Inverse 1,2,3-triazole-1-yl-ethyl substituted hydroxamates as highly potent matrix metalloproteinase inhibitors: (Radio)synthesis, in vitro and first in vivo evaluation

Article abstract of DOI:10.1021/jm4006753

Noninvasive imaging and quantification of matrix metalloproteinase (MMP) activity in vivo are of great (pre)clinical interest. This can potentially be realized by using radiolabeled MMP inhibitors (MMPIs) as positron emission tomography (PET) imaging agen

Full text of DOI:10.1021/jm4006753

Article
pubs.acs.org/jmc
Inverse 1,2,3-Triazole-1-yl-ethyl Substituted Hydroxamates as Highly
Potent Matrix Metalloproteinase Inhibitors: (Radio)synthesis, in Vitro
and First in Vivo Evaluation
Verena Hugenberg,*,† Burkhard Riemann,† Sven Hermann,‡ Otmar Schober,† Michael Schafers,‡,§
̈
Katrin Szardenings,∥ Artem Lebedev,∥ Umesh Gangadharmath,∥ 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: Noninvasive imaging and quantification of matrix metalloproteinase (MMP) activity in vivo are of great
(pre)clinical interest. This can potentially be realized by using radiolabeled MMP inhibitors (MMPIs) as positron emission
tomography (PET) imaging agents. Triazole-substituted MMPIs, discovered by our group, are highly potent inhibitors of
MMP-2, -8, -9, and -13. The triazole ring and its position contribute significantly to the potency of the MMP inhibitor. To
evaluate structure−activity relationships (SARs) of the initially discovered triazole-substituted MMPIs, an additional CH2-group
between the backbone of the molecule and the triazole core was inserted, and the triazole ring was “inversed” by switching the
alkyne and azide groups. Similar to the original triazole-substituted hydroxamates, the inverse triazole MMPIs are excellent
inhibitors with promising in vivo properties. Pharmacokinetic properties and metabolic stability of an 18F-labeled candidate in
mice were investigated.
applications. This can be realized through radiolabeled MMP
inhibitors (MMPIs), which can be used as radiotracers for the
detection of activated MMPs using modern molecular imaging
techniques, such as single photon emission computed
tomography (SPECT) and positron emission tomography
(PET). An alternative approach is to attach a fluorescent-dye to
the MMPI and use an optical imaging technique, such as
fluorescence reflectance imaging (FRI) and fluorescence
mediated tomography (FMT), for MMP detection.
A number of different synthetic MMPIs have been developed
and extensively explored in recent years.8 Examples of
nonpeptidic small molecule broad spectrum MMPIs are N-
sulfonyl amino acid hydroxamates CGS 25966 and CGS
27023A, which chelate the Zn2+ ion in the active site of the
INTRODUCTION
■
Matrix metalloproteinases (MMPs) are a family of structurally
and functionally related zinc- and calcium-dependent secreted
or membrane bound endopeptidases.1 They are involved in
many physiological processes such as the degradation of
extracellular matrix components and tissue remodeling but also
play a critical role in many pathophysiological mechanisms
promoting a wide range of diseases. Activated MMPs are tightly
controlled by endogenous tissue inhibitors of metalloprotei-
nases (TIMPs). In addition to other direct TIMP/MMP
interactions, the TIMPs also bind to the zinc atom of the active
site of the MMPs and as a result block their activity.
Dysregulated MMP expression and activation can be associated
with cancer,2 atherosclerosis,3 stroke, arthritis,4 periodontal
disease,5 multiple sclerosis,6 and cardiovascular disease.7
Therefore, noninvasive imaging of MMP activity in vivo is of
enormous interest in basic research as well as in clinical
Received: May 7, 2013
© XXXX American Chemical Society
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Figure 1. Lead structures 1a and 1b as well as a selection of radiolabeled analogs and a fluorescent photoprobe.
Figure 2. Novel class of 1,2,3-triazol-4-yl-methyl-substituted hydroxamate MMPIs.26
enzyme in a bidentate manner via the hydroxamate moiety.9,10
These hydroxamate lead structures with their ability to inhibit a
broad spectrum of MMPs were initially chosen in order to
check the possibility of MMP visualization with the
aforementioned imaging modalities. After a successful proof
of this concept, the development of labeled MMP-specific
tracers might be worthwhile.
exhibiting excellent MMP inhibition potencies for MMP-2,
MMP-8, MMP-9, and MMP-13 (IC50 = 0.006−58 nM) as well
as significantly increased hydrophilicities as compared to the
lead compounds. The compounds were tested against these
specific MMPs because of their distinguished impact in
cardiovascular diseases, one main focus of our research so
far.20−24 In this MMPI class, the benzyl- and the picolyl-
moieties of the CGS lead structures were systematically
substituted with a 1,2,3-triazol-4-yl-methyl unit (Figure 2).
Normally, these substituents reach into the S2′ enzyme pocket,
which is solvent-exposed and should tolerate some limited
structural changes.25 We propose that the triazole ring itself as
well as its orientation play an important role for MMP
inhibition. In particular, we discovered that MMPIs bearing the
1,2,3-triazole ring close to the hydroxamate moiety were
considerably more potent as compared to analogs with a benzyl
polyethylene glycol spacer between the backbone of the
molecule with a triazole unit attached at the end of the spacer.
Obviously the MMPIs with the 1,2,3-triazole ring close to the
hydroxamate moiety form additional attractive interaction(s)
and/or hydrogen bonds with the residues of the S2′ enzyme
pocket and/or the zinc ion of the active site mediated by the N-
atoms of the triazole unit. A radiolabeled representative
member of this new generation of triazole-substituted
hydroxamate-based MMPIs ([18F]3a) revealed increased
metabolic stability (63% parent in plasma after 30 min) in
mice in comparison to the original compound [18F]FEtO-CGS
25966 ([18F]1e, 37% parent after 20 min in plasma). No
nonspecific binding of the radiotracer in nonexcretory organs
and tissues nor defluorination was observed in vivo.
Fluorescent photoprobes and radiolabeled derivatives of the
CGS lead structures have already been developed by our group.
A Cy5.5-labeled photoprobe of CGS 25966 enabled the specific
imaging of MMPs in vitro, ex vivo, and in vivo.11,12 The
SPECT-compatible 123I-labeled CGS 27023A derivative (HO-
[
123I]I-CGS 27023A) was successfully used to image activated
MMPs in vivo in vascular lesions, which develop after carotid
artery ligation in apolipoprotein E-deficient (ApoE−/−)
mice.13,14 Next, we focused on the development of 18F-labeled
PET-compatible radiotracers. Radiosyntheses, in vitro and in
vivo evaluations of three different analogs ([18F]FEtO-CGS
27023A, [18F]FEtO-CGS 25966, and [18F]F-CGS 27023A)
have been described.15−18 The radiosynthesis of the most
promising candidate [18F]FEtO-CGS 25966 ([18F]1e in Figure
1) under GMP conditions has been enabled for first-in-man
studies.19
Initial human PET-scans with [18F]1e indicated the potency
of this compound to image activated MMPs in vivo but also
showed an extensive tracer accumulation in the intestine and
liver complicating for example imaging of atherosclerosis.
