Discovery of 1-(1H-Pyrazolo[4,3-c]pyridin-6-yl)urea Inhibitors of Extracellular Signal-Regulated Kinase (ERK) for the Treatment of Cancers

Article abstract of DOI:10.1021/acs.jmedchem.6b00708

The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway. Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing hydrogen bond interactions specific for ERK in the binding pocket identified 1-(1H-pyrazolo[4,3-c]pyridin-6-yl)ureas as a viable lead series. Sequential SAR studies led to the identification of highly potent and selective ERK inhibitors with low molecular weight and high LE. Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF^V600E xenograft model.

Full text of DOI:10.1021/acs.jmedchem.6b00708

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Article
Discovery of 1–(1H–Pyrazolo[4,3–c]pyridin–6–yl)urea Inhibitors of
Extracellular Signal–Regulated Kinase (ERK) for the Treatment of Cancers
Jongwon Lim, Elizabeth H. Kelley, Joey L Methot, Hua Zhou, Alessia Petrocchi, Hongmin Chen, Susan
E. Hill, Marlene C. Hinton, Alan Hruza, Joon O. Jung, John K. F. Maclean, My Mansueto, George
N. Naumov, Ulrike Philippar, Shruti Raut, Peter Spacciapoli, Dongyu Sun, and Phieng Siliphaivanh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00708 • Publication Date (Web): 22 Jun 2016
Downloaded from http://pubs.acs.org on June 22, 2016
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Journal of Medicinal Chemistry
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Discovery of 1−(1H−Pyrazolo[4,3−c]pyridin−6−yl)urea
Inhibitors of Extracellular Signal−Regulated Kinase
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(ERK) for the Treatment of Cancers
,†
†
†
†
†
Jongwon Lim,* Elizabeth H. Kelley, Joey L. Methot, Hua Zhou, Alessia Petrocchi, Hongmin
ǁ
⊥
#
∞
§
§
Chen, Susan E. Hill, Marlene C. Hinton, Alan Hruza, Joon O. Jung, John K. F. Maclean, My
ǁ
#
‡
ǁ
ǁ
ǁ
Mansueto, George N. Naumov, Ulrike Philippar, Shruti Raut, Peter Spacciapoli, Dongyu Sun and
†
Phieng Siliphaivanh
†
‡
ǁ
#
§
Departments of Chemistry, Oncology, In Vitro Pharmacology, In Vivo Pharmacology, Chemistry
⊥
Modeling and Informatics, Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and
∞
Structural Chemistry, Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115
Abstract
The ERK/MAPK pathway plays a central role in the regulation of critical cellular processes and is
activated in more than 30% of human cancers. Specific BRAF and MEK inhibitors have shown clinical
efficacy in patients for the treatment of BRAF−mutant melanoma. However, the majority of responses
are transient, and resistance is often associated with pathway reactivation of the ERK signal pathway.
Acquired resistance to these agents has led to greater interest in ERK, a downstream target of the
MAPK pathway. De novo design efforts of a novel scaffold derived from SCH772984 by employing
hydrogen bond interactions specific for ERK in the binding pocket identified
1
−(1H−pyrazolo[4,3−c]pyridin−6−yl)ureas as a viable lead series. Sequential SAR studies led to the
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identification of highly potent and selective ERK inhibitors with low molecular weight and high LE.
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V600E
Compound 21 exhibited potent target engagement and strong tumor regression in the BRAF
xenograft model.
KEYWORDS: ERK, MAPK, kinase inhibitors, 1−(1H−pyrazolo[4,3−c]pyridin−6−yl)urea, sequence
conservation.
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Introduction
The extracellular signal−regulated kinase (ERK) mitogen−activated protein kinase (MAPK) pathway
plays a central role in the regulation of critical cellular processes such as cell proliferation, migration,
1
and differentiation. Activation of RAS leads to the recruitment and activation of RAF, a
serine−threonine kinase. Activated RAF phosphorylates and activates MEK1/2, which then
phosphorylates and activates ERK1/2. When activated, ERK1/2 phosphorylates several downstream
targets involved in a multitude of cellular events including cytoskeletal changes and transcriptional
activation. The ERK/MAPK pathway is activated in more than 30% of human cancers, most frequently
via RAS and BRAF mutations, and thus has attracted significant interest as a therapeutic target for
2
cancer. Specific inhibitors of BRAF and its downstream effectors have been developed to target
BRAF− and RAS−mutant tumors. In particular, the BRAF inhibitors, vemurafenib and dabrafenib, and
the MEK inhibitor, trametinib, have been approved for the treatment of BRAF−mutant melanoma, and
3
−5
have shown clinical efficacy in patients.
However, the majority of responses are transient, and
6
−8
resistance is often associated with pathway reactivation of the ERK signal pathway. The combination
of the BRAF inhibitor dabrafenib with the MEK inhibitor trametinib has shown significant clinical
benefit in BRAF−mutant melanoma compared with BRAF or MEK inhibitor monotherapy. However,
drug−resistant tumor cells invariably emerged leading to disease progression. Acquired resistance to
9
these agents has led to greater interest in ERK, a downstream target of the MAPK pathway.
In 2013, researchers at Merck reported discovery and characterization of SCH772984 (1), a novel and
1
0
selective inhibitor of ERK1/2 (Table 1). Compound 1 has subnanomolar ERK2 potency and
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Journal of Medicinal Chemistry
nanomolar cellular potency in tumor cells with mutations in BRAF or RAS. Compound 1 inhibits not
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only the kinase activity of ERK (inhibition of pRSK) but also the phosphorylation of ERK by MEK
1
1,12
(inhibition of pERK) via a large shift of the glycine rich loop of ERK upon binding.
This unique
binding mode is believed to account for its excellent kinome selectivity (> 50% inhibition at 1 ꢀM by 7
out of 309 kinases). Compound 1 induced tumor regressions in the xenograft models at tolerated doses.
More importantly, 1 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK
inhibitor−resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and
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MEK inhibitors. These data support that pursuing ERK inhibition is a viable option for the treatment of
13,14
tumors refractory to BRAF and MEK inhibitors.
In fact, the discovery of novel ERK inhibitors has
been actively pursued by many research groups, and various classes of ERK inhibitors have been
reported.15−26
Although 1 presented high ERK potency, excellent kinome selectivity, and high in vivo efficacy, its
high molecular weight (MW 588) is suboptimal and may become a limitation. We pursued novel
scaffolds that exhibit improved drug−like properties such as low molecular weight and high ligand
2
7
efficiency (LE). Herein, we report de novo design of a novel scaffold derived from 1 and a series of
modifications
to
the
scaffold.
The
SAR
efforts
led
to
the
discovery
of
1
−(1H−pyrazolo[4,3−c]pyridin−6−yl)ureas as highly potent and selective ERK inhibitors with low
V600E
molecular weight and high LE. In vivo efficacy results of compound 21 in a BRAF
xenograft
model are also described.
Table 1. Structure and Key Profile of SCH772984 (1)
MW
588
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hERK2 IC (nM)
0.4
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LE
pRSK IC (nM)
0.29
57
50
pERK (A375) IC (nM)
9
5
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A375SM Prolif IC (nM)
39
5
0
No. of Kinases Tested
Kinase Selectivity
309
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7 kinases > 50% inhib @ 1 ꢀM
a
FaSSIF Solubility (pH 6.5, ꢀM)
38
a
FaSSIF = human fasted−state simulated intestinal fluid.
