Tuning transthyretin amyloidosis inhibition properties of iododiflunisal by combinatorial engineering of the nonsalicylic ring substitutions

Article abstract of DOI:10.1021/co5001234

Two series of iododiflunisal and diflunisal analogues have been obtained by using a two step sequential reaction solution-phase parallel synthesis. The synthesis combined an aqueous Suzuki-Miyaura cross-coupling and a mild electrophilic aromatic iodination step using a new polymer-supported iodonium version of Barluengas reagent. From a selected set of 77 noniodinated and 77 iodinated diflunisal analogues, a subset of good transthyretin amyloid inhibitors has been obtained with improved turbidimetry inhibition constants, high binding affinity to transthyretin, and good selectivity for TTR compared to other thyroxine binding proteins.

Full text of DOI:10.1021/co5001234

Research Article
pubs.acs.org/acscombsci
Tuning Transthyretin Amyloidosis Inhibition Properties of
Iododiflunisal by Combinatorial Engineering of the Nonsalicylic Ring
Substitutions
†
‡
‡
io Almeida, Alfredo Ballesteros,∥
§
Maria Vilaro, Joan Nieto, Juan Ramon La Parra, Maria Rosar
́
́
́
‡ ,†
†
Antoni Planas, Gemma Arsequell, and Gregorio Valencia*
†
Unit of Glycoconjugate Chemistry, I.Q.A.C.-C.S.I.C., 08034 Barcelona, Spain
‡
Laboratory of Biochemistry, Institut Químic de Sarria,
̀
Universitat Ramon Llull, 08022 Barcelona, Spain
IBMC-Instituto de Biologia Molecular e Celular and ICBAS-Instituto de Ciencias Biomedicas de Abel Salazar, Universidade do
Porto, 4150-180 Porto, Portugal
§
̂
́
∥
́
Instituto Universitario de Química Organometalica “Enrique Moles”, Universidad de Oviedo, 33071 Oviedo, Spain
*
S Supporting Information
ABSTRACT: Two series of iododiflunisal and diflunisal
analogues have been obtained by using a two step sequential
reaction solution-phase parallel synthesis. The synthesis
combined an aqueous Suzuki-Miyaura cross-coupling and a
mild electrophilic aromatic iodination step using a new polymer-
supported iodonium version of Barluenga’s reagent. From a
selected set of 77 noniodinated and 77 iodinated diflunisal
analogues, a subset of good transthyretin amyloid inhibitors has
been obtained with improved turbidimetry inhibition constants,
high binding affinity to transthyretin, and good selectivity for
TTR compared to other thyroxine binding proteins.
KEYWORDS: transthyretinkinetic stabilizers, TTR amyloidosis inhibitors, solution-phase parallel synthesis,
5
-aryl salicylic acid core libraries, iododiflunisal analogues library, polymer-supported iodonium reagent, Barluenga’s reagent,
aqueous Suzuki-Miyaura reaction
6
INTRODUCTION
FAP. Tafamidis (Vyndaqel) is a newly developed small
■
molecule having a benzoxazole scaffold that binds selectively
to TTR (Kd1 = 2 nM, Kd2 = 154 nM) and stabilizes the
tetrameric structures of both wild-type and variant TTRs.
Tafamidis was approved by the European Medicines Agency in
2011 for the treatment of early stage (stage I) FAP and by the
Japanese Pharmaceuticals and Medical Devices Agency in 2013
Transthyretin (TTR) related diseases are a group of rare
diseases affecting the peripheral nerves and other vital organs
by the formation of amyloid deposits of this protein. They
comprise senile systemic amyloidosis (SSA), familial amyloi-
1
dotic polyneuropathy ,3(FAP), and familial amyloidotic
2
cardiomyopathy (FAC).
7
for the treatment of FAP (any stage). Alternatively, the FDA
Many different families of small molecules are known to
stabilize TTR effectively thus preventing its aggregation in vitro.