Therefore, we aimed at the improvement of the pharmacoki-
netics of these hydroxamate-based radiotracers and recently
developed a new class of triazole-substituted CGS derivatives,
B
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a
Scheme 1. Synthesis of the Azidoethyl Substituted Hydroxamic Acids 9a−c
a
Reaction conditions: (a) 4-methoxybenzene-1-sulfonyl chloride (a), 4-methylbenzene-1-sulfonyl chloride (b), 4-(2-fluoroethoxy)benzene-1-
sulfonyl chloride16 (c), pyridine (5a: 82% 5b: 82%, 5c: 50%); (b) 2-azidoethyl 4-methylbenzenesulfonate,27 K2CO3, DMF, 60 °C (6a: 82%, 6b: 73%,
6c: 84%); (c) KSF clay, CH3CN, 84 °C (7a: 46%, 7b: 30%, 7c: 84%); (d) THPONH2, EDC, NMM, HOBT, DMF (8a: 97%, 8b: 86%, 8c: 88%);
(e) 4N HCl in dioxane, MeOH, dioxane (9a: 46%, 9b: 35%, 9c: 72%).
Interestingly, despite being structurally quite different, [18F]3a
and [18F]1e have very similar clearance characteristics. [18F]3a
represents a promising new radiotracer for noninvasive PET
imaging of activated MMPs in vivo, due to its high inhibition
potencies for MMP-2, -8, -9, and -13, metabolic stability and
adequate biodistribution properties.26
To further evaluate the structure−activity relationships
(SARs), particularly the optimal position of the triazole unit,
the initially discovered triazole-substituted MMPIs were
chemically modified by the introduction of an additional
CH2-group between the backbone of the molecule and the
triazole core and by an inverse arrangement of the nitrogens in
the triazole ring. These modifications resulted in a 1,2,3-
triazole-1-yl-ethyl substitution pattern at the sulfonamide
nitrogen with increased conformational flexibility. To inves-
tigate the relevance of the p-methoxy substituent at the
benzenesulfonyl moiety, which occupies the S1′ pocket of the
enzyme, a series of p-methyl and p-fluoroethoxy substituted
derivatives were prepared.
in DMF. Cleavage of the THP protecting groups was
performed in hydrochloric acid containing dioxane/methanol
mixtures, yielding the azide key intermediates 9a−c with
different p-substituents at the benzenesulfonyl moiety (9a:
OCH3, 9b: CH3, 9c: OCH2CH2F).
4-(2-Fluoroethoxy)benzene-1-sulfonyl chloride, required for
the synthesis of MMPI precursor 9c, was prepared according to
literature procedures.16
5-Fluoropent-1-yne, used as the alkyne building block for the
1,3-dipolar cycloaddition, was prepared starting from 4-
pentyne-1-ol via tosylation reaction and subsequent fluorination
with TBAF (Scheme 2).
a
Scheme 2. Synthesis of 5-Fluoropent-1-yne (11)
a
Reaction conditions: (a) 4-methylbenzene-1-sulfonyl chloride (82%);
(b) TBAF, water (46%).
Here, we describe the (radio)synthesis, in vitro and first in
vivo evaluation of this new group of hydroxamate-based
MMPIs with an inversely attached triazole moiety.
Copper(I)-catalyzed click reactions of the azido key
intermediates 9a−c with different alkyne building blocks were
accomplished with varying yields (11−71%), resulting in a
series of inverse 1,2,3-triazole-substituted hydroxamic acid
derivatives 12a−e, 13a−c, and 14a−e (Table 1). Applying
this strategy, the synthesis of three groups of fluorinated inverse
1,2,3-triazole-substituted MMPIs, bearing a p-methoxy (12a−
e), a p-methyl (13a−c), or a p-fluoroethoxy (14a−e) moiety at
the benzenesulfonyl ring, was successfully realized. In contrast
to the p-methoxy (12a−e) and p-methyl series (13a−c), the p-
fluoroethoxy analogs (14a−e) represent reference compounds
of radiofluorinated isotopomers with two different potential
positions for 18F. Additionally, our previous studies have shown
that the p-fluoroethoxy motif at this position should be well
tolerated without affecting the MMP inhibition.15,16
Furthermore, the set of the initially discovered highly potent
triazole-substituted MMPIs26 was further explored with
inhibitors carrying a p-fluoroethoxy moiety at the benzene-
sulfonyl ring (20a−c) to estimate their inhibition potencies
compared to the inverse triazoles. Therefore, a hydroxamate
intermediate containing a p-(2-fluoroethoxy)benzenesulfonyl
and a propargyl moiety was synthesized according to Scheme 3
(19), and click reactions with different azide building blocks
RESULTS AND DISCUSSION
■
Chemistry. To evaluate the SARs of these new triazole-
substituted hydroxamates, initially a small set of nonradioactive
fluorinated standard compounds was synthesized and tested in
enzymatic fluorogenic MMP inhibition assays.
The key step to prepare these inverse 1,2,3-triazole-1-yl-ethyl
substituted MMPIs was a copper(I) catalyzed Huisgen 1,3-
dipolar cycloaddition of azidoethyl substituted hydroxamic acid
derivatives with different fluorinated alkyne building blocks.
