Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR)

Article abstract of DOI:10.1021/jm3007933

A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity.

Full text of DOI:10.1021/jm3007933

Article
pubs.acs.org/jmc
Discovery of a Novel Class of Highly Potent, Selective,
ATP-Competitive, and Orally Bioavailable Inhibitors of the
Mammalian Target of Rapamycin (mTOR)
Craig S. Takeuchi, Byung Gyu Kim,*,† Charles M. Blazey, Sunghoon Ma, Henry W. B. Johnson,
Neel K. Anand, Arlyn Arcalas, Tae Gon Baik, Chris A. Buhr, Jonah Cannoy, Sergey Epshteyn,
Anagha Joshi, Katherine Lara, Matthew S. Lee, Longcheng Wang, James W. Leahy,*,‡ John M. Nuss,
Naing Aay, Ron Aoyama, Paul Foster, Jae Lee, Isabelle Lehoux, Narsimha Munagala, Arthur Plonowski,
Sharmila Rajan, John Woolfrey, Kyoko Yamaguchi, Peter Lamb, and Nicole Miller
Department of Drug Discovery, Exelixis, 169 Harbor Way, South San Francisco, California 94083, United States
ABSTRACT: A series of novel, highly potent, selective, and ATP-competitive
mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold
have been identified. Lead optimization resulted in the discovery of inhibitors with low
nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K
kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex
1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates.
Furthermore, this compound displayed good pharmacokinetics and oral exposure in
multiple species with moderate bioavailability. Oral administration of compound 28 to
athymic nude mice implanted with human tumor xenografts afforded significant and
dose-dependent antitumor activity.
inhibit both mTORC1 and mTORC2 may offer a clinical
advantage over rapalogues.
INTRODUCTION
■
The mammalian target of rapamycin (mTOR) is a large protein
kinase that integrates both extracellular and intracellular signals
of cellular growth, proliferation, and survival. Both extracellular
mitogenic growth factor signaling from cell surface receptors
and intracellular signals that convey hypoxic stress, energy, and
nutrient status converge at mTOR.1−4 mTOR exists in two
distinct multiprotein complexes: mTOR complex 1 (mTORC1)
and mTOR complex 2 (mTORC2). mTORC1 is a key mediator
of translation and cell growth, via its substrates p70S6 kinase
(p70S6K) and eIF4E binding protein 1 (4E-BP1), and promotes
cell survival via the serum and glucocorticoid-activated kinase
(SGK), whereas mTORC2 promotes activation of prosurvival
kinase AKT.5−8 mTORC1, but not mTORC2, can be inhibited
by an intracellular complex between rapamycin and FK506
binding protein (FKBP).9,10 However, rapamycin−FKBP may
indirectly inhibit mTORC2 in some cells by sequestering mTOR
protein and thereby inhibiting assembly of mTORC2.11
Given the role of mTOR signaling in cellular growth,
proliferation, and survival as well as its frequent deregulation
in cancers, several rapamycin analogues (rapalogues) that are
selective allosteric mTORC1 inhibitors have been extensively
evaluated in a number of cancer clinical trials. Demonstrated
clinical efficacy for rapalogues is currently limited to patients
with advanced, metastatic renal cell carcinoma (RCC) despite
extensive development efforts. This result is likely attributed
not only to a lack of inhibition of mTORC2 by rapalogues that
leads to upregulation of Akt through a negative feedback loop,
but also to only partial inhibition of mTORC1.12,13 Therefore,
ATP-competitive mTOR inhibitors that should simultaneously
As a key component of the phosphoinositide 3-kinase-related
kinase (PIKK) family, which is comprised of phosphoinositide
3-kinases (PI3Ks), DNA-PK, ATM, and ATR, mTOR shares
the highly conserved ATP binding pockets of the PI3K family
with sequence similarity of 25% in the kinase catalytic
domain.14 In light of this fact, it is not surprising that many
of the first reported ATP-competitive mTOR inhibitors such as
BEZ235 and GDC-0980 (Figure 1) also inhibited PI3Ks.15−18
PI3Ks are responsible for the production of 3-phosphoinositide
lipid second messengers such as phosphatidylinositol 3,4,5-
triphosphate (PIP3), which are involved in a number of critical
cellular processes, including cell proliferation, cell survival,
angiogenesis, cell adhesion, and insulin signaling. Therefore,
the development of ATP-competitive mTOR inhibitors that
are selective over PI3Ks may offer an improved therapeutic
potential relative to rapalogues as well as dual PI3K/mTOR
inhibitors. Recently, several selective ATP-competitive mTOR
inhibitors such as Torin 2 and AZD8055 (Figure 1) have been
reported with sufficient promise to warrant clinical trials.11,19−26
Herein we describe the identification, characterization, and lead
optimization of a structurally novel class of mTOR inhibitors
exemplified by compound 28, derived from a benzoxazepine
scaffold.
Received: June 6, 2012
© XXXX American Chemical Society
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Figure 1. Literature examples of dual PI3K/mTOR and selective ATP-competitive mTOR inhibitors.
Figure 2. Early benzoxazepine derivatives of HTS hit 1. Note 1: average IC50 (n ≥ 2).
topological polar surface area below 60 Å2, five rotatable bonds,
four hydrogen bond acceptors, and no hydrogen bond donors,
it falls within traditionally desirable druglike parameters. Given
these characteristics, we were therefore surprised that the
benzoxazepine scaffold has received relatively little attention in
drug discovery.
While reconfirming the activity of our initial lead, we also
performed a rapid assessment of simple perturbations around
the benzoxazepine scaffold which suggested that both sub-
stituents were critical. Analogues lacking the quinoline at the
7-position (1a) and the 4-ketobenzamide at the 4-position (1b)
displayed none of the activity of the original lead. Simplified
benzamide 1c, however, was only slightly less potent than 1
RESULTS AND DISCUSSION
■
Efforts directed toward the identification of mTOR-selective
inhibitors were initiated by high-throughput screening (HTS)
of our in-house compound library against mTOR. The
measurement of mTOR activity was performed in an ELISA
assay format following the phosphorylation of 4E-BP1 protein
in a 384-well format. We found a number of potential
candidates suitable for lead optimization studies, but one that
we found to be particularly attractive was benzoxazepine 1
(Figure 2). It was a potent inhibitor of mTOR (IC50 = 95 nM)
with 4-fold selectivity over PI3Kα (IC50 = 398 nM) and much
more pronounced selectivity over other kinases in a preliminary
screen. With a molecular weight below 500, a cLogP below 5, a
B
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Figure 3. (A) Proposed binding orientation of BEZ235 in PI3Kγ (PDB code 1E8Z).27 (B) Potential binding orientation of 1 where benzoxazepine
oxygen forms a hinge region hydrogen bond. (C) Potential binding orientation of 1 where quinoline nitrogen forms a hinge region hydrogen bond.
with modestly improved selectivity over PI3Kα. This provided
us with some confidence that further optimization was possible.
While we were encouraged by the potential of 1 as an initial
candidate, it was unclear exactly how it was interacting with the
target, and we did not have crystallographic mTOR assets to
provide any insight. An examination of how compounds such as
BEZ235 interact with the structurally similar PI3K suggested a
critical hydrogen bond between the core imidazoquinoline ring
and the hinge region valine and provided a schematic model
for the binding pocket (Figure 3).27 One possible binding
orientation for 1 involved the quinoline interacting in a similar
fashion to form a hydrogen bond at the same site. Alternatively,
the ring oxygen of the benzoxazepine core could be envisioned
to perform this function. The latter possibility represents a
binding orientation that has never been described, but seemed
plausible in light of the numerous morpholine oxygens such as
in GDC-0980 that have been reported to play a similar role.18
Furthermore, even though we were intrigued by the
promising characteristics of 1, we knew that there was plenty
of room for improvement. In addition to increasing potency
and selectivity, we needed to find analogues with cellular
activity and improved ADME (absorption, distribution,
metabolism, and excretion) properties such as poor mouse
liver microsome stability (23% remained after 30 min),
moderate kinetic solubility (Ksol = 40.1 μM), and poor cell
The more constrained indazole group provided analogues with
significantly greater mTOR activity and restored selectivity over
PI3Kα. We were also gratified to note that both indazole
analogues 6 and 7 exhibited submicromolar cellular activity
as well. The impact of functionality capable of serving as a
proton donor appears to be substantial, as methylated analogue
9 leads to a >20-fold loss in potency versus desmethyl derivative
8. Additionally, the drop in activity of the truncated pyrazole
analogue 10 demonstrated the value of the phenyl “spacer”.
The best group found from these initial efforts was the
benzimidazole (11 and 12), with 12 being the most potent in
our biochemical assay thus far. In fact, 12 was the first
subnanomolar mTOR inhibitor we found with greater than
100-fold selectivity over PI3Kα. The benzimidazole was a
particularly intriguing alternative, as the tautomeric nature
allows for multiple orientations that could serve as a proton
donor. We were pleased to note a corresponding increase in
cellular potency for the benzimidazoles. In addition, both 11 and
12 showed comparable stability in a mouse liver microsome
stability study with approximately 50% of both compounds
remaining after 30 min. These data translated to similar mouse
plasma exposure levels of both 11 (1.5 μM at 1 h and 0.4 μM
at 4 h) and 12 (1.0 μM at 1 h and 0.2 μM at 4 h) when
dosed orally at 100 mg/kg. We considered 11 to be slightly
superior to 12 due to increased cellular potency and higher oral
exposure in mouse. However, 11 suffered from poor ADME
characteristics such as low kinetic solubility (Ksol < 1.0 μM),
poor cell permeability (MDCK Papp = 9.8 nm/s), and inhibition
of <3 μM against CYP 2C9 and 3A4 isoforms. Additionally, we
were interested in compounds with a greater level of selectivity
against PI3Kα.
In light of the evidence that suggested the binding orienta-
tion depicted in Figure 3B, we were reasonably confident that
substitution at any other position around the aromatic portion
of the benzoxazepine core would interfere with mTOR binding.
All attempts to explore even the simplest analogues led to
a complete loss of activity and were quickly abandoned. We
therefore turned our attention to exploring substitution about
the oxazepine ring.
We anticipated that the trifluoromethyl ketone functionality
on our initial hit represented an opportunity for optimization of
the ADME characteristics. We therefore prepared a number of
amide derivatives using the benzimidazole as a standard sub-
stituent. Given the dramatic drop in activity we observed in our
initial SAR efforts when we removed the para substituent,
we decided to focus on those derivatives. We were pleased to
discover that a variety of substituents were well tolerated
(Table 2) with respect to mTOR activity. Extrusion of the
ketone carbonyl group (13) significantly improved mTOR
permeability (Madin−Darby canine kidney (MDCK) Papp
=
16.9 nm/s). Toward that end, in vitro cellular activity was
examined by assessing inhibition of phosphorylation of pS6
(S240/244), a downstream protein in mTORC1 signaling, in
a PC-3 cell line.28 Inhibition of growth-factor-stimulated
phosphorylation of AKT (S473), a downstream protein in
mTORC2 signaling, was also determined in the same cell line
for compounds with >100-fold selectivity and >200 nM IC50
against PI3Kα.
We initiated our structure−activity relationship (SAR)
efforts with the goal in mind of determining which of the
two likely binding orientations was responsible for our activity.
Naphthalene derivative 2 rapidly provided the answer (Table 1).
Retention of the mTOR activity of 2 effectively ruled out the
potential binding orientation depicted in Figure 3C and
prompted us to focus our attention on the prospects of the
novel binding orientation in Figure 3B. Furthermore, this simple
change afforded a substantial improvement in the selectivity for
mTOR activity over PI3Kα, and even the simple phenyl sub-
stituent 3 was reasonably active. Addition of polar functionality
has a dramatic impact on activity, especially when appended in
a slightly extended orientation. For example, installation of an
acetamide such as in 5 leads to a substantial increase in activity
against PI3Kα with virtually no impact on mTOR activity.
