Dimeric Macrocyclic Antagonists of Inhibitor of Apoptosis Proteins for the Treatment of Cancer

Article abstract of DOI:10.1021/acsmedchemlett.5b00091

A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimatel

Full text of DOI:10.1021/acsmedchemlett.5b00091

Letter
pubs.acs.org/acsmedchemlett
Dimeric Macrocyclic Antagonists of Inhibitor of Apoptosis Proteins
for the Treatment of Cancer
Yong Zhang,*,† Benjamin A. Seigal,‡ Nicholas K. Terrett,‡ Randy L. Talbott,† Joseph Fargnoli,†
Joseph G. Naglich,† Charu Chaudhry,† Shana L. Posy,† Ragini Vuppugalla,† Georgia Cornelius,†
Ming Lei,† Chunlei Wang,† Yingru Zhang,† Robert J. Schmidt,† Donna D. Wei,† Michael M. Miller,†
Martin P. Allen,† Ling Li,† Percy H. Carter,† Gregory D. Vite,† and Robert M. Borzilleri†
†Bristol-Myers Squibb Research, P.O. Box 4000, Princeton, New Jersey 08543, United States
‡Ensemble Therapeutics Corp., 99 Erie Street, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: A series of dimeric macrocyclic compounds were
prepared and evaluated as antagonists for inhibitor of apoptosis
proteins. The most potent analogue 11, which binds to XIAP and c-
IAP proteins with high affinity and induces caspase-3 activation and
ultimately cell apoptosis, inhibits growth of human melanoma and
colorectal cell lines at low nanomolar concentrations. Furthermore,
compound 11 demonstrated significant antitumor activity in the
A875 human melanoma xenograft model at doses as low as 2 mg/kg
on a q3d schedule.
KEYWORDS: Inhibitor of apoptosis proteins (IAPs), dimeric macrocyclic antagonists, cancer
he ability of tumor cells to evade apoptosis, or
programmed cell death, is a hallmark of human cancers.1
much higher binding affinity for the IAP proteins, were shown
to be significantly more potent at inducing apoptosis in
multiple cancer cell lines. Several Smac mimetics that bind
selectively to the BIR domains of XIAP and cIAPs have entered
clinical trials and shown encouraging results.11
T
In addition to cancer cell survival, defects in apoptotic pathways
(i.e., extrinsic and intrinsic) may also contribute to tumor
progression and chemo-resistance. The inhibitor of apoptosis
proteins (IAPs), which includes the X-linked inhibitor of
apoptosis protein (XIAP) and two cellular inhibitor of
apoptosis proteins (cIAP-1 and cIAP-2), are a family of pro-
oncogenic proteins that can inhibit apoptosis by regulating
initiator and effector caspases.2 XIAP directly binds to and
inhibits caspase-3, -7, and -9 through its second and third
baculovirus IAP repeat (BIR2 and BIR3) domains, resulting in
inhibition of apoptosis.3 While not direct inhibitors of caspases,
cIAPs are involved in NF-κB signaling and play important roles
in regulating caspase-8 activation.4 Elevated expression of XIAP
and cIAPs has been reported in many cancers and is associated
with poor prognosis.5
The antiapoptotic activities of the IAP proteins can be
neutralized by the mitochondrial protein Smac/Diablo (second
mitochondrial activator of caspases/direct IAP binding protein
with a low isoelectric point), which binds to the BIR domains
of the IAP proteins through its N-terminal AVPI tetrapeptide
binding motif.6 Importantly, it has been demonstrated that the
AVPI peptide itself, or small molecule AVPI mimetics, can
potently antagonize the IAP proteins and elicit antitumor
activity in vivo.7−10 Furthermore, dimeric AVPI mimetics, which
can bind to both the BIR2 and BIR3 domains and thus achieve
We recently reported the discovery of dimeric macrocyclic
IAP antagonist 1 (Table 1) via the generation of “milla-
molecular” libraries.12,13 Macrocyclization has been exploited
extensively as a means of conformationally restricting peptides
and thereby improving their proteolytic stability, target affinity,
and cell penetration.14−16 We have shown that compound 1
binds to both cIAP and XIAP proteins with high affinities and is
active in cellular assays despite its peptidic nature and high
molecular weight. One drawback of compound 1 is that it is
about 10-fold less potent in cell-based proliferation assays than
other nonmacrocyclic IAP antagonists we have recently
disclosed.17,18 As a result, significantly higher doses are required
to achieve similar levels of efficacy in mouse tumor xenograft
models. In an effort to improve the in vitro and in vivo potencies
of this millamolecular series with the ultimate goal of
identifying a clinical candidate, we systematically designed
and synthesized a series of analogues of 1. Herein, we report
Received: February 26, 2015
Accepted: May 27, 2015
© XXXX American Chemical Society
A
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a
Table 1. Biological Activities of IAP Antagonists
Figure 1. Binding model of compound 1 in the Bir2-3 domains of
XIAP protein. Carbon atoms of 1 are in green. Oxygen and nitrogen
atoms are highlighted in red and blue, respectively. The protein surface
is represented by electrostatic potential.
