Structure-Activity Relationships of Organofluorine Inhibitors of β-Amyloid Self-Assembly

Article abstract of DOI:10.1002/cmdc.201100569

A broad group of structurally diverse small organofluorine compounds were synthesized and evaluated as inhibitors of β-amyloid (Aβ) self-assembly. The main goal was to generate a diverse library of compounds with the same functional group and to observe general structural features that characterize inhibitors of Aβ oligomer and fibril formation, ultimately identifying structures for further focused inhibitor design. The common structural motifs in these compounds are CF₃-C-OH and CF₃-C-NH groups that were proposed to be binding units in our previous studies. A broad range of potential small-molecule inhibitors were synthesized by combining various carbocyclic and heteroaromatic rings with an array of substituents, generating a total of 106 molecules. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy for monitoring fibril formation, biotinyl Aβ_1-42 single-site streptavidin-based assays for observing oligomer formation, and atomic force microscopy for morphological studies. These assays revealed a number of structures that show significant inhibition against either Aβ fibril or oligomer formation. A detailed analysis of the structure-activity relationship of anti-fibril and -oligomer properties is provided. These data present further experimental evidence for the distinct nature of fibril versus oligomer formation and indicate that the interaction of the Aβ peptide with chiral small molecules is not stereospecific in nature.

Full text of DOI:10.1002/cmdc.201100569

MED
DOI: 10.1002/cmdc.201100569
Structure–Activity Relationships of Organofluorine
Inhibitors of b-Amyloid Self-Assembly
Bꢀla Tçrçk,*[a] Abha Sood,[a] Seema Bag,[a] Aditya Kulkarni,[a] Dmitry Borkin,[a]
Elizabeth Lawler,[a] Sujaya Dasgupta,[a] Shainaz Landge,[a] Mohammed Abid,[a]
Weihong Zhou,[a] Michelle Foster,[a] Harry LeVine, III,[b] and Marianna Tçrçk*[a]
A broad group of structurally diverse small organofluorine
compounds were synthesized and evaluated as inhibitors of b-
amyloid (Ab) self-assembly. The main goal was to generate a
diverse library of compounds with the same functional group
and to observe general structural features that characterize in-
hibitors of Ab oligomer and fibril formation, ultimately identify-
ing structures for further focused inhibitor design. The
common structural motifs in these compounds are CF3-C-OH
and CF3-C-NH groups that were proposed to be binding units
in our previous studies. A broad range of potential small-mole-
cule inhibitors were synthesized by combining various carbo-
cyclic and heteroaromatic rings with an array of substituents,
generating a total of 106 molecules. The compounds were
tested by standard methods such as thioflavin-T fluorescence
spectroscopy for monitoring fibril formation, biotinyl Ab1–42
single-site streptavidin-based assays for observing oligomer
formation, and atomic force microscopy for morphological
studies. These assays revealed a number of structures that
show significant inhibition against either Ab fibril or oligomer
formation. A detailed analysis of the structure–activity relation-
ship of anti-fibril and -oligomer properties is provided. These
data present further experimental evidence for the distinct
nature of fibril versus oligomer formation and indicate that the
interaction of the Ab peptide with chiral small molecules is not
stereospecific in nature.
Introduction
Protein deposits in the form of neurofibrillary tangles and amy-
loid plaques are the hallmarks of Alzheimer’s disease (AD).[1,2]
The major component of extracellular amyloid plaques is the
b-amyloid (Ab) peptide.[3] One of the suggested therapeutic
strategies for treating AD is inhibition of the amyloid cascade,[4]
and many inhibitors of Ab self-assembly have been identified.
These include small organic molecules, peptides, peptidomi-
metics, and proteins.[5] Recent studies have indicated that the
soluble oligomeric aggregates of Ab are more neurotoxic than
the fibrillar end-products of the process.[6] Therefore, it has
become imperative to distinguish between molecules that in-
hibit oligomerization, fibril formation, or both. Many small-mol-
ecule anti-amyloidogenic compounds have been categorized,
and the underlying oligomer structures characterized through
the use of conformation-specific antibodies.[7–9] Several ac-
counts[4,5,10] on the development of inhibitors active against fi-
brillogenesis and oligomer assembly serve as an excellent
source of information. However, one cannot overlook the fact
that the literature is far from systematic regarding the chemical
nature of inhibitors. Most original studies focus on a single
compound, or a small group of compounds with no clear indi-
cation as to why the compounds were selected. Moreover, ra-
tional extended structure–activity relationship studies outside
the pharmaceutical industry are quite rare.[11,12] Although the
target (fibrils, oligomers, etc.) is usually specified, frequently
there is little indication about the type of interaction that
occurs between the inhibitor and the peptide.
Our chemistry-based approach is intended to fill this gap.
While many approaches in the search for potential inhibitors
were discovery based, our design of a core structure was
based on published data. In an earlier study we described a
new class of organofluorine molecules as Ab fibrillogenesis in-
hibitors.[13] These compounds have been found to be active in
the disassembly of preformed fibrils as well.[14] In a study in-
cluding chiral isomer pairs of the same compounds, it was also
observed that individual chirality does not appear to result in a
significant difference in the action of these compounds.[15]
However, while providing interesting information and effective
anti-fibril compounds, these studies were limited in scope;
only a few compounds with closely related structural features
were included. In continuation of our work on anti-AD com-
pounds, we designed and synthesized a broad range of orga-
nofluorine molecules with one common motif (CF3-C-XH,
[a] Prof. B. Tçrçk, A. Sood, Dr. S. Bag, Dr. A. Kulkarni, Dr. D. Borkin, E. Lawler,
Dr. S. Dasgupta, Dr. S. Landge, Prof. M. Abid, W. Zhou, Prof. M. Foster,
Prof. M. Tçrçk
Department of Chemistry, University of Massachusetts Boston
100 Morrissey Boulevard, Boston, MA 02125 (USA)
E-mail: bela.torok@umb.edu
marianna.torok@umb.edu
[b] Prof. H. LeVine, III
Department of Molecular and Cellular Biochemistry
Chandler School of Medicine and Center on Aging
University of Kentucky, Lexington, KY 40536 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100569.
