Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR)

Article abstract of DOI:10.1021/jm400822z

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Full text of DOI:10.1021/jm400822z

Article
pubs.acs.org/jmc
Structure- and Reactivity-Based Development of Covalent Inhibitors
of the Activating and Gatekeeper Mutant Forms of the Epidermal
Growth Factor Receptor (EGFR)
Richard A. Ward,* Mark J. Anderton, Susan Ashton, Paul A. Bethel, Matthew Box, Sam Butterworth,
Nicola Colclough, Christopher G. Chorley, Claudio Chuaqui,† Darren A. E. Cross, Les A. Dakin,†
́
Judit E. Debreczeni, Cath Eberlein, M. Raymond V. Finlay, George B. Hill, Matthew Grist,
Teresa C. M. Klinowska, Clare Lane, Scott Martin, Jonathon P. Orme, Peter Smith,‡ Fengjiang Wang,†
and Michael J. Waring
AstraZeneca, Oncology & Discovery Sciences Innovative Medicines, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG,
United Kingdom
†AstraZeneca, Oncology Innovative Medicines, Gatehouse Park, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: A novel series of small-molecule inhibitors has been developed
to target the double mutant form of the epidermal growth factor receptor
(EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and
erlotinib. Our reported compounds also show selectivity over wild-type EGFR.
Guided by molecular modeling, this series was evolved to target a cysteine
residue in the ATP binding site via covalent bond formation and demonstrates
high levels of activity in cellular models of the double mutant form of EGFR.
In addition, these compounds show significant activity against the activating
mutations, which gefitinib and erlotinib target and inhibition of which gives
rise to their observed clinical efficacy. A glutathione (GSH)-based assay was
used to measure thiol reactivity toward the electrophilic functionality of the
inhibitor series, enabling both the identification of a suitable reactivity window
for their potency and the development of a reactivity quantitative structure-
property relationship (QSPR) to support design.
50% of these initially responsive patients, disease progression is
associated with the gain of a secondary mutation, T790M in
exon 20 of EGFR (typically referred to as the gatekeeper
mutation), in combination with an activating mutation.5
INTRODUCTION
■
Ligand-induced activation of receptor tyrosine kinases such as
the epidermal growth factor receptor (EGFR) promote the
subsequent activation of downstream signaling pathways, which
in turn drive pro-proliferative and survival cellular signals.1 In a
subset of non-small cell lung cancer (NSCLC) patients, EGFR
has gained activating mutations such as L858R and exon 19
deletion, which largely renders the receptor constitutively
activated, independent of ligand stimulation.2 This results in
sustained hyper-activation of signaling pathways downstream of
EGFR, such as AKT (protein kinase B) and ERK (extracellular
signal-regulated kinase), and promotes the oncogenic depend-
ency of the cell on these pro-proliferative and survival drives
resulting from the mutated EGFR. Therefore, inhibition of
activated EGFR using small-molecule kinase inhibitors prevents
its signaling and reduces the oncogenic drive, ultimately
resulting in the inhibition of tumor growth.
The specific mechanism by which the activating mutants
exert their effect is not entirely clear, but studies on the L858R
mutation have shown there to be a decreased affinity for ATP
(Figure 1).6 It has been proposed that the activating mutations
may favor the active conformation of the kinase to which
compounds such as gefitinib (1)4 and erlotinib (2)7 may bind
(Figure 2).8 Further reports have suggested additional and
alternative mechanisms of activation, including the differential
ability of the mutants to sample the active and inactive
conformations.9,10 However, the secondary mutation, T790M,
increases the affinity of the kinase for ATP.6,11 An increase in
the kinase domain’s ATP affinity reduces the inhibitory activity
of ATP-competitive inhibitors such as gefitinib and erlotinib
against EGFR. This amino acid is known as the gatekeeper
residue, and it is positioned at the entrance of what is typically
First generation EGFR tyrosine kinase inhibitors (TKIs)
such as gefitinib and erlotinib are now established therapies in
NSCLC patients having activating mutations in EGFR.3,4
Around 70% of patients respond initially but develop resistance
with a median time to progression of 10−16 months. In at least
Received: June 3, 2013
© XXXX American Chemical Society
A
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compounds are tolerated in this region of the binding site.
It is therefore also possible that there is a steric component
to the resistance mechanism against early EGFR inhibitors,
which specifically affects compounds such as gefitinib and
erlotinib that were identified by targeting the wild-type
threonine gatekeeper. In addition, it has been proposed that
the T790M mutation can lead to differential structural re-
arrangements of the protein across inhibitor classes,9 impact-
ing the potency and selectivity of compounds against the
mutations.
A number of second-generation irreversible inhibitors were
subsequently developed on the basis of the anilinoquinazoline
template of gefitinib. These inhibitors contained an acrylamide
functionality that covalently targets Cys-797, which is
positioned in the solvent channel of the kinase.14,15 Such
reported compounds include afatinib, 3,16 canertinib, 4,17 and
dacomitinib, 5,18 shown in Figure 2. The covalent mechanism is
thought to overcome the increase in ATP affinity of the double
mutant, resulting in the potent activity of these compounds in
cellular models. However, Cys-797 is present in all relevant
forms of EGFR; these second-generation compounds, there-
fore, not only have increased activity against the activating and
resistant mutations but also against wild-type EGFR. The
inhibition of wild-type EGFR is not thought to contribute to
their clinical efficacy but is thought to be responsible for the
side-effects of skin rash and diarrhea.19 As these side effects are
Figure 1. Location of activating (L858R and exon 19 deletion) and
resistant secondary mutations (T790M) in the kinase domain of
EGFR (PDB code 2ITY13).
referred to as the back pocket.12 The size and nature of this
residue has a significant effect on determining which
Figure 2. Selection of published EGFR inhibitors disclosed in the literature.
B
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a
Scheme 1. General Synthesis Approach to the Key Novel Compounds (Table 1) Reported in This Manuscript
a
Reagents and conditions: (i) Aniline displacement: excess NaHCO3, 2-pentanol or EtOH, 85 °C; suzuki coupling: Na2CO3, ArB(OR)2, Pd(PPh3)4,
MeCN, water, 100 °C; indole displacement: MeMgBr (1 equiv, 3.2 M in 2-methyl THF), indole (1 equiv), THF, 0−60 °C. (ii) Et3N (1.1 equiv),
butyl-vinyl-ether (1.1 equiv), Pd(OAc)2 (0.03 equiv), PEG 200, 80 °C. (iii) Either 1-aminopyridinium iodide (1 equiv), K2CO3 (2.5 equiv), DMA,
110 °C or 2-amino-pyridine (1 equiv), NBS (1.2 equiv), 1,4-dioxane, water, 85 °C. (iv) Either aniline (1.1 equiv), p-TSA (1.1 equiv), 2-pentanol,
80−120 °C, or aniline (1.1 equiv), 4 M HCl in 1,4-dioxane, TFE, 150 °C (μW), or aniline (1.1 equiv), Pd(OAc)2 (0.08 equiv), Xantphos
(0.1 equiv), Cs2CO3 (2 equiv), 1,4-dioxane, 80 °C. (v) Acetyl aniline (1 equiv), p-TSA (3 equiv), n-pentanol/NMP, 150 °C. (vi) 1-Methylpiperazine
(2.5 equiv), TFE, 120 °C. (vii) Either iron (3 equiv), NH4Cl (0.7 equiv), EtOH, water, 100 °C or Pt/C (1 equiv), ZnI2 (0.1 equiv), H2, MeOH,
EtOAc. (viii) Either acryloyl chloride (1M, THF, 1 equiv), DIPEA (1.1 equiv), THF, 0 °C, or (E)-4-(dimethylamino)but-2-enoic acid (1 equiv),
HATU (1.1 equiv), DIPEA (2.5 equiv), DCM, or (E)-4-bromobut-2-enoic acid (1.1 equiv), HATU (1.1 equiv), DIPEA (2.5 equiv), 0 °C then
Me2NH (20 equiv). (ix) Deprotection (where required): Either K2CO3 (3 equiv), THF, MeOH or Cs2CO3 (3 equiv), THF, MeOH, 0 °C. (x)
Zn(CN)2 (0.6 equiv), Zn (0.1 equiv), Pd2(dba)3 (0.1 equiv), dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine (0.2 equiv), DMA, 90 °C.
typically dose limiting in patients, it is possible that the activity
of these inhibitors against wild-type EGFR will limit their
achievable activity against the T790M mutation in patients.
Our initial aim was to identify a double-mutant-selective
template by exploiting the differences in the properties of
the respective threonine and methionine gatekeeper residues.
We hypothesized that exploiting the methionine gatekeeper
residue to leverage the increased potency might lead to a
selectivity profile in which the compounds are active against the
double mutant enzyme but with reduced activity against wild-
type EGFR. Ideally, compounds would also show greater
activity against the activating EGFR mutations than against
wild-type EGFR.
RESULTS AND DISCUSSION
■
Chemistry. Where possible, compounds were prepared
according to the general strategy outlined in Scheme 1 from
commercially available pyrimidines (A). Installation of the
reactive warhead, where present, at the last or penultimate step
was desirable because it was not expected to be robust enough
during further chemical manipulation. Although Scheme 1
captures the synthesis approach to the majority of the key
examples from this manuscript (shown in Table 1), because of
the structural diversity of the compounds reported, some examples
were not synthesized using this approach. However, the full
synthesis of all compounds can be found in the Experimental
Section or in the Supporting Information.
From the time that we initiated our efforts to identify
compounds with this preferred selectivity profile, other
compounds have been reported that exploit similar principles.
An early published example was from the Dana-Farber Cancer
Institute (WZ-4002, 6)20 and was based on a pyrimidine
template. More recently, additional covalent inhibitors from
Clovis Oncology (CO-1686) and Hanmi Pharmaceuticals
(HM61713) are reported to be in clinical studies, although
their chemical structures are yet to be disclosed. Also reported
is the reversible anaplastic lymphoma kinase (ALK) inhibitor
(AP26113) from ARIAD, with reported activity against the
double mutant form of EGFR. Further publications have also
been disclosed, from the time that we initiated this work,
reporting on other potential compound series active against the
EGFR double mutation.21−23
Installation of the 4-substituent of the pyrimidine was achieved
by direct regioselective displacement of 4-chloro under basic
conditions or by Suzuki coupling. Where necessary, the aryl group
was constructed from vinyl butyl ether intermediate B, which in
turn was synthesized from the 4-chloro-pyrimidine via Heck coupl-
ing. Subsequent cyclization reactions afforded aryl pyrimidines C.
The aniline addition to 2-chloropyrimidines C was achieved
via Buchwald−Hartwig coupling with substituted anilines or by
acid-catalyzed SNAr reaction, typically by heating with p-TSA in
2-pentanol. For compounds where R3 = H, commercial 5-nitro-
2-methoxy-aniline was used. Where R3 = F, the aniline was not
commercially available and was synthesized by nitration of
2-methoxy-4-fluoro-aniline prior to use. Amine substitution of
the fluorine by SNAr proceeded in fair to good yield by heating
with either an excess of the amine or using DIPEA as base.
C
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Table 1. Novel Compounds from This Manuscript Synthesized Using Scheme 1
Subsequent reduction of the nitro group and reaction with
acryloyl chloride (R4 = H) or HATU coupling with commercial
(E)-4-(dimethylamino)but-2-enoic acid yielded test com-
pounds F in moderate yield.
For compound 7, which does not contain an acrylamide, the
anilino substituent could be installed fully elaborated. This
portion was constructed from commercial starting materials, as
detailed in Scheme 2.
D
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a
Scheme 2. Synthesis of the Intermediate for Compound 7
a
Reagents and conditions: (i) 1-Acetyl piperazine (1.2 equiv), DIPEA (1 equiv), DMA, 90 °C, 18 h, 90%. (ii) Platinum oxide (5 mol %), EtOH,
hydrogen (1.3 bar), 3 h, 99%. (iii) Acetic anhydride (6.6 equiv), acetic acid (4.5 equiv), 1 h, 73%.
The full synthesis and characterization of the novel com-
pounds within this manuscript is detailed in the Experimental
Section (key compounds 7, 9, 15, and 58) and in the
Supporting Information for the remaining examples.
Activity Profile of Published EGFR Inhibitors. We
measured the cellular activity of a number of known inhibitors
against the different forms of EGFR (Table 2) using phosphorylation
Table 2. Cellular Activity Profile of Published EGFR
Inhibitors against the Double Mutant (DM), Activating
a
Mutant (AM), and Wild-Type (WT) Enzymes
DM cell
(μM)
AM cell
(μM)
WT cell
(μM)
DM/WT
margin
compound
Gefitinib (1)
Erlotinib (2)
Afatinib (3)
3.3
0.0087
0.0059
0.00057
0.00063
0.044
0.062
0.077
0.012
0.011
1.18
0.02
0.01
0.53
0.27
51
7.6
0.023
0.042
0.023
Dacomitinib (5)
WZ-4002 (6)
a
Activity data in this manuscript are quoted as the geometric mean
Figure 3. EGFR wild-type (WT) and T790M/L858R (DM)
biochemical inhibition data on a probe set of compounds. Red points
show reversible EGFR inhibitors, yellow points show covalent EGFR
inhibitors, and blue points show selected compounds from internal
AstraZeneca projects. Compound activity is expressed as a pIC50.
