Dual inhibitors of LSD1 and spermine oxidase

Article abstract of DOI:10.1039/c8md00610e

We have previously described the synthesis and evaluation of 3,5-diamino-1,2,4-triazole analogues as inhibitors of the flavin-dependent histone demethylase LSD1. These compounds are potent inhibitors of LSD1 without activity against monoamine oxidases A and B, and promote the elevation of H3K4me2 levels in tumor cells in vitro. We now report that the cytotoxicity of these analogues in pancreatic tumor cells correlates with the overexpression of LSD1 in each tumor type. In addition, we show that a subset of these 3,5-diamino-1,2,4-triazole analogues inhibit a related flavin-dependent oxidase, the polyamine catabolic enzyme spermine oxidase (SMOX) in vitro.

Full text of DOI:10.1039/c8md00610e

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M. Dunworth, T. Murray-Stewart, Y. Peterson, P. Burger, J. Kirkpatrick, H. Chen, R. Casero and P. M.
Woster, Med. Chem. Commun., 2019, DOI: 10.1039/C8MD00610E.
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DOI: 10.1039/C8MD00610E
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ARTICLE
Dual Inhibitors of LSD1 and Spermine Oxidase
Steven Holshouser,a Matthew Dunworth,b Tracy Murray Stewart,b Yuri K. Peterson,a Pieter Burger,a
Joy Kirkpatrick,a Huan-Huan Chen,a Robert A. Casero, Jr. *b§ and Patrick M. Woster *a§
Received 00th January 20xx,
Accepted 00th January 20xx
We have previously described the synthesis and evaluation of 3,5-diamino-1,2,4-triazole analogues as inhibitors of the flavin-
dependent histone demethylase LSD1. These compounds are potent inhibitors of LSD1 without activity against monoamine
oxidases A and B, and promote the elevation of H3K4me2 levels in tumor cells in vitro. We now report that the cytotoxicity
of these analogues in pancreatic tumor cells correlates with the overexpression of LSD1 in each tumor type. In addition, we
show that a subset of these 3,5-diamino-1,2,4-triazole analogues inhibit a related flavin-dependent oxidase, the polyamine
catabolic enzyme spermine oxidase (SMOX) in vitro.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Lysine and arginine residues on nucleosomal histone protein tails
undergo reversible mono-, di- and, in the case of lysine,
trimethylation that serves to regulate gene expression. Unlike
histone acetylation, which activates gene transcription, histone
methylation can either activate or silence gene expression,
depending on the specific chromatin mark involved. The primary
function of the flavin-dependent amine oxidase lysine-specific
demethylase 1, (LSD1, also known as KDM1A), is to remove
methyl groups from the activating chromatin marks monomethyl
histone 3 lysine 4 (H3K4me) and dimethyl histone 3 lysine 4
(H3K4me2). LSD1 is also known to demethylate lysine 370 of
the tumor suppressor p53, and has been shown to play a
regulatory role in a number of cancer and non-cancer disease
states.1 Over-expression of LSD1 has been observed in a variety
of tumor cell lines, and promotes the aberrant silencing of tumor
suppressor genes. For these reasons, LSD1 is regarded as an
attractive target for therapeutic intervention. A number of LSD1
inhibitors have been described (Figure 1), including oligoamines
such as verlindamycin 12-5 and related isosteric ureas and
thioureas,5, 6 tranylcypromine-based irreversible inhibitors such
as GSK2879552 (2)7 and ORY-1001 (3),8-10 reversible
benzohydrazide inhibitors such as SP-2509 (4)10 and triazole-
based reversible inhibitors such as 5.11 In addition,
dithiocarbamate-urea hybrid LSD1 inactivators such as 6,12 and
linear13-16 and cyclic17, 18 peptide inhibitors have been reported.
The most potent peptide inhibitor to date is 7 (LSD1 IC50 = 107
nM). Compounds 2, 3 and 4 are currently being evaluated in
human clinical trials.
Figure 1. Structures of known LSD1 inhibitors 1-7.
Our group previously described a series of 3,5-diamino-1,2,4-
triazoles related to 5 that are effective reversible inhibitors of
LSD1. Triazole 5, the most potent analogue in the initial series,
was shown to inhibit LSD1 with an IC50 of 1.2 M, and a Ki of
2.2 M.11 Compound 5 produced a cell type-specific cytotoxicity
in a panel of 5 tumor cell lines, and effectively increased cellular
levels of H3K4me2. Importantly, 5 did not inhibit monoamine
oxidases A and B at concentrations up to 100 M.
a. Department of Drug Discovery and Biomedical Sciences, Medical University of
South Carolina, 70 President St., Charleston, SC 29425, USA.
bSidney Kimmel Comprehensive Cancer Center, 1650 Orleans St. Room 551 Johns
Hopkins School of Medicine, Baltimore, MD 21287.
§ Co-corresponding authors.
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DOI: 10.1039/C8MD00610E
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ARTICLE
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Table 1. Inhibition of LSD1/CoREST at 10 M by compounds 5a, 14-43, 5b and 46-66. Tranylcypromine (TCP) and the known spermine oxidase inhibitor MDL 72,527 were used as
View Article Online
DOI: 10.1039/C8MD00610E
positive and negative controls, respectively. Where IC50 values are reported, they are derived from a curev with data points collected in triplicate, and which varied by 10% or less in
each case.
% Inhibition
Cmpd.
R1
R2
R3
R4
R5
X
of LSD1 (10
µM)
TCP
--
--
--
--
--
--
--
--
--
--
--
--
68.1 + 1.9
0.0
MDL 72527
(IC50 >100µM)
84.9 + 4.7
(IC50 1.19 µM)
38.6 ± 5.6
59.3 ± 6.1
56.3 ± 3.2
44.6 ± 5.1
58.2 + 8.8
67.9 + 6.5
44.9 + 4.6
Not
5a
H
H
H
H
H
Cl
14
15
16
17
18
19
20
H
H
H
H
H
CH3O
H
CH3
CF3
CF3O
CH3
H
H
t-butyl
H
H
H
CH3
H
H
CH3
CF3
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
isopropyl
H
H
H
H
H
21
H
H
CF3S
H
H
Cl
determined
(insoluble)
Not
22
23
24
Br
CF3
H
H
H
H
Br
Br
H
H
H
H
H
H
Cl
Cl
Cl
determined
(insoluble)
67.8 + 5.4
Not
CH3S
determined
(insoluble)
84.7 + 3.2
63.3 + 8.6
80.6 + 1.9
74.2 + 5.2
65.8 + 6.4
95.3 + 8.8
(IC50 0.23 µM)
92.9 + 12.8
(IC50 0.19 µM)
66.8 + 9.3
77.7 + 7.0
67.6 + 4.6
76.4 + 4.7
67.5 + 3.4
68.3 + 2.6
64.4 + 6.4
55.5 + 3.8
25
26
27
28
29
CH3O
H
CH3
CH3
H
H
CH3O
CH3
H
CH3
H
H
H
CH3O
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
-O-CH2-O-
30
31
-CH=CH-CH=CH-
H
H
H
H
H
Cl
Cl
H
CH=CH-CH=CH-
phenyl
32
33
34
35
36
37
38
39
H
H
H
H
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
ethoxy
CH3
H
F
CH3O
H
-NH-CH=CH-
H
H
H
CH3O
-CH2-CH2-CH2-
H
F
(Et)2-N
H
H
F
H
H
3-methoxy-
phenethyl
40
H
H
H
H
Cl
82.2 + 7.8
Not
41
Cl
H
Cl
H
H
Cl
determined
(insoluble)
51.3 ± 3.17
56.0 + 3.6
80.2 ± 2.6
73.8 + 3.5
58.4 ± 0.23
61.1 ± 3.56
5b
46
47
48
49
50
H
H
H
CH3O
H
CH3O
H
CH3
F
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-CH=CH-CH=CH-
ethoxy
F
H
H
H
(Et)2N
H
H
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51
52
53
54
55
56
57
58
59
60
61
62
H
CH3O
H
H
CH3
H
H
H
CH3O
CH3
CH3O
H
t-butyl
H
CH3O
CH3
H
CH3
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
56.6 +V5ie.3w Article Online
isopropyl
56.4 ± 2.85
DOI: 10.1039/C8MD00610E
H
H
CF3
H
ethoxy
H
CH3
H
57.9 ± 9.13
54.8 ± 3.39
58.3 ± 2.28
80.2 ± 0.11
73.8 + 7.82
57.0 ± 3.22
52.2 ± 8.17
52.1 ± 3.5
Br
CH3S
CH3
H
H
CH=CH-CH=CH-
H
H
H
H
phenyl
F
H
H
52.1 ± 8.17
54.6 ± 1.63
The closely related flavin-dependent amine oxidase known as
To determine whether the chlorine substituent was required for
spermine oxidase (SMOX) was discovered and first inhibitory activity, a novel synthetic route was developed
20
characterized by our group in 2001.19,
SMOX and its (Scheme 2) that allowed formation of compounds featuring the
peroxisomal homologue polyamine oxidase (PAOX) are key
components of the polyamine back-conversion pathway.21
SMOX has recently been implicated as a causative factor in
gastric cancer initiated by infection with Helicobacter pylori.22-
24 H. pylori infection has been shown to induce SMOX in gastric
epithelial cells, which generates hydrogen peroxide leading to
DNA damage and possibly apoptosis.23 Inhibition of SMOX by
the pan polyamine oxidase inhibitor MDL 72,52725 is capable of
reducing these effects. However, MDL 72,527 is an irreversible
inhibitor of SMOX with unacceptably low potency. To date, few
inhibitors of SMOX have been identified,26, 27 and no specific
inhibitors of the enzyme have been discovered. 11
Figure 2. Determination of the IC50 values for inhibition of LSD1/CoREST by
compounds 30 and 31. Each data point is the average of three determinations
+.SEM.
