Design, synthesis and biological evaluation of indane-2-arylhydrazinylmethylene-1,3-diones and indol-2-aryldiazenylmethylene-3-ones as β-amyloid aggregation inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.12.029

Biological screening of (hetero)aromatic compounds allowed the identification of some novel inhibitors of Aβ_1-40 aggregation, bearing indane and indole rings as common scaffolds. Molecular decoration of lead compounds led to inhibitors exhibiting a potency, measured by the Thioflavin T fluorimetric assay, ranging from high to low micromolar IC₅₀. The 2-(p-isopropylphenyldiazenylmethylene)indolone derivative 6c resulted as the most potent aggregation inhibitor exhibiting an IC₅₀ of 1.4?μM, with complete lack of fibril formation as confirmed by transmission electron microscopy. Structure-activity relationships suggested that binding to the Aβ peptide may be largely guided by π-stacking and hydrogen bond interactions.

Full text of DOI:10.1016/j.ejmech.2009.12.029

European Journal of Medicinal Chemistry 45 (2010) 1359–1366
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of
indane-2-arylhydrazinylmethylene-1,3-diones and
indol-2-aryldiazenylmethylene-3-ones as b-amyloid aggregation inhibitors
Marco Catto a, Rosaria Aliano a, Angelo Carotti a, Saverio Cellamare a, Fausta Palluotto a, Rosa Purgatorio a,
,
Angelo De Stradis b, Francesco Campagna a
*
a
`
Dipartimento Farmacochimico, Universita degli studi di Bari Aldo Moro, via Orabona 4, 70125 Bari, Italy
Istituto di Virologia Vegetale del CNR, Universita degli Studi di Bari, Via Amendola 165/A, 70126 Bari, Italy
b
`
a r t i c l e i n f o
a b s t r a c t
Article history:
Biological screening of (hetero)aromatic compounds allowed the identification of some novel inhibitors
of Ab1–40 aggregation, bearing indane and indole rings as common scaffolds. Molecular decoration of lead
compounds led to inhibitors exhibiting a potency, measured by the Thioflavin T fluorimetric assay,
Received 11 May 2009
Received in revised form
30 September 2009
Accepted 14 December 2009
Available online 23 December 2009
ranging from high to low micromolar IC50
derivative 6c resulted as the most potent aggregation inhibitor exhibiting an IC50 of 1.4
complete lack of fibril formation as confirmed by transmission electron microscopy. Structure–activity
.
The 2-(p-isopropylphenyldiazenylmethylene)indolone
m
M, with
relationships suggested that binding to the A
hydrogen bond interactions.
b
peptide may be largely guided by
p-stacking and
Keywords:
b
-Amyloid peptide
Ó 2009 Elsevier Masson SAS. All rights reserved.
Alzheimer’s disease
Amyloid aggregation inhibitors
1. Introduction
Over the years small organic molecules containing single or
multiple (hetero)aromatic residues very often with phenolic
hydroxyls, have been extensively tested for their ability to inhibit
Amyloid aggregates are the hallmarks of many neurodegener-
ative disorders, including Alzheimer’s (AD), Huntington’s and Par-
Ab fibrillization in vitro (Chart 1). The detected inhibitors include
kinson’s
diseases
[1].
Morphological and
biochemical
Congo red [9], furan [10], indole [11–13], and bis-styrylbenzene
derivatives [14], stilbenes [15], curcumin [16], quercetin [17], and
many other diverse synthetic and natural compounds [18–20].
Several studies have suggested that interactions between
aromatic units may play an important role in many areas of
chemistry and biochemistry, most notably in molecular recognition
and self-assembly [21–24]. The attractive non-bonded interactions
characterization of the plaques in AD indicated beta-amyloid
proteins Ab1–40 and Ab1–42 as their main constituents. The forma-
tion of neuronal plaques is a lengthy, albeit yet undefined,
biochemical process. The key steps of this process imply an initial
conformational transition from the natural unfolded structure to
a b-sheet, a nucleation process and the formation of low-oligomeric
aggregates which evolve into the high-oligomeric species proto-
fibrils and fibrils. The aggregation of fibrils with other biological
components finally leads to the formation of extracellular neuronal
plaques.
between planar aromatic rings are referred to as
p–p interactions
or -stacking. The steric constrains associated with the formation
p
of these ordered stacking structures play a fundamental role in self-
assembly processes leading to the formation of supramolecular
Diverse strategies have been envisaged to identify inhibitors of
structures [21–27]. The hypothetical role of
in amyloid fibril formation suggests that molecules capable of
blocking -stacking may be potential candidates for the control of
p-stacking interactions
b
-amyloid protein folding and aggregation, including the rational
design of synthetic peptides and peptidomimetics [2,3], the search
for natural A -binding proteins [4–7] and the biological screening
p
b
amyloid diseases [26].
of a large number of molecular libraries [8].
Selection and screening, by an in vitro assay with Thioflavin T
(ThT) [28], of (hetero)aromatic compounds synthesized in our
laboratory allowed the identification, among others, of 2-[(2-(4-
chlorophenyl) hydrazinyl) methylene]-1H-indene-1,3(2H)-dione 3f
[29] (Table 2) as
a novel inhibitor of amyloid aggregation
* Corresponding author. Tel.: þ39 080 5442780; fax: þ39 080 5442230.
E-mail address: campagna@farmchim.uniba.it (F. Campagna).
(IC50 ¼ 23 M). The good activity of this compound, originally
m
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.029

1360
M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
NH2
NH2
Cl
O
O
Cl
N
N
N
N
N
N
H
O
N
N
H
SO3Na
SO3Na
Congo red
N
H
R1
X
4
8
F3C OH
COOEt
R2
Fig. 1. Structure of compounds 4 and 8.
R3
N
R1
R3
R3
Intermediates 5 reacted with diverse arylhydrazines to yield the
dehydrogenated derivatives 6a–m instead of the expected arylhy-
drazines deriving from the nucleophilic substitution of the dime-
thylamino group. The structural assignments of 6a–m were
accomplished on the basis of elemental analysis, ESI-MS and 1H
NMR spectral data.
R2
R2
Indole derivatives
bis-Styryl derivatives
OH
O
O
O
OH
OH
OH
HO
O
HO
OH
OCH3
The oxidation reaction leading to compounds 6 might involve
the formation of the tautomeric intermediate 7(B) that in the air
might be oxidated to the C-2 hydroxylated derivative, which in turn
might easily dehydrate to yield final compounds 6 (Scheme 3).