The preparation of the azidoethyl substituted key inter-
mediates, possessing different p-substituents at the benzene-
sulfonyl moiety, is summarized in Scheme 1. Nucleophilic
substitution of the commercially available tert-butyl ester of D-
valine hydrochloride 4 with different p-substituted benzene-1-
sulfonyl chlorides in pyridine resulted in the sulfonamides 5a−
c. N-Alkylation with 2-azidoethyl 4-methylbenzenesulfonate27
under basic conditions yielded the carboxylic acid esters 6a−c,
which were converted to the carboxylic acids 7a−c in
acetonitrile with KSF clay under reflux. Preparation of 7a−c
into the corresponding hydroxamic acid esters 8a−c was
achieved by O-THP hydroxylamine, EDC, HOBT, and NMM
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Table 1. Synthesis of MMPIs via Copper(I)-Catalyzed Click Reaction
cpd
R1
OCH3
R2
yield (%)
12a
12b
12c
12d
12e
13a
13b
13c
14a
14b
14c
14d
14e
CH2CH2CH2F
38
71
42
53
30
11
30
58
38
40
48
20
17
OCH3
CH2(OCH2CH2)4OCH2CH2F
OCH3
CH2CH2CH2CH2O-(3-(2-fluoro-pyridinyl))
N-(4-fluoro-benzenesulfonamidyl)
N-methyl-N-(4-fluoro-benzenesulfonamidyl)
CH2CH2CH2F
OCH3
OCH3
CH3
CH3
CH2(OCH2CH2)4OCH2CH2F
CH3
CH2CH2CH2CH2O-(3-(2-fluoro-pyridinyl))
CH2CH2CH2F
OCH2CH2F
OCH2CH2F
OCH2CH2F
OCH2CH2F
OCH2CH2F
CH2(OCH2CH2)4OCH2CH2F
CH2CH2CH2CH2O-(3-(2-fluoro-pyridinyl))
N-(4-fluoro-benzenesulfonamidyl)
N-methyl-N-(4-fluorobenzenesulfonamidyl)
a
Scheme 3. Synthesis of MMPI 20a−c
a
Reaction conditions: (a) propargyl bromide, K2CO3, DMF (78%); (b) KSF clay, CH3CN (78%); (c) THPONH2, EDC, NMM, HOBT, DMF
(92%); (d) 4 N HCl in dioxane, MeOH, dioxane (59%); (e) CuSO4·5H2O, sodium ascorbate, DMF, H2O, 1-azido-2-fluoroethane,28 1-azido-2-(2-
(2-(2-fluoroethoxy)ethoxy)ethoxy)ethane,29 3-(2-(2-(2-(2-azidoethoxy)ethoxy)-ethoxy)ethoxy)-2-fluoropyridine30 (20a: 63%, 20b: 36%, 20c: 63%).
were performed yielding the corresponding 1,2,3-triazole-4-yl-
methyl-substituted derivatives 20a−c.
accomplished via click reactions with precursors 9a−c and
alkyne based radiosynthons, i.e., 5-[18F]fluoropent-1-yne, 1-
[18F]fluoro-3,6,9,12,15-pentaoxaoctadec-17-yne, 2-[18F]fluoro-
3-(hex-5-yn-1-yloxy)pyridine, 4-[18F]fluoro-N-(prop-2-yn-1-
yl)benzenesulfonamide, and 4-[18F]fluoro-N-methyl-N-(prop-
2-yn-1-yl)benzenesulfonamide, that are readily available via one
18F-labeling step. On the other hand, the propargyl key
intermediate 19 can be converted with azide based radio-
synthons, i.e., 1-azido-2-[18F]fluoroethane, 1-azido-2-(2-(2-(2-
[18F]fluoroethoxy)ethoxy)ethoxy)ethane and 3-(2-(2-(2-(2-
azidoethoxy)ethoxy)ethoxy)ethoxy)-2-[18F]fluoropyridine also
producible in one step. Additionally, the preparation of a p-
[18F]fluoroethoxy substitutent at the benzenesulfonyl unit is
In summary, three series of inverse triazole-substituted
hydroxamate MMPIs with different p-substituents at the
benzenesulfonyl moiety were prepared from azidoethyl
substituted key intermediates 9a−c by 1,3-dipolar cycloaddition
with alkyne building blocks. Additionally, click reactions from
the propargylic key intermediate 19 bearing a p-fluoroethoxy-
substituent at the benzenesulfonyl unit were performed yielding
the 1,2,3-triazole-substituted CGS-derivatives 20a−c. Fluori-
nated nonradioactive reference compounds of potential MMP-
targeted radioligands 12a−e, 13a−c, 14a−e, and 20a−c were
obtained to measure their MMP inhibition potencies by
enzymatic fluorogenic assays. 18F-labeling can potentially be
D
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Table 2. MMP Inhibition Potencies and clog D (log D) Values of Novel, (Inverse) Triazole-Substituted Hydroxamic Acids 12−
14, and 20
a
IC50 [nM]
log D values
b
compound
MMP-2
MMP-8
d
MMP-9
d
MMP-13
11 0.3
clog D
log D (exp)
1b10
1e17
2a26
2b26
2c26
3a26
3b26
3c26
3d26
9a
11
23
2
27
3.81
4.03
2.09
1.21
1.15
1.53
0.65
1.37
0.58
2.57
2.25
0.64
3.05
2.81
3.31
2.51
2.84
0.41
3.89
2.79
3.12
0.70
4.18
3.04
3.54
2.83
1.75
0.87
1.59
d
4
2
4
3
1
50 27
2.02 0.03
0.60 0.01
1
0.2 0.08
0.6 0.2
6
3
1
0.5
5
0.8
6
5
1
4
30 10
58
43
3
18
2
c
0.13 0.07
0.1 0.03
0.02 0.004
0.04 0.006
0.03 0.003
0.05 0.007
0.006 0.003
0.04 0.007
3
0.5
5
0.7
0.5 0.2
0.3
0.4 0.2
0.08
4
0.9
1
0.3
0.2 0.09
0.9 0.05
0.7 0.02
0.6 0.2
0.5 0.03
2
6
2
9
2
c
12a
12b
12c
12d
12e
9b
0.07 0.006
0.4
0.05 0.02
0.4
1.25 0.01
4
0.9
1
2
6
0.9 0.08
0.5 0.06
0.2 0.05
0.3 0.09
0.6 0.3
0.04 0.004
0.09 0.01
5
3
4
2
3
3
4
2
5
1
1
1
1
0.3
0.1
3
4
3
0.6
0.3
0.1
1
1
0.2
0.5
1
13a
13b
13c
9c
0.4
2
0.9 0.07
0.3 0.03
0.1 0.02
2
4
1
0.5
0.6
0.1
0.4 0.1
0.4
1
6
4
2
0.5
0.3
0.7
3
1
0.2
0.5
14a
14b
14c
14d
14e
19
0.4
2
0.4 0.07
35 24
0.07 0.002
10 10
7
1
0.4 0.1
0.05 0.02
0.7 0.04
0.8 0.1
0.07 0.01
0.1 0.01
4
9
1
2
3
0.4
1
5
1
11
1
1
0.8 0.2
0.3 0.1
0.5
0.07 0.009
0.04 0.01
0.7 0.04
0.6 0.4
20a
20b
20c
2
2
0.01
0.5
2
7
1
0.2 0.09
0.2 0.1
0.2 0.7
0.5 0.2
0.3 0.04
0.3 0.04
a
b
Values are the mean SD of three experiments. clog D values were calculated by ACD/Chemsketch version ACD/Laboratories 6.00 (log D = log
c
d
P at physiological pH (7.4)). log D (exp) value was determined for compounds [18F]3a and [18F]12a. Ki values, where SDs are not denoted.
potentially feasible by direct substitution of the corresponding
tosylate precursor with [18F]fluoride.
inhibition potencies against activated MMP-2, -8, -9, and -13.
