Synthesis of quinoline derivatives: Discovery of a potent and selective phosphodiesterase 5 inhibitor for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2012.12.009

Phosphodiesterase type 5 (PDE5) mediates the degradation of cGMP in a variety of tissues including brain. Recent studies have demonstrated the importance of the nitric oxide/cGMP/cAMP-responsive element-binding protein (CREB) pathway to the process of learning and memory. Thus, PDE5 inhibitors (PDE5Is) are thought to be promising new therapeutic agents for the treatment of Alzheimer's disease (AD), a neurodegenerative disorder characterized by memory loss. To explore this possibility, a series of quinoline derivatives were synthesized and evaluated. We found that compound 7a selectively inhibits PDE5 with an IC₅₀ of 0.27 nM and readily crosses the blood brain barrier. In an in vivo mouse model of AD, compound 7a rescues synaptic and memory defects. Quinoline-based, CNS-permeant PDE5Is have potential for AD therapeutic development.

Full text of DOI:10.1016/j.ejmech.2012.12.009

European Journal of Medicinal Chemistry 60 (2013) 285e294
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of quinoline derivatives: Discovery of a potent and selective
phosphodiesterase 5 inhibitor for the treatment of Alzheimer’s disease
Jole Fiorito a, Faisal Saeed a, Hong Zhang a, Agnieszka Staniszewski a, Yan Feng a, Yitshak I. Francis a,
,
a,
**
Sudha Rao a, Devarshi M. Thakkar a, Shi-Xian Deng b, Donald W. Landry b , Ottavio Arancio
*
a Department of Pathology and Cell Biology and Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, 630 W 168th St., New York,
NY 10032, USA
b Department of Medicine, Columbia University, 650 W 168th Street, BB 1029, New York, NY 10032, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphodiesterase type 5 (PDE5) mediates the degradation of cGMP in a variety of tissues including
brain. Recent studies have demonstrated the importance of the nitric oxide/cGMP/cAMP-responsive
element-binding protein (CREB) pathway to the process of learning and memory. Thus, PDE5 inhibi-
tors (PDE5Is) are thought to be promising new therapeutic agents for the treatment of Alzheimer’s
disease (AD), a neurodegenerative disorder characterized by memory loss. To explore this possibility,
a series of quinoline derivatives were synthesized and evaluated. We found that compound 7a selectively
inhibits PDE5 with an IC50 of 0.27 nM and readily crosses the blood brain barrier. In an in vivo mouse
model of AD, compound 7a rescues synaptic and memory defects. Quinoline-based, CNS-permeant
PDE5Is have potential for AD therapeutic development.
Received 19 August 2012
Received in revised form
29 November 2012
Accepted 4 December 2012
Available online 14 December 2012
Keywords:
PDE5 inhibitor
Alzheimer’s disease
Quinoline derivative
cGMP
Ó 2012 Elsevier Masson SAS. All rights reserved.
Memory
Long-term potentiation
1. Introduction
of erectile dysfunction (ViagraÒ, CialisÒ, and LevitraÒ) and subse-
quently pulmonary hypertension [8,9]. Recently, a number of
Phosphodiesterases (PDEs) are a superfamily of enzymes that
degrade the intracellular second messengers, cyclic adenine
monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP), leading to the modulation of several biological processes,
such as intracellular calcium level via Ca2þ-calmodulin signaling
pathway [1]. The PDEs superfamily is classified into 11 families
(PDE1 to PDE11) on the basis of tissue distribution, amino acid
sequences, regulatory properties, substrate specificities and phar-
macological properties [1e3]. Some PDEs specifically bind to cAMP
(PDE4, PDE7, and PDE8); PDE5, PDE6, and PDE9 are highly specific
for cGMP while the remaining PDEs have mixed specificity (PDE1,
PDE2, PDE3, PDE10, and PDE11) [2,3]. Among all PDEs, PDE5 is
widely expressed in a variety of tissues, such as brain, smooth
muscle, lung, platelets and kidney (see Gene Logic’s ASCENTA
System and [4e7]). Due to the PDE5 smooth muscle localization,
inhibitors of this enzyme were initially developed for the treatment
studies have shown a growing interest in PDE5 as a promising
target for the treatment of other diseases, including Alzheimer’s
disease (AD) [2,10,11].
AD is the most common form of dementia and is characterized
by aggregation of amyloid
b peptides (Ab) into fibrillar plaques that
lead to memory loss [12]. The nitric oxide/cGMP/cAMP-responsive
element-binding protein (CREB) pathway has been demonstrated
to play a crucial role in memory, neurotransmission, as well as
hippocampal long-term potentiation (LTP), a type of synaptic
plasticity thought to underline learning and memory [11,13e15]. In
fact, impairment of CREB phosphorylation has been shown to occur
as a result of Ab overproduction and accumulation [15]. Based on
these findings, elevation of cGMP levels through the inhibition of
PDE5 represents an alternative strategy for improving learning and
memory [4,16e18]. In this regard, the PDE5 inhibitor (PDE5I) sil-
denafil (ViagraÒ) has been previously demonstrated to re-establish
normal synaptic and cognitive function in AD mouse models with
a beneficial effect that outlasts the presence of the drug in the blood
stream by several months [4].
* Corresponding author. Tel.: þ1 212 305 5838; fax: þ1 212 305 8466.
** Corresponding author. Tel.: þ1 212 342 0533; fax: þ1 212 305 5498.
E-mail addresses: dwl1@columbia.edu (D.W. Landry), oa1@columbia.edu
(O. Arancio).
None of the PDE5Is were developed for the central nervous
system (CNS) penetration with the selectivity necessary for chronic
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.009

286
J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
administration. Among the commercial PDE5Is, likely due to PDE6
inhibition, sildenafil and vardenafil cause moderate-severe visual
disturbances [19], whereas tadalafil induces back pain as a side
effect, which is likely to be a concern for chronic use in senile
population (incidentally, it is not clear whether this side effect is
due to the inhibition of PDE11 for which the compound is not
selective or other targets) [20e23]. This prompted us to develop
novel selective molecules customized for the CNS. As a strategy, we
first searched for a valid scaffold from a pool of compounds known
to possess PDE5 inhibitory activity. Next, we modified its structure
in order to obtain potent and selective PDE5Is. Most importantly,
during the process of designing new compounds, we considered
physicochemical properties that are important for molecules to
penetrate the bloodebrain barrier (BBB), such as molecular weight,
polar surface area, and number of hydrogen-bond donors and
acceptors [24].
quinoline derivatives (Fig. 1C); 4) isoquinolinone, naphthyridinone
and pyridopyrazinone derivatives (Fig. 1D) [25e30]. Among them,
compounds based on a quinoline structure have demonstrated to
be potent inhibitors with a selectivity for PDE1 through 6 and
unknown selectivity for the remaining PDEs [28]. Therefore, we
decided to use the quinoline scaffold to continue the development
of potent and selective PDE5Is for the treatment of AD. Additionally,
we decided to maintain the cyano group at the C-7 position, the
hydroxymethyl group at the C-3 position, and the benzylamino
moiety of the quinoline ring because they have been shown to be
important for PDE5 potency and selectivity [28]. We, in turn,
focused on the modification of the C-8 position of the quinoline
ring in order to evaluate the influence of a variety of substituents on
PDE5 activity.
