8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01635

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me₃ and H3K4Me₃ demethylation in a cell-based assay.

Full text of DOI:10.1021/acs.jmedchem.5b01635

Article
pubs.acs.org/jmc
8‑Substituted Pyrido[3,4‑d]pyrimidin-4(3H)‑one Derivatives
As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1)
Histone Lysine Demethylase Inhibitors
Vassilios Bavetsias,* Rachel M. Lanigan, Gian Filippo Ruda, Butrus Atrash, Mark G. McLaughlin,
,†
†
‡,§
†
†
‡
,§
†
†
†
†
Anthony Tumber ,† N. Yi Mok, Yann-Vaï Le Bihan, Sally Dempster, Katherine J. Boxall,
‡,§
‡
‡
‡
Fiona Jeganathan, Stephanie B. Hatch, Pavel Savitsky, Srikannathasan Velupillai, Tobias Krojer,
‡
,§
†
†
†
†
Katherine S. England, Jimmy Sejberg, Ching Thai, Adam Donovan, Akos Pal,
Giuseppe Scozzafava,‡,§ James M. Bennett, Akane Kawamura, Catrine Johansson,
‡,§
∥
‡,⊥
‡
‡
‡
‡,#,∇
Aleksandra Szykowska, Carina Gileadi, Nicola A. Burgess-Brown, Frank von Delft,
‡
,⊥
○
○
†
◆
Udo Oppermann, Zoe Walters, Janet Shipley, Florence I. Raynaud, Susan M. Westaway,
◆
‡,§
‡,§
†
∥
†
Rab K. Prinjha, Oleg Fedorov, Rosemary Burke, Christopher J. Schofield, Isaac M. Westwood,
Chas Bountra, Susanne Mu
‡
†
,‡,§
,†
̈
ller, Rob L. M. van Montfort, Paul E. Brennan,* and Julian Blagg*
†
Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold Road, London SM2 5NG, U.K.
Structural Genomics Consortium (SGC), University of Oxford, ORCRB Roosevelt Drive, Oxford OX3 7DQ, U.K.
Target Discovery Institute (TDI), Nuffield Department of Medicine, University of Oxford, NDMRB Roosevelt Drive, Oxford OX3 7FZ, U.K.
Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
‡
§
∥
⊥
Botnar Research Centre, NIHR Oxford Biomedical Research Unit, Oxford OX3 7LD, U.K.
#
Diamond Light Source (DLS), Harwell Science and Innovation Campus, Didcot OX11 0DE, U.K.
∇
Department of Biochemistry, University of Johannesburg, Auckland Park 2006, South Africa
○
Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research, London SM2 5NG, U.K.
◆Epinova Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage SG1 2NY, U.K.
*
S Supporting Information
ABSTRACT: We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization,
guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone
N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety
allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker
gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1)
subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in
the Caco-2 assay, and, for 54k, inhibition of H3K9Me and H3K4Me demethylation in a cell-based assay.
3
3
Special Issue: Epigenetics
Received: October 18, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b01635
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Article
INTRODUCTION
role in breast and prostate cancer, respectively. KDM5B has
■
20,21
been implicated in several subtypes of breast cancer,
while
Post-translational histone modifications, which include lysine
or arginine methylation, play a pivotal role in maintaining
chromatin architecture and regulating transcription. Histone
lysine methylation is a reversible process; the removal of methyl
groups is catalyzed by two classes of histone lysine demethylases
KDMs): the flavin-dependent lysine specific demethylase
LSD/KDM1) family that demethylate mono- and dimethyl
lysine substrates, and the larger class of Fe(II) and 2-oxoglutarate
2OG)-dependent enzymes which can additionally convert
trimethyl lysine substrates to their dimethyl forms.
OG-dependent KDMs belong to the Jumonji C (JmjC) domain
containing subfamily of such enzymes and have a catalytic
site wherein methylated lysine substrates can be oxidized to
hemiaminal intermediates, which subsequently decompose to
KDM5C has been shown to be an independent prognostic factor
22
1
in prostate cancer. JmjC KDM activity is also associated with
23
diseases other than cancer; for example, KDM6B/A (JMJD3/
UTX)-mediated demethylation of H3K27 has been reported
as a critical determinant of pro-inflammatory gene activation
(
(
24
in human primary macrophages. Thus, JmjC KDMs have
emerged as important therapeutic targets, particularly for the
7
,23,25
treatment human malignancies.
(
2
−4
A range of small-molecule inhibitors of JmjC KDMs has
The
been reported, the majority of which are 2OG-competitive and
2
26−34
coordinate to Fe(II) in the catalytic site,
compounds 1−3
for example,
(Figure 1). Many of these early histone
demethylase modulators lack human 2OG oxygenase specific-
32−34
32
2
,4
ity and possess a carboxylic acid moiety which may limit cell
permeability. As a result, such compounds are suboptimal
chemical tools with which to better understand the biological
role of specific KDM4 subfamily enzymes in disease and normal
development, in particular, to study the importance of JmjC
domain KDM activity within the context of full length KDM4
proteins wherein the noncatalytic domains may play a role in
chromatin localization or scaffolding functions. In an attempt
to overcome the poor cell permeability of inhibitors bearing a
carboxylic acid, corresponding ester prodrugs have been
investigated in some cases; for example, for the KDM6-selective
inhibitor 4 (GSK-J1, Figure 1) where the corresponding ethyl
the corresponding demethylated lysine and formaldehyde.
Human JmjC histone demethylases comprise five sub-
families. The KDM4 (JMJD2) subfamily consists of six mem-
bers KDM4A−F; KDM4E and KDM4F are believed to be
pseudogenes although demethylase activity has been demon-
strated when KDM4E is ectopically expressed. KDM4A−C
contain PHD and Tudor domains as well as a single JmjC
domain that catalyzes demethylation of histone substrates
5
,6
1
,5,7,8
H3K9Me /Me , H3K36Me /Me , and H1.4K26Me .
3
2
3
2
3
KDM4D is distinguished from KDM4A−C in terms of its
domains (it lacks both the PHD and Tudor domains), its selec-
tivity for demethylation of H3K9Me /Me over H3K36Me /
3
2
3
5
,7
24
Me2, and also its ability to demethylate both the dimethylated
ester prodrug found utility in a cellular context.
7
,9
and trimethylated forms of H1.4K26.
Extensive interest in the KDM4/5 subfamilies in the pro-
gression of human cancers, and the well-defined catalytic
mechanism for JmjC KDMs, prompted us to initiate a program
to identify histone KDM inhibitors, and in particular potent,
cell permeable, and selective inhibitors of the KDM4 subfamily
with which to further explore the potential of such compounds
as anticancer agents. Herein, we report the identification of a
Emerging evidence suggests that KDM4 subfamily members
play an important role in cancer initiation and progression;
various studies have shown overexpression of KDM4A−C in a
range of human malignancies.
KDM4C are reported to be overexpressed in breast cancer;
KDM4B knockdown in ER-positive ZR-75-1 breast cancer cells
impaired growth in a human tumor xenograft model, and
KDM4B knockdown in human gastric cancer cells also sup-
pressed growth in a human tumor xenograft model. KDM4B
5
,7,10−12
For example, KDM4B and
10,11
11
4
-(pyridin-2-yl)thiazol-2-amine series as KDM inhibitors from
a high throughput screening (HTS) campaign and their sub-
sequent structure-based optimization into potent, cell permeable
dual inhibitors of the KDM4 and KDM5 subfamilies.
1
2
gene expression is high in rhabdomyosarcomas and has been
associated with poor prognosis in neuroblastoma.
1
3,14
A recent
report demonstrates a role for KDM4A in site-specific copy gain
CHEMISTRY
■
of chromosomal domains (e.g., 1q12) and rereplication of
15
regions amplified in tumors; furthermore, a coding single-
nucleotide polymorphism (SNP) in KDM4A was shown to
associate with worse outcome in non-small cell lung cancer
Synthesis of the HTS-derived series of N-(4-(pyridin-2-
yl)thiazol-2-yl)benzamides 7a−d (Scheme 1A, Table 1) was
achieved by EDCI/HOBt-mediated coupling of commercially
available benzoic acids 6 with 4-(pyridin-2-yl)thiazol-2-amine
(5). This procedure has recently been reported for the
preparation of 2-aminothiazole derivatives as antimycobacterial
16,17
patients and with increased sensitivity to mTOR inhibitors.
The KDM5 subfamily are histone H3 lysine 4 (H3K4)
demethylases and have been implicated in cancer progression in
18,19
35
several tumor types.
Along with members of the KDM4
and antiplasmodial agents. 4-(Pyridin-3-yl)thiazole derivatives
family, KDM5B and KDM5C have been shown to play a
9a,b (Scheme 1B, Table 2) were obtained from commercially
Figure 1. Pyridine-based histone demethylase inhibitors.
B
DOI: 10.1021/acs.jmedchem.5b01635
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Article
a
Scheme 1
N-Boc protecting group provided the primary amide 16. Access
to 2-benzamidothiazole 19 was achieved by coupling of benzoyl
chloride with 2-aminothiazole 17 followed by ester hydrolysis
under alkaline conditions (Scheme 2).
Preparation of 22 followed an alternative approach whereby
the thiazole ring was introduced directly via a Stille cross-
coupling reaction (Scheme 3). Access to 24, a key intermediate
for the preparation of 25, was achieved by bromination of
2
3 followed by 2-methylthiazole ring formation in a manner
analogous to the preparation of 14 (Scheme 3).
-Benzylaminomethylpyridine (30b) and 2-furanylmethyla-
2
36
mino derivative (30a) were prepared from pyridine-2,4-
dicarboxylate (26) in a four-step sequence (Scheme 4).
Esterification of 26, followed by DIBAL-mediated selective
reduction of the carboxylic acid ester in the 2-position of 27
afforded aldehyde 28. Reductive amination of aldehyde 28 with
either benzylamine or 2-aminomethylfuran in the presence of
sodium triacetoxyborohydride led to ester derivatives 29a,b,
which were subsequently hydrolyzed to the corresponding
carboxylates 30a,b with lithium hydroxide (Scheme 4).
a
Reagents and conditions: (a) EDCI, 1-hydroxybenzotriazole, CH Cl2
2
or CH Cl /DMF, room temp, 3.5−36 h, (7a = 33%, 7b = 25%,
7
2
2
c = 40%, 7d = 50%, 9a = 28%, 9b = 16%).
8
-Chloropyrido[3,4-d]pyrimidin-4(3H)-one (34), the key
intermediate for the synthesis of thiazole-C4 substituted
derivative 37, 2,4-disubstituted thiazole 40 and subsequent
SAR exploration (vide infra), was prepared in three steps from
methyl-3-amino-2-chloroisonicotinate (31) (Scheme 5). Ester
hydrolysis afforded 32 which was converted into the primary
amide 33 via the corresponding acid chloride; subsequent ring
formation was effected upon treatment with triethyl orthofor-
mate (Scheme 5). A SEM-protecting group was introduced by
reacting 34 with SEM-Cl in the presence of potassium carbonate
to give protected pyridopyrimidinone 35. Bromide 39, a key
intermediate for the preparation of 40, was obtained from 34
in a manner analogous to that described for 13 (Scheme 2).
2-Aminothiazole formation to afford 40 was achieved by reacting
available 4-(pyridin-3-yl)thiazol-2-amine (8) by a procedure
analogous to that described for the synthesis of 7a−d.
Syntheses of 2-aminothiazoles 15 and 16 and 2-benzamido-
thiazole 19 are shown in Scheme 2. Treatment of methyl
2-bromoisonicotinate (10) with stannane 11 under Pd-catalyzed
conditions gave methyl 2-(1-ethoxyvinyl)isonicotinate (12);
subsequent reaction with N-bromosuccinimide afforded bro-
mide 13. Thiazole ring formation to give the 2,4-disubstituted
thiazole 14 was effected by reacting 13 with N-Boc-thiourea in
the presence of trimethylamine, and subsequent removal of the
protecting groups provided 2-aminothiazole derivative 15.
Treatment of 2,4-disubstituted thiazole 14 with ammonia in
methanol at 80 °C followed by acid-mediated removal of the
b
Table 1. N-Substituted 4-(Pyridin-2-yl)thiazole-2-amine Derivatives
a
b
Results are from a single experiment. Results are mean values of two independent determinations or mean (±SD) for n > 2 unless specified
otherwise.
C
DOI: 10.1021/acs.jmedchem.5b01635
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Article
a
Table 2. N-Substituted 4-(Pyridin-3-yl)thiazole-2-amine Derivatives
a
Results are mean values of two independent determinations unless specified otherwise.
a
Scheme 2
a
Reagents and conditions: (a) Pd(PPh ) , 1,4-dioxane, reflux, 18 h, 65%; (b) N-bromosuccinimide, 10% water in THF, room temp, 1 h, 61%;
3
4
(
c) N-Boc-thiourea, Et N, EtOH, reflux, 1 h, 56%; (d) (i) 4 M HCl in dioxane, room temp, 1 h, (ii) THF/H O, NaOH, room temp, 1 h, 55%;
3
2
(
e) (i) 7 M NH in MeOH, 80 °C, 18 h, (ii) 4 M HCl in dioxane, room temp, 1 h, 91%; (f) 4 M HCl in dioxane, room temp, 1 h, 83%; (g) benzoyl
3
chloride, CH Cl , Et N, room temp, 2 h, 35%; (h) THF/MeOH, H O, NaOH, room temp, 1 h, 40%.
2
2
3
2
3
9 with thiourea in the presence of Et N. Compound 37 was
subsequent reductive amination with the requisite primary or
secondary amines gave the 8-aminomethyl derivatives 44a−g
(Scheme 7, Table 5).
Compounds 52a−g (Table 6), 53a−i (Table 7), and 54a−n
(Table 8) were prepared either from primary alcohol 47 by
3
prepared from 34 by a Stille cross-coupling reaction to introduce
the thiazole ring followed by removal of the SEM protecting
group under acidic conditions (Scheme 5).
8-Chloropyrido[3,4-d]pyrimidin-4(3H)-one (34) was also
employed for the synthesis of pyrazolyl derivatives 41a,b by a
Suzuki cross-coupling reaction with (1H-pyrazol-3-yl)boronic
acid or ((N-methyl)pyrazol-3-yl)boronic acid pinacol ester,
respectively (Scheme 6).
Suzuki cross-coupling reaction of 34 with trimethylboroxine
afforded the C8-methyl derivative 42 which was oxidized to
mesylate formation followed by S 2 displacement or from
N
carboxaldehyde 48 by reductive amination (routes A and B,
respectively, Scheme 8). The pyrazole moiety at C8 of the
pyrido[3,4-d]pyrimidin-4(3H)-one scaffold was introduced
using an S Ar reaction on key intermediate 35. Subsequent
N
acidic TBS deprotection on intermediate 46 afforded the alcohol
47. Compound 59 (Table 6) bearing a longer linker at C4 of the
4-oxo-3,4-dihydropyrido[3,4-d]pyrimidine-8-carbaldehyde (43);
D
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a
a
Scheme 3
Scheme 5
a
Reagents and conditions: (a) Pd(PPh ) , Ag O, DMF, 100 °C, 1 h,
3
4
2
6
4%; (b) MeOH/H O, NaOH, 1 h, 46%; (c) AcOH, 48% aqueous
2
HBr, Br , 0 °C to room temp for 1 h, then 75 °C for 1.5 h, 38%;
2
(
(
d) (i) thioacetamide, EtOH, Et N, reflux, 1 h, (ii) EtOH, conc HCl
3
2 drops), reflux, 20 min, 23%; (e) MeOH/H O, 1 M NaOH, room
2
temp, 50 min, 45%.
a
Scheme 4
a
Reagents and conditions: (a) MeOH/H O, 1 M NaOH, room temp,
2
4
5 min, 77%; (b) (i) SOCl , DMF, reflux, 2 h, (ii) ammonium
2
hydroxide, THF, room temp, 1.5 h, 89%; (c) CH(OEt) , reflux
3
2
4
4 h, 82%; (d) SEM-Cl, K CO , DMF, 60 °C, 4 h, 84%; (e)
2 3
-(tributylstannyl)thiazole, Pd(PPh ) , Ag O, DMF, 100 °C, 2 h, 32%;
3
4
2
(
f) THF, 6 M HCl, 50 °C, 4 h, 94%; (g) Pd(PPh ) , 1,4-dioxane,
3 4
reflux, 2 h, 64%; (h) 10% H O in THF, N-bromosuccinimide, room
2
temp, 1 h, 72%; (i) thiourea, Et N, EtOH, reflux, 1 h, 13%.