These inverse triazoles are still more potent as compared to the
MMPIs with a benzyl polyethylene glycol spacer between the
backbone of the molecule and the triazole unit 2a−c (IC50
values of 0.2−58 nM). Considering the various p-substituents
at the benzenesulfonyl moiety obvious differences in the MMP
inhibition potencies were not determined. Apparently,
variations of small-sized substituents at this position are
tolerated with respect to the investigated MMPs. This
observation was also made for the fluoroethoxy and 1,2,3-
triazole-4-yl-methyl containing MMPIs 20a−c. They also
showed potent MMP inhibition with IC50 values comparable
to those of the inverse triazole-substituted counterparts 14a−e.
By the introduction of hydrophilic groups, such as mini-PEG
units and triazole cores, in combination with fluorinated
building blocks a series of MMPIs with a wide range of
hydrophilicities were obtained while maintaining good MMP
inhibition. To evaluate the hydrophilic properties of the target
compounds, the corresponding calculated log D values (clog D)
of the synthesized hydroxamic acids are also highlighted in
Table 2. The inverse triazole-substituted hydroxamic acids
show clog D values ranging from 0.41 (MMPI 13b) to 4.18
(MMPI 14c).
In Vitro Enzyme Assays and clog D Values. The MMP
inhibition potencies of the key intermediates 6a−c, the inverse
triazole-substituted hydroxamates 12a−e, 13a−c, 14a−e, and
the triazole-substituted hydroxamates 20a−c bearing a
fluoroethoxy moiety against MMP-2, -8, -9, and -13 were
measured by fluorogenic in vitro enzymatic inhibition assays
using a previously described protocol.31 The resulting IC50
values of the investigated MMPs were compared to those of the
initially discovered trizaole-based MMPIs and the lead
compound 1e.
As displayed in Table 2, the new class of inverse triazole-
substituted hydroxamic acids revealed excellent MMP inhib-
ition potencies with IC50 values in the nanomolar and
picomolar range (0.04−35 nM), similar to the initially
discovered triazole-based MMPIs (IC50 values of 0.006−5
nM). Compared to the lead compound 1e (IC50 values of 2−50
nM), most of the inhibitors of the new series were more potent.
Inverse triazole-substituted MMPIs bearing an N-methyl-N-(4-
fluorobenzenesulfonamidyl) group at the triazole unit (12e and
14e) demonstrated slightly higher IC50 values (1−11 nM).
In summary, the formal introduction of an additional CH2-
group between the backbone of the molecule and the triazole
core as well as the inverse arrangement of the nitrogen atoms in
the triazole ring did not indicate any significant effect on the
Additionally, the partition coefficient (log D value) of the
radiofluorinated analog [18F]12a (see section Radiochemistry)
was determined experimentally (log D (exp) = 1.25 0.01).
E
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a
Scheme 4. Radiosynthesis of [18F]12a
a
Reaction conditions: (a) K(K222)[18F]F, K2CO3, CH3CN, 110 °C, 120 s ([18F]11: rcy: 72%, decay corrected); (b) CuSO4·5H2O, sodium ascorbate,
DMF, H2O, rt, 15 min ([18F]12a: overall rcy: 47 1%, decay corrected).
The log D (exp) differs from the clog D value (clog D (12a) =
2.25) in 1 unit. Compared to the radiofluorinated triazole
substituted analog [18F]3a (log D (exp) = 0.60
0.01) the
inverse triazole-substituted hydroxamic acid [18F]12a is
approximately 4.5 times more lipophilic.
Similar to the initially discovered 1,2,3-triazole-4-yl-methyl-
substituted MMPIs, the inverse 1,2,3-triazole-1-yl-ethyl-sub-
stituted MMPIs also revealed excellent MMP inhibition.
Neither the introduction of an additional CH2-group between
the backbone of the molecule and the triazole core nor the
inverse arrangement of the nitrogen atoms in the triazole ring
attenuated the in vitro potencies. Furthermore, different p-
substituents at the benzenesulfonyl moiety that occupies the
principal specificity S1′ enzyme pocket were tolerated without
changing the inhibition of MMP-2, -8, -9, and -13. This finding
may be explained by the similar shape of the hydrophobic S1′
pocket of the here investigated enzymes (MMP-2, -8, -9:
intermediate S1′ pocket; MMP-13: deep S1′ pocket), while the
binding behavior of the compounds against MMPs with shallow
S1′ pockets (e.g., MMP-1, -7) may differ.25,32 But also
important to potency and specificity of the MMPI besides
the S1′ pocket is the chelating moiety which interacts with the
catalytic zinc ion. The hydroxamate moiety is one of the most
potent zinc-binding groups, and its affinity for Zn2+ can
overwhelm the specific protease binding contributions of the
substitutents reaching the S1′ pocket.32,33 Anyhow, these results
encouraged us to radiosynthesize the representative 18F-labeled
MMP-targeted tracer [18F]12a for further in vitro and in vivo
evaluation.
Radiochemistry. To compare the inverse triazole-sub-
stituted MMPIs with the initially discovered triazole-substituted
MMPIs, compound 12a was chosen for the radiosynthesis of its
18F-labeled analog [18F]12a, because of the structural similarity
to [18F]3a.26 Analogue to the radiosynthesis of [18F]3a, a
semiautomated two-step procedure for the radiosynthesis of
[18F]12a was developed and optimized (Scheme 4). In the first
step, the labeling synthon 5-[18F]fluoropent-1-yne ([18F]11)
was prepared by nucleophilic substitution of the tosylate
precursor (solution of DMF) with anhydrous [18F]fluoride in
the presence of Kryptofix 2.2.2 (K222), heating of the reaction
mixture to 110 °C, and direct distillation of the immediately
formed [18F]11 into a cooled (−10 °C) flask within 2 min.
Figure 3. In vitro stability of [18F]12a (tR = 8.67 min) after incubation
in mouse blood serum at 37 °C after 30 min (top), 90 min (middle),
and 120 min (bottom) measured at analytical HPLC system A,
method A2, starting with 35% CH3CN in water (0.1% TFA) for 13
min, followed by a linear gradient from 35% to 90% CH3CN in water
(0.1% TFA) over 2 min, followed by a linear gradient from 90% to
35% CH3CN in water (0.1% TFA) over 3 min with a flow rate of 1
mL·min−1.
[18F]11 was isolated after 22
2 min in an average
radiochemical yield (rcy) of 72
1% (decay corrected, n =
sulfate pentahydrate and sodium ascorbate in 15 min under
stirring at room temperature (rt). After purification by
semipreparative HPLC, evaporation, and formulation, the
two-step radiosynthesis of the inverse 1,2,3-triazole [18F]12a
5). The second step consisting of the Huisgen 1,3-dipolar
cycloaddition was performed outside the automated radio-
synthesizer in DMF using aqueous solutions of copper(II)
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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, no accumulation of [18F]12a in
organs/tissues such as the brain, myocardium, lung, and
muscles, indicating unspecific binding, was observed.
