New tranylcypromine derivatives containing sulfonamide motif as potent LSD1 inhibitors to target acute myeloid leukemia: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2020.103808

Lysine-specific demethylase 1 (LSD1) is frequently elevated in acute myeloid leukemia (AML) and often leads to tumorigenesis. In recent years, numerous LSD1 inhibitors based on tranylcypromine (TCP) scaffolding have reached clinical trials. Most TCP derivatives were modified at the amino site of cyclopropane motif. Herein, we for the first time introduced a sulfonamide group in TCP benzene ring of series a compounds and performed a systematical study on structure and activity relationships by varying sulfonamide groups. The introduction of sulfonamide significantly increased the targeting capacity of TCP against LSD1. Moreover, we discovered that the Boc attached LSD1 inhibitors (labelled as series b compounds) substantially improved their anti-proliferation capacity towards AML cells. The intracellular thermal shift and LC-MS/MS results implied that Boc enhanced the drug lipophilicity and might be removed under the cancerous acidic environment to release the real pharmacophore, evidenced by the fact that a structurally similar but acidic inert pivaloyl to replace Boc dramatically dropped the cellular anti-proliferation effect. Finally, a benzyl group installed at the amino site to appropriately increase lipophilicity led to trans-4-(2-(benzylamino)-cyclopropyl)-N,N-diethylbenzenesulfonamide a10 that showed better anti-proliferation activity in AML cells and enzymatic inhibition against LSD1. Taken together, our work offers a novel TCP-based structure and provides a prodrug strategy for the discovery of potent LSD1 inhibitors by having appropriate lipophilicity.

Full text of DOI:10.1016/j.bioorg.2020.103808

BioorganicChemistry99(2020)103808
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New tranylcypromine derivatives containing sulfonamide motif as potent
LSD1 inhibitors to target acute myeloid leukemia: Design, synthesis and
biological evaluation
Liyun Lianga, Haiwen Wanga, Yongliang Dua, Bingling Luoa, Ning Menga, Meifeng Cenb,
⁎
Peng Huanga, A. Ganesanc, Shijun Wena,
a State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 651 Dongfeng East Road,
Guangzhou 510060, China
b Medical Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510120, China
c School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Keywords:
Lysine-specific demethylase 1 (LSD1) is frequently elevated in acute myeloid leukemia (AML) and often leads to
tumorigenesis. In recent years, numerous LSD1 inhibitors based on tranylcypromine (TCP) scaffolding have
reached clinical trials. Most TCP derivatives were modified at the amino site of cyclopropane motif. Herein, we
for the first time introduced a sulfonamide group in TCP benzene ring of series a compounds and performed a
systematical study on structure and activity relationships by varying sulfonamide groups. The introduction of
sulfonamide significantly increased the targeting capacity of TCP against LSD1. Moreover, we discovered that
the Boc attached LSD1 inhibitors (labelled as series b compounds) substantially improved their anti-proliferation
capacity towards AML cells. The intracellular thermal shift and LC-MS/MS results implied that Boc enhanced the
drug lipophilicity and might be removed under the cancerous acidic environment to release the real pharma-
cophore, evidenced by the fact that a structurally similar but acidic inert pivaloyl to replace Boc dramatically
dropped the cellular anti-proliferation effect. Finally, a benzyl group installed at the amino site to appropriately
increase lipophilicity led to trans-4-(2-(benzylamino)-cyclopropyl)-N,N-diethylbenzenesulfonamide a10 that
showed better anti-proliferation activity in AML cells and enzymatic inhibition against LSD1. Taken together,
our work offers a novel TCP-based structure and provides a prodrug strategy for the discovery of potent LSD1
inhibitors by having appropriate lipophilicity.
Lysine-specific demethylase 1
Inhibitor
Acute myeloid leukemia
Lipophilicity
Prodrug
1. Introduction
retinoic acid (ATRA) has been used to break the differentiation
blockade in AML [13]. Therefore, LSD1 is emerging as an essential
Aberrant epigenetic modification is a hallmark of hematological
malignancy and correlated with the leukemic transformation [1,2].
Simultaneously, epigenetic regulation is a highly dynamic process
manipulated by multiple enzymes, and such process can be used as a
potential drug target [3–6]. An epigenetic modification enzyme, lysine-
specific demethylase 1 (LSD1, also known as KDM1A), is a nuclear
amine oxidase homolog and specifically targets mono- and di-methy-
lated lysine 4 and lysine 9 of histone H3 (H3K4me1/2, H3K9me1/2)
using flavin adenine dinucleotide (FAD) as the coenzyme [7–11]. Evi-
dences suggest that LSD1 is often elevated in acute myeloid leukemia
(AML), particularly poor-differentiated AML harboring chromosomal
translocation [12]. A combination of LSD1 inhibitors with all-trans-
target for AML therapy.
In the past decades, numerous LSD1 inhibitors were developed and
could be divided into irreversible and reversible types according to
their action mechanisms [14–25]. Currently, irreversible LSD1 in-
hibitors based on tranylcypromine (TCP) scaffolding have achieved
good progress, illustrated by the two most advanced drug candidates
ORY-1001 and GSK2879552 (Fig. 1A) [26,27]. Their inhibition me-
chanism involves the formation of an amine cation radical to covalently
react with FAD to block the biological function of LSD1 (Fig. 1B). As far
as we know, ORY-1001 and GSK2879552 have entered into phase Ⅰ
clinal trials with enzymatic inhibition at a nanomolar level (Fig. 1C).
The development of irreversible LSD1 inhibitors often focuses on the
⁎ Corresponding author.
E-mail address: wenshj@sysucc.org.cn (S. Wen).
https://doi.org/10.1016/j.bioorg.2020.103808
Received 6 February 2020; Received in revised form 20 March 2020; Accepted 29 March 2020
Availableonline17April2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

L. Liang, et al.
BioorganicChemistry99(2020)103808
Fig. 1. (A) The structures, (B) inhibitory mechanism and (C) enzymatic activities of three representative irreversible LSD1 inhibitors based on TCP scaffolding.
alteration of the cyclopropane amino side, and there are relatively few
reports of modifications on the benzene ring [28–31]. The sulfonamide
group is frequently employed in the drug development [32] and it is
shaped in a tetrahedron three-dimension, very different from the planar
amides [33]. Thus, it was of our interest to install a sulfonamide on the
benzene ring of TCP to develop novel potent LSD1 inhibitors.
formation of compounds b1-b9. Meanwhile, deprotection of Boc using
HCl in methanol gave series a compounds a1-a9 in a hydrochloride salt
form (Scheme 1A). For the preparation of c9, compound a9 reacted
with pivaloyl chloride in presence of DIPEA to assemble c9
(Scheme 1B). Additionally, compound b10 was also synthesized from
b9 by introducing a benzyl group (Scheme 1C), and the Boc depro-
tection from b10 afforded compound a10. As a result, both alkyl and
aryl primary amines as well as both cyclic and acyclic secondary amines
were installed to construct three series of novel sulfonamide TCP de-
rivatives for further anticancer screening.
