Dual action Smac mimetics–zinc chelators as pro-apoptotic antitumoral agents

Article abstract of DOI:10.1016/j.bmcl.2016.08.065

Dual action compounds (DACs) based on 4-substituted aza-bicyclo[5.3.0]decane Smac mimetic scaffolds (ABDs) linked to a Zn²⁺-chelating moiety (DPA, o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone) through their 10 position are reported and characterized. Their synthesis, their target affinity (XIAP BIR3, Zn²⁺) in cell-free assays, their pro-apoptotic effects and cytotoxicity in tumor cells with varying sensitivity to Smac mimetics are described. The results are interpreted to evaluate the influence of Zn²⁺chelators on cell-free potency and on cellular permeability of DACs, and to propose novel avenues towards more potent antitumoral DACs based on Smac mimetics and Zn²⁺chelation.

Full text of DOI:10.1016/j.bmcl.2016.08.065

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4613–4619
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dual action Smac mimetics–zinc chelators as pro-apoptotic
antitumoral agents
Leonardo Manzoni a, Davide Gornati c, Mattia Manzotti c, Silvia Cairati a, Alberto Bossi a,b
,
Daniela Arosio a,b, Daniele Lecis d, Pierfausto Seneci c,
⇑
a Istituto di Scienze e Tecnologie Molecolari (ISTM), Consiglio Nazionale delle Ricerche (CNR), Via Golgi 19, I-20133 Milan, Italy
b SmartMatLab, Centre, Via Golgi 19, I-20133 Milan, Italy
c Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, I-20133 Milan, Italy
d Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42, I-20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Dual action compounds (DACs) based on 4-substituted aza-bicyclo[5.3.0]decane Smac mimetic scaffolds
(ABDs) linked to a Zn2+-chelating moiety (DPA, o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone) through
their 10 position are reported and characterized. Their synthesis, their target affinity (XIAP BIR3, Zn2+) in
cell-free assays, their pro-apoptotic effects and cytotoxicity in tumor cells with varying sensitivity to
Smac mimetics are described. The results are interpreted to evaluate the influence of Zn2+ chelators on
cell-free potency and on cellular permeability of DACs, and to propose novel avenues towards more
potent antitumoral DACs based on Smac mimetics and Zn2+ chelation.
Received 27 June 2016
Revised 18 August 2016
Accepted 20 August 2016
Available online 22 August 2016
Keywords:
Dual action compounds
Smac mimetics
Zinc chelation
Apoptosis
Ó 2016 Elsevier Ltd. All rights reserved.
Peptidomimetics
Cancer implies a variety of pathologically altered mechanisms,
and hundreds of putative molecular targets.1 Tumor cells become
resistant to candidates either by mutating their target (i.e., ima-
tinib2), or by effluxing them outside the cancer cell (i.e., taxol3).
A dual action compound (DAC) interferes with two validated cancer
targets, and should overcome resistance. We reported4 integrin
antagonist-Smac mimetic DACs, exploiting our experience with
anti-angiogenetic5 and pro-apoptotic6 agents.
Zinc is involved in the catalytic function and structural stability
of over 300 enzymes and proteins.7 Zinc ions prevent the autocat-
alytic conversion of procaspase-3 to active caspase-3 by interac-
tion with its ‘safety catch’ DDD sequence.8 Zinc chelation/
depletion induces rapid degradation of inhibitor of apoptosis pro-
teins (IAPs) via activation of caspase-3 and -9 in PC-3 cells.9
Smac-DIABLO is a mitochondrial protein that binds to IAPs,10
frees apoptosis-related caspases-3, -7 and -9,11 induces degradation
of cellular IAPs (cIAPs),12 and restores apoptosis in cancer cells.
Substituted aza-bicyclo[5.3.0]decane derivatives (ABDs) are potent,
cytotoxic Smac mimetics targeted against IAPs.6,13 We reasoned
that zinc depletion by Zn2+-chelating DACs built around ABDs
should activate caspases and restore apoptosis in cancer cells.
Benzoylated 4-alkylamine ABDs 1 and 2 are potent, cell perme-
able pro-apoptotic leads (Fig. 1).6 Their 10-benzhydrylamide sub-
stituent is essential for IAP binding through its pro-(S) phenyl
group.13 We replaced the IAP binding-irrelevant pro-(R)-phenyl
group with a Zn2+-chelating moiety.
