Pyrroloquinoxaline hydrazones as fluorescent probes for amyloid fibrils

Article abstract of DOI:10.1039/c1ob05288h

Here we describe the identification and preliminary characterization of a new class of pyrrolo(imidazo)quinoxaline hydrazones as flurescent probes for Aβ_1-42 fibrils. All the newly developed compounds were able to bind amyloid fibrils formed in

Full text of DOI:10.1039/c1ob05288h

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PAPER
Pyrroloquinoxaline hydrazones as fluorescent probes for amyloid fibrils†
Sandra Gemma,a,b Laura Colombo,a,c Gianluigi Forloni,a,c Luisa Savini,a,b Claudia Fracasso,a,c Silvio Caccia,a,c
Mario Salmona,a,c Margherita Brindisi,a,b Bhupendra P. Joshi,a,b Pierangela Tripaldi,a,b Gianluca Giorgi,d
Orazio Taglialatela-Scafati,a,e Ettore Novellino,a, f Isabella Fiorini,a,b Giuseppe Campiani*a,b and
Stefania Butinia,b
Received 22nd February 2011, Accepted 14th April 2011
DOI: 10.1039/c1ob05288h
Here we describe the identification and preliminary characterization of a new class of
pyrrolo(imidazo)quinoxaline hydrazones as flurescent probes for Ab1-42 fibrils. All the newly developed
compounds were able to bind amyloid fibrils formed in vitro and some of them displayed an increase of
their fluorescence upon binding. When tested on brain tissue preparations presenting Ab deposits, the
described hydrazones selectively stained amyloid structures and did not display aspecific binding. The
hydrazones did not show antifibrillogenic activity and electron microscopy analysis revealed that they
do not interfere with fibrils structure. The described pyrrolo(imidazo)quinoxalines could be useful for
studying amyloid structures in vitro. Moreover, their experimentally proven ability to cross the
blood–brain barrier in mouse opens the possibility of developing these compounds as potential
amyloid imaging agents for in vivo applications.
T (ThT, 1, Fig. 1) and Congo Red (2), have been widely used
Introduction
for studying amyloid fibrils. These compounds characteristically
A fluorescent probe is a molecular system able to produce a
stain amyloid-like deposits and specifically interact with the
detectable fluorescent signal upon interaction with a chemical
crossed-b-sheet structure of amyloid. ThT and similar fluorogenic
species. An extremely important field of application of fluorescent
probes is the detection of amyloid fibrils, highly organized
fibrillar aggregates characterized by a b-pleated sheet structure.
Amyloid fibrils are a fundamental feature of several disorders
collectively designated as protein misfolding disorders. Among
them, Alzheimer’s disease (AD) is characterized by the formation
of amyloid plaques, which are composed by fibrils of 40–42 amino
acid peptides termed b-amyloid (Ab). The detection of amyloid
deposits in affected brain tissues, as well as the evaluation of
fibrillogenesis and fibrillation kinetics in vitro are important areas
of intense research.1–3 Two fluorescent probes such as thioflavin
aEuropean Research Centre for Drug Discovery and Development (NatSyn-
Drugs), University of Siena, via Aldo Moro, 53100, Siena, Italy. E-mail:
campiani@unisi.it; Fax: 39 0577 234333; Tel: 39 0577 234172
bDip. Farmaco Chimico Tecnologica, University of Siena, via Aldo Moro,
53100, Siena, Italy. E-mail: campiani@unisi.it; Fax: 39 0577 234333;
Tel: 39 0577 234172
cIstituto di Ricerche Farmacologiche “Mario Negri” (IRFMN), Via la Masa
19, 20156, Milano, Italy
dDip. di Chimica, University of Siena, via Aldo Moro, 53100, Siena, Italy
eDip. di Chimica delle Sostanze Naturali, Universita` di Napoli Federico II,
via D. Montesano 49, 80131, Napoli, Italy
f
Dip. di Chimica Farmaceutica e Tossicologica, Universita` di Napoli Federico
II, via D. Montesano 49, 80131, Napoli, Italy
Fig. 1 Reference and title compounds. R, X and Y are defined in
Scheme 1.
† CCDC reference numbers 612697, 612698. For crystallographic data in
CIF or other electronic format see DOI: 10.1039/c1ob05288h
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compounds are broadly used for detecting cellular components
and structures in vitro but are not able to cross the blood–
brain barrier (BBB). On the other hand several uncharged agents
with the backbone structure of ThT and Congo Red have been
synthesized and evaluated for in vivo use as probes to image
amyloid plaques in AD brain.4–14
We have been recently interested in the field of amyloid binding
agents and in particular in developing fluorescent probes for
amyloid fibrils to be used for in vitro histochemical and cellular
characterization of amyloid structures. We recently proved that
specific 2-quinolylhydrazones typified by 3 (Fig. 1) were able to
bind amyloid fibrils in vitro and, after oral administration, stained
amyloid deposits in the brain of transgenic mice carrying the
human gene for b-APP.15 Moreover, detection of amyloid plaques
in mouse brain preparations was readily accomplished by the
distribution of compound-associated fluorescence. Although the
binding mode has not been defined at atomic level yet, linear dyes
such as ThT are believed to bind to extended channel-like motifs
along solvent-exposed surfaces of fibrils, in turn formed by side
chains of amino acids of the b-sheet structures. A striking feature
of ThT is the fluorescence enhancement which is observed upon
binding to fibrils.16,17 This feature has been intensively studied
suggesting that the fluorescence behaviour of ThT is related to
the rotation of the aniline and benzothiazolium rings around
their shared carbon-carbon bond. Although Dzwolak and co-
workers hypothesized that ThT is significantly twisted when bound
to amyloid,18 most studies suggest that ThT binding to amyloid
fibrils probably immobilizes the ThT aniline and benzothiazolium
rings in a flat conformation, resulting in a high quantum yield
of fluorescence.19 The uncharged compound 3, formed by two
aromatic systems conjugated through a hydrazone linker, is likely
to have a binding mode to amyloid fibrils similar to that of ThT.
With the aim of improving the binding affinity of 3 for amyloid
fibrils, we decided to replace the 4-methylquinolyl system of 3
with a pyrroloquinoxaline moiety (compound 4a, Fig. 1) in order
to increase the contact surface of the resulting compound within
the channel. During this study we discovered that introduction
of the pyrroloquinoxaline system strongly favored an almost flat
conformation of the whole system due to extensive conjugation
of the two heteroaromatic moieties through the hydrazone linker.
Starting from 4a, a small set of analogues was synthesized in order
to preliminarly evaluate the effect of differently substituted poli-
cyclic scaffolds for Ab binding. In particular, different substituents
at the benzofused ring (4a–e) were introduced, the benzo-fused
ring of 4a was replaced by a pyridine (4f), the pyrrole-fused ring
by an imidazole (4g,h) and the 4-quinoline by a 2-isoquinoline
moiety (4i). Here we report the synthesis, structural analysis,
amyloid binding properties, and fluorescent characterization in
the presence of amyloid fibrils of compounds 3 and 4a–i (Scheme
1).
