Design, synthesis and evaluation of monovalent Smac mimetics that bind to the BIR2 domain of the anti-apoptotic protein XIAP

Article abstract of DOI:10.1016/j.bmcl.2011.05.049

We report the systematic rational design and synthesis of new monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Characterization of compounds in vitro (including 9i; ML101) led to the determination of key structural requirements for BIR2 binding affinity. Compounds 9h and 9j sensitized TRAIL-resistant breast cancer cells to apoptotic cell death, highlighting the value of these probe compounds as tools to investigate the biology of XIAP.

Full text of DOI:10.1016/j.bmcl.2011.05.049

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4332–4336
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of monovalent Smac mimetics that bind
to the BIR2 domain of the anti-apoptotic protein XIAP
a,b,
9
Marcos González-Lopez
, Kate Welsh a, , Darren Finlay a, Robert J. Ardecky a,b, Santhi Reddy Ganji a,b
,
Ying Su b, Hongbin yuan b, Peter Teriete a,b, Peter D. Mace a, Stefan J. Riedl a, Kristiina Vuori a,b
,
John C. Reed a,b, Nicholas D. P. Cosford a,b,
⇑
a Cancer Research Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
b Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the systematic rational design and synthesis of new monovalent Smac mimetics that bind
preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Characterization of compounds
in vitro (including 9i; ML101) led to the determination of key structural requirements for BIR2 binding
affinity. Compounds 9h and 9j sensitized TRAIL-resistant breast cancer cells to apoptotic cell death, high-
lighting the value of these probe compounds as tools to investigate the biology of XIAP.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 27 April 2011
Revised 14 May 2011
Accepted 16 May 2011
Available online 24 May 2011
Keywords:
Apoptosis
XIAP
Monovalent Smac mimietics
Probe compounds
Programmed cell death plays an essential role in development,
typically occurring in animal species by apoptosis.1 Defects in
apoptosis are associated with many diseases characterized by
either insufficient cell death (e.g., cancer) or excessive cell death
(e.g., degenerative diseases).2,3 The inhibitor of apoptosis proteins
(IAPs) contain ꢀ70 amino acid motifs termed baculovirus IAP
repeat (BIR) domains.4,5 These BIR domains are primarily responsi-
ble for the anti-apoptotic activity of IAPs due to their ability to bind
and inhibit distinct caspases, cysteine–aspartyl proteases that are
critical for the initiation and execution phases of apoptosis.6 Hu-
man X-linked inhibitor of apoptosis protein (XIAP) is the most po-
tent caspase inhibitor in the IAP family.7 XIAP contains three BIR
domains (designated 1, 2, and 3) which exhibit specificity for dif-
ferent caspases. A short linker peptide in the BIR2 region of XIAP
mediates the interaction with the effector caspases-3 and -7,
whereas the third BIR domain (BIR3) targets the initiator cas-
pase-9.8–10 All apoptotic signaling, triggered via either the intrinsic
or the extrinsic pathway, converges on caspases-3 or -7 and this
points to the importance of developing novel chemical entities
with preferential affinity for the BIR2 domain of XIAP.
intermembrane space of the mitochondria into the cytosol perpet-
uates the apoptotic signal by competing with caspases for binding
to XIAP.11 The four hydrophobic amino acids Ala-Val-Pro-Ile (AVPI)
at the N-terminus of mature Smac bind to the surface groove on
the BIR3 domain of XIAP, removing caspase-9 inhibition, and bind
with lower affinity to the BIR2 domain, alleviating inhibition by
caspase-3 and -7.12 Up-regulation of XIAP expression in tumors
causes resistance to current chemotherapeutic agents, and thus
inhibition of the protein–protein interaction between XIAP and
caspases-3, -7 and -9 represents a promising approach for the
treatment of cancer.13
In the past few years substantial efforts have focused on small
molecule Smac mimetics that target the BIR3 domain of XIAP.14
Conversely, there have been only a few reports on the design and
synthesis of compounds that effect inhibition by binding to the
BIR2 domain of XIAP. Examples include bivalent dimers, macrocy-
clic peptides, polyphenylureas and the natural product delaquini-
um.15 While these compound classes exhibit interesting
properties, including cellular activity, their utility as potential drug
leads is limited by high molecular weight, low potency, poor solu-
bility or other disadvantageous physicochemical properties. The
present study was performed within the framework of the Molec-
ular Libraries Probe Production Centers Network (MLPCN; http://
mli.nih.gov/mli/mlpcn/) with the goal of developing novel BIR2-
selective probes. Herein we report the rational design and SAR of
low molecular weight tripeptide derivatives that bind to the BIR2
domain of XIAP with high affinity. We further demonstrate that
In the intrinsic cell death pathway, apoptotic signaling is regu-
lated by the mitochondrial protein Smac, an endogenous dimeric
proapoptotic antagonist of XIAP. The release of Smac from the
⇑
Corresponding author.