C
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Table 1. mTOR Activity, PI3Kα Activity, Cellular Activity, MLM, and CYP Inhibition Data for Right-Hand-Side Modifications
a
of 1
a
Notes: (1) average IC50 (n ≥ 2); (2) pS6 (S240/244) in PC-3; (3) percent remaining after 30 min; (4) Not determined.
Table 2. mTOR Activity, PI3Kα Activity, Cellular Activity, MLM, and CYP Inhibition Data for Right-Hand-Side Modifications
of 11
CYP inhibition (μM)
a
a
b
a
c
compd
X
mTOR IC50 (nM)
PI3Kα IC50 (nM)
cell IC50 (nM)
MLM
2C8
2C9
2D6
3A4
11
13
14
15
16
17
COCF3
CF3
2.3
3.5
2.9
3.5
4.4
3.6
89
1092
763
151/−
49
74
64
64
82
72
6.2
3.6
0.8
1.6
1.3
1.1
3.0
1.7
4.0
3.1
4.3
2.3
8.1
3.3
2.3
1.9
6.3
2.8
3.8
2.2
306/680
1801/1672
451/812
342/805
343/1077
SO2NH2
SO2NHMe
SO2Me
SO2Et
13.9
1.6
1090
670
>20
5.0
2172
a
b
c
Average IC50 (n ≥ 2). pS6 (S240/244)/pAKT (S473) in PC-3. Percent remaining after 30 min.
D
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Table 3. mTOR Activity, PI3Kα Activity, Cellular Activity, MLM, and CYP Inhibition Data of Substituted Modifications of 16
CYP inhibition (μM)
a
a
b
a
c
compd
R1
R2
mTOR IC50 (nM)
PI3Kα IC50 (nM)
cell IC50 (nM)
MLM
2C8
2C9
2D6
3A4
16
18
19
20
21
22
23
24
25
26
H
H
H
H
H
H
H
H
Me
F
4.4
5.7
670
>3000
>3000
>3000
>3000
705
343/805
422/1007
233/689
1184/3302
nd/nd
82
51
50
87
nd
58
45
68
94
>20
5.4
3.0
2.9
5.4
nd
8.1
9.3
>20
nd
3.8
4.1
2.0
nd
Me
Et
5.2
5.6
d
n-Pr
NHEt
Cl
28.0
204
23.2
5.2
nd
nd
5.4
3.4
nd
3.4
3.1
nd
nd
nd
337/1062
343/453
591/1219
329/437
152/277
9.0
4.2
nd
9.2
9.2
nd
3.5
3.0
nd
Br
693
H
6.6
459
Me
Et
1.4
2344
6.4
2.2
9.6
11.5
3.4
2.6
F
2.9
>3000
59
a
b
c
d
Average IC50 (n ≥ 2). pS6 (S240/244)/pAKT (S473) in PC-3. Percent remaining after 30 min. Not determined.
selectivity over PI3Kα without any substantial loss of activity in
our primary assays. In addition, this simple change conveyed
some stability upon exposure to mouse liver microsomes,
although it did not improve upon the CYP inhibition of 11.
A number of analogues were identified that demonstrated
>100-fold selectivity for mTOR over PI3Kα, which allowed us
to focus on those with improved ADME profiles. Sulfonamides
14 and 15, for example, suggested that the poor permeability
(MDCK Papp = 42 nm/s) of the former correlated well with
a lack of cellular activity. Among the best of these initial
congeners were the alkyl sulfones. Methyl sulfone 16 presented
an improvement in CYP inhibition and mouse liver microsome
stability compared to 11 and exhibited significantly higher
mouse plasma exposure (7.8 μM (at 1 h) and 1.0 μM (at 4 h))
when dosed orally at 100 mg/kg. We felt that these attributes
made 16 more attractive than ethyl sulfone 17 despite the
increased mTOR selectivity of the latter compound.
Using the methyl sulfone as a reference, we initiated an
exploration of additional substitution around the benzamide
ring. The introduction of small alkyl groups ortho to the
carbonyl such as in 18 and 19 conferred additional selectivity
for mTOR over PI3Kα (Table 3). The erosion of activity of
n-propyl derivative 20 suggests a spatial limitation to
perturbations at this position, and the more significant loss of
activity of the isosteric 21 infers that the potential internal
hydrogen bond between the NH and the carbonyl is not
favored. Other types of ortho substituents such as the
halogenated analogues 22 and 23 were tolerated, but did not
lead to any discernible improvement. Likewise, monosubstitu-
tion at the position ortho to the sulfonyl group such as in 24
failed to impart any benefit over 16. However, substitution at
both positions proved beneficial. For example, fluorinated
analogues 25 and 26 are both more than 1000-fold selective for
mTOR over PI3Kα. Additionally, the mouse liver microsome
stability of 25 was particularly attractive, as >90% remained
after incubation for 30 min.
microsome profile for the aminopyridine analogue is a
considerable improvement over that of the original benzimida-
zole, even though that improvement comes with approximately
a 3-fold loss in potency. We reasoned that the use of the
optimized amide would restore some of that lost activity, which
was confirmed by comparison of benzimidazole 25 to
aminopyridine 28 (XL388).29 It should be noted at this point
that our biochemical assay proved to be reasonably robust.
Over the course of 35 different IC50 determinations for 28, we
observed a range between 14.7 and 4.3 nM with a standard
deviation of 2.8. We were delighted to see that 28 was not only
very active and selective in our biochemical and cellular assays,
but exhibited an attractive in vitro ADME and physicochemical
profile. Our initial screen of CYP isoform inhibition did not
reveal significant inhibition below a concentration of 10 μM,
but did reveal a reasonable mouse liver microsome stability
of 75% after incubation for 30 min along with acceptable
permeability (MDCK Papp = 151 nm/s) and kinetic solubility
(Ksol = 232 μM).
In light of the promising in vitro characteristics of 28, we
undertook an investigation of its in vivo parameters. The mean
plasma protein binding of 28 in human, monkey, dog, rat, and
mouse plasma was evaluated at 5 μM and was determined to
be 86%, 90%, 89%, 85%, and 84%, respectively, suggesting a
significant free concentration of 28 in the plasma. An initial
examination in a mouse study revealed substantial and sustained
plasma exposure (22.0 μM at 1 h and 17.8 μM at 4 h) following
an oral dose at 100 mg/kg. Further examination of the liver
microsomal stability of 28 in a mammalian panel revealed that
93%, 65%, 82%, and 78% for human, monkey, dog, and rat,
respectively remained after 30 min. Gratifyingly, this promising
stability data translated to reasonable oral exposure in expanded
pharmacokinetic studies (Table 5). The dog pharma-
cokinetic profile of 28 dosed as a solution at 3 mg/kg is
shown in Figure 4.
To ascertain the mode of inhibition of mTOR by 28, we
determined the IC50 values at varying ATP concentrations.30
These results revealed that 28 acts in an ATP-competitive
manner, with a linear increase in IC50 values with increasing
ATP concentration (Figure 5). Additionally, 28 demonstrated
rapidly reversible inhibition of mTOR activity. A solution of
Equipped with an improved amide alternative, we revisited
the benzimidazole standard with the intent of improving the
CYP inhibition profile. After extensive evaluation, the amino-
pyridine proved to be the best alternative. Comparison of 16
with 27 (Table 4) shows that the CYP and mouse liver
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a
Table 4. Summarized Data of Advanced Compounds
a
Notes: (1) average IC50 (n ≥ 2); (2) pS6 (S240/244)/pAKT (S473) in PC-3; (3) percent remaining after 30 min.
Table 5. PK Properties of 28
species
Cmax, iv/po
Tmax, po
AUC0−∞ (μM·h), iv/po
T1/2 (h), iv
CL (L/kg/h), iv
Vss (L/kg), iv
F (%), po
a
mouse (10 mg/kg)
9.14/1.30
4.12/0.707
2.28/1.66
0.95/0.25
0.5
0.5
1.0
2.0
8.00/2.51
3.72/0.952
11.0/7.66
1.35
0.45
6.11
0.86
2.75
1.27
0.598
5.05
1.96
1.27
3.15
6.42
31.4
25.6
69.6
53.4
a
rat (3 mg/kg)
a
dog (3 mg/kg)
b
cyno (3 mg/kg)
1.31/0.70
a
b
Formulation EPW (5% EtOH/45% PEG400/water + 1:2 HCl (m/m). Formulation EPW (5% EtOH/45% PEG400/water + 1:1.5 HCl (m/m).
Figure 4. Pharmacokinetic profile of 28 in dogs. Compound 28 was dosed at 3 mg/kg in male beagles. Data are reported as the mean SD (n = 3).
mTOR saturated with 28 was diluted with a mixture containing
1 mM ATP, revealing rapid and complete recovery of enzyme
activity (t1/2 < 2 min) compared to an enzyme control in the
absence of inhibitor. In addition, 28 showed no cross-reactivity
(<3000 nM) with members of the PI3KK lipid kinase super-
family as well as a panel of 141 protein kinases tested in house
or by Millipore (Table 6). The hERG (manual Patchclamp,
15.9 μM), P-glycoprotein (>20 μM), and PXP CYP3A4 induction
(50 μM) of 28 was also characterized. Collectively, the attractive
in vitro and in vivo ADME profile along with high mTOR bio-
chemical and cell potency of 28 provided validation that it could
serve as a viable selective and ATP-competitive mTOR inhibitor.
To assess the pharmacodynamic effects of our inhibitor on
the mTOR pathway signaling, athymic nude mice bearing PC-3
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and homogenized with buffer. Tumor lysates from each animal
(n = 5) were then pooled for each group and analyzed by
immunoblot for levels of phosphorylated p70S6K, S6, 4E-BP1,
and AKT. Strong inhibition of both mTORC1 readout
(p-p70S6K, p-S6, p-4E-BP1) and mTORC2 readout (p-AKT
(S473)) was achieved 4−8 h following administration of 28,
which diminished to near-basal levels of phosphorylation by
16 h postdose (Figure 6). Modest inhibition (39−45%) of
phosphorylation of the PI3K target AKT (T308) was also
observed 4−8 h postdose, returning to basal levels of phos-
phorylation by 16 h postdose. Rapamycin resulted in strong
inhition of the mTORC1 biomarkers but had no effect on the
mTORC2 biomarker as expected. Similar levels of pharmaco-
dynamic effects of 28 on the mTOR signaling pathway were
also observed in athymic nude mice bearing MCF-7 xenograft
tumors when dosed orally at 100 mg/kg.31 Maximal inhibition
(85−96%) of phosphorylation of mTORC1 readout (p70S6K,
S6, 4E-BP1) as well as mTORC2 readout (AKT (S473)) was
achieved 1 h following administration of 28 and was sustained
through 8 h postdose (Figure 7). Significantly, moderate to strong
Figure 5. IC50 for 28 as a function of the ATP concentration. Error
bars represent the standard error of the mean on four IC50 values at
each ATP concentration.
prostate tumors were dosed orally at 100 mg/kg of 28.
