Design, synthesis and in vitro evaluation of stilbene derivatives as novel LSD1 inhibitors for AML therapy

Article abstract of DOI:10.1016/j.bmc.2018.10.037

LSD1 is implicated in a number of malignancies and has emerged as an exciting target. As part of our sustained efforts to develop novel reversible LSD1 inhibitors for epigenetic therapy of cancers, in this study, we reported a series of stilbene derivatives and evaluated their LSD1 inhibitory activities, obtaining several compounds as potent LSD1 inhibitors with IC₅₀ values in submicromolar range. Enzyme kinetics studies and SPR assay suggested that compound 8c, the most active LSD1 inhibitor (IC₅₀ = 283 nM), potently inhibited LSD1 in a reversible and FAD competitive manner. Consistent with the kinetics data, molecular docking showed that compound 8c can be well docked into the FAD binding site of LSD1. Flow cytometry analysis showed that compound 8c was capable of up-regulating the expression of the surrogate cellular biomarker CD86 in THP-1 human leukemia cells, suggesting the ability to block LSD1 activity in cells. Compound 8c showed good inhibition against THP-1 and MOLM-13 cells with IC₅₀ values of 5.76 and 8.34 μM, respectively. Moreover, compound 8c significantly inhibited colony formation of THP-1 cells dose dependently.

Full text of DOI:10.1016/j.bmc.2018.10.037

Accepted Manuscript
Design, synthesis and in vitro evaluation of stilbene derivatives as novel LSD1
inhibitors for AML therapy
Yingchao Duan, Wenping Qin, Fengzhi Suo, Xiaoyu Zhai, Yuanyuan Guan,
Xiaojuan Wang, Yichao Zheng, Hongmin Liu
PII:
S0968-0896(18)31296-3
https://doi.org/10.1016/j.bmc.2018.10.037
BMC 14596
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
18 July 2018
17 October 2018
29 October 2018
Please cite this article as: Duan, Y., Qin, W., Suo, F., Zhai, X., Guan, Y., Wang, X., Zheng, Y., Liu, H., Design,
synthesis and in vitro evaluation of stilbene derivatives as novel LSD1 inhibitors for AML therapy, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.037
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Design, synthesis and in vitro evaluation of stilbene derivatives as
novel LSD1 inhibitors for AML therapy
Yingchao Duana, Wenping Qina, Fengzhi Suob, Xiaoyu Zhaia, Yuanyuan Guana,
Xiaojuan Wangc, Yichao Zhengb, *, Hongmin Liub,*
a School of Pharmacy, Xinxiang Medical University, Xinxiang, Henan 453003, China.
b Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China; Co-
innovation Center of Henan Province for New Drug R & D and Preclinical Safety; Institute of Drug
Discovery and Development; School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue
Avenue, Zhengzhou, Henan 450001, China.
c College of Chemistry and Chemical Engineering, Shangqiu Normal University, Shangqiu, Henan
476000, China.
* Corresponding authors
E-mail address: yichaozheng@zzu.edu.cn (Y.C. Zheng)
E-mail address: liuhm@zzu.edu.cn (H.M. Liu)
Abstract
LSD1 is implicated in a number of malignancies and has emerged as an exciting
target. As part of our sustained efforts to develop novel reversible LSD1 inhibitors for
epigenetic therapy of cancers, in this study, we reported a series of stilbene derivatives
and evaluated their LSD1 inhibitory activities, obtaining several compounds as potent
LSD1 inhibitors with IC50 values in submicromolar range. Enzyme kinetics studies and
SPR assay suggested that compound 8c, the most active LSD1 inhibitor (IC50 = 283nM),
potently inhibited LSD1 in a reversible and FAD competitive manner. Consistent with
the kinetics data, molecular docking showed that compound 8c can be well docked into
the FAD binding site of LSD1. Flow cytometry analysis showed that compound 8c was
capable of up-regulating the expression of the surrogate cellular biomarker CD86 in
THP-1 human leukemia cells, suggesting the ability to block LSD1 activity in cells.
Compound 8c showed good inhibition against THP-1 and MOLM-13 cells with IC50
values of 5.76 and 8.34 μM, respectively. Moreover, compound 8c significantly
inhibited colony formation of THP-1 cells dose dependently.
Key words: Lysine-specific demethylase 1; AML; stilbene; synthesis
1. Introduction
Lysine-specific histone demethylase 1 (LSD1, also known as KDM1A) was the
first histone demethylase identified by the group of Yang Shi in 2004. LSD1 utilizes
the flavin adenine dinucleotide (FAD) as a cofactor to specifically demethylate mono-
and di-methylated histones H3K4 and H3K9 and serves as a transcriptional repressor
or activator depending on the target cell context 1-3. LSD1 functions as an integral
subunit of several protein complexes, such as MLL transcription complex 4, 5, CoREST
3
6
and NuRD , and is involved in a number of cellular processes, including cell
proliferation, epithelial-mesenchymal transition (EMT), stem cell biology, and cell
differentiation 7, 8. Furthermore, LSD1 also demethylates several nonhistone substrates
including p53 9, E2F1 10, DNA methyltransferase1 (DNMT1) 11 and myosin light-chain
phosphatase 1 (MYPT1) 12, and further regulates their biological functions and stability.
Aberrant expression of LSD1 is believed to be responsible for acute myeloid leukemia
(AML) 13, 14, prostate cancer 2, 15, lung cancer 16, estrogen receptor (ER)-negative breast
17
18
19
cancer , colon cancer , and neuroblastoma . Moreover, its high expression is

closely correlated with poor prognosis in several malignancies, such as prostate cancer
20
21
15, breast cancer and non-small cell cancer . Notably, more and more evidences
support that LSD1 overexpression plays a crucial role in sustaining the self-renewal and
oncogenic transcriptional programmes of leukemic stem cells in multiple types of AML
14, 22. Knockdown of LSD1 or pharmacologic inactivation of LSD1 resulted in induction
of differentiation in both murine and primary human MLL-fusion AML cells and
inhibited the cell colony growth 14, 23-25. Co-treatment with LSD1 inhibitors and other
agents, including histone deacetylases (HDACs) inhibitor, NEDD8-activating enzyme
inhibitor, EZH2 inhibitor and all-trans-retinoic acid, showed promising results and
displayed synergistic activity against AML 26-29. Therefore, inhibition of LSD1 has
become an attractive therapeutic target in treating AML as well as other solid tumors
30, 31
.
To date, a variety of LSD1 inhibitors with several scaffolds, including
35
tranylcypromine(1, 2) 25, 32, benzohydrazide (3) 33, 34, thienopyrrole (4) , amidoxime
(5) 36, (4-cyanophenyl)glycine (6)37, pyridine (7) 38 and others 39-43, have been reported
by different research groups, representatively shown in Fig. 1. The most extensively
studied and potent LSD1 inhibitors are irreversible, and were mainly derived from
tranylcypromine (2-PCPA). Among them, ORY-1001 (1) 14, 44, 45, GSK2879552 (2) 46,
INCB059872 (undisclosed structure) 47 and IMG-7289 (undisclosed structure) 48, four
2-PCPA derivatives, developed by Oryzon Genomics, GSK, Incyte and Imago
BioSciences, respectively, are in phase I/II clinical trials for treatment of AML and
myelofibrosis. We have previously reported several novel LSD1 inhibitors, including
triazole-dithiocarbamate based compounds 49, pyrimidine-thiourea based compounds 41,
2-PCPA based compounds 50 and resveratrol derivatives either 51.
Fig. 1. Representative LSD1 inhibitors described in the literatures
Although several irreversible LSD1 inhibitors have recently entered into clinical
trials for the treatment of AML, the development of effective reversible inhibitors has
proved more challenging. Extensive interest in the LSD1 in the progression of AML
prompted us to identify novel reversible LSD1 inhibitors for AML therapy. Herein, a
series of stilbene derivatives designed based on the resveratrol-based LSD1 inhibitors,
51
reported by our group recently , were synthesized and evaluated as potent LSD1
inhibitors to treat AML. The results showed that these LSD1 inhibitors possessed
moderate to potent activity against AML and deserved further structural modification
to find more effective cancer therapeutics.
2. Results and discussion
2.1. Chemistry
Compounds 1-4 and 5-8 were synthesized according to the procedures outlined in
Schemes 1 and 2. Commercially available boronic acid compounds were reacted with
various substituted 2-bromopyridine or 2-bromopyrimidine following Suzuki coupling

reaction procedures, to give compounds 1 and 5. Compound 1 or 5 was treated with
different substituted diethyl benzylphosphonate in dry DMF, in the presence of t-BuOK,
to produce the stilbene derivatives 2a-l and 6a-g, respectively. Demethylation of 2a-e
or 6a-d with BBr3 in dry CH2Cl2 provided compounds 3a-e and 7a-d, respectively. The
reduction of 2f-l and 6e-g using stannous chloride in ethyl acetate or ethanolprovided
amines 3f-m and 7e-g, which were then reacted with BBr3 in dry CH2Cl2 to yield 4f-m
and 8e-g. Compounds 3a-c and 7a-c were treated with hydroxylamine in the presence
of Et3N to produce amidoxime compounds 4a-c and 8a-c. All of the compounds were
confirmed by 1H NMR, 13C NMR and HRMS (ESI) spectra.
Scheme 1. Synthesis of compounds 1-4. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, toluene,
EtOH, 95℃, 4-6 h; (b) substituted diethyl benzylphosphonate, t-BuOK, dry DMF, 0℃-rt, 0.5-3 h;
(c) BBr3, dry CH2Cl2, -35℃-rt, overnight; (d) NH2OH.HCl, Et3N, CH3OH, reflux, 6h; (e) SnCl2,
EtOAc or C2H5OH, reflux, 3-6 h; (f) BBr3, dry CH2Cl2, -35℃-rt, overnight.
Scheme 2. Synthesis of compounds 5-8. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, toluene,
EtOH, 95℃, 4-6h; (b) substituted diethyl benzylphosphonate, t-BuOK, dry DMF, 0℃-rt, 0.5-3h; (c)
BBr3, dry CH2Cl2, -35℃ -rt, overnight; (d) NH2OH.HCl, Et3N, CH3OH, reflux, 6h; (e) SnCl2,
C2H5OH, reflux, 3-6h; (f) BBr3, dry CH2Cl2, -35℃-rt, overnight.
2.2. Enzyme inhibition of target compounds against LSD1
All the target compounds synthesized in this study were investigated for their
inhibitory activities against LSD1 in a biochemical in vitro assay that was previously

51
described . ORY-1001 was used as a positive control. The data are summarized in
Table 1. Apparently, most of the tested compounds possessed a reasonable inhibitory
potency against LSD1 with the exception of compounds 3f, 3h, 4i and 8f. Among them,
compounds 4a-4h and 8a-e showed single-digit or submicromolar IC50 values, and
compound 8c was found most active against LSD1 with an IC50 value of 283 nM. These
results demonstrated that the target compounds were novel highly potent LSD1
inhibitors. Compounds 4f and 4h, with a amino group, retained anti-LSD1 activities
comparable to that of 4a and 4b, but hydroxy substitution (3d, 3e and 7d) resulted in a
dramatical decrease in activity, thus revealing that the amidoxime or amino moiety in
the benzene ring were preferable. Introducing different substituents in pyridine ring (4j-
4m) generally gave remarkable reduced activity compared with compounds 4f-4g,
showing substituents at this position is not favorable. The amino or amidoxime moiety
located at the meta position on the benzene ring resulted in the best activity: compounds
4f, 4h and 8a were more potent against LSD1 than the corresponding para-substituted
derivatives. Compounds bearing a methoxy group at the 2-position of the arylbenzene
moiety (3f and 3h) exhibited no activities against LSD1 compared with compounds 4f
and 4h, indicating the significance of the hydroxy group in retaining their activities.
Table 1 Inhibitory Activity (IC50 or percentage of inhibition at 10 μM) against LSD1
LSD1
Compounds Structures
IC50(μM)a
Inhibition % at 10μM
HO
OH
3d
4.24 ± 0.89
85.09%
N
HO
N
OH
3e
3f
N.T.b
20.18%
N.I.c
N
O
NH2
NH2
N.T.
N
O
N
3h
4a
4b
N.T.
N.I.
N
OH
HO
N
0.72 ± 0.12
1.29 ± 0.07
97.84%
87.50%
NH2
OH
N
HO
N
N
NH2
N
HO
N
4c
4f
0.92 ± 0.04
94.29%
98.37%
N
N
OH
NH2
HO
NH2
0.301 ± 0.03
N
HO
4g
4h
3.57 ± 0.31
94.32%
97.66%
N
NH2
NH2
HO
N
0.859 ± 0.07
N

HO
N
N
4i
4j
9.55 ± 1.01
N.T.
51.31%
35.43%
NH2
HO
N
O
O
O
NH2
HO
N
NH2
4k
4l
N.T.
43.89%
30.28%
89.30%
31.63%
97.69%
HO
N
F
NH2
N.T.
HO
HO
NH2
4m
7d
8a
1.47 ± 0.24
N.T.
N
N
OH
OH
OH
N
N
OH
0.364 ± 0.03
NH2
N
8b
0.764 ± 0.03
97.57%
N
OH
OH
NH2
OH
N
8c
8e
8f
0.283 ± 0.02
2.96 ± 0.74
9.03 ± 0.66
11.78 ± 1.09
99.23%
94.80%
54.14%
50.11%
F
OH
N
NH2
N
OH
NH2
N
F
OH
NH2
OH
N
8g
F
NH2
NH2
ORY-
1001
2HCl
11.26 ± 0.72 (nM) N.T.
N
H
a IC50 values are expressed as mean ± SD from at least three independent experiments.
b N.T.: Not tested.
c N.I.: No Inhibition.
The inhibitory effects of four selected compounds (4a, 4f, 8a and 8c) against
LSD1’s homologies MAO-A and MAO-B were also tested to assess their selectivity,
clorgyline and R-(-)-deprenyl were chosen as positive control for MAO-A and MAO-
B, respectively. As shown in Table 2, the selected four compounds demonstrated
excellent selectivity for LSD1 over both MAO-A and MAO-B (IC50 >50μM).
Table 2 In vitro Inhibition of LSD1, MAO-A and MAO-B of selected compounds
Compounds IC50(μM)

