Discovery of a Potent and Selective Oral Inhibitor of ERK1/2 (AZD0364) That Is Efficacious in Both Monotherapy and Combination Therapy in Models of Nonsmall Cell Lung Cancer (NSCLC)

Article abstract of DOI:10.1021/acs.jmedchem.9b01295

The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC₅₀ = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.

Full text of DOI:10.1021/acs.jmedchem.9b01295

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Drug Annotation
Discovery of a potent and selective oral inhibitor of ERK1/2
(AZD0364) that is efficacious in both monotherapy and combination
therapy in models of non-small cell lung cancer (NSCLC)
Richard A Ward, Mark J Anderton, Paul A. Bethel, Jason Breed, Calum Cook, Emma Davies,
Andrew Dobson, Zhiqiang Dong, Gary Fairley, Paul Farrington, Lyman J. Feron, Vikki Flemington,
Francis D Gibbons, Mark A. Graham, Ryan David Robert Greenwood, Lyndsey Hanson, Philip
Hopcroft, Rachel Howells, Julian Hudson, Michael James, Clifford D. Jones, Christopher Jones,
Yongchao Li, Scott Lamont, Richard James Lewis, Nicola A. Lindsay, James Francis McCabe,
Thomas M. McGuire, Philip B. Rawlins, Karen Roberts, Linda Sandin, Iain Simpson, Steve
Swallow, Jia Tang, Gary Tomkinson, Michael Tonge, Zhenhua Wang, and Baochang Zhai
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01295 • Publication Date (Web): 11 Nov 2019
Downloaded from pubs.acs.org on November 11, 2019
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Discovery of a potent and selective oral inhibitor of ERK1/2 (AZD0364) that is
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efficacious in both monotherapy and combination therapy in models of non-small cell
lung cancer (NSCLC)
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Richard A. Ward* , Mark J. Anderton , Paul Bethel , Jason Breed , Calum Cook , Emma J.
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Zhenhua Wang , Baochang Zhai .
1Oncology and Discovery Sciences R&D, AstraZeneca, Darwin Building, 310 Cambridge
Science Park, Milton Rd, Cambridge, CB4 0WG and AstraZeneca, Hodgkin Building, c/o
Darwin Building, 310 Cambridge Science Park, Milton Rd, Cambridge, CB4 0WG, UK.
2Chemical Development, Pharmaceutical Technology & Development, AstraZeneca,
Macclesfield Campus, SK10 2NA.
3Bioscience, Oncology R&D. AstraZeneca, Alderley Park, Macclesfield, SK10 4TG, UK.
4
Former employee of AstraZeneca.
5Pharmaron Beijing Co., Ltd., 6 Taihe Road BDA, Beijing, 100176, P.R. China.
6DMPK, Oncology R&D. AstraZeneca, Waltham, Massachusetts, USA.
7Medicinal Chemistry, Respiratory, Inflammation and Autoimmune (RIA),
BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
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Keywords
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ERK1/2; inhibitors; RAS/RAF/MEK/ERK signaling pathway; structure-based drug design;
SBDD; oncology.
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Abstract
The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in
approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes.
The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within
this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by
the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF
V600E/K metastatic melanoma, however resistance frequently develops. Importantly, ERK1/2
inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK
inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF
V600E/K melanoma. We describe our structure-based design approach leading to the
discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364
exhibits high cellular potency (IC = 6 nM) and excellent physico-chemical and absorption,
5
0
distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging
anti-tumour activity in pre-clinical models.
Introduction
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The MAPK (RAS/RAF/MEK/ERK) pathway has been intensively studied for many years and
is known to play a key role in cell cycle progression, protein synthesis, differentiation,
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migration, survival and senescence. Receptor tyrosine kinases (RTKs), G protein-coupled
receptors (GPCRs) and cytokine receptors can activate the pathway which subsequently causes
the activation of KRAS, HRAS and NRAS. When this pathway is activated signals are
transmitted downstream through the RAF family of kinases (BRAF, CRAF and ARAF) and
MEK1/2. Both ERK1 and ERK2 can be activated by MEK1 or MEK2 through
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phosphorylation of the T-E-Y motif in the kinase activation loop. Due to its relative
positioning on the MAPK pathway, both ERK1 and ERK2 control the output by activating
their (numerous) nuclear and cytoplasmic substrates. There is a clear rationale for targeting
this pathway by small molecule inhibitors, where a high frequency of mutations has been
3
identified in diseases such as melanoma, thyroid and colorectal cancer. In addition, members
of the RAS family are mutated in colorectal, melanoma and pancreatic tumours. Multiple drug
approvals, such as vemurafenib and dabrafenib, have now been reported which target the
4
-6
V600E mutation of BRAF. However, resistance to these treatments occur rapidly from
MAPK pathway re-activation and a combination approach of BRAF and MEK inhibitors (such
as trametinib) is now commonly used in melanoma.7-8
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Cl
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Figure 1 – Selection of published ERK1/2 inhibitors, 1 (SCH772984), 2 (SCH900353), 3
Ulixertinib/BVD-523), 4 (GDC-0994), 5 (LY-3214996) and 6 (CC-90003).
