Nonpeptidic and potent small-molecule inhibitors of cIAP-1/2 and XIAP proteins

Article abstract of DOI:10.1021/jm100487z

A series of compounds were designed and synthesized as antagonists of cIAP-1/2 and XIAP based upon our previously identified lead compound SM-122 (1). The most potent of these (7) binds to XIAP, cIAP-1, and cIAP-2 proteins with K_i values of 36, <1, and <1.9 nM, respectively. Consistent with its potent binding affinities to IAPs, 7 effectively antagonizes XIAP in a cell-free caspase-9 functional assay, efficiently induces cIAP-1 degradation in cells at concentrations as low as 10 nM, and triggers activation of caspases and PARP cleavage in the MDA-MB-231 breast cancer cell line. Compound 7 potently inhibits cell growth in the MDA-MB-231 cancer cell line with an IC₅₀ value of 200 nM and is 9 times more potent than compound 1.

Full text of DOI:10.1021/jm100487z

J. Med. Chem. 2010, 53, 6361–6367 6361
DOI: 10.1021/jm100487z
Nonpeptidic and Potent Small-Molecule Inhibitors of cIAP-1/2 and XIAP Proteins
Haiying Sun,† Jianfeng Lu,† Liu Liu,† Han Yi,† Su Qiu,† Chao-Yie Yang,† Jeffrey R. Deschamps,‡ and Shaomeng Wang*,†
†Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry, University of Michigan,
1500 E. Medical Center Drive, Ann Arbor, Michigan 48109, and ‡Naval Research Laboratory, 4555 Overlook Avenue, Washington, D.C. 20375
Received April 20, 2010
A series of compounds were designed and synthesized as antagonists of cIAP-1/2 and XIAP based upon
our previously identified lead compound SM-122 (1). The most potent of these (7) binds to XIAP, cIAP-1,
and cIAP-2 proteins with Ki values of 36, <1, and <1.9 nM, respectively. Consistent with its potent
binding affinities to IAPs, 7 effectively antagonizes XIAP in a cell-free caspase-9 functional assay,
efficiently induces cIAP-1 degradation in cells at concentrations aslow as10 nM, and triggers activation of
caspases and PARP cleavage in the MDA-MB-231 breast cancer cell line. Compound 7 potently inhibits
cell growth in the MDA-MB-231 cancer cell line with an IC50 value of 200 nM and is 9 times more potent
than compound 1.
Introduction
constrained, nonpeptidic Smac mimetic.20 It potently binds to
XIAP, cIAP-1, and cIAP-2, efficiently inhibits cell growth,
and induces cell death in a number of cancer cell lines, but the
subsequent pharmacokinetic (PK) studies indicated that it
has a less-than-ideal PK profile. In order to improve the
PK property, we modified its [8,5] bicyclic core structure to
produce SM-337 (Figures 1 and 2),25 in which carbon 5 of the
eight-membered ring of 1 was replaced with a nitrogen atom
to which a phenylacetyl group was attached through an amide
bond. Compound 2 binds to XIAP, cIAP-1, and cIAP-2
proteins with higher affinities than 1 and is more potent than
1 in cell growth inhibition in the MDA-MB-231 cell line.25 It
also has an improved pharmacokinetic profile and is orally
bioavailable,25 suggesting that modifications of the eight-
membered ring in 1 can produce new Smac mimetics with
improved in vitro and in vivo properties. In this paper, we
report the design, synthesis, and evaluation of a series of new
Smac mimetics inwhich a substituted group was introduced to
the eight-membered ring in 1. Our efforts have led to novel
Smac mimetics that bind to cIAP-1/2 and XIAP with high
affinities and are more potent than compound 1 in inhibition
of cancer cell growth and in induction of apoptosis.
Inhibitor of apoptotic proteins (IAPsa) are a class of key
regulators of apoptosis characterized by one to three baculo-
virus IAP repeat domains.1,2 Among these, cIAP-1 and cIAP-
2 inhibit death receptor mediated apoptosis3,4 while XIAP, by
binding to effectors caspase-3 and -7 and an initiator caspase-
9 and inhibiting the activities of these three caspases, blocks
both death receptor-mediated and mitochondria-mediated
apoptosis.5 These three IAPs each have three baculoviral
IAP repeat (BIR) domains. The BIR3 domain of XIAP binds
to caspase-9 and the BIR2 domain together with the linker
preceding it binds to caspase-3 and -7.6 Biological studies
haveindicated thatcIAP-1/-2and XIAPconferon cancercells
resistance to various anticancer drugs and, as a consequence,
strategies targeting these IAPs have potential as novel anti-
cancer therapies.7
The second mitochondria derived activator of caspases
(Smac) is an endogenous inhibitor of these IAPs8,9 and
interacts with IAP proteins via its N-terminal AVPI tetra-
peptide motif.10,11 Smac antagonizes cIAP-1 and cIAP-2
by binding to the BIR3 domain of these two proteins and
inducing their rapid degradation.12,13 In comparison, Smac
protein, in a dimeric form, binds concurrently to both the
BIR2 and BIR3 of XIAP and removes the inhibition of XIAP
to caspase-9 and caspase-3/7.14,15 The AVPI tetrapeptide of
Smac has been used as a lead structure for the design of both
peptidic and nonpeptidic small-molecule Smac mimetics as
antagonists of IAP proteins.16-21 The design and synthesis of
bivalent Smac mimetics to mimic Smac dimer have also been
reported.22,23
Results and Discussion
Our data for compound 2 suggest that introduction of a
polar group into the eight-membered ring region in com-
pound 1 may have significant effect on binding affinities of the
resulting compounds to XIAP, cIAP-1, and cIAP-2 proteins,
as well as on their cellular activities and in vivo properties. To
further explore the structure-activity relationship in this
region, we have therefore introduced a hydroxyl group on
the eight-membered ring in compound 1 totest the influencein
binding and cellular activity of the resulting compounds.
