C-5-disubstituted barbiturates as potential molecular probes for noninvasive matrix metalloproteinase imaging

Article abstract of DOI:10.1021/jm049145x

Studies have demonstrated a positive correlation between inflammation, metastasis, or atherosclerosis and the unbalanced or culminated expression of matrix metalloproteinases (MMPs). The molecular imaging of locally upregulated MMP activity in vivo is a c

Full text of DOI:10.1021/jm049145x

3400
J. Med. Chem. 2005, 48, 3400-3409
C-5-Disubstituted Barbiturates as Potential Molecular Probes for Noninvasive
Matrix Metalloproteinase Imaging
Hans-Jo¨rg Breyholz,*,† Michael Scha¨fers,† Stefan Wagner,† Carsten Ho¨ltke,† Andreas Faust,† Helmut Rabeneck,‡
Bodo Levkau,§ Otmar Schober,† and Klaus Kopka†
Department of Nuclear Medicine, University Hospital of the Westfa¨lische Wilhelms-Universita¨t, Mu¨nster, Germany, Insitute of
Inorganic and Analytical Chemistry, Westfa¨lische Wilhelms-Universita¨t, Mu¨nster, Germany, and Institute of Pathophysiology,
Center of Internal Medicine, University Hospital, Essen, Germany
Received October 26, 2004
Studies have demonstrated a positive correlation between inflammation, metastasis, or
atherosclerosis and the unbalanced or culminated expression of matrix metalloproteinases
(MMPs). The molecular imaging of locally upregulated MMP activity in vivo is a clinical
challenge. Actually, radioligands based on nonpeptidyl MMP inhibitors (MMPIs) are currently
in development as putative radiopharmaceutical agents for the noninvasive in vivo assessment
of activated MMPs. Nonpeptidyl MMPIs bind to the zinc active site of the activated enzyme
via mono- (e.g. carboxylate) or bidentate (e.g. hydroxamate) complexation thereby exhibiting a
broad-spectrum MMP binding potency. Thus, these mentioned endopeptidase inhibitors should
be useable lead compounds for the redevelopment as diagnostic MMPI radiotracers. Recently,
the non-hydroxamate C-5-disubstituted pyrimidine-2,4,6-triones were disclosed as subgroup-
selective MMP inhibitors. We here describe a set of fine-tuned barbiturates as a new class of
MMPI radiotracers for the noninvasive in vivo visualization of activated MMPs using
scintigraphic techniques such as SPECT or PET.
Introduction
also known as ‘deep pocket’ MMPIs. Nonpeptidyl MMPI
classes with a zinc chelating functionality (e.g. carboxyl-
ic or hydroxamic acids) have previously been de-
scribed.9,10
The zinc- and calcium-dependent matrix metallopro-
teinases (MMPs) represent a subfamily of the metzincin
superfamily and have been implicated in the proteolytic
degradation of extracellular matrix (ECM) components.
To date more than 20 human MMPs have been char-
acterized. On the basis of their specificity, the MMPs
are classified into collagenases, gelatinases, stromely-
sins, and matrilysins. Another subclass of MMPs is
represented by the membrane-type MMPs (MT-MMPs)
that additionally contain a transmembrane and intra-
cellular domain, a membrane linker domain, or are
membrane associated.1 The MMPs are secreted as
inactive zymogens (pro-MMPs). Once activated via the
‘cysteine switch’ mechanism, a zinc active site is pro-
vided that initiates its proteolytic activity.2 MMP activ-
ity is tightly controlled by several endogenous inhibitors,
e.g. the plasma inhibitor R2-macroglobulin, four tissue
inhibitors of metalloproteinases (TIMPs),3 or the re-
cently described plasma-membrane-anchored cell sur-
face receptor RECK (reversion-inducing cysteine-rich
protein with kazal motifs).4
Recently, the radioiodinated nonpeptidyl N-sulfonyl
amino acid hydroxamate, HO-[123I]I-CGS 27023A,11
which is based on the broad-spectrum MMPI CGS
27023A,12 was evaluated by our group as a feasible
radioligand for the in vivo imaging of activated MMPs
in diseases with localized MMP overexpression.
Currently, several research groups are working on the
development of 123I-, 11C-, and 18F-labeled ligands based
on nonpeptidyl MMPIs with carboxylate or hydroxamate
moieties for zinc complexation that could function as in
vivo MMP imaging agents for the noninvasive diagnosis
of metastatic cancer.11,13-22 The nonpeptidyl MMPIs
radiolabeled so far are based on the corresponding drugs
with broad-spectrum MMP acitivity that recently have
been evaluated in clinical trials.23-24
Grams et al. identified the pyrimidine-2,4,6-triones
(barbiturates) as non-hydroxamate MMPIs that exhibit
specific activities for a subgroup of secreted MMPs
comprising the gelatinases A (MMP-2) and B (MMP-9),
as well as the membrane bound MMPs MT-1-MMP
(MMP-14) and MT-3-MMP (MMP-16).25 In contrast to
the bidentate zinc(II) binding moiety of the hydrox-
amates the pyrimidine-2,4,6-triones bindseach as an
enolic tautomersin an almost tridentate manner to the
zinc(II) active site.26,27 In the work presented here we
suggest the C-5-disubstituted pyrimidine-2,4,6-triones
as a new class of potential MMPI radiotracers with
improved MMP potency for the molecular imaging of
MMPs in vivo by means of the scintigraphic techniques
single photon emission computed tomography (SPECT)
or positron emission tomography (PET).
In pathophysiological processes an increased cytokine
and growth-factor-stimulated MMP gene transcription,
followed by a pro-MMP secretion and zymogen activa-
tion, might become prevalent, triggering the progression
of inflammation, cancer, or cardiovascular diseases.3,5-8
Numerous MMP-inhibiting strategies in experimental
models of cancer and clinical trials have led to several
synthetic nonpeptidomimetic MMP inhibitors (MMPIs),
* To whom correspondence should be addressed. Phone: +49-251-
83-47362. Fax: +49-251-83-47363. E-mail: breyholz@uni-muenster.de.
† University Hospital of the Westfa¨lische Wilhelms-Universita¨t.
‡ Westfa¨lische Wilhelms-Universita¨t.
§ Center of Internal Medicine, University Hospital.
