A new generation of radiofluorinated pyrimidine-2,4,6-triones as MMP-targeted radiotracers for positron emission tomography

Article abstract of DOI:10.1021/jm201142w

Radiolabeled C-5-disubstituted pyrimidine-2,4,6-triones have recently been suggested by our group as a class of potent matrix metalloproteinase (MMP) targeted radiotracers that can noninvasively visualize activated MMPs by means of positron emission tomography (PET). MMPs belong to the zinc- and calcium-dependent endopeptidases which are involved in the proteolytic degradation of components of the extracellular matrix (ECM) but also are capable of processing and releasing bioactive molecules such as growth factors, proteinase inhibitors, and cytokines. Locally increased levels of activated MMPs modulate and contribute to the progression of various diseases, such as cancer, atherosclerosis, stroke, arthritis, and others. Therefore, activated MMPs are suitable biological targets for the specific and noninvasive visualization of aforementioned pathologies in vivo. On the basis of our recent results, we here describe a series of new fluorinated pyrimidine-2,4,6-triones of the second generation with maintained MMP inhibition potencies (IC₅₀ = 4-605 nM), which are fine-tuned toward more hydrophilic versions, and show the improved biodistribution behavior of one selected radiofluorinated pyrimidine-2,4,6-trione by means of small-animal PET.

Full text of DOI:10.1021/jm201142w

Article
pubs.acs.org/jmc
A New Generation of Radiofluorinated Pyrimidine-2,4,6-triones as
MMP-Targeted Radiotracers for Positron Emission Tomography
Daniela Schrigten,†,∥ Hans-Jorg Breyholz,† Stefan Wagner,† Sven Hermann,§ Otmar Schober,†
̈
Michael Schafers,§,∥ Gunter Haufe,‡ and Klaus Kopka*,†,∥
̈
̈
†Department of Nuclear Medicine, University Hospital Munster, Albert-Schweitzer-Campus 1, Building A1, D-48149 Munster,
̈
̈
Germany
‡Organisch-Chemisches Institut, Westfalische Wilhelms-Universitat, Corrensstrasse 40, D-48149 Munster, Germany
̈
̈
̈
§European Institute for Molecular Imaging, University of Munster, Mendelstrasse 11, D-48149 Munster, Germany
̈
̈
∥Interdisciplinary Centre of Clinical Research (IZKF), Munster, Germany
̈
S
* Supporting Information
ABSTRACT: Radiolabeled C-5-disubstituted pyrimidine-2,4,6-triones have recently been suggested by our group as a class of
potent matrix metalloproteinase (MMP) targeted radiotracers that can noninvasively visualize activated MMPs by means of
positron emission tomography (PET). MMPs belong to the zinc- and calcium-dependent endopeptidases which are involved in
the proteolytic degradation of components of the extracellular matrix (ECM) but also are capable of processing and releasing
bioactive molecules such as growth factors, proteinase inhibitors, and cytokines. Locally increased levels of activated MMPs
modulate and contribute to the progression of various diseases, such as cancer, atherosclerosis, stroke, arthritis, and others.
Therefore, activated MMPs are suitable biological targets for the specific and noninvasive visualization of aforementioned
pathologies in vivo. On the basis of our recent results, we here describe a series of new fluorinated pyrimidine-2,4,6-triones of the
second generation with maintained MMP inhibition potencies (IC50 = 4−605 nM), which are fine-tuned toward more
hydrophilic versions, and show the improved biodistribution behavior of one selected radiofluorinated pyrimidine-2,4,6-trione by
means of small-animal PET.
e.g., the plasma inhibitor α2-macroglobulin, four tissue
inhibitors of metalloproteinases (TIMPs),5 or the plasma-
membrane anchored cell surface receptor RECK (reversion-
inducing-cysteine-rich protein with kazal motifs).6
In pathophysiological processes an increased cytokine and
growth factor-stimulated MMP gene transcription, followed by
an abnormal pro-MMP secretion and zymogen activation, might
become prevalent, triggering the progression of inflammation,
cancer, or especially cardiovascular diseases like atherosclerosis
and arterial aneurysm.7,8
There are several groups that are currently working on the
development and evaluation of MMP inhibitor (MMPI) based
radiotracers targeting MMPs in their activated forms and on the
noninvasive imaging of MMP-associated diseases by means of
single photon emission computed tomography (SPECT) or
positron emission tomography (PET).9−12
INTRODUCTION
■
The visualization of locally up-regulated and activated
extracellular matrix (ECM) degrading peptidases in vivo
(such as matrix metalloproteinases (MMPs)) is a clinical
challenge. MMPs are involved in the proteolytic degradation of
ECM components but also are capable of modulating and
initiating the release of bioactive molecules such as growth
factors, proteinase inhibitors, and cytokines.1 To date, more
than 20 human MMPs have been characterized. On the basis of
their specificity, the MMPs are classified into collagenases,
gelatinases, stromelysins, and matrilysins. Another subclass of
the MMPs is represented by the membrane-type MMPs (MT-
MMPs) that additionally contain a transmembrane and
intracellular domain, a membrane linker domain or are
membrane associated.2,3
MMPs are secreted as inactive zymogens (pro-MMPs). Once
activated via the “cysteine switch” mechanism, a zinc active site
is provided that initiates its proteolytic activity.4 Activated
MMPs are tightly controlled by several endogenous inhibitors,
Received: August 26, 2011
Published: November 28, 2011
© 2011 American Chemical Society
223
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
Recently, the pyrimidine-2,4,6-triones (or barbiturates) were
identified as potent MMPIs that exhibit specific activities by
binding to the zinc(II) active site of a subgroup of activated
MMPs comprising the gelatinases A (MMP-2) and B (MMP-9),
neutrophil collagenase (MMP-8), and the membrane bound
MMPs MT-1-MMP (MMP-14) and MT-3-MMP (MMP-16).13
The C-5-disubstituted pyrimidine-2,4,6-trione derivative 1 (RO
28-2653) was found to exhibit a strong antitumoral and anti-
angiogenic efficacy and thus is able to target tumor tissues.14
These prerequisites support the approach of using the
barbiturates as molecular MMP-targeted imaging agents.15 In
contrast to the bidentate zinc(II) binding moiety of the
radiofluorinated hydroxamate-based MMP-targeted radiotracers
recently introduced by our group,16−18 it is speculated that the
enolic tautomer of the pyrimidine-2,4,6-triones either binds in
an almost tridentate manner to the zinc active site19 or chelates
in a monodentate pattern via a nitrogen whereupon the
flanking oxygen atoms occupy the second coordination shell.20
We lately have introduced a first radiofluorinated C-5-
disubstituted prototype MMP-targeted radiotracer, a potent
and moderately lipophilic MMPI radiotracer based on the lead
structure 1 (experimental log D of 2.15) showing one- to two-
digit nanomolar IC50 values especially for the gelatinases (MMP-2,
IC50 = 23 nM; MMP-9, IC50 = 7 nM) and which is radiolabeled
with the prominent PET-compatible positron emitter 18F
(Figure 1).21
1,2,3-triazole building blocks in combination with known
(radio)fluorinated prosthetic groups. As a result, a set of new
MMPIs with very high MMP inhibition potencies covering a
wide range of lipophilicities were synthesized. Hereupon we
identified a radiofluorinated, more hydrophilic pyrimidine-
2,4,6-trione of the second generation and discovered, by means
of small-animal PET imaging, its significantly accelerated in
vivo excretion characteristics compared with the radiofluori-
nated MMP-targeted radiotracer of the first generation.