Compounds 3 and 4 in which a hydroxyl group is attached
to carbon 6 of the eight-membered ring in compound 1 were
therefore synthesized. In a fluorescence polarization (FP)
based binding assay, 3 and 4 are equipotent in binding to
XIAP, cIAP-1, and cIAP-2 BIR3 proteins and only slightly
less potent than 1, showing that the introduction of a hydroxyl
We have previously reported the design, synthesis, and
evaluation of SM-122 (1 in Figure 1) as a conformationally
*To whom correspondence should be addressed. Phone: (734) 615-
0362. Fax: (734) 647-9647. E-mail: shaomeng@umich.edu.
a Abbreviations: IAP, inhibitor of apoptotic protein; XIAP, X-linked
IAP; cIAP, cellular inhibitor of apoptotic protein; Smac, second mito-
chondria derived activator of caspases; BIR, baculoviral inhibitor of
apoptotic protein repeat; PK, pharmacokinetic; PARP, poly(ADP-ribose)
polymerase; FP, fluorescence polarization; mP, millipolarization units.
r
2010 American Chemical Society
Published on Web 08/04/2010
pubs.acs.org/jmc

6362 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Sun et al.
group to carbon 6 of the eight-membered ring of 1 is not
detrimental to the binding to these IAP proteins (Table 1).
We then designed compounds 5 and 6 by introducing a phenyl-
acetyl group at carbon 6 with two different configurations at
the new chiral center. In the FP-based binding assay, the cis
isomer 5 has Ki values of 108, 1.6, and 4.2 nM to XIAP, cIAP-
1, and cIAP-2, respectively, and is thus equipotent with 2 and
2-5 times more potent than 1. The trans isomer 6 is 2-3 times
less potent than 1, indicating that the cis configuration is more
favorable for binding to the BIR3 domains of these IAPs.
On the basis of the more potent compound 5, we then
synthesized 7 by replacement of the diphenylmethyl group in 5
with an R-1-tetrahydronaphthyl group. Previously, we have
found that this modification can improve both binding and
cellular activities.20 Indeed, 7 binds to XIAP with a Ki value of
36 nM and is 5 times more potent than 1. It also binds potently
to cIAP-1 and cIAP-2 with Ki < 1 nM and Ki < 1.9 nM,
respectively, being 10 times more potent than 1. Thus, com-
pound 7 is the most potent compound in this series.
Compounds 5, 6, and 7wereevaluatedtogetherwith1 and 2
for their ability to antagonize XIAP in a cell-free caspase-9
functional assay (Figure 2). In this assay, XIAP BIR3 pro-
tein dose-dependently inhibits the activity of caspase-9 and, at
500 nM, achieves 80% inhibition. All of these new Smac
mimetics 5, 6, and 7 can dose-dependently restore the activity
of caspase-9. Compound 5 is as potent as 1 and 2, restoring
60% of caspase-9 activity at 5 μM, while 6 is about 3 times less
potent than 1, 2, or 5. Consistent with its more potent binding
affinity to XIAP BIR3, 7 is 3 times more potent than 1,
restoring 60% of caspase-9 activity at 1.5 μM.
Our previous study showed that 1 and 2 can effectively
inhibit cell growth and induce apoptosis in the MDA-MB-231
human breast cancer cell line.25 Therefore, compounds 5, 6,
and 7, together with 1 and 2, were evaluated for their ability to
inhibit cell growth in the MDA-MB-231 cancer cell line
(Figure 3). It was found that in this assay, 5 is as potent as 2
and 3 times more potent than 1. Although 6 is less potent than
1 in binding to XIAP, cIAP-1, and cIAP-2, it is slightly more
Figure 1. Structures of Smac mimetics.
Figure 2. Smac mimetics antagonize XIAP BIR3 in a cell-free
caspase-9 functional assay. 500 nM XIAP BIR3 protein achieves
80% inhibition of caspase-9 activity in Caspase-Glo 9 assay kit, and
Smac mimetics dose-dependently restore the activity of caspase-9.
Caspase-9 activity was measured after incubation with the caspase-9
specific substrate for 1 h.
Figure 3. Inhibition of cell growth by Smac mimetics in the MDA-
MB-231 cancer cell line. Cells were seeded in 96-well flat-bottom cell
culture plates at a density of (3-4) ꢀ 102 cells/well and grown
overnight, then incubated with Smac mimetics for 4 days. Cell
growth was determined using a WST-based assay.
