Synthesis and biophysical evaluation of arylhydrazono-1H-2-indolinones as β-amyloid aggregation inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.11.015

A series of isatin-3-arylhydrazones were synthesized and evaluated in vitro as inhibitors of Aβ_1-40 aggregation using a thioflavin T fluorescence method. An exploration of the effects on Aβ_1-40 aggregation of a number of diverse subs

Full text of DOI:10.1016/j.ejmech.2010.11.015

European Journal of Medicinal Chemistry 46 (2011) 275e284
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biophysical evaluation of arylhydrazono-1H-2-indolinones as
-amyloid aggregation inhibitors
b
,
a
a
a
a
Francesco Campagna a , Marco Catto , Rosa Purgatorio , Cosimo D. Altomare , Angelo Carotti ,
*
Angelo De Stradis b, Gerardo Palazzo c
a Dipartimento Farmaco Chimico, Università degli Studi di Bari” Aldo Moro”, Via Orabona 4, 70125 Bari, Italy
b Istituto di Virologia Vegetale del CNR, Università degli Studi di Bari” Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy
c Dipartimento di Chimica e CSGI, Università degli Studi di Bari” Aldo Moro”, Via Orabona 4, 70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of isatin-3-arylhydrazones were synthesized and evaluated in vitro as inhibitors of Ab1e40
aggregation using a thioflavin T fluorescence method. An exploration of the effects on Ab1e40 aggregation
of a number of diverse substituents at phenylhydrazone group and 5,6- positions of the indolinone
nucleus led us to single out some new anti-aggregating compounds with IC50 values in the low micro-
molar range. The most active compounds carry methoxy- or hydroxy- substituents in the indolinone
5,6-positions and lipophilic groups such as iPr and Cl at 40- and 30-position, respectively, of the phe-
Received 4 August 2010
Received in revised form
10 November 2010
Accepted 10 November 2010
Available online 18 November 2010
nylhydrazone moiety. Two derivatives are noteworthy, namely 18 (IC50 ¼ 0.4
m
M) and 42 (IC50 ¼ 1.1
mM).
Keywords:
Isatin-3-arylhydrazones
Alzheimer’s disease
The in vitro effects of the highly active, water soluble, compound 42 on the temporal evolution of Ab1e40
fibrils formation were further investigated by circular dichroism spectroscopy, transmission electron
microscopy and dynamic light scattering studies, which clearly showed that this compound delayed and
lowered the amyloid fibril formation.
b
-Amyloid aggregation inhibitors
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
types of intermediate conformations observed for the unfolded
monomer that: 1) can aggregate directly into fibrils without
Protein misfolding and aggregation are common hallmarks in
neurodegenerative diseases such as Alzheimer’s (AD), Parkinson’s,
Huntington’s and prion diseases [1]. Although different strategies
for tackling protein aggregation and related toxic events are
intensively investigated, adequate therapeutic approaches are still
missing in the treatment of such invalidating pathologies [2].
previous association into oligomers, 2) does not favour the
assembly into either oligomers or fibrils, 3) supports oligomer
formation but is incompetent for assembly in fibrils [6]. Since the
toxicity and neurodegeneration in AD are attributed to soluble
intermediate oligomers and to a lesser extent to amyloid fibrils [7],
agents that prevent or reverse the oligomerization and/or fibrilli-
AD is characterized by the aggregation of amyloid
) into fibrillar plaques in selected areas of brain [3,4]. The main
constituents Ab1e40 and Ab1e42, normally present as soluble species
in extracellular fluids, polymerize through a -sheet arrangement
in ordered structures, oriented in parallel and anti-parallel direc-
tion, giving rise to specific self-assemblies termed cross-strand
ladders [5].
b
peptides
zation of Ab may have potential therapeutic application in the
(Ab
treatment of AD. Therefore, inhibitory compounds could be clas-
sified in three distinct groups: inhibitors of fibrillization but not of
oligomerization; inhibitors of oligomerization but not of fibrilliza-
tion; inhibitors of both aggregation pathways.
b
A number of efforts in searching for inhibitors of Ab aggregation
has demonstrated that small molecules, containing single or
multiple (hetero)aromatic rings often bearing hydroxy groups, can
inhibit proteineprotein interactions [8]. A not exhaustive list of
these structurally diverse inhibitors includes indole derivatives
such as polysubstituted fluorinated indoles [9], melatonin [10],
hydroxyindoles [11], as well as polyphenols [12,13], such as cur-
cumin [14] and resveratrol [15] (Chart 1).
A
b aggregation is indeed a multistep process involving the
formation of several intermediate species, including oligomers and
protofibrillar aggregates. The existence of oligomeric and fibrillar
assembly states, and the finding that oligomers are not obligate
intermediates in the fibril formation, are consistent with three
Several studies suggested that the driving forces governing the
A
b
assembly may include aromatic packing, hydrophobic forces
* Corresponding author. Tel.: þ39 080 5442780; fax: þ39 080 5442230.
E-mail address: campagna@farmchim.uniba.it (F. Campagna).
and electrostatic interactions [16e19]. The interactions between
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.015

276
F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
assess quantity, shape and dimension of fibrils [6], and finally with
dynamic light-scattering (DLS), aimed at characterizing the
temporal evolution of intermediate species in the pathway of
Ab1e40 free aggregation [34] and in the coincubation assay with
inhibitor 42.
2. Chemistry
Various arylhydrazines, commercially available as hydrochloride
salts, were condensed with unsubstituted, 5- and 5,6-substituted
isatins in methanol, affording the corresponding isatin-3-arylhy-
drazone derivatives 2e45 (Scheme 1). The non-commercial isatins
1aeb, and 1c, previously reported by us [35], were prepared via the
classical Sandmeyer reaction [36] involving conversion of
substituted anilines with hydroxylamine and chloral hydrate into
isonitrosoacetanilides, followed by cyclization in concentrated sul-
phuric acid, as shown in Scheme 1.
Chart 1. Selected known amyloid aggregation inhibitors.
aromatic units, referred to as pep interactions or p-stacking, may
play an important role in many areas of chemistry and biochem-
istry, most notably in molecular recognition and self-assembly
[20e24]. The hydrophobic interactions between Ab side chains,
leading to A fibrillization, could be blocked by small molecules
b
competing with such an association, ultimately leading to inhibi-
tion of fibril formation [25]. These interactions could be reinforced
by hydrogen bond (HB) formation between hydroxy or methoxy
groups on aromatic rings of the inhibitor and HB donor-acceptor
The synthesis and characterization of hydrazones 2, 4, 7, 8,11,12,
31 have been already reported [28,30,35,37e40].
The spectroscopic data of compounds 2e45, in good agreement
with previous studies on isatin-b-arylhydrazones [30], showed that
groups of Ab1e40
.
these compounds exist in the hydrazone planar form, stabilized by
a strong intramolecular hydrogen bond N-H$$$O forming a pseudo
six-membered ring. Therefore, the C]N double bond of the phe-
nylhydrazone moiety is in Z configuration.
Finally, mono- and poly-methyl ethers 10, 12e15, 18, 21, 24e29
were demethylated in high yield using BBr3 [41] to give the cor-
responding hydroxyl derivatives 33e45 (Scheme 1).
Our recent studies in the field of low-molecular weight inhibi-
tors of Ab1e40 aggregation led us to identify two series of
N-terminus benzamides of glycine-based symmetric oligopeptides
[26] and 1H-3-indolinone derivatives [27]. As a continuation of our
investigation in the field we synthesized a large number of isatin-3-
arylhydrazones (2e45), as structural analogs of previously reported
indol-2-aryldiazenylmethylene-3-ones [27] (Chart 2), and evalu-
ated their activity as inhibitors of Ab1e40 aggregation using a thio-
flavin T (ThT) fluorescence assay.
3. Inhibition of amyloid aggregation
It is worth noting that isatin-3-arylhydrazones are compounds
with interesting pharmacological profile, some of them showing
pharmacological activity as antineoplastics [28], cyclin-dependent
kinase 2 inhibitors [29], protein tyrosine phosphatase-2 inhibitors
[30] and antimicrobials [31].
In this study the in vitro effects of the highly active, water
soluble, compound 42 on the temporal evolution of Ab1e40 fibrils
formation was studied by ThT fluorescence spectroscopy, which
furnishes a direct information on fibril formation [6,32], and further
investigated by circular dichroism (CD) spectroscopy, which allows
one to investigate conformational changes of the monomeric,
In vitro inhibition of Ab1e40 aggregation was assessed following
a previously reported, improved ThT fluorescence-based method
[27], with the use of 2% HFIP as aggregation enhancer. Samples of
Ab were coincubated with test molecules at 100 mM concentration
and anti-aggregating activities were measured after 2 h of incu-
bation at 25 ꢀC. For the most active compounds (ꢁ80% Ab1e40
aggregation inhibition) IC50s were determined under the same
assay conditions.
4. Transmission electron microscopy (TEM)
unordered Ab1e40 to
[33]. The characterization of the inhibitory behaviour of compound
42, throughout the A polymerization process, was then investi-
b-sheet ordered amyloidogenic intermediates
TEM analysis was performed at 60000-fold magnification for
compound 42, compared with a free incubation sample of Ab.
