Synthesis and anti-prion activity evaluation of aminoquinoline analogues

Article abstract of DOI:10.1016/j.ejmech.2010.07.054

Transmissible spongiform encephalopathies form a group of neurodegenerative diseases that affect humans and other mammals. They occur when the native prion protein is converted into an infectious isoform, the scrapie PrP, which aggregates, leading to neurodegeneration. Although several compounds were evaluated for their ability to inhibit this conversion, there is no effective therapy for such diseases. Previous studies have shown that antimalarial compounds, such as quinolines, possess anti-scrapie activity. Here, we report the synthesis and evaluate the effect of aminoquinoline derivatives on the aggregation of a prion peptide. Our results show that 4-amino-7-chloroquinoline and N-(7-chloro-4-quinolinyl)-1,2-ethanediamine inhibit the aggregation significantly. Therefore, such aminoquinolines might be considered as candidates for the further development of therapeutics to prevent the development of prion diseases.

Full text of DOI:10.1016/j.ejmech.2010.07.054

European Journal of Medicinal Chemistry 45 (2010) 5468e5473
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and anti-prion activity evaluation of aminoquinoline analogues
Bruno Macedo a, Catherine H. Kaschula b, Roger Hunter b, Juliana A.P. Chaves a, Johannes D. van der
,
Merwe b, Jerson L. Silva c, Timothy J. Egan b, Yraima Cordeiro a
*
a Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Av Carlos Chagas Filho, Rio de Janeiro 21941-902, Brazil
b Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
c Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Transmissible spongiform encephalopathies form a group of neurodegenerative diseases that affect
humans and other mammals. They occur when the native prion protein is converted into an infectious
isoform, the scrapie PrP, which aggregates, leading to neurodegeneration. Although several compounds
were evaluated for their ability to inhibit this conversion, there is no effective therapy for such diseases.
Previous studies have shown that antimalarial compounds, such as quinolines, possess anti-scrapie
activity. Here, we report the synthesis and evaluate the effect of aminoquinoline derivatives on the
aggregation of a prion peptide. Our results show that 4-amino-7-chloroquinoline and N-(7-chloro-4-
quinolinyl)-1,2-ethanediamine inhibit the aggregation significantly. Therefore, such aminoquinolines
might be considered as candidates for the further development of therapeutics to prevent the devel-
opment of prion diseases.
Received 19 April 2010
Received in revised form
22 July 2010
Accepted 28 July 2010
Available online 6 August 2010
Keywords:
Prion
Aggregation
Antimalarials
Quinoline
Ó 2010 Elsevier Masson SAS. All rights reserved.
Inhibitor
Neurodegeneration
1. Introduction
A great variety of compounds and also synthetic peptides have
been evaluated for their ability to prevent the conversion of PrPC to
The prion protein (PrP) is an unusual infectious pathogen that is
responsible for neurodegenerative diseases that affect humans and
other animals e the transmissible spongiform encephalopathies
[1]. Conversion of the innocuous, native PrP (PrPC, which is rich in
PrPSc and to inhibit PrP aggregation [7e9]. Polyamines [10], sulfated
glycans [11,12] and cyclic tetrapyrroles, such as porphyrins [13], are
effective in vitro. However, not all of these presented prophylactic
activity in vivo [14]. Several antimalarial compounds, such as acri-
dines and quinolines, were also investigated as effectiveinhibitors of
proteinase-resistant PrP forms in neuroblastoma cells (N2a) that
had been infected with PrPSc [8,15e19]. One antimalarial drug,
mefloquine (a quinoline compound) showed high activity against
scrapie agent-infected cells [18]. However, when administered to
mice that had been inoculated with a scrapie strain, it did not delay
the onset of clinical symptoms [18]. Quinacrine, an important
inhibitor [16], did not increase survival in a murine model of prion
disease [20,21], and high doses of this compound lead to liver
damage without therapeutic efficacy in humans affected with
Creutzfeldt-Jakob disease [22]. A recent observational study repor-
ted that 300 mg per day of quinacrine was tolerated reasonably, but
it did not affect the clinical course of the prion disease significantly
[23]. Other antimalarials that have been proposed for prion therapy
include the 4- and 8-aminoquinolines. The results presented so far
indicate that at least two fused aromatic rings are necessary for PrP
binding and anti-scrapie activity [16,17,24]. In a recent high-
throughput assay that evaluated PrPSc reduction in prion-infected
neuroblastoma cell lines (ScN2a), 2-aminothiazoles were identified
a
-helical structures) into an abnormal isoform, the PrP scrapie
(PrPSc, which is rich in
b
-sheet structures) is the most important
step leading to the development of these diseases [1]. PrPC is
soluble and does not aggregate. In contrast, PrPSc is protease-
resistant, highly insoluble in aqueous solution and aggregates
readily [2]. In vitro, ex vivo and transgenic animal studies suggest
that the conversion is aided by other biological molecules that
lower the energetic barrier preventing the spontaneous conversion
of PrPC to PrPSc, acting as cofactors [3e6]. Among such cofactors,
nucleic acids have been shown to interact with PrPC, presenting
both catalytic and protective effects on the conversion of PrP into
a scrapie-like conformation (depending on the PrP:NA molar ratio)
[4,6]. Despite huge efforts from the scientific community, there is
no effective therapy to prevent or reduce the progression of the
disease to date.