Chemistry
a
Scheme 1. Synthesis of the 1−(1H−Pyrazolo[4,3−c]pyridin−6−yl)ureas
Trt
Trt
Trt
H
Cl
Cl
NH2
N
N
N
Cl
N
a, b
c
d
N
N
N
N
N
N
N
N
I
R1
R1
2
3
4
e, g
or f, g
h, g
H
N
H
O
N
N
N
HN
R2
R1
5
c, g
Trt
Trt
Trt
H
H
CO Me
N
2
CO H
NH2
N
O
N
2
N
N
b, i
j
e or f
N
N
N
N
N
N
N
N
HN
R2
Br
Br
Br
Br
6
7
8
a
1
1
(a) I , KOH, DMF; (b) TrtCl, NaH, THF; (c) R −boronic acid or R −boronic ester, PdCl (dppf),
2
2
o
K CO , 1,4−dioxane/water, 80 C; (d) LiHMDS, Pd (dba) , 2−(dicyclohexylphosphino)biphenyl, THF,
2
3
2
3
o
2
2
o
6
5 C; (e) CDI, imidazole, DCM; R NH ; (f) R NCO, 1,4−dioxane, 60 C; (g) TFA, triethylsilane,
2
2
o
o
DCM; (h) R NHCONH , BrettPhos precatalyst, Cs CO , DMA, 100 C; (i) 1 N KOH, MeOH, 50 C; (j)
2
2
3
o
DPPA, TEA, 1,4−dioxane, 80 C; 1 N KOH.
Synthetic routes for the 1−(1H−pyrazolo[4,3−c]pyridin−6−yl)ureas (5) are outlined in Scheme 1.
Intermediate 2 was prepared starting from 6−chloro−1H−pyrazolo[4,3−c]pyridine via iodination at the
C−3 position followed by protection of the 1H−pyrazole with a trityl group. Various substituents were
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Journal of Medicinal Chemistry
2
8
introduced to the C−3 position by employing Suzuki reaction to afford 3. The C6−Cl was then
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29
converted to the amino group via palladium mediated coupling conditions. The amino group of
intermediates 4 was reacted with either an array of amines in the presence of CDI or various isocyanates
at an elevated temperature followed by sequential trityl group deprotection with TFA to afford ureas 5.
Alternatively, intermediates 3 were directly converted to 5 by employing palladium mediated coupling
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conditions with alkylureas followed by deprotection. For a rapid derivatization at the C−3 position,
intermediate 7 was converted to ureas 8 before various substituents were introduced to the C−3 position.
Intermediate 7 was prepared via sequential protection with a trityl group, saponification, and Curtius
rearrangement starting from methyl 3−bromo−1H−pyrazolo[4,3−c]pyridine−6−carboxylate.
Results and Discussions
Sequence Conservation in the ERK2 Binding Pocket.
Protein kinases regulate cellular signaling pathways by catalyzing phosphorylation of serine, threonine,
and tyrosine residues of proteins in eukaryotic cells. Thus, protein kinases have emerged as promising
3
1
therapeutic targets for many human diseases. Small molecule protein kinase inhibitors have been
widely used to elucidate the cellular and organismal roles of protein kinases. However, the ATP binding
active site of human protein kinase domains is highly conserved even among distal human kinome
families. Therefore, designing selective ATP−competitive inhibitors of a target kinase continues to be a
daunting challenge.
Understanding the structural biology of the active site of a protein kinase is crucial for designing highly
specific inhibitors for the kinase. To this end, we examined the sequence alignment of the ATP binding
pocket of ERK compared to those of other kinases. We envisioned that we could improve kinase
selectivity for ERK by implementing either favorable interactions between an inhibitor and residues
distinct to ERK or unfavorable interactions between an inhibitor and residues common to off−target
kinases. In the ATP binding pocket, there are several residues found in ERK with low frequency of
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occurrence versus the kinome including Gln103 (1%), Lys112 (6%), Asp109 (7%), Tyr62 (10%), and
Ser151 (11%) (Figure 1A). Especially, a combination of the gatekeeper Gln103 and Lys112 is only
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found in ERK1/2 out of 491 human kinases, which presents an excellent opportunity for designing
ERK selective inhibitors.
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We pursued novel scaffolds derived from 1 that exhibit improved drug−like properties such as low
27
molecular weight and high ligand efficiency. First, we replaced the long side chain at the 5−position of
the indazole, which takes up two−thirds of the molecular weight, with an amide group (9 in Figure 1B).
When 9 (R = Me) was docked to the ERK crystal structure, it was predicted to bind to the hinge and
Lys112 in the same mode as 1 binds to the pocket (9 in cyan in Figure 1C). However, 9 lacks the key
interactions that the side chain of 1 makes, especially the π−π interaction between the terminal
pyrimidine group of 1 and Tyr62 with concomitant shift of the glycine rich loop. The unique shift of the
glycine rich loop and the π−π interaction with Tyr62 are believed to account for excellent kinome
selectivity of 1. Not surprisingly, 9 suffered significant erosion in kinase selectivity compared to 1 in a
broad panel of kinome (data not shown).
We envisioned that we could overcome the loss in kinase selectivity by designing novel short side
chains that are capable of making additional interactions with ERK residues that are not employed by 1.
Gln as a gatekeeper is very rare, occurring in only 1% of human kinases (vide supra), thus making a
favorable interaction with Gln103 could significantly improve kinase selectivity for ERK. While 1 lacks
1
1
a direct hydrogen bond interaction with Gln103, other ERK inhibitors are known to employ a direct
1
5,16,18
hydrogen binding to Gln103 in the literature.
Docking of 9 to the ERK crystal structure suggests
that a hydrogen bond donor substituted at the 6−position, instead of the 5−position, would be pointing
toward Gln103 within a hydrogen bond distance. To explore the gatekeeper interaction, we designed the
5
−azaindole core with a urea group installed at the 6−position (10 in Figure 1B). Compound 10 (R = Me)
docks to the binding pocket in the same mode as 9 does (10 in yellow in Figure 1C) making hydrogen
bond interactions with the hinge and Lys112. Furthermore, the urea of 10 is positioned to make two
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Journal of Medicinal Chemistry
additional interactions with the enzyme, one with gatekeeper Gln103 and the other with catalytic Lys52.
In this de novo design, the intramolecular hydrogen bonding between the 5−aza nitrogen and the urea
nitrogen γ to the core is important as it would fix the urea bond α to the core to s−trans and direct the
urea carbonyl group toward Lys52.
1
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A)
B)
H
N
H
H
H
N
N
6
5
N
6
O
R
O
N
N
N
N
O
R
N
HN
N
N
H
N
N
HN
N
O
1
N
N
N
9
10
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C)
1
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Figure 1. (A) Sequence conservation in the ERK2 binding pocket, (B) de novo design of the
1
−(1H−pyrazolo[4,3−c]pyridin−6−yl)ureas, (C) docking of 9 (cyan) and 10 (yellow) to the binding
pocket of ERK2.
Initial Exploration at C−6 of the 5−Azaindazole.
To test the hypothesis, we introduced a urea and other hydrogen bond donating groups at the 6−position
of the 5−azaindazole. It was found that 2−methyl group on the pyridine at the 3−position afforded
ameliorated CYP inhibitory profile of the molecules compared to the unsubstituted pyridine (data not
shown), thus 2−methylpyridin−4−yl group was employed for our initial SAR studies (Table 2).
Gratifyingly, the benzylurea analogue 11 was as potent as 1 in the ERK2 enzymatic assay, hinting that
1
1 might be making the envisioned hydrogen bond interactions with Gln103 and Lys52. The
benzenepropanamide and the benzyl carbamate analogues (12 and 13), lacking the intramolecular
hydrogen bond and favoring s−cis conformation of the amide and carbamate α to the core, were less
potent than 11 by greater than 3 and 4 log units, respectively. The benzyl carbamate on the indazole core
(14) was not tolerated at this position, either. The potency loss of these analogues confirmed the
importance of the intramolecular hydrogen bond for efficient binding to ERK. The thiourea and the
sulfamide analogues (15 and 16) suffered from loss of potency, albeit both would have the
intramolecular hydrogen bond. Aminothiazoles at the 6−position were also pursued (17 and 18), where
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Journal of Medicinal Chemistry
the sulfur atom would have a favorable electrostatic interaction with the 5−aza nitrogen and the nitrogen
of the thiazole ring would be positioned toward Lys52. However, compounds 17 and 18 displayed either
greater than 60−fold loss of potency or a complete loss of potency, respectively.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
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3
3
3
4
4
4
4
4
4
4
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4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Initial SAR at C−6 of the Azaindazole
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hERK2
mERK2
X
R
a
b
IC (nM)
IC (nM)
5
0
50
11
N
0.9
0.2
c
c
c
1
2
3
N
N
ND
ND
ND
12
420
1
> 3,000
> 3,000
14
CH
N
c
1
5
ND
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Journal of Medicinal Chemistry
Page 10 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
1
6
N
2,400
ND
c
c
1
1
7
8
N
N
56
ND
ND
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
> 3,000
a
b
c
human ERK2 IC . mouse ERK2 IC . ND = not determined.
