Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 1. 3-Amino-4-pyridine Carboxylate Derivatives

Article abstract of DOI:10.1021/acs.jmedchem.5b01537

Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC₅₀ ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC₅₀ = 6-8 μM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC₅₀ = 100-125 nM).

Full text of DOI:10.1021/acs.jmedchem.5b01537

Article
pubs.acs.org/jmc
Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone
Lysine Demethylases. 1. 3‑Amino-4-pyridine Carboxylate Derivatives
Susan M. Westaway,*,† Alex G. S. Preston,*,† Michael D. Barker,† Fiona Brown,‡ Jack A. Brown,†
Matthew Campbell,† Chun-wa Chung,‡ Hawa Diallo,† Clement Douault,† Gerard Drewes,§ Robert Eagle,‡
Laurie Gordon,‡ Carl Haslam,‡ Thomas G. Hayhow,† Philip G. Humphreys,† Gerard Joberty,§
Roy Katso,‡ Laurens Kruidenier,†,# Melanie Leveridge,‡ John Liddle,† Julie Mosley,‡ Marcel Muelbaier,§
Rebecca Randle,‡ Inma Rioja,† Anne Rueger,§ Gail A. Seal,† Robert J. Sheppard,†,∥ Onkar Singh,‡
Joanna Taylor,‡ Pamela Thomas,‡ Douglas Thomson,§ David M. Wilson,†,∥ Kevin Lee,†,⊥
and Rab K. Prinjha†
†Epinova Discovery Performance Unit, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage SG1 2NY, U.K.
‡Platform Technology and Science, Medicines Research Centre, GlaxoSmithKline R&D, Stevenage SG1 2NY, U.K.
§Cellzome GmbH, a GSK Company, Meyerhofstrasse 1, 69117 Heidelberg, Germany
S
* Supporting Information
ABSTRACT: Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-
4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the
KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family
biochemical (RFMS) assays with ≥50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay
(IC50 = 6−8 μM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100−125 nM).
that leads to gene repression.4 The Jumonji C (JMJ-C) family
INTRODUCTION
■
of KDMs consists of iron and 2-oxoglutarate (2-OG)
Epigenetic post-translation modifications of the N- and C-
dependent dioxygenases that demethylate Nε-methylated lysine
terminal tails of the histone proteins that form the core of
residues present in histone tails. This is thought to proceed
nucleosomes, the basic units of DNA packaging in eukaryotic
through methyl hydroxylation followed by hydrolysis of the
cell nuclei, play a vital role in the regulation of gene
intermediate hemiaminal, then loss of formaldehyde.4,6 Our
transcription in both healthy and disease states.1−4 One of
interest in understanding how inhibition of members of the
these modifications is methylation of the side chain amino
KDM4 (also known as JMJD2) family of H3K9Me3
group (Nε) of lysine residues contained within the histone tails.
demethylases affects a range of potentially disease relevant
This is a dynamic process controlled by a range of histone
phenotypes led us to undertake a research program aimed at
lysine methyl transferases (KMTs)4,5 and histone lysine
the discovery and development of inhibitors of this family.
demethylases (KDMs)4,6 that act in a site specific manner to
A selection of reported inhibitors of the KDM4 family is
add and remove up to three methyl groups to and from each
shown in Figure 1. Many of these have been shown to be
lysine. Methylation of specific histone lysine residues may be
associated with the formation of euchromatin that is transcrip-
tionally active or poised for activation, e.g., the histone-3-lysine-
Special Issue: Epigenetics
4 trimethyl (H3K4Me3) mark. Alternatively, the H3K9Me3 and
H3K27Me3 marks are strongly associated with heterochromatin
Received: October 1, 2015
© XXXX American Chemical Society
A
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Figure 1. Examples of published compounds with KDM4 (JMJD2) family inhibitory activity.
Figure 2. Structure and biochemical screening data for HTS hit 12: X-ray crystal structure of 12 (cyan, PDB code 5FP3) bound to KDM6B with the
structure of NOG, 2 overlaid (magenta, from an X-ray structure in KDM6B19).
competitive with the natural cofactor 2-OG 1, and one of the
earliest inhibitors N-oxalylglycine (NOG), 2,7 is a catalytically
inert analogue of 1. Inhibitors such as 38 and 49 are elaborated
analogues of 2, and the pyridine carboxylates 5−710−12
maintain at least one of the carboxylate groups present in 2.
Binding of the 5-carboxy-8-hydroxyquinoline 813 results in a
shift in the active site iron atom when compared with 1, while
the acylhydrazide 914 demonstrates a novel binding mode
whereby the compound interacts with the iron through both
the carbonyl and terminal dialkylamino moieties. Furthermore,
inhibitors that combine 2-OG competitive elements with motifs
that are proposed to interfere with peptide substrate binding
have also been described, e.g., 10a15 and 10c,16 as well as sulfur
and selenium based inhibitors that act through extrusion of a
structural zinc atom present in many members of the KDM4
family.17 Inhibitors that contain carboxylate moieties generally
rely on an ester prodrug approach to enable cellular
penetration, and levels of cell activity are typically modest;
10b (MethylStat), the ester prodrug of pan-KDM inhibitor 10a,
is one of the most potent of this type with reported cellular
activity at IC50 < 10 μM.14 Non-carboxylate containing 8-
hydroxyquinoline, ML324, 11, has been reported as a chemical
probe with submicromolar biochemical activity and promising
antiviral cellular activity in the micromolar range.18
At GSK we initiated a program to discover and develop
inhibitors of the KDM4 family with a combination of
biochemical activity and physicochemical properties that
would lead to cellular activity, ideally in the submicromolar
range to enable exploration of phenotypic effects resulting from
inhibition of the KDM4 family. In this report we describe the
discovery and optimization of a series of 3-aminopyridine-4-
carboxylate based inhibitors that led to the identification of cell
penetrant analogues with single digit micromolar activity in a
KDM4C cellular mechanistic assay.
RESULTS AND DISCUSSION
■
A high throughput screen of the GlaxoSmithKline screening
collection against KDM6B (JMJD3) was undertaken as part of
our efforts toward the discovery of novel inhibitors of this
demethylase, and from this we identified compound 12 as a hit
B
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(Figure 2). In our routine KDM6B SAR assay that utilized the
RapidFire mass spectrometry (RFMS) platform,19 this
compound was shown to possess micromolar activity (pIC50
= 5.9) for its inhibition of demethylation of an
H3(20−36)K27Me3 peptide analogue by a truncated construct
of KDM6B. We also found through cross-screening against
KDM4C and KDM4D in equivalent assays utilizing an
H3K9Me3 peptide20 that 12 possessed 3- to 5-fold higher
activity at these targets (Figure 2). An homogeneous time-
resolved fluorescence (HTRF) assay format was used to assess
inhibitory activity against the more distantly related 2-OG
utilizing dioxygenase enzyme, the prolyl hydroxylase EGLN3
(also known as PHD3).21 In this assay 12 was less potent than
in the KDM6B assay with pIC50 ≤ 4.5. Compound 12 contains
the pyridine-4-carboxylate core also found in previously
disclosed compounds 5 and 7 but lacks the second carboxylate
group in the 2-position, thus making it a potentially more
attractive starting point for lead optimization targeting cellular
penetrant molecules than these dicarboxylates. We therefore
initiated a program to explore the SAR of this hit for inhibition
of KDM6B and the KDM4 family of histone lysine
demethylases.
An X-ray crystallography structure of 12 bound into KDM6B
revealed that the pyridine nitrogen formed a key interaction
with the metal atom in the active site. The 4-carboxylate group
forms a network of interactions with K1381, T1387, and N1400
at the back of the cofactor binding pocket, in a manner similar
to that observed for 2 (Figure 2). These interactions of the
pyridine carboxylate core of 12 are also similar to those
observed for compounds 5 and 6 bound to KDM4A.10,11
Initially, the effects of changing the length of the linker
between the pyridine carboxylate core and the terminal phenyl
ring were investigated (Table 1). Compounds 12−15 were
Scheme 1. Synthesis of 3-Amido-4-pyridinecarboxylic Acids
12−15
a
a
i
Conditions: (a) RCOCl, Pr2NEt, pyr, DMF, rt, 3.5 d or RCOCl,
DMF, 0 °C to rt, 1 h; (b) RCOCl, K2CO3, DCM/H2O, rt, 16 h; (c)
LiOH.H2O, THF/H2O, rt, 2 h.
conformation with good complementarity to a pocket formed
by two β strands lying at the entrance to the active site. The
variation in affinity with alkyl chain length may be explained by
the position in which the terminal phenyl ring can be placed.
For odd numbers of carbons in the chain, 12 and 14, the
phenyl ring can be placed in shape-complementary parts of the
pocket. For even numbers or no carbons 13 and 15, the phenyl
would be directed toward the wall of the pocket (toward P1388
and T1330, respectively) and therefore require a strained
conformation in order to bind. In KDM4D this pocket has a
different shape and the phenyl group in 12 is not bound within
the pocket but is directed toward the surface of the enzyme
(Figure 3) and therefore the same pattern of variation for the
SAR of these compounds is not observed.
We also profiled the fragment core of this series of molecules,
3-amino-4-pyridinecarboxylic acid 16, and were very interested
to find that it represented a highly ligand efficient fragment that
was selective for the KDM4 family over KDM6B and EGLN3
(Table 1). The low level of activity of this fragment in our
KDM6B assay adds to the SAR evidence described above in
which an appropriately positioned side chain is required for
good potency at this target. Given the encouraging profile of
the phenyl analogue 15, we continued to investigate the
potential for this template to provide selective KDM4 family
inhibitors. From this point, the data from the KDM4C RFMS
assay only will be reported for most compounds, as this is
representative of the levels of inhibitory activity/SAR observed
against other KDM4 family members. However, the full profiles
against KDM4A, KDM4C, KDM4D, and KDM4E will be
detailed for key compounds.
A series of amine analogues 17−25 was also prepared from
ethyl-3-amino-4-pyridine carboxylate or 3-amino-4-pyridine-
carboxylic acid via reductive amination with the appropriate
aldehyde or via nucleophilic substitution of 3-fluoropyridine-
carboxylic acid by the appropriate primary amine as shown in
Scheme 2.
