A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment

Article abstract of DOI:10.1021/jm101505d

We report the discovery and characterization of SM-406 (compound 2), a potent and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins (IAPs). This compound binds to XIAP, cIAP1, and cIAP2 proteins with K_i of 66.4, 1.9, and 5.1 nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free functional assay, induces rapid degradation of cellular cIAP1 protein, and inhibits cancer cell growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-human primates, and dogs, is highly effective in induction of apoptosis in xenograft tumors, and is capable of complete inhibition of tumor growth. Compound 2 is currently in phase I clinical trials for the treatment of human cancer.

Full text of DOI:10.1021/jm101505d

ARTICLE
pubs.acs.org/jmc
A Potent and Orally Active Antagonist (SM-406/AT-406) of Multiple
Inhibitor of Apoptosis Proteins (IAPs) in Clinical Development for
Cancer Treatment
||
Qian Cai,†,^,|| Haiying Sun,†, Yuefeng Peng,†,#,|| Jianfeng Lu,† Zaneta Nikolovska-Coleska,†
Donna McEachern,† Liu Liu,† Su Qiu,† Chao-Yie Yang,† Rebecca Miller,† Han Yi,† Tao Zhang,‡
Duxin Sun,‡ Sanmao Kang,§ Ming Guo,§ Lance Leopold,§ Dajun Yang,§ and Shaomeng Wang*,†
†Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry, University of
Michigan, Ann Arbor, Michigan 48109, United States
‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States
§Ascenta Therapeutics, 101 Lindenwood Drive, Malvern, Pennsylvania 19355, United States
ABSTRACT: We report the discovery and characterization of SM-406 (compound 2), a potent
and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins
(IAPs). This compound binds to XIAP, cIAP1, and cIAP2 proteins with Ki of 66.4, 1.9, and 5.1
nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free
functional assay, induces rapid degradation of cellular cIAP1 protein, and inhibits cancer cell
growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-
human primates, and dogs, is highly effective in induction of apoptosis in xenograft tumors, and is capable of complete inhibition of
tumor growth. Compound 2 is currently in phase I clinical trials for the treatment of human cancer.
’ INTRODUCTION
also influence a multitude of other cellular processes, such as ubiquitin
(Ub) dependent signaling events that regulate activation of nuclear
factor κB (NFκB), which in turn drive the expression of genes
important for inflammation, immunity, cell migration, and cell
survival.10 IAPs also modulate signaling events that promote the
activation of cell motility kinases and metastasis,11 and they regulate
mitogenic kinase signaling, proliferation, and mitosis. Many of these
cellular processes are frequently deregulated in cancer and contribute
directly or indirectly to disease initiation, tumor maintenance, and/or
progression.12,13 IAP proteins are therefore attractive new cancer
therapeutic targets.12ꢀ14
Smac/DIABLO (second mitochondria-derived activator of
caspases or direct IAP binding protein with low pI) has been
identified as an endogenous antagonist of XIAP, cIAP1, and
cIAP2.21,22 It nullifies the inhibition by XIAP of caspase-9 by
binding to the BIR3 domain in XIAP through its Ala-Val-Pro-Ile
(AVPI) tetrapeptide binding motif (1 in Figure 1) and directly
competing with a similar tetrapeptide, Ala-Thr-Pro-Phe (ATPF)
motif, in caspase-9.15,16,23,24 It has been proposed that Smac
protein binds to XIAP BIR2 via its AVPI motif, preventing the
binding of XIAP to caspase-3/caspase-7.25 cIAP1 interacts with
Smac through its BIR3 domain but not other BIR domains.26
Interestingly, in HeLa cells, expression of Smac protein selec-
tively induces degradation of cIAP1/2 but not XIAP.27
Apoptosis is a cellular process critical to the normal development
and homeostasis of multicellular organisms. Dysfunction of the
apoptosis machinery has been linked to many human diseases,
including cancer, inflammation, and neurological conditions.1ꢀ3
Defects in the apoptosis machinery confer on cancer cells resistance
to therapeutic agents and are known to compromise current antic-
ancer therapies, leading ultimately to their failure.2ꢀ4 Avoidance of
apoptosis is a hallmark of human cancer,4 and targeting key apop-
tosis regulators with the goal of overcoming the evasion of apoptosis
by tumor cells is an exciting therapeutic strategy for the treatment of
human cancer.1
The inhibitors of apoptosis proteins (IAPs) are a class of key
apoptosis regulators.5ꢀ7 Extensive studies have shown that although
their role is not limited to regulation of apoptosis,9,10 X-linked IAP
(XIAP) and cellular IAP 1 and IAP 2 (cIAP1 and cIAP2) are in central
positions as inhibitors of death signals that proceed through a number
of pathways.5ꢀ7 XIAP functions as a potent apoptosis inhibi-
tor by directly binding to and effectively inhibiting three caspases,
caspase-3 and -7, and -9.5ꢀ8 The third BIR domain (BIR3) of XIAP
selectively targets caspase-9,15,16 while the BIR2 domain, together with
the linker preceding it, inhibits both caspase-3 and caspase-7.17ꢀ19
By inhibiting these three caspases, XIAP plays a central role in the
inhibition of apoptosis in both death receptor-mediated and mito-
chondria-mediated pathways.5ꢀ7 cIAP1 and cIAP2 were originally
identified through their ability to interact directly with tumor necrosis
factor associated factor 2 (TRAF2).20 Through their interactions with
TRAF2, cIAP1 and cIAP2 are recruited to TNF-receptor-1- and TNF-
receptor-2-associated complexes where they suppress caspase-8 acti-
vation and death-receptor-mediated apoptosis.5 Furthermore, IAPs
The interaction of Smac with these IAP proteins involves the
AVPI tetrapeptide motif in Smac and a well-defined groove in the
IAP proteins, and there is considerable interest in the design of
Received: November 22, 2010
Published: March 28, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of Smac AVPI peptide, a potent and orally active Smac mimetic 2, its inactive control 3, a biotinylated analogue 4, and a
fluorescently tagged analogue 5.
nonpeptidic small molecules as a new class of anticancer drugs
that mimic the Smac AVPI peptide and antagonize these IAP pro-
teins.28,29 Employing a structure-based design strategy based upon
the crystal structure of Smac in a complex with XIAP BIR3 protein,
we have designed and synthesized several classes of conformationally
constrained non-peptide Smac mimetics.30ꢀ38 Our efforts have led to
the discovery of SM-406 (2 in Figure 1) as a potent and orally bio-
available Smac mimetic that possesses all the desirable properties as a
drug candidate for clinical development, which has subsequently
been advanced into phase I clinical trials. In this paper, we describe
the design, synthesis, and in vitro and in vivo evaluations of com-
pound 2 as a potent and orally active Smac mimetic.
Alternatively, condensation of the ammonium salt with L-NR-Boc-
NR-methyltryptophan and subsequent removal of the Boc protecting
group yielded the designed inactive analogue 3.
For the synthesis of biotinylated analogue 4, condensation of
compound 9 with N-carbobenzoxy-ε-aminocaproic acid (Z-6-ami-
nohexanoic acid) furnished an amide, mesylation of which, followed
by substitution of the mesylate with sodium azide, provided the
azide 10. Condensation of 7 with S-1-phenylprop-2-yn-1-amine
yielded an amide. Removal of the Boc protecting group from this
amide followed by condensation of the resulting ammonium salt
with L-N-Boc-N-methylalanine afforded amide 11. Cycloaddition of
11 with 10 under the influence of CuSO4 and sodium L-ascorbate
furnished a triazole. Removal of the Cbz protecting group in this
triazole followed by condensation of the resulting amine with (þ)-
biotin N-hydroxysuccinimide ester yielded a biotinylated amide,
from which removal of the Boc protecting group gave the biotiny-
lated analogue compound 4.
For the synthesis of fluorescently tagged analogue 5, con-
densation of compound 9 with benzyl chloroformate furnished a
carbamate, for which mesylation of the hydroxyl group and substitu-
tion of the mesylate with sodium azide provided an azide 12.
