SPION-Smac mimetic nano-conjugates: Putative pro-apoptotic agents in oncology

Article abstract of DOI:10.1016/j.bmcl.2014.03.048

Non-covalent (NP-1/3) and covalent (NP-A-1/3) pro-apoptotic SPION-Smac mimetic nano-conjugates antitumor agents are reported. The solution synthesis of key Smac mimetics, their support onto SPIONs through non-covalent adsorption (NP-1/3) or APTES-mediated

Full text of DOI:10.1016/j.bmcl.2014.03.048

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2374–2378
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SPION-Smac mimetic nano-conjugates: Putative pro-apoptotic
agents in oncology
a
a
b
b
a
Pierfausto Seneci a, , Mattia Rizzi , Luigi Ballabio , Daniele Lecis , Annalisa Conti , Claudio Carrara ,
⇑
Emanuela Licandro a,
⇑
a Università degli Studi di Milano, Dipartimento di Chimica, Via Golgi 19, I-20133 Milan, Italy
b Fondazione IRCCS Istituto Nazionale dei Tumori, Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Via Amadeo 42, 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:
Non-covalent (NP-1/3) and covalent (NP-A-1/3) pro-apoptotic SPION-Smac mimetic nano-conjugates
antitumor agents are reported. The solution synthesis of key Smac mimetics, their support onto SPIONs
through non-covalent adsorption (NP-1/3) or APTES-mediated covalent binding (NP-A-1/3), the analyti-
cal characterization of SPION-Smac mimetic conjugates, their target affinity in cell-free assays, and their
cytotoxicity against tumor cells are thoroughly described.
Received 27 January 2014
Revised 16 March 2014
Accepted 17 March 2014
Available online 25 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
SPION nanoparticles
Smac mimetics
Pro-apoptotics
Oncology
Peptidomimetics
Nanoparticles are popular tools in cancer detection and treat-
ment, due to major advancements in nanosynthesis, bioengineer-
ing and imaging technology.1 Super Paramagnetic Iron Oxide
Nanoparticles (SPIONs) are particularly attractive as diagnostics
(e.g. biomarker-targeted magnetic resonance imaging/MRI
contrast agents) and therapeutics (e.g. magnetic nanoparticle-
enhanced hyperthermia).2 Their large surface areas to conjugate
targeting ligands and load therapeutic agents,3 their unique
magnetic properties,4 their biocompatibility5 and low toxicity6
make them suitable technology platforms in oncology.
We introduced 4-substituted, aza-bicyclo [5.3.0] decane (ABD)-
based N-AVPI mimetics as potent, pro-apoptotic cytotoxic agents
(see Fig. 1 for ABD structure and numbering). Monomers such as
1 (Smac136, Fig. 1) are potent, orally available BIR3 binders with
moderate cytotoxicity.11 Dimers such as 2 (Smac83, Fig. 1) bind
IAPs on both BIR domains, resulting in a stronger cytotoxic activity,
but possess a sub-optimal PK profile and cannot be administered
orally.12
We reasoned that SPION-Smac mimetic nano-conjugates may
take advantage of nanoparticle-specific access to cytosolic com-
partments (e.g. receptor-mediated endocytosis, macropinocyto-
sis).13 The spatial arrangement of monomeric Smac mimetics on
the surface of SPIONs may allow, or even promote, the binding of
two SPION-grafted Smac mimetics to a single IAP molecule
(dimer-like binding). Finally, a single SPION-Smac mimetic nano-
conjugate may bind several IAPs (multi-presentation mode). Thus,
we decided to synthesize and biologically characterize a small
library of SPION-Smac mimetic nano-conjugates.
4-CH2OR and 4-CH2NR1R2-substituted ABD Smac mimetics pos-
sess strong BIR/IAP affinity.11 N-Boc-protected hydroxyacid 4 and
N-Boc-protected aminoamide 5 were prepared from the tricyclic
intermediate 314 as previously reported11a (Scheme 1), although
several reaction conditions were optimized. 4-Connected
Smac-linker carboxylate constructs (6, ester connection, and 7,
amide connection) were obtained in good yields respectively from
compounds 4 and 5 (Scheme 1).
Anti-apoptotic Inhibition of Apoptosis Proteins (IAPs)7 bind
Cysteine ASPartic acid-specific proteASEs (caspases, CASP),8 the
major apoptotic effectors, through their baculovirus inhibitor re-
peat (BIR) domains. CASP-IAP interactions inactivate caspases
and block apoptosis. The endogenous Smac protein9 (Second Mito-
chondria-derived Activator of Caspases) binds to IAPs through its
N-terminus AVPI sequence. The tetrapeptide binds to BIR3, the
CASP-9 binding domain of IAPs, and prevents linker-BIR2-CASP-
3/7 interactions through a weaker interaction.10 Thus, Smac is a
pro-apoptotic IAP ligand that restores caspase-dependent apoptosis
in cancer cells.
⇑
Corresponding authors. Tel.: +39 02 5031 4060; fax: +39 02 5031 4072.
E-mail addresses: pierfausto.seneci@unimi.it (P. Seneci), emanuela.licandro@
unimi.it (E. Licandro).
http://dx.doi.org/10.1016/j.bmcl.2014.03.048
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

