Synthesis and structural analysis of halogen substituted fibril formation inhibitors of Human Transthyretin (TTR)

Article abstract of DOI:10.3109/14756366.2016.1167048

Transthyretin (TTR), a β-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile

Full text of DOI:10.3109/14756366.2016.1167048

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and structural analysis of halogen
substituted fibril formation inhibitors of Human
Transthyretin (TTR)
Lidia Ciccone, Susanna Nencetti, Armando Rossello, Enrico Adriano Stura &
Elisabetta Orlandini
To cite this article: Lidia Ciccone, Susanna Nencetti, Armando Rossello, Enrico Adriano Stura
& Elisabetta Orlandini (2016): Synthesis and structural analysis of halogen substituted fibril
formation inhibitors of Human Transthyretin (TTR), Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2016.1167048
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1167048
Published online: 11 Apr 2016.
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Date: 19 April 2016, At: 06:23

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1167048
!
RESEARCH ARTICLE
Synthesis and structural analysis of halogen substituted fibril formation
inhibitors of Human Transthyretin (TTR)
Lidia Ciccone1,2, Susanna Nencetti1, Armando Rossello1, Enrico Adriano Stura2, and Elisabetta Orlandini1
1
2
Dipartimento di Farmacia, Universita di Pisa, Pisa, Italy and CEA, iBiTec-S, Service d’Ingenierie Moleculaire des Proteines (SIMOPRO), Gif-sur-Yvette,
`
´
´
´
France
Abstract
Keywords
Transthyretin (TTR), a b-sheet-rich tetrameric protein, in equilibrium with an unstable
amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils,
is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis,
familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic
strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril
formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted
9-fluorenyl- and di-benzophenon-based ligands and their three-dimensional crystallographic
analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer
with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode
prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichloro-
fluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid
(1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen
substitutions.
Fibril formation inhibitors, fluorenyl,
transthyretin, X-ray TTR-ligand complexes
History
Received 15 January 2016
Revised 11 March 2016
Accepted 14 March 2016
Published online 11 April 2016
Introduction
epithelial cells and the choroid plexus, where the mutated TTR
continues to be synthesized. A different non-invasive therapy to
prevent fibril formation or to destroy already formed fibrils will
need to be developed to alleviate unsolved ocular dysfunction and
Transthyretin (TTR), is a 55 kDa plasma homotetrameric b-sheet-
rich protein, composed by 127-amino acids1. The main function
of the protein is the transport of thyroxine (T4) and retinol,
through its association with retinol binding protein (RBP), in
cerebrospinal fluid (CSF) and plasma2,3. The major sites of
protein biosynthesis are the liver and the choroid plexus of the
brain4. It is also produced in small amounts in the retina4 and in
human placenta5, so that TTR is present in human plasma and in
CSF at different concentrations6. TTR circulates in plasma as a
soluble protein but in some individuals, under unknown condi-
tions it can form amyloid fibrils. In approximately 20% of the
population over the age of 70, wild-type TTR becomes
amyloidogenic and leads to the onset of senile amyloidosis
(SSA)7,8. The amyloidogenic potential of TTR is enhanced by
single point mutations that can be associated with TTR hereditary
amyloidoses, such as familial amyloid polyneuropathy FAP and
familial cardiomyopathy FAC (http://amyloidosismutations.com).
Until a few years ago, the only therapy available to prevent the
formation of additional amyloid deposits was to remove the
source of TTR amyloidogenic variants through transplantation of
a liver secreting the less amyloidogenic wild-type TTR9.
Unfortunately, this therapeutic approach has no effect on retinal
CNS symptoms10–12
.
The TTR monomer is constituted by 2 four-stranded antipar-
allel b-sheets and a short a-helix. A stable dimer is generated
through a network of hydrogen-bond interactions between the
two-edge b-strands H and F of each monomer. Two dimers
associate back to back to build a tetramer traversed by a central
cavity in which two molecules of T4 bind2. The T4 hormone binds
in two identical funnel-shaped sites located at dimer–dimer
interface that is bisected by a two-fold axis. Although the TTR
tetramer presents two symmetric binding sites, the binding of T4
in solution shows strong negative cooperativity13,14
.
Starting from the knowledge that only 1% of TTR binding sites
in CSF and in plasma are occupied by T415, Kelly and
coworkers16,17 proposed a strategy to ameliorate TTR amyloidosis
by binding small molecules to the remaining 99% of active sites
left unoccupied. Consequently, a large number of TTR ligands
have been studied for their ability to bind the T4 binding sites and
stabilize the TTR tetramer18. Among these, the non-steroidal anti-
inflammatory drug diflunisal and tafamidis (VindaquelÕ) were
found to be able to stabilize TTR tetramers, reduce fibril
formation and slow the neurological impairment due to TTR
amyloidosis19–21. Tafamidis was approved for the treatment of
familial amyloid polineuropathy (FAP) by the European Medicines
Agency in 2011 and by the Japanese Pharmaceuticals and Medical
Devices Agency in 2013.
Address for correspondence: Dr Susanna Nencetti, Dipartimento di
`
Farmacia, Universita di Pisa, via Bonanno 6, Pisa 56126, Italy. Tel: +39
050 2219559. E-mail: susanna.nencetti@farm.unipi.it
´
Dr Enrico Adriano Stura, CEA, iBiTec-S, Service d’Ingenierie
Moleculaire des Proteines (SIMOPRO), Gif-sur-Yvette, F-91191,
In our previous work, we evaluated fluorenyl-based inhibitors
with only one unsubstituted aromatic portion linked through a
´
´
France. Tel: +33 (0)1 69 08 4302. E-mail: estura@cea.fr