tional reasons. Compounds with the acid moieties may be able
to more easily adopt the conformation required for binding
simutaneously to the BIR2 and BIR3 proteins. Consistent with
this hypothesis, replacing one or both of the carboxylic acid
groups with similarly acidic cyclopropyl acylsulfonamide or
tetrazole moieties was well tolerated. The bis-cyclopropyl
acylsulfonamide 4 is equipotent to 1 in both biochemical
(XIAP BIR2-3 IC50 = 1.8 nM) and cellular antiproliferation
assay (A875 IC50 = 79 nM), whereas monocyclopropyl
acylsulfonamide analogue 5 gave similar biochemical potency
but improved cellular potency (A875 IC50 = 39 nM).20
On the basis of these promising results, we decided to
determine if the acid isosteres had improved pharmacokinetic
(PK) properties. As shown in Table 2, following a 1 mg/kg IV
Table 2. Pharmacokinetic Parameters of Select Compounds
ab
,
in Mice Following a 1 mg/kg IV Dose
cmpd
T1/2 (h)
CL (mL/min/kg)
Vss (L/kg)
AUC0−7 (nM h)
1
4
5
0.85
0.90
1.0
7.1
3.6
1.5
0.3
0.3
0.1
1550
2350
5850
a
IC50 values are an average of three independent experiments unless
a
b
Vehicle: 12% hydroxypropyl-β-cyclodextrin saline solution. Data
reported as an average of two male mice.
b
c
otherwise noted. N = 1. Inhibition of cell growth in A875 cancer cell
line in the presence of TNF.
the synthesis, structure−activity relationship, and in vivo
evaluation of a series of macrocyclic dimeric IAP antagonists
with improved potency and pharmacokinetic properties.
bolus injection, bis-cyclopropyl acylsulfonamide 4 demonstra-
ted reduced clearance and enhanced exposure (AUC0−7 = 2350
nM h) relative to compound 1. Monocyclopropyl acylsulfona-
mide 5 provided lower clearance, lower steady state volume of
Compound 1 is a potent antagonist of XIAP BIR2-3 protein
(IC50 = 1.4 nM, Table 1) and inhibits the proliferation of A875
human melanoma cells with an IC50 of 73 nM. On the basis of
our predicted binding model and previous SAR, we
hypothesized that compound 1 occupies the same binding
pocket as the AVPI peptide on the surface of the BIR2-3
protein (Figure 1). In this model, the C-terminal carboxylic
acids are solvent exposed and do not contribute significantly to
binding potency. In contrast to this prediction, however, the
mono- and bis-methyl esters analogues 2 and 3 are significantly
less potent than 1 in both the XIAP BIR2-3 FRET binding
assay19 and the A875 antiproliferation assay (IC50 = 310 and
690 nM, respectively, Table 1). Several additional analogues of
1, where the carboxylic acids were replaced with nonacidic
primary or secondary amide groups, also gave poor biochemical
and cellular activities (data not shown). These results lead us to
postulate the acid moieties may be important for conforma-
distribution, and higher exposure than both 1 and 4 (AUC0−7
=
5850 nM h) at the same dose. Thus, in addition to maintaining
an optimal level of cellular potency, the acylsulfonamide acid
isosteres also benefited from improved PK properties relative to
the initial lead 1.
We next sought to evaluate the effect of the triazole linkers
on cellular potency, using monoacylsulfonamide 5 as reference.
