3-(Piperidin-4-ylmethoxy)pyridine Containing Compounds Are Potent Inhibitors of Lysine Specific Demethylase 1

Article abstract of DOI:10.1021/acs.jmedchem.5b01361

Methylation of histone lysine residues plays important roles in gene expression regulation as well as cancer initiation. Lysine specific demethylase 1 (LSD1) is responsible for maintaining balanced methylation levels at histone H3 lysine 4 (H3K4). LSD1 is a drug target for certain cancers, due to important functions of methylated H3K4 or LSD1 overexpression. We report the design, synthesis, and structure-activity relationships of 3-(piperidin-4-ylmethoxy)pyridine containing compounds as potent LSD1 inhibitors with K_i values as low as 29 nM. These compounds exhibited high selectivity (>160×) against related monoamine oxidase A and B. Enzyme kinetics and docking studies suggested they are competitive inhibitors against a dimethylated H3K4 substrate and provided a possible binding mode. The potent LSD1 inhibitors can increase cellular H3K4 methylation and strongly inhibit proliferation of several leukemia and solid tumor cells with EC₅₀ values as low as 280 nM, while they had negligible effects on normal cells.

Full text of DOI:10.1021/acs.jmedchem.5b01361

Article
pubs.acs.org/jmc
3‑(Piperidin-4-ylmethoxy)pyridine Containing Compounds Are
Potent Inhibitors of Lysine Specific Demethylase 1
Fangrui Wu,†,§ Chao Zhou,†,§ Yuan Yao,† Liping Wei,† Zizhen Feng,† Lisheng Deng,†
and Yongcheng Song*,†,‡
‡
†Department of Pharmacology, and Dan L. Duncan Cancer Center, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas
77030, United States
S
* Supporting Information
ABSTRACT: Methylation of histone lysine residues plays
important roles in gene expression regulation as well as cancer
initiation. Lysine specific demethylase 1 (LSD1) is responsible
for maintaining balanced methylation levels at histone H3
lysine 4 (H3K4). LSD1 is a drug target for certain cancers, due
to important functions of methylated H3K4 or LSD1
overexpression. We report the design, synthesis, and
structure−activity relationships of 3-(piperidin-4-ylmethoxy)-
pyridine containing compounds as potent LSD1 inhibitors with Ki values as low as 29 nM. These compounds exhibited high
selectivity (>160×) against related monoamine oxidase A and B. Enzyme kinetics and docking studies suggested they are
competitive inhibitors against a dimethylated H3K4 substrate and provided a possible binding mode. The potent LSD1 inhibitors
can increase cellular H3K4 methylation and strongly inhibit proliferation of several leukemia and solid tumor cells with EC50
values as low as 280 nM, while they had negligible effects on normal cells.
SET domain, which cannot methylate H3K4.11 Rather, the
fusion partners can recruit DOT1L, a histone H3 lysine 79
INTRODUCTION
■
Post-translational modifications of the ε-amino group of
(H3K79) methyltransferase, which causes aberrant H3K79
histone lysine residues, such as acetylation and methylation,
methylation at MLL target gene loci and overexpression of
play important roles in gene expression regulation in normal
these genes, eventually leading to the initiation of the
physiology as well as diseases.1−4 Small molecule modulators of
leukemia.20−22 Pharmacological inhibition of DOT1L has
histone modifying enzymes are therefore chemical probes for
been found to block proliferation of MLL-rearranged leukemia
biological studies and potentially useful therapeutics for such
diseases.5−7 Histone H3 lysine 4 (H3K4) methylation,
including mono-, di-, and trimethylation (H3K4me1, -2, and
cells, showing effectiveness of the targeted epigenetic
therapy.23,24
LSD1 is a flavin adenine dinucleotide (FAD) dependent
-3) has been found to be generally a biomarker for active gene
transcription.8,9 Mixed lineage leukemia (MLL), a large,
oxidase, related to monoamine oxidase A and B (MAO-A and
-B).13 Figure 1A schematically shows the mechanism of
multidomain protein containing ∼4000 amino acids, can
methylate H3K4, with its C-terminal SET domain being
catalysis of LSD1. Upon binding to LSD1, the methyl group
catalytically active.9−11 In addition, cellular H3K4 methylation
of a methylated histone lysine residue (e.g., H3K4me2) is
oxidized by the cofactor FAD, followed by hydrolysis to give
levels are tightly regulated because of their important functions.
Lysine specific demethylase 1 (LSD1, also known as KDM1a),
formaldehyde and demethylated lysine. FADH2, the reduced
form of FAD, is oxidized by O2 in the solvent to give H2O2 and
has been found to be a member of an MLL transcription
FAD to complete a catalytic cycle.25 A number of LSD1
complex. LSD1 catalyzes an oxidative demethylation reaction
inhibitors with several chemotypes, including cyclopropyl-
from H3K4me1 and -2 (but not H3K4me3) and thus
counteracts the enzyme function of MLL.12,13 Both MLL and
amine, propargylamine, hydrazine, and triazole dithiocarba-
mate, have been reported in journals and patents,26,27 as
LSD1 are essential for hematopoiesis, showing a balanced
representatively shown in Figure 1B. Most LSD1 inhibitors are
cellular H3K4 methylation is crucial for normal hematopoi-
esis.11,14 In addition, H3K915 and other proteins such as
cyclopropylamine containing compounds, derived from tranyl-
DNMT116 were reported as a substrate of LSD1.
