Identification of the KDM2/7 histone lysine demethylase subfamily inhibitor and its antiproliferative activity

Article abstract of DOI:10.1021/jm400624b

Histone N^ε-methyl lysine demethylases KDM2/7 have been identified as potential targets for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits KDM2A, KDM7A, and KDM7B, with IC₅₀s of 6.8, 0.2, and 1.2 μM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.

Full text of DOI:10.1021/jm400624b

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Identification of the KDM2/7 Histone Lysine Demethylase Subfamily
Inhibitor and its Antiproliferative Activity
Takayoshi Suzuki,*,†,‡ Hiroki Ozasa,§ Yukihiro Itoh,† Peng Zhan,† Hideyuki Sawada,† Koshiki Mino,∥
Louise Walport,⊥ Rei Ohkubo,∥ Akane Kawamura,⊥ Masato Yonezawa,# Yuichi Tsukada,▽
Anthony Tumber,○ Hidehiko Nakagawa,§ Makoto Hasegawa,∥ Ryuzo Sasaki,∥ Tamio Mizukami,*,∥
Christopher J. Schofield,⊥ and Naoki Miyata*,§
†Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 13 Taishogun Nishitakatsukasa-Cho, Kita-ku, Kyoto
603-8334, Japan
‡PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
§Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
∥Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829,
Japan
⊥Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
#Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, 4-6-1 #34
Komaba, Meguro-ku, Tokyo 153-8904, Japan
▽Division of Molecular Immunology, Research Center for Infectious Diseases, Medical Institute of Bioregulation, Kyushu University,
Higashi-ku, Fukuoka 812-8582, Japan
○Structural Genomics Consortium, University of Oxford, Headington OX3 7DQ, U.K.
S
* Supporting Information
ABSTRACT: Histone Nε-methyl lysine demethylases KDM2/7 have been identified as potential targets
for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of
hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits
KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2, and 1.2 μM, respectively. While inhibitors of
KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted
antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.
Several JmjC-domain containing demethylases such as
KDM2, KDM4, KDM5, and KDM7 have been implicated in
tumorigenesis.5 For example, it has been reported that KDM7B
(also known as JHDM1F, PHF8, KIAA1111) is associated with
proliferation of prostate cancer cells and osteosarcoma cells.6
Therefore, KDM2/7-subfamily inhibitors are of interest, both
as tools for probing the biological functions of KDM2/7
subfamily and also as candidate anticancer agents. Several types
of KDM inhibitors have been found so far by us and others,5a,7
including N-oxalylglycine (NOG, 1), 2,4-pyridinedicarboxylic
acid (PCA, 2), NCDM-32 (3), daminozide (4), and GSK-J1
(5) (Chart 1). Among these, daminozide (4) has been reported
as a KDM2/7-subfamily selective inhibitor. Daminozide (4)
was once widely used as a plant growth retardant but later
withdrawn because of genotoxic concerns.8 The genotoxicity of
daminozide (4) is proposed to be derived from its 1,1-
dimethylhydrazine structure.9 Therefore, it is desirable to
INTRODUCTION
■
Nε-Methylation of lysine residues on histone tails is a pivotal
“epigenetic mark” that is involved in defining both transcrip-
tionally active and inactive chromatin.1 For instance, methyl-
ation at lysine 4 of histone 3 (H3K4) is associated with actively
transcribed gene loci, whereas methylation at H3K9 and
H3K27 leads to reduction in transcription.2 Methylation of
histone lysine residues is reversible and is mediated by histone
lysine methyltransferases and histone lysine demethylases
(KDMs).3
KDMs fall into two classes as defined by their structure and
mechanisms.3 One class comprises the KDM1s, which are
homologues of the flavin-containing amine oxidases.3a,b The
other class comprises Jumonji C (JmjC)-domain containing
demethylases, which belong to the family of Fe(II)/α-
ketoglutarate-dependent oxygenases.3c,d Human JmjC-domain
containing demethylases can be divided into five subfamilies,
i.e., the KDM2/7, KDM3, KDM4, KDM5, and KDM6
subfamilies, according to sequence and structural similarities.4
Received: April 20, 2013
Published: August 21, 2013
© 2013 American Chemical Society
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Chart 1. Examples of Reported Small Molecule KDM Inhibitors
identify novel KDM2/7-inhibitors without the 1,1-dimethylhy-
drazine structure for use as functional probes and for evaluation
in cancer therapies. Here we describe the identification of a
novel KDM2/7 subfamily inhibitor that shows antiproliferative
activity.
RESULTS AND DISCUSSION
■
With the aim of identifying potent KDM2/7 inhibitors,
hydroxamate derivatives, which have been previously prepared
in work on KDM4 inhibitors,7d were screened for KDM7B-,
KDM4A (also known as JMJD2A, JHDM3A)-, KDM4C (also
known as JMJD2C, JHDM3C, GASC1)-, and KDM5A (also
known as JARID1A, RBP2)-inhibitory activity. We selected
KDM4A, KDM4C, and KDM5A for the primary inhibition
selectivity studies because they are relatively similar to KDM7B
in that they have a plant homeodomain.4 Compounds 1−4
were evaluated as positive controls for inhibition. In our
enzyme assays, compound 4 selectively inhibited KDM7B in
preference to KDM4 and KDM5 as reported before (Table 1).4
We also found that compounds 17a and 27b did not inhibit
KDM7B (at 250 μM), and compound 2 showed selectivity for
the KDM4-subfamily over KDM7B as for the reported KDM4
inhibitor 37d under the conditions tested. Among the tested
compounds, compound 6 was found to inhibit KDM7B
relatively selectively although its potency was moderate (IC50
= 24 μM).