910
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 910 – 919

Ab Self-Assembly Inhibitors
MED
where X=O,N), yet considerable structural diversity. This func-
tionality was found to be crucial for activity; removal of either
the CF3 or OH group resulted in completely inactive com-
pounds.[13] The compounds were evaluated in fibril and oligo-
mer inhibition and disassembly assays. Herein we describe a
broad structure–activity relationship study of these organo-
fluorine compounds as potential anti-AD agents.
lyzed hydroxyalkylation[18,20] to confirm the effect of inhibitor
stereochemistry on anti-aggregation potency, especially be-
cause only the anti-fibril effect was described in our earlier
report.[15]
For the synthesis of trifluoromethyl amino acid esters and
additional larger compounds, multistep methods were ap-
plied.[21,22] These procedures are summarized in Scheme 2. The
syntheses provided the products in good to excellent yields,
and in the case of the amino acid esters, the optical purities of
the products were also high (up to 98% ee). Notably, several of
these synthesized molecules are new compounds, whereas
others were previously reported by our research group.[16–21]
Therefore, other than the few compounds that appear in our
previous publication on anti-fibrillogenesis compounds, none
have been tested and described in Ab aggregation inhibition
studies until now.
Results
Various substituted/unsubstituted monocyclic/bicyclic aromat-
ic/heteroaromatic molecules such as benzene, pyrrole, furan,
and indole were derivatized by using commercially available
trifluoromethyl hydroxyalkylating agents: ethyl trifluoropyru-
vate (TFP), ethyl trifluoroacetoacetate (TFAA), hexafluoroace-
tone (HFA), and trifluoroacetaldehyde ethylhemiacetal (TFAE).
The basic synthetic procedures for the preparation of these
compounds are summarized in Scheme 1.
Overall we completed the synthesis of 106 structurally relat-
ed compounds. All these products possess a CF3-C-OH or CF3-
C-NH unit as a common feature; otherwise the compounds are
structurally diverse. It was our intention to observe the role
that aromatic/heteroaromatic groups and their substituents
may play in the course of fibril formation. In addition, the
effect of double substitution was also tested, namely, whether
the presence of two CF3-C-OH units in one compound is bene-
ficial or disadvantageous for the biological effect.
A common feature to all syntheses in Scheme 1 is that every
process occurs in one step, with commercially available hy-
droxyalkylating agents. In some cases, we used our own meth-
ods reported earlier;[16–18] for the remaining products, analo-
gous published methods were applied.[19] A few selected chiral
compounds were also synthesized by a cinchona-alkaloid-cata-
Following the syntheses, initial biochemical tests were car-
ried out. Because both fibrillar aggregates and soluble oligo-
meric species of Ab are neurotoxic, the inhibitory activity of
the compounds was determined against both forms of self-as-
sembly products. Our goal was to use Ab1–40, as it is the most
abundant form of the peptide and readily forms fibrils. Howev-
er, Ab1–42 was used for oligomers, because Ab1–40 forms oligo-
mers poorly at the low concentration (10 nm) used to avoid
fibril formation unless a stimulant is applied. The efficacy of
the inhibitors against fibrillogenesis was evaluated by the com-
monly applied quantitative thioflavin-T (THT) fluorescence
spectroscopy assay.[23–25] The calculated intensity values are
based on maximum fluorescence intensities in the l 480–
490 nm emission region (lex =435 nm) after subtracting the
background fluorescence of the starting solutions (0 h). The
samples were incubated for four days, THT measurements
were made at the plateau phase of fibril assembly, and the
data obtained with the inhibitor-containing samples were
compared with those of inhibitor-free controls. The assays
were carried out using an Ab/inhibitor ratio of 0.1 at an Ab
concentration of 100 mm; thus the original inhibitor concentra-
tion was at 1 mm (except compounds 89–93, which were
tested at a 1:1 molar ratio, thus at 100 mm, due to solubility
problems). The data along with the compound structures are
summarized in Figure 1. The anti-oligomer activities of the
compounds were also determined using the quantitative bio-
tinyl-Ab1–42 single-site streptavidin-based assay.[26,27] Samples
were incubated for 30 min in the assays. These assays were
carried out at an Ab/inhibitor ratio of 0.0002, with Ab at
0.01 mm. The efficacy of the compounds was determined at
these given concentrations. The measured intensities of the in-
hibitor-containing samples (Isample) were normalized to the con-
Scheme 1. One-step syntheses of a diverse series of aryl CF3-C-OH group-
containing compounds from commercially available reagents.
ChemMedChem 2012, 7, 910 – 919
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
911

B. Tçrçk, M. Tçrçk, et al.
MED
in which ITHT is the fluorescence intensity of the inhib-
itor-containing sample expressed as a percentage of
control, P is the inhibitor/Ab molar ratio, EC50 is the
median inhibitor constant, and ECmax is the maximum
inhibition. A double-reciprocal plot of Equation (2)
allows the determination of EC50. Because inhibitor/
Ab molar ratios were applied in the formula, the EC50
values were obtained as a ratio as well. Multiplying
the obtained ratio with the Ab concentration of
100 mm provided the values in concentration units
(mm).
The most active compounds and their EC50 data
are listed in Table 1. The data show that Group I and
II compounds are the most effective fibrillogenesis in-
hibitors, whereas inhibitors of oligomer assembly
appear in several groups. A detailed analysis is pro-
vided in the Discussion section below to point out
structural similarities among the active compounds.
Atomic force microscopy (AFM) was also used to con-
firm the THT assay data. Several illustrative AFM
images of a control sample and inhibitor samples are
depicted in Figure 2.
The data observed in the AFM images are in good
agreement with the data from the THT fluorescence
measurements (Figure 1). The control sample
(Figure 2, control) shows the expected, well-devel-
oped network of mature fibrils. Images obtained in
the presence of 1, 5, 44, and 46, individually, indicate
extensive fibril formation and hence little inhibition.
This is exactly what was observed in the quantitative
assay; these compounds have practically no effect on
fibril formation. Indeed the highest value, observed
with 1 (11% inhibition), is still negligible. Compounds
44 and 46 were found to be promoters of fibril for-
mation, resulting in visually denser fibrillar morpholo-
gy.
Scheme 2. Syntheses of aryl trifluoromethyl amino acid esters and larger compounds
with CF3-C-XH group (X=O,N) by multistep approaches.
Compounds 2, 3, and 4 possess similar structure;
all of them are in Group I (Scheme 2, Figure 1). These
compounds exhibited significant inhibition in the
trol sample (Icontrol) containing Ab only. The percent values by
which a compound decreased the expected signal (control) are
listed in Figure 1 as percent inhibition.