10.1021/jm049145x CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/12/2005

Probes for Noninvasive MMP Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3401
Scheme 1. Synthesis of Barbiturate Derivatives 8a-ea
a Reagents: (a) K2CO3, DMF; (b) S8, morpholine; (c) CH3CO2H, H2O, H2SO4; (d) benzyl bromide, KOH, EtOH, H2O; (e) SOCl2, MeOH;
(f) H2SO4, MeOH; (g) benzyl trichloroacetimidate, CF3SO3H, CH2Cl2, cyclohexane; (h) NaH, dimethyl carbonate, dioxane; (i) urea, NaOEt,
EtOH; (j) thiourea, NaOEt, EtOH.
Compound 10a, 5-[4-(4-bromophenoxy)-phenyl]-5-[4-
(2-hydroxyethyl)-piperazin-1-yl]-pyrimidine-2,4,6-tri-
one, was chosen as the lead compound which is a potent
non-hydroxamate MMPI with the phenoxyphenyl resi-
due (P1′ substituent) reaching into the narrow S1′ pocket
of the MMP and the hydroxyethyl piperazine residue
(P2′ substituent) oriented to the S2′ site of the enzyme.25
The two substituents at the C-5-position of the pyrimi-
dine trione ring offer the feasibility of fine-tuning the
potency of this class of MMPIs. The gelatinase-selective
non-hydroxamate MMPI 10a was especially modified at
the C-5 position of the pyrimidine trione ring for further
evaluating the structure-activity relationships (SAR)
with regard to the gelatinases A and B to create a
potential MMPI radioligand. Thus, a first model MMPI,
radiolabeled with iodine-125, a radionuclide relevant for
in vitro and ex vivo evaluation studies, has been
successfully prepared. In the next steps potential can-
didates will be resynthesized radiochemically with the
clinically relevant radionuclides iodine-123, carbon-11,
and fluorine-18 to achieve SPECT- or PET-compatible
radiotracers for the in vivo imaging of diseases associ-
ated with locally elevated MMP-2 and MMP-9 activity
(i.e. MMP-9: vulnerable atherosclerosis;5 i.e., MMP-2
and MMP-9: cancer and metastases23).
Results
Chemistry. The pyrimidine-2,4,6-trione lead com-
pound 10a as well as the structurally modified barbi-
turate analogues 10b-p were prepared. Compounds
10a and 10c were previously described in the literature
and found to exhibit high inhibition efficiency and
selectivity for the subgroup of the membrane bound MT-
1-MMP (MMP-14) and MT-3-MMP (MMP-16), and for
the gelatinases (MMP-2 and MMP-9).25 To examine the
SAR between the potential MMPI and the enzyme, the
prepared compounds were modified in the pattern of
aromatic substitution of the P1′ residue (10b and 10c)
and also in the nature of the piperazine substituent (P2′
residue) of the pyrimidine-2,4,6-trione core (10e and
10g-p). These compounds may serve as potential
precursors (10a, 10e, 10g, 10i, 10k, 10o) of the corre-
sponding radiolabeled analogues, or as the nonradioac-
tive references (10b, 10c, 10h, 10l, 10n, 10p) of
potential radioactive counterparts.
The barbiturate derivatives 10a-d, 10g-j, and
10k-m were synthesized using an eight-step synthesis
(Scheme 1 and Scheme 2). The C-5-monosubstituted key
intermediates 8a-e (Scheme 1) were accessed over six
steps and converted into the barbiturate derivatives
10a-d, 10g-j, and 10k-m similar to the procedure
described previously.25 The pyrimidine-2,4,6-triones

3402 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Scheme 2. Synthesis of Barbiturate Derivatives 10a-pa
Breyholz et al.
a Reagents: (k) NBS, dibenzoylperoxide, CCl4; (l) HBr, Br2, H2O; (m) R′H, MeOH; (n) [125I]NaI, 2,5-dihydroxybenzoic acid, ascorbinic
acid, CuSO4‚5H2O, EtOH, H2O; (o) CBr4, PPh3, CH3CN; (p) (CH3O)2SO2, NaOH, H2O, ((CH3)2CH)2O (q) N-succinimidyl-3-(tri-n-
butylstannyl)-benzoate, DMF; (r) N-succinimidyl-3-iodobenzoate, DMF; (s) ICl, MeOH.
Table 1. MMP Inhibition of Barbiturate Derivatives 8a,
8a-d as well as 2-thioxo-dihydro-pyrimidine-4,6-dione
10a-p
8e were brominated either with N-bromosuccinimide/
IC50 (MMP-2) IC50 (MMP-9)
[nM] [nM]
MMP-9
dibenzoyl peroxide or with bromine/hydrobromic acid to
yield the bromo compounds 9a-e, that were subse-
quently reacted with the piperazine derivatives R′H
(Scheme 2) to form the final barbiturate derivatives
10a-d, 10g-j, and 10k-m. Compound 10c was con-
verted into 10f with an excess of dimethyl sulfate.
Compound 10e was prepared from 10a by heating with
carbon tetrabromide and triphenylphosphine in aceto-
nitrile. Compound 10k was converted into 10n via
iododesilylation with iodine monochloride in methanol.
Compounds 10o and 10p were prepared by reacting 10g
either with N-succinimidyl-3-(tri-n-butylstannyl)-ben-
zoate or with N-succinimidyl 3-iodobenzoate28 in reflux-
ing DMF. N-Succinimidyl-3-(tri-n-butylstannyl)-ben-
zoate was synthesized by treatment of N-succinimidyl
3-iodobenzoate with hexabutylditin and tetrakis(tri-
phenylphosphine)-palladium(0) in refluxing toluene.
compd
selectivitya log Db
8a
3700
n.d.
0.41
10a
10b
10c
10d
10e
10f
9
4
2.3
3.5
1.5
3.42
3.68
1.79
3.85
4.67
0.95
3.04
4.47
0.78
0.81
8.32
6.11
6.27
7.22
12.4
5.86
7
9
2
6
721
30
n.d.
26
1.2
inactive
5
inactive
2
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
2.5
26
0.71
3.3
2.7
0.63
0.28
8.6
26
1
10
14
85
26
1500
12
550
19
16
57
86
10
n. d.
15
n. d.
20
0.75
a IC50 (MMP-2)/IC50(MMP-9) ratio. b log D values calculated by
ACD/LogD Suite (log D ) log P at physiological pH 7.4 with
consideration of charged species).
