Discovery and synthesis of novel indole derivatives-containing 3-methylenedihydrofuran-2(3H)-one as irreversible LSD1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.04.065

Lysine-specific demethylase 1 (LSD1), demethylase against mono- and di - methylated histone3 lysine 4, has emerged as a promising target in oncology. More specifically, it has been demonstrated as a key promoter in acute myeloid leukemia (AML), and several LSD1 inhibitors have already entered into clinical trials for the treatment of AML. In this paper, a series of new indole derivatives were designed and synthesized based on a lead compound obtained by a high-throughput screening with our in-house compound library. Among the synthetic compounds, 9e was characterized as a potent LSD1 inhibitor with an IC₅₀ of 1.230 μM and can inhibit the proliferation of THP-1 cells effectively. And most importantly, this is the first irreversible LSD1 inhibitor that is not derived from monoamine oxidase inhibitors. Hence, the discovery of 9e may serve as a proof of concept work for AML treatment.

Full text of DOI:10.1016/j.ejmech.2019.04.065

Accepted Manuscript
Discovery and synthesis of novel indole derivatives-containing 3-
methylenedihydrofuran-2(3H)-one as irreversible LSD1 inhibitors
Hong-Min Liu, Feng-Zhi Suo, Xiao-Bo Li, Ying-Hua You, Chun-Tao Lv, Chen-Xing
Zheng, Guo-Chen Zhang, Yue-Jiao Liu, Wen-Ting Kang, Yi-Chao Zheng, Hai-Wei Xu
PII:
S0223-5234(19)30389-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.065
EJMECH 11300
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2019
Revised Date: 24 April 2019
Accepted Date: 25 April 2019
Please cite this article as: H.-M. Liu, F.-Z. Suo, X.-B. Li, Y.-H. You, C.-T. Lv, C.-X. Zheng, G.-C. Zhang,
Y.-J. Liu, W.-T. Kang, Y.-C. Zheng, H.-W. Xu, Discovery and synthesis of novel indole derivatives-
containing 3-methylenedihydrofuran-2(3H)-one as irreversible LSD1 inhibitors, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.065.
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ACCEPTED MANUSCRIPT

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Discovery and synthesis of novel indole
derivatives-containing
3-methylenedihydrofuran-2(3H)-one as irreversible
LSD1 inhibitors
Hong-Min Liu a, Feng-Zhi Suo a, Xiao-Bo Li a, Ying-Hua You a, Chun-Tao Lva, Chen-Xing Zhenga,
Guo-Chen Zhang a, Yue-Jiao Liu a, Wen-Ting Kang a, Yi-Chao Zheng a,b,#, Hai-Wei Xu a,#
a
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, School
of Pharmaceutical Science, Institute of Drug Discovery and Development, Zhengzhou
University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China
b
National Center for International Research of Micro-nano Molding Technology of Henan
Province, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001,
Corresponding Authors: Dr. Hai-Wei Xu & Dr. Yi-Chao Zheng
E-Mail: xhwei01@126.com (Hai-Wei Xu) & yichaozheng@zzu.edu.cn (Yi-Chao Zheng)
Abstract: Lysine-specific demethylase 1 (LSD1), demethylase against mono- and di -
methylated histone3 lysine 4 , has emerged as a promising target in oncology. More
specifically, it has been demonstrated as a key promoter in acute myeloid leukemia
(AML), and several LSD1 inhibitors have already entered into clinical trials for the
treatment of AML. In this paper, a series of new indole derivatives were designed and
synthesized based on a lead compound obtained by a high-throughput screening with
our in-house compound library. Among the synthetic compounds, 9e was
characterized as a potent LSD1 inhibitor with an IC50 of 1.230 µM and can inhibit the
proliferation of THP-1 cells effectively. And most importantly, this is the first
irreversible LSD1 inhibitor that is not derived from monoamine oxidase inhibitors.
Hence, the discovery of 9e may serve as a proof of concept work for AML treatment.
Keywords: LSD1; irreversible; inhibitor; indole; lactone
1. Introduction
Epigenetic is the study of heritable changes in gene function that does not involve
changes in DNA sequence. Example of mechanisms that produce such changes is
histone modification. Histones, where multiple signaling pathways converge on, are
small basic proteins in eukaryotic cell nucleus that package and order the DNA into
nucleosomes, which are the fundamental units of eukaryotic chromatin [1, 2]. It can
be subjected to a diverse array of post-translational modifications, such as methylation,
acetylation, phosphorylation and ubiquitination, and these modifications contribute to
dysregulation of target genes by modeling chromatin structure precisely [1, 3].
Among these modifications, methylated histone can be demethylated by specific
erasers, such as LSD1, which is the first identified histone demethylase with flavin
adenine dinucleotide (FAD) as a cofactor to demethylate mono- and di- methylation
of H3K4 and H3K9 [4, 5], and it plays a vital role in several kinds of disease, such as
cancer [6, 7].
As reported, LSD1 is overexpressed in a variety of tumors, such as lung cancer[8],

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neuroblastoma [9], gastric cancer [10], retinoblastoma [11], breast cancer [12], and
leukemia [13], and it has been considered as a potential cancer therapeutic target.
Hence, many medicinal chemists tried to design and synthesize potent and selective
LSD1 inhibitors. As LSD1 is a homology of monoamine oxidase (MAO) and shares
similar catalytic mechanisms as MAO [14], MAO inhibitors, such as
trans-2-phenylcyclopropylamine (TCP), were used as scaffolds for further
development. Until now, tens of TCP based LSD1 inhibitors have been identified, but
only six of TCP based LSD1 inhibitors have entered into clinic trials alone or in
combination with other therapeutics, including ORY-1001 (Fig. 2A), ORY2001,
GSK2879552 (Fig. 2A), CC-90011, IMG-7289 and INCB059872 [15, 16]. And all of
them are applied for the treatment of AML as well as myelofibrosis as LSD1 is a
central regulator of hematopoietic stem cell and progenitor cell [17], and it can also
regulate the expression of a variety of leukemia subtype oncogenes [18-23]. Hence,
extensive study on irreversible inhibitors of LSD1 with different backbone is urgently
needed. Here, we reported the first irreversible LSD1 inhibitor 9e, that is not derived
from monoamine oxidase inhibitors. 9e is the most potent one among the series of
new indole derivatives we got and can inactivate LSD1 with IC50 value of 1.230 µM.
In THP-1 cells, compound 9e blurred cell membrane boundaries and shrank
chromatin, inhibited the intracellular activity of LSD1, meanwhile, compound 9e
inhibited cell proliferation ability and colony formation dose dependently. In a
nutshell, compound 9e is a potent irreversible inhibitor of LSD1 with fully novel
skeleton, which can provide a promising strategy for the development of irreversible
LSD1 inhibitors for AML treatment.
2. Results and discussion
2.1. Chemistry
Fig.1 The structure of compound 1a.
Recent years, our groups focused on the synthesis and bioactive research of
butenolide derivatives [24, 25]. Based on our previous work on LSD1, an in-house
compound library screening against LSD1 was done to discover LSD1 inhibitor.
Compound 1a, an indole derivative including butenlide moiety was obtained with IC50
of 20.50 µM against LSD1. With compound 1a as a hit, series of novel indole
derivatives were designed, synthesized to study the SAR by modification at the P1, P2,
and P3 on compound 1a (Fig 1).

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O
H
R
a
b
O
N
H
N
N
H
H
2
4
3
c
d
O
O
R
Cl
N
O
O
N
N
OH
OH
5
1a~1f
a: R= H; b: R= 4-F; c: R= 2-CF3; d: R= 2-F; e: R= 4-Cl; f: R= 3,4-diCl
Reagents and conditions: (a) DMF, POCl3, 0°C, 98%; (b) NaH, MeCN, Benzyl
chloride derivatives, r.t. 50% ~ 90%; (c) THF, Zn, NH4Cl, 3 compound 6, r.t.,
68%~85%. (d) (1) NaH, MeCN, 2-chloro-5- (chloromethyl)pyridine, r.t.; (2) THF, Zn,
NH4Cl, compound 6, r.t. 66% for two steps.
Scheme 1 Synthesis of compounds1a~1f.
Reagents and conditions: (a) Me3PhN+Br3, 1,4-dioxane, r.t. 15h, 87.6%; (b)
Li2CO3/LiBr, TBAB, MeCN, 85°C, 48.2%.
Scheme 2 Synthesis of intermediate 6.
Firstly, compounds 1b-f were designed to investigate the evaluation of benzyl groups
of P1 at N1. Compounds 1b~1f with substituted phenyl groups at N1 were
synthesized by the formylation reaction, N-benzylation reaction and Blaise reaction
with indole as starting material as shown in Scheme 1. The key intermediate
bromolactone 6 was obtained from the commercially available tulipalin (7) by
bromination reaction followed by regioselective elimination (Scheme 2).
ID
9a
R
ID
9l
R
9b
9c
9d
9m
9n
9o
9e
9f
9p
10a
10b
9g
9h
9i
10c
10d

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9j
9k
10e
10f
Reagents and conditions: (a) DMF, POCl3, 0°C, 95%; (b) NaH, MeCN, aryl or alkyl
methylene chloride, r.t. 50%~90%; (c) THF, Zn, NH4Cl, compound 6, r.t. 43~76%; (d)
NaH, MeCN, acyl chloride, r.t. 60%~85%; (e) THF, Zn, NH4Cl, compound 6, r.t.
69%~80%.
Scheme 3. Synthesis of compounds 9a~9p and compounds 10a~10f.
Compounds 5 and 9a~9p were designed to compare the heterocyclic methylene
substitution with benzyl groups at N1. Compounds 9a~9o were prepared by the same
method to prepare compound 1 with 5-choloride indole as material(Scheme 3).
In order to compare the methylene and formyl groups for P1, compounds 10a~10g
were designed and synthesized. Similarly, the N-acylation group was introduced at N1
on indole to yield compounds 10a~10g (Scheme 3).
Keeping the P1 and P3, compounds 16a~16i were designed to evaluate the
contribution of various substitution such as chlorine, fluorin, and methoxyl groups at
different position of indole phenyl group of P2. They were synthesized with
substituted indole as raw materials by the reactions used as below (Scheme 4).
Reagents and conditions. (a) DMF, POCl3, 0°C, >90%; (b) NaH, MeCN, PTSC, r.t.
76.14%; (c) THF, Zn, NH4Cl, compound 6, r.t. 69%; (d) NaH, MeCN, 4-Chlorobenzyl
chloride, r.t. 50%-90%; (e) THF, Zn, NH4Cl, compound 6, r.t. 60% ~ 83%.
Scheme 4 Synthesis of compounds 15,16a~i.
Compound 15 was designed and synthesized to prove whether the sulfonyl group with
tetrahedral pose is better than that of formyl groups with plane trigonometry to link to
N1 for the LSD1 inhibition (Scheme 4).
In order to test the importance of the 4-(1-hydroxyethyl)-3-methylene dihydrofuran
-2(3H)-one group at C3 (P3) on core compound 1a, compounds 21, 24, 25 and 26, in
which the P3 was displaced with phenyl, furan, pyridinyl groups and so on, were
designed and synthesized as shown in Scheme 5.

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ID
R
ID
R/X
21a
CH3
21g
21b
21c
21h
21i
21d
24a
24b
O
S
21e
21f
Reagents and conditions: (a) NaH, MeCN, r.t. 93.96%; (b) CH2Cl2, AlCl3, Acyl
chloride derivatives, r.t. 70%~85%; (c) (1) CH2Cl2, AlCl3, Acetyl chloride, r.t. 78%; (2)
MeOH, NaOH (aq), 2-Furfural or 2-Thenaldehyde, r.t. 68%; (d) MeOH, NaBH4, r.t.
70%; (e) EtOH/Pyridine, NH2OH·HCl, 80°C, 90%.
Scheme 5 Synthesis of compounds 21a~i, 24a, 24b, 25 and 26
2.2 Inhibitory Activity against LSD1 Recombinant
Table 1 LSD1 inhibition of synthetic compounds
ID
IC50/µM
ID
IC50/µM
ID
IC50/µM
20.497±1.312
4.467±0.650
>10
>10
>10
>10
1a
1b
9l
16g
16h
9m
8.959±0.952
5.849±0.767
9.469±0.976
6.580±0.818
5.903±0.771
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1c
1d
1e
1f
9n
16i
>10
9o
21a
21b
21c
21d
21e
21f
21g
21h
21i
>10
9p
>10
10a
10b
10c
10d
10e
10f
15
>10
5
>10
9a
9b
9c
9d
9e
9f
>10
>10
>10
>10
>10
>10
1.230±0.159
>10
1.784±0.249
>10
16a
16b
16c
24a
24b
25
>10
>10
9g
9h
>10
>10

