C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4)inhibitors, compound profiling in cell-based target engagement assays

Article abstract of DOI:10.1016/j.ejmech.2019.05.041

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between

Full text of DOI:10.1016/j.ejmech.2019.05.041

Accepted Manuscript
C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification
of potent, cell penetrant Jumonji C domain containing histone lysine demethylase
4
subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement
assays
Yann-Vaï Le Bihan, Rachel M. Lanigan, Butrus Atrash, Mark G. McLaughlin,
Srikannathasan Velupillai, Andrew G. Malcolm, Katherine S. England, Gian Filippo
Ruda, N. Yi Mok, Anthony Tumber, Kathy Tomlin, Harry Saville, Erald Shehu,
Craig McAndrew, LeAnne Carmichael, James M. Bennett, Fiona Jeganathan, Paul
Eve, Adam Donovan, Angela Hayes, Francesca Wood, Florence I. Raynaud, Oleg
Fedorov, Paul E. Brennan, Rosemary Burke, RobL.M. van Montfort, Olivia W.
Rossanese, Julian Blagg, Vassilios Bavetsias
PII:
S0223-5234(19)30451-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.041
EJMECH 11351
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 January 2019
Revised Date: 14 May 2019
Accepted Date: 14 May 2019
Please cite this article as: Yann.-Vaï. Le Bihan, R.M. Lanigan, B. Atrash, M.G. McLaughlin, S. Velupillai,
A.G. Malcolm, K.S. England, G.F. Ruda, N.Y. Mok, A. Tumber, K. Tomlin, H. Saville, E. Shehu, C.
McAndrew, L. Carmichael, J.M. Bennett, F. Jeganathan, P. Eve, A. Donovan, A. Hayes, F. Wood,
F.I. Raynaud, O. Fedorov, P.E. Brennan, R. Burke, R.M. van Montfort, O.W. Rossanese, J. Blagg, V.
Bavetsias, C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent,
cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors,
compound profiling in cell-based target engagement assays, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.041.
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ACCEPTED MANUSCRIPT
H276
H188
K206
HN
N
Y132
E190
N
O
D311
N
N
N
V313
1
9a
Y175
KDM4A Ki = 0.004 µM, EC = 4.7 µM
5
0
KDM5B Ki = 0.007 µM, EC = 13.4 µM
5
0

ACCEPTED MANUSCRIPT
C8-Substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of
potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4
subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement
assays
1
,┴
1,┴
1
1
Yann-Vaï Le Bihan,
Rachel M. Lanigan,
Butrus Atrash, Mark G. McLaughlin,
2
1
2,3
Srikannathasan Velupillai, Andrew G. Malcolm, Katherine S. England, Gian Filippo
2
1
2,3
1
1
1
Ruda, N. Yi Mok, Anthony Tumber, Kathy Tomlin, Harry Saville, Erald Shehu, Craig
1
1
2,3
1
1
McAndrew, LeAnne Carmichael, James M. Bennett, Fiona Jeganathan, Paul Eve, Adam
1
1
1
1
2,3
Donovan, Angela Hayes, Francesca Wood, Florence I. Raynaud, Oleg Fedorov, Paul E.
2
,3
1
1
1
Brennan, Rosemary Burke, Rob L. M. van Montfort, Olivia W. Rossanese, Julian
,
1
,1
Blagg,* Vassilios Bavetsias*
*
corresponding author
┴
authors contributed equally
1
2
Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London.
Structural Genomics Consortium (SGC), University of Oxford, ORCRB Roosevelt Drive,
Oxford OX3 7DQ, UK.
3
Target Discovery Institute (TDI), Nuffield Department of Medicine, University of Oxford,
NDMRB, Roosevelt Drive, Oxford OX3 7FZ, UK.
1

ACCEPTED MANUSCRIPT
Abstract
Residues in the histone substrate binding sites that differ between the KDM4 and KDM5
subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-
one series was designed to rationally exploit these residue differences between the histone
substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-
subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were
targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-
substituent was investigated. These approaches yielded potent and cell-penetrant dual
KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007
µM, respectively). Compound cellular profiling in two orthogonal target engagement assays
revealed a significant reduction from biochemical to cell-based activity across multiple
analogues; this decrease was shown to be consistent with 2OG competition, and suggest that
sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-
d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
Keywords:
pyridopyrimidinones, KDM4 subfamily, KDM5 subfamily, KDM inhibitors, histone
demethylases
2