To confirm the proposed oxidation mechanism we carried out the
reaction of 5b with 3-chlorophenylhydrazine, under an argon
atmosphere, in an ethanol/acetic acid 97/3 solvent mixture previ-
ously degassed with helium. Under these experimental conditions,
TLC monitoring (light petroleum ether/ethyl acetate 65:35 v/v as
eluent mixture)showed onlyone spot atRf 0.50compared with 6k(Rf
0.80). However, the work-up of the reaction always led to 6k instead
of the corresponding and readily oxidizable arylhydrazine 7k.
A sample of 7k was isolated by condensing 5-bromo-3-hydroxy-
1H-indole-2-carbaldehyde, prepared by hydrolysis of the enamine
5b with KOH [36], with 3-chlorophenylhydrazine in an ethanol/
acetic acid 97/3 solvent mixture under an argon atmosphere. ESI-
MS and 1H NMR spectral data were in good agreement with the
proposed arylhydrazine structure.
OCH3
Quercetin
Curcumin
Chart 1. Known amyloid aggregation inhibitors.
reported as a benzodiazepine receptor ligand [29–32], prompted us
to design and synthesize a series of arylhydrazine derivatives – 2, 3
(Scheme 1) and 4 (Fig. 1) – and the indole analogues 6a–m (Scheme
2) and 8 (Fig. 1).
2. Chemistry
Compounds 2 and 3a–f were prepared starting from 1,3-cyclo-
pentanedione and 1,3-indanedione respectively, according to the
synthetic pathway shown in Scheme 1.
1,3-diketones were refluxed in N,N-dimethylformamide dime-
thylacetal (DMFDMA) to yield the corresponding dimethylamino-
methylene intermediates which were then reacted with suitable
arylhydrazines in an ethanol/acetic acid solvent mixture at 0 ꢀC to
produce 2 and 3a–f. Hydrazone 4 (Fig. 1) was obtained by
condensing ninhydrin with 4-chlorophenylhydrazine hydrochlo-
ride in tetrahydrofuran at room temperature.
Oxidation of 7k to 6k was monitored in acetone-d6 solution by
recording 1H NMR spectra over time (Fig. 2).
Indole compounds 6a–m might exist in two tautomeric forms,
namely hydrazone-imine form C and diazenyl-enamine form D
(Scheme 4). Their 1H NMR spectra in DMSO-d6 suggested a high
Indole derivatives 6a–m were prepared starting from commer-
cially available 3-acetoxyindoles according to the sequence of
reactions shown in Scheme 2.
prevalence of tautomeric form D. In fact, signals at
8.00 ppm could be attributed to 20 and 60 protons, and doublet close
to 7.10 ppm might be assigned to the H-7 proton, easily distin-
guishable from the H-4 signals in the 5-Br derivatives 6g–m.
Moreover, singlets close to 7.4 and 11 ppm could be assigned to
d
¼ 7.80–
The reaction of 3-acetoxyindoles with DMFDMA yielded the
(2Z)-2-(dimethylaminomethylene)-indol-3-ones 5a–b [33]. The Z
configuration was assigned to intermediates 5a–b on the basis of 1H
NMR spectral data. Unlike 2-aminomethylene-1,3-dicarbonyl
derivatives 1a–b, the 1H NMR spectra of 5a–b showed only one
signal (singlet) for the two methyl groups of the dimethylamino
moiety (Table 1), that are magnetically equivalent owing to their
free rotation around the C–N(Me)2 single bond. The hindered
rotation around the same bond in compounds 1a–b determined
a different pattern of signals for the two methyls of the dimethy-
lamino group, whose 1H NMR spectra showed indeed two separate
singlets, in full agreement with literature data [34,35].
d
d
d
CH and NH protons respectively. This assignment was in full
agreement with the 1H NMR data reported in the literature for
highly similar compounds [37].
In order to further discriminate between the hydrazone-imine
and the diazenyl-enamine forms we decided to run a phase-sensi-
tive 2D-NOESY experiment on a DMSO-d6 solution of compound 6b.
Interestingly, a strong cross-peak was seen between NH and the
OCOCH3
R'
Cl
O
H
N
N
N
H
O
H
O
O
i
O
O
R'
O
i
N
ii
2
O
N
H
N
R'
O
O
ii
N
H
N
R
N
H
N
H
N
1a,b
R
5a,b
6a-m
3a-f
Scheme 1. Reagents and conditions: (i), DMFDMA, reflux; (ii), R–C6H4NHNH2, ethanol,
acetic acid, 0 ꢀC.
Scheme 2. Reagents and conditions: (i), DMFDMA, reflux, 1 h; (ii), R–C6H4NHNH2,
ethanol, acetic acid, 0 ꢀC. 5a, R0 ¼ H; 5b, R0 ¼ Br.

M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
1361
Table 1
Identificative chemical shifts (1H NMR, DMSO-d6) of enaminoketones 1a–b and 5a–b.
O
H
N
W
CH3 (E)
X
CH3 (Z)
Compound
W
–
X
d
C]C–H
d
CH3 (E)
d
CH3 (Z)
d
N–H
1a
C]O
7.40
3.50
3.60
–
1b
5a
C]O
7.54
3.37
3.67
–
NH
6.97
7.02
3.16
3.18
8.80
9.07
Br
5b
NH
indole H-7 at
d
7.13, and this may occur only in the diazene–enamine
modified using DMSO (10%) as a co-solvent to allow poorly water-
soluble compounds to be tested even at high concentrations (up to
200 mM). 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was also used as
form. In addition, a weaker cross-peak was also observed between
NH and the arylhydrazinic CH at 7.83 (see Supporting Information).
d
The latter would be consistent with a Z geometry of the exocyclic
enaminic bond which would justify, at the same time, the strong
downfield shift observed for NH (d 10.98) most probably due to the
formation of a six-membered intramolecular hydrogen bond.
Finally, compound 8 (Fig. 1), which is structurally related to 6f
and 6l, was prepared by reacting 5-methoxyindole-3-carbaldehyde
with 4-chlorophenylhydrazine hydrochloride.
a co-solvent (2% v/v), because it dramatically accelerates the
aggregation process, as already reported [39]. The modified
experimental conditions allowed us to perform medium-
throughput screening of small chemical libraries, either available
commercially or originating from our laboratories, in much less
time compared with the co-solvent-free ThT assay. Anti-aggre-
gating activities were measured after 2 h of incubation at 25 ꢀC
instead of the 2–3 weeks required in co-solvent-free experiments.
