Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors

Article abstract of DOI:10.1021/jm0708735

To develop potent transthyretin (TTR) amyloidogenesis inhibitors that also display high binding selectivity in blood, it proves useful to systematically optimize each of the three substructural elements that comprise a typical inhibitor: the two aryl rings and the linker joining them. In the first study, described herein, structural modifications to one aryl ring were evaluated by screening a library of 2-arylbenzoxazoles bearing thyroid hormone-like aryl substituents on the 2-aryl ring. Several potent and highly selective amyloidogenesis inhibitors were identified that exhibit minimal thyroid hormone nuclear receptor and COX-1 binding. High resolution crystal structures (1.3-1.5 ?) of three inhibitors (2f, 4f, and 4d) in complex with TTR were obtained to characterize their binding orientation. Collectively, the results demonstrate that thyroid hormone-like substitution patterns on one aryl ring lead to potent and highly selective TTR amyloidogenesis inhibitors that lack undesirable thyroid hormone receptor or COX-1 binding.

Full text of DOI:10.1021/jm0708735

260
J. Med. Chem. 2008, 51, 260–270
Biochemical and Structural Evaluation of Highly Selective 2-Arylbenzoxazole-Based
Transthyretin Amyloidogenesis Inhibitors
Steven M. Johnson,† Stephen Connelly,‡ Ian A. Wilson,‡ and Jeffery W. Kelly*,†
Departments of Chemistry and Molecular Biology, and The Skaggs Institute of Chemical Biology, The Scripps Research Institute,
BCC 265, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed July 19, 2007
To develop potent transthyretin (TTR) amyloidogenesis inhibitors that also display high binding selectivity
in blood, it proves useful to systematically optimize each of the three substructural elements that comprise
a typical inhibitor: the two aryl rings and the linker joining them. In the first study, described herein, structural
modifications to one aryl ring were evaluated by screening a library of 2-arylbenzoxazoles bearing thyroid
hormone-like aryl substituents on the 2-aryl ring. Several potent and highly selective amyloidogenesis
inhibitors were identified that exhibit minimal thyroid hormone nuclear receptor and COX-1 binding. High
resolution crystal structures (1.3–1.5 Å) of three inhibitors (2f, 4f, and 4d) in complex with TTR were
obtained to characterize their binding orientation. Collectively, the results demonstrate that thyroid hormone-
like substitution patterns on one aryl ring lead to potent and highly selective TTR amyloidogenesis inhibitors
that lack undesirable thyroid hormone receptor or COX-1 binding.
Introduction
initially.7–9 In addition to the requirement for life-long immune
suppression, another drawback of this strategy is that WT-TTR
deposition often continues post-transplantation in the heart,
leading to cardiomyopathy.10 Liver transplantation is not
envisioned to be effective for the treatment of SSA because
this disease is caused by WT-TTR deposition. Because of these
limitations, an alternative, generally applicable, oral small
molecule therapy for all the TTR-based amyloid diseases is
highly desirable.4
Transthyretin (TTRa) is one of more than 25 disease-
associated secreted human proteins that misfolds and misas-
sembles into a variety of extracellular aggregate morphologies,
including fibrillar cross-ꢀ-sheet structures known as amyloid.
The native, tetrameric structure of TTR must first dissociate
for the resulting monomers to partially unfold and misassemble
into aggregates, including amyloid.1,2 Substantial genetic and
biochemical evidence link the process of TTR amyloid forma-
tion (amyloidogenesis), involving numerous assembly interme-
diates of variable morphology, to the tissue degeneration,
including neurodegeneration, prevalent in senile systemic amy-
loidosis (SSA), familial amyloid cardiomyopathy (FAC), fa-
milial amyloid polyneuropathy (FAP), and central nervous
system selective amyloidosis (CNSA).1,3–6 FAP, FAC, and
CNSA are familial diseases caused by the aberrant amyloido-
genesis of one of >100 destabilized TTR mutants. These
diseases can present as early as the second decade of life but
typically present in the fourth to fifth decade. In contrast, SSA
is a late onset disease associated with aggregation of wild type
transthyretin (WT-TTR), typically presenting in the seventh to
eighth decade. Without treatment, the TTR amyloidoses are fatal.
Transthyretin transports the vitamin A-retinol binding protein
(RBP) complex and thyroxine (T4) in blood. Because of the
high concentration of TTR and the presence of two other
thyroxine carrier proteins (thyroxine binding globulin and
albumin), <1% of the thyroxine binding sites in the TTR
tetramers are bound to T4. TTR is composed of 127-amino acid,
ꢀ-sheet-rich subunits that associate into a homotetrameric
quaternary structure exhibiting two unique dimer–dimer inter-
faces (Figure 1).4,11–15 The energetically weaker interface creates
two funnel-shaped thyroxine binding pockets. Allosteric com-
munication between the two binding sites causes thyroxine to
bind with negative cooperativity, the structural basis of which
is not entirely clear.4,16,17 Each T4 binding site contains three
pairs of symmetric hydrophobic depressions, referred to as the
halogen binding pockets (HBPs), wherein the iodine atoms of
T4 reside (Figure 1).18 One pair is located in each of the smaller
inner and larger outer binding cavities, while the third is found
at the interface between the two cavities.
The liver secretes TTR into the blood, which later is
envisioned to undergo amyloidogenesis. The only treatment
currently available for FAP is gene therapy mediated by liver
transplantation, whereby the FAP mutant TTR/WT-TTR secret-
ing liver is replaced by a WT-TTR/WT-TTR secreting liver.
This procedure dramatically decreases plasma mutant TTR
levels and halts disease progression in most patients, at least
Numerous small molecules, typically composed of two
differentially substituted aromatic rings connected by linkers
of variable chemical composition (Figure 2), are known to avidly
bind to the unoccupied T4 sites within TTR (displaying either
positive-, non-, or negative-cooperativity).4 One aryl ring
typically bears polar substituents that enable electrostatic
interactions with the Lys-15 ꢀ-NH3+ and/or the Glu-54 carboxy
groups positioned at the periphery of the outer cavity of the T4
binding site. Alternatively, these groups can hydrogen bond with
the Ser-117 or Thr-119 hydroxyls in the inner cavity when
bound in the opposite binding orientation. The other aryl
substructure generally displays halogen or alkyl substituents that
* To whom correspondence should be addressed. Tel.: 858-784-9605.
Fax: 858-784-9610. E-mail: jkelly@scripps.edu.
†
Department of Chemistry.
Molecular Biology.
‡
aAbbreviations: TTR, transthyretin; COX-1, cyclooxygenase-1; SSA,
senile systemic amyloidosis; FAC, familial amyloid cardiomyopathy; FAP,
familial amyloid polyneuropathy; CNSA, central nervous system amyloi-
dosis; RBP, retinol binding protein; HBP, halogen binding pocket; PGE2,
prostaglandin E2; T4, thyroxine; T3, triiodothyronine; NSAID, nonsteroidal,
anti-inflammatory drug. PDB ascession codes: 2QGB, 2QGC, 2QGD,
2QGE.
