4-Hydroxy-3-methylbenzofuran-2-carbohydrazones as novel LSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127109

Histone lysine specific demethylase 1 (LSD1 or KDM1A) is a potential therapeutic target in oncology due to its overexpression in various human tumors. We report herein a new class of benzofuran acylhydrazones as potent LSD1 inhibitors. Among the 31 compounds prepared, 14 compounds exhibited excellent LSD1 inhibitory activity with IC₅₀ values ranging from 7.2 to 68.8 nM. In cellular assays, several compounds inhibited the proliferations of various cancer cell lines, including PC-3, MCG-803, U87 MG, PANC-1, HT-29 and MCF-7. This opens up the opportunity for further optimization and investigation of this class compounds for potential cancer treatment.

Full text of DOI:10.1016/j.bmcl.2020.127109

Journal Pre-proofs
4-Hydroxy-3-methylbenzofuran-2-carbohydrazones as novel LSD1 inhibitors
Xingrui He, Yuan Gao, Zi Hui, Guodong Shen, Shuo Wang, Tian Xie, Xiang-
Yang Ye
PII:
S0960-894X(20)30198-0
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127109
BMCL 127109
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
15 January 2020
23 February 2020
12 March 2020
Please cite this article as: He, X., Gao, Y., Hui, Z., Shen, G., Wang, S., Xie, T., Ye, X-Y., 4-Hydroxy-3-
methylbenzofuran-2-carbohydrazones as novel LSD1 inhibitors, Bioorganic & Medicinal Chemistry Letters
(2020), doi: https://doi.org/10.1016/j.bmcl.2020.127109
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1
4-Hydroxy-3-methylbenzofuran-2-carbohydrazones as novel
LSD1 inhibitors
Xingrui Hea,b,c,d,#, Yuan Gaoa,e,#, Zi Hui a,b,c, Guodong Shend, Shuo Wangd, Tian
Xie*,a,b,c, Xiang-Yang Ye*,a,b,c
ORCID ID’s of all contributors:
Xingrui He: 0000-0002-7591-8022; Yuan Gao: 0000-0003-2254-6381; Hui Zi:
0000-0002-3211-8025; Tian Xie: 0000-0001-7066-1443; Xiang-Yang Ye: 0000-0003-3739-0930
aKey Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province,
bEngineering Laboratory of Development and Application of Traditional Chinese Medicine
c
from Zhejiang Province, Holistic Integrative Pharmacy Institutes (HIPI), School of Medicine,
Hangzhou Normal University, Hangzhou, Zhejiang 311121, P.R.China. dSchool of Pharmacy,
School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, Shandong
e
252000, P.R.China. School of Clinical Medicine, Guangdong Pharmaceutical University,
Guangzhou, Guangdong 510000, P.R.China.
#These two authors contributed equally
*Address correspondence to these authors at the Holistic Integrative Pharmacy Institutes
(HIPI), School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China;
Tel: +86-571-28860236 (Xiang-Yang Ye); E-mail: xyye@hznu.edu.cn (Xiang-Yang Ye);
Tel: +86-571-28860237 (Tian Xie); E-mail: xbs@hznu.edu.cn (Tian Xie);
ABSTRACT
Histone lysine specific demethylase 1 (LSD1 or KDM1A) is a potential therapeutic target
in oncology due to its overexpression in various human tumors. We report herein a new class
of benzofuran acylhydrazones as potent LSD1 inhibitors. Among the 36 compounds prepared,
15 compounds exhibited excellent LSD1 inhibitory activity with IC50 values ranging from 3.5
to 89 nM. In cellular assays, several compounds inhibited the proliferations of various cancer
cell lines, including PC-3, MCG-803, U87 MG, PANC-1, HT-29 and MCF-7. This opens up
the opportunity for further optimization and investigation of this class compounds for
potential cancer treatment.
OH
O
R
O
HN N
Novel benzofuran acylhydrazone LSD1 inhibitors
11ab
11ag
11al
11am
: LSD1 IC50: 14 nM (R=2,4-di-OH)
: LSD1 IC50: 15 nM (R=2-OH-6-F)
: LSD1 IC50: 27 nM (R=2-OH-4-Br)
: LSD1 IC50: 24 nM (R=2-OH-5-Me)