ACCEPTED MANUSCRIPT
1
. Introduction
Human Jumonji C-domain (JmjC) histone lysine demethylases (KDMs) are Fe(II) and 2-
oxoglutarate (2OG)-dependent oxygenases that demethylate mono-, di-, and trimethyl histone
lysine substrates [[1], [2]]. They comprise five subfamilies varying in domain architecture but
sharing a well conserved catalytic site [3]. Since the reporting of the first JmjC histone lysine
demethylase in 2006 [[4], [5]], the JmjC KDMs have emerged as important players in
maintaining chromatin architecture and regulating transcription [6].
The KDM4 subfamily consists of six members (A-F) that demethylate histone tail substrates,
most notably di-, and tri-methylated lysine 9 (H3K9Me /Me ) and lysine 36
3
2
(
H3K36Me /Me ) on histone 3 [[6], [7]]. As well as the JmjC catalytic domain, KDM4
3 2
subfamily enzymes contain a JmjN domain [6]; KDM4A-C also possess PHD and Tudor
methyl-lysine reader domains. It has been suggested that PHD and Tudor domains could
stimulate KDM-driven demethylation via the recognition of histone substrates [[8], [9]].
KDM4 subfamily members have been implicated in a number of human cancers, including
promoting growth and cell survival in acute myeloid leukemia [10] and as regulators of ER-
dependent breast cancer cell growth [[11], [12]]. High expression of KDM4B in MYCN-
amplified neuroblastomas is associated with poor prognosis [13], and overexpression of
KDM4A is reported to promote site-specific copy number gain (e.g. 1q12) [14]. In addition,
KDM4A is reported to promote prostate cancer initiation through the transcription factor
ETV1 [15].
The human KDM5 subfamily, which consists of four members (A-D), is structurally the most
closely related to the KDM4 subfamily and also contains a JmjN domain. KDM5 enzymes
catalyse demethylation of H3K4Me /Me histone lysine substrates and are implicated in
3
2
various types of human cancers [[16], [17], [18]]. A requirement for KDM5A has been
3

ACCEPTED MANUSCRIPT
reported in drug tolerant human tumor cells [19], while KDM5B and KDM5C have been
implicated in breast [[20], [21]] and prostate cancers [22], respectively. Also Dalvi et al
recently reported that taxane-platin resistant lung cancer cells showed hypersensitivity to JIB-
0
4 (a pan-selective JmjC KDM inhibitor) and the KDM5/KDM6 subfamily inhibitor GSK-J4
[
23].
Figure 1: Reported KDM inhibitors
These findings have generated considerable interest in small-molecule inhibitors of KDM
demethylase functions [24]. Early inhibitors of JmjC KDMs, such as the pyridine carboxylic
acid-based analogues 1 and 2 (Figure 1) [[25], [26]] display pan - JmjC histone demethylase
inhibitory activity, and feature a carboxylic acid which may hinder cell permeability [27].
4

ACCEPTED MANUSCRIPT
More recently, a range of structurally diverse JmjC KDM inhibitors has been reported with
evidence of selective inhibition of specific KDM subfamilies. For example, the
pyridopyrimidinone derivatives 3 and 4 (Figure 1) show selective inhibition of both KDM4
and KDM5 subfamilies over members of KDM2, 3, or 6 demethylases [[28], [29]].
Compounds 5, 6, and 7 (Figure 1) selectively inhibit KDM5 subfamily members [[30], [31],
[
32]], and compound 8 (Figure 1) has recently been reported as a potent KDM4 subfamily
inhibitor with promising selectivity [33]. In addition, a chemoproteomics approach revealed
compound 9 as a selective KDM4 subfamily inhibitor [34]. Covalent inhibitors of KDM5
subfamily have also been recently reported [[35], [36]]. In addition, a rhodium(III) complex
has been reported as a selective inhibitor of KDM5A [37].
We previously reported the discovery of C8-substituted pyridopyrimidinones (e.g. 4) as a
potent, cell penetrant dual KDM4 and KDM5 inhibitors [29]. Determination of the crystal
structures of the mono m-Cl counterpart of 4 in complex with both KDM4A and KDM5B
allowed us to identify residue differences in the histone substrate binding site that could be
exploited for selective KDM4 subfamily inhibition. Herein, we report our efforts to gain
selectivity for KDM4 over KDM5 subfamily inhibition by targeting residues V313
(KDM4A) and E169 (KDM4A). We also report our attempts to reduce the conformational
flexibility of the C8-substituent to improve KDM inhibitory activity and investigate the
impact of such changes on KDM4 versus KDM5 selectivity. Finally, we demonstrate cell-
based target engagement for exemplar compounds from the 2-OG-competitive
pyridopyrimidinone series and reveal a consistent drop off from in vitro biochemical to cell-
based activity.
5