Cycloaddition of 11 with 12 in the presence of CuSO4 and sodium
L-ascorbate furnished a triazole. Removal of the Cbz protecting group
in this triazole followed by condensation of the resulting amine with
5-carboxyfluorescein N-succinimidyl ester yielded an amide. Removal
of the Boc protecting group from this amide gave the fluorescently
tagged analogue compound 5.
’ CHEMISTRY
The syntheses of 2, an inactive analogue 3 (SM-428), a
biotinylated analogue 4 (BL-SM-406), and a fluorescently tagged
compound 5 (FL-SM-406) are outlined in Scheme 1, and their
chemical structures are shown in Figure 1.
For the synthesis of all these compounds, the common key
intermediate compound 6 was prepared using our previously
reported method.35 Condensation of 6 with isovaleryl chloride
followed by hydrolysis of the methyl ester gave the free acid 7 and
condensation of which with diphenylmethylamine yielded the amide
8. Removal of the Boc protecting group from 8 gave an ammo-
nium salt, which upon condensation with L-N-Boc-N-methylalanine
followed by removal of the Boc protecting group gave compound 2.
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Scheme 1. Synthesis of compounds 2ꢀ5a
a Reagents and conditions: (a) i. isovaleryl chloride, N,N-diisopropylethylamine, CH2Cl2; ii. Two N LiOH, 1,4-dioxane, 82% over two steps; (b)
aminodiphenylmethane, EDC, HOBt, N,Ndiisopropylethylamine, CH2Cl2, 83%; (c) i. Four N HCl in 1,4-dioxane, methanol; ii. L-N-Boc-N-methylalanine,
EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2; iii. Four N HCl in 1,4-dioxane, methanol, 74% over three steps; (d) i. Four N HCl in 1,4-dioxane, methanol; ii.
L-Boc-Na-methyl-tryptophan, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2; iii. Four N HCl in 1,4-dioxane, methanol, 63% over three steps; (e) i. Z-6-
aminohexanoic acid, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2; ii. MsCl, Et3N, CH2Cl2; iii. NaN3, DMF, 110 ꢀC, 49% over three steps; (f) i. S-1-
phenylprop-2-yn-1-amine, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2; ii. Four N HCl in 1,4-dioxane, methanol; iii. L-N-Boc-Nmethyl-alanine, EDC,
HOBt, N,N-diisopropylethylamine, 69% over three steps; (g) i. 10, CuSO4, sodium L-ascorbate, t-BuOH-H2O 1:1; ii. H2, 10% PdꢀC, methanol; iii. (þ)-biotin
N-hydroxysuccinimide ester, N,N-diisopropylethylamine, CH2Cl2; iv. Four N HCl in 1,4-dioxane, MeOH, 43% over four steps; (h) i. CbzCl, Et3N, CH2Cl2;
ii. MsCl, Et3N, CH2Cl2; iii. NaN3, DMF, 66% over three steps; i. 11, CuSO4, sodium L-ascorbate, t-BuOH-H2O 1:1; ii. H2, 10% PdꢀC, methanol;
iii. 5-Carboxyfluorescein N-succinimidyl ester, N,N-diisopropylethylamine, CH2Cl2; iv. Four N HCl in 1,4-dioxane, MeOH, 34% over four steps.
’ RESULTS AND DISCUSSION
Compound 2 was designed to mimic the interaction of the
Smac AVPI peptide with XIAP BIR3 protein. The binding of the
Smac AVPI peptide complexed with XIAP BIR3 in the crystal
structure23 of Smac protein and the predicted binding model of 2
in complex with XIAP BIR3 are shown in Figure 2. Compound 2
appears to mimic closely the AVPI peptide in both hydrogen
bonding and hydrophobic interactions with XIAP but has addi-
tional hydrophobic contacts with W323 of XIAP. On the basis of
this model, the terminal methylamino group in 2 inserts into a
small, hydrophobic binding pocket associated with W310 in
XIAP BIR3. Hence, replacement of the methyl group in 2 with a
bulky 3-methyl-1H-indole group disrupts the optimal interaction
of 2 with XIAP BIR3, which results in an inactive compound 3.
To probe the cellular targets for our designed Smac mimetics,
we have designed the biotinylated analogue 4. Modeling suggests
that the pro-(S)-phenyl group in compound 2, without contact
with the XIAP protein, is exposed. Accordingly, this phenyl group
was replaced by a triazole group. The biotinylated analogue 4 was
synthesized using a highly efficient “click chemistry” method.39
To accurately determine the binding affinities of our Smac
mimetics, we have designed and synthesized a fluorescently
Figure 2. Structure-based design of conformationally constrained Smac
mimetics. (a) Crystal structure of Smac in complex with XIAP BIR3. For
clarity, only the AVPI peptide in Smac is shown. (b) Superposition of the
predicted binding model of compound 2 in complex with BIR3 on the
crystal structure of Smac AVPI peptide complexed with XIAP BIR3. The
carbon atoms of AVPI peptide and compound 2 are shown in yellow
and green, respectively, and the nitrogen and oxygen atoms in both
ligands are shown in blue and red, respectively. The protein is repre-
sented as its solvent accessible surface and colored by atom types
(carbon, gray; nitrogen, blue; oxygen, red). Hydrogen bonds are shown
in dashed light blue lines.
tagged compound 5 for further optimization of fluorescence
polarization (FP) assays.
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Figure 3. Saturation curves of fluorescently tagged compound 5 to (A) XIAP BIR3 protein, (B) cIAP1 BIR3 protein, and (C) cIAP2 BIR3 protein. The
standard deviation for the Kd value to each protein was calculated from three independent experiments.
Figure 4. Determination of the binding affinities of compounds 2, 3, and the Smac AVPI peptide to (A) XIAP BIR3, (B) cIAP1 BIR3, and (C) cIAP2
BIR3 using sensitive and quantitative fluorescence polarization assays. The standard deviation for the IC50 value to each protein was calculated from
three independent experiments.
Saturation experiments showed that the fluorescently tagged
compound 5 has Kd of 4.4 nM to XIAP BIR3 protein, 0.7 nM to
cIAP1 BIR3 protein, and 1.9 nM to cIAP2 BIR3 protein (Figure 3).
Furthermore, the dynamic range (ΔmP) for each protein is more
than 100, providing a robust signal for the development of
competitive FP assays for accurate and quantitative determination
of the binding affinities of designed Smac mimetics.
By use of compound 5 as a tracer, a set of competitive FP
assays were developed and optimized for XIAP BIR3, cIAP1
BIR3, and cIAP2 BIR proteins. The binding affinities of com-
pounds 2 and 3 and the Smac AVPI peptide to these IAP proteins
were determined using these assays (Figure 4). Compound 2
binds to XIAP BIR3, cIAP1 BIR3, and cIAP2 BIR3 proteins with
Ki of 66.4, 1.9, and 5.1 nM, respectively. The designed inactive
control 3 has minimal binding to these IAP proteins at 10 μM.
Under the same assay conditions, the Smac AVPI peptide binds
to XIAP BIR3, cIAP1 BIR3, and cIAP2 BIR3 proteins with Ki of
3.6 μM, 184 nM, and 316 nM, respectively. Hence, compound 2
binds to these three IAP proteins with 50ꢀ100 times higher
affinities than the Smac AVPI peptide.
Figure 5. Functional antagonism of compound 2 against XIAP BIR3 to
restore caspase-9 activity in a cell-free assay. Compound 3 was used as
the control compound.
concentration, 2 completely antagonizes XIAP and restores the
activity of caspase-9, consistent with its high binding affinity to
XIAP BIR3. The inactive control, 3, at concentrations as high as
100 μM fails to antagonize XIAP and to promote caspase-9 activity.
Compound 2 is therefore a potent antagonist of XIAP BIR3 protein
in this cell-free system.
