Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant

Article abstract of DOI:10.1002/anie.201603736

The treatment of non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations. Third-generation EGFR inhibitors have been designed to overcome resistance through covalent binding to the Cys 797 residue of the enzyme, and these inhibitors are effective against most clinically relevant EGFR mutants. However, the high dependence of these recent EGFR inhibitors on this particular interaction means that additional mutation of Cys 797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients. A new generation of irreversible and reversible mutant EGFR inhibitors was developed with strong noncovalent binding properties, and these compounds show high inhibitory activities against the cysteine-mutated L858R/T790M/C797S EGFR.

Full text of DOI:10.1002/anie.201603736

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Communications
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International Edition: DOI: 10.1002/anie.201603736
German Edition: DOI: 10.1002/ange.201603736
Kinase Inhibitors
Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against
a Therapy-Resistant L858R/T790M/C797S Mutant
Marcel Gꢀnther+, Michael Juchum+, Gerhard Kelter, Heiner Fiebig, and Stefan Laufer*
Abstract: The treatment of non-small-cell lung cancer
(NSCLC) with epidermal growth factor receptor (EGFR)
inhibitors is made challenging by acquired resistance caused by
somatic mutations. Third-generation EGFR inhibitors have
been designed to overcome resistance through covalent binding
to the Cys797 residue of the enzyme, and these inhibitors are
effective against most clinically relevant EGFR mutants.
However, the high dependence of these recent EGFR inhibitors
on this particular interaction means that additional mutation of
Cys797 results in poor inhibitory activity, which leads to tumor
relapse in initially responding patients. A new generation of
irreversible and reversible mutant EGFR inhibitors was
developed with strong noncovalent binding properties, and
these compounds show high inhibitory activities against the
cysteine-mutated L858R/T790M/C797S EGFR.
response to an anti-EGF therapy develop acquired drug
resistance through different mechanisms. In approximately
50% of initially responding patients, a secondary somatic
mutation, the gate-keeper mutation T790M, is the underlying
cause of drug resistance.[7] The mutation at the gate-keeper
Thr790 residue is a single amino acid exchange in the catalytic
domain of EGFR.[8] Third-generation EGFR inhibitors
(Rociletinib, Osimertinib, and WZ4002) have aimed to
overcome T790M resistance through covalent inhibition of
EGFR by binding to a cysteine side chain (Cys797) through
Michael addition with a suitable electrophile attached to the
inhibitor molecule.[9] Recently, several studies have revealed
the development of resistance to these new third-generation
TKIs.[10] A recent study exposed the occurrence of the tertiary
point mutation C797S in 40% of the patients treated with
AZD9291 (a current mutant-selective FDA-approved third-
generation EGFR inhibitor).[11] Loss of the covalent inter-
action results in a marked decrease in inhibition, and selection
pressure then leads to resistance development These new
findings suggest that the development of EGFR inhibitors
with new structural features as well as unique selectivity
profiles for different EGFR mutations is necessary to study
and overcome resistance against third-generation EGFR
inhibitors.
Non-small cell lung cancer (NSCLC) is among the most
deadly cancer types worldwide.[1] Even after early diagnosis,
most patients that receive surgery or platinum-based chemo-
therapy suffer from the development of drug resistance and
tumor progression. As a member of the ERBB family, the
receptor tyrosine kinase EGFR has been identified as one of
the most promising drug targets for the treatment of
NSCLC.[2] However, at least one so-called activating mutation
in the tumor genome must be present for significant patient
response.[3] The two most frequently observed activating
mutations are the single amino acid exchange L858R, caused
by a somatic mutation in exon 21, and the delE746-A750
deletion in exon 19.[4] While treatment of NSCLC patients
harboring these activating mutations with first-generation
EGFR inhibitors generally leads to rapid and massive tumor
shrinkage,[5] patients harboring the wild-type (wt) EGFR
show little response to small-molecule anti-EGF drugs.[6] The
two approved amino quinazoline tyrosine kinase inhibitors
(TKIs) Gefitinib and Erlotinib show very potent and selective
inhibition profiles with respect to these activating mutations.