Our synthetic strategy required access to multi-gram quantities
of N-Boc-protected 10-carboxy ABDs 3 and 4 (Fig. 1). We optimized
their synthesis by improving yields, reducing side reactions and
simplifying experimental protocols. Scheme 1 reports the 10-step,
optimized synthesis of compound 3. Its ꢀ50% yield is a significant
increase with respect to published un-optimized routes to 3.6,14
Similar improvements were obtained also within the synthetic
route to compound 4.15
The Zn2+ Kd of known polypyridyl Zn2+ chelators16 varies
between nano- (tri-dentate 2,20-dipicolylamine, DPA, 5) and femto-
molar values (hexa-dentate N,N,N0,N0,-tetrakis(2-pyridylmethyl)
ethylenediamine, TPEN, 6, Fig. 2). We reasoned that a small,
nanomolar Zn2+ chelating moiety coupled to a potent Smac
mimetic should ensure Zn2+ chelation in the vicinity of caspases
and IAPs. A DPA-like moiety should also reduce the risk of aspecific
Zn2+ chelation elsewhere, and should limit the molecular weight of
the resulting DACs.
We selected three DPA-based reagents to be coupled with the
10-COOH group of compound 3. DPA itself was coupled to 3 by
⇑
Corresponding author. Tel.: +39 0250314060.
E-mail address: pierfausto.seneci@unimi.it (P. Seneci).
http://dx.doi.org/10.1016/j.bmcl.2016.08.065
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

4614
L. Manzoni et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4613–4619
pro
(R)
OH
O
pro
(S)
NH
N
O
N
O
O
O
NH
( )n
O
N
H
NH
O
( )n
n = 1
3
4
N
H
N
n = 2
O
BOC
n = 1, IC50, XIAP BIR3 = 121 nM
n = 2, IC50, XIAP BIR3 = 120 nM
1
2
NH
Figure 1. Structure of pro-apoptotic Smac mimetics 1 and 2, and of key synthetic intermediates 3 and 4.
OMe
O
OMe
O
OtBu
N
N
N
O
a, b
c, d
e, f
O
O
O
N
N
N
BOC
H
HO
O
O
OH
O
OMe
O
OMe
O
N
N
N
O
g, h
i, j
O
O
NH
O
HN
3
O
N
N
BOC
H
H
N3
N3
O
N
N
BOC
BOC
a, TFA, Et3SiH, CH2Cl2, rt; b, SOCl2, MeOH, 0°C to rt, 98% (2 steps); c, H2, Pd/C, EtOH/H2O 85/15, 0.7 mL/min,
85°C, 10 bar; d, Boc2O, TEA, CH2Cl2, rt, 77% (2 steps); e, MsCl, TEA, CH2Cl2, rt; f, NaN3, DMF, 80°C, 95% (2
steps); g, TFA, DCM, rt; h, N-Me,N-Boc-(S)-2-aminobutyric acid, TBTU, HOBt, DIPEA, CH2Cl2/DMF 8/1, rt, 81%
(2 steps); i, Me3P, PhCOOH, PyS-SPy, toluene, 0° to 55°C; j, LiOH, THF/H2O 3/1, rt, 88% (2 steps).
Scheme 1. Synthesis of key intermediate 3.
means of standard peptide coupling conditions (Scheme 2). The
synthesized ‘negative control’ amide 8 does not contain the 10
(S)-phenyl group, and should lose the chelation ability of its triden-
tate DPA moiety due to amide bond formation.17
DPA was condensed with N-Boc (S)-phenylglycine 9, and after
N-deprotection the amine 11 was coupled to 3 in standard peptide
coupling conditions (Scheme 3, steps a–c).18 The resulting amide
13 contains a 10 (S) phenyl group, that should ensure IAP

L. Manzoni et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4613–4619
4615
N
N
N
N
N
N
HN
N
N
Kd Zn2+ = 0.3 fM
6
Kd Zn2+ = 210 nM
5
Figure 2. Structure of polypyridyl Zn2+ chelating agents 5 (DPA) and 6 (TPEN).
binding/inhibition. The chelation ability of its tridentate DPA moi-
ety should still be inactivated by the amide bond.
its coupling with BPEN, as observed during N-Boc (S)-phenyl-
glycine coupling with DPA in the synthesis of 13a,b. Reverse phase
purification produced two batches of pure 18a and 18b, with >90%
diastereomeric excess (Scheme 4). We will verify the effectiveness
of optimized experimental conditions (steps a0, a00, Scheme 3) to
synthesize a strongly enriched batch of 10 (S) diastereomer 18a.
