Design, synthesis and identification of N, N-dibenzylcinnamamide (DBC) derivatives as novel ligands for α-synuclein fibrils by SPR evaluation system

Article abstract of DOI:10.1016/j.bmc.2020.115358

PET imaging of α-synuclein (α-syn) deposition in the brain will be an effective tool for earlier diagnosis of Parkinson's disease (PD) due to α-syn aggregation is the widely accepted biomarker for PD. However, the necessary PET radiotracer for imaging is clinically unavailable until now. The lead compound discovery is the first key step for the study. Herein, we initially established an efficient biologically evaluation system well in high throughput based on SPR technology, and identified a novel class of N, N-dibenzylcinnamamide (DBC) compounds as α-syn ligands through the assay. These compounds were proved to have high affinities against α-syn aggregates (K_D < 10 nM), which well met the requirement of binding activity for the PET probe. These DBC compounds were firstly reported as α-syn ligands herein and the preliminary obtained structure has been further modified into F-labeled ones. Among them, a high-affinity tracer (5–41) with 1.03 nM (K_D) has been acquired, indicating its potential as a new lead compound for developing PET radiotracer.

Full text of DOI:10.1016/j.bmc.2020.115358

Journal Pre-proofs
Design, Synthesis and Identification of N, N-Dibenzylcinnamamide (DBC)
Derivatives as Novel Ligands for α-synuclein Fibrils by SPR Evaluation Sys-
tem
Yanfei Chen, Jiang Bian, Peng Zhang, Lulu Bu, Yan Shen, Wenbo Yu,
Xiuhong Lu, Xin Lin, De-Yong Ye, Jian Wang, Yong Chu
PII:
S0968-0896(20)30149-8
https://doi.org/10.1016/j.bmc.2020.115358
BMC 115358
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 October 2019
22 January 2020
31 January 2020
Please cite this article as: Y. Chen, J. Bian, P. Zhang, L. Bu, Y. Shen, W. Yu, X. Lu, X. Lin, D-Y. Ye, J. Wang,
Y. Chu, Design, Synthesis and Identification of N, N-Dibenzylcinnamamide (DBC) Derivatives as Novel Ligands
for α-synuclein Fibrils by SPR Evaluation System, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115358
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Design, Synthesis and Identification of N, N-Dibenzylcinnamamide (DBC)
Derivatives as Novel Ligands for α-synuclein Fibrils by SPR Evaluation System
Yanfei Chena, 1, Jiang Biana, 1, Peng Zhanga, Lulu Bub, Yan Shenb, Wenbo Yub,
Xiuhong Lub, Xin Lina, De-Yong Yea,*, Jian Wangb,*, Yong Chua,*
aDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University,
Shanghai 201203, China
bDepartment of Neurology and National Clinical Research Center for Aging and
Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China
1These authors contributed equally to this work
* Correspondence to: Dr. Yong Chu, Phone/Fax: 86-21-51980125, E-mail:
cy110@fudan.edu.cn; Dr. Jian Wang, Phone: 86-13321934789, Fax: 86-21-52888163,
E-mail: wangjian336@hotmail.com; Prof. De-Yong Ye, Phone/Fax: 86-21-51980117,
E-mail: dyye@shmu.edu.cn
Abbreviations: PD, Parkinson's disease; α-syn, α-synuclein; Aβ, amyloid-beta; ThT,
Thioflavin T; PET, Positron emission tomography; SPR, Surface plasmon resonance,
HBTU, O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;
DIPEA, N,N-diisopropylethylamine, DCM, dichloromethane; THF, tetrahydrofuran;
EA, ethyl acetate.
1

