A potent bivalent Smac mimetic (SM-1200) achieving rapid, complete, and durable tumor regression in mice

Article abstract of DOI:10.1021/jm400216d

We have designed, synthesized, and evaluated a series of new compounds based upon our previously reported bivalent Smac mimetics. This led to the identification of compound 12 (SM-1200), which binds to XIAP, cIAP1, and cIAP2 with K_i values of 0.5, 3.7, and 5.4 nM, respectively, inhibits cell growth in the MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines with IC₅₀ values of 11.0 and 28.2 nM, respectively. Compound 12 has a much improved pharmacokinetic profile over our previously reported bivalent Smac mimetics and is highly effective in induction of rapid and durable tumor regression in the MDA-MB-231 xenograft model. These data indicate that compound 12 is a promising Smac mimetic and warrants extensive evaluation as a potential candidate for clinical development.

Full text of DOI:10.1021/jm400216d

Article
pubs.acs.org/jmc
A Potent Bivalent Smac Mimetic (SM-1200) Achieving Rapid,
Complete, and Durable Tumor Regression in Mice
Rong Sheng,†,∥ Haiying Sun,† Liu Liu,† Jianfeng Lu,† Donna McEachern,† Guanfeng Wang,‡,§
Jianfeng Wen,‡,§ Ping Min,‡,§ Zhenyun Du,‡,§ Huirong Lu,‡,§ Sanmao Kang,‡,§ Ming Guo,‡,§
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
‡Yasheng Biomedical Inc., Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, China
§Ascentage Pharma (Jiangsu), Medical City Avenue, QB3 Building First Floor, Taizhou, Jiangsu Province, China
ABSTRACT: We have designed, synthesized, and evaluated a
series of new compounds based upon our previously reported
bivalent Smac mimetics. This led to the identification of
compound 12 (SM-1200), which binds to XIAP, cIAP1, and
cIAP2 with Ki values of 0.5, 3.7, and 5.4 nM, respectively, inhibits
cell growth in the MDA-MB-231 breast cancer and SK-OV-3
ovarian cancer cell lines with IC50 values of 11.0 and 28.2 nM,
respectively. Compound 12 has a much improved pharmacoki-
netic profile over our previously reported bivalent Smac mimetics
and is highly effective in induction of rapid and durable tumor regression in the MDA-MB-231 xenograft model. These data
indicate that compound 12 is a promising Smac mimetic and warrants extensive evaluation as a potential candidate for clinical
development.
domains. As a consequence, bivalent Smac mimetics are much
more effective than monovalent Smac mimetics in antagonizing
XIAP containing both BIR2 and BIR3 domains in cell-free
functional assays.34−36 In addition to their ability to antagonize
XIAP, both monovalent and bivalent Smac mimetics potently
bind to cIAP1 and cIAP2 and induce rapid degradation of
cIAP1/2 in cells.15,16 While both types of Smac mimetics
effectively kill tumor cells in some human cancer cell lines,
bivalent Smac mimetics can be >100 times more potent than
their corresponding monovalent Smac mimetic analogues.30−36
One major advantage for monovalent Smac mimetics as
therapeutic agents is their favorable pharmaceutical properties
stemming from their low molecular weights.14−17 Indeed, well
designed monovalent Smac mimetics, including those in Figure
1, can achieve oral bioavailability.14−17 In comparison, bivalent
Smac mimetics such as those shown in Figure 2, have molecular
weights at least twice that of monovalent Smac mimetics and
are not orally bioavailable.
INTRODUCTION
■
X-linked inhibitors of apoptosis protein (XIAP) and cellular
IAP1 (cIAP1) and cIAP2 are critical regulators of apoptosis and
attractive new cancer therapeutic targets.1−8 The second
mitochondria-derived activator of caspases (Smac), also
known as the direct IAP binding protein with low pI
(DIABLO), is an endogenous antagonist of IAP proteins.9,10
Crystal structures11−13 reveal that the interaction between
Smac and the IAP proteins XIAP, cIAP1, and cIAP2 is
mediated by the AVPI tetra-peptide binding motif in Smac
protein and a well-defined surface binding groove in these IAP
proteins. In the past few years, various laboratories, including
ours, have intensely pursued the design of small molecules
(Smac mimetics), which can mimic the interaction between
Smac and these IAP proteins, as new cancer therapeutic
agents.14−36
Two types of Smac mimetics, monovalent and bivalent Smac
mimetics, have been designed.14−17 Monovalent Smac mimetics
are designed to mimic a single AVPI binding motif, while
bivalent Smac mimetics contain two small-molecule mimics of
the AVPI binding motif, tethered through a linker.14−17 While
monovalent Smac mimetics can effectively and potently
antagonize the inhibition by XIAP BIR3 protein of caspase-9
activity, they are less effective in antagonizing the inhibition of
caspase-3 by XIAP protein containing both BIR2 and BIR3
domains.34−36 In comparison, bivalent Smac mimetics are much
more potent than the corresponding monovalent Smac
mimetics in binding to XIAP containing both BIR2 and BIR3
To date, four monovalent and two bivalent Smac mimetics
have been advanced into clinical development for the treatment
of human cancers.14,15 For example, the orally active
monovalent Smac mimetic, SM-406/AT-406 designed in this
laboratory, is in phase I/II clinical trials as a new anticancer
drug.18
Received: February 11, 2013
Published: May 7, 2013
© 2013 American Chemical Society
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Figure 1. Chemical structures of previously reported monovalent Smac mimetics.
Figure 2. Chemical structures of previously reported bivalent Smac mimetics.
Because bivalent Smac mimetics are much more effective
than monovalent Smac mimetics in antagonizing XIAP and
inducing apoptosis against tumor cells, we have sought to
develop bivalent Smac mimetics for cancer treatment and have
designed, synthesized, and evaluated a series of bivalent Smac
mimetics.30−34 Although a number of highly potent Smac
mimetics were obtained from our previous efforts, none of
them was found to be suitable for advanced preclinical
development. For example, while 8 (SM-164) effectively
inhibited tumor growth and in fact induced partial tumor
regression at its maximum tolerated dose (MTD) in the MDA-
MB-231 xenograft model in mice, it failed to achieve complete
tumor regression at its MTD.34 Our pharmacokinetic (PK)
study of compound 8 with intravenous administration in rats
also revealed that it has a very low AUC (area-under-curve) in
the plasma, indicating a poor PK profile (Table 1). These data
indicated that further improvements are clearly needed toward
identification of a suitable bivalent Smac mimetic for clinical
development.
