Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent and Selective Inhibitor of Histone Methyltransferase EZH2, Suitabl

Article abstract of DOI:10.1021/acs.jmedchem.6b01315

Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis

Full text of DOI:10.1021/acs.jmedchem.6b01315

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Article
Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-
yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-
yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent
and Selective Inhibitor of Histone Methyltransferase EZH2,
Suitable for Phase I Clinical Trials for B-Cell Lymphomas
Rishi G Vaswani, Victor S Gehling, Les A. Dakin, Andrew Cook, Christopher G. Nasveschuk,
Martin Duplessis, Priyadarshini Iyer, Srividya Balasubramanian, Feng Zhao, Andrew C
Good, Robert Campbell, Christina Lee, Nico Cantone, Richard T. Cummings, Emmanuel
Normant, Steven F. Bellon, Brian K. Albrecht, Jean-Christophe P. Harmange, Patrick Trojer,
James E. Audia, Ying Zhang, Neil Justin, Shuyang Chen, Jon Wilson, and Steve Gamblin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01315 • Publication Date (Web): 14 Oct 2016
Downloaded from http://pubs.acs.org on October 15, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Identification of (R)ꢀNꢀ((4ꢀMethoxyꢀ6ꢀmethylꢀ2ꢀ
oxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylꢀ1ꢀ(1ꢀ
(1ꢀ(2,2,2ꢀtrifluoroethyl)piperidinꢀ4ꢀyl)ethyl)ꢀ1Hꢀ
indoleꢀ3ꢀcarboxamide (CPIꢀ1205), a Potent and
Selective Inhibitor of Histone Methyltransferase
EZH2, Suitable for Phase I Clinical Trials for BꢀCell
Lymphomas
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Rishi G. Vaswani,δ,* Victor S. Gehling, * Les A. Dakin,‡ Andrew S. Cook,∇ Christopher G.
Nasveschuk,† Martin Duplessis,† Priyadarshini Iyer,∥ Srividya Balasubramanian, Feng Zhao,
Andrew C. Good,⊥ Robert Campbell,§ Christina Lee,# Nico Cantone, Richard T. Cummings,
Emmanuel Normant,○ Steven F. Bellon,∂ Brian K. Albrecht,◆ JeanꢀChristophe Harmange,◆
Patrick Trojer, James E. Audia.*
Ying Zhang,≠ Neil Justin,≠ Shuyang Chen,≠ Jon R. Wilson,≠ Steve J. Gamblin,≠,*
Constellation Pharmaceuticals, Inc. 215 First Street, Suite 200, Cambridge, MA 02142, United
States.
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KEYWORDS
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Enhancer of Zeste Homolog 2 (EZH2); Histone Methyltransferase; Polycomb Repressive
Complex 2 (PRC2); Embryonic Ectoderm Development (EED); H3K27me3; Epigenetics;
KARPASꢀ422; SꢀadenosylꢀLꢀmethionine (SAM); SAMꢀCompetitive Inhibitor; Indole; Pyridone;
CPIꢀ1205.
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ABSTRACT
Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional
silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of
PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic
engine of the PRC2 complex and thus represents a key candidate oncology target for
pharmacological intervention. Here we report the optimization of our indole based EZH2
inhibitor series that led to the identification of CPIꢀ1205, a highly potent (biochemical IC50
=
0.002 µM, cellular EC50 = 0.032 µM) and selective inhibitor of EZH2. This compound
demonstrates robust antiꢀtumor effects in a Karpasꢀ422 xenograft model when dosed at 160
mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the coꢀcrystal
structure of our inhibitor series bound to the human PRC2 complex.
Introduction
It is well established that triꢀmethylation of lysine 27 on histone 3 (H3K27) contributes to the
modification of chromatin structure which serves to repress transcription.1–3 The addition of triꢀ
methyl ‘marks’ on H3K27 is generally catalyzed by the multimeric protein complex polycomb
repressive complex 2 (PRC2), through its enzymatic subunit enhancer of zeste homolog 2
(EZH2). EZH2 catalyzes the transfer of a methyl group from the coꢀfactor SꢀadenosylꢀLꢀ
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methionine (SAM) to the εꢀNH2 group of H3K27 culminating in trimethylation of H3K27
(H3K27me3) and subsequent silencing of targeted genes.
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Dysregulation of mechanisms that alter chromatin structure has been implicated in a variety of
disease processes, particularly oncogenesis.2 EZH2 is frequently overexpressed in a broad
spectrum of solid and hematological cancers such as prostate, breast, kidney, lung, myeloma, and
lymphoma.2,4 Elevated EZH2 transcript and protein levels in these cancers usually correlate with
greater levels of H3K27me3, advanced stages of disease, and poor prognosis.5,6 Additionally,
somatic recurrent mutations within the catalytic domain of EZH2 (the suppressor of variegation,
enhancer of zeste, trithorax (SET) domain) have been identified in diffuse large Bꢀcell
lymphoma (DLBCL), follicular lymphoma and melanoma.7 These mutations alter the substrate
specificity of EZH2 culminating in an increase in global levels of H3K27me3.4,8–10
Consequently, the increase in levels of H3K27me3, either by overexpression of EZH2 or its
altered function through mutations, in cancer tissues may reinforce the silencing of target genes
that promote differentiation and restrain proliferation.11 Alternatively, EZH2 may serve to
silence genes not targeted in normal cells to afford growth and survival advantage in the
malignant setting. Collectively, these observations offer a compelling argument for the inhibition
of EZH2 as a potential therapeutic approach for the treatment of cancer.
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Figure 1. Reported EZH2 inhibitors.
The development of small molecule inhibitors of EZH2 has been an intense area of research.
Several potent EZH2 inhibitors demonstrating promising antiꢀtumor activity in preclinical
species have been disclosed. These EZH2 inhibitors include EPZꢀ005687,12 EPZꢀ6438,13,14 EPZꢀ
011989,15 GSKꢀ343,16 GSKꢀ126,17 UNCꢀ1999,18 EI1,19 and our previously disclosed probes CPIꢀ
36020 and 1 (CPIꢀ169).21 Although these inhibitors originated from independent high throughput
screening efforts, they all share a common structural feature, a pyridone core. The prevalence of
a pyridone motif embedded within these inhibitors highlights the importance of this functionality
for high affinity binding. While a novel series of 4ꢀaminoꢀ2,2’,6,6’ꢀtetramethyl piperidine
analogues has also been identified as SAMꢀcompetitive inhibitors of EZH2, these analogues
demonstrated inferior cellular potency when compared to the pyridoneꢀbased inhibitors.22,23
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Currently, Phase I clinical trials of three different pyridone based EZH2 inhibitors tazemetostat
(EPZꢀ6438) (NCT01897571, NCT02601937, NCT02601950), GSKꢀ126 (NCT02082977), and
13 (CPIꢀ1205) (NCT02395601) have been reported.