Abstract:
PET imaging of α-synuclein (α-syn) deposition in the brain will be an effective
tool for earlier diagnose of Parkinson's disease (PD) due to α-syn aggregation is the
widely accepted biomarker for PD. However, the necessary PET radiotracer for
imaging is clinically unavailable until now. The lead compound discovery is the first
key step for the study. Herein, we initially established an efficient biologically
evaluation system well in high throughput based on SPR technology, and identified a
novel class of N, N-dibenzylcinnamamide (DBC) compounds as α-syn ligands
through the assay. These compounds were proved to have high affinities against α-syn
aggregates (KD < 10 nM), which well met the requirement of binding activity for the
PET probe. These DBC compounds were firstly reported as α-syn ligands herein and
the preliminary obtained structure has been further modified into F-labeled ones.
Among them, a high-affinity tracer (5-41) with 1.03 nM (KD) has been acquired,
indicating its potential as a new lead compound for developing PET radiotracer.
Key Words:
α-synuclein, Parkinson's disease, SPR, PET tracer, N, N-dibenzylcinnamamide,
F-labeled stable isotope tracer
1. Introduction
Parkinson's disease (PD) is a widespread type of central nervous system
degenerative disease.1 In its pathogenesis and progression, α-synuclein (α-syn) plays
an critical role and now accepted as the most valuable biomarker for early diagnosis
of PD.2-4 The α-syn fibrous aggregates formed by misfolding in the brain are the main
component of Lewy body,5 which is the typical pathological hallmark of PD.6 The
2

most important thing is the process especially lasts several years from the formation
of Lewy body to the onset of PD symptoms, and so it is recognized to be an important
stage for early intervention for PD therapy. In the patients, the gradual death of
dopaminergic neurons in the basal striatum and substantia nigra makes them fail to
produce enough dopamine in the brain,7 which leads to various mobility disorders and
mental changes. To explain the mechanism of abnormal death of dopaminergic
neurons, different hypotheses such as mitochondrial dysfunction, oxidative stress, and
inflammatory response are proposed.8 All of these mechanisms are closely related to
α-syn accumulation9 caused by the ubiquitination-proteasome degradation of
intracellular denaturing proteins.10
Positron Emission Tomography (PET) is an extremely efficient and powerful
imaging technology with advantages of real-time, accurate, and direct observation of
the distribution and dynamics of the corresponding target in the living body.
Moreover, it is the only one to be capable of imaging for function and metabolism
nowadays. Currently, PET imaging has been widely used in the research and
clinically diagnosis of brain amyloid diseases such as Alzheimer's disease, PD.11-13
Obviously, PET imaging of the abnormal α-syn aggregates is greatly powerful to
make a distinguish of PD syndrome and deepen the understanding of the pathogenesis.
For PET imaging, it is necessary to use the specific radioligands that can strongly and
selectively bind to α-syn aggregates as the PET tracers. However, there is still no
commercial PET tracer available for imaging α-syn. It is actually an extremely
challenging task to find a suitable α-syn ligand. The ligand should primarily have a
very high affinity against α-syn, normally with a KD value below 50 nM,14 because of
much low content of α-syn in the brain.
As well known, a suitable lead structure is a basis for the development of PET
tracer. However, little small molecular ligands of α-syn are reported until now (Figure
1). Two lead compounds of SIL5 and WC58a were reported as representatively
high-affinity candidates identified by a Thioflavin T (ThT) competition method 15,16
.
The assay indirectly gave a detection of the fluorescence changes when compounds
3

competed with the α-syn-ThT conjugate. However, due to its inherent inadequacy
such as time-consuming, complicated experimental steps, fluorescence quenching and
limited ThT competitive action manner, the ThT assay is not well suitable for the
evaluation on large-scale and for different action modes of small molecules.
NO2
O
S
NO2
O
N
H
N
F
O
SIL5
KD= 32.1 nM
WC58a
KD= 2 nM
Figure 1. Representative α-syn ligands reported
Therefore, there is an urgent need for an efficient biologically evaluation system
to seek new chemicals that can serve as lead compounds for PET radiotracer. Surface
plasmon resonance (SPR) technology is a powerful tool characterized by high
sensitivity and throughput, and already has a wide range of applications in the
discovery of new drugs,17 such as screening for small molecular ligands of proteins
and structural optimization.18 To our best knowledge, though SPR technology has
been widely applied in drug discovery, no SPR assay has been reported for the
discovery of small molecular ligands of α-syn. Herein, we initially constructed an
efficient SPR system and evaluated our newly designed small molecules as α-syn
ligands. We describe the design and synthesis of a novel class of compounds, the N,
N-dibenzylcinnamamide (DBC) derivatives, as well as their in vitro evaluation by our
established SPR biological system. These compounds were identified to have a strong
binding potency against α-syn aggregates (KD <10 nM), which well meets the target
activity requirement for the PET probe. Furthermore, the F-labeled DBC analogs were
synthesized and identified as a potential stable isotope tracer (5-41) with an increasing
affinity (KD = 1.03 nM), indicating its potential as a novel lead compound for
developing PET radiotracer.
4

