Design, Synthesis, and Functional Evaluation of CO-Releasing Molecules Triggered by Penicillin G Amidase as a Model Protease

Article abstract of DOI:10.1002/anie.201502445

Protease-triggered CO-releasing molecules (CORMs) were developed. The viability of the approach was demonstrated through the synthesis of compounds consisting of an η⁴-oxydiene-Fe(CO)₃ moiety connected to a penicillin G amidase (PGA)-cleavable unit through a self-immolative linker. The rate of PGA-induced hydrolysis was investigated by HPLC analysis and the subsequent CO release was quantitatively assessed through headspace gas chromatography. In an in vitro assay with human endothelial cells, typical biological effects of CO, that is, inhibition of the inflammatory response and the induction of heme oxygenase-1 expression, were observed only upon co-administration of the CORM and PGA. This work forms a promising basis for the future development of protease-specific CORMs for potential medicinal applications.

Full text of DOI:10.1002/anie.201502445

.
Angewandte
Communications
International Edition: DOI: 10.1002/anie.201502445
German Edition: DOI: 10.1002/ange.201502445
CO-Releasing Molecules
Design, Synthesis, and Functional Evaluation of CO-Releasing
Molecules Triggered by Penicillin G Amidase as a Model Protease**
Nikolay S. Sitnikov, Yingchun Li, Danfeng Zhang, Benito Yard, and Hans-Günther Schmalz*
Abstract:
Protease-triggered
CO-releasing
molecules
CORMs)[4] and pH-sensitive CORMs[5] have been developed.
Recently, Zobi and co-workers produced a photo-CORM
with improved pharmacological properties by conjugating
CORM-1 ([Mn2(CO)10]) to vitamin B12.[6]
As a different approach to the development of tissue- or
cell-specific CORMs, we recently introduced h4-acyloxycy-
clohexadiene–Fe(CO)3 complexes as enzyme-triggered
CORMs (ET-CORMs).[7] These compounds release their
CO load after activation by a hydrolytic enzyme. The
proposed mechanism of CO release is depicted in Scheme 1.
(CORMs) were developed. The viability of the approach was
demonstrated through the synthesis of compounds consisting
of an h4-oxydiene–Fe(CO)3 moiety connected to a penicillin G
amidase (PGA)-cleavable unit through a self-immolative
linker. The rate of PGA-induced hydrolysis was investigated
by HPLC analysis and the subsequent CO release was
quantitatively assessed through headspace gas chromatogra-
phy. In an in vitro assay with human endothelial cells, typical
biological effects of CO, that is, inhibition of the inflammatory
response and the induction of heme oxygenase-1 expression,
were observed only upon co-administration of the CORM and
PGA. This work forms a promising basis for the future
development of protease-specific CORMs for potential medic-
inal applications.
F
or a long time, carbon monoxide (CO) was considered to
be just a toxic gas that inhibits oxygen transport by red blood
cells. However, in the past decade, CO has been identified as
an essential biological signaling molecule, which is endoge-
nously produced in humans mainly in the course of heme
oxygenase (HO)-catalyzed heme degradation.[1] Given its
pronounced anti-inflammatory, cytoprotective, and anti-
hypertensive activity, CO has great therapeutic potential.[2]
However, the pharmacological use of gaseous CO is ham-
pered by a high risk of intoxication and a lack of tissue
selectivity.[2f,g] To circumvent these problems, CO-releasing
molecules (CORMs), mainly based on transition-metal car-
bonyl complexes, have emerged as potential tools for the
in vivo administration of CO.[2a,3] While the first generation
CORMs liberate CO (more or less spontaneously) through
ligand exchange, there is a demand for CORMs that release
CO in vivo in a controlled (preferentially triggered) fashion.
As a possible solution, photoactivated CORMs (photo-
Scheme 1. Proposed activation mechanism of enzyme-triggered CO-
releasing molecules (ET-CORMs) of type 1.
Upon enzymatic cleavage by an esterase, a dienyl ester
complex (of type 1; FG = acyl) is converted into a highly
labile dienol–Fe(CO)3 complex intermediate 2, which dis-
integrates even under slightly oxidative conditions to afford
up to three molecules of CO, as well as a ferric cation (Fe3+)
and the dienone ligand 4.
