Synthesis and biological evaluation of novel (E)-N′-(2,3-dihydro-1H-inden-1-ylidene) benzohydrazides as potent LSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.06.054

Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine methylation at residues K4 and K9 on histone H3 and is recognized as an attractive therapeutic target in multiple malignancies. In this study, a series of novel (E)-N′-(2,3-dihydro-1H-inden-1-ylidene) benzohydrazides were synthesized and biologically evaluated for their potential LSD1 inhibitory effect. Among them, compounds 5a and 5n showed the most potent LSD1 inhibitory activity with IC₅₀values of 1.4 and 1.7?nM, respectively, which were about 10 times more potent compared with (E)-N-(1-(5-chloro-2-hydroxyphenyl) ethylidene)-3-(morpholinosulf-only) benzohydrazide (J. Med. Chem. 2013, 56, 9496–9508; as reference compound). Compounds 5a and 5n also exhibited marked anti-proliferation activities against cancer cell lines that highly expressed LSD1. These results suggest that these optimized compounds might be served as promising LSD1 inhibitors against cancer, which merit further study.

Full text of DOI:10.1016/j.bmcl.2015.06.054

Accepted Manuscript
Synthesis and biological evaluation of novel (E)-N’-(2, 3-dihydro-1H-inden-1-
ylidene) benzohydrazides as potent LSD1 inhibitors
Yang Zhou, Yan Li, Wen-Jing Wang, Pu Xiang, Xin-Mei Luo, Li Yang, Sheng-
Yong Yang, Ying-Lan Zhao
PII:
S0960-894X(15)00647-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.054
BMCL 22844
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2015
27 May 2015
11 June 2015
Please cite this article as: Zhou, Y., Li, Y., Wang, W-J., Xiang, P., Luo, X-M., Yang, L., Yang, S-Y., Zhao, Y-L.,
Synthesis and biological evaluation of novel (E)-N’-(2, 3-dihydro-1H-inden-1-ylidene) benzohydrazides as potent
LSD1 inhibitors, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.06.054
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Synthesis and biological evaluation of novel (E)-N'-(2,
3-dihydro-1H-inden-1-ylidene) benzohydrazides as potent LSD1 inhibitors
Yang Zhou1, Yan Li1, Wen-Jing Wang1, Pu Xiang1, Xin-Mei Luo1, Li Yang1，Sheng-Yong Yang1, Ying-Lan
Zhao*, 1
1State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China
Hospital, West China Medicinal School, Sichuan University, Chengdu 610041, Sichuan, China
* Author to whom correspondence should be addressed; E-Mail: zhaoyinglan@scu.edu.cn (Y.-L.Z); Tel.:
+86-28-8550-3628; Fax: +86-28-8550-3628.
Abstract: Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine
methylation at residues K4 and K9 on histone H3 and is recognized as an attractive therapeutic target in
multiple malignancies. In this study, a series of novel (E)-N'-(2, 3-dihydro-1H-inden-1-ylidene)
benzohydrazides were synthesized and biologically evaluated for their potential LSD1 inhibitory effect.
Among them, compounds 5a and 5n showed the most potent LSD1 inhibitory activity with IC50 values of
1.4 and 1.7 nM respectively, which were about 10 times more potent compared with
(E)-N-(1-(5-Chloro-2-hydroxyphenyl) ethylidene)-3-(morpholinosulf -only) Benzohydrazide (Sorna, V.,
et al., 2013; as reference compound). Compounds 5a and 5n also exhibited marked anti-proliferation
activities against cancer cell lines that highly expressed LSD1. These results suggest that these optimized
compounds might be served as promising LSD1 inhibitors against cancer, which merit further study.
Keywords: Histone methyltransferase, Lysine specific demethylase 1 (LSD1), Conformational constraint,
Anticancer.
Epigenetic disregulation contributes to the aberrant gene expression by chromatin modifications,
which directly causes oncogenesis and development of cancer.1 Lysine-specific demethylase（LSD1）, a
member of the flavin monoamine oxidase family, is the first discovered histone demethylase, which can
reversibly catalyze the oxidative demethylation of mono- and dimethylated histone H3 at lysine 4
(H3K4me1 and H3K4me2) and lysine 9 (H3K9me1 and H3K9me2) to mediate changes in gene
expression.2, 3 Overexpression of LSD1 is observed in a variety of cancers and is associated with the
progression, invasion and recurrence of these cancers including breast cancer, colon cancer, prostate
cancer, lung cancer and gastric cancer.4-8 Knocking down of LSD1 with small-interfering RNAs (siRNAs)
resulted in reactivation of tumor suppressor genes through increasing methylation at H3K4, thus
suppressed the proliferation and metastasis of cancer cells. Therefore, LSD1 is considered as a promising
anticancer drug target.

