Benzazepinones and benzoxazepinones as antagonists of inhibitor of apoptosis proteins (IAPs) selective for the second baculovirus iap repeat (BIR2) domain

Article abstract of DOI:10.1021/jm400731m

XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.

Full text of DOI:10.1021/jm400731m

Article
pubs.acs.org/jmc
Benzazepinones and Benzoxazepinones as Antagonists of Inhibitor
of Apoptosis Proteins (IAPs) Selective for the Second Baculovirus IAP
Repeat (BIR2) Domain
Andrew F. Donnell,*,† Christophe Michoud,† Kenneth C. Rupert,† Xiaochun Han,† Douglas Aguilar,‡
Karl B. Frank,§ Adrian J. Fretland,§ Lin Gao,‡ Barry Goggin,∥ J. Heather Hogg,† Kyoungja Hong,∥
Cheryl A. Janson,‡ Robert F. Kester,† Norman Kong,† Kang Le,† Shirley Li,‡ Weiling Liang,†
Louis J. Lombardo,† Yan Lou,† Christine M. Lukacs,‡ Steven Mischke,† John A. Moliterni,†
Ann Polonskaia,∥ Andrew D. Schutt,∥ Dave S. Solis,‡ Anthony Specian,† Robert T. Taylor,§
Martin Weisel,† and Stacy W. Remiszewski†
‡
§
∥
†Departments of Discovery Chemistry, Discovery Technologies, Non-clinical Safety, Early ADME, and Discovery Oncology,
Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110, United States
S
* Supporting Information
ABSTRACT: XIAP is a key regulator of apoptosis, and its overexpression in cancer cells
may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR
domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are
selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways
activated upon cIAP binding contribute to the function of these compounds. Inhibitors
selective for XIAP should exert pro-apoptotic effects through competition with the terminal
caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective
benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to
the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which
potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.
AVPI peptide sequence.12−15 This four-peptide motif has
INTRODUCTION
■
inspired substantial interest in compounds that target the
Apoptosis is a cell death program that is essential for normal
IAPs,16−19 which has resulted in the discovery of several
physiological function, and the ability to avoid apoptosis is one
compounds that have entered clinical trials, those illustrated in
of the characteristics of cancer cells.1 The inhibitor of apoptosis
Figure 1, SM-406 (1),20 GDC-0152 (2),21 and LCL161
proteins (IAPs) are key regulators of the apoptosis pathways
and are often overexpressed in cancer cells, rendering them
(3),22,23 as well as one additional monovalent inhibitor and
two bivalent inhibitors.24 These compounds are pan-IAP
potential targets for cancer therapy.2−5 Members of the IAP
inhibitors that bind to XIAP, cIAP1, and cIAP2. They bind
family include X-linked IAP (XIAP), cellular IAP 1 (cIAP1),
cellular IAP 2 (cIAP2), and others.
There are two major pathways for apoptotic signal
transduction. The intrinsic pathway is activated by intracellular
stress, such as that caused by chemotherapy and radiation, and
leads to the activation of caspase 9. The extrinsic pathway is
with high affinity to the BIR3 domain of these proteins and, in
some cases, are selective for BIR3 over BIR2. In other cases, the
BIR2 affinity has not been disclosed. It has been shown that
they cause degradation of the cIAPs, leading to production of
the cytokine TNFα via NF-κB pathways, and it has been
suggested that their activity depends primarily on this
activated in response to extracellular signals mediated through
process.25−27 We expected that competition with the terminal
the death receptors and leads to the activation of caspase 8.
caspases by selective inhibitors of XIAP would present a distinct
Both pathways converge by causing the activation of the
and attractive mechanism of action, and this could potentially
effector caspases 3 and 7. XIAP regulates both pathways by
be achieved by selectively targeting the BIR2 domain of XIAP.
directly binding to and inhibiting caspases 3, 7, and 9 via two of
its three baculovirus IAP repeat (BIR) domains.6 The BIR3
One recent paper describes a series of Smac-mimetic
domain of XIAP binds to and inhibits caspase 9,7,8 and the
compounds with up to 7-fold selectivity for XIAP BIR2 versus
XIAP BIR3.28 Herein, we report the discovery of a novel class
of inhibitors that are highly selective for the BIR2 domain of
XIAP over the BIR3 domain, and we describe the iterative
BIR2 domain is involved with caspase 3 and 7 binding.9−11
Second mitochondria-derived activator of caspases (Smac,
also known as direct IAP-binding protein with a low pI,
DIABLO) is an endogenous mitochondrial protein that binds
with high affinity to the BIR3 domain of XIAP and the cIAPs
and with lower affinity to the BIR2 domain via its N-terminal
Received: May 15, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
CHEMISTRY
■
Our studies of the benzazepinone scaffold included the
exploration of a range of N-substituents of the azepinone
amide. We also explored some modifications to the alanine
group. The chemistry to prepare these analogues is outlined in
Scheme 1. Starting with the amino benzazepinone 6, amide
a
Scheme 1. Synthesis of Benzazepinones 12−23
a
Reagents and conditions: (a) R1CO2H, HBTU, HOBt, Et3N, DMF,
rt, 47%−quant; (b) R2CH2X, Cs2CO3, NaI, DMF, 60 °C; (c) HCl,
MeOH, Et2O, rt or TFA, CH2Cl2, rt, 24−60% (2 steps).
coupling with either BOC-N-methyl-L-alanine or the amino
acid of interest gave 7−11. These compounds were then
alkylated with the appropriate alkyl halide or mesylate in the
presence of cesium carbonate. Under these conditions, we
observed exclusive alkylation of the aniline amide. The BOC
group was then removed to provide the final compounds 12−
23. The compounds were typically isolated and tested as either
the HCl or TFA salts.
Construction of the benzoxazepinone scaffold commenced
with the SNAr reaction of (S)-2-(tert-butoxycarbonylamino)-3-
hydroxypropanoic acid (24) and 1-fluoro-2-nitrobenzene to
yield ether 25 (Scheme 2). Reduction of the nitro group,
Figure 1. IAP inhibitors.
synthetic explorations and structure-based design efforts leading
to a highly potent and selective analogue that has good oral
bioavailability. We show that this compound is 78-fold selective
for the BIR2 versus the BIR3 domain of XIAP and it is 7-fold
selective for XIAP versus cIAP1. We also demonstrate that it
affects apoptotic signaling without promoting cIAP-related NF-
κB activation, supporting the utility of selective XIAP inhibition
via the BIR2 domain as a potential approach for cancer therapy.
A high-throughput screen of our corporate compound library
led to the identification of benzazepinone 4 (Figure 2) as a
a
Scheme 2. Synthesis of Benzoxazepinone 27
Figure 2. HTS hit and initial lead evolution.
BIR2-selective XIAP inhibitor. The relatively small size (MW
261) and low lipophilicity (cLogP 0.62) of 4 made it an
appealing starting point for a medicinal chemistry campaign.
Preliminary exploration of the SAR around this scaffold led to
compounds such as 5, where benzazepinones and benzox-
azepinones had comparable BIR2 potency, N-methylation of
the L-alanine moiety was preferred, and there was room for
expansion around the azepinone N-substituent. In particular,
substituents containing aromatic groups were preferred in this
position.
a
Reagents and conditions: (a) NaH, 1-fluoro-2-nitrobenzene, DMF, 0
°C, 97%; (b) H2, 10% Pd/C, EtOH, rt, 95%; (c) EDCI, DMF, rt, 62%;
(d) TFA, CH2Cl2, 0 °C, quant; (e) BOC-N-methyl-L-alanine, HBTU,
HOBt, Et3N, DMF, rt, 95%; (f) 6-bromo-1-(chloromethyl)-2-
methoxynaphthalene, Cs2CO3, NaI, DMF, 60 °C, 54%; (g) TFA,
CH2Cl2, 0 °C, 56%.
B
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of Spirocyclic Benzoxazepinone Analogues 33−40
a
Reagents and conditions: (a) LDA, THF; dihydro-2H-pyran-4(3H)-one, −78 °C to rt, 50%; (b) KOH, H2O, MeOH, 60 °C, 92%; (c) 1-fluoro-2-
nitrobenzene, KHMDS, THF, rt, 92%; (d) Zn, NH4Cl, MeOH, 65 °C, quant; (e) EDCI, HOBt, DMF, rt, 74%; (f) H2, Pd(OH)2/C, MeOH, rt,
quant; (g) BOC-N-methyl-L-alanine, HBTU, HOBt, Et3N, DMF, rt, separated diastereomers, 37%; (h) RCH2X, Cs2CO3, NaI, DMF, rt−70 °C; (i)
for 33−35, 37, 38, HCl, MeOH, Et2O, rt; for 39, 40, TFA, CH2Cl2, 0 °C, 44−93% (2 steps); (j) 6-bromo-1-(chloromethyl)-2-methoxynaphthalene,
Cs2CO3, NaI, DMF, 60 °C, 54%; (k) CO, MeOH, Pd(OAc)2, Xantphos, Et3N, 70 °C, 67%; (l) LiOH·H2O, THF−H2O, rt, 88%; (m) TFA, CH2Cl2,
rt, 83%.
cyclization under amide-coupling conditions, and acidic
removal of the BOC group provided the amino benzoxazepi-
none core 26, which was subjected to chemistry similar to that
described in Scheme 1 above to prepare compound 27.
We also prepared benzoxazepinones with a spirocyclic
tetrahydropyran substituent (Scheme 3). The synthesis of
these analogues began with the aldol condensation of
dibenzylglycine ethyl ester (28) and dihydro-2H-pyran-
4(3H)-one, which was followed by hydrolysis of the ester to
provide acid 29. The ether linkage of 30 was formed by the
reaction with 1-fluoro-2-nitrobenzene. Chemoselective reduc-
tion of the nitro group, cyclization, and removal of the benzyl
protecting groups gave the amino benzoxazepinone scaffold 31.
The amino acid moiety was installed and the diastereomers
were separated using SFC to provide 32, and then the targeted
analogues 33−40 were made via the same alkylation−
deprotection sequence described above. This proved to be a
versatile method for preparing spirocyclic benzoxazepinones,
and we synthesized several novel analogues starting from a
variety of cyclic ketones (not shown). Analogue 36, which has a
carboxylic acid at the 6-position of the naphthalene, was
prepared from the corresponding aryl bromide by palladium-
catalyzed carbonylation and hydrolysis of the resulting methyl
ester.
bromo-2-methoxynaphthyl 19. None of these compounds had
any significant activity at XIAP BIR3 at up to 54 μM.
The 6-bromo-2-methoxynaphthyl analogue 19 displayed
moderate clearance in our in vitro rat liver microsome and
hepatocyte assays (17 and 38 mL/min/kg, respectively),
however, when we advanced it into rat PK studies (single-
compound dosing, 2 mg/kg iv), we observed a very high in vivo
clearance rate of 116 mL/min/kg, approximately two times
hepatic blood flow. To help determine which structural features
contributed to this clearance rate, we conducted an in vivo
metabolite identification study in rats by administering a 4 mg/
kg iv dose of 19 and analyzing urine and plasma samples, where
we observed significant quantities of the material derived from
hydrolysis of the alanine amide.
Initially, we sought to stabilize the amide against hydrolysis
by modulating the steric properties of the amino acid
substituent (Table 2). To efficiently profile the effect of these
changes on the clearance values, we employed cassette dosing
in our PK studies using groups of 3−4 compounds per study.
Selected compounds were tested in both cassette and single-
compound PK studies, confirming that each method provided
comparable values.29 The D-alanine analogue 20 was 10-fold
less potent than the natural isomer. Some larger substituents
were tolerated, such as those of the α-aminobutyric acid
analogue 21 and the threonine analogue 22, although these
groups did not improve the clearance, and in the case of the 22
it was considerably worse. Larger substituents were significantly
less active at BIR2, as were analogues with a quaternary α-
carbon (data not shown). Some modifications at the N-
terminus were tolerated. For example, the methyl group could
be replaced by ethyl (23), but this did not affect the clearance
rate.
RESULTS AND DISCUSSION
■
Our efforts commenced with a survey of azepinone N-
substituents with the aim of improving the XIAP BIR2 potency
while retaining selectivity over XIAP BIR3. Results are
displayed in Table 1. The starting point was benzyl analogue
12. Extended linkers, such as that of 13, were disfavored.
Naphthyl 14 was approximately equipotent with 12, while the
analogue 15 that was attached at the 1-position of the naphthyl
was more potent. We explored a range of substitutions on the
naphthyl ring, and analogues such as 16 provided a modest 2-
fold improvement in potency compared with 15. Substitution at
the 2-position of the naphthyl was preferred, and the 2-
methylnaphthyl analogue 17 had an IC50 of 0.489 μM. Further
improvements were realized by replacing the methyl with a
methoxy group (18), and the most promising analogue was 6-
These efforts were complicated by a continued lack of
correlation between the clearance in rat liver microsomes or
hepatocytes and that observed in vivo. Despite conducting the
PK studies using cassette dosing, we still lacked sufficient
throughput to rapidly build up the structure−property
relationships necessary to address the clearance issue, so we
aimed to develop an alternate in vitro screening protocol. We
profiled selected compounds in several systems, including rat
C
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. XIAP Activity for N-Substituted Benzazepinone
Analogues
Table 2. XIAP Activity for Benzazepinone Amino Acid
Analogues
a
All compounds were HCl salts except 21 which was a TFA salt.
b
Values that are the result of multiple determinations are reported as:
c
mean SD (number of determinations). Cassette dosing, 0.5 mg/kg
iv. Not tested, the compound was not evaluated in this assay.
d
synthetically tractable benzoxazepinone template. Many of the
substitution patterns we examined brought improvements to
blood stability, and the spiro-tetrahydropyran 33 was the most
promising (Table 3). It had high blood stability, and when we
profiled 33 in PK studies it had a substantially lower clearance
than 27, although at 60 mL/min/kg its rate was still close to rat
liver blood flow.
