Discovery of (R,E)-N-(7-chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (EGF816), a novel, potent, and WT sparing covalent inhibitor of oncogenic (L858R, ex19del) and resistant (T790M) EGFR mutants for the treatment of EGFR mutant non-small-cell lung cancers

Article abstract of DOI:10.1021/acs.jmedchem.5b01985

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

Full text of DOI:10.1021/acs.jmedchem.5b01985

Article
pubs.acs.org/jmc
Discovery of (R,E)‑N‑(7-Chloro-1-(1-[4-(dimethylamino)but-2-
enoyl]azepan-3-yl)‑1H‑benzo[d]imidazol-2-yl)-2-methylisonicotinamide
(EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of
Oncogenic (L858R, ex19del) and Resistant (T790M) EGFR Mutants for
the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers
Gerald Lelais,*,† Robert Epple,† Thomas H. Marsilje,† Yun O. Long,† Matthew McNeill,† Bei Chen,†
́
Wenshuo Lu,† Jaganmohan Anumolu,‡ Sangamesh Badiger,§ Badry Bursulaya,† Michael DiDonato,†
Rina Fong,†,∥ Jose Juarez,† Jie Li,† Mari Manuia,† Daniel E. Mason,† Perry Gordon,† Todd Groessl,†
Kevin Johnson,† Yong Jia,† Shailaja Kasibhatla,† Chun Li,† John Isbell,† Glen Spraggon,† Steven Bender,†
and Pierre-Yves Michellys†
†Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, California 92121, United States
‡Aurigene Discovery Technologies, Bollaram Road, Miyapur, Hyderabad 500 049, India
§Aurigene Discovery Technologies, 39-40, Electronic City Phase 2, Bangalore 560 100, India
S
* Supporting Information
ABSTRACT: Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung
cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the
gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these
therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach
toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on
both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective
high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants
EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good
tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.
afatinib (3)7 (Figure 1), dacomitinib (4)8,9 (Figure 1), and
INTRODUCTION
■
ceritinib.10 Their mode of action (MoA) relies on the inactiva-
Inhibitors of epidermal growth factor receptor (EGFR), such
as erlotinib (1), gefitinib (2), and afatinib (3) (Figure 1) deliver
high objective response rates and increased overall survival
(OS) for non-small-cell lung cancer (NSCLC) patients with
tion of EGFR by forming a covalent bond between an
electrophilic Michael acceptor and a conserved cysteine
(Cys797) near the ATP binding domain,12 leading to a more
complete and sustained inhibition of the target. Although 3
oncogenic EGFR mutation cancers (i.e., exon 19 deletions
showed promise in preclinical models of T790M,7 it is lacking
or exon 21 L858R single-point mutation).1−3 However, upon
efficacy in the clinic as a single agent for patients with T790M
continuous treatment, patients become resistant and, in 50−60%
mutation13 and is unable to achieve an efficacious concentration
of cases, develop a secondary T790M mutation at the gatekeeper
residue of EGFR.4,5 Evidence suggests that this modification
due to dose-limiting WT EGFR-driven toxicities (e.g., GI-tox
and rash).7,14
does not block inhibitor binding but rather confers resistance by
increasing affinity to ATP.6
More recently, a new class of mutant-selective irreversible
EGFR inhibitors containing an anilinopyrimidine core has been
To overcome drug resistance caused by the T790M
mutation, several irreversible pan-EGFR inhibitors belonging
to the same structural class as their reversible counterparts
Received: December 22, 2015
(4-anilinoquinazolines) have been developed.7−11 These include
© XXXX American Chemical Society
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Figure 1. Structures of selected EGFR inhibitors: (a) reversible pan-EGFR inhibitors; (b) irreversible pan-EGFR inhibitors; (c) irreversible mutant-
selective EGFR inhibitors.
Figure 2. (a) High throughput screening (HTS) approach that culminated in the identification of both allosteric and ATP-competitive EGFR hits.
Biochemical IC50 (μM) of ATP-competitive hit 7 as average of at least duplicate measurements assessed at 10 μM ATP concentration for either
mutants (L858R and L858R-T790M) and WT EGFR constructs. (b) Docking model for the binding of 7 in the ATP-pocket of EGFR T790M-
AEE788 cocrystal structure (PDB code 2jiu).6
described in the literature.15−22 Importantly, these inhibitors
have low nanomolar activity on EGFR-T790M mutant cells
and demonstrate good selectivity toward WT EGFR. On
November 13, 2015, osimertinib (AZD9291, 5)20,23 (Figure 1)
was granted accelerated approval by FDA24 and rociletinib
(CO-1686, 6)25,26 (Figure 1) was in pivotal registration trials.
Although more clinical data are required, both compounds have
the potential to overcome EGFR-T790M resistance mutations
while reducing WT EGFR-driven toxicities.23,25−28
In this article we report the discovery of 47 (EGF816,
nazartinib), a potent covalent mutant-selective EGFR inhibitor
with a unique structural motif, currently being evaluated in a
number of clinical trials for the treatment of NSCLC.29−31
competitive hits. This communication focuses on the identifica-
tion of ATP-competitive hits, and the latter ones will be discussed
in a separate publication. To further assess the compound’s
selectivity, we used biochemical counterscreens against EGFR
L858R and WT EGFR kinase constructs. Compounds with
good activity on EGFR L858R-T790M and selectivity against
WT EGFR were considered to be good starting points for
further optimization. Activity on EGFR L858R was deemed
beneficial but not necessary for hit selection, as we hoped to
increase EGFR L858R activity during the hit-to-lead process
(vide infra). Among the identified hits, compound 7 displayed
an interesting profile, with submicromolar activity on the
L858R-T790M construct and selectivity against WT EGFR
(Figure 2), and its rather uncommon, but not unprece-
dented,33−36 hinge binding motif further stimulated our interest.
In fact, although there are a number of aminobenzimidazoles
targeting IRAK433,34 and ITK,35 analogue 7 was the only mutant
EGFR inhibitor not derived from an aminopyrimidine core,
making it a unique structural motif in the EGFR field. Docking
of 7 into the active site of EGFR T790M (PDB code 2jiu)6
revealed a similar binding mode to what had been reported in
the literature for IRAK-4 and ITK kinases. It showed a hydrogen
bond between the amide carbonyl group of 7 and the hinge-NH
of Met793, with the meta-trifluorobenzamide group extending
RESULTS AND DISCUSSION
■
Hit Identification and Modeling. Our strategy for identify-
ing mutant-selective EGFR inhibitors started with a HTS
campaign against the EGFR L858R-T790M kinase domain using
a homogeneous time-resolved fluorescence (HTRF) based
biochemical assay.32 To achieve maximal inhibitory sensitivity,
the compounds were profiled at 1 μM ATP concentrations
(ATP Km ≈ 2 μM) in single point inhibition. The hits were then
reconfirmed with IC50 curves at both 1 μM and 1000 μM ATP
allowing for the identification of both ATP and non-ATP
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a
Scheme 1
a
Reagents and conditions: (a) DMF, DIPEA, 120 °C; (b) Pd/C, H2, MeOH; (c) CNBr, MeCN/H2O/MeOH 55 °C; (d) 3-(trifluoromethyl)benzoic
acid, HATU, DIPEA, DMF or 3-(trifluoromethyl)benzoic acid, HOBt, EDCI, Et3N, DMF.
a
Scheme 2
a
Reagents and conditions: (a) R1 = Boc: DMF, base (DIPEA or K2CO3), 110−130 °C; R1 = H: neat or DMF, 130−150 °C. (b) For p, q, s,
R1 = H: Boc2O, Et3N, dioxane. (c) Pd/C, H2, MeOH; (d) CNBr, MeCN/H2O/MeOH 45−50 °C; (e) 3-(trifluoromethyl)benzoic acid, HATU,
DIPEA, DMF; (f) TFA, CH2Cl2; (g) acryloyl chloride, with or without Et3N, CH2Cl2, 0−20 °C.
toward the methionine gatekeeper residue (Met790) and the
cyclohexanol group projecting toward the lip of the ATP-
pocket’s Cys797 (Figure 2), a residue that had been successfully
targeted by irreversible EGFR inhibitors.7,11,37,38 With this
information in hand, we evaluated the structure−activity relation-
ship (SAR) of reversible analogs with the goal of identifying
potent ATP-competitive EGFR inhibitors. At the same time,
we rationalized that converting mutant-selective ATP-compet-
itive hits into targeted covalent inhibitors would provide
compounds with retained selectivity against WT EGFR, with
the added benefit of more complete target inhibition as it had
been first demonstrated with the identification of the mutant-
selective EGFR inhibitor N-(3-((5-chloro-2-((2-methoxy-4-(4-
methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-
acrylamide (WZ4002).15
amines or appropriately protected diamines followed by nitro
reduction and cyclization with cyanogen bromide afforded
2-aminobenzimidazole intermediates 18−22, 30a−s, 37, or 38
in moderate yields (14−85% over several steps). Amide coupling
of 18−22, 30a−s, or 37 with 3-(trifluoromethyl)benzoic acid
provided 23−27 (Scheme 1) or intermediates 31a−s and 39,
respectively. The latter were deprotected and transformed to the
desired products 32a−s (Scheme 2) or 43 and 44 (Scheme 3),
in acceptable yields (17−71%). Amide coupling of 37 and 38
with 2-substituted isonicotinic acids afforded 40−42 that were
deprotected and further coupled with (E)-4-(dimethylamino)-
but-2-enoic acid to provide 45−47 in moderate yields (35−61%,
Scheme 3).
In Vitro SAR. The docking of 7 in the kinase domain
revealed the solvent exposed nature of the ethoxymethyl group.
The lack of stabilizing interactions with the protein provided
a rationale for investigating simpler benzimidazole core sub-
stitutions as shown in Table 1. As expected, the unsubstituted
Chemistry. Compounds were prepared from commercially
available 1-fluoro-2-nitroarenes according to the general pro-
cedures described in Schemes 1−3. SNAr reaction with primary
C
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a
Scheme 3
a
Reagents and conditions: (a) neat, DMA or DMF, DIPEA, 110−140 °C. (b) For 35: Pd/C, H2, MeOH. For 36: Zn, AcOH, room temperature.
(c) CNBr, MeCN/H2O/MeOH 55 °C; (d) acid, HATU, Et3N, CH2Cl2 or acid, HATU, DIPEA, DMF; (e) TFA, CH2Cl2 or HCl, dioxane/MeOH.
(g) For 43: acryloyl chloride, CH2Cl2, 0−20 °C. For 44−47: (E)-4-(dimethylamino)but-2-enoic acid hydrochloride, HOBt, EDCI, Et3N, DMF or
(E)-4-(dimethylamino)but-2-enoic acid, HATU, Et3N, CH2Cl2.
Table 1. Structure−Activity Relationship of Benzimidazole
of adding a well-placed Michael acceptor capable of reacting
with Cys797 and result in a targeted covalent inhibitor that
could demonstrate improved potency on the mutant EGFR but
retain WT EGFR selectivity. As such, compounds of Table 2
were synthesized and a broad variety of lengths and geometries
around the reactive thiol of Cys797 were interrogated.
a
Core
The inhibitory activity of compounds 32a−s was assessed
against the L858R-T790M biochemical construct at 0 and 90 min
enzyme/compound preincubation. We employed 90 min pre-
incubation times based on time dependent inhibition studies
of irreversible EGFR inhibitors to ensure maximal inhibition of
covalent inhibitors (data not shown). These assays were carried
out in the presence of 1 mM ATP to simulate physiological
intracellular levels of ATP. Importantly, striking differences in
EGFR L858R-T790M inactivation were observed, with few
linkers (compounds 32b, 32e, 32g, 32l, 32m, 32q, and 32r)
capable of inhibiting the enzyme at nanomolar ranges after a
90 min preincubation and only compound 32g, with a racemic
3-substituted azepane linker, demonstrating submicromolar
activity without preincubation. With these data in hand, we
further profiled the most active compounds in various cellular
target modulation assays of oncogenic (e.g., H3255, HCC827),
resistance (e.g., H1975), and WT (e.g., HaCaT) EGFR settings
as shown in Table 3. The cellular potency on H1975 was in
good agreement with the biochemical inhibitory concentra-
tion on the L858R-T790M construct, with 32g demonstrating
compd
R
L858R-T790M
L858R
WT
23
24
25
26
27
H
0.26 0.07
1.37 0.56
0.14 0.10
0.28 0.12
0.09 0.01
2.00 0.41
11.75 5.22
>8.48
>50
>50
4-Me
5-Me
6-Me
7-Me
>11.78
>16
>50
0.68 0.07
5.27 2.42
a
Biochemical IC50 (μM) assessed at 10 μM ATP concentration for
either mutants (L858R and L858R-T790M) and WT EGFR
constructs. All data are an average of at least duplicate measurements.
derivative 23 was well tolerated, demonstrating 2-fold improved
biochemical activity on the L858R-T790M construct. Com-
parable potency was observed with the 6-methyl analogue (26),
while slight potency improvements were noted with com-
pounds that had a methyl substitution at positions 5 and 7
(25 and 27). Conversely, as predicted by modeling, compound
24 was less potent, likely due to the methyl group pointing
toward the hinge binding domain of EGFR. Importantly, in all
cases, selectivity against WT EGFR was maintained, and hints
of activity on the oncogenic L858R mutant were observed with
23 and 27.
superior activity (IC50 = 0.006 μM), followed by 32e (IC50
=
0.02 μM; R-enantiomer). In addition, compound 32g was highly
potent across all mutant EGFR lines and displayed acceptable
WT EGFR selectivity (≥50-fold).
Intrigued by the initial observation that 7-methyl derivative
27 displayed a preferred substitution pattern, we prepared both
enantiomers of the 7-methyl analogue of 32g. Interestingly,
the R-enantiomer 43 demonstrated very good activity
across mutants cell lines with acceptable WT EGFR selectivity
(≥25-fold, Table 4), whereas the S-enantiomer was >200-fold
Unfortunately, all attempts to further improve the potency of
these ATP-competitive hits resulted in little success. A number
of amide and cyclohexanol replacements were prepared, none
of which resulted in potency improvements (data not shown).
Nonetheless, the exquisite WT EGFR selectivity observed in
the biochemical assays prompted us to investigate the possibility
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Table 2. Structure−Activity Relationship of the Linker Region Aimed at Identifying the Optimal Length and Geometry for
a
Covalent Bond to Cys797
compd
0 min
>50
90 min
>50
compd
0 min
90 min
>50
32a
32b
32c
32d
32e
32f
32g
32h
32i
32k
32l
>50
>50
>50
>50
>50
>50
>50
>50
0.05 0.008
>50
0.004 0.001
0.06 0.05
>50
>50
32m
32n
32o
32p
32q
32r
32s
>50
>50
>50
0.02 0.002
16.46 7.86
<0.002
>50
>37.67
0.83 0.14
>50
>32.77
0.004 0.002
0.01 0.003
>50
>50
22.31 24.75
>50
>50
6.81 1.88
>50
32j
>50
a
Biochemical IC50 (μM) assessed with or without a 90 min preincubation of compound and EGFR L858R-T790M enzyme at 1 mM ATP
concentrations using HTRF assay as described in the methods section. All data are an average of at least duplicate measurements.
WT EGFR constructs. The structures of 43 bound to either
WT EGFR or the T790M single mutant were determined
and exhibit an overall structure consistent with a kinase in the
active conformation, DFG-in and intact salt bridge to helix-C.
Comparison of the two 43-bound structures revealed nearly
identical binding site residues and compound conformations
(Figure 3B) and hence did not provide any clues that could
explain the observed selectivity toward the T790M mutant
versus the WT enzyme. However, examining the apo structures
of both the WT enzyme (PDB code 2gs2)39 and the T790M
mutant (PDB code 2jit)6 revealed a network of water molecules
bound to the hinge region of the WT enzyme that is not
present in the apo structure of the T790M mutant (Figure 3A).