Structure-Activity Relationships (SAR). The
potencies of the modified barbiturate analogues 10b-p
were measured with MMP-2 and MMP-9 using fluoro-
genic in vitro inhibition assays.11 The resulting MMP
inhibitions were compared to the parent compound 10a
as IC50 values, resulting from a nonlinear regression fit
of the concentration-dependent reaction rates. The
results are shown in Table 1.
Table 1 also contains the calculated log D values of
the synthesized pryrimidine-2,4,6-trione derivatives to
estimate the changes of the lipophilicities, caused by the
chemical modifications of the lead compound 10a.
Initially, the lead compounds 10a (X ) Br) and 10c
(X ) OCH3) were prepared to serve as reference

Probes for Noninvasive MMP Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3403
compounds in the enzyme assays. According to Grams
et al.25 (10a: IC50 (MMP-2) ) 2 nM, IC50 (MMP-9) )
1.2 nM; 10c: IC50 (MMP-2) ) 8 nM, IC50 (MMP-9) )1.3
nM), both compounds were very potent MMP inhibitors
(10a: IC50 (MMP-2) ) 9 nM, IC50 (MMP-9) ) 4 nM;
10c: IC50 (MMP-2) ) 9 nM, IC50 (MMP-9) ) 6 nM). The
iodo analogue 10b (X ) I) also showed low nanomolar
potencies, that were comparable to those of 10a and 10c.
The calculated lipophilicity of 10c (log D ) 1.79) is
reduced by 1.6-1.9 log units as compared to 10a (log D
) 3.42) or 10b (log D ) 3.68). The series 10a-c
possesses a moderate selectivity for MMP-9: The iodo
derivative 10b exhibited the highest MMP-9 selectivity
(IC50 (MMP-2)/IC50(MMP-9) ratio 3.5), followed by the
bromo compound 10a (IC50 (MMP-2)/IC50(MMP-9) ratio
2.3) and the methoxy derivative 10c (IC50 (MMP-2)/IC50-
(MMP-9) ratio 1.5).
substituent. All nonradioactive representatives of fea-
sible radiotracers (10l, 10n, 10p) exhibited acceptable
gelatinase potencies (range 10-86 nM, Table 1). The
exchange of the hydroxyethylpiperazine residue in 10a
for a substituted phenyl or benzoylpiperazine group is
obviously associated with a certain loss of potency, but
apart from 10k all these analogues retain a good
inhibitory potency. According to the calculated values
(log D g 5.9), 10k-n possess an elevated lipophilicity
in comparison with the other representatives in the
library. The trimethylsilyl derivative 10k (IC50 (MMP-
2)/IC50(MMP-9) ratio 2.7) and the iodo derivative 10n
(IC50 (MMP-2)/IC50(MMP-9) ratio 8.6) displayed an
increased selectivity for MMP-9, whereas the fluoro
compound 10l (IC50 (MMP-2)/IC50(MMP-9) ratio 0.63),
the nitro compound 10m (IC50 (MMP-2)/IC50(MMP-9)
ratio 0.28), and the iodo compound 10p (IC50 (MMP-2)/
IC50(MMP-9) ratio 0.75) were slightly more MMP-2
selective.
Next, the effect of exchanging the hydroxy group of
the C-5-hydroxyethylpiperazine (S2′-residue) for another
functional group was explored by keeping the 5-(4-
bromophenoxy-phenyl) substituent of 10a fixed as S1′-
residue. Following this approach, five corresponding
derivatives (10e and 10g-j) were prepared, containing
an amino (10i), carboxy (10j), and bromo group (10e)
instead of the hydroxy function. Furthermore, the
isopropylpiperazine derivative 10h without any further
functionality and the unsubstituted piperazine deriva-
tive 10g were prepared. Compared to 10a, all these
variations influenced the inihibitory effectiveness of the
respective compounds only moderately (Table 1): sub-
stitution of hydroxy for amino in 10i (IC50 (MMP-2) )
10 nM, IC50 (MMP-9) ) 14 nM) reduced the MMP-9
potency only slightly (3.5-fold compared to 10a), whereas
the MMP-2 potency was not affected. Substitution of
hydroxy for carboxy in 10j (IC50 (MMP-2) ) 85 nM, IC50
(MMP-9) ) 26 nM) reduced the potency for MMP-9 by
6-7-fold, and for MMP-2 by 9-10-fold (each compared
to 10a). Exchange of hydroxy for bromo in 10e (IC50
(MMP-2) ) 30 nM, IC50 (MMP-9) ) 26 nM) led to slight
decreases in potency for MMP-2 (ca. 3-fold) and MMP-9
(6-7-fold). Changing the hydroxyethylpiperazine moiety
in 10a for a piperazine (10g) or isopropylpiperazine
(10h) residue led to two very potent MMPIs. Actually
we found that the piperazine derivative 10g (IC50
(MMP-2) ) 5 nM, IC50 (MMP-9) ) 2 nM) was the most
potent inhibitor of both gelatinases. Compound 10g
displayed even lower IC50 values than the lead com-
pound 10a (factor ∼2 for both, MMP-2 and MMP-9). The
isopropylpiperazine compound 10h (IC50 (MMP-2) ) 26
nM, IC50 (MMP-9) ) 1 nM) was the most potent
compound with respect to MMP-9. Like 10a-c, com-
pounds 10e (IC50 (MMP-2)/IC50(MMP-9) ratio 1.2), 10g
(IC50 (MMP-2)/IC50(MMP-9) ratio 2.5) and 10j (IC50
(MMP-2)/IC50(MMP-9) ratio 3.3) displayed a moderately
elevated MMP-9 selectivity. As a main result 10h (IC50
(MMP-2)/IC50(MMP-9) ratio 26) was found to be the
most MMP-9 selective MMP inhibitor in the compound
series presented here. In contrast, the selectivity of the
amino derivative 10i seems to be slightly more shifted
toward MMP-2 (IC50 (MMP-2)/IC50(MMP-9) ratio 0.71).
Intermediate 8a is only monosubstituted with a
4-bromophenoxyphenyl residue at the C-5 position. This
compound (IC50 (MMP-2) ) 3.7 µM) displayed a ∼400-
fold decreased potency for MMP-2, compared to 10a.
