Discovery of highly potent, selective, and efficacious small molecule inhibitors of ERK1/2

Article abstract of DOI:10.1021/jm501921k

Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.

Full text of DOI:10.1021/jm501921k

Article
pubs.acs.org/jmc
Discovery of Highly Potent, Selective, and Efficacious Small Molecule
Inhibitors of ERK1/2
Li Ren,*,† Jonas Grina,† David Moreno,† James F. Blake,† John J. Gaudino,† Rustam Garrey,†
Andrew T. Metcalf,† Michael Burkard,† Matthew Martinson,† Kevin Rasor,† Huifen Chen,§ Brian Dean,§
Stephen E. Gould,§ Patricia Pacheco,§ Sheerin Shahidi-Latham,§ Jianping Yin,§ Kristina West,§
Weiru Wang,§ John G. Moffat,§ and Jacob B. Schwarz§
†Array BioPharma Inc, 3200 Walnut Street, Boulder, Colorado 80301, United States
§Genentech Inc., 1 DNA Way, South San Francisco, California 94080-4990, United States
S
* Supporting Information
ABSTRACT: Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to
the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft
models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.
ation, survival, angiogenesis, and differentiation, all hallmarks of
the cancer phenotype.7 Thus, it may be beneficial to target the
ERK proteins directly as a more effective way to block the MAPK
signaling pathway. Furthermore, an ERK inhibitor may have
utility in combination with other MAPK inhibitors. Recently,
researchers at Genentech and Merck independently reported
that dual inhibition of MEK and ERK by small molecule
inhibitors acted to overcome acquired resistance to MEK
inhibitors, providing further rationale for developing selective
ERK1/2 inhibitors.8 Herein, we describe the medicinal chemistry
efforts leading to the discovery of (S)-14k, a potent, selective, and
efficacious inhibitor of ERK1/2.
INTRODUCTION
■
The Ras/Raf/MEK/ERK (MAPK) signal transduction pathway
is widely activated in a large subset of human cancers and thus has
attracted significant interest as a therapeutic target for cancer.1
Constitutive activation of the MAPK pathway by overexpression
of growth factors, as well as oncogenic Ras and Raf mutations, are
frequently observed in tumor types such as colon, lung,
pancreatic, ovary, and kidney.2 The downstream consequence
of disregulation of this pathway is over and/or constitutive
production of phosphorylated ERK1/2 (pERK). Activity of this
pathway has been linked to disease progression and clinical
outcome in several malignancies.2
Small molecule inhibitors of B-Raf and MEK have shown
promising activities in the clinic for a variety of solid tumors. The
approval of Zelboraf3 (vemurafenib), Telfinlar4 (Dabrafenib),
and Mekinist5 (trametinib) as treatments for metastatic
melanoma validate the approach of targeting the MAPK pathway
as an effective way of treating cancer. In comparison to B-Raf and
MEK inhibitors, the development of small molecule inhibitors of
ERK1/2 has lagged behind with only few reports of preclinical
development.6 ERK1 and 2, which show 89% sequence identity
within the kinase domain, are pivotal in the pathway downstream
of Ras, Raf, and MEK, acting as a central node where multiple
signaling pathways converge to drive transcription. ERK1/2 are
activated by MEK1/2 through phosphorylation of both a
threonine and a tyrosine residue, namely Thr202 and Tyr204
of ERK1 and Thr173 and Tyr185 of ERK2. Once activated,
ERK1/2 phosphorylates serine/threonine residues of more than
50 downstream substrates and activates both cytosolic and
nuclear proteins that are responsible for cell growth, prolifer-
STRUCTURE-BASED DESIGN
■
Previously, we disclosed a novel series of ATP competitive
ERK1/2 inhibitors, exemplified by 1a and 1b (Figure 1) built
upon a piperidinopyrimidine urea template.9 On the basis of
excellent potency and good in vitro ADME properties (medium
predicted CL in hepatocytes and moderate plasma protein
binding),9 the pharmacokinetics of 1a was evaluated in mice, rats,
beagle dogs, and cynomolgous monkeys. Unfortunately,
compound 1a showed high clearance and low oral bioavailability
in multiple species, a profile shared with most analogues from the
series.9
To understand the underlying reason for the poor PK across
the series, in vitro metabolic profilings were performed on
representative analogues. Consistently, major metabolites were
Received: December 11, 2014
© XXXX American Chemical Society
A
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Figure 1. Stuctures of 1a and 1b from the piperidinopyrimidine urea series.
Scheme 1. Structure-Based Design: From Piperidinopyrimidine Ureas to Pyridones
identified that resulted from oxidation of the benzylic C-5 and C-
8 positions on the core (Scheme 1). In addition to high clearance,
1a exhibited low permeability (Papp AB (10−6 cm/s) = 1.3) and
efflux (efflux ratio BA/AB = 7.8), limiting its oral absorption.
These inherent PK liabilities of the piperidinopyrimidine urea
template prompted us to search for an alternative core.
First of all, modeling studies (Figure 2) suggested that the
pyridone carbonyl mimics the urea oxygen and should make the
same hydrogen bond interactions to both Lys54 and the bound
water molecule that is complexed to Gln105, Glu71, and Asp167.
Second, an overlap of the pyridone and the piperidinopyrimidine
urea templates revealed that the left-hand-side pyrimidine and
right-hand-side benzyl moieties occupy similar space and should
maintain critical contacts with the hinge and the glycine-rich
loop. It was reasoned that the pyridone series could have
improved metabolic stability due to removal of the two metabolic
soft spots present in the piperidinopyrimidine scaffold (C-5 and
C-8). Another salient feature of the pyridone design is
elimination of the urea linker. Although the urea oxygen is
necessary for activity, the urea NH does not make any specific
interaction with the protein and removal of the unnecessary
hydrogen bond donor could positively impact permeability and
absorption.
In designing a new template, it was highly desirable to maintain
all the critical interactions between 1a and the ERK2 protein
while removing the metabolic liability inherent in the
piperidinopyrimidine core. As can be seen in the X-ray cocrystal
structure of 1b bound to ERK2 (Figure 2A), the pyrimidine ring
accepts a hydrogen bond from the backbone NH of Met108
while the amino group at the 2-position donates a H-bond to the
carbonyl oxygen of the same residue. The oxygen of the
tetrahydro-2H-pyran is hydrogen-bonded to the side chain of
Lys114, although it appears that the bulk of the potency realized
from this group is derived via hydrophobic interactions near the
hinge region. Additionally, the urea oxygen accepts two hydrogen
bonds: one from the side chain of Lys54 and another from a
bound water molecule that is complexed to Gln105, Glu71, and
Asp167 (Figure 2B). The latter H-bond is believed to be critical
to ERK1/2 and a key feature of our strategy to achieve selectivity
against other members of the closely related CMGC family of
kinases. The (S)-CH2OH group is in the vicinity of Asp167 and
Asn154 and can make a hydrogen bond interaction to either the
side chain carboxylate of Asp167 or the amide carbonyl of
Asn154. It is important to note that the stereochemistry of this
chiral center is critical for potency, as observed by us9 and
others.6 The 3-Cl-4-F phenyl moiety points toward the glycine-
rich loop, making largely hydrophobic contacts. The metabol-
ically labile piperidine acts mainly as a spacer and a conforma-
tional control and could potentially be replaced. It was
hypothesized that a 4-pyridone could serve as effective
replacement for the piperidine urea linker, as shown in Scheme
1, based on the following considerations.
CHEMISTRY
■
Pyrimidine pyridones 6 were prepared in a four-step procedure
as described in Scheme 2.10 Suzuki coupling of commercially
available 2,4-dichloropyrimidine and 2-fluoropyridin-4-ylboronic
acid produced 3. Replacing the 2-Cl with tetrahydro-2H-pyran-4-
amine (THP amine) gave aminopyrimidine 4, which was
converted to pyridone 5 under acidic conditions. Chemo-
selective N-alkylation with various benzyl bromides afforded 6a−
6f.
Hydroxylated analogues such as 14 required a slightly different
synthesis as illustrated in Scheme 3.10 Suzuki coupling of
commercially available 4-bromo-2-(methylthio)pyrimidine and
2-fluoropyridin-4-ylboronic acid produced 7, which was
converted to the key pyridone intermediate 8 via acidic
hydrolysis. Chemoselective N-alkylation with mesylate 10
(prepared via aryl Grignard addition to commercially available
TBS protected glycolaldehyde) gave racemic methyl sulfide 11,
B
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Figure 2. (A) Pyridone 6a (orange) docked into the X-ray structure of 1b (green) with ERK2 (PDB code 4O6E, solved at 1.95 Å resolution). Hydrogen-
bonding interactions are illustrated with red dashed lines. (B) Same model as in (A), rotated to illustrate key water mediated H-bond interactions.
a
Scheme 2. Preparation of Compounds 6a−6f
a
Reaction conditions: (a) 5% Pd(dppf)Cl2(DCM), Na2CO3, 1,4-dioxane/H2O (4:1), 80 °C, 3 h, 96%; (b) tetrahydro-2H-pyran-4-amine, N-ethyl-N-
isopropylpropan-2-amine, 2-butanol, 100 °C, 16 h, 49%; (c) 2 N HCl, 100 °C, 16 h, 94%; (d) benzyl bromide, K2CO3, DMF, 25 °C, 65%.
which was oxidized to sulfone 12. Installation of the THP amine
followed by TBS deprotection afforded 14a−14o.
The synthetic route shown in Scheme 3 to produce racemic
compounds was modified to obtain the desired (S)-enantiomers
with high ee (Scheme 4).10 It was hypothesized that the key
pyridone N-alkylation between 18 and 8 would occur via a SN2
mechanism with complete inversion at the newly created chiral
center (19) and thus would require a precursor such as 16. The
absolute (R)-stereochemistry of the chiral center in diol 16 could
be set via the well-known Sharpless dihydroxylation by using AD-
C
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a
Scheme 3. Preparation of Compounds 14a−14o
a
Reaction conditions: (a) 5% Pd(dppf)Cl2(DCM), Na2CO3, 1,4-dioxane/H2O (4:1), 80 °C, 2 h, 90%; (b) 2 N HCl, 100 °C, 2 h, 50%; (c) THF, 0
°C, 1 h, 93%; (d) MsCl, NEt3, DCM, 0 °C, 1 h, 96%; (e) KHMDS, THF, 65 °C, 30 h, 65%; (f) mCPBA, DCM, 0 °C, 2 h, 89%; (g) tetrahydro-2H-
pyran-4-amine, 2-butanol, 120 °C, 4 h, 90%; (h) TBAF, THF, 25 °C, 16 h, 90%.
a
Scheme 4. Preparation of Compounds (S)-14k and 22a−22i
a
Reaction conditions: (a) NaH, methyltriphenylphosphonium bromide, THF, 25 °C, 16 h, 47%; (b) AD-mix-β, t-BuOH/H2O (1:1), 25 °C, 16 h,
64%; (c) TBSCl, imidazole, DCM, 0 °C, 1 h, 58%; (d) MsCl, NEt3, DCM, 0 °C, 1 h, 96%; (e) KHMDS, THF, 65 °C, 30 h, 65%; (f) mCPBA, DCM,
0 °C, 2 h, 89%; (g) R4NH2, 120 °C, 4 h, 90%; (h) TBAF, THF, 25 °C, 16 h, 90%.
mix-β.11 Indeed, treatment of styrene 15 with AD-mix-β afforded
16 with 95% ee (determined from 17 by chiral chromatography).
TBS protection of the primary alcohol (17) followed by
mesylation of the benzylic hydroxyl group gave 18. With
KHMDS as a base, displacement of mesylate by intermediate 8
occurred predominantly on nitrogen to afford 19. Although the
enantiomeric excess of 19 was not measured, it was inferred to be
>95% because no ee loss was observed during the transformation
from 17 (95% ee) to (S)-14k (96% ee, determined by chiral
chromatography). The absolute stereochemistry of (S)-14k was
D
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confirmed by X-ray crystallography (Figure 3), indicating that
the N-alkylation to produce 19 occurred with clean inversion.
analogues such as 6d and 6e remained similarly active, extending
the pyridone side chain from benzyl to phenylethyl (6f) resulted
in substantial lost in cellular potency.