Table 1. Binding Affinities of Smac Mimetics to XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3 Proteins
XIAP BIR3 cIAP-1 BIR3
cIAP-2 BIR3
compd
IC50 ( SD (nM)
Ki ( SD (nM)
IC50 ( SD (nM)
Ki ( SD (nM)
IC50 ( SD (nM)
Ki ( SD (nM)
1
2
3
4
5
6
7
635.7 ( 50.7
369.7 ( 187.3
1068 ( 89.3
1102 ( 98.3
361.0 ( 86.0
1017 ( 95.0
125.0 ( 9.6
190.7 ( 15.2
110.9 ( 56.2
320.3 ( 26.8
330.6 ( 29.5
108.0 ( 26.1
305.2 ( 28.5
36.0 ( 2.9
46.2 ( 8.3
9.0 ( 2.8
6.7 ( 1.2
1.3 ( 0.4
7.9 ( 0.6
6.6 ( 0.5
1.6 ( 0.6
12.2 ( 2.4
<1
74.7 ( 12.1
26.3 ( 6.3
91.8 ( 13.2
99.6 ( 12.3
19.1 ( 6.2
195.8 ( 10.4
5.3 ( 1.5
18.3 ( 2.9
6.3 ( 1.5
22.5 ( 3.1
24.8 ( 2.9
4.2 ( 1.7
47.0 ( 2.5
<1.9
54.5 ( 4.1
45.5 ( 3.4
10.9 ( 4.2
84.1 ( 16.6
2.6 ( 0.3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6363
Figure 4. Induction of cIAP-1 degradation, cleavage of PARP, and processing of caspase-3 by compounds 1, 2, 5, and 7 in the MDA-MB-231
cell line. Cells were treated with different concentrations of the compounds for 24 h, and levels of cIAP-1, cleaved PAPR (CL PARP), and
cleaved caspase-3 (CL C3) were probed by Western blot analysis.
Scheme 1. Synthesis of Key Intermediatesa
a Reagents and conditions: (a) 9-BBN, THF, then H2O2 (35% in water), 3 N NaOH; (b) Dess-Martin periodinane, CH2CI2, 18% for 12, and 66% for
13; (c) Dess-Martin periodinane, CH2CI2, 86%; (d) NaBH3CN, MeOH, H2SO4 (cat.), 89%.
potent than 1 in this cell growth assay with an IC50 of 1.1 μM.
The most potent compound 7 achieves an IC50 of 0.2 μM and
is 9 times more potent than 1.
analysis (Figure 5). Oxidation of the mixture of the other three
isomers by Dess-Martin periodinane yielded two ketones, 12
and 13, which can be separated by chromatography. Oxida-
tion of 9 under the same conditions yielded 12, thus confirming
the structures of both ketones. Reduction of the ketone 12 by
NaBH3CN under the influence of a catalytic amount of H2SO4
furnished alcohol 10 as a single isomer.
Hydrogenolysis of the benzyl ester group in 9 and 10
followed by condensation of the resulting two acids with
aminodiphenylmethane afforded 15 and 16 (Scheme 2). Re-
moval of the Boc protecting group from 15 or 16 followed by
condensation of the resulting ammonium salts with L-N-Boc-
N-methylalanine afforded two amides. Removal of the Boc
protecting groups in these two amides yielded 3 and 4.
Condensation of the acid from hydrogenolysis of the benzyl
ester group in 10 with (R)-1,2,3,4-tetrahydronaphthyl-1-
amine furnished 17. Transformation of the hydroxyl group
in 15, 16, and 17 to an azido group by a Mitsunobu reaction
gave three corresponding azides, 18, 19, and 20. Removal
of the Boc protecting group from these azides followed by
condensation of the resulting ammonium salts with L-N-Boc-
N-methylalanine afforded three azido amides, reducing of the
Several reports have shown that Smac mimetics induce
rapid cIAP-1 degradation in cancer cells and that cIAP-1 is
a direct and critical cellular target for Smac mimetics.12,13,24
We evaluated 1, 2, 5, and 7 for their ability to induce cIAP-1
degradation and cleavage of caspase-3 and poly(ADP-ribose)
polymerase (PARP) in the MDA-MB-231 cell line (Figure 4).
We found that 5 and 7 at 10 nM effectively induce cIAP-1
degradation and are as potent as 2 and more potent than 1.
Compounds 5 and 7 also induce robust cleavage of PARP and
processing of caspase-3, two biochemical markers of apopto-
sis, within 24 h and are more effective than 1 (Figure 4).
Synthesis of these new Smac mimetics is shown in Schemes
1 and 2. Compound 8 was prepared by a published method.26
Hydroboration of the C-C double bond in 8 by reaction with
9-BBN followed by oxidation of the resulting borane by
alkaline H2O2 gave a mixture of four alcohols, two 5-hydroxy
epimers (9 and 10), and two 6-hydroxy epimers (11). The
alcohol 9 was separated from the other three isomers by
chromatography, and its structure was confirmed by X-ray

6364 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Sun et al.
binds to XIAP, cIAP-1, and cIAP-2 with low to subnano-
molar affinities. Compound 7 induces cIAP-1 degradation in
cancer cells with concentrations as low as 10 nM and anta-
gonizes XIAP in a cell-free functional assay. Compound 7
effectively induces processing of caspase-3 and PARP clea-
vage, has an IC50 value of 200 nM in inhibition of cell growth
in the MDA-MB-231 breast cancer cell line, and is 9 times
more potent than the initial lead compound 1. On the basis of
these findings, additional in vitro and in vivo studies for
compound 7 are underway, and the results will be reported
in due course.
Experimental Section
Chemistry. General. NMR spectra were measured at 300
MHz. H chemical shifts are reported relative to HDO (4.79
1
ppm) as the internal standard. Final products were purified
using a C18 reverse phase semipreparative HPLC column with
solvent A (0.1% of TFA in water) and solvent B (0.1% of TFA
in CH3CN) as eluents. All the target compounds (3, 4, 5, 6, and
7) have purities of >95% based upon elemental analysis.
Benzyl (3S,6S,9R,10aR)-6-((tert-Butoxycarbonyl)amino)-9-
hydroxy-5-oxodecahydropyrrolo[1,2-a]azocine-3-carboxylate (9).