cypromine (1), an inhibitor of MAO-A and -B as well as an
FDA-approved antidepression drug. Mechanistically, the α-C
atom of the cyclopropyl ring of these compounds is oxidized,
causing opening of the constrained ring followed by covalent
Dysregulated histone lysine methylations may lead to a
disease, such as cancer.2 For example, ∼75% acute leukemia in
infants and 10% of that in adults contain an MLL gene
translocation and this subtype of leukemia shows a poor
prognosis.17−19 All MLL translocations identified to date
Received: September 3, 2015
produce an onco-MLL fusion protein without the C-terminal
© XXXX American Chemical Society
A
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Figure 1. (A) Mechanism of catalysis for LSD1. (B) Structures of representative LSD1 inhibitors, among which compounds in the upper panel are
irreversible inhibitors and those in the lower panel are reversible.
link to FAD (Supporting Information Figure S1).25 Reversible
Chart 1. Inhibitor Design Leading to the Finding of New
LSD1 inhibitors have also been reported.26b
a
LSD1 Inhibitors
LSD1 has been suggested to be a drug target for several types
of cancers (e.g., prostate and breast) due to its overexpression
in these malignancies.28−30 In addition, LSD1 was recently
found to be required for an MLL-rearranged leukemia.31
Knockdown of LSD1 can abrogate the transforming ability of
the onco-MLL fusion gene and increase the H3K4me2 levels.
Using a cyclopropylamine based LSD1 inhibitor also showed
activity against the MLL-rearranged leukemia in cell and mouse
models.31 However, this compound was reported to have
severe toxicities. Nonetheless, these previous studies have
indicated LSD1 is a validated drug target and more chemotypes
of inhibitors are therefore needed.32,33
Here, we report the discovery, synthesis, and structure−
activity relationships (SARs) of 3-(piperidin-4-ylmethoxy)-
pyridine containing compounds as potent LSD1 inhibitors.
Enzyme kinetics and molecular modeling studies were used to
investigate the possible mechanism of inhibition as well as the
binding mode of these compounds. Molecular and cell biology
experiments were performed to characterize the biological
activities of selected compounds. During the revision of this
manuscript, we noticed that scientists at GlaxoSmithKline
found a structurally similar LSD1 inhibitor and presented its
preliminary data at the 2013 American Association of Cancer
Research annual meeting.34,26b,27i
RESULTS AND DISCUSSION
■
a
Inhibitor Design and Discovery. On the basis of the
IC50 values of compound 1 against MAO-A and -B are from ref 35.
results from previous27a and our own studies on cyclopropyl-
amine containing inhibitors of LSD1, it is clear that a second
basic N-substituent, such as the piperidin-4-ylmethyl group (in
red dashed circle, Chart 1) in compound 2, can significantly
increase the inhibitory activity as well as selectivity for LSD1.
For example, compound 2 (IC50 = 62 nM) is ∼250-fold more
active against human recombinant LSD1 than compound 1
(IC50 = 15.7 μM 31) and, moreover, the piperidin-4-ylmethyl
group enhances the enzyme selectivity for LSD1 by >1500-fold.
Similar features have been observed by us and others27a for
compounds S1 and S2 for the (4-methylpiperazin-1-yl)-
carbonylmethyl group in compound S2 (Supporting Informa-
tion Figure S2). These data strongly support an important role
of these basic amine groups in inhibitor recognition and/or
binding to LSD1. However, there has been no structural
information as to how these groups bind to LSD1, since X-ray
crystallography might not be suited for this study because these
groups are to be eliminated upon completion of the reaction
(Figure S1). Nevertheless, given its important role in inhibitory
potency and selectivity for LSD1, a basic amine group such as
B
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Figure 2. Docking studies of potential LSD1 inhibitors, using an LSD1 crystal structure (PDB code 2V1D) as the docking template. For clarity, only
key residues (in light blue) in the active site of LSD1 are shown, together with the cofactor FAD (light blue tube model). Hydrogen bonds are shown
as orange dashed lines. (A) Lwest-energy docking structure of compound 3 (tube model with C atoms in green), suggesting favorable interactions
between its pyridine ring and FAD and Y761. (B) Lowest-energy docking structure of 4 (in yellow), showing introducing an additional 5-phenyl
group could be favorable.
piperidin-4-ylmethyl should be included for the design of new
LSD1 inhibitors.
Scheme 1. General Synthetic Method for Pyrazine
Containing Inhibitors
a
We wished to find competitive, druglike inhibitors of LSD1
that do not covalently bind to FAD or the enzyme. Compound
3 (Chart 1) was designed in which the piperidin-4-ylmethyl and
the phenyl group in the potent inhibitor 2 are retained, while
the reactive cyclopropylamine moiety is replaced by a
chemically stable pyridine core. Our modeling (docking)
study (Figure 2A) predicted that the protonated piperidine
amino group (at the physiological pH) could form a hydrogen
bond with Asp555 and the pyridine ring could have favorable
electrostatic and hydrophobic interactions with the flavin ring
of FAD as well as Tyr761. Compound 3 was found to be
inactive presumably due to the large active site of LSD1.