Incubation conditions were suitably modified [27], without use of
HFIP and with ethanol as the co-solvent (10%) in pH 8.0 phosphate
buffer and incubation at 37 ꢀC, in order to achieve the complete
aggregation within 7 days.
b
gated with transmission electron microscopy (TEM), in order to
100.00
80.00
60.00
40.00
20.00
0.00
5. CD spectroscopy
CD spectra were recorded in the spectral range 195e250 nm,
following the conformational random coil-
revealed by the increase of the negative peak of CD signal at
215 nm. Reference A and A /compound 42 coincubation samples
b-sheet transition as
b
b
were prepared as above described for TEM studies.
6. DLS studies
0
1
2
3
4
5
6
7
8
days
Dynamic light scattering measurements were detected through
a 4 mW He-Ne laser (633 nm wavelength) and fixed detector angle
of 173ꢀ; the samples were prepared as in TEM studies. Data of
scattered light were analyzed in order to evaluate the size distri-
bution of particles and their z-averaged hydrodynamic diameter
Fig. 1. Time course of kinetics of
percentage of the final value of CD ellipticity at 215 nm (full line, diamonds) and ThT
fluorescence (dotted line, squares), respectively, attained at the plateau of the fibril-
lization reaction of Ab1e40 incubated alone (full symbols) and coincubated with 5
42 (open symbols).
A
b1e40 aggregation. Results are expressed as
mM

F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
277
Fig. 2. Effects of fibrillization inhibitor 42 on Ab1e40 aggregation. Ab1e40 (50
m
M) was incubated under fibrillization conditions alone (AeC) or in the presence of 5
m
M 42 (DeF).
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 42: (D) at time point 0; (E) at day 3; (F) after 7 days. The black scale bars are 100 nm.
(dh) through cumulant analysis and subsequent application of
StokeseEinstein equation.
donating properties of the OH groups. The introduction of 30-Cl
(and not 20- or 40-Cl) and 40-iPr substituents (cpds 42 and 44) on the
phenylhydrazone moiety of the 5,6-dihydroxy derivatives gave rise
to a more than two-fold increase in the inhibition potency, sug-
gesting that a suitable combination of lipophilic properties of the R
substituents with the OH groups at the 5,6-positions can improve
7. Results and discussion
The inhibitory effects on the Ab1e40 aggregation of compounds
2e45, which share a common arylhydrazono-indolin-2-one core
structure, was assessed by a ThT fluorescence spectroscopy assay
[26,27]. For all the synthesized compounds we measured the
the A
indolinones. Compounds 42 and 44, with IC50 values of 1.1 and
1.6 M, respectively, are worth of noting.
As for the 40-iPr derivatives, while the 5,6-dimethoxy compound
28 (IC50 ¼ 4.4 M) was less active than the corresponding dihy-
droxy compound 44 (IC50 ¼ 1.6 M), quite surprisingly the 5-OCH3
derivative 18 (but not the 6-OCH3 one, 24) proved to be the most
active one with IC50 value in the submicromolar range (0.4 M).
b anti-aggregating properties of the arylhydrazono-1H-2-
m
percent of aggregation inhibition at 100 mM concentration, whereas
m
for the most active compounds (ꢁ80% Ab1e40 aggregation inhibi-
tion) we determined the half maximal inhibitory concentration
(IC50). The inhibition data are summarized in Table 1.
m
m
This data suggest that the hydrophobicity of alkyl substituents, like
the iso-propyl group, at the para-position of the phenylhydrazone
moiety should play a major role in improving the Ab1e40 anti-
aggregating properties. The activity is however decreased by the
steric hindrance of R alkyl substituents bulkier than the iso-propyl
group such as tert-butyl (19). Similarly, steric hindrance can explain
the sharp decrease in anti-aggregating potency of the 40-benzyloxy
derivative 22 compared to the corresponding 40-OCH3 derivative 21
7.1. Structure-activity relationships (SARs)
Within the limits of the examined property space (i.e., R, R1 and
R2 substituents varying in their electronic, steric, lipophilic and HB
properties) Ab1e40 aggregation data clearly proved that the aryl-
hydrazono-indolin-2-one derivatives lacking (or bearing substitu-
ents different from) hydroxyl or methoxy groups (2e9, 11, 30e32)
are inactive (11) or poor inhibitors (ꢂ80% inhibition at 100
mM
(IC50 ¼ 3.7
mM).
concentration).
Among the most active compounds, showing IC50 values in the
low (sub) micromolar range, we selected for further in-depth
biophysical investigation compound 42, due to its high water
solubility.
As a matter of facts, within the subset of analogs lacking
substituents on the indoline-2-one moiety (R1 ¼ R2 ¼ H; cpds. 2e10
and 33), only the derivatives bearing 40-OCH3 (10) and 40-OH (33)
groups showed IC50 values in the low micromolar range (13 and
9.3 mM, respectively). All the others did not exhibit any effect of
interest. The most potent anti-aggregating molecules were found
among the derivatives bearing OH and/or OCH3 groups at C-5 (R1)
and/or C-6 (R2) of the indoline-2-one moiety. Interestingly, the 5,6-
7.2. Kinetic studies of Ab aggregation inhibitory activity of 42
7.2.1. ThT fluorescence and CD spectroscopy studies
dihydroxy derivatives 40e44 (IC50 ¼ 4.3, 11, 1.1, 17, 1.6
m
M) showed
In order to achieve a reliable biophysical study on this class of
compounds, 42 was preferred because of its good solubility in
aqueous buffer. Incubation samples were prepared as above
higher activities compared to the respective 5,6-dimethoxy
congeners 14 and 25e28, suggesting that the interaction of the
examined compounds with Ab1e40 can be reinforced by the HB
described for TEM analysis. Aliquots, containing Ab1e40 (50
mM)

278
F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
10
8
a 1.792 Days
(A) 0.178 day
(B) 1.82 day
(C) 2.76 days
Aβ+42
b 2.87 Days
C
Aβ only
15
10
5
a
6
C
b
4
A
B
2
0
0
1
10
100
1000
1
10
100
1000
hydrodynamic diameter /nm
hydrodynamic diameter /nm
250
Aβ
Aβ+42
200
150
100
50
C
B
b
A
0.5
a
0
0.0
1.0
1.5
2.0
2.5
3.0
3.5
time /days
Fig. 3. Kinetics of aggregation of A
b
50
m
M solutions followed in DLS. Lower panel: time course of the average hydrodynamic diameter (obtained from the cumulant analysis of the
peptide and 5 M compound 42 (open symbols; lines are mere guide for the eye). Left upper panel: intensity
size distributions obtained by inverse Laplace transformation of the ACF of particles formed by A peptide alone at three different incubation times. The corresponding average
hydrodynamic diameters are labelled by the capital letters in the lower panel. Right upper panel: intensity size distributions obtained by inverse Laplace transformation of the ACF
ACF) for solutions of A
b
protein only (full symbols) and mixtures of A
b
m
b
of particles formed by A
lower panel.
b protein in presence of 42 at two different incubation times. The corresponding average hydrodynamic diameters are labelled by the small letters in the
alone or incubated with 42, were removed at various time points
during aggregation and analyzed by ThT fluorescence and CD
spectroscopy (Fig. 1). The ThT fluorescence curve exhibited a char-
acteristic sigmoidal shape, with an initial lag phase of two days and
an exponential growth going to completion between days 3 and 4.
The exponential phase was lowered and delayed in the presence of
for free incubation sample a stepwise increment from random coil
to -sheet content, measured as the absolute value of the typical
negative peak of ellipticity at 215 nm. It is worth noting that the
coincubated sample with 5 M 42 gave the same increment in the
first two days, but no further extension of -sheet content across
the following days. Also for CD analysis, raise of the concentration
of 42 up to 100 M resulted in the complete loss of -sheet struc-
b
m
b
5
mM 42 (Fig. 1) and completely zeroed at 25 and 100
mM concen-
m
b
tration (data not shown). Similarly, evolution of CD spectra showed
ture and in a stable random coil arrangement of peptide (data not
shown).
7.2.2. TEM studies
R
R
TEM results confirmed the conformational changes observed in
ThT fluorescence spectroscopy and CD studies above described
(Fig. 2). Ab1e40 did not form any detectable aggregates at time point
0 (Fig. 2A) but evolved to isolated fibrils with diameter of 8e10 nm
by day 3 (Fig. 2B), coexisting with small amorphous aggregates
(30e40 nm in size). These filaments gave rise after 7 days to an
extensive formation of long and curly fibrils (Fig. 2C). The effects of
compound 42 on the Ab1e40 assembly are depicted in Fig. 2DeF. At
time point 0 (Fig. 2D) Ab1e40 showed no aggregation; after 3 day of
H
N
NH
O
N
N
H
N
R1
R'
N
R2
O
H
indol-2-aryldiazenylmethylene-3-ones
Chart 2. Title isatines (right) and already published indole derivatives (left).
isatin-3-phenylhydrazones

F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
279
R
O
NH
O
N
R1
R1
iii, iv
R1
i, ii
O
N
H
R2
N
R2
NH2
R2
H
1a: R1 = H, R2 = OCH3
1b: R1 = R2 = OCH3
1c: R1 = F, R2 = Cl
R1 = R2 = H
R1 = F,
R2 = Cl
2 - 45
R1 = H, R2 = OCH3
R1 = R2 = OCH3
R1 = Br, R2 = H
R1 = NO2, R2 = H
R1 = OCH3, R2 = H
Scheme 1. Reagents and conditions: (i), CCl3CH(OH)2, Na2SO4ꢃ10H2O, H2O, conc. HCl, NH2OHꢃHCl, reflux; (ii), conc. H2SO4, 80 ꢀC; (iii), ReC6H4NHNH2ꢃHCl, MeOH, room
temperature; (iv), BBr3, CH2Cl2, ꢄ70 ꢀC.