RESULTS AND DISCUSSION
■
Chemistry. The pyrimidine-2,4,6-trione 1 was chosen as
lead compound, and modifications were made in the piperazinyl
moiety with the aim to improve the clearance characteristics and
to enable 18F-labeling while retaining MMP inhibition potencies
of the resulting derivatives. This approach involves the introduc-
tion of short PEG chains, which are known to increase the
hydrophilicity and the water solubility, directing the tracer to a
predominant renal excretion route. Compared to a non-PEGylated
derivative the mini-PEG tracer should therefore lead to a higher
tracer-related signal-to-noise-ratio in the target tissue.22−24 In
addition we recently observed that by introduction of mini-PEG
units as linkers at the piperazinyl moiety, the MMP inhibition
potencies of modified barbiturate derivatives are maintained.21,25
The preparation of the key intermediates 8−12 is
summarized in Scheme 1. Pyrimidine-2,4,6-triones 9, 11, and
12 were synthesized from the corresponding piperazines as
previously reported by our group.21,25 According to this
procedure, the alkyne derivative 8 was prepared by reaction of
5-(4-phenoxyphenyl)pyrimidine-2,4,6-trione 326 with propargyl-
amine. Similarly, the piperazine 5 yielded the alkyne derivative
10 with an elongated PEG chain. To obtain the piperazine
derivative 5 from tetraethylene glycol (TEG) (Scheme 2), a
“Williamson type” coupling reaction27 using potassium tert-
butoxide (t-BuOK) in tert-butanol (t-BuOH) was applied
affording the PEG8 derivative 16 in 83% yield.
Next, a variety of fluorinated building blocks were incorporated
into the pyrimidine-2,4,6-trione structure to give access to products
with a wide range of lipophilicities and to fathom the tolerance to
heterocycles in this position, in particular to 1,2,3-triazoles and
fluoropyridinyl substituents. The lipophilic MMPI structures 13
and 14 were obtained as outlined in Scheme 1. Compound 14 was
prepared in 78% yield via coupling with N-succinimidyl-4-fluoro-
benzoate28 in the presence of triethylamine.
A set of more hydrophilic pyrimidine-2,4,6-triones were achieved
from alkyne- and azido-substituted intermediates 8−11 by employ-
ing copper(I) catalyzed 1,3-dipolar cycloadditions.29 The fluori-
nated triazoles 28−36 were prepared in yields of 27−72%
using copper(II) sulfate and sodium ascorbate in dimethylforma-
mide (DMF) (Scheme 3). With an addition of the Cu(I)-
stabilizing tris(benzyltriazolyl)methylamine ligand (TBTA), no
improvement of reaction yields was observed.
These reaction pathways provided a novel series of pyri-
midine-2,4,6-triones including nonradioactive reference com-
pounds 13, 14, and 28−36 of potential 18F-fluorinated radioli-
gands as well as precursors 8−12 applicable for 18F-labeling in
two steps using the corresponding radiofluorinated prosthetic
groups, e.g., N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)
for amino precursor 12, 2-[18F]fluoroethylazide for alkyne
precursors 9 and 10, and 2-[18F]fluoro-3-(hex-5-ynyloxy)-
pyridine ([18F]FPy5yne) for azide precursor 11.
Figure 1. Structures of 1 (RO 28-2653) and radiofluorinated
pyrimidine-2,4,6-trione of the first generation 2.21
As expected, the preliminary biodistribution studies using
the quadHIDAC small-animal PET device showed that this
18F-labeled pyrimidine-2,4,6-trione does not specifically accu-
mulate in tissues of nondiseased wild-type mice. The
wholebody dynamic small-animal PET scan over 120 min
indicated that the tracer uptake in nontarget organs, such as the
brain, lung, heart, and muscle, was low over all time points.
There was also a notable uptake in the bladder, indicative of a
predominant renal excretion with some hepatobilliary clear-
ance. In the case of occurring MMP-associated diseases the
visualization of dysregulated MMP activity in possible target
tissues such as brain, heart, lung, and tumors might be feasible.
However, the clearance of any 18F-labeled radiotracer candidate
from a living subject should correspond with or be less than the
physical half-life of the applied PET radionuclide (18F, T1/2
=
109.7 min), and thus, an excretion behavior of the radiotracer is
desired that is more rapid compared with the 18F-labeled
prototype pyrimidine-2,4,6-trione to finally obtain high signal-to-
noise ratios of detected target tissues within a shorter time frame.
We here present a series of new fluorinated pyrimidine-2,4,6-
triones, fine-tuned with mini-polyethylene glycol (PEG) and
In Vitro Enzyme Assays and log D Values. The IC50
values of the target compounds 13, 14, and 28−36 against
224
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
a
Scheme 1. Syntheses of Phenoxyphenylpyrimidine-2,4,6-triones 8−14
a
Reaction conditions: (a) (1) NBS, DMF, 5−10 °C; (2) propargylamine or piperazines 4−7, K2CO3, room temp, 18 h; (b) (1) PPh3, THF, room
temp, 16 h; (2) H2O, room temp, 3 h; (c) N-succinimidyl-4-fluorobenzoate, Et3N, CH2Cl2, room temp, 7.5 h.
a
Scheme 2. Synthesis of Piperazine 5
a
Reaction conditions: (a) TEG, t-BuOK, t-BuOH, reflux, 18 h; (b) propargyl bromide, NaH, THF, 0 °C, then room temp, 26 h; (c) TsOH·H2O,
MeOH, reflux, 20 h; (d) TsCl, Et3N, CH2Cl2, 0 °C, then room temp, 18 h; (e) 1-Boc-piperazine, Et3N, CH3CN, reflux, 24 h; (f) TFA, CH2Cl2, room
temp, 19 h.
activated MMP-2, -8, -9, and -13 were measured by fluorogenic
in vitro inhibition assays following the procedure previously
described.30 The assays were validated using the nonradioactive
versions of the MMP inhibitors. IC50 values were obtained from
nonlinear regression analysis of the concentration-dependent
reaction rates.
As displayed in Table 1, the target compounds revealed, similar
to the previously reported (radio)ligand 2, MMP inhibition
potencies in the nanomolar range (IC50 of 4−605 nM). These
results clearly demonstrate a wide tolerance to modifications at
the piperazinyl moiety, which orients into the S2′ subsite of
the activated MMP. The modifications include insertion of
1,2,3-triazoles and fluoropyridinyl substituents. Moreover, as a
new finding, replacement of the piperazinyl moiety gave
MMPIs 28 and 29 with inhibition potencies comparable to
those of derivatives comprising this group. We have selected
compounds 28 and 29, avoiding the piperazinyl approach but
mimicking the nitrogen−nitrogen distance given in the
piperazinyl moiety.
Because of the fact that the hydrophilicity of a radioligand
has a significant impact on the biodistribution characteristics of
any radiotracer, an evaluation of the hydrophilic properties of
the target compounds is required. Therefore, the corresponding
calculated log D values (clogD) were tabulated along with the
inhibition potencies (Table 1). The obtained new generation of
pyrimidine-2,4,6-triones shows clogD values ranging from
−0.45 (MMPI 31) to 4.27 (MMPI 13). The differing log D
values display the influence of the incorporation of structural
features such as PEG units and 1,2,3-triazoles in combination
with fluorinated building blocks.
Additionally, the partition coefficient (log D) of the radio-
fluorinated analog [18F]30 (see section Radiochemistry) was
determined experimentally (log D (exp) = 0.78 0.02). The
experimental log D is comparable to the calculated value (clogD
(30) = 0.99). The pyrimidine-2,4,6-trione 30 is therefore
approximately 100 times more hydrophilic than the first
generation radioligand 2. The desired increased hydrophilicity
of pyrimidine-2,4,6-trione 30 is indeed realized while main-
taining its in vitro MMP inhibition characteristics, as shown by the
double-digit nanomolar IC50 values. This encouraged us to further
improve the radiosynthesis of the 18F-labeled isotopolog [18F]30,
as follows, aiming at initial in vivo experiments using this new
MMPI radiotracer.