To a solution of compound 8 (1.25 g, 3 mmol) in 50 mL of dry
THF was added 12 mL of 9-BBN solution (0.5 M in THF,
6 mmol). After the solution was stirred under N2 at room tempe-
rature for 12 h, 2 mL of 3 M NaOH solution and 3 mL of H2O2
solution (35% in water) were added dropwise at 0 °C. The mix-
ture was warmed to room temperature and stirred for 2 h before
being extracted with ethyl acetate. The combined organic layer
was dried over Na2SO4 and then condensed. The residue was
purified by chromatography to give compound 9 (320 mg, 25%)
and a mixture of three other isomers 10 and 11 (580 mg, 45%).
Figure 5. Crystal structure of the key intermediate 9.
Scheme 2. Synthesis of Smac Mimetics 3-7a
Chemical data for compound 9: [R]2D0 -21.5 (c 1, CHCl3). H
1
NMR (300 MHz, CDCl3): δ 7.40-7.28 (m, 5H), 5.43 (brd, J =
8.0 Hz, 1H), 5.28, 5.18 (ABq, J = 12.2 Hz, 2H), 4.67 (t, J = 9.3
Hz, 1H), 4.65 (m, 1H), 4.20 (m, 1H), 3.96 (m, 1H), 2.45-1.60 (m,
10H), 1.38 (brs, 9H). 13C NMR (75 MHz, CDCl3): δ 172.38,
170.83, 155.13, 135.39, 128.58, 128.40, 128.34, 79.70, 70.63, 67.14,
60.13, 56.16, 50.70, 45.22, 32.67, 31.70, 28.35, 27.34. ESI MS:
m/z 433.3 (M þ H)þ. HR ESI MS for C23H33N2O6 required,
433.2339; found, 433.2339.
Benzyl (3S,6S,10aR)-6-((tert-Butoxycarbonyl)amino)-5,9-dioxo-
decahydropyrrolo[1,2-a]azocine-3-carboxylate (12) and Benzyl
(3S,6S,10aR)-6-((tert-Butoxycarbonyl)amino)-5,8-dioxodeca-
hydropyrrolo[1,2-a]azocine-3-carboxylate (13). To a solution of
the mixture of 10 and 11 obtained above (570 mg, 1.3 mmol) in
15 mL of CH2Cl2 was added Dess-Martin periodinane (660 mg,
1.56 mmol) at room temperature. The mixture was stirred at the
same temperature for 2 h and then condensed. The residue was
purified by chromatography togive compounds12 (102 mg, 18%)
and 13 (370 mg, 66%). Compound9 can be oxidized to compound
12 using the same method. Chemical data for compound 12: [R]D20
-246.6 (c 1, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.40-7.28
(m, 5H), 5.42 (brd, J = 8.2Hz, 1H), 5.28, 5.18 (ABq, J = 12.2 Hz,
2H), 4.62 (dd, J = 9.0, 8.2 Hz, 1H), 4.37 (m, 2H), 3.13 (m, 1H),
3.02 (t, J = 12.0 Hz, 1H), 2.50-1.98 (m, 6H), 1.83 (m, 1H), 1.60
(m, 1H), 1.38 (brs, 9H). 13C NMR (75 MHz, CDCl3): δ 211.37,
172.20, 170.94, 154.92, 135.23, 128.56, 128.41, 128.22, 79.81,
67.17, 60.53, 56.06, 53.39, 52.88, 36.79, 32.36, 30.18, 28.22,
27.00. ESI MS: m/z 431.3 (M þ H)þ. HR ESI MS for C23H31-
N2O6 required, 431.2182; found, 431.2170. Chemical data for
a Reagents and coditions: (a) (i) H2, 10% Pd-C, methanol; (ii) amino-
diphenylmethane, EDC, HOBt, N,N-diisopropylethylamine, CH2CI2;
(b) (i) 4 N HCI in 1,4-dioxane, methanol; (ii) L-N-Boc-N-methylalanine,
EDC, HOBt, N,N-diisopropylethylamine, CH2CI2; (iii) 4 N HCI in 1,4-
dioxane, methanol. (c) (i) H2, 10% Pd-C, methanol; (ii) (R)-1,2,3,4-
tetrahydronaphthyl-1-amine, EDC, HOBt, N,N-diisopropylethylamine,
CH2CI2, 79% over two steps; (d) (i) diethyl azodicarboxylate, DPPA,
PPh3, THF; (e) (i) 4 N HCI in 1,4-dioxane, methanol; (ii) L-N-Boc-
N-methylalanine, EDC, HOBt, N,N-diisopropylethylamine, CH2CI2;
(iii) PPh3, THF-H2O 1:1; (iv) phenylacetyl chloride, N,N-diisopropyl-
ethylamine, CH2CI2; (v) 4 N HCI in 1,4- dioxane, methanol.
azido group in which with triphenylphosphine in 1:1 THF-
H2O yielded the corresponding amines which were condensed
with phenylacetyl chloride to furnish the phenylacetyl amides.
Removal of the Boc protecting group from these amides
provided our designed compounds 5, 6, and 7.
