Discovery, design, and synthesis of indole-based EZH2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.06.056

The discovery and optimization of a series of small molecule EZH2 inhibitors is described. Starting from dimethylpyridone HTS hit (2), a series of indole-based EZH2 inhibitors were identified. Biochemical potency and microsomal stability were optimized during these studies and afforded compound 22. This compound demonstrates nanomolar levels of biochemical potency (IC₅₀ = 0.002 μM), cellular potency (EC₅₀ = 0.080 μM), and afforded tumor regression when dosed (200 mpk SC BID) in an EZH2 dependent tumor xenograft model.

Full text of DOI:10.1016/j.bmcl.2015.06.056

Accepted Manuscript
Discovery, design, and synthesis of indole-based EZH2 inhibitors
Victor S. Gehling, Rishi G. Vaswani, Christopher G. Nasveschuk, Martin
Duplessis, Priyadarshini Iyer, Srividya Balasubramanian, Feng Zhao, Andrew
C. Good, Robert Campbell, Christina Lee, Les A. Dakin, Andrew S. Cook,
Alexandre Gagnon, Jean-Christophe Harmange, James E. Audia, Richard T.
Cummings, Emmanuel Normant, Patrick Trojer, Brian K. Albrecht
PII:
S0960-894X(15)00649-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.056
BMCL 22846
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 May 2015
11 June 2015
15 June 2015
Please cite this article as: Gehling, V.S., Vaswani, R.G., Nasveschuk, C.G., Duplessis, M., Iyer, P., Balasubramanian,
S., Zhao, F., Good, A.C., Campbell, R., Lee, C., Dakin, L.A., Cook, A.S., Gagnon, A., Harmange, J-C., Audia, J.E.,
Cummings, R.T., Normant, E., Trojer, P., Albrecht, B.K., Discovery, design, and synthesis of indole-based EZH2
inhibitors, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.056
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Discovery, design, and synthesis of indole-
based EZH2 inhibitors.
Leave this area blank for abstract info.
Victor S. Gehling, Rishi G. Vaswani, Christopher G. Nasveschuk, Martin Duplessis, Priyadarshini Iyer,
Srividya Balasubramanian, Feng Zhao, Andrew C. Good, Robert Campbell, Christina Lee, Les A. Dakin,
Andrew S. Cook, Alexandre Gagnon, Jean-Christophe Harmange, James E. Audia, Richard T. Cummings,
Emmanuel Normant, Patrick Trojer, Brian K. Albrecht.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery, design, and synthesis of indole-based EZH2 inhibitors.
Victor S. Gehling*, Rishi G. Vaswani, Christopher G. Nasveschuk, Martin Duplessis, Priyadarshini Iyer,
Srividya Balasubramanian, Feng Zhao, Andrew C. Good, Robert Campbell, Christina Lee, Les A. Dakin,
Andrew S. Cook, Alexandre Gagnon, Jean-Christophe Harmange, James E. Audia, Richard T. Cummings,
Emmanuel Normant, Patrick Trojer and Brian K. Albrecht.
Constellation Pharmaceuticals, Inc., 215 First Street, Cambridge, MA, 02142, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
The discovery and optimization of a series of small molecule EZH2 inhibitors is described.
Starting from dimethylpyridone HTS hit (2), a series of indole-based EZH2 inhibitors were
identified. Biochemical potency and microsomal stability were optimized during these studies
and afforded compound 22. This compound demonstrates nanomolar levels of biochemical
potency (IC50 = 0.002 µM), cellular potency (EC50 = 0.080 µM), and afforded tumor regression
when dosed (200 mpk SC BID) in an EZH2 dependent tumor xenograft model.
Revised
Accepted
Available online
Keywords:
EZH2
2009 Elsevier Ltd. All rights reserved
.
