Structure-based design and discovery of potent and selective KDM5 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.03.083

Histone lysine demethylases (KDMs) play a key role in epigenetic regulation and KDM5A and KDM5B have been identified as potential anti-cancer drug targets. Using structural information from known KDM4 and KDM5 inhibitors, a potent series of pyrazolylpyridines was designed. Structure-activity relationship (SAR) exploration resulted in the identification of compound 33, an orally available, potent inhibitor of KDM5A/5B with promising selectivity. Potent cellular inhibition as measured by levels of tri-methylated H3K4 was demonstrated with compound 33 in the breast cancer cell line ZR-75-1.

Full text of DOI:10.1016/j.bmcl.2018.03.083

Accepted Manuscript
Structure-based design and discovery of potent and selective KDM5 inhibitors
Zhe Nie, Lihong Shi, Chon Lai, Shawn M. O'Connell, Jiangchun Xu, Ryan K.
Stansfield, David J. Hosfield, James M. Veal, Jeffrey A. Stafford
PII:
S0960-894X(18)30288-9
https://doi.org/10.1016/j.bmcl.2018.03.083
BMCL 25737
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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14 February 2018
27 March 2018
29 March 2018
Please cite this article as: Nie, Z., Shi, L., Lai, C., O'Connell, S.M., Xu, J., Stansfield, R.K., Hosfield, D.J., Veal,
J.M., Stafford, J.A., Structure-based design and discovery of potent and selective KDM5 inhibitors, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.083
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Bioorganic & Medicinal Chemistry Letters
Structure-based design and discovery of potent and selective KDM5 inhibitors
Zhe Nie,a,* Lihong Shi,a Chon Lai,a Shawn M. O'Connell,a,c Jiangchun Xu,a Ryan K. Stansfield,a,d David J.
Hosfield,b James M. Veala,d and Jeffrey A. Stafforda,d
aCelgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, CA 92121
bBen May Department for Cancer Research, University of Chicago, 929 East 57th Street, Chicago, IL 60637
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Histone lysine demethylases (KDMs) play a key role in epigenetic regulation and KDM5A and
KDM5B have been identified as potential anti-cancer drug targets. Using structural information
from known KDM4 and KDM5 inhibitors, a potent series of pyrazolylpyridines was designed.
Structure-activity relationship (SAR) exploration resulted in the identification of compound 33,
an orally available, potent inhibitor of KDM5A/5B with promising selectivity. Potent cellular
inhibition as measured by levels of tri-methylated H3K4 was demonstrated with compound 33 in
the breast cancer cell line ZR-75-1.
Keywords:
Epigenetics
Histone lysine demethylase
KDM5
2018 Elsevier Ltd. All rights reserved.
Tri-methylated H3K4
Structure-based design
Aberrant expression of histone-modifying enzymes results in
epigenetic alterations and has been implicated in tumorigenesis.1
Reversible methylation and demethylation of histone lysine
residues are facilitated by two enzyme families, histone lysine
methyltransferases (KMTs) and histone lysine demethylases
(KDMs), that have recently emerged as promising drug targets
for cancer treatment.2,3
some contain groups that could be strong metal chelators.
Recently, researchers have identified potent and selective KDM5
inhibitors using high-throughput screening and structure-based
drug design.12-15 Our goal was to identify potent, selective and
cell-permeable KDM5 inhibitors to use as tool compounds to
interrogate the disease functions of the catalytic activities of
KDM5 enzymes.
The KDM5 family of histone demethylases are Fe(II)-
dependent enzymes which utilize 2-oxoglutarate (2-OG) as a co-
substrate and remove a methyl group from the activating marks
of H3K4me3 and H3K4me2.4 Overexpression of KDM5A/5B
(also known as JARID1A/1B) has been reported in a variety of
malignant tumors.5 Previous studies have demonstrated that loss
of the retinoblastoma binding protein
2 (RBP2) histone
demethylase (aka KDM5A or JARID1A) suppresses
tumorigenesis in mice lacking Rb1 or Men1, and the authors of
the study concluded that RBP2-inhibitory drugs would have anti-
cancer activity.6 The KDM5 family is also implicated in the
Scheme 1. Design of initial pyrazolylpyridine core
4'-((2-Aminoethyl)carbamoyl)-[2,2'-bipyridine]-4-carboxylic
acid (compound 1) is a sub µM 2-OG competitive inhibitor of
KDM4E.16 The co-crystal structure of 1 in complex with
KDM4A (PDB code 3PDQ) reveals that both pyridinyl nitrogens
of 1 form key interactions with the active site metal through a
bidentate chelation. As shown in Scheme 1, we designed a
pyrazolylpyridine core, where the pyrazole ring was used as a
less basic replacement for pyridine in order to mitigate its metal
chelation strength and the possible toxicities associated with
strong metal chelation. By modelling, the pendant phenyl
substitution off the pyrazole ring would extend into peptide
binding region lined by residues I71, Q73, N86, Y132, A134,
D135, Y177, A186 and K241 (KDM4A residues). Despite high
sequence homology between KDM4A and KDM5B, there are
survival of drug-tolerant persister cancer cells (DTPs).7-9
A
KDM5 specific inhibitor, CPI-455, reduces survival of DTPs,
indicating the potential of targeting KDM5 to overcome
treatment-induced resistance.10 Furthermore, in-house gene
knockdown of KDM5A/5B in cancer-derived organoids inhibits
colony formation (data not shown), suggesting KDM5A/5B
inhibitors may have efficacy in the treatment of cancer.
Early inhibitors of the Jumonji family KDMs, such as 2,4-
pyridine dicarboxylic acid (2,4-PDA), 2,2-bipyridines and
hydroxyquinolines, are mostly 2-OG analogs.11 These compounds
in general are non-selective, have poor cell permeability, and