2
Keywords: Epigenetics; Lysine-specific demethylase 1; benzofuran; N-acylhydrazone (NAH);
anti-tumor activity;
Epigenetic modifications, including modifications on DNA (such as methylation), and
histones (such as acetylation, phosphorylation, and methylation), play pivotal roles in
regulating carcinogenesis.1-3 Among these modifications on histones, acetylation and
phosphorylation have been well studied for a long time. In contrast, histones methylation was
considered to be an irreversible process before 2004, and did not gain great attention until the
discovery of histone lysine-specific demethylase 1 (LSD1). LSD1 is a flavin-dependent
monoamine oxidase demethylating mono- and dimethylated lysines, specifically histone 3
lysine 4 and histone 3 lysine 9 (i.e. H3K4 and H3K9).4 The confirmation of the reversibility
of histones methylation/demethylation process, opened up a revolutionary new field had
attracted extensive studies in the past 10 years.5, 6
LSD1 uses flavin adenine dinucleotide (FAD) as a cofactor to specifically remove mono
or dimethylated histone 3 lysine 4 (H3K4) and histone 3 lysine 9 (H3K9).7,8 Overexpression
of this enzyme was found in numerous types of cancer, including lung and bladder cancers,9
neuroblastoma,10 retinoblastoma11 and breast cancer.12 Mechanistic studies at the molecular
level suggest that LSD1 involves in promoting cell proliferation, migration, invasion, and
metastasis by epithelial mesenchymal transition (EMT) induction.13 Most recently, scientists
unveiled that the inhibition of LSD1 could enhance the immunogenicity of the tumor and
could promote T-cell infiltration.14 Such findings spark great interests in seeking potential
combination therapeutics usefulness for tumor treatment with anti-PD-1 immunotherapy. Like
intervention on histones deacetylation (HDAC), intervention/modifications on histones
methylation such as LSD1 are expected to generate therapeutic drugs in the near future.15, 16
There are many LSD1 inhibitors reported in literature.17-22 Among them, the following
three small molecules, all belonging to irreversible tranylcypromine derivatives, were
advanced into the clinical stage: ORY-1001 (1),23 GSK-2879552 (2),24, 25 and CC-90011 (3)
(Figure 1).26 These three inhibitors are currently being evaluated in humans alone or in
combination with other therapeutic agents. Despite the great interests and deepened
knowledge of LSD1 field from recent years,5, 27 there is no LSD1 inhibitor approved as drug
up to date. Continuous seeking for new inhibitors targeting LSD1 is undoubtedly necessary
and important.
F
NC
O
NH2
H
N
N
H
N
O
N
HO
N
H2N
NH
2HCl
O
GSK-2879552 2
( )
ORY-1001 1
( )
CC-90011 3
( )
F
Figure 1. Clinical candidates of LSD1 inhibitors

