Discovery and optimization of tetramethylpiperidinyl benzamides as inhibitors of EZH2

Article abstract of DOI:10.1021/ml400494b

The identification and development of a novel series of small molecule Enhancer of Zeste Homologue 2 (EZH2) inhibitors is described. A concise and modular synthesis enabled the rapid development of structure-activity relationships, which led to the identification of 44 as a potent, SAM-competitive inhibitor of EZH2 that dose-dependently decreased global H3K27me3 in KARPAS-422 lymphoma cells.

Full text of DOI:10.1021/ml400494b

Letter
pubs.acs.org/acsmedchemlett
Discovery and Optimization of Tetramethylpiperidinyl Benzamides
as Inhibitors of EZH2
Christopher G. Nasveschuk,*,† Alexandre Gagnon,†,‡ Shivani Garapaty-Rao, Srividya Balasubramanian,
Robert Campbell, Christina Lee, Feng Zhao, Louise Bergeron, Richard Cummings, Patrick Trojer,
James E. Audia, Brian K. Albrecht, and Jean-Christophe P. Harmange
Constellation Pharmaceuticals, 215 1st Street, Suite 200, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: The identification and development of a novel
series of small molecule Enhancer of Zeste Homologue 2
(EZH2) inhibitors is described. A concise and modular
synthesis enabled the rapid development of structure−activity
relationships, which led to the identification of 44 as a potent,
SAM-competitive inhibitor of EZH2 that dose-dependently
decreased global H3K27me3 in KARPAS-422 lymphoma cells.
KEYWORDS: methyltransferase, PRC2, EZH2, KARPAS-422, tetramethylpiperidine, diphenylether
istone lysine methyltransferases (HKMTs; HMTs when
referred to both lysine and arginine methyltransferases)
H
contribute to the organization of chromatin structure, and thus
play a role in the regulation of gene expression. Enhancer of
Zeste Homologue 2 (EZH2) catalyzes methylation of histone
H3 lysine 27 (H3K27) and functions as part of a multisubunit
complex termed Polycomb Repressive Complex 2 (PRC2) with
known application in regulating cell identity.1 PRC2/EZH2 has
been widely implicated in cancer progression, largely due to its
prevalent overexpression, which correlates with significant
increases in H3K27 methylation, disease stage, and poor
prognosis.2,3 The identification of recurrent gain of function
mutations within the EZH2 catalytic domain provides a
powerful argument that certain cancers might be dependent
on EZH2 catalytic activity. All identified mutated residues,
Y641, A677, and A687, alter substrate specificity, facilitating the
conversion from H3K27 dimethylated (me2) to trimethylated
(me3) states,4−8 while wild-type (wt) EZH2 preferentially
converts H3K27me1 to H3K27me2. To date, EZH2 gain of
function mutations have been found exclusively in germinal
center B-cell like diffuse large B-cell lymphoma (GCB-DLBCL)
and Follicular Lymphoma (FL) but not in activated B-cell
DLBCL (ABC-DLBCL) or any other lymphoma subtype,
suggesting that EZH2 addiction might be restricted to certain
lymphoma subtypes.
Multiple small molecule inhibitors of EZH2 catalytic activity,
bearing a common pyridone-amide motif, have recently been
disclosed (Figure 1).9−16 It is of interest to note that the
similarities between these compounds is not a result of
deliberate differentiation strategies for generating novel
inhibitors but rather an indication of the relative paucity of
commercial and corporate compound collections for chemical
matter with the appropriate structural features to inhibit this
enzyme. Several pyridone-amides have been used to unveil new
biology linked to the catalytic activity of EZH2. On target in
Figure 1. Disclosed EZH2 inhibitors.
vitro and in vivo efficacy has been demonstrated with GSK-126,
EPZ-6438, and UNC-1999; where EPZ-6438 has recently
progressed into phase 1 trials for the treatment of lymphoma.
