Parallel synthesis, evaluation, and preliminary structure-activity relationship of 2,5-diamino-1,4-benzoquinones as a novel class of bivalent anti-prion compound

Article abstract of DOI:10.1021/jm100882t

A library of 11 entries, featuring a 2,5-diamino-1,4-benzoquinones nucleus as spacer connecting two aromatic prion recognition motifs, was designed and evaluated against prion infection. Notably, 6-chloro-1,2,3,4-tetrahydroacridine 10 showed an EC₅₀ of 0.17 μM, which was lower than that displayed by reference compound BiCappa. More importantly, 10 possessed the capability to contrast prion fibril formation and oxidative stress, together with a low cytotoxicity. This study further corroborates the bivalent strategy as a viable approach to the rational design of anti-prion chemical probes.

Full text of DOI:10.1021/jm100882t

J. Med. Chem. 2010, 53, 8197–8201 8197
DOI: 10.1021/jm100882t
Parallel Synthesis, Evaluation, and Preliminary Structure-Activity Relationship
of 2,5-Diamino-1,4-benzoquinones as a Novel Class of Bivalent Anti-Prion Compound
Salvatore Bongarzone,†,‡,# Hoang Ngoc Ai Tran,¥,# Andrea Cavalli, ,§ Marinella Roberti,§ Paolo Carloni,†,‡,ꢀ,^
Giuseppe Legname,*,¥,§ and Maria Laura Bolognesi*,§
†Statistical and Biological Physics Sector, Neurobiology Sector, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Via Bonomea 265,
34136 Trieste, Italy, ‡Italian Institute of Technology, SISSA-ISAS Unit, 34151 Trieste, Italy, §Department of Pharmaceutical Sciences,
Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Department of Drug Discovery and Development,
Italian Institute of Technology, 16163 Genova, Italy, and ^CNR-INFM-DEMOCRITOS Modeling Center for Research in Atomistic Simulation,
34151 Trieste, Italy. # These authors contributed equally to the experimental work. ¥ Laboratory of Prion Biology, Neurobiology Sector,
Scuola Internazionale Superiore di Studi Avanzati (SISSA), Area Science Park, Basovizza, 34151 Trieste, Italy. ꢀ German Research School for
Simulation Sciences GmbH, Forschungzentrum Ju€lich GmbH, RWTH Aachen University, Aachen, Germany.
Received July 14, 2010
A library of 11 entries, featuring a 2,5-diamino-1,4-benzoquinones nucleus as spacer connecting two
aromatic prion recognition motifs, was designed and evaluated against prion infection. Notably,
6-chloro-1,2,3,4-tetrahydroacridine 10 showed an EC50 of 0.17 μM, which was lower than that dis-
played by reference compound BiCappa. More importantly, 10 possessed the capability to contrast prion
fibril formation and oxidative stress, together with a low cytotoxicity. This study further corroborates
the bivalent strategy as a viable approach to the rational design of anti-prion chemical probes.
Introduction
novel drugs.7 Most of the anti-prion molecules that have been
identified so far are derived from screening approaches.
Structurally, diverse chemical compounds covering a broad
range of the chemical space have been identified.8 Intrigu-
ingly, most of them share a common bivalent structure. This is
the case of the natural product curcumin,9 the bis-acridine
analogues,10,11 the diphenylmethane derivative (GN8),12
bebeerine,13 bisepigallocatechin digallate,13 2,20-bisquinolines,14
4,5-dianilinophthalimide,15 analogues of Congo red,16 and
diketopiperazines (DKP).17
Although a structure-activity relationship is not easy
to discern from such chemically unrelated compounds, we
envisaged that bivalent ligands bearing lipophilic bi- or
tri(hetero)cyclic scaffolds connected by a central core might
possess anti-prion activity. Bivalency, and multivalency in
general, is a well-known and efficient strategy widely used by
medicinal chemists to enhance binding efficacy in molecular
recognition processes.18 Multivalent chemical probes, featur-
ing multiple copies of an amyloid binding motif connected by
a spacer, have been developed with the aim to simultaneously
bind to several binding sites or several amyloid peptides, thus
achieving higher potency.19 In prion research, by joining two
quinacrine moieties through a piperazine spacer, May et al.