Removal of the carbonyl oxygen from 15 to afford the
benzylamine analogue 17 gave enhanced potency at KDM4C
and KDM6B but preserved the 60-fold selectivity for the
former (Table 2). The linker length as well as replacement of
the terminal aromatic group was therefore investigated in this
series. Increasing the length of the alkylene chain, 18−20
resulted in small decreases in KDM4C activity and increases in
KDM6B activity. The terminal aromatic group could be
replaced with alkyl groups 21−23 to give compounds with
Table 1. pIC50 Values for 12−16 vs Selected KDM Enzymes
and EGLN322
KDM6B
pIC50
KDM4C
pIC50
KDM4D
pIC50
EGLN3
pIC50
compd
R
12
13
14
15
16
-(CH2)3Ph
-(CH2)2Ph
-CH2Ph
-Ph
5.9
4.4
6.4
6.5
6.5
5.9
6.2
6.6
6.4
6.7
5.8
5.8
4.5, <4.3
4.5
5.4
<4.3
<4.0
<4.3
<4.3
a
4.2
a
4.2, n = 12; <4.0, n = 12.
prepared from either 3-amino-4-pyridinecarboxylic acid 16 or
the corresponding ethyl ester and the appropriate acyl chloride
using standard amide coupling conditions, followed by ester
hydrolysis as required (Scheme 1). Interestingly, reduction of
the alkylene linker length from three to two atoms, compound
13 resulted in a significant drop in potency for KDM6B but not
for KDM4C and KDM4D. The benzyl analogue 14 showed
increased activity at KDM6B when compared with 13, whereas
the phenyl analogue 15 was at least 60-fold selective for
KDM4C and KDM4D over KDM6B.
The X-ray crystal structure of 12 bound to KDM6B (Figure
3) showed the phenylpropyl moiety bound in an extended
C
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Figure 3. X-ray crystal structures of HTS hit 12 bound to KDM6B (cyan, PDB code 5FP3) and KDM4D (green, PDB code 5FP4).
possessed submicromolar KDM4C activity. However, this
potency was still lower than that of propyl analogue 22,
indicating that the amino group is not forming an energetically
productive interaction with the protein. For all of these
compounds, activity against EGLN3 (PHD3) had been reduced
to below detectable levels (pIC50 < 4.3).
Scheme 2. General Methods for Synthesis of 3-
Aminopyridinecarboxylic Acids 17−43
Given the interesting overall profile of 17, we further profiled
the compound against other members of the KDM4 family as
shown in Table 3. Compound 17 showed high levels of
biochemical activity at KDM4A, KDM4D, and KDM4E in
addition to KDM4C.
Table 3. Inhibition Profile of 17 vs KDM4 Family Members
in RFMS Assays22
compd KDM4A pIC50 KDM4C pIC50 KDM4D pIC50 KDM4E pIC50
17
6.9
7.0
7.2
7.4
Table 2. Activity of Benzylamine Derivatives 17−25 vs
A crystal structure of 17 bound to KDM4D shows the
pyridine carboxylate core of this series anchors compounds in a
conserved manner within the 2-OG site (Figure 4a). The
flexible linker of 17 allows the terminal phenyl to lie against a
pocket flanked by hydrophobic residues such as Y136, A138 on
one side, while a water mediated interaction between D139 and
K245 closes off another edge.
KDM4C and KDM6B22
compd
R
KDM4C RFMS pIC50 KDM6B RFMS pIC50
To evaluate cellular activity, we utilized a mechanistic, high
content imaging assay: U2OS cells were subjected to a
Bacmam-mediated transfection with full-length HALO-tagged
KDM4C in the presence of test compound, incubated at 37 °C
for 24 h, then fixed and stained.
Those cells that expressed the Halo-tagged KDM4C in the
nuclear region above a threshold level were analyzed to
determine the level of global H3K9Me3 demethylation. A signal
window for demethylation was established using DMSO as a
negative vehicle control and the iron chelator desferroxamine
(DFO) as a positive control. On testing in this assay,
compound 17 demonstrated a modest potency, pIC50 = 4.4
(IC50 = 40 μM), which was some 400-fold lower than its
biochemical potency and too low for use as a probe molecule
for exploration of potential phenotypic responses associated
with KDM4 family inhibition.
a
17
18
19
20
21
22
23
24
25
-Ph
7.0
6.4
6.7
6.8
6.5
6.9
6.8
4.2
6.3
5.2
5.6
-CH2Ph
-(CH2)2Ph
-(CH2)3Ph
-CH2CH3
6.1
6.2
5.4
-(CH2)2CH3
-CH(CH3)2
-CH2NH2
-(CH2)2NH2
5.6
5.7
<4.0
<4.0
a
5.2, n = 8; <4.0, n = 1.
IC50 less than1 μM at KDM4C but with only a 13- to 20-fold
selectivity window over KDM6B.
Interestingly, the introduction of a polar group in the form of
a terminal primary amine resulted in low, unmeasurable activity
at KDM6B, but the activity at KDM4C depended on the length
of the alkylene linker. The ethylene linked side chain 24 was
not well tolerated, whereas the propylene linked analogue 25
We also assessed a prodrug approach analogous to that
successfully used for our previously described pyridylpyrimidine
inhibitors of the KDM6 family (JMJD3/UTX).19 Compound
D
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Figure 4. X-ray crystal structure of 17 (magenta stick format, PDB code 5FP7) bound to KDM4D (magenta lines, white surface). (a) Overlaid with
12 (green; PDB code 5FP4). (b) Phenyl ring of 17 sits in a pocket created by a water bridged interaction between K245 and D139 lined on one side
by hydrophobic residues of A138 and Y136.
Table 4. Biochemical and Cellular Activity of Benzylamine Derivatives 17−43 vs KDM4C22
compd
X
R
KDM4C RFMS pIC50
KDM4C cell pIC50
4.4
ΔpIC50 (RFMS-cell)
cLogP
ChromLogDpH7.4
17
26
27
28
18
20
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CHMe
CMe2
CHPh
Ph
7.0
6.2
2.6
1.9
1.7
>1.7
1.9
1.6
1.6
1.5
2.4
2.0
2.0
2.0
2.5
>2.9
2.0
1.9
1.9
1.5
2.0
3.4
4.4
4.3
5.3
4.1
5.0
4.6
4.1
2.9
3.1
3.1
2.6
1.8
1.5
3.6
3.6
3.5
4.4
3.7
4.1
4.8
0.7
1.7
1.7
2.2
1.3
2.0
1.7
1.3
0.1
0.5
0.6
0.1
−0.5
−0.7
1.1
1.2
1.1
1.8
1.2
1.4
2.3
a
2,5-Me2Ph
2,3-Me2Ph
3-biphenyl
CH2Ph
4.3
b
6.5
4.8
5.7
6.4
6.8
6.9
6.7
6.9
7.2
7.2
7.1
7.0
6.9
6.7
7.0
7.1
5.9
6.2
5.8
<4.0
c
4.5
(CH2)3Ph
c-hexyl
5.2
5.3
5.2
4.5
5.2
5.2
5.1
4.5
<4.0
4.7
5.1
5.2
4.4
4.2
nd
c-pentyl
c-propyl
2-thienyl
3-thienyl
2-furyl
4-thiazolyl
5-pyrazolyl
5-Me-2-thienyl
4-Me-2-thienyl
3-Me-2-thienyl
2-benzothienyl
Ph
Ph
Ph
5.2
nd
a
b
c
4.3, n = 1; <4.0, n = 1. 6.5, n = 3; <4.0, n = 1. 4.5, n = 6; <4.0, n = 2.
20 possessed submicromolar biochemical potency at KDM6B,
but when its ethyl ester (20E) (Supporting Information Figure
1) was tested for its ability to inhibit TNF production by LPS-
stimulated human primary macrophages, no inhibition was
observed (data not shown). It is possible that the lack of
cellular activity of putative prodrug 20E is a result of less
efficient hydrolysis of this more hindered ester in the cell, or
potentially reduced cellular penetration, although 20E is more
lipophilic (cLogP = 5.4) than the previously described KDM6
family prodrug ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azepin-
3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoate
(GSK-J4)19 (cLogP = 4.8). This result, combined with a desire
not to restrict the potential utility of these inhibitors to
phenotypic investigations in esterase-containing cell types, led
us to prepare a range of analogues (26−43, Scheme 2) of lead
benzylamine compound 17 with the aim of determining if
improved cellular potency could be achieved through further
modulation of biochemical activity and physicochemical
properties.
From the data presented in Table 4 it can be seen that
KDM4C RFMS potency of less than 100 nM (pIC50 ≥ 7.0)
could be achieved with a number of five-membered heteroaryl
replacements of the terminal phenyl group (32−35, 38, 39).
From the examples explored, replacement of the phenyl group
with cycloalkyls 29−31 was not detrimental to potency at
KDM4C. When steric constraints were pushed further, a more
E
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Table 5. Further Profiling of Cell Active Monocarboxylic Acid Inhibitors 34 and 39 with Inactive Control Analogues 44 and
4522
KDM4A RFMS
pIC50
KDM4C RFMS
pIC50
KDM4D RFMS
pIC50
KDM4E RFMS
pIC50
KDM6B RFMS
pIC50
KDM5C RFMS
pIC50
KDM4C cell
pIC50
KDM5C cell
pIC50
compd
a
34
39
44
45
7.0
7.0
7.1
7.1
7.0
7.0
7.4
7.3
4.9
5.3
7.0
6.9
5.1
5.2
4.2
4.6
<4.0
<4.0
b
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
nt
<4.0
<4.0
<4.0
<4.0
<4.0
a
b
4.2, n = 3; <4.0, n = 4. nt = not tested.
significant drop in potency occurred; for example, the biphenyl
28 and benzothienyl 40 analogues showed decreases of 12- to
20-fold. Substitution at the benzylic carbon atom also resulted
in reduced potency (41−43) with the hindered dibenzylidine
analogue 43 showing a 60-fold decrease compared to 17. Taken
together these data indicate that the region of the active site
that is occupied by this substituent is relatively open to a range
of substitution patterns, although bulky substituents in the
vicinity of the core appear to be less favored.
Examining the effects of these substituents on cellular activity
revealed that there is a significant downward potency shift
when moving into a whole cell assay as may be expected for
compounds containing a carboxylate moiety. The lipophilicity
data for compounds 17−40 are presented in terms of both
calculated log P (cLogP) and measured chromatographic log D
at pH 7.4 (ChromLogDpH7.4).23 There is a reasonable
correlation between the drop in potency from biochemical to
cell assay [expressed as ΔpIC50 (RFMS-cell)] and cLogP or
ChromLogD with the more lipophilic compounds tending to
show a smaller decrease, thus indicating higher cell penetration
(Table 4 and Supporting Information Figure 2). Compounds
20, 27, 29, 30, and 40 showed a 50-fold or lower decrease
(ΔpIC50 ≤ 1.7) and possessed cLogP values higher than 4. A
number of these analogues also possessed single digit
micromolar potency (pIC50 > 5) in the cell assay (20, 29,
30). This level of cell activity could also be achieved with
compounds that are less lipophilic (cLogP = 2.9−3.6) but
possess increased biochemical potency, typically pIC50 ≥ 7
(IC50 ≤ 100 nM) (32−34, 38, 39). The interesting level of cell
activity demonstrated by these carboxylic acids led us to further
profile exemplars 34 and 39 across the KDM4 family and at
KDM6B and KDM5C as shown in Table 5. Compounds 34
and 39 were also tested for their activity against prolyl
hydroxylase EGLN3 and found to possess negligible activity
(pIC50 ≤ 4.1).
