4-Connected azabicyclo[5.3.0]decane Smac mimetics-Zn2+ chelators as dual action antitumoral agents

Article abstract of DOI:10.1016/j.bmcl.2017.04.032

Putative dual action compounds (DACs 3a–d) based on azabicyclo[5.3.0]decane (ABD) Smac mimetic scaffolds linked to Zn²⁺-chelating 2,2′-dipicolylamine (DPA) through their 4 position are reported and characterized. Their synthesis, their target a

Full text of DOI:10.1016/j.bmcl.2017.04.032

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Connected azabicyclo[5.3.0]decane Smac mimetics-Zn2+ chelators as
dual action antitumoral agents
Leonardo Manzoni a, Alessandro Samela b, Stefano Barbini b, Silvia Cairati a, Marta Penconi a,c
,
Daniela Arosio a,c, Daniele Lecis d, Pierfausto Seneci b,
⇑
a Istituto di Scienze e Tecnologie Molecolari (ISTM), Consiglio Nazionale delle Ricerche (CNR), Via Golgi 19, I-20133 Milan, Italy
b Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, I-20133 Milan, Italy
c SmartMatLab Centre, 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:
Putative dual action compounds (DACs 3a–d) based on azabicyclo[5.3.0]decane (ABD) Smac mimetic
scaffolds linked to Zn2+-chelating 2,20-dipicolylamine (DPA) through their 4 position are reported and
characterized. Their synthesis, their target affinity (cIAP1 BIR3, Zn2+) in cell-free assays, their pro-apop-
totic effects, and their cytotoxicity in tumor cells with varying sensitivity to Smac mimetics are described.
A limited influence of Zn2+ chelation on in vitro activity of DPA-substituted DACs 3a–d was sometimes
perceivable, but did not lead to strong cellular synergistic effects. In particular, the linker connecting
DPA with the ABD scaffold seems to influence cellular Zn2+-chelation, with longer lipophilic linkers/
DAC 3c being the optimal choice.
Received 24 February 2017
Revised 8 April 2017
Accepted 11 April 2017
Available online xxxx
Keywords:
Dual action compounds
Smac mimetics
Zinc chelation
Apoptosis
Ó 2017 Published by Elsevier Ltd.
Peptidomimetics
Multiple pathophysiological mechanisms are connected with
the development and the progression of cancer.1 The ability of
cancer cells to develop resistance to chemotherapeutics and tar-
get-directed therapies2 demands for more effective treatments.3
A dual action compound (DAC) should interfere with two validated
mechanisms against cancer, and should thus increase efficacy and
minimize resistance.4
4 and 10 of their bicyclic core (Fig. 1) can be substituted to increase
their IAP affinity, and their drug-like profile.11 We reasoned that
zinc depletion by Zn2+-chelating DACs built around ABDs
should cause synergistic pro-apoptotic effects. We reported12
4-amidoalkyl ABDs substituted in position 10 with a Zn2+-chelating
moiety (i.e., 2,20-dipicolylamine, DPA in 1, Fig. 1). A (S)-PheGly
linker connected DPA with Smac mimetic ABD scaffolds (i.e.,
4-benzamidomethyl Smac mimetic 2,11 Fig. 1).
Smac-DIABLO is
a mitochondrial protein with multiple
pro-apoptotic effects in cancer cells. It binds to inhibitor of
apoptosis proteins5 (IAPs), frees caspases-3, -7 and -9,6 and induces
degradation of cellular IAPs (cIAPs).7 Zinc ions show anti-apoptotic
effects in cancer cells. They prevent the autocatalytic conversion of
procaspase-3 to active caspase-3 by interaction with its ‘‘safety
catch” DDD sequence,8 and their chelation causes serine
protease-dependent depletion of anti-apoptotic X-linked IAP
(XIAP).9
10-Connected Smac mimetic- Zn2+-chelator DACs showed
cell-free potency against IAPs and Zn2+-chelating properties. Their
cytotoxicity was moderate, and Zn2+ chelation-dependent
effects in cellular assays were not observed.12 We then targeted
DPA-containing, ABD-based DACs connected through position 4,
bearing an IAP affinity-best diphenylmethylamide substitution in
position 10 (generic structure 3, Fig. 2). In particular, we aimed
to evaluate the influence of the linker between DPA and ABD scaf-
folds on cell-free properties, on biological activity and bioavailabil-
ity in cancer cells.