5
0
50
Crystal Structure of Compound 11.
A)
B)
Figure 2. X−ray crystal structure of 11 bound to rat ERK2: (A) electrostatic surface view of the ERK2
active site bound to 11. (B) hydrogen−bonding pattern in the structure of 11 bound to ERK2. Hydrogen
bond distances are in Å.
The X−ray crystal structure of 11 bound to rat ERK2 confirmed the modeling hypothesis for the urea
substitution interactions with the gatekeeper Gln103 (Figure 2). The 5−azaindazole moiety of 11
hydrogen bonds to the backbone carbonyl and amide nitrogen of hinge residues Asp104 and Met106,
1
1
mimicking the indazole of 1. Lys112 sidechain forms a hydrogen bond to the nitrogen of
methylpyridine as seen in 1. The urea group forms an extensive hydrogen bonding network to the
gatekeeper Gln103 sidechain, the side chain of the catalytic Lys52, and the sidechain of Glu69.
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Page 11 of 35
Journal of Medicinal Chemistry
Compounds 11 and 1 have a conserved water, which is located in the back by the gatekeeper involved in
the extensive hydrogen bonding network. Additionally, the urea nitrogen γ to the core makes an
intramolecular hydrogen bond with the 5−aza nitrogen of the core, producing a nearly planar extension
from the 5−azaindazole core. The benzyl group extends toward the glycine rich loop and does not
disrupt the π−π stacking between Tyr34 and Tyr62 as found in the apo−form of ERK2, nor is there any
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
major rearrangements of the C−helix or the DFG motif, unlike in the crystal structure of 1.
Exploration of the Urea Substituents.
Encouraged by high ERK potency of the novel 5−azaindazole with a urea group at the 6−position and
novel hydrogen bond interactions confirmed by X−ray crystallography, we next focused on substituents
on the urea. Throughout the SAR studies, key compounds were screened against a broad panel of
kinases (101 or 265 kinases) to assess their kinome selectivity. When compound 11 was screened
against a panel of 101 kinases, it showed greater than 100−fold selectivity against 95% of the kinases
(Table 3). In the kinase counter−screening assays, coupled assays were employed for MEK1, RAF1,
COT, and BRAF, where activity of the downstream kinase ERK was measured. Kinase selectivity of 11
is excellent considering that MEK1, RAF1, COT, and BRAF are 4 out of 5 off−targets inhibited by11
with less than 100−fold selectivity.
While phenylurea 19 suffered from greater than 100−fold loss of the enzymatic potency, phenethylurea
2
0 displayed high potency comparable to 11 both in the enzymatic assay and in the cell biochemical
assay, inhibition of phosphorylation of RSK (pRSK), downstream of ERK. Compound 20, however,
showed significant erosion in kinome selectivity (> 100−fold selective against 87% of the kinases).
Major improvements in ERK potency were observed with methyl substituent at the benzylic position of
the urea. Compared to 11, (1R)−1−phenylethylurea 21 not only offered 3−fold improvement in cell
potency (pRSK IC = 70 nM) but also maintained excellent kinase selectivity (> 100−fold selective
5
0
against 97% of kinases in the panel of 265 kinases). It was found that stereochemistry at the benzylic
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Journal of Medicinal Chemistry
Page 12 of 35
position was important, as the (S)−enantiomer 22 was greater than 20−fold less potent than 21. The
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
indane analogue 23, tethering the benzylic methyl group to the benzene, showed greater than 5−fold loss
of potency even if the more potent enantiomer of 23 was assumed to exclusively contribute to the
potency.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. SAR of the Urea Substituents
No. of
Kinases
Tested
% of
hERK2
pRSK IC
5
0
R
a
Kinases >
IC (nM)
(nM)
5
0
b
100×
1
1
0.9
220
101
−
95
c
19
130
> 10,000
ND
d
e
2
0
0.5
110
101
87
97
21
22
0.2
70
265
d
e
c
38
1,800
−
ND
c
2
3
5
750
−
ND
a
b
c
d
From A375SM cells. Fold selectivity against invitrogen ERK2 IC . ND = not determined. From
5
0
e
mERK2 IC (nM). From A375 cells.
5
0
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Journal of Medicinal Chemistry
Exploration of the C−3 Substituents.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Having identified promising (1R)−1−phenylethylurea substituent at the 6−position of the 5−azaindazole,
we next explored various substituents at the 3−position of the 5−azaindazole core. Compared to 21, the
analogues with alkyl substituents such as n−propyl, cyclopropyl, and cyclopentyl groups at the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
−position (24, 25, and 26, respectively) suffered from loss of the cell potency by 5−11 fold (Table 4).
Loss of the cell potency with the alkyl substituents, presumably due to absence of the interaction with
Lys112, directed us toward exploring aromatic groups that could employ hydrogen bond interaction
with Lys112. The 4−fluorophenyl, 2−methoxypyridin−4−yl, and 6−methylpyridazin−4−yl analogues
(28, 31, and 32, respectively) also showed a loss of potency in the cell assay. On the other hand, the
1
−methyl−1H−pyrazol−4−yl, 2,6−dimethylpyridin−4−yl, and 2−fluoropydin−4yl analogues (27, 29, and
30, respectively) offered comparable enzymatic and cell potency to 21. While 27 displayed slight
erosion in kinase selectivity, both 29 and 30 showed excellent selectivity in the broad panel of kinases.
While our SAR studies were primarily driven by on−target potency and kinome selectivity, activities
32
against other off−targets such as hERG and CYPs were closely monitored in parallel. Compounds 21,
27, 29, and 30 offered satisfactory hERG activity profile (all IC > 20,000 nM) in addition to their
5
0
excellent ERK potency and kinase selectivity. Compounds 27 and 29, however, showed submicromolar
activities in CYP3A4 inhibition (IC = 380 nM and 780 nM, respectively) while 21 and 30 offered
5
0
more favorable CYP3A4 inhibitory activities (IC = 3,900 nM and 29,000 nM, respectively).
5
0
Table 4. SAR of Substituents at the C−3 Position
No. of
Kinases
Tested
% of
hERK2
IC (nM)
pRSK IC
50
R
a
Kinases >
(nM)
50
b
100×
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
1
0.2
70
265
97
24
25
26
2
2
4
350
550
810
101
101
96
95
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
−
ND
2
7
8
0.3
0.8
110
360
101
94
96
2
101
29
0.3
0.3
0.9
0.9
130
97
101
101
−
99
96
30
31
32
c
c
300
810
ND
−
ND
a
b
c
From A375SM cells. Fold selectivity against invitrogen ERK2 IC . ND = not determined.
5
0
Cell Activity and PK Profile of 21.
Compared to 1, compound 21 inhibits ERK with the same potency level in pRSK and A375SM
V600E
(BRAF
) proliferation assays (IC = 43 nM) with a fraction of molecular weight (MW 372 vs 588),
50
27
thereby offers much higher ligand efficiency (LE 0.46 vs 0.29) (Table 5). One stark difference
between 1 and 21 is that 1 inhibits phosphorylation and activation of ERK (pERK IC = 9 nM) while
50
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Journal of Medicinal Chemistry
2
1 does not (pERK IC > 10,000 nM). Compound 21 showed excellent kinome selectivity comparable
50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to 1, hitting only 8 kinases with less than 100−fold selectivity out of 265 kinases tested. Compound 21
was weakly active in the wild−type CHL−1 proliferation assay (IC = 1,300 nM), confirming its high
5
0
selectivity for ERK/MAPK pathway. Compound 21 displayed PK properties in rats (Cl = 35
p
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mL/min/kg, F = 12%) suitable for in vivo studies. While its polarity is within a good range judged by
cLogD value, its modest solubility is suboptimal.