* Corresponding author.
E-mail address: yraima@pharma.ufrj.br (Y. Cordeiro).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.054

B. Macedo et al. / European Journal of Medicinal Chemistry 45 (2010) 5468e5473
5469
as potent anti-prion compounds [25]. However, the mechanism of
action of all the cited compounds has not yet been understood fully.
In view of these prior results, we studied the effect of novel
4-aminoquinoline derivatives (Table 1) on the aggregation of PrP.
The selected compounds act as antimalarials, and inhibit the
spontaneous formation of hemozoin (a malarial pigment that is
generated upon heme polymerization in the Plasmodium) [26]. The
mechanism of antimalarial action of such compounds is believed to
operate via direct interaction with heme, blocking the formation of
hemozoin and leading to free heme accumulation in the food
vacuole of Plasmodium. This results in the death of the parasite, as
free heme is toxic to these infectious agents [27]. We investigated
compounds that contain a chlorine atom at position 7 on the
quinoline ring in this work, as it was verified previously that this
substitution plays an important role in the inhibition of hemozoin
crystal formation. The substitution is, however, not a determinant
for the binding of the compound to heme [28].
the aggregation by 50% at approximately 0.2 and 0.1
(Table 1). These values are somewhat better than those obtained
using the antimalarial drug, mefloquine, which inhibits the aggre-
m
M, respectively
gation by 50% ate1
mM [18]. However, the activity of mefloquine was
evaluated in cells that expressed PrP scrapie, and the compounds
that we tested might also have different inhibitory concentrations in
this model. Using fluorescence anisotropy measurements (Fig. 3), we
observed that compounds 1 and 4 were able to bind to the prion
domain at the concentrations that we tested.
3.2. Inhibitory 4-aminoquinolines reduce the aggregation of the
ShaPrP peptide into amyloid-like structures
We also evaluated the binding of the fluorescent probe thio-
flavin T (Thio-T) to the peptide in the presence or absence of the
aminoquinolines. Thioflavin T is a benzothiazole dye that is
commonly used to diagnose amyloid fibrils [33,34]. When free in
aqueous solution, its fluorescence emission is negligible. However,
on binding to highly ordered, beta-sheet-rich structures, such as
amyloid fibrils, its fluorescence emission at 482 nm is increased
markedly [33,34]. We observed that Thio-T binds to the peptide (in
the absence of aminoquinolines or urea) which aggregates, forming
amyloid-like structures. The binding of Congo red was also evalu-
ated, confirming the results obtained with Thio-T (not shown).
To monitor whether the inhibition of aggregation that is medi-
ated by compounds 1 and 4 was related to a decrease in the
formation of such structured aggregates, we incubated Thio-T with
ShaPrP109e149 in the presence of the selected aminoquinolines and
measured its fluorescence emission (Fig. 4). Thio-T binding was
reduced significantly in the presence of compounds (1), (2), (3), (4)
and (7) (Fig. 4). However, Thio-T binding was reduced more for
compounds 1 and 4, which were the only aminoquinolines that
2. Chemistry
The compounds 4-amino-7-chloroquinoline (1), 7-chloro-4-
methylaminoquinoline (2), 2-(7-chloro-4-quinolinyl)-aminoethanol
(3), N-(7-chloro-4-quinolinyl)-1,2-ethanediamine (4), N2-(7-chloro-
4-quinolinyl)-N1,N1-dimethyl-1,2-ethanediamine (5) and 4-(cyclo-
pentylamino)-7-chloro-quinoline (7) were synthesized as reported
previously [28,29]. Compound 6, N2-(7-trifluoromethylthio-4-qui-
nolinyl)-N1,N1-diethyl-1,2-ethanediamine, is a new aminoquinoline
derivative and was prepared using a six-step synthesis, as described
in the Experimental section.
3. Results
3.1. ShaPrP peptide aggregation is inhibited by 4-aminoquinolines
inhibited ShaPrP109e149 aggregation significantly at 1.0
mM. All of
the data was corrected for the emission of Thio-T in buffer alone,
and in the presence of the aminoquinolines but not the peptide.
To verify whether the aminoquinolines investigated in this work
would modulate the aggregation of prion peptides, we evaluated
the aggregation of the peptide in the presence of the selected
compounds. To perform the aggregation kinetic assays, we chose
a model peptide encompassing the Syrian hamster PrP (ShaPrP)
residues 109e149 (PrP109e149). This domain corresponds to a loop
4. Discussion
The search for compounds that stabilize cellular PrP or prevent
its conversion into PrPSc remains important for the control of such
devastating diseases, where the formation of PrPSc is characteristic
of the transmissible spongiform encephalopathies. Therefore, the
identification of compounds that inhibit PrP aggregation in vitro is
an initial step in testing for in vivo and ex vivo anti-scrapie activity.
