Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development

Article abstract of DOI:10.1021/acs.jmedchem.6b00389

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes

Full text of DOI:10.1021/acs.jmedchem.6b00389

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Drug Annotation
Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-
(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-
one (GDC-0994), an Extracellular Signal-Regulated Kinase
1/2 (ERK1/2) Inhibitor in Early Clinical Development
James F Blake, Michael Burkard, Jocelyn Chan, Huifen Chen, Kang-Jye Chou, Dolores Diaz, Danette
A. Dudley, John J. Gaudino, Stephen E. Gould, Jonas Grina, Thomas Hunsaker, Lichuan Liu, Matthew
Martinson, David Moreno, Lars Mueller, Christine Orr, Patricia Pacheco, Ann Qin, Kevin Rasor,
Li Ren, Kirk Robarge, Sheerin K. Shahidi-Latham, Jeffrey Stults, Francis Sullivan, Weiru Wang,
Jianping Yin, Aihe Zhou, Marcia Belvin, Mark Merchant, John G. Moffat, and Jacob B. Schwarz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00389 • Publication Date (Web): 26 May 2016
Downloaded from http://pubs.acs.org on May 26, 2016
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Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-
2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-
yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one
(GDC-0994), an Extracellular Signal-Regulated
Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical
Development
James F. Blake1, Michael Burkard1, Jocelyn Chan2, Huifen Chen2, Kang-Jye Chou2, Dolores
Diaz2, Danette A. Dudley2, John J. Gaudino1, Stephen E. Gould2, Jonas Grina1, Thomas
Hunsaker2, Lichuan Liu2, Matthew Martinson1, David Moreno1, Lars Mueller2, Christine Orr2,
Patricia Pacheco2, Ann Qin2, Kevin Rasor1, Li Ren1, Kirk Robarge2, Sheerin Shahidi-Latham2,
Jeffrey Stults2, Francis Sullivan1, Weiru Wang2, Jianping Yin2, Aihe Zhou2, Marcia Belvin2, Mark
Merchant2, John Moffat2, and Jacob B. Schwarz2,*
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1Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301 USA
2Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 USA
ABSTRACT
The extracellular signalꢀregulated kinases ERK1/2 represent an essential node within the
RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in
BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF
and MEK inhibitors has proven effective clinically, resistance frequently develops through
reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as
MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for
acquired resistance. Described herein is the discovery and characterization of GDCꢀ0994 (22), an
orally bioavailable small molecule inhibitor selective for ERK kinase activity.
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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 oncogenic RAS and RAF mutations
as well as activation or overexpression of Growth Factor Receptors results in sustained activation
of the central effector kinases ERK1 and ERK2 (MAPK3 and MAPK1). ERK1/2 phosphorylates
more than 50 downstream substrates responsible for cell growth, proliferation, survival,
angiogenesis, and differentiation.2,3,4
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Small molecule inhibitors of BRAF and MEK have shown promising activities in the clinic for a
variety of solid tumors. The approval of vemurafenib,5 dabrafenib,6 trametinib,7 and
cobimetinib8 as treatments for BRAFꢀmutant metastatic melanoma validate the approach of
targeting the MAPK pathway as an effective way of treating cancer. However, these agents can
show limitations in duration of efficacy for some patients due to the acquisition of pathwayꢀ
reactivating mutations.9 Further, outside of this indication, the efficacy of these agents has been
disappointing, despite apparent MAPK pathway activation and clear therapeutic rationale in
BRAF or RAS mutant cancers. Negative feedback loops within the MAPK pathway enable
pathway reactivation when targeted with inhibitors of single nodes, driving innate insensitivity of
RAS mutant tumors to MEK inhibitors, and some BRAF mutant tumors (e.g. colorectal) to
BRAF or MEK inhibitors.10 Even when tumors respond to therapy, acquired resistance to
receptor tyrosine kinase (RTK) BRAF or MEK inhibitors can limit durable responses, many of
which involve bypass or reactivation of the drug target and restoration of persistent MAPK
activation resulting in high ERK activity.11
Downstream of MEK, the ERK kinases represent the final single node that would be expected to
suppress MAPK signaling. ERK1/2 are effector kinases for the MAPK pathway that are
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responsible for phosphorylating multiple targets to drive gene transcription and protein
translation to enable cell growth. Targeting ERK is therefore expected to directly suppress the
effector node of the MAPK pathway and potentially address acquired or innately resistant
tumors.12 ERK inhibitors could further combine with drugs targeting upstream nodes in the
MAPK pathway to drive deeper pathway suppression and reduce the incidence of acquired
resistance.13 Herein we report the optimization of preclinical potency, selectivity and
pharmacology parameters resulting in the discovery of (S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀ
hydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone (22),
which was advanced to human clinical trials.
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ERK has been a longstanding target in the pharmaceutical industry, and several highly optimized
inhibitors have been reported.14 One of the first examples of potent, selective ERK inhibitors
was a pyrroleꢀbased series from Vertex that appeared in 2007.15 A compound that emerged from
this work, BVDꢀ523 1, is currently being studied in early clinical development by Biomed
Valley Discoveries (Figure 1).16 Cyclized variants of 1, represented by 2, were recently
disclosed by researchers at Novartis.17 Merck reported in 2013 that the linear indazole 3
(SCH772984) displayed behaviors typical of both type I and type II kinase inhibitors.18 More
recently, covalent ERK2 inhibitors such as 4 have been disclosed by AstraZeneca.19
Figure 1. Highly optimized ERK inhibitors.
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We have previously reported the discovery of novel, potent and selective ERK1/2 inhibitors
based on piperidinopyrimidine20 and pyridone21 scaffolds, as typified by compounds 5 and 6.
While compound 6 in particular possessed a number of desirable attributes, some critical features
were identified for further optimization. The human dose projection for 6 based on PK/PD
modeling of the mouse xenograft data and allometric scaling was considered to be unacceptably
high (>1 gram/day),22 and therefore a strategy to reduce the predicted compound load was
developed. Two specific areas for optimization were targeted: improved potency and improved
exposure. For the former, recourse to structureꢀbased drug design was available for enhancing
potency against ERK1/2. For the latter, we initiated the investigation by considering the
metabolic fate of the lead pyridone 6.