afforded the first bivalent antiprion ligand BiCappa (Figure 1SI),
which was 100 times more potent than monomeric quinacrine.10
Heterodimers incorporating recognition elements taken from
quinacrine itself and imipramine with a piperazine unit were
shown toimprovetheanti-prion efficacy ofquinacrineup to a
low nanomolar range.20 Furthermore, assembling multiple
acridine or curcumin moieties to a cyclopeptide scaffold has
emerged as a promising strategy for the development of
inhibitors against amyloid formation.21,22
Transmissible spongiform encephalopathies, or prion diseases,
are rapidly progressive, fatal, and untreatable neurodegenera-
tive syndromes that can affect humans and animals. A key
pathogenic event in prion diseases is the conversion of a host-
encoded cellular prion protein, termed PrPC,a into its abnormal
amyloidogenic isoform, PrPSc, predominantly in the central
nervous system of the infected host.1,2 The cellular and molec-
ular biology of the prion protein (PrP) remains enigmatic.3
Several lines of evidence suggest that PrPSc acts as a conforma-
tional template by which PrPC is converted to a new molecule of
PrPSc, which in turn has a strong tendency to aggregate into
insoluble amyloid fibrils.2 The PrPC f PrPSc conversion is
interrelated with multiple molecular mechanisms involving
reactive oxygen species (ROS) production, increased oxidation
of lipids, proteins, and DNA and an imbalance of metal ions
finally leading to neuronal death.4,5 These mechanisms are
found also in other similar conformational diseases.6
Despite the numerous efforts aimed at identifying com-
pounds useful against prion diseases, there are still no thera-
pies on the market. Therefore, it is of continued importance to
identify chemical scaffolds to be exploited for the design of
*To whom correspondence should be addressed. For G.L.: phone,
þ39 040 375 6515; fax, þ39 040 375 6502; e-mail, giuseppe.legname@
sissa.it. For M.L.B.: phone, þ39 051 209 9718; fax, þ39 051 209 9734;
e-mail, marialaura.bolognesi@unibo.it.
a Abbreviations: BQ, 2,5-diamino-1,4-benzoquinones; CoQ, coenzyme Q;
DKP, diketopiperazines; NQO1, NAD(P)H/quinone oxidoreductase 1;
GT1, mouse hypothalamus cells; OS, oxidative stress; PPIs, protein-protein
interactions; PrP, prion protein; PrPC, normal cellular prion protein; PrPSc,
infectious conformational form of prion protein; moPrP, recombinant
mouse prion protein; PK, proteinase K; ROS, reactive oxygen species;
ScGT1, scrapie-infected mouse hypothalamus cells; SFP, sulforaphane;
TBARS, thiobarbituric acid-reactive substances; THA, 1,2,3,4-tetrahy-
droacridine.
Building on the bivalent approach, we have recently
reported that a 2,5-diamino-1,4-benzoquinone (BQ) linked to
r
2010 American Chemical Society
Published on Web 11/03/2010
pubs.acs.org/jmc

8198 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Bongarzone et al.
Scheme 1a
a Reagents and conditions: (a) phenol, NaI, 120 ꢀC (1 h), followed by addition of amine, 5 h, 120 ꢀC; (b) EtOH, 5 h, 60 ꢀC (38-88% yield); (c) EtOH,
80 ꢀC (1 h), followed by addition of amine, 5 h, 50 ꢀC (40%). A = 6-chloro-2-methoxyacridine; B = 7-chloroquinoline; C = 1,2,3,4-tetrahydroacridine;
D = 6-chloro-1,2,3,4-tetrahydroacridine; E = 6-methoxyquinoline.
two phenylalanine residues displayed remarkable anti-prion
activity in a cellular model of prion replication.23 In this
compound, because of a resonance effect of the BQ ring, a
hydrophobic and planar system is generated, which might
interfere, through hydrophobic interactions, with aromatic
residues critical to fibril formation.23 This hypothesis is
corroborated by the key role of planarity as a major determi-
nant for anti-prion activity in a recently synthesized series of
bivalent DKP.17
Encouraged by the good yields and the straightforward work-
up associated with this reaction, we decided to synthesize the
designed bivalent 1-11 using a solution phase parallel synthe-
sis approach. Eleven N-substituted polyamines (13-23,
Scheme 1) were loaded with 2,5-dimethoxy-1,4-benzoquinone
into different vessels of a carousel workstation. After heating
at 50 ꢀC for 5 h, the desired products formed in moderate
to good yields (38-88%). Monovalent 12 was obtained by
Michael reaction starting from naftoquinone and amine 17
(40%). The preparation of intermediates 13-23 was easily
achieved treating in parallel fashion commercially available
polyamines 24-26 with heteroaryl halides 27-31. Com-
pounds 13-23 were obtained in 25-67% yield by reacting a
large excess of the polyamine with the corresponding hetero-
aryl halide (27:1) in phenol and using NaI as a catalyst
(Scheme 1). In these conditions we were able to obtain
selective monosubstitution at the terminal primary amino
group of the polyamine, obviating the need for protection/
deprotection of the other amino functionalities. Furthermore,
we overcame the low-yield of common SNAr reactions and
the use of costly reagents of Pd-catalyzed amination meth-
odologies.