The excretion of [18F]12a predominantly via the hepatobili-
ary pathway was found to be similar to the original triazole-
substituted tracer [18F]3a.26
Biostability and Metabolism of [18F]12a. Three 10-
month-old female ICR (CD1) wild type mice were examined at
30 min p.i. to determine the biostability of [18F]12a.
Representative radio HPLC traces are shown in Figure 6.
The %ID/g values of the total organs are shown in Figure 7
and listed in Table 3 for 30 min time points. The retention time
of unchanged [18F]12a tracer was between 10.3−13.0 min. A
total of five more polar metabolites were detected, with
retention times (tR) of 1.9 min (metabolite 1), 5.9 min
(metabolite 2), 7.5 min (metabolite 3), 8.6 min (metabolite 4),
and 9.9 min (metabolite 5).
Figure 4. In vivo biodistribution of radioactivity in an adult C57/Bl6
mouse after intravenous injection of [18F]12a. Maximum intensity
projections of selected time frames p.i. demonstrate elimination of
radioactivity from the blood primarily via the liver and less
pronounced via the kidneys.
was accomplished with an overall radiochemical yield of 47
1% (decay corrected, n = 5) in 97 2 min from the end of
radionuclide production. [18F]12a was isolated in radio-
chemical purities of >98% with specific activities in the range
of 9−46 GBq/μmol at the end of the synthesis. The radioligand
was formulated in phosphate-buffered saline (PBS) to
determine the log D (exp) value and to study its in vitro
stability in mouse blood serum at 37 °C.
In Vitro Stability. An in vitro stability study was carried out
using mouse blood serum. During long-term incubation for up
to 120 min at 37 °C, [18F]12a revealed high serum stability. As
shown in Figure 3, only the parent compound [18F]12a was
detected by radio HPLC. Significant radiometabolites or
decomposition products could not be observed.
The muscle %ID/g of [18F]12a at 30 min was 0.50% with no
other metabolites present. In plasma, the %ID/g was 0.33% at
30 min, and the radioactivity was partitioned between 35%
(0.12% ID/g) of the parent tracer and 65% (0.21% ID/g) of
four metabolites: 9% metabolite 1 (0.03% ID/g), 25%
metabolite 3 (0.08% ID/g), 23% metabolite 4 (0.07% ID/g)
and 8% metabolite 5 (0.03% ID/g). In the liver sample at 30
min 12% parent (0.35% ID/g) were present, while the
remaining 88% (2.56% ID/g) of activity were distributed
between four metabolites (metabolites 2, 3, 4 and 5). The main
metabolites in the liver were metabolite 3 with 61% (1.77% ID/
g) and metabolite 4 with 20% (0.58% ID/g). Examination of
the small intestine and the gall bladder both showed 14% (8.6%
ID/g for small intestine, 12.4% ID/g for gall bladder) parent
compound, while 86% (53.1% ID/g for small intestine, 76.3%
ID/g for gall bladder) of the remaining activity were portioned
between the five mentioned metabolites. Similar to the liver,
the main compounds were metabolite 3 with 49% (30.2% ID/g
for small intestine, 43.5% ID/g for gall bladder) and metabolite
4 with 18% (11.1% ID/g for small intestine, 16.0% ID/g for gall
bladder). In the kidney homogenate, the parent tracer was
present at higher amounts compared to other tissues at 92%
In Vivo Biodistribution Study. Representative coronal
whole body images 0−1, 1−5, 5−10, and 90−120 min after
injection of [18F]12a in C57/BL6 mice are shown in Figure 4.
Overall, [18F]12a is cleared very fast and efficiently from the
body through hepatic and renal elimination with no significant
tracer remaining in nonexcretion organs 90−120 min p.i.
Because of the fast clearance MMP imaging with [18F]12a is
realistic within the first minutes p.i. in C57/Bl6 mice but not
within several hours p.i. in contrast to the radioiodinated HO-
[
123I]I-CGS 27023A that showed significantly higher lesional
uptake in ApoE−/− mice compared to blocked mice at imaging
time points ≥80 min p.i.14
Figure 5. In vivo biodistribution of radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]12a. Time-activity curves illustrate
tracer dynamics in selected regions of interests (ROI). %ID: percentage injected dose.
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Figure 6. Representative radio HPLC traces for 30 min p.i. of the metabolism study of [18F]12a. The radiochemical purity of [18F]12a was >98%
before injection. The samples were analyzed by radio-HPLC using a γ-detector (Raytest GmbH/Agilent). For the HPLC analysis, a Phenomenex
C18 column (250 × 4.6 mm) was used and a gradient method with acetonitrile and water (both modified with 0.05% TFA).
(10.3% ID/g) along with two metabolites: metabolite 1 (2%,
0.22% ID/g) and metabolite 3 (6%, 0.67% ID/g). In urine, 67%
(43.7% ID/g) of the parent compound was observed at 30 min.
The five mentioned metabolites were also present, exposing
metabolite 3 again as the main metabolite with 21% (13.7%
ID/g).
The tracer and the metabolites were predominantly cleared
through liver, gall bladder, and small intestine. The activity
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Table 3. % ID/g Values of the Metabolites of [18F]12a in Muscle, Plasma, Kidney, Urine, Liver, Small Intestine, and Gall
Bladder
percent injected dose per gram (% ID/g)
30 min p.i.
muscle
plasma
kidney
urine
liver
small intestine
gall bladder
[18F]12a: tR ∼ 10.9 min
metabolite 1: tR ∼ 1.9 min
metabolite 2: tR ∼ 5.9 min
metabolite 3: tR ∼ 7.5 min
metabolite 4: tR ∼ 8.6 min
metabolite 5: tR ∼ 9.9 min
0.50
0.00
0.00
0.00
0.00
0.00
0.12
0.03
0.00
0.08
0.07
0.03
10.28
0.22
0.00
0.67
0.00
0.00
43.74
1.96
3.26
1.30
13.71
1.31
0.35
0.00
0.12
1.77
0.58
0.09
8.65
4.32
12.42
6.21
4.94
7.10
30.27
11.12
2.47
43.48
15.97
3.55
Figure 7. % ID/g values of the total organs at 30 min post injection of [18F]12a.
remaining in the liver at 30 min p.i. was four times lower than
in the kidneys. The amount of activity excreted by the liver into
the gall bladder and the small intestine was two times higher
than the activity in urine excreted by the kidneys. Therefore this
tracer appears to be cleared faster through the liver into small
intestine and gall bladder than through the kidneys into urine.
At 30 min p.i. 35% of the plasma radioactivity in mice was
confirmed as nonmetabolized [18F]12a, whereas 65% resulted
from metabolites more polar than [18F]12a (Table 3).