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Compound 6 was incubated with human, mouse, dog, rat, and cynomolgus monkey hepatocytes
for 3 hours and the metabolite profile examined by LCꢀMS/MS (Figure 2). In all species
metabolism occurred primarily on the tetrahydropyran (THP) ring, and the major product was
determined to be hydroxy acid 7. In vivo metabolism studies with 6 in rat, dog, and monkey
confirmed 7 to be the primary circulating species in plasma as well as urine. Clearly,
substitution or outright replacement of the THP moiety would be key to any strategy to enhance
the metabolic stability. A number of saturated aliphatic and cyclic THP replacements of 6 were
reported previously,20 but unfortunately none of these resulted in improved metabolic stability.
Heteroaromatic replacements were considered to eliminate the methylene soft spots of 6.
Importantly, the hydrogen bond accepting nature of the THP oxygen atom was a key potency
driver for 6 and would need to be preserved in order to maintain the crucial hydrogen bond
interaction with Lys114 of ERK2, thereby considerably limiting the number of suitable candidate
motifs.
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Figure 2. UV traces from incubation of 6 with hepatocytes at t = 3 h (M3 = compound 7)
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ah = human, m = mouse, r = rat, d = dog, c = cynomolgus monkey. The most abundant metabolites M1ꢀM6 are
labeled. The first peak common to all five traces represents an internal standard.
THP replacement analogs were prepared by the methods previously described.20,23 Hence, TBSꢀ
protected sulfone 8 was subjected to SNAr displacement aided by deprotonation of or heating
with the appropriate amino heterocycle (Figure 3). Subsequent deprotection of the silyl
protecting group with TBAF or HCl in organic solvent afforded compounds 9ꢀ28 for evaluation.
The first THP replacement that was investigated was 2ꢀmethylpyridine 9. The pyridine nitrogen
atom was expected to form a hydrogen bond with the Lys114 side chain. The methyl group was
incorporated to thwart potential metabolism at the pyridyl nitrogen atom. Although the
mechanistic cell potency of the compound (inhibition of ERKꢀdependent p90RSK
phosphorylation, pRSK) was outstanding, the compound was a potent CYP3A4 inhibitor (IC50 =
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0.34 ꢀM). In addition, compound 9 showed high clearance and low bioavailability in rat,
thereby eliminating it from consideration for further development. Utilization of alternate
pyridine substitution patterns, or adding or reorganizing heteroatoms in the ring did not afford
any useful attenuation of CYP3A4 activity without concomitant loss in activity (data not shown).
As a result, we turned our attention toward THP replacement by fiveꢀmembered ring motifs.
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Figure 3. Synthesis of THP replacement analogs 9ꢀ28.
Conditions (a) for 4ꢀaminopyrazoles: i. HetꢀNH2, sꢀBuOH, 120 °C; ii. TBAF, THF. Conditions (b) for 5ꢀ
aminopyrazoles: i. NaH, DMF, rt or 150 °C; HCl, EtOAc.
The first replacement of the aminopyridine in 9 to be studied was 4ꢀaminopyrazole. Based on
molecular modeling, 4ꢀpyrazoles would present a hydrogen bond acceptor in an ideal orientation
to interact with the Lys114 side chain while also providing vectors suitable for substitution to
improve selectivity. A number of analogs with subtle alkyl modifications were synthesized and
tested (Table 1). Compounds were screened for enzyme potency against ERK1 and ERK2,
mechanistic potency in HepG2 cells, and metabolic stability in the presence of human
microsomes. The simple Nꢀmethylated pyrazole 10 showed enzyme and cell potency similar to
6, but adding a second methyl to the adjacent position as in 11 resulted in a sharp decline in
activity. However, if the second methyl was added to the other available carbon as in 12, not
only was potency restored but an enhancement of selectivity against the cell cycle kinase CDK2
was observed. CDK2 was chosen as the primary antiꢀtarget kinase to screen based on its close
homology in the binding site to ERK2 (vide infra) and the observation that it was consistently
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more strongly inhibited than other members of the CDK family in kinase selectivity profiling
(not shown). The enhanced selectivity of 12 against CDK2 may be attributed to the Cꢀ3 methyl
group which was predicted by docking to point toward the ERK2 Leu107 and insult the larger
Phe82 side chain in CDK2. Extension of the Cꢀmethyl group of 12 as in 13 resulted in loss of
potency due to steric clash with the protein, although extension of the Nꢀmethyl group (14) was
tolerated. Deletion of the Cꢀmethyl group of 14 as in 15 brought back potency but imparted loss
of selectivity, allowing for the conclusion that the Cꢀmethyl substituent was a key selectivity
driver. Bulking up the Nꢀsubstitution of 15 to isopropyl in 16 resulted in lower potency. In
contrast, when the Nꢀsubstituent of 12 was removed altogether, a potent and selective compound
17 was obtained. Having two Cꢀmethyl groups on the pyrazole as in 18 was not tolerated, which
was also the case for trifluoromethyl substituted 19. Noteworthy was the fact that the pyrazoles
did not demonstrate potent inhibition of the various CYP enzymes, unlike pyridine 9 (data not
shown).
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Table 1. Pyridine and 4ꢀaminopyrazole derivatives.