Scheme 1 Synthesis of hydrazones4a–i. a) i. POCl3, N,N-dimethylaniline,
120 ◦C, 2–4 h (from 6: POCl3, N,N-dimethylaniline, 120 ◦C, 4 h, then
methanol, 25 ◦C, 5 min); ii. NH2NH2·H2O, ethanol, 90 ◦C, 2–4 h; b) quino-
lin-4-carboxaldehyde, ethanol, 90 ◦C, 4 h; c) quinolin-2-carboxaldehyde,
ethanol, 90 ◦C, 6 h.
Scheme 2 Synthesis of lactam 6. a) Dimethoxytetrahydrofuran, AcOH,
120 ◦C, 1 h; b) Pd/C, H2 (1 atm), 1 : 1 EtOAc/MeOH, 14 h; c) triphosgene,
toluene, reflux, 4 h.
and these latter compounds were reacted with hydrazine mono-
hydrate in refluxing ethanol to afford hydrazino-derivatives 7a–h.
In the last step of the synthesis, a mixture of hydrazines 7a–h
and quinolin-4-carboxaldehyde or quinolin-2-carboxyaldheyde in
ethanol was heated under reflux to furnish the desired hydrazones
4a–i in good overall yield. The final compounds were purified by
means of flash-chromatography followed by crystallization from
ethanol.
Lactam 6, necessary for the synthesis of hydrazone 4e was
prepared starting from the carboxylic acid 8 (Scheme 2) that was
converted into the corresponding pyrrole 9 applying the Clauson–
Kaas protocol. The nitro-group of 9 was reduced by catalytic
hydrogenation in the presence of palladium on carbon as the
catalyst and the aniline 10 thus obtained was finally cyclized to
lactam 6 by treatment with triphosgene in toluene.
Results and discussion
Chemistry
The synthesis of the target compound 4a–i was performed as
depicted in Schemes 1 and 2. Lactams 5a–d,f–h20 and 6 (Scheme 1)
were converted into the corresponding chlorides by treatment with
phosphorous oxychloride in the presence of N,N-dimethylaniline
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Structural analysis
NMR investigation of the target compounds revealed that their
proton spectra, performed in DMSO-d6 presented two different
sets of signals in almost 1 : 1 ratio. To investigate the structures
of these hydrazones, we decided to analyze a representative
compound both in solid phase (through X-ray diffraction) and
in solution (through NMR techniques). Depending upon the
crystallization solvent, compound 4a crystallized in two poly-
morphic structures (4aa from EtOH and 4ab, from MeOH).
Both structures (Fig. 2A,B) showed a single bond between the
two pendant nitrogen atoms and a double bond between N15
and the quinoxaline ring (tautomer A, Fig. 3). This caused the
formation of an intramolecular hydrogen bond N(1)–H ◊ ◊ ◊ N(16)
˚
with H ◊ ◊ ◊ N distances of 2.36 and 2.24 A for 4aa and 4ab,
respectively. The two polymorphic structures showed quite similar
geometrical and conformational parameters with the exception of
the orientation of the methoxy group. The dihedral angles formed
by the pyrroloquinoxaline and the quinoline rings are 10.58(1)◦
and 12.65(11)◦ for 4aa and 4ab, respectively.
Fig. 3 Prototropic tautomers A and B exemplified for compound 4a.
Observed NOE effects are indicated by double arrows. Numbering from
Fig. 2.
of signals observed in the NMR spectra of all compounds could
be due to the co-existence in solution of the two equilibrating
tautomers A and B (indicated in Fig. 3 for compound 4a).
To test this hypothesis, we first assigned the two series of 1H
NMR signals in DMSO-d6 for the mixture of tautomers of
4a through interpretation of 2D NMR spectra (COSY, HSQC,
HMBC). Two different exchangeable protons were evident in
the 1H NMR spectrum at dH 10.70 and 11.80, respectively,
assignable to the N-linked protons present in tautomers A and
B. Cross-peaks exhibited by these key signals in the 2D NMR
ROESY spectrum (see Fig. 3) were then utilized to obtain their
unambiguous structural localization, thus also supporting the
structures proposed for the two different tautomers. In particular,
the signal at dH 10.70 showed ROESY cross-peak with H-12 (in
turn spatially coupled to the methoxy group at C-11) and was
therefore assigned to NH-1, in agreement with the structure of
tautomer A. On the other hand, the exchangeable signal at dH
11.80 showed ROESY cross-peak with the imine proton at dH 9.32
(H-17) and was therefore assigned to NH-15, in agreement with
the structure of tautomer B.
The existence of tautomers A and B was also observed by
RP-HPLC analysis revealing the presence of two peaks for each
compound after isocratic elution with methanol or acetonitrile.
Isolation of the two peaks was not possible due to rapid
equilibration in solution.
Differently from compounds 4a–i, the quinoline derivative 3 did
not show tautomeric equilibrium, as indicated by the single peak
displayed during HPLC analysis and by the single set of signals
in the proton NMR spectrum recorded in DMSO-d6. It can be
argued that the presence of the pyrrole ring in structures 4a–i has
the effect of weakening the aromatic stability of the condensed six-
membered ring, thus rendering energetically feasible the existence
of the tautomer with exocyclic double bond.
Fig. 2 X-ray crystal structures of 4a in the two polymorphic forms. A.
Crystallization from EtOH (4aa); B. Crystallization from MeOH (4ab) with
the disordered methanol omitted for clarity.
Although it is described that heterocyclic hydrazones can
present different prototropic tautomeric equilibria involving their
acidic hydrogen,21,22 stable imino-derivatives of heterocyclic hydra-
zones are not common features of these class of compounds and
only a few examples have been previously reported in the literature
including a neutral phthalazinylhydrazone reported by us,23 and
the imino tautomers of 2-quinolylhydrazone derivatives.24,25
Taking into consideration the above results and the observation
that the NMR signal splitting was particularly evident only for
protons of the quinoxaline ring, we hypothesized that the two set
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Table 1 Calculated logP for compounds 4a–i and 3, effect of treatment of pre-formed fibers of Ab1-42 with compounds 3, 4a–i on the fluorescence signal
of ThT and quantification of their anti-aggregation properties
CLogPa
Reduction of ThT
fluorescent signal (%)
Reduction of preformed
Compd
TA
TB
UAF (Ex = 420 – Em = 480)b
Ab fibrils (%)c
ThT, 1
4a
4b
4c
4d
4e
4f
—
—
356.0 11.5
103.3 8.5
49.4 6.1
—
71
86
79
55
2
44
43
57
40
60
—
—
—
0
34
—
6
10
11
—
—
6.2
5.8
5.5
6.7
6.4
4.7
5.2
5.0
6.4
6.5
4.3
4.1
4.3
4.5
4.5
4.4
4.4
4.5
4.7
—
74.9 8.1
159.2 8.3
347.3 5.9
198.0 10.9
203.2 16.8
154.1 13.5
181.1 3.6
143.9 16.4
4g
4h
4i
3
a ACD/logP, version 11.00, Advanced Chemistry Development, Inc., Toronto, Canada). b ThT Fluorescence signal intensity measured at the excitation
and emission wave lengths of 420 and 480 nm, respectively after incubation with the tested compound for 5 min. The fluorescence values of the samples
containing only tested compounds was determined and subtracted from the values of the samples containing Ab42 fibrils and compounds. c Quantification
of the amount of aggregated Ab peptide after exposure to the tested compounds was performed by HPLC.