Compared to [18F]3a, which shows radioactive metabolites in
plasma in a fraction of about 37% at 30 min p.i., the inverse
triazole substituted radioligand [18F]12a was less stable in vivo.
Obviously, the more flexible conformation of the triazole
moiety and/or rather the additional CH2-group between the
backbone of the molecule and the triazole core cause increased
metabolic instability in mice.
additional CH2-group and the inverse arrangement of the
nitrogen atoms in the triazole ring, nor the different p-
substituents at the benzenesulfonyl moiety negatively affected
MMP inhibition. In conclusion, these results suggest that the
introduction of a heteroaromatic system (e.g., the triazole)
provide additional potential hydrogen bond acceptors,
increasing the binding potencies of the inhibitors to the
enzyme active site. One promising MMPI lead, [18F]12a, with
moderate hydrophilicity compared to tracer 3a,26 was
successfully radiolabeled with an overall radiochemical yield
of 47
1% (decay corrected). The radiofluorinated MMPI
[18F]12a showed excellent serum stability in vitro and rapid
clearance in vivo in mice with a clear preference for the
hepatobiliary over the urinary excretion pathway, similar to
previous observations in the noninverted triazole-substituted
[18F]3a. Compared to [18F]3a, the inverse triazole-substituted
radiotracer [18F]12a appears to be less stable. However, no
defluorination or nonspecific binding of the radioligand in
nonexcretory organs were observed in vivo.
CONCLUSION
■
Our previous results with triazole-substituted MMPIs indicate
that the triazole ring greatly influences enzyme inhibition. To
further evaluate the SAR and the relevance of the position of
the triazole unit, we herein present the synthesis and in vitro
characterization of a chemically modified series of fluorinated
inverse triazole-substituted hydroxamate-based MMPIs. These
modifications result in a 1,2,3-triazole-1-yl-ethyl substitution
pattern at the sulfonamide nitrogen with an increased
conformational flexibility of this moiety and different p-
substitutents at the benzenesulfonyl moiety to estimate the
relevance of the original p-methoxy substitutent. Similar to the
original triazole-substituted MMPI series the inverse triazole
hydroxamates are excellent inhibitors of MMP-2, -8, -9, and -13
(IC50 values of 0.04−35 nM) and show calculated log D values
ranging from 0.41 to 4.18. Thus, neither the introduction of an
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 (mp) are uncorrected
and were determined in capillary tubes on a Stuart Scientific SMP3
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
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nm). NMR spectra were recorded in CDCl3, CD3OH, or DMSO-d6 on
a Bruker ARX300, a 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 a Varian Unity plus 600 (1H NMR,
600 MHz, 13C NMR, 151 MHz) spectrometer. TMS (1H), CDCl3,
DMSO-d6, CD3OH (13C), and CFCl3 (19F) were used as internal
standards and all chemical shift values 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: Two Smartline 1000 pumps and a
Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γ-
heated to reflux for 3 h. After complete conversion, as indicated by
TLC, the reaction mixture was filtered and washed with EA (2 × 20
mL). The combined organic layers were dried over anhydrous
magnesium sulfate, and the solvent was removed under reduced
pressure. Column chromatographic purification (silica gel, CH/EA
2:1) yielded the product as colorless oil (2.05 g, 5.8 mmol, 46%).
HRMS-ES-EM: m/z = 379.1045 [(M + Na)+] calcd for
C14H20N4O5SNa+: 379.1047.
(2R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-3-meth-
yl-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (8a). To a solution
of the carboxylic acid 7a (3.25 g, 9.1 mmol) in DMF (25 mL) HOBT
(1.47 g, 10.9 mmol, 1.2 equiv), NMM (3.0 mL, 27.3 mmol, 3.0 equiv),
THPONH2 (3.3 g, 28.2 mmol, 3.1 equiv) and EDC (2.45 g, 12.8
mmol, 1.4 equiv) were added. After being stirred at rt for 16 h, the
reaction mixture was diluted with water (150 mL) and extracted with
EA (3 × 50 mL). The combined organic phases were washed with
water, 5% aqueous KHSO4, saturated aqueous NaHCO3, and brine.
After being dried over magnesium sulfate, the solvent was removed
under reduced pressure. The product was purified by column
chromatography (silica gel, CH/EA 4:1) yielding a diastereomeric
mixture of THP-protected hydroxamic ester 8a as a colorless wax
(4.02 g, 8.8 mmol, 97%). HRMS-ES-EM: m/z = 478.1725 [(M +
Na)+] calcd for C19H29N5O6SNa+: 478.1731. HRMS-ES-EM: m/z =
454.1762 [(M − H)−] calcd for C19H28N5O6S−: 454.1766.
(R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-N-hy-
droxy-3-methylbutanamide (9a). (2R)-2-(N-(2-Azidoethyl)-4-me-
thoxyphenylsulfonamido)-3-methyl-N-((tetrahydro-2H-pyran-2-yl)-
oxy)butanamide (8a, 2.25 g, 4.9 mmol) was dissolved in dry dioxane
(3.0 mL). 4 N hydrochloric acid in dioxane (4.9 mL, 19.7 mmol, 4.0
equiv) and dry MeOH (3.0 mL) were added. After being stirred for 1.5
h at rt, the reaction mixture was diluted with EA (20 mL). The organic
layer was washed with water and dried over magnesium sulfate, and
the solvent was removed under reduced pressure yielding the
hydroxamic acid 9a as beige solid (835 mg, 2.3 mmol, 46%); mp
147 °C. HRMS-ES-EM: m/z = 394.1156 [(M + Na)+] calcd for
C14H21N5O5SNa+: 394.1146.
General Procedure for the Preparation of Triazoles 12a−e,
13a−c, 14a−e, and 20a−c. To a solution of the azide 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 alkyne (0.032−2.0 mmol, 1.0−3.7
equiv) in sequence. After being stirred at rt, 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
(magnesium sulfate). After evaporation of the solvent, the residue
was purified by silica gel column chromatography. Experimental and
spectroscopic data of triazoles 12b−e, 13a−c, 14a−e, and 20a−c are
listed in the Supporting Information.
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 Isotopenmessgerate 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 35% CH3CN in water (0.1% TFA) for 13 min,
followed by a linear gradient from 35% to 90% CH3CN in water (0.1%
TFA) over 2 min, followed by a linear gradient from 90% to 35%
CH3CN in water (0.1% TFA) over 3 min with a flow rate of 1 mL·
min−1. HPLC system B: Two K-1800 pumps and an S-2500 UV
detector (Herbert Knauer GmbH), 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 Eurospher column (100
C18, 250 mm × 20 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 90% to 10% CH3CN in water
(0.1% TFA) over 2 min, with a flow rate of 7.0 mL·min−1. N-
[(Methoxyphenyl)sulfonyl]-D-valine tert-butyl ester (5)9 2-azidoethyl
4-methylbenzenesulfonate,27 1-azido-2-fluoroethane,28 1-azido-2-(2-
(2-(2-fluoroethoxy)ethoxy)ethoxy)ethane,29 3-(2-(2-(2-(2-
azidoethoxy)ethoxy)-ethoxy)ethoxy)-2-fluoropyridine,30 2-fluoroethyl
4-methylbenzenesulfonate (10),16 1-fluoro-3,6,9,12,15-pentaoxaocta-
dec-17-yne,26 2-fluoro-3-(hex-5-yn-1-yloxy)pyridine,30 4-fluoro-N-
(prop-2-yn-1-yl)benzenesulfonamide34 and 4-fluoro-N-methyl-N-
(prop-2-yn-1-yl)benzenesulfonamide35 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.