P. Seneci et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2374–2378
2375
H
O
N
N
O
H
N
10
H
N
N
N
H
4
HN
N
O
OH
N
O
3
O
O
N
NH
F C
3
2
N
H
1
6
5
8
7
4
9
N
1
3
10
2
N
HO
O
O
N
N
azabicyclo[5.3.0]decane
(ABD) scaffold
N
H
N
N
H
O
N
H
O
Figure 1. Monomeric and dimeric Smac mimetics.
O
O
4 steps (trans-
esterification,
COOH
N
H
O
N
O
N
a-c
reduction, amide
coupling and
O
O
N
H
N
H
hydrolysis, Ref.11a*)
HO
O
NH
6
N
H
3
Boc
O
+
t
4
HCOO-
COO Bu
N
O
HO
N
O
O
O
Ph
N
H
O
3
N
N
N
H
O
6 steps (hydrolysis,
amidation, reduction,
amide coupling,
O
d,e
O
N
H
N
H
H2N
HN
O
7
azidation and
azide reduction, Ref.11a*)
NH
N
H
3HCOO-
Boc
+
5
O
*: see Supplementary Information for
optimized reaction conditions
HO
a) Ph2CHNH2.HCl, EDC.HCl, HOBt, DIPEA, dry CH2Cl2, N2, rt, 24hrs, 51%; b) succinic anhydride, cat. DMAP, DIPEA,
dry CH2Cl2, N2, rt, 20hrs, 85%; c) neat HCOOH, rt, 3hrs, 75%; d) as in a), 53%; e) as in c), quantitative.
Scheme 1. Synthesis of 4-connected Smac-linker carboxylate constructs 6 and 7.
COOH
H
H
N
COOH
N
O
N
a-c
O
O
O
O
N
H
N
H
HO
HO
N
H
Boc
H3N
Cl –
4
8
+
a) (R)-PheGlyOMe, EDC.HCl, HOBt, DIPEA, dry CH2Cl2, N2, rt, 24hrs, 48%; b) 3N HCl in MeOH, rt, 20hrs, 95%;
c) LiOH, H2O/1,4-dioxane, rt, 3hrs, then 2N aq. HCl, quantitative.
Scheme 2. Synthesis of C terminus-connected Smac-linker carboxylate construct 8.
BIR3 affinity is also preserved when the C terminus diphenyl-
methyl amide of Smac mimetic 1 is replaced by an (R)-phenylgly-
cine amide. N-protected hydroxyacid 4 was used to prepare in
good yields the C terminus-connected Smac-linker carboxylate
construct 8 (Scheme 2).
Smac-linker constructs 6–8 and all previously unreported inter-
mediates were analytically and spectroscopically characterized
(LC–MS, 1H and 13C NMR).
The carboxy group of Smac-linker constructs 6–8 is used to
non-covalently adsorb the compounds onto surface-exposed OH