Several recent studies have demonstrated that Smac mimetics
induce rapid degradation of cIAP1 and that such degradation is a key
early event in apoptosis induction by Smac mimetics as single agents
in cancer cells.40ꢀ44 Since 2 binds to cIAP1 with Ki = 1.9 nM, we eva-
luated its ability to induce cIAP1 degradation in the MDA-MB-231
Since XIAP BIR3 binds to and inhibits caspase-9,16 we
determined if 2 can effectively antagonize XIAP BIR3 and so
promote the activity of caspase-9 (Figure 5). In a cell-free functional
assay, addition of cytochrome c and dATP into the MDA-MB-231
cell lysates robustly activates caspase-9, as measured with Z-LEHD-
AFC, a substrate specific to caspase-9. Addition of 500 nM XIAP
BIR3 effectively suppresses caspase-9 activity, and compound 2
dose-dependently restores it. At 1 μM, twice that of the XIAP BIR3
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Figure 6. Compound 2 induces rapid cIAP-1 degradation in the MDA-MB-231 cancer cell line. Cells were treated with compound 2 as indicated, and
Western blot analysis was performed using specific antibody against cIAP1 and actin.
Figure 7. Biotinꢀstreptavidin pull-down experiments to probe the binding of compounds 2 and 3 to cellular XIAP and cIAP1 in the MDA-MB-231
breast cancer cell lysates using biotinylated compound 4.
Figure 8. Inhibition of cell growth by compounds 2 and 3 in the MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines, as determined in a
water-soluble tetrazolium (WST) cell proliferation assay. Four independent experiments were performed for each compound in each cell line.
cancer cells and the associated kinetics, with the results shown in
Figure 6. Compound 2 effectively induces cIAP1 degradation at
concentrations as low as 10 nM, consistent with its very high binding
affinity to cIAP1. Induction of cIAP1 degradation by 2 is also very
rapid; treatment of MDA-MB-231 cells by 2 at 100 nM for 15 min
causes complete degradation of the cIAP1 protein. Thus, com-
pound 2 is highly potent and effective in induction of rapid cIAP1
degradation.
To probe the cellular targets for 2, we have designed and
synthesized compound 4, a biotinylated analogue of 2. Com-
pound 4 binds to recombinant XIAP, cIAP1, and cIAP2 BIR3
proteins with Ki of 25.9, <1, and 1.7 nM, respectively. We have
performed streptavidinꢀbiotin pull-down experiments with 4
and competitive assays to probe the cellular targets of 2 in the
MDA-MB-231 cell lysates, and the results are given in Figure 7.
In the MDA-MB-231 cells that have high levels of XIAP and
cIAP1 proteins but very low levels of cIAP2 protein, biotinylated
compound 4 dose-dependently pulls down the cellular XIAP and
cIAP1 proteins. In the competitive experiment, 2 blocks the
interaction of cellular XIAP/cIAP1 with compound 4 in a dose-
dependent manner while the inactive control 3 fails to do so.
These pull-down experiments show that, consistent with the FP-
based binding data using recombinant IAP proteins, both 2 and 4
bind to cellular XIAP and cIAP1 proteins.
Screening of 2 against more than 100 human cancer cell lines
revealed that it is effective in inhibition of cell growth in approxi-
mately 15% of these cell lines and its activity is not limited to a
single tumor type. For example, in a 4-day WST assay, 2 potently
inhibits cell growth in the MDA-MB-231 breast and SK-OV-3
ovarian cancer cell lines with IC50 of 144 and 142 nM, respec-
tively (Figure 8). The inactive control 3 has IC50 > 10 μM in the
same assay, demonstrating the specificity for compound 2.
Compound 2 effectively induces cell death in a time- and dose-
dependent manner (Figure 9A and Figure 9B). More than 50% of
the MDA-MB-231 cancer cells underwent cell death upon
treatment with compound 2 at 1 μM for 24 h. Western blot
analysis showed that at 1.5 μM, 2 induces robust activation of
caspase-3 and cleavage of PARP in the MDA-MB-231 cancer
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Figure 9. Analysis of induction of cell death and apoptosis by compound 2 in the MDA-MB-231 cell line. MDA-MB-231 cells were treated with
compound 2 at (A) different concentrations for 24 h (B) with 1 μM concentration for different time points. (C) Western blot analysis of caspase
processing and cleavage of poly ADP-ribose polymerase (PARP). MDA-MB-231 cells were treated with 1.5 μM concentration of compound 2 for 24 h
and cleaved PARP, pro-caspase-3 and cleaved caspase-3 were probed with specific antibodies and actin was used as a loading control.
Figure 10. Induction of cell death by compound 2 in (A) normal-like human breast epithelial MCF-12F cells and (B) primary human normal prostate
epithelial cells. Cells were treated with different concentrations of compound 2 for 48 h, and cell viability was determined using the trypan blue
exclusion assay.
cells in as little as 12 h (Figure 9C). Hence, compound 2 is a
potent activator of caspases and effectively induces apoptosis in
the MDA-MB-231 breast cancer cell line.
An ideal anticancer drug should be selectively toxic against
cancer cells with minimal toxicity to normal cells. We have
evaluated the toxicity against normal or normal-like human
epithelial cells using the trypan blue cell death assay. Our data,
presented in Figure 10, show that 2 has minimal toxicity against
these normal or normal-like human epithelial cells at concentra-
tions as high as 10 μM.
(17 μΜ), T1/2 = 27 h, AUC = 198.2 μM h/mL at 10 mg/kg oral
dosing and an oral bioavailability of 55%.
3
To investigate its mechanism of action in vivo, compound 2
was tested in MDA-MB-231 xenograft tumors in severe combined
immune deficiency (SCID) mice. Mice bearing the MDA-MB-231
tumors were given a single oral dose of 2, vehicle control, or intra-
venous Taxotere (TXT). The levels of cIAP1, procaspase-8, and
cleaved poly ADP-ribose polymerase (PARP) in xenograft tissues
were analyzed by Western blotting with the results given in Figure 11.
At 100 mg/kg, 2 has effectively induced cIAP1 degradation in tumor
tissues at the 2 h time-point, and this effect persists for 16 h. It also
effectively induces processing of procaspase-8 at the 2 and 6 h time-
points, as well as cleavage of the apoptosis marker, PARP, at the 2- h
time-point. PARP cleavage reaches a maximum level at the 6-h time
point and is still evident after 16 h. In comparison, TXT induces the
processing of procaspase-8 but has a minimal effect on cIAP1
degradation and PARP cleavage at the 24 h time-point. Thus, 2 is
Compound 2 was evaluated for its pharmacokinetic (PK)
properties in mice, rats, non-human primates, and dogs. It has an
excellent PK profile and good oral bioavailability in each of these four
species (Table 1). For example, in non-human primates (NHP), 2
has Cmax = 2.2 μg/mL (∼4 μM) in the plasma, T1/2 = 5.4 h, area-
under-the-curve (AUC) = 13.5 μg h/mL at 5 mg/kg oral dosing,
3
and oral bioavailability of 42%. In dogs, 2 achieves Cmax = 8.5 μg/mL
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Table 1. Pharmacokinetic Properties of Compound 2 in Four Different Species
species
route
dose (mg/kg)
AUC(0ꢀt) (h μg/mL)
Cmax (μg/mL)
T1/2 (h)
3.0
2.3
1.4 ( 0.2
1.8 0.3
6.3 ( 0.4
5.4 ( 1.0
16.3 ( 3.8
27.1 ( 13.7
F (%)
3
mice
iv
po
iv
po
iv
po
iv
10
30
5
25
2.5
5
3.8
4.3
1.2
38
45
42
55
rats
1.1 ( 0.1
2.3 ( 0.7
17.9 ( 11.0
13.5 ( 7.9
81.6 ( 17.3
90.3 ( 23.5
0.66 ( 0.27
2.2 ( 0.6
8.5 ( 2.5
NHP
dogs
5
10
po
Figure 11. Evaluation of compound 2 on cIAP1, caspase-8 processing, and PARP cleavage in the MDA-MB-231 xenograft tissues. Mice bearing the
MDA-MB-231 tumors were treated with a single oral dose of compound 2, intravenous Taxotere (TXT), or intravenous vehicle. cIAP1, XIAP, pro-
caspase-8 (Pro-C8), and cleaved PARP (CL-PARP) were examined by Western blotting.
effective in induction of cIAP1 degradation, processing of caspase-8,
and PARP cleavage in the MDA-MB-231 xenograft tissues.