Here, we have investigated the effect of novel 4-aminoquino-
lines on the aggregation profile of a prion hydrophobic domain.
Evaluating the aggregation kinetics of this peptide (which corre-
sponds to part of the N-terminal PrP domain and to the first alpha-
helix of the Syrian hamster prion protein) [30,31] is a valuable tool
for the study of anti-aggregating compounds.
Several approaches to developing new drugs for prion disease
therapy involve screening drugs that are already marketed for other
applications. The advantage is that this can bypass some clinical
stages in drug approval. Most methods for such screening are based
on the “curative” effect on cell lines that are naturally scrapie-
infected [8,10,11,16e19] i.e., by evaluation of the loss of proteinase-
K resistance of PrP extracted from the cells when treated with an
effective compound.
and the first
implicated in the
a
-helix of the N-terminal region of PrP, which is
-sheet conversion [30,31]. It was previously
b
shown that this PrP domain promptly aggregates when diluted in
aqueous solutions under denaturing conditions [4,32]. Briefly, we
monitored the aggregation of the peptide by the increase in light
scattering at 450 nm as a function of time after dilution in MES
buffer at pH 5.0. On dilution in aqueous buffer, aggregation occurs
within a few seconds (Fig. 1) and is dependent on the concentration
of the peptide [4,32]. We tested whether the compounds could
inhibit aggregation of the prion peptide. The peptide is maintained
in a 6 M urea stock solution at pH 5.0; in this condition, it is partially
unfolded and does not aggregate [4]. To study the aggregation
kinetics, we diluted the peptide in buffer containing increasing
concentrations of the compounds and monitored the aggregation
by following the changes in light scattering as a function of time.
Our results show that 4-amino-7-chloroquinoline (1) and N-(7-
chloro-4-quinolinyl)-1,2-ethanediamine (4) inhibit the aggregation
of ShaPrP109e149 significantly (Table 1). Both compounds lead toe90%
inhibition at 1.0
m
M. At the same concentration, 7-chloro-4-meth-
Some antimalarial compounds have been evaluated for anti-
scrapie activity and were found to be effective inhibitors of
proteinase-resistant forms of PrP in neuroblastoma cells (N2a) that
were infected with PrPSc [8,16e19]. However, when tested in vivo, the
results were not as satisfactory as hoped. One promising compound,
quinacrine [16], showed no efficacy in a murine model of prion
disease [20,21]. When administered to humans suffering from
ylaminoquinoline (2) and N2-(7-trifluoromethylthio-4-quinolinyl)-
N1,N1-diethyl-1,2-ethanediamine (6) inhibited the aggregation by
60% and 50%, respectively. No significant inhibition of aggregation
was mediated by the other 4-aminoquinolines at the same concen-
tration (1.0
mM) (Fig. 2). Compounds 1 and 4 were the most active
compounds against prion peptide aggregation, and these inhibited

5470
B. Macedo et al. / European Journal of Medicinal Chemistry 45 (2010) 5468e5473
Table 1
The anti-aggregating activity of 4-aminoquinolines.
a
Compound
% Aggregation with 1
m
M
C50%
NH2
(1)
4-amino-7-chloroquinoline
8.4%
w0.2 mM
Cl
N
CH3
HN
(2)
(3)
(4)
7-chloro-4-methylaminoquinoline
38.6%
>0.9 mM
Cl
N
OH
HN
2-(7-chloro-4-quinolinyl)-aminoethanol
97%
>1 mM
Cl
N
NH2
HN
N-(7-chloro-4-quinolinyl)-1,2-ethanediamine
7.9%
w0.1 mM
Cl
N
CH3
N
HN
CH3
(5)
N2-(7-chloro-4-quinolinyl)-N1,N1-dimethyl-1,2-ethanediamine
82%
>1
m
M
Cl
N
CH3
N
CH3
HN
N
(6)
N2-(7-trifluoromethylthio-4-quinolinyl)-N1,N1-diethyl-1,2-ethanediamine 54%
>1 mM
F3CS
N
H
(7)
4-(cyclopentylamino)-7-chloro-quinoline
88%
>1 mM
Cl
N
a
The concentration of the compound where 50% of aggregation was achieved for the peptide at 0.5 mM final concentration.