2. Results and discussion
2.1. Chemistry
Numerous LSD1 inhibitors were often modified at the cyclopropane
amino side. Structure-activity relationship studies (SARs) on the ben-
zene ring were relatively scant, for example, a reported LSD1 inhibitor
T-3775440 has a para-positioned carboxamide [34]. For the first time,
we introduced the sulfonamide group at the benzene ring side of TCP to
develop novel LSD1 inhibitors (Fig. 2). As a result, a series of com-
pounds defined as series a was designed. Simultaneously, in our pre-
vious work, compounds with Boc group contributed to an increased
cellular uptake, and it was of our interest to further explore its ad-
vantage. Thus, Boc group was newly introduced at the cyclopropane
amino side to obtain series b compounds. Meanwhile, c9 with a
structurally similar pivaloyl was also designed.
2.2. Preliminary anti-proliferation screening of modified TCP derivatives at
the cellular level
The selection of appropriate cancer cell lines to evaluate the anti-
proliferation effect of inhibitors is essential for acquiring realistic re-
sults. Several reports have studied drug sensitivity to LSD1 inhibitors in
numerous cell lines of hematopoietic malignancy, and acute myeloid
leukemia MV4-11 was regarded as the drug-sensitive cell line towards
LSD1 inhibition [36,37]. Before the preliminary screening of our syn-
thetic compounds, we wanted to confirm the reported results firstly.
LSD1 was significantly elevated in acute myeloid leukemia via ana-
lyzing patient-derived data from the Oncomine database (Fig. S1A).
After ORY-1001 and GSK2879552 were confirmed to inhibit LSD1 at
nanomolar concentrations (Fig. 1C), these two compounds and TCP
were employed to screen AML cell lines treated with each drug at 5 μM
for 8 days. Among six tested AML cell lines, MV4-11 was found to be
most sensitive towards LSD1 inhibition, which was consistent with lit-
erature reports (Fig. 3A and Fig. S1B-F) [36]. Although both ORY-1001
and GSK2879552 quickly reached 40% inhibition of cancer cell growth
at low concentrations (< 1 μM), they could not inhibit cell growth
further at higher concentrations (Fig. 3B). The results suggested that the
development of more potent LSD1 inhibitors to increase their cellular
anti-proliferation capacity would be highly favorable.
All the racemic compounds a1-a10, b1-b10 and c9 were synthe-
sized via the routes described in Scheme 1. Commercially available 2-
phenyloxirane (labeled as 1) was reacted with triethyl phosphonoace-
tate in the presence of n-BuLi to acquire trans-cyclopropyl ester 2 [33].
Following a procedure of Huntress’ work, [35] 2 was treated with
chlorosulfonic acid in anhydrous dichloromethane, and
a chlor-
osulfonyl group was successfully installed in the para position of the
benzene ring to afford compound 3. Then, a series of different amino
groups were added respectively to get 4 with varying sulfonamides.
Series b compounds b1-b9 were then obtained after three steps. Firstly,
4 were hydrolyzed into acids 5 under subjection to LiOH, and the fol-
lowing coupling reaction with DPPA at room temperature provided acyl
azide 6. Curtius rearrangement in the refluxing t-BuOH resulted in the
Compounds a1-a9 and b1-b9 were screened for their anti-pro-
liferation capacity using MV4-11 cell line at three gradient
2

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BioorganicChemistry99(2020)103808
Fig. 2. The racemic compounds designed from tranylcypromine. (A) Series a compounds with a free amine. (B) Series b compounds with Boc at the cyclopropane
amino site. (C) Series c compound with a pivaloyl.
concentrations (1 μM, 10 μM and 30 μM) via MTS assay, as shown in
Fig. 3C-E. At a low concentration of 1 μM, most of the synthetic com-
pounds did not prevent cancerous cell growth obviously. While the
dosage increased (10 μM and 30 μM), some compounds especially b9
started to show substantial anti-proliferation capacity, which was sig-
nificantly better than the three typical positive compounds (TCP, ORY-
1001 and GSK2879552). Generally, series b compounds with Boc group
exhibited better anti-proliferation activity than the corresponding series
a compounds with only free amines at 10 μM and 30 μM (Fig. 3F).
Compared to the simple TCP, the lipophilic alkylsulfonamides see-
mingly increased the anti-proliferation potency of many of series b
compounds. Notably, the best compound b9 almost fully inhibited the
cell growth of MV4-11, much better than three well-known LSD1
inhibitors. CLogP is a classic parameter representing lipid solubility,
and its range between 1 and 5 is optimal for the drug development. To
study the potential relationship between lipophilicity and anti-pro-
liferation capacity of the synthetic compounds, CLogP values were
calculated by ChemDraw and the correlation analysis was conducted
through GraphPad Prism. Series a compounds exhibited a lower CLogP
value (< 2.4), likely leading to the worse cellular uptake.
Consequently, the cell growth inhibitory rate of a1-a9 at 30 μM were
below 50% (Table S1). Introduction of Boc group improved the lipo-
philicity of compounds b1-b9, and the inhibition rates were elevated as
a result. Especially, while the ClogP reached the range of between 3.0
and 4.0, the anti-proliferation capacity of compounds was enhanced
significantly. Collectively, there was an apparent positive correlation
Scheme 1. Synthesis of the designed compounds. Reagents and conditions: (a) triethyl phosphonoacetate, n-BuLi, 1,4-dioxane, r.t., then microwave at 135 °C for
60 min; (b) chlorosulfonic acid, anhydrous CH2Cl2, 0 °C-r.t., 3 h; (c) DIPEA, anhydrous CH2Cl2, 0 °C-r.t., 2 h; (d) THF/H2O (3:1), LiOH, r.t., 3 h; (e) Et3N, DPPA, THF,
r.t., 4 h; (f) t-BuOH, reflux, 12 h; (g) HCl/MeOH, r.t., 2 h; (h) DIPEA, anhydrous CH2Cl2, r.t., 2 h; (i) NaH, benzyl bromide, THF, r.t., 2 h. Note: the compounds are
racemic; DIPEA: N,N-diisopropylethylamine, DPPA: diphenyl phosphoryl azide.
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BioorganicChemistry99(2020)103808
Fig. 3. Primary cellular anti-proliferation screening of our synthetic compounds. (A) Cell counting after treatment with three typical TCP-derived LSD1 inhibitors at
5 μM for 8 days. (B) Cell viability after treatment with three inhibitors at gradient dosages for 6 days, determined by MTS assay. (C-E) Preliminary anti-proliferation
effect screening of TCP derivatives in MV4-11 cell line via MTS assay at three gradient concentrations, (C) 1 μM, (D) 10 μM and (E) 30 μM. (F) Quantitative anti-
proliferation comparison of three known inhibitors, series a and series b compounds. (G) Correlation analysis of anti-proliferation capacity at 30 μM against CLogP
n.s.
values. All data are representative of 3 independent experiments and shown as mean
SD of triplicates.
no significance, *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001 by unpaired, 2-tailed Student’s t test.
between cell inhibition at 30 μM dosage and lipophilicity as shown in
Fig. 3G, implying that series b compounds might increase their cellular
anti-proliferation capacity via an elevated lipophilicity mediated by the
Boc group.
Table 1
Enzymatic inhibitory activity of the synthetic compounds (IC50).