PAC-1 (procaspase activating compound 1, 19, Fig. 3) was iden-
tified as a procaspase 3 activator in a HTS campaign.20 The
o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone moiety in PAC-1
The HPLC chromatogram of N-protected amide 12 showed two
close, equimolar peaks with the expected MW. We attributed them
to racemization of N-Boc (S)-phenylglycine 9 during its coupling
with DPA (step a). Reverse phase purification after N-deprotection
produced two batches of diastereomers 13a and 13b, each with
>90% diastereomeric excess (Scheme 3).
A revised experimental protocol based on mixed anhydride cou-
pling between DPA and N-Boc (S)-phenylglycine 9 (steps a0, a00),
lower reaction temperature and hindered sym-collidine as a base
(step c0, Scheme 3) was set up after extensive optimization. It pro-
duced a strongly enriched batch of 10 (S)-diastereomer 13a (92%
de).
activates caspase 3 by chelation of procaspase-bound Zn2+ 21
We
.
synthesized a PAC-1-based DAC 20 (DAC–PAC-1), bridging the o-
hydroxy, m-allyl, N-acyl (E)-phenylhydrazone moiety with the 10
COOH group of Smac mimetic 4 (Scheme 5).17
The N-Boc protecting group in compound 9 was converted to
N-Teoc (steps a, b, compound 21) to prevent acid degradation of
PAC-1-like acylhydrazones. The o-hydroxy, m-allyl, N-acyl (E)-
phenylhydrazone moiety was built in a two step sequence (steps
c and d), using conditions known to minimize epimerization and
to decrease the formation of the unwanted (Z)-hydrazone. Teoc
deprotection (step e) gave the aminoacyl hydrazone 22. The Smac
mimetic 4 was N-deprotected (step f, compound 23), to avoid an
acylhydrazone-harmful acid deprotection. Coupling of 23 with 22
(step g, Scheme 5) provided, after purification, enantiomerically
pure DAC 20 in moderate yield.
N,N,-Bis(2-pyridylmethyl)ethylenediamine 14 (BPEN)19 was
condensed with N-Boc (S)-phenylglycine, and after N-deprotection
amine 16 was coupled to 3 in standard peptide coupling conditions
(Scheme 4). The resulting amide 18 contains a 10 (S)-phenyl group,
that should ensure IAP binding/inhibition. The inserted ethylenedi-
amine spacer should preserve the chelation ability of its tridentate
DPA moiety. Thus, 18 should be a Smac mimetic—Zn2+ chelator
DAC (DAC–DPA).17
We observed by HPLC two equimolar diastereomeric DACs 18a,
b, most likely due to N-Boc (S)-phenylglycine racemization during
N
N
N
O
N
N
O
N
N
N
3
+
5
O
7
O
8
b
a
NH
NH
HN
HN
O
O
O
O
N
NH
BOC
a, HBTU, HOBt, dry DIPEA, dry CH2Cl2/DMF 8/1, N2, rt, 85%;
b, TFA, CH2Cl2, 0°C to rt, reverse phase HPLC, 87%.
Scheme 2. Synthesis of ‘negative control’ DPA amide 8.

4616
L. Manzoni et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4613–4619
N
N
N
b
a
5
+
OH
a', a"
HN
BOC
N
H2N
HN
BOC
N
O
N
O
O
9
11a,b
10a,b
N
N
H
N
N
O
N
O
N
3
O
12a,b
c
a:b ≈1:1 from step a-c
a:b ≈96:4 from step a'-c'
+
N
H
c'
N
NH
N
11a,b
HN
O
O
N
O
O
d
13a,b
N
O
a: (S)-PheGly
b: (R)-PheGly
BOC
NH
HN
O
O
NH
a, TBTU, HOBt, dry DIPEA, dry CH2Cl2/DMF 8/1, N2, rt, 92%; a', N-Boc-(S)-PheGly, iBuOCOCl, 4-Me-
morpholine, dry THF, N2, -15°C; a", 5, dry THF, N2, -30°C, 70% (2 steps from a'); b, TFA, CH2Cl2, 0°C to rt; c,
TBTU, HOBt, dry DIPEA, dry DMF, N2, 0°C to rt, 85% (2 steps from b); c', HBTU, HOBt, sym-collidine, dry DMF,
N2, 0°C to rt, 85% (2 steps from b); d, TFA, CH2Cl2, 0°C to rt, reverse phase HPLC, 76%.