Isample
Icontrol
Table 1. EC50 data for the most active compounds in Ab fibrillogenesis
and oligomer assembly assays.[a]
ð1Þ
% Inhibition ¼ 100ꢀ
ꢁ 100
Compound
EC50 fibril [mm]
EC50 oligomer [mm]
In some cases compounds promoted self assembly, thus
sample >Icontrol, and therefore negative percent inhibition values
2
3
4
380ꢂ1.8
250ꢂ4.7
190ꢂ0.07
>1000
50[13]
>100
N/A
N/A
53ꢂ3.5
>100
>100
I
are listed. Compounds with significant activity in the screening
assay were titrated, and the EC50 values were determined. The
EC50 calculations were carried out as previously described.[13]
Fluorescence intensity versus molar ratio functions were used
to determine the relative potency of inhibitors using a simple
equation, similar to the analysis of Michaelis–Menten kinetics
or ligand binding to macromolecules [Eq. (2)]:[13]
22
29
30
31
43
64
79
90
92
20[13]
30[13]
>1000
N/A
N/A
N/A
>100
28ꢂ2.8
15ꢂ1.4
60ꢂ10.6
19ꢂ5.1
23ꢂ4.9
N/A
ECmax
EC50 þ P
P
ð2Þ
ITHT ¼ 100ꢀ
[a] N/A: either no inhibition or promoter.
912
www.chemmedchem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 910 – 919

Ab Self-Assembly Inhibitors
MED
Figure 1. General structure of the compounds used in the current study and their activity in Ab aggregation assays (FI: fibril inhibition; OI: oligomer inhibi-
tion).
Discussion
THT assays in increasing order (2: 82%, 3: 93%, 4: 100%), and
this trend can be followed in Figure 2 as well. Ever fewer fibrils
appear in the images from 2 to 3, and there are practically no
fibrils in the presence of compound 4. Other compounds (8
and 9) showed moderate inhibition (34–47%), and this is re-
flected in the AFM images. The dense fibrillar network charac-
teristic of the control is less frequent with these compounds;
however, a significant amount of fibrils is still present in the
images. Visual analysis of images obtained with other inhibi-
tors also indicate strong inhibition (compounds 37, 42, 67, 69,
80, and 81). It also reveals that in the presence of inhibitors,
aggregates with various morphologies can form. In many cases
the obtained fibrils are nearly identical to those observed in
the control sample, although less dense in appearance. In
some cases (Figure 2: 3, 67, and 69) only a few identifiable fi-
brils are present. In other cases, however, the aggregates
appear short (Figure 2: 42 and 80) or emerge in the form of
round-shaped deposits (Figure 2: 81), indicating that strong
polymorphism can occur from the presence of these inhibitors.
Analysis of the above data indicates that several compounds in
the synthesized compound library show strong activity against
the formation of either fibrils or oligomers. This phenomenon
appears to support earlier suggestions that not all stable oligo-
mers are obligatory precursors to the fibrils and that the two
processes can occur in parallel pathways. Therefore, a given
compound may affect one process or the other, and not neces-
sarily both.[7–9] The structure–activity relationships are discussed
separately for fibril and oligomer inhibition.
Fibrillogenesis inhibitors
The most potent fibril inhibitors were compounds 2–4 and 6
from Group I, bearing the (CF3)2-C-OH motif, whereas several
CF3(COOEt)-C-OH-containing compounds (31, (S)-31, and (R)-
31) showed the strongest and nearly identical activity. Other
functional groups typically imparted weaker inhibitory poten-
tial, with inhibition values up to ~50%. These data are consis-
tent with our previous hypothesis that the acidity of the inhibi-
ChemMedChem 2012, 7, 910 – 919
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
913

B. Tçrçk, M. Tçrçk, et al.
MED
Figure 2. Atomic force microscopy images of Ab1–40 samples incubated without (control) and with the indicated test compounds (see Figure 3) for four days.
tors is a crucial factor in the mode of action of these com-
pounds; the more acidic the OH group, the greater the poten-
cy. The order of acidic strength and, in parallel, inhibitor activi-
ty of the various motifs used is illustrated in Figure 3.
ocyclic aryl group, particularly indole over carbocyclic rings.
Notably, whereas the carbocyclic derivatives of Group I (16–18)
showed weak inhibitory effects, similar compounds from all
other groups were found to be self-assembly promoters (44–
48 and 75–78). In summary, indole-based inhibitors showed
strong fibril-inhibition properties, while other aromatic units
were modest inhibitors at best, and often acted as promoters
of fibrillogenesis.
The role of the aromatic groups also appears important. In
every group of compounds tested, the indolyl derivatives were
found to be the most active followed by the pyrrole-based
compounds. The weakest (or no) effect was consistently ob-
served for the simple carbocyclic molecules (benzene deriva-
tives). This observation highlights the significance of the heter-
Based on this observation, our discussion on the role of indi-
vidual substituents is focused on indole derivatives. Group I
and II compounds are the most promising fibril inhibitors; the
general activities of other compounds, while consistent, show
a steady decrease as a function of the acidity of the CF3-C-XH
group (X=O,N; Figure 3). For Group II compounds, the 5-halo-
gen-substituted (R3 substituent) indoles were found to be the
best inhibitors, in the order of F<Cl<Br<I. The same order
was observed in the case of the newly synthesized Group I
molecules. Compound 4 (R3 =I) showed 100% fibril inhibition
Figure 3. The rank order of acid strength of the major types of inhibitors
used.
914
www.chemmedchem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 910 – 919

Ab Self-Assembly Inhibitors
MED
under the experimental conditions, whereas the Br- and Cl-
substituted inhibitors showed decreasing but still high efficacy
(93 and 82%). Therefore, it appears that the presence of a
larger halogen atom at the R3 position of the indole ring in-
creases the inhibitory activity toward fibril formation. However,
substitution of the halogens with bulky electron-withdrawing
groups such as -COOMe, -CN, and -CONH2 at the R3 position of
indoles decreases the ability of these compounds to inhibit the
formation of fibrils. Although substituents at the other posi-
tions show minor effects, they do not appear to significantly
alter inhibitor activity.
tion of Ab assembly, given the experimental error of the fibril
growth and analytical processes. Chiral compounds in our cur-
rent set of molecules are included in Groups II and V. The re-
sults obtained with these compounds support our earlier con-
clusions.[15] The differences between the inhibitory effect of
enantiomers and racemic mixtures mostly fall within a 0–20%
range (29–5%, 30–7%, 31–2%, 82–13%, and 83–18%), al-
though in a few cases it is greater than that. Notably, similar
observations were made in the inhibition of both fibrillogene-
sis and oligomer assembly. Although more data using structur-
ally diverse compounds are clearly needed to provide a defi-
nite answer to this problem, the results presented herein sug-
gest that the interaction of chiral small molecules with Ab is
most likely not stereo- or enantiospecific.
