Design, Synthesis, and Pharmacological Evaluation of Second Generation EZH2 Inhibitors with Long Residence Time

Article abstract of DOI:10.1021/acsmedchemlett.0c00045

Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27 - a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.

Full text of DOI:10.1021/acsmedchemlett.0c00045

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Letter
Design, Synthesis and Pharmacological Evaluation of Second
Generation EZH2 Inhibitors with Long Residence Time
Avinash Khanna, Alexandre Côté, Shilpi Arora, Ludivine Moine, Victor S Gehling, Jehrod Brenneman,
Nico Cantone, Jacob I Stuckey, Shruti Apte, Ashwin Ramakrishnan, Kamil Bruderek, William D.
Bradley, James E. Audia, Richard T. Cummings, Robert J. Sims, Patrick Trojer, and Julian R. Levell
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00045 • Publication Date (Web): 26 Mar 2020
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Design, Synthesis and Pharmacological Evaluation of Second
Generation EZH2 Inhibitors with Long Residence Time
Avinash Khanna‡, Alexandre Côté‡, Shilpi Arora, Ludivine Moine, Victor S. Gehling, Jehrod
Brenneman, Nico Cantone, Jacob I. Stuckey, Shruti Apte, Ashwin Ramakrishnan, Kamil Bruderek,
William D. Bradley, James E. Audia, Richard T. Cummings, Robert J. Sims III, Patrick Trojer, Julian R.
Levell
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Constellation Pharmaceuticals 215 First St. Suite 200, Cambridge MA, 02142
KEYWORDS: Residence time, EZH2, thiomethyl pyridone, CYP induction
ABSTRACT: Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of
histone H3K27 - a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic
malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. 1st
generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a 2nd generation EZH2 inhibitor could further
build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign,
we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time.
Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced anti-tumor activity and improved physiochemical
properties, which have the potential to expand the clinical use of EZH2 inhibition.
Polycomb repressive complex
responsible for the tri-methylation of lysine 27 on histone 3
(H3K27), modification which serves to repress
2
(PRC2) is
H3K27Me3 that result from EZH2 upregulation reinforce the
silencing of target genes, which promote differentiation and
restrain proliferation in normal cells[10]. Clinical EZH2
inhibitors (Figure 1) are currently being evaluated as
monotherapy and in combination in both hematological
malignancies and solid tumors, including specific genomic
contexts such as mutations in EZH2 and components of
SWItch/Sucrose non-fermentable (SWI/SNF) – a nucleosome
remodeling complex (NCT03213665). Initial results in the
clinic have further validated the role of EZH2 catalytic activity
in the context of follicular lymphoma, where patients with
activating mutations respond at a higher rate to EZH2 inhibitors
than those with wild-type EZH2 [11].
a
transcription[1]. Enhancer of zeste homologue 2 (EZH2), a
subunit of PRC2, catalyzes the transfer of a methyl group from
the co-factor S-adenosyl-L-methionine (SAM) to the -NH2
group of H3K27 culminating in trimethylation of H3K27
(H3K27Me3)[2] and subsequent silencing of targeted genes[3].
The dysregulation of target genes by the “trimethylation marks”
has been implicated in a variety of disease processes,
particularly oncogenesis[4, 5].
EZH2 has been reported to be mutated or
overexpressed in a broad spectrum of oncology indications such
as prostate cancer[6], lung cancer[7], myeloma, and
lymphoma[8]. Furthermore, activating mutations in EZH2 have
been shown to be a key driver of diffuse large B-cell lymphoma
and follicular lymphoma[9], while resistance to hormone
therapy has been shown to arise from upregulation of EZH2
expression in prostate cancer[6]. The increased levels of
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EZH2 inhibitors in the clinic have been reported to exhibit
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the active site as the SAM co-factor (Figure 1) [16] [17]. In
particular, the amino acid moiety of either SAM or S-
adenosylhomocysteine (SAH), and the pyridone motif of the
EZH2 inhibitors overlap (highlighted in Figure 1). The
pyridone competes with the co-factor for the H-bond donor-
acceptor interaction with tryptophan 624. Specifically, the N-H
of the pyridone serves as an H-bond donor for the peptide-
backbone’s carbonyl oxygen. Accordingly, we surmised that
favoring the compound’s tautomer population towards the
pyridone over the hydroxypyridine would lead to more potent
EZH2 inhibitors. This analysis suggested to us that the
electronic nature of the pyridone, electron-rich or -poor, would
be highly critical for the inhibitor’s potency for EZH2.