In the present study, we initiated hit-to-lead optimization
studies intended to reveal new 3,5-diamino-1,2,4-triazole-based
LSD1 inhibitors with improved potency and selectivity
compared to the parent compound 5. Our initial goal was to
identify inhibitors selective for LSD1, and with little or no
activity against MAO A and B, SMOX or PAOX. During the
course of our enzyme specificity determinations, we discovered
that a subset of these compounds had significant activity against
SMOX that was superior to the currently used agent MDL
72,527, and that have potential for use as chemopreventive
agents. The synthesis of these molecules and the corresponding
biological evaluation studies are described below.
dechlorinated 3,5-diamino-1,2,4-triazole scaffold, and that
supported the rapid synthesis of analogues. In this way, we were
able to produce the penultimate intermediate in bulk, and rapidly
synthesize new analogues in the final step. Thus, the 1,3-
dihalogenated-2-cyanobenzene 42 was reduced in the presence
of LiAlH4 to yield the corresponding primary amine 43.
Compound 43 was then appended to 12 (see Scheme 1) to afford
intermediate 44, which was cyclized in the presence of hydrazine
to form 45. At this step, intermediate 45 was derivatized in small
portions to complete the addition of a variety of phenols 8a-s,
resulting in the rapid formation of a library of analogues related
to 5b. Unexpectedly, conversion of 45 to the final product 5b
yielded the corresponding dechlorinated product. We postulate
that this effect is due to stabilization of the aromatic ring via
anchimeric assistance that promotes the elimination of the
chlorine prior to attack of the phenol on fluorine. Coupling of 45
with phenols 8a-s then afforded the dechlorinated analogue 5b
and the previously unreported 3,5-diaminotriazoles analogues
46-62, shown in Table 1.
Results
Synthetic procedures
To generate a library of analogues related to the parent 3,5-
diamino-1,2,4-triazole 5, we used our previously published
synthetic route,11 as shown in Scheme 1. Condensation of
phenols 8a-ac and 2-cyano-3-chlorofluorobenzene 9 (K2CO3,
microwave, 190oC, 6 min) resulted in the phenoxyphenyl
intermediates 10a-ac. The cyano group in compounds 10a-ac
was then reduced (LiAlH4) to afford primary amine 11a-ac,
which was reacted with 12 (microwave, 40oC, 5 min) to yield
13a-ac. Intermediates 13a-ac were then treated with hydrazine
(microwave, 90oC, 10 min) to produce the desired 3,5-
diaminotriazole 5a and the previously unreported 3,5-
diaminotriazole analogues 14-41 (Table 1).
Enzyme inhibition studies
Compounds 5a, 5b, 14-41 and 46-62 were evaluated for the
ability to inhibit recombinant LSD1/CoREST using
a
commercially available assay kit (#700120, Cayman Chemical,
Ann Arbor, MI). The results of this assay are summarized in
Table 1. The most potent analogues, 30 and 31, were found to
have IC50 values of 0.23 and 0.19 M, respectively (Figure 2).
4 | MedChemComm., 2018, 00, 1-3
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ARTICLE
LSD1 QSAR
from the polyamine metabolic pathway, SMOX and N1-
View Article Online
29
DOI: 10.1039/C8MD00610E
acetylpolyamine oxidase (PAOX), as previously described.
The most effective SMOX inhibitors were compounds 30, 31, 55
and 61 (Table 2), with IC50 values of 147.9, 162.7, 120.96 and
25.71 µM, respectively. By contrast, the parent analogue 5
possessed an IC50 value against SMOX of 177.9 µM.
Importantly, compound 61 is significantly more potent against
SMOX than the known polyamine oxidase inhibitor MDL72527
(IC50 = 89.7 M, Table 2), retains good activity against LSD1
(Table 1), and is thus the most potent SMOX inhibitor reported
to date. It is noteworthy that the naphthalene-containing
compounds 30, 31 and 60 are significantly less potent against
SMOX, suggesting that the positioning of the aromatic rings and
their ability to rotate freely are important in terms of binding to
the SMOX active site. Additional experimentation will be
required to test this hypothesis, and to develop structure/activity
parameters for SMOX inhibition. None of the analogues tested
had inhibitory activity against PAOX.
Using an integrated feature of Molecular Operating Environment
(MOE. Chemical Computing Group, Montreal), computational
methods were employed to generate a traditional QSAR model
to predict changes in substituents that would increase compound
efficacy. New and previously reported small molecules known
to be reversible inhibitors of LSD1 were used to build a QSAR
model based on positive and negative descriptors within a
defined chemical space. This model was developed using
chemical descriptors such as size, weight, ClogP, aromaticity,
hydrogen donors/acceptors, molar refractivity, number of rigid
bonds and radius of substituents versus potency towards LSD1.
Some specific computational positive descriptors that were used
were SlogP_VSAO, opr_brigid, weinerPol and zagrab. Negative
descriptors were vsa_acc, opr_nrot, SMR_VSA4 and radius.
Computational analysis was then performed using compounds
developed in our laboratories as well as compounds described in
the literature, as long as they fit our selection criteria and had
defined IC50 values toward LSD1. Results were plotted as a
linear XY plot having experimentally derived in-vitro pIC50
values along the x-axis versus computationally predicted pIC50
LSD1 and SMOX expression in pancreatic tumor cell lines and
response to inhibition
In order to determine whether there was a relationship between
LSD1 or SMOX expression levels in pancreatic tumor cell lines
and cytotoxicity, we determined the expression level of these
enzymes in the BxPc-3, Miapaca2 and PANC-1 pancreatic tumor
cell lines, and in the CRL7869 human intestinal cell line as a
reference standard, by western blotting (Figure 4). All cell lines
were purchased from ATCC (Manassas, VA). There were no
significant differences in LSD1 expression among the three
tumor cell lines tested (data not shown). However, the levels of
SMOX in the BxPc-3 pancreatic tumor cell line were 4.6-fold
higher than in the MiaPaca-2, PANC-1 or CRL7869 cell lines.