Toward our goal of identifying a suitable bivalent Smac
mimetic for clinical development, we report herein the design,
synthesis, and evaluation of a series of new bivalent Smac
mimetics. This study has led to the identification of a promising
new Smac mimetic (12, SM-1200). Compound 12 has a much
improved PK profile over compound 8 and is capable of
achieving rapid, complete, and durable tumor regression at a
well-tolerated dose-schedule in mice.
CHEMISTRY
■
The synthesis of the newly designed compounds 12−20 is
shown in Scheme 1.
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Table 1. Binding Affinities to XIAP, cIAP1, and cIAP2 and Cell Growth Inhibition in the MDA-MB-231 and SK-OV-3 Cell
a
Lines of Our Designed Smac Mimetics
binding affinities
cell growth inhibition
XIAP (linker-BIR2-BIR3)
cIAP1 (BIR3)
cIAP2 (BIR3)
IC50 (nM) Ki (nM)
compd
IC50 (nM)
Ki (nM)
IC50 (nM)
Ki (nM)
IC50 (nM) (MDA-MB-231)
IC50 (nM) (SK-OV-3)
10
11
12
13
14
15
16
17
18
19
20
21
22
11.5 3.2
24 13
2
0.6
2
17.4 1.9
12.2 1.7
21.7 2.4
41.7 5.8
27.4 3.0
28.4 1.6
10.1 0.9
30.6 1.3
32.7 4.3
22.9 3.8
48.6 8.8
28.5 2.5
74.6 2.1
2.9 0.4
3.6 0.3
3.7 0.6
7.4 1.1
4.7 0.6
4.9 0.3
1.5 0.2
5.4 0.3
5.8 0.8
3.9 0.7
8.7 1.7
5.0 0.4
13.7 0.4
52
43
6
5
14
11
2
1
3.4 0.6
9.6
5
50.8 8.5
180
4.2 1.9
9.6 1.9
4.1 1.3
5.4 0.8
1.1 0.1
9.5 0.8
19.7 4.4
4.6 0.4
4.1 1.5
7.3 1.6
8.5 1.4
<1
23.3 3.7
48.9 2.0
31.4 4.0
39.5 1.6
11.1 0.6
39.8 0.4
43.4 1.7
30.1 1.4
51.7 2.0
32.2 1.0
94.8 1.4
5.4 1.0
12.6 0.5
7.7 1.1
9.9 0.4
2.1 0.2
10.0 0.1
11.0 0.4
7.4 0.4
13.3 0.6
7.9 0.2
25.3 0.4
11
55
12
3
2
7
4
1
28
4
1.7 0.5
<1
176
8
65 11
35 11
0.7 0.2
<1
0.9 0.2
25
460 90
11
3
1.7 0.2
4.5 1.3
<1
4
73 15
670 90
18
5
4
1
51
18
9
2
<1
1.3 0.4
1.5 0.3
380 113
222 50
759 179
406 11
a
Standard deviation (SD) was obtained from 3−5 independent experiments. Data for compounds 10 and 11 were taken from ref 36.
a
Scheme 1. Synthesis of Bivalent Smac Mimetics 12−20
a
Reagents and conditions: (a) (i) bis-isoyanate, CH2Cl2 or DMF, N,N-diisopropylethyl amine; (ii) 4N HCl in 1,4-dioxane, methanol.
a
Scheme 2. Synthesis of Bivalent Smac Mimetics 21 and 22
a
Reagents and conditions: (a) (i) 1,4-benzenediacetic acid or 1,4-phenylenedipropionic acid, EDC, HOBt, CH2Cl2, N,N-diisopropylethyl amine; (ii)
4N HCl in 1,4-dioxane, methanol.
a
Table 2. Pharmacokinetics of Compounds 8, 10, 12, and 16 in Rats with Intravenous Administration
compd
dosing (iv) (mg/kg)
AUC (h·μg/L)
T1/2 (h)
Vz (L/kg)
CL (L/h/kg)
8
1
221 58
885 275
3771 216
457 19
0.14 0.05
0.85 0.14
4.9 3.9
1.0 0.6
1.5 0.7
0.5 0.3
2.0 0.2
4.7 1.3
1.2 0.5
0.6 0.1
5.4 0.2
10
12
16
1
2.5
2.5
1.4 0.1
a
AUC, area-under-curve; T1/2, half elimination time; Vz, apparent volume of distribution during terminal phase; CL, apparent total body clearance of
the drug from plasma.
The key intermediate 23 can be synthesized according to our
previously reported method.36 Reaction of 23 with bis-
isocyanates furnished a series of ureas, and removal of the
Boc protecting groups from these ureas provided our designed
Condensation of 23 with 1,4-benzenediacetic acid or 1,4-
phenylenedipropionic acid yielded the corresponding bisa-
mides. Removal of the Boc protecting group in these amides
gave compounds 21 and 22.
bivalent Smac mimetics 12−20.
The synthesis of the newly designed compounds 21 and 22 is
RESULTS AND DISCUSSION
■
We previously reported the design of a class of bivalent Smac
mimetics based upon our monovalent Smac mimetic 2, as
shown in Scheme 2.
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Figure 3. Chemical structures of compound 11 and new bivalent Smac mimetics.
exemplified by compound 1036 (Figure 2). Compound 10
binds to XIAP, cIAP1, and cIAP2 with high affinities, (Ki = 2.0,
2.9, and 14 nM, respectively) and potently inhibits cell growth
in the MDA-MB-231 breast cancer cell line and other cancer
cell lines sensitive to Smac mimetics. Pharmacokinetic
evaluation of compound 10 showed that it has an improved
PK profile over compound 8 (Table 2), suggesting that further
modifications of compound 10 may yield a promising bivalent
Smac mimetic for advanced preclinical development and
clinical trials.