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8.344±0.921
8.410±0.925
>10
>10
>10
9i
9j
16d
16e
26
0.024±0.001
GSK-LSD1
>10
>10
9k
16f
The LSD1 inhibitory activities of all synthesized compounds were evaluated by the
previously reported high-throughput method of our group [10]. Some of them showed
inhibition against LSD1 (Table 1). Among them, compounds 1b~1f, 5, 9e, 9i, 9j
containing benzyl groups or pyridinyl methyl group at N1 of indole with IC50 of from
9.5µM to 1.2µM were more active than hit compound 1a, of which 9e with 5-chloro
and N-(4-F-benzyl) indole was the best one with IC50 of 1.23 µM. However,
compounds 9 and 10 with 5-chloro and N-aryl or alkyl methyl indole or 5-chloro and
N-aryl or alkyl formyl indole showed more weak LSD1 inhibitory activity than
compound 9e. Compound 15 with 1-tosyl indole derivative, an analog of compounds
10, showed similar LSD1 inhibition activity with IC50 of 1.78µM. Keeping the P3,
compounds 16 with introducing different substitutions such as chlorine, fluorin, and
methoxyl groups resulted in the decreasing of LSD1 inhibition of hit compound 1a.
Unfortunately, all the compounds 21, 24, 25 and 26 without the lactone moiety P3 at
C-3 position of indole compare to compound 2b showed no inhibition against LSD1
at 10µM. Based on the above, compound 9e was chosen for further study.
The SAR suggests that the benzyl groups at N1 and the 4-(1-hydroxyethyl)-
3-methylenedihydrofuran -2(3H)-one group at C3 play important roles for their LSD1
inhibition of title compounds. The 4-(1-hydroxyethyl)-3-methylenedihydrofuran
-2(3H)-one group at C3 could not be replaced by other groups. The compounds with
benzyl groups or benzenesulfonyl group at N1 showed better LSD1 inhibition than
that of benzoyl and heterocyclic formyl substitution derivatives, which tell us that the
phenyl groups and the tetrahedral configuration of the link atom to N1 from phenyl
groups are crucial for their activities.
2.3 Compound 9e can directly bind and irreversibly inactivate LSD1
To further understand the binding model of 9e to LSD1 (Fig. 2B), several biochemical
assay and biophysical experiment were performed. As shown in Figure 2C, compound
9e inactivated LSD1 with IC50 = 1.230 µM (Hillslope = 1.138) , while GSK LSD1
was used as a positive control with IC50 = 21.840 nM (Hillslope = 1.211) (Fig. 4A).
To characterize the binding model of compound 9e to LSD1, including the binding
reversibility of compound 9e to LSD1, a dilution assay was performed (Fig. 2D). We
found that not only can the irreversible positive compound GSK-LSD1 inactivate
LSD1 even after being diluted by 80 folds, compound 9e can also. This result
indicates that compound 9e may be an irreversible inhibitor. In order to further
confirm the inhibition behavior of compound 9e, we used the ultrafiltration assay,
which also showed that compound 9e may inhibit LSD1 in an irreversible manner
(Fig. 2E).
To further confirm the direct interaction between compound 9e and LSD1, surface
plasmon resonance (SPR) was applied. As shown in Figure 2F, compound 9e
introduced a high signal value for the binding curve which indicated that this inhibitor
can directly interact with LSD1 strongly with KD of 2.03E-04 M. And positive control
GSK-LSD1 got a similar affinity with KD of 4.25E-04 M (Fig. 4B). For the further
test of the target engagement of compound 9e to LSD1, protein thermal shift assay,
which has been extensively used in drug screening [26-28], was performed. The

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results showed that Tm, which is the temperature at protein half denatured to show the
protein thermal stabilization, in sample groups (containing LSD1 and compound 9e)
was higher than the reference group (containing LSD1 and DMSO) in a concentration
dependent manner (Fig. 2G). As we know, a higher Tm represents a more stable
protein, indicating an interaction between protein and compound. So, the result
implied that compound 9e can bind well to LSD1. When performing the same assay
on GSK-LSD1, it also showed a concentration dependent manner to increase Tm
value (Fig. 4C).
Fig. 2 Properties of compound 9e against LSD1 recombinant. (A) Representative
inhibitors for LSD1 that have entered into clinical studies; (B) Structure of compound
9e; (C) LSD1 inactivation curve of compound 9e; (D) Dilution assay of compound 9e
to LSD1 activity, GSK-LSD1 was used as a control; (E) Ultrafiltration assay of
compound 9e to LSD1 activity, GSK-LSD1 was used as a control; (F) SPR
sensorgrams of the interaction between LSD1 and compound 9e with indicated

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concentrations; (G) △Tm caused by different concentrations of compound 9e with
protein thermal shift assay, △Tm=Tmsample - Tmreference. Data are the mean ± SD. P <
0.01 was considered statistically significant. All experiments were carried out at three
times.
2.3 Evaluation of biological activity at cellular level
LSD1 inhibitors were reported to be effective against AML cell proliferation [13, 19,
21]. Moreover, overexpression of LSD1 is highly relevant to the poor prognosis of
human mixed lineage leukemia (MLL) translocated AML [29-31]. Therefore, THP-1,
which is an MLL-AF9 translocate AML cell line, is used to evaluate LSD1 inhibitory
activity of compound 9e at cellular level. First, THP-1 cells were treated with
compound 9e to test the anti-proliferation activity. Compound 9e inhibited the
proliferation of THP-1 cells apparently after 5 days incubation in a dose dependent
manner with IC50 as 1.204 µM (Hillslope = 1.383). Next, colony formation assay
indicated that compound 9e and GSK-LSD1 can both suppress tumor colony
formation in vitro even at lower concentration (Fig. 3A, Fig. 4D). The photographs
implied that compared to the control group, cell populations in 9e and GSK-LSD1
treated groups were smaller in a concentration dependent manner. In addition,
hematoxylin and eosin staining (HE staining) was applied to monitor the cellular
morphology change. After 3 days incubation of compound 9e at indicated
concentrations, significant morphological changes were observed, including blurred
cell membrane boundaries and chromatin shrinkage with dose dependently (Fig. 3B).
GSK-LSD1 also got a similar result in Figure 4E.
As reported, abrogation of LSD1 lead to differentiation of AML cells [13, 21], hence,
we further focused on CD86, a type of myeloid differentiation marker whose
expression level can be considered as a surrogate cellular biomarker of LSD1 [32, 33].
Flow cytometry assay was performed to compare the expression levels of CD86. The
results showed that compound 9e can significantly increase the expression level of
CD86 in a concentration dependent manner in THP-1 cells (Fig. 3C). Moreover, 4 µM
compound 9e performed similar potency to induce the expression of CD86 as
GSK-LSD1 (Fig. 4F). So, this indicates compound 9e may inactivate LSD1 in THP-1
cells and regulate myeloid differentiation by modulating the expression of CD86.

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Fig. 3 Inhibitory effect of compound 9e against LSD1 in THP-1 cells. (A) Colony
formation assay for THP-1 cells treated with compound 9e for 2 weeks; (B) HE
staining of THP-1 cells treated with compound 9e for 3 days; (C) CD86 expression in
THP-1 cells treated with compound 9e for 3 days. Data are mean ± SD. **p<0.01 was
considered statistically highly significant. All experiments were carried out at least
three times.

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Fig. 4 Properties of GSK-LSD1 against LSD1 recombinant and LSD1 in THP-1 cells. (A) LSD1
inactivation curve of GSK-LSD1; (B) SPR sensorgrams of the interaction between LSD1 and
GSK-LSD1 with indicated concentrations; (C) △Tm caused by different concentrations of
GSK-LSD1 with protein thermal shift assay, △Tm=Tmsample - Tmreference. (D) Colony formation
assay for THP-1 cells treated with GSK-LSD1 for 2 weeks; (E) HE staining of THP-1 cells treated
with GSK-LSD1 for 3 days; (F) CD86 expression in THP-1 cells treated with GSK-LSD1 for 3
days. Data are the mean ± SD. P < 0.01 was considered statistically significant. All experiments

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were carried out at three times.
3. Conclusion
In summary, we have synthesized a series of novel indole derivatives based on the
screening of a compound library constructed by ourselves for LSD1 activity and have
identified them as a novel class of LSD1 inhibitor. Some of the indole derivatives,
especially compound 9e with 5-chloro and N-(4-F-benzyl) indole can inhibit LSD1
effectively. Dilution assay and ultrafiltration assay suggested that compound 9e is an
irreversible LSD1 inhibitor. Importantly, these indole derivatives are the first series of
LSD1 targeting irreversible inhibitors that are not derived from monoamine oxidase
inhibitors. Moreover, in AML cells, which with high expression of LSD1, compound
9e significantly inhibited cell proliferation and increased the expression level of CD86
in THP-1 cells, which means compound 9e promoted myeloid differentiation. So, our
findings indicated that these indole derivatives could be served as a good starting
point to design and synthesize new irreversible inhibitors targeting LSD1.
4. Experimental section
4.1 General information
Reagents and solvents were purchased from Bide Pharmatech Ltd, Aladdin,
1
Sinopharm Chemical Reagent Co, Ltd. with purities of at least 97%. H NMR (400
MHz)and 13C NMR (100 MHz) spectra were recorded with a Bruker spectrometer. All
reactions were monitored by thin-layerchromatography (TLC) on 25.4 mm × 76.2 mm
silica gel plates (GF-254) and UPLC-Mass on Waters ACQUITY UPLC H-Class or
Q-Tof Micro HRMS on waters. Melting points were determined on a Beijing Keyi
XT4A apparatus. The silica gel used for column chromatography was 200-300 mesh
or recrystallization with solvents specified in the corresponding experiments.
4.2 General Synthetic Procedure for the compounds
Synthesis of compound 3：POCl3 (13.09 g, 85.36 mmol) was slowly added dropwise
to DMF (10 mL) under ice bath. The mixture was stirred at ice bath for 30 minutes.
Then, DMF (6.24 g, 85.36 mmol) solution of indole (5 g, 42.68 mmol) was added
dropwise to the reaction system. Keeping the reaction at room temperature for 3 hours,
ice water and 10% NaOH aq. were added into the reaction system to pH of 7-8 and
continue to stir to a lot of white solid precipitation. The solid was recrystallized from
ethyl acetate and petroleum ether to obtain compound 3 in yields of 97.6%.
General Synthesis Procedure for 1-benzyl-1H-indole-3-carbaldehydes (4) To the
solution of compound 3 (1 equiv) in MeCN was added NaH (3 equiv) and the Benzyl
chloride derivatives (1.5 equiv) at room temperature. The reaction mixture was stirred
at room temperature for 4 h. The mixture was extracted with ethyl acetate, washed
with brine, dried (Na2SO4), and filtered. The filtrate was concentrated in vacuo, and
the residue was purified by column chromatography (silica gel, ethyl
acetate/Petroleum ether) to give the desired product.
Synthesis of 4-((1-((6-chloropyridin-3-yl)methyl)- 1H-indol-3-yl)(hydroxy)methyl)
-3-methylenedihydrofuran-2(3H)-one (5): To the solution of compound 3 (200 mg,
1.38 mmol) in MeCN (5 mL) was added NaH (99.19 mg, 4.13 mmol) and 2-chloro-5-
(chloromethyl) pyridine (334.83 mg, 2.07 mmol) at room temperature. The reaction
mixture was stirred at room temperature for 4 h. The mixture was extracted with ethyl
acetate, washed with brine, dried (Na2SO4), and filtered. The filtrate was concentrated
in vacuo, and the residue was purified by column chromatography (silica gel, ethyl

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acetate/Petroleum ether) to give the desired product. It mixed with
α-bromomethyl-γ-butene lactone (6) (78.46 mg, 443.27 µmol) and Zn powder (120.75
mg, 1.85 mmol) in THF. And then, a saturated aqueous solution of ammonium
chloride was added dropwise from a constant pressure low liquid funnel. The reaction
mixture was stirred at room temperature for 4 h. After suction filtration, the mixture
was evaporated under reduced pressure to move THF. EtOAc (100 mL) was added,
and the resulting solution was washed with brine water (3 × 50mL). The organic
phase was dried over Na2SO4, filtered, and concentrated. The product was purified on
a silica gel column eluting with EtOAc, to give compound 5 as light yellow solid in
1
66.06% yield. m.p.: 55.2-55.7℃. H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.1 Hz,
1H), 7.77 (d, J = 7.8 Hz, 1H), 7.38–7.30 (overlap, 2H), 7.27 –7.24(overlap, 2H), 7.22
– 7.18 (m, 1H), 7.14 (s, 1H), 6.39 (d, J = 2.1 Hz, 1H), 5.90 (d, J = 1.5 Hz, 1H), 5.32 (s,
2H), 5.04 (d, J = 8.0 Hz, 1H), 4.29 – 4.21 (m, 1H), 4.18 – 4.12 (m, 1H), 3.73 – 3.67
(m, 1H); 13C NMR (100 MHz, CDCl3) δ 170.74, 151.23, 148.09, 137.30, 136.70,
135.36, 131.59, 126.14, 125.93, 125.32, 124.65, 123.15, 120.55, 119.86, 116.17,
109.90, 69.94, 67.95, 47.03, 44.76. HRMS (ESI) calcd. C20H18ClN2O3 [M+H]+m/z:
369.1000/371.0971, found: 369.1002/371.0973.
Synthesis of 3-bromo-3-(bromomethyl)dihydrofuran-2(3H)-one (8): α-methylene
-γ- butyrolactone (7) (1 g, 10.19 mmol) was added to a solution of compound 6 (4.29
g, 11.42 mmol) in dioxane (5 mL). The reaction mixture was stirred at room
temperature for 2 h. After suction filtration, EtOAc (100 mL) was added, and the
resulting solution was washed with brine water (3 × 50mL). The organic phase was
dried over Na2SO4, filtered, and concentrated. The product was purified on a silica gel
column eluting with EtOAc, to give compound 8 as a red solid in 87.6% yield
.
Synthesis of 3-(bromomethyl)furan-2(5H)-one (6): Compound 8 (2.65 g,
10.27mmol) was added to a solution of tetrabutylammonium bromide (TBAB)
(331.24 mg, 1.03 mmol), Li2CO3/LiBr (1: 1, 51.37 mmol) in MeCN (30 mL). The
reaction mixture was stirred at room temperature for 20h. After suction filtration,
MeCN was evaporated under reduced pressure. EtOAc (100 mL) was added, and the
resulting solution was washed with brine water (3 × 100mL). The organic phase was
dried over Na2SO4, filtered, and concentrated. The product was purified on a silica gel
column eluting with EtOAc, to give compound 6 (877mg) as a red liquid in 48.22%
1
yield. H NMR (400 MHz, CDCl3) δ 7.69 –7.43(m, 1H), 4.94 – 4.82(m, 2H), 4.18–
4.07(m, 2H).
Synthesis of Compounds 1a~1f with 4-((1-benzyl-1H-indol-3-yl) (hydroxy)methyl)
-3-methylenedihydrofuran-2(3H)-one (1a) as a sample: Compound 6 (541.63 mg,
3.06 mmol) mixed with a solution of intermediate compound 4a (600mg, 2.55 mmol)
and Zn powder (833.62 mg, 12.75mmol) in THF. And then, a saturated aqueous
solution of ammonium chloride was added dropwise from a constant pressure low
liquid funnel. The reaction mixture was stirred at room temperature for 4 h. After
suction filtration, THF was evaporated under reduced pressure. EtOAc (100 mL) was
added, and the resulting solution was washed with brine water (3 × 50mL). The
organic phase was dried over Na2SO4, filtered, and concentrated. The product was
purified on a silica gel column eluting with EtOAc, to give compound 1a as a white
1
solid in 85.8% yield. m.p.: 43.6-43.9℃. H NMR (400 MHz, CDCl3) δ 7.75 (d, J =
7.9 Hz, 1H), 7.38 – 7.29 (m, 4H), 7.27 – 7.22 (m, 1H), 7.21 – 7.16 (m, 1H), 7.16 –
7.09(m, 3H), 6.36 (d, J = 2.0 Hz, 1H), 5.88 (d, J = 1.3 Hz, 1H), 5.29 (s, 2H), 4.96 (d, J
13
= 8.1 Hz, 1H), 4.24 – 4.17 (m, 1H), 4.08 (m, 1H), 3.69 – 3.62 (m, 1H); C NMR