While the spiro-tetrahydropyran may have effectively blocked
the extrahepatic amide hydrolysis, other degradation pathways
would also need to be addressed to bring the clearance of 33 to
an acceptable range for in vivo applications. To determine
potential sites of CYP-mediated oxidative metabolism, we
incubated 33 with both rat and human hepatocytes and
analyzed the metabolites formed. We detected two major
metabolites: one derived from demethylation of the N-methyl-
L-alanine moiety and the other from demethylation of the
methoxy group of the naphthalene. To address the latter issue,
we reexamined the naphthyl moiety (Table 4). Replacing the
methoxy group with methyl caused a 10-fold loss of potency
(compound 34). Attempts to stabilize the methoxy group with
analogues such as the difluoromethoxy 35 likewise resulted in
an unacceptable loss of potency. We did not evaluate the PK
properties of these less potent analogues. In a different
approach, we looked at other changes such as replacing the
bromine with a carboxylic acid (36). The BIR2 potency of this
compound was improved, but its clearance was worse. We
prepared several heterocyclic analogues to modulate the
electronic properties of the ring system. Quinoline 37 had an
acceptable level of BIR2 activity, but its clearance was about 3-
fold higher than that of 33. Benzisoxazole 38 was significantly
less active. To aid in our design of new analogues, we obtained
a cocrystal structure of the carboxylic acid analogue 36 bound
to the XIAP BIR2 domain (Figure 3). This also revealed some
of the features that may contribute to the selectivity for BIR2
versus BIR3.30 For example, the fused benzene ring of the
a
b
All compounds were HCl salts. Values are reported as: mean SD
(number of determinations).
liver and kidney S9 fractions, plasma, and whole blood, and we
observed a correlation between in vivo clearance and stability in
rat whole blood. Compounds were incubated in rat blood for 4
h, and the percent of the compound remaining was quantitated.
All compounds with <50% remaining displayed unacceptably
high in vivo clearance. For compounds with >50% remaining,
we observed a range of in vivo clearance values, suggesting that
we were addressing one of multiple potential clearance
pathways. Nevertheless, the blood stability assay proved to be
a valuable filter to ease our reliance on resource-intensive PK
studies. As we continued to modify the scaffold, only those
analogues that passed the 50% blood-stability cutoff were
profiled in vivo.
Another approach we explored for blocking hydrolysis of the
amide involved the introduction of substituents onto the
azepinone ring. The benzazepinone and benzoxazepinone
templates typically provided equivalent activity and carried
similar metabolic liabilities (compare 19 and 27, Table 3), so
most of these changes were evaluated using the more
D
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. Comparison of in Vitro CL and Blood Stability with in Vivo CL across Scaffolds
19
27
33
a
a
XIAP BIR2 IC50 (μM)
XIAP BIR3 IC50 (μM)
0.194 0.124 (6)
0.101 0.011 (2)
0.065 0.042 (4)
>54 (6)
17
>54 (2)
27
23.5 10.3 (4)
rat liver microsomes CL (mL/min/kg)
rat liver hepatocytes CL (mL/min/kg)
rat blood stability (% remaining at 4 h)
28
21
88
b
38
n.t.
40
7
c
rat iv PK, CL (mL/min/kg)
155
521
60
a
b
c
Values are reported as: mean SD (number of determinations). Not tested, the compound was not evaluated in this assay. Cassette dosing, 0.5
mg/kg iv.
benzoxazepinone scaffold is situated in close proximity to
His223. This is a larger tryptophan residue in BIR3 that would
clash with the benzene ring. The naphthyl ring system extends
into a hydrophobic pocket between the side chains of Lys206
and Lys208. This pocket is absent in BIR3, which has glycine
and threonine at these positions. Additionally, the methoxy
group of the naphthyl is in the cleft between His223 and
Phe224; this cleft is much more restricted in BIR3 where these
residues are tryptophan and tyrosine. The structural data
indicated that relatively bulky groups could be added to the
solvent-exposed edge of the naphthyl moiety, and we
hypothesized that a 2-cyanophenyl group extending from the
bicyclic system could accept a hydrogen bond from the
backbone NH of Lys206. After modeling several possible
arrangements, we expected a 5,6-bicycle such as indazole to
optimally position this group (Figure 4). To test this, we
prepared 39 and found that it did improve BIR2 potency
compared to 38 (Table 4). Notably, this change also brought
an improvement to the clearance. Because 2-position
substituents on the naphthyl ring generally enhanced the
BIR2 affinity, we prepared the analogous indole system of 40
with a nitrile in that position. This compound exhibited
excellent BIR2 potency while maintaining an acceptable
clearance rate. Interestingly, this compound also showed a
greater level of XIAP BIR3 inhibition than the other analogues,
but it was still 78-fold selective for BIR2. The PK properties of
this compound were comparable in both rat and mouse and it
had good oral exposure (Table 5). We felt that the potency and
selectivity of this compound, coupled with its favorable PK
profile, would allow us to more fully evaluate the biological
properties of this class of XIAP BIR2 inhibitors.
To further understand the selectivity profile of 40, we
measured its activity against the BIR2 and BIR3 domains of
cIAP1 (Table 6). This compound did not have any significant
activity at the cIAP1 BIR3 domain, but it did have a
submicromolar IC50 for cIAP1 BIR2 inhibition. Still, it was 7-
fold selective for XIAP BIR2. Table 6 also shows the IC50 values
for the known BIR3-selective pan-IAP inhibitor 3 for
comparison.31
To assess the functional competence of BIR2-selective
compounds, we profiled 40 in an in vitro caspase reactivation
assay (Table 6). Cellular lysates were treated with cytochrome c
and ATP, resulting in activation of caspases. The caspase
activity was monitored by the cleavage of the fluorogenic
substrate Ac-DEVD-AFC, which is specific for caspase 3/7.
Addition of full-length XIAP inhibited the caspase activity and
addition of 40 restored it with an EC50 of 0.140 μM. This was
similar behavior to that observed for 3.
Compound 40 was screened for cell-based activity against
several cancer cell lines. No single-agent activity was observed,
but a significant effect on cell viability was observed in
combination with the DR5 antibody conatumumab,32 which
provides apoptotic pressure mediated through the extrinsic
pathway. In that regard, its activity was compared to that of 3.
Parts A and B of Figure 5 show the dose−response curves for
conatumumab in a 5 day MTS assay in SW620 cells in
combination with two concentrations of test compound. In the
absence of test compound, conatumumab had little effect on
cell viability at any of the tested concentrations. Modest
sensitization was observed in the presence of 1.1 μM of test
compound (Figure 5A) and greater sensitization was observed
at 3.3 μM (Figure 5B). At the latter concentration, 40 was more
effective than 3. Similar levels of activity were observed in other
cancer cell lines (data is summarized in Figure 6; see
Supporting Information Figure S1 for tabulated EC50 values).
To confirm that this antiproliferative activity correlated with
increased apoptotic signaling, we measured the caspase 3/7
activity in SW620 cells after treatment with conatumumab in
combination with either 3 or 40 (Figure 7). Slightly elevated
caspase 3/7 levels were observed upon treatment with
conatumumab alone, and this was potentiated in a dose-
dependent manner by both 3 and 40. Again, 40 promoted
greater levels of activity than 3.
The BIR3-selective compound 3 exerts single agent activity
in MDA-MB-231 cells that is dependent on cIAP binding and
degradation, which leads to increased TNFα levels.33 Because
40 has some affinity for the BIR2 domain of cIAP1, we
examined whether it would display a similar activity profile in
this system. We treated MDA-MB-231 cells with either 3 or 40
and measured TNFα levels after 19 h (Figure 8). TNFα levels
were significantly elevated in cells treated with 3, while cells
treated with 40 were not substantially different from vehicle at
up to 10 μM, confirming that 40 does not induce TNFα in this
system.
To further understand the activity of our inhibitor, we
conducted an in vivo study using LOX xenograft-bearing nude
mice. The mice were treated with a single dose of
conatumumab (1 mg/kg ip), 3 (100 mg/kg po), or 40 (200
E
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 4. XIAP Activity of N-Substituted Derivatives of Spirocyclic Benzoxazepinone Core
a
b
All compounds were HCl salts except 36, which was a TFA salt and 39 and 40 which were free bases. Values that are the result of multiple
c
d
determinations are reported as mean SD (number of determinations). Cassette dosing, 0.5 mg/kg iv. Not tested, the compound was not
evaluated in this assay.
mg/kg po), or a combination of conatumumab with 3 or 4.
After 8 h, the tumors were removed, and caspase 3/7 activity
was determined using a fluorescence assay based on cleavage of
DEVD-AMC (Figure 9). Additionally, the effect on protein
levels was examined by Western blot analysis (Figure 10). The
caspase activity of conatumumab, 3, or 40 alone was not
significantly different from vehicle, whereas in combination
with the DR5 antibody, both 3 and 40 promoted substantial
F
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 6. Biochemical Properties of 40 Compared to 3
a
a
property
value for 40
value for 3
XIAP activity
BIR2 IC50 (μM)
BIR3 IC50 (μM)
cIAP1 activity
BIR2 IC50 (μM)
BIR3 IC50 (μM)
0.039 0.010 (2)
3.06 0.28 (2)
1.05 0.31 (2)
0.030 0.009 (2)
0.271 0.115 (2)
>55 (2)
2.72 0.81 (2)
0.058 0.004 (2)
XIAP-caspase reactivation
EC50 (μM) 0.140
0.283
a
Values that are the result of multiple determinations are reported as
mean SD (number of determinations).
substantial levels of cIAP1 degradation, while this was not
observed for 40. XIAP levels were not significantly impacted by
either compound.
Further profiling of 40 revealed significant in vitro safety
liabilities. This compound was both a potent inhibitor and time-
dependent inhibitor of CYP3A4, and it also exhibited
significant CYP3A4 induction. These liabilities limited the
clinical utility of 40, however, we felt that we could apply some
of the SAR lessons from this investigation to overcome these
liabilities using a related benzodiazepinone scaffold. Those
optimization efforts are the subject of a subsequent report.34
Despite its liabilities, 40 is a potent and selective XIAP BIR2
inhibitor with a PK profile suitable for in vivo studies, and it
proved to be a valuable tool for investigating the biology of
BIR2-selective XIAP inhibitors. In a separate report, we will
describe additional studies of the biological utility of 40,
including a demonstration of in vivo efficacy in a tumor
xenograft study.35 These studies support the usefulness of
targeting the BIR2-domain to achieve selectivity for XIAP and
impact apoptotic pathways in a manner mechanistically
differentiated from BIR3-selective pan-IAP inhibitors.
Figure 3. X-ray cocrystal structure of 36 bound to the peptide-binding
groove of the BIR2 domain of XIAP, represented with an electrostatic
surface. Hydrogen-bonding interactions between the ligand and the
protein are indicated by dotted lines.
EXPERIMENTAL SECTION
■
Chemistry. General. All commercially available reactants, reagents,
and solvents were used as received. Reactions using air- or moisture-
sensitive reagents were performed under an atmosphere of Ar or N2.
Reactions were monitored by LC-MS or TLC. Flash chromatography
was performed with Isco CombiFlash Companion or AnaLogix
IntelliFlash chromatography systems using prepacked silica gel
columns (40−60 μm particle size RediSep or 20−40 μm spherical
silica gel RediSep Gold columns purchased from Teledyne Isco or
comparable products from other vendors). Preparative HPLC was
performed using a Waters HPLC system consisting of a 2767 sample
manager, 2525 binary gradient module, and 2996 photodiode array
detector with a SunFire Prep C18 OBD column (5 μm, 30 mm × 100
mm) eluted with a 10−100% MeCN−H2O linear gradient with 0.1%
v/v TFA at a flow rate of 30 mL/min or using similar methods on a
comparable system. NMR spectra were measured on Bruker 300 or
400 MHz spectrometers. Chemical shifts are reported in ppm
downfield from TMS using residual nondeuterated solvent as an
internal standard (CHCl3, 7.26 ppm; DMSO, 2.50 ppm; MeOH, 3.31
ppm). Data are reported in the form: chemical shift (multiplicity,
coupling constants, integration). Multiplicities are recorded by the
following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad. HRMS data were recorded on a ThermoFisher
OrbiTrap FTMS system using flow injection with electrospray
ionization in positive mode. Analytical HPLC and LC-MS analyses
were performed using either Waters or Agilent LC-MS systems. The
Waters system was comprised of a ZQ mass spectrometer using
multimode (ES/APCI) ionization with alternating switching and an
ES Industries Chromegabond WR C18 column (3 μm, 120 Å, 30 mm
× 3.2 mm) eluted with a 10−90% MeCN−H2O linear gradient with
Figure 4. The energy-minimized structure of 39 (green) overlaid with
the crystal structure of 36 (yellow) bound to XIAP BIR2. The energy
minimization of 39 was performed with MOE (Chemical Computing
Group Inc., version 2011.10) using the MMFF94x forcefield.
Table 5. PK Properties of Compound 40
a
b
c
property
rat, iv
mouse, iv
mouse, po
AUC (ng·h/mL)
Cmax (ng/mL)
t1/2 (h)
196
190
1.7
4.1
41
543
495
6.9
8.6
33
2858
647
5.0
Vss (L/kg)
CL (mL/min/kg)
F (%)
53
a
b
Cassette dosing, 0.5 mg/kg iv. Single-compound dosing, 1 mg/kg iv.
c
Single-compound dosing, 10 mg/kg po.
increases in caspase 3/7 activity. Western blot analysis showed
cleavage of PARP and caspase 3 upon treatment with
conatumumab in combination with either 3 or 40, consistent
with the induction of apoptosis. Compound 3 promoted
G
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. (A,B) Comparison of the activity of 3 and 40 in combination with the DR5 antibody conatumumab in SW620 cells in a 5 day MTS cell
proliferation assay. Each graph shows dose−response curves for conatumumab in the presence of the indicated concentration of test compound.