ACCEPTED MANUSCRIPT
2
. Chemistry
a
Reagents and conditions: (a) (i) methanesulfonic anhydride, anhydrous CH Cl , 0 °C, 15
2
2
min, work-up, (ii) triethylamine, C4-substituted piperidine, anhydrous DMF, 50 °C, 15 h; (b)
C4-substituted piperidine, anhydrous 1,2-dichloroethane or CH Cl , NaBH(OAc) , room
2
2
3
temp.; (c) 6 M HCl, THF, 50 to 60 °C, 3 to 8 h or 4 M HCl in dioxane, H O, 50 °C.
2
Compounds 16b-m (Table 1), 17a-g (Table 2), 18b-c (Table 3), and 19a-d (Table 4) were
prepared from key intermediates 10 and 11 via methods A and B, respectively as previously
described for the synthesis of closely related analogues (Scheme 1) [29].
6

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) aryl/heteroaryl halide, Pd(dppf)Cl , DME, 1 M aqueous
2
Na CO , microwave, 120 °C, 45 min; (b) (i) Pd(OH) on carbon, EtOH, 1 M HCl, H , room
2
3
2
2
temp 1 h or 10% Pd/C, EtOH, H , room temp, 2 h, (ii) when R = Boc: 4 M HCl in dioxane,
2
room temp, 2 h or when R = Cbz: AcOH, 10% Pd/C, H , room temp, 4 to 6 h.
2
The key C4-substituted piperidine intermediates required for the S 2 displacement or
N
reductive amination reactions to prepare 12b-m, 13a-g, 14b-c, and 15a-d (see Scheme 1)
were commercially available or obtained as shown in Scheme 2. N-Protected
tetrahydropyridine-4-boronic acid pinacol ester was reacted with appropriately substituted
aryl/heteroaryl halides under Pd-catalysed conditions to provide intermediates 22e-h, 22j,
2
2l,m, 23a, 23e-g and 24b,c (Scheme 2). Appropriately substituted aryl/heteroaryl halides
were commercially available or readily synthesised via standard transformations as detailed
in the experimental procedure (see Supporting Information). Reduction of the double bond
followed by the removal of the N-protecting group afforded the desired C4-substituted
piperidines (Scheme 2).
7

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) (i) LiN(SiMe ) (1M solution in THF), THF, 0 °C, 45 min, (ii)
3
2
tert-butyl bis(2-chloroethyl)carbamate, 0 °C, 2 h; (b) 10% Pd/C, EtOH, H , room temp, 2 h;
2
(c) 4 M HCl in dioxane, room temp, 2 h.
The spirocyclopiperidine 30 required for the synthesis of 19b (Table 4) was accessed by
reacting 4,7-dimethyl-1H-indene with lithium bis(trimethylsilyl)amide and tert-butyl bis(2-
chloroethyl)carbamate followed by the hydrogenation of the double bond in 29, and finally,
removal of the Boc protecting group under acidic conditions (Scheme 3).
a
Reagents and conditions: (a) aldehyde, NaBH(OAc) , DMF, room temp, stirring up to 6 h;
3
(
b) Cs CO , anhydrous MeCN, 8-chloro-3-((2-(trimethylsilyl)ethoxy)methyl)pyrido[3,4-
2 3
d]pyrimidin-4(3H)-one, reflux, 18 h; (c) 6 M HCl, THF, 50 to 60 °C, 3 to 8 h.
8

ACCEPTED MANUSCRIPT
Access to conformationally constrained pyrazole-C4 compounds 34b-h (Scheme 4, Table 5)
was achieved by reductive amination of the relevant aldehyde by piperidine derivative 31
followed by the introduction of the substituted pyrazole moiety 32 at the C8-
pyridopyrimidinone position via a S Ar displacement reaction, and removal of the SEM
N
protecting group under acidic conditions (Scheme 4).
Scheme 5a
H
H
N
N
H
N
N
N
b
N
a
Br
N
N
35
36
37
a
Reagents and conditions: (a) (i) 2.5 M nBuLi in hexane, THF, -78 °C to room temp (1.5 h)
then cool to -78 °C, (ii) 1-methylpiperidin-4-one, warm up to room temp, 1.5 h, then
AcOH/TFA, 90 °C, 4 h; (b) 10% Pd/C, EtOH, 1 M HCl, H , 16 h.
2
Preparation of 1-methyl-4-(1H-pyrazol-4-yl)piperidine (37), required for the synthesis of 34a
(Table 5) was carried out using an alternative approach whereby 4-bromopyrazole (35) was
reacted with nBuLi followed by the addition of 1-methylpiperidin-4-one. Subsequent
dehydration of the addition product under acidic conditions followed by hydrogenation of the
double bond in 36 afforded 37 (Scheme 5). The incorporation of the piperidine 37 at the C8-
pyridopyrimidinone position was achieved via a S Ar displacement reaction as described for
N
analogues 33b-h (Scheme 4), and the SEM protecting group was removed by treatment with
TBAF in THF.
9

