A new class of highly potent matrix metalloproteinase inhibitors based on triazole-Substituted hydroxamates: (Radio)synthesis and in vitro and first in vivo evaluation

Article abstract of DOI:10.1021/jm300199g

In vivo imaging of MMPs is of great (pre)clinical interest and can potentially be realized with modern three-dimensional and noninvasive in vivo molecular imaging techniques such as positron emission tomography (PET). Consequently, MMP inhibitors (MMPIs) radiolabeled with positron emitting nuclides (e.g., ¹⁸F) represent a suitable tool for the visualization of activated MMPs with PET. On the basis of our previous work and results regarding radiolabeled and unlabeled derivatives of the nonselective MMPIs, we discovered a new class of fluorinated MMPIs with a triazole-substituted hydroxamate substructure. These novel MMPIs are characterized by an increased hydrophilicity compared with the lead structures and excellent MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13 (IC₅₀ = 0.006-107 nM). Therefore, one promising fluorinated triazole-substituted hydroxamate (30b) was selected and resynthesised as its ¹⁸F-labeled version to yield the potential PET radioligand [¹⁸F]30b. The biodistribution behavior of this novel compound was investigated with small animal PET.

Full text of DOI:10.1021/jm300199g

Article
pubs.acs.org/jmc
A New Class of Highly Potent Matrix Metalloproteinase Inhibitors
Based on Triazole-Substituted Hydroxamates: (Radio)Synthesis and
in Vitro and First in Vivo Evaluation
Verena Hugenberg,*,† Hans-Jorg Breyholz,† Burkhard Riemann,† Sven Hermann,‡ Otmar Schober,†
̈
Michael Schafers,‡,§ Umesh Gangadharmath,∥ Vani Mocharla,∥ Hartmuth Kolb,∥ Joseph Walsh,∥
̈
Wei Zhang,∥ Klaus Kopka,†,§,⊥ and Stefan Wagner†,⊥
†Department of Nuclear Medicine, University Hospital Munster, Albert-Schweitzer-Campus 1, Building A1, D-48149 Munster,
̈
̈
Germany
‡European Institute for Molecular Imaging, University of Munster, Mendelstrasse 11, D-48149 Munster, Germany
̈
̈
§Interdisciplinary Centre of Clinical Research (IZKF), Albert-Schweitzer-Campus 1, Building D3, D-48149 Munster, Germany
̈
∥Siemens Medical Solutions USA, Inc., 6100 Bristol Parkway, Culver City, California 90230, United States
S
* Supporting Information
ABSTRACT: In vivo imaging of MMPs is of great (pre)clinical interest and can potentially be realized with modern three-
dimensional and noninvasive in vivo molecular imaging techniques such as positron emission tomography (PET). Consequently,
MMP inhibitors (MMPIs) radiolabeled with positron emitting nuclides (e.g., 18F) represent a suitable tool for the visualization of
activated MMPs with PET. On the basis of our previous work and results regarding radiolabeled and unlabeled derivatives of the
nonselective MMPIs, we discovered a new class of fluorinated MMPIs with a triazole-substituted hydroxamate substructure.
These novel MMPIs are characterized by an increased hydrophilicity compared with the lead structures and excellent MMP
inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13 (IC50 = 0.006−107 nM). Therefore, one promising fluorinated
triazole-substituted hydroxamate (30b) was selected and resynthesised as its 18F-labeled version to yield the potential PET
radioligand [18F]30b. The biodistribution behavior of this novel compound was investigated with small animal PET.
endopeptidases with a catalytic domain containing two Zn2+
INTRODUCTION
In 1962 Jerome Gross and Charles M. Lapier
■
and two or three Ca2+ ions. The Ca2+ ions and one of the Zn2+
̀
e published their
ions are called structural and are responsible for the
stabilization of the domain structure.6−8 The second Zn2+ ion
participates in the catalytic process and is located directly in the
active site of MMPs. It is coordinated by three histidine
residues that are part of the Zn2+-binding consensus sequence
HExxHxxGxxH (where x represents a variable amino acid)
which is characteristic for proteolytically active metzincins.9 To
date, 23 members of the human MMP family are described that
are in the majority of cases excreted as inactive proenzymes
characterized by an N-terminal prodomain that shields the
studies about the collagenolytic activity in amphibian tissues.
They investigated the metamorphosis in tadpole tissues and
observed the degradation of collagen by a proteinase that
operated at neutral pH and physiological temperature.1 Six
years later, a corresponding enzyme was isolated in its proform
from human skin by Eisen et al.2 The proteinase was initially
called interstitial collagenase and was later renamed as matrix
metalloproteinase 1 (MMP-1) representing the first member of
the matrix metalloproteinase (MMP) enzyme family. Similar to
a disintegrin and metalloproteinases (ADAMs) and the
ADAMs with a thromospondin motif (ADAMTs), MMPs are
a subfamily of the ubiquitously expressed metzincins enzyme
superfamily.3−5 MMPs are multidomain Zn2+-dependent
Received: February 14, 2012
Published: April 30, 2012
© 2012 American Chemical Society
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catalytic site from potential substrate molecules. After
activation, namely, delocalization of the prodomain, via the
so-called cysteine-switch mechanism, MMPs normally possess a
broad substrate specificity.10−12 They degrade on the one hand
extracellular matrix (ECM) and basement membrane (BM)
proteins and are on the other hand involved in the processing
of nonconventional substrates such as cytokines, chemokines,
growth factors, serine proteinase inhibitors, cell receptors, and
other MMPs.13 According to their substrate recognition and
cleavage mechanism, MMPs can be grouped in collagenases,
gelatinases, stromelysins, matrilysins, membrane-associated
MMPs, and MMPs with no group designation.12 MMPs are
inhibited and regulated by the common protease inhibitor α2-
macroglobulin, the angiogenesis inhibitor thrombospondin 1
(TSP-1), and the four tissue inhibitors of metalloproteinases
(TIMPs).14 The enzymes are involved in physiological
processes such as wound healing,15 ovulation,16 muscle
homeostasis,17 bone remodeling,18 apoptosis,19 and neuro-
genesis.20 In contrast, overexpression and overactivity of MMPs
are observed in pathological situations including cancer,21
rheumatoid arthritis,22 osteoarthritis,23 multiple sclerosis,
chronic obstructive pulmonary disease,24 and cardiovascular
disease.25
Consequently, the molecular imaging of activated MMPs in
vivo, preferably with noninvasive approaches in the field of
nuclear medicine such as positron emission tomography (PET)
and single photon emission computed tomography (SPECT)
as well as the optical techniques fluorescence reflectance
imaging (FRI) and fluorescence mediated tomography (FMT),
is of great (pre)clinical interest because it would represent a
powerful tool for the diagnosis and therapy control of MMP
associated diseases. A potential approach to achieve this aim is
the application of radiolabeled or fluorescence dye-labeled
MMP inhibitors (MMPIs) functioning as molecular imaging
probes for the visualization of activated MMPs.26
Figure 1. Lead structures 1a and 1b and a selection of radiolabeled
analogues.
CGS 27023A) was synthesized and successfully used for
specific scintigraphic imaging of MMP activity in the arterial
wall of mice in vivo.37,38 In our subsequent work we focused on
the development of a second radiotracer generation charac-
terized by the introduction of the most prominent positron
emitter 18F. Three different analogues ([18F]1d ([18F]FEtO-
CGS 27023A), [18F]1e ([18F]FEtO-CGS 25966), and [18F]1f
([18F]F-CGS 27023A)) were radiosynthesized and evaluated in
vitro and in vivo.39−42
The most promising and accessible candidate [18F]1e that
represents a hydrophobic member of the series shown in Figure
1 (calculated log D (clogD) of 4.03; experimental log D
(logD(exp)) of 2.02) was selected for a good manufacturing
practices (GMP) compliant fully automated radiosynthesis43
aiming at first-in-man studies.
To further improve the pharmacokinetics of these hydrox-
amate-based radiotracers, we aimed to shift the main clearance
route from the hepatobiliary system to the kidneys by
increasing the hydrophilicity of the new radiotracer class
compared to [18F]1e.44 A faster excretion of radiotracer that is
not specifically bound to the target tissue results in a desired
high signal-to-noise ratio of detected target tissues within a
shorter time frame. Therefore, a new class of MMPIs was
developed that consists of additional typical hydrophilic
moieties,45−48 such as minipolyethylene glycol (mini-PEG)
units and/or triazole subcores, at ring B (Figure 1) or that
contains these hydrophilic structural elements instead of ring B.
Ring B was chosen for these modifications because this residue
is located in the S2′ enzyme pocket that is solvent-exposed and
should tolerate limited structural variations.49 The work
presented here describes the (radio)synthesis and in vitro and
first in vivo evaluation of a new class of triazole-substituted
hydroxamate-based MMPIs displaying remarkable in vitro
properties with elevated inhibition potencies and moderate to
high hydrophilicities, respectively.