Series a
IC50 (μM)
Series b
IC50 (μM)
a1
a2
a3
a4
a5
a6
a7
a8
a9
0.3691
0.2006
0.4927
0.4973
0.2922
0.8413
0.1994
0.2382
0.1229
b1
b2
b3
b4
b5
b6
b7
b8
b9
> 1
> 1
> 1
> 1
> 1
> 1
> 1
> 1
> 1
2.3. Comparison between series a and b compounds at the enzymatic level
Following the preliminary cellular anti-proliferation screening re-
sults, we further look at the enzymatic inhibition activity of these two
series compounds. Targeting ability against LSD1 was determined by a
commercially available LSD1 Inhibitor Screening Assay Kit (#700120,
Cayman Chemical, USA), and IC50 values in the enzymatic inhibition
level were presented in Table 1. Enzymatic curves were shown in Fig.
S2A-L. All series a compounds were better than the parent inhibitor
The IC50 value indicated the concentration that achieved half inhibition of
enzymatic activity.
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BioorganicChemistry99(2020)103808
Fig. 4. The intracellular LSD1 targeting capacity of series b compounds with the Boc group. (A-B) Cellular thermal shift assay of LSD1 in MV4-11 cells treated for 24 h
with (A) a9, b9 and (B) c9 at 5 μM. The level of LSD1 was determined by Western blot. (C) Dose-response curves for the cell viability of MV4-11 cells treated with
each drug for 6 days, determined by MTS assay. (D-E) LC-MS/MS detection of a9 in MV4-11 cells treated with each drug at 10 μM for (D) 3 days and (E) 6 days. (F)
Potential conversion mode from b9 to a9 within cancerous acid environment. All data are representative of 3 independent experiments and shown as mean
SD.
**p < 0.01, ***p < 0.001, ****p < 0.0001 by One-way ANOVA, Dunnett test.
TCP (IC50, 22 μM) with their IC50 values at nanomolar levels, indicating
that the introduction of sulfonamide groups significantly enhanced
their LSD1 targeting ability. Surprisingly, no significant LSD1 inhibition
was observed in all series b compounds at 1000 nM (Fig. S2M), which
was counterintuitive. It seemed that other factors including lipophili-
city might play a crucial effect on the ani-proliferation capacity of series
b compounds.
validate our hypothesis, the Boc in b9 was changed to a structurally
similar pivaloyl group that is not easily removed even under strong acid
environment. Thus, compound c9 was designed and synthesized as
shown in Scheme 1B. Cellular thermal shift experiments showed that c9
did not stabilize LSD1 significantly and moreover its cellular anti-pro-
liferation effect dropped dramatically (Fig. 4B and 4C). Meanwhile, c9
did not inhibit the enzymatic activity of LSD1 in vitro (Fig. S3A). Based
on these findings, the Boc might serve as a prodrug functional group in
TCP derivatives to increase cell permeability and achieve a more ef-
fective anti-proliferation effect.
2.4. The Boc in series b compounds served as a prodrug functional group
Furthermore, LC-MS/MS was used to investigate the potential in-
tracellular conversion of b9 to a9 into a real LSD1 inhibitor. Firstly,
such conversion was tested in the culture medium without cells in vitro
(Fig. S3B). The mass ion signal of a9 could be detected at pH 6.5 and it
substantially increased at pH 6, indicating a possibility that series b
compounds were able to convert to series a compounds under such
weak acid conditions. To further validate the in vitro results, MV4-11
cells were treated with 10 μM of either a9, b9 or c9 for 3 days and
6 days, and then the cells were lysed and extracted using a solvent
system (chloroform : methanol : water = 6 : 3 : 1). The extract was
dried via nitrogen blower concentrator and then re-dissolved in 50%
MeOH/H2O solution for further LC-MS/MS analysis. The data as shown
in Fig. 4D and 4E, clearly demonstrated that a9 could be detected in
both samples treated with a9 and b9. The signal of a9 detected in b9-
treated cell samples was significantly higher than in a9-treated cell
samples. Moreover, cellular concentration of a9 converted from b9
steadily sustained high while the a9-treated samples contained less a9
Due to the potential lability of Boc to acids, we hypothesized that
Boc might be removed within cancerous acid environment to release
the real LSD1 inhibitors from series b compounds that did not show
detectable inhibition on recombinant LSD1 as shown in Table 1.
Therefore, the intracellular LSD1 targeting ability was further tested via
thermal shift experiments within cells that were pretreated with each
compound. The results, as shown in Fig. 4A, indicated that both a9 and
b9 could stabilize LSD1 protein almost to the same level, confirming
that series b compounds were able to target cellular LSD1. The action
mechanism of TCP compounds to inhibit LSD1 is associated with the
generation of amino cation radicals to covalently form an adduct with
FAD (Fig. 1B) [26,27]. The Boc at the amino site might decrease the
nitrogen electronegativity to prevent a cation radical from formation,
which was the reason why b9 did not show apparent enzymatic in-
hibition of recombinant LSD1. Meanwhile, these results might support
our hypothesis that Boc in b9 might be removed intracellularly to
generate the free amine a9 to act as a real LSD1 inhibitor. To further
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BioorganicChemistry99(2020)103808
Fig. 5. Compounds a9 and b9 induced myeloid differentiation. (A, B) Flow cytometry analysis of CD11b on MV4-11 cells treated for (A) 24 h and (B) 96 h with each
drug at 10 μM. (C) Statistic quantitative analysis of the mean CD11b fluorescence intensity from Fig. 5A and B. (D, E) Flow cytometry analysis of CD11b of HL-60 cells
treated for (D) 24 h and (E) 96 h with each drug at 10 μM. (F) Statistic quantitative analysis of the mean CD11b fluorescence intensity from Fig. 5D and 5E. All data
n.s.
are representative of 3 independent experiments and shown as mean
test.
SD.
no significance, ***p < 0.001, ****p < 0.0001 by unpaired, 2-tailed Student’s t
Table 2
Enzymatic activity (IC50), cell growth inhibition and CLogP of TCP, a9, and a10.
Compounds
Structure
IC50 (μM)
Cell Growth
LSD1
MAO-A
MAO-B
CLogP
TCP
a9
> 30
> 30
22a
0.2691
0.5067
1.5
0.1229
0.0290
0.8067
0.5255
0.8975
0.7017
1.7
3.9
a10
15.4
The IC50 value indicated the concentration that achieved half inhibition of either cell growth or enzymatic activity. CLogP values were analyzed via ChemDraw.
a
Reported in the commercial LSD1 Inhibitor Screening Assay Kit.
after 6 days, likely resulting from the continuous consumption of a9 by
covalently binding to LSD1 co-enzyme FAD as shown in Fig. 1B.
Meanwhile, a9 was rarely detected in the c9-treated samples as ex-
pected. Taken together, the continuous and steady supply of cellular a9
from b9 might contribute to its better anti-proliferation capacity, de-
monstrating the potential of Boc as a prodrug group (Fig. 4F).
results were also validated in another AML cell line, HL-60 (Fig. 5D-F).
