Synthesis and biological evaluation of coumarin-1,2,3-triazole- dithiocarbamate hybrids as potent LSD1 inhibitors

Article abstract of DOI:10.1039/c4md00031e

Two series of coumarin-1,2,3-triazole-dithiocarbamate hybrids were designed, synthesized and evaluated for their inhibitory activity towards lysine specific demethylase 1 (LSD1). Compounds 8a, 8d-8f, 8i-8l presented potent activity against lysine specific demethylase 1. Among them, compound 8k showed potent and reversible inhibition against lysine specific demethylase 1 with an IC₅₀ value of 0.39 μM, which was 74-fold more potent than that of tranylcypromine (2-PCPA). Besides, compound 8k displayed excellent selectivity against lysine specific demethylase 1 without inhibition against monoamine oxidases (MAOs) A and B. Further investigation revealed that compound 8k was active at both recombinant and cell levels by upregulating the expression of H3K4me1, H3K4me2 and H3K9me2. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4md00031e

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Synthesis and biological evaluation of coumarin–
1,2,3-triazole–dithiocarbamate hybrids as potent
LSD1 inhibitors†
Cite this: Med. Chem. Commun., 2014,
5, 650
Xian-Wei Ye,‡a Yi-Chao Zheng,‡a Ying-Chao Duan,b Meng-Meng Wang,a Bin Yu,a
a
a
a
a
*
Jing-Li Ren, Jin-Lian Ma, En Zhang and Hong-Min Liu
Two series of coumarin–1,2,3-triazole–dithiocarbamate hybrids were designed, synthesized and evaluated
for their inhibitory activity towards lysine specific demethylase 1 (LSD1). Compounds 8a, 8d–8f, 8i–8l
presented potent activity against lysine specific demethylase 1. Among them, compound 8k showed
potent and reversible inhibition against lysine specific demethylase 1 with an IC50 value of 0.39 mM,
which was 74-fold more potent than that of tranylcypromine (2-PCPA). Besides, compound 8k displayed
excellent selectivity against lysine specific demethylase 1 without inhibition against monoamine oxidases
(MAOs) A and B. Further investigation revealed that compound 8k was active at both recombinant and
cell levels by upregulating the expression of H3K4me1, H3K4me2 and H3K9me2.
Received 23rd January 2014
Accepted 1st March 2014
DOI: 10.1039/c4md00031e
www.rsc.org/medchemcomm
Coumarin-containing molecules have attracted great interest
because of their diverse biological activities, such as anticancer,14
antioxidant, anti-inammatory,15 antimicrobial,16 and enzymatic
Introduction
Histone modications, including methylation, acetylation,
phosphorylation and hydroxylation, play an important role in
the epigenetic control of gene expression. Among these modi-
cations, histone lysine methylation is reversibly regulated by
histone lysine methyltransferases (HLMTs) and demethyla-
ses(HDMs). Lysine specic demethylase 1 (LSD1), the rst
characterized histone lysine demethylase discovered in 2004,
removes the methyl groups from mono-, di-methylated Lys4 and
Lys9 of histone H3 (H3K4 and H3K9) through avin adenine
dinucleotide (FAD) dependent enzymatic oxidation.1 LSD1
could also demethylate p53,2 DNA methyltransferase 1,3 E2F
transcription factor 1 (E2F1),4 and regulate their cellular func-
tions. Besides, the downregulation of LSD1 expression or inhi-
bition of its activity can inhibit cancer progression.5–7 Hence
LSD1 has been considered as an ideal target for the treatment of
cancer. LSD1 is a member of the monoamine oxidase (MAO)
family, which shows homology with monoamine oxidases
(MAOs) A and B (17.6% identity). As reported, MAO inhibitors
(Fig. 1), such as tranylcypromine (2-PCPA), phenelzine and
pargyline, have been evaluated as inhibitors of LSD1.8 However,
more novel LSD1 inhibitors have rarely been studied.9–13
inhibition.17,18 Particularly, some coumarins were described as
monoamine oxidase inhibitors.17 In our previous work, we repor-
ted the synthesis and biological activities of a series of 1,2,3-tri-
azole–dithiocarbamate hybrids (Fig. 2, I). Several compounds
showed an excellent broad spectrum of anticancer activity and
good anti-LSD1 activities.19–21 The preliminary structure–activity
relationship (SAR) studies revealed that the tert-butyloxycarbonyl
group attached to the piperazine ring and only one carbon length
between the triazole ring and the phenyl ring were critical for their
inhibitory activity. So in this study, these two biologically impor-
tant groups are retained. Another intriguing nding was that
substituents on the phenyl ring displayed marked impact on its
anti-LSD1 activity. In continuation with our efforts toward the
discovery of novel anti-LSD1 agents,21 and inspired by the signi-
cant activities of coumarins against MAO,17 we herein design novel
coumarin–1,2,3-triazole–dithiocarbamate hybrids by introducing
a coumarin scaffold and further evaluate their anti-LSD1 activity.