Therefore, using our CPI-1205 indole scaffold as a starting
point we generated a variety of substituted pyridones, including
compounds 1-5 (Figure 2). The biochemical potencies of these
inhibitors were found to be in the range of 30 nM to 0.2 nM,
and their comparison showed that heteroatom substitution was
1
2
3
4
5
6
7
8
favorable safety profiles and display anti-tumor activity,
particularly in hypersensitive contexts, such as lymphoma, and
in combination [12]. Initial dose escalation studies have
resulted in recommended phase 2 doses of >800 mg BID[13].
Even though clinical agents have thus far been found to be well-
tolerated, significant reduction in exposure is observed after
multiple dosing in humans [12, 14], consistent with induction
of CYP3A4 leading to faster metabolism of the drug [11],[15].
The high dose and CYP induction present potential limitations
when including first generation EZH2 inhibitors as part of a
combination therapy. Furthermore, our preclinical data showed
that prolonged treatment is necessary to trigger a decrease in
H3K27Me3, in agreement with the slow turnover kinetics of
histone H3K27Me3 modification. To maximize the oncology
application potential of EZH2 inhibition in the clinic, we
initiated a 2nd generation program that sought to achieve
increased and more durable target coverage.
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Figure 1. Left: Clinical inhibitors of EZH2 and S-adenosyl-methionine, the endogenous co-factor of EZH2. Right: Overlay of SAH (S-
adenosylhomocysteine) in cyan bound to EZH2 (grey) and a CPI-1205 analog (green), with the structures of the ligands depicted above.
We initiated our medicinal chemistry campaign with an
analysis of inhibitor bound EZH2 co-crystal structures, which
preferred at the 4-position over the 2-position, with the 4-
thiomethyl-pyridone exhibiting the lowest IC50. The rank
10000
O
EZH2 IC50
N
N
H
H3K27Me3 EC50
1000
100
10
40
Residence Time
F
F
F
N
20
0
1
0.1
2
3
CPI-1205
4
5
1
O
O
O
O
O
O
HN
HN
HN
HN
HN
HN
Cl
O
O
S
Cl
showed that the pyridone moiety occupies the same pocket of
ordering of pyridone biochemical potencies also correlated well
Figure 2. TR-FRET and MSD based EZH2 inhibition assay, and TR-FRET based residence time characterization.
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O
with their activity in the cellular H3K27Me3 level reduction
assay. Inhibitors 2 and 3, which feature a 2-chloro and a 2-
methoxy pyridone, respectively, were found to have cellular
S
10
11
19 (6)
>304
>304
1
2
3
4
Me
O
S
4.1 (0.4)
O
Table 1: Optimization of 4-thioether pyridone
Me
5
6
7
8
O
O
a Average with the standard deviation reported in parentheses,
where n > 2.
HN
N
N
H
R
F
N
F
EC50s of >1 µM. Whereas, the inhibitors that feature
heteroatoms at the 4-position, such as 5, 1 and CPI-1205 were
found to exhibit cellular EC50’s of <100 nM. In particular, the
4-thiomethyl pyridone 1 was found to be the most potent in both
the biochemical and the cellular H3K27Me3 mark reduction
assays (EC50 = 2.5 nM). Due to the over-performance of 1 in the
cellular assay, we sought to characterize the residence time of
the more active analogs, 4, 5, 1 and CPI-1205. We found that,
although their biochemical potencies were in a similar range
(180 pM - 800 pM), the thiomethyl pyridone 1 exhibited a
distinctly longer residence time. More starkly, 1 exhibits nearly
a 10-fold greater residence time than its oxygen analog, CPI-
1205 (Figure 1), in agreement with a 10-fold improvement in
both the H3K27Me3 EC50 and the GI50 (KARPAS-422
lymphoma cells).[18] Note, we also tested the 2-thiomethyl, 4-
methyl substituted pyridone analog and found it to be inactive in
the biochemical assay, similar to compound 3.