IC50 of at least three independent measurements. The DM/WT
margin is calculated by dividing the mean wild-type IC50 by the mean
double-mutant IC50.
end points. These included the wild-type enzyme (WT) with a
human LoVo cell line and the exon 19 deletion activating
mutation (AM) using a PC9 cell line. Activity against the
L858R/T790M double-mutant enzyme (DM) was measured
using a NCI-H1975 cell line. Although much of this data set was
already in the public domain from a number of different groups, it
was useful to measure their activities in consistent assays to allow
an accurate comparison of their profile across the mutants.
As expected, gefitinib and erlotinib were shown to be active
against wild-type EGFR and to have a greater activity against
the activating mutant but significantly reduced cellular activity
against double-mutant EGFR (3.3 and 7.6 μM, respectively).
Afatinib (3) and dacomitinib (5) have increased activity against
wild-type and activating-mutant EGFR along with increased
activity against the double mutant compared to gefitinib and
erlotinib. However, the potency of these compounds against the
double mutant is lower than the potency against wild-type EGFR
(i.e., double mutant to wild-type margin of <1). WZ-4002 (6) is
more active against the double-mutant enzyme and maintains
activity against the activating mutant but with a margin over the
wild-type enzyme broadly consistent with the published data.24
Identification of Double-Mutant-Selective Templates.
To identify mutant-selective templates, a set of compounds was
selected and tested against the EGFR wild-type and double-
mutant enzymes using biochemical assays at an external CRO25
(Figure 3). This compound set included some reversible EGFR
inhibitors from our compound collection (red) along with a
selection of published irreversible EGFR inhibitors (yellow) for
reference. Additional examples of compounds from our collec-
tion were added that have shown activity against kinases with
methionine gatekeepers during previous AstraZeneca drug-
discovery projects (blue). The reversible EGFR inhibitors
showed increased activity against the wild-type enzyme when
compared to the double mutant, consistent with the described
activity profiles of gefitinib and erlotinib. The irreversible anilino-
quinazoline-based EGFR inhibitors (including 3, 4, and 5)
showed significant activity in both assays, consistent with an
increase in activity from the formation of the covalent bond.
Note that it is difficult to accurately compare the relative
potencies of the reversible versus irreversible compounds in
these assays because of their different mechanism of action,
with irreversible inhibitors often demonstrating time-dependent
inhibition.26,27 However, the set of compounds that was of
particular interest were the examples from internal AstraZeneca
projects that had shown activity against kinases with a
methionine gatekeeper.
Interestingly, these compounds were active against double-
mutant EGFR with selectivity over wild-type, a common feature
of which was a pyrimidine core. A representative example, 7
(Table 3), contains an indole headgroup with an IC50 of 0.009 μM
against the double mutant and with an 88-fold selectivity over
wild-type EGFR. Although this reversible inhibitor was active in
this biochemical double-mutant assay, we observed a large
E
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covalent bond. The meta position of the aniline appeared to be
the most suitable location, and such examples were prioritized.
A range of different substituents were modeled to estimate their
potential to target Cys-797, and from this work we decided to
incorporate an acrylamide-based side chain. A particular
example of interest was compound 8, which showed improved
cellular activity in the activating- and double-mutant cell assays
with selectivity over wild-type (Table 4).
Table 3. Identified Double-Mutant (DM)-Selective
Reversible EGFR Enhibitor
From a set of further analogues synthesized, an example with
an ortho-methoxy group at R1 (compound 9) showed increased
biochemical and cellular activity. Further examples were
synthesized with the acrylamide attached to different regions
of the molecule. Interestingly, moving the acrylamide to the para
position (compound 10), which is also likely to be positioned
near Cys-797, appears not to result in covalent inhibition (Table 5).
Compound 10 showed only moderate activity in the double-
mutant biochemical assay (0.3 μM) and minimal activity in cellular
assays. The acrylamide was also appended to a dihydroindole
ring; however, this compound (11) was less potent in the
double-mutant assays (0.052 and 2.1 μM vs enzyme and cell,
respectively). Furthermore, nitrile-based compound 12 was synthe-
sized in an attempt to form a reversible covalent interaction
resulting in a thioimidate adduct, as precedented in cysteine
and serine protease inhibitors.30 Although this compound was
active in the double-mutant biochemical assay (IC50 0.065 μM),
12 showed minimal cellular activity (IC50 2.8 μM), suggesting
that this functional group was perhaps not suitable for the
cysteine in EGFR or that a suitable geometry had not been
achieved for the covalent bond to form.
To gain further confidence that our early compounds would
be able to form the desired covalent bond, 9 was prioritized for
crystallization in an EGFR wild-type structural system, which
was available via an external CRO.31 The binding mode of
compound 9 was subsequently solved (PDB code 4LI5), as
shown in Figure 5, demonstrating that a covalent bond was
formed to Cys-797. In this case, the binding mode of this
template in the EGFR receptor was consistent with the binding
mode found in our earlier modeling work.
DM enzyme
WT enzyme
DM/WT
selectivity
DM cell
compound
(μM)
(μM)
(μM)
7
0.009
0.79
88
0.77
enzyme-to-cell drop-off of around 90-fold when it was tested in
our double-mutant cellular assay (IC50 0.77 μM), which is likely
driven by the high ATP affinity of this enzyme and the high
ATP concentration present in the cellular environment.28
Therefore, we aimed to convert compound 7 into a covalent
inhibitor to partially overcome the effect of the increased
affinity of ATP for the mutant kinase while hoping to maintain
the intrinsic selectivity over the wild-type enzyme.
Structure-Based Design Targeting Cys-797. A predicted
binding mode of compound 7 was modeled using GLIDE,29
utilizing a public domain crystal structure of the EGFR-T790M
mutation (PDB code 2JIT6) following protocols detailed in the
Supporting Information. Although this structure does not
contain an activating mutation, we hoped that it would be
adequate for the modeling of potential binding modes that we
could exploit by structure-based design.
Of the various potential binding modes suggested by the
modeling, we decided to progress with the hypothesis that the
pyrimidine core of the molecule formed a hydrogen bond to
the kinase hinge, specifically to the backbone of Met-793
(Figure 4). In this binding mode, the chlorine substituent is
Focus on Reducing Lipophilicity. One of the key issues
with our initial compounds described above was their high
lipophilicity (LogD7.4 >4.3). This level of lipophilicity can often
translate into poor physicochemical properties and pharmaco-
kinetics (PK), hindering later development.32,33 We were also
keen to ensure low activity against the hERG potassium ion
channel. A key aim of the evolution of this series was therefore
to reduce the overall lipophilicity, indicated in subsequent tables
by the experimental LogD7.4 measurements, while retaining the
existing levels of EGFR potency and the favorable selectivity
profile. The activity and wild-type selectivity of subsequent
compounds was monitored using in-house cellular assays. We
no longer routinely measured the biochemical activity of our
compounds.
To improve the physical properties of compounds within this
series, we decided to modify what we referred to as the head
group. In addition, we decided to assess the effect of the
addition of a basic group to the acrylamide (Table 6). Out of
these head groups, the pyrollopyridine system (compound 13)
was especially interesting, maintaining reasonable potency in
our double- and activating-mutant cell assays (IC50 0.033 and 0.15 μM)
but with an improved margin to wild-type EGFR (390-fold).
Furthermore, the basic acrylamide-based side chains of
template B (examples 14 and 15), although showing a small
decrease in activity of 2 to 3 fold compared to the neutral
Figure 4. Modeled binding mode of compound 7 in a published
EGFR crystal structure (PDB code 2JIT) that contains a T790M
mutation.
directed toward the methionine gatekeeper. The indole group is
positioned adjacent to the region of the kinase referred to as the
back pocket. The anilino group of the inhibitor is oriented
toward the solvent channel of the ATP pocket and was
predicted to form an additional hydrogen bond to the hinge.
Assuming this modeled binding mode, we attempted to design
compounds with a reactive substituent positioned in a manner
that would enable targeting Cys-797 via the formation of a
F
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Table 4. Activity Data for Synthesized Inhibitors Targeting Cys-797 Using the Acrylamide Functionality
compound
R1
DM enzyme (μM)
AM cell (μM)
DM cell (μM)
WT cell (μM)
DM/WT selectivity
LogD7.4
8
9
H
0.053
0.27
0.081
0.022
3.5
43
25
>4.3
>4.3
OMe
0.0063
0.029
0.55
Table 5. Activity Data for Synthesized Compounds Exploring SAR around the Covalent Bond Interaction
equivalents, did show a marked reduction in LogD7.4 to a level
at which meaningful experimental values could be measured.
On the basis of the overall potency, selectivity, and physical
property profile of 15, this compound was selected as a key in
vivo probe compound for this template. These compounds also
began to populate a more favorable area of physicochemical
property space, with a LLE (ligand lipophilicity efficiency) of
3.4 for example 15 using the double-mutant cell assay and
experimental LogD7.4.
Investigating the Effect on 5-Position Substituents on
the Pyrimidine. The 5-position of our template was predicted
by our modeling to be positioned adjacent to the gatekeeper
residue, so we thought that varying this position may have some
interesting effects on the potency and selectivity of compounds
within our series. Our initial hit contained chlorine in this
position, which is relatively lipophilic; therefore, varying this
group offered the potential for compound lipophilicity to be
Figure 5. Protein crystal structure of compound 9 bound in wild-type
EGFR. The described covalent bond to Cys-797 is shown (PDB code
4LI5).
G
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Table 6. Activity and Lipophilicity Data for Compounds Targeting an Improved Physicochemical Property Space
a
LLE (DM) = pIC50 DM (cell) − LogD7.4.
Table 7. Activity and Lipophilicity Data of Compounds Exploring the Effect of the 5-Substituent Position (R1) on the
Pyrimidine
compound
R1
AM cell (μM)
DM cell (μM)
WT cell (μM)
DM/WT selectivity
LogD7.4
LLE (DM)
15
16
17
18
19
Cl
H
0.40
0.39
0.38
0.25
1.3
0.096
0.25
0.22
0.29
15.2
23
237
80
3.6
2.6
3.3
3
3.4
4
20
F
19
85
3.4
3.5
3.5
Me
24
82
S(=O)Me
>30
>2
1.3
reduced.34 A range of 5-substituted compounds was sub-
sequently prepared and assessed across our in-house cellular
assays (Table 7).
The range of R1 substituents that we investigated varied in
both size and lipophilicity. Although potency varied across the
double-mutant cell assay, the LLE (using the double-mutant
cell data) remained constant at around 3.4 to 3.5 except for
R1H where it was increased to 4. This was an interesting
result because removing the chlorine of compound 15 (giving
compound 16) reduced LogD7.4 by an order of magnitude
H
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a
Table 8. Glutathione (GSH) Reactivity of 6-Membered Ring Aromatic Model Compounds
compound
GSH t1/2 (min)
Log kGSH* (M‑1 s‑1)
R1
R2
R3
R4
R5
R1 σmeta
R1 σI
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
816
−2.51
−2.11
−3.07
−2.24
−2.08
−2.23
−2.32
−1.17
−1.76
−2.16
−0.94
−1.12
< −3.54
< −3.60
< −3.60
< −3.60
4-aminophenyl
4-fluorophenyl
benzyl
H
H
H
C
H
H
H
H
H
H
H
H
H
H
H
H
C
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C
−0.006
0.12
0.052
0.13
0
323
2940
436
−0.08
0.06
phenyl
0.12
0.12
0.119
0.11
0.194
0.16
0.1
299
phenyl
H
H
H
H
H
H
H
H
H
H
H
H
0.06
428
2-methylphenyl
4-methoxyphenyl
2-cyanophenyl
3-fluorophenyl
4-methylphenyl
2-pyridyl
0.07
524
0.05
37.2
0.18
145
0.15
361
0.06
22
0.33
0.25
0.175
0.05
0.12
0.12
0.12
32.9
4-cyanophenyl
tert-butyl-phenyl
phenyl
0.221
−0.043
0.06
>8640
>10 000
>10 000
>10 000
phenyl
0.06
phenyl
H
0.06
a
The full list of the explicit structures for compounds in this table can be found in Table S1 of the Supporting Information. Note that the rate
constant, k, was derived using [GSH] 4.61 mM. σ values were determined using ACD/Laboratories software (version 12.01). All compounds with an
absolute rate constant showed a more polar product peak of mass parent +307 except for example 22 where no products were observed (reflecting a
much slower reaction compared to other compounds coupled to the detection limits of LC−MS).
(3.6 compared to 2.6) and might allow the physicochemical
properties of this series to be improved further. Although the
double-mutant activity was reduced for compound 16, compared
to 15 (0.25 vs 0.096 μM) the activity against the activating-mutant
form was unchanged (0.39 vs 0.40 μM). The double-mutant
selectivity of 16 was reduced to 80-fold, from 237-fold, for
compound 15, potentially resulting from chlorine interacting more
effectively with the methionine gatekeeper residue. However, our
collective data on these compounds suggested that it might be
possible to achieve reasonable potency in the EGFR double and
activating mutants while leveraging the selectivity over wild-type
without a chlorine in the 5-position. We postulated that removing
the chlorine might improve the physicochemical properties via the
reduction in overall lipophilicity of these molecules.