Scheme 3. Synthesis of PheGly-DPA amides 13a,b.
We measured the affinity of compounds 1, 8, 13a,b, 18a,b, 19
spectrum of DAC–DPA 18a (panel A, Fig. S3, Supporting Informa-
tion) clearly indicated Zn2+ complexation.
and 20 for the BIR3 domain of XIAP (Table 1), using a fluorescence
polarization-based assay.22
The lack of a basic bridge nitrogen among the pyridine rings in 8
(Fig. S4, Supporting Information) and 13a (panel B, Fig. S3, Support-
ing Information) should prevent Zn2+ chelation. Accordingly, fluo-
rescence emission intensity and emission shape in both spectra
Compounds either lacking the 10 phenyl substitution (8) or
bearing the ‘wrong’ 10 (R)-phenyl group (13b, 18b) do not show
affinity for XIAP BIR3. Conversely, compounds bearing the 10 (S)-
phenyl group retain an affinity for XIAP BIR3 either comparable
(13a, 18a), or reduced (20) with respect to standard compound 1
(Table 1). Thus, the addition of a Zn2+ chelating moiety at the 10
position of ABDs does not necessarily impair their Smac mimetic
nature. As expected, PAC-1 19 does not interact with XIAP BIR3.
We examined the Zn2+ chelating ability of compounds 5, 8, 13a,
18a and 20 by measuring changes of their fluorescence spectra in
presence of Zn2+. Each compound was titrated with increasing con-
centrations of Zn2+, and the corresponding spectra were recorded
after equilibration (15 min, see Fig. S1 and text, Supporting
Information). Increasing [Zn2+] caused a measurable increase of
intensity in the fluorescence emission of standard DPA 5 (Fig. S2,
Supporting Information). The growth of a 500 nm band in the
were unchanged by Zn2+
.
The o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone moiety in
DAC–PAC-1 20 caused an increase of Zn2+-dependent fluorescence
intensity and UV–visible absorbance (panels C and D, Fig. S3,
Supporting Information). The spectroscopic properties of DAC–
PAC-1 20 allow a thorough characterization of free and bound Zn
species in terms of fluorescence quantum efficiency and radiative
lifetimes (Table S1, Supporting Information).
Thus, DACs containing various Zn2+ chelators may be built on
the 10-amide group of Smac mimetics, preserving both pro-apop-
totic effects when connected with suitable spacers.
We then determined the effects of compounds 1, 8, 13a,b, 18a,b,
19 and 20 on apoptosis-connected cellular processes. Western

L. Manzoni et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4613–4619
4617
N
N
N
N
N
b
9
a
N
N
NH
N
NH
H2N
N
NH2
HN
BOC
16a,b
O
14
15a,b
O
c
3
N
N
N
N
N
N
H
N
H
N
O
O
17a,b
d
NH
O
NH
O
a:b ≈1:1
N
N
O
O
18a,b
a: (S)-PheGly
b: (R)-PheGly
NH
NH
HN
HN
O
O
O
O
NH
N
BOC
a, TBTU, HOBt, dry DIPEA, dry CH2Cl2/DMF 8/1, N2, rt, 91%; b, TFA, CH2Cl2, 0°C to rt, 98%; c, TBTU,
HOBt, sym-collidine, dry DMF, N2, 0°C to rt, 86%; d, TFA, CH2Cl2, 0°C to rt, reverse phase HPLC, 64%.
Scheme 4. Synthesis of Smac mimetic—Zn2+ chelating DAC amides 18a,b.
DPA 18a, significant for 13a and DAC–PAC 20, and negligible for
other compounds. The levels of XIAP (lane 2, Fig. 4) are less
affected by our compounds, excluding a strong Zn2+ chelation-
mediated effect of DACs 18a and 20 on XIAP.