Figure 1. Chemical structures of Pargyline, TCP and TCP'S derivatives
To date, a handful of small molecule inhibitors of LSD1 have been reported (Figure1), such as
Monoamine oxidase (MAO) inhibitors tranylcypromine (TCP), pargyline (A and B) and several
derivatives of these scaffolds (C – E).9-14 But these small molecule inhibitors presented sorts of drawbacks,
like the poor selectivity, low potency and in vivo toxicity.15 For these reasons, the further contribution of
LSD1 in anticancer exploration has been impeded. Recently, a series of N′-(1-phenylethylidene)-
benzohydrazides had been described as potent LSD1inhibitors with a novel scaffold.15 Especially, the
novel compound (E)-N-(1-(5-Chloro-2-hydroxyphenyl) ethylidene)-3- (morpholinosulf -only)
Benzohydrazide exerted several advantages of superior selectivity and reversibility.15 However, its
inhibitory activity was still insufficient with the IC50 value of 13nM since the IC50 value of the most potent
LSD1 inhibitor that had been reported was 5nM.9 Therefore, it is needed to optimize this novel compound
to improve the inhibitory activity against LSD1. In this study, we set (E)-N-(1-(5-Chloro-
2-hydroxyphenyl) ethylidene)-3-(morpholinosulf -only) Benzohydrazide as the reference compound and
take the optimization as following.
Scheme 1. Initial LSD1 inhibitors
As we know that the use of conformational constraint is a general strategy with which to improve
ligand selectivity for a molecular target.16 We noticed that the conformation of acetophenone moiety of the
reference compound could be restricted on one plane if acetophenone moiety was replaced by 2,
3-dihydro-1H-inden-1-one (Scheme 1). Besides, the previous study had demonstrated the hydroxyl group
of the reference compound was essential and the inhibitory activity would be improved slightly with

adding the chlorine atom at the o-position of the hydroxyl group.15 Herein, we designed and synthesized
compound 5a ((E)-N'-(7-hydroxy-2,3-dihydro-1H-inden-1-ylidene)-3-(morpholinosulfonyl)
benzohydrazide), 5n ((E)-N'-(4-chloro-7-hydroxy-2,3-dihydro-1H-inden-1-ylidene)-3-(morpholinosul-
fonyl)benzohydrazide) and a series of derivatives (5b-m and 5o-z) by replacing variety of sulfamides
based on 5a and 5n. As shown in Table 1, there were several compounds with ring-like amino substituents
showed preferable inhibitory activity against LSD1 (5a-c, 5n-p and 5m). Especially, 5a presented an
excellent potent inhibitory activity against LSD1 with an IC50 value of 1.4 nM, which was about 10 times
higher than that of the reference compound. 5b, 5c and 5n-p were also more potent than the reference
compound with IC50 values in the single- or double-digit nanomolar range. This result demonstrated that
using conformational constraint strategy could indeed improve the inhibitory activity of the reference
compound against LSD1. Moreover,
a
series of (E)-N'-(2, 3-dihydro-1H-inden-1-ylidene)
benzohydrazides (5a-c, 5n-p and 5m) were also found as potent inhibitors.