B. Macedo et al. / European Journal of Medicinal Chemistry 45 (2010) 5468e5473
5471
Fig. 1. The inhibition of PrP peptide aggregation by 4-amine-7-chloroquinoline (1), as
monitored by changes in the light scattering (LS). The aggregation was followed by
measuring the LS (excitation 450 nm, emission 450 nm). We diluted ShaPrP109e149 that
had been previously unfolded in a 6 M urea solution at pH 5.0 to a final concentration
of 0.5 mM in 50 mM MES buffer, in the absence or presence of increasing concentra-
tions of compound 1. The light scattering of the solutions was monitored as a function
of time. The graph shows the aggregation of the peptide in the absence of compound 1
(black trace); in 6 M urea (red trace/dashed black trace); with 0.1
m
M (1) (green trace/
M (1)
dark gray trace), with 0.25 M (1) (blue trace/light gray trace) and with 1.0
m
m
(gray trace/dashed gray trace). The arrow indicates the moment at which the peptide
was diluted in the buffer, and this was w50 s after initiating acquisition of the baseline.
(For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
Creutzfeldt-Jakob disease, no therapeutic efficacy was obtained e
even when the subjects were treated with high doses [22]. Therefore,
identifying and developing new anti-prion disease drugs that would
be more effective and less toxic is important. Although the mecha-
nism of action of most anti-prion compounds has not yet been
understood, an interesting report showed that two structurally
unrelated anti-prion drugs, 6-aminophenanthridine and guanabenz,
inhibit the RNA-mediated ribosomal protein folding activity, without
affecting protein synthesis [35]. Both compounds interact with
ribosome in an RNA-dependent manner [35], evidencing the
possible role of RNA in prion physiopathology [6].
We have identified two novel 4-aminoquinolines (compounds 1
and 4) that inhibited the aggregation of
a ShaPrP peptide
Fig. 3. Evaluation of the binding of ShaPrP109e149 to the quinoline compounds using
fluorescence anisotropy. ShaPrP109e149 was titrated into a solution containing the
quinoline compounds (1) or (4) at pH 5.0, and the fluorescence anisotropy of the
compounds was recorded. A) 4-amino-7-chloroquinoline (1) at 0.5
360 nm, emission: 410 nm; B) N-(7-chloro-4-quinolinyl)-1,2-ethanediamine (4) at
0.5 M; excitation: 390 nm, emission: 450 nm.
mM; excitation:
m
significantly. This raises the possibility that they might serve as lead
compounds with anti-prion activity.
A detailed structure-activity analysis is not possible, given the small
number of quinolines that were investigated in this study. However,
several features are worth noting. The most active compounds (1 and
4) contain primary amino groups. Interestingly, the activity does not
appear to relate to the basicity of the amine, as the primary alkyl amine
4 is much more basic than the aromatic primary amine 1. While this
might suggest that the hydrogen bonding ability of the group is
required for its activity, the effect appears to be more complex, as
compound 3 (which contains an eOH group that can act as either
a hydrogen bond donor or acceptor) is almost inactive. Attaching
a methyl group to the aromatic amino function results in intermediate
activity, whereas the much more bulky cyclopentyl group almost
abolishes the activity. Finally, the intermediate activity of 6 (relative to
Fig. 2. The extentof inhibition of theaggregation is shown forcompounds (1), (2), (3), (4),
(5),(6)and(7).Themaximumlightscattering(at450nm) obtainedfromeach kinetictrace
was measured and normalized relative to the control (peptide aggregation in the absence
of urea or the compounds), which was taken to represent 100% aggregation (0% inhibi-
tion). This was taken as the highest LS value when the peptide was diluted in 6 M urea.
Each data point is the mean of three independent measurements (the means ꢀ S.E.M.).

5472
B. Macedo et al. / European Journal of Medicinal Chemistry 45 (2010) 5468e5473
has been reported in detail previously (in a description of their
antimalarial activities) [28]. The synthesis of 4-(cyclopentylamino)-
7-chloro-quinoline (7) has also been reported recently (in connection
with its antischistosomal activity) [29]. Compound 6, N2-(7-tri-
fluoromethylthio-4-quinolinyl)-N1,N1-diethyl-1,2-ethanediamine, is
a new compound and was prepared using a six-step synthesis:
A mixture of 3-trifluoromethylthioaniline (1.0 g, 5.3 mmol) and
diethyl methoxymethylene malonate (1.3 g, 5.8 mmol) was
refluxed in ethanol (100 ml) for 3 h, after which the solvent was
removed in vacuo. The resulting solid was recrystallized from
ethanol and dried to give ethyl 2-ethoxycarbonyl-3-(3-tri-
fluoromethylthioanilino)propenoate (6a), (1.9 g, 99%), mp 69 ꢂC; dH
(400 MHz, CDCl3) 1.29 (3H, t, J ¼ 7.0 Hz, CH3), 1.32 (3H, t, J ¼ 7.0 Hz,
CH3), 4.22 (2H, q, J ¼ 7.0 Hz, CH2), 7.15 (1H, m, Ar-H), 7.32 (3H, m, Ar-
H), 8.38 (1H, d, J ¼ 13.6 Hz, vinylic H), 10.97 (1H, bd, J ¼ 13.2 Hz,
NH); dC (100.6 MHz, CDCl3) 14.2 (CH3), 60.3 (CH2), 60.6 (CH2), 95.1
(C]CH), 119.2 (C-6), 124.4 (C-4), 126.4 (C-3), 129.3 (q, JC-
¼ 308.2 Hz, SCF3), 130.8 (C-5), 132.0 (C-2), 140.4 (C-1), 151.2 (C]
Fig. 4. The binding of thioflavin T to ShaPrP109e149 is reduced in the presence of
F
compounds (1) and (4). ShaPrP109e149 (1.0
m
M) was incubated with the selected ami-
M) and Thio-T was added after 10 min at a final concentration of
M. The excitation was set at 450 nm and the emission was recorded from 470 to
520 nm. The plot shows the area of the Thio-T fluorescence emission spectra (in the
presence of ShaPrP109e149
after subtraction of the probe emission at the same
conditions and without the addition of peptide. Each data point is the mean of three
experiments (the means S.E.M.). *, ** and ***, denote significant differences
CH), 165.5 (C]O), 168.9 (C]O); MS (HRMS), Found: m/z 363.07429,
requires Mþ: 363.07521; CHNS, Found: C, 49.40; H, 4.82; N, 3.87; S,
8.8%. Requires for C15H16F3NO4S: C, 49.5; H, 4.4; N, 3.8; S, 8.8%.
