Design and synthesis of novel azapeptide activators of apoptosis mediated by caspase-9 in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2014.05.095

A set of azapeptides was designed based on the Ala-Val-Pro-Ile peptide (derived from Smac protein) to activate caspase-9 and induce apoptosis in breast cancer cells. The diversity-oriented synthesis of the aza-peptides 5-9 was accomplished by alkylation o

Full text of DOI:10.1016/j.bmcl.2014.05.095

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3361–3365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel azapeptide activators of apoptosis
mediated by caspase-9 in cancer cells
Carine B. Bourguet a, Pierre-Luc Boulay b, , Audrey Claing b, William D. Lubell a,
⇑
a Département de Chimie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal H3C 3J7 QC, Canada
b Département de Pharmacologie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal H3C 3J7 QC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of azapeptides was designed based on the Ala-Val-Pro-Ile peptide (derived from Smac protein) to
activate caspase-9 and induce apoptosis in breast cancer cells. The diversity-oriented synthesis of the
aza-peptides 5–9 was accomplished by alkylation of the aza-residue of aza-Gly-Pro dipeptide 15 using
potassium tert-butoxide and a range of different alkyl halides. The resulting protected aza-dipeptide
building blocks were then introduced into mimics 5–9 using standard coupling conditions. Biological
evaluation of 5–9 was performed in MDA-MB-231 breast cancer cells, and indicated that the aza-Gly
and aza-Phe analogs 5 and 7 were most efficient in inducing cell death by a caspase-9 mediated apoptotic
pathway. Revealing a relationship between azabicycloalkanone and aza peptide mimics, novel AVPI mim-
ics were synthesized which exhibit utility for studying structure–activity relationships to develop leads
for activating apoptosis in cancer cells.
Received 30 April 2014
Revised 26 May 2014
Accepted 28 May 2014
Available online 14 June 2014
Keywords:
Azapeptides
Peptide mimic
Beta-turn
Caspase-9 activator
Apoptosis
Ó 2014 Elsevier Ltd. All rights reserved.
Anti-cancer drug
Apoptosis is a controlled sequence by which cells signal their
own termination. Programmed cell death is crucial for normal tis-
sue development and homeostasis,1,2 because it permits elimina-
tion of aging and abnormal cells to maintain organism integrity.
In contrast to normal cells, cancer cells ‘switch off’ apoptosis,
develop and divide indefinitely.3,4
Programmed cell death is a multistep process highly regulated
and primarily under the control of two type of proteins: the cas-
pase enzyme family and their modulators, so-called IAPs (inhibi-
tors of apoptosis proteins).5 Among the latter, X-linked IAPs
(XIAPs) are key regulators of death-receptor- and mitochondria-
mediated apoptosis pathways. They also constitute attractive
targets because of their roles in the resistance of cancer cells to
apoptosis.6,7 The XIAP contains three baculoviral IAP repeat
(BIR1–3) domains, which bind and inhibit the activity of caspas-
es-3, -7 and -9.
tetra-peptide have thus been targeted to inhibit the XIAP-BIR3
interaction and induce apoptosis.12–16 Among the constrained ana-
logs, those containing fused 6,5-, 7,5- and 8,5- bicyclic turn surro-
gates as replacements of the Val-Pro dipeptide have exhibited
notable potency (e.g., 2–4, Fig. 1) and have demonstrated that a
b-turn is preferable for affinity and activity (Fig. 1).12–16 Although
such azabicyclo[X.Y.0]alkan-2-one amino acid analogs represent
an important class of Smac mimics, their synthesis demands multi-
ple steps restricting analog development.
Semicarbazides are aza-amino acids. In peptides, aza-amino
acid residues can exert conformational constraints due to the urea
and hydrazine, which induce turn conformations. Moreover, the
introduction of an aza-residue into
a
peptide can improve
O
( )n
O
O
H
H
N
H2N
N
H2N
N
Smac (second mitochondria-derived activator of caspase) is a
potent pro-apoptotic protein released from mitochondria in
response to apoptotic stimuli.8,9 Smac promotes apoptosis in cells
by binding to the BIR3 domain of XIAP primarily via its N-terminal
four residues Ala-Val-Pro-Ile,10,11 and liberating consequently cas-
pase-9. Mimics based on a constrained version of the Smac AVPI
N
Ph
N
H
N
H
NH2
CH3
O
CH3
O
O
O
Ph
2 : n = 1 ; Ki = 2.33
M
Smac-derived AVPI-NH2
3 : n = 2 ; Ki = 60 nM
4 : n = 3 ; Ki = 14 nM
1 Ki = 0.58
M
Aza-Gly-Pro Smac Analogs
5 : R = H
O
R
H
N
H2N
N
N
6 : R = propargyl
7 : R = benzyl
Ph
N
H
⇑
CH3
O
Corresponding author. Tel.: +1 514 343 7339; fax: +1 514 343 7586.