Table 5. Profile of 21
MW
372
hERK2 IC (nM)
0.2
5
0
LE
pRSK IC (nM)
0.46
77
5
0
pERK IC (nM)
> 10,000
5
0
A375SM Prolif IC (nM)
43
5
0
CHL−1 Prolif IC (nM)
1,300
265
5
0
No. of Kinases Tested
BRAF, ERK1, FLT4, MEK1,
a
D842V
Kinases < 100×
MEK2, MELK, PDGFRA
,
V561D
PDGFRA
Rat PK Properties
b
AUCnorm (ꢀM⋅h/mpk)
1.3
35
b
Cl (mL/min/kg)
p
b
V (L/kg)
1.7
0.7
12
dss
b
t (h)
1
/2
c
Bioavailability (%)
Physical Properties
d
cLogD (pH 7.4)
2.2
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Journal of Medicinal Chemistry
Page 16 of 35
Rat plasma protein binding (%)
98.8
18
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
e
FaSSIF Solubility (pH 6.5, ꢀM)
a
b
Fold selectivity against invitrogen ERK2 IC . Dosed IV at 0.5 mg/kg as a solution in PEG400:H O
5
0
2
c
d
(60:40 (v/v)). Dosed PO at 1 mg/kg as a solution in PEG400:H O (60:40 (v/v)). At pH 7.4 from ACD
Labs v10. FaSSIF = human fasted−state simulated intestinal fluid.
2
e
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vivo Studies of 21.
On the basis of its excellent ERK cell potency both in pRSK and proliferation assays, clean off−target
kinase activity profile, and PK properties suitable for oral dosing in rats, compound 21 was chosen for
in vivo proof−of−mechanism studies. In this in vivo xenograft model, mice bearing A375SM tumor
xenograft were dosed with either vehicle or compound 21 orally at 25 mg/kg BID, 50 mg/kg BID, and
1
1
00 mg/kg QD for 20 days. After the final dose on day 20, blood samples from the vehicle arm and the
00 mg/kg QD arm were obtained at various time points, and exposure of 21 and pRSK as well as
pERK levels were measured (Figure 3A). The mice dosed with 21 exhibited potent and sustained
inhibition of pRSK in response to the exposure level of 21, while durable inhibition of pERK was not
observed. These results confirm the inhibitory activity of ERK by 21 in vivo. Compared to the mice
treated with vehicle, the mice treated with 21 showed either tumor growth inhibition (82% at 25 mg/kg
BID) or tumor regression (47% at 50 mg/kg BID; 42% at 100 mg/kg QD) in a dose dependent manner
(
Figure 3B). The plasma exposure of 21 on day 20 displayed a strong correlation between the exposure
and the efficacy (Table 6). The strong efficacy of 21 in this mouse model supports that inhibition of
ERK activity without inhibition of ERK activation, as judged by sustained inhibition of pRSK and lack
of durable inhibition of pERK (Figure 3A), could be a viable option for the treatment of cancers.
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Journal of Medicinal Chemistry
1
A)
Exposurea 20
B)
2
1
1
1
1
00
80
60
40
20
5.2
6.7
1.6 0.1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pRSK
pERK
100
8
6
0
0
40
20
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21 100 mpk QD
Figure 3. Effect of 21 in A375SM xenograft model. Compound 21 was dosed orally as a solution in
PEG300:H O (50:50 (v/v)). (A) PK/PD of the 100 mpk QD arm of 21 compared to the vehicle treated
2
a
arm on day 20. Exposure of 21 in ꢀM from the 100 mg/kg QD arm after the final dose on day 20. (B)
Effect of 21 in tumor volume compared to the vehicle treated arm. tumor growth inhibition. tumor
regression.
b
c
Table 6. Exposure of 21 on day 20 in A375SM xenograft model.
Dosing
AUC
(ꢀM⋅h)
c
d
Regimen of
Cmax (ꢀM)
T
max (h)
b
a
21
2
5
5 mpk BID
0 mpk BID
95
12
23
27
11
11
240
320
1
00 mpk QD
4
a
c
b
Compound 21 was dosed orally as a solution in PEG300:H O (50:50 (v/v)). Area under the curve.
Peak plasma concentration. Time to reach Cmax.
2
d
Conclusions
A novel 1−(1H−pyrazolo[4,3−c]pyridin−6−yl)urea scaffold was successfully designed by installing a
urea group at the 6−position of the 5−azaindazole core. In addition to the hydrogen bonds with the hinge
and Lys52, the scaffold employs hydrogen bond interactions with gatekeeper Gln103 and Lys112,
which are unique to ERK out of 491 human kinases. The interactions with the residues specific to ERK
in otherwise highly conserved binding pocket of kinases account for excellent kinome selectivity of
1
−(1H−pyrazolo[4,3−c]pyridin−6−yl)ureas. SAR studies on the 6−position of the 5−azaindazole core
led to identification of (1R)−1−phenylethylurea as an optimal substituent. Subsequent exploration of the
C−3 substituents identified both 2−methylpyridin−4−yl and 2−fluoropyridin−4−yl groups that offered
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Journal of Medicinal Chemistry
Page 18 of 35
high ERK potency as well as low off−target activities. Compound 21 is highly potent in the target
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
engagement and the proliferation assays in a pathway specific manner, and highly selective for ERK in
V600E
the kinome panel. In the BRAF
xenograft model, 21 exhibited potent target engagement and strong
tumor regression in a dose dependent manner, demonstrating a potential of ERK inhibitors as an
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
anti−cancer agent.
Experimental
Chemistry. Commercial reagents were obtained from reputable suppliers and used as received. All
solvents were purchased in septum−sealed bottles stored under an inert atmosphere. All reactions were
sealed with septa through which an argon atmosphere was introduced unless otherwise noted. Liquid
reagents and solvents were transferred under a positive pressure of nitrogen via syringe. Reactions were
conducted in microwave vials or round bottomed flasks containing Teflon−coated magnetic stir bars.
Microwave reactions were performed with a Biotage Initiator Series Microwave (fixed hold time
setting; reaction temperatures monitored by the internal infrared sensor).
Reactions were monitored by thin layer chromatography (TLC) on pre−coated TLC glass plates (silica
gel 60 F254, 250 µm thickness) or by LC/MS (30 mm x 2 mm 2 ꢀm column + guard; 2 ꢀL injection;
3% to 98% MeCN/water + 0.05% TFA gradient over 2.3 minutes; 0.9 mL/min flow; ESI; positive ion
mode; UV detection at 254 nm). Visualization of the developed TLC chromatogram was performed by
fluorescence quenching. Flash chromatography was performed on an automated purification system
1
using pre−packed silica gel columns. H NMR spectra were recorded on either a 500 or a 600 MHz
Varian spectrometer; chemical shifts (δ) are reported relative to residual proton solvent signals. Data for
NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, brs = broad
singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, td = triplet of doublets, m =
multiplet), coupling constant (Hz), integration.
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Page 19 of 35
Journal of Medicinal Chemistry
All tested compounds reported are of at least 95% purity, as judged by LCAP (150 mm x 4.6 mm ID, 5
m column; 5 ꢀL injection; 10−100% MeCN/H O + 0.05% TFA gradient over 6.75 min; 1 mL/min
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ
2
flow; ESI; positive ion mode; UV detection at 254 nm, Bw8).
