Discovery and Optimization of Dibenzodiazepinones as Allosteric Mutant-Selective EGFR Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00381

Allosteric kinase inhibitors represent a promising new therapeutic strategy for targeting kinases harboring oncogenic driver mutations in cancers. Here, we report the discovery, optimization, and structural characterization of allosteric mutant-selective EGFR inhibitors comprising a 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one scaffold. Our structure-based medicinal chemistry effort yielded an inhibitor (3) of the EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S) mutants with an IC₅₀ of a?10 nM and high selectivity, as assessed by kinome profiling. Further efforts to develop allosteric dibenzodiazepinone inhibitors may serve as the basis for new therapeutic options for targeting drug-resistant EGFR mutations.

Full text of DOI:10.1021/acsmedchemlett.9b00381

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Letter
Discovery and Optimization of Dibenzodiazepinones
as Allosteric Mutant-selective EGFR Inhibitors
Dries J. H. De Clercq, David E Heppner, Ciric To, Jaebong Jang, Eunyoung
Park, Cai-Hong Yun, Mierzhati Mushajiang, Bo Hee Shin, Thomas W
Gero, David A Scott, Pasi A. Jänne, Michael J. Eck, and Nathanael S Gray
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 22 Oct 2019
Downloaded from pubs.acs.org on October 22, 2019
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ACS Medicinal Chemistry Letters
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Discovery and Optimization of Dibenzodiazepinones as
Allosteric Mutant-selective EGFR Inhibitors
Dries J. H. De Clercq,a,b,‡ David E. Heppner,a,b,‡ Ciric To,c,d,e Jaebong Jang,a,b Eunyoung Park,a,b Cai-
Hong Yun,a,b,f Mierzhati Mushajiang,c Bo Hee Shin,c Thomas W. Gero,a,b David A. Scott,a,b Pasi A.
Jänne,c,d,e,g Michael J. Eck,a,b,* and Nathanael S. Gray a,b,*
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aDepartment of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
bDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215, USA
cLowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
dDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
eDepartment of Medicine, Harvard Medical School, Boston, MA 02215, USA
fDepartment of Biochemistry and Biophysics, Peking University Health Science Center, Beijing 100191, China.
gBelfer Center for Applied Cancer Science, Boston, MA 02215, USA
KEYWORDS: EGFR, kinase inhibitor, allosteric inhibitor, dibenzodiazepinone, mutant-selective, non-small cell lung cancer
ABSTRACT: Allosteric kinase inhibitors represent a promising new therapeutic strategy for targeting kinases harboring oncogenic
driver mutations in cancers. Here, we report the discovery, optimization, and structural characterization of allosteric mutant-
selective EGFR inhibitors comprising a 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one scaffold. Our structure-based
medicinal chemistry effort yielded an inhibitor (3) of the EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S) mutants with
an IC50 of ~10 nM and high selectivity, as assessed by kinome profiling. Further efforts to develop allosteric dibenzodiazepinone
inhibitors may serve as the basis for new therapeutic options for targeting drug-resistant EGFR mutations.
(e.g. WZ4002, osimertinib) that irreversibly form a covalent
N
NH
S
O
S
O
bond with C797 at the edge of the ATP binding site.5-6
Tumors acquire resistance to these inhibitors, in ~20-25% of
cases, by the further acquisition of a C797S mutation
rendering these inhibitors ineffective by preventing the
formation of the potency-conferring covalent bond.7-8
Therefore, continued development of inhibitors is required to
address mutations that confer resistance to first- and third-
generation EGFR targeting TKIs.
N
N
N
N
N
N
H
H
O
O
R'
HO
R
F
EAI001
JBJ-04-125-02
R' = R = H
R' = OH; R = F
EAI045
1
2
R' = F; R =
R' = F; R =
N
N
NH
NH
O
N
Accordingly, we sought to discover small-molecule
inhibitors of activating EGFR mutations that act through an
alternative, allosteric binding mode.9 To that end, we
O
O
R'
R
N
H
3
R' = F; R =
N
N
recently reported
a mutant-selective EGFR allosteric
EAI002
R' = H; R = F
DDC4002
R' = F; R = H
inhibitor (EAI001 Fig. 1) which binds a pocket adjacent to
the ATP-binding site, affording exquisite selectivity for the
mutant kinase compared to WT EGFR.10 Initial optimization
of this hit produced EAI045 (Fig. 1), which exhibited 1000-
fold selectivity for L858R/T790M EGFR compared to WT.