In this study we report the synthesis and PDE activity of a series
of quinoline derivatives. Pharmacokinetic (PK) studies of
compound 7a (the most potent PDE5I of our quinoline analogs)
were also performed. Further, we investigated the effect of 7a on
synaptic dysfunction and cognitive abnormalities in two mouse
models of AD, a transgenic model (the APP/PS1 mouse) and a non-
An extensive literature analysis shows a large variety of chem-
ical structures as PDE5Is that can be grouped, on the basis of
their structural similarity, as follows: 1) cGMP-based derivatives,
represented by sildenafil and vardenafil (Fig. 1A); 2)
b-
carbolines-derived molecules, represented by tadalafil (Fig. 1B);
transgenic model in which amyloid-b is infused into dorsal
3) pyrazolopyridine, phthalazine, pyrazolopyridopyridazine and
hippocampi.
Fig. 1. PDE5 inhibitors: A) cGMP-based derivatives; B)
b-carbolines-derived molecules; C) pyrazolopyridine, phthalazine, pyrazolopyridopyridazine and quinoline derivatives; D)
isoquinolinone, naphthyridinone and pyridopyrazinone derivatives.

J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
287
2. Results and discussion
2.2. Biological analysis
2.1. Chemistry
2.2.1. PDE5 inhibitory activity
The PDE5 inhibitory activity of all compounds was determined
by in vitro enzymatic assay (PBS PDE assay kits). All substituents at
C-8 position provided potent PDE5Is (7aef) with potency in the low
nanomolar range (Table 1). Compounds 7a, 7b, and 7f were the
most potent, exceeding sildenafil, vardenafil and tadalafil, with the
least active within two orders of magnitude. The two most potent
compounds, 7a and 7b showed an excellent selectivity against
a panel of all eleven PDEs (Table 2). Importantly, the selectivity of
7a and 7b against PDE6 and PDE11 was much improved compared
to sildenafil, vardanafil and tadalafil. Encouraged by these in vitro
results, 7a was chosen for further evaluation including assessment
of in vivo activity, PK evaluation, electrophysiological analysis and
behavioral studies.
The quinoline derivatives 7aef were synthesized from
commercially available 4-amino-3-bromobenzonitrile 1, which
was condensed with ethoxymethylenemalonate by refluxing in
toluene to yield cluster 2 (Scheme 1). Cyclization of 2 in refluxing
diphenyl ether afforded oxoquinoline 3. The chlorination of 3
with POCl3 provided 4-chloroquinoline 4 in good yield. The
reaction of 4-chloroquinoline 4 with (3-chloro-4-methoxyphenyl)
methanamine hydrochloride in the presence of DIPEA afforded the
quinoline ester 5. Organometallic reactions in the presence of
palladium were used to obtain quinolines 6aef. In particular, the
Suzuki coupling reaction of 5 with cyclopropylboronic acid, in the
presence of Cs2CO3 and a catalytic amount of Pd(PPh3)4 yielded 6a.
Quinolines 6bef were synthesized by BuchwaldeHartwing
coupling with Pd(OAc)2, (R)-BINAP and Cs2CO3, under refluxing
condition. Finally, the reduction of quinoline esters 6aef
with lithium tri(tert-butoxy)aluminum hydride afforded 3-
hydroxymethyl derivatives 7aef.
2.2.2. Hippocampal cGMP levels
To confirm the in vitro data, we measured cGMP levels in adult
mice after treatment with compound 7a. In a series of preliminary
experiments we demonstrated that foot shock induces an
Scheme 1. Synthesis of compounds 7aef. Reagents and conditions: (i) diethyl ethoxymethylenemalonate, toluene, reflux, overnight; (ii) Ph2O, reflux, 2 h; (iii) POCl3, reflux, 48 h;
(iv) (3-chloro-4-methoxyphenyl)methanamine hydrochloride, DIPEA, n-propanol, 2.5 h; (v) cyclopropylboronic acid, Pd(PPh3)4, Cs2CO3, toluene, reflux, overnight; or amines,
Pd(OAc)2, (R)-BINAP, Cs2CO3, toluene, reflux, overnight; (vi) LiAlH(OtBu)3 in THF, reflux, overnight.

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J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
Table 1
compound 7a at different time points are shown in Fig. 3. The data
in Table 3 indicates that 7a is rapidly absorbed as illustrated by the
peak plasma concentration occurring at 0.5 h after dosing. More-
over, the Tmax values in the brain and plasma were similar, indi-
cating that the distribution of 7a to the brain is also fast. Finally, the
amount of 7a in the brain was lower than that in the plasma with an
AUC0-t ratio of 0.41 and the elimination half-lives of 7a in the brain
and plasma were 1.04 and 1.33, respectively.
PDE5 inhibitory activity of compounds 7aef compared to sildenafil.
2.2.4. Electrophysiological and behavioral studies
Natural oligomers of human Ab42, in the absence of monomers
and fibrils, markedly reduce memory and LTP [31e36]. We therefore
determined whether compound 7a could attenuate LTP and memory
defects following elevation of Ab42 in the presence of oligomeric
A
b42, or vehicle. For LTP, 200 nM Ab42 or vehicle were perfused
through the bath solution for 20 min prior to the tetanus. For
memory, 200 nM Ab42 (in a final volume of 1 l over 1 min) or vehicle
were bilaterally infused, 15 min prior to fear memory induction
through a foot shock, into the hippocampus of the mouse that had
been pre-implanted with cannulas the week before. Ab42 reduced
Compd
R8
PDE5 IC50 (nM)
m
7a
7b
0.277
0.40
LTP (144.65
ꢀ
9.99 potentiation in Ab42-treted slices vs.
