Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy

Article abstract of DOI:10.1016/j.bmc.2009.07.009

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimizati

Full text of DOI:10.1016/j.bmc.2009.07.009

Bioorganic & Medicinal Chemistry 17 (2009) 5834–5856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Rational design, synthesis and characterization of potent, non-peptidic Smac
mimics/XIAP inhibitors as proapoptotic agents for cancer therapy
b
Pierfausto Seneci a,b, , Aldo Bianchi , Cristina Battaglia b,c, Laura Belvisi a,b, Martino Bolognesi d,
*
Andrea Caprini b, Federica Cossu d, Elena de Franco b, Marilenia de Matteo b, Domenico Delia e,
Carmelo Drago b, Amira Khaled b, Daniele Lecis e, Leonardo Manzoni b,f, Moira Marizzoni b,
Eloise Mastrangelo d, Mario Milani d, Ilaria Motto b, Elisabetta Moroni b, Donatella Potenza a,b
Vincenzo Rizzo b, Federica Servida g, Elisa Turlizzi h, Maurizio Varrone h, Francesca Vasile b,
Carlo Scolastico a,b
,
a Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, I-20133 Milan, Italy
b Centro Interdisciplinare di Studi Biomolecolari e Applicazioni Industriali (CISI), Università degli Studi di Milano, Via Fantoli 16/15, I-20138 Milan, Italy
c Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate (MI), Italy
d Dipartimento di Scienze Biomolecolari e Biotecnologie e CNR-INFM, Università degli Studi di Milano, Via Celoria 26, I-20133 Milan, Italy
e Dipartimento di Oncologia Sperimentale, Istituto Nazionale dei Tumori (INT), Via Venezian 1, I-20133 Milan, Italy
f Istituto di Scienze e Tecnologie Molecolari (ISTM), Consiglio Nazionale delle Ricerche (CNR), Via Fantoli 16/15, I-20138, Milan, Italy
g Fondazione Matarelli, Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi di Milano, Via Vanvitelli 32, I-20129 Milan, Italy
h SienaBiotech S.p.A., Via Fiorentina 1, I-53100, Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized.
Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class
of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP
BIR3 and linker-BIR2–BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been
performed, identifying one lead for further in vitro and in vivo evaluation.
Received 21 May 2009
Revised 1 July 2009
Accepted 5 July 2009
Available online 10 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
XIAP
Smac
Apoptosis
Oncology
Rational drug design
Crystallography
NMR
Medicinal chemistry
1. Introduction
In such a heterogeneous and complex scenario, hitting more
than one of the putative significant targets with a given lead should
The apoptotic process of programmed cell death (PCD) and dys-
functions in its regulation in a variety of human pathologies,
among which cancer1,2 and neurodegenerative diseases,3 have be-
come the focus of extensive pharmaceutical research. A variety of
pathways have been reported as leading to PCD. Among them,
the extrinsic or death receptor-dependent path4,5 and the intrinsic
or mitochondrial path,4,6 both of them caspase-dependent; and a
variety of caspase-independent pathways, such as apoptosis induc-
ing factor (AIF)-dependent apoptosis.7
maximize the chances of therapeutic success. Thus, we focused on
targeting molecular entities involved both in the extrinsic and
intrinsic caspase-dependent pathways.
The family of Inhibitor of Apoptosis Proteins (IAPs)8,9 is charac-
terized 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,10 IAPs were considered putative targets
for pro-apoptotic drugs in oncology. The assumption of a common
anti-apoptotic mechanism for IAPs through interference with cas-
pase-dependent pathways11 has been proven only partially correct
for some human IAPs,12,13 but their interest as oncology targets re-
lated to PCD has not been seriously challenged.
* Corresponding author. Tel.: +39 02 50320914; fax: +39 02 50320945.
E-mail address: pierfausto.seneci@unimi.it (P. Seneci).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.009

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5835
Surely, the most caspase-connected human IAP is the X-Inhibi-
tor of Apoptosis Protein14,15 (XIAP). XIAP is capable of binding
caspase 9 (the initiator caspase) and both caspases 3 and 7 (the
executioner caspases).16 XIAP binding prevents activation of
caspases and, consequently, prevents cells from entering PCD. An-
other protein released from mitochondria, the Second Mitochon-
dria-derived Activator of Caspases (Smac)17/Direct IAp Binding
protein with LOw pI (DIABLO)18 is able to bind XIAP as a dimer19
on the same binding sites of caspase 9 (BIR3 domain).20–22 Smac
interferes also with the XIAP binding site of caspases 3 and 7 (lin-
ker-BIR2 domain),23 thus promoting both the extrinsic and intrin-
sic PCD paths. The delicate balance of this binding equilibrium is
altered in various diseases; for example, several tumour cell lines
show overexpression of XIAP and, consequently, a caspase-depen-
dent resistance to enter PCD.24,25 Thus, XIAP inhibition via Smac
mimics’ binding is considered a validated mechanism for interven-
tion in cancer therapy;26 moreover, structurally related human IAP
family members such as cIAP127 and cIAP2,28 other popular oncol-
ogy targets,29 can also be targeted by Smac mimics.
Recently several authors have shown how small monomeric
Smac mimics/XIAP inhibitors such as 1a and 1b, inspired by the
Smac N-terminal AVPI sequence and built on the 1-aza-2-oxo-3-
aminobicyclo[5.3.0]decane-10-carboxylic acid scaffold 2, can bind
XIAP on its BIR3 domain with sub-micromolar potency, and have
an effect on apoptosis in tumour cells.30,31 Here we report a struc-
ture-driven approach to potent, monomeric Smac mimics/XIAP
inhibitors of general formula I.32
The introduction of an appropriate 4-substitution on the 1-aza-
2-oxobicyclo[5.3.0]decane scaffold (i.e., (m)X = CH2OH as in top
left, and (m)X = CH2NH2 as in top right, Fig. 1), when compared
with known 1a,b30,31 and other 4-unsubstituted analogues, estab-
lishes novel molecular interactions with the binding sites on XIAP
and contributes to an overall improvement in the ‘drug-like’ profile
(i.e., solubility, penetration through biological membranes) of our
Smac mimics/XIAP inhibitors. Computational, NMR and X-ray data,
and the resulting structural information acquired, have contrib-
uted to the establishment of a preliminary SAR for XIAP inhibitors,
and to the design of optimized analogues.
2. Results and discussion
2.1. Computational studies
Some of us recently reported33,34 an innovative synthesis strat-
egy to access compounds of general formula I, which differ from
reported structures for an additional 4-substitution on the 1-aza-
2-oxobicyclo[5.3.0]decane scaffold. Having secured synthetic ac-
cess to such structures, we decided to ascertain if appropriate sub-
stituents on the 4-position could be reconduced to advantageous
interactions with members of the IAP family, and with XIAP in par-
ticular. A structure-based approach, based on a computational
model of the AVPI-binding sites of the BIR3 domain of XIAP and
on data from NMR and X-ray crystallography, was conceived to
understand and optimize the effects of 4-substitutions on the 1-
aza-2-oxobicyclo[5.3.0]decane nucleus.
A docking approach has been set up starting from the high res-
olution crystal structure of the Smac/BIR3 complex (PDB code
1G73).21 The Glide35 docking protocol reproduced the crystallo-
graphic binding mode of AVPI in BIR3, and the measured BIR3
binding affinity trend of a previously reported30 training set of
Smac mimics including 1a. The validated docking protocol was ap-
plied to a small library of ligands of general formula I (most prom-
ising structures shown in Fig. 1).
Nitrogen and/or oxygen-containing functional groups in posi-
tion 4 (e.g., amines, alcohols, amides and ureas) and aromatic lipo-
Figure 1. Prioritized 4-substitutions.

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P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
philic extensions on the 4-arm (e.g., phenyl ester and amide, ben-
zyl amine or urea, Fig. 1) resulted to be privileged substitutions. In
particular, 4-substituted compounds in Figure 1 showed an AVPI-
like binding mode and additional hydrophobic contacts with the
protein involving the azabicycloalkane scaffold and the phenyl
rings. Furthermore, most of them seemed to establish additional
interactions through their 4-side chains, such as hydrogen bonds
through the interaction with the Thr308 and/or the Asp309 BIR3
residues (shown for the hydroxy compound, Fig. 2).
as hydrochloride salts (overall unoptimized yield: 35%, 3a and
8%, 4a).
Recently it was reported28 that the N-methyl 4-unsubstituted
compound 1b retains similar potency on cell-free XIAP binding as-
says, while largely improving its cytotoxicity against cancer cells
with respect to 1a. We thus prepared the N-methylated 4-CH2OH
and 4-CH2NH2 compounds 3b and 4b from 5 (overall unoptimized
yield: 39%, 3b and 6%, 4b) as seen for compounds 3a and 4a, simply
replacing BocNHAbuOH with BocN(Me)AbuOH and going through
intermediates 6b–7b (3b) and 6b–10b (4b, Scheme 1). Due to the
instability of 9b in acidic Boc deprotection conditions, 4b was ob-
tained via Boc deprotection (10b) and final azide reduction (H2,
Pd/C).
These encouraging theoretical findings prompted us to synthe-
size
a medium-sized array of 4-substituted 1-aza-2-oxobicy-
clo[5.3.0]decanes of general formula II, where X is a substituted
nitrogen atom or a substituted oxygen atom. Thus, we constantly
kept the (S)-ethylglycine and the diphenylamino ‘best’ substitu-
tions on the 3-NH2 and 10-COOH groups in scaffold 2, targeting
compounds of formula IIa.
Compounds 7a, 8a and 9a were used to access other 4-substi-
tuted Smac mimics. Most compounds were obtained as pure mate-
rials after final preparative HPLC purification. The synthesis of
compounds prepared from N-protected alcohol 7a and N-protected
azide 8a is reported in Scheme 2.
The N-protected alcohol 7a was acylated (RCOCl, DMAP) to give
N-Boc protected 11a,b, and deprotected (TFA, CH2Cl2) to provide
the esters 12a,b as TFA salts (overall unoptimized yield: 46%, 12a
and 85%, 12b). The N-protected azide 8a was deprotected (HCl,
MeOH) to provide the azide 10a as a hydrochloride salt in 45%
unoptimized yield. It was also reacted in ‘click chemistry’ condi-
tions (1-pentynyl-3-ol, Cu(OAc)2, sodium ascorbate) to give the
triazolyl alcohol 14a as a hydrochloride salt after deprotection of
N-Boc protected 13a (HCl, MeOH) in overall 58% unoptimized
yield. Finally, it was guanylated (guanylating agent, LiOH, Ph3P)
to yield the guanidino compound 16a after deprotection of N-Boc
protected 15a (HCl, MeOH) in overall 31% unoptimized yield.
The N-protected primary amine 9a was used to obtain several
classes of compounds (Schemes 3 and 4).
Secondary amines 18a,b were prepared (overall unoptimized
yield: 71%, 18a and 76%, 18b) by reductive amination (RCHO, NaB-
H(OAc)3) to give N-Boc protected compounds 17a,b, and deprotec-
tion (HCl, MeOH). Both N-Boc protected secondary amines were
also converted to tertiary amines 20a,b (from 17b, overall unop-
timized yield from 9a: 70%, 20a and 68%, 20b) and 20c (from
17a, overall unoptimized yield from 9a: 47%) by reductive amina-
tion (RCHO, NaBH(OAc)3) and deprotection (HCl, MeOH) (Scheme
3).
2.2. Synthesis—structures IIa
At first, we prepared the 4-substituted primary alcohol 3a and
primary amine 4a (Scheme 1). Briefly, compound 534 was transe-
sterified (HCl, MeOH), hydrogenated (Pd/C, 5 atm) and coupled
with BocNHAbuOH (HOBt, EDC, DIPEA) to give 6a. Ester hydroly-
sis (LiOH) and coupling with diphenylaminomethane (HOBt, EDC,
DIPEA) gave 7a. The N-protected 4-CH2OH compound 7a was
then transformed into the 4-CH2NH2 compound 9a via mesylation
(MsCl, TEA), azide displacement (NaN3) to give the N-protected 4-
CH2N3 compound 8a, and Staudinger reduction. N-Boc protected
compounds 7a and 9a were deprotected (HCl, MeOH) to give,
after preparative HPLC purification, pure compounds 3a and 4a
Carboxylic amides 22a–c were prepared (overall unoptimized
yield: 61%, 22a , 88%, 22b and 71%, 22c) either from carboxylic
acids (22a: RCOOH, EDC, HOBt, DIPEA) or from acyl chlorides
(22b,c: RCOCl, pyridine), after final deprotection of N-Boc pro-
tected compounds 21a–c (HCl, MeOH) (Scheme 4). Similarly, sul-
fonamides 24a,b were prepared (overall unoptimized yield: 67%,
24a and 74%, 24b) from sulfonyl chlorides (RSO2Cl, K2CO3), after fi-
nal deprotection of N-Boc protected compounds 23a,b (HCl,
MeOH). N,N0-Substituted ureas 26a,b (overall unoptimized yield:
81%, 26a and 93%, 26b) and thioureas 26c,d (overall unoptimized
yield: 87%, 26c and 83%, 26d) were prepared by the addition of
the corresponding isocyanates or isothiocyanates (RCNX, DIPEA),
followed by deprotection of N-Boc protected compounds 25a–d
(HCl, MeOH). Finally, the N0-unsubstituted urea 28a (overall 66%
unoptimized yield) and thiourea 28b (overall 56% unoptimized
yield) were prepared by addition of the corresponding benzoyliso-
cyanate or isothiocyanate (PhCONCX, DIPEA), followed by basic
(K2CO3, MeOH) and acidic (HCl, MeOH) deprotection of N-Boc pro-
tected compounds 27a,b (HCl, MeOH).
2.3. Biophysical characterization—structures IIa
Figure 2. Docking best pose of the 4-CH2OH compound (cyan) into the crystallo-
graphic (1G73) BIR3 binding site (protein residues involved in hydrogen bond
interactions are labelled; C atoms in green, N in blue and O in red) overlaid on the
AVPI-bound conformation (magenta). Non polar hydrogens hidden for better visual
representation, intermolecular hydrogen bonds visible (black dashed lines).
In parallel with our synthetic efforts, we investigated the influ-
ence of the 4-substitution in Smac mimics/IAPs inhibitors of gen-
eral formula IIa by using some popular biophysical methods.