6
−Chloro−3−iodo−1−trityl−1H−pyrazolo[4,3−c]pyridine (2). Step 1. A flask was charged with
6−chloro−1H−pyrazolo[4,3−c]pyridine (3.0 g, 19.5 mmol), iodine (13.1 g, 51.7 mmol), KOH (3.29 g,
8.6 mmol), and DMF (60 mL). The mixture was heated to 40 °C for 16 h and then additional iodine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
(7.8 g, 30.7 mmol) and KOH (1.6 g, 28.4 mmol) were added. The mixture was heated to 70 °C for 3 h,
treated with 1N Na S O solution, and extracted with EtOAc (3×). The combined organics were washed
2
2
3
with water and brine, dried over sodium sulfate, and concentrated. DCM was added to the residue and
the solid was collected by filtration to afford 6−chloro−3−iodo−1H−pyrazolo[4,3−c]pyridine (3.2 g,
+
1
1.5 mmol, 59% yield) as a yellow solid. LRMS (ESI) calcd for (C H ClIN ) [M + H] 280, found 280.
6
4
3
1
H NMR (600 MHz, DMSO−d ) δ 14.00 (s, 1H), 8.63 (s, 1H), 7.68 (s, 1H).
6
Step 2. To a stirred solution of 6−chloro−3−iodo−1H−pyrazolo[4,3−c]pyridine (1.87 g, 6.71 mmol) in
THF (20 mL) was added NaH (60% in mineral oil, 0.40 g, 10.1 mmol) at 0 °C and the mixture was
stirred for 50 min at 0 °C. Trityl chloride (2.24 g, 8.05 mmol) was added at the same temperature and
the mixture was stirred at room temperature for 16 h, treated with saturated NH Cl solution, and
4
extracted with EtOAc (3×). The combined organics were washed with water and brine, dried over
sodium sulfate, concentrated, and purified by flash chromatography to afford 2 (3.26 g, 6.25 mmol, 93%
+
1
yield) as a light yellow solid. LRMS (ESI) calcd for (C H ClIN ) [M + H] 522, found 522. H NMR
2
5
18
3
(
600 MHz, CDCl ) δ 8.54 (s, 1H), 7.14−7.33 (m, 15H), 6.01 (s, 1H).
3
1
6−Chloro−3−(2−methylpyridin−4−yl)−1−trityl−1H−pyrazolo[4,3−c]pyridine
−methylpyridinyl). A mixture of 2 (2.47 g, 4.72 mmol), 2−methylpyridine−4−boronic acid (0.97 g,
7.05 mmol), PdCl (dppf)−CH Cl adduct (0.58 g, 0.71 mmol), and K CO (1.96 g, 14.2 mmol) in
(3,
R
=
2
2
2
2
2
3
1
,4−dioxane (10 mL) and H O (2.5 mL) was purged with nitrogen for 5 min, heated to 80 °C overnight,
2
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Page 20 of 35
cooled to room temperature, and diluted with EtOAc. The reaction mixture was filtered through Celite
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and then partitioned between EtOAc and saturated NaHCO solution. The organic layer was extracted
3
with EtOAc (3×) and the combined organics were dried over sodium sulfate and concentrated. The
1
residue was purified by flash chromatography to afford 3 (R = 2−methylpyridinyl) (1.02 g, 2.09 mmol,
+
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4% yield). LRMS (ESI) calcd for (C H ClN ) [M + H] 487, found 487. H NMR (600 MHz,
31 24 4
DMSO−d ) δ 9.42 (s, 1H), 8.56 (d, J = 4.8 Hz, 1H), 7.81 (s, 1H), 7.69 (d, J = 5.4 Hz, 1H), 7.19−7.41
6
(m, 15H), 6.09 (s, 1H), 2.56 (s, 3H).
1
3
−(2−Methylpyridin−4−yl)−1−trityl−1H−pyrazolo[4,3−c]pyridin−6−amine
(4,
R
=
2−methylpyridinyl). Pd (dba) (0.19 g, 0.21 mmol), 2−(dicyclohexylphosphino)biphenyl (0.073 g, 0.21
2
3
1
mmol), and 3 (R = 2−methylpyridinyl) (1.02 g, 2.09 mmol) were placed in a microwave vial under
nitrogen and dissolved in THF (11 mL). The reaction mixture was purged with nitrogen for 5 min,
treated with LiHMDS (1 M in THF, 2.51 mL, 2.51 mmol), and heated to 65 °C overnight. The mixture
was cooled to room temperature and treated with additional amounts of Pd (dba) (0.096 g, 0.1 mmol),
2
3
2
−(dicyclohexylphosphino)biphenyl (0.036 g, 0.1 mmol) and LiHMDS (1 M in THF, 1.25 mL, 1.25
mmol). The mixture was purged with nitrogen for 5 min, heated to 80 °C for 3 h, cooled to room
temperature, and treated with 1N HCl. The mixture was concentrated and partitioned between DCM and
1
N NaOH. The organic layer was extracted with DCM (3×) and the combined organics were dried over
1
sodium sulfate and concentrated. The residue was purified by flash chromatography to afford 4 (R =
2
−methylpyridinyl) (549 mg, 2.09 mmol, 56% yield) as a solid. LRMS (ESI) calcd for (C H N ) [M +
3
1
26
5
+
1
H] 468, found 468. H NMR (600 MHz, DMSO−d ) δ 8.93 (d, J = 1.2 Hz, 1H), 8.49 (d, J = 4.8 Hz,
6
1
1
3
H), 7.68 (s, 1H), 7.58 (dd, J = 5.4, 1.2 Hz, 1H), 7.20−7.38 (m, 15H), 5.84 (s, 2H), 5.38 (d, J = 1.2 Hz,
H), 2.53 (s, 3H).
−Bromo−1−trityl−1H−pyrazolo[4,3−c]pyridine−6−carboxylic acid (6). Step 1. To a brown
suspension of methyl 3−bromo−1H−pyrazolo[4,3−c]pyridine−6−carboxylate (5.0 g, 19.5 mmol) in
DMF (50 mL) was added NaH (60% in mineral oil, 1.17 g, 29.3 mmol) in portions at 0 °C. The reaction
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Journal of Medicinal Chemistry
mixture was stirred at that temperature for 30 min, and a solution of trityl chloride (6.53 g, 23.4 mmol)
in DMF (50 mL) was added dropwise. The reaction mixture was slowly warmed to room temperature
and left to stir overnight. The reaction mixture was treated with water and extracted with EtOAc (3×).
The combined organics were washed with brine, dried over sodium sulfate, concentrated, and purified
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
by
flash
chromatography
to
afford
methyl
3
−bromo−1−trityl−1H−pyrazolo[4,3−c]pyridine−6−carboxylate as a white solid (6.0 g, 12.0 mmol, 62%
+
1
yield). LRMS (ESI) calcd for (C H BrN O ) [M + H] 498, found 498. H NMR (600 MHz, CDCl ) δ
27 21
3
2
3
8
.99 (s, 1H), 7.16−7.34 (m, 15H), 6.92 (s, 1H), 3.89 (s, 3H).
Step 2. To a suspension of methyl 3−bromo−1−trityl−1H−pyrazolo[4,3−c]pyridine−6−carboxylate (6.0
g, 12.0 mmol) in MeOH (60 mL) was added 1N KOH (42.1 mL, 42.1 mmol). The reaction mixture was
o
heated to 50 C for 1 h. The mixture was cooled to room temperature and acidified to pH 4 with 1N
HCl. The precipitate was filtered, washed with water, and dried to afford 6 (5.3 g, 11 mmol, 91% yield).
+
1
LRMS (ESI) calcd for (C H BrN O ) [M + H] 484, found 484. H NMR (600 MHz, DMSO−d ) δ
26
19
3
2
6
13.16 (brs, 1H), 9.02 (s, 1H), 7.15−7.39 (m, 15H), 6.92 (s, 1H).