OH
H
N
N
N
Their effects on moderately Smac mimetic-resistant BT-549
cells (Fig. S5, Supporting Information) were weaker in potency,
but similar to what observed in MDA-MB231 cells. Standard 1
and DAC–DPA 18a reduced cIAP1 levels and weakly activated cas-
pase-3 and PARP, while DAC–PAC-1 20 reduced cIAP1 levels. As to
fully Smac mimetic-resistant MDA-MB543 cells (Fig. S6, Support-
ing Information), compounds 1, 13a and DACs 18a and 20 caused
cIAP1 degradation, but did not activate caspase-3 or PARP. The
levels of XIAP were not markedly affected in both cell lines.
We then determined the cytotoxicity of compounds 1, 8, 13a,b,
18a,b, 19 and 20 on the same cell lines (Table 2) after 24 h
exposure.
N
O
19
Figure 3. Structure of pro-apoptotic Zn2+ chelator 19 (PAC-1).
blots from sensitive (MDA-MB231), partially resistant (BT-549)
and fully Smac mimetic-resistant breast carcinoma tumor cell lines
(MDA-MB453) are shown in Figures 4, S5 and S6.
Their effects on cIAP1 degradation in Smac mimetic-sensitive
MDA-MB231 cells are shown on lane 1, Figure 4. cIAP1 degradation
was almost complete for DAC–DPA 18a, significant for standard 1,
13a and DAC–PAC-1 20, and negligible for other compounds, in
agreement with their affinity for BIR3 XIAP. The activation of apop-
tosis, quantified through the cleaved forms of caspase-3 (lane 4)
and PARP proteins (lane 3), is stronger for standard 1 and DAC–
XIAP-inactive compounds 13b and 18b were not cytotoxic
against Smac mimetic-sensitive MDA-MB-231 cells. Good cytotox-
icity (<5
and with DAC–PAC-1 20. Surprisingly, nanomolar IAP inhibitors
13a and DAC–DPA 18a showed only moderate, >10
lM) was observed with standard compounds 1 and 19,
lM

4618
L. Manzoni et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4613–4619
c-e
a, b
OH
H
N
OH
OH
H2
N
N
HN
BOC
HN
TEOC
O
O
O
22
9
21
OH
O
OH
O
N
N
f
O
O
NH
NH
NH
O
NH
O
23
4
NH
O
N
O
BOC
O
H
N
OH
N
N
N
H
g
23
+
O
O
N
22
O
H
NH
20
O
N
H
a, TFA, CH2Cl2, rt; b, Teoc-OSu, Na2CO3, THF/H2O 2/1, 0°C to rt, 96% (2 steps); c, N2H4.H20, EDC, HOBt,
CH3CN, rt; d, 3-allylsalicylaldehyde, cat. HCl, EtOH, reflux, 49% (2 steps); e, TBAF, THF, 70°C; f, TFA, CH2Cl2, rt,
95%; g, HATU, HOAt, sym collidine, dry DMF, N2, 0°C to rt, reverse phase HPLC, 63%.
Scheme 5. Synthesis of Smac mimetic—Zn2+ chelating DAC N-acylhydrazone 20.
Table 1
XIAP-BIR3 binding affinity
In conclusion, we have prepared pro-apoptotic DACs built by
joining a Zn2+-chelating moiety (DPA—18a, PAC-1—20) with a
Smac mimetic through its 10 amide group. DACs mostly preserve
the biological activity of both components, but do not appear to
IC50, nanomolar
16
260
8
>10,000
370
>10,000
91
>10,000
>10,000
1090
13a
13b
18a
18b
19
cIAP1
20
XIAP
cytotoxicity. We presume that the tridentate basic nature of DPA-
derived Zn2+ chelating moieties in 13a and 18a severely hindered
their permeability through cell membranes.
Cl PARP
Cl Caspase-3
PAC-1 19 and DAC–PAC-1 20 showed cytotoxicity against tumor
cell lines partially or totally resistant to Smac mimetics (Table 2).