3
a
Scheme 6
a
Reagents and conditions: (a) PTSA, EtOH, 80 °C, 79%; (b) DIBAL,
THF, PhMe, −78 °C, 39%; (c) benzylamine or 2-aminomethylfuran,
NaBH(OAc) , AcOH, CH Cl , (29a = 99%, 29b = 93%); (d) LiOH
3
2
2
(
aq), MeCN, (30a = quant, 30b = quant).
pyrazole moiety was also prepared according to route A
described in Scheme 8.
a
Reagents and conditions: (a) Pd(PPh ) , Na CO , DMA,
3
4
2
3
(1H-pyrazol-3-yl)boronic acid hydrochloride or ((N-methyl)pyrazol-
3
-yl)boronic pinacol ester, 100 °C, 1 h, (41a = 12%, 41b = 21%).
RESULTS AND DISCUSSION
■
The discovery of chemical starting points for JmjC histone
KDM inhibition was based upon an HTS campaign of a 150k
compound collection tested at 30 μM versus human
recombinant KDM4B (amino acid residues 1−500 with an
N-terminal GST tag). The format of the HTS was the same
KDM4B AlphaScreen assay described in the Methods section;
this assay was also used to monitor the activity of compounds
reported herein. This screening campaign provided N-substituted
-(pyridin-2-yl)thiazole-2-amine derivatives exemplified by 7c as
promising hits (Scheme 1, Table 1). The KDM4B inhibitory
activity of 7c was confirmed with a resynthesized sample (IC =
0.9 μM) and triggered a medicinal chemistry program to identify
more potent, cell permeable inhibitors of the KDM4 subfamily.
The methylsulfonyl analogue 7a, p-SO Et isomer 7b, and
2
benzamido analogue 7d displayed similar KDM4 inhibitory
activities to 7c (Table 1); all four analogues (7a−d) were more
potent inhibitors of KDM4B relative to KDM5B by at least 3-fold
under the standard assay conditions (Table 1). However, the
commercially available fragment-like scaffold 4-(pyridin-2-yl)-
thiazol-2-amine (5) demonstrated very weak inhibition of
KDM4B (30% inhibition at 100 μM) and no activity against
KDM5B at 100 μM, indicating an important role for the primary
amine N-substituent.
4
50
1
E
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Journal of Medicinal Chemistry
Article
a
Scheme 7
The commercially available phenyl analogue of 5, lacking the
pyridyl nitrogen (4-phenylthiazol-2-amine), was also inactive
against KDM4A and KDM4B at 100 μM compound concen-
tration (data not tabulated).
Attempts to elucidate the binding mode of 7a or close
analogues by cocrystallization with KDM4A or by soaking of
apo-KDM4A crystals with 7a were unsuccessful. Docking studies
of the fragment-like scaffold 5 using the cocrystal structure of 2 in
33
KDM4A (PDB code 3PDQ) suggested that 5 could bind to the
active site metal through bidentate coordination of the pyridyl
and thiazolyl nitrogen atoms (Figure 2A,B). Furthermore, the
docked pose of 5 overlaid well with the binding mode of
the bipyridyl-based analogue 2-bound KDM4A cocrystal
33
structure (PDB code 3PDQ). This observation prompted
the introduction of a carboxylate moiety para to the pyridine
nitrogen in 5 to interact with K206 and Y132 (KDM4A
residue numbering) in a manner analogous to that observed for
the cosubstrate 2OG, NOG, and literature pyridyl-based
a
Reagents and conditions: (a) Pd(PPh ) , K CO , trimethylboroxine,
32,33,37
3
4
2
3
analogues 1 and 2.
Pleasingly, 15 (Table 3) demonstrated
DME, 90 °C, 1 h, 59%; (b) SeO , 1,4-dioxane, 90 °C, 8 h, 14%;
2
potent inhibition of KDM4A and KDM4B (IC50 = 0.200 and
.083 μM respectively, Table 3), KDM5B (IC50 = 0.012 μM,
Table 3) as well as KDM3A (IC = 0.057 μM) while inhibiting
(
1
c) NaBH(OAc) , R R NH, MeOH/CH Cl , room temp, 1−3 h,
3
1
2
2
2
0
2−55%.
50
KDM2A and KDM6B to a lesser extent (IC = 1.2 and 1.0 μM,
50
We set out to establish the binding mode of the 4-(pyridin-2-
yl)thiazol-2-amine series to facilitate further medicinal chemistry
design. We postulated that the pyridine nitrogen in 7a−d
Table 1) would be a major contributor to KDM4A/B binding
respectively). Interestingly, introduction of a carboxylate at the
equivalent position para to the pyridine nitrogen in the
N-(thiazol-2-yl)benzamido derivative 7d to give compound 19
(Table 3) resulted in demethylase activity similar to 7d (KDM4B
IC = 17.0 μM, Table 3) consistent with the docking predictions
that binding of extended analogues at the C2 thiazole position
could be compromised due to the steric clash of the benzamido
substituent with the protein (Figure 2B).
(
via coordination to the metal in the JmjC-domain catalytic site
analogous to the binding mode of previously reported pyridine-
50
2
4,32−34
based KDM inhibitors such as 1−4 (Figure 1).
Consistent with this notion, and also with previous reports that
suggested a significant drop in potency for pyridin-3-yl
Protein−ligand crystallographic analysis was mainly per-
24
regioisomers of active compounds, the 4-(pyridin-3-yl)thiazole
derivatives 8, 9a, and 9b were considerably less potent or
inactive relative to their pyridin-2-yl counterparts (Tables 1 and 2).
formed using KDM4A which has a similar JmjC catalytic site
38
to that of KDM4B. A crystal structure of 15 bound to KDM4A
confirmed the proposed binding mode involving interactions of
a
Scheme 8
a
Reagents and conditions: (a) Cs CO , anhydrous MeCN, reflux, 18 h, 71%; (b) 1 M HCl, MeOH, 0 °C, 5 min, 99%; (c) Dess−Martin periodinane,
2
3
anhydrous CH Cl , room temp; (d) (i) methanesulfonic anhydride, Et N, anhydrous CH Cl , 0 °C, 15 min, workup, (ii) Cs CO , R R NH,
2
2
3
2
2
2
3
1 2
anhydrous DMF, 90 °C, 15 h or Et N, anhydrous DMF, 50 °C, 15 h, 17−84%; (e) R R NH, anhydrous 1,2-dichloroethane, NaBH(OAc) , room
3
1
2
3
temp, overnight, 24−69%; (f) 6 M HCl, THF, 50−60 °C, 3 to 8 h or 4 M HCl in dioxane, dioxane/H O, 50 °C, 16−97%; (g) pyrazole, Cs CO ,
2
2
3
anhydrous MeCN, reflux, 3 h, 80%; (h) THF, 6 M HCl, 50 °C, 4 h, (56 = 70%, 57 = 75%).
F
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Figure 2. Overlay of a view from crystal structure of 2 (light-blue ball-
and-sticks) bound to KDM4A (displayed in gray lines or gray surface;
33
PDB code 3PDQ) and a model of 5 (magenta) docked into the
protein structure from 3PDQ. (A) Interaction network. (B) Surface
representation. Ni(II) shown in place of the active site Fe(II).
the carboxylate moiety with K206 and Y132 and bidentate
coordination of both the pyridyl and thiazolyl nitrogens to the
metal. A deviation from coplanarity of the pyridyl and thiazolyl
aromatic ring systems (torsional angle = 17.4°) and a rotation of
the plane of the carboxylate relative to the plane of the pyridine
ring was also observed (28.6°, Figure 3A). These observations
are broadly consistent with the binding mode of an analogue
34
of the triazolopyridine 3 (Figure 1) and of aminomethyl
36
substituted pyridines 30a and 30b (Table 4). The carboxylic
acid in 30b interacts with K206 and Y132 of KDM4A and the
carboxylic acid in 30a with corresponding residues of KDM4D;
furthermore, both ligands exhibit bidentate metal coordination
Figure 3. Views from crystal structures of 15, 16, and 30b bound to
KDM4A and of 30a bound to KDM4D. (A) KDM4A-bound 15 (gray),
showing a close-up view of ligand−metal coordination and interaction
with Y132 and K206. (B) Overlay of KDM4A-bound 15 (gray) and 16
(
3
Figure 3D,E). The aminomethyl substituent in both 30a and
0b is rotated away from the plane of the biaryl ring scaffold in 15
to interact with Y175 in KDM4A and the equivalent residue in
KDM4D (Y179) (Figure 3D,E); additionally, the furan oxygen
of 30a interacts with the hydroxyl moiety of Y181 in KDM4D
(
(
green). (C) 2D interaction map of 15 with key residues in KDM4A.
D) Overlay of KDM4A-bound 15 (gray) and 30b (purple for chain A,
(
equivalent residue Y177 in KDM4A) (Figure 3E).
The 15-bound KDM4A structure also revealed π-stacking
cyan for chain B of the asymmetric unit). (E) Overlay of KDM4A-bound
5 (gray) and KDM4D-bound 30a (orange). Indicated residues
1
of the pyridyl ring with the side chain of residue F185, an
correspond to KDM4A numbering. Zn(II) and Ni(II) are shown at
interaction of the thiazole C2-NH with E190, and interaction of
the active site.
2
this exocyclic NH to both D191 and N290 via a mediating water
molecule (Figure 3B,C). Consistent with the latter observation,
the desamino analogue 22 was a less potent KDM4 inhibitor
2
significant drop in KDM4A and KDM4B potency (500- and
400-fold, respectively), highlighting the importance of the
carboxylate−K206 ionic interaction for potent histone KDM
inhibition. A crystal structure of 16 bound to KDM4A showed a
very similar binding mode to that of 15. Interestingly, the primary
amide of 16 and the carboxylic acid of 15 overlay well, further
reinforcing the significant benefit of the carboxylate−K206
compared to 15 (KDM4A IC50 = 0.90 μM, KDM4B IC50
.35 μM; Table 3), and a further drop in potency was observed
with the thiazole C2-Me analogue 25 (KDM4A IC = 16.5 μM,
=
0
5
0
KDM4B IC = 2.3 μM; Table 3). Conversion of the carboxylic
50
acid in 15 to the corresponding primary amide 16 led to a
b
Table 3. 2-(2-Aminothiazol-4-yl)isonicotinic Acid Derivatives
a
b
Results are from a single experiment. Results are mean values of two independent determinations or mean (±SD) for n > 2 unless specified
otherwise.
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b
Table 4. 2-(Aminomethyl)pyridine-4-carboxylate Derivatives
a
b
Results are from a single experiment. Results are mean values of two independent determinations or mean (±SD) for n > 2 unless specified
otherwise. Compounds tested as their lithium salts.
b
Table 5. C8-Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatization
a
b
Results are from a single experiment. Results are mean values of two independent determinations or mean (±SD) for n > 2 unless specified
otherwise.
H
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charge interaction (Figure 3B). Although the ionizable carboxylic
acid appeared beneficial for ligand affinity, we considered it
a significant contributing factor to the observed poor cellular
permeability for 15 (Caco-2 A to B flux <0.8 × 10− cm/s).
Therefore, we focused on pyridine-4-carboxylic acid isosteres
with the potential to coordinate to the active site metal, make
strong interactions with K206/Y132 (KDM4A) and to be only
partially ionized at physiological pH to improve cellular
permeability while maintaining potent enzyme binding. Our
attention was drawn to the pyrido[3,4-d]pyrimidin-4(3H)-one
compared to 15 and 58. A similar trend was observed with the
2-desamino analogue 37 (KDM4B IC50 = 3.1 μM, Table 5).
Compound 37 proved a more potent inhibitor of KDM3A
(IC50 = 0.89 μM) and KDM5B (IC50 = 0.31 μM; Table 5)
and showed weaker inhibition of KDM2A (59% inhibition at
100 μM) and KDM6B (31% inhibition at 100 μM), consistent
with the profile of compound 15. Crystal structures of 40 and 37
bound to KDM4A show bidentate coordination to the metal as
well as the predicted interactions with K206/Y132 (Figure 4B).
However, interaction of K241 with N1 of the pyridopyrimidi-
none scaffold is no longer observed for C8-substituted
pyridopyrimidinones 37 and 40, which may contribute to their
lower potency compared to 58 (Figure 4A,B). We postulate that
steric bulk of the thiazole C8 substituent in 37 and 40 hinders
interaction of the scaffold with K241. Furthermore, we note that
metal coordination of the thiazole nitrogen in 37 and 40 may
result in suboptimal interactions of the pyridopyrimidinone
CONH moiety with K206/Y132 owing to the planar bicyclic
scaffold which precludes the 28.6° dihedral angle between the
pyridine and carboxylate observed in our 15-bound KDM4A
structure (Figures 3A, 4A,B). Interestingly, the torsion angles
between the planes of the pyridopyrimidinone and the thiazole
ring systems in 37 and 40 are similar to that observed between
the pyridine and the thiazole in 15 (4.7° and 7.2° difference with
respect to 15, respectively), rendering the metal-coordination
geometry similar for 15, 37, and 40 (Figure 4B). Although
introduction of a C8-thiazole substituent led to a slight decrease
in KDM4A and KDM4B potency compared to 58, we envisaged
that potency for this class of compounds could be improved by
introducing favorable interactions with D135 and other residues
in the histone peptide binding site, a tactic previously reported
to yield potent JmjC histone demethylase modulators in the
6
39
scaffold (58, Table 5, Figure 4) kindly disclosed to us by GSK
Figure 4. Comparison of binding modes of 15, 37, 40, and 58 to
KDM4A and 44a bound to KDM4D. (A) Overlay of KDM4A-bound 15
(gray) and 58 (yellow). (B) Overlay of KDM4A-bound 15 (gray), 37
(light-green), and 40 (orange). (C) Overlay of KDM4A-bound 15
(gray) and KDM4D-bound 44a (light blue). The indicated residue
33
bipyridyl scaffold exemplified by 2 (Figure 2A). Access to the
histone peptide binding pocket required derivatization through
a position equivalent to the sulfur atom in 37 (see Figures 2A,B
and 4A,B) and necessitated the introduction of alternative
heterocyclic rings at the C8 position of the pyrido[3,4-d]pyrimidin-
4(3H)-one scaffold (Table 5).
