Discovery of selective irreversible inhibitors for EGFR-T790M

Article abstract of DOI:10.1016/j.bmcl.2010.12.036

Targeting the epidermal growth factor receptor kinase (EGFR) with ATP-competitive kinase inhibitors results in dramatic but short-lived responses in patients with EGFR mutant non small cell lung cancer. A series of novel covalent EGFR kinase inhibitors with selectivity for the clinically relevant T790M 'gatekeeper' resistance mutation relative to wild-type EGFR were discovered by library screening. A representative compound 3i was obtained through a systematic SAR study guided by mutant EGFR-dependent cellular proliferation assays.

Full text of DOI:10.1016/j.bmcl.2010.12.036

Bioorganic & Medicinal Chemistry Letters 21 (2011) 638–643
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of selective irreversible inhibitors for EGFR-T790M
Wenjun Zhou a, Dalia Ercan b, Pasi A. Jänne b,c, Nathanael S. Gray a,
⇑
a Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Department of Cancer Biology, Dana Farber Cancer Institute,
Boston, MA 02115, United States
b Lowe Center for Thoracic Oncology, and Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02115, United States
c Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Targeting the epidermal growth factor receptor kinase (EGFR) with ATP-competitive kinase inhibitors
results in dramatic but short-lived responses in patients with EGFR mutant non small cell lung cancer.
A series of novel covalent EGFR kinase inhibitors with selectivity for the clinically relevant T790M
‘gatekeeper’ resistance mutation relative to wild-type EGFR were discovered by library screening. A rep-
resentative compound 3i was obtained through a systematic SAR study guided by mutant EGFR-depen-
dent cellular proliferation assays.
Received 22 October 2010
Revised 1 December 2010
Accepted 6 December 2010
Available online 10 December 2010
Keywords:
EGFR
Ó 2010 Elsevier Ltd. All rights reserved.
Kinase inhibitor
T790M
Mutant selective
Protein kinases, particularly receptor tyrosine kinases, are esti-
mated as the second largest class of therapeutic targets. There are
approximately 518 protein kinase genes encoded in the human
genome. Many of these have been observed to become constitu-
tively activated by amplification or mutation, thus becoming onco-
genic. A phenomenon that has been termed ‘oncogene-addiction’
renders some cancer cells to be exquisitely sensitive to the inhibi-
tors targeting that particular oncogene.1 For example, patients
with non small cell lung cancer (NSCLC) that harbor activating
mutations in the EGFR kinase domain such as L858R and exon 19
deletions exhibit excellent objective response rates when treated
with EGFR kinase inhibitors such as Gefitinib or Erlotinib.
The epidermal growth factor receptor (EGFR) family of recep-
tor tyrosine kinases regulates cell proliferation, survival, adhesion,
migration and differentiation. It consists of EGFR/ErbB-1, HER2/
ErbB-2, Her3/Erb-3 and Her4/Erb-4. Among them, the aberrant
activity of EGFR and ErbB-2 have been shown to play an impor-
tant role in the development and growth of tumor cells. The
blockade of EGFR and ErbB-2 has been clinically validated as a
leading approach to selectively target cancer cells. Currently
two small molecule kinase inhibitors, ZD1839 (Iressa, gefitinib)
and OSI774 (Tarceva, erlotinib), are in clinical use for the treat-
ment of lung cancer. There are more than ten EGFR inhibitors cur-
rently being tested in clinical trials. Among them are several
covalent inhibitors such as HKI-357,2 HKI-272,2 EKB569,2
BIBW2992,3 and PF29980444 which target a unique cysteine
797 residue located at the lip of the EGFR ATP binding site.5
Point mutations in the kinase domain of EGFR as well as up-
regulation of by-pass signaling pathways are frequently observed
resistance mechanisms in patients treated with gefitinib and
erlotinib.6,7 A single point mutation at the gatekeeper position,
T790M in EGFR kinase domain accounts for approximately 50% of
acquired resistance and results in relapse of disease with a median
progression free survival of 10–14 months. Inspection of inhibitor-
kinase complex structures suggests that replacement of threonine
with bulkier methionine would be expected to sterically interfere
with inhibitor binding and would result in loss of a key hydro-
gen-bond. However binding assays have demonstrated that the
T790M mutation appears to exert its effects primarily by increas-
ing the affinity for ATP which leads to higher concentrations of
the competitive inhibitors being required to effectively suppress
kinase activity.8 We focused our efforts on potential covalent com-
pounds because effective targeting of the T790M EGFR mutant
with a reversible inhibitor is estimated to require a Kd of
0.05 nM.8 Although several C797 directed covalent inhibitors are
reported to be active against T790M EGFR,2 the concentrations re-
quired to inhibit this mutant are considerably higher and unlikely
to be achieved in patients being treated with these agents. To date,
there have been no clinical reports of responses of T790M patients
to first-generation covalent inhibitors. As the gatekeeper position
is known to be one of the most important selectivity determinants
for kinase inhibitors, we reasoned that a fundamentally different
scaffold relative to the common quinazoline or quinoline nitrile-
type EGFR scaffold might be better suited for inhibition of
⇑
Corresponding author. Tel.: +1 617 582 8590.