(
As an additional potentially ‘druggable’ target on the pathway, the direct inhibition of ERK1/2
may provide valuable therapeutic options for cancers driven by BRAF or RAS mutations.
Alternatively, ERK1/2 inhibitors could provide an alternative treatment for patients who have
9
progressed on RAF and or MEK inhibitor treatment due to pathway reactivation. Such interest
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has resulted in a number of small molecule ERK1/2 inhibitors being reported in recent years
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(
Figure 1), including 1 (SCH772984), 2 (SCH900353), 3 (Ulixertinib/BVD-523), 4 (GDC-
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0994) , 5 (LY-3214996) and 6 (CC-9003) . In addition, there are reports of ERK1/2
inhibitors being evaluated for clinical utility where their chemical structures are yet to be
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disclosed, including examples from Novartis, Kura Oncology, Astex and Asana. The
identification of high quality, potent and selective inhibitors of ERK1/2 therefore continues to
be an area of great interest for drug discovery efforts in oncology.
We have previously reported the discovery of a novel, potent and selective inhibitor of ERK1/2
(7), optimised from a promiscuous chemical start point informed by structure-based drug
19
design. This inhibitor binds to the ATP pocket and is active via both inhibition of catalysis
(IOC) and prevention of activation (POA) mechanisms and has demonstrated anti-tumour
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activity in pre-clinical models of cancer. The IOC activity of ERK1/2 inhibitors can be
measured by the impact on levels of phospho-RSK and POA activity can be evaluated through
changes in phospho-ERK. In this manuscript we report on our efforts to further develop this
series to a molecule with suitable properties for clinical candidate evaluation. Our primary
design objectives were to increase the ERK1/2 potency whilst maintaining, or possibly
improving, the kinase selectivity and ADME properties to enable a low predicted human dose.
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ERK2 Biochemical,
k ATP IC (µM)
<0.0003
M
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ERK2 Biochemical, High ATP IC (µM)
0.0005
0.012 /
5
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Cell MOA A375 pERK/pRSK IC (µM)
5
0
0
.062
Log D7.4a
2.5
Aqueous Solubility (µM)b
>480
9
Kinase Selectivity
N>80% out of 128 Kinase panel)
c
(
Table 1 – Profile of lead compound (7). All IC data are reported as µM and are the mean
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a
of at least three independent measurements. Distribution coefficient between 1-octanol and
b
aqueous phosphate buffer at pH 7.4. Solubility of compounds in aqueous phosphate buffer
c
at pH 7.4 after 24 h at 25 °C (μM). Kinase selectivity data measured at 1 µM compound
concentration, two of these 9 kinases were ERK1 and ERK2 with three being coupled assays
with ERK (MEK1, BRAF and CRAF).
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Results and discussion
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Scaffold hopping. Having reported on the optimisation of our early pyrololactam-based series
and then the subsequent move to a pyrrolopyrazinone core we decided to identify additional
scaffolds which may enable us to improve target potency and provide potential for improved
kinase selectivity. A selection of cores were evaluated using the quantum mechanical (QM)
computational approach described in our previous manuscript to ensure that the appropriate
binding mode conformation could be fulfilled with minimal energetic penalty. One addition
was made to this protocol due to the observation that a methyl group on the 5-position of the
pyrimidine consistently increased potency. Therefore, conformational calculations were also
carried out with this substituent to understand if it was likely to be tolerated. The most
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interesting potential core from this analysis was the dihydroimidazopyrazinone core (B, figure
o
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) where the binding mode appeared to be more energetically favoured over the 180 flipped
conformation when compared to our previous pyrrolopyrazinone core (A). Using the higher
level B3LYP/6-31G* basis set it can be observed that the binding mode (at zero degrees) is
favoured by around 7-8 kcal/mol for the dihydroimidazopyrazinone (B) core, with little impact
of the additional methyl group. This profile can be rationalised by a potential clash of lone
pairs between the imidazole and pyrimidine groups in the higher energy conformation. In
addition, the methyl group has a preference to be positioned adjacent to the imidazole whereas
it would be expected to be less complimentary with the C-H in the alternative conformation.
This compares to the pyrrolopyrazinone (A) where this difference was around 2 kcal/mol
o
between the expected binding mode and 180 flipped conformation.
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Figure 2 – QM torsion profile of an alternative dihydroimidazopyrazinone ring system (B)
compared to pyrrolopyrazinone core (A) with and without methyl group substituent. Gas
phase energy reported in kcal/mol calculated in Jaguar (Maestro version 2016-1,
Schrodinger) using a relaxed coordinate scan with PM3 and B3LYP/6-31G* basis sets.