Compound 10d (IC50 (MMP-2) ) 721 nM) was modified
by an exchange of the C-2 carbonyl for a C-2 thiocar-
bonyl group. This modification resulted in a 80-fold loss
in MMP-2 potency. Compound 10f represents the 1,3-
N,N-dimethylated methoxyethyl piperazine analogue of
10c. This modification resulted in a complete loss of
gelatinase potency.
Radioiodinated Model Compound. The 125I-la-
beled model radiotracer [125I]10b was obtained using no-
carrier-added [125I]NaI and the bromo precursor 10a via
a Cu(I)-assisted aromatic iododebromination technique
(Scheme 2).29,30 Compound 10b was used as the nonra-
dioactive reference compound of [125I]10b to prove its
identity. Compound [125I]10b was isolated in 49% aver-
age radiochemical yield. The chemical and radiochemi-
cal purities were >90%. The calculated specific activity
of [125I]10b was 0.146 GBq/µg (2.176 Ci/µmol) at the end-
of-synthesis (EOS).
Discussion
The 123I-, 11C-, and 18F-labeled MMPI radioligands
synthesized so far are based on nonpeptidyl MMPIs with
carboxylate or hydroxamate moieties as zinc binding
functionalities. The model tracer HO-[123I]I-CGS 27023A
which was evaluated by our group exemplifies the first
designed radiotracer with moderate lipophilicity (cal-
culated log D ) 1.60 (pH 7.4)) that specifically ac-
cumulates in atherosclerotic MMP-9-rich lesions of
Apolipoprotein E-deficient (ApoE-/-) mice.31 The specific
accumulation can be observed despite lower potencies
(experimental data: IC50(MMP-9) ) 153 nM) in com-
parison with the parent compound CGS 27023A (ex-
perimental data: IC50(MMP-9) ) 60 nM).11 The MMP-9
potency of HO-[123I]I-CGS 27023A corresponds to that
of other recently suggested MMPI radiotracers, e.g.
(2R)-3-methyl-2-[[4-[4-([11C]methoxybenzoyl)amino]ben-
zenesulfonyl]amino]butanoic acid18 (IC50(MMP-2) ) 110
nM and IC50(MMP-9) ) 200 nM), or (2R)-2-[4-(6-[18F]-
fluorohex-1-ynyl)-benzenesulfonyl-amino]-3-methylbu-
tyric acid [18F]SAV0316 (IC50(MMP-2) ) 1.9 µM). The
micromolar MMPI radiotracer [18F]SAV03 was signifi-
With the series of barbiturates 10k-p we prepared
some derivatives with a para-substituted phenylpipera-
zine residue (10k-n) or a meta-substituted benzoylpi-
perazine residue (10o and 10p) serving as the S2′

3404 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Breyholz et al.
cantly detected in Ehrlich tumor-bearing mice by whole
body autoradiographic studies.16 In contrast to the
promising in vivo results stated above, micro-PET
imaging studies using the potent hydroxamate-based
MMPI radiotracer [11C]CGS 25966 (IC50(MMP-2) ) 11
nM and IC50(MMP-9) ) 27 nM) as well as the carboxy-
late-based radioligand [11C]MSMA (IC50(MMP-2) ) 3
nM and IC50(MMP-9) ) 2.2 µM) in breast cancer
athymic mice (MDA-MB-435) was not successful.22
(MMP-2)/IC50(MMP-9) ratio 0.71). As a major finding,
the isopropyl derivative 10h exhibits a notably high
MMP-9 selectivity (IC50 (MMP-2)/IC50(MMP-9) ratio 26),
which is the highest in the whole library. The bromo
compound 10e is a precursor for a feasible radiofluori-
nated PET tracer which would result from a substitu-
tion of bromine by [18F]fluoride.35 Future work will
concentrate on preparing the fluoro analogue of 10e as
the nonradioactive reference of this putative PET ra-
diotracer.
Thus, to date no clinical application for imaging MMP
activity in vivo is available, and the need of alternative
imaging strategies is obvious. This is the aim of the
current study which was initiated to investigate pyrimi-
dine-2,4,6-triones25-27,32 as a new class of potential
MMPI radiotracer with superior MMP potency, i.e.,
characterized by IC50 values in the lower nanomolar
range. Although well-known as barbiturates (sedatives,
hypnotics, narcotics, and antiepileptic agents), the 5,5-
disubstituted pyrimidine-2,4,6,-triones presented here
exhibit neither toxicity nor sedative effects in CD-1
mice.32 To our knowledge, compounds bearing this
functionality have not yet been used to design new
SPECT or PET radiotracers for MMP imaging. Due to
their superior MMP inhibition efficiency and specifity,
barbiturate-based MMPI radiotracers may result in
successful MMP imaging in vivo. Furthermore, different
pharmacokinetic characteristics can be conducted in
comparison with the hydroxamate-based broad-spec-
trum MMPIs which additionally are known to show side
effects such as musculoskeletal syndrome and maculo-
papular rash if therapeutically applied (e.g. peptidomi-
metic batimastat (BB94) and marimastat (BB-2516),9
e.g. nonpeptidomimetic CGS 27023A33). Recently, the
structurally related pyrimidine trione derivative Ro-28-
2653, an inhibitor selective for the MMPs expressed by
tumor and/or stromal cells (e.g. MMP-1, -2, -7, -14, -15,
-16, and to a slight extent MMP-9) was found to exhibit
a strong antitumoral and antiangiogenic efficacy with-
out displaying these undesireable side effects.34 The fact
that Ro-28-2653 is able to target tumor tissues further
supports the here presented idea of using the barbitu-
rates as molecular imaging agents.
We prepared a library of 15 derivatives of the potent
lead compound 10a. Four complete pairs of precursors
with the corresponding nonradioactive references of
potential SPECT and PET radiotracers (10a/10b, 10g/
10h, 10k/10n, 10o/10p) have already been established.
The series 10a-c, representing the derivatives with the
highest potency (IC50 values < 10 nM), was found to be
slightly selective for MMP-9 ((IC50 (MMP-2)/IC50(MMP-
9) ratio 1.5-3.5). The variation of potencies by an
exchange of bromine in 10a for iodine (10b) or methoxy
(10c) at the C-5-phenoxyphenyl residue was marginal.