Compound 4 was next designed with the rationale that an
additional phenyl group could increase the binding affinity to
LSD1, as suggested by docking (Figure 2B). The use of the
pyrazine ring in 4 was intended for quick synthesis (see the
Synthesis section below). Compound 4 was found to be a weak
inhibitor with an apparent Ki value of 47.8 μM. We next
performed a small scale of SAR studies (described below) based
on compound 4, which yielded a good inhibitor 5 with a Ki
value of 2.3 μM. Enzyme kinetics studies (described below)
suggested that compound 5 is a competitive inhibitor of LSD1
against a dimethylated H3K4 peptide substrate. These findings,
together with its druglike chemical structure, show that
compound 5 represents a promising new scaffold for further
inhibitor optimization.
a
Reagents and conditions: (i) 2-aminoacetamide, NaOH; (ii) POCl3;
(iii) BOC protected 4-(hydroxymethyl)piperidine, NaH; (iv) HCl in
1,4-dioxane; (v) Pd(PPh3)4, Zn(CN)2.
chromatography to give, upon deprotection, compounds 8−10
(described below).
Scheme 2 shows a general synthesis of 3-(piperidin-4-
ylmethoxy)pyridine containing LSD1 inhibitors (such as
Scheme 2. General Synthetic Method for Pyridine
Containing Inhibitors
a
Synthesis. Scheme 1 illustrates a general method for
synthesis of pyrazine containing inhibitors. A commercially
available benzil compound was reacted with an equal molar
amount of 2-aminoacetamide in the presence of sodium
hydroxide to give 2-hydroxy-5,6-diphenylpyrazine. Upon treat-
ment with POCl3, its 2-hydroxy group was converted to a -Cl.
The compound thus obtained was subjected to a substitution
reaction using a sodium salt of tert-butyloxycarbonyl (BOC)
protected 4-(hydroxymethyl)piperidine. Pyrazine containing
inhibitors (such as compound 4) were synthesized after
deprotection of the BOC group. In addition, treatment of the
BOC protected intermediate compound (R = Br) with zinc
cyanide in the presence of a catalytic amount of Pd(PPh3)4 to
produce a mixture of monocyano and dicyano substituted
compounds, which can be carefully separated by column
a
Reagents and conditions: (i) BOC protected 4-(hydroxymethyl)-
piperidine, PPh3, diisopropyl azodicarboxylate; (ii) R5-B(OH)2,
Pd(PPh3)4, Na2CO3, 80 °C; (iii) R6-B(OH)2, Pd(PPh3)4, K3PO4,
140 °C; (iv) HCl in 1,4-dioxane.
C
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compound 5). A Mitsunobu reaction between 5-bromo-6-
chloropyridin-3-ol and BOC protected 4-(hydroxymethyl)-
piperidine introduces a BOC-protected 3-(piperidin-4-ylme-
thoxy) R3 substituent. A selective Suzuki coupling reaction was
performed to give R5-substituted compound, which underwent
a second Suzuki coupling reaction with its 6-Cl group to add a
R6-substituent. Upon deprotection of the BOC group, 3-
(piperidin-4-ylmethoxy)pyridine containing compounds 5, 11−
40 can be obtained. In addition, by use of similar methods,
related compounds 41−45 can be readily synthesized
(Supporting Information Experimental Section).
The pyridine core structure, in which the two substituents
can be modified separately, was used for SAR studies. Although
compound 11 (Chart 2, which corresponds to the pyrazine
compound 4) is inactive, compound 12 with a Ki value of 4.0
μM is a good LSD1 inhibitor and stronger than the
corresponding pyrazine compound 8. This showed the pyridine
core can be a good scaffold for activity optimization.
The 6-position of the pyridine ring was optimized, and the
results are summarized in Table 1. Compound 5 with an
unsubstituted 6-phenyl group was found to be slightly more
active (Ki = 2.3 μM) than 12. Compound 13 having a 4-
fluorophenyl group exhibited potent inhibitory activity with a Ki
of 220 nM, ∼10× more active than the parent compound 5.
Adding an additional -F at the 3- or 2-position in compound 14
(Ki = 570 nM) or 15 (Ki = 1.2 μM), respectively, resulted in a
∼2.5× or 5.5× activity reduction, suggesting that 3- or 2-
substitution of the phenyl ring seems to be disfavored. The 4-
trifluoromethyl group in compound 16 considerably enhanced
the inhibitory potency to 58 nM. Change to a 4-methyl group
in compound 17 (Ki = 29 nM) further caused a ∼2-fold activity
increase. Move of the methyl group to 3-position in compound
18 (Ki = 800 nM) drastically decreased the activity by ∼27-fold,
showing again substitution at the 4-position of the 6-phenyl
ring is favorable. Compounds 19 and 20 with a 4-ethyl and 4-
isopropyl group, respectively, were found to be ∼7× and 3×
less active (Ki = 200 and 88 nM) than 17 with a 4-methyl,
showing a bigger alkyl group at this position is less favorable. As
compared to the most potent inhibitor 17, while compound 21
(Ki = 740 nM) having a 4-methoxy group showed a significant
activity reduction, compound 22 with a 4-trifluoromethoxy
substituent exhibited a similar inhibitory activity (Ki = 46 nM).
Activities of compounds 23−26 indicated that a 6-pyridinyl
ring is less favorable than a phenyl ring. Compounds 23 and 24
(Ki = 5.0 and 3.2 μM) with a pyridin-4-yl and -3-yl ring,
respectively, are slightly less active than is compound 5 (Ki =
2.3 μM). More noticeably, compound 25 (Ki = 1.8 μM) with a
4-fluoropyridin-3-yl substituent exhibited a ∼8-fold activity
reduction as compared to the corresponding phenyl compound
13. Although the 4-methyl group in compound 26 resulted in a
significant activity increase (Ki = 380 nM, as compared to 3.2
μM for compound 24), compound 26 is still ∼13× less active
than the corresponding phenyl compound 17. In addition,
introducing a 3-thiophenyl ring to the 6-position produced
compound 27, which exhibited a comparable inhibitory activity
(Ki = 2.4 μM) to that of the phenyl compound 5.