Interference with ThT binding and electron-microscopy analysis
with the compound concentration. After standardization of the
experimental conditions for compound 3, the other compounds
were tested at a single time and at the higher concentration of
10 mM. All compounds presented a certain level of fluorescence
that is strongly (Fig. 6) or modestly altered (Fig. 7) when mixed
with Ab1-42 fibrils similarly to ThT (Fig. 5A).
Compounds 4a–i were tested for their ability to interfere with ThT
staining of pre-formed Ab1-42 fibrils. Therefore, the compounds
at a concentration of 10 mM were incubated for 30 min in the
presence of Ab1-42 fibrils before the ThT binding was measured.
As illustrated in Table 1, all compounds, with the exception of 4e,
were able to interfere with ThT fluorescence. The extent of the
fluorescent signal reduction ranged from a maximum of 87% for
4a to a minimum of 40% for 4i.
Staining of amyloid deposits in CRND8 Tg mice
The ability of compounds 3, 4a,b and 4f,g to bind amyloidogenic
structures was tested in brain sections from CRND8 transgenic
mice. These animals carry human APP with double mutations and
accumulate Ab deposits in brain parenkyma and at cerebrovas-
cular level.26 Five-micrometre-thick serial sections of paraffin-
embedded blocks from frontal cortex were used for staining. Tissue
sections were covered with a solution of EtOH 96% with the
different compounds at the concentration of 0.5 mM. Fluorescent
sections were viewed using fluoromicroscope equipped with filters
UV, FITC and TRITC. The binding of fluorescent compounds
to amyloid deposits was compared to the staining obtained with
Congo Red and thioflavin S (ThS), that specifically bind amyloid
structures in adjacent sections. With the exception of 3, for all the
other compounds (4a,b,f,g) the intensity of the fluorescent signals
was similar to those obtained with Congo red or ThS either in the
parenchyma (Fig. 8) or on the vascular walls (Fig. 9). These results
suggested that the newly developed compounds recognize the b-
pleated sheet structure of amyloid fibrils similarly to standard dyes,
like ThS or Congo Red and that they are not involved in aspecific
binding to tissue preparations.
Electron microscopy (EM) analysis performed on fibers exposed
to compounds 3 and 4a,b allowed a direct examination of self-
assembling capacity of the peptide. EM microphotographs (Fig.
4) showed structured amyloid fibrils obtained with Ab1-42 after
an incubation period of 5 days (Panel A). The presence of
compounds 3, 4a,b (Panels B–D) during the incubation time
induced some changes but substantially did not alter the structure
of the fibrils. Thus, the observed reduction of fluorescent signal of
ThT is likely due to displacement of the staining by compounds
3, 4a,b and cannot be ascribed to an anti-fibrillogenic capacity
of pyrroloquinoxaline hydrazones 4a,b and quinolylhydrazone 3.
The observation that binding to the fibers was not associated
with anti-aggregation capacity of the examined compounds was
further investigated for 4c,d,f,g by the direct quantitative determi-
nation of Ab1-42 peptide by HPLC in the pellet after five days
of incubation and the centrifugation of the peptide solution.
The co-incubation of Ab1-42 (in the proportion 1 : 1) with the
different compounds did not significantly reduce the amount
of the peptide found in the pellet with respect to the control
(Table 1).
Brain-to-plasma distribution studies
Fluorescence measurements
Crossing of the BBB is a key aspect to develop potential tools for
diagnosis. Brain permeability studies were carried out to evaluate
the potential application of these non-charged amyloid probes
as imaging agents in PET techniques. Hydrazones examined in
this study were characterized by high calculated logP values as
indicated in Table 1, predictive of their ability to cross the BBB.
In order to experimentally prove the brain permeability of this
The fluorescence behaviour of the compounds in the presence
and in the absence of amyloid fibrils was also analyzed. The
spectrum of 3 was accurately analyzed (Fig. 5) at different
incubation times (Fig. 5C) or concentrations (Fig. 5D) with or
without fibrils. The incubation time did not substantially affect
the results while the spectra alteration progressively increased
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Fig. 4 Electron microscope microphotographs structured amyloid fibrils obtained with Ab1-42 (Panel A) and after co-incubation with 3 (Panel B), 4a
(Panel C), and 4b (Panel D).
class of amyloid fluorescent probes, a preliminary evaluation
of the distribution between plasma and brain of compound 4c
was performed in an animal model. The doses of compound
4c (1.25–10 mg kg-1, dissolved in PEG-400 : DMSO : ethanol)
and route of administration (intraperitoneal) were derived from
previous studies with structurally related derivatives.27 In plasma,
compound 4c reached the highest mean concentrations at 30 min;
these concentrations appeared to be dose proportional between
1.25 (173 13 ng mL-1) and 2.5 mg kg-1 (506 39 ng mL-1) but
slightly less than proportional at higher doses (540 303 and 737
211 ng mL-1, at 5 and 10 mg kg-1, respectively), possibly reflecting
dose related variability in the rate or extent of absorption from the
injection site. At the lower doses these plasma concentrations were
low and variable and rapidly fell below the limit of quantitation
after 2 h (<125 ng mL-1, using 0.2 mL of mouse plasma). At
the 10 mg kg-1 dose, mean plasma concentrations at 2 h were
less than one-third those at 30 min (720 ng mL-1 or about
2 mM), and at 8 h they were below the limit of quantitation of
the analytical procedure (125 ng mL-1, using 0.2 mL of mouse
plasma).
In brain, the compound concentrations were about three times
higher than plasma concentrations regardless of the dose and
sampling time (435 59, 1211 214, 1413 663 and 1522
477, at 1.25, 2.5, 5 and 10 mg kg-1). As shown in Fig. 10 there was
a linear relationship between plasma and brain concentrations
30 min after dosing (brain = 233 + 1.96 plasma; r2 = 0.85). This
linear relationship further suggests that compound 4c crosses the
BBB by passive diffusion, with plasma concentrations consistently
reflecting the whole brain concentrations. These preliminary data
make this class of compounds promising lead structures for
further development as amyloid imaging agents to be used as
diagnostic tools. Their prospective development as diagnostic
imaging agents is further justified by the promising results ob-
tained with the fluorine analogue 4c that could be labelled for PET
application.
