Rational design, synthesis and characterization of potent, drug-like monomeric Smac mimetics as pro-apoptotic anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2012.01.098

A set of phenyl-substituted Smac mimetics/IAP inhibitor analogues of lead compound 2a was synthesized, aiming to retain its strong cell-free potency while increasing its bioavailability. Seventeen compounds 2b-r were prepared and characterized in vitro, u

Full text of DOI:10.1016/j.bmcl.2012.01.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2204–2208
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rational design, synthesis and characterization of potent, drug-like monomeric
Smac mimetics as pro-apoptotic anticancer agents
Aldo Bianchi a, Marcello Ugazzi b, Luca Ferrante a, Daniele Lecis c, Cinzia Scavullo d, Eloise Mastrangelo e,
Pierfausto Seneci a,b,
⇑
a CISI scrl, Via Fantoli 16/15, I-20138 Milan, Italy
b Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, I-20133 Milan, Italy
c Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42, I-20133 Milan, Italy
d Fondazione Matarelli, Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi di Milano, Via Vanvitelli 32, I-20129 Milan, Italy
e Dipartimento di Scienze Biomolecolari e Biotecnologie e CNR-INFM, Università degli Studi di Milano, Via Celoria 26, I-20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of phenyl-substituted Smac mimetics/IAP inhibitor analogues of lead compound 2a was synthe-
sized, aiming to retain its strong cell-free potency while increasing its bioavailability. Seventeen com-
pounds 2b–r were prepared and characterized in vitro, using cell-free and cellular assays. Among
them, the p-CF3 substituted analogue 2m showed the best permeability through cell membranes, and
was selected for further in vitro and in vivo studies due to its strong, sub-micromolar cellular potency.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 December 2011
Revised 24 January 2012
Accepted 25 January 2012
Available online 2 February 2012
Keywords:
IAP inhibitors
Smac mimetics
Apoptosis
Oncology
Peptidomimetics
The apoptotic process of Programmed Cell Death (PCD) is dys-
functional in its regulation in a variety of human pathologies,
among which cancer,1–3 inflammation4,5 and neurodegeneration.6,7
A variety of pathways have been reported as leading to PCD, and
have become the focus of extensive pharmaceutical research.
Among them the caspase-dependent extrinsic,8,9 or death recep-
tor-dependent, and intrinsic,8,10 or mitochondrial, paths.
Chances of therapeutic success should be maximized by hitting
more than one putative significant target with a lead. Thus, we
decided to target molecular entities involved in several apoptotic
pathways, rather than extremely targeted agents.
The most caspase-connected human IAP is the X-Linked Inhibi-
tor of Apoptosis Protein20,21 (XIAP). XIAP binds caspase 9 (the ini-
tiator caspase) and both caspases 3 and 7 (the executioner
caspases).22 XIAP binding prevents activation of caspases and, con-
sequently, prevents cells from entering PCD.
Another protein released from mitochondria is the Second
Mitochondria-derived Activator of Caspases (Smac)23/Direct IAp
Binding protein with LOw pI (DIABLO).24 It binds XIAP as a dimer25
on the binding site of caspase 9 (BIR3 domain).26–28 Smac inter-
feres also with the XIAP binding site of caspases 3 and 7 (linker-
BIR2 domain),29 thus promoting both the extrinsic and intrinsic
PCD paths. The balance of this binding equilibrium is altered in
various diseases; for example, several tumor cell lines show
over-expression of XIAP and, consequently, a resistance to enter
caspase-dependent PCD.30,31 Thus, XIAP inhibition via Smac
mimetics’ binding is a validated mechanism for intervention in
cancer therapy.32–34
Smac binding to therapeutically relevant cellular Inhibitor of
Apoptosis Protein 135 and 236 (cIAP-1 and cIAP-2) through their
BIR domains is also proven. Thus, Smac mimetics may modulate
the pathological action of XIAP and cIAPs,37–39 although the com-
plex network of IAP-dependent interactions still awaits full eluci-
dation in order to understand the implications for the use of
Smac mimetics in therapy.40
Inhibitor of Apoptosis Proteins (IAPs)11,12 are characterized by
the presence of one or more Baculovirus IAP Repeat (BIR) domains.
Since the discovery of the first viral iap gene and its linkage with
apoptosis,13 IAPs were considered putative targets for pro-apoptotic
drugs in oncology.14 The initial assumption of a common anti-
apoptotic mechanism for the whole IAP target family through inter-
ference with caspase-dependent pathways15,16 was proven to be
partially correct for other IAP proteins,17,18 but their interest as
oncology targets related to PCD has not been seriously challenged.19
⇑
Corresponding author. Tel.: +39 02 50314060; fax: +39 02 50314075.