ACCEPTED MANUSCRIPT
(100MHz, CDCl3) δ 171.12, 137.04, 135.41, 128.91, 127.91, 126.90, 126.53, 126.00,
125.35, 122.60, 120.03, 119.56, 115.33, 110.30, 69.91, 68.20, 50.11, 44.64. HRMS
(ESI) calcd. C21H20NO3 [M+H]+m/z: 334.1438, found: 334.1439.
Synthesis of 4-((1-(4-fluorobenzyl) -1H-indol-3-yl)(hydroxy)methyl) -3-methylene
dihydrofuran-2(3H)-one (1b): Compound 4b (600 mg, 2.37 mmol) was converted to
compound 1b (625 mg, 75.08%) as a white solid by the same procedure as described
for compound 1a. m.p.: 265.2-266.1℃. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.9
Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.26 – 7.22(overlap, 2H), 7.21 – 7.15 (overlap, 2H),
7.10 (s, 1H), 6.90 (dd, J = 8.3, 2.0 Hz, 1H), 6.37 (d, J = 1.9 Hz, 1H), 5.87 (d, J = 1.3
Hz, 1H), 5.24 (s, 2H), 5.00 (d, J = 8.0 Hz, 1H), 4.28 – 4.19 (m, 1H), 4.14 – 4.09 (m,
1H), 3.71 – 3.64 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 170.87, 137.04(d, J=42Hz),
135.32, 133.09, 132.06, 130.94, 128.67, 126.22, 126.03(d, J=4.6Hz) 125.40, 123.00,
120.40, 119.74, 115.87, 110.05, 69.98, 68.06, 49.08, 44.69. m.p.: HRMS (ESI) calcd.
C21H19FNO3 [M+H]+m/z: 352.1343, found: 352.1344.
Synthesis of 4-(hydroxy(1-(2-(trifluoromethyl)benzyl) -1H-indol-3-yl)methyl)-3-
methylenedihydrofuran-2(3H)-one (1c): Compound 1c (580 mg, 73.04%) as a
white solid was prepared from compound 4c (600mg, 1.98mmol) by the same
1
procedure as described for compound 1a. m.p.: 133.9-134.7℃. H NMR (400 MHz,
DMSO‑d6) δ 7.84 – 7.80 (m, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.55–7.45(m, 2H), 7.41 (s,
1H), 7.21 (d, J = 8.0 Hz, 1H), 7.15–7.03 (overlap, 2H), 6.51 (d, J = 6.3 Hz, 1H), 6.07
(d, J = 1.0 Hz, 1H), 5.67 – 5.60 (overlap, 3H), 5.56 (s, 1H), 5.10 (t, J=5.5Hz, 1H),
4.37 (t, J = 8.7 Hz, 1H), 4.29 (dd, J = 9.2, 4.0 Hz, 1H), 3.67 – 3.59 (m, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 171.07, 137.14, 136.69, 136.25, 133.42, 128.36, 127.92(d,
J=5.3Hz), 126.73, 126.44, 126.16, 123.81, 122.31, 120.36, 119.76, 116.88, 110.22,
69.01, 68.56, 46.08, 44.71. HRMS (ESI) calcd. C22H19F3NO3 [M+H]+m/z: 402.1312,
found: 402.1310.
Synthesis
of
4-((1-(2-fluorobenzyl)-
1H-indol-3-yl)
(hydroxy)methyl)
-3-methylene dihydrofuran-2(3H)-one (1d): Compound 1d (610 mg, 73.28%) was
synthesize from compound 4d (600 mg, 2.37 mmol) as a yellow solid by the same
1
procedure as described for compound 1a. m.p.: 54.1-54.7℃. H NMR (400 MHz,
CDCl3) δ 7.77 (d, J = 7.9 Hz, 1H), 7.31 – 7.27 (overlap, 2H), 7.26–7.22 (overlap, 2H),
7.21 – 7.16 (m, 1H), 7.14-7.08 (m, 2H), 7.02–6.96 (m, 1H), 6.38 (d, J=1.6Hz, 1H),
5.91–5.87 (m, 1H), 5.28 (s, 2H), 5.01 (d, J = 8.0 Hz, 1H), 4.28 – 4.21 (m, 1H), 4.14 –
13
4.11 (m, 1H), 3.72 – 3.66 (m, 1H); C NMR (100 MHz, CDCl3) δ 170.91, 145.00,
139.08(d, J=3Hz), 136.96, 135.38(d, J=54Hz), 130.21(d, J=6.6Hz), 128.13, 126.90,
126.33, 125.99, 124.92, 122.86, 120.65, 120.25, 119.64, 115.74, 110.13, 70.01 (d,
J=5.3Hz), 68.10, 49.57, 44.70. HRMS (ESI) calcd. C21H19FNO3 [M+H]+m/z:
352.1343, found: 352.1344.
Synthesis of 4-((1-(4-chlorobenzyl) -1H-indol-3-yl)(hydroxy)methyl) -3-methylene
dihydrofuran-2(3H)-one (1e): Compound 4e (600 mg, 2.22 mmol) was converted to
compound 1e (620 mg, 75.77%) as a light-yellow solid by the same procedure as
described for compound 1a. m.p.: 106.3-106.7℃. 1H NMR (400 MHz, CDCl3) δ 7.74
(d, J = 7.9 Hz, 1H), 7.30 – 7.26 (overlap, 3H), 7.26 – 7.21 (m, 1H), 7.19 – 7.14 (m,
1H), 7.09 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.34 (d, J = 2.0 Hz, 1H), 5.84 (d, J = 1.3
Hz, 1H), 5.25 (s, 2H), 4.97 (d, J = 7.9 Hz, 1H), 4.24 – 4.14(m, 1H), 4.14 – 4.10 (dd, J

ACCEPTED MANUSCRIPT
= 5.7, 3.7 Hz, 1H), 3.70 – 3.60 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 171.15,
136.85, 135.63, 135.33, 133.62, 129.04, 128.21, 126.44, 126.07, 125.33, 122.69,
120.13, 119.66, 115.57, 110.18, 69.85, 68.24, 49.47, 44.66. HRMS (ESI) calcd.
C21H19ClNO3 [M+H]+m/z: 368.1048/370.1018, found: 368.1049/370.1050.
Synthesis
of
4-((1-(3,4-dichlorobenzyl)-1H
-indol-3-yl)(hydroxy)methyl)
-3-methylene dihydrofuran-2(3H)-one (1f): Compound 4f (300 mg, 2.22 mmol)
was converted to compound 1f (280 mg, 68.44%) as a yellow solid by the same
1
procedure as described for compound 1a. m.p.: 60.9-61.3℃. H NMR (400 MHz,
CDCl3) δ 7.76 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.25–7.21 (m, 1H), 7.19 –
7.14 (m, 1H), 7.12–7.06 (overlap, 3H), 7.05–6.97 (overlap, 2H), 6.39 (d, J = 2.0 Hz,
1H), 5.93 (d, J = 1.4 Hz, 1H), 5.27 (s, 2H), 4.99 (dd, J = 8.2, 2.3 Hz, 1H), 4.25–4.19
13
(m, 1H), 4.12 – 4.07 (m, 1H), 3.73 – 3.66 (m, 1H); C NMR (100 MHz, CDCl3) δ
170.80, 163.59, 161.14, 136.94, 135.45, 132.59(d, J=3.2Hz), 128.57(d, J=8.2Hz),
126.20, 125.95, 125.38, 122.81, 120.23, 119.62, 115.99, 115.77, 115.42, 110.19,
70.04, 67.99, 49.51, 44.69. HRMS (ESI) calcd. C21H18Cl2NO3 [M+H]+m/z: 402.0658,
found: 402.0657.
Synthesis of 5-chloro-1H-indole-3-carbaldehyde (12): compound 11 (5.00 g,
32.98mmol) was converted to compound 12 (5.58 g, 94.72%) as a white solid by the
same procedure as described for 3.
General procedure for Synthesis of 5-chloro-1- methylene -1H-indole-3-
carbaldehyde (13): Compound 13 was synthesized from compound 12 used the same
reaction for the preparation of compound 4.
Synthesis of 4-((5-chloro-1-((5-chlorothiophen-2-yl) methyl)-1H-indol-3-yl)
(hydroxy)methyl) -3-methylenedihydrofuran-2(3H)-one (9a): compound 13a (700
mg, 2.37 mmol) was converted to compound 9a (520mg, 56.52%) as a white solid by
1
the same procedure as described for compound 1a. m.p.: 113.2-113.8℃. H NMR
(400 MHz, DMSO-d6) δ 7.69 (d, J = 1.9 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.39 (s,
1H), 7.09 (dd, J = 8.7, 2.0 Hz, 1H), 6.92 (d, J = 3.8 Hz, 1H), 6.89 (d, J = 3.8 Hz, 1H),
5.95 (d, J = 1.1 Hz, 1H), 5.55 (d, J = 4.7 Hz, 1H), 5.47 (s, 2H), 5.29 (s, 1H), 4.94 (t, J
= 5.3 Hz, 1H), 4.26 (t, J=8.7Hz, 1H), 4.13(dd, J=9.1, 4.0Hz, 1H), 3.51 – 3.38 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ170.55, 139.77, 135.58, 134.38, 128.08, 127.51,
127.24, 126.50, 126.30, 123.99, 123.19, 121.47, 119.00, 116.44, 111.79, 68.27, 68.23,
44.18, 43.98. HRMS (ESI) calcd. C19H15Cl2NO3SNa [M+Na]+m/z: 430.0042, found:
430.0049.
Synthesis
of
4-((5-chloro-1-((2-chlorothiazol-5-yl)
methyl)-1H-indol-3-yl)
(hydroxy) methyl) -3-methylenedihydrofuran-2(3H)-one (9b): compound 13b (700
mg, 2.24 mmol) was converted to compound 9b (485mg, 52.76%) as a white solid by
1
the same procedure as described for compound 1a. m.p.: 135.3-135.6℃. H NMR
(400 MHz, DMSO-d6) δ 7.77 (t, J = 5.3 Hz, 1H), 7.72 (s, 1H), 7.63 (d, J = 8.8 Hz,
1H), 7.49 (s, 1H), 7.18 (dd, J = 8.7, 1.8 Hz, 1H), 6.03 (s, 1H), 5.70 – 5.61 (overlap,
3H), 5.37 (s, 1H), 5.04 (t, J = 5.2 Hz, 1H), 4.35 (t, J=8.7Hz, 1H), 4.23(dd, J=9.1,
13
3.8Hz, 1H), 3.53 (s, 1H). C NMR (101 MHz, DMSO-d6) δ 170.56, 150.10, 140.12,
138.03, 135.55, 134.29, 127.91, 127.30, 124.19, 123.22, 121.65, 119.10, 116.73,
111.69, 68.27, 68.21, 43.98, 41.51. HRMS (ESI) calcd. C18H14Cl2N2O3SNa
[M+Na]+m/z: 430.9994, found: 431.0004.