10.1021/jm0708735 CCC: $40.75
2008 American Chemical Society
Published on Web 12/21/2007

Transthyretin Amyloidogenesis Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 261
Figure 2. Schematic depiction of one of the two T4 binding pockets
within TTR occupied by a typical small molecule TTR aggregation
inhibitor. Y represents a linker of variable chemical structure (e.g., NH,
O, CHdCH, C(O)NH, etc.) joining the two aryl rings, which typically
bear a combination of alkyl, carboxyl, halide, trifluoromethyl, or
hydroxyl substituents (X and Z). Library 1, being the focus of this
manuscript, explores the SAR of the aryl-X substructure.
there is good reason to be optimistic that small molecule TTR
kinetic stabilizers will be efficacious against TTR amyloid
disease. This strategy, called interallelic trans-suppression, is
observed in compound heterozygotes having one allele coding
for the disease-associated V30M-TTR variant (associated with
high FAP penetrance) and the other allele coding for T119M-
TTR. Inclusion of T119M subunits into heterotetramers other-
wise composed of V30M subunits kinetically stabilizes TTR,
preventing tetramer dissociation, amyloidogenesis, and the onset
of FAP symptoms.4,19,21–23 Besides demonstrating the efficacy
of kinetic stabilization for ameliorating transthyretin amyloid
disease, this data provides strong evidence for the amyloid
hypothesis as the basis for pathology, that is, the notion that
TTR dissociation, misfolding, and misassembly cause the TTR
amyloidoses.23
The efficacy of candidate small molecule transthyretin
amyloidogenesis inhibitors is typically evaluated by using a
simple turbidity assay (turbidity increases with protein aggrega-
tion). Screening and structure-based drug design have enabled
the evaluation of more than 650 candidate TTR amyloidogenesis
1
inhibitors.4,18,20,24–36 Of these, ∼ /3 are potent inhibitors of acid-
mediated aggregation of TTR in vitro (described in more detail
below), that is, they allow <10% of the aggregation (>90%
inhibition) exhibited by inhibitor-free control samples over a
72 h time course. Potent inhibition of TTR amyloidogenesis in
vitro is necessary, but not sufficient, for efficacy in humans:
the small molecule TTR kinetic stabilizers must also bind
selectively to TTR over all of the other proteins in blood.37 Of
the ∼200 compounds exhibiting >90% inhibition of TTR
Figure 1. Crystal structure (2ROX) of thyroxine (T4) bound within the two
pockets created by the weak dimer–dimer interface of tetrameric TTR (the
two dimer–dimer interfaces are represented by the dashed lines).18 The
expanded view of one site shows T4 bound in its symmetry-related binding
modes (green and yellow), with the binding site surface shown in gray (figure
adapted from ref 18). Primed amino acids or halogen binding pockets (HBPs)
refer to symmetry-related monomers of TTR. Two structural series of inhibitors
are shown above, the bisaryloxime ethers and biphenyls (R ) variable
substitution patterns of fluorine, chlorine, trifluoromethyl, carboxyl, or hydroxyl
substituents), many of which are excellent TTR kinetic stabilizers that display
high plasma TTR binding selectivity.25
1
amyloidogenesis in vitro, only ∼ /4 of these display high binding
selectivity in plasma (>1.0 out of a maximum of 2 equiv of
inhibitor bound per tetramer).37
To develop an effective clinical candidate, it would be
advantageous to have a number of structurally diverse, potent,
and highly selective TTR kinetic stabilizers. The majority of
the potent and selective TTR kinetic stabilizers are found in
three structural families: bisaryloxime ethers, biphenyls, and
1-aryl-4,6-biscarboxydibenzofurans (∼20, 30, and 40%, respec-
tively); the remaining 10% are based on 2-phenylbenzoxazole
or biphenylamine substructures.18,25,27,31–33,36 Unfortunately,
many of the most potent and selective bisaryloxime ethers have
lower than desirable chemical stability,25 leaving the biphenyls
and dibenzofuran inhibitors as primary candidates. The lack of
selective, structurally diverse clinical candidates is in large part
due to the fact that there has been no systematic optimization
of the three structural elements composing a typical small
molecule TTR amyloidogenesis inhibitor, that is, the two
variably substituted aromatic rings and the linker joining them
(Figure 2). Consequently, we have developed three small
complement the hydrophobic HBPs positioned in the inner or
outer cavities of the T4 binding sites.
Small molecule binding to the T4 sites can noncovalently
bridge neighboring monomeric subunits via specific hydrophobic
and electrostatic interactions, stabilizing the weaker dimer–dimer
interface (Figure 1). This imposes kinetic stability on the native
quaternary structure of TTR, precluding rate-limiting tetramer
dissociation and, hence, amyloidogenesis from commen-
cing.4,13,15,19,20 Because genetic studies have uncovered a
mechanistically analogous strategy that prevents human disease,

262 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Johnson et al.
Figure 3. Inhibition of TTR aggregation and stoichiometry of benzoxazole bound to TTR in human blood plasma. Percent (%) values represent
the extent of in vitro WT-TTR fibril formation in the presence of inhibitor (7.2 µM inhibitor, 3.6 µM TTR, pH 4.4, 37 °C, 72 h) relative to
aggregation in the absence of inhibitor (100%), with the best values shown in red (<20% aggregation; errors are typically less than (5 percentage
points). The average stoichiometries of the most potent aggregation inhibitors bound to TTR in human blood plasma ex vivo are shown in italics
(10.8 µM inhibitor, 1.8–5.4 µM TTR; maximum binding stoichiometry 2) with those exhibiting exceptional binding selectivity to TTR boxed
(errors are typically less than (0.1).
molecule libraries to systematically optimize these substructures
to create potent and especially highly selective TTR amyloido-
genesis inhibitors. The structure–activity relationship (SAR) data
from these libraries will ultimately be utilized to predict the
structures of diverse, potent, and highly selective TTR amy-
loidogenesis inhibitors, which should increase the chances of
developing a successful clinical candidate.
is impressive considering that potent inhibition generally requires
two differentially substituted aryls connected by a linker.4,18,20,24–36
It is also known that incorporation of the 3,5-dihalo-4-
hydroxyphenyl substructure into bisaryloxime ether and biphe-
nyl-based inhibitors (Figure 1, bottom) yields potent TTR
amyloidogenesis inhibitors that exhibit selective binding to
plasma TTR, almost irrespective of the substituents on the other
aryl ring.25,32 Collectively, these data suggest that it is likely
that appending a 3,5-dihalo-4-hydroxyphenyl aryl ring, or a
mimetic thereof, to a variety of molecules could render them
potent and selective TTR kinetic stabilizers; hence, we focused
on similar substituents and substitution patterns (Figure 3). Aryl
rings substituted in this fashion were envisioned to interact
within the inner cavity of the T4 binding site (much like the
analogous aryl ring in thyroxine, Figure 1). Optimization of the
composition of the aryl-X ring will enable these aryl-X
substructures to be utilized in future studies that seek ideal aryl-Z
ring and linker-Y substructures (Figure 2). Data generated in
this study can be combined with future analogous data on
optimal aryl-Z and like substructures to predict the structures
of exceptionally potent and selective TTR amyloidogenesis
inhibitors.