Results and discussion
The synthetic routes to coumarin–1,2,3-triazole–dithiocarba-
mate hybrids 8a–l and 9a–b are outlined in Schemes 1–3. Key
aNew Drug Research & Development Center, School of Pharmaceutical Sciences,
Zhengzhou University, No. 100, Avenue Kexue, Zhengzhou 450001, P.R. China.
E-mail: liuhm@zzu.edu.cn; Fax: +86 371 67781739; Tel: +86 371 67781739
bSchool of Pharmacy, Xinxiang Medical University, Jinsui Road, Xinxiang 453003, P.R.
China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00031e
‡ These authors contributed equally.
Fig. 1 MAO inhibitors that inhibit LSD1.
650 | Med. Chem. Commun., 2014, 5, 650–654
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Fig. 2 Designed structures of coumarin–1,2,3-triazole–dithiocarbamate hybrids.
Scheme 1 Synthesis of the azides (4a–k and 7a–b). Reagents and conditions: (a) ethyl 4-chloroacetoacetate, 70% H2SO4, 0 ꢀC; (b) NaN3, CH3CN
or acetone–H2O; (c) CH3CH2COONa, (CH3CH2CO)2O, Et3N, reflux; (d) NBS, AIBN, CCl4, reflux.
Scheme
2 Synthesis of the coumarin–1,2,3-triazole–dithiocarbamate hybrids 8a–k and 9a–b. Reagents and conditions: (a) CS2,
Na3PO4$12H2O, propargyl bromide, acetone, rt; (b) CuSO4$5H2O, sodium ascorbate, THF–H2O (1/1), rt.
Scheme 3 Synthesis of the coumarin–1,2,3-triazole–dithiocarbamate hybrid (8l). Reagents and conditions: (a) DMF, K2CO3, CH3I, 80 ꢀC.
intermediate 2 was efficiently prepared by following our azides through the Huisgen 1,3-dipolar cycloaddition.
previous described method.19 Compounds 4a–k were obtained Compound 8l was synthesized through methylation of 8k in the
by reaction of sodium azide with 3a–k that were synthesized presence of K2CO3.
from phenols and ethyl 4-chloroacetoacetate by using Pechman
In order to determine the inhibitory activity of the synthe-
condensation conditions. Condensation of substituted salicy- sized compounds against LSD1, we generated LSD1 recombi-
laldehyde with propanoic anhydride in reuxing propionic nant expressing vector containing human LSD1 cDNA. The
anhydride gave compounds 5a–b in modest yield.22 Compounds expression of recombinant LSD1 was then induced in Escher-
7a–b were synthesized from compounds 6a–b employing ichia coli (E. coli) and puried according to the reported
similar conditions for the synthesis of compounds 4a–k. method.24 The demethylase activity of the recombinant LSD1
Compounds 6a–b were generated from the AIBN mediated was further determined by a uorescence-based method, using
bromination of 5a and 5b with NBS.23 Finally, compounds 8a–k synthesized H3K4me2 as a substrate.25 The emission wave-
and 9a–b were obtained from alkyne 2 and corresponding length for LSD1 inhibitor screening was 590 nm and the
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Fig. 3 Fluorescence scanning of compound 8k at the 530 nm excitation wavelength.
excitation wavelength was 530 nm. To eliminate the possible were also more potent than 2-PCPA. The substituents on
artifacts caused by the uorescent nature of these compounds, coumarins had a profound effect on the LSD1 inhibitory
we did an experiment about the uorescence scanning of activity. Specically, the incorporation of chloro and methyl
compound 8k. As shown in Fig. 3, there was no uorescence groups into the 7-position of the coumarin nucleus (8a and 8f)
absorption at around 590 nm (the detection wavelength), indi- showed improved inhibition against LSD1 with the IC50 values
cating that the uorescent nature of compound 8k had no effect in the nanomolar range. By contrast, compounds 8b and 8g with
towards its uorescence absorption at around 590 nm.