Binding mode consistent with zero degrees dihedral angle on these charts.
In addition to the energetic rationale above we also speculated that the introduction of a
hydrogen bond acceptor in this position on the core may enable an improved complementarity
with the gatekeeper residue in ERK1/2 (Gln-105), which is rare across the kinome (figure 3).21
The binding mode of a selected example from our previous disclosure (8) shows how a key
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water molecule forms a bridge between the carbonyl group on the inhibitor (2.7 Å) and Gln-
05 (3.5 Å). The distance between Gln-105 and the position of the introduced nitrogen on the
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core was expected to be around 4 Å using the 5NHP crystal structure as reference. Although
the distance between this nitrogen on the core and Gln-105 is potentially too far for a direct
interaction it is possible that the inhibitor and or gatekeeper residue could move closer to each
other if an appropriate interaction could be formed. Alternatively, and perhaps more likely,
this change may modulate the existing water mediated interactions between ligand and the
protein.
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Figure 3 – ERK2 x-ray crystal structure of series exemplar (8) bound in ERK1 (PDB Code
NHP) highlighting the potential to target Gln-105.
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In our previous scaffold hopping efforts, we initially synthesised a simplified version of novel
cores for evaluation against our original pyrrololactam scaffold. However, in this case with
greater confidence in our approach, we decided to initially synthesise more elaborated
examples, using pyrrolopyrazinone 9 as a suitable reference molecule. Compound 10 proved
to be an interesting lead molecule as the biochemical and pRSK cell MOA activity was broadly
comparable to 9, but Log D was significantly reduced from 3.5 to 2.5. This modulation of
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.4
Log D is consistent with the observed increase in aqueous solubility (112 versus 2 µM) and
7
.4
−1
reduced human liver microsome (HLM) intrinsic clearance (CL ) (13 versus 32 μL min
nt
−
1
mg ). A particular attraction of this reduction in Log D was that it would enable the use of
7
.4
different SAR learnt on the original pyrololactam and pyrrolopyrazinone cores, in particular
SAR enabling increased potency, but which previously resulted in excessively high overall
lipophilicity. Compound 10 also showed improved kinase selectivity to 9 (11 hits >80%
compared to 22), suggesting our efforts to modulate the gatekeeper interaction may have
proven beneficial. One further interesting difference between these two molecules was that 10
demonstrated a dual IOC/POA (i.e. pRSK ~ pERK) profile as opposed to 9 where the data
suggests an IOC only profile (i.e. pRSK > pERK).
ACS Paragon Plus Environment

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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
Cl
N
N
O
O
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
N
N
N
H
N
N
N
H
N
9
9
10
10
ERK2 Biochemical,
k ATP IC (µM)
<0.0003
<0.0003
M
50
ERK2 Biochemical,
High ATP IC (µM)
0.00013
0.00034
5
0
Cell MOA A375
pERK/pRSK IC50
(µM)
>14 /
0.01
0.0047 /
0.012
Log D7.4a
3.5
2
2.5
Aqueous Solubility
112
b
(µM)
In vitro Clearance
HLM
32
22
13
11
(μL/min/mg protein)
Kinase Selectivity
(
N>80% out of 140
Kinase panel)
c
Table 2 – Scaffold hopping to dihydroimidazopyrazinone core. All IC data are reported as
5
0
a
µM and are the mean of at least three independent measurements. Distribution coefficient
c
between 1-octanol and aqueous phosphate buffer at pH 7.4. Solubility of compounds in
aqueous phosphate buffer at pH 7.4 after 24 h at 25 °C (μM). Kinase selectivity data
ACS Paragon Plus Environment

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Journal of Medicinal Chemistry
measured at 1 µM compound concentration, one of these kinases was ERK2 with three being
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
coupled assays with ERK (MEK1, BRAF and CRAF).
Scaffold elaboration. As we speculated that the SAR around the dihydroimidazopyrazinone
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
core may be broadly consistent in terms of potency with our earlier cores we utilised a Free-
2
2
Wilson approach to rapidly identify preferred substituents based on our previously
synthesised compounds. Using this approach, substituents were computationally stripped from
the respective cores using in-house software and exhaustively enumerated producing 9,432
potential virtual target structures (figure 4). The biochemical (high ATP) and cellular activity
(pRSK) was then estimated on the enumerated set by Free-Wilson theory and used to prioritise
compounds along with prediction of Log D (1.5-3.5) and intrinsic permeability (CaCo2 A
7
.4
-6
23
to B P >5 x 10 cm/s). These models were quantitative structure property relationship
app
(QSPR) based models using a range of 2D descriptors and trained on internal data. Whilst
prioritising potential target molecules we initially focused on examples with the preferred
methyl in the 5-position of the pyrimidine and the specified pyrazolo solvent channel group.
In addition, some groups were deprioritised based on wider data generated such as
pharmacokinetic (PK), kinase selectivity or hERG data.