2.6. Further lipophilic benzyl installation led to a10 with potent LSD1
inhibition and improved anticancer capacity
Based on our observation, appropriate lipophilicity was crucial for
TCP-based LSD1 inhibitors to enter cancer cells to enact their in-
tracellular biological function. Compared with series a compounds,
series b compounds with a Boc had a properly high CLogP values and
displayed better anticancer capacity, likely due to their improved in-
tracellular uptake. Therefore, a lipophilic functional group could be
installed at the amino site to increase lipophilicity while remaining
enzymatic LSD1 inhibitory potency. Thus, compound a9 with the best
enzymatic LSD1 inhibition was selected to install a benzyl group at the
cyclopropane amino site, leading to a new compound a10 with an
elevated CLogP. The introduction of the lipophilic benzyl group sur-
prisingly improved the cell growth inhibition and LSD1 targeting ca-
pacity (Table 2 and Fig. S4). Notably, a10 showed better selectivity
against LSD1 over other homologous enzymes MAO-A and MAO-B.
2.5. Compounds a9 and b9 promoted myeloid differentiation
Many examples in the literatures demonstrated that the anticancer
effect of LSD1 inhibitors is often involved in the induction of cell dif-
ferentiation in AML [26]. Since CD11b is regarded as a myeloid dif-
ferentiation marker, we detected the level of CD11b to compare the
effect of compounds a9, b9 and c9. The treatment with each of these
three compounds for 24 h did not show any impact on MV4-11 cells.
However, after 96 h treatment, both a9 and b9 significantly induced
myeloid differentiation while c9 did not (Fig. 5A-C). Meanwhile,
compound b9 induced more differentiation than a9, which might result
in the better anti-proliferation activity of series b compounds. Similar
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BioorganicChemistry99(2020)103808
Fig. 6. Compound a10 caused cell cycle blockade, induced myeloid differentiation and exhibited a synergistic effect with the clinical drug ATRA in MV4-11 cell line.
(A) Western blot of LSD1 methylated markers treated with a10 at various concentrations for 24 h. (B) Statistical analysis of the methylation levels in A (Student’s t
test, unpaired, 2-tailed). (C) Thermal shift assay of intracellular LSD1 with a10 at 5 μM for 24 h. (D) Flow cytometry analysis of cell cycle after 24 h treatment with
each drug at 10 μM (One-way ANOVA, Dunnett test). (E) Flow cytometry analysis of CD11b after treatment with each drug at 10 μM for 24 h and 96 h (One-way
ANOVA, Dunnett test). (F) Dose-response curves of cells co-treated with a10 at various concentrations and ATRA at indicated concentrations for 6 days. (G)
Combination index (CI) of combinations of a10 and ATRA, analyzed by Calcusyn Software. CI < 1 means a synergistic effect. All data are representative of 3
n.s.
independent experiments and shown as mean
SD.
no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Meanwhile, the cellular anti-proliferation capacity of b10 dropped
compared to the parental compound b9, implying that too high lipo-
philicity (CLogP > 5) might impair cell permeability of b10 although
b10 was able to bind to cellular LSD1 as demonstrated in the cellular
thermal shift experiment (Table S2 and Fig. S5A). Since a10 showed the
best potency against recombinant LSD1 although it inhibited MV4-11 at
a slightly higher IC50 concentration compared to b10 (Fig. S5B), a10
was chosen for further biological studies. Cell counting experiments
confirmed that a10 significantly blocked cell growth at 5 μM after
6 days of treatment (Fig. S5C). Moreover, two other AML cell lines
Kasumi-1 and BaF3/ITD also displayed good anti-proliferation re-
sponses to the treatment of a10 (Fig. S5D). The direct outcome of LSD1
inhibition was an elevation of H3K4 and H3K9 methylation levels. In-
deed, our experiment confirmed that the degrees of H3K4me1,
H3K4me2 and H3K9me2 increased significantly after a treatment with
a10, especially at 10 μM (Fig. 6A and 6B). Thermal shift experiment
also verified that a10 could bind to cellular LSD1 to prevent its thermal
destabilization (Fig. 6C).
Since a10 inhibited LSD1 both extracellularly and intracellularly,
the consequent effect on cell cycle and cell differentiation was tested.
After 24 h treatment, a10 blocked the cell cycle at G0/G1 phase along
with the proportional decrease at S and G2/M phase (Fig. 6D). Three
reported LSD1 inhibitors required longer time to cause a substantial cell
cycle arrest (Fig. S5E), whereas a10 already caused more AML cells
death. CD11b, a marker of myeloid differentiation, was also examined.
The four compounds did not exhibit an obvious impact on CD11b ex-
pression in MV4-11 cells treated for a short 24 h time. However, after
96 h treatment, a10 substantially increased the level of CD11b, better
than the other three known LSD1 inhibitors (Fig. 6E). The induction of
myeloid differentiation was also validated in HL60 cell line (Fig. S5F).
Taken together, compound a10 targeted LSD1 and subsequently pre-
vented cell growth via cell cycle blockade and myeloid differentiation
promotion.
The combination of two drugs is often employed to increase their
pharmacological efficacy [38,39]. Examples in the literature have de-
monstrated that LSD1 inhibitors in a combination with the clinical drug
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BioorganicChemistry99(2020)103808
ATRA exhibited better anticancer effects [13,37]. Our synthetic com-
pound a10 was further investigated in a co-treatment with ATRA, and
indeed such a combination achieved a synergistic anti-proliferation
effect towards MV4-11 cells via MTS assay (Fig. 6F, 6G and Fig. S5G).
procedure.
Compound 3 (1.0 equiv) was sufficiently dissolved in anhydrous
CH2Cl2, then each type of amine (1.2 equiv) and DIPEA (2.0 equiv) was
added at 0 °C respectively. The reaction was stirred at room tempera-
ture for 2 h to obtain 4. Compound 4 (1.0 equiv) was dissolved in THF/
H2O (3:1) and then LiOH (2.0 equiv) was added. The reaction continued
for 12 h at room temperature to get 5. DPPA (1.2 equiv) and Et3N (1.2
equiv) were added dropwise at 0 °C into the solution of 5 (1.0 equiv) in
anhydrous THF. The reaction mixture was stirred for 2.5 h at room
temperature, and extracted with EtOAc and purified with column
chromatography to get 6. The solution of 6 (1.0 equiv) in t-BuOH was
refluxed at 110 °C for 12 h to finally obtain series b compounds b1-b9.
Deprotection of Boc group under an acidic condition gave com-
pounds a1-a9. Series b compound was weighed into the bottle, and a
HCl solution in methanol was added at 0 °C carefully. The reaction
mixture was stirred at room temperature for 2 h to acquire series a
compound.
3. Conclusions
In our present work, a new series of TCP-based LSD1 inhibitors were
developed and evaluated against AML. The introduction of sulfonamide
groups in TCP benzene ring site substantially increased the LSD1 in-
hibition potency. Meanwhile, our work demonstrated that an appro-
priate lipophilicity and potent LSD1 inhibition contributed to the im-
proved anti-proliferation effect of LSD1 inhibitors towards AML cell
lines. The lipophilic Boc group installed in the polar TCP derivatives
benefited the drug cellular uptake and could be removed to release the
real LSD1 inhibitors under acidic microenvironment in cancer cells.