DPA-based 13a and 18a were inactive, possibly also due to above
mentioned permeability issues. Zn2+ chelation by PAC-1 19 caused
cytotoxicity against BT-549 and especially MDA-MB453 cells. The
slightly lower cytotoxic effects of DAC–PAC-1 20 are likely due to
stronger o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone-driven
Zn2+ chelation.
Acꢀn
MDA-MB231
Figure 4. Pro-apoptotic effects of 1, 8, 13a,b, 18a,b, 19 and 20 on Smac mimetic-
sensitive MDA-MB231 cells. C, control; ⁄, unrelated compound/experiment.

L. Manzoni et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4613–4619
4619
Table 2
8. Truong-Tran, A. Q.; Grosser, D.; Ruffin, R. E.; Murgia, C.; Zalewski, P. D. Biochem.
Pharmacol. 2003, 66, 1459.
Cytotoxicity of compounds 1, 8, 13a,b, 18a,b, 19 and 20
9. Makhov, P.; Golovine, K.; Uzzo, R. G.; Rothman, J.; Crispen, P. L.; Shaw, T.; Scoll,
B. J.; Kolenko, V. M. Cell Death Differ. 2008, 15, 1745.
MDA-MB231*
BT-549*
MDA-MB543*
10. Du, C.; Fang, M.; Li, Y.; Wang, X. Cell 2000, 102, 33.
1
8
2.3
>25
>25
>25
>25
>25
>25
14.3
21.7
>25
>25
>25
>25
>25
>25
2.0
11. Chai, J.; Du, C.; Wu, J.-W.; Kyin, S.; Wang, X.; Shi, Y. Nature 2000, 406, 855.
12. Varfolomeev, E.; Blankenship, J. W.; Wayson, S. M.; Fedorova, A. V.; Kayagaki,
N.; Garg, P.; Zobel, K.; Dynek, J. N.; Elliott, L. O.; Wallweber, H. J.; Flygare, J. A.;
Fairbrother, W. J.; Deshayes, K.; Dixit, V. M.; Vucic, D. Cell 2007, 131, 669.
13. Sun, H.; Nikolovska-Coleska, Z.; Lu, J.; Meagher, J. L.; Yang, C.- Y.; Qiu, S.;
Tomita, Y.; Ueda, Y.; Jiang, S.; Krajewski, K.; Roller, P. P.; Stuckey, J. A.; Wang, S.
J. Am. Chem. Soc. 2007, 129, 15279.
14. Bianchi, A.; Ugazzi, M.; Ferrante, L.; Lecis, D.; Scavullo, C.; Mastrangelo, E.;
Seneci, P. Bioorg. Med. Chem. Lett. 2012, 22, 2204.
15. The detailed, optimized Scheme to compound
Supplementary Information.
21.0
18.5
>25
16.0
>25
4.7
13a
13b
18a
18b
19
20
4.1
6.2
*
IC50, micromolar. Measured by CellTiter-Glo (Promega).
4 is reported in the
16. Zuo, J.; Schmitt, S. M.; Zhang, Z.; Prakash, J.; Fan, Y.; Bi, C.; Kodanko, J. J.; Dou, Q.
P. J. Cell. Biochem. 2012, 113, 2567.
17. The experimental protocol to compounds 8, 13b, 18a,b and 20, and their
analytical characterization are reported in the Supplementary Information.
18. Experimental protocol to DAC–DPA 18a:
provide synergistic effects in cellular assays. In particular,
DPA-based moieties are detrimental to cytotoxicity due to poor
permeability through cell membranes.
We plan to continue our investigation on Smac mimetic—Zn2+
chelator DACs by exploring their connection through the 4 position
of Smac mimetics; by studying the influence of linkers between
units; and by employing Zn2+ chelating moieties with more suit-
able physico-chemical properties for cellular permeation. These
studies will be reported in due time.
