Synthesis of 2,5-diaryl-substituted thiophenes as helical mimetics: Towards the modulation of islet amyloid polypeptide (IAPP) amyloid fibril formation and cytotoxicity

Article abstract of DOI:10.1002/chem.201303928

A range of 2,5-diarylated thiophenes were synthesised as small molecule mimetics of the α-helix to modulate the amyloidogenesis and cytotoxic effect of islet amyloid polypeptide (IAPP). 3-Substituted thiophene-2-carboxylic acids were used as key intermediates and functionalised by palladium decarboxylative cross-coupling and direct C=H activation successively with overall yields ranging from 23 to 95 %. The effect of the ligands on IAPP amyloid fibril formation was evaluated with the thioflavina T (ThT) fluorescence-based assay. Furthermore, the capacity of these compounds to inhibit the cytotoxic effect of IAPP was assessed using β-pancreatic cells. A twist manipulated: A range of 2,5-diarylated thiophenes was synthesised as small molecule mimetics of the α-helix to modulate the amyloidogenesis and cytotoxic effect of islet amyloid polypeptide (IAPP; see scheme). The effect of the ligands on IAPP amyloid fibril formation was evaluated. Furthermore, the capacity of these compounds to inhibit the cytotoxic effect of IAPP was assessed using β-pancreatic cells. Copyright

Full text of DOI:10.1002/chem.201303928

DOI: 10.1002/chem.201303928
Full Paper
&
Peptidomimetics
Synthesis of 2,5-Diaryl-Substituted Thiophenes as Helical
Mimetics: Towards the Modulation of Islet Amyloid Polypeptide
(IAPP) Amyloid Fibril Formation and Cytotoxicity
Avid Hassanpour,[a] Carole Anne De Carufel,[b] Steve Bourgault,*[b] and Pat Forgione*[a]
Abstract: A range of 2,5-diarylated thiophenes were syn-
thesised as small molecule mimetics of the a-helix to modu-
late the amyloidogenesis and cytotoxic effect of islet amy-
loid polypeptide (IAPP). 3-Substituted thiophene-2-carboxylic
acids were used as key intermediates and functionalised by
palladium decarboxylative cross-coupling and direct CꢀH ac-
tivation successively with overall yields ranging from 23 to
95%. The effect of the ligands on IAPP amyloid fibril forma-
tion was evaluated with the thioflavin T (ThT) fluorescence-
based assay. Furthermore, the capacity of these compounds
to inhibit the cytotoxic effect of IAPP was assessed using b-
pancreatic cells.
Introduction
were recently recognised as the most cytotoxic proteospecies
of the amyloidogenic cascade,[6] suggesting a potential draw-
back to this approach.
The aberrant assembly of polypeptides into insoluble protein
aggregates, including amyloid fibrils, is the hallmark of several
diseases, such as Alzheimer’s, type II diabetes (DM-2) and sys-
temic amyloidosis.[1] These protein misfolding/aggregation dis-
orders differ by the identity of the protein that misassembles
and by the tissue subjected to protein deposition and cellular
degeneration.[2] For instance, in patients afflicted by DM-2, the
islet amyloid polypeptide (IAPP) deposits in the pancreas, lead-
ing to the degeneration of the islets of Langerhans.[3] IAPP is
a 37-amino acid C-terminally a-amidated peptide that is co-se-
creted with insulin by pancreatic b-cells. IAPP is an unusual ag-
gregation-prone peptidic hormone that readily forms amyloid
fibrils.[3] The IAPP amyloidogenic process observed in the pan-
creas is believed to accelerate DM-2 pathogenesis by exacer-
bating b-cell degeneration and ultimately compromising insu-
lin secretion.[3] When applied to isolated pancreatic b-cells in
culture, IAPP is cytotoxic by mechanisms that are still not clear-
ly understood.[4] Over the last two decades, several compounds
have been reported to inhibit IAPP aggregation in vitro by in-
terfering with the later stages of fibrillogenesis, that is, through
the destabilisation of the ordered cross b-sheet quaternary
structure of the amyloid fibrils.[4b,5] However, this mechanism
does not prevent the formation of prefibrillar oligomers that
IAPP exhibits a conformational ensemble mainly populated
by disordered conformations in its non-aggregated soluble
state, although it diverges from an absolute random coil by
the presence of local and transient ordered structure.[7] Recent
mechanistic studies have suggested that this pro-amyloidogen-
ic peptide undergoes a random coil to a-helix conformational
conversion during the initial phase of self-assembly and that
the helical intermediates could be on-pathway to amyloid for-
mation.[7,8] According to this model, a-helix formation and self-
association of helical segments are linked and accelerate self-
assembly,[8] with similar driving forces to those of coiled coil
motif formation. Consequently, this accelerated self-assembly
generates a high local concentration of the amyloidogenic
domain of IAPP (segment 20–29), which has a high propensity
to adopt a b-structure, favouring the formation of cross-b-
sheet assemblies and en route to amyloid formation. Consis-
tently, IAPP was shown to adopt a helical structure spanning
approximately residues 8 to 19 when the peptide was bound
to model membranes[9] or glycosaminoglycans (GAGs),[4a] and
these interactions accelerate the rate of IAPP amyloid fibril
formation.