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) triethylamine, anhydrous DMF, 50 °C, 15 h; (b) 6 M HCl, THF,
5
0 °C, 3 h.
Access to 41, a matched-pair inactive control for 19a, was achieved upon treatment of the
mesylate 38, prepared as previously described [38], with spirocyclopiperidine 39 and
triethylamine in DMF followed by the removal of the SEM protecting group of 40 under
acidic conditions (Scheme 6).
3
. Results and Discussion
We recently reported a series of C8-substituted pyridopyrimidinone derivatives, with the
most advanced analogues, such as 4 (Figure 1) and 16a (Table 1), demonstrating selective
and equipotent inhibition of KDM4 and KDM5 over the KDM2, KDM3, and KDM6
subfamilies [29].
1
0

ACCEPTED MANUSCRIPT
Figure 2: Overlay of crystal structures of 16a bound to KDM4A (light orange), and KDM5B
(cyan). Compound 16a is shown in ball and stick representation and protein backbone chains
are represented as cartoon tubes, with key amino acid side-chains displayed in line
representation. Metal ions are shown as spheres. Labels highlight residues that are different
between KDM4A (black) and KDM5B (cyan). KDM5B loop 91-100 is not displayed due to
its construct-derived artefactual position as discussed in Figure S1.
To identify potential interactions that could increase KDM4 subfamily potency and
selectivity over KDM5 subfamily enzymes, we determined the crystal structures of 16a in
complex with KDM4A and KDM5B (Figure 2). In the KDM4A-bound structure, the C4-
pyrazole substituent of 16a extends into the histone substrate binding site and towards the
surface of the enzyme (Figure S2); the m-Cl substituent on the phenyl group makes
hydrophobic contact with V313 of KDM4A (Figure 2), a residue that is conserved across all
1
1

ACCEPTED MANUSCRIPT
KDM4 subfamily members (Figure S3). We postulated that this favourable hydrophobic
interaction with V313 contributes to the increased KDM4 inhibitory profile of 16a compared
to the KDM5-preferring profile of earlier analogues in this series [29]. In addition, 16a is a
more potent inhibitor of both KDM4A and KDM4B than its unsubstituted phenyl counterpart
(compound 54g in reference 29) consistent with the observed hydrophobic interaction of the
m-Cl substituent. Furthermore, the crystal structure of 16a bound to KDM5B indicated no
residue equivalent to KDM4A V313 due to a different protein fold (Figure 2). This
observation further supports the hypothesis that the hydrophobic contact of the m-Cl
substituent in 16a with V313 increases KDM4 inhibitory activity leading to a balanced
KDM4/5 inhibitory profile.
Figure 3: A) Overlay of crystal structures of 1 (2,4-PDCA, blue, PDB 2VD7) and 16a
(beige) bound to KDM4A. B) Overlay of crystal structures of 1 (2,4-PDCA, orange, PDB
5
A3W) and 16a (cyan) bound to KDM5B. Metal ions are shown as spheres. Protein
backbone chains are represented as cartoon tubes, key residues are displayed in line
representation. Compounds 1 (2,4-PDCA) and 16a are shown in ball and stick representation.
1
2

ACCEPTED MANUSCRIPT
KDM5B loop 91-100 is not displayed due to its construct-derived artefactual position as
discussed in Figure S1.
A more detailed study of the 16a-bound KDM5B crystal structure revealed additional
residues close to the m-chlorophenyl moiety of the bound inhibitor that could be exploited to
enhance KDM4-subfamiliy selectivity (Figure 2). For example, C480 is a serine residue in all
other KDM5 subfamily members, and a glutamate in the KDM4 subfamily (E169 in
KDM4A, Figures 2, S3 and S4). This suggests a potential for differential KDM4 versus
KDM5 targeting although we recognised that exploiting selective interaction with the side
chain of E169 (KDM4A numbering) might be challenging due to its solvent exposed location
and high mobility; for example, the side chain of E169 could be not associated to any
electron density in the 16a-bound KDM4A crystal structure. W486 in KDM5B is conserved
across the KDM5 subfamily whilst the equivalent residue in the KDM4 subfamily is a
tyrosine (Y175 in KDM4A, Figures 2, S3 and S4). However, the potential to exploit this
difference is limited due to the similarity of tryptophan and tyrosine residues. We also
observed that A430 in KDM5B is an asparagine residue in KDM4A (N137, Figure 2).
However, these residues are not conserved within their respective subfamilies. N137 in
KDM4A corresponds to S138 in KDM4B, S141 in KDM4D, and N139 in KDM4C (Figure
S3), whilst A430 in KDM5B corresponds to S414 in KDM5A, and to H445/H435 in KDM5C
and KDM5D, respectively (Figure S4), rendering exploitation of such differences for KDM4
over KDM5 subfamily selective inhibition challenging.
Based on these structural insights, we decided to further explore the favourable hydrophobic
contact with V313 (KDM4A) to increase affinity and selectivity for the KDM4 over KDM5
subfamily. As an alternative approach, we also considered the introduction of a basic
substituent at either the m- or p-position of the phenyl ring in 16a to target E169 (KDM4A).
We recognised that achieving selective KDM4- over KDM5-subfamily inhibition may be
1
3