Previous investigations of dimeric IAP antagonists revealed that
the length, rigidity, and polarity of the linker can have a
profound effect on biochemical binding and cell permeabil-
ity.21,22 The relatively polar and rigid triazole linker was
selected in the initial optimization to take advantage of the
efficiency of constructing macrocycles using azide−alkyne click
chemistry.23 However, our predicted binding model indicated
that both triazole groups in 1 occupy hydrophobic pockets on
the surface of the XIAP BIR2-3 proteins (Figure 1), suggesting
B
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that a less polar and nonaromatic linker may be tolerated. We
explored ring closing metathesis (RCM), which is compatible
with acylsulfonamide-containing peptide substrates to provide
access to analogues 7 and 8, where one or both of the triazoles
were replaced with less polar but similarly rigid propenyl
groups. Both 7 and 8 displayed comparable biochemical and
cellular activities to the bis-triazole analogue 5, thus
demonstrating the triazole linker was not required for binding
or cell permeability. Macrocyclization was also shown to be
essential for potent in vitro activity, as the macrocycle 8 was
greater than 20-fold more potent than the corresponding
uncyclized compound 9 in the biochemical binding and
antiproliferation assays (see Table 3).
select a compound for full in vitro and in vivo characterization.
In particular, we aimed to identify a compound with sufficient
aqueous solubility compatible with intravenous administration.
We found that in this series, aqueous solubility correlates well
with lipophilicity and overall charge of the peptide. Compounds
that are more lipophilic and net neutral are in general less
soluble. Accordingly, the most lipophilic compound 10, while
among the most potent compounds tested in biochemical and
cellular assays, has greatly diminished aqueous solubility (<0.01
mg/mL at pH 7.4) relative to compounds 1 and 11 (0.13 and
0.05 mg/mL, respectively, at pH 7.4). On the basis of its in vitro
potency and aqueous solubility, compound 11 was selected for
further characterization in additional biological assays.
We previously described a solid-phase synthesis of
compound 1 using an on-resin cyclization promoted by a Cu-
mediated azide−alkyne click reaction.13 Synthesis of compound
11 on resin, however, was significantly more challenging,
especially on the scale required to provide sufficient material for
additional profiling. Since the key RCM reaction could only be
performed in solution and the alkene-containing amino acid
building blocks required to assemble the linear peptide
precursors were difficult to access, a solution based method
was developed to prepare compound 11. Starting from
commercially available 2-naphthyl alanine 12, coupling to the
known 4-substituted prolines 13a and 13b furnished
compounds 14a and 14b. Successive incorporation of tert-
leucine and N-methylalanine building blocks using EDC-HOAt
as the coupling reagents furnished 15a and 15b after hydrolysis.
HATU-mediated amide bond coupling of 15a and 15b with
substituted tyrosine derivatives 16 and 18 provided pentapep-
tides 17 and 19, respectively. Cycloaddition of 17 and 19 in the
presence of CuSO4, followed by RCM and TFA deprotection,
furnished compound 8. Compound 8 was then converted to 11
via a straightforward hydrogenation reaction. Multiple batches
of compound 11 were prepared on the 400−500 mg scale using
this approach (see Scheme 1).
a
Table 3. Biological Activities of IAP Antagonists
Compound 11 was evaluated in additional FRET-based
binding assays and a cell-free caspase-3 rescue assay24 (Table
4). FRET assays confirmed binding of compound 11 to the
isolated BIR3 domain of the XIAP protein as well as the BIR2-3
domain of cIAP-1 protein with IC50 values in the low single-
digit nanomolar range. Consistent with the mechanism of
action of antagonizing XIAP to relieve inhibition of effector
caspases, 11 was found to increase caspase-3 activity in a dose-
dependent manner, with an IC50 of 9.4 nM.
Compound 11 was further profiled to establish its in vitro
safety and ADME properties (Table 4). Despite the increased
lipophilicity of the acylsulfonamide substituent and alkyl linker,
compound 11 possesses a favorable inhibitory profile toward
human cytochrome P450 (CYP) isoforms, has very low
potential for CYP induction based on the human PXR
transactivation assay, and showed almost no propensity for
hERG inhibition. Plasma protein-binding of 11 was measured
by equilibrium dialysis. No significant difference in plasma
protein binding was observed in mouse, rat, dog, or human
plasma. Additionally, compound 11 demonstrated excellent
metabolic stability in liver microsomes (t1/2 = 84 min in rat and
>120 min in mouse, dog, and human liver microsomes).
Finally, PK properties of compound 11 were assessed in mouse,
rat, and dog following a single IV dose. Consistent with the
predicted hepatic clearance from liver microsome metabolic
stability assays, compound 11 demonstrated short to moderate
half-lives and very low clearance across species.
a
IC50 values are an average of three independent experiments unless
b
c
otherwise noted. N = 1. Inhibition of cell growth in A875 cancer cell
line in the presence of TNF.