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Medicinal Chemistry and Structure–Activity Relationships
We recently disclosed 1, a potent indole based EZH2 inhibitor that showed robust antiꢀtumor
activity and pharmacodynamic (PD) target engagement in a KARPASꢀ422 lymphoma xenograft
model in mice.21 This compound, however, suffered from limited oral bioavailability (0.09% F
and 0.12% F observed in rats and dogs, respectively).) As part of our ongoing drug discovery
and development efforts we continued to optimize the indole based scaffold toward clinical
candidate selection. Herein we report the optimization of the indole based EZH2 inhibitor series
that led to the identification of 13, a potent and selective inhibitor of EZH2 currently under
evaluation in Phase I clinical trials. In addition, we report the coꢀcrystal structure of a similar
pyridone containing inhibitor (10) bound to human PRC2. This structure has provided a context
for the molecular nature of the interaction between our chemical series and its target.
In an attempt to improve upon the physical properties of 1, we began the investigation of our
structure–activity relationship (SAR) with different Nꢀsubstituents on the piperidine ring. We
rationalized that the pyridoneꢀindole core was previously optimized with respect to biochemical
potencies (against both wildꢀtype and mutant EZH2) as evidenced by NꢀH piperidine (2) and Nꢀ
Me piperidine (3) analogues (Table 1). Unfortunately both 2 and 3 suffered from considerable
loss in cellular potency in the HeLa H3K27me3 mechanism of action (MOA) assay.
Derivatization of the NꢀH piperidine to a variety of amides, ureas, carbamates, and sulfonamides
yielded biochemically potent analogues. However, these analogues displayed less than ideal
cellular potencies when examined in the MOA assay. For example, substitution of the piperidine
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Nꢀatom to give the corresponding isobutyl amide (4), isopropyl carbamate (5), and NꢀMeꢀurea
(6) afforded approximately fiveꢀ, twoꢀ, and elevenꢀfold loss in cellular potencies relative to 1.
Table 1. Piperidine Structure–Activity Relationships.
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R
N
O
N
H
HN
N
O
O
HeLa
H3K27me3
(µM) b
EZH2 WT
EZH2 Y641N
Calc. pKa c
Entry
R
(µM)a
MT (µM) a
<0.0010
0.0028
0.080
ꢀ
1
0.0020
0.0039
0.0017
1.1 d
0.47
9.7
9.7
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3
<0.0010
0.0025
<0.0010
0.0033
0.016
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0.0089
0.0017
0.0025 d
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0.11
0.93
0.15
0.26 d
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ꢀ
<0.0010
<0.0010 d
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0.0023
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<0.0010
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0.0016
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Biochemical data generated via scintillation proximity assay (SPA) with PRC2, H3K27me3
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activator peptide and biotinylated oligonucleosomes and [H]ꢀSAM. IC50 values reported as an
b
average ≥2 determinations.
The mechanism of action cellular assay measured global
c
H3K27me3 levels in HeLa cells; see supporting information for further details. pKa values
generated via ChemDraw version 15.0 d IC50 value from a single determination.
In addition to the disparate trends in potencies (i.e., poor translation into cellular assays), Nꢀ
acylated derivatives (e.g., amides, ureas, carbamates, and sulfonamides) generally suffered from
high microsomal clearance or rapid clearance in vivo (Figure 2). In contrast, a variety of basic
amines derived from piperidine 2 showed acceptable ADME properties (low Clint and low CYP
inhibition). As such, we subsequently sought to improve the physiochemical properties of
piperdine 2 to attain the desired in vitro and in vivo potencies.
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Figure 2. General Trends in Structure–Activity Relationships.
We found that attenuation of the basicity and hence perturbation of the piperidine pKa had
profound effects on the cellular potency, selectivity, toxicity, bioavailability, and PK
properties.24 For example, oxetane 7 (ChemDraw calculated pKa was 7.6) demonstrated a 10ꢀ
fold improvement in the cellular potency when compared to piperidine 2 (ChemDraw calculated
pKa was 9.7).25 Other electronꢀwithdrawing substituents, such as acetate 8 also afforded
improvement in cellular potencies.
We synthesized nꢀtrifluoropropyl piperidine analogue 9 (calculated pKa ~8.2) and observed an
over threeꢀfold increase in potency (EC50 of 0.29 ꢀM) relative to piperidine 2 when tested in the
cellular MOA assay (Table 1). While introduction of the nꢀtrifluoropropyl motif was a step in the
right direction, we believed that cellular potency could be further enhanced. To examine the
effect of keeping the three carbon linker fixed, and moving the fluorine atoms to the 2ꢀposition
of the alkyl chain, we subsequently synthesized the 2,2ꢀdifluoropropyl containing analogue (10).
The pKa was calculated to decrease by half a log unit relative to analogue 9 (Chemdraw
calculated pKa 7.6 versus 8.2, respectively), but gratifyingly even this modest decrease in
basicity resulted in a tenꢀfold gain in cellular potency (EC50 of 0.020 ꢀM) over the linear nꢀ
trifluropropyl analogue. The addition of fluorine atoms in the βꢀcarbon (with respect to the
piperidine nitrogen) appeared optimal for attenuation of basicity of the piperidine and increase in
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cellular potency. As such a series of analogues that embedded the β,βꢀdifluoroethyl motif were
synthesized and further examined. The tetrafluoropropyl (11), difluoroethyl (12), and
trifluoroethyl (13) piperidines were prepared and these fluorinated analogues were comparable in
potency (EC50 of 0.038 ꢀM, 0.039 ꢀM, and 0.032 ꢀM, respectively) to 10.
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CoꢀCrystal of Ligand Bound to PRC2 Complex
In order to understand the mechanism of action within this lead series, we were able to exploit
the crystallization system developed for our recent structure of the human PRC2 catalytic
complex.26 Coꢀcrystals were obtained with the 2,2ꢀdifluoropropyl analogue (10) that diffracted
to 3.5 Å and the structure determined by molecular replacement (PDB code: 5LS6). Electron
density corresponding to compound 10 was identified in a pocket at the interface of the EZH2
SET domain, the SAL region of the EZH2 Nꢀterminus and EED (Figure 3A). The electron
density was improved by fourꢀfold averaging, so that even at 3.5 Å it is remarkably wellꢀdefined
and the position and orientation of the inhibitor could be unambiguously determined (Figure 3B).