In summary, we have designed and synthesized a series of
new Smac mimetics based on a previously identified nonpeptidic
Smac mimetic compound, 1. The most potent compound 7
1
compound 13: [R]2D0 -66.4 (c 1, CHCl3). H NMR (300 MHz,
CDCl3): δ 7.40-7.20 (m, 5H), 5.49 (brd, J = 7.7 Hz, 1H), 5.17
(s, 2H), 5.09 (m, 1H), 4.52 (t, J = 8.5 Hz, 1H), 4.22 (m, 1H), 3.08
(dd, J = 12.7, 4.5 Hz, 1H), 2.92 (m, 1H), 2.60 (m, 2H), 2.36-1.72
(m, 6H), 1.43 (brs, 9H). 13C NMR (75 MHz, CDCl3): δ 207.72,
170.93, 170.15, 154.74, 135.58, 128.37, 128.30, 128.14, 80.00,
66.67, 60.10, 59.74, 52.13, 48.52, 39.65, 34.18, 32.36, 28.21,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6365
26.90. ESI MS: m/z 431.3 (M þ H)þ. HR ESI MS for C23H31-
N2O6 required, 431.2182; found, 431.2177.
EDC (115 mg, 0.6 mmol), HOBt hydrate (80 mg, 0.6 mmol), and
0.5 mL of N,N-diisopropylethylamine. The mixture was stirred at
room temperature overnight and then condensed. The residue was
purified by chromatography to furnish 17 (186 mg, 79% for two
steps). To a solution of 17 in 20 mL of THF was added diethyl
azodicarboxylate (170 mg, 1 mmol), PPh3 (260 mg, 1 mmol),
and diphenylphosphorylazide (350 mg, 1.3 mmol). The mixture
was stirred at room temperature overnight and then condensed.
The residue was purified by chromatography to yield compound
20 (155 mg, 79%). [R]D20 54.1 (c 1, CHCl3). 1H NMR (300 MHz,
CDCl3): δ 7.32 (m, 1H), 7.15 (m, 2H), 7.06 (m, 1H), 6.77 (brd, J =
8.3 Hz, 1H), 5.43 (brd, J= 8.2 Hz, 1H), 5.16 (m, 1H), 4.54 (m, 1H),
4.48 (t, J = 7.5 Hz, 1H), 4.25 (m, 1H), 3.52 (m, 1H), 2.80 (m, 2H),
2.48-1.50 (m, 14H), 1.42 (brs, 9H). 13C NMR (75 MHz, CDCl3):
δ 171.02, 170.07, 154.94, 137.11, 136.33, 128.98, 128.76, 127.20,
126.23, 79.76, 61.17, 60.35, 56.99, 49.85, 47.47, 42.20, 32.74, 29.84,
29.14, 29.01, 28.25, 26.08, 19.76. ESI MS: m/z 497.3 (M þ H)þ.
HR ESI MS for C26H37N6O4 required, 497.2876; found, 497.2883.
(3S,6S,9R,10aR)-N-Benzhydryl-9-hydroxy-6-((S)-2-(methyl-
amino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide (3). HCl (4 N) in 1,4-dioxane (2 mL) was added
to a solution of 13 (120 mg, 0.23 mmol) in MeOH (5 mL). The
solution was stirred at room temperature overnight and then
concentrated to give an ammonium salt. L-N-Boc-N-methylala-
nine (60 mg, 0.3 mmol), EDC (57 mg, 0.3 mmol), HOBt hydrate
(45 mg, 0.3 mmol), and 0.3 mL of N,N-diisopropylethylamine
were added to a mixture of this salt in CH2Cl2 (10 mL). The mix-
ture was stirred at room temperature overnight and then concen-
trated. The residue was purified by chromatography to yield
an amide to a solution of which in MeOH (5 mL) was added
4 N HCl in 1,4-dioxane (2 mL). The solution was stirred at room
temperature overnight, then concentrated. The residue was puri-
fied by semipreparative HPLC to give 3 as a trifluoroacetate salt.
The gradient used ran from 75% of solvent A and 25% of solvent
B to 55% of solvent A and 45% of solvent B in 40 min. The purity
of the product was confirmed by analytical HPLC to be over 98%.
1H NMR (300 M Hz, D2O) δ 7.35-7.15 (m, 10H), 5.90 (s, 1H),
4.65 (m, 1H), 4.45-4.28 (m, 2H), 4.05 (m, 1H), 3.82 (m, 1H), 2.55
(s, 3H), 2.35-1.45 (m, 10H), 1.42 (d, J=7.2Hz, 3H). ESIMS:m/z
Benzyl (3S,6S,9S,10aR)-6-((tert-Butoxycarbonyl)amino)-9-
hydroxy-5-oxodecahydropyrrolo[1,2-a]azocine-3-carboxylate (10).
To a solution of compound 12 (160 mg, 0.37 mmol) in 15 mL of
methanol was added NaBH3CN (120 mg, 1.9 mmol) and 3 drops
of H2SO4 (98%) at -15 °C. After the mixture was stirred at the
same temperature for 4 h, 10 mL of water was added and the
mixture was extracted with ethyl acetate (30 mL ꢀ 4). The com-
bined organic layers were dried over Na2SO4 and then condensed.