Values are the mean of four determinations SD.
Figure 7. Caspase 3/7 activity in SW620 cells 4 h after treatment with
Figure 6. Cell line sensitivity to 40 and 3 in combination with DR5
the test compounds. Cells were incubated with conatumumab, 3, and/
agonistic antibody conatumumab. Each bar represents the EC50 of
or 40 at the indicated concentrations (combinations used 0.5 μg/mL
conatumumab, either alone (blue bars) or in the presence of 3 (3.3
conatumumab with the indicated concentrations of 3 or 40) for 4 h,
μM) (red bars) or 40 (3.3 μM) (green bars) in a 5 day MTS assay
and then they were lysed and the caspase 3/7 activity was measured by
measuring viability of the indicated cell line (for LOX cells, the assay
the cleavage of DEVD-AMC. Values are the mean of two
was performed in a 3 day format).
determinations SD.
0.1% v/v HCO2H. The Agilent system was comprised of a 6140 mass
spectrometer using multimode (ES/APCI) ionization with alternating
switching and an Agilent Zorbax SB C-18 column (3.5 μm, 30 mm
× 2.1 mm) eluted with a 10−90% MeCN−H2O linear gradient with
0.1% v/v HCO2H. All tested compounds were evaluated on one of
these analytical HPLC systems and determined to be ≥95% pure.
General Procedure A: Amino Acid Coupling. To a rt solution of 6,
25, or 30 (1 equiv) in DMF (0.1−0.2 M) was added the appropriate
amino acid (1.0−1.2 equiv), followed by Et3N (3 equiv), HBTU (1.2
equiv), and HOBt (1.2 equiv). The reaction was stirred at rt until
complete, then it was diluted with H2O and extracted with EtOAc.
The combined organic layers were washed with 1:1 satd aq NaHCO3−
satd aq NaCl and then satd aq NaCl, dried over Na2SO4, filtered, and
concentrated in vacuo. The crude material was purified by flash
chromatography to provide product.
General Procedure B: Alkylation of Core. To a rt solution of above
material in DMF was added the appropriate alkyl halide or mesylate
Figure 8. Presence of TNFα in the supernate from MDA-MB-231
cells 19 h after treatment with the indicated concentration of BIR3-
selective compound 3 or BIR2-selective compound 40 in comparison
with TNFα standards. Values are the mean of two determinations
SD.
(1.4 equiv), Cs2CO3 (1.5 equiv), and NaI (1.5 equiv). The reaction
was stirred at 60 °C overnight, then cooled to rt, diluted with H2O,
and extracted with EtOAc. The combined organic layers were washed
with satd aq NaCl, dried over Na2SO4, filtered, and concentrated in
vacuo. The crude material was purified by flash chromatography to
provide product.
H
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
tert-Butyl Methyl((R)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1H-
benzo[b]azepin-3-ylamino)propan-2-yl)carbamate (8). According
to general procedure A, racemic 6 (0.50 g, 2.8 mmol) and BOC-N-
methyl-D-alanine (692 mg, 3.4 mmol) were converted to 8 (0.409 g,
40%) as a white solid (the diastereomers were separated during flash
1
chromatography). H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H),
7.88 (d, J = 7.86 Hz, 1H), 7.23−7.31 (m, 2H), 7.10−7.15 (m, 1H),
7.00 (d, J = 7.7 Hz, 1H), 4.32−4.68 (m, 1H), 4.15−4.24 (m, 1H), 2.70
(s, 3H), 2.63−2.77 (m, 2H), 2.20−2.33 (m, 1H), 2.02−2.14 (m, 1H),
1.39 (s, 9H), 1.13−1.21 (m, 3H).
tert-Butyl Methyl((S)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1H-
benzo[b]azepin-3-ylamino)butan-2-yl)carbamate (9). According to
general procedure A, 6 (81.1 mg, 0.460 mmol) and (S)-2-(tert-
butoxycarbonyl(methyl)amino)butanoic acid (100 mg, 0.460 mmol)
1
were converted to 9 (121 mg, 70%) as a white foam. H NMR (400
MHz, DMSO-d6) δ 9.81 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.23−7.32
(m, 2H), 7.09−7.16 (m, 1H), 7.00 (d, J = 7.4 Hz, 1H), 4.10−4.48 (m,
2H), 2.68 (s, 3H), 2.64−2.75 (m, 2H), 2.20−2.33 (m, 1H), 2.02−2.13
(m, 1H), 1.73−1.87 (m, 1H), 1.46−1.61 (m, 2H), 1.38 (s, 9H), 0.76−
0.88 (m, 3H). MS m/z 376 [M + H]+.
tert-Butyl (2S,3S)-3-Hydroxy-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahy-
dro-1H-benzo[b]azepin-3-ylamino)butan-2-ylcarbamate (10). Ac-
cording to general procedure A, except 6 equiv Et3N were used, 6 (123
mg, 0.698 mmol) and BOC-L-allo-threonine dicyclohexylamine (336
mg, 0.838 mmol) were converted to 10 (263 mg, quant) as a white
foam. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.02 (d, J = 7.5
Hz, 1H), 7.23−7.33 (m, 2H), 7.11−7.18 (m, 1H), 7.01 (d, J = 7.7 Hz,
1H), 6.72 (d, J = 8.7 Hz, 1H), 4.88 (d, J = 4.0 Hz, 1H), 4.11−4.21 (m,
1H), 3.68−3.92 (m, 2H), 2.63−2.79 (m, 2H), 2.26−2.39 (m, 1H),
1.99−2.09 (m, 1H), 1.30−1.40 (m, 9H), 1.03 (d, J = 6.0 Hz, 3H). MS
m/z 378 [M + H]+.
Figure 9. Caspase 3/7 activity in LOX melanoma xenografts. Tumor-
bearing nude mice were treated with a single dose of conatumumab (1
mg/kg ip), 40 (100 mg/kg po), 3 (200 mg/kg po), or a combination.
After 8 h, xenografts were removed and caspase 3/7 activity was
determined with a fluorescence assay measuring cleavage of DEVD-
AMC. Mean SD is indicated for each group (n = 3).
General Procedure C: Deprotection. To a rt solution of above
material in MeOH was added 2.0 M HCl in Et2O. The reaction was
stirred at rt until complete, then concentrated in vacuo and lyophilized
from MeCN−H2O to provide product.
tert-Butyl Methyl((S)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1H-
benzo[b]azepin-3-ylamino)propan-2-yl)carbamate (7). According
to general procedure A, 6 (0.419 g, 2.38 mmol) and BOC-N-
methyl-L-alanine (579 mg, 2.85 mmol) were converted to 7 (0.808 g,
94%) as a white foam. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H),
7.87 (d, J = 7.9 Hz, 1H), 7.22−7.31 (m, 2H), 7.09−7.16 (m, 1H), 7.00
(d, J = 7.5 Hz, 1H), 4.29−4.62 (m, 1H), 4.10−4.24 (m, 1H), 2.69 (s,
3H), 2.66−2.75 (m, 2H), 2.19−2.36 (m, 1H), 2.00−2.14 (m, 1H),
1.36 (s, 9H), 1.22 (d, J = 7.2 Hz, 3H). MS m/z 362 [M + H]+.
tert-Butyl Ethyl((S)-1-oxo-1-((S)-2-oxo-2,3,4,5-tetrahydro-1H-
benzo[b]azepin-3-ylamino)propan-2-yl)carbamate (11). According
to general procedure A, 6 (203 mg, 1.15 mmol) and (S)-2-(tert-
butoxycarbonyl(ethyl)amino)propanoic acid (0.25 g, 1.2 mmol) were
1
converted to 11 (0.205 mg, 47%) as a white foam. H NMR (300
MHz, DMSO-d6) δ 9.86 (s, 1H), 7.65−7.79 (m, 1H), 7.22−7.32 (m,
2H), 7.09−7.17 (m, 1H), 7.00 (d, J = 7.7 Hz, 1H), 4.08−4.58 (m,
Figure 10. Western blot analysis of LOX melanoma xenograft homogenates after 8 h treatment with a single dose of the DR5 antibody
conatumumab (1 mg/kg ip) or a combination of conatumumab (1 mg/kg ip) with 3 (100 mg/kg po) or 40 (200 mg/kg po). Each lane is the result
of a single xenograft.
I
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 3.01−3.27 (m, 2H), 2.60−2.80 (m, 2H), 2.23−2.39 (m, 1H),
1.94−2.12 (m, 1H), 1.37 (s, 9H), 1.24 (d, J = 7.0 Hz, 3H), 1.02 (t, J =
6.9 Hz, 3H). MS m/z 398 [M + Na]+.
(m, 2H), 1.95−2.09 (m, 1H), 1.61 (d, J = 6.9 Hz, 3H). HRMS m/z
calcd for C26H30O2N3 [M + H]+ 416.23325, found 416.23212.
(S)-N-((S)-1-((2-Methoxynaphthalen-1-yl)methyl)-2-oxo-2,3,4,5-
tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)propanamide
Hydrochloride (18). According to general procedures B and C, 7 (0.10
g, 0.277 mmol) and 1-(chloromethyl)-2-methoxynaphthalene (0.086
g, 0.415 mmol) were converted to 18 (55.0 mg, 42% 2 steps) as a
light-yellow solid. 1H NMR (300 MHz, methanol-d4) δ 8.24 (d, J = 8.5
Hz, 1H), 7.68−7.76 (m, 2H), 7.42−7.55 (m, 2H), 7.28−7.36 (m, 1H),
7.23 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 9.1 Hz, 1H), 7.04 (t, J = 7.4 Hz,
1H), 6.90−6.95 (m, 1H), 6.04 (d, J = 14.5 Hz, 1H), 5.38 (d, J = 14.5
Hz, 1H), 4.25−4.38 (m, 1H), 3.88 (q, J = 6.9 Hz, 1H), 3.77 (s, 3H),
2.62 (s, 3H), 1.96−2.42 (m, 4H), 1.62 (d, J = 6.9 Hz, 3H). HRMS m/z
calcd for C26H30O3N3 [M + H]+ 432.22817, found 432.22678.
(S)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2-
oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)-
propanamide Hydrochloride (19). According to general procedures B
and C, 7 (220 mg, 0.609 mmol) and 6-bromo-1-(chloromethyl)-2-
methoxynaphthalene (261 mg, 0.913 mmol) were converted to 19
(190 mg, 57% 2 steps) as a white solid. 1H NMR (400 MHz, DMSO-
d6) δ 8.95 (d, J = 7.9 Hz, 1H), 8.87 (br s, 2H), 8.17 (d, J = 9.1 Hz,
1H), 8.09 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.52−7.60 (m,
2H), 7.25−7.33 (m, 2H), 7.04−7.09 (m, 1H), 6.98−7.02 (m, 1H),
5.94 (d, J = 14.5 Hz, 1H), 5.24 (d, J = 14.5 Hz, 1H), 4.15−4.24 (m,
1H), 3.80 (q, J = 6.9 Hz, 1H), 3.71 (s, 3H), 2.43 (s, 3H), 2.19−2.29
(m, 1H), 2.05−2.17 (m, 2H), 1.90−2.02 (m, 1H), 1.42 (d, J = 6.9 Hz,
3H). HRMS m/z calcd for C26H29O3N3Br [M + H]+ 510.13868, found
510.13901.
(R)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2-
oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)-
propanamide Hydrochloride (20). According to general procedure B,
8 (0.20 g, 0.55 mmol) and 6-bromo-1-(chloromethyl)-2-methox-
ynaphthalene (261 mg, 0.913 mmol) were reacted to provide tert-butyl
(R)-1-((S)-1-((6-bromo-2-methoxynaphthalen-1-yl)methyl)-2-oxo-
2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylamino)-1-oxopropan-2-yl-
(methyl)carbamate (174 mg, 52%). A portion of this material (60 mg,
0.098 mmol) was reacted according to general procedure C to provide
20 (50.0 mg, 93%) as a white solid. 1H NMR (400 MHz, methanol-d4)
δ 8.21 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 9.1
Hz, 1H), 7.49−7.56 (m, 2H), 7.26 (t, J = 7.9 Hz, 1H), 7.22 (d, J = 9.1
Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 6.7 Hz, 1H), 6.01 (d, J =
14.5 Hz, 1H), 5.37 (d, J = 14.5 Hz, 1H), 4.26 (dd, J = 11.8, 6.95 Hz,
1H), 3.82−3.91 (m, 1H), 3.75 (s, 3H), 2.71 (s, 3H), 2.14−2.37 (m,
3H), 1.96−2.08 (m, 1H), 1.46 (d, J = 6.9 Hz, 3H). HRMS m/z calcd
for C26H29O3N3Br [M + H]+ 510.13868, found 510.13852.
(S)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2-
oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(methylamino)-
butanamide 2,2,2-trifluoroacetate (21). According to general
procedure B, 9 (121 mg, 0.322 mmol) and 6-bromo-1-(chlorometh-
yl)-2-methoxynaphthalene (138 mg, 0.483 mmol) were converted to
tert-butyl (S)-1-((S)-1-((6-bromo-2-methoxynaphthalen-1-yl)methyl)-
2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylamino)-1-oxobutan-
2-yl(methyl)carbamate (88 mg, 44%). To a rt solution of this material
in CH2Cl2 (2 mL) was added TFA (0.4 mL). The reaction was stirred
at rt for 30 min, then concentrated in vacuo and lyophilized from
MeCN−H2O to provide 21 (76.5 mg, 85%) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 8.99 (d, J = 7.8 Hz, 1H), 8.86 (br s, 1H),
8.74 (br s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 2.0 Hz, 1H), 7.79
(d, J = 9.4 Hz, 1H), 7.52−7.59 (m, 2H), 7.23−7.34 (m, 2H), 7.04−
7.09 (m, 1H), 6.99−7.02 (m, 1H), 5.93 (d, J = 14.5 Hz, 1H), 5.26 (d, J
= 14.5 Hz, 1H), 4.20−4.31 (m, 1H), 3.68−3.74 (m, 1H), 2.39−2.46
(m, 3H), 1.70−2.29 (m, 6H), 0.99 (t, J = 7.4 Hz, 3H). HRMS m/z
calcd for C27H31O3N3Br [M + H]+ 524.15433, found 524.15430.