CHEMISTRY
■
The routes used for the synthesis of compounds 7−13 (Chart
2), which were prepared for this study, are shown in Schemes 1
Chart 2. Structures of Compounds 7−13
To improve the potency and selectivity of 6 for KDM7B, we
investigated the binding mode of 6 with KDM7B by modeling
(Figure 1).10 An inspection of the simulated KDM7B/
compound 6 complex indicates that the hydroxamate group
of compound 6 coordinates to the Fe(II) in a bidentate
manner, via its carbonyl and hydroxyl groups, and that its
carboxylate group will bind to form hydrogen bonds/
electrostatic interactions with Asn 189, Thr 244, and Lys 264
at the KDM7B active site, in a similar manner to the C-5
carboxylate of 2-oxoglutarate. Most importantly, it is also
suggested that the alkyl chain of compound 6 binds to a
hydrophobic pocket formed by the side chains of Ile 191, Tyr
234, Leu 236, Phe 250, Val 255, Tyr 257, and Phe 359, which
we propose is unique to KDM2 and KDM7 (Supporting
Information Figures S1−S6).
On the basis of the structure of the modeled KDM7B/
compound 6 complex, we designed and synthesized com-
pounds 8−13 (Table 2) in which the isopropyl group of
compound 6 was replaced with various hydrophobic groups
that might interact with hydrophobic amino acid residues more
efficiently than compound 6. Compound 7 was prepared to
investigate the importance of the carboxylic acid of the
inhibitor.
and 2. Scheme 1 shows the preparation of compounds 8−13.
Michael addition of O-benzylhydroxylamine to tert-butyl
acrylate 14 afforded amine 15. 9-Bromononanoic acid sodium
salt 16 was converted to alkyl carboxylic acids 19−22 by
treatment with an alkyl magnesium bromide 17 and dilithium
tetrachlorocuprate. 9-Phenylnonanoic acid 23 was obtained by
the treatment of 16 with phenyl magnesium bromide and ferric
acetylacetonate in the presence of tetramethylethylenediamine
and hexamethylenetetramine in THF. Carboxylic acids 18−23
were treated with amine 15 in the presence of EDCI and HOBt
to give amides 24−29. The benzyl group of compounds 24−29
was removed by hydrogenation to give hydroxamates 30−35.
Removal of the tert-butyl group of 30−35 using hydrochloric
acid afforded the desired compounds 8−13.
Scheme 2 shows the synthesis of compound 7. Michael
addition of O-benzylhydroxylamine to methyl acrylate 36
afforded amine 37. 10-Methylundecanoic acid 38 was prepared
using the procedure described for the synthesis of 19−22.
Amine 37 was treated with 38 in the presence of EDCI and
HOBt to yield compound 39. Removal of the benzyl group of
compound 39 gave compound 7.
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a
Scheme 1. Synthesis of Compounds 8−13
a
Reagents and conditions: (a) O-benzylhydroxylamine, Et3N, 1,4-dioxane, reflux, 44%; (b) RMgBr (17), Li2CuCl4, THF, −10 °C, 74−91%; (c)
PhMgBr, Fe(acac)3, TMEDA, HMTA, THF, 0 °C, 67%; (d) EDCI, HOBt, DMF, room temp, 24−72%; (e) H2, Pd/C, AcOEt, room temp, 34−95%;
(f) HCl, CH2Cl2, room temp, 23−59%.
a
Scheme 2. Synthesis of Compound 7
a
i
Reagents and conditions: (a) O-benzylhydroxylamine, Et3N, 1,4-dioxane, reflux, 59%; (b) PrMgBr, Li2CuCl4, THF, −10 °C, 29%; (c) EDCI,
HOBt, DMF, room temp, 65%; (d) H2, Pd/C, AcOEt, room temp, 61%.
Table 1. In Vitro KDM7B-, KDM4A-, KDM4C-, and
As shown in Table 2, the conversion of carboxylic acid (6) to
a methyl ester (7) significantly reduced the KDM7B-inhibitory
activity, supporting the proposed binding mode of compound
6. Compounds 8−13 were found to be KDM7B inhibitors. In
particular, a pronounced inhibitory effect (IC50 = 1.2 μM) was
observed with the cyclopropyl-containing compound 9, which
was 20-fold more active than 6 in KDM7B in MALDI assays.
Docking of compound 9 in the active site of KDM7B suggested
that the cyclopropyl group of compound 9 interacts with the
phenyl-group of Phe 250 through CH−π or cyclopropyl−π
interactions (Figure 2). Furthermore, compound 9 inhibited
KDM2A (also known as JHDM1A, FBXL11), KDM7A (also
known as JHDM1D, KIAA1718), and KDM7B, demonstrating
inhibition of all KDM2/7 subfamily members tested (Tables 2
and 3). Compound 9 displayed selectivity for KDM2/7 over
KDM4A (IC50 > 120 μM), KDM4C (IC50 = 83 μM), KDM5A
KDM5A-Inhibitory Activities of Compounds 1−4 and
a
Screening Hit Compound 6
(IC50 = 55 μM), and KDM6A (also known as UTX) (IC50
>
100 μM); note however different assays conditions were used
(see Experimental Section). Thus, compound 9 was the most
potent KDM7B inhibitor identified in the enzyme assays. In
addition, compound 13 showed comparatively high selectivity
for KDM2A over KDM7B and the other isozymes tested.
Because the molecular modeling suggest that the space around
a
Values are means of two experiments that in all cases varied by 19%
b
or less. The IC50 of 4 for KDM7B was reported to be 0.55 μM in
AlphaScreen assays.4
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Figure 1. View of the conformation of compound 6 (ball-and-stick)
docked into the KDM7B active site.
Figure 2. View of the conformation of compound 9 (ball-and-stick)
docked into the KDM7B active site.
the cyclopropane ring of 9 is not so large in the hydrophobic
pocket of KDM7B (Figure 2), it may be difficult for compound
13 bearing a phenyl ring to have a conformation which can
efficiently interact with Phe of the pocket. On the other hand,
Table 2. In Vitro KDM7B-, KDM2A-, KDM4A-, KDM4C-, KDM5A-, and KDM6A-Inhibitory Activities of Compounds 4 and 6−
a
13
a
b
Values are means of two experiments that in all cases varied by 30% or less. The IC50 of 4 for KDM7B was reported to be 0.55 μM in AlphaScreen
assays.4 Not determined.
c
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Table 3. Enzyme-Inhibitory Activities and Antiproliferative
a
Activities of Compounds 1−3 and 9
IC50 (μM)
GI50 (μM)
compd KDM2A KDM7A KDM7B KDM4C KYSE150 HeLa
1
2
3
9
45
4.1
>250
15
>250
>250
19
>250
8.2
2.2
83
>240
>240
>470
16
>710
>240
>470
40
0.20
6.8
1.6
0.20
1.2
a
Values are means of two experiments that in all cases varied by 30%
or less.