Radiochemistry. The 2-[18F]fluoroethylazide-conjugated
pyrimidine-2,4,6-trione [18F]30 was achieved by a semi-
automated two-step procedure consisting of the nucleophilic
radiofluorination of 2-azidoethyl-4-methylbenzenesulfonate31
and subsequent copper(I) catalyzed cycloaddition with the
alkyne precursor 9 (Scheme 4). The radiosynthesis of click
chemistry building block 2-[18F]fluoroethylazide ([18F]24) was
carried out according to a procedure previously described by
Glaser and Årstad.32 [18F]24 could be isolated by distillation in
68 8% radiochemical yields (decay corrected, n = 6). The
click labeling was performed outside the automated synthesizer.
Investigations of the impact of reaction time, temperature, and
the catalytic system on the radiochemical yield of [18F]30
revealed the optimal conditions to be 8 min of reaction time at
room temperature in the presence of 2 equiv of copper sulfate
relative to the alkyne precursor 9. This successfully provided
the 1,4-disubstituted 1,2,3-triazole [18F]30 in radiochemical
225
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
a
Scheme 3. Syntheses of 1,2,3-Triazoles 28−36
a
Reaction conditions: (a) CuSO4·5H2O, sodium ascorbate, DMF, room temp, 2.5−72 h.
Table 1. IC50 and log D of Novel Pyrimidine-2,4,6-triones 13, 14, and 28−36
a
IC50 (nM)
log D
b
compd
MMP-2
MMP-8
MMP-9
MMP-13
clogD
log D (exp)
2
23
9
138 12
129 28
7
2
645 17
48 13
2.88
4.27
3.24
1.07
1.80
0.99
−0.45
0.82
3.09
2.09
2.59
3.23
2.15 0.0221
13
14
28
29
30
31
32
33
34
35
36
203 47
9
2
5
2
15
48
6
4
4
0.2
2
16
87
9
3
7
64
7
41
10
27
29 10
58 14
49 36
58
1
1
c
3
6
51 11
94 58
605 16
0.78 0.02
44
202 25
22
13 0.2
3
150 21
107 38
74 19
56 25
9
27
19
1
1
23
20
7
7
3
7
1
23
21
2
7
12
52
1
4
15 0.2
1
116 17
60 20
40 13
a
b
Values are the mean SD of three experiments. clogD values were calculated by ACD/Chemsketch, version 6.00, ACD/Laboratories (log D =
c
log P at physiological pH (7.4)). log D value was determined for compound [18F]30. Value is the mean SD of two independent experiments.
yields of 63 10% (decay-corrected, n = 6). After purification
by semipreparative HPLC, concentration by rotary evaporation,
and formulation, the two-step radiosynthesis of [18F]30 was
accomplished with an overall radiochemical yield of 43
9% (decay corrected, n = 6) in 140 min from the end of
radionuclide production. The radiochemical purity of [18F]30
was >97%, and the determined specific activities were 11−20
GBq·μmol−1 at the end of radiosynthesis. The radioligand was
formulated in phosphate buffered saline (PBS) to determine
log D7.4 values and to study its in vitro stability in human serum
at 37 °C.
In Vitro Stability. An in vitro stability study was carried
out using human serum. During long-term incubation for up
to 120 min at 37 °C, [18F]30 revealed an excellent stability.
As shown in Figure 2, only parent compound [18F]30 can be
detected by radio-HPLC. Radiometabolites cannot be observed.
226
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
Scheme 4. Radiosynthesis of 2-[18F]Fluoroethylazide-
(CT), were performed throughout the studies and recon-
structed into a series of images reflecting the radiotracer
distribution at different time frames after injection of [18F]30
(Figure 3).
a
Conjugated Pyrimidine-2,4,6-trione [18F]30
a
Reaction conditions: (a) [18F]fluoride (potassium 222 cryptate),
K2CO3, CH3CN, 84 °C, 15 min; (b) CuSO4·H2O, sodium ascorbate,
DMF, room temp, 8 min.
Figure 3. Biodistribution of radioactivity in adult C57/BL6 mouse
after intravenous injection of [18F]30 visualized by small-animal PET.
Images represent maximum intensity projections at selected time
frames. Labeled structures are as follows: liver (lv), gallbladder (gbl),
bladder (bl), kidneys (kd), small bowel (sb), lung (lu).
A similar pharmacokinetic behavior was observed compared
to radioligand 2 which has been described earlier by our
group.21 The tracer uptake in nontarget organs, such as brain,
heart, and lung, was low over all time points. Furthermore, low
accumulation of [18F]30 in the skeleton suggests that the extent
of tracer defluorination and generation of free [18F]fluoride was
negligible. [18F]30 is notably excreted via the kidneys and the
liver as indicated by the tracer accumulation in the urinary bladder
and the gallbladder/intestine, respectively. For tissues involved
in metabolic and excretory processes (liver, gallbladder, intestine,
kidney, bladder) and for potential regions for diagnostic MMP-
PET, CT-guided volumes of interest (VOIs) were defined and
time−activity curves were determined (Figure 4).
Initially, a high level of radioactivity is observed in the liver
which decreases to background level in 10−15 min pi. In
comparison, model tracer 2 features a much slower clearance
from the liver in 90−120 min pi. Furthermore, by comparison
of the renal elimination of model tracer 2 with the more
hydrophilic radioligand [18F]30, the latter presents a faster
clearance from the kidneys. Hence, alteration of the chemical
structure by introducing a mini-PEG and a triazole unit led to a
favorable pharmacokinetic behavior for in vivo PET-imaging.
CONCLUSION
■
Extending our recent work on the development of MMP-
targeted radioligands, we herein present the synthesis and in
vitro characterization of a new series of fluorinated pyrimidine-
2,4,6-trione-based MMPIs derived from lead structure 1. All
compounds were evaluated as potent inhibitors of MMP-2, -8,
-9, and -13 with calculated log D values ranging from −0.45 to
4.27. This allows a systematic investigation of the effect of
hydrophilicity on biodistribution patterns of respective radio-
fluorinated PET tracers together with the exploration of their in
vivo stability. One promising MMPI, [18F]30, with an increased
hydrophilicity compared to model tracer 2 described earlier by our
group,21 was successfully radiolabeled with good radiochemical
yields of 43 9%. This radiofluorinated pyrimidine-2,4,6-trione
[18F]30 of the new generation revealed an excellent serum
Figure 2. Stability of [18F]30 (tR = 6.5 min; analytical HPLC system II,
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, 1.5 mL·min−1) after incubation in
human serum in vitro at 37 °C after 20 min (top), 90 min (middle),
and 120 min (bottom).
In Vivo Biodistribution Study. Biodistribution character-
istics of radiotracer [18F]30 were studied in adult Balb/c mice.
Dynamic PET scans, co-registered to computed tomography
227
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
Figure 4. Representative biodistribution of radioactivity in adult C57/Bl6 mouse after intravenous injection of [18F]30: (A) time−activity curves of
the blood, brain, lung, and muscle; (B) time−activity curves of blood, kidney, bladder, liver, and small bowel; ROI, region of interest; % ID, percent
injected dose.
column (250 mm × 4.6 mm). The recorded data were processed by
stability in vitro and more rapid clearance characteristics, as
shown by in vivo biodistribution studies in wild type mice.
Thus, [18F]30 represents a promising MMP-targeted radiotracer
for the noninvasive PET imaging of activated MMPs in vivo,
which is putatively more favorable compared to compound 2
with respect to the physical half-life of the PET-radionuclide 18F.
