Synthesis, structure-activity relationship studies and biological characterization of new [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.02.039

The histone lysine specific demethylase 1 (LSD1/KDM1A) is implicated in the development of cancers, targeting LSD1 has been recognized as a promising strategy for cancer therapy. To date, some small-molecule inhibitors are currently being investigated in clinical trials. Herein we report the design, synthesis and biochemical characterization of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new LSD1 inhibitors. Of these compounds, compound C26 inhibited LSD1 in a reversible manner (IC₅₀ = 1.72 μM) and showed selectivity to LSD1 over MAO-A/B. Besides, compound C26 displayed FAD-competitive binding to LSD1. Interestingly, C26 did not inhibit horseradish peroxidase (HRP) and quench H₂O₂, thus excluding the possibility that LSD1 inhibition by C26 was due to the HRP inhibition and consumption of H₂O₂. In LSD1 overexpressed A549 cells, compound C26 concentration-dependently induced accumulation of H3K4me1/me2 and H3K9me2 and showed cellular target engagement to LSD1. Additionally, compound C26 significantly inhibited migration of A549 cells in a concentration-dependent manner, further western blot analysis showed that C26 increased expression levels of epithelial cell markers E-Cadherin and Claudin-1, down-regulated mesenchymal cell marker N-Cadherin and the upstream transcription factors Snail and Slug. Docking studies were also performed to rationalize the potency of C26 toward LSD1. To conclude, the [1,2,4]triazolo[1,5-a]pyrimidine could serve as a promising scaffold for the development of new LSD1 inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.02.039

Accepted Manuscript
Synthesis, structure-activity relationship studies and biological characterization of new
[1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors
Shuai Wang, Zhong-Rui Li, Feng-Zhi Suo, Xiao-Han Yuan, Bin Yu, Hong-Min Liu
PII:
S0223-5234(19)30153-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.039
EJMECH 11131
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2018
Revised Date: 13 January 2019
Accepted Date: 10 February 2019
Please cite this article as: S. Wang, Z.-R. Li, F.-Z. Suo, X.-H. Yuan, B. Yu, H.-M. Liu,
Synthesis, structure-activity relationship studies and biological characterization of new
[1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.039.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, structure-activity relationship studies and biological characterization
of new [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors
Shuai Wang a,c,d,#, Zhong-Rui Li a,c,d,#, Feng-Zhi Suo a,c,d, Xiao-Han Yuan a,c,d, Bin Yu
a,b,c,d,*, and Hong-Min Liu a,c,d,*.
a
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
China;
b State key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, Jiangsu, People’s Republic of China.
c
Key Laboratory of Advanced Drug Preparation Technologies (Ministry of
Education), Zhengzhou University, Zhengzhou 450001, China;
d
Co-Innovation Center of Henan Province for New Drug R & D and Preclinical
Safety, Zhengzhou University, Zhengzhou 450001, China;
* Corresponding authors:
Dr. Bin Yu (zzuyubin@hotmail.com)
Prof. Hong-Min Liu (liuhm@zzu.edu.cn)
# Shuai Wang and Zhong-Rui Li contributed equally to this work.
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Abstract: The histone lysine specific demethylase 1 (LSD1/KDM1A) is implicated in
the development of cancers, targeting LSD1 has been recognized as a promising
strategy for cancer therapy. To date, some small-molecule inhibitors are currently
being investigated in clinical trials. Herein we report the design, synthesis and
biochemical characterization of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new
LSD1 inhibitors. Of these compounds, compound C26 inhibited LSD1 in a reversible
manner (IC50 = 1.72 µM) and showed selectivity to LSD1 over MAO-A/B. Besides,
compound C26 displayed FAD-competitive binding to LSD1. Interestingly, C26 did
not inhibit horseradish peroxidase (HRP) and quench H2O2, thus excluding the
possibility that LSD1 inhibition by C26 was due to the HRP inhibition and
consumption of H2O2. In LSD1 overexpressed A549 cells, compound C26
concentration-dependently induced accumulation of H3K4me1/me2 and H3K9me2
and showed cellular target engagement to LSD1. Additionally, compound C26
significantly inhibited migration of A549 cells in a concentration-dependent manner,
further western blot analysis showed that C26 increased expression levels of epithelial
cell markers E-Cadherin and Claudin-1, down-regulated mesenchymal cell marker
N-Cadherin and the upstream transcription factors Snail and Slug. Docking studies
were also performed to rationalize the potency of C26 toward LSD1. To conclude, the
[1,2,4]triazolo[1,5-a]pyrimidine could serve as a promising scaffold for the
development of new LSD1 inhibitors.
Keywords: [1,2,4]triazolo[1,5-a]pyrimidines; LSD1 inhibitors; Antiproliferative
activity; Migration inhibition;
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1. Introduction
Histone lysine specific demethylase LSD1 (also known as KDM1A) was first
identified in 2004 by Professor Shi Yang [1]. As a monoamine oxidase homologue,
LSD1 specifically removes methyl groups of H3K4/H3K9, thereby causing activation
or suppression of genes [2]. LSD1 is highly expressed in many tumor cells [3, 4].
Abnormally high expression of LSD1 promotes tumorigenesis by regulating
chromatin (such as chromatin remodeling and aggregation) [5]. In addition, high
expression of LSD1 can lead to inhibition of p53 function by inhibiting the interaction
between p53 and TP53BP1 (p53 binding protein 1) [6], which then affects the cancer
cell cycle [7], promotes tumor growth, invasion and metastasis by affecting the
methylation/demethylation process [8-10]. Therefore, targeting LSD1 is becoming an
important therapeutic strategy for cancer therapy [11]. To date, a large number of
natural and synthetic LSD1 inhibitors [12-23] have been identified, of which 2-PCPA,
phenelzine, and pargyline [24-27] were initially found to be able to inhibit LSD1
irreversibly. Subsequent modifications based on the 2-PCPA scaffold have generated
numerous covalent LSD1 inhibitors. Encouragingly, GSK-2879552 [28, 29],
ORY-1001 [30], IMG-7289, CC-90011, INCB059872 [31, 32] and ORY-2001 have
advanced into clinical trials. Based on our previous work on the identification of
LSD1 inhibitors [33-41], herein we report the design, synthesis and biological
characterization of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new LSD1
inhibitors, of which compound C26 inhibited LSD1 in a reversible manner and was
also on target in A549 cells.