However, almost all patients that initially show a dramatic
Based on selectivity screening of the highly potent
reversible p38 inhibitor 1, we identified EGFR inhibition as
an off-target effect of this compound (Figure 1).[12] High
potency, as well as moderate physicochemical properties and
cellular activity against p38, lead us to pick this compound as
a first lead structure for further improvements in terms of the
inhibition of EGFR mutants. In general pyridinyl imidazoles
can potentially inhibit cytochrome P450 (CYP) enzymes,
however this toxic side effect is not linked to the entire
[*] M. Gꢀnther,[+] M. Juchum,[+] Prof. Dr. S. Laufer
Eberhard Karls Universitꢁt Tꢀbingen
Institute of Pharmaceutical Sciences
Pharmaceutical and Medicinal Chemistry, Auf der Morgenstelle 8,
72076 Tꢀbingen (Germany)
E-mail: stefan.laufer@uni-tuebingen.de
Dr. G. Kelter, Prof. Dr. H. Fiebig
Cell Biology & Compound Screening Oncotest GmbH
Am Flughafen 12–14, 79108 Freiburg (Germany)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201603736.
Figure 1. Lead structures from screening data. IC50 values [nm] in a wt
EGFR enzyme assay.
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structural class. In the past, we have successfully designed
several inhibitors against different kinases (e.g., p38)[13] based
on this scaffold with low hepatotoxicity/CYP interaction. We
examined approximately 2000 proprietary compounds that
were structurally similar or related to 1 in an activity screen
against wt EGFR and identified 2 as a promising lead
structure for further investigation. In order to minimize the
vulnerability of the compounds toward Cys797 mutations, we
applied a two-pronged approach. On the one hand, we
designed reversibly binding inhibitors with the ability to
overcome T790M resistance. On the other hand, we synthe-
sized covalent irreversible inhibitors with strong reversible
binding properties. The irreversible binding was not utilized
to overcome T790M resistance, but rather to tune target
residence time and activity. Both approaches yielded com-
pounds with high activity against the L858R/T790M/C797S
mutant that is resistant to the covalent attack by irreversible
third-generation EGFR inhibitors (Scheme 1).
Figure 2. Molecular docking of 1a (A) and 2c (B) in a T790M X-ray
structure (PDB ID: 2JIU) employing glide-induced fit (A) and glide
covalent docking (B) plugins. Proposed H-bonds are indicated as
orange dashed lines.
the acrylamide and the imidazole NH can be seen for 2c
(Figure 2B). This hydrogen bond might lock the inhibitor
molecule in a conformation that has a perfect orientation of
the Michael acceptor toward Cys797, thus enabling covalent
binding.
We first tested 1a, 1b, and 2a–d in an enzyme assay
against EGFR wt, L858R, and L858R/T790M (Table 1). We
Table 1: Kinase inhibitory activities of the new EGFR inhibitors. *
indicates IC50 values below the resolution limit of the assay.
IC50 [nm] against EGFR
wt
L858R
L858R/T790M
1a
1b
2a
2b
2c
2d
<0.5*
<0.5*
<0.5*
<0.5*
<0.5*
<0.5*
1.3Æ0.2
<0.5*
<0.5*
4.2
6.6Æ0.1
160Æ61
<0.5*
7.1Æ0.8
211Æ2.2
<0.5*
<0.5*
0.65Æ0.01
17.1
34Æ0.2
<0.5*
<0.5*
Staurosporine
WZ4002
AZD9291
1.4Æ0.02
1.1Æ0.62
Scheme 1. A rational approach to the design of two compound series
derived from 1 and 2, supported by computer-aided drug design.
<0.5*
<0.5*
<0.5*
Driven by molecular docking of several derivatives of
1 with a T790M EGFR mutant, we identified 1a and 1b,
which should form beneficial hydrogen bonds to Asn842
(Figure 2A), thereby giving higher inhibitory activity. Fur-
thermore, we identified position 3 of the aniline moiety of 2 as
an appropriate position to attach a Michael acceptor to target
Cys797 covalently, yielding 2a. Compound 2c was decorated
with an additional methoxy group in position 6 to provide
gentle steric hindrance against the free rotation around the
obtained IC50 values below the resolution limit of 0.5 nm with
1a and 1b with wt and L858R. However, both compounds
showed a slight decrease in activity in the double-mutant
assay, with IC50 values of 6.6 and 160 nm. Nevertheless, 1a is
a highly potent single-digit nanomolar reversible T790M
inhibitor. Our second approach to synthesize covalent EGFR
inhibitors yielded compounds 2a and 2c. Both compounds
showed extraordinarily high activities in all three kinase
assays. The reversible counterpart 2d, which is decorated with
an additional methoxy group, proved to be a highly active
reversible EGFR inhibitor, with IC50 values of 0.65 nm, less
than 0.5 nm, and 34 nm against wt, L858R and L858R/T790M,
respectively. The high activity of 2d indicates that the
covalent interaction is only a minor contributor to the IC50
value of 2c, which is in distinct contrast to reported third-
generation irreversible EGFR inhibitors.