Conclusions
A new series of pyrroloquinoxaline hydrazones characterized
by affinity for both Ab1-42 fibrils aggregated in vitro and Ab
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Fig. 5 Fluorescent spectroscopy of ThT and 3 in the presence or the absence of b-amyloid fibrils A) ThT at various temperature, B) 3 at 10 mM with
and without fibrils; C) 3 and fibrils at various concentrations; and D) 3 at different incubation times.
Fig. 7 Fluorescence spectroscopy of compounds 4b,d,g,i (10 mM) in the
absence (red) or in the presence of Ab1-42 fibrils (green). The addition of
fibrils slightly altered the emission spectra of the compounds.
Fig. 6 Fluorescence spectroscopy of compounds 4a,c,f,h (10mM) in the
absence (red) or in the presence of Ab1-42 fibrils (green). The addition of
fibrils changed significantly the emission spectra of the compounds.
Experimental section
plaques in brain tissue preparations was identified. The analyzed
compounds showed a fluorescence behaviour similar to that of
classic amyloid fluorescence probes such as ThT and Congo Red.
It is also worth noticing that selected compounds 4a–c do not
alter the Ab fibrils stability as assessed by EM analysis and by
quantitative measurement of aggregated Ab1-42 after treatment
with these staining agents. These compounds represent a novel
chemotype to be used as fluorescent probes for in vitro study of
amyloid structures. Moreover, due to their ability to cross the BBB
and concentrate in the mouse brain, a prospective application of
these compounds could be as potential amyloid imaging agents.
General information
Reagents were purchased from Aldrich and were used as received.
Reaction progress was monitored by TLC using Merck silica gel
60 F254 (0.040–0.063 mm) with detection by UV. Merck silica
gel 60 (0.040–0.063 mm) was used for column chromatography.
Melting points were determined in Pyrex capillary tubes using an
Electrothermal 8103 apparatus and are uncorrected. 1H NMR and
13C NMR spectra were recorded on Bru¨ker 200 MHz or Varian
300 MHz or Varian 500 MHz spectrometers by using the residual
signal of the deuterated solvent as internal standard. Splitting
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Fig. 9 Amyloid deposits in vascular walls sections from temporal cortex
of Tg CRND8 mice incubated with compounds ThS (A), 2 (B), 3 (C), 4a
(D), 4b (E), 4f (F), 4g (G) at 0.5 mM. Fluorescent sections were viewed
using fluoromicroscope UV, FITC, and TRITC.
Elmer 240C elemental analyser and the results were within 0.4%
of the theoretical values, unless otherwise noted. Intermediates
7a–c,f–h were prepared as previously described.20,28,29 Compound 3
was prepared as previously described15 and here has been reported
its full characterization.
Fig. 8 Amyloid plaques in parenkyma sections from temporal cortex of
Tg CRND8 mice incubated with compounds ThS (A), 2 (B), 3 (C), 4a (D),
4b (E), 4f (F), 4g (G) at 0.5 mM. Fluorescent sections were viewed using
fluoromicroscope UV, FITC, and TRITC.
3-Nitro-4-(1H-pyrrol-1-yl)benzoic
acid
(9). 2,5-Dimeth-
oxytetrahydrofuran (3.5 mL, 27.7 mmol) was added to a solution
of 4-amino-3-nitrobenzoic acid 8 (5.0 g, 27.4 mmol) in glacial
acetic acid (25 mL) heated to 120 ◦C. After 1 h, the reaction
mixture was cooled to 25 ◦C, the volatiles were evaporated and
the residue was purified by column chromathography (1 : 20
MeOH/CHCl3) to afford acid 9 as a yellow low melting solid (6.0
g, 94%): IR nmax (CHCl3; cm-1) 1690; dH (300 MHz, DMSO-d6)
13.69 (s, 1H), 8.43 (d, J = 1.8 Hz, 1H), 8.24 (dd, J = 8.4, 1.9 Hz,
1H), 7.75 (d, J = 8.4 Hz, 1H), 7.13–6.87 (m, 2H) and 6.43–6.19 (m,
2H); dC (100 MHz, DMSO-d6) 165.7, 144.5, 136.9, 134.8, 130.5,
128.5, 126.6, 122.0 and 112.1; m/z (ESI) 231 (M-H)-; m/z HRMS
(ESI) [M - H]- calcd 231.0406 for C11H7N2O4, found 231.0411;
Elemental calcd. C, 56.90; H, 3.47; N, 12.06 for C11H8N2O4; found
C, 57.10; H, 3.78; N, 12.12.
patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), and broad (br); the value of chemical shifts (d) are given in
ppm and coupling constants (J) in Hertz (Hz). ESI-MS spectra
were performed by an Agilent 1100 Series LC/MSD spectrometer.
HPLC analysis were performed by a LaPrep P130 apparatus using
a P311 UV detector. Yields refer to purified products and are not
optimised. All moisture-sensitive reactions were performed under
argon atmosphere using oven-dried glassware and anhydrous
solvents. All the organic layers were dried using anhydrous sodium
sulfate. Purity of tested compounds is ≥95% as determined by
elemental analysis. Elemental analyses were performed in a Perkin-
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resulting chloride was dissolved in ethanol (10 mL). Hydrazine
monohydrate (0.11 mL, 2.3 mmol) was added to the resulting
mixture, and after heating under reflux for 2 h, the reaction
mixture was cooled to room temperature. The precipitated solid
was collected by filtration and washed with diethyl ether to afford
5d as a white solid (171 mg, 73%): mp 280 ◦C (hexane); IR
nmax (CHCl3; cm-1) 3615 and 1533; dH (200 MHz, DMSO-d6)
7.75–7.74 (m, 1H), 7.49–7.46 (m, 2H), 6.68–6.61 (m, 2H), 6.23
(bs, 1H), 4.22 (s, 2H), 2.38 (s, 3H) and 2.34 (s, 3H); m/z (ESI)
227(M+H)+; m/z HRMS (ESI) [M+H]+ calcd 227.1297 for
C13H15N4, found 227.1299; Elemental calcd. C, 69.00; H, 6.24; N,
24.76 for C13H14N4; found C, 68.84; H, 6.62; N, 24.66.
4-Hydrazino-7-carbomethoxypyrrolo[1,2-a]quinoxaline (7e).
