Design, synthesis, and evaluation of oxazole transthyretin amyloidogenesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.12.022

Ten oxazoles bearing a C(4) carboxyl group were synthesized and evaluated as transthyretin (TTR) amyloid fibril inhibitors. Substituting aryls at the C(2) position of the oxazole ring reveals that a 3,5-dichlorophenyl substituent significantly reduced amy

Full text of DOI:10.1016/j.bmcl.2004.12.022

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1075–1078
Design, synthesis, and evaluation of oxazole transthyretin
amyloidogenesis inhibitors
Hossein Razavi, Evan T. Powers, Hans E. Purkey, Sara L. Adamski-Werner,
Kyle P. Chiang, Maria T. A. Dendle and Jeffery W. Kelly*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, BCC265,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 10 November 2004; revised 6 December 2004; accepted 9 December 2004
Available online 13 January 2005
Abstract—Ten oxazoles bearinga C(4) carboxyl group were synthesized and evaluated as transthyretin (TTR) amyloid fibril inhib-
itors. Substitutingaryls at the C(2) position of the oxazole ringreveals that a 3,5-dichlorophenyl substituent significantly reduced
amyloidogenesis. The efficacy of these inhibitors was enhanced further by installing an ethyl, a propyl, or a CF3 group at the C(5)
position. The CF3 substitution at C(5) also improves the TTR bindingselectivity over all the other proteins in human blood.
Ó 2004 Elsevier Ltd. All rights reserved.
Transthyretin (TTR) is a homotetrameric protein com-
posed of 127-amino acid subunits that functions as the
primary transporter of L-thyroxine (T4) in cerebral
To date, hundreds of structurally different compounds
in several distinct classes have been identified as inhibi-
tors of TTR fibril formation. Most inhibitors have two
aromatic substructures connected by a variety of linkers.
Generally, one aromatic ringbears hydrophobic substit-
uents (e.g., halogens), while the other ring is substituted
with hydrophilic moieties (e.g., carboxylic acid or hy-
droxyl groups). Examples of efficacious TTR amyloido-
genesis inhibitors include substituted biaryl amines,9
biaryl ethers,10 anthranilic acids,11 bivalent inhibitors,12
N-phenyl phenoxazines,13 and analogs of the non-steroi-
dal anti-inflammatory drugs flufenamic acid,14 diclofe-
nac,15 and diflunisal.16 In our attempt to identify novel
classes of TTR inhibitors with enhanced potency and
improved bindingselectivity to TTR over other plasma
proteins, we have examined aryl oxazoles bearinga car-
boxyl group at the C(4) position.
spinal fluid, as well as a carrier of T4 and holo retinol
1,2
bindingprotein in plasma.
Although TTR is a stable
protein under physiological conditions (pH 7.2), it disso-
ciates to monomers under partial denaturingconditions,
which is typically rate-limitingfor amyloidoegnesis.
However, dissociation is not sufficient for amyloidogen-
esis; the folded monomer has to partially denature in or-
der to misassemble into aggregates including amyloid
fibrils.3 Soluble aggregates of TTR and/or amyloid fi-
brils have been implicated as the causative agents in dis-
eases such as senile systemic amyloidosis,4 familial
amyloid cardiomyopathy,5 and familial amyloid
polyneuropathy.6
Since it is not yet clear whether the soluble aggregates or
the amyloid fibrils themselves lead to neuropathology,
the most conservative strategy is to stabilize the native
The oxazole core can be accessed via the oxidation of
oxazolines. For the construction of oxazolines, we relied
on the method reported by Wipf and Miller which uti-
lizes [(methoxycarbonyl)sulfamoyl]triethylammonium
hydroxide inner salt (Burgess Reagent) as an agent for
cyclodehydratingpeptides and N-acyl amino esters.17
7
tetramer utilizingsmall organic molecules.
Molecules
that bind TTR and selectively stabilize the tetramer over
the dissociative transition state raise the kinetic barrier
dramatically
dissociation.8
slowingor
preventingtetramer
Startingmaterials 1a-1d (Scheme 1; Supplementary
data), prepared by benzoylation of L-threonine benzyl
ester, were treated with Burgess Reagent in THF at
reflux for 2 h. This method provides oxazolines via
SN2 intramolecular cyclization with inversion of
Keywords: Amyloidogenesis inhibitors; Aryl oxazole; Inhibitor binding
selectivity; Transthyretin.
*
Correspondingauthor. Tel.: +1 858 784 9880; fax: +1 858 784
9899; e-mail: jkelly@scripps.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.022