High flexibility of their linkers is a characteristic common to
compounds 8 and 10, and this may affect their pharmacoki-
netics in animals. We have therefore synthesized compound
1136 (Figure 3), which has a rigid linker. This compound binds
to XIAP, cIAP1, and cIAP2 with Ki = 5, 3.6, and 11 nM,
respectively (Table 1). Although compound 11 has Ki values
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similar to those of compound 10, 11 has IC50 values of 51 and
139 nM in inhibition of cell growth in the MDA-MB-231 breast
cell line under the same assay conditions (Table 1), being >10
times less potent than 10. Therefore, the cellular activity of
compound 11 needed to be improved. Since our previous study
showed that the linker in bivalent Smac mimetics plays a key
role in cellular activity by modulating the cell permeability,35 we
have focused our modifications on the linker in compound 11
in an attempt to improve its cellular potency. The binding
affinities of our newly synthesized compounds to XIAP, cIAP1,
and cIAP2 were determined using our optimized competitive
fluorescence-polarization (FP) assays.35−37 Their cell growth
inhibitory activity was assessed against the MDA-MB-231
breast cancer and SK-OV-3 cell lines, which are sensitive to
Smac mimetics.35,36 The data obtained from binding and cell
growth experiments are provided in Table 1.
In a first step, both of the amide groups in the linker of
compound 11 were replaced with a urea group, which resulted
in compound 12. Compound 12 binds to XIAP, cIAP1, and
cIAP2 with Ki values of <1, 3.7, and 5.4 nM, respectively, and is
thus more potent than 11. Compound 12 has IC50 values of 11
and 28 nM, respectively, against the MDA-MB-231 breast
cancer and SK-OV-3 ovarian cancer cell lines and is 5−6 times
more potent than 11 in these cancer two cell lines.
Upon the basis of these promising data for compound 12, we
next made additional modifications on the linker to further
explore the structure−activity relationship in both binding
affinities and cellular activity (Figure 3 and Table 1). Moving
the linker from 1,4 positions on the phenyl ring in 12 to 1,3
positions led to compound 13, which binds to XIAP, cIAP1,
and cIAP2 proteins with high affinities but is only half as potent
as compound 12 and 5−6 times less potent than compound 12
in inhibition of cell growth in the MDA-MB-231 and SK-OV-3
cancer cell lines. Extension of the linker in compound 13 by
two extra carbon atoms resulted in compound 14, which is as
potent as compound 12 in binding to XIAP, cIAP1, and cIAP2
as well as in inhibition of cell growth in the MDA-MB-231 and
SK-OV-3 cancer cell lines. Replacement of the phenyl group in
compound 12 with 4,4′-oxidibenzyl yielded compound 15,
which binds to these IAP proteins with the same affinities as
compound 12. This compound (15) is 4 times more potent
than 12 in inhibition of cell growth in the MDA-MB-231 cell
line, but both 15 and 12 are essentially equipotent in the SK-
OV-3 cell line. Replacement of the phenyl group in compound
12 with 4,4′-1,2-diphenylethane resulted in compound 16,
which has a higher binding affinity to XIAP than compounds 12
and 15 based upon their IC50 values but has the same high
binding affinities to cIAP1 and cIAP2 as compared to 12 and
15. Compound 16 has an IC50 value of 0.9 nM in inhibition of
cell growth in the MDA-MB-231 cell line and is thus >10 times
more potent than 12 but is only 2 times more potent than 12 in
the SK-OV-3 cell line.
To further investigate if the conformational flexibility of the
linker plays a role in binding and/or cellular activity, we
designed and synthesized compound 19, in which a linear and
conformationally flexible butyl group was used to the replace
the 1,4-disubstituted rings in 11−13. Compounds 19 and 12
have the same binding affinities to XIAP and cIAP1/2 proteins
and essentially the same IC50 values in inhibition of cell growth
in the MDA-MB-231 and SK-OV-3 cancer cell lines, suggesting
that the flexible linker in compound 19 is not detrimental to
binding to these IAP proteins and to the activity in cell growth
inhibition. However, compounds with flexible linkers may not
have good pharmacokinetics in animals.
In view of the high binding affinities to these IAP proteins
and potent cellular activity for compound 19, we next
synthesized compound 20 with an n-octyl linker, four carbons
longer than the linker in 19. Compound 20 has the same high
binding affinities as 12 and 19 to the three IAP proteins.
However, compound 20 is 3 and 2 times more potent than
compound 19 in inhibition of cell growth in the MDA-MB-231
and SK-OV-3 cancer cell lines, respectively. The cellular data
for compounds 19 and 20 are consistent with our previous
results that more hydrophobic linkers in general improve the
cellular activity of bivalent Smac mimetics.35
To confirm that the urea groups in the linker indeed improve
the cell permeability with respect to the amide groups, we
synthesized compounds 21 and 22 in which the linker in
compound 11 has been extended by extra two or four carbons
to make the linker more hydrophobic. Although compounds 21
and 22 bind to the three IAP proteins with high affinities and
are only slightly less potent than compound 12, they are >10
times less potent than 12 in inhibition of cell growth in both
the MDA-MB-231 and SK-OV-3 cancer cell lines. These data
show that compounds with the urea groups in the linker indeed
have superior cell permeability to similar analogues with the
amide groups.
Our previous study has shown that bivalent Smac mimetics
are much more potent in antagonizing XIAP than monovalent
Smac mimetics. We evaluated compounds 12 and 16 for their
functional antagonism against XIAP containing linker-BIR2-
BIR3 domains and included our monovalent Smac mimetic 1
and bivalent Smac mimetic 8 as the controls. Our data showed
that bivalent Smac mimetics 8, 12, and 16 all potently and
effectively antagonize XIAP in both caspase-9 and caspase-3
functional assays (Figure 4). While compounds 8, 12, and 16
have similar potencies, they are much more potent than
monovalent Smac mimetic 1 in antagonizing XIAP both
caspase-9 and caspase-3 functional assays, consistent with our
previous data.
We investigated the cellular mechanism of action of
compound 12 and 16 by Western blot analysis, with compound
8 included as a positive control compound (Figure 5).