Oligomerization inhibitors
Analysis of the molecular features that result in effective oligo-
mer inhibitors leads to the observation that, in contrast to fi-
brillogenesis inhibition, the acidity of the CF3-C-OH motif does
not appear to be of primary importance. Interestingly, the typi-
cally good fibril inhibitors (Groups I and II) show poor perfor-
mance in oligomer inhibition assays, providing further evi-
dence in support of the earlier findings that fibrils and stable
oligomers do not form via the same pathways.[7,8] In fact, in
certain cases these compounds promote oligomer formation.
Effective oligomer inhibitors were found in all groups except
Group I ((CF3)2-C-OH derivatives). Interestingly, from the groups
of typical fibril inhibitors (I and II) only one compound showed
significant oligomer inhibition (22, EC50 =53 mm), and this com-
pound is a weak fibril inhibitor. The most active inhibitors of
oligomer assembly are those with multiple active CF3-C-OH
substituents (43, 90, and 92), showing better EC50 values (19–
25 mm). Similar to the fibrillogenesis inhibitors, compounds
with carbocyclic (benzene) rings are inactive in the inhibition
of oligomer assembly. All effective inhibitors possess heterocy-
clic rings. Based on the molecular structures of compounds
listed in Table 1 for oligomer inhibitors, a single, well-defined
relationship cannot be made. However, the double CF3-C-OH
units, and in certain cases the larger size or more aromatic
rings, suggest that for oligomer inhibition the presence of aro-
matic groups and the possibility of p–p interactions[28–30] is
more likely to be a decisive feature than the presence of acidic
groups. It is also supported by data obtained with the carbocy-
clic compounds. These compounds (44–48 and 75–78) mostly
promoted fibril formation, in contrast to their mild-to-moder-
ate (76–28% and 77–40%, respectively) inhibition of oligomer
assembly, highlighting the importance of p–p stacking.[28–30]
The effect of chirality on the inhibition of Ab self-assembly
has also attracted considerable attention.[15,28,31,32] Earlier results
obtained with peptide-based[28] versus small-molecule inhibi-
tors[15,31] appear to be controversial. While the individual
(amino acid) chirality is of key importance for peptide inhibi-
tors,[28,32] it did not appear to be so for small-molecule inhibi-
tors. In a recent work it was stated that chirality is an impor-
tant feature for methoxytacripyrines as inhibitors.[33] Analysis of
the published data, however, led us to the conclusion that the
activity differences between the enantiomers or racemic mix-
tures, while considerably high for human acetylcholinesterase
(hAChE) inhibition, are rather insignificant (<10%) for inhibi-
Conclusions
In summary, the synthesis and activity evaluation of a set of
106 structurally diverse compounds with the same motif (CF3-
C-X; X=OH,NH2) resulted in valuable information for the fur-
ther design of Ab self-assembly inhibitors. An earlier observa-
tion regarding the importance of the acidity of the OH group
in fibrillogenesis inhibition was confirmed, and new lead com-
pounds 2–4 were identified. It was also observed that acidity is
a relatively unimportant characteristic of the compounds in
the inhibition of oligomer assembly. Instead, active oligomer
inhibitors feature dual binding groups and more electron-rich
aromatic units, emphasizing that ability to participate in p–p
interactions is also a dominant aspect of these compounds.
Experimental Section
Chemistry
General information: The cinchona alkaloids were purchased from
Fluka and used without further purification. Indole derivatives and
ethyl 3,3,3-trifluoropyruvate, hexafluoroacetone trihydrate, trifluor-
oacetaldehyde ethylhemiacetal, substituted anilines, and benzalde-
hydes were obtained from Aldrich. CDCl3 used as a solvent (99.8%)
for NMR studies was from Cambridge Isotope Laboratories. The
19F NMR reference compound CFCl3 was purchased from Aldrich.
Other solvents used in synthesis, with minimum purity of 99.5%,
were purchased from Fisher. K-10 montmorillonite, a solid acid
used as catalyst, was obtained from Fluka. Mass spectrometric
identification of the products was carried out with an Agilent 6850
gas chromatograph – 5973 mass spectrometer system (70 eV elec-
tron impact ionization) using a 30-m-long DB-5 column (J&W Sci-
1
entific). H, 13C, and 19F NMR spectra were obtained on a 300 MHz
superconducting Varian Gemini 300 NMR spectrometer, in CDCl3
solvent with tetramethylsilane and CCl3F as internal standards. The
temperature was kept at 25ꢂ18C by a Varian temperature control
unit. Determination of enantiomeric excess was carried out by
chiral HPLC analysis using a Jasco PU-2080 HPLC coupled with a
PU-2075 UV/Vis detector. The samples were analyzed in hexane/
iPrOH (95:5) mobile phase using a Chiralcel OJ-H (Daicel) analytical
column at l 260 nm.
Synthesis of inhibitor candidates: Compounds used in this study
were synthesized using published methods[16–22] or as described
below. In each case, compounds were purified by flash chromatog-
ChemMedChem 2012, 7, 910 – 919
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
915

B. Tçrçk, M. Tçrçk, et al.
MED
raphy or preparative thin-layer chromatography (TLC). The identifi-
cation and purity determination were carried out by GC–MS and
NMR spectroscopy (1H, 13C, and 19F when applicable). The known
compounds showed NMR and MS characteristics identical to pub-
lished data (see Supporting Information).
(0.0375 mmol) were placed into a glass reaction vessel and Et2O
(3 mL) was added. The mixture was stirred at ꢀ88C (salt–ice cool-
ing bath) for 30 min. Ethyl 3,3,3-trifluoropyruvate (0.75 mmol) was
then added, and the mixture was stirred at ꢀ88C (salt–ice cooling
bath) for an additional 3 h, and the progress was monitored by
TLC. After reaction completion, the solvent and excess TFP were re-
moved by evaporation. The mixture was then dissolved in Et2O,
and the cinchona catalyst was removed by a treatment with K-10
montmorillonite (500 mg; a solid acid). After treatment, the alka-
loid–K-10 complex was removed by filtration, and the solvent was
evaporated. A colorless solid was obtained in 98% yield. The enan-
tiomeric excess of the product was determined by HPLC (see
below). The product purity was >98% (86–93% ee).
General procedure for the synthesis of 1,1,1,3,3,3-hexafluoro-2-
(indol-3-yl)propan-2-ols 1–12: A microwave reaction vial contain-
ing molecular sieves (4 ꢂ, 200 mg) was charged with indole
(1 mmol) and HFA·3H2O (1.5 mmol, 205 mL). The contents of the
vial were irradiated in a CEM Discover microwave reactor for
10 min at 1008C. Reaction progress was monitored by GC–MS.
