Optimization of Fused Bicyclic Allosteric SHP2 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01725

SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.

Full text of DOI:10.1021/acs.jmedchem.8b01725

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Article
Optimization of Fused-Bicyclic Allosteric SHP2 Inhibitors
Jeffrey Bagdanoff, Zhuoliang Chen, Michael G Acker, Ying-Nan P Chen, Homan Chan,
Michaël Doré, Brant Firestone, Michelle Fodor, Jorge Fortanet, Murphy Hentemann, Mitsunori
Kato, Robert Koenig, Laura R. LaBonte, Shumei Liu, Movarid Mohseni, Rukundo Ntaganda,
Patrick Sarver, Troy Smith, Martin Sendzik, Travis Stams, Stan Spence, Christopher
S. Towler, Hongyun Wang, Ping Wang, Sarah L. Williams, and Matthew J. LaMarche
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01725 • Publication Date (Web): 28 Jan 2019
Downloaded from http://pubs.acs.org on January 28, 2019
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Optimization of Fused-Bicyclic Allosteric SHP2 Inhibitors
a
a
b
b
b
b
e
Jeffrey T. Bagdanoff, Zhouliang Chen, Michael Acker, Ying-Nan Chen, Homan Chan,
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Michael Dore, Brant Firestone, Michelle Fodor, Jorge Fortanet, Murphy Hentemann,
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Mitsunori Kato, Robert Koenig, Laura R. LaBonte, Shumei Liu, Movarid Mohseni,
a
a
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Rukundo Ntaganda, Patrick Sarver, Troy Smith, Martin Sendzik, Travis Stams, Stan
b
e
b
b
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Spence, Christopher Towler, Hongyun Wang, Ping Wang, Sarah L. Williams, and
Matthew J. LaMarche*,a
aGlobal Discovery Chemistry, bOncology Disease Area, cChemical Biology &
d
Therapeutics, Pharmacokinetic Sciences, Novartis Institutes for Biomedical Research,
and eChemical and Pharmaceutical Profiling, Novartis Pharmaceuticals, 250
Massachusetts Avenue, Cambridge, Massachusetts, 02139, United States
ABSTRACT
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SHP2 is a non-receptor protein tyrosine phosphatase (PTP) within the MAPK
pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also
participates in the programed cell death pathway (PD-1/PD-L1) governing immune
surveillance. Small molecule inhibition of SHP2 has been widely investigated including
our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding
site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional
hit finding, evaluation, and structure-based scaffold morphing. These studies, reported
here in the first of 2 papers, led to the identification of multiple 5,6-fused bicyclic scaffolds
which bind to the same allosteric tunnel as 2. These studies demonstrate the structural
diversity permitted by the tunnel pharmacophore, and culminated in the identification of
pyrazolopyrimidinones (e.g., SHP389, 1), that modulate MAPK signaling in vivo. These
studies also served as the basis for further scaffold morphing and optimization, detailed
in the following manuscript.
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H
H N
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SHP2 Allosteric Pocket
Pharmacophore Model
INTRODUCTION
Activation of SHP2 phosphatase, encoded by the gene PTPN11, is genetically
associated with multiple cancer types, including juvenile myelomonocytic leukemia, B-cell
1
acute lymphoblastic leukemia, and acute myeloid leukemia. SHP2 activating mutations
also occur in solid tumors including lung adenocarcinoma, colon cancer, neuroblastoma,
melanoma, and hepatocellular carcinoma.2 SHP2 is a non-receptor protein tyrosine
phosphatase composed of a C-terminal domain, a PTP domain, and two N-terminal Src
homology 2 (SH2) domains. In its basal state, SHP2 adopts an autoinhibited
conformation which associates the SH2 and PTP domains and restricts substrate access
to the catalytic site. Activation of SHP2 occurs via binding of bis-phosphotyrosyl peptides
(
e.g., IRS-1) to the SH2 domains, disrupting the SH2-PTP association. The resulting
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conformational change exposes the catalytic site and activates the enzyme by relieving
auto-inhibition, promoting cancer dependent phosphatase activity.3
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SHP2 is involved in numerous oncogenic cell signaling cascades, including the
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canonical RAS-ERK, PI3K-AKT, and JAK-STAT pathways. The role of SHP2 in growth
signaling via RAS/ERK/MAPK is more precisely defined, as SHP2 was reported to bind
and dephosphorylate RAS and increase RAS–RAF association, activating downstream
proliferative signaling.5 Furthermore, the role of SHP2 in the T-cell programmed cell
death/checkpoint pathway (PD–L1/PD–1) contributing to immune evasion is under
investigation.6,7 The PD-1/SHP2/STAT1/T-bet signaling axis mediates the suppressive
effects of PD-1 on Th1 tumor immunity. Inhibition of PD-1 or SHP2 is therefore expected
to restore Th1 immunity and T-cell activation, countering immunosuppression within the
tumor microenvironment. Given recent clinical success of anti-PD-1 and PD-L1
8
therapeutics, small molecule SHP2 inhibitors for cancer immunotherapy are highly