We introduced a variety of five-membered heterocycles at C8
of the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold 58, including
pyrazol-3-yl and pyrazol-1-yl moieties (41a and 56, respectively,
Table 5) which exhibited similar potency versus KDM4B and
KDM5B. We also investigated whether methylamino substitu-
ents at C8 could provide access to the pocket occupied by the
numbers correspond to KDM4A numbering. Zn(II) and Ni(II) are
shown in place of the active site Fe(II).
and also reported in the patent literature as a template for JmjC
4
0
KDM inhibition. The bicyclic aromatic scaffold 58 is a highly
efficient ligand for both KDM4 and KDM5 subfamily members
(
KDM4B IC = 1.0 μM, LE = 0.76, and KDM5B IC = 1.3 μM,
50 50
LE = 0.75; Table 5) while a less potent inhibitor of KDM3A
IC50 = 26.2 μM), KDM2A (29% inhibition at 100 μM), and
(
KDM6B (28% inhibition at 100 μM). Pleasingly, 58 also dis-
played high cellular permeability (Caco-2 A to B flux = 42.61 ×
1
−6
0
cm/s) with a measured pK for the pyridopyrimidinone
trimethylated lysine of the H3K9Me peptide substrate, as
3
a
41
amide moiety of 8.23.
previously described for 30a and 30b in the pyridine-4-carboxylic
acid series (Table 4; Figure 3D,E). Compounds 44a and 44b
were both less potent inhibitors of KDM4B and KDM5B
compared to their pyridine-4-carboxylate counterparts 30b and
30a, respectively (Tables 4, 5). We confirmed the binding mode
of 44a to KDM4D by cocrystal structure determination, which
proved consistent with that of the parent compound (compare
Figure 3D,E and Figure 4C). Interestingly, both aminomethyl-
substituted pyridopyrimidinones 44a and 44b display a
preference for KDM5B/C inhibition over KDM4A/B. Tertiary
amine analogues 44c−g were designed to rescue KDM4 potency
by maintaining bidentate metal chelation while also extending
into space occupied by the C8-thiazole of 37 or 40 and benefiting
from interactions with Y175 and Y177 in KDM4A (Figure 4).
At best, the tertiary aminomethyl compounds were comparable
in potency to simple C8-heterocyclic substituted derivatives
41a,b and 56 (Table 5). Considering the data presented in
Table 5, and cognizant of synthetic tractability, we focused our
The 58-bound KDM4A structure demonstrates simultaneous
coordination of the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold
to the active site metal via the pyridine nitrogen (position 7) and
interaction of the pyrimidinone CONH moiety with K206/
Y132; in addition, N1 of 58 is positioned to interact with the side
chain of K241. This network of interactions is consistent with the
highly ligand efficient inhibition profile of 58 versus KDM4A
and KDM4B, which are very similar in their JmjC catalytic sites.
Superposition of the 15-bound and 58-bound KDM4A
structures (Figure 4A) suggested that the pyrido[3,4-d]-
pyrimidin-4(3H)-one scaffold 58 may provide a suitable
replacement for the pyridine-4-carboxylate, whereby derivatiza-
tion at the C8 position of 58 could lead to bidentate metal
coordination and access to the histone peptide binding site (vide
infra). To test this hypothesis, the hybrid structures 37 and 40
were synthesized (Scheme 5, Table 5). Compound 40 displayed
lower KDM4B inhibitory activity (IC50 = 5.0 μM, Table 5)
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b
Table 6. 8-(1H-Pyrazol-1-yl)pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives
a
b
Results from a single experiment. n.d. = not determined Results (IC s) are mean values of two independent determinations or mean (±SD) for
50
n > 2 unless specified otherwise.
effort on elaboration of the pyrazol-1-yl analogue 56 by targeting
residues in the histone substrate binding site including D135
that the compound bound in multiple conformations, with the
two most predominant binding modes depicted in Figure 5. In
conformation A, the pyrazole-C4 substituent adopts an extended
conformation with the p-fluorobenzyl group oriented toward the
solvent accessible area; this induces movement of the side chain
of Y175 and displaces the nearby loop (residues 165−175).
However, no interaction between 52d and the induced side chain
conformation of Y175 is observed (minimal distance of 4.7 Å
between a carbon of the Y175 aromatic ring and a carbon of
the p-fluorophenyl group of 52d). In conformation B, the
p-fluorobenzyl group of the pyrazole-C4 substituent oriented
toward an asparagine/glutamine-rich pocket (residues 73, 84,
and 86, Figure 5). In conformation B, the basic nitrogen of the
pyrazole-C4 substituent is proximal to D135 with a minimum
interaction distance of 2.9 Å (distances between 2.9 and 3.2 Å
depending upon the chain in the asymmetric unit), slightly
shorter compared with the equivalent distance in conformation A
(
KDM4A) (Table 6).
Substitution at the pyrazole-C4 position with a hydroxyethyl
group (57) was tolerated and resulted in a similar KDM4/5
inhibitory profile to 56 (Tables 5 and 6). Introduction of a basic
nitrogen at the pyrazole-C4 substituent, exemplified by com-
pounds 52a, 52b, and 52c, resulted in at least a 4-fold improve-
ment in KDM4A/B inhibitory activity (Table 6); notably, all
three compounds were more potent inhibitors of KDM5B/C
compared to KDM4A/B. Introduction of a p-fluorobenzyl group
(52d), gave a more potent inhibitor of KDM4B, KDM5B,
and KDM5C (IC50 = 0.39, 0.042, and 0.078 μM, respectively,
Table 6). Pleasingly, 52d displayed a weaker inhibitory activity
against KDM2A (IC = 13.4 μM), KDM3A (IC = 13.9 μM),
5
0
50
and KDM6B (IC50 = 26.3 μM) relative to KDM4/5 subfamily
members. A crystal structure of 52d bound to KDM4A revealed
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Pleasingly, 52d displayed cellular permeability (Caco-2 A to B
flux = 9.98 × 10− cm/s, Table 6) and moderate HLM and MLM
stability (40% and 42% metabolized after a 30 min incubation,
respectively). Surprisingly, we found that 52d had high in vivo
plasma clearance (iv clearance in mouse of 1640 mL/min/kg,
following a 6 mg/kg iv compound administration), which we
suspected may be due to aldehyde oxidase (AO)-mediated
6
42
metabolism. We subsequently identified the C2 position of the
pyrido[3,4-d]pyrimidin-4(3H)-one template as the major site of
43
AO-mediated metabolism.
Figure 5. (A) Overlay of views from crystal structure of KDM4A−37
The 3,4-dichlorobenzyl analogue 52e also demonstrated
cellular permeability (Caco-2 A to B flux = 21.21 × 10− cm/s,
Table 6); however, attempts to gain additional KDM4A/B
affinity by introduction of hydrogen bond interactions with
N86, Q73, or Q84 (52f and 52g; Figure 5) were unsuccessful
(
light-green) and −52d (two main conformations: dark-brown for
6
conformation A and light-brown for conformation B). (B) Overlay of
2d bound to KDM4A (brown) and KDM5B (cyan), the indicated
5
residue numbers correspond to KDM5B numbering. Zn (II) or Ni(II)
are shown in place of the active site Fe (II).
(
Table 6). Homologation of the pyrazole-C4 substituent by one
(
3.4−4.3 Å). Attempts to cocrystallize compounds presented in
methylene unit (59) also failed to improve KDM4A or KDM4B
inhibition (Table 6).
this study with KDM5B proved more challenging relative to
KDM4A crystallography; however, we were successful in resolv-
ing a crystal structure of 52d bound to KDM5B (Figure 5B),
which shows coordination of both the pyridyl and pyrazole
N2 nitrogen to the active site metal and interaction of the
pyridopyrimidinone CONH moiety with K517/Y425 (equiv-
alent to residues K206/Y132 in KDM4A). The electron density
of the p-fluorobenzyl group in this crystal structure was poor,
precluding accurate positioning of this moiety. However, the
binding mode of the well-defined ligand scaffold to KDM5B
was similar to that observed in the structure of 52d bound to
KDM4A.
The multiple ligand conformations observed in the 52d-bound
KDM4A structure suggested that no single binding mode was
favored and is consistent with multiple degrees of freedom in
the ligand conformation. Thus, we next attempted to constrain
the pyrazole-C4 substituent by introduction of a piperidine ring.
This approach led to the synthesis of 53a, a potent and cell
permeable (Caco-2 A to B flux = 6.86 × 10− cm/s) KDM4A and
KDM4B inhibitor (IC50 = 0.126 and 0.050 μM, respectively)
6
which also strongly inhibited KDM5B and KDM5C (IC50
=
0.014 and 0.023 μM, respectively; Table 7). This KDM4 and
KDM5 subfamily profile differentiates 53a from 52d, which is
a
Table 7. C8-Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatization
a
Results (IC s) are mean values of two independent determinations unless specified otherwise.
50
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approximately 9-fold more potent in inhibiting KDM5B
compared to KDM4B (Table 6). Pleasingly, 53a maintained
selectivity for KDM4 and KDM5 subfamily inhibition versus
KDM2A (IC50 = 8.6 μM), KDM3A (IC50 = 4.8 μM), and
KDM6B (IC = 48.5 μM). The crystal structure of 53a bound to
KDM4A confirmed a single binding mode in which the ligand
extends toward the histone peptide binding site similar to
conformation A of 52d (Figures 5 and 6). The bulky pyrazole-C4
substituent of 53a induces movement of the Y175 side chain
and the nearby loop (residues 165−175) as observed in con-
formation A of 52d. We also observed a hydrophobic interaction
between the piperidine ring of 53a with the side chain of Y175
KDM4A/B and KDM5B/C inhibition profile (see 54h and 54i,
Table 8). The thiophene derivative (54i) was a less potent
inhibitor of KDM3A (IC = 8.3 μM), KDM6B (25% inhibition at
50
100 μM), and KDM2A (IC50 = 2.4 μM). Moderate HLM and
MLM stability was observed for 54i (49% and 62% metabolized
after a 30 min incubation, respectively), and cellular permeability
was maintained (Caco-2 A to B flux = 17.83 × 10− cm/s).
Notably, introduction of a 3,5-dichlorophenyl, compound 54j
gave the most potent inhibitor of KDM4 and KDM5 subfamily
members in this series, with KDM4A/B and KDM5B/C potencies
indicating a balanced inhibition profile (KDM4A/B IC = 0.080
50
6
5
0
and 0.017 μM, KDM5B/C IC50 = 0.014 and 0.051 μM,
respectively, Table 8). Pleasingly, 54j showed cellular permeability
(Caco-2 A to B flux = 6.34 × 10− cm/s) and a selective inhibition
profile versus KDM3A (IC = 6.1 μM), KDM6B(4% inhibitionat
(
3.5 Å between proximal piperidine carbon atoms and the
6
aromatic ring of Y175, Figure 6). Notably, the distance between
the piperidine nitrogen and the carboxylate of D135 is 4 Å, longer
than the equivalent distance observed in a crystal structure of
50
100 μM), and KDM2A (IC = 2.4 μM). The m-Cl analogue 54k
50
52d (conformation B) bound to KDM4A (Figures 5 and 6).
displayed a similar profile to that of 54j (Table 8) and benefited
from low MLM and HLM turnover (13% and 17% metabolized
respectively after a 30 min incubation). Compound 54k also
displayed a selective inhibition profile versus KDM2A (IC50
=
1
2.9 μM), KDM3A (IC = 5.3 μM), and KDM6B(15% inhibition
50
at 100 μM). Furthermore, profiling of 54k in a 50-kinase panel,
44
representative of the human kinome, at a concentration of 1 μM
revealed no significant kinase inhibitory activity (Supporting
Information, Table S2). Analogues 54l−n also demonstrated a
broadly balanced KDM4B and KDM5B inhibition profile and
showed good cellular permeability in the Caco-2 assay, reinforcing
the SAR trend in this 4-phenyl piperidine subseries (Table 8).
Cellular activity of 54k was assessed in HeLa cells transiently
overexpressing wild-type (WT) KDM4A or KDM5B or the
respective mutants (MUT) lacking catalytic activity. Cells were
treated with the inhibitor, and changes in the global methylation
levels were quantified by high content immunofluorescence assay
after 24 h. Global hypomethylation was observed in cells over-
expressing either WT KDM4A or WT KDM5B as determined
by reduction in the levels of methyl−lysine antibody staining
(relative to cells overexpressing the corresponding MUT
demethylase or nontransfected cells). Treatment with 54k
resulted in a concentration-dependent increase of the respective
Figure 6. Binding mode of 53a in KDM4A. Zn(II) is shown in place of
the active site Fe(II).
Additionally, the p-chlorobenzyl group points toward the histone
peptide binding site and the solvent accessible area creating a
new hydrophobic interaction with V313 (3.9 Å between the
p-chlorobenzyl of 53a and the nearest side chain carbon of V313).
Considering these structural observations, including the single
observed conformation of the pyrazole-C4 substituent in 53a,
we further explored the hydrophobic interactions with V313
45
(
(
Table 7). Replacement of p-Cl with H (53b), F (53c), CF3
53d), or introduction of an additional meta substituent (53e and
53g) resulted in a KDM4A/B and KDM5B/C inhibitory profile
methyl mark (H3K9Me
cells overexpressing WT demethylase. At high concentrations
of 54k (>50 μM), H3K9Me levels approached those in HeLa
3
for KDM4A, H3K4Me for KDM5B) in
3
broadly similar to that of 53a (Table 7). The m-methoxybenzyl
analogue (53f) was a less potent inhibitor of KDM4A relative to
3
5
3a although KDM5B/C potency was maintained (Table 7).
cells overexpressing MUT KDM4A, consistent with cell-based
inhibition of KDM4A demethylase activity; similarly, at high
Replacement of the p-chlorophenyl in 53a with heterocyclic
moieties failed to improve KDM4A/B or KDM5B/C inhibitory
activities (compounds 53h and 53i, Table 7).
In a further attempt to restrain conformational freedom in
the pyrazole side chain of 53a, we attached the p-chlorophenyl
moiety directly to the piperidine 4 position (54a); this tactic
broadly maintained KDM4A/B and KDM5B/C inhibitory
activities (KDM4B IC = 0.086 μM, KDM5B IC = 0.030 μM;
concentrations of 54k, H3K4Me levels approached those in
3
HeLa cells overexpressing MUT KDM5B, consistent with cell-
based inhibition of KDM5B demethylase activity. Occasionally,
at the highest concentrations, an effect on the methylation
mark was also observed in cells overexpressing MUT KDM,
pointing to inhibition of either the endogenous enzyme(s) or an
unspecific effect (Supporting Information, Figure S1).
