Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2

Article abstract of DOI:10.1021/acs.jmedchem.1c00905

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Full text of DOI:10.1021/acs.jmedchem.1c00905

pubs.acs.org/jmc
Article
Discovery of ASTX029, A Clinical Candidate Which Modulates the
Phosphorylation and Catalytic Activity of ERK1/2
Tom D. Heightman, Valerio Berdini, Luke Bevan, Ildiko M. Buck, Maria G. Carr, Aurélie Courtin,
Joseph E. Coyle, James E. H. Day, Charlotte East, Lynsey Fazal, Charlotte M. Griffiths-Jones,
Steven Howard, Justyna Kucia-Tran, Vanessa Martins, Sandra Muench, Joanne M. Munck,
David Norton, Marc O’Reilly, Nicholas Palmer, Puja Pathuri, Torren M. Peakman, Michael Reader,
David C. Rees, Sharna J. Rich, Alpesh Shah, Nicola G. Wallis, Hugh Walton, Nicola E. Wilsher,
Alison J.-A. Woolford, Michael Cooke, David Cousin, Stuart Onions, Jonathan Shannon, John Watts,
and Christopher W. Murray*
Cite This: J. Med. Chem. 2021, 64, 12286−12303
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: Aberrant activation of the mitogen-activated protein
kinase pathway frequently drives tumor growth, and the ERK1/2
kinases are positioned at a key node in this pathway, making them
important targets for therapeutic intervention. Recently, a number of
ERK1/2 inhibitors have been advanced to investigational clinical trials
in patients with activating mutations in B-Raf proto-oncogene or Ras.
Here, we describe the discovery of the clinical candidate ASTX029
(15) through structure-guided optimization of our previously published
isoindolinone lead (7). The medicinal chemistry campaign focused on
addressing CYP3A4-mediated metabolism and maintaining favorable
physicochemical properties. These efforts led to the identification of
ASTX029, which showed the desired pharmacological profile
combining ERK1/2 inhibition with suppression of phospho-ERK1/2
(
pERK) levels, and in addition, it possesses suitable preclinical
pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I−II clinical trial in
patients with advanced solid tumors.
INTRODUCTION
upstream pathway proteins and upregulation of expression of
MAP kinase phosphatases (DUSPs).
Anticancer drugs targeting the MAPK pathway have shown
promising success, including inhibitors of B-Raf proto-
■
(
4
Activation of the Ras-RAF-MEK-ERK signaling pathway
mitogen-activated protein kinase (MAPK) pathway) is one
of the major mechanisms driving tumor growth, occurring
through activating mutations in upstream growth factor
oncogene (BRAF) and mitogen activated protein kinase
5,6
(
MEK) which are approved for mutant BRAF melanomas.
1
receptors, in the GTPase Ras, or in RAF kinases. Activation
However, the breadth and duration of efficacy of these agents
are limited through both innate and drug-induced resistance
mechanisms that release negative feedback loops to increase
of RAF by these mechanisms triggers phosphorylation of
MEK1 and 2, which in turn phosphorylates the activation loop
of the extracellular related kinases ERK1 and 2 (also known as
mitogen activated protein kinases MAPK1 and 2) on the
7
activation of ERK1/2. This has provided strong motivation to
seek selective ERK1/2 inhibitors which might show improved
efficacy alone or in combination with agents targeting other
pathway components.
2
threonine and tyrosine residues in a TEY motif. Activation of
ERK1 and 2 leads to not only phosphorylation of substrates in
the cytoplasm such as ribosomal S6 kinase (RSK) but also
promotes nuclear translocation leading to activation of
transcription factors regulating expression of numerous genes
Received: May 19, 2021
Published: August 13, 2021
3
involved in cell cycle progression. The ERK1/2 kinases hence
form a particularly important node in the signaling pathway
and are subject to tight regulation through negative feedback
mechanisms including direct inhibitory phosphorylation of
©
2021 American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00905
1
2286
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. ERK1/2 inhibitors in clinical trials with published structures.
The last decade has seen the development of a variety of
RESULTS AND DISCUSSION
■
ERK1/2 inhibitors with promising preclinical efficacy that have
Metabolic Studies on the Lead Compound. In the
preceding paper, we characterized the lead 7 as a highly potent
and selective inhibitor of ERK kinase activity and a modulator
of ERK phosphorylation (pERK), with an IC50 of 3.5 nM
8
been progressed into clinical trials. Those with disclosed
9
structures (Figure 1) include ulixertinib (BVD-523, 1),
10
11
ravoxertinib (GDC-0994, 2), ATG-017 (also known as
1
2
13
AZD0364, 3), and LY3214996 (4), which are classical type
21
1
4
versus pRSK and 2.3 nM versus pERK in A375 cells.
1
inhibitors of ERK1/2 catalytic activity; CC-90003 (5),
15
Compound 7 showed promising pharmacokinetics in mice,
with low in vivo clearance of 13.2 mL/min/kg and oral
bioavailability of 59%, which translated to efficacy in mutant
BRAF Colo205 xenograft studies at either 50 mg/kg once daily
or 25 mg/kg twice daily. However, despite this promising
profile, detailed metabolic studies demonstrated oxidative
metabolism of compound 7 mediated by CYP3A4 with a
short in vitro half-life (<5 min). We considered this result to
present a potential risk of first pass metabolism, limiting oral
which is type 1 covalent inhibitor; and MK-8353 (6), which
not only inhibits ERK catalytic activity but also profoundly
suppresses ERK1/2 phosphorylation by MEK. Additional
1
6
17
18
clinical compounds include KO-947, LTT462, ASN007,
1
9
and HH2710 for which structures are yet to be disclosed.
We and others hypothesized that inhibitors with a dual-
mechanism blocking phosphorylation of ERK1/2 and its
catalytic activity might have potential to show greater efficacy
than type 1 inhibitors, especially in cells with more strongly
activated negative pathway loops such as BRAF or MEK
2
3
exposure in humans. Identification of metabolites of
compound 7 present in rat bile using LC/MS/MS indicated
significant levels of oxidative metabolism at both ends of the
molecule, on the tolyl methyl group and the oxan (THP) ring
20
inhibitor-resistant mutant BRAF cells and mutant Ras cells.
We recently described the fragment-based discovery of the
isoindolinone lead 7 (Table 1, compound 27 in ref 21),
representing a novel series of ERK1/2 inhibitors with
promising oral pharmacokinetics and showing dual inhibition
of ERK1/2 catalytic activity and its activation through
phosphorylation by MEK. In this paper, we disclose our lead
optimization strategy, focusing on enhancing the predicted
pharmacokinetics in humans and culminating in the discovery
of the clinical candidate ASTX029 (compound 15). Detailed
pharmacological profiling of ASTX029 has been published
(
data not shown). An optimization strategy was therefore set,
making changes to reduce the metabolism by directly blocking
the vulnerable sites with replacement for more stable isosteres,
by conformational rigidification, and by modulating the overall
physicochemical properties of the compounds. In vitro assays
used in the screening cascade are described in the
Experimental Procedures section and included an ERK2
biochemical assay (time-resolved fluorescence, TRF), anti-
proliferative cellular assays (BRAF mutant A375 and/or
2
2
elsewhere.
1
2287
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 1. Biochemical and Cellular SARs for Benzylamine Analogues in the Benzolactam ERK1/2 Inhibitor Series
Colo205 cells), microsomal intrinsic clearance (mouse and
human), and CYP3A4 phenotyping, together with physi-
ochemical assays including ChromLogD7.4 and kinetic
solubility (NMR). Selected compounds were also assessed
for pRSK and pERK IC s (mesoscale discovery (MSD) and
ELISA assays in A375 cells, respectively), low-dose mouse PK,
PD, and efficacy studies.
Structure−Activity Relationship Studies to Modulate
the Metabolic Profile. Structure-based drug design can be
used to narrow down the number of plausible options in lead
50
1
2288
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. (a) X-ray crystal structure of lead compound 7 in complex with ERK2 (PDB code: 6G9N). Filling both the secondary pocket (upper part
of the figure) and the hinge region (lower part of the figure) was believed to be important for the desired pharmacology. (b) Chemical structure of
compound 7 indicating potential sites for chemical modification that might modulate the metabolic profile without affecting the binding mode.
Figure 3. X-ray bound (PDB code: 6G9K) and solution conformations of the unsubstituted phenyl analogue 8.
optimization, and the crystal structure of the lead 7 in complex
with ERK2 showed a number of potential sites for chemical
modification to modulate the metabolic profile, while
maintaining potency (Figure 2). For example, the metabolic
studies discussed above suggested replacements of the oxan
ring. The metabolic studies also motivated modifications to the
tolyl group by alternative substitutions of the ring and
heteroatom insertion or annulation of the secondary amide
and/or benzylic carbon. Additionally, the overall lipophilicity
of the molecule could be modified by substitution of the
acetamide linker or replacement of the chlorine substituent on
the aminopyrimidine. Finally, it was hypothesized that the
isoindoline might prove to be a metabolic soft spot, so
substitution or heteroatom insertion to the 3- or 4-positions of
the isoindoline core was explored.
was required to elicit the desired dual pharmacological profile.