KDM2A has a hydrophobic pocket more spacious than
KDM7B (Supporting Information Figure S2), in which the
phenyl ring can efficiently interact with hydrophobic amino
acid residues of KDM2A. These may be the reason that
compound 13 shows selectivity for KDM2A over KDM7B.
To investigate whether compound 9 is active as an inhibitor
of KDM7A and KDM7B in cells, we performed a cellular assay
with Western blot analysis. Because KDM7 is known to
catalyze the demethylation of H3K27me2,10,11 the methylation
level of H3K27 in cells was analyzed. In this study, we used N2a
cells because it has been reported that KDM7 is expressed in
the cells.11 As Figure 3 shows, the level of H3K27me2 was
Figure 4. Growth-inhibitory activity of compound 9 toward N2a cell
lines.
proliferation,7a however, the KDM4C inhibitors 2, 3 (Table 3),
and their methyl ester prodrugs (GI50 > 500 μM) did not show
any effects on the growth inhibition of tested cancer cells
although they are cell membrane permeable.7d,13 These results
suggest that the demethylase activity of KDM4C is not directly
associated with cancer cell growth, at least in some cell types.
On the other hand, cell growth suppression by the KDM2/7-
subfamily inhibitor 9 was observed for KYSE150 and HeLa cell
lines (Figure 5). Furthermore, compound 9 caused H3K27
Figure 3. Western blot detection of H3K27me2 levels in N2a cells
after 24 h incubation with dimethyl ester prodrug of NOG (DMOG)
and compound 9.
dose-dependently elevated in the presence of 9. Although the
interpretation of changes in global histone N-methylation status
can be complex, the elevation in the H3K27me2 levels is
consistent with KDM7 inhibition. These results suggest that
H3K27me2 demethylation is inhibited by compound 9 in cells,
and it looks to be useful as a tool for probing the biological role
of KDM7.
Although it has been reported that RNAi-mediated knock-
down of KDM7B suppresses the growth of some cancer cells,6
it remains unclear whether the demethylase function of
KDM7B is responsible for the suppression because RNAi-
mediated knockdown of KDM7B should cause loss of not only
the demethylase function but also other functions of KDM7B,12
including those relating to noncatalytic binding domains.
Initially, we investigated the N2a cell growth inhibition activity
of compound 9. N2a cell growth suppression by compound 9
was observed (GI50 = 86 μM) (Figure 4) at the concentration
range in which distinct H3K27 methylation was detected on
Western blot analysis (Figures 3). Thus, this may suggest the
demethylase function of KDM7 is involved in this cell growth,
however, more potent and selective compounds will be needed
to fully elucidate this fact. Next, we carried out cell growth
inhibition assays of compound 9 as well as prodrugs of 1, 2, and
3 using HeLa cells and KYSE150 cells (Table 3). It has also
been reported that knockdown of KDM4C decreases cell
Figure 5. Growth-inhibitory activity of compound 9 toward KYSE150
and HeLa cell lines.
methylation both in HeLa cells and in KYSE150 cells at the
concentration range in which the cell growth inhibition was
observed (Figure 6). The data shown in Table 3 indicate that
KDM2/7 inhibitors are worthy of evaluation as candidate
anticancer agents.
A recent study reported that KDM7B activates the
transcription of the E2F1 transcription factor in HeLa cells,
which promotes cell cycle progression.14 Because compound 9
decreases the growth of HeLa cells with H3K27me2
accumulation (Table 3; Figures 5 and 6), we examined whether
compound 9 down-regulates the expression of E2F1 in HeLa
cells by quantitative RT-PCR. As shown in Figure 7, compound
9 significantly decreases the mRNA level of E2F1 at 80 μM in
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Figure 6. Western blot detection of H3K27me2 levels in HeLa cells
and KYSE150 cells after 24 h incubation with compound 9.
Figure 8. Induction of cell cycle arrest in HeLa cells and KYSE150
cells by compound 9.
Figure 7. Change of E2F1 gene expression in HeLa cells by compound
9.
spectra were recorded on a JEOL JMS-SX102A mass spectrometer.
Purity tests by analytical HPLC used a Shimazu instrument equipped
with a ODS-3 (4.6 mm × 150 mm, GL Science) and eluted at 1 mL/
min with Milli-Q water and CH3CN, and all values ≥95% purity.
Preparative HPLC was performed with a Jasco instrument equipped
with a Inertsil ODS-3 (20 mm × 250 mm, GL Science) and eluted at
10 mL/min with Milli-Q water and CH3CN. Gradient conditions of
HPLC were as follows: (A is CH3CN containing 0.1% TFA
(CF3CO2H), B is Milli-Q water containing 0.1% TFA, C is CH3CN
containing 0.1% FA, D is Milli-Q water containing 0.1% FA
(HCOOH)); gradient (I), A 50% (0−2 min), A 50% to A 80% (2−
20 min), A 80% (20− 30 min), A 80% to A 50% (3− 40 min) water B;
gradient (II), C 60% (0− 2 min), C 60% to C 80% (2−20 min), C
80% (20−30 min), C 80% to C 50% (30−40 min) water D; gradient
(III), C 50% (0−2 min), C 50% to C 80% (2−20 min), C 80% (20−
30 min), C 80% to C 50% (30−40 min) water D. Reagents and
solvents were purchased from Aldrich, Tokyo Kasei Kogyo, Wako
Pure Chemical Industries, and Kanto Kagaku and used without
purification. Flash column chromatography was performed using Silica
Gel 60 (particle size 0.046−0.063 mm) supplied by Merck.
3-(9-Cyclopropyl-N-hydroxynonanamido)propanoic Acid (9).