A cell-screening assay was used to test anti-prion activity
across the library of synthesized compounds. Prior, we deter-
mined the effects of library compounds on cell viability by
calcein-AM assay, measuring viable ScGT1 cells after incuba-
tion in the drug-doped medium with various compound
concentrations of 10 nM to 10 μM for 5 days (Table 1). Then
their ability to reduce PrPSc in ScGT1 cells was determined by
Western blot densitometry of the PK-resistant PrPSc, in
comparison with BiCappa, used as a reference compound.
For entries BiCappa, 2, 5, 6, and 10, we also calculated the
EC50 values, which represent the effective concentrations for
half-maximal inhibition. Cell viability at EC50 values were
expressed as an average percentage of viable cells versus
untreated control. In addition, for BiCappa, 2, 5, 6, and 10,
the capability of inhibiting prion fibril formation was studied
in vitro by using a previously reported amyloid seeding assay
(Table 1, Figures 1 and 3SI).24
Altogether these results allowed us to propose that the
planar BQ scaffold may be considered as a privileged motif
in modulating protein-protein interactions (PPIs) and as a
promising spacer in the search for more effective bivalent anti-
prion chemical probes. On this basis, we have here designed a
small combinatorial library of bivalent ligands whose general
structure is depicted in Figure 2SI. The ligands feature the BQ
nucleus as a central core, with two linkers in positions 2 and 5
connecting two terminal moieties. As linkers, we selected three
polyamine chains (24-26, Scheme 1) that would allow ex-
ploring different lengths and chemical composition for the
different molecules. This is of particular importance, since
linker length has been shown by May et al. to be very critical
against PrPSc formation for the bivalent acridines series.10 As
terminal moieties, starting from the consideration that aro-
matic groups provided the best activity in the previous series
of BQ compounds,23 we selected aromatic prion recogni-
tion motifs, such as 6-chloro-2-methoxyacridine (as in 1-3,
Scheme 1), 7-chloroquinoline (4-6), and 1,2,3,4-tetrahydro-
acridine (THA) (7-9). Several acridine and quinoline
derivatives have been shown to inhibit PrPSc formation in
infected cells and to bind PrP.14,24,25 Given that the analo-
gous THA-9-amine is active against yeast prion,26 derivatives
7-9 were also designed. As a second step, on the basis of
the remarkable profile shown by 5, three other derivatives
(10-12) were designed with the aim of further optimizing
activityintheexistingseriesofcompounds. Herein, wepresent
a solution-phase parallel synthesis of a library of bivalent BQ
derivatives, which were chosen for their anti-prion activity in
ScGT1 cells, together with their capability of inhibiting PrPSc
aggregation and of reducing oxidative stress (OS).