ACCEPTED MANUSCRIPT
Synthesis
of
4-((1-((2-aminothiazol-5-yl)methyl)-
5-chloro-1H-indol-3-yl)
(hydroxy) methyl)-3-methylenedihydrofuran-2(3H)-one (9c): compound 13c
(700mg, 2.40 mmol) was converted to compound 9c (576mg, 61.58%) as an oily
1
yellow liquid by the same procedure as described for compound 1a. H NMR (400
MHz, DMSO-d6) δ 7.59 (d, J=1.9Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.22 (s, 1H), 6.98
(dd, J=8.7, 2.0Hz, 1H), 6.78 (s, 2H), 6.17 (s, 1H), 5.92 (d, J = 1.3 Hz, 1H), 5.45 (d, J
= 4.7 Hz, 1H), 5.30 (s, 1H), 4.98 (s, 2H), 4.87 – 4.81 (m, 1H), 4.18 (t, J=8.7Hz, 1H),
13
4.03 (dd, J=9.2, 4.0Hz, 1H), 3.42 – 3.32 (m, 1H). C NMR (100 MHz, DMSO-d6) δ
170.64, 168.81, 147.90, 135.55, 134.75, 128.62, 126.92, 123.51, 121.05, 118.70,
115.43, 111.94, 103.16, 68.29, 56.63, 55.80, 45.92, 44.04.HRMS (ESI) calcd.
C18H16ClN3O3SNa [M+Na]+m/z: 412.0493/414.0464, found: 412.0498/414.0496.
Synthesis of 4-((5-chloro-1-((3,5-dimethylisoxazol-4-yl) methyl)-1H-indol-3-yl)
(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one (9d): compound 13d
(700mg, 2.41 mmol) was converted to compound 9d (456mg, 48.71%) as a light
1
yellow oily liquid by the same procedure as described for compound 1a. H NMR
(400 MHz, DMSO-d6) δ 7.77 (d, J = 4.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.32 (s,
1H), 7.16 (dd, J = 8.8, 2.0 Hz, 1H), 6.04 (s, 1H), 5.59 (d, J = 4.7 Hz, 1H), 5.50 (s, 1H),
5.21 (s, 2H), 5.03 – 4.98 (m, 1H), 4.30 (t, J = 8.7 Hz, 1H), 4.18 (dd, J = 9.2, 4.0 Hz,
1H), 3.60 – 3.50 (m, 1H), 2.37 (s, 3H), 1.97 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 170.50, 166.67, 159.04, 135.76, 134.58, 127.96, 127.06, 123.83, 123.29, 121.43,
119.04, 115.92, 111.51, 110.36, 68.25(m), 55.79, 44.06, 38.11, 32.05, 29.56 (d, J =
4.4 Hz).HRMS (ESI) calcd. C20H19ClN2O4Na [M+Na]+m/z: 409.0926/411.0896,
found: 409.0932/411.0912.
Synthesis of 4-((5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl) (hydroxy)methyl)
-3-methylenedihydrofuran-2(3H)-one (9e): compound 13e (700mg, 2.59 mmol)
was converted to compound 9e (720mg, 76.70%) as a white solid by the same
1
procedure as described for compound 1a. m.p.: 152.4-152.8℃. H NMR (400 MHz,
DMSO-d6) δ 7.77(d, J=1.9Hz, 1H), 7.53 – 7.47(m, 2H), 7.27 – 7.21(m, 2H), 7.19 –
7.08(overlap, 3H), 6.04(s, 1H), 5.63(d, J=4.6Hz, 1H), 5.40(s, 2H), 5.03(t, J=5.4Hz,
1H), 4.35(t, J=8.7Hz, 1H), 4.23(dd, J=9.2Hz, 3.9Hz, 1H), 3.59 – 3.49(m, 1H).13C
NMR (100 MHz, DMSO-d6) δ 175.82, 166.69(d, J=242Hz), 140.91, 139.77, 139.38
(d, J=3.0Hz ), 134.31(d, J=8.3Hz), 133.88, 132.43, 129.01, 128.52, 126.58, 124.17,
121.15, 120.65, 120.43, 117.10, 73.51(d, J=11.5Hz), 53.60, 49.30. HRMS (ESI) calcd.
C21H17ClFNO3Na [M+Na]+m/z: 408.0773/410.0744, found: 408.0775/140.0735.
Synthesis of 4-((5-chloro-1-(pyridin-4-ylmethyl)- 1H-indol-3-yl)(hydroxy) methyl)
-3-methylenedihydrofuran-2(3H)-one (9f): compound 13f (700mg, 2.43 mmol) was
converted to compound 9f (643mg, 67.42%) as a white solid by the same procedure as
1
described for compound 1a. m.p.: 64.3-64.7℃. H NMR (400 MHz, CDCl3) δ 8.31 –
8.19 (m, 2H), 7.74 (d, J = 1.3 Hz, 1H), 7.15 – 7.10(overlap, 2H), 7.10–7.03 (m, 1H),
6.81(d, J=4.9Hz, 2H), 6.32 (s, 1H), 5.81 (s, 1H), 5.25 (s, 2H), 4.98 (d, J = 7.5 Hz, 1H),
13
4.24 (t, J=8.8Hz, 1H), 4.18 – 4.07(m, 1H), 3.67–3.57 (m, 1H). C NMR (100MHz,
CDCl3) δ 170.90, 149.80, 146.31, 135.26, 135.17, 127.58, 127.14, 126.14, 125.22,
123.26, 121.32, 119.54, 116.26, 110.96, 69.58, 68.07, 49.06, 44.72.RMS (ESI) calcd.
C20H17ClN2O3Na [M+Na]+m/z: 391.0820/393.0790, found: 391.0825/393.0821.

ACCEPTED MANUSCRIPT
Synthesis of 4-((5-chloro-1- ((5-methylpyridin-3-yl) methyl)-1H-indol-3-yl)
(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one (9g): compound 13g
(700mg, 2.46 mmol) was converted to compound 9g (613mg, 65.13%) as an oily
1
yellow liquid solid by the same procedure as described for compound 1a. H NMR
(400 MHz, DMSO-d6) δ 8.31 (s, 2H), 7.76 (d, J = 1.8 Hz, 1H), 7.52 (d, J = 10.2 Hz,
2H), 7.36 (s, 1H), 7.12(dd, J=8.7, 2.0Hz, 1H), 6.03 (s, 1H), 5.62 (d, J = 4.6 Hz, 1H),
5.41 (s, 3H), 5.04 (t, J = 5.3 Hz, 1H), 4.35(t, J=8.7Hz, 1H) 4.24 (dd, J=9.1, 3.8Hz,
13
1H), 3.59 – 3.51 (m, 1H), 2.23 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 170.52,
149.15, 145.63, 135.70, 134.99, 134.50, 132.86(d, J=5Hz), 128.58, 127.23, 123.87,
123.15, 121.44, 118.98, 116.02, 111.77, 68.32, 68.13, 46.59, 44.10, 29.53(d, J=5Hz)
17.73. HRMS (ESI) calcd. C21H19ClN2O3Na [M+Na]+m/z: 405.0976/407.0947, found:
405.0981/407.0952.
Synthesis of 4-((5-chloro-1-((5-chloropyridin-2-yl) methyl)-1H-indol-3-yl)
(hydroxy) methyl) -3-methylenedihydrofuran-2(3H)-one (9h): compound 13h
(700mg, 2.29 mmol) was converted to compound 9h (653mg, 70.59%) as a white
1
solid by the same procedure as described for compound 1a. m.p.: 163.1-163.8℃. H
NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 2.4 Hz, 1H), 7.87 (dd, J=8.4, 2.5Hz 1H),
7.78 (d, J = 1.9 Hz, 1H), 7.49 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.11 (dd, J = 8.7, 2.0
Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.06 (d, J = 1.2 Hz, 1H), 5.65 (d, J = 4.7 Hz, 1H),
5.51 (s, 2H), 5.44 (s, 1H), 5.04 (t, J = 5.3Hz, 1H), 4.35 (t, J=8.7Hz, 2H), 4.23 (dd,
J=9.2, 3.9Hz, 1H), 3.59–3.51(m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 170.61,
155.74, 147.77, 136.83, 135.57, 134.78, 129.96, 128.89, 127.18, 123.88, L.123.40,
122.57, 121.43, 118.93, 116.08, 111.80, 68.28 (d, J = 8.5 Hz), 55.79, 50.41,
44.05.HRMS (ESI) calcd. C20H16Cl2N2O3Na[M+Na]+m/z: 425.0430, found:
425.0435.
Synthesis of 4-((5-chloro-1-((6-chloropyridin-3-yl)methyl)- 1H-indol-3-yl)
(hydroxy) methyl)-3-methylenedihydrofuran-2(3H)-one (9i): compound 13i
(700mg, 2.29 mmol) was converted to compound 9i (596mg, 64.43%) as a white solid
1
by the same procedure as described for compound 1a. m.p.: 172.2-172.8℃. H NMR
(400 MHz, DMSO-d6) δ 8.35 (d, J = 1.9 Hz, 1H), 7.77 (s, 1H), 7.60 (dd, J = 8.3, 2.3
Hz, 1H), 7.57–7.52 (d, overlap, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.14 (dd, J = 8.7, 1.8
Hz, 1H), 6.03 (s, 1H), 5.63 (d, J = 4.5 Hz, 1H), 5.46 (s, 2H), 5.40 (s, 1H), 5.03 (t, J =
5.1 Hz, 1H), 4.34(t, J=8.6Hz, 1H), 4.22(dd, J=9.1, 3.7Hz, 1H), 3.59–3.49 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ 170.54, 149.41, 148.65, 138.51, 135.67, 134.44,
133.09, 128.51, 127.27, 124.24, 124.00, 123.25, 121.54, 119.03, 116.32, 111.78, 68.28,
68.16, 45.85, 44.04.HRMS (ESI) calcd. C20H16Cl2N2O3Na [M+Na]+m/z: 425.0430,
found: 425.0437.
Synthesis of 4-((5-chloro-1-((4-(3-methoxypropoxy)-3- methylpyridin-2-yl)
methyl)-1H-indol-3-yl)(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one
(9j): compound 13j (700mg, 1.88 mmol) was converted to compound 9j (425mg,
48.07%) as a white solid by the same procedure as described for compound 1a. m.p.:
71.2-71.4℃. 1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 5.6 Hz, 1H), 7.74 (d, J =
1.7 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.31 (s, 1H), 7.08 (dd, J = 8.7, 1.9 Hz, 1H),
6.93 (d, J = 5.7 Hz, 1H), 6.02 (s, 1H), 5.57 (d, J = 4.8 Hz, 1H), 5.46 (s, 2H), 5.43 (s,
1H), 5.01 (t, J = 5.3 Hz, 1H), 4.33(t, J=8.7Hz, 1H), 4.20(dd, J=9.1, 4.0Hz, 1H) 4.08(t,
J=6.2Hz, 2H), 3.56 –3.50 (m, 1H), 3.47 (t, J = 6.2 Hz, 2H), 3.23 (s, 3H), 2.12 (s, 3H),
2.01 –1.91 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.62, 162.69, 154.82,