In this context two slightly different concepts are followed in
our group:
(1) The first strategy is aimed at the specific imaging of
discrete subclasses of MMPs. In this case we utilize a
pyrimidine-2,4,6-trione (barbiturate) derivative (RO 28-2653)
as lead structure for labeled analogues. This compound is
described as an MMPI selective for the gelatinases (MMP-2,
MMP-9), neutrophil collagenase (MMP-8), and two of the
membrane-associated MMPs (MMP-14, MMP-16).27 Different
labeled analogues of this lead structure were synthesized and
evaluated partly in vitro and in vivo. Representative barbiturate-
based MMPIs were labeled with the γ-emitters 125I and the
SPECT-compatible 123I, with the positron emitting 124I, 18F,
and 68Ga and with the fluorescence cyanine dye Cy5.5,
resulting in a series of tracers with fine-tuned pharmacokinetics
for different imaging modalities.28−32
(2) The second approach is based on the development of
labeled small-molecule non-peptide MMPIs bearing broad-
spectrum inhibition potency without explicit specificity for
MMP-subgroups. Here, the hydroxamate-based MMPIs 1a
(CGS 27023A) and 1b (CGS 25966) were chosen as lead
compounds for the design of labeled derivatives (Figure 1). In
contrast to the barbiturate analogues, chelating the Zn2+ ion of
the enzyme active site in a tridentate manner, the CGS
analogues coordinate to the Zn2+ ion in a bidentate mode using
the hydroxamate moiety.33−35 In addition to the Cy5.5-labeled
photoprobe 1b that shows specific MMP binding in vitro and
ex vivo,36 the 123I-labeled hydroxamate [123I]1c (HO[123I]I-
RESULTS AND DISCUSSION
■
Chemistry. To evaluate the MMP inhibition potencies of
the new mini-PEG and/or triazole-substituted hydroxamates,
the corresponding nonradioactive fluorinated target com-
pounds were synthesized and tested by in vitro fluorogenic
MMP assays.
Building blocks used for the synthesis of the MMPIs are
represented by the mini-PEG alkyne derivatives 7 and 8
(Scheme 1). Monosubstitution of tetraethylene glycol with
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a
Scheme 1. Syntheses of the Mini-PEG Compounds 7 and 8
a
Reaction conditions: (a) propargyl bromide, NaH, THF (41%); (b) MsCl, Et3N, CH2Cl2 (93%); (c) 4-hydroxybenzaldehyde, Cs2CO3, DMF
(59%); (d) NaBH4, MeOH (93%); (e) (TFA)2O, THF (100%); (f) LiBr, THF (100%); (g) 2-fluoroethyl 4-methylbenzenesulfonate,40 NaH, THF
(90%).
a
Scheme 2. Syntheses of the MMPIs 16a−c
a
Reaction conditions: (a) 4-methoxybenzene-1-sulfonyl chloride, pyridine (75%); (b) K2CO3, DMF, 7 (79%); (c) HCl (gas), CH2Cl2 (95%); (d) O-
(tetrahydro-2H-pyran-2-yl)hydroxylamine, EDC, NMM, HOBT, DMF (84%); (e) 4 N HCl in dioxane, methanol/dioxane, 1:1 (89%); (f)
CuSO4·5H2O, sodium ascorbate, DMF, H2O, 1-azido-2-fluoroethane (15a),51 1-azido-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)ethane (15b)30 or 2-
deoxy-2-fluoro-β-glucopyranosylazide (15c)52 (16a, 21%; 16b, 27%; 16c, 6%).
propargyl bromide and subsequent mesylation gave the
PEGylated alkyne 3. Nucleophilic substitution of the mesyl
moiety with 4-hydroxybenzaldehyde and reduction with
sodium borohydride yielded the PEGylated benzyl alcohol 5,
which was converted to the benzyl bromide 7 in two steps.
Compound 7 was obtained in overall chemical yield of 21%.
Derivative 8 was obtained with a chemical yield of 37% over
both steps by nucleophilic substitution of the monosubstituted
mini-PEG alkyne intermediate 2 with 2-fluoroethyl 4-tosylate40
using sodium hydride.
The synthetic strategy of the 1b based derivatives 16a−c
with mini-PEG and triazole units is depicted in Scheme 2. The
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a
Scheme 3. Synthesis of the MMPI 24
a
Reaction conditions: (a) NaBH(OAc)3, C2H4Cl2 (90%); (b) 4-methoxybenzene-1-sulfonyl chloride, pyridine (92%); (c) K2CO3, DMF, 7 (66%);
(d) HCl (gas), CH2Cl2 (73%); (e) O-THP hydroxylamine, EDC, NMM, HOBT, DMF (92%); (f) 4 N HCl in dioxane, dioxane/MeOH 1:1 (86%);
(g) CuSO4·5H2O, sodium ascorbate, DMF, H2O, 8 (6%).
sulfonamide 10 was derived from the commercially available
tert-butyl ester of D-valine hydrochloride 9 and 4-methox-
ybenzene-1-sulfonyl chloride in the presence of pyridine. N-
Alkylation of 10 with benzyl bromide 7 gave the carboxylic acid
ester 11, which was transformed to the carboxylic acid 12 using
gaseous hydrochloric acid in dichloromethane. Conversion of
12 into the corresponding hydroxamic acid ester 13 was
achieved by O-THP hydroxylamine, EDC, HOBT, and NMM
in DMF. Cleavage of the THP protecting group was performed
in a hydrochloric acid containing dioxane/methanol mixture,
yielding the alkyne key intermediate 14. Copper(I) catalyzed
Huisgen 1,3-dipolar cycloadditions with different fluorinated
azide building blocks (15a−c) were employed,50 providing the
1,2,3-triazole substituted, mini-PEGylated and fluorinated
hydroxamic acids 16a−c with overall chemical yields of 9%
for 16a, 11% for 16b, and 3% for 16c.
The azido key intermediate 23 was previously synthesized
according to a literature procedure published by our group
starting from the sulfonamide 10 and the benzyl bromide 19
(Scheme 3).36 This synthetic approach was improved in this
work because an alternative and shorter synthesis sequence
utilizing the azidobenzaldehyde 17 instead of 19 as mini-PEG
building block was developed (Scheme 3). Compared to the
synthesis of 19, the preparation of 17 saved at least three
reaction steps.36 Starting from the tert-butyl ester of D-valine 9
and the benzaldehyde 17, reductive amination with sodium
triacetoxyborohydride in dichloroethane provided the N-
benzylated D-valine tert-butyl ester 18. Substitution reaction
with 4-methoxybenzene-1-sulfonyl chloride in pyridine at 60 °C
gave the carboxylic acid ester 20. After acidic removal of the
ester protective group, conversion of the corresponding
carboxylic acid 21 into the THP-protected hydroxamic acid
ester 22, and cleavage of the THP protecting group with HCl in
dioxane, the key intermediate 23 was obtained in 48% overall
yield. Copper(I) catalyzed click reaction with the mini-
PEGylated fluoroalkyne 8 provided the double mini-PEGylated
1,2,3-triazole substituted hydroxamic acid 24.
Compared to the lead structures, hydroxamates 16a−c and
24 are characterized by the additionally introduced mini-PEG
and 1,2,3-triazole units. An advanced approach for the
modification of the pharmacokinetics of the lead structure
deals with the replacement of ring B by a 1,2,3-triazole unit
(Figure 1). The 1,2,3-triazole ring system is endowed with
unique chemical and biological stability. In general 1,4-
disubstituted 1,2,3-triazoles are putatively metabolically inert
and contain H-bond acceptor sites capable of replacing the H-
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a
Scheme 4. Syntheses of the MMPIs 30a−h
a
Reaction conditions: (a) propargyl bromide, K2CO3, DMF (100%); (b) HCl (gas), CH2Cl2 (99%); (c) t-BuONH2, EDC, NMM, HOBT, CH2Cl2
(88%); (d) HCl (gas), C2H4Cl2 (50%); (e) CuSO4·5H2O, sodium ascorbate, DMF, H2O, 2-azidoethyl 4-methylbenzenesulfonate (29a),57 15a,51 2-
(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzene-sulfonate (29b),30 15b,30 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane
(29c),36 side product (2R,2′S)-2,2′-(((1,1′-(((oxy-bis(ethane-1,2-diyl))bis(oxy))bis(ethane-1,2-diyl))bis(1H-1,2,3-triazole-1,4-diyl))bis-
(methylene))bis(((4-methoxyphenyl)sulfonyl)azanediyl))bis(N-hydroxy-3-methylbutanamide) (30h) was isolated from the reaction with 28, 3-
(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (29d),31 or 15c52 (30a, 88%; 30b, 31%; 30c, 73%; 30d, 53%; 30e, 37%; 30f, 18%;
30g, 22%).
Table 1. MMP Inhibition Potencies, clogD, and logD(exp) of Novel Hydroxamic Acids
a
IC50 (nM)
b
compd
MMP-2
MMP-8
MMP-9
MMP-13
34 12
clogD
logD(exp)
1a41
1b35
1e41
14
3
1
2
0.4
6
3
2.30
3.81
4.03
2.86
2.09
2.09
1.15
0.85
2.60
2.58
1.53
1.87
0.65
0.77
1.37
0.58
1.76
d
d
d
11
4
23
2
27
3
1
50 27
11 0.3
2.02 0.03
0.35 0.1
0.11 0.01
0.2 0.08
0.37 0.03
0.6 0.2
0.09 0.03
16a
16b
16c
24
2
4
1
6
3
1
0.5
5
0.8
6
5
1
4
30 10
58
3
43
3
3
18
1
2
4
5
2
1
2
0.3
0.4
0.2
0.2
3
28
14
8
2
2
10
5
30a
30b
30c
30d
30e
30f
30g
30h
107 42
0.7
7
0.02
c
0.13 0.07
0.07 0.03
0.1 0.03
1.2 0.1
0.02 0.004
0.03 0.001
0.04 0.006
0.03 0.003
0.06 0.02
0.05 0.007
0.9 0.05
0.006 0.003
0.02 0.0002
0.04 0.007
0.60 0.01
9
5
1
6
1
1
3
0.5
0.7
4
0.9
0.3
0.2 0.09
0.5 0.2
0.6 0.2
0.5 0.03
>100 μM
>100 μM
>100 μM
>100 μM
a
b
Values are the mean SD of three experiments. clogD values were calculated by ACD/Chemsketch, version ACD/Labs 6.00 (log D = log P at
c
d
physiological pH 7.4). log D value was determined for compound [18F]30b. Ki values, where SDs are not denoted.
bond acceptor basicity of a peptide bond.53−56 The synthetic
route to a variety of 1,2,3-triazole substituted and mini-
PEGylated hydroxamic acid derivatives is depicted in Scheme 4.