Substituted azabicyclo[5.3.0]decane Smac mimetics (ABDs) are
potent, cytotoxic Smac mimetics targeted against IAPs.10 Positions
Our synthetic strategy required access to gram quantities
of N3-Boc-protected 4-amino-10-diphenylmethylamido ABD
4
(Scheme 1). We optimized its synthesis from tricyclic butyl ester
⇑
Corresponding author.
E-mail address: pierfausto.seneci@unimi.it (P. Seneci).
http://dx.doi.org/10.1016/j.bmcl.2017.04.032
0960-894X/Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

2
L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
4
10
1
10
4
2
Fig. 1. Structure of ABD-based compounds 1 and 2.
4
LINKER
10
3
Fig. 2. General structure of 4-connected Smac mimetic- Zn2+-chelator DACs 3.
5,11 available in large quantity in our lab, by improving yields and
simplifying known experimental protocols.11 Scheme 1 shows the
10-step optimized synthesis of amine 4 in an overall ꢀ20% yield
from 5.
DPA-containing synthon 6 was prepared following a published
procedure.13 Nucleophilic substitution on DPA
with ethyl
7
2-chloroacetate 8 in mild conditions (step a, Scheme 2) led to ester
9, then hydrolyzed in aqueous KOH and neutralized (step b) to acid
6 in a good, overall ꢀ60% yield. Coupling of carboxylate 6 with
amine 4 in basic peptide coupling conditions (step c) led to
N-Boc-protected 10a, that was then deprotected in acidic condi-
tions, yielding target ‘‘no linker” DAC 3a (step d, Scheme 2) in a
good ꢀ76% yield from 4.14
We selected N,N-bis(2-pyridinylmethyl)glycine
6 as a key
DPA-containing synthon (boxed structure, Fig. 3), and we coupled
it to key Smac intermediate 4 either as such (compound 3a,
LINKER = CH2, ‘‘no linker”) or through three linkers with varying
length and lipophilicity. Namely, b-alanine (compound 3b,
LINKER = CH2CONH(CH2)2, ‘‘short lipophilic”), 11-aminounde-
We reasoned that time- and cost-expensive amine 4should be
used as late as possible in a synthetic scheme, to minimize its con-
sumption. Thus, target DACs 3b and 3c, where the Zn2+-chelating
moiety and the Smac mimetic portion are connected by lipophilic
linkers, were prepared by coupling of pre-formed acid DPA-linker
constructs 11a,b with amine 4 (Scheme 3). Commercially available
canoic acid (compound 3c, LINKER = CH2CONH(CH2)10
, ‘‘long
lipophilic”), and 8-amino-3,6-dioxaoctanoic acid (compound 3d,
LINKER = CH2CONH(CH2)2O(CH2)2OCH2, ‘‘hydrophilic”) were used
respectively as short lipophilic, long lipophilic and long hydrophilic
linkers (Fig. 3).
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
a, b
c, d
5
e, f
a, TFA, Et3SiH, dry CH2Cl2, N2, rt; b, Ph2CHNH2-HCl, EDC.HCl,
HOBt, dry DIPEA, dry CH2Cl2, N2, rt, 71% (2 steps); c, H2, Pd/C,
EtOH/H2O 85/15, 0.7 mL/min, 85°C, 10 bar; d, Boc2O, dry TEA, dry
CH2Cl2, N2, rt, 75% (2 steps); e, MsCl, dry TEA, dry CH2Cl2, N2, rt; f,
NaN3, dry DMF, N2, 80°C, 71% (2 steps); g, TFA, DCM, rt; h, N-
Me,N-Boc-(S)-2-aminobutyric acid (from N-methylation, step i),
TBTU, HOBt, dry DIPEA, dry CH2Cl2/DMF 8/1, N2, rt, 74% (3 steps);
j, Me3P, dry CH2Cl2, N2, rt, then 0.1M HCl, 68%.