2 Establishment of SPR evaluation system
Human wild-type α-syn monomer was prepared by the method described in the
literature,19 and then to form the polymeric protein by shaking the monomer at 37 °C
for hours. The obtained polymeric protein was fixed on the SPR chip performed on
GE Biacore T200.20 The reported compound SIL5 was selected as a positive reference
and was synthesized according to the reported method.21 Its structure was confirmed
by 1H-NMR and mass spectrometry. The KD value was measured by our SPR assay to
be 27.95 nM, which was well consistent with the data (KD = 32.1±1.3 nM) reported in
the literature.15 The result confirmed our SPR biological evaluation system reliable.
3. Chemistry
3.1 Compounds design
At present, the binding sites in α-syn aggregations have not been clear, so it is
difficult to design a new ligand scaffold utilizing the strategy of structure-based drug
design. Because of the structurally high similarity of aggregations between α-syn and
Aβ in amyloid family,22 we carefully considered to get information from the reported
structures of Aβ aggregate for our α-syn ligands design.
In the aggregated Aβ fibrils, it was found that 19-position phenylalanine folded
into the β-sheet to form a hydrophobic center, making the 18-position amino acid
residue exposed to the protein surface. A "trench" was formed between the amino acid
and the 20-position phenylalanine.23 One recent work based on cryo-electron
microscopy also confirmed the existence of the "trench".24 According to the results of
literature, we had a hypothesis that the "trench" may be the binding site of aromatic
fluorescent dyes such as ThT.
More reported works validated our hypothesis. According to the literature,25 the
site of ThT likely binding to in AS is similar to that in Aβ. On this basis, we examined
5

the complex of α-syn monomer and the binding proteins β -wrapins.26 Unlike the
"parallel" ladder structure formed by Phe 20 and Val 18 in Aβ,27 the α-syn monomer
underwent a certain degree of distortion due to the formation of a Tyr39-His50
hydrogen bond. According to the above information, we thought the good α-syn
ligands should be characterized by an open chain with approximately planar polycycle
in lower flexibility, which would be helpful for ligands inserting into that "trench"
site.
Curcumin was already reported to have a weak ability to prevent the formation of
α-syn aggregates in a dose-dependent manner with concentration ranging from 10-5 to
10-1 mM.28 One of analogs FMeC1 was reported to bind to Aβ.29 Based on the
analysis above and the structure-activity relationship analysis of curcumin analogs,
we audaciously designed a novel of N, N-dibenzylcinnamamide (DBC) compounds as
new α-syn imaging probes (Figure 2).
O
O
O
O
O
R1
N
R3
HO
OH
OCF3
HO
OH
OCH3
R2
OCF3
OCH3
O
OCH3
FMeC1
curcumin
DBC
Figure 2. Reference lead compounds and our DBC scaffold
3.2 Synthesis of DBC objects
The general synthesis of DBC compounds is outlined in Scheme 1. The synthesis
of intermediates 3 was performed by the reductive amination of various substituted
aromatic amines or fatty amines with substituted benzaldehydes under the action of
sodium triacetoxyborohydride. Followed by the condensation of obtained 3 with lots
of substituted cinnamic acids 4 under the catalysis of HBTU at room temperature, the
DBC derivatives 5-1 ~ 5-29 were successfully obtained from recrystallization or
column chromatography in moderate to good yields. Hydrolysis of 5-3 and 5-28 could
6