212.35 ꢀ 18.62 in vehicle-treated slices) and contextual memory
(w10% freezing in Ab42-infused mice vs. w30% in vehicle-treated
mice) (Fig. 4A and B). In turn, compound 7a (50 nM, for 10 min
7c
4.3
prior to the q-burst in the LTP experiments; 3 or 10 mg/kg, p.o.,
immediately after training in the behavioral experiments) rescued
the LTP (192.63 ꢀ 8.91 potentiation; F(1,11) ¼ 6.073, p ¼ 0.0314
comparing Ab42-treated slices vs. Ab42 þ 7a treated slices) and
behavioral defects (w30% freezing in mice treated with both 3 and
10 mg/kg 7a þ Ab42; p ¼ 0.113 or p ¼ 0.018 comparing 3 and 10 mg/kg
7a þ Ab42 treated mice vs. Ab42-treated mice, respectively; Fig. 4A
and B). Collectively, these experiments suggest that 7a can rescue the
damage to synaptic plasticity and memory caused by Ab42 elevation.
Our next goal was to determine whether compound 7a was
capable of rescuing synaptic and memory defects in APP/PS1 mouse
model, an amyloid-depositing animal that represents a more
“physiological” approach than exogenous application of Ab42. We
induced LTP or fear memory in 3e4 month old transgenic animals
and their wild-type (WT) littermates either treated with 7a or
vehicle. As previously shown [37,38], vehicle-treated APP/PS1 mice
showed a reduction in LTP and contextual memory (Fig. 5A and B).
Compound 7a (50 nM, through the bath perfusion, for 10 min prior
to the tetanus for LTP; 3 mg/kg, i.p, for 3 weeks in behavioral
experiments), in turn, rescued the defect in LTP and memory in the
transgenic animals. We found 180.85 ꢀ 11.46 potentiation in slices
from APP/PS1 mice treated with compound vs. 145.84 ꢀ 7.78 in
slices from APP/PS1 mice treated with vehicle (F(1,13) ¼ 8.958,
7d
7e
15.0
4.1
7f
0.63
3.4
Sidenafil
immediate increase in cGMP levels in the hippocampus (Fig. 2).
Compound 7a (3 mg/kg, i.p., 30 min prior to electric shock) further
enhanced cGMP levels at different time points (10 s, 1 min, and
3 min) (0.83 ꢀ 0.059, 0.69 ꢀ 0.055, and 0.89 ꢀ 0.0786 after 10 s, 60 s
and 3 min, respectively) as compared to vehicle (0.318 ꢀ 0.0145,
0.41 ꢀ 0.028, and 0.46 ꢀ 0.0385 after 10 s, 60 s and 3 min,
respectively) (Fig. 2).
2.2.3. Pharmacokinetics
The PK study of compound 7a (50 mg/kg, p.o.) was conducted in
male BALB/c mice. The plasma and brain concentrations of
Table 2
PDE5 selectivity of compounds 7a and 7b compared to sildenafil, vardenafil and tadalafil.
Compd
7aa
PDE1
PDE2
PDE3
PDE4
PDE5
PDE6
339
1256
5100
PDE7
PDE8
PDE9
PDE10
PDE11
IC50 (nM)
PDEX/PDE5
IC50 (nM)
PDEX/PDE5
IC50 (nM)
PDEX/PDE5
IC50 (nM)
PDEX/PDE5
IC50 (nM)
PDEX/PDE5
>104
>104
>104
>104
1500
682
>104
>104
>104
>104
>104
>104
3100
3100
>104
>104
>104
>104
>104
>104
>104
>104
580
>104
>104
>104
>104
>104
>104
3800
3800
9200
7667
0.27
1
0.40
1
2.20
1
1.0
1
>104
>104
>104
>104
>104
>104
1900
1900
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
5600
2545
680
>104
>104
>104
>104
6800
3091
880
>104
>104
>104
>104
6100
2773
240
240
10
8
7ba
12,750
Sildenafilb
Vardenafilb
Tadalafilb
9.5a
4
300
11.0c
11
300
580
680
880
>104
>104
>104
>104
1.2
1
5200d
>104
>104
>104
>104
4333
a
Data obtained by BPS Bioscience, CA, USA.
G.L. Card et al., Structural basis for the activity of drugs that inhibit phosphodiesterases, structure 12 (2004) 2233e2247.
I. Saenz de Tejada et al., The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil, Intern. J. Impot. Res. 13
b
c
(2001) 282e290.
d
A. Daugan et al., The discovery of Tadalafil, a novel and highly selective PDE5 inhibitor. 2: 2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
analogues, J. Med. Chem. 46 (2003) 4533e4542.

J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
289
w30% of the time compared to w10% in vehicle-treated transgenic
mice (p ¼ 0.067, Fig. 5E) and made w1 error in the 2-day RAWM
test compared to w4 in vehicle-treated transgenic mice
(F(1,26) ¼ 4.454, p ¼ 0.045, Fig. 5F). Thus, synaptic and cognitive
improvements persist beyond the administration of the inhibitor.
3. Conclusions
A series of quinoline derivatives were synthesized and evaluated
for PDE5 inhibitory activity. Among the synthesized derivatives,
compound 7a showed to possess higher potency and selectivity
compared to sildenafil, vardenafil and tadalafil. Compound 7a
increased the level of cGMP in mouse hippocampus and rescued
the defects in synaptic plasticity and memory. The in vivo activity of
7a, along with its good pharmacokinetics profile, supports the
potential of PDE5Is for the treatment of AD and encourages us to
pursue the drug optimization of this class of molecules.
Fig. 2. Hippocampal cGMP levels measurement. Concentration of cGMP was measured
by enzyme immunoassay. Basal represents cGMP levels without foot shock. Values are
the mean of duplicate determinations. Errorbars show S.E.M. (n ¼ 3 per group); *p < 0.01.
4. Experimental section
p ¼ 0.0104, Fig. 5A). Transgenic mice treated with the compound
froze w30% of the time when they were exposed to the same
context in which they had received an electric shock compared to
w10% in vehicle-treated APP/PS1 mice (p ¼ 0.011, Fig. 5B). Addi-
tionally, the beneficial effect of compound 7a could be extended to
spatial short-term memory, a type of memory that can be assessed
in transgenic mice using the 2-day radial arm water maze (RAWM)
[39]. APP/PS1 mice treated with the compound made less than 2
errors in the 2-day RAWM test vs. w3 errors in vehicle-treated APP/
PS1 mice (F(1,28) ¼ 12.21, p ¼ 0.002, Fig. 5C). Thus, findings obtained
4.1. Chemistry
4.1.1. Materials and methods
Solvents and reagents were obtained from commercial suppliers
and were used without further purification. Flash chromatography
purification was performed on a Merck silica gel 60 0.040e
0.063 mm (230e400 mesh) stationary phase. 1H NMR and 13C
NMR spectra were recorded using Varian INOVA (300 MHz for 1H
and 75 MHz for 13C) and Agilent-NMR-vnmrs400 (400 MHz for 1H)
spectrometers in CDCl3, DMSO-d6 or acetone-d6. TMS was used as
an internal standard. Coupling constants (J) are reported in hertz.