desired clinical assets.9
Previously, we reported the identification of two new allosteric binding sites for SHP2
inhibition.10,11 Initial medicinal chemistry efforts identified a moderately potent, selective, and
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orally bioavailable inhibitor, SHP099 (2, Figure 1A).12 2 stabilizes an inactive conformation by
concurrent binding to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine
phosphatase domains. The SHP099-inhibited conformation of SHP2 resembles the published
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inactive apo structure (PDB code 2SHP) in which the N-terminal SH2 domain blocks the active
site, resulting in autoinhibition of PTP activity. The continued optimization of the aminopyrazines
will be described at a later time, separately from our published patent applications.14 We have also
described a second allosteric binding modality and found that dual small molecule allosteric
1
1
binding via both mechanisms was possible. Taken together, these pivotal publications and patent
application disclosures have stimulated wide interest from the scientific community.15 Both
academic institutions16 and biopharmaceutical companies17 have disclosed drug discovery
programs, exploiting related chemical matter which utilizes the initial allosteric modality. In this,
the first of two articles, we report the design rationale and SAR studies which led to the
identification of inhibitors across multiple fused-bicyclic chemical series, including the
potent and selective pyrazolopyrimidinones, e.g., SHP389 (1). These studies also served
as the basis for continued scaffold evolution and optimization.18
RESULTS AND DISCUSSION
As part of a comprehensive program directed at allosteric SHP2 inhibitors, we
conducted a structure-based prioritization of chemical matter identified through high-
throughput screening.10 Comparison of the binding pose of our previously disclosed
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SHP2 inhibitor 2 with the SHP2-cocrystal structure of HTS hits 3 and 4 (PDB codes 5EHR,
6MD9, 6MDA, respectively) revealed several conserved protein-ligand interactions
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(Figure 1). For example, all three ligands engage a Pi-cation interaction between the
haloarene and the guanidinium of R111. The haloarene of each ligand is also framed by
Van der Waals interactions with the lipophilic sidechains P491 and L254. In the case of
pyrazolopyridine 4, the pendant carboxylate accesses an additional water-mediated
interaction with R111 and a direct polar interaction with N217. Both 2 and 4 individually
form an additional polar interaction with the back-bone carbonyl of E250 with aniline and
pyrazole proton donors, respectively, while 3 lacks the required H-bond donor. Of the
three structures, only 2 contained a hydrogen bond donor contacting the F113 backbone
carbonyl.
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a.
b.
H2N
O
N
N
Cl
N
N
SHP2 IC50 = 0.070 M
SHP2: -31% @ 40 M
NH2
N
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o
DSF T = 0.67 C
O
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E E2 52 05 0
E250
L254
L254
Q257
H114
Q257
R111
R111
F113
F113
c.
H
N
N
N
SHP2: -30% @ 40 M
DSF T = 0.62 C
o
m
O
4
Br
OH
N217
H114
P491
E250
P491
E250
N217
H114
L254
Q257
Q257
R111
Figure 1. SHP2 binding interactions with (a) 2, (b) 3 and (c) 4. PDB codes: 5EHR, 6MD9,
MDA, respectively.
6
A pharmacophore model adopted from 2, 3 and 4 (Figure 2A) broadly
characterized SHP2 allosteric pocket ligands as being composed of: an H-bond donor to
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the backbone carbonyl of E250; an H-bond acceptor to the guanidinium of R111; an H-
bond donor to F113; a Pi-cation interaction with R111; Van der Waals interactions
between the pendant arene and lipophilic residues P491 and L254. Accounting for all the
interactions implied by this model, pyrazolopyrimidinone 5 was envisioned which
combines the chloroarene and amine features of 2, the 5,6-fused, bicyclic core of 3 and
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, and the E250 binding N-H donor present in 2 and 4. Consistent with our design
hypothesis, pyrazolopyrimidinone 5 was found to potently inhibit the biochemical activity
of SHP2 (Figure 2B, IC = 0.067 M), and inhibited SHP2 mediated phosphorylation of
50
ERK kinase (IC50 = 0.746 M) and demonstrated a modest antiproliferative effect in a
KYSE520 cell line (IC = 4.76 M). Analysis of the co-crystal structure of 5 in the SHP2
50
allosteric binding pocket (PDB code 6MDB, Fig 2C) confirmed engagement of all
interactions implicated in the pharmacophore model (vide supra). In addition to these
predicted interactions, the pyrimidinone carbonyl participated in a water-mediated
interaction with N127 and T219 while the tertiary amine donated a proton in an H-bond to
E249. Compound 5 also had an acceptable pharmacokinetic profile in mouse (Cl 34
mL/min/kg, 25% oral bioavailability), supporting the further optimization of the
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pyrazolopyrimidinone class for improved potency and selectivity over the hERG channel
(
IC = 0.200 M).