To remove the potentially labile allyl ether functionality,
reduction of the alkene groups provided bis- or monopropyl-
linked analogues 10 and 11. Binding data showed that despite
increased conformational flexibility, both 10 and 11 bind to
XIAP BIR2-3 proteins with IC50 values in the low single-digit
nanomolar range. Both compounds also displayed approx-
imately a 5-fold improvement in cellular potency relative to
compound 1 (A875 IC50 = 15 and 19 nM, respectively).
Encouraged by the excellent cellular potency of compounds
10 and 11, we evaluated their physiochemical properties to
C
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Scheme 1. (a) EDCI, HOAt, NMM, DMF; (b) TFA, DCM; (c) Boc-t-L-Leucine, EDCI, HOAt, NMM, DMF; (d) TFA, DCM;
(e) Boc-N-methyl-L-Alanine, EDCI, HOAt, NMM, DMF; (f) aq. LiOH, THF; (g) HATU, NMM, DMF; (h) CuSO4, sodium
ascorbic acid, THF/tBuOH/H2O; (i) Hoveyda-Grubbs II catalyst, DCE, 70 °C; (j) TFA, DCM; (k) Pd/C, H2, MeOH
Table 4. Summary of IAP Biological Data and ADME
a
Properties for Compound 11
assay
results
3.5 nM
XIAP BIR3 IC50
XIAP BIR2-3 IC50
cIAP-1 BIR2-3 IC50
Caspase-3 Rescue EC50
0.8 nM
3.0 nM
9.4 nM
human CYP 1A2, 2B6, 2C8, 2C9, 2D6, 3A4 IC50
PXR-TA EC50
>20 μM
>50 μM
hERG inhibition at 30 μM
9.0%
protein binding (% free): mouse, rat, dog, human
5.8, 1.9, 2.4, 7.5
IV PK: T1/2 (h), CL (mL/min/kg), AUC0−7 (nM h)
mouse (1 mg/kg):
1.7, 2.1, 4490
1.0, 6.2, 1710
4.7, 0.4, 8430
Figure 2. Antitumor activity of 11 in the A875 xenograft model in
mice. Compounds were administered intraperitoneally (i.p.) every 3
days for six doses or weekly for two doses beginning on day 10.
rat (1 mg/kg):
dog (0.3 mg/kg):
a
IC50 and EC50 values are an average of three experiments.
observed. These results compare favorably to those of 1, where
a 50 mg/kg dose on a more frequent q3d schedule was required
to achieve similar efficacy.
In vivo efficacy studies were conducted with 11 in A875
human tumor xenografts implanted in athymic mice. Gratify-
ingly, compound 11 demonstrated robust antitumor activity
(Figure 2). Intraperitoneal administration of 11 at 2 mg/kg
every 3 days for six doses resulted in 67% tumor growth
inhibition (TGI). Complete tumor stasis (>100% TGI) was
observed throughout dosing at 5 mg/kg. Compound 11 was
also efficacious when administered twice on a weekly schedule
at 5 mg/kg (TGI = 80%). Importantly, compound 11 was well
tolerated. No overt toxicity (weight loss or morbidity) was
Compound 11 was further profiled against a panel of human
colorectal cancer cell lines, including those that harbor the
multidrug-resistant (MDR) phenotype. Significantly, com-
pound 11 displayed robust activities in these tumor cell lines
regardless of their MDR status. For example, compound 11 was
highly potent at inhibiting cell growth in both the parental
HCT116 cell line and its MDR positive variant HCT116/
VM4625 (IC50 = 92 and 11 nM, respectively). This is in
D
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contrast to several previously reported nonmacrocyclic IAP
antagonists reported by us and others, which were shown to be
susceptible to the MDR phenotype.17,26
REFERENCES
■
(1) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: The next
generation. Cell 2011, 144, 646−674.
In summary, a potent dimeric macrocyclic IAP antagonist 11
with improved in vitro and in vivo activities was identified
through systematic optimization of the carboxylic acid and
linker regions of compound 1. Despite its high molecular
weight and large number of lipophilic aromatic rings,
compound 11 demonstrated desirable in vitro pharmacology,
safety, and PK parameters. Compound 11 was found to be
efficacious at a much lower dose compared to compound 1
when evaluated in the A875 xenograft model. Furthermore,
given the large number of tumors that are susceptible to the
MDR phenotype, the intriguing potency of this novel series of
IAP antagonists in MDR-positive cell lines may provide a
distinct advantage for this target class. Further profiling of
compound 11 in additional MDR-positive cell lines both in
vitro and in vivo is ongoing and will be reported in due course.