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5837
Scheme 1. Reagents and conditions: (i) 3 N HCl in MeOH, rt; (ii) H2, Pd/C, 5 atm, rt; (iii) BocNHAbuOH (a) or BocN(Me)AbuOH (b), HOBt, EDCꢀHCl, DIPEA, CH2Cl2, rt; (iv) LiOH,
1,4-dioxane, rt; (v) Ph2CHNH2, HOBt, EDCꢀHCl, DIPEA, CH2Cl2, rt; (vi) MsCl, TEA, CH2Cl2, rt; (vii) NaN3, DMF, 80 °C; (viii) Me3P, H2O/CH2Cl2, rt; (ix) H2, Pd/C, H2O/1,4-dioxane
1:1, rt.
Namely, we submitted a few compounds (together with the stan-
dard compound 1a30) to extensive NMR and X-ray characterization
in presence of either full length XIAP, and some of its BIR domains
responsible for caspase and Smac binding.
average, relative to 3a) towards the Asp309 residue, with concom-
itant loss of hydrogen bonds and hydrophobic interactions. The
shift is mostly due to the appearance of a novel hydrogen bond/salt
bridge interaction between the positively charged 4-CH2NH2 group
in 4a and the highly mobile Asp309 residue36 (Fig. 3).
As to crystallography, while co-crystals with 1a were not ob-
tained, both compounds 3a and 4a provided good co-crystals with
the BIR3 domain of XIAP. Up to now, we could not obtain any co-
crystal of either 1a, 3a or 4a with the BIR2 XIAP domain. The
BIR3-inhibitor crystals of 3a and 4a were thoroughly characterized
through X-ray crystallography (3D structures refined at 2.7 and
2.5 Å resolution, respectively).36 The compounds bind to the AVPI
site, establishing specific hydrogen bonds and contacts. The pri-
mary alcohol 3a shows a binding mode with minimal changes with
respect to the BIR3–AVPI complex, although an additional hydro-
gen bond stems from the interaction of the 4-CH2OH group with
Thr308 of BIR337 (data not shown). Remarkably, the binding mode
of the primary amine 4a with BIR3 shows an overall shift (1.5 Å on
As to NMR, saturation transfer difference (STD-NMR)38 was
used to highlight binding interactions between 1a, 3a, 4a, 16a
and 18b as small ligands, and recombinant XIAP BIR3 (primary
Smac binding site, caspase 9 binding site), the construct linker-
BIR2–BIR3 (including the secondary Smac binding site linker-
BIR2, caspases 3 and 7 binding site) and full length XIAP as targets.
Using STD, only the ligand protons interacting with the target are
visible in the NMR spectrum (see formula IIa for proton number-
ing), thus identifying the ligand binding regions. While we could
not obtain and use the linker-BIR2 construct per se, we reasoned
that any additional proton–ligand interaction observed for a given
small ligand in presence of the construct linker-BIR2–BIR3 (or of

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P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
Scheme 2. Reagents and conditions: (i) RCOCl, DMAP, CH2Cl2, rt; (ii) TFA, CH2Cl2, rt; (iii) 3 N HCl in MeOH, rt; (iv) 1-pentynyl-3-ol, sodium ascorbate, Cu(OAc)2, H2O/t-BuOH,
rt; (v) N,N0-bis-Boc-1-guanylpyrazole, LiOH, Ph3P, H2O/THF, rt.
Scheme 3. Reagents and conditions: (i) RCHO, NaBH(OAc)3, CH2Cl2, rt; (ii) 3 N HCl in MeOH, rt; (iii) RCHO, NaBH(OAc)3, AcOH, CH2Cl2, rt.

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5839
Scheme 4. Reagents and conditions: (i) RCOOH, EDC, HOBt, DIPEA, CH2Cl2, rt; (ii) RCOCl, pyridine, CH2Cl2, rt; (iii) 3 N HCl in MeOH, rt; (iv) RSO2Cl, K2CO3, CH2Cl2, rt; (v) RCNX,
DIPEA, CH2Cl2, rt; (vi) PhCOCNX, DIPEA, CH2Cl2, rt; (vii) K2CO3, MeOH, rt.
nylamido (7.3 ppm-Ar amide, 6.0 ppm-H18, strong interaction) and
the ethylglycine moiety (0.9 ppm-H15) both interact with BIR3
(lane A), while no interaction with any scaffold proton is detect-
able. Conversely, both the construct linker-BIR2–BIR3 (lane B)
and full length XIAP (lane C) show a strong interaction with the
4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold itself (e.g.,
4.0 ppm-H7, 3.6 ppm-H11/4-substitution), likely due to the linker-
BIR2 domain.
Primary amine 4a, when compared with 3a, shows only a weak
interaction of the ethylglycine moiety with BIR3 (data not shown)
most likely due to a shift in the BIR3 binding site in agreement
with previously described X-rays evidence. Guanidine 16a (data
not shown) does not bind at all with BIR3, while benzylamine
18b (Fig. 6) has an interaction profile with BIR3 comparable with
3a (lane A). Namely, we observe interactions with Ar amide
(7.3 ppm), H18 (6.0 ppm) and H15 (0.9 ppm) protons; for the first
time we also observe an interaction between the BIR3 domain
and the lipophilic part of the 4-substituent (7.4 ppm-benzyl Ar).
Interactions between the N-benzyl 4-substitution of 18b and XIAP
BIR3 could also be observed within our computational experi-
Figure 3. X-ray crystal structure of 4a (blue skeletal model) and XIAP BIR3 (grey
ments (i.e., proximity of the benzyl in 18b with the side chain
ribbon): the interaction between the 4-CH2NH2 group and Asp309 is visible in the
of Thr308, Fig. 7; favourable van der Waals contacts with the side
upper part. N- and C-terminal ends of the BIR3 domain are labeled.
chain of Asp309, Lys311 or Trp323 in other docking poses, data
not shown).
full length XIAP), and absent in presence of the BIR3 domain alone
should indicate an interaction targeted to the linker-BIR2 binding
site.
The spectra of reference compound 1a (Fig. 4) in presence of
BIR3 (lane A) and of full length XIAP (lane B) show a similar inter-
action pattern, including protons in the bicyclic scaffold (e.g., H3
and H10), on the diphenylamide moiety (e.g., the aromatic protons)
and on the ethylglycine moiety (e.g., H13, H15).
4-Unsubstituted 1-aza-2-oxobicyclo[5.3.0]decanes, such as 1a,
seem to interact with the two XIAP-caspase binding sites similarly.
Compounds 3a, 4a, 16a and 18b behave as 1a in terms of binding
with the linker-BIR2–BIR3 construct, sometimes showing addi-
tional interactions with 4-substituents. Namely, alcohol 3a
(3.6 ppm-H11/4-substitution, lane B, Fig. 5) and benzylamine 18b
(7.4 ppm-benzyl Ar, lane B, Fig. 6) show such interactions, while
small, charged 4-substituents (primary amine 4a, guanidine 16a)
do not interact with linker-BIR2–BIR3. Conversely, substitution in
4 appears to prevent the interactions of scaffold protons and to
modulate the interactions of the ethylglycine moiety with BIR3
The spectra of 3a (Fig. 5) in presence of various binding partners
suggest different binding interactions of 4-substituted 1-aza-2-
oxobicyclo[5.3.0]decanes with BIR2 and BIR3 domains. The diphe-

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P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
Figure 4. STD-NMR spectra of compound 1a with BIR3 (A), full length XIAP (B) and as such (C).
Figure 5. STD-NMR spectra of compound 3a with BIR3 (A), linker BIR2–BIR3 (B), full length XIAP (C) and as such (D).
(3a,18b > 4a > 16a), thus differentiating our small ligands in terms
of binding properties on different caspase binding domains. Ongo-
ing and future STD–NMR experiments should clarify the impact of
differential BIR2 and BIR3 binding modes for our small ligands on
their biological activity, and to establish a robust SAR for further 4-
substitutions.

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5841
Figure 6. STD-NMR spectra of compound 18b with BIR3 (A), linker-BIR2–BIR3 (B) and as such (C).
2.4. Synthesis, computational and biophysical
characterization—elongated structures
In accordance with suggestions from crystallography, we first
aimed to primary amine (R = CH2CH2NH2) 32a and alcohol
(R = CH2CH2OH) 34a, and to their N-methylated analogues 32b
and 34b, whose synthesis is reported in Scheme 5.
Briefly, compounds 7a or 7b were homologated to elongated
amines 32a,b via mesylation (MsCl, TEA) and cyanide displacement
(nBu4NCN, DMF) to give N-Boc nitriles 29a,b, hydrogenation (H2,
Ni-Raney) to give N-Boc amines 30a,b and N-Boc deprotection
(HCl, MeOH; overall unoptimized yield: 25%, 32a and 41%, 32b).
N-Boc deprotection of 29a (HCl, MeOH) yielded also pure nitrile
31a in quantitative yield (deprotection of 29b was not attempted).
N-Boc amines 30a,b were converted to primary alcohols 34a,b via
nitrosation (NaNO2, citric acid) and N-Boc deprotection (HCl,
MeOH), although with poor yields (overall unoptimized yield:
12%, 34a and 9%, 34b).
Compounds 30a,b were also used to access the 1-aza-2-oxobi-
cyclo[5.3.0]decane amides 36a,b (overall unoptimized yield: 50%,
36a and 55%, 36b), ureas 38a,b (overall unoptimized yield: 31%,
38a and 89%, 38b), ethylamines 40a,b (overall unoptimized yield:
24%, 40a and 21%, 40b) and benzylamines 40c,d (overall unoptim-
ized yield: 41%, 40c and 48%, 40d) (Scheme 6). Reaction conditions
were similar to the ones used for the synthesis of secondary
amines, amides and ureas in Schemes 3 and 4. Most compounds
needed a final preparative HPLC purification to be obtained as pure
materials.
Compound 32a was then fully characterized with our structure-
based, biophysical engine.
As to crystallography, we have recently obtained co-crystals of
32a with the BIR3 domain of XIAP.37 The XIAP BIR3/32a crystal
structure reported in Figure 8 (superimposition between crystals’
data and docking models of 32a in XIAP BIR3 active site) is in
Figure 7. Docking best pose of 18b (cyan) into the crystallographic (1G73) BIR3
binding site (protein residues involved in hydrogen bond interactions are labeled; C
atoms are shown in green, N in blue, and O in red). Intermolecular hydrogen bonds
are indicated as black dashed lines. Non polar hydrogens of Thr308 side chain and of
the 4-CH2NHCH2Ph group in 18b are shown to display van der Waals contacts (i.e.,
dashed red line, H–H distance = 3.0 Å).
Crystallographic data suggested a modest elongation of the
scaffold’s arm in 4a to optimize the interaction between the 4-
CH2NH2 and Asp309, without causing the shift of the ligand mole-
cule observed for 4a with associated loss of stabilizing interac-
tions.36 Thus, 4-elongated compounds with an additional CH2
unit between position 4 on the scaffold and an X substituent,
where X is a substituted nitrogen atom or a substituted oxygen
atom, were targeted.

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P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
Scheme 5. Reagents and conditions: (i) MsCl, TEA, CH2Cl2, rt; (ii) nBu4NCN, DMF, 80 °C; (iii) H2, Ni-Raney; (iv) 3 N HCl in MeOH, rt; (v) NaNO2, citric acid, THF, 0 °C to rt.
Scheme 6. Reagents and conditions: (i) PhCOCl, DIPEA, CH2Cl2, rt; (ii) 3 N HCl in MeOH, rt; (iii) PhCH2NCO, DIPEA, CH2Cl2, rt; (iv) EtCHO, NaBH(OAc)3, CH2Cl2, rt; (v) PhCHO,
NaBH(OAc)3, CH2Cl2, rt.
agreement with our predictions. The interactions between the 4-
CH2CH2NH2 arm and Asp309, and the prevention of ligand mole-
cule shift observed for 4a confirm the validity of the design criteria
adopted.

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5843
at a comparable level with 3a. This indicates, once again in accor-
dance with crystallographic evidence, the non-shifted, 3a-like
binding mode of elongated 32a with BIR3.
2.5. In vitro biology profiling
Reference compounds 1a30 and 1b,31 non-elongated com-
pounds 3a–31a (see Fig. 10) and elongated compounds 32a–40d
(see Fig. 11) were tested for their in vitro binding to XIAP BIR3
and linker-BIR2–BIR3 domains, using two reported assay
formats.39,40
Their IC50 values on both fragments of full length XIAP are re-
ported in Table 1. We derive for standard compound 1a a
Ki = 200 nM, definitely higher than the reported 25 nM value.30
As to 1b, we obtain a Ki = 230 nM versus the reported 61 nM va-
lue.31 These discrepancies may stem from different environmental
parameters and from Ki calculation methods, as discussed in de-
tails elsewhere.36
The data show how the introduction of a 4-substitution with N-
or O-containing functional groups on the 1-aza-2-oxobicy-
clo[5.3.0]decane scaffold is often beneficial in terms of affinity for
BIR3. For example, 4-CH2X-subst. compounds bearing hydroxyl
groups (3a), secondary amines (i.e., 18a and 18b), amides (i.e.,
22c) and ureas/thioureas (i.e., 26a and 26b) show around a twofold
increase in potency, when compared to 1a and 1b. Interestingly,
aromatic lipophilic extensions on the 4-arm, as in benzyl amine
18b, benzamide 22c and benzyl urea 26b, were recommended by
modelling studies. Furthermore, it is significant to observe that
the 4-CH2NH2 substituted 4a shows a reduced affinity for BIR3
Figure 8. X-ray crystal structure of 32a and XIAP BIR3. Superposition of the lowest
free energy XIAP BIR3/32a docked model (32a light blue, protein light grey) on the
XIAP BIR3/32a crystal structure (32a cyan, protein grey). Residues involved in the
main stabilizing interactions are shown.
As to NMR, the STD–NMR spectra of 32a (Fig. 9) in presence of
various binding partners confirms different binding interactions of
4-substituted 1-aza-2-oxobicyclo[5.3.0]decanes with BIR2 and
BIR3 domains. As seen for 3a (Fig. 4), the diphenylamido and the
ethylglycine moiety interact with BIR3 (lane A, 7.3 ppm-Ar amide,
6.0 ppm-H18, 0.9 ppm-H15, Fig. 9), while the construct linker-BIR2–
BIR3 shows a putative linker-BIR2 interaction with the 4-substi-
tuted 1-aza-2-oxobicyclo[5.3.0]decane scaffold itself (lane B,
4.0 ppm-H7, Fig. 9), although less intense than observed for 3a.
Interestingly, although the 4-CH2CH2NH2 moiety (H11 ꢁ 1.8 ppm,
H19 ꢁ 2.9 ppm) in 32a does not interact with any of the targets,
as seen previously for 4a and 16a, the interaction between its eth-
ylglycine moiety and BIR3 (weak for 4a, absent for 16a) is restored
(IC50 ffi 1
lM), while its 4-elongated homologue 32a is more potent
(IC50 = 250 nM). These results substantiate the crystallographic
prediction of loss of potency for 4a (overall shift towards the
Asp309 residue, with concomitant loss of hydrogen bonds and
hydrophobic interactions) and of increased potency for 32a
(optimization of the interaction between 4-CH2NH2 and Asp309,
Figure 9. STD-NMR spectra of compound 32a with BIR3 (A), linker BIR2–BIR3 (B) and as such (C).