g-i
j
4
Cl-
Scheme 1. Synthesis of key Smac amino ABD intermediate 4.
b -alanine 12a and 11-aminoundecanoic acid 12b were esterified
in acid conditions (step a, Scheme 3). Aminoesters 13a15 and
13b16 were acylated with bromoacetylbromide in mild basic con-
ditions (step b). Nucleophilic substitution of DPA 7 with bro-
moesters 14a17 and 14b16 (step c) led to methyl ester DPA-linker
constructs 15a, b. Aqueous basic hydrolysis (step d) led to target
acid DPA-linker constructs 11a, b with moderate (11b, ꢀ21%) to
good (11a, ꢀ44%) un-optimized overall yields from aminoacids
12a, b. Coupling of acid DPA-linker constructs 11a, b with amine
4 in basic peptide coupling conditions (step e) led to N-Boc-pro-
tected 10b, c, that were then deprotected in acidic conditions,
yielding target DACs 3b, c (step f, Scheme 3) in a moderate (‘‘long
lipophilic” 3c,18 ꢀ27%) to good (‘‘short lipophilic” 3b,14 ꢀ66%) un-
optimized overall yields from 4.
The synthesis of DAC 3d, connected by a hydrophilic linker, took
advantage of the commercial availability of N-CBz protected
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

4
L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
4
10
6
3a
4
10
3b n = 1 short lipophilic
3c n = 9 long lipophilic
4
2
10
3d
Fig. 3. Structure of DPA-containing synthon 6, and of 4-connected, ABD-based DACs 3a–d.
aminoacid 16 (Scheme 4). Its coupling with amine 4 in basic
peptide coupling conditions (step a) led to N-protected linker-
Smac construct 17. Deprotection of 17 in hydrogenolytic condi-
tions (step b) produced the free amino construct 18, that was then
coupled with the DPA-containing synthon 6 in basic peptide
coupling conditions (step c). Acidic deprotection of compound 19
(step d, Scheme 4) yielded target ‘‘hydrophilic” DAC 3d in a mod-
erate, un-optimized ꢀ31% overall yield from 4.14
We determined the effects of compounds 1, 3a–d and 2 on
apoptosis-connected cellular processes. Western blots from sensi-
tive (MDA-MB231) and Smac mimetic-resistant breast carcinoma
tumor cell lines (MDA-MB453) are respectively shown in Figs. 4
and 5.
Effects on cIAP1 degradation in Smac mimetic-sensitive
MDA-MB231 cells are shown on lane 1, Fig. 4. cIAP1 degradation
was almost complete for 10-connected DAC 1, still significant for
4-connected DACs 3a–c and standard 2, and lower for 4-connected
DAC 3d, possibly due to limited cell permeability. The activation of
apoptosis, quantified through the cleaved forms of caspase-3 (lane
4) and PARP proteins (lane 3), was stronger for 4-connected
DACs 3a and 3c and standard 2, still significant for 4-connected
DAC 1, and limited for 4-connected DACs 3b and 3d. Cellular
pro-apoptotic effects could be influenced by cell permeability
and linker length/lipophilicity. The levels of XIAP (lane 2, Fig. 4)
should mirror putative Zn2+ chelation-mediated effect of DACs on
XIAP degradation. Compound 3c showed significant XIAP degrada-
tion, while other compounds did not affect XIAP degradation. We
hypothesize that a long lipophilic linker may be needed to exert
We measured the affinity of 10-connected DAC 1, of 4-con-
nected DACs 3a-d, and of standard 4-benzamidomethyl Smac
mimetic 211 for the BIR3 domain of cIAP1 (Table 1, column 2),
using a fluorescence polarization-based assay.19
The low nM affinity of Smac mimetics such as 2 is maintained
either by 10-connected and 4-connected DACs (Table 1). The small
decrease in affinity observed for lipophilic 3c may be due either to
a limited hindrance caused by its long lipophilic linker, or to
aspecific aggregation of the more lipophilic DAC. Nevertheless,
the addition of a Zn2+ chelating moiety at either the 10- or the
4-position of ABD-based DACs does not impair their Smac mimetic
nature.