E-mail address: ncosford@sanfordburnham.org (N.D.P. Cosford).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.049

9
M. González-Lopez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4332–4336
4333
these small molecule probes are effective tools for investigating
the biology of XIAP in cells.
P1 P2
P3
R2
H
N
O
Monovalent Smac mimetics based on the AVPI tetrapeptide
have previously been shown to inhibit the interaction of XIAP with
caspase-9 by binding to the XIAP BIR3 domain. For our studies we
used compounds exemplified by the structures shown in Figure 1
as a starting point and employed a rational design approach to
investigate the structural requirements for XIAP BIR2 versus BIR3
domain potency.16
N
R
N
H
R4
O
R1
O
N
H
N-Terminus
C-Terminus
Figure 2. Initial tripeptide binding model.
At the outset we developed a putative binding model based on
the structures of 1a and 1b (Fig. 2). Our approach consisted of
retaining the common structural features of 1a and 1b, namely
the N-methyl alanine moiety at the P1 position and the proline res-
idue at P3, while investigating the effects of varying the P2 position
and the C-terminal substituent. Thus, our preliminary synthetic ef-
forts were focused on: (1) investigating the optimal R2 and R4 sub-
stituents; and (2) studies to determine the effect of amino acid
stereochemistry at the P2 and P3 positions on the potency of inhib-
itors at the BIR2 and BIR3 domains of XIAP.
The general procedure for the synthesis of analogs is summa-
rized in Scheme 1. Condensation of amino acid derivative 2 with
proline derivative 3, followed by acidic cleavage of the N-terminal
Boc protecting group afforded dipeptide 4. Condensation of 4 with
alanine derivative 5 followed by hydrogenolysis of the benzyl ester
afforded tripeptide acid 6. Finally, condensation of 6 with the
appropriate amine followed by removal of the Boc group provided
the target XIAP inhibitors 7.17
OBn
R2
R2
N
H
3
N
O
Boc
OH
H2N
N
H
O
a, b
OBn
O
O
2
4
Me
N
O
a, c
Boc
OH
R2
Me
5
R4NH2
a, b
H
N
R2
Me
N
O
O
N
N
Boc
N
H
6
Me
N
H
7
R4
O
O
Me
OH
Me
N
H
O
O
Scheme 1. General synthetic route for the synthesis of small molecule XIAP
inhibitors. Reagents and conditions: (a) EDC, HOBt, N-methylmorpholine, DMF (b)
TFA, CH2Cl2 (c) H2, Pd/C, MeOH.
All of the Smac mimetics synthesized were evaluated for their
ability to bind the BIR2 or BIR3 domains of XIAP by employing fluo-
rescence-polarization (FP) competition assays.18 The results are ex-
pressed as competitive inhibition constants (Ki), derived from the
corresponding IC50 values by application of a mathematical equa-
tion developed by Wang and coworkers.19
In the initial phase of SAR studies we investigated the effects of
different substituents at the P2 position on BIR2 and BIR3 potency.