Modulating Compound Reactivity. The potency of
covalent inhibitors is influenced by both the reversible binding
properties of the compound to the protein (Ki) and the ability
of the compounds to covalently bind to a suitable residue in the
active site (kinact). As such, it is important to aim for compounds
of sufficient chemical reactivity toward an active-site residue (in
this example cysteine) while balancing against unwanted off-
target binding that could lead to high compound clearance and
or idiosyncratic toxicity.35 One method of optimizing covalent
compounds in this way is through the measurement of the
kinact/Ki values for compounds within a series. This approach,
however, proved technically challenging using our assays, so we
were unable to exploit it for driving SAR routinely.26 Our alter-
native approach was to establish a suitable ‘reactivity window’
for our compounds and to develop an understanding of how to
synthesize compounds that were more likely to fall within the
intended range. As a measure of thiol reactivity, we have used
an assay based on determining the second-order rate constant
(kGSH) for the reaction of our compounds with glutathione
(GSH).36−38
To determine the structural and electronic features that
influenced the chemical reactivity of our compounds, a set of
six-membered aromatic ring acrylamide-containing models
were initially investigated in the glutathione assay (Table 8).
These model acrylamides have relatively good aqueous solubility
and thus we avoid any complication of reaction kinetics that
might have arisen because of precipitation issues. All com-
pounds investigated were stable in phosphate buffer alone at
pH 7.4. In the presence of glutathione, a simple addition
reaction (Scheme 3) was observed, generating the more polar
glutathione adduct, which is seen by liquid chromatography−
mass spectrometry (LC−MS) as a mass increase of 307 units.
The Hammett plots in Figure 6 enable a quantitative under-
standing of the relationship between the electronic effects
exerted through the R1 position of the acrylamide, expressed as
σ values, and glutathione reactivity. Consequently, this
knowledge can be applied to design elaborated compounds
that sit in the desired reactivity window using appropriate R1 σ
values. From our results set, the rate of the reaction appeared to
be dependent on the electron-withdrawing ability of groups in
the R1 position (Figure 6) of the acrylamide. In particular, the
inductive effect of the R1 group (represented either as σmeta
or σind) was seen to determine thiol reactivity with ρ values of
5.56 and 9.23, respectively. The addition of further electronic
descriptors such as σpara or σmeso to the Hammett equations (1)
and (2) in Figure 6 did not appear to be significant. Such
inductive electron withdrawal at the R1 position would be
consistent with enhanced polarization of the alkene bond via
carbonyl conjugation and also serves to stabilize the enolic
transition state. The introduction of methyl groups onto either
the α or β carbon of the model acrylamides (R3 and R4)
resulted in no reaction being observed with glutathione, ruling
out these substituents as options on elaborated compounds.
A similar decrease in thiol reactivity following methylation at
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Scheme 3. Addition Reaction of Model Compounds with Glutathione
Figure 6. Hammett plot of σmeta and σind properties versus compound reactivity (Log kGSH) across the model compound set.
the α and β carbon has been observed by other workers in their
investigations with α,β-unsaturated aldehydes, ketones, esters,
acids, and amides.39−42 Engels et al. calculated via quantum
mechanical/molecular modeling (QM/MM) an increased
energy barrier to methyl thiolate attack on α,β-unsaturated
phenyl ketones with an elevated energy of the enolate
intermediate upon methylation at the α and β positions,
suggesting steric effects as a possible reason for such a reactivity
change.41 Although other workers have similarly suggested
steric effects as an explanation for decreased reactivity for α and
β methylation,39 Kerske et al. argue that reactant stabilization
by hyperconjugation of α and β methyls and not steric
hindrance is the reason for the lowered reactivity.42
Table 9 captures the glutathione reactivity constant data of
more elaborated compounds. This compound set consists of
examples containing pyrimidine, anilinoquinazoline, and
anilinoquinoline scaffolds. To increase the diversity of the
structures within this data set, we have also included examples
from an additional template we identified on the basis of a bis-
anilino-pyrimidine scaffold, some examples from which have
since been disclosed in the literature.43
0.43 in Log kGSH (factor 2.7) on substituting hydrogen for
methoxy in the R4′ position of the aniline (matched pairs
36/14 and 43/44). In contrast, varying the substituent at R5′
(cyano, methyl, hydrogen, and chlorine) on the pyrimidine had
no significant effect on reaction rate, with the largest difference
in Log kGSH being 0.13 (factor 1.3). This is consistent with the
increased distance of R5′ from the acrylamide group, thereby
exerting minimal electronic effect. Similarly, varying the R4′
position in the quinoline series (core iv, examples 53 and 54)
produced no significant effect on the reaction rate. There are,
however, clear outliers on the Hammett plot in Figure 7.
Compound 46 shows a significantly faster reaction than was
expected from an R1 inductive effect alone. Analysis of the
reaction products of 46 using LC−MS indicates the substitution
of the morpholine group for glutathione (Scheme 4). The
increased reaction rate is believed to be due to intramolecular
base catalysis by the α-morpholine group, which is able to form
a six-membered hydrogen-bond ring in the transition state,
facilitating nucleophilic attack. Two mechanisms can be
postulated to occur, namely, a two step addition/elimination
with the morpholine stabilizing the enolate transition state
(Scheme 4a) or a concerted reaction where the morpholine
base activates the glutathione by removal of a proton (Scheme 4b).
Identifying which of these mechanisms is dominant is not clear
on the basis of the present data. In work by Tsou et al.
that looked at quinazoline acrylamides, the activation of
glutathione by α-morpholine has been proposed as a mecha-
nism to explain the observed reactivity in a competition assay in
THF/H2O/Methanol.44
With the more diverse elaborated compounds (Figure 7), the
dependence of reactivity on the inductive effect at R1 as
expressed by R1 σmeta is observed across a variety of R1
templates and fits the same Hammett line as seen with the
model systems. This suggests that the learning from our model
systems can be applied to more leadlike molecules.
Looking across the anilinopyrimidine-containing compounds
(core i, Table 9), there is a consistent drop in reactivity of
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Compound 41 (core ii) also appeared to react more quickly
than expected from the σmeta value. The reason for this is
unclear, but the reaction represents the only example in this
data set involving a five-membered heterocyclic substituent
attached to the acrylamide. Also of interest was 38, which
proved to be highly unstable in pH 7.4 buffer alone. The
introduction of the dimethylaminobenzyl group into the
acrylamide phenyl ring facilitated rapid intramolecular attack
by the amine to form a stable cyclic quaternary ion (Scheme 5
and Figure S1, Supporting Information).
Substitution at the acrylamide α and β positions, R1′ and R2′,
with a cyclic piperidine (47 and 48, Figure 8) resulted in no
measurable reaction with glutathione. This indicates that the
electronic activation afforded by the electron-withdrawing
partially protonated amine groups (pKa of 7.9 and 7.4,
respectively) toward the α and β carbons is insufficient to
overcome the steric barrier from the ring.
Interestingly, other workers have shown that the introduction
of electron-withdrawing groups cyano and hydroxyl at the α
and β positions of alkenals and acrylates affords an enhanced
reaction rate with thiols.39,45 When the dimethyl amine group is
introduced as a pendant substituent at the β-carbon in 15 (pKa 7.8),
a measurable reaction with glutathione is observed (Table 8).
Unfortunately, the matched pair compound of 15 without
the pendant amine group was too insoluble to measure a
glutathione reaction constant, but the matched pair of 44 and
45 reveals that the pendant amine group does not offer a
significant enhancement of the reaction rate over the
unsubstituted compound (factor of 2). This is borne out by
Figure 7, where compounds with (+) and without (open
squares) a pendant base (β substituent R1′) both sit on the
same Hammett equation line. Significantly enhanced reactivity
resulting from the pendant base (+) would have been seen as
points sitting above the line. Because an electronically
significant difference would not be expected in terms of the
amine-group activation of the β carbon between 15 and 48, this
suggests that the unconstrained pendant base in 15 offers less
steric resistance to glutathione attack than 48. The uncon-
strained β-pendant base in 15 would also be expected to more
readily able to undertake intramolecular base catalysis than 48,
but no significantly enhanced reaction rate is observed. This
contrasts with earlier studies by Tsou et al. who showed a
significant enhancement when a β-pendant base was added to
quinazoline acrylamides in a competition assay.44 It is suggested
that this difference may be due to solvent effects.
Alternative thiol reactive groups to acrylamides, namely,
acetylenes and ethene sulphonamides have also been
investigated in the glutathione assay (49, 50, 51, and 52,
shown in Table 10).
a
Table 9. Glutathione (GSH) Reactivity Data of Elaborated Acrylamide-Containing Compounds
K
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Table 9. continued
a
The full list of the explicit structures for compounds in this table can be found in Table S2 of the Supporting Information. *R1 σmeta is defined in the
scheme in Table 8 and all other substituents are defined in the figure above.
Comparing the matched pairs 44/51 and 42/52 indicates
that both acetylenes and ethene sulphonamide groups are
significantly more reactive to glutathione than acrylamides
(26.4 and 26.9 times more reactive, respectively). Interestingly,
when a pendant dimethyl amine base is added to the acetylene
group in 49, the compound is >58.7 times more reactive than
the acrylamide matched pair 15, suggesting that the basic group
has a significant activating effect not observed with acrylamides.
This is also supported by a comparison of 49 with 51, which
both share the same R1 σmeta/σI values but show >5.8 fold
difference in glutathione reactivity when the pendant base is
added to the acetylene. 52 also shares the same σmeta/σI values
as 49 and 51, indicating that the ethene sulphonamide group
has comparable glutathione reactivity to the acetylene
group (cf. 51). As with the acrylamides, the substitution of
a methyl group at the β position of the acetylene (50)
deactivates the acetylene to thiol reaction. Comparing
compound 50 to acrylamide 55, which share the same R1
σmeta/σI values (differing in their R1 structure by a distal Br
for Me change on aniline R3 substituent), shows that the
methyl acetylene is 2.6 times less reactive to glutathione than
the acrylamide.
A selection of compounds from both the model and
elaborated series covering a range of reactivities with
glutathione were also studied in the presence of the human
glutathione-S-transferase enzyme (Supporting Information,
L
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glutathione reactivity for the data set discussed in this section.
As we hoped, there is not a strong correlation because the
initial target noncovalent binding (Ki) will play a key part in
defining potency (R2 0.52 and RMS 0.53). The noncovalent
contribution to the binding is essential for driving the selectivity
of these compounds over other proteins with accessible cysteine
residues. However, there is a general trend toward more
reactive compounds showing increased activity, and com-
pounds with pIC50 values >7 for the most part tend to have
glutathione half-lives <400 min. As such, we took a glutathione
half-life of 400 min as a guide for a suitable lower limit of
reactivity. Afatinib (3) is currently in phase III clinical studies,
and the reported clinical side effects of the compound appear to
be linked to the inhibition of wild-type EGFR rather than off-
target effects resulting from covalent binding. As such, this
seemed to be a good compound to benchmark in our
glutathione assay, generating a half-life of 25 min. Therefore,
we used a half-life of 25 min as the higher limit to the window
of reactivity that we aimed to work within.
Figure 7. Reactivity data for elaborated acrylamide compounds
overlaid onto Log kGSH versus σmeta Hammett plot for model systems.
Data on the model-system compounds (Table 8) are shown in red and
elaborated compounds (Table 9) are shown in green. The data is
further classified by a selection of the β substituents (R1′) defined in
Table 9 (not defined for compounds 47 and 48 because of a lack of
observed reactivity).
Further Optimizing of the Lead Series. Our aim was to
increase further the potency of our series by exploiting
additional interactions with the protein along with the learning
from our exploration of compound reactivity. As part of this, we
synthesized a set of compounds with a piperazine substituent
on the para position of the aniline (Table 11). We expected
Table S3). This revealed that the glutathione reaction was not
catalyzed by the glutathione-S-transferase enzyme.
In seeking to define a suitable ‘reactivity window’, the
elaborated compounds studied for glutathione reactivity were
simultaneously analyzed in the double-mutant cell assay.
Figure 9 reveals the relationship between the target potency and
Scheme 4. Postulated Mechanisms for the Nucleophilic Substitution Reaction Observed for Compound 46 with Glutathione
a
Scheme 5. Proposed Intramolecular Reaction of Compound 38 in Buffer at pH 7.4
a
Formation of cyclic quaternary confirmed by MSMS m/z = 478.18.
M
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Figure 8. Novel compounds reported in this manuscript containing alpha substitutions (R2′).
a
Table 10. Selection of Alternative Covalent Modifying Groups to Acrylamide Synthesized as Part of This Work
a
The R1 substituent is defined in Table 8.
that the addition of the piperazine adjacent to the acrylamide
would effect the conformation and geometry of both
substituents, so we were keen to assess whether this com-
bination was compatible with good potency on our template.
We combined this change with the 5-position SAR described
in the previous section using both our indole and pyrollo-
pyridine head groups.
The data on this compound set suggests a general improve-
ment in potency with the addition of the piperazine substituent.
Compound 56 is more potent than 15 in both primary cell
assays (0.14 vs 0.4 μM in the activating-mutant and 0.019 vs
0.096 μM in the double-mutant assays), maintaining a good
selectivity for the double mutant over wild-type of 620-fold.