PRC2
Histone methyltransferase
Epigenetics
Pyridone
———
* Corresponding author. Tel.: +1-617-714-0540; fax: +1-617-577-0472; e-mail: victor.gehling@constellationpharma.com

Post-translational modifications of histones are important for
the dynamic regulation of chromatin structure and transcription
of genetic material.1 Improper regulation of histone “marks” play
an important role in the initiation and progression of certain
diseases, in particular cancer. In this context, the histone lysine
methyltransferase Enhancer of Zeste Homolog 2 (EZH2), the
catalytic subunit of the Polycomb Repressive Complex 2 (PRC2),
is involved in transcriptional silencing of targeted genes by
catalyzing the methylation of the ε-NH2 motif of histone H3
lysine 27 (H3K27).2 Studies have shown that EZH2 is
significantly overexpressed in a wide range of human cancers
whereby the relative EZH2 expression level correlates well with
cancer stage and poor prognosis.3,4 Additionally, recurrent mono-
allelic gain of function mutations of residues, such as Y641,
A677 and A687, within the EZH2 catalytic domain have been
observed in germinal center B-cell like diffuse large B-cell
lymphoma (GCB-DLBCL) and follicular lymphoma.5-9 It follows
that the inhibition of EZH2 activity holds promise as a potential
treatment for cancer.10
Figure 2. Merge of compound 1 with HTS hit 2.
Given the potency disparity between 1 and 3, it was clear that the
structure
activity
relationships
(SAR)
from
the
tetramethylpiperidinyl benzamide series could not be applied to
the emerging dimethylpyridone series. A brief screen of linkers
between the two phenyl rings revealed that a carbon-bridged
linker provided a 3-fold boost in potency (5, Table 1). In order to
lower the logP and move the series into more drug-like chemical
property space, polarity was introduced on the central ring.21 This
effort afforded pyridone 6 which retained similar activity to
carbon-linked 5. We then contracted the pyridone to a pyrazole
(7) which led to a ~6-fold decrease in activity and using a 3-
indolyl group (8) resulted in further loss of potency. However,
inclusion of a 2-methyl group on indole 9 achieved a 126-fold
improvement in potency to provide a compound with sub-100
nM activity against both wild-type (wt) and mutant EZH2. As
methyl substitution at the 2-position of the indole afforded such a
large potency increase, we investigated replacing the methyl
group with other small substituents. A variety of functional
groups spanning a range of physical properties were tested, but
none of the replacements were tolerated.22 Relaxed coordinate
scan derived conformation energy profiles suggest that inclusion
of the 2-methyl group impacts the rotational flexibility of the
amide, significantly biasing the system towards one rotamer. In
addition, the 2-methyl serves to tilt the amide bond out of the
plane of the indole; these conformational effects appear essential
to achieving potent inhibition of EZH2.23
In this context we and others embarked on the discovery and
optimization of inhibitors of the catalytic activity of EZH2.
Through these efforts a variety of small molecule inhibitors of
EZH2 have been described which produce robust phenotypes
both in vitro and in vivo (Figure 1).11-19
Table 1. Genesis of the indole scaffold.
Figure 1. Reported EZH2 inhibitors.
EZH2 wt IC50
EZH2 Y641N
IC50 (µM)a
Compound
5
Structure
(µM)a
We previously reported 1 as an effective tool compound that
characteristic EZH2 inhibitory phenotype in
lymphoma Unfortunately, the
cells.20
produced
a
KARPAS-422
0.153 ± 0.013
0.094b
0.795b
1.35b
pharmacokinetic (PK) profile for 1 was not suitable for further
evaluation of EZH2 inhibition in rodent disease models. Herein
we describe our efforts towards the successful identification of a
small molecule suitable for the evaluation of EZH2 inhibition in
vivo.