−Bromo−1−trityl−1H−pyrazolo[4,3−c]pyridin−6−amine (7). To a suspension of 6 (1.95 g, 4.03
mmol) in 1,4−dioxane (20 mL) was added a solution of diphenylphosphoryl azide (DPPA) (1.047 mL,
3
4
.83 mmol) and TEA (0.842 mL, 6.04 mmol) in 1,4−dioxane (10 mL) dropwise over 5 min. The clear
o
solution was heated to 80 C for 2 h and treated with 1N KOH (20.1 mL, 20.1 mmol) at that
o
temperature. The reaction mixture was heated to 80 C overnight, cooled to room temperature, and
extracted with EtOAc (3×). The combined organics were washed with brine, dried over sodium sulfate,
concentrated, and purified by flash chromatography to afford 7 (0.61 g, 1.3 mmol, 33% yield) as an
+
1
off−white solid. LRMS (ESI) calcd for (C H BrN ) [M + H] 455, found 455. H NMR (600 MHz,
2
5
20
4
DMSO−d ) δ 8.28 (s, 1H), 7.18−7.35 (m, 15H), 5.97 (s, 2H), 5.31 (s, 1H).
6
2
1
−(3−bromo−1−trityl−1H−pyrazolo[4,3−c]pyridin−6−yl)−3−[(1R)−1−phenylethyl]urea (8, R
=
(1R)−1−phenylethyl). To a flask were added 7 (500 mg, 1.10 mmol), CDI (356 mg, 2.20 mmol),
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Journal of Medicinal Chemistry
Page 22 of 35
imidazole (150 mg, 2.20 mmol), and DCM (11 mL). The mixture was stirred at room temperature for 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
h, treated with (1R)−1−phenylethanamine (272 mg, 2.25 mmol), and left to stir for 1 h. The mixture was
2
purified by flash chromatography to afford 8 (R = (1R)−1−phenylethyl) (582 mg, 0.966 mmol, 88%
+
1
yield) as a white solid. LRMS (ESI) calcd for (C H BrN O) [M + H] 602, found 602. H NMR (600
34 29
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, CDCl ) δ 9.59 (s, 1H), 8.52 (s, 1H), 7.02−7.31 (m, 21H), 5.33 (s, 1H), 5.01 (m, 1H), 1.50 (d, J =
3
7
.2 Hz, 3H).
(R)−1−(1−Phenylethyl)urea. A mixture of (R)−1−phenylethylamine (1.00 g, 8.25 mmol) in water (4
mL) was treated with HCl (1 N, 8.5 mL, 8.5 mmol) followed by potassium cyanate (3.35 g, 41.2 mmol).
The reaction mixture was warmed to 80 °C for 1 hour and cooled to room temperature. The mixture was
neutralized with saturated NaHCO solution and extracted with EtOAc and 2−methyltetrahydrofuran.
3
The combined organics were dried over sodium sulfate, concentrated, and purified by flash
chromatography to afford (R)−1−(1−Phenylethyl)urea (1.32 g, 8.04 mmol, 97% yield) as a white solid.
+
1
LRMS (ESI) calcd for C H N O) [M + H] 165, found 165. H NMR (600 MHz, DMSO−d ) δ
9
13
2
6
7
1
1
.23−7.28 (m, 4 H), 7.17 (t, J = 6.7 Hz, 1 H), 6.38 (d, J = 7.9 Hz, 1 H), 5.38 (br s, 2 H), 4.65 (m, 1 H),
.26 (d, J = 7.1 Hz, 3 H).
−Benzyl−3−[3−(2−methylpyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]urea (11). Step 1. To a
1
stirred solution of CDI (520 mg, 3.21 mmol) in DCM (3 mL) was added a solution of 3 (R =
2−methylpyridinyl) (300 mg, 0.642 mmol) and imidazole (131 mg, 1.93 mmol) in DCM (3 mL). The
mixture was left to stir overnight and treated with benzylamine (344 mg, 3.21 mmol). The reaction
mixture was left to stir for 2 h and purified by flash chromatography to afford a solid, which was
directly dissolved in DCM (20 mL) and treated with triethylsilane (75 mg, 0.64 mmol) and TFA (4 mL).
The reaction mixture was left to stir overnight, concentrated, diluted with saturated sodium bicarbonate
solution, and extracted with chloroform:i−PrOH (3:1, 3×). The combined organics were dried over
sodium sulfate, concentrated, and purified by flash chromatography to afford 11 (167 mg, 0.47 mmol,
+
1
7
3% yield). HRMS (ESI) calcd for (C H N O) [M + H] 359.1620, found 359.1623. H NMR (600
20 19 6
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Journal of Medicinal Chemistry
MHz, DMSO−d ) δ 13.5 (s, 1H), 9.28 (s, 1H), 9.23 (s, 1H), 8.55 (d, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.81
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(d, J = 5.4 Hz, 1H), 7.71−7.79 (m, 2H), 7.25−7.37 (m, 5H), 4.39 (d, J = 6.0 Hz, 2H), 2.58 (s, 3H).
1
−[3−(2−Methylpyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]urea
(21). Prepared in analogy to that described for 11 in 83% yield. HRMS (ESI) calcd for (C H N O) [M
2
1
21
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
+
H] 373.1777, found 373.1779. H NMR (600 MHz, DMSO−d ) δ 13.49 (s, 1H), 9.24 (s, 1H), 9.13 (s,
6
1H), 8.55 (d, J = 4.8 Hz, 1H), 7.89 (s, 1H), 7.81 (d, J = 4.2 Hz, 1H), 7.35−7.56 (m, 2H), 7.24−7.39 (m,
5
H), 4.90 (m, 1H), 2.58 (s, 3H), 1.43 (d, J = 7.2 Hz, 3H).
1
−(2,3−Dihydro−1H−inden−1−yl)−3−[3−(2−methylpyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−y
l]urea trifluoroacetate (23). Prepared in analogy to that described for 11 in 23% yield. HRMS (ESI)
+
1
calcd for (C H N O) [M + H] 385.1777, found 385.1787. H NMR (600 MHz, DMSO−d ) δ 14.00 (s,
2
2
21
6
6
1H), 9.37 (s, 1H), 9.23 (s, 1H), 8.77 (d, J = 6.6 Hz, 1H), 8.41 (s, 1H), 8.33 (brs, 1H), 7.94 (s, 1H), 7.55
brs, 1H), 7.22−7.32 (m, 4H), 5.26 (m, 1H), 2.77 (s, 3H), 1.28−2.97 (m, 4H).
(
N−[3−(2−Methylpyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−phenylpropanamide (12). To
1
a stirred mixture of 3 (R = 2−methylpyridinyl) (53.2 mg, 0.114 mmol), 3−phenylpropionic acid (27.3
mg, 0.182 mmol) and 2−chloro−1−methylpyridinium iodide (49 mg, 0.19 mmol) in DCE (1 mL) was
added TEA (0.048 mL, 0.34 mmol). The reaction mixture was heated to 70 °C for 1 h, cooled to room
temperature, diluted with EtOAc and saturated sodium bicarbonate solution, and extracted with EtOAc
(3×). The combined organics were dried over sodium sulfate and concentrated. The residue was
dissolved in DCM (1.5 mL), treated with triethylsilane (0.018 mL, 0.11 mmol) and TFA (0.15 mL), and
left to stir overnight. The mixture was concentrated and purified by preparative−HPLC. The collected
fractions were partitioned between EtOAc and saturated sodium bicarbonate solution and extracted with
EtOAc (3×). The combined organics were dried over sodium sulfate and concentrated to afford 12 (17
+
mg, 0.048 mmol, 42% yield). HRMS (ESI) calcd for (C H N O) [M + H] 358.1668, found 358.1670.
2
1
20
5
1
H NMR (600 MHz, DMSO−d ) δ 13.66 (s, 1H), 10.72 (s, 1H), 9.30 (s, 1H), 8.57 (d, J = 5.4 Hz, 1H),
6
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Journal of Medicinal Chemistry
Page 24 of 35
8
.29 (s, 1H), 7.91 (s, 1H), 7.85 (d, J = 4.8 Hz, 1H), 7.18−7.30 (m, 5H), 2.93 (t, J = 7.8 Hz, 2H), 2.76 (t,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
J = 7.8 Hz, 2H), 2.59 (s, 3H).
1
−Benzyl−3−[3−(2−methylpyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]thiourea
(15).