Table 1.Effect of Different Sulfamides on LSD1 Inhibition
Inhibition(%)
Inhibition(%)
d
d
Cmpd#a R3
-NR1R2
IC50 (µM)
Cmpd# R3
-NR1R2
IC50 (µM)
50µM 10µM
50µM 10µM
H
H
99
99
99
0.0014 0.0003
0.0049 0.0011
Cl
Cl
99
77
99
0.0017 0.0007
0.0092 0.0006
5a
5b
5n
5o
100
100
H
H
99
100
N.I.
0.0023 0.0003
>100
Cl
Cl
100
55
100
31
0.0046 0.0006
>10
5c
5p
5q
N.I.b
5d
H
H
H
H
H
48
22
9
33
6
>50
>100
>100
>100
>10
Cl
Cl
Cl
Cl
Cl
20
28
23
46
38
8
>100
>100
>100
>50
5e
5f
5r
5s
5t
7
N.I.
4
N.I.
23
22
5g
5h
5i
18
64
5u
5v
62
>50

H
H
H
H
-
1
N.I.
17
2
>100
>10
Cl
Cl
Cl
Cl
81
47
>10
5j
5k
5w
5x
5y
5z
52
6
11
11
>100
>100
>100
>100
N.I.
N.I.
N.I.
N.I.
5l
92
99
59
99
2.9000 0.2000
0.0130 0.0010
5m
RCc
-
aCmpd# = compound number
bN.I. = No. Inhibition
cRC = reference compound
dValues are means of at least three measurements.

The synthetic route of designed compounds is provided in Scheme 2. Commercially available
3-(chlorosulfonyl) benzoic acid 1 was reacted with amino-compound in THF to give intermediate 2.
Intermediate 2 was reacted with MeOH to give ester 3. The ester 3 was reacted with N2H4 in MeOH to
give hydrazide compounds 4. Then hydrazide compounds 4 was reacted with 7-hydroxy-2,3-dihydro-
1H-inden-1-one or 4-chloro-7-hydroxy-2,3-dihydro-1H-inden-1-one under the catalysis of AcOH to give
the target compounds 5a – z.
Scheme 2. Synthetic route of the designed compounds. Reagents and conditions (a) THF, rt, overnight,
52%-60%; (b) conc H2SO4, MeOH, 65℃, 12h, 90%; (c) hydrazine, MeOH, MW, 130℃,30min, 50%-
60%; (d) AcOH, EtOH, 80℃, overnight, 40%-60%.
To further determine whether the sulfonamide replacement by other types of substituents or
sulfonamide position alteration might affect the compounds’ binding of LSD1, we carried out the
following structure-activity relationship (SAR) studies. First, we replaced the sulfamide of 5a and 5n
with the amide respectively. As shown in Table 2, the modified compounds (6a – n) displayed no
inhibitory activity against LSD1, suggesting that the amide group could not improve the inhibitory
activity of 5a and 5n against LSD1.
Table 2.Effect of Different Amides on LSD1 Inhibition
Inhibition(%)
50µM 10µM
N.I.b
N.I.
Inhibition(%)
Cmpd#a
R3
-NR1R2
IC50(µM)
Cmpd#
R3
-NR1R2
IC50(µM)
50µM
25
10µM
H
>100
Cl
N.I.
>100
6a
6h

H
H
H
N.I.
N.I.
32
N.I.
N.I.
N.I.
>100
>100
>100
Cl
Cl
Cl
N.I.
N.I.
N.I.
N.I.
N.I.
N.I.
>100
>100
>100
6b
6c
6d
6i
6j
6k
H
H
H
50
5
N.I.
N.I.
N.I.
>100
>100
>100
Cl
Cl
Cl
23
10
>100
>100
>100
6e
6f
6l
6m
6n
N.I.
16
N.I.
N.I.
6
6g
aCmpd# = compound number
bN.I. = No. Inhibition
Next, we investigated the effect of sulfamide position alteration on the inhibitory activity of 5a
and 5n against LSD1. The position of the sulfamide was moved from 3-position to 4-position of the
benzohydrazide moiety. As shown in Table 3, the modified compounds (7a - v) had little inhibitory
activity against LSD1. Moreover, it could be confirmed that the sulfamide at 3-position of
benzohydrazide moiety was essential to retain the inhibitory activity of 5a and 5n against LSD1.
Table 3.Effect of the Position of Sulfonamide on LSD1 Inhibition
Inhibition(%)
Inhibition(%)
Cmpd#a R3
-NR1R2
IC50(µM) Cmpd# R3
-NR1R2
IC50(µM)
50µM 10µM
50µM 10µM
H
12
31
N.I.b
>100
>100
Cl
Cl
N.I.
N.I.
N.I.
N.I.
>100
>100
7a
7l
H
H
H
N.I.
7b
7c
7d
7m
18
3
3
>100
>100
Cl
Cl
21
15
9
1
>100
>100
7n
7o
N.I.