However, EAI045 showed minimal cellular activity owing
to this compounds inability to bind the allosteric pocket of
the active state, where the αC-helix is positioned inward, on
the receiver subunit of the active EGFR asymmetric kinase
dimer.11 EAI045 was rendered effective at regressing
L858R/T790M/C797S tumors in vivo upon co-treatment
with the EGFR monoclonal antibody cetuximab, which
disrupts the formation of active EGFR dimers.10 Further
efforts produced a more potent analog (JBJ-04-125-02, Fig.
Fig. 1. Chemical structures of EAI001, EAI045, JBJ-04-125-
02 and 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one
allosteric EGFR inhibitors described in this work.
Activating mutations of the epidermal growth
factor receptor (EGFR), e.g. L858R and in-frame exon 19
deletions, give rise to non-small cell lung cancer and confer
sensitivity to EGFR-targeted tyrosine kinase inhibitors
(TKIs) such as gefitinib and erlotinib.1-2 Acquired resistance
to these TKIs occurs predominantly by the acquisition of a
T790M ‘gatekeeper’ mutation, which enhances ATP binding
to the EGFR kinase.3-4 Selective inhibition of T790M-
positive tumors is accomplished with third-generation TKIs
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1), which incorporates a phenylpiperazine on the C6 position recent EAI045
Page 2 of 7
crystal
structure
bound
to
1
2
3
4
5
6
7
8
of the EAI045 isoindolinone moiety and is capable of acting
as a single-agent inhibitor in EGFR L858R/T790M/C797S
cells and genetically engineered mouse models.12
Furthermore, dual targeting of EGFR with JBJ-04-125-02
and osimertinib was found to be more effective in vitro and
in vivo than either single agent alone.12
T790M/C797S/V948R in the absence of AMP-PNP shows
limited variance in EAI045 binding mode indicating that
allosteric inhibitor binding to EGFR is structurally agnostic
to the presence of ATP binding.15
To swiftly access the selectively substituted fused
[6-7-6] tricyclic core, we streamlined our original route
(Route A, Scheme 1) to a versatile, concise 2-step synthesis
In this study, we sought to evaluate a second series
of allosteric mutant-selective EGFR inhibitors. As
previously described, EAI001 was discovered in a screen for
involving
a tandem copper(I)-catalyzed intramolecular
Ullmann condensation (Route B, Scheme 1).16 Based on the
binding mode of DDC4002 (Fig. 2B), we hypothesized that
functionalization at the C2 position would be capable of
enhancing biochemical potency of these inhibitors, in a
similar manner to that observed for EAI045 and JBJ-04-
125-02.12 Following the latter example, coupling of a 4-
(piperazinyl)phenyl substituent to C2 (1, Scheme 1)
productively enhanced the potency of this scaffold.
Structure-activity relationships revealed that engineering
flexibility at this position via an Ullmann biaryl ether linkage
(2 and 3, Scheme 1) modestly improved the effectiveness of
these inhibitors, with 3 exhibiting biochemical potencies
similar to EAI045 against L858R/T790M and
L858R/T790M/C797S.
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mutant-selective inhibitors of EGFR(L858R/T790M).10,
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The same screen also yielded EAI002 comprising of a 5,10-
dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one
scaffold
(Fig. 1), which selectively inhibited L858R/T790M with a
biochemical IC50 of 52 nM compared to >1000 nM for WT.
Subtle optimization of this hit compound via a fluorine shift
afforded DDC4002 (Fig. 1), which exhibited mutant-
selective nanomolar biochemical IC50 values against
L858R/T790M and L858R/T790M/C797S compared to WT
(Table 1). Intriguingly, DDC4002 resembles previously
reported ATP-competitive, selective checkpoint kinase 1
(Chk1) inhibitors,14 however, selective benzylation of the
core amide precludes active site binding by abrogating
critical hydrogen bonding contacts to the hinge likely
favoring redirection to the allosteric site.
To establish that these compounds inhibit EGFR
through an allosteric mechanism, biochemical IC50 values
were measured at varying ATP concentrations spanning 1 to
1000 µM (Table S1). Indeed, compounds 2 and 3 showed no
significant variance with [ATP] consistent with allosteric
To define the inhibitor binding mode, we
determined crystal structures of EGFR(T790M/V948R) in
complex with EAI002 and DDC4002 (Fig. 2 & S1-4).