There is also a small increase in reactivity with glutathione for
compound 56 (t1/2 188 min) compared to 352 min for com-
pound 15, which is consistent with the small increase in
electron-withdrawing ability of the group attached to the
acrylamide nitrogen (σmeta 0.09 compared to σmeta 0.07). A
number of factors could influence this increase in activity,
including the preference for a basic group in this region of the
protein, the increased compound reactivity, and the effect on
acrylamide geometry. The LLE of these compounds is generally
improved, with 56 showing an increased LLE of 4.1 compared
to 3.4 for 15. Removal of the chlorine (compound 57) showed
a reduction in double-mutant cellular activity, 0.071 μM com-
pared to 0.019 μM, but no effect on activating-mutant activity,
as observed previously. The transferring of this learning back to
the indole head group identified compounds that were sig-
nificantly more potent against the activating and double mutant.
Example 58 is a particularly potent compound, with an LLE of 4.6.
N
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In Vivo Efficacy of Compound 15. Compound 15 was
identified as a suitable in vivo proof-of-concept compound from
our scoping work. To build further confidence in the covalent
mechanism of action, the equivalent nonreactive propionamide
was synthesized (compound 60, Figure S2, Supporting Information),
showing a lower double-mutant biochemical activity (0.018 μM)
and significantly reduced cellular activity (2.3 μM). Although
not the most potent (or wild-type selective) example synthesized,
the overall balance of properties identified 15 as having an
acceptable combination of activity and pharmacokinetic (PK)
properties to validate our approach (Table 12). In addition, 15
demonstrated encouraging antiproliferative activity when tested
in our H1975 (double mutant), PC9 (activating mutant), and
Calu3 (wild-type) cell lines with an EC50 of 0.099, 0.14, and
1.78 μM respectively. This profile is consistent with our
described cellular data showing significant activity in the double
and activating mutants with selectivity over the wild-type
enzyme. Subsequently, 15 was orally dosed in an in vivo
efficacy study using an H1975 double-mutant xenograft model in
immune-compromised mice (Figure 10). Briefly, tumor cells were
inoculated subcutaneously, and the mice were randomized into
groups of 4−10 mice prior to compound dosing when tumors
reached approximately 0.2−0.4 cm3. Tumor growth was measured
from the start of treatment, assessed by comparison of the mean
change in the tumor volume for the control and treated groups,
and represented as tumor growth inhibition (where >100% TGI is
a regression from the starting size). Statistical significance was
evaluated using a one-tailed t test. Compound 15 showed
significant efficacy of 105% total growth inhibition (TGI) after 7
Figure 9. Compound reactivity measured by Log kGSH (M−1 s−1)
plotted against EGFR double-mutant (DM) cellular pIC50 for the
elaborated compounds reported in this manuscript. Dashed lines
indicate the ‘reactivity window’ in which the design of novel
compounds was subsequently focused.
The data on these compounds is also consistent with the early
data on this head group, with the indole-containing compounds
showing a reduced margin with wild-type EGFR (140-fold and
19-fold for 58 and 59, respectively), albeit with greater absolute
double-mutant activity. Whether a more potent EGFR double-
and activating-mutant inhibitor with a smaller margin to wild-type
is preferable over a less potent inhibitor with a greater margin to
wild-type will require further assessment.
Table 11. Activity and Lipophilicity Data on Compounds with the Addition of a Piperazine Group
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Table 12. Overview of Activity, Physicochemical, and PK Data of Compound 15
AM cell
(μM)
DM cell
(μM)
WT cell
(μM)
solubility
a
PPB (% free)
b
Human Heps Clint
Rat Heps Clint
hERG
(μM)
Rat PK
c
compound
(μM)
(M/R/H)
(μL min−1 × 106 cells) (μL min−1 × 106 cells)
CL/F
15
0.40
0.096
23
1.6
1.2/1.2/0.8
<3 18
4.2
8.1/45%
a
b
Solubility was evaluated under thermodynamic conditions using crystalline solid. Plasma protein binding (PPB) was assessed by equilibrium
c
dialysis in the appropriate species (mouse/rat/human) at 37 °C. Free and bound concentrations were quantified by LC−MS. Intravenous clearance
(mL min−1 kg−1) was assessed in male Han Wistar rats when dosed with a 3 μmol kg−1 solution. Oral bioavailability was observed with a 6 μmol kg−1
solution dosed in male Han Wistar Rats.
b
Table 13. Summary of the Efficacy Data from Xenograft Studies on Compound 15 Compared to Gefitinib (1)
compound
15
Gefitinib (1)
oral dose (mg/kg/day)
60
H1975 (% TGI)
PC9 (% TGI)
A431 (% TGI)
105 (p < 0.001)
134 (p < 0.01)
142 (p < 0.001)
46 (p < 0.05)
79 (p < 0.001)
a
6.25
8 (p NS)
a
b
Data from a 100 mg/kg/day oral dose of gefitinib. Statistical significance (p values in brackets) was calculated from compound-treated and vehicle
groups using a one-tailed t-test. NS indicates that the % TGI was not significant compared to the vehicle-treated group.
The compound showed >75% inhibition for 10 kinases, which
were IGF1R, insulin receptor (IR), CAMKKbeta, CHK2, PBK,
FLT1, MAP3K11, smMLCK, BTK, and MELK. However, out
of these kinases, only BTK has a cysteine in the same position
as EGFR, so the mode of inhibition is likely to be via a
reversible mechanism toward the majority of these targets. The
full selectivity profile of compound 15 can be viewed in the
Supporting Information (Table S4). We do not believe these
off-target activities contributed to the efficacy in the reported
xenograft models; however, information on the optimization of
the selectivity profile of this series will be reported in future
disclosures.
CONCLUSIONS
■
We have reported the development of a series of potent
inhibitors of the activating and resistant mutants of EGFR with
varying degrees of selectivity over the wild-type enzyme. This
activity profile across the mutants would appear to be suitable
to demonstrate activity against the activating and resistant
mutants of EGFR but with an improved margin to toxicity
associated with wild-type activity. As part of this approach, we
simultaneously optimized the covalent and noncovalent
elements of binding into a suitable range to identify compounds
with satisfactory potency and intrinsic reactivity. This approach
may be exploited more generally to optimize covalent inhibitors
across a broader range of targets. Also reported is encouraging
antitumor efficacy in H1975 double-mutant and PC9 activating-
mutant models with compound 15. This compound was also
observed to have significantly lower activity in the A431 model
used here as a measure of EGFR wild-type activity. However,
the relatively poor solubility (1.6 μM) and the hERG IC50 of
4.2 μM demonstrated the need for further development efforts
for this compound and across the scaffold more generally.
Further evolution of this series will be reported at a later date; in
particular, this will focus on improving the potency, kinase
selectivity, and properties of the series toward a clinical candidate.
Figure 10. Antitumor efficacy study data on compound 15 across the
double (H1975), activating (PC9), and wild-type (A431) EGFR
xenograft models. Tumor growth inhibition (% TGI) was calculated
between the compound treated and vehicle groups, with a % TGI >
100 indicating tumor regression from the initial tumor size.
days at an oral dose of 60 mg/kg/day (formulated as the mesylate
salt). This dose and formulation was chosen because we had
observed good exposure in the nude (Cmax of 7.1 μM and AUC0−t
48.2 μM h) and severe-combined immunodeficiency (SCID)
(Cmax 8.1 μM and AUC0−t 119.3 μM h) mice that are used in
these models, which also compared well with the rat PK reported
in Table 12 (Cmax of 5.32 μM and AUC0−t 92.7 μM h).
Compound 15 was subsequently taken into an efficacy study
in a PC9 activating-mutant cell line and a wild-type A431 cell
line in immunocompromised mice. The activity against the
PC9 xenograft model at the same dose (134% TGI, resulting in
tumor regression) was also encouraging, and the compound
showed reduced activity against the A431 xenograft model
(46% TGI), as was hoped from the wild-type margin demon-
strated from the in vitro cellular profile.
As a comparison, an oral dose of gefitinib of 6.25 mg/kg/day
(representative of the clinically relevant human dose), showed
142% TGI in the PC9 xenograft model and 79% TGI in the
A431 xenograft model after 7 days (Table 13). Gefitinib
showed little activity in the H1975 xenograft model, even at the
higher oral dose of 100 mg/kg/day, which resulted in an 8%
TGI (not significant) after 7 days.
Kinase Selectivity of Compound 15. Compound 15 was
submitted to a broad panel of >70 diverse kinases at The
University of Dundee,46 where it was tested at a concentration
of 1 μM in biochemical assays to assess wider kinase selectivity.
EXPERIMENTAL SECTION
■
Biochemical and Cellular Activity Assays. Compounds were
tested at a range of concentrations. The mean data values for each
concentration along with untreated control wells and 100% inhibition/
competition control wells were used to derive a plot of inhibition/
competition against concentration. Origin software was used to
interpolate the IC50 values by nonlinear regression.
Kinase Inhibition Assay Using HTRF (Homogenous Time-
Resolved Fluorescence). The inhibitory activity of compound
against the kinases EGFR (T790M/L858R) was determined with
P
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CisBio HTRF (homogenous time-resolved fluorescence) KinEASE
TK (#62TKOPEC). The enzyme reaction contains recombinant
N-terminal GST-tagged human EGFR (T790M/L858R), which
phosphorylates the HTRF tyrosine kinase biotinylated substrate.
The sequence of the substrate is proprietary to CisBio. Compounds
were serially diluted in 100% (v/v) DMSO before being acoustically
dispensed from an Echo 555 (Labcyte) into black Corning 1536-well
assay plates. Kinase activity assays were performed in a total reaction
volume of 3 μL per well. A 1.5 μL enzyme reaction consisted of
1.6 nM EGFR (T970M, L858R), 1 mM DTT, and 10 mM MgCl2. A
1.5 μL substrate mix consisted of 1 μM TK substrate, 30 μM ATP,
1 mM DTT, and 10 mM MgCl2. Following a 50 min incubation, 3 μL
of stop mix was added, which consisted of 250 nM Strep-XL665 and
TK Ab-Cryptate diluted in kit detection buffer. The plates were
incubated for 1 h before being read on Pherastar using standard HTRF
settings. N-terminal GST-tagged recombinant human EGF receptor,
with amino acids 696-end containing the T790M and L858R muta-
tions, was obtained from Millipore.
from the total number of cells, and the live cell number was plotted to
determine EC50 values.
X-ray Crystallography. The kinase domain of the wild-type
EGFR protein was expressed in Sf9 insect cells as a GST-fusion
protein with a thrombin cleavage site.47 After affinity purification and
cleavage of the GST tag, a gel-filtration step was performed, and the
resulting protein was concentrated to 6 mg/mL. Apo crystals were
prepared using the vapor-diffusion technique by drops equilibrated
against 0.2 M NH4Cl and 1.2 M Na−K-tartrate buffered with 10 mM
acetate at pH 4.6 and 0.15 M hepes, pH 7.0. Protein−ligand complex
crystals were generated by soaking apo-crystlas with 2 mM of the
compound (dissolved in DMSO at 100 mM). The Free Mounting
System (Proteros biostructures31) was used for cryo cooling.
Diffraction data were collected at the Swiss Light Source at 100 K
on a Pilatus6M detector, and the data was processed using XDS and
XSCALE.48 The crystallographic model was completed in Coot49 and
refined with Refmac5.50 Crystallographic data and refinement
parameters are summarized in Table 14.
Measurement of Cellular Phosphorylation Assay. For EGFR
(T790M/L858R), the human lung cell line NCI-H1975 was obtained
from the American Type Culture Collection. For EGFR (Exon 19
deletion), the human lung cell line PC9 was obtained from the Akiko
Hiraide from Preclinical Sciences R&D AZ Japan. The growth media
was RPMI 1640 containing 10% fetal calf serum and 2 mM glutamine.
For EGFR (wild type), the human colon adenocarcinoma cell line
LoVo was obtained from the European Collection of Cell Cultures.
LoVo growth media was RPMI 1640 containing 3% charcoal-stripped
fetal calf serum and 2 mM glutamine. All cells were used from assay-
ready frozen cryo banks. Assays to measure cellular phosphorylation of
endogenous p-EGFR in cell lysates were carried out according to the
protocol described in the R&D Systems DuoSet IC Human Phospho-
EGFR ELISA (R&D Systems, no. DYC1095). Following thawing and
resuspension, 40 μL of cells were seeded (10 000 cells/well for NCI-
H1975 and PC9 or 15 000 cells/well for LoVo) in growth medium in
Corning black, clear-bottomed 384-well plates and incubated at 37 °C
with 5% CO2 overnight. The cells were acoustically dosed using an
Echo 555 with compounds serially diluted in 100% DMSO (v/v). The
cells were incubated for a further 2 h and following aspiration of
medium, 40 μL of 1× lysis buffer was added to each well. LoVo were
stimulated with EGF (25 ng/mL) for 10 min before lysis. Greiner
black high-bind 384-well plates were coated with capture antibody and
then blocked with 3% BSA. Following the removal of the blocking
solution, 15 μL of lysate was transferred to the Greiner black high-bind
384-well plates and incubated for 2 h. Following aspiration and
washing of the plates with PBS/A, 20 μL of detection antibody was added
and incubated for 2 h. Following aspiration and washing of the plates with
PBS/T, 20 μL of QuantaBlu fluorogenic peroxidase substrate (Thermo-
Fisher Scientific, no. 15169) was added and incubated for 1 h. Twenty
microliters of QuantaBlu stop solution was added to the plates, and
fluorescence was read on an Envision plate reader using an excitation
wavelength of 352 nm and an emission wavelength of 460 nm.