The results are summarized in Table 1. The previously reported
compound 1a16a was synthesized and tested as a benchmark
against which the new analogs were compared. Incorporation of
cyclohexyl, phenyl, benzyl or ethyl as the R2 substituent provided
compounds 8a–d which exhibited moderate affinity for BIR2 and
good BIR3 binding affinity. A valine residue at the P2 position
(8e) improved potency against both BIR2 and BIR3. Compounds
Table 1
Binding affinities to BIR2 or BIR3 domains of XIAP for analogs 8a–8k
R2
H
N
O
N
Me
N
H
H
Me
O
N
O
8
Compds
R2
BIR2 Kia
(
l
M)
BIR3 Kia
(lM)
1a
8a
8b
8c
8d
8e
8f
8g
8h
8i
iso-Propyl
Cyclohexyl
Phenyl
1.36 ( 0.02)
3.58 ( 0.11)
8.06 ( 0.28)
9.07 ( 3.24)
3.12 ( 0.69)
1.84 ( 0.96)
>56
>56
>56
0.25 ( 0.03)
0.66 ( 0.11)
0.66 ( 0.06)
1.05 ( 0.31)
0.70 ( 0.01)
0.44 ( 0.13)
>39
>39
29.88 ( 0.17)
8f–8h that incorporate an amino acid with unnatural
D stereo-
chemistry were essentially inactive, demonstrating the importance
Benzyl
of employing amino acids with natural L stereochemistry at P1, P2
Ethyl
and P3. Other R2 substituents such as iso-butyl, sec-butyl or pro-
pylamide (compounds 8i–8k) did not improve on the BIR2 potency
exhibited by 8e with Val at P2.
iso-Propyl
iso-Propylb
iso-Propylc
iso-Propyld
iso-Butyl
sec-Butyl
We next investigated the effect of varying the C-terminal sub-
stituent (R4) on BIR2 and BIR3 potency and selectivity. For this
phase the P1-P2-P3 tripeptide was retained as NMeAla-Val-Pro
and the C-terminal was varied to determine the optimal R4 substi-
tuent. Compounds 9a–9j were synthesized and their BIR2 and BIR3
potencies determined in the FP assays (Table 2). The approximate
1.67 ( 0.11)
4.41 ( 0.31)
2.33 ( 0.45)
0.35 ( 0.01)
0.80 ( 0.01)
0.65 ( 0.02)
8j
8k
Propylamide
a
Values were determined in duplicate and reported as the Ki , standard devi-
ation which is given in parentheses.
b
D
D
D
Valine at P2.
Proline at P3.
Alanine at P1.
c
d
O
BIR2:BIR3 selectivity for each compound is shown as a ratio in the
last column of Table 2.
As shown in Table 2, the benzylamine derivative 9a exhibited
similar BIR2 and BIR3 potency (Ki = 3.36 lM and 0.59 lM) to the
benzhydryl derivative 8e, while the isomeric pyridinylmethan-
amine derivatives 9b–9d were significantly less potent than 9a at
both BIR2 and BIR3. However, despite the loss of potency, the
BIR2 versus BIR3 selectivity ratio improved to 1:1 for 9c and 9d.
H2N
N
O
N
H
H
N
N
O
Me
NH
Me
N
H
O
O
Me
N
H
O
1a
1b
Figure 1. Examples of monovalent tripeptide XIAP BIR3 inhibitors.

9
4334
M. González-Lopez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4332–4336
Table 2
Table 3
Binding affinities to BIR2 or BIR3 domains of XIAP for selected tripeptide analogs 9a–
Binding affinities to BIR2 or BIR3 domains of XIAP for selected heterocyclic analogs
9j
9k–9t
Compds R4
BIR2 Kia
(l
M) BIR3 Kia
(l
M)
BIR2 versus BIR3
selectivity ratio
H
N
O
N
Me
N
H
R4
Me
9kb
7.20 ( 1.06)
56.13 ( 10.73) 8:1
Me
O
N
H
O
9
N
9l
6.07 ( 0.08)
4.94 ( 0.15)
2.55 ( 0.13)
1.40 ( 0.10)
37.06 ( 7.88)
13.78 ( 0.64)
16.21 ( 1.22)
4.07 ( 0.07)
6:1
3:1
6:1
3:1
N
Compds R4
BIR2 Kia
(l
M) BIR3 Kia
(l
M)
BIR2 versus BIR3
selectivity
9m
9n
N
Me
9a
9b
3.36 ( 0.96)
9.70 ( 1.32)
10.93 ( 0.96)
0.59 ( 0.03)
5.62 ( 0.44)
8.18 ( 0.45)
1:6
1:2
N
N
N
9o
9p
N
N
9c
1:1
1:1
1.29 ( 0.05)
1.50 ( 0.15)
1:1
9d
20.07 ( 1.71) 26.15 ( 0.96)
Me
N
N
9q
9r
1.29 ( 0.01)
0.99 ( 0.08)
0.54 ( 0.01)
0.42 ( 0.03)
1:2
1:2
9e
9f
2.01 ( 0.16)
1.37 ( 0.11)
4.97 ( 0.04)
0.44 ( 0.02)
0.12 ( 0.02)
1:17
N
N
0.055 ( 0.011) 1:25
Me
9s
9t
4.75 ( 0.32)
0.74 ( 0.08)
6.10 ( 0.10)
5.5 ( 0.09)
1:1
7:1
9g
9h
1.40 ( 0.22)
1.51 ( 0.13)
1:4
3:1
N
H
a
Values are determined in duplicate
parentheses.