Expectedly, both inhibitors are bound to the kinase domain
in an allosteric pocket adjacent to the ATP-binding site (Fig.
2A), as observed previously for EAI00110 and JBJ-04-125-
0212. The 7-membered diazepinone ring of DDC4002 is
puckered inward toward the αC-helix (Fig. 2B) with the 8-
fluorobenzene ring bound within the hydrophobic back
pocket and the unsubstituted benzene ring along the αC-helix
positioned out toward solvent. The benzyl substituent
extends toward the kinase N-lobe, bound in between AMP-
PNP and side chains of K745, L788, and the T790M
gatekeeper mutation. While the inhibitor mostly forms
hydrophobic interactions, the diazepinone N-H forms a H-
bond with the backbone carbonyl of F856 in the DFG motif
(red dotted line Fig. 2B). Additionally, the crystal structure
of EAI002 contains four EGFR chains in the asymmetric
unit all with EAI002 bound in the allosteric site, but with
only one AMP-PNP and three AMP bound in the ATP site,
presumably from AMP-PNP hydrolysis. The binding of
either AMP-PNP or AMP does not impact the EAI002
binding mode.
inhibition.
This
further
supports
that
the
dibenzodiazepinones are effective inhibitors of mutant
EGFR, operating in an allosteric mechanism as characterized
crystallographically (Fig. 2).
We additionally determined the impact of EGFR
inhibition on cellular proliferation in transformed murine
Ba/F3 cells. All compounds were found to be ineffective at
limiting Ba/F3 proliferation due to EGFR dimer-induced
resistance as we have observed previously (Table S2).10 Co-
treatment with cetuximab (1 μg/mL) establishes this to be
the case and leads to productive inhibition of L858R/T790M.
Consistent
with
their
biochemical
potencies,
dibenzodiazepinones with biaryl ether moieties (2 and 3)
exhibit the best effect in combination with cetuximab with
IC50 of
~ 0.2-0.4 µM against L858R/T790M and
L858R/T790M/C797S cells. Compounds 1-3 were not
amenable to crystallographic characterization, but we expect
that the phenylpiperazine group extends along the αC-helix
to enhance the potency of 1-3 relative to DDC4002 in a
manner analogous to that observed for JBJ-04-125-02.12
While these dibenzodiazepinone-based inhibitors are
effective mutant-selective inhibitors of EGFR in a cellular
context, they are still critically reliant on co-treatment with
cetuximab.
Additionally, we determined a crystal structure of
EGFR(T790M/V948R) in complex with the phenylglycine
EAI045 (Fig. S1B and S4). Similar to EAI00110 and JBJ-
04-125-02,12 EAI045 binds exclusively in the
R
configuration. Structural alignment of the kinase domains
reveals that, despite distinct chemical structures, the binding
mode of the dibenzodiazepinone inhibitors has significant
overlap with that of the phenylglycines (Fig. 2C and S3).
Specifically, both scaffolds exhibit H-bonding to the
backbone carbonyl of F856 as well as fluorobenzene
moieties positioned toward the hydrophobic cleft at the back
of the pocket. These conserved interactions confirm that
these apparently unrelated scaffolds are anchored to the
allosteric site through conserved interactions. Additionally, a
We next sought to assess the selectivity of the best
performing inhibitor (3) against a panel of 468 kinases via
KINOMEscan profiling (DiscoverX). At a concentration of
10 μM, 3 displayed excellent selectivity across the human
kinome with S-Score(35) = 0.01 (Fig. S5, Table S4). While
the KINOMEscan shows binding of 3 to EGFR WT and
mutants, the results from activity and cellular assays indicate
more reliable and robust selectivity for the oncogenic mutant
targets (Table 1). Except for the expected WT EGFR and
EGFR mutants, only two additional targets, SLK and
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ACS Medicinal Chemistry Letters
opportunity to discover additional chemical series as
KIT(V559D/V654A), were identified. We confirmed these
hits to be false positives of with SLK and
KIT(V559D/V654A) (Kd 10 μM; KINOMEscan
KdELECT, DiscoverX) and confirmed no impact on SLK
enzymatic activity (IC50 10 µM; Invitrogen,
1
2
3
4
5
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7
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3
allosteric EGFR inhibitors. Our structure-based medicinal
chemistry effort yielded an inhibitor (3) of the
EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S)
mutants with an IC50 of ~10 nM and high selectivity, as
assessed by kinome profiling. Co-treatment with cetuximab
resulted in antiproliferative activity in EGFR-mutant Ba/F3
>
>
LanthaScreen). Although it is a valuable survey tool for
assessing kinase selectivity, in our experience,
KINOMEScan profiling does not correlate with enzymatic
and cellular potencies as we have recently shown in the case
of JBJ-04-125-02.12
cells.