The synthesis and evaluation of the first library, designed to
optimize the aryl-X ring (Figure 2), is presented herein. In this
exercise, the 2-aryl ring of an otherwise unsubstituted 2-aryl-
benzoxazole scaffold is modified with various substituents to
evaluate the influence of thyroid hormone-like aryl substructures
(3,4,5-substituted aryls, Figure 3) on transthyretin amyloido-
genesis and selective binding to TTR in blood plasma. Because
amelioration of transthyretin amyloidosis (SSA, FAP, and FAC)
is envisioned to require life-long therapy with TTR small
molecule kinetic stabilizers, it is essential that these drugs
minimally interfere with other biological processes to mitigate
toxicity. Therefore, we have further evaluated the potent TTR
amyloidogenesis inhibitors identified here for their ability to
bind to the nuclear thyroid hormone receptor, as well as inhibit
cyclooxygenase-1 (COX-1) activity. Effective clinical candidates
should be neither thyroid hormone agonists/antagonists, nor
should they inhibit COX-1 enzymatic activity because FAP/
FAC patients typically have amyloid deposition in the kidneys
and gastro-intestinal tract, which compromises function and can
be exacerbated by COX-1 inhibition.38
In 2003, we demonstrated that 2-arylbenzoxazole-based
compounds are potent TTR kinetic stabilizers in vitro that
display the full range of binding selectivity in human plasma,
affording stoichiometries from near zero to the theoretical
maximum of two, depending on the substituents and substitution
patterns on the 2-phenyl and benzoxazole substructures.33
Therefore, substituting a variety of aryls at the 2-position of an
otherwise unsubstituted benzoxazole scaffold should enable
optimization of the aryl-X ring, as discussed above. A 42-
member library of 2-arylbenzoxazole compounds, with variation
in the 2-aryl ring, was synthesized as described previously
(Scheme 1, Figure 3).33,39 Compounds were characterized by
Herein, we report potent and highly selective TTR amyloido-
genesis inhibitors bearing thyroid hormone-like substitution
patterns on the aryl-X ring that, in general, do not bind to the
COX-1 enzyme or the thyroid hormone receptor. Further
evaluation by X-ray crystallography provides interesting insight
into the structural basis for the ability of several of these potent
inhibitors to bind to TTR.
Design and Synthesis. Previous studies reveal that 2,4,6-
triiodophenol is a potent TTR amyloidogenesis inhibitor, which

Transthyretin Amyloidogenesis Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 263
Scheme 1. General Synthesis of the Thyroid Hormone-Like
excess of ∼1 equiv bound per tetramer (4c-f), with the
remainder displaying <0.5 equiv bound.
2-Arylbenzoxazolesa
Inhibition of COX-1 Enzymatic Activity by the Potent
TTR Amyloidogenesis Inhibitors. The 13 potent TTR ag-
gregation inhibitors were further evaluated for their ability to
inhibit the COX-1 enzyme (analyses that were contracted out
to the Cerep laboratories in Redmond, WA). Compound analyses
were performed using assay catalog reference #777-1 hr, which
uses procedures developed by Glaser et al.42 Briefly, the COX-1
enzyme (∼2 µg in 250 µL of buffer) is preincubated in the
absence or presence of test compound (10.0 µM) for 20 min at
22 °C. Arachidonic acid (4 µM) is then added, which undergoes
catalytic conversion by COX-1 to afford prostaglandin-E2
(PGE2). After 10 min, the reaction is quenched by addition of
2 M HCl followed by 1 M Tris-HCl (pH 7.8) and cooling to 4
°C. PGE2 is then quantified using an EIA detection kit. Results
represent the % inhibition of arachidonic acid conversion to
PGE2 due to competitive binding of test compound to COX-1
(Figure 4, lower, black values). Of the 13 compounds evaluated,
all but three display <5% inhibition of COX-1 activity, which
is important to mitigate toxicity in the FAP/FAC patients for
which inhibition of COX-1 enzymatic activity (anti-inflamma-
tory agents) is contraindicated. Compounds 2d, 2f, and 4e
display 11–18% COX-1 inhibition.
a (A) (a) pyridine, xylenes, rt, 1 h, then p-TsOH·H2O added and refluxed.
(B) (b) p-TsOH·H2O, refluxing xylenes; (c) (i) N,N′-carbonyldiimidazole
with ArCO2H in THF, 15 min, rt, then 2-aminophenol added and refluxed;
(ii) p-TsOH·H2O, refluxing xylenes. X1 ) H or X2; X2 ) H, F, Cl, Br, I,
CH3, CF3, OH, OCH3; and X3 ) H, OH, OCH3.
1H and 13C NMR spectroscopy, by mass spectrometry, and by
RP-HPLC (all display >95% purity).
Binding of TTR Amyloidogenesis Inhibitors to the
Thyroid Hormone Nuclear Receptor. Because the compounds
under investigation bear thyroid hormone-like 2-aryl substruc-
tures, the potent TTR amyloidogenesis inhibitors were further
evaluated for their ability to interact with the thyroid hormone
receptor (analyses that were contracted out to the Cerep
laboratories in Redmond, WA). Compound analyses were
performed using assay catalog reference #855, which uses
procedures developed by Inoue et al.43 Briefly, liver membrane
homogenates (100 µg protein in 500 µL of buffer) were
incubated for 18 h at 4 °C with 0.1 nM 125I-labeled triiodot-
hyronine ([125I]T3, the primary thyroid hormone, with a binding
IC50 ) 0.38 nM) in the absence or presence of test compound
(10.0 µM). The samples were vacuum filtered through glass
fiber filters, which were then rinsed several times with ice-cold
buffer, dried, and counted for radioactivity. Nonspecific binding,
determined in the presence of 1 µM T3, is subtracted from the
Results
Evaluating 2-Arylbenzoxazole-Mediated Inhibition of
WT-TTR Amyloidogenesis. The ability of candidate 2-aryl-
benzoxazoles to inhibit WT-TTR amyloidogenesis was evalu-
ated using the established acid-mediated aggregation assay.40
Briefly, physiologically relevant concentrations of WT-TTR (3.6
µM) preincubated with a candidate inhibitor (7.2 µM) were
subjected to partial denaturation under conditions that enable
∼90% of WT-TTR to aggregate in the absence of inhibitor after
72 h (pH 4.4, 37 °C). The extent of aggregation is monitored
by measuring sample turbidity, which has previously been
demonstrated to be equivalent to monitoring amyloidogenesis
by thioflavin-T fluorescence.41 Inhibitor potency is expressed
as % aggregation at the 72 h time point (compared to
aggregation in the absence of inhibitor) with 0% aggregation
equating to 100% inhibition. For the purposes here, we have
defined a potent inhibitor as allowing <20% TTR aggregation;
13 compounds proved to be potent inhibitors of WT-TTR
aggregation by this definition (Figure 3, red data). Although
the series 1, 5, and 6 compounds are poor kinetic stabilizers of
TTR, several compounds in the 2-4 series proved to be potent
TTR amyloidogenesis inhibitors.
Binding Selectivity of Amyloidogenesis Inhibitors to
TTR in Human Blood Plasma. The 13 potent 2-arylbenzox-
azole aggregation inhibitors were further evaluated for their
ability to bind selectively to TTR in the blood using the
published ex vivo TTR plasma binding selectivity assay.37
Briefly, the candidate 2-arylbenzoxazole (10.8 µM) is incubated
in human blood plasma in the dark at 37 °C for 24 h.
Transthyretin, with any bound inhibitor, is then captured by a
resin-conjugated anti-TTR antibody and any unbound material
is washed away (including weakly or nonspecifically bound
inhibitors). The captured TTR ·(inhibitor)n complex is then
dissociated from the antibody under alkaline conditions and
quantified by RP-HPLC. The results represent the average
stoichiometry of inhibitor bound per TTR tetramer (Figure 3,
lower italicized values), the theoretical maximum being 2, owing
to the two thyroxine binding sites. Of the 13 potent aggregation
inhibitors evaluated, four display binding stoichiometries in
[
125I]T3 binding results. Results represent the % displacement
of [125I]T3 due to competitive binding of test compound to the
thyroid hormone receptor (Figure 4, red, italicized values). Of
the 13 compounds evaluated, all but one display insignificant
(<5%) inhibition of T3 binding to the thyroid hormone nuclear
receptor, important for avoiding toxicity in the intended long-
term use. Compound 4e displays 17% inhibition of T3 binding,
a compound also displaying COX-1 binding capacity.
X-ray Crystallographic Analysis of WT-TTR Bound to
Inhibitors 2f, 4d, and 4f. Crystal structures of apo-WT-TTR
and TTR in complex with compounds 2f, 4d, and 4f were
determined to 1.4, 1.45, 1.5, and 1.3 Å resolutions, respectively.