A
1
suspension of 3 (R = 2−methylpyridinyl) (25 mg, 0.05 mmol) in toluene (1 mL) was treated with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(isothiocyanatomethyl)benzene (15 mg, 0.1 mmol) and the mixture was heated to 100 °C overnight. The
mixture was cooled to room temperature and concentrated. The residue was dissolved in DCM (0.8
mL), treated with TFA (0.2 mL) and triethylsilane (0.008 mL, 0.05 mmol), left to stir for 5 h,
concentrated, and purified by preparative−HPLC to afford 15 trifluoroacetate (7 mg, 0.014 mmol, 27%
+
1
yield) as a yellow solid. HRMS (ESI) calcd for (C H N S) [M + H] 375.1392, found 375.1402. H
20
19
6
NMR (600 MHz, DMSO−d ) δ 11.95 (br s, 1H), 10.77 (s, 1H), 9.35 (s, 1H), 8.69 (d, J = 5.0 Hz, 1H),
6
8.27 (s, 1H), 8.18 (br s, 1H), 7.55−7.24 (m, 5H), 7.14−7.10 (m, 1H), 4.92 (d, J = 5.3 Hz, 2H), 2.67 (s,
3
H).
3
−(2−Methylpyridin−4−yl)−N−(1,3−thiazol−2−yl)−1H−pyrazolo[4,3−c]pyridin−6−amine
1
trifluoroacetate (17). A suspension of 3 (R = 2−methylpyridinyl) (42 mg, 0.090 mmol) in 1,4−dioxane
(0.5 mL) was treated with sodium hydride (60%, 18 mg, 0.45 mmol), left to stir for 10 min, and treated
with 2−chlorothiazole (0.012 mL, 0.14 mmol). The mixture was heated to 90 °C for 3.5 h, cooled to
room temperature, diluted with EtOAc, washed with saturated sodium bicarbonate solution, and
concentrated. The residue was dissolved in DCM (0.8 mL), treated with of TFA (0.2 mL) and
triethylsilane (0.014 mL, 0.090 mmol), and left to stir for 2 h. The mixture was concentrated and
purified by preparative−HPLC to afford 17 trifluoroacetate (22 mg, 0.052 mmol, 58% yield) as a yellow
+
1
solid. HRMS (ESI) calcd for (C H N S) [M + H] 309.0922, found 309.0923. H NMR (600 MHz,
1
5
13
6
DMSO−d ) δ 13.96 (s, 1H), 11.33 (brs, 1H), 9.54 (s, 1H), 8.79 (d, J = 6.6 Hz, 1H), 8.49 (s, 1H), 8.38
6
(brs, 1H), 7.40 (d, J = 3.6 Hz, 1H), 7.37 (s, 1H), 7.03 (d, J = 3.6 Hz, 1H), 2.80 (s, 3H).
3
−(2−Methylpyridin−4−yl)−N−(5−phenyl−1,3−thiazol−2−yl)−1H−pyrazolo[4,3−c]pyridin−6−amine
trifluoroacetate (18). Prepared in analogy to that described for 17 in 32% yield. HRMS (ESI) calcd for
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Journal of Medicinal Chemistry
+
1
(
C H N S) [M + H] 385.1235, found 385.1242. H NMR (600 MHz, DMSO−d ) δ 14.05 (s, 1H),
2
1
17
6
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
11.47 (brs, 1H), 9.62 (s, 1H), 8.81 (d, J = 6.0 Hz, 1H), 8.54 (s, 1H), 8.43 (d, J = 6.0 Hz, 1H), 7.82 (s,
1
3
H), 7.59 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.32 (s, 1H), 7.27 (t, J = 7.5 Hz, 1H), 2.82 (s,
H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1−[3−(4−fluorophenyl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]urea (28). Step
1
1
. To a vial were added 3 (R = 4−fluorophenyl) (73 mg, 0.15 mmol), (R)−1−(1−phenylethyl)urea (37
mg, 0.23 mmol), Cs CO (120 mg, 0.367 mmol), BrettPhos precatalyst (11.7 mg, 0.015 mmol), and
2
3
DMA (1.1 mL). The mixture was evacuated and purged with nitrogen six times and heated to 100 °C for
5 min. The mixture was diluted with EtOAc, washed with water and brine, dried over sodium sulfate,
5
concentrated,
and
purified
by
flash
chromatography
to
afford
(R)−1−(3−(4−fluorophenyl)−1−trityl−1H−pyrazolo[4,3−c]pyridin−6−yl)−3−(1−phenylethyl)urea
(85
+
mg, 0.14 mmol, 92% yield) as a yellow solid. LRMS (ESI) calcd for (C H FN O) [M + H] 618,
40
32
5
1
found 618. H NMR (600 MHz, DMSO−d ) δ 9.06 (s, 1H), 8.97 (s, 1H), 7.22−7.96 (m, 25H), 6.80 (s,
6
1H), 4.79 (m, 1H), 1.35 (d, J = 7.2 Hz, 3H).
Step
2.
To
a
stirred
solution
of
(R)−1−(3−(4−fluorophenyl)−1−trityl−1H−pyrazolo[4,3−c]pyridin−6−yl)−3−(1−phenylethyl)urea
(61
mg, 0.10 mmol) in DCM (0.5 mL) were added TFA (0.5 mL) and triethylsilane (0.05 mL, 0.3 mmol).
The mixture was stirred at room temperature for 10 min, concentrated, and purified by
preparative−HPLC to afford 28 trifluoroacetate (40 mg, 0.083 mmol, 83% yield) as a yellow solid.
+
1
HRMS (ESI) calcd for (C H FN O) [M + H] 376.1573, found 376.1582. H NMR (600 MHz,
2
1
19
5
DMSO−d ) δ 13.24 (s, 1H), 9.12 (s, 1H), 9.06 (s, 1H), 8.04 (dd, J = 8.8, 5.5 Hz, 2H), 7.75 (s, 1H), 7.65
6
(s, 1H), 7.39−7.27 (m, 6H), 7.22−7.18 (m, 1H), 4.86 (m, 1H), 1.39 (d, J = 6.9 Hz, 3H).
1−(3−Cyclopropyl−1H−pyrazolo[4,3−c]pyridin−6−yl)−3−[(1R)−1−phenylethyl]urea (25). Prepared
+
in analogy to that described for 28 in 70% yield. HRMS (ESI) calcd for (C H N O) [M + H]
1
8
20
5
1
3
22.1668, found 322.1674. H NMR (600 MHz, DMSO−d ) δ 12.57 (s, 1H), 8.98 (s, 1H), 8.74 (s, 1H),
6
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Journal of Medicinal Chemistry
Page 26 of 35
7
.88 (brs, 1H), 7.53 (s, 1H), 7.22−7.35 (m, 5H), 4.88 (m, 1H), 2.28 (m, 1H), 1.41 (d, J = 8.4 Hz, 3H),
0.95−1.02 (m, 4H).
−[3−(1−Methyl−1H−pyrazol−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]ur
ea (27). Prepared in analogy to that described for 28 in 50% yield. HRMS (ESI) calcd for (C H N O)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
9
20
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
[
M + H] 362.1729, found 362.1730. H NMR (600 MHz, DMSO−d ) δ 12.91 (s, 1H), 9.06 (s, 1H),
6
9.02 (s, 1H), 8.44 (s, 1H), 8.01 (s, 1H), 7.86 (brs, 1H), 7.61 (s, 1H), 7.24−7.36 (m, 5H), 4.90 (m, 1H),
3
.92 (s, 3H), 1.42 (d, J = 8.4 Hz, 3H).
1
−[3−(2,6−Dimethylpyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]ure
a trifluoroacetate (29). Prepared in analogy to that described for 28 in 52% yield. HRMS (ESI) calcd
+
1
for (C H N O) [M + H] 387.1933, found 387.1946. H NMR (600 MHz, CD OD) δ 9.33 (brs, 1H),
2
2
23
6
3
8.23 (s, 2H), 7.51 (brs, 1H), 7.18−7.35 (m, 5H), 3.28 (m, 1H), 2.79 (s, 6H), 1.51 (s, 3H).