The residue was purified by chromatography to give compound 10
1
(142 mg, 89%). [R]2D0 -66.5 (c 1, CHCl3). H NMR (300 MHz,
CDCl3): δ 7.30 (brs, 5H), 5.45 (brd, J = 8.2 Hz, 1H), 5.20, 5.10
(ABq, J = 12.0 Hz, 2H), 4.85 (m, 1H), 4.48 (m, 2H), 4.13 (m, 1H),
3.16 (brs, 1H), 2.42-1.45 (m, 10H), 1.40 (brs, 9H). 13C NMR
(75MHz, CDCl3):δ172.30, 171.03, 155.06, 135.43, 128.49, 128.27,
128.15, 79.36, 67.60, 66.87, 59.74, 54.00, 51.65, 43.62, 32.12, 31.82,
29.30, 28.29, 27.11. ESI MS: m/z 433.3 (M þ H)þ. HR ESI MS for
C23H33N2O6 required, 433.2339; found, 433.2340.
tert-Butyl ((3S,6S,9R,10aR)-9-Azido-3-(benzhydrylcarbamoyl)-
5-oxodecahydropyrrolo[1,2-a]azocin-6-yl)carbamate (18). To a solu-
tion of compound 10 (430 mg, 1 mmol) in 20 mL of methanol was
added 100 mg of 10% Pd-C. After the mixture was stirred under
H2 overnight, the catalyst was removed and the filtration was
condensed to give an acid. To a solution of this acid in 20 mL of
CH2Cl2 was added aminodiphenylmethane (220 mg, 1.2 mmol),
EDC (230 mg, 1.2 mmol), HOBt (160 mg, 1.2 mmol), and 1 mL
of N,N-diisopropylethylamine. The mixture was stirred at room
temperature overnight and then condensed. The residue was
purified by chromatography to furnish 16 (416 mg, 82% for
two steps). To a solution of 16 (254 mg, 0.5 mmol) in 20 mL
of THF was added diethyl azodicarboxylate (170 mg, 1 mmol),
PPh3 (260 mg, 1 mmol), and diphenylphosphorylazide (350 mg,
1.3 mmol). The mixture was stirred at room temperature over-
night and then condensed. The residue was purified by chromato-
graphy to yield compound 18 (228 mg, 86%). [R]2D0 34.3 (c 0.85,
CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.52 (brd, J = 8.6 Hz,
1H), 7.42-7.18 (m, 5H), 6.21 (d, J = 8.6 Hz, 1H), 5.40 (brd, J =
8.2 Hz, 1H), 4.71 (t, J = 6.7 Hz, 1H), 4.50 (m, 1H), 4.22 (m, 1H),
3.50 (m, 1H), 2.51 (m, 1H), 2.24 (m, 1H), 2.08 (m, 2H), 1.95-1.75
(m, 2H), 1.48-1.20 (m, 13H). 13C NMR (75 MHz, CDCl3): δ
171.50, 169.56, 155.02, 141.58, 141.30, 128.62, 127.52, 127.45,
127.38, 127.15, 79.96, 64.14, 60.55, 60.20, 57.20, 57.08, 49.96,
42.25, 32.83, 29.23, 28.32, 25.03. ESI MS: m/z 533.3 (M þ H)þ.
HR ESI MS for C29H37N6O4 required, 533.2876; found,
533.2874.
tert-Butyl ((3S,6S,9S,10aR)-9-Azido-3-(benzhydrylcarbamoyl)-
5-oxodecahydropyrrolo[1,2-a]azocin-6-yl)carbamate (19). Com-
pound 15 was synthesized using the same method as that
for 16 from compound 9 (85% for two steps), and compound
19 was synthesized using the same method as that for 18 from
15. Chemical data for 19: [R]2D0 3.2 (c 1, CHCl3). 1H NMR
(300 MHz, CDCl3): δ 7.76 (brd, J = 8.6 Hz, 1H), 7.38-7.19
(m, 5H), 6.20 (d, J = 8.6 Hz, 1H), 5.55 (brd, J = 7.7 Hz, 1H),
4.70 (t, J = 7.1 Hz, 1H), 4.60 (m, 1H), 4.31 (m, 1H), 3.35 (m,
1H), 2.61 (m, 1H), 2.20-1.96 (m, 4H), 1.82-1.43 (m, 3H), 1.42
(brs, 9H), 1.41-1.28 (m, 2H). 13C NMR (75 MHz, CDCl3): δ
171.48, 169.50, 154.89, 141.68, 141.21, 128.62, 128.59, 127.52,
127.46, 127.39, 127.14, 79.72, 59.57, 59.10, 57.23, 54.73, 51.68,
39.98, 32.77, 31.51, 28.35, 28.23, 23.95. ESI MS: m/z 533.3
(M þ H)þ. HR ESI MS for C29H37N6O4 required, 533.2876;
found, 533.2879.
493.3 (M þ H)þ. Anal. (C28H36N4O4 1.5CF3COOH) C, H, N.
3
(3S,6S,9S,10aR)-N-Benzhydryl-9-hydroxy-6-((S)-2-(methyl-
amino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-car-
boxamide (4). Compound 4 was synthesized from intermediate 14
by the same method used for 3. The purity was confirmed by
1
analytical HPLC as over 98%. H NMR (300 M Hz, D2O) δ
7.35-7.18 (m, 10H), 5.95 (s, 1H), 4.70 (m, 1H), 4.45-4.32 (m,
2H), 4.05 (m, 1H), 3.85 (m, 1H), 2.58 (s, 3H), 2.32-1.55(m, 10H),
1.42 (d, J = 7.2 Hz, 3H). ESI MS: m/z 493.3 (M þ H)þ. Anal.
(C28H36N4O4 1.3CF3COOH) C, H, N.
3
(3S,6S,9R,10aR)-N-Benzhydryl-6-((S)-2-(methylamino)-
propanamido)-5-oxo-9-(2-phenylacetamido)decahydropyrrolo-
[1,2-a]azocine-3-carboxamide (5). To a solution of 16 (106 mg,
0.2 mmol) in MeOH (5 mL) was added HCl solution (4 N in 1,4-
dioxane, 2 mL). The solution was stirred at room tempera-
ture overnight and then concentrated to give an ammonium salt.