Also included for comparison are compounds 44 and 45 that
are the KDM4 family inactive isomers of 34 and 39, wherein
the pyridine nitrogen atom has been translocated relative to the
carboxylate. This renders the nitrogen no longer able to interact
with the active site iron given the expected binding mode of
these molecules within KDM4 enzymes, as was confirmed by
crystallography of the 4-methylthienyl analogue 38 within
KDM4D (Figure 5).
Interestingly, while compounds 34 and 39 are equipotent
across the KDM4 family and selective vs KDM6B and EGLN3,
we found that they are potent inhibitors of the H3K4Me3/2
demethylase KDM5C (JARID1C). This activity translated into
Figure 5. X-ray crystal structure of 38 (blue stick format, PDB code
5FP8) bound to KDM4D (blue lines, white surface) overlaid with 17
(magenta stick, PDB code 5FP7). The conserved binding of the two
molecules is apparent, with the 4-methylthiophene of 38 occupying
the same space as the phenyl ring of 17.
the cellular context utilizing a similar mechanistic, high content
imaging assay to that described earlier for KDM4C although
the levels of activity in the KDM5C cell assay were lower than
for KDM4C. The reasons for this reduced cellular activity at
KDM5C when compared to KDM4C are not clear. The assay
uses the same U20S cells and protocol, but it is plausible that
differences in the efficiency of expression of the two targets in
the cells and/or differences in the affinity of the two enzymes
for the natural cofactor 2-OG, with which these inhibitors
compete, may play a part. Compounds 34 and 39 were also
profiled in our in-house selectivity panel of nonrelated drug and
liability targets. Compound 34 showed measurable activity in
3/48 assays and compound 39 in 5/49 assays (Supporting
Information Tables 1 and 2). All potencies were ≥25 μM
except for that of compound 39 in the OATP1B1 transporter
inhibition assay where it possessed an IC50 value of 8 μM.
Compound 34 and KDM4C “inactive” analogues 44 and 45
had no measurable activity in this assay (IC50 > 50 μM).
CONCLUSION
■
In summary, we have optimized a pyridine monocarboxylate
KDM6B HTS hit to a series of potent inhibitors of the
KDM4A, KDM4C, KDM4D, KDM4E, and KDM5C histone
lysine demethylases.24 Furthermore, exemplars such as 34 and
39 possessed interesting activity in a target specific, cellular
F
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min 100% B at a flow rate of 1 mL/min. For the TFA method, elution
was with 0.1% v/v solution of trifluoroacetic acid in water (solvent A)
and 0.1% v/v solution of trifluoroacetic acid in acetonitrile (solvent B)
using the following elution gradient: 0−1.5 min 3−100% B, 1.5−1.9
min 100% B, 1.9−2.0 min 100−3% B at a flow rate of 1 mL/min. High
resolution mass spectra (HRMS) were acquired as profile data using a
Thermo Scientific LTQ Orbitrap mass spectrometer, equipped with an
ESI interface, over a mass range of 120−1000 Da at a mass resolution
of 100 000. A commercial calibration solution (Pierce LTQ ESI
positive ion calibration solution) was used to externally calibrate the
instrument prior to analysis. Ionization was achieved with a spray
voltage of 4 kV, a capillary voltage of 45 V, tube lens voltage of 110 V,
with sheath and auxiliary gas flows of 40 and 20 (arbitrary units),
respectively. The capillary temperature was maintained at 300 °C. The
elemental composition was calculated using Xcalibur (version 2.0.7)
for the [M + H]+ and the mass error quoted as ppm; an error of <5
ppm indicates that the measured mass is consistent with the proposed
formula. Melting point analysis was carried out using a Stuart SMP40
melting point apparatus, and melting points are uncorrected. The
purity of all compounds screened in the biological assays was found to
be ≥95% by LCMS analysis unless otherwise specified.
mechanistic assay against overexpressed KDM4C with IC50
values of <10 μM. Activity was also observed against
overexpressed KDM5C albeit with lower potency. In the
limited set of biochemical assays utilized to assess selectivity
during this program of work, compounds 34 and 39 showed
low levels of activity vs KDM6B and EGLN3. Further profiling
to assess the selectivity of compound 34 against a broader
subset of 2-OG dependent dioxygenases, including other KDM
enzymes, was undertaken using a chemical proteomics based
approached developed in our laboratories, and this will be
published in due course.25
These compounds may have utility in exploring the
phenotypic effects of KDM4 and KDM5 family inhibition
when used in combination with negative control molecules
such as 44 and 45. However, we were keen to explore whether
we could maintain or improve on the levels of activity observed
in this series with less acidic and thus potentially more cell
penetrant compounds, targeting cellular activity with pIC50 ≥ 6
(IC50 ≤ 1 μM), and our findings are reported in the
accompanying article.26
3-(4-Phenylbutanamido)isonicotinic Acid, 12. To 3-amino-4-
pyridinecarboxylic acid (500 mg, 3.62 mmol) in DMF (10 mL) were
added N-ethyl-N-isopropylpropan-2-amine (1264 μL, 7.24 mmol) and
pyridine (293 μL, 3.62 mmol). The reaction was stirred, and 4-
phenylbutanoyl chloride (596 μL, 3.62 mmol) was added. The
reaction mixture was stirred at room temp for 3.5 days. The solid was
collected by filtration and washed with EtOAc and MeOH, then dried
in vacuo. MeOH (15 mL) was added to the crude, and the resulting
solution was filtered and loaded onto an SCX column. The column
was washed with MeOH, and the product was eluted using 2 M NH3
in MeOH. The fractions were concentrated to afford 12 as a white
solid (555 mg, 54%). LCMS (TFA method) retention time 0.67 min,
[M + H]+ = 285. 1H NMR (600 MHz, DMSO-d6) δ ppm 1.93 (quin, J
= 7.3 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H), 7.18
(t, J = 7.3 Hz, 1H), 7.23 (d, J = 7.0 Hz, 2H), 7.29 (t, J = 7.8 Hz, 2H),
7.76 (d, J = 4.8 Hz, 1H), 8.30 (d, J = 5.1 Hz, 1H), 9.55 (s, 1H).
3-(2-Phenylacetamido)isonicotinic Acid, 14. Phenylacetyl
chloride (0.240 mL, 1.810 mmol) was added dropwise to a stirred
suspension of 3-aminonicotinic acid (250 mg, 1.810 mmol) in DMF (5
mL) at 0 °C under N2. After stirring at 0 °C for 5 min, the flask was
removed from the cooling bath and allowed to stir at rt for 1 h by
which time a solution had formed. H2O (20 mL) was added, and the
resultant suspension was stirred for 15 min. The suspension was
filtered, then washed with H2O (100 mL), followed by MeOH (100
mL) and finally Et2O (100 mL). The solid was then dried in a vacuum
oven at 40 °C for 12 h to give 14 as a white solid (323 mg, 70%).
LCMS (formic method) retention time 0.51 min, [M + H]+ = 257. 1H
NMR (400 MHz, DMSO-d6) δ ppm 3.79 (s, 2H), 7.25−7.31 (m, 1H),
7.36 (d, J = 4.5 Hz, 4H), 7.74 (d, J = 5.1 Hz, 1H), 8.41 (d, J = 5.1 Hz,
1H), 9.48 (s, 1H), 10.70 (br s, 1H).
EXPERIMENTAL SECTION
■
Chemistry. All commercial chemicals and solvents are reagent
grade and were used without further purification unless otherwise
specified. Reactions were monitored by thin-layer chromatography on
0.2 mm silica gel plates (POLYGRAM SIL G/UV254, Macherey-
Nagel) and were visualized with UV light. Compounds were typically
purified by automated flash silica chromatography (Biotage SP4),
manual chromatography on prepacked cartridges (SPE), or mass
directed autopreparative chromatography (MDAP). Where specifically
indicated, the following MDAP methods were used. For the formic
method, the HPLC analysis was conducted on a Sunfire C18 column
(150 mm × 30 mm i.d., 5 μm packing diameter) at ambient
temperature, eluting with 0.1% formic acid in water and 0.1% formic
acid in acetonitrile using an elution gradient. The UV detection was an
averaged signal from wavelength of 210 to 350 nm. The mass spectra
were recorded on a Waters ZQ mass spectrometer using alternate-scan
positive and negative electrospray. Ionization data were rounded to the
nearest integer. For the high pH method, the HPLC analysis was
conducted on an XBridge C18 column (100 mm × 30 mm, i.d. 5 μm
packing diameter) at ambient temperature, eluting with 10 mM
ammonium bicarbonate in water adjusted to pH 10 with ammonia
solution, and acetonitrile using an elution gradient. The UV detection
was an averaged signal from wavelength of 210 to 350 nm. The mass
spectra were recorded on a Waters ZQ mass spectrometer using
alternate-scan positive and negative electrospray. Ionization data were
rounded to the nearest integer. 1H and 13C NMR spectra were
recorded on either a Bruker DPX-400 spectrometer at 400 and 126
MHz, respectively, or a Bruker AV-600 spectrometer at 600 and 150
MHz, respectively. Chemical shifts are reported in parts per million
(ppm, δ units). Splitting patterns are designated as s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad; etc. LCMS
spectra were recorded on an Acquity UPLC BEH C18 column (50
mm × 2.1 mm i.d., 1.7 μm packing diameter) at 40 °C. The UV
detection was a summed signal from wavelength of 210 to 350 nm.
The mass spectra were recorded on a Waters ZQ mass spectrometer
using alternate-scan positive and negative electrospray. Ionization data
were rounded to the nearest integer. As specifically indicated, the
compounds were eluted by one of the following LCMS methods. For
the formic method, elution was with 0.1% v/v solution of formic acid
in water (solvent A) and 0.1% v/v solution of formic acid in
acetonitrile (solvent B) using the following elution gradient: 0−1.5
min 3−100% B, 1.5−1.9 min 100% B, 1.9−2.1 min 3% B at a flow rate
of 1 mL/min. For the high pH method, elution was with 10 mM
ammonium bicarbonate in water adjusted to pH 10 with ammonia
solution (solvent A) and acetonitrile (solvent B) using the following
elution gradient: 0−1.5 min 1−97% B, 1.5−1.9 min 97% B, 1.9−2.1
3-Benzamidoisonicotinic Acid, 15. Step 1. Benzoyl chloride (91
μL, 0.78 mmol) was added to a stirred solution of ethyl 3-amino-4-
pyridinecarboxylate (100 mg, 0.602 mmol) in DCM (3 mL) and sat.
aq K2CO3 (3 mL) at rt. The resultant biphasic solution was stirred
rapidly for 16 h, and then DCM (10 mL) and H2O (20 mL) were
added. The separated aqueous phase was extracted with DCM (2 × 20
mL); the combined organic phases were passed through a hydro-
phobic frit and evaporated under reduced pressure to give a yellow oil.
