Pyridones as Highly Selective, Noncovalent Inhibitors of T790M Double Mutants of EGFR

Article abstract of DOI:10.1021/acsmedchemlett.5b00428

The rapid advancement of a series of noncovalent inhibitors of T790M mutants of EGFR is discussed. The optimization of pyridone 1, a nonselective high-throughput screening hit, to potent molecules with high levels of selectivity over wtEGFR and the broader kinome is described herein.

Full text of DOI:10.1021/acsmedchemlett.5b00428

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Letter
Pyridones as Highly Selective, Noncovalent
Inhibitors of T790M Double-Mutants of EGFR
Marian C. Bryan, Daniel J. Burdick, Bryan K Chan, Yuan Chen, Saundra Clausen, Jennafer Dotson,
Charles Eigenbrot, Richard Elliott, Emily J. Hanan, Robert Andrew Heald, Philip Jackson, Hank
La, Michael Lainchbury, Shiva Malek, Sam E. Mann, Hans E. Purkey, Gabriele Schaefer, Stephen
Schmidt, Eileen Seward, Steve Sideris, Shumei Wang, Ivana Yen, Christine Yu, and Timothy P Heffron
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00428 • Publication Date (Web): 17 Dec 2015
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Pyridones as Highly Selective, Noncovalent Inhibitors of T790M
Double-Mutants of EGFR
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Marian C. Bryan,1* Daniel J. Burdick,1 Bryan K. Chan,1 Yuan Chen,1 Saundra Clausen,1 Jennafer
Dotson,1 Charles Eigenbrot,1 Richard Elliott, 2 Emily J. Hanan,1 Robert Heald,2 Philip Jackson,2
Hank La,1 Michael Lainchbury,2 Shiva Malek,1 Sam E. Mann,2 Hans E. Purkey,1 Gabriele Schaefer,1
Stephen Schmidt,1 Eileen Seward,2 Steve Sideris,1 Shumei Wang,1 Ivana Yen,1 Christine Yu,1 Timo-
thy P. Heffron1
1Genentech, South San Francisco, CA 94080, United States, 2Argenta, Early Discovery Charles River, 7/9 Spire Green
Centre, Flex Meadow, Harlow, Essex CM19 5TR, United Kingdom
KEYWORDS mutant EGFR, non-covalent, pyridone, T790M.
ABSTRACT: The rapid advancement of a series of noncovalent inhibitors of T790M mutants of EGFR is discussed. The
optimization of pyridone 1, a non-selective high-throughput screening hit, to potent molecules with high levels of selec-
tivity over wtEGFR and the broader kinome is described herein.
Epidermal growth factor receptor (EGFR), a member of the
ErbB family of tyrosine kinases, is involved in the activation
of numerous crucial cell signaling pathways that play critical
roles in cellular survival, proliferation, inhibition of apoptosis
and angiogenesis.1,2 EGFR overexpression has been found in
62% of non-small cell lung cancer (NSCLC) cases.3 While
EGFR overexpression is considered a prognosticator of poor
survival odds, NSCLCs harboring specific activating muta-
tions in the tyrosine kinase domain of EGFR, including the
point mutation L858R in exon 2 and deletions within exon 19
(e.g. residues 746-750), are known to confer hyper-sensitivity
to tyrosine kinase inhibitors (TKIs) such as erlotinib and
gefitinib.1 As inhibition of wild-type EGFR (wtEGFR) has
been linked to dose-limiting toxicities, the increased inhibi-
tion of mutant EGFR relative to wtEGFR by TKIs allows for
improved tolerability.4
generate a potent and selective molecule for analysis of selec-
tivity in cellular assays as well as develop SAR towards an in
vivo tool compound.
A high throughput screen identified pyridone 1 as an in-
hibitor of TMLR with a Ki of 65 nM and LE11 of 0.3 (Table 1).
Although relatively active and moderately ligand efficient,
the initial lead had several liabilities requiring attention.
Most importantly, compound
1 lacked selectivity over
wtEGFR. In addition, improvement of the potency and selec-
tivity over the kinome were essential as it showed greater
than 70% percent inhibition for 67 kinases out of a panel of
220 kinases when tested at a 1 µM compound concentration
(Supporting Information Table S1).