H
H
H
N.I.
4
N.I.
N.I.
N.I.
>100
>100
>50
Cl
Cl
Cl
1
N.I.
2
>100
>100
>100
7e
7f
7p
7q
7r
14
13
47
N.I.
7g
H
H
H
H
N.I.
16
N.I.
N.I.
16
>100
>100
>50
Cl
Cl
Cl
Cl
2
N.I.
N.I.
11
>100
>100
>50
7h
7i
7s
7t
N.I.
43
4
30
7j
7u
7v
N.I.
N.I.
>100
N.I.
>100
7k
aCmpd# = compound number
bN.I. = No. Inhibition
Based on the above results, it could be figured out that the conformational constraint of the
acetophenone moiety contributed to the inhibitory activity of the reference compound against LSD1.
Furthermore, a compound (10, see Figure 3) with a carbon chain extension at the acetophenone moiety
had been reported with an IC50 value of > 3μM.15 Therefore, we hypothesized that the low activity of
10 may be caused by sterically hindered, as the extend carbon chain blocked the maintenance of active
conformation. On the other side, compound 5n constrained the conformation on one plane showed an
IC50 value of 1.7nM, which probably due to maintaining at an active conformation and enhanced the
binding to LSD1. However, changing the said-chain resulted in a complete loss of inhibitory activity.
This may be that the spatial structures of the side chain mismatch the binding site of LSD1.
Furthermore, molecular docking was performed to understand the possible binding mode of
compound 5n with LSD1. Figure 2 shows a predicted binding mode of compound 5n and reference
compound in complex with LSD1 (PDB code: 2Z5U). As can be seen, the docking program positioned 5n
in the pocket in such a way that its hydroxyl group forms strong hydrogen bonding interactions with
residues Gly314 and its O atom on morphine forms hydrogen bonding interactions with residues Val590,
which is a very similar with the reference compound. At the same time, the acyl group of 5n forms a
hydrogen bond with Arg310, which is not seen in reference compound-LSD1 binding mode. This may be
the reason that 5n shows a higher inhibition.

Figure 2 Putative binding mode of 5n and Reference compounds in complex with LSD1 (PDB code:
2Z5U). (A) Putative binding mode of 5n with LSD1. 5n is colored in magenta; (B) Reference
compounds in complex with LSD1. Reference compound is colored in cyans. The H-bonding
interactions are depicted in yellow dashed lines.
Figure 3. The Effect of Sterically Hindered on LSD1 Inhibition
To further confirm the sensitivity of the inhibitory activity compounds against LSD1 at cellular
level, the anti-viability activity of compounds 5a and 5n that own highly LSD1 inhibitory activity were
investigated by MTT assay in tumor cell lines. Reference compound was used as control. All the 9 tumor
cell lines were picked up from diversity cancers and all of the cells had been demonstrated to bear a high
expression level of LSD1.15, 17, 18 As shown in Table 4, compounds 5a and 5n treatment resulted in
significantly cell viability inhibition with the IC50 values ranging from 200nM to 3µM. Among these