L-N-Boc-N-methylalanine (62 mg, 0.3 mmol), EDC (58 mg,
0.3 mmol), HOBt hydrate (44 mg, 0.3 mmol), and 0.3 mL of N,N-
diisopropylethylamine were added to a mixture of this salt in
CH2Cl2 (10 mL). The mixture was stirred at room temperature
overnight and then concentrated. The residue was purified by
chromatography to yield an amide. Triphenylphosphine (80 mg,
0.3 mmol) and H2O (3 mL) were added to a solution of this amide
in THF (10 mL). The mixture was stirred at room temperature
overnight and then partitioned between CH2Cl2 (60 mL) and
brine (20 mL). The organic layer was dried over Na2SO4 and then
concentrated. The residue was redissolved in CH2Cl2 (10 mL).
Phenylacetyl chloride (0.04 mL) and N,N-diisopropylethylamine
(0.1 mL) were added to this solution, and the mixture was stirred
at room temperature overnight, then concentrated. The residue
was purified by chromatography to furnish a phenylacetylamide.
tert-Butyl ((3S,6S,9R,10aR)-9-Azido-5-oxo-3-(((R)-1,2,3,4-
tetrahydronaphthalen-1-yl)carbamoyl)decahydropyrrolo[1,2-a]-
azocin-6-yl)carbamate (20). To a solution of compound 10
(215 mg, 0.5 mmol) in 20 mL of methanol was added 50 mg
of 10% Pd-C. After the mixture was stirred under H2 over-
night, the catalyst was removed and the filtrate was condensed to
give an acid. To a solution of this acid in 20 mL of CH2Cl2 was
added (R)-1,2,3,4-tetrahydronaphthyl-1-amine (90 mg, 0.6 mmol),

6366 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Sun et al.
HCl (4 N in 1,4-dioxane, 2 mL) was added to a solution of this
amide in MeOH (10 mL), and the solution was stirred at room
temperature overnight, then concentrated. The residue was puri-
fied by semipreparative HPLC to give compound 5 as a salt with
TFA. The gradient used ran from 70% of solvent A and 30% of
solvent B to 50% of solvent A and 50% of solvent B in 40 min.
The purity was confirmed by analytical HPLC to be over 98%.
1H NMR (300 M Hz, D2O) δ 7.10-6.90 (m, 10H), 5.85 (s, 1H),
4.55 (m, 1H), 4.34 (m, 1H), 4.22 (m, 1H), 3.80 (m, 1H), 3.65
(m, 1H), 3.30, 3.20 (ABq, J = 8.4Hz, 2H), 2.55 (s, 3H), 2.10-1.45
(m, 10H), 1.40 (d, J = 7.2 Hz, 3H). ESI MS: m/z 610.3 (M þ H)þ.
and cIAP-2 BIR3, respectively) and increasing concentrations of
proteins were added to each well to a final volume of 125 μL in the
assay buffer (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 2-3 h with gentle shaking to ensure equilibrium. The polariza-
tion 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).
Anal. (C36H43N5O4 1.7CF3COOH) C, H, N.
3
(3S,6S,9S,10aR)-N-Benzhydryl-6-((S)-2-(methylamino)-
propanamido)-5-oxo-9-(2-phenylacetamido)decahydropyrrolo-
[1,2-a]azocine-3-carboxamide (6). Compound 6 was synthesized
from 17 bythesamemethodusedfor5. The gradient used ran from
70% of solvent A and 30% of solvent B to 50% of solvent A and
50% of solvent B in 40 min. The purity was confirmed by analytical
HPLC to be over 98%. 1H NMR (300 M Hz, D2O) δ 7.35-7.18
(m, 10H), 5.92 (s, 1H), 4.70 (m, 1H), 4.55-4.35 (m, 2H), 3.90 (m,
1H), 3.79 (m, 1H), 3.50, 3.40 (ABq, J = 8.4 Hz, 2H), 2.58 (s, 3H),
2.35-1.55 (m, 10H), 1.40 (d, J = 7.2 Hz, 3H). ESI MS: m/z 610.3
For competitive experiments, the Ki value of the tested
compound was determined in a dose-dependent competitive
binding experiment. Mixtures of 5 μL of the tested compound
in different concentrations in DMSO and 120 μL of preincu-
bated protein/tracer complex in the fixed concentrations in the
assay buffer were added into assay plates and incubated at room
temperature for 2-3 h with gentle shaking. Final concentrations
of proteins and tracers in the competitive assays were 10 and
2 nM for XIAP BIR3, 3 and 1 nM for cIAP-1 BIR3, and 5 and
1 nM for cIAP-2 BIR3, respectively. Negative controls contain-
ing protein/tracer complex only (equivalent to 0% inhibition)
and positive controls containing only free tracers (equivalent to
100% inhibition) were included in each assay plate. FP values
were measured as described above. IC50 values were determined
by nonlinear regression fitting of the competition curves. The Ki
values of the tested compound to these IAP proteins were
calculated using the measured IC50 values, the Kd values of the
tracer to different IAP proteins, and the concentrations of the
proteins and tracers in the competitive assays.27
Cell-Free Caspase-9 Functional Assay. For caspase-9 activity
assay, the enzymatic activity of active recombinant caspase-9
from Enzo Life Sciences was evaluated by the Caspase-Glo 9
assay kit from Promega. An amount of 2.5 μL of compound
solution in caspase assay buffer (CAB, 50 mM HEPES, 100 mM
NaCl, 1 mM EDTA with 0.1% CHAPS, and 10% glycerol, pH
7.4) containing 20% DMSO was mixed with 7.5 μL of XIAP
BIR3 protein and preincubated for 15 min, followed by addi-
tion of 2.5 μL of active caspase-9 solution in CAB. This mixture
was incubated at room temperature for 15 min. Luminogenic
Z-LEHD substrate was added with 1:1 ratio to give final con-
centrations of XIAP and caspase-9 as 500 nM and 2.5 unit/
reaction (according to the manufacturer instruction), respec-
tively. Luminescence from the substrate cleavage was monitored
by Tecan Infinite M-1000 multimode plate reader for 1 h.
