Design, synthesis, and evaluation of potent, nonpeptidic mimetics of second mitochondria-derived activator of caspases

Article abstract of DOI:10.1021/jm801101z

A series of new Smac mimetics have been designed, synthesized, and evaluated. The most potent compound 10 binds to XIAP, cIAP-1, and cIAP-2 BIR3 proteins with Ki of 3.9, 0.37, and 0.25 nM, respectively. Compound 10 antagonizes XIAP in a cell-free function

Full text of DOI:10.1021/jm801101z

J. Med. Chem. 2009, 52, 593–596
593
Letters
Design, Synthesis, and Evaluation of Potent,
Nonpeptidic Mimetics of Second
Mitochondria-Derived Activator of Caspases
Wei Sun,† Zaneta Nikolovska-Coleska,‡ Dongguang Qin,‡
Haiying Sun,‡ Chao-Yie Yang,‡ Longchuang Bai,‡ Su Qiu,‡
You Wang,† Dawei Ma,*,† and Shaomeng Wang*,‡,§
State Key Laboratory of Bio-organic and Natural Products
Chemistry, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China,
and ComprehensiVe Cancer Center and Departments of Internal
Medicine, Pharmacology, and Medicinal Chemistry,
UniVersity of Michigan, 1500 E. Medical Center DriVe,
Ann Arbor, Michigan 48109, USA
ReceiVed September 3, 2008
Abstract: A series of new Smac mimetics have been designed,
synthesized, and evaluated. The most potent compound 10 binds to
XIAP, cIAP-1, and cIAP-2 BIR3 proteins with Ki of 3.9, 0.37, and
0.25 nM, respectively. Compound 10 antagonizes XIAP in a cell-free
functional assay and induces rapid cIAP-1 degradation in cancer cells.
Compound 10 inhibits cell growth in the MDA-MB-231 cancer cell
line with an IC50 of 8.9 nM.
Figure 1. Examples of monovalent (1-4) and bivalent (5 and 6) Smac
mimetics.
Smac (second mitochondria-derived activator of caspases) is
a potent proapoptotic protein and an endogenous antagonist of
IAP proteins.10,11 Previous studies have firmly established that
Smac interacts with XIAP and cIAP-1/2 proteins via its AVPI
tetrapeptide motif.6,12-15 Smac, in its dimeric form, interacts
with BIR2 and BIR3 domains in XIAP and effectively removes
the inhibition of XIAP to not only caspase-9 but also caspase-
3/-7.
In the past few years, intense research efforts have been
devoted to the design and development of small molecules to
mimic the AVPI binding motif as antagonists of IAP proteins
and as a new class of anticancer drugs.4,16-23 These small
molecules are called Smac mimetics. To date, two different types
Apoptosis is a critical cell process in normal development
and homeostasis of multicellular organisms to eliminate un-
wanted or damaged cells. Evasion of apoptosis is now recog-
nized as a hallmark of all cancers.1,2 Targeting key apoptosis
regulators with a goal to promote apoptosis in cancer cells is a
new strategy for anticancer drug design.3,4
Inhibitors of apoptosis proteins (IAPsa) are a class of key
apoptosis regulators.5,6 Among them, cellular IAP-1 (cIAP-1)
and cIAP-2 play a critical role in regulation of tumor necrosis
factor (TNF) receptor-mediated apoptosis,7 and X-linked IAP
(XIAP) is a central regulator of death-receptor-mediated and
mitochondria-mediated apoptosis pathways.8 XIAP inhibits
apoptosis through direct binding to and inhibition of three
cysteine proteases, an initiator caspase-9 and the two effectors
caspase-3 and -7.5,6 XIAP contains three baculoviral IAP repeats
(BIR) domains. While the third BIR domain (BIR3) of XIAP
selectively targets caspase-9, the BIR2 domain, together with
theimmediateprecedinglinker,inhibitscaspase-3andcaspase-7.6,8
Since these caspases play a critical role in the execution of
apoptosis, XIAP functions as an efficient inhibitor of apoptosis.6,8
Accordingly, these IAP proteins, especially XIAP, are consid-
ered as promising cancer therapeutic targets.8,9
of Smac mimetics have been reported, namely, monovalent and
4,16-23
bivalent Smac mimetics (Figure 1).
Monovalent Smac
mimetics such as 1-4 are designed to mimic a single AVPI
binding motif,16-19 whereas the bivalent compounds, such as 5
and 6, contain mimetics of two AVPI binding motifs tethered
together through a linker.4,20-23 Bivalent Smac mimetics can
achieve much higher affinities to XIAP and can be much more
potent than the corresponding monovalent Smac mimetics in
induction of apoptosis in tumor cells.20 However, because of
their ideal low molecular weight (∼500), monovalent Smac
mimetics may possess major advantages for drug development
compared to bivalent Smac mimetics.
Recent studies established that Smac mimetics induce rapid
cIAP-1 degradation and cIAP-1 is a key cellular target for Smac
mimetics.21-23 Furthermore, our recent study clearly showed
that in order to most efficiently induce apoptosis in cancer cells
by Smac mimetics, the apoptotic blockade by cIAP-1 and XIAP
needs to be concurrently removed.24 Thus, cIAP-1 and XIAP
are important cellular targets for Smac mimetics.
* To whom correspondence should be addressed. For D.M.: phone, +86
21 5492 5130; fax, +86 21 64166128; e-mail, madw@mail.sioc.ac.cn. For
S.W.: phone, +1, 734 6150362; fax, +1 734 6479647; e-mail,
shaomeng@umich.edu.
†
Shanghai Institute of Organic Chemistry.
Comprehensive Cancer Center and Department of Internal Medicine,
‡
University of Michigan.
Our laboratory has previously reported the structure-based
design, synthesis, and initial evaluation of a series of confor-
mationally constrained monovalent Smac mimetics.16-18,20
Among them, 7 (SM-122, Figure 2) binds to the XIAP BIR3
protein with a Ki of 26 nM.20 Our subsequent binding studies
showed that 7 also binds to cIAP-1 and to cIAP-2 proteins with
§
Departments of Pharmacology and Medicinal Chemistry, University
of Michigan.
a Abbreviations: IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP;
cIAP-1/-2, cellular IAP 1/2; Smac, second mitochondria-derived activator
of caspases; BIR, baculoviral IAP repeats (BIR) domain; BIR2/BIR3, the
second or third BIR domain; TNF, tumor necrosis factor; FP, fluorescence
polarization; NOE, nuclear Overhauser effect.
10.1021/jm801101z CCC: $40.75
2009 American Chemical Society
Published on Web 01/12/2009