Received: December 5, 2013
Accepted: January 14, 2014
Published: January 14, 2014
© 2014 American Chemical Society
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Table 2. Right-Hand Aryl Ring SAR
Figure 2. HTS hit compound 6.
Table 1. Linker SAR
Since all genuine inhibitors of the catalytic activity of EZH2
described to date belong to one chemical series, the pyridone-
amides, the identification of structurally unrelated inhibitors
enhances the chemical biology toolbox for further exploration
of the function of this enzyme. Herein we describe our initial
efforts in this area utilizing high throughput screening (HTS),
biochemical hit triage, and hit optimization, which led to the
identification of structurally distinct EZH2 inhibitors capable of
suppressing EZH2 catalytic activity and globally reducing
H3K27me3 in a cellular setting.
Our HTS screen utilized a PRC2 enzymatic assay that
monitored 3H labeled methyl group transfer from [3H]-S-
adenosyl methionine (SAM) to oligonucleosome substrate and
identified 288 hits.17 These compounds were initially assessed
using a multistage process: hits bearing undesirable functional
groups, poor synthetic starting points, and promiscuous binders
were removed. The remaining hits were assessed in 10 point
concentration−response curves where the Hill coefficient (0.7−
1.5) was a prominent filter for further evaluation.18,19
did or did not shift in potency at cofactor and substrate
concentrations of 10-fold Km. According to the Cheng−Prusoff
relationship, an increase in substrate above Km should result in
predictable decreased inhibitor affinity for a competitive
compound, no effect for a noncompetitive compound, and
enhanced affinity for an uncompetitive one.17,20
After hit triage was complete, several relatively weak (IC50
>
20 μM) compounds remained, which met the criteria outlined
above. One of these, compound 6 (Figure 2), was of moderate
potency (EZH2wt IC50 = 51 μM), had a good Hill coefficient
(1.1), and LipE of 3.5.21,22 When funneled through our
secondary assays 6 shifted only with an increase in [SAM],
which was suggestive of a SAM competitive mechanism of
inhibiton.17 Additionally, 6 was found to be selective when
assessed against other HMTs in a biochemical assay.17 Given
these data and the potential for rapid synthesis of analogues, 6
was selected as a starting point for our initial efforts to develop
wild-type and disease-relevant mutant EZH2 inhibitors.
The aim of our initial medicinal chemistry campaign was to
rapidly navigate the structure−activity landscape of tetrame-
thylpiperidinyl-containing lead compound 6 (Table 1). We first
addressed the linker of the bis-phenylether by extending the
compound one methylene unit (−CH2O−, 7), which
Compounds were then assessed in three additional
enzymatic assays where titrations were run with (1) 10-fold
enzyme, (2) 10-fold Km SAM, or (3) 10-fold Km oligonucleo-
some substrate. These assays allowed another check for
nonideal behavior and a preliminary categorization of
compounds based on potential mechanism of inhibition
(MOI). This MOI assessment was based on whether they
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Table 3. Central Core SAR
Table 4. Further Modification of Right-Hand Aryl Ring
a
Y641N mutant EZH2 used.
Table 5. Tetramethylpiperidine SAR
maintained activity. The converse methylene insertion
(−OCH2−, 8) led to a loss of EZH2 affinity. Conversion of
ether 6 to thioether 9 maintained potency; however, oxidation
of the sulfur led to ablation of activity (Table 1, 10 and 11).
Interestingly, ketone 12 was tolerated, while the corresponding
racemic alcohol 13 was not. At this point, we postulated that
the ether, thio, and ketone linkages were imparting a
conformational bias to the system, which led to enhanced
potency over the sulfoxide, sulfone, and benzylic alcohol.