À
N C bond, thereby directing the electrophilic warhead
toward Cys797. We synthesized the reversibly binding
analogues 2b and 2d for comparison. Molecular modelling
indicated bidentate hinge binding for both 1a and 2c to
Met793 (Figure 2). For 1a, an additional hydrogen bond to
Asn842 can be observed. In this modelling study, an intra-
molecular hydrogen bond between the carbonyl O atom of
2
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Kinetic studies revealed low Kd values in the single-digit
Cys797. Our most promising compounds showed IC50 values
nanomolar range. There were no significant differences in the
Kd values for 2c used undiluted (dark gray bars) and 30-fold
diluted (light gray bars, Figure 3). By contrast, the reversible
of 90 and 20 nm, which implies that this new generation of
EGFR inhibitors with a very strong reversible binding pattern
can tolerate cysteine mutation better than the irreversible
third-generation EGFR inhibitors.
We also tested the most promising compounds in a set of
tumor-relevant cancer cell lines with different EGFR muta-
tions in a direct comparison to the most recent gold standard
treatments (Table 3). Inhibitor 1a revealed no significant
Table 3: Inhibitory activities in different NSCLC cell proliferation assays.
IC50 [mm] in different NSCLC cell lines
LXFA 526
wt
LXFA PC-9
Exon 19 del
LXFA NCl-H1975
L858R/T790M
1a
2c
Gefitinib
WZ4002
AZD9291
3.568
14.034
12.838
5.847
2.976
1.050
0.005
0.037
0.138
0.019
2.488
0.018
12.672
0.056
0.011
activity in the cell lines. Surprisingly, lower IC50 values were
observed in the mutant cells, with a slight selectivity over the
wt cells. Compound 2c is a very potent inhibitor of the
clinically relevant cell lines H1975 (18 nm) and LXFA PC-9
(5 nm); these values were in the same range or better than the
most potent and recently FDA-approved third-generation
inhibitor AZD9291. Furthermore, 2c showed high selectivity
(> 700 fold) towards the double-mutant cell line LXFA NCl-
H1975 over the wt cell line LXFA 526. A kinome scan of 2c,
which included 410 kinases, demonstrated a good selectivity
profile with a selectivity score of 0.039 (Table 4). Additionally,
this tyrosine kinase inhibitor was a very potent inhibitor of all
clinically relevant EGFR mutants. An interesting off-target
hit was the tyrosine kinase BRK (2% residual activity), which
has been identified in different cancer types, including
NSCLC.[14]
Figure 3. Binding kinetics of 2c and 2d (scanKINETIC). Kd values
displayed as 300 nm were actually measured to be >300 nm.
counterpart 2d showed a tremendous Kd shift after dilution,
thus supporting the strong assumption that 2c is an irrever-
sible inhibitor. Another experiment without dilution but with
an equilibration time of 6 hours (see the Supporting
Information) showed 3 to 5-fold lower Kd values. This
outcome indicates slow association kinetics of these com-
pounds, meaning equilibrium formation is not complete after
one hour of incubation.
The two most potent inhibitors, 1a and 2c, showed very
strong reversible binding to the EGFR active site, so we
tested them in an EGFR assay against the triple mutant
L858R/T790M/C797S, which plays a crucial role in the
development of drug resistance in lung cancer patients who
had previously been treated with third-generation inhibitors
(Table 2). As expected, the third-generation compound
WZ4002 exhibited very low potency against this mutant
kinase (> 7 mm), since most of its binding affinity relies on
covalent bond formation, which cannot take place without
Compounds 1a and 1b were synthesized by using the
optimized Stille cross-coupling reported by Selig et al.
(Figure 4).[12] The imidazoles 3a and 3b were obtained
through Radziszewski imidazole synthesis. The nitrile of 3b
was reduced to an amine followed by triple BOC protection
to give 4b. These protected imidazoles were stannylated using
nBuLi and Sn(Bu)3Cl. Stille cross-coupling with the previosly
synthesized
4-chloro-1-(methoxymethyl)-2-phenyl-1H-
pyrrolo[2,3-b]pyridine using a 1:3 mixture of Pd(OAc)2/X-
Table 4: Inhibitory activities of 2c in mutant EGFR enzyme assays
Table 2: Kinase inhibitory activities (single point) of the most potent new
compounds in a triple-mutant EGFR enzyme assay.