LSD1
MAO-A
>50
>50
MAO-B
>50
>50
>50
>50
a
Values are
of two
4a
0.72 ± 0.12
0.301 ± 0.03
0.364 ± 0.03
0.283 ± 0.02
bN.D.
the
mean
4f
experiments.
8a
8c
>50
b N.D.: Not
>50
detected
a 0.0031
N.D.
a0.071
Clorgyline
R(-)-deprenyl
N.D.
N.D.
2.3. Enzyme Kinetics
Studies
As shown in Table 1, compound 8c is the most potent LSD1 inhibitor we have
obtained. Furthermore, the reversibility of compound 8c against LSD1 was also
investigated using the dilution assay. As shown in Fig. 2A, 80-fold dilution of the
LSD1/compound 8c complex resulted in more than 80% recovery of LSD1 activity,
while the positive control GSK2879552 failed to recover the activity of LSD1. These
results indicated that compound 8c may interact with LSD1 in a reversible manner.
Additionally, with classic Lineweaver-Burk plots, compound 8c was characterized as a
cofactor (FAD) competitive inhibitor over LSD1 (Fig. 2B), which suggests that
compound 8c may penetrate into the cavity of LSD1 where FAD stands in order to
inactivate LSD1. Compound 8c was tested by the surface plasmon resonance (SPR),
where the binding response increased with the increasing concentration (Fig. 2C),
suggesting the tight binding between compound 8c and LSD1 (KD = 5.49×10-6M) is in
a manner of reversible model with fast association (Ka = 1.82×103 M-1·s-1) and slow
dissociation (Kd = 1.02×10-2 s-1). Consistent with the dilute assay, these results indicate
that compound 8c is a potent and reversible LSD1inhibitor.
Fig. 2. Inhibitory effect of compound 8c on LSD1 activity in vitro. (A) The reversibility of
compound 8c toward LSD1 activity was determined by the dilution assay, GSK2879552 was used
a control; (B) Lineweaver-Burk plots demonstrated that compound 8c was competitive with cofactor
FAD; (C) SPR based sensorgram of compound 8c. Bars represent means ± SD of three independent
experiments.
2.4. In vitro antiproliferative assay
The synthesized compounds 4a, 4f, 8a and 8c, with the most potent LSD-

inhibitory activities, were evaluated for antiproliferative activities against three human
acute monocytic leukemia cell lines MOLM-13, THP-1 and MV-4-11 in vitro, ORY-
1001 was used as a positive control. Data showed that all the four compounds showed
medium to good anti-proliferative activity with IC50 values ranging from 4.71 μM to
22.59 μM (Table 3). Compound 8c with the highest LSD1 enzyme inhibitory potency
exhibited the best antiproliferative activity against MOLM-13 and THP-1 with IC50
values of 5.76-8.34 μM. Compound 4a showed fairly good antiproliferative activity
against MV-4-11 with an IC50 value of 4.71 μM, which is more potent than compound
8c (IC50 of 7.49 μM). Overall, these antitumor activities suggested that this class of
LSD1 inhibitors could be potentially useful cancer therapeutics and further structural
modification is therefore warranted.
Table 3 In vitro antiproliferative activity of selected compounds in three cancer cell lines
IC50(μM)a
a
IC50 values are
Compounds
expressed as mean ± SD
from at least three
independent experiments.
b N.D.: Not detected
MOLM-13
THP-1
MV-4-11
4a
4f
8a
8c
22.59 ± 2.33
12.51 ± 1.06
9.05 ±0.99
8.34 ± 0.64
N.D.b
7.89 ± 0.71
10.51 ± 1.44
13.72 ± 1.08
5.76 ± 0.55
> 20 μM
4.71 ± 0.89
10.94 ± 2.03
15.85 ± 1.52
7.49 ± 1.17
N.D.
2.5. Cellular Activity of
Compound 8c
ORY-1001
To further evaluate the cellular activity of compound 8c, we evaluated whether LSD1
inhibitors may alter the colony-forming capacity of THP-1 cells with soft agar assay.
As indicated in Fig. 3A, treatment of compound 8c for 14 days significantly inhibited
colony formation of THP-1 cells does dependently. As B7-2 (CD86), one of type I
transmembrane proteins that was originally identified as a ligand for CD28/CTLA-4, is
52
a bona fide cellular biomarker of LSD1 activity , THP-1 cells were treated with
compound 8c at different concentrations to evaluate its effect on membrane expression
of CD86 with flow cytometry. As indicated in Fig. 3B, increased expression of CD86
was observed, suggesting that compound 8c may inactivate LSD1 in the cellular level.
To further determine whether target compounds are cell-active LSD1 inhibitors,
the effects of selected compound 8c on the methylation levels of LSD1 substrates
H3K4me1 and H3K4me2 were analyzed. After treatment of THP-1 cells for 5 days
with compound 8c at different concentrations (0, 1.25, 2.5 and 5.0 M), the amounts of
H3K4me1 and H3K4me2 were dose dependently elevated, which supported the target
engagement of compound 8c in THP-1 cells (Fig. 3C).
In addition, hematoxylin and eosin stain (HE stain) was applied to monitor the
cellular morphology. After 3 days incubation with compound 8c at indicated
concentrations, characteristic morphological changes were identified by microscope,
including blurred cell membrane boundaries and chromatin shrinkage with dose
dependently manner (Fig. 3D).

Fig. 3. Cellular activity of compound 8c in THP-1 cells. (A) Representative images of THP-1 cells
after treatment with DMSO and compound 8c for 14 days; (B) Flow cytometry analysis of the
expression of CD86 in THP-1 cells with indicated treatment for 3 days; (C) Western blot analysis
of the effect of histone methylation in THP-1 cells with indicated treatment for 5 days; (D) HE
staining of THP-1 cells treated as indication for 3 days. Bars represent means ± SD of three
independent experiments; **p < 0.01, compared to control.
2.6. Molecular docking
To explore the binding mode of compound 8c and LSD1 and explain the observed
biological activity of compound 8c, molecular docking study was performed using the
software MOE 2015.10. In this study, the crystal structure of LSD1 (PDB ID: 2V1D)
was selected as the docking receptor, which contains a free cofactor FAD, a substrate
of H3K4 mimetic peptide, and a corepressor of CoREST. Based on the structural
51
similarity of compound 8c with the compounds reported in the literatures36, , the
position occupied by the cofactor FAD was selected as the docking site.
As displayed in Fig. 4, compound 8c can well docked into the FAD-binding site
of LSD1. The amine of the amidoxime moiety formed a hydrogen bond with the
carbonyl group of the backbone of Thr624 and an electrostatic interaction with the O-
of Glu801, respectively, meanwhile its imine nitrogen atom formed a hydrogen bond
with the carbonyl group of the backbone of Glu801. Additionally, the phenyl group
connecting with the amidoxime formed arene-H interaction with Val288. The phenyl
group connecting with the pyridine ring was found to be situated in a hydrophobic
cavity surrounded by Gly330, Tyr761, Leu329, Trp751, Lys661, Leu659 and Trp810,
and formed arene-H interaction with Trp751. The hydrogen bonds, arene-H,
electrostatic and hydrophobic interactions made the compound 8c bind steadily to
LSD1.

Fig. 4. Docking results of compound 8c in the FAD-binding site of LSD1 (PDB ID: 2V1D). (A)
Interactions between compound 8c and the residues of LSD1 and FAD. Compound 8c and FAD are
shown as yellow and green sticks, respectively; the associated residues are shown as gray lines, and
hydrogen bonds are shown as gray dash lines. (B) Two-dimensional schematics of the protein-ligand
interaction of compound 8c to LSD1. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
3. Conclusions
In summary, a series of novel stilbene derivatives were synthesized and identified
as LSD1 inhibitors. Most of these compounds were evaluated as robust inhibitors
against LSD1 recombinant. Especially compound 8c, the most potent inhibitor,
performed excellent LSD1 inhibition activity (IC50 = 283 nM). Dilution assay and SPR
study indicated that compound 8c bound to LSD1 in a manner of reversible model with
fast association and slow dissociation. Enzyme kinetics study and molecular docking
suggested that compound 8c maybe a competitive inhibitor against the LSD1 cofactor
FAD. Treatment of THP-1 cells with compound 8c lead to the increasing of CD86 dose
dependently, showing target engagement activity in cells. Compound 8c demonstrated
good antiproliferative activities against THP-1 and MOLM-13 human leukemia cells
with IC50 values of 5.76 and 8.34μM, respectively. Furthermore, compound 8c was able
to inhibit the colony formation in THP-1 cells in a concentration-dependent manner.
Encouraged by the interesting biological data of this stilbene-based reversible LSD1
inhibitors, further structural optimization is going on.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
Reagents and solvents were obtained from commercial sources, when necessary,
were purified and dried by standard methods. Melting points were determined on an X-
5 micromelting apparatus and are uncorrected. All compounds were characterized by
13
1H NMR and C NMR spectra on a Bruker Avance III HD 400 MHz and 100 MHz
spectrometer at room temperature respectively, with chemical shifts in parts per million
(δ) downfield from TMS, the internal standard. Spin multiplicities were described as s
(singlet), d (doublet), dd (doublet of doublets), t (triplet), br (broad signal), or m
(multiplet). Coupling constants were reported in hertz (Hz). ESI-HRMS were recorded
on a Bruker MicrOTOF-Q III Micro mass spectrometer.
4.1.2. General procedure for synthesis of compounds 1 and 5.

To a 50 mL two-necked flask equipped with magnetic stirrer and condenser was
added 2-bromopyridine (1.0 eq), Pd(PPh3)4 (5mol%), K2CO3 solution (2.0 eq) and
toluene under N2 at room temperture. After reacted for 15 minutes, a solution of the
boronic acid (1.2 eq) in EtOH was then added. The reaction mixture was then heated to
95℃ and reacted for 4 hours. After cooling to room temperature, to the reaction
mixture aqueous NH4Cl was added and extracted three times with EtOAc. The organic
extracts were then combined, washed with brine, dried with MgSO4 and then
concentrated under reduced pressure. The crude product was then purified by silica gel
column chromatography (Petroleum ether/EtOAc) to give compounds 1 and 5.
4.1.2.1. 4-methoxy-3-(pyridin-2-yl)benzaldehyde (1a)
1
White solid, yield: 87.5%, Mp:52-54℃. H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H),
8.74-8.72 (m, 1H), 8.31 (d, 1H, J = 2.4 Hz), 7.96 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz),
7.78-7.87 (m, 1H), 7.75 (td, 1H, J1 = 1.6 Hz, J2 = 7.2 Hz), 7.25-7.28 (m, 1H), 7.13 (d,
1H, J = 8.4 Hz), 3.96 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 190.99, 161.71, 154.63,
149.58, 135.94, 134.21, 131.15, 130.02, 129.55, 125.03, 122.33, 111.59, 56.05. HRMS
(ESI) calcd for C13H12NO2 [M + H]+: 214.0863, Found: 214.0870.
4.1.2.2. 4-methoxy-3-(pyrimidin-2-yl)benzaldehyde (1b)
White solid, yield: 87.1%, Mp:80-82℃.1H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H),
8.88 (d, 2H, J = 4.8 Hz), 8.26 (d, 1H, J = 2.4 Hz), 8.00 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8
Hz), 7.29 (t, 1H, J = 4.8 Hz), 7.16 (d, 1H, J = 8.8 Hz), 3.96 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 190.61, 164.71, 162.41, 157.17, 134.47, 132.52, 129.64, 128.77, 119.23,
112.05, 56.44. HRMS (ESI) calcd for C12H10N2NaO2 [M + Na]+: 237.0634, Found:
237.0639.
4.1.2.3. 3-(4-fluoropyridin-2-yl)-4-methoxybenzaldehyde(1c)
White solid, yield 77.1%, Mp: 73-75℃. 1H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H),
8.70 (dd, 1H, J1= 5.6 Hz, J2 = 8.8 Hz), 8.39 (t, 1H, J = 2.0 Hz), 7.98 (dd, 1H, J1= 1.2
Hz, J2 = 8.8 Hz), 7.63 (d, 1H, J = 10.4 Hz), 7.16 (d, 1H, J = 8.4 Hz), 7.05-7.01 (m,
1H), 3.99 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 190.88, 168.57 (d, JC, F = 259.4 Hz),
161.69, 157.33 (d, JC, F = 8.2 Hz), 151.58 (d, JC, F = 7.4 Hz), 134.23, 131.67, 130.04,
128.12 (d, JC, F = 3.4 Hz), 112.96 (d, JC, F = 18.4 Hz), 111.74, 110.33 (d, JC, F = 16.6
Hz), 56.11. HRMS (ESI) calcd for C13H10FNNaO2 [M + Na]+: 254.0588, Found:
254.0582 .
4.1.2.4. methyl 2-(5-formyl-2-methoxyphenyl)isonicotinate (1d)
White solid, yield: 70.1%, Mp:117-118℃. 1H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H),
8.84 (d, 1H, J = 4.8 Hz), 8.38 (s, 1H), 8.33 (d, 1H, J = 2.4 Hz), 7.97 (dd, 1H, J1 = 2.4
Hz, J2 = 8.4 Hz), 7.80 (dd, 1H, J1 = 2.0 Hz, J2 = 4.8 Hz), 7.14 (d, 1H, J = 8.4 Hz), 3.98
(s, 6H). 13C NMR (101 MHz, CDCl3) δ 190.86, 165.84, 161.79, 155.77, 150.30, 137.39,
134.10, 131.69, 130.08, 128.78, 124.33, 121.41, 111.68, 56.18, 52.79. HRMS (ESI)
calcd for C15H13NNaO4 [M + Na]+: 294.0737, Found: 294.0741.
4.1.2.5. 4-methoxy-3-(5-methoxypyridin-2-yl)benzaldehyde (1e)
White solid, yield: 69.3%, Mp: 91-92℃. 1H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H),
8.43 (d, 1H, J = 2.8 Hz), 8.31 (d, 1H, J = 2.0 Hz), 7.93 (dd, 1H, J1 = 2.0 Hz, J2 = 8.8
Hz), 7.79 (d, 1H, J = 8.8 Hz), 7.29-7.25 (m,1H), 7.12 (d, 1H, J = 8.8 Hz), 3.97 (s, 3H),
3.93 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 191.12, 161.54, 154.73, 146.94, 137.17,
133.93, 130.57, 130.06, 129.29, 125.22, 120.36, 111.53, 56.03, 55.68. HRMS (ESI)
calcd for C15H12NNaO2 [M + Na]+: 266.0788, Found: 266.0790.
4.1.2.6. 2-(2-methoxyphenyl)isonicotinaldehyde(5a)
White solid, yield: 73.6%, Mp: 51-53℃. 1H NMR (400 MHz, CDCl3) δ 10.12 (s, 1H),
8.93 (d, 1H, J = 4.8 Hz), 8.27 (t, 1H, J1 = 1.2 Hz), 7.84 (dd, 1H, J1 = 2.0 Hz, J2 = 7.6
Hz), 7.61 (dd, 1H, J1 = 1.2 Hz, J2 = 4.8 Hz), 7.45-7.39 (m, 1H), 7.11 (td, 1H, J1 = 1.2