Because preliminary SAR on the phenyl ring tracked well with
the piperidinopyrimidine ureas,9 we decided to focus on the
hydroxymethyl substituent at the benzylic position as we9 and
others6 have shown that this group is near optimal in terms of
both potency and kinase selectivity (Table 2). Although the
cellular IC50 of racemic 14a remained unchanged in comparison
to 6a at first glance, it was important to realize that potency
should improve by 2-fold if only the active (S)-enantiomer was
obtained.
To expedite the SAR exploration of other regions of the
molecule, we elected to proceed with testing racemic material
due to ease of synthesis. Once an optimal racemic compound was
identified, the more active (S)-enantiomer was prepared and
profiled. Using this strategy, SAR of the terminal phenyl group
was further expanded (Table 2). Again, exploring chlorine
substitution on the aryl ring quickly revealed that 3-Cl (14d vs
14b and 14c) was favored in both biochemical and cellular assays.
Further SAR exploration at this position showed 3-F (14e), 3-Me
(14f), and 3-CF3 (14g) were less potent in the cellular assay and
polar substituents were not tolerated (14h and 14i), likely due to
unfavorable interaction with the largely lipophilic P-loop. On the
basis of these results, we prepared dihalo analogues 14j−14o.
Most of these compounds retained cellular activity in
comparison to 14d, with the exception of 14m, which is 2×
less potent in the cellular assay.
Because of the combination of good cellular potency and
CDK2 selectivity, the active (S)-enantiomer of 14d, 14e, 14k,
and 14l were obtained by asymmetric synthesis as described in
Scheme 4. As expected, all of the (S)-enantiomers were potent
and selective (>70× over CDK2). These compounds showed
reasonable in vitro ADME properties, such as medium metabolic
stability as predicted by mouse liver microsomes and medium to
high permeability as measured in the MDCK cell line, and were
advanced to mouse (CD-1 strain) pharmacokinetic studies
(Table 3). Among these compounds, the one bearing the 3-F-4-
Cl substitution pattern ((S)-14k) showed the best overall mouse
PK profile with low clearance and high bioavailability and was
chosen as the preferred piece on the right-hand-side of the
molecule.
Having chosen the 3-F-4-Cl-Ph as the preferred right-hand-
side substituent, central pyridone ring SAR was investigated next
(Table 4). Again, we elected to proceed with testing racemic
material due to ease of synthesis. Only small substitutions,
especially at the 3- and 5-position of the pyridone were
considered, as conformational analysis suggested that the
pyrimidine and the pyridone core would prefer to be coplanar
when bound. 3-F substitution (23a) retained potency, albeit with
a loss of selectivity over CDK2. Substitutions at other positions
were not well tolerated, and 5-F (23b), 5-Me (23c), and 6-Me
(23d) were substantially less active. Changing the pyridone
template to either the pyrimidinone (23e) or the pyridazinone
(23f) resulted in a drop in potency. The decrease in activity of
23e and 23f was likely due to the increased polarity (decreased
lipophilicity) of the linker.
RESULTS AND DISCUSSION
■
Compounds were screened for potency against ERK2 enzymatic
activity, Cdk2/Cyclin A binding, and inhibition of ERK-
dependent p90RSK Serine 380 phosphorylation in HepG2
cells. To enable determination of subnanomolar enzyme
potencies, ERK2 kinase activity was assayed in the presence of
2 mM ATP (80× the ATP Km(apparent) of 25 μM) and Ki values
derived by the Cheng−Prusoff formula.12 Given that the residues
in the ATP site are conserved between ERK1 and ERK2,
compounds were not routinely tested against ERK1, however,
follow-up testing of several compounds confirmed that the ERK1
activity (Ki or IC50 values) were consistently within 3-fold of
ERK2.13 Cyclin-dependent kinase 2 (CDK2) was employed as a
representative kinase selectivity assay due to its relatively similar
sequence homology with ERK2 in the ATP binding pocket and
previous experience that this kinase was a frequent and
biologically significant off-target.9 Pyridone 6a, with an
unsubstituted benzyl group, showed promising activity with an
enzyme Ki = 0.6 nM and cellular IC50 = 120 nM. This
encouraging result confirmed the validity of our docking
experiments and gave us further confidence in using this model
to drive SAR. We first proceeded to introduce small, mostly
lipophilic substituents onto the phenyl ring to optimize
interaction with the glycine-rich loop (Table 1). In general,
halogens at the 3- and/or 4-position afforded a modest increase
in potency and selectivity over CDK2. 3-Chlorophenyl (6b) was
favored over 4-chlorophenyl (6c), affording a 4-fold improve-
ment in cellular activity over 6a. While 3,4-disubstituted
Table 1. SAR of the Right-Hand-Side Phenyl Ring
phospho-
ERK2
CDK2
p90RSK
compd
no.
enzyme K enzyme K
IC50
CDK2
a,
b i c d
b
,
b,d
R1
(nM)
(nM)
(nM)
Kd/ERK2 Ki
6a
6b
−CH2Ph
0.6 0.3
0.4 0.1
21 11
20 13
120 26
30 12
35
67
−CH2-3-
Cl-Ph
6c
−CH2-4-
0.8 0.1
0.4 0.1
49 22
37 12
85
29
6
6
61
93
Cl-Ph
6d
−CH2-3-
Cl-4-F-
Ph
6e
6f
−CH2-3-
F-4-Cl-
Ph
0.3 0.1
1.5 0.3
21
9
34 10
70
69
−CH2
CH2-3-
F-4-Cl-
Ph
103 32
615
9
a
Ki values were determined by carrying out ERK2 enzymatic assays in
SAR of the pyrimidine ring was examined next (Table 5).
Other hinge binding templates such as pyridine (24a) as well as
an isomeric pyrimidine (24b) were both less effective.
Substitution at the 5-position of the pyrimidine ring with
fluorine (24e) or chlorine (24d) largely retained cellular
potency, albeit with a loss of selectivity over CDK2. The loss
(app)
the presence of ATP at 80× Km
and applying Cheng−Prusoff
b
correction to convert IC50 to Ki. All assay results represent the mean
c
of at least three independent determinations. Kd values were
determined by competitive displacement of an ATP-analogue probe.
Cellular assay in PMA-stimulated HepG2 cells. See Supporting
Information for details.
d
E
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Table 2. Expanded SAR of the Right-Hand-Side Phenyl Ring
a,
b
b,
c
b,d
compd no.
R3
ERK2 enzyme Ki (nM)
CDK2 enzyme Kd (nM)
phospho-p90RSK IC50 (nM)
CDK2 Kd/ERK2 Ki
14a
14b
14c
14d
14e
14f
H
0.7 0.1
1.1 0.2
1.5 0.1
0.4 0.1
0.1 0.03
1.3 0.1
1.3 0.1
1.6 0.2
2.3 0.3
0.1 0.01
0.6 0.1
0.5 0.1
0.4 0.1
0.3 0.02
0.5 0.04
36 19
108 15
69 15
43 15
148 18
249 43
98 43
39 16
79 23
100 31
82 31
227 62
484 149
58 31
49 15
37 21
83 34
51
98
2-Cl
4-Cl
46
3-Cl
108
190
119
136
112
95
3-F
19
4
3-Me
155 129
177 116
179 124
219 127
14g
14h
14i
3-CF3
3-OMe
3-CN
14j
3,4-diF
3-F-4-Cl
3-Cl-4-F
3,5-diF
3,5-diCl
3-F-5-Cl
16
8
160
95
14k
14l
57 29
70 28
140
55
14m
14n
14o
22
3
40 16
27
9
133
37 13
41 19
74
a
Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to
b
c
convert IC50 to Ki. All assay results represent the mean of at least three independent determinations. Kd values were determined by competitive
displacement of an ATP-analogue probe. Cellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details.
d
Table 3. In Vitro ADME Properties and Mouse PK Parameters of (S)-14d, (S)-14e, (S)-14k and (S)-14l
in vivo CL
compd
no.
ERK2 enzyme CDK2 enzyme phospho-p90RSK
CDK2
in vitro predicted CL permeability
(mL/min/kg)
F
a,
b
b
,
c
b,
d
e
f
g
h
Ki (nM)
Kd (nM)
16
23 19
IC50 (nM)
27
42 24
Kd/ERK2 Ki
(mL/min/kg)
Papp AB
IV
AUC (μM·h)
(%)
(S)-14d
(S)-14e
(S)-14k
(S)-14l
0.2 0.03
0.1 0.03
0.3 0.02
0.2 0.03
2
7
80
230
70
71
59
60
66
8.5
5.5
114
39
0.59
1.72
3.05
2.18
38
36
66
76
21
8
17
4
11.3
33
31
6
38
8
165
4.6
63
a
Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to
b
c
convert IC50 to Ki. All assay results represent the mean of at least three independent determinations. Kd values were determined by competitive
d
e
displacement of an ATP-analogue probe. Cellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details. Mouse hepatic
f
g
CL predicted from liver microsomes. Permeability measurement determined in MDCK cell line, in units of 10−6 cm/s. IV clearance measured at 1
h
mg/kg for CD-1 mouse, dosed as a solution in 40% PEG400/60% H2O. CD-1 mouse PO PK at 5 mg/kg, dosed as a solution in 40% PEG400/60%
of 10% HP-b-CD in H2O.
Table 4. SAR of the Central Pyridone Ring
a,
b
b,
c
b,d
compd no.
X
Y
Z
ERK2 enzyme Ki (nM)
CDK2 enzyme Kd (nM)
phospho-p90RSK IC50 (nM)
CDK2 Kd/ERK2 Ki
14k
23a
23b
23c
23d
23e
23f
C−H
C−F
C−H
C−H
C−H
N
C−H
C−H
C−F
C-Me
C−H
C−H
C−H
C−H
C−H
C−H
C−H
C-Me
C−H
N
0.6 0.1
57 29
49 15
82 35
95
33
0.3 0.03
10
4
41
7
723 179
643 229
758 262
202 38
92 42
>5000
18
3.3 0.8
6.8 2.2
2.4 0.2
1.7 0.4
687 122
1619 213
697 156
230 58
195
111
84
C−H
54
a
Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to
convert IC50 to Ki. All assay results represent the mean of at least three independent determinations. Kd values were determined by competitive
displacement of an ATP-analogue probe. Cellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details.
b
c
d
F
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Table 5. SAR of the Pyrimidine Ring
a,
b
b
,
c
b,d
compd no.
A
B
ERK2 enzyme Ki (nM)
CDK2 enzyme Kd (nM)
phospho-p90RSK IC50 (nM)
CDK2 Kd/ERK2 Ki
14k
24a
24b
24c
24d
24e
N
C−H
C−H
N
0.6 0.1
1.8 0.3
14 1.0
2.2 0.2
0.2 0.02
0.1 0.01
57 29
567 274
763 209
136 63
4.2 1.4
1.8 0.8
49 15
385 119
4428 990
801 116
116 60
30 15
95
315
55
C−H
C−H
N
C-Me
C−Cl
C−F
62
N
21
N
18
a
Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to
convert IC50 to Ki. All assay results represent the mean of at least three independent determinations. Kd values were determined by competitive
displacement of an ATP-analogue probe. Cellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details.
b
c
d
of activity with a larger methyl group (24c) was attributed to an
unfavorable change in the dihedral angle between the pyrimidine
and pyridone rings, resulting in increased strain energy upon
binding.
3B) forms a H-bond to the pyridone carbonyl (2.85 Å), which
also interacts with the side chain of Lys54 (3.01 Å). The methyl
hydroxyl substituent interacts with the carboxylate side chain of
Asp167 (2.70 Å). The 3-F-4-Cl phenyl group occupies a small
hydrophobic pocket under the P-loop that is formed when Tyr36
is displaced toward the C-helix.