Cell Growth Inhibition Assay. The MDA-MB-231 breast
cancer cell line was purchased from the American Type Culture
Collection (ATCC). Cells were seeded in 96-well flat bottom cell
culture plates at a density of (3-4) ꢀ 102 cells/well and grown
overnight, then incubated with or without Smac mimetics for
4 days. Cell growth inhibition was determined with a LDH
based WST-8 assay (WST-8; Dojindo Molecular Technologies
Inc., Gaithersburg, Maryland). Briefly, when treatment of cells
was finished, WST-8 was added to each well to a final concen-
tration 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. Concentration of the com-
pounds that inhibited cell growth by 50% (IC50) was calculated
by comparing the absorbance in the untreated cells and the cells
treated with the compounds.
(M þ H)þ. Anal. (C36H43N5O4 1.4CF3COOH) C, H, N.
3
(3S,6S,9R,10aR)-6-((S)-2-(Methylamino)propanamido)-5-oxo-
9-(2-phenylacetamido)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-
yl)decahydropyrrolo[1,2-a]azocine-3-carboxamide (7). Com-
pound 7 was synthesized from the intermediate 18 using the same
method as that for 5 and purified by HPLC using the same gra-
dient. The purity was confirmed by analytical HPLC to be over
1
98%. H NMR (300 M Hz, D2O) δ 7.35-7.05 (m, 9H), 4.85
(m, 1H), 4.45 (m, 1H), 4.25 (m, 1H), 3.95-3.80 (m, 3H), 3.50, 3.40
(ABq, J = 8.5 Hz, 2H), 2.70 (m, 2H), 2.60 (m, 3H), 2.30-1.50 (m,
14H), 1.42 (d, J = 7.2 Hz, 3H). ESI MS: m/z 574.3 (M þ H)þ.
Anal. (C33H43N5O4 1.2CF3COOH) C, H, N.
3
Fluorescence Polarization Based Binding Assays for XIAP,
cIAP-1, and cIAP-2 Proteins. A set of sensitive and quantitative
fluorescence polarization (FP) based assays were used to deter-
mine the binding affinities of our designed Smac mimetics to
XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3 proteins.
Protein Expression and Purification. Human XIAP BIR3
(residues 241-356) was cloned into a pET28 vector (Novagen)
containing an N-terminal 6xHis tag. Protein was produced in
E. coli BL21(DE3) cells grown at 37 °C in 2xYT 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), ZnOAc (50 μM), 0.1% βME, and
leupectin/aprotin protease inhibitors. Protein was purified from
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), ZnOAc (50 μM), and dithiothreitol (DTT,
1 mM). After purification, DTT was added to a final concentra-
tion of 10 mM. Human cIAP-1 BIR3-only (residues 253-363)
and cIAP-2 BIR3-only (residues 238-349) werecloned intopHis-
TEV vector, produced, and purified using the same method as for
the XIAP protein.
FP-Based Binding Assays. A fluorescently labeled Smac
mimetic (Smac-2F) was used as the tracer in FP assays to
determine the binding affinities of our designed Smac mimetics
to XIAP, cIAP-1, and cIAP-2 proteins. The Kd values of Smac-
2F to XIAP BIR3, cIAP-1 BIR3, and cIAP-2 BIR3 were
determined to be 4.4 ( 0.7, 0.7 ( 0.5, and 1.9 ( 1 nM by
monitoring the total fluorescence polarization of mixtures
composed with fluorescent tracer at a fixed concentration and
different IAP proteins with increasing concentrations up to full
saturation. Fluorescence polarization values were measured
using the Infinite M-1000 plate reader (Tecan U.S., Research
Triangle Park, NC) in Microfluor 2 96-well, black, round-bottom
plates (Thermo Scientific). In the saturation experiments, Smac-2F
(2, 1, and 1 nM for experiments with XIAP BIR3, cIAP-1 BIR3,
Western Blot Analysis. Treated 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 13 000 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

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6367
transferred to PVDF membranes. Following blocking in 5%
milk, the membranes were incubated with a specific primary
antibody, washed three times, and incubated with horseradish
peroxidase-linked secondary antibody (Amersham). The signals
were visualizedwitha chemiluminescent HRP antibody detection
reagent (Denville Scientific). When indicated, the blots were
stripped and reprobed with a different antibody. Primary anti-
bodies against cleaved caspase 3, PARP, and β-actin were
purchased from Cell Signaling Technology, and primary anti-
body against cIAP-1 was purchased from R&D System.
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C.- Y.; Qiu, S.; Tomita, Y.; Ueda, Y.; Jiang, S.; Krajewski, K.;
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Acknowledgment. We are grateful for financial support
from the National Cancer Institute, National Institutes of Health
(Grant R01CA109025), the Breast Cancer Research Founda-
tion, the Susan G. Komen Foundation, and the University of
Michigan Cancer Center (Grant P30CA046592).
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