Synthesis and in vitro evaluation of α-synuclein ligands

Article abstract of DOI:10.1016/j.bmc.2012.06.023

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K_i = 32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K_i ≈ 50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.

Full text of DOI:10.1016/j.bmc.2012.06.023

Bioorganic & Medicinal Chemistry 20 (2012) 4625–4634
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of a-synuclein ligands
Lihai Yu a, Jinquan Cui a, Prashanth K. Padakanti a, Laura Engel b, Devika P. Bagchi b,
Paul T. Kotzbauer b, Zhude Tu a,
⇑
a Department of Radiology, Washington University School of Medicine, 510 South Kingshighway Boulevard, St. Louis, MO 63110, USA
b Department of Neurology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 February 2012
Revised 5 June 2012
Accepted 12 June 2012
Available online 19 June 2012
Accumulation of misfolded
Parkinson’s disease (PD). To identify ligands having high binding potency toward aggregated
a
-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of
-synuclein,
we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant
-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the
in vitro data suggest that compound 11b has high affinity to -synuclein fibrils (Ki = 32.10 1.25 nM)
and compounds 11d, 16a and16b have moderate affinity to
-synuclein fibrils (Ki ꢀ 50–100 nM). Further
optimization of the structure of these analogues may yield compounds with high affinity and selectivity
for aggregated -synuclein.
a
a
a
a
Keywords:
Parkinson’s disease
Lewy bodies
a
Ó 2012 Elsevier Ltd. All rights reserved.
a-Synuclein
Phenothiazine
Fluorescence
1. Introduction
than the amount of aggregated
The formation of -synuclein aggregates may result in synaptic
dysfunction and neuronal cell death.17–20 Quantifying
-synuclein
aggregation in vivo will be very useful to improve early diagnosis
of PD and to monitor disease progression. Furthermore, the ability
to quantify aggregated a-synuclein in vivo may be useful to mon-
itor therapeutic efficacy in early clinical studies of disease-modify-
ing treatments. Therefore, small molecules that have suitable
pharmaceutical properties as fibrillar a-synuclein ligands and that
a
-synuclein inside LBs and LNs.16
a
Parkinson’s disease (PD) is the second most common neurode-
generative disease after Alzheimer’s disease (AD). Its main clinical
features include rest tremor, bradykinesia, rigidity and loss of pos-
tural reflexes.1 The motor symptoms of PD are caused by degener-
ation of dopaminergic neurons in the substantia nigra, which is
accompanied by Lewy bodies (LBs) and Lewy neuritis (LNs). In
addition to motor symptoms, individuals with PD also have a high
risk of dementia2 and postmortem analysis has demonstrated a
correlation between dementia and LBs in cortical neurons.3–6 To
date, the correlation between the density of LBs/LNs in substantia
nigra and the severity of these diseases is not clear.7 Mechanisms
responsible for initiation and progression of pathogenic processes
a
can be labeled with position emission tomography (PET) radionuc-
lides such as C-11 and F-18, will have great opportunity to serve as
PET probes for quantifying
addition, inhibition of the progress of
a
-synuclein aggregation in the brain. In
-synuclein protein aggrega-
a
tion may be a potential strategy for treating PD and its associated
in PD are still not entirely understood. Aggregated
a
-synuclein is
diseases. Thus, investigators have attempted to identify highly po-
the major component of LBs and LNs.8–10
a-Synuclein, a presynap-
tent ligands for
developing highly potent
a
-synuclein fibrils.21–24 To achieve the goal of
tic terminal protein containing 140 amino acids,11 plays an impor-
tant function in the central nervous system (CNS) by regulating
synaptic vesicle recycling and synthesis, vesicular storage and re-
lease of neurotransmitter.12–15 One analysis approach suggests
a-synuclein ligands, we focused on
exploring the derivatives of phenothiazine. Herein, we report our
initial work on the synthesis of new analogues of phenothiazine
and the analysis of their binding affinity toward
Our current work was inspired by (1) to date, no small molecular
ligand for -synuclein fibrils has been reported to have the capabil-
ity to prevent -synuclein accumulation in vivo; (2) no suitable
PET tracer has been reported that can be used to assess aggregated
-synuclein accumulation in the brain in vivo; (3) phenothiazine
a-synuclein fibrils.
that aggregated a-synuclein outside LBs and LNs is 10-fold higher
a
a
Abbreviations: PD, Parkinson’s disease; AD, Alzheimer’s disease; LBs, Lewy
bodies; LNs, Lewy neuritis; CNS, central nervous system; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; ThT, thioflavin T; AFU, arbitrary fluorescence units.
a
structure has been proposed as a promising pharmacophore for
the therapy of neurodegenerative diseases25,26 by protecting the
dopaminergic neurons against oxidative stress; the representative
⇑
Corresponding author. Tel.: +1 314 362 8487; fax: +1 314 362 8555.
E-mail address: tuz@mir.wustl.edu (Z. Tu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.023

4626
L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
H3C
N
N
O
NH
H3C
H
N
N
NH
N
2HCl
H3C
N
S
S
Cl
S
1
2
3
Figure 1. Three chemical structures reported previously for neuronal protection.
structures include phenothiazine (1), N-acylaminophenothiazine
(2) and N-alkylphenothiazine (3) (Fig. 1). Furthermore, it was re-
Analogues 12, 13a–c, 14a–b, 15 and 16a–c were synthesized as
shown in Scheme 3. Compound 12 was directly obtained by the
N-methylation of 11b using methyl iodide in DMF in the presence
of sodium hydride. Compound 13a was generated by reducing
compound 12 via hydrogenation under hydrogen atmosphere in
the presence of 10% palladium on activated carbon in ethanol. Fur-
ther N-methylation of 13a using one or two equivalents of methyl
iodide in acetonitrile yielded compounds 13b and 13c. Compounds
14a and 14b were obtained via N-acetylation of compound 11b or
11c using acetyl chloride. Removing the methyl group from 14a
with boron tribromide afforded the corresponding phenol ana-
logue 15. O-Alkylation of compound 15 using 1-bromo-2-fluoroe-
thane, 3-bromopropyne or 3-bromo-1-iodopropene, followed by
hydrolysis in the presence of 3 N HCl aqueous solution gave the
compounds 16a, 16b and 16c. To obtain enough quantity of target
compounds for in vitro studies, some reactions discussed above
were repeated and scaled-up. However, in the current work, we
did not optimize the reaction conditions and determine the scala-
bility of reactions. Compound 1 was purchased from Sigma-Aldrich
Co., and compounds 2 and 3 were synthesized following the liter-
ature reported procedures28,29 and converted them to correspond-
ing oxalate salts.
ported that phenothiazines inhibit a-synuclein filament assembly
with IC50 values in the low micromolar range.21
2. Results and discussion
2.1. Chemistry
Based on our structure–activity relationship analysis, we opti-
mized the structure of phenothiazine and synthesized a series of
new analogues as shown in Schemes 1–3.
Using copper iodide as catalyst, in the presence of L-proline and
potassium carbonate, compound 4 was reacted with 1-bromo-4-
methoxybenzene in DMSO to afford bis(4-methoxyphenyl)amine
(5) following Ullman reaction (Scheme 1). The target compound
3,7-dimethoxy-10H-phenothiazine (6) was obtained by cyclization
of intermediate 5 with sulfur at 150 °C using dichlorobenzene as
solvent and iodine as the catalyst.