Thin-layer chromatography (TLC) was performed on silica gel plates
with a fluorescence indicator of F254 (0.2 mm, Merck); the spots
were visualized by UV light. The mass spectra were recorded on
Shimadzu LCMS-2010A Liquid Chromatography Mass Spectrom-
eter. Elemental analyses were obtained by Atlantic Microlab (www.
atlanticmicrolab.com).
with the Ab preparation are valid also in transgenic mice.
Our preliminary studies with sildenafil have demonstrated that
PDE5 inhibition has a prolonged beneficial effect on synaptic and
cognitive abnormalities in APP/PS1 mice that persists beyond the
administration of the inhibitor. This finding suggests the possibility
of using this class of compounds to interfere with the progression of
the memory deficits. Does this important therapeutic possibility
occur with 7a? In these experiments, both APP/PS1 and WT mice of
3 months of age were i.p. injected with 3 mg/kg/day 7a for 3 weeks,
then the treatment was stopped for 9e12 weeks prior to testing.
Mice were next subjected to fear conditioning and 2-day RAWM.
Finally, they were sacrificed for electrophysiological and
biochemical studies. We found that slices from transgenic mice
treated with the compound had 180.90 ꢀ 17.91 potentiation
compared to 112.29 ꢀ 9.12 in slices from vehicle-treated transgenic
mice (F(1,14) ¼ 12.32, p ¼ 0.0035, Fig. 5D). In the contextual memory
experiments, transgenic mice treated with the compound froze
4.1.2. Synthesis of compounds 2e5 and 6a
4.1.2.1. Diethyl 2-[(2-bromo-4-cyanophenylamino)methylene]malo-
nate (2). Diethyl ethoxymethylenemalonate (8.23 g, 38.1 mmol)
was added to a solution of 1 (5.00 g, 25.4 mmol) in 30 mL of toluene.
The mixture was then heated to refluxovernight with the condenser
open to the air. The resulting solution was cooled down to room
temperature and poured into 100 mL of hexanes. The white
precipitate was collected and washed with hexanes (3 ꢁ 30 mL) to
yield 11.9 g of an off-white solid as the desired product. MS ESI (m/z)
367 (M þ 1)þ; 1H NMR (CDCl3, 300 MHz):
11.44 (d,1H, J ¼ 12.6 Hz),
d
1200
8.44 (d, 1H, J ¼ 12.9 Hz), 7.86 (d, 1H, J ¼ 1.8 Hz), 7.63 (dd, 1H,
J1 ¼1.8 Hz, J2 ¼ 8.7 Hz), 7.33 (d,1H, J ¼ 8.4 Hz), 4.35 (q, 2H, J ¼ 6.9 Hz),
4.27 (q, 2H, J ¼ 6.9 Hz), 1.38 (t, 3H, J ¼ 6.9 Hz), 1.33 (t, 3H, J ¼ 6.9 Hz).
1000
Plasma
800
4.1.2.2. Ethyl 8-bromo-6-cyano-4-hydroxyquinoline-3-carboxylate
(3). 100 mL of diphenyl ether was heated to reflux followed by
addition of 2 (5.00 g, 13.6 mmol) in portions over 30 min. The
resulting brown solution was refluxed for another hour and then
cooled down to room temperature. The precipitate was collected
and washed with hexanes (3 ꢁ 15 mL) to give 5.69 g of a light brown
solid as the desired product. MS ESI (m/z) 321 (M þ 1)þ; 1H NMR
Brain
600
400
200
0
(DMSO-d6, 300 MHz): d 11.9 (s, 1H), 8.52 (s, 1H), 8.45 (s, 1H), 8.42 (s,
1H), 4.21 (q, 2H), 1.25 (t, 3H).
0
1
2
3
4
Time (h)
4.1.2.3. Ethyl 8-bromo-4-chloro-6-cyanoquinoline-3-carboxylate (4).
The mixture of 3 (4.0 g, 12.45 mmol) and 50 mL of POCl3 was heated
to reflux for 48 h. The solvent was removed in vacuum and co-
Fig. 3. Concentrationetime curve of 7a in mouse brain tissue and plasma (n ¼ 3 mice
per group).

290
J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
Table 3
and purified by flash chromatography (Hex:AcOEt 4:1) to yield
366 mg of a yellow solid as the desired compound. MS ESI (m/z) 436
Pharmacokinetic parameters of compound 7a in mouse brain tissue and plasma.
(M þ 1)þ, 1H NMR (CDCl3, 400 MHz):
9.56 (t,1H, J ¼ 5.6 Hz), 9.27 (s,
d
Parameters
Compound 7a
1H), 8.30 (d,1H, J ¼ 1.6 Hz), 7.38 (d,1H, J ¼ 2.0 Hz), 7.24e7.22 (m, 2H),
6.95 (d,1H, J ¼ 8.4 Hz), 4.83 (d, 2H, J ¼ 5.6 Hz), 4.37 (q, 2H, J ¼ 7.2 Hz),
3.91 (s, 3H), 3.12e3.05 (m, 1H), 1.39 (t, 3H, J ¼ 7.2 Hz), 1.21e1.16 (m,
2H), 0.82e0.77 (m, 2H).
Brain
Plasma
Ratioa
Tmax
Cmax
AUC0-t
AUC0-N
T1/2
(h)
0.5
0.5
e
(ng/mL or ng/g)
(ng h/mL or ng h/g)
(ng h/mL or ng h/g)
(h)
385
418
420
1.04
1.66
1022
1014
1133
1.33
1.61
0.38
0.41
0.37
e
4.1.3. General synthetic procedure for BuchwaldeHartwing
coupling reactions (6bef)
MRT
(h)
e
a
Ratio ¼ brain/plasma; 50 mg/kg, p.o.; vehicle for 7a is 0.5% methylcellulose
To a solution of quinoline bromide 5 (1.0 mmol) in toluene
anhydrous was added palladium (II) acetate (0.02 mmol), (R)-BINAP
(0.01 mmol), Cs2CO3 (0.5 mmol), and the amine (0.5 mmol). After
the mixture was refluxed overnight and under nitrogen, the
precipitate was removed by filtration. The filtrate was concentrated
and purified by flash chromatography.