SAR Studies. Upon obtaining compound 4 with an
apparent Ki value of 47.8 μM, we synthesized several analogs
of 4 shown in Chart 2. Compound 6 with 5,6-di-(4-tolyl) and
Chart 2. Structures and Ki Values (in Parentheses) of
Compounds 5−12
Next, compounds 28−40 were synthesized and tested for
LSD1 inhibition for SAR studies of the 5-position of the most
potent inhibitor 17. As summarized in Table 1, the 4-cyano
substituent of the 5-phenyl ring has turned out to be critically
important for LSD1 inhibition. Compound 28 with a 4-ethynyl
group was surprisingly found to suffer from a complete
(>1700×) activity loss, despite a similar size of the R5 groups.
This could be due to different electrostatic properties between
the ethynyl (in 28) and the cyano group (in 17). Compounds
29 and 30 with a 4-carboxy and 4-hydroxymethyl group in the
5-phenyl ring also completely lost inhibitory activities. In
addition, compounds 31−34, which contain a 4-amide group
with a range of steric, hydrophobic, and electronic properties,
were found to be inactive. Compounds 35−38 having a 4-
aminomethyl group retained some inhibitory activities against
LSD1. Compound 35 with a smaller ethylaminomethyl group is
a weak inhibitor with a Ki of 22.3 μM, while compounds 36 and
37 having a bulkier piperidin-1-ylmethyl and morpholin-4-
compound 7 with 5,6-di-(4-bromophenyl) substituents are
more active (Ki = 32.2 and 26.2 μM, respectively), showing the
two methyl or bromo groups are favorable. Synthesized from 7
using a Pd-catalyzed reaction, compound 8 with 5,6-di-(4-
cyanophenyl) was found to be considerably more active with a
Ki of 9.1 μM. In addition, monocyano substituted compounds 9
and 10 were also synthesized, which can be separated by
column chromatography. One isomer 9 exhibited potent
inhibitory activity against LSD1 with a Ki value of 540 nM,
while the other isomer 10 (Ki = 16.5 μM) is less active than
compound 8. This indicated that 5- and 6-substituent in
compounds 4, 6−10 should be individually optimized for LSD1
inhibition. However, this is not synthetically feasible for
compounds with a pyrazine core.
D
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Table 1. Structures and Activity of Compounds 5, 12−40
ylmethyl substituent, respectively, are almost inactive. Interest-
ingly, compound 38 with a dibasic piperazin-1-ylmethyl group
is a fairly good inhibitor with a Ki of 7.6 μM, although it is
>250× less active than compound 17 having the corresponding
-CN substituent. Moreover, compared to 17, adding a -F into
the 2- and 3-position of the 5-phenyl ring in compounds 39 and
40 (Ki = 220 and 150 nM), respectively, resulted in ∼8× and
5× activity reductions.
Finally, several compounds shown in Chart 3 were
synthesized for the SAR study of the 3-(piperidin-4-
ylmethoxy)pyridine core structure. Compound 41 (Ki = 4.9
μM) with a benzene core structure is ∼170-fold less potent
than compound 17, suggesting the pyridine core is of
importance for the activity. Although it is a racemic mixture,
the 650 nM Ki value of compound 42 shows that the piperidin-
3-yl substituent is significantly less favorable than the piperidin-
4-yl group in 17. Greatly reduced activity of compound 43 (Ki
= 1.2 μM) also indicates that change of an -O- linkage in
compound 17 to a -NH- is highly disfavored. Loss of activity for
compounds 44 (with a 3-OH group) and 45 (with a 3-
benzyloxy group) clearly shows the critical role of the basic
piperidine side chain in LSD1 inhibition.
Enzyme Kinetics Studies. A steady-state enzyme kinetics
study of compound 5, which is a representative compound for
this novel series of LSD1 inhibitors, was performed to
determine the possible mechanism of action. Since the cofactor
FAD has a very high affinity to LSD1, as evidenced by its
presence in the enzyme even after a lengthy expression and
purification process, the kinetics study was performed against a
dimethylated H3K4 peptide substrate with a sequence of
ARTK(Me2)QTARKSTGGKAPRKQKA. Initial velocities of
the LSD1 catalyzed reaction were measured with increasing
concentrations of compound 5 and the substrate. The program
SigmaPlot was used for enzyme kinetics modeling. The reaction
rate and ligand concentration data were imported into the
program and fitted to either competitive, uncompetitive, or
noncompetitive inhibition models. The best model, as judged
by R2 and AICc values, would show the mode of action for the
inhibitor. Thus, compound 5 was found to be a competitive
inhibitor of LSD1 against the substrate because the kinetics
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energies tightly clustered to one another and having similar
binding features (Figure 4A). The pyridine core, tolyl (R6), 4-
cyanophenyl (R5), and piperidin-4-ylmethoxy (R3) groups of
these 20 structures are located in the same site or pocket in
LSD1. Figure 4B and Figure 4C show two views of the binding
conformation of 17 with the lowest docking energy in the
active site of LSD1. Its central pyridine ring is predicted to have
favorable hydrophobic as well as electrostatic interactions with
the flavin ring, Tyr761, Ala809, Thr810, and Ala539. The tolyl
R6 group was found to be located in a mostly hydrophobic
pocket surrounded by flavin, Phe538, Val333, Tyr761, Met332,
Leu659, Lys661, and Trp695. The 4-cyanophenyl R5 group
occupies a pocket surrounded by with Ala539, Phe538, Trp695,
His564, and Thr335 and has favorable interactions with these
residues. The protonated amine of the piperidin-4-ylmethoxy
group was found to have a hydrogen bond as well as
electrostatic interactions with the negatively charged side
chain of Asp555. In addition, the skeleton of the piperidin-4-
ylmethoxy group possesses hydrophobic interactions with
Ala809, Pro808, Ala539, and Asn540.