The synthesis of compounds 11a–e was achieved as shown in
Scheme 2. Hydrolysis of compound 8a in the presence of potassium
hydroxide afforded 2-amino-5-methoxybenzothiol, which was
coupled with either 4-chloro-3-nitrobenzonitrile (7a) or 1-
chloro-2,4-dinitrobenzene (7b) to obtain the substituted diphenyl-
sulfide intermediate 9a or 9b under mild acidic condition. Acetic
anhydride was used to protect the amino group in 9a and 9b to af-
ford acetamides 10a and 10b which were cyclized via Smile rear-
rangement27 under the strong basic conditions to afford the
target compounds 11a and 11b. Following the similar synthetic
procedure followed for 9a and 9b, compound 9c was obtained
starting with 7b and 2-amino-benzothiol (8b) in the presence of
sodium hydroxide. Compound 9c was acylated with acetic anhy-
dride to obtain 10c. Compound 10c was treated with N-bromosuc-
cinimide (NBS) in dimethylformamide (DMF) or iodine
monochloride (ICl) in acetic acid to afford 10d or 10e, respectively.
Cyclization of 10c, 10d and 10e followed the similar procedure de-
scribed for 11a to obtain target compounds 11c, 11d and 11e.
To test how the proton on the nitrogen of the middle ring affects
2.2. Thioflavin T fluorescence assay for a-synuclein fibrils
At present, no existing standard protocol can be used to mea-
sure the binding affinity of compounds for -synuclein fibrils.
a
Determining the binding affinities of compounds toward proteins
with radioligands has higher accuracy and sensitivity than with
fluorescent probes. Nevertheless, until today, no radioligand was
reported to be suitable for determining binding affinities of com-
pounds toward aggregated
has been used to determine the binding affinity of compounds to-
ward b-amyloid, tau and
-synuclein aggregates.30–33 In the cur-
rent work, we used fluorescent methodology to determine the
relative binding potency of compounds toward -synuclein fibrils
a-synuclein. Moreover, thioflavin S
a
a
and to identify potential ligands that can be radiolabeled with I-
125 or H-3 to further characterize their binding properties.
Thioflavin T (ThT) is a fluorescent dye with an identified
chemical structure. It is weakly fluorescent in the presence of
the binding potency of analogues to
a-synuclein fibrils, a methyl
group or an acetyl group was substituted on the nitrogen.
H
H
N
a
b
NH2
N
H3CO
S
OCH3
H3CO
H3CO
OCH3
6
4
5
Reagents and conditions:
a) 1-bromo-4-methoxybenzene, CuI, L-proline, K2CO3, DMSO, 90°C; b) S, I2, 1,2-dichlorobenzene, 150°C
Scheme 1. Synthesis of compound 6.

L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
4627
AcHN
S
R1
R1
H2N
S
R1
H3CO
S
N
a
c
NH2
Cl
OCH3
OCH3
NO2
NO2
NO2
7a: R1 = CN
7b
8a
9a:
9b:
10a:
10b:
R1 = CN
R1 =NO2
R1 = CN
R1 = NO2
: R1 = NO2
O2N
AcHN
H2N
S
O2N
O2N
NH2
SH
b
c
Cl
S
R2
NO2
NO2
NO2
9c
7b
8b
10c: R2 = H
10d: R2 = Br
d
e
10e:
R2 = I
Ac
N
H
N
f
10a-e
R1
S
R2
R1
R2
NO2
S
11a:
11b:
R1 = CN, R2 = OCH3
R1 = NO2, R2 = OCH3
11c: R1 = NO2, R2 = H
11d: R1 = NO2, R2 = Br
11e:
R1 = NO2, R2 = I
Reagents and conditions:
a) i: KOH/H2O, reflux, overnight; ii: AcOH, EtOH/H2O, ambient temperature, 2h; b) NaOH, EtOH/H2O, ambient
temperature, 2h; c) pyridine, Ac2O, ambient temperature, 3h; d) NBS, DMF, 100°C, overnight; e) ICl, AcOH, reflux,
3h; f) KOH, Acetone/EtOH, reflux, 2h;
Scheme 2. Synthesis of compounds 11a–e.
monomeric
the presence of
a
-synuclein or in an
a
-synuclein fibril-free system. In
Differences in Kd values may be explained by differences in
a-syn-
a-synuclein fibrils, ThT fluorescence intensity in-
uclein fibril preparations and incubation conditions.34,35 Impor-
creases by several orders of magnitude at the emission wavelength
tantly, we observed consistent a-synuclein fibril binding affinity
when using different batches of fibrils in our experimental
maximum (kem = 483 nM).31 To determine the binding affinity of
compounds toward
emission spectrum was confirmed to be consistent with previously
reported data (Fig. 2, left); second, we incubated ThT with -synuc-
a-synuclein fibrils, first, the ThT fluorescent
protocol.
a
2.3. In vitro evaluation of binding affinities of phenothiazine
lein fibrils and measured ThT’s maximum fluorescent emission
wavelength (kem = 485 nM) under its excitation wavelength
(kex = 440 nM). No increase in fluorescent emission was observed
compounds to
a-synuclein fibrils
Compounds were incubated with
a-synuclein fibrils to deter-
when ThT was incubated in the presence of monomeric
lein fibrils or in -synuclein fibril-free buffer. Furthermore, the ra-
tio of ThT’s fluorescence intensity in the presence of -synuclein
fibrils to ThT’s fluorescence intensity in either monomeric -syn-
uclein fibrils or -synuclein fibril-free buffer is about 30-fold.
Therefore, the fluorescence intensity of ThT in either monomeric
-synuclein or -synuclein free buffer is negligible and unlikely
a
-synuc-
mine whether any fluorescence emission was present at
kem = 485 nM, which would have permitted binding affinity (Kd)
values to be directly assessed using the same procedure as ThT.
All of the analogues reported here had no significant fluorescence,
a
a
a
a
and their binding affinity for
using an indirect ThT competition assay, in which the competition
of ThT binding to -synuclein fibrils was determined at various
a-synuclein fibrils was determined
a
a
a
to interfere with the measurement of
affinity for compounds.
a
-synuclein fibril binding
compound concentrations. This approach enabled IC50 and Ki val-
ues of the new compounds to be determined and compared. Re-
sults of the competitive binding assays for each compound are
shown in Supplementary Figure 1 and Supplementary Table 1.
The corresponding Ki values are listed in Table 1. Although, fluores-
cence quenching can potentially interfere with measurement of
competitive binding, the data for the individual compounds closely
fit a competitive binding model. Absorbance spectra were mea-
sured at the IC50 concentration for each compound. Absorbance
To determine the binding affinity for new compounds toward
-synuclein fibril, we first evaluated the saturation binding curve
a
of ThT fluorescence intensity in the presence of a-synuclein fibrils,
from which 60 min was chosen as the optimized incubation time
(Fig. 2, right). In our system, we obtained the Kd value of
948 271 nM for ThT binding to
a
-synuclein fibrils, which is
different from other reported values, 15
l
M and 588 2 nM.31,32

4628
L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
CH3
N
CH3
N
a
b
R1
H3CO
S
N
H3CO
S
NO2
R2
12
13a:
R1 = R2 = H
H
N
c
13b: R1 = CH3 R2 = H
13c:
R
1 = R2 = CH3
R
S
NO2
11b:
R = OCH3
COCH3
N
11c: R = H
COCH3
N
e
d
R1
S
NO2
HO
S
NO2
15
14a: R = OCH3
14b: R = H
H
N
f,g
R
15
O
S
NO2
16a: R = CH2CH2F
16b:
R = CH2CH=CHI
16c: R = CH2C≡ CH
Reagents and conditions:
a) NaH, CH3I, DMF, 0°C, overnight; b) H2, Pd-C (10%), 90 psi, ambient temperature, overnight, c) CH3I, Na2CO3,
MeCN, ambient temperature, overnight; d) CH3COCl, DCM, ambient temperature, overnight; e) BBr3/DCM (1M),
DCM, -78°C, overnight. f) 1-bromo-2-fluroethane, 3-bromopropyne or 3-bromo-1-iodopropene, Na2CO3, MeCN; g)
HCl (3 M), H2O, reflux, 5h;
Scheme 3. Synthesis of N-substituted analogues 12, 13a–c, 14a–b, 15, 16a–c.