2376
P. Seneci et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2374–2378
O
O
O
N
N
H
O
N
O
N
H
O
O
a
N
H
N
NP-1, X=O
H
X
X
NH
H N
3
NP-2, X=NH
3
O
+
O
+
HCOO-
HCOO-
O
6,7
HO
O
COOH
H
H
N
O
O
N
H
N
b
O
O
N
H
O
O
O
NP-3
O
N
H
HO
N
H
H N
Cl
3
HO
8
+
–
NH3+Cl-
–
Cl
a) Naked SPIONs, H2O, sonication, 60°C, 4hrs, loading 0.272 mmol/gSPION (NP-1), 0.485 mmol/gSPION (NP-2);
b) Naked SPIONs, dry toluene, sonication, 60°C, 4hrs, loading 0.168 mmol/gSPION
.
Scheme 3. Synthesis of non-covalent SPION-Smac mimetic nano-conjugates NP-1/3.
a
EtO
O
+
EtO Si
EtO
NH2
O Si
O
NH2
NP-A
γ
-Fe2O3
SPIONs
O
O
O
N
H
O
N
H
N
O
N
O
O
O
b
NP-A-1, X=O
N
H
+
N
H
X
NP-A-2, X=NH
X
H3N
Cl –
NH3
O
O
+
HCOO-
6,7
O
Si O
O
HN
HO
COOH
H
N
H
O
O
H
H
N
O
N
N
H
O
b
O
O
N
O
Si O
O
NP-A-3
O
N
HO
H
N
H
+
H3N
HO
H3N
8
+
Cl–
Cl –
a) Naked SPIONs, dry toluene, N2, sonication, 60°C, 4hrs, loading 0.836 mmol/gSPION ; b) NP-A,
EDC.HCl, dry CH3 CN, N2, sonication, 60°C, 4hrs, loading 0.167 mmol/gSPION(NP-A-1), 0.308
mmol/gSPION(NP-A-2), 0.718 mmol/gSPION(NP-A-3).
Scheme 4. Synthesis of covalent SPION-Smac mimetic nano-conjugates NP-A-1/3.
groups of commercial SPIONs. Non-covalent nano-conjugates
NP-1/3 are obtained with moderate to good Smac mimetic loadings
(Scheme 3), as judged by elemental analysis.
loading SPION-APTES-NH2 construct NP-A (Scheme 4). NP-A is
then covalently coupled with the carboxy group of Smac-linker
constructs 6–8. Covalent nano-conjugates NP-A-1/3 are obtained
with moderate to good Smac loadings (Scheme 4) in unoptimized
reaction conditions.
Commercially available- maghemite
c-Fe2O3 SPIONs may be re-
acted with 3-aminopropyl triethoxysilane (APTES) to give the high