Scheme 1. Synthetic routes facilitating diversification
of pyridone scaffold.a
While treatment of NSCLC harboring EGFR mutations
with erlotinib or gefitinib offers initial strong clinical re-
sponses, these are followed by acquired resistance. Approxi-
mately 50-60% of such cases arise from a single secondary
mutation within the kinase domain where the threonine
gatekeeper residue, Thr790, is exchanged for methionine
(T790M).1,5 This single point mutation, found along with an
activating mutation, alters the binding pocket and renders
erlotinib and gefitinib less effective.5-7 The compelling nature
of the T790M EGFR mutants as therapeutic targets, along
with an understanding of the relationship between wtEGFR
inhibition and dose-limiting toxicities, led us to initiate ef-
forts to identify inhibitors of the major resistance mutations
of EGFR, namely T790M/L858R (TMLR) and T790M/del(746-
750) (TMdel), with selectivity over wtEGFR. In addition, a
noncovalent inhibitor profile was selected to prevent the
possible resistance mechanism known for covalent inhibi-
tors, such as those against TMLR currently in the clinic.8-10
Herein we describe our efforts beginning with pyridone 1 to
aReagents and conditions: (a) 2-methyl-2-butene, NaClO2,
NaPO4H2-H2O; (b) HBTU or oxalyl or thionyl chloride,
NH2R1; (c) HCl; (d) NHR2R3, Δ; (e) R1C1NH2C2NH2, MeOH, Δ.
In order to address liabilities and test key hypotheses, a
synthetic route was developed allowing for rapid exploration
at C3 and C4 (Scheme 1). For diversification at C4, pyridine 3
was first oxidized to carboxylic acid 4. Amide bond formation
using various coupling conditions installed R1 at C3 and de-
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methylation gave the pyridone. Final step diversification at
ring (ie replacing the phenethylamine with an aniline)
should effectively achieve this goal.
C4 with R2 and R3 occurred via nucleophilic aromatic substi-
1
2
3
4
5
6
7
8
9
tution (SNAr) to give 1-2, 5-22 and 38-39.
Indeed, truncating phenethyl (5) to the aniline (7) led to
the first compound with selectivity over wtEGFR (Table 1),
while the benzyl (6) was equipotent, yet not selective over
wtEGFR. Compound 7’s loss in potency for wtEGFR while
maintaining TMLR potency is presumably due to the short-
ened chain, leaving solvent-exposed hydrophobic space not
filled by the ligand in wtEGFR. This space is occupied by the
Met790 sidechain in the T790M protein. Removal of the
phenyl group altogether (8) decreased TMLR potency by less
than 3-fold while maintaining selectivity over wtEGFR.
Diversification at C3 followed similar steps to those de-
scribed above for compounds 23-26 and 28-36. From inter-
mediate 4, demethylation followed by SNAr at C4 gave the
pyridone late stage intermediate. The amide bond was then
formed via the acid chloride. Benzimidazoles 27 and 37 were
formed upon condensation of 3 with the required diamine
prior to pyridone formation and coupling to HNR2R3.
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An X-ray structure of compound 2 bound to TMLR (Figure
1B) informed our strategy to improve potency and selectivity
over wtEGFR. The pyridone of compound 2 binds the protein
hinge region through residues Met793 and Gln791. Two in-
tramolecular hydrogen bonds, one between the amide NH
and the pyridone carbonyl and a second between the amide
carbonyl and the pyridone NHR, position the phenyl amide
and the pyridone in a co-planar conformation, directing the
phenyl-piperazine toward solvent. The C4 phenethyl substit-
uent is then positioned toward a back pocket above the
Met790 gatekeeper.
Table 1. Effects of C4 modification on potency and
wtEGFR selectivity.
H
O
N
H
N
R1
O
N
N
wtEGFR
Ki
TMLR Ki
(µM)a
TMLR
Cmpd
1
R1
Select.b
(µM)a
0.065
0.018
<1
2
5
6
NH(CH2)2-4-Cl-Ph
NH(CH2)2Ph
NHCH2Ph
0.262
0.117
0.350
0.037
0.049
0.349
<1
<1
1
7
8
9
NHPh
NH2
0.144
0.346
0.082
>2.54
>2.54
1.480
>18
>7
18
NH-2-Cl-Ph
10
11
NH-3-Cl-Ph
NH-4-Cl-Ph
0.155
0.629
0.061
0.019
>2.06
0.108
0.095
2.520
>2.54
2.340
1.090
>2.54
>2.54
>2.54
16
>4
38
12
13
14
16
17
NH-2-Me-Ph
NH-2-OMe-Ph
NMe-2-OMe-Ph
NH-2-pyridyl
NH-2,4-pyrimidyl
58
Figure 1. A) Crystal structure of the apo-TMLR double mu-
tant (PDB 5EDP). The pocket surface is shown in grey. Com-
pound 2 is overlaid. B) X-ray structure of compound 2 with
the TMLR double mutant. Hinge residues Met793 and Gln791
are shown. The pocket surface is shown in grey. C) Overlay
of compound 2 (cyan) with the erlotinib crystal structure
with wtEGFR (orange) showing the pocket created by Thr790
(grey surface) (PDB 1M17). D) Structure of erlotinib with bio-
chemical potency in wtEGFR and TMLR.
