Identification of novel lysine demethylase 5-selective inhibitors by inhibitor-based fragment merging strategy

Article abstract of DOI:10.1016/j.bmc.2019.02.006

Histone lysine demethylases (KDMs) have drawn much attention as targets of therapeutic agents. KDM5 proteins, which are Fe(II)/α-ketoglutarate-dependent demethylases, are associated with oncogenesis and drug resistance in cancer cells, and KDM5-selective inhibitors are expected to be anticancer drugs. However, few cell-active KDM5 inhibitors have been reported and there is an obvious need to discover more. In this study, we pursued the identification of highly potent and cell-active KDM5-selective inhibitors. Based on the reported KDM5 inhibitors, we designed several compounds by strategically merging two fragments for competitive inhibition with α-ketoglutarate and for KDM5-selective inhibition. Among them, compounds 10 and 13, which have a 3-cyano pyrazolo[1,5-a]pyrimidin-7-one scaffold, exhibited strong KDM5-inhibitory activity and significant KDM5 selectivity. In cellular assays using human lung cancer cell line A549, 10 and 13 increased the levels of trimethylated lysine 4 on histone H3, which is a specific substrate of KDM5s, and induced growth inhibition of A549 cells. These results should provide a basis for the development of cell-active KDM5 inhibitors to highlight the validity of our inhibitor-based fragment merging strategy.

Full text of DOI:10.1016/j.bmc.2019.02.006

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of novel lysine demethylase 5-selective inhibitors by inhibitor-
based fragment merging strategy
Yuka Miyakea, Yukihiro Itoha, Atsushi Hatanakab, Yoshinori Suzumab, Miki Suzukia,
⁎
Hidehiko Kodamab, Yoshinobu Araib, Takayoshi Suzukia,c,
a Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamo-hangi-cho, Sakyo-ku, Kyoto 603-0823, Japan
b Nippon Pharmaceutical Chemicals Co., Ltd., 2-8-18 Higashiosaka-shi, Osaka 577-0056, Japan
c CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Histone lysine demethylases (KDMs) have drawn much attention as targets of therapeutic agents. KDM5 pro-
teins, which are Fe(II)/α-ketoglutarate-dependent demethylases, are associated with oncogenesis and drug re-
sistance in cancer cells, and KDM5-selective inhibitors are expected to be anticancer drugs. However, few cell-
active KDM5 inhibitors have been reported and there is an obvious need to discover more. In this study, we
pursued the identification of highly potent and cell-active KDM5-selective inhibitors. Based on the reported
KDM5 inhibitors, we designed several compounds by strategically merging two fragments for competitive in-
hibition with α-ketoglutarate and for KDM5-selective inhibition. Among them, compounds 10 and 13, which
have a 3-cyano pyrazolo[1,5-a]pyrimidin-7-one scaffold, exhibited strong KDM5-inhibitory activity and sig-
nificant KDM5 selectivity. In cellular assays using human lung cancer cell line A549, 10 and 13 increased the
levels of trimethylated lysine 4 on histone H3, which is a specific substrate of KDM5s, and induced growth
inhibition of A549 cells. These results should provide a basis for the development of cell-active KDM5 inhibitors
to highlight the validity of our inhibitor-based fragment merging strategy.
JHDM
KDM
Histone methylation
Epigenetics
Inhibitor design
1. Introduction
diseases.23–26 However, no JHDM inhibitors are being clinically tested.
Thus, drug discovery studies of JHDM inhibitors are relatively delayed.
Histone lysine residues are enzymatically and post-translationally
modified in cells, e.g., they are acylated and methylated.1–3 These
modifications control epigenetic gene expression independently of the
DNA sequence, and are involved in various biological events, such as
cell cycle, differentiation, and oncogenesis.4–8 Therefore, the enzymes
that modulate these modifications are attractive as targets of biological
tools and therapeutic agents to chemical biologists and medicinal
chemists in the field of epigenetics.9–12
JHDMs have different subfamilies, including KDM2–8.27 Among the
JHDM family proteins, KDM5s have been viewed as targets for cancer
therapy.28,29 The KDM5 family is composed of four members
(KDM5A–D) that catalyze the demethylation of tri- and dimethylated
lysine 4 on histone H3 (H3K4me3/me2).28,29 KDM5A is a member of
polycomb complexes and is deeply involved in gene repression.30
KDM5A is overexpressed in several human cancer cells, such as lung
and breast cancer cells, and is associated with cancer cell proliferation
and drug resistance.31–33 KDM5B is also overexpressed in human cancer
cells, and is involved in the proliferation of cancer cells through the
E2F/RB pathway.34 KDM5C and KDM5D are overexpressed in prostate
cancer cells and may play critical roles in the cell proliferation.35–37
Based on these backgrounds, KDM5-selective inhibitors that are active
in cells or animals are required for both chemical biology studies of
KDM5 and new cancer therapy.
Histone lysine demethylases (KDMs) are classified into two groups:
FAD-dependent demethylases (lysine-specific demethylases, LSDs; also
known as KDM1s) and Fe(II)/α-ketoglutarate-dependent demethylases
(jumonji C domain-containing histone demethylases, JHDMs).13–16
Both enzymes are attractive as therapeutic targets for various diseases.
LSDs are overexpressed in several cancer cells and associated with
oncogenesis.17–20 Indeed, some LSD1 inhibitors are currently being
tested in clinical trials.21,22 JHDMs are also regarded as targets for the
therapy of some diseases, such as cancer and inflammatory
Some KDM5 inhibitors have been reported.38–43 In 2015, we iden-
tified NCDM-81a (1a) and NCDM-82a (2a) (Fig. 1) as KDM5 inhibitors,
⁎ Corresponding author at: Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamo-hangi-cho, Sakyo-ku, Kyoto 603-0823,
Japan.
E-mail address: suzukit@koto.kpu-m.ac.jp (T. Suzuki).
https://doi.org/10.1016/j.bmc.2019.02.006
Received 25 December 2018; Received in revised form 23 January 2019; Accepted 1 February 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Miyake,Y.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.02.006

Y. Miyake et al.
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Phthalimide was reacted with 1,2-dibromoethane and subsequently
with N-hexylmethylamine to obtain compound 14. Phthalimide 14 was
converted into diamine 15 in the presence of hydrazine. Oxidation of
4,4′-dimethyl-2,2′-bipyridine in the presence of potassium permanga-
nate and esterification under the acidic condition gave diester 17.
Monohydrolysis of 17 under the basic condition yielded 18. Con-
densation of 15 and 18 afforded 19. Compound 19 was treated with
NaOH solution to give 7.
Compound 8 was prepared from N-(3-bromopropyl)phthalimide
and 2,4-pyridinedicarboxylic acid by using a similar route to the one
used for the preparation of 7 (Scheme 2). Compound 21 was synthe-
sized by an SN2 reaction of N-(3-bromopropyl)phthalimide with N-
hexylmethylamine, and this was followed by the removal of the
phthalimide scaffold. Compound 23 was obtained by the diesterifica-
tion of 2,4-pyridinedicarboxylic acid and the subsequent mono-
hydrolysis of diester 22. Condensation of 21 and 23 yielded 24. Hy-
drolysis of 24 under the basic condition gave 8.
Compound 9 was prepared from quinoxaline-2,3(1H,4H)-dione by
the procedure outlined in Scheme 3. The reaction of quinoxaline-
2,3(1H,4H)-dione with ethyl bromoacetate followed by treatment with
bromo-3-chloropropane in the presence of sodium hydride in DMF gave
compound 26. Then, 26 was allowed to react with N-hexylmethylamine
to give compound 27. Finally, the ethyl ester of 27 was converted into
carboxylic acid to obtain compound 9.
Figure 1. Structures of representative KDM5 inhibitors.