N-Alkylation of the sulfonamide 10 with propargyl bromide
under basic conditions, acidic hydrolysis of the ester 25 with
gaseous hydrochloric acid, and conversion of the carboxylic acid
26 with tert-butylhydroxylamine, EDC, HOBT, and NMM gave
the hydroxamic acid ester 27. After the cleavage of the tert-butyl
group with hydrochloric acid gas in dichloroethane the
propargylic hydroxamic acid 28 was obtained with a high
overall yield of 44%. Copper(I) catalyzed click reactions of 28
with different azidoalkyl and azido mini-PEGylated compounds
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a
Scheme 5. Radiosynthesis of 18F-Labeled [18F]30b
a
Reaction conditions: (a) K(K222)[18F]F, K2CO3, CH3CN, 110 °C, 100 s (58%); (b) CuSO4·5H2O, sodium ascorbate, DMF, rt, 10 min (55%).
were accomplished with varying yields (18−88%), resulting in a
small series of 1,2,3-triazole containing hydrophilic hydroxamic
acid derivatives 30a−g. The 1,2,3-triazoles 30a−g were
prepared using copper(II) sulfate and sodium ascorbate in
DMF. A second 1,3-dipolar cycloaddition of compound 30e
with the fluoro-PEG alkyne 8 yielded the desired click reaction
product, but purification was impossible because of decom-
position of the target compound during the purification
procedures.
In summary, a novel class of triazole and/or mini-PEG
containing hydroxamate MMPIs were prepared from the alkyne
and azido substituted key intermediates 14, 23, and 28 by 1,3-
dipolar cycloaddition. This reaction strategy yielded fluorinated
nonradioactive reference compounds of potential MMP
radioligands 16a−c, 24, 30b, 30d, 30f, and 30g, whose MMP
inhibiton potencies can be measured by in vitro fluorogenic
assays, as well as precursors for 18F-labeling 14, 23, 28, 30a, and
30c. Precursors 14, 23, and 28 can potentially be 18F-labeled via
two-step procedures using radiosynthons, i.e., [18F]15a,
[18F]15b, or [18F]15c for the alkyne precursors 14 and 28
and [18F]8 for the azido compound 23. Nucleophilic
substitution of the tosylate precursors 30a and 30c with
[18F]fluoride can potentially yield the radiofluorinated ana-
logues [18F]30b and [18F]30d in one step.
In Vitro Enzyme Assays and clogD Values. The MMP
inhibition potencies of the hydroxamic acids 14, 16a−c, 24, 28,
and 30a−h against activated MMP-2, -8, -9, and -13 were
measured by fluorogenic in vitro inhibition assays following the
procedure previously described.58 The resulting IC50 values for
the investigated MMPs were compared to those of the parent
compounds 1a, 1b, and 1e.
As displayed in Table 1, the new generation of mini-PEG
and/or 1,2,3-triazole substituted hydroxamic acids revealed
excellent MMP inhibition potencies with IC50 values in the
nanomolar to picomolar range (0.006−107 nM). In compar-
ison to the potent MMPI 1e (IC50 values of 2−50 nM) the
inhibition potencies of 30b−g were significantly increased with
IC50 values in the picomolar to low nanomolar range (0.006−5
nM). The 1,2,3-triazole ring appears to play an important role
regarding the enzyme binding potencies, and also its position in
the molecule seems to be of significance. In particular,
compounds 30b−d and 30g, with the 1,2,3-triazole moiety
positioned close to the hydroxamate group that chelates the
Zn2+ ion of the enzyme active site in a bidentate manner, show
considerably higher inhibition potencies (IC50 = 0.006−0.6
nM) than compounds 16a−c and 24 with a benzyl poly-
ethylene glycol spacer between the backbone of the molecule
and the 1,2,3-triazole unit that still represent potent MMPIs
(IC50 = 0.2−58 nM).
The aforementioned excellent binding potencies for 30b−d
and 30g could be due to additional attractive interactions
between the hydrophilic triazole nitrogen atoms and the
Zn2+ion of the enzyme active site or other functional groups in
the enzyme. In contrast, compound 30h with two triazole
substituted hydroxamates linked via a mini-PEG chain (side
product of the reaction of 28 with 29c, formal click product of
30e and 28, structure not shown in Scheme 4) was completely
inactive in the in vitro assay.
The lead structures based on 1a−b and 1e were modified by
the introduction of hydrophilic groups such as mini-PEG units
and 1,2,3-triazole moieties in combination with fluorinated
building blocks. Therefore, an evaluation of the hydrophilic
properties of the target compounds is required. Table 1 also
displays the calculated log D values (clogD) of the synthesized
hydroxamic acids to indicate the changes of the lipophilicities
caused by the structural modifications of the lead compounds
1a,b and the fluorinated analogue 1e. Compared to 1e (clogD
= 4.03), the new polyethylene glycol and/or 1,2,3-triazole
substituted hydroxamic acid derivatives (clogD = 0.58−2.86)
show a considerably increased hydrophilicity, as desired.
Additionally, the log D value of the radiofluorinated analogue
[18F]30b (see section Radiochemistry) was determined
experimentally (logD(exp) = 0.60
0.01). The logD(exp)
differs from the clogD (clogD(30b) = 1.53) by 1 unit.
Compared to the radiofluorinated analogue [18F]1e (logD
(exp) = 2.02 0.03), the triazole substituted hydroxamic acid
[18F]30b is approximately 26 times more hydrophilic.
The new MMPI class of mini-PEG and/or 1,2,3-triazole
substituted hydroxamates is characterized by at least one major
improvement. All new MMPIs showed an increased hydro-
philicity compared to the lead structure 1b. Furthermore
backbone triazole substituted MMPIs represented by com-
pounds 30b−d and 30g turned out to be the most potent
MMPI class developed by our group so far. These results
encouraged us to radiosynthesize a representative 18F-labeled
isotope for further in vitro and initial in vivo evaluation.
Radiochemistry. Because of the excellent MMPI potency
and high hydrophilicity of compound 30b, this derivative was
chosen for the radiosynthesis of its 18F-labeled analogue
[18F]30b. For this purpose a semiautomated two-step
procedure was developed and optimized (Scheme 5). The
preparation of the [18F]fluoroethyl-1,2,3-triazole substituted
inhibitor [18F]30b consisted of the nucleophilic radiofluorina-
tion of 2-azidoethyl 4-methylbenzenesulfonate and subsequent
copper(I) catalyzed cycloaddition with the alkyne precursor 28.
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According to a procedure previously described by Glaser and
Årstad, the radiosynthesis of 1-azido-2-[18F]fluoroethane
([18F]15a) was achieved.51 An improvement of the literature
procedure could be realized by direct separation of the
immediately formed [18F]15a from the reaction mixture by
destillation. By this we gained a savings of time of at least 15
min compared to the literature procedure. 1-Azido-2-[18F]-
fluoroethane could be isolated after 20 min in an average
radiochemical yield of 58 4% (decay corrected, n = 6). The
click labeling was performed outside the automated radio-
synthesizer. The optimal reaction conditions proved to be a
reaction time of 10 min under stirring at room temperature,
using aqueous solutions of copper(II) sulfate pentahydrate (1.4
equiv relative to the alkyne precursor 28) and sodium ascorbate
(1.8 equiv relative to 28). This reaction step successfully
provided the 1,2,3-triazole [18F]30b in an average radio-
chemical yield of 55
7% (decay corrected, n = 5). After
purification by semipreparative HPLC and concentration by
rotary evaporation and formulation, the two-step radiosynthesis
of [18F]30b was accomplished with an overall radiochemical
yield of 30 3% (decay corrected, n = 5) in 110 10 min.
[18F]30b was isolated in radiochemical purities of >98% with
specific activities in the range of 14−57 GBq/μmol at the end
of the synthesis. The radioligand was formulated in phosphate-
buffered saline (PBS) to determine the log D values and to
study its in vitro stability in human serum at 37 °C.
In Vitro Stability. By use of human blood serum, an in vitro
stability study was carried out. During long-term incubation for
up to 120 min at 37 °C, [18F]30b revealed excellent stability. As
shown in Figure 2, only the parent compound [18F]30b was
observed by radio-HPLC. Significant decomposition products
or radiometabolites could not be detected. Because of the fact
that first in vivo PET experiments were carried out with mice,
the blood serum stability measurement of [18F]30b was
additionally performed in mouse blood serum. Likewise, no
significant decomposition of products or radiometabolites were
detected (see Supporting Information).
In Vivo Biodistribution Study. Representative coronal
whole body images 0−1, 1−5, 5−10, and 90−120 min after
tracer injection in C57/BL6 mice are shown in Figure 3.
Overall, [18F]30b is cleared quickly and efficiently from the
body through hepatic and renal elimination with no significant
tracer remaining in nonexcretion organs 90−120 min pi.
Immediately upon injection of [18F]30b high levels of
radioactivity were observed in the liver and the kidneys.
Figure 2. Stability of [18F]30b in human blood serum: tR = 8.70 min;
analytical HPLC system A, method A2, starting with 30% CH3CN in
water (0.1% TFA) for 15 min, followed by a linear gradient from 30%
to 90% CH3CN in water (0.1% TFA) over 3 min, followed by a linear
gradient from 90% to 30% CH3CN in water (0.1% TFA) over 2 min
with a flow rate of 1 mL·min−1 after incubation in human serum in
vitro at 37 °C for 30 min (top), 90 min (middle), and 120 min
(bottom).