This approach necessarily drives toward inhibitors with
molecular weight in excess of 500 Da, creating challenges in
achieving suitable physicochemical properties for good oral
exposure. Hence, before embarking on broad-ranging sub-
stituent structure−activity relationship (SAR) studies, we
explored whether there might be opportunities for conforma-
tional rigidification of the series in order to make potency gains
without significant addition of molecular weight. Detailed
NMR and computational studies on the unsubstituted
analogue 8 suggested a largely extended conformation that
was similar to the X-ray bound conformation (Figure 3). The
acetamide linker showed a dominant preference for a trans
conformation in solution, evidenced by a strong NOE between
CH (23) and NH(28). However, the weak NOE correlation
2
A key element in the optimization strategy was the desire to
keep the molecular weight of analogues to a minimum. As
described previously, our hypothesis was that occupation of
both the hinge region and the secondary pocket (Figure 2a)
between NH(28) and the benzylic CH(29) and J−J couplings
of ∼6 Hz suggested that the hydroxymethylbenzyl region of 8
underwent more or less free rotation in solution. This
prompted us to explore rigidification of this part of the
1
2289
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
molecule, guided by the X-ray crystal structures of the ERK2
complexes with compounds 7 and 8. Among the approaches
attempted, annulating the benzylic carbon to the phenyl ring at
the relative ortho-position to give the indane 9 was well
tolerated in terms of potency (Table 1). An overlay of the
crystal structure of the ERK2 complex of 9 with that of 8
showed an almost identical bound conformation, ratifying the
design (Figure 4b); however, the indane 9 showed no benefit
conformation of Tyr36, while Tyr36 moved into the undesired
“out” conformation (Figure 4d).
Next, we explored the effects of substitution and/or
heteroatom insertion to the terminal phenyl of compound 7.
As mentioned above, metabolic studies identified the methyl
group as a potential site of oxidative metabolism. Replacement
of the methyl group with methoxy (compound 14) was
tolerated in terms of potency but gave no advantage in
metabolic stability in the CYP3A4 assay (Table 1). The crystal
structure of the 14-ERK2 complex showed space for a small
substituent in the 5-position such as a fluoro, leading to the
design of compound 15. Potency was maintained in this 3,5-
disubstituted analogue, as well as observing improved CYP3A4
metabolic stability, with a half-life of 35 min (Table 1). The
22
crystal structure of the 15-ERK2 complex confirmed the
designed binding mode, in which the methoxy group contacted
the aryl side chain of Tyr64 and the fluoro group occupied a
small space between methylenes in the side chains of Tyr64
and Arg67, forming a close contact (2.9 Å) with the hydroxyl
side chain of Thr68 (Figure 5a). A wide range of additional
monosubstituted and disubstituted analogues were also
prepared, but generally showed less promising stability in the
CYP3A4 assay, exemplified by the 3,5-difluoro analogue 16
with a half-life of less than 5 min. Incorporation of a
heteroatom was tolerated, as exemplified by the methoxypyr-
idine 17, which showed a potency of 18 nM in A375 cells and
a modest half-life of 15 min in the CYP3A4 assay. The crystal
structure of the 17-ERK2 complex showed a similar binding
mode to 16, although Tyr64 shifted away from the
methoxypyridine ring presumably to avoid an unfavorable
interaction between the nitrogen lone pair of 17 and the
electron-rich pi system of Tyr64 (Figure 5b). Noting that the
3
-position of this terminal aryl ring provided a vector toward
the solvent, we finally explored the effects of appending basic
groups to modulate physicochemical properties. Attachment of
a dimethylaminoethoxy group to the ring (compound 18)
attenuated the cellular potency while bringing no advantage in
terms of metabolic stability (Table 1). The phenylpiperazine
Figure 4. ERK2 costructure of 8 (yellow, PDB code: 6G9K) overlaid
with costructures of related conformationally constrained analogues.
(
a) Lead 7 (cyan, PDB code: 6G9N) shows a minimal change with
1
9 and pyridylpiperazine 20 both showed promising activity in
the addition of Me to the acetyl linker. (b) Indane 9 (magenta) shows
a similar bound conformation despite the potential clash between the
methylene of the indane ring and the carbonyl of the linker. (c)
Asymmetric benzazepine 10 (purple) also shows a similar bound
conformation with a minor rearrangement of Arg67 to accommodate
loss of the NH and OH. (d) Symmetrical benzazepine 11 (green)
shows a different bound conformation in which the fused phenyl ring
folds over the isoindoline and mimics Tyr36 in its “in” conformation,
while Tyr36 adopts an “out” conformation occupying a similar
position to the phenyl of 8.
cells and were progressed into in vivo PK studies (see below).
The crystal structure of compound 20 (Figure 5c) confirmed
the design hypothesis with the piperazine being accommo-
dated in the ERK protein with minimal disruption to the
binding mode.
Overall, from the SAR exploration of the terminal aryl group,
the 3-fluoro-5-methoxy derivative demonstrated the most
favorable balance of cellular potency and metabolic stability,
and this group was therefore used to probe changes in other
parts of the molecule.
Inspection of the crystal structure of the 15-ERK2 complex
(Figure 5a) suggested a small space that might accommodate
an additional methyl group on the alpha carbon of the alcohol.
Hence, the secondary alcohol 21 was designed in which it was
anticipated that this additional methyl group might block
potential metabolism of the hydroxy group. In the event, 21
showed similar potency to 15 but demonstrated higher
CYP3A4-mediated metabolism (Table 1).
in terms of metabolic stability, and hence, the additional
synthetic complexity to explore further indane analogues was
not warranted. An alternative annulation involved linking the
terminal phenyl ring to the amide nitrogen to give the isomeric
benzazepines 10 and 11. Previous SAR had shown that
methylation of the amide nitrogen reduced potency in cells
(
matched pair compounds 12 and 13, Table 1), and indeed,
both benzazepines were less potent in cells than their simpler
benzylamine counterpart, compound 12. Notably, while 10
adopted a similar bound conformation to 8 (Figure 4c), the
symmetrical benzazepine isomer 11 bound to ERK2 in a
folded conformation, in which the phenyl ring of the
benzazepine moiety occupied a similar position to the “in”
The SAR for replacements of the oxan group is summarized
in Table 2. A range of other groups were also tolerated if a
hydrogen-bond acceptor was suitably positioned to interact
with Lys114. The oxetane 22 showed a reasonable balance of
cellular activity and metabolic stability. Among the aromatic
heterocycles explored, the methylpyrazole 23 showed slightly
1
2290
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 5. Compound 15 (orange) ERK2 costructure (PDB code: 7AUV) overlaid with costructures of related analogues. (a) Lead 7 (cyan, PDB
code: 6G9N), showing minimal changes apart from slight adjustment of Tyr64 reflecting additional contacts. (b) Methoxypyridine 17 (light
purple) shows a very similar structure: Tyr64 is pushed away from the pyridyl lone pair and the MeO changes conformation to fill the resulting
space. (c) Piperazine analogue 20 (light blue) shows almost identical binding, with additional contacts between the hydrophobic edge of the
piperidine ring and the π-system of Tyr64. (d) Triazole 24 (light green) adopts a similar binding mode to 15, with slight adjustment of Lys114 to
allow hydrogen bonding to the triazole N.
weaker activity and was less stable, while the isosteric 2-methyl
,2,3-triazole and 3-methoxy-4-pyridyl analogues 24 and 25
addition, the reduction in lipophilicity (ChromLogD7.4 values
28: 3.18 cf. 15: 3.75) did not elicit an increase in human
metabolic stability. The analogue 29 was designed with a
methyl to block potential metabolism at the isoindolinone 3-
position; the structure of the 15-ERK2 complex suggested that
positioning this methyl in the (S)-configuration should be
sterically accommodated by the protein, while the methyl on
the acetamide linker was removed to avoid an intramolecular
clash. The design was ratified by good cellular potency;
however, this analogue was less metabolically stable than 15 in
the CYP3A4 assay. The analogue 30 explored the effect of
removing the acetamide methyl and led to a compound with a
similar in vitro profile to 15, suitable for further character-
ization.
1
both showed promising potency and stability. Smaller acyclic
groups were also tolerated in this position, such as the hydroxy
isopropyl 26, which possessed promising in vitro properties.
The crystal structures for one of the better analogues, 24,
showed the expected interactions with minor movements of
Lys114 to facilitate hydrogen bonding (Figure 5d). Overall,
these oxan replacements provided a range of compounds for
study in vivo, although it was apparent that none were as
potent nor appeared to provide a significant advantage over 15
in terms of metabolic stability.
Finally, we explored smaller changes in the core of 15 (Table
3
), guided by the crystal structure of its complex with ERK2
(
Figure 5a). The analogue 27 lacking the chloro group on the
In Vivo Pharmacokinetics. During the optimization
phase, in vivo mouse pharmacokinetic data were collected on
a range of compounds (data not shown), and the lipophilicity
range for achieving good oral exposure was assessed. As
mentioned above, the targeted pharmacological profile
necessitated molecular weights in excess of 500, and we, like
others, found that relatively high lipophilicity was required to
achieve good permeability as reflected in the cell-based assays
and in the oral exposure. This however needed to be controlled
aminopyrimidine ring was ∼6-fold less potent in cells, in
keeping with initial findings during the fragment optimization
2
1
stage, while metabolic stability was similar to 15. The aza
analogue 28 was designed to reduce lipophilicity, positioning
the nitrogen lone pair facing toward the solvent to avoid
impairing binding to the protein. Compound 28 was slightly
weaker than 15 in cellular assays, possibly due to the increase
in polar surface area which might compromise permeability. In
24
1
2291
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 2. Biochemical and Cellular SARs for Oxan Replacements in the Benzolactam ERK1/2 Inhibitor Series
Table 3. Biochemical and Cellular SARs for Changes to the Core in the Benzolactam ERK1/2 Inhibitor Series
prolifn IC /nM (n)
T1/2/min
5
0
Compound
R1
R2
R3
X
ERK2 IC /nM (n)
A375
Colo205
Mic mur
46
Mic hum
CYP3A4
50
1
2
2
2
3
5
7
8
9
0
Me
Me
Me
H
H
H
H
Me
H
Cl
H
Cl
Cl
Cl
CH
CH
N
CH
CH
2.7 (3)
1.8 (2)
2.8 (2)
1.1 (1)
1.1 (3)
3.4 (8)
21 (2)
12 (2)
5.9 (2)
4.0 (2)
3.6 (3)
15 (2)
8 (2)
35
62
36
22
31
35
49
23
7.3
H
86
because higher lipophilicity is often associated with general
trends for poorer metabolic stability, poorer solubility, and an
increased likelihood for off-target effects such as hERG activity.