E-mail address: pierfausto.seneci@unimi.it (P. Seneci).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.098

A. Bianchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2204–2208
2205
H
N
N
H
N
X
( )n
4
3
X
5
6
O
O
8
10
7
1
O
9
2
NH
N
H
N
O
N
H
N
N
H
O
R
O
AA
N
H
N
H
O
NH
O
2a
=
X heteroatom,
2
=
X CH2
1a
1b
=
AA aminoacid,
= )
X N-CO-CH2-CH CH3
2
(
=
R alkyl subst.
Figure 1. Monomeric Smac mimetics based on aza-containing bicycloalkane
carboxylate scaffolds.
Small monomeric Smac mimetics/IAP inhibitors 1a,b (Fig. 1, left),
inspired by the Smac N-terminal AVPI sequence and built on aza-
containing bicycloalkane scaffolds, bind to the BIR domains of XIAP,
cIAP-1 and cIAP-2 with low nanomolar potency. They also induce
rapid cIAP-1 degradation, and promote apoptosis in tumor cells
either as single agents, and when given in combination with other
cytotoxic agents.41,42 We43 reported a structure-driven approach
to potent, monomeric Smac mimetics/IAP inhibitors of general
formula 2 (Fig. 1, middle). 4-Substitution on the 1-aza-2-oxobicy-
clo[5.3.0]decane scaffold establishes novel molecular interactions
with the linker-BIR2 and/or BIR3 sites.44 A best-in-class representa-
tive 2a43,45,46 (Fig. 1, right) showed strong cell-free potency and a
quantifiable, although significantly reduced cellular potency.
We45 observed in the crystal structure of the 2a-cIAP-1 BIR3
complex (pdb: 3OZ1) that the 4-phenethylaminoethyl substituent
(red arrow, Fig. 2) does not participate to any significant binding
interaction. It rather appears disordered in the solvent, and its con-
tribution to the 2Fo–Fc difference Fourier map (magenta cage, con-
Figure 2. 2a-cIAP-1 BIR3 complex.45
R
HN
o
HN
N
N
H
O
O
NH
2b-r
toured at 1r, Fig. 2) is barely noticeable. We reasoned that phenyl
Figure 3. Phenyl-substituted analogues of lead compound 2a.
functionalization on the 4-substituent should not affect the bind-
ing potency of 2a, but could positively influence its physico-chem-
ical properties.
We report here the synthesis and in vitro characterization of a
small library of mono- and di-substituted phenyl analogues 2b–2r
(Fig. 3). Among them, we selected 2m as a bioavailable, cell-active
monomeric IAP inhibitor for in vivo biological characterization.
and cIAP-1 (column 5, Table 1), and their cytotoxicity on breast
cancer MDA-MB-231 cells (column 6, Table 1) were determined.
Assay conditions were as previously reported.43
Introduction of substituents on the phenyl ring does not affect
significantly the binding to BIR3 domains (columns 4 and 5). The
high binding efficiency of 2a is generally maintained, as expected,44
as is the stronger affinity observed for all compounds for cIAP-1
BIR3 when compared with XIAP BIR3. Namely, binding to XIAP
BIR3 varies between 41 nM (2j) and 198 nM (2k), while binding
to cIAP-1 BIR3 varies between <2 nM (2p,2q) and 3.3 nM (2n).51
Compounds 2a–r show different effects in cells. Their penetra-
tion through cell membranes should influence their cytotoxicity,
together with compound-specific mechanistic factors. The IC50 of
permeable compounds among 2a-r on MDA-MB-231 cells should
be comparable to their cell-free target binding—IC50 on the BIR3
domain of target IAPs.
We estimated the influence of 4-phenethyl ring substitution on
2a–r cell permeability, and on cytotoxicity, by measuring the ratio
IC50 MDA-MB-231/IC50 BIR3-XIAP (column 7, Table 1). We realize
that cIAP binding and subsequent degradation is a major mecha-
nism of action for Smac mimetics, but binding affinities for cIAP1
approached the sensitivity limit for the assay. In general, the sim-
ilar structure-activity trend observed for 2a–r on BIR3 domains
from the two IAP proteins supports our decision.
Compounds
2 are prepared from Boc-protected elongated
amine 3, obtained from accessible intermediate 447 through a
reported procedure43 (Scheme 1). Sub-optimal yields in the last
two steps were due to intra-molecular, OMs-mediated N3–O4 shift
of ethylglycine in 7, and to partial degradation of nitrile 8 in harsh
hydrogenation conditions.