This oil was purified by MDAP (high pH method) to give a yellow
solid (114 mg). The solid was further purified by MDAP (formic
method) to give a pale yellow solid. The solid was dissolved in EtOH
(10 mL) and loaded onto an amino propyl column (2 g) that had been
prewashed with EtOH. The column was washed with EtOH, the
fractions were combined and evaporated under reduced pressure to
give ethyl 3-benzamidoisonicotinate as a pale yellow solid (72 mg,
44%). LCMS (formic method) retention time 1.01 min, [M + H]+ =
1
271. H NMR (400 MHz, CDCl3) δ ppm 1.50 (t, J = 7.2 Hz, 3H),
4.52 (q, J = 7.0 Hz, 2H), 7.53−7.67 (m, 3H), 7.90 (d, J = 5.0 Hz, 1H),
G
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Article
8.07−8.09 (m, 1H), 8.10 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 10.29 (s,
1H), 11.71 (br s, 1H)
amino]-4-pyridinecarboxylate as a colorless oil (95 mg, 58%). LCMS
(formic method) retention time 0.94 min, [M + H]+ = 271. 1H NMR
(400 MHz, CDCl3) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 3.02 (t, J = 7.3 Hz,
2H), 3.57 (td, J = 7.2, 5.6 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 7.25−7.31
(m, 3H), 7.35 (t, J = 8.0 Hz, 2H), 7.46 (m, 1H), 7.64 (d, J = 5.1 Hz,
1H), 7.92 (d, J = 5.1 Hz, 1H), 8.27 (s, 1H).
Step 2. Lithium hydroxide monohydrate (19 mg, 0.44 mmol) was
added in a single portion to a stirred solution of ethyl 3-
benzamidoisonicotinate (60 mg, 0.222 mmol) in THF (3 mL) and
H2O (1 mL) at rt. After stirring at rt for 2 h, 2 M HCl (aq) (2 mL)
was added dropwise and the resultant suspension filtered. The filtered
solid was washed with Et2O (20 mL) and then dried under vacuum to
give 15 as a white solid (31 mg, 58%). LCMS (formic method)
Step 2. Lithium hydroxide monohydrate (30 mg, 0.715 mmol) was
added in a single portion to a stirred solution of ethyl 3-[(2-
phenylethyl)amino]-4-pyridinecarboxylate (95 mg, 0.351 mmol) in
THF (5 mL) and water (1.667 mL) at rt. The resultant suspension
was stirred for 16 h. The reaction was then warmed to 50 °C for 30
min. Upon cooling, 2 M HCl (aq) (5 mL) was added and the solvent
was evaporated under reduced pressure to give a yellow oil. The oil
was suspended in MeOH (10 mL) and loaded onto an SCX column (5
g) that had been prewashed with MeOH. The column was washed
with MeOH, then 2 M NH3 in MeOH. The methanolic ammonia
fractions were combined and evaporated under reduced pressure to
give 18 as a yellow solid (74 mg, 87%). LCMS (formic method)
retention time 0.56 min, [M + H]+ = 243. 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.88 (t, J = 7.3 Hz, 2H), 3.39 (t, J = 7.3 Hz, 2H),
7.21 (td, J = 5.8, 2.5 Hz, 1H), 7.29−7.33 (m, 4H), 7.54 (d, J = 4.8 Hz,
1H), 7.70 (d, J = 4.8 Hz, 1H), 7.99 (s, 1H).
3-((3-Phenylpropyl)amino)isonicotinic Acid, 19. Step 1.
Sodium triacetoxyborohydride (383 mg, 1.805 mmol) was added
portionwise over 0.5 min to a stirred solution of ethyl 3-amino-4-
pyridinecarboxylate (250 mg, 1.504 mmol), 3-phenylpropanal (0.218
mL, 1.655 mmol), and trifluoroacetic acid (0.695 mL, 9.03 mmol) in
isopropyl acetate (3 mL) at rt under N2. After stirring at rt for 30 min,
sat. NaHCO3 (aq) was added slowly over 5 min. The biphasic solution
was then diluted with sat. NaHCO3 (aq) and extracted with EtOAc.
The organic phase was passed through a hydrophobic frit and
evaporated to give an orange oil. This oil was purified using silica gel
column chromatography eluting with a gradient of 0−20% EtOAc/
cyclohexane to give ethyl 3-[(3-phenylpropyl)amino]-4-pyridinecar-
boxylate as a yellow oil (278 mg, 65%). LCMS (formic method)
retention time 1.03 min, [M + H]+ = 285.
Step 2. Lithium hydroxide monohydrate (74 mg, 1.764 mmol) was
added in a single portion to a stirred solution of ethyl 3-[(3-
phenylpropyl)amino]-4-pyridinecarboxylate (250 mg, 0.879 mmol) in
THF (5 mL) and water (1.7 mL) at rt. The resultant suspension was
stirred at rt for 16 h, and then 2 M HCl(aq) (5 mL) was added. The
solvent was evaporated under reduced pressure to give a yellow solid.
The solid was dissolved in MeOH (10 mL) and loaded onto an SCX
column (5 g) that had been prewashed with MeOH. The column was
then washed with MeOH, followed by 2 M NH3 in MeOH. The
methanolic ammonia fractions were combined and evaporated under
reduced pressure to give 19 as a yellow solid (112 mg, 50%). LCMS
(formic method) retention time 0.66 min, [M + H]+ = 257. 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.88 (m, 2H), 2.70 (t, J = 7.6 Hz, 2H),
3.15 (t, J = 7.0 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 7.23 (d, J = 7.2 Hz,
2H), 7.29 (t, J = 7.2 Hz, 2H), 7.54 (d, J = 4.8 Hz, 1H), 7.69 (d, J = 4.8
Hz, 1H), 7.90 (s, 1H).
3-((4-Phenylbutyl)amino)isonicotinic Acid, 20. 4-Phenylbutan-
1-amine (1.972 mL, 12.47 mmol) was added to a microwave vial
containing 3-fluoroisonicotinic acid (800 mg, 5.67 mmol). The vial
was sealed and then heated in a microwave at 150 °C for 2 h. The vial
was allowed to stand at rt for 12 h. The viscous solution was dissolved
in IPA (50 mL) and loaded onto an aminopropyl column (70 g) that
had been prewashed with IPA. The column was washed with IPA and
then 10% 2 M aq. HCl in IPA. The appropriate fractions were
combined and evaporated under reduced pressure to give 20 as a
yellow solid (1.23g, 70%). LCMS (formic method) retention time 0.72
min, [M + H]+ = 271. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.59−
1.73 (m, 4H), 2.64 (t, J = 7.2 Hz, 2H), 3.37 (t, J = 6.7 Hz, 2H), 7.16
(d, J = 7.2 Hz, 1H), 7.21 (d, J = 7.2 Hz, 2H), 7.28 (t, J = 7.4 Hz, 2H),
7.96 (d, J = 5.6 Hz, 1H), 8.04 (d, J = 5.8 Hz, 1H), 8.39 (s, 1H).
3-(Propylamino)isonicotinic Acid, 21. 3-Fluoroisonicotinic acid
(240 mg, 1.701 mmol) and propan-1-amine (0.156 mL, 1.871 mmol)
were dissolved in NMP (1 mL) in a microwave vial. The mixture was
then irradiated at 150 °C in a microwave for 4 h and then purified
1
retention time 0.51 min, [M + H]+ = 243. H NMR (400 MHz,
DMSO-d6) δ ppm 7.62 (t, J = 7.8 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H),
7.87 (d, J = 5.0 Hz, 1H), 7.99 (d, J = 7.8 Hz, 2H), 8.51 (d, J = 5.0 Hz,
1H), 9.69 (s, 1H), 11.66 (br s, 1H).
3-(3-Phenylpropanamido)isonicotinic Acid, 13. Compound
13 was prepared in a similar manner to compound 15. LCMS (formic
1
method) retention time 0.60 min, [M + H]+ = 271. H NMR (400
MHz, DMSO-d6) δ ppm 2.73 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 7.5 Hz,
2H), 7.16−7.22 (m, 1H), 7.24−7.32 (m, 4H), 7.75 (d, J = 5.1 Hz,
1H), 8.42 (d, J = 5.1 Hz, 1H), 9.38 (s, 1H), 10.61 (br s, 1H).
3-(Benzylamino)isonicotinic Acid, 17. Step 1. Sodium triacetox-
yborohydride (383 mg, 1.805 mmol) was added portionwise over 0.5
min to a stirred suspension of ethyl 3-amino-4-pyridinecarboxylate
(250 mg, 1.504 mmol), benzaldehyde (0.167 mL, 1.655 mmol), and
trifluoroacetic acid (0.232 mL, 3.01 mmol) in isopropyl acetate (3 mL)
at rt under N2. After stirring at rt for 30 min, sat. NaHCO3 (aq) (10
mL) was added slowly over 5 min. The biphasic solution was then
diluted with sat. NaHCO3 (aq) (20 mL) and EtOAc (20 mL). The
separated aqueous phase was extracted with EtOAc (2 × 20 mL); the
combined organics were passed through a hydrophobic frit and
evaporated under reduced pressure to give an orange oil. This oil was
purified by silica gel column chromatography using a gradient of 0−
20% EtOAc/cyclohexane to give ethyl 3-(benzylamino)isonicotinate as
a pale yellow oil (300 mg, 78%). LCMS (formic method) retention
time 0.92 min, [M + H]+ = 257. 1H NMR (400 MHz, CDCl3) δ ppm
1.42 (t, J = 7.1 Hz, 3H), 4.38 (q, J = 7.3 Hz, 2H), 4.54 (d, J = 5.6 Hz,
2H), 4.73 (d, J = 5.8 Hz, 1H), 7.29−7.35 (m, 1H), 7.36−7.42 (m,
4H), 7.68 (d, J = 5.1 Hz, 1H), 7.86 (br s, 1H), 7.94 (d, J = 5.1 Hz,
1H), 8.22 (s, 1H).
Step 2. Lithium hydroxide monohydrate (85 mg, 2.029 mmol) was
added in a single portion to a stirred solution of ethyl 3-
(benzylamino)isonicotinate (260 mg, 1.014 mmol) in THF (5 mL)
and water (1.7 mL) at rt. The resultant suspension was stirred at rt for
16 h by which time a solution had formed. 2 M HCl (aq) (5 mL) was
added, and the resultant suspension was stirred for 5 min. The
suspension was filtered; the collected solid was washed with H2O (50
mL) and Et2O (50 mL) and dried under vacuum to give 17 as a pale
yellow solid (44 mg, 19%). LCMS (formic method) retention time
1
0.51 min, [M + H]+ = 229. H NMR (400 MHz, DMSO-d6) δ ppm
4.58 (s, 2H), 7.24−7.30 (m, 1H), 7.35−7.39 (m, 4H), 7.58 (d, J = 5.1
Hz, 1H), 7.83 (d, J = 5.1 Hz, 1H), 8.15 (s, 1H).