This method was validated by satisfactorily reproducing inhibition
data of known anti-aggregating molecules [28].
3. Fluorescence spectroscopy
In vitro inhibition of Ab1–40 aggregation was studied following
a recently reported improved fluorescence-based method [28]. The
ThT fluorimetric assay [38] in phosphate-buffered saline was
4. Transmission electron microscopy (TEM)
TEM analysis was performed for the most active compound 6c,
compared with a free incubation sample of Ab. Incubation condi-
O
O
tions were suitably modified [40], without use of HFIP and with
ethanol as co-solvent (10%) in pH 8 phosphate buffer and incuba-
tion at 37 ꢀC, in order to achieve the complete aggregation within 7
days.
R
H
N
R
R'
R'
N
N
H
N
H
N
N
H
H
7(A)
7(B )
[O]
5. Results and discussion
O
O
R
R
The anti-aggregating activities of the examined compounds on
R'
R'
OH
OH
N
N
N
N
H
A
b1–40 fibril formation by the ThT fluorimetric assay are listed in
Tables 2 and 3 as IC50 or, for less active compounds, as the
percentage of aggregation inhibition at 100 M. Even though we
N
H
N
H
m
- H2O
are aware about the higher aggregating propensity and neurotox-
icity of Ab1–42 protein, we performed our inhibition experiments on
the more studied and readily accessible Ab1–40. Quercetin, a known
O
R'
inhibitor of
as the reference compound. Under our experimental conditions an
IC50 of 0.8 M was measured for quercetin, a value quite similar to
those reported in the literature derived from traditional ThT assays
(0.5–2.0 M) [17, 28]. The effects of indanedione derivatives 3a–f
b-amyloid aggregation [17], was included in our assay
N
N
H
m
N
6
m
R
and 4 and cyclopentanedione analogue 2 on Ab1–40 aggregation are
Scheme 3. Possible pathway of the air oxidation of 7 to 6.
reported in Table 2.

1362
M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
O
H
Br
N
N
H
Cl
N
H
A
7k
B
C
O
Br
N
N
H
D
N
Cl
6k
10.0
9.0
8.0
7.0
ppm (t1)
Fig. 2. Air oxidation of 7k (A) to 6k (D) in CD3COCD3 after 4 (B) and 30 days (C).
Analysis of biological data from Table 2 revealed that all the
examined compounds inhibited, to a different extent, the A
aggregation process. Compounds 3c–f displayed good IC50 values
equal to 18, 32, 18 and 23 M, respectively. Removal or substitution
with a methyl group of either lipophilic chlorine or isopropyl para-
substituents yielded compounds 3a–b with reduced inhibitory
activity.
Removal of the aromatic moiety of indanedione (see compound
2) or shortening of the linker (see compound 4) also induced
a significant lessening of activity compared to compounds 3c–f.
These findings revealed that the distance between the two
aromatic nuclei is an essential determinant of high activity.
To elaborate structure–activity relationships and learn more
a considerable increase of the activity with the increase of
substituent lipophilicity (6c > 6b > unsubstituted 6a). Since no
other evidences can be proposed to explain the low activity of 6f, l,
m, further studies will be undertaken by exploring a larger series of
analogs. Moreover, to carry on through the biological data discus-
sion, the substitution with bromine at position 5 of the indole
moiety decreased inhibitory activity of the unsubstituted and para-
alkylsubstituted compounds (compare 6g–i vs. 6a–c). Finally,
compound 8, containing a p-chlorophenylhydrazonomethylene
substituent at C-3 of the indole ring, showed a significantly
enhanced inhibitory potency compared to the analog 6f (IC50 > 100
b
m
and 23
Although our in vitro ThT fluorimetric assay does not provide
any experimental evidence about which step leading to A aggre-
gation is inhibited by our compounds, it seems reasonable to
hypothesize that prevention of cross- -sheet strand formation and/
or disruption might occur. In either case, efficient binding at the A
mM for compounds 6f and 8, respectively).
about the nature of binding interactions with the A
b
peptide, indole
b
derivatives 6a–m and 8 were prepared and tested (Table 3). Bio-
logical data indicated that simply altering the position of the chloro
group (6d–f and 6j–m), determined noteworthy effects on potency.
In particular, ortho and meta-monosubstitution (6d–e and 6j–k) led
to highly active inhibitors, whereas the para-chlorosubstituted
derivatives 6f, l, m resulted unexpectedly inactive. On the contrary
b
b
peptide guided by
p-stacking and hydrogen bond interactions
Table 2
compounds
para-monoalkylsubstituted
6b–c
showed
Inhibition data of Ab1–40 fibrillogenesis of indane and cyclopentane derivatives 2,
3a–f, 4.
Ma (% inhibition @
M ꢁ SEM)
O
O
Compound
R
IC50
100
, m
m
R'
R'
H
N
H
N
2
(44 ꢁ 5)
(49 ꢁ 2)
(44 ꢁ 4)
18
32
18
N
N
H
3a
3b
3c
3d
3e
3f
4
H
H N
N
4-CH3
4-CH(CH3)2
2-Cl
3-Cl
4-Cl
R
R
23
(38 ꢁ 5)
Hydrazone-imine form C
Diazenyl-enamine form D
a
SEMs of IC50s are within ꢁ15% of the reported values.
Scheme 4. Tautomeric equilibria of compounds 6.

M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
1363
Table 3
aggregates after 7 days (Fig. 3C). The effects of compound 6c on the
Inhibition data of Ab1–40 fibrillogenesis of indole derivatives 6a–m and 8.
A
A
b1–40 assembly are depicted in Fig. 3D–F. At time point 0 (Fig. 3D)
b1–40 showed no aggregation; after 3 day of incubation (Fig. 3E) 6c
Compound
R
R0
IC50
100
,
m
m
Ma (% inhibition @
M ꢁ SEM)
induced only a very reduced formation of isolated protofibrils. Data
obtained after 7 days of incubation (Fig. 3F) were consistent with
the inhibition of the extended fibril formation.