1
>24
>27
19 NH-3-NH2-2,4-pyrimidyl 0.004
0.850
>1.23
213
>8
20
NH-3-NH2-4-pyridyl
0.145
aKi data are an average of at least two independent experi-
ments. bRatio of wtEGFR Kiapp over EGFR(TMLR) Kiapp.
We next explored the aniline SAR. Substitution with chlo-
rine at C4 negatively impacted potency relative to C2 and C3
(11 versus 9 and 10). Alternate C2 substitution led to increas-
es in both potency and selectivity for tolyl 12 and methoxy 13,
with 13 possessing 58-fold selectivity over wtEGFR. Compar-
ing the crystal structures of compound 13 soaked into either
TMLR (cyan) or wtEGFR (orange, Supporting Information
Figure S1A) reveals the phenyl group leaving the large back
pocket of wtEGFR free of ligand. In TMLR, the smaller pock-
et allows for hydrophobic contacts between the phenyl ring
and the Met790 side chain, and directs the methyl group of
the methoxy along the side of the back pocket where it may
achieve Van der Waals contacts (Supporting Information
Figure S1B). When the amine linker was substituted with a
methyl group (tertiary amine 14), measureable potency for
both the mutant and wild type protein was lost, presumably
Comparison of the apo crystal structure of TMLR with that
of 2 bound to TMLR shows an induced pocket above the Met
gatekeeper upon binding 2 (Figures 1A, 1B). This region is
similar to the crystal structure of erlotinib with wtEGFR
(Figure 1C, orange with surface) where a large pocket above
the gatekeeper is occupied by the phenyl alkyne.12 Overlay of
2 into the erlotinib wtEGFR crystal structure shows that the
phenethyl group of 2 could occupy this space. This pocket
appears important to activity for erlotinib, which is signifi-
cantly more potent for wtEGFR than the double mutant
(Figure 1D).13,14 By not inducing the “wtEGFR-like” pocket,
and through effectively occupying the back pocket observed
in the TMLR apo structure, we hypothesized selectivity
would be gained over wtEGFR. Modeling suggested that by
eliminating the ethylene linker between C4 and the phenyl
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due to loss in planarity and hydrogen bonding with the am-
ide.
Replacement of the phenyl ring with 6-membered het-
al restriction of the amide led to benzimidazole 27 which
1
2
3
4
5
6
7
8
maintained selectivity over wtEGFR with only a modest de-
crease in potency relative to compound 13. The isopropyl
group on the phenyl ring was predicted to occupy the lipo-
philic space within the ribose pocket.9
eroaryls was then undertaken. 2-Pyridine 16 and 2,4-
pyrimidine 17 were equipotent to phenyl 7. Addition of an
amine to the C3 position of 17, designed to target polar inter-
actions with the side chains of catalytic Lys745 and Glu762
(Supporting Information Figure S2), generated the first single
digit nanomolar compound of the series, with >200x selectiv-
ity over wtEGFR (compound 19). However, removal of one of
the pyrimidine nitrogens of 19 erased this gain in potency
(pyridine 20) potentially due to the diaminopyridine being
positively charged at physiological pH, unlike the pyrimi-
dine, and clashing with Lys745.15
Focusing on the piperazine, we discovered several changes
were tolerated (Table 2). Extension of the linker between the
aryl ring and the piperazine (28), replacement of the linking
nitrogen with carbon (29) and increase in the ring size (30)
were all tolerated by TMLR. Interestingly, replacement of the
terminal nitrogen with carbon (31) was not tolerated but
replacement with a sulfone (32) was, with less than 2x loss in
potency relative to 13, suggesting that polarity was beneficial
in this region but a positive charge was not required.