Chart 3. Structures and Ki Values (in Parentheses) of
Compounds 41−45
Of interest is that the docking results could be used to
rationalize our experimental SAR. For example, the predicted
strong interactions between the protonated piperidin-4-
ylmethoxy group and Asp555 (Figure 4B) could explain the
importance of this group for LSD1 inhibition. As comparof ied
to compound 41 with a benzene core structure, possibly
enhanced interactions between the pyridine core in 17 and
flavin as well as Tyr761 (Figure 4B) could account for the
increased activity of 17. For SAR on the R6 group, as compared
to compound 5, the increased activity of 17 could be due to the
favorable interactions of the methyl group in 17 with Phe538,
Val333, Met332, and Lys661 (Figure 4B and Figure 4C). In
addition, the methyl is predicted to range from ∼3.3 Å away
from Met332 to ∼4.8 Å from Trp695 by the docking study.
This limited space might account for somewhat decreased
activities of compounds 19 and 20 with a bigger -Et and -i-Pr
group, respectively.
data for 5 were best fitted to the competitive mode of action
(Figure 3), as compared to the uncompetitive and non-
competitive fitting (Figure S3).
Docking Studies. We carried out molecular modeling
(docking) studies to predict how the most potent inhibitor 17
binds to LSD1. A number of X-ray crystal structures of LSD1 in
complex with covalent inhibitors,25,36 a reversible inhibitor,37
and substrate-like peptide38,39 are available for the study. The
docking program Glide in Schrodinger Suite molecular
̈
modeling software package was used for docking.40 The crystal
structure (PDB code 2V1D) of human LSD1 in complex with a
H3K4 mimetic peptide39 (with a K4M mutation) was used as a
docking template, which was prepared by adding hydrogen
atoms to the structure, removing the peptide and water
molecules, while keeping FAD as an integrated component of
the protein. Compound 17 can be well docked into the active
site of LSD1, with its 20 docking structures with the lowest
Enzyme Selectivity. LSD1 belongs to a family of FAD-
dependent monoamine oxidases including MAO-A and -B. Due
to an important role of MAO-A and -B in degradation of
neurotransmitters in the central nervous system, selective
inhibition of LSD1 is highly desirable. We investigated enzyme
selectivity of several representative 3-(piperidin-4-ylmethoxy)-
Figure 3. Lineweaver−Burk plot of competitive inhibition model for compound 5 with variable concentrations of the dimethylated H3K4 peptide
substrate, with an enlarged figure in the left panel showing details near the origin. The Ki was determined to be 1.8 μM in this experiment.
F
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Figure 4. Docking results of compound 17, using an LSD1 structure (PDB code 2V1D) as the docking template: (A) 20 lowest-energy docking
structures of compound 17, with the most stable conformation shown as an orange tube model; (B) lowest-energy docking structure of 17, showing
the interacting key residues (in light cyan) of LSD1 and FAD (light cyan tube model); (C) lowest-energy docking structure of 17 (as a space-filing
model) in the active site of LSD1 (shown as a meshed electrostatic potential surface model).
pyridine containing LSD1 inhibitors against MAO-A and -B.
Thus, compounds 5, 16, 17, and 22 were tested for their
activity against recombinant human MAO-A and -B and their
selectivity indices were also calculated. These results are
summarized in Table 2. All four potent LSD1 inhibitors did
Cell Activity Testing. The most potent compound 17 was
tested to see whether it can change the cellular H3K4
methylation level in a leukemia cell line MV4-11. Thus, cells
were treated with 17 at 0.2 and 1 μM and histones were then
collected, separated by electrophoresis, and probed by a specific
antibody of H3K4me2, as well as that of histone H3 as input
controls. As can be seen in Figure 5, compound 17 appeared to
Table 2. Inhibitory Activity (Ki, μM) against MAO-A and -B
and Selectivity Index for LSD1
compd
LSD1
MAO-A
MAO-B
selectivity index
5
2.3
>50
>50
>50
>50
10.6 2.8
9.7 2.9
18.7 6.9
2.6 1.2
>4.6
>160
>640
>56
16
17
22
0.058
0.029
0.046
Figure 5. Treatment of MV4-11 cells with potent LSD1 inhibitor 17
caused an increased cellular level of H3K4me2.
increase the cellular level of H3K4me2 at 1 μM, suggesting it is
cell membrane permeable and inhibits LSD1 in cells. It is noted
because this method is semiquantitative. More quantitative
investigation could be useful to characterize how compound 17
affects cellular histone methylations globally and at specific
gene loci.
not significantly inhibit MAO-A with Ki > 50 μM, showing
excellent selectivity. For MAO-B, compounds 5, 16, and 17 had
only weak activities with Ki values of 9.7−18.7 μM. Due to its
relatively low activity against LSD1, compound 5 showed 4.6×
selectivity against MAO-B. However, potent LSD1 inhibitors
16 and 17 exhibited very high selectivity of >160- and >640-
fold against MAO-B, respectively. Moreover, despite having a
relatively good activity against MAO-B (Ki = 2.6 μM),
compound 22 is still a highly selective LSD1 inhibitor with a
selectivity index of >56 against MAO-B, because of its potent
inhibition of LSD1. Structurally, the 4-CF3, -Me, and -OCF3 of
the 6-phenyl ring in compounds 16, 17, and 22, respectively,
account for the high selectivity against MAO-B because as
compared to the parent compound 5, these substituents have
less impact (<4×) on MAO-B inhibition but increase the
inhibitory potency against LSD1 by >39-fold.