Compound 2 was evaluated for its in vivo antitumor activity as
an oral agent in the MDA-MB-231 xenograft model in mice. In our
toxicity evaluation, compound 2 was found to be well tolerated in
mice at 200 mg/kg, daily for 5 days a week for 2ꢀ3 weeks via oral
gavage. In this efficacy experiment, each treatment or control group
consisted of eight to nine mice, each mouse bearing one tumor.
Treatment started on day 25 when the mean tumor size reached 150
mm3. Compound 2 was given via oral gavage daily, 5 days a week for
2 weeks at 10, 30, and 100 mg/kg. Vehicle alone was administered
daily, 5 days a week for 2 weeks, to a control group of animals. TXT
was given intravenously at 7.5 mg/kg once a week for 2 weeks. The
results, shown in Figure 12, indicate that while 2 has no significant
antitumor activity at 10 mg/kg, it strongly inhibits tumor growth at
30 and 100 mg/kg and completely inhibits tumor growth during the
treatment with 100 mg/kg. The T/C values for 2 at 100 mg/kg on
days 36 and 50 are 0.26 and 0.30, respectively, when the control
tumors reach 500 and 1100 mm3. Hence, in this model, compound 2
is a highly active agent, according to the National Cancer Institute
(NCI) criteria (T/C< 0.42). The tumor growth inhibition on day 36
at both 30 and 100 mg/kg is statistically highly significant with p of
0.0083 and 0.0012, respectively, compared to the vehicle control
group. The positive control, TXT, also achieves a strong antitumor
activity in the MDA-MB-231 xenografts, similar to that of 2 at 100
mg/kg, but it causes significant weight loss in animals during the
treatment period (p = 0.01 at day 36), compared to the vehicle-
treated animals. These results indicate that compound 2 is very
effective in inhibition of tumor growth in the MDA-MB-231
xenograft model and has minimal toxicity to animals.
To gain further insights into the PK of compound 2 in both
plasma and tumor tissues, we performed an analysis with SCID mice
bearing the MDA-MB-231 tumors. SCID mice were given a single
dose of compound 2 either intravenously (iv) or via oral gavage
(po), and the concentration of compound 2 was determined in both
Figure 12. In vivo antitumor activity of compound 2 in the MDA-MB-
231 xenograft model. Taxotere (TXT) was used as the control: (A)
tumor volume; (B) animal weight.
plasma and tumor tissues. The data are shown in Figure 13 and
Table 2. At 10 mg/kg iv, 10 mg/kg po, 30 mg/kg po, or 100 mg/kg
po dosing, compound 2 achieves Cmax of 3.0 μg/mL (5.0 μM),
0.9 μg/mL (1.5 μM), 1.2 μg/mL (2.0 μM), and 5.9 (9.9 μM),
respectively, and AUC(0ꢀt) of 3.8, 1.2, 4.3, and 17.4 h μg/mL in
3
the plasma. In comparison, at 10 mg/kg iv, 10 mg/kg po, 30 mg/kg
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Figure 13. Analysis of pharmacokinetics of compound 2 in plasma and in tumor tissues in SCID mice bearing the MDA-MB-231 xenograft tumors.
Compound 2 was dosed via either intravenously (iv) or oral gavage (po).
Table 2. Pharmacokinetics of Compound 2 in Mice in Plasma and in Tumor Tissuesa
dose (mg/kg) (route)
AUC(0ꢀt) (h μg/mL)
Cmax (μg/mL)
Tmax (h)
T1/2 (h)
F (%)
3
Plasma
3.0
10 (iv)
3.8
1.2
3.0
2.4
2.3
5.4
10 (po)
30 (po)
100 (po)
0.9
1
31
38
46
4.3
1.2
2.0
0.25
17.4
5.9
Tumor Tissues
10 (iv)
8.3
1.1
3.6
0.26
2.4
0.25
1.0
10 (po)
30 (po)
100 (po)
9.6
2.0
57.3
10.8
2.0
a Compound 2 was dosed via either intravenously (iv) or oral gavage (po).
po, or 100 mg/kg po dosing, compound 2 achieves Cmax of 3.6
μg/mL (6.0 μM), 0.26 μg/mL (0.5 μM), 2.4 μg/mL (3.9 μM), and
10.9 μg/mL (18.2 μM), respectively, and AUC(0ꢀt) values of
interaction with cytochrome P450s, does not inhibit hERG ion
channel at concentrations as high as 30 μM, and is well tolerated
in dogs (data not shown). Taken together, compound 2 repre-
sents an excellent compound for clinical development. Com-
pound 2 (renamed AT-406 by Ascenta Therapeutics) is now
being evaluated in phase I clinical trials as a new therapy for the
treatment of solid and hematological human tumors.
8.3, 1.1, 9.6, and 57.3 h μg/mL in the tumor tissues. These data
3
indicate that compound 2 has an excellent tumor tissue penetration.
Since compound 2 is effective in inhibition of tumor growth in the
MDA-MB-231 xenograft model at 30 mg/kg oral dosing and com-
pletely inhibits tumor growth at 100 mg/kg oral dosing, these data
support the dose levels and drug exposures needed for compound 2
to achieve strong antitumor activity in vivo.
’ EXPERIMENTAL SECTION
Chemistry. General Methods. 1H NMR spectra were measured at
300 MHz. H chemical shifts are reported with CHCl3 (7.27 ppm),
In summary, we have designed and synthesized compound 2, a
nonpeptidic Smac mimetic. Our data demonstrate that com-
pound 2 binds to XIAP, cIAP1, and cIAP2 with nanomolar
affinities and is a potent antagonist against these IAP proteins. It
also achieves an excellent oral bioavailability in rodents and non-
rodents and has good aqueous solubility (>50 mg/mL). As a
single agent, it effectively inhibits cancer cell growth in a subset of
human cancer cell lines and is highly effective in inhibition of
tumor growth with oral dosing while causing no signs of toxicity
in mice. On the basis of these data, it was selected for further
evaluation as a candidate for clinical development. Investiga-
tional new drug (IND) enabling studies under FDA guidelines
were conducted. These IND-enabling studies demonstrated
that compound 2 has good metabolic stability, has a minimal
1
DHO (4.79 ppm), and CD3OH (3.34 ppm) as internal standards. Final
products were purified by HPLC on a C18 reverse phase semiprepara-
tive column with solvent A (0.1% of TFA in water) and solvent B (0.1%
of TFA in CH3CN) as eluents, and the purity of the final products was
checked by analytical HPLC using the C18 reverse phase column to be
over >95%.
tert-Butyl ((5S,8S,10aR)-8-(Benzhydrylcarbamoyl)-3-(3-methylbuta-
noyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocin-5-yl)carbamate (8).
Isovaleryl chloride (0.2 mL) and N,N-diisopropylethylamine (0.4 mL)
were added to a solution of 6 (340 mg, 1 mmol) in CH2Cl2 (20 mL). The
solution was stirred at room temperature for 3 h and then concentrated.