3-(Phenethylamino)isonicotinic Acid, 18. Step 1. Sodium
triacetoxyborohydride (191 mg, 0.903 mmol) was added in a single
portion to a stirred solution of ethyl 3-amino-4-pyridinecarboxylate
(100 mg, 0.602 mmol) in trifluoroacetic acid (0.6 mL, 7.79 mmol) at
rt. After stirring at rt for 10 min, a solution of phenylacetaldehyde
(0.074 mL, 0.662 mmol) in DCM (1.2 mL) was added dropwise over
1 min. The resultant solution was stirred at rt for 30 min, and sodium
triacetoxyborohydride (191 mg, 0.903 mmol) was added. The
resultant solution was stirred at rt for 10 min, and then a solution
of phenylacetaldehyde (0.074 mL, 0.662 mmol) in DCM (1.2 mL)
was added dropwise over 1 min. The resultant solution was stirred at rt
for 1 h. The solution was diluted with DCM and poured into sat.
NaHCO3 (aq) (50 mL). The separated aqueous phase was extracted
with DCM; the combined organics were passed through a hydro-
phobic frit and evaporated under reduced pressure to give a yellow oil.
The oil was purified by MDAP (formic method) to give a yellow oil.
The oil was dissolved in EtOH (10 mL) and loaded onto an
aminopropyl column (5 g) that had been prewashed with EtOH. The
product was eluted with EtOH and the combined fractions were
evaporated under reduced pressure to give ethyl 3-[(2-phenylethyl)-
H
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Article
using MDAP (formic method) to yield the 21 as a pale yellow powder
(109 mg, 36%). LCMS (formic method) retention time 0.38 min, [M
+ H]+ = 181. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.96 (t, J = 7.3
Hz, 3H), 1.62 (m, 2H), 3.25 (t, J = 7.0 Hz, 2H), 7.55 (d, J = 5.1 Hz,
1H), 7.82 (d, J = 5.1 Hz, 1H), 8.24 (s, 1H).
propyl)amino)isonicotinic acid (227 mg, 0.769 mmol, 27.1% yield) as
a yellow oily solid. LCMS (TFA method) retention time 0.54 min, [M
1
+ H]+ = 296. H NMR (400 MHz, DMSO-d6) δ ppm 1.37−1.40 (m,
9H), 1.69 (t, J = 7.1 Hz, 2H), 2.70 (s, 1H), 2.77 (m, 1H), 3.03 (q, J =
6.7 Hz, 2H), 7.54 (d, J = 4.8 Hz, 1H), 7.76 (d, J = 5.1 Hz, 1H), 8.10 (s,
1H).
3-(Butylamino)isonicotinic Acid, 22. To 3-fluoroisonicotinic
acid (223 mg, 1.580 mmol) under nitrogen was added butan-1-amine
(470 μL, 4.74 mmol), and the mixture was heated to 180 °C in a
microwave for 4 h. The reaction mixture was diluted with DMSO and
purified by MDAP (high pH method) to give 22 as a cream solid (10.4
mg, 3%). LCMS (formic method) retention time 0.47 min, [M + H]+
Step 2. To a solution of 3-((3-((tert-butoxycarbonyl)amino)-
propyl)amino)isonicotinic acid (45 mg, 0.152 mmol) in DCM (3
mL) was added trifluoroacetic acid (0.1 mL, 1.298 mmol), and the
mixture was stirred at room temperature for 16 h. The solvent was
removed under reduced pressure and the residue dissolved in MeOH.
The solution was applied to a 5 g Isolute SCX cartridge, washing with
MeOH (25 mL) and eluting with 2 M NH3 in MeOH (25 mL). The
solvent was removed under reduced pressure to give 25 (21 mg, 70.6%
yield) as a pale yellow solid. LCMS (TFA method) retention time 0.21
min, [M + H]+ = 196. 1H NMR (400 MHz, D2O) δ = 2.06−1.93 (m,
2H), 3.09 (t, J = 7.6 Hz, 2H), 3.39−3.30 (m, 2H), 7.54 (d, J = 4.8 Hz,
1H), 7.86 (d, J = 4.8 Hz, 1H), 8.07 (s, 1H).
1
= 195. H NMR (400 MHz, DMSO-d6) δ ppm 0.93 (t, J = 7.3 Hz,
3H), 1.40 (m, 2H), 1.60 (quin, J = 7.2 Hz, 2H), 3.25−3.28 (m, 2H),
7.54 (d, J = 5.1 Hz, 1H), 7.81 (d, J = 5.1 Hz, 1H), 8.22 (s, 1H).
3-(Isobutylamino)isonicotinic Acid, 23. Step 1. Sodium
triacetoxyborohydride (478 mg, 2.257 mmol) was added portionwise
over 0.5 min to a stirred solution of ethyl 3-amino-4-pyridinecarbox-
ylate (250 mg, 1.504 mmol), 2-methylpropanal (0.151 mL, 1.655
mmol), and trifluoroacetic acid (1.500 mL, 19.47 mmol) in isopropyl
acetate (3 mL) at rt under N2. After stirring at rt for 3 h, the reaction
mixture was left to stand for 65 h. Saturated NaHCO3 (aq) (10 mL)
was added slowly over 5 min. The biphasic solution was then diluted
with sat. NaHCO3(aq) (20 mL) and EtOAc (20 mL). The separated
aqueous phase was extracted with EtOAc (2 × 20 mL); the combined
organic phases were passed through a hydrophobic frit and evaporated
under reduced pressure to give a dark yellow oil. The oil was purified
by silica gel column chromatography eluting with a gradient of 0−20%
EtOAc/cyclohexane to give a yellow oil. The oil was further purified by
MDAP (formic method) to give ethyl 3-[(2-methylpropyl)amino]-4-
pyridinecarboxylate as a yellow oil (105 mg, 31%). LCMS (formic
3-((2,5-Dimethylbenzyl)amino)isonicotinic Acid Hydrochlor-
ide, 26. Step 1. To 3-fluoroisonicotinic acid (200 mg, 1.417 mmol)
and N-ethyl-N-isopropylpropan-2-amine (619 μL, 3.54 mmol) was
added (2,5-dimethylphenyl)methanamine (230 mg, 1.701 mmol) in a
microwave vial. The reaction mixture was irradiated in a microwave for
4 h at 160 °C. The reaction mixture was diluted with 4 mL of DMSO
and purified by MDAP (high pH method) to give 3-((2,5-
dimethylbenzyl)amino)isonicotinic acid (178 mg, 49%) as a white
solid. LCMS (high pH method) retention time 0.72 min, [M + H]+ =
1
257. H NMR (400 MHz, DMSO-d6) δ = 2.23 (s, 3H), 2.28 (s, 3H),
4.47 (s, 2H), 7.01 (d, J = 7.6 Hz, 1H), 7.10 (s, 1H), 7.10 (d, J = 7.6
Hz, 2H), 7.58 (d, J = 5.1 Hz, 1H), 7.85 (d, J = 5.1 Hz, 1H), 8.19 (s,
1H).
1
method) retention time 0.87 min, [M + H]+ = 223. H NMR (400
MHz, CDCl3) δ ppm 1.05 (d, J = 6.8 Hz, 6H), 1.42 (t, J = 7.1 Hz,
3H), 2.00 (m, 1H), 3.12 (dd, J = 6.6, 5.8 Hz, 2H), 4.37 (q, J = 7.2 Hz,
2H), 7.50 (br s, 1H), 7.64 (d, J = 5.3 Hz, 1H), 7.90 (d, J = 5.1 Hz,
1H), 8.24 (s, 1H).
Step 2. To a solution of 3-((2,5-dimethylbenzyl)amino)isonicotinic
acid (49.9 mg, 0.195 mmol) in 1,4-dioxane (4 mL) was added 2 M
HCl in diethyl ether (0.097 mL, 0.195 mmol), and the solution was
blown down to give 26 (56 mg, 49%). LCMS (high pH method)
retention time 0.71 min, [M + H]+ = 257. 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.23 (s, 3H), 2.29 (s, 3H), 7.03 (d, J = 7.8 Hz, 1H),
7.08 (s, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.97 (q, J = 5.6 Hz, 2H), 8.27
(s, 1H).
3-((2,3-Dimethylbenzyl)amino)isonicotinic Acid Hydrochlor-
ide, 27. Step 1. To 3-fluoroisonicotinic acid (200 mg, 1.417 mmol)
and N-ethyl-N-isopropylpropan-2-amine (619 μL, 3.54 mmol) was
added (2,3-dimethylphenyl)methanamine (230 mg, 1.701 mmol) in a
microwave vial. The reaction was irradiated in a microwave for 4 h at
160 °C. The reaction was diluted with DMSO and purified by MDAP
(high pH method) to give 3-((2,3-dimethylbenzyl)amino)isonicotinic
acid (139.2 mg, 38%) as a white solid. LCMS (high pH method)
retention time 0.71 min, [M + H]+ = 257, 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.22 (s, 3 H), 2.28 (s, 3 H), 4.52 (s, 2 H),
7.02−7.14 (m, 3 H), 7.58 (d, J = 5.1 Hz, 1 H), 7.84 (d, J = 5.1 Hz,
1H), 8.19 (s, 1 H).
Step 2. To a solution of 3-((2,3-dimethylbenzyl)amino)isonicotinic
acid (51.1 mg, 0.199 mmol) in 1,4-dioxane (4 mL) was added 2 M
HCl in 1,4-dioxane (0.100 mL, 0.199 mmol). The solution was blown
down and dried under vacuum to give 27 (55.2 mg, 95%) as a light
yellow solid. LCMS (high pH method) retention time 0.71 min, [M +
H]+ = 257. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.22 (s, 3H), 2.28
(s, 3H), 4.56 (s, 2H), 7.06 (t, J = 7.5 Hz, 1H), 7.10−7.14 (m, 2H),
7.88 (d, J = 5.6 Hz, 1H), 7.95 (d, J = 5.6 Hz, 1H), 8.27 (s, 1H).
3-(([1,1′-Biphenyl]-3-ylmethyl)amino)isonicotinic Acid, 28.