Step 1: Preparation of tert-Butyl 3-(benzyloxyamino)propanoate
(15). A solution of tert-butyl acrylate (11.5 g, 90 mmol),
benzylhydroxylamine hydrochloride (3.6 g, 26 mmol), and triethyl-
amine (TEA) (6.0 mL) in dioxane (60 mL) was stirred with reflux for
19 h. The reaction mixture was poured into water and extracted with
AcOEt. The organic layer was separated and washed with brine and
dried over Na2SO4. Filtration, concentration in vacuo, and purification
by flash column chromatography (AcOEt/n−hexane = 1/4) gave 3.3 g
(44%) of 15 as yellow oil. 1H NMR (CDCl3, 500 MHz, δ; ppm) 7.35
(5H, m), 5.83 (1H, s), 4.70 (2H, s), 3.17 (2H, m), 2.50 (2H, m), 1.44
(9H, s).
which the growth of HeLa cells was affected. These data suggest
that the KDM7B-mediated regulation of E2F1 gene expression
may be one of the mechanisms of growth regulation in some
cancer cells.
We also investigated the effect of compound 9 on cell cycle
progression by FACS analysis. HeLa cells and KYSE150 cells
incubated with 10 or 100 μM of compound 9 for 24 h showed a
dose-dependent reduction in G2−M phase, whereas there was a
dose-dependent increase in G0−G1 phase (Figure 8). These
results revealed that HeLa cells and KYSE150 cells cultured
with compound 9 arrested in the G0/G1 phase of the cell cycle,
which is consistent with the down-regulation of E2F1 by
compound 9 (Figure 7).15
CONCLUSIONS
■
We have identified a novel KDM2/7 subfamily inhibitor 9,
which should be useful as a lead structure in the development
of more potent and selective KDM2/7 inhibitors. Such
inhibitors are candidates for anticancer agents as well as tools
for studying the biological roles of KDM2/7 subfamily in cells.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a Yanagimoto
micro melting point apparatus or a Buchi 545 melting point apparatus.
■
̈
Proton nuclear magnetic resonance spectra (1H NMR) and carbon
nuclear magnetic resonance spectra (13C NMR) were recorded using a
JEOL JNM-LA500, JEOL JNM-A500, or BRUKER AVANCE600
spectrometer in solvent as indicated. Chemical shifts (δ) are reported
in parts per million relative to the internal standard tetramethylsilane.
Elemental analysis was performed with a Yanaco CHN CORDER NT-
5 analyzer, and all values were within 0.4% of the calculated values,
which indicates >95% purity of the tested compounds. High-resolution
mass spectra (HRMS) and fast atom bombardment (FAB) mass
Step 2: Preparation of Cyclopropanenonanoic Acid (19). To the
suspension of 9-bromononanoic acid sodium salt (16) (400 mg, 1.5
mmol) in anhydrous THF (3 mL) was added the solution of Li2CuCl4
in THF (200 μL, 0.02 mmol) at −10 °C. Then, cyclopropyl
magnesium bromide (4.6 mL, 2.3 mmol) was added dropwise with
vigorous stirring. The color of the mixture changed from orange
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through green and blue to white. After addition of the Grignard
reagent, the temperature was kept at −10 °C for another 2 h. The
reaction mixture was then diluted with water and extracted three times
with Et2O, and the organic extracts were washed by saturated aqueous
solution of Na2CO3. The water layer was then acidified by HCl and
washed three times by Et2O, and the combined organic layers were
washed with water and dried over Na2SO4. Filtration and
3-(9-Cyclohexyl-N-hydroxynonanamido)propanoic Acid (11).
Compound 11 was prepared from 16 (2.0 g 7.7 mmol) using the
procedure described for 9 (steps 2−5): yield 21%; a white solid; mp
1
94−96 °C. H NMR (CDCl3, 500 MHz, δ; ppm) 3.94 (2H, m), 2.81
(2H, m), 2.43 (2H, m), 1.69−1.67 (7H, m), 1.26−1.17 (16H, m), 0.86
(2H, m). 13C NMR (CDCl3, 125 MHz, δ; ppm) 174.35, 168.35, 44.57,
44.33, 37.68, 37.55, 33.47, 32.20, 31.36, 31.04, 29.94, 29.53, 29.37,
26.86, 26.78, 26.47, 25.31, 24.63. MS (FAB) m/z 328 (MH+). HRMS
calcd for C18H34O4N, 328.24878, found 328.25015. HPLC tR = 21.86
min (gradient (I), purity 97.8%).
1
concentration in vacuo gave 19 (226 mg, 74%) as a white solid. H
NMR (CDCl3, 500 MHz, δ; ppm) 2.35 (2H, t, J = 7.5 Hz), 1.66−1.59
(2H, m), 1.32−1.21 (12H, m), 1.18−1.14 (2H, m), 0.67−0.60 (1H,
m), 0.41−0.35 (2H, m).
3-(9-Cycloheptyl-N-hydroxynonanamido)propanoic Acid (12).
Compound 12 was prepared from 16 (400 mg, 1.5 mmol) using the
procedure described for 9 (steps 2−5): yield 13%; a white solid; mp
Step 3: Preparation of tert-Butyl 3-(N-Benzyloxy-9-
cyclopropylnonanamido)propanoate (25). A mixture of 19 (221
mg, 1.10 mmol), 15 (232 mg, 1.10 mmol), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) (320 mg, 1.70 mmol),
and HOBt·H2O (226 mg, 1.70 mmol) in DMF (5 mL) was stirred at
80 °C for 15 h. The mixture was poured into water and extracted with
AcOEt. The organic layer was washed with brine and dried over
Na2SO4. Filtration, concentration in vacuo, and purification by silica
gel flash column chromatography (AcOEt/CHCl3 = 1/10) gave 115
1
86−87 °C. H NMR (CDCl3, 500 MHz, δ; ppm) 3.94 (2H, m), 2.79
(2H, m), 2.41 (2H, m), 1.68−1.55 (8H, m), 1.49−1.37 (5H, m),
1.30−1.25 (10H, m), 1.18−1.11 (4H, m). 13C NMR (CDCl3, 125
MHz, δ; ppm) 174.01, 168.27, 60.43, 44.57, 44.25, 39.28, 38.24, 34.68,
32.17, 31.32, 31.04, 29.96, 29.55, 29.37, 28.58, 27.42, 26.59, 25.29,
24.63. MS (FAB) m/z 342 (MH+). HRMS calcd for C19H36O4N,
342.26443, found 342.26507. HPLC tR = 21.64 min (gradient (III),
purity 98.9%).