Within these families, compounds that display in vitro
transthyretin (TTR) amyloid inhibitory activity usually bind
and stabilize the tetrameric form of TTR by mimicking the
action of thyroid hormones. These compounds differentially
stabilize the native tetrameric structure of TTR over the
dissociative transition state, raising the kinetic barrier, imposing
kinetic stabilization on the tetramer, preventing TTR
amyloidogenesis. Already identified families of inhibitors
belong to classes of compounds as diverse as tetrahydroquino-
lines, dihydropyridines, benzodiazepines, phenoxazines, stil-
benes, and benzoxazoles. From all of them, only two TTR
tetramer stabilizers, tafamidis and the FDA registered NSAID,
registered NSAID diflunisal has spawned a surge of interest and
is currently being tested in clinical trials in FAP patients for its
efficacy to ameliorate peripheral neuropathy resulting from
8
TTR depositions.
In our hands the biological properties of diflunisal were
9
improved by iodination on its salicylic moiety. The rational was
to imitate the unique structural feature of thyroid hormones of
presenting iodine atoms because these hormones are the
natural ligands of the TTR binding site where halogen binding
4
10
pockets have been defined. The strategy rendered one of our
5
Received: August 7, 2014
Revised: October 10, 2014
Published: November 13, 2014
diflunisal, were shown to slow down disease progression of
©
2014 American Chemical Society
32
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ACS Combinatorial Science
Research Article
The main feature for the preparation of the second library
was the use of Barluenga’s reagent, the iodonium complex
bis(pyridine)iodonium(I) tetrafluoroborate (IPy BF ), a versa-
2
4
16
tile and mild reagent to perform a wide range of reactions.
Toward upgrading the application of this versatile reagent for
1
7,18
combinatorial chemistry procedures,
here we describe the
lead compounds, iododiflunisal (IDIF), a small molecule with
negligible thyromimetic activity (affinity to thyroid hormone
nuclear receptors) and cyclooxygenase-1 (COX-1) activities
preparation and the first example of its use in parallel synthesis.
Briefly, after testing different immobilization materials and
methods, a simple method such as suspending a slurry of
poly(4-vinylpyridine) beads (PV-Py) in a solution of
Barluenga’s reagent afforded the functional material with high
iodonium load (PV-Py-I-Py in Scheme 1).
19
(
see Annex II in the Supporting Information). A first
optimization of our lead compound iododiflunisal (IDIF) was
conducted by systematic modifications on the functional groups
11
12
of its salicylic ring. Recently, we have also shown that
halogenation of diflunisal gradually improves binding up to one
order of magnitude from fluorine (see FDIF in Figure 1) to
Scheme 1. Synthesis of the Polymer Supported Iodonium
Reagent
Figure 1. (A) Our lead compound iododiflunisal (IDIF) and other
halogenated diflunisal derivatives. (B) Iododiflunisal modifications
proposed in this work on the nonsalicylic acid ring (inside the dashed
line).
The reactivity pattern of the polymer supported iodonium
reagent against aromatic standard substrates does not
significantly deviate from its soluble counterpart, but the
polymeric version facilitates product purification and avoids the
usual aqueous thiosulfate extractions required. The library of
diflunisal analogues (5-aryl salicylic acids) in the first step
readily reacted in a second reaction step with either soluble
IPy BF reagent or with the polymer-supported version of the
reagent. In this latter case, the reaction also proceeded
satisfactorily in ethanol or methanol rendering clear crude
reaction mixtures because the excess of supported reagent do
not readily decomposes into iodine as does the soluble
counterpart which always yields dark-brown solutions.
The two steps in Scheme 2 were used to obtain a discrete
library of both diflunisal and iododiflunisal analogues by parallel
synthesis. First, 108 commercial aryl boronic acids were
selected, having in common a single six membered aromatic
ring and showing a variety of mono-, di-, and trisubstitution
patterns resulting from the combination of groups such as
fluoro, chloro, bromo, methyl, trifluoromethyl, tert-butyl, ethyl,
isopropyl, phenyl, benzyl, silyl, methoxy, benzyloxy, phenoxy,
carboxyl, hydroxyl, amino, and acetamido (see Figure S3 in the
Supporting Information).
iodine substitution (see IDIF in Figure 1) through interactions
that can be interpreted as a suboptimal halogen bonding (XB)
or as rather optimized van der Waals contacts or as a mixture of
2
4
1
3
both. In this work, we report a close examination of the
substitution pattern of the 2,4-difluorophenyl unit on
iododiflunisal in order to fine-tune the amyloidogenic inhibition
properties of iododiflunisal (Figure 1).