A
mixture of 6 (0.5 g, 2.19 mmol), POCl3 (4.5 mL) and N,N-
dimethylaniline (0.83 mL, 6.6 mmol) was heated to 115 ◦C for 3 h,
◦
afterward the reaction mixture was cooled to 0 C. The reaction
mixture was stirred in the presence of MeOH and then it was
poured into water. The aqueous phase was extracted with EtOAc,
the organic extracts were washed with a saturated solution of
NaHCO3 and dried over Na2SO4. The solvent was removed and
the crude product was purified by column chromathography to
afford the intermediate chloride (0.24 g, 45%) as a white low
melting solid: dH (300 MHz, CDCl3) 8.57 (d, J = 1.8 Hz, 1H),
8.19 (dd, J = 8.6, 1.9 Hz, 1H), 7.99 (dd, J = 2.8, 1.3 Hz, 1H), 7.85
(d, J = 8.6 Hz, 1H), 7.09 (dd, J = 4.1, 1.3 Hz, 1H), 6.94 (dd, J =
4.0, 2.8 Hz, 1H) and 3.97 (s, 3H). The above chloride was treated
with hydrazine monohydrate (0.19 mL, 3.9 mmol) in refluxing
methanol. After 2 h the reaction mixture was cooled to 25 ◦C,
the solvent was removed and the resulting solid was recrystallized
from MeOH to obtain 7e as a pale yellow solid (60%): mp 185 ◦C
(methanol); IR nmax (CHCl3; cm-1) 3682 and 1718; dH (300 MHz,
DMSO-d6) 8.93 (bs, 1H), 8.26 (s, 1H), 8.14 (d, J = 8.5 Hz, 1H),
8.06 (d, J = 1.7 Hz, 1H), 7.73 (dd, J = 8.4, 1.7 Hz, 1H), 7.06 (d, J =
3.2 Hz, 1H), 6.84–6.66 (m, 1H), 4.60 (br, 2H) and 3.86 (s, 3H). m/z
(ESI) 279 (M+Na)+; m/z HRMS (ESI) [M+Na]+ calcd 279.0858
for C13H12N4O2Na, found 279.0863; Elemental calcd. C, 60.93;
H, 4.72; N, 21.86 for C13H12N4O2; found C, 61.21; H, 4.54; N,
21.61.
Fig. 10 Relationship between whole brain and plasma concentrations of
compound 4c after intraperitoneal doses of 1.25–10 mg/kg.
3-Amino-4-(1H-pyrrol-1-yl)benzoic acid (10). A mixture of 9
(2.0 g, 8.6 mmol) and a catalytic amount of 10% palladium on
carbon in a 1 : 1 EtOAc/MeOH (20 mL) was stirred at 25 ◦C
under an H2 atmosphere for 14 h. The reaction mixture was filtered
through Celite and the filtrate was concentrated. The pale yellow
low melting solid thus obtained (10, 1.6 g, 92%) was used in the
next step without further purification: IR nmax (CHCl3; cm-1) 3609,
3330 and 1685; dH (300 MHz, DMSO-d6) 12.75 (bs, 1H), 7.47 (d,
J = 1.8 Hz, 1H), 7.19 (dd, J = 8.1, 1.8 Hz, 1H), 7.10 (d, J = 8.1
Hz, 1H), 6.95 (t, J = 2.1 Hz, 2H), 6.25 (t, J = 2.1 Hz, 2H) and
5.10 (bs, 2H); m/z (ESI) (M-H)- 201; m/z HRMS (ESI) [M+H]+
calcd 203.0821 for C11H11N2O2, found 203.0827; Elemental calcd.
C, 65.34; H, 4.98; N, 13.85 for C11H10N2O2; found C, 65.48; H,
4.71; N, 14.04.
7-Carboxypyrrolo[1,2-a]quinoxalin-4(5H)one (6). A mixture
of aniline 10 (1.0 g, 4.34 mmol) and triphosgene (0.42 g, 1.43
mmol) in dry toluene (20 mL) was stirred under reflux. After 4 h the
reaction mixture was cooled to 25 ◦C and bubbled with a stream
of nitrogen overnight. The resulting solid was washed with EtOAc
to afford 6 (0.74 g, 75%) as white prisms after recrystallization
(hexane): mp > 300 ◦C (hexane); IR nmax (CHCl3; cm-1) 1782 and
1715; dH (300 MHz, DMSO-d6) 13.04 (s, 1H), 11.42 (s, 1H), 8.23
(d, J = 1.3 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 1.6
Hz, 1H), 7.73 (dd, J = 8.5, 1.6 Hz, 1H), 7.06 (dd, J = 3.8, 1.0 Hz,
1H) and 6.88–6.60 (m, 1H); m/z (ESI) 227 (M-H)-; m/z HRMS
(ESI) [M+H]+ calcd 229.0613 for C12H9N2O3, found 229.0612;
Elemental calcd. C, 63.16; H, 3.53; N, 12.28 for C12H8N2O3; found
C, 63.40; H, 3.65; N, 11.96.
2-[(Quinolin-4-yl)methylene]-1-(7-methoxy-4-methylquinolin-2-
yl)hydrazine (3). An equimolar mixture of 7-methoxy-4-methyl-
2-hydrazinoquinoline30 and quinoline-4-carbaldehyde was dis-
solved in ethanol and heated under reflux for 3 h. The title
compound was isolated in 86% yield by crystallization from
ethanol: mp 265 ◦C (ethanol); IR nmax (CHCl3; cm-1) 1605 and
1518; dH (300 MHz, DMSO-d6) 11.57 (s, 1H), 8.92 (d, J = 4.6 Hz,
1H), 8.79 (s, 1H), 8.54 (dd, J = 8.4, 1.1 Hz, 1H), 8.07 (dd, J = 8.3,
1.1 Hz, 1H), 7.93 (d, J = 4.6 Hz, 1H), 7.87–7.66 (m, 3H), 7.41 (d,
J = 0.9 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 6.99 (dd, J = 9.0, 2.6 Hz,
1H) and 3.87 (s, 3H). m/z (ESI) 343 (M+H)+; m/z HRMS (ESI)
[M+H]+ calcd 343.1559 for C21H19N4O, found 343.1562; Elemental
calcd. 73.67; H, 5.30; N, 16.36 for C21H18N4O; found C, 73.53; H,
5.26; N, 16.00.
4-Hydrazino-7,8-dimethylpyrrolo[1,2-a]quinoxaline
(7d).