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
8
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
8.8
3.6
1.4
13
4-CH3
4-CH(CH3)2
2-Cl
3-Cl
4-Cl
2.5
6. Conclusions
(33 ꢁ 1)
H
35
The ThT fluorescence and TEM analysis results clearly indicated
4-CH3
4-CH(CH3)2
2-Cl
3-Cl
4-Cl
51
23
7.8
4.4
(20 ꢁ 3)
(23 ꢁ 2)
23
that indane and indole derivatives inhibit fibrillization of Ab1–40
.
Structural optimization led to novel and potent Ab1–40 aggre-
gation inhibitors 2–4, 6a–m and 8, showing IC50 values in the
micromolar range. Some of them exhibited considerable potency
comparable to the reference compound quercetin.
3-Cl, 4-Cl
QUR
0.8
Structure-activity relationships suggested that both distance
and substitution of the two aromatic moieties of the examined
compounds play a key role, along with the possible hydrogen bond
a
SEMs of IC50s are within ꢁ15% of the reported values. QUR:quercetin.
formation, in binding to the Ab peptide.
should play a key role. Moreover, the highest activity of compound
6c, which contains the isopropyl substituent on the phenyl-
hydrazine ring, suggests that additional hydrophobic interactions
Though in-depth biochemical and biophysical studies are
necessary to clarify the effect of our compounds on Ab1–40 fibrilli-
zation and then establish their anti-aggregating mechanism, this
work highlights new chemical entities eliciting high inhibitory
activity against amyloid aggregation.
could significantly strengthen binding to the Ab peptide.
Since fibrils ultimately form after extended incubation times, we
performed a TEM assay to qualitatively validate the inhibitory
potency of most active compound 6c (Fig. 3). To ensure the highest
reproducibility in comparison with other literature data, we
decided to shift the incubation conditions to those described by
Bartolini et al. [40], where fibrillization process is achieved in 10%
ethanol/phosphate buffer pH 8 at 37 ꢀC (ethanol was preferred to
DMSO as co-solvent). Aliquots were removed at various time points
during aggregation and analyzed by TEM assay at 60,000-fold
magnification. Ab1–40 was not aggregated at time point 0 (Fig. 3A)
but formed protofibrils with a diameter as small as 10–20 nm by
7. Experimental
7.1. Chemistry
Melting points (mp) were taken on a Gallenkamp MFB 595010
M apparatus and are uncorrected. Elemental analyses were per-
formed on a Carlo Erba 1106 analyser for C, H, N; experimental
results agreed to within ꢁ0.40% of the theoretical values. IR spectra
were recorded using potassium bromide disks on a Perkin Elmer
day
3 (Fig. 3B). These filaments gave amorphous amyloid
Fig. 3. Effect of fibrillization inhibitor 6c on Ab1–40 aggregation. Ab1–40 was incubated under ‘‘fibrillization conditions’’ alone (A–C) or in the presence of 50 mM 6c (D–F). Aliquots of
each reaction were assayed by TEM (60.000-fold magnification) at various time points. In the control reaction: (A) at time point 0; (B) at day 3; (C) after 7 days. In the presence of 6c:
(D) at time point 0; (E) at day 3; (F) after 7 days. The black scale bars are 100 nm.

1364
M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
Spectrum One FT-IR spectrophotometer, only the most significant
and diagnostic absorption bands being reported. 1H NMR spectra
were recorded in DMSO-(d6), unless otherwise specified, on a Var-
ian Mercury 300 spectrometer. Chemical shifts are expressed in
(d, 1H, J ¼ 7.8 Hz, H-30), 7.60–7.70 (br, 1H, CH–N), 7.70–7.80 (m, 4H,
H-4, H-5, H-6 and H-7), 8.60 (s,1H, NH), 10.90 (br, 1H, NH). MS (ESI),
m/z ¼ 296.9 (100%) [M ꢂ H]ꢂ, 298.8 (35%) [M ꢂ H]ꢂ þ 2. IR (KBr):
3240, 1700, 1650 cmꢂ1. Anal. calcd for C16H11ClN2O2: C, 64.33; H,
3.71; N, 9.38. Found: C, 64.02; H, 3.83; N, 9.12. mp 207–9 ꢀC from
ethanol.
d
(ppm) and the coupling constants J in Hz. The following abbre-
viations were used: s, singlet; d, doublet; t, triplet; ep, eptuplet; dt,
double triplet; m, multiplet. Exchange with deuterium oxide was
used to identify OH and NH protons, which in some cases gave
broad signals (br) widely spread on the base line and thus very
difficult to detect. Chromatographic separations were performed
on silica gel 63-200 (Merck). Commercial reagents and solvents
were purchased from Sigma–Aldrich.
ESI-MS was performed with an electrospray interface and an ion
trap mass spectrometer (1100 Series LC/MSD Trap System Agilent,
Palo Alto, CA). The sample was infused via a KD Scientific syringe
7.1.1.5. 2-((2-(3-Chlorophenyl)hydrazinyl)methylene)-1H-indene-
1,3(2H)-dione 3e. Yield: 65%; 1H NMR 6.65–6.75 (m, 2H, H-20 and
d
H-60), 6.80–6.90 (m,1H, H-40), 7.23 (t,1H, J ¼ 8.0 Hz, H-50), 7.57–7.65
(br, 1H, CH–N),7.66–7.75 (m, 4H, H-4, H-5, H-6 and H-7), 9.00 (s, 1H,
NH), 10.90 (br, 1H, NH). MS (ESI), m/z ¼ 296.9 (100%) [M ꢂ H]ꢂ,
298.8 (34%) [M ꢂ H]ꢂ þ 2. IR (KBr): 3240, 1700, 1645 cmꢂ1. Anal.
calcd for C16H11ClN2O2: 64.33; H, 3.71; N, 9.38. Found: C, 64.20; H,
3.74; N, 9.30. mp 192–4 ꢀC from ethanol.
pump at a rate of 10 mL/min. Ionization was achieved in the negative
ion mode. The pressure of the nebulizer gas was 15 psi. The drying
gas was heated to 350 ꢀC at a flow of 5 L/min. Full-scan mass spectra
were recorded in the mass/charge (m/z) range of 50–800 amu.
Compounds 1a, b and 3a were prepared according to Schenone
et al. [34]. Compound 3f was previously described in ref. [29].
Indole enaminone 5b was prepared as described for 5a [33],
which had yields and spectroscopic data in very good agreement
with those reported; both were used without further purification.