As we needed gram amounts of 3, its synthesis was revised
(Scheme 2). N3-Boc protection of 9 followed by cyanide introduc-
tion to give 10 before N3-functionalization with ethylglycine led to
a threefold yield improvement.48
We also optimized the previously reported43 synthesis of lead
compound 2a. One-pot imine formation and reduction with
NaBH3X (X = CN, OAc) was slow at rt, and led to bis-alkylation/ter-
tiary amine formation upon heating (step a, Scheme 3). Thus, we
set up a TLC-monitored two-step procedure (steps a0,b0, Scheme
3) with imine formation prior to faster reduction using NaBH4 as
a stronger reducing agent. Acidic deprotection was improved by
careful determination of optimal reaction times and acid concen-
trations (see conditions and yields for b and c0, Scheme 3). The yield
increase and the absence of any side products allowed the prepara-
tion of an array of analogues of 2a.
Lead compound 2a shows a 73.5 ratio, due to an IC50 = 8.08 lM
on MDA-MB-231 cells46 and to an IC50 = 110 nM on the BIR3-XIAP
domain. Lower ratios would indicate an easier access to the intra-
cellular site of action of IAP inhibitors, while higher ratios would
indicate compounds whose penetration through cell membranes
is more difficult than for lead 2a.
Sixteen compounds 2b–q were prepared with moderate to good
yields as in a0–c0, Scheme 3 (columns 1–3, Table 1).49 The p-NH2
analogue 2r was prepared from the p-NO2, N-Boc protected com-
pound 12p as shown in Scheme 4.50
Compounds 2a–r were characterized as Smac mimetics. Their
binding affinity to the BIR3 domain of XIAP (column 4, Table 1)
Compounds bearing polar substituents—for example, 2o, 2p and
2q—may have a reduced permeation, with ratios close to 150. The

2206
A. Bianchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2204–2208
Ph
Ph
HN
O
COOt-Bu
O
COOMe
N
N
O
c-e
a,b
N
O
O
NH
53% -
3 steps
90% -
2 steps
HO
N
NH2
HO
O
Ph
6
N
5
Boc
4
quantitative
HN
f
Ph
Ph
Ph
Ph
Ph
Ph
HN
O
HN
N
O
N
N
O
O
h
g
O
O
NH
MsO
NH
NH
48 %
51%
O
N
O
H2N
O
N
Boc
N
N
3
8
Boc
Boc
7
Scheme 1. Un-optimized synthesis of Boc-protected elongated amine 3.
Ph
Ph
Ph
Ph
COOt-Bu
NH
O
NH
O
N
O
a-c
d-f
N
O
N
O
O
85 %
81 %
3 steps
73 %
2 steps
N
3 steps
NHBoc
Ph
NH2
HO
N
g,h
10
4
9
Ph
NH
Ph
Ph
NH
O
N
O
Ph
N
O
O
i
NH
N
NH
66 %
O
H2N
11
O
N
Boc
N
3
Boc
Scheme 2. Optimized synthesis of Boc-protected elongated amine 3.
Ph
Ph
Ph
Ph
Ph
Ph
HN
HN
HN
O
b - 50%
c' - 85%
N
O
O
a - 48%
O
O
N
N
O
O
O
O
a',b' - 81%
HN
HN
HN
N
Boc
N
Boc
NH
NH
H2N
NH
Ph
Ph
12a
3
2a
Scheme 3. Chemical routes to lead compound 2a.
positively charged p-NH2 analogue 2r, as expected, shows almost no
cytotoxicity due to its lack of permeation through the lipophilic cell
membrane. Most of the analogues bearing substituents with lower
polarity—that is, 2c–2g and 2j—show similar ratios to 2a. A few oth-
ers—that is, 2b, 2h, 2i, 2k and 2n, see bold values in Table 1—show
ratios between 6 and 20, possibly due also to higher permeability.
The latter compounds include ortho-(2b,2h), meta-(2i), para-(2n)
and di-(2k) substitutions, and a clear SAR depending on substitution
patterns does not emerge.
