Design, synthesis and biological activity of N-(3-substituted-phenyl)benzenesulfonamides as selective and reversible LSD1 inhibitors

Article abstract of DOI:10.1007/s00044-016-1706-8

Lysine specific demethylase 1?plays a crucial role in regulating histone methylation at residues K4 and K9 on histone H3 and over-expresses in a variety of cancers. Here we designed, synthesized and evaluated a series of N-(3-substituted-phenyl)benzenesul

Full text of DOI:10.1007/s00044-016-1706-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1706-8
ORIGINAL RESEARCH
Design, synthesis and biological activity of N-(3-substituted-phenyl)
benzenesulfonamides as selective and reversible LSD1 inhibitors
1,2
Liming Wu1,2,3 Siyuan Xu4 Fangxia Zou1,2 Jiayue Xi1,2
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Xiaoming Zha
Tianfang Ma1,2 Rongfeng Liu5 Yu-Chih Liu5 Dawei Deng2 Yueqing Gu2
●
Jinpei Zhou3 Fei Lan4
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Received: 26 March 2016 / Accepted: 1 August 2016
© Springer Science+Business Media New York 2016
Abstract Lysine specific demethylase 1 plays a crucial role
in regulating histone methylation at residues K4 and K9 on
histone H3 and over-expresses in a variety of cancers.
Here we designed, synthesized and evaluated a series of
N-(3-substituted-phenyl)benzenesulfonamides as reversible
lysine specific demethylase 1 inhibitors. All the compounds
exhibited lysine specific demethylase 1 inhibition with the
half maximal inhibitory concentration (IC50) values
between 7.5 and 68 μM. Three most active compounds 2a,
2c and 2i displayed only modest effect on flavin adenine
dinucleotide-dependent enzymes mono-amine oxidases
A and B, and their reversibilities of lysine specific
demethylase 1 inhibition were confirmed. Molecular
docking was also carried out to predict the binding mode of
2a into the active site of lysine specific demethylase 1.
Taken together, N-(3-substituted-phenyl)benzenesulfona-
mides including 2a represent a new class of selective and
reversible lysine specific demethylase 1 inhibitors with
pharmaceutical research.
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●
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Keywords LSD1 Small molecule inhibitors Selective
●
Reversible Molecular modeling
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1706-8) contains supplementary material,
which is available to authorized users.
Abbreviations
TEA
Triethylamine
DIPEA
EDCI
N,N-Diisopropylethylamine
1-Ethyl-3-(3-dimethylaminopropyl) carbodii-
mide hydrochloride
N-Hydroxybenzotriazole
Room temperature
Benzotriazol-1-yl-
Xiaoming Zha, Liming Wu and Siyuan Xu contributed equally to this
article.
* Xiaoming Zha
HOBt
rt
PyBOP
xmzha@cpu.edu.cn
* Jinpei Zhou
jpzhou668@163.com
oxytripyrrolidinophosphonium
4-Dimethylaminopyridine
* Fei Lan
fei_lan@fudan.edu.cn
DMAP
1
Jiangsu Key Laboratory of Drug Screening, State Key Laboratory
of Natural Medicines, Nanjing, People’s Republic of China
2
Department of Biochemical Engineering, Nanjing, People’s
Introduction
Republic of China
3
Department of Medicinal Chemistry, China Pharmaceutical
Histone lysine methylation can be dynamically regulated by
two counteracting families of enzymes, the histione
methyltransferase and the histone demethylase. Histone
lysine-specific demethylase 1 (LSD1, also known as
KDM1A, AOF2, BHC110 or KIAA0601) was the first
histone demethylase discovered in 2004 (Shi et al., 2004),
which is a highly conserved flavin adenine dinucleotide
University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic
of China
4
Laboratory of Epigenetics, Institute of Biochemical Sciences,
Fudan University, 131 Dong’An Road, Shanghai 200032,
People’s Republic of China
5
Shanghai ChemPartner Co. Ltd., Zhangjiang Hi-Tech Park,
Shanghai 201203, People’s Republic of China

Med Chem Res
(FAD)-dependent oxidative enzyme containing a homo-
logous domain to amine oxidase, catalyzing the demethy-
lation reaction. LSD1 plays important roles in normal
processes and malignant transformation, mainly as a tran-
scription regulator. It demethylates mono- and dimethylated
histone H3 lysine 4 (H3K4me1 and H3K4me2) and H3
lysine 9 (H3K9me1 and H3K9me2) (Shi et al., 2004;
Metzger et al., 2005) as well as p53, DNA methyltransfer-
ase 1 (DNMT1) and E2F transcription factor 1(E2F1),
which further regulates their downstream cellular function
(Lynch et al., 2012).
class have shown significant selectivity towards LSD1 over
MAO-A and B, the possibilities to affect other FAD
dependent enzymes in vivo has not been fully investigated
(Hitchin et al., 2013). Thus it is of great importance for the
development of reversible inhibitors. For the reversible
LSD1 inhibitors, E, F and G only moderately inhibit LSD1
at micromolar level (Hazeldine et al., 2012; Zheng et al.,
2013; Zhou et al., 2015). GSK354 (H), disclosed as a potent
and reversible inhibitor of LSD1 with high selectivity
against MAO-A (IC50 > 200 mM), yet the further biological
data has not been disclosed. Notably, SP2509 (I) was a
potent and specific LSD1 inhibitor, however, was found to
be cytotoxic in THP1 cells dosed for 24 h at concentrations
≥ 2 μM, and such discrepancy was not fully understood yet
(Hitchin et al., 2013; Dhanak, 2013). However, the core N
′-(2-hydroxybenzylidene)hydrazide motif of I has been
previously identified as a pan-assay interference compound
(PAINS), limiting its further clinical potential (Hitchin
et al., 2013; Sorna et al., 2013; Baell and Holloway, 2010).
Hence, the discovery of drug-like LSD1 inhibitors is
necessary for the treatment of LSD1 overexpressing cancer
as well as the elucidation of LSD1 function in cancer.