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
7
b
a
6
9
8
a, NaHCO3SiH, dry MeCN, N2, 85°C; b, NaOH,
3/1 THF/H2O, rt, then 0.1M HCl, 60% (2 steps);
c, 4, EDC.HCl, HOBt, dry TEA, dry DMF, N2, rt,
86%; d, TFA, CH2Cl2, rt, 89%.
c
10a
d
3a
Scheme 2. Synthesis of DPA-containing acid 6, and of 4-connected, ABD-based DAC 3a.
cellular pro-apoptotic Zn2+-chelation, possibly to elongate DPA
away from bulky ABD scaffold (no/short linker-containing 3a,b:
inactive).
Good cytotoxicity (ꢁ5
lM) could not be observed with 10-
connected DACs12 such as 1 on Smac mimetic-sensitive MDA-
MB231 cells. Conversely, in agreement with pro-apoptotic
effects shown in Fig. 4, compounds 3a and in particular 3c
showed good cytotoxicity. As to the latter, we suggest better
cell permeability and better Zn2+ accessibility for the DPA moi-
ety due to the long lipophilic linker as reasons for its sub-
micromolar cytotoxicity on MDA-MB231 cells. Unfortunately,
none among tested DACs showed cytotoxicity against MDA-
MB453 cells. We suggest that observed effects on cIAP1 and
XIAP degradation by DACs 3a, 3c and 3d are not strong enough
to induce significant cytotoxicity on Smac-resistant MDA-MB453
cells.
Effects on Smac mimetic-resistant MDA-MB453 cells (Fig. 5) are
weaker, and sometimes different from the ones observed on MDA-
MB231 cells. 10-Connected DAC 1 and standard 2 did not show any
pro-apoptotic effect in MDA-MB453 cells. 4-Connected DACs 3a–d
caused cIAP1 degradation (lane 1, Fig. 5), but did not activate
caspase-3 or PARP (lanes 3 and 4). Lipophilic compound 3c shows
limited XIAP degradation; hydrophilic 3d shows a slightly higher
XIAP degradation (lane 2). The pro-apoptotic activity on MDA-
MB453 of 3d vs. its inactivity on MDA-MB231 cells may be the
result of different permeability-availability for hydrophilic com-
pounds in the two cell lines.
We examined the Zn2+ chelating ability of DACs 3a–d, and of
DPA 7 by fluorescence spectroscopy. Changes in their emission
spectra in presence of increasing amounts of Zn2+ were measured
by excitation at 273 nm (3a–d) or at 270 nm (7), using a published
protocol.12 The fluorescence titration of 3c is shown in Fig. 6,
We then determined the cytotoxicity of compounds 1, 2 and
3a–d on the same cell lines (lanes 3 and 4, Table 1) after 24 h
exposure. The inhibition curves of compounds 1, 3a-d and 2 on
MDA-MB231 cells are shown in Fig. S1, Supporting Information.
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

6
L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
a
b
14a,b
12a n = 1
12b n = 9
13a,b
c
d
15a,b
11a,b
a, SOCl2, MeOH, rt to 80°C; b, bromoacetyl bromide, aq. K2CO3, CH2Cl2, rt
(66%, 14a; 46%, 14b; 2 steps); c, 7, NaHCO3, dry MeCN, N2, rt (79%, 15a;
51%, 15b); d, 1M aq. NaOH, THF, rt (84%, 11a; 89%, 11b); e, 4,
EDC.HCl, HOBt, dry TEA, dry CH2Cl2, N2, rt (73%, 10b; 42%, 10c); f, TFA,
e
CH2Cl2, rt (91%, 3b; 67%, 3c).
10b n = 1
10c n = 9
f
3b,c
Scheme 3. Synthesis of acid DPA-linker constructs 11a, b, and of lipophilic 4-connected, ABD-based DACs 3b, c.
while the others are shown in Figs. S2–S5, Supporting
Information.
lane 2, Figs. 4 and 5) might be ascribed to in cellulo availability of
their DPA moiety.