ACCEPTED MANUSCRIPT
147.74, 135.52, 135.29, 128.93, 126.90, 123.51, 123.30, 121.06, 119.64, 118.67,
115.41, 111.99, 106.46, 68.34, 68.29, 65.04, 57.92, 49.80, 44.03, 28.64, 9.85. HRMS
(ESI) calcd. C25H28ClN2O5 [M+H]+m/z: 471.1681/473.1652, found: 471.1685/
473.1655.
Synthesis of 4-((5-chloro-1-(pyrimidin-2-ylmethyl)-1H-indol-3-yl) (hydroxy)
methyl)-3-methylenedihydrofuran-2(3H)-one (9k): compound 13k(700mg, 2.58
mmol) was converted to compound 9k (538mg, 56.47%) as an oily yellow liquid solid
by the same procedure as described for compound 1a. 1H NMR (400 MHz, DMSO-d6)
δ 8.25 (dd, J=5.6Hz, 1H), 8.02–7.85 (m, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 2.0
Hz, 1H), 7.73 – 7.67 (m, 1H), 7.55 – 7.48 (m, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.08 (dd,
J = 8.7, 2.0 Hz, 1H), 6.05 (d, J = 1.2 Hz, 1H), 5.69 (s, 2H), 5.64 (d, J = 4.9 Hz, 1H),
5.48(s, 1H), 5.07 (t, J = 5.3 Hz, 1H), 4.43 – 4.23 (m, 2H), 3.62–3.48 (m, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 208.89, 185.24, 169.67, 159.94, 148.82, 143.08,
136.09, 134.53, 127.90 (d, J = 7.8 Hz), 127.26, 125.74 (d, J = 5.5 Hz), 123.60, 122.51,
120.02, 116.70, 113.06, 68.58, 55.63, 52.74.HRMS (ESI) calcd.C19H17ClN3O3
[M+H]+m/z: 370.0953/372.0923, found: 370.0957/372.0933.
Synthesis of 4-((5-chloro-1-((4-methylquinazolin-2-yl)methyl) -1H-indol-3-yl)
(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one (9l): compound 13l
(700mg, 2.08 mmol) was converted to compound 9l (589mg, 65.12%) as a
yellow solid by the same procedure as described for compound 1a. m.p.: 59.8-60.4℃.
1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 4.9 Hz, 2H), 7.77 (d, J = 2.0 Hz, 1H),
7.44 (s, 1H), 7.43–7.36 (overlap, 2H), 7.09 (dd, J = 8.7, 2.0 Hz, 1H), 6.05 (d, J = 1.4
Hz, 1H), 5.68 – 5.56 (overlap, 3H), 5.43 (s, 1H), 5.04(t, J=5.4Hz, 1H), 4.36 (t,
J=8.7Hz, 1H), 4.22(dd, J=9.1, 4.0Hz, 1H)3.59 – 3.44 (m, 1H), 3.36 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 170.69, 165.70, 157.61, 135.37, 135.29, 129.33, 127.03,
123.62, 123.49, 121.21, 120.24, 118.73, 115.79, 111.75, 68.49, 68.40, 68.33, 56.63,
55.80, 51.80, 43.96, 32.06, 29.57, 29.52. MS (ESI) calcd. C24H20ClN3O3Na
[M+Na]+m/z: 456.1085/458.1056, found: 456.1081/458.1052.
Synthesis of 4-((1-(benzo[d]oxazol-2-ylmethyl) -5-chloro-1H-indol-3-yl) (hydroxy)
methyl)-3-methylenedihydrofuran-2(3H)-one (9m): compound 13m (700mg, 2.25
mmol) was converted to compound 9m (396mg, 43.00%) as a yellow solid by the
same procedure as described for compound 1a. m.p.: 67.2-67.4℃. 1H NMR (400
MHz, DMSO-d6) δ 7.62 (d, J = 1.8 Hz, 1H), 7.61–7.55 (m, 1H), 7.40–7.36(m, 1H),
7.33 (d, J = 8.8 Hz, 1H), 7.30 – 7.21 (overlap, 2H), 7.19 (s, 1H), 7.15 (dd, J = 8.8, 1.9
Hz, 1H), 6.29 (d, J = 2.0 Hz, 1H), 5.75 (d, J = 1.4Hz, 1H), 5.40 (s, 2H), 4.87 (d, J =
7.8 Hz, 1H), 4.16 (t, J=9.4Hz, 1H), 4.04(dd, J=9.6, 4.2Hz, 1H) 3.56–3.48 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ165.60, 155.25, 145.63, 135.26, 130.15, 129.76,
122.48, 121.85, 121.19, 120.64, 120.30, 119.66, 118.24, 115.11, 114.04, 110.93,
105.87, 105.63, 64.41, 62.82, 39.26, 38.65. MS (ESI) calcd. C22H17ClN2O4Na
[M+Na]+m/z: 431.0769/433.0740, found: 431.0768/433.0743.
Synthesis of 4-((1-(benzo[d]thiazol-2-ylmethyl)-5-chloro -1H-indol-3-yl) (hydroxy)
methyl)-3-methylenedihydrofuran-2(3H)-one (9n): compound 13n (700mg, 2.14
mmol) was converted to compound 9n (508mg, 55.82%) as a yellow solid by the
same procedure as described for compound 1a. m.p.: 71.2-71.4℃. 1H NMR (400
MHz, CDCl3) δ 7.98 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 1.7 Hz,
1H), 7.51–7.43 (m, 1H), 7.37 (t, J = 7.4 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.22 (s, 1H),

ACCEPTED MANUSCRIPT
7.18 (dd, J = 8.8, 1.8 Hz, 1H), 6.36(d, J = 1.9 Hz, 1H), 5.84 (d, J = 1.3 Hz, 1H), 5.61
(s, 2H), 4.96 (d, J =7.7 Hz, 1H), 4.29 –4.21 (m, 1H), 4.13(dd, J=9.6, 4.3Hz, 1H), 3.66
– 3.56 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 170.80, 166.88, 152.84, 135.27,
135.05, 134.96, 127.53, 127.21, 126.58, 126.53, 125.77, 125.54, 123.54, 123.23,
121.85, 119.39, 116.43, 111.18, 69.72, 68.04, 48.67, 44.56. MS (ESI) calcd.
C22H18ClN2O3S [M+H]+m/z: 425.0721/427.0692, found: 425.0721/427.0691.
Synthesis of 4-((1-((1H-benzo[d]imidazol-2-yl)methyl) -5-chloro-1H-indol-3-yl)
(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one (9o): compound 13o
(700mg, 2.26 mmol) was converted to compound 9o (639mg, 69.33%) as a
yellow solid by the same procedure as described for compound 1a. m.p.: 58.6-59.0℃.
1H NMR (400 MHz, DMSO-d6) δ 7.77 (d, J=1.8Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H),
7.56–7.50(overlap, 1H), 7.48(s, 1H) 7.20 – 7.11 (overlap, 3H), 6.03 (s, 1H), 5.66 –
5.62 (m, 1H), 5.60 (s, 2H), 5.44 (s, 1H), 5.03 – 4.98 (m, 1H), 4.59–4.49 (m, 1H),
4.35(t, J=8.7Hz, 1H), 4.20 (dd, J=9.1Hz, 1H), 3.56-3.48 (m, 1H). 13C NMR (101
MHz, DMSO-d6) δ 170.66, 150.12, 135.51, 135.01, 128.67, 127.11, 123.90, 123.51,
121.43, 118.85, 116.22, 111.88, 68.68, 68.49, 68.37, 68.27, 56.63, 55.80, 43.98, 43.85,
32.06, 29.57.MS (ESI) calcd. C22H18ClN3O3Na [M+Na]+m/z: 430.0929/432.0899,
found: 430.0927/432.0898.
Synthesis of 4-((5-chloro-1-(cyclopropylmethyl) -1H-indol-3-yl) (hydroxy)
methyl)-3-methylenedihydrofuran-2(3H)-one (9p): compound 13p (700mg, 3.00
mmol) was converted to compound 9p (716mg, 72.04%) as an oily yellow liquid solid
by the same procedure as described for compound 1a. 1H NMR (400 MHz, CDCl3) δ
7.70 (d, J = 1.9 Hz, 1H), 7.30 – 7.26 (m, 1H), 7.23 (s, 1H), 7.19 (dd, J = 8.7, 1.9 Hz,
1H), 6.37 (d, J = 2.1 Hz, 1H), 5.89 (d, J = 1.5 Hz, 1H), 4.92 (d, J = 8.2 Hz, 1H), 4.26
– 4.16 (m, 1H), 4.07 (dd, J = 9.6, 4.4 Hz, 1H), 3.93 (dd, J = 6.8, 3.7 Hz, 2H), 3.67–
3.58 (m, 1H), 2.16 (s, 1H), 0.68–0.58 (m, 2H), 0.38–0.33 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 170.86, 135.35, 135.30, 127.00, 126.73, 125.54, 125.40, 122.59,
118.94, 114.27, 111.01, 69.88, 68.06, 50.91, 44.67, 11.07, 4.18, 4.15.MS (ESI) calcd.
C18H18ClNO3Na [M+Na]+m/z: 354.0867/356.0838, found: 354.0863/356.0837.
General Procedure for 1-formyl-5-chloro-1H-indole-3-carbaldehyde (14): To the
compound 12 (1 equiv) in MeCN was added NaH (3 equiv) and Acyl chloride
derivatives (1.5 equiv) at room temperature. The reaction mixture was stirred at room
temperature for 4 h and was then adjust with HCl (1 M) until pH 6-7. The mixture
was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and filtered. The
filtrate was concentrated in vacuo, and the residue was purified by column
chromatography (silica gel, ethyl acetate/Petroleum ether) to give the desired product.
Synthesis of 4-((5-chloro-1-(3-methylthiophene-2-carbonyl) -1H-indol-3-yl)
(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one (10a): compound 14a
(600mg, 1.98 mmol) was converted to compound 10a (586mg, 73.82%) as a white
solid by the same procedure as described for compound 1a. m.p.: 194.8-195.6℃. 1H
NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 8.8 Hz, 1H), 8.00 – 7.89 (overlap, 2H),
7.55 (s, 1H), 7.42 (dd, J = 8.8, 2.0 Hz, 1H), 7.17 (d, J = 5.0 Hz, 1H), 6.07 (d, J = 1.6
Hz, 1H), 5.84 (d, J = 5.1 Hz, 1H), 5.39 (d, J = 0.9 Hz, 1H), 5.07 (t, J = 5.3 Hz, 1H),
4.40 (t, J=8.7Hz, 1H), 4.27(dd, J=9.2, 3.7Hz, 1H), 3.66 – 3.48 (m, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 170.46, 162.05, 143.78, 135.13, 134.23, 131.39, 131.37,
129.97, 128.22, 128.10, 126.69, 124.75, 123.58, 122.45, 119.90, 117.08, 68.37, 67.81,

ACCEPTED MANUSCRIPT
43.29, 15.43.MS (ESI) calcd. C20H16ClNO4[M+Na] +m/z: 424.0381/426.0351, found:
424.0382/426.0351.
Synthesis of 4-((5-chloro-1-(furan-2-carbonyl)-1H-indol-3-yl) (hydroxy)methyl)
-3-methylenedihydrofuran-2(3H)-one (10b): compound 14b (600mg, 2.19 mmol)
was converted to compound 10b (653mg, 80.12%) as a yellow solid by the same
procedure as described for compound 1a. m.p.: 65.3-65.9℃. 1H NMR (400 MHz,
CDCl3) δ 8.33 (d, J = 8.9 Hz, 1H), 7.97 (s, 1H), 7.68(d, J=0.9Hz, 1H), 7.62 (d, J = 2.0
Hz, 1H), 7.42 (d, J = 3.5 Hz, 1H), 7.23–7.26 (m, 1H), 6.64 (dd, J = 3.6, 1.7 Hz, 1H),
6.34 (d, J = 2.1 Hz, 1H), 5.75 (d, J = 1.6 Hz, 1H), 4.95 (d, J = 7.2 Hz, 1H), 4.36 –
4.25 (m, 1H), 4.20(dd, J=9.6, 3.9Hz, 1H), 3.62–3.53 (m, 1H).13C NMR (100 MHz,
CDCl3) δ 170.83, 156.22, 146.94, 146.36, 135.32, 134.44, 129.84, 128.98, 125.75,
125.42, 121.86, 121.69, 119.32, 117.98, 112.70, 69.39, 68.12, 43.94, 29.20.MS (ESI)
calcd. C19H14ClNO5K [M+K] +m/z: 410.0192/412.0163, found: 410.0192/412.0161.
Synthesis of 4-((5-chloro-1-(3,5-dimethylisoxazole-4-carbonyl) -1H-indol-3-yl)
(hydroxy)methyl)-3-methylenedihydrofuran-2(3H)-one (10c): compound 14c
(600mg, 1.97 mmol) was converted to compound 10c (560mg, 70.60%) as a
light-yellow solid by the same procedure as described for compound 1a. m.p.:
145.8-146.2℃. 1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 8.8 Hz, 1H), 7.93 (d, J
= 1.9 Hz, 1H), 7.53 (s, 1H), 7.44 (dd, J = 8.8, 2.0 Hz, 1H), 6.09 (d, J = 1.1 Hz, 1H),
5.81 (t, J = 5.4 Hz, 1H), 5.57 (s, 1H), 4.99 (t, J = 5.9 Hz, 1H), 4.36(t, J=8.7Hz, 1H),
4.22(dd, J=9.2, 3.9Hz, 1H),, 3.67 – 3.55 (m, 1H), 2.44 (s, 3H), 2.29 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 171.29, 170.40, 160.85, 158.78, 135.32, 133.89, 130.43,
128.27, 126.38, 124.85, 123.89, 122.96, 120.03, 117.23, 111.63, 68.12, 67.71, 43.31,
12.58, 10.29. MS (ESI) calcd. C20H17ClN2O5Na [M+Na] +m/z: 423.0718/425.0689,
found: 423.0716/425.0688.
Synthesis of 4-((5-chloro-1-cinnamoyl-1H-indol-3-yl)(hydroxy) methyl)-3-
methylenedihydrofuran-2(3H)-one (10d): compound 14d (600mg, 1.94 mmol) was
converted to compound 10d (589mg, 74.55%) as a yellow solid by the same
procedure as described for compound 1a. m.p.: 177.5-178.2℃. 1H NMR (400 MHz,
DMSO-d6) δ 8.50 (d, J = 8.9 Hz, 1H), 8.37 (s, 1H), 8.00–7.53 (overlap, 3H), 7.91 (d,
J = 2.1 Hz, 1H), 7.79 (d, J = 15.4 Hz, 1H), 7.54 – 7.47 (m, 3H), 7.41 (dd, J = 8.8, 2.0
Hz, 1H), 6.13 (d, J = 1.5 Hz, 1H), 5.91 (d, J = 4.9 Hz, 1H), 5.57 (s, 1H), 4.99 (t,
J=8.7Hz, 1H), 4.42(t, J=8.7Hz, 1H), 4.27(dd, J=9.3, 3.8Hz, 1H), 3.73 – 3.55 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ 170.47, 164.08, 146.65, 135.25, 134.36, 134.26,
130.89, 130.21, 129.06, 128.86, 127.98, 125.44, 124.68, 124.02, 122.57, 119.69,
117.88, 117.39, 68.23, 67.79, 43.32. MS (ESI) calcd. C23H18ClNO4 [M+Na]+m/z:
430.0817/432.0787, found: 430.0815/432.0789.
Synthesis
of
4-((5-chloro-1-(2-chloronicotinoyl)-1H-indol-3-yl) (hydroxy)
methyl)-3- methylenedihydrofuran-2(3H)-one (10e): compound 14e (600mg, 1.88
mmol) was converted to compound 10e (610mg, 77.76%) as a white solid by the same
1
procedure as described for compound 1a. m.p.: 74.2-74.6℃. H NMR (400 MHz,
DMSO-d6) δ 8.48 (dd, J = 4.9, 1.9 Hz, 1H), 8.17 (s, 1H), 8.09 (dd, J = 7.6, 1.9 Hz,
1H), 7.75 (d, J = 2.1 Hz, 1H), 7.49 (dd, J = 7.6, 4.9 Hz, 1H), 7.28 (dd, J = 8.8, 2.1 Hz,
1H), 6.95 (s, 1H), 5.83 (d, J = 1.8 Hz, 1H), 5.59 (d, J=5.4Hz, 1H), 5.15 (s, 1H), 4.77(t,
J=5.5Hz, 1H), 4.14(t, J=8.7Hz, 1H), 3.98 (dd, J=9.2, 3.7Hz, 1H), 3.39 –3.23 (m, 1H).
13C NMR (100 MHz, DMSO-d6) δ 170.44, 164.01, 151.96, 145.88, 138.76, 134.95,