The series 10e, 10g-j shows a slightly decreased
inhibitory activity in comparison with the lead structure
10a (IC50 values in the range 1-85 nM). Anyhow, the
isopropyl derivative 10h is the most potent compound
in the whole library with respect to MMP-9, and
piperazine derivative 10g is the most potent compound
with respect to both gelatinases. Apart from 10i, all
other representatives in the series 10e, 10g-j feature
a slight MMP-9 selectivity. An inversion of this selectiv-
ity was ascertained for the amino derivative 10i, which
obviously shows a slight preference for MMP-2 (IC50
In the series of aryl piperazine derivatives 10k-p,
the trimethylsilyl derivative 10k and the stannyl de-
rivative 10o were synthesized as precursor compounds
of the corresponding radioiodinated analogues of 10n
(iododesilylation) and 10p (iododestannylation), respec-
tively. Compound 10l can act as the nonradioactive
reference of the corresponding radiofluorinated PET
tracer. Future work will concentrate on preparing a
suitable precursor to produce the radiofluorinated ana-
logue of 10l. As nucleophilic radiofluorination of aro-
matic systems is restricted only to strong electron-
deficient systems, 10m is not a suitable precursor. If
the nitro group of 10m is reduced to an amino function,
the introduction of [18F]fluoride should be possible by
fluoro-dediazoniation. An effective method for such a
decomposition of aromatic diazonium salts in the pres-
ence of [18F]fluoride has been described.36
Considering previously described SAR studies on
barbiturate derivates,25,32 compounds 10k-p show the
expected lower inhibitory potencies. However, all rel-
evant representatives, i.e., 10l, 10n, and 10p retain a
potency, which should be sufficient for prospective PET
or SPECT radiotracer candidates.
In the series of N-phenylpiperazine derivatives 10k-n
the gelatinase-selectivity might be influenced by the
electronic effect of the relevant substituent: compounds
substituted by a -I or -M substituent (10l and 10m)
were more selective for MMP-2. Derivatives equipped
with a +I substituent or a substituent displaying a low
negative inductive effect (10k and 10n) were more
selective for MMP-9. Besides 10h, compound 10n is the
second derivative that displays a significant shift toward
MMP-9 selectivity.
Compared to 10a, derivatives 8a and 10d show a 400-
and 80-fold decrease of potency because of loss of
tetrahedral environment at C-5, due to enolization (8a)
or poor hydrogen bond acceptor ability (10d). These
effects weaken the ligand-enzyme interaction. A com-
plete reduction of potency was achieved with compound
10f, due to removal of the amide hydrogens, that are
essential for the ligand to bind to the MMP active site.
Therefore, 10f had obviously lost all its tendency to
function as an MMP inhibitor and thus might serve as
a negative control for evaluation studies of the future
MMPI radioligands.
Significance
The aim of the study presented here is the molecular
imaging of MMP activity in vivo with a new class of
barbiturate-based MMPI radioligand. Chemical modi-
fications of the lead compound 10a resulted in six direct
precursors and six nonradioactive references of feasible
radiotracers for the in vivo imaging of locally elevated
MMP activity by means of single photon emission

Probes for Noninvasive MMP Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3405
(M+, 66), 278 (16), 277 (97), 276 (14), 275 (100), 168 (22). Anal.
(C14H11BrO2) C, H.
1-[4-(4-Iodo-phenoxy)-phenyl]-ethanone 3b. Yield: 48.8
g (144 mmol, 80%). Mp: 99-101 °C. MS (EI): m/e (intensity
%): 339 (M + H+, 13), 338 (M+, 87), 324 (16), 323 (100), 212
(20), 197 (46), 168 (9), 139 (7), 76 (5), 43 (3). Anal. (C14H11IO2)
C, H.
1-[4-(4-Methoxy-phenoxy)-phenyl]-ethanone 3c. Yield:
39.8 g (164 mmol, 91%). Mp: 58-59 °C. MS (EI): m/e
(intensity %): 242 (M+, 100), 227 (91), 199 (3), 171 (4), 113
(6), 92 (2), 43 (12). Anal. (C15H14O3) C, H.
1-[4-(4-Benzyloxy-phenoxy)-phenyl]-ethanone 3d. Yield:
42.4 g (133 mmol, 74%). Mp: 99-100 °C. MS (EI): m/e
(intensity %): 318 (M+, 81), 283 (4), 277 (97), 227 (7), 171 (6),
104 (3), 91(100). Anal. (C21H18O3) C, H.
General Procedure for the Preparation of the 2-(4-
Phenoxy-phenyl)-1-morpholin-4-yl-ethanethiones 4a-d.
A mixture of 1.0 equiv of phenoxy-phenyl-ethanone 3a-d, 2.5
equiv of sulfur and 2.0 equiv of morpholine was heated to 150
°C for 2.5 h. After being cooled in an ice bath, the mixture
was treated with ethanol for a period of 30-60 min. The
precipitated bright yellow solid was collected by suction
filtration and recrystallized from ethanol. The sufur could not
completely be removed from the products by recrystallization.
In cases, where the product possessed a low solubility in
ethanol the purification process was modified in the following
manner: The crude product was treated with ethanol and
heated to reflux. Undissolved material was dissolved or
separated by treatment with ethyl acetate followed by the
filtration of the hot suspension.
2-[4-(4-Bromo-phenoxy)-phenyl]-1-morpholin-4-yl-ethane-
thione 4a. Yield: 92% of a bright green, crystalline solid.
Mp: 89-92 °C. MS (EI): m/e (intensity %): 394 (M + H+, 20),
393 (M+, 100), 392 (M + H+, 21), 391 (M+, 93), 306 (34), 304
(35), 277 (13), 275 (13), 263 (16), 261 (18), 130 (85), 86 (27).
2-[4-(4-Iodo-phenoxy)-phenyl]-1-morpholin-4-yl-ethane-
thione 4b. Yield: 88% of a mustard yellow solid. Mp: 123-
127 °C. MS (EI): m/e (intensity %): 441 (M + 2H+, 20), 440
(M + H+, 26), 439 (M+, 100), 423 (12), 352 (24), 335 (10), 313
(11), 309 (19), 130 (30), 86 (9).
computed tomography (SPECT) or positron emission
tomography (PET).
Four complete pairs of precursors with the corre-
sponding references have been established. In fact, most
of the chemical modifications did not cause serious
decreases in the MMP binding potency. The derivatives
10b and 10g exhibit even higher potencies compared
with the lead compound 10a. A significant fine-tuning
of gelatinase-selectivity was also possible. Compounds
10b, 10h, and 10n proved to possess an increased
selectivity toward MMP-9 in comparison with the lead
compound 10a.