F
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H3K27Me3
EC50 a (nM)
Cmpd
R
IC50 a (nM)
S
1
0.18 (0.06)
2.5 (0.54)
Me
S
6
7
0.73 (0.04)
0.49 (0.04)
45.3
Et
S
48.5
S
8
9
0.62 (0.2)
3.8 (0.5)
181
-
Ph
S
Bn
Table 2. Optimization of piperidine sidechaina
O
O
HN
N
N
H
R
S
H
H
H
H
R
=
O
O
OH
F
N
N
N
O
F
F
N
N
HN
N
O
F
OMe
F
Cmpd
EZH2 IC50
(nM)
H3K27Me3
EC50 (nM)
CYP3A4
UpReg @ 10
M
1
12
13
14
15
16
17
0.18 (0.06)
2.5 (0.54)
0.067 (0.01)
26
0.12 (0.007)
5.1
0.18 (0.03)
5.9 (2.0)
0.13 (0.02)
6.7 (2.81)
0.16 (0.01)
2.4 (0.58)
0.059 (0.01)
2.0 (0.49)
25X (3.5)
-
6.4X (1.0)
45X (1.9)
5X (0.79)
3X (0.18)
2X (0.12)
cLogD
 (h)
3.8
2.9
3.2
42.5 (0.45)
3.8
3.0
2.3
3.3
46.3 (2.1)
7.52 (0.45)
29.1 (0.05)
138 (5.0)
40.9 (4.5)
141 (8.8)
a Average with the standard deviation reported in parentheses, where n > 2.
Table 3. Optimization of piperidine sidechain and indole-core
O
O
O
O
HN
N
H
HN
N
H
N
H
H
Het
S
S
N
N
O
R
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1
2
3
N
N
N
N
N
O
O
CF3
O CF2H
O
Me
N
N
N
N
F
N
Cl
F
4
5
6
7
Cmpd
17
0.059
(0.01)
18
0.14
(0.02)
19
0.10
(0.03)
20
0.072
(0.05)
21
0.057
(0.009)
22
0.063
(0.01)
23
0.10
(0.01)
24
0.092
(0.04)
25
0.060
(0.0004)
EZH2 IC50
a (nM)
H3K27Me
8
9
3 EC
2.0 (0.49)
1.8 (0.01)
0.7 (0.13)
3.5
1.8 (0.5)
3.4
6.3 (0.9)
3.5 (0.5)
1.8 (0.4)
50
(nM)
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CYP3A4
Up-Reg @ 2X (0.12)
10 µM
41X
(0.03)
17X
(0.01)
2.2X
(0.002)
1.6X
(0.01)
32X (0.4)
4.0
-
-
12X (0.3)
3.2
cLogD
3.2
3.6
3.5
3.2
3.6
2.8
3.7
KARPAS-
422
33
32
-
12 (1.3)
211 (29)
13
12
32
29
37 (4.4)
24 (3.4)
GI (nM)
50
141 (8.8)
289 (22)
57.5 (5.2)
100 (2.7)
51.9 (1.9)
64.2 (5.2)
65.8 (1.2)
 (h)
a Average with the standard deviation reported in parentheses, where n > 2.
1500
1000
500
0
2000
1500
1000
500
0
Vehicle (PO)
Vehicle (SC)
Vehicle
17 50 MPK BID-SC
Tazmetostat 160 MPK BID-PO
1 50 MPK BID-SC
17 50 MPK BID-SC
0
5
10
15
20
25
0
5
10
15
20
25
a)
b)
Day
Day
Figure 3. a) Evaluation of 1 and 17 in KARPAS-422 lymphoma xenograft mouse model. b) Evaluation of 17 in 22Rv1 prostate cancer xeno-
graft mouse model
Having identified a novel pyridone motif that
engenders increased residence time and cellular potency, we
generated a series of 4-thioalkyl substituted pyridones, 6-11
(Table 1). We found that increasing the substituent size (6-9)
led to a decrease in potency. Likewise, oxidation of the
thioether to a methylsulfinyl or a methylsulfone led to an even
greater decrease in potency. Given the large drop in the
biochemical potency, we did not characterize the residence time
for this series. The 4-thiomethyl substituent, exemplified in 1,
was found to be the most potent EZH2 inhibitor. We have
applied this finding to many other clinical EZH2 scaffolds by
replacing their pyridone with the 4-thiomethylpyridone of 1 and
Being conscious of the previously reported CYP3A4
induction, we sought to characterize the propensity of 1 to
induce expression of the cytochrome p450 enzyme CYP3A4.