O
Ph
8 : R = allyl
E-mail address: Lubell@chimie.umontreal.ca (W.D. Lubell).
Current address: Rosalind and Morris Goodman Cancer Center, 1160 Pine Avenue,
Montreal H3A 1A3 QC, Canada.
9 : R = methyl
Figure 1. AVPI and peptide mimic activators of caspase-9.
http://dx.doi.org/10.1016/j.bmcl.2014.05.095
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3362
C. B. Bourguet et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3361–3365
metabolic stability, because the urea is more resistant to chemical
and enzymatic degradation than its amide counterpart.17 Recently,
we have demonstrated the relationship between the azabicy-
clo[4.3.0]alkan-2-one amino acid (indolizidinone amino acid,
I2aa) and aza-amino acyl proline residues as turn mimics.18,19 For
example, replacement of the Gly33-Pro34 moiety in [D31, P34, F35-
]calcitonin gene-related peptide27–37 by AzaGly33-Pro34 and
I2aa33–34 gave respectively 10- and 7-fold increases in antagonist
potency.18 Subsequently, in our efforts to develop molecules,
which can inhibit uterine contractions and delay preterm labor,
we have recently reported that replacing the central I2aa residue
Pro-Ot-Bu dipeptide 15 (Scheme 2).21 Treatment of aza-dipeptide
15 with tert-butoxide and alkylation with different alkyl halides
(propargyl bromide, benzyl bromide, allyl bromide and methyl
iodide) afforded the corresponding aza-amino acyl proline dipep-
tides 16a–d in 56–84% yields.21 The benzhydrylidene protection
was then removed by treatment with hydroxylamine hydrochloride
in pyridine and Fmoc-Ala was coupled to the resulting semicarbaz-
ides using iso-butyl chloroformate and N-methyl morpholine to give
the aza-tripeptides 17a–d in 45–77% yields. Cleavage of the tert-
butyl ester with trifluoroacetic acid in dichloromethane, coupling
to diphenyl methyl amine with the same coupling conditions, and
Fmoc group removal with a solution of 20% piperidine in DMF
yielded Smac mimics 6–9 in 40–65% overall yields from 18a–d
(Scheme 2). In addition, Ala-Val-Pro-Ile-NH2 was prepared as a posi-
tive control by standard Fmoc-based solid-phase peptide synthesis
on Rink resin in 25% overall yield (see Experimental section).22
The pro-apoptotic potential of Smac mimics 6–9 was examined
in MDA-MB-231 breast cancer cells at two different concentra-
of the prostaglandin F2
a modulator PDC113.824 by the azaGly-
Pro dipeptide can maintain activity and efficacy.19 Considering
the potential to explore the importance of valine for binding and
activity by the preparation of aza-analogs of AVPI, we report the
design and synthesis of a new series Smac mimics and their biolog-
ical evaluation as caspase-9 activators that induce apoptosis in
breast cancer cell lines (e.g., 5–9, Fig. 1).
The synthesis of azaGly-Pro dipeptide 11 and its use for the syn-
thesis of Smac mimic 5 were previously reported (Scheme 1).20
Acylation of proline with the activated benzhydrylidine carbazate
generated from benzophenone hydrazone and p-nitrophenyl chlo-
roformate gave azaGly-Pro dipeptide 11, which was coupled to
diphenyl methyl amine. Synthesis of azaGly-Pro Smac mimic 5
was then completed by removal of the benzophenone protection,
acylation with Boc-Ala, and Boc group removal. Aza-peptide 5
was thus obtained in five steps and 35% overall yield.20
tions: 50 lM and 100 lM (Fig. 2). At 100 lM, AVPI-NH2 and Smac
mimics 5–9, all induced cell death at levels (ꢀ50%) similar to the
topoisomerase II inhibitor Etoposide, which blocks DNA replica-
tion.23 At 50
l
M, the relative activities of Smac mimics 5–9 became
apparent. Relative to vehicle (DMSO), the parent peptide AVPI-NH2
weakly induced cell death (11.5%) at 50 M. Aza-propargyl and
l
aza-allylglycine analogs 6 and 8 exhibited no activity, and aza-ala-
nine analog 9 was only slightly more active relative to vehicle. In
contrast, aza-glycine and aza-phenylalanine analogs 5 and 7 pro-
moted cell death more effectively than the parent peptide at the
same concentration.