Final areas of SAR investigation focused on exploring other
amino groups at the 2-position of the pyrimidine. This area is
solvent exposed and can potentially be used to design inhibitors
with improved physiochemical properties (Table 6). To
maintain potency, substituents capable of interacting with the
side chain of Lys114 (as the oxygen of the THP moiety) were
evaluated. In this exercise, we proceeded with enantiomerically
enriched material because large quantities of intermediate 20 had
become available at this stage of the project. A variety of
structural changes were explored, such as varying the ring size
(22a, 22b, 22c), ring substitution (22e), THP oxygen atom
replacement (22d, 22f) as well as ring opening (22g, 22h, 22i),
without improving upon the potency of (S)-14k. Although the
activity of oxetane 22c looked promising initially, it was
chemically unstable toward acid (pH = 1) and was not advanced.
To confirm our earlier hypothesis that the (S)-enantiomer was
critical for potency, the opposite epitope (R)-14k was obtained
(by chiral separation) and profiled (Table 7). As anticipated, (R)-
14k was 25-fold less active in the cellular assay in comparison to
(S)-14k. Importantly, the more active enantiomer also
maintained CDK2 selectivity. When compared to our previous
lead 1a, (S)-14k has equivalent cellular potency with improved
CDK2 selectivity. With an optimal combination of activity,
selectivity, and mouse pharmacokinetics, (S)-14k emerged as a
promising lead. The kinase selectivity profile of compound (S)-
14k was determined by screening against a panel of 170 kinases.
No kinase showed >70% inhibition other than ERK1 and ERK2
at a test concentration of 100 nM (Supporting Information).
Furthermore, (S)-14k showed no significant inhibition when
screened against a panel of 40 receptors at a concentration of 10
μM except for mild adenosine (64% inhibition) A1 agonism
(Supporting Information).
In Vitro ADME and Pharmacokinetics. The in vitro
ADME properties of (S)-14k were determined (Table 8). Liver
hepatocytes predicted low to medium stability across species,
similar to what was observed with our pervious lead 1a.
Permeability measurement in the MDCK cell line showed high
permeability without efflux, a significant improvement over 1a,
suggesting better absorption potential. Other properties such as
solubility (45 μg/mL @ pH 7.4) and human plasma protein
binding (95.8%) were deemed sufficient although less desirable
than 1a. Compound (S)-14k displayed no inhibition of
cytochrome P450 enzymes (IC50 > 10 μM for CYP3A4,
CYP1A2, CYP2C9, CYP2C19, CYP2D6) and was negative in
the time-dependent CYP inhibition (TDI) assay for major CYP
isoforms (CYP3A4, CYP1A2, CYP2C9, CYP2C19, and
CYP2D6). In addition, minimal hERG channel inhibition was
observed with an IC50 = 14.5 μM.
On the basis of the combination of good potency, selectivity,
and in vitro ADME profile, the PK of (S)-14k was evaluated in
nude mice (Table 9). The clearance was modest (CL = 40 mL/
min/kg) at 1 mg/kg IV, and oral bioavailability was excellent at 5
mg/kg (F = 139%). More extensive PK evaluations were
performed in rats, dogs, and cynomolgous monkeys. The results
of these experiments were summarized in Table 9.
The overall PK profiles of (S)-14k looked quite promising
across species. For example, the IV clearances were modest in all
three species, an improvement over the previous lead 1a (near
hepatic blood flow CL in rat, dog, and cyno).9 These
observations confirmed our hypothesis that by removing
metabolic soft spots in the previous template, the pyridones
should have improved metabolic stability over the piperidinopyr-
imidine ureas. Furthermore, oral bioavailability of (S)-14k in rat
(F = 44%) and dog (F = 46%) at 5 mg/kg were superior to 1a
(11% F in rat and 26% in dog),9 partly due to improved
absorption. In vitro ADME profiling revealed that while 1a was
an efflux substrate (efflux ratio BA/AB = 7.8) with low intrinsic
permeability (Papp AB (10−6 cm/s) = 1.3), (S)-14k was a highly
permeable compound without efflux. This was attributed to
replacing the urea linker with a pyridone template, eliminating an
X-ray Crystal Structure. An X-ray structure of (S)-14k
bound to ERK2 at 2.58 Å resolution was obtained (Figure 3).
The crystal structure confirms earlier modeling conclusions that
the 2-aminopyrimidine interacts via a pair of H-bonds with the
hinge region of ERK2 at Met108. The 4-THP interacts with
Lys114 via the ether oxygen, although it appears that the bulk of
the potency is derived from hydrophobic interactions in this
region. As we hypothesized above, a key water molecule (Figure
G
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Table 6. SAR of the Amino-pyrimidine
a
Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to
b
c
convert IC50 to Ki. All assay results represent the mean of at least three independent determinations. Kd values were determined by competitive
displacement of an ATP-analogue probe. Cellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details.
d
unnecessary hydrogen bond donor in the process. Importantly,
our new lead also performed well in dose-escalation PK studies in
nude mice. When dosed orally at 30, 100, and 150 mg/kg, a
proportional increase in AUC was observed (Table 9). At the
lower dose of 30 mg/kg, the drug level in the plasma covered the
free fraction adjusted cellular IC50 (pRSK IC50/Fu = 944 nM) for
more than 8 h (Figure 4). Upon the basis of these results, (S)-14k
was progressed into a PK/PD study (75 mg/kg, QD) in nude
mice bearing subcutaneous human HCT116 colorectal cancer
cell xenografts.
Significant pRSK activity was observed at the 75 mg/kg dose,
with the corresponding pRSK knockdown at the 2 h time point
observed to be ∼70% (Figure 5). Interestingly, at the 12 h time
point, there was an apparent rebound in hyperphosphorylation
of RSK, probably as a result of compensational feedback. Similar
results were observed from other cell lines (A375 and Colo205,
data not shown). On the basis of these results, (S)-14k was
evaluated in a proof-of-concept tumor growth inhibition (TGI)
study.
(S)-14k was administered twice daily (BID) orally in nude
mice bearing HCT116 colorectal cancer xenografts for 21 days
(Figure 6). At the 75 mg/kg dose, significant tumor growth
inhibition (70%) was obtained and the treatment was well
tolerated. Body weight losses were within an acceptable range
(<5%) throughout the study (Supporting Information Figure 1).
In comparison, our prior lead 1a was not as active at the 75 mg/
kg dose in this model and only showed 69% TGI with 150 mg/kg
dose after 28 days of BID dosing.
CONCLUSIONS
■
In summary, a series of potent and selective ERK1/2 inhibitors
were developed. These inhibitors are based on a novel pyridone
template, which evolved through structure-based design from a
piperidinopyrimidine urea lead 1a. Comprehensive and system-
H
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Article
(triplet), q (quartet), m (multiplet), dd (doublet of doublet), dt
(doublet of triplet), br (broad). Coupling constants are reported in hertz
(Hz). The purity of each compound that was synthesized and tested for
biological activity was ≥95% via HPLC analysis.
For experiments involving the use of aminals, all procedures were
carried out in compliance with the guidelines on animal studies outlined
in the ACS Ethical Guidelines.
Table 7. Activities of (R)-14k, (S)-14k, and 1a
2-Chloro-4-(2-fluoropyridin-4-yl)pyrimidine (3). Sodium car-
bonate (4.91 g, 46.3 mmol) was added to 2-fluoropyridin-4-ylboronic
acid (2.61 g, 18.5 mmol) and 2,4-dichloropyrimidine (2.30 g, 15.4
mmol) in dioxane/water (77 mL, 4:1), and the suspension was purged
with argon. To this was added PdCl2(dppf)·DCM (0.630 g, 0.772
mmol), and the mixture was heated at 80 °C under argon. After 3 h, the
reaction mixture was diluted with water (100 mL), and the resulting
solid was collected by vacuum filtration to yield 2-chloro-4-(2-
fluoropyridin-4-yl)pyrimidine (3.12 g, 96% yield). 1H NMR (400
MHz, CDCl3) δ 8.80 (d, J = 5.2 Hz, 1H), 8.42 (d, J = 5.0 Hz, 1H), 7.87−
7.78 (m, 1H), 7.71 (d, J = 5.4 Hz, 1H), 7.65−7.60 (m, 1H); m/z (APCI-
pos) M + 1 = 210.0, 212.1
ERK2
CDK2
enzyme K
enzyme K
phospho-p90RSK
CDK2
a,
b i
b,
c d
b,d
compd no.
(nM)
(nM)
IC50 (nM)
49 15
433 159
Kd/ERK2 Ki
(rac)-14k
(R)-14k
(S)-14k
1a
0.6 0.1
2.8 0.7
0.3 0.02
0.1 0.02
57 29
95
95
70
41
267 57
21
8
17
20
4
3
4.1 1.8
a
Ki values were determined by carrying out ERK2 enzymatic assays in
the presence of ATP at 80× Km
correction to convert IC50 to Ki. All assay results represent the mean
(app)
and applying Cheng−Prusoff
b
c
of at least three independent determinations. Kd values were
4-(2-Fluoropyridin-4-yl)-N-(tetrahydro-2H-pyran-4-yl)-
pyrimidin-2-amine (4). N-Ethyl-N-isopropylpropan-2-amine (0.496
mL, 2.86 mmol) was added to tetrahydro-2H-pyran-4-amine (0.265 g,
2.62 mmol) and 2-chloro-4-(2-fluoropyridin-4-yl)pyrimidine (0.50 g,
2.39 mmol) in 2-butanol (2 mL) in a sealed tube (microwave vial). The
vial was sealed and heated at 100 °C in an oil bath overnight. The
reaction mixture was evaporated, and the dark residue was treated with
EtOAc (50 mL) and water (50 mL) and filtered through Celite, and the
layers were separated. The EtOAc layer was washed with brine, dried
over MgSO4, filtered, and evaporated. The crude product was purified by
chromatography, eluting with 2:1 EtOAc/hexanes to give 4-(2-
fluoropyridin-4-yl)-N-(tetrahydro-2H-pyran-4-yl)pyrimidin-2-amine
(0.32 g, 49% yield). 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 5.1 Hz,
1H), 8.34 (d, J = 6.0 Hz, 1H), 7.76−7.72 (m, 1H), 7.55−7.53 (m, 1H),
6.99 (d, J = 5.1 Hz, 1H), 5.38 (br, s, 1H), 4.21−4.11 (m, 1H), 4.07−3.98
(m, 2H), 3.64−3.54 (m, 2H), 2.16−2.06 (m, 2H), 1.68−1.53 (m, 2H);
m/z (APCI-pos) M + 1 = 275.1.
4-(2-((Tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-
pyridin-2(1H)-one (5). HCl (1.0 M, 35 mL, 35 mmol) was added to 4-
(2-fluoropyridin-4-yl)-N-(tetrahydro-2H-pyran-4-yl)pyrimidin-2-
amine (0.32 g, 1.2 mmol), and the mixture was heated at reflux
overnight. The cooled reaction mixture was neutralized with solid
NaHCO3. The resulting solid was collected by vacuum filtration, washed
into a flask with EtOAc/MeOH, evaporated, and dried to afford 4-(2-
((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one
(0.30 g, 94% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.74 (br s, 1H),
8.40 (d, J = 5.0 Hz, 1H), 7.48 (d, J = 6.5 Hz, 1H), 7.33 (d, J = 7.5 Hz,
1H), 7.11 (d, J = 5.0 Hz, 1H), 6.99 (s, 1H), 6.85−6.76 (m, 1H), 4.04−
3.92 (m, 1H), 3.92−3.85 (m, 2H), 3.45−3.36 (m, 2H), 1.90−1.81 (m,
2H), 1.59−1.48 (m, 2H); m/z (APCI-pos) M + 1 = 273.1.
1-Benzyl-4-(2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-
4-yl)pyridin-2(1H)-one (6a). A mixture of 4-(2-((tetrahydro-2H-
pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (0.030 g, 0.110
mmol), (bromomethyl)benzene (0.0144 mL, 0.121 mmol), and K2CO3
(0.0305 g, 0.220 mmol) in DMF (1.10 mL) was stirred at room
temperature for 12 h. The mixture was worked up with DCM (50 mL)
and water (50 mL). The organics were extracted with DCM twice,
washed with brine, dried with Na2SO4, and concentrated. The crude
product was purified on reverse phase HPLC to give 1-benzyl-4-(2-
((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one
(0.0332 g, 66% yield). 1H NMR (400 MHz, CDCl3) δ 9.85 (br s, 1H),
8.18 (s, 1H), 7.46 (d, J = 7.2 Hz, 1H), 7.39−7.31 (m, 6H), 7.04 (d, J = 6.0
Hz, 1H), 6.75 (d, J = 7.0 Hz, 1H), 5.21 (s, 2H), 4.22−4.18 (m, 1H),
4.06−4.00 (m, 2H), 3.58−3.50 (m, 2H), 2.04−2.00 (m, 2H), 1.82−1.78
(m, 2H); m/z (APCI-pos) = 363.2.
determined by competitive displacement of an ATP-analogue probe.