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F113
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NH2
N
Cl
O
L254
5
SHP2 IC50
=
0.067 M
p-ERK IC50 = 0.746 M
antiprolif. IC50 = 4.76 M
R111
hERG IC50
=
0.20 M
c.
T219
E250
N217
P491
T219
P491
E250
N217
R111
L254
L254
Q257
T253
F113
E249
F113
E110
T108
Figure 2. (a) Pharmacophore model derived from 2, 3 and 4. Blue is H-bond donor. Red
is H-bond acceptor. Green is lipophilic. (b) In vitro profile of designed ligand 5. (c)
Cocrystal of 5 in SHP2 allosteric pocket (PDB code 6MDB).
The synthesis of pyrazolopyrimidinones began with the reaction of 6-chlorouracil
6
with hydrazine hydrate to provide intermediate 7 after condensation with 4-
methoxybenzaldehyde. Alternatively, the benzyl protected intermediate 8 was accessed
by a similar sequence using benzaldehyde. Piperidine catalyzed cyclocondensation of 7
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with an aromatic aldehyde completed the carbon framework present in intermediate 9.
Intermediate 8 was derivatized by parallel synthetic strategies, one of which required
generation of the halide 10. Intermediates 8, 9 and 10 were further advanced by
installation of aliphatic amines under peptide coupling or SnAr conditions to provide after
deprotection, final pyrazolopyrmidinone products 11-25.
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H
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H
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PMB
N
O
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N
N
PMB N
N
N
H
N
d, e
H
2 3
a, b
a, f
c
N
O
NR R
Cl
O
R1
N
O
O
N
7
N
N
9
R1
H
O
Bn
N
N
O
N
c, d,
g
1
1-25
6
N
N
O
Cl
N
Bn
N
O
N
8
c, h
R1
i, g
10
Scheme 1. General synthesis of pyrazolopyrimidinones.a
a
o
Reagents and conditions: (a) H NNH -H O, EtOH, 80 C; (b) 4-methoxybenzaldehyde,
2
2
2
1
o
2
3
MeOH; (c) R COH, piperidine, DMF, i-PrOH, 90 C; (d) R R NH , BOP, DMF (e) TFA,
2
o
+ -
DCE, 70 C; (f) benzaldehyde, MeOH; (g) t-BuONa, DMSO, O ; (h) POCl , Me4N I , 100
2
3
o
2
3
o
C; (i) R R NH , DIPEA, NMP, -wave, 120 C.
2
We observed water with variable weak density across co-crystal structures in the
vicinity of the basic amine of 5 (e.g., Figure 2C). Elaboration of the amine substituent20
provided a handle for modulating the biochemical and cellular potency of prototype 5
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2
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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
(
Table 1). Extending the basic amine in order to displace the proposed water, returned a
modest improvement in SHP2 biochemical inhibition with improvement of hERG
selectivity (e.g., compound 11). However, the modest biochemical improvement did not
clearly benefit either the p-ERK or KYSE proliferation cellular assays. Cyclization to the
spiro[4.5]-amine (12) provided little additional benefit to SHP2 biochemical potency but
provided a >10-fold improvement in the p-ERK and KYSE antiproliferation cellular assays.
This was likely due to an increase in affinity, as the biochemical IC ranged from 0.009
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
50
to 0.045 M (the average of three experiments shown in table 1, 0.031 M).
Tetrahydrofuran analog 13 was designed to mitigate amine basicity, but was less potent
than 12 by all measures. 13 presented an improved lipophilic efficiency (LipE), owing to
a one log reduction in lipophilicity. The expected reduction in lipophilicity and basicity
provided by ether 13 produced only a minor benefit to hERG inhibition. In an attempt to
restore cellular potency by rebalancing lipophilicity, the spirocyclic ether was methylated
to provide 14. This maneuver improved both the p-ERK modulation (IC = 0.012 M)
50
and enhanced by 10-fold the antiproliferative activity against KYSE520 cells (IC = 0.167
50
M).