(Fig. 1) Reference Dhanak (2015) is cite in the text but not
provided in the reference list. Please provide this reference
in the list or delete rom the text.
Emerging evidences indicate LSD1 as a potential ther-
apeutic target for the treatment of various cancers. Over-
expression of LSD1 in the human malignancies has been
observed in a variety of tumors including ER-negative breast
cancer, prostate cancer, non-small cell lung cancer, neuro-
blastoma (Kahl et al., 2006; Lim et al., 2010; Yoshimatsu
et al., 2011; Zhao et al., 2012), particularly acute myeloid
leukemia (AML) and bladder cancer (Lynch et al., 2012).
To date, a large number of LSD1 inhibitors have been
developed. As a member of monoamine oxidase (MAO)
family, LSD1 shares high level of sequence homology to
MAO-A and MAO-B at the active sites, and requires FAD
as its cofactor to catalyze the oxidation, and consequently,
the removal of the methyl groups from its substrates (Shi
et al., 2004). Therefore, MAO inhibitors were investigated
as potential inhibitors of LSD1. Tranylcypromine (TCPA,
A), a MAO inhibitor originally developed as antidepressant
agent in the 1960s, was identified as an irreversible and
poor selective LSD1 inhibitor with moderate inhibitory
effect. Since then, a variety of TCPA-based compounds
have been reported with enhanced level of potency and
increased selectivity (Mimasu et al., 2010; Ueda et al.,
2009; Binda et al., 2010; Ortega et al., 2011; Guibourt et al.,
2010; McCafferty and Pollock, 2013; Liang et al., 2013).
OG-L002 (B), a TCPA-based compound, shows potent
inhibition against LSD1 at 20 nM (https://www.
clinicaltrialsregister.eu/ctr-search/trial/2013-002447-29/ES).
ORY-1001(C), inducing cell differentiation and sig-
nificantly reducing the clonogenic potential of primary
human MLL-AF9 AML cells, was granted orphan drug
status by EMA in September 2013 and is currently in phase
I/IIa trial study for AML (https://www.oryzon.com/en/
therapeutic-programs/our-pipeline). GSK2879552 (D) is a
selective, mechanism-based irreversible LSD1 inhibitor
under the phase I trial studies for the treatment of relapsed/
refractory small cell lung carcinoma (http://clinical
trials.gov/ct2/show/NCT02034123?term=lysine+specific
+demethylase&rank=2). Those TCPA based agents are
irreversible inhibitors and form covalent bond with the
cofactor, FAD, at the LSD1 active site (Ueda et al., 2009;
Binda et al., 2010; Mimasu et al., 2008). Although some of
the more recently reported compounds belonging to this
Recently,
N-(substituted-phenyl)benzenesulfonamides
have drawn increasing attention because of their numerous
biological activities, especially the anticancer activity (Levin
et al., 1999; Kawai et al., 2006; Comess et al., 2004;
Sheppard et al., 2006). In this paper, we utilized its
core motif to sucessfully design and synthesize a series
of N-(3-substituted-phenyl)benzenesulfonamides derived
LSD1 specific inhibitors. All the compounds exhibited
LSD1 inhibition with the IC50 values in the range of 7.5–68
μM. Three most active compounds 2a, 2c and 2i displayed
only modest effect on the homogulous enzymes MAO-A
and MAO-B, indicating good selectivity for LSD1 inhibi-
tion. Furthermore, their reversible effects toward LSD1 were
further comfirmed experimentally. Molecular docking was
also carried out to predict the binding mode of 2a at the
active site of LSD1, providing future opportunities for fur-
ther optimization. Taken together, our work discovered N-
(3-substituted-phenyl)benzenesulfonamides as a novel class
of selective and reversible LSD1 inhibitors, and particularly,
the 2a represents a lead compound for further research.
Results and discussion
Chemistry
The general route for the synthesis of the N-(substituted-
phenyl)benzenesulfonamides (2a–2i) is depicted in

Med Chem Res
Fig. 1 Representative structures of reported LSD1 inhibitors.
a Tranylcypromine (TCPA) IC50 = 20.7 uM, b OG-L002 IC50 = 20
nM, c ORY-1001 IC50 = 20 nM, d GSK-2879552 IC50 = 20 nM,
e IC50 = 16.8 uM, f IC50 = 2.41 uM, g IC50 = 2.11 uM, h GSK-354
IC50 = 90 nM, i SP-2509 IC50 = 13nM
Scheme 1 Synthesis of
compounds 2a–2f. Reagents and
conditions: i m-aminobenzoic
acid, Na2CO3, H2O, rt,4 h;
ii EDCI, HOBt, CH2Cl2, rt, 12 h
Schemes 1 and 2. p-Benzenesulfonyl chlorides were
reacted with m-aminobenzoic acid at room temperature to
afford the intermediates 1a–1c. Then p-phenethylamines
were reacted with 1a–1c and EDCI/HOBt[(3-dimethyla-
Biochemical evaluation
Biochemical activity of the candidate compounds against
LSD1 and the preliminary structure–activity relationship
(SAR) analysis
minopropyl)ethyl-carbodiimid
monohydrochloride/N-
Hydroxybenzotriazole] at room temperature to synthesize
the target compounds 2a–2f, respectively. Similarly, the
reaction of p-benzenesulfonyl chlorides with m-phenyle-
nediamine in THF was utilized to make the intermediates
1a–1c, and p-hydroxyphenylacetic acid was then reacted
with 1d–1f using DIPEA (N,N-Diisopropylethylamine)
as the base to give the corresponding target compounds
2g–2i. At this step, we used PyBOP (Benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate) as
the condensating agent instead of EDCI/HOBt because of
its weak condensating effect.