Key residues that define the inhibitor pocket include EZH2 SET domain Tyr661, Phe665,
Tyr658 and Phe686, the EZH2 SAL region Tyr111 and Met110 and EED residues His213 and
Asp237. Although the inhibitor binding site partially overlaps with the pocket for SAH
(superimposed from PDB ID: 5HYN) it then extends in the opposite direction and is therefore
distinct from both the substrate and cofactor binding sites (Figure 3B and 3C). The partial
overlap between the 2,2ꢀdifluoropropyl piperidine 10 and the SAH carboxylic acid is consistent
with a SAM competitive mechanism of inhibition.
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Figure 3. (A) Surface representation of the coꢀcrystal structure of 10 (yellow spheres) bound to
the PRC2 complex (EED (grey), SUZ12 (pink), EZH2 (pale blue at Nꢀterminus to blue at Cꢀ
terminus) (PDB ID: 5LS6). (B) Electron density (NCS averaged Fourier Map (2FoꢀFc map
contoured at 2σ)) surrounding compound 10. Main interactions are between pyridone of 10 and
key residues from the SETꢀI and SAL domains of EZH2 and from EED (PDB ID: 5LS6). (C)
The PRC2ꢀcompound 10 complex (colored as above) superimposed on the PRC2 complex with
SAH cofactor and peptide (green). (PDB ID: 5HYN). (D) The pyridone and indole are
constrained in a binding site composed of residues from both the SET domain (blue) and SAL
region (pale blue) of EZH2 (PDB ID: 5LS6).
Overall 10 adopts a zigꢀzag like shape, which is defined by both the constraints of the pocket,
as well as the preꢀorganized conformation induced by the chiral methyl proximal to the indole
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and the torsional angle defined by the C3ꢀindole amide connecting to the pyridone. The indole
and piperidine are tightly constrained by the narrow hydrophobic channel and by a putative
hydrogen bond from the EED Asp237 sideꢀchain to one of the fluorine atoms. Beyond the
piperidine the pocket widens considerably, which is consistent with the diverse array of
substituents tolerated in this region (Table 1). At the opposite end of the molecule the pyridone is
surrounded by the side chains of Phe665, Phe686, and Trp624 and has the potential to form two
hydrogen bonds with the protein backbone of Trp624 (Figure 3D). Recently, a coꢀcrystal
structure of a human/chameleon hybrid PRC2 construct was reported that also contained a
pyridone based EZH2 inhibitor, (PDB codes: 5IJ7 and 5IJ8).28 Both pyridone orientation and
binding is consistent between the two structures.
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A major difference between the inhibitor complex structure and that obtained with
peptide/SAH occurs at the active site of the EZH2 SET domain (Figure 4). In the absence of
substrate peptide the Cꢀterminus of the SET domain occupies the histone binding groove with the
side chains of Tyr728 and Phe667 located to the target lysine channel. In the inhibitor complex
beyond residue Ser729 the Cꢀterminus is disordered. Similarly, in the structure of the isolated
apo EZH2, there is a pronounced rearrangement of this region.29 The alternate conformation of
the EZH2 Cꢀterminus observed in these structures are indicative of its inherent flexibility. It is
not clear to what extent the conformation observed in the inhibitor complex structure is due to
the binding of inhibitor or reflects the absence of cofactor and/or histone substrate.
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Figure 4. Comparison of the EZH2 Cꢀterminus with the SET domain active site. (A) Human
PRC2 complex with compound 10. (PDB ID: 5LS6) (B) Equivalent region of the human PRC2ꢀ
H3K27MꢀSAH complex, (PDB ID: 5HYN) with cofactor (orange) and histone H3 peptide (red).
(C) Overlay of residues forming the SET domain lysine channel for both complexes, highlighting
that the position of the Cꢀterminus conformation observed in the compound complex is
incompatible with substrate binding (red). (D) and (E) Surface representations showing that the
lysine binding channel is closed in the PRC2ꢀcompound 10 complex (PDB ID: 5LS6) but
accessible in the SAH / substrate complex (PDB ID: 5HYN). (F) Surface representation of EZH2
observed in the PRC2ꢀcompound 10 complex but with the H3 peptide overlaid from the PRC2ꢀ
H3K27MꢀSAH complex, confirming that substrate binding is incompatible with the EZH2
conformation in the PRC2ꢀcompound 10 complex.
In Vitro ADME and In Vivo Pharmacokinetics of Fluorinated Analogues
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Having established sufficient activity in the MOA assay, we subsequently profiled 9, 10, 12,
and 13 in an ADME panel (Table 2). Relative to 1, these fluorinated analogues generally
displayed higher plasma protein binding and in vitro clearance, which is presumably a function
of their increased lipophilicity (cLogP). Interestingly, there were significant interspecies
differences observed with regards to the in vitro microsomal clearances and PPB. The highest in
vitro microsomal clearance (>100 µL/min/mg protein) and plasma protein binding (>97% bound)
were uniformly observed in mice for 10, 12, and 13. Additionally, the in vitro clearance
measured from rat derived microsomes closely mirrored those derived from humans for these
analogues.
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
Table 2. ADME of Fluorinated Analogues 9, 10, 12, and 13.
Compound
cLogPa
9
10
12
13
3.39
3.1
3.71
4.0
3.19
4.2
3.41
4.1
LiPEb
Mouse
Rat
61.3
25.7
35.3
35.4
99.7
97.4
96.3
95.9
5.0
193
86.1
68.9
83.8
99.4
92.4
93.6
94.7
0
131
159
99.6
73.9
104
97.9
89.9
90.3
92.8
0
Clint
71.3
52.4
73.6
97.8
83.1
64.3
83.3
25.2
(µL/min/mg
Dog
protein)c
Human
Mouse
Rat
In Vitro
ADME
PPB
(% bound)d
Dog
Human
3A4
CYP Inh.
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7
8
9
(%at 10
µM)e
2D6
2C8
1A2
2C9
17.2
23.1
19.4
11.6
7.7
50.9
0
26.1
18.9
28.6
9.5
41
15
11.1
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
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41
42
43
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50
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5.0
25.4
a
b
ClogP values were generated with ChemDraw Professional 15.0.
LiPE = lipophilic
c
efficiency = pEC50 − cLogP (ChemDraw 15.0). Intrinsic clearance (Clint) of compounds in
d
mouse / rat / dog / human liver microsomes (expressed in µL/min/mg protein). Measured in
vitro binding of compounds to mouse / rat / dog / human plasma protein bindings (expressed in
% bound). e In vitro inhibition of cytochrome P450 isoforms at single concentration (10 ꢀM).