5844
P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
Figure 10. Chemical structures of 4-unsubstituted and non-elongated compounds 1a–31a.
without binding-detrimental overall shifts). The same trend is ob-
served on 4 N-substituted, non-elongated/elongated couples (18a/
40a, 18b/40c, 22c/36a, 26b/38a), where the increase in potency
with elongation varies between 2 (18a/40a, 22c/36a, 26b/38a)
and 6 (18b/40c).
Our results on the XIAP linker-BIR2–BIR3 construct are even
more surprising. It has been shown41,42 that dimeric Smac mimet-
ics based on scaffold 2 have a low nanomolar-high picomolar IC50
on the bivalent binding construct, most likely due to their intramo-
lecular binding to both BIR domains of the same biological target
molecule.43 Reference monomers 1a and 1b show similar IC50 val-
ues (around 4–500 nM) for both BIR3 and linker-BIR2–BIR3 con-
structs from XIAP. This is to be expected, as BIR3 is the high
affinity binding site (Kd around 500 nM) for the Smac N-terminal
AVPI peptide, while BIR2 is the lower affinity binding site (Kd
This trend is not observed with non-elongated (3a) and elon-
gated (34a) alcohols, which show similar potencies. This is once
again in accordance with our structural data, which show no over-
all binding-detrimental shift in the active site pocket of BIR3 for 3a.
N-methylation on the ethylglycine residue in 4-unsubstituted, 1-
aza-2-oxobicyclo[5.3.0]alkane-based Smac mimics only marginally
decreases their affinity for the BIR3 domain of XIAP, while being
beneficial in terms of cytotoxicity on cancer cells.31 Comparing
IC50 data on BIR3 for 8 NH/N-Me couples presented in this work
(3a/b, 4a/b, 32a/b, 34a/b, 36a/b, 38a/b, 40a/b, 40c/d), we confirm
that N-methylated Smac mimics are potent BIR3 binders. Surpris-
ingly, they are consistently better than their NH analogues, up to
one order of magnitude (4a/b; in this case, N-methylation could
influence the overall binding-detrimental shift in the active site
pocket of BIR3 observed for 4a), with the single exception of the
40c/d couple.
around 10 lM) for the same peptide. Thus, adding the BIR2 domain
to the XIAP binding construct should not change the measured Kd
or IC50 for a monomer, while simultaneous binding to BIR2 and
BIR3 by a dimeric molecule causes a significant increase in potency
and mimicks the biological mode of action and potency of full
length Smac. An indirect confirmation of this hypothesis is given
by the similar potency of monomeric and dimeric Smac mimetics
on the XIAP BIR3 single domain.41,43 Most of our compounds show
a significant increase in potency when tested on the linker-BIR2–
BIR3 construct. For example, compounds 4a, 16a, 18b, 20b, 20c,
26c, 26d and 28b show a ꢂ4-fold increase in potency; and most

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5845
Figure 11. Chemical structures of elongated compounds 32a–40d.
of other 4-substituted, N-containing functional groups show at
least a twofold increase. Interestingly, most compounds containing
an O-functional group on the 4-arm (i.e., 3a, 3b, 12a and 34b) show
a much smaller potency increase. Although further experiments
are needed to fully evaluate the extent and the relevance of this
observation, we could now hypothesize that the different behav-
iour observed in STD–NMR studies by our Smac mimics, rather
than pointing towards a reduction in affinity for the primary
BIR3 binding site, indicates a gain of interactions with the second-
ary/linker-BIR2 binding site, leading to an overall increase in affin-
ity for the linker-BIR2–BIR3 construct and, hopefully, for XIAP in
cells and in animal models.
and the IC50 on linker-BIR2–BIR3 (linker-BIR2–BIR3 potency, LBBP)
is reported for each compound tested on MBA-MB-231 cells in
Table 3. A large PC/LBBP value indicates a potent compound with
poor potency in cells, most likely due to limited access to its intra-
cellular target/poor cell penetration; a value close to 1 indicates a
potent compound both in cell-free and cellular assays.
The largest PC/LBBP ratio appears for reference compound 1a,
confirming its reported poor bioavailability in cells. This ratio is de-
creased ꢁ1000 times, without increasing LBBP, just by methylating
its amino group as in 1b.31 Unfavourable PC/LBBP ratios are mea-
sured for 4-substituted compounds bearing small charged groups,
that is, NH2 (4a = 54.8; 32a = 150; 4b = 10; 32b = 27.1). As already
mentioned, beneficial effects are provided by N-methylation.
Unexpectedly, a ꢁ3-fold increase of PC/LBBP ratio in elongated,
more lipophilic compounds (4a vs 32a, 4b vs 32b) was also ob-
served. Favourable PC/LBBP ratios are obtained by replacing the
charged NH2 with polar, non-charged OH (3a = 3.55; 3b = 1.18;
34b = 1.73). A PC/LBBP decrease is also observed by functionaliza-
tion of primary amines in 4, either as a non-charged amide
(22c = 3.86) and as charged secondary amines (18a = 3.11;
18b = 1.38). N-Methylation (positive effect, 3b vs 3a, 34b vs 34a,
36b vs 36a, 40b vs 40a, 40d vs 40c) and elongation (negative effect,
3a vs 34a, 3b vs 34b, 18a vs 40a, 18b vs 40c, 22c vs 36a) influence
the ratio as seen for primary amines 4a,b and 32a,b. Benzyl ureas
(26b = 35.1, 38a = 144, 38b = 20) show a surprisingly high PC/LBBP
value, which cannot be explained by LBBP-related factors, as the
compounds bind potently both BIR3 and linker-BIR2–BIR3. In par-
ticular, N-alkylation on position 4 elicits similar beneficial effects
on cytotoxicity/cell penetration as N-methylation on the ethylgly-
cine moiety (4-CH2NH2 4a = 54.8 vs 4-CH2NHEt 18a = 3.11 vs 4-
CH2NHCH2Ph 18b = 1.38), and the two effects are additive (4-
CH2CH2NH2 32a = 150 vs N-Me,4-CH2CH2NH2 32b = 27.1 and 4-
A smaller set of potent compounds was tested on a panel of tu-
mour cell lines including MDA-MB-231 cells (breast), HL-60 cells
(leukemia) and PC-3 cells (prostate). Their IC50 are reported in
Table 2.
N-Methylation of the ethylglycine residue, as expected, gives
beneficial effects on cytotoxicity, although not as much as ob-
served for the 4-unsubstituted standard couple 1a/b (ꢁ1000 in-
crease) on MDA-MB-231 cells.31 The
8 NH/N-Me couples
characterized here span from a 4–5-fold increase (3a/b) to a >10-
fold increase (4a/b) on MDA-MB-231 cells. More resistant cell lines
provide comparable cytotox results for the standard couple 1a/b
and for our compounds, never surpassing a twofold increase (1a/
b and 4a/b, PC-3; 36a/b and 38a/b, HL-60). A comparison between
elongated (4-CH2CH2X, E) and non-elongated (4-CH2X, NE) com-
pounds provides unexpected results. Elongation causes a slight
but consistent reduction in cytotoxicity in almost all NE/E couples
(3a/34a, 3b/34b, 4a/32a, 4b/32b, 18a/40a, 18b/40c, 22c/36a, 26b/
38a), reaching a 4-fold maximum for 4b/32b on MDA-MB-231
cells. An opposite trend was expected, due to affinity-driven (no
4a-like shift in the active site) and lipophilic (one CH2 more)
contributions.
CH2CH2NHCH2Ph
40c = 11.4,
vs
N-Me,4-CH2CH2NHCH2Ph
Cytotoxicity depends on the nature and the lipophilicity of 4-
substituents. The ratio between the IC50 (potency in cells, PC)
40d = 5.2); is more favourable for activity than N-acylation (18b-
benzyl amine vs 22c-benzamide: slightly better cell-free potency,

5846
P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
Table 1
Table 2
Experimentally determined IC50 of compounds 1a, 1b, 3a–40d (fluorescence polar-
ization-based assays) on XIAP BIR3 and ligand-BIR2–BIR3
Experimentally determined IC50
cell lines
(lM) of compounds 1a, 1b and 3a–40d on tumour
Compound
IC50 BIR3a,b
RSD (%)
IC50 linker-BIR2–BIR3a,b
RSD (%)
Compound
MDA-MB-231a,b
HL-60a,b
PC-3a,b
1a
1b
3a
3b
4a
4b
460 (Ki = 25)c
530 (Ki = 61)d
270
230
970
100
450
400
600
370
4400
240
320
650
440
370
430
330
260
360
510
300
240
330
380
340
220
320
250
140
280
130
120
120
7
11
7
12
5
14
15
8
26
8
54
10
4
4
12
4
11
18
12
11
81
12
9
3
6
1
14
6
360
420
290
200
110
54
240
320
150
220
390
92
42
31
31
21
21
48
25
59
27
23
30
16
20
22
21
25
25
27
30
29
31
30
22
16
18
24
21
26
31
22
33
36
23
26
31
29
30
38
31
53
1a
1b
3a
3b
4a
4b
>100(>10)c
0.146 0.007(0.1)d
1.03 0.68
0.237 0.003/
6.03 1.54
0.542 0.002
0.286 0.007
0.099 0.003
0.425 0.012
3.4 0.7
12.9 0.3
2.3 1.2
1.8 0.03
0.173 0.002
0.93 0.02
0.21 0.01
10.8 0.1
1.2 0.01
8.0 0.9
24.5 1.3
20.2 1.5
6.5 2.4
>100
56.9 1.1
>100
>100
>100
51.7 1.1
>100
24.1 1.8
80.1 0.5
>100
85.2 1.3
>100
>100
>100
>100
>100
>100
83.5 6.2
>100
>100
39.2 9.8
7.8 0.3
26.1 1.8
69.3 3.55
51.8 0.06
84.0 4.0
13.1 2.2
36.0 7.0
51.0 0.01
25.0 1.8
31.0 1.3
>100
74.8 5.3
50.7 0.2
32.6 5.0
83.3 9.6
52.2 0.1
62.8 0.2
76.4 11.3
15.4 8.6
5.2 0.2
10a
12a
12b
14a
16a
18a
18b
20a
20b
20c
22a
22b
22c
24a
24b
26a
26b
26c
26d
28a
28b
31a
32a
32b
34a
34b
36a
36b
38a
38b
40a
40b
40c
40d
18a
18b
22c
26b
32a
32b
34a
34b
36a
36b
38a
38b
40a
40b
40c
40d
72
150
110
110
150
190
110
240
160
190
97
83
54
240
54
240
86
85
70
100
58
55
75
60
56
70
1.3 0.1
0.4 0.007
0.141 0.001
a
IC50, see Section 4 for details.
Average value from 2 experiments, each point done in triplicate.
Value in brackets from Ref. 30.
b
c
d
Value in brackets from Ref. 31.
2
7
minary early ADMET assay panel, including solubility, metabolic
stability and permeability, so to determine their appropriateness
as cell-permeable, metabolically stable and soluble leads (Table 5).
Some interesting observations can be made, despite the small
number of assays and the limited set of tested compounds. In
terms of metabolic stability (human CYP3A4), most compounds
are reasonably stable, with some warnings for 4-N elongated
amides 36a,b and ureas 38a,b. Alcohols, primary and secondary
amines are at least as stable as 4-unsubstituted standards 1a,b.
In terms of solubility (acidic and neutral pH), both N-methylation
of the ethylglycine residue and introduction of 4-N or 4-O contain-
11
17
16
16
15
14
9
16
12
24
140
100
110
86
49
110
35
27
a
Nanomolar values, see Section 4 for details.
Average value from four measurements.
Ki in brackets from Ref. 30.
b
c
d
Ki in brackets from Ref. 31.
Table 3
Experimentally determined ratios between IC50 on linker-BIR2–BIR3 (LBBP) and IC80
on MDA-MB-231 cells (PC) for compounds 1a, 1b and 3a–40d
better PC/LBBP ratio); and favours bulky, lipophilic vs small alkyl
groups (18b-benzyl amine vs 18a-ethyl amine: slightly better
cell-free potency, better PC/LBBP ratio).
The cytotoxic activity of 1-aza-2-oxobicyclo[5.3.0]alkane-based
Smac mimics on HL-60 and PC-3 cells is much lower, as mirrored
by higher PC1/LBBP (HL-60) and PC2/LBBP (PC-3) ratios, reported
in Table 4.
Best ratios are obtained for HL-60 cells with alcohol 3a (PC1/
LBBP = 22.4) and for PC-3 cells with benzyl amine 40d (PC2/
LBBP = 288.9), and in general with 4-unsubstituted standard 1b
(PC1/LBBP = 48.1 and PC2/LBBP = 135.5). Most trends observed
for MDA-MB-231 cells are also applicable here (18b better than
18a and 22c; elongated compounds slightly worse than their
non-elongated counterparts), while others are not (N-alkylation
on ethylglycine either neutral or negative for cytotoxicity, see for
example 3a vs 3b, 32a vs 32b). It remains to be seen if the moder-
ate activity of our Smac mimics on HL-60 and PC-3 cells, probably
due to a mix of target-related and permeability-related factors, can
be improved.
Compound
CFPa
PCb
PC/LBBP
1a
1b
3a
3b
4a
4b
360
420
290
200
110
54
92
72
110
97
86
85
70
100
58
55
75
60
56
70
>100
0.146
1.03
0.237
6.03
0.542
0.286
0.099
0.425
3.4
12.9
2.3
1.8
0.173
0.93
0.21
10.8
1.2
>280
.348
3.55
1.18
54.8
10.0
3.11
1.38
3.86
35.1
150
27.1
25.7
1.73
16.0
3.82
144
18a
18b
22c
26b
32a
32b
34a
34b
36a
36b
38a
38b
40a
40b
40c
40d
20
8.0
1.3
0.4
0.141
142.9
18.6
11.4
5.2
35
27
We have shown how 4-substitution on 1-aza-2-oxobicy-
clo[5.3.0]decanes is suitable to modulate their potency and phys-
ico-chemical properties (i.e., lipophilicity and solubility). As to
the latter, some promising compounds were submitted to a preli-
a
Nanomolar values, see Table 1 and Section 4 for details.
Micromolar values, see Table 2 and Section 4 for details.
b