ACCEPTED MANUSCRIPT
133.86, 130.42, 130.31, 128.86, 125.76, 125.38, 123.99, 123.71, 123.62, 120.20,
117.37, 68.38, 67.69, 43.07.MS (ESI) calcd.C20H14Cl2N2O4K[M+K]+m/z: 454.9962,
found: 455.1000.
Synthesis of 4-((5-chloro-1-(cyclopropanecarbonyl)-1H-indol-3-yl) (hydroxy)
methyl) -3- methylenedihydrofuran-2(3H)-one (10f): compound 14f (600mg, 2.42
mmol) was converted to compound 10f (581mg, 69.36%) as a light-yellow solid by
1
the same procedure as described for compound 1a.mp: 75.3-75.9℃. H NMR (400
MHz, DMSO-d6) δ 8.34 (d, J = 8.9 Hz, 1H), 8.16 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H),
7.36 (dd, J = 8.8, 2.1 Hz, 1H), 6.11 (d, J = 1.4 Hz, 1H), 5.91 (d, J = 5.0 Hz, 1H), 5.49
(s, 1H), 5.05 (t, J = 5.5 Hz, 1H), 4.42 (t, J=8.7Hz, 1H), 4.27(dd, J=9.2, 3.8Hz,
1H)3.69–3.49 (m, 1H), 2.80 –2.64 (m, 1H), 1.23 (s, 2H), 0.91 – 0.75 (m, 2H). MS
(ESI)
384.0400/386.0372.
calcd.C18H16ClNO4K[M+K]+
m/z:
384.0399/386.0370,
found:
General Procedure for synthesis of substitued-1H-indole-3-carbaldehydes (18a-i):
compounds 18a-i was prepared from compounds 17a-i by the same procedure as
described for compound 3.
Synthesis of 5-chloro-1-tosyl-1H-indole-3-carbaldehyde (19): To the solution of
compound 18f (200mg, 1.11 mmol) in MeCN(5mL) was added NaH (80.17mg, 3.34
mmol) and paratoluensulfonyl chloride (318.44mg, 1.67 mmol) at room temperature.
The reaction mixture was stirred at room temperature for 4 h. The mixture was
extracted with ethyl acetate, washed with brine, dried (Na2SO4), and filtered. The
filtrate was concentrated in vacuo, and the residue was purified by column
chromatography (silica gel, ethyl acetate/Petroleum ether) to give the product
compound 19 (283mg, 76.14%).
synthesis of 4-((5-chloro-1-tosyl-1H-indol-3-yl)(hydroxy)methyl) -3-methylene
dihydrofuran-2(3H)-one (15): compound 19 (600mg, 2.42 mmol) was converted to
compound 15(581mg, 69.36%) as a white solid by the same procedure as described
for compound 1a. m.p.: 185.3-185.7℃. 1H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J
= 8.9 Hz, 1H), 7.93 – 7.85(overlap, 3H), 7.66 (s, 1H), 7.40 (dd, J = 8.9, 2.1 Hz, 1H),
7.15 – 7.11 (m, 1H), 7.11 – 7.08 (m, 1H), 5.98 (d, J = 1.6 Hz, 1H), 5.86 (d, J = 5.1 Hz,
1H), 5.06 – 4.99 (m, 2H), 4.34 (t, J=8.7Hz, 1H), 4.21(dd, J=9.2, 3.7Hz, 1H), 3.80 (s,
13
3H), 3.55–3.44 (m, 1H). C NMR (100 MHz, DMSO-d6) δ 170.40, 163.93, 135.03,
133.17, 129.89, 129.11, 128.02, 127.99, 125.52, 124.85, 123.53, 123.28, 120.29,
115.05, 114.87, 68.32, 67.71, 55.90, 43.00. MS (ESI) calcd. C21H18ClNO5SK
[M+K]+m/z: 470.0226/472.0196, found: 470.0227/472.0194.
General Procedure for synthesis of substituted-1-(4-chlorobenzyl)- 1H-indole
-3-carbaldehydes (20a-i): To the solution of compounds 18a-i (1 equiv) in MeCN
was added NaH (3 equiv) and 4-Chlorobenzyl chloride (1.5 equiv) at room
temperature. The reaction mixture was stirred at room temperature for 4 h. The
mixture was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and
filtered. The filtrate was concentrated in vacuo, and the residue was purified by
column chromatography (silica gel, ethyl acetate/Petroleum ether) to give the desired
product.

ACCEPTED MANUSCRIPT
Synthesis of 4-((1-(4-chlorobenzyl)-4-methoxy-1H-indol-3-yl) (hydroxy)methyl)-
3-methylenedihydrofuran-2(3H)-one (16a): compound 20a (200mg, 667.21 µmol)
was converted to compound 16a (165mg, 62.16%) as a white solid by the same
procedure as described for compound 1a. m.p.: 134.5-135.1℃. 1H NMR (400 MHz,
DMSO-d6) δ 7.38 (d, J=8.4Hz, 2H), 7.26 (s, 1H), 7.19 (s, 1H), 7.17 (s, 1H), 7.04 –
6.97 (overlap, 2H), 6.57 – 6.47 (t, J=1.0Hz, 1H), 5.94 (s, 1H), 5.39 (d, J=5.1Hz, 1H),
5.36 (s, 2H), 5.29 (t, J = 4.9 Hz, 1H), 4.94 (s, 1H), 4.42(t, J=8.5Hz, 1H), 4.31(dd,
J=9.0, 3.5Hz, 1H), 3.84 (s, 3H), 3.55 – 3.44 (m, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 170.89, 153.72, 137.60, 137.31, 135.04, 131.86, 128.82, 128.42, 125.88, 122.67,
122.31, 116.77, 115.61, 103.65, 99.72, 69.10, 68.78, 55.16, 48.38, 44.77.HRMS (ESI)
calcd. C22H20ClNO4Na [M+Na]+m/z: 420.0973/422.0944, found: 420.0977/422.0935.
Synthesis of 4-((1-(4-chlorobenzyl)-5-methoxy- 1H-indol-3-yl)(hydroxy) methyl)
-3-methylenedihydrofuran-2(3H)-one (16b): compound 20b (200mg, 667.21 µmol)
was converted to compound 16b (186mg, 70.07%) as a yellow solid by the same
procedure as described for compound 1a. m.p.: 62.4-62.6℃. 1H NMR (400 MHz,
DMSO-d6) δ 7.36 (s, 2H), 7.34(s, 1H), 7.27 (d, J = 8.9 Hz, 1H), 7.21 – 7.10 (overlap,
3H), 6.74 (dd, J=8.9, 2.3Hz, 1H), 6.05 (d, J = 1.2 Hz, 1H), 5.59 (d, J = 4.3 Hz, 1H),
5.46 (s, 1H), 5.33 (s, 2H), 5.02 (t, J=5.4Hz, 1H), 4.35 (t, J = 8.7 Hz, 1H), 4.24 (dd,
J=9.2, 4.0Hz, 1H), 3.75 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 170.66, 153.36,
137.39, 135.78, 131.84, 131.23, 128.73, 128.40, 127.57, 126.55, 123.28, 115.60,
111.44, 110.89, 101.54, 68.46, 68.19, 55.38, 48.39, 43.97.HRMS (ESI) calcd.
C22H20ClNO4Na [M+Na]+m/z: 420.0973/422.0944, found: 420.0976/422.0940.
Synthesis of 4-((1-(4-chlorobenzyl)-6-methoxy-1H-indol -3-yl)(hydroxy)methyl)
-3-methylenedihydrofuran-2(3H)-one (16c): compound 20c (200mg, 667.21 µmol)
was converted to compound 16c (189mg, 71.20%) as a deep yellow solid by the same
procedure as described for compound 1a. m.p.: 59.5-60.3℃. 1H NMR (400 MHz,
CDCl3) δ 7.60 (d, J = 8.7 Hz, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 7.02 (s, 1H), 7.00 (s,
1H), 6.95 (s, 1H), 6.80 (dd, J = 8.7, 2.2 Hz, 1H), 6.68 (d, J = 2.1 Hz, 1H), 6.34 (d, J =
2.2 Hz, 1H), 5.88 (d, J = 1.3 Hz, 1H), 5.18 (s, 2H), 4.92 (d, J = 7.5 Hz, 1H), 4.23 –
4.15 (m, 1H), 4.12– 4.07 (m, 1H), 3.79 (s, 3H), 3.68– 3.57 (m, 1H).HRMS (ESI)
calcd. C22H20ClNO4Na [M+Na]+m/z: 420.0973/422.0944, found: 420.0978/422.0950.
Synthesis of 4-((1-(4-chlorobenzyl)-7-methoxy-1H -indol-3-yl)(hydroxy) methyl)
-3-methylenedihydrofuran-2(3H)-one (16d): compound 20d (200mg, 667.21 µmol)
was converted to compound 16d (181mg, 68.18%) as a yellow solid by the same
procedure as described for compound 1a. m p : 70.1-70.6℃. 1H NMR (400 MHz,
DMSO-d6) δ 7.32 (d, J=8.4Hz, 2H), 7.28 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.09 (d, J
= 5.1 Hz, 2H), 6.92 (t, J = 7.9 Hz, 1H), 6.64 (d, J = 7.7 Hz, 1H), 6.00 (d, J = 1.4 Hz,
1H), 5.51 (d, J = 6.6 Hz, 2H), 5.34 (s, 1H), 4.99 (d, J = 5.9 Hz, 1H), 4.34 (t, J=8.7Hz,
1H), 4.24(dd, J=9.2, 3.8Hz, 1H), 3.77 (s, 3H), 3.54 – 3.50 (m, 1H).13C NMR (100
MHz, DMSO-d6) δ 170.80, 146.99, 138.81, 135.35, 131.55, 128.49, 128.33, 128.23,
127.97, 125.34, 123.47, 119.94, 116.22, 112.24, 103.02, 68.42, 68.23, 55.22, 50.97,
43.99. HRMS (ESI) calcd. C22H20ClNO4Na [M+Na]+m/z: 420.0973/422.0944, found:
420.0974/422.0945.
Synthesis of 4-((4-chloro-1-(4-chlorobenzyl)- 1H-indol-3-yl) (hydroxy) methyl)-
3-methylenedihydrofuran-2(3H)-one (16e): compound 20e(200mg, 657.53 µmol)
was converted to compound 16e (190mg, 71.83%) as a white solid by the same