3
Benzofuran moiety is one of the important structural units widely found in natural
products and synthetic compounds in drug discovery. Compounds bearing benzofuran moiety
generally possess certain biological activities such as anti-proliferation activity through
multiple mechanisms.28-31 For instance, Moracin related compounds are bioactive benzofuran
derivatives isolated from species of the moraceae family. Moracin O (4) was reported for their
ability to inhibit HIF-1 activation, with IC50 value of 0.14 M,31, 32. BNC-105P (5), a tubulin
34
polymerization inhibitor, is being developed in clinical trials.33,
Fruquintinib (6), an
inhibitor of vascular endothelial growth factor receptors/VEGFRs, has been approved for
fighting against various cancers in clinics.35 As a key functional moiety, benzofuran appears
in the structures of many natural products and approved drugs (Figure 2).36
O
O
O
O
OH
OH
H
O
O
N
O
HO
O
O
N
O
O
O
Na
O
O
N
O
P
Na
O
O
Moracin O 4
( )
BNC-105P 5
( )
Fruquintinib 6
( )
Figure 2. Representative structures of benzofuran derivatives.
On the other hand, N-acylhydrazone (NAH) is one of the most ubiquitous functional
groups in medicinal chemistry.37 A large number of hits and lead compounds bearing such
functional group are found to exhibit a wide range of biological activities targeting a variety
of molecular proteins.38-40 The synthesis of NAH is simple and practical, and can be achieved
via a condensation reaction between corresponding aldehydes or ketones with hydrazides.
The approved drugs containing NAH are: nitrofurazone (7), nitrofurantoinb,41
carbazochrome,42 testosterone 17-enanthate 3-benzilic acid hydrazine,43 nifuroxazide (8),44
dantrolene,45 and azumolene.46 PAC-1 is an NAH-based therapeutic agent that entered clinical
trials in 2015.47, 48 Recently, Sunil Sharma et al. performed an in silico screen of 20,500 small
molecules and identified NAH-based LSD1 inhibitor hits with the IC50 ranging from 200-400
nM. Further SAR optimization led to the discovery of compound 9 with IC50 value of 13 nM
(Figure 3).49
OH
O
O
O
O
S
O
NO2
N
N
O2N
N
O
N
N
Cl
N
O
N
NH2
H
O
H
H
HO
Nitrofurazone
7
( )
Nifuroxazide
8
( )
9
Figure 3. Representative structures bearing NAH functional group.
Previously we reported a class of compounds 10 (Figure 4) as new elicitor for plant to
combat insects.50, 51 Coupled with our interest on benzofuran core as natural product-like
scaffold,52 we set up to synthesize compounds 11, in which the acylhydrazone portion was
moved from 5- position to 2- position of the benzofuran scaffold. The original idea for doing
such swapping was to test our hypothesis that combination of two biologically active

4
fragments (i.e. benzofuran and NAH) in different orientation might generate new compounds
with different class of biological activities. The first analog 11a was then synthesized and
tested against a set of list of biological targets. To our delight, 11a showed 66% inhibitory
activity against LSD1 enzyme at 5 M concentration. Following the literature searching, we
noticed that compound 9 was reported by Sunil Sharma et al as LSD1 inhibitors.49 The
sulfonamide in 9 was claimed to be the crucial group to LSD1 activity. It is interesting to note
that compound 11a is active against LSD1 enzyme, despite its lack of sulfonamide portion.
OH
A
swapping from 5- to 2-
O
OH
HN
O
N
R
N
2
N
5
5
H
2
O
O
11a-11an
10
potent LSD1 inhibitor
this work
novel elicitor for plant
references 49 & 50
Figure 4. Medicinal chemistry design of LSD1 inhibitors 11.
Compound 11a was synthesized using the reactions presenting in Scheme 1. The
condensation reaction of cyclohexane-1,3-dione (12) with ethyl 2-chloroacetoacetate in basic
condition produces ethyl 3-methyl-4-oxo-4,5,6,7-tetrahydrobenzofuran-2-carboxylate (13) in
65% yield. Upon aromatization with NBS/AIBN, compound 13 was converted to compound
14 in 55% yield. Hydrazine exchange with ester functional group of compound 14 took place
smoothly in methanol to produce a good yield of compound 15. The final step to synthesize
11a can be simply achieved by mixing 15 and benzaldehyde in refluxed ethanol overnight.
O
O
OH
O
O
b
a
c
O
OEt
O
OEt
O
14
13
12
OH
OH
O
HN N
A
O
d
O
O
HN NH2
A
O
15
11
16
Scheme 1. Reagents and conditions: (a) KOH, ethyl 2-chloroacetoacetate, H2O/MeOH (6:1), RT, 5 d,
~65%; (b) NBS, AIBN, CCl4, 80 °C, 5 h, 55 %; (c) N2H4.H2O, MeOH, 80 °C, 5 h, 85 %; (d) EtOH,
85 °C, 12 h.
With the synthetic route developed for 11a (Scheme 1), we first quickly surveyed the
scope of phenyl portion (i.e. “A” group) to explore the requirements of “A” group. Phenyl
group in 11a was replaced with 2-furanyl (11b), 2-thiophenyl (11c), or pyridyl (11d-11f)
(Table 1). All compounds appear to be comparable in inhibiting LSD1 enzyme at 5 M
concentration. 3-Pyridyl analog 11e appears to have the highest percentage inhibition among
all compounds. However, 11e was found to have very limited cellular activity in
anti-proliferation experiment against cancer cell line (will discuss later).