5
0
50
Table 8). Notably, 54a demonstrated selectivity for KDM4B and
KDM5B versus KDM2A (IC50 = 6.74 μM) and also displayed
cellular permeability (Caco-2 A to B flux = 2.66 × 10 cm/s,
Table 8). Replacement of thep-Cl substituent in54a with F (54b),
In studies to assess the quality of promising analogues as
potential lead compounds, we noted chemical instability of
54j and 54l when left in air at room temperature for prolonged
periods; mass spectrometry analysis suggested an oxidation-
mediated degradation. However, compound 54j was stable for
>8 weeks as solution in DMSO when stored under a nitrogen
atmosphere. These observations prompted us to resynthesize 54j
and study its stability as a solution in DMSO kept at room
temperature with no protection from air and also in solid form
kept at room temperature under a nitrogen atmosphere. LCMS
and NMR (solid sample) monitoring indicated no degradation
for up to 8 weeks. Given these observations, we recommend that,
−6
OMe (54c), SO Me (54d), or CN (54e) resulted in a similar
2
KDM4A/B inhibitory profile to that of 54a. Interestingly,
balanced inhibition of KDM4 and KDM5 subfamily members
was observed for 54a, 54b, and 54c (Table 8). Analogue 54f
bearing an o-chlorophenyl substituent and the unsubstituted
phenyl derivative 54g exhibited weaker KDM4A and KDM4B
inhibition compared to that of 54a (Table 8). Heterocyclic
replacements for the p-chlorophenyl moiety in 54a gave a similar
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b
Table 8. C8-Pyrido[3,4-d]pyrimidin-4(3H)-one Modifications
a
b
Results are from a single determination. n.d. = not determined Results (IC s) are mean values of two independent determinations or mean
50
(±SD) for n > 2 unless specified otherwise.
as a precaution, compounds of this class are stored under a
nitrogen atmosphere.
respectively). However, in the 54a- and 54j-bound structures,
the piperidine nitrogen resides at a minimum distance of 3.9 and
3.8 Å from the carboxylate of D135, respectively, which is
longer than the equivalent distance in the 52d−KDM4A
complex (conformation B). Notably, the p-chlorophenyl and
m-dichlorophenyl groups of 54a and 54j both engage in
hydrophobic interactions with V313 of KDM4A, albeit with a
slightly different orientation of the aromatic ring (Figure 7). We
observe a closer interaction of one m-Cl of 54j with the side chain
of V313 (distance of 3.5 Å, Figure 7B) compared to the aromatic
ring of 54a (distance of 4 Å), potentially explaining the enhanced
KDM4A/B inhibition of 54j; indeed, this hydrophobic V313
Crystal structures of 54a and 54j bound to KDM4A showed
that both compounds adopt an extended conformation con-
sistent with that observed for 52d and 53a (Figure 7A). The
orientation and position of the piperidine ring in 54a and 54j is
perpendicular to the aromatic ring of Y175 in KDM4A and is
rotated by nearly 90° compared to the piperidine ring of 53a
(
compare Figures 6 and 7A). The piperidine ring in 54a or 54j
maintains hydrophobic interactions with the side chain of Y175
distances of 3.5 and 3.1 Å between the nearest carbon atom of
the piperidine and the aromatic ring of Y175 for 54a and 54j,
(
M
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C8-pyrazolo-pyrido[3,4-d]pyrimidin-4(3H)-one scaffold to the
active site metal and additional beneficial interactions of the
4
-phenylpiperidine substituent with D135 and Y175 as well as a
further hydrophobic interaction with V313 of KDM4A (KDM4A
residue numbering), which we postulate is important to the
balanced KDM4 and KDM5 activity profile of these compounds.
Importantly, incorporation of the conformationally constrained
4
-phenylpiperidine in derivatives 54j and 54k results in selective
inhibition of the KDM4 and KDM5 subfamily demethylases
over representative exemplars of the KDM2, KDM3, and KDM6
subfamilies, cellular permeability in the Caco-2 assay, and, for
54k, we demonstrate inhibition of H3K9Me and H3K4Me
Figure 7. (A) Overlay of KDM4A-bound 54a (orange) and 54j (cyan).
(
B) 2D interaction diagram of 54j showing interactions with key
3
3
residues in KDM4A. Zn(II) is shown for the active site Fe(II).
demethylation in a cell-based assay. We propose that compounds
such as 54k could be utilized for the study of KDM4/KDM5
subfamilies in a cellular context. As both KDM4 and KDM5
family members have been implicated in breast, prostate, and
other cancer contexts, such tool compounds may be particularly
useful in studying the merits of dual inhibition of these
family members in cell-based assays. In addition, the SARs and
the ligand−protein interactions presented in this study could
provide valuable leads for the further exploring the design and
synthesis of KDM4 or KDM5 subfamily selective compounds
with which to elucidate the role of the JmjC catalytic domain
activity of these subfamilies in human disease. We are continuing
our research toward the identification of potent, cell permeable,
and selective inhibitors of the KDM4 subfamily with which to
study the role of KDM4 enzyme function in cancer biology and
to further explore the potential of such compounds as anticancer
agents. We will report this work in future communications.
interaction may contribute to the balanced KDM4 and KDM5
activity profile of these compounds through enhancing KDM4
subfamily affinity.
CONCLUSION
■
An HTS campaign versus the JmjC histone lysine demethylase
KDM4B resulted in the discovery of N-substituted 4-(pyridin-2-
yl)thiazole-2-amine derivatives as promising hit compounds.
Comparison with previously reported pyridine-based JmjC
KDM inhibitors generated a binding mode hypothesis, and
subsequent incorporation of an appropriately positioned
carboxylic acid moiety led to the discovery of 15, a potent
inhibitor of the KDM3, KDM4, and KDM5 KDM subfamilies
but which lacked cellular permeability. A crystal structure of 15
bound to KDM4A confirmed the proposed binding mode
involving interactions of the carboxylate moiety with K206 and
Y132 as well as bidentate coordination of both the pyridyl and
thiazolyl nitrogens to the active site metal. Interestingly, the
corresponding primary amide 16 maintained this binding mode;
however, loss of the carboxylate−K206 interaction led to a >400-
fold reduction in potency, illustrating the dominance of this ionic
interaction in the buried KDM4 catalytic site. Replacement of the
pyridine-4-carboxylate scaffold in 15 with the less acidic, bicyclic
EXPERIMENTAL SECTION
■
KDM4A and KDM4B AlphaScreen Biochemical Assays. Protein
production and purification: Baculovirus containing residues 1−500 of
KDM4B with an N-terminal GST tag were generated according to
Bac-to-Bac protocol. Following infection of Sf 9 insect cells, protein was
purified by GSH affinity chromatography followed by gel filtration
(Superdex 200). Purification of KDM4A is described in the crystallo-
graphy section. For the purpose of biochemical assays, the His tag was
not removed.
pyrido[3,4-d]pyrimidin-4(3H)-one isostere 58 (pK = 8.23) gave
a
cell-permeable KDM inhibitors with maintained affinity,
presumably due to ionization of the weakly acidic scaffold in
the KDM4 active site. A crystal structure of 58 bound to KDM4A
demonstrated simultaneous coordination to the active site metal
via the pyridine nitrogen, interaction with K206/Y132 via the
pyrimidinone CONH moiety, and interaction with the side chain
of K241 through N1 of the scaffold. Subsequent structure-guided
optimization at C8 of the pyrido[3,4-d]pyrimidin-4(3H)-one
allowed extension into the histone peptide binding site by
virtue of a C8-pyrazole moiety. This tactic favored bidentate
coordination to the active site metal through both the pyridine
and pyrazole N2 nitrogen as well as concomitant additional
pyrazole substitution to gain beneficial interactions with
D135 and Y175 (KDM4A residue numbering) as exemplified
by a crystal structure of 52d bound in KDM4A. However, this
Enzyme activity was measured using an AlphaScreen assay that
monitored the demethylation of a biotinylated trimethylated H3K9
peptide using a H3K9 dimethyl specific antibody and appropriate donor
46
and acceptors beads from PerkinElmer Life Sciences. For each
compound, a 10 mM stock concentration in 100% DMSO was used.
Compounds were dispensed using an ECHO 550 acoustic dispenser
(Labcyte Inc., Sunnyvale, CA, USA) to generate 8 pt dilution
curves directly into 384-well Proxiplates (no. 6008289, PerkinElmer,
Waltham, MA, USA) to give final assay concentrations in the range
0
.0005−100 μM in 2% (v/v) DMSO as appropriate. The enzyme
reaction was performed in assay buffer which consisted of HEPES
(
(
50 mM, pH 7.5), BSA (0.1% w/v), sodium ascorbate (100 μM), 2OG
2 μM for KDM4B or 10 μM for KDM4A), ammonium iron(II) sulfate
hexahydrate (1 μM), and Tween 20 (0.01% v/v). Compounds were
preincubated for 10 min with 2.5 μL enzyme N-terminal GST KDM4B
prepared in-house (0.5 nM final concentration) or His tagged KDM4A
prepared in-house (1.5 nM final concentration) before the addition of
52d-bound crystal structure also revealed alternative bound
conformations of the flexible pyrazole C4 substituent, suggesting
that conformational restriction may be required to further
benefit from interactions in the histone peptide binding site.
Incorporation of a rigidifying piperidine linker into pyrazole C4
substituent and subsequent positioning of a meta-substituted
phenyl ring at the piperidine 4-position, resulted in the discovery
of potent JmjC histone demethylase inhibitors such as 54j,
2.5 μL of peptide (ARTKQTARK(Me )STGGKAPRKQLA-GGK-
3
biotin, Genecust, 30 nM final concentration). The plate was sealed
and centrifuged at 1000 rpm for 1 min before being left for 20 min at
room temperature. The reaction was stopped with the addition of 2.5 μL
of EDTA (30 mM final concentration) before the addition of 2.5 μL of
AlphaScreen detection reagents (no. 6760606M, PerkinElmer) and
antibody. The detection reagents containing IgG mouse acceptor beads,
streptavidin donor beads (20 μg/mL final concentration), and an
antidimethyl H3K9 antibody (no. Ab1220, 0.5 nM final concentration)
were preincubated for 1 h in detection buffer containing HEPES
5
4k, and 54i. A crystal structure of 54j bound in KDM4A
demonstrates a conserved bidentate binding mode of the
N
DOI: 10.1021/acs.jmedchem.5b01635
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Journal of Medicinal Chemistry
Article
4
9
(
50 mM, pH 7.5), BSA (0.1% w/v), and Tween 20 (0.01% v/v) prior to
Ligands were prepared using LigPrep, applying the OPLS_2005 force-
field with possible tautomeric and ionization states within pH range
5.0−9.0 generated using Epik metal binding state settings. Using Extra
Precision (XP) settings, flexible docking of ligands was constrained to
the coordinates of the core, defined as the terminal pyridine ring of the
cocrystallized ligand in PDB 3PDQ, with an RMSD tolerance of 1.0 Å.
The docked poses with the lowest RMSD and state penalties were
selected as the preferred solutions.
addition to the plate. Plates were incubated at room temperature for 1 h
in the dark before being read on an EnVision multilabel reader
(PerkinElmer Life Sciences), using an AlphaScreen format. The signal
was expressed in counts per second. All data analysis was carried out
using the Studies package from Dotmatics (Bishops Stortford, UK). The
percentage inhibition was calculated relative to blank wells (containing
no enzyme and 2% DMSO) and total wells (containing all reagents and
2
% DMSO). IC s were generated using a four-parameter logistics fit.
KDM5B and KDM5C AlphaScreen Biochemical Assays. All
Crystal Structure Determinations. For KDM4A bound to
compounds 15, 16, 37, 40, 52d, 53a, 54a, 54j, and 58: A previously
established N-terminally His-tagged KDM4A construct (residues
50
buffer components were from Sigma-Aldrich unless otherwise stated
and were of the highest purity available. Bovine serum albumin fraction
V was essentially free from fatty acids and globulins (Sigma-Aldrich,
code A7030). 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid (HEPES) buffer was from Life Technologies (code BI8181).
The biotinylated peptide ligand substrate used for the KDM5 assay
3
7
1−359) was produced in Escherichia coli and purified by nickel affinity
chromatography, followed by tag removal with TEV protease, reverse
nickel affinity, and gel filtration. The protein was stored at −80 °C at
10 mg/mL concentration in a buffer containing 10 mM HEPES pH 7.5
and 200 mM NaCl. Purified KDM4A was crystallized in the apo form at
18 °C using the hanging-drop vapor-diffusion method. The crystal-
lization drops were composed of 1.5 μL of protein (7 mg/mL) and
1.5 μL of reservoir solution containing 12−16% (w/v) PEG4000 and
0.1 M BTP pH 7.5, placed over 800 μL of reservoir solution. Plate-like
crystals typically grew in a week and were then soaked by addition
of 0.75 μL of compound at 10−200 mM in DMSO directly to the
drops, followed by 4−48 h incubation at 18 °C. Crystals were briefly
transferred to cryoprotectant solution containing 14% (w/v) PEG4000,
75 mM BTP pH 7.5, and 25% (v/v) glycerol prior to freezing in liquid
nitrogen.
(
H3(1−21)K4-Me -GGK Biotin) was from Anaspec (code 64192).
3
Anti H3K4Me antibody was from Cell Signaling Technology (code
2
9
725S), and AlphaScreen General IgG detection kit was from
PerkinElmer. AlphaScreen assay buffer (50 mM HEPES pH 7.5, 0.1%
BSA, 0.01% Tween-20) was prepared fresh each week, filter sterilized
through a 0.2 μm filter, and stored at 4 °C. Ferrous ammonium sulfate
(
2
FAS) was dissolved fresh each day to 400 mM (156.856 mg/mL) in
0 mM HCl and diluted to 1 mM in deionized water. 2OG was prepared
fresh each day by dissolving to 10 mM (1.901 mg/mL) in deionized
water. L-Ascorbic acid (L-AA) was dissolved to 50 mM (8.806 mg/mL)
in deionized water. Compound dispensing was performed using an
ECHO-550 acoustic dispenser (Labcyte), and all reagent dispenses
were performed using a multidrop combi reagent dispenser (Thermo
Scientific) equipped with a small tube dispensing cassette (Thermo
Scientific) unless otherwise stated. All KDM5 AlphaScreen assays
were carried out in white 384-well proxiplates plus (PerkinElmer, code
For KDM4A bound to compound 30b: The same KDM4A protein
37
was produced, purified, and stored as described previously. A complex
of KDM4A with compound 30b was formed by mixing 30 mg/mL
protein in 20 mM HEPES pH 7.5, 5% (v/v) glycerol, 500 mM NaCl, and
0.5 mM TCEP with 100 mM compound 30b in DMSO to a final
concentration of 5 mM. Then, cocrystallization was performed in sitting
drop plates at 4 °C by mixing 100 nL of protein−ligand complex with
50 nL of reservoir solution containing 0.1 M Bis-Tris pH 5.9, 0.15 M
ammonium sulfate, 13% (w/v) PEG3350, placed over 20 μL of reservoir
solution. Plate-like crystals typically grew in a week. Prior to data
collection, crystals were briefly transferred to a cryoprotectant solution
consisting of the precipitation buffer supplemented with 25% (v/v)
ethylene glycol and were subsequently flash cooled in liquid nitrogen.
For structures of KDM4D bound to compounds 30a and 44a: DNA
encoding for KDM4D (residues 1−342) was cloned into an in-house
6
008280). Compounds for IC50 determination were dissolved in 100%
DMSO to a concentration of 10 mM and an 11-point, 3-fold dilution of
each compound for IC50 was dry dispensed (100 nL) into 384-well
proxiplates using an ECHO-550 acoustic dispenser. The final assay
concentration range was 0.0006−100 μM. Then 100 nL of DMSO was
dispensed into each well of column 12 and 2,4-PDCA (100 mM) was
dispensed into each well of column 24. KDM5B and KDM5C were
diluted to 4 nM in assay buffer, and 5 μL of KDM5 enzyme dispensed
into each well of a 384-well proxiplate. Plates were sealed with an
aluminum foil, centrifuged at 1000 rpm for 5 s, and compound allowed
to preincubate with enzyme for 15 min at room temperature before
addition of peptide substrate. Substrate was prepared in assay buffer
5
0
pET-based vector pNIC28-Bsa4 using ligation-independent
cloning. The resulting construct expressed the desired proteins with
an N-terminal His -tag and tobacco etch virus (TEV) protease cleavage
6
(
200 μM L-AA, 20 μM FAS, 0.2 μM H3K4Me peptide, 10 μM 2OG)
site (extension MHHHHHHSSGVDLGTENLYFQ*SM). The protein
was produced in E. coli and purified by nickel affinity chromatography,
followed by Tag removal with TEV protease, reverse nickel affinity, and
gel filtration. The protein was stored at −80 °C at 11 mg/mL in a buffer
containing 10 mM HEPES pH 7.5, 0.5 M NaCl, 5% glycerol, and 0.5 μM
TCEP.