2. Results and Discussion
2.1. Chemistry
The synthetic routes for the synthesis of compounds C1-C32. As shown in Scheme
1, 4H-1,2,4-triazol-3-amine reacted with different β-ketoesters in acetic acid
generated compounds A1-A4, which were then subjected to the chlorination reaction
in POCl3, affording compounds B1-B4. Subsequent substitution reactions of B1-B4
with 4-(4-methylpiperazino)aniline yielded compounds C1-C4. Starting from
5-amino-4H-1,2,4-triazole-3-thiol, the intermediates D1-D22 were obtained through
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the substitution reactions with various alkyl halides. Compounds D1-D22 then reacted
with diverse β-ketoesters to generate compounds A5-A29. Chlorination of compounds
A5-A29 in POCl3 formed compounds B5-B29, which then reacted with commercially
available amines to form the corresponding compounds C5-C32.
Scheme 1. Synthesis of compounds C1-C32. Reagents and conditions: (a) Acetic acid,
o
120 oC, 2-15 h, reflux. (b) POCl3, 90 C, 3-5 h, reflux; (c) Et3N, EtOH, rt, 5 h; (d)
Na2CO3, acetone, 60 oC, 3-5 h, reflux.
2.2. SARs investigations
Based on our previously established screening methods against LSD1 [40], we
screened our in-house library using GSK2879552 as a control compound (LSD1 IC50
= 24 nM) [42], leading to the discovery of hit compound C1, which inhibited LSD1
with an IC50 value of 31.35 µM (Fig. 1). Initial modifications focused on the
replacement of the methyl group with more steric ethyl and phenyl groups, generating
compounds C2 and C3 with decreased anti-LSD1 activity (IC50 = 38.13 and >50 µM,
respectively). Besides, the cyclopentane fused compound C4 was also inactive against
LSD1 (IC50 > 50 µM). The results suggest that the less steric group (e.g. –Me) was
preferred for the activity.
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Fig. 1. Initial modifications based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold.
With compound C1 in hand, we then performed further SARs studies as shown in Fig.
2. Aniline and –NH2 substituted compounds C30 and C31 exhibited decreased
activity (IC50 > 50 µM) compared to the hit compound C1, indicating the importance
of the hydrophilic N-methylpiperazine group for the activity. This finding is consistent
with previous reports [12, 27, 43-47], namely, the hydrophilic groups such as
piperidine, piperazine, etc. always exist in some highly potent and selective LSD1
inhibitors (e.g. ORY-1001, GSK-2879552, etc.). Intriguingly, the introduction of the
benzyl mercapto group to the [1,2,4]triazolo[1,5-a]pyrimidine scaffold led to the
increase of the anti-LSD1 activity by a fact of about 2.5. Compound C5 inhibited
LSD1 with an IC50 value of 12.39 µM. Compounds C19 and C20 bearing the
–(CH2)2Ph and –(CH2)3Ph moiety, respectively displayed significantly decreased
anti-LSD1 activity compared to C1, underscoring the importance of the length of the
side chain for the activity. To our surprise, compounds C28 and C29 possessing
additional methyl and n-pentyl groups were found to be inactive against LSD1 (IC50 >
50 µM).
Figure 2. Further optimization based on the hit compound C1.
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Molecular fine-turning on the phenyl ring of C5 was further carried out, generating
compounds C6-C18 and C26-C27. As shown in Fig. 3, the incorporation of halo atom
into the phenyl ring caused decrease of the anti-LSD1 activity. Compounds C6-C10,
C14-C16, and C18 showed moderate inhibition against LSD1 with the IC50 values
ranging from 18 to 30 µM, slightly less potent than C5. While the –NO2 containing
compounds C11 and C12 displayed significantly decreased activity with the IC50
value of 32.56 and >50 µM, respectively. The –OMe and –Me containing compounds
C13 and C17 also exhibited decreased activity with the IC50 values of 17.38 and
25.78 µM, respectively. Compound C18 bearing the –CF3 group showed comparable
inhibitory activity with C5. Interestingly, replacement of the phenyl ring in C5 with
benzimidazole and benzothiophene formed C26 and C27 (Fig. 4), of which C26
inhibited LSD1 with an IC50 value of 1.72 µM. To our surprise, removal of
N-methylpiperazine group in compound C26 generated compound C32, which
showed with significantly decreased anti-LSD1 activity (IC50 > 50 µM), the data
unveiled the importance of the hydrophilic N-methylpiperazine group for the activity.
We also found that replacement of the benzyl ring with other aliphatic groups caused
decrease of the activity (Fig. 4). Compound C23 bearing a terminal alkyne group
showed comparable activity (IC50 = 14.84 µM) with C5.
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Figure 3. Structural modifications on the phenyl ring of C5.
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Figure 4. Structures and biochemical potency of compounds C5 and C21-C27.
2.3. Biochemical characterization of C26
The assay procedure used in this work couples the production of H2O2 from the LSD1
reaction with horseradish peroxidase (HRP), the product of which is then captured by
Amplex Red. While the drawback of this commonly used assay procedure is that
compounds with aromatic character often act as HRP inhibitors, giving a false
positive reading [48]. This interaction can make it appear that a compound inhibits
LSD1, when in fact it only inhibits HRP. We first detected the inhibitory effect of C26
on HRP, as shown in Fig. 5A, C26 did not inhibit HRP even at 10 µM, thus excluding
false positive results caused by HRP inhibition. Additionally, compounds that quench
H202 could also falsely show LSD1 inhibition [49]. We next examined whether the
thioether C26 could quench H202 using a structurally similar but inactive compound
C32 (IC50 > 50 µM) as a control. As shown in Fig. 5B, both compounds could not
quench H2O2, thus excluding the possibility that LSD1 inhibition by C26 was due to
the consumption of H2O2. In view of the acceptable potency of C26 against LSD1, we
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first examined inhibitory effects of compound C26 against MAO-A/B as LSD1 shares
similar amino acid sequence with MAO-A/B [50, 51]. We found that compound C26
inactivated MAO-A 40% at 10 µM and 13% at 1 µM (Fig. 5C). Meanwhile, we also
observed that compound C26 inactivated MAO-B 34% at 10 µM and 18% at 1 µM.
These results indicated that compound C26 had certain selectivity to LSD1 over
MAO-A/B. Next, a dilution assay was performed to examine the reversibility of
compound C26 against LSD1 using GSK2879552 as the control compound [28],
which is a potent, selective, mechanism-based LSD1 inactivator (ClinicalTrials.gov
Identifier: NCT02929498). As shown in Fig. 5D, 80-fold dilution of the
LSD1/compound C26 mixture resulted in the recovery of LSD1 activity, suggesting
reversible binding of C26 to LSD1. In contrast, the covalent LSD1 inhibitor
GSK2879552 failed to recover the anti-LSD1 activity after dilution. It is well
recognized that LSD1 inhibitors could competitively bind to LSD1 with FAD or
histone substrates (e.g. H3K4me2) [52]. Herein we performed the competitive
analysis of compound C26 within the active site of LSD1. As depicted in non-linear
regression analysis (Figs. 5E and 5F), compound C26 competitively bound to LSD1
with FAD (Fig. 5F), while it showed a noncompetitive binding with H3K4me2
(Figure Fig. 5E). These results indicated that compound C26 may occupy the binding
pocket that FAD stands, finally leading to decrease of the demethylase activity of
LSD1. To conclude, compound C26 was a reversible and FAD-competitive LSD1
inhibitor.
