Isatin derivatives, a novel class of transthyretin fibrillogenesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.03.004

The isatin core structure was found to be a novel chemical scaffold in transthyretin (TTR) fibrillogenesis inhibitor design. Among the series of isatin analogues prepared and tested, the nitro compound 1,3-dihydro-3-[(4-nitrophenyl)imino]-2H-indol-2-one (2r) is as potent as triiodophenol, which is one of the most active known TTR inhibitors. The E/Z stereochemistry of these molecules in solution, elucidated by ¹H NMR, does not influence their biological activity. The compounds do not bind to the native tetrameric TTR suggesting that their inhibitory action is independent of the protein binding and stabilization.

Full text of DOI:10.1016/j.bmcl.2009.03.004

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5270–5273
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isatin derivatives, a novel class of transthyretin fibrillogenesis inhibitors
Asensio González a, Josefina Quirante a, Joan Nieto b, Maria Rosário Almeida c, Maria Joao Saraiva c,
Antoni Planas b, Gemma Arsequell d, Gregorio Valencia d,
*
a Laboratori de Química Orgànica, Facultat de Farmàcia, Institut de Biomedicina, (IBUB), Universitat de Barcelona, Barcelona, Spain
b Laboratory of Biochemistry, Bioengineering Department, Institut Quimic de Sarria, Universitat Ramon Llull, Barcelona, Spain
c Molecular Neurobiology Unit, IBMC-Instituto de Biologia Molecular e Celular e Departamento de Biologia Molecular, ICBAS—Instituto de Ciências Biomédicas Abel Salazar,
Universidade do Porto, Portugal
d Institut de Química Avançada de Catalunya, Consejo Superior de Investigaciones Científicas (IQAC-CSIC), Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The isatin core structure was found to be a novel chemical scaffold in transthyretin (TTR) fibrillogenesis
inhibitor design. Among the series of isatin analogues prepared and tested, the nitro compound 1,3-dihy-
dro-3-[(4-nitrophenyl)imino]-2H-indol-2-one (2r) is as potent as triiodophenol, which is one of the most
active known TTR inhibitors. The E/Z stereochemistry of these molecules in solution, elucidated by 1H
NMR, does not influence their biological activity. The compounds do not bind to the native tetrameric
TTR suggesting that their inhibitory action is independent of the protein binding and stabilization.
Ó 2009 Published by Elsevier Ltd.
Received 12 January 2009
Revised 2 March 2009
Accepted 3 March 2009
Available online 6 March 2009
Keywords:
Isatins
Transthyretin
Amyloidosis
Inhibitors
Isatin (1H-indole-2,3-dione), an endogenous compound identi-
fied in many organisms, shows a wide range of biological activi-
ties.1–3 Owing to these properties, derivatives of isatin have been
developed for therapeutic applications. A recent example is the
compound SU11248 (Sutent), a 5-fluoro-3-substituted-2-oxoin-
dole that has received FDA approval for the treatment of gastroin-
testinal stromal tumors4 and advanced renal cell carcinoma.5 Other
halogenated derivatives of isatin, including the 5,6,7-tribromoisat-
in,6 a number of 5,7- dibromoisatins,7,8 and a series of 5-bromo-(2-
oxo-3- indolinyl)thiazolidine-2,4-diones,9 have also been reported
to exhibit potential as anticancer drugs when assayed in various
cancer cell lines. Examples of the diverse biological activities of
isatin derivatives are the 3-arylimino-2-indolones, which are po-
tent and selective galanin GAL3 receptor antagonists10 and DNA
gyrase inhibitors.11
Among the already known families of isatin analogs, the isatin-
3-arylimines show a basic structure of two bridged aromatic rings
that can readily accommodate different functional groups. This
simple structural feature is present in many pharmacophores.
Thus, several families of transthyretin (TTR) fibrillogenesis inhibi-
tors12 with structural patterns as diverse as tetrahydroquinolines,
dihydropyridines, benzodiazepines, phenoxazines, stilbenes and
benzoxazoles have in common this two bridged aromatic system.13
Moreover, TTR small molecule inhibitors usually bear halogen sub-
stituents in one ring and hydrophilic functions in the other. To the
best of our knowledge, in spite of these apparent structural analo-
gies, isatin derivatives have not yet been tested as potential TTR
fibrillogenesis inhibitors.