KDM4D was crystallized in sitting drop plates at 20 °C by mixing
100 nL of protein complex with 50 nL of reservoir solution containing
0.1 M Bis-Tris pH 5.9, 0.15 M ammonium sulfate, and 11% (w/v)
PEG3350, placed over 20 μL of reservoir solution. The crystals were
soaked with compound 30a or 44a by mixing 0.5 μL of 10 mM
compound (in 9 parts ethylene glycol/1 part DMSO) with 1.5 μL of
reservoir solution and adding it to the crystals. The crystals were then
flash frozen in liquid nitrogen after incubating them for 3 h.
3
and 5 μL dispensed into each well to initiate the enzyme reaction. Plates
were sealed with an aluminum foil centrifuged at 1000 rpm for 15 s, and
the enzyme reaction incubated at room temperature for 20 min. Enzyme
reactions were stopped after 20 min by addition of 5 μL of assay buffer
containing EDTA (30 mM) and NaCl (1.2 M). AlphaScreen detection
reagent was prepared by diluting AlphaScreen streptavidin donor and
protein A acceptor beads in assay buffer (0.08 mg/mL with respect to
each bead) containing anti-H3K4Me antibody (1:750) and preincu-
bated for 1 h. Product H3K4Me methyl mark was detected by addition
2
2
of 5 μL of detection reagent followed by 2 h incubation at room
temperature. After 2 h, plates were read in a BMG Labtech plate reader
equipped with an AlphaScreen 680 nm/570 nm optic module. Data
were normalized to the no enzyme (2,4-PDCA) control and IC50
determined from nonlinear regression curve fitting using Graphpad
Prism 5.0.
For KDM5B bound to compound 52d: A KDM5B construct
encoding regions Phe26−Ile770 was amplified from an Origene cDNA
clone and cloned into a pFastBac-derived vector (pFB-LIC-Bse)
containing a tobacco etch virus (TEV) protease cleavable N-terminal
KDM2A, KDM3A, and KDM6B AlphaScreen Biochemical
3
4
Assays. IC50 values were determined as previously described.
Computational Chemistry. An enzyme−substrate cocrystal struc-
His -tag. The recombinant KDM5B (residues 26−770) construct was
6
33
ture of KDM4A (PDB 3PDQ) was prepared for modeling using
expressed in Sf9 cells, with generation of recombinant baculo viruses,
insect cell culture, and infections being performed according to the
manufacturer’s instructions (Invitrogen). The cells were harvested 72 h
post infection, lysed in a buffer containing 50 mM HEPES pH 7.5,
500 mM NaCl, 10 mM imidazole, 5% glycerol, 0.5 mM TCEP, and a
protease inhibitor cocktail (Calbiochem) and purified using nickel
affinity chromatography using a stepwise gradient of imidazole. The
47
Protein Preparation Wizard in Maestro, and all water molecules not
coordinating to the catalytic metal were removed. To propose predicted
binding modes of the hit series, Glide (Grid-based Ligand Docking with
48
Energetics) was used for the docking experiments. The receptor grid
3
was defined by a grid box of 30 × 30 × 30 Å with a default inner box
3
(
10 × 10 × 10 Å ) centered on the cocrystallized ligand in PDB 3PDQ.
O
DOI: 10.1021/acs.jmedchem.5b01635
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Journal of Medicinal Chemistry
Article
eluted protein was then incubated with TEV protease at 4 °C overnight
followed by size-exclusion chromatography (Superdex 200). The TEV
protease and uncleaved protein were removed using nickel affinity
chromatography, and protein was concentrated to 8.1 mg/mL and
stored at −80 °C. Protein was crystallized at 4 °C using the sitting drop
vapor diffusion method. KDM5B was preincubated with 4 mM MnCl2
before the protein was transferred to crystallization plates. Crystals were
obtained in drops consisting of 100 nL of protein mix (8.1 mg/mL),
(Life Technologies). Four hours after transfection, the cells were treated
with serial dilutions of compound 54k for 24 h. The cells were then fixed
and stained with a mouse monoclonal anti-Flag (Sigma F3165) and
with either a rabbit anti-H3K9me3 1 h at RT (Abcam ab8898) or a
rabbit anti-H3K4me3 overnight at 4 °C (Diagenode C15410003).
The secondary antibodies were goat antirabbit Alexa Fluor 488 (Life
Technologies A11034) and goat antimouse Alexa Fluor 568 (Life Tech-
nologies A21124). Image acquisition was conducted using the Operetta
high content imaging system (PerkinElmer), and image analysis
was performed with the Columbus software (PerkinElmer). After the
DAPI-stained nuclei were automatically identified, a minimum thresh-
old for anti-Flag intensity was set so that only cells highly expressing the
demethylase were analyzed.
2
00 nL of a precipitant consisting of 1.6 M Na/K phosphate, 0.1 M
HEPES pH 7.5, and 20 nL of KDM5B seeds of crystals obtained from
the same condition. Compound was soaked into apo crystals of KDM5B
for 5 min at a concentration of 5 mM. Crystals were cryoprotected with
mother liquor supplemented with 25% ethylene glycol before they were
flash-frozen in liquid nitrogen.
Microsomal and Aldehyde Oxidase (AO) Clearance Studies.
CYP450 and AO-mediated metabolism of KDM4 inhibitor compounds
were assessed by incubation of compound (1 μM concentration) in
mouse and human liver microsomes (MLM, HLM) and mouse liver
Data Collection, Processing, and Refinement. X-ray data were
collected in-house at the Institute of Cancer Research, London, UK, on a
Rigaku FRX-AFC11-VariMax Cu-VHF-Pilatus300 K, at the Struc-
tural Genomics Consortium, Oxford, UK, on a Bruker Microstar
generator equipped with an APEX II detector, at Diamond Light Source,
Oxfordshire, UK, at beamlines I02, I03, I04, and I04-1, and at the
European Synchrotron Ray Facility, Grenoble, France, at beamline
63
cytosol (0.5 mg/mL) (Tebu-bio) respectively in 0.1 M PBS at 37 °C.
Microsomal incubations were initiated by addition of NADPH and
UDPGA for phase I and II reactions. Cytosol incubations were initiated
by KDM4 inhibitor compounds in the presence and absence of AO
inhibitor raloxifene. Compound levels were measured over time (0, 5,
10, 15, and 30 min) to calculate clearance by removing aliquots of the
incubate at specific time points and quenching into acetonitrile with
internal standard (IS) and centrifugation at 3000 rpm for 30 min at 4 °C.
Supernatant was diluted for LC-MS/MS analysis.
ID23. Crystals of KDM4A belonged to the space group P12 1 (or
1
P2 2 2 for KDM4A bound to compound 30b) and diffracted to
1
1
resolutions between 2.05 and 2.65 Å. Crystals of KDM4D belonged to
the space group P4 2 2 and diffracted to resolutions between 1.41 and
3
1
1
.88 Å. The KDM5B crystal belonged to the space group P6 22 and
5
diffracted to a resolution of 2.35 Å. All data sets were integrated with
XDS, scaled, and merged with AIMLESS, except for data sets
collected on the Bruker system which were integrated with SAINT
Chemistry. Commercially available starting materials, reagents, and
anhydrous solvents were used as supplied. Flash column chromatog-
raphy was performed using Merck silica gel 60 (0.025−0.04 mm). Thin
layer chromatography was performed using Merck Millipore TLC silica
gel 60 F254 aluminum sheets and visualized by UV (254 and 280 nm),
51
52
(
version 8.3, Bruker AXS Inc., 2013) and scaled with SADABS (version
2
012/1, Bruker AXS Inc., 2012). The structures were solved by mole-
53,54
cular replacement using PHASER,
and publicly available KDM4A,
iodine, and KMnO . Column chromatography was also performed on a
4
KDM4D, and KDM5B structures (PDB codes 2OQ7, 3DXT, and 5A1F,
respectively) with ligand and water molecules removed were used as
FlashMaster personal unit using isolute Flash silica columns or a Biotage
Isolera purification system using Biotage KP-SNAP cartridges. Ion
37
molecular replacement models. The protein−ligand structures were
exchange chromatography was performed using acidic Isolute Flash
55
56
1
manually corrected and rebuilt in COOT and refined with BUSTER,
SCX-II cartridges or basic Isolute Flash NH cartridges. H NMR
2
5
7
58
Phenix, or REFMAC 5 in iterative cycles. Ligand restraints were
spectra were recorded on either a Bruker Avance-500 or Bruker Avance-
400 NMR machine. Samples were prepared as solutions in a deuterated
solvent and referenced to the appropriate internal nondeuterated
solvent peak or tetramethylsilane. Chemical shifts were recorded in ppm
(δ) downfield of tetramethylsilane.
LC-MS Analysis. Analysis was performed on a Waters LCT with
a Waters Alliance 2795 separations module and Waters 2487 dual
wavelength absorbance detector coupled to a Waters/Micromass LCT
time-of-flight mass spectrometer with ESI source.
Method A: Analytical separation was carried out at 30 °C on a Merck
Purospher STAR column (RP-18e, 30 mm × 4 mm) using a flow rate of
1.5 mL/min in a 4 min gradient elution with detection at 254 nm. The
mobile phase was a mixture of methanol (solvent A) and water (solvent
B), both containing formic acid at 0.1%. Gradient elution was as follows:
10:90 (A/B) to 90:10 (A/B) over 2.5 min, 90:10 (A/B) for 1 min, and
then reversion back to 10:90 (A/B) over 0.3 min, finally 10:90 (A/B) for
0.2 min.
9
60
generated with GRADE and MOGUL6. The quality of the struc-
1,62
tures was assessed with MOLPROBITY.
The data collection and
refinement statistics are presented in Supporting Information, Table S1.
Caco-2 Permeability. Papp (apparent permeability) was determined
in the Caco-2 human colon carcinoma cell line. Cells were maintained
(
DMEM with 10% fetal bovine serum, penicillin, and streptomycin) in
a humidified atmosphere with 5%CO /95% air for 10 days. Cells were
2
plated out onto a cell culture assembly plate (Millipore, UK), and
monolayer confluency was checked using a TEER electrode prior to the
assay. Media was washed off and replaced in the appropriate apical and
basal wells with HBSS buffer (pH7.4) containing compound (10 μM,
1% DMSO). The Caco-2 plate was incubated for 2 h at 37 °C, and
Lucifer Yellow was used to confirm membrane integrity after the assay.
Samples from the apical and basolateral chambers were analyzed using
Waters TQ-S LC-MS/MS.
4
5
Cell-Based Assays. Full length cDNA for KDM4A (JMJD2A,
O75164) and KDM5B (JARID1B, Q9UGL1) were amplified by PCR
and cloned into the pDONR-221 vector using a Gateway BP reaction.
To produce catalytically inactive KDMs, residues involved in iron
coordination were mutated to alanine (KDM5B, H499A/E501A;
KDM4A, H188A/E190A). Mutations were introduced into the full
length KDM Gateway entry clones using 15 cycles of the QuikChange II
PCR protocol (Agilent Technologies). Mammalian expression con-
structs encoding N-terminal 3×FLAG were constructed by a Gateway
LR recombination reaction between pCDNA5-FRT/TO-3FLAG
destination vector (PMID: 24412199) and the wild-type or mutated
KDM Gateway entry clone.
Analysis was also performed on an Agilent 1200 series HPLC and
diode array detector coupled to a 6210 time-of-flight mass spectrometer
with dual multimode APCI/ESI source.
Method B: Analytical separation was carried out at 30 °C on a Merck
Purospher STAR column (RP-18e, 30 mm × 4 mm) using a flow rate of
1.5 mL/min in a 4 min gradient elution with detection at 254 nm. The
mobile phase was a mixture of methanol (solvent A) and water (solvent
B), both containing formic acid at 0.1%. Gradient elution was as follows:
10:90 (A/B) to 90:10 (A/B) over 2.5 min, 90:10 (A/B) for 1 min, and
then reversion back to 10:90 (A/B) over 0.3 min, finally 10:90 (A/B) for
0.2 min.
The human cervical carcinoma cell line HeLa was obtained from the
American Type Cultures Collection and maintained in Eagle’s Minimal
Essential Media (Sigma-Aldrich) supplemented with 10% heat-
inactivated FBS (Life Technologies). Cells were transiently transfected
with either the Flag-tagged demethylase or an inactive version
containing mutations in the catalytic site using Lipofectamine 3000
Method C: Analytical separation was carried out at 40 °C on a Merck
Purospher STAR column (RP-18e, 30 mm × 4 mm) using a flow rate of
3 mL/min in a 2 min gradient elution with detection at 254 nm. The
mobile phase was a mixture of methanol (solvent A) and water (solvent
B), both containing formic acid at 0.1%. Gradient elution was as follows:
10:90 (A/B) to 90:10 (A/B) over 1.25 min, 90:10 (A/B) for 0.5 min,
P
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Journal of Medicinal Chemistry
Article
and then reversion back to 10:90 (A/B) over 0.15 min, finally 10:90
glacial acetic acid (solvent B). Gradient elution was as follows: 95:5
(A/B) over 0.5 min, 95:5 (A/B) to 5:95 (A/B) over 2 min, 5:95 (A/B)
over 1 min, then reversion back to 95:5 (A/B) over 0.1 min and 95:5
(A/B) over 1.4 min.
(
A/B) for 0.1 min.
Method D: Analytical separation was carried out at 40 °C on a Merck
Chromolith Flash column (RP-18e, 25 mm × 2 mm) using a flow rate of
1
.5 mL/min in a 2 min gradient elution with detection at 254 nm. The
Method L: Analytical separation was carried out on a Waters sunfire
C18 column (4.6 mm × 150 × 4.6 mm, 100 A) using a flow rate of 1 mL/
min in a 20 min gradient run. The mobile phase was a mixture of 99.99%
water and 0.01% CF CO H (solvent A) and 99.99% acetonitrile and
mobile phase was a mixture of methanol (solvent A) and water (solvent B),
both containing formic acid at 0.1%. Gradient elution was as follows: 5:95
(A/B) to 100:0 (A/B) over 1.25 min, 100:0 (A/B) for 0.5 min, and then
3
2
reversion back to 5:95 (A/B) over 0.05 min, finally 5:95 (A/B) for 0.2 min.
Method E: Analytical separation was carried out at 30 °C on a Merck
Chromolith Flash column (RP-18e, 25 mm × 2 mm) using a flow rate of
0.01% CF CO H (solvent B). Gradient elution was as follows: 100:0
3 2
(A/B) over 2 min, 100:0 (A/B) to 0:100 (A/B) over 16 min and 0:100
(A/B) over 2 min. All compounds submitted for biological testing were
determined to be >95% pure by Methods A−L unless stated otherwise.
LC-HRMS Analysis. LC-HRMS was performed using either an
Agilent 1200 series HPLC and diode array detector coupled to a 6210
time-of-flight mass spectrometer with dual multimode APCI/ESI source
(methods B and E) or a Waters Acquity UPLC and diode array detector
coupled to a Waters G2 QToF mass spectrometer fitted with a
multimode ESI/APCI source (methods G and I) or on an Agilent
1290 uHPLC system coupled with an Agilent ESI-QTOF 6530 mass
spectrometer (method J). LC-HRMS methods B and E referenced
0.75 mL/min in a 4 min gradient elution with detection at 254 nm. The
mobile phase was a mixture of methanol (solvent A) and water (solvent
B), both containing formic acid at 0.1%. Gradient elution was as follows:
5
:95 (A/B) to 100:0 (A/B) over 2.5 min, 100:0 (A/B) for 1 min, and
then reversion back to 5:95 (A/B) over 0.1 min, finally 5:95 (A/B) for
0
.4 min.