According to the helical intermediates hypothesis described
above, an alternative strategy to control the formation of IAPP
amyloid fibrils would be to design molecules that target and
stabilise the transient helical segment 8–19 of IAPP, modulat-
ing the helix-assembly process. This approach could inhibit the
formation of oligomeric and fibrillar aggregates by over-stabil-
ising the helical intermediates, not allowing the propagation of
the b-sheet conformation from the 20–29 domain of IAPP.
Recent studies using membrane models have shown that,
indeed, IAPP can be trapped in a non-amyloid prone helical
conformation.[8,9b,10] Alternatively, as reported for lipids and
GAGs, helical targeting ligands could potently accelerate the
[a] A. Hassanpour, Prof. P. Forgione
Concordia University
Department of Chemistry and Biochemistry, Pharmaqam
7141 rue Sherbrooke O., Montrꢀal, QC H4B 1R6 (Canada)
E-mail: pat.forgione@concordia.ca
[b] C. A. De Carufel, Prof. S. Bourgault
Pharmaqam, Department of Chemistry
Universitꢀ du Quꢀbec ꢁ Montrꢀal
C.P. 8888, Succursale Centre-Ville Montrꢀal, QC H3C 3P8 (Canada)
E-mail: bourgault.steve@uqam.ca
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303928.
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self-assembly of IAPP into b-sheet-rich amyloid fibrils by initial-
ly shifting the conformational equilibrium towards the a-helix,
without overly stabilising the helical motif. Considering that
oligomers are the most potent cytotoxic proteospecies,[6c] both
pathways will decrease the toxicity induced by the amyloido-
genic process of IAPP, either by blocking the formation of pre-
fibrillar assemblies (a-helix over-stabilisation) or by accelerating
the structural conversion of oligomers into less cytotoxic amy-
loid fibrils.