594 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Letters
Figure 4. Cell growth inhibition by Smac mimetics in the MDA-MB-
231 cancer cell line. Cells were treated for 4 days, and cell growth
was determined by a WST-8 assay.
Figure 2. Chemical structures of designed Smac mimetics.
BIR3.25 Compounds 8 and 9 were determined to bind to XIAP
BIR3 with a Ki value of 14 nM and 209 nM, respectively, in a
competitive FP-based binding assay.25 Their binding affinities
to cIAP-1 and cIAP-2 BIR3 proteins were also subsequently
determined in newly established competitive binding assays
(Table 1, Supporting Information). Compound 8 binds to cIAP-1
and cIAP-2 with Ki of 0.68, and 1.0 nM, respectively. In
comparison, compound 9 has Ki values of 29, and 132 nM to
cIAP-1 and cIAP-2, respectively. Hence, compound 8 is
substantially more potent than 9 to each of these three IAP
proteins.
Table 1. Binding Affinities of Smac Mimetics to XIAP, cIAP-1, and
cIAP-2 BIR3 Proteinsa
Ki ( SD (nM)
compd
XIAP BIR3
cIAP-1 BIR3
cIAP-2 BIR3
7
8
9
10
11
26 ( 5
0.80 ( 0.19
0.68 ( 0.15
29 ( 6
0.37 ( 0.25
1.27 ( 1.05
1.84 ( 0.62
1.00 ( 0.04
132 ( 17
0.25 ( 0.07
2.09 ( 0.78
14 ( 5
209 ( 73
3.9 ( 1.8
31 ( 15
a Ki and standard deviation (SD) values were calculated on the basis of
three to five independent experiments.
Consistent with their binding affinities, 8 is 4 times more
potent than 7 in inhibition of cell growth in the MDA-MB-231
cancer cell line and has an IC50 of 73 nM (Figure 4). In
comparison, 9 is less potent than 7 and 8 and has an IC50 of
1800 nM (Figure 4).
Our predicted binding model showed that there is a hydro-
phobic pocket on the surface of XIAP BIR3, between W323
and Y324 (Figure 3). We next designed and synthesized 10, in
which a benzyl group is appended to the five-membered ring,
to test if targeting this hydrophobic pocket can further improve
the binding affinities to IAP proteins and cellular activity.
Modeling predicted that the pro-(S) configuration is preferred.
Accordingly, we have developed a stereoselective synthetic
method for 10 (Scheme 1). To test if the conformation of the
eight-membered ring is critical for binding to IAP proteins, we
have also synthesized 11, which has a double bond in its eight-
membered ring.
Compound 10 binds to XIAP, cIAP-1, and cIAP-2 with very
high affinities and has Ki of 3.9, 0.37, and 0.25 nM to these
three proteins, respectively (Table 1). In comparison, 11 has Ki
of 31, 1.27, and 2.09 nM to XIAP, cIAP-1, and cIAP-2 proteins,
respectively, significantly less potent than 10.
Figure 3. Predicted binding models of compounds 7-10 in complex
with XIAP BIR3 protein. Predicted binding models are superimposed
on the crystal structure of Smac in complex with XIAP BIR3. Protein
is shown in surface with key binding residues shown and labeled.
Compounds 7-10 and AVPI peptide are shown in stick. Carbon atoms
in AVPI are shown in yellow, and carbon atoms in 7-10 are shown in
green. Oxygen and nitrogen atoms are shown in red and blue,
respectively.
Consistent with its high affinities to IAP proteins, 10 potently
inhibits cell growth in the MDA-MB-231 cancer cell line and
has an IC50 of 8.9 nM and is >25 times more potent than the
initial lead 7 (Figure 4).
These Smac mimetics were evaluated for their ability to
antagonize XIAP in a cell-free functional assay (Figure 5 and
Supporting Information). While the XIAP BIR3 protein ef-
fectively inhibits the activity of caspase-3/-7, these Smac
mimetics dose-dependently antagonize the inhibition of XIAP
to caspase activity. Consistent with their binding affinities to
XIAP BIR3, compounds 8 and 10 are the most potent
antagonists of XIAP in this cell-free functional assay while 9
is the least potent antagonist against XIAP BIR3.
Recent studies showed that Smac mimetics induce cIAP-1
degradation in cells, which is crucial for apoptosis induction
by Smac mimetics.21-23 Indeed, Western blotting analysis
showed that 7, 8, and 10 effectively and dose-dependently
induce the degradation of cIAP-1 (Figure 6). Compounds 8 and
10 are capable of inducing marked cIAP-1 degradation at
concentrations as low as 10 nM, consistent with their high
high affinities (Table 1). Compound 7 potently inhibits cancer
cell growth and effectively induces apoptosis in cancer cells.20
These data indicated that 7 is a promising lead compound for
further optimization. We report herein the design, synthesis, and
evaluation of new analogues of 7. These efforts led to the
discovery of highly potent monovalent Smac mimetics and
yielded new insights into their structure-activity relationship.
The predicted binding model of 7 in complex with XIAP
BIR3 showed that while the pro-(R)-phenyl group in 7 inserts
into a hydrophobic pocket in XIAP, the pro-(S)-phenyl group
does not have specific interactions with the protein (Figure 3).
We have thus investigated if replacement of the diphenylmethyl
group by a conformationally constrained 1-tetrahydronaphtha-
lenyl group, which was previously used for the design of Smac
peptidomimetics,17 could further improve the binding to XIAP.
Modeling predicted that the compound with the (R)-configu-
ration is preferred for binding to XIAP BIR3 (Figure 3).
Compounds 8 and 9 with the (R)- or the (S)-configuration were
synthesized and evaluated for their binding affinities to XIAP