E-mail address: nathanael_gray@dfci.harvard.edu (N.S. Gray).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.036

W. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 638–643
639
O
O
O
T790M. In order to rapidly survey a variety of scaffolds we pre-
pared a small library consisting of frequently used heterocyclic
cores that all contained an acrylamide functionality. Molecular
modeling of the presumed binding mode was used to guide selec-
tion of where to install the acrylamide to allow covalent bond for-
mation with C797.
NH2
NO2
NH
N
N
(c)
(a)
(b)
Cl
+
O
NO2
NO2
NH2
A1
The first class of irreversible compounds was based on purines
and was synthesized as shown in Scheme 1.
Cl
N
The aniline A1 was obtained in three-steps starting with acyla-
tion using propionyl chloride, methylation with methyl iodide and
hydrogenation of the 4-nitroaniline. Tetrahydropyran (THP)
protected 2,6-dichloropurine was regioselectively aminated at
the 6-position to afford intermediate A2. Buchwald coupling with
A1 in the presence of a carbene ligand provided A3, which was con-
verted into 1a through acidic deprotection of THP, followed by
acylation with acryloyl chloride. The purine analogs of 1a were
synthesized in an analogous manner using the appropriate amines
and anilines.
The compounds listed in Table 1 were evaluated for their anti-
proliferative effects in cellular assays using cancer cell lines that
are known to be dependent on specific EGFR genotypes. Specifi-
cally, PC9 cells (EGFR E746_A750) were used to model an activated
allele of EGFR while PC9 growth-resistant GR cells (EGFR
E746_A750/T790M) were used to model the T790M resistance
allele.9 To control for non-specific cytotoxicity, compounds were
evaluated against A549 lung adenocarcinoma cells (K-Ras depen-
dent). Evaluation of purine and pyrrolopyrimidine acrylamide
BocN
NH
N
N
(d)
(e)
N
N
N
N
N
H
Cl
Cl
THP
A2
O
N
BocN
NH
N
NH
N
(f)
N
N
N
N
N
N
N
O
O
N
N
H
N
H
H
THP
A3
1a
Scheme 1. Synthesis of purine compound 1a. Reagents and conditions: (a)
pyridine, CH2Cl2; (b) NaH, MeI, DMF; (c) Pd/C, H2, MeOH (d) 3,4-2H-dihydropyran;
1-Boc-3-aminopiperidine, DIEA, BuOH, 60 °C; (e) Pd2(dba)3, 1,3-bis(2,6-diisopropy-
phenyl)imidazonium chloride, NaOtBu, 1,4-dioxane, 95 °C; (f) (i) HCl, EtOH; (ii)
acryloyl chloride, DIEA, CH2Cl2.
Table 1
Cellular antiproliferative data
R1
R1
R2
O
R2
N
N
N
N
H
N
H
N
N
N
H
1
2
Compd
R1
R2
Cellular antiproliferative activity (EC50, l
M)a
EGFR del
Del/T790M
Iressa
PF299804
0.017
0.006
7
5
N
N
N
N
N
1a
1b
1c
1d
7
1
10
N
H
O
O
O
O
O
N
N
5
10
6
N
H
N
N
O
10
0.90
N
H
O
N
N
O
O
N
1e
2a
0.30
0.50
0.60
N
N
H
O
0.50
N
N
H
3-
(continued on next page)

640
W. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 638–643
Table 1 (continued)
Compd
R1
R2
Cellular antiproliferative activity (EC50, l
M)a
EGFR del
Del/T790M
O
N
H
2b
6
8
3-
N
O
O
O
O
2c
6
6
6
N
H
3-
4-
2d
6
N
N
N
H
4-
N
H
2e
10
10
a
n P 2.
sub-libraries revealed that several compounds such as 1e and 2a
possessed submicromolar cellular potency which compared
favorably with reference inhibitors such as Iressa and PF299804
(Table 1), while they did not show inhibition on proliferation of
A549 cells. The SAR also revealed that superior potency could be
obtained when the C6-position of the purine was substituted with
an aryl versus a cycloalkyl group. Variation of water solubility-
enhancing groups at the 2-position of purine did not exert a dra-
matic influence on the antiproliferative activities.