1
mg (24%) of 25 as a colorless oil. H NMR (CDCl3, 500 MHz, δ;
ppm) 7.38 (5H, m), 4.82 (2H, s), 3.94−3.88 (2H, m), 2.53 (2H, t, J =
7.0 Hz), 2.35 (2H, t, J = 7.0 Hz), 1.60−1.54 (4H, m), 1.41 (9H, s),
1.30−1.24 (10H, m), 1.19−1.15 (2H, m), 0.66−0.63 (1H, m), 0.40−
0.36 (2H, m).
3-(N-Hydroxy-9-phenylnonanamido)propanoic Acid (13). Step 1:
Preparation of 9-Phenylnonanoic Acid (23). To a solution of 9-
bromononanoic acid sodium salt (16) (1.0 g, 3.9 mmol), Fe(acac)3
(67 mg, 0.19 mmol), tetramethylethylenediamine (57 μL, 0.38 mmol),
and hexamethylenetetramine (27 mg, 0.19 mmol) in dry THF (6.0
mL) was added phenylmagnesium bromide (4.87 mL, 5.0 mmol) at 0
°C. The reaction mixture was then stirred for 2.5 h, after which it was
diluted with water and extracted three times with Et2O. The organic
extracts were washed with saturated aqueous solution of Na2CO3. The
aqueous layer was then acidified by HCl and extracted three times with
Et2O, and the combined organic layers were washed with water and
dried over Na2SO4. Filtration and concentration in vacuo gave 23 (610
mg, 67%) as a yellow oil. 1H NMR (CDCl3, 500 MHz, δ; ppm) 7.29−
7.25 (2H, m), 7.18−7.16 (3H, m), 2.60 (2H, t, J = 7.3 Hz), 2.34 (2H,
m), 1.64−1.61 (4H, m), 1.39−1.28 (8H, m).
Step 4: Preparation of tert-Butyl 3-(9-Cyclopropyl-N-
hydroxynonanamido)propanoate (31). To a solution of 25 (116
mg, 0.27 mmol) in 3 mL of AcOEt was added 50 mg of 5% Pd/C. The
mixture was stirred under H2 at room temperature for 1 h and then
filtered through Celite. The filtrate was concentrated in vacuo and
purified by flash column chromatography (AcOEt/CHCl3 = 3/1) to
give 72 mg (78%) of 31 as a white solid. 1H NMR (CDCl3, 500 MHz,
δ; ppm) 8.36 (1H, broad s), 8.13 (1H, broad s), 3.89−3.83 (2H, m),
2.65 (2H, t, J = 6.0 Hz), 2.46−2.40 (2H, m), 1.60−1.54 (4H, m), 1.46
(9H, s), 1.40−1.25 (10H, m), 1.19−1.15 (2H, m), 0.65−0.62 (1H, m),
0.40−0.36 (2H, m).
Step 5: Preparation of 3-(9-Cyclopropyl-N-hydroxynonanamido)-
propanoic Acid (9). To a solution of 31 (69 mg, 0.20 mmol) in DCM
(1 mL) was added 4N HCl in dioxane (1 mL) in a dropwise fashion
with ice-bath cooling; the mixture was stirred at room temperature for
16 h. Concentration in vacuo gave a colorless solid. The solid was
recrystallized from AcOEt to give 13 mg (23%) of 9 as colorless
Steps 2, 3, and 4: Preparation of 3-(N-Hydroxy-9-
phenylnonanamido)propanoic Acid (13). Compound 13 was
prepared from 23 (604 mg, 2.6 mmol) using the procedure described
1
for 9 (steps 3−5): yield 19%; a white solid; mp 87−88 °C. H NMR
(CDCl3, 500 MHz, δ; ppm) 7.27 (3H, m), 7.18−7.17 (2H, m), 3.93
(2H, m), 2.80 (2H, m), 2.59 (2H, t, J = 7.6 Hz), 2.46−2.41 (2H, m),
1.60 (4H, m), 1.31 (8H, m). 13C NMR (CDCl3, 125 MHz, δ; ppm)
177.15, 174.53, 142.88, 128.40, 128.23, 125.57, 44.40, 35.96, 31.83,
31.47, 29.34, 29.27, 29.16, 25.28, 24.68, 22.65. MS (FAB) m/z 322
(MH+). HRMS calcd for C18H28O4N, 322.20183, found 322.20252.
HPLC tR = 15.19 min (gradient (I), purity 96.1%).
1
crystals: mp 86−87 °C. H NMR (CDCl3, 500 MHz, δ; ppm) 3.94
(2H, m), 2.79 (2H, m), 2.42 (2H, m), 1.64 (4H, m), 1.37−1.29 (10H,
m), 1.19−1.15 (2H, m), 0.64 (1H, m), 0.40−0.36 (2H, m). 13C NMR
(CDCl3, 125 MHz, δ; ppm) 176.88, 173.92, 44.62, 44.26, 38.06, 34.75,
29.63, 29.47, 29.36, 26.50, 25.27, 24.63, 10.96, 4.36. MS (FAB) m/z
286 (MH+). HRMS calcd for C15H28O4N, 286.20183, found
286.20242. HPLC tR = 10.27 min (gradient (III), purity 96.1%).
3-(N-Hydroxydecanamido)propanoic Acid (8). Compound 8 was
prepared from 18 using the procedure described for 9 (steps 3−5):
yield 14% from 18 (800 mg, 4.6 mmol); a pale-pink solid; mp 84−85
Methyl 3-[Hydroxyl(10-methylundecanoyl)amino]propanoate
(7). Step 1: Preparation of Methyl 3-(Benzyloxyamino)propanoate
(37). Compound 37 was prepared from methyl acrylate (36) (4.4 g, 51
mmol) using the procedure described for 9 (step 1): yield 59%; a
yellow oil. 1H NMR (CDCl3, 500 MHz, δ; ppm) 7.37−7.28 (5H, m),
5.82 (1H, broad s), 4.68 (2H, s), 3.67 (3H, s), 3.20 (2H, t, J = 6.4 Hz),
2.60 (2H, t, J = 6.4 Hz).