The residue was purified by chromatography to give an amide. Then 2 N
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LiOH solution (2.0 mL) was added to a solution of this amide in 1,4-
dioxane (3.0 mL). AfterTLCshowedall of theamide hadbeenconsumed,
the mixture was neutralized with 1 N HCl to pH 5 and then extracted with
EtOAc (3 ꢁ 10 mL). The combined organic layers were dried over Na2SO4
and then concentrated to give the acid 7 (340 mg, 82% over two steps),
which was used directly, without purification, in the next step. To a solution
of 7 (105 mg, 0.25 mmol) in CH2Cl2 (10 mL) were added aminodiphe-
nylmethane (55 mg, 0.3 mmol), EDC (58 mg, 0.3 mmol), HOBt (45 mg,
0.33 mmol), and N,N-diisopropylethylamine (0.5 mL) at 0 ꢀC. The mixture
was stirred at room temperature for 6 h and then concentrated. The residue
stirred at room temperature for 6 h and then concentrated. The residue was
purified by chromatography to give an amide. To a solution of this amide in
MeOH (15 mL) was added HCl solution (4 N in 1,4-dioxane, 2 mL). The
solution was stirred at room temperature overnight and then concentrated to
give an ammonium salt. To a mixture of this salt in CH2Cl2 (20 mL) was
added L-N-Boc-N-methylalanine (124 mg, 0.6 mmol), EDC (120 mg, 0.6
mmol), HOBt (83 mg, 0.6 mmol), and N,N-diisopropylethylamine (1 mL)
at 0 ꢀC. The mixture was stirred at room temperature for 6 h and then
concentrated. The residue was purified by chromatography to give 11 (210
mg, 69% over three steps). 1H NMR (300 MHz, CDCl3, TMS): δ 7.58 (m,
1H), 7.54ꢀ7.49 (m, 2H), 7.36ꢀ7.20 (m, 4H), 5.95 (dd, J= 8.5, 2.4 Hz, 1H),
4.70 (m, 1H), 4.60ꢀ4.45 (m, 2H), 3.95 (m, 1H), 3.92ꢀ3.80 (m, 2H), 2.79
(s, 3H), 2.70ꢀ2.35 (m, 5H), 2.35ꢀ1.75 (m, 7H), 1.50 (brs, 9H), 1.33 (d, J=
7.2 Hz, 3H), 1.05ꢀ0.95 (m, 6H). ESI MS: m/z 610.3 (M þ H)þ.
1
was purified by chromatography to give 8 (120 mg, 82%). H NMR
(CDCl3): δ 7.70 (brd, J = 7.0 Hz, 1H), 7.35ꢀ7.16 (m, 10H), 6.25 (d, J =
7.5 Hz, 1H), 5.90 (brd, J = 7.0 Hz, 1H), 4.70 (m, 1H), 4.45 (m, 1H),
4.08ꢀ3.70 (m, 3H), 2.80ꢀ2.50 (m, 3H), 2.42 (m, 1H), 2.30ꢀ1.50 (m, 7H),
1.47 (brs, 9H), 1.02 (m, 6H). ESI MS: m/z 577.3 (M þ H)þ.
(5S,8S,10aR)-5-((S)-2-(Methylamino)propanamido)-3-(3-methylbuta-
noyl)-6-oxo-N-((S)-(1-(10-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thi-
eno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)decyl)-1H-1,2,3-tria-
zol-4-yl)(phenyl)methyl)decahydropyrrolo[1,2-a][1,5]diazocine-8-carboxa-
mide (4). Z-6-Aminohexanoic acid (790 mg, 3 mmol), EDC (590 mg, 3
mmol), HOBt (395 mg, 3 mmol), and triethylamine (2 mL) were added to a
solution of 10-amino-1-decanol (9, 520 mg, 3 mmol) in CH2Cl2 (15 mL) at
0 ꢀC. The mixture was stirred at room temperature for 6 h and then
concentrated. The residue was purified by chromatography to furnish an
amide. Methanesulfonyl chloride (0.3 mL) and triethylamine (1 mL) in
CH2Cl2 (20 mL) were added to a solution of this amide at 0 ꢀC. The solution
was stirred at room temperature for 6 h and then concentrated. The residue
was purified by chromatography to yield a mesylate. Sodium azide (200 mg)
was added to a solution of this mesylate in DMF (10 mL), and the mixture was
stirred at 110 ꢀC overnight and then partitioned between EtOAc (50 mL) and
brine (30 mL). The organic layer was dried over Na2SO4 and then
concentrated. The residue was purified by chromatography to provide 10
(650 mg, 49% over three steps). CuSO4 (25 mg, 0.1 mmol) and sodium L-
ascorbate (100 mg, 0.5 mmol) in water (5 mL) were added to a solution of 10
(122 mg, 0.2 mmol) and 9 (90 mg, 0.2 mmol) in acetonitrile (3 mL) and tert-
butanol (3 mL). The mixture was stirred at room temperature overnight and
then partitioned between EtOAc (30 mL) and brine (20 mL). The organic
layer was dried over Na2SO4 and then concentrated. The residue was purified
by chromatography to give a triazole which, mixed with 10% PdꢀC (100 mg)
in MeOH (20 mL), was stirred under H2 overnight and then filtered through
Celite. The filtration was concentrated to yield an amine. To a solution of this
amine in CH2Cl2 (20 mL) was added (þ)-biotinN-hydroxysuccinimide ester
(78 mg, 0.2 mmol) and N,N-diisopropylethylamine (0.5 mL). The solution
was stirred at room temperature overnight and then concentrated to give a
residue which was purified by chromatography to afford a biotinylated amide.
To a solution of this amide in MeOH (10 mL) was added HCl solution (4 N
in 1,4-dioxane, 2 mL). The mixture was stirred overnight and then concen-
trated to furnish a crude product which was purified by a semipreparative
HPLC using a reverse phase C18 column to give pure compound 4 (salt with
TFA, 98 mg, 43% over four steps). The gradient ran from 70% of A and 30% of
B to 40% of A and 60% of B in 30 min. The purity of the compound was
determined by an analytical HPLC using a reverse phase C18 column to be
over 95%. 1H NMR (D2O): δ 7.70 (brs, 1H), 7.35ꢀ7.05 (m, 5H), 6.05 (brs,
1H), 4.70 (m, 1H), 4.50ꢀ4.30 (m, 2H), 4.30ꢀ4.05 (m, 3H), 4.05ꢀ3.60 (m,
2H), 3.60ꢀ2.80 (m, 5H), 2.80ꢀ2.50 (m, 6H), 2.40ꢀ1.05 (m, 44H), 0.80
(m, 6H). ESI MS: m/z 1047.6 (M þ H)þ.
(5S,8S,10aR)-N-Benzhydryl-5-((S)-2-(methylamino)propanamido)-
3-(3-methylbutanoyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocine-
8-carboxamide (2). HCl (4 N in 1,4-dioxane, 1 mL) was added to a
solution of 8 (58 mg, 0.1 mmol) in MeOH (10 mL). The solution was
stirred at room temperature overnight and then concentrated to give an
ammonium salt. To a mixture of this salt in CH2Cl2 (10 mL) was added
L-N-Boc-N-methylalanine (30 mg, 0.15 mmol), EDC (29 mg, 0.15
mmol), HOBt (22 mg, 0.16 mmol), and N,N-diisopropylethylamine
(0.3 mL) at 0 ꢀC. The mixture was stirred at room temperature for 6 h
and then concentrated. The residue was purified by chromatography to
furnish an amide. To a solution of this amide in 5 mL of MeOH was
added HCl solution (4 N in 1,4-dioxane, 1 mL). The solution was stirred
at room temperature overnight and then concentrated to give crude
product which was purified by HPLC to give pure compound 2 (salt with
TFA, 48 mg, 74% over three steps). The gradient ran from 70% A and
30% B to 50% A and 50% B in 30 min. The purity was determined by
1
reverse analytical HPLC to be over 95%. H NMR (CD3OD, 300 M
Hz): δ 8.97 (d, J = 7.5 Hz, 1H), 7.38ꢀ7.25 (m, 10H), 6.16 (d, J = 7.5 Hz,
1H), 4.80 (m, 1H), 4.58 (m, 1H), 4.25 (m, 1H), 3.96 (m, 3H), 3.47 (m,
1H), 2.72 (s, 3H), 2.49 (d, J = 7.2 Hz, 2H), 2.37ꢀ2.32 (m, 1H),
2.15ꢀ2.00 (m, 6H), 1.84ꢀ1.80 (m, 1H), 1.56 (d, J = 5.4 Hz, 3H), 1.00
(d, J = 6.0 Hz, 6H). ESI-MS: m/z 562.3 (M þ H)þ.