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Figure 5. Biochemical characterization of compound C26 toward LSD1 activity in
vitro. (A) The effect of C26 on HRP activity. (B) H2O2 quantitation curve of C26 and
C32 was measured by ferrous ion oxidation-xylenol orange (FOX) method. (C)
Selectivity of compound C26 to LSD1 over MAO-A/B. (D) The reversibility of
compound C26 (40 µM) to LSD1 was determined by the dilution assay, GSK2879552
(1.6 µM) was used as a control. (E, F) The non-linear regression analysis
demonstrated that compound C26 is noncompetitive with the histone H3 substrate (E)
and competitive with the LSD1 cofactor FAD (F). Data are mean ± SD. P < 0.01 was
considered statistically highly significant. All experiments were carried out at least
three times.
2.4. Cellular effects of compound C26 in LSD1 overexpressed A549 cells
It has been reported that LSD1 is highly expressed in human lung cancer cells [28, 51,
53]. It is well known that LSD1 can catalytically remove the methyl groups of LSD1
substrates H3K4me1/2 and H3K9 me1/2 [3]. Herein we investigated the effects of
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compounds C26, C32 (inactive control) and GSK-LSD1 (positive control) on the
methylation of histone substrates H3K4me1/2 and H3K9 me1/2 in LSD1
overexpressed A549 cells. As demonstrated in Figs. 6A and 6C, compounds C26 and
GSK-LSD1 concentration-dependently induced accumulation of H3K4me1/2 and
H3K9 me1/2 in A549 cells after treatment for 96 h. In contrast, the inactive compound
C32 was ineffective in modulating methylation of H3 peptide substrates in A549 cells
(Fig. 6B). These results proved that C26 was cellularly active.
Figure 6. Histone methylation in A549 cells after treatment with C26 (A), C32 (B)
and GSK-LSD1 (C) for 4 days. The expressions of H3K4me1/2 and H3K9me1/2
were determined by western blot. The total levels of histone (H3) were used as
loading control. (*) P < 0.05 was considered statistically significant. (**) P < 0.01
was considered statistically highly significant. All experiments were carried out at
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least three times.
In order to further validate the cellular target engagement of C26 in A549 cells, we
employed the cellular thermal shift assay (CETSA) [54] to examine whether C26
treatment could increase thermal stability of LSD1 using GSK-LSD1 as a positive
control (Fig. 7). As expected, GSK-LSD1 increased the stability of cellular LSD1 at
49 and 52 oC at 10 µM (Fig. 7C). As shown in Fig. 7A, treatment with C26 (10 and 20
µM) at 46 and 49 oC increased stability of cellular LSD1. Particularly, compound C26
o
significantly increased cellular thermal stability of LSD1 in A549 cells at 52 C
compared to the group treated with DMSO, while the cellular LSD1 protein was
denatured at 52 °C in A549 cells with DMSO treatment. In contrast, after treatment
with the negative compound C32 (10 µM), the thermal stability of cellular LSD1 was
similar to that of the DMSO group (Fig. 7B). This data suggested that C26 was on
target to LSD1 in A549 cells.
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Figure 7. Cellular target engagement of C26 in A549 cells. (A) C26 (10, 20 µM), (B)
C32 (10 µM) and (C) GSK-LSD1 (10 µM) were used in cellular thermal shift assay
(CETSA). A549 cells were treated with compound C26 for 1 h, followed by heating at
different temperatures for 3 min. Protein levels of LSD1 were examined by the
western blotting analysis. GAPDH was used as a loading control. (∗) P < 0.05 was
considered statistically significant. (∗∗∗) P < 0.01 was considered statistically highly
significant. All experiments were carried out at least three times.
In view of the acceptable potency against LSD1 and cellular target engagement of
C26 in A549 cells, we also evaluated the effect on the cell viability of A549 cells. As
shown in Fig. 8, GSK-LSD1 had no effect on the cell viability even at high
concentration, while compound C26 concentration-dependently reduced the cell
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viability of A549 cells with an IC50 value of 23.76 µM. The negative compound C32
did not affect the cell viability of A549 cells at < 40 µM, C32 at higher concentration
caused significantly decreased cell viability. The decreased cell viability induced by
C26 may be partly due to the LSD1 inhibition.
Figure 8. The effect of C26, C32 and GSK-LSD1 on the cell viability of A549 cells.
2.5. Migration inhibition of compound C26 against A549 cells
As reported previously, LSD1 was involved in epithelial-mesenchymal transition
(EMT), which contributed to the migration and invasion of cancer cells [55, 56].
Therefore, the high content analysis was performed to examine the effect of C26 on
the migration of A549 cells using GSK-LSD1 and C32 as the controls. As shown in
Fig. 9A, compound C26 significantly inhibited migration of A549 cells in a
concentration-dependent manner, especially at 16 µM. We also examined the
expression of the typical proteins of EMT process (Fig. 9B), showing that compound
C26 up-regulated the epithelial cells biomarkers, E-Cadherin and claudin-1, while the
mesenchymal cells biomarker N-Cadherin was down-regulated correspondingly. Then
the upstream transcription factors Snail and Slug were also down-regulated after
treatment with compound C26, indicating that compound C26 may regulate the
biomarkers of the EMT process through inhibiting the expression of the transcription
factors and then cause the interference of EMT process ultimately. At the same time,
typical proteins of the EMT process were also examined after treatment with C32 and
GSK-LSD1. As shown in Fig. 9C, C32 had no effect on the expression of these
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proteins in A549 cells. Like C26, GSK-LSD1 also inhibited the EMT process,
accompanied by up-regulation of E-Cadherin and claudin-1 as well as
down-regulation of N-Cadherin, Snail, and Slug (Fig. 9D). As expected, the inactive
compound C32 did not affect the expression levels of the EMT-related proteins. We
also found that C26, C32 and GSK-LSD1 did not affect expression of LSD1.
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Figure 9. The effect of compounds C26, C32 and GSK-LSD1 on the migration of
A549 cells and expression levels of key proteins. (A) Migration ability assay.
Treatment of compound C26 resulted in the migration inhibition. The expressions of
E-Cadherin, Claudin-1, N-Cadherin, Slug, Snai1 and LSD1 after treatment with
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compound C26 (B), C32 (C) and GSK-LSD1 (D) for 96 h. GAPDH was used as
loading control. (∗) P < 0.05 was considered statistically significant. (∗∗) P < 0.01
was considered statistically highly significant. All experiments were carried out at
least three times.