1
°C. H NMR (CDCl3, 500 MHz, δ; ppm) 3.96−3.90 (2H, m), 2.80−
2.76 (2H, m), 2.47−2.42 (2H, m), 1.70−1.50 (2H, m), 1.30−1.27
(12H, m), 0.88 (3H, t, J = 7.0 Hz). 13C NMR (CDCl3, 125 MHz, δ;
ppm) 177.20, 174.61, 44.50, 32.42, 31.88, 31.60, 29.46, 29.37, 29.28,
25.31, 24.66, 22.67, 14.11. MS (FAB) m/z 260 (MH+). HRMS calcd
for C13H26O4N, 260.18618, found 260.18719. HPLC tR = 10.37 min
(gradient (I), purity 96.8%).
Steps 2, 3, and 4: Preparation of Methyl 3-[Hydroxyl(10-
methylundecanoyl)amino]propanoate (7). Compound 7 was
prepared from 37 (3.1 g, 15 mmol) using the procedure described
1
for 9 (steps 2−4): yield 11%; a colorless oil. H NMR (CDCl3, 500
MHz, δ; ppm) 3.94 (2H, m), 3.73 (3H, s), 2.75 (2H, m), 2.50−2.44
(2H, m), 1.62−1.56 (4H, m), 1.51 (1H, sep, J = 6.7 Hz), 1.30−1.25
(8H, m), 1.20−1.10 (2H, m), 0.86 (2H, d, J = 6.7 Hz). 13C NMR
(CDCl3, 125 MHz, δ; ppm) 52.39, 44.59, 39.04, 32.57, 29.88, 29.54,
29.39, 27.98, 27.39, 25.30, 24.68, 22.66. MS (EI) m/z 301 (M+).
HRMS calcd for C16H31O4N, 301.22531, found 301.22442. HPLC tR =
11.81 min (gradient (II), purity 97.9%).
3-(9-Cyclopentyl-N-hydroxynonanamido)propanoic Acid (10).
Compound 10 was prepared from 16 (400 mg, 1.5 mmol) using the
procedure described for 9 (steps 2−5): yield 14%; a white solid; mp
1
101−102 °C. H NMR (CDCl3, 500 MHz, δ; ppm) 3.93 (2H, m),
2.79 (2H, m), 2.42 (2H, m), 1.72 (4H, m), 1.60−1.55 (3H, m), 1.53−
1.48 (2H, m), 1.30−1.26 (12H, m), 1.06−1.04 (2H, m). 13C NMR
(CDCl3, 125 MHz, δ; ppm) 176.76, 174.28, 44.62, 44.23, 40.18, 36.26,
32.74, 32.44, 32.17, 31.32, 31.05, 29.90, 29.53, 29.38, 28.79, 25.21,
24.62. MS (FAB) m/z 314 (MH+). HRMS calcd for C17H32O4N,
314.23313, found 314.23329; Anal. Calcd for C17H31NO4: C, 65.14;
H, 9.97; N, 4.47. Found: C, 64.75; H, 9.95; N, 4.53.
Molecular Modeling. The X-ray structure of KDM7B (PDB code
3KV4) was used as a model for docking. Protein preparation, receptor
grid generation, and ligand docking were performed using the software
Glide 3.5. Compounds 6 and 9 were docked into the active site of the
protein. The standard precision mode of Glide was used to determine
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favorable binding poses, which allowed the ligand conformation to be
flexibly explored while holding the protein as a rigid structure during
docking.
KDM7A Inhibition Assay. The KDM7A assay was run using
MALDI and K27me2 peptide as a substrate under the same conditions
as used in ref 4.
Biology. KDM7B Inhibition Assay. KDM7B (0.5 mg/mL) was
incubated with 150 mM KCl, 2.5% glycerol, 0.5 mM dithiothreitol,
0.05 mM PMSF, 2.5 mM glutathione reduced form, 20 μM
(+)-Fe(II)-L-ascorbic acid, 20 μM ZnCl2, 0.5 mM ascorbic acid, 0.5
mM 2-oxoglutarate, and 5 μM H3K4me3K9me2 (ART(Kme3)QTAR-
(Kme2)STGGKAPRKQL-Cys) for 1 h at 37 °C in 8 μL of 10 mM
Tris-HCl buffer (pH 8.0). The reaction was stopped by adding 75 μL
of matrix-solution (5 mg/mL α-cyano-4-hydroxycinnamic acid, 37%
acetonitrile, and 0.12% trifluoroacetic acid) and then sonicated for 30
s. Then 1 μL of the reaction mixture was spotted on the sample plate,
dried, and analyzed by matrix-assisted laser desorption ionization time-
of-flight mass spectrometry (MALDI-TOF MS) using Voyager-DE
PRO (Applied Biosystems). The KDM7B inhibition activity of the test
compounds was calculated from the remaining amount of H3K9me2.
The 50% inhibitory concentration (IC50) of the test compounds was
calculated as the concentration at which the half amount of H3K9me2
was removed compared to that removed when the enzyme was added
(Supporting Information Figure S7).