Rapamycin was also administered intraperitoneally at 5 mg/kg
as a reference. Plasma and tumor samples were collected at 1, 4,
8, 16, 24, and 32 h for 28 and at 4 h for rapamycin after dosing
Table 6. Kinase Profile of 28
a
kinase
IC50 (nM)
b
b
b
b
,
,
,
,
c
c
c
c
PI3Kα
>3000
>5000
>5000
>5000
PI3Kβ
PI3Kγ
PI3Kδ
b
DNA-PK
8831
b
,
c
c
VPS34
>3500
>3500
>10000
b
,
AKT1; ALK; CDC7; CHK1; cRAF1; EGFR; ERBB2; FAK; FLT1; FLT3; HSP90; IRK; JAK2; KDR; KIT; MEK1; MET; p70S6K; PDGFRβ; PDK1; PIM1; SRC
b,c
EPH4; FGFR1; FGFR2; FGFR3; FGFR4; IPFK2; RSK2
b,d
ABL; ACK1; ARG; AMPK; ASK1; AKT2; AKT3; Aurora-A; AXL; BLK; BRK; BRSK1; CaMKI; CaMKIV; CDK3/CyclinE; CDK5/p25; CHK2; CK1δ; CK2; >10000
CLK2; CSK; DAPK2; DDR2; DYRK2; EPHA2; EPHB1; FES; GCK; GRK5; GSK3α; GSK3β; Haspin; HCK; HIPK2; IGF-1R; IKKα; IKKβ; IRAK1; ITK;
JNK1α1; JNK2α2; JNK3; LIMK1; LKB1; LOK; LYN; MAPK1; MAPK2; MAPKAP-K3; MARK1; MER; MINK; MKK4; MKK6; MKK7β; MLK1; MNK2;
MSK1; MSSK1; MST1; MuSK; NEK2; NEK7; NLK; PAK3; PAK6; PASK; PDK1; PKA; PKCα; PKCβI; PKCβII; PKCγ; PKCδ; PKCε; PKCη; PKCι; PKCμ;
PKCθ; PKCζ; PKD2; PKG1β; PLK1; PLK3; PRAK; PRK2; PYK2; RET; RIPK2; ROCK-I; ROCK-II; ROS; RSE; RSK1; RSK3; RSK4; SGK; SGK2; SGK3;
STK33; TAK1; TAO1; TBK1; TSSK1; ULK2; WNK2; VRK2; YES; ZAP-70; ZIPK
a
b
c
d
Average IC50 (n ≥ 2). Kinases tested in house. Highest concentration tested. Kinases tested by Millipore (Charlottesville, VA).
Figure 6. Inhibition (%) of phosphorylation of p70S6K, S6, 4E-BP1, and AKT in PC-3 xenograft tumors and exposure data after a single oral dose
(100 mg/kg).
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Figure 7. Inhibition (%) of phosphorylation of p70S6K, S6, 4E-BP1, and AKT in MCF-7 xenograft tumors and exposure data after a single oral dose
(100 mg/kg).
Figure 8. In vivo efficacy of 28 in MCF-7 xenograft tumors.
(36−69%) inhibition of phosphorylation was still observed
through 16 h postdose before returning to basal levels by the
24 h time point.
once daily also for 14 days. Compound 28 resulted in complete
inhibition of MCF-7 xenograft tumor growth at both doses
(Figure 8), with significant tumor regression of 22% and
40% below pretreatment values at the 50 and 100 mg/kg doses,
respectively. Furthermore, 28 does not appear to show
significant toxicity. Mice in the 50 mg/kg cohort over the
course of the study gained on average 2.2% additional mass,
while those in the 100 mg/kg cohort lost on average 2.8% of
their body mass. These results correlate well with the
pharmacodynamic activity of 28 and taken in conjunction
Having identified a potent and selective inhibitor of mTOR
with strong pharmacodynamic effects and good mouse oral
exposure, we decided to assess its antitumor efficacy.
Specifically, 28 was administered to animals implanted with
MCF-7 xenograft tumors to determine its effects on tumor
growth. Rapamycin was used as a control to compare the
results to those of a selective mTORC1 inhibitor. Tumors were
established in female athymic nude mice and staged when
the average tumor mass reached 102 17.7 mg. Compound 28
was orally administered as a solution/fine suspension in
water (with 1:1 molar ratio of HCl) once daily (qd) at 50
and 100 mg/kg for 14 days. Rapamycin, formulated at 5 mg/kg
in 12.5 ethanol, 12.5 cremophor, and 75% Hank’s balanced
salt solution (HBSS), was administered intraperitoneally (ip)
with its inhibition of cell proliferation (proliferation IC50
=
1.37 μM in MCF-7 cell line) suggest that blockade of mTOR
signaling is a viable target for antitumor activity. Rapamycin
also resulted in complete inhibition of tumor growth, although
significant tumor regression below pretreatment values was not
achieved at 5 mg/kg.
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THF/MeOH afforded methyl 2-bromo-4-(methylsulfonyl)-
benzoate (32) (Scheme 2). Silver-promoted Suzuki−Miyaura
coupling of 32 with ethylboronic acid followed by hydrolysis
provided benzoic acid 33.34 In a similar manner, n-propyl
derivative 33 was prepared. Buchwald−Hartwig amination of
32 with ethylamine in the presence of xantphos gave the
corresponding anthranilate,35 which was hydrolyzed to yield
acid 35.
CHEMISTRY
■
We recognized from the outset that benzoxazepine 29 was
going to be a critical synthetic intermediate in this project.
While we initially accessed this compound via the previously
reported synthesis,32 subsequently a new synthetic route was
designed which was more amendable to a larger scale prepara-
tion of the intermediate in Scheme 1. We envisioned that
The synthesis of tetrasubstituted carboxylate 37 began with
bromination of 2,3-difluorotoluene followed by selective
metal−halogen exchange of the bromide and trapping with
CO2 to give acid 36 (Scheme 3). Selective displacement of the
a
Scheme 1
a
Scheme 3
a
Reagents and conditions: (a) 2-aminoethanol, NaBH4, MeOH, Boc2O;
(b) (Ph)3P, DIAD, THF, (c) n-BuLi, THF, triisopropyl borate, −78 °C,
HCl.
a
i
Reagents and conditions: (a) Fe, Br2, CHCl3; (b) PrMgCl, THF,
CO2; (c) NaSCH3, DMF, Oxone, NaOH, NaHCO3, acetone/H2O
(1:1), 0 °C.
the benzoxazepine core could be accessible from intermediate
30, which could be generated from commercially available
5-bromosalicylaldehyde. Reductive amination of 5-bromosalicyl-
aldehyde with 2-aminoethanol followed by Boc protection
provided 30, which was then converted to benzoxazepine 29
through a Mitsunobu reaction. Particularly noteworthy about
this new route is that we could routinely prepare over 200 g
of benzoxazepine 29 in a single batch. Metal−halogen exchange
of 29 with n-BuLi at −78 °C in the presence of triisopro-
poxyborate afforded benzoxazepinylboronic acid 31, another
useful synthetic intermediate.
fluoride para to the carboxylate with sodium thiomethoxide and
subsequent oxidation with Oxone afforded 3-fluoro-2-methyl-4-
(methylsulfonyl)benzoic acid (37). A similar approach was
used for the preparation of ethyl derivative 39, but we required
a convenient source of the requisite 2,3-difluoroethylbenzene.
Borane reduction of the corresponding commercially available
acetic acid produced the anticipated primary alcohol (Scheme 4),
which was efficiently converted to 38 via tosylation, bromination,
and reductive dehalogenation. With the appropriate ethyl starting
material in hand, acid 39 was generated without incident
following the previously developed protocol.
We also required a synthetic route that would provide access
to noncommercially available 4-(methylsulfonyl)benzoic acid
derivatives. Toward that end, treatment of 2-bromo-4-(methyl-
sulfonyl)benzoic acid33 with (trimethylsilyl)diazomethane in
Our plan from the outset was to install the left-hand-side
functionality via a Suzuki−Miyaura coupling. For coupling
partners where the desired boronic acid was commercially
a
Scheme 2
a
Reagents and conditions: (a) (TMS)CHN2, THF/MeOH (2:1), 0 °C; (b) RB(OH)2, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium-
(II) complex with dichloromethane, K2CO3, Ag2O, THF, 85 °C; (c) MeOH, 1 N NaOH, 45 °C; (d) ethylamine, Pd2(dba)3, xantphos, Cs2CO3,
1,4-dioxane, 95 °C; (e) CH2Cl2/MeOH (1:1), 1 N NaOH, 45 °C.
I
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a
a
Scheme 4
Scheme 6
a
Reagents and conditions: (a) BH3−SMe2, THF; (b) TsCl, Et3N,
a
CH2Cl2, LiBr, acetone; (c) Mg, Et2O, BrCH2CH2Br, aqueous NH4Cl;
(d) Fe, Br2, CHCl3; (e) PrMgCl, THF, CO2; (f) NaSCH3, DMF,
Reagents and conditions: (a) 6-bromo-1H-indazole, 1,1′-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) complex with
dichloromethane, triethylamine, 1,4-dioxane, H2O, 85 °C; (b) 4 N
HCl, 1,4-dioxane, rt; (c) N,N-diisopropylethylamine, HATU, DMF,
4-(2,2,2-trifluoroacetyl)benzoic acid, rt.
i
Oxone, NaOH, NaHCO3, acetone/H2O (1:1), 0 °C.
available, direct coupling with bromide 29 formed the desired
carbon−carbon bond such as for 40 in Scheme 5. Standard Boc
a
models. Furthermore, an efficacy study in mice bearing MCF-7
xenograft tumors revealed complete tumor growth inhibition.
On the basis of these results, compound 28 was selected for
drug candidate (DC) status and was subjected to further
preclinical evaluation.
Scheme 5
EXPERIMENTAL SECTION
■
All reagents and solvents employed were purchased commercially and
used without further purification unless otherwise indicated. Melting
points were determined on a Melt-Temp melting point apparatus,
model II, and are uncorrected. NMR spectra were recorded on a
Varian Mercury Plus 400 MHz instrument. 13C NMR spectra are
assigned with peaks “as observed” with no comment on splitting
patterns. Reported spectra may appear to contain superfluous signals
due to the existence of rotamers and/or carbon−fluorine coupling.
Chemical shifts are reported in parts per million relative to an internal
standard of tetramethylsilane in deuterated dimethyl sulfoxide
(DMSO-d6), deuterated methanol (CD3OD), or deuterated chloro-
form (CDCl3). Positive ion fast atom bombardment (FAB) high-
resolution mass spectrometry was conducted by Analytical Instrument
Group, Inc., Raleigh, NC. All final compounds were purified to ≥95%
purity as assayed by analytical HPLC (YMC-Pack Pro 150 × 4.6 mm,
5 μm C18 column, Shimadzu LC-10AT VP system equipped with a
Shimadzu SPD-M10A VP diode array detector) at a 1.5 mL/min flow
rate with a gradient of 5−95% acetonitrile (containing 0.1% TFA) in
0.1% aqueous TFA for 25 min and a total run time of 27 min.
Pharmacokinetic, liver microsome assay, CYP inhibition, MDCK, and
Ksol data, reported as average IC50 (n ≥ 2), were carried out as
previously described.36,37
tert-Butyl 7-Bromo-2,3-dihydrobenz[f ][1,4]oxazepine-4(5H)-
carboxylate (29). To a stirred solution of 2-aminoethanol (6.11 g,
100 mmol) in MeOH (60 mL) was added 5-bromosalicylaldehyde
(20.1 g, 100 mmol). The reaction mixture was stirred at ambient
temperature for 2 h. To this mixture was added sodium borohydride
(1.51 g, 40.0 mmol) portionwise with vigorous stirring. After being
stirred for an additional 30 min, the reaction mixture was diluted with
6 N NaOH (60 mL). To this mixture was added di-tert-butyl
dicarbonate (32.7 g, 150 mmol) slowly. After the reaction mixture was
stirred for 30 min, an additional amount of di-tert-butyl dicarbonate
(10.9 g, 50.0 mmol) was added, and the reaction mixture was stirred
for an additional 30 min. Most of the methanol was removed on a
rotary evaporator, and the residual solid was suspended with H2O
(50 mL). To this suspension was added 1 N HCl slowly with vigorous
stirring until the reaction mixture reached pH 4−5. The white
precipitates were collected by filtration and washed with H2O. The
precipitates were then suspended with EtOAc (50 mL) and filtered
to provide 30 (25.8 g, 75%) as a white solid. MS (EI): m/z for
C14H20BrNO4, found 346.0 (MH+).
a
Reagents and conditions: (a) quinolin-3-ylboronic acid, [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex
with dichloromethane, triethylamine, 1,4-dioxane, H2O, 85 °C; (b)
4 N HCl, 1,4-dioxane, rt; (c) N,N-diisopropylethylamine, HATU,
DMF, 4-(2,2,2-trifluoroacetyl)benzoic acid, rt.
deprotection in acid followed by HATU (O-(7-azabenzotriazol-
1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)
coupling with 4-(2,2,2-trifluoroacetyl)benzoic acid afforded
compound 1. Compounds 1a−c, 2−5, and 10 were synthesized
in a similar manner. Alternatively, the Suzuki−Miyaura
coupling reaction could be performed using the boronic acid
of the benzoxazepine previously described. For example,
reaction of 31 with 6-bromo-1H-indazole smoothly formed
the anticipated product 41 (Scheme 6), which was then
subjected to the same deprotection/esterification sequence to
yield 6. This latter route was used for the synthesis of 7−9 and
11−28 and was sufficiently scalable that it was used to prepare
over 100 g of 28 in a single batch.