Based on their combination of potency and in vitro profile 12 and 13 were subsequently
evaluated for their in vivo metabolic profile and systemic exposure in mice in advance of planned
pharmacodynamic (PD) and efficacy studies (Table 3). Both 12 and 13 showed excellent oral
bioavailability demonstrating significant improvement over 1. Surprisingly, 12 displayed a poor
overall PK profile when compared to 13. When dosed in mice at 1 mg/kg intravenous (iv) and
100 mg/kg per os (po), 12 displayed high clearance of 4.45 L/h/kg (82% liver blood flow), low
volume of distribution (1.10 L/kg), short halfꢀlife (0.40 h), and excellent bioavailability (~92%
F). In contrast 13 exhibited moderate clearance of 2.16 L/h/kg (40% liver blood flow), a halfꢀlife
of ~1.6 h, similar volume of distribution (1.4 L/kg), and excellent bioavailability (100% F). As
part of the comparison we also examined total exposure (AUC) and unbound exposure
(AUCunbound) since we were interested in maintaining free levels of compound significantly
above the measured HeLa EC50. Both 12 and 13 achieved unbound exposures well above their
respective cellular potencies, however only the unbound exposure for 13 remained well above
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the cellular EC50 up to 4 h (Figure 5). Overall the in vivo metabolic profile of 13 provided a
compelling argument for further evaluation in a mouse xenograft model.
7
8
9
Table 3. Mouse Pharmacokinetic Profiles for Fluorinated Analogues 12 and 13.a
10
11
12
13
14
15
16
17
18
19
in
IV
PO
vitro
i
Cmax
AUC0ꢀinf
[AUCunbound
(ꢀM*h)
CL
f
g
j
t1/2
Vss
tmax
Fk
[Cmaxꢀ
h
d
%Qe
l
]
[CLunbound
]
Compound
fu
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
]
(h) (L/kg)
(h) (%)
unbound
(L/h/kg)
(ꢀM)
4.45
[202.3]
2.16
42.01
[0.924]
88.88
52.14
[1.15]
67.36
[1.41]
12b
13c
82 0.402 1.10
0.250 91.6 0.022
40
1.63
1.36
ꢀ
100 0.021
[102.9]
[1.87]
a Based on an intravenous (iv) dose of 1 mg/kg and a per os (po) dose of 100 mg/kg in fasted
b
male Balb/C mice (except where indicated). Compound was formulated in 5:25:70 DMA /
PEG400 / 20% SBECD for iv dosing and in 0.5% methyl cellulose (adjusted to pH 3) for po
dosing. c Compound was formulated in 5:60:35 DMA/PEG400/20% SBECD for iv dosing and in
d
0.5% methyl cellulose (adjusted to pH 3ꢀ4) for po dosing. CL = total clearance. CLunbound
=
e
f
CL/free fraction. Q = percent of liver blood flow, based on 5.4 L/h/kg (mouse). Plasma halfꢀ
life. g VSS = volume of distribution at steady state. Extrapolated total exposure following single
h
i
dose. Calculated unbound exposure is in parentheses. Maximum plasma concentration
j
k
l
achieved. Time at which maximum plasma concentration was achieved. Oral bioavailability.
fu = Fraction unbound to plasma protein.
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Figure 5. Mouse Pharmacokinetic Profile of 13.
Synthesis of Indole Piperidine Analogues
Many of the analogues profiled in Table 1 were prepared by similar synthetic routes, with
appropriately substituted building blocks. We identified indole 14 as a key scaffold for the
construction of Nꢀsubstituted piperidines. Synthetically, the alkylation of 2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀ
carboxylate with branched alkyl electrophiles under a variety of basic conditions failed to deliver
desired indole 14. The lackluster behavior of 2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxylate toward various
alkylation conditions was presumably a consequence of deactivation of the indole Nꢀatom by the
3ꢀcarboxylate moiety and additional steric constraints imposed by the 2ꢀmethyl substitution. As
such, we looked to devise an alternative strategy towards a more convergent, robust, and scalable
synthesis of indole 14.
Scheme 1. Palladium Mediated Intramolecular C–N Arylation to Indole 14.a
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aReagents and Conditions: (a) AcOH, tꢀBuOH, reflux (76% yield); (b) RuPhos precatalyst
(generation III) (2 mol%), RuPhos (3 mol%), NaOMe (1.5 equiv), 1,4ꢀdioxane, 100 °C, followed
by HCl (4M in 1,4ꢀdioxane) (81% yield over twoꢀsteps).
We were drawn to the possibility of utilizing haloꢀaryl enamines as latent precursors for a
palladiumꢀmediated intramolecular C–N bond construction of indoles.30 We rationalized that
generation of enamines from βꢀketo esters would allow for the introduction of a variety of
amines and this modular approach would serve as a diversity generating element for our drug
discovery efforts. To that end, the construction of indole 14, began with the condensation
reaction between βꢀketo ester30 15 and chiral amine31,32 16 under mildly acidic conditions to
deliver enamine 17, predominantly as the Zꢀisomer, in 76% yield. Paramount to the success of
this intramolecular C–N arylation was a systemic investigation of palladium catalysts and
reactions conditions.30 We were gratified to find that treatment of chiral enamine 17 with
Buchwald’s RuPhos preꢀcatalyst system in the presence of sodium methoxide cleanly induced
intramolecular C–N bond arylation to yield chiral indole piperidine 18 while maintaining the
stereochemical integrity of the chiral center. Subsequent deprotection of NꢀBoc piperidine 18
with anhydrous hydrochloric acid produced piperidine 14 in 81% yield over twoꢀsteps.
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Scheme 2. Synthesis of 10 and 13.a
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
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60
aReagents and Conditions: (a) 2,2,2ꢀTrifluoroacetic anhydride (1.4 equiv), iꢀPrNEt2 (2.5 equiv),
CH2Cl2, 0 °C; (b) BH3•THF (2.2 equiv), THF, 70 °C; (c) 6N aq. NaOH (6.0 equiv), EtOH, 85
°C; (d) CDI (1.3 equiv), THF, 60 °C
The piperidine 14, a common and versatile intermediate, was subsequently utilized for the
construction of a variety of analogues, in particular 13, via a series of similar chemical
transformations. The synthesis of 13 commenced with amidation of piperidine 14 (in free base
form) with trifluoroacetic anhydride in the presence of Hünig’s base to provide amide 19 in near
quantitative yield. Subsequent reduction of amide 19 with borane THF complex and heating
delivered Nꢀtrifluoroethyl piperidine 20. Saponification of the pendant methyl ester (20) with 6N
sodium hydroxide gave the penultimate acid 21 in 99% yield. The requisite amide bond was
forged in a CDI mediated union between acid 21 and pyridone amine 2233 to yield the Nꢀ
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trifluoroethyl piperidine analogue (13) in 79% yield. The current synthetic scheme enabled
generation of enantiomerically pure 13 on over 100 g scales for in vivo profiling.