(Figure 4A). We next obtained unidentified pancreatic tissue
slices from malignant pancreatic and non-cancerous patients
from our in-house tissue bank, and measured SMOX levels by
immunohistochemistry (Figure 4B). The levels of SMOX in the
pancreatic tumor tissue (left) was significantly higher than that
in the normal pancreatic tissue (right). Using gemcitabine (a
nucleoside-based chain terminator that is a first-line treatment
for pancreatic cancer), the potent LSD1 inhibitors 5 and 30, and
the two most potent SMOX inhibitors 55 and 61, we then
determined the IC50 values for growth inhibition in the Miapaca2
and BxPc-3 cell lines. In the Miapaca cell line, 5 and gemcitabine
were essentially equipotent, exhibiting IC50 values of 2.66 and
1.86 M, respectively, while compounds 30, 55 and 61 had IC50
values between 30 and 200 M (Figure 4C). By contrast, in the
BxPc-3 cell line, gemcitabine exhibited and IC50 of 5.71 M, but
growth inhibition plateaued at 30% inhibition (Figure 4D). Our
most potent SMOX inhibitor, 61, inhibited cell growth with an
IC50 of 61.5 M. The remaining compounds, including the potent
LSD1 inhibitors 5 and 30 and the weak SMOX inhibitor 55, had
IC50 values well above 100 M. (Figure 4D). These data suggest
that cytotoxicity of these 3,5-diamino-1,2,4-triazoles in the
MiaPaCa2 cell line may be mediated by an as yet unknown
mechanism, and may correspond with the expression level of
SMOX in the BxPc-3 cell line. Additional compounds and in
8
7
6
pIC50
(predicted)
5
4
3
3
4
5
6
7
8
pIC50
(experimental)
Figure 3. Predictive QSAR model for LSD1 inhibitor library. New and previously
reported small molecule competitive inhibitor IC50 values were entered into MOE
to develop a predictive QSAR model. The model used various chemical descriptors
vs IC50 values to allow the prediction of future inhibitors.
values on the y-axis (Figure 3). The resulting graph revealed an
R2 value of 0.71 and a cross-validated predictive strength
(XPRED) of 0.798. Efforts were made to increase the R2 value
to greater than 0.80, but there was insufficient inhibition data
among the small molecules that fall within the parameters for
specific inhibition of LSD1. This model will be used for the
design of second-generation 3,5-diamino-1,2,4-triazoles related
to 5.
Inhibition of related flavin-dependent oxidase enzymes
In order to assess the specificity/selectivity of 5a, 5b, 14-41 and 46-
68 for LSD1, selected analogues were evaluated for the ability to
inhibit monoamine oxidases (MAO) A and B using a commercial
assay kit (#V1401, Promega Biosciences, Madison, WI). The results
of this assay are displayed in Table 2. TCP inhibited both isoforms
of MAO within the range of previously published IC50 values.28
As a further monitor of specificity for LSD1, selected
compounds were evaluated for the ability to inhibit two enzymes
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Table 2. Inhibition of LSD1/CoREST, MAO-A, MAO-B, SMOX and PAOX at 10 M by compounds 5a, 30, 31, 5b and 52-62. Tranylcypromine (TCP) and the known spermine oxidase
DOI: 10.1039/C8MD00610E
inhibitor MDL 72,527 were used as controls. Where IC50 values are reported, they are derived from a curev with data points collected in triplicate, and which varied by 10% or less
in each case.
%
%
%
%
%
Inhibition
of LSD1 (10
M)
Inhibition
of MAO-A
(10 M)
Inhibition
of MAO-B
(10 M)
Inhibition
of SMOX
(10 M)
Inhibition
of PAOX
(10 M)
Cmpd.
TCP
R1
R2
R3
R4
R5
Y
X
(IC50 = 4.2
M)
0.0
(IC50
(IC50= 5.8
M)
0.0
(IC50
>200M)
--
--
--
--
--
--
--
--
--
--
--
--
--
--
68.1 + 2.0
0
0
0
4.8 + 9.6
(IC50 89.7
µM)
MDL
72527
32.1 +
13.2
>200M)
28.0 +
14.2
(IC50 177.9
µM)
19.0 +
10.9
(IC50 147.9
µM)
84.9 + 4.7
(IC50 1.19
M)
0.0
(IC50
>200M)
0.0
(IC50
>200M)
5a
30
H
H
H
H
H
H
H
H
O
O
Cl
Cl
0
95.3 + 8.8
(IC50 0.19
µM)
0.0
(IC50
>200M)
7.12
(IC50
>200M)
-CH=CH-CH=CH-
ND
ND
92.9 + 12.8
(IC50 0.25
µM)
0.0
(IC50
>200M)
0.0
(IC50
>200M)
23.6 + 3.9
(IC50 162.7
µM)
31
5b
52
53
54
H
CH=CH-CH=CH-
H
H
H
H
H
H
H
O
O
O
O
O
Cl
H
H
H
H
0
0
(IC50 >200
M)
IC50 = 50.2
± 1.0 M
18.77 ±
4.77
H
H
H
H
H
51.3 ± 3.2
56.4 ± 2.9
(IC50 >200
M)
13.9 + 3.7
(IC50 >200
M)
0
(IC50 = 55.5
± 11.3
49.73 ±
9.78
isopropyl
CH3
H
(IC50 >200
M)
0
0
57.9 ±
9.139
(IC50 = 61.5
± 1.1
32.34 ±
7.46
H
H
H
t-butyl
H
(IC50 >200
M)
(IC50 >200
M)
0
0
IC50 = 91.4
± 11.8 M)
83.41 ±
3.15
CH3
CH3
54.8 ± 3.4
58.3 ± 2.3
(IC50 >200
M)
0 + 13.49
(IC50
120.96
µM)
(IC50 >200
M)
0
(IC50 = 3.0 ±
1.2 M)
90.08 ±
2.34
55
CF3
H
Br
H
H
O
H
(IC50 >200
M)
0
0
(IC50 >200
M)
IC50 = 38.4
± 5.6
31.66 ±
7.51
56
58
59
60
H
H
H
CH3S
H
H
CH3O
H
H
H
H
H
O
O
O
O
H
H
H
H
80.2 ± 0.1
57.0 ± 3.2
52.2 ± 8.2
52.1 ± 3.5
(IC50 >200
M)
0.0
(IC50
>200M
0
(IC50
>200M
0
0
17.06 ±
27.61
CH3O
CH3
(IC50 >200
M)
(IC50 >200
M)
0
0
68.61 ±
5.43
CH3
H
H
(IC50 >200
M)
(IC50 >200
M)
0
0
IC50 = 79.2
± 1.1 M)
81.58 ±
0.96
CH=CH-CH=CH-
H
(IC50 >200
M)
(IC50 >200
M)
25.7 ±
11.4
(IC50 25.7
µM)
0
IC50 = 50.0
± 4.2 M)
26.35 ±
9.80
61
62
H
H
H
H
phenyl
F
H
H
H
H
O
O
H
H
52.11 ± 8.2
54.58 ± 1.6
(IC50 >200
M)
(IC50
=
0
0
40.7 ±
6.14
89.33 ±
1.16 M)
(IC50 >200
M)
(IC50 >200
M)
This journal is © The Royal Society of Chemistry 20xx
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View Article Online
DOI: 10.1039/C8MD00610E
A
B
SMOX Expression
6
4
2
0
***
BXPC3
Miapaca 2
FOLD
INCREASE
PANC-1
CRL7869
3
2
1
9
-
6
C
a
c
C
8
7
P
N
a
X
L
A
p
B
R
a
P
i
C
M
Cell Type
C
D
BxPC-3
Miapaca-2
100
50
0
100
50
0
% cell
viability
% cell
viability
5a
gemcitabine
5a
30
55
61
gemcitabine
30
55
61
0.1
1
10
100
1
10
100
[I], uM
[I], uM
Figure 4. Growth inhibition by SMOX in the Miapaca2 and BxPC-3 pancreatic tumor cell lines is related to the degree of overexpression of SMOX. Panel A: Expression of SMOX
in 3 pancreatic tumor cell lines and in CRL7869 intestinal epithelial cells as determined by western blot. Each data point is the average of 3 determinations + SEM; Panel B:
Immunohistochemical staining of human pancreatic tissue slices from cancerous (left) and non-cancerous patients; Panel C: Response of Miapaca2 pancreatic tumor cells to 5,
gemcitabine, 30, 55 and 61; Panel C: Response of BxPC-3 pancreatic tumor cells to 5, gemcitabine, 30, 55 and 61. For panels C and D, each data point is the average of three
determinations + SEM.