Hz, J2 = 7.6 Hz), 7.04 (dd, 1H, J1 = 0.8 Hz, J2 = 8.4 Hz), 3.90 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 192.15, 157.86, 157.07, 150.62, 141.48, 131.16, 130.75, 127.80, 124.57,
121.18, 119.51, 111.41, 55.64. HRMS (ESI) calcd for C13H12NO2 [M+H]+: 214.0863,
Found: 214.0866.
4.1.2.7. 2-(5-fluoro-2-methoxyphenyl)isonicotinaldehyde(5b)
Yellowish solid, yield: 83.4%, Mp: 72-74℃. 1H NMR (400 MHz, CDCl3) δ 10.13 (s,
1H), 8.93 (dd, 1H, J1 = 1.2 Hz, J2 = 4.8 Hz), 8.34 (t, 1H, J = 1.2 Hz), 7.67-7.63 (m,
2H), 7.13-7.08 (m, 1H), 6.97 (dd, 1H, J1 = 4.4 Hz, J2 = 8.8 Hz), 3.89 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 191.88, 156.90 (d, JC, F = 237.6 Hz), 156.47 (d, JC, F = 2.0 Hz),
153.37 (d, JC, F = 2.1 Hz), 150.68, 141.63, 128.84 (d, JC, F = 7.2 Hz), 124.31, 120.07,
117.58 (d, JC, F = 24.5 Hz), 116.73 (d, JC, F = 22.9 Hz), 112.61 (d, JC, F = 8.0 Hz), 56.24.
HRMS (ESI) calcd for C13H11FNO2 [M+H]+: 232.0768, Found: 232.0770.
4.1.3. General procedure for synthesis of compounds 2 and 6.
To a solution of t-BuOK (3.0 equiv) in dry DMF was added a substituted diethyl
benzylphosphonate (1.1 eq). After the mixture was stirred at 0℃ under nitrogen for 05.
h, compound 1 or 5 (1.0 eq) in dry DMF was added dropwise to the solution. The
reaction mixture was warmed to room temperature and stirred for 0.5-2 h. Ice-water
was added, and the precipitate was filtered to afford the crude product. Purification of
the crude product by recrystallization from ethyl acetate or by silica gel column
chromatography provided the target products 2 and 6.
4.1.3.1. (E)-3-(4-methoxy-3-(pyridin-2-yl)styryl)benzonitrile(2a)
Colorless oil, yield: 57.8%. 1H NMR (400 MHz, CDCl3) δ 8.74 (ddd, 1H, J1 = 0.8 Hz,
J2 =1.6 Hz, J3 = 4.8 Hz), 7.99 (d, 1H, J = 2.4 Hz), 7.85 (dt, 1H, J1 = 1.2 Hz, J2 =8.0
Hz), 7.75-7.67 (m, 3H), 7.53-7.48 (m, 2H), 7.43 (t, 1H, J = 8.0 Hz), 7.26-7.23 (m, 1H),
7.16 (d, 1H, J = 16.4 Hz),7.04-6.99 (m, 2H), 3.90 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 157.21, 155.56, 149.51, 138.95, 135.78, 130.61, 130.32, 130.30, 129.65, 129.44,
128.65, 125.22, 124.58, 122.00, 118.93, 112.84, 111.68, 55.80. HRMS (ESI) calcd for
C21H16N2NaO [M + Na]+: 335.1155, Found: 335.1151.
4.1.3.2. (E)-3-(4-methoxy-3-(pyrimidin-2-yl)styryl)benzonitrile (2b)
1
Colorless oil, yield: 58.6%. H NMR (400 MHz, CDCl3) δ 8.89 (d, 2H, J = 4.8 Hz),
7.93 (d, 1H, J = 2.4 Hz), 7.74 (t, 1H, J = 1.6 Hz), 7.69 (dt, 1H, J1 = 1.6 Hz, J2 = 8.0
Hz), 7.58 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 7.50 (dt, 1H, J1 = 1.6 Hz, J2 = 7.6 Hz),
7.45 (t, 1H, J = 7.6 Hz), 7.27 (t, 1H, J = 4.8 Hz), 7.15 (d, 1H, J = 16.4 Hz), 7.06 (d, 1H,
13
J = 8.8 Hz), 7.02 (d, 1H, J = 16.4 Hz), 3.92 (s, 3H). C NMR (101 MHz, CDCl3) δ
165.54, 157.86, 157.09, 138.84, 130.38, 130.36, 130.29, 130.06, 129.71, 129.67,
129.44, 129.14, 128.64, 124.73, 118.92, 118.88, 112.87, 112.22, 56.26. HRMS (ESI)
calcd for C20H15N3NaO [M + Na]+: 336.1107, Found: 336.1100.
4.1.3.3. (E)-4-(4-methoxy-3-(pyrimidin-2-yl)styryl)benzonitrile (2c)
White solid, yield: 60.3%, Mp:142-144℃. 1H NMR (400 MHz, CDCl3) δ 8.91 (d, 2H,
J = 4.8 Hz), 7.94 (t, 1H, J = 2.0 Hz), 7.63-7.53 (m, 5H), 7.29-7.18 (m, 2H), 7.08-7.00
(m, 2H), 3.94 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.52, 158.04, 157.11, 142.13,
132.49, 131.50, 130.25, 129.85, 129.09, 128.70, 126.63, 125.27, 119.15, 118.94,
112.25, 110.17, 56.26. HRMS (ESI) calcd for C20H15N3NaO [M + Na]+: 336.1107,
Found: 336.1108.
4.1.3.4. (E)-2-(2-methoxy-5-(3-methoxystyryl)phenyl)pyridine(2d)
Colorless oil, yield: 54.2%. 1H NMR (400 MHz, CDCl3) δ 8.75-8.72 (m, 1H), 7.96 (d,
1H, J = 2.4 Hz), 7.83 (dt, 1H, J1 = 1.2 Hz, J2 = 8.0 Hz), 7.72 (td, 1H, J1 = 2.0 Hz, J2 =
8.0 Hz), 7.51 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz), 7.28-7.21 (m, 2H), 7.13 (d, 1H, J =
16.4 Hz), 7.08-6.98 (m, 4H), 6.79 (dd, 1H, J1 = 2.4 Hz, J2 = 8.0 Hz), 3.88 (s, 3H), 3.84

(s, 3H) .13C NMR (101 MHz, CDCl3) δ 159.88, 156.68, 155.85, 149.49, 139.15, 135.72,
130.35, 129.61, 129.30, 128.34, 128.17, 127.00, 125.21, 121.88, 119.06, 113.05,
111.65, 111.42, 55.79, 55.24. HRMS (ESI) calcd for C21H19NNaO2 [M +
Na]+:340.1308, Found :340.1314.
4.1.3.5. (E)-2-(2-methoxy-5-(3-methoxystyryl)phenyl)pyrimidine(2e)
1
Colorless oil, yield: 83.8%. H NMR (400 MHz, CDCl3) δ 8.89 (d, 2H, J = 4.8 Hz),
7.91 (d, 1H, J = 2.4 Hz), 7.57 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 7.28-7.23 (m, 2H),
7.11 (d, 1H, J = 16.4 Hz), 7.08 (d, 1H, J = 7.2 Hz), 7.05-7.00 (m, 3H), 6.80 (dd, 1H, J1
13
= 2.4 Hz, J2 = 8.4 Hz), 3.90 (s, 3H), 3.84 (s, 3H). C NMR (101 MHz, CDCl3) δ
165.74, 159.88, 157.33, 157.07, 139.04, 130.06, 129.89, 129.62, 129.32, 128.47,
128.09, 127.17, 119.06, 118.82, 113.06, 112.17, 111.49, 56.23, 55.24. HRMS (ESI)
calcd for C20H18N2NaO2 [M + Na]+: 341.1260, Found: 341.1266.
4.1.3.6. (E)-2-(2-methoxy-5-(3-nitrostyryl)phenyl)pyridine (2f)
Yellow solid, yield: 67.5%, Mp:108-110℃.1H NMR (400 MHz, CDCl3) δ 8.76-8.73
(m, 1H), 8.34 (t, 1H, J = 2.0 Hz), 8.08-8.05 (m, 1H), 8.02 (d, 1H, J = 2.0 Hz), 7.86 (dt,
1H, J1 =1.2 Hz, J2 = 8.0 Hz), 7.78-7.71 (m, 2H), 7.54 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4
Hz), 7.50 (t, 1H, J = 8.0 Hz), 7.27-7.23 (m, 2H), 7.10 (d, 1H, J =16.4 Hz), 7.03 (d, 1H,
13
J = 8.4 Hz), 3.91 (s, 3H). C NMR (101 MHz, CDCl3) δ 157.29, 155.55, 149.53,
148.75, 139.54, 135.76, 132.02, 131.07, 129.52, 129.49, 129.46, 129.38, 128.72,
125.19, 124.50, 121.99, 121.63, 120.66, 111.71, 55.81. HRMS (ESI) calcd for
C20H17N2O3 [M + H]+: 333.1234, Found: 333.1242.
4.1.3.7. (E)-2-(2-methoxy-5-(4-nitrostyryl)phenyl)pyridine (2g)
Yellow solid, yield: 93.3%, Mp: 82-84℃. 1H NMR (400 MHz, CDCl3) δ 8.75-8.73 (m,
1H), 8.20 (d, 2H, J = 8.8 Hz), 8.03 (d, 1H, J = 2.0 Hz), 7.86 (dt, 1H, J1 = 1.2 Hz, J2 =
8.0 Hz), 7.74 (td, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 7.60 (d, 2H, J = 8.8 Hz), 7.55 (dd, 1H,
J1 = 2.4 Hz, J2 = 8.8 Hz), 7.31-7.24 (m, 2H), 7.11 (d, 1H, J = 16.4 Hz), 7.03 (d, 1H, J
= 8.4 Hz), 3.91 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 157.53, 155.42, 149.51, 146.43,
144.29, 135.79, 132.70, 129.75, 129.48, 129.28, 128.94, 126.55, 125.21, 124.66,
124.17, 122.05, 111.71, 55.81. HRMS (ESI) calcd for C20H17N2O3 [M + H]+: 333.1234,
Found: 333.1238.
4.1.3.8. (E)-2-(2-methoxy-5-(3-nitrostyryl)phenyl)pyrimidine (2h)
Yellow solid, yield: 52.2%, Mp:130-132℃. 1H NMR (400 MHz, CDCl3) δ 8.90 (d, 2H,
J = 4.8 Hz), 8.34 (t, 1H, J = 2.0 Hz), 8.08-8.05 (m, 1H), 7.96 (d, 1H, J = 2.4 Hz), 7.76
(dt, 1H, J1 = 1.6 Hz, J2 = 7.6 Hz), 7.60 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz), 7.50 (t, 1H,
J = 8.0 Hz), 7.27 (t, 1H, J = 4.8 Hz), 7.24 (d, 1H, J = 16.4 Hz), 7.10-7.05 (m, 2H), 3.93
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.55, 157.95, 157.11, 148.75, 139.43, 132.01,
130.82, 130.13, 129.80, 129.53, 129.07, 128.68, 124.65, 121.70, 120.69, 118.93,
112.26, 56.27. HRMS (ESI) calcd for C19H15N3NaO [M + Na]+: 356.1006, Found:
3
356.1004.
4.1.3.9. (E)-2-(2-methoxy-5-(4-nitrostyryl)phenyl)pyrimidine(2i)
Yellow solid, yield: 87.1%, Mp:174-176℃.1H NMR (400 MHz, CDCl3) δ 8.92 (d, 2H,
J = 4.8 Hz), 8.22 (d, 2H, J = 8.8 Hz), 7.98 (d, 1H, J = 2.0 Hz), 7.64-7.60 (m, 3H), 7.31-
13
7.28 (m, 2H), 7.10 (d, 1H, J = 16.8 Hz), 7.09 (d, 1H, J = 8.4 Hz), 3.95 (s, 3H). C
NMR (101 MHz, CDCl3) δ 165.47, 158.21, 157.13, 146.50, 144.18, 132.43, 130.38,
130.02, 128.98, 128.73, 126.59, 124.82, 124.18, 118.97, 112.26, 56.27. HRMS (ESI)
calcd for C19H16N3O3 [M + H]+: 334.1186, Found: 334.1188.
4.1.3.10. (E)-methyl 2-(2-methoxy-5-(4-nitrostyryl)phenyl)isonicotinate (2j)
Yellow solid, yield: 42.0%, Mp:193-195℃. 1H NMR (400 MHz, CDCl3) δ 8.88 (d, 1H,
J = 4.8 Hz), 8.42 (t, 1H, J = 1.2 Hz), 8.21 (d, 2H, J = 8.8 Hz), 8.04 (d, 1H, J = 2.0 Hz),
7.80 (dd, 1H, J1 = 1.2 Hz, J2 = 4.8 Hz), 7.62-7.56 (m, 3H), 7.31-7.27 (m, 1H), 7.11 (d,