2.6. Molecular docking studies
In order to rationalize the potency of compound C26 against LSD1, the crystal
structure (PDB code: 2v1d) of LSD1 in complex with CoREST and a substrate-like
peptide inhibitor [57] was downloaded from the RCSB database and used as a
receptor for the docking studies. As shown in Fig. 10A, compound C26 (colored in
green) occupied the hydrophobic tabular cavity of LSD1, showing overlap with FAD
(colored in yellow). The predicted modeling further confirmed FAD-competitive
binding mode as shown in the Lineweaver−Burk plots (Figs. 5E and 5F). The binding
models indicate that the 3D geometry of C26 may play critical roles in the anti-LSD1
activity. As shown in Fig. 10B, the aromatic triazolo[1,5-a]pyrimidine ring laying at
the central of the pocket formed two π-H interactions, one of the nitrogen atom of the
pyrimidine ring also had an interaction with Val288. The NH attached to the
triazolo[1,5-a]pyrimidine ring formed an hydrogen bond with Arg316. These
interactions may be important in maintaining the preferred 3D geometry. Additionally,
the benzimidazole NH and the protonated N-Me piperazine NH had an H-bond
interaction with Glu308 and Met4, respectively. The predicted binding models may
provide structural basis for further structure-based modifications.
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Figure 10. Molecular docking studies of C26 in the active site of LSD1. (A)
Three-dimensional binding models of C26 in the active site of LSD1 (PDB code:
2v1d). FAD and C26 were colored in yellow and green, respectively. The
hydrophobic region of LSD1 was colored in green, while the hydrophilic region was
colored in purple; (B) Two-dimensional (2D) interaction diagram of C26 with
surrounding residues, the 2D diagram was generated with the MOE software. Green
dashed lines indicate hydrogen bonds, blue dashed line represents backbone acceptor.
3. Conclusions
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A
new
series
of
4-(N-methylpiperazin-1-yl)aniline
substituted
[1,2,4]triazolo[1,5-a]pyrimidine derivatives were synthesized and evaluated for their
anti-LSD1 activity. Based on the initially identified hit compound, further medicinal
chemistry efforts led to the discovery of compound C26, which inhibited LSD1 (IC50
= 1.72 µM) in a reversible manner, showed selectivity to LSD1 over MAO-A/B, but
did not inhibit HRP or quench H2O2, excluding the false positive results. Besides,
compound C26 competitively bound to LSD1 with FAD, while it showed a
noncompetitive binding with H3K4me2. In LSD1 overexpressed A549 cells,
compound C26 concentration-dependently induced accumulation of LSD1 substrates
- H3K4me1/me2 and H3K9me2 and showed cellular target engagement to LSD1.
Additionally, compound C26 significantly inhibited migration of A549 cells in a
concentration-dependent manner, further western blot analysis showed that C26
increased expression levels of epithelial cell markers E-Cadherin and Claudin-1,
down-regulated mesenchymal cell marker N-Cadherin and the upstream transcription
factors Snail and Slug. Docking studies were also performed to rationalize the
potency of C26 toward LSD1, suggesting that C26 occupied FAD binding site of
LSD1. Taken together, the [1,2,4]triazolo[1,5-a]pyrimidine could serve as a promising
scaffold for the development of new LSD1 inhibitors.
4. Experimental section
4.1. General Information.
Reagents and solvents were purchased from commercial sources and used without
further purification. Thin-layer chromatography (TLC) was carried out on glass plates
coated with silica gel (Qingdao Haiyang Chemical Co., G60F-254) and visualized by
UV light (254 nm). The products were purified by column chromatography over silica
gel (Qingdao Haiyang Chemical Co., 200–300 mesh). Melting points were
determined on an X-5 micromelting apparatus and are uncorrected. All the NMR
spectra were recorded with a Bruker DPX 400 MHz spectrometer with TMS as
internal standard in CDCl3 or DMSO-d6. Chemical shifts are given as δ ppm values
relative to TMS. High-resolution mass spectra (HRMS) were recorded on a Waters
Micromass Q-T of Micromass spectrometer by electrospray ionization (ESI).
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4.2. General method for the synthesis of D1-D22.
Compounds D1-D22 were prepared following the previously reported method [37].
To a solution of 3-amino-5-mercapto-1,2,4-triazole (1.0 g, 8.61 mmol) in acetone (30
mL) were added sodium carbonate (1.37 g, 12.92 mmol), sodium iodide (129.06 mg,
o
0.861 mmol) and alkyl halide (9.47 mmol). The reaction mixture was stirred at 60 C
for 3-6 h before cooling to room temperature. Na2CO3 and NaI were removed via
filtration. The residue was concentrated under vacuum and then dissolved in EtOAc.
The organic layer was washed with water (2 × 10 mL) and brine (2 × 20 mL) and then
dried over anhydrous MgSO4. After removal of the solvent, the resulting residue was
subjected to column chromatography, giving the corresponding products D1-D22.
Compound D1, white solid, yield: 73 %. 1H NMR (400 MHz, DMSO-d6) δ 7.41-7.18
(m, 5H), 4.34 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 152.19, 147.08, 136.55,
128.79, 128.43, 127.48, 35.41. HRMS (ESI): m/z calcd for C9H9N4S (M-H)-,
205.0548; found, 205.0548.
4.3. General method for the synthesis of A1-A29.
Compounds A1-A29 were prepared following the previously reported method [37] as
described for A5. D1 (1g, 4.85 mmol) and ethyl acetoacetate (630.94mg, 4.85mmol)
o
was stirred in AcOH (10 mL) at 120 C. 3-6 h later, the reaction mixture was cooled
to room temperature and the white precipitate formed was filtered and washed using
water to afford the desired compound A5. Compound A5, white solid, yield: 85 %.
m.p.: 240-245 oC. 1H NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H), 7.44 (d, J = 7.1 Hz,
2H), 7.32 (t, J = 7.3 Hz, 2H), 7.26 (d, J = 7.2 Hz, 1H), 5.80 (s, 1H), 4.43 (s, 2H), 2.29
(s, 3H). 13C NMR (100MHz, DMSO-d6) δ 161.92, 154.75, 151.09, 150.63, 137.35,
128.77, 128.38, 127.22, 98.46, 34.48, 18.43. HRMS (ESI): m/z calcd for C13H11N4OS
(M-H)-, 271.0654; found, 271.0660.
4.4. General method for the synthesis of B1-B29.
Compounds B1-B29 were prepared following the previously reported method [37] as
described for the synthesis of B5. A5 was stirred in POCl3 (excess) at 90 oC for 3-5h.
The resultant oil was added dropwise to ice cold bath. The resulting aqueous mixture
was extracted with CH2Cl2 (4 × 30 mL) and the organic layers were washed by
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saturated aqueous NaHCO3 (3×10 mL). The organic layers were dried over MgSO4
and concentrated under reduced pressure to give the orange solid B5, which was used
directly without further purification. B5 was unstable enough and therefore only
characterized by HRMS. HRMS (ESI): m/z calcd for C13H12ClN4S (M+H)-, 291.0471;
found, 291.0463.