Cellular assay in PMA-stimulated HepG2 cells. See Supporting
Information for details.
d
atic SAR investigation led to the identification of (S)-14k, a
highly potent, selective, and efficacious ERK1/2 inhibitor. In
comparison to our previous lead 1a, (S)-14k showed much
improved PK across species, which translated into better TGI in
xenograft studies, especially at lower doses. These improvements
were attributed to the design of a superior template by replacing
the piperidine urea linker with a pyridone core, which led to the
elimination of PK liabilities (metabolic soft spots and an
unnecessary hydrogen bond donor) associated with the previous
scaffold. On the basis of its in vivo efficacy and preliminary safety
profile, (S)-14k was selected for further preclinical evaluation.
The results from these studies will be reported in due course.
EXPERIMENTAL SECTION
■
The reactions described herein were generally conducted under a
positive pressure of nitrogen or argon or with a drying tube in
commercial anhydrous solvents unless otherwise stated, and the
reaction flasks were typically fitted with rubber septa during the
introduction of reagents via a syringe or cannula. Glasswares were oven
and/or heat dried. All reagents and solvents were used without further
purification unless otherwise stated. Reaction progress was monitored
by analytical TLC, HPLC, and LCMS. Analytical TLC was performed
using glass plates precoated with silica gel from EMD (60 F254, 250 μm).
TLC visualization was performed with UV light or by staining with
Hanessian’s stain14 or basic KMnO4 solution. Flash column
chromatography was performed on a Biotage model SP1 purification
system running SPX software with prepacked silica gel or C18 columns
and UV detection at 220 and 254 nm. Analytical HPLC was performed
on an Agilent 1200 (column YMC ODS-AQ, 3 μm, 120 Å, 3.0 mm × 50
mm; run gradient 5−95% 0.01% HFBA/1% IPA/water/acetonitrile; UV
detection 220 and 254 nm). Analytical LCMS was performed on an
Agilent 1200 HPLC coupled with an Agilent 6120 quadruple LC/MS
spectrometer using ESI or APCI ionization sources (column YMC
ODS-AQ, 4.6 mm × 50 mm, 3 μm, 120 Å; run gradient 5−95% B over
4.5 min, holding 95% B for 1 min, followed by equilibration for 1 min;
flow rate 2 mL/min; solvent A water/10 mM ammonium acetate/1%
IPA; solvent B acetonitrile/10 mM ammonium acetate/1% IPA).
Melting points were recorded on an electrothermal melting point
1
apparatus, model 9100. H NMR spectra were recorded in CDCl3,
MeOH-d4 or DMSO-d6 solvents on a Varian Mercury (400 MHz) NMR
spectrometer or on a Bruker AV III (400 or 500 MHz) spectrometer.
Chemical shifts are expressed in parts per million (ppm, δ scale) using
tetramethylsilane as the reference standard. When peak multiplicities are
reported, the following abbreviations are used: s (singlet), d (doublet), t
1-(3-Chlorobenzyl)-4-(2-((tetrahydro-2H-pyran-4-yl)amino)-
pyrimidin-4-yl)pyridin-2(1H)-one (6b). 1H NMR (400 MHz,
CDCl3) δ 8.85 (br s, 1H), 8.24 (s, 1H), 7.42 (d, J = 7.0 Hz, 1H),
7.34−7.22 (m, 5H), 6.99 (d, J = 5.3 Hz, 1H), 6.78 (d, J = 7.0 Hz, 1H),
5.16 (s, 2H), 4.20−4.16 (m, 1H), 4.06−3.98 (m, 2H), 3.59−3.51 (m,
I
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Figure 3. (A) X-ray structure of (S)-14k (green) bound to ERK2, solved at 2.58 Å resolution (PDB code 4XJ0). Hydrogen-bonding interactions are
illustrated with red dashed lines. (B) X-ray structure rotated to illustrate key water mediated H-bond interactions.
Table 8. In Vitro ADME Properties of (S)-14k and 1a
in vitro predicted CL
a
(mL/min/kg)
PPB % bound
b
c
compd
human
rat
mouse
permeability Papp AB
efflux ratio BA/AB
solubility (μg/mL) @ pH 7.4
human
rat
mouse
(S)-14k
4
3
19
26
36
22
11.3
0.84
7.8
45
95.8
82.4
96.7
98.2
89.5
1a
1.3
526
82.8
a
b
c
Hepatic CL predicted from liver hepatocytes. Permeability measurement determined in MDCK cell line, in units of 10−6 cm/s. Thermodynamic
solubility.
2H), 2.08−2.00 (m, 2H), 1.78−1.70 (m, 2H); m/z (APCI-pos) = 397.2,
399.2.
1-(4-Chlorobenzyl)-4-(2-((tetrahydro-2H-pyran-4-yl)amino)-
pyrimidin-4-yl)pyridin-2(1H)-one (6c). 1H NMR (400 MHz,
CDCl3) δ 9.82 (br s, 1H), 8.18 (s, 1H), 7.46 (d, J = 7.02 Hz, 1H),
7.36−7.26 (m, 5H), 7.04 (d, J = 6.0 Hz, 1H), 6.76 (d, J = 7.0 Hz, 1H),
5.16 (s, 2H), 4.22−4.18 (m, 1H), 4.06−3.98 (m, 2H), 3.58−3.50 (m,
2H), 2.06−1.98 (m, 2H), 1.84−1.76 (m, 2H); m/z (APCI-pos) = 397.2,
399.2.
1-(3-Chloro-4-fluorobenzyl)-4-(2-((tetrahydro-2H-pyran-4-
1
yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (6d). H NMR (400
MHz, CDCl3) δ 9.38 (br s, 1H), 8.21 (s, 1H), 7.46−7.38 (m, 2H), 7.28−
J
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7.20 (m, 1H), 7.14−7.08 (m, 2H), 7.01 (d, J = 5.6 Hz, 1H), 6.80−6.76
(m, 1H), 5.15 (s, 2H), 4.21−4.17 (m, 1H), 4.07−4.01 (m, 2H), 3.58−
3.50 (m, 2H), 2.06−2.00 (m, 2H), 1.82−1.76 (m, 2H); m/z (APCI-pos)
= 415.2, 417.2.
Table 9. Key Pharmacokinetic Parameters for (S)-14k
a
b
b
b
species
dose PO
mouse
rat
5
dog
5
cyno
5
5
30
100
150
4-(2-Fluoropyridin-4-yl)-2-(methylthio)pyrimidine (7). A sus-
pension of 4-bromo-2-(methylthio)pyrimidine (7.00 g, 34.1 mmol), 2-
fluoropyridin-4-ylboronic acid (5.05 g, 35.8 mmol), Na2CO3 (10.9 g,
102 mmol), and PdCl2(dppf)·DCM (1.40 g, 1.71 mmol) in dioxane/
H2O (100 mL; 1:1) was heated to 85 °C under an Ar balloon for 2 h.
The reaction mixture was cooled to room temperature and
concentrated. The residue was diluted with EtOAc (200 mL) and
water (100 mL). The layers were separated, and the aqueous layer was
extracted with EtOAc (1×). The organics were dried, filtered, and
concentrated. The crude product was purified via column chromatog-
raphy, eluting with hexanes/EtOAc (3:1) to give 4-(2-fluoropyridin-4-
(mg/kg)
max (μM)
1/2 (h)
C
T
3.12
3.52
5.40
139
20.2
2.33
46.8
169
38.9
1.71
129
139
67.7
1.97
175
127
1.49
1.16
2.15
44
2.91
1.55
4.87
46
0.98
2.39
3.30
22
AUC (μM·h)
F (%)
CL
40
39
18
12
(mL/min/kg)
c
IV
a
Dosed as a suspension in 40% PEG400/60% of 10% HP-b-CD in
H2O in nude mouse. Dosed as a solution in 40% PEG400/60% of
10% HP-b-CD in H2O. IV clearance measured at 1 mg/kg for nude
mouse and rat, at 2 mg/kg for dog and cyno dosed as a solution in
40% PEG400/60% H2O.
b
c
1
yl)-2-(methylthio)pyrimidine (6.83 g, 90%) as a solid. H NMR (400
MHz, DMSO-d6) δ 8.85 (d, J = 5.2 Hz, 1H), 8.46 (d, J = 5.2 Hz, 1H),
8.13−8.09 (m, 1H), 7.96 (d, J = 5.2 Hz, 1H), 7.92 (s, 1H), 2.62 (s, 3H);
m/z (APCI-pos) M + 1 = 222.1.
4-(2-(Methylthio)pyrimidin-4-yl)pyridin-2(1H)-one (8). A sus-
pension of 4-(2-fluoropyridin-4-yl)-2-(methylthio)pyrimidine (6.83 g,
30.9 mmol) in 2 N HCl (100 mL) was heated to reflux for 2 h. The
reaction mixture was cooled to room temperature and placed in an ice
bath. The pH was adjusted to about 7 with 2N NaOH (about 100 mL).
The resulting solids were collected by filtration, washed with water, and
dried to give 4-(2-(methylthio)pyrimidin-4-yl)pyridin-2(1H)-one (5.07
g) as a solid. This material was placed in the thimble of a Soxhlet
apparatus and was attached to a 1 L flask charged with EtOAc (500 mL).
The material was continuously extracted for 3 days. The resulting white
precipitate from the EtOAc layer was collected by filtration to give 4-(2-
1
(methylthio)pyrimidin-4-yl)pyridin-2(1H)-one (3.3 g, 49% yield). H
NMR (400 MHz, DMSO-d6) δ 11.85 (br, s, 1H), 8.75 (d, J = 5.0 Hz,
1H), 7.79 (d, J = 5.0 Hz, 1H), 7.54 (d, J = 7.0 Hz, 1H), 7.13 (s, 1H), 6.86
(d, J = 7.0 Hz, 1H), 2.58 (s, 3H); m/z (APCI-pos) M + 1 = 220.0.
2-(tert-Butyldimethylsilyloxy)-1-(3-chlorophenyl)ethanol
(9d, R3 = 3-Cl). A solution of 2-(tert-butyldimethylsilyloxy)-
acetaldehyde (2.00 g, 10.3 mmol) in dry THF (40 mL) was placed in
an ice bath and to this was added 0.5 M (3-chlorophenyl)magnesium
bromide (24.8 mL, 12.4 mmol) in THF dropwise via a syringe. The
reaction mixture was stirred at 0 °C for 1 h and then carefully quenched
by dropwise addition of water (5 mL). The reaction mixture was
concentrated, and the residue was partitioned between EtOAc (100 mL)
and saturated NH4Cl (50 mL). The layers were separated, and the
organics were dried, filtered, and concentrated. The crude product was
purified by column chromatography, eluting with hexanes/EtOAc
(20:1) to give 2-(tert-butyldimethylsilyloxy)-1-(3-chlorophenyl)ethanol
(2.55 g, 86%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.39 (s,
1H), 7.29−7.21 (m, 3H), 4.75−4.69 (m, 1H), 3.79−3.73 (m, 1H),
3.55−3.48 (m, 1H), 2.95 (d, J = 1.8 Hz, 1H), 0.90 (s, 9H), 0.07 (s, 3H),
0.06 (s, 3H).
Figure 4. Dose-escalation of (S)-14k in nude mice. Three nude mice
were dosed orally with (S)-14k at doses of 30, 100, and 150 mg/kg in
10%HP-b-CD/PEG400 suspension. At different time intervals, a blood
sample was taken from each animal and plasma concentration was
determined. Reported values were the average of three animals. The free
fraction adjusted cellular IC50 was calculated by dividing the cellular IC50
by the unbound fraction in mouse.