In the WT apo structure, the Thr790 side chain mediates a
hydrogen bonding interaction to two water molecules that are
also coordinated to the backbone of several residues in the
hinge region (Ala743, Gln791, and Leu788). In addition, there
is a third water molecule coordinated to the backbone amide of
Met793. These three water molecules are absent in the apo
T790M mutant structure and are instead replaced by two
loosely coordinated water molecules that interact with the DFG
aspartic acid side chain, Asp855 (Figure 3A). In the compound-
bound structures (Figure 3B), analogue 43 is covalently linked
to Cys797 and makes a hydrogen bonding interaction to the
backbone amide NH of Met793. Binding of the compound to
the WT enzyme requires the displacement of the three strongly
coordinated water molecules present in this region. We hypo-
thesize that the cost of displacing these water molecules that are
Table 3. Activity Profiles of 32b, 32e, 32g, 32l, 32m, 32q,
32r, and Reference Compounds 5 and 6 from Patient-
Derived Cell Lines H1975 (EGFR L858R-T790M), H3255
(EGFR L858R), and HCC827 (EGFR Del E746-A750) and
the Immortalized Human Keratinocyte Cell Line HaCaT
a
(WT EGFR)
compd
H1975
H3255
>10000
HCC827
HaCaT
>10000
32b
525 126.4
24.7 5.3
6.1 2.0
1653 717
153 29.4
15.9 1.9
ND
b
32e
421 139.3
74.9 31.9
543 0.195
>3716
>10000
32g
4330 779
>10000
c
32l
41.4 8.7
409 338
134.8 29.3
181 45.3
2.5 0.7
c
32m
ND
>10000
32q
32r
5
1041 327
660 169.1
4.1 1.4
272.1 48.5
267.5 48.1
2.5 0.5
9.5 0.9
>10000
>10000
73.7 16.6
262.3 79.9
6
4.1 1.4
13.8 3.2
a
Target modulation IC50 values (nM) were determined after a 3 h
compound treatment to the cells. All data are an average of at
b
least duplicate measurements. Cellular IC50 values for the active
R-enantiomer, obtained after chiral HPLC separation (see Supporting
c
Information). Data for 32l and 32m are from engineered NIH-3T3
cells of the corresponding L858R-T790M, L858R, or WT-EGFR
mutants.
less potent across assays (H1975 IC50 = 401 nM, H3255 IC50
=
3125 nM, HCC827 IC50 = 863 nM, HaCaT IC50 ≥ 10 000 nM).
To gain confidence that these analogues were indeed binding
to EGFR L858R-T790M via a covalent bond to Cys797, we
performed crystallization studies with a variety of mutant and
E
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Table 4. In Vitro and in Vivo Profile of Select Mutant-Selective EGFR Inhibitors
43
44
45
46
47
a
H1975 IC50 (nM)
1.39 0.03
2.18 0.75
1.51 0.01
2.65 0.07
1.48 0.56
72.0 15.5
4.59
2.11 0.17
3.05 0.14
1.01 0.33
68.0 22.8
4.83
4.18 0.03
a
H3255 IC50 (nM)
14.84 0.91
45.90 15.78
6.11 0.08
a
HCC827 IC50 (nM)
3.93 2.03
12.45 5.91
1.52 0.08
a
HaCaT IC50 (nM)
249.6 94.0
351.0 240.4
3.13
160.6 12.8
H1975 LipE
3.65
<2
4.30
>175
>25
7
HT-sol., pH 6.8 (μM)
10.5
12.7
>175
a
CYP3A4 IC50
3.5
>25
3.4
83
22.2
>25
>25
a
CYP2D6 IC50
16.7
6.1
>25
a
CYP2C9 IC50
17.6
>25
>25
>25
34
b
human CLint
49
71
26
b
mouse CLint
245
54.0
808
6
233
96
62
66
mouse iv CL (mL min−1 kg−1
)
57.2
62.5
68.5
31.6
12789
60
c
mouse po AUC (h·nM)
mouse F (%)
4391
40
2839
3527
27
34
a
b
All IC50 data given in μM unless otherwise noted and are an average of at least duplicate measurements. CLint is intrinsic clearance in liver
c
microsomes, μL min−1 mg−1. AUC from 20 mg/kg oral dose to male balb/c mice. 43 and 45 were formulated in 0.5% methylcellulose and 0.5%
Tween 80 suspension. 44 was formulated in solution containing 75% PEG300 and 25% D5W (5% dextrose in water). 46 was formulated in solution
containing 5% ethanol, 30% PEG300, and 65% Solutol (20%), and 47 was formulated in a solution containing 5% ethanol, 25% PEG300, and 70%
D5W (5% dextrose in water).
human liver microsomes showed that oxidation of 7-methyl
groups and oxidation of acrylate groups are the two main
metabolic pathways (Figure S1 in Supporting Information).
To modulate the compound’s reactivity, we first investigated
a number of Michael acceptors similar to those reported in
the literature.37,38 Amino-substituted crotonamides had been
identified as optimal groups for a number of covalent pan-
EGFR inhibitors (e.g., afatinib, dacomitinib, neratinib). The
dimethylamino crotonamide analogue 44 retained comparable
activity to 43 in H1975, but it was consistently a few fold less
potent toward cell lines with oncogenic mutations (H3255 and
HCC827). Conversely, it demonstrated measurable improve-
ments in solubility and metabolic stability (Table 4) and resulted
in increased oral exposure and bioavailability in mice. Addition-
Figure 3. Comparison of apo and drug bound hinge region. (A)
Superposition of apo WT EGFR (white sticks, PDB code 2gs2)39 and
EGFR T790M mutant (cyan sticks, PDB code 2jit).6 Water molecules
associated with the WT structure are shown in gray spheres; those
associated with the T790M structure are shown in cyan spheres. Polar
interaction is depicted by magenta dotted line. (B) Superposition of
the apo WT EGFR (white sticks, PDB code 2gs2),39 WT EGFR-43
complex (green sticks, PDB code 5fed), and EGFR T790M-43
complex (cyan sticks, PDB code 5fee). Polar interactions are depicted
by yellow dotted line. In both drug bound structures, 43 is shown in
yellow sticks.
ally, the amino-substituted crotonamide resulted in improved
CYP inhibition profiles with IC50 > 10 μM for CYP3A4 and 2C9.
Next, we sought to improve the compound’s lipophilic
efficiency (LipE)41 and explore opportunities to increase activity
on the oncogenic mutations while maintaining good WT EGFR
selectivity. As the only difference between mutants resides at
the gatekeeper residue, we investigated the SAR of the trifluor-
obenzamide group. In general, (hetero)alkyl and (hetero)-
cycloalkyl groups led to complete loss in activity (data not
shown), whereas heteroaromatic groups were tolerated. In par-
ticular, the trifluoromethylpiridyl 45 demonstrated single digit nM
activity on H1975 and was nearly equipotent on the activating
mutations, resulting in a 10-fold potency improvement com-
pared to 44. It is important to note that increased potency was
also observed on HaCaT but to a lesser extent than the L858R
and L858R-T790M mutant cell lines. From docking and X-ray
cocrystallization structures (vide infra) we believe the reason
for improved activity on oncogenic and WT EGFR cell lines
results from the displacement of one of the conserved water
molecules by the pyridine group, with the nitrogen serving as
not present in the T790M apo structure may lead to the
observed selectivity profile of 43.
Although 43 demonstrated promising cellular activity, it had
very low aqueous solubility (<2 μM) and poor ADME and PK
properties. It is a moderate inhibitor of CYP3A4 and 2C9 with
an IC50 of ∼3.5 μM. It also has high intrinsic microsomal
clearance,40 especially in rodents, which correlated with high in
vivo clearance (CL) in mice. The high in vitro liver metabolism
and poor solubility resulted in only 6% oral bioavailability in
mice (Table 4). In vitro metabolism study in mouse, rat, and
F
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Figure 4. (a) Mouse H1975 abbreviated efficacy study. Compounds were dosed po q.d. for 5 days. Δ % body weights were recorded on day 5, and
percent mean tumor volume of the drug-treated group versus mean tumor volume of the control group (T/C) was measured at end of study (day 6).
(b) End of study free plasma exposure of compounds 45−47 for 50 mg/kg dose. Plasma PK samples were collected on day 5 at 30 min, 3 h, 7 h, and
24 h postdosing.
H-bond acceptor and interacting with Thr854. The difference
in the extent of increased activity toward oncogenic versus
WT EGFR cell lines may be due to the difference in ATP Km
between enzymes and because the oncogenic mutations are
constitutively active, whereas WT EGFR is in equilibrium between
active and inactive forms.6,42
GSH adducts (<5%). In contrast, afatinib showed high level of
GSH conjugate (60%). The much reduced reactivity of 43−47
toward GSH suggests a lower risk of nonspecific covalent
binding to proteins.
In Vivo Efficacy Studies for Compound Selection. An
integrated approach evaluating PK, tolerability, and efficacy was
incorporated into the in vivo studies for compound selection.
As such, 45−47 were profiled in a short-term in vivo efficacy
study using a H1975 mouse xenograft model and compared to
afatinib as positive control (Figure 4). In short, H1975 tumor
cells were inoculated subcutaneously in Foxn1 female nude
mice. Mice were then randomized into groups of 5 animals
and dosed orally once daily for 5 days at 50 and 20 mg/kg or
25 mg/kg once tumors reached 150−200 mm3. Afatinib was
dosed at maximum tolerated dose of 25 mg/kg. Body weight
was measured daily and change in tumor volume compared to
control (T/C) was assessed on days 4 and 6. Plasma samples
were collected after last dose on day 5, and exposures of each
compound were determined. Afatinib demonstrated tumor
growth inhibition with 38% T/C, consistent with what was
reported in the literature.7 All other compounds demonstrated
dose-dependent efficacy with near complete regression at the
highest dose tested (50 mg/kg), and free drug concentrations
in plasma were well above their in vitro IC50 values for at least
14 h within each dosing interval. Compound 47 was very well
tolerated with body weight gains comparable to vehicle at both
doses. On the other hand, 45 and 46 displayed up to 5% body
weight loss.
Although solubility was slightly improved, 45 was still
relatively insoluble (12.7 μM HT-sol. at pH 6.8). Replacing the
trifluoromethyl functionality by a methyl group resulted in 46, a
compound with comparable potency across mutants EGFR and
selectivity against WT EGFR but with much improved water
solubility (>175 μM HT-sol. at pH 6.8). Both derivatives had
relatively high in vivo clearance in mice and low to moderate
oral bioavailability (Table 4). As mentioned above, one of the
main metabolic soft spots was the 7-methyl group. Replacing
this residue by a chloro group led to analogue 47. Gratifyingly,
47 had single digit activity on the mutant cell lines (4, 6, 2 nM
IC50 in H1975, H3255, and HCC827, respectively) with less
than 2-fold loss of potency on H3255 compared to 46. At the
same time, it displayed reduced activity on HaCaT (0.18 μM),
providing similar WT EGFR selectivity (∼36-fold). Impor-
tantly, 47 demonstrated improved ADME and PK properties as
compared to other compounds.
The chemical reactivity of 43−47 and potential for nonspecific
covalent binding to proteins were assessed in comparison to
afatinib. Upon incubation for 1 h with 10 mM glutathione
(GSH) or with human liver microsomes in the presence of
NADPH and GSH, compounds 43−47 formed only minor
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Article
nominated as our clinical candidate after confirming adequate
safety profile (Tables S2−S4).
Table 5. In Vivo PK Parameters of 47 in Mouse, Rat, and Dog
mouse
rat
dog
Compound 47 and Close Analogue 46 Covalently
Bind to Cys797. Attempts to cocrystallize 47 with various
EGFR constructs have not yet been successful. Instead, we
obtained cocrystals of the close analogue 46 with the EGFR
T790M construct that confirmed the covalent bond to Cys797
(Figure 5a). The covalent nature of 47 with EGFR L858R and
EGFR T790M-L858R mutant constructs was assessed by mass
spectrometry (Figure 5b,c). Incubation of 47 with both EGFR
constructs resulted in one main product per protein that had
an average mass 495 Da higher than the unmodified protein.
This was consistent with addition of one molecule of 47 to the
protein on average. LC/MS/MS of the enzymatic digests iden-
tified adducts of 47 that localized to Cys797 and Cys775. Spec-
tral counting was used for relative comparison of adduction; 96%
of MS/MS acquired for Cys797 was identified as modified with
47 (96 out of 100 spectra), while only 0.3% of Cys775 was mod-
ified (1 out of 324 spectra). The relative number of 47-modified
to total spectra for the two cysteines suggested that Cys797 is the
main site of 47 modification.
a
a
a
a
b
c
parameter
iv
po
20
iv
po
10
iv
po
10
dose (mg/kg)
CL (mL min−1 kg−1
Vss (L/kg)
MRT (h)
5
3
1
)
31.6
2.8
15.7
2.4
43.7
35.2
13.8
783
1.5
2.6
AUC (h·nM)
Cmax (nM)
Tmax (h)
5343
12786
2938
0.83
60
6527
11951
987
3.0
5230
435
2.3
F (%)
55
67
a
Free base formulated in a solution of 5% ethanol, 25% PEG300, and
70% D5W (5% dextrose in water). HCl salt formulated in a solution
of 100% D5W (5% dextrose in water). HCl salt formulated in a
solution of 0.5% (w/v) aqueous methylcellulose and 0.5% Tween 80.
b
c
For this reason, compound 47 was further profiled in pre-
clinical in vivo PK studies in mouse, rat, and dog (Table 5).
The compound showed moderate volume of distribution and
low to moderate clearance in rodents (30% and 35% of rat and
mouse liver blood flow, respectively). In the dog, 47 showed
high clearance and high volume of distribution. The high
clearance in dog was consistent with higher in vitro dog micro-
somal clearance and higher fraction unbound in dog plasma
as compared to other species. In contrast, the human liver
microsomal CL of 47 is relatively low, and the compound is
predicted to have favorable human PK profile with low to
moderate clearance and good oral exposure.
CONCLUSIONS
■
We have described the identification of a novel, covalent,
mutant-selective EGFR inhibitor with nearly equipotent activity
on both oncogenic (L858R and ex19del) and T790M-resistant
mutations and good selectivity over WT EGFR. From a high-
throughput screen, we converted a reversible mutant-selective
EGFR hit (7) into an irreversible inhibitor lead (43) by carefully
investigating the linker length and geometry necessary to engage
On the basis of these results, analogue 47 was selected for
further profiling as described by Jia et al.43 and was ultimately
Figure 5. Compounds 46 and 47 bind irreversibly to EGFR constructs: (a) cocrystal structure of EGFR T790M-46 complex; (b) deconvoluted mass
spectrum of EGFR L858R-T790M after in vitro incubation with 47; (c) MS/MS of EGFR peptide covalently modified with 47 at Cys797.
H
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Journal of Medicinal Chemistry
Article
by chiral HPLC or chiral SFC and demonstrated to have at least
95% ee.
Cys797. Molecular docking and metabolite identification experi-
ments were used to increase potency on cell lines with oncogenic
mutations while retaining adequate WT EGFR selectivity.
Finally, we employed an abbreviated in vivo efficacy study to
select 47 for further profiling and ultimately as the clinical
candidate. Compound 47 is currently being tested in numerous
clinical trials,29−31 and it has already demonstrated encourag-
ing efficacy in NSCLC patients with T790M mutations. As
previously disclosed, efficacy was seen across all dose levels with
an overall response rate of 60% and a disease control rate of
93% in early assessments.44 Full results from clinical trials will be
reported in due course.