4.5. General method for the synthesis of C1-C32.
The general method was described for the synthesis of C1. Compound B1 (150 mg,
0.889 mmol) was dissolved in ethanol (2 mL), followed by addition of the
corresponding amine or ammonium hydroxide (0.889 mmol) and triethylamine
(180.08 mg, 1.78 mmol) . The reaction mixture was stirred at room temperature for 5
h. The resultant mixture was concentrated under reduced pressure to the residue,
which was then purified by column chromatography to give C1.
o
1
Compound C1, white solid, yield: 48 %. m.p.: 162-166 C. H NMR (400 MHz,
DMSO-d6) δ 7.03 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 5.84 (s, 1H), 3.16 –
13
3.06 (m, 5H), 2.48 – 2.41 (m, 4H), 2.22 (d, J = 3.5 Hz, 6H). C NMR (100 MHz,
DMSO-d6) δ 161.34, 156.47, 152.96, 147.82, 124.56, 116.28, 88.26, 54.69, 48.55,
45.74, 24.48. HRMS (ESI): m/z calcd for C17H22N7 (M+H)+, 324.1936; found,
324.1907.
o
1
Compound C2, white solid, yield: 61 %. m.p.: 153-156 C. H NMR (400 MHz,
DMSO-d6) δ 8.48 (s, 1H), 7.27 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 8.6 Hz, 2H), 6.17 (s,
1H), 3.19 (s, 4H), 2.66 (dd, J = 14.8, 7.3 Hz, 2H), 2.26 (s, 3H), 1.18 (t, J = 7.5 Hz,
3H). 13C NMR (100 MHz, DMSO-d6) δ 154.34, 125.88, 115.85, 54.43, 47.80, 45.56,
30.97, 12.98. HRMS (ESI): m/z calcd for C18H24N7 (M+H)+, 338.2093; found,
338.2059.
o
1
Compound C3, white solid, yield: 58 %. m.p.: 176-179 C. H NMR (400 MHz,
DMSO-d6) δ 8.59 (s, 1H), 7.99 (dd, J = 6.4, 2.9 Hz, 2H), 7.58 – 7.47 (m, 3H), 7.43 (d,
J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 6.74 (s, 1H), 3.87 (s, 2H), 3.47 (s, 2H), 3.17
(s, 4H), 2.82 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.48, 155.88, 155.01,
147.94, 147.15, 137.45, 130.35, 128.80, 128.56, 127.13, 125.88, 116.64, 85.68, 55.99,
21 / 39

ACCEPTED MANUSCRIPT
52.06, 45.36, 41.94, 18.52. HRMS (ESI): m/z calcd for C22H24N7 (M+H)+, 386.2093;
found, 386.2092.
o
1
Compound C4, white solid, yield: 65 %. m.p.: 137-142 C. H NMR (400 MHz,
DMSO-d6) δ 7.14 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 3.22 – 3.08 (m, 4H),
2.80 (t, J = 7.7 Hz, 2H), 2.49 – 2.41 (m, 4H), 2.23 (s, 3H), 2.14 (t, J = 7.2 Hz, 2H),
13
1.98 – 1.80 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 173.08, 155.62, 153.81,
149.55, 146.34, 142.51, 127.93, 127.68, 114.77, 102.24, 55.99, 54.52, 47.88, 45.69,
34.09, 30.66, 28.46, 22.95, 18.52.
HRMS (ESI): m/z calcd for C19H24N7 (M+H)+, 350.2093; found, 350.2064.
o
1
Compound C5, white solid, yield: 52 %. m.p.: 155-158 C. H NMR (400 MHz,
DMSO-d6) δ 7.49 (d, J = 7.3 Hz, 2H), 7.30 (m, 5H), 7.10 (d, J = 8.9 Hz, 2H), 6.15 (s,
1H), 4.55 (s, 2H), 3.84 (s, 2H), 3.60 – 3.43 (m, 2H), 3.19 (d, J = 9.8 Hz, 4H), 2.81 (s,
3H), 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.43, 163.68, 155.77, 147.98,
145.57, 138.02, 128.90, 128.50, 128.42, 127.20, 126.03, 116.61, 88.93, 51.99, 45.36,
41.89, 34.50, 24.63. HRMS (ESI): m/z calcd for C24H28N7S (M+H)+, 446.2126; found,
446.2094.
o
1
Compound C6, white solid, yield: 43 %. m.p.: 182-184 C. H NMR (400 MHz,
DMSO-d6) δ 7.52 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.8 Hz,
2H), 7.10 (d, J = 8.9 Hz, 2H), 6.14 (s, 1H), 4.52 (s, 2H), 2.81 (s, 3H), 2.35 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 164.14, 163.76, 155.77, 148.00, 145.55, 137.43,
131.78, 130.78, 128.44, 128.33, 126.05, 116.61, 88.96, 52.07, 45.43, 41.98, 33.58,
24.62. HRMS (ESI): m/z calcd for C24H27FN7S (M+H)+, 464.2032; found, 464.1995.
o
1
Compound C7, white solid, yield: 58 %. m.p.: 172-175 C. H NMR (400 MHz,
DMSO-d6) δ 7.54 (dd, J = 8.6, 5.6 Hz, 2H), 7.30 (d, J = 8.9 Hz, 2H), 7.19 – 7.06 (m,
4H), 6.14 (s, 1H), 4.53 (s, 2H), 2.82 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 164.27, 163.74, 160.10, 155.77, 147.98, 145.55, 134.49, 130.95, 130.87,
128.47, 126.03, 116.62, 115.26, 115.05, 88.94, 52.10, 45.45, 42.00, 33.56, 24.63.
HRMS (ESI): m/z calcd for C24H27ClN7S (M+H)+, 480.1737; found, 480.1697.
o
1
Compound C8, white solid, yield: 62 %. m.p.: 162-165 C. H NMR (400 MHz,
DMSO-d6) δ 7.48 (q, J = 8.5 Hz, 4H), 7.30 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.9 Hz,
22 / 39

ACCEPTED MANUSCRIPT
2H), 6.14 (s, 1H), 4.51 (s, 2H), 3.27 (s, 8H), 2.77 (s, 3H), 2.35 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 164.12, 163.74, 155.77, 148.09, 145.57, 137.86, 131.24, 131.13,
128.37, 126.04, 120.29, 116.56, 88.95, 52.20, 45.55, 42.18, 33.66, 24.63. HRMS
(ESI): m/z calcd for C24H27BrN7S (M+H)+, 524.1232; found, 524.1182.
o
1
Compound C9, white solid, yield: 41 %. m.p.: 142-146 C. H NMR (400 MHz,
DMSO-d6) δ 7.72 (d, J = 6.6 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.33 (dd, J = 14.9, 8.0
Hz, 3H), 7.23 (t, J = 7.0 Hz, 1H), 7.10 (d, J = 8.8 Hz, 2H), 6.15 (s, 1H), 4.62 (s, 2H),
2.80 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.00, 163.79, 155.80,
148.03, 145.59, 136.92, 132.72, 131.42, 129.59, 128.43, 127.90, 126.06, 124.02,
116.60, 89.00, 52.05, 45.41, 41.97, 35.23, 24.64. HRMS (ESI): m/z calcd for
C24H27BrN7S (M+H)+, 524.1232; found, 524.1192.