Cellular protein thermal shift experiments further verified that the best
anticancer Boc-attached TCP derivative b9 stabilized intracellular LSD1
although it did not inhibit the recombinant enzyme. Moreover, the mass
spectroscopy further confirmed the intracellular release of the real
LSD1 inhibitor a9 from b9. Finally, a novel compound a10 bearing an
appropriate lipophilicity was developed, and it substantially inhibited
LSD1 and exhibited a good anticancer capacity in several AML cell
lines. a10 may provide a new structural scaffolding to develop more
potent LSD1 inhibitors for the therapeutic treatment of acute myeloid
leukemia.
4.1.3. The procedure for synthesis of c9
Compound c9 was obtained from a9. To a solution of a9 (138.4 mg,
1.0 equiv) in anhydrous CH2Cl2 (2 mL), DIPEA (276.8 μL, 3.5 equiv)
was added at 0 °C carefully. After stirring for 5 min, pivaloyl chloride
(83.9 μL, 1.5 equiv) was added dropwise and the reaction continued at
room temperature for 2 h. The mixture was extracted with EtOAc and
the organic phases were washed with brine, dried over Na2SO4 and
finally purified with column chromatography (PE/EtOAc = 3:1) to
obtain c9 as a white solid (43 mg, 27% yield).
4. Experimental section
4.1.4. The procedures for synthesis of a10 and b10
4.1. Chemistry
Compounds a10 and b10 were obtained from b9. To a solution of
b9 (50 mg, 1.0 equiv) in anhydrous THF at 0 °C was added NaH (8 mg,
1.5 equiv). The reaction mixture was stirred for 30 min, and then benzyl
bromide (24 μL, 1.2 equiv) was added. The reaction mixture was then
warmed to room temperature and stirred for 2 h. The mixture was
extracted with EtOAc and the organic phases were washed with brine,
dried over Na2SO4 and finally purified with column chromatography
(PE/EtOAc = 8:1) to obtain b10 as a white solid (30 mg, 48% yield).
Compound b10 (25 mg, 1.0 equiv) was weighed into the bottle, and a
HCl solution in methanol was added at 0 °C carefully. The reaction
mixture was stirred at room temperature for 2 h to acquire a10 as a
yellow solid (10 mg, 46% yield) after the solvent was removed under a
vacuum.
4.1.1. General information
Reagents and solvents were commercially available and used
without further purification. All nuclear magnetic resonance (NMR)
spectra, including 1H NMR and 13C NMR spectra, were recorded on a
Bruker Avance 400 MHz and 100 MHz spectrometer with TMS as an
internal standard. Chemical shifts were exhibited in parts per million (δ,
ppm) and referenced to CDCl3 with 7.26 for 1H and 77.16 for 13C, and
to methanol‑d4 with 3.31 for 1H and 49.00 for 13C. High-resolution
mass spectra (HRMS) were recorded on a Shimadzu Micromass Q-T of
Micromass spectrometer by electrospray ionization (ESI). Agilent 1100
HPLC system equipped with an Eclipse XDB-C18 column was used to
determine the purity of all final compounds. The flow rate of HPLC was
set at 1 mL/min and the gradient elution was run for 15 min, from 10%
MeOH/H2O to 90% MeOH/H2O for 8 min and 90% MeOH/H2O for
another 7 min. In addition, 1‰ formic acid was added to the methanol
to elevate the solubility of free amine compounds.
4.1.4.1. 4-(2-Aminocyclopropyl)-N-ethylbenzenesulfonamide
(a1). Yellow solid, 91% yield, 96% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.78 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H),
3.00–2.93 (m, 1H), 2.87 (q, J = 7.2 Hz, 2H), 2.51 (m, 1H), 1.59–1.49
(m, 1H), 1.43 (m, 1H), 1.04 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
methanol‑d4) δ 145.1, 140.3, 128.4, 128.1, 39.0, 32.4, 22.4, 15.2, 14.5.
HRMS (ESI): m/z calcd for C11H17N2O2S (M + H)+, 241.1005; found,
241.1000.
4.1.2. The procedures for synthesis of series a and b compounds
The appropriate triethyl phosphonoacetate (4.11 g, 1.1 equiv) was
dissolved in 8 mL of anhydrous 1,4-dioxane (8 mL), and then n-BuLi
(1.23 g, 1.2 equiv) was added at 0 °C carefully. After stirring for 30 min,
2-phenyloxirane (2.0 g, 1.0 equiv) was added, and then the reaction
mixture was subjected to microwave at 135 °C for 1 h. The reaction was
stopped using aqueous saturated NH4Cl, and the mixture was extracted
with EtOAc. The organic phases were washed with brine, dried over
Na2SO4 and finally purified with column chromatography (PE/
EtOAc = 50:1) to obtain 2 as a red oil (2.87 g, 91% yield). Then
chlorosulfonic acid (12.5 equiv) was dissolved in anhydrous CH2Cl2
(20 mL), and then 2 (2.87 g, 1.0 equiv) in anhydrous CH2Cl2 (5 mL) was
dropped into the mixture over a period of 1 h at 0 °C. The reaction
continued for another 2 h at room temperature. The mixture was finally
extracted with CH2Cl2 three times and purified via column chromato-
graphy (PE/EtOAc = 5:1) to obtain 3 as a yellow oil (2.79 g, 64%
yield). Then a series of amines were introduced at the sulfonyl group
side and all compounds shared the following general synthetic
4.1.4.2. Tert-butyl (2-(4-(N-ethylsulfamoyl)phenyl)cyclopropyl)carbamate
(b1). White solid, 72% yield, 99% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.72 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H),
2.86 (q, J = 7.3 Hz, 2H), 2.72 (m, 1H), 2.10–2.02 (m, 1H), 1.43 (s, 9H),
1.25 (t, J = 7.0 Hz, 2H), 1.04 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
methanol‑d4) δ 159.0, 148.2, 139.1, 128.0, 127.6, 39.0, 34.6, 28.7,
25.7, 17.1, 15.2. HRMS (ESI): m/z calcd for C16H23N2O4S (M−H)-,
339.1384; found, 339.1377.
4.1.4.3. 4-(2-Aminocyclopropyl)-N-phenethylbenzenesulfonamide
(a2). Yellow solid, 92% yield, 98% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.70 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H),
7.18 (m, 2H), 7.11 (m, 1H), 7.06 (d, J = 6.9 Hz, 2H), 3.03–2.96 (m,
2H), 2.95–2.89 (m, 1H), 2.70–2.63 (m, 2H), 2.52 (m, 1H), 1.58–1.50
8

L. Liang, et al.
BioorganicChemistry99(2020)103808
(m, 1H), 1.41–1.32 (m, 1H). 13C NMR (100 MHz, methanol‑d4) δ 145.1,
140.3, 139.9, 129.7, 129.5, 128.3, 128.1, 127.4, 45.6, 37.1, 32.4, 22.4,
14.5. HRMS (ESI): m/z calcd for C17H21N2O2S (M + H)+, 317.1318;
found, 317.1333.