ERK1 IC50 ERK2 IC50
CDK2
pꢀRSK IC50 HLM CLhep
Cpd
R
nM
nM
/ERK2b
(nM)c
(mL/min/kg)d
(95% CI)a
(95% CI)
2.7
(2.4, 3.0)
1.1
(0.77, 1.6)
16
14
49
9
6
3.9
(3.2, 4.7)
0.94
(0.54, 1.6)
8
11
9
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7.9
4.1
14
14
54
28
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14
44
530
64
11
14
16
18
18
15
10
11
12
13
14
15
(7.3, 8.5)
(2.7, 5.9)
6
7
8
9
N
N
62
(53, 71)
29
(18, 47)
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11
12
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17
18
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13
(11, 15)
5.9
(4.5, 7.8)
43
(36, 50)
23
(19, 28)
230
100
34
18
(10, 30)
7.7
(2.9, 20)
5.0
(4.0, 6.1)
1.8
(0.92, 3.6)
19
(18, 21)
10
(5.5, 18)
9.5
127
17
16
N
N
3.8
(1.9, 7.6)
0.9
(0.7, 1.0)
65
38
9
17
18
510
240
3.5
5,000
12
(350, 720) (190, 290)
550
290
ꢀꢀ
3,500
8
19
(430, 710) (180, 470)
aEnzyme potency against ERK1 (n > 2 with 95% confidence interval). bRatio of CDK2 IC50 to ERK2 IC50.
cInhibition of ERK dependent p90RSK Serine 380 phosphorylation in PMAꢀstimulated HepG2 cells. dHuman
hepatic clearance predicted from liver microsomes.
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Given the similarity in vitro of compound 12 to the previous lead compound THP 6, a PK study
was performed in CDꢀ1 mice. Although the clearance was similar for both compounds
(extraction ratio ~ 30%), pyrazole 12 showed greater oral bioavailability (95% vs. 66% for 6),
rationalizing further investment in this series to increase potency. Armed with this promising
result, a series of substituted 5ꢀaminopyrazoles was investigated (Table 2). The SAR generally
tracked with the 4ꢀaminopyrazole series, but the direct comparator to 12 (compound 24) was
roughly twofold less potent in cells. There was, however, a clear standout from the series in
terms of cell potency which was Nꢀmethylpyrazole 22. It was tenfold more potent in cells than
the next most potent compound (26) in the series. It was also fivefold more potent in cells than
compound 12. In addition, it was moderately stable when incubated with human liver
microsomes. As a result we decided to more fully profile compound 22 as a potential
development candidate.
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Table 2. 5ꢀAminopyrazole derivatives.
ERK1 IC50
nM
ERK2 IC50
nM
(95% CI)
CDK2 pꢀRSK IC50 HLM CLhep
Cpd
R
/ERK2b
(nM)c
(mL/min/kg)d
(95% CI)a
9,610
(8,390,
10,990)
5,650
(4,270,
7,480)
ꢀꢀ
5,000
2
20
85
(67, 110)
37
(28, 49)
N
50
298
6
21
N
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3.1
25
107
20
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247
129
2500
132
5000
635
8
9
22
23
24
25
26
27
28
(5.0, 7.4)
(1.8, 5.5)
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7
8
9
26
(18, 37)
16
(8.9, 31)
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20
(17, 23)
13
(7, 25)
10
13
15
17
8
150
(110, 210)
89
(51, 150)
19
27
(26, 27)
17
(9.8, 28)
17
1,070
490
2.7
7.2
(580, 1,980) (430, 550)
74
(54, 100)
52
(36, 74)
aEnzyme potency against ERK1 (n > 2 with 95% confidence interval). bRatio of CDK2 IC50 to ERK2 IC50.
cInhibition of ERK dependent p90RSK Serine 380 phosphorylation in PMAꢀstimulated HepG2 cells. dHuman
hepatic clearance predicted from liver microsomes.
To fully characterize the binding interactions, we determined a coꢀcrystal structure of 22 and
ERK2. The overall binding mode is consistent with the predicted docking pose, largely
resembling that of 6 (PDB:4XJ0).21 The 2ꢀaminopyrimidine interacts with the hinge region of
ERK2 at Met108 and Leu107 by forming a pair of Hꢀbonds (Figure 4A). The pyridone carbonyl
makes water mediated Hꢀbonds with gatekeeper Gln105 and catalytic Lys54.
The
hydroxymethyl group is within Hꢀbond distance from the side chains of Asp167 and Asn154.
The fluoroꢀchlorophenyl terminal group binds to a hydrophobic pocket under the glycineꢀrichꢀ
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loop that involves the sidechain of Tyr36. The 5ꢀamimopyrazole accepts an Hꢀbond from
Lys114, providing the same interaction as the THP oxygen. Comparison with a crystal structure
of CDK2 in complex with 22 helped to illustrate the origin of selectivity within the pyrazole
series (Figure 4B).24 This improved selectivity against CDK2 may be attributed to the Nꢀmethyl
group which was predicted to point toward the Phe82 residue in CDK2, which is the counterpart
of a less sterically demanding Leu107 in ERK2. The Nꢀmethyl group on the pyrazole insults the
Phe82 side chain, but is better tolerated by ERK2.25 Additionally, the ERK2 gatekeeper residue
Gln105 (which is very rare among kinases) is replaced by Phe80 in CDK2 rendering CDK2
devoid of the polar interactions near the gatekeeper. The structure of 22 also shifts toward the
bulk solvent region in CDK2 structure relative to its position in ERK2, thereby compromising
the interactions around the hydroxymethyl linker and the fluoroꢀchlorophenyl terminal group.
This positional shift of 22 also affects its interaction with Lys89 of CDK2, the equivalent residue
to Lys114 in ERK2. The sidechain of Lys89 adopts a rotamer that avoids clashing into the
ligand, but loses the hydrogen bond with the 5ꢀaminopyrazole. We reason that these multiple
critical distinctions in 22 interactions with ERK2 versus CDK2 collectively contribute to the
overall selectivity.
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Figure 4. Crystal structures of 22 bound to ERK2 (brown) and CDK2 (purple).a
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A
B
Leu107
Met108
Phe82
Gln105
Phe80
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Lys54
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Asp167
Lys33
Tyr15
Asn154
Lys89
Lys114
Tyr36
aFigure A: Compound 22 bound to ERK2. Figure B: superposition of ERK2 and CDK2 coꢀcrystal structures with
compound 22. Red dotted lines = hydrogen bonds, red spheres = water molecules.