Together
with
the
previously
reported
dibenzodiazepine-based inhibitors of PAK1, which bind a
closely related but distinct allosteric pocket, these
compounds potentially indicate a broader application of
benzodiazepine compounds as allosteric kinase inhibitors.17
Additionally, dibenzodiazepinone compounds represent new
additions to the growing list of allosteric inhibitors for kinase
targets, as previously explored for MEK,18-19 BCR-ABL1,20
and others.21-22 We plan to further optimize physicochemical
and pharmacokinetic properties to produce more effective
mutant-selective allosteric EGFR inhibitors, which is the
subject of on-going efforts.
9
In conclusion, we have discovered and optimized
an allosteric mutant-selective EGFR inhibitor based on the
dibenzodiazepinone scaffold. As the presently established
mutant-selective allosteric EGFR inhibitors consist of a
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phenylglycine scaffold,10, the compounds described here
12
demonstrate that diverse chemical scaffolds are capable of
acting as mutant-selective EGFR inhibitors while preserving
essential structural elements that anchor the inhibitors to the
allosteric pocket. Therefore, this discovery expands the
Fig. 2. Structure and binding mode of a dibenzodiazepinone EGFR allosteric inhibitor. A) Overall view of the structure of
EGFR(T790M/V948R) bound to DDC4002 and AMP-PNP (PDB 6P1D). The V948R mutation enables the kinase domain to
crystallize in the inactive state. DDC4002 is shown in CPK spheres with green carbon atoms. B) Detailed view of DDC4002 bound
to the allosteric pocket with AMP-PNP. P-loop and A-loop segments are hidden for clarity. C) View of DDC4002 (green) and
EAI045 (PDB 6P1L, white) from the overlay of crystal structures.
Scheme 1. Synthetic routes (A,B) for synthesis of 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-ones DDC4002 and
compounds 1-3a
I
Route A:
F
I
R2
O
N
O
R
O
N
F
N
N
Br
Br
Br
ii
iii
F
N
O
H
N
O2N
O2N
R
H
5
R = NO2 ( )
R = OH
R = Cl
4
i
R2 = Boc ( )
iv
9
6
R = NH2 ( )
x
1
H ( )
ix
v
(R1 = Br)
Route B:
O
O
O
O
I
N
N
R
R
vi
vii
xi
(R1 = OH)
F
R1
F
O
HO
N
H
N
H
N
I
H
R1 = H (
N
8
DDC4002
)
R = Br ( )
R = OMe (
13
)
10
R3 = Boc (
)
7
Br ( )
N
xii
R3
2
H ( )
11
OMe (
)
viii
3
Me ( )
12
OH (
)
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aReagents and conditions: [i] SOCl2, DMF, Δ; [ii] 5-fluoro-2-iodoaniline, Et3N, CH2Cl2, 0 °C to RT, 81 %, two steps; [iii]
BnBr, NaH, THF, 0 °C to 40 °C; [iv] Fe, NH4Cl, THF/MeOH/H2O, 50 °C, 72 %, two steps; [v] CuI, K2CO3, DMSO, 135 °C,
64 %; [vi] benzylamine, EDC.HCl, HOBt, DIEA, DMF, 87 %; [vii] 4-fluoro-2-iodoaniline, CuI, K2CO3, DMSO, 80 °C to
135 °C, 44 %; [viii] BBr3, CH2Cl2, -20 °C to RT, 85 %; [ix] tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)piperazine-1-carboxylate, PdCl2(dppf), XPhos, 2 N Na2CO3, 1,4-dioxane, 100 °C; [x] TFA, CH2Cl2, 45 %, two
steps; [xi] 1-(4-iodophenyl)-4-methylpiperazine (3) or tert-butyl 4-(4-iodophenyl)piperazine-1-carboxylate (13), CuI, L-
Proline, K2CO3, DMSO, 80 °C, (3 36 %); [xii] TFA, CH2Cl2, 30 %, two steps.