Results and Discussion
Chemistry and Biology
Preliminarily, the possible toxicity of 1-9 was assessed in
ScGT1 cells. At 1 μM, the toxicity profiles among the library
members varied from 1.5% to 114.8% (Table 1). Treatment
with 1 and 3 decreased cell viability to 18.2% and 1.5%,
We have previously reported how a disubstitution reaction
of diamines with 2,5-dimethoxy-1,4-benzoquinone provides
easy access to a variety of 2,5-diamino-1,4-benzoquinones.27,28

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8199
Table 1. Cell viability and anti-prion activity on ScGT1 cells of library compounds
% of viable cells
at 1 μMb
75.6 ( 7.1a
% of PrPSc inhibition
% of viable cells
b
at EC50
92.4 ( 6.2a
Cpd
at 1 μMc
EC50 (μM)c
0.32 ( 0.03a
Lag Phase (hours)d
BiCappa
1
102.1 ( 2.7a
ND
55 ( 7*
18.2 ( 1.2
80.1 ( 6.3 (at 0.2 μM)
65.5 ( 5.6
1.5 ( 0.2
3.1 ( 0.3 (at 0.2 μM)
89.7 ( 5.1
ND
2
3
0.68 ( 0.05
75.2 ( 8.4
45 ( 10#
65.8 ( 4.6 (at 0.2 μMb)
114.8 ( 7.9
100.4 ( 3.6
105.0 ( 7.4
108.0 ( 8.4
104.4 ( 5.6
95.4 ( 7.4
5.4 ( 0.4 (at 0.2 μMc)
6.2 ( 0.6
4
5
85.5 ( 3.9
49.1 ( 2.2
7.1 ( 0.9
3.6 ( 0.4
3.4 ( 0.2
105.3 ( 5.5
4.7 ( 0.3
2.9 ( 0.1
0.73 ( 0.03
1.2 ( 0.1
99.6 ( 2.7
91.3 ( 4.2
53 ( 5#
6
40 ( 10*
7
8
9
10
11
12
78.6 ( 5.2
87.2 ( 5.8
94.3 ( 3.8
0.17 ( 0.01
101.5 ( 3.6
57 ( 6#
a Values are the mean of three experiments, standard deviations are given. b ScGT1 cells were cultured in DMEM with 10% FBS, plated 25000 cells
in each well of 96-well plates. The compounds were dissolved in DMSO (100%) and diluted in PBS 1X before adding various concentrations (10 nM -
10 μM) and incubated for 5 days at 37 ꢀC, 5% CO2. The results were developed by calcein-AM fluorescence dye and read by microplate reader. c The
effect of library compounds on inhibition of scrapie prion replication. ScGT1 cells were cultured in DMEM with 10% FBS, split 1:10 into Petri dishes
and incubated for 2 days at 37 ꢀC and 5% CO2. Then, various compound concentrations (10 nM - 2 μM), being non-toxic for the cells, were added to the
plates. After a 5-day incubation, proteins of cells were extracted, quantified, digested with proteinase K (PK), and western-blotted. d Prion fibril
formation inhibitory activity in vitro (Control 45 ( 4 h). Statistical analysis was done by analysis of Student’s t-test (n = 4); (*) p e 0.05, (#) p e 0.01.
respectively. Because of the toxicity shown, 1 and 3 were
studied for their anti-prion activity at a lower concentration
(0.2 μM), whereas the other library members were assayed at
1 μM. The synthesized 2, 4-9 were found to cover a broad
range of activity against PrPSc formation, with inhibition
spanning from 3.4% to 89.7% (Table 1).
Compounds 1-3, bearing an acridine moiety, displayed a
general higher toxicity in the cell viability assay. 2 turned out
to be the most active compound, with a submicromolar EC50
(0.68 ( 0.05 μM) and a percentage of viable cells at EC50 of
75.2%. A different toxicity profile was observed for quinoline
derivatives 4-6, which were not toxic to ScGT1 cells (cell
viability of >100% at 1 μM). Intriguingly, 5 and 6 showed
also remarkable submicromolar EC50 values (0.73 ( 0.03 μM,
and 1.2( 0.1 μM, respectively;Figure3SI) comparabletothat
of BiCappa (0.32 ( 0.03 μM). To note, a series of bisquino-
lines with a polyamine linker have been already designed and
tested in ScN2a cell line but showed a lower activity against
prion infection (in the one-digit micromolar range).25 This
might confirm the design rationale, indicating that the pres-
ence of a BQ core is critical for activity. The replacement
of the 2,6-disubstited acridine ring of 1-3 with the unsubsti-
tuted THA, as in 7-9, resulted in a complete loss of activity,
pointing out to a possible role for the aromatic substituents in
the recognition process. Interestingly, these latter compounds
did not show toxicity. For all the three series (1-3, 4-6, and
7-9), data from the cell-screening assay suggest that a linker
length of three methylenes is important for optimal activity.
Intriguingly, a similar trend was observed by May et al. in
their series of analogous bivalent ligands.10 Altogether, these
preliminary results suggest that a specific length of the linker
and the presence of a chlorine substituent on the prion
recognition motifs might contribute to activity against PrPSc
formation. Regarding toxicity, the presence of the acridine
ring seems to be a major determinant, in line with the reported
DNA intercalation properties of this heterocycle.10 Conver-
sely, quinoline and THA moieties do not confer cytotoxicity.