In vivo Efficacy Studies
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
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41
42
43
44
45
46
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50
51
52
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Having sufficient quantities in hand, we evaluated the performance of inhibitor 13 for tumor
pharmacodynamic effects and antiꢀtumor efficacy in a KARPASꢀ422 Bꢀcell lymphoma xenograft
model in mice. KARPASꢀ422 xenograft cells harbor a recurrent, monoallelic mutation (Y641N)
within the EZH2 catalytic domain.17 These mutations alter the EZH2 substrate specificity, and
thus represent a context of constitutive EZH2 pathway activation. Informed by previous in vivo
studies, 13 was dosed at 160 mg/kg orally twice daily (po BID) for twentyꢀfive days in tumor
bearing female CBꢀ17 SCID mice (Figure 6). Upon treatment of tumor bearing CBꢀ17 SCID
mice with 13, tumor regression was observed within two weeks. By the end of day twentyꢀfive,
significant tumor growth inhibition was recorded (>97% TGI relative to vehicle, see Figure 6A).
Inhibitor 13 was well tolerated for repeat dosing as demonstrated by the absence of significant
body weight loss (Figure 6B). To allow for analysis of tumor tissues at the end of the study,
treatment was suspended at day twentyꢀfive. Tumor samples were harvested 1 h post last dose
and their analysis revealed considerable reduction of H3K27me3 (47% reduction in
H3K27me3/global H3 ratio relative to vehicle control, see Figure 6C). Analysis of plasma and
tumor PK at 1 h post last dose on day twentyꢀfive shows sufficient plasma and tumor tissue
concentrations of 13, 11,388 ng/mL [22 ꢀM] versus 5,286 ng/g [10 ꢀM], respectively (Figure
6D).
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Figure 6. Tumor xenograft with 13. A) Effect of dosing analogue 13 at 160 mpk BID for 25 days
on KARPASꢀ422 tumor growth in mouse xenograft. B) Corresponding body weight change
during the course of the study. C). Pharmacodynamic effect of 13 on reduction of H3K27me3
normalized to total H3. D). Analysis of plasma and tumor concentrations of 13 at 1 h post last
dose on day 25.
Selectivity and Additional Profiling
On the basis of the successful KARPASꢀ422 efficacy, we selected 13 for further in vivo and in
vitro profiling. Additional PK data on 13 in rats and dogs was collected (Table 4). Analogue 13
shows relatively high clearance in both rats and dogs (3.19 L/h/kg and 1.41 L/h/kg, respectively),
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but demonstrates good oral bioavailability in both species (44.6% F in rats and 46.2% F in dogs).
As part of the characterization of 13, its activity against a number of other targets were
evaluated. Inhibitor 13 showed a clean selectivity profile when tested against thirty other histone
or DNA methyltransferases.34 Additionally compound 13 demonstrated modest selectivity
(EZH1 IC50 of 52 ± 11 nM) when tested against enhancer of zeste homolog 1 (EZH1), a
methyltransferase highly related to EZH2. Examination of the sequence similarity between
EZH1 and EZH2 in the context of the coꢀcrystal structure reveals that the residue positioned in
close proximity to the inhibitor, Cys663, is one of only four residues within the EZH2 SET
domain that are not conserved in EZH1. The equivalent EZH1 residue is Ser664. Cys663 makes
van der Waals contact with the bound inhibitor and is predicted to be a key selectivity
determinant with respect to EZH1 (Figure 3B). In order to test this hypothesis we measured the
potency of 10 and 13 in the context of an EZH2 version with a single amino acid substitution
from cysteine to serine at position 663 (C663S) incorporated into reconstituted PRC2. As
expected the potency of the inhibitors decreased in the mutated EZH2 C663S, which is
consistent with the reduction in potency observed in EZH1 enzymatic assays. The fact that this
single mutation does not fully recapitulate the EZH2/EZH1 potency differences may reflect a
subtle effect of other more distant residue differences between the two proteins.
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
Table 4. Pharmacokinetic data of 13 in rats and dogs.
in
iv
po
vitro
CL
h
e
f
i
Fj
Cmax
t1/2
Vss
tmax
AUC0ꢀinf
c
k
[CLunbound
]
%Qd
fu
Species
g
[AUCunbound
]
(h) (L/kg)
(ꢀM)
(h) (%)
(L/h/kg)
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(ꢀM*h)
5
6
7
8
9
3.19
[31.6]
1.41
7.06
5.88
[0.593]
1.24
Rat a
97 0.653 2.22
0.50 44.6 0.098
1.67 46.2 0.089
[0.713]
3.19
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
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50
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53
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Dog b
76
1.84
1.60
[14.5]
[0.309]
[0.120]
a Based on an intravenous (iv) dose of 1 mg/kg and a per os (po) dose of 25 mg/kg in male
SpragueꢀDawley rats. Compound was formulated in 5:60:35 DMA / PEG400 / 20% SBECD for
b
iv dosing and in 0.5% methyl cellulose (adjusted to pH 3–4) for po dosing. Based on an
intravenous (iv) dose of 1 mg/kg and a per os (po) dose of 5 mg/kg in male beagle dogs.
Compound was formulated in 5:25:70 DMA / PEG400 / 20% SBECD for iv dosing and in 0.5%
methyl cellulose for po dosing. c CL = total clearance. CLunbound = CL/free fraction. d Q = percent
e
f
of liver blood flow, based on 3.3 L/h/kg (rat) and 1.85 L/h/kg (dog). Plasma halfꢀlife. VSS
=
g
volume of distribution at steady state. Extrapolated total exposure following single dose.
h
i
Calculated unbound exposure is in parentheses. Maximum plasma concentration achieved.
j
k
Time at which maximum plasma concentration was achieved. Oral bioavailability. fu =
Fraction unbound to plasma protein.
Further in vitro profiling of 13 showed no timeꢀdependent inhibition (TDI) of the cytochrome
P450 enzymes 1A2, 2C9, 2C8, 2D6 and 3A4. Additionally, 13 was evaluated for secondary
pharmacology against a panel of fiftyꢀfour physiologically relevant receptors, transporters, and
ion channels at 10 ꢀM. As such, 13 did not inhibit any target more than 50%. Finally, 13 was
examined for any potential cause of cardiac arrhythmias associated with delayed ventricular
repolarization (QT interval prolongation). When tested within an in vitro hERG binding assay at
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concentration ranges of 45 nM to 100 ꢀM, 13 showed an IC50 of 21.3 ꢀM. This concentration is
well above the freeꢀconcentration predicted to be achieved in patients.
