqueous environment,
while values of 2 and 4 were considered for the ligand-enzyme
complex. The electrostatic potentials were calculated using a
grid spacing of 0.25 A. The interior of the solutes was defined as
the volume inaccessible to a solvent probe sphere of radius 1.4 A.
The nonpolar contribution (ΔGsnoolnpolar) was calculated using a
linear dependence with the solvent-accessible surface.107 Final-
ly, entropy changes upon complexation were assumed to cancel
out in the comparison of the binding affinities of 20 and 25, as
the number of rotatable bonds is the same in both compounds.
However, since the spacer of 27 is larger by two methylene units,
its binding affinity was corrected for the entropic penalty
associated with the freezing of the two extra internal degrees
of freedom using an empirical correction of 0.6 kcal/mol
per rotatable bond.108,109
ꢀ
for C.G. (IBUB) and O.H. (MCT) are acknowledged.
Supporting Information Available: Experimental procedures,
spectral and analytical data of synthesized compounds
(except for 9-14, 17c,e, and 28c,e, 18, and 27, herein described);
solution stability study with 23; table with pKa values of
selected compounds; tables and figures with additional data
on docking and molecular dynamics; PAMPA-BBB studies.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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