incubation (Fig. 2E) 42 induced only the formation of above
described aggregates. Data obtained after 7 days of incubation
(Fig. 2F) were consistent with the inhibition of the extended fibril
formation. In both cases the resulting fibrils displayed a regular
helical pitch of 46 nm with diameters of 4 nm (node) and 8 nm
(internode).
the first day of incubation at 37 ꢀC corresponding to the lag-time,
the optical features of the solution were typical of small molecules
solutions: the scattering was very low (not shown) and the analysis
of DLS indicated the presence of particles with a hydrodynamic
diameter (dh) of about 14 nm, in very good agreement with the
hydrodynamic size reported for freshly prepared Ab1e40 [42]. Such
small particles are supposed to be micelle-like oligomers of Ab1e40
peptide [43]. The corresponding size distribution function is
essentially monomodal as shown in the left upper panel of Fig. 3
(curve A). After about 30 h (corresponding to the increase of CD
7.2.3. DLS studies
The time evolution of the hydrodynamic size probed by dynamic
light scattering is shown in the lower panel of Fig. 3 for the solution
of Ab1e40 peptide in presence (open circles) and in absence (closed
symbols) of compound 42. For the solution of Ab1e40 peptide alone,
the kinetics can be roughly divided in three temporal regimes. In
signal of b-sheet structure) there was a drastic increase in the size
of the particles that rapidly grew in size reaching a dh of about
40 nm. The size distribution remained monomodal although some
Table 1
Inhibition data of Ab1e40 fibrillogenesis of 3-arylhydrazonoindolin-2-one derivatives 2e45.
a
a
cpd
R1
R2
R
IC50
,
% Inhibition
cpd
R1
R2
R
IC50
,
% Inhibition
m
M
@ 100
m
Ma
m
M
@ 100 m
Ma
ꢅSEM
ꢅSEM
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
3-F
2Cl
3-Cl
61 ꢅ 1
52 ꢅ 4
44 ꢅ 5
39 ꢅ 4
24 ꢅ 3
57 ꢅ 3
29 ꢅ 1
64 ꢅ 4
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
H
H
H
H
4-CH(CH3)2
2-Cl
3-Cl
7.1
17
OCH3
OCH3
OCH3
OCH3
OCH3
F
Br
NO2
H
OH
OH
OH
OH
H
68 ꢅ 2
71 ꢅ 4
4-Cl
4-Cl
4-CH(CH3)2
4-OCH3
4-Cl
4-Cl
4-Cl
4-OH
H
3-Cl
4-CH(CH3)2
4-OH
H
4-CH(CH3)2
H
4.4
22
4-OCF3
3-NO2
4-CH(CH3)2
4-OCH3
H
H
H
H
3-Cl
28 ꢅ 3
63 ꢅ 4
12 ꢅ 2
H
13
NO2
OCH3
H
0
9.3
52 ꢅ 4
62 ꢅ 3
5.8
H
H
H
18
21
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
68 ꢅ 2
72 ꢅ 1
43 ꢅ 2
29 ꢅ 3
61 ꢅ 2
4-Cl
OH
OH
OH
OH
OH
OH
OH
OH
22
4-CH3
4-CH(CH3)2
4-C(CH3)3
3-C(CH3)3
4-OCH3
4-OCH2Ph
3-OCH2Ph
H
6.0
4.3
11
1.1
17
1.6
24
H
H
H
H
H
H
0.4
OH
OH
OH
OH
OH
OH
15
3.7
2-Cl
3-Cl
4-Cl
4-CH(CH3)2
OH
56 ꢅ 2
20 ꢅ 1
44 ꢅ 4
a
Data are means of three independent experiments; SEM of IC50 values are < 10%.

280
F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
very large particles are detectable in the size distributions (see
curve B of the left upper panel of Fig. 3). At the same time the
intensity of light scattered increased by more than one order of
magnitude. As a whole these results indicated the formation of
aggregates, whose size did not change for about one day. After 2.5
days of incubation a sudden raise in the average hydrodynamic size
was observed (well above 200 nm of average dh). Note that the dh
on the ordinate of the lower panel of Fig. 3 is an average diameter.
Actually, the size distribution obtained from the autocorrelation
function (ACF) collected after 2.2 days are markedly bimodal (in
agreement with TEM analysis, see Fig. 2B) with the coexistence of
small particles (dh around 40 nm) and very large particles with dh of
about 400e500 nm (see trace C in the left upper panel of Fig. 3 for
a representative example). Such huge dh corresponds (according to
the equations proposed by Lomakin [42]) to rigid rods of about
in the field of the small molecule inhibitors of amyloid aggregation,
which is involved in neurodegenerative diseases.
9. Experimental
9.1. Chemistry
Melting points (mp) were determined by the capillary method on
a Stuart Scientific SMP3 electrothermal apparatus and are uncor-
rected. Elemental analyses were performed on the EuroEA 3000
analyzerforC,H, and N;experimentalresultsagreedtowithinꢅ0.40%
of the theoretical values. IR spectra were recorded using potassium
bromide disks on a PerkineElmer Spectrum One FT-IR spectropho-
tometer; only the most significant IR absorption bands are reported.
1H NMR spectra were recorded in DMSO-(d6), unless otherwise
specified, on a Varian Mercury 300 spectrometer. Chemical shifts
3 mm in length. Thus it seems that the aggregation in native Ab1e40
solution proceeded through the formation, after a 24 h lag time, of
intermediate aggregates, leading after about 3 days of incubation to
mature (mm in length) fibrils.
The kinetics in presence of the inhibitor 42 was markedly
different. Already few minutes after the complete dissolution of
are expressed in d (ppm) and the coupling constants J in Hz. The
following abbreviations were used: s, singlet; d, doublet; t, triplet;
ep, septuplet; dd, double doublet; 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 s). Chro-
matographic separations were performed on silica gel 63e200
(Merck). Commercial reagents and solvents were purchased from
SigmaeAldrich and Alfa Aesar.
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
the solid Ab peptide the DLS probed the presence in solution of
discrete aggregates of about 30 nm in hydrodynamic diameter
(see the size distribution a in the right upper panel of Fig. 3).
Differently from the case of solutions without inhibitor, in pres-
ence of 42 the size of the particles in solution did not change for
about 3 days (see lower panel in Fig. 3). These aggregates were
somehow different from those observed in solutions of Ab1e40
without inhibitor. Indeed they formed immediately, were smaller
in size and delayed somehow the formation of fibrils by 12 h. It is
only after almost three days of incubation that a marked increase
in the average hydrodynamic size was observed (lower panel in
Fig. 3). Also in this case the size distributions are all bimodal and
show the coexistence of the small aggregates (dh w30 nm) and
large aggregates (dh w400 nm).
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 50e800 amu.
Compounds 1a [44],1b [45],1c [35], 2 [38], 4 [28], 7 [35], 8 [30],11
[38] and 12 [39] have been prepared according to quoted references.
9.1.1. General procedure for the synthesis of arylhydrazono-indolin-
2-ones 2e32
Suitable indoline-2,3-dione 1aec (1.0 mmol) was dissolved in
methanol (5 mL) with gentle heating, then arylhydrazine
(2.4 mmol) was slowly added with stirring at room temperature.
After stirring overnight, the resulting precipitate was filtered and
crystallized or the reaction mixture was evaporated to dryness and
the residue suitably purified.
8. Conclusions
The isatin-3-phenylhydrazone proved to be a scaffold for a new
class of low-molecular weight inhibitors of Ab1e40 aggregation.
Hydroxyand methoxysubstituents at 5,6-positions of the indoline-2-
one moietyand lipophilicgroupsoflimited sizeatthe meta-and para-
position of the phenylhydrazone (i.e., 30-Cl and 40-iPr, respectively)
increase the anti-aggregating potency, suggesting a significant role of
HB and hydrophobic interactionsinthe bindingof thearylhydrazono-
1H-2-indolinone derivatives to the Ab1e40 peptide.
As a major outcome of this study, three compounds, namely 18,
42 and 44, were found to inhibit in vitro Ab1e40 aggregation with
IC50 values in the low micromolar range (0.4, 1.1 and 1.6
respectively), as assessed by a ThT fluorescence spectroscopy assay.
Further biophysical kinetic studies, based on TEM and DLS, carried
out on the most water soluble compound 42, allowed us to reveal
a delayed and lowered fibril formation in Ab1e40 coincubated with
the test compound, compared with Ab1e40 alone. In particular, DLS
studies clearly showed that, in presence of 42, the intermediate
9.1.1.1. (3Z)-1H-indole-2,3-dione 3-[(3-fluorophenyl)hydrazone] 3.
Yield: 92%; 1H NMR (CDCl3)
d
6.72 (dt, 1H, Jm ¼ 1.9 Hz, Jo ¼ 7.8 Hz,
H-40), 6.89 (d, 1H, JoHeF ¼ 8.1 Hz, H-20), 7.01 (dd, 1H, Jm ¼ 2.2 Hz,
Jo ¼ 8.0 Hz, H-7), 7.07e7.30 (m, 4H, H-5, H-6, H-50 and H-60), 7.63 (d,
1H, Jo ¼ 7.7 Hz, H-4), 7.69 (s, 1H, NH), 12.66 (s, 1H, NNH). MS (ESI),
m/z ¼ 253.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3260,1670,1590, 1555, 1485,
1458 cmꢄ1. Anal. calcd for C14H10FN3O: C, 65.88; H, 3.95; N, 16.46.