TTR is a plasma protein involved in the transport of thyroid hor-
mones and, by interacting with retinol binding protein, of vitamin
A. This protein self-assembles as a homotetramer of 55 kDa leaving
a central hydrophobic channel with two symmetrical binding sites.
Being an amyloid prone protein, TTR is linked to a distinctive group
of amyloid diseases. Deposits of wild type TTR in the heart and
peripheral nerves appear to cause senile systemic amyloidosis,
whereas most of the one hundred already identified TTR mutants
result in two groups of diseases known as familial amyloidotic pol-
yneuropathy and familial amyloidotic cardiomyopathy.14 The
aggregation pathway of TTR into amyloid fibrils is not yet well
characterized but most fibrillogenesis models postulate several
steps, including dissociation of the tetramer, changes in monomer
conformation, aggregation of conformationally modified mono-
mers into non-fibrillar oligomers that later form protofibrils and
further elongate into mature fibrils.15
This mechanism, along with the fact that binding of thyroid hor-
mones to TTR results in tetramer stabilization, suggests that amy-
loid fibril formation can be inhibited by small molecule
compounds structurally similar to thyroid hormones. Indeed, this
hypothesis has been confirmed by the identification of several fam-
ilies of compounds that bind to TTR and stabilize the ground state
of the protein proportionally to the dissociation constants.16 Such a
* Corresponding author.
E-mail address: gregorio.valencia@iqac.csic.es (G. Valencia).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.03.004

A. González et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5270–5273
5271
Table 1
stabilizing effect of the tetrameric TTR is sufficient to arrest or re-
tard the cascade of events leading to the formation of aggregates
and protein precipitation that can be observed in vitro when the
soluble TTR is challenged by an acidic pH. Thus, in vitro
experiments of TTR aggregation in the presence of inhibitors that
measure the rate and amount of protein precipitation by
turbidimetry constitute the standard test for the identification
and characterization of novel TTR fibrillogenesis inhibitors.17,18
Binding of inhibitors to TTR has also been demonstrated to oc-
cur ex vivo in human and transgenic animal blood plasma samples
and in vivo using cell cultures.19,20 Thus, TTR tetramer stabilization
is considered to be one of the most promising approaches for the
discovery of the first effective pharmacological intervention for
TTR amyloid diseases. However, in spite of this evidence, a com-
pound that prevents in vivo deposition of TTR aggregates after
being administered to an animal model of these diseases has still
not been identified.
TTR fibrillogenesis inhibition properties of isatin-imines measured in vitro by using
the kinetic turbidimetric assaya
O
2
3
4
2
N
R
3
5
R
2
R
O
O
N
R
6
N
R
1
7
1
1
1a-b
2a-s
R1
H
R2
R3
IC50
(lM)
RAc (%)
b
Compound
1a
1b
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2°
2p
2q
2r
2s
TIP
T4
H
H
H
H
I
H
H
H
H
H
H
H
>50
6.3
>50
11.0
>50
>50
6.3
n.a. (24)d
100
31
CH3
H
CH3
H
77
1
H
H
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
I
I
I
I
I
n.a. (14)d
8.6
100
81
CH3
H
CH3
H
8.1
7.7
The isatin-3-arylimines here studied were prepared in
a
straightforward manner using already known methods21 following
the reaction depicted in Scheme 1. Commercially available isatin,
N-methylisatin or 5-substituted isatins were reacted with different
commercial 3-substituted anilines in refluxing ethanol. Direct crys-
tallization of reaction mixtures by spontaneous solvent evapora-
tion afforded the isatin imines of Table 1 in good yields.