Diversification of the aza-glycine residue was accomplished by a
route featuring alkylation of benzophenone-protected azaGly-
1. 4-nitrophenylchloroformate
Ph
H2NCH(Ph)2, TBTU,
Ph
CH2Cl2,
C
H
HOBt, DIEA, CH2Cl2
2. Pro-OH, DIEA, CH2Cl2
NH2
N
N
Ph
N
Ph
N
CO2H
O
10
11 (58%)
HCl (1N), THF,
C, 24h
Ph
H
N
H
N
H
N
H
N
N
Ph
N
2N
Ph
Ph
N
O
O
Ph
O
O
Ph
12 (96%)
13 (99%)
O
Boc-Ala, TBTU,
HOBt, DIEA, CH2Cl2
H
H
RHN
N
N
N
Ph
N
H
CH3
O
O
Ph
1) 25% TFA/CH2Cl2, 1h
2) HCl (1N), freeze dry
14: R = Boc (65%)
HCl (99%)
5
Scheme 1. Synthesis of azaGly-Smac mimic 5.
1. 4-nitrophenylchloroformate
1. t-BuOK
Ph
Ph
CH2Cl2,
C
C
H
N
2. Pro-Ot-Bu, DIEA, CH2Cl2
NH2
2. R-X
N
Ph
N
Ph
N
CO2t-Bu
O
10
15 (51%)
1. NH2OH HCl,
O
R
N
Ph
R
N
C 12h
FmocHN
N
N
N
H
Ph
N
2. Fmoc-Ala-OH,
i-BuOCOCl,
C
CO2t-Bu
CO2t-Bu
16a-d (56-84%)
O
O
17a-d (45-77%)
O
R
N
R
1. TFA:CH2Cl2 (9:1)
H
N
PHN
N
Ph
a: -CH2C CH
b: -CH2Ph
c: -CH2CH CH2
d: -CH3
N
H
2. (Ph)2CHNH2,
i-BuOCOCl,
C
O
O
Ph
18a-d : P = Fmoc (64-92%)
6-9 : P = H (40-65%)
20% piperidine
DMF
Scheme 2. Synthesis of Smac mimic 6–9.

C. B. Bourguet et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3361–3365
3363
mimics induced cell death at the higher concentration, the two
aza-analogs 5 and 7 proved to be more effective than AVPI-NH2
A
MDA-MB-231
60
50
40
30
20
10
0
at 50 lM.
***
***
The activation of caspase-9 and caspase-3 via proteolytic cleav-
age is crucial for the regulation of the apoptotic program. There-
fore, we examined conversion of their pro-forms into active
proteins by Western blotting (Fig. 3). High binding affinity between
Smac protein and XIAP is contingent on the interaction of its AVPI
peptide with the BIR3 domain of XIAP, which induces caspase-9
activity. Thus, we expected Smac mimicry would increase levels
of caspase-9 in the Western blot (Fig. 3). Moreover, caspase-3
activation is induced by caspase-9, thus intensity of the band for
caspase-3 should correlate with caspase-9 and Smac mimic
activity.
*
O
5
1
6
2
3
8
4
9
7
Smac mimetics
compounds
50
M
At 100 lM concentration, Smac mimics 5–9 as well as AVPI-NH2
MDA-MB-231
***
and Etoposide, all enhanced formation of active caspases-3 and -9
(Fig. 3B). However, relative effectiveness of the AVPI analogs was
better indicated at 50 lM. At this concentration, only aza-phenyl-
B
60
50
40
30
20
10
0
alanine analog 7 produced detectable amounts of caspases-3 and -
9. In addition, Etoposide resulted only in caspase-3 activation at
this concentration, likely due to the short exposure to the drug.
In fact, previous publications reported that a 48 h treatment was
required to activate caspase-3 in MDA-MB-231 cells.24
Dose–response experiments were subsequently performed to
determine EC50 values (Fig. 4). The EC50 values of AVPI-NH2, and
aza-glycine and aza-phenylalanine analogs 5 and 7 were respec-
O
5
1
2
7
3
8
4
9
6
tively, 72.66 lM, 73.37 lM and 50.18 lM. In sum, at high concen-
Smac mimetics
compounds
100
M
tration, AVPI-NH2 and azapeptides 5–9, all exhibited a pro-
apoptotic potential through the activation of caspases-3 and -9.
Among the aza-analogs, aza-phenylalanine derivative 7 exhibited
the highest efficacy in activating caspases-3 and -9.