Best results were obtained with the p-CF3 analogue 2m—see
italic values in Table 1. Compound 2m has an IC50 = 111 nM
against the BIR3-XIAP domain, almost identical to parent com-

A. Bianchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2204–2208
2207
Table 1
Phenyl-substituted analogues 2a–r: synthesis and biological characterization
b
b
Compounda
Yield %, a0,b0
Yield %, c0
IC50 BIR3-XIAPc
IC50 BIR3-cIAP1c,d
IC50 MDA-MB-231e
Ratio IC50 MDA-MB-231/IC50 BIR3-XIAP
2a (R = H)
81
61
75
90
55
61
86
77
62
98
63
70
85
86
89
77
82
24f
85
89
87
84
89
82
77
93
82
77
85
92
57
54
92
82
76
47
110
58
52
57
78
47
47
53
44
41
198
65
111
181
57
58
57
52
<2 (0.9)
<2 (0.3)
<2 (0.3)
<2 (0.3)
<2 (0.8)
<2 (1.1)
<2 (0.6)
<2 (0.4)
<2 (0.3)
<2 (0.4)
2.6
<2 (0.2)
<2 (0.3)
3.3
<2 (0.4)
<2 (0.1)
<2 (0.1)
<2 (1.4)
8.08
0.40
3.77
3.70
6.27
2.72
3.14
0.92
0.43
2.79
1.44
10.2
0.23
1.53
10.1
11.2
9.24
>50
73.5
6.90
72.5
64.9
80.4
57.9
66.8
17.4
9.77
68.0
7.27
156.9
2.07
8.45
177.2
193.1
162.1
ꢁ1000
2b (R = o-Me)
2c (R = m-Me)
2d (R = p-Me)
2e (R = o-Cl)
2f (R = m-Cl)
2g (R = p-Cl)
2h (R = o-F)
2i (R = m-F)
2j (R = p-F)
2k (R = 3,5-diCl)
2l (R = 3,5-diF)
2m (R = p-CF3)
2n (R = p-tBu)
2o (R = p-OMe)
2p (R = p-NO2)
2q (R = p-COOMe)
2r (R = p-NH2)
a
b
c
d
e
f
R as in Figure 3.
a’, b’, c’ as in Scheme 3.
IC50, nM.
Lower sensitivity limit for the assays. Values in brackets are given as indications of affinity trends among the most potent compounds.
IC50 M.
,
l
Un-optimized yield of NO2 reduction to NH2.
Ph
HN
Ph
Ph
HN
O
Ph
N
O
N
O
O
O
a,b
12p
O
HN
HN
82%
2 steps
N
Boc
N
H2N
NH
Boc
c
24%
O2N
Ph
Ph
Ph
Ph
HN
O
HN
O
N
N
O
O
d
O
O
HN
HN
47%
Figure 4. Docking of 2m in cIAP-1 BIR3.52 Five docked conformations with lower
binding energy (full molecule, green conformation; 4-substituents, pink, magenta,
cyan and orange conformation) are shown.
N
Boc
N
H
NH
NH
H2N
H2N
Scheme 4. Synthesis of compound 2r.
2r
unbalance. Its pharmacokinetic evaluation and in vivo pharmaco-
logical characterization will be reported in due time.
12r
Acknowledgments
pound 2a, but shows an impressive IC50 = 230 nM on MDA-
MB-231 cells. The resulting 2.07 ratio suggests better
permeation through the cell membrane for 2m, when compared
to its analogues.
a
The authors are thankful to ‘FONDAZIONE CARIPLO’ for financ-
ing through the ‘Ricerca Biomedica 2009’ fund and the ‘Inhibitors
of Apoptosis Proteins (IAPs) as anticancer agents’ approved project.
We docked in silico 2m into the cIAP-1 BIR3 model, using Auto-
Dock4,52 to evaluate the preferred orientations of its 4-substituent
(Fig. 4). The five lower energy conformations, ranging between
ꢀ10.46 and ꢀ9.43 Kcal/mol, suggest that the 4-substituent is
highly mobile and does not establish any significant binding inter-
action. Thus, it is unlikely that the p-CF3 group strongly influences
the binding affinity of 2m for cIAP-1 BIR3.
Supplementary data
Supplementary data (synthetic procedures and full analytical
characterization of compounds 3 and 2a–r, and in vitro assay pro-
cedures) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2012.01.098.
Currently compound 2m is profiled in a number of in vitro cel-
lular tests on tumor cells showing an IAP-dependent apoptotic

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A. Bianchi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2204–2208
37. Chen, D. J.; Huerta, S. Anti-Cancer Drugs 2009, 20, 646.
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At room temperature, a solution of N-protected amines 12 (1 equiv) was
dissolved in 3N HCl (40 equiv) in MeOH. The mixture was stirred at room
temperature and the reaction was monitored by LC-MS. After reaction
completion (1–2 h), the solvent was evaporated under reduced pressure.
Crude products 2 were purified by semi-preparative HPLC, conditions: 15 min
gradient starting from 70% of H2O (+0.1%TFA) and 30% of CH3CN (+0.1%TFA) to
40% H2O (+0.1%TFA) and 60% of CH3CN (+0.1%TFA).
50. The synthesis of compound 2r using the chemical route shown in Scheme 4 is
thoroughly described in the Supplementary data.
51. The lower sensitivity in the assay is 2 nM. IC50 values were calculated from
three independent experiments performed in duplicate. Affinity trends for the
most active compounds are provided in Table 1 (values in brackets, column 5).
52. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comp. Chem. 1998, 19, 1639.
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