1H, J = 16.4 Hz), 7.06 (d, 1H, J = 8.8 Hz), 3.99 (s, 3H), 3.94 (s, 3H). HRMS (ESI)
calcd for C22H18N2NaO5 [M + Na]+: 413.1108, Found: 413.1111.
4.1.3.11. (E)-5-methoxy-2-(2-methoxy-5-(3-nitrostyryl)phenyl)pyridine(2k)
Yellow solid, yield: 58.4%, Mp:142-144℃. 1H NMR (400 MHz, CDCl3) δ 8.44 (d, 1H,
J = 2.8 Hz), 8.33 (t, 1H, J = 2.0 Hz), 8.07-8.04 (m, 1H), 8.00 (d, 1H, J =2.4 Hz), 7.82
(dd, 1H, J1 = 0.8 Hz, J2 = 8.4 Hz), 7.76 (dt, 1H, J1 = 1.2 Hz, J2 = 8.0 Hz), 7.51 -7.47
(m, 2H), 7.27-7.22 (m, 2H), 7.09 (d, 1H, J = 16.4 Hz), 7.01 (d, 1H, J = 8.4 Hz), 3.92
(s, 3H), 3.90 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 157.12, 154.54, 148.74, 147.89,
139.58, 137.01, 132.01, 131.19, 129.50, 129.34, 129.19, 129.12, 128.14, 125.36,
124.36, 121.58, 120.64, 120.35, 111.66, 55.80, 55.68. HRMS (ESI) calcd for
C21H19N2O4 [M + H]+: 363.1339, Found: 363.1339.
4.1.3.12. (E)-4-fluoro-2-(2-methoxy-5-(3-nitrostyryl)phenyl)pyridine(2l)
Yellow solid, yield: 82.8％, Mp:97-98℃. 1H NMR (400 MHz, CDCl3) δ 8.71 (dd, 1H,
J1 = 5.6 Hz, J2 = 9.2 Hz), 8.36 (t, 1H, J = 2.0 Hz), 8.12 (d, 1H, J = 2.4 Hz), 8.10-8.06
(m, 1H), 7.78 (dt, 1H, J1 = 1.2 Hz, J2 = 8.0 Hz), 7.70 (dd, 1H, J1 = 2.8 Hz, J2 = 10.8
Hz), 7.57 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz), 7.52 (t, 1H, J = 8.0 Hz), 7.24 (d, 1H, J =
16.4 Hz), 7.09 (d, 1H, J = 16.4 Hz), 7.03 (d, 1H, J = 8.8 Hz), 7.01-6.98 (m, 1H), 3.93
(s, 3H). HRMS (ESI) calcd for C20H16FN2O3 [M + H]+: 351.1139, Found: 351.1142.
4.1.3.13. (E)-3-(2-(2-(2-methoxyphenyl)pyridin-4-yl)vinyl)benzonitrile(6a)
White solid, yield: 88.3%, Mp: 90-92℃.1H NMR (400 MHz, CDCl3) δ 8.71 (dd, 1H, J1
= 0.8 Hz, J2 = 5.2 Hz), 7.87 (t, 1H, J = 1.2 Hz), 7.82 (t, 1H, J = 2.0 Hz), 7.77 (d, 1H, J
= 1.6 Hz), 7.75 (d, 1H, J = 1.6 Hz), 7.59 (dt, 1H, J1 = 1.6 Hz, J2 = 8.0 Hz), 7.49 (t, 1H,
J = 8.0 Hz), 7.42-7.37 (m, 1H), 7.32 (dd, 1H, J1 = 1.6 Hz, J2 = 5.2 Hz), 7.27 (d, 1H, J
= 16.4 Hz), 7.15-7.07 (m, 2H), 7.03 (dd, 1H, J1 = 1.2 Hz, J2 = 8.0 Hz), 3.89 (s, 3H).
13C NMR (101 MHz, CDCl3)δ 156.93, 156.90, 149.81, 143.42, 137.66, 131.62, 131.18,
131.02, 130.33, 130.29, 130.18, 129.68, 129.34, 128.81, 122.90, 121.10, 118.78,
118.56, 113.17, 111.44, 55.74. HRMS (ESI) calcd for C21H17N2O [M + H]+: 313.1335,
Found: 313.1337.
4.1.3.14. (E)-4-(2-(2-(2-methoxyphenyl)pyridin-4-yl)vinyl)benzonitrile(6b)
White solid, yield: 59.2%, Mp:120-122℃.1H NMR (400 MHz, CDCl3) δ 8.70 (dd, 1H,
J1 = 0.8 Hz, J2 = 5.2 Hz), 7.88 (t, 1H, J = 0.8 Hz), 7.77 (dd, 1H, J1 = 1.6 Hz, J2 = 7.6
Hz), 7.67 (d, 2H, J = 8.4 Hz), 7.63 (d, 2H, J = 8.4 Hz), 7.42-7.37 (m, 1H), 7.33-7.27
(m, 2H), 7.18 (d, 1H, J = 16.4 Hz), 7.10 (td, 1H, J1 = 1.2 Hz, J2 = 7.6 Hz), 7.03 (dd,
13
1H, J1 = 1.2 Hz, J2 = 8.4 Hz), 3.89 (s, 3H). C NMR (101 MHz, CDCl3) δ 156.98,
156.91, 149.91, 143.24, 140.80, 132.60, 131.16, 130.71, 130.38, 130.17, 128.88,
127.39, 122.91, 121.11, 118.79, 111.61, 111.44, 55.75. HRMS (ESI) calcd for
C21H17N2O [M + H]+: 313.1335, Found: 313.1338.
4.1.3.15. (E)-3-(2-(2-(5-fluoro-2-methoxyphenyl)pyridin-4-yl)vinyl)benzonitrile (6c)
White solid, yield: 80.3%, Mp:185-187℃.1H NMR (400 MHz, CDCl3) δ 8.69 (d, 1H, J
= 5.2 Hz), 7.93 (t, 1H, J = 1.2 Hz), 7.83 (t, 1H, J = 1.6 Hz), 7.77 (dt, 1H, J1 = 1.6 Hz,
J2 = 8.0 Hz), 7.61-7.55 (m, 2H), 7.50 (t, 1H, J = 7.6 Hz), 7.34 (dd, 1H, J1 = 2.0 Hz, J2
= 5.2 Hz), 7.27 (d, 1H, J =16.4 Hz), 7.13 (d, 1H, J = 16.0 Hz), 7.11-7.05 (m, 1H), 6.96
(dd, 1H, J1 = 4.4 Hz, J2 = 8.8 Hz), 3.88 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 157.33
(d, JC, F = 237.5 Hz), 155.58 (d, JC, F = 1.9 Hz), 153.20 (d, JC, F = 2.1 Hz), 149.96, 143.56,
137.58, 131.69, 131.04, 130.44, 130.34, 130.04 (d, JC, F = 7.4 Hz), 129.70, 129.18,
122.77, 119.18, 118.55, 117.75 (d, JC, F = 24.1 Hz), 116.08 (d, JC, F = 22.8 Hz), 113.18,
112.68 (d, JC, F = 8.0 Hz), 56.40. HRMS (ESI) calcd for C21H16FN2O [M + H]+:
331.1241, Found: 331.1241.
4.1.3.16. (E)-2-(2-methoxyphenyl)-4-(3-methoxystyryl)pyridine (6d)
Colorless oil, yield: 82.5%.1H NMR (400 MHz, CDCl3) δ 8.65 (d, 1H, J = 5.2 Hz),

7.84 (t, 1H, J = 1.2 Hz), 7.75 (dd, 1H, J1 = 1.6 Hz, J2 = 7.6 Hz), 7.40-7.35 (m, 1H),
7.32-7.25 (m, 3H), 7.14 (dt, 1H, J1 = 1.2 Hz, J2 = 7.6 Hz), 7.11-7.04 (m, 3H), 7.06 (d,
1H, J =8.4 Hz), 6.87 (dd, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 3.87 (s, 3H), 3.85 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 159.96, 156.95, 156.78, 149.73, 144.29, 137.83, 132.70,
131.17, 129.98, 129.83, 129.23, 126.98, 122.69, 121.06, 119.72, 118.75, 114.24,
112.25, 111.44, 55.75, 55.33. HRMS (ESI) calcd for C21H19NNaO2 [M + Na]+ :
340.1308, Found: 340.1305.
4.1.3.17. (E)-2-(2-methoxyphenyl)-4-(4-nitrostyryl)pyridine (6e)
White solid, yield: 70.7%, Mp:116-118℃.1H NMR (400 MHz, CDCl3) δ 8.71 (d, 1H,
J = 5.2 Hz), 8.25 (d, 2H, J = 8.4 Hz), 7.90 (d, 1H, J = 1.6 Hz), 7.78 (dd, 1H, J1 = 2.0
Hz, J2 = 6.4 Hz), 7.69 (d, 2H, J = 8.4 Hz), 7.42-7.33 (m, 3H), 7.24 (d, 1H, J = 16.4
Hz), 7.10 (t, 1H, J = 7.2 Hz), 7.03 (d, 1H, J = 8.0 Hz), 3.90 (s, 3H).13C NMR (101
MHz, CDCl3) δ 157.05, 156.93, 149.96, 147.40, 143.13, 142.76, 131.23, 131.17,
130.26, 130.21, 128.84, 127.49, 124.22, 122.99, 121.13, 118.83, 111.45, 55.76.
HRMS (ESI) calcd for C20H17N2O3 [M + H] +: 333.1234, Found: 333.1233.
4.1.3.18. (E)-2-(5-fluoro-2-methoxyphenyl)-4-(4-nitrostyryl)pyridine (6f)
Yellowish solid, yield: 63.1%, Mp:140-142℃.1H NMR (400 MHz,CDCl3) δ 8.71 (d,
1H, J = 5.2 Hz), 8.26 (d, 2H, J = 8.8 Hz), 7.96 (d, 1H, J = 1.6 Hz), 7.70 (d, 2H, J = 8.8
Hz), 7.58 (dd, 1H, J1 = 3.2 Hz, J2 = 9.6 Hz), 7.38-7.34 (m, 2H), 7.24 (d, 1H, J = 16.4
Hz), 7.11-7.06 (m, 1H), 6.97 (dd, 1H, J1 = 4.4 Hz, J2 = 9.2 Hz), 3.88 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 157.36 (d, JC, F = 237.5 Hz), 155.70 (d, JC, F = 1.7 Hz), 153.22 (d,
JC, F = 2.1 Hz), 150.01, 147.46, 143.37, 142.65, 131.02, 130.49, 130.00 (d, JC, F = 7.2
Hz), 127.52, 124.23, 122.89, 119.27, 117.67 (d, JC, F = 24.3 Hz), 116.16 (d, JC, F = 22.8
Hz), 112.74 (d, JC, F = 8.0 Hz), 56.42. HRMS (ESI) calcd for C20H15FN2NaO3 [M +
Na]+: 373.0959, Found: 373.0959.
4.1.3.19. (E)-2-(5-fluoro-2-methoxyphenyl)-4-(3-nitrostyryl)pyridine (6g)
Yellow solid, yield: 52.8%. Mp:133-135℃. 1H NMR (400 MHz,CDCl3) δ 8.70 (d, 1H,
J = 5.2 Hz), 8.42 (t, 1H, J = 2.0 Hz), 8.18-8.15 (m, 1H), 7.96 (t, 1H, J = 1.2 Hz), 7.85
(dt, 1H, J1 = 1.2 Hz, J2 = 8.0 Hz), 7.60-7.55 (m, 2H), 7.39-7.33 (m, 2H),7.22 (d, 1H, J
= 16.4 Hz), 7.11-7.06 (m, 1H), 6.97 (dd, 1H, J1 = 4.4 Hz, J2 = 9.2 Hz), 3.89 (s, 3H).
HRMS (ESI) calcd for C20H16FN2O3 [M + H]+: 351.1139, Found: 351.1140.
4.1.4. General procedure for synthesis of compounds 3a-e and 7a-d.
To a solution of compounds 2a-e or 6a-d (1.0 equiv) in dry CH2Cl2 was added BBr3
(6.0 equiv) in dry CH2Cl2 dropwise at -35℃ under nitrogen. The resulting mixture was
allowed to warm to room temperature and stirred overnight. The solution was poured
into ice-water, and a precipitate was formed and collected by filtration to provide the
crude product, which was purified by flash column chromatography on silica gel.
4.1.4.1. (E)-3-(4-hydroxy-3-(pyridin-2-yl)styryl)benzonitrile(3a)
Yellow solid, yield: 56.5%, Mp:116-118℃. 1H NMR (400 MHz, DMSO-d6) δ 14.36 (s,
1H), 8.67-8.65 (m, 1H), 8.34 (d, 1H, J = 8.4 Hz), 8.30 (d, 1H, J = 2.0 Hz), 8.10 (td, 1H,
J1 = 2.0 Hz, J2 = 8.0 Hz), 8.04 (t, 1H, J = 2.0 Hz), 7.89 (dt, 1H, J1 = 1.2 Hz, J2 = 8.0
Hz), 7.70 (dt, 1H, J1 = 1.2 Hz, J2 = 7.6 Hz), 7.62-7.57 (m, 2H), 7.50-7.47 (m, 1H), 7.44
(d, 1H, J = 16.4 Hz), 7.28 (d, 1H, J = 16.4 Hz), 6.98 (d, 1H, J = 8.4 Hz).13C NMR
(101MHz, DMSO-d6) δ 159.92, 156.95, 146.79, 139.43, 139.17, 131.19, 131.12, 130.74,
130.42, 130.22, 129.76, 128.07, 126.25, 124.24, 123.03, 120.46, 119.43, 119.34,
118.92, 112.35. HRMS (ESI) calcd for C20H15N2O [M + H]+: 299.1179, Found:
299.1179.
4.1.4.2. (E)-3-(4-hydroxy-3-(pyrimidin-2-yl)styryl)benzonitrile (3b)