In the next step preclinical PET/CT studies in disease models
with known MMP up-regulation (e.g., tumor, Lewis lung
carcinoma bearing mice; atherosclerotic plaques, apolipoprotein
E-deficient mice) will be assessed.
the GINA Star software (Raytest Isotopenmessgerate GmbH). The
̈
HPLC system I 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). N-Boc
piperazine, pyrimidine-2,4,6-triones 4, 6, and 12, and PEG derivatives
15 and 21 were prepared as previously described by our group.21,25
The syntheses of 5-(4-phenoxyphenyl)pyrimidine-2,4,6-trione,26
N-succinimidyl-4-fluorobenzoate,25 2-azidoethyl-4-methylbenzenesul-
fonate,31 2-fluoroethylazide (24),32 propargyl 4-fluorobenzoate
(25),33 and 4-fluoro-N-methyl-N-(prop-2-ynyl)benzenesulfonamide
(26b)34 were carried out according to literature procedures. 4-(6-
Fluoropyridin-2-yl)piperazin-1-ium trifluoroacetate (7) was prepared
from 4-(6-fluoropyridin-2-yl)piperazine-1-carboxylic acid tert-butyl
ester35 via deprotection with trifluoroacetic acid (TFA). 4-Fluoro-3-
(hex-5-ynyloxy)pyridine (27) was synthesized as described by Inkster
et al.36 According to this procedure, 3-(2-{2-[2-(2-azidoethoxy)-
ethoxy]ethoxy}ethoxy)-2-fluoropyridine (23) was prepared from
toluene-4-sulfonic acid 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl
ester21 (see Supporting Information). All animal experiments were
conducted in accordance with local institutional guidelines for the care
and use of laboratory animals.
EXPERIMENTAL SECTION
■
General. All chemicals, reagents, and solvents were analytical
grade, purchased from commercial suppliers, and used without further
purification unless otherwise specified. Melting points were measured
on a Stuart Scientific SMP3 capillary melting point apparatus and are
uncorrected. 1H NMR, 13C NMR, and 19F NMR spectra were
recorded in CDCl3 or in DMSO-d6 on Bruker AV400, Bruker AV300,
or Varian Unity plus 600 spectrometer with the corresponding solvent
signals as an internal standard. For 19F NMR shift values, CFCl3 was
the internal standard. Chemical shifts are reported in δ (ppm). Exact
mass analyses were conducted on a Bruker MicroTof apparatus.
Reactions were monitored by thin layer chromatography (TLC,
performed on silica gel coated polyester backed TLC plates, SIL
G/UV254, Macherey-Nagel) using solvent mixtures of cyclohexane
(CH), ethyl acetate (EtOAc), methanol (MeOH), and triethylamine
(TEA). The ≥95% purities of each new nonradioactive compound
were assessed by elementary analysis or analytical reversed phase
HPLC on HPLC system I: two Smartline 1000 pumps and a Smartline
UV detector 2500 (Knauer), a GabiStar γ-detector (Raytest Isoto-
General Procedure for the Preparation of Pyrimidine-2,4,6-
triones 8, 10, and 13. Compounds 8, 10, and 13 were synthesized
according to previously described procedures.25,26 A solution of 5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione (2−10 mmol, 1 equiv) in
DMF (2 mL/mmol) was cooled to 10 °C, and a solution of NBS (2−
10 mmol, 1 equiv) in DMF (1 mL/mmol) was added dropwise. After
the mixture was stirred at 5−10 °C for 20 min, propargylamine
(10 mmol, 1 equiv), compound 5 (2 mmol, 1 equiv), or compound 7
(2.5 mmol, 1 equiv) was added in one portion, followed by potassium
penmessgerate GmbH), and a Nucleosil Eurosphere 100-5 C-18
̈
228
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
carbonate (4−20 mmol, 2 equiv). After being stirred for 1 h at 5−
10 °C, the mixture was allowed to warm to room temperature and
then stirred overnight.
The mixture was stirred at room temperautre for 7.5 h and evaporated
to dryness. The crude product was purified by silica gel
chromatography (EtOAc/MeOH (9/1) + 2% TEA) to give pure 14
(143 mg, 78%). Mp: 124−126 °C. 1H NMR (300 MHz, DMSO-d6): δ
2.41−2.57 (m, 10H, N−CH2), 3.35−3.53 (m, 14H, CH2), 7.00−7.06
(m, 4H, HAryl), 7.14−7.20 (m, 1H, HAryl), 7.24−7.32 (m, 2H, HAryl),
7.37−7.44 (m, 4H, HAryl), 7.88−7.96 (m, 2H, HAryl), 8.55 (t, 1H, 3J =
5.5 Hz, NH), 11.62 (br s, 2H, NH) ppm. 13C NMR (75.5 MHz,
DMSO-d6): δ 43.37, 47.29, 53.72, 57.11, 68.11, 68.88, 69.64, 69.74,
5-(4-Phenoxyphenyl)-5-prop-2-ynylaminopyrimidine-2,4,6-
trione (8). For the preparation of 8, 5-(4-phenoxyphenyl)pyrimidine-
2,4,6-trione (2.96 g, 10.0 mmol) and propargylamine (640 μL, 551 mg,
10.0 mmol) were subjected to the general procedure. For workup, the
reaction mixture was poured onto ice−water and adjusted to pH 4,
using dilute hydrochloric acid (6 N). The resulting precipitate was
filtered off, stirred in diethyl ether, and dried in vacuo, giving 8 as an
off-white solid (1.73 g, 50%). Mp: 248−249 °C (dec). 1H NMR (300
MHz, DMSO-d6): δ 3.17 (t, 1H, 4J = 1.7 Hz, CCH), 3.34 (br s, 1H,
NH), 3.38−3.46 (m, 2H, NH−CH2), 6.99−7.05 (m, 4H, HAryl), 7.14−
7.20 (m, 1H, HAryl), 7.37−7.46 (m, 4H, HAryl), 11.65 (br s, 2H, NH)
ppm. 13C NMR (75.5 MHz, DMSO-d6): δ 33.54, 68.78, 75.40, 81.64,
118.31, 119.15, 124.00, 128.11, 130.18, 132.42, 149.73, 155.93, 157.29,
170.64 ppm. HRMS-ESI: calcd for C19H15N3O4Na ([M + Na]+),
372.0955; found 372.0955. The purity of 8 was determined by
analytical HPLC to be 96%, tR = 7.90 0.02 min (n = 3).
2
73.95, 115.17 (d, J = 21.6 Hz), 118.07, 119.29, 124.07, 129.65, 129.69,
129.82 (d, 3J = 9.0 Hz), 130.19, 130.87 (d, 4J = 2.8 Hz), 149.44, 155.81,
1
157.39, 163.82 (d, J = 248 Hz), 165.18, 169.98 ppm. 19F NMR (282
MHz, DMSO-d6): δ −109.64 ppm. HRMS-ESI: calcd for C35H40FN5O8
([M + H]+), 678.2934; found 678.2935. Anal. Calcd for C35H40FN5O8: C
62.03, H 5.95, N 10.33. Found: C 61.63, H 5.85, N 10.31
General Procedure for the Preparation of Triazoles 28−36. To
a solution of the acetylene compound (0.5−1.0 mmol, 1 equiv) in
DMF (8 mL·mmol−1) were added aqueous CuSO4·5H2O (30−50 mol %),
aqueous sodium ascorbate (40−60 mol %), and the corresponding
azide (0.5−1.2 mmol, 1.0−1.2 equiv) in sequence. After the mixture
was stirred at room temperature, the solvents were removed and the
residue was purified by silica gel chromatography (EtOAc + 2% TEA →
5-(4-Phenoxyphenyl)-5-{4-[2-(2-{2-[2-(2-{2-[2-(2-prop-2-ynyloxy-
ethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethyl]piper-
azin-1-yl}pyrimidine-2,4,6-trione (10). For the preparation of 10,
5-(4-phenoxyphenyl)pyrimidine-2,4,6-trione (593 mg, 2 mmol) and 5
(1.18 g, 2 mmol) were subjected to the general procedure. For
workup, the reaction mixture was diluted with EtOAc, followed by
addition of aqueous citric acid (0.1 mmol mL−1). After separation of
the organic phase, the aqueous phase was extracted with EtOAc (2×).