2-(tert-Butyldimethylsilyloxy)-1-(3-chlorophenyl)-
ethylmethanesulfonate (10d, R3 = 3-Cl). To a cold (0 °C) solution
of 2-(tert-butyldimethylsilyloxy)-1-(3-chlorophenyl)ethanol (1.40 g,
4.88 mmol) in DCM (30 mL) was added triethylamine (1.02 mL,
7.32 mmol), followed by methanesulfonyl chloride (0.453 mL, 5.86
mmol). The reaction mixture was stirred at 0 °C for 1 h and then
queched with water (5 mL). The layers were separated, and the organics
were, dried, filtered, and concentrated to give 2-(tert-butyldimethylsi-
lyloxy)-1-(3-chlorophenyl)ethylmethanesulfonate (1.70 g, 95%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.38 (s, 1H), 7.37−7.26 (m,
3H), 5.54−5.49 (m, 1H), 3.96−3.89 (m, 1H), 3.83−3.77 (m, 1H), 2.95
(s, 3H), 0.88 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H).
Figure 5. PK/PD in HCT116 xenograft tumors for compound (S)-14k
at 2, 4, 8, and 12 h time points dosed orally at 75 mg/kg QD. Mean and
sd for n = 3 animals are plotted. Green triangles represent total plasma
concentration, red squares represent total tumor concentration.
1-(2-(tert-Butyldimethylsilyloxy)-1-(3-chlorophenyl)ethyl)-4-
(2-(methylthio)pyrimidin-4-yl)pyridin-2(1H)-one (11d, R3 = 3-
Cl). To a cooled (0 °C) suspension of 4-(2-(methylthio)pyrimidin-4-
yl)pyridin-2(1H)-one (0.556 g, 2.54 mmol) in THF (25 mL) was added
1.0 M of KHMDS (3.30 mL, 3.30 mmol) in THF. The reaction mixture
was left at 0 °C for 10 min before 2-(tert-butyldimethylsilyloxy)-1-(3-
chlorophenyl)ethylmethanesulfonate (1.76 g, 4.82 mmol) was added as
a solution in THF (10 mL). The cold bath was removed, and the
7.20 (m, 2H), 7.16−7.12 (m, 1H), 7.01 (d, J = 5.2 Hz, 1H), 6.80−6.76
(m, 1H), 5.13 (s, 2H), 4.20−4.16 (m, 1H), 4.06−4.00 (m, 2H), 3.58−
3.50 (m, 2H), 2.06−2.00 (m, 2H), 1.80−1.72 (m, 2H); m/z (APCI-pos)
= 415.2, 417.2.
1-(4-Chloro-3-fluorobenzyl)-4-(2-((tetrahydro-2H-pyran-4-
1
yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (6e). H NMR (400
MHz, CDCl3) δ 9.32 (br s, 1H), 8.22 (s, 1H), 7.44−7.38 (m, 2H), 7.28−
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Figure 6. HCT116 colorectal tumor xenograft treated with compound (S)-14k orally at 75 mg/kg BID for 21 days. Cubic spline fitted tumor volume
curves are shown for each group in red. Individual tumor volume traces are shown in gray, traces terminating in an X indicate animals sacrificed when
tumors reached maximum permissible volume. The cubic spline fit for the vehicle group is shown in the (S)-14k treated group for comparison (green
dashed line).
reaction mixture was heated to 55 °C for 40 h. The reaction mixture was
cooled to room temperature and concentrated. The residue was taken
up in EtOAc (100 mL) and washed with water (2 × 50 mL). The
organics were dried, filtered, and concentrated. The crude product was
purified via column chromatography, eluting with hexanes/EtOAc (3:1)
to give 1-(2-(tert-butyldimethylsilyloxy)-1-(3-chlorophenyl)ethyl)-4-
residue was taken up with EtOAc (50 mL) and water (20 mL). The
layers were separated, and the organic layer was washed with water (1×).
The combined organics were dried, filtered, and concentrated. The
crude product was purified via column chromatography, eluting with
EtOAc to give 1-(1-(3-Chlorophenyl)-2-hydroxyethyl)-4-(2-(tetrahy-
dro-2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (0.40 g,
67%). 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 5.0 Hz, 1H), 7.42−
7.35 (m, 2H), 7.35−7.30 (m, 2H), 7.27−7.23 (m, 1H), 7.19 (s, 1H),
6.89 (d, J = 5.0 Hz, 1H), 6.81−6.73 (m, 1H), 6.29−6.18 (m, 1H), 5.15
(d, J = 8.0 Hz, 1H), 4.38−4.27 (m, 2H), 4.16−4.04 (m, 1H), 4.03−3.96
(m, 2H), 3.61−3.50 (m, 2H), 2.65−2.55 (m, 1H), 2.13−2.00 (m, 2H),
1.66−1.50 (m, 2H); m/z (APCI-pos) M + 1 = 427.1, 429.1.
1
(2-(methylthio)pyrimidin-4-yl)pyridin-2(1H)-one (0.79 g, 64%). H
NMR (400 MHz, CDCl3) δ 8.65 (d, J = 5.0 Hz, 1H), 7.52−7.45 (m,
2H), 7.37−7.27 (m, 4H), 6.87−6.82 (m, 1H), 6.28−6.24 (m, 1H),
4.41−4.33 (m, 1H), 4.26−4.21 (m, 1H), 2.64 (s, 3H), 0.88 (s, 9H), 0.06
(s, 3H), 0.01 (s, 3H); m/z (APCI-pos) M + 1 = 488.1, 490.1.
1-(2-(tert-Butyldimethylsilyloxy)-1-(3-chlorophenyl)ethyl)-4-
(2-(methylsulfonyl)pyrimidin-4-yl)pyridin-2(1H)-one (12d, R3 =
3-Cl). To a solution of 1-(2-(tert-butyldimethylsilyloxy)-1-(3-
chlorophenyl)ethyl)-4-(2-(methylthio)pyrimidin-4-yl)pyridin-2(1H)-
one (0.79 g, 1.6 mmol) in 20 mL of DCM was added mCPBA (1.00 g,
4.0 mmol). The mixture was stirred at room temperature for 2 h and
then quenched with saturated Na2S2O3 (20 mL). The organics were
washed with NaHCO3 (1×), dried over Na2SO4, filtered, and
evaporated. The crude product was purified via column chromatog-
raphy, eluting with hexanes/EtOAc (1:1) to give 1-(2-(tert-butyldime-
thylsilyloxy)-1-(3-chlorophenyl)ethyl)-4-(2-(methylsulfonyl)-
pyrimidin-4-yl)pyridin-2(1H)-one (0.73 g, 87%). 1H NMR (400 MHz,
CDCl3) δ 9.06 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 5.4 Hz, 1H), 7.57 (d, J =
7.5 Hz, 1H), 7.46 (s, 1H), 7.36−7.29 (m, 3H), 6.94−6.89 (m, 1H),
6.27−6.21 (m, 1H), 4.40−4.34 (m, 1H), 4.27−4.20 (m, 1H), 3.45 (s,
3H), 0.88 (s, 9H), 0.06 (s, 3H), 0.01 (s, 3H); m/z (APCI-pos) M + 1 =
519.1, 521.1.
1-(2-Hydroxy-1-phenylethyl)-4-(2-(tetrahydro-2H-pyran-4-
1
ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14a). H NMR (400
MHz, CDCl3) δ 8.37 (d, J = 5.1 Hz, 1H), 7.46−7.30 (m, 6H), 7.19 (s,
1H), 6.87 (d, J = 5.2 Hz, 1H), 6.78−6.71 (m, 1H), 6.35−6.27 (m, 1H),
4.39−4.30 (m, 2H), 4.15−4.04 (m, 1H), 4.04−3.95 (m, 2H), 3.60−3.50
(m, 2H), 2.84 (br, s, 1H), 2.09−2.01 (m, 2H), 1.65−1.51 (m, 3H); m/z
(APCI-pos) M + 1 = 393.2.
1-(1-(2-Chlorophenyl)-2-hydroxyethyl)-4-(2-(tetrahydro-2H-
pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14b). 1H
NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 5.0 Hz, 1H), 7.64−7.63
(m, 1H), 7.51−7.48 (m, 2H), 7.41−7.33 (m, 3H), 7.14 (d, J = 5.2 Hz,
1H), 7.09 (s, 1H), 6.84 (d, J = 7.2 Hz, 1H), 6.17−6.14 (m, 1H), 5.40−
5.37 (m, 1H), 4.08−3.97 (m, 3H), 3.89−3.86 (m, 2H), 3.43−3.37 (m,
2H), 1.87−1.83 (m, 2H), 1.58−1.48 (m, 2H); m/z (APCI-pos) M + 1 =
427.1, 429.1.
1-(1-(4-Chlorophenyl)-2-hydroxyethyl)-4-(2-(tetrahydro-2H-
pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14c). 1H
NMR (400 MHz, CDCl3) δ 8.37 (d, J = 4.6 Hz, 1H), 7.41−7.28 (m,
5H), 7.17 (s, 1H), 6.87 (d, J = 5.2 Hz, 1H), 6.79−6.71 (m, 1H), 6.27−
6.19 (m, 1H), 5.18 (d, J = 6.8 Hz, 1H), 4.35−4.26 (m, 2H), 4.14−4.04
(m, 1H), 4.04−3.94 (m, 2H), 3.61−3.50 (m, 2H), 2.89 (br, s, 1H),
2.10−2.01 (m, 2H), 1.64−1.50 (m, 2H); m/z (APCI-pos) M + 1 =
427.1, 429.1.
1-(1-(3-Fluorophenyl)-2-hydroxyethyl)-4-(2-((tetrahydro-2H-
pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (14e). 1H
NMR (400 MHz, CDCl3) δ 8.37 (d, J = 5.0 Hz, 1H), 7.40−7.33 (m,
2H), 7.17−7.01 (m, 4H), 6.87 (d, J = 5.2 Hz, 1H), 6.78−6.76 (m, 1H),
6.28−6.25 (m, 1H), 5.20 (d, J = 8.2 Hz, 1H), 4.32−4.31 (m, 2H), 4.13−
4.06 (m, 1H), 4.02−3.98 (m, 2H), 3.59−3.52 (m, 2H), 3.11−3.08 (m,
1H), 2.07−2.04 (m, 2H), 1.63−1.53 (m, 2H); m/z (APCI-pos) M + 1 =
411.1.
1-(1-(3-Chlorophenyl)-2-hydroxyethyl)-4-(2-(tetrahydro-2H-
pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14d). Step
1. A solution of 1-(2-(tert-butyldimethylsilyloxy)-1-(3-chlorophenyl)-
ethyl)-4-(2-(methylsulfonyl)pyrimidin-4-yl)pyridin-2(1H)-one (0.73 g,
1.4 mmol) and tetrahydro-2H-pyran-4-amine (0.21 g, 2.1 mmol) in sec-
BuOH (4 mL) was heated to 120 °C for 4 h in a sealed tube. The
reaction mixture was cooled to room temperature and concentrated.
The crude 1-(2-(tert-butyldimethylsilyloxy)-1-(3-chlorophenyl)ethyl)-
4-(2-(tetrahydro-2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-
one (13d, R3 = 3-Cl) was used without purification in Step 2. m/z
(APCI-pos) M + 1 = 541.2, 542.2.
Step 2. A solution of 1-(2-(tert-butyldimethylsilyloxy)-1-(3-
chlorophenyl)ethyl)-4-(2-(tetrahydro-2H-pyran-4-ylamino)pyrimidin-
4-yl)pyridin-2(1H)-one (0.76 g, 1.40 mmol) in THF (20 mL) was
treated with 1.0 M of TBAF (1.69 mL, 1.69 mmol) in THF at room
temperature for 30 min. The reaction mixture was concentrated, and the
L
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1-(2-Hydroxy-1-m-tolylethyl)-4-(2-(tetrahydro-2H-pyran-4-
6.0 Hz, 1H), 5.94−5.91 (m, 1H), 5.36−5.33 (m, 1H), 4.21−4.14 (m,
1H), 4.09−3.95 (m, 2H), 3.89−3.86 (m, 2H), 3.43−3.37 (m, 2H),
1.88−1.84 (m, 2H), 1.58−1.49 (m, 2H); m/z (APCI-pos) M + 1 =
461.1, 463.1.