6a (1.0 g, 2.7 mmol) was refluxed in diphenyl ether (10 ml) for
30 min. The flask was opened during the first 10 min of the reaction
to allow the ethanol that was formed to escape. When the reaction
mixture was cooled to room temperature, a white precipitate
formed. This was filtered and washed with petroleum ether, ethyl
acetate and methanol to remove all diphenyl ether and other
impurities to give ethyl 1,4-dihydro-4-oxo-7-(trifluoromethylthio)
quinoline-3-carboxylate (6b) (0.6 g, 75%) after drying, mp
265e266 ꢂC; nmax/cmꢁ1 (KBr) 3150, 1637 (CO); dH (200 MHz, d6-
DMSO) 1.30 (3H, t, J ¼ 7.2 Hz, CH3), 4.25 (2H, q, J ¼ 7.2 Hz, CH2), 7.60
(1H, d, J ¼ 4.6 Hz, H-6), 7.98 (1H, s, H-8), 8.25 (1H, d, J ¼ 4.6 Hz, H-5),
8.67 (1H, s, H-2); MS, Found: m/z 317, requires Mþ: 317; CHNS,
Found: C, 49.1; H, 3.1; N, 4.4; S, 9.7%. Requires for C13H10F3NO3S:
C, 49.2; H, 3.1; N, 4.4; S, 10.1%.
noquinolines (10
20
m
m
)
ꢀ
(*p < 0.05, **p < 0.01 and ***p < 0.001, respectively).
the virtual inactivity of 5) hints that changes at the 7-position on the
quinoline ring may affect the activity. It is noteworthy that the eSCF3
group in 6 is considerably more electron-withdrawing than the eCl
group present in 5. However, the investigation of a substantially larger
set of compounds would be needed to confirm these apparent trends.
In conclusion, we believe that this new class of compounds
shows promise for the future development of anti-scrapie
compounds, and that assays in neuroblastoma cells that have been
infected with PrPSc should be undertaken to address the pharma-
cology of such therapeutic candidates.
A mixture of 6b (0.9 g, 2.8 mmol) and 10% NaOH (10 ml) was
heated at 120 ꢂC for 2 h. During this time, all of the solid dissolved.
The aqueous phase was then carefully neutralized with 10% H2SO4
to avoid the formation of the water-soluble quinolinium ion. A
white solid precipitated, and this was filtered off and washed with
cold water. The solid was dried over P2O5 under vacuum to yield
1,4-dihydro-4-oxo-7-(trifluoromethylthio)quinoline-3-carboxylic
acid (6c) (0.5 g, 70%), mp 233e236 ꢂC; nmax/cmꢁ1 (KBr) 2900, 1620;
dH (300 MHz, d6-DMSO) 7.80 (1H, dd, J ¼ 8.4, 1.5 Hz, H-6), 8.16 (1H,
d, J ¼ 1.5 Hz, H-8), 8.37 (1H, d, J ¼ 8.4 Hz, H-5), 8.98 (1H, s, H-2),
13.44 (1H, bs, COOH), 14.85 (1H, bs, Ar-OH); dC (75.5 MHz, d6-
DMSO) 108.5 (C-3), 125.6 (Ar-CH), 126.3 (C-5), 126.8 (C-8), 129.3 (q,
JC-F ¼ 308.3 Hz, SCF3), 129.5 (C-4a), 131.3 (C-6), 139.5 (C-8a), 146.3
(C-2), 165.7 (COOH), 177.8 (C-4); MS (HRMS), Found: m/z
289.00182; Requires for C11H6F3NO3S: 289.00205.