POCl3 (3.6 mL, 23.7 mmol) and N,N-dimethylaniline
(0.35 mL, 2.70 mmol) were added to 5d (0.20 g, 1.08 mmol) and
the resulting mixture was heated to 120 ◦C for 4 h. Thereafter,
the reaction mixture was cooled to room temperature, poured
into ice-water (10 mL), and extracted with ethyl acetate (3 ¥
20 mL). The combined organic layers were washed with brine,
dried over Na2SO4 and evaporated. The residue was purified
by flash-chromatography (10% ethyl acetate in hexane) and the
2-[(Quinolin-4-yl)methylene]-1-(7-methoxypyrrolo[1,2-a]quinox-
alin-4-yl)hydrazine (4a). Quinoline-4-carbaldehyde (40 mg, 0.25
mmol) was added to a solution of 7a (46 mg, 0.23 mmol) in ethanol
(6 mL) and the resulting mixture was heated under reflux. After
4 h it was cooled to room temperature and the volatiles were
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evaporated. The residue was chromatographed (2% methanol in
chloroform) to afford 4a as a yellow solid (72.8 mg, 94%): mp
201 ◦C (ethanol). Rt 8.6, 10.6 min [MeCN]. IR nmax (CHCl3;
cm-1) 3680, 3372, 1622, 1610 and 1541; dH (500 MHz, DMSO-
d6) Tautomer A 10.70 (bs, 1H, NH-1), 9.32 (s, 1H, H-17), 9.02
(d, 1H, J = 8.2 Hz, H-26), 8.65 (d, 1H, J = 8.5 Hz, H-20), 8.50 (d,
1H, J = 8.2 Hz, H-27), 8.09 (d, 1H, J = 8.2 Hz, H-23), 7.97 (d, 1H,
J = 7.5 Hz, H-9), 7.83 (dd, 1H, J = 8.2, 7.5 Hz, H-21), 7.71 (dd, 1H,
J = 8.2, 7.5 Hz, H-22), 7.42 (s, 1H, H-12), 6.80 (d, 1H, J = 7.5 Hz,
H-10), 7.07 (bs, 1H, H-6), 6.70 (bs, 2H, H-4, H-5) and 3.87 (s, 3H,
11-OMe); Tautomer B 11.80 (bs, 1H, NH-15), 9.32 (s, 1H, H-17),
9.02 (d, 1H, J = 8.2 Hz, H-26), 8.65 (d, 1H, J = 8.5 Hz, H-20),
8.50 (d, 1H, J = 8.2 Hz, H-27), 8.13 (d, 1H, J = 7.5 Hz, H-9), 8.09
(d, 1H, J = 8.2 Hz, H-23), 7.83 (dd, 1H, J = 8.2, 7.5 Hz, H-21),
7.71 (dd, 1H, J = 8.2, 7.5 Hz, H-22), 7.40 (s, 1H, H-12), 7.06 (d,
1H, J = 7.5 Hz, H-10), 7.05 (bs, 1H, H-6), 6.70 (bs, 2H, H-4, H-5)
and 3.86 (s, 3H, 11-OMe). dH (300 MHz, CDCl3) 9.29 (s, 1H), 9.21
(s, 1H), 8.97 (d, J = 4.4 Hz, 1H), 8.48 (d, J = 8.3 Hz, 1H), 8.16
(d, J = 8.4 Hz, 1H), 7.95 (d, J = 4.2 Hz, 1H), 7.80–7.69 (m, 2H),
7.64–7.59 (m, 2H), 7.53 (d, J = 9.0 Hz, 1H), 6.80–6.64 (m, 3H) and
3.86 (s, 3H); m/z (ESI) 368 (M+H)+; m/z HRMS (ESI) [M+H]+
calcd 368.1511 for C22H18N5O, found 368.1515; Elemental calcd.
C, 71.92; H, 4.66; N, 19.06 for C22H17N5O; found C, 71.64; H, 4.49;
N, 19.31.
(M+H)+; m/z HRMS (ESI) [M+H]+ calcd 366.1719 for C23H20N5,
found 366.1725; Elemental calcd. C, 75.59; H, 5.24; N, 19.16 for
C23H19N5; found C, 75.70; H, 4.92; N, 19.28.
2-[(Quinolin-4-yl)methylene]-1-(7-carbomethoxypyrrolo[1,2-
a]quinoxalin-4-yl)hydrazine (4e). Starting from 7e the title com-
pound was prepared following the above described procedure and
was obtained as a yellow solid: mp 227 ◦C (ethanol). IR nmax
(CHCl3; cm-1) 1722, 1625, 1551, 1524 and 1433; dH (200 MHz,
CDCl3) 9.44 (s, 1H), 9.18 (s, 1H), 8.92 (d, J = 4.5 Hz, 1H), 8.39
(d, J = 8.4 Hz, 1H), 8.12 (dd, J = 8.5, 0.8 Hz, 1H), 7.90 (d, J =
4.5 Hz, 1H), 7.85 (s, 1H), 7.81–7.66 (m, 2H), 7.56 (dd, J = 18.8,
8.6 Hz, 3H), 7.24 (d, J = 3.4 Hz, 1H), 6.78–6.65 (m, 1H) and
3.92 (s, 3H); m/z (ESI) 396 (M+H)+; m/z HRMS (ESI) [M+H]+
calcd 396.1460 for C23H18N5O2, found 396.1455; Elemental calcd.
C, 69.86; H, 4.33; N, 17.71 for C23H17N5O2; found C, 70.13; H,
4.37; N, 17.41.
2-[(Quinolin-4-yl)methylene]-1-(pyrido[3,2-e]pyrrolo[1,2-a]pyra-
zin-6-yl)hydrazine (4f). Starting from 7f (54 mg, 0.27 mmol),
the title compound was prepared following the above described
procedure and was obtained as a yellow solid (60 mg, 65%): mp
236 ◦C (ethanol). Rt 9.4, 12.0 [MeCN]. IR nmax (CHCl3; cm-1) 3623,
1620 and 1532; dH (300 MHz, CDCl3) 9.29 (s, 1H), 9.12 (s, 1H),
9.00 (d, J = 4.5 Hz, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.28–8.07 (m,
2H), 7.95 (d, J = 3.4 Hz, 1H), 7.82–7.73 (m, 1H), 7.70–7.58 (m,
1H), 7.58–7.43 (m, 1H), 7.38–7.21 (m, 3H) and 6.86–6.67 (m, 1H);
m/z (ESI) 339 (M+H)+; m/z HRMS (ESI) [M+H]+ calcd 339.1358
for C20H15N6, found 339.1360; Elemental calcd. C, 70.99; H, 4.17;
N, 24.84 for C20H14N6; found C, 71.25; H, 3.98; N, 24.63.
2-[(Quinolin-4-yl)methylene]-1-(pyrrolo[1,2-a]quinoxalin-4-yl)
hydrazine (4b). Starting from 7b, the title compound was pre-
pared as above described and it was obtained in 89% yield as
a yellow solid: mp 226 ◦C (ethanol). Rt 9.1, 11.4 min [MeCN].