ACCEPTED MANUSCRIPT
challenging due to protein flexibility in the histone substrate binding site, as exemplified by
induced-fit movement observed in C8-substituted pyrido[3,4-d]pyrimidin-4-one-bound
KDM4A and KDM5B crystal structures compared with small 2OG-competitive inhibitors
such as 1 (Figure 3). For example, the pyrazole C4-substituent in 16a induces movement of
Y175 and nearby loop 160-171 of KDM4A, such that residues 162-169 become too mobile to
be observed in the electron density map. In the KDM5B-16a crystal structure, W486 and
nearby loop 426-435 are displaced relative the corresponding compound 1-bound structure,
and an alternative conformation is observed for residues 479-482. We also recognised that
conformational flexibility of the ligand pyrazole C4-substituent may adversely impact KDM
inhibition and subfamily selectivity.
a
Table 1. 4-Phenylpiperidine derivatisation: m-Phenyl substitution
Compound
X
KDM4A
KDM4B
KDM5B
KDM5C
Caco-2
-
6
IC (µM) IC (µM) IC (µM) IC (µM) (x10
5
0
50
50
50
cm/s)
b
1
6a
Cl
0.102±
.058
0.084±
.019
0.031±
0.012
0.032±
0.016
0.023
0.065
11.80
0
1
6b
F
0.029
0.096
11.63
4.67
0
t
c
c
1
6c
Bu
0.141
0.049±
0.025
0.063
0
.041
1
6d
OMe
0.212±
0.067±
0.036
0.101
0.023
0.086
not
tested
10.77
0.013
c
c
1
6e
6f
0.178
0.051
0.066
0.156
0.213
c
c
c
c
1
0.282
0.145
not
tested
1
6g
0.136
0.033±
0.042
0.079
not
0
.012
tested
1
4

ACCEPTED MANUSCRIPT
c
c
c
c
1
6h
0.056
0.027±
.014
0.017
0.059
0.054
< 1.73
<1.52
0
1
6i
0.088±
0.047±
0.024
0.021
0.046
1
6j
0
0
.083±
.049
0.019±
0.013
0.012
0.030
<0.76
1
6k
c
c
0.259
0.063±
0.048
0.113
not
0
.023
tested
c
c
c
c
1
6l
0.059
0.015
0.012
0.006
0.035
0.014
<0.92
1
6m
0.038±
0.008
<1.52
0
.006
a
Results are mean values of two independent determinations or mean (±SD) for n>2 unless
specified otherwise. Data taken from reference 29. Results are from a single experiment.
b
c
Both fluoro and tert-butyl substitutions (compounds 16b and 16c, respectively) resulted in
similar KDM inhibitory activities to those observed for the m-Cl counterpart 16a (Table 1).
The methoxy and isopropyl derivatives 16d and 16e proved marginally inferior KDM4
subfamily inhibitors compared to 16a, and a similar trend was also observed with the
introduction of a bulkier alkoxy substituent (16f, Table 1).
1
5

ACCEPTED MANUSCRIPT
Figure 4: Overlay of crystal structures of 16m (green) and 16a (beige) bound to KDM4A.
Protein backbone chains are represented as cartoon tubes, key residues are displayed in line
representation. Compounds 16m and 16a are shown in ball and stick representation. The two
methyl groups in 16m have not been modelled due to too weak density linked to the high
mobility of the corresponding atoms. Metal ions are shown as spheres.
The introduction of 6-membered heterocyclic substituents (compounds 16g-i) was tolerated
but with no evidence for selective KDM4 subfamily inhibition (Table 1). Some of the most
active inhibitors incorporated a basic substituent at the m-position of the phenyl ring; notably
pyrrolidine and dimethylamino derivatives 16j and 16m, respectively (Table 1). Consistent
with previous findings [29], these m-substituted analogues retained selectivity over other
KDM subfamilies; for example, 16j displayed weaker inhibition of KDM2A (IC = 1.97
5
0
1
6