(R)-2-(N-(2-(4-(3-Fluoropropyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-me-
thoxyphenylsulfonamido)-N-hydroxy-3-methylbutanamide (12a).
12a was obtained from 9a (100 mg, 0.27 mmol) and 5-fluoropent-
1-yne (11, ca. 1.0 mmol) after 2 h of stirring at rt. Column
chromatographic purification (silica gel, EA) gave a colorless solid (47
mg, 0.10 mmol, 38%), mp 54 °C. 1H NMR (300 MHz, CDCl3) δ 9.47
(s, OH, 1H), 7.74 (d, ArH, 3JH,H = 8.9 Hz, 2H), 7.46 (s, CCHN, 1H),
6.97 (d, ArH, 3JH,H = 8.9 Hz, 2H), 4.72 (m, CH2CH2N, 1H), 4.58 (m,
CH2CH2N, 1H), 4.50 (dt, 2JH,F = 47.2 Hz, 3JH,H = 5.9 Hz, CH2F, 2H),
Synthesis of MMPI Precursor 9a. (R)-tert-Butyl 2-(4-methox-
yphenylsulfonamido)-3-methylbutanoate (5a). A white solid, yield:
75%, mp 120.3 °C, analytical data see ref 9.
(R)-tert-Butyl 2-(N-(2-azidoethyl)-4-methoxyphenylsulfonami-
do)-3-methylbutanoate (6a). To a solution of (R)-tert-butyl 2-(4-
methoxyphenylsulfonamido)-3-methylbutanoate (5a) (7.96 g, 23.2
mmol) in DMF (ca. 65 μmol/mL, 360 mL) 2-azidoethyl 4-
methylbenzenesulfonate27 (5.60 g, 23.2 mmol) and potassium
carbonate (32.1 g, 232 mmol) were added. The resulting suspension
was stirred at 50 °C for 2 days. The mixture was diluted with water
(400 mL) and extracted with EA (3 × 100 mL). The combined
organic phases were washed with brine and dried over magnesium
sulfate, and the solvent was removed under reduced pressure. The
crude product was purified by column chromatography (silica gel,
CH/EA 9:1). The product was obtained as gray oil (7.85 g, 19.0
mmol, 82%). HRMS-ES-EM: m/z = 435.1671 [(M + Na)+] calcd for
C18H28N4O5SNa+: 435.1673.
3.97 (m, CH2CH2N, 1H), 3.86 (s, OCH3, 3H), 3.62 (d, NCH, 3JH,H
=
10.9 Hz, 1H), 3.47 (m, CH2CH2N, 1H), 2.93−3.78 (m,
CH2CH2CH2F, 2H), 2.21 (m, CH(CH3)2, 1H), 2.10 − 1.97 (m,
3
CH2CH2F, 2H), 0.84 (d, CH(CH3)2, JH,H = 6.6 Hz, 3H), 0.37 (d,
3
CH(CH3)2, JH,H = 6.5 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
168.03 (CONH), 163.46 (qArCOCH3), 146.86 (CCHN), 130.51
(qArCSO2), 129.42 (ArCH), 122.11 (CCHN), 114.44 (ArCH), 83.09
1
(d, CH2F, JC,F = 47.2 Hz), 62.97 (NCH), 55.69 (OCH3), 49.35
2
(CH2CH2N), 44.31 (CH2CH2N), 29.92 (d, CH2CH2F, JC,F = 20.0
(R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-3-methyl-
butanoic Acid (7a). To a solution of (R)-tert-butyl 2-(N-(2-
azidoethyl)-4-methoxyphenylsulfonamido)-3-methylbutanoate (6a,
5.16 g, 12.5 mmol) in CH3CN (25 mL) montmorillonite KSF clay
(4.17 g, ca. 1.0 g/3 mmol ester) was added, and the suspension was
3
Hz), 26.58 (CH(CH3)2), 21.24 (d, CH2CH2CH2F JC,F = 5.7 Hz),
19.37 (CH(CH3)2), 18.57 (CH(CH3)2). 19F NMR (282 MHz,
2
3
CDCl3) δ −220.45 (tt, JH,F = 47.2, JH,F = 25.8 Hz, 1F). HRMS-
ES-EM: m/z = 480.1684 [(M + Na)+] calcd for C19H28FN5O5SNa+:
J
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480.1687. HPLC system B, method B1: tR = 18.05 min (>99%).
HPLC system A, method A1: tR = 7.88 min (>99%).
were calculated by nonlinear regression analysis using the Grace 5.1.8
software (Linux).
Determination of the Partition Coefficient (log D (exp)). The
lipophilicity of radioligand [18F]12a was assessed by determination of
the water−octanol partition coefficient following a published
procedure.36 In brief, approximately 20 kBq of [18F]12a 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 rt. The
tubes were centrifuged (3000 rpm, 2 min), and three samples of 100
μL of each layer were counted in a gamma counter (Wallac Wizard,
Perkin-Elmer Life Science). The partition coefficient was determined
by calculating the ratio cpm (octanol)/cpm (PBS) and expressed as
log D (exp) log(cpmoctanol/cpmPBS)). Two independent experiments
were performed in triplicate, and data were provided as mean values
standard deviation.
Stability in Mouse Serum. The serum stability of radioligand
[18F]12a was evaluated by incubation in mouse serum at 37 °C for up
to 120 min. An aliquot of the PBS-formulated [18F]12a (20 μL, 5
MBq) was added to a sample of mouse 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
MeOH/CH2Cl2 (1:1 (v/v), 100 μL) followed by centrifugation for 2
min. The organic layer was analyzed by analytical radio-HPLC A
(method A2, tR = 8.67 min).
Biostability and Metabolism Study. Approximately 11.1 MBq
of [18F]12a (in a maximum volume of 200 μL) was injected into three
mice via tail vein injection. The animals were sacrificed at 30 min p.i.
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.