Consistent with their high binding affinities to cIAP1 and
potent cell growth inhibitory activity, compounds 8, 12, and 16
each effectively induce the degradation of cIAP1 in the MDA-
MB-231 cell line at very low concentrations. Compound 12 at
concentrations as low as 1 nM and compounds 8 and 16 at
concentrations as low as 0.3 nM induce clear cIAP1
degradation and also dose-dependently induce cleavage of
caspase-8, caspase-3, and PARP (poly ADP ribose polymerase),
indicative of robust apoptosis induction by these Smac
mimetics.
Changing the phenyl group in compound 12 into a 1,4-trans-
cyclohexyl group yielded compound 17. Compound 17 is
approximately 2-fold less potent than compound 12 in binding
to XIAP, cIAP1, and cIAP2 and is also 2 times less potent than
12 in inhibition of cell growth in both the MDA-MB-231 and
SK-OV-3 cancer cell lines. Compound 18, with a shorter linker
than that in 12, is 5 times less potent than 12 in binding to
XIAP and both 12 and 18 have similar binding affinities to
cIAP1 and cIAP2. Compound 18 is >20 times less potent than
12 in inhibition of cell growth in both the MDA-MB-231 and
SK-OV-3 cancer cell lines.
Compounds 8, 12, and 16 were evaluated for their ability to
induce cell death by trypan blue assay in the MDA-MB-231
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Figure 6. Induction of cell death by compounds 8, 12, and 16 in the
MDA-MB-231 breast cancer cell line. MDA-MB-231 cancer cells were
treated with different concentrations of compounds 8, 12, and 16 for
24 h, and cell death was analyzed by trypan blue assay. * (p < 0.05), **
(p < 0.01), *** (p < 0.001), **** (p < 0.001).
compound 8 at 10 nM, 12 at 10 nM, and 16 at 3 nM induced
>75% of the MDA-MB-231 cells to undergo cell death.
Among these new compounds, compound 16 is the most
potent based upon its activity in inhibition of cell growth in the
MDA-MB-231 and SK-OV-3 cell lines, whereas compound 12
has a rigid linker and also potent cellular activity. We therefore
selected these two compounds for evaluation of their
pharmacokinetics (PK) in rats. While compound 12 has a
significantly improved PK profile over compound 8 based upon
their AUC values and clearance rates (p < 0.01), compound 16
has the worst pharmacokinetics among these four compounds
(Table 2).
Figure 4. Functional antagonism of Smac mimetics against XIAP
(linker-BIR2-BIR3 domains) in cell-free, caspase-9, and caspase-3
functional assays. (a). Caspase-9 functional assay. XIAP L-BIR2-BIR3
effectively inhibited caspase-9 activity, and Smac mimetics dose-
dependently restored the activity of caspase-9. (b). Caspase-3
functional assay. XIAP L-BIR2-BIR3 effectively inhibited caspase-3
activity and Smac mimetics dose-dependently restored the activity of
caspase-3.
Upon the basis of the encouraging PK data, we next
evaluated the antitumor activity of compound 12 in the MDA-
MB-231 xenograft model in immunodeficient mice and
included docetaxel as a positive control agent (Figure 7).
Docetaxel at 7.5 mg/kg effectively inhibited tumor growth but
failed to achieve tumor regression. In contrast, compound 12 at
both 3 and 10 mg/kg weekly dosing induced rapid tumor
regression. After a single dose, the tumors regressed on average
by 55% with 3 mg/kg of compound 12 and by 88% with 10
mg/kg of compound 12. After four weekly doses, the tumors
regressed by 93% on average with 3 mg/kg of compound 12
and by 99% with 10 mg/kg of compound 12. After treatment
with compound 12 at 10 mg/kg weekly for 4 weeks, 6 out of 7
mice had no measurable tumor. The tumor regression was also
durable. At day 76, 27 days after the last dose, 5 out of the 7
mice treated with 10 mg/kg of 12 remained tumor-free and 2 of
the 7 mice treated with 12 at 3 mg/kg were tumor-free. Our
statistical analysis showed that compound 12 at both doses was
statistically more effective than docetaxel at 7.5 mg/kg (Table
3). Importantly, compound 12 at both doses was well tolerated
and caused minimal weight loss in mice. In comparison, our
previous study showed that while compound 8 effectively
inhibited tumor growth in the MDA-MB-231 xenograft model
in mice at 5 mg/kg (iv), 5 days a week for 2 weeks, it failed to
achieve complete and durable tumor regression.34 These data
showed that compound 12 is highly effective in induction of
rapid tumor regression at well-tolerated doses.
Figure 5. Induction of cIAP1 degradation, cleavage of PARP, and
processing of caspase-3, caspase-9, and caspase-8 by compounds 8, 12,
and 16 in the MDA-MB-231 cell line. Cells were treated with different
concentrations of compounds at the indicated concentrations for 24 h.
cIAP1, cleaved PARP (CL PARP), caspase-8 (C8) and cleaved
caspase-8 (CL-C8), caspase-9 (C9) and cleaved caspase-9 (CL-C9),
and caspase-3 (C3) and cleaved caspase-3 (CL-C3) were probed by
Western blot analysis.
To elucidate the underlying in vivo mechanism of action for
compound 12, we performed a pharmacodynamic study in
mice bearing the MDA-MB-231 xenograft tumors (Figure 8).
Western blot analysis showed that a single dose of compound
12 at 5 mg/kg induced rapid, complete, and sustained
degradation of cIAP1 in tumor tissues. At the 2 h time-point
after dosing, the levels of cIAP1 protein in tumor tissues
breast cancer cell line (Figure 6). All these three compounds
potently and effectively induce cell death in the MDA-MB-231
cancer cells in a dose-dependent manner. In a 24 h treatment,
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Figure 8. Pharmacodynamic studies of compound 12 in the MDA-
MB-231 xenograft tumors in mice. SCID mice bearing MDA-MB-231
xenograft tumors were treated with compound 12 at 5 mg/kg
intravenously. Mice were sacrificed at indicated time-points, and tumor
tissue lysates were examined by Western blot analysis with specific
antibodies against cIAP1, XIAP, caspase-3 (C3), PARP, and cleaved
PARP. GAPDH was used as the loading control.
of full-length of XIAP occurs after robust activation of caspase-
3,38 caspase-3 is probably responsible for the decreased levels of
full-length XIAP. Because XIAP is an efficient inhibitor of
caspase-9 and caspase-3/7, the decreased levels of XIAP can
further enhance the activation of caspases, leading to a positive
feedback on apoptosis induction by compound 12.