Therefore, we treated the immortalized human hepatpocytes,
Fa2N-4 cells with 10 M of 1 and found nearly a 25-fold
increase in the expression of CYP3A4 [21]. With the 4-
thiomethyl-pyridone motif in place, we sought to optimize the
piperidine sidechain to further enhance potency and to limit
CYP3A4 upregulation. We found that introduction of beta-
oxygenation led to a decrease in CYP3A4 upregulation (12 and
13), however this also led to a decrease in biochemical potency
(Table 2). The potency was enhanced by replacing the
piperidine with an exocyclic amine, as in 14 and 17. Further
characterization of compound mediated CYP3A4 upregulation
showed that 17 only upregulated CYP3A4 2-fold, in comparison
to 25-fold with 1. SAR of the piperidine sidechain showed a
correlation between cLogD and CYP3A4 upregulation, where
the compounds with lower cLogD exhibited limited CYP3A4
upregulation (Table 2)[22]. We determined that a 2-fold
increase of CYP3A4 expression, under our assay conditions of
10 M compound treatment, would be sufficient for
progressing the program given that we do not anticipate
found a similar increase in inhibitor residence time (8
- 23
fold)[19]. We hypothesize that the superiority of the 4-
thiomethyl substitution is due to a combination of multiple
factors, including: i) pyridone:hydroxypyridine tautomeric
ratio; ii) the larger van der Waal’s radius of sulfur which leads
to a larger hydrophobic buried surface area; iii) the tighter
methyl contact with the protein due to larger C-S bond lengths;
and possibly iv) a positive S- interaction with the electron rich
sidechains of phenylalanines 665 and 686.[20]
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achieving such high concentrations in vivo. As a result, by
supplemental). Meanwhile, Tazmetostat was dosed out to day
24 to ensure the benchmark compound also achieved
regression.
1
2
3
elaborating from 1 to 17, we improved biochemical potency,
retained cellular activity, enhanced residence time and limited
CYP3A4 upregulation.
We then characterized the efficacy of 17 in the 22Rv1
prostate cancer xenograft model in mice. 22Rv1 cells have been
shown to express high levels of EZH2 when compared to other
prostate cancer cell models, and they are dependent on
androgen receptor signaling for growth [24]. The 22Rv1 cell-
line is not sensitive to androgen-degrader therapies such as
Eznalutamide because it expresses the AR-V7 isoform of the
androgen-receptor, which lacks the ligand-binding domain[25].
We found that a 50 mg/kg BID - SC dose of 17 led to ~ 55%
TGI.
4
5
6
7
8
9
Table 4: In vitro and in vivo characterization of 1, and 21
21
0.057
1.8
Assay
1
EZH2 IC50 (nM)
H3K27Me3 EC50 (nM)
cLogD (StarDrop)
0.18
2.5
3.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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3.1
Although 17 achieved in vivo efficacy in both models,
CYP3A4 UpReg
@ 10 µM
2.2X
25X
it was not orally bioavailable, therefore we next sought to
improve upon the inhibitor’s physiochemical properties. We
hypothesized that the methoxy substituent was not lipophilic
enough to mask to heightened basicity of azetidine 17, this
potentially led to decreased absorption[16]. We replaced the
methyl group of the azetidinol with substituents that would
mitigate the basicity of the exocyclic amine, such as
fluoroalkyls 18, and 19. We also replaced the methyl group of
the azetidinol with a cyclopropane in 20 to increase the
lipophilicity, and in-turn improve permeability. Through these
modifications we found that the methyl to cyclopropyl
replacement resulted in a 3-fold improvement in GI50 (17 vs.
20). Although these modifications led to increased potency,
particularly in the cellular proliferation assays, as seen with 19
and 20, they also resulted in an increase of CYP3A4
upregulation, potentially due to their higher clogD. To combat
CYP3A4 upregulation, we retained the cyclopropoxy sidechain
of 20 and introduced heteroatoms within the central indole-core
to increase polarity. In particular, we introduced a nitrogen
within the central core to generate aza-indole 21, which retained
many of the desirable properties of 17 such as cellular potency
and decreased CYP3A4 upregulation (~5 fold 20 vs. 21).