All the compounds synthesized in this study were evaluated
for their in vitro inhibitory effect on LSD1, as reported in
our previous study (Zhou et al., 2015). A known LSD1
inhibitor, RN-1, was chosen as a positive control (Rivers
et al., 2014). The results are summarized in Table 1. All the
test compounds showed IC50s at low micromolar range
(7.5–68 μM) in vitro. The LSD1 AlphaLISA assay was
carried out using recombinant LSD1 protein and H3K4me2
peptide as substrate. Importantly, nitro group in the R1
postion was the favorable substituent such as compound

Med Chem Res
Scheme 2 Synthesis of
compounds 2g–2i. Reagents and
conditions:
i m-phenylenediamine, TEA
(triethylamine), THF, rt, 9 h;
ii p-hydroxyphenylacetic acid,
PyBoP, DIPEA, DMF, rt, 24 h
2a (IC50 = 7.5 μM), 2c (IC50 = 11.4 μM) and 2i (IC50
8.6 μM), while the methyloxy and hydroxy groups in the
same position decreased the inhibitory effect.
=
value −44.75 kj/mol. These results may further explain why
compound 2a possesses more potent inhibition against
LSD1 than compound 2g.
Evaluation of the in vitro inhibitory effect of compounds 2a,
2c and 2i against LSD1’s homologies MAO-A and MAO-B
Conclusion
Based on the in vitro results of LSD1 inhibition of the tested
compounds (Table 1), the three most potent compounds 2a,
2c and 2i were further selected to be evaluated on their
potential cross inhibitory effect toward the homologuous
enzymes MAO-A and MAO-B. The reuslts are summarized
in Table 1. Significantly, 2a, 2c and 2i only exhibited
modest inhibition against MAOs with the IC50 of 90.0, 63.9
and 109.2 μM for MAO-A, 226.1, 297.5 and 344.4 μM for
MAO-B, respectively. Taken together, 2a, 2c and 2i showed
around 10 and 30 folds of selective inhibitions for LSD1
over MAO-A and MAO-B, respectively.
In summary, we synthesized and evaluated a series of N-(3-
substituted-phenyl)benzenesulfonamides which exhibited
low micromolar IC50s of LSD1 inhibition in the range of
7.5–68 μM. Three most active compounds 2a, 2c and 2i,
also showed good selectiveties against the homogulous
enzymes, MAO-A and MAO-B Furthermore, 2a, 2c and 2i
were further confirmed as LSD1 reversible inhibitiors.
Docking study was also taken to predict the binding mode
of 2a with LSD1 which shows lower CDOCKER energy
and two hypothetic hydrogen bonds. Taken together, we
have provided preliminary evidences that N-(3-substituted-
phenyl)benzenesulfonamides may be a novel class of
selective and reversible LSD1 inhibitors that deserve further
investigation on their therapeutic potentials.
Reversiblity of the inhibition by 2a, 2c and 2i toward LSD1
Given that the most potent compounds 2a, 2c and 2i
displayed micromolar inhibition against LSD1 (IC50
=
7.5–10 μM), we further carried out a jump dilution analysis
to investigate the reversibility of the inhibition of these three
compounds. The results are summarized in Scheme 3. All
three compounds displayed great reversibility at 87, 67 and
84 %, respectively, indicating they were indeed reversible
LSD1 inhibitors.
Experimental section
Chemistry
All chemicals (reagent grade) were purchased from Sino-
pharm Chemical Reagent Co. Ltd. (Shanghai, China).
Reactions were followed by thin-layer chromatography
(TLC) on silica gel GF254 plates (Qingdao Haiyang Che-
mical Plant, Qingdao, Shandong, China) and the spots were
visualized using a UV lamp (λ = 254 nm). Chromatographic
separations were performed on silica gel columns (90–150
μm; Qingdao Marine Chemical Inc., Qingdao, Shandong,
China) by flash chromatography. Melting points were
measured on an XT-4 micromelting point instrument (Beij-
ing Tech Instrument Corp., Beijing, China) and uncorrected.
IR (KBr-disk) spectra were recorded by Bruker Tensor
Docking analyses of potential binding modes of
compound 2a and 2g binding to LSD1
To understand the initial binding mode and stability of
ligand-receptor complexes, molecular modeling studies
(CDOCKER docking analysis) were performed utilizing 2a
and 2g and depicted in Scheme 4. In the case of 2a, the
oxygen atom of sulfonamide formed a hydrogen bond with
Arg316 residue and a hydrogen bond was observed between
the oxygen of sulfur atom and Ser289 residue. Moreover, it
entirely occupied FAD-binding fold region, which could
interact with the active site thoroughly with the CDOCKER
energy value −46.23 kj/mol. Comparedly, only one oxygen
atom of sulfonamide of 2g interacted with Ser289 via H-
bond and it occupied the active site with CDOCKER energy
1
27 spectrometer (Bruker Corp., Billerica, MA, USA). H
NMR spectra (300 MHz) and 13C NMR (75 MHz) were
acquired on a Bruker ACF-300 spectrometer (Bruker Corp.,
Billerica, MA, USA) at 25°C and referenced to tetra-
methylsilane (TMS). Chemical shifts were reported in ppm
(δ) using the residue solvent line as internal standard.

Med Chem Res

Med Chem Res
Scheme 3 Compounds 2a, 2c
and 2i are reversible LSD1
inhibitors. The reversibility of a
2a, b 2c and c 2i
Scheme 4 Representation of the
theoretical binding mode of
compounds 2a a and 2g b with
LSD1(PDB id: 2Z5U)
A
B
Splitting patterns are designed as s, singlet; d, doublet; t,
triplet; m, multiplet. The identity of final compounds was
confirmed by acquiring mass spectra on an Agilent 1100
Series (Agilent Technologies, Santa Clara, CA, USA) LC/
MSD high performance ion trap mass spectrometer and a
Mariner ESI-TOF spectrometer. Compound purity was
evaluated by reverse phase chromatography using a
Shimadzu Shim-Pack VP-ODS (150 × 4.6 mm i.d., 5 µm
particle size, Shimadzu Corp., Kyoto, Japan) column and
DAD detection. Satisfactory chromatographic analyses
were obtained for all new compounds, confirming >95 %
purity (see Supporting Information).
7.4 Hz, 2H), 7.62 (d, J = 5.6 Hz, 1H), 7.38 (m, 4H), 2.36 (s,
3H) (Deng and Mani, 2006).