Having demonstrated the feasibility of the ET-CORM
concept with either esterases/lipases or phosphatases as
triggers for CO liberation, the development of ET-CORMs
activated by specific proteases (peptidases) remained an
important challenge since such compounds would enable
selective CO delivery to cells displaying enhanced expression
of specific proteases.[8] Notably, the up-regulation of proteo-
lytic enzymes is known to be associated with diseases like
cancer, rheumatoid arthritis, neurodegenerative disorders,
and cardiovascular disorders,[9] all of which are potential
targets for CO-based therapy.[2b,f]
[*] Dr. N. S. Sitnikov, Prof. Dr. H.-G. Schmalz
Department für Chemie, Universität zu Kçln
Greinstrasse 4, 50939 Kçln (Germany)
E-mail: schmalz@uni-koeln.de
Dr. N. S. Sitnikov
Department of Organic Chemistry
Nizhni Novgorod State University
Gagarina av. 23, 603950 Nizhni Novgorod (Russia)
The direct attachment of an enzyme-cleavable amide
bond to the diene–Fe(CO)3 moiety didn’t appear too promis-
ing because of the difficult synthetic access and the expected
instability of such structures. Moreover, since proteases are
much more substrate-specific in comparison to esterases, we
reasoned that a close spatial proximity of the amide bond to
the bulky and hydrophobic organometallic fragment might
hamper hydrolytic cleavage. Therefore, we propose a general
Y. Li, D. Zhang, Prof. Dr. B. Yard
Medizinische Klinik, Universitätsmedizin Mannheim
Theodor-Kutzer-Ufer 1–3, 68167 Mannheim (Germany)
[**] This research was supported by the University of Cologne within the
DFG Excellence Program (UoC Forum). N.S. thanks the Alexander
von Humboldt Foundation for a postdoctoral fellowship.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502445.
12314
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 12314 –12318

Angewandte
Chemie
tripartite structure for the protease-activated ET-CORMs
comprising 1) a peptidase-specific (oligo-) peptide (the
“specifier”), 2) a self-immolative linker,[10] and 3) an oxy-
cyclohexadiene–Fe(CO)3 moiety (Figure 1). After enzymatic
Figure 2. Target complexes rac-9 to rac-12, which contain a PGA-
cleavable moiety.
Figure 1. General design of protease-activated ET-CORMs.
cleavage of the specified peptide bond, spontaneous linker
elimination would generate the dienol–Fe(CO)3 complex
intermediate (2), which then undergoes oxidation concom-
itant with the release of CO. Besides offering the possibility to
employ different specifiers, this architecture has the advant-
age that the CO-release kinetics can be fine-tuned through
variation of either the self-immolative linker or the diene–
Fe(CO)3 unit. Moreover, the remote positioning of the
enzyme-cleavable bond relative to the organometallic
moiety should prevent disturbances of the enzymatic hydrol-
ysis step and also suppress kinetic resolution of the “racemic”
iron complex unit during the enzymatic reaction. The
necessity to control the absolute configuration of the planar
chiral diene–Fe(CO)3 moiety can thus be eliminated.
For the proof-of-concept studies reported herein, we
selected a p-aminobenzyl (PABA)[11] and a “diamine”[12]
linker, which differ both in the mechanism of self-immolation
(elimination or cyclization) and in the functional group (ether
or carbamate) connected to the oxydiene–Fe(CO)3 moiety
(Scheme 2).
Scheme 3. Synthesis of starting complexes rac-13 and rac-14 according
to Ref. [7c]. a) LDA, THF, À788C, 1 h; then (iPr)3SiOTf, À788C to RT,
1 h. b) [Fe2(CO)9], Et2O, reflux, 16 h. c) LiHMDS, TPPA, THF, À788C,
1 h; then (iPr)3SiOTf, À788C to room temperature, 1 h. LDA=lithium
diisopropylamide, THF=tetrahydrofuran, LiHMDS=lithium hexame-
thyldisilazane, TPPA=tripyrrolidinophosphoric acid triamide.