The combined organic extracts were washed with water, dried
(MgSO4), filtered, and concentrated. The residue was purified by
silica gel chromatography (EtOAc/MeOH, 4/1) to afford 10 as a waxy
oil (1.23 g, 80%). 1H NMR (400 MHz, CDCl3): δ 2.43 (t, 1H, 4J = 2.4
Hz, CCH), 2.74−2.81 (m, 10H, N−CH2), 3.56−3.69 (m, 30H,
1
EtOAc/MeOH (4/1) + 2% TEA). H, 13C, and 19F NMR data of
triazoles 28−36 are listed in the Supporting Information.
5-{[1-(2-{2-[2-(2-Fluoroethoxy)ethoxy]ethoxy}ethyl)-1H-
1,2,3-triazol-4-ylmethyl]amino}-5-(4-phenoxyphenyl)-
pyrimidine-2,4,6-trione (28). For the preparation of 28, 8 (349 mg,
1 mmol), CuSO4·5H2O (75 mg, 0.3 mmol, 30 mol %, in 1 mL of H2O),
sodium ascorbate (79 mg, 0.4 mmol, 40 mol %, in 1 mL of H2O), and 21
(221 mg, 1 mmol) were subjected to the general procedure. After the
mixture was stirred for 2.5 h, the reaction yielded 28 as an off-white foam
(413 mg, 72%). Mp: 117 °C. HRMS-ESI: calcd for C27H31FN6O7 ([M +
Na]+), 593.2130; found 593.2138. Anal. Calcd for C27H31FN6O7: C
56.84, H 5.48, N 14.73. Found: C 56.47, H 5.43, N 14.53.
4
CH2), 4.18 (d, 2H, J = 2.4 Hz, CH2−CCH), 6.94−6.96 (m, 2H,
HAryl), 7.00−7.02 (m, 2H, HAryl), 7.11−7.16 (m, 1H, HAryl), 7.32−7.36
(m, 2H, HAryl), 7.46−7.48 (m, 2H, HAryl). 13C NMR (101 MHz,
CDCl3): δ 46.95, 53.64, 57.40, 58.49, 67.54, 69.18, 70.41, 70.43, 70.47,
70.50, 70.51, 70.53, 70.56, 70.58, 70.59, 70.60, 70.64, 74.31, 74.76,
79.78, 118.36, 119.66, 124.05, 128.83, 130.01, 130.12, 149.40, 156.40,
158.38, 169.70 ppm. HRMS-ESI: calcd for C39H54N4O12 ([M + H]+),
771.3811; found 771.3809. The purity of 10 was determined by
analytical HPLC to be 98%, tR = 8.12 0.01 min (n = 3).
5-({1-[2-(2-{2-[2-(2-Fluoropyridin-3-yloxy)ethoxy]ethoxy}-
ethoxy)ethyl]-1H-1,2,3-triazol-4-ylmethyl}amino)-5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione (29). For the prepara-
tion of 29, 8 (349 mg, 1 mmol), CuSO4·5H2O (100 mg, 0.4 mmol,
40 mol %, in 1 mL of H2O), and sodium ascorbate (99 mg, 0.5 mmol,
50 mol %, in 1 mL of H2O) and 23 (314 mg, 1 mmol) were subjected
to the general procedure. After the mixture was stirred for 23 h, the
reaction yielded 29 as a light yellow solid (438 mg, 66%). Mp: 104 °C.
HRMS-ESI: calcd for C32H34FN7O8Na ([M + Na]+), 686.2345; found
686.2346. The purity of 29 was determined by analytical HPLC to be
96%, tR = 8.30 0.03 min (n = 3).
5-(4-{2-[2-(2-{2-[1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-
ylmethoxy]ethoxy}ethoxy)ethoxy]ethyl}piperazin-1-yl)-5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione (30). For the prepara-
tion of 30, 9 (595 mg, 1 mmol), CuSO4·5H2O (75 mg, 0.3 mmol,
30 mol %, in 1 mL of H2O), sodium ascorbate (79 mg, 0.4 mmol,
40 mol %, in 1 mL of H2O), and a solution of 24 (107 mg, 1.2 mmol)
were subjected to the general procedure. After stirring for 72 h, the
reaction yielded 30 as a white solid (440 mg, 64%). Mp: 118 °C.
HRMS-ESI: calcd for C33H42FN7O8 ([M + H]+), 684.3152; found
684.3168. Anal. Calcd for C33H42FN7O8: C 57.97, H 6.19, N 14.34.
Found: C 57.55, H 6.18, N 14.04.
5-[4-(2-{2-[2-(2-{2-[2-(2-{2-[1-(2-Fluoroethyl)-1H-1,2,3-tria-
zol-4-ylmethoxy]ethoxy}ethoxy)-ethoxy]ethoxy}ethoxy)-
ethoxy]ethoxy}ethyl)piperazin-1-yl]-5-(4-phenoxyphenyl)-
pyrimidine-2,4,6-trione (31). For the preparation of 31, 10 (385 mg,
0.5 mmol), CuSO4·5H2O (37 mg, 0.15 mmol, 30 mol %, in 0.5 mL of
H2O), sodium ascorbate (40 mg, 0.2 mmol, 40 mol %, in 0.5 mL of H2O)
and a solution of 24 (53 mg, 0.6 mmol) were subjected to the general
procedure. After the mixture was stirred for 24 h, the reaction yielded 31
as a colorless oil (152 mg, 35%). HRMS-ESI: calcd for C41H59FN7O12
([M + H]+), 860.4200; found 860.4182. The purity of 31 was determined
by analytical HPLC to be 95%, tR = 7.62 0.01 min (n = 3).
5-[4-(6-Fluoropyridin-2-yl)piperazin-1-yl]-5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione (13). For the prepara-
tion of 13, 5-(4-phenoxyphenyl)pyrimidine-2,4,6-trione (741 mg,
2.50 mmol) and 7 (736 mg, 2.50 mmol) were subjected to the general
procedure. For workup, the reaction mixture was diluted with EtOAc,
followed by an addition of aqueous citric acid (0.1 mmol mL−1). After
separation of the organic phase, the aqueous phase was extracted with
EtOAc (2×). The combined organic extracts were washed with water,
dried (MgSO4), filtered, and concentrated. The residue was purified by
silica gel chromatography (CH/EtOAc, 2/1) and the concentrated
eluent was subsequently stirred in diethyl ether to afford 13 as a white
solid (812 mg, 68%). Mp: 235 °C. 1H NMR (600 MHz, DMSO-d6): δ
2.67−2.69 (m, 4H, N−CH2), 3.45−3.47 (m, 4H, N−CH2), 6.26 (dd,
1H, 3J = 7.7 Hz, 4J = 2.8 Hz, HPyridinyl), 6.63 (dd, 1H, 3J = 8.3 Hz, 4J =
2.5 Hz, HPyridinyl), 7.03−7.09 (m, 4H, HAryl), 7.17−7.20 (m, 1H, HAryl),
7.40−7.47 (m, 4H, HAryl), 7.60−7.69 (m, 1H, HPyridinyl), 11.66 (br s,
2H, NH) ppm. 13C NMR (75.5 MHz, DMSO-d6): δ 45.29, 47.29,
2
4
74.17, 95.34 (d, J = 37 Hz), 103.48 (d, J = 3.2 Hz), 118.20, 119.40,
124.17, 129.44, 129.67, 130.23, 142.62 (d, 3J = 8.0 Hz), 149.41,
3
1
155.74, 157.60, 157.88 (d, J = 15.9 Hz), 162.02 (d, J = 233 Hz),
169.98 ppm. 19F NMR (282 MHz, DMSO-d6): δ −68.70 ppm. HRMS-
ESI: calcd for C25H22FN5O4 ([M + H]+), 476.1729; found 476.1724.