three key amino acid differences in this region: I71 to R86, A186
to C497 and K241 to L552, providing a rationale that further
substitution on phenyl ring could provide selectivity between the
KDM5 and KDM4 families.
proximal to Y177 and is observed to have two conformers in a
tetrameric crystal. In 3 molecules of the asymmetric unit, the
inhibitor phenol is directed away from D135 and is H-bonded to
a structural water. In the fourth unit, the phenol forms a direct
H-bond to the side chain of D135. Compound 9, with both 4-Me
and 3-OH groups, is the most potent and selective inhibitor in the
series. Further substitutions on the phenyl ring does not make
significant improvement on inhibiting KDM5B enzymatic
activity.
Table 1. SAR of representative compounds with phenyl
substitutions
KDM5B
IC50 (µM)
KDM4C
IC50 (µM)
0.49
Compd
R
2
4-F
0.24
3
4
5
6
7
8
3-F
4-H
4-Me
4-SO2Me
4-OH
3-OH
0.50
4.4
0.2
0.66
6.2
1.1
2.7
0.35
0.32
0.12
0.45
0.41
Scheme 2. Design of benzyloxypyrazolylpyridine core
1.1
9
3-OH, 4-Me
3-OMe, 4-Me
3-OMe, 4-Cl
2,4-diF
0.076
0.54
0.23
0.17
0.37
Analysis of the co-crystal structure of KDM4A with a potent
inhibitor from a different series (compound 14, PDB code 6CG1,
Figure 2 in Supplemental Data,)17 reveals that the benzyl group
of 14 extends further into the peptide binding pocket and forms
productive -stacking interaction with Y132 and a hydrophobic
1.8
10
11
12
13
2.9
0.38
0.56
3,4-diF
interaction with I71. As shown in Scheme 2,
a
benzyloxypyrazolylpyridine core was thus designed so that the
benzyl ether group (Pink) could overlap with the benzylamine of
compound 14 (Blue) as shown in Figure 2.
Figure 1. Co-crystal structure of compound 8 in complex with
KDM4A (PDB code: 6CG2)-major conformer.
Our initial effort toward discovery of potent and selective
KDM5 inhibitors focused on various substitutions on the phenyl
ring off the pyrazolylpyridine core as shown in Table 1. Small
substitutions on 2-, 3-, and 4-positions improve KDM5B
enzymatic inhibition, with 4-Me substitution giving the best
potency and selectivity over KDM4C. The 3-OH analog is more
potent than both 4-OH and 3-OMe, which indicates that it might
be involved in a polar interaction with water or protein residues
nearby. Due to difficulties in obtaining a KDM5B co-crystal
structure at the time, KDM4A was used as a surrogate. To
confirm the binding mode, a co-crystal structure of compound 8
in complex with KDM4A was solved to 2.34 Å. As shown in
Figure 1 (electron density of refined active site structure is
shown in Figure 1, Supplemental Data), the metal Ni2+ ion (Fe
surrogate) is coordinated to both nitrogens on the pyridyl and
pyrazolyl rings through a bidentate chelation. The pyridyl ring -
stacks with F185 and the 4-carboxyl group is anchored through
its interactions with K206 and Y132. The phenol moiety is
Figure 2. Model of compound 15 overlaid with compound 14 in
KDM4A active site.
Compound 15 demonstrated improved enzymatic inhibition
against KDM5B by at least 5 fold compared to compound 8. SAR
of pyrazolylpyridine analogs with various substitutions on the
benzyloxy ring was carried out. These inhibitors were all 2-OG
competitive inhibitors, and the IC50 of tested compounds were not
affected by the presence of different concentrations of Fe(II)
(data not shown). As shown in Table 2, the 4-F compound 16
was a potent inhibitor for KDM5A and KDM5B, whereas 3-F
compound 17 and 2-F compound 18 were only weak inhibitors of
KDM5A, which shows that a single substitution change can
result in dramatic change in enzymatic inhibitions. Compounds
with electron-withdrawing groups (20, 21 and 22) or small alkyl
groups (23 and 24) at para-position showed stronger inhibitory
activities against KDM5A, whereas compounds bearing para
electron donating group (19) and large alkyl groups (25 and 26)