6c (0.8 g, 2.9 mmol) was added to diphenyl ether (8 ml) and the
mixture was refluxed in a preheated oil bath. After 30 min, when
the solid had entirely dissolved, the reaction mixture was allowed
to cool. A precipitate formed, and this was filtered, and washed
with petroleum ether and cold ethyl acetate to remove all diphenyl
ether to yield 7-(trifluoromethylthio)quinoline-4-one (6d) (0.4 g,
68%), which was recrystallized from methanol and ethyl acetate,
mp 214e215 ꢂC; nmax/cmꢁ1 (KBr) 2943, 1630; dH (300 MHz, d6-
DMSO) 6.12 (1H, J ¼ 7.2 Hz, H-3), 7.52 (1H, dd, J ¼ 8.4, 1.2 Hz, H-6),
7.92 (1H, d, J ¼ 1.2 Hz, H-8), 7.99 (1H, d, J ¼ 7.2 Hz, H-2), 8.19 (1H, d,
J ¼ 8.4 Hz, H-5), 11.89 (1H, bs, Ar-OH); dC (100.6 MHz, d6-DMSO)
109.6 (C-3), 125.6 (C-6), 126.6 (C-7/C-4a), 126.6 (C-7/C-4a), 126.7
(C-5), 128.7 (C-8), 129.4 (q, JC-F ¼ 308.2 Hz, SCF3), 140.1 (C-8a), 140.2
(C-2), 176.1 (C-4); MS (HRMS), Found: m/z 245.01228; Requires for
C10H6F3NOS: 245.01222.
5. Experimental
5.1. Reagents and the peptide model
All the reagents used were of analytical grade. Thioflavin T was
purchased from SigmaeAldrich (St. Louis, MO, USA). The Syrian
hamster prion protein peptide (109e149) was acquired from Gen-
emed Synthesis, Inc. (San Antonio, TX, USA), where it was made
using solid phase synthesis and purified by RP-HPLC (>90% purity).
The identity of the peptide was confirmed using mass spectrometry,
according to the Genemed Quality Control Certificate. The peptide
was solubilized just before use in 50 mM MES (4-Morpholinoe-
thanesulfonic acid) buffer at pH 5.0 with 6 M urea and 10 mM
sodium dodecyl sulfate (SDS). The peptide stock solution was
centrifuged at 10,000 rpm for 10 min, and the concentration of the
supernatant was measured using a molar extinction coefficient of
12,490 Mꢁ1 cmꢁ1 at 280 nm. This value was calculated from the
amino acid sequence using the software ProtParam (http://us.
expasy.org/tools/protparam.html). The sequence of the prion
peptide was: 109 MKHMAGAAAAGAVVGGLGGWMLGSAMSRPMMH
FGNDWEDRY 149.
5.2. The source or synthesis methods of the compounds
The synthesis of compounds 4-amino-7-chloroquinoline (1),
7-chloro-4-methylaminoquinoline (2), 2-(7-chloro-4-quinolinyl)-
aminoethanol (3), N-(7-chloro-4-quinolinyl)-1,2-ethanediamine (4),
N2-(7-chloro-4-quinolinyl)-N1,N1-dimethyl-1,2-ethanediamine (5)

B. Macedo et al. / European Journal of Medicinal Chemistry 45 (2010) 5468e5473
5473
A mixture of 6d (0.4 g, 1.4 mmol), phosphorous oxychloride
Acknowledgments
(1 ml, 11.4 mmol) and phosphorous pentachloride (0.3 g, 1.6 mmol)
was refluxed under N2 at 120 ꢂC for 30 min. The reaction mixture
was then poured into cold aq. NaOH (1 M, 50 ml) and the white
precipitate that formed as a result was filtered and washed with
water. The solid was then dissolved in ethyl acetate (100 ml), dried
(MgSO4) and the solvent removed in vacuo. Recrystallization from
petroleum ether gave 4-chloro-7-trifluoromethylthioquinoline (6e)
(0.3 g, 88%), mp 69 ꢂC; dH (400 MHz, CDCl3) 7.57 (1H, d, J ¼ 4.7 Hz,
H-3), 7.83 (1H, dd, J ¼ 9.0, 1.7 Hz, H-6), 8.28 (1H, d, J ¼ 9.0 Hz, H-5),
8.48 (1H, d, J ¼ 1.7 Hz, H-8), 8.85 (1H, d, J ¼ 4.7 Hz, H-2); dC
(75.5 MHz, CDCl3) 122.8 (C-3), 122.9 (C-7), 125.7 (C-5), 127.6 (C-4a),
129.6 (q, JC-F ¼ 308.6 Hz, SCF3), 133.2 (C-6), 138 (C-8), 142.9 (C-4),
149.0 (C-8a), 151.3 (C-2); MS (HRMS), Found: m/z 262.97808;
Requires for C10H5ClF3NS: 262.97833.
We thank Prof. Luis Mauricio T. R. Lima and Dr. Carolina A. C. A.
Braga for critical reading of the manuscript and Prof. Marcus F.