6
7c
0.604 ± 0.453
1.64b
5.55 ± 1.34
>10b
As part of our ongoing program to discover and develop
clinically relevant EZH2 inhibitors, we assessed other
chemotypes identified through diversity screening such as
compound 2, which was found to be a SAM-competitive
inhibitor of PRC2 methyltransferase activity (Figure 2). The
commonality of the benzoyl amide motif between 1 and 2 led us
to test the interchangeability of the tetramethylpiperidine (TMP)
with the dimethylpyridone as in compound 3. While significant
potency was lost relative to 1, amalgamated compound 3 had
improved potency relative to 2. While the chloro, cyano, and
pyridazine substituents are important for potency in the TMP
scaffold (1), removal of these substituents, to afford biphenyl
ether 4, enhanced potency in the dimethylpyridone series.
8
9
0.013 ± 0.008
0.062 ± 0.011
a IC50 values reported as an average ≥2 determinations with
standard deviation reported (SD). b Single measurement, no
standard deviation reported. cSingle unknown stereoisomer.
Subsequent SAR efforts focused on substitution of the indole
nitrogen. Initially we separated the two enantiomers of 9 to afford
10 and 11 (Table 2). Both compounds were quite potent with IC50
values less than 50 nM against wild-type EZH2. The R
enantiomer (10) was modestly preferred and provided
a
a
compound with submicromolar cell potency in HeLa
H3K27me3 mechanism of action (MOA) assay. Further profiling
of compound 10 in our in vitro ADME assays showed high
microsomal metabolism, perhaps attributable to its lipophilicity
(ClogP = 3.824) of this compound. Further efforts were focused

on increasing EZH2 potency with concomitant improvement in
the physical properties of the series.25
carbon to afford sec-butyl compound 15 which had micromolar
cell activity and improved ADME compared to 10.
Our efforts to modify the physical properties of these
compounds focused on the indole side chain. Initially, the benzyl
group of 10 was replaced with a variety of heterocycles,
including pyrazole (12) and 2-pyridine (13). While this effort
afforded compounds with improved microsomal stability relative
to 10, in general heteroaromatic substitutions were poorly
tolerated and resulted in potency losses of approximately 5 fold
in our biochemical assay. We next simplified the side chain to
afford isopropyl derivative 14, however this modification led to
significant losses in biochemical potency. Potency could be
regained by extending the chain of the isopropyl group by one
Polar saturated systems were next investigated, including
amide 16 and methoxy ether 17 (Table 2). Amide 16 had limited
activity against EZH2 while compound 17 demonstrated similar
biochemical and cellular potency to 10. With methoxy ether 17
we had successfully lowered the ClogP to an acceptable range
(ClogP = 2.2) while maintaining activity, but despite this
improvement in physical properties compound 17 still displayed
high microsomal metabolism. Further efforts to increase the
stability of our EZH2 inhibitors focused on investigation of
cyclic
saturated
systems.
Table 2: Indole side-chain SAR.
EZH2
Y641N
(µM)a
PPB (m/r/d/h)
(% bound)c,d
EZH2 wt
(µM)a
Clint (m/r/d/h)
(µL/min/mg)c
Compound
R
HeLa (µM)b
0.013 ±
0.008
0.062 ±
0.011
9
n.t.
n.t.
n.t.
0.007 ±
0.004
0.034 ±
0.012
10
0.484e
953/755/n.t./400
98/98/n.t./99
0.022 ±
0.002
0.097 ±
0.085
11
n.t.
n.t.
n.t.
0.073 ±
0.020
0.388 ±
0.051
12
13
n.t.
n.t.
229/92/41/84
349/47/n.t./72
86/80/77/98
88/88/n.t./98
0.076 ±
0.032
0.358 ±
0.126
14
15
0.159e
0.512e
n.t.
966/156/64/116
154/235/n.t./209
95/92/91/97
0.014 ±
0.013
0.118 ±
0.054
0.929e
97/94/n.t./99
16
17
1.76e
>10e
n.t.