Found: C, 66.27; H, 3.98; N, 16.55. mp 226e228 ꢀC from ligroin.
mM,
9.1.1.2. (3Z)-1H-indole-2,3-dione 3-[(3-chlorophenyl)hydrazone] 5.
Yield:77%; 1H NMR
d
6.90 (d, 1H, Jo ¼ 7.4 Hz, H-7), 7.00e7.06 (m, 2H,
H-5 and H-40), 7.24 (dt,1H, Jm ¼ 1.1 Hz, Jo¼ 7.4 Hz, H-6), 7.34e7.38 (m,
2H, H-60 and H-50), 7.52e7.53 (m,1H, H-20), 7.57 (d,1H, Jo ¼ 7.4 Hz, H-
4), 11.04 (s, 1H, NH), 12.64 (s, 1H, NNH). MS (ESI), m/z ¼ 269.8 (100%)
[M ꢄ H]ꢄ, 271.8 (33%) [M ꢄ H]ꢄ þ2. IR (KBr): 3183, 1678, 1555, 1235,
1172 cmꢄ1. Anal. calcd for C14H10ClN3O: C, 61.89; H, 3.71; N, 15.47.
Found: C, 61.55; H, 4.07; N, 15.61. mp 233e234 ꢀC from ethanol.
aggregates, immediately formed in the mixture with Ab peptide,
were more stable than those formed with Ab1e40 alone. Further in
vitro and in vivo biological studies (e.g., citotoxicity studies) should
better clarify the nature and toxicity of the observed intermediate
aggregates. This evaluation is even more important, when consid-
ering such intermediate aggregates as actual responsible of path-
ological events in AD.
9.1.1.3. (3Z)-1H-indole-2,3-dione 3-[(4-chlorophenyl)hydrazone] 6.
Yield: 96%; 1H NMR
d
6.90 (d, 1H, Jo ¼ 7.6 Hz, H-7), 7.03 (dt, 1H,
In summary, the new chemical entities identified in this study,
albeit still requiring chemical optimization, add to our knowledge
Jm ¼ 1.1 Hz, Jo ¼ 7.6 Hz, H-5), 7.23 (dt,1H, Jm ¼ 1.1 Hz, Jo ¼ 7.6 Hz, H-
6), 7.38 (d, 2H, J ¼ 9.2 Hz, H-30 and H-50), 7.45 (d, 2H, J ¼ 9.2 Hz, H-20

F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
281
and H-60), 7.53 (d, 1H, Jo ¼ 7.6 Hz, H-4), 11.02 (s, 1H, NH), 12.68 (s,
1H, NNH). MS (ESI), m/z ¼ 269.9 (97%) [M ꢄ H]ꢄ, 271.9 (35%)
[M ꢄ H]ꢄ þ2. IR (KBr): 3173,1676,1557,1463, 1245,1166 cmꢄ1. Anal.
calcd for C14H10ClN3O: C, 61.89; H, 3.71; N, 15.47. Found: C, 61.52; H,
4.05; N, 15.59. mp 266e267 ꢀC (dec.) from ethanol.
(d, 2H, J ¼ 8.5 Hz, H-30 and H-50), 7.32 (d, 2H, J ¼ 8.5 Hz, H-20 and H-
60), 10.78 (s, 1H, NH), 12.76 (s, 1H, NNH). MS (ESI), m/z ¼ 280.0
(100%) [M ꢄ H]ꢄ. IR (KBr): 3435, 1674, 1549, 1248, 1196 cmꢄ1. Anal.
calcd for C16H15N3O2: C, 68.31; H, 5.37; N, 14.94. Found: C, 67.92; H,
5.37; N, 14.63. mp 240e242 ꢀC from ethanol.
9.1.1.4. (3Z)-1H-indole-2,3-dione 3-[(4-isopropylphenyl)hydrazone]
9.1.1.11. (3Z)-5-Methoxy-1H-indole-2,3-dione 3-[(4-isopropylphenyl)
9. Yield: 94%; 1H NMR
d
1.17 (d, 6H, J ¼ 7.0 Hz, CH(CH3)2), 2.84 (ep,
hydrazone] 18. Yield: 70%; 1H NMR
d
1.18 (d, 6H, J ¼ 7.0 Hz, CH
1H, J ¼ 7.0 Hz, CH(CH3)2), 6.89 (d, 1H, Jo ¼ 7.7 Hz, H-7), 7.02 (t, 1H,
Jo ¼ 7.7 Hz, H-5), 7.18e7.24 (m, 3H, H-6, H-30 and H-50), 7.33 (d, 2H,
J ¼ 8.4 Hz, H-20and H-60), 7.51 (d, 1H, J ¼ 7.7 Hz, H-4), 10.99 (s, 1H,
NH), 12.73 (s, 1H, NNH). MS (ESI), m/z ¼ 277.9 (100%) [M ꢄ H]ꢄ. IR
(KBr): 3129, 2944, 1668, 1550, 1244, 1174 cmꢄ1. Anal. calcd for
C17H17N3O: C, 73.10; H, 6.13; N, 15.04. Found: C, 73.47; H, 6.45; N,
15.43. mp 194e196 ꢀC dec. from ethanol.
(CH3)2), 2.84 (ep, 1H, J ¼ 7.0 Hz, CH(CH3)2), 3.75 (s, 3H, 5-OCH3),
6.79e6.80 (m, 2H, H-6 and H-7), 7.09 (s, 1H, H-4), 7.22 (d, 2H,
J ¼ 8.6 Hz, H-30 and H-50), 7.34 (d, 2H, J ¼ 8.6 Hz, H-20 and H-60),
10.79 (s, 1H, NH), 12.78 (s, 1H, NNH). MS (ESI), m/z ¼ 307.9 (100%)
[M ꢄ H]ꢄ. IR (KBr): 3158, 2957, 1668, 1546, 1483, 1164, 1031,
785 cmꢄ1. Anal. calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58.
Found: C, 69.52; H, 6.17; N, 13.45. mp 166e168 ꢀC from ethanol.
9.1.1.5. (3Z)-1H-indole-2,3-dione 3-[(4-methoxyphenyl)hydrazone] 10.
9.1.1.12. (3Z)-5-Methoxy-1H-indole-2,3-dione
phenyl)hydrazone] 19. Yield: 77%; 1H NMR
3-[(4-tert-butyl-
1.27 (s, 9H, C(CH3)3),
Yield: 88%; 1H NMR
d
3.73 (s, 3H, OCH3), 6.89 (d, 1H, Jo ¼ 7.5 Hz, H-7),
d
6.95 (d, 2H, J ¼ 8.8 Hz, H-30 and H-50), 7.01 (dt, 1H, Jm ¼ 1.1 Hz,
Jo ¼ 7.5 Hz, H-5), 7.20 (dt,1H, Jm ¼ 1.1 Hz, Jo ¼ 7.5 Hz, H-6), 7.37 (d, 2H,
J ¼ 8.8 Hz, H-20 and H-60), 7.50 (d, 1H, Jo ¼ 7.5 Hz, H-4), 10.95 (s,1H,
NH), 12.75 (s, 1H, NNH). MS (ESI), m/z ¼ 265.9 (100%) [M ꢄ H]ꢄ. IR
(KBr): 3162, 1682, 1555, 1497, 1233, 1165, 1037, 746 cmꢄ1. Anal. calcd
for C15H13N3O2$H2O: C, 63.15; H, 5.30; N, 14.73. Found: C, 62.78; H,
5.27; N, 15.12. mp 189e190 ꢀC from ethanol.
3.76 (s, 3H, 5-OCH3), 6.81e6.82 (m, 2H, H-6 and H-7), 7.09e7.10 (m,
1H, H-4), 7.36e7.37 (m, 4H, H-20, H-30, H-50 and H-60), 10.80 (s, 1H,
NH), 12.78 (s, 1H, NNH). MS (ESI), m/z ¼ 321.9 (100%) [M ꢄ H]ꢄ. IR
(KBr): 3162, 2960, 1670, 1552, 1484, 1248, 1184, 1163, 782 cmꢄ1
.
Anal. calcd for C19H21N3O2: C, 70.57; H, 6.55; N, 12.99. Found: C,
70.33; H, 6.57; N, 13.14. mp 203e206 ꢀC from ethanol.
9.1.1.13. (3Z)-5-Methoxy-1H-indole-2,3-dione 3-[(3-tert-butylphenyl)
9.1.1.6. (3Z)-6-Methoxy-1H-indole-2,3-dione 3-(phenylhydrazone)
hydrazone] 20. Yield: 100%; 1H NMR
d 1.28 (s, 9H, C(CH3)3), 3.75 (s,
13. Yield: 35%; 1H NMR
d
3.77 (s, 3H, 6-OCH3), 6.46 (s,1H, H-7), 6.61
3H, 5-OCH3), 6.80e6.81 (m, 2H, H-6 and H-7), 7.06 (dd, 1H
Jm ¼ 1.8 Hz, Jo ¼ 8.8 Hz, 1H, H-40), 7.11 (s, 1H, H-4), 7.28e7.30 (m,
2H, H-50 and H-60), 7.37 (br s, 1H, H-20), 10.83 (s, 1H, NH), 12.78 (s,
1H, NNH). MS (ESI), m/z ¼ 321.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3152,
2954, 1670, 1552, 1484, 1186, 1161, 807 cmꢄ1. Anal. calcd for
C19H21N3O2: C, 70.57; H, 6.55; N, 12.99. Found: C, 70.93; H, 6.66; N,
13.25. mp 186e187 ꢀC from ethanol.