While the activity in the kidney decreased (Tmax = 30 s, T1/2
=
13 min pi) in parallel to the activity in the blood, the liver first
showed a further accumulation of [18F]30b (Tmax = 4 min, T1/2
= 18 min pi) before clearance into the gallbladder and finally
into the intestine (Figure 4).
Defluorination of the radioligand in vivo potentially
impairing image interpretation (indicated by bone uptake of
[18F]fluoride ions) was not observed in the entire dynamic
imaging study. Furthermore, accumulation of [18F]30b in
organs/tissues such as the brain, myocardium, lung, and
muscles, indicating unspecific binding, was not observed.
Interestingly, in comparison to [18F]1e, compound [18F]30b
does not show any significant difference in the biodistribution
characteristics. The overall shapes of the time−activity curves of
[18F]30b reveal similar radiotracer dynamics as observed in
studies using [18F]1e (see Figure 4). Only the clearance of
[18F]30b from kidneys and liver is slightly delayed compared to
Figure 3. In vivo biodistribution of radioactivity in an adult C57/Bl6
mouse after intravenous injection of [18F]30b. Maximum intensity
projections of selected time frames after injection demonstrate
elimination of radioactivity from the blood primarily via the liver
and less pronounced via the kidneys. Bone uptake of [18F]fluoride ions
is not observed.
[18F]1e (for kidney, Tmax = 1.5 min and T1/2 = 3 min pi; for
liver, Tmax = 2.5 min and T1/2 = 7 min pi).
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Figure 4. In vivo biodistribution of radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]30b. Time−activity curves illustrate
tracer dynamics in selected regions of interests (ROI). % ID is percentage injected dose.
Obviously, the structural modification of [18F]1e directed
toward a higher hydrophilicity via the substitution of ring B by
a triazole moiety in variant [18F]30b did not significantly
influence the biodistribution behavior. In future experiments
radiolabeled triazole-substituted hydroxamates with an addi-
tional hydrophilic subunit (e.g., [18F]30d with a mini-PEG
chain) will be investigated to examine the influence of an
additional shift to higher tracer hydrophilicity on the
biodistribution characteristics. Potentially, by the additional
introduction of a mini-PEG chain, this structural modification
would lead to a radiotracer with the desired increased renal
clearance characteristics.
The muscle % ID/g of [18F]30b at 30 min was 0.21% with
no other metabolites present. In plasma, at 30 min, the
radioactivity was partitioned between 63% (0.29% ID/g) of the
parent tracer and 37% (0.17% ID/g) of metabolite 1. No other
metabolites were found in plasma. In the liver sample at 30 min,
26% parent (0.78% ID/g) was present while the remaining 74%
of activity was distributed between three metabolites, 4%
metabolite 1 (0.12% ID/g), 44% metabolite 3 (1.32% ID/g),
and 26% metabolite 4 (0.78% ID/g). In the kidney
homogenate, the parent tracer was present at higher amounts
compared to other tissues at 89% (4.6% ID/g) along with three
metabolites: 5% metabolite 1 (0.26% ID/g), 4% metabolite 3
(0.21% ID/g), 2% metabolite 4 (0.1% ID/g). The tracer and
the metabolites are predominantly cleared through the kidneys
and through urine.
Biostability and Metabolism of [18F]30b. Three 10-
month-old female ICR (CD1) wild type mice were examined at
30 min pi to determine the biostability of [18F]30b.
Representative radio-HPLC traces are shown in Figure 5.
The % ID/g values of the total organs are listed in Table 2
for 30 min time points. The retention time of unchanged
[18F]30b tracer was between 14.1 and 14.3 min. A total of four
metabolites were detected, with retention times of 3.8 min
(metabolite 1), 11.1 min (metabolite 2), 13.1 min (metabolite
3), and 16.1 min (metabolite 4).
As shown in Table 2, although [18F]30b metabolizes to
about 37% to a more polar compound in vivo in mice, in
plasma no other metabolites were detected. It appears that the
tracer clears through urine via kidneys. Though there is some
activity remaining in muscle, this might be due to the
experimental limitation to exclude blood from muscle.
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Figure 5. Representative radio-HPLC traces for 30 min pi of the metabolism study of [18F]30b. The radiochemical purity of [18F]30b was >98%
before injection. The samples were analyzed by HPLC, using a γ-detector (Raytest GmbH/Agilent). The HPLC was done on a Phenomenex C18
column (250 mm × 4.6 mm) using a gradient method with acetonitrile and water (both having 0.05% TFA).
Table 2. % ID/g of the Metabolites of [18F]30b in Muscle,
derivative 30b was successfully realized in a two-step procedure
with an overall radiochemical yield of 30
3% (decay
Kidney, Liver, Plasma, and Urine
corrected). The radiofluorinated triazole-substituted hydrox-
amate MMPI [18F]30b showed an excellent serum stability in
vitro and a rapid clearance, as shown by in vivo biodistribution
studies in wild type mice. Furthermore, undesired unspecific
binding of the radiotracer in nonexcretion organs was not
observed, and compared to the more lipophilic analogue
[18F]1e, compound [18F]30b does not possesses any significant
difference concerning its biodistribution pattern. In any case
[18F]30b exhibits a promising MMP-targeted radiotracer for
the noninvasive PET imaging of activated MMPs in vivo. In
future steps compound [18F]30b will be evaluated in preclinical
PET/CT studies using murine disease models that are
characterized by up-regulated levels of activated MMPs (e.g.,
for atherosclerotic plaques, apolipoprotein E-deficient mice; for
tumor, Lewis lung carcinoma bearing mice). In parallel, the
radiosynthesis of [18F]30d will be established to examine the
influence of an additional shift to higher tracer hydrophilicity in
vivo.
percent injected dose per gram (% ID/g), 30
min pi
muscle kidney liver plasma urine
[18F]30b: tR ≈ 14.2 min
0.21
0.00
0.00
0.00
0.00
4.60
0.26
0.00
0.21
0.10
0.78
0.12
0.00
1.32
0.78
0.29
0.17
0.00
0.00
0.00
27.86
2.32
0.33
2.32
0.33
metabolite 1: tR ≈ 3.8 min
metabolite 2: tR ≈ 11.1 min
metabolite 3: tR ≈ 13.1 min
metabolite 4: tR ≈ 16.1 min
CONCLUSION
■
On the basis of our previous results with radiolabeled and
unlabeled derivatives of the lead MMPIs 1a and 1b, the
synthesis and in vitro characterization of a new MMPI series of
fluorinated triazole-substituted hydroxamates are described.
The novel compounds possess moderate to high hydro-
philicities with clogD values ranging from 0.58 to 2.86 and
represent potent inhibitors of MMP-2, -8, -9, and -13 with IC50
values between 0.006 and 107 nM. In particular, inhibitors
30b−d and 30g that are characterized by the substitution of the
phenyl or pyridyl group by a triazol unit at the ring B position
(compare Figure 1) displayed excellent inhibition potencies
with IC50 values in the picomolar range (0.006−0.6 nM).
Compounds 30b−d and 30g turned out to be the most potent
MMPI class developed so far by our group. The radiosynthesis
of the 18F-labeled counterpart [18F]30b of the promising
EXPERIMENTAL SECTION
■
General. All chemicals, reagents, and solvents for the synthesis of
the compounds were analytical grade, purchased from commercial
sources, and used without further purification unless otherwise
specified. All air- and moisture-sensitive reactions were performed
under argon atmosphere. Solvents were purified and dried by literature
methods where necessary. The melting points are uncorrected and
were determined in capillary tubes on a Stuart Scientific SMP3
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capillary melting point apparatus. Column chromatography was
performed on Merck silica gel 60 (0.040−0.063 mm). Thin layer
chromatography (TLC) was carried out on silica gel coated polyester
backed TLC plates (Polygram, SIL G/UV254, Macherey-Nagel) using
solvent mixtures of cyclohexane (CH), ethyl acetate (EA), and
methanol (MeOH). Compounds were visualized by UV light (254
nm). NMR spectra were recorded in CDCl3, CD3OH, or DMSO-d6 on
Bruker ARX300, Bruker DPX300 (1H NMR, 300 MHz; 13C NMR, 75
MHz; 19F NMR, 282 MHz), a Bruker AMX 400 (1H NMR, 400 MHz;
13C NMR, 100 MHz), and Varian Unity Plus 600 (1H NMR, 600
MHz; 13C NMR, 151 MHz) spectrometers. TMS (1H), CDCl3,
DMSO-d6, CD3OH (13C), and CFCl3 (19F) were used as internal
standards, and all chemical shifts were recorded in ppm (δ). Exact
mass analyses were conducted on a Bruker MicroTof apparatus. The
chemical purities of each new nonradioactive compound were ≥95%
and assessed by analytical gradient reversed-phase HPLC system A or
B (λ = 254 nm). HPLC system A consisted of two Smartline 1000
pumps and a Smartline UV detector 2500 (Herbert Knauer GmbH), a
2-(4-Methoxyphenylsulfonamido-N-(prop-2-yn-1-yl))-3-
methylbutanoic Acid (26). A stirred solution of the carboxylic acid
ester 25 (22.0 g, 57.67 mmol) in dichloromethane (100 mL) was
cooled to 0 °C. Hydrochloric acid gas was bubbled through the
solution. TLC was used to monitor the reaction progress (EA). After
complete conversion the solvent was removed under reduced pressure
to give 26 as a colorless wax (18.6 g, 57.23 mmol, 99%). MS-ES-EM
m/z = 348.0877 [(M + Na)+] calcd for C15H19NO5SNa+: 348.0876.