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Page 13 of 46
Journal of Medicinal Chemistry
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9
H
N
N
N
Cl
R
N
Cl
O
SHP2
IC50 (M)
p-ERK antiproliferation
IC50 (M)
hERG
IC50 (M)
Compound
R
cLogP/LipE
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
IC50 (M)
H2N
1
1
2
0.034
0.031
0.355
13.49
3.1/3.1
0.98
0.25
N
N
H2N
1
0.030
0.465
3.4/3.2
H2N
H2N
1
3
4
0.050
0.028
0.123
0.012
1.73
2.3/4.7
2.9/4.0
0.93
0.29
N
N
O
O
Me
1
0.167
Table 1. Optimization of the spirocyclic amine for potency and hERG selectivity.
In light of the promising biochemical and cellular data, we sought to mitigate the
hERG liability initially observed in the dofetilide binding assay and later confirmed by Q-
Patch (14: IC = 0.70 M). Bearing in mind our SHP2 allosteric pocket pharmacophore
50
model (Figure 2A), we modeled potential binding interactions for various alternative
heterocyclic cores within the SHP2 allosteric pocket. Our structural analysis indicated
that retraction of the H-bond acceptor carbonyl presented by the pyrazolopyrimidinone
core, as exemplified by pyrazolopyrazine 15 (Table 2), would retain the native donor-
acceptor SHP2 interactions. A docking analysis of 15 in the SHP2 allosteric pocked
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Journal of Medicinal Chemistry
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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
further supported this hypothesis; the pyrazine nitrogen modeled within Van der Waals
radius for interaction with R111 (Figure 3). Consistent with the elevated lipophilicity, 15
was a remarkably potent SHP2 inhibitor (biochemical IC = 0.006 M) with substantial
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
50
cellular potency (p-ERK IC = 0.031 M, KYSE520 antiproliferative IC = 0.46 M), but
50
50
also potently inhibited the hERG channel (IC = 0.004 M). While the pyrazole N-H of
50
the pyrazolopyrimidine heterocycle was deemed important due to the E250 backbone
carbonyl contact, other heteroatoms in the core formed no H-bond acceptor interactions
and were removed. The resulting pyrrolopyrazine, 16, was moderately potent in
biochemical (IC = 0.039 M) and p-ERK KYSE520 cellular (IC = 0.473 M) assays.
50
50
Unfortunately, neither 15 nor 16 improved the selectivity over the hERG channel.
SHP2
IC50 (M)
p-ERK
IC50 (M)
hERG
IC50 (M)
antiproliferation
Compound
Structure
cLogP/LipE
IC50 (M)
H
N
H N
2
N
Cl
Cl
N
1
5
6
0.006
0.039
0.031
0.484
0.46
--
3.9/4.3
0.004
0.007
N
N
Cl
Cl
N
N
O
O
H
N
H2N
N
1
4.1/3.3
Table 2 Core 5,6-fused bicycle SAR.
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Page 15 of 46
Journal of Medicinal Chemistry
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7
8
9
P491
E250
L254
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
T108
R111
Q257
E110
F113
Figure 3. 15 modeled into the SHP2
allosteric binding pocket preserves R111
interaction. (Docking model built from
PDB code 6MDB)
After optimizing the spirocyclic amine for biochemical and cellular potency and
deprioritizing core heterocycle modifications as a means for improving hERG inhibition,
we next focused our SAR study at the pendant chloroarene. Substituting the
dichlorophenyl subunit in 11-16 with dichloropyridine 17 (Table 3) moderately mitigated
hERG inhibition (IC50 = 1.2 M). The heteroatom insertion substantially reduced
lipophilicity by ~1.5 log units (vs. matched pair 14, Table 1) resulting in improved lipophilic
efficiency (17: LipE = 4.9). Compound 17 had moderate p-ERK cellular potency (p-ERK
IC = 0.093 M), and the antiproliferative effects were blunted (antiproliferative IC = 2.6
50
50
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Journal of Medicinal Chemistry
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1
1
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1
1
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1
1
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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

M). The net benefits of tuning polarity and lipophilicity via the pendant arene prompted
further investigation.