In a 10 mL vial, 3-fluoro-4-pyridinecarboxylic acid (100 mg, 0.70
mmol) and 3-phenylbenzylamine (129 mg, 0.070 mmol) were taken
up in THF (0.5 mL) and stirred at −78 °C under N2. LiHMDS (1 M
in THF) (3.9 mL, 3.90 mmol) was added, and the reaction was stirred
at −78 °C for 15 min and then at rt for 16 h. The reaction was treated
with 2 M NaOH (2 mL) and stirred at rt for 24 h. The reaction was
concentrated and triturated with ether, and the resulting solid was
purified by HPLC to yield 28 (14 mg, 6%). LCMS (formic method)
retention time 0.78 min, [M + H]+ = 305. 1H NMR (400 MHz,
DMSO-d6) δ 4.60 (s, 2H), 7.35 (t, J = 4 Hz, 2H), 7.40 (q, J = 8 Hz,
Step 2. Lithium hydroxide monohydrate (28 mg, 0.667 mmol) was
added in a single portion to a stirred solution of ethyl 3-[(2-
methylpropyl)amino]-4-pyridinecarboxylate (75 mg, 0.337 mmol) in
THF (5 mL) and water (1.7 mL) at rt. The solution was stirred at rt
for 21 h. 2 M HCl (aq) (5 mL) was added, and the resultant solution
was stirred at rt for 10 min. The solvent was removed under reduced
pressure to give a yellow oil. The sample was dissolved in MeOH and
loaded onto a SCX-2 cartridge (5 g) that had been prewashed with
methanol. The column was washed with MeOH and then eluted with
2 M NH3 in MeOH. The appropriate fractions were combined and
concentrated to give 23 as a yellow solid (53 mg, 81%). LCMS (formic
1
method) retention time 0.50 min, [M + H]+ = 195. H NMR (400
MHz, MeOH-d4) δ ppm 1.05 (d, J = 6.8 Hz, 6H), 1.97 (dt, J = 13.3,
6.6 Hz, 1H), 3.08 (d, J = 6.8 Hz, 2H), 7.77 (q, J = 5.1 Hz, 2H), 8.02 (s,
1H).
3-((2-Aminoethyl)amino)isonicotinic Acid, 24. 3-Fluoroisoni-
cotinic acid (255 mg, 1.807 mmol) was suspended in ethylenediamine
(1.5 mL, 22.44 mmol) and irradiated in a microwave at 150 °C for 2 h.
The mixture was loaded on to a 5 g flash NH2 column, then eluted
with IPA (30 mL) and 2 M HCl (25 mL). The acidic fractions were
evaporated to dryness, loaded on to a 10 g SCX cartridge, and eluted
with water/MeOH (1:1) followed by 2 M NH3 in MeOH. The basic
fractions were evaporated to a white solid, and this was triturated with
MeOH, filtered, and dried under reduced pressure to give 24 as a
white solid (140 mg, 40%). LCMS (TFA method) retention time 0.18
min, [M + H]+ = 182. 1H NMR (400 MHz, D2O) δ ppm 3.27 (t, J =
5.9 Hz, 2H), 3.62 (t, J = 5.8 Hz, 2H), 7.56 (d, J = 5.1 Hz, 1H), 7.90 (d,
J = 5.1 Hz, 1H), 8.09 (s, 1H).
3-((3-Aminopropyl)amino)isonicotinic Acid, 25. Step 1. To a
solution of 3-fluoroisonicotinic acid (400 mg, 2.83 mmol) in 1,4-
dioxane (3 mL) was added tert-butyl (3-aminopropyl)carbamate (494
mg, 2.83 mmol), and the mixture was irradiated at 120 °C in a
microwave for 36 h. The mixture was concentrated and the residue
was purified by silica gel chromatography, eluting with a gradient of 0−
10% MeOH in DCM to give 3-((3-((tert-butoxycarbonyl)amino)-
I
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Journal of Medicinal Chemistry
Article
2H), 7.55 (t, J = 4 Hz, 2H), 7.60 (d, J = 12.0 Hz, 2H), 7.68 (s, 2H),
7.8 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H).
furan-2-ylmethanamine (157 μL, 1.701 mmol) in a microwave vial.
The reaction mixture was irradiated in a microwave for 4 h at 160 °C.
The reaction mixture was diluted with DMSO and purified by MDAP
(high pH method) to give a white solid (31.1 mg). This solid was
dissolved in 1,4-dioxane (2 mL), and 2 M HCl in Et2O (71.3 μL, 0.142
mmol) was added to the solution. The solution was concentrated and
dried to give 34 as a yellow solid (36.7 mg, 10%). LCMS (high pH
method) retention time 0.49 min, [M + H]+ = 219. HRMS:
C11H11N2O3 requires (M + H)+ 219.0764, found 219.0758 (error −2.7
3-((Cyclohexylmethyl)amino)isonicotinic Acid, 29. A micro-
wave vial was charged with 3-fluoroisonicotinic acid (200 mg, 1.417
mmol), cyclohexylmethanamine (0.276 mL, 2.126 mmol), and N-
ethyl-N-isopropylpropan-2-amine (0.370 mL, 2.126 mmol) in IPA (4
mL). The reaction was irradiated in a microwave at 150 °C for 5 h.
After cooling, the solvent was evaporated affording an orange oil. The
sample was purified using silica gel column chromatography, eluting
with a gradient of 0−50% EtOAc/cyclohexane followed by 0−20%
MeOH/DCM to give crude title compound as an orange solid. This
was taken up in MeOH and a precipitate formed which was removed
by filtration and dried to give an orange solid. The crude orange solid
was further purified by MDAP (formic method) to give 29 (7 mg, 2%)
as a yellow solid. LCMS (formic method) retention time 0.66 min, [M
1
ppm). H NMR (400 MHz, DMSO-d6) δ ppm 4.64 (br s, 2H), 6.41
(d, J = 5.1 Hz, 2H), 7.62 (br s, 1H), 7.91 (br. d, J = 4.8 Hz, 1H), 7.99
(m, 1H), 8.45 (br s, 1H). 13C NMR (126 MHz, DMSO-d6) δ ppm
169.3, 152.5, 144.6, 143.0, 136.4, 136.4, 123.7, 116.4, 110.9, 107.9,
39.4. Mp 242−245 °C.
3-((Thiazol-4-ylmethyl)amino)isonicotinic Acid, 35. 3-Amino-
isonicotinic acid (30 mg, 0.217 mmol) was dissolved in MeOH (1.5
mL). To this was added thiazole-4-carbaldehyde (49 mg, 0.434 mmol),
and the reaction mixture was stirred at rt for 10 min. NaCNBH3 (27
mg, 0.434 mmol) was then added, and the reaction mixture was stirred
for a further 18 h. The solvent was then evaporated and the residue
was purified by reverse phase column chromatography, eluting with a
gradient of acetonitrile in water (0−100%) to give 35 (10 mg, 20%) as
a white solid. LCMS (high pH method) retention time 0.31 min, [M +
1
+ H]+ = 235. H NMR (400 MHz, DMSO-d6) δ ppm 1.00 (m, 2H),
1.11−1.29 (m, 3H), 1.52−1.61 (m, 1H), 1.64 (d, J = 10.5 Hz, 1H),
1.67−1.73 (m, 2H), 1.78 (d, J = 13.2 Hz, 2H), 3.10 (d, J = 6.6 Hz,
2H), 7.54 (d, J = 4.9 Hz, 1H), 7.76 (d, J = 4.9 Hz, 1H), 8.14 (s, 1H).
3-((Cyclopentylmethyl)amino)isonicotinic Acid, 30. To 3-
fluoroisonicotinic acid (200 mg, 1.417 mmol) under nitrogen were
added N-ethyl-N-isopropylpropan-2-amine (619 μL, 3.54 mmol, IPA
(619 μL), and cyclopentylmethanamine hydrochloride (231 mg, 1.701
mmol), and the mixture was irradiated in a microwave at 180 °C for 4
h. The reaction mixture was diluted with DMSO and purified by
MDAP (high pH method) to give 30 (32 mg, 9%) as a yellow solid.
LCMS (high pH method) retention time 0.58 min, [M + H]+ = 221.
1H NMR (400 MHz, DMSO-d6) δ ppm 1.22−1.32 (m, 2H), 1.51−
1.59 (m, 2H), 1.62 (d, J = 6.6 Hz, 2H), 1.73−1.82 (m, 2H), 2.18 (m,
1H), 3.19 (d, J = 7.1 Hz, 2H), 7.54 (d, J = 5.1 Hz, 1H), 7.80 (d, J = 5.1
Hz, 1H), 8.21 (s, 1H).
1
H]+ = 236. H NMR (400 MHz, DMSO-d6) δ ppm 4.67 (s, 2H),
7.59−7.51 (m, 2H), 7.83 (d, J = 5.0 Hz, 1H), 8.28 (s, 1H), 9.09 (d, J =
2.0 Hz, 1H), 13.39 (b. s, 1H).
3-(((1H-Pyrazol-5-yl)methyl)amino)isonicotinic acid, 36. In a
microwave vial, to 3-fluoroisonicotinic acid (200 mg, 1.417 mmol) and
(1H-pyrazol-5-yl)methanamine dihydrochloride (289 mg, 1.701
mmol) was added N-ethyl-N-isopropylpropan-2-amine (617 μL, 3.54
mmol). The reaction was irradiated in a microwave for 4 h at 160 °C.
The reaction mixture was diluted with DMSO and purified by MDAP
(high pH method) to give 36 as a yellow solid (28 mg, 9%). LCMS
(high pH method) retention time 0.35 min, purity 86%, [M + H]+ =
219. 1H NMR (400 MHz, DMSO-d6) δ ppm 4.47 (s, 2H), 6.20 (d, J =
2.0 Hz, 1H), 7.55 (d, J = 4.9 Hz, 1H), 7.61 (br s, 1H), 7.81 (d, J = 4.9
Hz, 1H), 8.24 (s, 1H).
3-(((5-Methylthiophen-2-yl)methyl)amino)isonicotinic Acid
Hydrochloride, 37. To 3-fluoroisonicotinic acid (200 mg, 1.417
mmol) and N-ethyl-N-isopropylpropan-2-amine (0.619 mL, 3.54
mmol) was added (5-methylthiophen-2-yl)methanamine (0.219 mL,
1.701 mmol) in a microwave vial. The reaction mixture was irradiated
in a microwave for 4 h at 160 °C. The reaction mixture was diluted
with DMSO and purified by MDAP (high pH method) to give a white
solid (74.2 mg). This solid was dissolved in 1,4-dioxane (3 mL), and 2
M HCl in Et2O (0.149 mL, 0.298 mmol) was added. The mixture was
blown down and dried under reduced pressure to give 37 as a yellow
solid (82.4 mg, 20%). LCMS (high pH method) retention time 0.62
min, [M + H]+ = 249. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.39 (s,
3H), 4.74 (s, 2H), 6.67 (d, J = 2.3 Hz, 1H), 6.92 (d, J = 3.3 Hz, 1H),
8.00 (s, 2H), 8.39 (s, 1H).
3-((Cyclopropylmethyl)amino)isonicotinic Acid, 31. To 3-
fluoroisonicotinic acid (199 mg, 1.410 mmol) were added cyclo-
propylmethanamine (147 μL, 1.692 mmol) and N-ethyl-N-isopropyl-
propan-2-amine (614 μL, 3.53 mmol). The mixture was irradiated at
180 °C in the microwave for 4 h. The reaction was diluted with
MeOH and purified by MDAP (formic method) to give 31 (67 mg,
95%) as a pale yellow solid. LCMS (formic method) retention time
1
0.40 min, [M + H]+ = 193. H NMR (400 MHz, DMSO-d6) δ ppm
0.25−0.30 (m, 2H), 0.50−0.55 (m, 2H), 2.33 (s, 1H), 3.16 (d, J = 6.8
Hz, 2H), 7.55 (d, J = 5.0 Hz, 1H), 7.82 (d, J = 5.0 Hz, 1H), 8.23 (s,
1H).