Compounds 10–13 were synthesized as outlined in Scheme 4. 5-
Amino-1H-pyrazole-4-carbonitrile was reacted with diethyl 2-iso-
propylmalonate to obtain compound 28. Dichlorination of compound
28 and a subsequent reaction with sodium hydroxide furnished com-
pound 29. Oxidation of 2-(4-bromophenyl)ethanol by Dess-Martin
periodinane gave aldehyde 30. Compounds 31 were prepared from
aldehyde 30 or 4-bromothiophene-2-carbaldehyde by reductive ami-
nation. Pinnacol borates 32 were prepared by SN2 reactions of benzyl
bromides with N-hexylmethylamine (for compounds 32a and 32b) or
coupling reactions in the presence of palladium catalyst of bis(pinaco-
late)diboron with bromides 31 (for compounds 32c and 32d). Suzuki
coupling of compound 29 with pinacol borates 32 provided desired
compounds 10–13.
although their IC50 values were in the micromolar range and KDM5
selectivities were moderate.44 In 2016, three potent and selective KDM5
inhibitors, CPI-455 (3), KDM5-C49 (4a), and GSK467 (5), were re-
ported (Fig. 1).45,46 Furthermore, in 2018, N71 (6a) was identified as a
covalent KDM5 inhibitor.47 However, their cellular activities were ex-
tremely weak and a high concentration was needed for cellular study
even if their prodrug forms, namely, NCDM-81b (1b), NCDM-82b (2b),
KDM5-C70 (4b), and N73 (6b) (Fig. 1), were used. These KDM5-se-
lective inhibitors warrant improvements. In this study, we attempted to
identify novel KDM5-selective inhibitors that are active in cells at low
concentrations. To this end, we designed compounds by strategic
fragment merging of two fragments for competitive inhibition with α-
ketoglutarate and for KDM5-selective inhibition, focusing on the
structures of the reported KDM5 inhibitors. Through this, we identified
highly potent and selective KDM5 inhibitors 10 and 13. We also found
that 10 and 13 induced the trimethylation of H3K4 in cells and more
strongly inhibited the growth of cancer cells than CPI-455 (3). Herein,
we present our drug discovery study of KDM5-selective inhibitors.
3. Results and discussion
3.1. Inhibitor design
Thus far, we have identified several KDM inhibitors consisting of a
hydroxamate, a carboxylate, and an alkyl chain group.44,48,49 The
hydroxamate group and the carboxylate group are essential for the
competitive inhibition with α-ketoglutarate, which is a co-enzyme of
JHDMs: the hydroxamate works as a Fe(II) chelator in the KDM
catalytic site, and the carboxylate corresponds to the carboxylate group
of α-ketoglutarate (Fig. 2A). However, these two groups decrease the
2. Chemistry
The compounds tested in this study are described in Fig. 3 and
Table 2. The compounds were prepared by the synthetic routes shown
in Schemes 1–4. Scheme 1 shows the preparation of compound 7.
Scheme 1. Reagents and reaction conditions: (a) 1,2-dibromoethane, K2CO3, DMF, room temperature, 2 h, 34%; (b) N-hexylmethylamine, K2CO3, DMF, 100 °C, 3 h,
46%; (c) N2H4·H2O, EtOH, reflux, 2 h, 91%; (d) KMnO4, H2O, reflux, 6 h, 99%; (e) SOCl2, MeOH, reflux, overnight, 91%; (f) NaOH, THF, MeOH, H2O, room
temperature, overnight, 84%; (g) EDCI·HCl, HOBt, Et3N, DMF, room temperature, overnight, 12% (from 18); (h) NaOH, THF, MeOH, H2O, room temperature,
overnight, 60%.
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Scheme 2. Reagents and reaction conditions: (a) N-hexylmethylamine, K2CO3, MeCN, reflux, 1 h, 84%; (b) N2H4·H2O, EtOH, reflux, 2.5 h, 75%; (c) SOCl2, MeOH,
reflux, 5 h, 89%; (d) KOH, MeOH, reflux, 1 h; (e) EDCI·HCl, HOBt, Et3N, DMF, 70 °C, 4.5 h, 32% (from 21); (f) KOH, MeOH, reflux, 1 h, 87%.
active KDM5 inhibitors would be obtained by merging N-hexyl-N-me-
thyl amino chain with a scaffold that is expected to compete with α-
ketoglutarate and not to give rise to high polarity and low membrane
permeability. As shown in Fig. 2B, we planned to use bipyridine,
picolinamide, and quinoxalinedione moieties for the Fe(II) chelator part
as the scaffold, and to conjugate them with carboxylate. We also at-
tempted to use 3-cyano pyrazolo[1,5-a]pyrimidin-7-one, which works
as a monodentate Fe(II) chelator and a carboxylate mimic (Fig. 2B). The
reason why we focused on these structures was that these have been
already used in KDM5 inhibitors or Fe(II) chelators; bipyridine has been
used in several KDM inhibitors;50 picolinamide and quinoxalinedione
can function as a bidentate metal ion chelator51,52 the 3-cyano pyrazolo
Scheme 3. Reagents and reaction conditions: (a) Ethyl bromoacetate, NaH,
DMF, room temperature, 2 h, 25%; (b) 1-bromo-3-chlroropropane, NaH, DMF,
room temperature, 2 h, 54%; (c) N-hexylmethylamine, K2CO3, MeCN, reflux,
5 h, 82%; (d) NaOH, THF, MeOH, H2O, room temperature, 2 h, 75%.
[1,5-a]pyrimidin-7-one scaffold has been used in KDM5 inhibitors, such
as CPI-455 (3).45 The linker structures were designed based on the
superposition of NCDM-82a (2a) on each α-ketoglutarate mimic
(Fig. 3). The conformation of NCDM-82a (2a) docked to KDM5A, which
was previously reported by our group,44 was superimposed on the bi-
pyridine, picolinamide and quinoxalinedione, and 3-cyano pyrazolo
[1,5-a]pyrimidin-7-one moieties as their α-ketoglutarate mimic parts
were located on the same place. Based on these superimposed struc-
tures, the linkers were designed so that the nitrogen atom of the N-
hexyl-N-methyl amino chain is positioned as close to that of NCDM-82a
(2a) as possible; a two-methylene linker for compound 7; a three-me-
thylene linker for compounds 8 and 9; and a one-methylene linker for
compound 10. Thus, we designed compounds 7–10 by an inhibitor-
based fragment merging strategy.
cellular activity of the KDM inhibitors because they are highly polar
functional groups that result in the low membrane permeability of the
KDM5 inhibitors. On the other hand, the alkyl chain is a key structure
for KDM subfamily selectivity. In the case of KDM5-selective inhibitors,
such as NCDM-81a (1a) and NCDM-82a (2a), an amino alkyl chain with
a methyl group and a long alkyl group (n-pentyl or n-hexyl group) is
preferred (Fig. 2A).44 Furthermore, the linker length between the Fe(II)
chelator part and the nitrogen atom is important for KDM5 selectivity
(Fig. 2A).44 Based on these backgrounds, we hypothesized that cell-
Scheme 4. Reagents and reaction conditions: (a) NaOEt, EtOH, MW 160 °C, 0.5 h, quant.; (b) POCl3, MW 170 °C, 2 h, 26%; (c) NaOH, THF, H2O, reflux, 0.75 h, 72%;
(d) Dess-Martin periodinane, CH2Cl2, room temperature, 1 h, 62%; (e) N-hexylmethylamine, NaBH(OAc)3, 1,2-DCE, room temperature, 5 h, 53% for 31a or 79% for
31b; (f) N-hexylmethylamine, K2CO3, MeCN, reflux for 32a or room temperature for 32b, 1 h for 32a or overnight for 32b, 69% for 32a or 82% for 32b; (g) bis
(pinacolate)diboron, PdCl2(dppf)·CH2Cl2, AcOK, dioxane, MW 140 °C, 0.5 h, 26% for 32c or 85% for 32d; (h) PdCl2(dppf)·CH2Cl2, Na2CO3, DME, H2O, MW 160 °C,
0.5 h, 20% for 10, 7% for 11, 20% for 12, or 18% for 13.
3

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Figure 2. (A) Our previous work. (B) This work.
Figure 3. Drug design based on fragment merging of α-ketoglutarate mimic scaffolds and N-hexyl-N-methyl amino group.
3.2. Enzyme assay
KDM5A strongly, and its inhibitory activity was comparable or slightly
superior to that of CPI-455 (3) (Table 1, entries 2 and 3). On the other
hand, the inhibitory activities of picolinamide 8 and quinoxalinedione
9 were inferior to that of NCDM-81a (1a) (Table 1, entries 1, 4, and 5).
Interestingly, 3-cyano pyrazolo[1,5-a]pyrimidin-7-one 10 exhibited the
most potent inhibitory activity among the tested compounds.
Initially, we evaluated the KDM5A inhibitory activities of synthetic
compounds 7–10 by conducting an AlphaLISA screen assay. We used
NCDM-81a (1a) and CPI-455 (3) as positive controls, and tested the
compounds at 1 or 25 μM. As shown in Table 1, bipyridine 7 inhibited
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Table 1
part of 10 (Table 2). We changed the alkyl amino chain in 10 from
para-position to meta-position. The KDM5A inhibitory activity of
meta-substituted compound 11 was three times lower than that of 10
(Table 2, entries 4 and 5), although it was high compared to the KDM5A
inhibitory activity of 7. We also replaced the para-substituted benzene
ring in 10 with a 2,4-substituted thiophene ring to yield 12. However,
the KDM5A inhibitory activity of 12 was 8 times lower than that of 10
In vitro KDM5A inhibitory activities of NCDM-81 (1a), CPI-455 (3), and com-
pounds 7–10.
a
Entry
Compound
% Inhibition
at 1 μM
at 25 μM
1
2
3
4
5
6
1a
3
30.8
68.9
87.0
N.D.