Figure 7. Antitumor activity of compound 12 in the MDA-MB-231
breast cancer xenograft model in mice. Tumors were grown to an
average size of 100 mm3, and compound 12 or docetaxel (TXT) was
administered intravenously at the indicated dose and schedule. (A)
Tumor growth. (B) Animal body weight.
SUMMARY
■
With the goal of identifying a potent and efficacious bivalent
Smac mimetic for advanced preclinical development and
clinical trials, we have performed modifications on compound
11, a bivalent Smac mimetic we had previously reported, with a
focus on the linker region. Our results showed that replacement
of the two amide groups in the linker region with urea groups
yielded new bivalent Smac mimetics with high binding affinities
to these IAP proteins and potent cellular activity. For example,
compound 16 binds to these IAP proteins with Ki values of
0.5−2.1 nM and inhibits cell growth in the MDA-MB-231
breast cancer and SK-OV-3 ovarian cancer cell lines with IC50
values of 0.9 and 11.1 nM, respectively. Compound 12 binds to
XIAP, cIAP1, and cIAP2 with Ki values of 0.5, 3.7, and 5.4 nM,
respectively and inhibits cell growth in the MDA-MB-231
breast cancer and SK-OV-3 ovarian cancer cell lines with IC50
values of 11.0 and 28.2 nM, respectively. Although compound
12 is less potent than compound 16 in inhibition of cell growth
in the MDA-MB-231 and SK-OV-3 cancer cell lines, compound
12 has a much better PK profile than 16, as well as compounds
8 and 10, and was subjected to extensive evaluations in animals.
Our in vivo testing showed that compound 12, administered
weekly is highly effective in induction of rapid and durable
tumor regression in the MDA-MB-231 xenograft model.
Compound 12, with 10 mg/kg weekly dosing for 4 weeks,
achieved 99% of tumor regression and 6 out of 7 mice
remained tumor free 27 days after the final dose. In
comparison, while our previous reported bivalent Smac
compound 8 was very effective in inhibition of tumor growth
in the MDA-MB-231 xenograft model and in fact induced
partial tumor regression with daily dosing for 2 weeks, it failed
to achieve complete tumor regression. Although these two
Table 3. Statistical Analysis of the Efficacy Data Shown in
Figure 7
P value (t test)
P value (t test)
median tumor volume in the median tumor volume in the
control group reached 750
mm3 (day 44)
TXT treated group reached
750 mm3 (day 76)
control vs 12
0.0004
0.0001
0.0005
0.1719
(3 mg/kg)
control vs 12
(10 mg/kg)
control vs
TXT
12 (3 mg/kg)
vs 12 (10
mg/kg)
0.1247
12 (3 mg/kg)
0.0667
0.018
0.0107
0.003
vs TXT
12 (10
mg/kg) vs
TXT
became undetectable and the effect persisted for at least 24 h.
Robust cleavage of both caspase-3 and PARP was observed
starting from the 4 h time-point, indicative of strong apoptosis
induction. Although the amount of cleaved PARP was less at
the 24 h time-point than at the 4 and 8 h time-points, the levels
of full-length PARP became very low, suggesting that strong
apoptosis was still occurring at the 24 h time-point.
Interestingly, there was a clear decrease in the levels of XIAP,
starting at 8 h time-point and lasting through the 24 h time-
point. Because active caspase-3 cleaves XIAP and the decrease
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2.14−2.02 (m, 6H), 1.80−1.73 (m, 4H), 1.56 (d, J = 6.9 Hz, 6H). ESI
MS: m/z 1207.3 (M + H)+.
compounds have similar potencies in induction of cell death in
the MDA-MB-231 cell line, compound 12 is much more
efficacious than compound 8 in vivo. Our in vivo pharmacody-
namic study showed that compound 12 induces rapid and
persistent cIAP1 degradation, consistent with its mechanism of
action as a Smac mimetic. In line with its ability to induce rapid
tumor regression, robust induction of apoptosis was evident
with a single dose of compound 12 at 5 mg/kg in the MDA-
MB-231 tumor tissues based upon strong cleavage of PARP and
caspase-3. Taken together, these data indicate that compound
12 is a promising bivalent Smac mimetic and warrants extensive
evaluations as a candidate for clinical development.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(Methylenebis(4,1-
phenylene))bis(N8-benzhydryl-5-((S)-2-(methylamino)-
propanamido)-6-oxooctahydropyrrolo[1,2-a][1,5]diazocine-
3,8(4H)-dicarboxamide) (16). Yield 51% over two steps. Purity was
1
determined by reverse phase analytical HPLC to be over 95%. H
NMR (300 MHz, CD3OD): δ 7.48 (d, J = 8.1 Hz, 4H), 7.33 (m,
20H), 7.11 (d, J = 8.1 Hz, 4H), 6.17 (s, 2H), 4.82 (m, 2H), 4.63 (m,
2H), 4.25 (m, 2H), 4.08−4.03 (m, 6H), 3.88 (s, 2H), 3.30−3.20 (m,
4H), 2.70 (s, 6H), 2.34 (m, 2H), 2.20−2.04 (m, 6H), 1.75−1.60 (m,
4H), 1.55 (d, J = 6.9 Hz, 6H). ESI MS: m/z 1206.4 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(Cyclohexane-1,4-diyl)bis-
(N8-benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxooctahydropyrrolo[1,2-a][1,5]diazocine-3,8(4H)-dicarboxamide)
(17). Yield 43% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz,
CD3OD): δ 7.34−7.23 (m, 20H), 6.14 (s, 2H), 4.92 (m, 2H), 4.70 (m,
4H), 4.08−3.86 (m, 8H), 3.59 (m, 2H), 3.16−3.05 (m, 4H), 2.70 (s,
6H), 2.36 (m, 2H), 2.10−1.92 (m, 10H), 1.79−1.71 (m, 4H), 1.53−
1.45 (m, 8H). ESI MS: m/z 1121.7 (M + H)+.