Table 14. Crystallographic Data Collection and Refinement
Summary
data collection statistics
Cell: a = b = c, α = β = γ (Å, deg)
resolution (Å)
unique reflections
multiplicity
completeness (%)
Rsym (%)
mean (I)/sd
144.89, 90
2.64 (2.96−2.78)
15 026 (2185)
24.4 (25.5)
a
a
a
a
99.9 (100.0)
a
9.2 (44.5)
a
32.02 (9.75)
refinement statistics
number of reflections (working/test)
R/Rfree (%)
14 487/539
16.5/20.3
number of atoms
protein
water
2245
69
ligand
30
ions
1
deviation from ideal geometry
bond lengths (Å)
bond angles (deg)
bonded B’s (Å2)
Ramachandran plot
most favored region
additional allowed region
generously allowed region
disallowed region
0.007
1.04
2.10
92.1
7.9
0
0
a
Data in parentheses refer to the highest resolution shell.
Experimental Procedure for Cellular Proliferation Experi-
ments. Cell lines were plated in 384−well plates at between 500 and
1000 cells per well, depending on the cell line, in 70 μL per well of
RPMI media containing 10% fetal calf serum, 2 mM L-glutamine, and
1% penicillin/streptomycin. The cells were allowed to attach overnight
at 37 °C under 5% CO2. The following day, titrations of test
compound were added to the assay plates using an Echo Liquid
Handler Labcyte, and the treated cells were incubated for a further
72 h at 37 °C under 5% CO2. Each compound was tested as an
11-point dose response, with a top concentration of 10 μM using 1:3
dilutions. Following a 72 h incubation of the compound-treated plates,
5 μL of 2 μM SYTOX Green Nucleic Acid Stain, Life Technologies
was added per well, and the plates were incubated at room
temperature for 1 h. The number of fluorescent cells per well was
measured with an Acumen TTP LabTech Ltd., with this number
representing the dead cell count. Ten microliters of 0.25% saponin was
added per well, and the plates were incubated overnight at room
temperature. The total number of fluorescent cells per well was
acquired with the Acumen. The number of dead cells was subtracted
Synthesis and Characterization of Key Compounds 7, 9, 15,
and 58. General Chemistry Statement. Microwave reactions were
performed using one of the following reactors: Biotage Initiator,
Personal Chemistry Emrys Optimizer, Personal Chemistry Smithcrea-
tor, or CEM Explorer. Work-up procedures were carried out using
traditional phase-separating techniques or by using strong cation-
exchange (SCX) chromatography using an Isolute SPE flash SCX-2
column (International Sorbent Technology Limited); when necessary,
organic solutions were dried over anhydrous MgSO4 or Na2SO4. Flash
column chromatography (FCC) was performed on Merck Kieselgel
silica (Art. 9385), on Silicycle cartridges (40−63 μm silica, 4−330 g
weight), or on Grace resolv cartridges (4−120 g) either manually or
automated using an Isco Combi Flash Companion system. Preparative
reverse-phase HPLC (RP HPLC) was performed with C18 reversed-
phase silica, for example, with a Waters Xterra or XBridge preparative
reversed-phase column (5 μm silica, 19 mm diameter, 100 mm length)
or with a Phenomenex Gemini axia preparative reversed-phase column
(5 μm silica, 110A, 21.1 mm diameter, 100 mm length) using
decreasingly polar mixtures as eluent (e.g., 1−5% formic acid or 1−5%
Q
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aqueous ammonium hydroxide (d = 0.88)) as solvent A and
acetonitrile as solvent B or MeOH/MeCN 3:1. The intermediates
were not necessarily fully purified, but their structures and purity were
assessed by TLC, NMR, HPLC, and mass spectral techniques, and
they are consistent with the proposed structures. The purities of
compounds used for biological testing were assessed by NMR, HPLC,
and mass spectral techniques, and they are consistent with the
proposed structures; the purity was ≥95%. FT 1H and 13C NMR
spectra were obtained either with a 400 MHz (1H, 400 MHz; 13C, 101
MHz), 500 MHz (1H, 500 MHz; 13C, 126 MHz), or 700 MHz (1H,
4-Fluoro-2-methoxy-1-nitrobenzene (62).
Potassium carbonate (44 g, 318.27 mmol) and 5-fluoro-2-
nitrophenol (50 g, 318.27 mmol) dissolved in acetone (750 mL)
were stirred under argon for 30 min, and methyl iodide (19.81
mL, 318.27 mmol) was added dropwise. The resulting solution
was stirred at 60 °C overnight, allowed to cool to room
temperature, and concentrated to dryness. Water (500 mL) was
added, and the resulting mixture was extracted with EtOAc (3 ×
200 mL). The organic layers were combined, washed with a 1
M aqueous solution of NaOH (2 × 200 mL) and brine (200
mL), dried (MgSO4), and concentrated to afford the crude
product as an oil, which was crystallized to give 4-fluoro-2-
methoxy-1-nitrobenzene (53.8 g, 99%) as a clear beige solid. 1H
NMR (DMSO-d6) δ 3.93 (3H, s), 6.93−6.98 (1H, m), 7.28−
7.31 (1H, m), 7.99−8.02 (1H, m). No ionization in LC−MS.
1-[4-(3-Methoxy-4-nitrophenyl)piperazin-1-yl]ethanone (63).
1
700 MHz; 13C, 176 MHz) Bruker spectrometer. H and 13C shifts are
given in ppm and are measured relative to the internal residual solvent
peak (CDCl3 7.25 ppm/77 ppm). Peak multiplicities are expressed as
follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br s,
broad singlet; and m, multiplet. Analytical HPLC was performed on
C18 reverse-phase silica with a Phenomenex Gemini reversed-phase
column (5 μm silica, 110 A, 2 mm diameter, 50 mm length) using
decreasingly polar mixtures as eluent (e.g., decreasingly polar mixtures
of water containing 0.1% formic acid or 0.1% ammonia as solvent A
and acetonitrile as solvent B or MeOH/MeCN 3:1), with a flow rate of
about 1 mL/min. Detection was by electrospray mass spectrometry
and by UV absorbance at a wavelength of 254 nm. Electrospray mass
spectral data were obtained using a Waters ZMD or Waters ZQ
LC/mass spectrometer acquiring both positive and negative ion data,
and generally, only ions relating to the parent structure are reported.
Accurate mass and MSMS fragmentation data were obtained using a
ThermoScientific hybrid LTQ−FT mass spectrometer with an Agilent
1100 quaternary pump with PDA and Autosampler. FIve microliters of
sample dissolved in 50:50 acetonitrile/water in 0.1% formic acid was
injected onto a Thermo Scientific Hypersil Gold 50 × 2.1 mm, 5 μm
particle LC column. The gradient was 5−100% B over 17 min, with a
3 min re-equilibration time at 5% B. The flow rate was 0.5 mL/min,
with A being 0.1% formic acid in water and B being 0.1% formic acid
in acetonitrile. The MS and MSMS spectra were obtained in ESI +ve
mode in both the ion trap and ion cyclotron resonance (ICR) cell
using helium as the collision gas at a normalized collision energy of
35 eV. The ICR cell was run at resolution settings of 25 000 in MS
mode and 12 500 in MSMS mode.
1-Acetylpiperazine (12.36 g, 96.42 mmol) and DIPEA (18.32
mL, 105.19 mmol) were added to a stirred solution of 4-fluoro-
2-methoxy-1-nitrobenzene (62) (15 g, 87.65 mmol) dissolved
in DMA (45 mL) under argon. The resulting solution was
stirred at 90 °C overnight. The reaction mixture was allowed to
cool to room temperature and quenched with water (500 mL).
The aqueous layer was extracted with EtOAc (×3). The
combined organic layers were washed with water (×2) and
brine, dried (MgSO4), and concentrated to afford 1-(4-(3-
methoxy-4-nitrophenyl)piperazin-1-yl)ethanone (22.0 g, 90%)
1
as a yellow solid. H NMR (CDCl3) δ 2.15 (3H, s), 3.43 (4H,
Synthesis of Compound 7. 3-(2,5-Dichloropyrimidin-4-yl)-1H-
indole (61).
dt), 3.67 (2H, t), 3.80 (2H, t), 6.33 (1H, d), 6.42 (1H, dd),
8.00 (1H, d); ES+ m/z: (M + H)+ 280.35.
1-[4-(4-Amino-3-methoxyphenyl)piperazin-1-yl]ethanone (64).
Methylmagnesium bromide (3.2 M in 2-methyltetrahydrofuran,
3.37 mL, 10.79 mmol) was added dropwise over 10 min to a
solution of indole (1.28 g, 10.79 mmol) in THF (6 mL) at
0 °C. The solution was stirred at 0−2 °C for 30 min. 2,4,5-
Trichloropyrimidine (1 g, 5.40 mmol) was added dropwise,
resulting in a yellow solution. The ice bath was removed, and
the solution was stirrred at ambient temperature for 1 h,
resulting in a red solution. The temperature was elevated to
60 °C, and the mixture was stirred at 60 °C for 1.5 h. The
mixture was cooled to 25 °C, and acetic acid (634 μL, 11.06
mmol) was added dropwise. Water (9.90 mL) and THF
(2 mL) were added, and the mixture was stirred for 20 min at
60 °C, resulting in a biphasic solution. The layers were
partitioned, and heptane (11 mL) was added to the organic
solution, resulting in the crystallization of a solid. The solid was
collected by filtration, washed with heptane (2 mL), and dried
in a vacuum oven to yield 3-(2,5-dichloropyrimidin-4-yl)-1H-
indole (1.015 g, 66%) as a yellow solid. 1H NMR (DMSO-d6) δ
7.24−7.32 (2H, m), 7.55−7.58 (1H, m), 8.52−8.55 (1H, m),
8.71−8.73 (2H, m), 12.24 (1H, s); ES+ m/z: (M + H)+ 264/266.
A suspension of 1-(4-(3-methoxy-4-nitrophenyl)piperazin-1-
yl)ethanone (7 g, 25.06 mmol) and platinum oxide (0.398 g,
1.75 mmol) in EtOH (200 mL) at 25 °C was stirred under 1.3
bar hydrogen at 25 °C for 3 h. The resulting suspension was
filtered through Dicalite Speed Plus, and the filtrate was con-
centrated to dryness to afford 1-(4-(4-amino-3-methoxy-
phenyl)piperazin-1-yl)ethanone (6.19 g, 99%) as a purple
1
solid. H NMR (400 MHz, DMSO-d6) δ 2.03 (3H, s), 2.87
(2H t, J = 5.2 Hz), 2.94 (2H t, J = 5.2 Hz), 3.53−3.57 (4H, m),
3.75 (3H, s), 4.28 (2H, s), 6.30−6.33 (1H, m), 6.52−6.54 (2H,
m); ES+ m/z: (M + H)+ 250.27.
N-[4-(4-Acetylpiperazin-1-yl)-2-methoxyphenyl]acetamide (65).
1-(4-(4-Amino-3-methoxyphenyl)piperazin-1-yl)ethanone (64)
(3 g, 12.03 mmol) was added to a stirred mixture of acetic
R
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anhydride (7.49 mL, 79.42 mmol) and acetic acid (3.10 mL,
54.15 mmol). The resulting deep-blue solution was stirred at
25 °C for 1 h. The reaction mixture was then diluted in Et2O
(500 mL) under stirring. The precipitate was collected by
filtration and washed with Et2O to give N-(4-(4-acetylpiper-
azin-1-yl)-2-methoxyphenyl)acetamide (2.55 g, 73%) as a clear
purple solid. 1H NMR (DMSO-d6) δ 2.02 (3H, s), 2.06 (3H, s),
3.09 (4H, ddd), 3.58 (4H, m), 3.81 (3H, s), 6.45 (1H, d), 6.63
(1H, s), 7.61 (1H, d), 8.92 (1H, s); ES+ m/z: (M + H)+
292.36.
8.49−8.58 (2H, m), 8.62 (1H, s), 8.93 (1H, d), 11.93 (1H, s); ES
+ m/z: (M + H)+ 396.13.
N′-[5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]-4-methoxyben-
zene-1,3-diamine (67).
1-[4-[4-[[5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-3-
methoxyphenyl]piperazin-1-yl]ethanone (7).
5-Chloro-4-(1H-indol-3-yl)-N-(2-methoxy-5-nitrophenyl)-
pyrimidin-2-amine (66) (1.2 g, 3.03 mmol), iron (1.016 g,
18.19 mmol), and ammonium chloride (0.114 g, 2.12 mmol)
were heated in ethanol (15 mL) and water (5 mL) at reflux for
3 h. The mixture was cooled and concentrated to a thick slurry.