(2S,3S)-2-Amino-N-((S)-1-((6-bromo-2-methoxynaphthalen-1-yl)-
methyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-3-hy-
droxybutanamide hydrochloride (22). According to general
procedures B and C, 10 (104 mg, 0.276 mmol) and 6-bromo-1-
(chloromethyl)-2-methoxynaphthalene (102 mg, 0.358 mmol) were
converted to 22 (61 mg, 41% 2 steps) as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 8.80 (d, J = 7.9 Hz, 1H), 8.16 (d, J = 9.3 Hz, 1H),
8.09 (d, J = 1.8 Hz, 1H), 7.96 (br s, 3H), 7.80 (d, J = 9.3 Hz, 1H), 7.57
(S)-N-((S)-1-Benzyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-
3-yl)-2-(methylamino)propanamide Hydrochloride (12). According
to general procedures B and C, 7 (0.10 g, 0.279 mmol) and benzyl
bromide (0.071 g, 0.415 mmol) were converted to 12 (46.6 mg, 48% 2
steps) as a white solid. 1H NMR (300 MHz, methanol-d4) δ 8.71 (d, J
= 6.9 Hz, 1H), 7.30−7.40 (m, 2H), 7.16−7.27 (m, 7H), 5.29 (dd, J =
14.8, 2.4 Hz, 1H), 4.81−4.86 (m, 1H), 4.32−4.45 (m, 1H), 3.78−3.90
(m, 1H), 2.62 (d, J = 2.1 Hz, 3H), 2.54 (d, J = 5.4 Hz, 2H), 2.36 (d, J
= 9.7 Hz, 1H), 2.01−2.20 (m, 1H), 1.49−1.63 (m, 3H). HRMS m/z
calcd for C21H26O2N3 [M + H]+ 352.20195, found 352.20105.
(S)-2-(Methylamino)-N-((S)-2-oxo-1-phenethyl-2,3,4,5-tetrahy-
dro-1H-benzo[b]azepin-3-yl)propanamide Hydrochloride (13). Ac-
cording to general procedure B, except the sodium iodide was omitted,
and general procedure C, 7 (0.13 g, 0.36 mmol) and (2-bromoethyl)-
benzene (0.10 g, 0.54 mmol) were converted to 13 (52.4 mg, 36% 2
1
steps) as a white solid. H NMR (300 MHz, DMSO-d6) δ 9.09−9.33
(m, 1H), 8.81−8.95 (m, 1H), 8.65−8.79 (m, 1H), 7.33−7.41 (m, 2H),
7.08−7.32 (m, 7H), 4.30−4.50 (m, 1H), 4.05−4.23 (m, 1H), 3.71−
3.89 (m, 2H), 2.63−2.91 (m, 2H), 2.35−2.59 (m, 5H), 2.09−2.25 (m,
1H), 1.87−2.03 (m, 1H), 1.27−1.41 (m, 3H). HRMS m/z calcd for
C22H28O2N3 [M + H]+ 366.21760, found 366.21683.
(S)-2-(Methylamino)-N-((S)-1-(naphthalen-2-ylmethyl)-2-oxo-
2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydro-
chloride (14). According to general procedure B, except the sodium
iodide was omitted, and general procedure C, 7 (0.10 g, 0.279 mmol)
and 2-(bromomethyl)naphthalene (0.092 g, 0.415 mmol) were
1
converted to 14 (72.6 mg, 60% 2 steps) as a white solid. H NMR
(300 MHz, DMSO-d6) δ 9.34 (br s, 1H), 8.97 (d, J = 7.6 Hz, 1H),
8.84 (br s, 1H), 7.76−7.89 (m, 3H), 7.70 (s, 1H), 7.43−7.51 (m, 3H),
7.30−7.38 (m, 2H), 7.14−7.25 (m, 2H), 5.44 (d, J = 15.1 Hz, 1H),
5.00 (d, J = 15.1 Hz, 1H), 4.22−4.37 (m, 1H), 3.75−3.86 (m, 1H),
3.45−3.63 (m, 1H), 2.47−2.60 (m, 1H), 2.42−2.46 (m, 3H), 2.03−
2.33 (m, 2H), 1.41 (d, J = 7.0 Hz, 3H). MS m/z 402 [M + H]+.
(S)-2-(Methylamino)-N-((S)-1-(naphthalen-1-ylmethyl)-2-oxo-
2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)propanamide Hydro-
chloride (15). According to general procedures B and C, 7 (0.10 g,
0.279 mmol) and 1-(chloromethyl)naphthalene (0.073 g, 0.415
mmol) were converted to 15 (40.9 mg, 33% 2 steps) as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 9.28 (br s, 1H), 9.00 (d, J =
7.6 Hz, 1H), 8.82 (br s, 1H), 8.21 (dd, J = 6.2, 3.5 Hz, 1H), 7.91 (dd, J
= 6.2, 3.2 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H),
7.49−7.55 (m, 2H), 7.28−7.38 (m, 3H), 7.09−7.15 (m, 2H), 5.94 (d, J
= 15.1 Hz, 1H), 5.21 (d, J = 15.4 Hz, 1H), 4.24−4.36 (m, 1H), 3.75−
3.86 (m, 1H), 3.35−3.48 (m, 1H), 2.41−2.47 (m, 3H), 1.97−2.38 (m,
3H), 1.42 (d, J = 6.6 Hz, 3H). HRMS m/z calcd for C25H28O2N3 [M +
H]+ 402.21760, found 402.21646.
(S)-2-(Methylamino)-N-((S)-1-((4-methylnaphthalen-1-yl)-
methyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-
propanamide Hydrochloride (16). According to general procedures B
and C, 7 (0.10 g, 0.277 mmol) and 1-(chloromethyl)-4-methylnaph-
thalene (0.079 g, 0.415 mmol) were converted to 16 (30.5 mg, 24% 2
steps) as a white solid. 1H NMR (300 MHz, methanol-d4) δ 8.22 (d, J
= 7.9 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.44−7.57 (m, 3H), 7.28−
7.36 (m, 1H), 7.23−7.27 (m, 1H), 7.06−7.19 (m, 3H), 5.97 (d, J =
15.1 Hz, 1H), 5.17 (d, J = 15.1 Hz, 1H), 4.36−4.47 (m, 1H), 3.86 (q, J
= 6.7 Hz, 1H), 2.63 (s, 3H), 2.60 (s, 3H), 2.21−2.43 (m, 3H), 2.01−
2.15 (m, 1H), 1.59 (d, J = 6.9 Hz, 3H). HRMS m/z calcd for
C26H30O2N3 [M + H]+ 416.23325, found 416.23218.
(S)-2-(Methylamino)-N-((S)-1-((2-methylnaphthalen-1-yl)-
methyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-
propanamide Hydrochloride (17). According to general procedures B
and C, 7 (0.10 g, 0.277 mmol) and 1-(chloromethyl)-2-methylnaph-
thalene (0.079 g, 0.415 mmol) were converted to 17 (63.7 mg, 51% 2
steps) as a white solid. 1H NMR (300 MHz, methanol-d4) δ 8.15−8.25
(m, 1H), 7.73−7.81 (m, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.37−7.46 (m,
2H), 7.07−7.20 (m, 4H), 7.01−7.06 (m, 1H), 5.91 (d, J = 14.8 Hz,
1H), 5.42 (d, J = 14.8 Hz, 1H), 4.24−4.34 (m, 1H), 3.86 (q, J = 6.9
Hz, 1H), 2.62 (s, 3H), 2.41 (s, 3H), 2.35−2.50 (m, 1H), 2.12−2.31
J
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(dd, J = 9.1, 2.0 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 9.3 Hz,
1H), 7.26 (t, J = 7.1 Hz, 1H), 7.04−7.09 (m, 1H), 6.98−7.02 (m, 1H),
5.92 (d, J = 14.5 Hz, 1H), 5.51 (d, J = 4.4 Hz, 1H), 5.26 (d, J = 14.5
Hz, 1H), 4.12−4.25 (m, 2H), 3.81 (d, J = 4.1 Hz, 1H), 3.71 (s, 3H),
1.90−2.28 (m, 4H), 1.15 (d, J = 6.7 Hz, 3H). HRMS m/z calcd for
C26H29O4N3Br [M + H]+ 526.13360, found 526.13416.
2.72 (m, 3H), 1.38 (s, 9H), 1.24 (d, J = 6.9 Hz, 3H). MS m/z 386 [M
+ Na]+.
Step 2. According to general procedure B, tert-butyl methyl((S)-1-
oxo-1-((S)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-
ylamino)propan-2-yl)carbamate (1.1 g, 3.03 mmol) and 6-bromo-1-
(chloromethyl)-2-methoxynaphthalene (951 mg, 3.33 mmol) were
converted to tert-butyl (S)-1-((S)-5-((6-bromo-2-methoxynaphthalen-
1-yl)methyl)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ylami-
no)-1-oxopropan-2-yl(methyl)carbamate (1.00 g, 54%) as a light-
(S)-N-((S)-1-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-2-
oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-2-(ethylamino)-
propanamide Hydrochloride (23). According to general procedures B
and C, 11 (205 mg, 0.545 mmol) and 6-bromo-1-(chloromethyl)-2-
methoxynaphthalene (234 mg, 0.818 mmol) were converted to 23
1
yellow foam. H NMR (300 MHz, DMSO-d6) δ 8.18 (d, J = 8.2 Hz,
1H), 7.99−8.07 (m, 2H), 7.78 (d, J = 9.1 Hz, 1H), 7.46−7.54 (m,
2H), 7.33 (d, J = 9.4 Hz, 1H), 7.02−7.17 (m, 2H), 6.89 (d, J = 7.6 Hz,
1H), 5.86 (d, J = 14.8 Hz, 1H), 5.24 (d, J = 14.5 Hz, 1H), 4.35−4.75
(m, 2H), 4.22−4.31 (m, 1H), 4.10−4.20 (m, 1H), 3.80 (s, 3H), 2.70
(s, 3H), 1.37 (s, 9H), 1.27 (d, J = 6.9 Hz, 3H).
1
(206 mg, 67% 2 steps) as a light-yellow solid. H NMR (400 MHz,
DMSO-d6) δ 9.49 (br s, 1H), 9.00 (d, J = 7.8 Hz, 1H), 8.78 (br s, 1H),
8.17 (d, J = 9.4 Hz, 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.79 (d, J = 9.0 Hz,
1H), 7.58 (dd, J = 9.0, 2.0 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.24−
7.32 (m, 2H), 7.03−7.08 (m, 1H), 6.97−7.01 (m, 1H), 5.93 (d, J =
14.5 Hz, 1H), 5.24 (d, J = 14.5 Hz, 1H), 4.14−4.23 (m, 1H), 3.83−
3.93 (m, 1H), 3.71 (s, 3H), 2.71−2.86 (m, 2H), 1.90−2.29 (m, 4H),
1.44 (d, J = 6.64 Hz, 3H), 1.14 (t, J = 7.2 Hz, 3H). HRMS m/z calcd
for C27H31O3N3Br [M + H]+ 524.15433, found 524.15466.
Step 3. To a 0 °C solution of tert-butyl (S)-1-((S)-5-((6-bromo-2-
methoxynaphthalen-1-yl)methyl)-4-oxo-2,3,4,5-tetrahydrobenzo[b]-
[1,4]oxazepin-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate (52
mg, 0.085 mmol) in CH2Cl2 (1 mL) was added TFA (1 mL). The
reaction was stirred at 0 °C for 1 h, then concentrated in vacuo and
triturated with Et2O to provide 27 (30 mg, 56%) as a light-brown
(S)-2-tert-Butoxycarbonylamino-3-(2-nitrophenoxy)propionic
Acid (25). To a 0 °C suspension of NaH (60% w/w in mineral oil, 2.9
g, 72 mmol) in DMF (25 mL) was added a solution of (S)-2-(tert-
butoxycarbonylamino)-3-hydroxypropanoic acid (7 g, 34 mmol) in
DMF (25 mL). After 2 h, a solution of 1-fluoro-2-nitrobenzene (5.29
g, 37.5 mmol) in DMF (25 mL) was added and the resulting mixture
was stirred at 0 °C for 3 h. The mixture was poured into 0 °C H2O
(200 mL), acidified to pH 5.0 with 1 N aq HCl, and extracted with
EtOAc. The combined organic layers were dried over Na2SO4, filtered,
and concentrated in vacuo. The crude material was purified by flash
chromatography to provide 25 (10.9 g, 97%). MS m/z 349 [M + Na]+.
(S)-3-Amino-2,3-dihydrobenzo[b][1,4]oxazepin-4(5H)-one (26).