afford different acids, which were transformed into other DBC compounds 5-30 ~
5-39 by condensation with varied amines.
O
O
R1
CHO
HO
N
a
R3
4
R1
R1NH2
R3
R2
N
R2
R2
H
b
3
1
2
5-1~29
O
O
N
c
N
d
5-3, 5-28
R3
R2
5-30~39
COOH
R2
(a) NaBH(OAc)3, HOAc, DCE, rt, overnight; (b) acids, HBTU, DIPEA, DCM, rt, 12
h.(c) LiOH, THF, MeOH, H2O, rt, 4h; (d) amines, HBTU, DIPEA, DCM, rt, 12 h.
Scheme 1 Synthesis of N, N-dibenzylcinnamamide derivatives
Table 1 Structure of N, N-dibenzylcinnamamide derivatives and their binding affinity
O
R1
N
R3
R2
Synthetic Target
intermedi compou
R1
R2
R3
Bindin KD
(nM)
g
ates
3-1
3-2
3-2
nds
5-1
5-2
5-3
5-4
5-5
5-6
(RU)a
264.5
121.9
120.2
61.8
0.4
2-MeO-Ph
2-NO2
H
111.6
N.D.
N.D.
N.D.
N.D.
N.D.
Bn
Bn
H
2-NO2
2-NO2
2-NO2
H
NH2
COOMe
COOMe
COOMe
NO2
H
3-2
Bn
2-NO2
190.0
7

3-2
3-2
3-5
3-3
3-4
3-16
5-7
Bn
2-NO2
2-NO2
2-NO2
2-NO2
2-NO2
CN
243.1
34.0
199.8
2.7
105.3
N.D.
N.D.
N.D.
N.D.
N.D.
5-8
Bn
tBuCONH
CN
5-9
4-CH3O-Bn
cyclohexyl
cyclopropyl
5-10
5-11
5-12
CN
CN
2.0
4-(1-BOC-Pipe 2-NO2
ridinyl)
CN
19.7
3-6
5-13
5-14
5-15
5-16
5-17
5-18
5-19
5-20
5-21
5-22
5-23
5-24
5-25
5-26
5-27
5-28
5-29
5-30
5-31
Et2N(CH2)2
2-NO2
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
9.0
N.D.
8.21
3-7
2-FuranylCH2 2-NO2
380.8
236.6
240.9
153.9
216.9
168.4
17.1
3-8
Ph
2-NO2
2-NO2
2-NO2
2-NO2
2-NO2
2-NO2
2-NO2
2-NO2
439.2.
115.5
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
565.3
N.D.
N.D.
106.9
N.D.
N.D.
N.D.
344.0
N.D.
3-9
Ph(CH2)2
Ph(CH2)3
4-F-Bn
3-Cl-Bn
4-Cl-Bn
4-Br-Bn
4-CH3-Bn
Bn
3-10
3-11
3-12
3-13
3-14
3-15
3-17
3-22
3-16
3-18
3-19
3-20
3-21
3-2
114.6
183.8
273.9
192.2
2.7
4-MeO CN
Bn
4-NO2
CN
4-piperidinyl 2-NO2
CN
Bn
Bn
Bn
Bn
Bn
Bn
H
CN
243.0
191.6
105.7
162.2
274.0
2-Cl
3-Cl
2-CF3
2-NO2
3-Cl
CN
COOMe
CN
tBuNHCO
3-20
(4-Me-1-piperazinyl 23.9
)CO
3-20
3-20
5-32
5-33
Bn
Bn
3-Cl
3-Cl
(1-piperidinyl)CO
110.3
N.D.
(1-morpholinyl)CO 307.5
871.8
8