Next, the broad kinase selectivity of 22 was evaluated at 1 ꢀM against the Invitrogen
Selectscreen panel containing 279 kinases (Figure 5). The top hits were subjected to follow up
IC50 generation. For all seven kinases identified as potential selectivity flags, at least fiftyꢀfold
selectivity was observed relative to ERK1 IC50 (Table 3).
Figure 5. Activity of 22 against 279 kinases at 1 ꢀM.
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Table 3. Kinase selectivity of compound 22.
Kinase
PrKX
Fold selectivity vs ERK1
50
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GSK3α
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217
CHK2
HIPK4
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P38δ
Cdk2/CyA
Aurora A
Having identified compound 22 as having the best mix of potency and selectivity, we set out to
evaluate its DMPK properties. Compound 22 demonstrated kinetic solubility and permeability
in MDCK cells similar to previous lead compound 6 (29.7 ꢀg/mL, AB = 3.9 x 10ꢀ6 cm/s).
Taking into account that the free fraction in mouse plasma was determined to be 5.8%, the target
coverage for efficacy in a mouse xenograft study (pRSK IC50/fu) was calculated to be ca. 200
nM total concentration in plasma. In CDꢀ1 mice, a 10 mg/kg oral dose of 22 was sufficient to
achieve the desired target coverage for at least 8 hours (Figure 6).26 For comparison, a plasma
concentration of ca. 2 ꢀM for THP 6 would be required for 8 hours to achieve the same target
coverage, which was only achievable using a 60 mg/kg PO dose.21
Figure 6. Compound exposure vs. time in a multiꢀdose mouse PK study with compound 22.a
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aFormulated in 40% PEG400/ 60% (10%HPβCD) water.
Next, IV/oral crossover PK was surveyed in rat, dog, and cynomolgus monkey (Table 4).
Interestingly, the in vivo clearance measured across species was well predicted by in vitro
incubation with hepatocytes whereas liver microsomes tended to overꢀpredict the clearance for
compound 22. Higher clearance was observed exclusively in cynomolgus monkey (extraction
ratio ~ 80%). Crossꢀspecies metabolite identification in hepatocytes showed very little
metabolism except in cynomolgus monkey, where the major product was shown to be
glucuronidation (93% vs. 5% in human). With the exception of cynomolgus monkey, the in vivo
clearance values were significantly lower than those observed for compound 6. Given the high
predictive value of in vitro clearance hepatocytes for compound 22, clearance in human was
projected to be low (< 5 mL/min/kg).
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Table 4. Pharmacokinetic parameters of compound 22.
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6
7
8
In vitro predicted
CL
Liver microsomes
(mL/min/kg)
Hepatocytes
(mL/min/kg)
Rat
23
Mouse
48
Dog
15
Cyno
23
Human
7.9
9
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22
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14
19
5.4
35
4.7
PPB (fu)
0.044
0.058
0.098
0.056
0.080
In vivo PK
CLp (mL/min/kg)
15
1.8
2.0
35
19
1.6
1.3
39
6.4
2.1
6.8
112
3.8
31
2.5
2.4
15
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
V (L/kg)
ss
t
(hr)
1/2, iv
F (%)
AUC/D (kg*hr/L)
0.41
0.54
0.09
po
As a result of the promising PK data, 22 was progressed into in vivo PK/PD and efficacy studies.
The HCT116 human colorectal cancer xenograft model was selected as representative of KRASꢀ
mutant cancer, which is both a ‘higher bar’ and more representative of the likely clinical
indication than BRAFꢀmutant melanoma. A PK/PD study (15, 30, and 100 mg/kg, QD) was
carried out in nude mice bearing subcutaneous HCT116 tumors. Significant inhibition of pRSK
was observed at the 30 and 100 mg/kg doses, with the corresponding pRSK knockdown at the 2
h time point observed to be 65% and 87% (Figure 7). As was observed previously for
compound 6,21 there was an apparent rebound to above baseline levels in phosphorylation of
p90RSK at 16 and 24 hr for 22 (Figure 7). Pathway reactivation and rebound transiently above
basal levels correlate with decreased drug exposure and are consistent with rebound effects
observed with other MAPK inhibitors. Next, compound 22 was evaluated in a proofꢀofꢀconcept
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tumor growth inhibition (TGI) study. The compound was administered once daily (QD) orally
for 21 days in nude mice bearing HCT116 human colorectal cancer subcutaneously implanted
xenograft tumors (Figure 8). At doses greater than 30 mg/kg, there was significant tumor growth
inhibition with 30, 60 and 100 mg/kg resulting in 49%, 57%, and 80% tumor growth inhibition
(TGI), respectively. In comparison, efficacy with the MEK inhibitor, cobimetinib (GDCꢀ0973),
at a clinically relevant dose of 7.5 mg/kg (PO, QD) resulted in 58% TGI. There was no observed
body weight loss with treatment by 22 in this study.
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Figure 7. HCT116 study PK/PD analysis with compound 22.a
aPK/PD data for 22 in the HCT116 mouse xenograft model. HCT116 tumors were established in nude mice to a
tumor volume of 400ꢀ600 mm3. Mice were were treated with a single oral dose of 22 at 15, 30 or 100 mg/kg versus
vehicle control alone (40% PEG400/60% (10% HPβCD)) followed by tumor and plasma collection at 2, 8, 16 and
24 hr postꢀdose. Tumor levels of phosphorylated p90RSK (pRSK) relative total p90RSK (tRSK) were measured by
quantitative western blot and were normalized to vehicle control at 2 hr postꢀdose (set to 100%). Plasma and tumor
concentrations were measured by LC/MS.
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Figure 8. HCT116 mouse xenograft data with compound 22.
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Preclinical safety findings for compound 22 were consistent with those of other MEK and ERK
inhibitors, namely phosphorous dysregulation with soft tissue mineralization, decreased albumin
levels and skin toxicity in rats; and gastrointestinal toxicity in dogs.27 On the basis of the
favorable efficacy and tolerability profile reported herein, compound 22 (GDCꢀ0994) was
advanced into human clinical trials.