2
L. Ciccone et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
flexible chain to a carboxylic acid22. The most active compound Turbidimetric assay
7BD shows a heteroatomic (C¼NO–CH –CH ) flexible linker
2
2
The percentage of fibril formation was determined by observing
the turbidity of wt-TTR at pH 4.4 in absence and in the presence
of inhibitory compounds22. Commercially available Prealbumin
from Human Plasma was purchased from Calbiochem (Merck
Millipore, Darmstadt, Germany). 7.2 mM wt-TTR in 10 mM
phosphate buffer (100 mM KCl, 1.7 mM EDTA, pH 7.6) was
preincubated with 2 molar equivalents of aminoxyacetic acid
derivatives (1a–e, 2a–e, 3) dissolved in DMSO or for negative and
positive controls, DMSO and 2 molar equivalents of diflunisal,
respectively. The assay was performed in wells of a 96-well
microplate (Corning Incorporated, New York, NY)24. After
incubation for 30 min at room temperature, an equal volume of
200 mM acetate buffer, 100 mM KCl, 1.7 mM EDTA, pH 4.2 was
added to each well in order to shift the pH to that for optimal
fibrillogenesis. After acidification, the 96-well microplate was
incubated at 37 ꢀC for 72 h without stirring. After a 3-day
incubation, the plate was vortexed in double orbital mode for 15 s,
after which the optical density (OD) was measured at 400 nm
using a SPECTROstarNano (200–1000 nm) UV/Vis spectropho-
tometer (Ortenberg, Germany). The assay was repeated in
triplicate. The OD ratio was calculated from the OD for each
inhibitor and compared to that of a sample prepared in absence of
any inhibitors multiplied by 100% to give the percentage of fibril
formation25. Before the assay, the solubility of each inhibitor was
evaluated by verifying the absence of absorbance at 400 nm prior
to mixing with the protein to ensure that the turbidity depended
only on TTR amyloid fibril formation.
between the fluorenyl and the carboxylic moiety (Figure 1).
The X-ray structure of the TTR-inhibitor crystal complex
shows that the fluorenyl derivative 7BD binds the TTR active
sites in both the forward and reverse modes. The same result was
obtained for the acetic analog ES8 even though the fluorenyl ring
occupies an alternative hydrophobic area in the TTR binding
sites23. The flexibility of the oxime–ether linker enables these
small ligands to better tailor their binding along the TTR tunnel.
The acid moieties of 7BD and ES8 can mediate interactions
with the key TTR binding site residues: Ser117, Thr119 and
Lys15. Based on ES8, a new series of halogen or nitro substituted
fluorenyl derivatives 1a–e were synthesized in order to evaluate
the influence of such substituents on the positioning of the
aromatic moiety and the carboxylic groups within the TTR
binding cavity. To achieve a better understanding the spatial
relationship between the aromatic and the carboxylic portion, we
synthesized a series of benzophenone (2a–e) and indanone (3a)
derivatives. Their simplified aromatic portions are less bulky and
have different rigidity than the 9-fluorenyl derivatives.
Here we report the synthesis, in vitro evaluation using the
turbidimetric UV-Vis assay and three-dimensional crystallo-
graphic analysis of TTR complex with the active ligands.
We discuss the finding that the dual binding mode, prevalent in
the absence of substitutions on the fluorenyl ring, is replaced by a
clear preference for a single orientation in the crystal structures of
the halogen substituted analogs, (2,7-dichloro-fluoren-9-ylidenea-
minooxy)-acetic acid (1c), (2,7-dibromo-fluoren-9-ylideneami-
nooxy)-acetic acid (1b) and (E/Z)-2-((3,4-dichlorophenyl)
(phenyl)methyleneaminooxy)acetic acid (2c) (Figures 2 and 3).
Crystal preparation and structure determination
Lyophilized human TTR (Calbiochem, Merck Millipore,
Darmstadt, Germany) was dissolved, 1 mg in 100 mL 0.02%
(w/v) NaN3, and dialyzed against 0.1M NaCl, 50 mM sodium
acetate, pH 5.5, overnight. The (2,7-dibromo-fluoren-9-ylidenea-
Methods
Chemistry
The 9-fluorenylaminooxyacetic (1a–e), the benzophenoneami- minooxy)-acetic acid (1b), (2,7-dichloro-fluoren-9-ylideneami-
nooxyacetic (2a–e) and indanoneaminooxyacetic (3) derivatives nooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-
were prepared as shown in Scheme 1, Scheme 2 and Scheme 3, methyleneaminooxy)-acetic acid (2c) were dissolved at 10mM
respectively. The appropriate ketones were treated with hydro- concentration in DMSO. The ligand-TTR complexes were prepared
xylamine hydrochloride to give the corresponding oximes (4a–e, from TTR at 5 mg/mL to which the ligand was added in a
6a–e and 8, respectively) which, after reaction with ethyl volumetric ratio of 1:6. This gives a molar ratio of 30 ligands per
bromoacetate, yielded the corresponding esters (5a–e, 7a–e and protein monomer and a ligand concentration of 1.3 mM. The
9, respectively). The esters were hydrolyzed with a stoichiometric crystals were grown from a 1 mL TTR-ligand solution and 1 mL
amount of 1M NaOH solution to yield the corresponding acids precipitant from the reservoir (500mL), by sitting drop vapor
(1a–e, 2a–e and 3, respectively).
diffusion using CrysChem plates (Corning Incorporated, New
Figure 1. Chemical structures of acetic acid
compounds.

DOI: 10.3109/14756366.2016.1167048
Synthesis and structural analysis of halogen substituted fibril formation inhibitors of TTR
3
Figure 2. Electron density and binding modes
for acetic acid compounds 1b, 1c and 2c. (A)
Ligand 1c and its electron density represented
as a multi-wire mesh to highlight the electron
dense bromine atoms is shown in the context
of the tetramer formed by TTR molecule A
(left) and B (bottom) and their symmetry
related molecules (top). (B) The bromine
atoms occupy two of the halogen-specific
binding sites with the carboxylate oxygens
(O1 and O2) making polar contacts with
˚
˚
Ser117 (O2—OG ¼ 2.9 A; O2—O ¼ 3.1 A)
˚
and Thr119 (O1—OG ¼ 2.4 A; O2—
˚
OG ¼ 2.9 A). Lys15, shown in the back-
ground, does not participate in binding to the
carboxylate, characterizing the ligand as
reverse mode binder. (C) Ligand 1b also
binds to a single site in good electron density.
(D) The binding mode is similar but not
identical to that of 1c, the differences being
characterized by longer H-bond distances
˚
with Ser117 (O1—OG ¼ 3.1 A; O2—
˚
O ¼ 3.1 A) and a single interaction with
˚
Thr119 (O1—OG ¼ 2.8 A). (E) There is good
electron density for ligand 2c in both TTR
binding sites. The carboxylate points towards
Lys15 defining the ligand as a forward mode
binder. (F) The salt bridge distances are:
˚
˚
O1—NZ ¼ 2.7 A and O2—NZ ¼ 3.2 A.
York, NY) that were equilibrated in a cooled incubator at 20ꢀC. cryoprotectant solution enriched with extra ligand before flash
The reservoir solutions were mixed using various combinations of cooling by dipping into liquid nitrogen at 100 K (Table 1). The
the commercial working solutions (WS) from the Stura Footprint data sets for the ligands-TTR complexes were collected at
Screen (Molecular Dimensions Ltd., Newmarket, UK)26. The synchrotron facilities (ESFR beamlines ID23–1 and ID23–2 in
reservoir conditions used for growing the crystals analyzed in this Grenoble, France). Data processing was carried out using the
study consisted of 50–60% of (PEG_4D: 30% or PEG_4C: 20% automated system available at the ESRF synchrotron facility or on
PEG-4000, 0.2 M imidazole malate, pH 6.0) with 40–50% of laboratory computers using XDS with the ‘‘xdsme’’ script
(PEG_6C: 22.5% PEG-10000, 0.1 M ammonium acetate, pH 4.5) (https://github.com/legrandp/xdsme) to optimize data quality.
(Table 1). The choice of high molecular weight polyethylene Unfortunately, the initial data set collected on ID23–1 for the 2c
glycols (PEG) has been discussed elsewhere27. Whenever crystals complex could not be refined with good statistics, probably
did not appear spontaneously after few hours, the drops were because of superposition of diffraction from microcrystals of the
streak seeded28. In the absence of crystals after overnight storage, poorly solubilized ligand. It was recollected on beamline
a booster solution (50 mL of 5 M NaCl with or without acetic acid) Proxima-2A35 at the Soleil storage ring in St. Aubin, France. To
was added to the reservoir27 followed by streak seeding if crystals recollect the data, crystals that had dried out after 18-month
did not appear within a few hours.
storage were re-hydrated by the addition of 300 mL of water to the
The crystallization follows the strategy of reverse screening34 sitting drop reservoir. The well was then resealed and the crystals
making use of different combinations of premixed working allowed to rehydrate over four hours. The re-hydrated crystals
solutions to achieve small variation in pH and precipitating power were transferred to a cryoprotectant solution as described in Table
with greater precision than can be achieved by mixing 1 and flash cooled in liquid nitrogen. To avoid the problem
concentrated precipitant and buffer solutions. This strategy has encountered previously, the ligand-solubilizing cryoprotectant
allowed us to obtain in a short time and using only six drops, solution SM129 was used instead of CM29 (Table 1). The data
crystallographic data at high resolution from crystals of TTR in were collected over 348.2 mm of a long crystal with 1ꢀ angular
complex with compounds 1b, 1c and 2c. For data collection, the range for a total of 300 images using the helical scan method in
crystals were cryoprotected by quick immersion into
a
which the crystal is translated in conjunction with each rotation.