aqueous solution; AUC: area under curve; MRT: mean residence time.
distilled with CHCl3 (50 mL) and toluene (2 ꢁ 50 mL). The resulting
dark brown syrup was dissolved in 50 mL of CH2Cl2 and treated
with Et3N until pH > 10. The dark-red solution was then allowed to
go through a silica gel pad (3 cm ꢁ 4 cm). The silica pad was washed
with 100 mL of CH2Cl2. The filtrates were collected and concen-
trated to obtain an off-white solid as the desired product. MS ESI
4.1.3.1. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(N,N-
dimethylamino) quinoline-3-carboxylate (6b). Flash chromatog-
raphy (Hex:AcOEt 2:1); yellow solid, yield: 32%; MS ESI (m/z) 439
(m/z) 321 (M þ 1)þ; 1H NMR (CDCl3, 300 MHz):
d 9.41 (s, 1H), 8.78
(M þ 1)þ; 1H NMR (CDCl3, 300 MHz):
9.42 (t,1H, J ¼ 5.4 Hz), 9.16 (s,
d
(s, 1H), 8.31 (s, 1H), 4.52 (q, 2H, J ¼ 6.9 Hz), 1.47 (t, 3H, J ¼ 6.9 Hz).
1H), 8.00 (d,1H, J ¼ 1.5 Hz), 7.38 (d,1H, J ¼ 2.1 Hz), 7.27e7.23 (m,1H),
7.12 (d,1H, J ¼ 1.8 Hz), 6.96 (d,1H, J ¼ 8.4 Hz), 4.81 (d, 2H, J ¼ 5.7 Hz),
4.38 (q, 2H, J ¼ 7.2 Hz), 3.92 (s, 3H), 3.09 (s, 6H),1.41 (t, 3H, J ¼ 7.2 Hz).
4.1.2.4. Ethyl 8-bromo-4-[(3-chloro-4-methoxybenzyl)amino]-6-
cyanoquinoline-3-carboxylate (5). Compound 4 (4.5 g, 13.2 mmol),
3.12 g of (3-chloro-4-methoxyphenyl)methanamine hydrochloride
(15 mmol), and 7.74 g of diisopropylethylamine were dissolved in
50 mL of n-propanol. The resulting mixture was refluxed for 2.5 h
and then poured to 100 mL of ice-water. The precipitate was
collected by filtration and washed with H2O (2 ꢁ 30 mL) and
ethanol (2 ꢁ 30 mL) to give 5.0 g of a yellow solid as the title
compound. MS ESI (m/z) 474 (M þ 1)þ, 1H NMR (CDCl3, 300 MHz):
4.1.3.2. Ethyl
4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-
(6c).
(N,N-dimethylethane-1,2-diamimo)quinoline-3-carboxylate
Flash chromatography (AcOEt:MeOH 10:1); yellow solid, yield:
60%; MS ESI (m/z) 482 (M þ1)þ; 1H NMR (CDCl3, 400 MHz):
d 9.47
(t, 1H, J ¼ 5.2 Hz), 9.06 (s, 1H), 7.69 (d, 1H, J ¼ 1.6 Hz), 7.37 (d, 1H,
J ¼ 2.4 Hz), 7.24e7.22 (m, 1H), 6.94 (d, 1H, J ¼ 8.4 Hz), 6.69 (t, 1H,
J ¼ 4.8 Hz), 6.66 (d, 1H, J ¼ 1.6 Hz), 4.85 (d, 2H, J ¼ 5.2 Hz), 4.34 (q,
2H, J ¼ 7.2 Hz), 3.90 (s, 3H), 3.30 (q, 2H, J ¼ 5.8 Hz), 2.67 (t, 2H,
J ¼ 6.2 Hz), 2.31 (s, 6H), 1.38 (t, 3H, J ¼ 7.2 Hz).
d
9.89 (br s, 1H), 9.32 (s, 1H), 8.49 (d, 1H, J ¼ 1.5 Hz), 8.15 (d, 1H,
J ¼ 1.8 Hz), 7.39 (d, 1H, J ¼ 2.1 Hz), 7.25 (dd, 1H, J1 ¼ 2.1 Hz,
J2 ¼ 8.1 Hz), 6.97 (d, 1H, J ¼ 8.7 Hz), 4.87 (d, 2H, J ¼ 5.4 Hz), 4.38 (q,
2H, J ¼ 7.2 Hz), 3.92 (s, 3H), 1.40 (t, 3H, J ¼ 7.2 Hz).
4.1.3.3. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(eth-
ylamino)quinoline-3-carboxylate
(6d). Flash
chromatography
4.1.2.5. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-
cyclopropylquinoline-3-carboxylate (6a). To a mixture of 5 (475 mg,
1 mmol) in 5 mL of toluene anhydrous 129 mg of cyclopropylboronic
acid (1.5 mmol), 58 mg of tetrakis(triphenylphosphine)palladium(0)
(0.05 mmol) and 815 mg of Cs2CO3 (2.5 mmol) were added. The
mixture was refluxed overnight under nitrogen and then the
precipitate was removed by filtration. The filtrate was concentrated
(Hex:AcOEt 2:1); yellow solid, yield: 76%; MS ESI (m/z) 439
(M þ 1)þ; 1H NMR (CDCl3, 300 MHz):
9.50 (t, 1H, J ¼ 5.4 Hz), 9.02
d
(s, 1H), 7.68 (d, 1H, J ¼ 1.5 Hz), 7.38 (d, 1H, J ¼ 2.4 Hz), 7.23 (d, 1H,
J ¼ 2.1 Hz), 6.95 (d, 1H, J ¼ 8.4 Hz), 6.66 (s, 1H), 6.29 (t, 1H,
J ¼ 5.1 Hz), 4.85 (d, 2H, J ¼ 5.7 Hz), 4.36 (q, 2H, J ¼ 7.2 Hz), 3.92 (s,
3H), 3.28 (m, 2H), 1.39 (t, 6H, J ¼ 7.2 Hz).
Fig. 4. Beneficial effect of compound 7a on Ab42-induced synaptic and cognitive dysfunction. A) 7a (50 nM through the bath solution for 10 min prior to the q-burst) ameliorates the LTP
deficit in Ab42-treated slices. The graph represents the average of the last 5 min of recording at 60 min after the tetanus; n represents the number of slices per group. B) 7a (3 or 10 mg/kg,
p.o., immediately after the electric shock in the behavioral experiments) ameliorates the contextual fear memory deficit in Ab42-infused mice; n represents the number of mice per group.