N.D.
97.9
2.1
89.9
100
0.9
(IC50 of 12: 179
12 nM), and was nearly the same as that of 7
3.3
0.43
3.0
(Table 2, entries 3 and 6). Next, we extended the methylene linker
between the benzene ring and the nitrogen atom conjugating the me-
thyl and n-hexyl groups. Compound 13 with a two-methylene linker
exhibited potent KDM5A inhibitory activity: its activity was 5 times and
116 times greater than that of 10 and CPI-455 (3), respectively
(Table 2, entries 2, 4, and 7). Thus, compound 13 was the strongest
KDM5A inhibitor among the tested compounds.
7
10.7
96.1
12.0
16.5
N.D.
b
b
8
1.4
9
9.9
b
10
0.5
a
Values represent means
standard deviation of at least three experi-
b
ments. No data.
Then, to investigate the selectivity of 10 and 13 for KDM5 enzymes,
we evaluated the inhibitory activities of compounds 10, 13, NCDM-81a
(1a), and CPI-455 (3) against KDM5B and KDM5C by AlphaLISA screen
assay. The IC50 values of NCDM-81a (1a) for KDM5B and KDM5C were
Table 2
In vitro KDM5A inhibitory activities of compounds NCDM-81 (1a), CPI-455 (3),
7, and compounds 10–13.a
9240
704 nM and 28900
1720 nM, respectively, which indicate
that NCDM-81a (1a) is a relatively KDM5A-selective inhibitor (Table 3,
entry 1). CPI-455 (3) inhibited KDM5A and KDM5B strongly compared
to KDM5C (Table 3, entry 2). On the other hand, 10 showed low
KDM5C selectivity (Table 3, entry 3, KDM5A IC50 = 22.7
3.2 nM;
KDM5B IC50 = 51.1
5.3 nM; KDM5C IC50 = 5.84
0.65 nM). Fur-
Entry
Compound
R
IC50 (nM)
thermore, 13 exhibited potent inhibitory activity against all the three
1
2
3
4
1a
3
–
2700
506
210
enzymes with IC50 values of 1.34–6.24 nM (Table 3, entry 4, KDM5A
Ph
–
38
12
IC50 = 4.37
IC50 = 6.24
0.19 nM; KDM5B IC50 = 1.34
0.16 nM; KDM5C
7
170
0.70 nM). The results of the KDM5 inhibition profiling
10
22.7
3.2
of 10 and 13 suggest that the slight difference in the amino alkyl chain
is responsible for the selectivity differences for KDM5s: optimization of
the chain structure may lead us to the identity of KDM5-isozyme-se-
lective inhibitors, although we need further studies of the structure-
activity or selectivity relationships.
5
6
11
12
65.3
179
8.6
12
Finally, we confirmed the selectivity of 10 and 13 for other KDM
family proteins (KDM2A, KDM3A, KDM4A, KDM6B, and KDM7B). As
shown in entries 3 and 4 of Table 3, the inhibitory activities of 10 and
13 at 25 μM and 50 μM against the five enzymes were not high; we
estimated that the IC50 values of 10 for KDM4A and 13 for KDM3A
should be between 25 and 50 μM, and that the other IC50 values were
higher than 50 μM. Thus, 10 and 13 have significantly higher KDM5
inhibitory activities than the other KDM subfamily proteins, and we
conclude that they are highly potent KDM5-selective inhibitors.
7
13
4.37
0.19
a
Values represent means
standard deviation of at least three experi-
ments.
Compound 10 at 1 μM almost completely inhibited KDM5A (Table 1,
entry 6). Given the results, we determined the IC50 values of 7 and 10
for KDM5A inhibition (Table 2). The IC50 values of 7 and 10 were lower
than those of reference compounds NCDM-81a (1a) and CPI-455 (3)
(Table 2, entries 1 and 2). Especially, 10 had 22 times stronger KDM5A
inhibitory activity than CPI-455 (3).
3.3. Molecular docking
We performed docking simulation to understand the structural basis
of KDM5A inhibition by most potent KDM5 inhibitor 13. Compound 13
was docked to the three-dimensional structure of KDM5A (PDB ID:
5CEH) using Molegro Virtual Docker 6.0 software. As shown in Fig. 4,
13 was docked to the KDM5A active site. As is the case with CPI-455 (3)
Subsequently, we performed the structure optimization of the linker
Table 3
In vitro KDM inhibitory activities of NCDM-81 (1a), CPI-455 (3), 7, and compounds 10 and 13.a
Entry
Compound
IC50 (nM)
% inhibition at 50 μM (upper) and at 25 μM (bottom)
KDM5A
KDM5B
9240
337
KDM5C
28900
3010
KDM2A
KDM3A
KDM4A
KDM6B
KDM7B
1
2
3
4
1a
3
2700
506
210
704
144
1720
1235
0.65
51.7
N.D.
25.7
N.D.
13.4
N.D.
38.4
N.D.
3.4
2.2
5.7
6.9
30.1
N.D.
31.9
N.D.
61.5
13.0
15.9
N.D.
4.9
3.1
25.4
N.D.
61.1
35.9
32.5
N.D.
71.0
32.4
3.0
17.8
N.D.
N.D.
69.1
29.1
N.D.
22.7
N.D.
2.3
N.D.
N.D.
N.D.
N.D.
N.D.
12.1
N.D.
11.8
38
2.5
5.2
3.5
2.8b
0.4
10
13
22.7
4.37
3.2
0.19
51.1
5.3
5.84
4.8
1.4
5.9
2.9
5.5
1.34
0.16
6.24
0.70
3.1
3.1
3.1
a
b
Values represent means
standard deviation of at least three experiments. IC50 = 14300
2250 nM.
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Figure 5. Western blots showing H3K4me3, H3K9me3, H3K27me3, and H3
levels in A549 cells treated with CPI-455 (3), 10, and 13. The methylation
levels were analyzed after 48 h incubation with the inhibitors.
detected H3K4me3 levels by western blot analysis because H3K4me3 is
a specific substrate of KDM5s.28,29 As shown in Fig. 5, 10 and 13 at 1, 5,
and 25 μM increased H3K4me3 levels in a dose-dependent manner like
CPI-455 (3). These results indicate that compounds 10 and 13 inhibited
KDM5s in A549 cells. In addition, we analyzed the influence of the
inhibitors on H3K9me3 and H3K27me3, which are substrates of KDM4s
and KDM6s, respectively.13 Compounds 10 and 13 did not affect the
methylation levels of H3K9me3 and H3K27me3 (Fig. 5). These results
suggest that 10 and 13 selectively inhibit KDM5s in preference to other
KDMs in A549 cells.
Finally, we tested the growth inhibitory activity of 10 and 13
against A549 cells because KDM5A is involved in the proliferation of
A549 cells.31 Treatment of A549 cells with 50 μM each of compounds
10 and 13 inhibited the growth of A549 cells, whereas CPI-455 (3) at
the same concentration did not inhibit it strongly (Fig. 6A). Further-
more, we determined the half-maximal growth inhibition concentration
(GI50) values of 10 and 13. As shown in Fig. 6B, 10 and 13 inhibited cell
growth in a dose-dependent manner, and the GI50 values were 40.0 μM
for 10 and 29.6 μM for 13. The GI50 value of CPI-455 (3) was higher
Figure 4. Docking simulation of 13 to KDM5A (PDB ID: 5CEH). (A) View of the
conformation of 13 (ball-and-stick, purple carbons) docked into the KDM5A
active site. (B) Schematic diagram of binding of 13 docked into the KDM5A
active site. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
(Supplementary Figure S1), the cyano group of 13 could coordinate to
the catalytic Fe(II) ion and the pyrazole carbonyl group could interact
with Lys501/Asn575 through hydrogen bonds. The hexyl group of 13
was located in a hydrophobic space surrounded by Lys113, Ile114, and
Val573. Interestingly, a C-H group that is conjugated with the amino
group was located in a position where it could form a N+-C-H···O hy-
drogen bond53 with the hydroxyl group of Tyr409. We assume that this
kind of hydrogen bond is not formed between compound 10 and the
hydroxyl group of Tyr409 because the methylene spacer of compound
10 is shorter than that of compound 13. This simulation suggests that
the hydrophobic interaction and the N+-C-H···O hydrogen bond for-
mation are important for the KDM5 inhibitory activity of 13.