4
L. Ciccone et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 3. Comparison of ligand binding in the
forward and reverse mode. (A) Ligands 1b
and 1c show only minor differences in their
positioning in the TTR binding site. The
differences in radii between the two halides is
responsible for the changes. (B) Ligands 1c
and 2c show differences in the binding mode,
in the halogen pocket that is used for their
binding but occupy the same overall volume,
when the superposition with its symmetry
related molecule is considered. (C) The
binding of ligand 1c is better compared to
ligand 7BD23, shown in salmon sticks, that to
ligands ES823 shown in panel (D) Since the
addition of the halogen atoms precludes a
perpendicular binding mode similar to ES8, it
must adopt a reverse mode binding similar to
7BD but shifted due to the presence of the
halogen atoms. (E) Ligand 2c binds to both
TTR binding sites with only minor differ-
ences. (F and G) These minor binding
differences appear amplified when 2c is
compared to ES8 in the two sites. (H) The
superimposed ES8 binding to TTR show the
same qualitative variation observed for 2c
and for 1b relative to 1c where the carb-
oxylate changes its orientation.
Scheme 1. Synthesis of fluorenyl derivatives 1a–e.
Scheme 2. Synthesis of benzophenone compounds 2a–e.
The final dataset at 1.5A could be refined with good statistics COOT37 and the inhibitor built using the monomer library sketcher
(Table 1). The structures were solved by rigid body refinement from CCP4 program suite38 was placed in the difference electron
using REFMAC536 starting with a TTR model (PDB entry 4PM127 density. The structure was subjected to at least three cycles of
without inhibitor. The electron density maps were viewed in rebuilding and refinement with REFMAC536 and phenix.refine33.
˚

DOI: 10.3109/14756366.2016.1167048
Synthesis and structural analysis of halogen substituted fibril formation inhibitors of TTR
5
Scheme 3. Synthesis of indanone derivatives 3.
Table 1. Statistics for data collection, processing and refinement on TTR-ligand complexes.
PDB code Ligand
5E23 LiC (1b)
5E4A Lic3 (1c)
5E4O (2c)
Crystallization
Cryoprotectant
60% PEG_4D 40% PEG_6C
40% CM29*, 25% MPEG 5000,
100 mM AABy
60% PEG_4D 40% PEG_6C
40% CM29*, 25% MPEG 5000,
100 mM AABy
50% PEG_4D 50% PEG_4C (re-hydrated crystal)
40% SM1*, 25% MPEG 5000,
100 mM AABy buffer 60%
buffer 60% A/40% B) 1 mM
compound 1b.
buffer 60% A/40% B) 1 mM
compound 1c.
A/40% B) 1 mM compound 2c.
Data collection
Source
˚
Wavelength (A)
ESRF ID23-1
0.8701
ESRF ID23-1
0.8701
Soleil Proxima-2
0.9801
method
Space group
È rotation only
P21212
È rotation only
P21212
Helical scan
P21212
˚
Unit-cell (A)
Molec./asym.
43.2, 86.0, 63.3
2
43-1.41/1.46-1.41
100.0/68.0
17.3/1.75
43.2, 86.0, 63.8
2
43-1.33/1.36-1.33
99.9/57.1
43.5 84.9 63.9
2
51-1.5/1.49
99.9/49.9
5.5/1.56
5.1/145.7
4.9/129.6
99.4/98.0
4.93/4.67
˚
Resolution (A)
CC1/2
5I/s(I)4z
14.92/1.22
7.6/197.2
7.2/186.4
99.9/99.9
9.1/9.2
Rmerge (%)
Rp.i.m.(%)
8.8/127.2
8.3/119.8
99.9/100.0
9.1/8.9
Completeness (%)
Multiplicity
Refinement
Resolution (A)
No. of reflections
Rwork (%)
Rfree (%)
˚
43-1.41/1.44-1.41
46271/2551
17.1/24.8
43-1.33/1.36-1.33
55377
14.3/33.7
51-1.5/1.54-1.5
38403
19.3/33.7
20.7/31.6
19.7/29.1
19.3/35.4
R.M.S. deviations
˚
Bond lengths (A)
Bond angles (ꢀ)
0.007
1.296
0.028
2.452
0.017
2.022
Ramachandranô
Favored (%)
Outliers (#)
99.0
1
98.1
0
98.0
0
*Cryoprotection: CM29: 25% diethylene glycol, 12.5% MPD, 37.5% 2,3-butanediol, 12.5% 1,4-dioxane. SM1: 12.5% diethylene glycol + 12.5%
glycerol + 12.5% 1,2-propanediol + 25% DMSO + 25% 1,4-dioxane29. Cryoprotectant solution is formulated with 40% v/v mixed compounds (CM),
50% v/v precipitant and 10% v/v buffer30
.
yBuffers are at 100 mM. PCTP: sodium propionate, sodium cacodylate, Bis-Tris-propane (component A pH 4; B pH 9.5)31
.
zCC1/2: Data quality correlation coefficient32
.
ôFrom phenix.refine output33
.
Results
analogs 2,7-dibromo and 2,7-dichloro substituted compounds (1b
and 1c) effectively reduced fibril formation to 34% and 37% FF,
respectively. The 2-fluoro substituted fluorenyl derivative (1a)
Biological assay
The percentage of TTR fibril formation (FF) was evaluated by a was found to be a less effective WT-TTR aggregation inhibitor.
doublet concentration of synthesized compounds 1a–e, 2a–e and The 2-nitro (1e) substituted compound showed poor inhibitory
3 to determine their potential as inhibitors (Table 2).
activity and the 2,7-di-nitro substitution (1d) abolished all
The 7.2 mM, the concentration used in the tests represents activity. Among the dibenzophenone derivatives the ((3,4-
twice the TTR concentration in plasma (3.6 mM). Diflunisal (9% dichloro-phenyl)-phenyl-methyleneaminooxy)-acetic acid (2c)
FF) was used as the reference drug (positive control) against inhibited TTR fibril formation (only 15% FF) with an effective-
which the ability of the newly synthesized compounds to reduce ness comparable with diflunisal, while the 2-chloro substituted
fibril formation was compared. The negative control, TTR compound 2b showed a lower inhibition potency. Decreased
without inhibitor under acid conditions (pH 4.2) is used to inhibitory activity was recorded for 2a (unsubstituted) and 2d
determine the 100% FF mark.
(3,30-fluoro substitution) and falls to zero for 2e (4,40-fluoro
Among the 11 compounds tested, three molecules showed substitution). Similarly the indanone-based compound 3 was
better than 50% FF inhibition (1b–c and 2c). The fluorenyl completely devoid of any activity.