J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
291
Fig. 5. Beneficial effect of compound 7a on synaptic and cognitive deficits in APP/PS1 mice. A) Residual potentiation recorded during the last 5 min of a 2 h recording following
tetanic stimulation of the Schaffer collateral fibers at the CA3-CA1 connection. Compound 7a (50 nM through the bath solution for 10 min prior to the -burst) rescued the LTP
q
defect in transgenic slices, whereas had no effect onto WT slices. B) Daily treatment with compound 7a (3 mg/kg, i.p.) for 3 weeks at the age of 3e4 months re-established normal
freezing in a test for contextual fear memory. C) Daily treatment with compound 7a (3 mg/kg, i.p.) for 3 weeks at the age of 3e4 months reduced the number of errors with the 2-
day radial arm water maze. D) Residual potentiation recorded during the last 5 min of a 2 h recording following tetanic stimulation of the Schaffer collateral fibers at the CA3-CA1
connection. Daily treatment with compound 7a (3 mg/kg, i.p.) for 3 weeks at the age of 3e4 months re-established normal potentiation when slices were recorded at 6e7 months of
age. E) Daily treatment with compound 7a (3 mg/kg, i.p.) for 3 weeks at the age of 3e4 months re-established normal freezing in a test for contextual fear memory when mice were
examined at 6e7 months of age. F) Daily treatment with compound 7a (3 mg/kg, i.p.) for 3 weeks at the age of 3e4 months reduced the number of errors with the 2-day radial arm
water maze when mice were examined at 6e7 months of age.
4.1.3.4. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-(N-
cyclopropylamino) quinoline-3-carboxylate (6e). Flash chromatog-
raphy (Hex:AcOEt 8:2); yellow solid, yield: 80%; MS ESI (m/z) 451
30 min at room temperature. The mixture was poured into a sepa-
ratory funnel, followed by addition of 150 mL of CH2Cl2 and 50 mL
of 1 N NaOH, the organic layer was separated, washed with 1 N
NaOH (50 mL) and dried over MgSO4. The solid was filtered off and
the filtrate was concentrated to give the final compound.
(M þ 1)þ; 1H NMR (CDCl3, 300 MHz):
9.53 (t, 1H, J ¼ 5.1 Hz), 8.99
d
(s, 1H), 7.74 (s, 1H), 7.38 (d, 1H, J ¼ 1.8 Hz), 7.23 (s, 1H), 7.12 (s, 1H),
6.95 (d, 1H, J ¼ 8.4 Hz), 6.59 (s, 1H), 4.85 (d, 2H, J ¼ 5.4 Hz), 4.35 (q,
2H, J ¼ 7.2 Hz), 3.91 (s, 3H), 2.53e2.50 (m, 1H), 1.39 (3H, J ¼ 7.2 Hz),
0.89e0.83 (m, 2H), 0.66e0.61 (m, 2H).
4.1.4.1. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-cyclopropyl-3-
(hydroxymethyl) quinoline-6-carbonitrile (7a). White solid, yield:
62%; MS ESI (m/z) 394 (M þ 1); 1H NMR (DMSO-d6, 300 MHz):
4.1.3.5. Ethyl
4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-
d
8.69 (d, 1H, J ¼ 1.2), 8.48 (s, 1H), 7.42 (t, 1H, J ¼ 7 Hz), 7.37 (d, 1H,
(morpholin-4-yl) quinoline-3-carboxylate (6f). Flash chromatogra
phy (Hex:AcOEt 8:2); yellow solid; yield: 70%; MS ESI (m/z)
J ¼ 2.1 Hz), 7.33 (d, 1H, J ¼ 1.2 Hz), 7.21 (dd, 1H, J1 ¼ 8.4 Hz,
J2 ¼ 2.1 Hz), 7.08 (d, 1H, J ¼ 8.4 Hz), 5.38 (t, 1H, J ¼ 5.1 Hz), 4.79 (d,
2H, J ¼ 7 Hz), 4.43 (d, 2H, J ¼ 5.1 Hz), 3.79 (s, 3H), 3.09e3.14 (m, 1H),
1.02e1.08 (m, 2H), 0.72e0.87 (m. 2H); 13C NMR (CDCl3, 75 MHz)
154.01, 150.61, 149.30, 148.67, 145.00, 134.76, 128.44, 126.72, 123.09,
121.62, 120.26, 120.09, 116.56, 113.56, 109.98, 107.05, 107.05, 56.88,
51.45, 48.40, 10.97. Anal. Calcd. for C22H20ClN3O2: C, 67.09; H, 5.12;
N, 10.67. Found: C, 66.92; H, 5.06; N, 10.58.
481(M þ 1)þ; 1H NMR (CDCl3, 300 MHz):
d
9.49 (t, 1H, J ¼ 5.1 Hz),
9.15 (s, 1H), 8.08 (s, 1H), 7.36 (s, 1H, J ¼ 2.4 Hz), 7.22 (dd, 1H,
J1 ¼ 2.4 Hz, J2 ¼ 8.7 Hz), 7.15 (s, 1H), 6.95 (d, 1H, J ¼ 8.7 Hz), 4.80 (d,
2H, J ¼ 5.4 Hz), 4.35 (q, 2H, J ¼ 7.2 Hz), 3.99 (s, 4H), 3.90 (s, 3H), 3.35
(s, 4H), 1.38 (t, 3H, J ¼ 7.2 Hz).
4.1.4. General procedure for the reduction of 3-ethylquinoline esters
(7aef)
Lithium tri(tert-butoxy) aluminum hydride 1 M sol. in THF
(2.2 mmol) was added to a solution of 3-ethyl-quinoline ester
(0.43 mmol) in THF anhydrous. The resulting solution was refluxed
overnight and then quenched with 1 mL of methanol and stirred for
4.1.4.2. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(N,N-dimethyla-
mino)-3-(hydroxymethyl) quinoline-6-carbonitrile (7b). Yellow
solid, yield: 27%; MS ESI (m/z) 397 (M þ 1); 1H NMR (DMSO-d6,
400 MHz):
d
8.37 (s, 1H), 8.28 (d, 1H, J ¼ 1.2 Hz), 7.34 (d, 1H,
J ¼ 2.4 Hz), 7.20e7.15 (m, 2H), 7.07e7.05 (m, 2H), 5.31 (t, 1H,

292
J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
J ¼ 5.2 Hz), 4.72 (d, 2H, J ¼ 6.8 Hz), 4.41 (d, 2H, J ¼ 5.2 Hz), 3.77 (s,
3H), 2.99 (s, 6H). Anal. Calcd. for C21H21ClN4O2: C, 63.55; H, 5.33; N,
14.12. Found: C, 63.42; H, 5.41; N, 13.55.
liquid nitrogen. Levels of cGMP were quantitated by Enzyme
Immunoassay procedure (Cayman Chemical Company, Item no.