Step 1. A mixture of 25 (7.20 g, 22.1 mmol) and 10% w/w palladium
on carbon (720 mg, 0.677 mmol) in EtOH (250 mL) was stirred
under hydrogen (1 atm) for 4 h. The reaction was filtered through
Celite, and the filtrate was concentrated in vacuo to provide (S)-3-(2-
aminophenoxy)-2-(tert-butoxycarbonylamino)propionic acid (6.2 g,
1
solid. H NMR (300 MHz, DMSO-d6) δ 9.06 (d, J = 7.9 Hz, 1H),
8.67−8.90 (m, 2H), 8.00−8.07 (m, 2H), 7.79 (d, J = 9.1 Hz, 1H),
7.47−7.57 (m, 2H), 7.34 (d, J = 9.4 Hz, 1H), 7.05−7.20 (m, 2H), 6.92
(d, J = 6.6 Hz, 1H), 5.87 (d, J = 14.5 Hz, 1H), 5.26 (d, J = 14.5 Hz,
1H), 4.67−4.79 (m, 1H), 4.12−4.31 (m, 2H), 3.81−3.90 (m, 1H),
3.79 (s, 3H), 2.45−2.48 (m, 3H), 1.42 (d, J = 6.9 Hz, 3H). HRMS m/z
calcd for C25H27O4N3Br [M + H]+ 512.11795, found 512.11705.
2-(Dibenzylamino)-2-(4-hydroxytetrahydro-2H-pyran-4-yl)acetic
Acid (29). Step 1. To a −78 °C solution of ethyl 2-(dibenzylamino)-
acetate (10 g, 35 mmol) in THF (100 mL) was added LDA (2.0 M
solution, 38.8 mL, 77.6 mmol). After 30 min, a solution of dihydro-
2H-pyran-4(3H)-one (3.91 mL, 42.3 mmol) in THF (20 mL) was
added. The reaction was warmed to rt and stirred for 1 h, then
quenched with satd aq NH4Cl, concentrated in vacuo, poured into
H2O (300 mL), and extracted with EtOAc (2 × 250 mL). The
combined organic layers were washed with H2O (2 × 200 mL) and
satd aq NaCl (100 mL), dried over Na2SO4, filtered, and concentrated
in vacuo. The crude material was purified by flash chromatography to
provide ethyl 2-(dibenzylamino)-2-(4-hydroxytetrahydro-2H-pyran-4-
1
95%) as a white solid. H NMR (300 MHz, DMSO-d6) δ 7.44 (d, J =
8.5 Hz, 1H), 6.63−6.76 (m, 2H), 6.56−6.62 (m, 1H), 6.42−6.50 (m,
1H), 4.38−4.45 (m, 1H), 4.24 (dd, J = 9.5, 4.7 Hz, 1H), 3.94−4.03
(m, 1H), 1.40 (s, 9H). MS m/z 297 [M + H]+.
1
yl)acetate (6.72 g, 50%) as a thick yellow oil. H NMR (400 MHz,
Step 2. To a rt mixture of (S)-3-(2-aminophenoxy)-2-(tert-
butoxycarbonylamino)propionic acid (5.8 g, 19.6 mmol) in DMF
was added EDCI (3.75 g, 19.6 mmol). The reaction was stirred at rt
for 1.5 h then diluted with EtOAc, washed with H2O and satd aq
NaCl, dried over Na2SO4, filtered, and concentrated in vacuo. The
crude material was triturated with hexanes to provide (S)-tert-butyl 4-
oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-ylcarbamate (3.4 g,
DMSO-d6) δ 7.32−7.38 (m, 8H), 7.22−7.30 (m, 2H), 4.57 (s, 1H),
4.10−4.29 (m, 4H), 3.36−3.58 (m, 6H), 3.09 (s, 1H), 1.77−1.86 (m,
1H), 1.49−1.59 (m, 1H), 1.38−1.48 (m, 1H), 1.29 (t, J = 7.0 Hz, 3H),
1.19−1.25 (m, 1H). MS m/z 384 [M + H]+.
Step 2. To a rt solution of ethyl 2-(dibenzylamino)-2-(4-
hydroxytetrahydro-2H-pyran-4-yl)acetate (6.72 g, 17.5 mmol) in
MeOH (150 mL) was added potassium hydroxide (5.9 g, 105
mmol) in H2O (50 mL). The mixture was stirred at 60 °C for 15 h,
then cooled to rt and diluted with H2O (200 mL). The pH was
adjusted to ∼4 by the addition of satd aq KHSO4, and the mixture was
extracted with EtOAc (2 × 200 mL). The combined organic layers
were washed with H2O (2 × 200 mL) and satd aq NaCl (100 mL),
dried over Na2SO4, filtered, and concentrated in vacuo to provide 29
(5.72 g, 92%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ
12.65 (br s, 1H), 7.32−7.38 (m, 8H), 7.23−7.29 (m, 2H), 4.12−4.21
(m, 2H), 3.46−3.58 (m, 4H), 3.29−3.41 (m, 2H), 3.01 (s, 1H), 1.76−
1.85 (m, 1H), 1.47−1.57 (m, 1H), 1.34−1.44 (m, 1H), 1.26−1.33 (m,
1H). MS m/z 356 [M + H]+.
2-(Dibenzylamino)-2-(4-(2-nitrophenoxy)tetrahydro-2H-pyran-4-
yl)acetic Acid (30). To a rt solution of 29 (5.72 g, 16.1 mmol) in THF
(90 mL) was added KHMDS (1.0 M solution in THF, 35.4 mL, 35.4
mmol), followed by 1-fluoro-2-nitrobenzene (1.87 mL, 17.7 mmol).
The reaction was stirred at rt for 2 h, then additional KHMDS (10 mL,
10 mmol) and 1-fluoro-2-nitrobenzene (1 mL, 9.5 mmol) were added
and the reaction was stirred for an additional 2 h. The reaction was
diluted with H2O (200 mL), acidified with satd aq KHSO4, and
extracted with EtOAc (2 × 250 mL). The combined organic layers
were washed with H2O (2 × 300 mL) and satd aq NaCl (200 mL),
dried over Na2SO4, filtered, and concentrated in vacuo. The crude
1
62%) as a white solid. H NMR (300 MHz, DMSO-d6) δ 9.92 (s,
1H), 7.04−7.18 (m, 5H), 4.20−4.39 (m, 3H), 1.36 (s, 9H). MS m/z
301 [M + Na]+.
Step 3. To a 0 °C solution of (S)-tert butyl 4-oxo-2,3,4,5-
tetrahydrobenzo[b][1,4]oxazepin-3-ylcarbamate (3.4 g, 7.2 mmol) in
CH2Cl2 (20 mL) was added TFA (20 mL). The reaction was stirred
for 1 h, then concentrated in vacuo. The residue was diluted with satd
aq NaHCO3 and extracted with CH2Cl2 (4×). The combined organic
layers were dried over Na2SO4, filtered, and concentrated in vacuo to
provide 26 (2.2 g, quant). 1H NMR (300 MHz, DMSO-d6) δ 9.88 (s,
1H), 7.02−7.11 (m, 4H), 4.28 (dd, J = 10.4, 6.2 Hz, 1H), 4.01 (t, J =
10.6 Hz, 1H), 3.60 (dd, J = 10.6, 6.3 Hz, 1H), 1.81 (br s, 2H). MS m/z
179 [M + H]+.
(S)-N-((S)-5-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-4-
oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-2-
(methylamino)propanamide 2,2,2-Trifluoroacetate (27). Step 1.
According to general procedure A, 26 (2.2 g, 12 mmol) and BOC-N-
methyl-L-alanine (2.48 g, 12.2 mmol) were converted to tert-butyl
methyl((S)-1-oxo-1-((S)-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]-
oxazepin-3-ylamino)propan-2-yl)carbamate (4.2 g, 95%) as a light-
1
yellow foam. H NMR (300 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.04
(d, J = 8.2 Hz, 1H), 7.05−7.16 (m, 4H), 4.24−4.71 (m, 4H), 2.68−
K
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
material was purified by flash chromatography to provide 30 (7.06 g,
92%) as a yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 13.00 (br s,
1H), 7.77 (dd, J = 8.2, 1.6 Hz, 1H), 7.33−7.41 (m, 9H), 7.26−7.32
(m, 2H), 7.16 (t, J = 7.6 Hz, 1H), 6.73 (d, J = 8.6 Hz, 1H), 4.08−4.17
(m, 2H), 3.89 (s, 1H), 3.60−3.69 (m, 2H), 3.30−3.50 (m, 2H), 3.14−
3.22 (m, 1H), 2.98−3.08 (m, 1H), 2.04−2.15 (m, 2H), 1.84−1.94 (m,
1H), 1.43−1.52 (m, 1H). MS 477 [M + H]+.
3-Amino-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-
2,4′-pyran]-4(5H)-one (31). Step 1. A mixture of 30 (7.06 g, 14.8
mmol), ammonium chloride (1.59 g, 29.6 mmol), and zinc (15.0 g,
230 mmol) in MeOH (300 mL) was stirred at 65 °C for 15 h. The
reaction was cooled to rt, filtered through Celite, concentrated in
vacuo, then partitioned between satd aq NaOAc (400 mL) and EtOAc
(400 mL). The aqueous layer was separated and extracted with EtOAc
(2 × 200 mL). The combined organic layers were washed with satd aq
NaCl (3 × 200 mL), dried over Na2SO4, filtered, and concentrated in
vacuo to provide 2-(4-(2-aminophenoxy)tetrahydro-2H-pyran-4-yl)-2-
(dibenzylamino)acetic acid (6.75 g, quant) as a brown foam. 1H NMR
(400 MHz, DMSO-d6) δ 7.35−7.48 (m, 4H), 7.14−7.30 (m, 6H),
6.68−6.76 (m, 2H), 6.50−6.59 (m, 1H), 6.34 (t, J = 7.4 Hz, 1H), 5.52
(br s, 2H), 4.11−4.41 (m, 2H), 3.71−3.97 (m, 3H), 3.35−3.46 (m,
1H), 3.10−3.23 (m, 1H), 2.84−2.98 (m, Hz, 1H), 2.34−2.46 (m, 1H),
2.24 (d, J = 8.6 Hz, 1H), 1.81−1.95 (m, 2H), 1.41−1.54 (m, 1H). MS
m/z 447 [M + H]+.
(S)-N-((S)-5-((6-Bromo-2-methoxynaphthalen-1-yl)methyl)-4-
oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-
2,4′-pyran]-3-yl)-2-(methylamino)propanamide Hydrochloride
(33). According to general procedure B, 32 (332 mg, 0.766 mmol)
and 6-bromo-1-(chloromethyl)-2-methoxynaphthalene (328 mg, 1.15
mmol) were converted to tert-butyl (S)-1-((S)-5-((6-bromo-2-
methoxynaphthalen-1-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-
spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-
yl(methyl)carbamate (281 mg, 54%). According to general procedure
C, a portion of this (92.3 mg, 0.135 mmol) was converted to 33 (67.7
1
mg, 81%) as an off-white solid. H NMR (400 MHz, DMSO-d6) δ
9.10−9.19 (m, 1H), 9.08 (d, J = 9.29 Hz, 1H), 8.76−8.85 (m, 1H),
8.01−8.06 (m, 2H), 7.79 (d, J = 9.0 Hz, 1H), 7.56 (dd, J = 8.0, 1.3 Hz,
1H), 7.47 (dd, J = 9.0, 2.0 Hz, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.13−
7.18 (m, 1H), 7.05−7.10 (m, 1H), 6.84−6.88 (m, 1H), 5.90 (d, J =
14.8 Hz, 1H), 5.27 (d, J = 14.6 Hz, 1H), 4.54 (d, J = 9.3 Hz, 1H),
3.93−4.02 (m, 1H), 3.83 (s, 3H), 3.79−3.86 (m, 1H), 3.64−3.78 (m,
2H), 3.44 (t, J = 11.2 Hz, 1H), 2.45−2.49 (m, 3H), 2.11 (dt, J = 12.9.
5.3 Hz, 1H), 1.48−1.63 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.34−1.38
(m, 1H). HRMS m/z calcd for C29H33O5N3Br [M + H]+ 582.15981,
found 582.15938.
(S)-2-(Methylamino)-N-((S)-5-((2-methylnaphthalen-1-yl)-
methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]-
oxazepine-2,4′-pyran]-3-yl)propanamide Hydrochloride (34). Ac-
cording to general procedure B, except the reaction was carried out at
rt, and general procedure C, 32 (100 mg, 0.231 mmol) and 1-
(chloromethyl)-2-methylnaphthalene (66.0 mg, 0.346 mmol) were
Step 2. A solution of 2-(4-(2-aminophenoxy)tetrahydro-2H-pyran-
4-yl)-2-(dibenzylamino)acetic acid (6.75 g, 15.1 mmol), HOBt (3.24
g, 21.2 mmol), and EDCI (2.9 g, 15.1 mmol) in DMF (100 mL) was
stirred at rt for 1 h, then diluted with EtOAc (500 mL), washed with
1:1 satd aq NaHCO3−satd aq NaCl (3 × 200 mL) and satd aq NaCl
(200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The
crude material was purified by flash chromatography to provide 3-
(dibenzylamino)-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]-
1
converted to 34 (58.4 mg, 48% 2 steps) as a light-yellow solid. H
NMR (400 MHz, DMSO-d6) δ 9.13−9.23 (m, 1H), 9.10 (d, J = 8.9
Hz, 1H), 8.76−8.88 (m, 1H), 8.19−8.26 (m, 1H), 7.77 (dd, J = 6.3,
3.5 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.38−7.43 (m, 3H), 7.21 (d, J =
8.2 Hz, 1H), 7.00−7.08 (m, 2H), 6.89−6.94 (m, 1H), 5.97 (d, J = 15.2
Hz, 1H), 5.37 (d, J = 15.2 Hz, 1H), 4.55 (d, J = 9.0 Hz, 1H), 3.94−
4.04 (m, 1H), 3.67−3.89 (m, 3H), 3.45−3.59 (m, 1H), 2.49−2.51 (m,
3H), 2.46−2.49 (m, 3H), 2.06−2.19 (m, 1H), 1.66 (d, J = 13.3 Hz,
1H), 1.54 (td, J = 13.2, 5.7 Hz, 1H), 1.41 (d, J = 7.0 Hz, 3H), 1.35−
1.39 (m, 1H). MS m/z 488 [M + H]+.