3-((4-Methoxyphenyl)sulfonamido)benzoic acid (1b)
According to the preparation procedure of 1a, 1b was
provided as light white solid (0.815, 72.8 %) without further
purification. 1H NMR (300 MHz, DMSO-d6, ppm): δ 13.01
(s, 1H), 10.37 (s, 1H), 7.72–7.69 (m, 3H), 7.59 (d, J = 6.2
Hz, 1H), 7.40–7.45 (m, 2H), 7.07 (d, J = 8.8 Hz, 2H), 3.80
(s, 3H).
3-(Phenylsulfonamido)benzoic acid (1c)
3-((4-Methylphenyl)sulfonamido)benzoic acid (1a)
According to the preparation procedure of 1a, 1c was pro-
vided as light white solid as light white solid (0.796 g,
1
To a solution of 3-aminobenzoic acid (500 mg, 3.65 mmol)
and tosyl chloride (695 mg, 3.65 mmol ) in 15 mL H2O was
added with an aqueous solution of 1 mol/L Na2CO3 drop-
wise to adjust the pH to 9. The mixture was stirred at room
temperature for about 4 h. After the completion of the
reaction monitored by TLC, the mixture was transferred to
100 mL beaker. Then 1 mol/L HCl was added dropwise to
adjust the pH to 2 in ice water. The precipitate was collected
and the product was afforded as light white solid (0.778 g,
78.8 %) without further purification. H NMR (300 MHz,
DMSO-d6 ppm): δ 13.98 (s, 1H), 10.52 (s, 1H), 7.77 (d, J =
7.8 Hz, 2H), 7.70 (s, 1H), 7.61–7.54 (m, 4H), 7.38–7.36
(m, 2H).
3-((4-Methylphenyl)sulfonamido)-N-(4-nitrophenethyl)
benzamide (2a)
To a solution of 3-((4-methylphenyl)sulfonamido)benzoic
1
73.4 %) without further purification. H NMR (300 MHz,
acid (400 mg, 1.37 mmol)
,
4-nitrophenethylamine
DMSO-d6, ppm): δ 10.51 (s, 1H), 7.74 (s, 1H), 7.69 (d, J =
hydrochloride (185.5 mg, 0.92 mmol), EDCI (228 mg,

Med Chem Res
1.19 mmol) and HOBt (161 mg, 1.18 mmol) in dichlor-
omethane (15 mL) was added with triethylamine (383 μL,
2.75 mmol). The reaction mixture was stirred overnight at
room temperature. After completion of the reaction
monitored by TLC, the solvent was removed under
vacuum and the crude mixture was dissolved by EtOAc
(15 mL). The solution was washed by NaHCO3 and
saturated NaCl solution (15 mL *3), then dried over
anhydrous Na2SO4. Purification of the crude product with
flash column chromatography (PE:EA = 2:1, v:v) gave
342 mg of 2a as a pale yellow solid, yield 85 %. Mp:
167–168 °C. 1H NMR (300 MHz, CDCl3): δ 10.37 (s,
SO2NH, 1H), 8.53–8.51 (m, CONH, 1H), 8.17 (d, J = 8.0
Hz, Ar–H, 2H), 7.66 (d, J = 8.1 Hz, Ar–H,2H), 7.56 (s,
1H), 7.53 (d, J = 8.4 Hz, Ar–H, 2H) 7.52 (s, Ar–H, 1H),
7.41 (d, J = 7.5 Hz, Ar–H, 1H), 7.35 (d, J = 7.7 Hz, Ar–H,
2H), 7.29 (d, J = 7.5 Hz, Ar–H, 1H), 7.22 (d, J = 8.9 Hz,
Ar–H, 1H), 3.56–3.49 (m, CH2CH2NHCO, 2H),
3.01–2.96 (m, CH2CH2NHCO, 2H), 2.34 (s, Ar–CH3,
3H). 13C NMR (75 MHz, DMSO-d6): 168.88 (CONH),
151.05(Ar–C), 149.13 (Ar–C), 146.39 (Ar–C), 141.08
(Ar–C), 139.63 (Ar–C), 138.68 (Ar–C), 133.09 (Ar–C),
132.74 (Ar–C), 132.08 (Ar–C), 129.71 (Ar–C), 126.40
(Ar–C), 125.25 (Ar–C), 122.15 (Ar–C), 43.14
(CH2CH2NH), 37.77 (CH2CH2NH), 23.97 (Ar–CH3). IR
(KBr, cm−1): 3408, 3160, 1639, 1541, 1517, 1347, 1161.
MS (m/z): 438.1 ([M–H]+). HRMS (ESI): calcd for
C22H21N3O5S, [M+H]+ m/z, 440.1275; found, 440.1265.
HPLC purity: 99.306 %.
(m, 1H), 8.17 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 7.0 Hz, 2H),
7.63–7.52 (m ,6H), 7.43 (d, J = 7.8 Hz, 1H), 7.34–7.28 (m,
1H), 7.23 (d, J = 8.1 Hz, 1H), 3.56–3.49 (m, 2H), 3.01–2.96
(m, 2H). 13C NMR (75 MHz, DMSO-d6): 168.88, 151.03,
149.12, 142.48, 140.97, 138.72, 135.99, 133.06, 132.30,
132.10, 129.66, 126.38, 125.51, 125.44, 122.41, 43.15,
37.79. IR (KBr, cm−1): 3384, 3133, 1649, 1547, 1517,
1344, 1158. MS (m/z): 424.1 ([M–H]). HR–MS (ESI): calcd
for C21H19N3O5S, [M+H]+ m/z, 426.1118; found,
426.1111. HPLC purity: 99.244 %.
N-(3,4-dimethoxyphenethyl)-3-((4-methylphenyl)
sulfonamido)benzamide (2d)
According to the preparation procedure of 2a, 2d was pre-
pared as a pale yellow solid, yield 87.3 %. Mp: 183–185 °C.