were weaker KDM5A inhibitors. Interestingly, KDM5B
enzymatic inhibition was less sensitive to these substitutions.
Compound 19, with an electron donating 4-substitution showed
selectivity for inhibition of KDM5B over KDM5A. Cellular
inhibition of KDM5 demethylases was determined using an
immuno-blotting assay measuring tri-methylated H3K4 levels in
the breast cancer cell line ZR-75-1.18 Although the data is limited,
compounds with strong enzymatic inhibition against both
KDM5A and KDM5B demonstrated better cellular efficacy (16
vs. 15 and 17).
Compd
R
KDM5Ab KDM5Bb KDM4Cb
IC50 (µM) IC50 (µM) IC50 (µM)
ZR-75-1
H3K4me3
EC50 (µM)
0.42
1.3
0.09
0.11
1.0
--
0.8
21
32
33
34
35
36
37
H
F
0.014
0.018
0.013
0.012
0.019
0.004
0.004
0.002
0.001
0.004
0.006
0.004
0.054
--
0.041
0.038
0.078
0.28
Table 2. Benzyloxypyrazolopyridine initial SAR
Me
OMe
OEt
OCH2cPr 0.075
OBn 0.032
0.074
bValues derived from biochemical assays with pre-incubation
Compd
R
KDM5Ab KDM5Bb KDM4Cb
IC50 (µM) IC50 (µM) IC50 (µM)
ZR-75-1
H3K4me3
Table 5. Selectivity data of compound 33 for other KDM family
members
KDM
EC50 (µM)
7
0.84
12
--
--
0.94
0.42
0.86
0.99
1.3
Compd 33
IC50 (µM)
>40
KDM
Compd 33
IC50 (µM)
0.035b
0.11
15
16
17
18
19
20
21
22
23
24
25
26
H
0.55
0.038
0.43
0.27
0.001
0.004
0.022
0.042
0.004
0.0006
0.004
0.002
0.004
0.003
0.008
--
0.005
0.024
--
4-F
3-F
2-F
4-OMe 1.7
4-CN
4-Cl
4-Br
4-Me
4-Et
4-cPr
4-CF3
KDM1A
KDM2A
KDM2B
KDM3A
KDM4A
KDM4B
KDM4C
KDM4D
KDM4E
KDM5A
KDM5B
KDM6B
0.82
0.63
2.3
0.099
0.17
--
0.16
0.068
0.014
0.054
--
1.1
3.6
0.013b
0.002b
2.3b
0.004
0.014
0.006
0.032
0.044
0.25
bValues derived from biochemical assays with pre-incubation
--
--
--
--
0.24
bValues derived from biochemical assays with pre-incubation
Further SAR exploration of the 4-fluoro and 4-chloro
benzyloxy pyrazolylpyridine cores as shown in Table 3 and 4
resulted in the identification of compound 33, a potent and
selective inhibitor of KDM5A/5B. It showed strong cellular
inhibition of KDM5A/5B which was indicated by induction of
H3K4me3 up to 10 fold in the ZR-75-1 breast cancer cell line
with EC50 below 0.1 µM. Compound 33 was highly selective
against other KDM family members, with the exception of
KDM4 family members (Table 5). The co-crystal structure of 33
bound to KDM5A was recently solved by an academic group at
Yale (PDB code 5IWO, KDM5-N19)19 which confirmed the
proposed binding mode and is useful to guide future discovery of
KDM5A/5B inhibitors.
Scheme 3. Reagents and conditions: (a) NH2NH2·H2O, 1-butanol/THF, 60-
100 oC; (b) Ethyl (2E)-3-(dimethylamino)prop-2-enoate, AcOH, EtOH, 90 oC;
(c) t-BuOK, EtOH, r.t.; (d) ROH, PPh3, DIAD, THF, r.t.; (e) NaOH, EtOH, 90
oC.
A general method18 to prepare benzyloxypyrazolylpyridines is
provided in Scheme 3. Chloroisonicotinonitrile (38) is treated
with hydrazine hydrate in a mixture of 1-butanol and THF at
elevated temperature (60-100 °C) to give intermediate 39, which
reacts with ethyl (2E)-3-(dimethylamino)prop-2-enoate in ethanol
in the presence of acetic acid to give intermediate 40. Upon
treatment with a strong base, such as potassium t-butoxide, the
hydroxypyrazole 41 is obtained. Via a Mitsunobu reaction, a
mixture of O-alkylation and N-alkylation products are obtained,
which could be separated by flash column chromatography to
give the desired O-alkylation product 42, which is then
hydrolyzed to the acid final product.
Table 3. 4-Fluorobenzyloxypyrazolopyridine SAR
Compd R1, R2
KDM5Ab KDM5Bb KDM4Cb ZR-75-1
IC50 (µM) IC50 (µM) IC50 (µM) H3K4me3
EC50 (µM)
The pharmacokinetic parameters of compound 33 were
determined in female CD-1 mice. After intravenous dose
administration (5 mg/kg), compound 33 displayed a low systemic
clearance of 2.1 mL/min/kg and a low volume of distribution of
0.16 L/kg. Compound 33 was readily absorbed after oral
administration (10 mg/kg) with an AUC of 44,000 ng/mL*hr and
an oral bioavailability of 55%. It has high plasma protein binding
(99.1%) in mouse, which is typical for a compound bearing a
carboxylic acid group. It has no issues in CYP, hERG, MDR1,
and it has good kinetic solubility.
27
28
29
30
31
R1=Me
R2=Me
R2=Et
0.18
0.077
0.029
0.041
0.054
0.024
0.001
0.068
0.31
2.9
0.26
0.019
>2.0
--
--
--
4.6
--
R2=CH2CH2NMe2 0.003
R2=CH2CH2OH 0.08
bValues derived from biochemical assays with pre-incubation
Table 4. 4-Chlorobenzyloxypyrazolopyridine SAR