A firstꢀinꢀhuman, phase 1 dose escalation study was conducted in patients with locally advanced
or metastatic solid tumors. Compound 22 (mesylate) was administered orally once daily as
powder in capsule on a 21ꢀday on / 7 day off schedule. The plasma concentration vs. time PK
profiles of 50, 100, and 200 mg (3 patients each) at steady state are shown in Figure 9. In
general, exposures increased with dose from 50 to 200 mg.
Figure 9. Mean 22 (± SD) human PK concentrations at steady state by dose.
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In summary, targeting the RASꢀRAFꢀMEKꢀERK pathway has given rise to a number of FDAꢀ
approved medicines to treat various cancers. Inhibition of ERK1/2 has not been as well explored
as inhibition of RAFs and MEK. Nonetheless, targeting the ERK node in the MAPK pathway is
rationalized by ERKs acting as the effector kinases for the MAPK pathway and ERK
phosphorylation being a central feature in therapeutic resistance. The addition of potent and
selective ERK inhibitors further enables the rational targeting of multiple nodes in the MAPK
pathway in order to achieve maximal efficacy. Previously, we reported compound 6 to have
many desirable traits as an ERK inhibitor, but the projected human dose based on preclinical PK
and efficacy studies was unacceptably high. Hence, a campaign to lower the predicted dose
using a combination of improved potency and/or PK was launched. Replacement of the
metabolically labile THP ring of 6 with an Nꢀmethylpyrazole as in compound 22 led to a
compound requiring approximately tenfold lower total concentration and projected human dose
relative to 6 to achieve target coverage. On the basis of preclinical PK/PD and efficacy
experiments, coupled with a relatively safe profile in tolerability studies, compound 22 was
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advanced into human clinical trials. Human PK data with 22 support the predicted low clearance
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and feasibility of attaining target coverage at achievable doses.
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Experimental
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General Methods: Melting points were determined with a DSC Q100 instrument, and HRMS
with a Dionex LC Ultimate3000 and ThermoScientific Q Exactive orbitrap mass spectrometer.
Samples were prepared in a mixture of MeOH/acetonitrile/water (4:4:1 v/v/v) with 200 ꢀM
concentration for high resolution LCMS analysis. Samples of 5 ꢀL were injected into the LCMS
system and were analyzed by 10ꢀmin gradient HPLC and HRMS with electrospray ionization.
Bruker instruments (400 and 500 MHz) were used for NMR characterization. Compounds in
Table 1 were prepared by the method previously described for 6.21 All final compounds were
purified to >95% chemical purity, as assayed by HPLC (Waters Acquity UPLC column, 21 mm
× 50 mm, 1.7 ꢁm) with a gradient of 0−90% acetonitrile (containing 0.038% TFA) in 0.1%
aqueous TFA, with UV detection at λ = 254 and 210 nm, and with CAD detection with an ESA
Corona detector. Protein Expression, purification, and crystallization of CDK2 and ERK2 were
performed by the methods previously described.21,23
General procedure for preparation of compounds in Table 2: To a solution of compound 8 (1
equiv) and the pyrazole (2 equiv) in DMF (0.03 M) was added sodium hydride (2.5 equiv, 60%
dispersion in mineral oil). The mixture was stirred at room temperature or heated in a sealed
tube until the addition was complete. The mixture was partitioned between EtOAc and water,
the phases were separated, the aqueous phase was extracted with EtOAc, the combined organic
phases were dried (Na2SO4), and concentrated. To the residue in EtOAc (0.02 M) was added a
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saturated solution of HCl in EtOAc. The mixture was stirred until complete (ca. 15 min),
concentrated and purified by HPLC.
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(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(2ꢀmethylpyridinꢀ4ꢀylamino)pyrimidinꢀ
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1
4ꢀyl)pyridinꢀ2(1H)ꢀone 9. H NMR (400 MHz, DMSOꢀd6) δ 10.16 (s, 1H), 8.72 (d, J = 5.1 Hz,
1H), 8.26 (d, J = 5.6 Hz, 1H), 7.98 (d, J = 7.3 Hz, 1H), 7.61 (ddd, J = 16.2, 9.5, 4.4 Hz, 4H), 7.45
(dd, J = 10.6, 1.7 Hz, 1H), 7.20 (dd, J = 20.6, 5.1 Hz, 2H), 6.97 (dd, J = 7.3, 2.0 Hz, 1H), 5.99
(dd, J = 7.8, 5.6 Hz, 1H), 5.34 (s, 1H), 4.19 (dd, J = 11.7, 8.1 Hz, 1H), 4.05 (d, J = 5.6 Hz, 1H),
2.42 (s, 3H). m / z (APCIꢀpos) M + 1 = 452.2.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀ
1
ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 10. H NMR (400 MHz, DMSOꢀd6) δ 9.61 (s, 1H),
8.55 (d, J = 5.0 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.87 (s, 1H), 7.64 – 7.52 (m, 2H), 7.45 (dd, J =
10.6, 1.9 Hz, 1H), 7.31 (d, J = 5.1 Hz, 1H), 7.21 – 7.14 (m, 2H), 6.93 (dd, J = 7.3, 1.8 Hz, 1H),
5.99 (dd, J = 7.7, 5.5 Hz, 1H), 5.34 (t, J = 5.3 Hz, 1H), 4.23 – 4.13 (m, 1H), 4.10 – 4.01 (m, 1H),
3.82 (s, 3H). m / z (APCIꢀpos) M + 1 = 441.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(1,5ꢀdimethylꢀ1Hꢀpyrazolꢀ4ꢀ
1
ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 11. H NMR (400 MHz, DMSOꢀd6) δ 8.87 (s, 1H),
8.47 (d, J = 4.9 Hz, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.57 (dd, J = 17.1, 9.0 Hz, 2H), 7.49 – 7.38
(m, 1H), 7.27 (d, J = 5.1 Hz, 1H), 7.19 – 7.05 (m, 2H), 6.84 (t, J = 15.6 Hz, 1H), 5.97 (dd, J =
7.8, 5.6 Hz, 1H), 5.32 (t, J = 5.3 Hz, 1H), 4.15 (ddd, J = 13.4, 7.9, 5.7 Hz, 1H), 4.03 (dt, J = 21.4,
7.5 Hz, 1H), 3.71 (d, J = 9.7 Hz, 3H), 2.17 (d, J = 8.2 Hz, 3H). m / z (APCIꢀpos) M + 1 = 455.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(1,3ꢀdimethylꢀ1Hꢀpyrazolꢀ4ꢀ
1
ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 12. H NMR (400 MHz, DMSOꢀd6) δ 8.90 (s, 1H),
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8.50 (d, J = 5.1 Hz, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.80 (s, 1H), 7.64 – 7.53 (m, 1H), 7.43 (dt, J =
19.1, 9.5 Hz, 1H), 7.28 (dd, J = 9.7, 5.1 Hz, 1H), 7.21 – 7.07 (m, 2H), 6.89 (d, J = 5.9 Hz, 1H),
6.04 – 5.92 (m, 1H), 5.34 (t, J = 5.2 Hz, 1H), 4.24 – 4.10 (m, 1H), 4.10 – 3.97 (m, 1H), 3.80 –
3.70 (m, 3H), 2.11 (s, 3H). m / z (APCIꢀpos) M + 1 = 455.1.