1H NMR (300 MHz, DMSO-d6): δ 10.35 (s, 1H), 8.47–8.42
(m, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.57 (s, 1H), 7.43 (d, J =
7.2 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.0 Hz,
1H), 7.21 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.80
(s, 1H), 6.72 (d, J = 9.6 Hz, 1H), 3.71 (s, 3H), 3.70 (s, 3H),
3.45–3.38 (m, 2H), 2.77–2.72 (m, 2H), 2.33 (s, 3H).
13C NMR (75 MHz, DMSO-d6): 168.69, 151.66, 150.28,
146.39, 141.10, 139.65, 138.84, 134.99, 132.75, 132.03,
129.71, 125.23, 123.51, 122.25, 115.57, 114.98, 58.56,
58.38, 44.07, 37.59, 23.29. IR (KBr, cm−1): 3362, 3136,
1638, 1585, 1547, 1515, 1344, 1126. MS (m/z): 477.1
([M+Na]+). HRMS (ESI): calcd for C24H26N2O5S, [M+H]+
m/z, 455.1635; found, 455.1631. HPLC purity: 99.158 %.
3-((4-Methoxyphenyl)sulfonamido)-N-(4-nitrophenethyl)
benzamide (2b)
N-(3,4-dimethoxyphenethyl)-3-(phenylsulfonamido)
benzamide (2e)
According to the preparation procedure of 2a, 2b was pre-
pared as a pale yellow solid, yield 85.6 %. Mp: 154–155 °C.
1H NMR (300 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.54–8.50
(m, 1H), 8.17 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H),
7.57 (s, 1H), 7.53 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 7.7 Hz,
1H), 7.33–7.28 (m ,1H), 7.23 (d, J = 8.1 Hz, 1H), 7.07 (d,
J = 8.8 Hz, 2H), 3.80 (s, 3H), 3.56–3.50 (m, 2H), 3.01–2.97
(m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 168.89, 151.03,
149.10, 141.19, 138.66, 134.07, 133.06, 132.05, 131.88,
126.38, 125.22, 125.17, 122.13, 117.42, 58.63, 43.14. IR
(KBr, cm−1): 3411, 3187, 1642, 1596, 1545, 1512, 1342,
1157. MS (m/z): 454.1([M–H]+). HR–MS (ESI): calcd for
C22H21N3O6S, [M+H]+ m/z, 456.1224; found, 456.1211.
HPLC purity: 99.497 %.
According to the preparation procedure of 2a, 2e was pre-
pared as a pale yellow solid, yield 88 %. Mp: 186–187 °C.
1H NMR (300 MHz, DMSO-d6): δ 10.43 (s, 1H), 8.47–8.43
(m, 1H), 7.76 (d, J = 7.3 Hz, 2H), 7.63–7.52 (m, 4H), 7.44
(d, J = 7.7 Hz, 1H), 7.32–7.27 (m, 1H), 7.21 (d, J = 9.8
Hz,3H), 6.87–6.80 (m, 2H), 6.73 (d, J = 8.0 Hz,1H), 3.70
(s, 3H), 3.69 (s, 3H), 3.45–3.38 (m, 2H), 2.77–2.72 (m, 2H).
13C NMR (75 MHz, DMSO-d6): 168.65, 151.66, 150.27,
142.47, 140.96, 138.87, 135.98, 134.99, 132.30, 132.03,
129.65, 125.43, 125.38, 123.50, 122.45, 115.57, 114.97,
58.54, 58.36, 44.08, 37.59, 17.10. IR (KBr, cm−1): 3351,
3134, 1732, 1638, 1586, 1515, 1340, 1167. MS (m/z): 463.1
([M+Na]+). HRMS (ESI): calcd for C23H24N2O5S, [M+H]+
m/z, 441.1479; found, 441.1474. HPLC purity: 99.303 %.
N-(4-nitrophenethyl)-3-(phenylsulfonamido)benzamide (2c)
N-(3,4-dimethoxyphenethyl)-3-((4-methoxyphenyl)
sulfonamido)benzamide (2f)
According to the preparation procedure of 2a, 2c was pre-
pared as a pale yellow solid, yield 83 %. Mp: 160–162 °C.
1H NMR (300 MHz, DMSO-d6): δ 10.44 (s, 1H), 8.54–8.51
According to the preparation procedure of 2a, 2f was pre-
pared as a pale yellow solid, yield 88 %. Mp: 189–190 °C.

Med Chem Res
1H NMR (300 MHz, DMSO-d6): δ 10.40 (s, 1H), 8.56–8.58
(m, 1H), 7.80 (d, J = 8.6 Hz, 2H), 7.69 (s, 1H), 7.55 (d, J =
7.6 Hz, 2H), 7.43–7.38 (m, 1H), 7.32 (d, J = 8.1 Hz, 1H),
7.17 (d, J = 8.6 Hz, 2H), 6.98–6.91 (m, 2H), 6.84 (d, J =
8.2 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H),
3.58–3.50 (m, 2H), 2.88–2.83 (m, 2H). 13C NMR (75 MHz,
DMSO-d6): δ 168.71, 151.65, 150.27, 141.21, 138.83,
134.99, 134.09, 132.00, 131.89, 125.19, 125.14, 123.50,
122.21, 117.43, 115.56, 114.96, 58.63, 58.54, 58.36, 44.07,
37.59. IR (KBr, cm−1): 3336, 3133, 1635, 1594, 1542,
1515, 1327, 1157. MS (m/z): 493.1 ([M+Na]+). HR–MS
(ESI): calcd for C24H26N2O6S, [M+H]+ m/z, 471.1584;
found, 471.1574. HPLC purity: 96.882 %.