9
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12
13
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19
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Table 2. Effects of C3 modification on TMLR potency.
Heteroaromatic replacements at C3 were also evaluated to
reduce lipophilicity of compound 13 (LogD7.4 = 3.5) (Table 2).
While pyridine 34 suffered a modest reduction in activity
relative to compound 13, it did improve overall lipophilicity
(LogD7.4 = 2.6) and kinetic solubility (65 µM vs 4 µM for
compound 13). Pyridine 33 was not active, presumably due to
a clash of the nitrogen lone-pair electrons with either the
backbone carbonyl of Met793 (Figure 1) or the carbonyl of
the amide depending on its conformation.
TMLR Ki wtEGFR TMLR
Cmpd
23
R2
R1
A
B
(µM)a
Ki (µM)a Select.b
CONH2
>0.688
>2.5
0.043
1.210
>3.6
With C3 and C4 SAR in hand, we sought to understand if
these changes improved selectivity over the broader kinome
by examining the kinase selectivity against an abridged panel
of those screened for compound 1. Kinases were selected
based on either strong inhibition seen with compound 1,
diversity or kinases known to show potential safety liabilities
(Supporting Information, Table S2). While compounds 13
and 19 showed improved wtEGFR selectivity relative to 1,
they maintained similar kinome selectivity and were potent
inhibitors of JAK2 at a 1 µM concentration. In a full dose-
response assay, 13 inhibited JAK2 with a 9 nM Ki (Table 3)
and 10-fold selectivity relative to JAK1 (Ki 172 nM). That com-
pounds with modest to high selectivity over wtEGFR would
lack JAK selectivity is unsurprising as both TMLR and the
JAK family contain a Met gatekeeper. Key amino acid differ-
ences between EGFR and numerous other kinases, including
JAK family members, appeared to be more accessible from
C3. This led us to hypothesize further changes in this region
would have greater impact on the kinome, our second selec-
tivity goal.
24
0.066
0.330
1
25
CONHPh
B
2
(Me)2N
NH
26
B
0.290
0.757
1
O
H
N
27
A
0.058
2.400
42
N
28
29
A
A
0.051
0.015
1.200
0.634
22
43
30
31
A
A
A
0.012
0.625
0.037
0.705
>2.5
>1.3
57
>4.1
>34
32
In an effort to improve kinase selectivity, we focused on
substitution of the phenyl ring to target key residue Leu792
in TMLR as has successfully been utilized for other kinase
inhibitors seeking selectivity over JAK.16 This residue is a Tyr
or Phe in the JAK family (Figure S3). By directing a methyl
(35) or ethyl (36) substituent in this direction, TMLR potency
and wtEGFR selectivity were comparable to 13, but markedly
increased selectivity against JAK1 was realized, with a modest
improvement in JAK2 selectivity for compound 36 (Table 3).
Figure S3 shows the phenyl ring of 13 at the edge of the sur-
face of an aligned JAK2 crystal structure. Substitution at C3
of the phenyl ring would clash with the surface of the JAK2
binding pocket. If the substitution occupied the alternate
side of the phenyl ring, the piperazine ring would need to
shift, and this in turn would clash with the surface. Either
would result in a loss in affinity for JAK as is reflected in 36
and 35 to a lesser extent. The disconnect in selectivity be-
tween JAK1 and JAK2 may be due to plasticity of the hinge
33
34
A
A
>2.06
0.078
>2.5
>2.5
1
>33
aKi data are an average of at least two independent exper-
iments. bRatio of wtEGFR Kiapp over EGFR(TMLR) Kiapp.
As we turned our attention to optimization of C3, the 2-
methoxyaniline of compound 13 was retained due to its com-
bination of potency and selectivity over wtEGFR. In order to
understand the minimal motifs necessary for inhibition, a
number of abridged amides were generated (Table 2). Trun-
cation to the primary amide gave significant loss in activity
(23) when compared to 13. Similar reductions in potency
were found with the unsubstituted phenyl ring analog (25)
and the dimethylaniline (26), which lost 4 to 5x when com-
pared to fully elaborated 24. Efforts to enforce conformation-
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region for these two proteins due to their sequence variabil-
when comparing pEGFR inhibition in the TMLR cell line
ity.14
H1975 vs. the wtEGFR-driven cell line H292 meeting the goal
of a selective tool compound with improved kinase selectivi-
ty for evaluation in cellular assays. In addition, 29 showed
desireable physicochemical properties, improved solubility,
and moderate in vitro stability highlighting the promise of
this series for further development.