After reaction completion, CH2Cl2 was added to the mixture. The
content of the vial was filtered into a round-bottom flask and con-
centrated in vacuo. The products were isolated as crystals or oils
and purified by flash chromatography if the purity by GC–MS was
>98%.
General procedure for the synthesis of ethyl 3,3,3-trifluoro-2-hy-
droxy-2-phenylpropanoates 44–48: Arene (1 mmol) and
HFA·3H2O (1.5 mmol) were added to a pressure tube containing
CH2Cl2 (3 mL) under N2 atmosphere. Trifluoromethanesulfonic acid
(2 equiv) was added to the reaction mixture dropwise. The reaction
mixture was stirred for 16 h, after which the contents were poured
into 5 mL H2O and extracted into CH2Cl2. After the evaporation of
solvents, the residue was purified by flash chromatography to give
the final product.
General procedure for the synthesis of 1,1,1,3,3,3-hexafluoro-2-
(1H-pyrrol-2-yl)propan-2-ols 13–15: A microwave reaction vial
containing molecular sieves (4 ꢂ, 200 mg) was charged with pyr-
role (1 mmol) and HFA·3H2O (1.5 mmol, 205 mL). The content of
the vial was irradiated in a CEM Discover microwave reactor for
10 min at 808C. Reaction progress was monitored by GC–MS. After
reaction completion, CH2Cl2 was added to the mixture. The content
of the vial was filtered into a round-bottom flask and concentrated
in vacuo. The products were isolated as crystals or oils and purified
by flash chromatography if the purity by GC–MS was >98%.
General procedure for the synthesis of ethyl 4,4,4-trifluoro-3-hy-
droxy-3-(1H-indol-3-yl)butanoates 49–59: A solution of indole
(0.5 mmol), ethyl 4,4,4-trifluoro-3-oxobutanoate (TFAA, 0.5 mmol)
and K-10 montmorillonite was mixed in toluene (3 mL) in a Teflon
screw-cap pressure tube. The reaction mixture was heated and
stirred at 608C, and reaction progress was monitored by TLC. After
satisfactory conversion, the product mixture was separated from
catalyst by filtration. The product was purified by flash chromatog-
raphy and isolated as crystals or oils.
General procedure for the synthesis of 1,1,1,3,3,3-hexafluoro-2-
phenylpropan-2-ols 16–18: Arene (1 mmol) and TFP (1.5 mmol)
were added to a dry pressure tube containing CH2Cl2 (3 mL) under
N2 atmosphere. Trifluoromethanesulfonic acid (50 mol%) was
added to the reaction mixture dropwise. The reaction mixture was
stirred for 16 h, after which the contents were poured into 5 mL
H2O and extracted with CH2Cl2. After evaporation of solvents, the
residue was purified by flash chromatography to give the final
product.
General procedure for the synthesis of ethyl 4,4,4-trifluoro-3-hy-
droxy-3-(1H-pyrrol-2-yl)butanoates 60–66: A solution of pyrrole
(0.5 mmol), TFAA (0.5 mmol) and K-10 montmorillonite was mixed
in toluene (3 mL) in a Teflon screw-cap pressure tube. The reaction
mixture was heated and stirred at 608C, and reaction progress was
monitored by TLC. After satisfactory conversion, the product mix-
ture was separated from catalyst by filtration. The product was pu-
rified by flash chromatography and isolated as crystals or oils.
General procedure for the synthesis of 3,3,3-trifluoro-2-hydroxy-
2-(indol-3-yl)propionic acid ethyl esters 19–34: The compounds
were synthesized based on
a
published method.[16] Indole
(0.75 mmol), ethyl 3,3,3-trifluoropyruvate (TFP, 1.125 mmol), and K-
10 montmorillonite (500 mg) were mixed in toluene (3 mL) in a
Teflon screw-cap pressure tube. The reaction mixture was heated
and stirred at 608C, and reaction progress was monitored by TLC.
After satisfactory conversion, the product mixture was separated
from catalyst by filtration. The solvent and excess TFP were re-
moved under vacuum. The products were isolated as crystals or
oils and purified by flash chromatography. Pyrrole derivatives 35–
43 were prepared by the same method.
General procedure for the synthesis of 1-(1H-indol-1-yl)-2,2,2-tri-
fluoroethanols 67–73: A solution of indole (0.5 mmol), TFAE
(2 mmol), and Et3N (0.05 mmol) in DMF (0.25 mL) was irradiated in
a CEM Discover microwave reactor for 20 min at 1508C. The reac-
tion mixture was then quenched with 10 mL H2O, and the product
was extracted with EtOAc (3ꢃ10 mL). The combined organic ex-
tracts were dried over anhydrous Na2SO4, evaporated, and the
product was isolated and purified by preparative TLC or column
chromatography.
General procedure for the synthesis of ethyl 3,3,3-trifluoro-2-hy-
droxy-2-(1H-pyrrol-2-yl)propanoates 35–43: Pyrrole (0.5 mmol)
and ethyl 3,3,3-trifluoropyruvate (TFP, 0.51 mmol) were mixed in a
round-bottom flask. The reaction mixture was stirred at room tem-
perature, and reaction progress was monitored by GC. After satis-
factory conversion, the product mixture was extracted into CH2Cl2.
The solvent and excess TFP were removed under vacuum. The
product was then purified by flash chromatography.
Synthesis of 1-(5-bromo-1H-indol-3-yl)-2,2,2-trifluoroethanol
(74): A mixture of 5-bromoindole (0.5 mmol) and trifluoroacetalde-
hyde methylhemiacetal (1 mmol) was irradiated in a CEM Discover
microwave reactor for 10 min at 1008C. The reaction mixture was
then directly subjected to preparative TLC for purification and
product isolation. Isolated yield: 72%; colorless solid, mp: 113–
115 8C; 1H NMR (300.128 MHz, CDCl3): d=7.76 (m, 1H), 7.33–7.37
(m, 3H), 6.57 (d, J=3.3 Hz, 1H), 6.09 (p, J=4.8 Hz, 1H), 3.88 ppm
(d, J=4.8 Hz, 1H); 13C NMR (75.474 MHz, CDCl3): d=130.68, 125.69,
125.58, 123.82, 120.30, 116.96, 114.13, 111.16, 104.23, 76.65 ppm (q,
J=36 Hz); 19F NMR (300.128 MHz, CDCl3): d=ꢀ77.63 (d, J=4.8 Hz);
MS C10H7BrF3NO (294) m/z (%): 293 ([M]+, 100), 295 ([M]+, 98), 214
(30), 175 (25).