RESULTS AND DISCUSSION
■
Toward this goal, a solution-phase parallel synthesis of two
libraries of in vitro fibrillogenesis inhibitory compounds was
envisaged to explore the chemical space around this ring by a
two steps retrosynthetic scheme (Figure 2).
To conduct the first step of the synthetic route, this is the
preparation of the first library of diflunisal analogues, an
aqueous Suzuki-Miyaura type of reaction was tested for the
14a,b
diflunisal analogues by adapting already known procedures.
The Suzuki-Beletskaya cross-coupling reaction in water at room
temperature using Pd(OAc) proved to be superior than the
Appropriate amounts of Pd(OAc) and individual boronic
2
2
organic solvent based alternatives already described for these
acids were weighed, and aliquots of aqueous and dioxane
solutions of the base (Na CO ) and 5-iodosalicylic acid,
14c,d
compounds
because of its milder conditions and avoidance
2
3
of a saponification step (see Scheme S1 in Supporting
Information).
respectively, were delivered and reacted in a Multiple Organic
Synthesizer. By including duplicated reactions of diflunisal
1
5
Figure 2. Retrosynthetic analysis of the libraries.
3
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ACS Combinatorial Science
Research Article
Scheme 2. (A) Aqueous Suzuki-Miyaura Cross-Coupling Reaction and (B) Electrophilic Aromatic Iodination Reaction Effected
by the Polymer-Supported Reagent (PV-Py-I-Py)
synthesis spread through the library series, accuracy and
reproducibility of the instrument were thus tested.
Table 1. Turbidimetric Kinetic Assay [RA(%) and IC
(μM)] Data for the Best 10 TTR Inhibitors (1−10)
5
0
a
HPLC analyses of the 108 compounds of the library showed
that 77 compounds were obtained with conversions in the
range of 80−100%. After discarding the other 31 low yielding
reactions, half of the mass of each of the 77 crude reaction
products was treated with an excess of the polymer-supported
iodonium reagent in methanol using the synthesizer. Most of
the HPLC conversions of the iodination reactions were within
the range of 60−80% (see Table S3 in the Supporting
Information).
compound
RA (%)
IC50
1
2
3
4
5
6
7
8
9
1
(200, IDIF)
(810)
92
77
92
87
99
92
87
95
87
81
90
83
4.1
5.7
5.5
5.2
3.3
5.0
6.1
5.6
9.5
8.9
5.4
19
(811)
(803)
(804)
(813)
(809)
(801)
The biological activity of the products of such library
composed of 77 noniodinated and 77 iodinated analogues has
been examined by testing their fibrillogenesis inhibition
properties on a high throughput screening turbidimetric kinetic
(807)
0 (805)
b
tafamidis
b
20
diflunisal (201)
assay that monitors TTR acid-induced fibrillogenesis (for full
data see Table S4 in the Supporting Information).
a
See Table S8 in Supporting Information. IC50 values are the median
of triplicate experiments with errors in the 7−10% experimental range.
Turbidimetric data on tafamidis and diflunisal are added for
Structure−Activity Relationships. Several trends can be
b
extracted from inspection of the IC values of all the tested
5
0
comparison purposes (in last two rows of the table).
compounds (see the Supporting Information):
(
a) Monosubstituted compounds at the nonsalicylic ring
(
with substituents such as halogens, acids, alcohols, ethers,
both good inhibitors and are selected in the group of the 10 top
inhibitors in the screening (Table 1). (d) Methoxy
substitutions on the nonsalicylic ring render poor inhibitors,
with no improvement upon iodination. If an halogen
substitution is also present, moderate inhibitors are obtained,
with the 2′-methoxy-5′-chloro compound 7 being in the group
of the 10 top inhibitors in the screening (Table 1).