(S)-N-((S)-5-((2-(Difluoromethoxy)naphthalen-1-yl)methyl)-4-
oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-
2,4′-pyran]-3-yl)-2-(methylamino)propanamide Hydrochloride
(35). To a rt solution of 32 in which the diastereomers had not
been separated (118 mg, 0.272 mmol) was added (2-
(difluoromethoxy)naphthalen-1-yl)methyl methanesulfonate (98.7
mg, 0.327 mmol) and Cs2CO3 (115 mg, 0.354 mmol). The reaction
was stirred at rt for 20 h, then diluted with EtOAc (25 mL), washed
with H2O (25 mL) and satd aq NaCl (25 mL), dried over Na2SO4,
filtered, and concentrated in vacuo. The crude material was purified by
flash chromatography then by SFC to separate the diastereomers and
provide tert-butyl (S)-1-((S)-5-((2-(difluoromethoxy)naphthalen-1-
yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]-
oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate
(52.6 mg, 30%) as a white foam. This material was reacted according
to general procedure C to provide 35 (46.3 mg, 98%) as a white solid.
1H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 9.06 Hz, 1H), 8.78 (br
s, 2H), 8.19 (d, J = 9.1 Hz, 1H), 7.82−7.93 (m, 2H), 7.43−7.56 (m,
3H), 7.27 (d, J = 8.7 Hz, 1H), 7.25 (t, J = 73.3 Hz, 1H), 7.04−7.18 (m,
2H), 6.87 (d, J = 7.2 Hz, 1H), 6.06 (d, J = 15.1 Hz, 1H), 5.27 (d, J =
15.1 Hz, 1H), 4.56 (d, J = 9.1 Hz, 1H), 3.90−4.02 (m, 1H), 3.63−3.85
(m, 3H), 3.43 (t, J = 11.9 Hz, 1H), 2.47−2.49 (m, 3H), 2.04−2.18 (m,
1H), 1.65 (d, J = 13.6 Hz, 1H), 1.46−1.59 (m, 1H), 1.39 (d, J = 6.4
Hz, 3H), 1.34−1.38 (m, 1H). HRMS m/z calcd for C29H32O5N3F2 [M
+ H]+ 540.23045, found 540.22913.
6-Methoxy-5-(((S)-3-((S)-2-(methylamino)propanamido)-4-oxo-
2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-
5(4H)-yl)methyl)-2-naphthoic Acid 2,2,2-Trifluoroacetate (36). Step
1. A mixture of tert-butyl (S)-1-((S)-5-((6-bromo-2-methoxynaph-
thalen-1-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo-
[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)-
carbamate (0.17 g, 249 μmol), Pd(OAc)2 (2.24 mg, 9.96 μmol), 4,5-
bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (11.5 mg,
19.9 μmol), MeOH (0.10 mL, 2.5 mmol), and Et3N (0.5 mL) was
1
oxazepine-2,4′-pyran]-4(5H)-one (4.77 g, 74%) as a beige solid. H
NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 7.30−7.36 (m, 8H),
7.22−7.29 (m, 2H), 7.18 (dd, J = 7.8, 1.2 Hz, 1H), 6.92−7.06 (m,
3H), 3.89 (d, J = 12.9 Hz, 2H), 3.65−3.74 (m, 1H), 3.57 (d, J = 13.3
Hz, 2H), 3.41−3.50 (m, 2H), 3.31−3.39 (m, 1H), 3.28 (s, 1H), 2.08−
2.14 (m, 1H), 1.45−1.55 (m, 1H), 1.35−1.42 (m, 1H), 1.12−1.22 (m,
1H). MS m/z 429 [M + H]+.
Step 3. A mixture of 3-(dibenzylamino)-2′,3′,5′,6′-tetrahydro-3H-
spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-4(5H)-one (4.77 g, 11.1
mmol; equiv, 1.00) and 20% palladium hydroxide on carbon (2 g)
in MeOH (150 mL) was stirred under H2 overnight. The reaction was
treated with additional catalyst and H2 and stirred overnight, then
filtered through Celite and concentrated in vacuo to provide 31 (2.75
1
g, quant) as an off-white solid. H NMR (400 MHz, DMSO-d6) δ
10.00 (s, 1H), 7.02−7.18 (m, 4H), 3.84−3.92 (m, 1H), 3.75−3.83 (m,
2H), 3.61−3.70 (m, 1H), 3.25 (s, 1H), 2.63−3.00 (m, 2H), 1.93−2.07
(m, 2H), 1.36−1.50 (m, 2H). MS m/z 249 [M + H]+.
tert-Butyl Methyl((S)-1-oxo-1-((S)-4-oxo-2′,3′,4,5,5′,6′-hexahy-
dro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-
propan-2-yl)carbamate (32). A solution of HBTU (43.2 g, 114
mmol) and HOBt·H2O (17.4 g, 114 mmol) in DMF (300 mL) was
added to a mixture of 3-amino-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo-
[b][1,4]oxazepine-2,4′-pyran]-4(5H)-one hydrochloride (27 g, 94.8
mmol), BOC-N-methyl-L-alanine (23.1 g, 114 mmol), and Et3N (52.9
mL, 379 mmol) in DMF (300 mL). After 18 h, the mixture was
poured into satd aq NaCl (750 mL) and extracted with EtOAc. The
combined extracts were washed with 1:1 sat NaHCO3−satd aq NaCl
and satd aq NaCl, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography and the
diastereomers were separated by SFC (Daicel AD column (5 cm × 25
cm) eluted with 15% MeOH−CO2 at 200 mL/min; 220 nM
detection; 100 bar backpressure; 35 °C oven). The first peak to
elute from the column provided 32 (15.29 g, 37%) as an off-white
foam. 1H NMR (300 MHz, DMSO-d6) δ 10.14 (s, 1H), 7.06−7.24 (m,
4H), 4.60 (q, J = 7.2 Hz, 1H), 4.51 (d, J = 9.0 Hz, 1H), 3.62−3.95 (m,
4H), 2.74 (s, 3H), 2.01−2.06 (m, 1H), 1.50−1.61 (m, 3H), 1.38 (s,
9H), 1.20−1.30 (m, 3H). MS m/z 434 [M + H]+.
L
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
sparged with CO, sealed and stirred at 70 °C for 18 h. The reaction
was cooled to rt, diluted with EtOAc, washed with 1 N aq HCl, H2O,
and satd aq NaCl, dried over Na2SO4, filtered, and concentrated in
vacuo. The crude material was purified by flash chromatography to
provide methyl 5-(((S)-3-((S)-2-(tert-butoxycarbonyl(methyl)amino)-
propanamido)-4-oxo-2′,3′,5′,6′-tetrahydro-3H-spiro[benzo[b][1,4]-
oxazepine-2,4′-pyran]-5(4H)-yl)methyl)-6-methoxy-2-naphthoate
(S)-N-((S)-5-(Benzo[d]isoxazol-3-ylmethyl)-4-oxo-2′,3′,4,5,5′,6′-
hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-yl)-2-
(methylamino)propanamide Hydrochloride (38). To a rt solution of
32 in which the diastereomers had not been separated (156 mg, 0.360
mmol) was added 3-(bromomethyl)benzo[d]isoxazole (83.9 mg,
0.396 mmol), Cs2CO3 (141 mg, 0.432 mmol), and NaI (65 mg,
0.43 mmol). The reaction was stirred at rt for 18 h, then diluted with
EtOAc (25 mL), washed with H2O (25 mL) and satd aq NaCl (25
mL), dried over Na2SO4, filtered, and concentrated in vacuo. The
crude material was purified by flash chromatography then by SFC to
separate the diastereomers and provide tert-butyl (S)-1-((S)-5-
(benzo[d]isoxazol-3-ylmethyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-
spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-
yl(methyl)carbamate (54 mg, 27%) as a white foam. This material was
reacted according to general procedure C to provide 38 (42 mg, 92%)
as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 9.1 Hz,
1H), 8.92 (br s, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.59−7.74 (m, 3H),
7.23−7.39 (m, 3H), 7.11 (d, J = 7.2 Hz, 1H), 5.71 (d, J = 15.5 Hz,
1H), 5.35 (d, J = 15.5 Hz, 1H), 4.65 (d, J = 9.1 Hz, 1H), 3.95 (q, J =
6.4 Hz, 1H), 3.70−3.89 (m, 3H), 3.54 (t, J = 11.3 Hz, 1H), 2.47 (s,
3H), 2.11 (td, J = 12.9, 5.1 Hz, 1H), 1.43−1.66 (m, 3H), 1.35 (d, J =
6.8 Hz, 3H). HRMS m/z calcd for C25H29O5N4 [M + H]+ 465.21325,
found 465.21185.
1
(111 mg, 67%) as a light-yellow foam. H NMR (400 MHz, DMSO-
d6) δ 8.48 (s, 1H), 8.28−8.40 (m, 1H), 8.18 (d, J = 8.6 Hz, 1H), 8.02
(d, J = 9.0 Hz, 1H), 7.81 (d, J = 9.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H),
7.39 (d, J = 9.0 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.02−7.08 (m, 1H),
6.82 (d, J = 7.4 Hz, 1H), 5.93 (d, J = 14.8 Hz, 1H), 5.28 (d, J = 14.8
Hz, 1H), 4.63 (q, J = 7.0 Hz, 1H), 4.54 (d, J = 9.0 Hz, 1H), 3.88 (s,
3H), 3.86 (s, 3H), 3.59−3.80 (m, 3H), 3.34−3.45 (m, 1H), 2.75 (s,
3H), 2.01−2.16 (m, 1H), 1.59 (d, J = 13.3 Hz, 1H), 1.43−1.52 (m,
1H), 1.38 (s, 9H), 1.23−1.32 (m, 4H). MS m/z 662 [M + H]+.
Step 2. To a solution of methyl 5-(((S)-3-((S)-2-(tert-
butoxycarbonyl(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′-tetra-
hydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)-
methyl)-6-methoxy-2-naphthoate (111 mg, 0.167 mmol) in THF (3
mL) was added a solution of LiOH·H2O (35.0 mg, 835 μmol) in H2O
(4 mL). The reaction was stirred at rt for 18 h, then diluted with 1 M
aq HCl (20 mL) and extracted with EtOAc (2 × 15 mL). The
combined organic extracts were washed with H2O (2 × 20 mL) and
satd aq NaCl (20 mL), dried over Na2SO4, filtered, concentrated in
vacuo, and lyophilized from MeCN−H2O to provide 5-(((S)-3-((S)-2-
(tert-butoxycarbonyl(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′-
tetrahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)-
methyl)-6-methoxy-2-naphthoic acid (95.2 mg, 88%) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ 12.90 (br s, 1H), 8.44 (s, 1H),
8.30−8.39 (m, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H),
7.80 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 9.4 Hz,
1H), 7.13 (t, J = 7.6 Hz, 1H), 7.02−7.08 (m, 1H), 6.82 (d, J = 7.8 Hz,
1H), 5.93 (d, J = 14.8 Hz, 1H), 5.29 (d, J = 14.5 Hz, 1H), 4.63 (q, J =
7.4 Hz, 1H), 4.54 (d, J = 9.0 Hz, 1H), 3.86 (s, 3H), 3.59−3.81 (m,
3H), 3.41 (t, J = 11.5 Hz, 1H), 2.75 (s, 3H), 2.02−2.15 (m, 1H), 1.60
(d, J = 13.3 Hz, 1H), 1.43−1.52 (m, 1H), 1.38 (s, 9H), 1.21−1.32 (m,
4H). MS m/z 648 [M + H]+.
Step 3. To a rt solution of 5-(((S)-3-((S)-2-(tert-butoxycarbonyl-
(methyl)amino)propanamido)-4-oxo-2′,3′,5′,6′-tetrahydro-3H-spiro-
[benzo[b][1,4]oxazepine-2,4′-pyran]-5(4H)-yl)methyl)-6-methoxy-2-
naphthoic acid (36.5 mg, 0.056 mmol) in CH2Cl2 (1 mL) was added
TFA (0.2 mL). The reaction was stirred at rt for 1 h, then it was
concentrated in vacuo and lyophilized from MeCN−H2O to provide
36 (31.0 mg, 83%) as an off-white solid. 1H NMR (300 MHz, DMSO-
d6) δ 12.93 (br s, 1H), 9.11 (d, J = 9.2 Hz, 1H), 8.66−8.90 (m, 2H),
8.45 (d, J = 1.3 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 9.0 Hz,
1H), 7.80 (dd, J = 9.0, 1.5 Hz, 1H), 7.58 (dd, J = 7.9, 1.3 Hz, 1H), 7.37
(d, J = 9.2 Hz, 1H), 7.13−7.19 (m, 1H), 7.04−7.11 (m, 1H), 6.82−
6.88 (m, 1H), 5.94 (d, J = 14.7 Hz, 1H), 5.30 (d, J = 14.7 Hz, 1H),
4.55 (d, J = 9.2 Hz, 1H), 3.98 (d, J = 6.4 Hz, 1H), 3.86 (s, 3H), 3.63−
3.84 (m, 3H), 3.32−3.47 (m, 1H), 2.49−2.51 (m, 3H), 2.10 (td, J =
13.0, 5.3 Hz, 1H), 1.46−1.68 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.27−
1.37 (m, 1H). MS m/z 548 [M + H]+.