First, the isomeric TIPS-protected dienol–Fe(CO)3 build-
ing blocks rac-13 and rac-14 were prepared from cyclo-
hexenone (4) as previously described (Scheme 3).[7c]
The synthesis of rac-9 commenced with the conversion of
p-aminobenzylic alcohol (7) into the phenylacetamide 15 and
subsequent conversion of the benzylic alcohol into a bromide
(16) under Corey–Kim conditions[14] (Scheme 4). After pu-
As a model protease specifier, we chose the phenyl-
acetamide unit, which is reliably recognized and cleaved by
penicillin G amidase (PGA).[13] Based on these considera-
tions, four compounds (rac-9 to rac-12) bearing the oxy
substituent either at the “outer” or the “inner” position of the
diene–Fe(CO)3 unit were designed (Figure 2).
Scheme 4. Synthesis of rac-9. a) Phenylacetyl chloride, pyridine,
CH2Cl2/DMF (8:1, v/v), RT, 12 h. b) NBS, Me2S, CH2Cl2, 08C to room
temperature, 12 h. c) TBAF, NaH, THF, 08C, 10 min. d) THF, 08C to
RT, 1 h. DMF=N,N-dimethylformamide, NBS=N-bromosuccinimide,
TBAF=tetrabutylammonium fluoride.
rification, 16 was reacted with the dienolate–Fe(CO)3complex
rac-17, which was generated in situ from rac-13 with TBAF/
NaH in THF, to smoothly afford rac-9.
The synthesis of rac-10 as the second target structure was
attempted under the same conditions. However, when 16 was
reacted with the dienolate–Fe(CO)3 complex rac-18 (gener-
ated in situ from rac-14), only compounds 19–21 were formed
as major products, while no traces of rac-10 could be isolated
(Scheme 5). Other attempts to achieve the synthesis of rac-10
Scheme 2. General structures and cleavage pathways of peptidase-
activated ET-CORMs containing self-immolative linkers.
Angew. Chem. Int. Ed. 2015, 54, 12314 –12318
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 12315

.
Angewandte
Communications
The rates of PGA-mediated hydrolysis and linker self-
immolation for rac-9, rac-11, and rac-12 were investigated by
reversed-phase HPLC analysis. For this purpose, a solution of
the studied compound (1 mmol) in DMSO (100 mL) was added
along with PGA (4 mL for rac-9; 2 mL for rac-11 and rac-12) to
degassed Sorensenꢀs phosphate buffer (50 mm, pH 7.8,
1.9 mL) preheated to 378C. The reaction mixture was stirred
at 378C for 24 h and aliquots were taken occasionally for
analysis (see the Supporting Information). In control experi-
ments performed under identical conditions, none of the
compounds underwent any hydrolysis within 24 h in the
absence of the enzyme. In the case of rac-9, three peaks
corresponding to the starting material, the deacylated inter-
mediate rac-25, and the dienol–Fe(CO)3 complex rac-2 were
observed in the presence of PGA (Figure 3). This allowed us
to monitor the concentration changes for all three species in
a time-resolved manner (Figure 4). The half-life of rac-9 was
5 h under these conditions.
As shown in Figure 4, the rates of enzymatic hydrolysis
and linker self-immolation are comparable, with both con-
tributing to the rate of rac-2 formation from rac-9. Notably,
HPLC measurements with a chiral stationary phase indicated
only minor kinetic resolution during the enzymatic hydrolysis
of rac-9, with a maximum of 6% ee (9/ent-9) after 1 h.
In contrast to rac-9, both PGA-mediated hydrolysis and
subsequent linker immolation were found to be rapid for rac-
Scheme 5. Attempted synthesis of rac-10. a) TBAF, NaH, THF, 08C,
10 min. b) THF, 08C to RT, 24 h.
by using TPPA as a cosolvent or by employing different
electrophiles (such as the mesylate or the trichloroacetimi-
date derived from 15) also failed.[15]
Next, we turned our attention to the synthesis of the
carbamate target molecules rac-11 and rac-12. For this
purpose, N-methyl-ethylenediamine was first converted into
the amide 24 through regioselective monoacylation with
methyl phenylacetate in the presence of 1,5,7-triazabicyclo-
[4.4.0]dec-5-ene.[16] The target complexes rac-11 and rac-12,
were then obtained through the reaction of 24 with the mixed
carbonates rac-22 and rac-23 (generated from rac-13 and rac-
14), respectively, through desilylation with TBAF and trap-
ping the in situ formed dienolate complexes with p-nitro-
phenyl chloroformate. Both target compounds (rac-11 and
rac-12) were obtained in good yield in a three-step one-pot
sequence (Scheme 6).