A second sub-library of pyrrolopyrimdine-derived acrylamides
was synthesized as depicted in Scheme 2. Regioselective iodination
of 6-chlorodeazapurine was achieved with N-iodosuccinimide. The
resulting intermediate was protected with SEM. Selective Suzuki
coupling at the C5 position of B1 gave rise to B2. Intermediate B3
was obtained by nucleophilic substitution at the C4 position. Com-
pound 2a was obtained after acidic deprotection of B3, followed by
acylation with acryloyl chloride and sequential removal of
hydroxymethyl group.
2d). Larger substituents such as 4-methoxyphenyl substituent at
the 3-position appear to be too bulky to fit in the ATP binding site.
A third sub-library was prepared using six-membered het-
erocycles including 4,6-pyrimidines, triazine (data not shown)
and 5-chloro-1,3-pyrimidines The synthetic scheme of one typ-
ical compound 3h is shown in Scheme 3. 3-Nitrophenol was
regioselectively introduced to the C4 position of 2,4,5-trichloro-
pyrimidine to afford intermediate C1. Amination of C1 with
4-(4-methylpiperazin-1-yl) aniline was accomplished in the
presence of trifluoroacetic acid at 100 °C. The nitro group in
C2 was reduced by PtO2/H2 without concominant reduction of
the C5 chloride. Acylation of C3 by acryloyl chloride gave rise
to 3h. The pyrimidine analogs of 3h were obtained in a similar
manner using the appropriate phenols and anilines.
The compounds were evaluated for their antiproliferative activ-
ity against PC9 and PC9 GR cells (Table 2). Only compounds with
meta-substituted acrylamides are shown because the correspond-
ing para-substituted acrylamides were less active. We initially
investigated SAR for variation of substituents at the C2-aniline of
the pyrimidine, which we would expect to be directed towards sol-
vent based upon the co-structures of related 2,4-disubstituted
pyrimidine with kinases (e.g., PDB code 2JKK). The potency of sim-
ple C2-fluoroaniline compound 3a may have been limited by its
poor water solubility (Table 2). The replacement of the fluoro
group with functionality that would be expected to improve water
solubility such as morpholine 3c, 4-hydroxypiperidine 3d, N-meth-
ylpiperidine 3e, 3f, 3g, piperazine 3i, piperazine derivative 3j
resulted in 5–40-fold increases in potency. The analogs which pos-
sess amide groups at the para position of the aniline (3k, 3l, 3m,
3n, 3o) exhibited no significant improvement in activity relative
to fluoro or pyridinyl substituted compounds. We next explored
SAR at the ortho position of C2-aniline moiety. Replacement of
methoxy with CF3 (3u vs 3i) resulted in six-fold loss of potency.
Removal of the methoxy group (3h) improved potency to the single
digital nanomolar EC50 range. However, 3i is more selective for
EGFR than 3h10 when evaluated in biochemical and cellular assays
of other kinases. A one carbon extension of the piperazine analog
3q is slightly less active than 3h. Introduction of CF3 to the meta-
position of C2-aniline (3r) resulted in a significant decrease in cel-
lular potency. Two carbon extension of the piperazine analog 3t
had approximately the same potency as 3h.
In the pyrrolopyridine and quinoline series, the compounds
with an electrophile at the meta-position exhibited superior po-
tency relative to para-substituted compounds (compare 2a with
Cl
N
N
Cl
N
I
Cl
N
(b)
N
(a)
N
N
N
H
N
SEM
N
SEM
B2
B1
O
NHBoc
NH
(c)
N
(d)
N
O
N
O
N
N
N
N
N
H
SEM
2a
B3
Scheme 2. Synthesis of pyrrolopyrimidine compound 2a. Reagents and conditions:
(a) (i) NIS, CH2Cl2; (ii) NaH, SEM-Cl, DMF; (b) pyridine-3-boronic acid, Pd(dppf)Cl2,
dppf, Na2CO3(aq)/dioxane (1:2); (c) 3-Boc-aminophenol, K2CO3, DMSO, 100 °C; (d)
(i) TFA, CH2Cl2; (ii) acryloyl chloride, DIEA, CH2Cl2; (iii) NaHCO3(aq), THF.