(5S,8S,10aR)-5-((S)-3-(1H-Indol-3-yl)-2-(methylamino)propanamido)-
N-benzhydryl-3-(3-methylbutanoyl)-6-oxodecahydropyrrolo[1,2-a][1,5]-
diazocine-8-carboxamide (3). HCl (4 N in 1,4-dioxane, 1 mL) was added
to a solution of 8 (58 mg, 0.1 mmol) in MeOH (10 mL). The solution was
stirred at room temperature overnight and then concentrated to give an
ammonium salt. To a mixture of this salt in 10 mL of CH2Cl2 was added L-
NR-Boc-NR-methyltryptophan (48 mg, 0.15 mmol), EDC (30 mg, 0.15
mmol), HOBt (22 mg, 0.16 mmol), and N,N-diisopropylethylamine
(0.3 mL) at 0 ꢀC. The mixture was stirred at room temperature for 6 h
and then concentrated. The residue was purified by chromatography to
furnish an amide. To a solution of this amide in 5 mL of MeOH was added
HCl (4 N in 1,4-dioxane. 1 mL). The solution was stirred at room
temperature overnight and then concentrated to give crude product which
was purified by HPLC to give 3 (salt with TFA, 49 mg, 63% over three
steps). The gradient ran from 70% A and 30% B to 40% A and 60% B in 30
min. The purity was determined by reverse analytical HPLC to be >95%. 1H
NMR (CD3OD, 300 M Hz): δ 8.88 (d, J = 8.4 Hz, 1H), 7.33ꢀ7.08 (m,
13H), 6.95 (m, 2H), 6.10 (m, 1H), 4.40 (m, 1H), 4.34 (m, 1H), 4.27 (m,
1H), 4.23 (m, 1H), 3.97 (m, 2H), 3.83 (m, 2H), 3.44 (m, 1H), 3.10 (m,
2H), 2.73 (2S, 3H), 2.52 (m, 2H), 2.36 (m, 1H), 2.18ꢀ1.72 (m, 6H), 1.00
(m, 6H). ESI-MS: m/z 677.4 (M þ H)þ.
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-((10-(4-((S)-((5S,8S,10aR)-5-
((S)-2-(methylamino)propanamido)-3-(3-methylbutanoyl)-6-oxodecahy-
dropyrrolo[1,2-a][1,5]diazocine-8-carboxamido)(phenyl)methyl)-1H-1,2,3-
triazol-1-yl)decyl)carbamoyl)benzoic Acid (5). Benzyl chloroformate
(0.5 mL) and triethylamine (2 mL) were added to a solution of 10-
amino-1-decanol (10, 520 mg, 3 mmol) in CH2Cl2 (15 mL) at 0 ꢀC. The
mixture was stirred at room temperature for 6 h and then concentrated. The
residue was purified by chromatography to furnish a carbamate. Methane-
sulfonyl chloride (0.5 mL) was added dropwise at 0 ꢀC to a solution of this
tert-Butyl Methyl((S)-1-(((5S,8S,10aR)-3-(3-methylbutanoyl)-6-oxo-
8-(((R)-1-phenylprop-2-yn-1-yl)carbamoyl)decahydropyrrolo[1,2-a][1,5]-
diazocin-5-yl)amino)-1-oxopropan-2-yl)carbamate (10). S-1-Phenylprop-
2-yn-1-amine (79 mg, 0.6 mmol), EDC (116 mg, 0.6 mmol), HOBt (80 mg,
0.6 mmol), and N,N-diisopropylethylamine (1 mL) were added to a solution
of 7 (205 mg, 0.5 mmol) in CH2Cl2 (15 mL) at 0 ꢀC. The mixture was
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carbamate in CH2Cl2 (20 mL) with triethylamine (1 mL). The solution was
stirred at room temperature for 6 h and then concentrated. The residue
was purified by chromatography to yield a mesylate. Sodium azide (300 mg)
was added to a solution of this mesylate in DMF (10 mL). The mixture was
stirred at 110 ꢀC overnight and then partitioned between EtOAc (50 mL)
and brine (30 mL). The organic layer was dried over Na2SO4 and then
concentrated. The residue was purified by chromatography to provide 12
(650 mg, 66% over three steps). A mixture of CuSO4 (25mg, 0.1mmol) and
sodium L-ascorbate (100 mg, 0.5 mmol) in water (5 mL) was added to a
solution of 11 (122 mg, 0.2 mmol) and 12 (66 mg, 0.2 mmol) in acetonitrile
(3 mL) and tert-butanol (3 mL). The mixture was stirred at room
temperature overnight and then partitioned between EtOAc (30 mL) and
brine (20 mL). The organic layer was dried over Na2SO4 and then
concentrated. The residue was purified by chromatography to give a triazole.
A mixture of this triazole and 100 mg of 10% PdꢀC in MeOH (20 mL) was
stirred under H2 overnight and then filtered through Celite. The filtration
was concentrated to yield an amine. To a solution of this amine in CH2Cl2
(20 mL) was added 5-carboxyfluorescein N-succinimidyl ester (95 mg, 0.2
mmol) and N,N-diisopropylethylamine (0.5 mL). The solution was stirred
at room temperature overnight and then concentrated. The residue was
purified by chromatography to afford an amide. To a solution of this amide in
MeOH (10 mL) was added HCl (4 N in 1,4-dioxane, 2 mL). The mixture
was stirred overnight and then concentrated to furnish a crude product which
was purified by semipreparative HPLC using a reverse phase C18 column to
give compound 5 (salt with TFA, 72 mg, 34% over four steps). The gradient
ran from 70% of A and 30% of B to 40% of A and 60% of B in 30 min. The
purity of the compound was determined by analytical HPLC using a reverse
phase C18 column to be over 95%. 1H NMR (CD3OD, 300 MHz): δ 8.48
(s, 1H), 8.22 (m, 1H), 7.70 (s, 1H), 7.50ꢀ7.20 (m, 5H), 6.75 (brs, 2H),
6.70ꢀ6.50 (m, 4H), 6.30 (brs, 1H), 4.70 (m, 1H), 4.65ꢀ4.50 (m, 2H),
4.40ꢀ4.30 (m, 2H), 4.30ꢀ4.15 (m, 2H), 4.05ꢀ3.80 (m, 3H), 3.50ꢀ3.40
(m, 2H), 2.70 (brs, 3H), 2.52ꢀ2.01 (m, 7H), 2.01ꢀ1.60 (m, 7H),
1.60ꢀ1.50 (m, 3H), 1.50ꢀ1.20 (m, 11H), 0.95 (m, 6H). ESI MS: m/z
1066.5 (M þ H)þ.
the soluble fraction using Ni-NTA resin (QIAGEN) followed by gel filtration
on a Superdex 75 column in Tris, pH 7.5 (20 mM), NaCl (200 mM), ZnAc
(50 μM), and dithiothretal (DTT, 1 mM). After purification, DTT was
addedtoafinal concentration of 10 mM. Human cIAP1 BIR3-only (residues
253ꢀ363) and cIAP2 BIR3-only (residues 238ꢀ349) were cloned into
pHis-TEV vector, produced, and purified using the same method as for the
XIAP BIR3 protein.
The fluorescently tagged compound 5 was employed to develop a set
of new FP assays for determination of the binding affinities of our
designed Smac mimetics to XIAP, cIAP-1, and cIAP-2 BIR3 proteins. The
Kd value of compound 5 to each IAP protein was determined by titration
experiments using a fixed concentration of compound 5 and different
concentrations of the protein up to full saturation. Fluorescence polarization
values were measured using an Infinite M-1000 plate reader (Tecan U.S.,
Research Triangle Park, NC) in Microfluor 2 96-well, black, round-bottom
plates (Thermo Scientific). To each well, compound 5 (2, 1, and 1 nM for
experiments with XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3, respectively)
and different concentrations of the protein were added to a final volume of
125 μLintheassaybuffer (100 mM potassium phosphate, pH 7.5, 100 μg/mL
bovine γ-globulin, 0.02% sodium azide, Invitrogen, with 4% DMSO).
Plates were mixed and incubated at room temperature for 2ꢀ3 h with
gentle shaking. The polarization values in millipolarization units (mP)
were measured at an excitation wavelength of 485 nm and an emission
wavelength of 530 nm. Equilibrium dissociation constants (Kd) were
then calculated by fitting the sigmoidal dose-dependent FP increases as a
function of protein concentrations using Graphpad Prism 5.0 software
(Graphpad Software, San Diego, CA).