(S)-N-((S)-5-((1-(2-Cyanophenyl)-1H-indazol-3-yl)methyl)-4-oxo-
2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-
pyran]-3-yl)-2-(methylamino)propanamide (39). According to gen-
eral procedure B, 32 (55 mg, 0.127 mmol) and 2-(3-(bromomethyl)-
1H-indazol-1-yl)benzonitrile (78 mg, 0.25 mmol) were converted to
(S)-2-amino-N-((S)-5-((1-(2-cyanophenyl)-1H-indazol-3-yl)methyl)-
4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)propanamide (60
mg, 71%). To a 0 °C solution of this material in CH2Cl2 (1 mL) was
added TFA (1.5 mL). The reaction was stirred at 0 °C for 1 h, then
diluted with satd aq NaHCO3 and extracted with CH2Cl2. The
combined organic layers were concentrated in vacuo and the residue
was purified by preparative TLC to provide 39 (48 mg, 94%) as a
1
yellow solid. H NMR (300 MHz, DMSO-d6) δ 8.31 (d, J = 9.0 Hz,
1H), 8.09 (d, J = 7.7 Hz, 1H), 7.86−7.94 (m, 2H), 7.63−7.79 (m,
3H), 7.45−7.57 (m, 2H), 7.19−7.31 (m, 3H), 7.06−7.11 (m, 1H),
5.50 (s, 2H), 4.63 (d, J = 9.0 Hz, 1H), 3.70−3.90 (m, 3H), 3.60 (t, J =
11.4 Hz, 1H), 2.96−3.07 (m, 1H), 2.18 (s, 3H), 1.91−2.06 (m, 1H),
1.66 (d, J = 13.4 Hz, 1H), 1.38−1.57 (m, 2H), 1.10 (d, J = 7.0 Hz,
3H); HRMS m/z calcd for C32H33O4N6 [M + H]+ 565.25578, found
565.25580.
(S)-N-((S)-5-((2-Cyano-1-(2-cyanophenyl)-1H-indol-3-yl)methyl)-
4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-
2,4′-pyran]-3-yl)-2-(methylamino)propanamide (40). Step 1. To a rt
solution 32 (8.5 g, 19.6 mmol) and 3-(bromomethyl)-1-(2-
cyanophenyl)-1H-indole-2-carbonitrile (9.89 g, 29.4 mmol) in DMF
(20 mL) was added Cs2CO3 (9.58 g, 29.4 mmol). The reaction was
stirred at 70 °C for 1 h, then cooled to rt, diluted with satd aq NH4Cl,
and extracted with EtOAc. The combined organic layers were washed
with H2O, dried over Na2SO4, filtered, and concentrated in vacuo. The
crude material was purified by flash chromatography to provide tert-
butyl (S)-1-((S)-5-((2-cyano-1-(2-cyanophenyl)-1H-indol-3-yl)-
methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]-
oxazepine-2,4′-pyran]-3-ylamino)-1-oxopropan-2-yl(methyl)carbamate
(13 g, 96%) as a light-yellow solid. MS m/z 689 [M + H]+.
(S)-N-((S)-5-((3-Methoxyquinolin-4-yl)methyl)-4-oxo-
2′,3′,4,5,5′,6′-hexahydro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-
pyran]-3-yl)-2-(methylamino)propanamide Hydrochloride (37).
According to general procedures B, except that the reaction was
conducted at rt, and general procedure C, 32 (100 mg, 0.231 mmol)
and 4-(chloromethyl)-3-methoxyquinoline (71.9 mg, 0.346 mmol)
Step 2. To a 0 °C solution of tert-butyl (S)-1-((S)-5-((2-cyano-1-
(2-cyanophenyl)-1H-indol-3-yl)methyl)-4-oxo-2′,3′,4,5,5′,6′-hexahy-
dro-3H-spiro[benzo[b][1,4]oxazepine-2,4′-pyran]-3-ylamino)-1-oxo-
propan-2-yl(methyl)carbamate (4 g, 5.81 mmol) in CH2Cl2 (50 mL)
was added TFA (25 mL) dropwise over 2 h. The reaction was stirred
for 30 min at 0 °C, then diluted with 1:1 satd aq NaHCO3 and H2O.
The organic layer was separated, dried over Na2SO4, filtered, and
concentrated in vacuo to provide 40 (3.4 g, 97%) as a light-yellow
1
were converted to 37 (75.9 mg, 57% 2 steps) as a yellow solid. H
NMR (400 MHz, DMSO-d6) δ 9.25−9.34 (m, 1H), 9.08 (d, J = 9.4
Hz, 1H), 8.90 (s, 1H), 8.79−8.88 (m, 1H), 8.27 (d, J = 8.6 Hz, 1H),
8.03 (d, J = 8.6 Hz, 1H), 7.54−7.72 (m, 3H), 7.22 (t, J = 7.6 Hz, 1H),
7.11−7.17 (m, 1H), 6.93 (d, J = 7.8 Hz, 1H), 5.99 (d, J = 14.5 Hz,
1H), 5.31 (d, J = 14.8 Hz, 1H), 4.56 (d, J = 9.0 Hz, 1H), 3.95−4.01
(m, 1H), 3.95 (s, 3H), 3.65−3.82 (m, 3H), 3.43 (t, J = 11.3 Hz, 1H),
2.45 (t, J = 5.3 Hz, 3H), 2.08 (td, J = 13.0, 5.3 Hz, 1H), 1.45−1.59 (m,
2H), 1.39 (d, J = 6.6 Hz, 3H), 1.20−1.36 (m, 1H). HRMS m/z calcd
for C28H33O5N4 [M + H]+ 505.24455, found 505.24310.
1
solid. H NMR (400 MHz, CDCl3) δ 8.13−8.24 (m, 1H), 7.76−7.91
(m, 3H), 7.62−7.68 (m, 1H), 7.46−7.52 (m, 1H), 7.33−7.45 (m, 2H),
7.16−7.31 (m, 3H), 7.01−7.06 (m, 1H), 6.91−6.97 (m, 1H), 5.85−
5.94 (m, 1H), 5.11−5.18 (m, 1H), 4.76−4.83 (m, 1H), 3.60−3.94 (m,
4H), 3.06−3.15 (m, 1H), 2.47−2.51 (m, 3H), 1.94−2.07 (m, 1H),
M
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.55−1.78 (m, 3H), 1.31−1.35 (m, 3H). HRMS m/z calcd for
C34H33O4N6 [M + H]+ 589.25578, found 589.25482.
TNFα ELISA Assay. MDA-MB-231 cells (400,000 cells/well) were
seeded into 24 well plates and incubated for 24 h. Media was aspirated
from wells and replaced with either compound or media alone (400
μL/well) and further incubated at 37 °C with 5% CO2 for 19 h. Total
media volume was then collected and centrifuged (1000g) at 4 °C to
pellet debris. TNF of media was quantitated by ELISA (TNF Human
ELISA kit, R&D Systems cat. no. DTA00B), and the results were
plotted following subtraction of background values (media, no cells).
in Vivo Caspase 3/7 Assay and Western Blot Analysis. A single
dose of compound was administered as follows to female nude mice
with LOX xenografts: conatumumab 1 mg/kg ip, 3 100 mg/kg po, 40
200 mg/kg po, or a combination. Xenografts were removed 8 h
postdose, frozen (−80 °C), transferred to 15 mL conical tubes on dry
ice, and treated with 1× lysis buffer (20 mM Tris-HCl (pH 7.5), 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TX-100, 5 mM sodium
pyrophosphate, 1 mM β-glycerophosphate, 10 mM sodium fluoride, 1
mM Na3VO4, 1 mM benzamidine, EMD proteinase inhibitor cocktail
V). Tumors were homogenized at full speed, diluted with the same
volume of lysis buffer, and kept on ice for 20 min. A portion of the
homogenate (2 mL) was transferred to a microcentrifuge tube and
thawed at full speed for 20 min at 4 °C. The supernatant was
optionally centrifuged again. Protein was quantitated and samples were
prepared for caspase assay and Western blot analysis (50 μg of total
protein was used for caspase assay/60 μg of total protein was used for
Western blot).
Caspase 3/7 Assay. Tumor lysate (10 μL, 5 μg/μL) was added into
white solid 96 well plate. Caspase-3/7 assay buffer (final
concentration: 10 mM Tris, 5 mM DTT, 1 mM EDTA, 50 μM
DEVD-AMC, 20 mM NaCl, 1% NP-40) (90 μL) was added into each
well. The plate was incubated for 2 h at rt with shaking and read at
385/460 nM.
Western Blot Analysis. Proteins were electrophoresed on 4−12%
NuPAGE Bis-Tris midi-gel (Invitrogen) and transferred to PVDF
membrane (Invitrogen). Membranes were blocked in 5% milk,
incubated with primary antibodies, washed, and incubated with
horseradish peroxidase linked secondary antibody (Cell Signaling
Technology). The blots were stripped and reprobed. The following
primary antibodies were used in the study: anti-XIAP (R&D Systems),
anticaspase-3 (Santa Cruz), anti-cIAP1, anti-PARP.
Biology. XIAP BIR2 and BIR3 TR-FRET Assay. Ten nanomolar of
6× histidine-tagged BIR2 (amino acids 124−240) or BIR3 (amino
acids 241−356) domain of the XIAP protein was mixed with 20 nM of
the peptide AVPIAQKSEK (ε-biotin)−OH 1:2 TFA in the presence
of 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol
(DTT), and 0.1 mg/mL bovine serum albumin (BSA). Following a 45
min incubation at 37 °C, europium−streptavidin and allophycocyanin
conjugated anti-histidine antibody were added to a final concentration
of 1.5 and 15 nM, respectively. Time-resolved fluorescence resonance
energy transfer (TR-FRET) signals were measured after 1 h at rt.
Compound potency was assessed at 10 serially diluted concentrations.
Percentage of inhibition at each concentration was determined to
generate an IC50 value for each compound. Typically, Z′ = ∼0.9.36
measure of the overall variability of the assay is provided by the
A
positive control: BIR2 IC50 = 1.25 0.91 μM (n = 160), BIR3 IC50
=
0.039 0.025 μM (n = 168).
cIAP1 BIR2 and BIR3 TR-FRET Assay. Thirty nanomolar of 6×
histidine−thrombin−TEV tagged BIR2 (amino acids 174−256) or
BIR3 (amino acids 260−352) domain of the cIAP protein was mixed
with 50 nM of the peptide AVPIAQKSEK (ε-biotin)−OH 1:2 TFA in
the presence of 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM
dithiothreitol (DTT), and 0.1 mg/mL bovine serum albumin (BSA).
Following a 45 min incubation at 37 °C, europium−streptavidin and
allophycocyanin conjugated anti-histidine antibody were added to a
final concentration of 1.5 and 15 nM, respectively. Time-resolved
fluorescence resonance energy transfer (TR-FRET) signals were
measured after 1 h at rt. Compound potency was assessed at 10 serially
diluted concentrations. Percentage of inhibition at each concentration
was determined to generate an IC50 value for each compound.
Rat Blood Stability Assay. A 10 mM DMSO solution of test
compound was diluted to 200 μM with MeCN and then further
diluted to 1 μM (total volume 1.0 mL) with heparin-treated rat blood
and then incubated at 37 °C with 5% CO2 for 4 h. Aliquots (50 μL)
were removed at 0, 15, 60, and 240 min, then treated with 0.6 mL of
0.1% formic acid in MeCN with 2 mg/mL 7-hydroxycoumarin internal
standard, vortexed, and centrifuged (4000 rpm) for 10 min. The
supernatants (150 μL) were transferred to injection plates containing
150 μL of 0.1% formic acid in water, vortexed, and centrifuged (4000
rpm) for 5 min, then analyzed by LC-MS/MS.
XIAP-Caspase Reactivation Assay. The caspase reactivation assay
was performed in 20 mM HEPES buffer (pH 7.0), containing 1.5 mM
MgCl2, 5 mM KCl, 1 mM ethylene diamine tetraacetic acid (EDTA), 1
mM ethylene glycol tetraacetic acid (EGTA), 1 mM DTT, and 0.1
mg/mL BSA. S-100 cell extract served as the source of cellular
caspases. Then 2 μM cytochrome C and 19.2 μM dATP were added to
activate the caspases. The cleavage of a fluorogenic substrate specific
for caspase-3 and -7, Ac-DEVD-AFC at 10 μM, was monitored at 530
nm after 4 h of incubation at rt. The addition of 0.75 nM glutathione
S-transferase (GST)-tagged full length XIAP inhibited the caspase
activation by ∼85%. The effect of serially diluted compounds to
restore caspase activation was determined to generate an EC50 value
for each compound.
ASSOCIATED CONTENT
■
S
* Supporting Information
EC50 values for activity in multiple cell lines, synthetic
procedures for intermediates, crystallographic method, crystal-
lographic data refinement and statistics. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The PDB ID code for 36 bound to XIAP BIR2 is 4KJV.
AUTHOR INFORMATION
■
Corresponding Author
Cell-Based Assay. SW-620 cells (1500 cells/well) were seeded into
a 96 well plate one day prior to treatment. Cells were treated with
indicated compounds and/or antibody and further incubated at 37 °C
with 5% CO2 for 5 days. Cell Titer 96 Aqueous One solution (25 μL/
well, Promega cat. no. G3580) was added, and the plates were further
incubated for 2−3 h. Plate’s OD at 490 nm was determined using
Envision 2101 plate reader (PerkinElmer), and EC50 values were
determined for compounds or antibody. A similar protocol was used to
evaluate the other cell lines.
*E-mail: andrew.f.donnell@gmail.com. Phone: (609) 785-1304.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
in Vitro Caspase 3/7 Assay. SW620 cells were treated with
indicated compounds and/or antibody and incubated for 4 h. Cell
lysate (25 μg) and caspase-3/7 assay buffer (1% NP40, 50 μM DEVD-
AMC (Biomol)) were added to a solid white 96 well plate. Lysates
were incubated at rt for 20 min, and cleavage of DEVD-AMC substrate
was measured with a plate reader (Molecular Devices Spectramax M5)
at 385/460 nm.