6
L. Ciccone et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 2. In vitro acid-mediated wt-TTR (7.2 mM) percentage of fibril formation for compounds 1a–e, 2a–f and 3.
Compounds % Fibril formation
a
(7.2 µM inhibitor)
diflunisal
9
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3
57
37
34
100
80
84
58
15
80
100
98
aThe percentage fibril formation was evaluated by turbidity measurements at 400 nm, pH 4.4. The fibril formation in absence of inhibitor was assigned
to be 100%. The error in the fibril formation assay is 5%.
Crystallization and structure determination
pocket (Lys15). In the reverse mode, the carboxylic moiety
directed towards the inner binding pocket in the forward mode it
points towards the lysine (Figure 2B, D and F). While compound
2c binds in both TTR binding sites in the forward binding mode,
1b and 1c bind select the reverse binding mode, as can be seen
with the ligands superimposed in Figure 3.
The crystals of the TTR complexes with 1b, 1c and 2c belong to
the orthorhombic space group P21212 with cell parameters similar
to each other (Table 1) and comparable to other TTR depositions
in the PDB database. The diffraction limit for the three structures
˚
ranges from 1.5 to 1.33 A with relative good electron density for
the ligands positioned along the two-fold axis of the tetramer
(Figure 2) which, since subject to the symmetric crystallographic
operation that builds the tetramer, must have occupancies that Chemistry
Experimental
cannot exceed 0.5. The disordered portions of the protein are
limited to the region 99–104 as is commonly the case in many
˚
TTR structures. The re-hydrated crystal at 1.5 A diffracted to a
lower resolution than those with 1b or 1c but better than the
˚
previous 2c data set collected to only 1.8 A resolution. Of the
˚
seven re-hydrated crystals, three gave data better than 1.8 A and
¨
Melting points were determined on a Kofler (Arthur H. Thomas
Co., Philadelphia, PA) hot-stage apparatus and are uncorrected.
Mass spectrometry data were collected using a Hewlett Packard
5988A spectrophotometer (Palo Alto, CA), by direct introduction
at a nominal electron energy of 70 eV and a source temperature of
350 ꢀC. H-NMR and 13C-NMR spectra were determined with a
Bruker UltrashieldTM 400 MHz (Fallander, Switzerland) or Varian
¨
1
only one was severely disordered and showed strong anisotropy. It
can be noted that the b cell parameter for the re-hydrated dataset
Gemini 200 MHz (Mountain View, CA) spectrometer. Chemical
shifts (ꢀ) are reported in parts per million (ppm) downfield using
tetramethylsilane as the internal standard. Coupling constants
J are reported in Hertz; 13C-NMR spectra were fully decoupled.
The following abbreviations are used: singlet (s), doublet (d),
triplet (t), broad (br) and multiplet (m). Microwave assisted (MW)
reactions were run in a Discover LabMate Microwave Synthesizer
(CEM, Matthews, NC). Reactions were monitored by thin-layer
chromatography (TLC) on Merck (Darmstadt, Germany) 0.2 mm
pre-coated silica gel aluminum sheets (60 F-254) and visualized
under a UV light (254 nm). Evaporation was performed in vacuo
with a rotating evaporator (Naples, Italy). Sodium sulfate was
used as the drying agent. Analytical reagents, starting materials
and solvents were purchased from Sigma Aldrich (St. Louis, MO).
˚
is 1 A smaller than for the other two crystals, this cannot be
attributed without ambiguity to the dehydration/re-hydration
process. The solubilizing SM1 cryomix was used for this crystal
(Table 1) and the identical change was also observed when the
solubilizing SM5 cryomix was used with curcumin complexed
TTR crystals27, while the use of CM29 (Table 1) gave the identical
cell parameters as those for 1b and 1c cryoprotected with CM29.
Crystallographic analysis
The structures show important differences in terms of ligand
binding without majors changes in the protein structure. There is
electron density for only one of the two TTR molecules in the
asymmetric unit for the structures obtained with inhibitors 1b and
1c. The TTR molecules with or without ligand show only minor
differences and double conformations for some of the side chains
involved in ligand binding. The binding occurs in either the
reverse or forward mode depending on the compound, but not
both as previously observed with fluorenone derivatives without
substitutions (23). The binding mode might be determined by the
halogen atoms that occupy the classic halogen binding pockets
and by the carboxylic groups that point alternatively towards
the inner (Ser117–Thr119) and the outer part of the binding
General procedure for the synthesis of the fluoren-9-one
oximes (4a–e), benzophenone oximes (6a–e) and
5-chloro-1-indanone oxime (8)
A solution of appropriate carbonyl derivatives variously sub-
stituted (0.925 mmol) in DMSO (6 mL) was added drop-wise to
hydroxylamine hydrochloride (3.70 mmol) previously solubilized
in H2O. The reaction mixture was stirred at room temperature and
monitored by TLC (n-hexane/AcOEt 8:2). The resulting solution