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ARTICLE
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vitro assays will be performed to verify this hypothesis, and and compound 61 is 3.5-fold more potent against the purified
View Article Online
DOI: 10.1039/C8MD00610E
reported in a subsequent publication.
enzyme than MDL72527. As such, 61 is the most potent SMOX
inhibitor reported to date. In addition, the SMOX inhibitors
described in Table 2 are the only known SMOX inhibitors that
do not also inhibit PAOX. Thus, these compounds can serve as
chemical tools to study the effects of differential inhibition of
SMOX. We postulate that dual inhibitors of LSD1 and SMOX
could be useful, since they produce both antitumor and
chemopreventive activities. The synthesis and evaluation of
analogues in this class are an ongoing concern in our
laboratories.
Discussion
Based on data gathered for the compounds described in this
paper, it is possible to postulate a number of structure/function
relationships within the 3,5-diamino-1,2,4-triazole series of
LSD1 inhibitors. There was a decrease in LSD1 inhibitory
activity when single substituents were appended to the phenoxy
ring, as in compounds 14-41, with the exception of an ortho
methoxy group or an ortho ethoxy para methyl substitution
(compounds 25 and 33, respectively), where activity was
Experimental
retained. Conversion of the phenoxy substituent to a naphthyloxy Synthesis
moiety resulted in an increase in inhibitory activity, which was
All reagents and dry solvents were purchased from Aldrich
slightly larger for the 1-naphthyloxy analogue 30 (IC50 0.19 M)
as compared to the 2-naphthyloxy compound 31 (IC50 0.25 M).
Compounds 30 and 31 are thus roughly 5 times more active
against LSD1 than the parent compound 5a. This indicates that
the active site pocket surrounding the bound FAD cofactor can
accommodate additional bulk, and that the orientation of the
second aromatic moiety is also important. This may also explain
why activity is retained in compound 40, which bears a 3-
methoxyphenethyl substituent on the phenoxy ring that could
assume the same position as the naphthylene ring system.
Chemical Co. (Milwaukee, WI), Sigma Chemical Co. (St. Louis,
MO), VWR (Radnor, PA) or Fisher Scientific (Chicago, IL) and
were used without further purification except as noted below.
Triethylamine was distilled from potassium hydroxide and
stored in a nitrogen atmosphere. Dry methanol, ethyl acetate,
tetrahydrofuran, dimethyl formamide and hexane were prepared
using a Glass Contour Solvent Purification System (Pure Process
Technology, LLC, Nashua, NH). Preparative scale
chromatographic procedures were carried out using
a
CombiFlash Rf200 chromatography system (Teledyne-Isco,
Lincoln, NE) fitted with silica gel 60 cartridges (230-440 mesh).
Thin layer chromatography was conducted on Merck precoated
silica gel 60 F-254. Ion exchange chromatography was
conducted on Dowex1X8-200 anion exchange resin.
Removal of the chlorine substituent from the molecule resulted
in a reduction in LSD1 inhibitory activity across the board
(compare compounds 5a and 5b, compounds 30 and 47,
compounds 33 and 48 and compounds 31 and 60). The most
effective compounds in the dechloro series were 47 (dechloro
30), 56 and 57 (dechloro 33). In addition, many of the dechloro
analogues were found to have modest activity against MAO-A
and MAO-B. Thus, inclusion of the chlorine in this scaffold is
required for optimal activity. Additional analogues in this and
other series are now being made to refine our structure/activity
model.
All 1H- and 13C-NMR spectra were recorded on a Bruker Avance
400 MHz spectrometer, and all chemical shifts are reported as 
values referenced to TMS or DSS. Splitting patterns are
indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet;
br, broad peak. In all cases, 1H-NMR, 13C-NMR and MS spectra
were consistent with assigned structures. Mass spectra were
recorded by LC/MS on a Waters autopurification liquid
chromatography with a model 3100 mass spectrometer detector.
Prior to biological testing procedures, all compounds were
determined to be >95% pure by UPLC chromatography (95%
H O/5% acetonitrile to 20% H O/80% acetonitrile over 10
It was recently reported that the increased incidence of gastric
cancer in patients infected with Helicobacter pylori is mediated
by reactive oxygen species (ROS) produced from increased
expression of spermine oxidase (SMOX). In addition, the
polyamine oxidase inhibitor MDL72527 was able to protect
against ROS-mediated damage, potentially leading to a reduced
risk of gastric cancer.22, 23 It has now been demonstrated that pre-
treatment with compound 5a can effectively prevent the
formation of ROS in RAW 264.7 macrophages that have been
infected with H. pylori (unpublished observations). In these
studies, and as previously reported,11 5a is not cytotoxic in a
number of cell lines. These data suggest that 5 may be an
effective inhibitor of SMOX in vitro, and led us to test whether
analogues of 5a were inhibitors of purified SMOX. As shown in
2
2
minutes) using a Waters Acquity H-series ultrahigh-performance
liquid chromatograph fitted with a C18 reversed-phase column
(Acquity UPLC BEH C18 1.7 M, 2.1 X 50 mm). Compounds 5a
and 14-41 were synthesized according to the general
procedures11 described below. Compounds 5a and 14-29 have
been previously reported.11
General procedure for the synthesis of substituted 2-chloro-6-
aryloxybenzonitriles
Table 2 above, compounds 5a, 30, 31, 55 and 61 were all able to Synthesis of 2-chloro-6-phenyloxybenzonitrile, 10a. To a 20 mL
inhibit SMOX with IC50 values between 25.7 and 177.9 M. microwave vial containing a magnetic stir bar was added 0.78 g
While these inhibitory constants are modest, they compare (5.0 mmol) of 2-fluoro-6-chlorobenzonitrile 9, (1.04 g, 7.5
favorably to the known inhibitor MDL72527 (IC50 = 89.7 M), mmol) of K2CO3, 0.52 g (5.5mmol) of phenol 8a and 12.0 mL of
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ARTICLE
anhydrous DMSO. The vial was then sealed and stirred to 11s exactly as described above for compound 5a inV8ie5w%ArtiyclieeOldnlianes
1
DOI: 10.1039/C8MD00610E
distribute the contents evenly. The mixture was then microwaved a white, amorphous solid. H NMR (400 MHz, DMSO) δ 4.49
at 190oC for 6 min at high absorption to insure even heating. The (s, 2H), 6.67-6.80 (dd, 1H), 6.99-7.01 (d, 1H), 7.25 (d, 2H), 7.54-
reaction mixture was poured into a beaker containing 100 mL of 7.59 (m, 3H), 7.77-7.79 (dd, 1H), 7.99-8.01 (dd, 1H), 8.12-8.14
crushed ice to precipitate the product. The aqueous layer was (dd, 1H). UPLC retention time: 14.34 min. MS calculated
extracted with three 50 mL portions of diethyl ether, and the 365.10, found 366.21([M+1]+)
ether layer was washed with 25 mL of saturated NaCl, dried over
anhydrous Na2SO4, filtered, and the ether was removed in vacuo Synthesis of N3-(2-chloro-6-(naphthalen-2-yloxy)benzyl)-1H-
to yield 1.11 g of the desired diaryl ether 10a (97% yield). The 1,2,4-triazole-3,5-diamine, 31. Compound 31 was prepared from
crude product was pure enough to be used in the next reaction 11t exactly as described above for compound 5a in 80% yield as
1
without further purification.
a white powder. H NMR (400 MHz, CD3OD) δ 2.14 (s, 2H),
6.72 (dd, 1H), 6.81-6.86 (dd, 1H), 7.07 (d, 1H), 7.08 (s, 1H), 7.17
(d,1H), 7.32 (m, 3H), 7,51 (dd,1H), 8.19 (dd, 1H). UPLC
retention time: 14.53 min. MS calculated 365.82, found 366.21
([M+1]+).