IR nmax (CHCl3; cm-1) 3376, 1652, 1611, 1537 and 1433; dH (300
MHz, CDCl3) 9.34 (s, 1H), 9.28 (s, 1H), 9.00 (d, J = 4.6 Hz, 1H),
8.54 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.98 (d, J =
4.6 Hz, 1H), 7.82–7.73 (m, 1H), 7.73–7.69 (m, 1H), 7.69–7.61 (m,
2H), 7.31–7.14 (m, 4H) and 6.75–6.70 (m, 1H); m/z (ESI) 338
(M+H)+; m/z HRMS (ESI) [M+H]+ calcd 338.1406 for C21H16N5,
found 338.1400; Elemental calcd. C, 74.76; H, 4.48; N, 20.76 for
C21H15N5; found C, 74.38; H, 4.70; N, 20.52.
2-[(Quinolin-4-yl)methylene]-1-(imidazo[1,2-a]quinoxalin-4-yl)-
hydrazine (4g). To a solution of 7a (46 mg, 0.23 mmol) in
ethanol (6 mL) was added quinoline-4-carbaldehyde (40 mg,
0.25 mmol) to room temperature and then resulting mixture was
refluxed for 4h at 90 ◦C. Thereafter, the reaction mixture was
cooled to room temperature and evaporated. The residue was
chromatographed (2% methanol in chloroform) to afford 7a as
◦
a dark yellow solid (40 mg, 51%): mp 258 C (ethanol). IR nmax
2-[(Quinolin-4-yl)methylene]-1-(7-fluoropyrrolo[1,2-a]quinoxa-
lin-4-yl)hydrazine (4c). Starting from 7c (60 mg, 0.28 mmol),
the title compound was prepared following the above described
procedure and was obtained as a yellow solid (72 mg, 73%): mp
233 ◦C (ethanol). Rt 8.4, 10.6 [MeCN]. IR nmax (CHCl3; cm-1) 3508
and 1609; dH (300 MHz, CDCl3) 9.37 (s, 1H), 9.27 (s, 1H), 8.99
(d, J = 4.6 Hz, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 8.5 Hz,
1H), 7.95 (d, J = 4.4 Hz, 1H), 7.77 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H),
7.69–7.55 (m, 3H), 7.01–6.84 (m, 3H) and 6.76–6.69 (m, 1H); m/z
(ESI) 356 (M+H)+; m/z HRMS (ESI) [M+H]+ calcd 356.1311 for
C21H15FN5, found 356.1314; Elemental calcd. C, 70.98; H, 3.97;
N, 19.71 for C21H14FN5; found C, 71.19; H, 4.26; N, 19.45.
(CHCl3; cm-1) 1596; dH (300 MHz, CDCl3) 10.08 (s, 1H), 9.49 (s,
1H), 9.00 (d, J = 4.8 Hz, 1H), 8.46 (d, J = 8.5 Hz, 1H), 8.18 (d, J
= 8.3 Hz, 1H), 8.12–7.96 (m, 2H), 7.83–7.73 (m, 1H), 7.73–7.60
(m, 2H), 7.60–7.43 (m, 1H) and 7.41–7.20 (m, 3H). m/z (ESI) 339
(M+H)+; m/z HRMS (ESI) [M+H]+ calcd 339.1358 for C20H15N6,
found 339.1351; Elemental calcd. C, 70.99; H, 4.17; N, 24.84 for
C20H14N6; found C, 70.94; H, 3.83; N, 24.47.
2-[(Quinolin-4-yl)methylene]-1-(7-fluoroimidazo[1,2-a]quinoxa-
lin-4-yl)hydrazine (4h). Starting from 7b (0.16 g, 0.73 mmol),
the title compound was prepared following the above described
procedure and was obtained as a yellow solid (0.24 g, 90%): mp
286 ◦C (ethanol). Rt 7.2, 9.3 [MeCN]. IR nmax 1545 and 1510
(CHCl3; cm-1) 1545 and 1510; dH (300 MHz, CDCl3) 9.92 (s,
1H), 9.51 (s, 1H), 9.01 (d, J = 4.6 Hz, 1H), 8.95 (s, 1H), 8.61
(d, J = 8.3 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.08–7.98 (m,
2H), 7.84–7.64 (m, 3H), 7.23–7.16 (m, 1H) and 7.08–6.89 (m,
1H). m/z (ESI) 357 (M+H)+; m/z HRMS (ESI) [M+H]+ calcd
357.1264 for C20H14FN6, found 357.1260; Elemental calcd. C,
67.41; H, 3.68; N, 23.58 for C20H13FN6; found C, 67.56; H, 3.55; N,
23.81.
2-[(Quinolin-4-yl)methylene]-1-(7,8-dimethylpyrrolo[1,2-a]quin-
oxalin-4-yl)hydrazine (4d). Starting from 7d (250 mg, 1.44
mmol), the title compound was prepared following the above
described procedure and was obtained as a yellow solid (182 mg,
45%): mp 265 ◦C (ethanol). Rt 8.4, 10.6 [MeOH]. IR nmax (CHCl3;
cm-1) 3688 and 1603; dH (200 MHz, CDCl3) 9.24 (br, 2H), 8.95
(d, 1H, J = 5.0 Hz), 8.47 (d, 1H, J = 8.4 Hz), 8.15 (d, 1H, J = 8.2
Hz), 7.98 (d, 1H, J = 4.5 Hz), 7.79–7.58 (m, 4H), 7.39 (s, 1H), 7.12
(s, 1H), 6.72 (s, 1H), 2.35 (s, 3H) and 2.32 (s, 3H); m/z (ESI) 366
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2-[(Quinolin-2-yl)methylene]-1-(pyrrolo[1,2-a]quinoxalin-4-yl)-
hydrazine (4i). Starting from 7a and quinoline-2-
centrifuged for 10 min at 13,000 rpm, the pellets were resuspended
in 300 mL of 50 mM glycine–NaOH buffer, pH 9.6 containing a
solution of 2 mM ThT. The samples were incubated for 5 min and
the fluorescence was determined by spectrophotofluorimetry (LS
5OB Perkin Elmer Instr., Bekomfield, UK) at the excitation and
emission wave lengths of 420 and 480 nm, respectively. The ability
of test compounds to interfere with ThT fluorescence increase was
evaluated using preformed Ab1-42 fibers as positive controls.
carboxyaldehyde and following the above described procedure,
the title compound was obtained in 85% yield as a yellow solid:
mp 189 ◦C (ethanol). IR nmax (CHCl3; cm-1) 3676, 1628, 1536 and
1430; dH (200 MHz, CDCl3) 9.50 (s, 1H), 8.77 (s, 1H), 8.22–8.06
(m, 3H), 7.78 (d, J = 8.0 Hz, 1H), 7.74–7.65 (m, 1H), 7.62 (s,
1H), 7.58–7.45 (m, 2H), 7.29–7.22 (m, 1H), 7.20 (d, J = 4.0 Hz,
2H), 7.15–7.04 (m, 1H) and 6.75–6.58 (m, 1H). m/z (ESI) 338
(M+H)+; m/z HRMS (ESI) [M+H]+ calcd 338.1406 for C21H16N5,
found 338.1411; Elemental calcd. C, 74.76; H, 4.48; N, 20.76 for
C21H15N5; found C, 74.42; H, 4.40; N, 20.66.