N,N-diisopropylethylamine (DIPEA) as a catalyst. The
reaction mixture was stirred at room temperature for about
36 h. The reaction was monitored by TLC. After the com-
pletion of the reaction, the mixture was transfered to water
(50 mL) and stirred for 15 min. The organic layer was
washed by saturated brine (15 mL*3) and dried over
anhydrous Na2SO4. Purification of the crude product with
flash column chromatography (CH2Cl2:CH3OH = 40:1 v:v)
gave 2g as a pale white solid (368 mg, 38.6 %). Mp:
1
142–145 °C. H NMR (300 MHz, CDCl3): δ 10.25 (s, 1H),
9.82 (s, 1H), 9.11 (s, 1H), 7.75 (d, J = 7.2 Hz, 2H),
7.61–7.49 (m, 3H), 7.44 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H),
7.10–7.04 (m, 1H), 6.98 (d, J = 8.1 Hz, 2H), 6.71 (d, J =
7.7 Hz, 1H), 6.4 (d, J = 8.2 Hz, 2H), 2.77–2.72 (m, 2H),
2.51–2.48 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
170.53, 155.45, 139.87, 139.57, 138.00, 132.78, 131.12,
129.13, 129.00, 126.62, 115.02, 114.66, 114.46, 110.60,
38.38, 29.99. IR (KBr, cm−1): 3143, 2966, 2867, 1663,
1607, 1554, 1516, 1459, 1447, 1243, 1156. MS (m/z):
395.1. HR–MS (ESI): calcd for C21H20N2O4S, [M+H]+ m/z,
397.1217; found, 397.1208. HPLC purity: 98.194 %.
N-(3-aminophenyl)benzenesulfonamide (1d)
To a solution of benzenesulfonyl chloride (1.77 g,
10 mmol) in THF (10 mL), a solution of triethylamine
(2.79 mL, 20 mmol) in THF (5 mL) and m-phenylenedia-
mine (1.08 g, 10 mmol) in THF (5 mL) were added drop-
wise respectively. The reaction mixture was stirred for 12
h at room temperature. After the completion of the reaction
monitored by TLC, the mixture was filtered and condensed
under reduced pressure. The crude product can be used for
3-(4-Hydroxyphenyl)-N-(3-((4-methoxyphenyl)sulfonamido)
phenyl)propanamide (2h)
1
the next step without further purification. H NMR (300
MHz, CDCl3): 7.71 (d, J = 7.1 Hz, 2H), 7.65–7.61 (m,
1H), 7.56–7.51 (m, 2H), 7.08 (s, 1H), 7.04–6.99 (m, 1H),
6.69–6.65 (m, 2H).
According to the preparation procedure of 2g, 2h was pre-
pared as a pale white solid, yield 37.8 %. Mp: 137–139 °C.
1H NMR (300 MHz, DMSO-d6): δ 10.18 (s, 1H), 9.85 (s,
1H), 9.15 (s, 1H), 7.72 (d, J = 8.80 Hz, 2H), 7.45 (s, 1H),
7.25 (d, J = 7.8 Hz, 1H), 7.12–7.01 (m, 5H), 6.74 (d, J =
8.2 Hz, 2H), 6.68 (d, J = 8.3 Hz, 1H), 3.80 (s, 3H),
2.80–2.75 (m, 2H), 2.55–2.52 (m, 2H). 13C NMR (75 MHz,
DMSO-d6): 173.62, 158.56, 142.92, 141.65, 134.50,
134.22, 132.17, 132.11, 131.92, 118.12, 117.38, 117.32,
113.47, 58.63, 41.47, 33.09. IR (KBr, cm−1): 3374, 3299,
1656, 1597, 1610, 1553, 1322, 1155. MS (m/z): 425.1
([M+H]+). HR–MS (ESI): calcd for C22H22N2O5S,
[M+H]+ m/z, 427.1322; found, 427.1320. HPLC purity:
99.937 %.
N-(3-aminophenyl)-4-methoxybenzenesulfonamide (1e)
According to the preparation procedure of 1d, 1e was
afforded from 4-methoxybenzenesulfonyl chloride. The
crude product can be used without further purification.
1H NMR (300 MHz, CDCl3): δ 7.76 (d, J = 8.6 Hz, 2H),
7.29 (s, 1H), 7.02–6.97 (m, 1H), 6.92 (d, J = 8.7 Hz, 2H),
6.73 (s, 1H), 6.45 (s, 1H), 6.45–6.39 (m, 2H), 3.85 (s, 3H).
N-(3-aminophenyl)-4-methylbenzenesulfonamide (1f)
According to the preparation procedure of 1d, 1f was
3-(4-Hydroxyphenyl)-N-(3-((4-methylphenyl)sulfonamido)
phenyl)propanamide (2i)
afforded from the 4-toluene sulfonyl chloride. The crude
1
product can be used without further purification. H NMR
(300 MHz, CDCl3): δ 7.70 (d, J = 8.1 Hz, 2H), 7.26 (d, J =
8.1 Hz, 2H), 7.14–7.08 (m, 1H), 7.02–7.00 (m, 2H),
6.84–6.80 (m, 1H), 2.43 (s, 1H).
According to the preparation procedure of 2g, 2i was pre-
pared as a pale white solid, yield 38.4 %. Mp: 145–147 °C.
1H NMR (300 MHz, DMSO-d6): δ 10.20 (s, 1H), 9.88 (s,
1H), 9.17 (s, 1H), 7.70 (d, J = 7.80 Hz, 2H), 7.49 (s, 1H),
7.37 (d, J = 7.3 Hz, 2H), 7.29 (d, J = 7.2 Hz, 1H), 7.16–7.10
(m, 1H), 7.05 (s, J = 7.8 Hz, 2H), 6.77 (s, J = 8.8 Hz, 1H),
6.70 (d, J = 8.1 Hz, 2H), 2.83–2.78 (m, 2H), 2.55–2.53 (m,
2H). 2.37 (s, 3H). 13C NMR (75 MHz, DMSO-d6): 173.61,
158.52, 146.20, 142.93, 141.22, 139.78, 134.20, 132.64,
132.20, 132.09, 129.77, 118.10, 117.58, 117.33, 113.44,
3-(4-Hydroxyphenyl)-N-(3-(phenylsulfonamido)phenyl)
propanamide (2g)
1d (600 mg, 2.42 mmol), 3-(4-hydroxyphenyl)propanoic
acid (803 mg, 4.83 mmol), PyBOP (3.14 mmol, 1.63 g)
were dissolved in DMF (25 mL) in the presence of

Med Chem Res
41.45, 33.07, 23.99. IR (KBr, cm−1): 3374, 3307, 1655,
1610, 1555, 1496, 1323, 1157. MS (m/z): 409.1 ([M+H]+).