Following the above observations, a subset of the 10 top best
inhibitors from the screening, having very close IC50 activity
values in the range of 3.7−10.0 μM, was selected. These
compounds were individually resynthesized, purified by using
conventional procedures and fully characterized, and rechecked
for fibrillogenesis inhibition (Figure 3 and Table 1). Tafamidis
was also included as a reference compound in these assays for
comparison purposes.
The lead compound, iododiflunisal (IDIF, 1) but not
diflunisal was found among the selected compounds. Two
pairs of twin iodinated/non iodinated analogues (2 and 3, and
4 and 5) and four other unpaired noniodinated compounds are
also in the group (7−10). The finding of these noniodinated
highly active analogues among the best inhibitors supports the
nitro derivatives, amines, and acetamide, among others, at
different positions of the residue on the nonsalicylic ring) are in
general bad or poor inhibitors. In those compounds iodination
has not a significant effect, except for compound 10 selected in
the group of the 10 top inhibitors in the screening (Table 1).
The carboxylic acid function in the nonsalicylic ring of 10 may
result in a different binding mode than the reference diflunisal
21
and iododiflunisal. (b) Disubstituted compounds in positions
′,5′- and 2′,4′- are moderate inhibitors, but iodination
3
consistently improves their inhibition properties (except for
the 3′,5′-dichlorinated analogues 8 and 4 that are already good
inhibitors, in those compounds iodination has essentially no
additional effect). The 3′,5′-dimethylated compound 4 and its
iodinated derivative 5, the 3′,5′-dichlorinated compound 8, and
the reference iododiflunisal (1, IDIF) are among the 10 best
inhibitors selected from this screening (Table 1). (c)
Disubstituted compounds in positions 2′,5′- are in general
poor or moderate inhibitors, and iodination does not improve
their inhibition properties. However, the 2′,5′-difluorinated
compound 2 and its corresponding iodinated derivative 3 are
3
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ACS Combinatorial Science
Research Article
Figure 3. Selection of a subset of the best TTR inhibitors identified (in parentheses, compound numbering corresponds to the code in our database).
hypothesis that both iodination and the substitution pattern in
the nonsalicylic ring are a crucial factor for activity.
CONCLUSIONS
■
By using a two step solution-phase parallel synthesis, two
related libraries of 77 diflunisal (5-aryl salicylic acids) and 77
iododiflunisal analogues (3-iodo-5-aryl salicylic acids) have
been obtained. A new solid supported iodonium reagent
These pure compounds were further studied for their
binding potency to TTR by measuring displacement of
21
radiolabeled thyroxine (T ) from TTR, which shows the
4
affinity of the different compounds for TTR. Values of relative
binding affinities thus obtained (data in Table S8 in Supporting
Information) are plotted in Figure 4. Combined data on the 10
prepared from Barluengas reagent (IPy BF ) has been used for
́
2 4
first time for polymer-assisted solution-phase parallel synthesis
of a library of TTR amyloid inhibitors related to iododiflunisal.
Thus, starting from 5-iodosalicylic acid and a set of 108
commercially available aryl boronic acids, the synthesis was
conducted by combining an aqueous Suzuki cross-coupling
methodology and an electrophilic aromatic substitution step
affected by the immobilized iodonium version of Barluenga’s
reagent. A selected library of 77 noniodinated diflunisal
analogues coming from the first reaction step and 77 iodinated
diflunisal analogues coming from the second one indicated that
the inhibition potency of diflunisal can be enhanced by
substitution patterns on its difluorophenyl ring such as mono-,
di-, or tri-substitutions, presenting polar (−OH and −COOH)
and nonpolar groups which can be either halogen atoms (−F
and −Cl) or simple aliphatic groups (−CH ). Although no
3
straightforward structure−activity relationships can be deduced
from the present study, it is apparent that iodination does not
generally improve the inhibition properties except for the
compounds with 2′,4′- and 3′,5′- substitution in the non-
salicylic ring. In addition, by changing the relative position and
the number of the fluorine atoms on this ring, we have
discovered the 3′,4′,5′-trifluoro substituted iodinated diflunisal
analogue 6 (813) which is the best inhibitor in this study
because it combines high binding affinity as measured by
competition for T and low IC on the TTR fibrillogenesis
Figure 4. Values of relative binding affinities EC50 (T displacement
assay) and IC50 (turbidimetric kinetic assay) of selected TTR
4
inhibitors (in pale blue, IC ; in violet, EC rel).