Figure 2. Left: Fluorescence emission spectra of ThT in the corresponding buffer alone (blue), monomer (red) and fibrils (green) at kex = 440 nM; right: saturation curve of ThT
(3 M) for -synuclein fibrils (1.5 M) in Tris buffer (30 mM, pH 7.4) at different incubation times: 30 min (circle), 60 min (square), 90 min (triangle) at room temperature.
l
a
l
The Kd for ThioT binding to fibrils was 948 nM and the Bmax was 5672 afu.
was less than 0.001 in the range of 400–500 nM for all of the com-
pounds including 11b as shown in Supplementary data Figure 3,
indicating that absorbance at the excitation or emission wave-
lengths did not interfere with the fluorescence assay.
3.79 which suggests the lipophilicity of 11b is a little higher than
the desired range (range from 1 to 3), but it is still acceptable with
a high possibility of crossing the blood–brain-barrier.
To test the possibility of improving binding affinity of com-
Based on in vitro data generated by the protocol described in
the experimental section, it was found that the dimethoxy substi-
tuted phenothiazine analogue 6 had a Ki value of 121.8 5.1 nM.
When a cyano group was used to replace one of the methoxy
groups in 6, the binding affinity (Ki value) for 11a was decreased
2.8-fold to 346 37 nM; contrarily, when a nitro group was used
to replace one methoxy group in 6, the binding affinity (Ki value)
for 11b was increased about 3.8-fold to reach 32.1 1.3 nM. Com-
pound 11b is the most potent compound in our current work. In
addition, the calculated LogP value using ACD/log D program is
pounds to a-synuclein fibrils, compounds 11c–e were synthesized
by replacing the methoxy group in the structure of compound 11b
using H, Br and I, respectively. Compared to compound 11b
(Ki = 32.1 1.3 nM), the binding affinities of all three compounds
toward
a-synuclein fibrils, 11c, 11d and 11e were decreased; the
Ki values were decreased to 116.5 1.9, 75.3 8.4, 106.0 9.3 nM
for 11c, 11d and 11e, respectively. The order of binding potency
to
a
-synuclein fibrils is –OCH3 > –Br > –I > –H.
To confirm if the free proton on the nitrogen of the middle ring
is necessary for having high -synuclein fibril binding potency,
a

L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
4629
Table 1
greater than 90% inhibition of ThT fluorescence at a concentration
of 3 M, the lack of fibril dissociation indicates that changes in
fluorescence reflect competition for ThT binding.
Collectively, based on our structure–activity relationship stud-
ies, we found that: (1) there is a high possibility of identifying
Binding affinity (Ki SD, nM)a of synthesized compounds determined by ThT
l
competition assay
R3
N
highly potent ligands for imaging a-synuclein fibril aggregates by
optimizing the structure of phenothiazine; (2) the presence of an
electron withdrawing nitro group in one of the aromatic rings fa-
R1
S
R2
Compound # R1
R2
R3
H
Ki (nM)
LogPb
vors the
the amino group in the middle ring is essential for maintaining
high binding affinity of compounds toward -synuclein fibrils;
otherwise, the binding affinities of compounds will drop dramati-
cally. Compounds having moderate binding affinity for -synuclein
a-synuclein fibril binding affinity; (3) the free proton of
1
H
H
H
H
>500
4.15
2.29
2.85
2
H
N2C5OH11 >500
N3C7H16
a
3
H
>500
121.8 5.1 3.98
346 37
32.1 1.3
6
OCH3
OCH3
OCH3
H
Br
I
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
CN
H
H
H
H
H
H
CH3
CH3
CH3
a
11a
11b
11c
11d
11e
12
13a
13b
13c
14a
14b
15
3.50
3.79
fibrils (Ki < 60 nM) will be assessed for their in vitro binding affin-
ity toward Ab1–40/42, tau proteins or other neurotransmitters,
receptors, transporters, enzymes, and ion channels to determine
NO2
NO2
NO2
NO2
NO2
NH2
NHCH3
116.5 1.9 3.88
75.3 8.4
106.0 9.3
>500
>500
>500
>500
>500
>500
>500
4.65
4.91
4.14
3.12
3.78
4.52
1.86
2.76
1.93
4.02
5.72
3.83
their binding specificity for
a-synuclein fibrils in future studies.
Based on previous studies in the development of PET/SPECT ligands
for imaging Ab amyloid in vivo, we anticipate that compounds with
N(CH3)2 CH3
binding affinities less than 10 nM for
to be obtained.36,37 Further optimizing the phenothiazine ana-
logues to identify ligands that have high -synuclein potency
a-synuclein fibrils will need
NO2
NO2
NO2
NO2
COCH3
COCH3
COCH3
a
OH
(Ki < 10 nM) and specificity, suitable lipophilicity (LogP ranges
from 1 to 3) and other biological properties is necessary to achieve
16a
16b
16c
OCH2CH2F
OCH2CH@CHI NO2
OCH2C„CH NO2
H
H
H
49.0 4.9
57.9 2.7
>500
the goal of identifying a candidate PET/SPECT ligand for imaging
synuclein fibril aggregation in the brain.
a-
a
Ki values (mean SEM) were determined in at least three experiments and were
calculated using Kd of 948 nM for ThT binding to
a-synuclein fibrils as shown in
Figure 2.
b
3. Conclusion
Calculated value at pH 7.4 with ACD/Lab, version 7.0 (Advanced Chemistry
Development, Inc., Canada)
In this work, we successfully optimized the structure of pheno-
thiazine for binding -synuclein fibrils by synthesizing a series of
new analogues and assessing their -synuclein fibril binding affin-
ity in a ThT competition assay. Based on the in vitro binding affin-
ity screening data, several lead compounds, 11b, 11d, 16a and 16b
a
compounds 12, 13a–b, 14a–b, 15 were made; the competitive
binding data demonstrated that none of these compounds dis-
played higher a-synuclein fibril binding affinity (Ki > 500 nM) com-
pared to compounds 6 and 11a–e that have a free proton on the
a
were identified with high potency for
a-synuclein fibrils with
nitrogen of the middle ring.
Ki < 100 nM. Particularly, compounds 11b, 16a and 16b have Ki val-
ues as 32.10 1.25, 48.96 4.94 and 57.94 2.67 nM, respectively.
After further validating the binding specificity of these three com-
Since compound 11b displayed relatively high affinity
(32.10 1.25 nM), the methoxy was replaced by fluoroethoxy, (3-
iodoallyl)oxy and prop-2-yn-1-yloxy to synthesize compounds
16a–c. The in vitro data indicated that both compounds 16a and
16b had relatively high affinities with Ki value of 49.0 4.9 and
57.9 2.7 nM, respectively, which are comparable to the affinity
of compound 11b (32.1 1.3 nM. LogP = 3.79). However, com-
pound 16c displayed significantly lower affinity (Ki > 500 nM).
The structures of compound 16a and 16b contain fluorine or iodine
atom, providing positions for labeling with F-18 or I-125.