ACCEPTED MANUSCRIPT
µM), KDM3A (IC = 1.85 µM), and KDM6B (IC = 32.26 µM). Likewise, 16b displayed
5
0
50
selectivity over KDM2A (IC = 4.68 µM), KDM3A (IC = 6.42 µM), and KDM6B (IC >
5
0
50
50
1
00 µM). The crystal structure of 16m bound to KDM4A confirmed a direct but suboptimal
interaction between the amino group of the m-substituent and the side chain of residue N137
distance of 3.2Å, Figure 4), consistent with the improved KDM4A potency of 16m (0.038
µM) compared to the unsubstituted parent (54g in reference 29; KDM4A IC = 0.456 µM).
(
5
0
Overall, 16m shows a balanced KDM4/KDM5 subfamily inhibitory profile whereas the
unsubstituted parent compound (54g in reference 29; KDM5B IC = 0.058 µM) is KDM5B
5
0
preferring. However, direct interaction of the basic substituent with KDM4A E169 was not
observed by X-ray crystallography, likely due to the high mobility of the E169 side chain
observed in all crystal structures (no electron density detected). Compounds bearing a
lipophilic piperidine 4-substituent (16b, 16c, and 16e) displayed moderate cell permeability
(
Caco-2 assay, A to B flux) similar to that of 16a (Table 1). However, the introduction of a
heteroaromatic ring (16h and 16i), or the inclusion of an additional basic centre (16j, 16l, and
6m) had a detrimental effect on cell permeability (Table 1).
1
a
Table 2. 4-Phenylpiperidine derivatisation: p-Phenyl substitution
Compound
X
KDM4A
KDM4B
KDM5B
KDM5C
Caco-2
-
6
IC (µM) IC (µM) IC (µM) IC (µM) (x10
5
0
50
50
50
cm/s)
b
1
7a
0.218
0.131
0.153
0.146
0.896
not
tested
2.29
1
7b
0.024±
0.080
0.246
0
.018
1
7

ACCEPTED MANUSCRIPT
b
b
1
7c
0.068
0.032
0.127
0.549
<1.81
1
7d
0.188±
0.079±
0.033
not
tested
b
b
b
b
0.063
0.081
0.026
0.313
0.053
1
7e
7f
0.077±
.007
0.018±
0.006
<0.55
0
1
0.093
0.020±
0
0.012
0.041
0.048
0.108
<0.76
<1.52
.011
0.020±
.009
Results are mean values of two independent determinations or mean (±SD) for n>2 unless
1
7g
0.103
0
a
b
specified otherwise. Results are from a single experiment.
We subsequently investigated the effect of selected p-substituents on the 4-phenylpiperidine
moiety. The presence of a 6-membered heterocycle (17a-c, Table 2) or alkyl chains bearing a
basic centre (17e and 17f, Table 2) elicited a KDM4/5 inhibitory profile similar to their m-
substituted counterparts with no evidence of clear selective KDM4 over KDM5 inhibition
(Tables 1 and 2). However, selectivity for dual KDM4/5 inhibition over other KDM
subfamilies was maintained; for example 17f displayed a weaker inhibitory activity against
KDM2A (IC = 1.42 µM), KDM3A (IC = 2.02 µM), and KDM6B (IC = 52.41 µM).
5
0
50
50
Crystal structures of 17b, 17e and 17f bound to KDM4A confirmed that the phenyl p-
substituent points towards the solvent accessible region without making any specific contact
with the protein including the targeted residue E169 (Figure 5). More precisely, the p-
substituents of both 17b and 17e could not be associated with any electron density,
suggesting high mobility and lack of stabilisation by the protein. In the 17f-bound KDM4A
structure, the p-dimethylamino moiety points towards the solvent accessible region, but
without clear interactions with the protein (Figure 5). Similarly to the m-substituted
analogues (Table 1), p-substituted compounds displayed significantly lower permeability in
1
8

ACCEPTED MANUSCRIPT
the Caco-2 assay (A to B flux) relative to that of 16a (Table 2). This drop in cellular
permeability is consistent with increased polarity and/or the presence of a basic nitrogen.
Figure 5: Overlay of crystal structures of 17b (pink), 17e (magenta) and 17f (light-green)
bound to KDM4A. Zn(II) atoms are shown as grey spheres. Protein backbone chains are
represented as cartoon tubes, key residues are displayed in line representation. The pyridyl
ring in 17b has not been modelled due to too weak density of the corresponding atoms.
Compounds 17b, 17e and 17f are shown in ball and stick representation.
a
Table 3. 4-Phenylpiperidine derivatisation: 3,5-phenyl substitution
1
9