3.4. Cellular assay
Figure 6. Growth inhibitory activity of CPI-455 (3), 10, and 13 in A549 cells
after treatment for 72 h. (A) Treatment of the cells with 50 μM of the inhibitors.
(B) Dose-dependent curves of 10 and 13. Values were calculated from three
Compounds 10 and 13, which are potent KDM5-selective inhibitors,
were tested in cellular assays. As KDM5A is overexpressed in human
lung cancer cell line A549,31 we used A549 cells in this study. We in-
cubated A549 cells with compounds 10 and 13, and after 48 h, we
independent determinations. Bars represent means
dependent experiments.
SD from three in-
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than 50 μM (Fig. 6A). The GI50 values of CPI-455 (3), 10, and 13 (GI50
:
dropwise fashion, and the resulting suspension was stirred at room
temperature for 2 h. Then, the reaction was quenched with water and
extracted with n-hexane/AcOEt (2/1). The organic layer was separated
and concentrated in vacuo. The residue was purified by silica gel
column chromatography to give 2-(2-bromoethyl)isoindoline-1,3-dione
(1.73 g, 34%). A mixture of 2-(2-bromoethyl)isoindoline-1,3-dione
(158 mg, 0.624 mmol), N-hexylmethylamine (190 µL, 1.25 mmol), and
K2CO3 (104 mg, 0.749 mmol) in DMF (1.5 mL) was stirred at 100 °C for
3 h. The reaction mixture was cooled and concentrated in vacuo. The
residue was extracted with AcOEt, washed with water, and dried over
Na2SO4. Filtration, evaporation in vacuo, and purification by silica gel
column chromatography gave 14 (82.0 mg, 46%): 1H NMR (CDCl3,
300 MHz, δ; ppm), 7.85–7.68 (4H, m), 3.80 (2H, t, J = 6.6 Hz), 2.64
(2H, t, J = 6.6 Hz), 2.36 (2H, t, J = 7.5 Hz), 2.29 (3H, s), 1.37–1.17
(8H, m), 0.81 (3H, t, J = 6.3 Hz); 13C NMR (CDCl3, 75 MHz, δ; ppm),
168.4, 133.8, 132.2, 123.1, 57.8, 54.8, 42.2, 35.9, 31.7, 27.2, 27.0,
22.6, 14.0; MS (ESI) m/z 289.1 (MH+).
3 > 10 > 13) are consistent with their IC50 values in the in vitro
KDM5A assay (IC50: 3 > 10 > 13) (Table 3). We also investigated the
effects of CPI-455 (3), 10, and 13 on breast cancer MDA-MB-231 cells
where KDM5A expression level is lower than that in A549 cells
(Supplementary Figure S2A). The growth of MDA-MB-231 cells treated
with 50 μM of 3, 10, and 13 was higher than that of A549 cells
(Supplementary Figure S2B), which is consistent with difference in the
KDM5A expression level between MDA-MB-231 and A549 cells. The
results of cellular assays suggest that 10 and 13 inhibited KDM5s in
A549 cells and induced cell growth inhibition.
4. Conclusion
In conclusion, to identify novel KDM5-selective inhibitors, we de-
signed several compounds on the basis of our previous reports. By the
inhibitor-based fragment merging strategy, i.e., the conjugation of an
amino alkyl chain with a 3-cyano pyrazolo[1,5-a]pyrimidin-7-one
scaffold, we identified 10 and 13, which showed potent KDM5 in-
hibitory activity compared to the previously reported representative
KDM5 inhibitor CPI-455 (3). In particular, 13 strongly inhibited
KDM5A, KDM5B, and KDM5C, whereas 10 exhibited low KDM5C se-
lectivity. In addition, both inhibitors showed high selectivity for the
KDM5 subfamily over other KDMs. In cellular assays using A549 cells,
compounds 10 and 13 increased H3K4me3 levels without affecting
H3K9me3 and H3K27me3 levels, which indicates that 10 and 13 se-
lectively inhibit KDM5s in cells. Compounds 10 and 13 also inhibited
the growth of A549 cells more strongly than CPI-455 (3). We believe
that the compounds described here will be useful tools for probing the
biology of KDM5s and be candidate therapeutic agents for cancer, and
the fragment merging strategy will be applied to other targets.
5.1.2.2. Preparation of N1-hexyl-N1-methylethane-1,2-diamine (15). A
solution of 14 (100 mg, 347 µmol) and N2H4·H2O (35 µL, 270 µmol) in
EtOH (3.0 mL) was heated at reflux temperature for 2 h. The reaction
mixture was filtered and the filtrate was concentrated in vacuo. The
residue was suspended in 5 mL of CH2Cl2 and the insoluble material
was removed by filtration. The filtrate was concentrated to obtain 15
(50.0 mg, 91%): 1H NMR (CDCl3, 300 MHz, δ; ppm), 2.77 (2H, t,
J = 6.0 Hz), 2.40 (2H, t, J = 6.0 Hz), 2.32 (2H, t, J = 7.5 Hz), 2.21 (3H,
s), 1.79 (2H, br), 1.52–1.24 (8H, m), 0.88–0.94 (3H, m); 13C NMR
(CDCl3, 75 MHz, δ; ppm), 60.6, 58.1, 54.1, 42.2, 39.6, 29.7, 27.0, 22.6,
14.0; MS (ESI) m/z 159.1 (MH+).
5.1.2.3. Preparation of [2,2′-bipyridine]-4,4′-dicarboxylic acid (16). A
solution of 4,4′-dimethyl-2,2′-bipyridine (1.02 g, 5.43 mmol) and
potassium permanganate (3.20 g, 20.4 mmol) in H2O (35 mL) was
heated at reflux temperature for 6 h. The reaction mixture was
filtered through Celite and ether was added to the residue to remove
the unreacted starting material. The water layer was separated and
acidified with 6 N aqueous HCl solution. The precipitate was collected
by filtration and washed with water to give 16 (1.34 g, 99%) as a white
powder: mp > 300 °C; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.92
(2H, d, J = 4.2 Hz), 8.85 (2H, s), 7.91 (2H, d, J = 3.3 Hz); 13C NMR
(DMSO‑d6, 75 MHz, δ; ppm), 166.5, 156.0, 151.1, 140.0, 123.9, 120.0;
MS (ESI) m/z 244.1 (MH+).
5. Experimental section
5.1. Chemistry
5.1.1. General
Reagents and solvents were purchased from Aldrich, Merck, Nacalai
Tesque, Tokyo Kasei Kogyo, Wako Pure Chemical Industries, Kishida
Kagaku, and Kanto Kagaku, and were used as received. Flash column
chromatography was performed using silica gel supplied by Merck or
Toyota Silica gel. Proton nuclear magnetic resonance spectra (1H NMR)
and carbon nuclear magnetic resonance spectra (13C NMR) were re-
corded on a Bruker AVANCE 300 spectrometer in solvent as indicated.
Chemical shifts (δ) are reported in parts per million relative to internal
tetramethylsilane. Electrospray ionization (ESI) mass spectra were re-
corded on a Bruker HCTplus mass spectrometer or a Shimadzu LCMS-
2020 instrument. High-resolution mass spectra (HRMS) were recorded
on a Shimadzu LCMS-IT-TOF mass spectrometer. Melting points were
determined using a Yanaco Micro Melting Point apparatus. The purity
of all tested compounds was determined by HPLC using a Shimadzu
UFLC system (SPDM20A UV detector, DGU-20A3R degassing unit, LC-
20AD solvent delivery unit, and CBM-20A system) with a 5C18-AR-II
column (150 mm × φ4.6 mm, Cosmosil) (UV detection, λ = 220 or
254 nm; flow, 1 mL/min). Microwave reactions were carried out in a
Biotage microwave reaction kit (sealed vials) in an Initiator+ (Bio-
tage).
5.1.2.4. Preparation of dimethyl [2,2′-bipyridine]-4,4′-dicarboxylate
(17). Thionyl chloride (300 µL, 4.08 mmol) was added to
suspension of 16 (400 mg, 1.62 mmol) in MeOH (30 mL) in
a
a
dropwise fashion. The mixture was heated at reflux temperature
overnight. Then, the solvent was removed under reduced pressure
and the residue was partitioned between CH2Cl2 and water. The organic
layer was washed with saturated aqueous NaHCO3 solution and dried
over Na2SO4. Filtration, evaporation in vacuo, and recrystallization in
AcOEt gave 17 (400 mg, 91%): mp 200–202 °C; 1H NMR (CDCl3,
300 MHz, δ; ppm), 8.92 (2H, d, J = 4.2 Hz), 8.85 (2H, s), 7.91 (2H,
d, J = 3.3 Hz); 13C NMR (CDCl3, 75 MHz, δ; ppm), 166.5, 156.0, 151.1,
140.0, 123.9, 120.0; MS (ESI) m/z 273.0 (MH+).