R1
R3
R1
R3
R1
R3
N
*
N
N
O
O
O
*
N
N
*
*
N
N
*
R4
R4
N
N
N
R2
R2
N
N
N
N
N
N
N
N
H
H
H
Free-Wilson based
enumeration output
on preferred core
Filtered output
with preferred
substituents
Full dataset
A
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
Figure 4 – Representation of Free-Wilson analysis. A. Substructures of compounds in the
full dataset, versus the Free-Wilson enumeration on the novel core and filtered output using
preferred substituents. B. Representation of Free-Wilson analysis by R1-R3 coloured by
predicted biochemical pIC coefficient
5
0
.
F
F
F
F
F
O
F
Cl
F
R1=
Cl
R1
R2
N
O
N
7
N
Rank=
2
3
4
5
6
1
F
N
F
R2=
N
O
H
N
N
N
H
Rank=
1
2
3
4
Figure 5 – Selection of favoured substituents from Free-Wilson analysis enumerated on the
dihydroimidazopyrazinone core. Subsituents ordered using the free-wilson coefficient based
on biochemical activity as a ranking.
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Journal of Medicinal Chemistry
A selection of favoured R1 and R3 groups emerged from the Free-Wilson analysis (Figure 5)
are shown ranked by the Free-Wilson coefficient from the high ATP biochemical activity data.
Acceptable combinations of these were identified by the calculation of predicted properties
within the physicochemical property space highlighted therefore combinations were not
synthesised exhaustively. Although our preferred LogD7.4 range to target reasonable
physicochemical properties and favourable permeability was between 2 and 3, these
constraints were relaxed when filtering to account for model error. One of the most interesting
early dihydroimidazopyrazinone core examples synthesised was 11 (Table 2), with a pRSK
cellular activity of 0.031 µM. The specific SAR which was identified from the Free-Wilson
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
rd
approach was the 2,3-difluoro group (ranked 3 ), exemplified by pyrrolopyrazinone 12, which
binds in the glycine rich loop but when utilised on this core the molecule suffered from poor
aqueous solubility (7 µM), likely driven by high Log D (3.2). However, consistent with the
7
.4
initial observation around the potential for reduced lipophilicity of this core, the Log D of
7.4
1
1 was 2.2 with aqueous solubility of 277 µM. As had been observed with our first
dihydroimidazopyrazinone 10, improved kinase selectivity was seen with this novel core (6 vs
0 hits >80% inhibition). A second key piece of SAR which the Free-Wilson approach
2
highlighted was a methoxymethyl substituent on the pyrrolopyrazinone core (ranked 1st),
exemplified by 13, where our previous aim had been to exploit the glycine rich loop region
using our knowledge of the binding mode. Consistent with our design hypothesis, we observed
good pRSK cell activity of 0.06 µM, compared to 0.15 µM for the S-enantiomer. As for to the
2
,3-difluro group discussed previously, we believed this SAR could only be fully utilised on
the pyrrolopyrazinone core with intrinsically less potent (but less lipophilic) heterocyclic
groups in the adjacent position. However, the dihydroimidazopyrazinone core matched pairs
were predicted to be highly potent and in a reasonable Log D range therefore the methyl (14)
7
.4
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and methoxymethyl (15) substituted examples were both synthesised in the R-enantiomeric
form. 14 demonstrated a slightly increased potency in the cell MOA assay with IC 0.0034 vs
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
0
0.0091 µM in pERK compared to matched pair 11 lacking the additional methyl group, but
equivalent activity in pRSK (0.038 vs 0.031 µM). Whereas, compound 15 demonstrated a
particularly interesting profile with impressive pRSK potency of 0.0057 µM and 0.0017 µM
potency in pERK. In addition, the molecule had excellent physicochemical properties
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
aqueous solubility 468 µM), low HLM and hepatocyte CL , good intrinsic permeability in
int
-6
CaCo2 (P A to B 26.7 10 cm/s) with acceptable hERG (11 µM). Compound 15 was the
app
th
16 compound synthesised with the dihydroimidazopyrazinone core and approximately 50
were synthesised in total, 15 subsequently became our clinical candidate AZD0364. This speed
at which this molecule was identified on this novel core provides some validation as to the
value of the highlighted Free-Wilson approach. An additional observation of note with
compounds 13-15 is that the cellular activity data are consistent with a dual IOC and POA
profile. Additional potent and selective compounds were synthesised, in addition to examples
varying R2, but no improvement was observed over 15 in terms of the overall profile.