standard deviation which is given in
9i
1.91 ( 0.35) 12.71 ( 0.81)
7:1
3:1
N
b
In this case the residue employed at P2 was Abu instead of Val.
N
9j
H
0.39 ( 0.17)
1.08 ( 0.06)
a
Values are determined in duplicate and reported standard deviation which is
Structural modeling of 9t on the BIR2 domain was performed
based on binding modes observed in BIR3/SMAC and BIR2 com-
plexes8,12 and suggests a basis for the properties of the compound
(Fig. 3). Thus, compound 9t was first manually docked into the
Smac-binding pocket of BIR2 based on available BIR3-Smac and
BIR2-caspase-3 structures followed by energetic minimization.21
The model suggests that BIR2 residues Lys 206 and Gln 197 contrib-
ute to the binding of the indole moiety of 9t which can readily be
accommodated in the P4 pocket. In this binding mode the aliphatic
stem of Lys 206 is poised to assist binding of the aromatic ring struc-
ture, while Gln 197 is positioned to form a hydrogen bond with the
indole nitrogen. Comparison to the equivalent surface on BIR3 indi-
cates that in BIR3 Lys 206 is replaced by a Glycine and Gln 197 by a
Lysine residue. The resulting pocket is unlikely to accommodate the
indole as favorably, accounting for the increased BIR2 selectivity of
compound 9t.
To assess the activity of the compounds in a relevant cellular con-
text their ability to sensitize TRAIL-resistant cells to apoptosis was
investigated. Thus, MDA-MB-231 breast adenocarcinoma cells were
incubated with the respective compounds as described for 4 h be-
fore adding varying concentrations of TRAIL for 20 h, at which time
cell viability was assessed.22 Both 9h (BIR2 selective) and 9f (BIR3
selective) showed strong TRAIL sensitizing activity (Fig. 4). Com-
pound 9j (BIR2 selective) showed a similar trend but was slightly
less potent at the concentration used. An LD50 value could not be
achieved in MDA-MB-231 cells with TRAIL alone but values of
12.2 ng/mL (0.68 nM), 11.3 ng/mL (0.63 nM) and 24.6 ng/mL
given in parentheses.
The effect of a tetrahydronaphthyl-1-amine R4 substituent was
investigated in compounds 9e–9g by analogy to compound 1b.
As expected based on previous studies, these compounds exhibited
good potency versus BIR3, with 9f, containing the R-tetrahydro-
naphthyl-1-amine isomer, being especially potent against BIR3
(Ki = 0.055 lM). A breakthrough came when the tetrahydronaph-
thyl substituent was replaced with a 1-naphthyl group, as in 9h.
Compound 9h displayed a significant potency boost for BIR2
(BIR2 Ki = 0.44
Ki = 1.51 M). Interestingly, the quinolin-5-amine derivative 9i
was less potent (BIR2 Ki = 1.91 M; BIR3 Ki = 12.71 M) but
lM) and was three-fold selective (BIR3
l
l
l
showed improved selectivity for BIR2 versus BIR3 (7:1), suggesting
that heteroatom incorporation may favor BIR2 selectivity. A second
breakthrough occurred in the form of the phenylhydrazine deriva-
tive 9j. Thus, compound 9j was found to be potent (BIR2
Ki = 0.39
lM; BIR3 Ki = 1.08 lM) and three-fold selective for BIR2
versus BIR3.
