Discovery of new tranylcypromine derivatives as highly potent LSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.127993

Tranylcypromine (TCP)-based structural modifications lead to the discovery of new LSD1 inhibitors, of which compounds 26b and 29b effectively inhibit LSD1 with the IC₅₀ values of 17 and 11 nM, respectively and also show good selectivity over MAO-B. Mechanistic studies showed that compound 29b concentration-dependently induced H3K4me1/2 accumulation in LSD1 overexpressed MGC-803 cells and also inhibited metastasis of MGC-803 cells. Collectively, both compounds could be promising lead compounds for further investigation.

Full text of DOI:10.1016/j.bmcl.2021.127993

Bioorg. Med. Chem. Lett. 41 (2021) 127993
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new tranylcypromine derivatives as highly potent
LSD1 inhibitors
Ming-Jie Huanga, Jia-Wen Guoa, Yun-Dong Fua, Ya-Zhen Youa, Wen-Yu Xua, Ting-Yu Songa,
,
,*
Ran Lia, Zi-Tong Chena, Li-Hua Huanga *, Hong-Min Liub
a Green Catalysis Center, and College of Chemistry, Zhengzhou University, Zhengzhou 450001, China
b School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tranylcypromine (TCP)-based structural modifications lead to the discovery of new LSD1 inhibitors, of which
compounds 26b and 29b effectively inhibit LSD1 with the IC50 values of 17 and 11 nM, respectively and also
show good selectivity over MAO-B. Mechanistic studies showed that compound 29b concentration-dependently
induced H3K4me1/2 accumulation in LSD1 overexpressed MGC-803 cells and also inhibited metastasis of MGC-
803 cells. Collectively, both compounds could be promising lead compounds for further investigation.
Histone demethylase
Tranylcypromine
LSD1 inhibitor
Gastric cancer
The lysine-specific histone demethylase 1A (known as LSD1 or
KDM1A) is the first histone demethylase identified in 2004, which
specifically removes methyl groups of histone substrate H3 lysine 4
(H3K4) in flavin adenine dinucleotide (FAD)-dependent manner 1. LSD1
has fundamental roles in physiological processes, and its dysregulation
is closely associated with the occurrence and development of various
pathological conditions including cancers, virus infections, neurode-
generative diseases, etc. 2–6. Accumulating evidence have showed that
pharmacological inhibition of LSD1 by small molecules or genetic
knockdown is an effective strategy in controlling the pathological states
7–9. These findings suggest that LSD1 is a well-characterized therapeutic
epigenetic target 10. To date, numerous natural and synthetic LSD1 in-
hibitors have been reported in last decades, showing great promise in
cancer therapy 11–14. Particularly, irreversible LSD1 inhibitors including
ORY-1001, tranylcypromine (TCP), ORY-2001, GSK-2879552,
INCB059872, IMG-7289, and reversible LSD1 inhibitor CC-90011 have
advanced into clinical assessment for the treatment of cancers such as
acute myeloid leukemia (AML) and small lung cancer cells (SCLC)
(Fig. 1) 15,16. Some of these clinical candidates have also shown promise
for treating myelodysplastic syndromes (MDS), multiple sclerosis (MS),
myelofibrosis, and Alzheimer’s disease (AD) 17–19. The success of these
LSD1 inhibitors highlight the importance of TCP for designing covalent
LSD1 inhibitors. Previous studies have showed that modifications on the
TCP scaffold could alter the inhibitory activity against LSD1 and also the
selectivity over monoamine oxidases (MAO-A/B) 20–22. As demonstrated
by the clinical candidate ORY-2001, the 1,3,4-oxadiazole ring is linked
to the TCP scaffold. We propose that replacement of the 1,3,4-oxadiazole
ring in ORY-2001 with other bioisosteres (e.g. the triazole ring) may
give new LSD1 inhibitors23,24. Herein, we designed the title compounds
by introducing the triazole ring to the TCP scaffold in place of the 1,3,4-
oxadiazole ring. Additionally, it is well recognized that TCP-based LSD1
inhibitors could form covalent adducts with FAD through the single-
electron transfer mechanism25,26. Thus, we speculate that the elec-
tronic effect of substituents may have certain impact on the inhibitory
activity of the title compounds. In this work, we introduced two repre-
sentative groups, namely trifluoromethyl group (CF3) and methoxyl
group (OCH3), into the phenyl ring, aiming to examine the effect of
substituents with different electronic property on the anti-LSD1 activity.
The preliminary structure–activity relationship studies (SARs) of new