4-Connected DACs 3a-d chelate zinc ions similarly to
10-connected DACs,12 likely through the DPA moiety. A 1:1
Zn2+-DAC stoichiometry was observed for each compound. Thus,
the differences observed among compounds 3a-d in terms of
Zn2+ chelation-dependent biological effects (degradation of XIAP,
In conclusion, we attached DPA onto the ABD scaffold through a
4-methylamido substituent, using linkers with varying length and
lipophilicity. Pro-apoptotic DACs 3a-d act as Smac mimetics, and
show limited, cell-dependent synergistic effects in cellular assays
(lipophilic 3c in MDA-MB231 cells, hydrophilic 3d in MDA-
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
7
a, 4, EDC.HCl, HOBt, dry TEA, dry CH2Cl2, N2,
rt, 90%; b, 10% Pd-C, H2, MeOH, rt, 94%; c, 6,
EDC.HCl, HOBt, dry TEA, dry DMF, N2, rt,
54%; d, TFA, CH2Cl2, rt, 68%.
2
16
a
2
17
b
2
18
c
2
19
d
2
3d
Scheme 4. Synthesis of hydrophilic target 4-connected, ABD-based DAC 3d.
Table 1
cIAP1 binding (cell free), cytotoxicity on MDA-MB231 and MDA-MB453 cells.
a
b
b
Compound
cIAP1, IC50
MDA-MB231, IC50
MDA-MB453, IC50
112
2
3a
3b
3c
3d
1.09
1.42
1.52
1.35
6.08
1.66
14.9
4.1
6.5
>25
0.97
>25
>25
>25
>25
>25
>25
>25
a
nanomolar affinity.
IC50, micromolar, measured by CellTiter-Glo (Promega).
b
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

8
L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
MB453 cells). In particular, compound 3c is the first ABD-based,
DPA-containing DAC reaching sub-micromolar cytotoxicity on
MDA-MB231 cells.
cIAP1
We will continue our investigation on Smac mimetic – Zn2+
chelator DACs by further studying the cellular effects possibly
attributable to Zn2+ chelation by compounds 3c,d; and by studying
the influence of other Zn2+ chelating moieties on cytotoxic potency
and cellular permeation of novel Smac mimetic-Zn2+-chelator
DACs. Results will be reported in due time.
XIAP
Cleaved PARP
Cleaved
Caspase-3
Acꢀn
Acknowledgments
MDA-MB231
We gratefully acknowledge MIUR (Ministero dell’Università e
della Ricerca) for financial support (PRIN project 2015WW5EH:
Tumor-targeting peptidomimetics: synthesis and biomedical
applications). The use of instrumentation purchased through the
Regione Lombardia – Fondazione Cariplo joint project ‘‘SmartMa-
tLab Centre” (decreti 12689/13, 7959/13; Azione 1 e 2) is gratefully
acknowledged. M.P. thanks Dr. Alberto Bossi for the discussions on
Zn2+ chelating experiments.
Fig. 4. Pro-apoptotic effects of compounds 1, 3a–d and 2 (5 lM) on Smac mimetic-
sensitive MDA-MB231 cells. C, control.
cIAP1
XIAP
A. Supplementary data
Supplementary data (Experimental procedures for the synthesis
of compounds 4, 6, 3a, 3b, 3d. LC–MS and NMR characterization of
intermediates and compounds 4, 6, 3a–d. Experimental procedures
for cell-free testing/binding affinity determination of DACs and
standards in presence of BIR3 constructs from cIAP1. Experimental
procedures and Figures for Zn2+ titration of compounds 7, 3a–d.
Experimental procedures and Figures for mechanism of action
studies and cellular cytotoxicity testing of 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.2017.04.032.
Cleaved PARP
Cleaved
Caspase-3
Acꢀn
MDA-MB453
Fig. 5. Pro-apoptotic effects of compounds 1, 3a–d and 2 (5 lM) on Smac mimetic-
resistant MDA-MB453 cells. C, control.