Replacing the aza-indole in 21 with a fluoro-indole in 22 led to
diminished activity in the cellular assays, including the cell
proliferation assay. We also generated the pyrrolopyrimidine 23
in an effort to decrease the clogD, however this further led to
loss in cellular activity. 5-halosubstituted aza-indoles 24 and 25
were found to be less active in the cellular assays, and, in the
case of 25, led to a greater CYP3A4 upregulation than 21.
Unlike the clear correlation observed with our earlier sidechain
modifications (Table 2), we did not observe a strict correlation
of clogD with CYP3A4 upregulation in the case of compounds
20-25. Nevertheless, during this survey we identified 21, an
aza-indole which provided a balance of favorable properties
such as improved potency, and negligible CYP3A4
upregulation, while also exhibiting 15% oral bioavailability
(Table 4).
KARPAS-422 GI50 (8 days)
(nM)
12.2
57.5
3.35
N/A
46.3
0.56
 (h)
Mouse IV
Clearance (L/h/Kg)
Mouse oral bioavailability - F
(%)
15
40
46.5
AUClast/D (h*mg/mL)
414.4
87, 97
Plasma Protein Binding - m, h
99.8, 97
91
77
KARPAS-422 %TGI
@ 25 MPK
BID-PO
@ 50 MPK
BID-SC
Given the improvements in potency and limited
CYP3A4 upregulation, we sought to characterize 1 and 17 in the
KARPAS-422 lymphoma xenograft mouse model (Figure 3). A
160 mg/kg BID - PO dose of Tazemetostat, which has
previously been shown to cause regression in this model[23],
was used as a benchmark compound in the study. With the goal
of rank-ordering compounds for our medicinal chemistry
campaign, we kept the BID dosing regimen constant during our
in vivo studies and evaluated efficacy and kinetics of achieving
regression. We dosed inhibitors at 50 mg/kg BID, which we
hypothesized would provide a dynamic range of efficacy
between compounds. 1 and 17, which were both dosed SC,
began to cause regression at days 7 and 9, respectively, a
marked improvement over the benchmark. To ensure enough
tumor sample could be obtained for downstream
characterization, mice in the treatment arms of 1 and 17 were
sacrificed after 17 days (no body weight loss observed, see
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1000
Hela EC50
KARPAS-422 GI50
1
2
3
4
5
6
7
8
ns
****
1500
1000
500
0
Vehicle
30
100
21 25 MPK BID-PO
20
10
1
10
0
0.1
0
5
10
15
20
25
1 h
4 h 10 h
Plasma Day 1
Vehicle Day 14
21 25 MPK Day 14
a)
b)
c)
Day
9
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Figure 4. a) Evaluation of 21 in KARPAS-422. b) Pharmacokinetics of 21 at day 1 of treatment. c) Modulation of H3K27Me3
pharmacodynamic marker upon treatment for 14 days
Scheme 1. Synthesis of 4-thiomethyl aminopyridone Int1a
O
Na
O
a
b
c
CN
O
Me
O
S
HN
O
S
CN
Na
+
H2N
1.0 equiv
Me
S
S
S
Me
Int1a
Int1b
Int1c
O
O
O
Boc
HN
NH2
HN
N
d
e
f
HCl
HN
NH2
H
S
S
S
Me
Me
Int1d
Int1e
Int1
Me
aReagents and conditions: (a) t-BuONa (2 equiv), CS2 (1.0 equiv), toluene, 0 °C, 4 h; (b) MeI (2.0 equiv), MeOH, 70 °C, 1 h (42% yield over two steps); (c)
t-BuONa, t-BuOH, 80 °C, 12 h (75% yield); (d) BH3-Me2S (4.0 equiv), THF, 70 °C, 2 h; (e) Boc2O (3.0 equiv), Et3N (1.5 equiv), THF, 25 °C, 12 h (75% yield
over two steps); (f) HCl, MeOH, 70 °C, 2 h.