Figure 2. Smac mimic induced cell death in MDA-MB-231 cell line. (A) Cells were
serum starved for 6 h and then treated with DMSO, etoposide (50 M), AVPI-NH2
(50 M) or indicated Smac mimetic compounds for 16 h using a final concentration
of 50 M. After incubation, cells were trypsinized, stained with Trypan blue and
counted. (B) MDA-MB-231 cells were treated as in A and incubated with all
compounds at a final concentration of 100 M. Data are expressed as percentage of
l
l
In conclusion, we developed a new strategy for making Smac
mimics that promote cell death based on the synthesis of azapep-
tides. In particular, aza-glycine and aza-phenylalanine analogs 5
and 7 promoted caspase-3 and -9 activation and caused ultimately
cell death. Aza-phenylalanine 7 was 25% more potent than AVPI-
NH2 in the MDA-MB-231 breast cancer cell assays. The enhanced
activity of 7 may be a combination of its potential to mimic the
active turn conformation of the native peptide as well as better
ability to interact at the binding site in which valine is normally
accommodated.
l
l
cell death (%). These results are the mean SEM of three independent experiments.
⁄⁄⁄P < 0.001, ⁄P < 0.05 are values compared to the paired control DMSO condition
and P < 0.001 are values compared to the paired AVPI-NH2 condition.
Moreover, aza-phenylalanine analog 7 enhanced cell mortality
with similar efficacy as Etoposide. In summary, although all Smac
A
B
MDA-MB-231
MDA-MB-231
Smac mimetics
Smac mimetics
50 µM
100 µM
compounds
5 6 7 8
9
5
6
7
compounds8
9
4
O
1
2
3
4
O
1
2
3
IB: Caspase-9
IB: Caspase-9
IB: Caspase-9 cleaved
IB: Caspase-3
IB: Caspase-9 cleaved
IB: Caspase-3
IB: Caspase-3 cleaved
IB: Caspase-3 cleaved
Figure 3. Smac mimic promoted activation of caspase-3 and caspase-9 in MDA-MB-231 cells. (A) Cells were serum starved for 6 h, treated with DMSO, etoposide (50
lM),
AVPI-NH2 (50 M) and related synthetic compounds at a final concentration of 50 M, harvested and activation of caspase-3 and caspase-9 was assessed by Western blotting.
l
l
Quantifications are the mean SEM of three independent experiments. (B) Cells were treated as in A, where all compounds were used at a final concentration of 100
lM.
Quantification is the mean SEM of three independent experiments.

3364
C. B. Bourguet et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3361–3365
Smac mimic 5
AVPI
A
B
O
25 50 75 100 125
O
25 50 75 100 125
M
M
IB: Caspase-9
IB: Caspase-9
IB: Caspase-9 cleaved
IB: Caspase-9 cleaved
3
2
1
0
***
***
7
6
5
4
3
2
1
0
**
**
**
0
25
50
75
100
125 ( M)
0
25
50
75
100
125 ( M)
AVPI
Smac mimic 5
C
Smac mimic 7
O
12.5 25 37.5 50 75 100
M
IB: Caspase-9
IB: Caspase-9 cleaved
7
6
5
4
3
2
1
0
***
***
**
( M)
100
0
25
50
75
Smac mimic 7
Figure 4. Smac mimic promoted activation of caspase-3 and caspase-9 in MDA-MB-231 cells. (A) Cells were serum starved for 6 h and treated with AVPI-NH2 at indicated
concentrations for 16 h and then harvested. Activation of caspase-9 was measured by Western blotting (upper panel). Quantifications are the mean SEM of two independent
experiments (lower panel). (B and C) MDA-MB-231 cells were treated as in A and incubated respectively with 5 and 7. Quantification is the mean SEM of two independent
experiments. ⁄⁄⁄P < 0.001, ⁄⁄P < 0.01, ⁄P < 0.05 are values compared to the paired untreated (DMSO) condition.
Considering the utility of diversity-oriented methods for gener-
ating aza-peptides, they represent a promising new class of Smac
mimics for probing the structure–activity relationships at the AVPI
binding site towards the development of improved therapy for
treating cancer.
and Ms. Sylvie Bilodeau and Mr. Cedric Malveau of the Regional
High-Field NMR Laboratory. This research was supported by the
Natural Sciences and Engineering Research Council of Canada
(NSERC).
Supplementary data
Acknowledgments
Supplementary data (synthetic experimental detailed proce-
dures and analytical data for compounds 17a–d, 18a–d, 6–9 and
AVPI-NH2, and biological assay protocols) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2014.05.095.
The authors would like to thank Dr. Mohamed Atfani for his
precious help in NMR analyses, Dr. Alexandra Fürtös, Marie-Chris-
tine Tang, Karine Venne and Christophe Camy of the Université de
Montréal Mass Spectrometry facility for mass spectral analyses,

C. B. Bourguet et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3361–3365
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