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(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(3ꢀethylꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀ
1
ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 13. H NMR (400 MHz, DMSOꢀd6) δ 8.85 (s, 1H),
8.48 (t, J = 8.0 Hz, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.49 –
7.38 (m, 1H), 7.27 (t, J = 5.6 Hz, 1H), 7.21 – 7.05 (m, 2H), 6.88 (d, J = 6.6 Hz, 1H), 5.98 (dd, J
= 7.8, 5.5 Hz, 1H), 5.33 (t, J = 5.2 Hz, 1H), 4.16 (ddd, J = 13.4, 7.9, 5.6 Hz, 1H), 4.11 – 3.96 (m,
1H), 3.75 (d, J = 3.9 Hz, 3H), 2.54 (dd, J = 15.1, 7.5 Hz, 2H), 1.11 (dd, J = 8.8, 6.3 Hz, 3H). m /
z (APCIꢀpos) M + 1 = 469.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(1ꢀethylꢀ3ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀ
1
ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 14. H NMR (400 MHz, DMSOꢀd6) δ 8.89 (s, 1H),
8.50 (d, J = 5.1 Hz, 1H), 7.91 (t, J = 5.3 Hz, 1H), 7.84 (s, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.43 (dd,
J = 10.6, 1.9 Hz, 1H), 7.28 (d, J = 5.1 Hz, 1H), 7.15 (dd, J = 9.6, 7.9 Hz, 2H), 6.88 (d, J = 6.6
Hz, 1H), 5.97 (dd, J = 7.8, 5.4 Hz, 1H), 5.32 (t, J = 5.1 Hz, 1H), 4.21 – 4.12 (m, 1H), 4.06 – 3.99
(m, 3H), 2.12 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). m / z (APCIꢀpos) M + 1 = 469.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(1ꢀethylꢀ1Hꢀpyrazolꢀ4ꢀ
1
ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 15. H NMR (400 MHz, DMSOꢀd6) δ 9.59 (s, 1H),
8.62 – 8.51 (m, 1H), 7.97 – 7.88 (m, 2H), 7.59 (t, J = 8.1 Hz, 2H), 7.45 (dd, J = 10.5, 1.6 Hz,
1H), 7.30 (d, J = 5.1 Hz, 1H), 7.16 (t, J = 5.0 Hz, 2H), 6.93 (dd, J = 7.2, 1.6 Hz, 1H), 6.02 – 5.96
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(m, 1H), 5.33 (s, 1H), 4.23 – 4.02 (m, 4H), 1.36 (t, J = 7.3 Hz, 3H). m / z (APCIꢀpos) M + 1 =
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455.1.
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ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 16. H NMR (400 MHz, DMSOꢀd6) δ 9.58 (s, 1H),
8.55 (d, J = 5.0 Hz, 1H), 7.96 – 7.89 (m, 2H), 7.59 (t, J = 8.1 Hz, 2H), 7.45 (dd, J = 10.6, 1.7 Hz,
1H), 7.30 (d, J = 5.1 Hz, 1H), 7.22 – 7.13 (m, 2H), 6.92 (dd, J = 7.3, 1.8 Hz, 1H), 6.04 – 5.94 (m,
1H), 5.33 (t, J = 5.2 Hz, 1H), 4.46 (dt, J = 13.3, 6.6 Hz, 1H), 4.21 – 4.13 (m, 1H), 4.10 – 4.02
(m, 1H), 1.41 (d, J = 6.7 Hz, 6H). m / z (APCIꢀpos) M + 1 = 469.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(3ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀ
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ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 17. H NMR (400 MHz, DMSOꢀd6) δ 12.28 (d, J =
50.5 Hz, 1H), 8.87 (s, 1H), 8.49 (s, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.65 (dd, J = 64.2, 56.0 Hz,
2H), 7.43 (dd, J = 10.6, 2.0 Hz, 1H), 7.27 (d, J = 5.1 Hz, 1H), 7.21 – 7.06 (m, 2H), 6.87 (d, J =
6.6 Hz, 1H), 5.98 (dd, J = 7.7, 5.5 Hz, 1H), 5.32 (t, J = 5.3 Hz, 1H), 4.23 – 4.11 (m, 1H), 4.11 –
4.00 (m, 1H), 2.16 (s, 3H). m / z (APCIꢀpos) M + 1 = 444.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ(3,5ꢀdimethylꢀ1Hꢀpyrazolꢀ4ꢀ
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ylamino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 18. H NMR (400 MHz, DMSOꢀd6) δ 12.04 (s, 1H),
8.48 (d, J = 34.0 Hz, 2H), 7.88 (d, J = 6.9 Hz, 1H), 7.57 (t, J = 8.1 Hz, 1H), 7.42 (dd, J = 10.5,
1.9 Hz, 1H), 7.24 (d, J = 5.1 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.04 (s, 1H), 6.79 (s, 1H), 6.01 –
5.91 (m, 1H), 5.31 (t, J = 5.2 Hz, 1H), 4.14 (s, 1H), 4.08 – 3.99 (m, 1H), 2.13 – 1.93 (m, 6H). m
/ z (APCIꢀpos) M + 1 = 455.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀmethylꢀ3ꢀ(trifluoromethyl)ꢀ1Hꢀ
pyrazolꢀ4ꢀyl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 19. 1H NMR (400 MHz, DMSOꢀd6) δ
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8.90 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.07 (d, J = 10.3 Hz, 1H), 7.89 (d, J = 7.3 Hz, 1H), 7.57 (t,
J = 8.1 Hz, 1H), 7.42 (ddd, J = 23.6, 22.8, 8.8 Hz, 2H), 7.23 – 7.03 (m, 2H), 6.84 (t, J = 5.7 Hz,
1H), 5.98 (dd, J = 12.8, 7.3 Hz, 1H), 5.31 (s, 1H), 4.14 (d, J = 8.3 Hz, 1H), 4.05 (s, 1H), 3.97 –
3.85 (m, 3H). m / z (APCIꢀpos) M + 1 = 509.1.