3-((Thiophen-2-ylmethyl)amino)isonicotinic Acid, 32. In a
microwave vial, to 3-fluoroisonicotinic acid (200 mg, 1.417 mmol) and
N-ethyl-N-isopropylpropan-2-amine (619 μL, 3.54 mmol) was added
thiophen-2-ylmethanamine (194 μL, 1.701 mmol). The reaction was
irradiated in a microwave for 4 h at 160 °C. The reaction mixture was
diluted with DMSO and purified by MDAP (high pH method) to give
32 as a light yellow solid (146 mg, 42%). LCMS (high pH method)
1
retention time 0.53 min, [M + H]+ = 235. H NMR (400 MHz,
DMSO-d6) δ ppm 4.76 (s, 2H), 7.00 (m, 1H), 7.10 (d, J = 2.5 Hz,
1H), 7.41 (d, J = 5.1, 1H), 7.57 (d, J = 5.1 Hz, 1H), 7.85 (d, J = 5.1
Hz, 1H), 8.27 (s, 1H).
3-(((4-Methylthiophen-2-yl)methyl)amino)isonicotinic Acid,
38. In a microwave vial, to 3-fluoroisonicotinic acid (200 mg, 1.417
mmol) and (4-methylthiophen-2-yl)methanamine (216 mg, 1.701
mmol) was added N-ethyl-N-isopropylpropan-2-amine (617 μL, 3.54
mmol). The reaction was irradiated in a microwave for 4 h at 160 °C.
The reaction mixture was diluted with DMSO and purified by MDAP
(high pH method) to give the 38 as a light yellow solid (69.8 mg,
20%). LCMS (high pH method) retention time 0.61 min, [M + H]+ =
3-((Thiophen-3-ylmethyl)amino)isonicotinic Acid Hydro-
chloride, 33. In a microwave vial, to 3-fluoroisonicotinic acid (200
mg, 1.417 mmol) and N-ethyl-N-isopropylpropan-2-amine (619 μL,
3.54 mmol) was added thiophen-3-ylmethanamine (198 mg, 1.701
mmol). The reaction was irradiated in a microwave for 4 h at 160 °C.
The reaction mixture was diluted with DMSO and purified by MDAP
(high pH method) to give a white solid (83 mg). This solid was
dissolved in 1,4-dioxane (3 mL), and 2 M HCl in ether (178 μL, 0.356
mmol) was added; the solution was evaporated and the resultant solid
was dried under high vacuum to give 33 as a yellow solid (92.3 mg,
24%). LCMS (high pH method) retention time 0.55 min, [M + H]+ =
235. 1H NMR (400 MHz, DMSO-d6) δ ppm 4.62 (s, 2H), 7.13 (dd, J
= 4.8, 1.0 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 4.9, 2.9 Hz,
1H), 7.99 (q, J = 5.6 Hz, 2H), 8.33 (s, 1H).
1
249. H NMR (400 MHz, DMSO-d6) δ ppm 2.16 (s, 3H), 4.68 (s,
2H), 6.91 (s, 1H), 6.97 (s, 1H), 7.57 (d, J = 4.9 Hz, 1H), 7.84 (d, J =
5.1 Hz, 1H), 8.24 (s, 1H).
3-(((3-Methylthiophen-2-yl)methyl)amino)isonicotinic Acid,
39. To 3-fluoroisonicotinic acid (200 mg, 1.417 mmol) and N-ethyl-
N-isopropylpropan-2-amine (0.619 mL, 3.54 mmol) was added (3-
methylthiophen-2-yl)methanamine (216 mg, 1.701 mmol) in a
microwave vial. The reaction mixture was heated for 4 h at 160 °C
in a microwave. The reaction mixture was diluted with DMSO (2 mL)
and purified by MDAP (high pH method) to give 39 as a light yellow
solid (40 mg, 11%). LCMS (formic method) retention time 0.54 min,
3-((Furan-2-ylmethyl)amino)isonicotinic Acid Hydrochlor-
ide, 34. To 3-fluoroisonicotinic acid (200 mg, 1.417 mmol) and N-
ethyl-N-isopropylpropan-2-amine (619 μL, 3.54 mmol) was added
J
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Journal of Medicinal Chemistry
Article
[M + H]+ = 249. HRMS: C12H12N2O2S requires (M + H)+ 249.0692,
found 249.0692 (error 0.1 ppm). H NMR (400 MHz, DMSO-d6) δ
LCMS (formic method) retention time 0.48 min, [M + H]+ = 219.
HRMS: C11H11N2O3 requires [M + H]+ 219.0764, found 219.0764
(error 0.0 ppm). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.66 (s, 2H),
6.25 (d, J = 3.2 Hz, 1H), 6.37 (m, 1H), 6.68−6.56 (m, 1H), 7.57 (d, J
= 1.7 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.26 (d, J = 4.7 Hz, 1H), 8.35
(s, 1H), 13.14 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ ppm 169.3,
158.3, 153.6, 153.3, 142.6, 140.6, 112.1, 110.9, 107.1, 107.0, 37.6. Mp
197−202 °C.
2-(((3-Methylthiophen-2-yl)methyl)amino)nicotinic Acid, 45.
Step 1. Methyl 2-bromonicotinate (0.3 g, 1.38 mmol, 1.0 equiv) was
dissolved in 1,4-dioxane (5 mL) under nitrogen. To this was added (3-
methylthiophen-2-yl)methanamine (0.212g, 1.66 mmol), Xantphos
(0.161g, 0.28 mmol), Pd2(dba)3 (0.127g, 0.14 mmol), and Cs2CO3
(1.13g, 3.47 mmol). The reaction mixture was then heated to 90 °C
for 3 h. The reaction was diluted with EtOAc and washed with water.
The organic phase was dried over Na2SO4, filtered, and evaporated.
The residue was purified by column chromatography, eluting with 0−
100% EtOAc/cyclohexane to give methyl 2-(((methylthiophen-2-
yl)methyl)amino)nicotinate (282 mg, 78%).
Step 2. Methyl 2-(((methylthiophen-2-yl)methyl)amino)nicotinate
(282 mg, 1.08 mmol) was dissolved in THF (2 mL), and to this was
added a solution of NaOH (64 mg, 1.61 mmol) in water (2 mL). The
reaction mixture was stirred at room temperature for 18 h and then
acidified with 5% NaHSO4 (aq). The precipitate was collected by
filtration, washed with water and EtOAc, then dried to give 45 (101
mg, 38%) as a cream solid. LCMS (formic method) retention time
0.69 min, [M + H]+ = 249. HRMS: C12H12N2O2S requires [M + H]+
249.0692, found 249.0692 (error 0.1 ppm). 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.21 (s, 3H), 4.73 (s, 2H), 6.63 (m, 1H), 6.82 (d, J
= 5.0 Hz, 1H), 7.24 (d, J = 5.0 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 8.27
(d, J = 4.7 Hz, 1H), 8.38 (s, 1H), 12.96 (s, 1H). 13C NMR (126 MHz,
DMSO-d6) δ ppm 169.3, 158.1, 153.6, 140.6, 136.3, 134.0, 130.4,
123.4, 112.1, 106.8, 37.5, 13.8. Mp 223−226 °C.
1
ppm 2.24 (s, 3H), 4.69 (s, 2H), 6.89 (d, J = 4.9 Hz, 1H), 7.33 (d, J =
5.1 Hz, 1H), 7.83 (d, J = 5.1 Hz, 1H), 7.96 (d, J = 5.1 Hz, 1H), 8.31 (s,
1H). 13C NMR (126 MHz, DMSO-d6) δ ppm 169.9, 164.0, 144.7,
136.5, 136.2, 135.2, 134.0, 130.7, 124.3, 123.5, 121.8, 13.8. Mp 238−
241 °C.
3-((Benzo[b]thiophen-2-ylmethyl)amino)isonicotinic Acid,
40. 3-Aminoisonicotinic acid (20 mg, 0.153 mmol) was dissolved in
MeOH (1 mL). To this was added benzo(b)thiophene 7-carbaldehyde
(49 mg, 0.305 mmol) and NaCNBH3 (20 mg, 0.305 mmol), and the
reaction mixture was stirred for 18 h at rt. The solvent was evaporated
and the residue was purified by reverse phase column chromatography,
eluting with a gradient of acetonitrile in water (0−100%) to give 40
(14 mg, 32%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 4.69
(s, 2H), 7.32−7.22 (m, 2H), 7.33 (s, 1H), 7.52 (dd, J = 4.7, 1.1 Hz,
1H), 7.68 (dd, J = 4.7, 1.2 Hz, 1H), 7−75 (d, J = 7.9 Hz, 1H), 7.86 (d,
J = 7.9 Hz, 1H), 7.94 (s, 1H), 9.68 (br s, 1H).
3-((1-Phenylethyl)amino)isonicotinic Acid, 41. To 3-fluoroiso-
nicotinic acid (200 mg, 1.417 mmol) under nitrogen was added 1-
phenylethanamine (548 μL, 4.25 mmol), and the mixture was
irradiated at 180 °C in a microwave for 4 h. The reaction mixture
was diluted with DMSO and purified by MDAP (high pH method) to
give 41 as a cream solid (204 mg, 54%). LCMS (formic method)
1
retention time 0.56 min, [M + H]+ = 243. H NMR (400 MHz,
DMSO-d6) δ ppm 1.47 (d, J = 6.6 Hz, 3H), 4.73 (q, J = 6.6 Hz, 1H),
7.21 (t, J = 7.5 Hz, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.37 (d, J = 8.0 Hz,
2H), 7.55 (d, J = 4.8 Hz, 1H), 7.69 (d, J = 4.8 Hz, 1H), 7.79 (s, 1H).
3-((2-Phenylpropan-2-yl)amino)isonicotinic Acid, 42. To a
solution of 3-fluoroisonicotinic acid (100 mg, 0.709 mmol) in NMP
(0.25 mL) under nitrogen was added 2-phenylpropan-2-amine (105
mg, 0.780 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.149 mL,
0.850 mmol). The mixture was irradiated at 180 °C in the microwave
for 12 h. The reaction mixture was diluted with MeOH and purified by
MDAP (high pH method) to give 42 (9 mg, 4%) as a cream powder.
LCMS (formic method) retention time 0.59 min, [M + H]+ = 257. 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.65 (s, 6H), 7.24 (t, J = 7.5 Hz,
1H), 7.35 (t, J = 7.6 Hz, 3H), 7.45 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 4.6
Hz, 1H), 7.64 (d, J = 4.6 Hz, 1H), 8.19 (s, 1H).