Scheme 2. Synthesis of 17a
O
O
O
O
O
H2N
HBr
H2N
H2N
H
N
(a)
(b)
(c)
(d)
HN
Br
H
O
O
OH
O
OH
Br
OH
1.1 equiv
OH
26
27
28
29
O
O
O
HO
O
O
O
N
H
N
H
N
H
HN
O
(f)
(g)
(e)
N
H
O
6
N
N
N
7.5 equiv
O
30
31
32
33
O
O
O
F
1
:
8
F
F
O
O
O
F
O
F
HN
N
N
H
N
H
H
(h)
(i)
S
N
N
O
34
17
O
aReagents and conditions: (a) HBr (35% in AcOH), 100 °C, 12 h; (b) PtO2 (0.05 equiv), H2 (55 psi), AcOH (1.5 equiv), MeOH, 50 °C, 30 h; (c) MeONa (1.1
equiv), MeOH, 25 °C, 1 h then ketoester (1.1 equiv), AcOH (1.3 equiv), tBuOH, 85 °C, 12 h (7.6% yield over 3 steps); (d) RuPhos precatalyst (generation 2)
(10 mol %), dioxane, 100 °C, 13 h (88% yield); (e) Dess-Martin reagent (2.0 equiv), DCM, 10 °C, 16 h (77% yield); (f) iPr2NEt (8.3 equiv), MeOH, 25 °C, 1
h then 30 (1.0 equiv), THF, 25 °C, 2 h then LiBH4 (1.4 equiv), -70 °C, 15 min (45% yield for desired diastereomer); (g) NaOH (10.0 equiv), MeOH, H2O, 80
°C, 12 h; (h) perfluorophenyl 2,2,2-trifluoroacetate (1.5 equiv), pyridine (2 equiv), DCM, 25 °C; (i) Int1 (3.0 equiv), iPr2NEt (3.0 equiv), DMF, 25 °C, 12 h
(31% yield over 2 steps)
Next, we profiled 21 in the KARPAS-422 lymphoma
mouse model at 25 mg/kg BID – PO, which is nearly a 6-fold
lower dose than the benchmark in figure 3. In this study, we
found that the tumors began to regress at day 14 and resulted in
TGI of 75% by day 21, with no impact on mouse body weight
(see supplemental information). Unbound plasma levels of 21
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were found to be above H3K27Me3 mark reduction assay EC50
in the tumors for 4 hours. Tumor PD experiments showed that
H3K27Me3 mark was significantly decreased at day 14 (Figure
4). The oxidative metabolic stability for 21 was not
characterized in metabolic liability identification studies.
However, we did not observe 10 or 11 during mouse PK
experiments of 1.
carbonitrile (see supplemental). The synthesis of the 4-
thiomethylaminopyridone intermediate 1 commenced by
condensing carbon disulfide onto acetone to give sodium
bisthiolate Int1a. The thiolates were then capped with
methyliodide to provide thioacetal Int1b, which was subjected
to an aldol condensation reaction with 2-cyanoacetamide, that
results in eventual cyclization under thermal conditions to
provide cyanopyridone Int1c. The resulting nitrile was reduced
to the primary amine, which was protected with Boc anhydride
to simplify its purification and isolation (24% yield from
acetone). Subsequent protecting group removal with
hydrochloric acid resulted in a bench-top stable salt (>6
months) of 4-thiomethyl aminopyridone Int1.
1
2
3
4
5
6
7
8
In summary, the goal of our second generation EZH2
medicinal chemistry campaign was to improve target coverage,
and to limit CYP3A4 upregulation. Starting with a reassessment
of the binding interaction of the inhibitor and EZH2, we
identified the 4-thiomethyl substituent as a key modification for
the enhancement of potency and residence time in 1. We then
tackled the challenge of retaining the improved cellular potency
and limiting CYP3A4 upregulation through the identification of
an exocyclic azetidine sidechain in 17. As a result, we were able
to expand the utility of an EZH2 inhibitor to an enzalutamide-
resistant prostate cancer mouse model (22Rv1). Although 17
provided impressive efficacy, it was not orally bioavailable.