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
The unsubstituted pyridine 18 delivered superior biochemical potency (IC = 0.008
50
M) with no discernable hERG signal and high lipE (5.9). However, 18 did not deliver a
concomitant improvement in cellular potency, presumably due to the lower membrane
permeability resulting from a log unit reduction in lipophilicity relative to dichloropyridine
17. Replacement of the 2,3-dichloropyridine motif with 2-amino, 3-chloro aminopyridine
19 significantly reduced hERG activity and provided a new vector for synthetic
elaboration. Appending small lipophilic groups including methyl (20) and cyclopropyl (1)
improved SHP2 biochemical and cellular potency in trend with increasing lipophilicity.
Cyclobutane analog 21 resulted in a steep reduction in biochemical and cellular potency,
presumably due to increased steric bulk. Overall, we observed that hERG IC roughly
50
tracked with lipophilicity, where progressively lipophilic molecules displayed an
increasingly severe hERG liability.
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Page 17 of 46
Journal of Medicinal Chemistry
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4
5
6
7
8
9
H
N
H2N
N
N
N
N
R
O
O
SHP2
IC50 (M)
p-ERK antiproliferation
IC50 (M)
hERG
IC50 (M)
Compound
17
R
cLogP/LipE
1.5/4.9
IC50 (M)
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
Cl
Cl
0.028
0.008
0.093
2.60
1.2
N
18
0.129
0.103
1.26
1.20
0.3/5.9
>30
N
Cl
19
H2N
0.032
0.055
0.6/6.4
1.4/5.7
>30
>30
N
Cl
H
N
20
0.022
0.37
N
Cl
H
1
2
N
0.036
0.350
0.036
0.260
0.36
0.38
2.0/4.7
2.3/4.4
17.0
1.9
N
Cl
H
N
1
N
N
22
23
0.037
0.027
0.038
0.096
0.66
0.72
0.2/5.8
1.2/5.7
6.6
H2N
N
>30
H2N
S
Cl
Table 3. Optimization of the pyridine substituent for potency and hERG selectivity.
While the collection of heteroatoms presented by 2-amino, 3-chloropyridine might
conceivably access specific interactions within the SHP2 allosteric pocket, our SAR
suggested otherwise. For example, reconfiguration of the heteroatom presentation, as in
regioisomeric aminoyridine 22, also delivered promising biochemical potency that was
robustly reproduced in cells. That 22 was sufficiently permeable for cellular potency was
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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
unexpected given the low clogP. For comparison, 22 was substantially less lipophilic than
1
(cLogP = 1.2 vs 2.0, respectively), yet delivered equivalent cellular potency. However,
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
the lower lipophilicity of 22 did not fully relieve hERG promiscuity (IC = 6.6 M).
50
More dramatic variations of the pyrazolopyrimidinone substructure were also
anticipated to bind the SHP2 allosteric tunnel. For example, a docking study (Figure 4)
supported the hypothesis that substitution of the Ar-Ar bond present in 19 with the Ar-S-
Ar motif, as is present in the HTS screening hit 24, could satisfy the pharmacophore model
developed for the 5,6-fused bicyclic chemical series (Figure 2A). The structural overlay
demonstrated rough alignment of the core heterocycle and flanking arene substructure.
Bearing in mind these design considerations, 23 was characterized as a potent
biochemical inhibitor (IC = 0.027 M). Cellular characterization further demonstrated
50
the potency of 23 in the p-ERK assay (IC = 0.096 M) and the KYSE antiproliferation
50
assay (IC50 = 0.72 M). As previously observed, the 2-amino, 3-chlopropyridine motif
correlated with low hERG affinity (IC > 30 M).
50
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Page 19 of 46
Journal of Medicinal Chemistry
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9
N
SHP2 IC50 (M)= 47.0
cLogP / LipE= 4.9 / -0.6
NH2
N
H
Cl
S
N
Cl
24
P491
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
E250
L254
T108
R111
Q257
F113
E110
Figure 4. Overlay of 24 and 19 supports binding hypothesis of hybrid 23. PDB code of
SHP2-24 6MDD.
Considering the substantial structural changes made to the flanking substituents
of the root design 5 (Figure 2), we were motivated to better understand the nature of the
evolving protein-ligand interactions. To this end, a cocrystal structure of 1 was obtained
(
Figure 5). The major polar interactions included those anticipated by the pharmacophore
model (Figure 2A): a polar interaction between the pyrazolopyrimidinone carbonyl and
R111; a polar interaction between pyrazolopyrimidinone N-H and E250; a Pi-cation
interaction between the 2-amino, 3-chloropyridine heterocycle and R111; an H-bond
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Journal of Medicinal Chemistry
Page 20 of 46
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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
donor interaction between the spirocyclic amine and F113. Previously unobserved
interactions included water mediated interactions between the pyrazolopyrimidinone
carbonyl, T219, and N217, as well as polar interactions between the spirocyclic amine
and the F113, E110, and T108 triad. Conspicuously absent were polar interactions with
the 2-aminocyclopyropyl, 3-chloropyridine substructure present in 1. This observation
helped rationalize the equivalent potency of structurally diverse regioisomeric
aminopyridine 22. Lacking donor-acceptor interactions in this region of the SHP2
allosteric binding pocket, regioisomeric aminopyridines produced comparable
biochemical inhibition. A key observation from Table 2 is that the aminopyridine based
analogs delivered hERG selective SHP2 inhibitors.