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 595
Scheme 1. Synthesis of Compounds 10 and 11a
Figure 6. Western blot analysis of the levels of cIAP-1, XIAP, cleaved
caspase-8 (CL C8), pro- and cleaved caspase-3 (Pro-C3 and CL C3),
cleaved PARP (CL PARP). MDA-MB- 231 breast cancer cells were
treated with Smac mimetics for 24 h, and proteins were probed with
specific antibodies. GAPDH was used as the loading control.
subsequent steps. The stereochemistry of the benzyl group in
13 and 14 was confirmed by two-dimensional (2D) Nuclear
Overhauser Effect (NOE) data of 14 (Supporting Information)
and was found to be the same as that in the previous report.26
The carbonyl group in 14 was selectively reduced, followed by
acetylation and introduction of a syn-allyl group at the C-5
position via the N-acyliminium ion chemistry to afford 15.27
The Cbz protective group in 15 was selectively removed to
generate 16, which was coupled with (S)-N-Boc-allylglycine,
followed by cyclization using the olefin metathesis method to
yield the key intermediate 17. The syn stereochemistry between
the allyl and the benzyl groups of 16 was confirmed by 2D
NOE data of 16 and its stereoisomer 16i (Supporting Informa-
tion). Compound 17 was hydrolyzed and then coupled with (R)-
1,2,3,4-tetrahydronaphthalen-1-amine to give amide 18. Re-
moval of the Boc protecting group in 18 yielded an ammonium
salt, which was condensed with N-Boc-methyl-L-alanine to
generate 19. Removal of the Boc protecting group in 19
produced 11. Hydrogenation of the CdC double bond in 19
catalyzed by 10% Pd-C, followed by removal of the Boc
protecting group, afforded 10.
a Reagents and conditions: (a) LiHMDS, benzaldehyde, BF3 ·OEt2, THF,
-78 °C; (b) MsCl, TEA, DCM, room temp; (c) H2, Pd/C, MeOH, room
temp, 75% (three steps); (d) TFA, DCM, 0 °C; (e) LiHMDS, CbzCl, THF,
-78 °C to room temp, 80% (two steps); (f) Super-Hydrid, THF, -78 °C;
(g) Ac2O, TEA, DMAP, DCM, room temp; (h) Bu3SnAllyl, BF3 · OEt2,
toluene, -78 °C, 4,5-syn/anti 5.6:1, 55% (three steps); (i) BF3 ·OEt2, Me2S,
DCM, 0 °C; (j) aq sat. NaHCO3, 88% (two steps); (k) (S)-N-Boc-allylglycin,
ClCOOiBu, NMM, THF, -20 °C; (l) Grubbs catalyst (first generation),
DCM, reflux, 67% (two steps); (m) LiOH ·H2O, THF/H2O/MeOH, 0 °C;
(n) (R)-1,2,3,4-tetrahydronaphthalen-1- amine, EDCI, HOBt, DIPEA, DCM,
room temp, 95% (two steps); (o) HCl/MeOH, 0 °C; (p) Boc-N-Me-Ala-
OH, EDCI, HOBt, DIPEA, DCM, room temp; (q) H2, Pd/C, MeOH, room
temp; (r) HCl/MeOH, 0 °C, 85% (four steps) for compound 10 and 91%
(three steps) for compound 11.
In summary, we have designed and synthesized a number of
novel Smac mimetics based on lead compound 7 by modifica-
tions of two different regions. Our efforts led to the discovery
of arguably the most potent, nonpeptidic, monovalent Smac
mimetic and insights into their structure-activity relationship
in binding, functional and cellular levels. The most potent
compound 10 (DM-28) binds to cIAP-1, cIAP-2, and XIAP with
Ki values in subnanomolar and low nanomolar affinities, potently