Overall, our analysis suggested that ChromLogD7.4 values in
the range 3−4 were more likely to lead to favorable property
profiles. This empirical observation was used prospectively to
model combinations of modifications across the molecule such
that only those predicted to be in the desired lipophilicity
range were selected for synthesis.
Pharmacokinetics parameters for an illustrative selection of
compounds are summarized in Table 4. Both the cyclized
derivatives, indane 9 and benzazepine 11, showed reduced
levels of oral exposure compared with the lead 7 and the
comparator unsubstituted phenyl analogue 8. The fluoro-
methoxy analogue 15 showed good oral exposure, as did the
methoxypyridine 17; this latter compound showed significantly
reduced clearance in vivo, potentially due to the metabolic
stabilization in this region of the molecule. The fluorophenyl
piperazine 19 showed negligible oral exposure, driven by high
clearance. The pyridyl piperazine 20 fared better, with lower
clearance and an AUC similar to 15, although surprisingly the
volume of distribution of this compound was not increased
despite the weakly basic character of the compound.
Oral exposures for the oxan replacements 22−26 were
comparable to compound 15, although somewhat lower for the
methylpyrazole 23 and hydroxypropyl 26. The oxetane 22,
triazole 24, and methoxypyridine 25 showed lower clearance
which was reflected in higher exposures.
The analogue 27 lacking the chlorine substituent on the
aminopyrimidine showed significantly lower oral exposure and
bioavailability compared with its matched pair 15. The aza-
derivative 28 also showed higher clearance and lower oral
exposure compared with 15, somewhat surprising, given its
lower lipophilicity. Finally, the analogue of 15 lacking the
methyl substitution on the acetamide linker, compound 30,
also showed significantly higher clearance reflected in lower
oral exposure, consistent with previous observations suggesting
that this substitution stabilizes this part of the molecule to
21
metabolism.
1
2292
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 4. In Vitro and In Vivo Pharmacokinetic Parameters for Selected Compounds
i.v.
p.o.
T1/2 (h)
Compound
ChromLogD7.4
Cl (mL/min/kg)
Vdss (L/kg)
AUC (ng h/mL)
F (%)
7
8
9
3.9
3.0
3.6
4.7
3.8
3.0
3.6
3.3
3.3
3.3
3.7
4.2
3.1
3.2
3.2
3.3
13
11
35
19
22
6.8
124
8.7
9.4
13
8.3
4.6
23
0.6
3700
3000
960
0.8
2.5
0.89
1.4
2.9
1.5
nd
1.0
1.7
1.1
1.5
1.0
1.8
1.2
1.9
1.5
59
41
40
10
42
20
nd
15
30
13
64
23
9
0.55
0.91
0.45
0.68
0.26
3.8
0.76
0.71
0.52
0.46
0.56
1.4
1
1
1
1
2
2
2
2
2
2
2
2
3
1
5
7
9
0
2
3
4
5
6
7
8
0
440
1600
2400
18
1300
2647
824
6400
4100
310
17
37
54
1.0
1.9
1.4
320
500
150
7
23
10
a
Mice were administered a single 0.5 mg/kg dose intravenously or 5 mg/kg orally.
Figure 6. Compound 15 modulates pharmacodynamic markers of MAPK signaling and confers antitumor activity in a Colo205 subcutaneous
xenograft model. (a) Levels of pERK in Colo205 cells after 2 h treatment with the indicated concentrations of compound 15. (b) Mean tumor
levels of pERK and pRSK after single oral dose of compound 15 administered at 50 mg/kg to mice bearing Colo205 xenografts. Each data point
represents mean ± SD from three animals. (c) Inhibition of tumor growth following once daily (qd) oral dosing of compound 15 to mice bearing
Colo205 xenografts. Each data point represents mean ± SEM from eight animals. *p < 0.05 and ***p < 0.001.
Pharmacodynamics and Efficacy. Overall, a number of
compounds showed improved CYP3A4 half-life compared to
the advanced lead compound 7, and many of the compounds
had comparable or superior pharmacokinetics and in vitro
antiproliferative data. These compounds were selected for
progression into in vivo PD and efficacy studies. Levels of
phosphorylation of the ERK substrate pRSK were measured
using MSD, while levels of pERK were determined by ELISA.
Figure 6a gives a representative concentration response curve
for pERK in the Colo205 mutant BRAF cell line for compound
assays tracked closely with the GI50 values, reaching low nM
values for the most potent compounds, and for this reason
were not routinely collected (Table S1). In the in vivo PD
assays using the Colo205 xenograft, the 3-fluoro-5-methox-
yphenyl compound 15 clearly outperformed the analogues 17
and 20 with methoxy pyridine and piperazine-pyridyl groups,
respectively, while analogues containing oxan replacements,
including oxetane 22 and methoxypyridine 25, also gave
weaker responses than 15 (Figures 6b, S1). Triazole 24 also
gave a weaker response than 15 in the PD assay (Figures 6b,
S1), probably because at the 50 mg/kg dose; compound levels
1
5. In general, the IC s for both pRSK and pERK in cellular
50
1
2293
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 5. Cross Species In Vivo Pharmacokinetic Parameters for Compound 15
i.v.
p.o.
species
rat
dose (mg/kg)
0.50
Cl (mL/min/kg)
18
Vdss (L/kg)
dose (mg/kg)
1
T1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUC (ng h/mL)
F (%)
0.77
1.5
2.8
2.5
1.1
0.5
2.0
2.0
1.0
25.76
2743
15,740
3664
57.94
17,460
117,800
12,320
6.1
35
>100
91
5
0
1
00
0.5
dog
0.25
0.56
0.071
a
Scheme 1
a
(
a) From acid 50: Compounds 9 (HATU, Et N, and DCM/DMF, chiral prep, 26%); 10 (44%); 11 (66%); and 30 (71%). From acid 51:
3
Compounds 14 (HATU, DIPEA, and DMF, 76%); 15 (TBTU, Et N, and DMF, 64%); 16 (TBTU, DIPEA, and DCM, 71%); and 17 (HATU,
3
DIPEA, and DCM, 44%).
a
Scheme 2
a
a) NaH and DMF, ice bath. (b) (Bpin) , AcOK, Pd(dppf) Cl ·DCM, and dioxane, 90 °C. (c) Trichloropyrimidine, K CO , dioxane/water, and
2
2
2
2
3
Pd(PPh ) , 100 °C. (d) 4-Aminooxan, DIPEA, and dioxane, 22% over four steps.
3
4
were not dose linear (data not shown) relative to the low dose
PK shown in Table 4. We ascribed this to poor solubility
leading to formulation challenges at the higher dose for
compound 24.
models, compound 15 was selected for further character-
22
ization. For example, compound 15 was shown to have good
in vivo efficacy against six other xenografted cell lines.
Additionally, compound 15 demonstrated high selectivity
with only five kinases (excluding ERK1 and ERK2) which
were inhibited to greater than 50% at 100 nM, when screened
The performance in the PD assays translated into efficacy in
the Colo205 xenograft tumor growth models. Compound 15
elicited tumor growth inhibition dose dependently in the range
22
across a panel of 460 kinases.
22
2
5−75 mg/kg (Figure 6c). Analogues 17, 20, and 25 also
elicited tumor growth inhibition at their highest doses in the
5−100 mg/kg range and with a lesser overall effect than 15
Figure S2). Compound 15 was therefore chosen as the most
Broader selectivity was assessed using a Panlabs safety screen
panel of 44 targets, all of which showed <50% inhibition at 10
μM, confirming the high selectivity of compound 15 (data not
shown). Suspecting that highly active compounds in this series
were too potent for accurate IC50 quantitation in the
biochemical assay, we determined the binding constant using
7
(
promising candidate due to its superior efficacy and PD
response, together with its good pharmacokinetic parameters
and robust in vitro data.
ITC titration, giving a K value of 0.47 nM (Figure S3).
d
Further Characterization of ASTX029 (15). Given its
promising performance in the Colo205 PD and efficacy
Compound 15 demonstrated favorable pharmacokinetics in
preclinical species (Table 5). In rat, compound 15
1
2294
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 3
a
(
a) BOC O, DCM, and DIPEA, 31%. (b) 2,2-Dimethoxypropane, TsOH, and DCM, 84%. (c) Me N(CH ) OMs, NaH, and DMF, ice bath to
2
2
2
2
room temp, 74%. (d) TFA and DCM, quant. (e) 51, HATU, DIPEA, and DMF, 10%.
a
Scheme 4
a
(
a) BOC O, DCM, and DIPEA, 92%. (b) 2,2-Dimethoxypropane, TsOH, and DCM, 93%. (c) N-Methylpiperazine, Cs CO , Xantphos, and
2
2
3
toluene, 120 °C, 14%. (d) HCl and EtOAc, quant. (e) 51, TBTU, DIPEA, and DCM, 22%.
demonstrated supraproportional pharmacokinetics, possibly
due to a reduction in the gastrointestinal and/or hepatic
extraction ratio. In vitro to in vivo extrapolation (IVIVE) of
hepatic clearance from intrinsic clearance measurements
measured in microsomes and hepatocytes showed reasonable
concordance with measured in vivo total clearance (data not
shown). Human clearance was predicted to be low (<25% liver
blood flow) based on IVIVE, and the predicted human dose
based on exposures from mouse xenograft studies at 25 mg/kg
was estimated to be approximately 500 mg.