1
2
3
4
5
6
7
8
9
a
Table 1. Biochemical activities and antiproliferative activities of a panel of EGFR allosteric inhibitors. IC50 values
were measured from a single experiment in triplicate. ATP concentration was 100 µM. Errors are reported as ± standard
error. bIC50 values were measured from a single experiment with 3 replicates. Errors are reported as ± standard deviations.
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Antiproliferative Activity
EGFR Biochemical Activity
Ba/F3 + Cetuximab
IC50 (nM)a
Compound
ID
IC50 (µM)b
L858R/
T790M/
C797S
L858R/
T790M/
C797S
L858R/
T790M
L858R/
T790M
WT
> 1000
L858R
8.8±0.9
WT
>10
L858R
EAI045
2.0±0.5
4.7±0.3
0.84±0.7
>10
0.47±0.2
1.5±0.4
0.25±0.2
DDC4002
> 1000
> 1000
> 1000
> 1000
690±120
150±23
130±12
154±15
39±4
31±2
59±8
19±3
9.7±0.5
4.1±1
1.2 ± 0.3
0.93 ± 0.2
0.35 ± 0.2
0.20 ± 0.08
1
2
3
3.7±0.1
3.8±0.5
2.7±1
0.77 ± 0.1
0.35 ± 0.07
0.36 ± 0.2
12±0.9
11±2
23±4
4.0±1
13±0.8
3.2±0.8
ASSOCIATED CONTENT
Conflicts of Interest Disclosure
Supporting Information
P.A. Jänne reports receiving commercial research grants from
AstraZeneca, Boehringer Ingelheim, Astellas Pharmaceuticals,
PUMA, Eli Lilly, and Takeda Oncology, has ownership interest
(including stock, patents, etc.) in Gatekeeper Pharmaceuticals and
LOXO Oncology, is a consultant or advisory board member for
The Supporting Information is available free of charge on the
ACS Publications website.
Chemistry, biological assay, and X-ray crystallography data
(PDF)
AstraZeneca,
Boehringer
Ingelheim,
Daiichi
Sankyo,
Roche/Genentech, Pfizer, Merrimack Pharmaceuticals, Chugai
Pharmaceuticals, Araxes Pharmaceuticals, Mirati, Ignyta, and
LOXO Oncology, and has received other remuneration from
Labcorp. M.J. Eck reports receiving a commercial research grant
from Novartis Institutes for Biomedical Research, reports
receiving other commercial research support from Takeda, and
has been consultant for Novartis Institutes for Biomedical
Research. N.S. Gray reports receiving a commercial research
grant from Takeda and is a consultant or advisory board member
for C4, Syros, Soltego, and B2S Bio. No potential conflicts of
interest were disclosed by the other authors.
AUTHOR INFORMATION
Corresponding Author
* Nathanael S. Gray nathanael_gray@dfci.harvard.edu, Michael
J. Eck eck@crystal.harvard.edu.
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the
manuscript. / ‡These authors contributed equally.
Funding Sources
ACKNOWLEDGEMENT
This work was supported by the National Institutes of Health
grants R01CA201049 (to M.J.E., N.S.G, and P.A.J.),
R35CA242461 (to M.J.E.), R01CA116020 (to M.J.E.),
P01CA154303 (to M.J.E., N.S.G, P.A.J), R01CA135257 (to
P.A.J), and R35 CA220497 (to P.A.J). This work is based upon
research conducted at the Northeastern Collaborative Access
Team beamlines, which are funded by the National Institute of
General Medical Sciences from the National Institutes of Health
(P30 GM124165). The Pilatus 6M detector on 24-ID-C beam line
is funded by a NIH-ORIP HEI grant (S10 RR029205). This
research used resources of the Advanced Photon Source, a U.S.
Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National
Laboratory under Contract No. DE-AC02-06CH11357.
We acknowledge Dr. Yong Jia (Novartis) for conducting high-
throughput screening. We also thank Drs. Kunhua Li and Tyler S.
Beyett for insightful discussions.
ABBREVIATIONS
EGFR, epidermal growth factor receptor; EAI, EGFR allosteric
inhibitor; TKI, tyrosine kinase inhibitor. EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide.
Hydroxybenzotriazole. DIEA, N,N-Diisopropylethylamine. Dppf,
HOBt,
1,1’-Bis(diphenylphosphino)ferrocene.
XPhos,
2-
Dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl.
PNP, adenylyl-imidodiphosphate.
AMP-
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10. Jia, Y.; Yun, C.-H.; Park, E.; Ercan, D.; Manuia, M.;
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