Yellow solid, yield: 50.6%, Mp: 141-143℃. 1H NMR (400 MHz, DMSO-d6) δ 13.42
(s, 1H), 9.02 (d, 2H, J = 5.2 Hz), 8.64 (d, 1H, J = 2.4 Hz), 8.11 (t, 1H, J = 1.6 Hz), 7.94
(dt, 1H, J1 = 1.6 Hz, J2 = 8.0 Hz), 7.75 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz), 7.68 (dt, 1H,
J1 = 1.2 Hz, J2 = 7.6 Hz), 7.59-7.54 (m, 2H), 7.51 (d, 1H, J = 16.4 Hz), 7.18 (d, 1H, J
= 16.4 Hz), 7.04 (d, 1H, J = 8.4 Hz). 13C NMR (101 MHz, DMSO-d6) δ 163.90, 160.66,
157.50, 139.26, 131.64, 131.28, 131.02, 130.80, 130.31, 129.98, 128.24, 128.03,
124.43, 120.15, 119.34, 118.90, 118.83, 112.29. HRMS (ESI) calcd for C19H13N3NaO
[M + Na]+: 322.0951, Found: 322.0952.
4.1.4.3. (E)-4-(4-hydroxy-3-(pyrimidin-2-yl)styryl)benzonitrile (3c)
Yellow solid, yield: 66.3%, Mp:199-200℃.1H NMR (400 MHz, DMSO-d6) δ 13.28 (s,
1H), 8.85 (d, 2H, J = 4.8 Hz), 8.69 (d, 1H, J = 2.0 Hz), 7.65-7.57 (m, 5H), 7.31-7.24
(m, 2H), 7.08 (d, 1H, J = 8.4 Hz), 7.07 (d, 1H, J = 16.4 Hz).13C NMR (101 MHz,
DMSO-d6) δ 164.79, 161.21, 156.24, 142.29, 132.49, 131.96, 131.53, 128.17, 127.69,
126.57, 124.54, 119.21, 118.78, 118.76, 118.69, 110.02. HRMS (ESI) calcd for
C19H13N3NaO [M + Na]+: 322.0951, Found: 322.0946.
4.1.4.4. (E)-4-(3-hydroxystyryl)-2-(pyridin-2-yl)phenol(3d)
Yellow solid, yield: 54.1%, Mp:168-169℃.1H NMR (400 MHz, DMSO-d6) δ 14.35 (s,
1H), 9.44 (s, 1H), 8.66-8.64 (m, 1H), 8.37 (d, 1H, J = 8.4 Hz), 8.26 (d, 1H, J = 2.0 Hz),
8.08 (td, 1H, J1 = 1.6 Hz, J2 = 8.4 Hz), 7.60 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4 Hz), 7.48
(dd, 1H, J1 = 5.2 Hz, J2 = 6.8 Hz), 7.20-7.15 (m, 3H), 7.02 (dt, 1H, J1 = 1.2 Hz, J2 =
8.0 Hz), 6.98 (t, 1H, J = 2.0 Hz), 6.96 (d, 1H, J = 8.4 Hz), 6.68 (dd, 1H, J1 = 1.6 Hz,
J2 = 8.0 Hz). 13C NMR (101 MHz, DMSO-d6) δ 159.45, 158.09, 157.12, 146.69, 139.30,
139.13, 130.08, 129.88, 128.61, 128.34, 126.67, 125.85, 122.93, 120.52, 119.26,
118.81, 117.65, 114.85, 113.17. HRMS (ESI) calcd for C19H16NO2 [M + H]+: 290.1176,
Found: 290.1176.
4.1.4.5. (E)-4-(3-hydroxystyryl)-2-(pyrimidin-2-yl)phenol (3e)
Yellow solid, yield: 62.7%, Mp: 217-219℃. 1H NMR (400 MHz, DMSO-d6) δ 13.35
(s, 1H), 9.41 (s, 1H), 9.01 (d, 2H, J = 4.8 Hz), 8.58 (d, 1H, J = 2.4 Hz), 7.74 (dd, 1H,
J1 = 2.4 Hz, J2 = 8.4 Hz), 7.57 (t, 1H, J = 4.8 Hz), 7.21(d, 1H, J = 16.4 Hz), 7.16 (t,
1H, J = 8.0 Hz), 7.07-6.98 (m, 4H), 6.68 (dd, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz). 13C NMR
(101 MHz, DMSO-d6) δ 164.03, 160.20, 158.06, 157.51, 139.09, 131.50, 130.03,
128.78, 128.16, 127.45, 126.95, 120.08, 118.76, 117.76, 114.93, 113.39. HRMS (ESI)
calcd for C18H14N2NaO2 [M + Na]+: 313.0947, Found : 313.0950.
4.1.4.6. (E)-3-(2-(2-(2-hydroxyphenyl)pyridin-4-yl)vinyl)benzonitrile (7a)
White solid, yield: 83.8%, Mp:178-179℃.1H NMR (400 MHz, CDCl3) δ 14.27 (s, 1H),
8.49 (d, 1H, J = 5.2 Hz), 7.95 (t, 1H, J = 1.2 Hz), 7.88-7.84 (m, 2H), 7.79 (dt, 1H, J1 =
1.6 Hz, J2 = 8.0 Hz), 7.62 (dt, 1H, J1 = 1.6 Hz, J2 = 8.0 Hz), 7.53 (t, 1H, J =7.6 Hz),
7.36-7.30 (m, 3H), 7.15 (d, 1H, J = 16.4 Hz), 7.04 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz),
6.96-6.92 (m, 1H).13C NMR (101 MHz, CDCl3) δ 160.15, 158.51, 146.32, 145.37,
137.23, 132.01, 131.72, 131.49, 131.12, 130.46, 129.80, 128.59, 126.07, 118.85,
118.74, 118.72, 118.45, 116.84, 113.31. HRMS (ESI) calcd for C20H15N2O [M + H]+:
299.1179, Found: 299.1180.
4.1.4.7. (E)-4-(2-(2-(2-hydroxyphenyl)pyridin-4-yl)vinyl)benzonitrile(7b)
White solid, yield: 67.1%, Mp:189-190℃. 1H NMR (400 MHz, DMSO-d6) δ 14.19 (s,
1H), 8.63 (d, 1H, J = 5.2 Hz), 8.43 (s, 1H), 8.13 (dd, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 7.92
(d, 2H, J = 8.4 Hz), 7.88-7.83 (m, 3H), 7.65 (dd, 1H, J1 = 1.2 Hz, J2 = 5.2 Hz), 7.57 (d,
1H, J = 16.8 Hz), 7.36-7.31 (m, 1H), 6.99-6.92 (m, 2H). 13C NMR (101 MHz, DMSO-
d6) δ 159.77, 157.88, 147.14, 146.37, 141.25, 133.29, 133.03, 131.97, 129.94, 128.26,

127.43, 120.10, 119.29, 119.27, 119.24, 118.43, 117.56, 111.20. HRMS (ESI) calcd for
C20H14N2NaO [M + Na]+: 321.0998, Found: 321.0999.
4.1.4.8. (E)-3-(2-(2-(5-fluoro-2-hydroxyphenyl)pyridin-4-yl)vinyl)benzonitrile(7c)
White solid, yield: 48.3%, Mp:184-185℃. 1H NMR (400 MHz, DMSO-d6) δ 13.99 (s,
1H), 8.63 (d, 1H, J = 5.2 Hz), 8.44 (s, 1H), 8.16 (t, 1H, J = 1.6 Hz), 8.02-7.97 (m, 2H),
7.86-7.81 (m, 2H), 7.64 (t, 1H, J = 8.0 Hz), 7.61 (dd, 1H, J1 = 1.2 Hz, J2 = 5.2 Hz),
7.52 (d, 1H, J = 16.8 Hz), 7.22-7.17 (m, 1H), 6.95 (dd, 1H, J1 = 5.2 Hz, J2 = 9.2 Hz).
13C NMR (101 MHz, DMSO-d6) δ 156.65 (d, JC, F = 2.8 Hz), 155.97 (d, JC, F = 1.1 Hz),
155.66 (d, JC, F = 231.5 Hz), 147.37, 146.70, 137.90, 132.82, 132.50, 132.24, 130.86,
130.67, 128.62, 120.84, 119.73 (d, JC, F = 7.4 Hz), 119.53 (d, JC, F = 9.9 Hz), 119.07,
118.70 (d, JC, F = 23 Hz), 117.56, 113.16 (d, JC, F = 24.3 Hz), 112.57. HRMS (ESI) calcd
for C20H13FN2NaO [M + Na]+: 339.0904, Found: 339.0903.
4.1.4.9. (E)-2-(4-(3-hydroxystyryl)pyridin-2-yl)phenol (7d)
Yellow solid, yield: 74.8%, Mp:169-170℃. 1H NMR (400 MHz, DMSO-d6) δ 14.36 (s,
1H), 9.60 (s, 1H), 8.57 (d, 1H, J = 5.2 Hz), 8.40 (s, 1H), 8.16 (dd, 1H, J1 = 1.6 Hz, J2
= 8.0 Hz), 7.70 (d, 1H, J = 16.4 Hz), 7.63 (dd, 1H, J1 = 1.2 Hz, J2 = 5.6 Hz), 7.35-7.23
(m, 3H), 7.13 (d, 1H, J = 7.6 Hz), 7.08 (t, 1H, J = 2.0 Hz), 6.98-6.92 (m, 2H), 6.79 (dd,
1H, J1 = 1.6 Hz, J2 = 8.0 Hz). 13C NMR (101 MHz, DMSO-d6) δ 159.88, 158.21, 157.79,
147.18, 146.84, 137.89, 135.06, 131.87, 130.34, 127.45, 126.03, 119.76, 119.25,
119.20, 118.67, 118.40, 117.21, 116.61, 114.19. HRMS (ESI) calcd for C19H15NNaO2
[M + Na]+: 312.0995, Found: 312.0998.
4.1.5. General procedure for synthesis of compounds 3f-m and 7e-g.
To a suspension of compounds 2f-l or 6e-g (1.0 equiv) in EtOH or EtOAc was added
.
SnCl2 2H2O (5.0 eq) and heated to reflux for 4-6 h under N2. The ethanol was
evaporated under vacuum. The residue was dissolved in ethyl acetate, neutralised with
NaHCO3 and extracted three times with EtOAc. The organic extracts were then
combined, washed with brine, dried with anhydrous Na2SO4 and then concentrated
under reduced pressure. The crude product was then purified by silica gel column
chromatography to give 3f-m and 7e-g. Notably, If compound 2l was reduced in ethanol,
the fluorine atom will be substituted by an ethoxyl group to give compound 3m, while
reduced in EtOAC, the fluorine atom will be retained to give compound 3l.
4.1.5.1. (E)-3-(4-methoxy-3-(pyridin-2-yl)styryl)aniline (3f)
Yellow solid, yield: 50.3%, Mp:133-135℃. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (dt,
1H, J1 =1.2 Hz, J2 = 4.8 Hz), 7.90 (d, 1H, J = 2.4 Hz), 7.84-7.82 (m, 2H), 7.62 (dd,
1H, J1 = 2.0 Hz, J2 = 8.4 Hz), 7.36-7.32 (m, 1H), 7.16 (d, 1H, J = 8.4 Hz), 7.10 (d, 1H,
J = 16.4 Hz), 7.00 (t, 1H, J =8.0 Hz ), 6.97 (d, 1H, J = 16.4 Hz), 6.77-6.74 (m, 2H),
6.49-6.45 (m, 1H), 5.06 (s, 2H), 3.85 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 156.73,
155.48, 149.81, 149.28, 138.17, 136.40, 130.31, 129.57, 129.09, 129.03, 128.52,
127.93, 127.24, 125.31, 122.58, 114.90, 113.92, 112.74, 112.02, 56.25. HRMS (ESI)
calcd for C20H18N2NaO2 [M + Na]+: 325.1311, Found: 325.1318.
4.1.5.2. (E)-4-(4-methoxy-3-(pyridin-2-yl)styryl)aniline (3g)
Yellow solid, yield: 42.3%, Mp:148-150℃.1H NMR (400 MHz, CDCl3) δ 8.76 (dt, 1H,
J1 =1.2 Hz, J2 = 4.8 Hz), 7.92 (d, 1H, J = 2.4 Hz), 7.85 (d, 1H, J = 8.0 Hz), 7.75 (td,
1H, J1 = 2.0 Hz, J2 = 7.6 Hz), 7.50 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz), 7.34 (d, 2H, J =
8.4Hz,), 7.26 (m, 1H), 7.03-6.99 (m, 2H), 6.95 (d, 1H, J = 16.4 Hz), 6.69 (d, 2H, J =
8.4 Hz). 13C NMR (101 MHz, CDCl3) δ 156.10, 155.95, 149.28, 145.82, 135.83, 131.13,
129.02, 128.81, 128.39, 127.69, 127.50, 127.21, 125.23, 124.45, 121.80, 115.24,
111.66, 55.81. HRMS (ESI) calcd for C20H19N2O [M + H]+: 303.1492, Found: 303.1494.
4.1.5.3. (E)-3-(4-methoxy-3-(pyrimidin-2-yl)styryl)aniline (3h)