In conclusion, a series of structurally novel and highly potent
mTOR inhibitors with complete selectivity over the PI3Ks were
discovered. Starting from HTS hit 1, lead optimization efforts
on both the left- and right-hand sides of the benzoxazepine core
led to the identification of 28, which inhibits mTOR at low
nanomolar concentration while exhibiting exquisite selectivity
over a large panel of kinases tested. Compound 28 also
exhibited good in vitro multispecies liver microsome stability
and showed good oral exposure with moderate to good half-
lives in several species. Profound pharmacodynamic effects
were observed with 28 in both PC-3 and MCF-7 xenograft
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A stirred solution of 30 (17.3 g, 50.0 mmol) and triphenylphos-
phine (13.6 g, 52.0 mmol) in THF (100 mL) was cooled to 0 °C, and
diisopropyl azodicarboxylate (11.1 g, 55.0 mmol) was added slowly.
Upon completion of addition, the reaction mixture was stirred at
ambient temperature for 30 min and diluted with H2O (50 mL) and
Et2O (50 mL). The separated organic layer was washed with 1 N HCl
(50 mL), 1 N NaOH (50 mL), and brine, then dried over anhydrous
sodium sulfate, and concentrated on a rotary evaporator. The residue
was purified with a short silica gel plug (hexanes/ethyl acetate (20:1))
xantphos (18 mg, 0.03 mmol), Pd2dba3 (14 mg, 0.015 mmol), and
Cs2CO3 (326 mg, 1.00 mmol) in 1,4-dioxane (3 mL) was stirred in a
sealed tube at 95 °C for 15 h. The reaction mixture was cooled to
ambient temperature and filtered through Celite, and the filtrate was
concentrated on a rotary evaporator. The residue was purified by
flash chromatography (hexanes/ethyl acetate (4:1)) to afford methyl
2-(ethylamino)-4-(methylsulfonyl)benzoate (39 mg, 22%) as a yellow
1
solid. H NMR (400 MHz, CDCl3): δ 8.06 (d, J = 8.4 Hz, 1H), 7.88
(br s, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.04 (dd, J = 8.4, 2.0 Hz, 1H), 3.90
(s, 3H), 3.33−3.26 (m, 2H), 3.06 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H).
To a stirred solution of methyl 2-(ethylamino)-4-(methylsulfonyl)-
benzoate (39 mg, 0.15 mmol) in MeOH (1 mL) and CH2Cl2 (1 mL)
was added 1 N aqueous NaOH (1 mL), and the resulting mixture was
stirred at 45 °C for 1 h. The reaction mixture was cooled to ambient
temperature and concentrated on a rotary evaporator. The aqueous
residue was acidified to pH 4 with HCl and extracted with EtOAc
(10 mL × 2). The combined organic layers were dried over anhydrous
sodium sulfate and concentrated to afford 35 (37 mg, quantitative) as
a yellow solid. MS (EI): m/z for C10H13NO4S, found 242.0 (MH−).
3-Fluoro-2-methyl-4-(methylsulfonyl)benzoic Acid (37). To a
stirred mixture of 2,3-difluorotoluene (1.9 g, 15 mmol) and Fe (82.7 mg,
1.48 mmol) in chloroform (10 mL) was added bromine (760 μL,
14.8 mmol) slowly at ambient temperature, and the mixture was stirred
overnight. Water (10 mL) was added, and the reaction mixture was
extracted with Et2O (20 mL). The separated organic layer was washed
with aqueous sodium thiosulfate and brine, dried over anhydrous
sodium sulfate, and concentrated on a rotary evaporator. The residue
was purified by Kugelrohr distillation to give 1-bromo-3,4-difluoro-2-
methylbenzene (2.49 g, 81%) as a colorless oil.
A stirred solution of 1-bromo-3,4-difluoro-2-methylbenzene (2.49 g,
12.0 mmol) in THF (5 mL) was cooled to 0 °C, and a solution of
isopropylmagnesium bromide (9.0 mL, 18 mmol, 2 M in THF) was
added slowly so that the temperature was maintained. Upon
completion of the addition, the resulting stirred mixture was allowed
to warm to ambient temperature for 24 h. Carbon dioxide, generated
from dry ice, was introduced to the reaction mixture for 10 min, and
the resulting mixture was stirred for an additional 30 min. The reaction
mixture was quenched with H2O (20 mL), and the volatile solvents
were removed on a rotary evaporator. The resulting aqueous layer was
acidified with concentrated HCl to pH 1−2. The white precipitate that
formed was filtered, washed with water and cold hexanes, and dried in
vacuo to afford 36 (1.67 g, 81%). 1H NMR (400 MHz, DMSO-d6): δ
13.10 (br s, 1H), 7.72−7.68 (m, 1H), 7.33 (dd, J = 18.4, 8.8 Hz, 1H),
2.48 (s, 3H). MS (EI): m/z for C8H6F2O2, found 171.0 (MH−).
To a stirred solution of 36 (700 mg, 4.1 mmol) in DMSO (5 mL)
was added NaOH (164 mg, 4.1 mmol), and the mixture was stirred at
ambient temperature for 30 min. To this mixture was added sodium
thiomethoxide (342 mg, 4.9 mmol), and the resulting mixture was
warmed to 55−60 °C for 4 h. The reaction mixture was cooled to 0 °C
and quenched with H2O (10 mL). The resulting mixture was acidified
with concentrated HCl to pH 1−2. The white precipitate was collected
by filtration, washed with water, and dried in vacuo to afford 3-fluoro-
2-methyl-4-(thiomethyl)benzoic acid (879 mg, quantitative), which
was used in the next step without further purification. MS (EI): m/z
for C9H9FO2S, found 199.0 (MH−).
1
to give 29 (15.2 g, 93%) as a white powder. H NMR (400 MHz,
CDCl3): δ (rotamers are observed) 7.43−7.27 (m, 2H), 6.93−6.88
(m, 1H), 4.44 and 4.36 (s, 2H), 4.03 and 4.02 (d, J = 4.4 Hz, 2H), 3.79
and 3.78 (d, J = 4.4 Hz, 2H), 1.43 (s, 9H). MS (EI): m/z for
C14H18BrNO3, found 328.0 (MH+).
4-(tert-Butoxycarbonyl)-2,3,4,5-tetrahydrobenz[f ][1,4]-
oxazepin-7-ylboronic Acid (31). A stirred solution of 29 (10.0 g,
30.5 mmol) and triisopropyl borate (9.1 mL, 40 mmol) in THF
(100 mL) was cooled to −78 °C, and a solution of n-butyllithium
(25 mL, 40 mmol, 1.6 M in THF) was added dropwise such that the
temperature was maintained. Upon completion of addition, the
reaction mixture was stirred for an additional 30 min, then quenched
with 1 N aqueous HCl (35 mL), and allowed to warm to ambient
temperature. The reaction mixture was extracted with EtOAc (100 mL),
and the separated organic layer was dried over anhydrous sodium sulfate
and concentrated on a rotary evaporator. Hexanes were subsequently
added to the crude reaction mixture, which resulted in the formation of a
white solid. This slurry was stirred for 1 h at ambient temperature and
filtered to afford 31 (8.6 g, 95%). MS (EI): m/z for C14H20BNO3, found
194.0 (MH+ − Boc).
2-Ethyl-4-(methylsulfonyl)benzoic Acid (33). A stirred solution
of 2-bromo-4-(methylsulfonyl)benzoic acid (5.18 g, 18.6 mmol) in
THF (40 mL) and MeOH (20 mL) was cooled to 0 °C, and a solution
of (trimethylsilyl)diazomethane (12 mL, 24 mmol, 2.0 M in hexanes)
was added dropwise such that the temperature was maintained. After
being stirred for 2 h, the reaction mixture was concentrated on a rotary
evaporator to afford 32 (5.58 g, quantitative) as a colorless solid.
1H NMR (400 MHz, CDCl3): δ 8.24−8.23 (m, 1H), 7.94−7.92
(m, 2H), 3.99 (s, 3H), 3.09 (s, 3H).
A mixture of 32 (200 mg, 0.683 mmol), ethylboronic acid (55 mg,
0.75 mmol), potassium carbonate (283 mg, 2.05 mmol), Ag2O
(395 mg, 1.70 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II) complex with dichloromethane (50 mg, 0.068
mmol) in THF (5 mL) was stirred in a sealed tube at 80 °C for 18 h.
The reaction mixture was filtered through Celite, and the filtrate was
concentrated on a rotary evaporator. The residue was purified by flash
chromatography (hexanes/ethyl acetate (4:1)) to give methyl 2-ethyl-
4-(methylsulfonyl)benzoate (100 mg, 60%) as a colorless oil. 1H NMR
(400 MHz, CDCl3): δ 7.99 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 2.0 Hz,
1H), 7.81 (dd, J = 8.0, 2.0 Hz, 1H), 3.95 (s, 3H), 3.08 (s, 3H), 3.04
(q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H).
To a stirred solution of methyl 2-ethyl-4-(methylsulfonyl)benzoate
(94 mg, 0.39 mmol) in MeOH (1 mL) and CH2Cl2 (1 mL) was added
1 N aqueous NaOH (1 mL), and the resulting mixture was stirred
at 45 °C for 2 h. The reaction mixture was cooled to ambient
temperature and concentrated on a rotary evaporator. The aqueous
residue was acidified with 1 N aqueous HCl to pH 1−2 and extracted
with EtOAc (10 mL × 2). The combined organic layers were dried
over anhydrous sodium sulfate and concentrated to afford 33 (83 mg,
94%) as a colorless solid. MS (EI): m/z for C10H12O4S, found
227.0 (MH−).
To a stirred suspension of 3-fluoro-2-methyl-4-(thiomethyl)benzoic
acid (879 mg, 4.1 mmol) in acetone (10 mL) and H2O (10 mL) were
added NaOH (330 mg, 8.3 mmol) and sodium bicarbonate (680 mg,
8.1 mmol). The stirred mixture was cooled to 0 °C, and Oxone (4 g)
was added portionwise over 10 min. The reaction was monitored by
LC/MS. Upon the completion of the reaction, concentrated HCl was
added to acidify the mixture to pH 2−3. The white precipitate that
formed was collected by filtration, washed with water, and dried. The
precipitate was resuspended in water (10 mL), stirred vigorously at
ambient temperature for 1 h, filtered, washed with water and hexanes,
and dried in vacuo to afford 37 (886 mg, 94%) as a white powder. 1H
NMR (400 MHz, DMSO-d6): δ 13.74 (br s, 1H), 7.82−7.76 (m, 2H),
3.37 (s, 3H), 2.47 (s, 3H). MS (EI): m/z for C9H9FO4S, found
231.0 (MH−).
Compound 34 was prepared from 32 according to a procedure
similar to that described for the synthesis of 33.