(R)-N-(tert-Butoxy)-2-(4-methoxyphenylsulfonamido-N-
(prop-2-yn-1-yl))-3-methylbutanamide (27). To a solution of
carboxylic acid 26 (16.3 g, 49.94 mmol) in dichloromethane (0.06
mmol/mL, 830 mL) were added 1-hydroxybenzotriazole hydrate
(HOBT, 6.7 g. 49.94 mmol), 4-methylmorpholine (NMM, 27.5 mL,
249.70 mmol), O-tert-butylhydroxylamine hydrochloride (18.8 g,
149.82 mmol), and N-((dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (EDC, 12.4 g, 64.92 mmol). After being stirred
overnight at room temperature, the reaction mixture was diluted
with water and extracted with dichloromethane (3 × 200 mL). The
combined organic phases were washed with brine, dried over
magnesium sulfate, and concentrated to give 27 as a colorless wax
(17.4 g, 43.80 mmol, 88%). [α]2D0 +107.8 (c 0.98, CHCl3). MS-ES-EM
m/z = 419.1614 [(M + Na)+] calcd for C19H28N2O5SNa+: 419.1611.
(R)-N-Hydroxy-2-(4-methoxyphenylsulfonamido-N-(prop-2-
yn-1-yl))-3-methylbutanamide (28). The hydroxamic acid ester 27
(17.3 g, 43.67 mmol) was dissolved in dichloroethane (0.06 mmol/
mL, 400 mL) containing 1.0 equiv of ethanol (1.3 mL). The solution
was cooled to 0 °C, and hydrochloric acid gas was bubbled through it.
TLC was used to monitor the reaction progress (EA). After complete
conversion the solvent was removed under reduced pressure. Column
chromatography on silica gel (CH/EA, 1:1) yielded 28 as a white solid
(7.4 g, 21.75 mmol, 50%), mp 149 °C. MS-ES-EM m/z = 363.0986
[(M + Na)+] calcd for C15H20N2O5SNa+: 363.0985. HPLC system A,
method A1: tR = 7.32 min (99%).
GabiStar γ-detector (Raytest Isotopenmessgerate GmbH), and a
̈
Nucleosil 100-5 C-18 column (250 mm × 4.6 mm). The recorded data
were processed by the GINA Star software (Raytest Isotopenmessg-
erate GmbH). The HPLC method A1 started with a linear gradient
̈
from 10% to 90% CH3CN in water (0.1% TFA) over 9 min, followed
by a linear gradient from 90% to 10% CH3CN in water (0.1% TFA)
over 6 min, with a flow rate of 1 mL·min−1 (unless otherwise
specified). HPLC method A2 started with 30% CH3CN in water (0.1%
TFA) for 15 min, followed by a linear gradient from 30% to 90%
CH3CN in water (0.1% TFA) over 3 min, followed by a linear gradient
from 90% to 30% CH3CN in water (0.1% TFA) over 2 min with a
flow rate of 1 mL·min−1. HPLC system B consisted of two K-1800
pumps and an S-2500 UV detector (Herbert Knauer GmbH) and a
GabiStar γ-detector (Raytest Isotopenmessgerate GmbH). The
̈
recorded data were processed by the ChromGate HPLC software
(Herbert Knauer GmbH). HPLC method B1 using a Nucleosil 100-5
C18 column (250 mm × 4.6 mm) started with a linear gradient from
10% to 80% CH3CN in water (0.1% TFA) over 18 min, holding for 20
min and followed by a linear gradient from 80% to 10% CH3CN in
water (0.1% TFA) over 2 min, with a flow rate of 1.5 mL·min−1.
HPLC method B2 using a ACE 5 AQ column (250 mm × 10 mm)
started with a linear gradient from 10% to 80% CH3CN in water (0.1%
TFA) over 18 min, holding for 20 min and followed by a linear
gradient from 80% to 10% CH3CN in water (0.1% TFA) over 2 min,
with a flow rate of 5.5 mL·min−1. N-[(Methoxyphenyl)sulfonyl]-D-
valine tert-butyl ester (10),34 1-azido-2-fluoroethane (15a),51 1-azido-
2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)ethane (15b),30 2-deoxy-2-
fluoro-β-glucopyranosylazide (15c),52 2-azidoethyl 4-methylbenzene-
sulfonate (29a),57 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-
methylbenzenesulfonate (29b),30 1-azido-2-(2-(2-(2-azidoethoxy)-
ethoxy)ethoxy)ethane (29c),30 3-(2-(2-(2-(2-azidoethoxy)ethoxy)-
ethoxy)ethoxy)-2-fluoropyridine (29d),31 and 2-fluoroethyl 4-methyl-
benzenesulfonate40 were synthesized following literature procedures.
For some long chain compounds 13C NMR signals at δ ≈ 70 ppm do
have multiple intensities. All animal experiments were conducted in
accordance with local institutional guidelines for the care and use of
laboratory animals.
Synthesis of MMPI 30b. (R)-tert-Butyl 2-(4-Methoxyphenyl-
sulfonamido-N-(prop-2-yn-1-yl))-3-methylbutanoate (25). To a
solution of (R)-tert-butyl 2-(4-methoxyphenylsulfonamido)-3-methyl-
butanoate (10) (20.0 g, 58.2 mmol) in DMF (∼65 μmol/mL, 900
mL) were added propargyl bromide (80% in toluene, 58.2 mmol, 6.5
mL) and potassium carbonate (582 mmol, 80 g). The resulting
suspension was stirred at room temperature for 2 days. The mixture
was diluted with water (500 mL) and extracted with EA (3 × 200 mL).
The combined organic phases were washed with brine, dried over
magnesium sulfate, and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography
(silica gel; CH/EA, 6:1). The product was obtained as colorless
crystals (22.2 g, 58.1 mmol, 100%), mp 79 °C. MS-ES-EM m/z =
404.1507 [(M + Na)+] calcd for C19H27NO5SNa+: 404.1502.
General Procedure for the Preparation of Triazoles 16a−c,
24, and 30a−h. To a solution of the alkyne compound (0.032−2.0
mmol, 1.0 equiv) in DMF (8 mL/mmol) and H2O (2 mL/mmol)
were added CuSO4·5H2O (50 mol %), sodium ascorbate (60 mol %),
and the corresponding azide (0.032−2.0 mmol, 1.0−1.2 equiv) in
sequence. After being stirred at room temperature, the reaction
mixture was diluted with H2O (20 mL) and extracted with EA (3 × 15
mL). The combined organic layers were washed with brine and dried
(MgSO4). After evaporation of the solvent, the residue was purified by
silica gel column chromatography. Experimental and spectroscopic
data of triazoles 16a−c, 24, 30a, 30c−h are listed in the Supporting
Information section.
(R)-2-(N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-
methoxyphenylsulfonamido)-N-hydroxy-3-methylbutanamide
(30b). 30b was obtained from 28 (340 mg, 1.0 mmol) and 1-azido-2-
fluoroethane (15a, 1.0 mmol), prepared from 2-fluoroethyl 4-
methylbenzenesulfonate40 and sodium azide following the literature
procedure,51 after 5 h of stirring at room temperature. Column
chromatographic purification (silica gel, EA) gave a white solid (116
mg, 0.31 mmol, 31%), mp 125 °C (dec). 1H NMR (300 MHz, CDCl3)
δ/ppm 10.26 (s, OH, 1H), 7.81 (s, CCHN, 1H), 7.68 (d, ArH, 3JH,H
=
3
8.6 Hz, 2H), 6.83 (d, ArH, JH,H = 8.6 Hz, 2H), 4.99 (d, AB, NCH2,
2
2JH,H = 16.6 Hz, 1H), 4.87−4.65 (dm, CH2F, JH,F = 46.6 Hz, 2H),
3
4.66−4.53 (dm, NCH2CH2F, JH,F = 26.2 Hz, 2H), 4.55 (d, AB,
NCH2, 2JH,H = 16.5 Hz, 1H), 3.90 (d, NCH, 3JH,H = 10.8 Hz, 1H), 3.78
(s, OCH3, 3H), 2.34 (m, CH(CH3)2, 1H), 0.83 (d, CH(CH3)2, 3JH,H
=
3
6.4 Hz, 3H), 0.57 (d, CH(CH3)2, JH,H = 6.5 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ/ppm 167.72 (CONH), 163.06 (qArCOCH3), 145.13
(CCHN), 131.07 (qArCSO2), 129.44 (ArCH), 124.85 (CCHN),
1
114.16 (ArCH), 81.37 (d, CH2F, JC,F = 172.9 Hz), 63.62 (NCH),
2
55.59 (OCH3), 50.52 (d, CH2CH2F, JC,F = 20.8 Hz), 39.57 (NCH2),
27.36 (CH(CH3)2), 19.29 (CH(CH3)2), 19.07 (CH(CH3)2). 19F
NMR (282 MHz, CDCl3) δ/ppm −222.04 (m, 1F). HRMS-ES-EM
m/z = 452.1375 [(M + Na)+] calcd for C17H24FN5O5SNa+: 452.1374.
HPLC system A, method A2: tR = 8.58 min (100%).
Radiochemistry. General Methods. Radiofluorinations were
carried out on a modified PET tracer radiosynthesizer (TRACERLab
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FxFDG, GE Healthcare). The recorded data were processed by the
TRACERLab Fx software (GE Healthcare). Separation and
purification of the radiosynthesized compounds were performed on
the following semipreparative radio-HPLC system C (λ = 254 nm): K-
500 and K-501 pump, K-2000 UV detector (Herbert Knauer GmbH),
NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec), and an ACE 5 AQ
column (250 mm × 10 mm). The recorded data were processed by
the water−octanol partition coefficient following a published
procedure.59 In brief, an amount of approximately 20 kBq [18F]30b
was mixed with equal amounts (0.5 mL) of PBS (pH 7.4) and 1-
octanol, and the resulting biphasic system was mixed vigorously for 1
min at room temperature. The tubes were centrifuged (3000 rpm, 2
min), and three samples of 100 μL of each layer were counted in a γ
counter (Wallac Wizard, Perkin-Elmer Life Sciences). The partition
coefficient was determined by calculating the ratio cpm(octanol)/
cpm(PBS) and expressed as log D7.4 (log(cpmoctanol/cpmPBS)). Two
independent experiments were performed in triplicate, and data were
provided as mean values standard deviation.