Finally one promising 125I-labeled candidate, [125I]10b,
has been prepared as a potential model compound of a
prospective 123I-labeled SPECT radiotracer. Further
evaluations of radiosyntheses leading to potential PET
radiotracers are currently in progress.
Using the here-introduced class of barbiturate-based
MMPI radioligands as molecular imaging probes could
evolve into an innovative tool to noninvasively image
MMP activity in vivo in pathophysiological processes
such as inflammation, cancer, or atherosclerosis.
Experimental Section
General Methods. Chemistry. All chemicals, reagents,
and solvents for the synthesis of the compounds were analyti-
cal grade, purchased from commercial sources and used
without further purification, unless otherwise specified. Melt-
ing points were determined in capillary tubes on a Stuart
Scientific SMP3 capillary melting point apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on
a Bruker ARX 300 and AMX 400 spectrometer. Two-dimen-
sional NMR spectra were determined on a Varian 600 MHz
unity plus instrument. CDCl3 contained tetramethylsilane
(TMS) as an internal standard. Mass spectra were obtained
on a Varian MAT 212 (EI ) 70 eV) spectrometer and a Bruker
MALDI-TOF-MS Reflex IV (matrix: DHB) instrument. Exact
mass analyses were conducted on a Waters Quattro LC and a
Bruker MicroTof apparatus. Elemental analysis was realized
by a Vario EL III analyzer. Separation of the radiosynthesized
compounds from unlabeled byproducts and analyses of the
radiochemical yields and the radiochemical purity were per-
formed by gradient radio-HPLC each with a RP-HPLC Nucleo-
sil column 100 C-18 5 µ 250 × 4.6 mm2, a corresponding 20 ×
4.6 mm2 precolumn, using a Knauer K-500 and a Latek P 402
pump, a Knauer K-2000 UV-detector (wavelength 254 nm),
and a Crismatec Na(Tl) Scintibloc 51 SP51 gamma detector.
Sample injection was carried out using a Rheodyne injector
block (type 7125 incl. 200 µL loop). The recorded data were
processed by the NINA radio-HPLC software (GE Functional
Imaging GmbH, Germany). The following conditions were
used: eluent A: CH3CN/H2O/TFA (950/50/1); eluent B: CH3-
CN/H2O/TFA (50/950/1); gradient time-program: eluent B from
92% to 50% within 45 min and then from 50% to 92% within
10 min; flow rate: 1.5 mL/min.
General Procedure for the Preparation of the 1-(4-
Phenoxy-phenyl)-ethanones 3a-d. 4-Fluoroacetophenone
2 (25.0 g, 181 mmol) was dissolved in 180 mL of dimethylfor-
mamide, whereupon the appropriate phenol 1a-d (181 mmol)
and 30.0 g (217 mmol) of potassium carbonate were added.
The mixture was heated to reflux for approximately 7 h,
allowed to cool to ambient temperature, and diluted with
water. After extraction with methylene chloride or chloroform
(3 × 200 mL), the combined organic layers were washed with
water and dried (Na2SO4). The solvent was removed in vacuo
to provide the crude products. These brownish oily residues
were recrystallized from hexane/ethyl acetate (7:3) to yield the
pure compounds as off-white crystalline solids.
2-[4-(4-Methoxy-phenoxy)-phenyl]-1-morpholin-4-yl-ethane-
thione 4c. Yield: 64% of an off-white solid. Mp: 103-104 °C.
MS (EI): m/e (intensity %): 343 (M+, 100), 256 (30), 213 (36),
149 (8), 86 (11).
2-[4-(4-Benzyloxy-phenoxy)-phenyl]-1-morpholin-4-yl-ethane-
thione 4d. Yield: 99% of an off-white solid. Mp: 130-132
°C. MS (EI): m/e (intensity %): 419 (M+, 63), 318 (58), 392
(21), 227 (5), 130 (17), 91 (100), 43 (5).
General Procedure for the Preparation of the (4-Phen-
oxy-phenyl)-acetic Acids 5a-e.37 Morpholinyl ethanethione
4a-d was heated with a mixture of glacial acetic acid (ca. 10
equiv), water (ca. 7 equiv), and concentrated sulfuric acid (ca.
1.5 equiv) to 150 °C for 4.5-12 h. After being cooled to ambient
temperature, the reaction mixture was diluted with water (ca.
10 mL/mmol) and extracted with ethyl acetate (3×). The
combined organic layers were washed with water and dried
(Na2SO4), and the solvent was evaporated in vacuo.
[4-(4-Bromophenoxy)-phenyl]-acetic Acid 5a. 54.1 g
(138 mmol) of 4a, 104 mL of glacial acetic acid, 24 mL of water,
and 16 mL of sulfuric acid (reaction time: 4.5 h). Yield: 38.9
g (127 mmol, 92%) of an off-white solid. Mp: 115-119 °C.
[4-(4-Iodo-phenoxy)-phenyl]-acetic Acid 5b. 26.9 g (61.1
mmol) of 4b, 54 mL of glacial acetic acid, 12 mL of water, and
8 mL of sulfuric acid (reaction time: 12 h). Yield: 20.1 g (56.8
mmol, 93%) of an off-white solid. Mp: 148-150 °C. Anal.
(C14H11IO3) C, H.
[4-(4-Methoxy-phenoxy)-phenyl]-acetic Acid 5c. 33.0 g
(96.1 mmol) of 4c, 54 mL of glacial acetic acid, 12 mL of water,
and 8 mL of sulfuric acid (reaction time: 10 h). An analytical
sample was prepared by silica gel chromatography (hexane/
ethyl acetate 7:3). Yield: 23.9 g (92.6 mmol, 96%) of a brown
oil (slightly pink solid after chromatography). Mp: 92-94 °C.
MS (EI): m/e (intensity %): 258 (M+, 100), 244 (19), 213 (53),
170 (3), 121 (2), 77 (3), 43 (2). Anal. (C15H14O4) C, H.
1-[4-(4-Bromophenoxy)-phenyl]-ethanone 3a. Yield: 38.7
g (133 mmol, 73%). Mp: 78-79 °C. MS (EI): m/e (intensity
%): 293 (M + H+, 11), 292 (M+, 67), 291 (M + H+, 11), 290

3406 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Breyholz et al.