In competitive binding experiments for XIAP3 BIR3, a tested
compound was incubated with 20 nM XIAP BIR3 protein and 2 nM
compound 5 in the assay buffer (100 mM potassium phosphate, pH 7.5;
100 μg/mL bovine gamma globulin; 0.02% sodium azide, Invitrogen).
In competitive binding experiments for cIAP1 BIR3 protein, 3 nM
protein and 1 nM compound 5 were used. In competitive binding
experiments for cIAP2 BIR3, 5 nM protein and 1 nM compound 5
were used.
For each competitive binding experiment, polarization values were
measured after 2ꢀ3 h of incubation using an Infinite M-1000 plate
reader. The IC50 value, the inhibitor concentration at which 50% of the
bound tracer was displaced, was determined from the plot using non-
linear least-squares analysis. Curve fitting was performed using the
PRISM software (GraphPad Software, Inc., San Diego, CA). A Ki value
for each compound was calculated based upon the IC50 using a
previously reported algorithm and the associated computer program.48
Cell-Free Functional Assay against XIAP BIR3 Protein.
MDA-MB-231 cell lysates were prepared by solubilizing cells in ice cold
buffer containing KCl (50 mM), EGTA (5 mM), MgCl2 (2 mM), DTT
(1 mM), 0.2% CHAPS, and HEPES (50 mM, pH 7.5), containing
cocktail protease inhibitors, incubating on ice for 10 min, then freezing in
liquid nitrogen. Cytochrome c and dATP were added to the cell lysates,
which were then incubated at 30 ꢀC in a water bath for 60 min to activate
caspase-9. Addition of recombinant XIAP BIR3 protein dose-depen-
dently suppressed the activity of caspase-9. Different concentrations of a
tested Smac mimetic (1 nM to 100 μM) were added to determine the
restoration of the activity of these caspases.
Computational Docking Simulation. The crystal structure of
XIAP BIR3 in a complex with the Smac protein23 (PDB code 1G73) was
used to predict the binding models of XIAP BIR3 bound to compound 2.
The binding pose of compound 2 with XIAP BIR3 was predicted with
the GOLD program (version 3.1.1).46,47 In the docking simulation, the
center of the binding site was set at Thr308 of XIAP BIR3 and the radius
of the binding site was defined as 13 Å, sufficient to cover all the binding
pockets. For each genetic algorithm (GA) run, a maximum of 200 000
operations were performed on a population of 5 islands of 100
individuals. Operator weights for crossover, mutation, and migration
were set to 95, 95, and 10, respectively. The docking simulation was
terminated after 20 runs for compound 2. GoldScore, implemented in
Gold, was used as the fitness function to evaluate the docked conforma-
tions. The 20 conformations ranked highest by each fitness function
were saved for analysis of the predicted docking modes. The docking
protocol was validated using a series of Smac mimetics.45 The predicted
binding pose of compound 2 shown in Figure 2 is the highest ranked
conformation from the docking simulations.
Fluorescence Polarization Based Assays for XIAP, cIAP1,
and cIAP2 BIR3 Proteins. Sensitive and quantitative fluorescence
polarization (FP) based assays were used to determine the binding
affinities of our designed Smac mimetics to XIAP BIR3, cIAP1 BIR3, and
cIAP2 BIR3 proteins.
Human XIAP BIR3-only (residues 241ꢀ356) was cloned into a
pET28 vector (Novagen) containing an N-terminal 6ꢁ His tag. Protein
was produced in E. coli BL21(DE3) cells grown at 37 ꢀC in 2ꢁ YT
containing kanamycin to an OD600 of 0.6. Protein expression was induced by
0.4 mM IPTG at 27 ꢀC for 4 h. Cells were lysed by sonication in buffer
containing Tris, pH 7.5 (50 mM), NaCl (200 mM), ZnAc (50 μM), 0.1%
βME, and leupectin/aprotin protease inhibitors. Protein was purified from
For determination of caspase-9 activity, the caspase-9 substrate
Z-LEHD-AFC (25 μM) (BioVision Inc.) was added. Fluorescence detec-
tion of substrate cleavage by caspase-9 for the substrate was carried out on
an ULTRA READER using an excitation wavelength of 400 nm and an
emission wavelength of 505 nm. The reaction was monitored for 1ꢀ2 h.
BiotinꢀStreptavidin Pull-Down Experiment. MDA-MB-231
cells were lysed in NETN buffer (50 mM Tris (pH 8), 1 mM EDTA,
150 mM NaCl, 0.5% NP-40). Lysates were precleared with streptavi-
dinꢀagarose beads to remove nonspecifically adsorbed proteins. Pre-
cleared lysates were incubated with biotinylated compound 4 for 1 h,
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followed by incubation with new streptavidinꢀagarose beads for 2 h to
pull-down proteins bound to compound 4. Beads were washed with
NETN buffer, and proteins were eluted by boiling in SDS-PAGE sample
buffer and analyzed by Western blotting using specific antibodies.
DMSO alone was used as a control for nonspecific pull-down. In the
competition experiments, precleared cell lysates were incubated with
different concentrations of compound 2 for 30 min, followed by
incubation with biotinylated 4 and streptavidinꢀagarose beads.
mice (SCID mice from Charles River), two tumors (left and right sides)
per mouse. Mice bearing MDA-MB-231 xenograft tumors were admi-
nistered with a single dose of compound 2 in its HCl salt form at 100
mg/kg via oral gavage. Blood and tumor samples were collected from
each mouse by terminal cardiac puncture at 0.25, 0.5, 1, 2, 4, 6, 8, 24 h
postdose. Samples were taken from three mice at each time point. Blood
samples were collected into potassium heparin treated tubes and
centrifuged at 2000g and 4 ꢀC for 10 min. Plasma was collected and
stored at ꢀ80 ꢀC prior to analysis. Isolated tumor tissues were imme-
diately frozen and ground with a mortar and pestle in liquid nitrogen,
then stored at ꢀ80 ꢀC until analysis.
Cell Growth Inhibition and Cell-Death Assays. The MDA-
MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines were
purchased from the American Type Culture Collection (ATCC). Cells were
seeded in 96-well flat bottom cell culture plates at a density of (3ꢀ4) ꢁ 103
cells/well with compounds and incubated for 4 days. The rate of cell growth
inhibition after treatment with different concentrations of the inhibitors was
determined by assaying with (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophe-
nyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8;
Dojindo Molecular Technologies Inc., Gaithersburg, MD). WST-8 was
added to each well to a final concentration of 10%, and then the plates were
incubated at 37 ꢀC for 2ꢀ3 h. The absorbance of the samples was measured
at 450 nm using a TECAN ULTRA reader. Concentration of the compou-
nds that inhibited cell growth by 50% (IC50) was calculated by comparing
absorbance in the untreated cells and the cells treated with the compounds.
A trypan blue exclusion assay was used for determination of cell
viability. Cells were treated with compounds at different concentrations and
time-points. Cell viability was determined using the trypan blue exclusion
assay. Blue cells or morphologically unhealthy cells were scored as dead cells.
At least 100 cells from each treatment, performed in triplicate, were counted.
Western Blot Analysis. Cells and xenograft tumor tissues were
lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (PBS
containing 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium
dodecyl sulfate [SDS]) supplemented with 1 μM phenylmethylsulfonyl
fluoride and 1 protease inhibitor cocktail tablet per 10 mL on ice for 20 min,
and lysates were then cleared by centrifugation prior to protein concentra-
tion determination using the Bio-Rad protein assay kit according to the
manufacturer’s instructions. Proteins were electrophoresed onto 18% SDS-
PAGE gels (Invitrogen, Grand Island, NY) and transferred to PVDF
membranes. Following blocking in 5% milk, membranes were incubated
with a specific primary antibody, washed, and incubated with horseradish
peroxidase linked secondary antibody (Amersham, Piscataway, NJ). The
signals were visualized with the chemiluminescent HRP antibody detection
reagent (Denville Scientific, Metuchen, NJ). When indicated, the blots were
stripped and reprobed with a different antibody.