DOI: 10.3109/14756366.2016.1167048
Synthesis and structural analysis of halogen substituted fibril formation inhibitors of TTR
7
was dissolved in H2O/ice to obtain a precipitate. The crude solid Bis-(3-fluoro-phenyl)-methanone oxime (6d)
was collected by filtration, washed with H2O and filtered under
vacuum to give the oxime (4a–e, 6a–e and 8).
(E)-2-fluoro-fluoren-9-one oxime (4a)
The title compound was prepared from bis-(3-fluoro-phenyl)-
phenyl-methanone following the general procedure. The crude
product was recrystallized with n-hexane to give 6d as a white
solid (40% yield); m.p.: 66–67 ꢀC; 1H-NMR (200 MHz, CDCl3) d:
The title compound was prepared from 2-fluoro-9-fluorenone 7.74 (s, 1H); 7.48–7.07 (m, 8H).
following the general procedure. The crude product was triturated
with EtOH/H2O to give 4a as a yellow solid (88% yield); m.p.:
181–182 ꢀC; 1H-NMR (200 MHz, DMSO-d6) d: 11.85 (s, 1H);
8.33–8.03 (m, 2H); 7.65–7.00 (m, 5H).
Bis-(4-chloro-phenyl)-methanone oxime (6e)
The title compound was prepared from bis-(4-chloro-phenyl)-
methanone following the general procedure. The crude product
was recrystallized with n-hexane to give 6e as a white solid (66%
yield); m.p.: 65–66 ꢀC (lit. 68–69 ꢀC43); 1H-NMR (200 MHz,
CDCl3) d: 8.05 (s, 1H); 7.49–7.00 (m, 8H).
2,7-Dichloro-fluoren-9-one oxime (4b)
The title compound was prepared from 2,7-dichloro-9-fluorenone
following the general procedure. The crude product was triturated
with EtOH/H2O to give 1b as a yellow solid (91% yield); m.p.:
262–263 ꢀC; 1H-NMR (200 MHz, DMSO-d6) d: 12.18 (s, 1H);
8.19 (s, 2H); 7.47–7.17 (m, 4H).
Bis-(4-fluoro-phenyl)-methanone oxime (6f)
The title compound was prepared from bis-(4-fluoro-phenyl)-
methanone following the general procedure. The crude product
was recrystallized with n-hexane to give 6f as a white solid (52%
2,7-Dibromo-fluoren-9-one-oxime (4c)
1
yield); m.p.: 58–60 ꢀC (lit. 137–138 ꢀC44); H-NMR (200 MHz,
The title compound was prepared from 2,7-dibromo-9-fluorenone
following the general procedure. The crude product was triturated
with MeOH to give 4c as a yellow solid (89% yield); m.p.: 265–
266 ꢀC (lit. 243 ꢀC39); 1H-NMR (200 MHz, DMSO-d6) d: 8.45 (s,
2H); 7.94–7.64 (m, 4H).
CDCl3) d: 8.05 (s, 1H); 7.49–7.00 (m, 8H).
(E)-5-Chloro-indan-1-one oxime (8)
The title compound was prepared from 5-chloro-indan-1-one
following the general procedure. The crude product was
2,7-Dinitro-fluoren-9-one-oxime (4d)
The title compound was prepared from 2,7-dinitro-9-fluorenone recrystallized with n-hexane to give 8 as a white solid
following the general procedure. The crude product was triturated (58% yield); m.p.: 144–145 ꢀC (lit.144 ꢀC45); 1H-NMR
with EtOH/H2O to give 4d as a yellow solid (89% yield); m.p.: (200 MHz, CDCl3) d: 8.00 (s, 1H), 7.62–7.28 (m, 3H,);
1
297–298 ꢀC (lit. 288–289 ꢀC40); H-NMR (200 MHz, DMSO-d6) 3.05–3.02 (m, 4H).
ꢀ: 9.03 (s, 2H); 8.49–8.33 (m, 4H).
General procedure for the synthesis of the fluoren-9-one
(E/Z)-2-Nitro-fluoren-9-one-oxime (4e)
ethyl esters (5a–e), biphenyl ethyl esters (7a–e) and (5-
The title compound was prepared from 2,7-dinitro-9-fluorenone chloro-indan-1-ylideneaminooxy)-acetic acid ethyl ester (9)
following the general procedure. The crude product was
The esters derivatives (5a–e, 7a–e, 9) were synthesized following
triturated with EtOH/H2O to give 4e as a yellow solid (92%
the general procedure as reported in literature. To a suspension of
yield) in mixture (isomer E/Z ꢁ50:50), which was not separated;
the appropriate oximes (4a–e, 6a–e, 8) (0.617 mmol) in DMF
m.p.: 279–280 ꢀC (lit. 262.5–263 ꢀC41). 1H-NMR (200 MHz,
(0.4 mL) and CH3CN (2 mL) was added of K2CO3 (1.74 mmol),
DMSO-d6) d: 9.05 (s, 1H);8.99 (s, 1H), 8.43–8.14 (m, 8H),
tetrabutylammonium bromide (0.053 mmol) and ethyl bromoace-
7.77–7.59 (m, 4H).
tate (0.694 mmol). The suspension was submitted to microwave
irradiation at a power of 80 W, tmax 70 ꢀC, 100 psi and monitored
by TLC. The reaction mixture was poured into ice, extracted with
Benzophenone oxime (6a)
The title compound was prepared from benzophenone following Et2O and washed with an aqueous NaOH 1N solution. Organic
the general procedure. The crude product was recrystallized with layer dried and evaporated under vacuum gave exclusively the
n-hexane to give 6a as a white solid (70% yield); m.p.: 142– crude ethyl ester (5a–e, 7a–e, 9), which were used for the
1
145 ꢀC (lit. 141–142 ꢀC42); H-NMR (200 MHz, CDCl3) d: 11.33 successive reaction without purification.
(s, 1H); 7.49–7.42 (m, 5H); 7.41–7.34 (m, 3H); 7.29–7.26 (m, 2H).
(E)-2-(2-Fluoro-fluoren-9-ylideneaminooxy)-acetic acid
ethyl ester (5a)
Bis-(2-chloro-phenyl)-methanone oxime (6b)
The title compound was prepared from bis-(2-chloro-phenyl)-
The title compound was prepared from (E)-2-fluoro-fluoren-9-
methanone following the general procedure. The crude product
one oxime (4a) following the general procedure. Yellow oil (64%
was recrystallized with n-hexane to give 6b as a white solid (48%
yield). 1H-NMR (200 MHz, CDCl3) ꢀ: 8.37–7.02 (m, 7H); 4.95 (s,
yield); m.p.: 135–137 ꢀC; 1H-NMR (200 MHz, CDCl3) d: 8.42 (s,
2H); 4.30 (q, 2H, J ¼ 7.2 Hz); 1.33 (t, 3H, J ¼ 7.2 Hz).
1H); 7.52–7.28 (m, 8H).
2–(2,7-Dichloro-fluoren-9-ylideneaminooxy)-acetic acid
ethyl ester (5b)
(E/Z)-(3,4-Dichloro-phenyl)-phenyl-methanone oxime (6c)
The title compound was prepared from (3,4-dichloro-phenyl)-
phenyl-methanone following the general procedure. The crude
product was recrystallized with n-hexane to give 6c as a white
solid in mixture (isomer E/Z ꢁ50:50), which was not separated
(46% yield). 1H-NMR (400 MHz, CDCl3) d: 7.92 (s, 1H); 7.90 (s,
1H), 7.77–7.75 (m, 4H), 7.64–7.49 (m, 10H).
The title compound was prepared from 2,7-dichloro-fluoren-9-
one oxime (5b) following the general procedure. Yellow oil (57%
1
yield). H-NMR (200 MHz, CDCl3) d: 8.32–7.27 (m, 1H); 7.99–
7.91 (m, 2H); 7.68–7.53 (m, 3H); 4.94 (s, 2H); 4.29 (q, 2H,
J ¼ 7.2 Hz); 1.32 (t, 3H, J ¼ 7.2 Hz).