−[3−(2−Fluoropyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]urea
(29). Prepared in analogy to that described for 28 in 46% yield. HRMS (ESI) calcd for (C H FN O)
1
2
0
18
6
+
1
[
M + H] 377.1526, found 377.1531. H NMR (600 MHz, DMSO−d ) δ 13.66 (s, 1H), 9.27 (s, 1H),
6
9.16 (s, 1H), 8.36 (d, J = 6.0 Hz, 1H), 8.01 (d, J = 6.0 Hz, 1H), 7.79 (s, 1H), 7.75 (s, 1H), 7.70 (brs, 1H),
7
.23−7.36 (m, 5H), 4.90 (m, 1H), 1.42 (d, J = 8.4 Hz, 3H).
1
−[3−(2−Methoxypyridin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]urea
2
trifluoroacetate (31). To a vial were added 8 (R = (1R)−1−phenylethyl) (40 mg, 0.066 mmol),
(2−methoxypyridin−4−yl)boronic acid (21 mg, 0.14 mmol), PdCl (dppf)−CH Cl adduct (11 mg, 0.013
2
2
2
mmol), 1,4−dioxane (0.7 mL), and Na CO (2 M, 0.1 mL, 0.2 mmol). The mixture was purged with
2
3
nitrogen six times, heated to 80 °C for 3 h, and cooled to room temperature. The mixture was diluted
with EtOAc, washed with water and brine, dried over sodium sulfate, and concentrated. The residue was
dissolved in DCM (0.5 mL) and treated with TFA (0.5 mL) and triethylsilane (0.02 mL, 0.13 mmol).
The mixture was stirred at room temperature for 16 h, concentrated, and purified by preparative−HPLC
to afford 31 trifluoroacetate (14 mg, 0.028 mmol, 42% yield). HRMS (ESI) calcd for (C H N O ) [M
2
1
21
6
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Journal of Medicinal Chemistry
+
1
+
H] 389.1726, found 389.1736. H NMR (600 MHz, DMSO−d ) δ 13.58 (s, 1H), 9.24 (s, 1H), 9.19 (s,
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1H), 8.30 (d, J = 4.8 Hz, 1H), 7.75 (m, 2H), 7.63 (dd, J = 5.4, 1.2 Hz, 1H), 7.23−7.38 (m, 6H), 4.90 (m,
H), 3.93 (s, 3H), 1.43 (d, J = 7.2 Hz, 3H).
1
1−[3−(6−Methylpyridazin−4−yl)−1H−pyrazolo[4,3−c]pyridin−6−yl]−3−[(1R)−1−phenylethyl]urea
trifluoroacetate (32). Prepared in analogy to that described for 31 in 38% yield. HRMS (ESI) calcd for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
(
C H N O) [M + H] 374.1729, found 374.1729. H NMR (600 MHz, DMSO−d ) δ 13.79 (s, 1H),
20 20
7
6
9
.71 (s, 1H), 9.36 (s, 1H), 9.19 (s, 1H), 8.26 (s, 1H), 7.81 (s, 1H), 7.69 (brs, 1H), 7.25−7.36 (m, 5H),
.90 (m, 1H), 2.75 (s, 3H), 1.43 (d, J = 6.6 Hz, 3H).
4
hERK2 Assay. Activated ERK2 activity was determined in an IMAP−FP assay (Molecular Devices).
Using this assay format, the potency (IC ) of each compound was determined from a 10 point (1:3
50
serial dilution, 3 ꢀΜ starting compound concentration) titration curve using the following outlined
procedure. To each well of a black Corning 384−well plate (Corning Catalog #3575), 7.5 nL of
compound (3333 fold dilution in final assay volume of 25 ꢀL) was dispensed, followed by the addition
of 15 ꢀL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL
(0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound and enzyme
incubation, each reaction was initiated by the addition of 10 ꢀL kinase buffer containing 2.45 ꢀΜ ERK2
IMAP substrate peptides (2.25 ꢀΜ−unlabeled IPTTPITTTYFFFK−COOH and 200 nM−labeled
IPTTPITTTYFFFK−5FAM (5−carboxyfluorescein)−COOH ), and 75 ꢀΜ ATP. The final reaction in
each well of 25 ꢀL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled−peptide, and
30 ꢀΜ ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately
quenched by the addition of 60 ꢀL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer
(Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding
equilibration using Fluorescence Polarization protocol (Perkin Elmer).
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pRSK Assay. Phospho−RSK in a BRAF mutant melanoma cell A375SM was quantified using
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mesoscale electrochemiluminescent sandwich assay. The potency (IC ) of each compound was
5
0
determined from 11 points titration curve. Briefly, 10,000 cells were seeded into a 384 well plate and
o
incubated with compounds for 4 hours. Following removal of culture medium, cells were lysed at 4 C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for 20 minutes in 30 µL buffer containing 150 mM NaCl/20 mM Tris−HCl/1 mM EGTA/1 mM
EDTA/1% Triton X/10 mM NaF plus protease and phosphatase inhibitors. Twenty microliter of cell
lysates were transferred to a BSA−coated streptavidin−mesoscale plate. Biotin labeled anti−RSK1
(Enzo Life Science) and SULFO−TAG labeled anti−pRSK (pThr359/Ser363) (Epitomics) antibodies
were diluted in assay buffer (PBS/0.2% Tween 20/2% BSA), and 10 µL of the diluted antibodies were
o
added to the cell lysate. Following overnight incubation at 4 C, plates were washed 3 times with
PBS/0.05%Tween 20 buffer before read on MSD Sector S 600 in 1X MSD Read Buffer P.
Proliferation Assay. A375SM and CHL−1 cells suspended in DMEM/10 mM HEPES/10%
FBS/Hyclone SH30071.03 were added (40 ꢀL/well) to 384 Perkin Elmer Cultur plates subsequent to
o
compound addition. Plates were incubated at 37 C, 5% CO for 96 hours. After incubation plates were
2
equilibrated at room temperature for 15 minutes, proliferation was determined by addition of Cell
Titer−Glo (Promega G7573) using the reagent protocol. Plates were read with a Perkin Elmer
Spectrometer equipped with a luminescence protocol. Compound potency (IC ) was determined by
5
0
analysis of 10 point, 3−fold compound titration curves.
Supporting Information
Experimental procedures and spectral data for 2, 13, 14, 16, 19, 20, 22, 24, 26; kinome data for 11, 20,
1, 24, 25, 27−30; X−ray statistics for 11; PDB coordinates for docking poses of 9 and 10. This material
2
is available free of charge via the Internet at http://pubs.acs.org.
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Journal of Medicinal Chemistry
Accession Codes
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PDB accession code of the structure of 11 bound to rat ERK2 is 5KE0. Authors will release the atomic
coordinates and experimental data upon article publication.
Author Information
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Corresponding Author
*
Phone: 617−992−2012. E−mail: jongwon_lim@merck.com.
Notes
The authors declare no competing financial interest.
Acknowledgment
We thank Manami Shizuka for preparation of compound 21 for the in vivo studies, Melissa Chenard for
designing the in vivo studies, and Wilfredo Pinto for analytical spectra of key compounds.
Abbreviations Used
ATP, adenosine triphosphate; AUC, area under the curve; BrettPhos precatalyst,
chloro[2−(dicyclohexylphosphino)−3,6−dimethoxy−2',4',6'−triisopropyl−1,1'−biphenyl][2−(2−aminoeth
yl)phenyl]palladium(II);
CDI,
1,1′−carbonyldiimidazole;
Clp,
plasma
clearance;
dba,
dibenzylideneacetone; DPPA, phosphoric acid diphenyl ester azide; dppf, 1,1'− bis( diphenylphosphanyl)
ferrocene; ERK, extracellular signal−regulated kinase; hERG, human ether−a−go−go related gene; LE,
lignad efficiency; LiHMDS, lithium bis(trimethylsilyl)amide; MAPK, mitogen−activated protein kinase;
MW, molecular weight; PK, pharmacokinetic; SAR, structure−activity relationship; V , volume of
dss
distribution at steady state
References
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