7
8
9
Toxicology Study of 13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
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29
30
31
32
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40
41
42
43
44
45
46
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50
51
52
53
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59
60
To establish a safety window for repeat dosing of 13 in human clinical trials, a preclinical
safety study was conducted in two separate species. Compound 13 was orally administered in a
GLP compliant toxicity study for four weeks to both SpragueꢀDawley rats and beagle dogs
followed by a four week recovery period. The compound was administered by oral gavage at
single daily doses (QD) of 100, 300, and 600 mg/kg to rats for twentyꢀeight days and at twice
daily doses (BID) of 50, 150, and 500 mg/kg for twentyꢀeight days to dogs. In general, 13 was
well tolerated in the twentyꢀeight day GLP toxicology studies and any findings were reversible
over the recovery period. The GLP toxicology studies of 13 demonstrated an acceptable safety
profile and enabled selection of clinical doses.
Conclusion
In summary, we have reported the discovery of 13, a highly potent and selective small
molecule inhibitor of EZH2, suitable for introduction into Phase I clinical trials for the treatment
of Bꢀcell lymphomas (NCT02395601). Using 1, our previously disclosed EZH2 chemical probe
as a starting point, we embarked on a focused campaign to optimize the cellular potency and
physiochemical properties of our pyridoneꢀindole scaffold. Removal of the sulfonamide
functionality (embedded within 1) yielded analogues that retained biochemical potency but
lacked sufficient cellular activity in the HeLa H3K27me3 assay. Observing that attenuation of
the pKa of the piperidine Nꢀatom has a profound effect on the correlation between biochemical
and cellular potency, we ultimately discovered a series of fluorinated analogues with improved
cellular activity and good oral bioavailability.
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During the course of our investigations we successfully determined the coꢀcrystal structure of
compound 10 bound to human PRC2. The present crystal structure illustrates several distinct
binding features. The overall density reveals a zigꢀzag like shape of the inhibitor, likely a
consequence of conformational preꢀorganization induced by the chiral methyl and C3ꢀamide
substituents that conforms to the enzyme’s pocket. Additionally, the pyridone motif forms two
hydrogen bonds with the protein backbone of Trp624 and is constrained in an aromatic
environment created by Phe665, Phe686, and Trp624. These crucial interactions with the
pyridone motif explain the importance of this functional group for its high affinity binding, its
prevalence in a vast majority of other reported EZH2 inhibitors, and the difficulty in finding
suitable pyridone replacements.
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
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49
50
51
52
53
54
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57
58
59
60
After triaging these analogues through in vitro and in vivo experiments, Nꢀtrifluoroethyl
piperidine analogue 13 was evaluated in a KARPASꢀ422 lymphoma xenograft model.
Gratifyingly, 13 was well tolerated, proved efficacious, and achieved >97% TGI after treatment
for twentyꢀfive days. After further in vitro/in vivo characterization and safety studies, compound
13 was advanced into human clinical trials. The clinical impact of 13 on EZH2 inhibition in
oncology will be described in due course.
Experimental Methods
All commercial reagents and anhydrous solvents were purchased and used without purification,
unless specified. Column chromatography was performed using a Biotage chromatography
system on Biotage or Silicycle normal phase silica gel columns. NMR spectra were recorded on
a Varian Unity Inova (400 MHz) or an Oxford (Varian, 300 MHz) instrument. LCꢀMS were
recorded on an Agilent 1200 series LC connected to an Agilent 6120 MS or Agilent 1100 series
LC connected to an Agilent 1956B MS or a Shimadzu LCꢀMSꢀ2020 system. Preparatory HPLC
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were performed using a Gilson GXꢀ281 or P230 Gradient System (Elite). Chiral preparatory
HPLC were performed using Elite P230 Preparative Gradient System, Thar Prepꢀ80 and Thar
SFC Xꢀ5 systems. The purity of the final products was >95% as determined by HPLC/MS and
1H NMR.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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(R)ꢀ1ꢀ(1ꢀ(1ꢀ(Ethylsulfonyl)piperidinꢀ4ꢀyl)ethyl)ꢀNꢀ((4ꢀmethoxyꢀ6ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀ
dihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxamide (1). Prepared as previously
1
reported.21 LCꢀMS m/z 529 [M+H]+. H NMR (400 MHz, DMSOꢀd6) δ 11.60 (s, 1H), 7.82 –
7.66 (m, 2H), 7.62 (d, J = 7.8, 1H), 7.17 – 6.99 (m, 2H), 6.15 (s, 1H), 4.32 (d, J = 5.1 Hz, 2H),
4.25 – 4.15 (m, 1H), 3.84 (s, 3H), 3.73 – 3.65 (m, 1H), 3.45 – 3.36 (m, 1H), 2.98 (q, J = 7.4 Hz,
2H), 2.87 – 2.77 (m, 1H), 2.60 (s, 3H), 2.54 – 2.45 (m, 1H), 2.42 – 2.30 (m, 1H), 2.20 (s, 3H),
2.06 – 1.97 (m, 1H), 1.58 – 1.48 (m, 3H), 1.42 – 1.31 (m, 1H), 1.17 (t, J = 7.4 Hz, 3H), 1.13 –
1.00 (m, 1H), 0.83 – 0.73 (m, 1H).
tertꢀButyl (R)ꢀ4ꢀ(1ꢀ((3ꢀ(2ꢀbromophenyl)ꢀ4ꢀmethoxyꢀ4ꢀoxobutꢀ2ꢀenꢀ2ꢀyl)amino)ethyl)piperidineꢀ
1ꢀcarboxylate (17).30 A 2ꢀL three neck round bottom flask (fitted with a magnetic stir bar,
thermocouple, reflux condenser, and rubber septa) was charged with methyl 2ꢀ(2ꢀbromophenyl)ꢀ
3ꢀoxobutanoate (116.25 g, 428.80 mmol), EtOH (850 mL, ~7 mL/g), (R)ꢀtertꢀbutyl 4ꢀ(1ꢀ
aminoethyl)piperidineꢀ1ꢀcarboxylate (121.00 g, 529.93 mmol), and AcOH (29.50 mL, 515.31
mmol). The reaction vessel was heated over a heating mantle to 80 °C–85 °C for 18 h. After 18
h, the reaction mixture was cooled to ambient temperature and the tertꢀbutanol was removed in
vacuo. The resultant oil was diluted with EtOAc and subsequently poured over saturated aqueous
NaHCO3 while vigorously stirring. Once the evolution of CO2 (g) ceased, the biphasic solution
was transferred to a separatory funnel, the phases were separated. The aqueous phase was
extracted with additional EtOAc (2x). The combined organic phase was dried over MgSO4,
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filtered, and concentrated in vacuo to afford crude product. This material was preꢀabsorbed onto
silica gel (~100 g) and filtered through a fritted funnel with 20% EtOAc to 80% hexanes afford
(R)ꢀtertꢀbutyl 4ꢀ(1ꢀ((3ꢀ(2ꢀbromophenyl)ꢀ4ꢀmethoxyꢀ4ꢀoxobutꢀ2ꢀenꢀ2ꢀyl)amino)ethyl)piperidineꢀ
1ꢀcarboxylate (155.95 g, 76% yield). The material was used without further purification. LCꢀMS
m/z 481 [M+H]+.