8
L. Ciccone et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
2-(2,7-Dibromo-fluoren-9-ylideneaminooxy)-acetic acid
ethyl ester (5c)
(E)-2-(5-Chloro-indan-1-ylideneaminooxy)-acetic acid
ethyl ester (9)
The title compound was prepared from 2,7-dibromo-fluoren-9- The title compound was prepared from 5-Chloro-indan-1-one
one oxime (5c) following the general procedure. Yellow oil (76% oxime (8) following the general procedure. Violet oil (68% yield).
yield). 1H-NMR (200 MHz, DMSO-d6) d: 8.41–8.40 (m, 2H); 1H-NMR (200 MHz, CDCl3) d: 7.21–7.62 (m, 3H); 4.71 (s, 2H);
7.94–7.72 (m, 4H); 5.09 (s, 2H); 4.20 (q, 2H, J ¼ 7.2 Hz); 1.24 4.25 (q, 2H, J ¼ 7 Hz); 3.03 (m, 4H); 1.31 (t, 3H, J ¼ 7 Hz).
(t, 3H, J ¼ 7.2 Hz).
General procedure for the synthesis of the 9-fluorenone
2-(2,7-Dinitro-fluoren-9-ylideneaminooxy)-acetic acid
ethyl ester (5d)
acetic acid (1a–e), biphenyl acetic acid (2a–e), (5-chloro-
indan-1-ylideneaminooxy)-acetic acid (3)
The title compound was prepared from 2,7-dinitro-fluoren-9-one A solution of ethyl ester (1a–e, 2a–e, 3) (0.387 mmol) in H2O
oxime (5d) following the general procedure. Clear oil (61% yield). (2.22 mL) and THF (4.44 mL) at 0 ꢀC was added dropwise an
1H-NMR (200 MHz, DMSO-d6) d: 8.97–8.96 (m, 2H); 8.56–8.30 aqueous NaOH 1N (1.11 mL) solution. The mixture was stirred
(m, 4H); 5.23 (s, 2H); 4.24 (q, 2H, J ¼ 7.2 Hz); 1.27 (t, 3H, for 2 h at 0 ꢀC and then at room temperature until the ester was
J ¼ 7.2 Hz).
completely hydrolyzed into acid. The reaction was monitored by
TLC (n-hexane/AcOEt 8:2). After solvent evaporation, the
resultant solution was dissolved in H2O and washed with Et2O.
The water solution acidified with 10% HCl at pH 4, extracted with
CHCl3, dried and evaporated, gave the crude acetic acids (1a–e,
2a–e, 3).
(E/Z)-2-(2-Nitro-fluoren-9-ylideneaminooxy)-acetic acid
ethyl ester (5e)
The title compound was prepared from 2-nitro-fluoren-9-one
oxime (5e) following the general procedure. The crude product
was obtained as a mixture (isomer E/Z ꢁ 54:46), which was not
(E)-2-(2-fluoro-fluoren-9-ylideneaminooxy)-acetic acid
(1a)
1
separated. Clear oil (66% yield). H-NMR (200 MHz, DMSO-d6)
d: 8.99 (s, 1H); 8.98 (s, 1H); 8.44–8.04 (m, 8H); 7.73–7.48
(m, 4H); 5.15 (s, 2H); 5.12 (s, 2H); 4.23 (q, 2H, J ¼ 7.2 Hz); 1.24 The title compound was prepared from ethyl ester-(E)-(2-fluoro-
(t, 3H, J ¼ 7.2 Hz).
fluoren-9-ylideneaminooxy)-acetic acid (5a) following the gen-
eral procedure. The crude product was recrystallized with
n-hexane to give as a yellow solid (64% yield); m.p.: 234–
2-(Diphenylmethylenaminooxy)-acetic acid ethyl ester (7a)
1
235 ꢀC; H-NMR (200 MHz, DMSO-d6) d: 8.92 (d, 1H); 7.92–
The title compound was prepared from diphenyl-methanone
oxime (6a) following the general procedure. Clear oil (70% yield).
1H-NMR (200 MHz, CDCl3) d: 7.31–7.08 (m, 10H); 4.25 (s, 2H);
4.21 (q, 2H, J ¼ 7.2 Hz); 1.30 (t, 3H, J ¼ 7.2 Hz).
7.69 (m, 4H); 7.84–7.94 (m, 2H); 4.97 (s, 2H); 13C NMR
(400 MHz, DMSO-d6) d: 170.8, 152.0, 139.5, 136.6, 134.5, 132.4,
131.4, 130.0, 129.6, 128.5, 122.8, 122.5, 121.9, 118.8, 72.3. MS:
271 (M+, 67), 214 (24), 197 (100), 188 (23), 168 (11), 74 (5).
2-(Bis-(2-chloro-phenyl)-methyleneaminooxy)-acetic acid
ethyl ester (7b)
2-(2,7-Dichloro-fluoren-9-ylideneaminooxy)-acetic acid (1b)
The title compound was prepared from ethyl ester-(2,7-dichloro-
fluoren-9-ylideneaminooxy)-acetic acid (5b) following the gen-
eral procedure. The crude product was recrystallized with
n-hexane to give 1b as a yellow solid (50% yield); m.p.: 247–
248 ꢀC; 1H-NMR (200 MHz, DMSO-d6) d: 8.27–8.26 (d, 1H);
7.98–7.91 (m, 2H); 7.68–7.53 (m, 3H); 5.00 (s, 2H); 13C NMR
(400 MHz, DMSO-d6) d: 170.8, 150.7, 139.3, 138.4, 136.3, 134.9,
133.8, 131.9, 131.2, 124.5, 123.4, 123.3, 122.1, 122.0, 72.6. MS:
The title compound was prepared from bis-(2-chloro-phenyl)-
methanone oxime (7b) following the general procedure. Clear oil
(75% yield). H-NMR (200 MHz, CDCl3) d: 7.28–7.55 (m, 8H);
4.76 (s, 2H); 4.25 (q, 2H, J ¼ 6.8 Hz); 1.31 (t, 3H, J ¼ 6.8 Hz).
1
(E/Z)-2-((3,4-Dichloro-phenyl)-phenyl-methyleneami-
nooxy)-acetic acid ethyl ester (7c)
The title compound was prepared from (3,4-dichloro-phenyl)- 321 (M+, 56), 264 (30), 247 (100), 211 (77), 177 (22), 83 (78).
phenyl methanone oxime (7c) following the general procedure.
The crude product was obtained as a mixture (isomer E/Z 2–(2,7-Dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c)
1
ꢁ50:50). Clear oil (81% yield). H-NMR (200 MHz, CDCl3) d:
The title compound was prepared from ethyl ester-(2,7-dibromo-
7.28–7.62 (m, 16H); 4.74 (s, 2H), 4.73 (s, 2H); 4.26 (q, 4H,
fluoren-9-ylideneaminooxy)-acetic acid (5c) following the gen-
eral procedure. The crude product was recrystallized with
J ¼ 7.2 Hz); 1.32 (t, 6H, J ¼ 7.2 Hz).
n-hexane to give 1c as a yellow solid (75% yield); m.p.: 243–
1
2-(Bis-(3-fluoro-phenyl)-methyleneaminooxy)-acetic acid
ethyl ester (7d)
244 ꢀC; H-NMR (200 MHz, DMSO-d6) ꢀ: 8.41 (m, 1H); 7.95–
7.68 (m, 5H); 5.01 (s, 2H); 13C NMR (400 MHz, DMSO-d6) d:
The title compound was prepared from bis-(3-fluoro-phenyl)- 171.1, 150.9, 140.3, 139.4, 136.4, 135.0, 134.2, 133.9, 132.2,
methanone oxime (7d) following the general procedure. Clear oil 124.6, 123.9, 123.9, 122.8, 122.1,72.9. MS: 411 (M+, 69), 354
1
(73% yield). H-NMR (200 MHz, CDCl3) d: 7.56–7.08 (m, 8H); (20), 337 (100), 257 (41), 194 (15), 177 (17), 83 (17).
4.72 (s, 2H); 4.24 (q, 2H, J ¼ 7 Hz); 1.30 (t, 3H, J ¼ 7 Hz).
2-(2,7-Dinitro-fluoren-9-ylideneaminooxy)-acetic acid (1d)
2-(Bis-(4-fluoro-phenyl)-methyleneaminooxy)-acetic acid
The title compound was prepared from ethyl ester-(2,7-dinitro-
ethyl ester (7e)
fluoren-9-ylideneaminooxy)-acetic acid (5d) following the
The title compound was prepared from bis-(4-fluoro-phenyl)- general procedure. The crude product was recrystallized with
methanone oxime (7e) following the general procedure. Yellow n-hexane to give 1d as a yellow solid (50% yield); m.p.: 277–
1
oil (73% yield). 1H-NMR (200 MHz, CDCl3) d: 7.54–6.99 (m, 278 ꢀC; H-NMR (200 MHz, DMSO-d6) ꢀ: 8.99 (m, 1H); 8.52–
8H); 4.72 (s, 2H); 4.24 (q, 2H, J ¼ 7.2 Hz); 1.32 (t, 3H, 8.36 (m, 5H); 5.13 (s, 2H); 13C NMR (400 MHz, DMSO-d6) d:
J ¼ 7.2 Hz).
170.1, 150.7, 140.3, 138.3, 136.1, 134.5, 134.1, 133.9, 133.2,