(d, 1H, Jo ¼ 8.4 Hz, H-5), 6.96e7.00 (m, 1H, H-40), 7.30e7.37 (m, 4H,
H-20, H-30, H-50 and H-60), 7.44 (d, 1H, Jo ¼ 8.4 Hz, H-4), 10.96 (s, 1H,
NH), 12.52 (s, 1H, NNH). MS (ESI), m/z ¼ 265.9 (100%) [M ꢄ H]ꢄ. IR
(KBr): 3194, 2837, 1685, 1566, 1499, 1149, 1099 cmꢄ1. Anal. calcd for
C15H13N3O2$½H2O: C, 65.21; H, 5.11; N, 15.21. Found: C, 65.60; H,
4.88; N, 15.02. mp 202e205 ꢀC from ethanol.
9.1.1.7. (3Z)-5,6-Dimethoxy-1H-indole-2,3-dione 3-(phenylhydrazone)
9.1.1.14. (3Z)-5-Methoxy-1H-indole-2,3-dione 3-[(4-methoxyphenyl)
14. Yield: 60%; 1H NMR
d
3.76 (s, 3H, 5-OCH3), 3.77 (s, 3H, 6-OCH3),
hydrazone] 21. Yield: 43%; 1H NMR 3.73 (s, 3H, 40-OCH3), 3.75 (s,
d
6.53 (s, 1H, H-7), 6.98 (dt, 1H, Jm ¼ 1.5 Hz, Jo ¼ 8.4 Hz, H-40), 7.12 (s,
1H, H-4), 7.30e7.40 (m, 4H, H-20, H-30, H-50 and H-60), 10.76 (s, 1H,
NH), 12.58 (s, 1H, NNH). MS (ESI), m/z ¼ 295.9 (100%) [M ꢄ H]ꢄ. IR
(KBr): 3151, 1680, 1556, 1496, 1166, 1135 cmꢄ1. Anal. calcd for
C16H15N3O3: C, 64.64; H, 5.09; N, 14.13. Found: C, 64.27; H, 5.13; N,
13.92. mp 226e228 ꢀC from ethanol.
3H, 5-OCH3), 6.78e6.79 (m, 2H, H-6 and H-7), 6.95 (d, 2H,
J ¼ 9.0 Hz, H-30 and H-50), 7.08e7.09 (m, 1H, H-4), 7.38 (d, 2H,
J ¼ 9.0 Hz, H-20 and H-60), 10.76 (s, 1H, NH), 12.78 (s, 1H, NNH). MS
(ESI), m/z ¼ 295.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3169, 2951, 1667, 1556,
1466, 1163, 1034, 778 cmꢄ1. Anal. calcd for C16H15N3O3: C, 64.64; H,
5.09; N, 14.13. Found: C, 64.27; H, 5.10; N, 14.01. mp 193e195 ꢀC
from ethanol.
9.1.1.8. (3Z)-5-Methoxy-1H-indole-2,3-dione 3-[(3-chlorophenyl)hydra-
zone] 15. Yield: 83%; 1H NMR
d
3.76 (s, 3H, 5-OCH3), 6.81e6.82 (m,
9.1.1.15. (3Z)-5-Methoxy-1H-indole-2,3-dione
phenyl]hydrazone} 22. Yield: 33%; 1H NMR
3-{[4-(benzyloxy)
3.32 (s, 3H, 5-OCH3),
2H, H-6 and H-7), 7.02e7.05 (m, 1H, H-40), 7.16e7.17 (m, 1H, H-4),
7.34e7.36 (m, 2H, H-60 and H-50), 7.55 (d, 1H, Jm ¼ 1.8 Hz, H-20),
10.85 (s, 1H, NH), 12.69 (s, 1H, NNH). MS (ESI), m/z ¼ 299.9 (100%)
[M ꢄ H]ꢄ, 301.8 (34%) [M ꢄ H]ꢄþ2. IR (KBr): 3167, 1668, 1558, 1476,
1162 cmꢄ1. Anal. calcd for C15H12ClN3O2: C, 59.71; H, 4.01; N, 13.93.
Found: C, 59.40; H, 4.04; N, 13.68. mp 230e232 ꢀC from ethanol.
d
5.07 (s, 2H, OCH2), 6.78e6.82 (m, 2H, H-6 and H-7), 7.09 (s,1H, H-4),
7.29e7.46 (m, 5H, H-200, H-300, H-400, H-500 and H-600), 7.02 (d, 2H,
J ¼ 8.6 Hz, H-20 and H-60), 7.41 (d, 2H, J ¼ 8.6 Hz, H-30 and H-50),
10.70 (s, 1H, NH), 12.70 (s, 1H, NNH). MS (ESI), m/z ¼ 371.9 (15%)
[M ꢄ H]ꢄ. IR (KBr): 3154, 2932, 1660, 1548, 1482, 1189, 1031,
694 cmꢄ1. Anal. calcd for C22H19N3O3: C, 70.76; H, 5.13; N, 11.25.
Found: C, 70.40; H, 5.19; N, 11.02. mp 218e220 ꢀC from ethanol.
9.1.1.9. (3Z)-5-Methoxy-1H-indole-2,3-dione
hydrazone] 16. Yield: 76%; 1H NMR
3-[(4-chlorophenyl)
d
3.75 (s, 3H, 5-OCH3),
6.80e6.84 (m, 2H, H-6 and H-7), 7.12 (s, 1H, H-4), 7.38 (d, 2H,
J ¼ 8.8 Hz, H-30 and H-50), 7.47 (d, 2H, J ¼ 8.8 Hz, H-20 and H-60),
10.83 (s, 1H, NH), 12.72 (s, 1H, NNH). MS (ESI), m/z ¼ 299.9 (100%)
[M ꢄ H]ꢄ, 301.8 (38%) [M ꢄ H]ꢄþ2. IR (KBr): 3153, 1672, 1559, 1485,
1160 cmꢄ1. Anal. calcd for C15H12ClN3O2: C, 59.71; H, 4.01; N, 13.93.
Found: C, 59.37; H, 3.94; N, 13.77. mp > 250 ꢀC (dec.) from ethanol.
9.1.1.16. (3Z)-5-Methoxy-1H-indole-2,3-dione 3-{[3-(benzyloxy)phenyl]
hydrazone} 23. Yield: 37%; 1H NMR
d 3.76 (s, 3H, 5-OCH3), 5.13 (s,
2H, OCH2), 6.68 (dd, 1H, Jm ¼ 1.8 Hz, Jo ¼ 8.1 Hz, H-40), 6.80e6.81
(m, 2H, H-6 and H-7), 6.98 (dd, 1H, Jm ¼ 1.8 Hz, Jo ¼ 8.1 Hz, H-60),
7.12e7.14 (m, 2H, H-4 and H-20), 7.22e7.48 (m, 6H, H-200, H-300, H-
400, H-500, H-600 and H-50), 10.83 (s, 1H, NH), 12.71 (s, 1H, NNH). MS
(ESI), m/z ¼ 371.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3150, 2951, 1681, 1563,
1483, 1195, 1035, 686 cmꢄ1. Anal. calcd for C22H19N3O3: C, 70.76; H,
5.13; N, 11.25. Found: C, 70.39; H, 5.26; N, 11.06. mp 209e211 ꢀC
from ethanol.
9.1.1.10. (3Z)-5-Methoxy-1H-indole-2,3-dione 3-[(4-methylphenyl)
hydrazone] 17. Yield: 86%; 1H NMR
d
3.30 (s, 3H, 40-CH3), 3.75 (s,
3H, 5-OCH3), 6.79e6.82 (m, 2H, H-6 and H-7), 7.10 (s, 1H, H-4), 7.16

282
F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
9.1.1.17. (3Z)-6-Methoxy-1H-indole-2,3-dione 3-[(4-isopropylphenyl)
hydrazone] 24. Yield: 35%; 1H NMR
1680, 1559, 1465, 1243, 1164, 818 cmꢄ1
. Anal. calcd for
d
1.18 (d, 6H, J ¼ 7.0 Hz, CH
C14H8Cl2FN3O: C, 51.88; H, 2.49; N,12.96. Found: C, 51.85; H, 2.63; N,
(CH3)2), 2.85 (ep, 1H, J ¼ 7.0 Hz, CH(CH3)3), 3.76 (s, 3H, 6-OCH3),
6.46 (d, 1H, Jm ¼ 2.2 Hz, H-7), 6.60 (dd, 1H, Jm ¼ 2.2, Jo ¼ 8.4 Hz, H-
5), 7.20 (d, 2H, J ¼ 8.4 Hz, H-30 and H-50), 7.28 (d, 2H, J ¼ 8.4 Hz, H-20
and H-60), 7.42 (d, 1H, Jo ¼ 8.4 Hz, H-4), 10.93 (s, 1H, NH), 12.53 (s,
1H, NNH). MS (ESI), m/z ¼ 307.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3151,
12.75. mp > 250 ꢀC (dec.) from ethanol.