Methyl 4-(Methylsulfonyl)-2-propylbenzoate. Colorless oil.
1H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 8.0 Hz, 1H), 7.84 (d, J =
1.6 Hz, 1H), 7.81 (dd, J = 8.0, 2.0 Hz, 1H), 3.95 (s, 3H), 3.07 (s, 3H),
2.98 (t, J = 8.0 Hz, 2H), 1.69−1.63 (m, 2H), 0.99 (t, J = 7.6 Hz, 3H).
4-(Methylsulfonyl)-2-propylbenzoic Acid (34). MS (EI): m/z
for C11H14O4S, found 241.0 (MH−).
2-(Ethylamino)-4-(methylsulfonyl)benzoic Acid (35). A solu-
tion of 32 (200 mg, 0.683 mmol), ethylamine (200 μL, excess),
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2-Ethyl-3-fluoro-4-(methylsulfonyl)benzoic Acid (39). To a
stirred solution of 2,3-difluorophenylacetic acid (17.2 g, 100 mmol) in
THF (150 mL) was added borane−dimethyl sulfide complex solution
(75 mL, 150 mmol, 2 M in THF) over the course of 1 h using a
dropping funnel. The reaction mixture started to reflux spontaneously.
After being stirred for an additional 3 h, it was cooled to 0 °C, and
excess MeOH (20 mL) was added carefully to the reaction mixture.
The stirred mixture was allowed to warm to ambient temperature for
1 h and then concentrated on a rotary evaporator. The residue was
diluted with dichloromethane (100 mL), washed with aqueous sodium
bicarbonate (100 mL), dried over anhydrous sodium sulfate, and
concentrated on a rotary evaporator to give 2-(2,3-difluorophenyl)-
ethanol (15.6 g, 99%), which was used in the next step without further
purification. GC/MS: m/z for C8H8F2O, found 158 (M+).
To a stirred solution of 2-(2,3-difluorophenyl)ethanol (15.6 g, 98.6
mmol) and Et3N (20.6 mL, 148 mmol) in dichloromethane (100 mL)
was added TsCl (20.7 g, 109 mmol). The reaction mixture was stirred
for 5 h at ambient temperature and then diluted with dichloromethane
(200 mL). The resulting solution was washed with water (100 mL)
and brine (100 mL), dried over anhydrous sodium sulfate, and
concentrated on a rotary evaporator to afford (2,3-difluorophenyl)-
ethyl 4-methylbenzenesulfonate (29.6 g, 96%), which was used in the
next step without further purification.
To a stirred mixture of (2,3-difluorophenyl)ethyl 4-methylbenzene-
sulfonate (29.6 g, 94.8 mmol) in acetone (150 mL) was added
LiBr (42.8 g, 492 mmol), and the reaction was heated to reflux for 3 h.
The mixture was cooled to ambient temperature and concentrated on
a rotary evaporator. The residue was partitioned with water (200 mL)
and hexanes (200 mL). The separated aqueous layer was extracted
with hexanes (100 mL). The combined organic layers were then
washed with brine, dried over anhydrous sodium sulfate, concentrated
on a rotary evaporator, and purified by flash chromatography
(hexanes) to give 1-(2-bromoethyl)-2,3-difluorobenzene (19.4 g,
89%). GC/MS: m/z for C8H7BrF2, found 142 (M − Br).
The reaction mixture was concentrated on a rotary evaporator to give
7-(quinolin-3-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]oxazepine dihydrochlo-
ride (259 mg, quantitative), which was used without further purification.
7-(Quinolin-3-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]oxazepine (1b) was
obtained from basic workup of a sample with aqueous sodium bi-
carbonate that was extracted with EtOAc (2×). The combined organic
layers were dried over anhydrous sodium sulfate and concentrated on a
rotary evaporator, and the residue was purified by reverse-phase HPLC
(acetonitrile/aqueous ammonium acetate buffer solution, 20−100%
1
gradient) to give 1b as a white powder after lyophilization. H NMR
(400 MHz, CD3OD): δ 9.38 (d, J = 2.4 Hz, 1H), 9.04 (s, 1H), 8.23 (d,
J = 8.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.00−7.93 (m, 3H), 7.87−
7.85 (m, 1H), 7.36 (d, J = 8.8 Hz, 1H), 4.56 (s, 2H), 4.37−4.35 (m,
2H), 3.69−3.66 (m, 2H). MS (EI): m/z for C18H16N2O, found
277.0 (MH+).
A mixture of crude 7-(quinolin-3-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]-
oxazepine dihydrochloride (229 mg, 0.66 mmol), N,N-diisopropylethyl-
amine (0.575 mL, 3.3 mmol), and 4-(2,2,2-trifluoroacetyl)benzoic
acid (173 mg, 0.79 mmol) in DMF (5 mL) was treated with
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate (326 mg, 0.86 mmol). After being stirred for 30 min at
ambient temperature, the reaction mixture was diluted with saturated
sodium bicarbonate (20 mL) and was extracted with EtOAc (30 mLx2).
The combined organic layers were dried over anhydrous sodium sulfate
and concentrated on a rotary evaporator, and the residue was purified
by reverse-phase HPLC (acetonitrile/aqueous ammonium acetate
buffer solution, 20−100% gradient) to give 1 (211 mg, 67%) as a
1
white powder after lyophilization. H NMR (400 MHz, DMSO-d6): δ
(rotamers are observed) 9.27 and 9.10 (s, 1H), 8.66 and 8.38 (s, 1H),
8.09−7.64 (m, 8H), 7.45 and 7.32 (d, J = 7.2 Hz, 2H), 7.16 and 6.98
(s, 1H), 4.93 and 4.53 (s, 2H), 4.32 and 4.22 (br s, 2H), 4.07 and 3.77
(br s, 2H). MS (EI): m/z for C27H19F3N2O3, found 477.0 (MH+).
Compounds 1c, 2−5, and 10 were prepared from 29 and the
corresponding boronic acid or 4,4,5,5-tetramethyl-1,3,2-dioxaborolane
according to a procedure similar to that described for the synthesis of
compound 1. Compound 1a was prepared from 2,3,4,5-tetrahydro-1,4-
benzoxazepine and 4-(2,2,2-trifluoroacetyl)benzoic acid.
2,2,2-Trifluoro-1-(4-(2,3,4,5-tetrahydrobenz[f ][1,4]oxazepin-
4-ylcarbonyl)phenyl)ethanone (1a). 1H NMR (400 MHz, DMSO-
d6): δ (rotamers are observed) 8.13 and 8.10 (d, J = 8.0 Hz, 2H),
7.51−7.45 (m, 2H), 7.28−7.04 (m, 3H), 7.00−6.97 and 6.61−6.59 (m,
1H), 4.83 and 4.41 (s, 2H), 4.25 and 4.16 (t, J = 8.4 Hz, 2H), 4.04 and
3.75 (t, J = 8.4 Hz, 2H). MS (EI): m/z for C18H14F3NO3, found
350.0 (MH+).
Phenyl(7-(quinolin-3-yl)-2,3-dihydrobenz[f ][1,4]oxazepin-
4(5H)-yl)methanone (1c). 1H NMR (400 MHz, CDCl3): δ
(rotamers are observed) 9.20 and 9.03 (s, 1H), 8.35 and 8.16 (s,
1H), 8.14 (d, J = 8.8 Hz, 1H), 7.91−8.31 (m, 1H), 7.76−7.58 (m,
3H), 7.52−7.37 (m, 5H), 7.23−7.19 (m, 1H), 6.93 (s, 1H), 4.93 and
4.55 (s, 2H), 4.31 and 4.19 (br s, 2H), 4.10−3.87 (br s, 2H). MS (EI):
m/z for C25H20N2O2, found 381.0 (MH+).
2,2,2-Trifluoro-1-(4-(7-(naphthalen-2-yl)-2,3,4,5-
tetrahydrobenz[f ][1,4]oxazepin-4-ylcarbonyl)phenyl)-
ethanone (2). 1H NMR (400 MHz, CDCl3): δ (rotamers are
observed) 8.20 and 8.14 (d, J = 7.6 Hz, 2H), 7.93−7.74 (m, 4H), 7.63
and 7.61 (d, J = 2.4 Hz, 1H), 7.58−7.48 (m, 5H), 7.20 and 7.17 (d, J =
3.6 Hz, 1H), 6.91 (br s, 1H), 4.94 and 4.50 (s, 2H), 4.31 and 4.21 (t,
J = 4.0 Hz, 2H), 4.10 and 3.80 (t, J = 4.0 Hz, 2H). MS (EI): m/z for
C28H20F3NO3, found 476.0 (MH+).
To a stirred suspension of Mg (4.26 g, 175 mmol) in Et2O (50 mL)
was added 1,2-dibromoethane (0.755 mL, 8.76 mmol), and the
mixture was allowed to stir for 10 min at ambient temperature.
The flask was affixed with a reflux condenser, and a solution of
1-(2-bromoethyl)-2,3-difluorobenzene (19.4 g, 87.6 mmol) and 1,2-
dibromoethane (0.755 mL, 8.76 mmol) in Et2O (50 mL) was added
over 30 min such that the ether warmed to a steady reflux. The stirred
reaction was maintained at reflux for 1 h and then allowed to cool to
ambient temperature before being further cooled to 0 °C. Aqueous
ammonium chloride (50 mL) was carefully added over the course of
1 h to quench the reaction. The resulting mixture was diluted with
H2O (100 mL), and the separated aqueous layer was extracted with
Et2O (100 mL). The combined organic layers were washed with brine,
dried over anhydrous sodium sulfate, and filtered. Careful distillation
1
of the filtrate gave 38 (9.5 g, 76%) as a colorless oil. H NMR (400
MHz, CDCl3): δ 7.01−6.97 (m, 3H), 2.70 (q, J = 7.6 Hz, 2H), 1.23
(t, J = 7.6 Hz, 3H). GC/MS: m/z for C8H8F2, found 142 (M+).
Compound 39 was prepared from compound 38 according to a
procedure similar to that described for the synthesis of compound 37.
1H NMR (400 MHz, DMSO-d6): δ 13.73 (br s, 1H), 7.82−7.76 (m,
2H), 3.76 (s, 3H), 2.94 (q, J = 7.2 Hz, 2H), 1.81 (t, J = 7.2 Hz, 3H).
MS (EI): m/z for C10H11FO4S, found 245.0 (MH−).
2, 2, 2-Trifluoro-1-(4-(7-(quinolin-3-yl)-2, 3, 4, 5-
tetrahydrobenz[f ][1,4]oxazepin-4-ylcarbonyl)phenyl)-
ethanone (1). A mixture of 29 (327 mg, 1.00 mmol), quinolin-3-
ylboronic acid (200 mg, 1.15 mmol), potassium carbonate (414 mg,
3.0 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloro-
palladium(II) complex with dichloromethane (94 mg, 0.12 mmol)
in DME (5 mL) was heated to 80 °C for 2 h. The reaction mixture
was cooled to ambient temperature and concentrated on a rotary
evaporator, and the residue was purified by flash chromatography
(hexanes/ethyl acetate (2:1)) to give 40 (282 mg, 75%) as a white
solid. MS (EI): m/z for C23H24N2O3, found 377.1 (MH−).
2,2,2-Trifluoro-1-(4-(7-phenyl-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepin-4-ylcarbonyl)phenyl)ethanone (3). 1H NMR (400
MHz, DMSO-d6): δ (rotamers are observed) 7.72−7.66 (m, 5H), 7.53
and 7.51 (d, J = 2.4 Hz, 1H), 7.46−7.28 (m, 5H), 7.11−7.05 (m, 1H),
4.85 and 4.48 (s, 2H), 4.26 amd 4.16 (br s, 2H), 4.02 and 3.74 (br s,
2H). MS (EI): m/z for C24H18F3NO3, found 426.0 (MH+).