Figure 3. HPLC chromatogram of the PGA-catalyzed hydrolysis of rac-9
after incubation for 6 h. Peaks 1, 2, and 3 correspond to the
compounds rac-9, rac-25, and rac-2 respectively.
Scheme 6. Synthesis of complexes rac-11 and rac-12. a) TBAF, THF,
08C, 10 min; then 4-nitrophenyl chloroformate, 08C, 40 min. b) THF,
pyridine, 08C to RT, 12 h.
The final goal of our study was to probe the ability of the
prepared compounds (rac-9, rac-11, and rac-12) to release CO
upon activation by PGA. The enzyme-triggered reaction
cascade comprises three steps, which all are expected to
contribute to the overall rate of carbon monoxide generation.
These are 1) enzymatic hydrolysis of the phenylacetamide
unit, 2) linker self-immolation to form the sensitive dienol–
Fe(CO)3 intermediate, and 3) oxidation-induced disintegra-
tion of the latter to release CO.
Figure 4. Monitoring PGA-catalyzed hydrolysis and linker elimination
for compound rac-9 by RP-HPLC.
12316 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 12314 –12318

Angewandte
Chemie
11 and rac-12 under the standard conditions. For both of these
substrates, a complete conversion to rac-2 (or the respective
external isomer) was observed within 10 min and no inter-
mediate could be detected by HPLC analysis.
Quantitative assessment of CO release from rac-9, rac-11,
and rac-12 associated with cleavage of the amide bond was
performed by using headspace GC (for details, including the
calibration procedure, see the Supporting Information). To
ensure the build-up of sufficiently high concentrations of CO
in the gas phase, the reactions were performed at higher
concentrations.
A solution of the studied compound
Figure 6. Western blot analysis of VCAM-1 and HO-1 expression in
HUVEC cells incubated for 24 h in the presence (+) or absence (À) of
(10 mmol) in DMSO (100 mL) was added along with PGA
(40 mL for rac-9 and 20 mL for rac-11 or rac-12) to Sorensenꢀs
phosphate buffer (50 mm, pH 7.8, 1.9 mL) preheated to 378C
in a 10 mL GC vial. After sealing the vial with a rubber
septum, the mixture was stirred at 378C for 96 h and the gas
phase was periodically sampled for analysis.
TNF-a (50 ngmLÀ1), PGA (1 UmLÀ1; ca. 3 nm), and rac-9
(12.5 mmolLÀ1). To confirm equal protein loading, the membranes
were re-probed with a monoclonal antibody against b-actin.
In conclusion, with the aim of developing protease-
triggered CORMs, we designed and successfully synthesized
a first series of oxydiene–Fe(CO)3 complexes with a PGA-
cleavable side chain connected to the organometallic unit
through either a p-aminobenzyl (rac-9) or a N-methyl-1,2-
ethylenediamincarbonyl unit (rac-11, rac-12) as a self-immo-
lative linker. As a proof of concept, we demonstrated that
upon amidase-induced cleavage of the amide bond, these
model substrates indeed undergo the expected decay, thereby
leading to the release of CO. The PGA-induced liberation of
CO was also demonstrated for rac-9 in a cell-based assay. We
are therefore optimistic that the concept disclosed herein
could be exploited for the future development of enzyme-
triggered CO-releasing molecules (ET-CORMs) with more
advanced protease specifiers, which would enable tissue-
specific CO release in response to the presence of disease-
relevant proteases. In this context, different linkers (e.g.,
elongated diamine linkers[18] or PABA units with additional
substituents[19]) might also be used to fine-tune the rate of CO
release and other pharmacological properties.
While control experiments clearly showed that no CO was
generated in the absence of added enzyme, PGA-triggered
CO release could be clearly detected for all three substrates.
Interestingly, the release of CO from rac-9 proceeded at
a comparably slow rate compared to the substrates containing
a diamine linker (rac-11 and rac-12; Figure 5).
Keywords: carbon monoxide · carbonyl complexes ·
enzyme catalysis · iron · organometallic compounds
Figure 5. PGA-triggered CO release from compounds rac-9, rac-11, and
rac-12, as determined by headspace gas chromatography.