Based on the promising selectivity for BIR2 displayed by the
quinoline derivative 9i we synthesized a small library of tripeptide
derivatives with heteroaromatic (quinoline, isoquinoline or indole)
C-terminal substituents. The structures and data for these analogs
are shown in Table 3. Several of these compounds, including 9k–n,
showed good selectivity for BIR2 although with lower overall po-
tency at both BIR2 and BIR3. Other analogs, such as 9p–r, exhibited
good potency but were not as selective as 9i. However, incorpora-
tion of an indole moiety at the C-terminal position provided a com-
pound (9t20) that was both seven-fold selective for BIR2 and
(1.37 nM) were obtained with 5 lM 9h, 9f, and 9j, respectively. Most
interestingly, these compounds showed little to no toxicity as single
agents (data not shown) but rather sensitized the cells to TRAIL-in-
duced apoptosis. Thus these agents are cell permeable TRAIL sensi-
tizing agents that may serve as potential lead compounds in the
retained potency (BIR2 Ki = 0.74 lM).

9
M. González-Lopez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4332–4336
4335
3. Okouchi, M.; Ekshyyan, O.; Maracine, M.; Aw, T. Y. Antioxid. Redox Signal. 2007,
9, 1059.
4. Deveraux, Q. L.; Reed, J. C. Genes Dev. 1999, 1, 239.
5. LaCasse, E. C.; Mahoney, D. J.; Cheung, H. H.; Plenchette, S.; Baird, S.; Korneluk,
R. G. Oncogene 2008, 27, 6252.
6. Pop, C.; Salvesen, G. S. J. Biol. Chem. 2009, 284, 21777.
7. Schimmer, A. D.; Dalili, S.; Batey, R. A.; Riedl, S. J. Cell Death Differ. 2006, 13, 179.
8. Riedl, S. J.; Renatus, M.; Schwarzenbacher, R.; Zhou, Q.; Sun, C.; Fesik, S. W.;
Liddington, R. C.; Salvesen, G. S. Cell 2001, 104, 791.
9. (a) Chai, J.; Shiozaki, E.; Srinivasula, S. M.; Wu, Q.; Dataa, P.; Alnemri, E. S.; Shi,
Y. Cell 2001, 104, 769; (b) Huang, Y.; Park, Y. C.; Rich, R. L.; Segal, D.; Myszka, D.
G.; Wu, H. Cell 2001, 104, 781.
10. Srinivasula, S. M.; Hegde, R.; Saleh, A.; Datta, P.; Shiozaki, E.; Chai, J.; Lee, R.-A.;
Robbins, P. D.; Fernandes-Alnemri, T.; Shi, Y.; Alnemri, E. S. Nature 2001, 410,
112.
11. Shiozaki, E. N.; Shi, Y. Trends Biochem. Sci. 2004, 39, 486.
12. (a) Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.; Oost, T.;
Herrmann, J.; Wu, J. C.; Fesik, S. W. Nature 2000, 408, 1004; (b) Wu, G.; Chai, J.;
Suber, T. L.; Wu, J.-W.; Du, C.; Wang, X.; Shi, Y. Nature 2000, 408, 1008.
13. Dean, E. J.; Ranson, M.; Blackhall, F.; Dive, C. Expert Opin. Ther. Targets 2007, 11,
1459.
14. (a) Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Qian, D.; Lu, J.; Qiu, S.; Bai, L.;
Peng, Y.; Cai, Q.; Wang, S. Acc. Chem. Res. 2008, 41, 1264; (b) Mannhold, R.;
Fulda, S.; Carosati, E. Drug Discovery Today 2010, 15, 210; (c) Flygare, J. A.;
Fairbrother, W. J. Expert Opin. Ther. Pat. 2010, 20, 251.