5
Table 1. Inhibitory activity of compounds 11a-11q against LSD1 enzyme at 5 M concentration.a
Percentage
inhibition at
concentration of 5
M against LSD1
(%)
Compound
code
A
=
11a
11b
66.4±6.1
51.7±5.2
O
11c
73.6±3.7
S
11d
11e
11f
69.3±5.3
91.7±6.2
76.2±7.5
N
N
N
HO
11g
108.5±8.7
OH
11h
11i
11j
75.4±5.2
69.3±3.4
56.6±7.5
OH
O
O
11k
11m
11n
11o
11p
52.1±3.1
20.2±7.6
40.2±5.3
44.8±8.4
51.1±6.7
F
F
F
F3C

6
11q
12
CF3
-5.1±7.3
23.9±7.1
(see Scheme 2 for
structure)
aData are presented as the means ± SDs of three independent experiments. p < 0.05.
Next, we decided to install one substituent into the phenyl portion of 11a, i.e. focusing
on mono-substituent phenyl. SAR analysis suggested that the substitutent plays important role
in LSD1 activity. Installation of ortho-OH (11g) in phenyl portion gives the best inhibitory
activity among all compounds tested (11g vs 11a and others). Moving hydroxyl group from
ortho-position to others (i.e. meta-OH (11h) and para-OH (11i)) appears to decrease the
inhibitory activity. Replacement of hydroxyl group with other electron-rich group (i.e. 11j
and 11k) or electron-withdrawing groups (i.e. 11m-11q) resulted in decrease of inhibitory
activity in certain extent.
Apparently, 11g, a compound bearing ortho-OH group of phenyl in the right hand side
of molecule, gave the highest inhibitory activity at 5 M concentration. This prompted us to
examine the importance of other –OH group, the one on the 4-position of benzofuran ring.
Thus, compound 12 was designed and synthesized (Scheme 2).
O
OH
O
O
O
O
O
O
O
b
a
O
Br
O
O
13
14
15
16
c
O
d
O
HN NH2
O
HN
N
OH
17
12
Scheme 2. Reagents and conditions: (a) K2CO3, CH3CN, 65 °C, 18 h, ~75%; (b) K2CO3, DMF,
130 °C, 2 h, ~65%; (c) N2H4.H2O, MeOH, 80 °C, 5 h, ~85 %; (d) salicylaldehyde, EtOH, 85 °C, 12 h，
~80%.
At 5 M concentration, compound 12 gave only 23.9% inhibitory activity against LSD1
enzyme. By comparing with this result with compound 11a, the hydroxyl group at the
4-position of benzofuran plays piviotal role in generating LSD1 activity. Subsequently
literature searching suggested that compound 12 was known compound, previously reported
by Taha et al as alpha-amylase inhibitor.53
Based on the structure-activity relationship (SAR) from Table 1, it’s clear that both the
two hydroxyl groups of 11g (one at the 4-position of benzofuran core, the other at the
ortho-position of right hand phenyl) play pivotal roles in generating inhibitory activity against
LSD1 enzyme.
In order to further optimize the lead compound 11g, the two hydroxyl groups on the two
ends of the molecule were fixed, and the additional one or two substituent(s) was incorporated
into the right hand phenyl. All compounds were synthesized according to Scheme 1. The
results of inhibitory activity of those compounds at 5 M concentration were listed in Table 2，