tranylcypromine derivatives led to the discovery of compounds 26b and
29b as highly potent LSD1 inhibitors, both compounds effectively
inhibited LSD1 with the IC50 values less than 20 nM and could represent
promising lead compounds for further development.
The synthetic protocol of 1,4-disubstituted-1,2,3-triazole analogue
23–31 was based on the copper-catalyzed azide-alkyne cycloaddition
(CuAAC) 27, which required two building blocks, N-propargylamines 4
(Scheme 1) and phenyl azides 14–22 (Scheme 2). As shown in Scheme 1,
trans-aminocyclopropanes 1 reacted with Boc2O in the presence of
K2CO3, generating the corresponding Boc protected trans-amino-
cyclopropanes 2, further alkylation with propargyl bromide in the
presence of sodium hydride gave the corresponding Boc-protected
propargylamines 3 28. The obtained compounds 3 underwent the
* Corresponding authors.
E-mail addresses: hlh606@zzu.edu.cn (L.-H. Huang), liuhm@zzu.edu.cn (H.-M. Liu).
https://doi.org/10.1016/j.bmcl.2021.127993
Received 22 December 2020; Received in revised form 14 March 2021; Accepted 19 March 2021
Available online 26 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M.-J. Huang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127993
Fig. 1. TCP-based LSD1 inhibitors in clinical trials (TCP is highlighted in blue color).
Scheme 1. Preparation of compounds 4a-b. Reagents and conditions: i) 1. Boc2O, DCM, N2, 0 ◦C, 0.5 h; 0 ◦C to RT, 2 h; 2. K2CO3, RT, 2 h; ii) 1. NaH, DMF, N2, 0 ◦C;
0 ◦C to RT, 0.5 h; 2. Propargyl bromide, RT, 2 h; 3. Ice, RT, 15 min; iii) HCl, THF, 0 ◦C to RT, 2 h.
Scheme 2. Preparation of compounds 23–31. Reagents and conditions: i) 1. HCl, NaNO2, H2O, 0 ◦C; 2. NaN3, H2O, 0 ◦C to RT, 1 h; ii) N-propargylamine, CuSO4,
sodium ascorbate, THF/H2O, RT, 9 h.
deprotection of the N-Boc group with dilute hydrochloric acid (6%) to
afford the required building blocks N-propargylamines 4 (Scheme 2).
The synthesis of phenyl azides 14–22 started from the appropriate an-
ilines 29. Treatment of anilines with NaNO2 in acidic aqueous media,
followed by azidation in the presence of NaN3, gave compounds 14–22.
The title compounds 23–31 were synthesized by the copper-catalyzed
click reaction between propargylamines 4 and phenyl azides 14–22
(Scheme 2).
Particularly, compounds 26b and 29b potently inhibited LSD1 with the
IC50 values of 17 and 11 nM, respectively. The inhibition cures of the
most potent compounds 26b and 29b against LSD1 are shown in Fig. 2.
For compounds 27b and 31b with the –CF3 group, their inhibitory ac-
tivity against LSD1 significantly decreased. We also observed that the
substituent attached to the 2- or 4-position of the phenyl ring was
tolerated, while the one at the 3-position was less tolerated. For
example, compounds 24b and 26b showed comparable inhibitory ac-
tivity with compound 23b, while compound 25b displayed decreased
inhibitory activity with the IC50 value lower than that of compound 23b.
The trend was the same for other compounds (27b vs. 28b, 29b vs. 30b).
Besides, we also tested the inhibitory activity of the compounds against
MAO-A/B to examine the selectivity, and clorgyline and R-(-)-deprenyl
were used as the reference compounds 31. As depicted in Table 1, most of
With the compounds in hand, we next tested the inhibitory activity of
the compounds against LSD1 using the well-known ORY-1001 as the
control compound 30. As shown in Table 1, the compounds showed su-
perior potency against LSD1 with the IC50 values as low as 11 nM.
Evidently, except for compound 31b, the remaining compounds bearing
the fluorine atoms had better potency than their counterparts without
the fluorine atoms, underscoring the importance of the fluorine atom for
the potency toward LSD1. Generally, the SARs studies indicated that
compounds bearing the electron-donating groups exhibited improved
anti-LSD1 inhibitory activity than those substituted with electron-
deficient groups. Compared to compound 23b, compounds 24b, 25b,
26b, 28b, and 29b showed comparable or improved potency.
the compounds at 10 μM showed excellent inhibitory activity against
MAO-A with the inhibitory rates up to 100%, but with relatively lower
potency against MAO-B. The results suggest that the compounds may be
dual LSD1/MAO-A inhibitors. It has been documented that ORY-2001 is
a dual LSD1/MAO-B inhibitor and currently being assessed in clinical
trials for the treatment of mild to moderate Alzheimer’s disease
2