3-20
3-20
3-20
3-20
3-20
3-20
5-34
5-35
5-36
5-37
5-38
5-39
Bn
Bn
Bn
Bn
Bn
Bn
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
tBuNHCO
154.3
127.6
50.4
2.8
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
nBuNHCO
hexthylNHCO
PhNHCO
BnNHCO
42.3
237.1
Ph(CH2)2NHCO
(a)The tested compounds (50µM) were injected into the SPR evaluation system, and
the binding signals (RU) variation were noted, which reflected the combination
between the compounds and a-syn fibrils. High RU values reveal a strong
combination. Furthermore, the KD values of compounds with RU over 200 were
determined.
3.3 Synthesis of F-labeled stable isotope tracers
We successfully identified the novel cinnamamide scaffold as a new lead
structure for α-syn ligand through SPR biological evaluation. The compound 5-14
well met the activity requirement for the PET probe with a high binding affinity (KD =
8.21 nM). Therefore, we further synthesized stable isotope probes to evaluate their
potency. Since 18F usually used in PET radiotracer due to its long half-life time
(t1/2=110 min), we decided to introduce the stable isotope of fluorine atom into our
DBC structure to be a cold PET probe.
Normally, the heat labeling reaction condition for introducing the radioactive 18F
was very harsh,30 such as limited in short reaction time (<10 min),31 using high
polarity organic solvent and high reaction temperature (usually 60~120 °C or even
higher). Since the group in the para position of the electron-withdrawing group of the
benzene ring can be easily substituted by nucleophiles in that condition,32 we decided
to introduce the fluorine atoms into the para position of the nitro groups in ring A. In
addition, the substitution at para position of benzene ring in cinnamamide might be
beneficial for keeping the binding affinity. Hence, the fluorine atom was introduced
into this position in ring C as well (shown as the dotted circle in Figure 3).
9

O
NO2
A
Ar (ring B) =
n Ar (B)
N
O
F
C
F
OMe
CN
Figure 3. Potential sites for introduction of F atom
Finally, we designed and synthesized nine F-labeled DBC probes. The
intermediates 3-23~27 with F atom were synthesized by the Borch reaction, and the
target molecules were obtained by condensation of the intermediates with different
substituted cinnamic acids. The synthetic route is shown in Scheme 2.
COOH
NO2
NO2
O
NO2
CHO
N
R2
N
R3
H
R2 NH2
R2
R3
a
R1
R1
R1
b
3-23~27
5-40~47
Reagents and conditions: (a) HOAc, NaBH(OAc)3, DCE, RT, 24h; (b) HBTU, DIPEA,
DCM, RT, 12h.
Scheme 2 Synthesis of F-labeled probes
Table 2 Structure of F-labeled NBC probes and their binding affinity
NO2
O
N
R2
R3
R1
Synthetic
Target
Binding
(RU)
KD
R1
R2
R3
(nM)
484.6
1.03
intermediates compounds
3-23
3-24
3-24
3-23
3-25
3-26
5-40
5-41
5-42
5-43
5-44
5-45
F
F
F
F
H
F
Ph-
CN
F
420.2
570.8
19.8
furan-2-yl
furan-2-yl
Ph-
CN
F
N.D.
10.90
47.34
N.D.
539.6
594.8
42.5
Ph-
F
2-MeO-Ph-
CN
10

3-26
3-27
5-46
5-47
F
2-MeO-Ph-
furan-2-yl
F
F
449.2
34.0
331.7
N.D.
H
RU
4
2
0
-2
-4
-6
-40
-20
0
20
40
60
80
Tim e
s
Figure 4. Kinetic binding curve of 5-41 by SPR analysis
4. Results and discussion
We designed and synthesized 39 DBC derivatives and evaluated their
bioactivities using our established SPR system. Their binding against the α-syn
aggregates loaded on the SPR chip was firstly measured, and the KD values were
further determined by SPR kinetic analysis for nine compounds with RU over 200
(Table 1). The KD values were in a range of from 10-3 μM to 10-1 μM. The best
compound 5-14 showed a high affinity with a KD value of 8.21 nM. In addition, some
compounds with an ortho-nitro group in ring A, and/or a cyano group in ring C
(Figure 3) showed high binding ability, indicating that H-bond acceptor substituents
might be a key factor for keeping binding activity.
We following optimized the lowest energy conformation for the hits using the
software Gaussian g09w, and to calculate the dihedral angles between the three rings
and the distances between the ring center and the amide N atom by Cartes V1.3.5. In
most of high-affinity DBC compounds, the distances between the center of the rings
A and B and the amide N atom are around 3.8 Å (except compound 5-1), and the
distances between the center of the ring C and the amide N atom are uniformed to
11