9.1.1.24. (3Z)-5-Nitro-1H-indole-2,3-dione 3-[(4-chlorophenyl)hydra-
zone] 32. Yield: 65%; 1H NMR
d
7.08 (d, 1H, Jo ¼ 8.4 Hz, H-7), 7.41 (d,
2H, J ¼ 8.8 Hz, H-30 and H-50), 7.60 (d, 2H, J ¼ 8.8 Hz, H-20 and H-60),
8.15 (dd,1H, Jm ¼ 2.2 Hz, Jo ¼ 8.4 Hz, H-6), 8.31 (d,1H, Jm ¼ 2.2 Hz, H-
4), 11.65 (s, 1H, NH), 12.66 (s, 1H, NNH). MS (ESI), m/z ¼ 314.8 (100%)
[M ꢄ H]ꢄ, 316.8 (35%) [M ꢄ H]ꢄ þ2. IR (KBr): 3325, 3225, 1692,1568,
1340, 1161, 821 cmꢄ1. Anal. calcd for C14H9ClN4O3$½H2O: C, 51.63; H,
3.09; N,17.20. Found: C, 51.26; H, 3.04; N,16.89. mp 226e228 ꢀC from
acetonitrile.
2961, 1674, 1557, 1150, 1029 cmꢄ1
.
Anal. calcd for
C18H19N3O2$½H2O: C, 67.91; H, 6.33; N, 13.20. Found: C, 67.55; H,
6.00; N, 13.54. mp 213e215 ꢀC from ethanol.
9.1.1.18. (3Z)-5,6-Dimethoxy-1H-indole-2,3-dione 3-[(2-chlorophenyl)
hydrazone] 25. Yield: 62%; 1H NMR
d 3.77 (s, 3H, 5-OCH3), 3.78 (s, 3H,
6-OCH3), 6.55 (s, 1H, H-7), 7.00 (dt,1H, Jm ¼ 1.5, Jo ¼ 7.7 Hz, H-40), 7.17
(s,1H, H-4), 7.37 (t,1H, Jo ¼ 7.7 Hz, H-50), 7.46 (d,1H, Jo ¼ 7.7 Hz, H-60),
7.80 (dd, 1H, Jm ¼ 1.5 Hz, Jo ¼ 7.7 Hz, H-30), 10.90 (s,1H, NH), 12.92 (s,
1H, NNH). MS (ESI), m/z ¼ 329.8 (79%) [M ꢄ H]ꢄ, 331.8 (27%)
[M ꢄ H]ꢄ þ2. IR (KBr): 3168, 1685, 1557, 1159, 1138 cmꢄ1. Anal. calcd
for C16H14ClN3O3: C, 57.93; H, 4.25; N, 12.67. Found: C, 57.74; H, 4.39;
N, 12.45. mp 259e261 ꢀC from ethanol.
9.1.2. General procedure for the synthesis of hydroxy derivatives
33e45
Compounds 10, 12e15, 18, 21, 24e29 (1.0 mmol) were dissolved
in dichloromethane (20 mL) at the temperature of ꢄ70 ꢀC, and 4
equivalents of BBr3 were added dropwise. The mixture was allowed
to raise within 4 h to room temperature under stirring, then poured
onto ice; the precipitate was filtered or extracted with dichloro-
methane and suitably purified.
9.1.1.19. (3Z)-5,6-Dimethoxy-1H-indole-2,3-dione 3-[(3-chlorophenyl)
hydrazone] 26. Yield: 36%; 1H NMR
d 3.76 (s, 3H, 5-OCH3), 3.77 (s,
3H, 6-OCH3), 6.53 (s, 1H, H-7), 6.96e7.00 (m, 1H, H-40), 7.16 (s, 1H,
H-4), 7.30e7.34 (m, 2H, H-50 and H-60), 7.50e7.52 (m, 1H, H-20),
10.79 (s, 1H, NH), 12.52 (s, 1H, NNH). MS (ESI), m/z ¼ 329.8 (58%)
[M ꢄ H]ꢄ, 331.8 (21%) [M ꢄ H]ꢄ þ2. IR (KBr): 3166, 1681, 1493,
1464, 1162, 1139 cmꢄ1. Anal. calcd for C16H14ClN3O3: C, 57.93; H,
4.25; N, 12.67. Found: C, 57.73; H, 4.57; N, 12.78. mp 238e240 ꢀC
from ethyl acetate.
9.1.2.1. (3Z)-1H-indole-2,3-dione3-[(4-hydroxyphenyl)hydrazone]33.
Yield: 43%; 1H NMR
d
6.77 (d, 2H, J ¼ 8.8 Hz, H-30 and H-50), 6.89
(d, 1H, Jo ¼ 8.8 Hz, H-5), 7.00 (t, 1H, Jo ¼ 7.6 Hz, H-7), 7.18 (dt, 1H,
Jm ¼ 1.1 Hz, Jo ¼ 7.6 Hz, H-4), 7.25 (d, 2H, J ¼ 8.8, H-20 and H-60),
7.48 (d, 1H, Jo ¼ 7.6 Hz, H-6), 9.30 (s, 1H, 40-OH), 10.91 (s, 1H, NH),
12.76 (s, 1H, NNH). MS (ESI), m/z ¼ 251.9 (100%) [M ꢄ H]ꢄ. IR
(KBr): 3201, 1685, 1564, 1463, 1231 cmꢄ1
. Anal. calcd for
C14H11N3O2$H2O: C, 61.99; H, 4.83; N, 15.49. Found: C, 61.64; H,
4.51; N,15.10. mp 230e233 ꢀC from ethyl acetate/light petroleum
ether 65:35 (v/v).
9.1.1.20. (3Z)-5,6-Dimethoxy-1H-indole-2,3-dione 3-[(4-chlorophenyl)
hydrazone]27. Yield: 63%; 1H NMR
d 3.76 (s, 3H, 5-OCH3), 3.77 (s, 3H,
6-OCH3), 6.52 (s, 1H, H-7), 7.12 (s, 1H, H-4), 7.35 (d, 2H, J ¼ 8.9 Hz, H-
30 and H-50), 7.42 (d, 2H, J ¼ 8.9 Hz, H-20 and H-60), 10.77 (s, 1H, NH),
12.54 (s, 1H, NNH). MS (ESI), m/z ¼ 329.8 (37%) [M ꢄ H]ꢄ, 331.8 (13%)
9.1.2.2. (3Z)-5-Hydroxy-1H-indole-2,3-dione3-(phenylhydrazone) 34.
Yield: 71%; 1H NMR
d
6.64 (dd,1H, Jm ¼ 2.2 Hz, Jo ¼ 8.4 Hz, H-6), 6.71
[M ꢄ H]ꢄ þ2. IR (KBr): 3131, 2841, 1680, 1559, 1494, 1338, 1137 cmꢄ1
.
(d, 1H, Jo ¼ 8.4 Hz, H-7), 6.93 (d, 1H, Jm ¼ 2.2 Hz, H-4), 6.98e7.04 (m,
1H, H-40), 7.30e7.44 (m, 4H, H-20, H-30, H-50 and H-60), 9.13 (s, 1H, 5-
OH),10.71 (s,1H, NH),12.74 (s,1H, NNH). MS (ESI), m/z ¼ 251.9 (100%)
Anal. calcd for C16H14ClN3O3$½H2O: C, 56.39; H, 4.44; N, 12.33.
Found: C, 56.74; H, 4.22; N,12.39. mp 261e263 ꢀC from ethyl acetate.
[M ꢄ H]ꢄ. IR (KBr): 3356, 3163, 1672, 1555, 1243, 1191. 1164 cmꢄ1
.
9.1.1.21. (3Z)-5,6-Dimethoxy-1H-indole-2,3-dione 3-[(4-isopropyl-
Anal. calcd for C14H11N3O2: C, 66.40; H, 4.38; N, 16.59. Found: C,
66.16; H, 4.76; N, 16.25. mp > 250 ꢀC (dec.) from ethyl acetate/light
petroleum ether 40:60 (v/v).
phenyl)hydrazone] 28. Yield: 70%; 1H NMR
d
1.18 (d, 6H, J ¼ 7.0 Hz,
CH(CH3)2), 2.84 (ep, 1H, J ¼ 7.0 Hz, CH(CH3)2), 3.76 (s, 3H, 5-OCH3),
3.77 (s, 3H, 6-OCH3), 6.53 (s, 1H, H-7), 7.10 (s, 1H, H-4), 7.20 (d, 2H,
J ¼ 8.6 Hz, H-30 and H-50), 7.30 (d, 2H, J ¼ 8.6 Hz, H-20 and H-60),
10.75 (br s, 1H, NH), 12.60 (s, 1H, NNH). MS (ESI), m/z ¼ 337.9 (100%)
[M ꢄ H]ꢄ. IR (KBr): 3168, 2953, 1671, 1550, 1484, 1139 cmꢄ1. Anal.
calcd for C19H21N3O3: C, 67.24; H, 6.24; N, 12.38. Found: C, 67.05; H,
6.39; N, 12.21. mp 234e236 ꢀC from ethanol.