General procedure for the synthesis of enantiomeric 3,3,3-tri-
fluoro-2-hydroxy-2-(indol-3-yl)propionic acid ethyl esters 29–31
(S and R): The enantiomers were prepared by an earlier cinchona-
alkaloid-catalyzed organocatalytic method. Indole (0.5 mmol) and
cinchonidine (for S products) or cinchonine (for R products)
916
www.chemmedchem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 910 – 919

Ab Self-Assembly Inhibitors
MED
General procedure for the synthesis of 2,2,2-trifluoro-1-phenyle-
thanols 75–78: Arene (1 mmol) and trifuoroacetaldehyde hemiace-
tal (TFAE; 1.5 mmol) were added to a pressure tube containing
CH2Cl2 (3 mL) under N2 atmosphere. Trifluoromethanesulfonic acid
(2 equiv) was added to the reaction mixture dropwise. The reaction
mixture was stirred for 16 h, after which the contents were poured
into 5 mL H2O and extracted with CH2Cl2. After evaporation of sol-
vents, the residue was purified by flash chromatography to give
the final product.
(Pearlman’s catalyst; 75 mg). The mixture was stirred under H2
(5 bar) at room temperature for 12 h. After the reaction comple-
tion, the catalyst was separated by filtration. The resulting filtrate
was concentrated in vacuo and purified by column chromatogra-
phy.
Synthesis of 3,3,3-trifluoro-2-hydroxy-2-(5-benzyloxyindol-3-yl)-
propionic acid ethyl ester (89): A solution of 5-hydroxyindole
(66.5 mg, 0.5 mmol), benzyl bromide (65 mL, 0.55 mmol), and K2CO3
(276 mg, 2 mmol) in DMF (0.5 mL) was stirred for 24 h. TFP (192 mL,
1.4 mmol) was then added, and the mixture was stirred continu-
ously for another 24 h. The reaction was diluted with H2O (10 mL)
and extracted with CH2Cl2 (3ꢃ5 mL). Combined organic extracts
were dried over anhydrous Na2SO4, and the solvent was removed
in vacuo. The residue was then subjected to column chromatogra-
phy to afford pure product (170 mg, 87% yield).
General procedure for the synthesis of ethyl 2-amino-3,3,3-tri-
fluoro-2-(1H-indol-3-yl)propanoates 79–88: Step 1. General proce-
dure for the preparation of ethyl 3,3,3-trifluoro-2-(1-phenylethylimi-
no)propanoate: Montmorillonite K-10 (4 g) and toluene (20 mL)
were placed into a round-bottom flask equipped with a stir bar, a
reflux condenser, and a dry tube. TFP (6.24 mL, 0.047 mol) and a-
methylbenzylamine (5 mL, 0.039 mol; racemic, R, or S) was dis-
solved in toluene (5 mL), and this solution was added to the above
mixture. The reaction mixture was stirred at 1008C for 4 h, and re-
action progress was monitored by TLC. After the reaction comple-
tion, the resulting mixture was filtered through a sintered glass
funnel and washed with CH2Cl2. The solvent and excess TFP were
removed in vacuo to obtain a brown oil, which was later subjected
to column chromatography (hexane/EtOAc 90:10) to obtain a col-
orless liquid in 92% isolated yield.
General procedure for the synthesis of 1,3- and 1,4-phenylene-
bis(methylene)bis[3-(3-ethoxy-1,1,1-trifluoro-2-hydroxy-3-oxo-
propan-2-yl)-1H-indole-5-carboxylates] 90 and 93: A solution of
5-indole carboxylic acid (80.5 mg, 0.5 mmol), a,a’-dibromoxylene
(meta or para, 66 mg, 0.25 mmol), and K2CO3 (276 mg, 2 mmol) in
0.5 mL DMF was stirred for 24 h. TFP (192 mL, 1.4 mmol) was then
added and stirred for 24 h. The reaction was quenched with H2O
(10 mL), and the mixture was extracted with CH2Cl2 (3ꢃ5 mL). The
combined organic extracts were dried over anhydrous Na2SO4, and
the solvent was evaporated. The residue was purified by column
chromatography to afford pure 90 (130 mg, 68% yield) or 93
(135 mg, 71% yield).
General procedure for synthesis of racemic 3,3,3-trifluoro-2-(1H-
indol-3-yl)-2-(1-phenylethylamino)propanoates: Step 2. Racemic
trifluoroimine, synthesized in Step 1 (300 mg, 1.09 mmol) and
indole (0.98 mmol) were dissolved in 2 mL CH2Cl2. The reaction
vessel was placed into an ice bath, and the mixture was stirred at
08C for 5 min. Trifluoromethanesulfonic acid (0.40 mmol, 20% solu-
tion in CH2Cl2) was added dropwise to the reaction mixture over a
period of 15 min. After complete addition, the reaction mixture
was stirred at 08C for another 2 h, and reaction progress was fol-
lowed by TLC. After reaction completion, H2O (5 mL) was added,
and the reaction mixture was stirred at room temperature for
5 min to quench the acid. The resulting mixture was extracted
with CH2Cl2, and the organic layer was washed with H2O three
times. The organic layers were combined, dried over Na2SO4, and
filtered. The solvent was removed by evaporation, and the result-
ing crude mixture was purified by column chromatography.
General procedure for the synthesis of diethyl 2,2’-(5,5’-(1,3-
and
1,4-phenylenebis(methylene))bis(oxy)bis(1H-indole-5,3-
diyl))bis (3,3,3-trifluoro-2-hydroxypropanoates 91 and 92: A so-
lution of 5-hydroxyindole (66.5 mg, 0.5 mmol), a,a’-dibromoxylene
(meta or para, 66 mg, 0.25 mmol) and K2CO3 (276 mg, 2 mmol) in
0.5 mL CH3CN was stirred for 24 h. TFP (192 mL, 1.4 mmol) was
then added, and stirring was continued for another 24 h. The reac-
tion was diluted with 10 mL H2O and extracted with CH2Cl2 (3ꢃ
5 mL). The combined organic extracts were dried over anhydrous
Na2SO4, and the solvent was removed in vacuo. The crude products
were subjected to column chromatography to yield pure 91
(143 mg, 81% yield) or 92 (138 mg, 78% yield).