279/115/54/111
75/64/61/87
84/73/n.t./91
0.005 ±
0.003
0.043 ±
0.010
0.703e
534/404/n.t./114
(R)
a IC50 values reported as an average ≥2 determinations with standard deviation reported (SD). b The MOA cell assay
measures global H3K27me3 levels in HeLa cells; see supporting information. c m/r/d/h = mouse/rat/dog/human. d Plasma
protein binding e Single measurement, no standard deviation reported. n.t = not tested.
SAR studies on cyclic saturated systems26 began with THP 18
which demonstrated excellent biochemical and cellular potency,
but as with our other potent inhibitors, displayed high levels of
microsomal metabolism in vitro (Table 3). Searching for a stable
saturated system, a series of piperidine analogs were synthesized
(19 and 20). The N-methyl piperidine derivative (19) was
biochemically potent and had very low microsomal metabolism,
but performed poorly in our cellular assay. In contrast to
piperidine 19, sulfonamide 20 showed excellent biochemical and
cellular potency with an EC50 of less than 100 nM. However 20
still had high microsomal metabolism in our ADME assays
despite its reasonable ClogP (2.6). Since saturated heterocycles
18 and 20 afforded better physical properties but did not provide
improved microsomal stability, we turned our attention to
modification of the pyridone.
Initially, we replaced the dimethylpyridone of 18 with a
methoxypyridone to afford 21 (Table 3). Methoxypyridone 21
retained biochemical and cellular activity and provided a
significant improvement in microsomal stability relative to the
parent (18). The enhancement in microsomal stability imparted
by the methoxypyridone translated to the piperidine series, as
ethyl sulfonamide 22 also presented improved microsomal
stability while retaining cellular activity. We investigated several
other pyridone modifications, including an nPr-pyridone (23) and
a
trifluoroethoxypyridone (24) but both the nPr and

trifluoroethoxy pyridones led to diminished cellular potency
relative to the methoxypyridone-containing compounds.
Table 3: Pyridone-indole SAR.
H3K27me3
EZH2
Y641N
(µM)a
Clint (m/r/d/h)
(µL/min/mg)c
PPB (m/r/d/h)
(% bound)c,d
EZH2 wt
Compound
R1
R2
(µM)a
HeLa
(µM)b
18
19
Me
Me
<0.001e
0.004e
0.098
729/277/182/251
<7/<7/<7/<7
95/86/77/94
52/41/58/70
0.002 ±
0.0003
0.007 ±
0.003
0.568 ±
0.470
0.001 ±
0.0002
0.003 ±
0.002
0.059 ±
0.020
20
21
Me
428/303/77/254
166/138/106/110
91/35/31/59
95/91/76/95
83/66/n.t./90
94/84/75/92
0.002 ±
0.001
0.004 ±
0.002
0.075 ±
0.030
OMe
OMe
0.002 ±
0.0004
0.003 ±
0.002
0.080 ±
0.032
22
0.005 ±
0.004
0.040 ±
0.005
0.266 ±
0.009
23
24
nPr
1665/928/283/794
n.t.
99/97/95/98
n.t.
OCH2CF3
0.040e
0.485e
1.08e
0.001 ±
0.0004
0.001 ±
0.0002
0.020 ±
0.010
EPZ-643816
GSK-12613
-
-
-
-
-
-
-
-
0.001 ±
0.0004
0.002 ±
0.001
0.280 ±
0.080
a IC50 values reported as an average ≥2 determinations with standard deviation reported (SD). b Our MOA cell assay
measures global H3K27me3 levels in HeLa cells; see supporting information. c m/r/d/h = mouse/rat/dog/human. d
Plasma protein binding e Single measurement, no standard deviation reported. n.t = not tested.
Ethyl sulfonamide 22 was chosen for profiling in mice due to
its excellent cellular potency and reasonable ADME properties.