9.1.2.3. (3Z)-5-Hydroxy-1H-indole-2,3-dione
3-[(3-chlorophenyl)
hydrazone] 35. Yield: 88%; 1H NMR
d
6.65 (dd, 1H, Jm ¼ 2.2 Hz,
Jo ¼ 8.3 Hz, H-6), 6.70 (d, 1H, Jo ¼ 8.3 Hz, H-7), 6.96 (d, 1H,
Jm ¼ 2.2 Hz, H-4), 7.00e7.04 (m, 1H, H-40), 7.30e7.37 (m, 2H, H-60
and H-50), 7.48 (d, 1H, Jm ¼ 1.1 Hz, H-20), 9.15 (s, 1H, 5-OH), 10.74 (s,
1H, NH), 12.65 (s, 1H, NNH). MS (ESI), m/z ¼ 285.8 (100%) [M ꢄ H]ꢄ,
287.8 (33%) [M ꢄ H]ꢄþ2. IR (KBr): 3196, 1677, 1559, 1165 cmꢄ1. Anal.
calcd for C14H10ClN3O2·½H2O: C, 56.76; H, 3.74; N, 14.16. Found: C,
56.73; H, 3.64; N, 13.87. mp 270e272 ꢀC from ethyl acetate/light
petroleum ether 50:50 (v/v).
9.1.1.22. (3Z)-5,6-Dimethoxy-1H-indole-2,3-dione 3-[(4-methoxyphenyl)
hydrazone] 29. Yield: 62%; 1H NMR 3.72 (s, 3H, 40-OCH3), 3.76 (s, 3H,
d
5-OCH3), 3.77 (s, 3H, 6-OCH3), 6.52 (s, 1H, H-7), 6.92 (d, 2H, J ¼ 9.1 Hz,
H-30 and H-50), 7.09 (s, 1H, H-4), 7.34 (d, 2H, J ¼ 9.1 Hz, H-20 and H-60),
10.71 (s, 1H, NH), 12.59 (s, 1H, NNH). MS (ESI), m/z ¼ 325.8 (40%)
[M ꢄ H]ꢄ. IR (KBr): 3147, 1679, 1551, 1484, 1163, 1137 cmꢄ1. Anal. calcd
for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84. Found: C, 62.50; H, 5.43; N,
12.72. mp 196e198 ꢀC from ethanol.
9.1.2.4. (3Z)-5-Hydroxy-1H-indole-2,3-dione
3-[(4-isopropylphenyl)
hydrazone] 36. Yield: 97%; 1H NMR
d
1.17 (d, 6H, J ¼ 7.0 Hz, CH(CH3)2),
2.84 (ep, 1H, J ¼ 7.0 Hz, CH(CH3)2), 6.62 (dd, 1H, Jm ¼ 2.2 Hz,
Jo ¼ 8.2 Hz, H-6), 6.69 (d,1H, Jo ¼ 8.2 Hz, H-7), 6.91 (d, 1H, Jm ¼ 2.2 Hz,
H-4), 7.22 (d, 2H, J ¼ 8.6 Hz, H-30 and H-50), 7.29 (d, 2H, J ¼ 8.6 Hz, H-20
and H-60), 9.10 (s, 1H, 5-OH), 10.68 (s, 1H, NH), 12.74 (s, 1H, NNH). MS
(ESI), m/z ¼ 293.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3260, 1666, 1559, 1474,
1187, 1163 cmꢄ1. Anal. calcd for C17H17N3O2·½ H2O: C, 67.11; H, 5.92; N,
13.82. Found: C, 66.80; H, 5.71; N, 13.49. mp 265e268 ꢀC from ethyl
acetate/light petroleum ether 50:50 (v/v).
9.1.1.23. (3Z)-6-Chloro-5-fluoro-1H-indole-2,3-dione 3-[(4-chloro-
phenyl)hydrazone] 30. Yield: 70%; 1H NMR
d 7.00 (d, 1H,
JH-F ¼ 6.2 Hz, H-7), 7.39 (d, 2H, J ¼ 9.0 Hz, H-30and H-50), 7.51 (d,
2H, J ¼ 8.9 Hz, H-20 and H-60), 7.56 (d, 1H, J ¼ 8.9 Hz, H-40), 11.13 (s,
1H, NH), 12.68 (s, 1H, NNH). MS (ESI), m/z ¼ 321.8 (78%) [M ꢄ H]ꢄ,
323.8 (59%) [M ꢄ H]ꢄ þ2, 325.8 (13%) [M ꢄ H]ꢄ þ4. IR (KBr): 3165,

F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
283
9.1.2.5. (3Z)-5-Hydroxy-1H-indole-2,3-dione 3-[(4-hydroxyphenyl)
hydrazone] 37. Yield: 51%; 1H NMR
6.66 (dd, 1H, Jm ¼ 2.6 Hz,
3232, 1657, 1556, 1476, 1243, 1136 cmꢄ1
C14H10ClN3O3$H2O: C, 52.27; H, 3.76; N, 13.06. Found: C, 52.50; H,
3.81; N,13.18. mp 260e261 ꢀC (dec.) from chloroform/methanol 95:5
(v/v).
.
Anal. calcd for
d
Jo ¼ 8.4 Hz, H-6), 6.68 (d, 1H, Jo ¼ 8.4 Hz, H-7), 6.76 (d, 2H,
J ¼ 9.0 Hz, H-30 and H-50), 6.87e6.88 (m, 1H, H-4), 7.21 (d, 2H,
J ¼ 9.0 Hz, H-20 and H-60), 9.06 (s, 1H, 40-OH), 9.28 (s, 1H, 5-OH),
10.62 (s, 1H, NH), 12.76 (s, 1H, NNH). MS (ESI), m/z ¼ 267.9 (88%)
[M ꢄ H]ꢄ. IR (KBr): 3234, 1678, 1560, 1481, 1233, 1182 cmꢄ1. Anal.
calcd for C14H11N3O3$½H2O: C, 60.43; H, 4.35; N, 15.10. Found: C,
60.03; H, 4.24; N, 14.77. mp > 250 ꢀC (dec.) from ethyl acetate/light
petroleum ether 70:30 (v/v).
9.1.2.12. (3Z)-5,6-Dihydroxy-1H-indole-2,3-dione 3-[(4-isopropylphenyl)
hydrazone] 44. Yield: 84%; 1H NMR
d
1.17 (d, 6H, J ¼ 7.0 Hz, CH(CH3)2),
2.83 (ep, 1H, J ¼ 7.0 Hz, CH(CH3)2), 6.35 (s, 1H, H-7), 6.88 (s, 1H, H-4),
7.19e7.20 (m, 4H, H-20, H-30, H-50 and H-60), 8.64 (s, 1H, 5-OH), 9.30 (s,
1H, 6-OH), 10.51 (s, 1H, NH), 12.50 (s, 1H, NNH). MS (ESI), m/z ¼ 309.9
(100%) [M ꢄ H]ꢄ. IR (KBr): 3358, 2959, 1660, 1556, 1474, 1251,
1134 cmꢄ1. Anal. calcd for C17H17N3O3$½H2O: C, 63.74; H, 5.66; N,
13.12. Found: C, 63.42; H, 5.46; N, 12.80. mp 244e245 ꢀC from chlo-
roform/methanol 95:5 (v/v).
9.1.2.6. (3Z)-6-Hydroxy-1H-indole-2,3-dione3-(phenylhydrazone)38.
Yield: 23%; 1H NMR
d
6.35 (s, 1H, H-7), 6.43 (d, 1H, Jo ¼ 8.4 Hz, H-5),
6.94e6.97 (m, 1H, H-40), 7.31e7.32 (m, 5H, H-4, H-20, H-30, H-50 and
H-60), 9.80 (s, 1H, 6-OH), 10.80 (s, 1H, NH), 12.46 (s, 1H, NNH). MS
(ESI), m/z ¼ 251.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3201, 1668, 1559, 1144,
1106 cmꢄ1. Anal. calcd for C14H11N3O2$H2O: C, 61.99; H, 4.83; N,15.49.
Found: C, 62.32; H, 4.46; N, 15.06. mp > 250 ꢀC (dec.) from chloro-
form/methanol 95:5 (v/v).
9.1.2.13. (3Z)-5,6-Dihydroxy-1H-indole-2,3-dione
3-[(4-hydrox-
yphenyl)hydrazone] 45. Yield: 64% 1H NMR
d
6.34 (s, 1H, H-7), 6.73
(d, 2H, J ¼ 8.8 Hz, H-30 and H-50), 6.85 (s, 1H, H-4), 7.13 (d, 2H,
J ¼ 8.8 Hz, H-20 and H-60), 8.59 (s, 1H, 5-OH), 9.16 (s, 1H, 40-OH), 9.21
(s, 1H, 6-OH), 10.44 (s, 1H, NH), 12.49 (s, 1H, NNH). MS (ESI), m/
z ¼ 283.8 (100%) [M ꢄ H]ꢄ. IR (KBr): 3234, 1633, 1557, 1236,
1134 cmꢄ1. Anal. calcd for C14H11N3O4$H2O: C, 55.45; H, 4.32; N,
13.86. Found: C, 55.13; H, 4.33; N, 13.51. mp 205e206 ꢀC (dec.) from
ethyl acetate/light petroleum ether 97:3 (v/v).
9.1.2.7. (3Z)-6-Hydroxy-1H-indole-2,3-dione 3-[(4-isopropylphenyl)
hydrazone] 39. Yield: 23%; 1H NMR
d
1.17 (d, 6H, J ¼ 7.0, CH(CH3)2),
2.83 (ep,1H, J ¼ 7.0, CH(CH3)2), 6.34 (d,1H, Jm ¼ 1.8Hz, H-7), 6.43(dd,
1H, Jm ¼ 1.8 Hz, Jo ¼ 8.4 Hz, H-5), 7.18 (d, 2H, J ¼ 8.8 Hz, H-30 and H-
50), 7.24 (d, 2H, J ¼ 8.8 Hz, H-20 and H-60), 7.31 (d,1H, Jo ¼ 8.4 Hz, H-4),
9.79 (s, 1H, 6-OH), 10.80 (s, 1H, NH), 12.46 (s, 1H, NNH). MS (ESI), m/
z ¼ 293.9 (100%) [M ꢄ H]ꢄ. IR (KBr): 3424, 3221, 2900, 1660, 1462,
1145,1106 cmꢄ1. Anal. calcd for C17H17N3O2$½H2O: C, 67.09; H, 5.96;
N, 13.81. Found: C, 66.74; H, 5.81; N, 13.48. mp 240e242 ꢀC from
chloroform/methanol 95:5 (v/v).