General procedure for synthesis of chiral 3,3,3-trifluoro-2-(1H-
indol-3-yl)-2-(1-phenylethylamino)propanoates (R or S): Trifluoro-
methylated imine (300 mg, 1.09 mmol) and indole (0.98 mmol)
were dissolved in 2 mL CH2Cl2. The reaction vessel was placed into
a cooling bath (EtOH/dry ice mixture) and the mixture was stirred
at ꢀ408C for 15 min. Trifluoromethanesulfonic acid (0.40 mmol,
20% solution in CH2Cl2) was added dropwise to the reaction mix-
ture over a period of 15 min. After complete addition, the reaction
mixture was stirred at ꢀ408C for another 2 h, and reaction prog-
ress was followed by TLC. After the reaction completion, H2O
(5 mL) was added, and the reaction mixture was stirred at room
temperature for 5 min to quench the acid. The resulting mixture
was extracted into CH2Cl2, and the organic layer was washed three
times with H2O. The organic layers were combined, dried over
Na2SO4, and filtered. The solvent was removed by evaporation, and
the resulting crude mixture was purified by column chromatogra-
phy.
Biochemical assays
General information: Fibril assays: NaH2PO4, Na2HPO4, NaN3,
NaOH, NaCl, glycine, DMSO, and thioflavin-T were obtained from
Sigma–Aldrich. Lyophilized Ab1–40 peptide (purity >95%) was pur-
chased from AnaSpec (Fremont, CA, USA). Mica sheets for AFM
measurements were obtained from Alfa Aesar.
Oligomer assays: 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), DMSO,
fluorescamine, ultrapure Tween 20, tetramethylbenzidine (free
base), N,N-dimethylacetamide, tetrabutylammonium borohydride,
and H2O2 (30% w/w) were obtained from Sigma–Aldrich. N-a-Bio-
tinyl-Ab1–42 (bio-Ab42) was purchased from AnaSpec. Fatty-acid-
free fraction V bovine serum albumin was obtained from Boehring-
er–Mannheim. Streptavidin–HRP (SA–HRP) was obtained from
Rockland. NeutrAvidin (NA) was obtained from Pierce. High-bind-
ing 9018 ELISA plates were purchased from Costar.
Thioflavin-T fluorescence assay for the determination of inhibi-
tor activity in Ab fibrillogenesis: Assays were carried out using a
standard procedure.[23–25] The synthetic lyophilized Ab1–40 peptide
was dissolved in 100 mm NaOH to a concentration of 40 mgmLꢀ1
and diluted in 10 mm HEPES, 100 mm NaCl, 0.02% NaN3 (pH 7.4)
General procedure for synthesis of ethyl 2-amino-3,3,3-trifluoro-
2-(1H-indol-3-yl)propanoate (hydrogenolysis): Step 3. 3,3,3-Tri-
fluoro-2-(1H-indol-3-yl)-2-(1-phenylethylamino)propanoate (150 mg,
0.38 mmol) was dissolved in EtOH (2 mL) along with Pd(OH)2
ChemMedChem 2012, 7, 910 – 919
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
917

B. Tçrçk, M. Tçrçk, et al.
MED
buffer to a final peptide concentration of 100 mm. The use of NaOH
as an initial solvent ensures that the isoelectric point of Ab is by-
passed, and the peptide remains in monomeric form.[34,35] Inhibi-
tors were dissolved in DMSO to achieve a concentration of 0.15m
and added to the Ab samples in HEPES buffer (inhibitor/Ab=10)
to attain a final concentration of 1 mm. After vigorous vortexing
for 30 s, the solutions were incubated at 378C with gentle shaking
(77 rpm) for seven days, and the increase in fibril amount in each
sample was followed by thioflavin-T fluorescence using the pep-
tide without inhibitor as the control. The fluorescence measure-
ments were carried out using a Hitachi F-2500 fluorescence spec-
trophotometer. The incubated peptide solutions were briefly vor-
texed before each measurement, and then 3.5 mL aliquots of the
suspended fibrils were withdrawn and added into 700 mL 5 mm thi-
oflavin-T prepared freshly in 50 mm glycine-NaOH (pH 8.5) buffer.
The maximum fluorescence intensity of these mixtures was mea-
sured at lem 484ꢂ5 nm, with preset lex 435 nm. None of the inhibi-
tor compounds showed fluorescence intensity in this region. For
the purposes of a screening assay, the fibril signal generated under
the conditions of the assay in the presence of 1% DMSO (solvent
control) and absence of compound is taken as 100%. The EC50
values of potent compounds were determined as described earli-
er.[13]
under the conditions of the assay in the presence of 1% DMSO
(solvent control) and absence of compound was taken as 100%.
Acknowledgements
Financial support was provided by the University of Massachu-
setts Boston and US National Institutes of Health grants
R15AG025777-03A1 and R21AG028816-01 (to H.L.).
Keywords: Alzheimer’s disease · b-amyloid · chiral inhibitors ·
heterocycles · organofluorine compounds
[1] The Alzheimer’s Association, http://www.alz.org/ (accessed October
2011).
[2] C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones,
Lancet 2011, 377, 1019–1031.
[3] F. Chiti, C. M. Dobson, Nat. Chem. Biol. 2009, 5, 15–22.
[4] L. D. Estrada, C. Soto, Curr. Top. Med. Chem. 2007, 7, 115–126.
[5] C. Stains, K. Mondal, I. Ghosh, ChemMedChem 2007, 2, 1674–1692.
[6] D. M. Walsh, D. J. Selkoe, J. Neurochem. 2007, 101, 1172–1184.
[7] M. Necula, R. Kayed, S. Milton, C. G. Glabe, J. Biol. Chem. 2007, 282,
10311–10324.
[8] C. G. Glabe, J. Biol. Chem. 2008, 283, 29639–29643.
[9] C. Nerelius, J. Johansson, A. Sandegren, Front. Biosci. 2009, 14, 1716–
1729.
Atomic force microscopy of fibrils: The morphology of the incu-
bated peptide samples were studied using atomic force microsco-
py (AFM).[36,37] Aliquots (2 mL) were spotted on freshly cleaved mica
sheets and air dried. The buffer salts were washed off with deion-
ized H2O. AFM was carried out using a Quesant Q-Scope 250 mi-
croscope in non-contact mode.
[10] H. LeVine III, Amyloid 2007, 14, 185–197.
[11] B. Tçrçk, S. Dasgupta, M. Tçrçk, Curr. Bioact. Comp. 2008, 4, 159–174.
[12] S. L. Adamski-Werner, S. K. Palaninathan, J. C. Sacchettini, J. W. Kelly, J.
Med. Chem. 2004, 47, 355–374.
[13] M. Tçrçk, M. Abid, S. C. Mhadgut, B. Tçrçk, Biochemistry 2006, 45,
5377–5383.