7.1.1.6. (2Z)-2-{[(E)-Phenyldiazenyl]methylene}-1,2-dihydro-3H-
indol-3-one 6a. Yield: 80%; 1H NMR
d
6.99 (t, 1H, J ¼ 7.5 Hz, H-5),
7.12 (d, 1H, J ¼ 8.0 Hz, H-7), 7.39 (s, 1H, CH]N), 7.50–7.63 (m, 5H, H-
30, H-50, H-4, H-6 and H-40), 7.90 (d, 2H, J ¼ 8.2 Hz, H-20 and H-60),
11.0 (s, 1H, NH). MS (ESI), m/z ¼ 247.9 (100%) [M ꢂ H]ꢂ. IR (KBr):
3430, 2920, 1690, 1630 cmꢂ1. Anal. calcd for C15H11N3O: C, 72.28; H,
4.45; N, 16.86. Found: C, 72.38; H, 4.81; N, 16.72. MP 207–9 ꢀC from
ethyl acetate/light petroleum ether 20:80 (v/v).
7.1.1. General procedure for the synthesis of compounds 2, 3b–e
and 6a–m
7.1.1.7. (2Z)-2-{[(E)-(4-Methylphenyl)diazenyl]methylene}-1,2-dihy-
dro-3H-indol-3-one 6b. Yield: 81%; 1H NMR
d 2.38 (s, 3H, CH3), 6.98
Suitable arylhydrazine (12 mmol) was dissolved in ethanol
(10 mL) and slowly added with stirring at 0 ꢀC to a solution of
intermediates 1 [34] or 5 [33] (10 mmol) in ethanol (30 mL) and
acetic acid (1.5 mL). After stirring overnight, the resulting precipi-
tate was filtered and crystallized or the reaction mixture was
evaporated to dryness and the residue purified by column
chromatography.
(t, 1H, J ¼ 7.4 Hz, H-5), 7.12 (d, 1H, J ¼ 8.2 Hz, H-7), 7.36 (d, 2H,
J ¼ 8.2 Hz, H-30 and H-50), 7.37 (s, 1H, CH]N), 7.55–7.62 (m, 2H, H-4
and H-6), 7.82 (d, 2H, J ¼ 8.2 Hz, H-20 and H-60),10.97 (s,1H, NH). 13C
NMR (150 MHz, DMSO-d6)
d 21.01 (CH3), 112.1 (C-7), 120.0 (C-3a),
120.6 (C-5), 122.6 (C-60and C-20), 124.0 (CH–N), 124.4 (C-4), 129.8
(C-50 and C-30), 137.2 (C-6), 138.9 (C-40), 141.7 (C-10), 151.5 (C-2),
152.9 (C-7a), 187.6 (C]O). MS (ESI), m/z ¼ 261.9 [M ꢂ H]ꢂ. IR (KBr):
3350, 1686, 1590, 1115 cmꢂ1. Anal. calcd for C16H13N3O: C, 72.99; H,
4.98; N, 15.96. Found: C, 73.15; H, 5.07; N, 16.11. mp 199–201 ꢀC
from ethyl acetate/light petroleum ether 30:70 (v/v).
7.1.1.1. 2-((2-(4-Chlorophenyl)hydrazinyl)methylene)cyclopentane-
1,3-dione 2. Yield: 70%; 1H NMR
d 2.42 (s, 4H, 2CH2), 6.76 (d, 2H,
J ¼ 8.8 Hz, H-20 and H-60), 7.25 (d, 2H, J ¼ 8.8 Hz, H-30 and H-50), 7.62
(s, 1H, CH–N), 9.22 (br, 1H, NH; NH signal not detectable). MS (ESI),
m/z ¼ 248.9 (100%) [M ꢂ H]ꢂ, 250.8 (34%) [M ꢂ H]ꢂ þ 2. IR (KBr):
3230, 1690, 1640 cmꢂ1. Anal. calcd for C12H11ClN2O2: C, 57.50; H,
4.42; N, 11.17. Found: C, 57.56; H, 4.45; N, 11.04. mp 197–9 ꢀC from
chloroform/methanol 95:5 (v/v).
7.1.1.8. (2Z)-2-{[(E)-(4-Isopropylphenyl)diazenyl]methylene}-1,2-
dihydro-3H-indol-3-one 6c. Yield: 55%; 1H NMR
d 1.22 (d, 6H,
J ¼ 6.9 Hz, 2CH3), 2.48 (ep, 1H, J ¼ 6.9 Hz, CH), 6.97 (t, 1H, J ¼ 7.8 Hz,
H-5), 7.12 (d, 1H, J ¼ 8.0 Hz, H-7), 7.37 (s, 1H, CH–N), 7.42 (d, 2H,
J ¼ 8.2 Hz, H-30 and H-50), 7.54–7.62 (m, 2H, H-4 and H-6), 7.83 (d,
2H, J ¼ 8.2 Hz, H-20 and H-60), 10.96 (s, 1H, NH). MS (ESI), m/
z ¼ 289.9 (100%) [M ꢂ H]ꢂ. IR (KBr): 3221, 1678, 1623, 1594,
1120 cmꢂ1. Anal. calcd for C18H17N3O: C, 74.20; H, 5.88; N, 14.42.
Found: C, 73.92; H, 6.04; N, 14.28. mp 159–60 ꢀC from light
petroleum ether/ethyl acetate 70:30 (v/v).
7.1.1.2. 2-((2-p-Tolylhydrazinyl)methylene)-1H-indene-1,3 (2H)-dione
3b. Yield: 55%; 1H NMR
d
2.20 (s, 3H, CH3), 6.79 (d, 2H, J ¼ 8.3 Hz, H-
20 and H-60), 7.00 (d, 2H, J ¼ 8.3 Hz, H-30 and H-50), 7.50–7.80 (m, 5H,
H-4, H-5, H-6, H-7 and CH–N), 8.60 (s, 1H, NH), 10.90 (br, 1H, NH).
MS (ESI), m/z ¼ 276.9 (100%) [M ꢂ H]ꢂ. IR (KBr): 3265, 3200, 1700,
1650 cmꢂ1. Anal. calcd for C17H14N2O2: C, 73.37; H, 5.07; N, 10.07.
Found: C, 73.32; H, 5.14; N, 9.97. mp 187–9 ꢀC from ethanol.