Therefore, we replaced the indole-core with a set of
heterocycles, which led to the discovery of 21.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds containing the trifluoroehtylpiperidine
sidechain were synthesized using the general scheme
previously described [16] (see supplemental for synthesis of
pyridones). The synthesis of 17 (Scheme 2) began with
demethylation of the commercial (R)-p-methoxybenzylamine,
followed by its reduction, using PtO2 at 55 psi of H2, to
aminoalcohol 27. The aminoalcohol was then condensed with
the bromoketoester to generate enamine 28, and subsequently
cyclized to furnish the indole with the RuPhos precatalyst (Gen
II) in 88% yield. Cyclohexanol 29, was oxidized to ketone 30,
which also served as a key intermediate for 14-15, and 18-20.
The methoxyazetidine was appended via reductive
Aza-indole 21 was found to be a highly efficacious
EZH2 inhibitor, achieving >91% TGI in 25 days with the oral
dose of 25 mg/kg BID – PO. Pharmacokinetic (PK) and
pharmacodynamic (PD) profiling showed that the exposure
levels of 21 were sustained above cellular EC50 for 4 hours and
the level of H3K27Me3 mark reduction was also maintained.
The discovery of the 4-thiomethyl pyridone modification and
its ability to bestow durable target coverage was a key stepping-
stone to developing 2nd generation EZH2 inhibitors.
amination using a two-step procedure of imine formation and
reduction with lithium borohydride, which provided the desired
diastereomer in an 8:1 ratio (use of sodium cyanoborohydride
led to a 1:1 mixture of both diastereomers). The two
diastereomers were readily separated by column
chromatography. The desired diastereomer, 32, was hydrolyzed
and activated as a perfluoprophenyl (PFP) ester then directly
treated with the aminopyridone Int1 to give 17.
Chemistry
We took advantage of the well-precedented peptide
coupling of the aminopyridone and the indole acid to synthesize
most of the compounds included in this report[16]. The 4-
thioether amino-pyridones were synthesized by adapting the
route from scheme 1, while the 6-methoxy and 6-
chloropyridones were synthesized via the 6-hydroxypyridone
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Methods and Materials, Biochemical and Cellular Assay Procedures, Experimental Details for In Vivo Experiments, Synthetic
Experimental Details, and Compound Purity Assessment
AUTHOR INFORMATION
Corresponding Author
* Avinash Khanna: Avinash.khanna@constellationpharma.com. Tel. (617) 714-0556
Present Addresses
†Alexandre Côté (Schrödinger Inc., New York, NY), †Shilpi Arora (X-Chem Inc. Waltham, MA), †Ludivine Moine (Blueprint Medicines,
Cambridge, MA), †Jehrod Brenneman (KSQ Therapeutics, Cambridge, MA), †Jacob I. Stuckey (Third Rock Ventures, Boston, MA),
†Shruti Apte (X-Chem Inc. Waltham, MA), †Ashwin Ramakrishnan (BMS, Cambridge, MA), †Kamil Bruderek (Relay Therapeutics,
Cambridge, MA), †William D. Bradley (Cyteir Therapeutics, Lexington, MA), †James E. Audia (Third Rock Ventures, Boston, MA),
†Robert J. Sims III (Third Rock Ventures, Boston, MA)
Author Contributions
The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. /
‡These authors contributed equally. (match statement to author names with a symbol)
Funding Sources
Any funds used to support the research of the manuscript should be placed here (per journal style).
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ABBREVIATIONS
1
PTM, Post-translational modification; PRC2, polycomb repressive complex 2; H3K27, histone 3 lysine 27; EZH2, enhancer of zeste
homologue 2; SWI/SNF, SWItch/Sucrose non-fermentable; SAM, Sadenosyl-L-methionine; PD, pharmacodynamics; cLogD, calculated
LogD; SAR, structure activity relationships; PK, pharmacokinetic; SC, sub-cutaneous; IV, intravenous; PO, per os; F, bioavailability; DMA,
dimethylacetamide; PEG, polyethylene glycol; iPrNEt2, hunig’s base; CDI, carbonyldiimadazole; THF, tetrahydrofuran; TGI, tumor-growth
inhibition; BID, twice a day; QD, once a day; m, mouse; h, human; PtO2, platinum oxide; PFP, perfluoprophenyl
2
3
4
5
6
7
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Graphic entry for the Table of Contents (TOC):
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