7
and RN-154 was chosen as the positive control.
Table 2. Inhibitory activity of compounds 11aa-11an against LSD1 enzyme.a
Percentage inhibition
at the concentration of
5 M against LSD1 (%)
108.5±7.1
IC50 against
LSD1
Compound
A
code
=
(nM)
11g
11aa
11ab
11ac
11ad
11ae
11af
11ag
11ah
11ai
2-OH phenyl
2,3-di-OH phenyl
2,4-di-OH phenyl
2,5-di-OH phenyl
2,3,4-tri-OH phenyl
2-OH-3-F phenyl
2-OH-5-F phenyl
2-OH-6-F phenyl
2-OH-5-Cl phenyl
2-OH-6-Cl phenyl
2-OH-3,5-di-Cl phenyl
2-OH-3-Br phenyl
2-OH-4-Br phenyl
2-OH-5-Me phenyl
2-OH-5-NO2C6H3
(see ref. 53 for
68.8±5.2
28.1±6.4
14.4±4.8
55.0±6.2
7.2±3.1
31.8±6.4
58.08±4.3
14.78±6.2
31.3±5.4
25.9±6.3
59.3±3.8
52.1±7.5
27.0±4.2
23.7±7.2
-
103.6±9.4
111.1±6.5
109.9±3.6
105.0±7.5
99.2±8.3
102.4±5.2
103.4±7.7
102.7±8.8
99.4±4.3
11aj
11ak
11al
107.5±6.9
97.1±4.6
101.4±8.3
11am
11an
96.7±5.5
85.2±8.9
RN-1
3.1±5.3
structure)
a Data are presented as the means ± SDs of three independent experiments. p < 0.05.
To our delight, all compounds reach 85% inhibitory activity against LSD1 at 5 M
concentration level (Table 2). Further enzymatic experiment in dose-response mode produces
their IC50 value for all compounds except 11am. The results are very encouraging. This
further testifies the importance of 2-hydroxyl group of the phenyl ring. Incorporating
additional hydroxyl group onto the phenyl ring, regardless of the position, results in an
increase of LSD1 inhibition as suggested by their IC50 values (11aa, 11ab, 11ac, 11ad vs
11g). This is possibly due to stronger hydrogen bonding with LSD1 enzyme produced by the
additional hydroxyl group incorporated. The addition of halogen or nitro groups to phenyl
ring does not appear to produce significant effects in modulating IC50 values.
Next, the competitive analysis was performed in Autodock to evaluate the binding sites of
compound 11al on LSD1 enzyme (PBD code 5X60).55 The docking analysis suggests that the
hydroxyl on benzofuran forms critical hydrogen bond with residue ASP555. Additionally, the
carbonyl oxygen of compound 11al forms hydrogen bond to HIS564, while the oxygen atom
on benzofuran ring forms hydrogen bond to PHE560. These residues were also reported
essential residue for FAD-binding, so compound 11al may act as FAD competitor (Figure 5).

8
Figure 5. Compound 11ab (green color) was docking into LSD1-CoREST protein complex (PBD code
5X60) using Autodock. Only several key residues from protein complex were shown (grey color).
Hydrogen bonds were shown in green dot lines.
In view of the high expression of LSD1 in prostate cancer and relevant correlation with
androgen receptor, we first tested the inhibitory activity of these compounds against PC-3 cell
line proliferation (Table 3).56 Most of the 2-hydroxy compounds show good anti-proliferation
activity. Eight of them displayed IC50 less than 10 μM for PC-3 cell line, which generally
correlating to the enzymatic assay results. Surprisingly, despite of being the most active in
enzymatic assay, compound 11ad showed no cellular activity against PC-3 cell line (IC50 > 80
M). Too many hydroxyl groups presented in this molecule might increases molecular
polarity (cLogP = 2.2401), hence could decrease its ability penetrating the cell membrane
where LSD1 enzyme located.4
Table 3. Anti-proliferation activities against PC-3 cell line.a
Compound
11a
IC50 (μM)b
29.15±0.2
9.59±0.1
16.59±0.3
2.04±0.4
7.35±0.2
≥40
Compound
11q
IC50 (μM)b
≥40
11b
11aa
11ab
11ac
12.15±0.4
14.79±0.3
95.45±0.4
≥40
11c
11d
11e
11ad
11ae
11f
12.73±0.2
3.17±0.4
8.05±0.3
3.12±0.3
18.68±0.4
11g
8.35±0.1
17.64±0.3
21.7±0.3
≥40
11af
11h
11ag
11ah
11ai
11i
11j