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mg/kg induced H3K4me3 increases (Figure 3). The induction of
H3K4me3 level was over 2 fold at the 0.5 hour time point,
reaching a maximum level of over 4 fold of the vehicle control at
the 1 hour time point where the concentration of compound 33
also reached the highest level. The induction level of H3K4me3
was sustained until the 24 hour time point even though a very low
concentration of compound 33 was detected.
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In summary, we have identified
a novel series of
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crystal structure of compound 14 provided further guidance in
designing the benzyloxy pyrazolylpyridine series. Balancing
potency and physical properties resulted in the identification of
compound 33, an orally available, potent inhibitor of KDM5A/5B
with promising selectivity. Treatment with compound 33 caused
H3K4me3 increases both in vitro in a breast cancer cell line, ZR-
75-1, and in vivo in a MCF-7 breast cancer xenograft PK/PD
model.
*Corresponding author. Tel: +1-858-678-4010; e-mail:
znie@celgene.com
cPresent address: Pfizer Inc.
dPresent address: Jecure Therapeutics.
Acknowledgments
The authors would like to acknowledge the contributions of
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the syntheses of some of the inhibitors; Dongming Qian and team
at Viva Biotech for co-crystallization of inhibitors with KDM4A
and diffraction data collection. The author would also like to
thank Michael B. Wallace for reviewing and providing critical
comments for the manuscript.
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D.J.; Johns, M.A.; Fu, H.; Jadhav, A.; Vertino, P.M.; Yan, Q.; Cheng,

 KDM5A/5B have been identified as potential anti-cancer drug targets.
 Compound 33 is an orally available, potent inhibitor of KDM5A/5B with promising selectivity
 Compound 33 promotes H3K4me3 increases both in vitro and in vivo
 Compound 33 is a valuable tool compound to interrogate the biology of KDM5A/5B

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Structure-based design and discovery of
potent and selective KDM5 inhibitors
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Zhe Nie*, Lihong Shi, Chon Lai, Shawn M. O'Connell, Jiangchun Xu, Ryan K. Stansfield, David J. Hosfield,
James M. Veal and Jeffrey A. Stafford
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