Fig. 6. Fluorescence titration of ‘‘long lipophilic” DAC 3c with increasing [Zn2+].
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032

L. Manzoni et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
9
pressure, yielding bromoester 14b (384 mg, 1.14 mmol, 68% yield) as a light
brown oil that was used without further purification.
References
11-[2-(N,N-bispyridinylmethyl)aminoacetylamino]-undecanoic acid methyl
ester (15b). A solution of bromoester 14b (384 mg, 1.14 mmol, 1.1 eqs.) in
dry MeCN (1.0 mL) was added to a stirred suspension of DPA 7 (0.20 mL,
1.04 mmol, 1 eq.) and NaHCO3 (110 mg, 1.25 mmol, 1.2 eqs.) in dry MeCN
(1.1 mL) at rt under nitrogen. The reaction mixture was heated at 85 °C for 2 h
(TLC monitoring, eluant:9:1 CH2Cl2/MeOH). The reaction mixture was then
filtered and the solvent removed under reduced pressure. The crude solid
(480 mg) was purified by flash chromatography (eluant: 95:5 CH2Cl2/MeOH),
yielding pure DPA-linker ester construct 15b (240 mg, 0.53 mmol, 51% yield) as
a yellow oil.
1. Hanahan D, Weinberg RA. Cell. 2011;144:646.
2. Housman G, Bayler S, Heerboth S, et al. Cancers. 2014;6:1769.
3. Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Nat Rev Cancer.
2013;13:714.
4. O’Boyle NM, Meegan MJ. Curr Med Chem. 2011;18:4722.
5. Varfolomeev E, Vucic D. Future Oncol. 2011;7:633.
6. Mastrangelo E, Cossu F, Milani M, et al. J Mol Biol. 2008;384:673.
7. Varfolomeev E, Blankenship JW, Wayson SM, et al. Cell. 2007;131:669.
8. Truong-Tran AQ, Grosser D, Ruffin RE, Murgia C, Zalewski PD. Biochem
Pharmacol. 2003;66:1459.
9. Makhov P, Golovine K, Uzzo RG, et al. Cell Death Differ. 2008;15:1745.
10. Sun H, Nikolovska-Coleska Z, Yang C-Y, et al. Acc Chem Res. 2008;41:1264.
11. Seneci P, Bianchi A, Battaglia C, et al. Bioorg Med Chem. 2009;17:5834.
12. Manzoni L, Gornati D, Manzotti M, et al. Bioorg Med Chem Lett. 2016;26:4613.
13. Åstrand OAH, Aziz G, Farzand Ali S, Paulsen RE, Vidar Hansen T, Rongved P.
Bioorg Med Chem. 2013;21:5175.
14. The experimental protocol to intermediates 4 and 6, and to 4-connected DACs
3a, 3b and 3d, and their analytical characterization are reported in the
Supplementary Information.
15. Dekker FJ, Ghizzoni M, van der Meer N, Wisastra G, Haisma HJ. Bioorg Med
Chem. 2009;17:460.
16. Minazzi P, Lattuada L, Menegotto IG, Giovenzana GB. Org Biomol Chem.
2014;12:6915.
17. Trabocchi A, Pala N, Krimmelbein I, et al.
2015;30:466.
11-[2-(N,N-bispyridinylmethyl)aminoacetylamino]-undecanoic acid (11b).
1.0 M aqueous LiOHꢃH2O (0.41 mL, 0.41 mmol, 1.85 eqs.) was added to
a
solution of DPA-linker ester construct 15b (100 mg, 0.22 mmol, 1 eq.) in THF
(0.55 mL). The reaction mixture was stirred at rt for 39 h (TLC monitoring,
eluant: 9:1 CH2Cl2:MeOH). THF was then removed under vacuum, and 1 M
aqueous HCl was added until pH = 7. The mixture was then diluted with water
(2.5 mL) and extracted with CH2Cl2 (3 ꢂ 2.5 mL). The collected organic phases
were washed with brine (7 mL) and dried with Na2SO4. After solvent removal,
DPA-linker acid construct 11b (87 mg, 0.20 mmol, 89% yield) was obtained as a
red oil that was used without further purification.