General procedure for the synthesis of substituted 2-chloro-6-
aryloxybenzylamines
Synthesis of 2-chloro-6-aryloxybenzylamine, 11a. A 1.11 g
portion of 10a (4.8 mmol) was dissolved in 50 mL of anhydrous
diethyl ether, cooled to 0oC in an ice bath and stirred while
bubbling dry argon into the reaction mixture for 10 min. A 14.49
mL portion of 1.0 M LiAlH4 in THF (14.49 mmol) was then
added dropwise with stirring over 20 min. The resulting reaction
mixture was allowed to stir for 2 hrs at 0oC, and then warmed to
room temperature and allowed to stir overnight. The mixture was
cooled to 0oC, and the reaction was quenched by the slow
addition of Na2SO4•10 H2O. When the evolution of gas subsided,
the reaction was stirred for 30 min at room temperature, and the
mixture was filtered through a Celite pad. The filtrate was
concentrated to dryness to yield crude 2-chloro-6-
benzoxybenzylamine 11a. The crude product was pure enough
to be used in the next reaction without further purification.
Synthesis of N3-(2-([1,1'-biphenyl]-4-yloxy)-6-chlorobenzyl)-
1H-1,2,4-triazole-3,5-diamine, 32. Compound 32 was prepared
from 11u exactly as described above for compound 5a in 81%
yield as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 2.59
(s, 2H), 6.77-6.79 (d, 1H), 7.14 (m, 3H), 7.24 (d, 2H), 7.35 (d,
2H), 7.42 (d,2H), 7.54 (d, 2H), 7.59 (d, 2H). UPLC retention
time: 14.55 min. MS calculated 391.1, found 392.26 ([M+1]+)
3
Synthesis
of
N -(2-chloro-6-(2-methoxy-4-
methylphenoxy)benzyl)-1H-1,2,4-triazole-3,5-diamine,
33.
Compound 33 was prepared from 11v exactly as described above
for compound 5a in 40% yield as a white, amorphous solid. 1H
NMR (400 MHz, CD3OD) δ 2.40 (s, 3H), 3.75 (s, 3H), 6.54-6.57
(m, 3H), 6.83- 6.88 (d, 1H), 7.06-7.18 (m, 2H). UPLC retention
time 14.7 min. MS calculated 359.11, found 360.29 ([M+1]+)
3
Synthesis of N -(2-((1H-indol-4-yl)oxy)-6-chlorobenzyl)-1H-
General procedure for the synthesis of substituted N3-(2-chloro-6-
aryloxybenzyl)-1H-1,2,4-triazole-3,5-diamines
1,2,4-triazole-3,5-diamine, 34. Compound 34 was prepared from
11w exactly as described above for compound 5a in 72% yield
as a pale yellow solid. H NMR (400 MHz, CDCl3) δ 4.11 (s,
1
Synthesis of N3-(2-chloro-6-phenoxybenzyl)-1H-1,2,4-triazole-
3,5-diamine, 5a. A 0.935 g portion of benzylamine 11a (4.0
mmol) was dissolved in 12 mL of diethyl ether and added to a 20
mL microwave vial equipped with a magnetic stir bar. A 0.702 g
portion of dimethyl cyanodithioiminocarbonate (4.8 mmol) was
added and the vial was sealed. The contents were microwaved at
45oC for 5 min, cooled to room temperature, and the ether was
removed in vacuo to yield the intermediate 13a as a white to pale
yellow solid. A 0.192 g portion of hydrazine hydrate (6.0 mmol)
in 12 mL of dry ethanol was then injected, the vial was stirred to
break up the solid intermediate, and the resulting mixture was
microwaved at 90oC for 10 min at high absorption. The ethanol
was removed in vacuo, and the residue was purified by silica gel
chromatography (9% MeOH in CH2Cl2) to afford 1.07 g of pure
5a (85%) as an off-white, amorphous solid. 1H NMR (400MHz,
CD3OD) δ 4.21 (s, 2H), 6.77-6.80 (dd, 4H), 6.99-7.01 (d, 2H),
7.11-7.15 (t, 4H), 7.20-7.27 (m, 2H), 7.33-7.38 (t, 2H). UPLC
retention time: 12.1 min. MS calculated 359.08, found 316.33
([M+1]+).
2H), 6.46 (s, 1H), 6.85-6.87 (d, 1H), 6.86-6.88 (d, 1H), 7.01-7.07
(m, 2H), 7.24 (d,21H), 7.31-7.33 (d, 1H). UPLC retention time:
14.79 min. MS calculated 354.80, found 355.98 ([M+1]+)
3
Synthesis of N -(2-chloro-6-(4-fluorophenoxy)benzyl)-1H-
1,2,4-triazole-3,5-diamine, 35. Compound 35 was prepared from
11x exactly as described above for compound 5a in 68% yield.
1H NMR (400 MHz, CDCl3) δ 3.97 (s, 2H), 6.52-6.54 (d, 1H),
6.66-6.68 (d, 1H), 6.87-6.88 (d, 1H), 6.90-6.91 (d, 1H), 6.94-
6.96 (d, 1H), 6.98-7.00 (d, 1H), 7.08-7.09 (d, 1H). UPLC
retention time: 12.65 min. MS calculated 333.80, found 334.92
([M+1]+)
3
Synthesis of N -(2-chloro-6-(3,4,5-trimethoxyphenoxy)benzyl)-
1H-1,2,4-triazole-3,5-diamine, 36. Compound 36 was prepared
from 11y exactly as described above for compound 5a in 89%
yield. 1H NMR (400 MHz, CD3OD) δ 3.74 (s, 3H), 3.78 (s, 3H),
3.84 (s, 2H), 6.19 (s, 3H), 6.83-6.85 (d, 1H), 7.04-7.08 (m, 1H),
7.16-7.19 (d, 1H), 7.3-7.33 (d, 2H). UPLC retention time: 21.45
min. MS calculated 405.12, found 406.20 ([M+1]+)
3
Synthesis of N -(2-chloro-6-(naphthalen-1-yloxy)benzyl)-1H-
1,2,4-triazole-3,5-diamine, 30. Compound 30 was prepared from
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3
Synthesis
of
N -(2-chloro-6-((2,3-dihydro-1H-inden-4- through a Celite pad. The filtrate was then conVicewenAtrtricalteeOdnlitnoe
DOI: 10.1039/C8MD00610E
yl)oxy)benzyl)-1H-1,2,4-triazole-3,5-diamine, 37. Compound 37 dryness in vacuo. The crude material was purified on silica (0-
was prepared from 11z exactly as described above for compound 5% MeOH in dichloromethane), to produce the desired 2-chloro-
1
5a in 65 % yield as a white solid. 1H NMR (400 MHz, CDCl3) δ 6-fluorobenzylamine 43 (0.913 g, 89% yield). H NMR (400
2.71 (s, 2H), 2.84-2.87(M,2H), 2.97-3.0 (m, 4H), 6.83-6.85 (d, MHz, CDCl3) δ 4.88 (s, 2H), 7.09 (m, 1H), 7.28 (m, 2H).
1H), 6.92-6.95 (d, 1H), 6.99-7.03 (M, 1H), 7.08 (s, 1H), 7.12-
7.14 (d, 1H), 7.40-7.42 (d, 1H). UPLC retention time: 20.25 min. Synthesis of N -(2-chloro-6-fluorobenzyl)-4H-1,2,4-triazole-
MS calculated 355.83, found 357.23 ([M+1]+)
3
3,5-diamine, 45. A 0.913 g (5.72 mmol) of 2-chloro-6-
fluorobenzylamine 43 was dissolved in 12 mL of diethyl ether
3
Synthesis of N -(2-chloro-6-(3-(diethylamino)phenoxy)benzyl)- and added to a 20 mL microwave vial equipped with a magnetic
1H-1,2,4-triazole-3,5-diamine, 38. Compound 38 was prepared stirring bar. Dimethyl cyanodithioiminocarbonate 12 (1.0 g,
from 11aa exactly as described above for compound 5a in 68% 6.43 mmol)was then added and the vial was sealed. The contents
yield. 1H NMR (400 MHz, CD3OD) δ 1.15-1.18 (t, 6H), 2.98 (s, were microwaved at 45oC for 5 minutes, then allowed to cool to
2H), 3.31-3.36 (m, 4H), 6.12-6.14 (dd, 1H), 6.19-6.20 (m, 1H), room temperature, and the ether was removed in vacuo to yield
6.27-6.29 (dd, 1H), 7.04-7.08 (m,1H), 7.24-7.26 (d, 1H), 7.57- the intermediate 44 as a white solid. Hydrazine hydrate (0.275
7.59 (d, 1H), 8.01 (s, 1H). UPLC retention time: 0.46 min. MS g, 8.58 mmol) in 12 mL of ethanol was then injected into the vial,
calculated 386.16, found 387.55 ([M+1]+)
and the reaction was stirred to break up the intermediate solid.