General Procedure for the Synthesis of Compounds 13−17,
29a−o, 29r, 35, and 36. A solution of the desired 1-fluoro-2-
nitroarene 8−12 or 33 (1−1.5 equiv) in a suitable solvent, such as
DMF or DMA (0.2−0.8 M), or neat was treated with the requisite
amine (1 equiv) and a base such as DIPEA or K2CO3 (1.5−4 equiv),
and the reaction mixture was warmed to 110−140 °C for 2−16 h. The
reaction mixture was then cooled to room temperature and partitioned
between water and EtOAc. The aqueous layer was extracted twice
more with EtOAc, and the pooled extracts were washed consecutively
with water and brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by silica gel chromato-
graphy to afford the desired nitroanilines (13−17, 29a−o, 29r, 35,
and 36).
EXPERIMENTAL PROCEDURES
trans-4-((2-Nitrophenyl)amino)cyclohexan-1-ol (13). Yield, 69%.
■
trans-4-((3-Methyl-2-nitrophenyl)amino)cyclohexan-1-ol
Unless otherwise noted, materials were obtained from commercial
suppliers and were used without further purification. Removal of
solvent under reduced pressure or concentration refers to distillation
1
(14). Yield, 54%. H NMR (DMSO-d6, 400 MHz): δ 11.98 (s, 1H),
7.26 (t, J = 8.3 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.54 (d, J = 7.3 Hz,
1H), 6.08 (d, J = 7.9 Hz, 1H), 4.58 (m, 1H), 3.42 (m, 1H), 2.29
(s, 3H), 2.15 (m, 2H), 1.95 (m, 2H), 1.82 (m, 2H), 1.25 (m, 2H).
MS calculated for C13H17N2O3 (M − H)− 249.13, found 249.0.
trans-4-((4-Methyl-2-nitrophenyl)amino)cyclohexan-1-ol
using Buchi rotary evaporator attached to a vacuum pump (3 mmHg).
̈
Products obtained as solids or high boiling oils were dried under
vacuum (1 mmHg).
Purification of compounds by preparative RP-HPLC was achieved
using a Waters autopurification system consisting of a 2767 autosampler/
fraction collector, a 2525 binary gradient module, a 2487 UV detector,
and a ZQ mass spectrometer. Compounds were purified using flow
rate of 30 mL/min with a 50 mm × 20 mm i.d. Ultra 120 5 μm C18Q
column (Peeke Scientific, Novato, CA). A 7.5 min linear gradient
from 10% solvent A (acetonitrile with 0.035% trifluoroacetic acid) in
solvent B (water with 0.05% trifluoroacetic acid) to 30−90% A was
used, followed by a 2.5 min hold at 90% A. Silica gel chromatography
was performed by CombiFlash (separation system Sg. 100c, ISCO).
Elemental analyses were carried out by Midwest Microlabs LLC,
1
(15). Yield, 35%. H NMR (CDCl3, 400 MHz): δ 7.97 (s, 1H), 7.25
(d, J = 8.3 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 3.82 (m, 1H), 3.45
(m, 1H), 2.25 (s, 3H), 2.21 (m, 2H), 2.05 (m, 2H), 1.52 (m, 4H).
MS calculated for C13H17N2O3 (M − H)− 249.13, found 248.9.
trans-4-((5-Methyl-2-nitrophenyl)amino)cyclohexan-1-ol
(16). Yield, 68%. 1H NMR (CDCl3, 400 MHz): δ 8.05 (d, J = 7.5 Hz,
1H), 6.61 (s, 1H), 6.42 (d, J = 7.5 Hz, 1H), 3.78 (m, 1H), 3.45 (m,
1H), 2.35 (s, 3H), 2.15 (m, 4H), 1.55 (m, 4H). MS calculated for
C13H19N2O3 (M + H)+ 251.13, found 251.3.
trans-4-((2-Methyl-6-nitrophenyl)amino)cyclohexan-1-ol
1
(17). Yield, 68%. H NMR (DMSO-d6, 400 MHz): δ 7.78 (d, J =
1
Indianapolis, IN. H NMR spectra were recorded on Bruker XWIN-
7.4 Hz, 1H), 7.45 (d, J = 6.9 Hz, 1H), 6.89 (t, J = 7.8 Hz, 1H), 6.13 (d,
J = 9.8 Hz, 1H), 4.54 (d, J = 4.4 Hz, 1H), 3.38 (m, 1H), 3.33 (m, 1H),
2.32 (s, 3H), 1.77 (m, 4H), 1.19 (m, 4H). MS calculated for
C13H19N2O3 (M + H)+ 251.13, found 251.2.
tert-Butyl (2-((4-Methyl-2-nitrophenyl)amino)ethyl)-
carbamate (29a). Yield, 43%.
tert-Butyl (3-((4-Methyl-2-nitrophenyl)amino)propyl)-
carbamate (29b). Yield, 45%. MS calculated for C15H24N3O4
(M + H)+ 310.18, found 310.2.
NMR (400 MHz) spectrometer. Proton resonances are reported in
parts per million (ppm) downfield from tetramethylsilane (TMS).
1H NMR data are reported as multiplicity (s, singlet; d, doublet;
t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets;
br s, broad singlet). For spectra obtained in CDCl3, DMSO-d6,
CD3OD, CD3CN the residual protons (7.27, 2.50, 3.31, and 1.94 ppm,
respectively) were used as the internal reference. In general, peaks
associated with the azepane and (hetero)aromatic rings display two
distinct sets of peaks (approximately 50:50 intensity) in spectra
acquired at 300 K (atropoisomers). The peaks collapse to a single set at
higher temperature (383 K) (see Figures S2 and S3 for 1H and 13C NMR
spectra of 47). Optical rotations were measured at the sodium D line,
using an ATAGO POLAX-2L polarimeter and a 100 mm cell at 23 °C
and are reported as follows: concentration (c = g/100 mL), solvent.
Purity of Compounds. All final compounds were analyzed with a
Waters ZQ 2000 LC/MS system, which used an Agilent binary pump
or Shimadzu LC10 and Agilent photodiode array detector (PDA)
along with a Sedere 75 evaporative light scattering detector (ELSD).
Samples were injected from 96-well or 384-well plates with a Leap
Technologies HTS Pal autosampler. The mobile phases used were
(A) H2O + 0.05% TFA and (B) acetonitrile + 0.05% TFA. A gradient
HPLC method with flow of 1.0 mL/min started at 5% B, with a hold
of 0.1 min before a linear increase to 90% B at 2.60 min. At 2.71 min
the B mobile phase was increased to 100% and held until 2.98 min
before returning to 5% B at 2.99 min. Total run time was 3.0 min.
The column was re-equilibrated in the time between injections.
The column used was a Waters Atlantis dC18 2.1 mm × 30 mm, 3 μm.
The mass spectrometer was operated in positive mode, with a spray
voltage of 3.2 kV and cone voltage of 30 V. The source and desol-
vation temperatures were 130 and 400 °C, respectively, with 600 L/h
of nitrogen desolvation flow. The progress of reactions and the purity
of products were measured using 254 and 214 nm wavelengths and
electrospray ionization (ESI) positive or negative mode. Mass spectra
were obtained in ESI positive or negative mode. All of the final
compounds were found to be >95% when analyzed at either the
254 or 214 nm wavelengths. Chiral compounds were also analyzed
tert-Butyl 3-((4-Methyl-2-nitrophenyl)amino)azetidine-1-
1
carboxylate (29c). Yield, 56%. H NMR (DMSO-d6, 400 MHz):
δ 8.05 (s, 1H), 8.01 (s, 1H), 7.30 (d, J = 7.9 Hz, 1H), 6.44 (d, J =
8.3 Hz, 1H), 4.38 (m, 2H), 4.32 (m, 1H), 3.87 (m, 2H), 2.29 (s, 3H),
1.46 (s, 9H). MS calculated for C15H21N3O4 (M + H)+ 308.15, found
308.3.
tert-Butyl 3-((4-Methyl-2-nitrophenyl)amino)pyrrolidine-1-
carboxylate (29d). Yield, 66%.
tert-Butyl 3-((4-Methyl-2-nitrophenyl)amino)piperidine-1-
carboxylate (29e). Yield, 65%. MS calculated for C17H26N3O4
(M + H)+ 336.19, found 336.1.
tert-Butyl 4-((4-Methyl-2-nitrophenyl)amino)piperidine-1-
carboxylate (29f). Yield, 78%.
tert-Butyl 3-((4-Methyl-2-nitrophenyl)amino)azepane-1-car-
boxylate (29g). Yield, 93%. MS calculated for C18H28N3O4 (M + H)+
350.21, found 350.1.
tert-Butyl 4-((4-Methyl-2-nitrophenyl)amino)azepane-1-car-
1
boxylate (29h). Yield, 88%. H NMR (CDCl3, 400 MHz): δ 8.04
(d, J = 7.3 Hz, 1H), 7.98 (s, 1H), 6.70 (d, J = 8.8 Hz, 1H), 3.21−3.75
(m, 5H), 2.31 (s, 3H), 1.61−2.05 (m, 6H), 1.53 (s, 9H). MS
calculated for C18H28N3O4 (M + H)+ 350.20, found 350.2.
tert-Butyl (3-((4-Methyl-2-nitrophenyl)amino)cyclopentyl)-
carbamate (29i). Yield, 54%. MS calculated for C17H26N3O4
(M + H)+ 336.19, found 336.2.
tert-Butyl cis-(2-((4-Methyl-2-nitrophenyl)amino)-
cyclohexyl)carbamate (29j). Yield, 53%. 1H NMR (CDCl3, 400
MHz): δ 8.31 (d, J = 8.3 Hz, 1H), 7.98 (s, 1H), 7.22 (d, J = 8.8 Hz,
1H), 6.85 (d, J = 8.8 Hz, 1H), 4.55−4.75 (m, 1H), 3.98−4.18 (m, 1H),
I
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3.75−3.95 (m, 1H), 2.25 (s, 3H), 1.45−1.81 (m, 8H), 1.39 (s, 9H).
MS calculated for C18H28N3O4 (M + H)+ 350.20, found 350.2.
tert-Butyl trans-(2-((4-Methyl-2-nitrophenyl)amino)-
cyclohexyl)carbamate (29k). Yield, 54%. 1H NMR (CDCl3,
400 MHz): δ 8.17 (d, J = 7.4 Hz, 1H), 7.96 (s, 1H), 7.24 (d, J =
8.3 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 4.4−4.5 (m, 1H), 3.31−3.75 (m,
2H), 2.25 (s, 3H), 2.05−2.21 (m, 4H), 1.22−1.85 (m, 4H), 1.25 (s,
9H). MS calculated for C18H28N3O4 (M + H)+ 350.20, found 350.2.
tert-Butyl cis-(3-((4-Methyl-2-nitrophenyl)amino)-
cyclohexyl)carbamate (29l). Yield, 46%. 1H NMR (DMSO-d6,
400 MHz): δ 7.86 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.38 (d, J =
8.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 3.66−
3.64 (m, 1H), 3.41−3.37 (m, 1H), 2.22 (s, 3H), 2.11 (d, J = 11.2 Hz,
1H), 1.95 (d, J = 12.0 Hz, 1H), 1.80−1.71 (m, 2H), 1.37 (s, 9H),
1.23−1.05 (m, 1H). MS calculated for C18H28N3O4 (M + H)+ 350.21
found 350.1.
tert-Butyl trans-(3-((4-Methyl-2-nitrophenyl)amino)-
cyclohexyl)carbamate (29m). Yield, 34%. 1H NMR (CDCl3,
400 MHz): δ 7.86 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.38 (d, J =
8.3 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 3.55−
3.75 (m, 1H), 3.31−3.42 (m, 1H), 2.22 (s, 3H), 1.62−2.19 (m, 4H),
1.45 (s, 9H), 1.05−1.25 (m, 4H). MS calculated for C18H28N3O4
(M + H)+ 350.20, found 350.1.
tert-Butyl cis-(4-((4-Methyl-2-nitrophenyl)amino)-
cyclohexyl)carbamate (29n). Yield, 62%. 1H NMR (CDCl3,
400 MHz): δ 8.25 (br s, 1H), 8.15 (s, 1H), 7.25 (d, J = 9.3 Hz,
1H), 6.77 (d, J = 8.8 Hz, 1H), 4.45−4.61 (m, 1H), 3.51−3.79 (m,
2H), 2.26 (s, 3H), 1.65−1.95 (m, 8H), 1.45 (s, 9H).
tert-Butyl trans-(4-((4-Methyl-2-nitrophenyl)amino)-
cyclohexyl)carbamate (29o). Yield, 78%. 1H NMR (DMSO-d6,
400 MHz): δ 7.85 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.35 (d, J =
7.5 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H), 3.41−
3.58 (m, 1H), 3.19−3.31 (m, 1H), 2.21 (s, 3H), 1.75−2.15 (m, 4H),
1.41 (s, 9H), 1.21−1.45 (m, 4H).
tert-Butyl (2-((4-Methyl-2-nitrophenyl)amino)phenyl)-
carbamate (29p). Yield, 22%. MS calculated for C18H20N3O4
(M − H)− 342.15, found 342.1.
tert-Butyl (4-((4-Methyl-2-nitrophenyl)amino)phenyl)-
carbamate (29s). Yield, 31%. 1H NMR (DMSO-d6, 400 MHz):
δ 9.41 (br s, 1H), 9.24 (s, 1H), 7.91 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H),
7.31 (dd, J = 9.8 Hz, 2.0 Hz, 1H), 7.21 (d, J = 8.8 Hz, 2H), 6.99 (d, J =
8.8 Hz, 1H), 2.25 (s, 3H), 1.47 (s, 9H).
General Procedure for the Synthesis of Compounds 18−22,
30a−s, and 37. Synthesis of trans-4-(2-Amino-1H-benzo[d]-
imidazol-1-yl)cyclohexan-1-ol (18). A solution of 13 (350 mg,
1.48 mmol) in methanol (15 mL) was degassed with nitrogen, treated
with Pd/C (55 mg, 15.7 mol %), and then stirred under H2
atmosphere for 1 h. The reaction mixture was filtered through Celite,
the Celite rinsed with MeOH (40 mL), and the filtrate concentrated
under reduced pressure to afford a crude residue, which was dissolved
in water (1 mL) and added to a solution of cyanogen bromide (185 mg,
1.75 mmol) in MeOH/MeCN (3:1, 4 mL). The reaction mixture was
then warmed to 50 °C for 2 h, cooled to 0 °C, treated with saturated
aqueous Na2CO3 to pH 10, and then stirred for 30 min while warming
to room temperature. The resulting suspension was filtered and dried
under high vacuum to afford 18 (280 mg, 77%). 1H NMR (DMSO-d6,
400 MHz): δ 7.33 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 7.4 Hz, 1H), 6.91
(t, J = 7.4 Hz, 1H), 6.82 (t, J = 7.8 Hz, 1H), 6.34 (br s, 2H), 4.58−4.67
(m, 1H), 4.15−4.24 (m, 1H), 3.55−3.73 (m, 1H), 2.11−2.35 (m, 2H),
1.85−2.01 (m, 2H), 1.31−1.51 (m, 2H).
trans-4-( 2-Amino-4-methyl-1H-benzo[d]imidazol-1-yl)-
cyclohexan-1-ol (19). Yield, 26%.
trans-4-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
1
cyclohexan-1-ol (20). Yield, 91%. H NMR (DMSO-d6, 400 MHz):
δ 7.17 (d, J = 7.8 Hz, 1H), 6.91 (s, 1H), 6.63 (d, J = 8.3 Hz, 1H),
6.20 (br s, 2H), 4.65 (m, 1H), 4.15 (m, 1H), 2.65 (m, 1H), 2.28
(s, 3H), 2.21 (m, 2H), 1.85 (m, 2H), 1.65 (m, 2H), 1.41 (m, 2H).