30 were prepared by coupling the corresponding commercially
available amines with the acid intermediate 50 (compound 42
21
in Heightman et al. ) (Scheme 1). Compounds 14−17 were
prepared similarly from the respective commercial amines and
21
the acid intermediate 51 (compound 48 in Heightman et al. )
(
Scheme 1).
Compound 13 was prepared by an alternative, more
convergent route (Scheme 2). Alkylation of 6-bromoisoqui-
nolinone (53) with N-benzyl-2-chloro-N-benzylacetamide
(52) to give 54 was followed by palladium-catalyzed borylation
and Suzuki coupling to generate the dichloropyrimidine
Synthesis. Procedures for the preparation of compounds 7,
21
8
, and 12 were described previously. Compounds 9−11 and
1
2295
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 5
a
(a) N-Methylpiperazine and water, microwave 120 °C. (b) 51, TBTU, DIPEA, and DCM, 61%.
a
Scheme 6
a
t
i
(
a) (S)- Butyl sulfinamide, Ti(O Pr) , and THF, 48%. (b) Stage 1: 1-Bromo-3-fluoro-5-methoxybenzene, Mg, and Et O, 30 °C; stage 2, −78 °C,
4
2
addition to 70, 61%. (c) HCl and MeOH and then HCl, MeOH, H 5 bar, and Pd/C, 58%. (d) 50, TBTU, DIPEA, and DCM, 93%.
2
intermediate 56. This allowed nucleophilic aromatic sub-
stitution with 4-amino-oxan to give 13.
affected under microwave heating (Scheme 5). TBTU-
mediated coupling of the product gave 20.
The aminoalcohol for compound 21 was accessed using
chiral sulfoximine chemistry (Scheme 6). Titanium(IV)-
mediated condensation of (S)-2-methylpropane-2-sulfinamide
Compounds 18−20 required bespoke synthesis of the
corresponding amines prior to coupling with acid 51. The
dimethylaminoethoxy compound 18 was prepared according
to Scheme 3. BOC protection of commercial aminoalcohol 57
followed by acetonide formation gave the protected phenol 59
which was alkylated with 2-(dimethylamino)ethyl methanesul-
fonate to give the protected intermediate 60. Acidic
deprotection with TFA to give aminoalcohol 61 followed by
HATU-mediated coupling with acid 51 afforded 18. The
phenylpiperazine 19 was synthesized according to Scheme 4.
The bromofluorophenyl aminoalcohol 62 was subjected to a
similar BOC and acetonide protection to give 64, allowing
Buchwald−Hartwig coupling with N-methylpiperazine to give
(
69) with (S)-2-(benzyloxy)propanal gave the distereomeric
sulfinamide 70, which was subject to addition by the Grignard
reagent prepared from 1-bromo-3-ethoxy-5-fluorobenzene to
give 71. The desired aminoalcohol 72 was liberated by acidic
desulfinylation followed by hydrogenolytic debenzylation.
Finally, coupling with the acid 50 using TBTU gave 21 in
good yield.
Compounds bearing alternative groups to oxan were
assembled via alternative routes shown in Scheme 7. For the
aliphatic analogues 22 and 26, the chloropyrimidine acid
intermediate 74 was first coupled with (S)-2-amino-2-(3-
fluoro-5-methoxyphenyl)ethan-1-ol using TBTU to give the
6
5. Acidic deprotection with TFA and TBTU-mediated amide
coupling gave 19. Synthesis of the aminoalcohol intermediate
8 required for the pyridine analogue 20 did not require
amide 75, which was then subjected to S AR reaction with the
N
6
appropriate amine. For the methylpyrazole analogue 23, the
t
protection of the hydroxy and amino functions of the starting
aromatic amine was introduced by Bu-BrettPhos-catalyzed
material 67; instead, direct displacement of the fluoride was
Buchwald−Hartwig coupling, followed by acidic deprotection
1
2296
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 7
a
(
a) TFA and DCM, 95%. (b) Amine, TBTU, DIPEA, and DCM, 80%. (c) 22: oxetan-3-amine and EtOH, 80 °C, 90%; 26: L-alaninol and EtOH,
8
0 °C, 15%. (d) 1-Methyl-1H-pyrazol-5-amine, Cs CO , t-BuBrettPhos allyl (Pd-175), and dioxane, 50 °C, 80%. (e) TFA and DCM, 88%. (f)
2
3
Amine, TBTU, Et N, and DMF, 53%. (g) 78: 2-methyl-2H-1,2,3-triazol-4-amine, Cs CO , t-BuBrettPhos allyl (Pd-175), and DMF, 70 °C, 72%;
7
3
2
3
9: 2-methoxypyridin-4-amine, Cs CO , Xantphos, and DMF, 75 °C, 79%. (h) Amine, TBTU, DIPEA, and DMF; 24: 79%; 25: 82%.
2 3
a
Scheme 8
a
(
a) 2,4-Dichloropyrimidine, Na CO , Pd(PPh ) , and dioxane, 90 °C, 90%. (b) TFA and DCM, 98%. (c) Amine, TBTU, 4-methylmorpholine,
2 3 3 4
and DMF, 41%. (d) Oxan-4-amine and DIPEA, 90 °C, 37%.
of the acid and final amide coupling. The methyl triazole and
methoxypyridine analogues 24 and 25 were prepared similarly,
except that the Buchwald−Hartwig couplings were carried out
on the deprotected acid to avoid epimerization occurring alpha
to the ester group.
The des-chloro analogue 27 was prepared according to
Scheme 8. Suzuki coupling of the previously published
boronate 80 with 2,4-dichloropyrimidine followed by acidic
deprotection gave the acid intermediate 82; then, amide
coupling using TBTU followed by S AR reaction with oxan-4-
N
amine provided 27.
The aza-analogue 28 required a bespoke bicycle synthesis
(Scheme 9). Starting from the bromochloropyridine 84, a vinyl
group was inserted using Molander chemistry and ozonolyzed
to give the aldehyde 86. Reductive amination with the tert-
butyl ester of D-alanine gave the pyrrolopyridine intermediate
1
2297
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 9
a
(
a) H CCHBF K, Et N, PdCl (dppf) , and EtOH, 90 °C, 51%. (b) Ozone and DCM, −78 °C, then Me S, warm to room temp, 64%. (c) (R)-
2
4
3
2
2
2
tert-Butyl 2-aminopropanoate, DIPEA, and NaB(OAc) H, 40%. (d) (Bpin) , AcOK, Pd(dppf) Cl ·DCM, and dioxane, 90 °C, 82%. (e)
3 2 2 2
Trichloropyrimidine, Na CO , Pd(PPh ) , and dioxane, 90 °C. (f) Oxan-4-amine, DIPEA, and dioxane, 90 °C, 66% over two steps. (g) TFA and
2
3
3 4
DCM, 94%. (h) Amine, TBTU, DIPEA, and DMF, 33%.
a
Scheme 10
a
(
a) BOC O, DCM, and DIPEA, 95%. (b) CO, ([Ph P] CH CH ) , Pd(OAc) , and DMF, 90 °C. (c) TFA and DCM, 56% over two steps. (d)
2 2 2 2 2 2 2
t
BrCH CO Bu, NaH, and DMF, ice bath warms to room temp, 63%. (e) (Bpin) , AcOK, Pd(crotyl) (XPhos)Cl, and dioxane, 90 °C. (f) 98,
Na CO , Pd(PPh ) , and dioxane, 90 °C, 49% over two steps. (g) TFA and DCM, quant. (h) Amine, TBTU, Et N, and DCM, 64%.
2
2
2
2
3
3
4
3
8
7. Palladium-catalyzed borylation allowed Suzuki−Miyaura
The 3-methyl-substituted isoindolinone 29 also required
bespoke synthesis of the core (Scheme 10). The (R)-bromo-
chloro α-methylbenzylamine 92 was BOC-protected, and then,
palladium-catalyzed carbonylation and deprotection gave the
chiral 6-chloro-3-methylisoindolinone 95. Alkylation with tert-
coupling to generate the dichloropyridine 89, which was
subjected to S AR with oxan-4-amine and acidic deprotection
to afford acid 91. Finally, amide coupling with TBTU as before
gave 28.
N
1
2298
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
butyl bromoacetate gave intermediate 96, which was borylated
as before and then coupled with the oxanyl dichloropyrimidine
excitation at 340 nm, fluorescence was measured at 520 nm (A
counts) and 495 nm (B counts) using a Pherastar plate reader (BMG
Labtech, Ortenberg, Germany).
Cell Culture and Reagents. The human cell lines A375 and
Colo205 were purchased from the American Type Culture Collection
9
8 to give ester 99. Acidic deprotection and amide coupling
with TBTU as before gave 29.
(
ATCC, Manassas, VA, USA) and the European Collection of
CONCLUSIONS
■
Authenticated Cell Cultures (ECACC Salisbury, UK), respectively.