[4-(4-Hydroxy-phenoxy)-phenyl]-acetic Acid 5d. 65.9 g
(157 mmol) of 4d, 130 mL of glacial acetic acid, 30 mL of water,
and 20 mL of sulfuric acid (reaction time: 12 h). Yield: 13.4
g (54.9 mmol, 35%) of a slightly pink solid (petroleum ether/
ethyl acetate 1:1). Mp: 182 °C. MS (EI): m/e (intensity %):
244 (M+, 87), 199 (100), 171 (8), 128 (9), 89 (10), 65 (14). Anal.
(C14H12O4) C, H.
[4-(4-Benzyloxy-phenoxy)-phenyl]-acetic Acid 5e. Po-
tassium hydroxide (6.50 g, 116 mmol) was dissolved at ambient
temperature in 105 mL of ethanol and 13 mL of water, and
5d (13.0 g, 53.2 mmol) was added in portions to the stirred
solution. Benzyl bromide (9.10 g, 53.2 mmol) was added
dropwise over a period of 30 min, and the reaction mixture
was refluxed for 14 h. The mixture was treated with 15 mL of
10% aqueous KOH and refluxed for another 2 h. The ethanol
was evaporated in vacuo, and water (300 mL) was added to
the residue. The resulting solution was washed with ether (2
× 150 mL) and acidified with 37% hydrochloric acid. The
resulting precipitate was collected by filtration, washed with
water, and dried in vacuo. The crude product was recyristal-
lised from ethanol to give 13.3 g (39.8 mmol) of a colorless solid.
Yield: 75%. Mp: 138-139 °C. MS (EI): m/e (intensity %): 334
(M+, 24), 289 (1), 243 (10), 199 (4), 141 (2), 91 (100), 18 (7).
Anal. (C21H18O4) C, H.
General Procedure for the Preparation of the (4-Phen-
oxy-phenyl)-acetic Acid Methyl Esters 6a-d. A solution
of acetic acid 5a-c in methanol (ca. 2.5 mL/mmol) was cooled
to -10 °C, thionyl chloride (2.0 equiv) was added, and the
reaction mixture was heated to reflux for 1 h. After concentra-
tion the residue was redissolved in ether. The Etheral layer
was washed with water, dried (Na2SO4) and the solvent was
evaporated to yield viscous brown-red oils of 6a-6c.
The hydroxy derivative 6d was prepared by treatment with
catalytic concentrated sulfuric acid in methanol. The benzyloxy
derivative 6e was prepared as specified below.
[4-(4-Bromophenoxy)-phenyl]-acetic Acid Methyl Es-
ter 6a. Yield: 87%. MS (EI): m/e (intensity %): 323 (M + H+,
15), 322 (M+, 80), 321 (M + H+, 14), 320 (M+, 79), 264 (13),
263 (98), 262 (14), 261 (100), 181 (9), 166 (16), 154 (13), 107
(51). Anal. (C15H13BrO3) C, H.
solution were added 500 µL of cyclohexane, 1.17 g (4.63 mmol)
of benzyl trichloroacetimidate,38 and trifluoromethanesulfonic
acid (30 µL), and stirring was continued at ambient temper-
ature overnight. The precipitated trichloroacetimidate was
filtered off, and the filtrate was subsequently washed with 15
mL of saturated aqueous sodium hydrogencarbonate followed
by 15 mL of water and 15 mL of brine and finally dried (Na2-
SO4). The organic layer was concentrated, and the residue was
recrystallized from methanol, yielding 250 mg (718 µmol) of
an off-white solid. Yield: 31%. Mp: 79-80 °C. (further
analytical data: see above).
General Procedure for the Preparation of the (4-Phen-
oxy-phenyl)-malonic Acid Dimethyl Esters 7a-d. A sus-
pension of 2.0 equiv of NaH (60% suspension in paraffin oil
(paraffin oil was removed by repeated washings with petro-
leum ether) and 6.4 equiv of dimethyl carbonate in absol
dioxane (ca. 0.5 mL/mmol) was heated to 100-120 °C, and a
solution of acetic acid methyl ester 6a-c and 6e, in absol
dioxane (1.5-2.0 mL/mmol) was added dropwise over a period
of 1 h. Refluxing was continued for 3 h, and the reaction
mixture was allowed to come to ambient temperature over-
night. The mixture was poured onto ice water and extracted
with methylene chloride (3×). The combined organic layers
were washed with water and brine, dried (Na2SO4), and
concentrated.
[4-(4-Bromo-phenoxy)-phenyl]-malonic Acid Dimethyl
Ester 7a. Yield: 83% of a viscous brown-red oil. MS (EI): m/e
(intensity %): 381 (M + H+, 16), 380 (M+, 100), 379 (M + H+,
16), 378 (M+, 85), 323 (22), 322 (40), 321 (100), 320 (39), 319
(82), 294 (4), 293 (23), 292 (4), 291 (26), 278 (8), 277 (30), 276
(9), 275 (30), 264 (5), 263 (32), 262 (5), 261 (34), 179 (10), 148
(35), 89 (15). Anal. (C17H15BrO5) C, H.
[4-(4-Iodo-phenoxy)-phenyl]-malonic Acid Dimethyl
Ester 7b. Yield: 87% of a viscous brown-red oil. MS (EI): m/e
(intensity %): 427 (M + H+, 18), 426 (M+, 100), 367 (81), 354
(11), 339 (38), 323 (29), 309 (16), 300 (34), 241 (56), 213 (30),
197 (25), 148 (50), 89 (29), 59 (13).
[4-(4-Methoxy-phenoxy)-phenyl]-malonic Acid Dimeth-
yl Ester 7c. The raw material was purified by silica gel chro-
matography (petroleum ether/ethyl acetate 7:3). Yield: 59%
of a viscous orange oil. MS (EI): m/e (intensity %): 330 (M+,
100), 271 (57), 272 (73), 273 (16), 258 (89), 243 (31), 227 (27),
213 (58), 171 (7), 123 (15). Anal. (C18H18O6) C, H.
[4-(4-Benzyloxy-phenoxy)-phenyl]-malonicAcidDimeth-
yl Ester 7d. Yield: 90% of a colorless solid (methanol). Mp:
98-99 °C. MS (EI): m/e (intensity %): 406 (M+, 100), 348 (22),
315 (22), 257 (8), 223 (5), 120 (5), 91 (100), 31 (7). Anal.