Anal. Calcd for C25H22FN5O4: C 63.15, H 4.66, N 14.73. Found: C
62.96, H 4.80, N 14.46.
4-Fluoro-N-(2-{2-[2-(2-{4-[2,4,6-trioxo-5-(4-phenoxyphenyl)-
hexahydropyrimidin-5-yl]piperazin-1-yl}ethoxy)ethoxy]-
ethoxy}ethyl)benzamide (14). TEA (0.1 mL, 0.72 mmol) was
added to a solution of 12 (150 mg, 0.27 mmol) in CH2Cl2 (4 mL),
followed by N-succinimidyl-4-fluorobenzoate (70 mg, 0.30 mmol).
5-[4-(2-{2-[2-(2-{1-[2-(2-{2-[2-(2-Fluoropyridin-3-yloxy)-
ethoxy]ethoxy}ethoxy)ethyl]-1H-1,2,3-triazol-4-ylmethoxy}-
ethoxy)ethoxy]ethoxy}ethyl)piperazin-1-yl]-5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione (32). For the preparation
229
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
of 32, 9 (297 mg, 0.5 mmol), CuSO4·5H2O (37 mg, 0.15 mmol, 30 mol %,
in 0.5 mL of H2O), sodium ascorbate (40 mg, 0.2 mmol, 40 mol %), and
23 (157 mg, 0.5 mmol) were subjected to the general procedure. After
the mixture was stirred for 2.5 h, the reaction yielded 32 as a colorless oil
(284 mg, 62%). HRMS-ESI: calcd for C44H57FN8O12 ([M + H]+),
909.4153; found 909.4158. The purity of 32 was determined by analytical
HPLC to be 95%, tR = 8.23 0.01 min (n = 3).
4-Fluorobenzoic Acid 1-(2-{2-[2-(2-{4-[2,4,6-Trioxo-5-(4-
phenoxyphenyl)hexahydropyrimidin-5-yl]piperazin-1-yl}-
ethoxy)ethoxy]ethoxy}ethyl)-1H-1,2,3-triazol-4-ylmethyl Ester
(33). For the preparation of 33, 11 (582 mg, 1 mmol), CuSO4·5H2O
(100 mg, 0.4 mmol, 40 mol %, in 1 mL of H2O), sodium ascorbate
(99 mg, 0.5 mmol, 50 mol %), and 25 (178 mg, 1 mmol) were
subjected to the general procedure. After the mixture was stirred for 21
h, the reaction yielded 33 as a white foam (464 mg, 61%). Mp: 74 °C.
HRMS-ESI: calcd for C38H42FN7O9 ([M + H]+), 760.3101; found
760.3091. The purity of 33 was determined by analytical HPLC to be
99%, tR = 8.63 0.03 min (n = 3).
4-Fluoro-N-[1-(2-{2-[2-(2-{4-[2,4,6-trioxo-5-(4-
phenoxyphenyl)hexahydropyrimidin-5-yl]piperazin-1-yl}-
ethoxy)ethoxy]ethoxy}ethyl)-1H-1,2,3-triazol-4-ylmethyl]-
benzenesulfonamide (34). For the preparation of 34, 11 (349 mg,
0.6 mmol), CuSO4·5H2O (45 mg, 0.18 mmol, 30 mol %, in 0.5 mL
of H2O), sodium ascorbate (48 mg, 0.24 mmol, 40 mol %), and 26a
(128 mg, 0.6 mmol) were subjected to the general procedure. After the
mixture was stirred for 18 h, the reaction yielded 34 as a colorless oil
(128 mg, 27%). HRMS-ESI: calcd for C37H43FN8O9S ([M + H]+),
795.2931; found 795.2939. The purity of 34 was determined by
analytical HPLC to be 97%, tR = 8.01 0.01 min (n = 3).
4-Fluoro-N-methyl-N-[1-(2-{2-[2-(2-{4-[2,4,6-trioxo-5-(4-
phenoxyphenyl)hexahydropyrimidin-5-yl]piperazin-1-yl}-
ethoxy)ethoxy]ethoxy}ethyl)-1H-1,2,3-triazol-4-ylmethyl]-
benzenesulfonamide (35). For the preparation of 35, 11 (349 mg,
0.6 mmol), CuSO4·5H2O (45 mg, 0.18 mmol, 30 mol %, in 0.5 mL
of H2O), sodium ascorbate (48 mg, 0.24 mmol, 40 mol %), and 26b
(136 mg, 0.6 mmol) were subjected to the general procedure. After the
mixture was stirred for 7.5 h, the reaction yielded 35 as a colorless oil
(148 mg, 30%). HRMS-ESI: calcd for C38H45FN8O9S ([M + H]+),
809.3087; found 809.3064. The purity of 35 was determined by
analytical HPLC to be 98%, tR = 8.51 0.01 min (n = 3).
5-[4-(2-{2-[2-(2-{4-[4-(2-Fluoropyridin-3-yloxy)butyl]-1,2,3-
triazol-1-yl}ethoxy)ethoxy]ethoxy}ethyl)piperazin-1-yl]-5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione (36). For the prepara-
tion of 36, 11 (291 mg, 0.5 mmol), CuSO4·5H2O (62 mg, 0.25 mmol,
50 mol %, in 0.5 mL H2O), sodium ascorbate (59 mg, 0.3 mmol,
60 mol %), and 27 (106 mg, 0.55 mmol) were subjected to the general
procedure. After the mixture was stirred for 5 h, the reaction yielded
36 as a light yellow wax (236 mg, 61%). HRMS-ESI: calcd for
C39H47FN8O8 ([M + H]+), 775.3574; found 775.3565. The purity
of 36 was determined by analytical HPLC to be 99%, tR = 8.30
0.02 min (n = 3).
Radiochemistry. General Methods. Radiofluorinations were
carried out on a modified PET tracer radiosynthesizer (TRACERLab
FxFDG, GE Healthcare). The recorded data were processed by the
TRACERLab Fx software (GE Healthcare). Separation and purifi-
cation of the radiosynthesized compounds were performed on the
following semipreparative radio-HPLC system: two K-1800 pumps
and S-2500 UV detector (Knauer), GabiStar γ-detector (Raytest
QMA cartridge, preconditioned with 5 mL of 1 M K2CO3 and 10 mL
of water).
5-(4-{2-[2-(2-{2-[1-(2-[18F]Fluoroethyl)-1H-1,2,3-triazol-4-
ylmethoxy]ethoxy}ethoxy)ethoxy]ethyl}piperazin-1-yl)-5-(4-
phenoxyphenyl)pyrimidine-2,4,6-trione ([18F]30). No-carrier-
added [18F]fluoride (5.3−10.2 GBq) was eluted from a QMA-cartridge
with a solution of Kryptofix 2.2.2 (18 mg) and K2CO3 (40 μL, 1 M) in
acetonitrile/water (8:2, 1 mL). The aqueous solution was evaporated to
dryness in vacuo. According to the procedure described by Glaser and
Årstad,32 2-[18F]fluoroethylazide was prepared by addition of 2-
azidoethyl-4-methylbenzenesulfonate31 (6 μL, 30 μmol) in anhydrous
acetonitrile (0.3 mL) to the carefully dried [18F]fluoride (potassium 222
cryptate) residue and subsequent reaction occurred at 84 °C for 15 min.