5.1.2.5. Preparation
of
4′-(methoxycarbonyl)-[2,2′-bipyridine]-4-
carboxylic acid (18). A solution of NaOH (27.0 mg, 0.488 mmol) in
water (1 mL) was added to a solution of 17 (100 mg, 0.286 mmol) in
MeOH/THF (5 mL/5 mL). The mixture was stirred at room temperature
overnight. Then, the reaction mixture was concentrated in vacuo and
the residue was extracted with water. The water layer was washed with
AcOEt twice and acidified with 6 N aqueous HCl solution to adjust the
pH to 2. The white precipitate was collected and dried in vacuo to give
5.1.2. Synthesis of 4′-((2-(hexyl(methyl)amino)ethyl)carbamoyl)-[2,2′-
bipyridine]-4-carboxylic acid (7)
5.1.2.1. Preparation of 2-(2-(hexyl(methyl)amino)ethyl)isoindoline-1,3-
dione (14). A solution of phthalimide (2.94 g, 20.0 mmol) in DMF
(20 mL) was added to a suspension of 1,2-dibromoethane (1.85 mL,
29.6 mmol) and K2CO3 (5.52 g, 40.0 mmol) in DMF (40 mL) in a
7

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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
18 (80.0 mg, 84%): mp > 300 °C; 1H NMR (DMSO‑d6, 300 MHz, δ;
ppm), 8.92 (2H, m), 8.85 (2H, s), 7.93 (2H, m), 3.96 (3H, s); 13C NMR
(DMSO‑d6, 75 MHz, δ; ppm), 166.6, 165.6, 156.2, 156.0, 151.4, 151.3,
140.2, 138.9, 124.2, 124.1, 120.2, 119.9, 53.52; MS (ESI) m/z 258.2
(MH+).
Yield, 1.74 g, 89%; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.93 (1H,
dd, J = 6.9, 0.6 Hz), 8.383–8.378 (1H, m), 8.07 (1H, dd, J = 6.9,
1.5 Hz), 3.93 (3H, s), 3.92 (3H, s).
5.1.3.4. Preparation of methyl 2-((3-(hexyl(methyl)amino)propyl)
carbamoyl)isonicotinate (24). A mixture of 22 (137 mg, 0.70 mmol)
and KOH (43.2 mg, 0.77 mmol) in MeOH (2 mL) was heated at reflux
temperature for 1 h. Then, the reaction mixture was concentrated in
vacuo to give crude 23. Crude 23 was dissolved in DMF (2 mL) and a
solution of 21 in DMF (2 mL), Et3N (0.24 mL, 1.75 mmol), EDCI·HCl
(148 mg, 0.77 mmol), and HOBt (104 mg, 0.77 mmol) were added. The
resulting mixture was heated at 70 °C for 4.5 h. The reaction was
quenched with water and extracted with AcOEt. The organic layer was
separated and dried over Na2SO4. Filtration, evaporation in vacuo, and
purification by silica gel column chromatography gave 24 (66 mg,
32%): 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.99 (1H, t, J = 5.1 Hz),
8.85 (1H, dd, J = 5.1, 0.6 Hz), 8.40 (1H, s), 7.98 (1H, dd, J = 8.1,
1.8 Hz), 3.91 (3H, s), 3.26 (2H, m), 2.33 (2H, t, J = 6.9 Hz), 2.25 (2H, t,
J = 6.9 Hz), 2.12 (3H, s), 1.71–1.62 (2H, m), 1.36–1.34 (2H, m), 1.21
(6H, s), 0.82 (3H, t, J = 6.9 Hz).
5.1.2.6. Preparation of methyl 4′-((2-(hexyl(methyl)amino)ethyl)
carbamoyl)-[2,2′-bipyridine]-4-carboxylate (19). A solution of Et3N
(20.0 μL, 0.146 mmol) and EDCI·HCl (28.0 mg, 0.146 mmol) in DMF
(1 mL) was added to a solution of 18 (40.0 mg, 0.146 mmol), 15
(25.0 mg, 0.158 mmol), and HOBt (22.0 mg, 0.158 mmol) in DMF
(5 mL). The mixture was stirred at room temperature overnight.
Then, the solvent was removed under reduced pressure and the
residue was extracted with AcOEt. The organic layer was washed
with water and dried over Na2SO4. Filtration, evaporation in vacuo, and
purification by silica gel column chromatography gave 19 (7.02 mg,
12%): mp 86.5–88.0 °C; 1H NMR (CDCl3, 300 MHz, δ; ppm), 8.95 (2H,
m), 8.84 (2H, s), 7.91–7.84 (2H, m), 4.01 (3H, s), 3.76 (2H, t,
J = 6.0 Hz), 3.00 (2H, t, J = 4.5 Hz), 2.76 (2H, t, J = 7.5 Hz), 2.60
(2H, s), 1.34–1.27 (8H, m), 0.87 (3H, t, J = 6.9 Hz); 13C NMR (CDCl3,
75 MHz, δ; ppm): 165.8, 165.7, 156.6, 156.3, 150.2, 150.1, 142.3,
138.5, 123.1, 121.7, 120.6, 118.6, 57.63, 53.04, 52.68, 41.40, 36.27,
31.39, 29.68, 26.64, 22.43, 13.88; MS (ESI) m/z 399.3 (MH+).
5.1.3.5. Preparation of 2-((3-(hexyl(methyl)amino)propyl)carbamoyl)
isonicotinic acid (8). A mixture of 24 (65 mg, 0.19 mmol) and KOH
(25.1 mg, 0.38 mmol) in MeOH (3 mL) was heated at reflux temperature
for 1 h. The reaction mixture was concentrated in vacuo and purified by
5.1.2.7. Preparation of 4′-((2-(hexyl(methyl)amino)ethyl)carbamoyl)-
[2,2′-bipyridine]-4-carboxylic acid (7). A 6 N aqueous NaOH solution
silica gel column chromatography to give 8 (53 mg, 87%): mp
(7.00 µL, 0.420 mmol) was added to
a
solution of 19 (5.00 mg,
137–140 °C; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 9.01 (1H, t,
J = 5.4 Hz), 8.60 (1H, d, J = 4.8 Hz), 8.30 (1H, s), 7.65 (1H, dd,
J = 5.4, 1.8 Hz), 3.29 (3H, q, J = 5.4 Hz), 2.46 (2H, m), 2.41 (3H, t,
J = 7.5 Hz), 2.24 (3H, s), 1.77–1.68 (2H, m), 1.42 (2H, m), 1.21 (6H,
brs), 0.82 (3H, t, J = 6.9 Hz); 13C NMR (DMSO‑d6, 75 MHz, δ; ppm),
167.8, 165.3, 149.3, 142.0, 126.1, 121.5, 121.1, 57.41, 55.17, 42.13,
13.0 µmol) in MeOH (1 mL). The mixture was stirred at room
temperature overnight. The reaction mixture was concentrated in
vacuo and the residue was acidified with 4 N HCl in dioxane (1 mL).
Then, the insoluble materials were removed by filtration and the filtrate
was concentrated in vacuo to give 7 (3.00 mg, 60%): mp 135–137 °C;
1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 9.39 (1H, s), 8.88 (2H, q,
J = 5.1 Hz), 8.83 (2H, s), 7.99 (1H, d, J = 4.8 Hz), 7.91 (1H, d,
J = 4.8 Hz), 3.71 (2H, t, J = 4.8 Hz), 3.03 (2H, t, J = 7.5 Hz), 2.77
(3H, s), 1.67–1.65 (2H, m), 1.25–1.18 (8H, m), 0.83 (3H, t, J = 6.9 Hz);
13C NMR (DMSO‑d6, 75 MHz, δ; ppm), 165.5, 155.8, 155.7, 150.7,
150.6, 142.3, 138.5, 124.1, 122.4, 120.3, 118.9, 55.59, 54.15, 45.60,
38.19, 31.62, 26.95, 26.76, 26.70, 22.51, 14.35; LC-MS (ESI) m/z 321
(MH+); HRMS (ESI) calcd for C17H28N3O3
, 322.2125, found,
+
322.2123; HPLC tR = 8.93 min, purity 98.3% (HPLC conditions,
eluent A: H2O containing 0.1% TFA, eluent B: acetonitrile containing
0.1% TFA. Gradient, B: 0 to 20 min, 10–90%, 20 to 30 min, 90%).