The dispersed reaction mixture was microwaved at 90oC for 10
3
Synthesis of N -(2-chloro-6-(2,4-difluorophenoxy)benzyl)-1H- min on high absorption. The mixture was allowed to cool to
1,2,4-triazole-3,5-diamine, 39. Compound 39 was prepared from room temperature, and the ethanol was removed in vacuo to yield
11ab exactly as described above for compound 5a in 57% yield the crude product. The crude material was purified on silica
as an off-white, amorphous solid. 1H NMR (400 MHz, CD3OD) (10% MeOH in dichloromethane), to afford 1.27 g of pure 45 as
1
δ 4.07 (s, 2H), 6.63-6.65 (d, 1H), 7.02 (s, 1H), 7.32-7.33 (d,1H), a white, amorphous powder (92% yield). H NMR (400 MHz,
7.58-7.59 (d, 1H), 7.74-7.78 (m, 1H), 8.15-8.17 (d, 1H). UPLC CDCl3) δ 4.32 (s, 2H), 7.20-7.22 (t, 4H), 7.31-7.36 (m, 2H).
retention time: 0.56 min. MS calculated 351.07, found 352.42 UPLC retention time: 0.46 min. MS calculated 241.05, found
([M+1]+)
242.30 ([M+1]+)
3
Synthesis
of
N -(2-chloro-6-(2-(3- General procedure for the synthesis of substituted N3-(6-
methoxyphenethyl)phenoxy)benzyl)-1H-1,2,4-triazole-3,5-
diamine, 40. Compound 40 was prepared from 11ac exactly as
described above for compound 5a in 75% yield as an off-white
solid. 1H NMR (400 MHz, CD3OD) δ 4.43 (s, 2H), 4.84 (s, 2H),
5.02 (s, 2H), 6.55-6.58 (d, 1H), 6.94-6.95 (d, 1H), 6.98-7.01 (d,
1H), 7.04 (s, 1H), 7.06 (d, 2H), 7.09 (s, 1H), 7.10 (d, 1H), 7.11
(s, 1H), 7.13 (d, 1H), 7.18-7.19 (m, 3H), 7.37-7.38 (d, 1H).
UPLC retention time: 20.47 min. MS calculated 449.94, found
450.23 ([M+1]+)
phenyloxybenzyl)-4H-1,2,4-triazole-3,5-diamines
Synthesis of N3-(6-phenyloxybenzyl)-4H-1,2,4-triazole-3,5-
diamine 5b. To a 20 mL microwave vial equipped with a
3
magnetic stir bar was added N -(2-chloro-6-fluorobenzyl)-1H-
1,2,4-triazole-3,5-diamine (1.0 g, 4.14 mmol (0.86 g, 6.21 mmol)
K2CO3, (0.429 g, 4.55 mmol) phenol 8a, and 12 mL of anhydrous
dimethylsulfoxide. The vial was then sealed and stirred to
distribute contents evenly. The reaction was microwaved at
190⁰C for 6 min on high absorption, and the contents were
poured into a beaker containing 100 mL crushed ice. The
product was extracted with three 50 mL portions of diethyl ether,
and the ether layer was washed with 25 mL of saturated NaCl.
The organic layer was dried over Na2SO4, filtered, and the filtrate
was removed in vacuo to provide crude 5b. The crude material
was purified on silica (10% MeOH in dichloromethane), to
3
Synthesis of N -(2-chloro-6-(2,4-dichlorophenoxy)benzyl)-1H-
1,2,4-triazole-3,5-diamine, 41. Compound 41 was prepared from
11ad exactly as described above for compound 5a in 75% yield.
1H NMR (400 MHz, CD3OD) δ 3.75 (s, 2H), 6.52 (s, 1H), 6.56-
6.57 (m, 1H), 6.68-6.71 (d, 1H), 6.81-6.84 (d, 1H), 7.23-7.26 (dd,
2H). UPLC retention time: 21.45 min. MS calculated 384.65,
found 385.73 ([M+1]+)
1
afford pure1.14 g of pure 5b (98% yield) as a white solid. H
NMR (400 MHz, CD3OD) δ 4.61 (s, 2H), 6.80-6.82 (dd, 1H),
7.25-7.33 (d, 2H), 7.27-7.3 (m, 1H), 7.50-7.52 (m, 2H), 7.68-
7.77 (d, 1H), 7.95-7.00 (m, 1H), 8.1-8.2 (d, 1H). UPLC retention
time: 0.47 min. MS calculated 278.5, found 279.65 ([M+1]+)
Synthesis of 2-chloro-6-fluorobenzylamine, 43. 2-chloro-6-
fluorobenzonitrile 42 (1.0 g, 6.43 mmol)was dissolved in 50 mL
of diethyl ether and stirred at 0⁰C while bubbling argon through
the solution for 10 minutes, followed by the dropwise addition
of 15 mL (3.0 equivalents) of 1.0 M LiAlH4 in THF over 20 min.
Following addition of the reducing agent, the reaction was stirred
for 2 hours at 0⁰C, warmed to room temperature and allowed to
stir overnight. The reaction was cooled to 0⁰C and quenched by
slow addition of Na2SO4•10 H2O. When the evolution of gas
was complete, the reaction was warmed to room temperature and
allowed to stir for an additional 30 minutes, and then filtered
Synthesis of N3-(2-(3,4,5-trimethoxyphenoxy)benzyl)-4H-1,2,4-
triazole-3,5-diamine, 46. Compound 46 was prepared from 8b
exactly as described above for compound 5b in 58% yield as a
white solid. 1H NMR (400 MHz, CDCl3) δ 3.86 (s, 6H), 3.90 (s,
3H), 4.89 (s, 2H), 6.44 (s, 2H), 6.80-6.82 (dd, 1H), 7.22-7.25 (m,
10 | MedChemComm., 2018, 00, 1-3
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ARTICLE
1H), 7.76-7.81 (m, 1H), 7.83-7.85 (d, 1H). UPLC retention time: exactly as described above for compound 5b in 66% yield as a
View Article Online
0.42 min. MS calculated 371.16, found 372.70 ([M+1]+).