Mouse IV PK of 22 showed a clearance of 2.74 L/h/kg, a
reasonable volume (0.795 L/kg), and short half-life of 0.37 h
(Figure 3A). Given the clearance of 22 in mice we suspected that
even high doses of 22 given via oral administration (PO) would
not afford the exposure necessary to see target engagement in
vivo. As a means of achieving higher exposures and a longer
half-life we dosed compound 22 subcutaneously (SC). We were
pleased to see that when given to mice at 200 mpk SC compound
22 showed a prolonged half-life and excellent AUC with a free-
fraction above the cellular EC50 out to 16 hours.
422 PD experiment with 22 was run for 17 days with 200 mg/kg
BID dosing. After 17 days the tumors were harvested and the
level of H3K27me3 was analyzed at 1 hour post last dose and
compound concentrations were analyzed at 1, 6, and 12 hours
post last dose. Compound 22 showed ~84% reduction in
H3K27me3 mark relative to vehicle control, demonstrating a
profound and prolonged inhibition of EZH2 in vivo (Figure 3B).
This strong PD effect, as measured by the decrease in
H3K27me3 levels, was achieved by maintaining the plasma and
tumor concentrations of 22 over the cellular EC50 (Figure 3C).
Based on these encouraging results we subsequently advanced
compound 22 into our KARPAS-422 efficacy model.
With this result in hand we advanced sulfonamide 22 into our
KARPAS-422 pharmacodynamic (PD) model.27 The KARPAS-

Figure 3: PK data, KARPAS-422 PD, and KARPAS-422 efficacy data of compound 22.; A. Mouse PK data of 22.; B. Ratio of H3K27me3 to histone H3 in
the KARPAS-422 PD study for vehicle and treated arms.; C. Time course of tumor and plasma concentrations of compound 22 from the KARPAS-422 PD study
at 1, 6, and 12 h post last dose.; D. Dose-response from the KARPAS-422 efficacy study.; E. Body-weight curves of the individual arms from the efficacy study
5. Majer, C. R.; Jin, L.; Scott, M. P.; Knutson, S. K.; Kuntz, K. W.;
Keilhack, H.; Smith, J. J.; Moyer, M. P.; Richon, V. M.;
The in vivo efficacy of sulfonamide 22 was examined in a
KARPAS-422 mouse xenograft model (Figure 3). Compound 22
was dosed at 50 mg/kg, 100 mg/kg, and 200 mg/kg via SC
administration following a twice daily dosing schedule (BID).
Dosing of sulfonamide 22 for 28 days resulted in a dose-
dependent reduction in tumor volume with tumor growth
inhibitions (TGI) of 54%, 77%, and >100% (regression),
respectively when compared to the vehicle arm (Figure 3D).28
Treatment with 22 was tolerated at all doses studied with less
than 10% body-weight loss observed during the course of
treatment (Figure 3E). The high efficacy and low toxicity of
compound 22 make it a useful tool compound for studying the
effect of EZH2 inhibition in both in vitro and in vivo contexts.
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this report describes the identification of a series of indole-based
EZH2 inhibitors. Optimization of this series afforded
sulfonamide 22, a highly potent inhibitor of EZH2. Compound
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Heike; Cernak, Tim Angew. Chem. Int. Ed. 2013, 52, 2.
23. Gas phase conformational energies calculated using Jaguar
version 8.2, release 12, Schrödinger, Inc., New York, NY
(DFT(b3lyp) method with cc-pvtz(-f)++ basis set). Further details
can be found in the supporting information.
24. Calculated using Chemdraw 13.0.
25. Meanwell, N. A. Chem. Res. Toxicol. 2011, 24, 1420.
26. For synthetic procedures see the supporting information.
27. Further information on the KARPAS PD and efficacy models can
be found in the supporting information.
28. The body weight loss observed in the 50 mpk arm is the result of
three animals. These animals bore tumors showing ulceration
which may have resulted in loss of body fluid and compromised
their ability to move and feed leading to non-drug related weight
loss.