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Journal of Medicinal Chemistry
F
F
F
F
1
2
3
4
5
6
7
8
9
F
F
F
F
N
O
O
N
N
N
N
N
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
N
N
N
N
H
N
N
N
H
N
N
N
H
N
N
N
H
N
N
N
H
N
1
1
12
13
12
14
15
1
1
13
14
15
ERK2 Biochemical,
k ATP IC (µM)
0.00083
<0.0003
<0.0003
<0.0003
<0.0003
M
50
ERK2 Biochemical,
High ATP IC50
0.0026
0.00035
0.0006
0.00032
0.00066
(
µM)
Cell MOA A375
pERK/pRSK IC50
0.0091 /
0.031
ND /
0.02
0.03 /
0.06
0.0034 /
0.038
0.0017 /
0.0057
(
µM)
Log D7.4a
2.2
3.2
7
2.5
2.5
99
2.3
Aqueous Solubility
277
725
468
b
(
µM)
Kinase Selectivity
N>80% out of 140
6
20
6
7
5
(
c
Kinase panel)
In vitro Clearance
HLM (µL/min/mg
protein)
4.3
20.7
(10.9/ ND)
14.6
(12.7/<1)
8.3
(3.9/<1)
8.7
(7.1/ ND)
(10.2/<1)
(Rat/Human Heps
6
(
µL/min/x10
d
cells))
CaCo2 A to B P
+ inhibitors (10
cm/s)
ND
23
ND
ND
49
20
ND
23
26.7
11
app
-6
hERG IC50
(
µM)
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Table 2 – Dihydroimidazopyrazinone core examples synthesised guided by Free-Wilson
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
approach. All IC data are reported as µM and are the mean of at least three independent
5
0
a
measurements. Distribution coefficient between 1-octanol and aqueous phosphate buffer at
b
pH 7.4. Solubility of compounds in aqueous phosphate buffer at pH 7.4 after 24 h at 25 °C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
(
μM). Kinase selectivity data measured at 1 µM compound concentration, one of these
kinases was ERK2 with three being coupled assays with ERK (MEK1, BRAF and CRAF).
d
−1
−1
Human microsome (μL min mg ) and rat hepatocytes metabolism intrinsic clearance (μL
−1
−1
min million cells ). ND, not determined.
Chemistry. Synthesis of 15 was performed in a 14-step overall sequence as shown in Scheme
1
. Two key building blocks were required before converging for the remaining 6 steps.
Synthesis of the first building block was 5 steps starting with a SEM protection of
commercially available ethyl 1H-imidazole-2-carboxylate. This imidazole intermediate was
then brominated, converted to the boronic acid before a Suzuki-Miyaura cross coupling was
carried out with 2,4-dichloro-5-methylpyrimidine. A final SEM deprotection step then
afforded 15e. The second building block was a shorter 3 step sequence starting with reduction
of commercial (S)-2-((tert-butoxycarbonyl)amino)-3-methoxypropanoic acid, followed by
cyclisation and oxidation to afford the BOC-sulfamidate 15h. Ring opening of this BOC-
sulfamidate with the imidazole NH of 15e, followed by double deprotection afforded primary
amine 15k as a bis-hydrochloride salt. Treatment of the salt with methanolic ammonia
promoted cyclisation to the dihydroimidazopyrazinone core. Alkylation with 4-
(Bromomethyl)-1,2-difluorobenzene and a final Buchwald-Hartwig cross coupling with 2nd
generation XantPhos precatalyst afforded 15. Full compound synthesis experimental details
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Journal of Medicinal Chemistry
and characterisation for 15 can be found in the experimental section, and all compound
synthesis details can be found in the supporting information, S1.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OH
B
N
N
N
O
O
Br
N
N
O
O
N
N
O
O
Cl
N
N
O
O
HO
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
O
Cl
N
N
O
O
N
H
O
O
O
O
(i)
(iI)
(iii)
N
H
(iv)
(v)
Si
Si
Si
Si
1
5a
15b
15c
15d
15e
H
O
H
O
O
O
O
S
O
O
O
S
O
O
O
HN
(ix)
HN
O
N
O
N
O
O
O
(vii)
(viii)
(vi)
O
O
O
O
15f
15h
1
5g
N
N
N
N
O
O
O
Cl
N
N
N
O
N
N
N
N
N
N
N
Cl
Cl
Cl
O
O
O
N
N
NH
(xii)
(xi)
O
O
(x)
O
O
NH2 .2HCl
O
N
H
O
O
O
N
OH
O
S
O
15l
1
5k
1
5j
1
5i
(xiii)
N
F
N
F
HN
N
N
O
N
Cl
N
N
N
O
F
F
N
N
N
N
(xiv)
O
O
1
5m
15
Scheme 1 – Synthesis of 15.