While the observed electron density for inhibitor 2f was weaker,
the electron density for the other two inhibitor-containing
structures was clear and allowed unambiguous placement of the
ligands. Inhibitors 2f and 4f bind in what is referred to as the
“forward” mode, with their 3,5-dimethylphenyl and 3,5-dimeth-
yl-4-hydroxyphenyl substructures, respectively, occupying the
inner cavity of the thyroxine binding site (Figure 5). As such,
their methyl substituents extend into the two halogen binding
pockets located in the inner binding cavity (HBP-3 and 3′). In
the 4f structure, the Ser-117 and 117′ side chains of adjacent
TTR subunits are bridged through hydrogen bonding with the

264 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Johnson et al.
Figure 4. Thyroid hormone receptor binding and COX-1 inhibition data for the most potent TTR kinetic stabilizers. The extent of competitive
inhibitor binding to the thyroid hormone receptor is shown in red italics, representing the % displacement of 125I-labeled triiodothyronine (T3, the
primary thyroid hormone; errors are typically less than (2 percentage points). COX-1 inhibition results are shown below in black, with values
representing the % inhibition of COX-1 mediated conversion of arachidonic acid to prostaglandin-E2 by the test compounds (errors are typically
less than (6 percentage points).
4-OH substituent of inhibitor 4f. In the 2f structure, an ordered
water molecule replaces the missing phenolic group. In contrast
to the forward binding mode exhibited by inhibitors 2f and 4f,
inhibitor 4d binds in the “reverse” binding mode, whereby its
bromines extend into the outer cavity HBPs-1 and 1′ (Figure
5) and the phenolic substituent interacts with the Lys-15 and
may be a result of the 3,5-X2 substituents being able to
efficiently interact with two HBPs on adjacent TTR subunits
simultaneously, whereas mono-meta-substitution enables oc-
cupancy of only one HBP and is, therefore, less efficient at
stabilizing the weaker dimer–dimer interface.
The contribution of the 4-OH group (series 3 and 4) to high
binding affinity is emphasized by the increased potencies of
the series 3 and 4 compounds over the series 1 and 2
compounds. The phenolic group can make hydrogen bonding
interactions that bridge the Ser-117 and 117′ hydroxyls (Figure
5), as demonstrated in the 4f cocrystal structure. The H-bonding
enabled by 4f leads to higher affinity relative to 2f, which lacks
a phenolic substituent, and is consistent with the weaker electron
density for 2f that results in higher B-values (Table 1).
Interestingly, the isostructural compound 4d binds in an
orientation opposite to that of 4f. In the TTR·(4d)2 structure,
the phenolic group can now engage in electrostatic interactions
+
15′ ꢀ-NH3 groups. In all three structures, the 2-aryl ring is
bound rigidly in either the inner or outer cavities (2f/4f and 4d,
respectively), while occupancy by the benzoxazole rings show
greater variability. Although ordered water molecules are present
within each structure, these do not make any significant
interactions with any of the bound inhibitors.
Discussion
Aryl Substructures Leading to Potent WT-TTR Aggre-
gation Inhibitors. The specific interactions made by the rigidly
positioned substituted 2-aryl rings (aryl-X) in the 2d, 4d, and
4f amyloidogenesis inhibitor/TTR cocrystal structures likely
contribute the most to the binding affinity and specificity, while
the unsubstituted benzoxazole aromatic appears to contribute
less. Although the potentially unpredictable binding orientations
of the 2-arylbenzoxazoles complicate a detailed structure–ac-
tivity relationship (SAR) interpretation, several trends are evident
even in the absence of structural data for each ligand (Figure
3). Two dominant ligand-protein interactions contribute to
inhibitor potency: the hydrophobic/electrostatic interactions of
the 3,5-X2 substituents and the electrostatic interactions of the
4-OH group. The most potent inhibitors, containing the thyroxine-
like 3,5-X2-4-OH 2-aryl substitution pattern (series 4, Figure
3), enable both hydrophobic and electrostatic interactions.
The hydrophobicity of the 3,5-substituents is known to play
a role in the binding affinity of ligands to transthyretin; increased
hydrophobicity generally leads to increased binding affinity and,
therefore, increased inhibitor potency.44 Consistent with this
interpretation, hydrogen and fluorine substituents tend to afford
poorer aggregation inhibitors than their chloro, bromo, iodo,
and methyl counterparts due to less-efficient interactions with
the HBPs. Halogens are known to contribute more to ligand
binding affinity than would be expected based on their hydro-
phobicity alone.44,45 Formation of a charge-transfer complex
between the halogen and a neighboring oxygen atom could be
the basis for enhanced inhibitor binding.44,45 Bis-meta-substitu-
tion appears superior to mono-meta-substitution, as exemplified
by the increased potency of several of the series 2 (3,5-X2)
compounds over the series 1 compounds (3-X only). This effect
+
with the Lys-15 and 15′ ꢀ-NH3 groups. We posit that the
binding orientation differences of 4d and 4f are caused by the
different inductive effects of the bromine and methyl substit-
uents. With 4f, the electron-donating methyls result in a higher
pKa for the phenolic group, such that it is protonated (pKa calcd
to be 9.21; refer to Figure S2 in the Supporting Information),46
favoring hydrogen bonding with the Ser-117 and 117′ hydroxyls.
In contrast, the electron-withdrawing bromines of 4d appear to
afford the phenoxide ion (pKa calcd tothe importance of phenol
ionization. Interestingly, O-methylation of 4f to give analogue
6f does not cause such a dramatic decrease in activity, reflecting
the fact that our preliminary explanation may be oversimplistic.
Aryl Substructures Conferring Selective Binding to
TTR in Human Blood. 2-Arylbenzoxazoles that have the
highest affinity for recombinant TTR in buffer are not neces-
sarily the most selective TTR binders in the blood because
binding to other prominent plasma proteins, such as albumin,
is possible. An ideal clinical candidate should bind selectively
to TTR in the blood to mitigate off-pathway toxicity. Therefore,
the TTR binding selectivities of the 13 potent 2-arylbenzoxazole
inhibitors displaying <20% fibril formation were evaluated
using the previously established TTR binding selectivity assay.
Since only the binding stoichiometry of the inhibitor to TTR is
evaluated in this assay, we cannot comment on selectivity as it
relates to inhibitor binding to other specific plasma proteins,
which limits a detailed interpretation of the primary factors
governing TTR binding selectivity.

Transthyretin Amyloidogenesis Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 265
Figure 5. X-ray crystallographic structures of homotetrameric WT-TTR unliganded (PDB ascession code 2QGB) and cocrystallized with inhibitors 2f, 4f, and
4d (PDB ascession codes 2QGE, 2QGC, and 2QGD, respectively). (A) Three-dimensional ribbon diagram of apo-TTR (top) with zoomed images of inhibitors
2f, 4f, and 4d shown bound in one of the two symmetrical thyroxine binding sites. Individual TTR monomers have been colored red, yellow, green, and blue for
differentiation. Inhibitors are shown bound in their two symmetry-related binding modes (orange and grey) and appear superimposed on one another due to their
positioning on the crystallographic C2 axis. The ordered water molecule bridging the Ser117 and 117′ hydroxyls of adjacent TTR monomers in the TTR ·(2f)2
structure is shown as a red sphere. Important hydrogen bonding and electrostatic interactions between residue side chains and the ligands (or H2O as in the
TTR·(2f)2 structure) are indicated by arrows, with distance measurements in Å. (B) Schematic representation of the bound inhibitors as presented in panel A
shown as two-dimensional topology diagrams (generated using MOE (2006.08), Chemical Computing Group, Montreal, Canada). Inhibitors have been shown in
only one of their symmetry binding modes for clarity. A graphical legend for interpretation of key binding site characteristics is displayed below. Ligand exposure
indicates specific portions of the ligand structures that are solvent accessible (i.e., not completely buried within the binding pocket). Residue exposure indicates
those amino acids for which their side chains and/or peptide backbones are partially solvent accessible.