The cells lines were not passaged for more than 6 months after
authentication by the cell banks (DNA fingerprinting and cytogenetic
analysis or short-tandem repeat PCR). Both cell lines were grown in
DMEM culture medium, supplemented with 10% FBS (Thermo
Fisher Scientific, Waltham, Massachusetts, USA) and maintained at
Our optimization strategy was driven by knowledge of
metabolic weaknesses in the lead molecule 7 together with
multiparameter optimization based on empirical data collected
across analogues within the lead series. Structure-based drug
design was also employed to narrow down the number of
plausible options in the lead optimization campaign, and
crystal structures in complex with ERK2 identified potential
sites for chemical modification to modulate the metabolic
profile, while maintaining potency. The metabolism studies
highlighted the oxan ring as a key site of metabolism in lead
molecule 7; however, replacement with more metabolically
stable isosteres produced compounds with inferior solubility,
consistent with previous observations on the need to maintain
3
7 °C in an atmosphere of 5% CO2.
Cell Proliferation Assay. Cell proliferation assays were carried
out using Alamar Blue (Thermo Fisher Scientific, Waltham,
Massachusetts, USA) as described previously. Briefly, 5 × 10
32
3
cells were seeded in complete culture medium into flat-bottomed
96-well plates, 1 day before the drug treatment. Cells were incubated
with the compound in 0.1% (v/v) dimethyl sulfoxide (DMSO) for 96
h before viability was assessed using Alamar blue. IC50 values were
calculated using a sigmoidal dose response equation (Prism GraphPad
software, La Jolla, CA, USA).
3
25,26
sufficient sp character in drug molecules.
Finally, retaining
In Vitro Permeability. Permeability of test compounds was
the oxan group while replacing the tolyl group, the other key
site of metabolic instability, with the 3-fluoro-5-methoxyphenyl
group in 15 provided sufficient metabolic stabilization in in
vitro models while maintaining favorable potency and
physicochemical properties. Although the oxan ring has been
associated with metabolic instability due to a propensity for
oxidative metabolism in alpha position to the ring oxygen
assessed using the CacoReady system. Test and control compounds
(
concentration of 10 μM in duplicate to either the apical (180 μL) of
the monolayer to measure apical to basolateral transport (A > B)
across the cell barrier or to the basolateral side (750 μL) to measure
the basolateral to apical transport (B > A). For A > B spiking, test and
control compounds were diluted from 10 mM DMSO stocks to 10
propranolol, antipyrine, and vinblastine) were incubated at a final
2
7
atom, this issue appears to be context-dependent, and there
are now a significant number of drugs incorporating this group
that show good pharmacokinetics with long half-lives in
μM in Hanks’ balanced salt solution buffer with 100 mM CaCl2·
2
H O, 50 mM MgCl ·5H O, and 0.5 mg/mL Lucifer yellow made to
2 2 2
volume with sterile water (Lucifer yellow was used to determine the
integrity of the Caco-2 monolayer). For B > A compound spiking, test
and control compounds were prepared as for the A > B solutions
without the addition of Lucifer yellow. Test and control compounds
were incubated for 1 h at 37 °C in a highly humidified atmosphere of
28
29
humans, including venetoclax and gilteritinib.
A combination of cellular antiproliferative potency data and
total plasma exposure was sufficient to predict compounds
showing the strongest pharmacodynamic response, which
correlated with efficacy in tumor xenografts. Compound 15
possessed the most favorable balance of performance in
efficacy models of both BRAF and KRas mutant cancers with
the selectivity and preclinical pharmacokinetic profile, leading
to its selection as a clinical candidate. Compound 15 is
currently in a phase I−II clinical trial in patients with advanced
solid malignancies who are not candidates for approved or
9
5% air and 5% CO2.
Quantification of pRSK by MSD. A375 cells were seeded at 1.5
4
× 10 cells per well into 96-well plates and allowed to recover for 16
h, prior to the addition of compounds (in 0.1% DMSO v/v) and
incubation for a further 4 h. Cells were lysed by adding cell lysis buffer
(
Cell Signaling Technology, Massachusetts, USA) and incubating at
room temperature for 20 min. Custom MSD plates (Meso Scale
Discovery, Maryland, USA) precoated with the anti-pRSK antibody
(
Cell Signaling Technology, Massachusetts, USA) were blocked with
30
available therapies.
kit blocking buffer for 1 h at room temperature, prior to washing.
Equivalent amounts of protein lysate were added to the blocked plates
and incubated for 3 h at room temperature. After washing, plates were
incubated for 1 h at room temperature with sulfo-tag-conjugated anti-
RSK detection antibodies (R&D Systems, Minneapolis, USA). Plates
were washed, and read buffer was added before reading the plate on a
MESO QuickPlex SQ 120 (Meso Scale Discovery, Maryland, USA).
IC50 values were calculated using a sigmoidal dose response equation
(Prism GraphPad software, La Jolla, CA, USA). For pharmacody-
namic studies, Colo205 xenograft tumor lysates were prepared as
described above and transferred directly to Custom MSD pRSK plates
(Meso Scale Discovery, Maryland, USA) for analysis.
Quantification of pERK by ELISA. A375 cells were treated with
compounds and lysed as described above. Lysates were then cleared
by centrifugation, transferred to pERK ELISA plates (Cell Signaling
Technology, Massachusetts, USA), and analyzed according to the
manufacturer’s instructions. IC50 values were calculated using a
sigmoidal dose response equation (Prism GraphPad software, La Jolla,
CA, USA). For pharmacodynamic studies, Colo205 xenograft tumor
lysates were prepared as described above, transferred to pERK ELISA
plates (Cell Signaling Technology, Massachusetts, USA), and
analyzed according to the manufacturer’s instructions.
EXPERIMENTAL PROCEDURES
■
Protein Expression, Purification, and Crystallography. Full-
length human ERK2 (hERK2) was cloned into pET30a with a
noncleavable MAHHHHHH N-terminal tag. hERK2 was expressed in
Escherichia coli BL21(DE3), and nonphosphorylated hERK2 (con-
firmed by) was purified using sequential Ni-HiTRAP, desalt,
Resource-Q, and S75 26/60 column steps. hERK2 was crystallized
under conditions adapted from Aronov et al., and crystals were
soaked in a solution equivalent to the crystallization solution but also
containing 0.1−100 mM ligand, 10 mM DTT, and 10% DMSO final.
Crystals were cryo-protected using crystallization solution containing
5% 2KMPEG final. X-ray diffraction data were collected using both
in-house and synchrotron X-ray sources.
ERK2 Kinase Assay. ERK2 kinase activity was determined using a
TRF activity assay. The ERK2 enzyme (0.25 nM) was incubated with
the substrates ATF2-GFP (0.4 μM) and ATP (20 μM) in 50 mM Tris
31
3
pH7.5, 10 mM MgCl , 1 mM EGTA, 0.01% Triton X100, 1 mM
2
DTT, and 2.5% DMSO, with shaking at room temperature for 30
min. Reactions were stopped by the addition of a stop and detection
mix, containing 25 mM EDTA and 2 nM Tb-pATF2 antibody in TR-
FRET dilution buffer (Life Technologies, Paisley, UK), and the plate
was incubated with shaking at room temperature for 1 h. Upon
In Vivo Studies. The care and treatment of experimental animals
were in accordance with the United Kingdom Coordinating
1
2299
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3
3
Committee for Cancer Research guidelines and the United
Xenograft Studies. Colo205 xenografts were prepared by
subcutaneously injecting 5 × 10 cells suspended in serum-free
34
6
Kingdom Animals (Scientific Procedures) Act 1986. Animals had
access to food and water ad libitum throughout the acclimatization
and study period.
Pharmacokinetic Studies. Pharmacokinetic studies were per-
formed in male BALB/c mice, obtained from Harlan Laboratories Inc.
medium mixed 1:1 with Matrigel (BD Biosciences, San Jose, CA,
USA) into the right flank of each male BALB/c nude mouse. Tumors
were measured using caliper and tumor volumes calculated by
applying the formula for an ellipsoid. For efficacy studies, when the
3
(
Shardlow, UK). Pharmacokinetic studies in male rats (Sprague−
tumors reached an average of approximately 200 mm , mice were
Dawley and Hans Wistar) and male Beagle dogs were performed by
either Quotient Bioresearch (Rushden, UK) or Aptuit (Verona, Italy).
For intravenous administrations in mice, test compounds were
formulated in either 20% DMA/80% saline, 10% DMSO/20%
PEG400/70% water, or 10% DMSO/90% saline. For oral
administrations in mice, test compounds were formulated in either
randomized into groups of eight. Mice were dosed orally once a day at
a dose of 25−75 mg/kg compound 15, 100 mg/kg compound 17 and
25, and 75 mg/kg compound 20. Body weights were recorded daily,
and tumor volumes were measured every 3−4 days. For PK/PD
studies, a single dose of compound was administered orally to mice.
For oral administrations in PD or efficacy studies, test compounds
were formulated in either 20% PEG200/0.5% Methocel or 75%
PEG200/25% water or 10% NMP/15% PEG200/0.75% Methocel.
Following dosing, blood samples were drawn in tubes containing
potassium EDTA via either saphenous vein bleeding or cardiac
puncture at various time points, prior to centrifugation (2000g at 4
1
0% ethanol/10% cremaphor/10% PEG200/70% water, 20%
PEG400/80% HPMC, or 10% DMSO/20% cremaphor/70% saline.
Intravenous dosing at 0.5 mg/kg was administered via the lateral tail
vein at a dose volume of 5 mL/kg. Test compounds were
administered orally at 5 mg/kg by nasogastric gavage at a dose
volume of 10 mL/kg. Compound 15 was administered to rats either
intravenously at 0.5 mg/kg (n = 3) or via oral gavage at 1, 50, and 100
mg/kg (n = 3), at a dose volume of 5 mL/kg, formulated in 10%
DMSO and 90% saline or 10% NMP, 15% PEG200 and 75%
methylcellulose (1% aq, w/v), and 75% PEG 400 and 25% water or
°
C, 10 min). The resultant plasma was separated from the erythrocyte
pellets for analysis and stored at −20 °C. Tumors were immediately
excised and flash-frozen in liquid nitrogen.