MS calculated for C14H20N3O (M + H)+ 246.15, found 246.2.
trans-4-(2-Amino-6-methyl-1H-benzo[d]imidazol-1-yl)-
tert-Butyl Methyl(3-((4-methyl-2-nitrophenyl)amino)-
phenyl)carbamate (29r). Yield, 33%. 1H NMR (DMSO-d6,
400 MHz): δ 9.17 (s, 1H), 7.93 (s, 1H), 7.38−7.05 (m, 6H), 3.18
(s, 3H), 2.27 (s, 3H), 1.39 (s, 9H). MS calculated for C19H22N3O4
(M − H)− 356.16, found 356.2.
1
cyclohexan-1-ol (21). Yield, 67%. H NMR (DMSO-d6, 400 MHz):
δ 7.13 (s, 1H), 6.98 (d, J = 7.9 Hz, 1H), 6.71 (d, J = 7.9 Hz, 1H),
6.18 (br s, 2H), 4.68 (br s, 1H), 4.15 (m, 1H), 3.65 (m, 1H), 2.33
(s, 3H), 2.11 (m, 2H), 1.95 (m, 2H), 1.66 (m, 2H), 1.42 (m, 2H).
MS calculated for C14H20N3O (M + H)+ 246.15, found 246.1.
trans-4-(2-Amino-7-methyl-1H-benzo[d]imidazol-1-yl)-
(R)-tert-Butyl 3-((2-Methyl-6-nitrophenyl)amino)azepane-1-
carboxylate (35). Yield, 60%. 1H NMR (CDCl3, 400 MHz):
δ 7.93−7.87 (m, 1H), 7.37−7.31 (m, 1H), 6.91−6.79 (m, 1.5H),
6.50−6.47 (m, 0.5H), 3.88−3.76 (m, 2H), 3.57−3.52 (m, 1H), 3.22−
2.78 (m, 2H), 2.43−2.41 (m, 3H), 1.92−1.60 (m, 5H), 1.47−1.38 (m,
10H). MS calculated for C18H28N3O4 (M + H)+ 350.20, found 350.0.
1
cyclohexan-1-ol (22). Yield, 33%. H NMR (DMSO-d6, 400 MHz):
δ 6.95 (d, J = 7.9 Hz, 1H), 6.80 (t, J = 7.3 Hz, 1H), 6.61 (d, J = 7.4 Hz,
1H), 5.97 (br s, 2H), 4.55 (m, 1H), 3.63 (m, 1H), 2.57 (s, 3H), 2.26
(m, 2H), 1.95 (m, 2H), 1.72 (m, 2H), 1.35 (m, 2H). MS calculated for
C14H20N3O (M + H)+ 246.15, found 246.1.
23
[α]D +71.42° (c 0.420, MeOH).
(R)-tert-Butyl 3-((2-Chloro-6-nitrophenyl)amino)azepane-1-
carboxylate (36). Yield, 54%. 1H NMR (CDCl3, 400 MHz):
δ 8.00−7.91 (m, 1H), 7.58−7.49 (m, 1H), 7.02−6.51 (m, 2H),
4.31−4.03 (m, 1H), 3.84−2.98 (m, 4H), 1.98−1.60 (m, 5H), 1.46−
1.39 (m, 10H). MS calculated for C17H25ClN3O4 (M + H)+ 370.15,
tert-Butyl (2-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
ethyl)carbamate (30a). Yield, 80%. 1H NMR (DMSO-d6, 400 MHz):
δ 6.98 (d, J = 7.5 Hz, 1H), 6.94 (s, 1H), 6.85 (m, 1H), 6.65 (d, J =
7.5 Hz, 1H), 6.21 (br s, 2H), 3.98 (m, 2H), 3.18 (m, 2H), 2.31 (s, 3H),
1.35 (s, 9H).
tert-Butyl (3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
propyl)carbamate (30b). Yield, 84%. MS calculated for C16H25N4O2
(M + H)+ 305.20, found 305.2.
23
found 370.10. [α]D −95.06° (c 0.263, MeOH).
General Procedure for the Synthesis of Compounds 29p−q
and 29s. Synthesis of tert-Butyl (3-((4-Methyl-2-nitrophenyl)-
amino)phenyl)carbamate (29q). A 100 mL round-bottom flask was
charged with 1-fluoro-4-methyl-2-nitrobenzene (3.4 g, 22.08 mmol)
and 1,3-phenylenediamine (2.0 g, 18.40 mmol), and the neat mixture
was warmed to 150 °C for 3 h. The reaction mixture was dissolved
in CH2Cl2 and purified by silica gel chromatography: 0−30% EtOAc/
hexanes gradient. The resulting N1-(4-methyl-2-nitrophenylbenzene-
1,3-diamine was dissolved in 1,4-dioxane (10 mL) and treated with
(Boc)2O (4.66 g, 21.3 mmol) and Et3N (4.4 mL, 31.8 mmol). The red
solution was stirred for 24 h at room temperature and then diluted
with CH2Cl2 (100 mL) and washed with water. The resulting organic
layer was separated, dried over anhydrous Na2SO4, and concentrated
under reduced pressure to a viscous red residue, which was purified
by silica gel chromatography (0−30% EtOAc/hexanes gradient) to
afford tert-butyl (3-((4-methyl-2-nitrophenyl)amino)phenyl)carbamate
29q (1.5 g, 20%). MS calculated for C18H20N3O4 (M − H)− 342.15,
found 342.1.
tert-Butyl 3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
1
azetidine-1-carboxylate (30c). Yield, 81%. H NMR (DMSO-d6,
400 MHz): δ 7.12 (d, J = 7.8 Hz, 1H), 7.00 (s, 1H), 6.74 (d, J =
7.8 Hz, 1H), 6.31 (br s, 2H), 5.24 (m, 1H), 4.31 (m, 4H), 2.32
(s, 3H), 1.45 (s, 9H).
tert-Butyl 3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
1
pyrrolidine-1-carboxylate (30d). Yield, 78%. H NMR (DMSO-
d6, 400 MHz): δ 7.04 (d, J = 7.8 Hz, 1H), 6.96 (s, 1H), 6.66 (d, J =
7.9 Hz, 1H), 6.34 (br s, 2H), 4.98 (m, 1H), 3.62 (m, 4H), 2.21
(m, 1H), 2.29 (s, 3H), 2.13 (m, 1H), 1.45 (s, 9H).
tert-Butyl 3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
piperidine-1-carboxylate (30e). Yield, 64%. 1H NMR (CDCl3,
400 MHz): δ 7.24 (s, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.85 (d, J =
8.0 Hz, 1H), 4.64 (br s, 2H), 4.17 (t, J = 14.8 Hz, 2H), 3.99−3.93 (m,
1H), 3.32 (d, J = 11.6 Hz, 1H), 2.79 (t, J = 12.4 Hz, 1H), 2.41 (s, 3H),
2.38−2.37 (m, 1H), 2.34 (d, J = 3.2 Hz, 1H), 1.91 (d, J = 13.6 Hz, 1H),
J
DOI: 10.1021/acs.jmedchem.5b01985
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.69−1.61 (m, 1H), 1.47 (s, 9H). MS calculated for C18H27N4O2
(M + H)+ 331.21, found 331.0.
1H), 6.68−6.19 (m, 1H), 6.19 (s, 2H), 3.26 (s, 3H), 2.32 (s, 3H),
1.42 (s, 9H). MS calculated for C20H25N4O2 (M + H)+ 353.20, found
353.0.
tert-Butyl 4-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
1
piperidine-1-carboxylate (30f). Yield, 72%. H NMR (DMSO-d6,
tert-Butyl (4-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
phenyl)carbamate (30s). Yield, 80%. 1H NMR (DMSO-d6,
400 MHz): δ 9.64 (br s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.38 (m, 2H),
7.07 (s, 1H), 6.61−6.80 (m, 4H), 2.38 (s, 3H), 1.45 (s, 9H).
tert-Butyl (R)-3-(2-Amino-7-methyl-1H-benzo[d]imidazol-1-
400 MHz): δ 7.55 (d, J = 7.5 Hz, 1H), 7.41 (m, 2H), 7.07 (s, 1H),
6.86 (d, J = 8.3 Hz, 1H), 4.2−4.4 (m, 1H), 3.82−4.02 (m, 2H), 3.31−
3.3.45 (m, 1H), 2.79−3.01 (m, 1H), 2.34 (s, 3H), 2.19−2.38 (m, 1H),
1.78−1.99 (m, 2H), 1.43−1.61 (m, 1H), 1.1 (s, 9H). MS calculated for
C18H27N4O2 (M + H)+ 331.21, found 331.3.
1
yl)azepane-1-carboxylate (37). Yield, 86%. H NMR (DMSO-d6,
tert-Butyl 3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
400 MHz): δ 6.99 (dd, J = 7.8, 1.2 Hz, 1H), 6.83 (t, J = 7.6 Hz, 1H),
6.72−6.56 (m, 1H), 6.07 (d, J = 20.8 Hz, 2H), 4.84−4.58 (m, 1H),
3.85−3.61 (m, 2H), 3.59−3.47 (m, 1H), 3.36 (dt, J = 16.1, 5.8 Hz,
1H), 2.59 (d, J = 9.2 Hz, 3H), 2.31−2.14 (m, 1H), 1.97−1.69
(m, 5H), 1.44 (s, 5H), 1.32 (s, 4H). MS calculated for C19H29N4O2
1
azepane-1-carboxylate (30g). Yield, 91%. H NMR (DMSO-d6,
400 MHz): δ 7.15−7.12 (m, 1H), 6.93 (s, 1H), 6.66 (d, J = 7.6 Hz,
1H), 6.26 (s, 2H), 4.29 (br s, 1H), 3.81−3.75 (m, 1H), 3.55−3.36
(m, 3H), 2.29 (s, 3H), 2.08 (s, 2H), 1.86−1.68 (m, 5H), 1.40 (s, 9H).
MS calculated for C19H29N4O2 (M + H)+ 345.23, found 345.1.
tert-Butyl 4-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
azepane-1-carboxylate (30h). Yield, 94%. MS calculated for
C19H29N4O2 (M + H)+ 345.22, found 345.1.
tert-Butyl (3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
cyclopentyl)carbamate (30i). Yield, 77%. 1H NMR (DMSO-d6,
400 MHz): δ 12.55 (br s, 1H), 7.93 (br s, 2H), 7.42 (d, J = 8.2 Hz,
1H), 7.21 (d, J = 1.4 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 4.88−4.71
(m, 1H), 3.99−3.79 (m, 1H), 2.40 (s, 3H), 2.25−1.81 (m, 6H), 1.40
(s, 9H).
23
(M + H)+ 345.22, found 345.2. [α]D +74.07° (c 0.272, MeOH).
tert-Butyl (R)-3-(2-Amino-7-chloro-1H-benzo[d]imidazol-1-
yl)azepane-1-carboxylate (38). A mixture of 36 (7.5 g, 19.5 mmol)
and Zn (12.8 g, 195 mmol) in AcOH (22 mL) was stirred at room
temperature for 2 h. The reaction was basified with saturated aqueous
Na2CO3, filtered and the filtrate extracted with EtOAc (3 × 80 mL).
The combined organic extracts were washed with brine, dried over
Na2SO4, and concentrated in vacuo to afford tert-butyl (R)-3-((2-
amino-6-chlorophenyl)amino)azepane-1-carboxylate (6.62 g, 19.48
mmol), which was immediately dissolved in MeOH (200 mL) and
treated with a solution of cyanogen bromide (3.09 g, 29.22 mmol)
in H2O (75 mL) and MeCN (75 mL). The reaction mixture was
stirred at 50 °C for 6 h, concentrated under reduced pressure, basified
with saturated aqueous Na2CO3, and then extracted with EtOAc
(3 × 90 mL). The combined organic extracts were dried over Na2SO4
and concentrated under reduced pressure. The crude material was
purified by HPLC to afford 38 (3.3 g, 46%). 1H NMR (CDCl3,
400 MHz): δ 7.34−7.26 (m, 1H), 7.04−6.97 (m, 2H), 6.05−5.85 (m,
1H), 5.84−5.72 (m, 1H), 5.50−5.37 (m, 0.5H), 5.10−4.80 (m, 0.5H),
4.41−4.23 (m, 1H), 4.09−3.96 (m, 0.5H), 3.94−3.81 (m, 1H), 3.76−
3.57 (m, 1H), 3.22−3.14 (m, 0.5H), 2.84−2.63 (m, 1H), 2.34−2.17
(m, 1H), 2.07−1.84 (m, 1H), 1.82−1.64 (m, 2H), 1.53 (s, 9H), 1.48−
1.37 (m, 1H). MS calculated for C18H26ClN4O2 (M + H)+ 365.17,
tert-Butyl cis-(2-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-
yl)cyclohexyl)carbamate (30j). Yield, 77%. 1H NMR (CDCl3,
400 MHz): δ 7.19 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.82 (d, J =
7.8 Hz, 1H), 4.81 (br s, 2H), 4.21−4.39 (m, 1H), 4.01−4.19 (m, 1H),
2.47−2.61 (m, 1H), 2.38 (s, 3H), 1.35−2.12 (m, 6H), 1.05 (s, 9H).
MS calculated for C19H29N4O2 (M + H)+ 345.22, found 345.0.
tert-Butyl trans-(2-(2-Amino-5-methyl-1H-benzo[d]imidazol-
1
1-yl)cyclohexyl)carbamate (30k). Yield, 75%. H NMR (CDCl3,
400 MHz): δ 7.32 (d, J = 7.3 Hz, 1H), 7.26 (s, 1H), 7.02 (d, J =
7.8 Hz, 1H), 4.81−4.95 (m, 1H), 4.41−4.50 (m, 1H), 4.01−4.15 (m,
1H), 3.61 (m, 1H), 3.15−3.31 (m, 1H), 2.41 (s, 3H), 1.62−2.19 (m,
4H), 1.11−1.38 (m, 4H), 1.42 (s, 9H). MS calculated for C19H29N4O2
(M + H)+ 345.22, found 345.0.
tert-Butyl cis-(3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-
1
23
yl)cyclohexyl)carbamate (30l). H NMR (DMSO-d6, 400 MHz):
found 365.1. [α]D +71.17° (c 0.281, MeOH).
δ 7.12 (d, J = 8.0 Hz, 1H), 6.93 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.66
(t, J = 6.8 Hz, 1H), 6.26 (s, 2H), 4.21 (dd, J = 3.6 Hz, 1H), 3.52−3.31
(m, 1H), 2.28 (s, 3H), 2.07−1.94 (m, 2H), 1.81 (d, J = 10.4 Hz, 2H),
1.66 (d, J = 11.6 Hz, 1H), 1.36 (s, 9H), 1.28−1.20 (m, 3H).
MS calculated for C19H29N4O2 (M + H)+ 345.23, found 345.0.
tert-Butyl trans-(3-(2-Amino-5-methyl-1H-benzo[d]imidazol-
1-yl)cyclohexyl)carbamate (30m). Yield, 95%.