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
P491
N217
T219
E250
T219
P491
L254
E250
N217
L254
R111
F113
E110
R111
F113
Q257
T108
E110
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Page 21 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 5. Co-crystal structure of 1 in the SHP2 allosteric pocket (PDB code 6MDC).
On the basis of their overall in vitro profile, 1 and 14 were prioritized for additional
profiling, and were selective versus a panel of 30 GPCRs, ion channels, nuclear
receptors, transporters, enzymes and kinases (IC50 >30 M). While preliminary
evaluation of hERG binding by a dofetilide binding assay indicated 1 weakly inhibited
hERG (IC = 17 M) a more detailed evaluation by Q-Patch demonstrated that the
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
50
compound did not engage a functional hERG interaction (IC50 >30 M). The in vitro
metabolic clearance profile (mouse ER = 27%, rat ER = 46%) and clean CYP450 profile
(CYP 3A4, 2D6 and 2C9 IC50 >50 M) of 1 encouraged further in vivo evaluation.
Compound 1 was dosed IV/PO at 1/5 mg/kg, respectively, to male Sprague Dawley rats
in a crossover study design. Compound 1 demonstrated a moderate-high clearance
(~83% hepatic extraction) which was under predicted in vitro by rat liver microsomes (Cl
=
26.1 L/min/mg, T1/2 = 53.2 min) assuming a hepatic blood flow of 55 mL/min/kg. The
volume of distribution was moderate-high (3.9 L/kg) with a terminal half-life of 2.7 h.
Following oral administration, the compound had low oral bioavailability (~2 %F) which is
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Journal of Medicinal Chemistry
Page 22 of 46
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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
consistent with poor in vitro permeability (PAMPA). This limited exposure therefore
prevented further pharmacologic evaluation of 1 in a mouse tumor xenograft model.
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
We thus turned to 14, a potent compound (KYSE520 p-ERK IC50 = 0.093 M;
antiproliferative IC = 0.170 M) with a less selective profile (hERG IC = 0.29 M) in
50
50
order to characterize in vivo pharmacodynamic markers. In addition to its robust
antiproliferative effects, 14 had acceptable stability in mouse liver microsomes (Cl = 36.8,

L/min/mg, T = 60.2 min) and was sufficiently soluble (pH = 6.8 equilibrium solubility = 0.42
1/2
mM) to enable PK pharmacokinetic evaluation. Compound 14 was dosed IV/PO at 1 and
5
mg/kg, respectively, to male Sprague Dawley rats in a crossover study design.
Compound 14 demonstrated high clearance (> hepatic blood flow), a high volume of
distribution (10.5 L/kg), a moderate half-life (T1/2 = 4.6 h), and moderate oral bioavailability
(F = 51 %). Additionally, the PK and in vivo activity of 14 was assessed in mice
subcutaneously implanted with the KYSE520 carcinoma cell line. Tumor-bearing mice were
given a single oral dose of 14 at 10, 30 and 100 mg/kg. Free concentrations of 14 (89%
mouse plasma protein bound) correlated well with the MAPK pharmacodynamic markers, p-ERK
and DUSP6 (Figure 3). The maximal PD effect, as determined by p-ERK and DUSP6
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Page 23 of 46
Journal of Medicinal Chemistry
1
2
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9
concentrations in blood, was observed at 100 mg/kg for at least 8 hours, with PD rebound
correlating with clearance of 14.
1
1
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1
1
1
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0
Figure 3. PK/PD of 14 at 10, 30, and 100 mg/kg in mice bearing KYSE520 xenografts.