cancer cell lines, A549, H1299, A2780, HCT116, MCF-7，MDA-MB-231 and DU145 were sensitive to
compound 5a with IC50 < 1 µM and A549，H1299, A2780, HCT116，MCF-7, MDA-MB-231, PC-3 and
DU145 were sensitive to compound 5n with IC50 < 1 µM. Generally, both of 5a and 5n showed stronger
viability inhibition than reference compound. These results indicated that compounds 5a and 5n had
significant cellular potency against various tumor cells bearing a high expression level of LSD1.
Table 4. The Viability Inhibition in LSD1-overexpressed cell lines
Reference compound
a
5a
5n
a
a
IC50 (µM)
IC50 (µM)
IC50 (µM)
a
a
a
Cell line
Cancer type
IC50 (µM)
IC50 (µM)
IC50 (µM)
(72h)
(72h)
(72h)
（96h）
（96h）
（96h）
2.69 0.19 0.83 0.11 0.92 0.05 0.39 0.09 0.78 0.07 0.40 0.07
3.01 0.13 1.73 0.21 2.01 0.15 0.79 0.03 3.33 0.11 0.98 0.09
15.75 0.15 5.36 0.16 2.96 0.38 1.34 0.06 3.16 0.06 1.22 0.11
A549
Lung
Lung
H1299
SMMC-7721
Hepatocellular
2.14 0.24 0.77 0.14 0.81 0.04 0.34 0.02 1.13 0.10 0.79 0.04
0.65 0.15 0.33 0.03 0.36 0.04 0.25 0.04 0.48 0.07 0.27 0.02
0.44 0.12 0.19 0.08 0.30 0.02 0.22 0.03 0.41 0.01 0.30 0.08
1.40 0.28 0.75 0.14 0.68 0.08 0.41 0.02 0.71 0.03 0.46 0.05
A2780
HCT116
MCF-7
Ovarian
Colorectal
Breast
MDA-MB-231
19.72 0.17 4.45 0.34 3.11 0.11 1.00 0.02 2.45 0.12 0.76 0.04
13.08 0.52 0.62 0.04 2.49 0.28 0.22 0.03 2.95 0.09 0.18 0.07
PC-3
Prostate
DU145
a Values are means of at least three measurements.
Despite the abundant literature on LSD1 and the many studies testing LSD1 inhibition in cancer
cells, the effects of LSD1 inhibitors in ovarian cancer cells have little been investigated. Hence, in
addition to cell viability, the effects of compounds 5a and 5n on the methylation levels of H3K4 and
H3K9 were analyzed in A2780. It had been demonstrated that LSD1 could activate the transcription of
nuclear genes by demethylation of H3K4 and H3K9.17 In our study, A2780 was treated with vehicle or
0.01, 0.1, 1, 10µM of compounds 5a and 5n for 48 h respectively. The Western-Bolt results showed that
the amounts of H3K4me2 and H3K9me2 were dose dependently elevated after treatment with
compounds 5a and 5n in A2780 (Figure 4). This suggested that compounds 5a and 5n could inhibit
cancer cells viability by on-targeting histone methylation mediated through LSD1 inhibition at the
cellular level.

Figure 4.The methylation levels of H3K4 and H3K9 treated with 5a and 5n in A2780. (A) The
methylation levels of H3K4 and H3K9 treated with 5a in A2780. (B) The methylation levels of H3K4 and
H3K9 treated with 5n in A2780
In conclusion, we successfully used conformational constraint strategy to optimize and obtain novel
potent LSD1 inhibitors (5a-c and 5n-p). Among them, Compounds 5a and 5n showed much more potent
LSD1 inhibitory activity compared with the reference compound. Further, compounds 5a and 5n
displayed a significant anti-viability effect against LSD1-overexpressed cancer cells. Moreover, the
mechanism of anti-viability effect of compounds 5a and 5n was attributed to the decrease methylation of
H3K4 and H3K9 by LSD1 inhibition. The potent inhibitory activity of the optimized compounds may
contribute to the exploration of LSD1 in caner and the further study of these compounds is highly
deserved.
Acknowledgements
This work was supported by National S&T Major project (2012ZX09501001-003), Program for New
Century Excellent Talents in University (NSET-11-0360) and Project of the National Natural
Sciences Foundation of China (81272459 and 81322035).
Supplementary information
1
The H NMR spectroscopic characterization of (E)-N'-(2, 3-dihydro-1H-inden-1-ylidene) benzohydra-
zides can be found in the online version.
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