(2) Fulda, S.; Vucic, D. Targeting IAP proteins for therapeutic
intervention in cancer. Nat. Rev. Drug Discovery 2012, 11, 109−124.
(3) 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.
(4) Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Federova, 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 autoubiquitination of c-
IAPs,NF-κB activation, and TNFα-dependent apoptosis. Cell 2007,
131, 669−681.
(5) Tamm, I.; Kornblau, S. M.; Segall, H.; Krajewski, S.; Welsh, K.;
Kitada, S.; Scudiero, D. A.; Tudor, G.; Qui, Y. H.; Monks, A.; Andreeff,
M.; Reed, J. C. Expression and prognostic significance of IAP-family
genes in human cancers and myeloid leukemias. Clin. Cancer Res. 2000,
6, 1796−1803.
(6) 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.
ASSOCIATED CONTENT
■
S
* Supporting Information
(7) Li, L.; Thomas, R. M.; Suzuki, H.; De Brabander, J. K.; Wang, X.;
Harran, P. G. A small molecule Smac mimic potentiates TRAIL and
TNFα-mediated cell death. Science 2004, 305, 1471−1474.
(8) Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A. S.; Betz, S.
F.; Deckwerth, T. L.; Ding, H.; Elmore, S. W.; Meadows, R. P.;
Olejniczak, E. T. Discovery of potent antagonists of the antiapoptotic
protein XIAP for the treatment of cancer. J. Med. Chem. 2004, 47,
4417−4426.
Experimental procedures for the synthesis of all new analogues
and procedures for IAP binding assays, A875 proliferation
assay, caspase rescue assay, pharmacokinetic experiments, and
in vivo efficacy experiments. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acsmedchemlett.5b00091.
(9) 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,
1264−1277.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (609) 252-5055. E-mail: yong.zhang@bms.com.
(10) Flygare, J.; Fairbrother, W. Small-molecule pan-IAP antagonists:
a patent review. Expert Opin. Ther. Pat. 2010, 20, 251−267.
(11) 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.
(12) Bristol-Myers Squibb refers to cyclic and acyclic compounds
with a molecular weight between 600 and 6000 Da as millamolecules.
Millamolecules, especially macrocyclic millamolecules, are ideal for
pursuing the so-called “undruggable targets” such as complex protein−
protein interactions.
(13) Seigal, B.; Connors, W. H.; Fraley, A.; Borzilleri, R. M.; Carter,
P. H.; Emanuel, S. L.; Fargnoli, J.; Kim, K.; Lei, M.; Naglich, J. G.;
Pokross, M. E.; Posy, S. L.; Shen, H.; Surti, N.; Talbott, R.; Zhang, Y.;
Terrett, N. K. The discovery of macrocyclic XIAP antagonists from a
DNA-programmed chemistry library, and their optimization to give
lead compounds with apoptotic activity in vivo. J. Med. Chem. 2015, 58,
2855−2861.
(14) Marsault, E.; Peterson, M. L. Macrocycles Are Great Cycles:
Applications, Opportunities, and Challenges of Synthetic Macrocycles
in Drug Discovery. J. Med. Chem. 2011, 54, 1961−2004.
(15) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The
exploration of macrocycles for drug discovery  an underexploited
structural class. Nat. Rev. Drug Discovery 2008, 7, 608−624.
(16) Several macrocyclic IAP antagonists have been described in the
literature: Sun, H.; Liu, L.; Lu, J.; Qiu, S.; Yang, C.; Yi, H.; Wang, S.
Cyclopeptide Smac mimetics as antagonists of IAP proteins. Bioorg.
Med. Chem. Lett. 2010, 20, 3043−3046 and reference 11.
(17) Kim, K. S.; Zhang, L.; Willliams, D.; Perez, H. L.; Stang, E.;
Borzilleri, R. M.; Posy, S.; Lei, M.; Chaudhry, C.; Emanuel, S.; Talbott,
R. Discovery of tetrahydroisoquinoline-based bivalent heterodimeric
IAP antagonists. Bioorg. Med. Chem. Lett. 2014, 24, 5022−5029.