7.1.1.9. (2Z)-2-{[(E)-(2-Chlorophenyl)diazenyl]methylene}-1,2-dihy-
dro-3H-indol-3-one 6d. Yield: 66%; 1H NMR
d
7.01 (t, 1H, J ¼ 7.4 Hz,
H-5), 7.12 (d, 1H, J ¼ 7.8 Hz, H-7), 7.42 (s, 1H, CH–N), 7.45–7.70 (m,
5H, H-30, H-50, H-4, H-6 and H-40), 7.86 (d, 1H, J ¼ 7.4 Hz, H-60), 11.25
(s, 1H, NH). MS (ESI), m/z ¼ 281.9 (100%) [M ꢂ H]ꢂ, 283.8 (32%)
[M ꢂ H]ꢂ þ 2. IR (KBr): 3430, 1685, 1600 cmꢂ1. Anal. calcd for
C15H10ClN3O: C, 63.50; H, 3.55; N, 14.81. Found: C, 63.41; H, 3.44; N,
15.01. mp 182–4 ꢀC from ethanol.
7.1.1.3. 2-((2-(4-Isopropylphenyl)hydrazinyl)methylene)-1H-indene-
1,3(2H)-dione 3c. Yield: 40%; 1H NMR
d
1.13 (d, 6H, J ¼ 6.9 Hz,
2CH3), 2.78 (ep, 1H, J ¼ 6.9 Hz, CH), 6.69 (d, 2H, J ¼ 8.4 Hz, H-20
and H-60), 7.09 (d, 2H, J ¼ 8.4 Hz, H-30 and H-50), 7.68–7.71 (m,
5H, H-4, H-5, H-6, H-7 and CH–N), 8.59 (s, 1H, NH), 10.70 (br,
1H, NH). MS (ESI), m/z ¼ 304.9 (100%) [M ꢂ H]ꢂ. IR (KBr): 3235,
3168, 1645, 1468 cmꢂ1. Anal. calcd for C19H18N2O2: C, 74.49; H,
5.92; N, 9.14. Found: C, 74.36; H, 5.93; N, 9.08. mp 212–4 ꢀC
from ethanol.
7.1.1.10. (2Z)-2-{[(E)-(3-Chlorophenyl)diazenyl]methylene}-1,2-
dihydro-3H-indol-3-one 6e. Yield: 56%; 1H NMR
d 7.00 (t, 1H,
J ¼ 7.6 Hz, H-5), 7.12 (d,1H, J ¼ 8.0 Hz, H-7), 7.39 (s,1H, CH–N), 7.55–
7.63 (m, 4H, H-4, H-6, H-40 and H-50), 7.90 (d, 1H, J ¼ 7.1 Hz, H-60),
7.94 (s, 1H, H-20), 11.18 (s, 1H, NH). MS (ESI), m/z ¼ 281.8 (100%)
[M ꢂ H]ꢂ, 283.8 (35%) [M ꢂ H]ꢂ þ 2. IR (KBr): 3435, 1670, 1599,
1464 cmꢂ1. Anal. calcd for C15H10ClN3O: C, 63.50; H, 3.55; N, 14.81.
7.1.1.4. 2-((2-(2-Chlorophenyl)hydrazinyl)methylene)-1H-indene-
1,3(2H)-dione 3d. Yield: 60%; 1H NMR
d
6.82 (d, 1H, J ¼ 7.8 Hz,
H-60), 6.87 (t, 1H, J ¼ 7.8 Hz, H-40),7.22 (t, 1H, J ¼ 7.8, H-50) 7.36

M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
1365
Found: C, 63.75; H, 3.62; N, 14.49. mp 157–8 ꢀC from light petro-
leum ether/ethyl acetate 70:30 (v/v).
7.1.1.17. (2Z)-5-Bromo-2-{[(E)-(4-chlorophenyl)diazenyl]methylene}-
1,2-dihydro-3H-indol-3-one 6l. Yield: 80%; 1H NMR
7.10 (d, 1H,
d
J ¼ 8.5 Hz, H-6), 7.40 (s, 1H, CH–N), 7.62 (d, 2H, J ¼ 8.6 Hz, H-30 and
H-50), 7.71 (dd, 1H, Jm ¼ 2.2 Hz, Jo ¼ 8.5 Hz, H-6), 7.74 (d, 1H,
Jm ¼ 1.6 Hz, H-4) 7.90 (d, 2H, J ¼ 8.6 Hz, H-20 and H-60), 11.21 (s, 1H,
NH). MS (ESI) m/z ¼ 359.9 (74%) [M ꢂ H]ꢂ, 361.7 (100%)
[M ꢂ H]ꢂ þ 2, 363.7 (25%) [M ꢂ H]ꢂ þ 4. IR (KBr): 3317, 1686, 1602,
1463, 1264 cmꢂ1. Anal. calcd for C15H9BrClN3O: C, 49.69; H, 2.50; N,
11.59. Found: C, 49.57; H, 2.39; N, 11.20. mp 241–3 ꢀC from chlo-
roform/light petroleum ether 80:20 (v/v).
7.1.1.11. (2Z)-2-{[(E)-(4-Chlorophenyl)diazenyl]methylene}-1,2-
dihydro-3H-indol-3-one 6f. Yield: 60%; 1H NMR
d 7.00 (t, 1H,
J ¼ 7.3 Hz, H-5), 7.12 (d, 1H, J ¼ 6.8 Hz, H-7), 7.39 (s, 1H, CH–N), 7.55–
7.67 (m, 4H, H-30, H-50, H-4 and H-6), 7.91 (d, 2H, J ¼ 8.8 Hz, H-20
and H-60), 11.10 (s, 1H, NH). MS (ESI), m/z ¼ 281.9 (100%) [M ꢂ H]ꢂ,
283.9 (34%) [M ꢂ H]ꢂ þ 2. IR (KBr): 3380, 2925, 1700, 1605 cmꢂ1
.
Anal. calcd for C15H10ClN3O: C, 63.50; H, 3.55; N, 14.81. Found: C,
63.81; H, 3.75; N, 14.69. mp 207–9 ꢀC from chloroform/methanol
95:5 (v/v).