H
CH3
Br
I
NO2
H
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.8
32
48
42
24
0
0
H
H
H
H
CH3
H
H
CH3
Br
I
NO2
H
n.a. (7)d
16
The potential fibrillogenesis inhibitory properties of these com-
pounds were assessed using a high throughput screening in vitro
assay previously validated in our labs.17 The method measures
the ability of each individual compound to inhibit the turbidimetry
induced by acidification of transparent solutions of the TTR variant
Y78F, which is one of the most amyloidogenic mutants of TTR. Be-
cause protein aggregation rates are proportional to turbidimetry
rates, different kinetic aggregation parameters can be deduced
after testing a series of concentrations of each inhibitor. Most
meaningful are IC50 and RA (%). Values of these parameters for
the prepared isatin analogs and for thyroid hormone T4 (thyroxine)
and triiodophenol (TIP) as reference compounds are reported in
Table 1.
H
H
H
H
23
I
n.a. (7)d
90
NO2
NO2
CH3
H
6.3
3.2
10.5
86
80
95
a
Parameters obtained from fitting the data of ‘initial rates of fibril formation (v0)’
versus ‘inhibitor concentration ([I])’ to Eq. 1 (Supplementary data), except when
indicated as (d). IC50 and RA (%) values are the mean of three experiments.
b
Concentration of inhibitor at which the initial rate of fibril formation is half the
rate at [I] = 0.
c
Percentage of reduction of the fibril formation rate at a high (infinite) inhibitor
concentration relative to the rate at [I] = 0.
d
Inhibitor for which v0 versus [I] follows a linear dependence. RA (%) is not
Reduction of amyloidosis, RA (%), is defined as the percent of
reduction of the fibril formation rate at a high inhibitor concentra-
tion relative to the rate at zero concentration of the tested com-
pound. RA (%) values of 100% indicate that the inhibitor is able to
fully prevent fibril formation. On the other hand, IC50 is the inhib-
itor concentration at which the initial rate of fibril formation is half
the rate in the absence of the inhibitor. Since the TTR tetramer can
hold up to two molecules of inhibitor, the maximum binding stoi-
chiometry is 2:1. However, an optimal inhibitor is expected to re-
quire the lowest ratio of just 1:1. Accordingly and given that the
applicable (indicated by n.a.). In brackets, RA (%) at 40 mM: percentage of reduction
of fibril formation rate at 40 mM inhibitor concentration.
observed for both the methoxy (2f, IC50 = 8.1
(2r, IC50 = 2.8 M) derivatives. The excellent potency of the nitro
derivative was even superior to that of triiodophenol (TIP,
IC50 = 3.2 M), which is one of the most potent TTR fibrillogenesis
lM) and the nitro
l
TTR concentration in these assays was 7.2
would display a maximum response at half of the protein concen-
tration, this is, 3.6 M. This concentration would be the maximum
lM, a good inhibitor
l
inhibitors known to date.
l
In addition, when further examining the influence of a second
substitution at the C-5 position in one of these already active isat-
in-3-imines, namely compound 2f, it was found that all of them
(compounds 2h to 2k, R2 = CH3, Br, I and NO2, respectively) deplete
rather than potentiate the activity of the parent compound, sug-
gesting that this 5-position is crucial and should be unoccupied
for effective TTR inhibition. When the influence of a second substi-
tution at C-5 was examined in the inactive compound isatin-3-
value of IC50 for a given TTR aggregation inhibitor that acts exclu-
sively through a tetramer stabilization mechanism.
Isatin (1a) did not show significant inhibitory activity when as-
sayed at concentrations within the range 0–40 lM. No activity was
also recorded for the corresponding isatin-3-imine derivatives of
aniline and 4-methyl and 4-iodo substituted anilines (2a, 2d and
2l). However, good to excellent values of inhibitory potency were
R3
O
N
R2
R2
EtOH, Δ
R3
+
O
H2N
O
N
AcOH cat
N
R1
R1
Scheme 1. Synthesis of 3-arylimino-2-indolones. Reagents and conditions: isatin (0.5 mmol), aniline (0.5 mmol), EtOH, cat. CH3COOH, reflux, 45 min.

5272
A. González et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5270–5273
imine 2l, inactive compounds (2n–q, R2 = CH3, Br, I and NO2,
respectively) were also obtained. The same occurred when the
inactive isatin-3-imine 2a was converted into the 5-iodo derivative
2c.