Indeed, diphenyl ethers are known to adopt a twisted
orientation due to a 1,5-CH-aryl clash and optimized π-overlap
with the lone pairs on the ether oxygen.23,24 Diphenylamine is
known to adopt a similar conformation to benzophenone,
which is more coplanar than the conformation of diphenyl
a
b
Y641N mutant EZH2 used. CACO-2 permeability assay.
ethers;24,25 therefore, absence of activity for compounds 13 and
14 could be attributed to preference of EZH2 for H-bond
acceptors and not donors as a linking group in addition to an
optimum conformation between the phenyl rings. This
hypothesis was further supported by the lack of activity for
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Scheme 1. Synthesis of Compound 44
(a) K2CO3, DMSO, 140 °C, 3 h, 81% yield; (b) EDC, HOBT, TEA, DCM, 23 °C, 3 h, 30% yield; (c) 10 mol% Pd(PPh3)2Cl2, LiCl, toluene, 90 °C,
12 h, 60% yield.
position (26, LipE = 3.8). Further exploration revealed a 15-
fold boost in potency with the incorporation of a 2-cyano
substituent (27, LipE = 4.8), suggesting the cyano nitrogen was
making a favorable interaction with the binding site in EZH2.
The 2-cyano substituent was utilized as the preferred
substituent for further SAR development.
We next addressed elaboration of the central aromatic ring.
While substitution proximal to the amide linkage did not yield
an improvement in biochemical potency relative to 27 (Table
3, 28−30), modification of the central core to a naphthyl
system did provide a 4-fold enhancement (Table 3, 31). We
suspected that monosubstitution adjacent to the ether linkage
would stabilize the intrinsic twist of the biphenylether and thus
enhance potency. We were gratified to find that this was the
case as fluoro, methyl, and cyano substitution all led to a
minimum of a 3-fold improvement (Table 3, 32−34). Both
cyclopropyl (35) and chloro (36) substitution improved
potency 6-fold over 27 with 36 being the more efficient
modification (0.45 μM, LipE = 5.1) at this position and
provided a compound with meaningful levels of potency against
EZH2 Y641N mutant (36, 7.9 μM). Switching from chloro
(36) to bromo (37) increased lipophilicity leading to a modest
boost in potency with no increase in efficiency as indicated by
the static LipE (5.1). Disubstitution of the central core did not
provide an advantage (Table 3, 38 and 39). The chloro group
was selected for further optimization.
In an attempt to improve the biochemical potency of the
tetramethylpiperidinyl-class of inhibitors, we chose to further
evaluate the right-hand aryl ring (Table 4). Methyl and chloro
Figure 3. Activity of compound 44 in KARPAS-422 cells. (A) Viability
of KARPAS-422 cells with growth inhibition observed with varying
substitution alpha to the 2-cyano group improved binding to
[44] and days of exposure. (B) Compound 44 treatment results in
EZH2 wild-type and Y641N mutant 3- and 4-fold over 36,
dose- and time-dependent reduction in global H3K27me3 levels in
respectively (Table 4, 40 and 41). Introduction of a pyrazole
KARPAS-422 cells. Total H3 levels were determined and used for
alpha to the 2-cyano group (42) led to a modest improvement;
however, inclusion of a 6-membered heterocycle enabled sub-
100 nM potency against the wild-type enzyme (Table 4, 43−
45). Changing the nitrogen aryl substitution from 4-pyridyl
(43, LipE = 5.4) to pyridazine (44, LipE = 6.8) then pyrimidine
(45, LipE = 6.3) led to improvement in both affinity and
efficiency.
Compound 44 was evaluated in a CACO-2 assay and found
to have low passive permeability and a high efflux ratio,
indicative of a compound with the potential for poor oral
bioavailability.26,27 We therefore sought to address this liability
through modification of the tetramethylpiperidine (Table 5).
One approach was to attenuate the basicity the nitrogen via
inductive effects; unfortunately, N-ethanol 46 lost 10-fold
against EZH2 wild-type, while difluoropiperidine 47 was
inactive (compare to 36). Indeed, a strongly basic nitrogen is
preferred for activity as pyran 48 was 38-fold less potent than
normalization purposes. Experiments were carried out in triplicate
SD.
carbon linked compounds 16−18, where the phenyl rings
should adopt a near orthogonal orientation.