ACCEPTED MANUSCRIPT
Compou
nd
X
Y
H
KDM4A
KDM4B
KDM5B
KDM5C
Caco-2
-6
IC (µM) IC (µM) IC (µM) IC (µM) (x10
5
0
50
50
50
cm/s)
15.31
<0.76
b
1
1
8a
8b
CF3
CF3
0.128
0.071
0.039
0.018
0.017
0.070
0.038
0.033±
0
.015
1
8c
c
c
CF3
0.246±
0.102±
0.062
0.032
0.096
<0.76
0
.146
a
Results are mean values of two independent determinations or mean (±SD) for n>2 unless
b
c
specified otherwise. Data taken from reference 29. Results are from a single experiment.
Noting that both m-chloro, -fluoro, -trifluoromethyl [29] substituents, and alkyl chains
bearing basic centres at the m-position of the distal phenyl ring (Table 1) impart potent and
balanced KDM4/5 subfamily inhibition, and also that their binding modes are potentially
complementary (see overlay of 16a and 16m, Figure 4), we combined these two substituents
in a small series of 3,5-disubstituted phenyl derivatives (Table 3). Synthesis of the most
desired compounds with both m-Cl and m-alkylamino side chains proved challenging due to
competing dehalogenation upon reduction of the piperidine double bond (Scheme 2).
Nevertheless, we were able to combine a m-CF substituent with basic functionality in the
3
remaining m-position (18b,c; Table 3). This approach led to potent dual KDM4 and KDM5
inhibition but no additive inhibitory effect was observed (Table 3), and the KDM4/5
inhibitory profile was broadly similar to that observed with the corresponding mono-
substituted analogues (Tables 1 and 3). We subsequently solved the structure of 18a bound to
2
0

ACCEPTED MANUSCRIPT
KDM4A (Figure 6). This structure indicated that the pendant phenyl ring is rotated such that
the m-CF substituent does not form an interaction with V313 in contrast to its m-Cl
3
counterpart (compare Figures 2, 4 and 6). This observation could partly explain the lack of
additive SAR in compounds 18b and 18c, assuming that the m-CF substituent in these 3,5-
3
disubstituted phenyl derivatives occupies the same position as observed for 18a. Compounds
1
8b and 18c maintained a selective inhibition profile versus other KDM subfamily members;
for example, 18b displayed weaker inhibitory activity against KDM2A (IC = 3.77 µM),
5
0
KDM3A (IC = 5.68 µM), and KDM6B (IC = 23.97 µM). However, both 18b and 18c
5
0
50
displayed low Caco-2 permeability (A to B flux) in line with previous results obtained with
compounds bearing a basic substituent on the phenyl ring (Table 1 and 2).
2
1

ACCEPTED MANUSCRIPT
Figure 6: Overlay of crystal structures of 18a (brown) and 16a (beige) bound to KDM4A.
Zn(II) atoms are shown as grey spheres. Proteins backbone chains are represented as cartoon
tubes, key residues are displayed in line representation. Compounds 18a and 16a are shown
in ball and stick representation.
a
Table 4. Pyrazole C4-substituent: Conformational restriction
Compound
KDM4A
KDM4B
KDM5B
KDM5C Caco-2
-6
R
IC (µM) IC (µM) IC (µM) IC50
(x10
5
0
50
50
(
µM)
cm/s)
11.64
1
9a
0
0
.100±
.041
0.043±
0.021
0.038
0.123
1
9b
0
0
.143±
.056
0.045
0.057
0.114
0.214
18.06
1
9c
b
b
0
.184±
0.123
0.014
0.470
4.25
0
.069
1
9d
b
0
0
.107±
.020
0.029±
0.006
not
tested
13.33
a
Results are mean values of two independent determinations or mean (±SD) for n>2 unless
b
specified otherwise. Results are from a single experiment.
2
2

ACCEPTED MANUSCRIPT
We next turned our attention to constraining the C4-pyrazole substituent in order to maintain
contact with V313 whilst minimising the entropic cost associated with ligand binding. We
envisaged that the lipophilic contact with V313 (KDM4A) (Figure 2) could be optimised by
conformational restriction directing the phenyl ring towards V313. This hypothesis led to
synthesis of the spirocyclic analogue 19a which inhibited KDM4 and KDM5 subfamily
members with IC values similar to those observed with 16a (Tables 1 and 4). 19a also
5
0
displayed selective inhibition of KDM4/5 over other KDM subfamilies, inhibiting KDM2A,
KDM3A, and KDM6B with IC values of 4.50, 5.78 and 90.22 µM, respectively.
5
0
Figure 7: Overlay of crystal structures of 19a (grey) and 16a (beige) bound to KDM4A.
Zn(II) atoms are shown as spheres. Protein backbone chains are represented as cartoon tubes,
2
3