1
ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14f). H NMR (400
MHz, DMSO-d6) δ 8.41 (d, J = 5.0 Hz, 1H), 7.85 (d, J = 7.0 Hz, 1H),
7.34 (d, J = 7.2 Hz, 1H), 7.26−7.22 (m, 1H), 7.15−7.09 (m, 5H), 6.86−
6.84 (m, 1H), 6.03−5.99 (m, 1H), 5.22−5.20 (m, 1H), 4.17−4.11 (m,
1H), 4.03−3.97 (m, 2H), 3.89−3.86 (m, 2H), 3.43−3.37 (m, 2H), 2.28
(s, 3H), 1.87−1.84 (m, 2H), 1.57−1.49 (m, 2H); m/z (APCI-pos) M +
1 = 407.1.
1-(2-Hydroxy-1-(3-(trifluoromethyl)phenyl)ethyl)-4-(2-(tet-
rahydro-2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one
(14g). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 5.0 Hz, 1H), 7.93
(d, J = 7.2 Hz, 1H), 7.70−7.68 (m, 2H), 7.62−7.61 (m, 2H), 7.35 (d, J =
8.0 Hz, 1H), 7.16 (d, J = 5.2 Hz, 1H), 7.11 (s, 1H), 6.89 (d, J = 8.2 Hz,
1H), 6.07−6.03 (m, 1H), 5.35−5.33 (m, 1H), 4.25−4.19 (m, 1H),
4.12−4.06 (m, 1H), 4.02−3.95 (m, 1H), 3.89−3.86 (m, 2H), 3.43−3.37
(m, 2H), 1.87−1.84 (m, 2H), 1.58−1.49 (m, 2H); m/z (APCI-pos) M +
1 = 461.1.
1-(2-Hydroxy-1-(3-methoxyphenyl)ethyl)-4-(2-(tetrahydro-
2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14h).
1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 5.0 Hz, 1H), 7.87 (d, J
= 8.0 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.29−7.25 (m, 1H), 7.14 (d, J =
6.0 Hz, 1H), 7.09 (s, 1H), 6.89−6.84 (m, 4H), 6.03−6.01 (m, 1H),
5.23−5.20 (m, 1H), 4.18−4.11 (m, 1H), 4.04−3.96 (m, 2H), 3.89−3.86
(m, 2H), 3.73 (s, 3H), 3.43−3.37 (m, 2H), 1.87−1.84 (m, 2H), 1.58−
1.48 (m, 2H); m/z (APCI-pos) M + 1 = 423.1.
3-(2-Hydroxy-1-(2-oxo-4-(2-(tetrahydro-2H-pyran-4-
ylamino)pyrimidin-4-yl)pyridin-1(2H)-yl)ethyl)benzonitrile
(14i). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 5.0 Hz, 1H), 7.92
(d, J = 7.0 Hz, 1H), 7.84 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 7.2
Hz, 1H), 7.60−7.56 (m, 1H), 7.36−7.34 (m, 1H), 7.16 (d, J = 5.0 Hz,
1H), 7.10 (s, 1H), 6.89 (d, J = 7.0 Hz, 1H), 6.02−5.99 (m, 1H), 5.35−
5.33 (m, 1H), 4.22−4.16 (m, 1H), 4.10−4.04 (m, 1H), 4.02−3.95 (m,
1H), 3.89−3.86 (m, 2H), 3.43−3.37 (m, 2H), 1.87−1.84 (m, 2H),
1.57−1.49 (m, 2H); m/z (APCI-pos) M + 1 = 418.1.
1-(1-(3,4-Difluorophenyl)-2-hydroxyethyl)-4-(2-(tetrahydro-
2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14j). 1H
NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 5.0 Hz, 1H), 7.88 (d, J = 7.3
Hz, 1H), 7.50−7.34 (m, 3H), 7.18−7.14 (m, 2H), 7.10 (s, 1H), 6.87 (d,
J = 8.7 Hz, 1H), 5.99−5.96 (m, 1H), 5.31−5.29 (m, 1H), 4.18−4.12 (m,
1H), 4.05−3.96 (m, 2H), 3.89−3.87 (m, 2H), 3.43−3.37 (m, 2H),
1.87−1.84 (m, 2H), 1.58−1.49 (m, 2H); m/z (APCI-pos) M + 1 =
429.1.
1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((tetra-
hydro-2H-pyran-4-ylamino))pyrimidin-4-yl)pyridin-2(1H)-one
(14k). 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 5.0 Hz, 1H), 7.42−
7.38 (m, 2H), 7.22−7.18 (m, 2H), 7.10 (d, J = 8.2 Hz, 1H), 6.88 (d, J =
5.0 Hz, 1H), 6.80−6.76 (m, 1H), 6.22−6.18 (m, 1H), 5.16 (d, J = 8.0 Hz,
1H), 4.34−4.28 (m, 2H), 4.12−4.08 (m, 1H), 4.04−3.96 (m, 2H),
3.58−3.52 (m, 2H), 2.74 (br, s, 1H), 2.09−2.03 (m, 2H), 1.62−1.56 (m,
2H); m/z (APCI-pos) M + 1 = 445.1, 447.0.
1-(1-(3-Chloro-4-fluorophenyl)-2-hydroxyethyl)-4-(2-((tetra-
hydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one
(14l). 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 5.0 Hz, 1H), 7.47−
7.44 (m, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.29−7.24 (m, 1H), 7.18−7.14
(m, 2H), 6.88 (d, J = 8.7 Hz, 1H), 6.80−6.77 (m, 1H), 6.21−6.17 (m,
1H), 5.17−5.14 (m, 1H), 4.32−4.29 (m, 2H), 4.15−4.08 (m, 1H),
4.03−3.98 (m, 2H), 3.60−3.52 (m, 2H), 2.70−2.66 (m, 1H), 2.09−2.03
(m, 2H), 1.63−1.53 (m, 2H); m/z (APCI-pos) M + 1 = 445.1, 447.0.
1-(1-(3,5-Difluorophenyl)-2-hydroxyethyl)-4-(2-(tetrahydro-
2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (14m).
1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 6.2 Hz, 1H), 7.91(d, J
1-(1-(3-Chloro-5-fluorophenyl)-2-hydroxyethyl)-4-(2-(tetra-
hydro-2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one
(14o). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 5.2 Hz, 1H), 7.93
(d, J = 7.1 Hz, 1H), 7.42−7.34 (m, 2H), 7.25 (s, 1H), 7.20 (d, J = 9.4 Hz,
1H), 7.16 (d, J = 5.1 Hz, 1H), 7.11 (d, J = 1.9 Hz, 1H), 6.89 (d, J = 7.4
Hz, 1H), 5.97−5.94 (m, 1H), 5.35−5.32 (m, 1H), 4.20−4.14 (m, 1H),
4.07−3.97 (m, 2H), 3.90−3.86 (m, 2H), 3.43−3.37 (m, 2H), 1.87−1.85
(m, 2H), 1.58−1.49 (m, 2H); m/z (APCI-pos) M + 1 = 445.1, 447.1.
1-Chloro-2-fluoro-4-vinylbenzene (15). Sodium hydride (8.549
g, 213.7 mmol, 60% suspension in mineral oil) was added portionwise to
a cold (0 °C) solution of 4-chloro-3-fluorobenzaldehyde (26.07 g, 164.4
mmol) and methyltriphenylphosphonium bromide (70.48 g, 197.3
mmol) in THF (400 mL). The reaction mixture was allowed to warm up
to room temperature overnight. The solids were removed by filtration,
and the filter cake was washed with ether (400 mL). The filtrate was
concentrated (water bath about 20 °C), and the residue was suspended
in hexanes (200 mL) and stirred for 30 min. The solids (mostly PPh3O)
were removed by filtration, and the filter cake was washed with hexanes
(200 mL). The filtrate was concentrated, and the crude product was
purified by column chromatography, eluting with hexanes/EtOAc
(25:1) to give 1-chloro-2-fluoro-4-vinylbenzene (12.1 g, 47%) as an oil.
1H NMR (400 MHz, CDCl3) δ 7.35−7.27 (m, 1H), 7.22−7.14 (m, 1H),
7.12−7.06 (m, 1H), 6.67−6.57 (m, 1H), 5.74 (d, J = 17.4 Hz, 1H), 5.32
(d, J = 10.8 Hz, 1H).
(R)-1-(4-Chloro-3-fluorophenyl)ethane-1,2-diol (16). 1-
Chloro-2-fluoro-4-vinylbenzene (9.40 g, 60.0 mmol) was added to a
cold (0 °C) solution of AD-mix-β (84.2 g, 108 mmol) in t-BuOH/H2O
(700 mL; 1:1), and the mixture was allowed to warm up to room
temperature overnight. The next day, the reaction was placed in an ice
bath and quenched with 10% Na2S2O3 (200 mL). The mixture was
stirred for 1 h and then extracted with EtOAc (3 × 500 mL). The
combined organics were dried, filtered, and concentrated. The crude
product was purified via column chromatography, eluting with DCM/
EtOA (1:1) to give give (R)-1-(4-chloro-3-fluorophenyl)ethane-1,2-diol
as an oil. (7.35 g, 64%). 1H NMR (400 MHz, DMSO-d6) δ 7.59−7.42
(m, 1H), 7.38−7.29 (m, 1H), 7.23−7.18 (m, 1H), 5.45 (d, J = 4.8 Hz,
1H), 4.79−4.72 (m, 1H), 4.59−4.51 (m, 1H), 3.49−3.36 (m, 2H).
(R)-2-(tert-Butyldimethylsilyloxy)-1-(4-chloro-3-
fluorophenyl)ethanol (17). Imidazole (0.911 g, 13.4 mmol) was
added to a cold (0 °C) solution of (R)-1-(4-chloro-3-fluorophenyl)-
ethane-1,2-diol (1.02 g, 5.35 mmol) in DCM (20 mL), followed by
TBSCl (0.968 g, 6.42 mmol). The reaction mixture was stirred at 0 °C
for 1 h and then quenched with water (50 mL). The layers were
separated, and the organics were dried, filtered, and concentrated. The
crude product was purified via column chromatography, eluting with
hexanes/EtOAc (100:1) to give (R)-2-(tert-butyldimethylsilyloxy)-1-
(4-chloro-3-fluorophenyl)ethanol (0.95 g, 58%; 95% enantiomeric
excess (“ee”) by chiral HPLC (Chiral Tech, column IA, 4.6 mm × 250
mm, 5 μ, 10% ethanol/90% hexanes, 1 mL/min, retention time for (S)-
17, 7.5 min; retention time for (R)-17, 8.4 min). 1H NMR (400 MHz,
CDCl3) δ 7.38−7.34 (m, 1H), 7.22−7.18 (m, 1H), 7.10−7.06 (m, 1H),
4.73−4.69 (m, 1H), 3.77−3.73 (m, 1H), 3.51−3.47 (m, 1H), 2.96 (d, J =
2.6 Hz, 1H), 0.90 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H).
(R)-2-(tert-Butyldimethylsilyloxy)-1-(4-chloro-3-
fluorophenyl)ethylmethanesulfonate (18). Triethylamine (2.09
mL, 15.0 mmol) was added to a cold (0 °C) solution of (R)-2-(tert-
butyldimethylsilyloxy)-1-(4-chloro-3-fluorophenyl)ethanol (3.05 g,
10.0 mmol) in DCM (100 mL), followed by methanesulfonyl chloride
(0.929 mL, 12.0 mmol). The reaction mixture was stirred at 0 °C for 30
min and then quenched with water (50 mL). The layers were separated,
and the organic layer was washed with saturated NaHCO3, dried,
filtered, and concentrated. The crude product was purified via column
chromatography, eluting with hexanes/EtOAc (25:1) to give (R)-2-
(tert-butyldimethylsilyloxy)-1-(4-chloro-3-fluorophenyl)-
= 7.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.23−7.15 (m, 2H), 7.11 (s, 1H),
7.09−7.04 (m, 2H), 6.89 (d, J = 7.2 Hz, 1H), 5.99−5.96 (m, 1H), 5.34−
5.31 (m, 1H), 4.19−4.13 (m, 1H), 4.08−3.95 (m, 2H), 3.89−3.87 (m,
2H), 3.43−3.37 (m, 2H), 1.87−1.84 (m, 2H), 1.59−1.49 (m, 2H); m/z
(APCI-pos) M + 1 = 429.1.