a
Reagents and conditions: (i) SEM-Cl, K CO , acetone, 0°C, 85%; (ii) NBS, DMF, DCM,
2
3
0
°C, 51%; (iii) 2nd Generation XPhos precatalyst, 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-
dioxaborolane), KOAc, 1,4-dioxane, 25°C, quant; (iv) Pd(Ph P) 2,4-dichloro-5-
3
4,
methylpyrimidine, Cs CO 1,4-dioxane, water, 85°C, 70% (v) TFA, DCM, 25°C, 93%; (vi)
2
3,
(a) isobutyl chloroformate, N-methylmorpholine, THF, 0 °C, (b) NaBH4, H2O. 52%; (vii)
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imidazole, thionyl chloride, NEt , DCM, -78°C, 56%; (viii) sodium metaperiodate,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ruthenium(III) chloride, MeCN, water, 0°C, 80%; (ix) K CO , 18-crown-6, 1,4-dioxane,
2
3
100°C, 100%; (x) 1M HCl, EtOH, 50°C, 24% (xi) 4M HCl in 1,4-dioxane, 25°C, 85%; (xii)
M NH in MeOH, 25°C, 97%; (xiii) 4-(Bromomethyl)-1,2-difluorobenzene, NaH, DMF,
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5°C, 95%; (xiv) 2nd Generation XantPhos precatalyst, 1-methyl-1H-pyrazol-5-amine,
Cs CO 1,4-dioxane, 100°C, 64%.
2
3,
Kinase selectivity. The wider kinase selectivity of 15 was then evaluated with only 14 kinase
assays out of 353 in the larger panel showing % inhibition > 80% (including four separate
CDK2 assays), with a compound concentration of 1 µM. However, the MEK1, MEK2, COT,
BRAF and CRAF assays listed are all coupled pathway assays using ERK protein as part of
the assay cascade, therefore, an inhibitor of ERK1/2 will show activity in these assays. In the
same assay panel, the single-protein binding assays for MEK1, MEK2, BRAF and CRAF
showed only -2.5%, -2.2%, 5.8% and -3.5% binding, respectively; therefore, 15 is not
considered to be active against these kinases. A single-protein binding assay for COT was not
available. Follow-up testing in these assays showed that ERK7 binding affinity for AZD0364
was 0.03 µM, ARK5 had an IC50 of 0.4 µM and CDK5 had IC50s of 0.3 µM in both
CDK5/p25 and CDK5/p35 activity assays with a binding affinity for inactive CDK5 of 1.9
µM. Further biochemical testing was performed with in-house assays for both MEK1 and
CDK2. 15 was inactive against the in-house MEK1 assay at the concentration range tested
(
>10 µM) and had an IC of 1 µM against CDK2-cyclin E.
5
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Journal of Medicinal Chemistry
Kinase
% Kinase
inhibition/binding
1
2
3
4
5
6
7
8
9
(@ 1µM)
MAP2K1 (MEK1)
MAPK3 (ERK1)
MAP3K8 (COT)
BRAF
104
105
103
102
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MAPK1 (ERK2)
MAP2K2 (MEK2)
RAF1 (cRAF) Y340D
Y341D
MAPK15 (ERK7)
CDK2/cyclin O
CDK2/cyclin A1
102
101
101
99
95
91
(binding)
CDK2/cyclin E1
CDK2/cyclin A (activity)
CDK5/p25
89
87
81
80
NUAK1 (ARK5)
Table 3 – Kinase panel selectivity of 15. Kinases in the ThermoFisher 353 wild-type kinase
panel that showed greater than 80% inhibition/binding after 1 µM treatment with 15 are
shown (data combines two experiments - one using a sub-panel of 122 kinases and one using
the full 353 wild-type panel). Kinase assays which include inactive ERK2 protein as part of a
coupled cascade assay are highlighted in bold.
Binding mode elucidation. ERK2 x-ray crystallography confirmed the binding mode of 15
in the ATP pocket and was broadly consistent with that of our previously disclosed series in
terms of the key interactions and scaffold position. The pyrimidine group forms a key
interaction with Met-108 and the pyrazole group form a hydrogen bond to Lys-114. The 2-3-
difluoro group is directed into the glycine-rich loop of the kinase contacting residues such as
Val-39. The additional methoxymethyl group also makes contacts with Val-39 and
hydrophobic interactions with Ile-31, consistent with improved potency and selectivity. The
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1
9
commonly conserved water molecule is present and the additional nitrogen on the
dihydroimidazopyrazinone core has the potential to be involved in the interactions between
the bridged water molecule (3.5 Å) and gatekeeper Gln-105 (2.7 Å). This water molecule is
also in contact range of a second water molecule (2.6 Å) which is hydrogen bonded to Glu-71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2.7 Å).
Figure 5 – Binding mode of 15 elucidated in ERK2 by x-ray crystallography (PDB Code
SLG).
6
Further profiling of 15 was carried out to assess the wider properties of the compound for
evaluation in vivo and potential clinical candidate nomination. Gratifyingly, the excellent
cellular MOA activity of 15 translated well into antiproliferative effects in the A375 (GI 0.06
5
0
µM) and Calu-6 (GI 0.2 µM) cell lines. The A375 cell line contains a BRAF V600E mutation
5
0
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Journal of Medicinal Chemistry
and Calu-6 a KRAS Q61K mutation. The physicochemical properties of the initial amorphous
material were very good (aqueous solubility 468 µM), however identifying a suitable method
of obtaining crystalline material proved more challenging and will be covered in a later section.