allows for several significant synthetic advantages. The pres-
ence of the heteroatom in thiophene introduces changes in re-
activity that allows convenient chemo- and regioselective path-
ways that are unavailable in the synthesis of terphenyl com-
pounds. Specifically, a five-membered heteroaromatic core
scaffold (Figure 1b, compound 2) affords a flexible synthetic
approach in which substituent modifications can be made in
a modular manner while avoiding the long synthetic routes
that have been used previously for the synthesis of
terphenyls.[14]
Miranker and Hamilton groups recently developed small
molecules targeting the transient helical state of IAPP in order
to inhibit lipid-catalysed aggregation.[11] These polycarboxylate
ligands were developed on pyridyl, quinoline or peptoid scaf-
folds and were shown to inhibit lipid-induced IAPP aggrega-
tion, but to strongly accelerate IAPP fibrillogenesis in lipid-free
conditions. Alternatively, other synthetic templates have been
shown to be attractive scaffolds towards interacting/stabilising
the a-helical region of proteins.[12] Functionalised terphenyls[13]
(compound 1) represent one such scaffold with a reported ap-
plication as a mimic of the a-helix side chain of smooth
muscle myosin light chain kinase (smMLCK) to disrupt its inter-
action with calmodulin (CaM; Figure 1a).[14]
The synthesis of diaryl substituted heteroaromatics has been
previously accomplished through various palladium catalysed
cross-coupling reactions. The predominant strategies involve
the utilisation of organometallic precursors and/or result in the
formation of symmetrically substituted heteroaromatics.[15b,16]
For example, palladium catalysed CꢀH activation reactions
have emerged as attractive methods for the formation of
carbonꢀcarbon bonds between heteroarenes and aryl halides
without the use of organometallic derivatives.[17] However, the
main limitation of CꢀH functionalisation of 3-substituted thio-
phenes is the formation of mixed arylated products at the C2
and C5 positions.[18] To avoid this limitation yet still take ad-
vantage of a CꢀH arylation strategy while controlling the re-
gioselectivity of the products, commercially available 3-substi-
tuted thiophene 2-carboxylic acids have been used in combi-
nation with decarboxylative cross-coupling. Decarboxylative
cross-coupling reactions have been developed as a powerful
method for the formation of carbonꢀcarbon bonds between
aliphatic and aromatic carboxylic acids and aryl or vinyl sub-
strates.[19] Decarboxylative arylation processes circumvent the
requirement of organometallic building blocks[20] offering read-
ily available, inexpensive and easy to use coupling partners. In
this view, we performed palladium catalysed decarboxylative
cross-coupling reaction of thiophene carboxylic acids and vari-
ous aryl bromides.[18b,21] The combination of both the CꢀH ary-
lation and decarboxylative cross-coupling reactions allows for
a short, modular pathway through which a large library of a-
helix mimetic compounds can be readily synthesised. In the
current work, the molecules produced by this approach were
tested as modulators of the formation of IAPP fibrils as
a proof-of-concept. However, the general synthetic route can
be used for the preparation of molecules tailored with differ-
ent side-chain residues to stabilise and/or interact with the a-
helix of other proteins for various applications.[22]
Figure 1. A) 3,2’,2’’-Tris-substituted terphenyl template (compound 1), B) 2,5-
diaryl substituted thiophene template (compound 2), C) ribbon representa-
tion of IAPP a-helix (PDB ID: 2KB8).[25]
Binding of ligands to the helical motif largely results from
the interaction of the ligand with the amino acid side chains
projecting on one face of the a-helix and spaced three or four
residues away from each other, referred to as i, i+4 and i+7
(Figure 1). In the transient helical conformation of IAPP com-
prising residues 8–19, residues Arg11, Phe15 and His18 are ori-
ented on one face of the a-helix[9a] and represent the key
motif that will be targeted to stabilise the transient a-helix of
IAPP (Figure 1c). As hypothesised from coiled coil motifs for-
mation, the presence of hydrophilic side chains (Arg, His) will
provide the specificity of interaction whereas the hydrophobic
residue Phe will contribute to the thermodynamic stability of
the interaction by hydrophobic core packing.
Results and Discussion
Two pathways have been envisaged for the preparation of 2,5-
diarylated thiophenes (2), differing only in the order of the two
different coupling reactions (Scheme 1). As illustrated in
Scheme 1, route A utilises C5-arylation of the substituted thio-
phene methyl ester 3a resulting in aryl-thiophene intermediate
4 followed by saponification to provide carboxylic acid 5. De-
carboxylative cross-coupling of acid 5 results in the formation
of the desired 2,5-diaryl substituted thiophene 2. Alternatively,
initial saponification of ester 3a in pathway B provides the
thiophene carboxylic acid intermediate 6 that can undergo de-
Relying on the bioisosterism of thiophene and benzene,[15]
we have designed a library of compounds related to the ter-
phenyl scaffold. The replacement of benzene with thiophene
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The required thiophene carboxylic acids underwent decar-
boxylative cross-coupling with a variety of aryl-bromides to
produce the corresponding arylated thiophenes (Table 1).[18b]
The protocol efficiently transforms 3-substituted thiophene car-
boxylic acid derivatives to a range of 2-aryl-substituted hetero-
aromatics in moderate to good yields (Table 1). We examined
electron-poor and -rich aryl bromides and the results illustrate
that both types of substituents are well tolerated in the reac-
tion.