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H. Razavi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1075–1078
R
O
R
O
a glycine-derived thioimidate, a nitrile ylide synthon
used in the synthesis of a variety of heterocyclic com-
pounds such as oxazolines, oxazoles, and thiazoles.
We envisaged that the latter method could be applied
to synthesize a panel of C(5)-substituted oxazoles with
a glycine-derived thioimidate serving as an advanced
intermediate.
a
HO
O
Ph
HO
O
Ph
NH3X
HN
Ar
1a-1e
O
R
C(5)
R
O
O
O
b, c
d, or e
O
Ph
OH
O
N
N
Therefore, glycine methyl ester hydrochloride was con-
verted to methyl azidoacetate by means of a diazo trans-
fer reaction (Scheme 2).20 The resultingazide was
treated with diphenyl methylphosphine yieldingthe
phosphazene derivative, which was transformed in situ
to its correspondingthioimidate usinga method de-
scribed previously.21 The reaction of the glycine-derived
thioimidate with various acyl chlorides or anhydrides in
the presence of diisopropyl ethyl amine, followed by the
hydrolysis of the resultingproducts, generated the corre-
spondingoxazoles 4-8 in low to moderate overall yields
(Scheme 2, and see Fig. 1 for line drawings of the struc-
tures prepared; Supplementary data).
C(2)
Ar
Ar
3a-3e
2a-2e
Scheme 1. Reagents and conditions: (a) ArCOCl, CH2Cl2, DIEA
[X = Cl, C2H2O4 (hemioxalate)] (70–84%); (b) Burgess Reagent, THF,
reflux; (c) DBU, BrCCl3, CH2Cl2 (34–70%, over two steps); (d) H2,
Pd–C, MeOH (96–99%); (e) LiOH, THF, MeOH, H2O (98–99%). See
Supplementary data for a detailed description of all experimental
procedures. [R = Me, Ar = 3,5-difluorophenyl (3a); R = Me, Ar = 3-
(trifluoromethyl)phenyl (3b); R = Me, Ar = 3,5-dichlorophenyl (3c);
R = Me, Ar = phenyl (3d); R = H, Ar = 3,5-dichlorophenyl (3e)].
configuration at the amino acid b-carbon. However, the
stereochemical consequences of these reactions were
unimportant to this study, since the products were
immediately converted into oxazoles by mild oxidation
with BrCCl3/DBU.18 Subsequently, esters 2a-2d were
hydrogenated or hydrolyzed to their corresponding
acids 3a-3d. Compound 3e was synthesized from N-ben-
zoylated L-serine benzyl ester 1e, usingthe approach
outlined in Scheme 1 (see Fig. 1 for line drawings of
the products).
Our strategy for identifying oxazole-based inhibitors is
based on the synthesis and evaluation of a limited num-
ber of suitably substituted analogs depicted in Figure 1.
Putative suitable substructures are based on those found
in other potent TTR amyloidogenesis inhibitors includ-
ingaromatic carboxylic acids and halogenated aromat-
ics.9–16 We envisioned that the carboxylic acid moiety
that interacts with the ammonium group of Lys 15
and Lys 150 within TTR could be directly displayed on
the C(4) of the oxazole ringwhile the haloegnated
C(5)-substituted analogs were synthesized according to
a method described previously.19 This method employs
CH
N
H
N
CH
CH
CH
3
3
3
3
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
Cl
F
Cl
N
N
N
Cl
F
CF
Cl
3
3a
3b
3c
3d
3e
CH
CH
3
3
CH
O
CF
3
3
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
Cl
Cl
Cl
Cl
Cl
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
4
5
6
7
8
Figure 1. Oxazoles utilized in this study. Synthesis pathways, and procedures are described in Schemes 1 and 2, and in the Supplementary data
section, respectively.
C(5)
c, d
O
O
O
R
O
a
b
N3
XH3N
Ar
N
O
O
O
OH
4-8
O
N
S
C(2)
Ph
Ar
Scheme 2. Reagents and conditions: (a) CF3SO2N3, CH2Cl2, DIEA (44%); (b) MePh2P, ArCOCl (Ar = 3,5-dichlorophenyl), PhSH, DIEA (16%); (c)
RCOX, DIEA, CH2Cl2 [R = Et (4), n-Pr (5), n-Bu (6), CF3 (7), Ph (8); X = Cl, CF3CO2]; (d) LiOH, THF, MeOH, H2O (10–79% over two steps). See
Supplementary data for a detailed description of all experimental procedures.