Structurally similar 5,6-diaryl-3-(aminoalkyloxy)pyridine
compounds were reported to be potent inhibitors of Akt
kinase,41 which is also a drug target for cancer. We examined
the enzyme selectivity of the most potent compound 17 against
human Akt. Compound 17 was found to have a weak activity
against Akt with an IC50 value of 87.6 21.6 μM, showing a
high enzyme selectivity for LSD1. In addition, these
compounds were reported as nicotinic receptor ligands42 as
well as antituberculosis agents.43 Although these do not directly
relate to the antitumor purpose of this study, a comprehensive
enzyme selectivity profiling could be useful for future
development of these compounds.
Next, selected potent LSD1 inhibitors, including compound
S2 (which is a cyclopropylamine-containing inhibitor, Figure
S2) as a positive control, were tested for their ability to inhibit
proliferation of four tumor cell lines and a normal fibroblast cell
line WI-38. Among the four tumor cells, MV4-11 and Molm-13
are leukemia cell lines with an MLL gene translocation. Two
representative breast cancer cells MCF-7 (estrogen receptor
positive) and MDA-MB231 (estrogen receptor negative) were
also included in the study, since LSD1 is often overexpressed in
breast cancers. The results are summarized in Table 3, together
with the LSD1 inhibitory activity of these compounds. Of
interest is that the five competitive inhibitors exhibited strong
antitumor activities against the four tumor cells with EC50
values of 280 nM to 8.6 μM. However, these compounds had
weak or no activity against the growth of normal fibroblast cells
WI-38. Particularly, LSD1 inhibitors 16, 17, 20, and 22 showed
potent activity against proliferation of MV4-11 leukemia cells
with EC50 values ranging from 280 nM to 480 nM, while less
potent compound 13 exhibited a reduced activity (EC50 = 1.1
μM). These compounds were also found to have strong activity
against the growth of Molm-13 leukemia cells with EC50 values
of 0.84−4.4 μM. For MCF-7 breast cancer, more potent LSD1
G
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Table 3. Antiproliferative Activity (EC50, μM) of LSD1 Inhibitors against Cancer and Normal Cells
compd
LSD1 Ki (μM)
MV4-11
Molm-13
MCF-7
MDA-MB231
WI-38
>50
13
16
17
20
22
S2
0.22
1.1 0.12
4.4 1.1
0.84 0.18
3.4 1.2
3.2 1.1
3.6 0.5
0.32 0.1
6.9 1.8
3.6 1.5
3.6 1.5
3.3 0.8
5.3 2.2
6.6 2.0
8.6 2.4
6.9 1.9
5.6 1.0
7.6 2.9
7.5 2.2
8.9 2.5
0.058
0.029
0.088
0.046
0.28 0.03
0.36 0.13
0.30 0.02
0.48 0.12
0.084 0.026
30.0 9.2
26.6 6.9
20.0 4.6
17.5 4.8
>50
a
0.077
a
IC50 value for compound S2, which is a covalent inhibitor of LSD1.
inhibitors 16, 17, 20, and 22 appeared to be superior (EC50 of
3.3−3.6 μM) to compound 13 (EC50 of 6.9 μM). These
compounds also showed fairly good antiproliferative activity
against MDA-MB231 breast cancer cells with EC50 values of
5.6−8.6 μM. In addition, these five competitive LSD1 inhibitors
showed similar antitumor activities as compared to the covalent
inhibitor S2, which also exhibited strong antiproliferative
activity (EC50 of 0.084−8.9 μM) against the tumor cells,
while it was inactive against the normal cells. Overall, the strong
and selective antitumor activity suggested that this class of
LSD1 inhibitors could be potentially useful cancer therapeutics
and further efforts of compound development are therefore
warranted.
methylation levels of several other histone lysine residues
(including H3K4me3), presumably because these are not
cellular substrates of LSD1. Finally, five potent LSD1 inhibitors
were found to exhibit potent to good activity against
proliferation of two MLL-rearranged leukemia cells and two
breast cancer cells with EC50 values ranging from 0.28 to 8.6
μM, while no or very weak activity of these compounds against
the growth of normal fibroblast cells was observed.
EXPERIMENTAL SECTION
■
All chemicals for synthesis were purchased from Alfa Aesar (Ward Hill,
MA) or Aldrich (Milwaukee, WI). All compounds were characterized
by 1H NMR on a Varian (Palo Alto, CA) 400-MR spectrometer.
Identification of the most active compounds was confirmed by HRMS
using an Agilent 6550 iFunnel quadrupole-time-of-flight (Q-TOF)
mass spectrometer with electrospray ionization (ESI). The purities of
synthesized compounds were determined by a Shimadzu Prominence
HPLC with a Zorbax C18 column (4.6 mm × 250 mm) monitored by
UV at 254 nm. The purities of the reported compounds were found to
be >95%. HPLC−MS experiments were performed using a Shimadzu
Prominence HPLC connected to a LCMS-2020 MS system.