2,2,2-Trifluoro-1-(4-(7-(pyridin-3-yl)-2,3,4,5-tetrahydrobenz-
[f][1,4]oxazepin-4-ylcarbonyl)phenyl)ethanone (4). 1H NMR
(400 MHz, DMSO-d6): δ (rotamers are observed) 8.95 and 8.87 (s,
1H), 8.70 and 8.52 (br s, 1H), 8.07−8.03 (m, 1H), 7.79−7.26 (m,
6H), 7.11−7.06 (m, 1H), 6.94−6.90 (m, 1H), 4.86 and 4.73 (s, 2H),
To a stirred solution of 40 (282 mg, 0.75 mmol) in 1,4-dioxane (5 mL)
was added a solution of HCl (2 mL, 8 mmol, 4 M in 1,4-dioxane),
and the resulting mixture was stirred at ambient temperature overnight.
L
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4.26 and 4.18 (br s, 2H), 4.00 and 3.72 (br s, 2H). MS (EI): m/z for
C23H17F3N2O3, found 427.0 (MH+).
solution of 8 (41 mg, 0.1 mmol) in DMF (2 mL) was added K2CO3
(42 mg, 0.3 mmol) and iodomethane (0.1 mL, 1.6 mmol). The reaction
mixture was stirred at ambient temperature for overnight. The crude
mixture was directly purified by reverse phase HPLC (acetonitrile/
aqueous ammonium acetate buffer solution; 20−100% gradient) to
N-(3-(4-(4-(2,2,2-Trifluoroacetyl)benzoyl)-2,3,4,5-
1
tetrahydrobenz[f ][1,4]oxazepin-7-yl)phenyl)acetamide (5). H
NMR (400 MHz, DMSO-d6): δ (rotamers are observed) 10.06 and
10.02 (s, 1H), 7.85 and 7.83 (s, 1H), 7.73−7.64 (m, 3H), 7.60 and
7.58 (s, 1H), 7.47−7.06 (m, 5H), 7.30 and 6.71 (br s, 1H), 4.84 and
4.48 (s, 2H), 4.27 and 4.16 (br s, 2H), 4.02 and 3.74 (br s, 2H), 2.07
(s, 3H). MS (EI): m/z for C26H21F3N2O4, found 483.0 (MH+).
1-(4-(7-(1H-Pyrazol-4-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]-
oxazepin-4-ylcarbonyl)phenyl)-2,2,2-trifluoroethanone (10).
1H NMR (400 MHz, DMSO-d6): δ (rotamers are observed) 12.92
(s, 1H), 8.18−7.58 (m, 5H), 7.46−7.41 (m, 2H), 7.29−6.95 (m, 2H),
4.80 and 4.40 (s, 2H), 4.19 and 4.11 (br s, 2H), 4.00 and 3.71 (br s,
2H). MS (EI): m/z for C21H16F3N3O3, found 416.0 (MH+).
1-(4-(7-(1H-Indazol-6-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]-
oxazepin-4-ylcarbonyl)phenyl)-2,2,2-trifluoroethanone (6).
A stirred mixture of 31 (293 mg, 1.0 mmol), 6-bromo-1H-indazole
(197 mg, 1.0 mmol), triethylamine (348 μL, 2.5 mmol), and [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with
dichloromethane (82 mg, 0.10 mmol) in DME (5 mL) and H2O
(1 mL) was heated to 90 °C for 4 h. The reaction mixture was cooled
to ambient temperature, concentrated on a rotary evaporator, and
purified by flash chromatography (hexanes/ethyl acetate (1:1)) to
give 41 (288 mg, 79%). MS (EI): m/z for C21H23N3O3, found
366.0 (MH+).
To a solution of 41 (288 mg, 0.79 mmol) in 1,4-dioxane (5 mL)
was added a solution of HCl (2 mL, 8 mmol, 4 M in 1,4-dioxane) and
the resulting mixture was stirred at ambient temperature for overnight.
The reaction mixture was concentrated on a rotary evaporator to give
7-(1H-indazol-6-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]oxazepine dihydro-
chloride (269 mg, quant.) which was used in the next step without
further purification.
A mixture of crude 7-(1H-indazol-6-yl)-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepine dihydrochloride (269 mg, 0.79 mmol), N,N-diisopro-
pylethylamine (0.688 mL, 3.9 mmol) and 4-(2,2,2-trifluoroacetyl)-
benzoic acid (207 mg, 0.95 mmol) in DMF (5 mL) was treated with
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate (390 mg, 1.03 mmol). After stirring for 30 min at ambient
temperature, the reaction mixture was diluted with saturated sodium
bicarbonate (20 mL) and the aqueous layer was extracted with EtOAc
(30 mL × 2). The combined organic layers were dried over anhydrous
sodium sulfate and concentrated on a rotary evaporator, and the residue
was purified by reverse phase HPLC (acetonitrile/aqueous ammonium
acetate buffer solution; 20−100% gradient) to give 6 (272 mg, 74%) as
a white powder after lyophilization. 1H NMR (400 MHz, DMSO-d6): δ
(rotamers are observed) 13.13 (s, 1H), 8.10 (s, 1H), 7.85−7.51 (m,
7H), 7.43−7.32 (m, 2H), 7.18−7.08 (m, 1H), 4.89 and 4.53 (d, J =
11.2 Hz, 2H), 4.28 and 4.17 (br s, 2H), 4.04 and 3.76 (br s, 2H). MS
(EI): m/z for C25H18F3N3O3, found 466.0 (MH+).
1
give 9 (24 mg, 54%) as a white powder after lyophilization. H NMR
(400 MHz, DMSO-d6): δ (rotamers are observed) 8.06−8.00 (m, 3H),
7.92−7.40 (m, 6H), 7.26−6.97 (m, 2H), 4.91 and 4.55 (s, 2H), 4.31
and 4.17 (br s, 2H), 4.12 and 4.08 (s, 3H), 4.06 and 3.74 (br s, 2H),
2.61 (s, 3H). MS (EI): m/z for C26H23N3O3, found 426.0 (MH+).
1-(4-(7-(1H-Benz[d]imidazol-6-yl)-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepin-4-ylcarbonyl)phenyl)-2,2,2-trifluoroethanone
1
(11). H NMR (400 MHz, DMSO-d6): δ (rotamers are observed)
12.51 and 12.43 (s, 1H), 8.26 and 8.25 (s, 1H), 7.90−7.43 (m, 8H),
7.35−7.30 (m, 1H), 7.10−7.06 and 6.76−6.74 (m, 1H), 4.86 and 4.52
(br s, 2H), 4.26 and 4.16 (br s, 2H), 4.03 and 3.75 (br s, 2H). MS
(EI): m/z for C25H18F3N3O3: 466.0 (MH+).
2,2,2-Trifluoro-1-(4-(7-(2-methyl-1H-benz[d]imidazol-6-yl)-
2,3,4,5-tetrahydrobenz[f ][1,4]oxazepin-4-ylcarbonyl)phenyl)-
1
ethanone (12). H NMR (400 MHz, DMSO-d6): δ (rotamers are
observed) 12.30 (br s, 1H), 8.10 and 7.81 (br s, 1H), 7.71−7.65 (m,
4H), 7.57−7.42 (m, 3H), 7.33−6.74 (m, 2H), 4.86 and 4.52 (br s,
2H), 4.25 and 4.15 (br s, 2H), 4.04 and 3.75 (br s, 2H), 2.55 (s, 3H).
MS (EI): m/z for C26H20F3N3O3, found 480.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(4-(trifluoromethyl)phenyl)methanone (13)
(HCl Salt). 1H NMR (400 MHz, DMSO-d6): δ (rotamers are
observed) 9.49 and 9.45 (s, 1H), 8.03−7.62 (m, 8H), 7.49−6.96 (m,
2H), 4.91 and 4.58 (s, 2H), 4.33 and 4.19 (br s, 2H), 4.05 and 3.74
(br s, 2H). MS (EI): m/z for C24H18F3N3O2, found 438.0 (MH+).
4-(7-(1H-Benz[d]imidazol-6-yl)-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepin-4-ylcarbonyl)benzenesulfonamide (14). 1H NMR
(400 MHz, DMSO-d6): δ (rotamers are observed) 12.53 (br s, 1H),
8.26 (s, 1H), 7.91−7.46 (m, 10H), 7.30−6.81 (m, 2H), 4.88 and 4.56
(s, 2H), 4.28 and 4.16 (br s, 2H), 4.04 and 3.73 (br s, 2H). MS (EI):
m/z for C23H20N4O4S, found 449.0 (MH+).
4-(7-(1H-Benz[d]imidazol-6-yl)-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepin-4-ylcarbonyl)-N-methylbenzenesulfonamide
1
(15). H NMR (400 MHz, DMSO-d6): δ (rotamers are observed)
12.51 and 12.42 (s, 1H), 8.25 (br s, 1H), 7.93−7.47 (m, 9H), 7.31−
6.76 (m, 2H), 4.88 and 4.53 (s, 2H), 4.27 and 4.16 (br s, 2H), 4.05
and 3.73 (br s, 2H), 2.45−2.35 (m, 3H). MS (EI): m/z for
C24H22N4O4S, found 463.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(4-methylsulfonyl)phenyl)methanone (16).
1H NMR (400 MHz, DMSO-d6): δ (rotamers are observed) 12.52
and 12.44 (s, 1H), 8.25 and 8.24 (s, 1H), 8.02−7.90 (m, 2H), 7.74−
7.49 (m, 6H), 7.29−6.85 (m, 2H), 4.88 and 4.53 (s, 2H), 4.28 and
4.16 (br s, 2H), 4.05 and 3.72 (br s, 2H), 3.27 (br s, 3H). MS (EI):
m/z for C24H21N3O4S, found 448.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
Compounds 7, 11−28 were prepared according to a similar
procedure as described for the synthesis of compound 6. Compound 8
was prepared from 7-(1H-indazol-6-yl)-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepine dihydrochloride and 4-acetylbenzoic acid.
1
oxazepin-4(5H)-yl)(4-ethylsulfonyl)phenyl)methanone (17). H
NMR (400 MHz, CDCl3): δ 8.14−7.56 (m, 4H), 7.55−7.41 (m, 4H),
7.23 (s, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.33 (d, J = 2.0 Hz, 1H), 4.91
and 4.42 (s, 2H), 4.26−3.78 (m, 4H), 3.28 (q, J = 7.6 Hz, 2H), 2.12
and 2.02 (s, 1H), 1.38 (t, J = 7.6 Hz, 3H). MS (EI): m/z for
C25H23N3O4S, found 462.0 (MH+).
1-(4-(7-(1H-Indazol-5-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]-
1
oxazepin-4-ylcarbonyl)phenyl)-2,2,2-trifluoroethanone (7). H
NMR (400 MHz, DMSO-d6): δ (rotamers are observed) 13.12 and
13.08 (s, 1H), 8.08 and 8.07 (s, 2H), 7.83−7.49 (m, 6H), 7.43−7.14
(m, 2H), 7.11−6.91 (m, 1H), 4.88 and 4.52 (d, J = 11.2 Hz, 2H),
4.30−4.27 and 4.16−4.14 (m, 2H), 4.05−4.00 and 3.74−3.70 (m,
2H). MS (EI): m/z for C25H18F3N3O3, found 466.0 (MH+).