Figure 3. Model of compound 9t (magenta) bound to BIR2 (gray) with residues Lys
206 and Gln 197 depicted in yellow.
15. (a) Sun, H.; Liu, L.; Lu, J.; Bai, L.; Li, X.; Nikolovska-Coleska, Z.; McEachern, D.;
Yang, C. Y.; Qiu, S.; Yi, H.; Sun, D.; Wang, S. J. Med. Chem. 2011, 54, 3306; (b)
Sun, H.; Liu, L.; Lu, J.; Qiu, S.; Yang, C. Y.; Yi, H.; Wang, S. Bioorg. Med. Chem. Lett.
2010, 20, 3043; (c) Schimmer, A. D.; Welsh, K.; Pinilla, C.; Wang, Z.; Krajewska,
M.; Bonneau, M. J.; Pedersen, I. M.; Kitada, S.; Scott, F. L.; Bailly-Maitre, B.;
Glinsky, G.; Scudiero, D.; Sausville, E.; Salvesen, G.; Nefzi, A.; Ostresh, J. M.;
Houghten, R. A.; Reed, J. C. Cancer Cell 2004, 5, 25; (d) Orzaez, M.; Gortat, A.;
Sancho, M.; Carbajo, R. J.; Pineda-Lucena, A.; Palacios-Rodriguez, Y.; Perez-
Paya, E. Apoptosis 2011, 16, 460.
16. (a) Sun, H.; Nikolovska-Coleska, Z.; Chen, J.; Yang, C.-Y.; Tomita, Y.; Pan, H.;
Yoshioka, Y.; Krajewski, K.; Roller, P. P.; Wang, S. Bioorg. Med. Chem. Lett. 2005,
15, 793; (b) Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A.-S.; Betz, S. F.;
Deckwerth, T. L.; Ding, H.; Elmore, S. W.; Meadows, R. P.; Olejniczak, E. T.;
Oleksijew, A.; Oltersdorf, T.; Rosenberg, S. H.; Shoemaker, A. R.; Tomaselli, K. J.;
Zou, H.; Fesik, S. W. J. Med. Chem. 2004, 47, 4417.
17. All synthesized compounds were purified by either medium pressure silica gel
chromatography or preparative HPLC using a C18 column. Compound purity
was determined using analytical HPLC/MS and NMR (1H and 13C). The yields for
the first two steps ranged from 70 – 90% while the yields for the last step
ranged from 20 – 90%. The purity of each final product was greater than 95% as
determined by HPLC/MS analysis.
18. Fluorescence polarization assays were run in 20 lL volume in 384 well format
with black plates. The assay solution was 25 mM Hepes at pH 7.5/1 mM tris(2-
carboxyethyl)phosphine/0.005% Tween 20 and 20 nM AVPIAQK-rhodamine.
XIAP BIR2 was present at 1
within a range of 100 M to 6.1 nM using two fold dilutions starting at 100
generating a 16 point curve. Plates were read on an Analyst HT in fluorescence
polarization mode with excitation at 530 nm, emission at 580 nm and
lM or XIAP BIR3 at 0.2 lM. Compound was present
Figure 4. Apoptosis in TRAIL-resistant cells sensitized with compounds 9f, 9h or 9j.
l
lM
a
dichroic mirror at 565 nm. Resulting data in mP was fit in GraphPad Prism 5
with a non-linear regression using a sigmodial curve with variable slope.
19. Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P.
P.; Krajewski, K.; Saito, N.; Stuckey, J.; Wang, S. Anal. Biochem. 2004, 332, 261.
20. Spectroscopic data for selected compound 9t: 1H NMR (400 MHz, D2O) d 7.52 (d,
J = 1.8 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 3.1 Hz, 1H), 7.01 (dd, J = 9.1,
2.4 Hz, 1H), 6.43 (d, J = 3.1 Hz, 1H), 4.44 (d, J = 7.3 Hz, 1H), 4.39 (m, 1H), 3.83
(m, 2H), 3.65 (m, 1H), 2.56 (s, 3H), 2.05 (m, 2H), 1.94 (m, 2H), 1.38 (d, J = 6.7 Hz,
3H), 0.91 (d, J = 6.7 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6) d 169.6, 169.3, 132.6, 131.1, 127.4, 125.9, 114.5, 111.1, 110.4, 100.9, 60.2,
56.4, 55.9, 47.3, 38.2, 31.3, 30.0, 29.5, 24.7, 19.0, 18.2, 16.3. MS m/z = 414
(M+H).