Holding the diphenylether constant, we next evaluated the
right-hand aryl ring. Incorporation of a 2-methyl group (Table
2, 19) was nearly equipotent with 6, and the continued scan led
to progressive loss of potency at the 3- and 4-positions (Table
2, 20 and 21). Focusing on the 2-aryl position, 2-chloro (21)
provided a modest improvement, while 2-methoxy (23) was
equipotent to 6. Extension of the 2-methoxy to 2-benzyloxy
(24) or branching of the 2-methyl to 2-t-butyl (25) led to a 10-
fold boost in potency with no increase in LipE, illustrating that
this position was tolerant of diverse functionality and
potentially reinforced an optimal conformation. A modest
boost in LipE was coupled with smaller rigid groups at the 2-
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44. It is worthy of note that 48 no longer displays efflux (efflux
ration = 1.1) and was quite permeable (A to B 21.9 × 10−6 cm/
s), lending support to the hypothesis that poor permeability
and efflux were a direct result of the basic nitrogen of 44.
Further manipulation of the tetramethylpiperidine through
selective methyl group deletion also provided compounds with
a significant reduction in activity (49−50). In the face of an
apparent SAR cliff around the modification of the tetrame-
thylpiperidine, we attempted to improve the permeability of
this series by manipulating the amide linker between the
tetramethylpiperidine and central core. Both ester (51) and
ether (52) were tolerated displaying a 4-fold loss in potency,
while improving permeability and reducing efflux. The
improved permeability achieved with 52 did not translate
into an improved shift between biochemical potency and
reduction of H3K27me3 in HeLa cells (HeLa cell assay 44 = 7
μM;17 52 ≥ 15 μM); therefore, compound 44 was prioritized
for further evaluation as a tool compound for the study of
EZH2 disease biology.
The synthesis of 44 commenced with a SNAr reaction to
establish the diphenyl ether linkage (Scheme 1). EDC-
mediated peptide coupling furnished the tetramethylpiperidin-
yl-benzamide scaffold primed for exploring diversity on the
right-hand side aryl ring. The pyridazine was coupled using
standard Stille conditions to provide 44 in three chemical steps
and 15% overall yield.
Time and concentration-dependent exposure of KARPAS-
422 cells to 44 led to growth arrest (Figure 3A) as well as dose-
dependent reduction in global H3K27me3 levels (Figure 3B).
These results are consistent with previous reports and validate
this scaffold as a useful tool compound to interrogate the
inhibition of EZH2.9−16
authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Shuangyi Wan of WuXi App Tec for expediting the
characterization of compound 44. We also thank Mike Hewitt
for insightful conversations and assistance in preparation of this
manuscript.
ABBREVIATIONS
■
K2CO3, potassium carbonate; DMSO, dimethylsulfoxide; EDC,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlor-
ide; HOBT, 1-hydroxybenzotriazole hydrate; TEA, triethyl-
amine; DCM, dichloromethane; Pd(PPh3)2Cl2, bis-
(triphenylphosphine)palladium(II) dichloride; LiCl, lithium
chloride; EZH2, enhancer of zeste homologue 2; PRC2,
polycomb repressive complex 2; H3K27me3, histone 3 lysine
27 trimethyl
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This work described the identification and optimization of a
new class of inhibitors of the catalytic activity of EZH2/PRC2,
the tetramethylpiperidinyl benzamides. Compound 6 was
initially identified as a SAM-competitive compound with
acceptable LipE and a modular synthesis well suited for rapid
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures, characterization data, the synthesis of
compound 44, and KARPAS-422 assay information. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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constellationpharma.com.
Present Address
‡Dep
́
artment de Chimie, Universite
́
du Queb
l, QC H3C 3P8, Canada.
́ ̀ ́
ec a Montreal,
C.P. 8888, Succ. Centre-Ville, Montrea
́
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†These authors (C.G.N. and A.G.) contributed equally. The
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