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(S)ꢀ4ꢀ(2ꢀ((1Hꢀpyrazolꢀ5ꢀyl)amino)pyrimidinꢀ4ꢀyl)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀ
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hydroxyethyl)pyridinꢀ2(1H)ꢀone 20. H NMR (400 MHz, DMSOꢀd6) δ 8.95 (d, J = 5.2 Hz, 1H),
8.06 – 7.91 (m, 2H), 7.59 (t, J = 8.1 Hz, 1H), 7.44 (dd, J = 9.2, 6.1 Hz, 2H), 7.25 (d, J = 1.9 Hz,
1H), 7.18 (d, J = 8.4 Hz, 1H), 6.97 (dd, J = 7.3, 1.9 Hz, 1H), 6.72 (s, 2H), 6.04 – 5.94 (m, 1H),
5.46 (d, J = 1.6 Hz, 1H), 5.34 (t, J = 5.2 Hz, 1H), 4.24 – 4.12 (m, 1H), 4.11 – 4.00 (m, 1H). m / z
(APCIꢀpos) M + 1 = 427.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀmethylꢀ1Hꢀpyrazolꢀ3ꢀ
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yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 21. H NMR (400 MHz, DMSOꢀd6) δ 9.90 (s, 1H),
8.56 (d, J = 5.1 Hz, 1H), 7.92 (t, J = 9.9 Hz, 1H), 7.63 – 7.54 (m, 2H), 7.45 (dd, J = 10.6, 2.0 Hz,
1H), 7.37 (d, J = 5.1 Hz, 1H), 7.17 (dt, J = 4.9, 2.4 Hz, 2H), 6.92 (dd, J = 7.3, 2.0 Hz, 1H), 6.62
(d, J = 2.2 Hz, 1H), 5.99 (dd, J = 7.9, 5.5 Hz, 1H), 5.36 – 5.29 (m, 1H), 4.23 – 4.12 (m, 1H), 4.10
– 4.01 (m, 1H), 3.76 (s, 3H). m / z (APCIꢀpos) M + 1 = 444.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀ
23
yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 22, mp = 304.4 °C. [α]D +113.8 (c 0.29, MeOH);
1
IR 1660, 1582, 1557 cmꢀ1; H NMR (500 MHz, DMSOꢀd6) δ 9.69 – 9.52 (s, 1H), 8.69 – 8.49
(d, J = 5.1 Hz, 1H), 8.09 – 7.77 (d, J = 7.3 Hz, 1H), 7.61 – 7.56 (t, J = 8.1 Hz, 1H), 7.50 – 7.47
(d, J = 5.1 Hz, 1H), 7.46 – 7.42 (dd, J = 10.6, 2.0 Hz, 1H), 7.38 – 7.36 (d, J = 1.9 Hz, 1H), 7.22 –
7.08 (m, 2H), 6.98 – 6.79 (dd, J = 7.3, 2.1 Hz, 1H), 6.35 – 6.23 (d, J = 1.9 Hz, 1H), 6.06 – 5.90
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(dd, J = 8.1, 5.4 Hz, 1H), 5.40 – 5.23 (d, J = 5.2 Hz, 1H), 4.24 – 3.97 (m, 2H), 3.80 – 3.56 (s,
3H); 13C NMR (101 MHz, DMSOꢀd6) δ 162.18, 161.53, 160.88, 160.55, 157.59 (d, JCF = 246.83
Hz), 147.19, 140.09 (d, JCF = 6.47 Hz), 138.30, 137.75, 137.42, 131.20, 125.53 (d, JCF = 3.45
Hz), 119.29 (d, JCF = 17.45 Hz), 117.74, 116.61 (d, JCF = 21.68 Hz), 109.68, 103.34, 99.36,
61.24, 59.20, 35.93. HRMS (ESI): m / z [M + H]+ calcd. for C21H18ClFN6O2: 441.1242; found:
441.1230.