(18) Perez, H. L.; Chaudhry, C.; Emanuel, S. L.; Fargnoli, J.; Gan, J.;
Kim, K. S.; Lei, M.; Naglich, J. G.; Traeger, S. C.; Vuppugalla, R.; Wei,
D. D.; Vite, G. D.; Talbott, R. L.; Borzilleri, R. M. Discovery of potent
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Caroline Fanslau for generation of XIAP binding data
and Sarah Traeger, Gerry Evelof, and Celia D’Arienzo for
analytical support.
ABBREVIATIONS
■
IAP, inhibitor of apoptosis protein; XIAP, X-linked inhibitor of
apoptosis protein; cIAP, cellular inhibitor of apoptosis protein;
BIR, baculovirus inhibitor of apoptosis repeat; Smac, second
mitrochondria-derived activator of caspases; MDR, multidrug
resistance; CL, clearance; Vss, steady-state volume of
distribution; AUC, area under the curve; EDCI, N-(3-
(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride;
HOAt, 1-hydroxy-7-azabenzotriazole; NMM, N-methylmor-
pholine; Boc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid,
N-Boc-L-tert-leucine, (S)-2-((tert-butoxycarbonyl)amino)-3,3-
dimethylbutanoic acid; rt, room temperature; Boc-N-methyl-
L-alanine, (S)-2-((tert-butoxycarbonyl)(methyl)amino)-
propanoic acid; DMF, dimethylformamide; DCM, dichloro-
methane; tBuOH, tert-butanol; THF, tetrahydrofuran; DCE,
̀
dichloroethane; hERG, human ether-a-go-go-Related Gene;
PXR-TA, pregnane X receptor-transactivation; TGI, tumor
growth inhibition; PK, pharmacokinetic
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heterodimeric antagonists of inhibitor of apoptosis proteins (IAPs)
with antitumor efficacy. J. Med. Chem. 2015, 58, 1556−1562.
(19) Chaudhry, C.; Zhang, Y.; Posy, S.; Zhang, L.; Metzler, W.; Blat,
Y.; Talbott, R.; Borzilleri, R. M.; Building sensitive time resolved FRET
assays for thermodynamic and kinetic characterization of potent in
hibitors of an IAP target. Anal. Biochem. Manuscript in preparation.
(20) Conformation of the macrocycles can also affect cellular
activities through effects on permeability. See Rezai, H.; Yu, B.;
Millhaser, G. L.; Jacobson, M. P.; Lokey, R. S. J. Am. Chem. Soc. 2006,
128, 2510−2511.
(21) Sun, H.; Liu, L.; Lu, J.; Bai, L.; Li, X.; Nikolovska-Coleska, Z.;
McEachern, D.; Yan, C.; Qiu, S.; Yi, H.; Sun, D.; Wang, S. Potent
bivalent Smac mimetics: effect of the linker on binding to inhibitor of
apoptosis protein (IAPs) and anticancer activity. J. Med. Chem. 2011,
54, 3306−3318.
(22) Hennessy, E. J.; Adam, A.; Aquila, B. M.; Castriotta, L. M.;
Cook, D.; Hattersley, M.; Hird, A. W.; Huntington, C.; Kamhi, V. M.;
Laing, N. M.; Li, D.; MacIntyre, T.; Omer, C. A.; Oza, V.; Patterson,
T.; Repik, G.; Rooney, M. T.; Saeh, J. C.; Sha, L.; Vasbinder, M. M.;
Wang, H.; Whitston, D. Discovery of a novel class of dimeric Smac
mimetics as potent IAP antagonists resulting in a clinical candidate for
the treatment of cancer (AZD5582). J. Med. Chem. 2013, 56, 9897−
9919.
(23) White, C. J.; Yudin, A. Contemporary strategies for peptide
macrocyclization. Nat. Chem. 2011, 3, 509−524.
(24) Sun, W.; Nikolovska-Coleska, Z.; Qin, D.; Sun, H.; Yang, C.-Y.;
Bai, L.; Qiu, S.; Wang, Y.; Ma, D.; Wang, S. Design, Synthesis and
evaluation of potent, non-peptidic mimetics of second mitochondria-
derived activator of caspases. J. Med. Chem. 2009, 52, 593−596.
(25) Long, B. H.; Wang, L.; Lorico, A.; Wang, R. R. C.; Brattain, M.
G.; Casazza, A. M. Mechanisms of resistance to etoposide and
teniposide in acquired resistant human colon and lung carcinoma cell
lines. Cancer Res. 1991, 51, 5275−5284.
(26) See Table 1 in the Supporting Information for activities of a list
of IAP compounds in the HCT116/VM46 antiproliferation assay.
F
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