After addition of acetonitrile (0.2 mL), 2-[18F]fluoroethylazide was
distilled at 120 °C under a flow of helium into a 5 mL flask containing
DMF (0.5 mL) and cooled to 0 °C. The radiolabeling agent was
collected with a radiochemical yield of 68 8% (mean SD, decay
corrected, n = 6). A solution of copper(II) sulfate pentahydrate (60 μL,
0.4 M), a solution of sodium ascorbate (50 μL, 0.6 M), water (600 μL),
and a solution of 9 (7 mg, 12 μmol) in DMF (100 μL) were added. After
8 min at room temperature, the mixture was passed through a Waters
Sep-Pak Light C18 cartridge filled with quartz wool. The cartridge was
rinsed with DMF (0.3 mL), and the resulting mixture was evaporated to
dryness in vacuo, followed by dissolving in acetonitrile/water (3:7, 1 mL,
0.1% TFA). An amount of 200 μL was fractionized by semipreparative
radio-HPLC (conditions: λ = 254 nm; flow = 5.5 mL·min−1; 33%
CH3CN in water (0.1% TFA) over 25 min). The product fraction of
compound [18F]30 was evaporated to dryness in vacuo and formulated
in saline/ethanol (10:1, 0.5 mL) for further experimental use. The
radioligand [18F]30 was obtained in an overall radiochemical yield of
43 9% (mean SD, decay-corrected, n = 6) in 140 min from end of
radionuclide production. Radiochemical purities (>97%, tR = 18.0 min)
and specific radioactivities (11−20 GBq·μmol−1) were determined by
analytical radio-HPLC (conditions: λ = 254 nm; flow = 0.3 mL·min−1;
33% CH3CN in water (0.1% TFA)).
In Vitro Enzyme Inhibition Assays (Table 1). The inhibition
potencies of barbituric acid derivatives 13, 14, and 28−36 against
activated MMP-2, -8, -9, and -13 were assayed using the synthetic
fluorgenic 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.30 In short, 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
wavelength 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 Partition Coefficient (log D7.4). The lip-
ophilicity of radioligand [18F]30 was assessed by determination of the
water−octanol partition coefficient following a published procedure.37
In brief, approximately 20 kBq [18F]30 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.
Isotopenmessgerate GmbH) and an ACE-126-2510 column (250 mm ×
̈
10 mm). The recorded data were processed by the ChromGate HPLC
software (Knauer). Radiochemical purities and specific activities were
determined using an analytical radio-HPLC system composed of a
Syknm S1021 pump, a Knauer K-2501 UV detector, a Crismatec
NaI(TI) Scintibloc 51 SP51 γ-detector, a RP-HPLC Nucleosil 100-3
C-18 column (250 mm × 3 mm), and the GINA Star software
(Raytest Isotopenmessgerate GmbH). 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. To
recover the 18O-water, the batch of aqueous [18F]fluoride was passed
through an anion exchange resin (Sep-Pak Light Waters Accell Plus
Stability in Human Serum. The serum stability of radioligand
[18F]30 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
230
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
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 =
6.5 min; analytical HPLC system II, 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,
1.5 mL·min−1).
emission computed tomography; TEA, triethylamine; TEG,
tetraethylene glycol; TFA, trifluoroacetic acid; THF, tetrahy-
drofuran; TIMP, tissue inhibitor of matrix metalloproteinases;
TLC, thin layer chromatography; VOI, volume of interest
REFERENCES
■
(1) Folgueras, A. R.; Pendas, A. M.; Sanchez, L. M.; Lop
Matrix metalloproteinases in cancer: from new functions to improved
́
ez-Otín, C.
In Vivo PET Studies. PET experiments were carried out using the
high resolution (0.7 mm full width at half-maximum) quadHIDAC
small-animal PET scanner (Oxford Positron Systems, Weston-on-the-
Green, U.K.).38 Balb/c mice (19−27 g) were anesthetized with
isoflurane (1.5%/0.3 L min−1) and placed on a heating pad to maintain
body temperature for insertion of tail vein catheters (26G, BD
VasculonPlus). The radiotracer [18F]30 (5−8 MBq in 100 μL, 150 μL
saline flush) was injected 30 s after the start of acquisition. List-mode
data were acquired for 60 min and reconstructed into dynamic time
frames using an iterative resolution recovery reconstruction algorithm.
Subsequently, the scanning bed was transferred to the 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, U.S.). Three-dimensional 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).
inhibition strategies. Int. J. Dev. Biol. 2004, 48, 411−424.
́
(2) Overall, C. M.; Lopez-Otín, C. Strategies for MMP inhibition in
cancer: innovations for the post-trial era. Nat. Rev. Cancer 2002, 2,
657−672.
(3) Wagner, S; Breyholz, H.-J.; Faust, A.; Holtke, C.; Levkau, B.;
̈
Schober, O.; Schafers, M; Kopka, K. Molecular imaging of matrix
̈
metalloproteinases in vivo using small molecule inhibitors for SPECT
and PET. Curr. Med. Chem. 2006, 13, 2819−2838.
(4) Borkakoti, N. Structural studies of matrix metalloproteinases.
J. Mol. Med. 2000, 78, 261−268.
(5) Stamenkovic, I. Extracellular matrix remodelling: the role of
matrix metalloproteinases. J. Pathol. 2003, 200, 448−464.
(6) Rhee, J. S.; Coussens, L. M. ECKing MMP function: implications
for cancer development. Trends Cell Biol. 2002, 12, 209−211.
(7) Vihinen, P.; Ala-aho, R.; Kahari, V. M. Matrix metalloproteinases
as therapeutic targets in cancer. Curr. Cancer Drug Targets 2005, 5,
203−220.
(8) Newby, A. C. Dual role of matrix metalloproteinases (matrixins)
in intimal thickening and atherosclerotic plaque rupture. Physiol. Rev.
2005, 85, 1−31.
(9) Tekabe, Y.; Li, Q.; Luma, J.; Weisenberger, D.; Sedlar, M.; Harja,
E.; Narula, J.; Johnson, L. L. Noninvasive monitoring the biology of
atherosclerotic plaque development with radiolabeled annexin V and
matrix metalloproteinase inhibitor in spontaneous atherosclerotic
mice. J. Nucl. Cardiol. 2010, 17, 1073−1081.
(10) Razavian, M.; Zhang, J.; Nie, L.; Tavakoli, S.; Razavian, N.;
Dobrucki, L. W.; Sinusas, A. J.; Edwards, D. S.; Azure, M.; Sadeghi,
M. M. Molecular imaging of matrix metalloproteinase activation to
predict murine aneurysm expansion in vivo. J. Nucl. Med. 2010, 51,
1107−1115.
(11) Zhang, J.; Nie, L.; Razavian, M.; Ahmed, M.; Dobrucki, L. W.;
Asadi, A.; Edwards, D. S.; Azure, M.; Sinusas, A. J.; Sadeghi, M. M.
Molecular imaging of activated matrix metalloproteinases in vascular
remodeling. Circulation 2008, 118, 1953−1960.
(12) Zheng, Q. H.; Fei, X.; Liu, X.; Wang, J. Q.; Stone, K. L.;
Martinez, T. D.; Gay, D. J.; Baity, W. L.; Miller, K. D.; Sledge, G. W.;
Hutchins, G. D. Comparative studies of potential cancer biomarkers
carbon-11 labeled MMP inhibitors (S)-2-(4′-[11C]methoxybiphenyl-4-
sulfonylamino)-3-methylbutyric acid and N-hydroxy-(R)-2-{[(4′-
[11C]methoxyphenyl)sulfonyl]benzylamino}-3-methylbutanamide.
Nucl. Med. Biol. 2004, 31, 77−85.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and analytical data for compounds 5,
1
16−20, 22, 23 and H, 13C, and 19F NMR data of compounds
28−36. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +492518347351. Fax: +498325147363. E-mail: kopka@
uni-muenster.de.