EXPERIMENTAL SECTION
■
1
1. Chemistry. General Methods. H NMR spectra were acquired
at 300 MHz and 13C spectra at 75 MHz. 1H chemical shifts are
reported with CHD2OD (3.31 ppm) or HDO (4.70 ppm) as internal
standards. The final products were purified by C18 reverse phase
semipreparative HPLC column with solvent A (0.1% of TFA in H2O)
and solvent B (0.1% of TFA in CH3CN) as eluents. The purity of all
the final compounds was confirmed by analytical HPLC to be >95%.
General Synthesis of Bivalent Smac Mimetics. N,N-Diisopropy-
lethyl amine (3 equiv) was added to a solution of 23 (1 equiv) and
corresponding bis-isocyanate (0.5 equiv) in CH2Cl2 or DMF (15 mL
per mmol of 23). The solution was stirred at room temperature
overnight then concentrated, and the residue was purified by
chromatography to give a urea. HCl (4N in 1,4-dioxane, 2 mL per
mmol of bis-triazole) was added to a solution of this urea in MeOH (5
mL per mmol of bis-triazole). The solution was stirred at room
temperature overnight and then concentrated to furnish a crude
product, which was purified by C18 reversed phase semipreparative
HPLC to give a bivalent Smac mimetic.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-(Piperazine-1,4-dicarbonyl)bis-
(N-benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide) (18).
Yield 41% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 95%. H NMR (300 MHz, CD3OD): δ
7.33−7.21 (m, 20H), 6.12 (s, 2H), 5.11 (m, 2H), 4.84 (m, 2H), 4.56
(t, J = 8.4 Hz, 2H), 4.25 (m, 2H), 3.93 (m, 2H), 3.66−3.53 (m, 6H),
3.22−3.15 (m, 8H), 2.67 (s, 6H), 2.34 (m, 2H), 2.15−1.96 (m, 4H),
1.83−1.77 (m, 6H), 1.54 (d, J = 6.9 Hz, 6H). ESI MS: m/z 1093.7 (M
+ H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(Butane-1,4-diyl)bis(N8-
benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxooctahydropyrrolo[1,2-a][1,5]diazocine-3,8(4H)-dicarboxamide)
(19). Yield 63% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz, D2O): δ
7.40−7.20 (m, 10H), 5.99 (s, 2H), 4.75 (m, 2H), 4.45 (m, 2H), 4.10
(m, 2H), 3.95 (m, 2H), 3.80 (m, 2H), 3.65 (m, 2H), 3.25−3.05 (m,
8H), 2.62 (m, 6H), 2.30 (m, 2H), 2.20−1.70 (m, 12H), 1.45 (m, 2H),
1.40 (d, J = 7.2 Hz, 6H). ESI MS: m/z 1095.4 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(Octane-1,8-diyl)bis(N8-
benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxooctahydropyrrolo[1,2-a][1,5]diazocine-3,8(4H)-dicarboxamide)
(20). Yield 65% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz,
CD3OD): δ 7.37−7.24 (m, 20H), 6.16 (s, 2H), 4.72−4.60 (m, 4H),
4.10 (m, 2H), 4.00−3.85 (m, 6H), 3.25−3.04 (m, 8H), 2.69 (s, 6H),
2.34 (m, 2H), 2.14−2.03 (m, 6H), 1.77−1.48 (m, 8H), 1.54 (d, J = 6.9
Hz, 6H), 1.35 (m, 8H). ESI MS: m/z 1151.8 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-(2,2′-(1,4-Phenylene)bis-
(acetyl))bis(N-benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide) (21).
1,4-Benzenediacetic acid (10 mg, 0.051 mmol), EDC (25 mg, 0.13
mmol), HOBt (15 mg, 0.11 mmol), and 0.3 mL of N,N-
diisopropylethylamine were added to a solution of 23 (58 mg, 0.1
mmol) in 5 mL of CH2Cl2. The solution was stirred at room
temperature overnight and then concentrated. The residue was
purified by chromatography to yield an amide. HCl solution (4N in
1,4-dioxane, 2 mL) was added to a solution of this amide in MeOH (5
mL). The solution was stirred at room temperature overnight and then
concentrated to furnish a crude product which was purified by C18
reversed phase semipreparative HPLC to give 87 mg of 21 as a salt
with TFA, yield 65% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz, CD3OD)
7.35−7.17 (m, 24H), 6.15 (s, 2H), 4.82 (m, 2H), 4.59 (m, 2H), 4.25
(m, 2H), 4.01−3.75 (m, 10H), 3.45 (m, 2H), 3.22 (m, 2H), 2.66 (s,
6H), 2.31 (m, 2H), 2.05−1.78 (m, 10H), 1.52 (d, J = 7.2 Hz, 6H). ESI
MS: m/ z 1113.7 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-((1S,4S)-cyclohexane-1,4-
diyl)bis(N8-benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxooctahydropyrrolo[1,2-a][1,5]diazocine-3,8(4H)-dicarboxamide)
(12). Yield 63% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz,
CD3OD): δ 7.53 (s, 4H), 7.37 (m, 20H), 6.18 (s, 2H), 4.84 (m, 2H),
4.67 (t, J = 8.4 Hz, 2H), 4.27 (m, 2H), 4.09 (m, 6H), 3.30−3.20 (m,
4H), 2.71 (s, 6H), 2.37 (m, 2H), 2.24−2.06 (m, 6H), 1.81−1.72 (m,
4H), 1.56 (d, J = 6.9 Hz, 6H). ESI MS: m/z 1115.9 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(1,3-Phenylene)bis(N8-benz-
h y d r y l - 5 - ( ( S ) - 2 - ( m e t h y l a m i n o ) p r o p a n a m i d o ) - 6 -
oxooctahydropyrrolo[1,2-a][1,5]diazocine-3,8(4H)-dicarboxamide)
(13). Yield 56% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz,
CD3OD): δ 8.14 (s, 1H), 7.34−7.18 (m, 23H), 6.17 (s, 2H), 4.84 (m,
2H), 4.67 (t, J = 8.4 Hz, 2H), 4.22 (m, 2H), 4.07 (m, 6H), 3.24 (m,
4H), 2.73 (s, 6H), 2.34 (m, 2H), 2.14−2.04 (m, 6H), 1.77−1.66 (m,
4H), 1.57 (d, J = 6.9 Hz, 6H). ESI MS: m/z 1115.9 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(1,3-Phenylenebis-
(methylene))bis(N8-benzhydryl-5-((S)-2-(methylamino)-
propanamido)-6-oxooctahydropyrrolo[1,2-a][1,5]diazocine-
3,8(4H)-dicarboxamide) (14). Yield 62% over two steps. Purity was
1