Building on these considerations, we decided to synthesize a
second setofcompounds in which the effect of the substituents
on the heteroaromatic ring was investigated by synthesizing
Figure 1. (A) Western blot of protease-digested ScGT1 cell lysates
depicting the presence or absence of PrPSc after treatment with 10
before (up) or after (bottom) PK: Ctrl = control.
the 6-chloro-THA (10) and the 6-methoxyquinoline (11)
derivatives. Furthermore, to probe the bivalent mechanism
of action of 5, its corresponding monomeric derivative 12 was
designed.
From the biological studies (Table 1), as expected, quino-
line 11, lacking the chlorine atom, was not toxic against
ScGT1 cells while displaying negligible activity against prion
replication (inhibition of 4.7%). These results again point out
the critical role played by the chlorine substituent of the
aromatic ring. This speculation was further confirmed by
the outstanding activity shown by 10. In contrast to 8, which
does not carry the chlorine atom and is devoid of anti-prion
activity, 10 showed a remarkable EC50 of 0.17 μM, which is
the lowest among the present series of derivatives, even better
than that of BiCappa. Remarkably, 10 showed a concomitant
low toxicity (101.5% of viable cells at EC50 value) (Figures 1
and 3SI).
By comparing the dramatically different profiles shown by
monovalent 12 and bivalent 5 (2.9% vs 85.5% of inhibition),
we were able to provide the definitive proof of principle that
two proper substituted aromatic prion recognition motifs
connected by a BQ spacer are critical for activity.
To study the mechanism of action of the most active
compounds (2, 5, 6, and 10) at a molecular level, a fibrillation

8200 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Bongarzone et al.
antioxidant property of related BQ derivatives27,28 and CoQ
itself concerns mainly their reduced hydroquinone forms.
NQO1, an inducible enzyme that catalyzes the reduction of
quinones to hydroquinones, was shown to be responsible for
the production of the CoQ-reduced antioxidant forms, as well
as that of BQ derivatives.27,28 Therefore, since 2, 5, 6, and 10
share the same BQ nucleus, their antioxidant activity was also
evaluated in ScGT1, following exposure to t-BuOOH, and in
the absence or presence of pretreatment with sulforaphane
(SFP), an inducer of NQO1. Figure 2B clearly shows that 2, 5,
6, and 10 (at 1 μM) in their oxidized forms show a basal
antioxidant activity, but this activity was increased in cells
pretreated with SFP, confirming that NQO1 is involved in the
activation of BQ derivatives. As expected, the antioxidant
activity of BiCappa is not influenced by the overexpression of
NQO1.
Conclusion
A library of 11 symmetrical bivalent compounds was
synthesized by solution phase parallel synthesis and tested
against prion replication. Despite the small number of com-
pounds, four of them (2, 5, 6, and 10) were active against prion
replication in the submicromolar range, whereas monovalent
12 showed negligible activity. These results confirmed the
rationale for the design of bivalent anti-prion ligands.
7-Chloroquinolines (5 and 6) and 6-chloro-THA (10) deriva-
tives showed a concomitant encouraging low toxicity. Nota-
bly, the EC50 of 10 was even lower than that displayed by
BiCappa, which is a reference compound for prion diseases.10
Furthermore, 10 showed the largest correlation between the
cellular anti-prion activity and the capability of inhibiting PrP
fibril formation. Interestingly, for 10 we could also find
correspondence between anti-prion and antioxidant activities,
in contrast to the results obtained by Miyata et al. in a series of
very potent pyrazolone derivatives.33 Although its mechanism
of action is not fully disclosed (see docking studies in Support-
ing Information), we assume that the bivalent structure of 10
favors the interaction with prion recognition domains,
whereas the spacer actssimultaneouslyas a disrupting element
against PPIs and an effective antioxidant moiety. Remark-
ably, the 6-chloro-THA scaffold emerges as an effective and
completely novel prion recognition motif.
Figure 2. (A) Effect of 32, BiCappa, 2, 5, 6, and 10 (1 μM) on
ScGT1, evaluated by TBARS formation. Values are the mean ( SD
(n = 3). (B) Antioxidant activity of BiCappa, 2, 5, 6, and 10 in
ScGT1 cells against ROS formation induced by t-BuOOH. Experi-
ments were performed with ScGT1 cells treated or not with 2.5 μM
SFP: (/) p e 0.05 with respect to t-BuOOH treated samples; (#) p e
0.05 with respect to t-BuOOH þ SFP treated samples.
assay was used. Only 5, 10, and BiCappa, at 2 μM, exhibited
significant PrP amyloid fibril forming inhibitory activity. In
fact, they extended the lag phase to g53 h, showing signifi-
cantly slower kinetics than the control (45 h, Table 1). These
results, although preliminary, are in agreement with the starting
hypothesis that bivalent ligands might interact directly with
the recPrP to prevent its conversion to the misfolded PrPSc
isoform. Furthermore, the idea that hydrophobic and planar
molecular features are crucial for perturbing PPIs in the prion
fibrillogenesis processes seems confirmed.23 In addition, a key
molecular determinant seems to be the presence of a chlorine
substituent on the heteroaromatic terminal moieties.