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(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀethylꢀ1Hꢀpyrazolꢀ5ꢀ
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yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 23. H NMR (500 MHz, DMSOꢀd6) δ 8.64 – 8.52
(d, J = 5.1 Hz, 1H), 7.98 – 7.87 (d, J = 7.4 Hz, 1H), 7.66 – 7.52 (t, J = 8.1 Hz, 1H), 7.47 – 7.45
(d, J = 5.1 Hz, 1H), 7.45 – 7.41 (dd, J = 10.5, 2.0 Hz, 1H), 7.40 – 7.38 (d, J = 1.9 Hz, 1H), 7.21 –
7.05 (m, 2H), 6.92 – 6.77 (dd, J = 7.3, 2.0 Hz, 1H), 6.33 – 6.18 (d, J = 1.8 Hz, 1H), 6.03 – 5.90
(dd, J = 8.0, 5.5 Hz, 1H), 4.24 – 4.07 (dd, J = 11.8, 8.2 Hz, 1H), 4.11 – 3.93 (q, J = 7.0 Hz, 3H),
1.41 – 1.13 (t, J = 7.2 Hz, 3H). m / z (APCIꢀpos) M + 1 = 455.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1,3ꢀdimethylꢀ1Hꢀpyrazolꢀ5ꢀ
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yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 24. H NMR (400 MHz, DMSOꢀd6) δ 9.54 (s, 1H),
8.59 (d, J = 5.1 Hz, 1H), 7.92 (t, J = 9.6 Hz, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.45 (dd, J = 11.5, 7.9
Hz, 2H), 7.20 – 7.08 (m, 2H), 6.87 (d, J = 7.3 Hz, 1H), 6.04 (s, 1H), 6.02 – 5.92 (m, 1H), 5.34 (t,
J = 5.2 Hz, 1H), 4.24 – 4.10 (m, 1H), 4.10 – 4.00 (m, 1H), 3.59 (s, 3H), 2.12 (s, 3H). m / z
(APCIꢀpos) M + 1 = 455.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀethylꢀ3ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀ
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yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 25. H NMR (400 MHz, DMSOꢀd6) δ 9.44 (s, 1H),
8.57 (d, J = 5.1 Hz, 1H), 7.92 (d, J = 7.3 Hz, 1H), 7.58 (t, J = 8.1 Hz, 1H), 7.49 – 7.37 (m, 2H),
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7.14 (dd, J = 13.4, 4.8 Hz, 2H), 6.86 (d, J = 7.2 Hz, 1H), 6.03 (s, 1H), 6.00 – 5.90 (m, 1H), 5.31
(t, J = 5.2 Hz, 1H), 4.22 – 4.09 (m, 1H), 4.08 – 3.99 (m, 1H), 3.94 (q, J = 7.2 Hz, 2H), 2.14 (s,
3H), 1.25 (t, J = 7.2 Hz, 3H). m / z (APCIꢀpos) M + 1 = 469.1.
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(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((3ꢀethylꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀ
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yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 26. H NMR (400 MHz, DMSOꢀd6) δ 9.48 (s, 1H),
8.58 (d, J = 5.1 Hz, 1H), 7.92 (d, J = 7.3 Hz, 1H), 7.57 (t, J = 8.1 Hz, 1H), 7.49 – 7.37 (m, 2H),
7.15 (dd, J = 8.6, 6.0 Hz, 2H), 6.86 (dd, J = 7.3, 1.9 Hz, 1H), 6.07 (s, 1H), 5.97 (dd, J = 7.8, 5.6
Hz, 1H), 5.30 (s, 1H), 4.16 (s, 1H), 4.04 (d, J = 11.9 Hz, 1H), 3.60 (s, 3H), 2.52 (d, J = 6.1 Hz,
1H), 2.47 (s, 1H), 1.16 (t, J = 7.6 Hz, 3H). m / z (APCIꢀpos) M + 1 = 469.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1,4ꢀdimethylꢀ1Hꢀpyrazolꢀ5ꢀ
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yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 27. H NMR (400 MHz, DMSOꢀd6) δ 9.21 (s, 1H),
8.54 (d, J = 5.1 Hz, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.57 (t, J = 8.1 Hz, 1H), 7.41 (dd, J = 7.8, 3.4
Hz, 2H), 7.25 (s, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.07 (s, 1H), 6.79 (s, 1H), 6.02 – 5.88 (m, 1H),
5.30 (t, J = 5.1 Hz, 1H), 4.14 (dd, J = 15.2, 9.9 Hz, 1H), 4.08 – 3.96 (m, 1H), 3.58 (s, 3H), 1.83
(s, 3H). m / z (APCIꢀpos) M + 1 = 455.1.
(S)ꢀ1ꢀ(1ꢀ(4ꢀchloroꢀ3ꢀfluorophenyl)ꢀ2ꢀhydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀmethylꢀ3ꢀ(trifluoromethyl)ꢀ1Hꢀ
pyrazolꢀ5ꢀyl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone 28. 1H NMR (400 MHz, DMSOꢀd6) δ
9.90 (s, 1H), 8.65 (d, J = 5.1 Hz, 1H), 7.94 (d, J = 7.3 Hz, 1H), 7.57 (dd, J = 15.4, 6.7 Hz, 2H),
7.43 (dd, J = 10.6, 1.9 Hz, 1H), 7.16 (dd, J = 9.7, 1.6 Hz, 2H), 6.86 (dd, J = 7.3, 1.9 Hz, 1H),
6.74 (s, 1H), 5.97 (dd, J = 7.8, 5.6 Hz, 1H), 5.30 (t, J = 5.2 Hz, 1H), 4.17 (ddd, J = 13.4, 8.0, 5.7
Hz, 1H), 4.09 – 3.97 (m, 1H), 3.80 (s, 3H). m / z (APCIꢀpos) M + 1 = 509.1.
Supporting Information
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Experimental procedures for the crystal structure determination of ERK2 and CDK2 complexes
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Corresponding Author Information: 650ꢀ225ꢀ7732, schwarz.jacob@gene.com.
Abbreviations Used: MAPK, mitogenꢀactivated protein kinase; LCꢀMS, liquid
chromatographyꢀmass spectrometry; TBAF, tetrabutylammonium fluoride; RSK, ribosomal s6
kinase; CYP, cytochrome P450; CDK, cyclinꢀdependent kinase; PK, pharmacokinetic; PD,
pharmacodynamics; SAR, structureꢀactivity relationship; MDCK, MadinꢀDarby canine kidney;
PO, per os (by mouth); IV, intravenous; HCT, human colorectal carcinoma; DMF,
dimethylformamide; EtOAc, ethyl acetate; MeOH, methanol.
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