Table 1. Synthesis of monoaryl substituted thiophenes.
Entry
R2
R1
Product
Yield[a] [%]
1
2
3
4
5
6
N-MeMs
N-MeMs
N-MeMs
Me
Me
Me
CN
7a
7b
7c
7d
7e
7 f
58
68
66
69
75
61
OMe
CO2Et
CN
OMe
CO2Et
[a] Isolated yields. Condition: thiophene carboxylic acid (2 equiv), aryl bro-
mide (1 equiv), Pd[P(tBu)3]2 (0.05 equiv), nBu4N+Clꢀ (1 equiv), Cs2CO3
(1.5 equiv), anhydrous DMF, 8 min microwave irradiation at 1708C.
Fagnou and co-workers have developed CꢀH activation re-
action conditions utilising a wide range of hetroaromatics and
aryl bromides.[17q] The products from the decarboxylative cross-
coupling reaction were subjected to these CꢀH activation con-
ditions to effect a regioselective CꢀH activation at the C5 posi-
tion of the 2-arylthiophene to generate 2,5-diaryl substituted
thiophenes. A range of aryl bromides were utilised and moder-
ate to excellent yields were obtained (Table 2). Moreover, the
electron density of the existing substituent aryl group on the
thiophene did not affect the yields.
Scheme 1. Comparison of the two synthetic pathways.
All compounds were initially investigated for their capacity
of modulating IAPP amyloid fibril formation by means of the
thioflavin T (ThT) fluorescence assay. ThT is a dye that fluores-
ces upon its binding to protein aggregates with a cross-b-
sheet structure, mostly fibrillar in morphology.[23] IAPP amyloi-
dogenesis is a nucleation-dependent polymerisation process
that is characterised by a ThT-negative phase (lag-phase;
around 6 h), in which the high-energy nucleus is formed, fol-
lowed by a thermodynamically favourable elongation phase
that is characterised by the rapid growth of ThT-positive fibrils
(Figure 2A). According to the helical intermediates described
above, the random coil!a-helix conformational conversion
occurred during the initial stage of the lag phase. Analysis of
the aggregation kinetics obtained by ThT fluorescence gave us
early mechanistic insights about the effects of these substitut-
ed thiophenes on IAPP amyloidogenic pathway.
carboxylative cross-coupling to afford aryl-thiophene 7. This is
followed by C5-arylation to provide the desired 2,5-diaryl sub-
stituted thiophene 2.
Initially, in order to compare the efficiency of each pathway,
both routes were carried out using the same substituted aryl-
bromides (2- and 3-bromobenzonitrile). Interestingly, both the
C5-arylation and decarboxylative cross-coupling steps in
route A resulted in lower yields compared to route B, giving
overall yields of 2,5-diaryl substituted thiophene 2a of 12 and
42% (R1 =CN, R3 =CN), respectively. In order to examine
whether the superior efficiency of route B was general, other
functionalised aryl halides were also employed in both path-
ways. Scheme 1 shows one other example using 2-bromoben-
zaldehyde and 3-bromoanisole in which, once again, a higher
overall yield was observed with route B compared to route A
(59 vs. 29%, respectively, R1 =OMe, R3 =CHO). Route B was
therefore chosen for the preparation of the remaining
analogues.
Among all compounds prepared in the course of this study,
compound 2l (Table 2, entry 16) slowed the formation of ThT-
positive aggregates, as observed by the increase of the lag-
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Table 2. Synthesis of monoaryl substituted thiophenes.