The 3,5-X2-4-hydroxyphenyl substitution pattern (X ) Cl,
Br, I, and CH3) in the series 4 inhibitors enables highly selective
binding to TTR, with the exception of 4b. These exemplary
binding stoichiometries emphasize the synergistic interactions
between the 3,5-X2 and the 4-OH substituents, which enhance
inhibitor binding affinity for reasons discussed above. That
inhibitor binding affinity and selectivity can be further enhanced
by adding substituents on the benzoxazole is encouraging for
future lead development.33
Thyroid Hormone-Like 2-Arylbenzoxazoles Minimally
Interact with the COX-1 Enzyme and the Thyroid
Hormone Nuclear Receptor. To mitigate off-target toxicity,
TTR small molecule kinetic stabilizers should not bind to
COX-1 or the nuclear thyroid hormone receptor. Because many
of these compounds resemble nonsteroidal, anti-inflammatory
drugs (NSAIDs), which are contraindicated for treating FAP
patients, we evaluated their COX-1 activity. The majority of
the amyloidogenesis inhibitors display little, if any, inhibition
of COX-1 activity (Figure 4, lower black entries).42 The
exceptions are compounds 2d, 2f, and 4e, which display some
COX-1 activity. The lack of COX-1 enzymatic inhibition by
the majority of these compounds may be due to the absence of
a carboxylate substituent, which is generally thought to be
important for high affinity binding to the enzyme.47–50
Because the most potent 2-arylbenzoxazole TTR amyloido-
genesis inhibitors have the 3-X-4-OH and 3,5-X2-4-OH aryl
substructures, as found in tri-iodothyronine (T3, the primary
thyroid hormone) and thyroxine (the prohormone), respectively,
these were evaluated for their ability to interact with the nuclear
thyroid hormone receptor. The T3 competition binding assay
indicates that, with the exception of 4e (bearing the exact 3,5-
I2-4-hydroxyphenyl substitution pattern of thyroxine), the major-
ity of the most potent TTR amyloidogenesis inhibitors do not
interact significantly with the thyroid hormone receptor (Figure
4, red, italicized values).43 The minimal thyroid hormone
receptor binding is likely due to the lack of the carboxylic acid
side chain that is present in T3, which is important for polar
interactions within the thyroid hormone receptor.52–55 Addition-
ally, the 3,5-X2 aryl substitution pattern presumably decreases
ligand binding affinity due to steric interaction of the 5-sub-

266 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Johnson et al.
Table 1. Data Collection and Refinement Statistics for the Crystal Structures of TTR in apo-Form and in Complex with Inhibitors 2f, 4d, and 4f
apo-WT-TTR
WT-TTR/2f
SSRL 11–1
0.9795
1.45 (1.45–1.50)
P21212
WT-TTR/4d
SSRL 11–1
0.9195
1.50 (1.50–1.53)
P21212
WT-TTR/4f
SSRL 11–1
0.9795
1.30 (1.30–1.35)
P21212
Data Collection
beamline
wavelength (Å)
resolution (Å)
SSRL 11–1
0.9795
1.40 (1.40–1.43)a
P21212
space group
a, b, c (Å)
42.85, 85.40, 64.35
2
321624 (20703)
47065 (3090)
99.4 (100)
4.5 (45.9)
42.92, 85.78, 64.56
2
294859 (43361)
42964 (6318)
99.9 (100)
4.0 (56.7)
42.95, 85.00, 65.10
2
55736 (38439)
38438 (2650)
99.9 (100)
6.2 (67.0)
43.00, 85.41, 64.42
2
419806 (40887)
59153 (5841)
99.9 (100)
3.6 (55.8)
No. of molecules in the a.u.
No. of observations
No. of unique reflections
completeness (%)
Rsym (%)b
avg I/σ
redundancy
32.4 (4.2)
6.8 (6.7)
48.5 (3.4)
6.9 (6.8)
39.2 (4.5)
14.5 (14.5)
51.7 (3.8)
7.1 (7.0)
Refinement Statistics
resolution (Å)
1.40–64.42
44643 (3009)
2378 (174)
16.6 (17.4)
18.8 (23.3)
891884/0/166
1.45–64.55
40759 (2926)
2163 (182)
16.4 (16.1)
20.0 (23.6)
891884/34/148
1.50–65.09
36456 (2491)
1924 (122)
15.9 (15.0)
19.9 (23.8)
891884/34/142
1.30–64.42
56119 (4033)
2986 (214)
15.4 (18.5)
17.4 (21.2)
891884/36/196
No. of reflections (working set)
No. of reflections (test set)
Rcryst (%)c
Rfree (%)d
No. TTR/ligands/water atoms
Average B-Values
TTR
13.7
0
16.4
14.3
36.1
16.6
15.2
23.0
17.3
13.6
13.8
14.1
ligand
Wilson B-value
Ramachandran Plot
most favored (%)
additionally allowed (%)
generously allowed (%)
disallowed (%)
91.1
8.9
0
91.1
8.9
0
90.1
9.9
0
91.1
8.9
0
0
0
0
0
R.M.S. Deviations
bond lengths (Å)
angles (°)
0.019
1.83
0.019
1.78
0.019
1.76
0.019
1.72
a Numbers in parentheses are for highest resolution shell of data. b Rsym ) Σhkl|I - <I > Σ|hkl I. c Rcryst ) Σhkl|Fo - Fc|ΣhklFo. d Rfree is the same as Rcryst
,
but for 5% of data excluded from the refinement.
stituent with residues in the thyroid hormone receptor binding
site, as T3 has a hydrogen in this position (Figure 1).51–55
Structural Insights for Development of Optimized TTR
Amyloidogenesis Inhibitors. Although the thyroid hormone-
like 2-aryl rings were envisioned to interact solely within the
smaller inner cavity of the thyroxine binding sites, X-ray analysis
demonstrates that this is not necessarily the case (Figure 5).
Binding in either orientation permits energetically favorable
interactions that noncovalently bridge and stabilize the adjacent
TTR subunits across the weaker of the two dimer–dimer
interfaces. Even though compounds 4d and 4f are both highly
active and selective compounds, they exhibit strikingly different
interactions throughout the outer and inner cavities of the T4
binding site, respectively. When the TTR ·(4d)2 and TTR ·(4f)2
cocrystal structures are overlaid, the substituted phenyl sub-
structures of each are reminiscent of the recently reported
TTR·(PCB-OH)2 cocrystal structure (Figure 6) in which the
protein backbones, side chains, and the 3,5-X2-4-hydroxyphenyl
rings of the three structures superimpose nearly identically.32
To our knowledge, PCB-OH is the most potent inhibitor
discovered to date; it binds with the highest affinity (and
relatively high selectivity) to TTR and is the only compound
that displays positive binding cooperativity.4,19,32,56,57 Because
the 2-aryl rings of inhibitors 4d and 4f (i.e., the substituted
phenyl rings) preferentially adopt conformations analogous to
that observed in TTR ·(PCB-OH)2, these structures (TTR ·(4d/
4f)2) should be valuable as templates to design inhibitors with
optimal interactions in both the inner and outer cavities of the
thyroxine binding sites.
compounds potently inhibit TTR aggregation in vitro.