DCM (100 mL), MeOH (5 mL), and water were added, and
the mixture stirred for 15 min and filtered. The filtrate layers
were separated, and the aqueous layer was re-extracted with
DCM. The combined organics were dried (MgSO4) and
concentrated. The crude product was purified by flash silica
chromatography, eluting with 1−5% MeOH in DCM. Pure
fractions were evaporated to dryness to afford N1-(5-chloro-4-
(1H-indol-3-yl)pyrimidin-2-yl)-6-methoxybenzene-1,3-diamine
(0.825 g, 74%) as a white/pale-yellow solid. 1H NMR (DMSO-
d6) δ 3.71 (3H, s), 4.55 (2H, s), 6.36 (1H, dd), 6.80 (1H, d),
7.08−7.13 (1H, m), 7.17−7.23 (1H, m), 7.26 (1H, d), 7.49
(1H, d), 8.13 (1H, s), 8.39 (1H, s), 8.42 (1H, d), 8.49 (1H, d),
11.85 (1H, s); ES+ m/z: (M + H)+ 366.17
3-(2,5-Dichloropyrimidin-4-yl)-1H-indole (61) (0.25 g, 0.95
mmol), 1-(4-(4-amino-3-methoxyphenyl)piperazin-1-yl)-
ethanone (0.271 g, 0.95 mmol), and p-toluene sulfonic acid
monohydrate (0.552 mL, 2.84 mmol) in n-pentanol (4 mL)/
NMP (1 mL) were stirred at 150 °C for 1 h. The reaction
mixture was filtered and concentrated in vacuo, and the residue
was purified by reverse-phase HPLC (acetonitrile/water/0.1%
TFA) using an Atlantis Prep T3 OBD column to afford 1-[4-[4-
[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-3-
methoxyphenyl]piperazin-1-yl]ethanone (138 mg, 31%) as a
white solid and the deacetylated product as a white solid (42
1
mg, 10%). H NMR (DMSO-d6) δ 2.07 (3H, s), 3.13−3.39
N-[3-[[5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-4-
methoxyphenyl]prop-2-enamide (9).
(4H, m), 3.65 (4H, d), 3.73−3.88 (3H, m), 6.63 (1H, dd), 6.82
(1H, d), 7.01 (1H, t), 7.14−7.23 (1H, m), 7.47 (1H, d), 7.54
(1H, d), 8.28 (1H, d), 8.34 (1H, s), 8.54 (1H, d), 11.94 (1H, br
s); 13C NMR (DMSO-d6) δ 21.10, 40.70, 45.49, 49.02, 49.35,
55.46, 100.86, 107.30, 110.88, 111.55, 114.07, 120.36, 120.70,
122.24, 123.17, 125.71, 126.12, 130.71, 135.87, 149.04, 153.11,
157.11, 157.75, 159.39, 168.17; HRMS−ESI (m/z): [M + H]+
calcd for C25H25O2N6Cl, 477.17969; found, 477.18003.
Synthesis of Compound 9. 5-Chloro-4-(1H-indol-3-yl)-N-(2-
methoxy-5-nitrophenyl)pyrimidin-2-amine (66).
Acryloyl chloride (0.040 mL, 0.49 mmol) was added dropwise
to N′-[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]-4-methoxy-
benzene-1,3-diamine (67) (180.2 mg, 0.49 mmol) and
DIPEA (0.146 mL, 0.54 mmol) in THF (5 mL) at 0 °C
under nitrogen. The resulting suspension was stirred at 0 °C for
2 h and then allowed to warm to room temperature. The
reaction mixture was diluted with water (15 mL) and extracted
with DCM (40 mL). The organics were washed sequentially
with saturated Na2CO3 (20 mL) and saturated brine (20 mL).
The organic layer was dried (MgSO4) and evaporated to afford
the crude product. The crude product was purified by flash
silica chromatography, eluting with 1−8% MeOH in DCM.
Pure fractions were evaporated to dryness to afford N-[3-[[5-
chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-4-
methoxyphenyl]prop-2-enamide (125 mg, 60%) as a pale-
2-Methoxy-5-nitroaniline (0.971 g, 5.77 mmol), p-toluene sul-
fonic acid monohydrate (2.197 g, 11.55 mmol), and 3-(2,5-
dichloropyrimidin-4-yl)-1H-indole (61) (1.525 g, 5.77 mmol)
were heated at 120 °C in 2-pentanol (20 mL) for 24 h. The
reaction mixture was evaporated to dryness, redissolved in
DCM (50 mL) and MeOH (5 mL), and washed sequentially with
saturated NaHCO3 (25 mL), water (25 mL), and saturated brine
(25 mL). The organic layer was dried (MgSO4) and evaporated to
afford the crude product. The resultant solid was triturated with
DCM/MeOH (9:1), filtered, and washed with DCM to give 5-
chloro-4-(1H-indol-3-yl)-N-(2-methoxy-5-nitrophenyl)pyrimidin-
1
brown solid. H NMR (DMSO-d6) δ 3.80 (3H, s), 5.70 (1H,
dd), 6.22 (1H, dd), 6.40 (1H, dd), 6.99 (1H, t), 7.06 (1H, d),
7.16 (1H, t), 7.46 (1H, d), 7.55 (1H, dd), 8.05 (1H, d), 8.33
(1H, d), 8.40 (2H, s), 8.51 (1H, d), 9.97 (1H, s), 11.85 (1H, s);
13C NMR (DMSO-d6) δ 55.77, 110.74, 111.27, 111.61, 114.97,
115.71, 116.06, 120.53, 122.28, 123.00, 126.04, 128.16, 131.02,
131.90, 131.99, 135.93, 147.87, 157.14, 158.02, 158.70, 162.67;
1
2-amine (1.20 g, 53%) as a yellow solid (1.2 g, 53%). H NMR
(DMSO-d6) δ 4.01 (3H, s), 6.95−7.10 (1H, m), 7.21 (1H, t), 7.32
(1H, d), 7.50 (1H, d), 8.03−8.12 (1H, m), 8.32−8.42 (1H, m),
S
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HRMS−ESI (m/z): [M + H]+ calcd for C22H19O2N5Cl,
420.12198; found, 420.12218.
Synthesis of Compound 15. (E)-4-(2-Butoxyvinyl)-2,5-dichloro-
pyrimidine (68).
75.44 mmol) and 3-(2,5-dichloropyrimidin-4-yl)pyrazolo[1,5-
a]pyridine (20 g, 75.44 mmol) in 2-pentanol (300 mL). The
resulting mixture was stirred at 100 °C for 2 days. The resulting
precipitate was collected by filtration, washed with 2-pentanol
(5 mL), and dried in vacuo to give a yellow solid. The solid was
slurried in acetonitrile, filtered, and dried in vacuo to give
5-chloro-N-(2-methoxy-5-nitrophenyl)-4-(pyrazolo[1,5-a]-
pyridin-3-yl)pyrimidin-2-amine (22.97 g, 77%). 1H NMR
(DMSO-d6, 100 °C) δ 4.05 (3H, s), 7.15 (1H, t), 7.33 (1H,
d), 7.43 (1H, t), 8.04 (1H, dd), 8.45−8.51 (2H, m), 8.57 (1H,
s), 8.81 (1H, d), 8.93−8.95 (2H, m); ES+ m/z: (M + H)+ 397.
N1-[5-Chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-yl]-6-
methoxybenzene-1,3-diamine (71).
Triethylamine (80 mL, 572.45 mmol), butyl vinyl ether (74.1
mL, 572.45 mmol), and palladium(II) acetate (4.28 g, 19.08
mmol) were added to a stirred solution of 2,4,5-trichloropyr-
imidine (100 g, 545.2 mmol) in poly(ethylene glycol) 200
(600 mL, 545.2 mmol) under nitrogen. The mixture was stirred
at 80 °C for 70 min and then cooled to room temperature. The
mixture was extracted with Et2O (4 × 1 L). The combined
organics were washed with water (2 × 1 L) and brine (1 L).
The solution was then dried (MgSO4) and concentrated in
vacuo. Purification by flash silica chromatography and elution
with 0−40% DCM in isohexane afforded (E)-4-(2-butoxyvin-
yl)-2,5-dichloropyrimidine (68.2 g, 51%) as a light-yellow oil.
1H NMR (DMSO-d6) δ 0.91 (3H, t), 1.33−1.42 (2H, m),
1.62−1.69 (2H, m), 4.11 (2H, t), 6.05 (1H, d), 8.04 (1H, d),
8.62 (1H, s); ES+ m/z: (M + H)+ 247.30
5-Chloro-N-(2-methoxy-5-nitrophenyl)-4-(pyrazolo[1,5-a]-
pyridin-3-yl)pyrimidin-2-amine (22.9 g, 57.71 mmol), iron
(powder, 325 mesh, 19.34 g, 346.28 mmol), and an NH4Cl
solution (1.412 mL, 40.40 mmol) were added to a mixture of
EtOH (1145 mL) and water (382 mL). The mixture was then
heated at reflux for 1 h and then cooled and concentrated to a
thick slurry. This residue was triturated with EtOAc (450 mL)
and a small amount of saturated NaHCO3 and then filtered.
The isolated solid was washed with water (250 mL) and then
slurried with 10% MeOH/DCM (1 L) for 15 min. The mixture
was filtered, and the isolated solid was reslurried with 10%
MeOH/DCM (250 mL) and filtered. The combined filtrates
were separated from the residual water, dried (Na2SO4), and
concentrated in vacuo. The resulting solid was suspended in
MeOH (150 mL), filtered, washed with MeOH and Et2O, and
dried on the filter to provide N1-[5-chloro-4-(pyrazolo[1,5-
a]pyridin-3-yl)pyrimidin-2-yl]-6-methoxybenzene-1,3-diamine
(17.1 g, 81%) as a yellow powder. 1H NMR (DMSO-d6) δ 3.68
(3H, s), 4.63 (2H, s), 6.41 (1H, dd), 6.82 (1H, d), 7.06 (1H,
d), 7.13 (1H, td), 7.38−7.45 (1H, m), 8.39 (1H, s), 8.42 (1H,
s), 8.48 (1H, d), 8.84 (1H, d), 8.95 (1H, s); ES+ m/z: (M +
3-(2,5-Dichloropyrimidin-4-yl)pyrazolo[1,5-a]pyridine (69).
To a stirred solution of (E)-4-(2-butoxyvinyl)-2,5-dichloropyr-
imidine (68) (8 g, 32.37 mmol) in DMA (100 mL) was added
1-aminopyridinium iodide (7.19 g, 32.37 mmol) followed by
K2CO3 (11.19 g, 80.93 mmol). The mixture was stirred at 110
°C for 5 h. After cooling to room temperature, the mixture
became solid. The mixture was diluted with EtOAc and a small
amount of MeOH and was then washed with water followed by
saturated brine. The mixture was filtered to remove the solid
from the phase interface. This solid contained some desired
product. The solid was dissolved in EtOAc/MeOH and was
combined with the organic extract. Purification by flash silica
chromatography and elution with 20−70% EtOAc in heptane
provided 3-(2,5-dichloropyrimidin-4-yl)pyrazolo[1,5-a]pyridine
(2.86 g, 33%) as an orange solid. 1H NMR (DMSO-d6) δ 7.28
(1H, td), 7.73 (1H, ddd), 8.57 (1H, dt), 8.81 (1H, s), 8.97 (1H,
dt), 9.09 (1H, s); (1H, d), 9.54 (1H, t); ES+ m/z: (M + H)+
265.
H)+ 367.
(E)-N-{3-[5-Chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-yla-
mino]-4-methoxyphenyl}-4-(dimethylamino)but-2-enamide (15).
5-Chloro-N-(2-methoxy-5-nitrophenyl)-4-(pyrazolo[1,5-a]pyridin-
3-yl)pyrimidin-2-amine (70).
DIPEA (0.119 mL, 0.68 mmol), (E)-4-bromobut-2-enoic acid
(49.5 mg, 0.30 mmol), and HATU (114 mg, 0.30 mmol) were
added to a stirred solution of N1-[5-chloro-4-(pyrazolo[1,5-
a]pyridin-3-yl)pyrimidin-2-yl]-6-methoxybenzene-1,3-diamine
(71) (100 mg, 0.27 mmol) in DMF (10 mL) at 0 °C. The
mixture was stirred at 1 °C under nitrogen for 2 h, allowed to
warm to room temperature, and stirred for a further 2 h. The
mixture was then recooled to 0 °C, and dimethylamine (2 M in
THF, 2.73 mL, 5.45 mmol) was added. The mixture was stirred
at 1 °C for 5 min, allowed to warm to room temperature, and
4-Methylbenzenesulfonic acid hydrate (15.79 g, 82.99 mmol)
was added in one portion to 2-methoxy-5-nitroaniline (12.69 g,
T
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stirred for a further 3 h. The mixture was then diluted with
EtOAc (50 mL) and was washed with water (2 × 50 mL) and
saturated brine (50 mL). The solution was dried (MgSO4) and
concentrated in vacuo. Purification by flash silica chromatog-
raphy and elution with 0−10% MeOH in DCM (containing 1%
aqueous NH3) provided (E)-N-{3-[5-chloro-4-(pyrazolo[1,5-
a]pyridin-3-yl)pyrimidin-2-ylamino]-4-methoxyphenyl}-4-
(dimethylamino)but-2-enamide (30 mg, 23%) as an orange
solid. 1H NMR (DMSO-d6) δ 2.19 (6H, s), 3.06 (2H, d), 3.79
(3H, s), 6.23 (1H, d), 6.69 (1H, dt), 7.06−7.15 (2H, m), 7.29−
7.35 (1H, m), 7.56 (1H, dd), 7.96 (1H, d), 8.42 (1H, d), 8.46
(1H, s), 8.73 (1H, s), 8.86 (1H, d), 8.99 (1H, s), 9.97 (1H, s);
13C NMR (DMSO-d6) δ 42.05, 55.77, 56.82, 106.97, 111.52,
3-(2,5-Dichloropyrimidin-4-yl)-1H-indole (61) (7.5 g, 28.40
mmol), 4-fluoro-2-methoxy-5-nitroaniline (72) (5.55 g, 29.82
mmol), and p-toluenesulfonic acid monohydrate (5.94 g, 31.24
mmol) were heated at 120 °C in 2-pentanol (210 mL) for 18 h.