ACKNOWLEDGMENTS
■
We thank Dr. Pamela Carroll and Dr. David Heimbrook for
their support of this program. We also thank Charles Belunis
for protein purification and Santina Russo and Joachim Diez of
Expose for data collection at SLS.
N
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(19) Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Qian, D.; Lu, J.;
Qiu, S.; Bai, L.; Peng, Y.; Cai, Q.; Wang, S. Design of Small-Molecule
Peptidic and Nonpeptidic Smac Mimetics. Acc. Chem. Res. 2008, 41,
1264−1276.
ABBREVIATIONS USED
■
IAP, inhibitor of apoptosis protein; XIAP, X-linked inhibitor of
apoptosis; cIAP, cellular inhibitor of apoptosis protein; BIR,
baculovirus IAP repeat; Smac, second mitochondria-derived
activator of caspases; DIABLO, direct IAP-binding protein with
a low pI; DR5, death receptor 5; EDCI, N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride; HOBt, hydroxy-
benzotriazole, HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate; PARP, poly ADP ribose
polymerase
(20) Cai, Q.; Sun, H.; Peng, Y.; Lu, J.; Nikolovska-Coleska, Z.;
McEachern, D.; Liu, L.; Qiu, S.; Yang, C.-Y.; Miller, R.; Yi, H.; Zhang,
T.; Sun, D.; Kang, S.; Guo, M.; Leopold, L.; Yang, D.; Wang, S. A
Potent and Orally Active Antagonist (SM-406/AT-406) of Multiple
Inhibitor of Apoptosis Proteins (IAPs) in Clinical Development for
Cancer Treatment. J. Med. Chem. 2011, 54, 2714−2726.
(21) Flygare, J. A.; Beresini, M.; Budha, N.; Chan, H.; Chan, I. T.;
Cheeti, S.; Cohen, F.; Deshayes, K.; Doerner, K.; Eckhardt, S. G.;
Elliott, L. O.; Feng, B.; Franklin, M. C.; Reisner, S. F.; Gazzard, L.;
Halladay, J.; Hymowitz, S. G.; La, H.; LoRusso, P.; Maurer, B.; Murray,
L.; Plise, E.; Quan, C.; Stephan, J.-P.; Young, S. G.; Tom, J.; Tsui, V.;
Um, J.; Varfolomeev, E.; Vucic, D.; Wagner, A. J.; Wallweber, H. J. A.;
Wang, L.; Ware, J.; Wen, Z.; Wong, H.; Wong, J. M.; Wong, M.;
Wong, S.; Yu, R.; Zobel, K.; Fairbrother, W. J. Discovery of a Potent
Small-Molecule Antagonist of Inhibitor of Apoptosis (IAP) Proteins
and Clinical Candidate for the Treatment of Cancer (GDC-0152). J.
Med. Chem. 2012, 55, 4101−4113.
REFERENCES
■
(1) Hanahan, D.; Weinberg, R. A. Hallmarks of Cancer: The Next
Generation. Cell 2011, 144, 646−674.
(2) Fulda, S.; Vucic, D. Targeting IAP proteins for therapeutic
intervention in cancer. Nature Rev. Drug Discovery 2012, 11, 109−123.
(3) Gyrd-Hansen, M.; Meier, P. IAPs: from caspase inhibitors to
modulators of NF-κB, inflammation and cancer. Nature Rev. Cancer
2010, 10, 561−573.
(4) Dean, E. J.; Ranson, M.; Blackhall, F.; Dive, C. X-linked inhibitor
of apoptosis protein as a therapeutic target. Expert. Opin. Ther. Targets
2007, 11, 1459−1471.
(5) Hunter, A. M.; LaCasse, E. C.; Korneluk, R. G. The inhibitors of
apoptosis (IAPs) as cancer targets. Apoptosis 2007, 12, 1543−1568.
(6) Eckelman, B. P.; Salvesen, G. S.; Scott, F. L. Human inhibitor of
apoptosis proteins: why XIAP is the black sheep of the family. EMBO
Rep. 2006, 7, 988−994.
(7) Srinivasa, S. M.; Hegde, R.; Saleh, A.; Datta, P.; Shiozaki, E.; Chai,
J.; Lee, R.-A.; Robbins, P. D.; Fernandes-Alnemri, T.; Shi, Y.; Alnemri,
E. S. A conserved XIAP-interaction motif in caspase-9 and Smac/
DIABLO regulates caspase activity and apoptosis. Nature 2001, 410,
112−116.
(8) Shiozaki, E. N.; Chai, J.; Rigotti, D. J.; Riedl, S. J.; Li, P.;
Srinivasula, S. M.; Alnemri, E. S.; Fairman, R.; Shi, Y. Mechanism of
XIAP-Mediated Inhibition of Caspase-9. Mol. Cell 2003, 11, 519−527.
(9) Chai, J.; Shiozaki, E.; Srinivasula, S. M.; Wu, Q.; Dataa, P.;
Alnemri, E. S.; Shi, Y. Structural Basis of Caspase-7 Inhibition by
XIAP. Cell 2001, 104, 769−780.
(22) Houghton, P. J.; Kang, M. H.; Reynolds, C. P.; Morton, C. L.;
Kolb, E. A.; Gorlick, R.; Keir, S. T.; Carol, H.; Lock, R.; Maris, J. M.;
Billups, C. A.; Smith, M. A. Initial Testing (Stage 1) of LCL161, a
SMAC Mimetic, by the Pediatric Preclinical Testing Program. Pediatr.
Blood Cancer 2012, 58, 636−639.
(23) Chen, K.-F.; Lin, J.-P.; Shiau, C.-W.; Tai, W.-T.; Liu, C.-Y.; Yu,
H.-C.; Chen, P.-J.; Cheng, A.-L. Inhibition of Bcl-2 improves effect of
LCL 161, a SMAC, mimetic in hepatocellular carcinoma cells. Biochem.
Pharmacol. 2012, 84, 268−277.
(24) See the discussion in ref 2 for more details.
(25) Petersen, S. L.; Wang, L.; Yalcin-Chin, A.; Li, L.; Peyton, M.;
Minna, J.; Harran, P.; Wang, X. Autocrine TNFα Signaling Renders
Human Cancer Cells Susceptible to Smac-Mimetic-Induced Apopto-
sis. Cancer Cell 2007, 12, 445−456.
(26) Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Fedorova,
A. V.; Kayagaki, N.; Garg, P.; Zobel, K.; Dynek, J. N.; Elliott, L. O.;
Wallweber, H. J. A.; Flygare, J. A.; Fairbrother, W. J.; Deshayes, K.;
Dixit, V. M.; Vucic, D. IAP Antagonists Induce Autoubitquitination of
c-IAPs, NJ-κB Activation, and TNFα-Dependent Apoptosis. Cell 2007,
131, 669−681.
(10) Huang, Y.; Park, Y. C.; Rich, R. L.; Segal, D.; Myszka, D. G.;
Wu, H. Structural Basis of Caspase Inhibition by XIAP: Differential
Roles of the Linker versus the BIR Domain. Cell 2001, 104, 781−790.
(11) Riedl, S. J.; Renatus, M.; Schwarzenbacher, R.; Zhou, Q.; Sun,
C.; Fesik, S. W.; Liddington, R. C.; Salvesen, G. S. Structural Basis for
the Inhibition of Caspase-3 by XIAP. Cell 2001, 104, 791−800.
(12) Du, C.; Fang, M.; Li, Y.; Li, L.; Wang, X. Smac, a Mitochondrial
Protein that Promotes Cytochrome c-Dependent Caspase Activation
by Eliminating IAP Inhibition. Cell 2000, 102, 33−42.
(13) Verhagen, A. M.; Ekert, P. G.; Pakusch, M.; Silke, J.; Connolly,
L. M.; Reid, G. E.; Moritz, R. L.; Simpson, R. J.; Vaux, D. L.
Identification of DIABLO, a Mammalian Protein that Promotes
Apoptosis by Binding to and Antagonizing IAP Proteins. Cell 2000,
102, 43−53.
(27) Vince, J. E.; Wong, W. W.-L.; Khan, N.; Feltham, R.; Chau, D.;
Ahmed, A. U.; Benetatos, C. A.; Chunduru, S. K.; Condon, S. M.;
McKinlay, M.; Brink, R.; Leverkus, M.; Tergaonkar, V.; Schneider, P.;
Callus, B. A.; Koentgen, F.; Vaux, D. L.; Silke, J. IAP Antagonists
Target cIAP1 to Induce TNFα-Dependent Apoptosis. Cell 2007, 131,
682−693.
́ ́
(28) Gonzalez-Lopez, M.; Welsh, K.; Finlay, D.; Ardecky, R. J.; Ganji,
S. R.; Su, Y.; Yuan, H.; Teriete, P.; Mace, P. D.; Riedl, S. J.; Vuori, K.;
Reed, J. C.; Cosford, N. D. P. Design, synthesis and evaluation of
monovalent Smac mimetics that bind to the BIR2 domain of the anti-
apoptotic protein XIAP. Bioorg. Med. Chem. Lett. 2011, 21, 4332−
4336.
(29) For example, the clearance for 19 was 155 mL/min/kg in a
cassette dosing study versus 116 mL/min/kg in a single-compound PK
study.
(30) For additional discussion, see: Lukacs, C.; Belunis, C.;
Crowther, R.; Danho, W.; Gao, L.; Goggin, B.; Janson, C. A.; Li, S.;
Remiszewski, S.; Schutt, A.; Thakur, M. K.; Singh, S. K.; Swaminathan,
S.; Pandey, R.; Tyagi, R.; Gosu, R.; Kamath, A. V.; Kuglstatter, A. The
structure of XIAP BIR2: understanding the selectivity of the BIR
domains. Acta Crystallogr., Sect. D.: Biol. Crystallogr. 2013, 69, 1717−
1725.
(14) Wu, G.; Chai, J.; Suber, T. L.; Wu, J.-W.; Du, C.; Wang, X.; Shi,
Y. Structural basis of IAP recognition by Smac/DIABLO. Nature 2000,
408, 1008−1012.
(15) Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.;
Oost, T.; Herrmann, J.; Wu, J. C.; Fesik, S. W. Structural basis for
binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 2000,
408, 1004−1008.
(16) Condon, S. M. The Discovery and Development of Smac
MimeticsSmall-Molecule Antagonists of the Inhibitor of Apoptosis
Proteins. Annu. Rep. Med. Chem. 2011, 46, 211−226.
(31) The values for 3 were generated in-house.
(32) Kaplan-Lefko, P. J.; Graves, J. D.; Zoog, S. J.; Pan, Y.; Wall, J.;
Branstetter, D. G.; Moriguchi, J.; Coxon, A.; Huard, J. N.; Xu, R.;
Peach, M. L.; Juan, G.; Kaufman, S.; Chen, Q.; Bianchi, A.; Kordich, J.
J.; Ma, M.; Foltz, I. N.; Gliniak, B. C. Conatumumab, a fully human
agonist antibody to death receptor 5, induces apoptosis via caspase
(17) Straub, C. S. Targeting IAPs as an Approach to Anti-Cancer
Therapy. Curr. Top. Med. Chem. 2011, 11, 291−316.
(18) Flygare, J. A.; Fairbrother, W. J. Small-molecule pan-IAP
antagonists: a patent review. Expert Opin. Ther. Pat. 2010, 20, 251−
267.
O
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
activation in multiple tumor types. Cancer Biol. Ther. 2010, 9, 618−
631.
(33) Gaither, A.; Porter, D.; Yao, Y.; Borawski, J.; Yang, G.; Donovan,
J.; Sage, D.; Slisz, J.; Tran, M.; Straub, C.; Ramsey, T.; Iourgenko, V.;
Huang, A.; Chen, Y.; Schlegel, R.; Labow, M.; Fawell, S.; Sellers, W. R.;
Zawel, L. A Smac Mimetic Rescue Screen Reveals Roles for Inhibitor
of Apoptosis Proteins in Tumor Necrosis Factor-α Signaling. Cancer
Res. 2007, 67, 11493−11498.
(34) Kester, R. F.; Donnell, A. F.; Lou, Y.; Remiszewski, S. W.;
Lombardo, L. J.; Chen, S.; Le, N. T.; Lo, J.; Moliterni, J. A.; Han, X.;
Hogg, J. H.; Liang, W.; Michoud, C.; Rupert, K. C.; Mischke, S.; Le,
K.; Weisel, M.; Janson, C. A.; Lukacs, C. M.; Fretland, A. J.; Hong, K.;
Polonskaia, A.; Gao, L.; Li, S.; Solis, D. S.; Aguilar, D.; Tardell, C.;
Dvorozniak, M.; Tannu, S.; Lee, E. C.; Schutt, A. D.; Goggin, B.
Optimization of Benzodiazepinones as Selective Inhibitors of the X-
Linked Inhibitor of Apoptosis Protein (XIAP) Second Baculovirus IAP
Repeat (BIR2) Domain. J. Med. Chem. 2013, DOI: 10.1021/
jm400732v.
(35) Schutt, A.; Goggin, B. S.; Hong, K.; Polonskaia, A.; To, T.;
Tardell, C.; Dvorozniak, M.; Tannu, S.; Aguilar, D.; Solis, D.; Gao, L.;
Lukacs, C.; Janson, C.; Hilton, H.; Fretland, A.; Liang, Z.; Lee, K.;
Kester, R.; Donnell, A.; Mischke, S.; Han, X.; Chen, S.; Le, N.; Lou, Y.;
Remiszewski, S.; Packman, K.; Lee, E. C. Unpublished results.
(36) Zhang, J.-H.; Chung, T. D. Y.; Oldenburg, K. R. A Simple
Statistical Parameter for Use in Evaluation and Validation of High
Throughput Screening Assays. J. Biomol. Screening 1999, 4, 67−73.
P
dx.doi.org/10.1021/jm400731m | J. Med. Chem. XXXX, XXX, XXX−XXX