DOI: 10.3109/14756366.2016.1167048
Synthesis and structural analysis of halogen substituted fibril formation inhibitors of TTR
9
124.7, 124.2, 124.0, 122.9, 122.5, 72.4. MS: 344 (M+, 4), 269 114.9, 114.7, 70.6. MS: 27 (11), 216 (100), 201 (12), 122 (9),
(100), 255 (15), 239 (73), 197 (40), 164 (65), 83 (78), 75 (13).
113 (9), 95 (12), 75 (38).
2-(Bis-(4-fluoro-phenyl)-methyleneaminooxy)-acetic acid
(2e)
(E/Z)-2-(2-nitro-fluoren-9-ylideneaminooxy)-acetic acid
(1e)
The title compound was prepared from ethyl ester-(bis-(4-fluoro-
phenyl)-methyleneaminooxy)-acetic acid (7e) following the gen-
eral procedure. The crude product was recrystallized with
n-hexane to give 2e as a white solid (88% yield); m.p.: 115–
The title compound was prepared from ethyl ester-(E/Z)-(2-nitro-
fluoren-9-ylideneaminooxy)-acetic acid (5e) following the gen-
eral procedure. The crude product was recrystallized with
n-hexane to give 1e as a yellow solid (50% yield) as a mixture
(isomer E/Z ꢁ 50:50), which was not separated. 1H-NMR
(200 MHz, DMSO-d6) d: 9.00 (s, 1H); 8.99 (s, 1H); 8.50–8.05
(m, 8H); 7.79–7.45 (m, 4H); 5.06 (s, 2H); 5.04 (s, 2H); 13C NMR
(400 MHz, DMSO-d6) d: 170.8, 151.1, 150.9, 147.72, 147.65,
146.87, 145.75, 139.0, 138.1, 135. 8, 135.48, 132.6, 131.5, 131.0,
130.9, 130.6, 129.9, 129.6, 127.7, 126.3, 123.9, 122.8, 112.78,
122.2, 121.9, 121.7, 116.4, 72.7. MS: 289 (M+, 22), 248 (24), 224
(70), 194 (61), 165 (87), 137 (25), 83 (100), 74 (16).
1
116 ꢀC. H-NMR (200 MHz, CDCl3) d: 7.50–6.99 (m, 8H); 4.77
(s, 2H); 13C NMR (400 MHz, CDCl3) d: 174.5, 157.8, 137.2,
136.5, 134.8, 132.3, 131.8, 130.6, 129.5, 127.7, 127.6, 127.5,
70.8. MS: 291 (M+,12), 216 (100), 201 (17), 122 (15), 133 (15),
95 (10), 75 (69).
(E)-2-(5-Chloro-indan-1-ylideneaminooxy)-acetic acid (3)
The title compound was prepared from ethyl ester-(5-Chloro-
indan-1-ylideneaminooxy)-acetic acid (9a) following the general
procedure. The crude product was recrystallized with n-hexane to
give 3a as a white solid (70% yield); m.p.: 140–141 ꢀC; 1H-NMR
(200 MHz, CDCl3) d: 7.58–7.27 (m, 3H); 4.76 (s, 2H); 3.03 (m,
4H); 13C NMR (400 MHz, CDCl3) d: 175.16; 164.0, 150.3, 136.7,
133.9, 127.6, 125.8, 122.9, 70.4, 28.4, 26.8. MS: 240 (M+, 50),
229 (16), 164 (100), 137 (15), 130 (14), 103 (16), 75 (9).
2-(Diphenylmethylenaminooxy)-acetic acid (2a)
The title compound was prepared from ethyl ester-(benzhydryli-
deneaminooxy)-acetic acid (7a) following the general procedure.
The crude product was recrystallized with n-hexane to give 2a as
a white solid (50% yield); m.p.: 109–110 ꢀC; 1H-NMR (200 MHz,
CDCl3) d: 7.50–7.27 (m, 8H); 4.08 (s, 2H); 13C NMR (400 MHz,
CDCl3) d: 176.2, 164.3, 153.6, 131.3, 138.8, 129.3, 128.6, 128.4,
127.8, 77.5. MS: 255 (M+, 17), 216 (100), 201 (20), 122 (25), 113
(10), 95 (10), 75 (65).
Discussion
Human Transthyretin tetramer dissociation is the rate-limiting
step in TTR amyloidogenic process. One of the therapeutic
strategies to ameliorate TTR amyloidoses and slow the progres-
sion of the disease is tetramer stabilization by small molecules,
such as tafamidis and diflunisal. TTR stabilizing molecules are
typically composed of two aromatic portions directly connected
or joined together by a linker of variable composition. The
unsubstituted fluorenyl compounds 7BD and ES8 previously
studied by us presented only one aromatic portion connected to
the terminal carboxylic moiety by an propionic or acetic linker,
respectively (22, 23). These fluorenyl derivatives (7BD, ES8) are
efficient in stabilizing the tetramer by binding the TTR both in
forward and in reverse mode with the distinction that the aromatic
portion of the acetic derivative ES8 occupies a different
hydrophobic pocket with respect to 7BD23. The new fluorenyl
analogs, 1b and 1c, that are halogen substituted also show a low
percentage of fibril formations (Table 2), result supported by the
X-ray analysis which show that they interact with the TTR in
similar manner of 7BD but only in reverse mode (Figure 3A and
C). The structures of TTR in complexed with 1b and 1c have
ligands binding to a single of the two binding sites. Given that in
the co-crystallization experiment, the ligand concentration was in
the millimolar range with excess ligand to saturate both sites 30
times over, the lack of ligand at the second site is not due to
insufficient ligand. This result can be interpreted as another
example of negative cooperativity. The evidence for negative
cooperativity in ligand binding to TTR is mounting46, although
doubts and Skepticism will undoubtedly persist. Negative
cooperative behavior, in the case of two binding sites that are
physically well separated from each other, must be relayed via
induced conformational changes. The existence of asymmetry due
to structural fluctuations that affect the affinity of the two sites for
various ligands cannot be considered a cooperative behavior. The
asymmetry found in TTR has been characterized by only small
differences in the conformation of the cavity of the two binding
sites. This asymmetry could be derived from crystal packing
constraints (Figure 4A and B). To prove that ligand binding, and
not crystal packing influences the structure of TTR, the
2-(Bis-(2-chloro-phenyl)-methyleneaminooxy)-acetic acid
(2b)
The title compound was prepared from ethyl ester-(bis-(2-chloro-
phenyl)-methyleneaminooxy)-acetic acid (7b) following the gen-
eral procedure. The crude product was recrystallized with
n-hexane to give 2b as a white solid (50% yield); m.p.: 113–
1
115 ꢀC, H-NMR (200 MHz, CDCl3) d: 7.50–7.27 (m, 8H); 4.08
(s, 2H); 13C NMR (400 MHz, CDCl3) d: 173.1, 122.91, 133.9,
133.6, 132.8, 132.8, 132.1, 130.8, 130.8, 130.7, 130.6, 129.8,
126.8, 126.6, 70.9. MS: 323 (M+,1), 288 (2), 248 (13), 214 (34),
177 (4), 149 (4), 75 (100), 56 (25).
(E/Z)-2-((3,4-Dichloro-phenyl)(phenyl)methyleneami-
nooxy)-acetic acid (2c)
The title compound was prepared from ethyl ester-((3,4-dichloro-
phenyl)-(phenyl) methyleneaminooxy)-acetic acid (7c) following
the general procedure. The crude product was recrystallized with
n-hexane to give 2c as a yellow solid (76% yield) in a mixture
1
(isomer E/Z ꢁ 55:45); H-NMR (200 MHz, CDCl3) d: 7.61–7.27
(m, 8H); 4.79 (s, 2H), 4.78 (s, 2H). 13C NMR (400 MHz, CDCl3)
d: 174.9, 156.9, 156.7, 135.7, 134.8, 134.0, 133.5, 132.7, 132.6,
132.4, 131.7, 131.3, 130.3, 130.24, 130.2, 129.7, 129.6, 129.1,
128.8, 128.5, 128.4, 128.1, 127.3, 77.3, 77.2. MS: 323 (M+, 16),
248 (49), 214 (100), 177 (15), 164 (13), 127 (7), 105 (15), 74 (25).
2-(Bis(4-fluoro-phenyl)-methyleneaminooxy)-acetic acid
(2d)
The title compound was prepared from ethyl ester-(bis(4-fluoro-
phenyl)-methyleneaminooxy)-acetic acid (7d) following the
general procedure. The crude product was recrystallized with
n-hexane to give 2d as a white solid (54% yield); m.p.: 106–
1
108 ꢀC H-NMR (200 MHz, CDCl3) d: 7.47–7.11 (m, 8H); 4.79
(s, 2H); 13C NMR (400 MHz, CDCl3) d: 174.5, 156.5, 137.3,
134.1, 134.0, 129.9, 129.8, 129.7, 129.4, 124.7, 116.6, 116.3,