50
50
best compounds confirmed that the best compound was the
,5-dimethyl substituted analogue 5 (804) (3.3 μM), followed
3
by iododiflunisal (1-200, IDIF, 4.1 μM), and the trifluorinated
analogue 6 (813) (5.0 μM) (Figure 4 and Table S5 in the
Supporting Information).
4
50
The iodinated diflunisal analogues show far higher affinity for
TTR than the noniodinated. This is an indication that affinity
seems to be strongly regulated by the iodine atom on the
salicylic ring. In turn, this factor can be fine-tuned by the
relative position and number of fluorine atoms in the
difluorophenyl ring as shown by the trisubstituted fluorinated
analogue 6 (813) which presents the highest binding potency.
In an attempt to initially explore in a more realistic system
the interactions of these selected compounds with plasma
proteins, we have qualitatively studied their relative affinity for
inhibition test.
EXPERIMENTAL PROCEDURES
■
Compound Characterization. All compounds were
obtained in milligram quantities. Details of the synthesis and
characterization of each compounds is described in the
Supporting Information (Compound Characterization). Struc-
1
13
tures of all reported compounds were confirmed by H and C
NMR and their mass confirmed by HRMS. Purity was
determined by HPLC analysis. All reported compounds had
>95% purity. Selected spectra are included in the Supporting
Information (Annex I).
TTR, TBG, and ALB using human plasma in the T
4
2
2
displacement test. Interestingly, both iododiflunisal (1,
IDIF) and the trifluorinated derivative 6 show the same
pattern with good affinity for TTR, medium for TBG, and none
to ALB. The other compounds either have less affinity for TTR
or higher affinity for the other two proteins making them less
selective for TTR (see Table S9 of the Supporting
Information).
General Procedure for the Synthesis of 5-Aryl Salicylic
Acids from the Corresponding Aryl Boronic Acids and 5-
Iodosalicylic Acid (Aqueous Suzuki-Miyaura Coupling).
Under an argon atmosphere, 1.1 equiv of the arylboronic acid, 3
equiv of 2 M Na CO and 4-iodosalicylic acid (1 equiv) are
2
3
3
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ACS Combinatorial Science
Research Article
added to 10 mL of deaerated H O. The catalyst Pd(OAc) (1%
inhibitor prepared by mixing different volumes of a stock
2
2
mol) is added, and the reaction is stirred at rt for a period of 2−
solution of the compound in H O/DMSO (1:1) to give a range
2
4
h. The reaction is monitored by TLC and/or HPLC. The
of final compound concentrations of 0−40 μM and DMSO
content adjusted to a final 5% (v/v), where all ligands tested are
soluble. After 30 min incubation at 37 °C with 15 s shaking
every minute, 100 μL of 400 mM KAcO, 100 mM KCl, and 1
mM EDTA buffer at pH 4.2 were added to each well. The final
mixture, containing 0.4 mg/mL TTR, 0−40 μM ligand, and 5%
DMSO, was incubated at 37 °C with 15 s shaking every minute.
Absorbance at 340 nm was monitored for 1.5 h at 1 min
crude reaction is dissolved in 100−150 mL of H O or in a
2
H O/dioxane (2:1) mixture, filtered through Celite and
2
acidified to pH = 2 with a 1 N HCl solution until a white
precipitate is formed. The crude is separated either by
centrifugation or by extraction with ethyl acetate and dried in
a desiccator under P O . The crude is purified by silica gel
2
5
column chromatography.