■
ACKNOWLEDGMENTS
The authors thank Anne Kanzog, Christine Batza, Roman
Priebe, Wiebke Gottschlich, Katrin Reckmann, and Marlena
Brink for technical support and the staff members of the
Organic Chemistry Institute, University of Munster, Germany,
■
̈
̈
for spectroscopic and analytical investigations. These inves-
tigations were supported by the Interdisciplinary Centre of
(13) 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.
(14) Maquoi, E.; Sounni, N. E.; Devy, L.; Olivier, F.; Frankenne, F.;
Clinical Research (IZKF) Munster, Germany (Project Scha2/
̈
̈
020/09), the Deutsche Forschungsgemeinschaft (DFG),
Collaborative Research Center ((Sonderforschungsbereich)
656 “Molecular Cardiovascular Imaging” Projects A2, B1, and
Z5), University of Munster, Germany, and Siemens Medical
̈
Krell, H.-W.; Grams, F.; Foidart, J.-M.; Noel, A. Anti-invasive,
̈
Solutions (Molecular Imaging).
antitumoral, and antiangiogenic efficacy of a pyrimidine-2,4,6-trione
derivative, an orally active and selective matrix metalloproteinase
inhibitor. Clin. Cancer Res. 2004, 10, 4038−4047.
ABBREVIATIONS USED
■
(15) Breyholz, H.-J.; Schafers, M.; Wagner, S.; Holtke, C.; Faust, A.;
̈
̈
CH, cyclohexane; CT, computed tomography; DMF, dimethylfor-
mamide; ECM, extracellular matrix; MMP, matrix metalloprotei-
nase; MMPI, matrix metalloproteinase inhibitor; MT-MMP
membrane-type matrix metalloproteinase; NBS, N-bromosuccini-
mide; PBS, phosphate buffered saline; PEG, polyethylene
glycol; PET, positron emission tomography; RECK, reversion-
inducing-cysteine-rich protein with kazal motifs; ROI, region of
interest; SD, standard deviation; SPECT, single photon
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.
(16) 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
231
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232

Journal of Medicinal Chemistry
Article
the molecular imaging of activated MMPs with PET. J. Med. Chem.
2007, 50, 5752−5764.
(34) Ramenda, T.; Kniess, T.; Bergmann, R.; Steinbach, J.; Wuest, F.
Radiolabelling of proteins with fluorine-18 via click chemistry. Chem.
Commun. 2009, 48, 7521−7523.
(17) Kopka, K.; Breyholz, H.-J.; Wagner, S.; Law, M. P.; Riemann, B.;
(35) Prante, O.; Tietze, R.; Hocke, C.; Lober, S.; Hubner, H.;
Schroer, S.; Trub, M.; Guilbert, B.; Levkau, B.; Schober, O.; Schafers,
̈
̈
̈
̈
Kuwert, T.; Gmeiner, P. Synthejkjhmjhvhmnhmsis, radiofluorination
and in vitro evaluation of pyrazolo[1,5-a]pyridine based dopamine D4
receptor ligands: discovery of an inverse agonist radioligand for PET. J.
Med. Chem. 2008, 51, 1800−1810.
M. Synthesis and preliminary biological evaluation of new
radioiodinated MMP inhibitors for imaging MMP activity in vivo.
Nucl. Med. Biol. 2004, 31, 257−267.
(18) Schafers, M.; Riemann, B.; Kopka, K.; Breyholz, H.-J.; Wagner,
̈
́
(36) Inkster, J. A. H.; Guerin, B.; Ruth, T. J.; Adam, M. J.
S.; Schafers, K. P.; Law, M. P.; Schober, O.; Levkau, B. Scintigraphic
̈
Radiosynthesis and bioconjugation of [18F]FPy5yne, a prosthetic
group for the 18F labeling of bioactive peptides. J. Labelled Compd.
Radiopharm. 2008, 51, 444−452.
imaging of matrix metalloproteinase activity in the arterial wall in vivo.
Circulation 2004, 109, 2554−2559.
(19) Brandstetter, H.; Grams, F.; Glitz, D.; Lang, A.; Huber, R.;
́
(37) Prante, O.; Hocke, C.; Lober, S.; Hubner, H.; Gmeiner, P.;
̈
̈
Bode, W.; Krell, H. W.; Engh, R. A. The 1.8-Å crystal structure of a
Kuwert, T. Tissue distribution of radioiodinated FAUC113:
assessment of a pyrazolo[1,5-a]pyridine-based dopamine D4
receptor radioligand candidate. Nuklearmedizin 2006, 45, 41−48.
matrix metalloproteinase-8 barbiturate inhibitor complex reveals a
peviously unobserved mechanism for collagenase substrate
recognition. J. Biol. Chem. 2001, 276, 17405−17412.
(38) Schafers, K. P.; Reader, A. J.; Kriens, M.; Knoess, C.; Schober,
̈
(20) Tochowicz, A.; Maskos, K.; Huber, R.; Oltenfreiter, R.; Dive, V;
Yiotakis, A.; Zanda, M.; Bode, W.; Goettig, P. Crystal structures of
MMP-9 complexes with five inhibitors: contribution of the flexible
Arg424 side-chain to selectivity. J. Mol. Biol. 2007, 371, 989−1006.
O.; Schafers, M. Performance evaluation of the 32-module
̈
quadHIDAC small-animal PET scanner. J. Nucl. Med. 2005, 46,
996−1004.
(21) 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.
(22) Zang, W.; Kung, M. P.; Oya, S.; Hou, C.; Kung, H. F.
18F-labeled styrylpyridines as PET agents for amyloid plaque imaging.
Nucl. Med. Biol. 2007, 34, 89−97.
(23) 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.
(24) 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.
(25) Faust, A.; Waschkau, B.; Waldeck, J.; Holtke, C.; Breyholz, H.-J.;
̈
Wagner, S.; Kopka, K.; Heindel, W.; Schafers, M.; Bremer, C. Synthesis
̈
and evaluation of a novel fluorescent photoprobe for imaging matrix
metalloproteinases. Bioconjugate Chem. 2008, 19, 1001−1008.
(26) Hutchings, S.; Liu, W.; Radinov, R. Palladium-catalyzed arylation
of diisopropyl malonate applied to the efficient synthesis of the selective
MMP inhibitor 5-(4-phenoxyphenyl)-5-[4-(2-pyrimidinyl)-1-
piperazinyl]barbituric acid. Heterocycles 2006, 67, 763−768.
(27) Tahtaoui, C.; Parrot, I.; Klotz, P.; Guillier, F.; Galzi, J.-L.;
Hilbert, M.; Ilien, B. Fluorescent pirenzepine derivatives as potential
bitopic ligands of the human M1 muscarinic receptor. J. Med. Chem.
2004, 47, 4300−4315.
(28) Vaidyanathan, G.; Zalutsky, M. R. Labeling proteins with
fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate. Nucl. Med.
Biol. 1992, 19, 275−281.
(29) Rostovtsev, V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: copper(I)-catalyzed
regioselectoive “ligation” of azides and terminal alkynes. Angew. Chem.
2002, 114, 2708−2711; Angew. Chem., Int. Ed. 2002, 41, 2596−2599.
(30) 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.
(31) Demko, Z. P.; Sharpless, K. B. An intramolecular [2+3]
cycloaddition route to fused 5-heterosubstituted tetrazoles. Org. Lett.
2001, 3, 4091−4094.
(32) Glaser, M.; Arstad, E. “Click labeling” with 2-
[18F]fluoroethylazide for positron emission tomography. Bioconjugate
Chem. 2007, 18, 989−993.
(33) Vaidyanathan, G.; White, B. J.; Zalutsky, M. R. Propargyl 4-
[18F]fluorobenzoate: a putatively more stable prosthetic group for the
fluorine-18 labeling of biomolecules via click chemistry. Curr.
Radiopharm. 2009, 2, 63−74.
232
dx.doi.org/10.1021/jm201142w | J. Med. Chem. 2012, 55, 223−232