Western Blot Analysis. N2a cells, HeLa cells, or KYSE150 cells (5
× 105) were treated for 24 h with inhibitors at the indicated
concentrations in OPTI-MEM medium, then collected and extracted
with SDS buffer. Protein concentrations of the lysates were
determined using a Bradford protein assay kit (Bio-Rad Laboratories);
equivalent amounts of proteins from each lysate were resolved in 4−
20% SDS-polyacrylamide gel and then transferred onto nitrocellulose
membranes (Bio-Rad Laboratories). After having been blocked for 30
min with Tris-buffered saline (TBS) containing 3% skim milk, the
transblotted membrane was incubated overnight at 4 °C with
dimethylated H3K27 antibody (CST) (1:1000 dilution) or H3
antibody (Abcam) (1:100000 dilution) in TBS containing 3% skim
milk or TBS-Tween 20 (TBS-T). The membrane was probed with the
primary antibody, then washed twice with TBS or TBS-T, incubated
with goat antirabbit IgG-horseradish peroxidase conjugates (diluted
1:2500) for 1.5 h at room temperature, and again washed twice with
TBS and once with TBS-T. The immunoblots were visualized by
enhanced chemiluminescence.
KDM4C Inhibition Assay. The KDM4C-inhibitory activity was
measured using 0.6 mg/mL enzyme. The compounds were dissolved
in DMSO. The final concentrations of DMSO in the reaction mixtures
were less than 3.3%, and it was confirmed that 3.3% DMSO did not
affect the KDM4C activity. Reaction with DMSO alone was also done
as a control. Reaction mixtures (94.6 μL), containing all of the
materials except H3K9me3 peptide and 2-oxoglutarate, were
preincubated for 5 min. Then the reactions were started by the
addition of 5.4 μL of a solution of 0.93 mM H3K9me3 peptide and 3.7
mM 2-oxoglutarate . The enzyme activity was determined as described
above. The ratio of the enzyme activity measured in the presence of
inhibitor to that of the control was plotted against log [Inhibitor]. To
confirm that the reduction of the KDM4C activity by test compounds
was not due to inhibition of the coupled enzyme FDH, we examined
the effects of the test compounds on FDH activity. The reaction
mixture (0.1 mL) contained 20 mM HEPES-KOH, pH 7.5, 50 μM
formaldehyde, 1 mM 3-acetylpyridine adenine dinucleotide, 1 mM
reduced glutathione, 0.1 mg/mL BSA, 0.1 mg/mL FDH, and a fixed
concentration of 123 μM test compound. The FDH activity was
measured by monitoring APADH formation as described above. The
FDH activity in the presence of test compounds was similar to that in
the absence of the compounds.
KDM4A Inhibition Assay. The KDM4A activity was measured by
the FDH-coupled assay as described for KDM4C except that reactions
were performed in a final volume of 30 μL in 384-well plate (Nunc)
and a final concentration of the KDM4A was 0.37 mg/mL.
KDM5A Inhibition Assay. The KDM5A activity was measured by
the FDH-coupled assay as described for KDM4C except that reactions
were performed with H3K4me3 peptide in a final volume of 30 μL in
384-well plate (Nunc) and a final concentration of the KDM5A was
0.64 mg/mL.
MTT Assays. The cells were plated at initial densities of 5000 cells/
well (100 μL/well) in 96-well plates in RPMI 1640 with 10% fetal
bovine serum and allowed to attach overnight. The cells were exposed
to inhibitors for 48 h at 37 °C in 5% CO2 incubator. A solution (5 mg/
mL) of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) (Sigma) was added (10 μL/well) and incubated with the cells
for 3 h before solubilization buffer (0.04 mol/L HCl−2-propanol) was
added (100 μL/well) onto the cultured cells. The solubilized dye was
quantified by colorimetric reading at 560 nm using a reference
wavelength of 750 nm. Absorbance values of control wells (C) and test
wells (T) were measured. Moreover, absorbance of the test wells (T0)
was also measured at time 0 (addition of compounds). Using these
measurements, cell growth inhibition (percentage of growth) by a test
inhibitor at each concentration used was calculated as: % growth =
100[(T − T0)/(C − T0)], when T > T0 and % growth = 100[(T −
T0)/T], when T < T0. Computer analysis of the % growth values
afforded the 50% growth inhibition parameter (GI50). The GI50 was
calculated as 100[(T − T0)/(C − T0)] = 50.
RNA Isolation and Semi-qRT-PCR. HeLa cells were treated for 48 h
with 0.238% DMSO or compound 9 at the concentration of 30 and 80
μM, respectively. Total RNA was isolated from HeLa cells using
RNAzol (Molecular Research Center, Inc.) following the manufac-
turer’s protocol. First-strand cDNA synthesis from total RNA was
carried out using ReverTra Ace (TOYOBO). Resulting cDNA was
then analyzed by semiquantitative PCR (semi-qPCR) using 2720
thermal cycler (Applied Biosystems). Primers are specific for genes
tested, and their sequences are as follows:
GAPDH 450bp Primer(F): 5′-TCCACCACCCTGTTGCTGTA-3′
(20mer) Primer(R): 5′-ACCACAGTCCATGCCATCAC-3′ (20mer)
E2F1 435bp Primer(F): 5′-ACTCCTCGCAGATCGTCAT-
CATCT-3′(24mer) Primer(R): 5′-GGACGTTGGTGATGTCATA-
GATGCG-3′(25mer)
Cycle parameters were 94 °C for 2 min, followed by 28 (E2F1), 20
(GAPDH) cycles of 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 30 s,
with a final extension at 68 °C for 1 min.
KDM2A Inhibition Assay. The inhibitory activities of test
compounds against KDM2A was assayed according to the method
reported in ref 4.
KDM6A Inhibition Assay. The Epigenase JMJD3/UTX demethy-
lase activity/inhibition assay kit (Epigentek Group Inc.) was used for
KDM6A enzyme assay. The materials supplied with the kit, 100 μM of
test compounds and KDM6A (human, recombinant, BPS Bioscience,
Inc.) (300 ng/well) were added to wells coating trimethylated histone
substrate, according to the supplier’s protocol. The resulting mixtures
were incubated at 37 °C for 120 min. After enzyme reaction, each well
was reacted with capture antibody for 60 min and with detection
antibody for 30 min. Finally, developer solution and stop solution were
added to wells in sequence and the absorbance (450 nm) in each well
was measured with an ARVO X3 microplate reader. The KDM6A
inhibition activity of test compounds was calculated from the
absorbance readings.