Surprisingly, N-methylisatin (1b) was a good inhibitor, as were
its isatin-3-imine derivatives (2b, 2e, 2g and 2s, R2 = H, CH3, OCH3,
NO2 respectively), with the exception of the totally inactive 5-iodo-
aniline derivative (2m). The potencies of these analogues were in
the same range as the natural thyroid hormone T4. This is in sharp
contrast with the aforementioned total inactivity of isatin (1a) and
the isatin-3-imine derivatives of aniline (2a) and 4-methyl substi-
tuted aniline (2d).
This rough SAR analysis corroborates that the substitution pat-
tern of the isatin and isatin-3-arylimine derivatives here tested is
crucial for activity. The most potent compounds identified were
those exhibiting polar substituents at position 4 of the aniline moi-
ety, namely compounds 2r and 2f, while compounds with less po-
lar groups or lipophilic substituents, namely 2a, 2d and 2l were not
active. This may suggest that an electrostatic interaction (dipolar,
hydrogen bond, etc.) critical for activity may be at play. On the
other hand, given that compounds of considerable potency were
obtained when isatin (1a) and the isatinimine derivatives of aniline
(2a) and 4-methyl substituted aniline (2d) were transformed into
the corresponding N-methyl derivatives (1b, 2b, and 2e, respec-
tively), it could be hypothesized that the lipophilicity of the isatin
moiety at the 1 position is a positive contributing factor for
activity.
ble TTR even at concentrations as high as 40 lM. These results are
a strong indication that, in contrast with the most common TTR
amyloidosis inhibitors, the isatin analogs do not act by inducing ki-
netic stabilization of the tetramer by ligand binding. In turn, this
suggests that other possible interactions between isatins and TTR
soluble aggregates inhibit TTR amyloidogenic processes. Although
much work is required to elucidate the molecular phenomena
underlying the inhibitory activity of these compounds, the high po-
tency of compounds such as 2r make isatin a very promising new
scaffold for the design and development of potential drugs for TTR-
related amyloid diseases. In addition, since isatins act by a different
mechanism from that of the known families of tetramer stabilizing
inhibitors, which up to now have failed to show their effectivity in
animal models, the isatins here reported offer a new possibility for
achieving this goal.
In conclusion, a new class of TTR fibrillogenesis inhibitors based
on isatin is reported. This family comprises simple derivatives such
as N-methylisatine (1b) as well as 3-arylimino-2-indolones such as
1,3-dihydro-3-[(4-nitrophenyl)imino]-2H-indol-2-one (2r), with a
potency matching that of triiodophenol (TIP) but lacking TTR bind-
ing affinity properties. Furthermore, no correlation between the
biological activity and the stereochemistry of such compounds in
solution was found. By acting with a novel yet unknown mecha-
nism, the isatins may open new avenues for the discovery of inhib-
itors that are active in animal models. The study of this mechanism
may also provide new insights for the complete characterization of
TTR amyloid processes.
It is well known that 3-arylimino-2-indolones exist as E and Z
mixtures of isomers that can be analyzed by 1H NMR, but as
they readily interconvert in solution they cannot be separated
by chromatography.4,10 To investigate if the differences in activ-
ity are correlated to the stereochemistry of these compounds in
solution, an NMR study of the isatin-3-arylimines 2f, 2g, 2l, and
2m was conducted (see Supplementary data). Within this group,
the two methoxyphenyl-3-imino derivatives of isatin and N-
methylisatin (compounds 2f and 2g) were very potent fibrillo-
genesis inhibitors while the iodinated analogues (2l and 2m)
were totally inactive.
The stereochemistry of the imines was established unequivo-
cally by NMR techniques using 2D homonuclear (COSY and NOESY)
and heteronuclear (HSQC and HMBC) experiments. The E stereo-
chemistry was assigned to the major isomer in DMSO-d6 solutions
because the NOESY experiments show a neat correlation between
Acknowledgments
This work was financially supported by grants to projects POCI/
SAU-MMO/57321/2004 from FCT-Fundação para a Ciência e Tecn-
ologia (Portugal) and CTQ2006-02390/BQU and BIO2007-67904-
C02-02 from Ministerio de Ciencia y Tecnologia (Spain).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.004.
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