General Procedure for Conversion of 5-Aryl Salicylic
intervals. Initial rates of protein aggregation (v ) were obtained
0
Acids to the Corresponding 5-Aryl-3-Iodosalicylic Acids
from the linear plot absorbance vs time. The dependence of v
on inhibitor concentration is defined as
0
with (a) IPy BF4 or (b) with the Polymer-Supported
2
Iodonium Reagent (PV-Py-I-Py). a. With IPy BF . To a
2
4
v = A + B exp−
C[I]
solution of the 5-aryl salicylic acid (1 equiv) in 4−10 mL of
0
(1)
CH Cl , a solution of the iodonium reagent IPy BF (1.5
2
2
2
4
where v is the initial rate of fibril formation (in absorbance
0
equiv) in 2−4 mL of the same solvent was added. The mixture
is stirred at rt for 1.5−2 h and the reaction monitored by
HPLC. After completion, water is added to the crude and
extracted, and the organic phases are washed with sodium
thiosulfate and brine. The organic phase is evaporated under
reduced pressure, and the crude is dried under reduced
pressure. In some cases the crude is purified by silica gel
column chromatography.
−
1
units per hour, AU h ) and [I] the concentration of the
inhibitor (μM).
From the adjustable parameters, the IC50 (inhibitor
concentration at which the initial rate of protein aggregation
is half than that in absence of inhibitor) and RA (%)
percentage reduction of amyloidosis at high inhibitor
concentration) were calculated.
(
Competition with T and Binding Selectivity Assays.
4
b. With the Polymer-Supported Iodonium Reagent (PV-
Py-I-Py). To a solution of 5-aryl salicylic acids (1 mmol) in
methanol (10 mL), polymer-supported iodonium reagent (3
mmol) was added and the mixture was allowed to stir at 50 °C.
The progress of the reaction was followed by HPLC. When the
reaction was completed, the resin was filtered and washed
successively with methanol. The solvent was evaporated, and
the pure product was obtained in moderate to high yields (60−
Binding to TTR is not the exclusive factor in determining the
therapeutic potential of new fribrillogenesis inhibitory com-
pounds. Binding specificity to plasma proteins can be a limiting
factor for biodistribution, metabolism, activity, and toxicity
profiles of any potential drug. This is especially crucial in this
case because very strong plasma protein competitors of TTR
include thyroxine-binding globulin (TBG), which has an order
of magnitude higher affinity for thyroxine, and albumin (ALB)
which is at concentrations of 2 orders of magnitude higher than
TTR in plasma.
80% yield).
Representative Examples: Compound (8 or 801) and
(
4 or 803). 3′,5′-Dichloro-4-hydroxy-[1,1′-biphenyl]-3-car-
Binding Competition between Compounds and T4.
To evaluate the relative binding affinity of the compounds for
TTR, an already described procedure was followed. Wild type
boxylic Acid (8 or 801). From 3′,5′-dichlorophenylboronic acid
(
0.209 g, 1.1 mmol), Na CO (0.318 g, 3 mmol),
2 3
5
-iodosalicylic (0.264 g, 1 mmol), and Pd(OAc) (0.002 g,
2
125
recombinant TTR was incubated with trace amounts of I-T4
0.01 mmol) yielded 0.238 g (84%). HPLC (GEN-1) RT: 13.46
1
in the presence of increasing amounts of test compounds.
min. H NMR (500 MHz; DMSO-d6) δ (ppm): 8.05 (d, J =
Protein bound 1 I-T was separated from the media by gel
25
4
2
2
.5 Hz, 1H), 7.86 (dd, J = 2.5, 9.0 Hz, 1H), 7.65 (d, J = 1.5 Hz,
H), 7.51 (t, J = 2.0 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H). 13
filtration and measured by scintillation. Competition curves
allowed calculating the relative T4 displacement potencies
C
NMR (125.7 MHz; DMSO-d6) δ (ppm): 171.4, 161.3, 142.6,
34.6, 133.9, 128.5, 128.2, 126.3, 124.8, 117.9, 113.7. MS
defined as EC50 of T /EC50 of the test compound for each
4
1
−
−
−
inhibitor.
(
ESI ) m/z 281 (M − H) . HRMS (ESI ): calcd for
−
Binding Selectivity for Plasma T
Human plasma was incubated with labeled T ( I-T ) in the
presence of each inhibitor. After separation of plasma proteins
by native polyacrylamide gel electrophoresis (PAGE),
qualitative T displacement binding from TBG, ALB, and
Binding Proteins.
4
[
C H Cl O −H] , 280.9772; found, 280.9771.