10 L. Ciccone et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 4. Crystal packing and ligand binding.
(A) Crystal packing of TTR monomer A with
inhibitor 1b bound and, (B) packing of
monomer B without a ligand. The asymmet-
ric unit of the P21212 lattice comprises a
dimer A–B that forms a tetramer with A–B0
via a crystallographic symmetry operation.
Differences in crystal packing distinguish the
two monomers, A and B, where monomers A
and A0 pack tighter that B–B0. By convention,
ligands bind to monomer A while the empty
site is assigned to monomer B. The RMSD
deviations on Ca between two chains
involved in ligand binding; between chains
both not involved in ligand binding and
between chains with ligand compared to
chains without ligand to show the divergence
that exists between monomers involved and
not involved in ligand binding. (C) The
binding of 1b appears to induce a small
conformational change in order to better
accommodate the bromine atom in the halo-
gen pocket of molecule A. (D) Molecule B
(has been changed from green to yellow after
superimposition on molecule A) shows that in
order for the same ligand to bind to molecule
B, similar conformational changes would
need to be induced. (E) Without a change,
that would facilitate binding to molecule B,
positive cooperativity can be excluded. The
evidence for conformational changes that
would hinder ligand binding at site B are also
lacking, since the dimer interface is essen-
tially the same for the two molecules. (F) The
differences observed in the loop 94–104
between molecule B and A are common to
most TTR-inhibitor complexes, as shown
here for ligand 2c.
differences between monomers with and without ligand should be cooperation acts, a more detailed analysis would be required,
larger that the differences between A and B monomers. possibly with a TTR polymorph without a crystallographic two-
The RMSD between the TTR chain A with 1b compared with fold axis47. This criticism is difficult to dismiss since almost all
chain B without ligand is 1.179. Similarly, the same comparison the liganded structures are derived from a single crystal
for the 1c complex gives an RMSD of 1.230, while when the two polymorph with the protein always confined in the same
chains complexed to 2c are compared the value is only 0.591 manner. The affinity of ligands appears to be another factor that
(Figure 4B). When the various monomers with and without bound can determine different ability of TTR ligands to saturate the two
ligand are compared, a larger RMSD characterizes heterogeneous T4 binding sites of the tetrameric protein. Experimental factors,
couples. To understand the mechanism by which negative such as the poor aqueous solubility of the ligands and the

DOI: 10.3109/14756366.2016.1167048
Synthesis and structural analysis of halogen substituted fibril formation inhibitors of TTR 11
Figure 5. Superimposition of 2c and ES8 on
3,30-diiodo-L-thyronine. (A) The halogen
binding site exploited by 2b matches with
that used by 3,30-diiodo-L-thyronine. (B) The
second halogen binding site used by 3,30-
diiodo-L-thyronine at right angles to the first
one superimposes on the fluorenyl ring of
ES8.
persistent use of high-salt crystallization conditions also influence Conclusion
the crystallographic results. The crystals used in this study were
The X-ray structures of TTR in complex with the non-
obtained from polyethylene glycol, conditions in which many
ligands are more soluble.
substituted acetic derivative ES8 showed that a single atom in
the linker between the tricyclic ring and acidic moieties can
change the positioning of the tricyclic ring that is located in
the pocket previously described for 1-amino-5-naphthalene
sulfonate (5NS) complex with TTR. In the present work we
have explored the effect produced by the decoration of 9-
fluorenyl scaffold with halogen groups and their effects on the
binding mode. The crystallographic studies have highlighted
that in the 9-fluorenyl acetic derivatives of type 1b
(PDB:5E23) the presence of halogen atoms on the aromatic
moiety repositions the tricyclic ring in the hydrophobic pocket
identified for the unsubstituted 9-fluorenyl propionic deriva-
tives, orienting the molecule solely in reverse mode. The X-
ray analysis of 2,3-dichlorobenzophenone 2c (PDB: 5E4O)
shows a different spatial disposition of the aromatic rings and
uni-modal binding opposed to that of halogen substituted 9-
fluorenyl derivatives.
The potential of fluorenyl scaffold was revealed by our
previous study (23). With the development of these new inhibitors
we can now select a specific conformation. There is now an
opportunity to improve the potency of fibril inhibitor 2c
combining by exploiting the knowledge acquired from these
studies to synthesize new inhibitors with improved affinity and
without the side effects due to hormonal mimicry or the anti-
inflammatory activity typical of NSAIDs.
Among the benzophenone (2a–e) and indanone (3a) derivatives
in which the aromatic was simplified having less steric hindrance
and different rigidity than 9-fluorenyl derivatives, the more potent
fibril formation inhibitors was compound 2c. Derivative 2c, like
tafamidis, are able to occupy both sites. In those cases, as for ligand
1b, where minor conformational changes are induced on ligand
binding (Figure 4C–F) it is difficult to find evidence for the
transmission of these changes to the other subunit. The changes
observed may be connected to the packing on the molecules in the
lattice. We have recently obtained a new crystal form47, without a
crystallographic two-fold axis that could be used to re-analyze
ligands like 2c that show asymmetric binding. The presence of two
tetramers in the asymmetric unit of this new polymorph might be
useful to analyze whether conformational changes induced by
ligand binding at one site might explain the absence of a ligand at
the other site and the apparent negative cooperative behavior of
TTR to give structural support to solution studies that show a
concerted opening and closing of the dimer–dimer interface48,
possibly in the form of a see-saw mechanism49.
The binding of compound 2c, with a fibril inhibitory activity
similar to tafamidis, shows certain features that are reminiscent of
the binding of 3,30-diiodo-L-thyronine (Figure 5A) without it
occupying of the second halogen pocket used by this ligand. This
second pocket is exploited by the fluorenyl moiety of ES8, from
our previous study, that has a spacer between the hydrophobic and
carboxylate moieties of the same character. This suggests that a
combination of the two scaffolds might provide a manner to
achieve greater tetramer stabilization.
Acknowledgements
The help of the staff at the ESRF and Soleil synchrotrons and the grant of
beamtime are acknowledged with gratitude.

12 L. Ciccone et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
fibrillogenesis inhibitors. J Enzyme Inhib Med Chem 2015. [Epub
ahead of print]. DOI: 10.3109/14756366.2015.1070265.
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throughput screening of in vitro transthyretin amyloid fibrillogenesis
inhibitors. J Comb Chem 2005;7:246–52.
Declaration of interest
This work was partially supported by Ministero dell’Istruzione,
`
dell’Universita e della Ricerca of Italy (PRIN 20109MXHMR_007).
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