The quantitative LC/MS/MS analyses were conducted using an
Agilent 1200 HPLC system coupled to an API 3200 mass spectrometer
(Applied Biosystems, MDS Sciex Toronto, Canada) equipped with an
API electrospray ionization (ESI) source. Mobile phase A and mobile
phase B were water and methanol, and both contained 5 mM ammo-
nium acetate and 0.1% formic acid (v/v). The flow gradient was initially
90:10 v/v of A/B for 2 min, linearly ramped to 0:100 over 4 min, held at
0:100 for 2 min, and then returned to 90:10 over 0.1 min. This condition
was held for a further 5 min prior to the injection of another sample. The
mass spectrometer was operated in ESI positive ion mode, and detection
of the ions was performed in the multiple reaction monitoring (MRM)
mode, monitoring the transition of m/z 562.3 precursor ion [M þ H]þ
to the m/z 167.1 product ion for compound 2 and m/z 610.2 precursor
ion [M þ H]þ to the m/z 167.1 product ion for IS using an analogue of
compound 2 (SM-408). The ion spray voltage was set at 5500 V.
Ionization temperature was set at 700 ꢀC. The instrument parameters,
curtain gas, gas 1, and gas 2 (auxiliary gas), were set at 20, 60 and 60,
respectively. Compounds parameters, declustering potential (DP),
collision energy (CE), entrance potential (EP), collision entrance
energy (CEP), and collision exit potential (CXP), were 61, 45, 5, 26,
4 V and 61, 45, 9, 27.3, 4 V for compound 2 and the IS, respectively. Data
acquisition and quantitation were performed using Analyst software,
version 1.4.2 (Applied Biosystems, MDS Sciex Toronto, Canada).
Compound 2 was dissolved in 50% acetonitrile, and the IS solution
was prepared by dissolving the IS in acetonitrile to yield a final
concentration of 100 ng/mL. Plasma samples were prepared by mixing
50 μL of plasma with 10 μL of 50% acetonitrile and 140 μL of IS solution.
The solution was vortexed for 1 min at high speed and centrifuged at
13 000 rpm for 10 min to precipitate protein. The clear supernatants were
transferred to vial inserts for LC/MS/MS analysis. Ten calibration standard
solutions for constructing standard curve were prepared by mixing 10 μL of
compound 2 at 2.5, 5, 25, 50, 250, 500, 2500, 5000, 10000, 25000 ng/mL
with 50 μL of blank plasma and 140 μL of IS solution. To prepare tumor
samples, about 60ꢀ80 mg of ground tumor powder was weighed and mixed
with PBS to obtain final concentrations (w/v) of 200 mg/mL. Samples were
homogenized using a tissue homogenizer (Tissuemiser homogenizer, Fisher
Scientific) for 20 s. Then 10 μL of 50% acetonitrile and 90 μL IS solution
were added and vortexed for 1 min at high speed. The tubes were centrifuged
at 13 000 rpm for 10 min to precipitate protein. The clear supernatants were
transferred to vial inserts for analysis. Calibration standard solutions for
constructing standard curve were prepared by mixing 10 μL of compound
2 at 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 ng/mL with 20 μL of
blank tumor homogenate and 90 μL of IS solution. Plasma and tumor
concentrationꢀtime data were analyzed using noncompartmental pharma-
cokinetic methods with the WinNolin software package (Pharsight Cor-
poration, CA, U.S.). The PK experiments were performed under the
guidelines of the University of Michigan Committee for Use and Care of
Animals.
The following primary antibodies were used in the study: anti-
cleaved-caspase 8, anti-XIAP, anti-PARP (Cell Signaling Technology,
Beverly, MA), anti-cIAP1 (from R&D), anti-cIAP2 (R&D), anti-cas-
pase-3, anti-caspase-9, and anti-procaspase-8 (Stressgen Biotechnolo-
gies, Victoria, Canada) for Western blot analysis.
In Vivo Pharmacodynamic (PD) Studies. For in vivo PD
studies, the MDA-MB-231 xenograft tumor model was employed. To
develop xenograft tumors, 5 ꢁ 106 MDA-MB-231 cancer cells with
Matrigel were injected subcutaneously on the dorsal side of the severe
combined immunodeficient mice (SCID mice from Charles River), one
tumor per mouse. Mice bearing MDA-MB-231 xenograft tumors were
administered with a single dose of compound 2 in its HCl salt form at
100 mg/kg via oral gavage, Taxotere at 7.5 mg/kg intravenously, or
vehicle control. Tumor tissues were harvested at indicated time points.
Tumor tissues were analyzed using Western blotting to examine levels of
cIAP1 and XIAP, caspase-8 processing, and PARP cleavage in tumor
tissues. All animal experiments were performed under the guidelines of
the University of Michigan Committee for Use and Care of Animals.
In Vivo Pharmacokinetic Studies in Plasma and MDA-MB-
231 Tumor Tissues in SCID Mice. To develop xenograft tumors,
5 ꢁ 106 MDA-MB-231 cancer cells with Matrigel were injected subcuta-
neously on the dorsal side of the severe combined immunodeficient
In Vivo Antitumor Efficacy Study. SCID mice (8ꢀ10 per
group) bearing MDA-MB-231 xenograft tumors were treated with
different doses of compound 2, or 7.5 mg/kg of Taxotere or vehicle
control daily, 5 days a week for 2 weeks. Tumor sizes and animal weights
were measured 3 times a week during the treatment and twice a week
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Journal of Medicinal Chemistry
ARTICLE
after the treatment. Data are presented as mean tumor volumes ( SEM.
Statistical analyses were performed by two-way ANOVA and unpaired
two-tailed t test, using Prism (version 4.0, GraphPad, La Jolla, CA). P <
0.05 was considered statistically significant. The efficacy experiment was
performed under the guidelines of the University of Michigan Commit-
tee for Use and Care of Animals.
Pharmacokinetics of Compound 2 in Rats, Dogs, and Non-
Human Primates. Pharmacokinetic (PK) studies in male Spragueꢀ
Dawley rats, beagle dogs, and cynomolgus monkeys (non-human primates)
were performed by the Division of Pharmacokinetics and Metabolism,
Shanghai Medicilon Inc., Shanghai 201203, P. R. China.
Compound 2 in its hydrochloride salt form was used in pharmaco-
kinetic (PK) evaluations and was dissolved in saline to yield a final
concentration at 25 mg/mL (pH ≈ 7). The solution was administered to
animals on preparation. The concentration of compound 2 in dosing
solution was confirmed by HPLC.
For PK studies in rats, dogs, and monkeys, animals were randomly
assigned to the treatment groups and were carotid cannulated before the PK
studies. The LC system comprised an Agilent (Agilent Technologies Inc.,
U.S.) liquid chromatograph equipped with an isocratic pump (1100 series),
an autosampler (1100 series), and a degasser (1100 series). Mass spectro-
metric analysis was performed using an API3000 (tripleꢀquadruple) instru-
ment from AB Inc. (Canada) with an ESI interface. The data acquisition and
control system were created using Analyst 1.4 software from ABI Inc. The
concentrations in plasma below the limit of quantitation (LOQ = 5 ng/mL)
were designated as zero. The pharmacokinetic data analysis was performed
using noncompartmental analysis. Oral bioavailability was calculated as
F(%) = {[dose (oral) ꢁ AUC(0ꢀ¥) (oral)]/[dose (iv) ꢁ AUC(0ꢀ¥)
(iv)]} ꢁ 100.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone:þ1 734 6150362. Fax:þ1 734 6479647. E-mail: shaomeng@
umich.edu.
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Present Addresses
^Guangzhou Institute of Biomedicine and Health, Chinese
Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou Science
Park, Guangzhou 510663, China.
#Drug Design and Synthesis Section, Chemical Biology Research
Branch, National Institute on Drug Abuse, National Institutes of
Health, 5625 Fishers Lane, Bethesda, Maryland 20892, Unites States.
Author Contributions
These authors contributed equally.
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’ ACKNOWLEDGMENT
We are grateful for the financial support from the Breast
Cancer Research Foundation, the National Cancer Institute
(Grant R01CA109025), Ascenta Therapeutics, and University
of Michigan Cancer Center Core grant from the National Cancer
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