581021) following the manufacturer’s guidelines in duplicate.
cGMP levels were normalized with the protein concentration
calculated using BCA Protein Assay Reagent (Thermo Scientific).
4.1.4.3. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(N,N-dimethyl-
ethane-1,2-diamino)-3-(hydroxymethyl) quinoline-6-carbonitrile
(7c). Yellow solid, yield: 50%; MS ESI (m/z) 440 (M þ 1)þ; 1H
4.4. Pharmacokinetics
NMR (DMSO-d6, 400 MHz):
d 8.28 (s, 1H), 7.93 (s, 1H), 7.32 (d, 1H,
J ¼ 2.0 Hz), 7.22 (t, 1H, J ¼ 7.0 Hz), 7.17 (dd, 1H, J1 ¼ 2.2 Hz,
J2 ¼ 8.6 Hz), 7.05 (d, 1H, J ¼ 8.4 Hz), 6.72 (t, 1H, J ¼ 5.2 Hz), 6.67 (s,
1H), 5.30 (t, 1H, J ¼ 5.0 Hz), 4.74 (d, 2H, J ¼ 6.8 Hz), 4.37 (d, 2H,
J ¼ 5.2 Hz), 3.76 (s, 3H), 3.23 (q, 2H, J ¼ 5.6 Hz), 2.51 (t, 2H,
J ¼ 6.2 Hz), 2.16 (s, 6H). Anal. Calcd. for C23H26ClN5O2$½H2O: C,
61.53; H, 6.06; N, 15.60. Found: C, 61.57; H, 6.02; N, 14.95.
To determine the time course of compound 7a action in the
brain, we investigated the plasma pharmacokinetics and BBB
penetration capability of the inhibitor. In these experiments, 7a was
administered to mice p.o. at a dosage of 50 mg/kg. Blood and brain
samples were collected at six time points (0, 0.25, 0.5, 1.0, 2.0, and
4.0 h) from three animals at each time point. For plasma
measurements, blood (approximately 250
ml) was collected via
4.1.4.4. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-ethylamino-3-
(hydroxymethyl) quinoline-6-carbonitrile (7d). Yellow solid, yield:
72%; MS ESI (m/z) 497 (M þ 1)þ; 1H NMR (DMSO-d6, 400 MHz):
retro-orbital puncture into tubes containing sodium heparin anti-
coagulant. Plasma was separated via centrifugation (4 ꢂC,
3500 rpm, 10 min) and stored at ꢃ80 ꢂC. At the time of measure-
ment, frozen plasma samples were thawed at room temperature
d
8.27 (s, 1H), 7.91 (d, 1H, J ¼ 1.2 Hz), 7.32 (d, 1H, J ¼ 2.4 Hz), 7.22e
7.15 (m, 2H), 7.05 (d, 1H, J ¼ 8.8 Hz), 6.64 (d, 1H, J ¼ 1.2 Hz), 6.57 (t,
1H, J ¼ 5.6 Hz), 5.30 (t, 1H, J ¼ 4.8 Hz), 4.73 (d, 2H, J ¼ 7.2 Hz), 4.38
(d, 2H, J ¼ 5.2 Hz), 3.76 (s, 3H), 3.22 (m, 2H), 1.99 (t, 3H, J ¼ 7.2 Hz).
Anal. Calcd. for C21H21ClN4O2$½H2O: C, 63.55; H, 5.33; N, 14.12.
Found: C, 62.75; H, 5.28; N, 13.43.
and vortexed thoroughly. Plasma (25
a 1.5 mL Eppendorf tube. To each sample, 25
25 l of the internal standard were added, followed by the addition
of 100 l of methanol. The sample mixture was vortexed for
ml) was transferred into
ml of methanol and
m
m
approximately 1 min. After centrifugation at 11,000 g for 5 min, the
upper organic layer was transferred to a glass tube and evaporated
at 40 ꢂC under a gentle stream of nitrogen. Residues were dissolved
4.1.4.5. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(N-cyclo-
propylamino)-3-(hydroxymethyl)
quinoline-6-carbonitrile
(7e).
in 150
ml of the mobile phase and mixed using a Vortex mixer. A
Yellow solid, yield: 85%; MS ESI (m/z) 409 (M þ 1)þ; 1H NMR
20 l aliquot of the resulting solution was injected onto the liquid
m
((CD3)2CO, 400 MHz)
d
8.38 (S, 1H), 7.87 (s, 1H), 7.44 (d, 1H,
chromatography/tandem mass spectrometry (LC/MS/MS) system
for analysis. For measurement of brain concentrations, mice were
killed by cervical dislocation after blood harvest. Brains were
immediately excised, weighed, and rinsed by cold saline and then
frozen at ꢃ80 ꢂC until further processing for LC/MS/MS analysis. On
the day of the assay, frozen tissue samples were thawed unassisted
at room temperature. When completely thawed, each tissue sample
of 200 mg was weighed and placed into a plastic tube. Methanol
(1.0 mL) was added and homogenization conducted using a Fluko
F6/10 superfine homogenizer for approximately 1 min. Then, the
J ¼ 2.4 Hz), 7.33e7.30 (m, 1H), 7.07 (d, 1H, J ¼ 8.8 Hz), 7.01 (s, 1H),
6.77 (br s,1H), 6.59 (t,1H, J ¼ 6.8 Hz), 4.91 (d, 2H, J ¼ 7.2 Hz), 4.68 (d,
2H, J ¼ 5.6 Hz), 4.45 (t, 1H, J ¼ 5.6 Hz), 3.87 (s, 3H), 2.61e2.56 (m,
1H), 0.90e0.86 (m, 2H), 0.63e0.60 (m, 2H). Anal. Calcd. for
C22H21ClN4O2: C, 64.62; H, 5.18; N, 13.70. Found: C, 64.08; H, 5.44;
N, 13.72.