9
11k
11m
11n
11o
11p
≥40
≥40
11ag
11ak
11al
9.59±0.2
16.59±0.5
2.05±0.3
7.35±0.6
17.13±0.3
≥40
≥40
11am
11an
26.17±0.1
a IC50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear fit-curve
b
(log of compound concentration versus normalized response—variable slope). Data are presented as
the means ± SDs of three independent experiments. p < 0.05.
The important role of LSD1 in the occurrence and development of gastric cancer
prompted us to test compounds in MCG-803 cell line (Table 4).57 Most compounds showed
IC50 value less than 10 μM.
Table 4. Anti-proliferative activities against MCG-803 cell line. a
Compound
11g
IC50 (μM)b
10.62±0.3
5.03±0.2
7.33±0.3
18.1±0.5
8.35±0.3
5.23±0.2
Compound
11ag
IC50 (μM)b
10.84±0.3
4.16±0.4
4.36±0.5
14.17±0.2
5.46±0.1
5.99±0.3
11aa
11ah
11ab
11aj
11ad
11ak
11ae
11al
11af
11am
aIC50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear
b
fit-curve (log of compound concentration versus normalized response—variable slope). Data are
presented as the means ± SDs of three independent experiments. p < 0.05.
To verify the broad-spectrum anti-proliferation effects, we selected three compounds to
test in 4 additional cell lines: neuroglioma U87 MG, Pancreatic cancer PANC-1, Human
colon cancer HT-29, and breast cancer MCF-7. The results are presented in Table 5. All
compounds showed good activities against all 4 cell lines except compound 11ad.
Table 5. Anti-proliferative activities against additional four cell linesa
IC50 (μM)b
Compound
U-87 MG
5.96±0.3
9.27±0.2
4.39±0.3
0.02±0.002
MCF-7
17.06±0.1
1.75±0.3
2.08±0.4
0.02±0.001
HT-29
>20
PANC-1
>20
11ad
11ag
11ab
STSc
2.20±0.4
2.88±0.3
0.01±0.002
8.23±0.3
9.77±0.2
0.02±0.002
aIC50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear fit-curve
b
(log of compound concentration versus normalized response—variable slope). Data are presented as the
means ± SDs of three independent experiments. p < 0.05. cSTS (Staurosporine) was used as positive control.
In conclusion, we reported a series of benzofuran acylhydrazone compounds as LSD1
inhibitors. Among the 36 compounds synthesized, 15 compounds showed IC50 values ranging
from 3.5 to 89 nM against LSD1. SAR analysis pointed to the importance of both hydroxyl

10
groups at 4-position of benzofuran and ortho-position of right hand phenyl. In cellular assays,
the compounds inhibited the proliferation of various cancer cell lines, including PC-3,
MGC-803, U87, PANC-1, HT-29 and MCF-7. Additional efforts in characterizing leads in
both PK and efficacy studies will be another topic for future disclosure.
Acknowledgments
This work was supported by the Key Programs of National Natural Science Foundation of
China (81730108), Hangzhou Normal University startup fund (4125C5021920419), and the
Natural Science Foundation of Shandong Province (ZR2019BB046). We are grateful to
Professor Dr. Renren Bai (College of Pharmaceutical Science, Zhejiang University of
Technology) for helpful suggestion. We also thank Mr. Wengang Wang for performing
docking experiment for us.
Disclosure of interest
The authors declare no competing financial interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/xxxxx.
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Reference#:
BMCL-D-20-00071.
4-Hydroxy-3-methylbenzofuran-2-carbohydrazones as novel LSD1
inhibitors
Declaration of Interest Statement
The authors declare no competing financial interest.
Graphical Abstract
Leave this area blank for abstract info.
4-Hydroxy-3-methylbenzofuran-2-carbohydrazones
as novel LSD1 inhibitors
OH
A
swapping from 5- to 2-
O
OH
HN
O
N
R
N
2
N
5
5
H
2
O
O
11a-11an
10
potent LSD1 inhibitor
this work
novel elicitor for plant
references 49 & 50

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