H. Razavi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1075–1078
1077
Table 1. In vitro transthyretin bindingselectivity assay with human
blood plasma
aromatic ringcould be linked to the oxazole core at the
C(2) position.
Entry
Compounda
Molar equivalence boundb
The activity of the synthesized oxazoles was evaluated in
duplicate usingthe TTR amyloid fibril formation as-
say,10 and the human plasma binding-selectivity assay.22
The amyloid fibril formation assay revealed that simple
oxazole 3d was a poor inhibitor of TTR fibril formation
(Figs. 1 and 2A). However, the display of halogens on
the phenyl substituent attached at C(2) improves the
activity of the parent compound (Figs. 1 and 2A, cf.
3d vs 3b or 3c). The meta-CF3 and 3,5-dichloro aryl sub-
stituents appeared optimal for anti-amyloidogenesis
activity (Fig. 2A). This improvement in activity is likely
to be attributable to the favorable hydrophobic interac-
tion(s) between the halogenated aryl and the so-called
halogen binding pockets of TTR. In this panel of com-
pounds, the 3,5-dichloro substituted pattern that im-
parts the highest level of activity serves as a lead
compound for further optimization.
1
2
3
4
4
5
7
8
0.49 0.07
0
0.68 0.04
0
a Compounds identified as good inhibitors in the fibril formation assay
(Fig. 2).
b Bindingselectivity to transthyretin in human blood plasma (trans-
thyretin concentration ꢀ5.4 lM based on ELISA assay) after a 24 h
incubation period (37 °C) in the presence of inhibitor (10.8 lM), as
determined by reverse phase HPLC.22
with hydrogen severely diminished the activity of the
parent compound (Fig. 2B, cf. 3e vs 3c). The activity
of the lead compound is also improved upon introduc-
tion of a CF3 moiety (Fig. 2B, compound 7). These re-
sults suggested that a hydrophobic group of the
appropriate size is required at C(5) for optimal activity
of an oxazole-based TTR amyloid inhibitor.
The second group of compounds was synthesized to
probe the role of the C(5) substituent on activity relative
to the lead compound 3c. The activity of the inhibitors
increased when the alkyl chain at the C(5) position
was lengthened. Optimal activity is observed for the
ethyl and propyl analogs (Fig. 2B, cf. 4 or 5 vs 3c or
6, respectively). Replacement of the methyl substituent
The more potent amyloid inhibitors (<10% fibril forma-
tion at a concentration of 7.2 lM) were further sub-
jected to the immunoprecipitation, HPLC-based assay
to evaluate compound bindingstoichiometry to TTR
in human blood plasma. These data reveal that com-
pounds 4 and 7 were moderately selective for TTR in
plasma (Table 1). The results suggest that the affinity
of oxazole-based inhibitors for TTR over other plasma
proteins is sensitive to the size of the C(5) substituent
(Table 1, cf. 4 vs 5). The information gathered from
both assays indicates that the C(5) substituent is not
only important for activity, but also for bindingselec-
tively to TTR over all the other plasma proteins.
In conclusion, oxazoles containinga C(4) carboxylic
acid, a C(5) alkyl group and a C(2) halogenated aro-
matic pharmacophore were synthesized. Evaluation of
these compounds reveals that several compounds in this
class inhibit TTR amyloidogenesis in vitro and two bind
TTR in human plasma with modest selectivity. In par-
ticular, compounds 4 and 7 completely inhibit fibril for-
mation at 7.2 lM (TTR 3.6 lM) and bind to TTR in the
presence of other plasma proteins with a stoichiometry
of at least 0.5 (2 equiv beingmaximal).
Acknowledgements
We gratefully acknowledge financial support by the
NIH (DK046335), the Skaggs Institute for Chemical
Biology, and the Lita Annenberg Hazen Foundation.
H. R. acknowledges the Skaggs Institute for Chemical
Biology for a postdoctoral fellowship.
Figure 2. The influence of oxazole-based inhibitors on transthyretin
amyloid fibril formation. The relative fibril yield was determined after
a 72 h incubation period under partial denaturingconditions (pH 4.4,
37 °C) in the presence or absence of inhibitor. Yields were expressed
relative to that obtained for TTR (3.6 lM) in the absence of inhibitor
(speckled bars). (A) The influence of substituents at the C(2) position
of the oxazole ringon TTR amyloidogenesis. The respective inhibitors
were applied at a concentration of 3.6 lM (hatched bars) or 7.2 lM
(solid bars). (B) The influence of the substituents at the C(5) position of
the oxazole ringon TTR amyloidogenesis. All inhibitors were applied
at a concentration of 7.2 lM (solid bars). Compounds 3a–3e and 4–8
are substituted oxazole-4-carboxylic acids as depicted in Figure 1.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2004.12.022.

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11. Oza, V. B.; Petrassi, H. M.; Purkey, H. E.; Kelly, J. W.
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