Residual activity in %
with 2c [200 nm]
IC50 [nm] against EGFR
L858R/T790M/C797S
EGF-R d746-750
EGF-R d747-749/A750P
EGF-R d747-752/P753S
EGF-R d752-759
EGF-R G719C
EGF-R G719S
EGF-R L861Q
EGF-R T790M
7
7
5
9
3
7
4
5
1a
1b
2c
2d
21
336
90
167
1
7294
Staurosporine
WZ4002
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CH2Cl2,
and
the
resulting a-bromoke-
tone 6 was cyclicized
with S-methylthiop-
seudourea to yield
imidazole 7 in moder-
ate yields. Subsequent
SEM protection of the
deprotonated imida-
zoles initially yielded
a
60:40 mixture of
regioisomers. The
regioisomeric mixture
was converted into the
sterically
isomer 8 through heat-
ing with catalytic
favored
a
amount of SEM-Cl in
acetonitrile to give
pure
8 in excellent
yields.[15] High yielding
electrophilic bromina-
tion of the imidazole
core was performed
under very controlled
conditions using N-
bromosuccinimide in
Figure 4. Synthesis of compounds 1a and 1b. Boc=tert-butoxycarbonyl, DMAP=4-(dimethylamino)pyridine,
MOM=methoxymethyl, TIPS=triisopropylsilyl.
Phos in refluxing dioxane resulted in compounds 5a and 5b.
Deprotection of the coupled products in HCl/MeOH under
reflux led to the desired compounds 1a and 1b.
We synthesized compounds 2a–d by utilizing a modular
route, which allowed derivatization in the last step before
deprotection (Figure 5). The commercially available 4-fluo-
roacetophenone was alpha brominated using bromine in
acetonitrile. To minimize proto-deboronation, we used opti-
mized reaction conditions for the Suzuki cross-coupling of 9
and applied portion-wise addition of the boronic acid ester.
We achieved excellent yields of 10 with this procedure.
Deacetylation of 10 was performed under basic conditions
and elevated temperatures. We recently optimized a Buch-
wald amination for the synthesis of a series of dual JNK3/p38
Figure 5. Synthesis of compounds 2a–2d. DCM=Dichloromethane, NBS=N-Bromosuccinimide, SEM=2-(trimethylsilyl)ethoxymethyl, TFA=Tri-
fluoroacetic acid.
4
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inhibitors[16] and were able to use this procedure to yield 12a–
d in good to excellent yields using predecorated bromoani-
lines for the cross-coupling. SEM removal was performed
under mild conditions using TFA in CH2Cl2 at room temper-
ature to yield the final compounds 2a–d.
[1] R. L. Siegel, K. D. Miller, A. Jemal, Ca-Cancer J. Clin. 2015, 65,
In summary, we have successfully developed a highly
potent, reversible EGFR L858R/T790M inhibitor with
potency in the single-digit nm range that is not reliant on
a covalent bond formation for strong binding to the active
site. Furthermore, this compound is a strong inhibitor of the
recently discovered triple mutant that occurs in patients after
treatment with third-generation inhibitors like Osimertinib.
Furthermore, we developed an irreversible L858R/T790M
EGFR inhibitor with potency in the pM range that is
equipotent and more selective for wild type EGFR inhibition
in tumor cell lines compared to Osimertinib. Moreover, this
compound also displays a strong reversible binding compo-
nent and is able to inhibit the EGFR triple mutant in the low
nm range. We have shown that covalent binding can be
a useful tool to tune the potency and target-residence time,
although this interaction is not crucial for compound potency
in general. The attractive properties of these compounds
should allow them to serve as lead compounds for the further
development of highly potent L858R/T790M/C797S inhib-
itors.
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Acknowledgements
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We thank Dr. Ch. Schaechtele, ProQinase GmbH, Freiburg,
Germany and Prof. H. Weinmann, Bayer Pharma Aktienge-
sellschaft, Berlin for screening data.
Received: April 18, 2016
Revised: May 27, 2016
Keywords: antitumor agents · drug design · EGFR · kinases ·
inhibitors
Published online: && &&, &&&&
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Communications
Kinase Inhibitors
From reversible to irreversible and back:
A new class of trisubstitued imidazoles
(see scheme) was designed to overcome
drug resistance caused by the EGFR
mutation T790M in non-small-cell lung
cancer, predominantly through non-
covalent interactions. In contrast to third-
generation irreversible EGFR inhibitors,
these imidazoles are up to 350-fold less
vulnerable to Cys797 mutation and show
high cellular activity in the low double-
digit nanomolar range.
M. Gꢁnther, M. Juchum, G. Kelter,
H. Fiebig, S. Laufer*
&&&& — &&&&
Lung Cancer: EGFR Inhibitors with Low
Nanomolar Activity against a Therapy-
Resistant L858R/T790M/C797S Mutant
6
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