1
4
3
8
2
3
125
4
4
-Hydroxy-3′,5′-dimethyl-[1,1′-biphenyl]-3-carboxylic
Acid (4 or 803). From 3,5-dimethylphenylboronic acid (0.165
g, 1.1 mmol), Na CO (0.318, 3 mmol), 5-iodosalicylic acid
2
3
4
(
0.264 g, 1 mmol), and Pd(OAc) (0.002 g, 0.01 mmol)
2
1
TTR (main plasma T binding proteins) was measured either
4
yielded 0.178 g (73%). HPLC (GEN-1) RT: 11.16 min. H
by autoradiography or by phosphorimaging of the dried gel.
NMR (500 MHz; CD COCD ) δ (ppm): 8.12 (d, J = 2.5 Hz,
3
3
1
8
H), 7.81 (dd, J = 2.5, 8.5 Hz, 1H), 7.23 (m, 2H), 7.03 (d, J =
.5 Hz, 1H), 6.98 (m, 1H), 2.33 (s, 6H). 1 C NMR (125.7
3
ASSOCIATED CONTENT
■
MHz; CD COCD ) δ (ppm): 172.6, 162.3, 140.5, 139.1, 135.3,
1
calcd for [C H O −H] , 241.0865; found, 241.0853.
*
S
Supporting Information
3
3
−
33.3, 129.5, 129.1, 125.1, 118.6, 113.3, 21.4. HRMS (ESI ):
Synthesis and characterization of the combinatorial libraries of
diflunisal and iododiflunisal analogues, fibrillogenesis inhibition
parameters of compounds, data on TTR binding assays, and
selectivity assays of the best compounds; polymer-supported
iodonium reagent preparation and characterization; and
−
15
14
3
Kinetic Turbidimetric Assay. Inhibition of fibrillogenesis
was determined by the kinetic turbidimetric assay previously
2
0
reported. Briefly, in seven different wells of a 96-well
microplate, 20 μL of a 4 mg/mL TTRY78F solution in 20
mM potassium phosphate buffer, 100 mM KCl, and 1 mM
EDTA at pH 7.6 was mixed with 80 μL of a solution of
1
13
selected spectra (HPLC, H NMR, C NMR, HRMS) of the
best target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
3
6
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Research Article
misfolding energetics. Science 2003, 299, 713−716. (c) Wiseman, R.
L.; Johnson, S. M.; Kelker, M. S.; Foss, T.; Wilson, I. A.; Kelly, J. W.
Kinetic stabilization of an oligomeric protein by a single ligand binding
event. J. Am. Chem. Soc. 2005, 127, 5540−5551.
AUTHOR INFORMATION
Corresponding Author
Phone: + 34934006113. Fax: +34932045904. E-mail:
gregorio.valencia@iqac.csic.es.
■
*
(5) (a) Nencetti, S.; Orlandini, E. TTR fibril formation inhibitors: is
Notes
there a SAR? Curr. Med. Chem. 2012, 19, 2356−2379. (b) Connelly,
S.; Choi, S.; Johnson, S. M.; Kelly, J. W.; Wilson, I. A. Structure-based
design of kinetic stabilizers that ameliorate the transthyretin
amyloidoses. Curr. Opin. Struct. Biol. 2010, 20, 54−62. (c) Saraiva,
M. J. Hereditary transthyretin amyloidosis: molecular basis and
therapeutical strategies. Expert Rev. Mol. Med. 2002, 4, 1−11.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We acknowledge Dr. Jose
letting the use of the Multiple Organic Synthesizer for parallel
synthesis. This work was partially supported by grants, from
■
́
Ignacio Borrell (IQS-URL) for
(
d) Obici, L.; Merlini, G. An overview of drugs currently under
investigation for the treatment of transthyretin-related hereditary
amyloidosis. Expert Opin. Invest. Drugs 2014, 1−13. (d) Alhamadsheh,
M. M.; Connelly, S.; Cho, A.; Reixach, N.; Powers, E. T.; Pan, D. W.;
Wilson, I. A.; Kelly, J. W.; Graef, I. A. Potent kinetic stabilizers that
prevent transthyretin-mediated cardiomyocyte proteotoxicity. Sci.