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5847
Table 4
3. Conclusion
Experimentally determined ratios between IC50 on linker-BIR2–BIR3 (LBBP) and IC80
on HL60 (PC1) and PC-3 cells (PC2) for compounds 1b and 3a–40d
In this paper we report the synthesis of several 4-substituted 1-
aza-2-oxobicyclo[5.3.0]decanes as monomeric Smac mimics, intro-
ducing small and large, charged and neutral N- and O-substitutions
on non-elongated and elongated 4-side chains (respectively 1 and
2 carbon atoms between the scaffold and the heteroatom). The
compounds were rationally designed using theoretical and exper-
imental data obtained through computational methods, NMR and
crystallography techniques. Structural theories derived from these
data, presented and discussed here, rationalize the binding mode
of the compounds, account for most of significant target binding
differences, and will be used to further optimize their properties
in second generation analogues. The compounds were tested for
inhibition of BIR3 and linker-BIR2–BIR3, two fragments of the pro-
tein XIAP, a popular and validated target in apoptosis/oncology.
Several compounds exhibited low nanomolar activities on both
targets, with best results obtained for compounds bearing a lipo-
philic N-substituent in position 4 (e.g., benzamides 36a,b, benzyl
ureas 38a,b, secondary amines 40a–d). Cytotoxicity of Smac mim-
ics was measured on three tumour cell lines with varying sensitiv-
ity to XIAP-dependent apoptosis (MDA-MB-231, HL-60 and PC-3).
The compounds exhibited nanomolar (MDA-MB-231)/micromolar
(HL-60, PC-3) potencies, depending on their affinity for the target
and on their drug-like properties. The relationship between cyto-
tox (PC) and cell-free potency (LBBP) was elucidated also by means
of a preliminary set of results on ADMET assays. We suppose that a
compromise between in vitro potency, stability, good solubility
and measurable permeability should produce drug-like, bioavail-
able compounds: for example, standard compound 1b, 4-hydroxy
compound 3b and 4-benzylamine 40d satisfy these criteria and
are potent cytotoxic agents.
Compound
CFPa
PC1/PC2b
PC1,2/LBBPc
1b
3a
3b
4a
420
290
200
110
100
92
72
110
97
86
85
100
58
55
75
60
56
70
35
27
20.2/56.9
6.5/NDd
26.1/ND
69.3/ND
51.8/51.7
84.0/ND
13.1/24.1
36.0/80.1
51.0/ND
25.0/85.2
31.0/ND
74.8/ND
50.7/ND
32.6/ND
83.3/ND
52.2/83.5
62.8/>100
76.4/ND
15.4/39.2
5.2/7.8
48.1/135.5
22.4/ND
130.5/ND
630ND
4b
518/517
18a
18b
22c
26b
32a
32b
34b
36a
36b
38a
38b
40a
40b
40c
40d
913.0/ND
181.9/334.7
327.3/728.2
525.6/ND
290.7/990.7
364.7/ND
748/ND
874.1/ND
592.7/ND
1110.7/ND
870/1391.7
1121.4/ND
1091.4/ND
440/1120
192.6/288.9
a
Nanomolar values, see Table 1 and Section 4 for details.
Micromolar values, see Table 2 and Section 4 for details.
First value PC1/LBBP, second value PC2/LBBP.
ND, not detectable.
b
c
d
Table 5
Early ADMET characterization of compounds 1a, 1b and 3a–40d on a preliminary
eADME panel
Compound
Metab. Stab.a
Solubilityb
PAMPAa,c
1a
1b
3a
3b
4a
4b
18a
22c
26b
32a
32b
36a
36b
38a
38b
40b
40d
87
83
79
86
93
78
81
81
73
75
86
65
43
65
51
90
81
19/23
23
42
17
30
2
<1
<1
21
4
<1
<1
4.1
3
<1
<1
<1
4.3
248/175
111/183
210/172
138/168
233/>250
159/182
81/134
Although further optimization is ongoing, we believe that a
compound such as 40d (elongated N-Me,4-CH2CH2NHCH2Ph), with
low nanomolar potency on linker-BIR2–BIR3 and on MDA-MB-231
cells, and low micromolar potency on both HL-60 and PC-3 cells
represents an early lead which will be further characterized in
in vitro and in vivo relevant models. Interestingly, while submit-
ting this paper we became aware of novel potent, monomeric Smac
mimics44 which contain some of the features present in our ‘best’
4-substitutions, although being built on a different scaffold.
36/21
169/207
>250/207
185/244
179/243
145/216
148/194
184/191
>250/>250
4. Experimental
a
4.1. Chemical procedures
Human CYP3A4, see Section 4 for more details.
pH7.4/pH3, see Section 4 for details.
Parallel Artificial Membrane Permeability Assay (PAMPA), see Section 4 for
b
c
4.1.1. General methods
more details.
1H NMR spectra were recorded on Bruker Avance in CDCl3,
CD3OD or D2O as solvent at 400 or 600 MHz. 13C NMR spectra were
recorded in CDCl3, CD3OD or D2O as solvent at 100 or 125 MHz.
Coupling constants are given in hertz and are rounded to the near-
est 0.1 Hz. Purifications were carried out either by flash chroma-
ing substituents is beneficial. Interestingly, 40d shows an extre-
mely high solubility profile at both tested pHs, and is a promising
candidate for further biological profiling due to its balanced activ-
ity; conversely, ureas 26b and (to a lower extent) 38a,b have a low-
er solubility, which may partially justify their reduced cytotoxicity.
In terms of cellular permeability (surrogate artificial membrane
penetration, PAMPA), highest values are observed for 4-unsubsti-
tuted and 4-O substituted compounds. As to 4-N substitutions,
only a few compounds (amides 22c, 38a,b, urea 26b and benzyl-
amine 40d) have a measurable permeability. Rather than searching
for a quantitative relationship, we may safely say that compounds
with negligible passive permeability (<1, Table 5) have PC/LBBP ra-
tios ꢂ10.0, that is, do not show cellular activities comparable with
their cell-free potencies; weakly cytotoxic ureas 38a,b (PC/
LBBP = 144 and 20, respectively) are a typical example.
tography on silica gel (particle size 60
Kieselgel, or by BiotageTM flash chromatography [Biotage columns
Si-12-M (150 Â 12 mm; silica gel (40–63 m), flow rate 12 mL/
min); Si-25-M (150 Â 25 mm; silica gel (40–63 m), flow rate
25 mL/min)], or by BiotageTM C18 reverse phase chromatography
m),
lm, 230–400 mesh),
l
l
[Biotage column C18HS (150 Â 25 mm; KP-C18-HS (35–70
l
flow rate 25/mL/min)]. Final products were purified by C18 reverse
phase semi-preparative HPLC using either a Waters X-Terra RP18
OBD column (19 mm  10.0 cm, 5
lm) or a Supelco Ascentis C18
m). Solvents were distilled and
column (21.2 mm  15.0 cm, 5
l
dried according to standard procedures, and reactions requiring
anhydrous conditions were performed under nitrogen or argon.
Solvents for the reactions were used directly from the bottle if

5848
P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
not specified. Optical rotations ½a D20
Š
were measured in cells of 1 dm
4.1.3. General procedure B—synthesis of compounds 7
A 2 N aqueous LiOH solution (7.0 equiv) was slowly added to an
ice cooled, stirred solution of methyl ester 6 (1.0 equiv) in 1,4-diox-
ane (0.25 M concentration for 6). The reaction mixture was then
stirred at room temperature until complete hydrolysis of the start-
ing material. The cloudy solution was concentrated after 3 h, the
residue was taken up in CH2Cl2 and water, and acidified to pH 3
with aqueous 2 N HCl. The organic layer was dried over Na2SO4,
and then the solvent was removed under reduced pressure. The
crude carboxylic acid, obtained as amorphous white solids, did
not require further purification.
pathlength and 1 mL capacity with a Perkin Elmer 241 polarimeter.
LC–MS data were collected with an Agilent 1100 HPLC connected
to a Bruker Esquire 3000+ ion trap mass spectrometer through
an ES interface.
4.1.2. General procedure A—synthesis of compounds 6
Compound 5 (2.05 g, 5.3 mmol) was dissolved in 26.5 mL of a
3 N methanolic HCl solution. The resulting mixture was stirred
at room temperature for 48 h and then condensed under reduced
pressure. The crude product was redissolved in CH2Cl2 and washed
once with a saturated solution of NaHCO3. The organic layer was
dried over Na2SO4, filtered and the solvent was removed under re-
duced pressure. The crude methyl ester was purified by BiotageTM
C18 reverse phase chromatography. BiotageTM eluant conditions:
from 90 H2O and 10% CH3CN to 100% CH3CN. Yield 90% (1.65 g,
MW 344.17, 4.79 mmol) of pure methyl ester as an amorphous
4.1.3.1. Acid a. Quantitative yield (4.27 g, MW 427.49, 10.0 mmol),
starting from methyl ester 6a (4.41 g, 10.0 mmol). Analytical charac-
terization: ½a 2D0
Š
¼ À123:0 (c 0.61, MeOH); 1H NMR (400 MHz,
CDCl3): d: 7.76 (d, J = 6.5 Hz, 1H), 7.20 (br s, 1H), 5.33 (m, 1H),
4.56 (m, 2H), 4.09 (m, 1H), 3.91 (d, J = 7.5 Hz, 1H), 3.64 (d,
J = 11.6 Hz, 1H), 3.43 (d, J = 9.5 Hz, 1H), 2.26 (d, J = 6.5 Hz, 1H),
2.12–1.62 (m, 10H), 1.42 (s, 9H), 0.95 (t, J = 6.5 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d: 173.8, 173.5, 171.0, 156.0, 80.4, 64.3, 60.8,
60.4, 59.0, 56.5, 54.0, 41.2, 33.4, 32.9, 31.0, 28.2, 27.6, 25.7, 10.1.
ESI-MS: m/z 427.9 [M+H]+, 877.8 [2M+Na]+.
white solid. Analytical characterization: ½a D20
¼ À142:0 (c 1.15,
Š
MeOH); 1H NMR (400 MHz, CDCl3): d: 7.44 (d, J = 6.8 Hz, 2H),
7.35–7.23 (m, 3H), 4.74 (dd, J = 7.2, 4.4 Hz, 1H), 4.46 (d,
J = 13.6 Hz, 1H), 4.13 (dd, J = 9.2, 7.6 Hz, 1H), 3.87 (m, 1H), 3.74,
(s, 3H), 3.65 (d, J = 13.6 Hz, 1H), 3.52 (dd, J = 7.6, 6.0 Hz, 1H), 3.18
(d, J = 10.0 Hz, 1H), 2.78 (m, 1H), 2.30 (m, 1H), 2.14–2.01 (m,
3H), 1.95–1.63 (m, 4H); 13C NMR (100 MHz, CDCl3): d: 172.6,
168.8, 137.4, 129.0, 128.2, 127.2, 73.2, 71.4, 61.4, 59.9, 58.8, 52.4,
44.9, 33.9, 33.3, 32.2, 27.4; ESI-MS: m/z 344.9 [M+H]+, 711.0
[2M+Na]+.
Dry DIPEA (1.5 equiv) was added to a solution of carboxylic acid
(1.0 equiv), EDCꢀHCl (1.2 equiv),and HOBt (1.2 equiv) in dry CH2Cl2.
The solution was stirred for 10 min, then Ph2CHNH2 (1.2 equiv)
and dry DIPEA (2 equiv) in dry CH2Cl2 (final concentration of the
acid: 0.1 M) were added. The reaction mixture was stirred at room
temperature for 1 h, then diluted with CH2Cl2 and washed once
with 5% citric acid and saturated NaHCO3. The organic layer was
dried over Na2SO4 and evaporated under reduced pressure. The
residues were purified by BiotageTM C18 reverse phase
chromatography.
Simultaneous N–O hydrogenolytic cleavage and benzyl depro-
tection was performed using the H-CubeTM continuous-flow hydro-
genation reactor. The methyl ester (1.65 g, 4.79 mmol) was
dissolved in 85:15 EtOH/water (190 mL, ꢁ0.025 M final concentra-
tion) and reduced over 10% Pd/C catalyst (hydrogen pressure:
10 bar, T = 85 °C, flow: 0.7 mL/min). The reaction was monitored
by LC–MS. After reaction completion the solvent was evaporated
under reduced pressure. The crude aminoalcohol was obtained in
quantitative yield and used without any further purification. ESI-
MS: m/z 256.8 [M+H]+.
Dry DIPEA (2 equiv) was added to a solution of N-Boc protected
aminoacid (1.2 equiv), EDCꢀHCl (1.2 equiv) and HOBt (1.2 equiv) in
dry CH2Cl2 at room temperature and under a nitrogen atmosphere.
The solution was stirred for 10 min before adding a solution of the
aminoalcohol (1 equiv) in CH2Cl2 (final concentration of aminoal-
cohol: 0.1 M). The reaction mixture was stirred at room and mon-
itored by LC–MS. After 1 h, the solution was diluted with CH2Cl2
and then washed once with 5% citric acid and saturated NaHCO3.
The organic layer was dried over Na2SO4 and evaporated under re-
duced pressure. The crude product was purified by BiotageTM C18 re-
verse phase chromatography.
4.1.3.2. Compound 7a. Compound 7a was synthesized by gen-
eral procedure B, starting from acid a (4.27 g, 10.0 mmol). BiotageTM
eluant conditions: from 90 H2O and 10% CH3CN to 100% CH3CN.
Yield 76% (4.5 g, MW 592.73, 7.6 mmol) of pure 7a as an amor-
phous white solid. Analytical characterization: ½a D20
¼ À125:0 (c
Š
1.48, MeOH); 1H NMR (400 MHz, CDCl3): d: 7.97 (d, J = 8.4 Hz,
1H), 7.62 (d, J = 7.2 Hz, 1H), 7.35–7.15 (m, 10H), 6.21 (d,
J = 8.8 Hz, 1H), 5.17 (d, J = 7.2 Hz, 1H), 4.73 (d, J = 7.6 Hz, 1H),
4.49 (m, 1H), 4.10 (m, 1H), 3.77 (dd, J = 17.6, 8.5 Hz, 1H), 3.67 (d,
J = 10.8 Hz, 1H), 3.28 (dd, J = 12.0, 3.2 Hz, 1H), 2.38 (dd, J = 12.0,
6.8 Hz, 1H), 2.23 (m, 1H), 2.10–1.95 (m, 2H), 1.89–1.55 (m, 5H),
1.43 (s, 9H), 1.30–1.10 (m, 2H), 0.96 (t, 7.2 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d: 173.9, 171.7, 171.1, 169.6, 155.8, 142.0,
141.2, 128.6, 128.4, 127.4, 127.2, 126.9, 80.3, 64.2, 61.2, 58.9,
56.8, 53.9, 41.5, 34.0, 33.2, 31.1, 28.3, 25.8, 25.6, 21.0, 14.2, 10.2.
ESI-MS: m/z 593.1 [M+H]+, 615.1 [M+Na]+.
4.1.2.1. Compound 6a. Compound 6a was synthesized by gen-
eral procedure A, starting from compound 5 (2.05 g, 5.3 mmol).
BiotageTM eluant conditions: from 90 H2O and 10% CH3CN to 100%
CH3CN. Yield 70% (1.48 g, MW 441.52, 3.35 mmol) of pure 6a as
Full analytical characterization of acid b and of compound 7b is
reported in the Supplementary data.
4.1.4. General procedure C—synthesis of compounds 8
an amorphous white solid. Analytical characterization:
½
a 2D0
Š
¼
Dry TEA (4.0 equiv) and MsCl (4.0 equiv) were added to a stirred
solution of alcohol 7 (1.0 equiv) in dry CH2Cl2 (0.25 M concentra-
tion for 7) under argon atmosphere at 0 °C. The reaction mixture
was stirred at room temperature overnight. After reaction comple-
tion, the resulting mixture was diluted with CH2Cl2 and washed
once with saturated NH4Cl. The organic layer was dried over
Na2SO4, and then the solvent removed under reduced pressure.
The crude product was dissolved in dry DMF (0.1 M concentration)
under argon at room temperature, and then NaN3 (10 equiv) was
added. The reaction mixture was stirred at 80 °C overnight. After
reaction completion, the mixture was filtered on a Celite pad after
dilution with CH2Cl2. The solvent was then removed under reduced
À146:5 (c 0.71, MeOH); 1H NMR (400 MHz, CDCl3): d: 7.53 (d,
J = 3.5 Hz, 1H), 5.07 (d, J = 7.6 Hz, 1H), 4.56 (dd, J = 8.5, 4.0 Hz,
1H), 4.48 (dd, J = 10.0 Hz, 7.5, 1H), 4.03 (m, 1H), 3.89 (m, 1H),
3.73 (s, 3H), 3.66 (m, 1H), 3.31 (dd, J = 12.0, 3.0 Hz, 1H), 2.30–
2.23 (m, 1H), 2.15–1.97 (m, 4H), 1.90–1.75 (m, 4H), 1.69–1.58
(m, 2H), 1.43 (s, 9H), 0.95 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d: 173.9, 172.4, 170.2, 155.6, 80.2, 64.3, 60.6, 58.8, 56.6,
54.1, 52.3, 41.5, 32.8, 31.2, 28.2, 25.7, 10.1. ESI-MS: m/z 441.9
[M+H]+, 463.9 [M+Na]+.
Full analytical characterization of compound 6b is reported in
the Supplementary data.