General Procedure for the Synthesis of Compounds 23−27,
31a−s, and 39−42. Synthesis of N-(1-(trans-4-Hydroxycyclo-
hexyl)-1H-benzo[d]imidazol-2-yl)-3-(trifluoromethyl)-
benzamide (23). To a solution of 18 (100 mg, 0.433 mmol) and
3-(trifluoromethyl)benzoic acid (82 mg, 0.433 mmol) in DMF (5 mL)
were added HATU (197 mg, 0.519 mmol) and DIPEA (167 mg,
1.30 mmol), and the reaction mixture was stirred at room temperature
for 14 h. The reaction mixture was then diluted with water (20 mL),
extracted with CH2Cl2 (3 × 100 mL), and the pooled organics were
washed with water and then brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The material thus obtained
was purified by silica gel chromatography (0−2% MeOH in CH2Cl2
gradient) to afford 23 (110 mg, 64%). 1H NMR (DMSO-d6,
400 MHz): δ 12.91 (s, 1H), 8.52 (m, 2H), 7.93 (m, 1H), 7.80
(m, 1H), 7.75 (m, 1H), 7.61 (m, 1H), 7.27 (m, 2H), 4.79 (m, 2 H),
3.72 (m, 1H), 2.59 (m, 2H), 2.06 (d, J = 10.4 Hz, 2H), 1.84 (d, J =
12.4, 2H), 1.49 (m, 2H). MS calculated for C21H21F3N3O2 (M + H)+
404.16, found 403.9.
N-(1-(trans-4-Hydroxycyclohexyl)-4-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (24). Yield, 20%.
1H NMR (DMSO-d6, 400 MHz): δ 12.44 (s, 1H), 8.49 (m, 2H), 7.92
(m, 1H), 7.77 (m, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.20 (m, 1H), 7.09
(d, J = 8.0 Hz, 1H), 4.76 (m, 2H), 3.68 (m, 1H), 2.51 (m, 2H), 2.50
(s, 3H), 2.02 (d, J = 10.4 Hz, 2H), 1.81 (d, J = 11.2 Hz, 2H), 1.46 (m,
2H). MS calculated for C22H23F3N3O2 (M + H)+ 418.17, found 418.0.
N-(1-(trans-4-Hydroxycyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (25). Yield, 18%.
1H NMR (DMSO-d6, 400 MHz): δ 12.79 (s, 1H), 8.48 (m, 2H),
tert-Butyl cis-(4-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-
yl)cyclohexyl)carbamate (30n). Yield, 98%.
tert-Butyl trans-(4-(2-Amino-5-methyl-1H-benzo[d]imidazol-
1
1-yl)cyclohexyl)carbamate (30o). Yield, 95%. H NMR (DMSO-
d6, 400 MHz): δ 7.22 (d, J = 7.8 Hz, 1H), 6.92 (s, 1H), 6.81 (d, J =
7.5 Hz, 1H), 6.41 (d, J = 7.5 Hz, 1H), 6.25 (br s, 2H), 4.15 (m, 1H),
3.41 (m, 1H), 2.35 (s, 3H), 2.21 (m, 2H), 1.95 (m, 2H), 1.85 (m, 2H),
1.41 (s, 9H), 1.39 (m, 2H). MS calculated for C19H29N4O2 (M + H)+
345.2, found 345.2.
tert-Butyl (2-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
phenyl)carbamate (30p). Yield, 96%. 1H NMR (DMSO-d6,
400 MHz): δ 8.28 (s, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.45 (m, 1H),
7.27 (m, 2H), 7.02 (s, 1H), 6.63 (d, J = 7.9 Hz, 1H), 6.47 (d, J =
7.6 Hz, 1H), 5.99 (br s, 2H), 2.32 (s, 3H), 1.45 (s, 9H). MS calculated
for C19H23N4O2 (M + H)+ 339.15, found 339.3.
tert-Butyl (3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
phenyl)carbamate (30q). Yield, 93%. 1H NMR (CDCl3, 400 MHz):
δ 7.61(s, 1H), 7.48−7.39 (m, 2H), 7.25 (d, J = 10.4 Hz, 1H), 7.10 (t,
J = 1.6 Hz, 1H), 6.95−6.85 (m, 3H), 2.41 (s, 3H), 2.17 (s, 2H), 1.51
(s, 9H). MS calculated for C19H23N4O2 (M + H)+ 339.18, found
339.1.
tert-Butyl (3-(2-Amino-5-methyl-1H-benzo[d]imidazol-1-yl)-
1
7.89 (d, J = 7.2 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.58 (d, J = 8.4 Hz,
1H), 7.38 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 4.75 (m, 2H), 3.68
(m, 1H), 2.61 (m, 2H), 2.39 (s, 3H), 2.02 (d, J = 10.4 Hz, 2H),
phenyl)(methyl)carbamate (30r). Yield, 93%. H NMR (DMSO-
d6, 400 MHz): δ 7.55 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.37
(s, 1H), 7.24 (d, J = 7.2 Hz, 1H), 7.03 (s, 1H), 6.77 (t, J = 8.4 Hz,
K
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1.79 (d, J = 11.2 Hz, 2H), 1.43 (m, 2H). MS calculated for
C22H23F3N3O2 (M + H)+ 418.17, found 418.2.
N-(1-(trans-4-Hydroxycyclohexyl)-6-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (26). Yield, 54%.
1H NMR (DMSO-d6, 400 MHz): δ 12.79 (s, 1H), 8.48 (m, 2H), 7.90
(d, J = 7.2 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.45 (d, J =
8.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 4.77 (m, 1H), 4.74 (br s, 1H),
3.70 (m, 1H), 2.56 (m, 2H), 2.43 (s, 3H), 2.03 (d, J = 10.0 Hz,
2H), 1.79 (d, J = 10.8 Hz, 2H), 1.46 (m, 2H). MS calculated for
C22H23F3N3O2 (M + H)+ 418.17, found 418.1.
N-(1-(trans-4-Hydroxycyclohexyl)-7-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (27). Yield, 39%.
1H NMR (DMSO-d6, 400 MHz): δ 12.93 (s, 1H), 8.52 (s, 1H), 8.43
(d, J = 7.6 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H),
7.46 (d, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz,
1H), 4.76 (m, 1H), 4.74 (br s, 1H), 3.64 (m, 1H), 3.00 (q, J = 12.0 Hz,
2H), 2.71 (s, 3H), 2.03 (d, J = 10.4 Hz, 2H), 1.89 (d, J = 11.6 Hz, 2H),
1.41 (m, 2H). MS calculated for C22H23F3N3O2 (M + H)+ 418.17,
found 418.2.
tert-Butyl (2-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)ethyl)carbamate (31a). Yield, 68%.
1H NMR (DMSO-d6, 400 MHz): δ 12.69 (br s, 1H), 8.54 (d, J =
7.8 Hz, 1H), 8.48 (s, 1H), 7.88 (d, J = 7.3 Hz, 1H), 7.73 (t, J = 5.4 Hz,
1H), 7.71 (t, J = 7.9 Hz, 1H), 7.38 (m, 1H), 7.08 (d, J = 7.8 Hz, 1H),
6.86 (br s, 1H), 4.28 (m, 2H), 3.37 (m, 2H), 2.39 (s, 3H), 1.25 (s, 9H).
tert-Butyl (3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)propyl)carbamate (31b). Yield, 68%.
1H NMR (DMSO-d6, 400 MHz): δ 12.7 (br s, 1H), 8.53 (d, J =
7.6 Hz, 1H), 8.46 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz,
1H), 7.41 (d, J = 8.4 Hz, 1H), 7.36 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H),
6.85 (br s, 1H), 4.26 (t, J = 6.8 Hz, 2H), 3.29−2.98 (m, 2H), 2.40
(s, 3H), 1.92 (t, J = 7.2 Hz, 2H), 1.32 (s, 9H). MS calculated for
C24H28F3N4O3 (M + H)+ 477.21, found 477.2.
J = 8.4 Hz, 1H), 8.43 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.71 (d, J =
7.6 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.18−7.08 (m,
3H), 5.42 (t, J = 8.4 Hz, 1H), 4.01 (br s, 1H), 3.29 (s, 1H), 2.40−1.9
(m, 7H), 1.39 (s, 9H).
tert-Butyl (cis-2-(5-Methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate
1
(31j). Yield, 55%. H NMR (CDCl3, 400 MHz): δ 12.55 (br s, 1H),
8.56 (s, 1H), 8.43 (d, J = 7.3 Hz, 1H), 7.74 (d, J = 7.3 Hz, 1H), 7.58 (t,
J = 7.9 Hz, 1H), 7.21 (m, 1H), 7.14 (s, 1H), 7.08 (d, J = 7.8 Hz, 1H),
4.55 (m, 1H), 4.21 (m, 1H), 3.15 (m, 1H), 2.44 (s, 3H), 1.45−2.15
(m, 8H), 1.06 (s, 9H). MS calculated for C27H32F3N4O3 (M + H)+
517.23, found 517.3.
tert-Butyl (trans-2-(5-Methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate
(31k). Yield, 44%. 1H NMR (CDCl3, 400 MHz): δ 12.40 (s, 1H), 8.57
(s, 1H), 8.45 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 7.3 Hz, 1H), 7.58 (t, J =
7.8 Hz, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.12 (s, 1H), 7.08 (d, J = 8.3 Hz,
1H), 4.11−4.95 (m, 3H), 2.44 (s, 3H), 1.85−2.35 (m, 4H), 1.31−1.55
(m, 4H), 1.15 (s, 9H). MS calculated for C27H32F3N4O3 (M + H)+
517.23, found 517.4.
tert-Butyl (cis-3-(5-Methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate
1
(31l). Yield, 49%. H NMR (DMSO-d6, 400 MHz): δ 12.78 (s, 1H),
8.52 (d, J = 7.2 Hz, 1H), 8.46 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.76−
7.51 (m, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.38 (s, 1H), 7.08 (d, J =
8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 4.91−4.75 (m, 1H), 3.62−3.41
(m, 1H), 2.39 (s, 3H), 2.35−2.24 (m, 2H), 1.99−1.75 (m, 4H),
1.35 (s, 9H), 1.59−1.29 (m, 2H). MS calculated for C27H32F3N4O3
(M + H)+ 517.24, found 517.3.
tert-Butyl (trans-3-(5-Methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate
(31m). Yield, 70%.
tert-Butyl (cis-4-(5-Methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate
(31n). Yield, 32%. 1H NMR (CDCl3, 400 MHz): δ 12.49 (s, 1H), 8.55
(s, 1H), 8.49 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.3 Hz, 1H), 7.60 (d, J =
7.3 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.16 (s, 1H), 7.10 (d, J = 8.3 Hz,
1H), 4.61−4.85 (m, 2H), 3.81−3.99 (m, 1H), 2.41−2.59 (m, 2H),
2.42 (s, 3H), 2.01−2.19 (m, 2H), 1.65−1.95 (m, 4H), 1.41 (s, 9H).
MS calculated for C27H32F3N4O3 (M + H)+ 517.23, found 517.1.
tert-Butyl (trans-4-(5-Methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)cyclohexyl)carbamate
tert-Butyl 3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-1H-
benzo[d]imidazol-1-yl)azetidine-1-carboxylate (31c). Yield, 60%.
tert-Butyl 3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-1H-
benzo[d]imidazol-1-yl)pyrrolidine-1-carboxylate (31d). Yield,
1
54%. H NMR (DMSO-d6, 400 MHz): δ 12.83 (s, 1H), 8.50 (d, J =
7.9 Hz, 1H), 8.45 (s, 1H), 7.95 (m, 1H), 7.72 (m, 1H), 7.42 (m, 1H),
7.41 (s, 1H), 7.09 (d, J = 7.8 Hz, 1H), 5.62 (m, 1H), 3.95 (m, 2H),
3.85 (m, 2H), 3.41 (m, 1H), 2.39 (s, 3H), 2.35 (m, 1H), 1.45 (s, 9H).
tert-Butyl 3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-1H-
benzo[d]imidazol-1-yl)piperidine-1-carboxylate (31e). Yield,
1
(31o). Yield, 71%. H NMR (DMSO-d6, 400 MHz): δ 12.78 (s, 1H),
1
8.47 (s, 1H), 8.46 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 8.2 Hz,
1H), 7.64 (d, J = 8.2 Hz, 1H), 7.39 (s, 1H), 7.06 (d, J = 7.2 Hz, 1H),
6.86 (d, J = 7.2 Hz, 1H),4.75 (m, 1H), 3.45 (m, 1H), 2.35 (s, 3H),
1.75−2.05 (m, 4H), 1.45 (s, 9H), 1.3−1.51 (m, 4H). MS calculated for
C27H32F3N4O3 (M + H)+ 517.23, found 517.2.
tert-Butyl (2-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)phenyl)carbamate (31p). Yield, 71%.
1H NMR (DMSO-d6, 400 MHz): δ 12.93 (s, 1H), 8.98 (s, 1H), 8.26
(s, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.86 and 7.81 (2d, J = 7.8 Hz, 2H),
7.63 (t, J = 7.8 Hz, 1H), 7.53 (m, 2H), 7.43 (s, 1H), 7.31 (m, 1H),
7.01 (d, J = 8.3 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 2.41 (s, 3H),
1.24 (s, 9H). MS calculated for C27H26F3N4O3 (M + H)+ 511.19,
found 511.1.
54%. H NMR (CDCl3, 400 MHz): δ 12.46 (s, 1H), 8.58 (s, 1H),
8.46 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.58−7.55 (m, 1H),
7.28 (s, 1H), 7.15 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 4.60 (br s, 1H),
4.3 (br s, 2H), 3.79 (t, J = 8.0 Hz, 1H), 2.90−2.65 (m, 2H), 2.46
(s, 3H), 2.17−1.93 (m, 2H), 1.85−1.8 (m, 1H), 1.47 (s, 9H).
MS calculated for C26H30F3N4O3 (M + H)+ 503.23, found 503.0.
tert-Butyl 4-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-1H-
benzo[d]imidazol-1-yl)piperidine-1-carboxylate (31f). Yield,
1
66%. H NMR (CD3OD, 400 MHz): δ 8.50 (s, 1H), 8.47 (d, J =
7.5 Hz, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.42 (d,
J = 7.9 Hz, 1H), 7.35 (s, 1H), 7.36 (d, J = 7.5 Hz, 1H), 4.85−5.05 (m,
1H), 4.25−4.41 (m, 2H), 2.91−3.12 (m, 2H), 2.55−2.75 (m, 2H),
2.41 (s, 3H), 1.82−1.96 (m, 2H), 1.45 (s, 9H). MS calculated for
C26H30F3N4O3 (M + H)+ 503.22, found 503.4.
tert-Butyl 3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-1H-
benzo[d]imidazol-1-yl)azepane-1-carboxylate (31g). Yield, 80%.
MS calculated for C27H32F3N4O3 (M + H)+ 517.24, found 517.3.
tert-Butyl 4-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-1H-
benzo[d]imidazol-1-yl)azepane-1-carboxylate (31h). Yield, 51%.
1H NMR (DMSO-d6, 400 MHz): δ 12.80 (d, J = 15.0 Hz, 1H), 8.51
(d, J = 6.8 Hz, 1H), 8.46 (s, 1H), 7.89 (d, J = 7.4 Hz, 1H), 7.61−7.81
(m, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.39 (s, 1H), 7.10 (d, J = 8.3 Hz,
1H), 4.65−5.01 (m, 1H), 3.15−4.05 (m, 4H), 2.49 (s, 3H), 1.65−2.15
(m, 6H), 1.21 and 1.41 (2s, 9H).
tert-Butyl (3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)cyclopentyl)carbamate (31i). Yield,
64%. 1H NMR (DMSO-d6, 400 MHz): δ 12.78 (s, 1H), 8.55 (d,
tert-Butyl (3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)phenyl)carbamate (31q). Yield, 76%.