The mixture was cooled and filtered. The solid was washed with
MeOH and Et2O and dried on the filter to give 5-chloro-N-(4-
fluoro-2-methoxy-5-nitrophenyl)-4-(1H-indol-3-yl)pyrimidin-2-
1
amine (7.28 g, 62%). H NMR (DMSO-d6) δ 3.98 (3H, s),
6.97−7.05 (1H, m), 7.17−7.24 (1H, m), 7.40 (1H, d), 7.49
(1H, dd), 8.29 (1H, d), 8.49 (1H, s), 8.52 (1H, d), 8.68 (1H,
d), 8.71 (1H, s), 11.92 (1H, s); ES+ m/z: (M + H)+ 414.1.
5-Chloro-4-(1H-indol-3-yl)-N-[2-methoxy-4-(4-methylpiperazin-
1-yl)-5-nitrophenyl]pyrimidin-2-amine (74).
114.37, 114.77, 116.28, 116.66, 120.60, 126.88, 128.00, 129.49,
130.89, 131.62, 132.22, 139.33, 143.38, 148.54, 155.61, 157.80,
158.76, 161.48. HRMS−ESI (m/z): [M + H]+ calcd for
C24H24O2N7Cl, 478.17502; found, 478.17528.
(E)-N-{3-[5-Chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2-
ylamino]-4-methoxyphenyl}-4-(dimethylamino)but-2-enamide (15)
Mesylate Salt. (E)-N-{3-[5-Chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)-
pyrimidin-2-ylamino]-4-methoxyphenyl}-4-(dimethylamino)but-2-en-
amide (15) (50 mg, 0.104 mmol) was dissolved in hot MeCN (5 mL)
and combined with a solution of methanesulfonic acid (10 mg, 0.104
mmol) in MeCN (1 mL). The resultant mixture was then stirred at
room temperature for 24 h. The resulting off-white solid was collected
by filtration and analyzed by XRD and thermal methods. On analysis,
this solid gave a different X-ray powder diffraction pattern from that of
the starting material, indicating a new crystalline phase had been
formed. This phase was shown by differential scanning calorimetry to
have an onset of melting of around 235 °C.
1-Methylpiperazine (492 μL, 4.44 mmol) was added to a
suspension of 5-chloro-N-(4-fluoro-2-methoxy-5-nitrophenyl)-
4-(1H-indol-3-yl)pyrimidin-2-amine (73) (612 mg, 1.48
mmol), and the reaction was stirred at 120 °C for 1 h. The
reaction mixture was evaporated to dryness, redissolved in
DCM (25 mL), and washed sequentially with water (2 × 25
mL) and saturated brine (25 mL). The organic layer was dried
(MgSO4) and evaporated to afford the crude product. The
crude product was purified by flash silica chromatography, with
an elution gradient of 1−10% MeOH in DCM. Pure fractions
were evaporated to dryness, the resulting gum was dissolved in
EtOH (25 mL), and a solid precipitated. The solid was
collected by filtration and washed with EtOH and Et2O to give
5-chloro-4-(1H-indol-3-yl)-N-[2-methoxy-4-(4-methylpiperazin-1-
yl)-5-nitrophenyl]pyrimidin-2-amine (365 mg, 50%) as a yellow
Synthesis of Compound 58. 4-Fluoro-2-methoxy-5-nitroaniline
(72).
1
solid. H NMR (DMSO-d6) δ 2.26 (3H, s), 2.47−2.55 (4H, m),
3.07−3.13 (4H, m), 3.93 (3H, s), 6.85 (1H, s), 6.99 (1H, t),
7.16−7.22 (1H, m), 7.48 (1H, d), 8.26 (1H, d), 8.36 (1H, s), 8.42
(1H, d), 8.51 (1H, s), 8.54 (1H, s), 11.88 (1H, s); ES+ m/z: (M +
H)+ 494.5.
N′-[5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]-4-methoxy-6-(4-
methylpiperazin-1-yl)benzene-1,3-diamine (75).
4-Fluoro-2-methoxyaniline (2.4 g, 17.00 mmol) was added
portionwise to concd H2SO4 (15 mL) and cooled in a ice-water
bath, keeping the temperature below 15 °C. The mixture was
stirred until all of the solid that formed had dissolved.
Potassium nitrate (0.815 mL, 17.00 mmol) was added
portionwise such that the temperature was maintained below
10 °C. The mixture was stirred overnight and then poured onto
ice/water. The mixture was basified with concd NH4OH. The
solid was filtered off and then dissolved in DCM, washed with
water, dried (Na2SO4), and concentrated onto silica. The crude
product was purified by flash silica chromatography and eluted
with a gradient of 50−100% DCM in heptane. Pure fractions
were evaporated to dryness to afford 4-fluoro-2-methoxy-5-
1
nitroaniline (2.450 g, 77%) as a yellow crystalline solid. H
5-Chloro-4-(1H-indol-3-yl)-N-[2-methoxy-4-(4-methylpiperazin-1-
yl)-5-nitrophenyl]pyrimidin-2-amine (74) (350 mg, 0.71 mmol),
iron (237 mg, 4.25 mmol), and ammonium chloride (26.5 mg,
0.50 mmol) were heated in EtOH (24 mL) and water (8 mL)
at reflux for 2 h. The mixture was cooled and concentrated to a
thick slurry. DCM (100 mL) and MeOH (10 mL) were added,
and the mixture was stirred for 15 min and filtered. The filtrate
was dried (MgSO4) and concentrated. The crude product was
purified by flash silica chromatography, eluting with 1−5%
MeOH/NH3 in DCM. Pure fractions were evaporated to
dryness to afford N′-[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-
yl]-4-methoxy-6-(4-methylpiperazin-1-yl)benzene-1,3-diamine
NMR (DMSO-d6) δ 3.91 (3H, s), 5.21 (2H, s), 7.03 (1H, d),
7.35 (1H, d); ES+ m/z: (M + H)+ 187.4.
5-Chloro-N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1H-indol-3-
yl)pyrimidin-2-amine (73).
U
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Journal of Medicinal Chemistry
Article
1
immunodeficiency (SCID) mice (SCID/CB17; PC9). Animals were
randomized into treatment groups of 4−10 mice, with a group mean
tumor size of ∼0.2−0.4 cm3. Mice were then treated once daily by oral
gavage with either vehicle or inhibitor for up to 14 days. Tumor
growth inhibition (TGI) was calculated for compound-treated groups
compared to vehicle controls (p values provided), where a TGI >
100% indicates tumor regression from the size of the initial tumor.
Statistical significance was evaluated using a one-tailed t test.
(288 mg, 88%) as a pale-yellow foam. H NMR (DMSO-d6) δ
2.26 (3H, s), 2.47−2.56 (4H, m), 2.88 (4H, t), 3.70 (3H, s),
4.29 (2H, d), 6.72 (1H, s), 7.04 (1H, t), 7.14 (1H, s), 7.15−
7.22 (1H, m), 7.46 (1H, d), 8.18 (1H, s), 8.35 (2H, d), 8.48
(1H, d), 11.81 (1H, s); ES+ m/z: (M + H)+ 464.5.
N-[5-[[5-Chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-4-
methoxy-2-(4-methylpiperazin-1-yl)phenyl]prop-2-enamide (58).
ASSOCIATED CONTENT
* Supporting Information
■
S
Reaction of compound 38 in buffer, structure of compound 60,
chemical structures of the compounds in Tables 7 and 8,
reaction of compounds with glutathione with and without
human glutathione-S-transferase, and kinase selectivity of
compound 15. Methods of protein−ligand docking and full
compound synthesis and characterization. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acryloyl chloride (1 M in THF, 0.621 mL, 0.62 mmol) was
added dropwise to N′-[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-
yl]-4-methoxy-6-(4-methylpiperazin-1-yl)benzene-1,3-diamine
(75) (288 mg, 0.62 mmol) and DIPEA (0.119 mL, 0.68 mmol)
in THF (15 mL) at 0 °C under nitrogen. The resulting
suspension was stirred at 0 °C for 1 h and then allowed to
warm to room temperature. The reaction mixture was diluted
with water (15 mL) and reduced in vacuo. The resultant crude
product was dissolved in DCM (20 mL) and MeOH (5 mL)
and washed with water and saturated brine. The organic layer
was dried (MgSO4) and evaporated. The crude product was
purified by flash silica chromatography, with an elution gradient
of 1−8% MeOH/NH3 in DCM. Pure fractions were evaporated
to dryness. The product was dissolved in DCM, and a beige
solid precipitated from the solution. This solid was diluted with
Et2O and filtered, washed with further Et2O, and dried to give
N-[5-[[5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl]amino]-4-me-
thoxy-2-(4-methylpiperazin-1-yl)phenyl]prop-2-enamide (134
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +44 (0)1625 519045. E-mail: richard.a.ward@
astrazeneca.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank all of our colleagues at AstraZeneca, past and present,
who have contributed to this work. We also thank Proteros for
supplying the described X-ray crystal structure and Millipore
for providing the early biochemical data. Finally, we thank Dr.
Andy Barker who was instrumental in supporting the early
efforts on this project.
1
mg, 42%) as a beige solid. H NMR (DMSO-d6) δ 2.27 (3H,
s), 2.53−2.59 (4H, m), 2.87−2.94 (4H, m), 3.79 (3H, s), 5.70
(1H, d), 6.17 (1H, dd), 6.60 (1H, dd), 6.89 (1H, s), 7.01 (1H,
t), 7.16 (1H, t), 7.45 (1H, d), 8.20 (1H, s), 8.27 (1H, d), 8.35
(1H, s), 8.43 (1H, s), 8.49 (1H, d), 8.96 (1H, s), 11.81 (1H, s);
13C NMR (DMSO-d6) δ 45.74, 51.23, 54.77, 55.72, 103.74,
110.82, 111.53, 114.43, 120.14, 120.48, 122.20, 123.03, 123.42,
124.46, 125.90, 126.05, 130.85, 132.23, 135.86, 141.31, 149.80,
157.15, 157.86, 159.13, 162.76; HRMS−ESI (m/z): [M + H]+
calcd for C27H28O2N7Cl, 518.20636; found, 518.20658.
The full synthesis and characterization of the novel compounds is
detailed in the Supporting Information. Additional compounds
reported from the reactivity data set from the alternative templates
and alternative covalent functionality were synthesized by exploiting
the modular approaches reported in this manuscript in addition to
using routes subsequently disclosed in the literature.43,51 Compounds
used as model systems (Table 7) were either available in our
compound collection, purchased from commercial sources, or
synthesized from commonly available building blocks.
Measuring Compound Reactivity. Fifty micromolar of com-
pound was reacted with an excess of glutathione (4.6 mM GSH) in
phosphate buffer at pH 7.4 and 37 °C for 24 h.36,37 The reaction was
followed by monitoring the loss of the parent by LC−UV−MS, and
the data were fitted to first-order kinetics from which the pseudo-first-
order rate constant was produced and subsequently kGSH. Reaction of
para-nitrobenzylchloride (PNBC) with glutathione was used as a
positive control for the assay (Log kGSH −1.747 mol−1 s−1, SD 0.077,
and n = 24).
DEDICATION
■
‡This manuscript is dedicated to the late Peter Smith.
ABBREVIATIONS USED
■
EGFR, epidermal growth factor receptor; NSCLC, non-small
cell lung cancer; TKI, tyrosine kinase inhibitor; QSPR,
quantitative structure property relationship; GSH, glutathione;
PK, pharmacokinetics; WT, wild type; AM, activating mutant;
DM, double mutant; LLE, ligand lipophilicity efficiency; LC−
MS, liquid chromatography−mass spectrometry; QM/MM,
quantum mechanical/molecular modeling; SAR, structure−
activity relationships; CRO, contract research organization
REFERENCES
■
(1) Normanno, N.; De Luca, A.; Bianco, C.; Strizzi, L.; Mancino, M.;
Maiello, M. R.; Carotenuto, A.; De Feo, G.; Caponigro, F.; Salomon,
D. S. Epidermal growth factor receptor (EGFR) signaling in cancer.
Gene 2006, 366, 2−16.
(2) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal
growth factor receptor mutations in lung cancer. Nat. Rev. Cancer
2007, 7, 169−181.
(3) Pao, W.; Chmielecki, J. Rational, biologically based treatment of
EGFR-mutant non-small-cell lung cancer. Nat. Rev. Cancer 2010, 10,
760−774.
In Vivo Xenograft Antitumor Studies. All animal experiments
were conducted in full accordance with the U.K. Home Office Animal
(Scientific Procedures) Act 1986. Xenografts were established by
subcutaneous cell implantation into the left flank of either athymic
(Swiss nu/nu; Alpk; H1975 and A431) or severe-combined
(4) Maemondo, M.; Inoue, A.; Kobayashi, K.; Sugawara, S.; Oizumi,
S.; Isobe, H.; Gemma, A.; Harada, M.; Yoshizawa, H.; Kinoshita, I.;
Fujita, Y.; Okinaga, S.; Hirano, H.; Yoshimori, K.; Harada, T.; Ogura,
T.; Ando, M.; Miyazawa, H.; Tanaka, T.; Saijo, Y.; Hagiwara, K.;
Morita, S.; Nukiwa, T. Gefitinib or chemotherapy for non-small-cell
V
dx.doi.org/10.1021/jm400822z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
lung cancer with mutated EGFR. N. Engl. J. Med. 2010, 362, 2380−
2388.