1
Compound C10, white solid, yield: 59 %. m.p.: 163-169 oC. H NMR (400 MHz,
DMSO-d6) δ 7.80 (s, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.53 – 7.47 (m, 1H), 7.34 (d, J =
7.8 Hz, 1H), 7.32 – 7.26 (m, 2H), 7.07 (d, J = 9.0 Hz, 2H), 6.17 (s, 1H), 4.58 (s, 2H),
13
3.29 – 3.18 (m, 4H), 2.54 – 2.49 (m, 4H), 2.40 (s, 3H), 2.28 (s, 3H). C NMR (100
MHz, DMSO-d6) δ 163.99, 163.70, 155.80, 149.47, 145.68, 141.25, 131.63, 130.53,
130.01, 128.03, 127.20, 126.02, 121.43, 115.79, 88.89, 54.53, 47.89, 45.72, 33.57,
24.62. HRMS (ESI): m/z calcd for C24H27BrN7S (M+H)+, 524.1232; found, 524.1192.
1
Compound C11, white solid, yield: 86 %. m.p.: 172-175 oC. H NMR (400 MHz,
DMSO-d6) δ 8.18 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.8 Hz,
2H), 7.07 (d, J = 8.9 Hz, 2H), 6.13 (s, 1H), 4.65 (s, 2H), 3.35 (s, 4H), 2.96 (s, 4H),
2.58 (s, 3H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.75, 155.79, 148.63,
146.69, 146.55, 145.64, 130.17, 127.88, 126.06, 124.50, 123.48, 116.27, 88.99, 53.13,
46.48, 33.54, 24.61. HRMS (ESI): m/z calcd for C24H27N8O2S (M+H)+, 491.1977;
found, 491.1933.
1
Compound C12, white solid, yield: 76 %. m.p.: 167-175 oC. H NMR (400 MHz,
DMSO-d6) δ 8.13 – 8.04 (m, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.71 (m, 1H), 7.62 – 7.51
(m, 1H), 7.31 (d, J = 8.9 Hz, 2H), 7.10 (d, J = 8.9 Hz, 2H), 6.15 (s, 1H), 4.83 (s, 2H),
13
3.49 (s, 8H), 2.77 (s, 3H), 2.36 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.97,
163.83, 155.78, 148.15, 147.93, 145.59, 133.90, 133.63, 132.50, 129.01, 128.30,
23 / 39

ACCEPTED MANUSCRIPT
126.07, 124.99, 116.55, 89.03, 52.20, 45.56, 42.20, 31.79, 24.62. HRMS (ESI): m/z
calcd for C24H27N8O2S (M+H)+, 491.1977; found, 491.1929.
1
Compound C13, white solid, yield: 76 %. m.p.: 161-165 oC. H NMR (400 MHz,
DMSO-d6) δ 7.41 (d, J = 8.7 Hz, 2H), 7.30 (d, J = 8.9 Hz, 2H), 7.10 (d, J = 9.0 Hz,
2H), 6.88 (d, J = 8.7 Hz, 2H), 6.14 (s, 1H), 4.48 (s, 2H), 3.72 (s, 4H), 2.82 (s, 3H),
2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.56, 163.68, 158.46, 155.77,
147.95, 145.53, 130.13, 129.72, 128.52, 126.01, 116.63, 113.83, 55.04, 52.12, 45.47,
34.06, 24.64. HRMS (ESI): m/z calcd for C25H30N7OS (M+H)+, 476.2232; found,
476.2190.
1
Compound C14, white solid, yield: 59 %. m.p.: 158-167 oC. H NMR (400 MHz,
DMSO-d6) δ 7.60 (dd, J = 10.5, 1.7 Hz, 1H), 7.53 (t, J = 8.1 Hz, 1H), 7.42 – 7.36 (m,
1H), 7.29 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.14 (s, 1H), 4.53 (s, 2H), 3.19
(s, 8H), 2.73 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.91, 155.79,
148.23, 145.60, 140.39, 130.46, 126.07, 124.51, 117.42, 116.51, 88.98, 52.51, 45.84,
33.22, 24.62. HRMS (ESI): m/z calcd for C24H26ClFN7S (M+H)+, 498.1643; found,
498.1597.
1
Compound C15, white solid, yield: 67 %. m.p.: 172-175 oC. H NMR (400 MHz,
DMSO-d6) δ 7.78 (dd, J = 7.2, 2.1 Hz, 1H), 7.58 – 7.48 (m, 1H), 7.41 – 7.26 (m, 3H),
7.10 (d, J = 8.9 Hz, 2H), 6.14 (s, 1H), 4.52 (s, 2H), 3.21 (s, 8H), 2.74 (s, 3H), 2.36 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 163.97, 163.78, 157.55, 155.80, 155.10,
148.22, 145.59, 136.55, 130.98, 129.69, 129.62, 128.26, 126.08, 119.12, 116.86,
116.66, 116.51, 88.96, 52.39, 45.74, 42.46, 32.99, 24.63. HRMS (ESI): m/z calcd for
C24H26ClFN7S (M+H)+, 498.1643; found, 498.1600.
1
Compound C16, white solid, yield: 66 %. m.p.: 191-196 oC. H NMR (400 MHz,
DMSO-d6) δ 7.54 (d, J = 8.1 Hz, 2H), 7.44 – 7.36 (m, 1H), 7.25 (d, J = 8.9 Hz, 2H),
7.02 (d, J = 9.0 Hz, 2H), 6.15 (s, 1H), 5.76 (s, 1H), 4.83 (s, 2H), 3.20 – 3.12 (m, 4H),
13
2.49 – 2.44 (m, 4H), 2.37 (s, 3H), 2.23 (s, 3H). C NMR (100 MHz, DMSO-d6) δ
163.80, 155.81, 149.41, 145.74, 135.09, 132.23, 130.45, 128.79, 127.23, 125.92,
115.78, 89.00, 59.72, 54.87, 54.52, 47.88, 45.69, 31.49, 24.66, 20.72, 14.05. HRMS
(ESI): m/z calcd for C24H26Cl2N7S (M+H)+, 514.1347; found, 514.1304.