7.1.1.18. (2Z)-5-Bromo-2-{[(E)-(3,4-dichlorophenyl)diazenyl]-
methylene}-1,2-dihydro-3H-indol-3-one 6m. Yield: 60%; 1H NMR
7.1.1.12. (2Z)-5-Bromo-2-{[(E)-phenyldiazenyl]methylene}-1,2-dihy-
d
7.09 (d, 1H, J ¼ 8.2 Hz, H-7), 7.39 (s, 1H, CH–N), 7.71–7.89 (m, 3H,
dro-3H-indol-3-one 6g. Yield: 55%; 1H NMR
d
7.09 (d, 1H,
H-60, H-50 and H-6), 7.74 (br, 1H, H-4), 8.10 (s, 1H, H-20), 11.29 (s, 1H,
NH). MS (ESI), m/z ¼ 393.7 (51%) [M ꢂ H]ꢂ, 395.7 (100%)
[M ꢂ H]ꢂ þ 2, 397.6 (42%) [M ꢂ H]ꢂ þ 4, 399.6 (6%) [M ꢂ H]ꢂ þ 6. IR
(KBr): 3284, 1690, 1598, 1466 cmꢂ1. Anal. calcd for C15H8BrCl2N3O:
C, 45.37; H, 2.03; N, 10.58. Found: C, 45.18; H, 2.28; N, 10.25. mp
226–7 ꢀC from ethyl acetate/light petroleum ether 30:70 (v/v).
J ¼ 8.4 Hz, H-7), 7.40 (s, 1H, CH–N), 7.52–7.59 (m, 3H, H-30, H-40 and
H-50), 7.72 (d, 1H, J ¼ 8.4 Hz, H-6), 7.76 (br, 1H, H-4), 7.90 (d, 2H,
J ¼ 8.0 Hz, H-20 and H-60), 11.15 (s, 1H, NH). MS (ESI), m/z ¼ 325.9
(95%) [M ꢂ H]ꢂ, 327.7 (100%) [M ꢂ H]ꢂ þ 2. IR (KBr): 3310, 1685,
1625, 1600 cmꢂ1. Anal. calcd for C15H10BrN3O: C, 54.90; H, 3.07; N,
12.80. Found: C, 55.03; H, 3.25; N, 12.77. mp 200–1 ꢀC from
ethanol.
7.1.2. Synthesis of compounds 4, 7k and 8
7.1.2.1. 2-[(4-Chlorophenyl)hydrazono]indane-1,3-dione
4. Ninhydrin (0.089 g, 0.5 mmol) and 4-chlorophenylhydrazine
hydrochloride (0.072 g, 0.5 mmol) in tetrahydrofuran (3 mL) were
stirred at room temperature for 24 h. The precipitate was filtered
7.1.1.13. (2Z)-5-Bromo-2-{[(E)-(4-methylphenyl)diazenyl]methylene}-
1,2-dihydro-3H-indol-3-one 6h. Yield: 45%; 1H NMR
d 2.38 (s, 3H,
CH3), 7.08 (d, 1H, J ¼ 8.4 Hz, H-7), 7.35–7.38 (m, 2H, H-30 and
H-50),7.39 (s, 1H, CH–N), 7.71 (d, 1H, J ¼ 8.4 Hz, H-6), 7.75 (br, 1H,
H-4),7.81 (d, 2H, J ¼ 8.3 Hz, H-20 and H-60), 11.08 (s, 1H, NH). MS
(ESI), m/z ¼ 339.8 (98%) [M ꢂ H]ꢂ, 341.7 (100%) [M ꢂ H]ꢂ þ 2. IR
and crystallized (51% yield). 1H NMR
d
7.48 (d, 2H, J ¼ 9.0 Hz, H-20
and H-60), 7.66 (d, 2H, J ¼ 9.0 Hz, H-30 and H-50), 7.88 (br, 4H, H-4, H-
5, H-6 and H-7), 13.08 (s, 1H, NH). MS (ESI), m/z ¼ 282.8 (100%)
[M ꢂ H]ꢂ, 284.8 (31%) [M ꢂ H]ꢂ þ 2. IR (KBr): 3160, 1715, 1675,
1590 cmꢂ1. Anal. calcd for C15H9ClN2O2: C, 63.28; H, 3.19; N, 9.84.
Found: C, 63.59; H, 3.37; N, 9.80. mp 246–8 ꢀC (dec.) from ethanol.
(KBr): 3436, 2926, 1626, 1599, 1465 cmꢂ1
. Anal. calcd for
C16H12BrN3O: C, 56.16; H, 3.53; N, 12.28. Found: C, 55.88; H, 3.34;
N, 12.12. mp 193–5 ꢀC (dec.) from light petroleum ether/ethyl
acetate 70:30 (v/v).
7.1.2.2. 3-Hydroxy-1H-indole-2-carbaldehyde(3-chloro-phenyl)-
hydrazone 7k. 3-Chlorophenylhydrazine (0.17 g, 1.2 mmol) was
dissolved in ethanol (6.5 mL) and slowly added, under an argon
atmosphere, with stirring at 0 ꢀC to a solution of 5-bromo-3-
hydroxy-1H-indole-2-carbaldehyde [36] (0.26 g, 1.0 mmol) in
ethanol (3.7 mL) and acetic acid (0.21 mL). After stirring overnight,
the resulting precipitate was filtered to give the crude product (14%
7.1.1.14. (2Z)-5-Bromo-2-{[(E)-(4-isopropylphenyl)diazenyl]-
methylene}-1,2-dihydro-3H-indol-3-one 6i. Yield: 30%; 1H NMR
d
1.22 (d, 6H, J ¼ 6.9 Hz, 2CH3), 2.48 (ep, 1H, J ¼ 6.9 Hz, CH), 7.09 (d,
1H, J ¼ 8.4 Hz, H-7), 7.39 (s, 1H, CH–N), 7.43 (d, 2H, J ¼ 8.5 Hz, H-30
and H-50), 7.71 (d, 1H, J ¼ 8.4 Hz, H-6), 7.74 (br, 1H, H-4), 7.84 (d, 2H,
J ¼ 8.2 Hz, H-20 and H-60), 11.08 (s, 1H, NH). MS (ESI), m/z ¼ 367.9
(94%) [M ꢂ H]ꢂ, 369.8 (100%) [M ꢂ H]ꢂ þ 2. IR (KBr): 3335, 1682,
1620, 1587, 1465 cmꢂ1. Anal. calcd for C18H16BrN3O: C, 58.39; H,
4.36; N, 11.35. Found: C, 58.33; H, 4.74; N, 11.31. mp 195–7 ꢀC from
ethyl acetate/light petroleum ether 10:90 (v/v).