How to cite: Angew. Chem. Int. Ed. 2015, 54, 12314–12318
Angew. Chem. 2015, 127, 12489–12493
Having proven the general ability of rac-9, rac-11, and rac-
12 to act as PGA-triggered CO-releasing molecules, we next
probed whether such compounds also produce CO-associated
biological effects in vitro. For this purpose, human umbilical
vein endothelial cells (HUVECs) were stimulated with the
pro-inflammatory cytokine TNF-a and the ability of rac-9
(12.5 mL) to alter the expression of vascular cell adhesion
molecule-1 (VCAM-1) and heme oxygenase-1 (HO-1) with/
without the addition of PGA as a trigger enzyme was assessed
by western blot analysis.[17]
Effective inhibition of VCAM-1 expression as well as
induction of HO-1, was observed only in the presence of both
rac-9 and PGA, while in the control experiments (with TNF-
a alone or with TNF-a/rac-9 in the absence of PGA) a normal
inflammatory response was observed (Figure 6). Notably, rac-
9 did not display significant levels of cytotoxicity towards
HUVECs at the concentration used (half maximal effective
concentration (EC50) > 50 mm).
[1] a) R. Motterlini, R. Foresti, Antioxid. Redox Signaling 2014, 20,
1810 – 1826; b) P. A. Dennery, Antioxid. Redox Signaling 2014,
20, 1743 – 1753; c) S. W. Ryter, J. Alam, A. M. K. Choi, Physiol.
Rev. 2006, 86, 583 – 650; d) M. D. Maines, Annu. Rev. Pharmacol.
Toxicol. 1997, 37, 517 – 554.
[2] a) S. H. Heinemann, T. Hoshi, M. Westerhausen, A. Schiller,
Chem. Commun. 2014, 50, 3644 – 3660; b) L. Rochette, Y. Cottin,
M. Zeller, C. Vergely, Pharmacol. Ther. 2013, 137, 133 – 152; c) F.
Gullotta, A. di Masi, P. Ascenzi, Iubmb Life 2012, 64, 378 – 386;
d) S. Ghosh, J. Gal, N. Marczin, Ann. Med. 2010, 42, 1 – 12;
e) B. E. Mann, Top. Organomet. Chem. 2010, 32, 247 – 285; f) R.
Motterlini, L. E. Otterbein, Nat. Rev. Drug Discovery 2010, 9,
728 – 743; g) R. Foresti, M. G. Bani-Hani, R. Motterlini, Inten-
sive Care Med. 2008, 34, 649 – 658; h) L. Y. Wu, R. Wang,
Pharmacol. Rev. 2005, 57, 585 – 630.
[3] a) S. García-Gallego, G. J. L. Bernardes, Angew. Chem. Int. Ed.
2014, 53, 9712 – 9721; Angew. Chem. 2014, 126, 9868 – 9877; b) F.
Zobi, Future Med. Chem. 2013, 5, 175 – 188; c) C. C. Rom¼o,
Angew. Chem. Int. Ed. 2015, 54, 12314 –12318
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 12317

.
Angewandte
Communications
W. A. Blaettler, J. D. Seixas, G. J. L. Bernardes, Chem. Soc. Rev.
2012, 41, 3571 – 3583; d) B. E. Mann, Organometallics 2012, 31,
5728 – 5735; e) M. J. Alcaraz, M. I. Guillen, M. L. Ferrandiz, J.
Megias, R. Motterlini, Curr. Pharm. Des. 2008, 14, 465 – 472;
f) R. Alberto, R. Motterlini, Dalton Trans. 2007, 1651 – 1660.
[4] a) M. A. Gonzales, P. K. Mascharak, J. Inorg. Biochem. 2014,
133, 127 – 135; b) U. Schatzschneider, Inorg. Chim. Acta 2011,
374, 19 – 23.
Noack, P. -Y. Renard, A. Romieu, Org. Biomol. Chem. 2010, 8,
1777 – 1780.
[11] a) P. L. Carl, P. K. Chakravarty, J. A. Katzenellenbogen, J. Med.
Chem. 1981, 24, 479 – 480; b) J. A. Richard, Y. Meyer, V. Jolivel,
M. Massonneau, R. Dumeunier, D. Vaudry, H. Vaudry, P. Y.
Renard, A. Romieu, Bioconjugate Chem. 2008, 19, 1707 – 1718;
c) J. A. Richard, L. Jean, C. Schenkels, M. Massonneau, A.
Romieu, P. Y. Renard, Org. Biomol. Chem. 2009, 7, 2941 – 2957.