Entry
R3
R2
R1
Product
Yield[a] [%]
7
8
9
CN
CHO
CF3
CO2Et
CO2Et
CN
CF3
CHO
CF3
CN
H
CHO
CN
CF3
CO2Et
CHO
CF3
CN
CHO
CF3
Me
Me
Me
Me
Me
Me
Me
Me
OMe
OMe
OMe
OMe
CO2Et
CO2Et
CO2Et
CO2Et
CN
CN
CN
OMe
OMe
OMe
CN
2c
2d
2e
2 f
2g
2h
2i
2j
2k
2l
2m
2b
2n
2o
2p
2q
2r
2a
2s
2t
23
79
81
73
94
81
72
65
47
50
69
87
75
78
77
64
95
72
65
75
93
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Me
Me
Me
N-MeMs
N-MeMs
N-MeMs
N-MeMs
N-MeMs
N-MeMs
N-MeMs
N-MeMs
N-MeMs
N-MeMs
CN
CN
CN
CO2Et
CO2Et
CO2Et
CN
2u
[a] Isolated yields. Condition: heterocyclic thiophene (1 equiv), aryl halide
(2 equiv), Pd(OAc)2 (0.05 equiv), PCy3·HBF4 (0.1 equiv), PivOH (0.3 equiv),
K2CO3 (1.5 equiv), anhydrous DMF, 16 h thermal heating at 1008C.
phase period (Figure 2A) when the compound was used at an
equimolar ratio. Moreover, compound 2l showed concentra-
tion-dependence inhibition of the formation of IAPP ThT-posi-
tive aggregates, with a lag phase of 15 h at 8 molar equiva-
lents (100 mm; Figure 2B).
At 50 and 100 mm (4 and 8 equivalents, respectively) com-
pound 2l also decreased the final ThT fluorescence, suggesting
that a lower amount of IAPP amyloid fibrils were formed and/
or that these aggregates showed a less defined cross-b-sheets
quaternary structure. We also varied the concentration of ThT
fluorescent dye to confirm that this inhibitory effect was not
the result of a displacement of ThT binding to fibrillar aggre-
gates by compound 2l. Our results showed that in the pres-
ence of 10, 40 or 100 mm ThT, the increase of the lag-phase
period observed with 12.5 mm of compound 2l was very simi-
lar (data not shown), strongly suggesting that this molecule
was, indeed, slowing down the amyloidogenic process. The
mechanism by which this 2,5-diaryl substituted thiophene de-
celerates and partially inhibits IAPP amyloid formation is cur-
rently under investigation based on these interesting prelimi-
nary results. In contrast, most of the other molecules prepared
had little or no effect on the kinetics of IAPP amyloid forma-
tion, as represented by compound 2d (Figure 2A; see the Sup-
porting Information for additional results). Nonetheless, several
of these compounds (compounds 2i, 2j, 2n, 2o, 2p and 2t)
Figure 2. Effects of 2,5-diaryl substituted thiophenes on IAPP kinetics of
amyloid fibril formation monitored by ThT fluorescence. IAPP (12.5 mm) was
incubated in 20 mm Tris, pH 7.4, at 258C without agitation in the absence
&
*
(A, B and C; ) or in the presence of 12.5 mm of compound 2d (A; ),
~
12.5 mm compound 2l (A; ), increasing molar equivalent of compound 2l
~
&
(B), 12.5 mm of compound 9a (C; ) or 12.5 mm compound 9c (C; ). ThT
fluorescence (40 mm) was measured every 10 min over the course of 25 h,
with excitation at 440 nm and emission at 485 nm.
increased the final ThT fluorescence without affecting the lag
phase or the rate of amyloid fibrils formation (see the Support-
ing Information).
IAPP is a positively charged peptide that displays three posi-
tive charges at physiological pH, thus favouring electrostatic
interactions with negatively charged molecules. Particularly, we
designed several mono- and diacid aryl substituted thiophenes
to target one side of the transient IAPP helix that exhibits a hy-
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Table 3. Mono- and diacid aryl substituted thiophenes.
Entry
R3
R2
Product
28
29
30
31
CN
Me
Me
Me
N-MeMs
9b
9c
9d
9e
CF3
CHO
CF3
drophobic region (Phe15) surrounded by polar and/or charged
residues (Arg11 and His18; Table 3).