21. The specific details of model-generation to illustrate the putative binding
mode of compound 9t to XIAP BIR2 are as follows. The experimental basis for
the approach was the prototypical structure of a Smac peptide (AVPI) bound to
XIAP BIR3,12 and our previous crystal structure showing a ‘Smac-like’ binding
of the N-terminus of caspase-3 from a crystallographic neighbor to the Smac
binding groove on XIAP BIR2 as described in Ref. 8. The latter still represents
the only experimental structural data available for Smac-like binding by the
XIAP BIR2 domain. The BIR domains of both structures were overlaid using
fitting algorithms from COOT and Pymol, which resulted in an overlay of the
P1–P3 residues of bound SMAC (AVP) and caspase-3 (SGV) of excellent fidelity,
and allowed us to place the SMAC sequence AVPI into the BIR2 SMAC binding
groove. We utilized the close structural similarity of compound 9t and AVPI to
model compound 9t onto BIR2 with backbone architecture and sidechain-like
moieties of compound 9t in similar orientations to AVP, while the sidechain
bearing the P4 indole was placed in the orientation observed for the Ile residue
of AVPI. This procedure was followed by a final energetic and geometrical
minimization step using Phenix.23 Comparison of this model with AVPI and the
BIR3 SMAC binding groove pointed to a generally very similar putative binding
of AVPI/compound 9t between XIAP BIR2 and 3 but revealed a different
binding pocket composition in P4 as described in the text and illustrated in
Figure 3.
search for clinically relevant drugs to circumvent TRAIL-resistant
cancers.
In summary, using a rational design approach we have systemat-
ically optimized small molecule monovalent Smac mimetics that in-
hibit XIAP by binding preferentially to the BIR2 domain. Compounds
9h, 9j, and 9t bind to the BIR2 domain with submicromolar affinities
and are three- to seven-fold selective for BIR2 over BIR3. In addition,
the XIAP inhibitory potency observed in vitro translates into cell kill-
ing activity in breast cancer cells, suggesting that these compounds
are potentially useful tools for probingapoptosis signaling pathways
in cells. Further optimization of these compounds towards the dis-
covery of potent and selective drug-like analogs is currently in
progress.
Acknowledgments
This work was supported by NIH grant HG005033 and
R01AA017238 to S.J.R. The authors thank Andrey Bobkov for expert
technical assistance. M.G.L. acknowledges Fundación Ramón Are-
ces for a postdoctoral fellowship.
References and notes
1. Riedl, S. J.; Salvesen, G. S. Nat. Rev. Mol. Cell Biol. 2007, 8, 405.
2. Lowe, S. W.; Lin, A. W. Carcinogenesis 2000, 21, 485.

9
M. González-Lopez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4332–4336
4336
22. Cell viability was assessed using the CellTiter-GloÒ Luminescent Cell Viability
Assay (Promega Corp., Madison, WI). Briefly, cells were seeded at 5000 cells/
cellular lysis before luminescence measurement on a Biotek Synergy 2 plate
reader. All experiments were carried out in at least triplicate, at least three
times. MDA-MB-231 cells were maintained in DMEM with 10% (v/v) FBS, and
well in 100 lL complete medium and allowed to attach overnight. The medium
was aspirated and replaced with fresh media containing the specified drug at
the concentrations described before re-incubation of the cells at 37 °C for 4 h.
TRAIL was then added to the desired final concentration and the cells again
incubated at 37 °C for 20 h. The plates were removed to room temperature for
penicillin/streptomycin/L-Glutamine (Omega Scientific Inc, Tarzana, CA).
23. Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.;
Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.;
Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.;
Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr., Sec. D: Biol. Crystallogr. 2010, 66,
213.
10 min before addition of one half volume (50
lL) of freshly prepared
CellTiterGlo reagent. The plates were gently shaken to ensure complete