4.1.4.11. 4-(2-Aminocyclopropyl)-N-(pyridin-2-yl)benzenesulfonamide
(a6). Yellow solid, 89% yield, 95% purity. 1H NMR (400 MHz,
methanol‑d4) δ 8.25 (d, J = 5.7 Hz, 1H), 8.13 (t, J = 7.9 Hz, 1H),
7.88 (d, J = 8.1 Hz, 2H), 7.45–7.30 (m, 4H), 2.92 (m, 1H), 2.49 (m,
1H), 1.56–1.49 (m, 1H), 1.38 (dd, J = 14.0, 6.8 Hz, 1H). 13C NMR
(100 MHz, methanol‑d4) δ 150.3, 146.9, 141.7, 139.2, 128.5, 120.2,
117.6, 101.3, 32.5, 22.4, 14.7. HRMS (ESI): m/z calcd for C14H16N3O2S
(M + H)+, 290.0958; found, 290.0958.
4.1.4.4. Tert-butyl
(2-(4-(N-phenethylsulfamoyl)phenyl)cyclopropyl)
carbamate (b2). White solid, 64% yield, 100% purity. 1H NMR
(400 MHz, methanol‑d4) δ 7.70 (d, J = 8.4 Hz, 2H), 7.22–7.28 (m,
4H), 7.17 (d, J = 7.2 Hz, 1H), 7.10 (d, J = 7.0 Hz, 2H), 3.04 (t,
J = 7.5 Hz, 2H), 2.75–2.67 (m, 3H), 2.09–2.00 (m, 1H), 1.43 (s, 9H),
1.31–1.21 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 156.4, 146.5, 137.8,
137.3, 128.9, 127.3, 127.0, 126.9, 80.0, 44.3, 35.9, 28.5, 17.0. HRMS
(ESI): m/z calcd for C22H27N2O4S (M−H)-, 415.1697; found, 415.1689.
4.1.4.12. Tert-butyl (2-(4-(N-(pyridin-2-yl)sulfamoyl)phenyl)cyclopropyl)
carbamate (b6). Yellow solid, 50% yield, 99% purity. 1H NMR
(400 MHz, CDCl3) δ 8.32 (d, J = 5.9 Hz, 1H), 7.79 (d, J = 8.2 Hz,
2H), 7.64 (t, J = 7.3 Hz, 1H), 7.37 (d, J = 8.9 Hz, 1H), 7.17 (d,
J = 7.9 Hz, 2H), 6.78 (t, J = 6.4 Hz, 1H), 4.89 (s, 1H), 2.72 (m, 1H),
2.05 (m, 1H), 1.42 (s, 9H), 1.27–1.15 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 155.4, 145.8, 142.5, 140.2, 139.4, 127.0, 126.9, 115.4, 113.9,
33.1, 28.5, 25.3, 16.9. HRMS (ESI): m/z calcd for C19H24N3O4S
(M + H)+, 390.1482; found, 390.1468.
4.1.4.5. 4-(2-Aminocyclopropyl)-N-cyclohexylbenzenesulfonamide
(a3). Pale yellow solid, 76% yield, 99% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.80 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.97
(m, 1H), 2.50 (m, 1H), 1.65 (m, 4H), 1.57–1.50 (m, 2H), 1.44 (m, 1H),
1.24–1.05 (m, 6H). 13C NMR (100 MHz, methanol‑d4) δ 144.9, 141.9,
128.2, 128.0, 53.9, 34.9, 32.5, 26.3, 26.0, 22.4, 14.6. HRMS (ESI): m/z
calcd for C15H23N2O2S (M + H)+, 295.1475; found, 295.1483.
4.1.4.13. 2-(4-(Morpholinosulfonyl)phenyl)cyclopropan-1-amine
(a7). Pale yellow solid, 91% yield, 95% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.72 (d, J = 7.9 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H),
3.72–3.66 (m, 4H), 2.99 (m, 1H), 2.96–2.91 (m, 4H), 2.55–2.47 (m,
1H), 1.59–1.50 (m, 1H), 1.46 (dd, J = 13.8, 6.9 Hz, 2H). 13C NMR
(100 MHz, methanol‑d4) δ 146.0, 142.6, 129.4, 128.3, 67.2, 47.4, 32.5,
4.1.4.6. Tert-butyl
(2-(4-(N-cyclohexylsulfamoyl)phenyl)cyclopropyl)
carbamate (b3). White solid, 62% yield, 100% purity. 1H NMR
(400 MHz, CDCl3) δ 7.75 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 7.7 Hz,
2H), 4.94 (s, 1H), 4.68 (d, J = 7.5 Hz, 1H), 3.15–3.03 (m, 1H), 2.77 (m,
1H), 2.09 (m, 1H), 1.66–1.57 (m, 2H), 1.54–1.46 (m, 2H), 1.43 (s, 9H),
1.28–1.06 (m, 8H). 13C NMR (100 MHz, CDCl3) δ 146.2, 138.9, 127.1,
127.0, 80.0, 52.7, 34.0, 29.8, 28.5, 25.3, 24.7, 17.0. HRMS (ESI): m/z
calcd for C20H31N2O4S (M + H)+, 395.1999; found, 395.1984.
22.4, 14.7. HRMS (ESI): m/z calcd for C13H19N2O3S (M + H)+
,
283.1111; found, 283.1104.
4.1.4.14. Tert-butyl
(2-(4-(morpholinosulfonyl)phenyl)cyclopropyl)
carbamate (b7). Yellow solid, 37% yield, 97% purity. 1H NMR
(400 MHz, CDCl3) δ 7.63 (d, J = 7.9 Hz, 2H), 7.27 (d, J = 7.8 Hz,
2H), 4.92 (s, 1H), 3.76–3.68 (m, 4H), 2.99–2.91 (m, 4H), 2.77 (m, 1H),
2.11 (m, 1H), 1.44 (s, 9H), 1.25 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
156.3, 147.0, 132.6, 128.1, 127.1, 66.2, 46.1, 28.5, 17.0. HRMS (ESI):
m/z calcd for C18H27N2O5S (M + H)+, 383.1635; found, 383.1621.
4.1.4.7. 4-(2-Aminocyclopropyl)-N-(tert-butyl)benzenesulfonamide
(a4). Yellow solid, 97% yield, 96% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.81 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H),
3.31 (s, 1H), 2.99–2.91 (m, 1H), 2.48 (m, 1H), 1.58–1.47 (m, 1H), 1.43
(m, 1H), 1.16 (s, 9H). 13C NMR (100 MHz, methanol‑d4) δ 144.7, 144.0,
128.2, 127.8, 54.8, 32.4, 30.4, 22.4, 14.6. HRMS (ESI): m/z calcd for
4.1.4.15. 2-(4-(Pyrrolidin-1-ylsulfonyl)phenyl)cyclopropan-1-amine
(a8). Yellow solid, 94% yield, 97% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.72 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H),
3.16 (t, J = 6.7 Hz, 4H), 2.98–2.91 (m, 1H), 2.51 (m, 1H), 1.73–1.64
(m, 4H), 1.58–1.50 (m, 1H), 1.40 (m, 1H). 13C NMR (100 MHz,
methanol‑d4) δ 145.6, 136.6, 129.0, 128.2, 32.5, 26.2, 22.4, 14.6.
HRMS (ESI): m/z calcd for C13H19N2O2S (M + H)+, 267.1162; found,
267.1156.
C
13H21N2O2S (M + H)+, 269.1318; found, 269.1310.