1-(4-(7-(1H-Indazol-6-yl)-2,3,4,5-tetrahydrobenz[f ][1,4]-
oxazepin-4-ylcarbonyl)phenyl)ethanone (8). 1H NMR (400
MHz, DMSO-d6): δ (rotamers are observed) 13.14 and 13.11 (s,
1H), 8.09 and 8.08 (s, 1H), 8.06 (d, J = 7.6 Hz, 2H), 7.85−7.38 (m,
6H), 7.22−6.91 (m, 2H), 4.89 and 4.55 (s, 2H), 4.31 and 4.16 (br s,
2H), 4.04 and 3.73 (br s, 2H), 2.61 (s, 3H). MS (EI): m/z for
C25H21N3O3, found 412.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(2-methyl-4-(methylsulfonyl)phenyl)-
1
methanone (18). H NMR (400 MHz, CDCl3): δ 8.11−7.80 (m,
4H), 7.54−7.38 (m, 3H), 7.28−7.14 (m, 2H), 6.29 (s, 1H), 4.39−4.10
(m, 6H), 3.21 (s, 3H), 2.12 (s, 1H), 2.02 (s, 3H). MS (EI): m/z for
C25H23N3O4S, found 462.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(2-ethyl-4-(methylsulfonyl)phenyl)-
1
methanone (19). H NMR (400 MHz, CDCl3): δ (rotamers are
observed) 8.10 (s, 1H), 8.03−7.79 (m, 3H), 7.53 (dd, J = 8.0, 2.0 Hz,
1H), 7.43 (dd, J = 8.8, 1.6 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.26 (s,
1H), 7.15 (d, J = 8.4 Hz, 1H), 6.29 (d, J = 2.4 Hz, 1H), 4.38−4.20 (m,
6H), 3.21 (s, 3H), 2.54−2.44 and 2.29−2.20 (m, 2H), 2.12 (br s, 1H),
1-(4-(7-(1-Methyl-H-indazol-6-yl)-2,3,4,5-tetrahydrobenz[f ]-
[1,4]oxazepin-4-ylcarbonyl)phenyl)ethanone (9). To a stirred
M
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1.14 (t, J = 7.6 Hz, 3H). MS (EI): m/z for C26H25N3O4S, found
476.0 (MH+).
(7-(6-Aminopyridin-3-yl)-2,3-dihydrobenz[f ][1,4]oxazepin-
4(5H)-yl)(3-fluoro-2-methyl-4-(methylsulfonyl)phenyl)-
1
methanone (28). H NMR (400 MHz, DMSO-d6): δ (rotamers are
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
observed) 8.24 and 8.03 (d, J = 2.4 Hz, 1H), 7.77 and 7.72 (t, J = 7.6
Hz, 1H), 7.71−7.39 (m, 2H), 7.57 and 6.63 (d, J = 2.4 Hz, 1H), 7.28
and 7.19 (d, J = 7.6 Hz, 1H), 7.04 and 7.02 (d, J = 8.0 Hz, 1H), 6.52
and 6.46 (d, J = 8.8 Hz, 1H), 6.05 (br s, 2H), 4.93−4.31 (m, 2H),
4.28−3.56 (m, 4H), 3.37 and 3.34 (s, 3H), 2.12 and 1.77 (d, J = 1.6
Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 167.3, 167.2, 166.6,
166.6, 158.9, 158.9, 158.4, 158.4, 157.4, 157.2, 155.9, 155.8, 145.4,
145.1, 145.1, 144.0, 143.9, 135.0, 134.7, 132.9, 132.8, 129.4, 129.2,
128.2, 128.2, 128.1, 128.0, 127.0, 126.9, 126.8, 125.9, 125.6, 125.4,
123.6, 123.5, 123.3, 123.1, 122.8, 122.0, 122.0, 121.9, 121.9, 121.2,
120.7, 107.8, 107.8, 70.9, 70.8, 51.1, 51.1, 47.4, 46.5, 43.5, 43.5, 43.5,
43.4, 11.0, 10.9, 10.7, 10.6. IR (KBr pellet): 1623, 1487, 1457, 1423,
1385, 1314, 1269, 1226, 1193, 1144, 1133, 1054, 1031, 962, 821,
768 cm−1. Mp: 204−205 °C. MS (EI): m/z for C23H22FN3O4S, 456.0
(MH+). High-resolution MS (FAB MS using glycerol as the matrix):
m/z calcd for C23H22FN3O4S 456.13878, found 456.13943.
mTOR ELISA Assay. The measurement of mTOR enzyme activity
was performed in an ELISA format following the phosphorylation of
4E-BP1 protein. All experiments were performed in the 384-well
format. Generally, 0.5 μL of DMSO containing varying concentrations
of the test compound was mixed with 15 μL of the enzyme solution.
Kinase reactions were initiated with the addition of 15 μL of a solution
containing the substrate. The assay conditions were as follows: 0.2 nM
mTOR, 10 μM ATP, and 50 nM NHis-tagged 4E-BP1 in 20 mM
Hepes, pH 7.2, 1 mM DTT, 50 mM NaCl, 10 mM MnCl2, 0.02 mg/mL
BSA, 0.01% CHAPS, 50 mM β-glycerophophate. Following an
incubation of 120 min at ambient temperature, 20 μL of the reaction
mixture was transferred to a Ni-chelate-coated 384-well plate. The
binding step of the 4E-BP1 protein proceeded for 60 min, followed by
washing four times each with 50 μL of Tris-buffered saline solution
(TBS). Anti-phospho-4E-BP1 rabbit immunoglobulin G (IgG; 20 μL,
1:5000) in 5% BSA−TBST (0.2% Tween-20 in TBS) was added, and
the reaction mixuture was further incubated for 60 min. Incubation with
a secondary horseradish peroxidase (HRP)-tagged anti-IgG was similarly
performed after the primary antibody was washed off (four washes of
50 μL). Following the final wash step with TBST, 20 μL of SuperSignal
ELISA Femto (Pierce Biotechnology, 37075) was added and the
luminescence measured using an EnVision plate reader. Data are
reported as the mean (n ≥ 2).
PI3Kα Biochemical Assay. PI3Kα activity is measured as the
percentage of ATP consumed following the kinase reaction using
luciferase−luciferin-coupled chemiluminescence. Reactions were con-
ducted in 384-well white, medium-binding microtiter plates (Greiner).
Kinase reactions were initiated by combining test compounds, ATP,
substrate (PIP2), and kinase in a 20 μL volume in a buffer solution.
The standard PI3Kα assay buffer is composed of 50 mM Tris, pH 7.5,
1 mM EGTA, 10 mM MgCl2, 1 mM DTT, and 0.03% CHAPS.
The standard assay concentrations for enzyme, ATP, and substrate are
1.5 nM, 1 μM, and 10 μM, respectively. The reaction mixture was
incubated at ambient temperature for approximately 2 h. Following the
kinase reaction, a 10 μL aliquot of luciferase−luciferin mix (Promega
Kinase-Glo) was added and the chemiluminescence signal measured
using a Victor2 plate reader (Perkin-Elmer). Total ATP consumption
was limited to 40−60%, and the IC50 values of the control compounds
correlated well with literature references. Data are reported as the
mean (n ≥ 2).
pS6 (S240/244) ELISA. PC-3 cells (ATCC) were seeded onto
96-well plates (Corning, 3904) in DMEM (Cellgro) containing 10%
FBS (Cellgro), 1% NEAA (Cellgro), and 1% penicillin−streptomycin
(Cellgro) at 8 × 103 cells per well. The cells were incubated at 37 °C
and 5% CO2 for 48 h, and the growth medium was replaced with
serum-free DMEM. Serial dilutions of the test compound in 0.3%
DMSO (vehicle) were added to the cells and incubated for 3 h. To fix
the cells, medium was removed and 100 μL/well of 4% formaldehyde
(Sigma) in TBS was added to each well at ambient temperature for
30 min. The cells were washed three times with 200 μL of TBST and
quenched with 100 μL of 0.6% H2O2 (VWR International) in TBST
oxazepin-4(5H)-yl)(4-(methylsulfonyl)-2-propylphenyl)-
1
methanone (20). H NMR (400 MHz, CDCl3): δ (rotamers are
observed) 8.11 (br s, 1H), 7.99 (s, 1H), 7.97 (d, J = 2.0 Hz, 1H), 7.81
(dd, J = 18.8, 10.0 Hz, 1H), 7.53 (dd, J = 8.4, 2.4 Hz, 1H), 7.42 (dd,
J = 8.8, 1.6 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.15 (d, J =
8.0 Hz, 1H), 6.29 (d, J = 2.4 Hz, 1H), 4.38 (d, J = 15.6 Hz, 1H), 4.25
(d, J = 14.8 Hz, 1H), 4.23 (br s, 4H), 3.21 (s, 3H), 2.45−2.37 (m 1H),
2.24−2.16 (m, 1H), 2.12 (s, 1H), 1.70−1.61 (m 1H), 1.50−1.43
(m, 1H), 0.89 and 0.86 (t, J = 7.2 Hz, 3H). MS (EI): m/z for
C27H27N3O4S, found 490.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(2-(ethylamino)-4-(methylsulfonyl)phenyl)-
methanone (21). 1H NMR (400 MHz, CDCl3): δ 8.10 (s, 1H), 7.80
(d, J = 8.0 Hz, 1H), 7.52 (dd, J = 8.4, 2.4 Hz, 1H), 7.44−7.39 (m, 3H),
7.25−7.23 (m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 6.47 (s, 1H), 4.46 (br s,
2H), 4.24−4.18 (m, 5H), 3.19 (s, 3H), 2.94 (br s, 2H), 2.12 (s, 1H),
0.99 (t, J = 6.8 Hz, 3H). MS (EI): m/z for C26H26N4O4S, found
491.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(2-chloro-4-(methylsulfonyl)phenyl)-
methanone (22). 1H NMR (400 MHz, CDCl3): δ 8.22 (s, 1H), 8.18
(d, J = 1.6 Hz, 1H), 8.04 (dd, J = 8.0, 2.0 Hz, 1H), 7.81 (d, J = 8.4 Hz,
1H), 7.53 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (dd, J = 8.4, 1.6 Hz, 1H), 7.36
(d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.35 (d, J =
2.4 Hz, 1H), 4.45 (d, J = 15.6 Hz, 1H), 4.42−4.32 (m, 2H), 4.25 (d,
J = 15.6 Hz, 1H), 4.16−4.05 (m, 2H), 3.24 (s, 3H), 2.11 (s, 1H). MS
(EI): m/z for C24H20ClN3O4S, found 482.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(2-bromo-4-(methylsulfonyl)phenyl)-
1
methanone (23). H NMR (400 MHz, CDCl3): δ 8.34 (d, J = 1.6
Hz, 1H), 8.10 (d, J = 1.6 Hz, 1H), 8.08 (s, 1H), 7.80 (d, J = 8.0 Hz,
1H), 7.54 (dd, J = 8.4, 2.4 Hz, 1H), 7.42 (dd, J = 8.4, 2.0 Hz, 1H), 7.35
(d, J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.35 (s,
1H), 4.44 (d, J = 15.6 Hz, 1H), 4.37−4.32 (m, 2H), 4.26 (d, J = 15.6
Hz, 1H), 4.19−4.08 (m, 2H), 3.23 (s, 3H), 2.12 (s, 1H). MS (EI):
m/z for C24H20BrN3O4S, found 526.0, 528.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(3-methyl-4-(methylsulfonyl)phenyl)-
1
methanone (24) (HCl Salt). H NMR (400 MHz, DMSO-d6): δ
(rotamers are observed) 9.55 and 9.53 (s, 1H), 8.03−7.78 (m, 4H),
7.64−7.49 (m, 2H), 7.46 and 7.37 (d, J = 8.8 Hz, 1H), 7.31−7.02 (m,
3H), 4.90 and 4.56 (s, 2H), 4.34 and 4.18 (br s, 2H), 4.04 and 3.74
(br s, 2H), 3.25 and 3.17 (s, 3H), 2.65 and 2.46 (s, 3H). MS (EI): m/z
for C25H23N3O4S, found 462.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(3-fluoro-2-methyl-4-(methylsulfonyl)-
phenyl)methanone (25). 1H NMR (400 MHz, DMSO-d6): δ
(rotamers are observed) 12.49 and 12.00 (br s, 1H), 8.25 (d, J = 8.4
Hz, 1H), 7.83−7.49 (m, 5H), 7.30−6.77 (m, 3H), 4.98−4.85 (m, 1H),
4.53−4.36 (m, 1H), 4.36−3.57 (m, 4H), 3.34 (s, 3H, overlapping
with H2O), 2.13 and 1.78 (d, J = 2.4 Hz, 3H). MS (EI): m/z for
C25H22FN3O4S, found 480.0 (MH+).