M.-J. Huang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127993
activity against LSD1 overexpressed cancer cell lines including PC-3,
MCF-7, MGC-803, and SGC-7901. As shown in Table 2, compounds
Table 1
The inhibitory activity of the compounds against LSD1 and MAO-A/
26b and 29b (16 μM) were almost inactive against these cell lines with
the inhibitory rates less than 50%, indicating the potential low toxicity.
The results are consistent with those previously reported, namely some
highly potent and selective LSD1 inhibitors such as ORY-1001 and GSK-
B.
Compound
R’
X
IC50 [nM]a or inhibition [%]b
2879552 are nontoxic against some cancer cells 30,33
.
LSD1
MAO-Ac
MAO-Bc
Considering the favorable potency of compound 29b against LSD1,
additional cellular studies were further conducted to verify its cellular
effects in LSD1 overexpressed MGC-803 cells. Then the expressions of
two LSD1 substrates, H3K4me1 and H3K4me2, were evaluated in
MGC803 cells after exposure to compound 29b for 72 h. ORY-1001 was
used as the positive control. As shown in Fig. 3A, compound 29b
concentration-dependently induced accumulation of H3K4me1 and
H3K4me2, supporting that compound 29b could inhibit the LSD1 ac-
tivity in vitro. Then, the migration ability of MGC-803 cells was further
evaluated by the transwell and wound healing assays. As shown in
Fig. 3B, compound 29b suppressed the MGC-803 cell migration in a
concentration-dependent manner compared to control group. And
further wound healing assay (Fig. 3C) showed that for the untreated
group, MGC-803 cells filled almost all the wounded area after scratching
the cell monolayer, while compound 29b concentration-dependently
inhibited the wound healing obviously. All the data demonstrated that
compound 29b could block the metastasis of LSD1-overexpressed MGC-
803 cells.
23a
23b
H
F
111
24
102%
101%
76%
57%
24a
24b
H
F
120
21
91%
90%
44%
51%
25a
25b
H
F
106
42
93%
54%
65%
102%
26a
26b
H
F
41
17
99%
55%
64%
100%
27a
27b
H
F
110
101
101%
90%
94%
43%
28a
28b
H
F
132
26
101%
101%
86%
82%
In summary, we have performed the structural modifications of TCP,
leading to the discovery of compounds 26b and 29b, which inhibited
LSD1 potently with the IC50 values of 17 and 11 nM, respectively and
also exhibited good selectivity over MAO-B. The SARs studies revealed
the structural features for LSD1 inhibition. Besides, the compounds
exhibited weak antiproliferative activity against the tested cancer cells,
suggesting the low toxicity. Mechanistic studies showed that compound
29b concentration-dependently induced accumulation of LSD1 sub-
strates H3K4me1/2 in LSD1 overexpressed MGC-803 cells and also
inhibited metastasis of MGC-803 cells in the transwell and wound
healing assays. Taken together, compounds 26b and 29b are two
promising LSD1 targeting lead compounds for further development and
have therapeutic potentials.
29a
29b
H
F
35
11
97%
89%
58%
48%
30a
30b
H
F
110
66
101%
93%
67%
42%
31a
31b
H
F
282
664
92%
91%
88%
35%
ORY-1001
0.14
NDd
NDd
NDd
NDd
Clorgyline
1.1 nM
NDd
R-(-)-deprenyl
NDd
70 nM
Declaration of Competing Interest
(a) The IC50 values for LSD1 were calculated from 8 data points; (b) Percentage
of inhibition at 10 μM; all compounds are single enantiomers. (c) The inhibitory
The authors declare that they have no competing financial interests
activity of Clorgyline and R-(-)-deprenyl against MAO-A/B was examined at 10
different concentrations, and all data are the mean value of two independent
determinations. (d) ND means Not Determined.
Table 2
Cellular antiproliferative activity of compounds 26b and 29b against the tested
cancer cell lines.
(ClinicalTrials.gov Identifier: NCT03867253) 32. The therapeutic po-
tential of such compounds (e.g., 26b and 29b) may deserve further
investigation.
Compound
Inhibition [%]
PC-3
MCF-7
MGC-803
SGC-7901
26b
29b
25.17
21.84
37.26
33.65
16.30
24.54
41.95
27.68
Encouraged by the high potency of compounds 26b and 29b against
LSD1 and the selectivity over MAO-B, we also examined their inhibitory
Fig. 2. Inhibition cures of compounds 26b, 29b and ORY-1001 against LSD1.
3

M.-J. Huang et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127993
Fig. 3. Cellular effects of compound 29b against MGC803 cells. (A) Expression of LSD1 substrates, H3K4me and H3K4me2, after treatment with compound 29b for
72 h in MGC-803 cells. H3 was used as the loading control; (B) Migration assay; (C) Wound healing assay. Data were shown as mean ± SD. **p < 0.01 was considered
statistically significant compared with the control. All the experiments were performed at least three times and a representative result was shown.
or personal relationships that could have appeared to influence the work
reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127993.
Acknowledgments
This work was supported by The Key Scientific and Technological
Project of Henan Province (No. 182102310121) and the National Nat-
ural Science Foundation of China (Project No. 81773562).
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