Transl. Med. 2011, 3 (97), 97ra81. (e) Ferreira, N.; Saraiva, M. J.;
Almeida, M. R. Epigallocatechin-3-gallate as a potential therapeutic
drug for TTR-related amyloidosis: “in vivo” evidence from FAP mice
models. PLoS One 2012, 7 (1), e29933.
Fundacio
BIO2011-15163-E to A.P., and from CTQ2010-20517-C02-02
to G.A. from Ministerio de Ciencia e Innovacion (Spain). The
work was also partially supported by FEDER funds through
COMPETE and Fundaca para a Ciencia e Tecnologia (FCT)
Portugal) under the Project FCOMP-01-0124-FEDER-
́ ́
La Marato de TV3 (ref. 080530/31/32), from
́
̧
o
̃
̂
(
021281 (PTDC/SAU-ORG/116645/2010) to M.R.A.
(
6) (a) Coelho, T.; Maia, L. F.; da Silva, A. M.; Cruz, M. W.; Plante-
Bordeneuve, V.; Suhr, O. B.; Conceicao, I.; Schmidt, H. H.; Trigo, P.;
Kelly, J. W.; Labaudiniere, R.; Chan, J.; Packman, J.; Grogan, D. R.
́
DEDICATION
This paper is dedicated to Prof. Dr. Jose
birthday.
■
̧
́
Barluenga on his 74th
̀
Long-term effects of tafamidis for the treatment of transthyretin
familial amyloid polyneuropathy. J. Neurol. 2013, 260, 2802−2014.
(b) Bulawa, C. E.; Connelly, S.; Devit, M.; Wang, L.; Weigel, C.;
Fleming, J. A.; Packman, J.; Powers, E. T.; Wiseman, R. L.; Foss, T. R.;
Wilson, I. A.; Kelly, J. W.; Labaudiniere, R. Tafamidis, a potent and
̀
selective transthyretin kinetic stabilizer that inhibits the amyloid
cascade. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 9629−9634.
c) Nencetti, S.; Rossello, A.; Orlandini, E. Tafamidis (Vyndaqel): A
Light for FAP Patients. ChemMedChem 2013, 8, 1617−1619.
d) Razavi, H.; Palaninathan, S. K.; Powers, E. T.; Wiseman, R. L.;
Purkey, H. E.; Mohamedmohaideen, N. N.; Deechongkit, S.; Chiang,
K. P.; Dendle, M. T. A.; Sacchettini, J. C.; Kelly, J. W. Benzoxazoles as
Transthyretin Amyloid Fibril Inhibitors: Synthesis, Evaluation, and
Mechanism of Action. Angew. Chem., Int. Ed. 2003, 115, 2864−2867.
(7) (a) Assessment Report: Vyndaqel, European Medicines Agency:
London, September 22, 2011, http://www.ema.europa.eu/docs/en_
GB/document_library/EPAR_-_Public_assessment_report/human/
ABBREVIATIONS
■
ALB, albumin; AcOH, acetic acid; DMSO, dimethyl sulfoxide;
EDTA, ethylendiaminetetraacetic acid; EtOAc, ethyl acetate;
HRMS, high-resolution mass spectrometry; HPLC, high-
performance liquid chromatography; IDIF, iododiflunisal;
IPy BF , bis(pyridine)iodonium(I) tetrafluoroborate; KAcO,
(
2
4
−
potassium acetate; MS (ESI ), mass spectrometry (electrospray
ionization, negative mode); PV-Py, poly(4-vinylpyridine)
beads; PV-Py-I-Py, polymer-supported iodonium reagent; Rf,
(
retention factor; RT, retention time; T , thyroxine; TTR,
4
transthyretin; TBG, thyroxine-binding globulin; TFA, trifluoro-
acetic acid; TLC, thin layer chromatography; TTR, trans-
thyretin; Y78F, transthyretin mutant with Phe replacing Tyr at
position 78
0
02294/WC500117838.pdf (accessed July 26, 2013). (b) EPAR
Summary for the Public, http://www.ema.europa.eu/docs/en_GB/
document_library/EPAR_-_Summary_for_the_public/human/
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