4.1.4.6. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-(morpholin-4-yl)-
3-(hydroxymethyl)-quinoline-6-carbonitrile (7f). Yellow solid, yield:
70%; MS ESI (m/z) 439 (M þ 1)þ; 1H NMR (DMSO-d6, 400 MHz):
samples were vortexed for 1 min and a 25
into an Eppendorf tube. To each sample, 25
of the internal standard were added and the samples centrifuged at
11,000 g for 5 min. A 20 l aliquot of the supernatants was diluted to
60 l with the mobile phase, and a 10 l aliquot was injected onto
ml aliquot was transferred
d
8.40 (s, 1H), 8.37 (s, 1H), 7.33 (d, 1H, J ¼ 2.0 Hz), 7.25 (t, 1H,
ml of methanol and 25
ml
J ¼ 6.6 Hz), 7.18e7.14 (m, 2H), 7.05 (d, 1H, J ¼ 8.8 Hz), 5.31 (t, 1H,
J ¼ 5.0 Hz), 4.72 (d, 2H, J ¼ 6.8 Hz), 4.38 (d, 2H, J ¼ 5.2 Hz), 3.76 (s,
7H), 3.30e3.29 (m, 4H). Anal. Calcd. for C23H23ClN4O3: C, 62.94; H,
5.28; N, 12.77. Found: C, 62.95; H, 5.50; N, 12.10.
m
m
m
the LC/MS/MS system for analysis. Quantification of the drug
concentration in each aliquot was achieved by the internal standard
method using peak area ratios of the analyte to the internal stan-
dard in plasma and brain. Concentrations were calculated using
a weighted least-squares linear regression (W ¼ 1/ꢁ2).
4.2. PDE enzyme assay procedure
The enzymatic reactions were conducted at room temperature
for 60 min in a 50
FAM-cGMP or FAM-cAMP substrate, 0.125 ng PDE, and the test
compound. After the enzymatic reaction, 100 l of a binding solu-
ml volume containing PDE assay buffer, 100 nM
4.5. Cannula infusion techniques
m
tion (1:100 dilution of the binding agent with the binding agent
diluent) was added to each sample. After 60 min at room temper-
ature fluorescence intensity was measured at an excitation of
485 nm and an emission of 528 nm using a Tecan Infinite M1000
microplate reader.
Following anesthesia with 20 mg/kg Avertin, mice were
implanted with 26-gauge guide cannulas into the dorsal part of the
hippocampi (coordinates: P ¼ 2.46 mm, L ¼ 1.50 mm to a depth of
1.30 mm) [40]. The cannulas were fixed to the skull with acrylic
dental cement (made from Paladur powder). After 6e8 days, mice
were bilaterally infused with A
b (200 nM) or vehicle in a final
4.3. Hippocampal cGMP levels
volume of 1 l over 1 min with a microsyringe connected to the
m
cannulas via polyethylene tubing.
4.6. Electrophysiological studies
Transverse hippocampal slices (400
2e3 month old male and female mice (20e25 g; C57Bl6 mice)
were injected with 7a (3 mg/kg, 2% DMSO & 2% Tween, i.p.) or
Vehicle (2% DMSO & 2% Tween, i.p.). 30 min after administration of
vehicle or 7a, mice were subjected to foot shock and sacrificed 10 s,
1 min and 3 min after shock by cervical dislocation and decapita-
tion. The hippocampal samples were extracted and snap frozen in
m
m) were cut with a tissue
chopper (EMS, PA) and maintained in an interface chamber at 29 ꢂC
for 90 min prior to recording, as previously described [14]. The

J. Fiorito et al. / European Journal of Medicinal Chemistry 60 (2013) 285e294
293
extracellular bath solution consisted of 124.0 mM NaCl, 4.4 mM KCl,
1.0 mM Na2HPO4, 25.0 mM NaHCO3, 2.0 mM CaCl2, 2.0 mM MgSO4,
and 10.0 mM glucose, continuously aerated with 95% O2/5% CO2 to
a final pH of 7.4. Field extracellular postsynaptic responses (fEPSPs)
were recorded by placing the stimulating and recording electrodes
in CA1 stratum radiatum. A bipolar tungsten electrode (FHC,
Bowdoin, ME) was used as a stimulating electrode, and a glass
pipette filled with bath solution was used as a recording electrode.
Basal synaptic transmission was first assessed by plotting the
stimulus voltages (V) against slopes of fEPSP to generate inpute
output relations. A 15 min baseline was first recorded every
minute at an intensity that evoked a response at approximately 35%
of the maximum evoked response. LTP was induced using a theta-
burst stimulation (4 pulses at 100 Hz, with the bursts repeated at
5 Hz, and each tetanus consisting of 3 ten-burst trains separated by
15 s). Responses were measured as fEPSP slopes expressed as
percentage of baseline.
been located, the mouse was guided gently through the water by
placing a hand behind it to direct it towards the platform. The
mouse rested on the platform for 15 s. The goal platform location
was different for each mouse. On day 2, the same procedure was
repeated as on day 1 for all 15 trials using only the hidden platform.
For data analysis, averages for each mouse were calculated using
blocks of 3 trials.
4.8. Statistical analyses
Experiments were performed in blind. Results were expressed
as Standard Error of the Mean (SEM). Level of significance was set
for p < 0.05. Results were analyzed by student t-test/2-way ANOVA
for repeated measures. Planned comparisons were used for post-
hoc analysis.
Acknowledgments
4.7. Behavioral studies
This work was financially supported by Alzheimer’s Drug
Discovery Foundation and NIH-NIA (U01-AG032973). The authors
gratefully thank Karan Nagar for helping with some behavioral
experiments.
Fear conditioning was assessed as previously described
[37,38,41]. First, sensory perception of electric foot shock was
examined in different groups of mice through the threshold
assessment test. Briefly, animals were placed in the conditioning
chamber and the electric current (0.1 mA for 1 s) was increased at
30 s intervals from 0.1 mA to 0.7 mA. Threshold to flinching (first
visible response to shock), jumping (first extreme motor response),
and vocalized response were quantified for each animal by aver-
aging the shock intensity at which each animal showed the
behavioral response to that type of shock. Training of fear condi-
tioning was performed by placing the mouse in a conditioning
chamber for 2 min before the onset of a tone (Conditioned Stimulus
(CS), 30 s, 85 dB sound at 2800 Hz). In the last 2 s of the CS, mice
were given a 2 s, 0.7 mA mild foot shock (Unconditioned Stimulus,
(US)) through the bars of the floor. After the US, the mice were left
in the chamber for another 30 s. Freezing behavior, defined as the
absence of movements except for respiratory excursions, was
scored using Freezeview software (Med Associates, St. Albans, VT).
Contextual fear learning was evaluated 24 h after training by
measuring freezing responses for 5 min in the same chamber
where the mice were trained. Cued fear learning was evaluated 24 h
after contextual testing. The mice were placed in a novel context for
2 min (pre-CS test), after which they were given a CS for 3 min (CS
test), and freezing behavior was measured during the first 30 s that
mimic the CS-US conditioning and the remaining 2.5 min.
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