CONCLUSIONS
■
LSD1 plays an important role in maintaining a balanced
methylated H3K4, a key “histone code” for active gene
transcription. It has also been found to be overexpressed in
certain cancers such as breast cancer.28−30 Therefore, LSD1
represents a drug target for intervention and more chemotypes
of potent LSD1 inhibitors are needed.
Synthesis and characterization of compounds 4−45 can be found
in Supporting Information Experimental Section.
On the basis of previous SAR of cyclopropylamine
containing LSD1 inhibitors, a series of 3-(piperidin-4-
ylmethoxy)pyridine and related compounds were found to be
new LSD1 inhibitors. Structure guided molecular modeling and
medicinal chemistry studies have led to the identification of
several potent LSD1 inhibitors (e.g., 16, 17, 20, and 22) with Ki
values as low as 29 nM. SAR investigation of this series of
compounds showed that (1) a pyridine or pyrazine core
structure is superior to a benzene ring; (2) a basic 3-(piperidin-
4-ylmethoxy) R3 group is important to LSD1 inhibition; (3) 4-
cyanophenyl group is the best R5 substituent in this study, while
many other groups such as amide or alkyl-containing phenyl
groups significantly reduce the inhibitory activity; (4) a phenyl
R6 group is superior to a corresponding pyridine group; and (5)
among the substituted phenyl R6 groups, several para-
substituents (e.g., -Me, CF3, and -OCF3 in 17, 16, and 22)
can significantly enhance LSD1 inhibition (by as high as 79×),
while meta- or ortho-substituents are less effective.
Enzyme kinetics studies suggested that these compounds are
competitive inhibitors against the dimethylated H3K4 peptide
substrate. Consistent with the kinetics data, docking studies
showed that these compounds can be well docked into the
substrate binding site of LSD1. In addition, the docking results
could be used to rationalize many of the observed SARs.
Several representative LSD1 inhibitors were found to exhibit
very high selectivity against related MAO-A and -B enzymes.
This feature is desirable for this series of LSD1 inhibitors, due
to important functions of MAO-A and -B in the central nervous
system. The most potent LSD1 inhibitor 17 was found to
increase the cellular level of H3K4me2, showing on-target
activity in cells. However, it did not significantly affect the
LSD1 Inhibition. The catalytic domain (172−833) of human
LSD1 was cloned, inserted into pGEX-KG vector, and the DNA
sequence was verified by sequencing. The pGEX-KG-LSD1 plasmid
thus generated was transformed into E. coli BL21-CodonPlus strain
(Agilent) and cultured at 37 °C in LB medium containing ampicillin
(50 μg/mL) and chloramphenicol (34 μg/mL). When the optical
density of the bacterial culture approached ∼0.9 at 600 nm, LSD1
expression was induced by adding 0.2 mM isopropylthiogalactoside
(IPTG) at 25 °C for 20 h. Cells were next collected, lysed, centrifuged
for 20 min at 20 000 rpm. The resulting supernatant was subjected to
an affinity column chromatography with glutathione sepharose resins.
The recombinant GST-LSD1 fusion protein was obtained in ∼90%
purity (SDS−PAGE) by elution with 10 mM glutathione solution and
a secondary purification using a Superdex 200 gel filtration column
chromatography followed by concentration.
A mass spectrometry based biochemical assay for LSD1 was
developed, with the amount of demethylated product peptide being
quantitatively determined by HPLC−MS. Increasing concentrations of
a compound were incubated with LSD1 (150 nM) in 50 mM
phosphate buffer (pH = 7.0) containing 0.01% Brij-35 for 10 min at 25
°C, before initiation of the reaction by adding 10 μM of dimethylated
peptide substrate ARTK(Me2)QTARKSTGGKAPRKQKA. The total
volume of the reaction mixture was 60 μL. Reactions were terminated
after 30 min by adding 6 N formic acid (5 μL), and 20 μL of the
reaction mixture was subjected to HPLC−MS to separate and
determine the amount of the reaction product. HPLC was run using a
Phenomenex C18 column (250 mm × 4.6 mm, 5 μm) with
acetonitrile/water (40:60, containing 0.1% TFA) as an eluent at a
flow rate of 0.5 mL/min. A selected ion monitoring (SIM) for 1142
Da was used to detect and quantitate the amount of the product
ARTK(Me1)QTARKSTGGKAPRKQKA (parameters: interface volt-
age, 3.0 kV; detector voltage, 1.3 kV; nebulizing gas, 1.5 L/min; drying
gas, 15 L/min; desolvation line temperature, 300 °C; heat block
H
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Journal of Medicinal Chemistry
Article
temperature, 300 °C; Pirani gauge vacuum, 150 Pa; ion gauge vacuum:
5 × 10−4 Pa). To ensure an initial velocity was determined, ∼5% of the
dimethylated substrate was consumed before the reaction was stopped.
Therefore, monomethylated peptide was obtained almost as the only
product. No significant amount of the nonmethylated product was
detected.
Data were imported into Prism 5.0 (GraphPad), and the IC50 values
were determined using the sigmoidal dose response fitting in the
program. For compounds with IC50 > 1.5 μM (i.e., ≫[LSD1]), Ki
values were calculated using the Cheng−Prusoff equation Ki = IC50/(1
+ [S]/Km), where [S] is the concentration of the peptide substrate (10
μM) and Km is a reported value of 10 μM.27a For compounds with
IC50 < 1.5 μM, Ki values were calculated using the Morrison tight
inhibition modeling in Prism. The reported Ki values were the mean
values of at least three independent experiments.