Stability in Human Serum. The serum stability of radioligand
[18F]30b was evaluated by incubation in human serum at 37 °C for up
to 120 min. An aliquot of the PBS-formulated 18F-labeled compound
(20 μL, 5 MBq) was added to a sample of human serum (200 μL), and
the mixture was incubated at 37 °C. Samples of 20 μL each were taken
after periods of 10, 20, 30, 60, 90, and 120 min and quenched in
methanol/CH2Cl2 (1:1 (v/v), 100 μL) followed by centrifugation for
2 min. The organic layer was analyzed by analytical radio-HPLC (tR =
8.70 min; analytical HPLC system A, method A2, starting with 30%
CH3CN in water (0.1% TFA) for 15 min, followed by a linear gradient
from 30% to 90% CH3CN in water (0.1% TFA) over 3 min, followed
by a linear gradient from 90% to 30% CH3CN in water (0.1% TFA)
over 2 min with a flow rate of 1 mL·min−1).
Biostability and Metabolism Study. Approximately 11.1 MBq
[18F]30b (in a maximum volume of 200 μL) was injected into three
mice via tail vein injection. The animals were sacrificed at 30 min pi.
Whole blood was obtained, weighed, and centrifuged at 3000 rpm (3
min) to isolate plasma. Urine was also collected. The muscle, kidneys,
and liver were harvested, weighed, and homogenized in lysis buffer
(1% SDS in PBS buffer). An aliquot of each sample (400 μL) was
subsequently removed, mixed with 400 μL of acetonitrile and 100 μL
of 3% acetic acid in acetonitrile, vigorously mixed, and placed on dry
ice for 3 min. After thawing, the samples were centrifuged at 13000
rpm (8 min) to allow for the separation of supernatant from the pellet.
The supernatant was then removed and assayed for radioactivity in a
PerkinElmer Wizard γ-counter (20 s). A 2 μL aliquot from the dose
sample was counted along with the samples and was used to calculate
the % ID/g. The samples were analyzed by HPLC, using a γ-detector
(Raytest GmbH/Agilent). HPLC was done on a Phenomenex C18
column (250 mm × 4.6 mm) using a gradient method with acetonitrile
and water (both having 0.05% TFA).
the GINA Star software (Raytest Isotopenmessgerate GmbH).
̈
Radiochemical purities and specific activities were determined using
the analytical radio-HPLC system A and method A2. No-carrier-added
aqueous [18F]fluoride was produced on a RDS 111e cyclotron (CTI-
Siemens) by irradiation of a 1.2 mL water target using 10 MeV proton
beams on 97.0% enriched 18O-water by the 18O(p,n)18F nuclear
reaction.
(R)-2-(N-((1-(2-[18F]Fluoroethyl)-1H-1,2,3-triazol-4-yl)-
methyl)-4-methoxyphenylsulfonamido)-N-hydroxy-3-methyl-
butanamide ([18F]30b). In a computer controlled TRACERLab
FxFDG synthesizer aqueous [18F]fluoride ions (0.1−5.5 GBq) from the
cyclotron target were passed through an anion exchange resin (Sep-
Pak Light Waters Accell Plus QMA cartridge, preconditioned with 5
mL of 1 M K2CO3 and 10 mL of water for injection). [18F]Fluoride
ions were eluted from the resin with a mixture of 40 μL of 1 M K2CO3,
200 μL of water for injection, and 800 μL of DNA-grade CH3CN
containing 18 mg (48 μmol) of Kryptofix 2.2.2 (K222) in the reactor.
Subsequently, the aqueous K(K222) [18F]F solution was carefully
evaporated to dryness in vacuo. An amount of 12 μL (14 mg, 60
μmol) of precursor 2-azidoethyl 4-methylbenzenesulfonate57 in 300
μL of dry DMF was added, and the mixture was heated at 110 °C for
100 s. Meanwhile, the labeled product [18F]15a was distilled from the
reactor in a 5 mL flask that contained 400 μL of dry DMF and was
cooled to −10 °C. The radiosynthon [18F]15a was collected with a
radiochemical yield of 58
4% in 20 min (mean
SD, decay
corrected, n = 6). Then 60 μL of 0.4 M CuSO4·5H2O, 40 μL of water,
6 mg (30 μmol) of sodium ascorbate in 100 μL of water, and 6 mg
(17.6 μmol) of alkyne 28 in 100 μL of DMF were added. After 10 min
at ambient temperature, the mixture was passed through a Waters Sep-
Pak Light cartridge filled with quartz wool. The cartridge was rinsed
with DMF (0.2 mL). The eluate was diluted with 500 μL of water, and
the resulting mixture was purified by gradient-radio-HPLC system C
(flow = 5.5 mL/min; eluents, A, CH3CN/TFA, 1000/1, B, H2O/TFA,
1000/1; isocratic, A/B 18/82 (v/v); λ = 254 nm; column, ACE 5 AQ
(250 mm × 10 mm)). The product fraction of compound [18F]30b
(retention time tR([18F]30b) = 37 min) was evaporated to dryness in
vacuo and redissolved in 1 mL of 0.9% NaCl/EtOH (9/1 v/v).
Product compound [18F]30b was obtained in an overall radiochemical
Animals. Adult C57/BL6 mice (male, 21−24 g) were anesthetized
by isoflurane/O2, and one lateral tail vein was cannulated using a 27
Ga needle connected to 15 cm polyethylene catheter tubing. [18F]1e
or [18F]30b (250 kBq/g bodyweight) was injected as a bolus (100 μL
of compound flushed with 100 μL of saline) via the tail vein, and
subsequent PET scanning was performed. Experiments were
conducted according to German animal welfare guidelines.
yield of 30
3% (decay-corrected, based on cyclotron-derived
[18F]fluoride ions, n = 5) in 110
10 min from the end of
radionuclide production. [18F]30b was isolated in radiochemical
purities of >98% with specific activities in the range of 14−57 GBq/
μmol at the end of the synthesis.
Small Animal PET Scanning. PET experiments were carried out
using a submillimeter high resolution (0.7 mm full width at half-
maximum) small animal scanner (32 module quadHIDAC, Oxford
Positron Systems Ltd., Oxford, U.K.) with uniform spatial resolution
(<1 mm) over a large cylindrical field (165 mm diameter, 280 mm
axial length).60
List-mode data were acquired for 120 min and reconstructed into
dynamic time frames using an iterative reconstruction algorithm.
Subsequently, the scanning bed was transferred to the computed
tomography (CT) scanner (Inveon, Siemens Medical Solutions, U.S.)
and a CT acquisition with a spatial resolution of 80 μm was performed
for each mouse. Reconstructed image data sets were co-registered
based on extrinsic markers attached to the multimodal scanning bed
and the image analysis software (Inveon Research Workplace 3.0,
Siemens Medical Solutions, USA). Three-dimensional volumes of
interest (VOIs) were defined over the respective organs in CT data
sets, transferred to the co-registered PET data, and analyzed
quantitatively. Regional uptake was calculated as percentage of
injected dose by dividing counts per milliliter in the VOI by total
counts in the mouse multiplied by 100 (% ID/mL).
In Vitro Enzyme Inhibition Assays (Table 1). The inhibition
potencies of hydroxamic acid derivatives 14, 16a−c, 24, 28, and 30a−
h against activated MMP-2, -8, -9, and -13 were assayed using the
synthetic fluorogenic substrate (7-methoxycoumarin-4-yl)acetyl-Pro-
Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)Ala-Arg-
NH2 (R&D Systems) as described previously.58 Briefly, MMP-2, -8, -9,
or -13 (each at 2 nM) and test compounds at varying concentrations
(10 pM to 1 mM) in Tris-HCl (50 mM), pH 7.5, containing NaCl
(0.2 M), CaCl2 (5 mM), ZnSO4 (20 μM), and 0.05% Brij 35 were
preincubated at 37 °C for 30 min. An aliquot of substrate (10 μL of a
50 μM solution) was added to the enzyme−inhibitor mixture (90 μL),
and the fluorescence changes were monitored using a Fusion Universal
microplate analyzer (Packard Bioscience) with excitation and emission
wavelengths of 330 and 390 nm, respectively. Reaction rates were
measured from the initial 10 min and plotted as a function of inhibitor
concentration. From the resulting inhibition curves, the IC50 values
were calculated by nonlinear regression analysis using the Grace 5.1.8
software (Linux).
Determination of the Partition Coefficient (log D7.4). The
lipophilicity of radioligand [18F]30b was assessed by determination of
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Article
(7) Dhanaraj, V.; Ye, Q. Z.; Johnson, L. L.; Hupe, D. J.; Ortwine, D.
F.; Dunbar, J. B., Jr.; Rubin, J. R.; Pavlovsky, A.; Humblet, C.; Blundell,
T. L. X-ray structure of a hydroxamate inhibitor complex of
stromelysin catalytic domain and its comparison with members of
the zinc metalloproteinase superfamily. Structure 1996, 4, 375−386.
(8) Borkakoti, N. Structural studies of matrix metalloproteinases. J.
Mol. Med. 2000, 78, 261−268.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and analytical data for compounds 1−
8, 10−14, 18, 20−23 and NMR data of 25−28. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +492518347851. Fax: +498347363. E-mail:
hugenber@uni-muenster.de.
(9) Bode, W.; Gomis-Ruth, F. X.; Stockler, W. Astacins, serralysins,
̈
̈
■
snake venom and matrix metalloproteinases exhibit identical zinc-
binding environments (HEXXHXXGXXH and Met-turn) and top-
ologies and should be grouped into a common family, the
“metzincins”. FEBS Lett. 1993, 331, 134−140.