white solid. 1H NMR (400 MHz, CDCl3)DδO1I:.1207.1(0s3,99/CH8)M,D40.70061(0sE,
2H), 6,77-6.79 (d, 1H), 7.11-7.13 (d, 2H), 7.54-7.59 (d, 2H),
Synthesis of
N3-(2-(naphthalen-1-yloxy)benzyl)-4H-1,2,4- 7.57-7.59 (d, 1H), 7.73-7.77 (m 1H), 7.82-7.84 (m, 1H). UPLC
triazole-3,5-diamine 47. Compound 47 was prepared from 8c retention time: 0.71 min. MS calculated 337.19, found 338.36
exactly as described above for compound 5b in 56% yield as a ([M+1]+)
1
white solid. H NMR (400 MHz, CDCl3) δ 4.66 (s, 2H), 7.36-
7.38 (d, 2H), 7.53-7.57 (d, 1H), 7.63-7.64 (m, 1H), 7.68-7.70 (d, Synthesis of N3-(2-(3,5-dimethylphenoxy)benzyl)-4H-1,2,4-
1H), 7.84-7.85 (d, 1H), 7.91-7.93 (d, 2H), 8.15-8.16 (m, 2H), triazole-3,5-diamine, 54. Compound 54 was prepared from 8j
8.17-8.19 (m, 1H). UPLC retention time: 0.44 min. MS exactly as described above for compound 5b in 48% yield as a
1
calculated 331.14, found 332.43 ([M+1]+)
white solid. H NMR (400 MHz, CD3OD) δ 1.27 (s, 3H), 2.36
(s, 3H), 4.68 (s,2H), 6.78 (s, 1H), 6.80 (s, 1H), 6.93 (s, 1H), 7.74-
Synthesis of N3-(2-(2-ethoxy-4-methylphenoxy)benzyl)-4H- 7.78 (m, 1H), 7.82-7.84 (m, 1H), 8.16-8.17 (d, 1H), 8.22-8.24 (d,
1,2,4-triazole-3,5-diamine, 48. Compound 48 was prepared from 1H). UPLC retention time: 0.45 min. MS calculated 309.16,
8d exactly as described above for compound 5b in 68% yield as found 310.57 ([M+1]+)
1
a white solid. H NMR (400MHz, CDCl) δ 1.15-1.18 (t, 6H),
2.98 (s, 2H), 3.31-3.36 (m, 4H), 6.12-6.14 (dd, 1H), 6.19-6.20 Synthesis
of
N3-(2-(4-bromo-2-
(m, 1H), 6.27-6.29 (dd, 1H), 7.04-7.08 (m,1H), 7.24-7.26 (d, (trifluoromethyl)phenoxy)benzyl)-4H-1,2,4-triazole-3,5-
1H), 7.57-7.59 (d, 1H), 7.75-7.83 (m, 1H), 8.01 (s, 1H). UPLC diamine, 55. Compound 55 was prepared from 8k exactly as
retention time: 0.46 min. MS calculated 339.17, found 340.65 described above for compound 5b in 48% yield. 1H NMR (400
([M+1]+)
MHz, CDCl3) δ 3.18 (s, 2H), 6.81-6.83 (d, 1H), 6.91-6.93 (d,1H),
7.41-7.42 (d, 1H), 7.44 (s, 1H), 7.54-7.56 (dd, 1H), 7.71-7.78 (m,
Synthesis of N3-(2-(2,4-difluorophenoxy)benzyl)-4H-1,2,4- 1H), 8.15-8.17 (m,1H). UPLC retention time: 0.47 min. MS
triazole-3,5-diamine, 49. Compound 49 was prepared from 8e calculated 427.03, found 428.55 ([M+1]+)
exactly as described above for compound 5b in 42% yield as a
white solid. 1H NMR (400MHz, CDCl3) δ 4.07 (s, 2H), 6.63-6.65 Synthesis of N3-(2-(4-(methylthio)phenoxy)benzyl)-4H-1,2,4-
(d, 1H), 7.02 (s, 1H), 7.32-7.33 (d,1H), 7.58-7.59 (d, 1H), 7.74- triazole-3,5-diamine, 56. Compound 56 was prepared from 8l
7.78 (m, 1H), 7.86-7.88 (m, 1H), 8.15-8.17 (d, 1H). UPLC exactly as described above for compound 5b in 48% yield as a
1
retention time: 0.56 min. MS calculated 315.11, found 316.61 yellow solid. H NMR (400 MHz, CDCl3) δ 2.53 (s, 3H), 4.60
([M+1]+)
(s, 2H), 6.81-6.84 (d,1H), 7.21-7.22 (d, 2H), 7.42-7.44 (d, 2H),
Compound 50 was prepared from 8f exactly as described above 7.69-7.71 (d, 1H), 7.87-7.89 (m, 1H), 7.91-8.00 (m,1H). UPLC
1
for compound 5b in 68% yield as a white solid. H NMR (400 retention time: 0.89 min. MS calculated 327.12, found 328.26
MHz, CDCl3) δ 1.15-1.18 (t, 6H), 2.98 (s, 2H), 3.31-3.36 (m, ([M+1]+)
4H), 6.12-6.14 (dd, 1H), 6.19-6.20 (m, 1H), 6.27-6.29 (dd, 1H),
7.04-7.08 (m,1H), 7.24-7.26 (d, 1H), 7.57-7.59 (d, 1H), 7.75- Synthesis of N3-(2-(2-ethoxy-4-methylphenoxy)benzyl)-4H-
7.83 (m, 1H), 8.01 (s, 1H). UPLC retention time: 0.46 min. MS 1,2,4-triazole-3,5-diamine, 57. Compound 57 was prepared from
calculated 352.20, found 353.65 ([M+1]+).
8m exactly as described above for compound 5b in 74% yield.
1H NMR (400 MHz, CD3OD) δ 1.12-1.16 (t, 3H), 2.41 (s, 3H),
Synthesis of N3-(2-(3,4,5-trimethoxyphenoxy)benzyl)-4H-1,2,4- 3.97-4.01 (m, 2H), 4.68 (s, 2H), 6.61-6.67 (d, 1H), 7.09-7.11 (d,
triazole-3,5-diamine, 51. Compound 51 was prepared from 8g 1H), 7.57-7.61 (m, 1H), 7.68-7.72 (m, 1H), 7.76-7.78 (s, 1H),
exactly as described above for compound 5b in 58% yield as a 8.15-8.17 (d, 1H), 8.22-8.24 (d, 1H). UPLC retention time: 0.44
white solid. 1H NMR (400 MHz, CDCl3) δ 3.86 (s, 6H), 3.90 (s, min. MS calculated 339.17, found 340.60 ([M+1]+).
3H), 4.89 (s, 2H), 6.44 (s, 2H), 6.80-6.82 (dd, 1H), 7.22-7.25 (m,
1H), 7.76-7.81 (m, 1H), 7.83-7.85 (d, 1H). UPLC retention time: Synthesis of N3-(2-(3,5-dimethoxyphenoxy)benzyl)-4H-1,2,4-
0.42 min. MS calculated 371.40, found 371.70 ([M+1]+)
triazole-3,5-diamine, 58. Compound 58 was prepared from 8n
exactly as described above for compound 5b in 68% yield as a
Synthesis of N3-(2-(2-isopropyl-5-methylphenoxy)benzyl)-4H- brown solid. 1H NMR (400 MHz, CD3OD) δ 2.90 (s, 3H), 2.98
1,2,4-triazole-3,5-diamine, 52. Compound 52 was prepared from (s, 3H), 4.74 (s,2H), 6.33-6.34 (m, 1H), 6.38-6.39 (m, 1H), 6.88-
8h exactly as described above for compound 5b in 55% yield as 6.90 (d, 1H), 7.10-7.12 (d, 1H), 7.77-7.81(s,1H), 8.07 (s, 2H).
a white solid. 1H NMR (400 MHz, CDCl3) δ 1.21 (s, 3H), 1.23 UPLC retention time: 0.46 min. MS calculated 341.15, found
(s, 3H), 2.35 (s, 3H), 4.69 (s, 2H), 6.64 (d, 1H), 6.80 (s, 1H), 342.63 ([M+1]+)
7.10-7.12 (d, 1H), 7.32-7.34 (d, 1H), 7.54-7.57 (m, 1H), 7.73-
7.75 (d, 1H), 7.79-7.87 (m, 1H). UPLC retention time: 1.99 min. Synthesis of N3-(2-(2,3-dimethylphenoxy)benzyl)-4H-1,2,4-
MS calculated 337.43, found 338.66 ([M+1]+)
triazole-3,5-diamine, 59. Compound 59 was prepared from 8p
exactly as described above for compound 5b in 48% yield as a
1
Synthesis of N3-(2-(4-(tert-butyl)phenoxy)benzyl)-4H-1,2,4- white solid. H NMR (400 MHz, CD3OD) δ 2.14 (s, 3H), 2.38
triazole-3,5-diamine, 53. Compound 53 was prepared from 8i (s, 3H), 4.71 (s, 2H), 6.54-6.56 (d, 1H), 6.95-6.97 (d, 1H), 7.13-
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7.15 (d, 1H), 7.18-7.22 (m, 1H), 7.57-7.61(d, 1H), 7.69-7.73 (m, WI), as previously described.11 Compounds wereVidewisAsroticlvleeOdnliinne
DOI: 10.1039/C8MD00610E
1H), 7.71-7.81 (m, 1H). UPLC retention time: 0.55 min. MS DMSO and diluted to the desired concentration with PBS, using
calculated 309.16, found 310.38 ([M+1]+)
TCP as a positive control and 1% DMSO as a negative control.