Enzyme Kinetics Study. A steady-state kinetics study was
conducted by measuring the initial velocities of reactions catalyzed
by LSD1 while varying the concentrations of compound 5 (0, 2 μM, 4
μM, and 6 μM) and the peptide substrate (2 μM, 3 μM, 5 μM, 10 μM,
50 μM, and 100 μM). Data were imported into SigmaPlot and fitted
into the competitive, noncompetitive, and uncompetitive inhibition
models. The best kinetic model was determined by the highest R2 and
lowest AICc values. Lineweaver−Burk plots were generated by
SigmaPlot.
according to the manufacturer’s protocol. Equal amounts of histones
(2 μg) were separated on SDS−PAGE and transferred to PVDF
membranes. The blots were probed with primary antibodies against
H3K4me2 and -me3, H3K27me2, H3K36me2, H4K20me2, and H3
(Cell Signaling), followed by anti-rabbit IgG (Thermo Scientific)
secondary antibodies. Quantitation of these blots were performed
using the program ImageJ.
Cell Growth Inhibition. The antiproliferation assays were done
using our previous method.47,48 In brief, for leukemia cells, 106 cells
per well were added into 96-well plates and cultured with increasing
concentrations of a compound in RPMI-1640 medium supplemented
with 10% fetal bovine serum and penicillin (100 U/mL) and
streptomycin (100 μg/mL) at 37 °C in a 5% CO2 atmosphere with
100% humidity. For solid tumor and normal cells, 105 cells per well
were added into 96-well plates and cultured in Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10% fetal bovine serum
and penicillin (100 U/mL) and streptomycin (100 μg/mL) overnight
for cell attachment. After addition of increasing concentration of a
compound, plates were incubated for a given period of time. Cell
viability was assessed by using an XTT assay kit (Roche) for leukemia
cells or an MTT assay (Sigma) for attachment cells. Compound IC50
values were calculated from dose response curves using Prism 5.0.
ASSOCIATED CONTENT
■
Molecular Modeling. Docking studies were performed with our
previous published methods44−46 using Schrodinger suite (version
S
* Supporting Information
̈
2015),40 which includes all of the programs described below. The
crystal structure of LSD1 in complex with an H3K4 peptide (PDB
code 2V1D) was prepared using the module “Protein Preparation
Wizard” in Maestro (version 10.1) using the default protein
parameters. Hydrogen atoms were added, the H3K4 peptide ligand
and all water molecules were extracted, and FAD was retained in the
protein structure for docking. H-bonds were next optimized, the
partial charges for all atoms were assigned, and the protein was energy-
minimized using OPLS-2005 force field. A receptor grid, which is large
enough to contain the active site, was generated with the program
Glide without any constraints. Inhibitor compounds were built,
energy-minimized using OPLS-2005 force field in Maestro, and then
docked into the prepared protein structure using Glide (docking
parameters: standard-precision and dock flexibly).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01361.
Figures S1−S3 and Experimental Section showing
detailed compound synthesis and characterization
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 713-798-7415. E-mail: ysong@bcm.edu.
Inhibition of MAO-A/-B. Human recombinant MAO-A and -B
enzymes were purchased from Sigma, and their biochemical activity
and inhibition were determined using a MAO-Glo assay kit from
Promega. Following the manufacturer’s protocol, the assays were
performed in 384-well white plates by adding all reagents in the kit,
MAO-A or -B (100 nM) and increasing concentrations of inhibitors to
a final volume of 20 μL. After 60 min, the reactions were stopped by
adding a reconstituted luciferin detection reagent (20 μL/well). Upon
incubation for an additional 20 min, luminescence of each well was
determined using a Beckman DTX-880 microplate reader. Using Km
values provided in the kit, Ki values were similarly calculated as
described above.
Inhibition of Akt Kinase. The inhibitory activity of compound 17
against human recombinant Akt kinase was determined using a
commercially available ELISA kit (Enzo life Sciences, Farmingdale,
NY). Following the manufacturer’s protocol, Akt kinase (5 nM), ATP,
and increasing concentrations of 17 (0.01−100 μM) were added to a
final volume of 30 μL in a 96-well plate precoated with a substrate.
After a 60 min incubation in 30 °C, the reactions were stopped by
removal of all reactants. The primary antibody against the
phosphorylated substrate and a peroxidase conjugated secondary
antibody were added sequentially. The optical absorbance at 450 nM
of each well was developed with tetramethylbenzidine substrate
(TMB) and determined using a Beckman DTX-880 microplate reader.
The data were imported into Prism (version 5.0, GraphPad) and IC50
values were calculated by using a standard dose response curve fitting.
The reported value is the average of three independent experiments.
Western Blot. 106 MV4-11 cells/well were incubated with
compounds 17 and 28 at 0.2 and 1 μM for 3 days. Histone proteins
were extracted using EpiQuik total histone extraction kit (Epigentek)
Author Contributions
§F.W. and C.Z. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Grant RP150129 from Cancer
Prevention and Research Institute of Texas (CPRIT) and
Grant R01NS080963 from National Institute of Neurological
Disorders and Stroke (NINDS/NIH) to Y.S. We also thank Dr.
Feng Li of BCM Metabolomics Core for performing HRMS.
ABBREVATIONS USED
■
BOC, tert-butyloxycarbonyl; FAD, flavin adenine dinucleotide;
H3K4, histone H3 lysine 4; H3K4me1, -2, or -3, mono-, di-, or
trimethylated H3K4; LSD1, lysine specific demethylase 1;
MAO, monoamine oxidase; MLL, mixed lineage leukemia;
SAR, structure−activity relationship
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DOI: 10.1021/acs.jmedchem.5b01361
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