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5849
pressure, and the crude product was purified by flash
chromatography.
1H), 4.73 (d, J = 7.6, 1H), 4.67 (t, J = 8.4, 1H), 4.06 (m, 1H), 4.01
(m, 2H), 3.87 (dd, J = 17.6, 8.8 Hz, 1H), 2.44 (m, 1H), 2.26 (m, 1H),
2.06 (s, 3H), 1.88 (m, 4H), 1.80–1.60 (m, 3H), 1.55 (m, 1H), 1.47
(s, 9H), 1.22–1.10 (m, 1H), 0.97 (t, 7.6 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d: 172.1, 171.7, 170.6, 169.5, 142.1, 141.2,
128.7, 128.6, 127.4, 127.3, 127.2, 65.4, 61.0, 58.7, 56.7, 56.3, 52.9,
39.1, 34.3, 33.4, 31.5, 28.3, 25.6, 20.9, 10.8. ESI-MS: m/z 635.0
[M+H]+, 657.0 [M+Na]+.
4.1.4.1. Compound 8a. Compound 8a was synthesized by gen-
eral procedure C, starting from alcohol 7a (1.18 g, 2.0 mmol). Elu-
ant mixture: petroleum ether/EtOAc 30:70. Yield 51% (635 mg,
MW 617.74, 1.02 mmol) of pure 8a as an amorphous white solid.
Analytical characterization:
½
a 2D0
Š
¼ À109:9 (c 0.62, MeOH); 1H
NMR (400 MHz, CDCl3): d: 7.82 (d, J = 8.8 Hz, 1H), 7.30–7.13 (m,
10H), 7.21 (d, J = 6.4 Hz, 2H), 7.11 (d, J = 6.8 Hz, 1H), 6.14 (d,
J = 8.8 Hz, 1H), 4.92 (br d, J = 7.2 Hz, 1H), 4.64 (d, J = 7.6 Hz, 1H),
4.50 (dd, J = 10.0 Hz, 8.0, 1H), 3.96 (dd, J = 13.6, 7.2 Hz, 1H), 3.76
(dd, J = 17.6, 9.2 Hz, 1H), 3.45 (dd, J = 12.4, 3.6 Hz, 1H), 3.06 (dd,
J = 12.4, 9.2 Hz, 1H), 2.36 (dd, J = 12.4, 6.8 Hz, 1H), 2.18 (m, 1H),
1.95 (m, 1H), 1.82–1.44 (m, 6H), 1.39 (s, 9H), 1.30–1.1 (m, 2H),
0.89 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 172.4,
171.3, 169.4, 155.8, 142.0, 141.1, 128.7, 128.6, 127.3, 127.2, 80.3,
61.0, 58.9, 56.8, 56.4, 53.6, 53.5, 40.0, 34.2, 33.3, 32.0, 28.3, 25.5,
21.0, 10.2. ESI-MS: m/z 618.1 [M+H]+, 640.1 [M+Na]+.
Full analytical characterization of compound 11b is reported in
the Supplementary data.
4.1.6.2. Synthesis of 13a. A 0.9 M water solution of sodium
ascorbate (45
Cu(OAc)2 (65
solutions of compound 8a (62 mg, 0.10 mmol) and of 1-pentynyl-
3-ol (8.6 L).
L, 0.10 mmol) in a 1:1 mixture of H2O/tBuOH (300
lL, 0.4 mmol) and
a 0.3 M water solution of
lL, 0.02 mmol) were sequentially added to stirred
l
l
The reaction mixture was stirred overnight at room temperature
and then the solvent was removed under reduced pressure. The
crude product was purified by BiotageTM. BiotageTM eluant condi-
tions: 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH and 90% of
CH2Cl2. Yield 58% (41 mg, MW 701.85, 0.058 mmol) of pure 13a
as an amorphous white solid. Analytical characterization:
Full analytical characterization of compound 8b is reported in
the Supplementary data.
4.1.5. General procedure D—synthesis of compounds 9
A 1 N solution of (CH3)3P in toluene (1.5 equiv) was added to a
stirred solution of azide 8 (1.0 equiv) in dry CH2Cl2 (0.67 M concen-
tration for 8) under argon atmosphere at room temperature. After
2 h, an excess of 1 N aqueous HCl was added to the reaction mix-
ture, which was stirred at room temperature for further 10–
20 min. After reaction completion, the reaction mixture was ex-
tracted with CH2Cl2 (3 times), the organic layers were combined
and dried over Na2SO4, and the solvent was removed under re-
duced pressure. The crude product was used without further
purification.
½
a 2D0
Š
¼ À50:0 (c 0.88, CHCl3); 1H NMR (400 MHz, CDCl3): 7.75
(dd, J = 8.4, 2.8 Hz, 1H), 7.35–7.00 (m, 11H), 6.11 (d, J = 8.4 Hz,
1H), 4.99 (d, J = 6.4 Hz, 1H), 4.76 (br s, 1H), 4.63 (m, 2H), 4.43 (m,
1H), 4.08 (m, 1H), 3.92 (m, 1H), 2.71 (m, 1H), 2.32 (m, 1H), 2.12
(m ,1H), 2.05 (m, 1H), 1.95–1.45 (m, 10H), 1.32 (s, 9H), 1.18 (s,
3H), 1.15–1.05 (m, 1H), 1.00–0.85 (m, 6H); 13C NMR (100 MHz,
CDCl3): 172.9, 170.7, 169.3, 141.8, 141.3, 128.7, 127.5, 127.3,
127.1, 80.3, 68.4, 61.2, 58.8, 56.8, 56.3, 54.0, 52.2, 40.3, 33.8,
33.3, 31.7, 30.3, 29.7, 28.3, 25.7, 10.2, 9.8. ESI-MS: m/z 702.5
[M+H]+, 724.5 [M+Na]+.
4.1.6.3. Synthesis of 15a. N,N’-Bis-Boc-1-guanylpyrazole (47 mg,
0.15 mmol) in a mixture of THF (1 mL) and water (0.2 mL), a 1 M
aqueous LiOH solution (0.2 mL, 0.2 mmol) and triphenylphosphine
(79 mg, 0.30 mmol) were sequentially added to a stirred solution
of compound 8a (62 mg, 0.10 mmol). The reaction mixture was
stirred at room temperature for 48 h and then THF was removed
under reduced pressure. The residue was partitioned between
aqueous 10% citric acid and EtOAc. The aqueous layer was ex-
tracted with EtOAc (3 times) and then the organic layers were
combined, washed once with brine and dried over Na2SO4. Finally,
the crude product was purified by chromatography on a C18 re-
verse phase semi-preparative HPLC column. HPLC eluant condi-
tions: from 45% of H2O (0.1% TFA) and 55% of CH3CN to 30% of
H2O (0.1% TFA) and 70% of CH3CN, flow rate 20 mL/min, 10 min
runs. Yield 31% (26 mg, MW 834.01, 0.031 mmol) of pure trifluoro-
acetate salt of 15a as an amorphous white solid. Analytical charac-
4.1.5.1. Compound 9a. Compound 9a was synthesized by gen-
eral procedure D, starting from azide 8a (617 mg, 1.0 mmol). Yield
92% (578 mg, MW 628.20, 0.92 mmol) of pure hydrochloride salt of
9a as an amorphous white solid. Analytical characterization:
½
a 2D0
Š
¼ À68:5 (c 1.31, MeOH); 1H NMR (400 MHz, CDCl3): d: 8.39
(br s, 3H), 8.04 (br s, J = 7.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.40–
7.20 (m, 10H), 6.18 (d, J = 8.4 Hz, 1H), 5.12 (d, J = 6.4 Hz, 1H),
4.71 (d, J = 7.2 Hz, 1H), 4.49 (t, J = 8.0 Hz, 1H), 4.05 (q, J = 7.2 Hz,
1H); 3.74 (q, J = 8.4 Hz, 1H); 2.90 (br s, 2H); 2.40 (m, 1H), 2.21
(m, 1H), 2.05–1.55 (m, 7H), 1.47 (s, 9H), 1.33 (m, 1H), 1.22 (m,
1H), 0.99 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 175.9,
170.1, 169.4, 142.2, 141.1, 128.7, 127.5, 127.4, 127.3, 80.3, 61.3,
58.7, 57.1, 53.8, 42.8, 38.1, 34.0, 33.3, 32.0, 29.0, 28.3, 25.8, 25.0,
10.4. ESI-MS: m/z 592.1 [M+H]+, 614.1 [M+Na]+.
Full analytical characterization of compound 9b is reported in
the Supplementary data.
terization:
½
a 2D0
Š
¼ À84:2 (c 0.65, MeOH); 1H NMR (400 MHz,
CDCl3): d: 7.60 (m, 2H), 7.50 (m, 1H), 7.30–7.08 (m, 11H), 6.12
(d, J = 8.4 Hz, 1H), 4.94 (m, 1H), 4.64 (d, J = 7.2 Hz, 1H), 4.54 (m,
1H), 3.77 (m, 2H), 3.54 (m, 1H), 2.87 (m, 1H), 2.34 (m, 1H), 2.30–
1.51 (m, 9H), 1.47 (s, 9H), 1.34 (s, 9H), 1.20 (s, 9H), 1.10 (m, 1H),
0.93 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 172.4,
171.3, 169.4, 158.1, 156.0, 154.0, 149.0, 142.0, 141.1, 128.7,
128.6, 127.3, 127.2, 126.9, 80.3, 79.5, 79.2, 61.0, 58.9, 56.8, 56.4,
53.6, 53.5, 40.0, 34.2, 33.3, 32.0, 31.2, 28.3, 27.9, 25.5, 21.0, 10.2.
ESI-MS: m/z 834.2 [M+H]+.
4.1.6. General procedure E—synthesis of compounds 11
Pyridine (81 lL, 1.0 mmol), a carboxylic acid chloride RCOCl
(0.10 mmol) and DMAP (12 mg, 0.02 mmol) were sequentially
added to a stirred solution of 7a (59 mg, 0.10 mmol) in dry CH2Cl2
(2 mL). The reaction mixture was stirred at room temperature
overnight, the solvent was removed under reduced pressure and
the crude product was purified by BiotageTM.
4.1.6.1. Compound 11a. Compound 11a was synthesized by gen-
eral procedure E. BiotageTM eluant conditions: from 1% of MeOH and
99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 92% (58 mg,
MW 634.74, 0.092 mmol) of pure 11a as an amorphous white solid.
4.1.7. General procedure F—synthesis of compounds 17
NaBH(OAc)3 (26 mg, 0.13 mmol) was added to a stirred solution
of compound 9a (0.10 mmol) and of
a carbonyl compound
Analytical characterization: ½a D20
Š
¼ À105:0 (c 1.28, MeOH); 1H NMR
(0.11 mmol) in dry CH2Cl2 (2 mL) under nitrogen atmosphere.
The reaction mixture was stirred overnight at room temperature,
and then the solvent was removed under reduced pressure. The
(400 MHz, CDCl3): d: 7.93 (d, J = 8.8 Hz, 1H), 7.38–7.19 (m, 10H),
7.10 (d, J = 8.4, 1H), 6.23 (d, J = 8.8 Hz, 1H), 5.01 (d, J = 6.8 Hz,