approximately 6.0 Å. The regularity of the dihedral angle between the rings was not
clear.
We finally selected the best molecules of 5-1, 5-7, 5-14 and 5-16 as the lead
compounds to optimize the potential probes. Eight F-labeled analogs were
synthesized and their binding considerably increased only by the introduction of F
atoms (Table 2). The KD values were further measured for five high binding
compounds (RU over 400). The compound 5-41 got the best binding activity of 1.03
nM (KD) from the kinetic analysis (Figure 4).
5. Conclusion
Here we initially established a SPR biological evaluation system for discovery of
α-syn PET probes. The assay is convenient and practicable with advantages such as
low protein usage, good reproducibility, high sensitivity, and high throughput. It also
can simultaneously load other proteins like amyloid-beta (Aβ) and Tau protein in
different channels on the same chip, which is valuable for the evaluation of selectivity.
In one word, the assay can satisfy the evaluation of large quantities of samples for in
vitro binding potency and selectivity.
Based on the similarity of the aggregation structure of the amyloid family, we
investigated the crystal structure of the reported Aβ aggregates and the binding action
of the fluorescent dye ThT against Aβ aggregates. In Aβ aggregation, the coplanar
hydrophobic groove constituted of multiple Phe20 and Val18 is considered as the
binding site of ThT, which led us infer possible binding sites for the ligand in the
α-syn aggregate. The hydrogen bond formed by Tyr39-His50 in α-syn is the central
axis, which forms a twisted double hydrophobic groove with the Val on both sides.
Based on this results and the SAR of curcumin analogs, we designed and synthesized
39 DBC derivatives. By utilization of the established SPR assay, we identified their
binding affinity against α-syn aggregates and found a hit compound with good
potency (KD = 8.21 nM), which well met the binding requirement for PET imaging.
12

The structural characteristics and the corresponding SAR on the novel DBC
compounds were also analyzed. Based on obtained results, we further designed and
synthesized eight F-labeled DBC analogs. Among them, a more active compound
5-41 (KD = 1.03 nM) was found out successfully after the modification. In summary,
our study here established an efficient SPR methodology for high-throughput
evaluation of small molecules against a-syn fibrils and provided a class of novel DBC
compounds as high-affinity binders against a-syn in nM level (KD). They were further
identified to still remain excellent affinity after the introduction of F atom into the
DBC scaffold, which suggested the potential of developing to be an F-labeled probe
for PET imaging of α-syn aggregates.
4. Experimental section
4.1 Biology
Preparation of α-syn monomer: Human wild-type α-syn was expressed in BL21
(DE3) and purified by Bio-Rad NGC medium-pressure protein purification system
and confirmed by SDS-PAGE. The concentration of protein was calculated by the
BCA method through a standard curve. The BCA kit was a P0012 type of protein
concentration determination kit produced by Biyuntian Biotechnology Co., Ltd..
Preparation of α-syn fibrils: A free α-syn (10 μM) in a Tris-HCl buffer (30 mM,
pH=7.4) containing 5% hexafluoroisopropanol (HFIP) and NaCl (150 mM) was
incubated for 36 h at 37 °C in 180 rpm. After 20 μL of incubation solution was added
to 200 μL of 20 μM ThT solution, the mixture was placed in a cuvette and was
detected for the fluorescence (excitation at 440 nm/emission at 485 nm) with Spectra
Max M5 microplate reader.
Protein fixation on-chip: All experiments were performed on the GE Biacore T200
surface plasmon resonance protein analyzer using the Xantec CMD 500M chip.
Firstly, pre-enrichment experiments were carried out to find out the best NaAc buffer.
After carboxyl groups on the surface of the chip were activated by
13