(7-(1H-Benz[d]imidazol-6-yl)-2,3-dihydrobenz[f ][1,4]-
oxazepin-4(5H)-yl)(2-ethyl-3-fluoro-4-(methylsulfonyl)phenyl)-
1
methanone (26). H NMR (400 MHz, DMSO-d6): δ (rotamers are
observed) 12.48 and 12.39 (br s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.79−
7.51 (m, 5H), 7.30−6.79 (m, 3H), 5.03 and 4.83 (d, J = 15.2 Hz, 1H),
4.49 and 4.41 (d, J = 15.6 Hz, 1H), 4.34−3.59 (m, 4H), 3.36 (s, 3H,
overlapping with H2O), 2.69−2.04 (m, 2H), 1.10 and 0.99 (t, J = 7.6
Hz, 3H). MS (EI): m/z for C26H242FN3O4S, found 494.0 (MH+).
(7-(6-Aminopyridin-3-yl)-2,3-dihydrobenz[f ][1,4]oxazepin-
4(5H)-yl)(4-(methylsulfonyl)phenyl)methanone (27). 1H NMR
(400 MHz, DMSO-d6): δ (rotamers are observed) 8.23 (br s, 1H),
8.04−7.97 (m, 2H), 7.70−7.64 (m, 2H), 7.56 and 6.72 (s, 1H), 7.50−
7.40 (m, 2H), 7.05 and 7.01 (d, J = 8.8 Hz, 1H), 6.53 and 6.46 (d, J =
8.8 Hz, 1H), 6.05 (s, 2H), 4.84 and 4.47 (s, 2H), 4.24 and 4.12 (br s,
2H), 4.03 and 3.70 (br s, 2H), 3.28 and 3.26 (s, 3H). MS (EI): m/z for
C22H21N3O4S, found 424.0 (MH+).
N
dx.doi.org/10.1021/jm3007933 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
for 30 min at ambient temperature. The plates were washed three
times with 200 μL of TBST and blocked with 100 μL of 5% BSA
(Jackson ImmunoResearch) in TBST for 1 h at ambient temperature.
Anti-pS6 (S240/244) antibody (Cell Signaling Technology, 2215) or
anti-total-S6 antibody (Cell Signaling Technology, 2217) was diluted
1:500 in 5% BSA in TBST. A 50 μL volume of either primary antibody
solution was added to the plate to detect pS6 or total S6. After
incubation overnight at 4 °C, the plates were washed four times with
200 μL of TBST. Goat antirabbit secondary antibody (Jackson
ImmunoResearch) was diluted at 1:15000 in 5% BSA in TBST. A
100 μL volume of antibody solution was added to each well and
incubated for 1 h at ambient temperature. The plates were washed
three times with 200 μL of TBST and two times with 200 μL of TBS.
Chemiluminescent substrate (SuperSignal Elisa Femto) was prepared
at rt. A 100 μL volume of chemiluminescent substrate per well was
added, and then the plate was shaken for 1 min. Luminescence was
read immediately on a Wallac plate reader at a wavelength of 560 nm.
After normalization of the pS6 signal to a total S6 signal, IC50 values
were determined relative to the DMSO-treated control. Data are
reported as the mean (n ≥ 2).
pAKT (S473) ELISA. PC-3 cells (ATCC) were seeded onto 96-well
plates (Corning, 3904) in DMEM (Cellgro, 10-013-CV) containing
10% FBS (Cellgro, 35-016-CV), 1% NEAA (Cellgro, 25-025-CI), and
1% penicillin−streptomycin (Cellgro, 30-002-CI) at 8 × 103 cells per
well. The cells were incubated at 37 °C and 5% CO2 for 48 h, and the
growth medium was replaced with serum-free DMEM. Serial dilutions
of the test compound in 0.3% DMSO (vehicle) were added to the cells
and incubated for 2 h and 50 min. The cells were then stimulated for
10 min with 20 ng/mL EGF (US Biologicals, E3374-07A)) for PC-3
or with 1.2 μg/mL Long R3 IGF-1 (Sigma, I1271) for MCF-7. To fix
the cells, medium was removed and 100 μL/well of 4% formaldehyde
(Sigma, F8775) in TBS (20 mM Tris, 500 mM NaCl) was added to
each well at ambient temperature for 30 min. The cells were washed
three times with 200 μL of TBS containing 0.1% Tween 20 (Bio-Rad,
170-6351) (TBST) and quenched with 100 μL of 0.6% H2O2 (VWR
International, VW3742-1) in TBST for 30 min at rt. The plates were
washed three times with 200 μL of TBST and blocked with 100 μL
of 5% BSA (Jackson ImmunoResearch, 001-000-173) in TBST for 1 h
at rt. Anti-pAKT (S473) antibody (Cell Signaling Technology, 4058)
or anti-total-AKT antibody (Cell Signaling Technology, 9272) was
diluted 1:400 or 1:500, respectively, in 5% BSA in TBST. A 50 μL
volume of either primary antibody solution was added to the plate to
detect pAKT (S473) or total AKT. After incubation overnight at 4 °C,
the plates were washed four times with 200 μL of TBST. Goat
antirabbit secondary antibody (Jackson ImmunoResearch, 111-035-
003) was diluted at 1:15000 in 5% BSA in TBST. A 100 μL volume of
antibody solution was added to each well and incubated for 1 h at
ambient temperature. The plates were washed three times with 200 μL of
TBST and two times with 200 μL of TBS. Chemiluminescent substrate
(SuperSignal Elisa Femto) was prepared at rt. A 100 μL volume of
chemiluminescent substrate per well was added, and then the plate was
shaken for 1 min. Luminescence was read immediately on a Wallac plate
reader at a wavelength of 560 nm. After normalization of the pAKT signal
to a total AKT signal, IC50 values were determined relative to the DMSO-
treated control. Data are reported as the mean (n ≥ 2).
amino acids at 37 °C in a humidified 5% CO2 atmosphere. On day 0,
cells were harvested by trypsinization, and 3 × 106 cells (passages
10−14, >95% viability) in 0.1 mL of ice-cold Hank’s balanced salt
solution were implanted subcutaneously into the hindflank of 5−8 week
old male nude mice. When the tumors reached 200−500 mg in size, the
mice were administered a single dose of 28. Scheduled takedown was at
4 h after a single dose. PC-3 tumor lysate was pooled for each group.
Phosphor-AKT (S473), phosphor-AKT (T308), phosphor-p70S6K
(T389), phosphor-S6 (S240/244), and phosphor-4E-BP1 (T37/46)
levels were analyzed by Western immunoblotting and normalized to
α-tubulin. α-Tubulin levels were analyzed by Western immunoblotting
from the same membrane as the p-S6 and p-4E-BP1 blots.
MCF-7 Breast Adenocarcinama Model. MCF-7 human mammary
adenocarcinoma cells were cultured in vitro in DMEM (Cellgro)
supplemented with 10% fetal bovine serum (Cellgro), penicillin−
streptomycin, and nonessential amino acids at 37 °C in a humidified
5% CO2 atmosphere. On day 0, cells were harvested by trypsinization,
and 5 × 106 cells in 0.1 mL of a solution made of 50% cold Hank’s
balanced salt solution with 50% growth factor reduced matrigel
(Becton Dickinson) were implanted subcutaneously into the hindflank
of 5−8 week old female nude mice. When the tumors reached
40−150 mg in size, the mice were administered a single dose of 28.
Scheduled takedown was at 4 h after a single dose. MCF-7 tumor
lysate was pooled for each group. Phosphor-AKT (S473), phosphor-
AKT (T308), phosphor-p70S6K (T389), phosphor-S6 (S240/244),
and phosphor-4E-BP1 (T37/46) levels were analyzed by Western
immunoblotting and normalized to α-tubulin. α-Tubulin levels were
analyzed by Western immunoblotting from the same membrane as the
p-S6 and p-4E-BP1 blots.
Antitumor (MCF-7 Model) Efficacy Studies. Female athymic
nude mice (NCr) 5−8 weeks of age and weighing approximately
20−25 g were used. Prior to initiation of study, the animals were
allowed to acclimate for a minimum of 48 h. During these studies, the
animals were provided food and water ad libitum and housed in a
room conditioned at 70−75 °F and 60% relative humidity. A 12 h light
and 12 h dark cycle was maintained with automatic timers. MCF-7
tumors were established subcutaneously and staged when the average
tumor mass reached 100−200 mg. Compound 28 was orally
administered as a solution/fine suspension in water (with a 1:1 molar
ratio of 1 N HCl) qd at 50 and 100 mg/kg for 14 days. Tumor mass was
determined by measuring perpendicular diameters with a caliper, using
the following formula: tumor mass (mg) = [tumor volume = length
(mm) × width2 (mm2)]/2. During the period of study, tumor masses
were determined twice weekly and body masses were recorded daily.
All animals were examined daily for compound-induced or tumor-
related deaths.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 82-2-470-9602 (B.G.K.); (813) 974-4642 (J.W.L.).
E-mail: byunggyu72kim@gmail.com (B.G.K.); jwleahy@usf.
edu (J.W.L.).
Present Addresses
†Asan Institute of Life Science, Songpa-gu, Seoul 138-736,
South Korea.
In Vivo Pharmacokinetic Studies. Pharmacokinetic studies of
28 were determined in female athymic nude mice, female CD rats,
male beagle dogs, and male cynomolgus monkeys. Compound 28 was
administered intravenously and by oral gavage at 10 mg/kg as a
solution formulated in EPW (5% ethanol/45% PEG400/water +
1:2 HCl (m/m)) to mice, 3 mg/kg as a solution formulated in EPW
(5% ethanol/45% PEG400/water + 1:2 HCl (m/m)) to CD rats and
male beagle dogs, and 3 mg/kg as a solution formulated in EPW
(5% ethanol/45% PEG400/water + 1:1.5 HCl (m/m)) to male
cynomolgus monkeys. The plasma levels of 28 were monitored over a
24 h period.
‡Departments of Chemistry and Molecular Medicine and
Florida Center of Excellence for Drug Discovery & Innovation,
University of South Florida, 3720 Spectrum Blvd., Suite 305,
Tampa, FL 33612.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Siamak Dailami for analytical support, Annapurna
Sapre and Eric Brooks for conducting the biochemical assay,
Jing Ping Yang, Peter Kim, Qiang Wu, Torsten Trowe, Suzanne
Crawley, Keith Calkins, Eun Ok Kim, Suresh Selvaraj, and Loan
In Vivo Pharmacodynamic Studies. PC-3 Prostate Adenocar-
cinoma Model. PC-3 human prostate adenocarcinoma cells were
cultured in vitro in DMEM (Mediatech) supplemented with 20% fetal
bovine serum (Hyclone), penicillin−streptomycin, and nonessential
O
dx.doi.org/10.1021/jm3007933 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and Inhibits the Growth of Cancer Cells with Activating PI3K
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Xiang Wu for collecting the reported ADME data, and
Atulkumar Ramaiya, Scott Womble, Michael F. Yakes, Joshua
Tanguay, and Scott Lam for obtaining PK and conducting PD/
efficacy studies.
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ABBREVIATIONS USED
■
mTOR, mammalian target of rapamycin; PI3K, phosphoinosi-
tide 3-kinase; Akt, v-akt murine thymoma viral oncogene
homologue 1; DNA-PK, DNA-activated protein kinase; ATM,
ataxia telangiectasia mutated kinase; 4E-BP1, eukaryotic
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