ACCEPTED MANUSCRIPT
1
procedure as described for compound 1a. H NMR (400 MHz, CDCl3, this molecule
appears as conformers in a 1: 2 ratio) δ 7.34 –7.27(overlap, 2H), 7.26 –7.07(overlap,
4H), 7.06 – 6.98(overlap, 2H), 6.35(d, J=2.6Hz, 0.34H), 6.29(d, J=2.1Hz, 0.62H),
5.78 (d, J = 4.5 Hz, 0.32H), 5.68 (d, J = 6.4 Hz, 0.62H), 5.62 (d, J = 2.2 Hz, 0.34H),
5.44 (d, J = 1.1 Hz, 0.63H), 5.26 (s, 2H), 4.56(dd, J=9.4, 4.0Hz, 0.35H), 4.40 (t,
J=8.9Hz, 0.65H), 4.31 (dd, J = 9.4, 4.0 Hz, 0.64H), 4.25 (t, J = 8.9 Hz, 0.32H), 3.84 –
3.74 (m, 0.33H), 3.71 – 3.60 (m, 0.65H). HRMS (ESI) calcd. C21H17Cl2NO3Na
[M+Na]+m/z: 424.0478, found: 424.0485.
Synthesis of 4-((5-chloro-1-(4-chlorobenzyl)- 1H-indol-3-yl) (hydroxy) methyl)
-3-methylenedihydrofuran-2(3H)-one (16f): compound 20f (200mg, 657.53 µmol)
was converted to compound 16f (220mg, 83.17%) as a light-yellow solid by the same
procedure as described for compound 1a. m.p.: 144.8-145.2℃. 1H NMR (400 MHz,
DMSO-d6) δ 7.72 (s, 1H), 7.30 (s, 1H), 7.27 (s, 1H), 7.20 – 7.14 (overlap, 2H), 7.14 –
7.07 (m, 1H), 7.02 (s, 1H), 6.99 (s, 1H), 6.38 (d, J = 2.1 Hz, 1H), 5.88 (d, J = 1.5 Hz,
1H), 5.24 (s, 2H), 4.94(d, J=8.7Hz, 1H), 4.26 – 4.19(m, 1H), 4.10(dd, J=9.6, 4.2Hz,
1H), 3.67–3.58 (m, 1H).13C NMR (100 MHz, CDCl3) δ 170.63, 135.29, 135.21,
134.92, 133.99, 129.19, 128.08, 127.52, 126.94, 126.11, 125.45, 123.18, 119.24,
115.10, 111.24, 69.88, 67.87, 49.77, 44.64. HRMS (ESI) calcd. C21H17Cl2NO3Na
[M+Na]+m/z: 424.0478, found: 424.0480.
Synthesis
of
4-((6-chloro-1-(4-chlorobenzyl)-1H-indol-3-yl)(hydroxy)methyl)-3-methylenedihy
drofuran-2(3H)-one (16g): compound 20g (200mg, 657.53 µmol) was converted to
compound 16g (196mg, 74.10%) as a white solid by the same procedure as described
for compound 1a. m.p.: 150.6-150.8℃. 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J =8.5
Hz, 1H), 7.29 (s, 1H), 7.26(d, J=2.6Hz, 1H), 7.24 (d, J = 1.6 Hz, 1H), 7.10 (dd, J =
8.5, 1.8 Hz, 1H), 7.06 (s, 1H), 7.01 (s, 1H), 6.99 (s, 1H), 6.32 (d, J = 2.0 Hz, 1H),
5.81 (d, J = 1.4 Hz, 1H), 5.19 (s, 2H), 4.94 (d, J = 7.8 Hz, 1H), 4.23–4.16 (m, 1H),
4.13 – 4.04 (m, 1H), 3.64–3.54 (m, 1H).13C NMR (100 MHz, CDCl3) δ 170.85,
137.25, 135.14, 134.92, 133.89, 129.16, 128.79, 128.10, 126.91, 125.38, 124.54,
120.90, 120.61, 115.75, 110.08, 69.75, 67.98, 49.53, 44.69. HRMS (ESI) calcd.
C21H17Cl2NO3Na [M+Na]+m/z: 424.0478, found: 424.0480.
Synthesis of 4-((7-chloro-1-(4-chlorobenzyl)-1H-indol-3 -yl)(hydroxy)methyl)-
3-methylenedihydrofuran-2(3H)-one (16h): compound 20h (200mg, 657.53 µmol)
was converted to compound 16h (161mg, 60.87%) as a white solid by the same
procedure as described for compound 1a. m.p.: 131.2-131.5℃. 1H NMR (400 MHz,
CDCl3) δ 7.62 (d, J = 7.9 Hz, 1H), 7.28–7.25 (overlap, 2H), 7.24(s, 1H), 7.18 (d, J =
7.5 Hz, 1H), 7.07 (s, 1H), 7.04 (d, J = 7.8 Hz, 1H), 6.95 (s, 1H), 6.93 (s, 1H), 6.34 (d,
J = 2.1 Hz, 1H), 5.81 (d, J = 1.5 Hz, 1H), 5.76–5.62 (m, 2H), 4.98 (d, J = 7.8 Hz, 1H),
4.22(t, J=8.7Hz, 1H), 4.10 (dd, J=9.6, 4.2Hz, 1H).13C NMR (100 MHz, DMSO-d6) δ
165.52, 131.77, 129.85, 128.20, 126.90, 123.82, 123.76, 123.74, 122.35, 120.23,
119.40, 115.82, 113.12, 111.90, 110.76, 64.38, 62.72, 46.05, 39.36. HRMS (ESI)
calcd. C21H17Cl2NO3Na [M+Na]+m/z: 424.0478, found: 420.0485.
Synthesis of 4-((1-(4-chlorobenzyl)-5-fluoro-1H- indol-3-yl)(hydroxy)methyl)
-3-methylene dihydrofuran-2(3H)-one (16i): compound 20i (600mg, 695.12 µmol)
was converted to compound 16i (213mg, 79.42%) as an oily yellow liquid by the
same procedure as described for compound 1a. m.p.: 67.3-67.7℃. 1H NMR (400

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ 7.51 (s, 1H), 7.47 (dd, J = 10.1, 2.5 Hz, 1H), 7.41 (dd, J = 9.0, 4.5
Hz, 1H), 7.37(d, J=8.4Hz, 2H), 7.19(d, J=8.4Hz, 2H), 7.00 – 6.92(m, 1H), 6.06 (d, J =
1.3 Hz, 1H), 5.66 (d, J = 4.6 Hz, 1H), 5.46 (s, 1H), 5.40 (s, 2H), 5.03 (t, J=5.4Hz, 1H),
4.35 (t, J=8.7Hz, 1H), 4.26(dd, J=9.2, 3.9Hz, 1H), 3.62 – 3.56(m, 1H). 13C NMR (100
MHz, DMSO-d6) δ 170.65, 156.90 (d, J=230Hz), 137.02, 135.61, 132.71, 132.00,
128.89, 128.78, 128.46, 126.38 (d, J = 10 Hz), 123.41, 116.07 (d, J = 4 Hz), 111.26 (d,
J = 10 Hz), 109.59 (d, J = 26 Hz), 104.46 (d, J = 23 Hz), 68.38, 68.15, 48.49,
44.03.HRMS (ESI) calcd. C21H17ClFNO3Na [M+Na]+m/z: 408.0773/410.0744, found:
408.0776/410.0750.
Synthesis of 5-chloro-1-(4-fluorobenzyl)-1H-indole (23): To the solution of
5-chloroindole (17) (200mg, 1.11 mmol) in MeCN(3mL) was added NaH (80.17mg,
3.34 mmol). The resulting mixture was stirred at room temperature for 1h, then a
solution of 4-fluorobenzylchloride (22（) 241.49mg, 1.67 mmol）in MeCN(2mL) added
dropwise to the system. The reaction mixture was stirred at room temperature for 4 h.
The mixture was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and
filtered. The filtrate was concentrated in vacuo, and the residue was purified by
column chromatography (silica gel, ethyl acetate/Petroleum ether=12: 1) to give the
product 23(300mg, 93.63%).
Synthesis of (E)-1-(5-chloro-1- (4-fluorobenzyl)- 1H-indol-3-yl)-3- (furan-2-yl)
prop-2-en-1-one (24a): To the mixture of AlCl3 (205.36mg, 1.54mmol) in
CH2Cl2(5mL) was added acetyl chloride (120.90mg, 1.54 mmol). The resulting
mixture was stirred at room temperature for 1h, then a solution of compound 23
（200mg, 770.09 µmol）in CH2Cl2(1mL) added dropwise. The reaction mixture was
stirred at room temperature for 12 h. CH2Cl2 was evaporated in vacuo. The mixture
was extracted with ethyl acetate, washed with brine, dried (Na2SO4), and filtered. The
filtrate was concentrated in vacuo, and the residue was purified by column
chromatography (silica gel, ethyl acetate/Petroleum ether=1: 4) to give the
intermediate compound (180mg, 77.46%). It (100mg, 331.41 µmol) mixed with a
solution of 2-furfural(47.76mg, 497.11µmol) in EtOH (2mL), NaOH(aq)(30µl) at
room temperature. After workup, the resulting mixture was extracted with ethyl
acetate, washed with brine, dried (Na2SO4), and concentrated. The crude residue was
purified by flash chromatography on silica gel to give the product 24a(85mg,
67.53%). m.p.: 107.3-106.8℃. 1H NMR (400 MHz, Acetone-d6) δ8.67 (s, 1H), 8.50
(d, J = 2.0 Hz, 1H), 7.70 (dd, J = 13.0, 2.3 Hz, 1H), 7.56 (d, J = 15.3 Hz, 1H), 7.51 (d,
J = 8.7 Hz, 1H), 7.47–7.35 (overlap, 3H), 7.24 (dd, J = 8.7, 2.1 Hz, 1H), 7.17–7.04
(m, 2H), 6.86 (d, J = 3.4 Hz, 1H), 6.60 (dd, J = 3.3, 1.8 Hz, 1H), 5.60 (s, 2H).13C
NMR (100 MHz, Acetone-d6) δ183.80, 163.29(d, J= 243Hz ), 152.92, 145.64,
138.15, 136.51, 133.72(d, J=3Hz), 130.33 (d, J = 8 Hz), 129.23, 128.75, 128.11,
124.41, 122.80, 122.19, 118.14, 116.46(d, J=22Hz), 115.83, 113.46, 113.17,
50.75.HRMS (ESI) calcd. C22H15ClFNO2Na [M+Na]+m/z: 402.0668/404.0638, found:
402.0672/406.0640.
Synthesis of (E)-1-(5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl) -3-(thiophen-2-yl)
prop-2-en-1-one (24b): Compound 23 was converted to compound 24b as a yellow
solid by the same procedure as described for compound 24a. m.p.: 109.6-111.7℃. 1H
NMR (400 MHz, Acetone-d6) δ 8.72 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 7.90 (d, J =
15.3 Hz, 1H), 7.59 (d, J = 5.1 Hz, 1H), 7.55–7.47 (overlap, 2H), 7.46–7.36 (overlap,
3H), 7.25 (dd, J=8.7, 2.1Hz, 1H), 7.19–7.06 (overlap, 3H), 5.60 (s, 2H).13C NMR

ACCEPTED MANUSCRIPT
(100 MHz, Acetone-d6)δ183.74, 163.27(d, J=243Hz), 141.48, 138.27, 136.52, 134.17,
133.76 (d, J=3Hz), 132.22, 130.21 (d, J = 8 Hz), 129.25, 129.18(d, J=4Hz), 128.74,
124.40, 123.56, 122.79, 118.02, 116.56, 116.34, 113.19, 50.72. HRMS (ESI) calcd.
C22H16ClFNOS [M+H]+m/z: 396.0620/398.0590, found: 396.0628/398.0485.
Synthesis of 1-(5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)-1-ones (21a~21i) with
compound 21a as sample: To the solution of AlCl3 (205.36mg) in CH2Cl2 was added
acetyl chloride compounds (120.90mg, 1.54 mmol). The resulting mixture was stirred
at room temperature for 1h, then a solution of compound 23（200mg, 770.09 µmol）
in CH2Cl2 was added dropwise. The reaction mixture was stirred at room temperature
for 12 h. CH2Cl2 was evaporated in vacuo. The mixture was extracted with ethyl
acetate(3×50mL), washed with brine, dried (Na2SO4), and filtered. The filtrate was
concentrated in vacuo, and the residue was purified by column chromatography (silica
gel) to give the product compound 21a. m.p.: 118.2-120.1℃. 1H NMR (400 MHz,
DMSO-d6) δ 8.63 (s, 1H), 8.18 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.37 (dd, J = 8.6, 5.5
Hz, 2H), 7.26 (dd, J = 8.7, 2.1 Hz, 1H), 7.17 (t, J = 8.9 Hz, 2H), 5.50 (s, 2H), 2.47 (s,
13
3H)； C NMR (100 MHz, DMSO-d6) δ 192.33, 161.63(d, J=242Hz), 138.63, 134.91,
132.97, 129.46, 129.38, 126.99, 122.97, 120.63, 115.62, 115.41, 112.78, 49.08, 27.22.
HRMS (ESI) calcd. C17H13ClFNONa [M+Na]+m/z: 324.0562/326.0532, found:
324.0564/326.0622.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(cyclopropyl)methanone
(21b): Compound 23 was converted to compound 21b as a yellow solid by the same
procedure as described for compound 21a. m.p.: 95.5-96.8℃. 1H NMR (400 MHz,
DMSO-d6) δ 8.81 (s, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.42–
7.33 (overlap, 2H), 7.25 (dd, J = 8.8, 2.1 Hz, 1H), 7.21–7.13 (overlap, 2H), 5.51 (s,
13
2H), 2.70–2.60 (m, 1H), 1.02–0.95 (m, 2H), 0.94–0.86 (m, 2H)； C NMR (100 MHz,
DMSO-d6) δ 194.01, 161.64(d, J=242Hz), 138.07, 134.91, 132.96, 129.44 (d, J = 8
Hz), 126.94 (d, J = 4 Hz), 123.02, 120.77, 115.98, 115.66, 115.45, 112.74, 49.14,
17.36, 9.58. HRMS (ESI) calcd. C19H15ClFNONa [M+Na]+m/z: 350.0718/352.0689,
found: 350.0726/352.0718.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(furan-2-yl)methanone
(21c): Compound 23 was converted to compound 21c as a yellow solid by the same
procedure as described for compound 21a. m.p, 119.8-121.2℃. 1H NMR (400 MHz,
DMSO-d6) δ8.88 (s, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.05–8.03(overlap, 1H), 7.63 (d, J
= 8.8 Hz, 1H), 7.44 (d, J = 3.5 Hz, 1H), 7.43–7.37 (overlap, 2H), 7.31 (dd, J = 8.7, 2.1
Hz, 1H), 7.22–7.14 (overlap, 2H), 6.79 (m, 1H), 5.61 (s, 2H)；13C NMR (100 MHz,
DMSO-d6) δ175.02, 161.62(d, J=242Hz), 152.76, 146.58, 138.59, 134.56, 132.91(d,
J=3Hz), 129.41(d, J=8Hz), 128.30, 127.25, 123.34, 120.89, 116.91, 115.64, 115.43,
112.86(d, 19Hz), 112.32, 49.18. HRMS (ESI) calcd. C20H14ClFNO2 [M+H]+m/z:
354.0692/356.0662, found: 354.0698/356.0677.
Synthesis
of
(5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(3-methyl-2,5-
dihydrothiophen -2-yl)methanone (21d): Compound 23 was converted to
compound 21d as a white solid by the same procedure as described for compound 21a.
m.p.: 123.5-125.1℃. 1H NMR (400 MHz, Acetone-d6) δ8.37 (d, J = 2.0 Hz, 1H),
8.32 (s, 1H), 7.62 (d, J = 5.0 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.44 -7.38 (overlap,
2H), 7.28 (dd, J = 8.8, 2.1 Hz, 1H), 7.16-7.10 (m, 2H), 7.08 (d, J = 5.0 Hz, 1H), 5.63