5850
P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
crude product was purified either by chromatography on a C18 re-
verse phase semi-preparative HPLC column or by BiotageTM.
J = 8.0, 1H), 4.04 (q, J = 7.2, 1H) , 3.77 (m, 3H), 3.45 (m, 1H), 3.05
(m, 1H), 2.45 (m, 1H), 2.25 (m, 1H), 1.90–1.55 (m, 7H), 1.44 (s,
9H), 1.43 (s, 9H), 1.30–1.05 (m, 2H), 0.98 (t, J = 7.6 Hz, 3H); 13C
NMR (100 MHz, CDCl3): d: 173.3, 171.6, 169.6, 169.4, 165.7,
156.0, 142.1, 141.2, 128.6, 127.4, 127.3, 80.7, 79.8, 61.1, 58.6,
56.8, 55.5, 54.0, 44.2, 39.8, 34.4, 33.3, 31.9, 28.3, 25.7, 25.5, 10.9.
ESI-MS: m/z 749.5 [M+H]+, 771.6 [M+Na]+.
4.1.7.1. Compound 17a. Compound 17a was synthesized by gen-
eral procedure F. HPLC eluant conditions: from 80% of H2O (0.1%
NH3) and 20% of CH3CN to 20% of H2O (0.1% NH3) and 80% of
CH3CN. Yield 79% (49 mg, MW 619.79, 0.079 mmol) of pure 17a
as an amorphous white solid. Analytical characterization:
½
a 2D0
Š
¼ À88:7 (c 1.08, CHCl3); 1H NMR (400 MHz, CDCl3): 8.60 (d,
4.1.9. General procedure H—synthesis of compounds 21b,c
J = 7.2 Hz, 1H), 8.53 (br s, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.40–7.20
(m, 10H), 6.21 (d, J = 8.4 Hz, 1H), 5.04 (d, J = 6.4, 1H), 4.73 (d,
J = 7.2 Hz, 1H), 4.61 (t, J = 8.4 Hz, 1H), 4.08 (dd, J = 13.6, 6.8 Hz,
1H), 3.82 (dd, J = 17.2, 8.8 Hz, 1H), 3.00 (m, 2H), 2.80 (m ,1H),
2.64 (br d, J = 12.8 Hz, 1H), 2.43 (m, 1H), 2.22 (m, 1H), 1.95–1.60
(m, 8H), 1.45 (s, 9H), 1.31 (t, J = 7.2 Hz, 3H), 1.15 (m, 1H), 1.05 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): 173.8, 170.7, 169.5,
142.1, 128.7, 128.5, 127.4, 127.2, 80.3, 61.2, 58.5, 57.0, 56.5, 54.5,
51.8, 44.7, 38.0, 34.4, 33.2, 32.8, 28.1, 25.7, 12.2, 10.3. ESI-MS: m/
z 620.8 [M+H]+.
Pyridine (81 lL, 1.0 mmol) and a carboxylic acid chloride RCOCl
(0.10 mmol) were sequentially added to a stirred solution of 9a
(59 mg, 0.10 mmol) in dry CH2Cl2 (2 mL) under nitrogen atmo-
sphere. The reaction mixture was stirred at room temperature
overnight and then, after reaction completion, the solvent was re-
moved under reduced pressure. The crude product was purified by
BiotageTM.
4.1.9.1. Compound 21b. Compound 21b was synthesized by
general procedure H. BiotageTM eluant conditions: from 1% of MeOH
and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 91%
(58 mg, MW 633.78, 0.091 mmol) of pure 21b as an amorphous
Full analytical characterization of compound 17b is reported in
the Supplementary data.
white solid. Analytical characterization:
½
a 2D0
Š
¼ À64:0 (c 1.15,
4.1.8. General procedure G—synthesis of compounds 19
CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.91 (d, J = 8.8 Hz, 1H),
7.38–7.27 (m, 10H), 7.19 (d, J = 8.4 Hz, 1H) 7.05 (br s, 1H), 6.22
(d, J = 8.8 Hz, 1H), 4.98 (d, J = 6.4 Hz, 1H), 4.72 (d, J = 7.6 Hz, 1H),
4.49 (t, J = 8.0 Hz, 1H), 3.97 (dd, J = 13.2, 6.0, 1H), 3.80 (m, 1H),
3.55 (m, 1H), 3.05 (d, J = 12.0 Hz, 1H), 2.45 (m, 1H), 2.25 (m, 1H),
2.08 (s, 3H), 2.00–1.55 (m, 8H), 1.46 (s, 9H), 1.20–1.00 (m, 1H),
1.00 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 173.6,
171.7, 170.5, 169.3, 155.9, 142.0, 141.1, 128.6, 127.5, 127.3,
127.2, 80.5, 61.2, 59.0, 57.1, 56.8, 54.1, 53.9, 41.4, 40.2, 34.5,
33.1, 31.9, 28.3, 25.6, 25.2, 23.3, 10.3. ESI-MS: m/z 634.5 [M+H]+,
656.5 [M+Na]+.
NaBH(OAc)3 (26 mg, 0.13 mmol) and acetic acid (6
0.1 mmol) were sequentially added to a stirred solution of second-
ary amines 17 (0.10 mmol) and of carbonyl compound
lL,
a
(0.11 mmol) in dry CH2Cl2 (2 mL) under nitrogen atmosphere.
The reaction mixture was stirred overnight at room temperature,
and then the solvent was removed under reduced pressure. The
crude product was purified by BiotageTM.
4.1.8.1. Compound 19a. Compound 19a was synthesized by gen-
eral procedure G, starting from secondary amine 17b. BiotageTM elu-
ant conditions: from 1% of MeOH and 99% of CH2Cl2 to 10% of
MeOH and 90% of CH2Cl2. Yield 92% (64 mg, MW 695.89,
0.092 mmol) of pure 19a as an amorphous white solid. Analytical
Full analytical characterization of compound 21c is reported in
the Supplementary data.
characterization: ½a D20
Š
¼ À84:2 (c 1.36, CHCl3); 1H NMR (400 MHz,
4.1.10. General procedure I—synthesis of compounds 23
A 0.3 M water solution of K2CO3 (1.3 mL, 0.40 mmol) and a sul-
fonyl chloride RSO2Cl (0.22 mmol) were added to stirred solutions
of compound 9a (59 mg, 0.10 mmol) in CH2Cl2 (2 mL). The reaction
mixture was stirred at room temperature overnight and then, after
reaction completion, the solvent was removed under reduced pres-
sure. The crude products were purified by BiotageTM.
CDCl3): d: 9.10 (br s, 1H), 8.35 (br s, 1H), 7.40–7.00 (m, 15H),
6.21 (d, J = 8.4 Hz, 1H), 5.15 (d, J = 7.6 Hz, 1H), 4.79 (d, J = 7.2 Hz,
1H), 4.49 (dd, J = 9.2, 6.0 Hz, 1H), 4.03 (m, 1H), 3.93 (dd, J = 17.6,
9.2 Hz, 1H), 3.60 (br s, 1H), 3.29 (br s, 1H), 2.63 (br s, 1H), 2.47
(m, 1H), 2.26 (m, 2H), 2.16 (br s, 3H), 1.95–1.85 (m, 5H), 1.80–
1.50 (m, 3H), 1.44 (s, 9H), 1.25 (m, 1H), 0.92 (t, J = 7.6 Hz, 3H);
13C NMR (100 MHz, CDCl3): d: 172.2, 170.0, 142.2, 141.3, 128.6,
128.5, 128.4, 127.3, 127.2, 127.1, 79.6, 61.1, 58.3, 56.8, 35.0, 33.6,
33.4, 31.2, 28.4, 26.7, 25.5, 9.9. ESI-MS: m/z 696.6 [M+H]+.
Full analytical characterization of compounds 19b,c is reported
in the Supplementary data.
4.1.10.1. Compound 23a. Compound 23a was synthesized by
general procedure I. BiotageTM eluant conditions: from 1% of MeOH
and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 74%
(50 mg, MW 669.83, 0.074 mol) of pure 23a as an amorphous
white solid. Analytical characterization: ½a D20
¼ À101:0 (c 1.68,
Š
4.1.8.2. Synthesis of 21a. N-Boc glycine (18 mg, 0.10 mmol),
EDCꢀHCl (23 mg, 0.12 mmol), HOBt (16 mg, 0.12 mmol) and dry DI-
CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.74 (d, J = 8.4 Hz, 1H),
7.30–7.18 (m, 10H), 7.12 (d, J = 7.2 Hz, 1H), 6.13 (d, J = 8.8 Hz,
1H), 5.95 (d, J = 9.2 Hz, 1H), 4.92 (d, J = 6.8 Hz, 1H), 4.63 (t,
J = 8.0 Hz, 1H), 4.40 (t, J = 8.4, 1H), 3.92 (dd, J = 13.6, 7.6 Hz, 1H),
3.70 (dd, J = 13.6, 8.3, 1H), 3.05 (m, 2H), 2.79 (s, 3H), 2.41 (m,
1H), 2.28 (m, 1H), 1.90–1.55 (m, 8H), 1.37 (s, 9H), 1.15 (m, 1H),
0.91 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 173.5,
171.2, 171.1, 169.3, 155.8, 142.0, 141.1, 128.7, 127.5, 127.4,
127.3, 127.2, 80.4, 61.3, 58.9, 56.8, 53.5, 45.7, 40.0, 39.8, 34.3,
33.1, 31.7, 28.3, 25.7, 25.4, 10.2. ESI-MS: m/z 692.5 [M+Na]+.
Full analytical characterization of compound 23b is reported in
the Supplementary data.
PEA (70 lL, 0.40 mmol) were sequentially added to a stirred solu-
tion of 9a (59 mg, 0.10 mmol) in dry CH2Cl2 (2 mL) under nitrogen
atmosphere. The reaction mixture was stirred at room temperature
overnight and then, after reaction completion, the solvent was re-
moved under reduced pressure. The crude product was taken up
with CH2Cl2 and then washed once with saturated aqueous NH4Cl,
with saturated aqueous NaHCO3 and with brine. The organic layer
was dried over Na2SO4, the solvent was removed under reduced
pressure and the crude product was purified by BiotageTM. BiotageTM
eluant conditions: from 1% of MeOH and 99% of CH2Cl2 to 10% of
MeOH and 90% of CH2Cl2. Yield 89% (67 mg, MW 748.91,
0.089 mmol) of pure 21a as an amorphous white solid. Analytical
4.1.11. General procedure J—synthesis of compounds 25
characterization: ½a D20
Š
¼ À41:0 (c 1.50, CHCl3); 1H NMR (400 MHz,
RNCO or RNCS (neat, 0.12 mmol) and dry DIPEA (21
0.12 mmol) were added to stirred solution of 9a (59 mg,
0.10 mmol) in dry CH2Cl2 (2 mL). The reaction mixture was stirred
lL,
CDCl3): d: 7.91 (d, J = 7.2 Hz, 1H), 7.55–7.15 (m, 12H), 6.21 (d,
J = 8.8 Hz, 1H), 5.09 (d, J = 7.4, 1H), 4.71 (d, J = 7.2 Hz, 1H), 4.53 (t,
a

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5851
at room temperature overnight, the solvent was removed under re-
duced pressure and the crude products were purified by BiotageTM.
61.1, 58.9, 57.2, 56.8, 54.6, 43.2, 40.7, 34.5, 33.2, 32.3, 29.7, 28.3,
25.6, 25.2, 10.4. ESI-MS: m/z 635.1 [M+H]+, 657.1 [M+Na]+.
Full analytical characterization of acyl urea b and of compound
27b is reported in the Supplementary data.
4.1.11.1. Compound 25a. Compound 25a was synthesized by
general procedure J. BiotageTM eluant conditions: 1% of MeOH and
99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 89%
(59 mg, MW 662.82, 0.089 mmol) of pure 25a as an amorphous
4.1.13. General procedure L—synthesis of compounds 29
Dry TEA (4.0 equiv) and MsCl (4.0 equiv) were added to stirred
solutions of alcohol 7 (1.0 equiv) in dry CH2Cl2 under argon. The
reaction mixture was stirred at room temperature overnight. After
reaction completion, the resulting mixture was diluted with CH2Cl2
and washed once with saturated aqueous NH4Cl. The organic layer
was dried over Na2SO4, and then the solvent removed under re-
duced pressure. The crude mesylate was dissolved in dry DMF un-
der argon at room temperature, and then a solution of nBu4NCN
(8.0 equiv) in dry DMF was added. The reaction mixtures were
heated at 0 °C while stirring. After 40 h the solvent was removed
under reduced pressure, the crude product was diluted with
CH2Cl2 and washed with water. The organic layer was dried over
Na2SO4, and then the solvent removed under reduced pressure.
The crude product was purified by BiotageTM.
white solid. Analytical characterization:
½
a 2D0
Š
¼ À83:3 (c 1.11,
CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.86 (d, J = 7.4 Hz, 1H),
7.30–7.18 (m, 9H), 7.08 (d, J = 7.2 Hz, 2H), 6.13 (d, J = 8.8 Hz, 1H),
5.62 (br s, 1H), 4.93 (d, J = 6.8 Hz, 1H), 4.63 (d, J = 7.6 Hz, 1H),
4.46–4.37 (m, 2H), 3.89 (dd, J = 13.6, 7.6 Hz, 1H), 3.68 (dd,
J = 17.6, 9.2 Hz, 1H), 3.29 (m, 1H), 3.14 (m, 2H), 3.05 (br d,
J = 12 Hz, 1H), 2.36 (m, 1H), 2.15 (m, 1H), 1.85–1.71 (m, 5H),
1.65–1.56 (m, 3H), 1.40 (m, 1H),1.36 (s, 9H), 1.05 (t, J = 7.2 Hz,
3H), 0.89 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 173.1,
172.0, 169.3, 158.4, 156.0, 142.0, 141.3, 128.7, 128.6, 127.5,
127.2, 80.6, 61.2, 58.9, 57.1 56.8, 54.5, 42.6, 40.8, 35.2, 34.5, 33.3,
32.0, 31.5, 28.3, 25.5, 25.6, 15.6, 10.3. ESI-MS: m/z 663.6 [M+H]+,
685.6 [M+Na]+.
Full analytical characterization of compounds 25b–d is reported
in the Supplementary data.
4.1.13.1. Compound 29a. Compound 29a was synthesized by
general procedure L, starting from alcohol 7a (711 mg, 1.20 mmol).
BiotageTM eluant conditions: from 1% of MeOH and 99% of CH2Cl2 to
10% of MeOH and 90% of CH2Cl2. Yield 51% (368 mg, MW 601.74,
0.612 mmol) of pure 29a as an amorphous white solid. Analytical
4.1.12. General procedure K—synthesis of compounds 27
RCONCO or RCONCS (neat, 0.12 mmol) and dry DIPEA (21
lL,
0.12 mmol) were added to stirred solution of 9a (59 mg,
a
0.10 mmol) in dry CH2Cl2 (2 mL). The reaction mixtures were stir-
red at room temperature overnight and then the solvent was re-
moved under reduced pressure. The crude products were purified
by BiotageTM. Acyl urea a: BiotageTM eluant conditions: from 1% of
MeOH and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield
83% (61 mg, MW 738.87, 0.083 mmol) of pure N-Boc protected acyl
urea a as an amorphous white solid. Analytical characterization:
characterization: ½a D20
Š
¼ À69:7 (c 1.19, CHCl3); 1H NMR (400 MHz,
CDCl3): d: 7.82 (d, J = 8.8 Hz, 1H), 7.40–7.15 (m, 11H), 6.24 (d,
J = 8.8 Hz, 1H), 4.98 (br d, J = 7.2 Hz, 1H), 4.75 (d, J = 7.6 Hz, 1H),
4.54 (dd, J = 10.0, 8.0 Hz, 1H), 4.01 (m, 1H), 3.84 (dd, J = 17.6,
9.2 Hz, 1H), 2.65 (m, 1H), 2.45 (m, 1H), 2.35–2.10 (m, 3H), 2.15
(m, 1H), 2.00–1.55 (m, 5H), 1.48 (m, 10H), 1.15 (m, 1H), 0.93 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 173.0, 170.5, 169.2,
142.1, 141.0, 128.8, 128.6, 127.8, 127.4, 127.3, 127.2, 80.4, 61.0,
58.8, 56.8, 54.2, 37.7, 34.4, 34.0, 33.2, 28.3, 27.9, 26.0, 25.5, 25.2,
20.7, 10.3. ESI-MS: m/z 602.5 [M+H]+, 624.5 [M+Na]+.
½
a 2D0
Š
¼ À91:1 (c 1.33, CHCl3); 1H NMR (400 MHz, CDCl3): d: 8.82
(bt, J = 6.4 Hz, 1H), 8.66 (br s, 1H), 7.95 (d, J = 7.2 Hz, 3H), 7.64 (t,
J = 7.6 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.40–7.24 (m, 9H), 7.21
(m, 3H), 6.21 (d, J = 8.4 Hz, 1H), 5.57 (d, J = 8.8 Hz, 1H), 4.75 (d,
J = 7.2 Hz, 1H), 4.67 (dd, J = 10, 7.6, 1H), 4.20 (br s, 1H), 3.85 (dd,
J = 16.8, 9.2 Hz, 1H), 3.47 (m, 1H), 3.34 (m, 1H), 2.44 (m, 1H),
2.25 (m, 1H), 2.05–1.85 (m, 3H), 1.80–1.60 (m, 4H), 1.47 (s, 9H),
1.15 (m, 1H), 0.97 (t, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d: 172.9, 171.7, 169.5, 167.7, 153.4, 142.1, 141.2, 133.3, 132.2,
129.0, 128.9, 127.4, 127.3, 127.2, 80.1, 61.0, 58.8, 56.8, 56.4, 54.3,
42.2, 40.7, 34.5, 33.3, 32.3, 28.3, 10.2. ESI-MS: m/z 739.6 [M+H]+,
761.6 [M+Na]+.
Full analytical characterization of compound 29b is reported in
the Supplementary data.
4.1.14. General procedure M—synthesis of compounds 30
Nitriles 29 were converted to the corresponding amines 30 by
continuous flow hydrogenation using the H-CubeTM system (Thales
Nanotechnology). 1 M aqueous citric acid (10% of EtOH) was added
to nitrile 29 (1 equiv) in EtOH (0.1 M concentration for 29), and the
solution was flowed through a Raney Nickel catalyst cartridge
(hydrogen pressure 60 bar, temperature 60 °C, flow rate 0.5 mL/
min). After 3 h, the solvent was removed under reduced pressure.
The crude product was used without any further purification,
and only an analytical sample was purified (semi-preparative HPLC
or BiotageTM C18 reverse phase chromatography) and characterized
(NMR).
2 M aqueous K2CO3 (100 lL, 0.20 mmol) was added to a stirred
solution of an N-Boc protected acyl(thio)urea (0.10 mmol) in
MeOH (2 mL). The reaction mixture was stirred at room tempera-
ture overnight. After reaction completion, the solvent was removed
under reduced pressure. The residue was dissolved in CH2Cl2 and
washed once with water. The organic layer was dried over Na2SO4,
and the solvent was removed under reduced pressure. The crude
product was used without further purification.
4.1.14.1. Compound 30a. Compound 30a was synthesized by
general procedure M, starting from nitrile 29a (61 mg, 0.10 mmol).
HPLC eluant conditions: from 80% of H2O (0.1% NH3) and 20% of
CH3CN to 20% of H2O (0.1% NH3) and 80% of CH3CN. Yield 48%
(29 mg, MW 605.77, 0.048 mmol) of pure 30a as an amorphous
4.1.12.1. Compound 27a. Compound 27a was synthesized by
general procedure K. Yield 82% (52 mg, MW 634.77, 0.082 mmol)
pure 27a as an amorphous white solid. Analytical characterization:
½
a 2D0
Š
¼ À76:4 (c 1.10, MeOH); 1H NMR (400 MHz, CDCl3): 7.84 (d,
white solid. Analytical characterization:
½
a 2D0
Š
¼ À65:7 (c 1.25,
J = 8.8 Hz, 1H), 7.27–7.08 (m, 11H), 6.13 (d, J = 8.8 Hz, 1H), 4.96
(d, J = 7.2 Hz, 1H), 4.64 (d, J = 7.2 Hz, 1H), 4.46 (m, 1H), 3.88 (dd,
J = 14.0, 7.2 Hz, 1H), 3.68 (dd, J = 17.6, 9.2 Hz, 1H), 3.17 (m, 1H),
3.06 (m, 1H), 2.36 (m, 1H), 2.16 (m, 1H), 1.81–1.70 (m, 4H),
1.65–1.40 (m, 4H), 1.36 (s, 9H), 1.10–1.00 (m, 1H), 0.91 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): 173.1, 171.7, 169.3,
158.9, 141.8, 141.2, 128.7, 128.6, 127.6, 127.4, 127.3, 127.2, 80.8,
CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.92 (d, J = 8.0 Hz, 1H), 7.51
(d, J = 8.0, 1H), 7.40–7.15 (m, 10H), 6.16 (d, J = 8.8 Hz, 1H), 5.72
(d, J = 7.6 Hz, 1H), 4.67 (d, J = 5.6 Hz, 1H), 4.55 (t, J = 8.8 Hz, 1H),
4.26 (m, 1H), 3.86 (dd, J = 17.6, 9.2 Hz, 1H), 2.69 (m, 1H), 2.41 (m,
1H), 2.22 (m, 2H), 2.00–1.60 (m, 7H), 1.55–1.15 (m, 13H), 1.00 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 172.5, 171.3, 170.9,
156.3, 141.4, 128.8, 128.6, 127.6, 127.5, 127.2, 80.0, 61.5, 58.5,