tert-Butyl 2-(bis(pyridin-2-ylmethyl)amino)-2-oxo-1-(S)-phenylethyl carbamate
(10a): 4-Methylmorpholine (0.168 mL, 1.528 mmol, 3 equiv) was added
under nitrogen at ꢁ30 °C to a stirred solution of N-Boc-(S)-PheGly 9 (128 mg,
0.510 mmol, 1 equiv) and ClCO2iBu (0.09 mL, 0.713 mmol, 1.4 equiv) in 2.4 mL
of dry THF. After stirring for 30 min, di(2-picolyl)-amine (DPA, 5, 152.2 mg,
0.764 mmol, 1.5 equiv) was added at ꢁ30 °C to the colourless suspension. The
mixture was stirred at ꢁ30 °C, and monitored by TLC (eluant condition CH2Cl2/
MeOH 95:5). After 3 hours, 15 mL of water were added to the mixture. The
resulting colourless solution was extracted with AcOEt (2 ꢂ 20 mL). The
organic phases were washed with NH4Cl (3 ꢂ 20 mL) and with 30 mL of
brine. The organic phases were dried with Na2SO4, filtered and the solvent was
removed under reduced pressure. The crude product (284.1 mg) was purified
by reverse phase flash chromatography (eluant H2O/MeCN from 100:0 to
0:100), yielding 150.1 mg of compound 10a as a pale yellow oil (0.351 mmol,
69% yield).
Acknowledgments
We gratefully acknowledge MIUR – Italy (Ministero dell’Univer-
sità
e della Ricerca) for financial support (PRIN project
2010NRREPL: Synthesis and biomedical applications of tumour-
targeting peptidomimetics). The use of instrumentation purchased
through the Regione Lombardia—Fondazione Cariplo joint project
‘SmartMatLab Centre’ (decreti 12689/13, 7959/13; Azione 1 e 2)
is gratefully acknowledged; A.B. thanks Dr. Marta Penconi for the
collection of fluorescence data and discussions.
2-Amino-2-(S)-phenyl-N,N-bis(pyridin-2-ylmethyl)acetamide
(11a):
TFA
(3.48 mmol, 0.265 ml, 30 equiv) was added under stirring at 0 °C to
a
solution of 10a (50.0 mg, 0.116 mmol, 1 equiv) in 0.58 mL of CH2Cl2 [0.2 M].
The reaction mixture was stirred at 0 °C for 30 min, and at rt for 2 h while
monitoring by TLC (eluant CH2Cl2/MeOH 95:5). The solvent was removed
under reduced pressure. Two aliquots of toluene (2 ꢂ 2 mL) were then added,
and removed under reduced pressure. The residue was then dried under
vacuum yielding 78.3 mg of crude product 11a as a yellow solid, that was used
without further purification.
tert-Butyl
(S)-1-((3S,6S,7R,9aS)-7-(benzamidomethyl)-3-(2-(bis(pyridin-2-
Supplementary data
ylmethyl)amino)-2-oxo-1-phenylethylcarbamoyl)-5-oxooctahydro-1H-pyrrolo
[1,2-a]azepin-6-ylamino)-1-oxobutan-2-yl(methyl)carbamate (12a): Compound
11a (58.3 mg, 0.140 mmol, 1.2 equiv) and 2,4,6 trimethylpyridine (0.03 mL,
2 equiv) were dissolved under nitrogen atmosphere in 0.8 mL of anhydrous
DMF. The mixture was cooled at 0 °C, then HBTU (57.1 mg, 0.151 mmol,
1.3 equiv) and HOBt (23.5 mg, 0.174 mmol, 1.7 equiv) were added. After
10 min, a solution of compound 3 (78.3 mg, 0.116 mmol, 1 equiv) and sym-
collidine (0.047 mL, 3 equiv) in 0.4 mL of anhydrous DMF was added, the
reaction mixture was warmed up to rt and monitored by TLC (eluant DCM/
MeOH 95:5). After 12 h the solution was diluted with 20 mL of AcOEt and
washed with NH4Cl (3 ꢂ 20 mL). The organic phases were dried with Na2SO4,
filtered and the solvent was removed under reduced pressure. The crude
product (120.9 mg) was purified by flash chromatography (eluant DCM/MeOH
96:4), yielding 84.7 mg of compound 12a as a pale yellow solid (0.098 mmol,
85% yield, ꢀ92 de).
Supplementary data (experimental procedures for the synthesis
of compounds 3, 4, 8, 13a, 13b, 18b and DACs 18a and 20. LC–MS
and NMR characterization of compounds 3, 4, 8, 13a, 13b, 18a, 18b
and DACs 18a and 20. Experimental procedures for cell-free test-
ing/binding affinity determination of compounds, DACs and stan-
dards in presence of BIR3 constructs from XIAP. Experimental
procedures for Zn2+ titration of compounds 5, 8, 13a, and DACs
18a and 20. Experimental procedures for mechanism of action
studies and cellular cytotoxicity testing of compounds, DACs and
standards against human tumor cell lines) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmcl.2016.08.065.