1-(1-(3,5-Dichlorophenyl)-2-hydroxyethyl)-4-(2-((tetrahy-
dro-2H-pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one
(14n). 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 5.1 Hz, 1H), 7.95
(d, J = 7.5 Hz, 1H), 7.57 (s, 1H), 7.39 (d, J = 1.7 Hz, 2H), 7.35 (d, J = 7.5
Hz, 1H), 7.16 (d, J = 5.1 Hz, 1H), 7.11 (d, J = 1.5 Hz, 1H), 6.90 (d, J =
1
ethylmethanesulfonate (3.80 g, 99%) as an oil. H NMR (400 MHz,
CDCl3) δ 7.44−7.40 (m, 1H), 7.22−7.18 (m, 1H), 7.14−7.10 (m, 1H),
M
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Journal of Medicinal Chemistry
Article
5.52−5.48 (m, 1H), 3.93−3.90 (m, 1H), 3.82−3.78 (m, 1H), 2.98 (s,
3H), 0.88 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H).
2H), 3.58−3.52 (m, 2H), 2.74 (br, s, 1H), 2.09−2.03 (m, 2H), 1.62−
1.56 (m, 2H); m/z (APCI-pos) M + 1 = 445.1, 447.0.
(S)-1-(1-(3-Chlorophenyl)-2-hydroxyethyl)-4-(2-(tetrahydro-
2H-pyran-4-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one ((S)-
14d). Enantiomeric excess (“ee”) 88% by chiral HPLC (Chiral Tech,
column OD-H, 4.6 mm × 250 mm, 5 μ, 20% ethanol/80% hexanes, 1
mL/min, retention time for (S)-14d, 20.0 min; retention time for (R)-
14d, 25.1 min) 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 5.0 Hz, 1H),
7.42−7.35 (m, 2H), 7.35−7.30 (m, 2H), 7.27−7.23 (m, 1H), 7.19 (s,
1H), 6.89 (d, J = 5.0 Hz, 1H), 6.81−6.73 (m, 1H), 6.29−6.18 (m, 1H),
5.15 (d, J = 8.0 Hz, 1H), 4.38−4.27 (m, 2H), 4.16−4.04 (m, 1H), 4.03−
3.96 (m, 2H), 3.61−3.50 (m, 2H), 2.65−2.55 (m, 1H), 2.13−2.00 (m,
2H), 1.66−1.50 (m, 2H); m/z (APCI-pos) M + 1 = 427.1, 429.1.
(S)-1-(1-(3-Fluorophenyl)-2-hydroxyethyl)-4-(2-((tetrahydro-
2H-pyran-4-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one ((S)-
14e). Enantiomeric excess (“ee”) 86% by chiral HPLC (Chiral Tech,
column OD-H, 4.6 mm × 250 mm, 5 μ, 20% ethanol/80% hexanes, 1
mL/min, retention time for (S)-14e, 10.3 min; retention time for (R)-
14e, 13.1 min). 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 5.0 Hz, 1H),
7.40−7.33 (m, 2H), 7.17−7.01 (m, 4H), 6.87 (d, J = 5.2 Hz, 1H), 6.78−
6.76 (m, 1H), 6.28−6.25 (m, 1H), 5.20 (d, J = 8.2 Hz, 1H), 4.32−4.31
(m, 2H), 4.13−4.06 (m, 1H), 4.02−3.98 (m, 2H), 3.59−3.52 (m, 2H),
3.11−3.08 (m, 1H), 2.07−2.04 (m, 2H), 1.63−1.53 (m, 2H); m/z
(APCI-pos) M + 1 = 411.1.
(S)-1-(1-(3-Chloro-4-fluorophenyl)-2-hydroxyethyl)-4-(2-
((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)pyridin-
2(1H)-one ((S)-14l). Enantiomeric excess (“ee”) 82% by chiral HPLC
(Chiral Tech, column OJ-H, 4.6 mm × 250 mm, 5 μ, 15% ethanol/85%
hexanes, 1 mL/min, retention time for (S)-14l, 18.8 min; retention time
for (R)-14l, 22.6 min). 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 5.0
Hz, 1H), 7.47−7.44 (m, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.29−7.24 (m,
1H), 7.18−7.14 (m, 2H), 6.88 (d, J = 8.7 Hz, 1H), 6.80−6.77 (m, 1H),
6.21−6.17 (m, 1H), 5.17−5.14 (m, 1H), 4.32−4.29 (m, 2H), 4.15−4.08
(m, 1H), 4.03−3.98 (m, 2H), 3.60−3.52 (m, 2H), 2.70−2.66 (m, 1H),
2.09−2.03 (m, 2H), 1.63−1.53 (m, 2H); m/z (APCI-pos) M + 1 =
445.1, 447.0.
1-((S)-1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((R)-
tetrahydrofuran-3-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one
(22a). 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 5.2 Hz, 1H), 7.42−
7.38 (m, 2H), 7.21−7.09 (m, 3H), 6.88 (d, J = 5.0 Hz, 1H), 6.80 (d, J =
9.0 Hz, 1H), 6.21−6.19 (m, 1H), 5.48 (d, J = 7.2 Hz, 1H), 4.64−4.58 (m,
1H), 4.30 (s, 2H), 4.03−3.97 (m, 2H), 3.90−3.84 (m, 1H), 3.78−3.74
(m, 1H), 3.26 (s, 1H), 2.39−2.30 (m, 1H), 1.94−1.87 (m, 1H); m/z
(APCI-pos) M + 1 = 431.1, 433.1.
1-(S)-1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((S)-
tetrahydrofuran-3-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one
(22b). 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 5.2 Hz, 1H), 7.42−
7.36 (m, 2H), 7.22−7.09 (m, 3H), 6.88 (d, J = 5.0 Hz, 1H), 6.80−6.79
(m, 1H), 6.22−6.19 (m, 1H), 5.51 (d, J = 7.2 Hz, 1H), 4.64−4.58 (m,
1H), 4.30−4.29 (m, 2H), 4.02−3.96 (m, 2H), 3.90−3.84 (m, 1H),
3.76−3.73 (m, 1H), 3.34 (s, 1H), 2.38−2.30 (m, 1H), 1.94−1.87 (m,
1H); m/z (APCI-pos) M + 1 = 431.1, 433.1.
(S)-1-(2-(tert-Butyldimethylsilyloxy)-1-(4-chloro-3-
fluorophenyl)ethyl)-4-(2-(methylthio)pyrimidin-4-yl)pyridin-
2(1H)-one (19). KHMDS (1.0 M, 5.09 mL, 5.09 mmol) in THF was
added to a cold (0 °C) suspension of 4-(2-(methylthio)pyrimidin-4-
yl)pyridin-2(1H)-one (0.93 g, 4.24 mmol) in THF (20 mL). The
reaction mixture was stirred at 0 °C for 10 min before (R)-2-(tert-
butyldimethylsilyloxy)-1-(4-chloro-3-fluorophenyl)ethylmethane-
sulfonate (2.44 g, 6.36 mmol) was added as a solution in THF (5 mL).
The reaction was heated to reflux for 30 h and then cooled to room
temperature and concentrated. The residue was taken up in EtOAc (200
mL) and washed with water (100 mL). The organics were dried, filtered,
and concentrated. The crude product was purified via column
chromatography, eluting with hexanes/EtOAc (4:1) to give (S)-1-(2-
(tert-butyldimethylsilyloxy)-1-(4-chloro-3-fluorophenyl)ethyl)-4-(2-
(methylthio)pyrimidin-4-yl)pyridin-2(1H)-one (1.35 g, 63%) as a solid.
1H NMR (400 MHz, CDCl3) δ 8.66 (d, J = 5.0 Hz, 1H), 7.47−7.41 (m,
2H), 7.34 (d, J = 5.0 Hz, 1H), 7.32−7.28 (m, 2H), 7.18−7.14 (m, 1H),
6.87−6.83 (m, 1H), 6.26−6.22 (m, 1H), 4.37−4.33 (m, 1H), 4.25−4.21
(m, 1H), 2.65 (s, 3H), 0.88 (s, 9H), 0.03 (s, 3H), −0.03 (s, 3H); m/z
(APCI-pos) M + 1 = 506.1, 508.1.
(S)-1-(2-(tert-Butyldimethylsilyloxy)-1-(4-chloro-3-
fluorophenyl)ethyl)-4-(2-(methylsulfonyl)pyrimidin-4-yl)-
pyridin-2(1H)-one (20). mCPBA (7.1 g, 29 mmol) was added to a cold
(0 °C) solution of (S)-1-(2-(tert-butyldimethylsilyloxy)-1-(4-chloro-3-
fluorophenyl)ethyl)-4-(2-(methylthio) pyrimidin-4-yl)pyridin-2(1H)-
one (5.8 g, 11 mmol) in DCM (100 mL), and the mixture was stirred
for 2 h. The reaction mixture was washed with saturated Na2S2O3 (1×),
saturated NaHCO3 (1×), dried, filtered, and evaporated. The crude
product was purified via column chromatography, eluting with hexanes/
EtOAc (1:1) to give (S)-1-(2-(tert-butyldimethylsilyloxy)-1-(4-chloro-
3-fluorophenyl)ethyl)-4-(2-(methylsulfonyl)pyrimidin-4-yl)pyridin-
2(1H)-one (5.5 g, 89%) as a solid. 1H NMR (400 MHz, CDCl3) δ 9.06
(d, J = 5.2 Hz, 1H), 7.91 (d, J = 5.4 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H),
7.45−7.41 (m, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.29−7.25 (m, 1H), 7.17−
7.13 (m, 1H), 6.95−6.91 (m, 1H), 6.24−6.20 (m, 1H), 4.37−4.33 (m,
1H), 4.26−4.22 (m, 1H), 3.45 (s, 3H), 0.88 (s, 9H), 0.03 (s, 3H), −0.03
(s, 3H); m/z (APCI-pos) M + 1 = 538.1, 540.0.
(S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-
((tetrahydro-2H-pyran-4-ylamino))pyrimidin-4-yl)pyridin-
2(1H)-one ((S)-14k). Step 1. A suspension of (S)-1-(2-(tert-
butyldimethylsilyloxy)-1-(4-chloro-3-fluorophenyl)ethyl)-4-(2-
(methylsulfonyl)pyrimidin-4-yl)pyridin-2(1H)-one (5.77 g, 10.7
mmol) and tetrahydro-2H-pyran-4-amine (2.17 g, 21.4 mmol) in sec-
BuOH (30 mL) was heated to 120 °C for 4 h in a sealed tube. The
reaction mixture was cooled to room temperature and concentrated.
The crude (S)-1-(2-(tert-butyldimethylsilyloxy)-1-(4-chloro-3-
fluorophenyl)ethyl)-4-(2-(tetrahydro-2H-pyran-4-ylamino)pyrimidin-
4-yl)pyridin-2(1H)-one was used without purification in Step 2. m/z
(APCI-pos) M + 1 = 559.2, 560.2.
(S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-(ox-
etan-3-ylamino)pyrimidin-4-yl)pyridin-2(1H)-one (22c). 1H
NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 5.1 Hz, 1H), 8.10 (d, J =
5.9 Hz, 1H), 7.89 (d, J = 7.4 Hz, 1H), 7.60−7.56 (m, 1H), 7.44 (d, J =
12.4 Hz, 1H), 7.24 (d, J = 5.1 Hz, 1H), 7.16 (d, J = 8.6 Hz, 1H), 7.12 (s,
1H), 6.88 (d, J = 9.1 Hz, 1H), 5.99−5.96 (m, 1H), 5.33−5.31 (m, 1H),
4.99−4.94 (m, 1H), 4.81−4.77 (m, 2H), 4.57−4.54 (m, 2H), 4.19−4.13
(m, 1H), 4.07−4.02 (m, 1H); m/z (APCI-pos) M + 1 = 417.1, 419.1.