HR–MS (ESI): calcd for C22H22N2O4S, [M+H]+ m/z,
411.1373; found, 411.1371. HPLC purity: 98.213 %.
concentration is 100 nM) to the reaction mixtures and
incubated for 60 min at room temperature. The reactions
were terminated by the addition of the acceptor and donor
beads mix (PerkinElmer) and incubated for 60 min at room
temperature, and then read on EnSpire (PerkinElmer) in
Alpha Mode. The reactions without enzyme but all other
component served as background. The data was analyzed in
GraphPad Prism 5.
Computational methods
Preparation of protein structure
MAO-A and MAO-B assays The MAO-A and MAO-B is
purchased from Active Motif (Cat#31502, Cat#31503).
Biochemical Kit is purchased from Promega (MAO-Glo
Assay, V1402). Inhibition assay were carried out according
to the manufacturer’s protocol. Luminescence is read on
Gen5 BioTek Spectrometer and data is processed using
GraphPad Prism 5.
The crystal structure of LSD1 resolved with the FAD-trans-
2-phenylcyclopropylamine adduct was retrieved from the
Protein Data Bank (PDB code: 2Z5U) (Mimasu et al.,
2010). The complex was cleaned of all water molecules and
the remaining structure was prepared using the Prepare
Protein utility in Discovery Studio 3.0 (DS 3.0) to fix
missing atoms and adding CHARMm force field in a phy-
siological condition (Studio D, 2010). The dielectric con-
stant was set to 10 and ionic strength was set to 0.145.
Based on the protonation state of the titratable residues
missing hydrogen atoms were added at a pH of 7.4.
Reversibility assay Reversibility was determined using
jump dilution analysis. Briefly, Compounds 2a, 2c and 2i at
the concentrations of 10× their biochemical IC50s were
pre-incuabted with high concentration of LSD1 protein
(500 nM) for 15 min, and then diluted 100-fold by
H3K4me2 peptide solution. The data was plotted in
GraphPad Prism 5.
Preparing the ligand database
Compounds were sketched in ChemBioDraw14.0 and
converted into 3D structures followed by a local mini-
mization using CHARMm force field. The resulting struc-
tures were used for the following docking simulations.
Acknowledgments X. Z. thanks the financial support by the Natural
Science Foundation of Jiangsu Province (BK20161458), the Funda-
mental Research Funds for the Central Universities (JKPZ2013001),
the Graduate Innovative Foundation supported by Huahai Pharma-
ceuticals Co., Ltd. (CX14S-004HH) and the Open Project Program of
Jiangsu Key Laboratory of Drug Screening (JKLDS2015KF-03). F. L.
thanks the financial support by China “Thousand Youth Talents”
(KHH1340001) and NSFC grant (91419306).
CDOCKER docking procedure
The CDOCKER program was utilized as a grid-based
dynamics docking algorithm. Ten replicas for each resulting
compound was created as a spherical site with a diameter of
16 Å for molecular docking and centered on the FAD-
inhibitor adduct. Using the rigid protein and flexible ligand
to perform the simulated annealing. Random conformation
was set as 10 and related simulated annealing steps were set
as 1000. The remaining parameters were left at default.
Finally, ten top hit conformations were saved for further
analysis.
Compliance with ethical standards
Conflict of interest F. L. is a shareholder of Constellation Phar-
maceuticals Inc. as well as a consultant of Active Motif. The remaining
authors declare that they have no conflict of interests.
References
Baell JB, Holloway GA (2010) New substructure filters for removal of
pan assay interference compounds (PAINS) from screening
libraries and for their exclusion in bioassays. J Med Chem
53:2719–2740
Biochemical assays
Binda C, Valente S, Romanenghi M, Pilotto S, Cirilli R, Karytinos A,
Ciossani G, Botrugno OA, Forneris F, Tardugno M, Edmondson
DE, Minucci S, Mattevi A, Mai A (2010) Biochemical, structural,
and biological evaluation of tranylcypromine derivatives as
inhibitors of histone demethylases LSD1 and LSD2. J Am Chem
Soc 132:6827–6833
Comess K, Erickson S, Henkin J, Kalvin D, Kawai M, Kim K,
BaMaung N, Park C, Sheppard G, Vasudevan A (2004) Sulfo-
namides having antiangiogenic and anticancer activity. US Patent
2,004,006,801,2
LSD1 screening assay The LSD1 screening biochemical
assay was completed by Shanghai ChemPartner Co. Ltd and
the detailed protocol was presented as followed. The
AlphaLISA assay of LSD1 (BPS Bioscience) was per-
formed in modified Tris buffer (pH 7.5). Briefly, the com-
pounds in DMSO were transferred to assay plate by Echo
550, and then pre-incubated with enzyme (final concentra-
tion is 5 nM) for 15 min at room temperature. The reactions
were initiated by adding H3K4me2 peptide solution (final
Deng X, Mani NS (2006) A facile, environmentally benign sulfona-
mide synthesis in water. Green Chem 8:835–838

Med Chem Res
Dhanak D (2013) Drugging the cancer epigenome. In: Proceedings of
the 104th Annual Meeting of the American Association for
Cancer Research, AACR., Washington p 6–10
Guibourt N, Ortega-Munoz A, Castro-Palomino Laria J (2010) Pre-
paration of phenylcyclopropylamine derivatives as LSD1 selec-
tive and LSD1/MAO-B dual inhibitors in treating or preventing
cancer. PCT WO 2010084160
Mimasu S, Umezawa N, Sato S, Higuchi T, Umehara T, Yokoyama S
(2010) Structurally designed trans-2-phenylcyclopropylamine
derivatives potently inhibit histone demethylase LSD1/KDM1.