MS calculated for C27H26F3N4O3 (M + H)+ 511.20, found 511.1.
tert-Butyl Methyl(3-(5-methyl-2-(3-(trifluoromethyl)-
benzamido)-1H-benzo[d]imidazol-1-yl)phenyl)carbamate
1
(31r). Yield, 67%. H NMR (DMSO-d6, 400 MHz): δ 12.99 (s, 1H),
8.32 (d, J = 6.8 Hz, 2H), 7.84 (d, J = 7.2 Hz, 1H), 7.67−7.45 9 (m,
6H), 7.14 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 3.28 (s, 3H),
2.42 (s, 3H), 1.41 (s, 9H). MS calculated for C28H28F3N4O3 (M + H)+
525.21, found 524.9.
tert-Butyl (3-(5-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)phenyl)carbamate (31s). Yield, 60%.
1H NMR (DMSO-d6, 400 MHz): δ 12.94 (s, 1H), 9.67 (s, 1H), 8.32
(s, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.3 Hz, 1H), 7.45−7.75
L
DOI: 10.1021/acs.jmedchem.5b01985
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(m, 6H), 7.24 (s, 1H), 7.03 (s, 1H), 2.41 (s, 3H), 1.52 (s, 9H).
MS calculated for C27H26F3N4O3 (M + H)+ 511.19, found 511.3.
tert-Butyl (R)-3-(7-Methyl-2-(3-(trifluoromethyl)benzamido)-
1H-benzo[d]imidazol-1-yl)azepane-1-carboxylate (39). 1H
NMR (DMSO-d6, 500 MHz) δ 12.94 (s, 1H), 8.59−8.47 (m, 1H),
8.43 (t, J = 6.7 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.84−7.67 (m, 1H),
7.46 (d, J = 7.7 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 7.09−6.97 (m,
1H), 4.96−4.72 (m, 1H), 4.50−4.21 (m, 1H), 3.86 (d, J = 12.6 Hz,
1H), 3.83−3.73 (m, 1H), 3.26−3.13 (m, 1H), 2.83−2.74 (m, 1H),
2.71 (s, 3H), 2.07 (m, 2H), 1.96 (m, 1H), 1.78 (m, 1H), 1.45 (s, 9H),
1.23 (m, 1H). MS calculated for C27H32F3N4O3 (M + H)+ 517.24,
3.27−2.66 (m, 2H), 2.39 (s, 3H), 2.00−1.91 (m, 2H), 1.64−1.5 (m,
1H); MS calculated for C24H24F3N4O2 (M + H)+ 457.19, found 456.9.
N-(1-(2-Acrylamidoethyl)-5-methyl-1H-benzo[d]imidazol-2-
yl)-3-(trifluoromethyl)benzamide (32a). Yield, 45%. 1H NMR
(DMSO-d6, 400 MHz): δ 12.73 (s, 1H), 8.53 (d, J = 8.0 Hz, 1H), 8.47
(s, 1H), 8.26−8.23 (m, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.70 (t, J =
8.0 Hz, 1H), 7.38−7.32 (m, 2H), 7.08 (d, J = 7.8 Hz, 1H), 6.97 (d, J =
7.6 Hz, 2H), 5.43 (7, J = 8.0 Hz, 1H), 4.40−4.37 (m, 2H), 3.59−3.56
(m, 2H), 2.40 (s, 3H). MS calculated for C21H20F3N4O2 (M + H)+
417.15, found 416.8.
N-(1-(3-Acrylamidopropyl)-5-methyl-1H-benzo[d]imidazol-
1
23
2-yl)-3-(trifluoromethyl)benzamide (32b). Yield, 35%. H NMR
found 517.4. [α]D +121.95° (c 0.123, MeOH).
(DMSO-d6, 400 MHz): δ 12.74 (s, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.45
(s, 1H), 8.21 (d, J = 5.6 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.72−7.68
(m, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.37 (m, 1H), 7.10 (d, J = 7.2 Hz,
1H), 6.23−6.02 (m, 2H), 5.56 (d, J = 2.0, 8.0 Hz, 1H), 4.32−4.28 (m,
2H), 3.33 (s, 1H), 3.25−3.20 (m, 1H), 2.40 (s, 3H), 1.99−1.94 (m,
2H). MS calculated for C22H22F3N4O2 (M + H)+ 431.17, found 431.1.
N-(1-(1-Acryloylazetidin-3-yl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32c). Yield, 58%.
1H NMR (DMSO-d6, 400 MHz): δ 12.86 (s, 1H), 8.46−8.42 (m, 2H),
7.87 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 8.4 Hz,
1H), 7.40 (s, 1H), 7.13 (d, J = 7.8 Hz, 1H), 6.45 (dd, J = 10.2, 16.6 Hz,
1H), 6.24 (dd, J = 1.9, 17.1 Hz, 1H), 5.86−5.74 (m, 2H), 5.03−5.0
(m, 1H), 4.77−4.68 (m, 2H), 4.46 (t, J = 9.7 Hz, 1H), 2.40 (s, 3H).
MS calculated for C22H20F3N4O2 (M + H)+ 429.15, found 429.0.
N-(1-(1-Acryloylpyrrolidin-3-yl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32d). Yield, 28%.
1H NMR (DMSO-d6, 400 MHz): δ 12.83 (s, 1H), 8.51−8.48 (m, 1H),
8.42 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.69−7.64 (m, 1H), 7.48−7.39
(m, 2H), 7.10 (d, J = 8.4 Hz, 1H), 6.78−6.54 (m, 1H), 6.26−6.17 (m,
1H), 5.78−5.60 (m, 2H), 4.27−4.22 (m, 0.5H), 4.13−4.00 (m, 1.5H),
3.95−3.86 (m, 1H), 3.79−3.73 (m, 0.5H), 3.58−3.51 (m, 0.5H),
2.83−2.71 (m, 1H), 2.40 (s, 3H), 2.38−2.25 (m, 1H). MS calculated
for C23H22F3N4O2 (M + H)+ 443.13, found 442.8.
N-(1-(1-Acryloylpiperidin-4-yl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32f). Yield, 41%.
1H NMR (DMSO-d6, 400 MHz): δ 12.79 (s, 1H), 8.46−8.40 (m, 2H),
7.88 (d, J = 8.0 Hz, 1H), 7.70−7.63 (m, 1H), 7.51 (d, J = 8.4 Hz, 1H),
7.38 (s, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.96−6.89 (m, 1H), 6.22−6.18
(m, 1H), 5.75−5.69 (m, 1H), 5.04−4.97 (m, 1H), 4.73−4.65 (m, 1H),
4.34−4.29 (m, 1H), 3.30−3.28 (m, 1H), 2.87−2.81 (m, 1H), 2.68−
2.54 (m, 2H), 2.40 (s, 3H), 2.00−1.93 (m, 2H). MS calculated for
C24H24F3N4O2 (M + H)+ 457.19, found 457.3.
N-(1-(1-Acryloylazepan-3-yl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32g). Yield, 38%.
1H NMR (DMSO-d6, 400 MHz): δ 12.81 (d, J = 19.6 Hz, 1H), 8.54
(s, 1H), 8.48−8.44 (m, 1H), 7.90−7.51 (m, 3H), 7.39 (d, J = 4.8 Hz,
1H), 7.10 (d, J = 7.6 Hz, 1H), 6.87−6.81 (m, 1H), 6.25−6.17 (m,
1H), 5.76−5.50 (m, 1H), 4.88 (br s, 1H), 4.16−4.03 (m, 2H), 3.90−
3.63 (m, 1H), 3.37−3.22 (m, 1H), 2.40 (s, 4H), 2.05−1.85 (m, 4H),
1.43−1.40 (m, 1H). MS calculated for C25H26F3N4O2 (M + H)+
471.20, found 471.2.
N-(1-(1-Acryloylazepan-4-yl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32h). Yield, 41%.
1H NMR (DMSO-d6, 400 MHz): δ 12.74 (s, 1H), 8.45−8.37 (m, 2H),
tert-Butyl (R)-3-(7-Methyl-2-(2-(trifluoromethyl)-
isonicotinamido)-1H-benzo[d]imidazol-1-yl)azepane-1-car-
1
boxylate (40). Yield, 63%. H NMR (CDCl3, 400 MHz) δ 12.53 (s,
1H), 8.89 (d, J = 4.6 Hz, 1H), 8.54 (d, J = 7.1 Hz, 1H), 8.27 (d, J =
4.6 Hz, 1H), 7.21 (dt, J = 7.8, 21.3 Hz, 2H), 7.08 (t, J = 9.2 Hz, 1H),
5.12−4.85 (m, 1H), 4.34 (dd, J = 10.6, 13.6 Hz, 1H), 4.16−3.85 (m,
2H), 3.36−3.21 (m, 1H), 2.94−2.82 (m, 1H), 2.80 (s, 3H), 2.30−
2.15 (m, 1H), 2.15−2.00 (m, 2H), 2.00−1.81 (m, 1H), 1.48 (s, 9H),
1.43−1.36 (m, 1H). MS calculated for C26H31F3N5O3 (M + H)+
23
518.23, found 518.2. [α]D +99.60° (c 0.251, MeOH).
tert-Butyl (R)-3-(7-Methyl-2-(2-methylisonicotinamido)-1H-
benzo[d]imidazol-1-yl)azepane-1-carboxylate (41). Yield,
1
100%. H NMR (DMSO-d6, 400 MHz) δ 12.95 (s, 1H), 8.60 (t,
J = 4.6 Hz, 1H), 7.89 (d, J = 5.7 Hz, 1H), 7.81 (t, J = 4.9 Hz, 1H), 7.46
(d, J = 7.2 Hz, 1H), 7.14 (t, J = 7.7 Hz, 1H), 7.10−7.02 (m, 1H),
4.96−4.71 (m, 1H), 4.32 (dd, J = 10.5, 13.4 Hz, 1H), 3.88−3.78 (m,
1H), 3.78−3.65 (m, 1H), 3.29−3.17 (m, 1H), 2.83−2.74 (m, 1H),
2.71 (s, 3H), 2.57 (s, 3H), 2.14−2.02 (m, 2H), 2.02−1.91 (m, 1H),
1.89−1.71 (m, 1H), 1.45 (s, 9H), 1.39−1.33 (m, 1H); MS calculated
23
for C26H34N5O3 (M + H)+ 464.26, found 464.2. [α]D +35.46°
(c 0.282, MeOH).
tert-Butyl (R)-3-(7-Chloro-2-(2-methylisonicotinamido)-1H-
benzo[d]imidazol-1-yl)azepane-1-carboxylate (42). Yield,
1
100%. H NMR (CDCl3, 400 MHz): δ 12.81 (br s, 1H), 8.65−8.62
(m, 1H), 7.95−7.85 (m, 2H), 7.27−7.11 (m, 3H), 5.64−5.51 (m, 1H),
4.56−4.44 (m, 1H), 4.07−3.92 (m, 1H), 3.79−3.71 (m, 0.5H), 3.41−
3.35 (m, 0.5H), 3.29−3.23 (m, 1H), 2.71−2.59 (m, 1H), 2.65 (s, 3H),
2.22−2.00 (m, 3H), 1.93−1.80 (m, 1H), 1.51−1.45 (m, 1H), 1.50
(s, 3.5H), 1.41 (s, 5.5H). MS calculated for C25H31ClN5O3 (M + H)+
23
484.20, found 484.20. [α]D +89.29° (c 0.112, MeOH).
General Procedure for the Synthesis of Compounds 32a−s
and 43. Synthesis of N-(1-(1-Acryloylpiperidin-3-yl)-5-methyl-
1H-benzo[d]imidazol-2-yl)-3-(trifluoromethyl)benzamide
(32e). To a stirred solution of 31e (0.300 g, 0.59 mmol) in CH2Cl2
(25 mL) at room temperature was added TFA (0.170 g, 1.49 mmol),
and the reaction mixture was stirred for 2 h, and then the volatiles were
removed under reduced pressure. The residue was then partitioned
between saturated aqueous NaHCO3 (15 mL) and CH2Cl2 (20 mL),
and the aqueous layer was extracted once more with CH2Cl2 (20 mL).
The pooled organic extracts were washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pressure
to afford (S)-N-(5-methyl-1-(piperidin-3-yl)-1H-benzo[d]imidazol-2-
yl)-3-(trifluoromethyl)benzamide (0.200 g, 83%), which was used as
is without further purification. To a 0 °C solution of (S)-N-(5-methyl-
1-(piperidin-3-yl)-1H-benzo[d]imidazol-2-yl)-3-(trifluoromethyl)-
benzamide (0.150 g, 0.37 mmol) in CH2Cl2 (20 mL) was added
acryloyl chloride (0.050 g, 0.55 mmol), and the reaction mixture was
stirred for 30 min at 0 °C. The reaction mixture was partitioned
between water (10 mL) and CH2Cl2 (10 mL), and the layers were
separated. The aqueous layer was extracted with CH2Cl2 (2 × 20 mL),
and the combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered, concentrated under reduced pressure, and
the resulting residue was purified by column chromatography (50%
EtOAc/hexane gradient) to give 32e (0.120 g, 0.329 mmol, 70%).
1H NMR (DMSO-d6, 400 MHz): δ 12.84 (s, 1H), 8.46 (d, J = 8.0 Hz,
2H), 7.89 (d, J = 8.0 Hz, 1H), 7.75−7.63 (m, 2H), 7.39 (s, 1H), 7.09
(d, J = 8.4 Hz, 1H), 6.95−6.79 (m, 1H), 6.19−6.13 (m, 1H), 5.75−5.59
(m, 1H), 4.79−4.56 (m, 2H), 4.31−4.19 (m, 1H), 4.15−3.65 (m, 1H),
7.87 (d, J = 7.4 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.50 (d, J = 8.3 Hz,
1H), 7.38 (s, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.92−6.81 (m, 1H), 6.29−
6.24 (m, 1H), 5.78−5.67 (m, 1H), 4.90 (br s, 1H), 3.92−3.43 (m,
4H), 2.60−2.56 (m, 1H), 2.39 (s, 3H), 2.10−1.78 (m, 4H), 1.25−1.21
(m, 1H). MS calculated for C25H26F3N4O2 (M + H)+ 471.20, found
471.0.
N-(1-(3-Acrylamidocyclopentyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32i). Yield, 17%.
1H NMR (DMSO-d6, 400 MHz): δ 12.78 (s, 1H), 8.55 (d, J = 8.0 Hz,
1H), 8.45 (s, 1H), 8.40 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H),
7.75−7.73 (m, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.40 (s, 1H), 7.10 (d, J =
7.6 Hz, 1H), 6.27−6.20 (m, 1H), 6.11 (dd, J = 2.0, 14.4 Hz, 1H),
5.61 (d, J = 2.0 Hz, 1H), 5.59−5.42 (m, 1H), 4.35−4.29 (m, 1H),
M
DOI: 10.1021/acs.jmedchem.5b01985
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Journal of Medicinal Chemistry
Article
2.40 (s, 3H), 2.37−1.92 (m, 6H). MS calculated for C24H24F3N4O2
(M + H)+ 457.19, found 457.40.
N-(1-(cis-2-Acrylamidocyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32j). Yield, 45%.
1H NMR (CDCl3, 400 MHz): δ 12.61 (s, 1H), 8.91 (s, 1H), 8.55
(s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 5.8 Hz, 1H), 7.66−7.62
(m, 1H), 7.19−7.11 (m, 3H), 6.0 (d, J = 16.7 Hz, 1H), 5.60−5.52 (m,
1H), 5.20 (d, J = 10.2 Hz, 1H), 4.46−4.44 (m, 2H), 3.50−3.41 (m,
1H), 2.53−2.46 (m, 1H), 2.45 (s, 3H), 2.13−2.10 (m, 1H), 1.98−1.94
(m, 1H), 1.69−1.56 (m, 3H), 1.55−1.43 (m, 1H). MS calculated for
C25H26F3N4O2 (M + H)+ 471.20, found 471.8.
(m, 1H), 2.42 (s, 3H). MS calculated for C25H20F3N4O2 (M + H)+
465.15, found 465.2.