Yellowish solid, yield: 49.4%, Mp: 160-162℃. 1H NMR (400 MHz, DMSO-d6) δ 8.91
(d, 2H, J = 4.8 Hz), 7.75 (d, 1H, J = 2.4 Hz), 7.66 (dd, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz),
7.47 (t, 1H, J = 4.8 Hz), 7.16 (d, 1H, J = 8.8 Hz), 7.08 (d, 1H, J = 16.4 Hz), 7.03-6.96
13
(m, 2H), 6.76-6.73 (m, 2H), 6.49-6.46 (m, 1H), 5.05 (s, 2H), 3.79 (s, 3H). C NMR
(101 MHz, DMSO-d6) δ 165.53, 157.67, 157.18, 149.28, 138.15, 129.95, 129.57,
129.34, 129.27, 129.23, 128.08, 126.98, 119.91, 114.89, 113.94, 112.95, 112.01, 56.27.
HRMS (ESI) calcd for C20H18N2NaO2 [M + Na]+: 326.1264, Found: 326.1266.
4.1.5.4. (E)-4-(4-methoxy-3-(pyrimidin-2-yl)styryl)aniline (3i)
Yellow solid, yield: 69.6%, Mp: 199-200℃. 1H NMR (400 MHz, DMSO-d6) δ 8.90 (d,
2H, J = 4.8 Hz), 7.68 (d, 1H, J = 2.0 Hz), 7.58 (dd, 1H, J1 = 2.0 Hz, J2 = 8.8 Hz), 7.46
(t, 1H, J = 4.8 Hz), 7.26 (d, 2H, J = 8.4 Hz), 7.13 (d, 1H, J = 8.8 Hz), 6.97 (d, 1H, J1
= 16.4 Hz), 6.90 (d, 1H, J1 = 16.4 Hz), 6.55 (d, 2H, J = 8.4 Hz), 5.26 (s, 2H), 3.77 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 165.65, 157.65, 156.54, 148.93, 130.77, 129.22,
128.71, 128.49, 127.95, 127.85, 125.44, 122.48, 119.86, 114.37, 112.97, 56.26. HRMS
(ESI) calcd for C20H18N2NaO2 [M + Na]+: 326.1264, Found: 326.1269.
4.1.5.5. (E)-ethyl 2-(5-(4-aminostyryl)-2-methoxyphenyl)isonicotinate (3j)
Yellow solid, yield: 40.3%, Mp:123-125℃.1H NMR (400 MHz, DMSO-d6) δ 8.88 (d,
1H, J = 4.8 Hz), 8.29 (t, 1H, J = 1.2 Hz), 7.88 (d, 1H, J = 2.4 Hz), 7.76 (dd, 1H, J1 =
1.6 Hz, J2 = 5.2 Hz), 7.58 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 7.26 (d, 2H, J = 8.4 Hz),
7.16 (d, 1H, J = 8.4 Hz), 6.96 (d, 1H, J = 16.4 Hz), 6.91 (d, 1H, J = 16.4 Hz), 6.54 (d,
2H, J = 8.4 Hz), 5.26 (s, 2H), 4.37 (q, 2H, J = 7.2 Hz), 3.85 (s, 3H), 1.33 (t, 3H, J =
13
7.2 Hz). C NMR (101 MHz, DMSO-d6) δ 165.29, 156.73, 156.12, 150.95, 148.97,
137.48, 131.31, 128.43, 128.38, 128.04, 127.88, 125.40, 123.89, 122.57, 121.13,
114.36, 112.91, 62.08, 56.35, 31.17, 14.49. HRMS (ESI) calcd for C22H20N2NaO3
[M+Na]+: 397.1523, Found: 397.1528.
4.1.5.6. (E)-3-(4-methoxy-3-(5-methoxypyridin-2-yl)styryl)aniline (3k)
Yellowish solid, yield: 64.2%, Mp: 110-112℃.1H NMR (400 MHz, DMSO-d6) δ 8.40
(d, 1H, J = 2.8 Hz), 7.90 (d, 1H, J = 2.4 Hz), 7.83 (d, 1H, J = 8.8 Hz), 7.57 (dd, 1H,
J1= 2.0 Hz, J2 = 8.4 Hz), 7.44 (dd, 1H, J1 = 2.8 Hz, J2 = 8.4 Hz), 7.13 (d, 1H, J = 8.4
Hz), 7.09 (d, 1H, J = 16.4 Hz), 6.98 (t, 1H, J = 8.0 Hz), 6.96 (d, 1H, J = 16.4 Hz), 6.77-
6.74 (m, 1H), 6.74 (d, 1H, J = 7.2 Hz), 6.49-6.46 (m, 1H), 5.05 (s, 2H), 3.88 (s, 3H) ,
3.85 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 156.53, 154.70, 149.29, 147.64, 138.19,
137.38, 130.25, 129.57, 128.78, 128.68, 127.88, 127.79, 127.36, 125.59, 120.75,
114.88, 113.89, 112.68, 112.02, 56.22, 56.07. HRMS (ESI) calcd for C21H20N2NaO2
[M + Na]+: 355.1417, Found: 355.1416.
4.1.5.7. (E)-3-(3-(4-fluoropyridin-2-yl)-4-methoxystyryl)aniline (3l)
White solid, yield: 69.4%, Mp:173-174℃. 1H NMR (400 MHz, DMSO-d6) δ 8.72 (dd,
1H, J1 =5.6 Hz, J2 = 9.2 Hz), 7.99 (d, 1H, J = 2.4 Hz), 7.74 (dd, 1H, J1 = 2.4 Hz, J2 =
11.2 Hz) , 7.66 (d, 1H, J1 = 2.4 Hz, J2 = 8.8 Hz), 7.33-7.28 (m, 1H), 7.19 (d, 1H, J =
8.8 Hz), 7.10 (d, 1H, J = 16.8 Hz), 7.03-6.96 (m, 2H), 6.77-6.74 (m, 2H), 6.49-6.46 (m,
1H), 5.06 (s, 2H), 3.88 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.28 (d, JC, F = 256
Hz), 158.39 (d, JC, F = 7.8 Hz), 156.83, 152.38 (d, JC, F = 7.4 Hz), 149.29, 138.12, 130.35,
129.58, 129.14, 129.02, 128.14, 127.64 (d, JC, F = 3.4 Hz), 127.08, 114.91, 113.97,
112.88, 112.63 (d, JC, F = 17.9 Hz), 112.06, 110.43 (d, JC, F = 16.4 Hz), 56.37. HRMS
(ESI) calcd for C20H17FN2NaO [M + Na]+: 343.1217, Found: 343.1218.
4.1.5.8. (E)-3-(3-(4-ethoxypyridin-2-yl)-4-methoxystyryl)aniline (3m)
Yellow solid, yield: 53.6%, Mp: 88-90℃.1H NMR (400 MHz, DMSO-d6) δ 8.48 (d,
1H, J = 5.6 Hz), 7.93 (d, 1H, J = 2.4 Hz), 7.61 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4 Hz) , 7.37
(d, 1H, J = 2.8 Hz), 7.15 (d, 1H, J = 8.8 Hz), 7.09 (d, 1H, J = 16.4 Hz), 7.00 (t, 1H, J
= 7.6 Hz), 6.97 (d, 1H, J = 16.4 Hz), 6.93 (dd, 1H, J1 = 2.8 Hz, J2 = 5.6 Hz), 6.77-6.74

(m, 2H), 6.49-6.46 (m, 1H), 5.06 (s, 2H), 4.15 (q, 2H, J = 6.8 Hz), 3.86 (s, 3H), 1.37 (t,
3H, J = 6.8 Hz). 13C NMR (101 MHz, DMSO-d6) δ 164.67, 156.81, 156.71, 150.95,
149.29, 138.17, 130.18, 129.57, 129.05, 128.94, 128.47, 127.89, 127.25, 114.88,
113.91, 112.74, 112.02, 111.83, 108.99, 63.80, 56.26, 14.79. HRMS (ESI) calcd for
C22H22N2NaO2 [M + Na] +: 369.1573, Found: 369.1575.
4.1.5.9. (E)-4-(2-(2-(2-methoxyphenyl)pyridin-4-yl)vinyl)aniline(7e)
Yellow solid, yield: 68.6%, Mp: 142-144℃. 1H NMR (400 MHz, CDCl3) δ 8.61 (d, 1H,
J = 5.2 Hz), 7.78 (s, 1H), 7.72 (dd, 1H, J1 = 1.6 Hz, J2 = 7.6 Hz), 7.40-7.35 (m, 3H),
7.27-7.25 (m, 1H), 7.23 (d, 1H, J = 16.0 Hz), 7.07 (t, 1H, J = 7.6 Hz), 7.01 (d, 1H, J =
13
8.4 Hz), 6.87 (d, 1H, J = 16.0 Hz), 6.67 (d, 2H, J = 8.4 Hz), 3.87 (s, 3H). C NMR
(101 MHz, CDCl3) δ 156.93, 156.56, 149.52, 147.13, 145.16, 132.90, 131.16, 129.85,
129.40, 128.45, 126.88, 122.69, 122.30, 121.00, 118.45, 115.13, 111.41, 55.75. HRMS
(ESI) calcd for C20H19N2O [M + H]+: 303.1492, Found: 303.1497.
4.1.5.10. (E)-4-(2-(2-(5-fluoro-2-methoxyphenyl)pyridin-4-yl)vinyl)aniline (7f)
Yellow solid, yield: 60.3%, Mp: 152-154℃.1H NMR (400 MHz, DMSO-d6) δ 8.54 (d,
1H, J = 5.2 Hz), 7.90 (s, 1H), 7.54 (dd, 1H, J1 = 3.2 Hz, J2 = 10.0 Hz), 7.45 (dd, 1H, J1
= 1.2 Hz, J2 = 5.2 Hz), 7.38-7.34 (m, 3H), 7.28-7.22 (m, 1H), 7.20-7.16 (m, 1H), 6.93
(d, 1H, J = 16.0 Hz), 6.58 (d, 2H, J = 8.8 Hz), 5.51 (s, 2H), 3.86 (s, 3H) . 13C NMR
(101 MHz, DMSO-d6) δ 156.82 (d, JC, F = 234.5 Hz), 154.72 (d, JC, F = 1.6 Hz), 153.69
(d, JC, F = 1.6 Hz), 150.29, 149.92, 145.87, 134.27, 130.38 (d, JC, F = 7.2 Hz), 129.06,
124.11, 122.02, 120.55, 118.96, 117.11 (d, JC, F = 24 Hz), 116.34 (d, JC, F = 22.7 Hz),
114.24, 113.99 (d, JC, F = 8.1 Hz), 56.76. HRMS (ESI) calcd for C20H17FN2NaO [M +
Na]+: 343.1217, Found: 343.1225.
4.1.5.11 (E)-3-(2-(2-(5-fluoro-2-methoxyphenyl)pyridin-4-yl)vinyl)aniline (7g)
Yellow solid, yield: 53.1%, Mp: 109-111℃.1H NMR (400 MHz, DMSO-d6) δ 8.61 (d,
1H, J = 5.2 Hz), 7.98 (s, 1H), 7.56-7.52 (m, 2H), 7.40 (d, 1H, J = 16.4 Hz), 7.26 (td,
1H, J1 = 3.2 Hz, J2 = 9.2 Hz), 7.19 (dd, 1H, J1 = 4.4 Hz, J2 = 9.2 Hz), 7.12 (d, 1H, J =
16.4 Hz), 7.07 (t, 1H, J = 8.0 Hz), 6.85-6.83 (m, 2H), 6.58-6.55 (m, 1H), 5.17 (s, 2H),
3.86 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 156.82 (d, JC, F = 234.6 Hz), 154.92 (d,
JC, F = 1.8 Hz), 153.71 (d, JC, F = 1.7 Hz), 150.10, 149.45, 145.02, 137.06, 134.45,
130.19 (d, JC, F = 7.3 Hz), 129.72, 125.58, 122.62, 119.55, 117.12 (d, JC, F = 24 Hz),
116.48 (d, JC, F = 22.7 Hz), 115.60, 115.15, 114.01 (d, JC, F = 8.1 Hz), 112.65, 56.77.
HRMS (ESI) calcd for C20H17FN2NaO [M + Na]+: 343.1217, Found: 343.1222.
4.1.6. General procedure for synthesis of compounds 4a-c and 8a-c.
To a solution of compounds 3a-c or 7a-c (1.0 eq) in methanol was added Et3N (3.0 eq)
and NH2OH.HCl (3.0 eq), and the reaction mixture was refluxed for 3-8 h. The reaction
mixture was concentrated under vacuum, the residue was diluted with water and
extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4,
concentrated in vacuo and purified by flash column chromatography on silica gel, to
afford the pure products 4a-c and 8a-c.
4.1.6.1. N'-hydroxy-3-((E)-4-hydroxy-3-(pyridin-2-yl)styryl)benzimidamide(4a)
White solid, yield: 57.9％, Mp: 195-197℃. 1H NMR (400 MHz, DMSO-d6) δ 14.34 (s,
1H), 9.65 (s, 1H), 8.66 (d, 1H, J = 4.4 Hz), 8.38 (d, 1H, J = 8.0 Hz), 8.29 (s, 1H), 8.08
(t, 1H, J = 7.2 Hz), 7.91 (s, 1H), 7.63-7.54 (m, 3H), 7.48 (dd, 1H, J1 = 5.2 Hz, J2 = 7.6
Hz), 7.38 (t, 1H, J = 7.6 Hz), 7.31 (d, 1H, J = 16.4 Hz), 7.26 (d, 1H, J = 16.4 Hz), 6.97
(d, 1H, J = 8.8 Hz), 5.87 (s, 2H).13C NMR (101 MHz, DMSO-d6) δ 159.55, 157.09,
151.35, 146.73, 139.14, 137.82, 134.29, 129.95, 128.96, 128.54, 127.16, 126.27,