CONCLUSION
As part of a program directed at identifying allosteric SHP2 inhibitors, we
successfully merged the salient structural features of established SHP2 ligands (e.g., 2)
with new HTS hits (e.g., 3, 4, 25). This pharmacophore blending exercise produced
pyrazolopyrimidinone 5, which contains a 5,6-fused heterocycle that was confirmed by X-
ray crystallography to bind to the same allosteric tunnel site as 2. Extension and
conformational restriction of the amine substituent led to the identification of 14, which
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1
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2
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5
5
6
elicited a robust PK-PD correlation in a mouse tumor xenograft model. This result further
motivated us to improve the poor hERG selectivity associated with the 5,6-fused bicyclic
series. Substitution of the pyrazolopyrimidinone core with other 5,6-fused heterocycles
provided new potent allosteric scaffolds with similar binding modalities, but failed to
address the hERG selectivity. A more successful approach was found through
modification of the pendant chloroarene presented by 14. Decoration of the chloroarene
with nitrogen heteroatoms, as in aminopyridine 19, provided a firm handle for improving
hERG selectivity and a new vector for synthetic manipulation. Continued experimentation
demonstrated that cyclopropane 1 successfully balanced high biochemical and cellular
potency with impressive hERG selectivity (Q-Patch IC50 >30 M). While our in vitro
objectives were met in this study, compound 1 ultimately fell short of demonstrating
sufficiently robust oral bioavailability to support PK/PD and tumor efficacy studies. Taken
together, these investigations demonstrated the structural determinants governing
efficient, cellularly potent, allosteric SHP2 binders from various 5,6-fused bicyclic
frameworks, a clear strategy for effectively improving hERG selectivity, and ultimately
served as basis for further optimization.18
0
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EXPERIMENTAL SECTION
Compound synthesis and characterization. Compound purity was assessed by HPLC to
confirm >95% purity. All solvents employed were commercially available anhydrous grade, and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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1
2
3
4
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8
9
0
reagents were used as received unless otherwise noted. A Biotage Initiator™ Sixty system was
used for microwave heating. Flash column chromatography was performed on either an Analogix
Intelliflash 280 using Si 50 columns (32-63 μm, 230-400 mesh, 60Å) or on a Biotage SP1 system
(32-63 μm particle size, KP-Sil, 60 Å pore size). Preparative high pressure liquid chromatography
(HPLC) was performed using a Waters 2525 pump with 2487 dual wavelength detector and 2767
sample manager. Columns were Waters C18 OBD 5μm, either 50x100 mm Xbridge or 30x100
mm Sunfire. NMR spectra were recorded on a Bruker AV400 (Avance 400 MHz) or AV600
(Avance 600 MHz) instruments. Analytical LC-MS was conducted using an Agilent 1100 series
with UV detection at 214 nm and 254 nm, and an electrospray mode (ESI) coupled with a Waters
ZQ single quad mass detector. One of three methods was used: Method A) 5-95 %
acetonitrile/H O with 5 mM ammonium formate with a 2 min run, 3 μL injection through an
2
inertisil C8 3 cm x 5 mm x 3μm; Method B) 20-95 % acetonitrile/H O with 10 mM ammonium
2
hydroxide with a 2 min run, 3 μL injection through an inertisil C8 3 cm x 5 mm x μm; Method C)
5
-95 % acetonitrile/H O with 0.05 % trifluoroacetic acid with a 2 min run, 3 μL injection through
2
an Sunfire C18 3.5 μm 3.0 x 30 mm. Purity of all tested compounds was determined by LC/ESI-
MS Data recorded using an Agilent 6220 mass spectrometer with electrospray ionization source
and Agilent 1200 liquid chromatography. The mass accuracy of the system has been found to be
<
5 ppm. HPLC separation was performed at 75 mL/min flow rate with the indicated gradient
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5
6
within 3.5 min with an initial hold of 10 seconds. 10 mM ammonia hydroxide or 0.1 M TFA was
used as the modifier additive in the aqueous phase.
Synthesis of SHP389: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-(3-
chloro-2-(cyclopropylamino)pyridin-4-yl)-5-methyl-2,5-dihydro-4H-pyrazolo[3,4-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
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9
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1
2
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9
0
1
2
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9
0
d]pyrimidin-4-one (1). Hydrazine hydrate (9.17 mL, 187 mmol) was added to a solution of
6-chloro-3-methyluracil (10 g, 62.3 mmol) in ethanol (200 mL). The resulting mixture was
stirred at 80 °C. The slurry became increasingly thick over the course of 10 min. The
heating was continued for 1 h, then the heat bath was removed and the reaction mixture
was allowed to cool to room temperature for 1 h. The resulting suspension was filtered
and the filter cake was dried under vacuum. The intermediate 6-hydrazinyl-3-
methylpyrimidine-2,4(1H,3H)-dione was obtained as a white powder (9.7 g, 100% yield)
and was used directly in the next transformation.