31.18, 26.16, 23.60, 22.31, 14.24; MS (ESI) m/z 385.2 (MH+); HRMS
5.1.4. Synthesis of 2-(4-(3-(hexyl(methyl)amino)propyl)-2,3-dioxo-3,4-
dihydroquinoxalin-1(2H)-yl)acetic acid (9)
+
(ESI) calcd for
C
21H29N4O3
,
385.2234, found, 385.2237; HPLC
tR = 10.58 min, purity 96.6% (HPLC conditions, eluent A: H2O
containing 0.1% TFA, eluent B: acetonitrile containing 0.1% TFA.
Gradient, B: 0 to 20 min, 10–90%, 20 to 30 min, 90%).
5.1.4.1. Preparation of ethyl 2-(2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-
yl)acetate (25). 60% NaH in oil (88 mg, 2.2 mmol) was added to a
solution of quinoxaline-2,3-dione (324 mg, 2.0 mmol) in DMF (20 mL)
with cooling in an ice bath. Then, ethyl bromoacetate (0.21 mL,
1.9 mmol) was also added to the mixture and the resulting mixture
was stirred at room temperature for 2 h. The reaction was quenched
with water and extracted with AcOEt. The organic layer was washed
with brine and dried over Na2SO4. Filtration and evaporation in vacuo
gave a crude solid. The crude solid was suspended in CHCl3 (5 mL) and
the insoluble material was collected by filtration to give 25 (117 mg,
25%): 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 12.20 (1H, brs),
7.31–7.28 (1H, m), 7.23–7.14 (3H, m), 4.97 (2H, s), 4.16 (2H, q,
J = 7.2 Hz), 1.21 (3H, t, J = 7.2 Hz).
5.1.3. Synthesis
of
2-((3-(hexyl(methyl)amino)propyl)carbamoyl)
isonicotinic acid (8)
5.1.3.1. Preparation of 2-(3-(hexyl(methyl)amino)propyl)isoindoline-1,3-
dione (20). A mixture of N-(3-bromopropyl)phthalimide (268 mg,
1.0 mmol), K2CO3 (207 mg, 1.5 mmol), and N-hexylmethylamine
(0.17 mL, 1.1 mmol) in MeCN (5 mL) was heated at reflux
temperature for 1 h. Filtration, evaporation in vacuo, and purification
by flash column chromatography gave 20 (255 mg, 84%): 1H NMR
(DMSO‑d6, 300 MHz, δ; ppm), 7.88–7.79 (4H, m), 3.59 (2H, t,
J = 6.9 Hz), 2.29 (2H, t, J = 6.9 Hz), 2.19 (2H, t, J = 6.9 Hz), 2.05
(3H, s), 1.75–1.66 (2H, m), 1.28–1.19 (8H, m), 0.83 (3H, t, J = 6.9 Hz).
5.1.4.2. Preparation of ethyl 2-(4-(3-chloropropyl)-2,3-dioxo-3,4-
dihydroquinoxalin-1(2H)-yl)acetate (26). 60% NaH in oil (22 mg,
0.55 mmol) was added to a solution of 25 (115 mg, 0.46 mmol) in
DMF (5 mL) with cooling in an ice bath. Then, 1-bromo-3-
chloropropane (0.09 mL, 0.92 mmol) was added to the mixture and
the resulting mixture was stirred at room temperature for 2 h. The
reaction was quenched with water and extracted with AcOEt. The
organic layer was washed with brine and dried over Na2SO4. Filtration,
evaporation in vacuo, and purification by flash column
chromatography gave 26 (81 mg, 54%): 1H NMR (DMSO‑d6,
300 MHz, δ; ppm), 7.52 (1H, dd, J = 8.1, 1.2 Hz), 7.38–7.23 (3H, m),
5.00 (2H, s), 4.28 (2H, t, J = 7.5 Hz), 4.17 (2H, q, J = 7.2 Hz), 3.79
5.1.3.2. Preparation
of
N1-hexyl-N1-methylpropane-1,3-diamine
(21). Compound 21 was prepared from 20 by using
a similar
procedure to the one used for the preparation of 15. Yield, 107 mg,
75%; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 2.52 (2H, t, J = 7.2 Hz),
2.26 (2H, t, J = 7.2 Hz), 2.21 (2H, t, J = 7.2 Hz), 2.07 (3H, s),
1.48–1.34 (4H, m), 1.24 (6H, brs), 0.83 (3H, t, J = 6.6 Hz).
5.1.3.3. Preparation
of
dimethyl
pyridine-2,4-dicarboxylate
(22). Compound 22 was prepared from 2,4-pyridinedicarboxylic acid
by using a similar procedure to the one used for the preparation of 17.
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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(2H, t, J = 6.6 Hz), 2.15–2.04 (2H, m), 1.21 (3H, t, J = 7.2 Hz).
preparation of 20. Yield, 694 mg, 69%; 1H NMR (CDCl3, 300 MHz, δ;
ppm), 7.78 (2H, d, J = 7.8 Hz), 7.34 (2H, d, J = 7.8 Hz), 3.51 (2H, s),
2.20 (3H, s), 1.57–1.47 (2H, m), 1.36 (12H, s), 1.33–1.28 (6H, m), 1.28
(2H, t, J = 7.2 Hz), 0.90 (3H, t, J = 6.9 Hz).
5.1.4.3. Preparation of ethyl 2-(4-(3-(hexyl(methyl)amino)propyl)-2,3-
dioxo-3,4-dihydroquinoxalin-1(2H)-yl)acetate (27). Compound 27 was
prepared from 26 by using a similar procedure to the one used for the
preparation of 20. Yield, 83 mg, 82%; 1H NMR (DMSO‑d6, 300 MHz, δ;
ppm), 7.54 (1H, dd, J = 8.1, 1.5 Hz), 7.36 (1H, dd, J = 7.5, 1.8 Hz),
7.28 (2H, td, J = 7.5, 1.8 Hz), 5.00 (2H, s), 4.20–4.13 (4H, m),
3.30–3.26 (2H, m), 2.39 (2H, t, J = 7.5 Hz), 2.26 (3H, t, J = 7.5 Hz),
1.77 (2H, t, J = 7.5 Hz), 1.40 (2H, m), 1.25 (6H, m), 1.21 (3H, t,
J = 7.5 Hz), 0.85 (3H, t, J = 6.9 Hz).
5.1.5.4. Preparation of 5-(4-((hexyl(methyl)amino)methyl)phenyl)-6-
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
(10). A solution of 29 (118 mg, 0.50 mmol), 32a (182 mg, 0.55 mmol),
Na2CO3 (106 mg, 1.0 mmol), and PdCl2(dppf)·CH2Cl2 (37 mg,
0.050 mmol) in DME/H2O (3 mL/1 mL) was heated by MW at 160 °C
for 0.5 h. After the reaction mixture was filtered, the filtrate was
extracted with AcOEt and washed with brine. The organic layer was
separated and dried over Na2SO4. Filtration, evaporation in vacuo, and
purification by silica gel column chromatography gave a crude solid.
The crude solid was recrystallized in MeOH to give 10 (41 mg, 20%):
mp 228–230 °C; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.07 (1H, s),
7.53 (2H, d, J = 7.8 Hz), 7.44 (2H, d, J = 7.8 Hz), 4.23 (2H, brs), 2.98
(2H, brs), 2.69–2.65 (4H, m), 1.65 (2H, m), 1.28–1.23 (12H, m), 0.87
(3H, t, J = 6.6 Hz); 13C NMR (DMSO‑d6, 75 MHz, δ; ppm), 157.5, 148.5,
132.4, 131.3, 130.7, 128.9, 114.2, 111.2, 108.4, 98.15, 74.63, 57.12,
56.02, 31.21, 29.27, 26.98, 26.24, 25.41, 22.34, 20.98, 14.29; LC-MS
(ESI) m/z 405 (MH+); HRMS (ESI) calcd for C24H32N5O+, 406.2601,
found, 406.2605; HPLC tR = 13.10 min, purity 97.1% (HPLC
conditions, eluent A: H2O containing 0.1% TFA, eluent B: acetonitrile
containing 0.1% TFA. Gradient, B: 0 to 20 min, 10–90%, 20 to 30 min,
90%).
5.1.4.4. Preparation of 2-(4-(3-(hexyl(methyl)amino)propyl)-2,3-dioxo-
3,4-dihydroquinoxalin-1(2H)-yl)acetic acid hydrochloride (9·HCl). To a
solution of 27 (81 mg, 0.2 mmol) in THF/H2O (1.5 mL/1.5 mL) was
added NaOH (16 mg, 0.4 mmol) and the mixture was stirred at room
temperature for 2 h. Then, the reaction mixture was acidified with 1 N
aqueous HCl solution to adjust the pH to 3, and concentrated in vacuo.