chloro atoms and methyl atoms on the 6-position of coumarin
In our previous work,21 the preliminary SAR studies revealed showed no inhibitory activity. Compared with 8h, compound 8i,
that one carbon length between the triazole ring and the phenyl with the 5,7-dihydroxy group represented excellent inhibitory
ring was optimal. So the coumarin hybrids reported were activity towards LSD1 (IC50 ¼ 3.00 mM). Besides, compounds 8d
inactive against LSD1, and we retained the biologically impor- and 8k having a triazolyl group attached to the 4-position of the
tant group in this study. All the compounds synthesized were coumarin nucleus had great inhibitory activity towards LSD1
examined for their in vitro inhibitory effect on the LSD1 activity with the IC50 values of 10.33 and 0.39 mM, respectively. While for
and the results are summarized in Table 1. 2-PCPA was chosen compounds 9a and 9b, the activity was totally lost.
as a positive control. As shown in Table 1, most of the synthe-
As LSD1 belongs to the monoamine oxidase family, in order
sized compounds exhibited moderate to excellent inhibitory to evaluate the selectivity of the inhibitors, inhibitory effects of
activity towards LSD1 with IC50 values ranging from 0.39 to compound 8k to MAO-A and MAO-B were investigated, and 2-
102.56 mM. Among them, compound 8k showed the most potent PCPA was chosen as a positive control. As shown in Table 2,
activity against LSD1 (IC50 ¼ 0.39 mM), which was 74-fold more compound 8k had no inhibitory effects on MAO-A and MAO-B,
potent than 2-PCPA. Moreover, compounds 8a, 8d–8f and 8i–8l while compound 8k showed potent inhibition with the IC50
value of 0.39 ꢁ 0.15 mM (74-fold more potent than that of 2-
PCPA). The ndings indicated the high selectivity of compound
Table 1 Preliminary in vitro inhibitory activities of compounds 8a–l,
9a–b (IC50) toward LSD1
8k on LSD1 in vitro. Besides, the reversibility was also evaluated
with dilution assays and dialysis experiments.21 As shown in
Fig. 4, the results indicated the reversibility of compound 8k,
compared to 2-PCPA.
Comp.
R1
R2
LSD1 (mM)
To further evaluate the cell level LSD1 inhibitory effect,
human gastric cancer cell line MGC-803 histone was extracted
and subjected to western blot analysis with the treatment of
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
7-Cl
6-Cl
7-F
H
7-NH2
7-CH3
6-CH3
5-CH3,7-OH
5,7-diOH
7,8-diOH
7-OH
7-OCH3
—
—
—
—
—
—
—
—
—
—
—
—
—
H
0.67 ꢁ 0.29
>125
84.2 ꢁ 2.47
10.33 ꢁ 1.09
0.53 ꢁ 0.11
0.71 ꢁ 0.31
>125
Table 2 In vitro inhibitory activities of compound 8k to LSD1 and
MAO-A, and MAO-B
>125
3.00 ꢁ 1.32
0.83 ꢁ 0.23
0.39 ꢁ 0.15
0.81 ꢁ 0.40
>125
IC50 (mM)
Compounds
LSD1
MAO-A
MAO-B
9a
9b
2-PCPA
—
—
7-OH
—
102.56 ꢁ 5.23 8k
0.39 ꢁ 0.15
>1250
10.63 ꢁ 1.02
>1250
5.9 ꢁ 0.85
28.73 ꢁ 1.21 2-PCPA
28.73 ꢁ 1.21
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Fig. 4 The reversibility of compound 8k to the LSD1 activity was determined by dilution assays (A) and dialysis experiments (B).
Fig. 5 Histone methylation in MGC-803 cells after treatment with compound 8k for 48 h. (A) Expression levels of H3K4me1, H3K4me2,
H3K4me3, and H3K9me2 were determined by western blot; (B) densitometry quantitation of H3K4me1 with the indicated treatment; (C)
densitometry quantitation of H3K4me2 with the indicated treatment. (D) Densitometry quantitation of H3K9me2 with the indicated treatment.
Total amounts of histone 3 (H3) were used as loading controls.
compound 8k. As shown in Fig. 5(A–D), an elevated expression Teachers' Fund for Doctor Stations, Ministry of Education
of H3K4me1/2 and H3K9me2 could be found, which suggested (Project no. 20114101120013).
that the activity of LSD1 may be inhibited by compound 8k. But
no obvious change of H3K4me3 can be observed, which illus-
trated the selectivity of compound 8k. Meanwhile, the total
amount of histone 3 was not changed. The results strongly
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