In conclusion, the present series of molecules are chemical
probes that may facilitate the exploration of the molecular
mechanism underlying prion disease. We envisage that a
better understanding of the molecular framework of bivalent
ligands capable of inhibiting prion aggregation and OS would
facilitate the creation of new effective anti-prion agents.
The PrPSc infected cells are under OS, mainly caused by
mitochondrial dysfunction.29 In light of this, antioxidants
might be beneficial against prion diseases.30 Indeed, benzo-
quinones, such as coenzyme Q (CoQ), can scavenge ROS, and
CoQ treatment has been proposed for prion and other neuro-
degenerative diseases.4,31,32 Thus, we tested the antioxidant
potential of the most active BQ derivatives (2, 5, 6, and 10) in
ScGT1 cell lines by using the thiobarbituric acid reactive
substances (TBARS) assay and the a water-soluble deriva-
tive of vitamin E (6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid, 32) as a positive control.5 The assay measures
lipid hydroperoxides and aldehydes expressed as an average
percent of TBARS of treated cells versus untreated cells. As
shown in Figure 2A, 2, 5, and 6 displayed low antioxidant
activity (83-87%) at 1 μM while 10 behaves similarly to 32
(69% vs 71%, respectively). As expected, BiCappa, which
does not carry a BQ scaffold, did not show any antioxidant
capacity (93%). We have previously demonstrated that the
Experimental Section
Chemistry. All starting reagents were of the best grade avail-
able from Aldrich. Direct infusion ESI-MS spectra were re-
corded on a Waters ZQ 4000 apparatus. 1H NMR and 13C
NMR spectra were recorded at 200 MHz (1H) and 50.3 MHz
(13C) or at 400 MHz (1H) and 100 MHz (13C). Elemental
analysis was used to confirm g95% sample purity, and the
elemental compositions of the compounds agreed to within
(0.4% of the calculated value.
General Procedure b for the Synthesis of Library Members
1-11. In distinct reactors, 2,5-dimethoxybenzoquinone (1 equiv)
was suspended in EtOH (15 mL) and heated to 80 ꢀC until the solid
was completely dissolved. After the mixture was cooled to 50 ꢀC,
the appropriate amines 13-20 (2 equiv) were added to each
reaction mixture that became progressively clear and red. Each

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8201
mixture was heated at 50 ꢀC for 5 h. After the mixture was cooled,
precipitates formed, which were collected by filtration. The solids
were dissolved in diethyl ether (7-10 mL), and trifluoroacetic acid
(0.2 mL) was added to the solutions to obtain the corresponding
trifluoroacetate salts.
amyloids by 4,5-dianilinophthalimide and analogs. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 7159–7164.
(16) Sellarajah, S.; Lekishvili, T.; Bowring, C.; Thompsett, A. R.;
Rudyk, H.; Birkett, C. R.; Brown, D. R.; Gilbert, I. H. Synthesis
of analogues of Congo red and evaluation of their anti-prion
activity. J. Med. Chem. 2004, 47, 5515–5534.
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Lopez-Cobenas, A.; Bongarzone, S.; Biarnes, X.; Lopez-Alvarado,
P.; Cabezas, N.; Caramelli, M.; Carloni, P.; Menendez, J. C.;
Legname, G. Discovery of a class of diketopiperazines as antiprion
compounds. ChemMedChem 2010, 5, 1324–1334.
(18) Corson, T. W.; Aberle, N.; Crews, C. M. Design and applications
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Acknowledgment. This work was supported by grants
from the Fondazione Compagnia di San Paolo and by
Friuli-Venezia-Giulia Region through the Project “Search
for New Drugs” (SeND) (to G.L.), by University of Bologna
and MUR (to M.L.B.). The authors thank Gabriella Furlan
for editing and proofreading the manuscript. S.B. thanks
Professor C. Melchiorre for fruitful discussions.
Supporting Information Available: Chemical characterization
of 1-23 biological methods, and docking results. This material
is available free of charge via the Internet at http://pubs.acs.org.
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