(S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-
(3,3-difluorocyclobutylamino)pyrimidin-4-yl)pyridin-2(1H)-
one (22d). 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 5.2 Hz, 1H),
7.90−7.87 (m, 2H), 7.60−7.56 (m, 1H), 7.45−7.43 (m, 1H), 7.25 (d, J =
5.0 Hz, 1H), 7.17−7.13 (m, 2H), 6.90−6.89 (m, 1H), 6.00−5.96 (m,
1H), 5.33−5.31 (m, 1H), 4.28−4.12 (m, 2H), 4.07−4.02 (m, 1H),
3.02−2.92 (m, 2H), 2.74−2.61 (m, 2H); m/z (APCI-pos) M + 1 =
451.1, 453.1.
Step 2. A solution of (S)-1-(2-(tert-butyldimethylsilyloxy)-1-(4-
chloro-3-fluorophenyl)ethyl)-4-(2-(tetrahydro-2H-pyran-4-ylamino)-
pyrimidin-4-yl)pyridin-2(1H)-one (6.00 g, 10.7 mmol) in THF (20
mL) was treated with 1.0 M TBAF in THF (12.9 mL, 12.9 mmol) at
room temperature for 30 min. The reaction mixture was concentrated,
and the residue was taken up with EtOAc (50 mL) and water (50 mL).
The organic layer was washed with water (1×). The combined organics
were dried, filtered, and concentrated. The crude product was purified
via column chromatography, eluting with EtOAc to give (S)-1-(1-(4-
chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((tetrahydro-2H-pyran-
4-ylamino))pyrimidin-4-yl)pyridin-2(1H)-one as a solid (4.14 g, 87%
over 2 steps; 96% enantiomeric excess (“ee”) by chiral HPLC (Chiral
Tech, column OJ-H, 4.6 mm × 250 mm, 5 μ, 30% ethanol/70% hexanes,
1 mL/min, retention time for (S)-14k, 11.1 min; retention time for (R)-
14k, 14.9 min). 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 5.0 Hz, 1H),
7.42−7.38 (m, 2H), 7.22−7.18 (m, 2H), 7.10 (d, J = 8.2 Hz, 1H), 6.88
(d, J = 5.0 Hz, 1H), 6.80−6.76 (m, 1H), 6.22−6.18 (m, 1H), 5.16 (d, J =
8.0 Hz, 1H), 4.34−4.28 (m, 2H), 4.12−4.08 (m, 1H), 4.04−3.96 (m,
(S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-2-
(((R,S)-2-methyl-tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-
yl)pyridin-2(1H)-one (22e). Isolated as a mixture of two diaster-
N
DOI: 10.1021/jm501921k
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
eomers (formate salt). 1H NMR (400 MHz, CDCl3) δ 8.37 (br s, 1H),
7.45−7.32 (m, 2H), 7.24−7.04 (m, 3H), 6.94−6.73 (m, 2H), 6.22−6.18
(m, 1H), 5.57 (br s, 1H), 4.33 (d, J = 4.5 Hz, 3H), 3.91−3.87 (m, 1H),
3.78−3.74 (m, 2H), 2.03−1.52 (m, 4H), 1.21 (d, J = 6.1 Hz, 3H); m/z
(APCI-pos) M + 1 = 459.1, 460.1.
ionization; Et, ethyl; EtOAc, ethyl acetate; %F, oral bioavail-
ability; equiv, equivalent; FDA, Food and Drug Administration;
FRET, Forster resonance energy transfer; mp, melting point;
̈
hERG, human ether-a-go-go related gene; HPLC, high perform-
ance liquid chromatography; IC50, half-maximal inhibitory
concentration; h, hour; IPA, 2-propanol; KHMDS, potassium
bis(trimethylsilyl)amide; LCMS, liquid chromatography−mass
spectrometry; mCPBA, 3-chlorobenzoperoxoic acid; MsCl,
methanesulfonyl chloride; NMP, N-methylpyrrolidone; Papp
AB, apparent cell permeability in the apical to basolateral
direction; PEG, polyethylene glycol; Ph, phenyl; PK, pharma-
cokinetics; PD, pharmacodynamics; QD, once daily; rt, room
temperature; SAR, structure−activity relationship; TBAF,
tetrabutylammonium fluoride; TBSCl, tert-butyldimethylchlor-
osilane; THF, tetrahydrofuran; THP amine, tetrahydro-2H-
pyran-4-amine; TLC, thin-layer chromatography; Vss, steady-
state volume of distribution
1-((S)-1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-2-
(((R,S)-1,1-dioxidotetrahydrothiophen-3-yl)amino)pyrimidin-
4-yl)pyridin-2(1H)-one (22f). Isolated as a mixture of two
1
diastereomers. H NMR (400 MHz, CDCl3) δ 8.48−8.24 (m, 1H),
7.50−7.32 (m, 2H), 7.22−7.15 (m, 1H), 7.13−7.02 (m, 2H), 6.99−6.87
(m, 1H), 6.81−6.72 (m, 1H), 6.24−6.15 (m, 1H), 5.99−5.89 (m, 0.5H),
5.73−5.66 (m, 0.5H), 4.94−4.79 (m, 1H), 4.38−4.24 (m, 2H), 3.63−
3.51 (m, 1H), 3.48−3.27 (m, 2H), 3.26−3.08 (m, 2H), 2.72−2.58 (m,
1H), 2.47−2.34 (m, 1H); m/z (APCI-pos) M + 1 = 479.0, 480.1.
(S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-
(((S)-4-methoxybutan-2-yl)amino)pyrimidin-4-yl)pyridin-
1
2(1H)-one (22g). H NMR (400 MHz, CDCl3) δ 8.35 (br s, 1H),
7.45−7.32 (m, 2H), 7.22−7.18 (m, 2H), 7.12−7.08 (m, 1H), 6.83 (d, J =
5.4 Hz, 2H), 6.20 (t, J = 5.3 Hz, 1H), 5.51 (br s, 1H), 4.28 (d, J = 6.0 Hz,
3H), 3.62−3.40 (m, 2H), 1.96−1.67 (m, 2H), 1.28 (d, J = 6.6 Hz, 1H);
m/z (APCI-pos) M + 1 = 447.1, 449.1.
REFERENCES
(S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-
■
(1,3-difluoropropan-2-ylamino)pyrimidin-4-yl)pyridin-2(1H)-
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1
one (22h). H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 4.7 Hz, 1H),
7.46−7.35 (m, 2H), 7.23−7.14 (m, 2H), 7.10 (d, J = 8.5 Hz, 1H), 6.97
(d, J = 4.8 Hz, 1H), 6.80 (d, J = 7.0 Hz, 1H), 6.22−6.15 (m, 1H), 5.50
(br, s, 1H), 4.78−4.71 (m, 1H), 4.69−4.59 (m, 3H), 4.57−4.49 (m, 1H),
4.33−4.28 (m, 2H), 2.67 (br, s, 1H); m/z (APCI-pos) M + 1 = 439.1,
441.1.
1-((S)-1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((R)-
1-hydroxy-3-methoxypropan-2-ylamino)pyrimidin-4-yl)-
pyridin-2(1H)-one (22i). 1H NMR (400 MHz, MeOH-d4) δ 8.38 (d, J
= 4.3 Hz, 1H), 7.81 (d, J = 7.2 Hz, 1H), 7.51−7.43 (m, 1H), 7.34−7.27
(m, 1H), 7.24 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.11−7.07 (m, 1H), 7.03
(d, J = 7.0 Hz, 1H), 6.15−6.07 (m, 1H), 4.36−4.22 (m, 2H), 4.21−4.14
(m, 1H), 3.73−3.65 (m, 2H), 3.64−3.52 (m, 2H), 3.37 (s, 3H); m/z
(APCI-pos) M + 1 = 449.1, 451.1.
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Chapman, P. B. Inhibition of Mutated, Activated BRAF in Metastatic
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis of 6f, 23a−23f, and
24a−24e, biological assay information, kinase selectivity of (S)-
14k, Cerep panel of receptor selectivity of (S)-14k, protein
expression and purification of ERK2 protein constructs for
crystallography, crystal structure determination of ERK2
complex with (S)-14k. This material is available free of charge
via the Internet at http://pubs.acs.org.
(4) Rheault, T. R.; Stellwagen, J. C.; Adjabeng, G. M.; Hornberger, K.
R.; Petrov, K. G.; Waterson, A. G.; Dickerson, S. H.; Mook, R. A.;
Laquerre, S. G.; King, A. J.; Rossanese, O. W.; Arnone, M. R.;
Smitheman, K. N.; Kane-Carson, L. S.; Han, C.; Moorthy, G. S.; Moss,
K. G.; Uehling, D. E. Discovery of Dabrafenib: a selective inhibitor of Raf
kinase with antitumor activity against B-raf-driven tumors. ACS Med.
Chem. Lett. 2013, 4, 358−362.
(5) Flaherty, K. T.; Robert, C.; Hersey, P.; Nathan, P.; Garbe, C.;
Milhem, M.; Demidov, L. V.; Hassel, J. C.; Rutkowki, P.; Mohr, P.;
Dummer, R.; Trefzer, U.; Larkin, J. M. G.; Utikal, J.; Dreno, B.; Nyakas,
M.; Middleton, M. R.; Becker, J. C.; Casey, M.; Sherman, L. J.; Wu, F. S.;
Quellet, D.; Martin, A. M.; Patel, K.; Schadendorf, D. Improved survival
with MEK inhibition in BRAF-mutated melanoma. N. Engl. J. Med. 2012,
367, 107−114.
AUTHOR INFORMATION
Corresponding Author
* Phone: 1-303-386-1448. Email: li.ren@arraybiopharma.com.
■
Notes
The authors declare no competing financial interest.
(6) (a) Aronov, A. M.; Baker, G.; Bemis, G. W.; Cao, J.; Chen, G.; Ford,
P. J.; Germann, U. A.; Green, J.; Hale, M. R.; Jacobs, M.; Janetka, J. M.;
Maltais, F.; Martinez-Botella, G.; Namchuk, M. N.; Straub, J.; Tang, Q.;
Xie, X. Flipped out: structure-guided design of selective pyrazolylpyrrole
ERK inhibitors. J. Med. Chem. 2007, 50, 1280−1287. (b) Aronov, A. M.;
Tang, Q.; Martinez-Botella, G.; Baker, G.; Bemis, G. W.; Cao, J.; Chen,
G.; Ewing, N. P.; Ford, P. J.; Germann, U. A.; Green, J.; Hale, M. R.;
Jacobs, M.; Janetka, J. M.; Maltais, F.; Markland, W.; Namchuk, M. N.;
Nanthakumar, S.; Poondru, S.; Straub, J.; Harr, E.; Xie, X. Structure-
guided design of potent and selective pyrimidylpyrrole inhibitors of
extracellular signal-regulated kinase (ERK) using conformational
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ABBREVIATIONS USED
Ac, acetyl; ACN, acetonitrile; ADME, absorption, distribution,
metabolism, and excretion; aq, aqueous; AUC, area under curve;
■
BID, twice daily; BOC, tert-butoxycarbonyl; CL, clearance; Cmax
,
maximal concentration; CYP, cytochrome P-450; d, day; DCE,
dichloroethane; DCM, dichloromethane; DIEA, diisopropyle-
thylamine; efflux, efflux ratio is the (B to A)/(A to B) value
derived from MDCK cells transfected with human MDR1 (p-
GP); ER%, extraction ratio %; DMA, N,N-dimethylacetamide;
DMF, N,N-dimethylformamide; DMPK, drug metabolism and
pharmacokinetics; DMSO, dimethyl sulfoxide; EC50, half-
maximal effective concentration; ELISA, enzyme-linked immu-
nosorbent assay; ER%, extraction ratio %; ESI, electrospray
(7) (a) Meloche, S.; Pouyssegur, J. The ERK1/2 mitogen-activated
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