P. Seneci et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2374–2378
2377
O
N
O
O
O
O
H
N
H
N
H
N
H
COOMe
N
N
O
O
O
O
N
H
N
H
N
H
HN
HO
O
H2N
H2N
H2N
O
O
11
10
9
Figure 2. Reference Smac mimetics.
Non-covalent and covalent SPION-Smac mimetic nano-conju-
gates NP-1/3 and NP-A-1/3 were analytically and spectroscopically
characterized (FT-IR, elemental analysis). Single reaction runs for
each nanoconjugate often lead to good loadings (0.272–0.718
mmol/gSPION; NP-1, NP-2, NP-A-2, NP-A-3). Observed moderate
loadings (<0.200 mmol/gSPION) could be improved by repeating
the synthetic procedure for NP-3 and NP-A-1.
The affinity of the six SPION-Smac mimetic nano-conjugates for
mono-functional (BIR3 domains from X-linked IAP/XIAP, and from
cellular IAPs/cIAP1 and cIAP2) and bi-functional IAP constructs
(linker-BIR2-BIR3 multi-domain region from XIAP) was measured
in a cell-free assay format. Standard reference compounds 4-acet-
oxymethyl/9, 4-acetamidomethyl/10 and C terminus (R)-phenyl-
glycinamide methyl ester/11 (Fig. 2) were prepared in solution as
previously described.11a They were biologically tested as soluble
analogues respectively of non covalent/covalent SPION-Smac mi-
metic nano-conjugate pairs NP-1/A-1 (9), NP-2/A-2 (10) and
NP-3/A-3 (11).
cancer, HeLa cells). The nano-conjugates were generally inactive
against tumor cells, with slight signs of cytotoxicity at the highest
concentrations (ꢀ25
like cell free binding to IAP constructs, and reference monomers
9–11 show moderate cytotoxicity (IC50 between 10 and 25
lM). NP-1/2 and NP-A-2/3 show monomer-
lM
against MDA-MB-231 cells). Thus, the weak cytotoxicity of nano-
conjugates is likely due to a surprising lack of multimeric behavior
that prevents their expected, dimer-like interaction with multiple
bifunctional L-BIR2-BIR3 construct from XIAP.
In summary, we report here the synthesis, the spectroscopical
and biological characterization of non-covalent (NP-1/3) and
covalent (NP-A-1/3) SPION-Smac mimetic nano-conjugates as
IAP-targeted pro-apoptotic agents. SPION-Smac mimetic nano-
conjugates behave as their soluble monomer analogues, retaining
monomer-like cell free binding affinity for IAP targets, while being
almost inactive in cellular assays. Further efforts will aim to
achieve cellular and in vivo activity for SPION-Smac mimetic
nano-conjugates in suitable cellular apoptosis/oncology models.
Namely, we will vary the connection between Smac mimetics
and SPIONs (chemical bond, linker/spacer length, hydro/lipophilic-
ity, etc.), and we will decorate SPIONs with more potent mono-
meric and dimeric Smac mimetics.
The IC50 values determined for ABD-based Smac mimetics 9–11
and SPION-Smac mimetic non-covalent (NP-1/3) and covalent
(NP-A-1/3) nano-conjugates are listed in Table 1.
SPION-Smac mimetic nano-conjugates generally maintain the
nanomolar binding affinity of reference compounds 9–11 for single
BIR3 domains from IAPs. Both non-covalent adsorption and cova-
lent drafting of Smac mimetics onto SPIONs are compatible with
IAP binding (Table 1). For example, the non-covalent conjugate
NP-1 and the reference standard 9 show similar binding strength
in presence of BIR3 from XIAP and L-BIR2-BIR3 from XIAP; and
the same behavior is shown by covalent conjugate NP-A-3 and ref-
erence standard 11 (Table 1). Loading may influence affinity, as
NP-3 and NP-A-1 (loading <0.2 mmol/gSPION) show extremely re-
duced affinity for IAP proteins. Unfortunately, there is no evidence
of a dimer-like binding/increase of affinity (expected IC50 < 10 nM
for dimer-like compounds) for any nano-conjugate against the
bifunctional L-BIR2-BIR3 construct from XIAP.
Acknowledgments
We are grateful for the financial support from Cariplo Founda-
tion, Bando 2011, grant title: ‘Inter-cellular delivery, trafficking,
and toxicity of engineered magnetic nanoparticles in macrophages
and CNS cells’; and from Associazione Italiana Ricerca sul Cancro
(AIRC, MCO—9998, D.D.).
Supplementary data
Supplementary data (experimental procedures for the synthesis
in solution of Smac mimetics 6–8 and 11. LC–MS and NMR charac-
terization of Smac mimetics 6–8 and 11. Experimental procedures
for the preparation of non-covalent (NP-1/3) and covalent (NP-A-1/
3) SPION-Smac mimetic nano-conjugates. Elemental analysis and
FT-IR of NP-1/3 and NP-A-1/3. Experimental procedures for cell-
free testing/binding affinity determination of soluble Smac mimet-
ics and SPION-Smac mimetic nano-conjugates in presence of BIR
domains/constructs from IAPs. Experimental procedures for cellu-
lar cytotoxicity testing of SPION-Smac mimetic nano-conjugates
against human tumor cell lines) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2014.03.048.
The cytotoxicity of cell free-active NP-1/2 and NP-A-2/3
nano-conjugates was tested on three human tumor cell lines
(breast cancer, MDA-MB-231, ovarian carcinoma, IGROV-1, cervical
Table 1
Binding affinity on BIR domains, IC50 (nM)
Compd
BIR3 XIAP
BIR3 cIAP1
BIR3 cIAP2
L-BIR2-3 XIAP
9
400
360
>2000
330
560
230
760
>2000
580
NTa
38
>2000
NT
150
38
160
1400
270
NT
78
>2000
NT
62
320
300
>2000
190
240
120
190
410
410
NP-1
NP-A-1
10
NP-2
NP-A-2
11
NT
180
>2000
180
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NP-A-3
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