yield). 1H NMR (Acetone-d6)
d
6.74 (d, 1H, J ¼ 7.5 Hz, H-60), 7.00 (d,
1H, J ¼ 7.8 Hz, H-40), 7.19–7.28 (m, 4H, H-20, H-50, H-6 and H-7), 7.40
(s, 1H, CH–N), 8.10 (br, 2H, H-4 and NH), 9.62 (br, 1H, NH), 10.20 (br,
1H, NH), MS (ESI), m/z ¼ 361.8 (78%) [M ꢂ H]ꢂ, 363.7 (100%)
[M ꢂ H]ꢂ þ 2, 365.7 (23%) [M ꢂ H]ꢂ þ 4. IR (KBr): 3413, 1596,
7.1.1.15. (2Z)-5-Bromo-2-{[(E)-(2-chlorophenyl)diazenyl]methylene}-
1479 cmꢂ1
.
1,2-dihydro-3H-indol-3-one 6j. Yield: 83%; 1H NMR
d 7.10 (d, 1H,
J ¼ 8.5 Hz, H-7), 7.43 (s,1H, CH–N), 7.44–7.56 (m, 2H, H-30 and H-50),
7.64–7.79 (m, 3H, H-40, H-4 and H-6), 7.84 (dd, 1H, Jm ¼ 2.0 Hz,
Jo ¼ 7.7 Hz, H-60), 11.35 (s, 1H, NH). MS (ESI), m/z ¼ 359.8 (75%)
[M ꢂ H]ꢂ, 361.7 (100%) [M ꢂ H]ꢂ þ 2, 363.7 (25%) [M ꢂ H]ꢂ þ 4. IR
(KBr): 3370, 1690, 1620, 1600 cmꢂ1. Anal. calcd for C15H9BrClN3O: C,
49.69; H, 2.50; N, 11.59. Found: C, 49.43; H, 2.60; N, 11.61. mp 171–
7.1.2.3. 5-Methoxy-1H-indole-3-carbaldehyde (4-chlorophenyl)hy-
drazone 8. 5-methoxyindole-3-carbaldehyde (0.088 g, 0.5 mmol)
and 4-chlorophenylhydrazine hydrochloride (0.072 g, 0.5 mmol) in
methanol (3 mL) were stirred at room temperature for 48 h.
Evaporation of solvent gave 8 as a yellow solid (40% yield). 1H NMR
d
3.81 (s, 3H, CH3), 6.81 (dd, 1H, Jm ¼ 2.6 Hz, Jo ¼ 8.6 Hz, H-6), 7.00
3
ꢀC from ethanol.
(d, 2H, J ¼ 9.0 Hz, H-20 and H-60), 7.22 (d, 2H, J ¼ 9.0 Hz, H-30 and H-
50), 7.29 (d, 1H, J ¼ 8.6 Hz, H-7), 7.58 (d, 1H, J ¼ 2.6 Hz, H-2), 7.72 (d,
1H, J ¼ 2.6 Hz, H-4), 8.10 (s, 1H, CH]N), 9.95 (br, 1H, NH), 11.20 (s,
1H, NH). MS (ESI), m/z ¼ 297.9 (100%) [M ꢂ H]ꢂ, 299.8 (35%)
[M ꢂ H]ꢂ þ 2. IR (KBr): 3113, 1652, 1486, 1230 cmꢂ1. Anal. calcd for
C16H14ClN3O: C, 64.11; H, 4.71; N, 14.02. Found: C, 64.10; H, 4.75; N,
13.63. mp 202–3 ꢀC from ethanol.
7.1.1.16. (2Z)-5-Bromo-2-{[(E)-(3-chlorophenyl)diazenyl]methylene}-
1,2-dihydro-3H-indol-3-one 6k. Yield 90%; 1H NMR
7.10 (d, 1H,
d
J ¼ 8.5 Hz, H-7), 7.41 (s,1H, CH–N), 7.56–7.64 (m, 2H, H-40 and H-50),
7.74 (dd, 1H, Jm ¼ 2.0, Jo ¼ 8.5, H-6), 7.77 (d, 1H, Jm ¼ 2.0, H-4), 7.88
(dt, 1H, Jm ¼ 2.0, Jo ¼ 7.0, H-60), 7.94 (d, 1H, Jm ¼ 2.0, H-20), 11.30 (s,
1H, NH). MS (ESI), m/z ¼ 359.9 (74%) [M ꢂ H]ꢂ, 361.8 (100%)
[M ꢂ H]ꢂ þ 2, 363.7 (28%) [M ꢂ H]ꢂ þ 4. IR (KBr): 3436, 1685, 1601,
1465, 1171 cmꢂ1. Anal. calcd for C15H9BrClN3O: C, 49.69; H, 2.50; N,
11.59. Found: C, 49.66; H, 2.19; N, 11.23. mp 168–9 ꢀC from chlo-
roform/light petroleum ether 70:30 (v/v).
7.2. Inhibition of b-amyloid aggregation
A spectrofluorimetric method modified from that of LeVine
[38], based on fluorescence emission of ThT, was followed. In

1366
M. Catto et al. / European Journal of Medicinal Chemistry 45 (2010) 1359–1366
order to obtain batches of
Ab1–40 free from preaggregates,
Appendix. Supporting Information
commercial peptide (purity >95%; Anaspec, USA) was dissolved
in HFIP, lyophilized and stored at ꢂ20 ꢀC as described by Liu [41].
2D-NOESY and 13C spectra of compound 6b, and dose/response
inhibition curve of 6c, can be found in the online version at doi:10.
1016/j.ejmech.2009.12.029.
The solution of ThT (25 mM) used for fluorimetric measures was
prepared in phosphate buffer 0.025 M, pH 6.0, filtered through
0.45
m teflon filters and stored at 4 ꢀC. Inhibitors were first
tested at 100 M; test samples were prepared in phosphate-
buffered saline (PBS; 0.01 M, NaCl 0.1 M, pH 7.4), 30 M A
m
m
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This work was supported by a grant from MIUR (Rome,
Italy; FIRB project RBAU01LSCE). The contribution to the NMR
studies by prof. Vito Capriati and the technical assistance of
Mr. Antonio Palermo and Mr. Giovanni Dipinto are gratefully
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