125.90, 124.72, 123.51, 122.96, 120.54, 119.36, 118.87. HRMS (ESI) calcd for
C20H18N3O2 [M + H]+: 332.1394, Found: 332.1389.
4.1.6.2. N'-hydroxy-3-((E)-4-hydroxy-3-(pyrimidin-2-yl)styryl)benzimidamide (4b)
1
Yellowish solid, yield: 42.8%, Mp: 221-222℃ . H NMR (400 MHz, DMSO-d6) δ
13.36 (s, 1H), 9.65 (s, 1H), 9.02 (d, 2H, J = 4.8 Hz), 8.62 (d, 1H, J = 2.0 Hz), 7.92 (s,
1H), 7.76 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4 Hz), 7.62-7.56 (m, 3H), 7.40-7.35 (m, 2H), 7.16
(d, 1H, J = 16.4 Hz), 7.04 (d, 1H, J = 8.8 Hz), 5.88 (s, 2H).13C NMR (101 MHz, DMSO-
d6) δ 164.11, 160.31, 157.47, 151.28, 137.73, 134.25, 131.49, 128.93, 128.88, 128.77,
127.62, 127.28, 126.56, 124.78, 123.59, 120.03, 118.93, 118.81. HRMS (ESI) calcd for
C19H16N4NaO2 [M + Na]+: 355.1165, Found: 355.1169.
4.1.6.3. N'-hydroxy-4-((E)-4-hydroxy-3-(pyrimidin-2-yl)styryl)benzimidamide (4c)
Yellowish solid, yield: 44.3%, Mp: 237-238℃.1H NMR (400 MHz, DMSO-d6) δ 13.38
(s, 1H), 9.66 (s, 1H), 9.02 (d, 2H, J = 4.8 Hz), 8.62 (s, 1H), 7.75 (d, 1H, J = 8.0 Hz),
7.68 (d, 2H, J = 8.0 Hz), 7.62 (d, 2H, J = 8.0 Hz), 7.58 (t, 1H, J = 4.8 Hz), 7.37 (d, 1H,
J = 16.4 Hz), 7.15 (d, 1H, J = 16.4 Hz), 7.03 (d, 1H, J = 8.4 Hz), 5.82 (s, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 164.00, 160.32, 157.53, 151.05, 138.33, 132.40, 131.52,
128.89, 128.75, 127.61, 126.41, 126.17, 126.06, 120.11, 118.84, 118.81. HRMS (ESI)
calcd for C19H17N4O2 [M + H]+: 333.1346, Found: 333.1342.
4.1.6.4. N'-hydroxy-3-((E)-2-(2-(2-hydroxyphenyl)pyridin-4-yl)vinyl)benzimidamide
(8a)
White solid, yield: 69.8%, Mp: 185-187℃.1H NMR (400 MHz, DMSO-d6) δ 14.32 (s,
1H), 9.70 (s, 1H), 8.60 (d, 1H, J = 5.2 Hz), 8.42 (s, 1H), 8.17 (dd, 1H, J1 = 1.6 Hz, J2
= 8.0 Hz), 8.03 (t, 1H, J = 1.6 Hz), 7.82 (d, 1H, J = 16.8 Hz), 7.70-7.66 (m, 2H), 7.64
(dd, 1H, J1 = 1.6 Hz, J2 = 5.6 Hz), 7.46 (t, 1H, J = 7.6 Hz), 7.41 (d, 1H, J = 16.8 Hz),
7.35-7.31 (m, 1H), 6.98-6.92 (m, 2H), 5.92 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ
159.86, 157.83, 151.11, 147.07, 146.96, 136.44, 134.68, 134.51, 131.90, 129.20,
128.19, 127.46, 126.61, 126.40, 124.62, 119.84, 119.28, 119.23, 118.42, 117.20.
HRMS (ESI) calcd for C20H18N3O2 [M + H]+: 332.1394, Found: 332.1390.
4.1.6.5. N'-hydroxy-4-((E)-2-(2-(2-hydroxyphenyl)pyridin-4-yl)vinyl)benzimidamide
(8b)
1
Yellowish solid, yield: 65.6%, Mp: 205-206℃ . H NMR (400 MHz, DMSO-d6) δ
14.32 (s, 1H), 9.75 (s, 1H), 8.59 (d, 1H, J = 5.2 Hz), 8.41 (s, 1H), 8.16 (dd, 1H, J1 =
1.6 Hz, J2 = 8.0 Hz), 7.82-7.75 (m, 3H), 7.70 (d, 2H, J = 8.0 Hz), 7.63 (dd, 1H, J1 =
1.6 Hz, J2 = 5.6 Hz), 7.42 (d, 1H, J = 16.8 Hz), 7.36-7.31 (m, 1H), 6.99-6.93 (m, 2H),
5.88 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.85, 157.81, 150.87, 147.08, 146.95,
137.03, 134.28, 134.02, 131.90, 127.41, 126.64, 126.26, 119.81, 119.27, 119.23,
118.42, 117.20. HRMS (ESI) calcd for C20H17N3NaO2 [M + Na]+: 354.1213, Found:
354.1211.
4.1.6.6. 3-((E)-2-(2-(5-fluoro-2-hydroxyphenyl)pyridin-4-yl)vinyl)-N'-hydroxy
Benzimidamide (8c)
White solid, yield: 34.4%, Mp: 220-221℃.1H NMR (400 MHz, DMSO-d6) δ 14.13 (s,
1H), 9.70 (s, 1H), 8.62 (d, 1H, J = 5.6 Hz), 8.48 (s, 1H), 8.08-8.02 (m, 2H), 7.89 (d,
1H, J = 16.4 Hz), 7.70-7.64 (m, 3H), 7.46 (t, 1H, J = 7.6 Hz), 7.39 (d, 1H, J = 16.4 Hz),
7.21 (td, 1H, J1 = 2.8 Hz, J2 = 8.4 Hz), 6.95 (dd, 1H, J1 = 4.8 Hz, J2 = 8.8 Hz), 5.92 (s,
2H). 13C NMR (101 MHz, DMSO-d6) δ 156.66 (d, JC, F = 2.8 Hz), 156.06, 155.67 (d,
JC, F = 231.4 Hz), 154.51, 151.07, 147.25, 147.16, 136.40, 134.94, 134.52, 129.21,
128.16, 126.42, 124.61, 120.65, 119.71 (d, JC, F = 7.5 Hz), 119.50 (d, JC, F = 8.1 Hz),
118.65 (d, JC, F = 23.0 Hz), 117.38, 113.22 (d, JC, F = 24.4 Hz). HRMS (ESI) calcd for
C20H17FN3O2 [M + H]+: 350.1299, Found: 350.1306.

4.1.7. General procedure for synthesis of compounds 4f-n and 8e-g.
To a solution of compounds 3f-n or 7e-g (1.0 equiv) in dry CH2Cl2 was added BBr3
(6.0 equiv) in dry CH2Cl2 dropwise at -35℃ under nitrogen. The resulting solution was
slowly warmed to room temperature and stirred overnight. The reaction mixture was
added slowly to aqueous NaHCO3, and the precipitate was collected by filtration to
provide the crude product, which was purified by flash column chromatography on
silica gel.
4.1.7.1. (E)-4-(3-aminostyryl)-2-(pyridin-2-yl)phenol(4f)
Yellow solid, yield: 39.3%, Mp:133-135℃. 1H NMR (400 MHz, DMSO-d6) δ 14.31 (s,
1H), 8.65 (dd, 1H, J1 = 1.6 Hz, J2 = 5.2 Hz), 8.38 (d, 1H, J = 8.4 Hz), 8.23 (d, 1H, J =
2.4 Hz), 8.07 (td, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 7.58 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4 Hz),
7.47 (dd, 1H, J1 = 5.2 Hz, J2 = 7.6 Hz), 7.08 (s, 2H), 7.03 (t, 1H, J = 7.6 Hz), 6.94 (d,
1H, J = 8.4 Hz), 6.77-6.74 (m, 2H), 6.48 (dd, 1H, J1 = 2.4 Hz, J2 = 8.0 Hz), 5.12 (s,
2H). 13C NMR (101 MHz, DMSO-d6) δ 159.30, 157.15, 149.22, 146.70, 139.13, 138.40,
129.75, 129.59, 128.78, 127.50, 127.37, 125.70, 122.91, 120.56, 119.27, 118.80,
114.83, 113.85, 111.95. HRMS (ESI) calcd for C19H16N2NaO [M + Na]+: 311.1155,
Found: 311.1158.
4.1.7.2. (E)-4-(4-aminostyryl)-2-(pyridin-2-yl)phenol(4g)
Yellow solid, yield: 50.2%, Mp: 167-168℃.1H NMR (400 MHz, DMSO-d6) δ 14.16
(s, 1H), 8.65-8.63 (m, 1H), 8.34 (d, 1H, J = 8.4 Hz), 8.14 (d, 1H, J = 2.4 Hz), 8.06-
8.04 (m, 1H), 7.50 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4 Hz), 7.48-7.44 (m, 1H), 7.26 (d, 2H,
J = 8.4 Hz), 7.04 (d, 1H, J = 16.4 Hz), 6.93-6.88 (m, 2H), 6.57 (d, 2H, J = 8.4 Hz),
5.26 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.57, 157.25, 148.77, 146.71, 139.07,
129.56, 129.17, 127.65, 127.28, 125.67, 124.88, 123.06, 122.81, 120.45, 119.22,
118.70, 114.43. HRMS (ESI) calcd for C19H17N2O [M + H]+: 289.1335, Found:
289.1332.
4.1.7.3. (E)-4-(3-aminostyryl)-2-(pyrimidin-2-yl)phenol(4h)
Yellow solid, yield: 62.3%, Mp: 207-208℃.1H NMR (400 MHz, DMSO-d6) δ 13.32 (s,
1H), 9.01 (d, 2H, J = 4.8 Hz), 8.56 (d, 1H, J = 2.0 Hz), 7.72 (dd, 1H, J1 = 2.0 Hz, J2 =
8.8 Hz), 7.57 (t, 1H, J = 4.8 Hz), 7.12 (d, 1H, J = 16.4 Hz), 7.04-6.99 (m, 2H), 6.97 (d,
1H, J = 16.4 Hz), 6.79-6.77 (m, 2H), 6.50-6.47 (m, 1H), 5.06 (s, 2H). 13C NMR (101
MHz, DMSO-d6) δ 164.03, 160.06, 157.52, 149.27, 138.16, 131.42, 129.58, 128.92,
127.62, 127.29, 127.23, 120.06, 118.75, 114.83, 113.89, 112.12. HRMS (ESI) calcd for
C18H15N3NaO [M + Na]+: 312.1107, Found: 312.1108.
4.1.7.4. (E)-4-(4-aminostyryl)-2-(pyrimidin-2-yl)phenol(4i)
Yellow solid, yield: 58.8%, Mp:194-196℃.1H NMR (400 MHz, DMSO-d6) δ 13.22 (s,
1H), 9.00 (d, 2H, J = 4.8 Hz), 8.50 (d, 1H, J = 2.0 Hz),7.64 (dd, 1H, J1 = 2.4 Hz, J2 =
8.8 Hz), 7.56 (t, 1H, J = 4.8 Hz), 7.29 (d, 2H, J = 8.4 Hz), 6.99-6.94 (m, 3H), 6.56 (d,
2H, J = 8.4 Hz), 5.26 (s, 2H). HRMS (ESI) calcd for C18H15N3NaO [M + Na]+:
312.1107, Found: 312.1106.
4.1.7.5. (E)-ethyl 2-(5-(4-aminostyryl)-2-hydroxyphenyl)isonicotinate(4j)
Yellow solid, yield: 48.6%, Mp: 111-112℃.1H NMR (400 MHz, DMSO-d6) δ 13.06
(s, 1H), 8.85 (dd, 1H, J1 = 0.8 Hz, J2 = 5.2 Hz), 8.61 (t, 1H, J = 1.2 Hz), 8.10 (d, 1H, J
= 2.0 Hz), 7.84 (dd, 1H, J1 = 1.2 Hz, J2 = 5.2Hz), 7.58 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4
Hz), 7.27 (d, 2H, J = 8.4 Hz), 6.99-6.94 (m, 3H), 6.56 (d, 2H, J = 8.4 Hz), 5.26 (s, 2H),
4.43 (q, 2H, J = 7.2 Hz), 1.39 (t, 3H, J = 7.2 Hz). 13C NMR (101 MHz, DMSO-d6) δ
164.70, 157.73, 157.45, 148.52, 148.37, 139.06, 129.67, 128.64, 127.44, 127.07,
125.90, 125.32, 122.72, 120.90, 119.79, 119.71, 118.37, 114.08, 62.06, 14.23. HRMS
(ESI) calcd for C22H20N2NaO3 [M + Na]+: 383.1366, Found: 383.1370.

4.1.7.6. (E)-4-(3-aminostyryl)-2-(4-ethoxypyridin-2-yl)phenol (4k)
Yellow solid, yield: 65.4%, Mp: 103-105℃.1H NMR (400 MHz, DMSO-d6) δ 14.80
(s, 1H), 8.45 (d, 1H, J = 6.0 Hz), 8.23 (d, 1H, J = 2.0 Hz), 7.83 (d, 1H, J = 2.4 Hz),
7.57 (dd, 1H, J1 = 1.6 Hz, J2 = 8.4 Hz), 7.08-7.00 (m, 4H), 6.90 (d, 1H, J= 8.4 Hz),
6.77- 6.73 (m, 2H), 6.49-6.45 (m, 1H), 5.07 (s, 2H), 4.30 (q, 2H, J= 6.8 Hz), 1.42 (t,
13
3H, J = 6.8 Hz). C NMR (101 MHz, DMSO-d6) δ 166.73, 159.72, 158.97, 149.32,
147.97, 138.47, 129.56, 128.58, 127.59, 127.24, 125.92, 118.92, 118.82, 114.67,
113.73, 111.93, 110.03, 105.86, 64.53, 14.79. HRMS (ESI) calcd for C21H20N2NaO2
[M + Na]+: 355.1417, Found: 355.1421.
4.1.7.7. (E)-4-(3-aminostyryl)-2-(4-fluoropyridin-2-yl)phenol (4l)
Yellow solid, yield: 61.7%, Mp:173-174℃.1H NMR (400 MHz, DMSO-d6) δ 13.93 (s,
1H), 8.71 (dd, 1H, J1= 5.6 Hz, J2 = 8.8 Hz), 8.34 (dd, 1H, J1= 2.4 Hz, J2 = 11.6 Hz) ,
8.27 (d, 1H, J = 2.0 Hz), 7.59 (dd, 1H, J1 = 2.0 Hz, J2 = 8.4 Hz), 7.44-7.40 (m, 1H),
7.12 (d, 1H, J = 16.4 Hz), 7.10-7.00 (m, 2H), 6.96 (d, 1H, J = 8.8 Hz), 6.77-6.73 (m,
2H), 6.50-6.46 (m, 1H), 5.10 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.83 (d, JC,
= 258.4 Hz), 160.63 (d, JC, F = 8.4 Hz), 159.07, 150.27 (d, JC, F = 8.6 Hz), 149.31,
F
138.38, 130.53, 129.60, 129.00, 127.65, 127.26, 126.00, 118.88 (d, JC, F = 3.3 Hz),
118.83, 114.76, 113.86, 111.89, 111.05 (d, JC, F = 18.0 Hz), 108.19 (d, JC, F = 19.2 Hz).
HRMS (ESI) calcd for C19H15FNaN2O [M + Na]+: 329.1061, Found: 329.1065.
4.1.7.8. (E)-6-(5-(3-aminostyryl)-2-hydroxyphenyl)pyridin-3-ol (4m)
Yellow solid, yield: 63.9%, Mp: 233-234℃.1H NMR (400 MHz, DMSO-d6) δ 13.98
(s, 1H), 10.39 (s, 1H), 8.22 (d, 1H, J = 8.8 Hz), 8.19 (d, 1H, J = 3.2 Hz), 8.09 (d, 1H, J
= 2.0 Hz), 7.47 (td, 2H, J1 = 2.0 Hz, J2 = 8.0 Hz), 7.08-7.00 (m, 3H), 6.89 (d, 1H, J =
8.4 Hz), 6.76-6.71 (m, 2H), 6.47 (dd, 1H, J1 = 2.4 Hz, J2 = 8.0 Hz), 5.07 (s, 2H). HRMS
(ESI) calcd for C19H15N2O2 [M - H]-: 303.1139, Found: 303.1129.
4.1.7.9. (E)-2-(4-(4-aminostyryl)pyridin-2-yl)phenol (8e)
Yellow solid, yield: 46.4%, Mp:151-152℃.1H NMR (400 MHz, DMSO-d6) δ 14.52 (s,
1H), 8.49 (d, 1H, J = 5.2 Hz), 8.27 (s, 1H), 8.13 (dd, 1H, J1 = 1.2 Hz, J2 = 8.0 Hz), 7.60
(d, 1H, J = 16.4 Hz), 7.51 (dd, 1H, J1 = 1.2 Hz, J2 = 5.6 Hz), 7.39 (d, 2H, J = 8.8 Hz),
7.34-7.29 (m, 1H), 6.98 (d, 1H, J = 16.4 Hz), 6.97-6.90 (m, 2H), 6.61 (d, 2H, J = 8.4
Hz), 5.59 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.97, 157.56, 150.61, 148.26,
146.50, 135.77, 131.72, 129.24, 127.30, 123.97, 120.00, 119.29, 119.11, 119.05,
118.38, 116.23, 114.27. HRMS (ESI) calcd for C19H17N2O [M+H]+: 289.1335, Found :
289.1338.
4.1.7.10. (E)-2-(4-(4-aminostyryl)pyridin-2-yl)-4-fluorophenol(8f)
Yellowish solid, yield: 41.2%, Mp:180-181℃. 1H NMR (400 MHz, DMSO-d6) δ 14.34
(s, 1H), 8.50 (d, 1H, J = 5.6 Hz), 8.32 (s, 1H), 8.03 (dd, 1H, J1 = 3.2 Hz, J2 = 10.8 Hz),
7.65 (d, 1H, J = 16.4 Hz), 7.52 (dd, 1H, J1 = 0.8 Hz, J2 = 5.2 Hz), 7.38 (d, 2H, J = 8.4
Hz), 7.17 (td, 1H, J1 = 3.2 Hz, J2 = 8.8 Hz), 6.97 (d, 1H, J = 16.4 Hz), 6.93 (dd, 1H, J1
= 5.2 Hz, J2 = 9.2 Hz), 6.61 (d, 2H, J = 8.4 Hz), 5.60 (s, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 156.42 (d, JC, F = 2.7 Hz), 156.19 (d, JC, F = 0.9 Hz), 155.63 (d, JC, F
231.3 Hz), 150.66, 148.48, 146.68, 136.06, 129.24, 123.95, 119.81, 119.73 (d, JC, F
=
=
7.4 Hz), 119.43 (d, JC, F = 7.9 Hz), 118.45 (d, JC, F = 23 Hz), 116.47, 114.28, 113.08 (d,
JC, F = 24.2 Hz). HRMS (ESI) calcd for C19H16FN2O [M + H]+: 307.1241, Found:
307.1237.
4.1.7.11. (E)-2-(4-(3-aminostyryl)pyridin-2-yl)-4-fluorophenol(8g)
Yellowish solid, yield: 59.2%, Mp:184-185℃. 1H NMR (400 MHz, DMSO-d6) δ 14.20
(s, 1H), 8.58 (d, 1H, J = 5.6 Hz), 8.43 (s, 1H), 8.07 (dd, 1H, J1 = 3.2 Hz, J2 = 10.8 Hz),
7.68 (d, 1H, J = 16.4 Hz), 7.64 (dd, 1H, J1 = 1.2 Hz, J2 = 5.2 Hz), 7.22-7.14 (m, 2H),