ACCEPTED MANUSCRIPT
13
(s, 2H), 2.49 (s, 3H)； C NMR (100 MHz, DMSO-d6) δ 181.77, 161.63(d, J=243Hz),
142.14, 138.68, 134.93 (d, J = 16 Hz), 132.92, 131.90, 129.52 (d, J = 8 Hz), 123.42,
120.74, 115.64, 115.43, 112.99, 49.03, 15.81. HRMS (ESI) calcd. C17H16ClFNOS
[M+H]+m/z: 384.0620/386.0590, found: 384.0625/386.0672.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(phenyl)methanone (21e):
Compound 23 was converted to compound 21e as a light-yellow solid by the same
procedure as described for compound 21a. m.p.: 104.8-106.5℃. 1H NMR (400 MHz,
DMSO-d6) δ8.38 (s, 1H), 8.27 (d, J = 1.7 Hz, 1H), 7.83 (d, J = 7.4 Hz, 2H), 7.70–
7.50 (overlap, 4H), 7.41–7.34 (overlap, 2H), 7.31 (dd, J = 8.8, 2.1 Hz, 1H), 7.20–7.10
(overlap, 2H), 5.55 (s, 2H)；13C NMR (100 MHz, DMSO-d6)δ189.43, 161.61(d,
J=242Hz), 139.91, 134.95, 132.89(d, J = 3Hz ), 131.39, 129.42(d, J=8Hz), 129.23,
128.47 (d, J=6Hz), 128.17, 127.28, 123.36, 120.90, 115.59, 115.38, 113.86, 112.95,
49.12. HRMS (ESI) calcd. C22H16ClFNO [M+H]+m/z: 364.0899/366.0869, found:
364.0906/366.0825.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl) (4-fluorophenyl)
methanone (21f): Compound 23 was converted to compound 21f as a yellow solid by
the same procedure as described for compound 21a. m.p.: 107.4-109.8℃. 1H NMR
(400 MHz, DMSO-d6)δ8.40 (s, 1H), 8.26 (d, J = 2.1 Hz, 1H), 8.03 -7.97 (m, 1H),
7.95–7.88 (overlap, 2H), 7.61 (d, J = 8.8 Hz, 1H), 7.44–7.36 (overlap, 4H), 7.20–7.12
(overlap, 2H), 5.55 (s, 2H).13C NMR (100 MHz, DMSO-d6)δ187.98, 165.77(d,
J=111Hz), 161.07(d, J=136Hz), 160.39, 139.95, 136.39 (d, J=3Hz), 134.93, 132.86 (d,
J=3Hz), 132.06(d, 9Hz), 131.15(d, J=9Hz), 129.44(d, J=8Hz), 128.14, 127.29, 123.40,
120.84, 115.58(d, J=2Hz), 115.36(d, J=2Hz), 113.35(d, J=68Hz), 49.15. HRMS (ESI)
calcd. C22H15ClF2NO [M+H]+m/z: 382.0805/384.0775, found: 382.0809/384.0662.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(p-tolyl)methanone (21g):
Compound 23 was converted to compound 21g as a light-yellow solid by the same
procedure as described for compound 21a. m.p.: 126.0-129.1℃. 1H NMR (400 MHz,
DMSO-d6) δ 8.34 (s, 1H), 8.24 (d, J = 2.1 Hz, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.58 (d,
J = 8.8 Hz, 1H), 7.39- 7.32 (overlap, 4H), 7.28 (dd, J = 8.7, 1.9 Hz, 1H), 7.18–7.10
(overlap, 2H), 5.53 (s, 2H), 2.40 (s, 3H)；13C NMR (100 MHz, DMSO-d6)δ189.14,
161.59(d, J=242Hz), 141.49, 139.64, 137.20, 134.89, 129.44 (d, J = 8 Hz), 129.03,
128.59, 128.19, 127.15, 123.28, 120.86, 115.60, 115.38, 113.91, 112.93, 49.08, 21.03.
HRMS (ESI) calcd. C23H18ClFNO [M+H]+m/z: 378.1055/380.1026, found:
378.1066/380.1255.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(pyridin-4-yl)methanone
(21h): Compound 23 was converted to compound 21h as a white solid by the same
procedure as described for compound 21a. m.p.: 142.1-144.7℃. 1H NMR (400 MHz,
DMSO-d6) δ 8.81 (d, J = 1.6 Hz, 2H), 8.80(d, J=1.5Hz) 8.45 (s, 1H), 8.26 (d, J =
2.1Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.61 (d, J = 8.8 Hz,
1H), 7.42- 7.36 (overlap, 2H), 7.34(dd, J = 8.8, 2.2 Hz, 1H), 7.22–7.11 (overlap, 2H),
5.55 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 188.02, 161.63(d, J=242Hz), 150.24,
146.34, 140.98, 135.06, 132.69(d, J=3Hz), 129.50(d, J=8Hz), 127.73(d, J=8Hz),
123.71, 122.13, 120.79, 115.50(d, J=21Hz), 113.30 (d, J = 16 Hz), 49.25. HRMS (ESI)
calcd. C21H15ClFN2O [M+H]+m/z: 365.0851/367.0822, found: 365.0854/367.0844.

ACCEPTED MANUSCRIPT
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl) (2-chloropyridin-4-yl)
methanone (21i): Compound 23 was converted to compound 21i as a sandy beige
solid by the same procedure as described for compound 21a. m.p.: 124.6-127.0℃. 1H
NMR (400 MHz, Acetone-d6) δ8.58 (d, J = 5.0 Hz, 1H), 8.39 (d, J = 2.1 Hz, 1H),
8.37 (s, 1H), 7.75 (s, 1H), 7.72 (dd, J = 5.0, 1.3 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H),
7.44–7.36 (overlap, 2H), 7.32 (dd, J = 8.8, 2.1 Hz, 1H), 7.14–7.07 (overlap, 2H), 5.62
(s, 2H)；13C NMR(100 MHz, Acetone-d6)δ187.26, 163.30 (d, J=243Hz), 152.43,
151.37, 151.29, 141.32, 136.57, 133.43(d, J= 9Hz), 130.27 (d, J = 9 Hz), 129.43,
129.24, 124.94, 123.61, 122.38 (d, J = 10 Hz), 116.55, 116.33, 115.00, 113.64, 50.89.
HRMS (ESI) calcd. C21H13Cl2FN2ONa [M+Na]+m/z: 421.0281, found: 421.0288.
Synthesis of (5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(phenyl)methanol (25):
To a stirred solution of compound 21f (500mg, 1.37 mmol) in anhydrous MeOH
(4mL) and THF (1mL) was added dropwise NaBH4 in MeOH (1mL) and NaOH (1%,
1mL) at room temperature. The reaction mixture was stirred at room temperature for
2-5 h. After work up, the resulting mixture was extracted with ethyl acetate(3×50mL),
washed with brine, dried (Na2SO4), and concentrated. The crude residue was purified
by flash chromatography on silica gel (ethyl acetate/Petroleum ether=1: 8) to give the
product 25 (85mg, 67.53%). m.p.: 96.5-97.9℃. 1H NMR (400 MHz, Acetone-d6)
δ7.55 (d, J = 2.0 Hz, 1H), 7.52 (s, 1H), 7.51 (s, 1H), 7.37–7.28 (overlap, 3H), 7.27 (s,
1H), 7.25–7.18 (overlap, 3H), 7.08–7.06 (m, 1H), 7.06 - 7.02 (overlap, 2H), 6.09 (d, J
= 4.4 Hz, 1H), 5.37 (s, 2H), 4.69 (t, J = 5.9 Hz, 1H). HRMS (ESI) calcd.
C22H17ClFNONa [M+Na]+m/z: 388.0875/390.0845, found: 388.0881/390.1012.
Synthesis
of
(5-chloro-1-(4-fluorobenzyl)-1H-indol-3-yl)(phenyl)methanone
oxime (26): Hydroxylamine hydrochloride (954 mg, 13.74 mmol) was dissolved in
EtOH/pyridine(1: 1, 5mL). The reaction mixture was stirred at room temperature for
30 min. Compound 21f (500mg, 1.37mmol) was added to the reaction solution at
room temperature. After workup, insoluble material was removed by diatomite. The
resulting mixture was extracted with ethyl acetate(3×50mL), washed with brine, dried
(Na2SO4), and concentrated. The crude residue was purified by flash chromatography
on silica gel (acetone/Petroleum ether=1: 10) to give the product 26 (467mg, 89.70%).
m.p.: 179.6-181.5℃. 1H NMR (400 MHz, DMSO-d6) δ11.43(s, 1H), 8.19 (s, 1H),
7.55 (d, J = 8.7 Hz, 1H), 7.48–7.39 (overlap, 5H), 7.34–7.29 (overlap, 2H), 7.21–7.14
(overlap, 2H), 7.12 (dd, J = 8.8, 2.1 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 5.52 (s, 2H)；
13C NMR (100 MHz, DMSO-d6)δ 161.51(d, J= 242Hz), 149.71, 137.51, 134.61,
133.75, 133.65 (d, J=3Hz), 129.25(d, J =8Hz), 128.73, 128.26, 128.10, 127.73,
124.33, 121.44, 120.42, 115.55, 112.20, 106.32, 48.61. HRMS (ESI) calcd.
C22H17ClFN2O [M+H]+m/z: 379.1008/381.0978, found: 379.1011/381.1022.
4.3 Inhibit evaluation of compounds for LSD1
The activity of LSD1 was measured according to our previous publication [10].
Compounds were incubated with the recombinant LSD1 and H3K4me2. Hydrogen
peroxide that produced during the demethylation of LSD1 will be quantified by
Amplex Red to indicate the inhibition ability of compounds against LSD1. The data is
analyzed by GraphPad prism 6.0.
4.4 Dilution assay
Inhibitors with a concentration of 20 folds of IC50 were incubated with recombinant
LSD1. Then the mixture was diluted 80 folds to test the activity. If compounds inhibit

ACCEPTED MANUSCRIPT
the activity of LSD1 in a reversible manner, LSD1 activity will recovery after dilution.
The data is analyzed by GraphPad prism6.0.
4.5 Ultrafiltration assay
Inhibitors with a concentration of 20 folds of IC50 were incubated with recombinant
LSD1. Then the mixture was added to a 1K cut-off ultrafiltration tube, and then
subjected to 10000 rpm, 10 min centrifuge. The lower chamber solution is then taken
to test whether it can inhibit the activity of LSD1. If compounds inhibit the activity of
LSD1 in an irreversible manner, the compounds will remain firmly bound to the
protein and will still be in the upper chamber of the ultrafiltration tube. Then the
lower layer solution will not affect the LSD1 activity. The data is analyzed by
GraphPad prism6.0.
4.6 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 a 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.
4.7 Protein Thermal Shift assay
SYPRO Orange was used as the dye to indicate the folding and unfolding status of
LSD1. LSD1 recombinant, compound, dye, and HEPES buffer were mixed as the
sample group, while the reference group was the mixture of LSD1 recombinant,
DMSO, dye and HEPES buffer. Melting temperature (Tm) of sample group and
reference group is obtained by gradually increasing the temperature to unfold the
protein and measuring the fluorescence at each point. The fluorescence is monitored
by real-time PCR instrument (Thermo Fisher, USA) and analyzed by Protein Thermal
Shift Software 1.3 (Thermo Fisher, USA).
4.8 Cell cultures
The human acute myeloid leukemia cell THP-1 was obtained from Cell Bank of
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
(Shanghai, China). Cells are cultured in RPMI-1640 medium plus 10% fetal bovine
serum (FBS) and maintained in an incubator at 37ºC and 5% CO2.
4.9 Cell proliferation assay
4000 cells are seeded in a 96-well plate and then incubated for 5 days in standard
medium in the presence of compounds with different concentration. After the
incubation period, cells are quantified with MTT (Solarbio, China). The data is
analyzed by GraphPad prism6.0.
4.10 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)
4.11 Colony formation assays
Add 1 mL methylcellulose-based medium in a six-well plate, add 500 cells in every
well with different concentration of compounds, then pipet the medium up and down
gently to mix them thoroughly. Place the plate in an incubator at 37°C and 5% CO2
for 2 weeks. Pictures were taken by the light microscope (Leica, German).

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4.12 Flow Cytometry Assay
Cells were seeded in six-well plate, treated by 9e at 0, 0.25 µM, 1 µM, 4 µM for 3
days before analysis. PerCP-conjugated mouse anti-human CD86 monoclonal
antibody (Abcam, USA) is used for staining 40 min. Samples were acquired using BD
Calibur flow cytometer (BD, USA) according to the manufacturer’s instructions. Then
results were analyzed with FlowJo V7.6 software (TreeStar, USA).
Acknowledgment
This work was supported by National Natural Science Foundation of China (Project
No. 81602961, for Y.-C. Z.; No. 81430085 and No. 81773562 for H.-M.L.);
Outstanding Young Talent Research Fund of Zhengzhou University (No. 1521331002,
for Y.-C. Z.); Henan Natural Science Foundation (182300410367); National Key
Research Program of Proteins (No. 2016YFA0501800, for H.-M. L.); Key Research
Program of Henan Province (No. 161100310100, for H.-M. L.).
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