antagonizes XIAP in a cell-free functional assay, and effectively
induces cIAP-1 degradation, processing of caspase-8 and -3,
and cleavage of PARP in the MDA-MB-231 cancer cell line at
concentrations as low as 10 nM. Compound 10 potently inhibits
cell growth in the MDA-MB-231 cell line with an IC50 of 8.9
nM and is >25 times more potent than 7. Extensive in vitro
and in vivo studies are under way to further elucidate mechanism
of action and therapeutic potential of 10 as a new anticancer
agent and the results will be reported in due course.
Figure 5. Functional antagonism of Smac mimetics against XIAP BIR3
in a cell-free functional assay. Activation of caspase-3/7 in cell lysates
was achieved by adding dATP and cyctochrome c. Addition of
recombinant XIAP BIR3 protein at 500 nM, together with dATP and
cyctochrome c, effectively inhibited the casapse activity. Smac mimetics
dose-dependently recovered the activity of caspase-3/-7. The caspase
activity data at 60 min was used (Supporting Information).
binding affinities to cIAP-1. These compounds have minimal
effect on the levels of XIAP protein.
We further examined the processing of caspase-8 and -3 and
cleavage of poly(ADP-ribose) polymerase (PARP), several
biochemical markers of apoptosis (Figure 6). Compounds 7, 8,
and 10 effectively induce processing of caspase-8 and -3 and
cleavage of PARP in a dose-dependent manner (Figure 6).
Compounds 8 and 10 are capable of inducing processing of
caspase-3 and -8 and cleavage of PARP at concentrations as
low as 10 nM and are more effective than 7. These data
indicated that these Smac mimetics not only inhibit cell growth
but effectively and dose-dependently induce apoptosis in the
MDA-MB-231 cancer cell line.
Acknowledgment. We are grateful for financial support from
the Chinese Academy of Sciences and the National Natural
Science Foundation of China (Grants 20620120106, 20621062,
and 90713047), the U.S. National Cancer Institute, National
Institutes of Health (Grant R01CA109025), the Breast Cancer
Research Foundation, the Susan G. Komen Foundation, the
Prostate Cancer Foundation, the University of Michigan Cancer
Center Core Grant P30CA046592, and Ascenta Therapeutics,
Inc.
The syntheses of 10 and 11 are shown in Scheme 1. Briefly,
13 was prepared from the commercially available pyroglutamate
ester 12.25 The Boc protective group in 13 was replaced by a
Cbz to obtain 14 for achieving better stereoselectivity in

596 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Letters
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Supporting Information Available: Information on the chemi-
cal data for 8-10 and intermediates, structural assignments of 14,
16, and 16i by two-dimensional NOE data, FP competitive binding
assays for XIAP and cIAP-1/-2 BIR3 proteins, cell-free functional
assay of XIAP BIR3, cell growth assay, Western blotting analysis,
and molecular modeling. This material is available free of charge
via the Internet at http://pubs.acs.org.
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