As suspected, acid(s)-functionalised thiophenes showed pro-
found effects on IAPP amyloidogenesis at a 1:1 molar ratio. In-
terestingly, monoacid substituted thiophenes with a methyl
group at position R2 (compounds 9b, 9c and 9d) virtually
abolished the lag phase without significantly affecting the final
ThT fluorescence. This aggregation kinetic suggests that these
compounds induce the formation of IAPP aggregates with
lower ThT-binding capacities, indicative of non-fibrillar struc-
ture (Figure 2C). In sharp contrast, the diacid analogue (com-
pound 9a) reduced the lag phase and led to a significant in-
crease of the final ThT fluorescence (Figure 2C). This suggests
that a larger amount of amyloids was formed in the presence
of 1 equivalent of compound 9a and/or that these amyloid fi-
brils exhibit a better-defined cross-b-sheet quaternary struc-
ture. These possibilities are currently under investigation. To
probe if the accelerating effects of the mono- and diacid aryl
substituted thiophenes on IAPP amyloidogenesis are simply
a result of non-specific charge neutralisation effects, we used
benzoic acid as a control compound. The amyloids formation
kinetic data obtained in the presence of 12.5 mm (1 equivalent)
and 125 mm (10 equivalent) of benzoic acid are very similar to
the control (see the Supporting Information). Together, these
data indicate that the negative charge(s) on the thiophene
scaffold are crucial for the modulating activity and that the
nature and/or the position of other substituents also play
a key role, suggesting specific interactions. We are currently in-
vestigating the mechanisms by which these derivatives modu-
late the formation of amyloid fibrils.
Figure 3. Effects of 2,5-diaryl substituted thiophenes on IAPP-induced toxici-
ty on pancreatic b-cells. A) INS-1 cells were treated with concentrations of
IAPP ranging from 0 to 100 mm for 24 h and cell viability was measured by
the resazurin reduction assay and compared to cells treated with vehicle
only (100% cell viability). B) INS-1 cells were treated with 50 mm IAPP that
had been pre-incubated for 20 h in 20 mm Tris, pH 7.4, 258C, in the absence
or presence of one molar equivalent of the thiophene derivatives. After 24 h
incubation, cell viability was measured.
this compound stimulates the formation of poorly toxic IAPP
quaternary species, mostly fibrillar, according to the high ThT
fluorescence observed (Figure 3B). It is noteworthy that all
tested compounds were not toxic on b-pancreatic cells when
used at a concentration of 50 mm. In this study, the cytotoxicity
assays were performed with IAPP that has been pre-incubated
for 20 h without or with compounds, since we wanted to ini-
tially evaluate the cytotoxicity of the species (quaternary and/
or monomeric) that are present when the ThT fluorescence
plateau is reached. We are currently assessing the cytotoxicity
of intermediates that are generated during the different amy-
loidogenic phases (lag, growth and plateau) in the presence of
these thiophene derivatives. Although these results are prelimi-
nary and biophysical investigations are in progress to delineate
the mechanisms by which these molecules interfere with IAPP
amyloidogenic process, this study demonstrates that we can
modulate not only the kinetics of amyloid fibril formation of
an amyloidogenic peptide, but also its cytotoxicity with small
molecules that were designed to mimic/target the transient
helical motif.
Subsequently we analysed the cytotoxicity of IAPP species
that has been pre-incubated for 20 h in the absence or pres-
ence of selected 2,5-diarylthiophene derivatives. We and
others have previously reported that IAPP induces death of
pancreatic cells when the amyloidogenic peptide is directly
added to the cell culture medium.[4,24] In fact, IAPP pre-incubat-
ed for 20 h without compounds decreased pancreatic b-cells
viability in a concentration-dependant manner (Figure 3A).
When IAPP was pre-incubated with 1 molar equivalent of
either compound 2d, 2l or 9c, no changes in the proteotoxic
effects induced by 50 mm IAPP were observed (Figure 3B).