24 / 39

ACCEPTED MANUSCRIPT
1
Compound C17, white solid, yield: 75 %. m.p.: 188-192 oC. H NMR (400 MHz,
DMSO-d6) δ 7.36 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.9 Hz, 2H), 7.12 (d, J = 7.9 Hz,
2H), 7.01 (d, J = 9.0 Hz, 2H), 6.11 (s, 1H), 4.49 (s, 2H), 3.21 – 3.11 (m, 4H), 2.49 –
2.43 (m, 4H), 2.35 (s, 3H), 2.26 (s, 3H), 2.23 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 164.45, 163.61, 155.78, 149.42, 145.64, 136.40, 134.82, 128.97, 128.82, 127.23,
125.97, 115.78, 88.81, 54.52, 47.88, 45.69, 34.29, 24.62, 20.65. HRMS (ESI): m/z
calcd for C25H30N7S (M+H)+, 460.2283; found, 460.2260.
1
Compound C18, white solid, yield: 67 %. m.p.: 172-176 oC. H NMR (400 MHz,
DMSO-d6) δ 7.71 (dd, J = 23.9, 8.2 Hz, 4H), 7.30 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.9
13
Hz, 2H), 6.14 (s, 1H), 4.62 (s, 2H), 2.81 (s, 3H), 2.35 (s, 3H). C NMR (100 MHz,
DMSO-d6) δ 163.98, 163.78, 155.79, 148.03, 145.59, 143.41, 129.69, 128.43, 127.87,
127.56, 126.06, 125.24, 125.20, 122.86, 116.60, 88.99, 52.05, 45.40, 41.96, 33.72,
24.61. HRMS (ESI): m/z calcd for C25H27F3N7S (M+H)+, 514.2000; found, 514.1956.
1
Compound C19, white solid, yield: 58 %. m.p.: 161-165 oC. H NMR (400 MHz,
DMSO-d6) δ 7.31 (t, J = 4.3 Hz, 6H), 7.27 – 7.21 (m, 1H), 7.10 (d, J = 8.4 Hz, 2H),
6.13 (s, 1H), 3.67 – 3.48 (m, 10H), 3.05 (t, J = 7.5 Hz, 2H), 2.81 (s, 3H), 2.36 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 164.68, 163.60, 155.83, 147.99, 145.57, 140.00,
128.62, 128.52, 128.32, 126.31, 126.07, 116.62, 88.87, 52.12, 45.47, 42.03, 35.43,
31.88, 24.61. HRMS (ESI): m/z calcd for C25H30N7S (M+H)+, 460.2283; found,
460.2242.
1
Compound C20, white solid, yield: 77 %. m.p.: 152-156 oC. H NMR (400 MHz,
DMSO-d6) δ 7.24 (m, 7H), 7.01 (d, J = 9.0 Hz, 2H), 6.10 (s, 1H), 3.25 (t, J = 7.1 Hz,
2H), 3.20 – 3.11 (m, 4H), 2.82 – 2.69 (m, 2H), 2.50 – 2.43 (m, 4H), 2.34 (s, 3H), 2.23
(s, 3H), 2.05 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.68, 163.48, 155.80,
149.41, 145.65, 141.16, 128.32, 128.30, 127.27, 126.01, 125.84, 115.78, 88.76, 54.52,
47.88, 45.69, 33.99, 31.12, 30.20, 24.61. HRMS (ESI): m/z calcd for C26H32N7S
(M+H)+, 474.2439; found, 474.2399.
1
Compound C21, white solid, yield: 73 %. m.p.: 192-197 oC. H NMR (400 MHz,
DMSO-d6) δ 7.23 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 6.09 (s, 1H), 3.27 –
3.20 (m, 2H), 3.19 – 3.13 (m, 5H), 2.46 (d, J = 4.9 Hz, 4H), 2.34 (s, 3H), 2.23 (s, 3H),
25 / 39

ACCEPTED MANUSCRIPT
1.75 (dd, J = 14.4, 7.2 Hz, 2H), 1.01 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 155.80, 155.22, 126.03, 117.13, 115.78, 54.54, 47.90, 32.51, 24.61,
22.84, 13.07. HRMS (ESI): m/z calcd for C20H28N7S (M+H)+, 398.2126; found,
398.2094.
1
Compound C22, white solid, yield: 62 %. m.p.: 162-165 oC. H NMR (400 MHz,
DMSO-d6) δ 7.24 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 6.11 (s, 1H), 6.08 –
5.97 (m, 1H), 5.36 (dd, J = 16.9, 1.5 Hz, 1H), 5.13 (d, J = 10.0 Hz, 1H), 3.94 (d, J =
6.9 Hz, 2H), 3.23 – 3.11 (m, 4H), 2.49 – 2.43 (m, 4H), 2.35 (s, 3H), 2.23 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 164.15, 163.60, 155.82, 149.44, 145.67, 134.00,
127.23, 126.00, 118.02, 115.77, 88.79, 54.54, 47.90, 45.72, 33.25, 24.62. HRMS
(ESI): m/z calcd for C20H26N7S (M+H)+, 396.1970; found, 396.1935.
1
Compound C23, white solid, yield: 72 %. m.p.: 202-205 oC. H NMR (400 MHz,
DMSO-d6) δ7.31 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.9 Hz, 2H), 6.16 (s, 1H), 4.13 (d, J
= 2.5 Hz, 2H), 3.23 (t, J = 2.5 Hz, 1H), 2.81 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 163.89, 163.27, 155.82, 147.98, 145.63, 128.44, 125.98, 116.62, 89.07,
80.18, 73.90, 52.05, 45.40, 41.95, 24.66, 19.11, 18.50. HRMS (ESI): m/z calcd for
C20H24N7S (M+H)+, 394.1813; found, 394.1777.
1
Compound C24, white solid, yield: 38 %. m.p.: 158-165 oC. H NMR (400 MHz,
DMSO-d6) δ 6.99 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 5.81 (s, 1H), 2.90 (d, J
= 7.2 Hz, 4H), 2.20 (s, 1H), 2.03 (s, 3H), 0.92 (m, 1H), 0.33 – 0.19 (m, 2H), 0.02 (d, J
= 4.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 165.04, 163.53, 155.77, 147.97,
145.50, 128.50, 126.02, 116.57, 88.79, 51.98, 45.34, 41.89, 36.19, 24.62, 11.22, 5.64.
HRMS (ESI): m/z calcd for C21H28N7S (M+H)+, 410.2126; found, 410.2093.