1
2
3
4
5
6
Table 3. Selectivity over JAK family members.
7
8
9
In conclusion, the compelling nature of the T790M EGFR
mutants as therapeutic targets for oncology led us to initiate
efforts to identify inhibitors of the major resistance muta-
tions of EGFR, namely T790M/L858R (TMLR) and
T790M/del(746-750) (TMdel), with selectivity over wtEGFR.
Beginning with high throughput screening hit 1, which
lacked selectivity over wtEGFR, we identified a novel series of
non-covalent pyridone-based T790M EGFR inhibitors. Re-
placement of the phenethyl motif with an aniline substruc-
ture provided a significant improvement in selectivity over
wtEGFR and a desirable starting point for further optimiza-
tion. Optimization of C3 provided a framework for address-
ing broad kinase selectivity. Several of the optimized inhibi-
tors showed substantial improvement in biochemical poten-
cy, ligand efficiency selectivity across the kinome. Gratifying-
ly, this selectivity was recapitulated in a cellular format. Fur-
thermore, improvements in physicochemical properties and
kinetic solubility with maintained moderate in vitro meta-
bolic stability exemplify the promise of the scaffold. Further
optimization of this scaffold to achieve improved cellular
potency will be the focus of future reports.
TMLR
Ki
wtEGFR
Select.b
JAK1
JAK2
Cmpd
Select.c
Select.d
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
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60
(µM)a
0.019
0.004
0.026
0.031
0.058
0.088
13
19
35
36
27
37
57
213
16
9
3
0.5
2
42
104
2
0.9
4
>80
42
0.2
27
6
0.3
aKi data are an average of at least two independent exper-
b
iments. Ratio of wtEGFR Kiapp over EGFR(TMLR) Kiapp.
cRatio of JAK1 Kiapp over EGFR(TMLR) Kiapp. dRatio of JAK2
Kiapp over EGFR(TMLR) Kiapp.
Benzimidazole 27 itself was not selective for TMLR over
JAK family members but provided an alternate vector to-
wards L792. Methyl substitution at benzimidazole C7 mod-
estly improved selectivity over JAK1 to 6x (37) and, while
JAK2 selectivity was not improved, broader selectivity in an
abbreviated kinase panel was realized.
ASSOCIATED CONTENT
Table 4. Physicochemical properties and cellular po-
tency for key analogs
Supporting Information. Additional figures, tables and
examples syntheses located in Supporting Information. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Cmpd
1
13
29
LogD, TPSA, LE a
Kinetic sol. (µM)
TMLR Ki (µM)b
wtEGFR Select.c
pEGFR H1975
EC50 (µM)b
-, 100, 0.3 3.5, 89, 0.34 2.7, 86, 0.34
AUTHOR INFORMATION
Corresponding Author
11
4
61
0.065
<1x
0.019
58x
0.015
29x
* Marian C Bryan: bryan.marian@gene.com, One DNA Way,
South San Francisco CA 94080.
0.412
1.09
5x
0.433
>23x
pEGFR select. vs
H292d
ABBREVIATIONS
H/RLMe
11/31
14/41
12,24
11/29
8/25
wtEGFR, wild-type EGFR; TMLR, T790M/L858R mutation.
H/R CLhepf
13/35
aLE calculated using TMLR Ki. Ki and EC50 data are an
b
REFERENCES
c
average of at least two independent experiments. Ratio of
(1)
Remon, J.; Morán, T.; Majem, M.; Reguart, N.; Dalmau,
wtEGFR Kiapp over EGFR(TMLR) Kiapp. dRatio of pEGFR
EC50 in H292 cells over pEGFR EC50 in H1975 cells. eH/RLM
defined as predicted hepatic clearance in liver microsomes
for human (H) and rat (R). Reported in units of mL/min/kg.
fH/R CLhep defined as predicted hepatic clearance in
hepatocytes for human (H) and rat (R). Reported in units of
mL/min/kg.
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tency, wtEGFR selectivity and broader kinome selectivity, a
subset of pyridone inhibitors were progressed into cellular
assays (Table 4). Compounds 13 and 29 had gains in bio-
chemical potency, ligand efficiency and wtEGFR selectivity
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proved cellular activity, compounds 13 and 29 both showed
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