Next we optimized the heteroatom linkage at the pyrimidine
4-position. The ether linkage was replaced with sulfur (4a, 4b)
and NH (4c) (Table 3). Both replacements were tolerated as

W. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 638–643
641
Table 2
Cellular antiproliferative data
Table 2 (continued)
Compd R1
R2
Cellular antiproliferative activity
a
(EC50, lM)
O
NH
EGFR del
0.80
Del/T790M
1
N
N
N
3n
3o
3p
3q
H
O
N
O
O
O
N
H
R2
Cl
N
N
H
MeO
MeO
H
0.50
ND
0.80
>10
N
H
R1
3
N
H
Compd R1
R2
Cellular antiproliferative activity
a
(EC50, lM)
ND
0.03
N
N
EGFR del
NDb
Del/T790M
0.80
N
N
3a
3b
MeO
F
3r
m-CF3
ND
0.42
MeO
MeO
ND
ND
0.90
0.15
N
N
N
N
N
3s
3t
H
H
ND
ND
0.050
0.008
N
O
N
O
3c
N
3d
MeO
0.20
0.80
3u
CF3
ND
0.090
OH
N
a
3e
3f
MeO
MeO
0.10
0.10
0.20
0.04
n P 2.
Not detected.
b
N
Table 3
Cellular antiproliferative data
N
N
3g
3h
3i
MeO
H
ND
0.20
O
NH
R2
N
X
N
N
N
N
Cl
0.015
0.036
0.003
0.014
N
H
N
R1
N
N
4
MeO
Compd
X
R1
R2
Cellular antiproliferative activity (EC50, lM)
EGFR del
Del/T790M
N
3i
O
S
S
NH
O
O
O
OMe
OMe
H
OMe
OMe
OMe
OMe
H
H
H
H
2-Cl
2-F
3-F
0.036
0.020
0.006
0.040
>1
0.014
0.020
0.008
0.020
>1
4a
4b
4c
4d
4e
4f
3j
MeO
0.08
0.10
0.80
N
N
ND
ND
0.031
0.020
3k
H
0.80
>1
O
O
N
N
N
evidenced by 3i, 4a, and 4c. The same trend was observed for ana-
logs without substituents at the ortho position of C2-aniline (3i vs
4b). Introduction of a chloro group at the ortho position of oxygen
linkage (4d) resulted in a loss of activity. In contrast, introduction
of a smaller fluoro-substituent at the ortho or meta-positions (4e,
4f) maintained similar potency as 3i.
3l
MeO
MeO
>1
N
O
N
3m
0.50
O
N
H
We next investigated how various substituents at the pyrimi-
dine 5-position change their potency and selectivity against

642
W. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 638–643
NO2
NO2
Cl
N
OH
N
O
N
O
Cl
(a)
(b)
N
Cl
N
Cl
N
N
+
Cl
NO2
N
H
Cl
N
C1
O
C2
NH2
NH
(d)
(c)
N
O
N
N
O
N
N
Cl
N
Cl
N
N
N
H
N
H
C3
3h
Scheme 3. Synthesis of pyrimidine compound 3h. Reagents and conditions: (a) K2CO3, DMF; (b) 4-(4-methylpiperazin-1-yl) aniline, TFA, 2-BuOH, 100 °C; (c) PtO2, H2, MeOH;
(d) acryloyl chloride, DIEA, CH2Cl2.
Table 4
Cellular antiproliferative data
and an EC50 of 2 nM in E746_A750_del/T790M relative to an EC50
of >3300 nM in E746_A750_del/T790M/C797S EGFR-transformed
Ba/F3 cells. In addition the reversible analog of 3i, which has a sat-
urated C–C bond instead of C@C bond and therefore is unable to
O
NH
undergo a Michael addition reaction with C797 was synthesized.
Its EC50 value in cellular proliferation assays against E746_A750/
T790M EGFR Ba/F3 cells was 1290 nM, which is 645-fold less po-
tent than 3i (EC50 = 2 nM). Furthermore, analysis of recombinant
EGFR-T790M kinase incubated with 3h by electrospray mass
spectrometry revealed stoichiometric addition of one inhibitor
molecule to the protein. Analysis of a pepsin digest of the modified
protein by tandem mass spectrometry identified Cys797 as the site
of modification, thus verifying covalent bond formation between
3h and EGFR.10
In summary we successfully developed a series of covalent
inhibitors for EGFR-T790M12 by a structure-based drug design.
Those inhibitors exhibited great potency and mutant selectivity
against drug resistant EGFR-T790M. A representative compound
3i blocked proliferation of EGFR-T790M PC9 cells with an EC50 in
the two digit nanomolar range. The approach of combining cellular
screens against clinically relevant mutant kinases with structure-
based drug design, should be useful for numerous important can-
cer targets. Further work will be reported on the pharmacokinetic
properties and in vivo activity for EGFR inhibitors in due course.