determined by reverse phase analytical HPLC to be over 95%. H
NMR (300 MHz, CD3OD): δ 7.36−7.15 (m, 24H), 6.15 (s, 2H), 4.84
(m, 2H), 4.63−4.53 (m, 4H), 4.32−4.14 (m, 4H), 3.99−3.81 (m, 6H),
3.16−3.06 (m, 4H), 2.63 (s, 6H), 2.34 (m, 2H), 2.18−2.85 (m, 6H),
1.85−1.60 (m, 4H), 1.50 (d, J = 7.2 Hz, 6H). ESI MS: m/z 1143.67
(M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-N3,N3′-(Oxybis(4,1-phenylene))bis-
(N8-benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxooctahydropyrrolo[1,2-a][1,5]diazocine-3,8(4H)-dicarboxamide)
(15). Yield 49% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz,
CD3OD): δ 7.55 (d, J = 9.0 Hz, 4H), 7.36−7.24 (m, 20H), 6.91 (d, J =
9.0 Hz, 4H), 6.17 (m, 2H), 4.84 (m, 2H), 4.64 (t, J = 8.1 Hz, 2H),
4.23 (m, 2H), 4.09 (m, 6H), 3.21 (m, 4H), 2.71 (s, 6H), 2.34 (m, 2H),
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-(3,3′-(1,4-Phenylene)bis-
(propanoyl))bis(N-benzhydryl-5-((S)-2-(methylamino)-
propanamido)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-car-
boxamide) (22). 1,4-Phenylenedipropionic acid (11 mg, 0.05 mmol),
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EDC (25 mg, 0.13 mmol), HOBt (15 mg, 0.11 mmol), and 0.3 mL of
N,N-diisopropylethylamine were added to a solution of 23 (58 mg, 0.1
mmol) in CH2Cl2 (5 mL). The solution was stirred at room
temperature overnight and then concentrated. The residue was
purified by chromatography to yield an amide. HCl solution (4N in
1,4-dioxane, 2 mL) was added to a solution of this amide in MeOH (5
mL). The solution was stirred at room temperature overnight and then
concentrated to furnish a crude product which was purified by C18
reversed phase semipreparative HPLC to give 84 mg of 21 as a salt
with TFA, yield 61% over two steps. Purity was determined by reverse
phase analytical HPLC to be over 95%. 1H NMR (300 MHz, CD3OD)
7.35−7.07 (m, 24H), 6.15 (s, 2H), 4.73 (m, 2H), 4.51 (m, 2H), 4.22
(m, 2H), 3.95−3.63 (m, 6H), 3.52 (m, 2H), 3.22 (m, 2H), 3.05−2.84
(m, 6H), 2.71−2.64 (m, 8H), 2.27 (m, 2H), 1.95−1.61 (m, 10H), 1.53
(d, J = 7.2 Hz, 6H). ESI MS: m/ z 1141.7 (M + H)+
mice were injected subcutaneously with 5 × 106 MDA-MB-231 cells in
50% Matrigel per mouse, one tumor per mouse. Treatment started on
day 28 after inoculation of tumor cells when the tumors reached an
average volume of 100 mm3. Mice were treated with vehicle weekly
(nine mice per group), docetaxel (TXT) at 7.5 mg/kg weekly
intravenously (seven mice per group), compound 12 at 3 or 10 mg/kg
weekly intravenously (seven mice per group). Tumor sizes and animal
weights were measured 3 times a week during the treatment and twice
a week 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.
For pharmacodynamic experiments, xenograft tumors (one tumor
per mouse) were developed by injection of 5 × 106 MDA-MB-231
cancer cells with Matrigel, subcutaneously, on the dorsal side of the
SCID mice (from Charles River). When tumors grew to approximately
100 mm3 in volume, mice bearing tumors were given a single dose of
compound 12 at 5 mg/kg or vehicle intravenously. Mice were
sacrificed at different time points and tumor tissues were harvested for
Western blot analysis to determine levels of cIAP1 and XIAP, as well
as for the cleavage of PARP and caspase-3.
2. Fluorescence Polarization Based Assays for XIAP, cIAP1,
and cIAP2 Proteins. Different sensitive and quantitative fluorescence
polarization (FP) based assays were used to determine the binding
affinities of the designed Smac mimetics to XIAP (linker-BIR2-BIR3),
cIAP1 (BIR3), and cIAP2 (BIR3) proteins. These assays were
described in our previous publications.35,36
The in vivo experiments were performed under the guidelines of the
University of Michigan Committee for Use and Care of Animals.
8. Pharmacokinetic Studies in Rats. Pharmacokinetic (PK)
studies of bivalent Smac mimetics were performed in male Sprague−
Dawley rats (body weight: 250−270 g) by Medicilon Inc., Shanghai
201203, P. R. China. Before the pharmacokinetic studies, animals were
carotid cannulated.
3. Caspase-9 and Caspase-3 Functional Assays. Cell-free
functional assays were employed to determine the functional
antagonism of our designed Smac mimetics. These assays have been
described previously in detail.35
4. Cell Growth Inhibition Assay. The MDA-MB-231 and SK-
OV-3 cell lines were purchased from the American Type Culture
Collection. Cells were seeded in 96-well flat bottom cell culture plates
at a density of 3−4 × 103 cells/well and grown overnight, then
incubated with compounds at different concentrations. The rate of cell
growth inhibition after treatment with different concentrations of a
compound was determined by assaying with (2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
monosodium salt (WST-8; Dojindo Molecular Technologies Inc.,
Gaithersburg, Maryland) which was added to each well to a final
concentration of 10%, and then the plates were incubated at 37 °C for
2−3 h. The absorbance of the samples was measured at 450 nm using
a TECAN ULTRA reader. The concentrations of the compounds that
inhibited cell growth by 50% (IC50) were calculated by comparing
absorbance in the untreated cells and the treated cells.