4.1.4.8. Tert-butyl
(2-(4-(N-(tert-butyl)sulfamoyl)phenyl)cyclopropyl)
carbamate (b4). White solid, 44% yield, 99% purity. 1H NMR
(400 MHz, CDCl3) δ 7.76 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.1 Hz,
2H), 4.98 (s, 1H), 4.90 (s, 1H), 2.75 (m, 1H), 2.07 (m, 1H), 1.43 (s, 9H),
1.23 (m, 1H), 1.20 (m, 1H), 1.19 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
156.5, 145.9, 141.1, 127.1, 126.8, 80.0, 54.7, 33.3, 30.2, 28.5, 25.4,
16.9. HRMS (ESI): m/z calcd for C18H27N2O4S (M−H)-, 367.1697;
found, 367.1684.
4.1.4.16. Tert-butyl
(2-(4-(pyrrolidin-1-ylsulfonyl)phenyl)cyclopropyl)
carbamate (b8). White solid, 57% yield, 99% purity. 1H NMR
(400 MHz, CDCl3) δ 7.71 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.2 Hz,
2H), 3.21 (t, J = 6.7 Hz, 4H), 2.76 (m, 1H), 2.14–2.05 (m, 1H),
1.78–1.71 (m, 4H), 1.44 (s, 9H), 1.27–1.20 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 156.5, 146.4, 134.5, 127.8, 126.9, 80.0, 48.0,
4.1.4.9. 4-(2-Aminocyclopropyl)-N-phenylbenzenesulfonamide
(a5). White solid, 82% yield, 96% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.69 (d, J = 7.8 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H),
7.19 (t, J = 7.6 Hz, 2H), 7.09–7.01 (m, 3H), 2.94–2.87 (m, 1H),
2.46–2.38 (m, 1H), 1.52–1.45 (m, 1H), 1.39 (d, J = 6.9 Hz, 1H). 13C
NMR (100 MHz, methanol‑d4) δ 145.5, 139.4, 138.9, 130.1, 128.6,
127.9, 125.7, 122.2, 32.4, 22.4, 14.6. HRMS (ESI): m/z calcd for
28.5, 25.3, 16.9. HRMS (ESI): m/z calcd for C18H27N2O4S (M + H)+
,
367.1686; found, 367.1678.
4.1.4.17. 4-(2-Aminocyclopropyl)-N,N-diethylbenzenesulfonamide
(a9). Yellow solid, 58% yield, 99% purity. 1H NMR (400 MHz,
methanol‑d4) δ 7.71 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H),
3.18 (q, J = 7.1 Hz, 4H), 2.95–2.88 (m, 1H), 2.48 (m, 1H), 1.56–1.47
(m, 1H), 1.39 (m, 1H), 1.07 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 143.5, 138.9, 127.8, 127.6, 42.5, 14.5, 14.2. HRMS (ESI): m/z
calcd for C13H21N2O2S (M + H)+, 269.1318; found, 269.1310.
C
15H17N2O2S (M + H)+, 289.1005; found, 289.0995.
4.1.4.10. Tert-butyl
(2-(4-(N-phenylsulfamoyl)phenyl)cyclopropyl)
carbamate (b5). White solid, 26% yield, 99% purity. 1H NMR
(400 MHz, CDCl3) δ 7.64 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 7.7 Hz,
2H), 7.13 (m, 3H), 7.05 (d, J = 7.9 Hz, 2H), 6.73 (s, 1H), 4.86 (s, 1H),
2.73 (m, 1H), 2.04 (m, 1H), 1.42 (s, 9H), 1.24 – 1.16 (m, 2H). 13C NMR
(100 MHz, methanol‑d4) δ 159.0, 148.6, 139.0, 138.2, 130.1, 128.2,
127.4, 125.7, 122.2, 80.4, 34.6, 28.7, 25.7, 17.1. HRMS (ESI): m/z
calcd for C20H25N2O4S (M + H)+, 389.1530; found, 389.1531.
4.1.4.18. Tert-butyl
(2-(4-(N,N-diethylsulfamoyl)phenyl)cyclopropyl)
carbamate (b9). White solid, 47% yield, 99% purity. 1H NMR
(400 MHz, CDCl3) δ 7.68 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.2 Hz,
2H), 3.20 (q, J = 7.1 Hz, 4H), 2.75 (m, 1H), 2.13–2.03 (m, 1H), 1.44 (s,
9

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BioorganicChemistry99(2020)103808
three typical LSD1 inhibitors (TCP, ORY-1001 and GSK2879552) at
5 μM for 8 days. On the last day, the cells were resuspended thoroughly
and counted via the cell counter.
9H), 1.26–1.18 (m, 2H), 1.12 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 156.3, 146 0.0, 137.8, 127.2, 126.9, 80.0, 42.2, 33.2, 28.5,
25.3, 16.9, 14.4. HRMS (ESI): m/z calcd for C18H29N2O4S (M + H)+
,
369.1843; found, 369.1846.
4.2.3. LSD1 enzymatic inhibition measurement
4.1.4.19. N-(2-(4-(N,N-diethylsulfamoyl)phenyl)cyclopropyl)pivalamide
Enzymatic inhibition rate was measured by a commercially avail-
able LSD1 Inhibitor Screening Assay Kit (#700120, Cayman Chemical,
USA) according to the manufacturer’s instructions. In brief, the reagents
including diluted assay buffer, LSD1 protein, horseradish peroxidase
and fluorometric substrate were added into the plates successively.
Then gradient concentrations of our synthetic compounds were added.
Finally, the reaction was initiated by the addition of the peptide cor-
responding to the first 21 amino acids of N-terminal tail of histone H3
with a di-methylated lysine at residue 4. The plate was covered and
incubated at 37 °C for 30 min. At the end of the incubation, the plate
was read using an excitation wavelength of 530–540 nm and an emis-
sion wavelength of 585–595 nm. The enzymatic inhibition rates were
calculated by dividing the control group after deduction of the back-
ground effect.
(c9). White solid, 27% yield, 95% purity. 1H NMR (400 MHz, CDCl3) δ
7.69 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 5.90 (s, 1H), 3.20 (q,
J = 7.1 Hz, 4H), 2.88 (m, 1H), 2.05 (m, 1H), 1.29 (m, 2H), 1.20 (s, 9H),
1.12 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 180.0, 145.8,
137.9, 127.2, 127.2, 42.2, 38.7, 32.9, 27.6, 25.2, 16.4, 14.3. HRMS
(ESI): m/z calcd for C18H29N2O3S (M + H)+, 353.1893; found,
353.1885.
4.1.4.20. 4-(2-(Benzylamino)cyclopropyl)-N,N-diethylbenzenesulfonamide
(a10). Yellow solid, 46% yield, 97% purity. 1H NMR (400 MHz, CDCl3)
δ 7.65 (d, J = 7.8 Hz, 2H), 7.53 (s, 2H), 7.34 (m, 3H), 7.04 (d,
J = 7.9 Hz, 2H), 4.12 (m, 2H), 3.20 (q, J = 7.1 Hz, 4H), 2.75 (m, 1H),
2.55 (m, 1H), 1.25 (m, 2H), 1.11 (t, J = 7.1 Hz, 6H). 13C NMR
(125 MHz, CDCl3) δ 142.9, 139.1, 131.6, 130.0, 129.8, 129.6, 128.0,
127.7, 53.6, 42.5, 29.8, 14.5. HRMS (ESI): m/z calcd for C20H27N2O2S
(M + H)+, 359.1788; found, 359.1777.