Bioanalysis. All in vitro and in vivo samples were extracted by
protein precipitation with the acetonitrile-containing internal standard
(
1:3 v/v). For quantitative studies, calibration standards and quality
1
00% PEG400, respectively. In male Beagle dogs, compound 15 was
controls were prepared in the blank matrix and extracted under the
administered intravenously at 0.25 mg/kg or via oral gavage at 0.5
mg/kg, at a dose volume of 5 mL/kg, formulated in 10% NMP, 20%
PEG200 and 70% saline or 10% NMP, 15% PEG200 and 75% water,
respectively. A week wash-out period was included between dosing (n
same conditions. All samples were centrifuged at 3700 rpm at 4 °C for
20 min. Compound bioanalysis of all in vitro and in vivo samples was
performed using high-performance liquid chromatography mass
spectrometry (LC/MS). Test compounds and the internal standard
=
3). All doses were calculated as freebase equivalent per kg of
+
were ionized using positive-mode (ESI ) electrospray ionization.
bodyweight. Following dosing, blood samples were drawn into tubes
containing potassium EDTA via either saphenous vein bleeding or
cardiac puncture at various time points over 24 h using sparse
sampling (n = 3 per time point) in mice, via tail vein and cardiac
puncture in rats, or via jugular vein in dogs using serial sampling.
Blood samples were centrifuged (2000g at 4 °C, 10 min), and the
resultant plasma was separated from the erythrocyte pellets for
analysis and stored at −20 °C. Noncompartmental pharmacokinetic
Analytes were detected using multiple reaction monitoring. Test
compounds typically ran on a gradient HPLC method over 5 min
with 10 mM ammonium acetate and acetonitrile mobile phase at a
flow rate of 0.5 mL/min. Separation was typically achieved using an
Acquity HSS T3 1.8 μM 50 × 2.1 mm column maintained at 40 °C.
Prediction of Human Pharmacokinetics. Microsomal and
hepatocyte intrinsic clearance in preclinical species was scaled to total
35
hepatic clearance using the well-stirred model to generate a
prediction of human clearance. Human dose was predicted using
the target AUC and was taken from efficacy studies in mouse
xenograft models, using the average bioavailability determined in
preclinical studies.
(PK) analyses were performed using Phoenix 6.3.0.395 (Certara USA,
Inc.) software.
In Vitro Phenotyping. Test compounds were incubated (1 μM)
in human Supersomes expressing (0.25 mg/mL) CYP isozymes (20
pmol/mL; CYP3A4, CYP2D6, CYP2C9, CYP2C8, CYP1A2,
CYP2B6, and CYP2C19) in the presence of NADPH at +37 °C for
Synthesis. General Procedures. All solvents and commercially
available reagents were used as received. All reactions were followed
by thin-layer chromatography (TLC) analysis (TLC plates GF254,
Merck) or LC−MS (liquid chromatography mass spectrometry).
LC−MS analysis was performed with an Agilent or Shimadzu LC
system with variable wavelength UV detection using reverse-phase
40 min. Samples were taken at 0, 5, 10, 20, and 40 min, and protein
was precipitated with acetonitrile and centrifuged prior to bioanalysis.
In Vitro Microsomal Stability. The test compound and the
positive control, diclofenac, were incubated in duplicate at 1 μM in
male CD-1 mouse and mixed gender human liver microsomes at 37
chromatography with a CH CN and water gradient with a 0.02 or
3
°C for 40 min at a microsomal protein concentration of 0.25 mg/mL,
0.1% TFA modifier (added to each solvent) and using a reverse-phase
in the presence of NADPH and UDPGA. In addition, compound 15
was incubated in male Sprague−Dawley rat and male Beagle dog liver
microsomes under the same conditions. Negative control incubations,
in the absence of NADPH and UDPGA, were performed. Incubations
were sampled (50 μL) at 0, 5, 10, 20, and 40 min, except for the
negative controls which were sampled at 0 and 40 min.
column, for example, Thermo Hypersil Gold C18. MS was
determined using either PE Sciex 150EX LC−MS, Waters ZQ LC−
MS, or Agilent 6140 LC−MS Single Quadrupole instruments.
Column chromatography was performed on prepacked silica gel
columns (3090 mesh, IST) using a Biotage SP4 or similar. NMR
1
spectra are referenced as follows: H (400 MHz), internal standard
In Vitro Hepatocyte Stability. Compound 15 and the positive
controls, verapamil and umbelliferone, were incubated in duplicate at
TMS at δ = 0.00. Abbreviations for multiplicities observed in NMR
spectra are as follows: s; singlet; br s, broad singlet; d, doublet; t,
triplet; q, quadruplet; p, pentuplet; and m, multiplet. All compounds
reported are of at least 95% purity according to LC−MS unless stated
otherwise.
1
μM in male CD-1 mouse, male Sprague−Dawley rat, and male
Beagle dog and at 0.2, 1, and 10 μM in mixed gender human
cryopreserved hepatocytes at 37 °C for 60 min. Williams E media
supplemented with 2 mM L-glutamine and 25 mM HEPES and test
compounds (final DMSO concentration, 0.25%) were preincubated at
(
2R)-2-(6-(5-Chloro-2-[(oxan-4-yl)amino]pyrimidin-4-yl)-1-oxo-
2
,3-dihydro-1H-isoindol-2-yl)-N-[(1S)-1-(3-fluoro-5-methoxyphen-
3
7 °C prior to the addition of a suspension of cryopreserved
yl)-2-hydroxyethyl]propanamide (15). A stirred solution of (R)-2-
(6-(5-chloro-2-((oxan-4-yl)amino)pyrimidin-4-yl)-1-oxoisoindolin-2-
yl)propanoic acid (compound 48 in Heightman et al. ) (70 mg,
0.168 mmol), (S)-2-amino-2-(3-fluoro-5-methoxyphenyl)ethanol·
HCl (41 mg, 0.185 mmol), and triethylamine (0.094 mL, 0.672
mmol) in DMF (1 mL) was treated with TBTU (65 mg, 0.202 mmol)
and stirred at room temperature overnight. The mixture was diluted
6
hepatocytes (final cell density of 0.5 × 10 viable cells/mL in Williams
E media supplemented with 2 mM L-glutamine and 25 mM HEPES)
to initiate the reaction. The final incubation volume was 500 μL.
Aliquots (50 μL) were sampled at timed intervals (0, 5, 10, 20, 40,
and 60 min) from each incubation. Negative vehicle controls were
included.
21
1
2300
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
with ethyl acetate (20 mL), was washed successively with 1 M KHSO4
Charlotte M. Griffiths-Jones − Astex Pharmaceuticals,
Cambridge CB4 0QA, U.K.
(
1
10 mL), NaHCO (10 mL), brine (2 × 10 mL), and then water (4 ×
3
0 mL), was dried (MgSO ), and evaporated. The residue was
4
Steven Howard − Astex Pharmaceuticals, Cambridge CB4
purified by chromatography (SiO , 12 g column, 0−5% EtOH in
2
0QA, U.K.
EtOAc) to give a glass, which was triturated with ether (2 mL) to give
Justyna Kucia-Tran − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.
Vanessa Martins − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
a solid. The solid was collected by filtration, washed with ether (2 × 1
mL), and dried under vacuum at 50 °C overnight to give the title
1
compound (64.3 mg, 64.3%) as a cream solid. H NMR (DMSO-d ):
6
δ 8.54 (d, J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.04 (d, J = 1.6 Hz, 1H), 7.97
(
dd, J = 7.9, 1.7 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 5.6 Hz,
Sandra Muench − Astex Pharmaceuticals, Cambridge CB4
1
1
1
3
2
H), 6.76−6.66 (m, 3H), 4.99 (q, J = 7.2 Hz, 1H), 4.90 (t, J = 5.5 Hz,
H), 4.81 (q, J = 6.6 Hz, 1H), 4.75 (d, J = 18.1 Hz, 1H), 4.61 (d, J =
8.1 Hz, 1H), 3.99−3.88 (m, 1H), 3.86 (dt, J = 11.3, 3.4 Hz, 2H),
.76 (s, 3H), 3.61−3.49 (m, 2H), 3.44−3.33 (m, 2H), 1.91−1.78 (m,
0QA, U.K.
Joanne M. Munck − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
David Norton − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.; orcid.org/0000-0002-8680-4936
Marc O’Reilly − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.
1
3
H), 1.60−1.47 (m, 2H), 1.45 (d, J = 7.2 Hz, 3H). C NMR (101
0
MHz, DMSO-d ): δ 170.8, 166.9, 162.8 (d, J = 242.0 Hz), 161.4 (very
6
broad), 160.4 (d, J = 11.6 Hz), 160.0, 158.2 (very broad), 144.6 (d, J
0
=
8.9 Hz), 143.9, 135.9 (very broad), 132.0, 132.0, 123.4, 123.1,
14.7, 109.0 (d, J = 2.4 Hz), 105.6 (d, J = 22.2 Hz), 99.8 (d, J = 25.2
Hz), 66.0, 64.3, 55.5, 54.8 (d, J = 2.2 Hz), 49.6, 47.2, 47.1, 32.3, 16.2.
1
Nicholas Palmer − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Puja Pathuri − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
+
+
LCMS: [M + H] 584. HRMS (ESI-QTOF): m/z [M + H] calcd for
C H ClFN O 584.2069; found, 584.2065. Δ = −0.75 ppm.
29
31
5
5
Torren M. Peakman − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.
Michael Reader − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00905.