We also measured the binding affinities for three previously de-
scribed phenothiazine analogues shown in Figure 1. Compound 1
(phenothiazine) had low affinity (Ki > 500 nM) indicating that the
substitutions described in this study are important to confer bind-
ing affinity to the phenothiazine pharmacophore structure. Com-
pounds 2 and 3 also had low affinity (Ki > 500 nM), consistent
with the low affinities observed for other analogues with substitu-
tion on the nitrogen of the middle ring. To further evaluate the
specificity of the ThT competition assay, we tested eticlopride
hydrochloride, a known selective ligand to the dopamine D2 recep-
tor, and observed no significant competition for ThT in the assay
(Supplementary data). Finally, we evaluated the possibility that
the observed changes in ThT fluorescence in the binding assays
could result from fibril dissociation rather than competition for
pounds toward
a
-synuclein fibrils, the radioactive
[
11C]11b,
[
18F]16a will be synthesized for further study to test the feasibility
of assessing
a
-synuclein fibril aggregation in vivo; [125I]16b could
be used as radioactive ligand to establish radioactive methodology
to screen the binding affinity of other compounds toward the
a-
synuclein fibrils. In addition, the in vitro data reported here will
provide very useful SAR information to guide further design and
synthesis of new analogues to achieve the goal of identifying
highly potent small molecules that have high affinity and selectiv-
ity for a-synuclein fibrils.
4. Experimental
All reagents and chemicals were purchased from Sigma-Aldrich
Corporation (Milwaukee, WI) or VWR international, Inc. (Earthy
city, MO) and used without further purification unless otherwise
stated. The solvent ‘hexane’ means ‘n-hexane’ unless otherwise
stated. The air and water sensitive reactions were carried out un-
der nitrogen. The melting points of all the intermediates and final
compounds were determined on Hake-Buchler melting point appa-
ratus and are uncorrected. 1H NMR spectra were recorded on Var-
ian-300 MHz and 13C NMR spectra were recorded on Varian-
400 MHz which were maintained by the Chemistry Department
of Washington University in St. Louis. Spectra are referenced to
the deuterium lock frequency of the spectrometer. The chemical
shifts (in ppm) of residual solvents were found to be at 7.26 for
CHCl3 and at 2.50 for DMSO. The following abbreviations were
ThT binding to
to quantify -synuclein fibrils in SDS–PAGE gels following centrifu-
gation of fibrils at 100,000g and conversion to monomer by boiling
in SDS sample buffer. We observed no change in the mass of -syn-
uclein fibrils during incubation with each of compounds 11b, 16a,
or 16b at a concentration of 3 M. Since each compound produces
a-synuclein fibrils. We used SYPRO Ruby staining
a
a
l

4630
L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
used to describe peak patterns when appropriate: br s = broad sin-
glet, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
Elemental analysis or HPLC methods (>95%) were used to deter-
mine the purity of the target compounds.
Plate reader & software was used for fluorescence scan: TECAN
infinite M100 plate reader, i-control 1.7 TECAN software was used
to run plate reader. Plate reader & software used for binding assay:
Biotek synergy 2 plate reader, Gen 5 software was used to run plate
reader, fluorescence filters: Excitation 440/30, emission 485/20.
Optical setting top 50% and sensitivity = 60. Absorbance scans were
performed in quartz cuvettes in a Beckman Coulter DU 800
spectrophotometer.
4.28 (br s, 2H), 6.87 (m, 2H), 7.03 (d, J = 9.0 Hz, 1H), 7.42 (m, 2H),
8.17 (d, J = 9.0 Hz, 1H), 9.12 (s, 1H). mp 150.7–151.8 °C.
4.1.6. N-(2-((4-Cyano-2-nitrophenyl)thio)-4-methoxy-
phenyl)acetamide (10a)
Acetic anhydride (10 mL) and pyridine (2 mL) were added into a
flask containing compound 9a (0.5 g, 1.66 mmol). The solution was
stirred for 3 h at ambient temperature and quenched by pouring
into ice-cold water. The precipitate was filtrated and washed
with water to afford yellow solid (0.46 g, 81%) 1H NMR (CDCl3): d
2.91 (s. 3H), 3.82 (s, 3H), 6.89 (d, J = 8.4 Hz, 1H), 7.08 (s, 1H), 7.15
(d, J = 9.3 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.66 (br s, 1H). 8.32
(d, J = 9.0 Hz, 1H). 8.58 (s, 1H). mp 195.0–198.9 °C.
4.1. Chemistry
4.1.7. N-(2-((2,4-Dinitrophenyl)thio)-5-methoxyphenyl)-
acetamide (10b)
4.1.1. Bis(4-methoxyphenyl)amine (5)
4-Aminoanisole (300 mg, 2.5 mmol), 4-bromoanisole (360 mg,
2 mmol), CuI (75 mg, 0.4 mmol), L-proline (95 mg, 0.8 mmol) and
Synthetic procedure described for 10a was followed starting
with 9b (0.3 g, 0.93 mmol) to afford yellow solid (0.31 g, 95%). 1H
NMR (CDCl3): d 2.05 (s, 3H), 3.81 (s, 3H), 6.95 (d, J = 9.0 Hz, 1H),
7.09 (s, 1H), 7.16 (d, J = 9.0 Hz,1H), 7.65(br s, 1H), 8.19 (d,
J = 9.0 Hz,1H), 8.33 (d, J = 8.7 Hz, 1H), 9.14 (s, 1H). mp 124.6–
125.6 °C.
K2CO3 (1.1 g, 8 mmol) were placed in a 50 mL flask and the DMSO
(10 mL) was added. The reaction mixture was stirred and heated at
100 °C for 2 d. The reaction mixture was quenched by adding water
(50 mL) and extracted with ethyl acetate. The organic phase was
dried over anhydrous Na2SO4 and concentrated. The crude product
was purified on a silica gel column using ethyl acetate/hexane (1/4,
v/v) to yield white solid (0.15 g, 33%). 1H NMR (CDCl3): d 3.76 (s,
6H), 5.29 (br s, 1H), 6.81 (d, J = 9.0 Hz, 4H), 6.93 (d, J = 9.0 Hz,
4H). mp 92.4–95.0 °C.
4.1.8. N-(2-((2,4-Dinitrophenyl)thio)phenyl)acetamide (10c)
Synthetic procedure described for 10a was followed starting
with 9b (1.1 g, 3.8 mmol) to afford yellow solid (1.25 g, 99%). 1H
NMR (CDCl3): d 2.10 (s, 3H), 6.88 (d, J = 8.7 Hz,1H), 7.26 (t,
J = 6.3 Hz,1H), 7.59 (m, 2H), 7.94 (br s, 1H), 8.19 (d, J = 9.0 Hz,1H),
8.54 (d, J = 8.4 Hz,1H), 9.14 (s, 1H). mp 182.7–184.0 °C.
4.1.2. 3,7-Dimethoxy-10H-phenothiazine (6)
Compound 5 (150 mg, 0.655 mmol), sulfur (91 mg, 2.3 mmol)
and I2 (29 mg, 0.1 mmol) were added into 1,2-dichlorobenzene
(10 mL). The reaction mixture was heated at 150 °C for 12 h. the
reaction mixture was cooled down to room temperature and puri-
fied on a silica gel column using ethyl acetate/hexane (1/4, v/v) as
mobile phase to yield yellow solid (50 mg, 29%).1H NMR (CDCl3): d
3.64 (s, 6H), 6.57–6.61 (m, 6H), 8.14 (br s, 1H). 13C NMR (DMSO-
d6): d 55.8, 111.9, 113.6, 115.2, 117.4, 136.7, 154.7. Anal. Calcd
for C14H13NO2S: C, 64.84; H, 5.05; N, 5.40. Found: C, 64.57; H,
5.15; N, 5.21. mp 194.2–196.1 °C.
4.1.9. N-(5-Bromo-2-((2,4dinitrophenyl)thio)phenyl)acetamide
(10d)
Compound 10c (2.0 g, 6 mmol) and NBS (4.0 g, 24 mmol) was
dissolved in DMF (5 mL). The reaction mixture was heated at
100 °C overnight. The reaction solution was quenched in water
(100 mL). The precipitate was filtered and purified on silica gel col-
umn chromatography using ethyl acetate/hexane(1/2, v/v) as mo-
bile phase to get yellow solid (2.3 g, 90%). 1H NMR (CDCl3): d
2.11 (s, 3H), 6.91 (d, J = 9.0 Hz, 1H), 7.73 (m, 2H), 7.91 (br s, 1H),
8.25 (d, J = 9.0 Hz, 1H), 8.51 (d, J = 9.3 Hz, 1H), 9.16 (s, 1H). mp
193.7–195.7 °C.
4.1.3. 4-((2-Amino-5-methoxyphenyl)thio)-3-nitrobenzonitrile
(9a)
4.1.10. N-(2-((2,4-Dinitrophenyl)thio)-5-iodophenyl)-acetamide
(10e)
Compound 8a (500 mg, 2.8 mmol) was suspended in 10 mL of
aqueous KOH (50%) solution and refluxed overnight. The reaction
solution was cooled down to room temperature and added drop-
wise to the solution of 7a (510 mg, 2.8 mmol) in ethanol (20 mL)/
AcOH (50 mL) in ice-water bath. The reaction mixture was stirred
for additional 3 h. The precipitate was filtrated and washed with
water/ethanol (1/1, v/v) to afford red solid. (510 mg, 60%). 1H
NMR (CDCl3): d 3.77 (s. 3H), 3.99 (br s, 2H), 6.85 (d, J = 8.4 Hz,
1H), 6.98 (m, 3H), 7.58 (d, J = 8.7 Hz, 1H), 8.56 (s, 1H). mp 163.6–
165.1 °C.
Compound 10c (0.5 g, 1.5 mmol) was dissolved in acetic acid
(20 mL). ICl (1 M) (10 mL) was added into above solution under
nitrogen atmosphere. The mixture was refluxed for 3 d and
quenched in water (200 mL). After filtration, the residue was puri-
fied on silica gel column chromatography using ethyl acetate/hex-
ane (1/2, v/v) as mobile phase to yield yellow solid (220 mg, 32%).
1H NMR (CDCl3): d 2.08 (s, 3H), 6.90 (d, J = 9.3 Hz, 1H), 7.87 (m, 2H),
7.99 (br s, 1H), 8.23 (d, J = 8.7 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 9.11
(s, 1H). mp 224.1–226.0 °C.
4.1.4. 2-((2,4-Dinitrophenyl)thio)-5-methoxyaniline (9b)
Synthetic procedure described for 9a was followed starting with
8a (5 g, 28 mmol) and 7b (6.7 g, 28 mmol), yellow solid (7.3 g,
81%). 1H NMR (CDCl3): d 3.76 (s. 3H), 4.00 (br s, 2H), 6.85 (d,
J = 8.4 Hz, 1H), 6.96–7.06 (m, 3H), 8.18 (d, J = 9.0 Hz, 1H), 9.13 (s,
1H). mp 169.4–171.7 °C.
4.1.11. General procedure A
Potassium hydroxide (98 mg, 1.74 mmol)) was added into the
solution of compound 10a (300 mg, 0.87 mmol) in acetone
(20 mL) in portion under reflux. The reaction solution was refluxed
for 2 h and quenched in ice-cold water. The precipitate was fil-
trated and recrystalized in acetone/water (3/2, v/v) to achieve the
corresponding product 11a. Compounds 11b–11e were prepared
by the same procedure.
4.1.5. 2-((2,4-Dinitrophenyl)thio)aniline (9c)
Compound 7b (10 g, 49 mmol) in ethanol was added dropwise
into the solution of 8b (6.8 g, 54 mmol), NaOH (2.16 g, 54 mmol)
in ethanol (50 mL). The reaction mixture was stirred at ambient
temperature for 2 h. the precipitate was filtered and washed by
ethanol to obtain yellow solid (11.4 g, 88%). 1H NMR (CDCl3): d
4.1.12. 7-Methoxy-10H-phenothiazine-3-carbonitrile (11a)
Yellow solid (100 mg, 45%). 1H NMR (DMSO-d6): d 3.67 (s, 3H),
6.62 (m, 4H), 7.34 (m, 2H), 9.02 (br s, 1H). 13C NMR (DMSO-d6): d

L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
4631
55.8, 102.7, 112.1, 113.7, 114.3, 116.1, 117.0, 117.0, 119.5, 129.9,
132.6, 133.4, 146.7, 155.8. Anal. Calcd for C14H10N2OS: C, 66.12;
H, 3.96; N, 11.02. Found: C, 66.13; H, 3.86; N, 11.03. mp 198.0–
198.9 °C.
4.1.18. 7-Methoxy-10-methyl-10H-phenothiazin-3-amine (13a)
Compound 12 (500 mg, 1.7 mmol) and 10% Pd on activated car-
bon (20 mg) were suspended in ethanol (15 mL). Under H2 (90 psi),
the reaction mixture was stirred at ambient temperature over-
night. The reaction solution was filtrated and concentrated in vac-
uum. The product was purified on the silica gel column
chromatography using ethyl acetate/hexane (1/2, v/v) as mobile
phase to afford yellow solid (350 mg, 80%). 1H NMR (DMSO-d6): d
3.15 (s, 3H), 3.69 (s, 3H), 4.79 (br s, 2H), 6.42–6.45 (m, 2H), 6.64
(d, J = 9.3 Hz, 1H), 6.73–6.80 (m, 3H). 13C NMR (DMSO-d6): d 35.3,
55.8, 112.8, 112.9, 112.9, 113.4, 114.6, 115.2, 122.8, 123.9, 136.1,
140.4, 144.4, 154.6. Anal. Calcd for C14H14N2OS: C, 65.09; H, 5.46;
N, 10.84. Found: C, 65.29; H, 5.53; N, 10.59. mp 141.6–142.9 °C.
4.1.13. 3-Methoxy-7-nitro-10H-phenothiazine (11b)
Synthetic procedure described for 11a was followed starting
with 10b (100 mg, 280 mmol). Violet solid (61 mg, 79%) 1H NMR
(DMSO-d6): d 3.66 (s, 3H), 6.58–6.64 (m, 4H), 7.71 (s, 1H), 7.83
(d, J = 9.0 Hz, 1H), 9.39 (br s, 1H). 13C NMR (DMSO-d6): d 55.8,
112.0, 113.3, 113.8, 116.2, 116.6, 116.8, 122.1, 125.2, 132.2,
140.8, 148.4, 156.3. Anal. Calcd for C13H10N2O3S: C, 56.92; H,
3.67; N, 10.21. Found: C, 56.64; H, 3.54; N, 10.07. mp 168.8–
170.1 °C.
4.1.19. 7-Methoxy-N,10-dimethyl-10H-phenothiazin-3-amine
(13b) and 7-methoxy-N,N,10-trimethyl-10H-phenothiazin-3-
amine (13c)
4.1.14. 3-Nitro-10H-phenothiazine (11c)
Methyl iodide (280 mg, 2 mmol) in acetonitrile (2 mL) was
added into the solution of compound 13a (200 mg, 0.77 mmol) in
acetonitrile (10 mL) with Na2CO3 (210 mg, 2 mmol). The reaction
mixture was sealed under nitrogen atmosphere and stirred at
80 °C overnight. The reaction mixture was cooled to room temper-
ature and partitioned between ethyl acetate and aqueous phase.
The organic extract was purified by silica gel column chromatogra-
phy using ethyl acetate/hexane (1/3, v/v) as mobile phase to yield
two yellow solids, compound 13b (32 mg, 15%) and compound 13c
(57 mg, 26%). 13b: 1H NMR (DMSO-d6): d 2.58 (s, 3H), 3.66 (s, 3H),
3.73 (m, 4H), 6.39 (m, 2H), 6.74 (m, 4H). 13C NMR (DMSO-d6): d
30.6, 35.3, 55.8, 110.5, 111.1, 112.8, 112.9, 114.7, 115.2, 123.1,
123.9, 136.0, 140.4, 146.1, 154.7. Anal. Calcd for C15H16N2OS: C,
66.15; H, 5.92; N, 10.29. Found: C, 66.36; H, 6.09; N, 10.24. mp
170.9–171.7 °C. 13c: 1H NMR (DMSO-d6): d 2.80 (s, 6H), 3.70 (s,
3H), 3.76 (m, 3H), 6.62 (m, 2H), 6.80 (m, 4H). 13C NMR (DMSO-
d6): d 35.3, 41.1, 55.8, 111.9, 112.4, 112.8, 113.0, 114.8, 115.1,
123.1, 123.8, 136.7, 140.2, 147.0, 154.8. Anal. Calcd for
Synthetic procedure described for 11a was followed starting
with 10c (300 mg, 0.9 mmol). Violet product (160 mg, 73%). 1H
NMR (DMSO-d6): d 6.69 (m, 2H), 6.84 (t, J = 7.2 Hz, 1H), 6.93 (d,
J = 7.2 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 7.73 (s, 1H), 7.85 (d,
J = 8.7 Hz, 1H), 9.51 (br s, 1H). 13C NMR (DMSO-d6): d 113.8,
115.7, 115.8, 117.3, 122.0, 124.1, 125.0, 126.7, 128.5, 139.2,
141.4, 148.2. Anal. Calcd for C12H8N2O2S: C, 59.00; H, 3.30; N,
11.47. Found: C, 59.02; H, 3.26; N, 11.33. mp 219.1–219.6 °C.
4.1.15. 3-Bromo-7-nitro-10H-phenothiazine (11d)
Synthetic procedure described for 11a was followed starting
with 10d (240 mg, 0.6 mmol) to afford violet solid (87 mg, 45%).
1H NMR (DMSO-d6): d 6.60 (d, J = 8.4 Hz, 1H), 6.67 (d, J = 8.4 Hz,
1H), 7.17 (m, 2H), 7.73(s, 1H), 7.85(d, J = 9.0 Hz, 1H), 9.59 (br s,
1H). 13C NMR (DMSO-d6): d 114.0, 114.8, 116.7, 117.2, 118.4,
122.1, 125.2, 128.6, 131.0, 138.7, 141.7, 147.6. Anal. Calcd for
C12H7BrN2O2S: C, 44.60; H, 2.18; N, 8.67. Found: C, 44.54; H,
2.26; N, 8.56. mp >250 °C.
C16H18N2OS: C, 67.10; H, 6.33; N, 9.78. Found: C, 67.36; H, 6.25;
N, 9.79. mp 185.0–186.5 °C.
4.1.16. 3-Iodo-7-nitro-10H-phenothiazine (11e)
4.1.20. 1-(3-Methoxy-7-nitro-10H-phenothiazin-10-yl)ethanone
(14a)
Synthetic procedure described for 11a was followed starting
with 10e (100 mg, 0.22 mmol) to afford violet solid (290 mg,
36%). 1H NMR (DMSO-d6): d 6.45 (d, J = 8.7 Hz, 1H), 6.64 (d,
J = 9.3 Hz, 1H), 7.22 (s, 1H), 7.30 (d, J = 9.3 Hz, 1H), 7.69 (s, 1H),
7.82 (d, J = 8.7 Hz, 1H), 9.54 (br s, 1H). 13C NMR (DMSO-d6): d
86.0, 114.0, 116.9, 117.6, 118.4, 122.1, 125.1, 134.0, 136.9, 139.1,
Acetyl chloride (850 mg, 11 mmol) was added into the solution
of compound 11b (1 g, 3.6 mmol) in dichloromethane (20 mL). The
reaction mixture was stirred overnight at ambient temperature.
The solvent and excessive acetyl chloride was removed in vacuum.
The residue was dissolved into ethyl acetate and washed by water
and saturated NaCl solution. The organic extract was dried by
anhydrous Na2SO4 and purified on silica gel column chromatogra-
phy using ethyl acetate/hexane (1/2, v/v) as mobile phase to yield
yellow solid (0.4 g, 89%).1H NMR (CDCl3): d 2.23 (s, 3H), 3.83 (s,
3H), 6.90 (d, J = 9.0 Hz, 1H), 6.98 (s, 1H), 7.32 (d, J = 8.7 Hz, 1H),
7.72 (d, J = 8.7 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.29 (s, 1H). 13C
NMR (CDCl3): d 22.9, 55.7, 112.7, 114.0, 122.0, 122.9, 127.4,
127.9, 130.7, 133.2, 134.3, 144.7, 145.6, 158.5, 169.2. Anal. Calcd
for C15H12N2O4S: C, 56.95; H, 3.82; N, 8.86. Found: C, 56.72; H,
3.89; N, 8.70. mp 155.9–156.8 °C.
141.6, 147.6. HRMS (ESI) m/z Calcd for
C12H7IN2O2S [M]
369.9276. Found: 369.9269. HPLC purity: 97%. mp >250 °C.
4.1.17. 3-Methoxy-10-methyl-7-nitro-10H-phenothiazine (12)
Sodium hydride (60%) (29 mg, 0.73 mmol) was added into the
solution of compound 11b (100 mg, 0.36 mmol) in DMF (10 mL)
at 0 °C. the reaction mixture was stirred under nitrogen atmo-
sphere for 30 min and warmed to room temperature. CH3I
(103 mg, 0.73 mmol) in DMF (2 mL) was added to the above solu-
tion and stirred for additional 2 h. The reaction mixture was
quenched by adding water (100 mL) and extracted with ethyl ace-
tate. The combined organic extracts were dried over anhydrous
Na2SO4 and concentrated on rotary evaporator. The residue was
purified on the silica gel column chromatography using ethyl ace-
tate/hexane (1/2, v/v) as mobile phase to afford red solid (90 mg,
87%). 1H NMR (DMSO-d6): d 3.28 (s, 3H), 3.63 (s, 3H), 6.71–6.76
(m, 2H), 6.87–6.94 (m, 2H), 7.84 (s, 1H), 7.96 (d, J = 9.3 Hz, 1H).
13C NMR (DMSO-d6): d 36.3, 55.9, 112.9, 113.7, 114.0, 116.9,
122.1, 122.3, 124.7, 136.7, 141.7, 151.8, 156.5. Anal. Calcd for
4.1.21. 1-(3-Nitro-10H-phenothiazin-10-yl)ethanone (14b)
Synthetic procedure described for 14a was followed starting
with 11c (150 mg, 0.6 mmol) to afford yellow solid (110 mg,
62%). 1H NMR (DMSO-d6): d 2.18 (s, 3H), 7.36 (t, J = 7.2 Hz, 1H),
7.45 (t, J = 7.2 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz,
1H), 7.86 (d, J = 7.8 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.42 (s, 1H).
13C NMR (DMSO-d6): d 23.1, 122.7, 123.3, 127.7, 127.9, 128.3,
128.5, 128.6, 131.2, 134.3, 138.1, 144.5, 145.8, 168.7. Anal. Calcd
for C14H10N2O3S: C, 58.73; H, 3.52; N, 9.78. Found: C, 58.60; H,
3.61; N, 9.62. mp 144.0–145.8 °C.
C14H12N2O3S: C, 58.32; H, 4.20; N, 9.72. Found: C, 58.11; H, 4.11;
N, 9.75. mp 174.7–175.5 °C.

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L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
4.1.22. 1-(3-Hydroxy-7-nitro-10H-phenothiazin-10-yl)ethanone
(15)
Compound 15 (50 mg, 0.17 mmol), (E)-3-bromo-1-iodoprop-1-
ene (50 mg, 0.2 mmol) and Na2CO3 (200 mg, 0.2 mmol) was mixed
in DMF (10 mL) and stirred for 3 h at ambient temperature. The
reaction mixture was quenched in water (50 mL) and extracted
with ethyl acetate. The organic extract was concentrated in vac-
uum. The residue was suspended in HCl (3 M) /MeOH (1:1, v/v)
and refluxed for 5 h. the precipitate was filtrated to get violet so-
lid.16b (25 mg, 36%).1H NMR (DMSO-d6): d 4.41 (s, 2H), 6.61–
6.71 (m, 6H), 7.71(s, 1H), 7.83 (d, J = 9.0 Hz, 1H), 9.41 (br s, 1H).
13C NMR (DMSO-d6): d 69.9, 82.6, 113.0, 113.4, 114.7, 116.2,
116.6, 116.9, 122.1, 125.2, 132.6, 140.8, 141.2, 148.4, 154.7. Anal.
Calcd for C15H11IN2O3S: C, 42.27; H, 2.60; N, 6.57. Found: C,
42.46; H, 2.72; N, 6.38. mp >250 °C.
The solution of BBr3 (1 M) in dichloromethane (1 mL) was
added dropwise into the solution of compound 11a (100 mg,
0.32 mmol) in dichloromethane (10 mL) at À78 °C. the reaction
solution was stirred overnight at ambient temperature. The solvent
was removed in vacuum. The residue was partitioned between
ethyl acetate and water. The organic extract was dried by anhy-
drous Na2SO4 and purified on silica gel column chromatography
using ethyl acetate/hexane (1/2, v/v) as mobile phase to yield yel-
low solid (79 mg, 81%). 1H NMR (DMSO-d6): d 2.15 (s, 3H), 6.82 (d,
J = 9.0 Hz, 1H), 6.93 (s, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.82 (d,
J = 9.0 Hz, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.39 (s, 1H), 10.00 (br s,
1H). 13C NMR (DMSO-d6): d 23.0, 114.3, 115.3, 122.6, 123.2,
128.4, 128.5, 129.3, 132.3, 134.2, 145.1, 145.6, 156.7, 169.1. HRMS
(ESI) m/z Calcd for C14H10N2O4S [M+1] 303.0440. Found: 303.0435.
HPLC purity: 98%. mp 202.3–205.1 °C.
4.1.25. 3-Nitro-7-(prop-2-yn-1-yloxy)-10H-phenothiazine (16c)
Synthetic procedure described for 16b was followed starting
with 15 (70 mg, 0.23 mmol) and 3-bromopropyne (38 mg,
0.25 mmol) to afford violet solid (55 mg, 86%). 1H NMR (DMSO-
d6): d 3.58 (s, 1H), 4.71 (s, 2H), 6.62–6.67 (s, 4H), 7.73 (s, 1H),
7.84 (d, J = 9.0 Hz, 1H), 9.43 (br s, 1H). 13C NMR (DMSO-d6): d
56.2, 78.7, 79.5, 113.2, 113.5, 115.0, 116.2, 116.5, 116.8, 122.1,
125.2, 133.1, 148.4, 154.1. Anal. Calcd for C14H10N2O3S: C, 54.89;
H, 3.62; N, 9.15. Found: C, 54.87; H, 3.54; N, 8.92.mp 226.4–
227.0 °C.
4.1.23. 3-(2-Fluoroethoxy)-7-nitro-10H-phenothiazine (16a)
Compound 15 (120 mg, 0.4 mmol) was dissolved in anhydrous
DMF (10 mL), NaH (24 mg, 0.6 mmol) was added under 0 °C and
stirred for 30 min. 1-Bromo-2-fluoroethane (150 mg, 0.6 mmol)
was added and stirred overnight at room temperature. The reac-
tion mixture was quenched with water (100 mL) and extracted
with ethyl acetate. After removal of solvent, the residue was sus-
pended in aqueous HCl solution (3 M) in methanol/water (1:1)
and refluxed for 5 h. the reaction mixture was quenched in water
and extracted with ethyl acetate, the organic extract was dried
by anhydrous Na2SO4 and purified on silica gel column chromatog-
raphy using ethyl acetate/hexane (1.2, v/v) as mobile phase to get
violet solid (40 mg, 33%). 1H NMR (DMSO-d6): d 4.09 (t, J = 3.6 Hz,
1H), 4.19 (t, J = 3.6 Hz, 1H), 4.60 (t, J = 3.6 Hz, 1H), 4.76 (t,
J = 3.6 Hz, 1H), 6.64 (m, 4H), 7.72 (s, 1H), 7.83 (d, J = 9.3 Hz, 1H),
9.41 (br s, 1H). 13C NMR (DMSO-d6): d 67.8, 68.0, 81.6, 83.3,
112.8, 113.4, 114.6, 116.3, 116.6, 116.9, 122.1, 125.2, 132.6,
140.9, 148.4, 155.1. Anal. Calcd for C15H10N2O3S: C, 60.39; H,
3.38; N, 9.39. Found: C, 60.15; H, 3.50; N, 9.15. mp 189.3–191.4 °C.
4.2. Thioflavin T fluorescence assay for a-synuclein fibrils
4.2.1. Production of purified recombinant
-Synuclein recombinant protein was produced in Escherichia.
coli.38,39 BL21(DE3)RIL E. coli were transformed with a pRK172 bac-
terial expression plasmid containing the human -synuclein cod-
a-synuclein protein
a
a
ing sequence. Freshly transformed BL21 colonies were inoculated
into 2 L baffled flasks containing 250 mL sterilized TB (1.2% bacto-
tryptone, 2.4% yeast extract, 0.4% glycerol, 0.17 M KH2PO4, 0.72 M
K2HPO4) with 50 lg/ml ampicillin, and incubated overnight at
37 °C with shaking. Overnight cultures were pelleted by centrifu-
gation at 3900g for 10 min at 25 °C. Bacterial pellets were resus-
pended in 20 mL osmotic shock buffer (30 mM Tris–HCl, 2 mM
EDTA, 40% Sucrose, pH 7.2) by gentle vortexing and incubated at
room temperature for 10 min. The cell suspension was then centri-
fuged at 8000g for 10 min at 25 °C and the pellet was resuspended
4.1.24. (E)-3-(3-Iodoallyloxy)-7-nitro-10H-phenothiazine (16b)
Prop-2-yn-1-ol (0.7 g, 12 mmol), Pd(Ph3P)2Cl2 (42 mg,
0.06 mmol) and HSnBu3 (3 g, 10 mmol) was dissolved in anhy-
drous tetrahydrofuran and stirred at ambient temperature for
1 h. The solvent was removed in vacuum. The residue was purified
on the silica gel column using ethyl acetate/hexane (1/9, v/v) as
mobile phase to yield colorless liquid, (E)-3-(tributylstan-
nyl)prop-2-en-1-ol (1.2 g, 34%) as intermediate.
Iodine solution in chloroform (0.1 M) was added into the solu-
tion of (E)-3-(tributylstannyl)prop-2-en-1-ol (200 mg, 0.58 mmol)
in CHCl3 (10 mL) and stirred at ambient temperature for 2 h. The
reaction mixture was quenched by aqueous Na2S2O5 (5%) and ex-
tracted with ethyl acetate. The organic extract was concentrated
and purified on silica gel column using ethyl acetate/hexane (1/3,
v/v) as mobile phase to yield colorless liquid ((E)-3-iodoprop-2-
en-1-ol) 85 mg, 79%. 1H NMR (CDCl3): d 2.04 (t, J = 5.7 Hz, 1H),
4.08 (t, J = 5.7 Hz, 2H), 6.39 (d, J = 14.4 Hz, 1H), 6.68 (d,
J = 14.4 Hz, 1H).
Triphenylphosphine (133 mg, 0.51 mmol) was added into the
solution of (E)-3-iodoprop-2-en-1-ol in CH2Cl2 (10 mL) at 0 °C
and stirred for 1 h, following by the addition of CBr4 (186 mg,
0.56 mmol) and stirred for additional 2 h at ambient temperature.
The solvent was removed in vacuum. The residue was purified by
silica gel column chromatography using ethyl acetate/hexane (1/
9, v/v) as mobile phase to get colorless liquid, (E)-3-bromo-1-iod-
oprop-1-ene (40 mg, 35%). 1H NMR (CDCl3): d3.87 (d, J = 7.8 Hz,
2H), 6.54 (d, J = 14.4 Hz, 1H), 6.71 (d, J = 14.4 Hz, 1H).
in 22.5 mL cold H2O before adding 9.4 lL 2 M MgCl2 to each tube.
The suspension was incubated on ice for 3 min prior to centrifuga-
tion at 20,000g for 15 min at 4 °C. The supernatant was transferred
to a fresh tube, streptomyocin was added to a final concentration
of 10 mg/mL and then centrifuged at 20,000g for 15 min at 4 °C.
The supernatant from this step was collected and dithiothreitol
(DTT) and Tris–HCl were added to final concentrations of 1 mM
and 20 mM, respectively, before boiling for 10 min to precipitate
heat-sensitive proteins, which were pelleted at 20,000g for
15 min at 4 °C. The supernatant was collected and filtered through
a 0.45 lm surfactant free cellulose acetate filter (Corning) before
loading onto a 1 mL DEAE Sepharose column equilibrated in
20 mM Tris–HCl pH 8, 1 mM EDTA, and 1 mM DTT. The DEAE col-
umn was washed with 20 mM Tris–HCl pH 8, 1 mM EDTA, 1 mM
DTT before eluting
buffer with 1 mM EDTA, 1 mM DTT and 0.3 M NaCl. The purified
-synuclein protein was dialyzed overnight in 10 mM Tris–HCl,
pH 7.6, 50 mM NaCl, 1 mM DTT. Preparations contained greater
than 95% -synuclein protein as determined by SDS–PAGE and
a-synuclein protein in 20 mM Tris–HCl, pH 8,
a
a
BCA assay with a typical yield of 30 mg protein per 250 ml culture.
4.2.2. Fibril preparation
The purified, recombinant
a-synuclein monomer (2 mg/mL)
was incubated in Tris–HCl (20 mM), NaCl (100 mM) at 37 °C while
shaking at 1000 rpm in an Eppendorf Thermomixer in a 37 °C

L. Yu et al. / Bioorg. Med. Chem. 20 (2012) 4625–4634
4633
temperature-controlled room for 72 h. To determine the concen-
tration of fibrils, fibril reaction mixture (100 L) was centrifuged
at 18,000 xg for 10 min to separate fibrils from monomer. The con-
centration of -synuclein monomer in the supernatant was deter-
phenothiazine compound, (2) 3
pound 16a, and (4) 3
period the reactions (n=3 for control and each compound) were
centrifuged at 100,000g for 20 min at 4 °C to pellet -synuclein fi-
l
M compound 11b, (3) 3
l
M com-
l
lM compound 16b. Following the incubation
a
a
mined in a bicinchoninic acid (BCA) protein assay along with a
bovine serum albumin (BSA) standard curve. The measured de-
crease in monomer concentration was used to determine the con-
centration of fibrils in the 72 h fibril reaction mixture.
To prepare fibrils for binding assays, the fibril mixture prepared
above was centrifuged at 18,000g for 10 min. The supernatant was
discarded and the fibril pellet was resuspended in Tris–HCl buffer
(30 mM, pH = 7.4) to achieve the desired concentration of fibrils for
use in the assay.
brils. The supernatants were removed and the fibrils in the pellet
were resuspended in SDS–PAGE sample buffer (recipe), sonicated
in a (sonicator model) sonicator at room temperature, and then
incubated at 95 °C for 10 min to solubilize the fibrils by dissocia-
tion to monomeric a-synuclein. Fifteen microlitre of solubilized fi-
brils from each reaction were loaded onto an SDS–PAGE (Bio-Rad
Criterion) gel and electrophoresed for 1 h at 175 V. Protein was
quantified by staining with SYPRO Ruby protein gel stain (Invitro-
gen) and then imaged with a Kodak Imager Station 440 using UV
excitation. A standard curve was included on the gel to ensure that
4.2.3. Determination of maximum excitation/emission
the reactions samples contained levels of
linear range of SYPRO Ruby detection.
a-synuclein within the
wavelength of ThT-
The ThT solution (6.0
L) was added into each of three wells containing
fibrils suspension (3.0 M) in Tris–HCl buffer (30 mM, pH = 7.4,
40 L) in a 96 well plate for fluorescence detection. The mixture
a
-synuclein fibrils
M) in Tris–HCl buffer (30 mM, pH = 7.4,
-synuclein
l
40
l
a
Acknowledgments
l
l
The authors like to thank Dr. Robert H. Mach for his advice on
the project. Financial support for these studies was provided by
the National Institutes of Health under Grants NS061025 (Z.T.),
NS075527 (Z.T.), MH092797 (Z.T.) and Barnes-Jewish Hospital
Foundation/Washington University Institute of Clinical & Transla-
tional Sciences (P,K). Mass spectra were acquired at the NIH NCRR
Biomedical Mass Spectrometry Resource at Washington University
(NIH 2P41RR000954).
was incubated at room temperature for 1 h with gentle shaking.
The reaction plate was scanned by the excitation wavelength range
from 430 to 465 nM. The maximum excitation wavelength (kex
was determined according to the fluorescent intensity-excitation
wavelength curve. At kex, the emission wavelength was scanned
to get maximum emission wavelength (kem). kex and kem for the
)
free ThT and ThT-monomeric
a-synuclein was determined by the
same procedure described above.
Supplementary data
4.2.4. Measurement of the dissociation constant of ThT-
a-
synuclein fibrils (Kd)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.023.
ThT solutions of various concentration from 10 nM to 40
Tris–HCl buffer (30 mM, pH = 7.4, 40
well plate containing -synuclein fibrils (3.0
fer (30 mM, pH = 7.4, 40 L). The mixture was incubated at room
l
M in
l
L) were added into the 96
a
l
M) in Tris–HCl buf-
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(30 mM, pH = 7.4,) was added into a 96 well plate containing
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