3-(Benzhydrylamino)isonicotinic Acid, 43. 1,1-Diphenylme-
thanamine (1.526 mL, 8.86 mmol) was added in a single portion to
a stirred suspension of 3-fluoro-4-pyridinecarboxylic acid (250 mg,
1.772 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.619 mL, 3.54
mmol) at rt. The reaction was irradiated in a microwave at 150 °C for
4 h. The crude reaction was diluted in DMSO and purified by MDAP
(high pH method) to give 43 as a white solid (9 mg, 2%). LCMS
(formic method) retention time 0.76 min, [M + H]+ = 305. 1H NMR
(400 MHz, DMSO-d6) δ ppm 6.04 (d, J = 4.0 Hz, 1H), 7.27 (t, J = 7.3
Hz, 2H), 7.37 (t, J = 7.3 Hz, 4 H), 7.42 (d, J = 7.3 Hz, 4 H), 7.61 (d, J
= 5.1 Hz, 1H), 7.85 (d, J = 5.1 Hz, 1H), 8.07 (s, 1H), 8.31 (br s, 1H).
2-((Furan-2-ylmethyl)amino)nicotinic Acid, 44. Step 1. Methyl
2-bromonicotinate (300 mg, 1.38 mmol) was dissolved in 1,4-dioxane
(5 mL) under nitrogen. To this was added furan-2-ylmethanamine
(161 mg, 1.66 mmol), Xantphos (160 mg, 0.28 mmol), Pd2(dba)3
(127 mg, 0.14 mmol), and Cs2CO3 (1.13 g, 3.47 mmol). The reaction
mixture was then heated to 90 °C for 3 h. The reaction was diluted
with EtOAc and washed with water. The combined organics were then
dried over Na2SO4, filtered, and evaporated. The residue was then
purified by column chromatography, eluting with 0−100% EtOAc/
cyclohexane to give methyl 2-((furan-2-ylmethyl)amino)nicotinate
(233 mg, 73%).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01537.
Figure of putative prodrug 20E; comparison of differ-
ential activity between KDM4C RFMS biochemical and
cell imaging assays (ΔpIC50) with compound lip-
ophilicity; LCMS spectra and selectivity profiling data
for 34 and 39; methods for the KDM5C RFMS, EGLN3
HTRF, KDM4C cellular imaging and KDM5C cellular
imaging assays; X-ray crystallography methods and data
for 12 bound to KDM6B and KDM4D constructs and
for 17 and 38 bound to KDM4D (PDF)
Molecular formula strings (CSV)
Accession Codes
X-ray crystal structures have been deposited in the Protein Data
Bank as follows: compound 12 bound to KDM6B, PDB code
5FP3; compound 12 bound to KDM4D, PDB code 5FP4;
compound 17 bound to KDM4D, PDB code 5FP7; compound
38 bound to KDM4D, PDB code 5FP8.
Step 2. Methyl 2-((furan-2-ylmethyl)amino)nicotinate (233 mg,
1.00 mmol) was dissolved in THF (2 mL), and to this was added a
solution of NaOH (60 mg, 1.50 mmol) in water (2 mL). The reaction
mixture was stirred at room temperature for 18 h and then acidified
with 5% NaHSO4 (aq). This was then extracted with EtOAc, and the
combined organic phases were dried over Na2SO4, filtered and
evaporated. The residue was then purified by reverse phase column
chromatography, eluting with a gradient of 0−100% acetonitrile with
0.2% NH3 in water to give 44 (56 mg, 26% yield) as a beige solid.
AUTHOR INFORMATION
■
Corresponding Authors
*S.M.W.: e-mail, sue.m.westaway@gsk.com; telephone, +44 (0)
1438763469.
*A.G.S.P.: e-mail, alex.gs.preston@gsk.com; telephone +44 (0)
1438790152.
K
DOI: 10.1021/acs.jmedchem.5b01537
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
histone demethylases: combined nondenaturing mass spectrometric
screening and crystallographic approaches. J. Med. Chem. 2010, 53,
1810−1818.
Present Addresses
∥R.J.S. and D.M.W.: Oncology Innovative Medicines and Early
Development, AstraZeneca, Cambridge Science Park, Milton
Road, Cambridge CB4 OWG, U.K.
́
(10) Rose, N. R.; Ng, S. S.; Mecinovic, J.; Lienard, B. M. R.; Bello, S.
⊥K.L.: Bicycle Therapeutics Limited, Meditrina Building,
Babraham Research Campus, Cambridge CB22 3AT, U.K.
#L.K.: Takeda Pharmaceuticals, 10275 Science Center Drive,
San Diego, CA 92121, U.S.
H.; Sun, Z.; McDonough, M. A.; Oppermann, U.; Schofield, C. J.
Inhibitor scaffolds for 2-oxoglutarate-dependent histone lysine
demethylases. J. Med. Chem. 2008, 51, 7053−7056.
(11) Chang, K. H.; King, O. N.; Tumber, A.; Woon, E. C.;
Heightman, T. D.; McDonough, M. A.; Schofield, C. J.; Rose, N. R.
Inhibition of histone demethylases by 4-carboxy-2,2′-bipyridyl
compounds. ChemMedChem 2011, 6 (5), 759−764.
(12) Thalhammer, A.; Mecinovic, J.; Loenarz, C.; Tumber, A.; Rose,
N. R.; Heightman, T. D.; Schofield, C. J. Inhibition of the histone
demethylase JMJD2E by 3-substituted pyridine 2,4-dicarboxylates. Org.
Biomol. Chem. 2011, 9, 127−135.
(13) Hopkinson, R. J.; Tumber, A.; Yapp, C.; Chowdhury, R.; Aik,
W.; Che, K. H.; Li, X. S.; Kristensen, J. B. L.; King, O. N. F.; Chan, M.
C.; Yeoh, K. K.; Choi, H.; Walport, L. J.; Thinnes, C. C.; Bush, J. T.;
Lejeune, C.; Rydzik, A. M.; Rose, N. R.; Bagg, E. A.; McDonough, M.
A.; Krojer, T. J.; Yue, W. W.; Ng, S. S.; Olsen, L.; Brennan, P. E.;
Notes
The authors declare the following competing financial
interest(s): All authors were GlaxoSmithKline and Cellzome
full-time employees at the time this work was carried out.
ACKNOWLEDGMENTS
■
The authors thank Aymeric Bencib and Pradip Songara for
synthetic chemistry contributions; Michael Woodrow and Neil
Garton for medicinal chemistry logistical assistance; Shenaz
Bunally for ChromLogD determination; Bill Leavens for
HRMS analysis; Argyrides Argyrou, Anshu Bhardwaja, Angela
Bridges, Matthew Burns, Rachel Grimley, Michelle Heathcote,
Thau Ho, Jon Hutchinson, Sue Hutchinson, Emma Jones, John
Martin, Lisa Miller, Linda Myers, Kelvin Nurse, Steve Ratcliffe,
Mike Rees, Anthony Shillings, Kate Simpson, Penny Smee, Rob
Tanner, Laura Williams, and Huizhen Zhao for assay reagent
production, development, management, and data generation.
Oppermann, U.; Muller, S.; Klose, R. J.; Ratcliffe, P. J.; Schofield, C. J.;
̈
Kawamura, A. 5-Carboxy-8-hydroxyquinoline is a broad spectrum 2-
oxoglutarate oxygenase inhibitor which causes iron translocation.
Chem. Sci. 2013, 4, 3110−3117.
(14) Rose, N. R.; Woon, E. C.; Tumber, A.; Walport, L. J.;
Chowdhury, R.; Li, X. S.; King, O. N.; Lejeune, C.; Ng, S. S.; Krojer,
T.; Chan, M. C.; Rydzik, A. M.; Hopkinson, R. J.; Che, K. H.; Daniel,
M.; Strain-Damerell, C.; Gileadi, C.; Kochan, G.; Leung, I. K.;
Dunford, J.; Yeoh, K. K.; Ratcliffe, P. J.; Burgess-Brown, N.; von Delft,
F.; Muller, S.; Marsden, B.; Brennan, P. E.; McDonough, M. A.;
Oppermann, U.; Klose, R. J.; Schofield, C. J.; Kawamura, A. Plant
growth regulator daminozide is a selective inhibitor of human KDM2/
7 histone demethylases. J. Med. Chem. 2012, 55, 6639−6643.
(15) Luo, X.; Liu, Y.; Kubicek, S.; Myllyharju, J.; Tumber, A.; Ng, S.;
Che, K. H.; Podoll, J.; Heightman, T. D.; Oppermann, U.; Schreiber, S.
L.; Wang, X. A selective inhibitor and probe of the cellular functions of
Jumonji C domain-containing histone demethylases. J. Am. Chem. Soc.
2011, 133, 9451−9456.
ABBREVIATIONS USED
■
KDM, lysine demethylase; JMJD, Jumonji C domain-containing
protein; RFMS, RapidFire mass spectrometry; HTRF, homo-
geneous time-resolved fluorescence; JARID, Jumonji AT-rich
interactive domain; 2-OG, 2-oxoglutarate; EGLN, egg-laying
deficiency protein ninelike protein; PHD, prolyl hydroxylase
domain protein; SCX, strong cation exchange; IPA, isopropyl
alcohol
(16) Hamada, S.; Suzuki, T.; Mino, K.; Koseki, K.; Oehme, F.;
Flamme, I.; Ozasa, H.; Itoh, Y.; Ogasawara, D.; Komaarashi, H.; Kato,
A.; Tsumoto, H.; Nakagawa, H.; Hasegawa, M.; Sasaki, R.; Mizukami,
T.; Miyata, N. Design, synthesis, enzyme-inhibitory activity, and effect
on human cancer cells of a novel series of Jumonji domain-containing
protein 2 histone demethylase inhibitors. J. Med. Chem. 2010, 53,
5629−5638.
(17) Sekirnik, R.; Rose, N. R.; Thalhammer, A.; Seden, P. T.;
Mecinovic, J.; Schofield, C. J. Inhibition of the histone lysine
demethylase JMJD2A by ejection of structural Zn(II). Chem. Commun.
2009, 6376−6378.
(18) Rai, G.; Kawamura, A.; Tumber, A.; Liang, Y.; Vogel, J. L.;
Arbuckle, J. H.; Rose, N. R.; Dexheimer, T. S.; Foley, T. L.; King, O.
N.; Quinn, A.; Mott, B. T.; Schofield, C. J.; Oppermann, U.; Jadhav,
A.; Simeonov, A.; Kristie, T. M.; Maloney, D. J. Discovery of ML324, a
JMJD2 demethylase inhibitor with demonstrated antiviral activity.
Probe Reports from the NIH Molecular Libraries Program; National
Center for Biotechnology Information (US): Bethesda, MD, 2010−
2012.
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