The residue was purified by flash column chromatography to obtain a
solid. The solid was dissolved in 4 N HCl in AcOEt and concentrated in
vacuo. The residue was suspended in Et2O and the insoluble material
was collected by filtration to give 9 (56 mg, 75%): mp 110–112 °C; 1H
NMR (DMSO‑d6, 300 MHz, δ; ppm), 7.49–7.46 (1H, m), 7.29–7.20 (3H,
m), 4.46 (2H, s), 4.18–4.11 (2H, m), 2.57–2.56 (2H, m), 2.32 (3H, m),
2.17 (2H, s), 1.77 (2H, m), 1.42 (2H, s), 1.26 (6H, m), 0.85 (3H, m); 13
C
NMR (DMSO‑d6, 75 MHz, δ; ppm), 169.1, 154.1, 154.0, 127.2, 126.4,
124.6, 124.5, 115.9, 55.40, 52.77, 44.80, 31.10, 26.04, 23.59, 22.26,
21.98, 21.30, 14.23; LC-MS (ESI) m/z 375 (MH+-HCl); HRMS (ESI)
5.1.6. Synthesis
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (11)
5.1.6.1. Preparation of
of
5-(3-((hexyl(methyl)amino)methyl)phenyl)-6-
+
calcd for
C
20H30N3O4
,
376.2231, found, 376.2235; HPLC
tR = 10.6 min, purity 98.8% (HPLC conditions, eluent A: H2O
containing 0.1% TFA, eluent B: acetonitrile containing 0.1% TFA.
Gradient, B: 0 to 20 min, 10–90%, 20 to 30 min, 90%).
N-methyl-N-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)hexan-1-amine (32b). Compound 32b was
prepared from 2-(3-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane by using a similar procedure to the one used for the
preparation of 20. Yield, 542 mg, 82%; 1H NMR (DMSO‑d6, 300 MHz, δ;
ppm), 7.62 (1H, s), 7.54 (1H, d, J = 7.2 Hz), 7.40 (1H, d, J = 7.2 Hz),
7.32 (1H, t, J = 7.2 Hz), 3.43 (2H, s), 2.29 (2H, t, J = 7.2 Hz), 2.08 (3H,
s), 1.46–1.16 (8H, m), 1.28–1.23 (12H, m), 0.85 (3H, t, J = 6.9 Hz).
5.1.5. Synthesis
of
5-(4-((hexyl(methyl)amino)methyl)phenyl)-6-
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (10)
5.1.5.1. Preparation of 6-isopropyl-5,7-dioxo-4,5,6,7-tetrahydropyrazolo
[1,5-a]pyrimidine-3-carbonitrile
(28). A
solution
of
diethyl
isopropylmalonate (2.1 mL, 10 mmol), 3-amino-4-pyrazolecarbonitrile
(1.1 g, 10 mmol), and NaOEt (20% in EtOH, 10 mL) in EtOH (5 mL) was
heated by MW at 160 °C for 0.5 h. MeOH was added to the reaction
mixture and the reaction mixture was acidified with 4 N HCl in AcOEt
to adjust the pH to 1. The precipitate was collected by filtration and
washed with Et2O to give 28 (2.18 g, quant.): 1H NMR (DMSO‑d6,
300 MHz, δ; ppm), 8.21 (1H, s), 3.21 (1H, m), 1.21 (6H, d, J = 6.6 Hz).
5.1.6.2. Preparation of 5-(3-((hexyl(methyl)amino)methyl)phenyl)-6-
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
(11). Compound 11 was prepared from 29 and 32b by using a similar
procedure to the one used for the preparation of 10. Yield, 30 mg, 7%;
mp 272–274 °C; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.08 (1H, s),
7.53–7.41 (4H, m), 4.27 (2H, brs), 2.97 (2H, brs), 2.64 (3H, brs), 1.65
(2H, brs), 1.26–1.22 (13H, m), 0.86 (3H, t, J = 6.9 Hz); 13C NMR
(DMSO‑d6, 75 MHz, δ; ppm), 157.1, 144.6, 130.7, 130.6, 130.3, 129.8,
129.2, 128.7, 115.6, 111.6, 86.25, 81.41, 74.57, 59.36, 55.53, 31.16,
29.28, 26.23, 24.20, 22.31, 20.93, 14.25; LC-MS (ESI) m/z 405 (M+);
HRMS (ESI) calcd for C24H31N5O+, 406.2601, found, 406.2597; HPLC
tR = 13.1 min, purity 99.3% (HPLC conditions, eluent A: H2O
containing 0.1% TFA, eluent B: acetonitrile containing 0.1% TFA.
Gradient, B: 0 to 20 min, 10–90%, 20 to 30 min, 90%).
5.1.5.2. Preparation of 5-chloro-6-isopropyl-7-oxo-4,7-dihydropyrazolo
[1,5-a]pyrimidine-3-carbonitrile (29). A solution of 28 (1.09 g,
5.0 mmol) in POCl3 (20 mL) was heated by MW at 170 °C for 2 h.
Then, the reaction was quenched with water and extracted with AcOEt.
The organic layer was separated and dried over Na2SO4, Filtration,
evaporation in vacuo, and purification by silica gel column
chromatography gave the dichloro intermediate (335 mg, 26%). A
solution of the intermediate (335 mg, 1.31 mmol) and 1 N aqueous
NaOH solution (2.6 mL, 2.6 mmol) in THF (2.6 mL) was heated at reflux
temperature for 0.75 h. Then, the reaction was quenched with 4 N HCl
in AcOEt to adjust the pH to 1 and extracted with AcOEt. The organic
layer was separated and dried over Na2SO4, and this was followed by
filtration and evaporation in vacuo. The residue was collected by
filtration and washed with n-hexane/AcOEt (1/9) to give 29 (224 mg,
72%): 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.20 (1H, s), 3.27 (1H,
m), 1.28 (6H, d, J = 6.9 Hz).
5.1.7. Synthesis of 5-(5-((hexyl(methyl)amino)methyl)thiophen-2-yl)-6-
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (12)
5.1.7.1. Preparation
of
N-((4-bromothiophen-2-yl)methyl)-N-
methylhexan-1-amine (31a). A solution of 4-bromothiophene-2-
carboxyaldehyde (1.15 g, 6.0 mmol), N-hexylmethylamine (0.76 mL,
5.0 mmol), and NaBH(OAc)3 (1.86 g, 7.0 mmol) in 1,2-DCE (30 mL)
was stirred at room temperature for 5 h. The reaction was quenched
with water and extracted with CHCl3. The organic layer was separated,
washed with brine, and dried over Na2SO4. Filtration, evaporation in
vacuo, and purification by silica gel column chromatography gave 31a
(0.92 g, 53%): 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 7.54 (1H, d,
J = 1.5 Hz), 6.96 (1H, d, J = 1.5 Hz), 3.64 (2H, s), 2.32 (2H, d,
J = 6.9 Hz), 2.16 (3H, s), 1.45–1.38 (2H, m), 1.31–1.24 (6H, m), 0.86
5.1.5.3. Preparation
of
N-methyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)hexan-1-amine (32a). Compound 32a was
prepared from 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane by using a similar procedure to the one used for the
9

Y. Miyake et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(3H, t, J = 6.9 Hz).
purity 99.1% (HPLC conditions, eluent A: H2O containing 0.1% TFA,
eluent B: acetonitrile containing 0.1% TFA. Gradient, B: 0 to 20 min,
10–90%, 20 to 30 min, 90%).
5.1.7.2. Preparation
of
N-((4-bromothiophen-2-yl)methyl)-N-
methylhexan-1-amine (32c). A mixture of 31a (871 mg, 3.0 mmol), bis
(pinacolate)diboron (1.1 g, 4.5 mmol), AcOK (441 mg, 4.5 mmol), and
PdCl2(dppf)·CH2Cl2 (112 mg, 0.15 mmol) in dioxane (9 mL) was heated
by MW at 140 °C for 0.5 h. The reaction mixture was filtered through
Celite and the solvent was removed under reduced pressure. The
residue was purified by silica gel column chromatography to give
crude 32c (266 mg), which was used in the next step without further
purification.
5.2. Molecular docking
Docking study was performed using Molegro Virtual Docker 6.0
software. The structure of 13 bound to KDM5A (PDB code: 5CEH) was
constructed by MolDock, which is based on a heuristic search algorithm
that combines differential evolution with a cavity prediction algorithm.
The docking parameters were as follows: grid resolution: 0.30, max
iterations: 1500, population size: 50, energy threshold: 100.00, simplex
evolution: 300 (max steps) and 1.00 (neighbor distance factor), search
space: (X, Y, Z) = (130.00, 136.00, 110.00) with radius 15, distance
constraints: an N atom of cyano group of 13 as an Fe(II) chelator,
constraint center (X, Y, Z) = (131.49, 368.84, 109.93) with hard
constraint between minimum 0.0 to maximum 0.5.