Author Contributions
(10) Van Wart, H. E.; Birkedal-Hansen, H. The cysteine switch: a
principle of regulation of metalloproteinase activity with potential
applicability to the entire matrix metalloproteinase gene family. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 5578−5582.
⊥Both authors contributed equally to this work and share
senior authorship.
Notes
(11) Ra, H. J.; Parks, W. C. Control of matrix metalloproteinase
catalytic activity. Matrix Biol. 2007, 26, 587−596.
The authors declare no competing financial interest.
(12) Zitka, O.; Kukacka, J.; Krizkova, S.; Huska, D.; Adam, V.;
Masarik, M.; Prusa, R.; Kizek, R. Matrix metalloproteinases. Curr. Med.
Chem. 2010, 17, 3751−3768.
ACKNOWLEDGMENTS
The authors thank Christine Batza, Daniel Burkert, Anne
Kanzog, Roman Priebe, Wiebke Gottschlich, Sandra Schroer,
Katrin Reckmann, and Marlena Brink for technical support and
■
̈
(13) Butler, G. S.; Overall, C. M. Updated biological roles for matrix
metalloproteinases and new “intracellular” substrates revealed by
degradomics. Biochemistry 2009, 48, 10830−10845.
̈
the staff members of the Organic Chemistry Institute,
(14) Brew, K.; Nagase, H. The tissue inhibitors of metalloproteinases
(TIMPs): an ancient family with structural and functional diversity.
Biochim. Biophys. Acta 2010, 1803, 55−71.
University of Munster, Germany, for spectroscopic and
̈
analytical investigations. This work was supported by the
Deutsche Forschungsgemeinschaft (DFG), Collaborative Re-
search Center 656 (Molecular Cardiovascular Imaging Projects
(15) Gill, S. E.; Parks, W. C. Metalloproteinases and their inhibitors:
regulators of wound healing. Int. J. Biochem. Cell Biol. 2008, 40, 1334−
1347.
A2, Z5, C6), University of Munster, and the Interdisciplinary
̈
Center of Clinical Research (IZKF core unit SmAP), Munster,
̈
(16) Zhang, X.; Nothnick, W. B. The role and regulation of the
uterine matrix metalloproteinase system in menstruating and non-
menstruating species. Front. Biosci. 2005, 10, 353−366.
(17) Carmeli, E.; Moas, M.; Reznick, A. Z.; Coleman, R. Matrix
metalloproteinases and skeletal muscle: a brief review. Muscle Nerve
2004, 29, 191−197.
(18) Krane, S. M.; Inada, M. Matrix metalloproteinases and bone.
Bone 2008, 43, 7−18.
(19) Mathew, S.; Fu, L.; Hasebe, T.; Ishizuya-Oka, A.; Shi, Y. B.
Tissue-dependent induction of apoptosis by matrix metalloproteinase
stromelysin-3 during amphibian metamorphosis. Birth Defects Res., Part
C 2010, 90, 55−66.
(20) Agrawal, S. M.; Lau, L.; Yong, V. W. MMPs in the central
nervous system: where the good guys go bad. Semin. Cell Dev. Biol.
2008, 19, 42−51.
(21) (a) Fingleton, B. Matrix metalloproteinases: roles in cancer and
metastasis. Front. Biosci. 2006, 11, 479−491. (b) Kessenbrock, K.;
Plaks, V.; Werb, Z. Matrix metalloproteinases: regulators of the tumor
microenvironment. Cell 2010, 141, 52−67. (c) Gialeli, C.; Theocharis,
A. D.; Karamanos, N. K. Roles of matrix metalloproteinases in cancer
progression and their pharmacological targeting. FEBS J. 2011, 278,
16−27.
(22) Okamoto, H.; Hoshi, D.; Kiire, A.; Yamanaka, H.; Kamatani, N.
Molecular targets of rheumatoid arthritis. Inflamm. Allergy: Drug
Targets 2008, 7, 53−66.
(23) Troeberg, L.; Nagase, H. Proteases involved in cartilage matrix
degradation in osteoarthritis. Biochim. Biophys. Acta 2012, 1824, 133−
145.
(24) Muroski, M. E.; Roycik, M. D.; Newcomer, R. G.; Van den
Steen, P. E.; Opdenakker, G.; Monroe, H. R.; Sahab, Z. J.; Sang, Q. X.
Matrix metalloproteinase-9/gelatinase B is a putative therapeutic target
of chronic obstructive pulmonary disease and multiple sclerosis. Curr.
Pharm. Biotechnol. 2008, 9, 34−46.
Germany.
ABBREVIATIONS USED
■
ADAM, a disintegrin and metalloproteinase; ADAMT, a
disintegrin and metalloproteinase with a thromospondin
motif; BM, basement membrane; CH, cyclohexane; ECM,
e x t r a c e l l u l a r m a t r i x ; E D C , N ′ - e t h y l - N ′ - ( 3 -
dimethylaminopropyl)carbodiimide hydrochloride; EA, ethyl
acetate; EM, exact mass; FMT, fluorescence mediated
tomography; FRI, fluorescence reflectance imaging; GMP,
good manufacturing practices; HOBT, 1-hydroxybenzotriazole;
ICR, imprinting control region; K222, Kryptofix 2.2.2; log D,
log P at physiological pH 7.4; MMP, matrix metalloproteinase;
MMPI, matrix metalloproteinase inhibitor; NMM, 4-methyl-
morpholine; PEG, polyethylene glycol; PET, positron emission
tomography; pi, postinjection; ROI, region of interest; % ID,
percentage injected dose; SD, standard deviation; SPECT,
single photon emission computed tomography; THP, tetrahy-
dropyranyl; TIMP, tissue inhibitor of metalloproteinase
REFERENCES
■
(1) Gross, J.; Lapier
̀
e, C. M. Collagenolytic activity in amphibian
tissues: a tissue culture assay. Proc. Natl. Acad. Sci. U.S.A. 1962, 48,
1014−1022.
(2) Eisen, A. Z.; Jeffrey, J. J.; Gross, J. Human skin collagenase.
Isolation and mechanism of attack on the collagen molecule. Biochim.
Biophys. Acta 1968, 151, 637−645.
(3) White, J. M. ADAMs: modulators of cell−cell and cell−matrix
interactions. Curr. Opin. Cell Biol. 2003, 15, 598−606.
(4) Tang, B. L. ADAMTs: a novel family of extracellular matrix
proteases. Int. J. Biochem. Cell Biol. 2001, 33, 33−44.
(5) Nagase, H.; Woessner, J. F., Jr. Matrix metalloproteinases. J. Biol.
Chem. 1999, 274, 21491−21494.
(6) Stocker, W.; Bode, W. Structural features of a superfamily of zinc-
endopeptidases: the metzincins. Curr. Opin. Struct. Biol. 1995, 5, 383−
390.
(25) (a) Brauer, P. R. MMPs-role in cardiovascular development and
disease. Front. Biosci. 2006, 11, 447−478. (b) Papazafiropoulou, A.;
Tentolouris, N. Matrix metalloproteinases and cardiovascular diseases.
Hippokratia 2009, 13, 76−82.
(26) Wagner, S.; Breyholz, H.-J.; Faust, A.; Holtke, C.; Levkau, B.;
̈
Schober, O.; Schafers, M; Kopka, K. Molecular imaging of matrix
̈
4725
dx.doi.org/10.1021/jm300199g | J. Med. Chem. 2012, 55, 4714−4727

Journal of Medicinal Chemistry
Article
metalloproteinases in vivo using small molecule inhibitors for SPECT
and PET. Curr. Med. Chem. 2006, 13, 2819−2838.
the molecular imaging of activated MMPs with PET. J. Med. Chem.
2007, 50, 5752−5764.
(41) Wagner, S.; Breyholz, H.-J.; Holtke, C.; Faust, A.; Schober, O;
(27) Grams, F.; Brandstetter, H.; D’Alo, S.; Geppert, D.; Krell, H. W.;
Leinert, H.; Livi, V.; Menta, E.; Oliva, A.; Zimmermann, G.
Pyrimidine-2,4,6-triones: a new effective and selective class of matrix
metalloproteinase inhibitors. Biol. Chem. 2001, 382, 1277−1285.
̈
Schafers, M.; Kopka, K. A new 18F-labelled derivative of the MMP
̈
inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal
PET studies. Appl. Radiat. Isot. 2009, 67, 606−610.
(42) Schafers, M.; Schober, O.; Hermann, S. Matrix-metal-
̈
(28) Breyholz, H.-J.; Schafers, M.; Wagner, S.; Holtke, C.; Faust, A.;
̈
̈
loproteinases as imaging targets for inflammatory activity in
atherosclerotic plaques. J. Nucl. Med. 2010, 51, 663−666.
Rabeneck, H.; Levkau, B.; Schober, O.; Kopka, K. C-5-disubstituted
barbiturates as potential molecular probes for noninvasive matrix
metalloproteinase imaging. J. Med. Chem. 2005, 48, 3400−3409.
(29) Kopka, K.; Breyholz, H.-J.; Wagner, S.; Brandau, W.; Hermann,
(43) Wagner, S.; Faust, A.; Breyholz, H.-J.; Schober, O.; Schafers, M.;
̈
Kopka, K. The MMP inhibitor (R)-2-(N-benzyl-4-(2-[18F]-
fluoroethoxy)phenylsulfonamido)-N-hydroxy-3-methylbutanamide:
improved precursor synthesis and fully automated radiosynthesis. Appl.
Radiat. Isot. 2011, 69, 862−868.