1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide
(NHS) buffer, the protein sample was injected into the analyzer to couple on the
surface of the chip and the chemically non-bonded protein was washed out by the
mobile phase. This process was repeated several times until the amount of coupling
protein reached the expected level. Unreacted carboxyl groups on the surface of the
chip were blocked with ethanolamine hydrochloride solution.
Screen of small molecules by SPR: The tested compounds were accurately weighed
and dissolved in DMSO to be a 10 mM preservation solution. The mobile phase
buffer of SPR system was applied to make a plurality of concentration gradients for
the testing solution. Different concentrations of the compound were injected to the
SPR system for 1 min at a flow rate of 60 µL/min, followed with regeneration by
HBS buffer for 3 min. According to the kinetic binding curves under different
concentration gradients, the KD values were obtained by instrument fitting.
4.2 Chemistry synthesis
All solvents and reagents were purchased from commercial sources and used directly
without purification. The used TLC silica gel plate was manufactured by Huangwu
Silica Gel Development Test Factory in Zhifu District, Yantai City. The flash column
chromatography instrument was a CombiFlash Rf150 type manufactured by Teledyne
Tech. Inc., and the silica gel column used was a SepaFlash type of rapid separation
column manufactured by Santai Tech. Inc. 1H and 13C NMR spectra were recorded on
Bruker AMX-400 spectrometer (Bruker Bioscience, Billerica, MA, USA) with TMS
as an internal standard. The chemical shift (δ) was in ppm. The coupling constant was
in Hz, and the proton coupling expression was abbreviated as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m). Mass spectra were determined by an Agilent
1100s LC-MSD ESI model mass spectrometer, and HRMS were determined by an
AB 5600+ Q TOF mass spectrometer.
14

The representative synthesis was listed below and more compounds and their data in
detail can be available in Supporting Information.
General procedure for preparation of the DBC derivatives
To a solution of 1,2-dichloroethane was added 3 mmol of different amines (including
substituted aromatic amines and aliphatic amines), substituted benzaldehydes (3
mmol), and glacial acetic acid (540 mg, 9 mmol). The mixture was stirred at room
temperature overnight under nitrogen atmosphere. Then 1.91 g (9 mmol) of sodium
triacetoxyborohydride was added in portions under an ice bath and the mixture was
kept stirring at room temperature overnight under nitrogen atmosphere. After the
reaction was completed monitored by TLC, the mixture was diluted with methylene
chloride. The organic phase was washed successively with 1M aqueous hydrochloric
acid, a solution of sodium hydrogen carbonate and brine, dried over anhydrous
sodium sulfate. After removal of the drying agent by suction filtration, the organic
phase was concentrated under reduced pressure to afford intermediates 3, which were
directly used in the next reaction without further purification.
N-(2-methoxyphenyl)-N-(2-nitrobenzyl) cinnamamide (5-1)
A mixture of cinnamate (74 mg, 0.5 mmol), intermediate 3-1 (94 mg, 0.75 mmol),
HBTU (379 mg, 1 mmol) and DIPEA (162 mg, 1.25 mmol) in 10 mL of
dichloromethane was stirred at room temperature for 12 hours under nitrogen
atmosphere. After the reaction was completed, the mixture was diluted with
dichloromethane, and the organic phase was washed successively with 1M aqueous
hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and brine. The
organic phase was dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The residue was purified by flash column chromatography
1
(Petroleum ether: Ethyl ester = 5:1) to give 171 mg of white solid. Yield 88%; H
NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.1 Hz, 1H), 7.74 (dd, J = 21.6, 11.7 Hz, 2H),
7.35 (t, J = 7.6 Hz, 1H), 7.40-7.26 (m, 7H), 7.05-6.89 (m, 3H), 6.33 (d, J = 15.5 Hz,
1H), 5.60 (d, J = 16.3 Hz, 1H), 5.10 (d, J = 16.3 Hz, 1H), 3.78 (s, 3H); ESI-MS (+)
15