N3-Boc protected ‘‘long lipophilic” DAC 3c (10c). Dry TEA (0.14 mL, 1.02 mmol,
10 eqs.) was added to a stirred solution of DPA-linker acid construct 11b
(87 mg, 0.20 mmol, 1.95 eqs.), EDCꢃHCl (37 mg, 0.2 mmol, 1.95 eqs.) and HOBt
(26 mg, 0.2 mmol, 1.95 eqs.) in dry CH2Cl2 (2.1 mL) under nitrogen. After
20 min, a solution of amine 4 (66 mg, 0.102 mmol, 1 eq.) in dry CH2Cl2 (3.0 mL)
was added. The reaction mixture was stirred at rt for 22 h (TLC monitoring,
eluant: 9: 1 CH2Cl2/MeOH). The reaction was quenched with saturated aqueous
NH4Cl (25 mL). The organic phase was washed with saturated aqueous NH4Cl
(25 mL), saturated aqueous NaHCO3 (25 mL) and brine (30 mL). The organic
phase was dried with Na2SO4, filtered and the solvent was removed under
reduced pressure. The crude product (95 mg) was purified by reverse phase
flash chromatography (eluant: from 90:10 H2O/MeCN to pure MeCN), yielding
pure compound 10c (45 mg, 0.0438 mmol, 42% yield) as a white solid.
‘‘Long lipophilic” 3c. TFA (0.20 mL, 0.863 mmol, 20 eqs.) was added to a
solution of compound 10c (45 mg, 0.0434 mmol, 1 eq.) in CH2Cl2 (0.5 mL). The
reaction mixture was stirred for 4 h at rt (TLC monitoring, eluant: 9:1 CH2Cl2/
MeOH). The solvent was then removed under reduced pressure, repeatedly
stripping the residue with toluene aliquots. The residue was then dried under
vacuum yielding 0.043 g of crude solid, that was purified by reverse phase
HPLC (eluant: 90:10 H2O/MeCN to 20:80 H2O/MeCN). Pure ‘‘long lipophilic” 3c
was obtained as a white solid (40 mg, 0.029 mmol, 67% yield).
J Enzyme Inhib Med Chem.
18. Experimental protocol to ‘‘long lipophilic” 3c:
Methyl-11-aminoundecanoate (13b).16 Thionyl chloride (0.723 mL, 9.94 mmol,
2 eqs.) was added dropwise to a stirred suspension of 11-aminoundecanoic
acid 12b (1.0 g, 4.97 mmol, 1 eq.) in MeOH (5 mL) at 0 °C. After warming to rt,
stirring was continued for 24 h (TLC monitoring, eluant: 9:1 CH2Cl2:MeOH). A
20% aqueous K2CO3 solution was then added until pH 9, and MeOH was
removed under reduced pressure. The aqueous phase was extracted with EtOAc
(3 ꢂ 15 mL), the organic phases were dried with Na2SO4, filtered and evapo-
rated under reduced pressure. Aminoester 13b (728 mg, 3.38 mmol, 68% yield)
was obtained as a white solid that was used without further purification.11-(2-
Bromoacetylamino)-undecanoic acid methyl ester (14b).16 20% Aqueous K2CO3
(3.6 mL, 0.872 g, 6.68 mmol, 4 eqs.) and 2-bromoacetyl bromide (0.375 mL,
4.32 mmol, 2.6 eqs.) were sequentially added dropwise to
a solution of
aminoester 13b (350 mg, 1.67 mmol, 1 eq.) in CH2Cl2 (17 mL). The reaction
mixture was vigorously stirred at rt for 3 h (TLC monitoring, eluant: 9:1 CH2Cl2:
MeOH). The two phases were then separated and the organic phase was
washed with H2O (3 ꢂ 20 mL) and with brine (20 mL). The organic layer was
dried with Na2SO4, filtered and the solvent was removed under reduced
19. Zhang B, Nikolovska-Coleska Z, Zhang Y, et al. J Med Chem. 2008;51:7352.
Please cite this article in press as: Manzoni L., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.04.032