S.; Schober, O.; Levkau, B.; Bremer, C.; Schafers, M. Radioiodinated
̈
pyrimidine-2,4,6-triones as model probes for the in vivo molecular
imaging of activated MMPs. J. Labelled Compd. Radiopharm. 2007, 50,
S397.
(44) Mutschler, E. Arzneimittelwirkungen; Wissenschaftliche Verlags-
gesellschaft mbH: Stuttgart, Germany, 1996; pp 20−36.
(45) Zang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung, H.
F. F-18 polyethyleneglycol stilbenes as PET imaging agents targeting
Aβ aggregates in the brain. Nucl. Med. Biol. 2005, 32, 799−809.
(46) Zang, W.; Kung, M. P.; Oya, S.; Hou, C.; Kung, H. F. 18F-
Labeled styrylpyridines as PET agents for amoloid plaque imaging.
Nucl. Med. Biol. 2007, 34, 89−97.
(47) Wu, Z.; Li, Z. B.; Chen, K.; Cai, W.; He, L.; Chin, F. T.; Li, F.;
Chen, X. MicroPET of tumor integrin ανβ3 expression using 18F-labeld
PEGylated tetrameric RGD peptide (18F-FPRGD4). J. Nucl. Med.
2007, 48, 1536−1544.
(48) Chen, X.; Park, R.; Hou, Y.; Khankaldyyan, V.; Gonzales-
Gomez, I; Tohme, M; Bading, J. R.; Laug, W. E.; Conti, P. S.
MicroPET imaging of brain tumor angiogenesis with 18F-labeled
PEGylated RGD peptide. Eur. J. Nucl. Med. Mol. Imaging 2004, 31,
1081−1089.
(30) Breyholz, H.-J.; Wagner, S.; Faust, A.; Riemann, B.; Holtke, C.;
̈
Hermann, S.; Schober, O.; Schafers, M.; Kopka, K. Radiofluorinated
̈
pyrimidine-2,4,6-triones as molecular probes for noninvasive MMP-
targeted imaging. ChemMedChem. 2010, 5, 777−789.
(31) Schrigten, D.; Breyholz, H.-J.; Wagner, S.; Hermann, S.;
Schober, O.; Schafers, M.; Haufe, G.; Kopka, K. A new generation of
̈
radiofluorinated pyrimidine-2,4,6-triones as MMP-targeted radio-
tracers for positron emission tomography. J. Med. Chem. 2012, 55,
223−232.
(32) Faust, A.; Waschkau, B.; Waldeck, J.; Holtke, C.; Breyholz, H.-J.;
̈
Wagner, S.; Kopka, K.; Schober, O.; Heindel, W.; Schafers, M.;
̈
Bremer, C. Synthesis and evaluation of a novel photoprobe for imaging
matrix metalloproteinases. Bioconjugate Chem. 2008, 19, 1001−1008.
(33) Brandstetter, H.; Grams, F.; Glitz, D.; Lang, A.; Huber, R.;
Bode, W.; Krell, H. W.; Engh, R. A. The 1.8-Å crystal structure of a
matrix metalloproteinase-8 barbiturate inhibitor complex reveals a
previously unobserved mechanism for collagenase substrate recog-
nition. J. Biol. Chem. 2001, 276, 17405−17412.
(49) Pirard, B.; Matter, H. Matrix metalloproteinase target family
landscape: a chemometrical approach to ligand selectivity based on
protein binding site analysis. J. Med. Chem. 2006, 49, 51−69.
(50) Lopez, M.; Salmon, A. J.; Supuran, C. T.; Poulsen, S. A.
Carbonic anhydrase inhibitors developed through “click tailing”. Curr.
Pharm. Des. 2010, 16, 3277−3287.
(34) MacPherson, L. J.; Bayburt, E. K.; Capparelli, M. P.; Carroll, B.
J.; Goldstein, R.; Justice, M. R.; Zhu, L.; Hu, S. I.; Melton, R. A.; Fryer,
L.; Goldberg, R. L.; Doughty, J. R.; Spirito, S.; Blancuzzi, V.; Wilson,
D.; O'Byrne, E. M.; Ganu, V.; Parker, D. T. Discovery of CGS 27023A,
a non-peptidic, potent, and orally active stromelysin inhibitor that
blocks cartilage degradation in rabbits. J. Med. Chem. 1997, 40, 2525−
2532.
(51) Glaser, M.; Årstad, E. Click labeling with 2-[18F]fluoroethylazide
for positron emission tomography. Bioconjugate Chem. 2007, 18, 989−
993.
(52) Maschauer, S.; Prante, O. A series of 2-O-trifluoromethylsul-
fonyl-D-mannopyranosides as precursors for concomitant 18F-labeling
and glycosylation by click chemistry. Carbohydr. Res. 2009, 344, 753−
761.
(53) Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.; Obach,
R. S.; O’Donnell, J. P. Biotransformation reactions of five-membered
aromatic heterocyclic rings. Chem. Res. Toxicol. 2002, 15, 269−299.
(54) Horne, W. S.; Yadav, M. K.; Stout, C. D.; Ghadiri, M. R.
Heterocyclic peptide backbone modifications in an alpha-helical coiled
coil. J. Am. Chem. Soc. 2004, 126, 15366−15367.
(55) Appendino, G.; Bacchiega, S.; Minassi, A.; Cascio, M. G.; De
Petrocellis, L.; Di Marzo, V. The 1,2,3-triazole ring as a peptide-
olefinomimetic element: discovery of click vanilloids and cannabinoids.
Angew. Chem., Int. Ed. 2007, 46, 9312−9315.
(56) Vatmurge, N. S.; Hazra, B. G.; Pore, V. S.; Shirazi, F.;
Deshpande, M. V.; Kadreppa, S.; Chattopadhyay, S.; Gonnade, R. G.
Synthesis and biological evaluation of bile acid dimmers linked with
1,2,3-triazole and bis-beta-lactam. Org. Biomol. Chem. 2008, 6, 3823−
3830.
(57) Macleod, F.; Lang, S.; Murphy, J. A. The 2-(2-azidoethyl)-
cycloalkanone strategy for bridged amides and medium-sized amine
́
derivatives in the Aube−Schmidt reaction. Synlett 2010, 529−534.
(58) Huang, W.; Meng, Q.; Suzuki, K.; Nagase, H.; Brew, K.
Mutational study of the amino-terminal domain and human tissue
inhibitor of metalloproteinases 1 (TIMP-1) locates an inhibitory
region for matrix metallo proteinases. J. Biol. Chem. 1997, 272, 22086−
22091.
(35) Scozzafava, A.; Supuran, C. T. Carbonic anhydrase and matrix
metalloproteinase inhibitors: sulfonylated amino acid hydroxamates
with MMP inhibitory properties act as efficient inhibitors of CA
isozymes I, II, and IV, and N-hydroxysulfonamides inhibit both these
zinc enzymes. J. Med. Chem. 2000, 43, 3677−3687.
(36) Faust, A.; Waschkau, B.; Waldeck, J.; Holtke, C.; Breyholz, H.-J.;
̈
Wagner, S.; Kopka, K.; Schober, O.; Heindel, W.; Schafers, M.;
̈
Bremer, C. Synthesis and evaluation of a novel hydroxamate based
fluorescent photoprobe for imaging matrix metalloproteinases.
Bioconjugate Chem. 2009, 20, 904−912.
(37) Kopka, K.; Breyholz, H.-J.; Wagner, S.; Law, M. P.; Riemann, B.;
Schroer, S.; Trub, M.; Guilbert, B.; Levkau, B.; Schober, O.; Schafers,
̈
̈
M. Synthesis and preliminary biological evaluation of new radio-
iodinated MMP inhibitors for imaging MMP activity in vivo. Nucl.
Med. Biol. 2004, 31, 257−267.
(38) Schafers, M.; Riemann, B.; Kopka, K.; Breyholz, H.-J.; Wagner,
̈
S.; Schafers, K. P.; Law, M .P.; Schober, O.; Levkau, B. Scintigraphic
̈
imaging of matrix metalloproteinase activity in the arterial wall in vivo.
Circulation 2004, 109, 2554−2559.
(39) Breyholz, H.-J.; Wagner, S.; Levkau, B.; Schober, O.; Schafers,
̈
M.; Kopka, K. A 18F-radiolabelled analogue of CGS 27023A for
assessment of matrix-metalloproteinase activity in vivo. Q. J. Nucl. Med.
Mol. Imaging 2007, 51, 24−32.
(40) Wagner, S.; Breyholz, H.-J.; Law, M. P.; Faust, A.; Holtke, C.;
̈
Schroer, S.; Haufe, G.; Levkau, B.; Schober, O.; Schafers, M.; Kopka,
̈
̈
K. Novel fluorinated derivatives of the broad-spectrum MMP
inhibitors N-hydroxy-2(R)-{[(4-methoxyphenyl)sulfonyl](benzyl)-
and (3-picolyl)-amino}-3-methyl-butanamide as potential tools for
(59) Prante, O.; Hocke, C.; Lober, S.; Hubner, H.; Gmeiner, P.;
̈
̈
Kuwert, T. Tissue distribution of radioiodinated FAUC113: assess-
4726
dx.doi.org/10.1021/jm300199g | J. Med. Chem. 2012, 55, 4714−4727

Journal of Medicinal Chemistry
Article
ment of a pyrazolo[1,5-a]pyridine-based dopamine D4 receptor
radioligand candidate. Nuklearmedizin 2006, 45, 41−48.
(60) Schafers, K. P.; Reader, A. J.; Kriens, M.; Knoess, C.; Schober,
̈
O.; Schafers, M. Performance evaluation of the 32-module
̈
quadHIDAC small animal PET scanner. J. Nucl. Med. 2005, 46,
996−1004.
4727
dx.doi.org/10.1021/jm300199g | J. Med. Chem. 2012, 55, 4714−4727