1
Compound C25, white solid, yield: 68 %. m.p.: 198-204 oC. H NMR (400 MHz,
DMSO-d6) δ 7.27 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.9 Hz, 2H), 6.11 (s, 1H), 3.18 (d, J
= 6.8 Hz, 3H), 2.98 (d, J = 4.9 Hz, 5H), 2.58 (s, 3H), 2.35 (s, 3H), 1.85 (d, J = 12.2
Hz, 2H), 1.65 (dd, J = 29.6, 10.5 Hz, 4H), 1.28 – 0.95 (m, 7H). 13C NMR (100 MHz,
DMSO-d6) δ 165.14, 163.48, 155.72, 145.55, 128.65, 128.27, 128.01, 126.04, 116.26,
114.37, 88.77, 53.14, 46.38, 33.14, 31.81, 25.81, 25.43, 24.61. HRMS (ESI): m/z
calcd for C24H34N7S (M+H)+, 452.2596; found, 452.2554.
26 / 39

ACCEPTED MANUSCRIPT
1
Compound C26, white solid, yield: 83 %. m.p.: 168-172 oC. H NMR (400 MHz,
DMSO-d6) δ 7.77 (dd, J = 6.0, 3.1 Hz, 2H), 7.50 (dd, J = 6.1, 3.1 Hz, 2H), 7.36 (d, J =
8.7 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.17 (s, 1H), 5.03 (s, 2H), 3.87 (s, 2H), 3.51 (s,
2H), 3.19 (s, 4H), 2.83 (s, 3H), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
163.80, 162.68, 155.85, 150.89, 148.12, 145.69, 131.74, 128.16, 126.26, 125.37,
116.58, 114.07, 89.18, 51.98, 45.33, 41.87, 25.57, 24.43. HRMS (ESI): m/z calcd for
C25H28N9S (M+H)+, 486.2188; found, 486.2148.
1
Compound C27, white solid, yield: 78 %. m.p.: 156-161 oC. H NMR (400 MHz,
DMSO-d6) δ 8.10 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.95 (s, 1H), 7.42 (dd,
J = 8.6, 1.8 Hz, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.9 Hz, 2H), 6.15 (s, 1H),
4.82 (s, 2H), 3.47 (s, 8H), 2.81 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 164.17, 163.79, 155.85, 148.04, 145.61, 139.13, 138.23, 131.52, 129.50, 128.43,
126.10, 124.71, 124.54, 121.65, 116.60, 88.94, 52.10, 45.45, 42.01, 27.75, 24.65.
HRMS (ESI): m/z calcd for C26H27ClN7S2 (M+H)+, 536.1457; found, 536.1411.
1
Compound C28, white solid, yield: 82 %. m.p.: 210-215 oC. H NMR (400 MHz,
DMSO-d6) δ 7.42 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.9 Hz,
2H), 6.91 (d, J = 9.0 Hz, 2H), 4.41 (s, 2H), 3.16 – 3.08 (m, 4H), 2.49 – 2.44 (m, 4H),
13
2.43 (s, 3H), 2.22 (s, 3H), 1.80 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.85,
163.33, 154.22, 148.12, 142.47, 137.44, 131.71, 130.76, 130.64, 128.24, 124.09,
115.31, 101.08, 54.47, 48.14, 45.59, 33.52, 23.83, 13.67. HRMS (ESI): m/z calcd for
C25H29ClN7S (M+H)+, 494.1893; found, 494.1849.
1
Compound C29, white solid, yield: 67 %. m.p.: 169-173 oC. H NMR (400 MHz,
DMSO-d6) δ 7.33 (dd, J = 19.6, 8.4 Hz, 4H), 7.02 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.8
Hz, 2H), 4.33 (s, 2H), 3.10 (s, 4H), 2.46 (s, 6H), 2.40 – 2.32 (m, 2H), 2.22 (s, 3H),
1.33 – 1.19 (m, 3H), 1.13 (m, 2H), 1.05 – 0.95 (m, 2H), 0.76 (t, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 163.38, 163.10, 154.24, 148.73, 142.86, 137.48,
131.67, 130.75, 130.22, 128.19, 125.53, 115.26, 105.88, 54.87, 54.33, 48.16, 45.49,
33.40, 31.10, 28.03, 25.64, 23.34, 21.80, 13.90. HRMS (ESI): m/z calcd for
C29H37ClN7S (M+H)+, 550.2519; found, 550.2471.
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Compound C30, white solid, yield: 85 %. m.p.: 189-192 C . H NMR (400 MHz,
CDCl3) δ 7.71 (s, 1H), 7.51-7.44 (m, 4H), 7.36-7.27 (m, 5H), 7.26-7.20 (m, 1H), 6.30
(s, 1H), 4.54 (s, 2H), 2.51 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 166.54, 164.77,
155.92, 144.35, 137.57, 135.76, 130.02, 129.11, 128.53, 127.36, 126.92, 123.82,
89.01, 35.80, 25.41. HRMS (ESI): m/z calcd for C19H16N5S (M-H)-, 346.1126; found,
346.1135.
1
Compound C31, yellow solid, yield: 89 %. m.p.: 196-201 oC. H NMR (400 MHz,
DMSO-d6) δ 7.93 (s, 2H), 7.55-7.43 (m, 2H), 7.37-7.28 (m, 2H), 7.24 (dd, J = 8.4, 6.1
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Hz, 1H), 6.12 (s, 1H), 4.49 (s, 2H), 2.37 (s, 3H). C NMR (100 MHz, DMSO-d6) δ
164.29, 162.84, 155.87, 147.50, 138.07, 128.92, 128.38, 127.16, 90.25, 34.36, 24.37.
HRMS (ESI): m/z calcd for C13H12N5S (M-H)-, 270.0813; found, 270.0821.
o
Compound C32, white solid, yield: 79%. m.p.: 180-19 C. 1H NMR (400 MHz,
DMSO-d6) δ 10.15 (s, 1H), 7.50 (m, 6H), 7.33 (d, J = 7.0 Hz, 1H), 7.18 (dd, J = 6.0,
3.1 Hz, 2H), 6.34 (s, 1H), 4.79 (s, 2H), 2.38 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 164.03, 163.80, 155.80, 150.66, 144.94, 136.53, 129.48, 126.12, 124.44, 122.01,
114.75, 89.29, 28.00, 24.60. HRMS (ESI): m/z calcd for C20H16N7S (M-H)-, 386.1188;
found, 386.1196.
4.6. Cell proliferation assay
The MTT assay was carried out following our previously reported methods [58] to
evaluate the antiproliferative activity of C26.
4.7. LSD1 enzymatic assay
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
o
BL21 (DE). The recombinant protein was induced with 0.25 mM IPTG at 20 C and
purified following affinity chromatography, ion exchange chromatography and gel
filtration. Then the compounds were incubated with the 5 nM recombinant LSD1 and
25 µM H3K4me2 peptide in the present of FAD (50 nM), Amplex Red (20 nM) and
horseradish peroxidase (5.5 U/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.
4.8. HRP enzymatic assay
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