Analysis was also performed on a Waters Acquity UPLC and diode
array detector coupled to a Waters G2 QToF mass spectrometer fitted
with a multimode ESI/APCI source.
Method F: Analytical separation was carried out at 30 °C on a
Phenomenex Kinetex XB-C18 column (30 mm × 2.1 mm, 1.7 μ, 100 A)
using a flow rate of 0.5 mL/min in a 2 min gradient elution with
detection at 254 nm. The mobile phase was a mixture of methanol
+
+
to caffeine [M + H] 195.087652, reserpine [M + H] 609.280657
(method B), or hexakis (2,2-difluoroethoxy)phosphazene [M + H]+
622.02896 (methods B and E) and hexakis(1H,1H,3H-tetrafluoro-
+
pentoxy)phosphazene [M + H] 922.009798. LC-HRMS method+s
(
solvent A) and water (solvent B), both containing formic acid at 0.1%.
Gradient elution was as follows: 10:90 (A/B) to 90:10 (A/B) over
.25 min, 90:10 (A/B) for 0.5 min, and then reversion back to 10:90
G and I referenced to leucine enkephalin fragment ion [M + H]
397.1876. LC-HRMS method J referenced to hexakis (2,2-difluoro-
+
1
ethoxy)phosphazene [M + H] 622.02896 and hexakis(1H,1H,3H-
+
(
A/B) over 0.15 min, finally 10:90 (A/B) for 0.1 min.
Method G: Analytical separation was carried out at 30 °C on a
tetrafluoropentoxy)phosphazene [M + H] 922.009798.
General Procedure 1: Amine Displacement of Mesylate Using
Cesium Carbonate. Cesium carbonate (3 equiv) was added to a
mesylate intermediate freshly made from alcohol 47 (1 equiv) and
Phenomenex Kinetex XB-C18 column (30 mm × 2.1 mm, 1.7 μ, 100 A)
using a flow rate of 0.3 mL/min in a 4 min gradient elution with
detection at 254 nm. The mobile phase was a mixture of methanol
amine (2 equiv) in anhydrous DMF under N . The reaction mixture was
2
(
solvent A) and water (solvent B), both containing formic acid at 0.1%.
Gradient elution was as follows: 10:90 (A/B) to 90:10 (A/B) over
min, 90:10 (A/B) for 0.5 min, and then reversion back to 10:90 (A/B)
stirred for 15 h at 90 °C and monitored by LCMS. The reaction mixture
was diluted in water and extracted three times with EtOAc. The
combined organic layers were washed with saturated brine solution,
3
over 0.3 min, finally 10:90 (A/B) for 0.2 min.
dried over MgSO , and concentrated in vacuo to give the crude material
4
Method H: Analytical separation was carried out at 30 °C on a
Phenomenex Kinetex C18 column (30 mm × 2.1 mm, 2.6 μ, 100 A)
using a flow rate of 0.5 mL/min in a 2 min gradient elution with
detection at 254 nm. The mobile phase was a mixture of methanol
which was purified by Biotage column chromatography (see individual
compounds for details of the eluent used).
General Procedure 2: Amine Displacement of Mesylate Using
Trimethylamine. Triethylamine (2 equiv) was added to a solution of
mesylate intermediate freshly made from alcohol 47 (1 equiv) and
amine [or amine hydrochloride] (1.2 equiv) in anhydrous DMF under
(
solvent A) and water (solvent B), both containing formic acid at 0.1%.
Gradient elution was as follows: 10:90 (A/B) to 90:10 (A/B) over
.25 min, 90:10 (A/B) for 0.5 min, and then reversion back to 10:90
1
N
2
. The reaction mixture was heated at 50 °C for 15 h and monitored
by LCMS. When the reaction had gone to completion, the reaction
mixture was diluted in H O and extracted three times with EtOAc.
The combined organic layers were washed with saturated LiCl solution
and saturated brine solution, dried over MgSO , and concentrated
(
A/B) over 0.15 min, finally 10:90 (A/B) for 0.1 min.
Method I: Analytical separation was carried out at 30 °C on a
2
Phenomenex Kinetex C18 column (30 mm × 2.1 mm, 2.6 μ, 100 A)
using a flow rate of 0.3 mL/min in a 4 min gradient elution with
detection at 254 nm. The mobile phase was a mixture of methanol
4
in vacuo to give the crude material which was purified by Biotage column
chromatography (see individual compounds for details of the eluent used).
General Procedure 3: Reductive Amination. To a microwave
vial was added the required aldehyde (1 equiv) and corresponding
amine (1.5 equiv) and the flask purged with argon. Anhydrous 1,2-
dichloroethane (4 mL) was then added, the mixture was stirred to allow
dissolution followed by the addition of sodium triacetoxyborohydride
(1.6 equiv), the vial was capped, and the mixture was stirred at room
temperature overnight. Once the reaction was deemed complete by
LCMS analysis, the mixture was directly absorbed unto silica gel and
purified by flash column chromatography to afford the requisite amine
(workup procedure A). Alternatively, the mixture was concentrated to
afford an amorphous solid and passed through a plug of silica eluting
with 40% MeOH/CH Cl to afford an oil which was used without
(
solvent A) and water (solvent B), both containing formic acid at 0.1%.
Gradient elution was as follows: 10:90 (A/B) to 90:10 (A/B) over
min, 90:10 (A/B) for 0.5 min, and then reversion back to 10:90 (A/B)
3
over 0.3 min, finally 10:90 (A/B) for 0.2 min.
Analysis was also performed on a Waters system equipped with a
Waters 2545 binary gradient module, a Waters SQ Detector 2, Waters
2
489 UV/visible detector, and a Waters 2424 ELS detector.
Method J: Analytical separation was carried out on a Kinetex 5u EVO
C18 column (100 mm × 3.0 mm, 100 A) using a flow rate of 2 mL/min
in a 3 min gradient elution. The mobile phase was a mixture of 93% H O,
2
5% acetonitrile, and 2% of 0.5 M ammonium acetate adjusted to pH 6
with glacial acetic acid (solvent A) and 18% H O, 80% acetonitrile, and
2
2% of 0.5 M ammonium acetate adjusted to pH 6 with glacial acetic acid
2
2
(
solvent B). Gradient elution was as follows: 95:5 (A/B) 0.35 min, 95:5
further purification (workup procedure B).
(
A/B) to 5:95 (A/B) over 1 min, 5:95 (A/B) over 0.75 min, and then
General Procedure 4: SEM Deprotection with 6 M HCl/THF.
Hydrochloric acid (6 M, 60−90 equiv) was added to a solution of SEM
protected material (1 equiv) in THF (0.1 M). The reaction mixture was
stirred at 50−60 °C for between 3 and 8 h and monitored by LCMS.
Following completion of the reaction, the reaction mixture was
concentrated in vacuo and purified by Biotage column chromatography
on a KP-NH snap column unless otherwise stated (see individual
compounds for details of the eluent used). The product obtained from
reversion back to 95:5 (A/B) over 0.1 min and 95:5 (A/B) over 0.8 min.
Method K: Analytical separation was carried out on a Chromolith
Performance RP-18e column (100 mm × 2.0 mm) using a flow rate of
1
.5 mL/min in a 5 min gradient elution. The mobile phase was a mixture
of 93% H O, 5% acetonitrile, and 2% of 0.5 M ammonium acetate
adjusted to pH 6 with glacial acetic acid (solvent A) and 18% H O, 80%
acetonitrile and 2% of 0.5 M ammonium acetate adjusted to pH 6 with
2
2
Q
DOI: 10.1021/acs.jmedchem.5b01635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
column chromatography was triturated with Et O to give the pure
solution a saturated NaHCO solution (50 mL) was carefully added.
2
3
product.
The two layers were separated, and the organic layer was carefully
General Procedure 5: SEM Deprotection with HCl/1,4-
Dioxane. To a microwave vial was added the SEM protected starting
material (1 equiv), 1,4-dioxane (3 mL), and distilled water (1 mL)
followed by the dropwise addition of HCl in dioxane (4M, 25 equiv).
The vial was capped and the mixture stirred at 50 °C until analysis by
LCMS indicated complete conversion to the product. The mixture was
then concentrated and passed through an SCX-2 cartridge washing
washed with more saturated NaHCO solution (30 mL) and water
3
(20 mL), dried (Na SO ), and concentrated in vacuo. The liquid residue
2
4
was absorbed onto silica gel and placed on a 50 g Isolute silica II column
which was eluted with 5%, 10%, and finally 15% EtOAc in hexane to
provide in order of elution the dibrominated product, and the title
compound as an oily residue which solidified on standing at room
1
temperature (0.98 g, 38%). H NMR and LC-MS as reported above.
initially with MeOH and then NH /MeOH. The basic wash was
3
Methyl 2-(2-((tert-Butoxycarbonyl)amino)thiazol-4-yl)-
isonicotinate (14). A mixture of methyl 2-(2-bromoacetyl)-
isonicotinate (0.100 g, 0.387 mmol, 1 equiv), N-Boc-thiourea
(0.068 g, 0.387 mmol, 1 equiv), and triethylamine (0.04 g, 0.4 mmol,
concentrated and triturated with Et O to afford a solid. If necessary, the
2
solid was further purified by flash column chromatography on a SNAP
KP-NH column eluting with 0−40% EtOH/CH Cl .
2
2
3
-(Ethylsulfonyl)-N-(4-(pyridin-2-yl)thiazol-2-yl)benzamide
1
.05 equiv) was suspended in ethanol (10 mL, 0.04 M) and heated
(
7c). To a mixture of 3-(ethylsulfonyl)benzoic acid (0.098 g, 0.46 mmol,
under reflux for 1 h. The reaction was concentrated in vacuo and the
residue purified by flash silica gel column chromatography eluting with
1
1
equiv), 1-hydroxybenzotriazole hydrate (0.068 g, 0.50 mmol,
.1 equiv), EDCI (0.105 g, 0.55 mmol, 1.2 equiv), and anhydrous
5
% EtOAc in CH Cl to give the product as a yellow oil which solidified
2 2
CH Cl2 (4 mL, 0.12 M) was added 2-amino-4-(2-pyridyl)thiazole
1
2
on standing (0.072 g, 56%). H NMR (500 MHz, CDCl ) δ 1.50
3
(
0.085 g, 0.48 mmol, 1.05 equiv). The reaction mixture was stirred at
(
(
s, 9H), 3.98 (s, 3H), 7.76 (s, 1H), 7.78 (dd, J = 1.6, 5.0 Hz, 1H), 8.47
room temperature for 4 h under argon, then diluted with CH Cl2
2
s, 1H), 8.62 (br s, 1H), 8.76 (d, J = 4.9 Hz, 1H). LC-MS (method C;
(
50 mL) and washed with a saturated NaHCO solution (2 × 20 mL)
+
3
ESI, m/z) t = 1.56 min −336 (M + H) .
R
and saturated brine solution (20 mL), dried (Na SO ), and
2
4
2-(2-Aminothiazol-4-yl)isonicotinic Acid (15). A solution of
concentrated in vacuo. This residue was absorbed on silica gel and the
free running powder was placed on a 20 g Isolute silica II column which
was eluted with 30% EtOAc in CH Cl , 70% EtOAc in CH Cl , and
4
M HCl in dioxane (1 mL) was added to methyl 2-(2-((tert-
butoxycarbonyl)amino)thiazol-4-yl)isonicotinate (0.022 g, 0.066 mmol).
The mixture was stirred at room temperature for 1 h. Volatiles were
removed in vacuo, and the residue was passed through an isolute-NH2
2
2
2
2
finally 2% MeOH in EtOAc/CH Cl (v/v; 4:1). The title compound
2
2
1
was obtained as a white solid (0.068 g, 40%). H NMR (500 MHz,
cartridge eluting with methanol to afford a brown solid which was
DMSO-d ) δ 1.15 (t, J = 7.2 Hz, 3H), 3.42 (q, J = 7.2 Hz, 2H), 7.36 (ddd,
6
dissolved in THF (2 mL) and NaOH solution (3.3 M, 0.3 mL). After
stirring at room temperature for 1 h, acetic acid (0.06 g, 1.0 mmol) was
added and the solution was evaporated to dryness. Water (1 mL) was
J = 1.2, 4.8, 7.5 Hz, 1H), 7.87 (t, J = 7.9 Hz, 1H), 7.91 (td, J = 1.9, 7.8 Hz,
1H), 7.94 (s, 1H), 8.02 (d, J = 7.8 Hz, 1H), 8.15 (dt, J = 1.3, 7.8 Hz, 1H),
8
.44 (dt, J = 1.3, 7.8 Hz, 1H), 8.61−8.64 (m, 2H), 13.15 (s, 1H). LC-MS
+
added to the residue and a white powder separated out which was filtered
(
method B, ESI, m/z) t = 2.50 min −374 (M + H) . HRMS (method
R
1
+
and dried (8 mg, 55%). H NMR (500 MHz, DMSO-d
) δ 7.18 (s, 2H),
6
B): found. 374.0610; calculated for C H N O S (M + H) , 374.0628.
17
16
3
3 2
7
5
0
.31 (s, 1H), 7.66 (dd, J = 1.6, 5.0 Hz, 1H), 8.30 (s, 1H), 8.68 (d, J =
Methyl 2-(1-Ethoxyvinyl)isonicotinate (12). To a mixture of
methyl 2-bromoisonicotinate (0.80 g, 3.7 mmol, 1 equiv), tributyl(1-
ethoxyvinyl)stannane (1.44 g, 4.0 mmol, 1.1 equiv), and 1,4-dioxane
.0 Hz, 1H), 14.00 (br s, 1H). LC-MS (method C); ESI, m/z) t =
R
+
.56 min −222 (M + H) . HRMS (method B): found, 222.0333;
+
calculated for C H N O S (M + H) , 222.0332.
9
8
3
2
(
(
20 mL, 0.19 M) was added tetrakis(triphenylphosphine)palladium
0.300 g, 0.173 mmol, 5 mol %). The reaction mixture was heated at
3
-Amino-2-chloroisonicotinic Acid (32). To a solution of methyl
-amino-2-chloroisonicotinate (1.00 g, 5.36 mmol, 1 equiv) in methanol
13 mL, 0.4 M) was added 1 M aqueous sodium hydroxide (14.4 mL,
3
reflux (120 °C) for 18 h under nitrogen, cooled to room temperature,
and then concentrated in vacuo. The residue was purified by silica gel
column chromatography eluting with CH Cl :hexane (2:1) to afford the
(
1
4
4.4 mmol), and the reaction was stirred at room temperature for
5 min. Methanol was removed in vacuo, and the remaining aqueous
2
2
desired product as a yellow oil which solidified on standing (0.500 g,
1
solution was acidified to pH 5.5 by addition of 1 M HCl. A white
6
5%). H NMR (500 MHz, CDCl ) δ 1.34 (t, J = 7.0 Hz, 3H), 3.95
3
1
precipitate formed which was filtered and dried (0.715 g, 77%). H
(
s, 3H), 3.98 (q, J = 7.0 Hz, 2H), 4.41 (d, J = 2.1 Hz, 1H), 5.45 (d,
NMR (500 MHz, DMSO-d ) δ 6.80 (br s, 2H), 7.58 (d, J = 5.0 Hz, 1H),
J = 2.1 Hz, 1H), 7.73 (dd, J = 1.6, 5.0 Hz, 1H), 8.19 (t, J = 1.0 Hz, 1H),
6
7
.61 (d, J = 5.0 Hz, 1H), 13.80 (br s, 1H). LC-MS (method C; ESI, m/z)
8
−
.69 (dd, J = 0.8, 5.0 Hz, 1H). LC-MS (method C; ESI, m/z) t = 1.4 min
208 (M + H) .