5852
P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
57.4, 56.0, 54.8, 37.0, 35.8, 33.4, 33.2, 32.8, 30.2, 27.0, 26.3, 25.8,
10.2. ESI-MS: m/z 606.0 [M+H]+, 628.0 [M+Na]+.
Full analytical characterization of compound 30b is reported in
the Supplementary data.
stirring at room temperature. After 1 h, the solution was diluted
with CH2Cl2 and washed once with water. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The
crude product was purified by flash chromatography or by Bio-
tageTM C18 reverse phase chromatography.
4.1.15. General procedure N—synthesis of compounds 33
NaNO2 (5 equiv) and 50% aqueous citric acid (0.5 mL) were
sequentially added to a stirred solution of primary amine 30
(1 equiv) in THF (0.3 M concentration for 30) at 0 °C. The reaction
mixture was stirred at room temperature overnight. After reaction
completion, the resulting mixture was diluted with water and ex-
tracted with EtOAc (3 times). The combined organic layers were
dried over Na2SO4 and the solvent was removed under reduced
pressure. The crude product was purified by semi-preparative
HPLC.
4.1.17.1. Compound 37a. Compound 37a was synthesized by
general procedure P, starting from 30a (73 mg, 0.120 mmol). Elu-
ant mixture: EtOAc/MeOH 99: 1. Yield 34% (30 mg, MW 738.91,
0.040 mmol) of pure 37a as an amorphous white solid. Analytical
characterization: 1H NMR (400 MHz, CDCl3): d: 7.94 (d, J = 8.0 Hz,
1H), 7.32–7.16 (m, 15H), 6.20 (d, J = 8.0 Hz, 1H), 5.30–5.12 (m,
2H), 4.73 (d, J = 4.0 Hz, 1H), 4.57 (dd, J = 8.4 Hz, 1H), 4.33 (m, 2H),
4.13 (m, 1H), 3.82 (m, 1H), 3.07 (m, 1H), 1.80–1.78 (m, 5H),
1.61–1.27 (m, 8H), 1.34 (s, 9H), 0.93 (t, J = 6.4 Hz, 3H); 13C NMR
(400 MHz, CDCl3): d: 172.3, 171.9, 170.0, 158.5, 156.2, 141.6,
141.3, 139.8, 128.8, 128.6, 128.5, 128.4, 127.5, 127.4, 127.3,
127.2, 127.0, 80.0, 60.9, 58.4, 56.8, 56.1, 54.8, 44.2, 38.0, 37.1,
33.5, 33.4, 32.8, 32.5, 28.2, 26.2, 26.0, 10.2. ESI-MS: m/z 739.4
[M+H]+.
4.1.15.1. Compound 33a. Compound 33a was synthesized by
general procedure N, starting from 30a (183 mg, 0.30 mmol). HPLC
eluant conditions: from 80% of H2O, 20% of CH3CN and 0.2% AcOH
to 100% of CH3CN and 0.2% AcOH. Yield 16% (28 mg, MW 606.75,
0.048 mmol) of pure 33a as an amorphous white solid. Analytical
characterization: 1H NMR (400 MHz, CDCl3): d: 8.01 (d, J = 8.9 Hz,
1H), 7.31–7.19 (m, 10H), 7.16 (d, J = 6.9 Hz, 1H), 6.15 (d,
J = 8.9 Hz, 1H), 5.09 (d, J = 5.9 Hz, 1H), 4.65 (d, J = 6.9 Hz, 1H),
4.52 (t, J = 8.9 Hz, 1H), 3.90 (br s, 1H), 3.79 (br s, 1H), 3.55 (m,
1H), 3.45 (m, 1H), 2.37 (m, 1H), 2.18 (m, 1H), 1.85–1.50 (m, 8H),
1.37 (s, 9H), 1.32–1.30 (m, 2H), 1.08 (m, 1H), 0.92 (m, 3H); 13C
NMR (100 MHz, CDCl3): d: 172.4, 172.2, 169.6, 142.0, 141.3,
128.6, 128.6, 127.4, 127.3, 80.4, 61.1, 60.9, 58.6, 56.8, 54.9, 37.0,
35.6, 34.7, 34.4, 33.5, 28.3, 25.6, 25.5, 10.3. ESI-MS: m/z 607.4
[M+H]+, 629.4 [M+Na]+.
Full analytical characterization of compound 37b is reported in
the Supplementary data.
4.1.18. General procedure Q—synthesis of compounds 39
A
solution of primary amine 30 (1 equiv) and NaBH3CN
(10 equiv) in dry THF (0.1 M concentration for 30) under argon
atmosphere was treated with dry DIPEA (1.2 equiv) and, a few
minutes after, with an aldehyde (1.05 equiv) while stirring at room
temperature. After 18 h, the solution was concentrated under re-
duced pressure and the crude product was purified by semi-pre-
parative HPLC.
Full analytical characterization of compound 33b is reported in
the Supplementary data.
4.1.18.1. Compound 39a. Compound 39a was synthesized by
general procedure Q,starting from 30a (44 mg, 0.072 mmol).. HPLC
eluant conditions: from 80% of H2O, 20% of CH3CN and 0.2% AcOH
to 55% H2O, 45% of CH3CN and 0.2% AcOH. Yield 27% (13 mg, MW
693.87, 0.019 mmol) of pure acetate salt of 39a as an amorphous
white solid. Analytical characterization: 1H NMR (400 MHz, CDCl3):
7.75 (br s, 1H), 7.51 (br s, 1H), 7.33–7.22 (m, 10H), 6.21 (d,
J = 8.0 Hz, 1H), 5.41 (br s, 1H), 4.72–4.65 (m,1H), 4.56 (m, 1H),
4.03 (br s, 1H), 3.84 (m 1H), 3.01–2.80 (m, 2H), 2.39 (m, 1H),
2.27 (m, 1H), 1.87–1.65 (m, 9H), 1.45 (s, 9H), 1.36–1.10 (m, 5H),
0.90 (br s, 3H); 13C NMR (100.6 MHz, CDCl3): 175.4, 172.7, 171.6,
169.6, 159.6, 141.9, 141.0, 128.7, 128.6, 127.5, 127.4, 127.3,
127.2, 80.6, 61.0, 58.6, 56.9, 56.4, 54.4,45.7, 43.0, 37.2, 34.1, 33.3,
29.7, 29.3, 28.4, 26.0, 25.1, 20.9, 11.2, 10.5. ESI-MS: 634.3
(M+H+), 656.2 (M+Na+)
4.1.16. General procedure O—synthesis of compounds 35
Dry DIPEA (1.2 equiv) was added to a solution of primary amine
30 (1 equiv) in dry CH2Cl2 (0.1 M concentration in 30) under argon
atmosphere at room temperature. After stirring for 10 min, the
solution was treated with benzoyl chloride (1.2 equiv) and dry DI-
PEA (1.2 equiv). After 1 h, the solution was diluted with CH2Cl2 and
washed once with water. The organic layer was dried over Na2SO4,
filtered and concentrated under reduced pressure. The crude prod-
uct was purified by semi-preparative HPLC.
4.1.16.1. Compound 35a. Compound 35a was synthesized by
general procedure O, starting from 30a (69 mg, 0.113 mmol). HPLC
eluant conditions: from 50% of H2O, 50% of CH3CN and 0.2% AcOH
to 40% H2O, 60% of CH3CN and 0.2% AcOH. Yield 50% (40 mg, MW
709.87, 0.056 mmol) of pure 35a as an amorphous white solid.
Analytical characterization: 1H NMR (400 MHz, CDCl3): d: 7.93 (d,
J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.46–7.17 (m, 15H), 6.22
(d, J = 8.0 Hz, 1H), 5.13 (d, J = 8.0 Hz, 1H), 4.72 (d, J = 7.2 Hz, 1H),
4.65 (t, J = 9.2 Hz, 1H), 4.02 (m, 1H), 3.85 (m, 1H), 3.38 (m, 2H),
2.44–2.40 (m, 1H), 2.26–2.21 (m, 1H), 1.88–1.40 (m, 9H), 1.35 (s,
9H), 1.19 (m, 1H), 0.99 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3): 172.3, 172.2, 169.5, 167.5, 156.1, 141.9, 141.2, 134.7,
131.1, 128.8, 128.7, 128.6, 128.3, 127.5, 127.4, 127.3, 127.2,
127.1, 79.9, 60.9, 58.6, 56.7, 56.6, 54.6, 38.1, 37.7, 34.3, 33.6,
33.4, 32.3, 28.3, 25.7, 25.5, 10.4. ESI-MS: m/z 710.5 [M+H]+.
Full analytical characterization of compound 35b is reported in
the Supplementary data.
Full analytical characterization of compounds 39b–d is reported
in the Supplementary data.
4.1.19. General procedure R—synthesis of compounds 3a–40d
A 3 N solution of HCl in MeOH (50 equiv) was added to a stirred
solution of N-Boc protected compounds 3a–40d (varying amounts,
ranging between 0.018 and 0.090 mmol) in MeOH (0.1 M in N-Boc
protected compound), except for compounds 3b, 12a and 12b. The
reaction mixture was left stirring at room temperature overnight
and then concentrated under reduced pressure. When the crude
was not sufficiently pure, the residue was purified by chromatog-
raphy on a C18 reverse phase semi-preparative HPLC column and
then lyophilized.
4.1.19.1. Compound 3a. Compound 3a was synthesized by gen-
eral procedure R from 7a (30 mg, 0.050 mmol). HPLC eluant condi-
tions: from 80% of H2O (0.1% TFA) and 20% of CH3CN to 40% of H2O
(0.1% TFA) and 60% of CH3CN, flow rate 20 mL/min, 10 min runs.
Yield 73% (22 mg, MW 606.63, 0.036 mmol) of pure trifluoroace-
4.1.17. General procedure P—synthesis of compounds 37
A solution of primary amine 30 (1 equiv) and dry DIPEA
(1.2 equiv) in dry CH2Cl2 (0.1 M concentration in 30) under argon
atmosphere was treated with benzyl isocyanate (1.2 equiv) while