ACCEPTED MANUSCRIPT
key residues are displayed in line representation. Compounds 19a and 16a are shown in ball
and stick representation.
The crystal structure of 19a bound to KDM4A (Figure 7) revealed a binding mode similar to
that of 16a, with the phenyl ring of the spirocyclic system slightly closer to the side chain of
V313 than in 16a (closest phenyl carbon atom is 3.7Å from the side chain of V313 for 19a,
versus 4Å for the corresponding carbon in 16a). In the 19a-bound KDM4A crystal structure,
we also observed that a loop comprising KDM4A residues 308-313 folds over the
conformationally restricted spirocyclic phenyl ring to elicit favourable hydrophobic stacking
interactions with both Cα and Cβ atoms of D311. In addition, we also observed electron
density for the main chain and Cβ of E169 below the spirocyclic phenyl ring of 19a −
interestingly, E169 is not commonly visible due to both main chain and side chain flexibility.
Further, the pyrazole C4-substituent in 19a is associated with a stronger electron density than
for the corresponding 16a structure, and is well defined in all four chains of the asymmetric
unit with B factors significantly lower than for the corresponding atoms in 16a (average B
factors of the terminal phenyl in 19a is 0.8 times the average B factor for the whole structure,
while it was 1.3 times for 16a). These observations suggest that 19a is more stably bound in
the active site of KDM4A than 16a. Compounds 19b and 19c gave no improvement to
KDM4/5 inhibitory profiles relative to 19a (Table 4); however, comparison of the structures
of 19a and 16a bound to KDM4A (Figure 7) prompted us to introduce a methyl group at the
piperidine C4-position in 16a to restrict the conformation without a spirocyclic ring system.
Pleasingly, 19d (Table 4) exhibited a KDM4/5 inhibitory profile similar to that observed with
1
9a and the crystal structure of 19d bound to KDM4A revealed the m-Cl phenyl substituent
in an equatorial position with the Cl atom making contact with V313 (distance of 3.5 Å,
Figure 8). However, the pyrazole C4-substituent appeared less well stabilised than its
equivalent in 19a (the full substituent is seen only in one chain for 19d, and with B factors up
2
4

ACCEPTED MANUSCRIPT
to 1.3 times higher than the average of the structure). This may arise from freedom of rotation
around the piperidine C4-bond in 19d and 16a compared to the constrained spirocyclic
compound 19a. Satisfyingly, all analogues in this subseries displayed good permeability in
the Caco-2 assay (A to B flux; Table 4).
Figure 8: Overlay of crystal structures of 19d (pink) and 16a (beige) bound to KDM4A.
Zn(II) are shown as spheres. Proteins backbone chains are represented as a cartoon tubes, key
residues are displayed in line representation. Compounds 19d and 16a are shown in ball and
stick representation.
2
5

ACCEPTED MANUSCRIPT
a
Table 5. Pyrazole C4-substituent: Conformational restriction
Compound
R
KDM4A
KDM4B
KDM5B
KDM5C
Caco-2
-
6
IC (µM)
IC (µM) IC (µM)
IC (µM) (x10
5
0
50
50
50
cm/s)
3
4a
4b
4c
4d
Me
Et
2.06
0.82
0.156
0.163
<0.76
b
3
0.693±
.231
0.848±
.234
0.455±
0.037
0.504±
0.072
0.149
0.439
0.081
0.566
<0.76
0
3
not
tested
not
tested
0
3
b
b
1
.06
0.497
0.372
0.601
0.447
0.856
0.571
b
b
3
4e
0.756
<0.76
b
b
3
4f
0.559±
0.248±
0.048
0.302
0.134
0.202
0.132
5.46
2.12
0
.152
b
3
4g
0.613
0.068
0.131
b
b
3
4h
2.41
0.573
0.377
not
tested
a
Results are mean values of two independent determinations or mean (±SD) for n>2 unless
b
specified otherwise. Results are from a single experiment.
2
6

ACCEPTED MANUSCRIPT
Figure 9: Overlay of crystal structures of 34a (blue) and 16a (beige) bound to KDM4A.
Zn(II) atoms are shown as spheres. Protein backbone chains are represented as cartoon tubes,
key residues are displayed in line representation. Compounds 34a and 16a are shown in ball
and stick representation.
In a further attempt to restrict the conformation of the C4-pyrazole substituent, we directly
attached the N-methylpiperidin-4-yl moiety to C4 of the pyrazole to give 34a, a more potent
inhibitor of KDM5 relative to KDM4 subfamily enzymes by at least 5-fold (Table 5). A
crystal structure of 34a in complex with KDM4A (Figure 9) revealed electron density for the
pyrazole substituent in all four chains of the asymmetric unit, with the piperidine nitrogen
closer to the side chain carboxylate of D191 than with 16a (2.9 Å versus 4.5 Å for 16a). In
contrast to the 16a-KDM4A structure, the loop comprising residues 160-171 remained in the
2
7