To a suspension of 6-hydrazinyl-3-methylpyrimidine-2,4(1H,3H)-dione (5.26 g,
33.7 mmol) in MeOH (25 mL) was added 2,3-dichlorobenzaldehyde (5.50 g, 40.4 mmol).
The result mixture was stirred at room temperature for 30 min. The slurry became very
thick, and was diluted with methanol (25 mL) to facilitate stirring. The reaction was
maintained for an additional 30 min, at which point the reaction was complete by LCMS.
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The slurry was filtered and the filter cake was dried under vacuum for 16 h to provide 6-
((4-methoxybenzyl)diazenyl)-3-methylpyrimidine-2,4(1H,3H)-dione as a white solid (8.0
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
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5
5
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6
0
1
2
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9
0
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2
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9
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9
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5
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9
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4
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7
8
9
0
+
g, 87% yield). LCMS: m/z 280 (M+1) , R =0.43 min.
t
To
a
suspension of 6-((4-methoxybenzyl)diazenyl)-3-methylpyrimidine-
2,4(1H,3H)-dione (0.68 g, 2.479 mmol) in DMF (8 mL) and i-PrOH (4 mL) was added 2,3-
dichloroisonicotinaldehyde (0.436 g, 2.479 mmol) and piperidine (0.241 mL, 2.430 mmol).
The result mixture was stirred at 85 °C for 1 h. At completion, the reaction mixture was
partitioned between water (50 mL) and ethyl acetate (50 mL). The layers were separated
and the aqueous layer was washed with ethyl acetate (50 mL). The combined organics
were washed with brine (40 mL), dried with Na SO , filtered and concentrated. The crude
2
4
was purified by flash chromatography over silica gel (0-10% methanol/DCM eluent) to
afford
3-(2,3-dichloropyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-
d]pyrimidine-4,6(5H,7H)-dione as a yellow solid (420 mg, 39% yield, 90% purity). LCMS:
+
m/z 432 (M+1) , R = 1.29 min.
t
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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
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3
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6
To a suspension of 3-(2,3-dichloropyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2H-
pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (500 mg, 1.16 mmol) in DMSO (3 mL) was
added cyclopropanamine (330 mg, 5.78 mmol). The resulting mixture was microwave
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
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2
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5
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9
0
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2
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4
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9
0
o
heated to 140 C for 20 min. At completion, the reaction was partitioned between water
(15 mL) and ethyl aceate (15 mL). The organics were separated and the aqueous layer
was washed with ethyl acetate (2 x 10 mL). The combined organics were washed with
brine, dried over Na SO and concentrated. The crude was purified by flash
2
4
chromatography over silica gel (0-70% ethyl acetate/heptane eluent) to provide 3-(3-
chloro-2-(cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,7-dihydro-4H-
pyrazolo[3,4-d]pyrimidine-4,6(5H)-dione (280 mgs, 53% yield). LCMS: m/z 453.2 (M+1)+,
R =0.65 min.
t
To
a
solution
of
3-(3-chloro-2-(cyclopropylamino)pyridin-4-yl)-2-(4-
methoxybenzyl)-5-methyl-2,7-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4,6(5H)-dione (280
mgs,
0.618
mmol)
in
DMF
(2
mL)
was
added
(benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (820 mgs, 1.855 mmol).
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After 15 min, the mixture was treated with (3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decan-
4-amine (195 mgs, 0.804 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (753 mgs, 4.95
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
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9
0
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5
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9
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1
2
3
4
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9
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2
3
4
5
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8
9
0
mmol). At completion, the reaction was partitioned between water (15 mL) and ethyl
acetate (15 mL). The organics were separated and the aqueous phase was washed with
ethyl acteate (2 x 10 mL). The combined organics were washed with brine (10 mL), dried
over MgSO and concentrated. The crude was purified by flash chromatography over
4
silica gel (0-5% MeOH/DCM eluent) to provide 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-
azaspiro[4.5]decan-8-yl)-3-(3-chloro-2-(cyclopropylamino)pyridin-4-yl)-2-(4-
methoxybenzyl)-5-methyl-2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (170 mg, 45%
+
yield). LCMS: m/z 606.0 (M+1) , R =0.72 min.
t
A solution of 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-(3-
chloro-2-(cyclopropylamino)pyridin-4-yl)-2-(4-methoxybenzyl)-5-methyl-2,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (170 mg, 0.281 mmol) in TFA (3 mL) and DCE (3 mL) was
o
heated to 70 C. After 2 h, the reaction was concentrated, then dissolved in DCM, treated
with heptane and concentrated. The crude was purified by prep HPLC to provide 6-
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