[12] W. S. Saari, J. E. Schwering, P. A. Lyle, S. J. Smith, E. L.
Engelhardt, J. Med. Chem. 1990, 33, 97 – 101.
[5] T. S. Pitchumony, B. Spingler, R. Motterlini, R. Alberto, Org.
Biomol. Chem. 2010, 8, 4849 – 4854.
[6] F. Zobi, L. Quaroni, G. Santoro, T. Zlateva, O. Blacque, B.
Sarafimov, M. C. Schaub, A. Y. Bogdanova, J. Med. Chem. 2013,
56, 6719 – 6731.
[13] a) A. K. Chandel, L. V. Rao, M. L. Narasu, O. V. Singh, Enzyme
Microb. Technol. 2008, 42, 199 – 207.
[14] E. J. Corey, C. U. Kim, M. Takeda, Tetrahedron Lett. 1972, 13,
4339 – 4342.
[15] For a mechanistic rationale of this reaction outcome, see the
Supporting Information.
[16] C. Sabot, K. A. Kumar, S. Meunier, C. Mioskowski, Tetrahedron
Lett. 2007, 48, 3863 – 3866.
[17] Although compounds rac-11 and rac-12 showed fast PGA-
induced CO release (as assessed by headspace GC), initial
experiments with endothelial cells indicated that they inhibit
VCAM-1 expression in a partly PGA-independent manner.
Since the reason for this unexpected behavior is unknown (it is
currently being investigated), we focused on compound rac-9,
which produces the characteristic biological effects of CO-
release in a clearly PGA-dependent manner.
[18] F. M. H. de Groot, W. J. Loos, R. Koekkoek, L. W. A. van Ber-
kom, G. F. Busscher, A. E. Seelen, C. Albrecht, P. de Bruijn,
H. W. Scheeren, J. Org. Chem. 2001, 66, 8815 – 8830.
[19] K. M. Schmid, L. Jensen, S. T. Phillips, J. Org. Chem. 2012, 77,
4363 – 4374.
[7] a) S. Romanski, B. Kraus, U. Schatzschneider, J.-M. Neudoerfl, S.
Amslinger, H.-G. Schmalz, Angew. Chem. Int. Ed. 2011, 50,
2392 – 2396; Angew. Chem. 2011, 123, 2440 – 2444; b) S. Roman-
ski, H. Ruecker, E. Stamellou, M. Guttentag, J.-M. Neudoerfl, R.
Alberto, S. Amslinger, B. Yard, H.-G. Schmalz, Organometallics
2012, 31, 5800 – 5809; c) S. Romanski, B. Kraus, M. Guttentag,
W. Schlundt, H. Ruecker, A. Adler, J.-M. Neudoerfl, R. Alberto,
S. Amslinger, H.-G. Schmalz, Dalton Trans. 2012, 41, 13862 –
13875; d) S. Romanski, E. Stamellou, J. T. Jaraba, D. Storz, B. K.
Kraemer, M. Hafner, S. Amslinger, H.-G. Schmalz, B. A. Yard,
Free Radical Biol. Med. 2013, 65, 78 – 88; e) E. Stamellou, D.
Storz, S. Botov, E. Ntasis, J. Wedel, S. Sollazzo, B. K. Kramer, W.
van Son, M. Seelen, H.-G. Schmalz, A. Schmidt, M. Hafner,
B. A. Yard, Redox biol. 2014, 2, 739 – 748.
[8] a) K. Y. Choi, M. Swierczewska, S. Lee, X. Chen, Theranostics
2012, 2, 156 – 178; b) U. H. Weidle, G. Tiefenthaler, G. Georges,
Cancer Genomics Proteomics 2014, 11, 67 – 79; c) D. Gabriel,
M. F. Zuluaga, H. van den Bergh, R. Gurny, N. Lange, Curr.
Med. Chem. 2011, 18, 1785 – 1805.
[9] C. Lopez-Otin, J. S. Bond, J. Biol. Chem. 2008, 283, 30433 –
30437.
[10] a) F. Kratz, I. A. Muller, C. Ryppa, A. Warnecke, ChemMed-
Chem 2008, 3, 20 – 53; b) Y. Meyer, J.-A. Richard, B. Delest, P.
Received: March 16, 2015
Revised: April 24, 2015
Published online: June 2, 2015
12318 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 12314 –12318