Compounds were added to a mixture of substrate and enzyme,
Synthesis of
N3-(2-(naphthalen-2-yloxy)benzyl)-4H-1,2,4- and the plate was allowed to incubate for 1hr at RT. Data were
triazole-3,5-diamine 60. Compound 60 was prepared from 8q generated using a luminescent plate reader.
exactly as described above for compound 5b in 64% yield as a
1
white solid. H NMR (400 MHz, CDCl3) δ 4.70 (s, 2H), 6.81-
6.83 (d, 1H), 7.35-7.38 (dd, 1H), 7.52-7.55 (m, 1H), 7.56-7.59
(m, 2H), 7.75-7.77 (d, 1H), 7.79-7.83 (d, 1H), 7.87 (s, 1H), 7.91- Measurement of SMOX and PAOX activity29
7.93 (m, 1H), 7.90-7.99 (d, 1H), 8.15-8.17 (d, 1H). UPLC
retention time: 0.95 min. MS calculated 331.14, found 332.60 For the measurement of oxidase activity, the enzyme was diluted
([M+1]+).
to a concentration of 15 ng/L. Each reaction contained 62.2 L
of 0.5 M glycine buffer, 20 L of the enzyme solution, 17.8 µL
Synthesis of N3-(2-([1,1'-biphenyl]-4-yloxy)benzyl)-4H-1,2,4- of the inhibitor or vehicle, and 20 µL of 1.5 mM spermine or N1-
triazole-3,5-diamine, 61. Compound 61 was prepared from 8r acetylspermine for SMOX or PAOX activity, respectively (final
exactly as described above for compound 5b in 64% yield. H concentration = 250 µM) in a 0.5 mL reaction tube. As a control,
1
NMR (400 MHz, CD3OD) δ 4.74 (s, 2H), 6.85-6.87 (d, 1H), one tube contained enzyme that had been boiled for 5 minutes
7.26-7.28 (d, 2H), 7.38-7.42 (m, 1H), 7.47-7.51 (m, 2H), 7.62- prior to addition. Each reaction was incubated at 37ºC for one
7.64 (d, 2H), 7.68-7.71 (d, 2H), 7.77-7.81 (m, 1H), 7.85-7.88 (m, hour in a thermal cycler, then immediately extracted with acid
1H), 8.15-8.17 (d, 1H). UPLC retention time: 0.43 min. MS and labeled with dansyl chloride. The production of spermidine
calculated 357.16, found 358.37 ([M+1]+)
from spermine (or acetylated spermine) was then determined by
HPLC. Where indicated, inhibitors were added at the specified
concentrations 2 min prior to the addition of substrate. Per cent
N3-(2-(4-fluorophenoxy)benzyl)-4H-1,2,4-triazole-3,5-diamine,
Synthesis of 62. Compound 62 was prepared from 8s exactly as inhibition of SMOX and PAOX were calculated using the
described above for compound 5b in 44% yield as a white solid. following equation:
1H NMR (400 MHz, CDCl3) δ 4.17 (s, 2H), 6.52-6.54 (d, 1H),
6.70-6.72 (d, 2H), 7.23-7.25 (d, 1H), 7.57-7.59 (m, 1H), 7.84- [Spermidine produced (with inhibitor)/spermine produced (no
7.86 (d, 2H), 7.94-7.99 (m, 1H). UPLC retention time: 0.46 min. inhibitor] X 100.
MS calculated 299.12, found 300.50 ([M+1]+)
Western blotting
Determination of LSD1 activity
Compounds 5a, 5b, 14-41 and 46-62 were evaluated for the
Western blot analysis was performed according to BioRad
ability to inhibit recombinant LSD1/CoREST using
a
technical bulletin 6376. Cells were plated at 350,000 cells per
well in a 6 well plate in a total volume of 2.0 mL per well. Cells
were incubated at 37ºC in a humidified environment containing
commercially available assay kit (#700120, Cayman Chemical,
Ann Arbor, MI). The substrate and all compounds were
incubated in assay buffer from 30 min up to 4 h at 37 °C as
described in the commercial protocol. The volume of each
reaction well was 50 l, containing 5 l of a 200 M solution
of substrate peptide and 20 l of a 15 ng/l enzyme solution.
All compounds were diluted in 1% DMSO with assay buffer to
a final volume of 50 M. Fluorescence was measured at the
5% CO . For protein collection, wells were washed twice with
2
cold PBS and lysed with 200 L of cold RIPA buffer containing
1% Halt Protease & Phosphatase inhibitor (Thermo Fisher
Scientific) for 30 min on ice. Individual cell lysates were
º
centrifuged at 16,000  g for 20 min in a 4 C pre-cooled
centrifuge. The supernatant fractions were collected, and
proteins were measured using a Pierce BCA Protein Assay kit
(Thermo Fisher Scientific). SDS gel electrophoresis and
immunoblot analysis were performed using the methodology
previously described.30 Anti-LSD1 and anti-GAPDH
monoclonal primary antibodies were used with HRP-conjugated
secondary antibodies (Abcam). Bands were detected by a
chemiluminescence detection methodology with the use of a
SpectraMax M5 instrument (Molecular Devices) equipped with
SOFTmax PRO 4.0 software. The band size and intensity for
each signal were quantified using Image Studio Lite ver5.2 (LI-
recommended wavelengths of k = 530 nm, k = 590 nm. IC
ex em
50
determinations were performed using serial dilutions at 250, 125,
62.5, 31.25, 6.25, 3.125, 1.563, 0.781 and 0.390 M.
Tranylcypromine (TCP) and the known spermine oxidase
inhibitor MDL 72,527 were used as positive and negative
controls, respectively. All blanks contained 1% DMSO to
determine any solvent effects.
Measurement of MAO-A and MAO-B activity
In order to assess the specificity/selectivity of 5a, 5b, 14-41 and COR Biosciences) and normalized to the loading control
46-68 for LSD1, selected analogues were evaluated for the GAPDH. The relative density value of the MCF-10A control
ability to inhibit monoamine oxidases (MAO) A and B using a lysate was set as 1.
commercial assay kit (#V1401, Promega Biosciences, Madison,
12 | MedChemComm., 2018, 00, 1-3
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Cell viability assay
studies are required to determine the structural reqVuiierweAmrtiecnletOsnflioner
optimal binding of analogues in this serDieOsI:t1o0.e10it3h9e/rC8SMMDO00X610oEr
LSD1.
All cell lines (BxPc-3, Miapaca2 and PANC-1 pancreatic tumor
cell lines, and the CRL7869 normal human intestinal cell line)
were purchased from ATCC (Manassas, VA). For the (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-
sulfo- phenyl)-2H-tetrazolium) (MTS) reduction assay, 2000
cells per well were seeded in 50 L of complete medium per well
of a 96-well plate and the cells were allowed to attach overnight
at 37oC in a 5% CO2 atmosphere. The medium was aspirated and
cells were treated with 100 mL of fresh medium containing
appropriate concentrations of each inhibitor to be tested. The
cells were incubated for 72 h at 37oC in 5% CO2, after which 20
mL of the MTS reagent solution (Promega CellTiter 96 Aqueous
One Solution Cell Proliferation Assay) was added to the
medium. The cells were incubated for another 2 h at 37oC, and
absorbance was measured at 490 nm on a SpectraMax M5
instrument (Molecular Devices) equipped with SOFTmax PRO
4.0 software to determine cell viability. A reference wavelength
of 690 nm was used to subtract the background. Percent cell
death was calculated by the following equation: % Cell Death =
(Abs Control - Abs sample)/Abs Control X100. A dose response
curve was constructed for each inhibitor, and each data point was
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The research described in this manuscript was supported by
National Institutes of Health grants 1 RO1 CA149095 (P.M.W.)
and 1 RO1 CA204345 (R.A.C., P.M.W.), and the Samuel
Waxman Cancer Research Foundation (R.A.C.). We are grateful
to the MUSC Hollings Cancer Center Biorepository and Tissue
Analysis Shared Resource for providing pancreatic tissue slices.
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DOI: 10.1039/C8MD00610E
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