(5) Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Jaenne, P. A.; Kocher,
O.; Meyerson, M.; Johnson, B. E.; Eck, M. J.; Tenen, D. G.; Halmos, B.
EGFR mutation and resistance of non-small-cell lung cancer to
gefitinib. N. Engl. J. Med. 2005, 352, 786−792.
(6) Yun, C.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.; Greulich,
H.; Wong, K.; Meyerson, M.; Eck, M. J. The T790M mutation in
EGFR kinase causes drug resistance by increasing the affinity for ATP.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2070−2075.
(7) Shepherd, F. A.; Pereira, J. R.; Ciuleanu, T.; Tan, E. H.; Hirsh, V.;
Thongprasert, S.; Campos, D.; Maoleekoonpiroj, S.; Smylie, M.;
Martins, R.; van Kooten, M.; Dediu, M.; Findlay, B.; Tu, D.; Johnston,
D.; Bezjk, A.; Clark, G.; Santabarbara, P.; Seymour, L. Erlotinib in
previously treated non-small-cell lung cancer. N. Engl. J. Med. 2005,
353, 123−132.
(8) Zhang, Z.; Stiegler, A. L.; Boggon, T. J.; Kobayashi, S.; Halmos, B.
EGFR-mutated lung cancer: A paradigm of molecular oncology.
OncoTargets Ther. 2010, 1, 497−514.
(9) Sogabe, S.; Kawakita, Y.; Igaki, S.; Iwata, H.; Miki, H.; Cary, D.
R.; Takagi, T.; Takagi, S.; Ohta, Y.; Ishikawa, T. Structure-based
approach for the discovery of pyrrolo[3,2-d]pyrimidine-based EGFR
T790M/L858R mutant inhibitors. ACS Med. Chem. Lett 2013, 4, 201−
205.
(10) Gajiwala, K. S.; Feng, J.; Ferre, R.; Ryan, K.; Brodsky, O.;
Weinrich, S.; Kath, J. C.; Stewart, A. Insights into the aberrant activity
of mutant EGFR kinase domain and drug recognition. Structure
(Oxford, U. K.) 2013, 21, 209−219.
(11) Yun, C.; Woo, M. S.; Greulich, H.; Meyerson, M.; Eck, M. J.;
Wong, K. In Structural and mechanistic studies of lung cancer mutations
in the EGFR kinase reveal a novel mechanism of drug resistance, 236th
National Meeting of the American Chemical Society, Philadelphia, PA,
Aug 17−21, 2008; American Chemisal Society: Washington, DC,
2008; COMP 373.
(12) Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.;
Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P.
Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol.
2011, 29, 1046−1051.
(13) Yun, C.; Boggon, T. J.; Li, Y.; Woo, M. S.; Greulich, H.;
Meyerson, M.; Eck, M. J. Structures of lung cancer-derived EGFR
mutants and inhibitor complexes: Mechanism of activation and
insights into differential inhibitor sensitivity. Cancer Cell 2007, 11,
217−227.
(14) Lovly, C. M.; Horn, L. Strategies for overcoming EGFR
resistance in the treatment of advanced-stage NSCLC. Curr. Treat.
Options Oncol. 2012, 13, 516−526.
(15) Ou, S. I. Second-generation irreversible epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitors (TKIs): A better
mousetrap? A review of the clinical evidence. Crit. Rev. Oncol. Hematol.
2012, 83, 407−421.
(16) Miller, V. A.; Hirsh, V.; Cadranel, J.; Chen, Y.; Park, K.; Kim, S.;
Zhou, C.; Su, W.; Wang, M.; Sun, Y.; Heo, D. S.; Crino, L.; Tan, E.;
Chao, T.; Shahidi, M.; Cong, X. J.; Lorence, R. M.; Yang, J. C. Afatinib
versus placebo for patients with advanced, metastatic non-small-cell
lung cancer after failure of erlotinib, gefitinib, or both, and one or two
lines of chemotherapy (LUX-Lung 1): A phase 2b/3 randomised trial.
Lancet Oncol. 2012, 13, 528−538.
M. Targeting the EGFR pathway for cancer therapy. Curr. Med. Chem.
2006, 13, 3483−3492.
(20) Zhou, W.; Ercan, D.; Chen, L.; Yun, C.; Li, D.; Capelletti, M.;
Cortot, A. B.; Chirieac, L.; Iacob, R. E.; Padera, R.; Engen, J. R.; Wong,
K.; Eck, M. J.; Gray, N. S.; Janne, P. A. Novel mutant-selective EGFR
kinase inhibitors against EGFR T790M. Nature 2009, 462, 1070−
1074.
(21) Chang, S.; Zhang, L.; Xu, S.; Luo, J.; Lu, X.; Zhang, Z.; Xu, T.;
Liu, Y.; Tu, Z.; Xu, Y.; Ren, X.; Geng, M.; Ding, J.; Pei, D.; Ding, K.
Design, synthesis, and biological evaluation of novel conformationally
constrained inhibitors targeting epidermal growth factor receptor
threonine790 → methionine790 mutant. J. Med. Chem. 2012, 55,
2711−2723.
(22) Bai, F.; Liu, H.; Tong, L.; Zhou, W.; Liu, L.; Zhao, Z.; Liu, X.;
Jiang, H.; Wang, X.; Xie, H.; Li, H. Discovery of novel selective
inhibitors for EGFR-T790M/L858R. Bioorg. Med. Chem. Lett. 2012,
22, 1365−1370.
(23) Lee, H.; Schaefer, G.; Heffron, T. P.; Shao, L.; Ye, X.; Sideris, S.;
Malek, S.; Chan, E.; Merchant, M.; La, H.; Ubhayakar, S.; Yauch, R. L.;
Pirazzoli, V.; Politi, K.; Settleman, J. Noncovalent wild-type-sparing
inhibitors of EGFR T790M. Cancer Discovery 2013, 3, 168−181.
(24) Zhou, W.; Ercan, D.; Chen, L.; Yun, C.; Li, D.; Capelletti, M.;
Cortot, A. B.; Chirieac, L.; Iacob, R. E.; Padera, R.; Engen, J. R.; Wong,
K.; Eck, M. J.; Gray, N. S.; Janne, P. A. Novel mutant-selective EGFR
kinase inhibitors against EGFR T790M. Nature 2009, 462, 1070−
1074.
(25) Merck Millipore Home Page. www.millipore.com.
(26) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence
of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307−317.
(27) Rauh, D. In Targeting protein kinases with irreversible inhibitors,
241st National Meeting of the American Chemical Society, Anaheim,
CA, March 27−31, 2011; American Chemical Society: Washington,
DC,2011; MEDI 157.
(28) Singh, J.; Evans, E.; Hagel, M.; Labinski, M.; Dubrovskiy, A.;
Nacht, M.; Petter, R. C.; Prasad, A.; Sheets, M.; StMartin, T.; Tjin
Tham Sjin, R.; Westlin, W.; Zhu, Z. Superiority of a novel EGFR
targeted covalent inhibitor over its reversible counterpart in over-
coming drug resistance. MedChemComm 2012, 3, 780−783.
(29) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;
Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.;
Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: A new
approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739−1749.
(30) Ehmke, V.; Quinsaat, J. E. Q.; Rivera-Fuentes, P.; Heindl, C.;
Freymond, C.; Rottmann, M.; Brun, R.; Schirmeister, T.; Diederich, F.
Tuning and predicting biological affinity: Aryl nitriles as cysteine
protease inhibitors. Org. Biomol. Chem. 2012, 10, 5764−5768.
(31) Proteros Biostructures Home Page. www.proteros.de.
(32) Edwards, M. P.; Price, D. A. Role of physicochemical properties
and ligand lipophilicity efficiency in addressing drug safety risks. Annu.
Rep. Med. Chem. 2010, 45, 381−391.
(33) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6, 881−890.
(34) Leach, A. G.; Jones, H. D.; Cosgrove, D. A.; Kenny, P. W.;
Ruston, L.; MacFaul, P.; Wood, J. M.; Colclough, N.; Law, B. Matched
molecular pairs as a guide in the optimization of pharmaceutical
properties; a study of aqueous solubility, plasma protein binding and
oral exposure. J. Med. Chem . 2006, 49, 6672−6682.
(17) Allen, L. F.; Eiseman, I. A.; Fry, D. W.; Lenehan, P. F. CI-1033,
an irreversible pan-erbB receptor inhibitor and its potential application
for the treatment of breast cancer. Semin. Oncol. 2003, 30, 65−78.
(18) Kalous, O.; Conklin, D.; Desai, A. J.; O’Brien, N. A.; Ginther,
C.; Anderson, L.; Cohen, D. J.; Britten, C. D.; Taylor, I.; Christensen,
J. G.; Slamon, D. J.; Finn, R. S. Dacomitinib (PF-00299804), an
irreversible pan-HER inhibitor, inhibits proliferation of HER2-
amplified breast cancer cell lines resistant to Trastuzumab and
Lapatinib. Mol. Cancer Ther. 2012, 11, 1978−1987.
(35) Johnson, D. S.; Weerapana, E.; Cravatt, B. F. Strategies for
discovering and derisking covalent, irreversible enzyme inhibitors.
Future Med. Chem. 2010, 2, 949−964.
(36) Clarke, E. D.; Greenhow, D. T.; Adams, D. Metabolism-related
assays and their application to agrochemical research: Reactivity of
pesticides with glutathione and glutathione transferases. Pestic. Sci.
1998, 54, 385−393.
(37) MacFaul, P. A.; Morley, A. D.; Crawford, J. J. A simple in vitro
assay for assessing the reactivity of nitrile containing compounds.
Bioorg. Med. Chem. Lett. 2009, 19, 1136−1138.
(19) Johnston, J. B.; Navaratnam, S.; Pitz, M. W.; Maniate, J. M.;
Wiechec, E.; Baust, H.; Gingerich, J.; Skliris, G. P.; Murphy, L. C.; Los,
W
dx.doi.org/10.1021/jm400822z | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(38) Schwobel, J. A. H.; Madden, J. C.; Cronin, M. T. D.
Examination of Michael addition reactivity towards glutathione by
transition-state calculations. SAR QSAR Environ. Res. 2010, 21, 693−
710.
(39) Esterbauer, H.; Zollner, H.; Scholz, N. Reaction of glutathione
with conjugated carbonyls. Z. Naturforsch., C 1975, 30, 466−473.
(40) Bohme, A.; Thaens, D.; Paschke, A.; Schuurmann, G. Kinetic
glutathione chemoassay to quantify thiol reactivity of organic
electrophiles-application to alpha,beta-unsaturated ketones, acrylates,
and propiolates. Chem. Res. Toxicol. 2009, 22, 742−750.
(41) Paasche, A.; Schiller, M.; Schirmeister, T.; Engels, B.
Mechanistic study of the reaction of thiol-containing enzymes with
α,β-unsaturated carbonyl substrates by computation and chemoassays.
ChemMedChem 2010, 5, 869−880.
(42) Krenske, E. H.; Petter, R. C.; Zhu, Z.; Houk, K. N. Transition
states and energetics of nucleophilic additions of thiols to substituted
α,β-unsaturated ketones: substituent effects involve enone stabiliza-
tion, product branching, and solvation. J. Org. Chem. 2011, 76, 5074−
5081.
(43) Gray, N. S.; Zhou, W. WIPO Patent WO2011140338, 2011.
(44) Tsou, H.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B.
C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis,
R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.; Wissner, A. 6-
Substituted-4-(3-bromophenylamino)quinazolines as putative irrever-
sible inhibitors of the epidermal growth factor receptor (EGFR) and
human epidermal growth factor receptor (HER-2) tyrosine kinases
with enhanced antitumor activity. J. Med. Chem. 2001, 44, 2719−2734.
(45) Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen,
M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Froedin, M.;
Taunton, J. Reversible targeting of noncatalytic cysteines with
chemically tuned electrophiles. Nat. Chem. Biol. 2012, 8, 471−476.
(46) Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, C. J.;
McLauchlan, H.; Klevernic, I.; Arthur, J. S. C.; Alessi, D. R.; Cohen, P.
The selectivity of protein kinase inhibitors: a further update. Biochem.
J. 2007, 408, 297−315.
(47) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the
epidermal growth factor receptor kinase domain alone and in complex
with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 2002, 277, 46265−
46272.
(48) Kabsch, W. Software XDS for image rotation, recognition and
crystal symmetry assignment. Acta Crystallogr., Sect. D 2010, 66, 125−
132.
(49) Emsley, P.; Cowtan, K. Coot: Model-building tools for
molecular graphics. Acta Crystallogr., Sect. D 2004, 60, 2126−2132.
(50) Vagin, A. A.; Steiner, R. A.; Lebedev, A. A.; Potterton, L.;
McNicholas, S.; Long, F.; Murshudov, G. N. REFMAC5 dictionary:
Organization of prior chemical knowledge and guidelines for its use.
Acta Crystallogr., Sect. D 2004, 60, 2184−2195.
(51) Gray, N. S.; Zhou, W. WIPO Patent WO2011079231, 2011.
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