To separate the gall bladder the whole liver was first extracted, the bile
duct was pinched with a tweezers and cut. The gall bladder was put in
a glass vial, 500 μL of lysis buffer and 500 μL of 5% acetic acid in
acetonitrile were added, and the sample was vortexed for 5 min
followed by centrifugation (13000 rpm, 8 min). Approximately 0.1−
0.2 g of small intestine sample from each mouse (n = 3 mice) was
dissected clean from mesenteric fat, the luminal contents was squeezed
out with tweezers. 2.00 g of lysis buffer was added to the sample and
the sample was weighted. The sample was then homogenized using a
mechanical tissue homogenizer, 200−400 mg of the homogenate were
added to a preweighted counting tube and counted in the gamma
counter. The homogenate was then frozen in the dry ice and
subsequently thawed at room temperature. The homogenate was
diluted with 2.0 mL of 3% acetic acid in acetonitrile and mixed again
using the tissue homogenizer. A 400 μL sample of the resulting slurry
was centrifuged (13000 rpm, 8 min). 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). The HPLC was done on a Phenomenex C18 column (250 ×
4.6 mm) using a gradient method with acetonitrile and water (both
having 0.05% TFA). Tebia bone was extracted from the hind leg,
cleaned of the muscle, ligaments and parts of the joint placed in a
preweighted plastic tube, weighted and counted in a γ-counter.
Animals. Adult C57/BL6 mice (male, 21−24 g) were anaesthetised
by isoflurane/O2 and one lateral tail vein was cannulated using a 27 G
needle connected to 15 cm polyethylene catheter tubing. [18F]12a
(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.
Radiochemistry. General Methods. Radiofluorinations were
carried out on a modified PET tracer radiosynthesiser (TRACERLab
FxFDG, GE Healthcare). The recorded data were processed by the
TRACERLab Fx software (GE Healthcare). Separation and
purification of the radiosynthesised 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 GINA Star software (Raytest Isotopenmessgerate GmbH).
̈
Radiochemical purities and specific activities were determined using
the analytical radio-HPLC system A. No-carrier-added aqueous
[18F]fluoride was produced on an RDS 111e cyclotron (CTI-Siemens)
by irradiation of a 1.2 mL water target using 10 MeV proton beams on
97% enriched 18O-water by the 18O(p,n)18F nuclear reaction.
Extraction and Drying of [18F]fluoride. To recover the
[18O]water, the batch of aqueous [18F]fluoride was passed through
an anion exchange resin (Sep-Pak Light Waters Accell Plus QMA
cartridge, preconditioned with 5 mL 1 M K2CO3 and 10 mL of water).
[18F]Fluoride was 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 of Kryptofix 2.2.2 (K222). Subsequently, the
aqueous [18F]K(K222)F solution was carefully evaporated to dryness in
vacuo (2 min vacuum with helium stream, 1 min at 56 °C without
helium, then 8 min at 84 °C without helium).
Reaction of [18F]fluoride with Pentynyl Tosylate. A solution of
the tosylate precursor 10 (20 mg, 75 μmol) dissolved in DMF (0.5
mL) was added to the reaction vessel containing the dried
[18F]fluoride. The vessel was heated to approximately 110 °C. The
immediately formed [18F]fluoropentyne [18F]11 was distilled directly
from the reaction vessel into a 5 mL-flask with 400 μL of DMF
(cooled to −10 °C) within 2 min. The click labeling was performed
outside the automated radiosynthesiser. The azide precursor 9a (6 mg,
dissolved in 100 μL of DMF), CuSO4 (0.4 M, 60 μL) and sodium
ascorbate (8 mg, dissolved in 100 μL of H2O) were added to the
reaction mixture and the reaction was stirred at rt for 15 min. The
crude 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 33/67 (v/v). The product fraction of compound [18F]10a
(retention time tR([18F]12a) = 10.3−13.0 min) was evaporated to
dryness in vacuo and redissolved in 0.5 mL of NaCl/EtOH (9/1 v/v).
Product compound [18F]12a was obtained in an overall radiochemical
yield of 47
1% (decay-corrected, based on cyclotron-derived
[18F]fluoride ions, n = 5) in 97 2 min from the end of radionuclide
production. [18F]12a was isolated in radiochemical purities of >98%
with specific activities in the range of 9−46 GBq/μmol at the end of
the synthesis. Radiochemical purity and specific activity were
determined using the analytical radio-HPLC system A (method A2).
In Vitro Enzyme Inhibition Assays (Table 3). The inhibition
potencies of hydroxamic acid derivatives 12a−e, 13a−c, 14a−e, and
20a−c 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-diamino-propionyl)Ala-Arg-
NH2 (R&D Systems) as described previously.31 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 function of inhibitor
concentration. From the resulting inhibition curves, the IC50 values
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
K
dx.doi.org/10.1021/jm4006753 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Positron Systems Ltd., Oxford, UK) with uniform spatial resolution
(<1 mm) over a large cylindrical field (165 mm diameter, 280 mm
axial length).37
emission computed tomography; THP, tetrahydropyranyl;
TIMP, tissue inhibitors of metalloproteinase
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 coregistered
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 coregistered 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).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and analytical data for the synthesis of
p-(2-fluoroethoxy)phenylsulfonylchloride, compounds 10−11,
5b−9b, 5c−9c, 16−19, 12b−e, 13a−c, 14a−e, 20a−c and
NMR data of 5a − 9a. This material is available free of charge
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AUTHOR INFORMATION
Corresponding Author
*Phone: +492518345685. Fax: +498347363. E-mail: verena.
hugenberg@uni-muenster.de.
■
Present Address
⊥German Cancer Research Center (dkfz), Radiopharmaceutical
Chemistry (E030), Im Neuenheimer Feld 280, D-69120
Heidelberg, Germany.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
The authors thank Marlena Brink, Daniel Burkert, Claudia
Essmann, Sven Fatum, Wiebke Gottschlich, Sarah Koster,
Roman Priebe, Katrin Reckmann, and Sandra Hoppner for
technical support and the staff members of the Organic
Chemistry Institute, University of Munster, Germany, for
spectroscopic and analytical investigations. This work was
supported in part by the Deutsche Forschungsgemeinschaft
(DFG), Collaborative Research Center 656 (Molecular
Cardiovascular Imaging Projects A02, Z05 and C06),
■
̈
̈
̈
University of Munster, and the Interdisciplinary Centre of
̈
Clinical Research (IZKF core unit PIX), Munster, Germany
̈
and a cooperation agreement with Siemens Medical Solutions
USA, Inc.
ABBREVIATIONS USED
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CH, cyclohexane; EDC, N′-ethyl-N′-(3-dimethylaminopropyl)-
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FMT, fluorescence mediated tomography; FRI, fluorescence
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HOBT, 1-hydroxy-benzotriazole; ICR, imprinting control
region; K222, Kryptofix2.2.2; log D, log P at physiological pH
(7.4); MMP, matrix metalloproteinase; MMPI, matrix metal-
loproteinase inhibitor; NMM, 4-methyl-morpholine; PEG,
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