Notably, four of these bind with high selectivity to tran-
sthyretin in blood plasma ex vivo. The most potent and
selective inhibitors display little, if any, thyroid hormone
nuclear receptor or COX-1 binding, which is highly desirable
for transthyretin amyloidosis patients. The 3,4,5-substituted
thyroxine-like aryl ring, but not necessarily the 3,5-I2-4-
hydroxyphenyl thyroxine aryl ring itself, appears to be an
optimal solution for aryl-X ring composition for future
studies, where subsequent libraries will be used to optimize
the second ring (aryl-Z) and the linker for potency and
binding selectivity. While X-ray crystallographic analysis
demonstrates that incorporation of the 3,4,5-substituted aryl
ring system into the 2-position of benzoxazoles does not
necessarily guarantee a predictable binding orientation, we
are optimistic that further studies for optimization of the
linker and aryl-Z ring will provide more insight for predicting
inhibitor binding orientations that can be verified by crystal-
lographic analysis.
Experimental Section
General Synthetic Methods. Unless otherwise stated, all
chemicals were purchased from commercial suppliers and used
without further purification. Reaction progress was monitored by
thin-layer chromatography on silica gel 60, F254-coated glass plates
(EM Sciences). Flash chromatography was performed using 230–400
mesh silica gel 60 (EM Sciences). NMR spectra were recorded on
a Bruker 500 MHz spectrometer. Chemical shifts are reported in
parts per million downfield from the internal standard Me4Si (0.0
ppm) for CDCl3 solutions, and d6-DMSO solutions were calibrated
to the solvent peaks at 2.49 and 39.52 ppm for the 1H and 13C
NMR spectra, respectively. Reverse phase high performance liquid
chromatography (RP-HPLC) was performed using a Waters 600 E
multisolvent delivery system employing a Waters 486 tunable
Concluding Remarks. From this initial study that was
designed to optimize the aryl-X substituents for transthyretin
amyloidogenesis inhibitor potency and binding selectivity,
we evaluated a library of 2-arylbenzoxazoles, of which 13

Transthyretin Amyloidogenesis Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 267
5.75 mmol) was added and the reaction was stirred at reflux. After
4 h, the reaction was cooled, extracted into EtOAc (50 mL), and
washed with saturated NaHCO3 (2 × 25 mL) and brine (25 mL).
The organics were then dried over Na2SO4, filtered, and concen-
trated. Flash chromatographic purification over silica (9:1 hexanes/
EtOAc) afforded 2-(3-chlorophenyl)benzoxazole (1c) as a white
1
solid (192 mg, 73%). H NMR (500 MHz, CDCl3) δ 8.25 (t, J )
1.8 Hz, 1H), 8.13 (dt, J ) 1.4, 7.6 Hz, 1H), 7.75–7.80 (m, 1H),
7.56–7.61 (m, 1H), 7.50 (ddd, J ) 1.3, 2.1, 8.0 Hz, 1H), 7.45 (dt,
J ) 0.3, 7.8 Hz, 1H), 7.35–7.39 (m, 2H); 13C NMR (125 MHz,
CDCl3) δ 161.63, 150.79, 141.94, 135.09, 131.48, 130.22, 128.88,
127.62, 125.64, 125.54, 124.81, 120.24, 110.70; RP-HPLC 99%
and 99% pure.
Please refer to the Supporting Information for specific synthetic
details and characterization data for inhibitors prepared analogously.
Representative Procedure for the Coupling of 2-Aminophe-
nol with Substituted Benzoic Acids and Cyclodehydration to
their Corresponding 2-Arylbenzoxazoles: Method B. Prepara-
tion of 2-Phenylbenzoxazole (1a). 2-Phenylbenzoxazole (1a) was
obtained from commercial sources and was also synthesized for
method validation. Benzoic acid (111 mg, 0.909 mmol), 2-ami-
nophenol (82.8 mg, 0.759 mmol), and p-TsOH ·H2O (720 mg, 3.79
mmol) were stirred in refluxing xylenes (10 mL). After 4 h, the
reaction was cooled, extracted into EtOAc (50 mL), and washed
with saturated NaHCO3 (2 × 25 mL) and brine (25 mL). The
organics were then dried over Na2SO4, filtered, and concentrated.
Flash chromatographic purification over silica (9:1 hexanes/EtOAc)
afforded 2-phenylbenzoxazole (1a) as a white solid (109 mg, 74%).
1H and 13C NMR were identical to the commercial sample. 1H
NMR (500 MHz, CDCl3) δ 8.24–8.28 (m, 2H), 7.76–7.80 (m, 1H),
7.56–7.60 (m, 1H), 7.50–7.55 (m, 3H), 7.33–7.37 (m, 2H); 13C
NMR (125 MHz, CDCl3) δ 163.05, 150.76, 142.11, 131.53, 128.92,
127.62, 127.17, 125.11, 124.59, 120.02, 110.60; RP-HPLC >99%
and 99% pure.
Refer to the Supporting Information for specific synthetic details
and characterization data for inhibitors prepared analogously.
Representative Procedure for the Coupling of 2-Aminophe-
nol with Substituted Benzoic Acids and Cyclodehydration to
their Corresponding 2-Arylbenzoxazoles: Method C. Prepara-
tion of 2-(3,5-Dibromophenyl)benzoxazole (2d). Modified pro-
cedures from Kumar et al. were employed.39 3,5-Dibromobenzoic
acid (584 mg, 2.09 mmol) was added to a stirring solution of N,N′-
carbonyldiimidazole (341 mg, 2.10 mmol) in anhydrous THF (20
mL) at room temperature under an argon atmosphere. After 15 min,
the 2-aminophenol was added (194 mg, 1.78 mmol) and the reaction
was stirred at reflux. After 18 h, the reaction was concentrated,
then refluxed with p-TsOH·H2O (1.70 g, 8.94 mmol) in p-xylene
(18 mL). After 2 h, the reaction was cooled, extracted into EtOAc
(100 mL), and washed with saturated NaHCO3 (2 × 25 mL) and
brine (25 mL). The organics were then dried over Na2SO4, filtered,
and concentrated. Flash chromatographic purification over silica
(100% hexanes to 9:1 hexanes/EtOAc gradient elution) afforded
2-(3,5-dibromophenyl)benzoxazole (2d) as a pale yellow solid (208
Figure 6. (A) Superimposition of the TTR ·(4d)2 and TTR ·(4f)2 crystal
structures with the benzoxazole rings omitted for clarity. The protein
backbones, residue side chains, and 4d and 4f inhibitors are displayed
in blue and yellow, respectively. (B) Overlay of the resulting TTR·(4d/
f)2 hybrid and TTR ·(PCB-OH)2 crystal structures.32 The protein
backbones, residue side chains, and PCB-OH inhibitor of the TTR·(PCB-
OH)2 structure are displayed in grey for differentiation from the
TTR·(4d/f)2 hybrid structures as represented in panel A.
absorbance detector and 717 autosampler. Samples were chromato-
graphically separated using a ThermoHypersil-Keystone Betabasic-
18 column (model 71503–034630, 150 Å pore size, 3 µm particle
size), eluting with a H2O/CH3CN gradient solvent system. Linear
gradients were run from either 100:0, 80:20, or 60:40 A/B to 0:100
A/B (A ) 95:5 H2O/CH3CN, 0.25% trifluoroacetic acid (TFA); B
) 5:95 H2O/CH3CN, 0.25% TFA). Final compound purities were
additionally evaluated under distinct RP-HPLC conditions by
chromatographically separating samples using a Vydac-C4 column
(model 214TP5415, 300 Å pore size, 5 µm particle size), eluting
with a H2O/MeOH gradient solvent system. Linear gradients were
run from 100:0 to 0:100 C/D (C ) 99.75% H2O, 0.25% TFA; D )
100% MeOH). All mass spectrometry data were collected at The
Scripps Research Institute Center for Mass Spectrometry.