Oliveira for helpful suggestions. This work was supported by grants
from Conselho Nacional de Desenvolvimento Científico e Tecnoló-
gico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do
Estado do Rio de Janeiro (FAPERJ), the National Institute of Science
and Technology for Structural Biology and Bioimaging, the
Millennium Institute for Structural Biology in Biomedicine and
Biotechnology (CNPq Millennium Program), and by a grant from
ABC-UNESCO-L’Oréal to Y. C.
References
6e (0.2 g, 1.0 mmol) and N,N-diethylethylenediamine (3 ml)
were heated together in a sealed tube under N2 at 140 ꢂC for 4 h. Aq.
10% NaOH (2 ml) was added to the product and the resulting yellow
precipitate was then extracted into dichloromethane (3 ꢃ 100 ml).
The solvent was dried (MgSO4) and removed under reduced pres-
sure to furnish a residue which was chromatographed on silica gel
using methanol and dichloromethane (2:8) as the eluent to give 6
(0.4 g, 80%), mp 110e111 ꢂC; dH (400 MHz, CDCl3) 1.08 (6H, t,
J ¼ 7.3 Hz, 2ꢃ CH3), 2.61 (4H, q, J ¼ 7.3 Hz, 2ꢃ CH2), 2.83 (2H, t,
J ¼ 6.2 Hz, CH2 b), 3.28 (2H, q, J ¼ 6.2 Hz, CH2 a), 6.21 (1H, bs, NH),
6.43 (1H, d, J ¼ 5.1 Hz, H-3), 7.61 (1H, dd, J ¼ 9.0, 1.8 Hz, H-6), 7.72
(1H, d, J ¼ 9.0 Hz, H-5), 8.30 (1H, d, J ¼ 1.8 Hz, H-8), 8.58 (1H, d,
[1] S.B. Prusiner, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 13363e13383.
[2] A. Aguzzi, M. Polymenidou, Cell 116 (2004) 313e327.
[3] G.C. Telling, M. Scott, J. Mastrianni, R. Gabizon, M. Torchia, F.E. Cohen,
S.J. DeArmond, S.B. Prusiner, Cell 83 (1995) 79e90.
[4] Y. Cordeiro, F. Machado, L. Juliano, M.A. Juliano, R.R. Brentani, D. Foguel,
J.L. Silva, J. Biol. Chem. 276 (2001) 49400e49409.
[5] S. Supattapone, J. Mol. Med. 82 (2004) 348e356.
[6] J.L. Silva, L.M.T.R. Lima, D. Foguel, Y. Cordeiro, Trends Biochem. Sci. 33 (2008)
132e140.
[7] S.A. Priola, A. Raines, W.S. Caughey, Science 287 (2000) 1503e1506.
[8] D.A. Kocisko, G.S. Baron, R. Rubenstein, J. Chen, S. Kuizon, B. Caughey, J. Virol.
77 (2003) 10288e10294.
[9] S. Sellarajah, C. Boussard, T. Lekishvili, D.R. Brown, I.H. Gilbert, Eur. J. Med.
Chem. 43 (2008) 2418e2427.
[10] S. Supattapone, H.O. Nguyen, F.E. Cohen, S.B. Prusiner, M.R. Scott, Proc. Natl.
Acad. Sci. U.S.A. 96 (1999) 14529e14534.
[11] B. Caughey, G.J. Raymond, J. Virol. 67 (1993) 643e650.
[12] R. Gabizon, Z. Meiner, M. Halimi, S.A. Ben-Sasson, J. Cell. Physiol. 157 (1993)
319e325.
J ¼ 5.1 Hz, H-2); dC (100.6 MHz, CDCl3) 12.3 (CH2CH3), 39.9 (CH2-
a),
46.7 (CH2CH3), 50.8 (CH2- ), 100.3 (C-3), 120.3 (C-7), 121.2 (C-5),
b
125.2 (C-4a), 129.8 (q, JC-F ¼ 308.1 Hz, SCF3), 130.3 (C-6), 138.5 (C-8),
148.7 (C-4), 149.9 (C-8a), 152.5 (C-2); MS (HRMS), Found: m/z
344.14095; Requires for C16H21F3N3S (M þ H): 344.14082.
[13] W.S. Caughey, L.D. Raymond, M. Horiuchi, B. Caughey, Proc. Natl. Acad. Sci.
U.S.A. 95 (1998) 12117e12122.
[14] N.R. Cashman, B. Caughey, Nat. Rev. Drug Discov. 3 (2004) 874e884.
[15] K. Doh-ura, T. Iwaki, B. Caughey, J. Virol. 74 (2000) 4894e4897.
[16] C. Korth, B.C. May, F.E. Cohen, S.B. Prusiner, Proc. Natl. Acad. Sci. U.S.A. 98
(2001) 9836e9841.
5.3. Spectroscopic measurements
[17] I. Murakami-Kubo, K. Doh-ura, K. Ishikawa, S. Kawatake, K. Sasaki, J. Kira,
S. Ohta, T. Iwaki, J. Virol. 78 (2004) 1281e1288.
[18] D.A. Kocisko, B. Caughey, J. Virol. 80 (2006) 1044e1046.