■
*
David C. Rees − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Cellular pRSK and pERK IC50 values for selected
compounds; PK/PD data for compounds 17, 20, 22, 24,
and 25; antitumor efficacy data for compounds 17, 20,
and 25; ITC titration of compound 15; HPLC trace for
compound 15; information on reproducibility for the
ERK2 enzyme and A375 cell assays; and synthesis of
compounds 9−11, 13, 14, 16−30, and associated
intermediates (PDF)
Sharna J. Rich − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Alpesh Shah − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Nicola G. Wallis − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Hugh Walton − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Molecular formula strings (CSV)
Nicola E. Wilsher − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
AUTHOR INFORMATION
Corresponding Author
Christopher W. Murray − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.; orcid.org/0000-0003-3733-0700;
Phone: +44 (0) 1223 226228; Email: Chris.Murray@
astx.com; Fax: +44 (0) 1223 226201
■
Alison J.-A. Woolford − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.; orcid.org/0000-0002-6453-5819
Michael Cooke − Sygnature Discovery Ltd., Nottingham NG1
1GF, U.K.
David Cousin − Sygnature Discovery Ltd., Nottingham NG1
GF, U.K.
Stuart Onions − Sygnature Discovery Ltd., Nottingham NG1
GF, U.K.
1
Authors
1
Tom D. Heightman − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.
Jonathan Shannon − Sygnature Discovery Ltd., Nottingham
NG1 1GF, U.K.
Valerio Berdini − Astex Pharmaceuticals, Cambridge CB4
John Watts − Sygnature Discovery Ltd., Nottingham NG1
0
QA, U.K.
1GF, U.K.
Luke Bevan − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00905
Ildiko M. Buck − Astex Pharmaceuticals, Cambridge CB4
0
QA, U.K.
Maria G. Carr − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.
Aurélie Courtin − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.
Joseph E. Coyle − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.
James E. H. Day − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.
Charlotte East − Astex Pharmaceuticals, Cambridge CB4
QA, U.K.
Notes
0
The authors declare no competing financial interest.
Coordinates and structure factors for the ERK2-ligand complex
have been deposited with PDB with the following accession
codes: 9, 7NQQ; 10, 7NQW; 11, 7NR3; 17, 7NR9; 20,
0
0
7
NR8; and 24, 7NR5. Authors will release the atomic
coordinates and experimental data upon article publication.
0
ACKNOWLEDGMENTS
0
■
Lynsey Fazal − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
We would like to acknowledge Anne Cleasby for technical help
in preparing the X-ray structures for deposition.
1
2301
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
11) ATG-017: Safety and preliminary efficacy of atg-017
ABBREVIATIONS
■
monotherapy in advanced solid tumors and hematological malig-
nancies (eraser). https://ClinicalTrials.gov/show/NCT04305249.
(
Davies, E. J.; Dobson, A.; Dong, Z.; Fairley, G.; Farrington, P.; Feron,
L.; Flemington, V.; Gibbons, F. D.; Graham, M. A.; Greenwood, R.;
Hanson, L.; Hopcroft, P.; Howells, R.; Hudson, J.; James, M.; Jones,
C. D.; Jones, C. R.; Li, Y.; Lamont, S.; Lewis, R.; Lindsay, N.;
McCabe, J.; McGuire, T.; Rawlins, P.; Roberts, K.; Sandin, L.;
Simpson, I.; Swallow, S.; Tang, J.; Tomkinson, G.; Tonge, M.; Wang,
Z.; Zhai, B. Discovery of a potent and selective oral inhibitor of erk1/
BRAF, B-Raf proto-oncogene; DMSO, dimethyl sulfoxide;
DSF, differential scanning fluorimetry; ERK, extracellular
signal-related kinase; HATU, 1-[bis(dimethylamino)-
methylene]-1I-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-
fluorophosphate; HBSS, Hanks’ balanced salt solution;
HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-
uronium hexafluorophosphate; HPMC, hydroxypropyl meth-
ylcellulose; IVIVE, in vitro to in vivo extrapolation; MAPK,
mitogen-activated protein kinase; MEK, mitogen activated
protein kinase; MSD, mesoscale discovery; RAS, retrovirus-
associated DNA sequence; RSK, ribosomal S6 kinase; T3P,
12) Ward, R. A.; Anderton, M. J.; Bethel, P.; Breed, J.; Cook, C.;
2
(azd0364) that is efficacious in both monotherapy and combination
therapy in models of nonsmall cell lung cancer (nsclc). J. Med. Chem.
019, 62, 11004−11018.
13) Bhagwat, S. V.; McMillen, W. T.; Cai, S.; Zhao, B.; Whitesell,
2
(
2
,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-triox-
ide; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium tetrafluoroborate; TRF, time-resolved fluorescence
M.; Shen, W.; Kindler, L.; Flack, R. S.; Wu, W.; Anderson, B.; Zhai,
Y.; Yuan, X.-J.; Pogue, M.; Van Horn, R. D.; Rao, X.; McCann, D.;
Dropsey, A. J.; Manro, J.; Walgren, J.; Yuen, E.; Rodriguez, M. J.;
Plowman, G. D.; Tiu, R. V.; Joseph, S.; Peng, S.-B. Erk inhibitor
ly3214996 targets erk pathway-driven cancers: A therapeutic approach
toward precision medicine. Mol. Cancer Ther. 2020, 19, 325−336.
REFERENCES
■
(
1) Samatar, A. A.; Poulikakos, P. I. Targeting ras−erk signalling in
cancer: Promises and challenges. Nat. Rev. Drug Discovery 2014, 13,
(
14) Aronchik, I.; Dai, Y.; Labenski, M.; Barnes, C.; Jones, T.; Qiao,
928.
L.; Beebe, L.; Malek, M.; Elis, W.; Shi, T.; Mavrommatis, K.; Bray, G.
L.; Filvaroff, E. H. Efficacy of a covalent erk1/2 inhibitor, cc-90003, in
kras-mutant cancer models reveals novel mechanisms of response and
resistance. Mol. Cancer Res. 2019, 17, 642−654.
(
2) Payne, D. M.; Rossomando, A. J.; Martino, P.; Erickson, A. K.;
Her, J. H.; Shabanowitz, J.; Hunt, D. F.; Weber, M. J.; Sturgill, T. W.
Identification of the regulatory phosphorylation sites in pp42/
mitogen-activated protein kinase (map kinase). EMBO J. 1991, 10,
(
15) Boga, S. B.; Deng, Y.; Zhu, L.; Nan, Y.; Cooper, A. B.; Shipps,
8
(
85−892.
G. W.; Doll, R.; Shih, N.-Y.; Zhu, H.; Sun, R.; Wang, T.; Paliwal, S.;
Tsui, H.-C.; Gao, X.; Yao, X.; Desai, J.; Wang, J.; Alhassan, A. B.;
Kelly, J.; Patel, M.; Muppalla, K.; Gudipati, S.; Zhang, L.-K.; Buevich,
A.; Hesk, D.; Carr, D.; Dayananth, P.; Black, S.; Mei, H.; Cox, K.;
Sherborne, B.; Hruza, A. W.; Xiao, L.; Jin, W.; Long, B.; Liu, G.;
Taylor, S. A.; Kirschmeier, P.; Windsor, W. T.; Bishop, R.; Samatar, A.
A. Mk-8353: Discovery of an orally bioavailable dual mechanism erk
inhibitor for oncology. ACS Med. Chem. Lett. 2018, 9, 761−767.
3) Eblen, S. T. Chapter fourExtracellular-regulated kinases:
Signaling from ras to erk substrates to control biological outcomes. In
Advances in Cancer Research; Tew, K. D., Fisher, P. B., Eds.; Academic
Press, 2018; Vol. 138, pp 99−142.
(
4) Ryan, M. B.; Der, C. J.; Wang-Gillam, A.; Cox, A. D. Targeting
ras-mutant cancers: Is erk the key? Trends Cancer 2015, 1, 183−198.
5) Robert, C.; Grob, J. J.; Stroyakovskiy, D.; Karaszewska, B.;
(
Hauschild, A.; Levchenko, E.; Chiarion Sileni, V.; Schachter, J.;
Garbe, C.; Bondarenko, I.; Gogas, H.; Mandalá, M.; Haanen, J. B. A.
G.; Lebbé, C.; Mackiewicz, A.; Rutkowski, P.; Nathan, P. D.; Ribas,
A.; Davies, M. A.; Flaherty, K. T.; Burgess, P.; Tan, M.; Gasal, E.; Voi,
M.; Schadendorf, D.; Long, G. V. Five-year outcomes with dabrafenib
plus trametinib in metastatic melanoma. N. Engl. J. Med. 2019, 381,
(
16) KO-947: First-in-human study of ko-947 in nonhematological
malignancies. https://ClinicalTrials.gov/show/NCT03051035.
17) LTT462: A phase i clinical study with the investigational
(
compound ltt462 in adult patients with specific advanced cancers.
https://ClinicalTrials.gov/show/NCT02711345.
(18) ASN007: A study of asn007 in patients with advanced solid
tumors. https://ClinicalTrials.gov/show/NCT03415126.
(19) HH2710: A study to evaluate the safety, tolerability, and
pharmacokinetics of hh2710 in patients with advanced tumors.
https://ClinicalTrials.gov/show/NCT04198818.
6
(
26−636.
6) Caunt, C. J.; Sale, M. J.; Smith, P. D.; Cook, S. J. Mek1 and
mek2 inhibitors and cancer therapy: The long and winding road. Nat.
Rev. Cancer 2015, 15, 577.
7) Kidger, A. M.; Sipthorp, J.; Cook, S. J. Erk1/2 inhibitors: New
weapons to inhibit the ras-regulated raf-mek1/2-erk1/2 pathway.