The in vitro clearance appeared relatively low in rodent species (rat heps 8.7 μL/min/million
cells) suggesting this was a suitable example for pre-clinical in vivo evaluation of
pharmacokinetics (PK), pharmacodynamics (PD) and efficacy in anti-tumour models.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Target residence time. An additional interesting property of 15 was revealed when evaluated
using surface plasma resonance (SPR) (Table 4), where the molecule demonstrated equivalent
affinity to phosphorylated and unphosphorylated ERK2 (pK 10.3 and 10 respectively) but
d
with a relatively long residence time in both forms (t = 277 and 194 minutes respectively).
1
/2
Interestingly, when a broader set of ERK1/2 inhibitors were tested in the same way we
observed clear differences in their kinetic behaviour. 15 has greater affinity against both
phosphorylated and non-phosphorylated forms of ERK2 than our chemical start point for this
work 7, in addition to increased cellular activity and longer residence time. Compound 1
(SCH772984) also demonstrated high ERK2 affinity against both forms, high cellular potency
along with extended residence times against both forms with some similarities to 15. These
data suggest that such levels of potency (and inhibitor to target residence times) can be
achieved without need for the extended group present in SCH772984. However, 3 (BVD-523)
appears to have differential binding kinetics with higher affinity and a longer residence time
against the phosphorylated form of ERK2 than the non-phosphorylated form. Interestingly,
this example appears to have an IOC only profile with reduced cellular activity against pERK.
Compound 4 (GDC-0994) has reduced, but equivalent, affinity towards the phosphorylated
and non-phosphorylated forms of ERK2 and significantly shorter residence times which may
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be consistent with the weaker cellular activity of the compound. Overall, in this set of
compounds the association rate constants are broadly comparable, however they appear to
show differential behaviour in their dissociation rate constants with 15 and 1 having
particularly slow dissociation rate constants.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
pRSK pERK
ERK2
Binding
Mean
ERK2
kon
ERK2
koff
Mean
(1/s)
ERK2
t½ Mean
(min)
A375
Mean
IC50
A375
Mean
IC50
Mean
-
1 -1
(pKd )
(M .s )
(ph/non-ph)
(µM)
(µM) (ph/non-ph) (ph/non-ph) (ph/non-ph)
1
0.012 0.0025
10/10.3
1.3E06/
9.4E05
1.0E-04/
4.4E-05
127/291
(SCH772984)
3
(BVD-523)
(GDC-
0.15
3.1
9.5/8
1.3E06/
4.1E-04/
7.4E-03
1.6E-02/
9.9E-03
35/2
7
.5E05
4
0.086
0.031
8.8/8.8
9.8E06/
6.2E06
0.8/1.2
0
994)
7
0.044
0.012
9.8/9.1
10.3/10
1.9E06/
1.3E06
8.9E-05/
3.2E-04/
1.0E-03
4.5E-05/
6.0E-05
37/11.9
277/194
1
5
0.0057 0.0017
6
.5E05
Table 4 –Surface plasma resonance (SPR) data on a set of ERK1/2 inhibitors. All data
reported are a mean of at least three independent measurements. Ph=phosphorylated, non-
ph=non-phosphorylated.
Salt/co-crystal screening of compound 15. With a view of evaluating suitable formulations
for 15 in a clinical setting, x-ray powder diffraction had highlighted that early batches of 15
were non-crystalline. However, initial attempts to crystallise from a range of solvents yielded
amorphous or glassy materials. Such materials may be challenging to develop as they can be
chemically unstable, difficult to isolate in desired purity and yield and can be challenging to
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Journal of Medicinal Chemistry
2
4
formulate. A salt screening approach was therefore initiated using a large collection of
counter-ions containing a variety of functionalities (such as carboxylic acids, dicarboxylic
acids, sulfonic acids, disulfonic acids, aromatic acids, alcohols etc.) to increase the probability
of successful crystalline salt formation. From this evaluation crystalline hits were obtained for
the following acidic counter-ions: adipic, citric, ethanesulfonic, fumaric and hydrochloric and
phosphoric acids. Initial scale-up of HCl and phosphoric acid salts yielded poorly crystalline
materials that were not investigated further. Scale up of adipic and ethanesulfonic acid salts
yielded highly crystalline material with suitable melting points (185 ºC and 204 ºC
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
respectively). Given the weakly basic nature of 15 (pK ~2.6), the ethanesulfonic acid adduct
a
was considered likely to exist as a salt (proton transfer from acid to base) whereas the adipic
acid adduct was considered likely to exist as a cocrystal (no proton transfer from acid to base).
NMR analysis confirmed this was indeed the case, with the ethanesulfonate salt containing a
protonated basic nitrogen in 15 and an unprotonated basic nitrogen in the adipic acid cocrystal.