Representative Procedure for the Coupling of 2-Aminophe-
nol with Substituted Benzoic Acids and Cyclodehydration to
their Corresponding 2-Arylbenzoxazoles: Method A. Prepara-
tion of 2-(3-Chlorophenyl)benzoxazole (1c). 3-Chlorobenzoyl
chloride (0.18 mL, 1.42 mmol), 2-aminophenol (125 mg, 1.15
mmol), and pyridine (94.0 µL, 1.15 mmol) were stirred in xylenes
(10 mL) at room temperature for 1 h, then p-TsOH·H2O (1.09 g,
1
mg, 33%). H NMR (500 MHz, CDCl3) δ 8.34 (d, J ) 1.7 Hz,
2H), 8.16 (t, J ) 1.8 Hz, 1H), 7.77–7.80 (m, 1H), 7.58–7.61 (m,
1H), 7.37–7.43 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 160.10,
150.84, 141.76, 136.70, 130.38, 129.13, 125.98, 125.06, 123.53,
120.45, 110.82; ESI-TOF m/z [MH]+ calcd for C13H8Br2NO,
351.8967; found, 351.8966; RP-HPLC 98% and 97% pure.
Please refer to the Supporting Information for specific
synthetic details and characterization data for inhibitors prepared
analogously.
Representative Procedure for the Methylation of Phenols.
Preparation of 2-(3-Methoxyphenyl)benzoxazole (1i). 2-(3-
Hydroxyphenyl)benzoxazole (1h, 29.6 mg, 0.140 mmol), io-
domethane (17.5 µL, 0.281 mmol), and K2CO3 (39.3 mg, 0.284
mmol) were stirred in DMF (3 mL) at room temperature. After
18 h, the reaction was concentrated with silica to a powder. Flash
chromatographic purification over silica (2:1 hexanes/EtOAc)
afforded 2-(3-methoxyphenyl)benzoxazole (1i) as an amber solid
1
(25.2 mg, 80%). H NMR (500 MHz, CDCl3) δ 7.85 (ddd, J )

268 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Johnson et al.
1.0, 1.4, 7.7 Hz, 1H), 7.76–7.80 (m, 2H), 7.56–7.60 (m, 1H), 7.43
(t, J ) 8.0 Hz, 1H), 7.33–7.38 (m, 2H), 7.08 (ddd, J ) 0.9, 2.7,
8.3 Hz, 1H), 3.92 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 162.98,
159.96, 150.77, 142.08, 130.01, 128.36, 125.17, 124.61, 120.14,
120.04, 118.36, 111.92, 110.61, 55.53; ESI-TOF m/z [MH]+ calcd
for C14H12NO2, 226.0863; found, 226.0860; RP-HPLC 97% and
96% pure.
Refer to the Supporting Information for specific synthetic
details and characterization data for inhibitors prepared
analogously.
final supernatant, dissociation of the TTR and bound test compound
from the resin-bound antibody was achieved through addition of
155 µL of aqueous triethylamine (100 mM, pH ∼ 11.5) and rocking
(18 rpm) at 4 °C for 30 min. The suspension was then centrifuged,
and 135 µL of the supernatant, containing both TTR and test
compound, was analyzed by RP-HPLC to determine the stoichi-
ometry of small molecule bound to TTR (the test compound-TTR
complex dissociates and the small molecule and protein are
chromatographically separable under the HPLC buffer conditions).
Quantification of test compound and TTR is achieved by comparing
the integrated peak areas to standard curves: the ratio of the amount
of test compound to TTR yields the binding stoichiometry, of which
a theoretical maximum value of 2 is possible due to the two T4
binding sites per TTR tetramer. Analyses were performed in at least
triplicates of duplicates from three different blood plasma samples
(i.e., at least six analyses), with average values obtained presented
in Figure 3 (errors are typically less than (0.1).
Evaluating the Inhibition of COX-1 Enzymatic Activity by
the Potent TTR Amyloidosis Inhibitors. The evaluation of the
most potent TTR aggregation inhibitors (displaying <20% fibril
formation) at inhibiting COX-1 activity was contracted out to the
Cerep laboratories in Redmond, WA. Compound analyses were
performed using assay catalog reference #777-1 hr, which uses
procedures developed by Glaser et al.42 A brief experimental
protocol as provided by Cerep is outlined below. In this assay, the
enzyme (∼2 µg) is preincubated in the absence (water control) or
presence of test compound (10.0 µM) for 20 min at 22 °C in 250
µL of buffer (100 mM Tris-HCl, pH 8, 2 mM phenol, 1 µM
hematine). Arachidonic acid (4 µM) is then added to initiate the
reaction (no arachidonic acid added for basal control measurements).
After incubation at 22 °C for 10 min, the reaction is quenched by
addition of 2 M HCl and 1 M Tris-HCl (pH 7.8) and cooling at 4
°C. Prostaglandin-E2 (PGE2) quantification is performed using an
EIA detection kit, with measurements made using a microplate
reader. Average values of duplicate analyses are presented in Figure
4 (black values), which represent the % inhibition of arachidonic
acid conversion to PGE2 due to competitive binding of test
compound to COX-1 (errors of less than ( 6 percentage points
are representative of the data). Control analyses are performed
analogously with the standard inhibitory reference compound
diclofenac tested at several concentrations to obtain an inhibition
curve from which its IC50 is calculated (12 nM).
Representative Procedure for the Demethylation of
Anisoles. Preparation of 2-(3,5-Dihydroxyphenyl)benzoxazole
(2h). Boron tribromide (6.20 mL of 1 M BBr3 in hexanes, 6.20
mmol) was added to a stirring mixture of 2i (159 mg, 0.623 mmol)
in anhydrous CH2Cl2 (6.0 mL), and the reaction was stirred at room
temperature under an argon atmosphere. After 18 h, the reaction
was quenched with MeOH (5 mL), extracted into EtOAc (50 mL),
and washed with H2O (2 × 25 mL) and brine (25 mL). The organics
were then dried over Na2SO4, filtered, and concentrated. Flash
chromatographic purification over silica (1:1 hexanes/EtOAc to
100% EtOAc gradient elution) afforded 2-(3,5-dihydroxyphenyl-
)benzoxazole (2h) as an off-white solid (98.8 mg, 70%). 1H NMR
(500 MHz, d6-DMSO) δ 9.74 (s, 2H), 7.74–7.79 (m, 2H), 7.36–7.43
(m, 2H), 7.06 (d, J ) 2.2 Hz, 2H), 6.44 (d, J ) 2.2 Hz, 1H); 13C
NMR (125 MHz, d6-DMSO) δ 162.46, 159,03, 150.08, 141.45,
127.80, 125.37, 124.77, 119.73, 110.85, 106.10, 105.28; ESI-TOF
m/z [MH]+ calcd for C13H10NO3, 228.0655; found, 228.0665; RP-
HPLC 98% and 98% pure.
Refer to the Supporting Information for specific synthetic details
and characterization data for inhibitors prepared analogously.