5. Cell Death Analysis. The MDA-MB-231 cells were treated with
compounds at different concentrations for 24 h. 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.
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.
6. Western Blot Analysis. Cells were harvested and washed with
cold PBS. Cell pellets were lysed in double lysis buffer (DLB; 50
mmol/L Tris, 150 mmol/L sodium chloride (1 mmol/L EDTA, 0.1%
SDS and 1% NP-40)) in the presence of PMSF (1 mmol/L) and
protease inhibitor cocktail (Roche) for 10 min on ice, then centrifuged
at 13000 rpm at 4 °C for 10 min. Protein concentrations were
determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories).
Proteins were electrophoresed onto a 4−20% gradient SDS-PAGE
(Invitrogen) and then transferred to PVDF membranes. After blocking
in 5% milk, the membranes were incubated with a specific primary
antibody, washed, and incubated with horseradish peroxidase−linked
secondary antibody (Amersham). The signals were visualized with a
Chemiluminescent HRP antibody detection reagent (Denville
Scientific). When indicated, the blots were stripped and reprobed
with a different antibody. Primary antibody against cleaved caspase 3
was purchased from Stressgen Biotechnologies, primary antibodies
against cIAP1 and cIAP2 were purchased from R&D Systems, primary
antibody against XIAP was purchased from BD Biosciences, and
primary antibodies against PARP and β-actin from Cell Signaling
Technology.
A stock solution of a Smac mimetic was prepared by dissolving the
drug in methanol to yield a final concentration of 200 μg/mL. An
aliquot of this solution was diluted using methanol to get a series of
working solutions of 25, 5, 2.5, 0.5, 0.25, 0.05, and 0.025 μg/mL. Seven
calibration standard solutions containing 5000, 1000, 500, 100, 50, 10,
and 5 ng/mL were obtained by adding 20 μL of working solution
prepared above into seven Eppendorff tubes containing 100 μL of
blank plasma. QC samples were prepared by spiking 100 μL of blank
plasma with 20 μL of diluted solutions containing 20, 4, 0.04 μg/mL of
analyte to get the final concentration of 4000, 800, and 8 ng/mL.
Each compound was injected into the tail vein in a group of three
male rats with intravenous bolus injection. Blood samples were
collected from carotid vein at predose and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8,
and 24 h postdose. Plasma samples were stored at −20 °C until
bioanalysis. Plasma samples were processed by protein precipitation.
Plasma samples (0.1 mL) were transferred to Eppendorff tube, then 20
μL of methanol, 50 μL of IS solution (1 μg/mL) and 1 mL of
acetoacetic ester were added to it. After vortexing for 5 min and
centrifuging for 3 min at 15000 rpm, 1 mL of supernatant was
transferred to another glass tube and evaporated under vacuum
condition. The residue was reconstituted with 100 μL of mobile phase
then 5 μL for injection.
The LC system comprised an Agilent (Agilent Technologies Inc.
USA) liquid chromatograph equipped with an isocratic pump (1100
series), an autosampler (1100 series), and a degasser (1100 series).
Mass spectrometric analysis was performed using an API3000 (triple-
quadruple) instrument from AB Inc. (Canada) with an ESI interface.
Data acquisition and control system were created using Analyst 1.4
software from ABI Inc. Concentrations in plasma below the limit of
quantitation (LOQ = 5 ng/mL) were designated as zero.
Pharmacokinetic data analysis was performed using noncompartmental
analysis.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 734-615-0362. Fax: 734-647-9647. E-mail:
shaomeng@umich.edu.
Present Address
∥For R.S.: ZJU-ENS Joint Laboratory of Medicinal Chemistry,
Zhejiang University, Hangzhou 310058, China.
7. In Vivo Antitumor Efficacy and Pharmacodynamics
Studies. For the efficacy experiment, female nude immunodeficient
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Notes
The authors declare the following competing financial
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has licensed the technology described in this manuscript from
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(24) Sun, H.; Stuckey, J. A.; Nikolovska-Coleska, Z.; Qin, D.;
Meagher, J. L.; Qiu, S.; Lu, J.; Yang, C. Y.; Saito, N. G; Wang, S.
Structure-based design, synthesis, evaluation, and crystallographic
studies of conformationally constrained Smac mimetics as inhibitors of
the X-linked inhibitor of apoptosis protein (XIAP). J. Med. Chem.
2008, 51, 7169−7180.
(25) Peng, Y.; Sun, H.; Nikolovska-Coleska, Z.; Qiu, S.; Yang, C.-Y.;
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ACKNOWLEDGMENTS
■
We are grateful for the financial support from the Breast Cancer
Research Foundation, the National Cancer Institute, NIH
(R01CA109025 and R01CA127551), the Susan G. Komen
Foundation, Ascentage Pharma Group, and University of
Michigan Cancer Center Core grant from the National Cancer
Institute, NIH (P30CA046592), “Key New Drug Creation”
project of the major science and technology program, China
(2012ZX09401005), National “863” Grant, China
(2012AA020305), International Cooperation Project of the
Ministry of Science and Technology of China
(2010DFB34090), Jiangsu Provincial Science and Technology
Innovation Team Grant, China (BE2010760), and Jiangsu
Provincial Key Laboratory Project grant (BM2012114). We
thank Dr. G.W.A. Milne for his critical reading of the
manuscript and many useful suggestions and Karen Kreutzer
for her excellent secretarial assistance.
ABBREVIATIONS USED
■
IAP, inhibitor of apoptotic protein; XIAP, X-linked IAP; cIAP,
cellular IAP; Smac, second mitochondria-derived activator of
caspases; BIR, baculoviral IAP repeat; PARP, poly(ADPribose)
polymerase; FP, fluorescence polarization; mP, millipolarization
units
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