4.2.4. Cellular thermal shift assay
Exponential growth phase MV4-11 cells (1 × 106 cells/well) were
seeded in a 6-well plate and incubated with compounds either a9, b9,
c9 or a10 at 5 μM for 24 h in the 5% CO2 containing incubator at 37 °C.
Then, the cells were centrifuged at 1000 rpm for 3 min, washed three
times with PBS, resuspended with 1 mL PBS containing 1% protease
inhibitors, and divided equally into eight PCR tubes (100 μL cell solu-
tion per tube). The tubes were treated with gradient temperatures
(40 °C-58 °C) for 3 min in a PCR thermal cycler, placed at room tem-
perature for 3 min and then subjected to snap-freezing in liquid ni-
trogen for 3 min. The above cycles were repeated three times. The
thawed cell lysates were centrifuged at 20,000 g at 4 °C for 20 min, and
then the supernatants were collected carefully for further Western Blot
analysis.
4.1.4.21. Tert-butyl benzyl(2-(4-(N,N-diethylsulfamoyl)phenyl)cyclopropyl)
carbamate (b10). White solid, 48% yield, 96% purity. 1H NMR
(400 MHz, CDCl3) δ 7.66 (d, J = 8.3 Hz, 2H), 7.33–7.28 (m, 2H), 7.23
(t, J = 6.5 Hz, 3H), 7.16–7.09 (m, 2H), 4.62 (d, J = 15.2 Hz, 1H), 4.35
(d, J = 15.4 Hz, 1H), 3.20 (q, J = 7.1 Hz, 4H), 2.73–2.66 (m, 1H), 2.18
(m, 1H), 1.41 (s, 9H), 1.20 (m, 2H), 1.10 (t, J = 7.2 Hz, 6H). 13C NMR
(125 MHz, CDCl3) δ 146.2, 138.6, 137.8, 130.6, 128.7, 127.6, 127.3,
127.1, 126.7, 80.4, 42.1, 39.6, 28.5, 14.2. HRMS (ESI): m/z calcd for
C
25H35N2O4S (M + H)+, 459.2312; found, 459.2312
4.2. Biological assays
4.2.1. Cell culture
4.2.5. Detection of a9 in cell samples via LC-MS/MS
AML cell lines MV4-11, HL-60, THP-1, U937, ML1, MOLM-13 and
BaF3/ITD were obtained from American Type Culture Collection
(Rockville, MD, USA) and maintained in RPMI-1640 medium (Gibco,
Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS,
Biological Industries, Israel). Kasumi-1 cell line was kindly provided by
Stem Cell Bank, Chinese Academy of Sciences and cultured in RPMI
1640 medium containing 20% FBS, 1% GlutaMAX™-I (Gibco, Carlsbad,
CA, USA) and 1% sodium pyruvate (Gibco, Carlsbad, CA, USA). All cell
lines were incubated at 37 °C supplied with 5% carbon dioxide.
The cells were seeded in a 6-well plate and incubated with com-
pounds either a9, b9 or c9 at 10 μM for 3 days and 6 days, respectively.
At the end of the incubation, the cells were washed twice with cold
saline, lysed and extracted using the solution (chloroform : methanol :
water = 6 : 3 : 1). The extract was dried via nitrogen blower con-
centrator and then re-dissolved in 100 μL of 50% MeOH/H2O solution
for LC-MS/MS detection.
Chromatographic separation was performed using an Agilent 1290
Infinity II liquid chromatography system (Agilent, USA) with ZORBAX
Eclipse Plus C18 analytical column (2.1 × 50 mm, 1.8 µm particle size)
(Waters, USA). The flow rate of HPLC was set at 0.4 mL/min and the
gradient elution was run for 15 min, from 10% MeOH/H2O to 90%
MeOH/H2O for 10 min and 90% MeOH/H2O for another 5 min. The
6495 Triple Quad mass spectrometer (Agilent, USA) with an electro-
spray ionization (ESI) interface operated in positive mode was used for
the multiple reaction monitoring (MRM) LC-MS/MS analysis. The fol-
lowing precursor and product ion transitions were used for MRM: a9,
269.1 → 115.1, 269.1 → 162.2. MS data were processed using the
Agilent MassHunter Quantitative Analysis (Version B.07.00, Agilent,
USA).
4.2.2. Cell proliferation assays through MTS and cell counting
Cell viability was determined by MTS assay. MTS (3-(4,5-di-
methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)–2H-tetrazolium, inner salt) can be reduced by dehy-
drogenase enzymes in metabolically active cells into a colored for-
mazan product that is soluble in the medium and is directly detected by
visible light absorption at 490 nm with a 96-well plate reader. Briefly,
cells were plated at 5000 cells per well in round bottom 96-well plates
in triplicate. After 2 h of incubation at the 37 °C incubator, the cells
were exposed to gradient concentrations of typical LSD1 inhibitors or
our synthetic compounds for 6 days. At the end of the incubation, 20 μL
of MTS solution (2 mg/mL) was added into each well and the cells were
incubated at 37 °C supplied with 5% carbon dioxide for 4 h. Then the
optical density value (OD) was determined at 490 nm wavelength with
4.2.6. Flow cytometric analysis of cell differentiation and cell cycle
Cell differentiation was determined by flow cytometry using anti-
CD11b (#553311, BD biosciences, New Jersey, USA). In brief, cells
were seeded in the 24-well plate and incubated with different com-
pounds at 10 μM for 24 h and 96 h. At the end of the incubation, the
cells were centrifuged at 800 rpm for 5 min to remove dead cells, wa-
shed twice with 1 mL cold PBS and then incubated with a solution
containing CD11b antibody at 4 °C for 30 min. Then, the cells were
washed and resuspended with 300 μL cold PBS for final detection by
a
BioTek Gen5 microplate-reader (BioTek Instruments, Winooski,
Vermont, USA).
To directly quantify the cell proliferation, cell numbers were
counted using a cell counter (Cellometer Auto T4, Nexcelom Bioscience,
Lawrence, MA, USA). Briefly, cells were plated at 1 × 105 cells per well
in 6-well plates. After 2 h of incubation, the cells were subjected to
10

L. Liang, et al.
BioorganicChemistry99(2020)103808
flow cytometer.
Technology Program (2017A020215198), and Guangzhou Science and
Cell cycle was detected by staining DNA with PI/RNase solution
(#550825, BD biosciences, New Jersey, USA) using flow cytometry.
Briefly, the cells (1 × 105 for 24 h and 4 × 105 for 96 h treatment)
were seeded in the 24-well plate and incubated with either TCP, ORY-
1001, GSK2879552 or a10 at 10 μM for 24 h and 96 h. At the end of the
incubation, the cells were centrifuged at 1000 rpm for 3 min, washed
once with 1 mL cold PBS and then resuspended with 500 μL of 70%
MeOH/H2O solution for 4 h. After removal of methanol through cen-
trifugation at 1000 rpm for 3 min, the cells were incubated with 300 μL
PI/RNase solution, for 30 min at room temperature. At the end of the
incubation, the resuspended cell solution was directly tested by flow
cytometry.
Technology Program (201807010041).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103808.
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