[14] A. Sood, M. Abid, C. Sauer, S. Hailemichael, M. Foster, B. Tçrçk, M.
Tçrçk, Bioorg. Med. Chem. Lett. 2011, 21, 2044–2047.
[15] A. Sood, M. Abid, S. Hailemichael, M. Foster, B. Tçrçk, M. Tçrçk, Bioorg.
Med. Chem. Lett. 2009, 19, 6931–6934.
[16] M. Abid, B. Tçrçk, Adv. Synth. Catal. 2005, 347, 1797–1803.
[17] S. M. Landge, D. A. Borkin, B. Tçrçk, Tetrahedron Lett. 2007, 48, 6372–
6376.
Assay for inhibition of Ab oligomer assembly: Biotinyl-Ab1–42
,
stored as a 1 mgmLꢀ1 solution in HFIP at ꢀ758C, was dried and
treated with neat trifluoroacetic acid for 10 min at room tempera-
ture to disaggregate the peptide and dissolved to 500 nm (50ꢃ) in
DMSO as described.[26,27] Peptide (2 mL) was dispensed into each
well of a polypropylene 96-well plate, and PBS (100 mL) containing
the desired concentration of test compound and 1% DMSO were
added to initiate oligomer formation at room temperature. After
30 min, 0.3% v/v Tween 20 (50 mL) was added to stop oligomer as-
sembly. This mixture (50 mL) was then assayed for oligomer content
by single-site streptavidin-based assay.
[18] D. Borkin, S. M. Landge, B. Tçrçk, Chirality 2011, 23, 612–616.
[19] M. Sridhar, C. Narsaiah, B. C. Ramanaiah, V. M. Ankathi, R. B. Pawar, S. N.
Asthana, Tetrahedron Lett. 2009, 50, 1777–1779.
[20] B. Tçrçk, M. Abid, G. London, J. Esquibel, M. Tçrçk, S. C. Mhadgut, P.
Yan, G. K. S. Prakash, Angew. Chem. 2005, 117, 3146–3149; Angew.
Chem. Int. Ed. 2005, 44, 3086–3089.
[21] M. Abid, L. Teixeira, B. Tçrçk, Org. Lett. 2008, 10, 933–935.
[22] H. S. Ban, H. Minegishi, K. Shimizu, M. Maruyama, Y. Yasui, H. Nakamura,
ChemMedChem 2010, 5, 1236–1241.
[23] H. Naiki, K. Higuchi, M. Hosokawa, T. Takeda, Anal. Biochem. 1989, 177,
244–249.
[24] H. LeVine III, Protein Sci. 1993, 2, 404–410.
Biotinyl-Ab1–42 single-site streptavidin-based assay for determi-
nation of inhibitor activity in Ab oligomer formation:[26,27] NA
(50 mL, 1 mgmLꢀ1) in 10 mm NaPi (pH 7.5) was coated per well
overnight at 48C on Costar 9018 high-binding ELISA plates sealed
with adhesive plastic film. The plates were blocked by the addition
of 200 mL phosphate-buffered saline (PBS; 10 mm sodium phos-
phate [pH 7.5], 150 mm NaCl, 0.1% v/v Tween 20) at room temper-
ature for 1–2 h and stored at 48C. In the assay after removal of the
blocking solution, a sample containing a mixture of oligomers and
monomers of biotinylated peptide (50 mL containing up to 10 nm
Ab) was bound for 2 h at room temperature. The wells were
washed three times with TBST (20 mm Tris-HCl [pH 7.5], 34 mm
NaCl, and 0.1% v/v Tween 20) on a Biotek ELꢃ50 automated plate
washer. After washing, SA–HRP (50 mL 1:20000) in PBS+0.1% v/v
Tween 20 was added, the plate sealed, and incubation was contin-
ued for 1 h at room temperature. The plate was washed again with
TBST, 100 mL tetramethylbenzidine/H2O2 substrate solution was
added, and the plate was incubated at room temperature for 5–
10 min. The OD450 was determined on a Biotech Synergy HT plate
reader after stopping the reaction with 100 mL H2SO4 (1% v/v). For
the purposes of a screening assay, the oligomer signal generated
[25] M. R. Nilsson, Methods 2004, 34, 151–160.
[26] H. LeVine III, Anal. Biochem. 2006, 356, 265–272.
[27] H. LeVine III, Q. Ding, J. A. Walker, R. S. Voss, C. E. Augelli-Szafran, Neuro-
sci. Lett. 2009, 465, 99–103.
[28] K. L. Sciaretta, D. J. Gordon, S. C. Meredith, Methods Enzymol. 2006, 413,
273–312.
[29] T. Takahashi, H. Mihara, Acc. Chem. Res. 2008, 41, 1309–1318.
[30] A. H. Armstrong, J. Chen, A. F. McKoy, M. H. Hecht, Biochemistry 2011,
50, 4058–4067.
[31] S. A. Moore, T. N. Huckerby, G. L. Gibson, N. J. Fullwood, S. T. Turnbull,
B. J. Tabner, O. M. A. El-Agnaf, D. Allsop, Biochemistry 2004, 43, 819–
826.
[32] R. J. Chalifour, R. W. McLaughlin, L. Lavoie, C. Morisette, N. Tremblay, M.
Boule, P. Sarazin, D. Stea, P. Tremblay, J. Biol. Chem. 2003, 278, 34874–
34881.
918
www.chemmedchem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 910 – 919

Ab Self-Assembly Inhibitors
MED
[33] M. Bartolini, M. Pistolozzi, V. Andrisano, J. Egea, M. G. Lopez, I. Iriepa, I.
Moraleda, E. Galvez, J. Marco-Contelles, A. Samadi, ChemMedChem
2011, 6, 1990–1997.
[36] T. T. Ding, J. D. Harper, Methods Enzymol. 1999, 309, 510–25.
[37] O. N. Antzutkin, Magn. Reson. Chem. 2004, 42, 231–246.
[34] Y. Fezoui, D. M. Hartley, J. D. Harper, R. Khurana, D. M. Walsh, M. M. Con-
dron, D. J. Selkoe, P. T. Lansbury, A. L. Fink, D. B. Teplow, Amyloid 2000,
7, 166–78.
[35] M. Bourhim, M. Kruzel, T. Srikrishnan, T. Nicotera, J. Neurosci. Methods
2007, 160, 264–268.
Received: November 30, 2011
Revised: January 30, 2012
Published online on February 20, 2012
ChemMedChem 2012, 7, 910 – 919
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
919