Methyl 2-(2-Bromoacetyl)isonicotinate (13). A mixture of
R
+
+
t = 0.83 min −173, 175 [(M + H) Cl isotopic pattern].
R
3-Amino-2-chloroisonicotinamide (33). To a stirred solution of
3
-amino-2-chloroisonicotinic acid (0.750 g, 4.35 mmol) in thionyl
methyl 2-(1-ethoxyvinyl)isonicotinate (0.500 g, 2.41 mmol) and
chloride (10 mL) was added 2 drops of DMF and the reaction heated at
reflux under nitrogen for 2 h. Volatiles were removed and the residue
was suspended in anhydrous THF (10 mL) and was slowly added to
a solution of ammonium hydroxide (10 mL) cooled in an ice bath and
the reaction stirred at room temperature for 1.5 h. EtOAc (40 mL) and
1
-bromopyrrolidine-2,5-dione (0.429 g, 2.41 mmol) was suspended in
10% water in THF:H O (2:0.2 mL). The reaction was stirred at room
2
temperature for 1 h. Volatiles were removed in vacuo and the residue
purified by flash silica gel column chromatography eluting with 30%
hexane in dichloromethane. The pure fractions provided the desired
product as a pale-brown oil which solidified on standing (0.38 g, 61%).
CH Cl (10 mL) were added and the layers separated. The organic
2 2
1
H NMR (500 MHz, CDCl ) δ 4.00 (s, 3H), 4.84 (s, 2H), 8.07 (dd, J =
solution was dried and concentrated in vacuo. The residue obtained was
purified by flash silica gel column chromatography eluting with 20%
3
1
1
.7, 5.0 Hz, 1H), 8.59 (dd, J = 0.9, 1.7 Hz, 1H), 8.84 (dd, J = 0.7, 5.0 Hz,
+
hexane in ethyl acetate to afford the desired product as a white powder
H). LC-MS (method C; ESI, m/z) t = 1.25 min −258, 260 [(M + H) ,
R
1
(
0.664 g, 89%). H NMR (500 MHz, DMSO-d ) δ 6.76 (br s, 2H), 7.50
Br isotopic pattern]. For the synthesis of compound 25, this inter-
mediate was prepared from methyl 2-acetylpyridine-4-carboxylate as
follows: To a “paste like” mixture of methyl 2-acetylpyridine-4-
carboxylate (1.79 g, 10.0 mmol), acetic acid (12.5 mL), and 48%
aqueous HBr (1.12 mL, 10.0 mmol) cooled in an ice-bath, bromine
6
(d, J = 5.1 Hz, 1H), 7.61 (d, J = 5.1 Hz, 1H), 7.67 (br s, 1H), 8.18 (br s,
+
1H). LC-MS (method A; ESI, m/z) t
Cl isotopic pattern].
= 0.52 min −172, 174 [(M + H)
R
40
8-Chloropyrido[3,4-d]pyrimidin-4(3H)-one (34). 3-Amino-2-
chloroisonicotinamide (0.60 g, 3.5 mmol) in anhydrous triethoxy-
methane (15 mL) was heated at reflux (160 °C) for 18 h. The reaction
was cooled to room temperature, and the pale-white powder was filtered
(
0.57 mL, 11.0 mmol) was added dropwise. The ice-bath was removed,
and stirring was continued at room temperature for 1 h under argon,
then at 75 °C for 1.5 h under argon. The reaction mixture was cooled
to room temperature with the aid of an ice-bath, diluted with THF
and washed with Et
white solid (0.52 g, 82%). H NMR (500 MHz, DMSO-d ) δ 7.96 (d,
O (2 × 5 mL) then dried to give the product as a
2
1
(
12 mL), and stirred at room temperature for 20 min and allowed to
6
stand overnight at room temperature; the volatiles were then removed
under vacuo. The residue was diluted with EtOAc (130 mL), and to this
J = 5.1 Hz, 1H), 8.32 (s, 1H), 8.42 (d, J = 5.1 Hz, 1H), 12.85 (br s, 1H).
+
LC-MS (method H; ESI, m/z) t = 0.66 min −182, 184 [(M + H) Cl
R
R
DOI: 10.1021/acs.jmedchem.5b01635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
isotopic pattern]. HRMS (method I): found, 182.0125; calculated for
the product as a white solid (210 mg, 99%). H NMR (500 MHz,
+
C H N ClO (M + H) , 182.0121.
CDCl ) δ 0.01 (s, 9H), 0.96−1.00 (m, 2H), 2.86 (t, J = 6.3 Hz, 2H),
7
4
3
3
8
-Chloro-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
3.67−3.72 (m, 2H), 3.88 (t, J = 6.3 Hz, 2H), 5.46 (s, 2H), 7.81 (s, 1H),
pyrimidin-4(3H)-one (35). 8-Chloropyrido[3,4-d]pyrimidin-4(3H)-
8.07 (d, J = 5.1 Hz, 1H), 8.30 (s, 1H), 8.60 (s, 1H), 8.62 (d, J = 5.1 Hz,
one (1.87 g, 10.3 mmol, 1 equiv) was dissolved in dry DMF (15 mL,
1H), OH signal not observed. LC-MS (method H; ESI, m/z) t =
1.34 min −388 [(M + H) ]. HRMS (method I): found, 388.1798;
calculated for C H N O Si (M + H) , 388.1805.
8-(4-(2-((4-Fluorobenzyl) (methyl)amino)ethyl)-1H-pyrazol-
1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (49d). Methanesulfonic anhydride (94 mg,
0.542 mmol, 1.5 equiv) was added in one portion to a solution of alcohol
47 (140 mg, 0.361 mmol, 1 equiv) and triethylamine (0.1 mL,
R
+
0
.7 M). The solution was heated to 60 °C, and potassium carbonate
+
(
2.85 g, 20.6 mmol, 2 equiv) was added. After 5 min of stirring,
18
26
5
3
(
2-(chloromethoxy)ethyl)trimethylsilane (2.06 g, 12.36 mmol,
1.2 equiv) was slowly added. Stirring was continued at 60 °C for 4 h,
the reaction cooled to room temperature, and EtOAc (60 mL) was
added. The heterogeneous mixture was washed with water (2 × 60 mL),
dried (Na SO ), and concentrated in vacuo. The crude product was
2
4
purified by silica gel column chromatography eluting with 3% EtOAc in
0.717 mmol, 2 equiv) in anhydrous CH
under N . The reaction mixture was stirred for 15 min at 0 °C and
monitored by LCMS. When the reaction was complete, the reaction
mixture was quenched with saturated NaHCO (10 mL) solution and
extracted with CH Cl (3 × 15 mL). The combined organic layers were
washed with saturated brine solution (30 mL), dried over MgSO , and
2 2
Cl (4 mL, 0.09 M) at 0 °C
CH Cl to provide the title compound as a white powder (2.7 g, 84%).
2
2
2
1
H NMR (500 MHz, CDCl ) δ 0.01 (s, 9H), 0.97−1.00 (m, 2H), 3.67−
3
3.71 (m, 2H), 5.46 (s, 2H), 8.06 (d, J = 5.1 Hz, 1H), 8.34 (s, 1H), 8.49
3
(
d, J = 5.1 Hz, 1 H). LC-MS (method H; ESI, m/z) t = 1.4 min (100%
2
2
R
+
purity) −254, 256 [(M − SEM + TMS) , Cl isotopic pattern)]. This
compound was also prepared in lower yield (55 to 65%) by performing
the reaction at room temperature.
4
concentrated in vacuo to give 2-(1-(4-oxo-3-((2-(trimethylsilyl)-
ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1H-pyrazol-
4-yl)ethylmethanesulfonate as a pale-yellow oil. This was used in the
next step without further purification. H NMR (500 MHz, CDCl
4
-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1H-pyrazole (45).
1
tert-Butylchlorodimethylsilane (7.09 g, 47.0 mmol, 1.05 equiv) was
added to a solution of 1H-imidazole (3.35 g, 49.2 mmol, 1.1 equiv) and
3
) δ
0.01 (s, 9H), 0.96−1.01 (m, 2H), 2.99 (s, 3H), 3.07 (t, J = 6.7 Hz, 2H),
2-(1H-pyrazol-4-yl)ethanol (5.02 g, 44.8 mmol, 1 equiv) in DMF
3.68−3.72 (m, 2H), 4.44 (t, J = 6.7 Hz, 2H), 5.47 (s, 2H), 7.81 (s, 1H),
(50 mL, 0.9 M) at room temperature. The resultant solution was stirred
8.09 (d, J = 5.1 Hz, 1H), 8.31 (s, 1H), 8.64 (d, J = 5.1 Hz, 1H), 8.68 (s,
+
for 30 min at room temperature and monitored by LCMS. On
completion, the reaction mixture was diluted with water (100 mL) and
extracted three times with EtOAc (3 × 100 mL). The combined organic
layers were washed with saturated lithium chloride solution (150 mL)
1H). LC-MS (method H; ESI, m/z) t
R
= 1.34 min −466 [(M + H) ].
According to general procedure 1, cesium carbonate (340 mg,
1.044 mmol), 2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-
dihydropyrido[3,4-d]pyrimidin-8-yl)-1H-pyrazol-4-yl)-
ethylmethanesulfonate (162 mg, 0.348 mmol), and 1-(4-fluorophenyl)-
N-methylmethanamine (0.1 mL, 0.759 mmol) were reacted together in
anhydrous DMF (2 mL). Purification on a KP-Sil snap cartridge (2%
and saturated brine solution (150 mL) and dried over MgSO . The
4
filtrate was concentrated in vacuo to give the product as a clear, pale-
1
yellow oil (9.63 g, 95%). H NMR (500 MHz, CDCl ) δ 0.04 (s, 6H),
3
0
2
1
.91 (s, 9H), 2.73 (t, J = 6.8 Hz, 2H), 3.76 (t, J = 6.8 Hz, 2H), 7.47 (s,
EtOH in CH
29%). H NMR (500 MHz, CDCl
2
Cl
2
) gave the title product as a pale-yellow oil (27.7 mg,
) δ 0.01 (s, 9H), 0.95−1.01 (m, 2H),
2.28 (s, 3H), 2.69 (t, J = 7.4 Hz, 2H), 2.80 (t, J = 7.4 Hz, 2H), 3.54 (s,
2H), 3.68−3.72 (m, 2H), 5.47 (s, 2H), 6.96−7.01 (m, 2H), 7.28−7.31
(m, 2H), 7.78 (s, 1H), 8.06 (d, J = 5.1 Hz, 1H), 8.30 (s, 1H), 8.54 (s,
1H), 8.63 (d, J = 5.1 Hz, 1H). LC-MS (method C; ESI, m/z) t =
1.19 min −509 [(M + H) ]. HRMS (method B): found, 509.2514;
calculated for C H FN O Si (M + H) , 509.2496.
8-(4-(2-((4-Fluorobenzyl) (methyl)amino)ethyl)-1H-pyrazol-
1-yl)pyrido[3,4-d]pyrimidin-4(3H)-one (52d). According to general
procedure 4, 8-(4-(2-((4-fluorobenzyl) (methyl)amino)ethyl)-1H-
pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]-
pyrimidin-4(3H)-one (42.46 mg, 0.083 mmol) and hydrochloric acid
(6 M, 1 mL) were reacted together in THF (1 mL). Purification on a
KP-NH snap cartridge (25% EtOH in CH Cl ) gave the product as a
1
H), NH signal not observed. LC-MS (method C; ESI, m/z) t =
3
R
+
.46 min −227 [(M + H) ]. HRMS (method B): found, 227.1580;
+
calculated for C H N OSi (M + H) , 227.1574.
11
23
2
8
-(4-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1H-pyrazol-1-yl)-
3
4
-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-
R
+
(3H)-one (46). Note: when attempted at scale, these reactions were
+
carried out in batches, generally using ∼150 mg portions of 8-chloro-3-
26
34
6
2
(
(2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one
per microwave vial. Cesium carbonate (1.33 g, 4.09 mmol, 2 equiv)
and 4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1H-pyrazole (920 mg,
4.06 mmol, 2 equiv) were added to a microwave vial equipped with a
stirrer bar. This was sealed and evacuated and flushed with N2.
Anhydrous MeCN (14 mL, 0.15 M) was added to the vial and the
reaction mixture stirred for 20 min under N at room temperature. The
2
2
2
1
cap was then removed, 8-chloro-3-((2-(trimethylsilyl)ethoxy)methyl)-
white solid (23.6 mg, 75%). H NMR (500 MHz, MeOH-d ) δ 2.35 (s,
4
pyrido[3,4-d]pyrimidin-4(3H)-one (637 mg, 2.04 mmol, 1 equiv) was
3H), 2.73−2.78 (m, 2H), 2.83−2.88 (m, 2H), 3.66 (s, 2H), 7.02−7.08 (m,
added, the vial resealed, and evacuated and flushed with N . The reaction
2H), 7.34−7.39 (m, 2H), 7.75 (s, 1H), 8.04 (d, J = 5.2 Hz, 1H), 8.23 (s,
2
mixture was then stirred at reflux for 18 h. Solids were removed by
filtration and rinsed with CH Cl (3 × 15 mL). The filtrate was
1H), 8.55 (d, J = 5.2 Hz, 1H), 8.70 (s, 1H), NH signalnot observed. LC-MS
+
2
2
(method C; ESI, m/z) t = 0.68 min −379 [(M + H) ]. HRMS (method
R
+
concentrated in vacuo and purified by Biotage column chromatography
B): found, 379.1665; calculated for C H FN O (M + H) , 379.1677.
20
20
6
on a KP-Sil snap cartridge (30% EtOAc in cyclohexane) to give the title
product as a pale-yellow solid (724 mg, 71%). H NMR (500 MHz,
8-(4-(2-(4-(4-Chlorophenyl)piperidin-1-yl)ethyl)-1H-pyrazol-
1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (51a). According to general procedure 2,
2-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido-
[3,4-d]pyrimidin-8-yl)-1H-pyrazol-4-yl)ethylmethanesulfonate (96 mg,
0.206 mmol), 4-(4-chlorophenyl)piperidine (48 mg, 0.247 mmol), and
triethylamine (0.057 mL, 0.409 mmol) in DMF were reacted at 50 °C
overnight. After workup, the resulting oil was passed through a plug of
1
CDCl ) δ 0.01 (s, 9H), 0.06 (s, 6H), 0.91 (s, 9H), 0.95−1.00 (m, 2H),
3
2
.80 (t, J = 6.7 Hz, 2H), 3.67−3.71 (m, 2H), 3.84 (t, J = 6.7 Hz, 2H), 5.47
(
8
s, 2H), 7.79 (s, 1H), 8.05 (d, J = 5.1 Hz, 1H), 8.29 (s, 1H), 8.61 (s, 1H),
.63 (d, J = 5.1 Hz, 1H). LC-MS (method C; ESI, m/z) t = 1.79 min
R
+
−
502 [(M + H) ]. HRMS (method B): found, 502.2672; calculated for
+
C H N O Si (M + H) , 502.2670.