N-(5-Methyl-1-(3-(N-methylacrylamido)phenyl)-1H-benzo-
[d]imidazol-2-yl)-3-(trifluoromethyl)benzamide (32r). Yield,
44%. 1H NMR (DMSO-d6, 400 MHz): δ 13.01 (s, 1H), 8.30 (d,
J = 6.4 Hz, 2H), 7.84 (d, J = 7.6 Hz, 1H), 7.75−7.62 (m, 4H), 7.50 (d,
J = 1.6 Hz, 1H), 7.49−7.45 (m, 1H), 7.17−7.08 (m, 2H), 6.27−
6.16 (m, 2H), 5.58−5.55 (m, 1H), 3.34 (s, 3H), 2.42 (s, 3H).
MS calculated for C26H22F3N4O2 (M + H)+ 479.17, found 478.7.
N-(1-(4-Acrylamidophenyl)-5-methyl-1H-benzo[d]imidazol-
1
2-yl)-3-(trifluoromethyl)benzamide (32s). Yield, 48%. H NMR
(DMSO-d6, 400 MHz): δ 12.99 (s, 1H), 10.43 (s, 1H), 8.38 (s, 1H),
8.32 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 7.6 Hz, 2H), 7.83 (d, J = 7.6 Hz,
1H), 7.67−7.58 (m, 2H), 7.42 (m, 2H), 7.12−7.05 (m, 2H), 6.56−
6.48 (m, 1H), 6.35−6.32 (m, 1H), 5.82−5.80 (m, 1H), 2.42 (s, 3H).
MS calculated for C25H20F3N4O2 (M + H)+ 465.15, found 465.3.
(R)-N-(1-(1-Acryloylazepan-3-yl)-7-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (43). Yield, 71%.
1H NMR (DMSO-d6, 400 MHz): δ 12.80 (br s, 1H), 8.52 (s, 1H),
N-(1-(trans-2-Acrylamidocyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32k). Yield, 45%.
1H NMR (CDCl3, 400 MHz): δ 12.32 (s, 1H), 8.57 (s, 1H), 8.47 (d,
J = 7.3 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.62−7.58 (m, 1H), 7.42−
7.40 (m, 1H), 7.12−7.06 (m, 2H), 6.18 (br s, 1H), 5.91 (dd, J = 1.5,
17.1 Hz, 1H), 5.73−5.67 (m, 1H), 5.36 (dd, J = 1.5, 10.3 Hz, 1H),
5.01−4.96 (m, 1H), 4.56 (br s, 1H), 2.43−2.38 (m, 4H), 2.12−1.94
(m, 3H), 1.56−1.42 (m, 4H). MS calculated for C25H26F3N4O2
(M + H)+ 471.20, found 471.2.
8.42 (d, J = 8.0 Hz, 1H), 7.85−7.70 (m, 2H), 7.46 (d, J = 8.0 Hz,
1H), 7.13 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.82−6.75
(m, 1H), 6.15 (dd, J = 2.4, 14.0 Hz, 1H), 5.66 (br s, 1H), 5.0 (br s,
1H), 4.25−4.22 (m, 2H), 4.0 (br s, 1H), 3.50 (br s, 1H), 2.7−2.65
(m, 4H), 2.10−1.89 (m, 4H), 1.42−1.39 (m, 1H). MS calculated
N-(1-(cis-3-Acrylamidocyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32l). Yield, 26%.
1H NMR (DMSO-d6, 400 MHz): δ 12.79 (s, 1H), 8.52 (d, J = 8.0 Hz,
1H), 8.49 (s, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H),
7.76 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.38 (s, 1H), 7.09
(d, J = 8.4 Hz, 1H), 6.22−6.08 (m, 1H), 6.04 (d, J = 2.0 Hz, 1H), 5.55
(dd, J = 2.4, 8.0 Hz, 1H), 4.98−4.77 (m, 1H), 3.99−3.83 (m, 1H),
2.41 (s, 3H), 2.50−2.35 (m, 2H), 1.96−1.80 (m, 4H), 1.62−1.30 (m,
2H). MS calculated for C25H26F3N4O2 (M + H)+ 471.20, found 471.2.
N-(1-(trans-3-Acrylamidocyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32m). Yield, 20%.
1H NMR (CDCl3, 400 MHz): δ 12.39 (s, 1H), 8.55 (s, 1H), 8.40 (d,
J = 7.6 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H),
7.30−7.28 (m, 1H), 7.14 (s, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.99−6.20
(m, 2H), 5.88 (br s, 1H), 5.71 (d, J = 10.4 Hz, 1H), 5.06 (t, J =
12.8 Hz, 1H), 4.56 (br s, 1H), 2.55 (t, J = 12.4 Hz, 1H), 2.44−2.22
(m, 5H), 2.03−2.01 (m, 3H), 1.76−1.41 (m, 2H). MS calculated for
C25H26F3N4O2 (M + H)+ 471.20, found 471.2.
N-(1-(cis-4-Acrylamidocyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32n). Yield, 30%.
1H NMR (DMSO-d6, 400 MHz): δ 12.80 (s, 1H), 8.56 (d, J = 7.8 Hz,
1H), 8.43 (s, 1H), 8.27 (d, J = 5.9 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H),
7.73−7.64 (m, 2H), 7.40 (s, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.58−6.51
(m, 1H), 6.18−6.13 (m, 1H), 5.67−5.64 (m, 1H), 4.98−4.89 (m, 1H),
4.05 (br s, 1H), 3.34−3.28 (m, 2H), 2.40 (s, 3H), 2.06−2.02 (m, 2H),
1.83−1.68 (m, 4H). MS calculated for C25H26F3N4O2 (M + H)+
471.20, found 471.0.
23
for C25H26F3N4O2 (M + H)+ 471.20, found 471.3. [α]D +127.2°
(c 0.0786, MeOH).
General Procedure for the Synthesis of Compounds 44−46.
Synthesis of (R,E)-N-(1-(1-(4-(Dimethylamino)but-2-enoyl)-
azepan-3-yl)-7-methyl-1H-benzo[d]imidazol-2-yl)-2-methyl-
isonicotinamide (46). A solution of 41 (6.81 g, 14.7 mmol) in
MeOH (15 mL) was treated with 4 M HCl in dioxane (100 mL,
400 mmol) and stirred for 12 h. The volatiles were removed under
reduced pressure to afford (R)-N-(1-(azepan-3-yl)-7-methyl-1H-
benzo[d]imidazol-2-yl)-2-methylisonicotinamide, which was dissolved
in CH2Cl2 (70 mL) and Et3N (6.15 mL, 44.1 mmol). In a separate
flask, a mixture of (E)-4-(dimethylamino)but-2-enoic acid hydro-
chloride (2.60 g, 19.1 mmol) and HATU (7.34 g, 19.1 mmol) in
CH2Cl2 (50 mL) was treated with Et3N (3.07 mL, 22.0 mmol) and
stirred at room temperature for 30 min. This was then treated with
the CH2Cl2/Et3N solution of (R)-N-(1-(azepan-3-yl)-7-methyl-1H-
benzo[d]imidazol-2-yl)-2-methylisonicotinamide, and the transfer was
assisted with an additional volume of CH2Cl2 (20 mL). The reaction
mixture was stirred for 30 min and then washed with 1 M NaOH
(100 mL). The aqueous layer was extracted with CH2Cl2 (2 × 150 mL),
and the pooled organics were washed with 1 M NaOH (100 mL) and
then brine (100 mL), dried over Na2SO4, filtered, and concentrated
under reduced pressure. The crude material was purified by column
chromatography (0−70% (9:1:0.175N CH2Cl2/MeOH/NH3)/CH2Cl2
1
N-(1-(trans-4-Acrylamidocyclohexyl)-5-methyl-1H-benzo[d]-
imidazol-2-yl)-3-(trifluoromethyl)benzamide (32o). Yield, 44%.
1H NMR (DMSO-d6, 400 MHz): δ 12.80 (s, 1H), 8.46 (m, 2H),
8.10 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.78−7.65 (m, 2H),
7.40 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.28−6.21 (m, 1H), 6.16−6.11
(m, 1H), 5.61−5.58 (m, 1H), 4.87−4.81 (m, 1H), 3.98−3.89 (m, 1H),
3.34−3.28 (m, 2H), 2.40 (s, 3H), 2.08−2.02 (m, 2H), 1.90−1.82 (m,
2H), 1.57−1.43 (m, 2H). MS calculated for C25H26F3N4O2 (M + H)+
471.20, found 471.0.
gradient) to afford 46 (4.26 g, 61%). H NMR (CD3OD, 400 MHz)
δ 8.57 (d, J = 5.2 Hz, 1H), 8.06 (s, 1H), 7.98 (d, J = 5.2 Hz, 1H), 7.40
(d, J = 8.2 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 8.7 Hz, 1H),
6.99 (t, J = 15.8 Hz, 1H), 6.75 (m, 1H), 5.14 (m, 2H), 4.60 (m,
1H), 4.32 (d, J = 13.0 Hz, 1H), 3.98 (m, 3H), 3.71 (m, 1H), 3.16
(m, 1H), 3.03 (m, 1H), 2.93 (s, 6H), 2.77 (s, 3H), 2.53 (s, 3H), 2.22
(m, 1H), 2.12 (m, 2H). MS calculated for C27H35N6O2 (M + H)+
23
475.28, found 475.1. [α]D +72.46° (c 0.069, MeOH).
(R,E)-N-(1-(1-(4-(Dimethylamino)but-2-enoyl)azepan-3-yl)-7-
N-(1-(2-Acrylamidophenyl)-5-methyl-1H-benzo[d]imidazol-
methyl-1H-benzo[d]imidazol-2-yl)-3-(trifluoromethyl)-
1
1
2-yl)-3-(trifluoromethyl)benzamide (32p). Yield, 24%. H NMR
benzamide (44). Yield, 55%. H NMR (CDCl3, 400 MHz) δ 12.61
(DMSO-d6, 400 MHz): δ 12.94 (s, 1H), 9.61 (s, 1H), 8.25−8.22 (m,
2H), 8.15 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 7.8 Hz, 2H), 7.64−7.54
(m, 2H), 7.43 (s, 1H), 7.39−7.35 (m, 1H), 6.99 (d, J = 7.3 Hz, 1H),
6.72 (d, J = 7.8 Hz, 1H), 6.23−6.16 (m, 1H), 6.10−6.05 (m, 1H),
5.56−5.52 (m, 1H), 2.40 (s, 3H). MS calculated for C25H20F3N4O2
(M + H)+ 465.15, found 465.1.
(s, 1H), 8.66 (s, 1H), 8.44 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.2 Hz,
1H), 7.62 (t, J = 7.7 Hz, 1H), 7.18 (m, 2H), 7.03 (m, 1H), 6.92 (dt,
J = 6.1, 15.2 Hz, 1H), 6.59 (d, J = 15.2 Hz, 1H), 5.10 (m, 1H), 4.46
(m, 2H), 4.01 (dt, J = 7.3, 14.4 Hz, 1H), 3.66 (m, 1H), 3.25 (s, 2H),
2.97 (s, 1H), 2.80 (s, 3H), 2.41 (s, 6H), 2.08 (m, 3H), 1.58 (m, 2H).
MS calculated for C28H33F3N5O2 (M + H)+ 528.26, found 528.2.
23
N-(1-(3-Acrylamidophenyl)-5-methyl-1H-benzo[d]imidazol-
[α]D +106.38° (c 0.141, MeOH).
1
2-yl)-3-(trifluoromethyl)benzamide (32q). Yield, 35%. H NMR
(R,E)-N-(1-(1-(4-(Dimethylamino)but-2-enoyl)azepan-3-yl)-7-
(DMSO-d6, 400 MHz): δ 12.99 (s, 1H), 10.44 (s, 1H), 8.36 (s, 1H),
8.32 (d, J = 7.6 Hz, 1H), 8.19 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.75
(d, J = 8.0 Hz, 1H), 7.67−7.58 (m, 2H), 7.46−7.41 (m, 2H), 7.20−
7.09 (m, 2H), 6.49−6.30 (m, 1H), 6.29−6.25 (m, 1H), 5.80−5.75
methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluoromethyl)-
1
isonicotinamide (45). Yield, 55%. H NMR (CD3OD, 400 MHz)
δ 8.88 (d, J = 4.9 Hz, 1H), 8.56 (s, 1H), 8.34 (d, J = 4.9 Hz, 1H),
7.41 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.12 (m, 1H),
N
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Journal of Medicinal Chemistry
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6.86 (t, J = 13.8 Hz, 1H), 6.78 (m, 1H), 5.12 (m, 1H), 4.54 (dd, J =
10.8, 13.0 Hz, 1H), 4.27 (m, 1H), 4.02 (m, 1H), 3.65 (m, 3H), 2.92
(m, 1H), 2.78 (s, 3H), 2.67 (s, 6H), 2.22 (m, 1H), 2.08 (m, 3H), 1.49
(m, 1H). MS calculated for C27H32F3N6O2 (M + H)+ 529.25, found
529.2. Anal. Calcd for C27H31F3N6O2 (528.58): C 61.35, H 5.91,
N 15.90. Found: C 61.10, H 5.93, N 15.75. [α]D23 +153.06° (c 0.098,
MeOH).
column pre-equilibrated with lysis buffer. The bound protein was
washed with lysis buffer supplemented with 20 mM imidazole. Bound
protein was then eluted with lysis buffer supplemented with 300 mM
imidazole. Fractions containing protein were combined and desalted
into 20 mM Tris, pH 8.0, 50 mM NaCl, 2 mM TCEP using a PD10
column (GE Healthcare). To remove the 6xHIS tag, TEV protease
was added to the desalted fractions at a ratio of 1 mg of TEV/20 mg of
fusion protein and incubated at 4 °C overnight. Following overnight
digest, the cleaved protein was applied to Ni-NTA resin to remove
uncleaved protein, the flow through was collected and concentrated
to 6 mg/mL. Aliquots were flash frozen in liquid nitrogen and stored
at −80 °C.
Crystallization and Data Collection. Crystallization experiments
were carried out using the sitting-drop vapor diffusion setup. The
protein drug complex was formed by incubating the concentrated
protein (WT EGFR kinase domain or T790M mutant) with 2 mM
MgCl2 and 0.5 mM drug (either racemic 43 or 46 from 10 mM DMSO
stock). After addition of the inhibitor, the solution was incubated at
room temperature for 90 min prior to crystallization plate setup.
Crystallization plates were incubated at 20 °C. After 1−2 weeks, crystals
of the EGFR T790M-43 complex were visible in a well containing 1.2 M
potassium sodium tartrate, 0.1 M HEPES, pH 7.5. Crystals of the EGFR
T790M-46 complex were visible in a well containing 32% v/v PEG
3350, 0.2 M lithium sulfate, 0.1 M HEPES, pH 7.25. Crystals of the WT
EGFR-43 complex grew out of a well containing 1.35 M potassium
sodium tartrate, 0.1 M HEPES, pH 7.9. Crystals were cryopreserved in
reservoir solution supplemented with 20% v/v ethylene glycol and flash
frozen in liquid nitrogen prior to data collection. X-ray diffraction data
were collected from frozen crystals on ALS beamline 5.0.3.
Structure Determination and Refinement. Diffraction data
were processed using HKL2000,45 and structures were solved by
molecular replacement using PHASER.46 Structure refinement was
carried out in PHENIX47 alternated with manual fitting in Coot.48
Selection of TLS groups was carried out using the automated pro-
cedure implemented in PHENIX. Refinement statistics for cocrystals
with 43 and 46 are included in the Supporting Information.