Biochem 49:6494–6503
Ortega A, Castro-Palomino L, Fyfe, M (2011) Lysine specific deme-
thylase inhibitors and their use. PCT Int Appl WO 2011035941:
A12011
Hazeldine S, Pachaiyappan B, Steinbergs N, Nowotarski S, Hanson
AS, Casero Jr RA, Woster PM (2012) Low molecular weight
amidoximes that act as potent inhibitors of lysine-specific
demethylase 1. J Med Chem 55:7378–7391
Rivers A, Vaitkus K, Ruiz MA, Ibanez V, Kouznetsova T, DeSimone J,
Lavelle D (2014) RN-1, a potent and selective LSD1 inhibitor,
induces high levels of fetal hemoglobin (HbF) in anemic baboons
(P. anubis). Blood 124:336–336
Hitchin JR, Blagg J, Burke R, Burns S, Cockerill MJ, Fairweather EE,
Hutton C, Jordan AM, McAndrew C, Mirza A (2013) Develop-
ment and evaluation of selective, reversible LSD1 inhibitors
derived from fragments. Med Chem Comm 4:1513–1522
Kahl P, Gullotti L, Heukamp LC, Wolf S, Friedrichs N, Vorreuther
R, Solleder G, Bastian PJ, Ellinger J, Metzger E, Schule R,
Buettner R (2006) Androgen receptor coactivators lysine-
specific histone demethylase 1 and four and a half LIM domain
protein 2 predict risk of prostate cancer recurrence. Cancer Res
66:11341–11347
Kawai M, BaMaung NY, Fidanze SD, Erickson SA, Tedrow JS,
Sanders WJ, Vasudevan A, Park C, Hutchins C, Comess KM,
Kalvin D, Wang J, Zhang Q, Lou P, Tucker-Garcia L et al. (2006)
Development of sulfonamide compounds as potent methionine
aminopeptidase type II inhibitors with antiproliferative properties.
Bioorg Med Chem Lett 16:3574–3577
Sheppard GS, Wang J, Kawai M, Fidanze SD, BaMaung NY,
Erickson SA, Barnes DM, Tedrow JS, Kolaczkowski L,
Vasudevan A, Park DC, Wang GT, Sanders WJ, Mantei RA,
Palazzo F et al. (2006) Discovery and optimization of anthranilic
acid sulfonamides as inhibitors of methionine aminopeptidase-2:
a structural basis for the reduction of albumin binding. J Med
Chem 49:3832–3849
Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA,
Casero RA, Shi Y (2004) Histone demethylation mediated
by the nuclear amine oxidase homolog LSD1. Cell 119:
941–953
Sorna V, Theisen ER, Stephens B, Warner SL, Bearss DJ,
Vankayalapati H, Sharma S (2013) High-throughput virtual
screening identifies novel N′-(1-phenylethylidene)-benzohy-
drazides as potent, specific, and reversible LSD1 inhibitors.
J Med Chem 56:9496–9508
Levin JI, Du MT, Venkatesan AM, Nelson FC, Zask A, Gu Y (1999)
Preparation and use of ortho-sulfonamido aryl hydroxamic acids
as matrix metalloproteinase and tace inhibitors. US Patent
5,929,097, 27 Jul 1999
Liang Y, Quenelle D, Vogel JL, Mascaro C, Ortega A, Kristie TM
(2013) A novel selective LSD1/KDM1A inhibitor epigenetically
blocks herpes simplex virus lytic replication and reactivation
from latency. mBio 4:e00558–12
Lim S, Janzer A, Becker A, Zimmer A, Schule R, Buettner R, Kirfel J
(2010) Lysine-specific demethylase 1 (LSD1) is highly expressed
in ER-negative breast cancers and a biomarker predicting
aggressive biology. Carcinogenesis 31:512–520
Lynch JT, Harris WJ, Somervaille TC (2012) LSD1 inhibition: a
therapeutic strategy in cancer? Expert Opin Ther Targets 16:
1239–1249
McCafferty DG, Pollock, J (2013) Arylcyclopropylamines and meth-
ods of use. US Patent 838,958,0, 5, Mar 2013
Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, Peters AH,
Gunther T, Buettner R, Schule R (2005) LSD1 demethylates
repressive histone marks to promote androgen-receptor-
dependent transcription. Nature 437:436–439
Mimasu S, Sengoku T, Fukuzawa S, Umehara T, Yokoyama S (2008)
Crystal structure of histone demethylase LSD1 and tranylcypro-
mine at 2.25 A. Biochem Biophys Res Commun 366:15–22
Studio D (2010) Version 3.0, Accelrys Software Inc. San Diego
Ueda R, Suzuki T, Mino K, Tsumoto H, Nakagawa H, Hasegawa M,
Sasaki R, Mizukami T, Miyata N (2009) Identification of cell-
active lysine specific demethylase 1-selective inhibitors. J Am
Chem Soc 131:17536–17537
Yoshimatsu M, Toyokawa G, Hayami S, Unoki M, Tsunoda T, Field
HI, Kelly JD, Neal DE, Maehara Y, Ponder BA, Nakamura Y,
Hamamoto R (2011) Dysregulation of PRMT1 and PRMT6,
Type I arginine methyltransferases, is involved in various types of
human cancers. Int J Cancer 128:562–573
Zhao ZK, Yu HF, Wang DR, Dong P, Chen L, Wu WG, Ding WJ, Liu
YB (2012) Overexpression of lysine specific demethylase 1
predicts worse prognosis in primary hepatocellular carcinoma
patients. W orld J Gastroenterol 18:6651–6656
Zheng YC, Duan YC, Ma JL, Xu RM, Zi X, Lv WL, Wang MM,
Ye XW, Zhu S, Mobley D, Zhu YY, Wang JW, Li JF, Wang ZR,
Zhao W et al. (2013) Triazole-dithiocarbamate based selective
lysine specific demethylase 1 (LSD1) inactivators inhibit gastric
cancer cell growth, invasion, and migration. J Med Chem
56:8543–8560
Zhou C, Kang D, Xu Y, Zhang L, Zha X (2015) Identification of novel
selective lysine-specific demethylase 1 (LSD1) inhibitors using a
pharmacophore-based virtual screening combined with docking.
Chem Biol Drug Des 85:659–671