(m/z): 389.1 (M+1)+.
(E)-N-Benzyl-N-3-(4-aminophenyl)-(2-nitrobenzyl) acrylamide（5-2）
Intermediate 3-22 (244 mg, 0.5 mmol) was dissolved in 5 mL of dichloromethane,
then 2 mL of trifluoroacetic acid was added, and the mixture was stirred at room
temperature for 3 hours. After the reaction was completed, the pH was adjusted to 8
by adding 25% concentrated aqueous ammonia dropwise in an ice bath. The organic
layer was extracted with dichloromethane, and washed by brine. The organic phase
was dried over anhydrous sodium sulfate and concentrated under reduced pressure to
give 186 mg of yellow solid. Yield 96%. 1H NMR (400 MHz, CDCl3) δ8.16-8.02 (m,
1H), 7.79 (d, J = 12.0 Hz, 1H), 7.64-7.58 (m, 1H), 7.48-7.24 (m, 9H), 6.76-6.44 (m,
3H), 5.03 (d, J = 17.2 Hz, 2H), 4.70 (d, J = 8.6 Hz, 2H), 3.96 (br, 2H); ESI-MS (+)
(m/z): 388.0 (M+1)+.
(E)-4-(3-(Benzyl(2-nitrobenzyl)amino)-3-oxo-1-propenyl) benzoic acid tert-butyl
ester (5-30）
Compound 5-3 (430 mg, 1 mmol) and lithium hydroxide (72 mg, 3 mmol) were added
to a mixed solvent of tetrahydrofuran (30 mL), methanol (10 mL) and water (10 mL).
After the completion of the reaction, the solvent was evaporated under reduced
pressure, and the residue was dissolved in water. A concentrated hydrochloric acid
was added dropwise to pH 2 under an ice bath. After filtration, the filter cake was
washed with water to obtain 395 mg (0.95 mmol) of white solid. Yield 95%. The
white solid was dissolved in 10 mL of dichloromethane, then added tert-butylamine
104 mg (1.4 mmol), HBTU 720 mg (1.9 mmol), DIPEA 369 mg (2.85 mmol) at room
temperature under nitrogen atmosphere. After stirring for 12 hours, the reaction
mixture was diluted with methylene chloride and washed sequentially with 1M
hydrochloric acid, saturated aqueous sodium hydrogen carbonate and brine. The
organic phase was dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The residue was purified by flash column chromatography
1
(Petroleum ether: ethyl acetate = 4:1) to give 359 mg of white solid. Yield 80%. H
16

NMR (400 MHz, CDCl3) δ 8.20-8.05 (m, 1H), 7.89-7.85 (m, 1H), 7.73-7.62 (m, 3H),
7.53-7.32 (m, 8H), 7.26-7.24 (m, 1H), 7.03-6.72 (m, 1H), 5.95 (s, 1H), 5.08 (d, J =
17.6 Hz, 2H), 4.75 (s, 2H), 1.64 (s, 1H), 1.49 (t, J = 6.4 Hz, 9H)；ESI-MS (+) (m/z):
472.0 (M+1)+.
Acknowledgments
We gratefully acknowledge the financial support by the National Natural Science
Foundation of China (Grant No. 81373275, 81771372, 81571232, 81971194), and by
the Shanghai Committee of Science and Technology, China (Grant No. 19431900600),
as well as by the Project (2016YFC1306500) from Ministry of Science and
Technology of China, Shanghai Municipal Science and Technology Major Project
(No.2018SHZDZX01) and ZJLab.
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Highlights
 We have synthesized a series of novel N, N-dibenzylcinnamamide (DBC)
compounds with a high affinity for α-syn aggregates.
 We initially established an efficient biological system based on SPR technology
suitable for large-scale evaluation of small molecules.
 The F-labeled DBC compound 5-41 was approved to be a new lead compound for
developing PET radiotracer for PD diagnosis.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.
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