7.10 (t, 1H, J = 8.0 Hz), 6.94 (dd, 1H, J1 = 5.2 Hz, J2 = 8.8 Hz), 6.87-6.84 (m, 2H),
6.61-6.58 (m, 1H), 5.22 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.63 (d, JC, F
=
2.8 Hz), 156.11 (d, JC, F = 1.0 Hz), 155.66 (d, JC, F = 231.4 Hz), 149.53, 147.56, 146.96,
137.02, 136.12, 129.82, 124.97, 120.42, 119.71 (d, JC, F = 7.4 Hz), 119.46 (d, JC, F = 8.0
Hz), 118.59 (d, JC, F = 23.0 Hz), 117.37, 115.63, 115.44, 113.24 (d, JC, F = 24.2 Hz),
112.71. HRMS (ESI) calcd for C19H15FN2NaO [M + Na]+: 329.1061, Found: 329.1066.
4.2. LSD1, MAO-A and MAO-B Enzymatic Assay
Inhibitory effects of the target compounds against LSD1 were evaluated according to
49
our previously reported methods . Full length LSD1 cDNA encoding LSD1 was
obtained by RT-PCR and cloned into pET-28b (pET-28b-LSD1). Then the plasmid
pET-28b-LSD1 was transfected into BL21 (DE). The recombinant protein was induced
with 0.25 mM IPTG at 20 ℃ and purified following affinity chromatography, ion
exchange chromatography and gel filtration. Then the compounds were incubated with
the 5nM recombinant LSD1 and 25μM H3K4me2 peptide in the present of FAD
(50nM), Amplex Red (20nM) and horseradish peroxidase (5.5U/mL) for 30 min. After
that, the fluorescence was measured at excitation wavelength 530 nm and emission
wavelength 590 nm as reported in order to evaluate the inhibition rate of the candidate
compounds. MAO inhibitory activities were determined using a commercialized MAO-
Glo assay kit from Promega, according to the manufacturer’s protocol.
53
The dilution assay was done as published . Briefly, an amount of 2.5 μg of
recombinant LSD1 was incubated with high concentration of compound 8c,
GSK2879552, or DMSO. After 1 h later, 1.25 μg aliquots were removed from all
samples and diluted into HRP-assay solution containing substrate and coupling reagents
to a final volume of 100 μL. This represents an 80-fold dilution of the inhibitor
concentration, which is expected to yield the same inhibition rate for an irreversible
inhibitor or significant difference for a reversible inhibitor.
For the competitive analysis of candidate compound, demethylase activity of
LSD1 was assessed in the presence of different concentrations of the compound at a
fixed concentration of histone peptide but different concentrations of FAD. Assays
were performed triplicate, and kinetics values were obtained using Lineweaver−Burk
plots made by GraphPad 6.0.
4.3. Surface plasmon resonance (SPR) experiment
Studies of binding kinetics are performed on a Biacore S200 (GE Healthcare,
USA). LSD1 is aimed for an immobilization level of approximately 8000 RU with a
CM5 sensor chip, and the running buffer is PBS-P (0.2 M phosphate buffer 0.027 M
KCl, 1.37 M NaCl, 0.5%Surfactant P20, PH 7.4). The direct binding assay is tested
with buffer and sample in 2% DMSO. Then compounds with twofold dilution series
are injected for 60s and dissociated for 300s at a flow rate of 30 μL/min. The data is
analyzed by Biacore S200 Evaluation Software 1.0.
4.4 Western blotting
106 THP-1 cells/well were incubated with compound 8c (0, 1.25, 2.5, 5.0 μM) for
5 days. Histone proteins were extracted using EpiQuik total histone extraction kit
(Epigentek) according to the manufacturer's protocol. Equivalent amounts of cell

lysates were denatured, separated by SDS-PAGE and transferred onto nitrocellulose
membranes. After blocking with PBS containing 5% nonfat milk, the membranes were
incubated overnight at 4℃with specific primary antibodies, followed by incubation
with appropriate secondary antibodies. The immunoblots were visualized by enhanced
chemiluminescence detection kit from Thermo Fisher.
4.5. Cell Viability Assay
The MTS/PMS method was used to evaluate the inhibition rate of cancer cell
proliferation. Cells were treated with various concentrations of the test compounds.
After the incubation of 8 days, MTS assay was performed using CellTiter 96®
AQueous One Solution Cell Proliferation Assay (Promega, USA) according to the
manufacturer’s instructions. Each treatment had triplicates and at least three
independent experiments were performed for statistics. All data were represented as
mean ± SD.
4.6. Colony Formation Assay
THP-1 cells (500 cells/well) were seeded in 24-well plate, 1:1 mixture of RPMI
1640 containing 10% FBS (BI) and Methylcellulose-based medium as the culture
medium. Cells were treated with compound 8c at indicated concentrations for 14 days.
Then cells were stained with DAPI (Sigma) according to the manufacturer’s
instructions and washed with PBS. The colony formation was imaged using microscope
(TS-100, Nikon, Japan).
4.7. Flow-cytometry Analysis
After the incubation with indicated concentrations of compound 8c or 0.1%
DMSO, 1×106 THP-1 cells were treated in the dark with FITC-conjugated CD86
antibody (abcam, ab77131) at 4 °C for 30 minutes. The expression of CD86 was
analyzed by FACS BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA,
USA) and the flow cytometry data was analyzed using Flowjo.
4.8. Hematoxylin and Eosin stain
THP-1 cells are seeded in 6-well plate with different concentration of compounds
then incubated for 3 days in standard medium. After the incubation period, medium
contain cells are centrifuged to gather the cells. Resuspend the cells by PBS and apply
them evenly to low-absorbance glass. After followed steps contain staining,
dehydration， cleaning with xylene and covered by coverslips upon gum (Solarbio,
China)，slides are photographed by light microscope (Leica, German)
4.8. Molecular Docking
All molecular modeling studies were performed with MOE (The Molecular
Operating Environment) Version 2015.10. The crystal structure for LSD1 (PDB code:
4LXZ) was obtained from the RCSB protein data bank. The docking procedure
contained the preparation of protein and ligand and the operation of docking. The
preparation of protein structure was performed using the Quickprep module, which
contained the deletion of waters, the addition of hydrogen atoms, the protonation and

the repair of missing residues. The geometry optimization of ligand structure mainly
was executed by energy minimization and conformation search. Next, compound 8c
was docked into the LSD1. Default triangle matcher method was used for placement of
ligand and the final conformation was scored by GBVI/WSA dG. All these above
treatments were formed in Amber 10: EHT forcefield.
Acknowledgments
This work were supported by National Natural Science Foundation of China (Project
No. 81402793 and No. U1704184 for Y.-C. D.; Project No. 81430085 and No.
81773562 for H.-M.L.; Project No. 81602961, for Y.-C. Z.; Project No. U1504207,
for X.-J.W.;); Outstanding Young Talent Research Fund of Zhengzhou University
(Project No. 1521331002, for Y.-C. Z.); Key Scientific Research Project for Higher
Education by Department of Education of Henan Educational Committee (Project No.
15A350018, for Y.-C. Z.); National Key Research Program of Proteins (Project No.
2016YFA0501800, for H.-M. L.; Project No. 2017YFD0501401, for Y.-C. Z.); Key
Research Program of Henan Province (Project No. 161100310100, for H.-M. L.);
Science and Technology Tackling Key Project of Henan Province (Project No.
182102310214, for Y.-C. D ).
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Table 1 Inhibitory Activity (IC50 or percentage of inhibition at 10 μM) against LSD1
LSD1
IC50(μM)a
Compounds Structures
Inhibition % at 10μM
HO
OH
3d
3e
4.24 ± 0.89
85.09%
N
HO
N.T.b
20.18%
N
OH
N

O
N.I.c
NH2
3f
N.T.
N
O
N
NH2
3h
4a
4b
N.T.
N.I.
N
OH
HO
N
0.72 ± 0.12
1.29 ± 0.07
97.84%
87.50%
NH2
N
OH
HO
N
N
NH2
N
HO
N
4c
4f
0.92 ± 0.04
94.29%
98.37%
N
N
OH
NH2
HO
N
NH2
0.301 ± 0.03
HO
4g
3.57 ± 0.31
0.859 ± 0.07
9.55 ± 1.01
N.T.
94.32%
97.66%
51.31%
35.43%
43.89%
30.28%
89.30%
31.63%
N
NH2
NH2
HO
N
N
4h
4i
N
HO
N
NH2
HO
N
O
4j
O
NH2
HO
N
O
NH2
4k
4l
N.T.
HO
N
F
NH2
N.T.
HO
HO
NH2
4m
7d
1.47 ± 0.24
N.T.
N
N
OH
OH

OH
N
N
OH
8a
8b
0.364 ± 0.03
0.764 ± 0.03
97.69%
97.57%
NH2
N
N
OH
OH
NH2
OH
N
N
8c
8e
8f
0.283 ± 0.02
2.96 ± 0.74
9.03 ± 0.66
11.78 ± 1.09
99.23%
94.80%
54.14%
50.11%
F
OH
NH2
N
OH
NH2
N
F
OH
NH2
OH
N
8g
F
NH2
NH2
ORY-
1001
2HCl
11.26 ± 0.72 (nM) N.T.
N
H
a IC50 values are expressed as mean ± SD from at least three independent experiments.
b N.T.: Not tested.
c N.I.: No Inhibition.
Table 2 In vitro Inhibition of LSD1, MAO-A and MAO-B of selected compounds
IC50(μM)
Compounds
4a
aValues are
LSD1
MAO-A
>50
MAO-B
>50
the
mean
of
two
0.72 ± 0.12
0.301 ± 0.03
0.364 ± 0.03
0.283 ± 0.02
bN.D.
experiments.
4f
>50
>50
bN.D.:
Not
8a
8c
>50
>50
detected
>50
>50
a 0.0031
N.D.
a0.071
Table 3 In vitro
Clorgyline
antiproliferative activity of
selected compounds in
R(-)-deprenyl
N.D.
N.D.
three cancer cell lines
a
IC50(μM)a
IC50 values are
Compounds
expressed as mean ± SD
from at least three
independent experiments.
b N.D.: Not detected
MOLM-13
THP-1
MV-4-11
4a
22.59 ± 2.33
12.51 ± 1.06
9.05 ±0.99
8.34 ± 0.64
N.D.b
7.89 ± 0.71
10.51 ± 1.44
13.72 ± 1.08
5.76 ± 0.55
> 20 μM
4.71 ± 0.89
10.94 ± 2.03
15.85 ± 1.52
7.49 ± 1.17
N.D.
4f
8a
8c
ORY-1001

Highlights
 A series of stilbene derivatives were discovered as novel LSD1
inhibitors.
 Compound 8c potently inhibited LSD1 in a reversible and FAD
competitive manner with an IC50 of 283 nM.
 Compound 8c dose-dependently increased surrogate cellular
biomarker CD86 in THP-1 human leukemia cells.
 Compounds 8c significantly inhibited proliferation and colony
formation of THP-1 cells.