EGFR Biochemical Assays. Biochemical assays for WT EGFR and
each mutant were carried out using a homogeneous time-resolved
fluorescence (HTRF) assay as described previously.49 Assays were
optimized for each ATP concentration. Assays were carried out with or
without 90 min enzyme/compound preincubation step. Compound
IC50 values were determined by 12-point inhibition curves (from 50 to
0.000282 μM) in duplicate.
H1975, H3255, HCC827, and HaCaT Cellular EGFR Target
Modulation Assays. Tissue culture cells were maintained in 10%
FBS/RPMI supplemented with 100 μg/mL penicillin/streptomycin
(Hyclone no. SH30236.01). The cells were harvested with 0.25%
trypsin/EDTA (Hyclone no. SH30042.1), resuspended in 5% FBS/
RPMI Pen/Strep, and plated at 7500 cells per well in 50 μL of medium
in a 384-well black plate with clear bottoms (Greiner no. 789068G).
The cells were allowed to incubate overnight in a 37 °C, 5% CO2
humidified tissue culture incubator. The 12-point serial diluted test
compounds were transferred to the plate containing cells by using a
50 nL Pin Head device (PerkinElmer), and the cells were placed back
in the incubator for 3 h.
Phospho-EGFR (Y1173) target modulation assay HaCaT cells were
stimulated with 10 ng/mL EGF (Peprotech no. AF-100-15) for 5 min
at room temperature. Constitutively activated EGFR mutant cell
lines (H1975, H3255, and HCC827) were not stimulated with EGF.
The medium was reduced to 20 μL using a Bio-Tek ELx 405 Select
plate washer. Cells were lysed with 20 μL of 2× lysis buffer containing
protease and phosphatase inhibitors (2% Triton X-100, 40 mM Tris,
pH 7.5, 2 mM EDTA, 2 mM EGTA, 300 mM NaCl, 2× complete
cocktail inhibitor (Roche no. 11 697 498 001), 2× phosphatase
inhibitor cocktail set II and set III (Sigma no. P5726 and no. P0044)).
The plates were shaken for 20 min. An aliquot of 25 μL from each well
was transferred to prepared ELISA plates for analysis.
Synthesis of (R,E)-N-(7-Chloro-1-(1-(4-(dimethylamino)but-
2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methyl-
isonicotinamide (47). A solution of 42 (8.62 g, 16.4 mmol) in
MeOH (67 mL) was treated with HCl in dioxane (4 M, 67 mL), and
the mixture was stirred at room temperature for 7 h. The mixture was
then concentrated under reduced pressure to afford (R)-N-(1-(azepan-
3-yl)-7-chloro-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide
(7.2 g) as the HCl salt. The product was used in the next step without
further purification. A mixture of (E)-4-(dimethylamino)but-2-
enoic acid hydrochloride (2.76 g, 21.4 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (4.10 g, 21.4 mmol)
in DMF (80 mL) was treated with hydroxybenzotriazole (3.27 g,
21.4 mmol) and stirred at room temperature for 1 h. The resulting
mixture was added to a solution of (R)-N-(1-(azepan-3-yl)-7-chloro-
1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (6.1 g, 13.4 mmol)
in DMF (20 mL). Et3N (13.0 mL, 93.5 mmol) was then added, and the
mixture was stirred for 24 h. Water (2 mL) was added, and the mixture
was concentrated under reduced pressure. The residue was diluted with
water (250 mL) and extracted with EtOAc (3 × 200 mL). The
combined organic layers were washed with water (100 mL), NaHCO3
(100 mL), and brine (2 × 50 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude was purified by column
chromatography (0−100% (9:1:0.175N CH2Cl2/MeOH/NH3)/
CH2Cl2 gradient) to afford 47 (3.67 g, 56%). 1H NMR (CD3CN,
400 MHz): δ 12.55 (br s, 1H), 8.61 (d, J = 5.2 Hz, 1H), 7.98 (s, 0.6H),
7.95 (s, 0.4H), 7.88−7.85 (m, 1H), 7.53 (dd, J = 1.2, 8.0 Hz, 0.6H), 7.51
(dd, J = 1.2, 8.0 Hz, 0.4H), 7.33−7.23 (m, 2H), 6.81−6.72 (m, 1H),
6.60 (dt, J = 15.2, 1.6 Hz, 1H), 5.66−5.55 (m, 1H), 4.81−4.74 (m, 1H),
4.61−4.55 (m, 1H), 4.29−4.01 (m, 2H), 3.89−3.82 (m, 1H), 3.71−3.65
(m, 1H), 3.44−3.38 (m, 1H), 3.14 (d, J = 6.0 Hz, 1.2H), 2.92 (d, J =
6.0 Hz, 0.8H), 2.85−2.72 (m, 1H), 2.61 (d, J = 2.0 Hz, 3H), 2.27
(s, 6H), 2.16−2.00 (m, 1H), 1.52−1.41 (m, 1H). MS calculated for
C26H32ClN6O2 (M + H)+ 495.23, found 495.20. Anal. Calc. for
C26H31ClN6O2 (495.02): C 63.09, H 6.31, N 16.98; found: C 62.90,
23
H 6.21, N 16.90. [α]D +53.76° (c 0.186, MeOH).
Molecular Docking Experiments. Flexible ligand docking was
performed using Glide 6.5 (Schrodinger Inc., Portland, OR, 2014).
The protein coordinates were taken from cocrystal structure of
AEE788 with EGFR T790M mutant (PDB code 2jiu).6 The grid box
was centered on the cocrystallized ligand and extended 10 Å from the
center, with the outer box extending an additional 20 Å. The ligand
was docked using the standard precision (SP) algorithm, and during
docking, constraints were imposed, requiring at least one H-bonding
contact in the hinge region. The final pose was selected based on the
lowest docking score using GlideScore.
Cloning, Expression, and Purification. DNA encoding the kinase
domain of human WT EGFR (residues 696−1022) was amplified
by PCR from a cDNA template and cloned into pFastBACHTb
(Life Technologies) in-frame with the N-terminal 6xHIS tag and TEV
(Tobacco Etch Virus) protease cleavage site. The T790M mutation
was created using site-directed mutagenesis (QuikChange, Agilent
Technologies). Bacmids were created via standard methods and
transfected into Sf9 cells to generate recombinant baculovirus. Large
scale expression of protein was also carried out in Sf9 cells with
cultures being infected at a cell density of 1 × 106 cells/mL. The
culture was harvested by centrifugation when cell viability reached 50%
(∼3−4 days post-transfection), and cell pellets were stored at −80 °C.
To purify the proteins (WT or T790M mutant), the cell pellets
were resuspended in lysis buffer containing 20 mM Tris, pH 8.0,
150 mM NaCl, 2 mM TCEP, 5% v/v glycerol, and protease inhibitor
cocktail tablets without EDTA (Roche). Cells were lysed by
sonication, and the lysate was cleared by centrifugation at 20 000g at
4 °C for 45 min. The clarified lysate was applied to a 5 mL Ni-NTA
Phospho-EGFR (Y1173) ELISA solid white 384-well high-
binding ELISA plates (Greiner no. 781074) were coated with
5 μg/mL goat anti-EGFR capture antibody overnight in 50 mM
O
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Journal of Medicinal Chemistry
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carbonate/bicarbonate pH 9.5 buffer. Plates were blocked with
1% BSA (Sigma no. A7030) in PBS for 1 h at room temperature,
and washes were carried out with a Bio-Tek ELx405 Select using
4 cycles of 100 μL of TBS-T (20 mM Tris, 137 mM NaCl, 0.05%
Tween-20) per well. A 25 μL aliquot of lysed cell was added to each
well of the ELISA plate and incubated overnight at 4 °C with gentle
shaking. A 1:1000 anti-phospho-EGFR in 0.2% BSA/TBS-T was
added and incubated for 2 h at room temperature. After washing,
1:2000 anti-rabbit-HRP in 0.2% BSA/TBS-T was added and incubated
for 1 h at room temperature. Chemiluminescent detection was carried
out with SuperSignal ELISA Pico substrate. Signal was read on
EnVision plate reader using built-in UltraLUM setting.
In Vivo Efficacy Studies. Foxn1 nude mice bearing the H1975
tumors were randomized and used for efficacy studies. Compounds
were formulated in 0.5% MC, 0.5% Tween80 suspension formulation
and administered by oral gavage at a dosing volume of 10 μL/g of the
animal body weight. Animals in each group received one oral dose
of either vehicle (n = 6) or the different test compounds (n = 6 in each
dose group). Plasma samples were collected for PK measurements at
30 min, 3 h, 7 h, and 24 h after last dose on day 5. Body weight was
monitored daily, and the % change in body weight was calculated
as [(BWcurrent − BWinitial)/(BWinitial)] × 100. Data are presented as
percent body weight change from the day of treatment initiation.
Tumor sizes were assessed three times during the efficacy study for
5 days. Tumor sizes were determined by using caliper measurements.
Tumor volumes were calculated with the formula (length × width ×
width)/2.
Percent treatment/control (T/C) values for tumor were calculated
using the following formula: % T/C = 100 × ΔT/ΔC if ΔT > 0;
% regression = 100 × ΔT/Tinitial if ΔT < 0; where T is the mean tumor
volume of the drug-treated group on the final day of the study; ΔT is
mean tumor volume of the drug-treated group on the final day of the
study minus mean tumor volume of the drug-treated group on initial
day of dosing; Tinitial is the mean tumor volume of the drug-treated
group on initial day of dosing; C is the mean tumor volume of the
control group on the final day of the study; and ΔC is mean tumor
volume of the control group on the final day of the study minus mean
tumor volume of the control group on initial day of dosing.
for automatic peak selection, integration, and spectral deconvolution
with a mass range of 15 000−75 000 Da.
Reaction solutions were also processed for enzymatic digestion
and LC/MSMS. Samples were buffer exchanged to remove excess
compound using 10 kDa Amicon Ultra 0.5 mL spin filters and
recovered in 30 μL of 100 mM Tris, pH 8, 20 mM DTT and incubated
at 60 °C for 20 min. Iodoacetamide was added to 25 mM, and the
samples were incubated for 45 min at room temperature in the dark
prior to dilution to 150 μL with 100 mM Tris, pH 8, 10 mM DTT.
The samples were divided in two, and 1 μL of either 0.5 mg/mL
chymotrypsin or sequencing grade modified trypsin was added and
incubated overnight at 37 °C. Samples were acidified to 1% formic acid
and analyzed on a Q-Exactive mass spectrometer for accurate mass and
MS/MS. Samples were injected on a precolumn (360 μm × 100 μm,
4 cm Poros10R2) for desalting and separated on a 360 μm × 75 μm
analytical column and integrated emitter (10 cm, 5 μm Monitor C18)
with a gradient of 2−27% B in 45 min; 27−36% B in 10 min at a flow
rate of 300 nL/min (B, 0.1% formic acid, MeCN). The Q-Exactive
parameters were set for “sensitive” analysis as previously defined by
Kelstrup et al.50 Briefly, one full scan MS was acquired at 75 000
resolution followed by 12 MS/MS at 35 000 resolution of the top ions
in the mass range of 350−1500 m/z. An additional dedicated scan for
MS/MS of 898.7184 was added to continuously target the adduct
Cys797. The data were converted to Mascot generic format (MGF)
using Proteome Discoverer (Thermo) and searched using Mascot against
a custom database allowing for variable modification of methionine
oxidation, cysteine carbamidomethylation, and the custom modification
of Michael addition by 47 to cysteines. Enzyme specificity was trypsin or
no enzyme (chymotrypsin digest), and the peptide and fragment ion
tolerances were set to 25 ppm and 50 mmu, respectively. The data were
visualized in Scaffold 4.4.3 and filtered for 95% peptide confidence and
99% protein confidence.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b01985.
■
S
Refinement statistics of cocrystal structures WT-EGFR-
43, EGFR T790M-43 and EGFR T790M-46; biochem-
ical selectivity profiling of 47 against a panel of kinase
and non-kinase targets; enantiopurity analysis of chiral
compounds; analytical LC−MS methods for analysis of
intermediates; metabolite identification studies for 43;
1H and 13C NMR spectra of 47 at 300 and 383 K (PDF)
Molecular formula strings and some data (CSV)
All data were expressed as mean
standard error of the mean
(SEM). The Δ tumor volume and body weight were used for statistical
analysis. Between groups comparisons were carried out using a one-
way ANOVA followed by a post hoc Tukey or Dunn’s. For all
statistical evaluations the level of significance was set at p < 0.05.
Significance compared to the vehicle control group is reported unless
otherwise stated.
PK Studies. Rodent PK studies of 43−47 were conducted in male
balb/c mice and male Wistar rats. Plasma concentrations of compounds
were determined by liquid chromatography−tandem mass spectrom-
etry (Applied Biosystems, Foster City, CA, USA). The lower limit of
detection was 1 ng/mL. Pharmacokinetic parameters were calculated
by noncompartmental regression analysis using an in-house fitting
program.
Accession Codes
The crystallographic coordinates and structure factors have
been deposited in the Protein Data Bank, www.pdb.org (PDB
codes 5fee for EGFR T790M-43, 5fed for WT EGFR-43, and
5feq for EGFR T790M-46).
In Vitro Adducts of 47 with EGFR. Recombinant kinase domain
of EGFR L858R and T790M-L858R mutants were incubated with
47 to confirm covalent modification of EGFR and site of adduction.
Recombinant enzyme was incubated at room temperature with a
20-fold molar excess of compound in 40 mM Tris, pH 8, 500 mM
NaCl, 1% glycerol, 5 mM TCEP for 1 h. The reaction was quenched
by addition of dithiothreitol (DTT, 80-fold excess to compound) and
transfer to ice. A third of the reaction (10 μL) was processed for intact
MS by adding an equal volume of 6 M Guan HCl, 100 mM Tris, pH 8,
20 mM DTT, 10 mM TCEP and incubating at room temperature for
15 min. Intact MS analysis was performed on an Agilent 6520 QToF
mass spectrometer equipped with a dual spray ion source (IS of 4500 V,
fragmentor of 250 V, fas temp of 350 °C, and skimmer of 75 V). The
samples were injected onto a PLRP-S column (2.1 mm × 50 mm,
Agilent), heated to 60 °C, and desalted for 2 min at 500 μL/min and
3% B prior to elution with a fast gradient of 3−50% B in 3 min
(B, 0.1% formic acid, MeCN). The data were analyzed in MassHunter
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-858-332-4452; e-mail: glelais@gnf.org.
■
Present Address
∥R.F.: Genentech, 1 DNA Way, South San Francisco, CA
94080, U.S.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the contribution to this work of all
past and present members of the mutant EGFR team. A big
thank you goes also to all supporting functions including Paul
P
DOI: 10.1021/acs.jmedchem.5b01985
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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Hollenbeck and his team (analytical group), Lucas Westling
(chiral analysis and purifications), David Jones (NMR), Connie
Chen (protein crystallization), and Tove Tuntland and her
team (DMPK).
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McNamara, D. J.; Nelson, J. M.; Sherwood, V.; Smaill, J. B.; Trumpp-
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ABBREVIATIONS USED
■
MoA, mode of action; OS, overall survival; HT-sol, high-
throughput solubility; HTRF, homogeneous time-resolved
fluorescence; GSH, glutathione; CL, clearance; LipE, lipophilic
efficiency; T/C, treatment/control; Da, dalton; RP-HPLC,
reverse phase high-performance liquid chromatography; PDA,
photodiode array detector; ELSD, evaporative light scattering
detector; DIPEA, N,N-diisopropylethylamine
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an irreversible second-generation EGFR inhibitor. Mol. Cancer Ther.
2012, 11, 784−791.
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M.; Cortot, A. B.; Chirieac, L.; Iacob, R. E.; Padera, R.; Engen, J. R.;
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