9.2. Thioflavin T fluorescence spectroscopy analysis
A spectrofluorimetric method already published by some of us
[27], based on fluorescence emission of ThT, was followed. In order
to obtain batches of Ab1e40 free from preaggregates, commercial
peptide (purity >95%; EzBiolab, Carmel, USA) was dissolved in HFIP,
9.1.2.8. (3Z)-5,6-Dihydroxy-1H-indole-2,3-dione 3-(phenylhydrazone)
lyophilized and stored at ꢄ20 ꢀC. The solution of ThT (25
mM) used
40. Yield: 77%; 1H NMR
d 6.36 (s, 1H, H-7), 6.89 (s, 1H, H-4),
for fluorimetric measures was prepared in phosphate buffer
6.92e6.98 (m, 1H, H-40), 7.27e7.34 (m, 4H, H-20, H-30, H-50 and H-60),
8.65 (br s, 1H, 5-OH), 9.33 (br s, 1H, 6-OH), 10.53 (s, 1H, NH), 12.51 (s,
1H, NNH). MS (ESI), m/z ¼ 267.8 (100%) [M ꢄ H]ꢄ. IR (KBr): 3284,
0.025 M, pH 6.0, filtered through 0.45
ꢀC. Inhibitors were first tested at 100
prepared in phosphate buffered saline (PBS; 0.01 M, NaCl 0.1 M, pH
7.4), 30 MA peptide concentration, andcontained2% HFIPand10%
DMSO. Incubations were run in triplicate at 25 ꢀC for 2 h. Fluori-
metric measures were performed in a 700 L cuvette with a Per-
kineElmer LS55 spectrofluorimeter, using FLWinlab program.
470 L of ThT solution were mixed with 30 L of sample, and the
mm nylon filters and stored at
4
mM; test samples were
1663, 1561, 1476, 1243, 1164, 1134 cmꢄ1
. Anal. calcd for
m
b
C14H11N3O3·½H2O: C, 60.43; H, 4.32; N, 15.11. Found: C, 60.76; H,
4.51; N, 14.90. mp 230e232 ꢀC from ethyl acetate.
m
9.1.2.9. (3Z)-5,6-Dihydroxy-1H-indole-2,3-dione 3-[(2-chlorophenyl)
m
m
hydrazone] 41. Yield: 100%; 1H NMR
d 6.38 (s, 1H, H-7), 6.93 (s, 1H,
resulting fluorescence measured with parameters set as follows:
excitation at 440 nm (slit 5 nm); emission at 485 nm (slit 10 nm);
integration time 2 s. Biological activity was determined as percent of
inhibitory activity Vi for each compound according to the formula:
H-4), 6.94e6.99 (m,1H, H-40), 7.36 (t,1H, Jo ¼ 8.0 Hz, H-50), 7.44 (dd,
1H, Jm ¼ 1.1 Hz, Jo ¼ 8.0 Hz, H-60), 7.69 (d,1H, Jo ¼ 8.0 Hz, H-30), 8.71
(br s, 1H, 5-OH), 9.48 (br s, 1H, 6-OH), 10.65 (s, 1H, NH), 12.50 (s, 1H,
NNH). MS (ESI), m/z ¼ 301.8 (100%) [M ꢄ H]ꢄ, 303.7 (33%)
[M ꢄ H]ꢄþ2. IR (KBr): 3474, 3280, 1666, 1562, 1134, 1118 cmꢄ1. Anal.
calcd for C14H10ClN3O3: C, 55.37; H, 3.32; N, 13.84. Found: C, 54.97;
H, 3.23; N, 13.82. mp > 250 ꢀC from ethyl acetate.
Vi ¼ 100 ꢄ ½ðFi ꢄ FbÞ=F0ꢆ ꢇ 100
where Fi is the fluorescence value of the sample, Fb its blank value,
and F0 the fluorescence value for free aggregation of a sample of
A
b1e40 incubated in the same buffer/HFIP/DMSO system and in
9.1.2.10. (3Z)-5,6-Dihydroxy-1H-indole-2,3-dione 3-[(3-chlorophenyl)
hydrazone] 42. Yield: 84%; 1H NMR
d 6.35 (s, 1H, H-7), 6.92 (s, 1H, H-
absence of inhibitors. For most active inhibitors, IC50s were deter-
mined by testing in duplicate 5e7 concentrations, ranging from
4), 6.96 (dd,1H, Jm ¼ 2.0 Hz, Jo ¼ 8.4 Hz, H-40), 7.22e7.39 (m, 2H, H-50
and H-60), 7.40e7.44 (m, 1H, H-20), 8.67 (s, 1H, 5-OH), 9.43 (s, 1H, 6-
OH), 10.55 (s, 1H, NH), 12.43 (s, 1H, NNH). MS (ESI), m/z ¼ 301.8
(100%) [M ꢄ H]ꢄ, 303.8 (33%) [M ꢄ H]ꢄþ2. IR (KBr): 3239,1660,1561,
1244, 1133 cmꢄ1. Anal. calcd for C14H10ClN3O3$H2O: C, 52.27; H, 3.76;
N, 13.06. Found: C, 52.65; H, 3.50; N, 12.66. mp 233e234 ꢀC (dec.)
from chloroform/methanol 95:5 (v/v).
200 to 0.1 mM, in three independent experiments; statistics were
calculated within GraphPad PrismÒ v. 5 software.
9.3. TEM studies
Samples for TEM analysis were prepared in phosphate buffer
0.217 M pH 8.0 with 10% ethanol as co-solvent and incubated for up
9.1.2.11. (3Z)-5,6-Dihydroxy-1H-indole-2,3-dione 3-[(4-chlorophenyl)
to 7 days at 37 ꢀC. Final concentration of Ab1e40 was 50
m
M, while
M. For each sample,
mL) of incubated sample solution was applied to
hydrazone] 43. Yield: 78%; 1H NMR
d
6.34 (s, 1H, H-7), 6.88 (s, 1H, H-
compound 42 was tested at 100, 25 and 5
a little drop (20
carbon coated copper/rhodium grid (400 mesh; TAAB Laboratories
Equipment Ltd, Aldermaston, Berks, GB). The coated grid was
m
4), 7.31e7.32 (m, 4H, H-20, H-30, H-50 and H-60), 8.66 (s, 1H, 5-OH),
9.39 (s, 1H, 6-OH), 10.54 (s, 1H, NH), 12.47 (s, 1H, NNH). MS (ESI), m/
z ¼ 301.8 (100%) [M ꢄ H]ꢄ, 303.7 (32%) [M ꢄ H]ꢄþ2. IR (KBr): 3445,

284
F. Campagna et al. / European Journal of Medicinal Chemistry 46 (2011) 275e284
floated for 2 min on the sample drop and rinsed with 200
double distilled water. Negative staining was performed with
200 L of 2% w/v uranyl acetate solution (TAAB Laboratories
Equipment Ltd). After draining off the excess of staining solution by
means of a filter paper, the specimen was transferred for exami-
nation in a Philips Morgagni 282D transmission electron micro-
scope, operating at 60 kV. Electron micrographs of negatively
stained samples were photographed on Kodak electron microscope
film 4489 (Kodak Company, New York, USA).
m
L of
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DLS measurements were performed using a Zetasizer-Nano S
from Malvern operating with a 4 mW He-Ne laser (633 nm wave-
length) and a fixed detector angle of 173ꢀ (non invasive backscat-
tering geometry NIBSÔ) and with the cell holder maintained at
37 ꢀC by means of a Peltier element. The samples were prepared as
in TEM studies and filtered through Anotop 10 syringe filters
(Whatman) with a 20 nm cut-off directly into disposable micro-
volume cuvettes (Malvern ZEN0040).
Data were collected leaving the instrument free to optimize the
instrumental parameters (attenuator, optics position and number
of runs [46,47]). Usually the time autocorrelation function (ACF) of
scattered light intensity was the average of 12e16 consecutive runs
of 10 s each. Four intensity ACFs collected every 10 min have been
subsequently examined by the operator and averaged.
ꢀ
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The ACF of scattered light intensity was converted into the ACF
of scattered electric field. From this last quantity, the software
supplied by the producer evaluates the z-averaged hydrodynamic
diameter (dh) through cumulant analysis (II order) and subsequent
application of StokeseEinstein equation assuming the viscosity of
the buffer solution at 37 ꢀC calculated by the software implemented
by the manufacturer. dh represents an average size value; the
fraction of the light intensity scattered by particles of different size
(i.e. the size distribution by intensity) has been recovered by taking
the inverse Laplace transform of the ACF using the software
implemented by the manufacturer.
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Acknowledgements
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[47] Zetasizer Nano User Manual 0.317. Malvern Instruments Ltd, England,
2007.
This work was supported by a grant from MIUR (Rome, Italy;
PRIN project 20085HR5JK_005).