N
O
N
N
X
N
N
H
5
Compd
X
Cellular antiproliferative activity (EC50, lM)
EGFR del
Del/T790M
3h
5a
5b
5c
Cl
H
CF3
Br
0.015
0.08
0.70
0.80
0.003
>1
0.80
0.90
EGFR-T790M. The analogs of 3h with Cl replaced by H, Br, CF3 were
synthesized using the same route as depicted in Scheme 3. Among
them 3h is the most potent (Table 4) because it is likely that the
chloro group engages in a dipole interaction with the thioether of
the methionine at position 790 of EGFR.10 Compounds 3h and 3i
were profiled against a panel of 400 kinases using the Ambit Ki-
nome screening platform. In addition to EGFR, several kinases
demonstrated strong binding particularly those kinases possessing
cysteines at the same position as EGFR. Potent inhibition of 3i
against EGFR-T790M mutant was also observed using enzymatic
assays.10,11 To confirm whether the observed binding activity
translated into cellular activity, 3h and 3i were assayed against
Ba/F3 cells transformed with TEL fusions of Bmx, Blk, Jak2 and
Jak3. Compounds 3h and 3i both showed no inhibition of Jak2
while 3i, which possesses an ortho-methoxy group at C2-aniline
substituent, is much weaker at inhibiting Jak3, Blk, Bmx, and thus
more selective for EGFR than 3h.10
To investigate if our EGFR inhibitors bind irreversibly, we
constructed EGFR-transformed Ba/F3 cell lines where the reactive
cysteine residue is replaced with a serine (C797S). The C797S
mutation resulted in a dramatic 400- and 1650-fold reduced
potency for 3i in the L858R and E746_A750 backgrounds, respec-
tively. For example, 3i possesses an EC50 of 8 nM in L858R/
T790M relative to an EC50 of >3300 nM in L858R/T790M/C797S
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12. The synthetic procedure of lead compound 3i was reported in Ref. 10.
1d
J = 7.8 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.41 (q, 1H), 6,26 (dd, J = 1.8, 16.8 Hz,
1H), 5.76 (dd, J = 1.8, 10.2 Hz, 1H), 3.72 (s, 3H), 3.01 (m, 4H), 2.40 (m, 4H), 2.20
(s, 3H).
1H NMR 600 MHz (DMSO-d6) d 9.18 (s, 1H), 8.00 (s, 1H), 7.72 (d, J = 6.4 Hz, 2H),
7.12 (d, J = 6.4 Hz, 2H), 6.83 (m, 1H), 6.08 (q, 1H), 5.60 (dd, J = 10.8 Hz, 1H), 4.20
(m, 1H), 4.05 (m, 1H), 3.0 (s, 3H), 2.96 (m, 5H), 1.98 (m, 6H), 0.97 (t, J = 8.4 Hz,
3H).
4c
1H NMR 600 MHz (DMSO-d6) d 10.1 (s, 1H), 9.88 (s, 1H), 9.08 (s, 1H), 8.06 (s,
1H), 7.80 (s, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.20 (m, 2H),
6.60 (s, 1H), 6.40 (q, 1H), 6.28 (m, 2H), 5.70 (d, J = 8.4 Hz, 1H), 3.70 (s, 3H), 3.40
(m, 4H), 3.02 (m, 4H), 2.80 (s, 3H).
2a
1H NMR 600 MHz (DMSO-d6) d 12.60 (s, 1H), 10.35 (s, 1H), 9.00 (s, 1H), 8.52 (d,
J = 10.8 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.90 (s, 1H), 7.70 (s, 1H), 7.44 (m, 2H),
7.00 (d, 1H), 6.50 (q, 1H), 6.30 (d, 1H), 5.80 (d, 1H).
3i
4e
1H NMR 600 MHz (DMSO-d6) d 10.20 (s, 1H), 9.80 (s, 1H), 8.33 (s, 1H), 7.78 (s,
1H), 7.60 (s, 1H), 7.40 (m, 1H), 7.20 (d, J = 8.8 Hz, 1H), 6.6 (s, 1H), 6.40 (m, 1H),
6.28 (m, 1H), 5.80 (dd, J = 10 Hz, 2 Hz, 1H), 3.78 (s, 3H), 3.43 (m, 4H), 3.10 (m,
4H), 2.92 (s, 3H).
1H NMR 600 MHz (DMSO-d6) d 10.3 (s, 1H), 8.33 (s, 1H)), 8.09 (s, 1H)), 7.62 (d,
J = 7.8 Hz, 1H), 7.54 (m, 1H), 7.39 (t, 1H), 7.22 (d, J = 10.5 Hz, 1H), 6.95 (d,