Given the low pK of 15, an ethanesulfonate salt was considered likely to be physically
a
unstable in aqueous media and this was the case which readily disproportionates back to the
amorphous free base upon exposure to water. The adipic acid co-crystal however is physically
stable in aqueous suspension up to pH 6.8, had favourable aqueous solubility across a relevant
pH range and has appropriate physiochemical properties to ensure low risk delivery at scale
and minimal formulation risk. Therefore, the adipic acid cocrystal had aqueous solubility of
1
42 µM and was selected to support the further development of 15.
In vivo testing of compound 15. The first in vivo anti-tumour model to be used to evaluate
this inhibitor was a Calu-6 (KRAS Q61H) model as we had reported on with our previously
disclosed inhibitor 7 (Figure 6). As a monotherapy treatment, 15 demonstrated 93% tumour
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growth inhibition (TGI) at a dose of 15 mg/kg QD and tumour regression (49%) at 50 mg/kg
QD. A PK/PD relationship was established as the reduction in levels of pRSK observed were
consistent with the PK (and binding kinetics) of the inhibitor. In this study almost total
knockdown of pRSK was observed at both 0.5 and 6 hour timepoints after the final dose of the
compound, with the free concentration of 15 showing a reduction at 6 hours. In addition to the
monotherapy study, 15 was evaluated in combination with the MEK1/2 inhibitor selumetinib
in an anti-tumour study in a A549 (KRAS G12C mutant) cell line which appeared to be less
sensitive to monotherapy treatment with an ERK1/2 inhibitor (Figure 7). The combination of
15 and selumetinib demonstrated tumour regressions (28% tumour regression) when both
compounds were dosed at 25 mg/kg, with the monotherapy arm for each showing only modest
inhibition of tumour growth.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
Figure 6 – Monotherapy anti-tumour activity of 15 in a Calu 6 (KRAS Q61H, panel A)
mouse model and end of study PD/PK analysis (panel B).
Figure 7 – Monotherapy and combination anti-tumour activity of 15 and selumetinib in a
A549 (KRAS G12C) mouse model.
Based on these data, the pre-clinical species PK and the in vitro human data were utilised to
predict the human PK.25 A range of human Cl predictions was generated from human
int
microsomes and hepatocytes (including different formats of hepatocytes to include plated and
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suspension), this gave rise to a range of predictions of clearance from 0.5 to 6 mL/min/Kg.
The human plasma protein binding (PPB) differences tested over a concentration range were
also reflected in the range of predictions. From these data, a predicted oral human dose of
between 20 and 110 mgs was calculated based on the requirement for free cover for 12 hours
over the Calu-6 GI , using BID dosing (Table 5). This range of predicted doses is consistent
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
with one of our previously set out design aims to identify a high-quality clinical candidate with
a low predicted human dose.
15
Cell MoA A375 pRSK / pERK IC µM
0.0017 / 0.0057
0.06 / 0.2
50
Proliferation A375 / Calu 6 GI µM
5
0
Plasma Protein Binding (% free)
Mouse, rat, dog, human)
12, 21, 44, 6-27
(
In vivo Pharmacokinetics
Rat
38
Dog
12
CL (mL/min/kg)
V (L/kg)
1.5
2.3
30
1.2
5
ss
1
t (h)
1
/2
F (%)
100
Human dose prediction (PO)
20 – 110 mg BID
Table 5 – Broader data overview of compound 15. All IC data are reported as µM and are
5
0
the mean of at least three independent measurements (A375 BRAF V600E, Calu-6 KRAS
1
Q61K). Oral administration.
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Journal of Medicinal Chemistry
Conclusion
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We have reported our drug discovery efforts on the identification of 15 (AZD0364) a potent
and selective ERK1/2 inhibitor which prevents activation of ERK1/2 and inhibits activation
of downstream targets. The ADME properties of 15 are favourable and enabled a low predicted
oral dose in human of between 20 and 110 mg BID. This inhibitor has excellent kinase
selectivity and has also been shown to have high affinity to both phosphorylated and non-
phosphorylated forms of ERK. 15 is also efficacious in disease relevant anti-tumour mouse
xenograft models of cancer as a monotherapy and in combination with the MEK1/2 inhibitor
selumetinib. Further disclosures will be reported in due course with broader pre-clinical
efficacy data in additional cell lines and disease models. Clinical trials evaluating AZD0364
are planned to commence later this year.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental
Synthesis and characterisation of compound 15
General chemistry statement
Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker 300, 400 or 500 MHz
1
instrument at room temperature unless otherwise stated. All H NMR spectra were measured
in part per million (ppm) relative to the signals for residual dimethyl sulfoxide (DMSO) in
deuterated DMSO (2.50 ppm), or the signals for tetramethylsilane (TMS) added into the
1
deuterated chloroform (0 ppm). Data for H NMR were reported as: chemical shift,
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