(3S,6S,7R,9aS)-7-(Benzamidomethyl)-N-(2-(bis(pyridin-2-ylmethyl)amino)-2-
oxo-1-phenylethyl)-6-((S)-2-(methylamino)butanamido)-5-oxooctahydro-1H-
pyrrolo[1,2-a]azepine-3-carboxamide (13a): TFA (0.230 mL) was added
dropwise over a period of 30 min at 0 °C to a stirred solution of compound
12a (84.7 mg, 0.098 mmol, 1 equiv) in CH2Cl2 (0.5 mL). The reaction mixture
was stirred at rt and monitored by TLC (eluant DCM/MeOH 95:5). After 2 hours
the solvent was removed under reduced pressure. Two aliquots of toluene
(2 ꢂ 2 mL) were then added, and removed under reduced pressure. The
remaining residue was then dried under vacuum yielding 100.4 mg of crude
product as a brown viscous oil. The crude product was purified by HPLC (eluant
H2O/MeCN 100:0 to 0:100), yielding 74.6 mg of compound 13a as a white solid
References and notes
1. http://www.phrma.org/media/releases/nearly-1000-medicines-development-
help-patients-their-fight-against-cancer.
2. Mauro, M. J. Hematology 2006, 1, 219.
3. Sangrajrang, S.; Fellou, A. Chemotherapy 2000, 46, 327.
4. Mingozzi, M.; Manzoni, L.; Arosio, D.; Dal Corso, A.; Manzotti, M.; Innamorati,
F.; Pignataro, L.; Lecis, D.; Delia, D.; Seneci, P.; Gennari, C. Org. Biomol. Chem.
2014, 12, 3288.
(0.068 mmol, 71% yield), and 3.2 mg of compound 13b as
a white solid
(0.003 mmol).
19. Fukuoka, S.; Kida, T.; Nakajima, Y.; Tsumagari, T.; Watanabe, W.; Inaba, Y.;
Mori, A.; Matsumura, T.; Nakano, Y.; Takeshita, K. Tetrahedron 2010, 66, 1721.
20. Putt, K. S.; Chen, G. W.; Pearson, J. M.; Sandhorst, J. S.; Hoagland, M. S.; Kwon, J.-
T.; Hwang, S.-K.; Jin, H.; Churchwell, M. I.; Cho, M.-H.; Doerge, D. R.; Helferich,
W. G.; Hergenrother, P. J. Nat. Chem. Biol. 2006, 2, 543.
21. Peterson, Q. P.; Goode, D. R.; West, D. C.; Ramsey, K. N.; Lee, J. J. Y.;
Hergenrother, P. J. J. Mol. Biol. 2009, 388, 144.
22. Nikolovska-Coleska, Z.; Meagher, J. L.; Jiang, S.; Kawamoto, S. A.; Gao, W.; Yi,
H.; Qin, D.; Roller, P. P.; Stuckey, J. A.; Wang, S. Anal. Biochem. 2008, 374, 87.
5. Manzoni, L.; Belvisi, L.; Arosio, D.; Civera, M.; Pilkington-Miksa, M.; Potenza, D.;
Caprini, A.; Araldi, E. M. V.; Monferini, E.; Mancino, M.; Podestà, F.; Scolastico,
C. ChemMedChem 2009, 4, 615.
6. Seneci, P.; Bianchi, A.; Battaglia, C.; Belvisi, L.; Bolognesi, M.; Caprini, A.; Cossu,
F.; De Franco, E.; De Matteo, M.; Delia, D.; Drago, C.; Khaled, A.; Lecis, D.;
Manzoni, L.; Marizzoni, M.; Mastrangelo, E.; Milani, M.; Motto, I.; Potenza, D.;
Rizzo, V.; Servida, F.; Turlizzi, E.; Varrone, M.; Vasile, F.; Scolastico, C. Bioorg.
Med. Chem. 2009, 17, 5834.
7. Hooper, N. M. Zinc Metalloproteases in Health and Disease; Taylor & Francis:
London, 1996.