24
40
5
3
2
8
-(4-(2-Hydroxyethyl)-1H-pyrazol-1-yl)-3-((2-(trimethylsilyl)-
silica eluting with 30% MeOH/DCM to afford a yellow oil which was
1
ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one (47). Hydro-
used without further purification. H NMR (500 MHz, CDCl ) δ 0.01
3
chloric acid (1 M, 5.5 mL, 10 equiv) was added to a solution of
(s, 9H), 0.96−1.02 (m, 2H), 1.75−1.90 (m, 4H), 2.12−2.19 (m, 2H),
2.48−2.57 (m, 1H), 2.67−2.73 (m, 2H), 2.81−2.86 (m, 2H), 3.14 (br s,
1H), 3.16 (br s, 1H), 3.68−3.73 (m, 2H), 5.47 (s, 2H), 7.11 (d, J =
8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.80 (s, 1H), 8.08 (d, J = 5.1 Hz,
1H), 8.30 (s, 1H), 8.57 (s, 1H), 8.64 (d, J = 5.1 Hz, 1H). LC-HRMS
(method C) tR = 1.28 min, m/z −565.2515 Cl isotopic pattern
8
-(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1H-pyrazol-1-yl)-3-((2-
(
(
trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]pyrimidin-4(3H)-one
274 mg, 0.546 mmol, 1 equiv) in MeOH (5.5 mL, 0.1 M) at 0 °C. The
reaction was stirred at 0 °C for 5 min, then diluted with EtOAc (20 mL),
washed with saturated NaHCO solution (20 mL) and saturated brine
3
+
solution (20 mL), dried over MgSO , and concentrated in vacuo to give
[(M + H) ].
4
S
DOI: 10.1021/acs.jmedchem.5b01635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
8
-(4-(2-(4-(4-Chlorophenyl)piperidin-1-yl)ethyl)-1H-pyrazol-
1,4-dioxane (4M, 0.1 mL, 0.407 mmol) in 1,4-dioxane/water at 50 °C
1
-yl)pyrido[3,4-d]pyrimidin-4(3H)-one (54a). According to general
overnight. After workup and trituration with Et O, the title product was
2
1
procedure 5, 8-(4-(2-(4-(4-chlorophenyl)piperidin-1-yl)ethyl)-1H-pyr-
azol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]-
pyrimidin-4(3H)-one (73 mg, 0.129 mmol) was reacted with HCl in
,4-dioxane (4 M, 0.81 mL, 3.23 mmol) in 1,4-dioxane/water at 50 °C
overnight. After workup and trituration, the title product was obtained
obtained (9 mg, 51%) as a yellow solid. H NMR (500 MHz, DMSO-d )
6
δ 1.66 (dq, J = 12.3, 3.5 Hz, 2H), 1.76 (br s, 1H), 1.78 (br s, 1H), 2.09
(t, J = 10.4 Hz, 2H), 2.53−2.57 (m, 1H), 2.60 (t, J = 7.3 Hz, 2H), 2.72
(t, J = 7.3 Hz, 2H), 3.04−3.10 (m, 2H), 7.22−7.26 (m, 2H), 7.30−7.35
(m, 2H), 7.73 (s, 1H), 7.98 (d, J = 5.3 Hz, 1H), 8.29 (s, 1H), 8.42
(s, 1H), 8.56 (d, J = 5.3 Hz, 1H), 12.78 (br s, 1H). LC-HRMS (method I)
t = 2.14 min. HRMS: found, 435.1675; calculated for C H ClN O
1
1
(
16 mg, 29%) as an off-white solid. H NMR (500 MHz, DMSO- d ) δ
6
1
7
1
1
5
.65 (qd, J = 12.3, 3.5 Hz, 2H), 1.76 (d, J = 11.2 Hz, 2H), 2.11 (t, J =
.5 Hz, 2H), 2.53−2.65 (m, 3H), 2.72 (t, J = 7.5 Hz, 2H), 3.09 (d, J =
1.2 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 7.73 (s,
H), 7.98 (d, J = 5.1 Hz, 1H), 8.29 (s, 1H), 8.42 (s, 1H), 8.56 (d, J =
.1 Hz, 1H), 12.44 (br s, 1H). LC-HRMS (method I) t = 2.01 min, m/z
found, 435.1696; calculated for C H ClN O [(M + H) ], 435.1700 Cl
R
23 24
6
+
[(M + H) ], 435.1695.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b01635.
■
R
S
*
+
23
24
6
isotopic pattern.
-(4-(2-(4-(3,5-Dichlorophenyl)piperidin-1-yl)ethyl)-1H-pyra-
8
zol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (51j). According to general procedure 2,
triethylamine (0.06 mL, 0.411 mmol), 2-(1-(4-oxo-3-((2-(trimethyl-
silyl)ethoxy)methyl)-3,4-dihydropyrido[3,4-d]pyrimidin-8-yl)-1H-pyr-
azol-4-yl)ethylmethanesulfonate (95.7 mg, 0.206 mmol), and 4-(3,5-
dichlorophenyl)piperidine (95 mg, 0.411 mmol) were reacted together
in anhydrous DMF (1 mL) and purified on KP-Sil snap cartridge (4%
Experimental procedures for compounds 7a,b,d, 9a,b, 16,
2
4
5
1, 22, 24, 25, 17−19, 27, 28, 29a,b, 30a,b, 36−40, 41a,b,
2, 43, 44a−g, 48, 55−57, 49a−c, 49e−g, 52a−c, 52e−g,
9, and intermediates for the synthesis of 59, 50a−i, 53a−
i, 51b−i, 51l−n, 54b−i, and 54l−n, a summary of
crystallographic analysis of compounds 15, 16, 30a, 30b,
3
7, 40, 52d, 53a, 54a, 54j, and 58 , kinase profiling for
[
0.2 M NH in MeOH] in CH Cl ) to give the product as a pale-yellow
3 2 2
1
compound 54k, and cellular activity of 54k in HeLa cells
assessed by immunofluorescence assay (PDF)
Molecular formula strings (CSV)
oil (104 mg, 84%). H NMR (500 MHz, CDCl ) δ 0.00 (s, 9H), 0.95−
3
1
(
.00 (m, 2H), 1.78 (qd, J = 12.0, 3.1 Hz, 2H), 1.83−1.89 (m, 2H), 2.14
td, J = 11.8, 1.9 Hz, 2H), 2.45−2.54 (m, 1H), 2.66−2.71 (m, 2H),
2
5
1
5
6
5
.79−2.85 (m, 2H), 3.14 (br d, J = 11.4 Hz, 2H), 3.66−3.72 (m, 2H),
.46 (s, 2H), 7.11 (d, J = 1.9 Hz, 2H), 7.19 (t, J = 1.9 Hz, 1H), 7.80 (s,
H), 8.06 (d, J = 5.1 Hz, 1H), 8.30 (s, 1H), 8.57 (s, 1H), 8.62 (d, J =
Accession Codes
Atomic coordinates and structure factors for the crystal
structures of KDM4A with compounds 15, 16, 30b, 37, 40,
52d, 53a, 54a, 54j, and 58 can be accessed using PDB codes
5F2S, 5F2W, 5F5I, 5F39, 5F32, 5F3C, 5F3G, 5F3E, 5F3I, and
5F37, respectively. Atomic coordinates and structure factors for
the crystal structures of KDM4D with compounds 30a and 44a
can be accessed using PDB codes 5F5A and 5F5C, respectively.
Atomic coordinates and structure factors for the crystal
structures of KDM5B with compound 52d can be accessed
using PDB code 5FPI.
.1 Hz, 1H). LC-MS (method H; ESI, m/z) t = 1.32 min −599, 601,
03 [(M + H) , Cl isotopic pattern]. HRMS (method I): found,
99.2128; calculated for C H Cl N O Si (M + H) , 599.2124.
8
zol-1-yl)pyrido[3,4-d]pyrimidin-4(3H)-one (54j). According to
general procedure 4, 8-(4-(2-(4-(3,5-dichlorophenyl)piperidin-1-yl)-
ethyl)-1H-pyrazol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido-
3,4-d]pyrimidin-4(3H)-one (40.6 mg, 0.068 mmol) and hydrochloric
acid (6 M, 1 mL) were reacted together in THF (1 mL). Purification on
R
+
+
29
37
2
6
2
-(4-(2-(4-(3,5-Dichlorophenyl)piperidin-1-yl)ethyl)-1H-pyra-
[
a KP-NH snap cartridge (40% EtOH in CH Cl ) gave the product as a
white solid (16.4 mg, 52%). H NMR (500 MHz, DMSO-d ) δ 1.66 (qd,
2
2
1
6
AUTHOR INFORMATION
Corresponding Authors
*For V.B.: phone, +44 (0) 20 87224158; E-mail, vassilios.
bavetsias@icr.ac.uk.
J = 12.4, 3.5 Hz, 2H), 1.73−1.80 (m, 2H), 2.06 (t, J = 11.6, 1.6 Hz, 2H),
■
2
7
5
(
[
.54−2.61 (m, 3H), 2.71 (t, J = 7.5 Hz, 2H), 3.06 (br d, J = 11.3 Hz, 2H),
.33 (d, J = 1.9 Hz, 2H), 7.41 (t, J = 1.9 Hz, 1H), 7.73 (s, 1H), 7.98 (d, J =
.1 Hz, 1H), 8.29 (s, 1H), 8.41 (s, 1H), 8.55 (d, J = 5.1 Hz, 1H) 12.79
br s, 1H). LC-MS (method H; ESI, m/z) t = 1.02 min −469, 471, 473
(M + H) , Cl isotopic pattern]. HRMS (method I): found, 469.1316;
calculated for C H Cl N O (M + H) , 469.1310.
R
*For P.E.B.: phone, +44 (0) 1865 612932; E-mail, paul.
brennan@sgc.ox.ac.uk.
+
+
23
23
2
6
*
For J.B.: phone, +44 (0) 20 87224051; E-mail, julian.blagg@
icr.ac.uk.
8
-(4-(2-(4-(3-Chlorophenyl)piperidin-1-yl)ethyl)-1H-pyrazol-
-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-d]-
pyrimidin-4(3H)-one (51k). According to general procedure 2,
-(1-(4-oxo-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4-dihydropyrido-
3,4-d]pyrimidin-8-yl)-1H-pyrazol-4-yl)ethylmethanesulfonate (79 mg,
1
Notes
2
[
The authors declare no competing financial interest.
0
0
.170 mmol), 4-(3-chlorophenyl)piperidine hydrochloride (59 mg,
.255 mmol), and triethylamine (0.057 mL, 0.409 mmol) in DMF were
ACKNOWLEDGMENTS
■
This work was supported by Cancer Research UK [CUK] grant
no. C309/A11566. We thank the Avon Foundation for Women
for funding (Gift Agreement grant no. 02-2013-52), and we
thank Dr. Stephen H. Friend MD Ph.D. of Sage Bionetworks for
his support. The SGC is a registered charity (no. 1097737) that
receives funds from AbbVie, Bayer Pharma AG, Boehringer
Ingelheim, Canada Foundation for Innovation, Eshelman
Institute for Innovation, Genome Canada, Innovative Medicines
Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766],
Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry
of Economic Development and Innovation, Pfizer, Sao Paulo
̃
Research Foundation-FAPESP, Takeda, and Wellcome Trust
[092809/Z/10/Z]. We acknowledge NHS funding to the NIHR
Biomedical Research Centre at The Institute of Cancer Research
reacted at 50 °C for 3 days. After workup, the oil obtained was purified
by flash column chromatography to afford the product (23 mg, 24%) as a
yellow oil. H NMR (500 MHz, CDCl ) δ 0.02 (s, 9H), 0.96−1.01 (m,
2
1
3
H), 1.81−1.93 (m, 5H), 2.15−2.24 (m, 2H), 2.49−2.60 (m, 1H),
.69−2.77 (m, 2H), 2.83−2.91 (m, 2H), 3.15−3.21 (m, 2H), 3.67−3.73
2
(
7
m, 2H), 5.48 (s, 2H), 7.12−7.15 (m, 1H), 7.17−7.20 (m, 1H), 7.23−
.27 (m, 1H), 7.81 (br s, 1H), 8.08 (d, J = 5.1 Hz, 1H), 8.32 (s, 1H), 8.59
(
br s, 1H), 8.64 (br s, 1H). LC-HRMS (method I) t = 2.80 min. HRMS:
R
+
found, 565.2480; calculated for C H ClN O Si [(M + H) ], 565.2514
29
38
6
2
Cl isotopic pattern.
-(4-(2-(4-(3-Chlorophenyl)piperidin-1-yl)ethyl)-1H-pyrazol-
-yl)pyrido[3,4-d]pyrimidin-4(3H)-one (54k). According to general
8
1
procedure 5, 8-(4-(2-(4-(3-chlorophenyl)piperidin-1-yl)ethyl)-1H-pyr-
azol-1-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-pyrido[3,4-d]-
pyrimidin-4(3H)-one (23 mg, 0.041 mmol) was reacted with HCl in
T
DOI: 10.1021/acs.jmedchem.5b01635
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and the Royal Marsden NHS Foundation Trust. We thank
Dr. Amin Mirza, Meirion Richards, and Dr. Maggie Liu for their
assistance with NMR, mass spectrometry, and HPLC. We thank
Dr Nora Cronin and the staff of DIAMOND Light Source and
European Synchrotron Radiation Facility for their support
during crystallographic data collection.
J. JARID2 is a Direct Target of the PAX3-FOXO1 Fusion Protein and
Inhibits Myogenic Differentiation of Rhabdomyosarcoma Cells.
Oncogene 2014, 33, 1148−1157.
(14) Yang, J.; Al Tahan, A. M.; Hu, D.; Wang, Y.; Cheng, P.-H.;
Morton, C. L.; Qu, C.; Nathwani, A. C.; Shohet, J. M.; Fotsis, T.; Koster,
J.; Versteeg, R.; Okada, H.; Harris, A. L.; Davidoff, A. M. The Role of
Histone Demethylase KDM4B in Myc Signaling in Neuroblastoma. J.
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ABBREVIATIONS USED
■
(15) Black, J. C.; Manning, A. L.; Van Rechem, C.; Kim, J.; Ladd, B.;
Cho, J.; Pineda, C. M.; Murphy, N.; Daniels, D. L.; Montagna, C.; Lewis,
P. W.; Glass, K.; Allis, D. C.; Dyson, N. J.; Getz, G.; Whetstine, J. R.
KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and
Rereplication of Regions Amplified in Tumors. Cell 2013, 154, 541−
Boc, tert-butoxycarbonyl; DME, 1,2-dimethoxyethane; DIBAL,
diisobutylaluminum hydride; EDCI, N-(3-(dimethylamino)-
propyl)-N′-ethylcarbodiimide hydrochloride; ESI, electrospray
ionization; HPLC, high performance liquid chromatography;
HOBt, 1-hydroxybenzotriazole; HRMS, high resolution mass
spectrometry; JmjC, Jumonji C; LC, liquid chromatography; LE,
ligand efficiency; LSD, lysine specific demethylase; NOG,
N-oxalylglycine; 2OG, 2-oxoglutarate; PTSA, p-toluenesulfonic
acid; PDB, Protein Data Bank; SAR, structure−activity relation-
ship; SEM, 2-(trimethylsilyl)ethoxymethyl; TFA, trifluoroacetic
acid
5
(
55.
16) Van Rechem, C.; Black, J. C.; Greninger, P.; Zhao, Y.; Donado, C.;
Burrowes, P. D.; Ladd, B.; Christiani, D. C.; Benes, C. H.; Whetstine, J.
R. A Coding Single-Nucleotide Polymorphism in Lysine Demethylase
KDM4A Associates with Increased Sensitivity to mTOR Inhibitors.
Cancer Discovery 2015, 5, 245−254.
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Associates with Translation Machinery and Regulates Protein Synthesis.
Cancer Discovery 2015, 5, 255−263.
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