[19] R. Klingenstein, P. Melnyk, S.R. Leliveld, A. Ryckebusch, C. Korth, J. Med. Chem.
49 (2006) 5300e5308.
[20] S.J. Collins, V. Lewis, M. Brazier, A.F. Hill, A. Fletcher, C.L. Masters, Ann. Neurol.
52 (2002) 503e506.
[21] A. Barret, F. Tagliavini, G. Forloni, C. Bate, M. Salmona, L. Colombo, L.A. De,
L. Limido, S. Suardi, G. Rossi, F. Auvre, K.T. Adjou, N. Sales, A. Williams,
C. Lasmezas, J.P. Deslys, J. Virol. 77 (2003) 8462e8469.
[22] P. Follette, Science 299 (2003) 191e192.
[23] J. Collinge, M. Gorham, F. Hudson, A. Kennedy, G. Keogh, S. Pal, M. Rossor,
P. Rudge, D. Siddique, M. Spyer, D. Thomas, S. Walker, T. Webb, S. Wroe,
J. Darbyshire, Lancet Neurol. 8 (2009) 334e344.
[24] H. Cope, R. Mutter, W. Heal, C. Pascoe, P. Brown, S. Pratt, B. Chen, Eur. J. Med.
Chem. 41 (2006) 1124e1143.
[25] S. Ghaemmaghami, B.C.H. May, A.R. Renslo, S.B. Prusiner, J. Virol. 84 (2010)
3408e3412.
[26] T.J. Egan, J. Inorg. Biochem. 91 (2002) 19e26.
[27] T.J. Egan, D.C. Ross, P.A. Adams, FEBS Lett. 352 (1994) 54e57.
[28] T.J. Egan, R. Hunter, C.H. Kaschula, H.M. Marques, A. Misplon, J. Walden,
J. Med. Chem. 43 (2000) 283e291.
[29] J.B. Correa Soares, D. Menezes, M.A. Vannier-Santos, A. Ferreira-Pereira,
G.T. Almeida, T.M. Venancio, S. Verjovski-Almeida, V.K. Zishiri, D. Kuter,
R. Hunter, T.J. . Egan, M.F. Oliveira, PLoS Negl. Trop. Dis. 3 (2009) e477.
[30] Z. Huang, S.B. Prusiner, F.E. Cohen, Fold. Des. 1 (1996) 13e19.
[31] C. Govaerts, H. Wille, S.B. Prusiner, F.E. Cohen, Proc. Natl. Acad. Sci. U.S.A. 101
(2004) 8342e8347.
[32] Y. Cordeiro, L.M.T.R. Lima, M.P. Gomes, D. Foguel, J.L. Silva, J. Biol. Chem. 279
(2004) 5346e5352.
[33] H. Naiki, K. Higuchi, M. Hosokawa, T. Takeda, Anal. Biochem. 177 (1989)
244e249.
Light scattering and fluorescence anisotropy measurements
were recorded on a PC1 spectrofluorometer (ISS, Champaign, IL) or
on a Jasco FP 6300 (Jasco Corp., Tokyo, Japan) in the “L” geometry
(at 90ꢂ in relation to the excitation light). The aggregation was
monitored by measuring the light scattering (excitation 450 nm,
emission 450 nm). For the aggregation kinetic assays, the peptide,
which had previously been unfolded in 6 M urea supplemented
with 10 mM SDS at pH 5.0, was diluted in buffer and the light
scattering was monitored as a function of time. The maximum light
scattering obtained from each kinetic trace was measured and
normalized, considering the control to represent 100% aggregation.
Zero percent aggregation was assumed for the LS value obtained
when the peptide was diluted in 6 M urea, where it does not
aggregate. For the aggregation kinetic assays, we diluted the
peptide at least 100-fold in the test buffer. The binding to thioflavin
T was evaluated by exciting the probe at 450 nm and recording the
fluorescence emission from 470 to 520 nm.
5.4. Statistics
Statistical analyses were carried out using the program Sigma
Plot (version 10.0). For the data presented in Figs. 2e4, we present
the mean of triplicates, and the error bars indicate the standard
error (SEM). For the thioflavin T binding assay (Fig. 4), we used
[34] H. LeVine III, Meth. Enzymol. 309 (1999) 274e284.
[35] D. Tribouillard-Tanvier, S. Dos Reis, F. Gug, C. Voisset, V. Béringue, R. Sabate,
E. Kikovska, N. Talarek, S. Bach, C. Huang, N. Desban, S.J. Saupe,
S. Supattapone, J.Y. Thuret, S. Chédin, D. Vilette, H. Galons, S. Sanyal,
M. Blondel, PLoS One. 3 (2008) e2174.
a
two-sample t-Test to determine the statistical differences
between the control (100% aggregation) and the results that were
obtained in the presence of the compounds (*p < 0.05, **p < 0.005,
***p < 0.001).