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5853
tate salt of 3a as an amorphous white solid. Analytical characteriza-
CH3CN. Yield 50% (17 mg, MW 648.67, 0.026 mmol) of pure trifluo-
tion: ½a 2D0
Š
¼ À52:6 (c 0.46, H2O); 1H NMR (400 MHz, D2O): d: 7.34–
roacetate salt of 12a as an amorphous white solid. Analytical char-
7.28 (m, 10H), 6.05 (s, 1H), 4.65 (m, 1H), 4.54 (m, 1H), 4.01 (m, 1H),
3.85 (m, 1H), 2.25–1.50 (m, 11H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR
(100 MHz, D2O): d: 173.0, 171.1, 169.5, 141.0, 140.8, 128.7, 127.8,
127.4, 127.2, 63.2, 62.0, 58.4, 57.4, 54.0, 53.8, 39.0, 32.3, 31.1,
29.3, 27.9, 24.4, 8.5. ESI-MS: m/z 493.6 [M+H]+, 615.6 [M+Na]+.
Full analytical characterization of compounds 4a,10a–b,14a,
16a,18a–b,20a–c,22a–c,24a–b,26a–d,28a–b,31a,32a–b,34a–b,36a–
b,38a–b,40a–d is reported in the Supplementary data.
acterization: ½a D20
Š
¼ À43:8 (c 0.68, H2O); 1H NMR (400 MHz, D2O):
d: 7.40–7.26 (m, 10H), 6.02 (s, 1H), 4.71 (m, 1H), 4.51 (dd, J = 7.2,
6.0 Hz, 1H), 4.11 (dd, J = 11.0, 5.4 Hz, 1H), 4.06–3.96 (m, 2H), 2.21
(m, 1H), 2.13 (m, 1H), 2.03 (s, 3H), 2.00–1.65 (m, 9H), 1.57 (m,
1H), 0.96 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, D2O) d: 175.0,
173.0, 170.5, 169.3, 141.0, 140.8, 129.0, 128.9, 127.8, 127.4,
127.2, 66.1, 62.0, 58.8, 57.7, 54.3, 53.6, 37.0, 32.3, 31.0, 29.5,
27.9, 24.4, 20.4, 8.3. ESI-MS: m/z 535.0 [M+H]+, 557.0 [M+Na]+.
Full analytical characterization of compound 12b is reported in
the Supplementary data.
4.1.19.2. Synthesis of 3b. Formic acid (1 mL) was added to 7b
(50 mg, 0.082 mmol). The reaction mixture was stirred at room
temperature overnight and then concentrated under reduced pres-
sure. The crude product was treated with 1 M NH3 in MeOH (2 mL),
the reaction mixture was stirred at room temperature overnight
and then concentrated under reduced pressure. The residue was
purified by semi-preparative HPLC. HPLC eluant conditions: from
80% of H2O (0.1% HCOOH) and 20% of CH3CN (0.1% HCOOH) to
60% of H2O (0.1% HCOOH) and 40% of CH3CN (0.1% HCOOH), flow
rate 20 mL/min, 10 min runs. Yield 74% (20 mg, MW 552.66,
0.036 mmol) of pure formate salt of 3b as an amorphous white so-
4.2. Structural methods
4.2.1. Computational methods
A docking protocol has been set up starting from the high reso-
lution crystal structure of the Smac/BIR3 complex (PDB code
1G73)21 and using the Glide 2.7 (Grid-based Ligand Docking with
Energetics)45 docking tool. Glide uses a hierarchical series of filters
to search for possible locations of the ligand in the active-site re-
gion of the receptor. The shape and properties of the receptor are
represented on a grid by several different sets of fields that provide
progressively more accurate scoring of the ligand poses. To begin
the Glide calculation an enclosing box and a bounding box are de-
fined starting from the centre of the reference ligand. The starting
poses for the ligands to be screened are generated by placing the
centre of the ligand in random points of the bounding box. Glide
allows torsional flexibility in the ligand and maintains rigid the
protein. Conformational flexibility is handled in Glide via an exten-
sive conformational search, augmented by a heuristic screen that
rapidly eliminates conformations deemed unsuitable for binding
to a receptor. During the docking process, the cycles conformations
in the ligands are held fixed, whereas the other dihedral angles are
free to rotate. The refined poses are scored using Schrödinger’s pro-
prietary GlideScore scoring function. GlideScore is based on Chem-
Score46 but includes a steric-clash term and adds buried polar
terms to penalize electrostatic mismatches. Representative con-
formers derived from a conformational search47 in the free state
lid. Analytical characterization: ½a D20
¼ À83:3 (c 1.00, MeOH); H NMR
Š
(400 MHz, D2O): d: 7.40–7.28 (m, 10H), 6.06 (s, 1H), 4.69 (m, 1H),
4.55 (dd, J = 5.5, 8.3 Hz, 1H), 4.06 (m, 1H), 3.90 (dd, J = 5.3, 7.2 Hz,
1H), 3.59 (d, J = 4.9 Hz, 2H), 2.68 (s, 3H), 2.26 (m, 1H), 2.19 (m,
1H), 2.00–1.80 (m, 6H), 1.80–1.55 (m, 3H), 0.98 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, D2O): d: 173.0, 170.9, 168.3, 141.0, 140.8,
129.0, 128.9, 127.4, 127.2, 65.0, 63.2, 62.8, 62.0, 58.7, 57.7, 54.5,
38.9, 32.3, 31.5, 31.0, 29.2, 28.0, 23.4, 8.2. ESI-MS: m/z 507.4
[M+H]+, 529.4 [M+Na]+.
4.1.19.3. Synthesis of 4b. Azide 10b was converted to the corre-
sponding amine 4b by continuous flow hydrogenation using the H-
CubeTM system (Thales Nanotechnology). 1 M HCl (107
lL,
0.107 mmol) was added at room temperature to 10b (61 mg,
MW 568.11, 0.107 mmol) in 1:1 water/dioxane (21.4 mL), and
the solution was flowed through a Pd–C catalyst cartridge (hydro-
gen pressure 1 bar, temperature 25 °C, flow rate 1.0 mL/min). The
solvent was then evaporated under reduced pressure and the crude
product was purified by semi-preparative HPLC. HPLC eluant con-
ditions: from 80% of H2O (0.2% trifluoroacetic acid) and 20% of
CH3CN (0.2% trifluoroacetic acid) to 60% of H2O (0.2% trifluoroace-
tic acid) and 40% of CH3CN (0.2% trifluoroacetic acid). Yield 32%
(25.5 mg, MW 733.7, 0.027 mmol) of pure bis-trifluoroacetate salt
of 4b as an amorphous white solid. Analytical characterization: 1H
NMR (400 MHz, D2O): d: 7.40–7.21 (m, 10H), 5.70 (s, 1H), 4.65–
4.61 (m, 1H), 4.52–4.43 (m, 1H), 4.09–3.98 (m, 1H), 3.94–3.85
(m, 1H), 3.14–3.05 (d, J = 12.0 Hz, 1H), 2.91–2.80 (dd, J = 12.0,
11.2 Hz, 1H), 2.62 (s, 3H), 2.24–1.79 (m, 8H), 1.78–1.53 (m, 3H),
0.88 (t, J = 6.4 Hz, 3H); 13C NMR (100.6 MHz, D2O): d: 172.9,
169.2, 169.0, 140.9, 140.7, 129.0, 128.9, 127.8, 127.4, 127.2, 62.6,
61.9, 58.4, 57.8, 54.3, 40.7, 36.1, 32.2, 31.5, 29.9, 28.3, 28.1, 23.3,
8.1. ESI-MS: m/z 506.3 [M+H]+, 528.3 [M+Na]+.
49
(MC/EM,48 AMBER
,
with all atom charges option, water GB/SA50
)
*
were selected as starting conformations for the docking of recently
reported Smac mimics30 and of the new ligands of general formula
I (see also Fig. 1).
The XIAP BIR3 protein structure was setup for docking as fol-
lows. The Chain D from 1G73 was selected and the metal atom
was manually assigned as Zn2+. The co-crystallized Smac chain A
was truncated at the four initial residues Ala 1-Val 2-Pro 3-Ile 4.
Ile 4 was capped as N-methyl amide. Protein-charged groups that
were neither located in the ligand-binding pocket nor involved in
salt bridges were neutralized using the Schrödinger pprep script.
Hydrogens were added using the Schrödinger graphical user inter-
face Maestro and the resulting structure was optimized using the
Schrödinger impref script. Docking results were analyzed by com-
paring the poses with the crystallographic bound AVPI in BIR3. The
pose visual inspection was carried out by monitoring binding fea-
tures such as intermolecular hydrogen bonds and Van der Waals
contacts between the ligand and the protein. Analysis of GlideScore
and of its contributes was combined to geometrical pose estima-
tion for the selection of ligand representative poses.
4.1.20. General procedure S—synthesis of compounds 12
TFA (450 lL, 5.0 mmol) was added to a stirred solution of N-Boc
esters 11 (0.10 mmol) in CH2Cl2 (2 mL). The reaction mixture was
stirred at room temperature overnight and then concentrated un-
der reduced pressure. When the crude was not sufficiently pure,
the residues were purified by semi-preparative HPLC.
4.2.2. NMR—STD methods
All protein/ligand samples were prepared in a 1:90 protein/li-
gand ratio. Tipically, the final concentration of the samples was
5 mM in Smac mimic and 0.055 mM in proteins, and the final vol-
4.1.20.1. Compound 12a. Compound 12a was synthesized by
general procedure S from 11a (33 mg, 0.052 mmol). HPLC eluant
conditions: from 70% of H2O (0.1% trifluoroacetic acid) and 30%
of CH3CN to 40% of H2O (0.1% trifluoroacetic acid) and 60% of
ume was 200
lL. The buffer used for BIR3 and full length XIAP
samples was 100 mM NaCl, 10 mM deuterated Tris, 5 mM deuter-

5854
P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
ated DTT in D2O or H2O with 10% D2O, pH 7.3. The buffer used for
L-BIR2–BIR3 was 20 mM, 200 mM, 10 mM deuterated DTT in H2O
with 10% D2O, pH 6.5. 1H NMR STD experiments were performed
at 600 MHz on a Bruker Avance spectrometer. The probe tempera-
ture was maintained at 298 K using the STD sequence of the Bruker
library. The on-resonance irradiation of the protein was performed
at a chemical shift of À0.05 ppm. Off-resonance irradiation was ap-
plied at 20 ppm, where no protein signals were visible. Selective
presaturation of the protein was achieved by a train of Gauss-
shaped pulses of 21.22-ms length each. The total length of the sat-
uration train was 1.91 s.
binding experiment. After being mixed for 15 min on a shaker
and incubated 3 h at room temperature, fluorescent polarization
was measured by Ultra plate reader (Tecan). All SMAC-mimics
and the fluorescent peptide were stocked in DMSO.
4.3.4. Fluorescence polarization assay—cloning, expression and
purification of human XIAP linker-BIR2–BIR3
A pET28 vector (Novagen) with the cDNA coding for human
XIAP from residue 124 to 356 (linker-BIR2–BIR3), coding for BIR2
and BIR3 domains and the linker region preceding BIR2, was used
to transform Escherichia coli strain BL21. Protein expression was in-
duced by adding isopropyl-b-D-thiogalactopyranoside (IPTG) to a
4.2.3. Crystallographic methods
final concentration of 1 mM and 100 lM zinc acetate (ZnAc) for
Crystals of the different XIAP BIR3 complexes were grown by
vapour diffusion methods as co-crystallizations.36,37 The crystal
structures of the XIAP BIR3 complexes with compounds 3a, 4a
and 24a were solved by molecular replacement and refined at
2.7 Å, 2.5 Å and 3.0 Å, respectively (PDB codes 3CM7, 3CM2,
3EYL) as previously described.36,37
20 h at 20 °C. Bacteria grown in 2YT medium plus kanamycin were
harvested, resuspended in a buffer containing 50 mM Tris HCl pH
7.5, 200 mM NaCl and protease inhibitors, treated with 100 lg/
mL lysozyme for 30 min in ice and then lysed by sonication. After
elimination of debris by centrifugation, the recombinant protein
was purified using Ni-NTA (His-trap Ffcrude, Ge-Healthcare) fol-
lowed by gel filtration (Superdex 200, Ge-Healthcare). The linker-
BIR2–BIR3-His-tag was eluted with 250 mM imidazole and there-
after stored in 20 mM Tris pH 7.5, 200 mM NaCl and 10 mM
Dithiothreitol.
4.3. In vitro biology profiling
4.3.1. Fluorescence polarization assay—cloning, expression and
purification of human XIAP BIR3
A pET28 vector (Novagen) with the cDNA coding for human
XIAP BIR3 domain from residue 241 to 356 was used to transform
Escherichia coli strain BL21. Protein expression was induced by
4.3.5. Fluorescence polarization assay—linker-BIR2–BIR3—
saturation binding experiments
Fluorescent polarization experiments were performed in black,
flat-bottom 96-well microplates (PBI) and fluorescent polarization
was measured by Ultra plate reader (Tecan). Fluorescent-labelled
dimeric Smac peptide SMAC-1F (Nikolovska-Coleska et al., Analyt.
Biochem. 374:87, 2008) to a final concentration of 1 nM and
increasing concentration of linker-BIR2–BIR3-His-tag from 0 to
adding isopropyl-b-
D-thiogalactopyranoside (IPTG) to a final con-
centration of 1 mM and 100
lM zinc acetate (ZnAc) for 3 h at
37 °C. Bacteria grown in 2YT medium plus kanamycin were har-
vested, resuspended in a buffer containing 50 mM Tris HCl pH
7.5, 200 mM NaCl and protease inhibitors, treated with 100 lg/
mL lysozyme for 30 min in ice and then lysed by sonication. After
elimination of debris by centrifugation, the recombinant protein
was purified using Ni-NTA (His-trap Ffcrude, Ge-Healthcare) fol-
lowed by gel filtration (Superdex 200, Ge-Healthcare). BIR3-His-
tag was eluted with 250 mM imidazole and thereafter stored in
20 mM Tris pH 7.5, 200 mM NaCl and 10 mM Dithiothreitol.
2
l
M were added to an assay buffer. The final volume in each well
was 125 L, with the assay buffer consisting of 100 mM potassium
phosphate, pH 7.5; 100 g/mL bovine -globulin; 0.02% sodium
l
l
c
azide. After a 15 min shaking, the plate was incubated for 3 h at
room temperature. Fluorescence polarization was measured at an
excitation and emission wavelengths of 485 nm and 530 nm
respectively. The equilibrium binding graphs were constructed by
plotting millipolarization units (mP) as function of the XIAP lin-
ker-BIR2–BIR3 concentration. Data were analyzed using Prism 4.0
software (Graphpad Software).
4.3.2. Fluorescence polarization assay—BIR3—saturation
binding experiments
Fluorescent polarization experiments were performed in black,
flat-bottom 96-well microplates (PBI) and fluorescent polarization
was measured by Ultra plate reader (Tecan). Fluorescent-labelled
Smac peptide [AbuRPF-K(5-Fam)-NH2] (FITC-SMAC) to a final con-
centration of 5 nM and increasing concentration of BIR3-His-tag
4.3.6. Fluorescence polarization assay—linker-BIR2–BIR3—
competitive binding experiments
SMAC-mimic compounds were evaluated for their ability to dis-
place SMAC-1F probe from recombinant protein. 1 nM of SMAC-1F,
3 nM of XIAP linker-BIR2–BIR3-His-tag and serial dilutions of the
from 0 to 20
in each well was 125
100 mM potassium phosphate, pH 7.5; 100
l
M were added to an assay buffer. The final volume
L, with the assay buffer consisting of
g/mL bovine -globu-
l
l
c
SMAC-mimic compounds (concentrations ranging from 2
lM to
lin; 0.02% sodium azide. After a 15 min shaking, the plate was incu-
bated for 3 h at room temperature. Fluorescence polarization was
measured at an excitation and emission wavelengths of 485 nm
and 530 nm respectively. The equilibrium binding graphs were
constructed by plotting millipolarization units (mP) as function
of the XIAP BIR3 concentration. Data were analyzed using Prism
4.0 software (Graphpad Software).
0.4 nM) were added to each well to a final volume of 125
lL in
the assay buffer described above. After being mixed for 15 min
on a shaker and incubated 3 h at room temperature, fluorescent
polarization was measured by Ultra plate reader (Tecan). All
SMAC-mimics and the fluorescent peptide were stocked in DMSO.
4.3.7. Cellular cytotoxicity assays
4.3.7.1. Cytotoxicity—cell lines. Human cell lines MDA-MB-231
(breast epithelial adenocarcinoma), HL-60 (promieloblast cells)
and PC-3 (prostate adenocarcinoma cells) were purchased from
Istituto Zooprofilattico di Brescia (www.bs.izs.it). Reagents for cell
culture were purchased from Sigma, unless otherwise indicated.
Cells were grown on Plastic Petri dishes (Falcon) in RPMI 1640
4.3.3. Fluorescence polarization assay—BIR3—competitive
binding experiments
SMAC-mimic compounds were evaluated for their ability to dis-
place FITC-SMAC probe from recombinant protein. 5 mM of FITC-
SMAC, XIAP BIR3-His-tag and serial dilutions of the SMAC-mimic
compounds (concentrations ranging from 4
added to each well to a final volume of 125
l
M to 0.4 nM) were
medium supplemented with 2 mM
L-glutamine, Penicillin (100 U/
lL in the assay buffer
mL) /Streptomicin (100 g/mL), 10% Fetal Bovine Serum. A sub-cul-
l
described above. The concentration of BIR3-His-tag used was
tivation ratio of 1:4 was used. Cells were maintained at 37 °C in an
60 nM, able to bind more than 50% of the ligand in the saturation
atmosphere of 95% air and 5% CO2.

P. Seneci et al. / Bioorg. Med. Chem. 17 (2009) 5834–5856
5855
4.3.7.2. Cytotoxicity—experimental protocols. Cells were seeded
samples at 10 lM were incubated in triplicate at room tempera-
in 96-well flat bottom cell culture plates at a density of 5000 cells/
well in 100 lL of culture medium. HL-60 cells were immediately
stimulated with the indicated compounds for 96 h in the incubator.
MDA-MB-231 and PC-3 cells were allowed to adhere for 24 h. prior
to be exposed to the compounds for 96 h in the incubator. Cells
were exposed to the following concentrations of the compounds:
ture for 30 min and then dialysed against the same volume of buf-
fer solution (67 mM, pH 7.4) for 4 h at 37 °C at 20 rpm in a rotating
incubator. After dialysis, 3 volumes of acetonitrile were added to
precipitate proteins, followed by centrifugation at 4000 rpm for
15 min at 4 °C and dilution with 0.1% HCOOH. LC–ESI(+)-MSMS
measurements using a fast gradient were performed using an
API4000 triple quadrupole mass spectrometer (Applied Biosys-
tems) in the multiple reaction monitoring mode (MRM). The frac-
tion unbound was calculated as the ratio between the
concentration unbound ligand (buffer compartment concentra-
tion) and the total concentration (from the denaturation of plasma
compartment samples).
50 nM, 100 nM, 200 nM, 500 nM, 1
100 M. Each point was done in triplicate. The cellular growth
inhibitory effect of our compounds was evaluated using the MTT
assay (Sigma). After 96 h of treatment, 10 L of MTT reagent solu-
lM, 5 lM, 10 lM, 50 lM and
l
l
tion (5 mg of MTT powder/mL diluted in Phosphate Buffer Salt sal-
ine solution) were added in each well and allowed to react for 3 h
in the incubator. After 3 h cells were solubilized in Lysis Buffer
(10% SDS/0.1% HCl in water, 100 lL for each well) for 24 h at
Supplementary data
37 °C. Finally, the absorbance was measured at 570 nm using a
multiplate reader. Absorbance values were collected and IC50/IC80
values were determined using GraphPad Prism5 software. The
experiments were repeated twice.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.009.
References and notes
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the membrane filter. Product suspensions/solutions were then fil-
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4.4.2. Metabolic stability
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an incubation mixture in a 96-well microplate containing 20 pmol/
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