HPLC determination of Ab1-42
A solution of synthetic peptide Ab1-42 in Tris-HCl 100 mM, pH
7.4 (Merck, Darmstadt, Germany) was incubated for 5 d at 37 ◦C
in the presence of the examined compounds. After centrifugation
at 13,000 X rpm for 10 min at 4 ◦C, the pellets were dissolved
in 80 mL of 10% formic acid containing 0.1% trifluoroacetic acid
and analyzed by RP-HPLC.34 Parallel samples were run in the
absence of compounds. The results were calculated as percentage
of peptide present in this condition.
X-Ray crystallography of 4a
Single crystals of 4a were obtained by dissolving some mg in
ethanol or methanol and allowing the solution to concentrate
at room temperature. Two sets of polymorphic crystals were
obtained: from ethanol, lath orange crystals (4aa) of approximate
dimensions 0.2 ¥ 0.09 ¥ 0.02 mm were produced, while from
methanol slab pale orange crystals (4ab) of approximate dimen-
sions 0.1 ¥ 0.1 ¥ 0.03 mm were obtained. A crystal of each set was
submitted to X-ray diffraction by using a Bruker-Nonius FR591
rotating anode diffractometer with graphite monochromated Mo-
Electron microscopy
The synthetic peptide Ab1-42 was suspended in 100 mM Tris-HCl,
pH 7.4, at a concentration of 0.11 mM either in the presence
or in the absence of equimolar concentration of the compounds.
Following 5 d incubation at 37 ◦C, 5 mL aliquots of peptide
suspension were applied to Formvar-coated nickel grids, negatively
stained with 5% (w/v) uranyl acetate, and observed in an electron
microscope (EM 109, Zeiss, Germany) at 80 kV.
˚
Ka radiation (l = 0.71073 A). The structures were solved by
direct methods implemented in the SHELXS-97 program.31 The
refinements were carried out by full-matrix anisotropic least-
squares on F2 for all reflections for non-H atoms by using
the SHELXL-97 program.32 Absorption correction obtained by
SADABS33 was applied to both the data collections.
Sections staining
4aa: C22H17N5O ¥ H2O (mol wt 385.4). Crystal system: or-
thorhombic, space group P212121 (n 19); a = 4.8025(2), b =
Brains from Tg CRND8 mice encoding a double mutant form of
amyloid precursor protein 695 (KM670/671NL+V717F) under
the control of the PrP gene promoter26 were dissected. Parts
of the brain were fixed in Carnoy and embedded in paraffin.
Five-micrometre-thick serial sections of paraffin-embedded blocks
from the temporal cortex were used for staining. Paraffin sections
were first taken through five 10 min incubations in xylene, five
10 min incubations in 100% EtOH to completely deparaffinize.
Tissue sections were covered with a solution of EtOH 96%
with the different compounds at 0.5 mM. After 30 min of
incubation the sections were washed in EtOH 96% Thereafter,
the sections were incubated in EtOH 100% and xylene before
to apply the coverslip. Fluorescent sections were viewed using
fluoromicroscope (Olympus BX 61) with M-3204C CCD equipped
with filters UV (Ex 330–385 nm/Em > 420), FITC (Ex 450–
480 nm/Em > 515) and TRITC (Ex 530–550 nm/Em >590).
Because in vitro studies using compound bound to synthetic amy-
loid demonstrated that maximal emission of compound excited
at 370 nm occurred at 481 nm, the filters cube were appropriate
for examination of compounds. The staining of compounds were
compared to Thioflavin S and Congo red staining.35
3
˚
˚
11.5872(8), c = 33.050(2) A, V = 1839.17(19) A , Z = 4, Dc =
1.392 g cm-13. 28594 reflections were collected at -153 ◦C of which
4048 are unique (Rint = 0.10). Final refinement converged to R1 =
0.057, wR2 = 0.097 for I>2sI, goodness-of-fit = 1.03. Min. max
-3
˚
height in last Dr map of -0.21 and 0.20 eA .
4ab: C22H17N5O ¥ 0.58 H2O ¥ 0.42 CH3OH (mol wt 391.1).
Crystal system: monoclinic, space group C2/c (n. 15); a =
◦
˚
24.252(3), b = 9.3398(11), c = 17.6282(17) A, b = 102.784(5) ,
V = 3894.0(8) A , Z = 8, Dc = 1.324 g cm-13. 19860 reflections
3
˚
were collected at -153 ◦C of which 3420 are unique (Rint = 0.09).
Final refinement converged to R1 = 0.093, wR2 = 0.218 for I>2sI,
goodness-of-fit = 1.09. Min max height in last Dr map of -0.30
-3
˚
and 0.39 eA . The compound crystallized with a water molecule
having site occupation factor (s.o.f.) equal to 0.58(1) and with a
disordered methanol molecule (s.o.f. 0.42(1)).
Interference with ThT Binding
Synthetic Ab1 - 42 (Tecnogen, Caserta, Italy) was dissolved at a
concentration of 0.11 mM in H2O/MeCN (1 : 1 = v : v), and
aliquots of the solution containing 15 mg were lyophilized. Samples
were dissolved in 15 ml of 10 mM NaOH, mixed with 15 ml of
100 mM Tris-HCl buffer, pH 7.4 and incubated for 5 days at
Fluorescence spectroscopy
◦
37 C. Samples were then centrifuged at 13,000 rpm for 10 min,
To investigate the binding of the compounds to A1-42, aliquots of
15 mg of peptide were dissolved in 15 ml of NaOH 10 mM, added
with 15 ml of Tris-HCl buffer 100 mM (pH 7.4) and the samples
were incubated at 37 ◦C for one week. After this time the samples
the supernatants were discharged and the pellets resuspended in 30
mL of the same buffer containing the test compounds at the con-
centration of 110 mM. After 30 min of incubation the samples were
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were centrifuged at 13,000 rpm for ten minutes the supernatant
was discharged and the pellet of fibrils was resuspended in 300 ml
of 50 mM glycine-NaOH buffer, pH 9.6 including test compounds,
10 mM. Five minutes after dilution the emission spectra were
determined with a Perkin Elmer LS 50B spectrophotofluorimeter
in the range of 300–650 nm with excitation wavelengths of 390 nm.
In parallel the alone test compounds and the alone fibrils were
analyzed at the same concentration and conditions. To calculate
the binding to Ab1-42 the fluorescence of solutions of the test drugs
alone was subtracted from that of samples containing the fibrils
and the drug.
Acknowledgements
The Authors thank NatSynDrugs and MIUR (PRIN) for financial
support.
Notes and references
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