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Methyl (R)ꢀ2ꢀmethylꢀ1ꢀ(1ꢀ(piperidinꢀ4ꢀyl)ethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxylate hydrochloride (14).
Step 1. To a 2 L 3ꢀneck round bottom flask (fitted with a magnetic stir bar, thermocouple, reflux
condenser, and rubber septa) containing RuPhos precatalyst (generation III) (4.38 g, 5.24 mmol)
and dicyclohexyl(2',6'ꢀdiisopropoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀyl)phosphine (3.67 g, 7.86 mmol) was
transferred
(R)ꢀtertꢀbutyl
4ꢀ(1ꢀ((3ꢀ(2ꢀbromophenyl)ꢀ4ꢀmethoxyꢀ4ꢀoxobutꢀ2ꢀenꢀ2ꢀ
yl)amino)ethyl)piperidineꢀ1ꢀcarboxylate (126.11 g, 261.95 mmol) in 1,4ꢀdioxane (800 mL, ~6.4
mL/g). The resultant dark solution was evacuated and purged with N2 (g). To the mixture was
added NaOMe (21.23 g, 392.93 mmol) in 4ꢀportions, followed by addition of 1,4ꢀdioxane (50
mL, total amount of 850 mL, ~8 mL/g) to rinse the powder funnel. The resultant heterogeneous
mixture was subjected to three cycles of evacuation and purging with N2 (g). The resultant dark
brown/red suspension was heated over a heating mantle to 100 °C for 4–6 h. As the temperature
reached 30–40 °C, a visible change in the reaction color was observed and the mixture turned
dark green. After 6 h, LCꢀMS analysis indicated complete conversion of the enamine to desired
indole. The reaction mixture was cooled to ambient temperature and filtered over a pad made of
a bed of silica gel over a bed of Celite®. The filter cake was washed with EtOAc (3X) and the
resultant brown filtrate was concentrated to provide methyl (R)ꢀ1ꢀ(1ꢀ(1ꢀ(tertꢀ
butoxycarbonyl)piperidinꢀ4ꢀyl)ethyl)ꢀ2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxylate (104.00 g, 99% yield)
as an orange colored foam. LCꢀMS m/z 401 [M+H]+.
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Step 2. To a cooled (0 °C) solution of (R)ꢀmethyl 1ꢀ(1ꢀ(1ꢀ(tertꢀbutoxycarbonyl)piperidinꢀ4ꢀ
yl)ethyl)ꢀ2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxylate (104.0 g, 259.67 mmol) in MeOH (300 mL, ~3
mL/g) was added hydrogen chloride (80.0 mL, 320.0 mmol) in a dropwise manner. After
complete addition of HCl, the reaction was allowed to gradually warm to ambient temperatures.
After 24 h, LCꢀMS analysis indicated complete conversion of the carbamate to desired product.
The reaction mixture was filtered over a pad of Celite® and subsequently concentrated in vacuo.
(Note: The reaction mixture contained minor quantities of insoluble black solids assumed to be
Pd(0)) The resultant dark brown oil was further concentrated from acetone (2 x 50 mL). The oil
was diluted in acetone (~200 mL). The resultant solids were stirred for 2 h and filtered to give
59.58 g (68% recovery after first round) of (R)ꢀ4ꢀ(1ꢀ(3ꢀ(methoxycarbonyl)ꢀ2ꢀmethylꢀ1Hꢀindolꢀ1ꢀ
yl)ethyl)piperidinꢀ1ꢀium chloride as offꢀwhite solids. The filtrate was concentrated and
subsequently triturated again with acetone (50 mL). The second crop of solids yielded an
additional 10 g of (R)ꢀ4ꢀ(1ꢀ(3ꢀ(methoxycarbonyl)ꢀ2ꢀmethylꢀ1Hꢀindolꢀ1ꢀyl)ethyl)piperidinꢀ1ꢀium
chloride (Total mass: 70.0 g, 80% yield after two rounds of trituration) as offꢀwhite solids. LCꢀ
MS m/z 301 [M+H]+.
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(R)ꢀNꢀ((4ꢀMethoxyꢀ6ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylꢀ1ꢀ(1ꢀ(piperidinꢀ4ꢀ
yl)ethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxamide (2). Step 1. To a solution of (R)ꢀmethyl 1ꢀ(1ꢀ(1ꢀ(tertꢀ
butoxycarbonyl)piperidimꢀ4ꢀyl)ethyl)ꢀ2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxylate (5.0 g, 12.5 mmol) in
methanol (20 mL) and water (10 mL) was added sodium hydroxide (5.0 g, 125 mmol). The
reaction mixture was stirred at 80 °C for 12 h. The mixture was cooled to room temperature and
acidified to pH 5 with hydrochloric acid (2N). The acidic solution was extracted with EtOAc.
The combined organics layer was washed with brine, dried over Na2SO4, filtered, and
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concentrated to afford (R)ꢀ1ꢀ(1ꢀ(1ꢀ(tertꢀbutoxycarbonyl)piperidinꢀ4ꢀyl)ethyl)ꢀ2ꢀmethylꢀ1Hꢀ
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indoleꢀ3ꢀcarboxylic acid (4.5 g, 94% yield) as an offꢀwhite solid. LCꢀMS m/z 409 [M+Na]+.