However, pre-incubation of IAPP with the diacid substituted
thiophene (compound 9a) before cell treatment totally abol-
ished the cytotoxic effects of IAPP. This result suggests that
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Conclusion
Acknowledgements
A modular approach has been developed for the synthesis of
highly functionalised 2,5-diaryl substituted thiophene scaffolds
utilising palladium mediated cross-coupling reactions. The
strategy allows us to quickly construct ligands in an efficient
manner for screening towards the interaction with and stabili-
sation of a-helices. In this effort, the ligands were assessed for
their capacity to modulate IAPP amyloidogenesis and cytotox-
icity on b-pancreatic cells. The preliminary results demonstrat-
ed that some of the molecules could act as modulators of IAPP
amyloidogenesis by increasing or decreasing the lag-phase
period of IAPP amyloid fibril formation. Investigations are in
progress to better understand the mechanism by which these
molecules interact with IAPP. As several amyloidogenic natively
disordered (poly)peptides, including the amyloid-b peptide,
calcitonin and a-synuclein, populate helical intermediates
during the initial phase of fibril formation, these 2,5-diaryl sub-
stituted thiophenes could ultimately lead to the development
of novel therapeutics for protein amyloid-related diseases.
We would like to thank the Natural Sciences and Engineering
Research Council (NSERC) of Canada and Le Fonds de Recher-
che du Quꢃbec, Nature et Technologies (FQRNT) and Pharma-
qam for financial support. C.A.D.C. is a recipient of a student-
ship from the Groupe de Recherche Axꢃ sur la Structure des
Protꢃines (GRASP). Support was also kindly provided by Boeh-
ringer Ingelheim, Merck Frosst, Wyeth, and Concordia Universi-
ty. We thank Dr. Simon Woo for reading this manuscript and
helpful suggestions. We also thank Dr. Alexey Denisov and
Jean-Pierre Falgueyret for useful discussions and access to
equipment.
Keywords: amyloids
arylation · peptidomimetics · thiophenes
·
decarboxylative arylation
·
CꢀH
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Experimental Section
Procedure for decarboxylative cross-coupling
The procedure employed by Forgione and co-workers was used
with slight modifications.[18b] In a 2–5 mL, open to air, oven dried
microwave vial were added the heterocyclic carboxylic acid
(2 equiv), aryl bromide (1 equiv), tetra-n-butylammonium chloride
(1 equiv), cesium carbonate (1.5 equiv), bis(tri-tert-butylphosphine)-
palladium(0) (0.05 equiv) and anhydrous DMF (0.1m of the aryl bro-
mide solution). The vial was capped with a septum and the mix-
ture was pre-stirred for 30 s at 238C and submitted to microwave
heating at 1708C for 8 min with stirring and the high absorption
setting. The crude mixture was cooled to 238C and was filtered
over Celiteꢁ. The solution was then diluted with EtOAc and the or-
ganic layer was washed with a saturated NaCl aqueous solution
(3ꢂ), saturated NaHCO3 aqueous solution (2ꢂ), water (1ꢂ), and sa-
turated NaCl aqueous solution (1ꢂ). The aqueous phases were
combined and extracted with EtOAc. The combined organic
phases were dried over sodium sulfate, and after filtration the sol-
vent was evaporated to provide the crude compound.
Procedure for CꢀH activation
A procedure employed by Fagnou and co-workers was used with
slight modifications.[17q] An oven-dried vial equipped with a mag-
netic stir bar was charged with heterocycle (1 equiv), aryl bromide
(2 equiv), PCy3·HBF4 (0.1 equiv), PivOH (0.3 equiv), K2CO3 (1.5 equiv),
and palladium(II) acetate (0.05 equiv). Anhydrous DMA (0.08m of
the heterocycle solution) was added. Liquid aryl bromides were
added after the addition of solvent. The mixture was heated for
16 h at 1008C. After being cooled to 238C, the reaction mixture
was diluted with EtOAc and filtered through a pad of Celite. The
filtrate was washed with a saturated NaCl aqueous solution (3ꢂ),
saturated NaHCO3 aqueous solution (2ꢂ; unless otherwise stated),
water (1ꢂ), and saturated NaCl aqueous solution (1ꢂ). The aque-
ous phases were combined and extracted with EtOAc. The com-
bined organic phases were dried over sodium sulfate, and after fil-
tration the solvent was evaporated to provide the crude
compound.
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Received: October 7, 2013
Published online on February 2, 2014
Chem. Eur. J. 2014, 20, 2522 – 2528
www.chemeurj.org
2528
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim