Design, Synthesis, and Characterization of 3-(Benzylidene)indolin-2-one Derivatives as Ligands for α-Synuclein Fibrils

Article abstract of DOI:10.1021/acs.jmedchem.5b00571

A series of 3-(benzylidine)indolin-2-one derivatives were synthesized and evaluated for their in vitro binding to alpha synuclein (α-syn), beta amyloid (Aβ), and tau fibrils. Compounds with a single double bond in the 3-position had only a modest affinity

Full text of DOI:10.1021/acs.jmedchem.5b00571

Article
pubs.acs.org/jmc
Design, Synthesis, and Characterization of 3‑(Benzylidene)indolin-2-
one Derivatives as Ligands for α‑Synuclein Fibrils
Wenhua Chu,† Dong Zhou,† Vrinda Gaba,‡ Jialu Liu,‡ Shihong Li,† Xin Peng,† Jinbin Xu,†
Dhruva Dhavale,‡ Devika P. Bagchi,‡ Andre d’Avignon,§ Naomi B. Shakerdge,∥ Brian J. Bacskai,∥
́
Zhude Tu,† Paul T. Kotzbauer,‡ and Robert H. Mach*,†,∥
†Department of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, United States
‡Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, United States
§Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States
∥MassGeneral Institute of Neurodegenerative Disease, Massachusetts General Hospital, Charlestown, Massachusetts 02129, United
States
S
* Supporting Information
ABSTRACT: A series of 3-(benzylidine)indolin-2-one deriva-
tives were synthesized and evaluated for their in vitro binding to
alpha synuclein (α-syn), beta amyloid (Aβ), and tau fibrils.
Compounds with a single double bond in the 3-position had only
a modest affinity for α-syn and no selectivity for α-syn versus Aβ
or tau fibrils. Homologation to the corresponding diene analogues
yielded a mixture of Z,E and E,E isomers; substitution of the
indoline nitrogen with an N-benzyl group resulted in increased
binding to α-syn and reasonable selectivity for α-syn versus Aβ
and tau. Introduction of a para-nitro group into the benzene ring
of the diene enabled separation of the Z,E and E,E isomers and led to the identification of the Z,E configuration as the more
active regioisomer. The data described here provide key structural information in the design of probes which bind preferentially
to α-syn versus Aβ or tau fibrils.
normal controls.5 More recent studies in patients with familial
AD have clearly shown that PiB(+) Aβ plaque formation occurs
INTRODUCTION
■
It is generally accepted that many neurodegenerative disorders
early in the disease process and suggest amyloid plaques may
are characterized by the accumulation of misfolded proteins
represent an antecedent biomarker of AD.6,7 The subsequent
which lead to the formation of insoluble protein aggregates in
emergence of 18F-labeled Aβ imaging agents, including
the CNS.1,2 For example, Alzheimer’s disease (AD) is
[18F]fluorbetapir,8,9 [18F]florbetaben,10 and [18F]flumetamol,11
characterized by the formation of two different insoluble
has enabled the expansion of PET studies in AD patients to
protein aggregates: (1) beta amyloid (Aβ plaques, which
consists of aggregated beta amyloid protein, Aβ1−42) and (2)
neurofibrillary tangles (NFTs), which consist of aggregates of
imaging centers without an on-site cyclotron facility. Recently, a
number of 18F- and 11C-labeled agents for imaging tau deposits
in NFTs have been reported.12−14 These agents are expected to
hyperphosphorylated tau protein. The diagnosis of AD
provide valuable information on the temporal separation
historically relied on the cognitive assessment of patients with
between the formation of Aβ plaques (a possible antecedent
moderate to severe memory deficits. Although a progressive
biomarker) and NFT formation (thought to reflect neuronal
cognitive decline resulting in a severe impairment of daily
function was considered consistent with “Alzheimer-like
dementia”, the clinical diagnosis of AD could not be confirmed
until post-mortem analysis demonstrated the presence of Aβ
loss in AD).6 This ability to measure the neuropathological
time course of Aβ plaque and NFT formation in patients will be
important in the evaluation of new disease-modifying
therapeutics aimed at slowing the clinical progression of AD.
plaques and NFTs in the temporal and parietal cortex. A major
Another protein capable of forming insoluble aggregates in
breakthrough in the study of patients presenting with AD-like
symptoms emerged with the development of [11C]PiB, a
positron-labeled analogue of thioflavin-T (Thio-T), the
brain is alpha synuclein (α-syn), which is the principal species
found in Lewy bodies (LBs) and Lewy neurites (LNs).15,16 LBs
are a dense core of insoluble aggregates of α-syn found in the
fluorescent dye used to visualize Aβ plaques in post-mortem
samples of AD brain.3,4 Initial PET studies in AD patients and
cell bodies of neurons, principally located in the brain stem and
healthy controls demonstrated increased [11C]PiB retention in
the frontal cortex in the patients with a clinical diagnosis of a
progressive loss of cognitive function in comparison with
Received: April 20, 2015
© XXXX American Chemical Society
A
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Figure 1. Reported ligands for tau, α-synuclein, and Aβ.
subcortical regions of the CNS. LNs are abnormal axons or
dendrites that contain insoluble aggregates of α-syn and have a
more diffuse morphology than LBs. The formation of LBs and
LNs are the primary pathological features of a collection of
neurological disorders referred to as “synucleinopathies”, which
include Parkinson’s disease (PD), Dementia with Lewy Bodies
(DLB), multiple system atrophy (MSA), and Picks’ disease.17
In addition, 50% of familial and sporadic AD patients show
evidence of LBs at autopsy.18 Furthermore, AD patients with
concomitant LB pathology demonstrate an accelerated
cognitive decline and may represent a subset of AD patients.18
These data suggest that a PET radiotracer for imaging insoluble
α-syn aggregates should be useful in the study of the formation
of LBs and LNs in a diverse panel of CNS disorders.
Recently, a number of phenothiazine analogues, including
the tricyclic ligands SIL23, SIL5, and SIL26, the fluorescent
dyes LDS 798 and LDS 730, and the indolinone 5 (Figure 1),
have shown reasonable in vitro selectivity for α-syn versus Aβ
and tau fibrils.19−24 Although these compounds are noteworthy
as the first analogues shown to label insoluble α-syn aggregates,
their low in vitro stability and moderate affinity for α-syn
aggregates limits their utility as PET imaging agents. Therefore,
there is a need to identify new chemical scaffolds which can
serve as lead compounds for PET radiotracer development. In
this study, we describe the synthesis and in vitro evaluation of a
novel class of compounds, the indolinone-diene analogues,
having high affinity and selectivity for α-syn aggregates which
could serve as a second generation lead compound for PET
radiotracer development.
fragments of LDS 798 and LDS 730 were incorporated into
compound 5 to determine if these groups would improve the
affinity and selectivity of the new analogues for α-syn fibrils
versus Aβ and tau fibrils.
The synthesis of the 3-benzylidene-indolin-2-one analogues
is shown in Scheme 1. 3-(4-(Dimethylamino)benzylidene)-
indolin-2-one (5), 3-(4-(dimethylamino)benzylidene)-1H-
pyrrolo[2,3-b]pyridin-2(3H)-one (6), and 3-(4-
(dimethylamino)benzylidene)-5-hydroxyindolin-2-one (7)
were prepared by condensing indolin-2-one (1), 1,3-dihydro-
2H-pyrrolo[2,3-b]pyridin-2-one (2), 5-hydroxyindolin-2-one
(3), and dimethylaminobenzaldehyde (4) in acetic acid with
refluxing for 3 h. The nitrogen of 5 and 6 was alkylated by
treatment of 5 or 6 with sodium hydride in DMF at 0 °C,
followed by addition of select alkyl halides to give compounds
8−14. Similarly, indolin-2-one (1), 1,3-dihydro-2H-pyrrolo-
[2,3-b]pyridin-2-one (2), or 5-hydroxyindolin-2-one (3) were
condensed with a 4-substituted cinnamaldehyde (15−17) in
acetic acid with refluxing to give 3-(4-substituted-phenyl)-
allylidene)indolin-2-one (18−20), 3-(3-(4-substituted phenyl)-
allylidene)-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (21), or 3-(3-
(4-(dimethylamino)phenyl)allylidene)-5-hydroxyindolin-2-one
(22), respectively. The nitrogen of compound 18−21 were
alkylated by treatment of 18, 19, 20, or 21 with sodium hydride
in DMF at 0 °C, followed by the addition of select alkyl halides
to give compounds 23−32. The nitro group of 28 was reduced
to an amino group with iron powder and HCl in methanol to
afford compound 33. 1-Phenylindolin-2-one (34) was con-
densed with 4 or 17 in acetic acid with refluxing to afford 35 or
36, respectively (Scheme 1).
Compounds 5, 6, and 7 and their nitrogen alkylated
analogues (8−14, and 35) exist as either Z or E geometric
isomers depending on the characteristics of the substituent at
the C-3 position of the 3-substituted indolin-2-one or 3-
substituted 1H-pyrrolo[2,3-b]pyridin-2(3H)-one. The 3-(4-
substituted-phenyl)allylidene)indolin-2-one (18−20), 3-(3-(4-
substituted phenyl)allylidene)-1H-pyrrolo[2,3-b]pyridin-
2(3H)-one (21), and 3-(3-(4-(dimethylamino)phenyl)-
allylidene)-5-hydroxyindolin-2-one (22) and their analogues
(23−33 and 36) should theoretically have four geometric
isomers for the double bond of each compound with Z,Z, E,E,
Z,E, and E,Z configuration. Because only the trans (E isomer)
of 4-substituted cinnamaldehyde was used for the synthesis,
most of the compounds have only two geometric isomers: E,E
RESULTS
■
Chemistry. The first step in the design of a probe for
imaging α-syn aggregates involved identifying a suitable lead
compound for our structure−activity relationship (SAR)
studies. An earlier publication reported that compound 5
(Figure 1) had a modest affinity for α-syn fibrils but low
selectivity versus Aβ and tau fibrils.20 We synthesized a number
of indolinone analogues to determine if it would be possible to
improve the affinity of compound 5 for α-syn and reduce its
affinity for Aβ and tau fibrils by exploring substituents in the
benzylidene aromatic ring and alkylation the indolin-2-one
nitrogen atom. A recent publication additionally demonstrated
that the fluorescent dyes LDS 798 and LDS 730 labeled Lewy
bodies in post-mortem PD cases.21 Therefore, the diene
B
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a
Scheme 1. Synthesis of 3-(Benzylidene)indolin-2-one Derivatives
a
Reagents: (a) CH3COOH, reflux 3 h; (b) (1) NaH, DMF, (2) CH3I, R-C6H4CH2X (X = Cl, Br), or ClCO2C2H5; (c) 28, Fe, CH3OH, HCl, reflux
3 h.
or Z,E. We first attempted to separate the E,E and Z,E isomers
Similarly, compounds 39 and 42−46 were synthesized from
condensation of 1 with 4-nitrobenzaldehyde (37) and (E)-3-(4-
nitrophenyl)acrylaldehyde (40) to give 3-(4-nitrobenzylidene)-
indolin-2-one (38) and 3-(3-(4-nitrophenyl)allylidene)indolin-
2-one (41), followed by alkylation of 38 and 41 with various
alkyl halides (Scheme 2). Unlike other 4-substituted-
benzaldehyde and 4-substituted cinnamaldehyde condensed
analogues, the isomers of condensed products of 4-nitro-
benzaldehyde (37) and (E)-3-(4-nitrophenyl)acrylaldehyde
(40) and their derivatives could be separated by flash
chromatography with CH2Cl2−ethyl ether (3:1) to afford
38a−39a and 38b−39b, and 41a−46a and 41b−46b,
respectively. The stability of compounds 41a and 41b in
by HPLC for binding assays. As an example, although the two
isomers 20 and 27 could be separated by HPLC, the isolated
pure Z,E and E,E isomers each isomerized within 20 min to a
mixture of Z,E and E,E isomers. Because we were unable to
obtain pure Z,E and E,E isomers for the binding assay, a
mixture of the Z and E isomers for compounds 5−7 and their
analogues (8−14 and 35) and mixtures of Z,E and E,E isomers
of 18−22 and their analogues (23−32 and 36) were used to
measure binding affinities; the ratio of the Z and E, or Z,E and
1
E,E isomers was measured with HPLC and determined by H
NMR spectra.
C
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a
Scheme 2. Synthesis of 3-(Benzylidene)indolin-2-one Derivatives
a
Reagents: (a) CH3COOH, reflux 3 h; (b) (1) NaH, DMF, (2) CH3I, or R-C6H4CH2X (X = Cl, Br); (c) Fe, CH3OH, HCl, reflux 3 h.
solution was measured by HPLC. In a solvent mixture of
CH2Cl2−MeOH (1:1), 41a was stable for 20 h as evidenced by
the HPLC peak and retention time. In contrast, 41b was
unstable under the same conditions, showing 33% isomer-
ization to an unidentified isomer different from 41a
(Supporting Information, Figure 4−7).
The two isomers, forms Z,E and E,E of compound 41−46,
could be distinguished by 2D NMR spectra analysis.
Compounds 41a and 41b were used to determine configuration
structure by 2D NMR. The E,E configured compounds should
show a nuclear Overhauser effect (NOE) between the proton at
the C-4 position and the vinyl proton, like H4 and Hb, whereas
the Z,E configured compounds should not show a NOE effect
between the proton at the C-4 position and the vinyl proton(s)
in the C-3 substitution of the 3-substituted indolin-2-ones
(Figure 2). The proton 1H shift of compound 41b was assigned
by 1H NMR. In DMSO solution, the peak of H4 in C-4 position
of indole overlapped with the peak of Hα in the nitrophenyl
ring. After adding 10% benzene-d6, the two peaks is separated
Figure 2. Nuclear Overhauser effect in E,E configuration of compound
41.
very well (H4, 8.15 ppm, and Hα, 8.07 ppm, respectively). The
H4 and Hb show a strong NOE effect in the 2D NMR spectra
of 41b. Therefore, compound 41b is E,E configured and 41a is
Z,E configured (Supporting Information, Figures S1−S3). The
configurations of the remaining compounds 42a−46a and
1
42b−46b were assigned by comparison of their H NMR
D
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a
Scheme 3. Synthesis of Labeling Precursor 48 and Radiosynthesis of [18F]46a
a
Reagents: (a) (1) NaH, DMF, (2) BrCH2C6H4OCH2CH2Br; (b) AgOMs, CH3CN; (c) [18F]KF, K2CO3, K222, tert-amyl alcohol.
Table 1. Ki Values of 3-(Benzylidene)indolin-2-one Derivatives for α-Syn, Aβ, and Tau
a
compd
R1
X
R2
E:Z
α-syn (nM)
Aβ (nM)
tau (nM)
Log P
5
H
CH
N
H
75:25
26:74
67:33
84:16
83:17
52:48
77:23
62:38
66:34
69:31
94:6
84.4 13.5
124.3 46.1
407.1 98.6
507.6 10.0
144.4 53.2
114.5 53.9
149.8 45.7
342.6 114.4
350.2 201.3
79.5 22.1
91.6 18.5
40.0 22.3
94.8 22.4
155.1 8.8
260.4 78.5
251.5 52.0
87.1 34.1
139.9 70.1
190.2 84.8
113.3 2.6
179.2 71.3
261.5 4.9
191.3 39.5
212.4 110.9
617.6 182.0
797.8 70.2
431.7 123.3
364.0 121.1
1116.2 69.1
858.8 172.0
853.5 158.6
789.8 153.7
3.0
2.4
2.6
3.2
4.9
4.8
3.6
3.8
4.3
4.2
4.9
6
H
OH
H
H
7
CH
CH
CH
CH
CH
CH
N
H
8
CH3
PhCH2
9
H
10
11
12
13
14
35
H
p-CH3OC6H4CH2
p-PyridylCH2
CO2C2H5
H
H
H
PhCH2
H
N
p-CH3OC6H4CH2
Ph
H
CH
290.2 120.2
a
Calculated by ChemBioDraw Ultra 13.0.
spectrum with 41a and 41b. The chemical shifts of its particular
proton Hb (dd couple) is 8.81 ppm for Z,E configuration and
7.78 ppm for E,E configuration. Similarly, configurations of the
compounds 5−14 and 35 were assigned based on the chemical
shifts of particular protons, Ha, H-2, and/or H-6, by comparing
the reported chemical shifts for compound 5;25 configurations
of compounds 18−33 and 36 were assigned based on the
chemical shifts of particular proton, Hb.
Radiosynthesis. The synthesis of the mesylate precursor 49
and the 18F-labeling are shown in Scheme 3. Radiosynthesis of
[18F]46a was carried out manually starting from 49 under
conventional conditions using tert-amyl alcohol as the solvent
to afford [18F]46a with a yield of 19.3 6.3 mCi (n = 6) (decay
corrected) with specific activity ranging from 800 to 5000 mCi/
μmol at the end of synthesis. The radiochemical purity was
>99%.
Fibril Binding Assays. Thio-T competitive binding assays
were utilized to measure affinity and selectivity for α-syn fibrils.
Thio-T is a weakly fluorescent dye that displays a dramatic
increase in fluorescence when bound to amyloid fibrils. Thio-T
assays for α-syn fibrils were modified from our previous
studies22 to improve the consistency of binding results for
different classes of compounds. Furthermore, we developed
additional competitive binding assays with Aβ and tau fibrils in
order to evaluate selectivity. Under the conditions used for
these studies, we determined that the affinity constants (Kd) of
Thio-T for α-syn, Aβ, and tau fibrils were 1850, 32, and 2700
nM, respectively (Supporting Information Figure S5). The
competitive binding assays measured displacement of Thio-T
binding by increasing concentrations of each indolinone
compound, using fixed concentrations of fibrils and Thio-T.
E
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Table 2. Ki Values of 3-(Benzylidene)indolin-2-one Derivatives for α-Syn, Aβ, and Tau
a
compd
R1
X
R2
R3
EE:ZE
α-syn (nM)
Aβ (nM)
tau (nM)
log P
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
36
47
H
CH
CH
CH
N
H
H
77:23
78:22
41:59
38:62
47:53
0:100
84:16
63:37
65:35
52:48
206.7 8.7
61.1 9.6
40.7 8.7
11.5 2.0
54.0 17.9
226.9 64.0
72.5 22.6
63.7 29.6
40.9 25.2
54.6 11.4
4.2 0.8
266.8 136.5
125.8 42.6
27.6 4.8
522.1 152.6
169.0 22.3
53.7 9.7
3.2
3.1
3.5
2.9
3.1
5.2
5.0
3.7
5.5
5.3
5.5
4.1
4.4
4.8
4.7
4.7
5.4
2.4
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
N(CH3)2
N(CH3)2
N(CH3)2
H
H
H
15.3 5.5
35.1 12.3
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
37.1 13.6
703.3 214.7
741.0 254.6
56.0 14.8
357.9 161.7
129.6 41.4
92.4 21.9
45.9 19.4
84.0 35.0
85.1 23.0
107.0 18.9
98.9 12.1
286.8 142.2
50.8 18.5
48.0 17.0
CH2Ph
CH2Ph
CH3
CH2Ph
682.4 120.2
717.6 295.3
229.1 121.4
374.0 209.3
239.7 157.6
90.0 48.8
OCH3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
NH2
p-CH3OC6H4CH2
p-NO2C6H4CH2
p-pyridylCH2
CO2C2H5
65.35
50:50
51:49
80:20
87:13
55:45
29:71
92.0 11.2
45.3 9.0
68.3 31.2
29.8 6.6
43.6 20.0
68.1 33.0
44.8 15.6
188.0 68.0
263.2 110.5
257.0 116.7
204.3 26.7
131.1 51.5
465.1 94.6
38.3 28.7
CH2Ph
N
p-CH3OC6H4CH2
p-NH2C6H4CH2
Ph
CH
CH
H
H
a
Calculated by ChemBioDraw Ultra 13.0.
The Thio-T concentration was set at 150% of the Kd value
determined for each fibril species.
case E,E and Z,E isomers. Substitution of the para-position of
the pendant phenyl group of 18 with a methoxy group (19)
resulted in an increase in affinity for α-syn (Ki = 62 versus 206
nM), Aβ (Ki = 125 versus 267 nM), and tau fibrils (Ki = 169
versus 522 nM), with a slight preference in affinity for α-syn.
However, a similar substitution with a dimethylamino group
resulted in a compound (20) having a higher affinity for Aβ (Ki
= 27 nM) versus α-syn (Ki = 41 nM) and tau (Ki = 54 nM).
The corresponding aza-indolinone-diene analogue of 20 (i.e.,
21) had high affinity (Ki ∼ 12−35 nM), for all three fibrils.
Substitution of the indolinone nitrogen atom of 18 with a
benzyl group resulted in 23, which was primarily in the Z,E
configuration and did not lead to an improvement in affinity for
α-syn (Ki = 227 nM), Aβ (Ki = 700 nM), or tau (Ki = 680 nM)
fibrils. However, substitution of the para position of 24 with
either a methoxy group (27) or nitro group (28) resulted in an
improvement in affinity for α-syn fibrils versus Aβ and tau
fibrils, but it is not clear if this improvement in affinity was due
to the substituent effect or the preference of these analogues for
the E,E isomer. The other analogues in Table 2, which
consisted of a mixture of E,E and Z,E isomers, had modest
affinity for α-syn, Aβ, and tau fibrils. The most interesting
compound in this series was 28, which has a 4 nM affinity for α-
syn fibrils and 20-fold lower affinity for Aβ and tau fibrils.
Introduction of a para nitro group into the benzene ring of
the diene moiety enabled the isolation of both the E,E and Z,E
regioisomers. In general, the Z,E regioisomers were more
potent than the corresponding E,E configuration (Table 3).
Although compound 41a had a similar affinity for α-syn, Aβ,
All final compounds in the manuscript possess a purity of at
least 95% as determined by elemental analysis or HPLC
analysis. The competitive binding results for indolinone and
aza-indolinone analogues having a single double bond are
shown in Table 1. In our assay, lead compound 5, with an E:Z
ratio of 75:25, had a similar affinity for α-syn and Aβ fibrils (Ki
∼ 85 nM), and a 3-fold lower affinity for tau fibrils (Ki = 262
nM). The corresponding aza-indolinone analogue 6 had a
reversed E:Z ratio and a higher affinity for Aβ (Ki = 40 nM)
versus α-syn and tau fibrils (Ki = 125−190 nM). Substitution of
the 5-position of 5 with a hydroxyl group 7 resulted in a
reduction in affinity for α-syn fibrils (Ki = 407 nM) and no
change in affinity for Aβ and tau fibrils. Substitution of the
indolinone nitrogen atom (8−14, 35) had only a minor effect
on the affinity of this series for all three fibrils. Replacement of
the dimethyl amino group of 5 and 9 with a nitro group
permitted the isolation of Z and E regioisomers. The Z isomer
38a had a higher affinity for α-syn (Ki = 347 nM) than the
corresponding E isomer 38b (Ki = 620 nM), but the affinity
was too low to be useful as a PET probe for imaging α-syn, a
feature observed in all of the analogues having a single double
bond in the indolinone ring system (Tables 1 and 3).
The results of the in vitro binding data for the indolinone-
diene analogues are shown in Tables 2 and 3. As with the
analogues of 5, indolinone-diene analogues having either a
hydrogen, methoxy, or dimethylamino group in the pendant
aromatic ring consisted of a mixture of regioisomers, in this
F
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Table 3. Ki Values of 3-(Benzylidene)indolin-2-one Derivatives for α-Syn, Aβ, and Tau
a
b
Calculated by ChemBioDraw Ultra 13.0. Measured: 4.18 0.02 (n = 3).
and tau fibrils (Ki ∼ 15−35 nM), substitution of the indolinone
nitrogen with a benzyl group 42a resulted in increase in
selectivity for α-syn (Ki = 40 nM) versus Aβ (Ki = 214 nM) and
tau (Ki = 121 nM) fibrils. The most potent and selective
compound in the series was 46a (Ki = 2 nM) and a 70-fold
selectivity versus Aβ and 40-fold selectivity versus tau fibrils.
The 2-fluoroethoxy-group in the benzyl moiety enabled 18F-
labeling for direct binding assays with α-syn, Aβ, and tau fibrils.
Fluorescent Tissue Staining. The fluorescent properties
of compound 20 and its high affinity for α-syn, Aβ, and tau
fibrils (Figure 3) encouraged us to investigate in vitro
fluorescent staining of post-mortem brain samples from PD/
DLB and PD cases. Sections from midbrain tissue of PD/DLB
and temporal lobe tissue sections from neuropathologically
verified AD cases were incubated with 5 μM of compound 20
and imaged using fluorescence microscopy. Antibody labeling
was performed on adjacent sections to determine the protein
species labeled by the fluorescent probe. Compound 20 was
found to label both Lewy bodies and Aβ plaques, which was
anticipated given the high affinity of this compound for α-syn
and Aβ fibrils. In addition, the clear staining of LBs and Aβ
plaques showed good agreement between the in vitro binding
studies using the fibrillary protein and insoluble protein
aggregates found in post-mortem samples of PD/DLB and
AD brain samples.
Saturation Binding Assays to Measure the Affinity of
46a for α-Syn Fibrils. To further confirm the binding affinity
determined in Thio-T assays, we synthesized [18F]46a and
directly measured its binding affinity in saturation binding
assays with α-syn fibrils. We observed specific binding of [18F]
46a to α-syn fibrils with an average Kd of 8.9 nM (Figure 4a).
We observed consistent binding values for three independently
prepared fibril batches. Scatchard analysis indicates that the
binding fits a one-site model (Figure 4b). We also determined
binding affinity of 46a for Aβ and tau fibrils. The average Kd
values for Aβ and tau fibrils were 271 and 50 nM, respectively,
in two independent assays (Figure 5).
G
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hydroxy)propoxy]-2-(4-dimethylaminophenyl)quinoline),
[18F]THK-5117 (6-[(3-18F-fluoro-2-hydroxy)propoxy]-2-(4-
methylaminophenyl)quinoline), and [11C]PBB3 (2-((1E,3E)-
4-(6-(11C-methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]-
thiazol-6-ol) have shown promise in imaging NFTs in AD
patients.26 A logical next step in the evolution of PET
radiotracers for imaging neurodegeneration is the development
of a probe capable of imaging α-syn aggregates in Lewy bodies
and Lewy neurites in PD.
A key step in the process of developing PET radioligands is
the identification of a suitable lead compound for structure−
activity relationship studies. The lead compound for the Aβ
imaging agent [11C]PiB was Thio-T, a fluorescent dye used for
staining Aβ plaques in post-mortem samples of AD brain
tissue.3,27,28 The 18F-labeled compounds [18F]fluorbetapir and
[18F]fluorbetapen were developed using X-34, a structural
analogue of the fluorescent dye Congo Red as the lead
compound.29,30 Thio-T also labels α-syn aggregates in Lewy
bodies and Lewy neurites, but its low fluorescent staining of α-
syn and higher affinity for Aβ plaques (which led to the
development of [11C]PiB) suggest that it would not be a good
lead compound for a PET radiotracer to image insoluble α-syn
aggregates in PD. Although several tricyclic analogues (SIL23,
SIL26, SIL5) were recently reported as ligands that bind
aggregated α-syn in post-mortem PD brain tissue assays,22,23
their affinity and selectivity is not optimal for imaging α-syn
aggregates in vivo.
Figure 3. Fluorescent microscopy studies of compound 20 in post-
mortem samples of PD/DLB and AD brain. Note the high labeling of
both Lewy bodies and Aβ plaques, which was confirmed with
appropriate immunohistochemistry labeling.
The indolinone analogue 5 (Figure 1) was previously
identified as having modest binding affinity to α-syn in a
rapid throughput screening assay potential lead compounds as a
tau imaging agent for PET.20 We used this compound as the
basis of the SAR studies described in this study. The first series
of compounds focused on direct analogues of 5 and explored
the effect of different substitutions in the indolinone ring
system. Most of the compounds within this series had only
modest affinity for α-syn and no selectivity toward α-syn, Aβ, or
tau fibrils. Although most of the compounds in this series
consisted of a mixture of E and Z regioisomers, it was possible
to separate compounds 38 and 39 into their E and Z isomers;
the Z configuration was found to have a higher affinity for α-
syn, Aβ, and tau fibrils than the corresponding E isomer.
The presence of a diene moiety in the structure of the dyes
LDS 798 and LDS 730 encouraged us to add a second double
bond into the structure of 5. This change in structure resulted
in improved affinity for α-syn fibrils; this indolinone-diene
compound (20), which was also a mixture of E,E and Z,E
regioisomers, had a high affinity for both Aβ and tau fibrils.
Fortunately the strong fluorescence of compound 20 enabled
us to confirm that the indolinone-dienes could label both Lewy
bodies and Aβ plaques in post-mortem samples of PD and AD
brain. It was difficult to identify clear structure−activity
relationships because of the EE:ZE mixture for each compound,
which varied depending on the nature of the substitution of the
indolinone nitrogen. However, it was possible to reach some
conclusions by comparing structural congeners having a similar
EE:ZE ratio. Substitution of the indolinone nitrogen with bulky
substituents such as a benzyl or substituted benzyl group versus
an N-methyl group appeared to improve affinity and selectivity
for α-syn versus Aβ and tau. The 4-nitrobenzyl analogue 28 was
the most potent compound in this series, having an affinity for
α-syn of 4 nM and 20-fold selectivity for α-syn versus Aβ and
tau.
Figure 4. [18F]46a binds recombinant α-syn fibrils with an average Kd
of 8.9 nM. Fibrils prepared from recombinant α-syn were incubated
with increasing concentrations of [18F]46a. Nonspecific binding was
determined in parallel reactions utilizing 20 as the competitor or in
reactions containing no fibrils. A representative plot of specific binding
versus [18F]46a concentration is shown in (A). Data points represent
mean sd (n = 3). The data were analyzed by curve fitting to a one-
site binding model using nonlinear regression. Kd values were
determined by fitting the data to the equation Y = Bmax × X/(X +
Kd). Scatchard analysis of binding is shown in (B). Similar results were
obtained in more than three independent experiments.
DISCUSSION
■
The past decade has witnessed significant advances in the field
of the development of PET-based radiotracers for imaging
insoluble protein aggregates which occur in many neuro-
degenerative disorders. Radiotracers such as [11C]PiB, [18F]-
fluorbetapir, [18F]florbetaben, and [18F]flutemetamol are
widely used in clinical PET studies, and it is currently believed
that imaging Aβ plaques with these radiotracers is capable of
identifying patients for risk for developing AD.6,7 More recent
studies have focused on imaging tau; clinical PET studies
conducted with [18F]T807 (7-(6-18F-fluoropyridin-3-yl)-5H-
pyrido[4,3-b]indole), [18F]THK-5105 (6-[(3-18F-fluoro-2-
H
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Figure 5. Radioligand binding studies demonstrate that [18F]46a binds with lower affinity to synthetic Aβ1−42 fibrils or recombinant tau fibrils
compared to α-syn fibrils. Binding affinities of [18F]46a to Aβ (A) and tau (C) fibrils were determined in saturation binding studies. The average Kd
values of Aβ and tau fibrils were 271 and 50 nM, respectively. Data points represent mean sd (n = 3). Scatchard plots of binding values are shown
in (B) for Aβ and (D) for tau. Similar results were obtained in two independent experiments.
Introduction of the 4-nitrobenzene ring into the diene
moiety enabled the separation of stable Z,E and E,E
regioisomers and led to the identification of the Z,E
configuration as being the more active of the regioisomers. A
number of compounds were identified as having a high affinity
for α-syn and good selectivity for α-syn versus Aβ and tau
fibrils. The most noteworthy compound in this series was 46a,
which had a high affinity (2 nM) and excellent selectivity for α-
syn versus Aβ and tau fibrils. This was also confirmed via direct
binding studies with the corresponding 18F-labeled compound
in α-syn, Aβ, and tau fibrils. Unfortunately, the high log P value
of this compound (4.18) made it difficult to obtain reliable and
reproducible binding data from insoluble α-syn obtained from
PD brain (results not shown). The high log P and potential of
the nitro group to be reduced to the corresponding amino
group in vivo indicates that [18F]46a will not be a useful in vivo
PET tracer for imaging Lewy bodies and Lewy neurites in PD
brain. However, our SAR results are noteworthy because they
identified structural features leading to preferential binding to
α-syn versus Aβ and tau fibrils. Consequently, compound 46a
will serve as a secondary lead compound for further SAR
studies. Other analogues within this series can serve as potential
radioligands for in vitro binding assays for α-syn and represent
an improvement to the current assay which uses Thio-T as a
screening ligand which binds more potently to Aβ versus α-syn,
and tau fibrils (Supporting Information Figure S5).
diene fragments. Within the indolinone-diene series, the Z,E
configuration was the more stable of the two regioisomers;
substitution of the indolinone nitrogen with a benzyl moiety
improved both the α-syn affinity and selectivity versus Aβ and
tau fibrils. The results of this study have identified clear
structural requirements for preparing compounds having a
preferential affinity for α-syn versus Aβ and tau fibrils.
EXPERIMENTAL SECTION
■
General Methods and Materials. All chemicals were obtained
from standard commercial sources and used without further
purification. All reactions were carried out using standard air-free
and moisture-free techniques under an inert nitrogen atmosphere with
dry solvents unless otherwise stated. Flash column chromatography
was conducted using Scientific Adsorbents, Inc. silica gel, 60A, “40
Micron Flash” (32−63 μm). Melting points were determined by using
MEL-TEMP 3.0 apparatus and are uncorrected. Routine H and 13C
1
NMR spectra were recorded at 400 and 100 MHz on Agilent
Technologies spectrometers. All chemical shifts were reported as a part
per million (ppm) downfield from tetramethylsilane (TMS). All
coupling constants (J) are given in hertz (Hz). Splitting patterns are
typically described as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. Elemental analysis (C, H, N) was determined by Atlantic
Microlab, Inc., Norcross, GA. Purities of final products were
determined to be >95% by elemental analysis. [18F]Fluoride was
produced at Washington University by the 18O(p,n)18F reaction
through proton irradiation of enriched (95%) [18O] water in the
RDS111 cyclotron. HPLC analysis was performed with an UV
detector and a scintillation NaI (Tl) detector and associated
electronics for radioactivity detection. An Agilent SB-C18 250 mm
× 9.4 mm 5 μm semipreparative column and an Agilent SB-C18 250
mm × 4.6 mm 5 μm analytical column were used for preparative
purification and for postsynthesis analysis of chemical and radio-
chemical purity and specific activity, respectively. The semipreparative
HPLC conditions were: 62% acetonitrile and 38% ammonium formate
buffer (0.1 M, pH 4.5) with a flow rate of 4 mL/min and UV at 264
nM for purification; the mobile phase for QC analysis was 80%
acetonitrile and 20% ammonium formate buffer with a flow rate of 1.5
mL/min. Radio-TLC was accomplished using a Bioscan AR-2000
imaging scanner (Bioscan, Inc. Washington, DC..
CONCLUSION
■
In summary, a series of indolinone and indolinone-diene
analogues were synthesized and in vitro binding assays were
conducted for α-syn, Aβ, and tau fibrils. In general, the
indolinone-diene analogues had a higher affinity for all three
fibrils versus the corresponding indolinone congeners. The
presence of two different regioisomers made it difficult to
identify clear structure−activity relationships for the two series
of compounds, but this was overcome by introduction of a
para-nitro substituent into the benzene ring of the ene and
I
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Synthesis method 1: The 2-oxindole (20 mmol) and aldehyde (20
mmol) in acetic acid (50 mL) and 37% HCl (1 mL) were heated at
reflux for 3 h, then water (500 mL) was added at rt. The solids were
filtered out and recrystallized with methanol to afford the 3-
(substituted) indolin-2-one condensation product.
methoxybenzyl chloride to afford the desired product (10) as an
orange solid (61%), mp 154.8−155.5 °C. 1H NMR (400 MHz,
CDCl3) δ 8.44 and 7.68 (d, J = 8.8 Hz, 2H), 7.91(d, J = 7.6 Hz, 1H),
7.86 and 7.49 (s, 1H), 7.48 and 7.26 (d, J = 5.2 Hz, 1H), 7.28 (d, J =
8.8 Hz, 2H), 7.10 (t, J = 7.6 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.82 (d,
J = 8.8 Hz, 2H), 6.73 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 169.3, 166.6, 158.8, 151.9, 151.4, 142.6, 140.4, 139.1, 138.5,
134.9, 132.1, 128.9, 128.7, 128.5, 128.3, 125.7, 122.5, 122.1, 121.4,
121.3, 112.0, 117.8, 114.1, 114.0, 111.4, 111.2, 108.9, 108.4, 55.2, 43.1,
42.9, 40.1, 40.0. Anal. C25H24N2O2 Calcd: C, 78.10; H, 6.29; N, 7.29.
Found: C, 77.85; H, 6.47; N, 7.24.
3-(4-(Dimethylamino)benzylidene)-1-(pyridin-4-ylmethyl)-
indolin-2-one (11). Prepared by method 2 with compound 5 and 4-
(bromomethyl)pyridine to afford the desired product (11) as a red
solid (56%), mp 153.0−153.7 °C. 1H NMR (400 MHz, CDCl3) δ 8.55
(d, J = 4.8 Hz, 2H), 8.43 and 7.97 (d, J = 8.0 Hz, 1H), 7.89 and 7.55
(s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 5.2 Hz, 2H), 7.14 (t, J =
7.6 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H), 6.76 (d, J = 9.2 Hz, 2H), 6.63 (d,
J = 8.0 Hz, 1H), 5.02 (s, 2H), 3.09 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 169.4, 151.5, 150.2, 150.1, 145.5, 141.8, 139.9, 135.0, 132.3,
128.3, 122.3, 122.2, 122.0, 121.9, 121.8, 121.3, 118.0, 111.4, 111.2,
108.5, 42.6, 40.1, 40.0. Anal. C23H21N3O Calcd: C, 77.72; H, 5.96; N,
11.82. Found: C, 77.64; H, 6.12; N, 11.74.
Ethyl 3-(4-(Dimethylamino)benzylidene)-2-oxoindoline-1-
carboxylate (12). Prepared by method 2 with compound 5 and
ethyl chloroformate to afford the desired product (12) as a red solid
(59%), mp 121.7−123.1 °C. 1H NMR (400 MHz, CDCl3) δ 8.36 and
7.67 (d, J = 8.4 Hz, 2H), 7.98 and 7.89 (d, J = 7.6 Hz, 2H), 7.82 and
7.50 (s, 1H), 7.52 and 7.29 (t, J = 7.6 Hz, 1H), 7.17 and 7.07 (t, J = 7.6
Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 4.51 (q, J = 7.2 Hz, 2H), 3.09 (s,
6H), 1.48 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 167.3
151.7, 140.5, 140.1, 135.5, 132.3, 128.6, 123.7, 122.8, 121.7, 121.4,
117.5, 115.0, 114.8, 111.3, 111.1, 63.1, 40.1, 14.3. Anal. C20H20N2O3
Calcd: C, 71.41; H, 5.99; N, 8.33. Found: C, 71.57; H, 6.15; N, 8.33.
1-Benzyl-3-(4-(dimethylamino)benzylidene)-1H-pyrrolo[2,3-
b]pyridin-2(3H)-one (13). Prepared by method 2 with compound 6
and benzyl bromide to afford the desired product (13) as a red solid
(70%), mp 144.7−145.4 °C. 1H NMR (400 MHz, CDCl3) δ 8.41 and
7.61 (d, J = 8.8 Hz, 2H), 8.10 and 8.05 (d, J = 7.6 Hz, 1H), 7.90 and
7.41 (s, 1H), 7.49 (d, J = 7.6 Hz, 2H), 7.31−7.21 (m, 4H), 6.91 and
6.83 (dd, J = 7.6 Hz, 5.6 Hz, 1H), 6.73 and 6.70 (d, J = 8.8 Hz, 2H),
5.13 and 5.11 (s, 2H), 3.06 (s, 6H). 13C NMR (100 MHz, CDCl3) δ
168.9, 166.2, 155.7, 153.6, 152.3, 151.7, 146.5, 145.5, 140.7, 140.3,
137.4, 137.2, 135.3, 132.4, 128.5, 128.4, 128.3, 127.3, 127.2, 124.3,
122.1, 121.7, 120.4, 112.0, 117.5, 117.3, 116.9, 111.5, 111.2, 42.5, 42.3,
40.1, 40.0. Anal. C23H21N3O Calcd: C, 77.72; H. 5.96; N, 11.82.
Found: C, 77.86; H, 6.05; N, 11.81.
Synthesis method 2: The 3-(substituted) indolin-2-one (1.0 mmol)
in THF 5 mL) was added to 60% NaH (1.5 mmol) at 0 °C. After 15
min, the alkyl halide (3 mmol) was added. The reaction mixture was
stirred for 8 h, ethyl acetate (75 mL) was added, washed with water
(50 mL × 2) and saturated NaCl (50 mL), and dried over Na2SO4.
After evaporation of the ethyl acetate, the crude product was purified
by silica gel column chromatography eluting with hexane−CH2Cl2−
EtOAc (10:10:3, v/v/v) to afford the nitrogen alkylated compound.
3-(4-(Dimethylamino)benzylidene)indolin-2-one (5). Pre-
pared by method 1 with compound 1 and 4 to afford the desired
product (5) as a red solid (90%), mp 190.5−191.9 °C. 1H NMR (400
MHz, CDCl3) δ 9.20 and 8.74 (s, 1H), 8.40, and 7.69 (d, J = 8.8 Hz,
2H), 7.90 and 7.48 (d, J = 7.6 Hz, 1H), 7.79 and 7.47 (s, 1H), 7.18 and
7.00 (t, J = 7.6 Hz, 1H), 7.15 and 6.92 (t, J = 8.0 Hz, 1H), 6.94 and
6.87 (d, J = 7.6 Hz, 2H), 3.07 (s, 6H). 13C NMR (100 MHz, CDCl3) δ
171.4, 168.6, 151.4, 141.0,139.1, 138.9, 138.7, 134.9, 132.2, 128.5,
127.0, 126.6, 122.6, 122.4, 122.3, 122.1, 121.4, 121.2, 120.4, 118.1,
111.4, 111.2, 110.1, 109.3, 40.1, 40.0. Anal. C17H16N2O Calcd: C,
77.25; H, 6.10; N, 10.60. Found: C, 77.24; H, 6.29; N, 10.60.
3-(4-(Dimethylamino)benzylidene)-1H-pyrrolo[2,3-b]-
pyridin-2(3H)-one (6). Prepared by method 1 with compound 2 and
4 to afford the desired product (6) as a red solid (47%), mp 247.4−
248.6 °C. 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.40 and 7.64
(d, J = 8.8 Hz, 2H), 8.10 and 7.67 (d, J = 6.4 Hz, 2H), 7.87 and 7.42
(s, 1H), 6.95 and 6.88 (t, J = 7.2 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H),
3.10 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 169.5, 167.7, 156.6,
154.2, 152.6, 152.2, 147.0, 145.8, 140.6, 139.8, 135.6, 132.9, 128.8,
125.6, 122.3, 121.2, 121.1, 120.4, 118.4, 117.6, 117.4, 116.7, 112.1,
111.6, 40.2. Anal. C16H15N3O·0.25H2O Calcd: C, 71.82; H, 5.75; N,
15.70. Found: C, 71.89; H, 5.83; N, 15.58.
3-(4-(Dimethylamino)benzylidene)-5-hydroxyindolin-2-one
(7). Prepared by method 1 with compound 3 and 4 to afford the
1
desired product (7) as a red solid (77%), mp 221.1−222.4 °C. H
NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.89 (s, 1H), 7.58 (d, J
= 8.0 Hz, 2H), 7.43 (s, 1H), 7.26 (s, 1H), 6.78 (d, J = 8.8 Hz, 2H),
6.62 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 7.6 Hz, 1H), 2.99 (s, 6H). 13C
NMR (100 MHz, DMSO-d6) δ 169.9, 152.1, 151.7, 137.4, 135.0,
132.2, 123.6, 122.9, 121.6, 115.7, 111.9, 110.4, 109.8, 40.1. Anal.
C17H16N2O2·0.125H2O Calcd: C, 72.26; H, 5.80; N, 9.91. Found: C,
72.31; H, 5.80; N, 9.80.
3-(4-(Dimethylamino)benzylidene)-1-methylindolin-2-one
(8). Prepared by method 2 with compound 5 and CH3I to afford the
1
3-(4-(Dimethylamino)benzylidene)-1-(4-methoxybenzyl)-
1H-pyrrolo[2,3-b]pyridin-2(3H)-one (14). Prepared by method 2
with compound 6 and 4-methoxybenzyl chloride to afford the desired
desired product (8) as a red solid (55%), mp 100.3−101.5 °C. H
NMR (400 MHz, CDCl3) δ 8.42 and 7.67 (d, J = 8.0 Hz, 2H), 7.91
and 7.49 (d, J = 7.6 Hz, 1H), 7.80 and 7.46 (s, 1H), 7.24 (t, J = 7.2 Hz,
1H), 7.03 and 6.94 (t, J = 7.2 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.75
(d, J = 8.0 Hz, 2H), 3.29 (s, 3H), 3.07 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 169.4, 151.3, 143.5, 138.8, 138.3, 134.8, 132.0, 128.4, 122.3,
122.2, 122.1, 122.0, 121.4, 121.3, 111.4, 111.1, 107.9, 107.5, 40.1, 26.1.
Anal. C18H18N2O Calcd: C, 77.67; H, 6.52; N, 10.06. Found: C, 77.45;
H, 6.77; N, 9.83.
1
product (14) as a red solid (70%), mp 123.5−124.2 °C. H NMR
(400 MHz, CDCl3) δ 8.41 and 8.04 (d, J = 8.4 Hz, 1H), 8.11 (d, J =
7.6 Hz, 1H), 7.89 and 7.41 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.47 (d, J
= 8.8 Hz, 2H), 6.93−6.80 (m, 3H), 6.73 and 6.71 (d, J = 9.2 Hz, 2H),
5.06 and 5.04 (s, 2H), 3.75 (s, 3H), 3.07 (s, 6H). 13C NMR (100
MHz, CDCl3) δ 168.8, 166.1, 158.9, 158.8, 155.7, 153.6, 152.3, 151.7,
146.4, 145.4, 140.5, 140.1, 135.2, 132.3, 129.8, 129.7, 129.5, 128.3,
124.2, 122.1, 121.7, 120.4, 120.0, 117.6, 117.2, 116.8, 113.8, 111.4,
111.2, 55.2, 41.9, 41.7, 40.1, 40.0. Anal. C24H23N3O2 Calcd: C, 74.78;
H, 6.01; N, 10.90. Found: C, 75.04; H, 6.10; N, 10.86.
1-Benzyl-3-(4-(dimethylamino)benzylidene)indolin-2-one
(9). Prepared by method 2 with compound 5 and benzyl bromide to
afford the desired product (9) as an orange solid (82%), mp 111.0−
112.4 °C. 1H NMR (400 MHz, CDCl3) δ 8.44 and 7.69 (d, J = 8.8 Hz,
2H), 7.92 and 7.50 (d, J = 7.6 Hz, 1H), 7.87 and 7.51 (s, 1H), 7.35−
7.22 (m, 5H), 7.11 (t, J = 7.6 Hz, 1H), 6.90 (t, J = 7.2 Hz, 1H), 6.74
(d, J = 8.4 Hz, 2H), 6.71 (m, 1H), 5.01 (s, 2H), 3.06 (s, 6H). 13C
NMR (100 MHz, CDCl3) δ 169.4, 152.0, 151.4, 142.6, 140.3, 139.2,
138.6, 136.7, 136.4, 134.9, 132.1, 128.7, 128.6, 128.3, 127.4, 127.3,
126.9, 122.5, 122.2, 122.1, 122.0, 121.5, 121.4, 117.8, 111.4, 111.2,
108.9, 108.4, 43.7, 40.1, 40.0. Anal. C24H22N2O Calcd: C, 81.33; H,
6.26; N, 7.90. Found: C, 81.22; H, 6.34; N, 7.96.
3-(3-Phenylallylidene)indolin-2-one (18). Prepared by method
1 with compound 1 and 15 to afford the desired product (18) as an
orange solid (91%), mp 203.9−205.2 °C. 1H NMR (400 MHz,
CDCl3) δ 8.60−8.53 (m, 1H), 8.30 (s, 1H), 7.73−7.58 (m, 3H),
7.51−7.30 (m, 4H), 7.26−7.15 (m, 2H), 7.09−6.85 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ 170.3, 169.1, 144.5, 143.1, 141.0, 139.8,
136.5, 136.4, 136.0, 129.7, 129.3, 129.0, 128.8, 128.7, 127.8, 127.7,
125.7, 124.8, 124.4, 124.2, 123.7, 123.4, 123.0, 122.1, 121.8, 119.5,
110.2, 109.7. Anal. C17H13NO·0.125H2O Calcd: C, 81.82; H, 5.33; N,
5.61. Found: C, 81.73; H, 5.47; N, 5.48.
3-(4-(Dimethylamino)benzylidene)-1-(4-methoxybenzyl)-
indolin-2-one (10). Prepared by method 2 with compound 5 and 4-
J
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3-(3-(4-Methoxyphenyl)allylidene)indolin-2-one (19). Pre-
pared by method 1 with compound 1 and 16 to afford the desired
product (19) as an orange solid (85%), mp 226.1−226.8 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.46 and 10.41 (s, 1H), 7.93 (d, J = 7.2 Hz,
1H), 7.72 (d, J = 7.6 Hz, 1H), 7.60−7.50 (m, 2H), 7.29−7.07 (m,
3H), 6.97 (m, 3H), 6.81 (d, J = 7.2 Hz, 1H), 3.77 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 169.4, 168.8, 161.1, 160.8, 145.0, 142.7,
142.3, 141.2, 136.5, 135.6, 130.3, 129.5, 129.3, 129.2, 129.1, 129.0,
124.9, 124.5, 124.4, 124.3, 122.7, 122.3, 121.8, 121.5, 121.4, 120.1,
115.1, 114.8, 110.0, 109.8, 55.8. Anal. C18H15NO2 Calcd: C, 77.96; H,
5.45; N, 5.05. Found: C, 77.66; H, 5.60; N, 5.02.
121.4, 119.0, 114.5, 114.3, 109.1, 108.8, 55.4, 43.6. Anal. C25H21NO2
Calcd: C, 81.72; H, 5.76; N, 3.81. Found: C, 81.57; H, 5.93; N, 3.82.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1-methylindolin-
2-one (25). Prepared by method 2 with compound 20 and CH3I to
afford the desired product (25) as an orange solid (67%), mp 170.5−
1
171.5 °C. H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 7.2 Hz, 1H),
7.53−7.41 (m, 3H), 7.30 (d, J = 11.6 Hz, 1H), 7.24 and 7.21 (t, J = 7.6
Hz, 1H), 7.09 and 6.97 (d, J = 14.8 Hz, 1H), 7.07 and 7.00 (t, J = 8.0
Hz, 1H), 6.83 and 6.79 (d, J = 8.0 Hz, 1H), 6.70 and 6.68 (d, J = 8.8
Hz, 2H), 3.27 (s, 3H), 3.02 and 3.02 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 168.9, 167.7, 151.4, 151.2, 145.5, 144.3, 143.0, 141.8, 137.6,
137.4, 129.6, 129.4, 127.8, 127.6, 124.6, 124.1, 124.0, 122.9, 122.8,
122.2, 121.7, 121.5, 120.3, 119.0, 118.5, 112.0, 111.9, 107.8, 107.6,
40.2, 26.0, 25.6. Anal. C20H20N2O Calcd: C, 78.92; H, 6.62; N, 9.20.
Found: C, 78.73; H, 6.72; N, 9.29.
3-(3-(4-(Dimethylamino)phenyl)allylidene)indolin-2-one
(20). Prepared by method 1 with compound 1 and 17 to afford the
desired product (20) as an orange solid (82%), mp 227.8−229.4 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.37 and 10.33 (s, 1H), 8.18 and
1-Benzyl-3-(3-(4-(dimethylamino)phenyl)allylidene)indolin-
2-one (26). Prepared by method 2 with compound 20 and benzyl
bromide to afford the desired product (26) as an orange solid (85%),
mp 131.5−133.1 °C. H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 5.6
7.45 (dd, J = 15.6 Hz, 11.6 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.60 and
7.39 (d, J = 8.8 Hz, 2H), 7.47 and 7.24 (d, J = 12.0 Hz, 1H), 7.20 and
7.04 (d, J = 15.6 Hz, 1H), 7.14 and 7.09 (t, J = 7.6 Hz, 1H), 6.96 and
6.89 (t, J = 7.6 Hz, 1H), 6.80 and 6.75 (d, J = 7.2 Hz, 1H), 6.72 and
6.71 (d, J = 8.8 Hz, 2H), 2.97 and 2.95 (s, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 169.5, 168.9, 151.8, 151.6, 146.4, 144.3, 141.9, 140.7,
136.6, 130.3, 129.5, 128.5, 124.7, 124.2, 124.0, 123.2, 122.8, 122.4,
121.6, 121.6, 121.2, 119.8, 112.5, 112.3, 109.9, 109.7, 40.2. Anal.
C19H18N2O·0.125H2O Calcd: C, 77.99; H, 6.29; N, 9.57. Found: C,
77.95; H, 6.32; N, 9.56.
1
Hz, 1H), 7.84 and 7.58 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 12.4 Hz, 1H),
7.53−7.64 (m, 4H), 7.36−7.20 (m, 5H), 7.12 and 7.01 (d, J = 14.8 Hz,
1H), 6.95 and 6.90 (t, J = 5.6 Hz, 1H), 6.69 (d, J = 8.8 Hz, 2H), 5.08
(s, 2H), 3.05 and 3.03 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 168.4,
155.3, 151.6, 151.5, 146.9, 146.0, 139.7, 139.3, 137.3, 137.2, 130.0,
129.7, 129.0, 128.5, 128.4, 128.2, 127.3, 123.7, 120.3, 120.0, 118.3,
117.6, 117.5, 111.9, 42.4, 40.1. Anal. C26H24N2O Calcd: C, 78.71; H,
6.08; N, 11.02. Found: C, 78.27; H, 6.16; N, 10.87.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1-(4-
methoxybenzyl)indolin-2-one (27). Prepared by method 2 with
compound 20 and 4-methoxybenzyl chloride to afford the desired
product (27) as an orange solid (47%), mp 169.5−170.3 °C. 1H NMR
(400 MHz, CDCl3) δ 8.46 and 7.46 (dd, J = 15.4 Hz, 11.6 Hz, 1H),
7.70 and 7.41 (d, J = 7.6 Hz, 1H), 7.57 and 7.33 (d, J = 12.0 Hz, 1H),
7.52−7.46 (m, 2H), 7.28−7.24 (m, 2H), 7.14−6.94 (m, 3H), 6.84−
6.81 (m, 2H), 6.74−6.65 (m, 3H), 4.91 (s, 2H), 3.75 (s, 3H), 3.02 and
3.01 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 169.0, 167.7, 158.9,
151.4, 151.2, 145.7, 144.6, 142.1, 140.9, 137.9, 137.8, 129.7, 129.5,
128.7, 128.6, 128.5, 127.7, 127.5, 124.6, 124.1, 124.0, 123.1, 122.9,
122.0, 121.7, 121.5, 121.2, 120.3, 118.9, 118.6, 114.0, 112.0, 111.9,
108.9, 108.6, 55.2, 43.0, 42.7, 40.2, 40.1. Anal. C27H26N2O2 Calcd: C,
79.00; H, 6.38; N, 6.82. Found: C, 79.08; H, 6.51; N, 6.75.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1H-pyrrolo[2,3-
b]pyridin-2(3H)-one (21). Prepared by method 1 with compound 2
and 17 to afford the desired product (21) as a purple solid (74%), mp
1
273.6−274.4 °C. H NMR (400 MHz, DMSO-d6) δ 10.96 and 10.92
(s, 1H), 8.22 and 7.79 (d, J = 7.2 Hz, 1H), 7.99 and 7.96 (d, J = 4.8
Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.44−7.34 (m, 2H), 7.26 and 7.08
(d, J = 15.2 Hz, 1H), 6.95 and 6.91 (d, J = 7.4 Hz, 5.2 Hz, 1H), 6.70
(d, J = 8.8 Hz, 2H), 2.97 and 2.96 (s, 6H). 13C NMR (100 MHz,
DMSO-d6) δ 169.1, 156.0, 155.1, 152.0, 151.8, 147.9, 146.4, 145.8,
139.7, 138.6, 130.7, 130.4, 129.9, 126.3, 123.9, 123.8, 120.8, 119.7,
119.0, 118.5, 117.7, 117.5, 117.4, 112.5, 112.2, 40.1. Anal. C18H17N3O
Calcd: C, 74.20; H, 5.88; N, 14.42. Found: C, 73.92; H, 5.84; N, 14.32.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-5-hydroxyindo-
lin-2-one (22). Prepared by method 1 with compound 1 and 15 to
afford the desired product (22) as a purple solid (81%), mp >280 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.06 and 10.02 (s, 1H), 8.93 and
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1-(4-
nitrobenzyl)indolin-2-one (28). Prepared by method 2 with
compound 20 and 4-nitrobenzyl bromide to afford the desired
product (28) as an orange solid (83%), mp 204.2−205.6 °C. 1H NMR
(400 MHz, CDCl3) δ 8.17 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 7.2 Hz,
1H), 7.61 and 7.40 (d, J = 12.4 Hz, 1H), 7.54−7.45 (m, 5H), 7.17−
7.00 (m, 3H), 6.71−6.61 (m, 3H), 5.08 (s, 2H), 3.06 and 3.04 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 169.0, 167.6, 151.6, 147.4, 146.6,
8.91 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.38−6.88 (m, 5H), 6.71 (d, J =
7.2 Hz, 2H), 6.59−6.54 (m, 2H), 2.96 and 2.94 (s, 6H). 13C NMR
(100 MHz, DMSO-d6) δ 169.5, 169.1, 152.5, 152.4, 151.7, 151.5,
145.7, 144.0, 137.0, 136.0, 134.5, 133.4, 129.9, 129.4, 125.5, 124.3,
124.0, 123.8, 123.7, 123.2, 119.8, 118.5, 115.0, 112.6, 112.4, 111.1,
110.3, 110.1, 110.0, 106.9, 40.1. Anal. C19H18N2O2·0.25H2O Calcd: C,
73.41; H, 6.00; N, 9.01. Found: C, 73.50; H, 6.09; N, 8.72.
1-Benzyl-3-(3-phenylallylidene)indolin-2-one (23). Prepared
by method 2 with compound 18 and benzyl bromide to afford the
desired product (23) as an orange solid (90%), mp 124.6−125.1 °C.
1H NMR (400 MHz, CDCl3) δ 8.67 (dd, J = 15.8 Hz, 11.6 Hz, 1H),
7.62 (d, J = 7.2 Hz, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.39−7.25 (m, 9H),
7.14 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 16.0 Hz, 1H), 6.99 (t, J = 7.6 Hz,
1H), 6.71 (d, J = 7.6 Hz, 1H), 4.97 (s, 2H). 13C NMR (100 MHz,
CDCl3) δ 167.4, 143.0, 141.6, 136.4, 136.3, 129.3, 128.8, 128.7, 128.6,
127.8, 127.5, 127.3, 124.5, 124.4, 123.5, 121.9, 119.3, 108.9, 43.3. Anal.
C24H19NO Calcd: C, 85.43; H, 5.68; N, 4.15. Found: C, 85.22; H,
5.88; N, 4.13.
145.5, 144.0, 138.8, 138.6, 137.7, 129.8, 129.7, 128.0, 127.9, 127.7,
127.5, 124.0, 123.9, 123.1, 122.3, 122.1, 121.1, 120.1, 118.8, 118.7,
112.0, 111.9, 108.4, 108.1, 43.0, 42.7, 40.2. Anal. C26H23N3O3 Calcd:
C, 73.39; H, 5.45; N, 9.88. Found: C, 73.48; H, 5.63; N, 9.81.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1-(pyridin-4-
ylmethyl)indolin-2-one (29). Prepared by method 2 with
compound 20 and 4-(bromomethyl)pyridine to afford the desired
1
product (29) as a purple solid (51%), mp 194.1−195.1 °C. H NMR
(400 MHz, CDCl3) δ 8.53 (d, J = 4.4 Hz, 2H), 7.76 (d, J = 7.2 Hz,
1H), 7.61 (d, J = 12.4 Hz, 1H), 7.54−7.39 (m, 3H), 7.20−7.02 (m,
5H), 6.71 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 7.6 Hz, 1H), 5.00 (s, 2H),
3.06 and 3.03 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 169.0, 151.5,
150.1, 146.4, 145.5, 145.3, 141.4, 138.5, 129.8, 129.6, 127.8, 123.9,
123.1, 122.2, 122.0, 121.3, 118.7, 112.0, 111.9, 108.5, 42.6, 40.2. Anal.
C25H23N3O Calcd: C, 78.71; H, 6.08; N, 11.02. Found: C, 78.76; H,
6.27; N, 10.95.
1-Benzyl-3-(3-(4-methoxyphenyl)allylidene)indolin-2-one
(24). Prepared by method 2 with compound 19 and benzyl bromide to
afford the desired product (24) as an orange solid (78%), mp 167.3−
1
169.3 °C. H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.2 Hz, 1H),
7.59−7.51 (m, 3H), 7.35−7.25 (m, 5H), 7.17−7.11 (m, 2H), 7.04 and
6.99 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 9.2 Hz,
1H), 6.72 and 6.70 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 3.86 and 3.84 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 168.8, 161.0, 160.8, 144.3, 143.1,
142.5, 136.9, 136.7, 136.4, 136.2, 129.5, 129.3, 128.9, 128.7, 128.4,
128.2, 127.4, 127.3, 127.2, 123.9, 123.2, 122.7, 122.6, 122.0, 121.7,
Ethyl 3-(3-(4-(Dimethylamino)phenyl)allylidene)-2-oxoindo-
line-1-carboxylate (30). Prepared by method 2 with compound 20
and ethyl chloroformate to afford the desired product (30) as a red
solid (57%), mp 169.9−170.5 °C. 1H NMR (400 MHz, CDCl3) δ 8.39
and 7.53 (dd, J = 15.2 Hz, 12.0 Hz, 1H), 7.98 and 7.91 (d, J = 8.0 Hz,
K
DOI: 10.1021/acs.jmedchem.5b00571
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 7.77 and 7.46 (d, J = 7.2 Hz, 1H), 7.52 and 7.50 (d, J = 9.2 Hz,
2H), 7.37−7.02 (m, 4H), 6.70 and 6.67 (d, J = 8.8 Hz, 2H), 4.51 (q, J
= 7.2 Hz, 2H), 3.05 and 3.04 (s, 6H), 1.49 (t, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 165.5, 151.5, 147.3, 146.4, 139.3, 139.1,
137.3, 130.0, 129.8, 128.0, 127.9, 124.7, 124.3, 124.1, 124.0, 123.8,
122.4, 120.2, 119.2, 118.5, 118.1, 115.1, 115.0, 112.0, 111.9, 63.1, 40.1,
14.4. Anal. C22H22N2O3 Calcd: C, 72.91; H, 6.12; N, 7.73. Found: C,
73.18; H, 6.04; N, 7.80.
138.5, 135.0, 129.8, 129.5, 127.6, 127.5, 126.7, 124.6, 124.2, 122.1,
121.0, 120.5, 110.8, 112.0, 109.1, 40.2. Anal. C25H22N2O Calcd C:
81.94, H: 6.05, N: 7.74. Found C: 81.92, H: 6.25, N: 7.59.
(Z)-3-(4-Nitrobenzylidene)indolin-2-one (38a) and (E)-3-(4-
Nitrobenzylidene)indolin-2-one (38b). Prepared by method 1
with compound 1 and 37. The crude product was purified by silica gel
column chromatography, eluting with CH2Cl2−EtOAc (100:5, v/v);
compound 38a eluted first and was afforded as a red solid (41%), mp
1
247.5−248.6 °C. H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.8 Hz,
1-Benzyl-3-(3-(4-(dimethylamino)phenyl)allylidene)-1H-
pyrrolo[2,3-b]pyridin-2(3H)-one (31). Prepared by method 2 with
compound 21 and benzyl bromide to afford the desired product (31)
2H), 8.28 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H),
7.42 (s, 1H), 7.29 (t J = 7.2 Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 6.86 (d,
J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 167.2, 147.8,
142.0, 140.7, 133.9, 132.9, 131.5, 130.9, 130.6, 124.6, 124.4, 123.6,
121.8, 121.1, 110.1. Anal. C15H10N2O3·0.125H2O Calcd: C, 67.10; H,
3.85; N, 10.43. Found: C, 67.08; H, 3.81; N, 10.47. Compound 38b
1
as a purple solid (72%), mp 203.1−204.0 °C. H NMR (400 MHz,
CDCl3) δ 8.11 (d, J = 5.2 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.61 (d, J
= 12.0 Hz, 1H), 7.52−7.34 (m, 4H), 7.31−7.20 (m, 4H), 7.11 (d, J =
15.2 Hz, 1H), 6.94 (dd, J = 7.4 Hz, 5.2 Hz, 1H), 6.68 (d, J = 9.2 Hz,
2H), 5.01 (s, 2H), 3.03 and 3.02 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 168.4, 155.3, 151.6, 151.5, 146.9, 146.0, 145.9, 139.6, 139.3,
137.3, 137.2, 130.0, 129.7, 129.0, 128.5, 128.4, 128.2, 127.3, 125.0,
124.2, 123.7, 120.3, 120.0, 119.0, 118.7, 118.3, 117.6, 117.4, 111.9,
42.4, 42.1, 40.1. Anal. C25H23N3O Calcd: C, 78.71; H, 6.08; N, 11.02.
Found: C, 78.88; H, 6.21; N, 10.93.
1
eluted second as a red solid (48%), mp 252.6−252.9 °C. H NMR
(400 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.31 (d, J = 8.4 Hz, 2H), 7.93
(d, J = 8.0 Hz, 2H), 7.63 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.23 (t, J =
8.0 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.81 (t, J = 7.6 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 168.6, 147.9, 143.9, 141.9, 133.4,
131.5, 130.9, 130.5, 124.4, 123.3, 121.8, 120.7, 110.8. Anal.
C15H10N2O3 Calcd: C, 67.67; H, 3.79; N, 10.52. Found: C, 67.54;
H, 3.71; N, 10.60.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1-(4-methoxy-
benzyl)-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (32). Prepared by
method 2 with compound 21 and 4-methoxybenzyl chloride to afford
the desired product (32) as a purple solid (81%), mp 189.7−191.9 °C.
1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 5.6 Hz, 1H), 7.82 (d, J =
7.6 Hz, 1H), 7.60 (d, J = 12.8 Hz, 1H), 7.46 (d, J = 8.8 Hz, 2H), 7.44
(d, J = 8.0 Hz, 2H), 7.30 (dd, J = 14.8 Hz, 12.4 Hz, 1H), 7.11 (d, J =
14.8 Hz, 1H), 6.93 (dd, J = 7.2 Hz, 5.6 Hz, 1H), 6.81 (d, J = 8.8 Hz,
2H), 6.67 (d, J = 8.8 Hz, 2H), 5.01 (s, 2H), 3.75 (s, 3H), 3.03(s, 6H).
13C NMR (100 MHz, CDCl3) δ 168.3, 158.8, 155.3, 151.6, 146.8,
(Z)-1-Benzyl-3-(4-nitrobenzylidene)indolin-2-one (39a). Pre-
pared by method 2 with compound 38a and benzyl bromide to afford
the desired product (39a) as a red solid (76%), mp 200.4−201.1 °C.
1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 9.2 Hz, 2H), 8.28 (d, J =
8.4 Hz, 2H), 7.57 (s, 1H), 7.56 (d, J = 6.8 Hz, 1H), 7.32−7.22 (m,
6H), 7.06 (t, J = 7.2 Hz, 1H), 6.75 (d, J = 7.6 Hz, 1H), 4.98 (s, 2H).
13C NMR (100 MHz, CDCl3) δ 165.7, 148.0, 142.2, 139.8, 135.7,
133.5, 132.3, 130.2, 129.4, 128.8, 127.7, 127.3, 123.5, 123.4, 122.3,
119.8, 109.2, 43.7. Anal. C22H16N2O3·0.25H2O Calcd: C, 73.22; H,
4.61; N, 7.76. Found: C, 72.93; H, 4.50; N, 7.75.
(E)-1-Benzyl-3-(4-nitrobenzylidene)indolin-2-one (39b). Pre-
pared by method 2 with compound 38b and benzyl bromide to afford
the desired product (39b) as red solid (66%), mp 161.9−162.2 °C. 1H
NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.4 Hz, 2H), 7.87 (s, 1H),
7.81 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 7.2 Hz, 1H), 7.35−7.26 (m, 5H),
7.20 (t, J = 7.6 Hz, 1H), 6.86 (t, J = 7.6 Hz, 1H), 6.75 (d, J = 7.6 Hz,
1H), 5.00 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 167.9, 147.9,
143.9, 141.7, 135.6, 133.7, 130.9, 130.0, 129.7, 128.8, 127.7, 127.3,
124.0, 123.0, 122.2, 120.4, 109.6, 43.9. Anal. C22H16N2O3 Calcd: C,
74.15; H, 4.53; N, 7.86. Found: C, 74.14; H, 4.61; N, 7.84.
145.9, 139.2, 129.8, 129.7, 129.5, 129.0, 123.7, 120.1, 118.4, 117.6,
117.4, 113.8, 111.9, 55.2, 41.8, 40.1. Anal. C26H25N3O2 Calcd: C,
75.89; H, 6.12; N, 10.21. Found: C, 75.97; H, 6.13; N, 10.18.
1-(4-Aminobenzyl)-3-(3-(4-(dimethylamino)phenyl)-
allylidene)indolin-2-one (33). A mixture of 28 (425 mg, 1 mmol)
and Fe powder (560 mg, 10 mmol) in methanol (30 mL) and 37%
HCl (1 mL) was heated to reflux for 3 h. The solid was filtered out, the
filtrate was evaporated, ethyl acetate (75 mL) was added, and washed
with saturated Na2CO3 (50 mL), water (50 mL), and NaCl (50 mL),
and dried over Na2SO4. After removed of the solvent, the crude
product was purified by silica gel column chromatography eluting with
EtOAc−MeOH (10:1, v/v) to afford 263 mg (66%) of 33 as a red
solid, mp 221.0−222.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.91 (d,
J = 7.6 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.51−7.36 (m, 2H), 7.26 (d,
J = 14.4 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H),
6.97 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 7.6 Hz, 1H), 6.70 (d, J = 8.4 Hz,
2H), 6.46 (d, J = 8.4 Hz, 2H), 4.98 (s, 2H), 4.71 (s, 2H), 2.96 (s, 6H).
13C NMR (100 MHz, DMSO-d6) δ 168.1, 151.9, 148.4, 147.2, 142.2,
(Z)-3-((E)-3-(4-Nitrophenyl)allylidene)indolin-2-one (41a)
and (E)-3-((E)-3-(4-Nitrophenyl)allylidene)indolin-2-one (41b).
Prepared by method 1 with compound 1 and 40, the crude product
was purified by silica gel column chromatography, eluting with
CH2Cl2−EtOAc (100:5, v/v). Compound 41a eluted first and was
1
afforded as a red solid (41%), mp 256.2−257.2 °C. H NMR (400
MHz, DMSO-d6) δ 10.56 (s, 1H), 8.58 (dd, J = 15.6 Hz, 11.2 Hz, 1H),
8.22 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 7.6 Hz,
1H), 7.54 (d, J = 11.2 Hz, 1H), 7.22 (d, J = 15.6 Hz, 1H), 7.17 (t, J =
7.2 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 168.6, 147.5, 143.1, 142.0, 139.5,
134.6, 130.1, 128.6, 128.3, 128.0, 124.7, 123.7, 121.7, 120.9, 110.1.
Anal. C17H12N2O3 Calcd: C, 69.86; H, 4.14; N, 9.58. Found: C, 69.59;
H, 4.03; N, 9.51. Compound 41b eluted second and was afforded as a
137.5, 130.4, 128.8, 128.3, 124.0, 123.9, 123.6, 122.6, 122.1, 121.5,
118.6, 114.3, 112.3, 109.4, 42.7, 40.1. Anal. C26H25N3O·0.5H2O Calcd:
C, 77.20; H, 6.48; N, 10.39. Found: C, 77.74; H, 6.34; N, 10.31.
3-(4-(Dimethylamino)benzylidene)-1-phenylindolin-2-one
(35). Prepared by method 1 with compound 4 and 34 to afford the
desired product (35) as a yellow solid (88%), mp 171.1−172.8 °C. 1H
NMR (400 MHz, CDCl3) δ 7.99 (d, J = 6.8 Hz, 1H), 7.89 (s, 1H),
7.72 (d, J = 8.4 Hz, 2H), 7.53−7.40 (m, 5H), 7.16 (t, J = 7.2 Hz, 1H),
6.97 (t, J = 7.2 Hz, 1H), 6.84 (d, J = 6.8 Hz, 1H), 6.77 (d, J = 8.8 Hz,
2H), 3.01 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 168.7, 151.4,
143.3, 139.6, 135.0, 132.2, 129.5, 128.3, 127.7, 126.9, 122.2, 122.1,
121.9, 111.4, 109.2, 40.1. Anal. C23H20N2O Calcd: C, 81.15; H, 5.92;
N, 8.23. Found: C, 81.01; H, 6.04; N, 8.18.
3-(3-(4-(Dimethylamino)phenyl)allylidene)-1-phenylindolin-
2-one (36). Prepared by method 1 with compound 17 and 34 to
afford the desired product (36) as a purple solid (77%), mp 206.8−
207.9 °C. 1H NMR (400 MHz, CDCl3) δ 8.48 (dd, J = 15.2 Hz, 11.6
Hz, 1H), 7.54−7.45 (m, 7H), 7.39 (d, J = 12.0 Hz, 1H), 7.38 (t, J = 7.6
Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 7.00 (d, J =
15.6 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.64 (d, J = 8.8 Hz, 2H), 2.99
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 167.1, 151.4, 145.0, 141.6,
1
red solid (46%), mp 263.8−264.4 °C. H NMR (400 MHz, DMSO-
d6) δ 10.49 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 3H),
7.87 (dd, J = 15.0 Hz, 12.8 Hz, 1H), 7.42 (d, J = 14.8 Hz, 1H), 7.24 (d,
J = 12.4 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 6.2
(d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 169.1, 147.6,
143.1, 142.8, 141.8, 133.6, 130.2, 129.3, 128.4, 127.8, 125.1, 124.4,
122.2, 121.9, 110.3. Anal. C17H12N2O3·0.50H2O Calcd: C, 67.77; H,
4.35; N, 9.30. Found: C, 67.94; H, 4.73; N, 8.87.
(Z)-1-Benzyl-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one
(42a). Prepared by method 2 with compound 41a and benzyl bromide
to afford the desired product (42a) as a red solid (76%), mp 224.5−
1
225.8 °C. H NMR (400 MHz, DMSO-d6) δ 8.66 (dd, J = 15.6 Hz,
11.6 Hz, 1H), 8.25 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.70
L
DOI: 10.1021/acs.jmedchem.5b00571
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(d, J = 12.0 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.33−7.18 (m, 7H),
7.00 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.93 (s, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 166.8, 143.0, 140.3, 137.1, 135.5,
131.3, 130.1, 129.1, 128.7, 128.2, 127.8, 126.7, 124.7, 122.5, 120.8,
109.6, 40.6. Anal. C24H18N2O3·0.125H2O Calcd: C, 74.94; H, 4.78; N,
7.28. Found: C, 74.83; H, 4.85; N, 7.18.
(d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 7.2 Hz, 1H),
7.32 (d, J = 12.0 Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 7.05−7.01 (m, 2H),
6.87 (m, 2H), 6.80 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 7.6 Hz, 1H), 4.90
(s, 2H), 3.85 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 167.2, 149.3,
148.5, 147.5, 142.7, 142.2, 139.1, 134.3, 129.6, 128.5, 128.0, 127.1,
124.1, 123.0, 122.1, 119.8, 119.7, 111.1, 110.6, 109.1, 56.0, 55.9, 43.3.
Anal. C26H22N2O5 Calcd: C, 70.58; H, 5.01; N, 6.33. Found: C, 70.70;
H, 5.14; N, 6.26.
(E)-1-Benzyl-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one
(42b). Prepared by method 2 with compound 41b and benzyl
bromide to afford the desired product (42b) as a red solid (67%), mp
(E)-1-(3,4-Dimethoxybenzyl)-3-((E)-3-(4-nitrophenyl)-
allylidene)indolin-2-one (45b). Prepared by method 2 with
compound 41b and 3,4-methoxybenzyl bromide to afford the desired
1
237.1−237.9 °C. H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 8.4 Hz,
2H), 7.79 (dd, J = 15.2 Hz, 12.4 Hz, 1H), 7.72 (d, J = 8.4 Hz, 3H),
7.54 (d, J = 12.0 Hz, 1H), 7.323−7.17 (m, 7H), 7.07 (t, J = 7.6 Hz,
1H), 6.75 (d, J = 7.6 Hz, 1H), 4.99 (s, 2H). 13C NMR (100 MHz,
CDCl3) δ 168.3, 147.8, 143.3, 142.2, 140.6, 135.9, 134.1, 129.7, 128.7,
128.0, 127.7, 127.6, 127.3, 127.2, 124.3, 123.8, 122.3, 122.0, 109.5,
43.7. Anal. C24H18N2O3 Calcd: C, 75.38; H, 4.74; N, 7.33. Found: C,
75.30; H, 6.78; N, 7.28.
1
product (45b) as a red solid (51%), mp 201.3−201.9 °C. H NMR
(400 MHz, CDCl3) δ 8.27 (d, J = 7.2 Hz, 2H), 7.82−7.71 (m, 2H),
7.72 (d, J = 6.8 Hz, 2H), 7.44 (d, J = 12.0 Hz, 1H), 7.26−7.17 (m,
2H), 7.08 (t, J = 7.2 Hz, 1H), 6.86 (m, 2H), 6.80 (d, J = 7.2 Hz, 2H),
4.92 (s, 2H), 3.84 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 168.3,
149.3, 148.5, 147.8, 143.3, 142.2, 140.6, 134.1, 129.7, 128.4, 128.0,
127.7, 127.3, 124.3, 123.8, 122.3, 122.0, 119.7, 111.1, 110.6, 109.5,
55.9, 55.8, 43.6. Anal. C26H22N2O5 Calcd: C, 70.58; H, 5.01; N, 6.33.
Found: C, 70.76; H, 5.26; N, 6.26.
(Z)-1-(4-Methoxybenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)-
indolin-2-one (43a). Prepared by method 2 with compound 41a and
4-methoxybenzyl chloride to afford the desired product (43a) as a red
solid (55%), mp 182.6−183.3 °C. 1H NMR (400 MHz, CDCl3) δ 8.82
(dd, J = 15.6 Hz, 11.6 Hz, 1H), 8.21 (d, J = 8.0 Hz, 2H), 7.72 (d, J =
8.4 Hz, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.30 (d, J = 12.8 Hz, 1H), 7.28
(d, J = 9.2 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 7.03 (d, J = 16.8 Hz, 1H),
7.01 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 8.0 Hz, 2H), 6.75 (d, J = 7.6 Hz,
1H), 4.90 (s, 2H), 3.77 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
167.1, 159.1, 147.5, 142.7, 142.2, 139.0, 134.2, 129.6, 128.7, 128.5,
128.1, 128.0, 127.1, 124.1, 123.0, 122.1, 119.8, 114.2, 109.1, 55.2, 42.9.
Anal. C25H20N2O4 Calcd: C, 72.80; H, 4.89; N, 6.79. Found: C, 72.52;
H, 5.03; N, 6.67.
(E)-1-(4-Methoxybenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)-
indolin-2-one (43b). Prepared by method 2 with compound 41b and
4-methoxybenzyl chloride to afford the desired product (43b) as a red
solid (47%), mp 237.7−238.5 °C. 1H NMR (400 MHz, CDCl3) δ 8.27
(d, J = 8.0 Hz, 2H), 7.78 (dd, J = 15.0 Hz, 12.0 Hz, 1H), 7.71 (d, J =
8.0 Hz, 2H), 7.53 (d, J = 12.0 Hz, 1H), 7.26−7.21 (m, 4H), 7.18 (d, J
= 14.8 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 8.0 Hz, 2H), 6.77
(d, J = 8.4 Hz, 1H), 4.91 (s, 2H), 3.77 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 168.2, 159.1, 153.8, 147.8, 143.3, 142.2, 140.5, 134.0, 129.7,
128.7, 128.0, 127.8, 127.4, 124.3, 123.7, 122.2, 122.0, 114.1, 109.5,
55.2, 43.2. Anal. C25H20N2O4 Calcd: C. 72.80; H. 4.89; N, 6.79.
Found: C, 72.75; H, 5.01; N, 6.73.
(Z)-1-(3-Methoxybenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)-
indolin-2-one (44a). Prepared by method 2 with compound 41a and
3-methoxybenzyl bromide to afford the desired product (44a) as a red
solid (52%), mp 193.3−194.6 °C. 1H NMR (400 MHz, CDCl3) δ 8.81
(dd, J = 16.0 Hz, 11.6 Hz, 1H), 8.21 (d, J = 8.4 Hz, 2H), 7.72 (d, J =
8.4 Hz, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.32 (d, J = 12.0 Hz, 1H), 7.24
(t, J = 7.6 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.03 (d, J = 17.2 Hz, 1H),
7.01 (t, J = 7.2 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 6.86 (s, 1H), 6.80 (d,
J = 8.8 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.94 (s, 2H), 3.77 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 167.2, 159.9, 147.5, 142.7, 142.2,
139.1, 137.6, 134.3, 129.9, 129.6, 128.5, 128.0, 127.0, 124.1, 123.0,
122.1, 119.8, 119.5, 113.2, 112.7, 109.1, 55.2, 43.3. Anal. C25H20N2O4
Calcd: C, 72.80; H, 4.89; N, 6.79. Found: C, 72.80; H, 5.06;, N, 6.66.
(E)-1-(3-Methoxybenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)-
indolin-2-one (44b). Prepared by method 2 with compound 41b and
3-methoxybenzyl bromide to afford the desired product (44b) as a red
solid (59%), mp 237.7−239.0 °C. 1H NMR (400 MHz, CDCl3) δ 8.28
(d, J = 8.0 Hz, 2H), 7.82−7.67 (m, 3H), 7.62−7.45 (m, 1H), 7.33−
7.18 (m, 3H), 7.08−7.04 (m, 2H), 6.91−6.75 (m, 4H), 4.95 (s, 2H),
3.77 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 140.6, 134.1, 129.8,
128.0, 127.3, 124.3, 124.2, 123.7, 122.3, 119.5, 113.1, 112.8, 109.5,
55.2, 43.7. Anal. C25H20N2O4 Calcd: C, 72.80; H, 4.89; N, 6.79.
Found: C, 72.87; H, 4.91; N, 6.72.
(Z)-1-(4-(2-Fluoroethoxy)benzyl)-3-((E)-3-(4-nitrophenyl)-
allylidene)indolin-2-one (46a). Prepared by method 2 with
compound 41a and 4-(2-fluoroethoxy)benzyl bromide to afford the
1
desired product (46a) as a red solid (61%), mp 187.8−188.1 °C. H
NMR (400 MHz, CDCl3) δ 8.81 (dd, J = 16.0 Hz, 11.6 Hz, 1H), 8.21
(d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H),
7.31 (d, J = 11.6 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 7.19 (t, J = 7.6 Hz,
1H), 7.03 (d, J = 16.0 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.88 (d, J =
8.4 Hz, 2H), 6.74 (d, J = 7.6 Hz, 1H), 4.91 (s, 2H), 4.73 (dt, J = 47.2
Hz, 4.0 Hz, 2H), 4.17 (dt, J = 28.4 Hz, 4.0 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 167.2, 157.9, 147.5, 142.7, 142.1, 139.1, 134.3, 129.6,
128.8, 128.7, 128.5, 128.0, 127.1, 124.1, 123.0, 122.1, 119.8, 114.9,
109.1, 81.8 (d, J = 169.7 Hz), 67.1 (d, J = 20.1 Hz), 42.8. Anal.
C26H21FN2O4 Calcd: C, 70.26; H, 4.76; N, 6.30. Found: C, 70.46; H,
4.91; N, 6.25.
(E)-1-(4-(2-Fluoroethoxy)benzyl)-3-((E)-3-(4-nitrophenyl)-
allylidene)indolin-2-one (46b). Prepared by method 2 with
compound 41b and 4-(2-fluoroethoxy)benzyl bromide to afford the
1
desired product (46b) as a red solid (55%), mp 225.4−226.1 °C. H
NMR (400 MHz, CDCl3) δ 8.27 (d, J = 8.4 Hz, 2H), 7.78 (dd, J =
15.0 Hz, 12.0 Hz, 1H), 7.72 (d, J = 8.4 Hz, 3H), 7.53 (d, J = 12.0 Hz,
1H), 7.27−7.17 (m, 4H), 7.07 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 8.8 Hz,
2H), 6.77 (d, J = 8.0 Hz, 1H), 4.92 (s, 2H), 4.73 (dt, J = 47.6 Hz, 4.0
Hz, 2H), 4.18 (dt, J = 28.0 Hz, 4.0 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 168.2, 157.9, 147.8, 143.3, 142.2, 140.6, 134.1, 129.7, 128.7,
128.6, 128.0, 127.8, 127.3, 124.3, 123.8, 122.3, 122.0, 114.9, 109.4, 81.8
(d, J = 169.7 Hz), 67.1 (d, J = 20.1 Hz), 43.2. Anal. C26H21FN2O4
Calcd: C, 70.26; H, 4.76; N, 6.30. Found: C: 70.22, H: 4.68, N: 6.31.
3-(3-(4-Aminophenyl)allylidene)indolin-2-one (47). A mixture
of 41a (146 mg, 0.5 mmol) and Fe powder (280 mg, 5 mmol) in
methanol (15 mL) and 37% HCl (0.5 mL) was heated to reflux for 3
h. The solid was filtered out, the filtrate was evaporated, ethyl acetate
(75 mL) was added, washed with saturated Na2CO3 (50 mL), water
(50 mL), and NaCl (50 mL), and dried over Na2SO4. After removed
of the solvent, the crude product was purified by silica gel column
chromatography eluting with EtOAc−MeOH (10:1, v/v) to afford 98
1
mg (67%) of 47 as a red solid, mp 263.3−264.4 °C. H NMR (400
MHz, DMSO-d6) δ 10.36 and 10.31 (s, 1H), 8.14 and 7.39 (dd, J =
15.6 Hz, 12.0 Hz, 1H), 7.86 and 7.44 (d, J = 7.2 Hz, 1H), 7.46 (d, J =
8.8 Hz, 2H), 7.26 and 7.15 (d, J = 11.2 Hz, and 12.8 Hz, 1H), 7.13 and
7.08 (t, J = 8.0 Hz, 1H), 6.94 and 6.88 (t, J = 8.0 Hz, 1H), 6.80 and
6.75 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 8.0 Hz, 2H), 5.79 and 5.75 (s,
2H). 13C NMR (100 MHz, DMSO-d6) δ 169.6, 169.0, 151.7, 151.4,
147.0, 144.8, 141.8, 140.6, 137.7, 136.8, 130.6, 129.9, 128.4, 128.1,
124.7, 124.1, 123.9, 123.8, 123.2, 122.4, 122.0, 121.6, 121.2, 119.5,
119.1, 118.0, 114.4, 114.2, 109.8, 109.6. Anal. C17H14N2O·0.125H2O
Calcd: C, 77.18; H, 5.43; N, 10.59. Found: C, 77.34; H, 5.58; N, 10.37.
(Z)-1-(4-(2-Bromoethoxy)benzyl)-3-((E)-3-(4-nitrophenyl)-
allylidene)indolin-2-one (48). Prepared by method 2 with
compound 41a and 4-(2-bromoethoxy)benzyl bromide to afford the
(Z)-1-(3,4-Dimethoxybenzyl)-3-((E)-3-(4-nitrophenyl)-
allylidene)indolin-2-one (45a). Prepared by method 2 with
compound 41a and 3,4-dimethoxybenzyl bromide to afford the
1
desired product (45a) as a red solid (55%), mp 199.6−200.2 °C. H
NMR (400 MHz, CDCl3) δ 8.81 (dd, J = 15.4 Hz, 11.6 Hz, 1H), 8.21
M
DOI: 10.1021/acs.jmedchem.5b00571
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
desired product (48) as a red solid (78%), mp 192.3−193.1 °C. H
NMR (400 MHz, CDCl3) δ 8.81 (dd, J = 15.8 Hz, 11.6 Hz, 1H), 8.22
(d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 7.6 Hz, 1H),
7.32 (d, J = 12.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 7.194 (t, J = 7.6
Hz, 1H), 7.04 (d, J = 17.2 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.86 (d, J
= 8.8 Hz, 2H), 6.75 (7.6 Hz, 1H), 4.91 (s, 2H), 4.25 (t, J = 6.2 Hz,
2H), 3.61 (t, J = 6.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 167.2,
157.6, 147.5, 142.7, 142.1, 139.1, 134.3, 129.6, 128.9, 128.8, 128.5,
128.0, 127.1, 124.1, 123.0, 122.1, 119.8, 115.0, 109.1, 67.8, 42.8, 29.0.
2-(4-(((Z)-3-((E)-3-(4-Nitrophenyl)allylidene)-2-oxoindolin-1-
yl)methyl)phenoxy)ethylmethanesulfonate (49). A solution of
48 (127 mg, 0.5 mmol) and silver methanesulfonate (500 mg, 5
mmol) in acetonitrile (15 mL) was heated to reflux for 20 h. After
evaporation of the solvent, the crude product was purified by silica gel
column chromatography eluting with EtOAc−MeOH (10:1, v/v) to
afford 89 mg (68%) of 49 as red solid, mp 186.4−187.1 °C. 1H NMR
(400 MHz, CDCl3) δ 8.80 (dd, J = 15.4 Hz, 11.4 Hz, 1H), 8.21 (d, J =
8.8 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.31
(d, J = 11.6 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H),
7.03 (d, J = 16.4 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 8.4 Hz,
2H), 6.73 (d, J = 7.6 Hz, 1H), 4.90 (s, 2H), 4.54 (t, J = 6.2 Hz, 2H),
4.20 (t, J = 6.2 Hz, 2H), 3.06 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
167.1, 157.5, 147.5, 142.7, 142.0, 139.2, 134.4, 129.6, 129.2, 128.9,
128.4, 128.0, 127.0, 124.1, 123.0, 122.2, 119.9, 114.8, 109.0, 67.9, 65.8,
42.8, 37.8.
concentration in the supernatant obtained by centrifuging the fibril
reaction mix at 18000g for 15 min to separate fibrils from monomer.
The measured decrease in α-syn monomer concentration was used to
determine the concentration of fibrils in the 72 h fibril reaction
mixture.
Preparation of Aβ1−42 Fibrils. Synthetic Aβ1−42 peptide (1 mg)
(Bachem, Torrance, CA) was first dissolved in 50 μL of DMSO. An
additional 925 μL of ultrapure water was added. Finally, 25 μL of 1 M
Tris-HCl, pH 7.6, was added to bring the final peptide concentration
to 222 μM (1 mg/mL). The dissolved peptide was incubated for 30 h
at 37 °C with shaking at 1000 rpm in an Eppendorf Thermomixer.
Fibril formation was confirmed by Thio-T fluorescence. To determine
the concentration of fibrils, the fibril reaction mix was centrifuged at
18000g for 15 min to separate fibrils from monomer. The
concentration of Aβ monomer in the supernatant was determined in
a BCA protein assay using a BSA standard curve that contained
DMSO at a percentage equivalent to the samples.
Preparation of Recombinant Tau Fibrils. Purified recombinant tau
monomer (300 μg/mL) was incubated in 20 mM Tris-HCl, pH 8.0,
100 mM NaCl, 25 μM low molecular weight heparin, and 0.5 mM
DTT for 48 h at 37 °C with shaking at 1000 rpm in an Eppendorf
Thermomixer. To determine the concentration of fibrils, the fibril
reaction mix was centrifuged at 18000g for 15 min to separate fibrils
from monomer. The concentration of tau monomer in the supernatant
was determined in a BCA protein assay along with a BSA standard
curve. The measured decrease in monomer concentration was used to
determine the concentration of tau fibrils in the 48 h fibril reaction
mixture.
Preparation of α-Syn, Aβ1−42, and Tau Fibrils for Binding and
Competition Assays. The prepared fibril mixture was centrifuged at
18000g for 15 min to prepare fibrils for binding assays. The
supernatant was discarded and the fibril pellet was resuspended in
30 mM Tris-HCl pH 7.4, 0.1% BSA to achieve the desired
concentration of fibrils for use in the assay.
In Vitro Saturation Binding Studies to Determine Binding Affinity
of Thio-T for Fibrils. A fixed concentration (1 μM) of α-syn, Aβ, or tau
fibrils were incubated for 2 h at 37 °C with increasing concentrations
of Thio-T in 30 mM Tris-HCl, pH 7.4, 0.1% BSA in a reaction volume
of 150 μL. Nonspecific binding was determined in a duplicate set of
binding reactions containing the same concentration of Thio-T with
no fibrils. Fluorescence was determined in a Biotek plate reader using a
440/30 excitation filter and a 485/20 emission filter. All data points
were performed in triplicate. The dissociation constant (Kd) and the
maximal number of binding sites (Bmax) values were determined by
fitting the data to the equation Y = Bmax × X/(X + Kd) by nonlinear
regression using Graphpad Prism software (version 4.0).
Thio-T Competition Studies. Competition assays used a fixed
concentration of α-syn, Aβ, or tau fibrils (1 μM), consisting of 3 μM,
50 nM, and 4 μM of Thio-T, respectively. The competitor reaction
was diluted in 30 mM Tris-HCl, pH 7.4, 0.1% BSA and added to the
reactions in varying concentrations. To optimize the determination of
selectivity, assays for all three fibril species were set up in parallel using
a common set of intermediate compound dilutions for each analogue.
Reactions were incubated at room temperature for 1.5 h before
quantifying bound ligand as described above for the saturation binding
assay. Fluorescence was determined in a Biotek plate reader using a
440/30 excitation filter and a 485/20 emission filter. All data points
were performed in triplicate. Nonspecific fluorescence was measured
in parallel reactions containing Thio-T plus each concentration of
competitor but no fibrils, and these measurements were subtracted
from the reactions with fibrils to yield fibril-specific fluorescence. Data
were analyzed using Graphpad Prism software (version 4.0) to obtain
EC50 values by fitting the data to the equation Y = bottom + (top −
bottom)/(1 + 10(x − log EC50)). Ki values were calculated from EC50
values using the equation Ki = EC50/(1 + [radioligand]/Kd).
In Vitro Saturation Binding Studies of [18F]46a. α-Syn, Aβ, or tau
fibrils (1 μM) were incubated for 1 h at 37 °C with increasing
concentrations of [18F]46a in 30 mM Tris-HCl, pH 7.4, 0.1% BSA in a
reaction volume of 150 μL. A fixed ratio of labeled:unlabeled 46a was
used for all radioligand concentrations. The exact labeled:unlabeled
Radiosynthesis of [18F]46a. [18F]Fluoride (50−200 mCi) in
[18O]water (without treatment by trapping on quaternary methyl
ammonia cartridge) was transferred into a Pyrex screw cap tube (10
mL) containing K2CO3 (0.3 mg, 2.2 μmol) and Kryptofix 222 (K222
)
(2.2 mg, 5.9 μmol); the activity was dried by azeotropic distillation at
105 °C using MeCN (3 × 1 mL) under a gentle flow of N2 gas. To the
dried activity was added the mesylate precursor 49 (1.8 mg, 3.46
μmol) as solid with tert-amyl alcohol (300 μL) using a transfer pipet.
The tube was capped and shaken in an oil bath of 105 °C to dissolve
the precursor. The reaction mixture was heated at 105 °C for 10 min.
After cooling to room temperature, the mixture was treated by passing
through a silica column (5 mm × 20 mm); MeCN (6 × 0.5 mL) was
used to rinse the tube and the column. The combined eluents were
concentrated at 105 °C under a flow of N2 to almost dryness, and
HPLC mobile phase (3 mL) was added to the above residue for HPLC
purification. Under the specified conditions, [18F]46a eluted at 40 min.
The HPLC fraction was collected and diluted with water (50 mL), and
the diluted solution was passed through a Waters Oasis HLB plus
cartridge. The cartridge was rinsed with water (10 mL), and the final
product was eluted with DMF for binding studies.
Binding Assay Methods. Preparation of Recombinant α-Syn
and Tau Protein. Recombinant proteins were produced in Escherichia
coli and purified as previously described.23 Briefly, α-syn was extracted
from E. coli using an osmotic shock method then purified by a
combination of heat-precipitation and DEAE (diethylaminoethyl) ion
exchange chromatography. Purified α-syn protein was dialyzed
overnight in 10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1 mM DTT.
Preparations contained greater than 95% α-syn protein as determined
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and bicinchoninic acid (BCA) protein assay (Thermo
Scientific, Rockford, IL), with a typical yield of 30 mg protein per
250 mL culture.
Recombinant tau protein was produced in E. coli. BL21(DE3)RIL
The bacterial cultures were transformed with a pRK172 bacterial
expression plasmid encoding a human tau fragment containing the
four microtubule binding repeats (amino acids 243−375), provided by
Marc Diamond at Washington University. Cultures were inoculated
and grown overnight as above for α-syn protein production. Purified
tau protein was prepared using the previously described method and
dialyzed overnight in 100 mM sodium acetate pH 7.0.31
Preparation of Recombinant α-Syn Fibrils. Purified recombinant
α-syn monomer (2 mg/mL) was incubated in 20 mM Tris-HCl, pH
8.0, 100 mM NaCl for 72 h at 37 °C with shaking at 1000 rpm in an
Eppendorf Thermomixer as previously described.23 Fibril concen-
tration was determined by BCA protein assay to determine monomer
N
DOI: 10.1021/acs.jmedchem.5b00571
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
46a ratio was measured in each experiment by counting a 2 μL sample
of the radioligand preparation in a scintillation counter. Nonspecific
binding was determined in the parallel binding reactions containing 20
as the competitor or in reactions containing no fibrils, both of which
yielded similar nonspecific binding measurements. Bound and free
radioligand were separated by vacuum filtration through 1.0 μm glass
fiber filters in 96-well filter plates (Millipore), followed by three 200
μL washes with ice-cold assay buffer. Filters containing the bound
ligand were mixed with 150 μL of Optiphase Supermix scintillation
cocktail (PerkinElmer) and counted immediately. All data points were
performed in triplicate. The dissociation constant (Kd) and the
maximal number of binding sites (Bmax) values were determined by
fitting the data to the equation Y = Bmax × X/(X + Kd) by nonlinear
regression using Graphpad Prism software (version 4.0).
AUTHOR INFORMATION
Corresponding Author
*Phone: (215) 746-8233. Fax: (215) 746-0002. E-mail:
rmach@mail.med.upenn.edu.
■
Present Address
⊥Robert H. Mach, Ph.D., Britton Chance Professor of
Radiology, University of Pennsylvania, Chemistry Building,
231 South 34th Street, Philadelphia, Pennsylvania 19104.
Phone: +1 215 746 8233. Fax: +1 215 746 0002 rmach@mail.
med.upenn.edu
Notes
The authors declare no competing financial interest.
Human Tissue Acquisition. Post-mortem midbrain tissue from
DLB and PD cases was obtained from the Harvard Brain Tissue
Resource Center at McLean Hospital. Paraffin-embedded formalin-
fixed tissue from the temporal lobe of AD cases was obtained through
the Massachusetts Alzheimer’s Disease Research Center Brain Bank.
The human brain tissue provided was neuropathologically defined as
DLB tissue using standard criteria.
ACKNOWLEDGMENTS
■
This study was funded in part by the Michael J. Fox Alpha
Synuclein Imaging Consortium, Washington University In-
stitute of Clinical and Translational Sciences Grant CTSA406,
and the Charles and Joanne Knight Alzheimer’s Research
Initiative of the Washington University Alzheimer’s Disease
Research Center. This study made use of the NIH/NIGMS
Biomedical Mass Spectrometry Resource at Washington
University in St. Louis, MO, which is supported by National
Institutes of Health\ National Institute of General Medical
Sciences Grant # 8P41GM103422. We would like to thank
personnel of the Washington University Cyclotron Facility for
[18F]fluoride production and the Department of Chemistry
staff and the Washington University High Resolution NMR
Facility for assistance with NMR spectra. Purchase of the 400
MHz NMR instrument was partially supported by S10
RR027207 from the NIH Shared Instrument Grant program.
Labeling of Synuclein Pathology in Human DLB and PD Cases.
Blocks of midbrain tissue from human PD/DLB cases were fixed with
4% paraformaldehyde and stored at −80 °C. In preparation for
immunohistochemistry, the tissue block was deparaffinized with xylene
and rehydrated. Midbrain tissue was sliced into 40 μm thick sections
with a freezing sledge Leica SM 2000R microtome. Free-floating tissue
was permeabilized with 0.5% Triton-X 100, then incubated with
normal goat serum (NGS) at room temperature for 1 h with shaking.
Primary H3C antibody (1:5000 in 1.5% NGS) to α-syn was incubated
overnight at 4 °C with shaking and then washed with 1× PBS three
times. The tissue was then incubated for 2 h at room temperature with
a fluorescent secondary antibody labeled with Alexa Fluor 488 (1:200
in PBS). Tissue was then treated with the compound to be tested.
Each tissue section was incubated at room temperature for 30 min
with 5 μM of test compound dissolved in PBS. The tissue was washed
with 1× PBS three times and then mounted onto Adhesion SuperFrost
Plus slide and coverslipped. The tissue was imaged with an Olympus
BX51 upright microscope (Olympus America Inc., Melville, NY) using
standard excitation/emission filters for Alexa Fluor 488 and Cy5.
Staining of Plaques in Human Tissue from AD Cases. Paraffin-
embedded paraformaldehyde (4%) fixed sections of temporal lobe of
neuropathologically verified human AD cases were deparaffinized.
Tissue sections were stained with 0.05% Thioflavin S Thio-T (Sigma,
St. Louis, MO) in 50% ethanol for 5 min to visualize senile plaques.
Tissue was then incubated with 5 μM of compound in PBS for 20 min
at room temperature. Slides were rinsed in PBS, coverslipped, and
then imaged on an Olympus BX51 microscope using a 20× Plan
Neofluar objective (NA = 0.7) using standard excitation/emission
filters for AlexaFluor488 and Cy5.
ABBREVIATIONS USED
■
α-syn, alpha synuclein; BCA assay, bicinchoninic acid assay;
DEAE ion exchange, diethylaminoethyl ion exchange; DLB,
dementia with Lewy bodies; LB, Lewy body; LN, Lewy neurite;
MSA, multiple system atrophy; NFT, neurofibrillary tangles;
QC, quality control; Thio-T, thioflavin-T
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■
S
* Supporting Information
Methods for the 2D NMR study; NOE in E,E configuration of
1
41b and 3D structure of 41a and 41b; H NMR spectrum of
41b; 2D NMR spectrum of 41b; stability of 41a and 41 b in
dichloromethane/methanol solution; saturation binding studies
to determine binding affinity of Thio-T for α-syn, Aβ, and tau
1
fibrils; analytical HPLC of [18F]46a; H NMR of compounds
5−14, 18−25, 27−33, 35, 36, 38a,b, 39a,b, 41a,b, 42a,b, 43a,b,
44a,b, 45a,b, 46a,b, 47, 48, and 49 (PDF). Molecular formula
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acs.jmedchem.5b00571.
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