Evaluating Small Molecule-Mediated Inhibition of WT-
TTR Amyloidogenesis. WT-TTR was purified from an E. coli
expression system, as described previously.58 To a 495 µL aliquot
of 0.4 mg/mL WT-TTR (7.2µM, 10 mM phosphate, pH 7.0, 100
mM KCl, 1 mM EDTA) in a disposable cuvette was added 5 µL
of a 1.44 mM DMSO solution of test compound and the sample
was vortexed briefly. After incubating for 30 min at ambient
temperature, the pH of the sample was lowered to 4.4 by the
addition of 500 µL of acidic buffer (100 mM acetate, pH 4.2, 100
mM KCl, 1 mM EDTA), and the solution was briefly vortexed
and incubated in the dark for 72 h at 37 °C (final protein and
inhibitor concentrations were 3.6 and 7.2 µM, respectively). The
sample was then vortexed to evenly distribute any precipitate, and
the turbidity was measured at 400 nm on a Hewlett-Packard model
8453 UV–vis spectrophotometer. The extent of WT-TTR aggrega-
tion (% fibril formation, f.f.; % inhibition ) 100 - %f.f.) was
determined by comparing the sample turbidity in the presence of
small molecule as a percentage of control WT-TTR sample
incubated with 5 µL of pure DMSO (representing 100% aggrega-
tion, 0% inhibition). All samples were performed in at least
triplicate, with average values obtained presented in Figure 3 (errors
are typically less than ( 5 percentage points).
Evaluating the Binding Selectivity of Amyloidogenesis
Inhibitors to TTR in Human Blood Plasma. The procedure for
the antibody capture method to evaluate the stoichiometry of
inhibitor bound to TTR in human blood plasma has been described
in detail elsewhere.37 Briefly, 7.50 µL of a 1.44 mM DMSO solution
of test compound was incubated with human blood plasma (1 mL)
in a 2 mL eppendorf tube in the dark at 37 °C for 24 h on a rocker
(30 rpm). Then, 125 µL of a 1:1 v/v slurry of unfunctionalized
sepharose resin in TSA (10 mM Tris, pH 8.0, 140 mM NaCl) was
added, and the mixture was incubated for another hour at 4 °C on
a rocker (18 rpm). The sample was then centrifuged, the supernatant
was divided into 2 aliquots of 400 µL each, which were each added
to 200 µL of a 1:1 v/v slurry of sepharose resin conjugated to an
anti-TTR antibody in TSA, and the sample was incubated again at
4 °C for 20 min on a rocker (18 rpm). The sample was then
centrifuged, the supernatant removed, and the TTR bound resin
was washed three times (10 min each wash) with 1 mL of TSA/
0.05% saponin, then twice (10 min each wash) with 1 mL TSA at
4 °C on a rocker (18 rpm). After centrifugation and removal of the
Evaluating the Binding of the Potent TTR Amyloidosis
Inhibitors to the Thyroid Hormone Nuclear Receptor. The
evaluation of the most potent TTR aggregation inhibitors (displaying
<20% fibril formation) at binding to the thyroid hormone receptor
was contracted out to the Cerep laboratories in Redmond, WA.
Compound analyses were performed using assay catalog reference
#855, which uses procedures developed by Inoue et al.43 A brief
experimental protocol as provided by Cerep is outlined below. In
this assay, liver membrane homogenates (100 µg protein) are
incubated for 18 h at 4 °C with 0.1 nM 125I-labeled triiodothyronine
([125I]T3, the primary thyroid hormone) in the absence or presence
of test compound (10.0 µM) in 500 µL of buffer (20 mM Tris-
HCl, pH 7.6, 50 mM NaCl, 2 mM EDTA, 10% glycerol, and 5
mM ꢀ-mercaptoethanol). The samples are then vacuum filtered
through glass fiber filters (GF/B, Packard), rinsed several times with
ice-cold buffer (50 mM Tris-HCl and 150 mM NaCl), and the filters
are dried and counted for radioactivity in a scintillation counter
(Topcount, Packard) using a scintillation cocktail (Microscint 0,
Packard). Nonspecific binding, determined in the presence of 1 µM
T3, is subtracted from the [125I]T3 binding results. Average values
of duplicate analyses are presented in Figure 4 (red italicized
values), which represent the % displacement of [125I]T3 due to
competitive binding of test compound to the thyroid hormone
receptor (errors of less than ( 2 percentage points are representative
of the data). Control analyses are performed analogously with T3
tested at several concentrations to obtain a competition curve from
which its IC50 is calculated (0.38 nM).

Transthyretin Amyloidogenesis Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 269
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X-ray Crystallographic Analysis of WT-TTR in apo-Form
and Bound to Inhibitors 2f, 4d, and 4f. WT-TTR was purified
from an E. coli expression system, as described previously.58 The
WT-TTR was concentrated to 4 mg/mL in 10 mM NaPi, 100 mM
KCl, at pH 7.6 and cocrystallized at room temperature with
inhibitors 2d, 4d, and 4f at a 5 M excess using the vapor-diffusion
sitting drop method. Crystals were grown from 1.395 M sodium
citrate, 3.5% v/v glycerol at pH 5.5. The crystals were cryo-
protected with an inhibitor-free solution of 10% v/v glycerol. Data
were collected at beam line 11-1 at the Stanford Synchrotron
Radiation Laboratory (SSRL) at a wavelength of 0.9795 Å for the
apo, 2f, and 4f crystals and at 0.9195 Å, corresponding to the
bromine peak wavelength, for the 4d crystals. The data sets were
integrated and scaled using HKL2000.59 The crystals were indexed
in space group P21212 with two subunits per asymmetric unit with
unit cell dimensions a ) 84.9 Å, b ) 43.9 Å, and c ) 64.8 Å. The
four crystal structures were determined by molecular replacement
using the model coordinates of 2FBR60 in the program Phaser61 to
1.4, 1.45, 1.5, and 1.3 Å resolutions for the apo, 2f, 4d, and 4f
structures, respectively. Further model building and refinement were
completed using Refmac.62 Hydrogens were added during refine-
ment and anisotropic B-values were calculated. Final models were
validated using the JCSG quality control server incorporating
Molprobity,63 ADIT (http://rcsb-deposit.rutgers.edu/validate) WHA-
TIF,64 Resolve,65 and Procheck.66 Data collection and refinement
statistics are presented in Table 1.
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Protein Data Bank Accession Codes. Atomic coordinates have
been deposited in the RCSB Protein Data Bank (www.pdb.org) and
are available under accession codes 2QGB (apo-form WT-TTR),
2QGE (WT-TTR in complex with 2f), 2QGD (WT-TTR in complex
with 4d), and 2QGC (WT-TTR in complex with 4f).
Acknowledgment. We are grateful for the financial support
of the NIH DK 46335 (J.W.K), CA58896, and AI42266
(I.A.W.), as well as the Skaggs Institute of Chemical Biology
and the Lita Annenberg Hazen Foundation. Assistance with the
thyroid hormone receptor and COX-I binding assays by the
Cerep laboratories (Redmond, WA, and France) and Richard
Labaudiniere of FoldRx Pharmaceuticals (Boston, MA), as well
as the technical expertise of Ted Foss, M. T. Dendle, and Mike
Saure are also greatly appreciated. Portions of this research were
carried out at the Stanford Synchrotron Radiation Laboratory,
a national user facility operated by Stanford University on behalf
of the U.S. Department of Energy, Office of Basic Energy
Sciences. The SSRL Structural Molecular Biology Program is
supported by the Department of Energy, Office of Biological
and Environmental Research and by the National Institutes of
Health, National Center for Research Resources, Biomedical
Technology Program, and the National Institute of General
Medical Sciences. The authors would also like to thank Drs.
Robyn Stanfield, Xiaoping Dai, and Petra Verdino in the Wilson
laboratory for assistance in data collection and structure
determination.
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Supporting Information Available: Detailed synthesis and
characterization of the 2-arylbenzoxazoles and tabulation of com-
pound purity as determined by RP-HPLC. This material is available
free of charge via the Internet at http://pubs.acs.org.
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