5.1.7.3. Preparation of 5-(5-((hexyl(methyl)amino)methyl)thiophen-3-yl)-
6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
(12). Compound 12 was prepared from 29 and 32c by using a similar
procedure to the one used for the preparation of 10. Yield, 65 mg, 20%;
mp 173–175 °C; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 8.08 (1H, s),
7.56–7.41 (2H, m), 4.27 (2H, brs), 2.98 (2H, brs), 2.65 (3H, s), 1.65
(2H, brs), 1.26–1.22 (13H, m), 0.86 (3H, t, J = 6.9 Hz); 13C NMR
(DMSO‑d6, 75 MHz, δ; ppm), 159.7, 156.9, 144.8, 141.1, 138.2, 129.6,
126.7, 123.8, 116.2, 112.3, 74.60, 55.51, 54.07, 31.27, 29.08, 26.38,
24.95, 22.39, 21.02, 14.29; MS (ESI) m/z 411 (MH+); HRMS (ESI) calcd
for C22H30N5OS+, 412.2166, found, 412.2161; HPLC tR = 12.9 min,
purity 95.7% (HPLC conditions, eluent A: H2O containing 0.1% TFA,
eluent B: acetonitrile containing 0.1% TFA. Gradient, B: 0 to 20 min,
10–90%, 20 to 30 min, 90%).
5.3. Enzyme assay
NCDM-81a (1a) and CPI-455 (3) were prepared according to the
procedures reported by Itoh et al.44 and Vinogradova et al.,45 respec-
tively. KDM activity assays were performed by an AlphaLISA screen
assay system. All components are summarized in Supplementary Table
S1. Initially, 2.5 μL of assay buffer (50 mM HEPES pH 7.5, 0.1% w/v
BSA, 0.01% v/v Tween 20 containing 3% DMSO as control or blank) or
100X concentrated inhibitor solution (in assay buffer containing 3%
DMSO) was introduced into the corresponding wells of a white opaque
OptiPlateTM-384. Then, 5.0 μL of enzyme solution was added to each
well (For blank, 5.0 μL of assay buffer was added instead.). After that,
2.5 μL of peptide/2-OG (50 µM)/Fe(II) (5 µM)/Asc (100 µM) mix in
assay buffer was added to each well. The final concentrations of enzyme
and peptide substrate are summarized in Supplementary Table S2. The
mixture was incubated for 1 h at room temperature with gentle shaking
(250 rpm) (Protein A acceptor beads were pre-incubated with anti-
methyl mark antibody for 1 h prior to addition to the assay plate.).
Then, 5.0 μL of acceptor beads in epigenetic buffer (100 µg/mL) was
added to each well and incubation was carried out for 1 h at room
temperature with gentle shaking (250 rpm). Ten μL of donor beads in
epigenetic buffer (50 µg/mL) was added to each well and incubation
was carried out for 30 min at room temperature in the dark. Then, the
5.1.8. Synthesis
of
5-(4-(2-(hexyl(methyl)amino)ethyl)phenyl)-6-
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile (13)
5.1.8.1. Preparation of 2-(4-bromophenyl)acetaldehyde (30). A solution
of 2-(4-bromophenyl)ethyl alcohol (2.00 g, 10.0 mmol) and Dess-
Martin periodinane (4.20 g, 12.0 mmol) in CH2Cl2 (100 mL) was
stirred at room temperature for 1 h. The reaction was quenched with
1 M aqueous Na2S2O3 solution and extracted with CH2Cl2. The organic
layer was separated, washed with brine, and dried over Na2SO4,
Filtration, evaporation in vacuo, and purification by silica gel column
chromatography gave 30 (1.23 g, 62%): 1H NMR (DMSO‑d6, 300 MHz,
δ; ppm), 9.68 (1H, t, J = 1.5 Hz), 7.56–7.52 (2H, m), 7.22–7.18 (2H,
m), 3.79 (2H, d, J = 1.5 Hz).
5.1.8.2. Preparation of N-(4-bromophenethyl)-N-methylhexan-1-amine
(31b). Compound 31b was prepared from 30 by using a similar
procedure to the one used for the preparation of 31a. Yield, 1.46 g,
79%; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 7.48–7.44 (2H, m),
7.22–7.18 (2H, m), 2.72–2.67 (4H, m), 2.45 (2H, m), 2.29 (3H, s),
1.42–1.38 (2H, m), 1.26–1.15 (6H, m), 0.85 (3H, t, J = 6.9 Hz).
®
Alpha signal of the wells was measured by an Ensight reader from
PerkinElmer Ltd. with excitation set at 615 nm and emission detection,
at 655 nm, and % inhibition was calculated from the Alpha signal
readings of inhibited wells relative to those of control wells. The con-
centration of a compound that results in 50% inhibitory concentration
(IC50) was determined by plotting log[Inh] versus the logit function of
% inhibition. IC50 values were determined by regression analysis of the
concentration/inhibition data. For determination of IC50 values, at least
five concentrations of inhibitors were used.
5.1.8.3. Preparation
of
N-methyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenethyl)hexan-1-amine (32d). Compound 32d was
prepared from 31b by using a similar procedure to the one used for the
preparation of 32c. Yield, 0.88 g, 85%; 1H NMR (DMSO‑d6, 300 MHz, δ;
ppm), 7.57 (2H, d, J = 8.1 Hz), 7.22 (2H, d, J = 8.1 Hz), 2.70 (2H, t,
J = 7.2 Hz), 2.30 (2H, t, J = 7.2 Hz), 2.18 (3H, s), 1.37–1.15 (16H, m),
1.07 (6H, s), 0.85 (3H, t, J = 6.9 Hz).
5.4. Cellular assay
5.4.1. Cell cultures
Human lung cancer A549 cells (provided by RIKEN BRC through the
National Bio-Resource Project of MEXT, Japan) were cultured in
RPMI1640 containing 10% heat-inactivated fetal bovine serum (FBS),
penicillin, and streptomycin mixture at 37 °C in a humidified atmo-
sphere of 5% CO2 in air. Human breast cancer MDA-MB-231 cells
(provided by American type culture collection, ATCC, USA) were cul-
tured in Leibovitz's L-15 medium containing 2 mM of glutamine, 10%
FBS, and a penicillin and streptomycin mixture at 37 °C.
5.1.8.4. Preparation of 5-(4-(2-(hexyl(methyl)amino)ethyl)phenyl)-6-
isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile
(13). Compound 13 was prepared from 29 and 32d by using a similar
procedure to the one used for the preparation of 10. Yield, 77 mg, 18%;
mp 184–186 °C; 1H NMR (DMSO‑d6, 300 MHz, δ; ppm), 7.34–7.29 (4H,
m), 3.34 (2H, m), 3.05–2.99 (4H, m), 2.75–2.68 (4H, s), 1.64 (2H, m),
1.29–1.23 (12H, m), 0.87 (3H, t, J = 6.9 Hz); 13C NMR (DMSO‑d6,
75 MHz, δ; ppm), 158.6, 144.4, 128.8, 128.7, 128.6, 128.5, 114.4,
111.2, 107.0, 104.0, 74.60, 60.96, 56.96, 55.86, 31.22, 29.27, 26.23,
24.16, 22.36, 21.02, 14.30; MS (ESI) m/z 419 (MH+); HRMS (ESI) calcd
for C25H34N5O+, 420.2758, found, 420.2755; HPLC tR = 13.61 min,
5.4.2. Western blot analysis
A549 and MDA-MB-231 cells (5 × 105 cells/2 mL/well) were
treated for 48 h with the test compounds at the indicated concentrations
10

Y. Miyake et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
in the culture medium. The cells were collected and extracted with SDS
buffer. Protein concentrations of the lysates were determined using a
BCA protein assay. The equivalent amounts of protein from each lysate
were resolved in 5–20% SDS-polyacrylamide gels and transferred onto
PVDF membranes. After blocking with TBS-T containing 5% skimmed
milk, the transblotted membranes were probed with primary antibodies
(See Supplementary Table S3). The probed membranes were washed
three times with TBS-T, incubated with HRP-linked secondary antibody
(See Supplementary Table S3), and again washed three times with TBS-
T. The immunoblots were visualized by enhanced chemiluminescence
with chemiluminescent HRP substrate (See Supplementary Table S3).
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Acknowledgments
The authors would like to thank Prof. K. Akaji and Dr. Y. Hattori for
technical support in HRMS analysis. This work was supported by the
JST CREST program (JPMJCR14L2 to T. Suzuki).
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