FACS Analysis. Cells (5 × 105) were treated for 24 h with
compound 9 at the indicated concentrations in RPMI 1640 with 10%
fetal bovine serum, then harvested by trypsinization. The cells were
collected by centrifugation, fixed with ice-cold 70% ethanol, washed
with phosphate-buffered saline, and resuspended in 0.5 mL of
phosphate-buffered saline containing propidium iodide (10 μg/mL)
and RNase A (0.2 mg/mL). After a final incubation at 25 °C for 30
min, the cells were analyzed using a JSAN flow cytometer (Bay
Bioscience). A total of 30000 events were counted for each sample.
Data were analyzed using FlowJo software (Tree Star).
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Article
histone demethylase PHF8 overexpressed in prostate cancer with an
impact on cell proliferation, migration and invasion. Oncogene 2012,
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Grummt, I. PHF8 activates transcription of rRNA genes through
H3K4me3 binding and H3K9me1/2 demethylation. Nature Struct.
Mol. Biol. 2010, 17, 445−450.
ASSOCIATED CONTENT
* Supporting Information
View of the catalytic sites of KDM7B, KDM2A, KDM4A,
KDM4C, KDM5A, and KDM6A. KDM7B-inhibitory activity of
compound 9. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
(7) (a) Cloos, P. A.; Christensen, J.; Agger, K.; Maiolica, A.;
Rappsilber, J.; Antal, T.; Hansen, K. H.; Helin, K. The putative
oncogene GASC1 demethylates tri- and dimethylated lysine 9 on
histone H3. Nature 2006, 442, 307−311. (b) Rose, N. R.; Ng, S. S.;
AUTHOR INFORMATION
Corresponding Authors
*For T.S.: suzukit@koto.kpu-m.ac.jp.
*For T.M.: mizukami@nagahama-i-bio.ac.jp.
*For N.M.: miyata-n@phar.nagoya-cu.ac.jp.
■
́ ́
Mecinovic, J.; Lienard, B. M.; Bello, S. H.; Sun, Z.; McDonough, M.
A.; Oppermann, U.; Schofield, C. J. Inhibitor scaffolds for 2-
oxoglutarate-dependent histone lysine demethylases. J. Med. Chem.
2008, 51, 7053−7056. (c) Rose, N. R.; Woon, E. C.; Kingham, G. L.;
Notes
King, O. N.; Mecinovic, J.; Clifton, I. J.; Ng, S. S.; Talib-Hardy, J.;
́
Oppermann, U.; McDonough, M. A.; Schofield, C. J. Selective
inhibitors of the JMJD2 histone demethylases: combined non-
denaturing mass spectrometric screening and crystallographic
approaches. J. Med. Chem. 2010, 53, 1810−1818. (d) Hamada, S.;
Suzuki, T.; Mino, K.; Koseki, K.; Oehme, F.; Flamme, I.; Ozasa, H.;
Itoh, Y.; Ogasawara, D.; Komaarashi, H.; Kato, A.; Tsumoto, H.;
Nakagawa, H.; Hasegawa, M.; Sasaki, R.; Mizukami, T.; Miyata, N.
Design, synthesis, enzyme-inhibitory activity, and effect on human
cancer cells of a novel series of Jumonji domain-containing protein 2
histone demethylase inhibitors. J. Med. Chem. 2010, 53, 5629−5638.
(e) Luo, X.; Liu, Y.; Kubicek, S.; Myllyharju, J.; Tumber, A.; Ng, S.;
Che, K. H.; Podoll, J.; Heightman, T. D.; Oppermann, U.; Schreiber, S.
L.; Wang, X. A selective inhibitor and probe of the cellular functions of
Jumonji C domain-containing histone demethylases. J. Am. Chem. Soc.
2011, 133, 9451−9456. (f) Lohse, B.; Nielsen, A. L.; Kristensen, J. B.;
Helgstrand, C.; Cloos, P. A.; Olsen, L.; Gajhede, M.; Clausen, R. P.;
Kristensen, J. L. Targeting histone lysine demethylases by truncating
the histone 3 tail to obtain selective substrate-based inhibitors. Angew.
Chem., Int. Ed. 2011, 50, 9100−9103. (g) Woon, E. C.; Tumber, A.;
Kawamura, A.; Hillringhaus, L.; Ge, W.; Rose, N. R.; Ma, J. H.; Chan,
M. C.; Walport, L. J.; Che, K. H.; Ng, S. S.; Marsden, B. D.;
Oppermann, U.; McDomough, M. A.; Schofield, C. J. Linking of 2-
oxoglutarate and substrate binding sites enables potent and highly
selective inhibition of JmjC histone demethylases. Angew. Chem., Int.
Ed. 2012, 51, 1631−1634. (h) Kruidenier, L.; Chung, C. W.; Cheng,
Z.; Liddle, J.; Che, K.; Joberty, G.; Bantscheff, M.; Bountra, C.;
Bridges, A.; Diallo, H.; Eberhard, D.; Hutchinson, S.; Jones, E.; Katso,
R.; Leveridge, M.; Mander, P. K.; Mosley, J.; Ramirez-Molina, C.;
Rowland, P.; Schofield, C. J.; Sheppard, R. J.; Smith, J. E.; Swales, C.;
Tanner, R.; Thomas, P.; Tumber, A.; Drewes, G.; Oppermann, U.;
Patel, D. J.; Lee, K.; Wilson, D. M. A selective jumonji H3K27
demethylase inhibitor modulates the proinflammatory macrophage
response. Nature 2012, 488, 404−408.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mie Tsuchida and Miho Hosoi for their technical
support. This work was supported in part by JST PRESTO
program (T.S.), a Grant-in-Aid for Scientific Research from the
Japan Society for the Promotion of Science (T.S.), Takeda
Science Foundation (T.S.), Naito Foundation (T.S.), NOVAR-
TIS Foundation (Japan) for the Promotion of Science, the
Wellcome Trust, BBSRC (L.W.), and the Royal Society (A.K.).
ABBREVIATIONS USED
■
KDM, lysine demethylase; LSD1, lysine-specific demethylase 1;
JmjC, Jumonji C; NOG, N-oxalylglycine; PCA, 2,4-pyridinedi-
carboxylic acid
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