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Step 2. A 250 mL round bottom flask was charged with a magnetic stir bar, (R)ꢀ1ꢀ(1ꢀ(1ꢀ(tertꢀ
butoxycarbonyl)piperidinꢀ4ꢀyl)ethyl)ꢀ2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxylic acid (1.950 g, 5.05
mmol), 3ꢀ(aminomethyl)ꢀ4ꢀmethoxyꢀ6ꢀmethylpyridinꢀ2(1H)ꢀone hydrochloride (2.065 g, 10.09
mmol), DMF (25.2 mL, 5.05 mmol), Hunig’s base (3.52 mL, 20.18 mmol). The reaction mixture
was cooled to 0 °C and COMU (2.16 g, 5.05 mmol) was added. The reaction was allowed to stir
overnight to room temperature. The reaction mixture was diluted with water and extracted with
EtOAc. The combined organic extract was washed with brine, dried with MgSO4, filtered and
conc. in vacuo to afford the crude material which was purified via silica gel chromatography
(120 g) using MeOH/ethyl acetate (1:5) as eluent to afford tertꢀbutyl (R)ꢀ4ꢀ(1ꢀ(3ꢀ(((4ꢀmethoxyꢀ6ꢀ
methylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)carbamoyl)ꢀ2ꢀmethylꢀ1Hꢀindolꢀ1ꢀ
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yl)ethyl)piperidineꢀ1ꢀcarboxylate (1.86 g, 65% yield). LCꢀMS m/z 537 [M+H]+ 1H NMR (400
MHz, DMSOꢀd6) δ 11.83 – 11.71 (m, 1 H), 7.80 (br. s., 1 H), 7.73 (d, J = 7.6 Hz, 1 H), 7.62 (d, J
= 7.8 Hz, 1 H), 7.06 (td, J = 7.1, 14.4 Hz, 2 H), 6.21 (s, 1 H), 4.32 (br. s., 2 H), 4.16 (br. s., 1 H),
4.02 (br. s., 1 H), 3.85 (s, 3 H), 3.75 (br. s., 1 H), 2.70 (br. s., 1 H), 2.58 (s, 3 H), 2.37 (br. s., 1
H), 2.21 (s, 3 H), 1.90 (d, J = 12.9 Hz, 1 H), 1.53 (d, J = 6.9 Hz, 3 H), 1.35 (s, 10 H), 1.21 (br. s.,
1 H), 0.89 (d, J = 8.7 Hz, 1 H), 0.67 (d, J = 11.8 Hz, 1 H).
Step 3. A 250 mL round bottom flask was charged with a magnetic stir bar, (R)ꢀtertꢀbutyl 4ꢀ(1ꢀ
(3ꢀ((4ꢀmethoxyꢀ6ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methylcarbamoyl)ꢀ2ꢀmethylꢀ1Hꢀindolꢀ
1ꢀyl)ethyl)piperidineꢀ1ꢀcarboxylate (1.850 g, 3.45 mmol), MeOH (13.79 mL, 3.45 mmol), and
HCl (2.59 mL, 10.34 mmol) (4 N in dioxane). The reaction was allowed to stir at room
temperature for 6 h before being conc. in vacuo to afford 1 (1.65 g, 91 % yield). LCꢀMS m/z 437
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[M+H]+ 1H NMR (400 MHz, methanolꢀd4) δ 7.62 (d, J = 7.5 Hz, 1 H), 7.52 (d, J = 7.7 Hz, 1 H),
7.08 – 6.96 (m, 2 H), 6.19 (s, 1 H), 4.43 (s, 2 H), 4.15 (dd, J = 10.4, 7.1 Hz, 1 H), 3.85 (s, 3 H),
3.40 (d, J = 12.8 Hz, 1 H), 3.07 (d, J = 12.6 Hz, 1 H), 2.96 (dt, J = 13.0, 2.8 Hz, 1 H), 2.68 – 2.50
(m, 5 H), 2.23 (s, 3 H), 2.17 (d, J = 14.3 Hz, 1 H), 1.56 (d, J = 6.8 Hz, 3 H), 1.14 – 1.05 (m, 1 H),
0.94 (d, J = 13.5 Hz, 1 H), 0.80 (t, J = 6.8 Hz, 1 H).
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(R)ꢀNꢀ((4ꢀMethoxyꢀ6ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylꢀ1ꢀ(1ꢀ(1ꢀ
methylpiperidinꢀ4ꢀyl)ethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxamide (3). To a solution of (R)ꢀNꢀ((4ꢀmethoxyꢀ6ꢀ
methylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylꢀ1ꢀ(1ꢀ(piperidinꢀ4ꢀyl)ethyl)ꢀ1Hꢀindoleꢀ
3ꢀcarboxamide (0.1 g, 0.229 mmol) in THF (3 mL) was added formaldehyde (0.051 mL, 0.687
mmol). This solution was mixed for 1 h then sodium triacetoxyborohydride (0.146 g, 0.687
mmol) was added and the reaction was mixed at ambient temperature for 2.5 h. The crude
reaction was deposited onto silica gel and purified by silica gel chromatography (90:10:1
DCM:MeOH:NH4OH) to afford 2 (68 mg, 66% yield). LCꢀMS m/z 451 [M+H]+; 1H NMR (400
MHz, DMSOꢀd6) δ 11.58 (s, 1 H), 7.76 – 7.65 (m, 2 H), 7.59 (d, J = 7.6 Hz, 1 H), 7.11 – 6.99
(m, 2 H), 6.14 (s, 1 H), 4.31 (d, J = 5.1 Hz, 2 H), 4.13 (br. s., 1 H), 3.83 (s, 3 H), 2.83 (d, J = 10.0
Hz, 1 H), 2.61 – 2.52 (m, 5 H), 2.19 (s, 3 H), 2.09 (s, 4 H), 1.88 (d, J = 10.7 Hz, 2 H), 1.53 (d, J
= 6.7 Hz, 3 H), 1.34 (br. s., 1 H), 1.02 (d, J = 8.2 Hz, 1 H), 0.66 (br. s., 1 H).
(R)ꢀ1ꢀ(1ꢀ(1ꢀIsobutyrylpiperidinꢀ4ꢀyl)ethyl)ꢀNꢀ((4ꢀmethoxyꢀ6ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀ
yl)methyl)ꢀ2ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxamide (4): To a round bottomed flask was added (R)ꢀNꢀ
((4ꢀmethoxyꢀ6ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ3ꢀyl)methyl)ꢀ2ꢀmethylꢀ1ꢀ(1ꢀ(piperidinꢀ4ꢀ
yl)ethyl)ꢀ1Hꢀindoleꢀ3ꢀcarboxamide (60 mg, 0.137 mmol) and DCM ( 2 mL). The solution was
cooled to 0 °C before addition of Et3N (21.07 µL, 0.151 mmol) and isobutyryl chloride (15.84
µL, 0.151 mmol). After stirring for 1 h at 0 °C, the reaction mixture was quenched by addition of
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