Pharmacol. Ther. 2018, 187, 45−60.
8) Roskoski, R. Targeting erk1/2 protein-serine/threonine kinases
(
(20) Morris, E. J.; Jha, S.; Restaino, C. R.; Dayananth, P.; Zhu, H.;
Cooper, A.; Carr, D.; Deng, Y.; Jin, W.; Black, S.; Long, B.; Liu, J.;
DiNunzio, E.; Windsor, W.; Zhang, R.; Zhao, S.; Angagaw, M. H.;
Pinheiro, E. M.; Desai, J.; Xiao, L.; Shipps, G.; Hruza, A.; Wang, J.;
Kelly, J.; Paliwal, S.; Gao, X.; Babu, B. S.; Zhu, L.; Daublain, P.;
Zhang, L.; Lutterbach, B. A.; Pelletier, M. R.; Philippar, U.;
Siliphaivanh, P.; Witter, D.; Kirschmeier, P.; Bishop, W. R.; Hicklin,
D.; Gilliland, D. G.; Jayaraman, L.; Zawel, L.; Fawell, S.; Samatar, A.
A. Discovery of a novel erk inhibitor with activity in models of
acquired resistance to braf and mek inhibitors. Cancer Discovery 2013,
3, 742−750.
(21) Heightman, T. D.; Berdini, V.; Braithwaite, H.; Buck, I. M.;
Cassidy, M.; Castro, J.; Courtin, A.; Day, J. E. H.; East, C.; Fazal, L.;
Graham, B.; Griffiths-Jones, C. M.; Lyons, J. F.; Martins, V.; Muench,
S.; Munck, J. M.; Norton, D.; O’Reilly, M.; Palmer, N.; Pathuri, P.;
Reader, M.; Rees, D. C.; Rich, S. J.; Richardson, C.; Saini, H.;
Thompson, N. T.; Wallis, N. G.; Walton, H.; Wilsher, N. E.;
Woolford, A. J.-A.; Cooke, M.; Cousin, D.; Onions, S.; Shannon, J.;
Watts, J.; Murray, C. W. Fragment-based discovery of a potent, orally
bioavailable inhibitor that modulates the phosphorylation and
catalytic activity of erk1/2. J. Med. Chem. 2018, 61, 4978−4992.
(22) Munck, J. M.; Berdini, V.; Bevan, L.; Brothwood, J. L.; Castro,
J.; Courtin, A.; East, C.; Ferraldeschi, R.; Heightman, T. D.; Hindley,
C. J.; Kucia-Tran, J.; Lyons, J. F.; Martins, V.; Muench, S.; Murray, C.
(
in human cancers. Pharmacol. Res. 2019, 142, 151−168.
9) Sullivan, R. J.; Infante, J. R.; Janku, F.; Wong, D. J. L.; Sosman, J.
(
A.; Keedy, V.; Patel, M. R.; Shapiro, G. I.; Mier, J. W.; Tolcher, A. W.;
Wang-Gillam, A.; Sznol, M.; Flaherty, K.; Buchbinder, E.; Carvajal, R.
D.; Varghese, A. M.; Lacouture, M. E.; Ribas, A.; Patel, S. P.;
DeCrescenzo, G. A.; Emery, C. M.; Groover, A. L.; Saha, S.;
Varterasian, M.; Welsch, D. J.; Hyman, D. M.; Li, B. T. First-in-class
erk1/2 inhibitor ulixertinib (bvd-523) in patients with mapk mutant
advanced solid tumors: Results of a phase i dose-escalation and
expansion study. Cancer Discovery 2018, 8, 184.
(
10) Blake, J. F.; Burkard, M.; Chan, J.; Chen, H.; Chou, K.-J.; Diaz,
D.; Dudley, D. A.; Gaudino, J. J.; Gould, S. E.; Grina, J.; Hunsaker, T.;
Liu, L.; Martinson, M.; Moreno, D.; Mueller, L.; Orr, C.; Pacheco, P.;
Qin, A.; Rasor, K.; Ren, L.; Robarge, K.; Shahidi-Latham, S.; Stults, J.;
Sullivan, F.; Wang, W.; Yin, J.; Zhou, A.; Belvin, M.; Merchant, M.;
Moffat, J.; Schwarz, J. B. 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. J. Med. Chem. 2016, 59, 5650−5660.
1
2302
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
W.; Norton, D.; O’Reilly, M.; Reader, M.; Rees, D. C.; Rich, S. J.;
Richardson, C. J.; Shah, A. D.; Stanczuk, L.; Thompson, N. T.;
Wilsher, N. E.; Woolford, A. J.-A.; Wallis, N. G. Astx029, a novel dual-
mechanism erk inhibitor, modulates both the phosphorylation and
catalytic activity of erk. Mol. Cancer Ther. 2021, DOI: 10.1158/1535-
7
(
163.mct-20-0909.
23) Kato, M. Intestinal first-pass metabolism of cyp3a4 substrates.
Drug Metab. Pharmacokinet. 2008, 23, 87−94.
24) Waring, M. J. Defining optimum lipophilicity and molecular
(
weight ranges for drug candidatesmolecular weight dependent
lower logd limits based on permeability. Bioorg. Med. Chem. Lett.
2
(
009, 19, 2844−2851.
25) Lovering, F.; Bikker, J.; Humblet, C. Escape from flatland:
Increasing saturation as an approach to improving clinical success. J.
Med. Chem. 2009, 52, 6752−6756.
(26) Lamanna, C.; Bellini, M.; Padova, A.; Westerberg, G.; Maccari,
L. Straightforward recursive partitioning model for discarding
insoluble compounds in the drug discovery process. J. Med. Chem.
2
(
008, 51, 2891−2897.
27) Delorme, D.; Ducharme, Y.; Brideau, C.; Chan, C.-C.; Chauret,
N.; Desmarais, S.; Dubé, D.; Falgueyret, J.-P.; Fortin, R.; Guay, J.;
Hamel, P.; Jones, T. R.; Lépine, C.; Li, C.; McAuliffe, M.; McFarlane,
C. S.; Nicoll-Griffith, D. A.; Riendeau, D.; Yergey, J. A.; Girard, Y.
Dioxabicyclooctanyl Naphthalenenitriles as Nonredox 5-Lipoxygenase
Inhibitors: Structure−Activity Relationship Study Directed toward the
Improvement of Metabolic Stability. J. Med. Chem. 1996, 39, 3951−
3
(
970.
28) Roberts, A. W.; Davids, M. S.; Pagel, J. M.; Kahl, B. S.; Puvvada,
S. D.; Gerecitano, J. F.; Kipps, T. J.; Anderson, M. A.; Brown, J. R.;
Gressick, L.; Wong, S.; Dunbar, M.; Zhu, M.; Desai, M. B.; Cerri, E.;
Heitner Enschede, S.; Humerickhouse, R. A.; Wierda, W. G.;
Seymour, J. F. Targeting bcl2 with venetoclax in relapsed chronic
lymphocytic leukemia. N. Engl. J. Med. 2016, 374, 311−322.
(29) Smith, C. C.; Levis, M. J.; Litzow, M. R.; Perl, A. E.; Altman, J.
K.; Gill, S.; Yuen, G.; Bonate, P.; Kadokura, T.; James, A. J.; Liu, C.;
Nagase, I.; Fisniku, O.; Bahceci, E. Pharmacokinetics and
pharmacodynamics of gilteritinib in patients with relapsed or
refractory acute myeloid leukemia. J. Clin. Oncol. 2016, 34, 7026.
(
30) ASTX029: Study of astx029 in subjects with advanced solid
tumors. https://ClinicalTrials.gov/show/NCT03520075.
31) Aronov, A. M.; Tang, Q.; Martinez-Botella, G.; Bemis, G. W.;
(
Cao, J.; Chen, G.; Ewing, N. P.; Ford, P. J.; Germann, U. A.; Green, J.;
Hale, M. R.; Jacobs, M.; Janetka, J. W.; Maltais, F.; Markland, W.;
Namchuk, M. N.; Nanthakumar, S.; Poondru, S.; Straub, J.; ter Haar,
E.; Xie, X. Structure-guided design of potent and selective
pyrimidylpyrrole inhibitors of extracellular signal-regulated kinase
(
6
erk) using conformational control. J. Med. Chem. 2009, 52, 6362−
368.
32) Squires, M. S.; Feltell, R. E.; Wallis, N. G.; Lewis, E. J.; Smith,
(
D.-M.; Cross, D. M.; Lyons, J. F.; Thompson, N. T. Biological
characterization of at7519, a small-molecule inhibitor of cyclin-
dependent kinases, in human tumor cell lines. Mol. Cancer Ther. 2009,
8
(
, 324−332.
33) Workman, P.; Aboagye, E. O.; Balkwill, F.; Balmain, A.; Bruder,
G.; Chaplin, D. J.; Double, J. A.; Everitt, J.; Farningham, D. A. H.;
Glennie, M. J.; Kelland, L. R.; Robinson, V.; Stratford, I. J.; Tozer, G.
M.; Watson, S.; Wedge, S. R.; Eccles, S. A. Guidelines for the welfare
and use of animals in cancer research. Br. J. Cancer 2010, 102, 1555.
(
34) Hollands, C. The animals (scientific procedures) act 1986.
Lancet 1986, 328, 32−33.
35) Wilkinson, G. R.; Shand, D. G. Commentary: A physiological
approach to hepatic drug clearance. Clin. Pharmacol. Ther. 1975, 18,
77−390.
(
3
1
2303
https://doi.org/10.1021/acs.jmedchem.1c00905
J. Med. Chem. 2021, 64, 12286−12303