(C24H22O6) C, H.
[4-(4-Iodo-phenoxy)-phenyl]-acetic Acid Methyl Ester
6b. Yield: 76%. MS (EI): m/e (intensity %): 369 (M + H+,
17), 368 (M+, 100), 309 (89), 310 (13), 243 (11), 242 (57), 183
(99), 128 (13), 64 (11), 51 (3). Anal.(C15H13IO3) C, H.
[4-(4-Methoxyphenoxy)-phenyl]-acetic Acid Methyl Es-
ter 6c. Yield: 94%. MS (EI): m/e (intensity %): 272 (M+, 100),
256 (44), 213 (77), 160 (10), 128 (13), 77 (4), 64 (10).
[4-(4-Hydroxy-phenoxy)-phenyl]-acetic Acid Methyl
Ester 6d. A few drops of concentrated sulfuric acid were added
to a solution of 1.43 g (5.85 mmol) of 5d in 30 mL of methanol.
The mixture was heated to reflux for 4 h and then cooled to
ambient temperature. The solvent was evaporated, and the
residue was redissolved in ether, washed with water (2 × 20
mL), and dried (Na2SO4). After evaporation of the solvent, the
residue was recrystallized from a chloroform/petroleum ether
mixture (1:1). Yield: 69%.
[4-(4-Benzyloxy-phenoxy)-phenyl]-acetic Acid Methyl
Ester 6e. Method A: Esterification of [4-(4-Benzyloxy-
phenoxy)-phenyl]-acetic Acid 5e. A few drops of concen-
trated sulfuric acid were added to a solution of 3.31 g (9.36
mmol) of 5e in 30 mL of methanol. The mixture was heated
to reflux for 1.5 h and then cooled to ambient temperature.
After concentration, the residue was redissolved in ether,
washed with water (2 × 50 mL), and dried (Na2SO4). After
evaporation of the solvent, the residue was recrystallized from
a chloroform/petroleum ether mixture (1:1) to give 2.61 g (7.49
mmol) of a colorless solid. Yield: 80%. Mp: 85 °C. MS (EI):
m/e (intensity %): 348 (M+, 41), 257 (16), 199 (16), 141 (3), 91
(100), 39 (2). Anal. (C22H20O4) C, H.
General Procedure for the Preparation of the 5-(4-
Phenoxy-phenyl)-pyrimidine-2,4,6-triones 8a-d and 5-(4-
Phenoxy-phenyl)-2-thioxo-dihydro-pyrimidine-4,6-di-
one 8e. Sodium (2 equiv) was dissolved in ethanol (ca. 10 mL/
mg), and urea/thiourea (1.7 equiv) was added to this solution.
A solution of malonic ester 7a-d in ethanol was added
dropwise, and the reaction mixture was heated to reflux for 6
h. After being cooled to ambient temperature, the mixture was
poured onto ice water and adjusted to pH 2, using dilute
hydrochloric acid. The precipitate was collected by suction
filtration and dried in vacuo.
The reaction products were obtained as amorphous solids,
which were insoluble in most organic solvents. Only polar
solvents, such as water, acetonitrile, methanol, ethanol, di-
methyl sulfoxide, or mixtures of these solvents, dissolved
certain amounts of these compounds, which exist as a mixture
of the keto and the enol forms (e.g. in a DMSO solution). The
keto/enol ratio of approximately 3:2 found by us reflected the
literature values.39,40
5-[4-(4-Bromo-phenoxy)-phenyl]-pyrimidine-2,4,6-tri-
one 8a. Yield: 82% of a mustard yellow solid. Mp: 231-233
°C. MS (MALDI-TOF) m/e: 397 (M + Na)+, 375 (M + H)+.
5-[4-(4-Iodo-phenoxy)-phenyl]-pyrimidine-2,4,6-trione
8b. Yield: 22% of a yellow solid (methanol/acetonitrile 1:1).
Mp: 285-286 °C (decomposition). MS (ESI-EM) m/e: 421.9711
(M)+ Calcd for C16H11IN2O4 421.9728.
Method B: Reaction of [4-(4-Hydroxy-phenoxy)-phen-
yl]-acetic Acid Methyl Ester 6d with Benzyl Trichloro-
acetimidate. Methyl ester 6d (600 mg, 2.32 mmol) was
dissolved in 10 mL of methylene chloride. To the stirred

Probes for Noninvasive MMP Imaging
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3407
5-[4-(4-Methoxy-phenoxy)-phenyl]-pyrimidine-2,4,6-
trione 8c. A polymeric material was formed, while collecting
the precipitated raw product, as well as on drying in vacuo at
50-100 °C. This polymeric material was treated with an
ethanol/water mixture (2:1) and heated to reflux. The resulting
suspension, containing a small amount of undissolved mate-
rial, was stored at 0 °C. The precipitate, formed on cooling,
was collected by suction filtration and washed with ethanol.
Yield: 25% of a slight gray solid. Mp: 262-263 °C (decomposi-
tion). MS (MALDI-TOF) m/e: 349 (M + Na)+, 327 (M + H)+.
Anal. (C17H14N2O5) C, H, N.
Mp: 247-248 °C. MS (MALDI-TOF) m/e: 506 (M + H)+, 505
(M)+, 504 (M + H)+. 503 (M)+. Anal. (C22H23BrN4O5‚0.2H2O)
C, H, N.
5-[4-(2-Hydroxy-ethyl)-piperazin-1-yl]-5-[4-(4-iodo-phen-
oxy)-phenyl]-pyrimidine-2,4,6-trione 10b. Yield: 67%.
Mp: 255-257 °C. MS (MALDI-TOF) m/e: 573 (M + Na)+, 551
(M + H)+. Anal. (C22H23IN4O5) C, H, N.
5-[4-(2-Hydroxy-ethyl)-piperazin-1-yl]-5-[4-(4-methoxy-
phenoxy)-phenyl]-pyrimidine-2,4,6-trione 10c. Yield: 58%.
Mp: 232 °C (decomposition). MS (MALDI-TOF) m/e: 455 (M
+ H)+. Anal. (C23H26N4O6‚0.9 H2O) C, H, N.
5-[4-(4-Benzyloxy-phenoxy)-phenyl]-pyrimidine-2,4,6-
trione 8d. Yield: 88% of a luminous yellow solid. Mp: 215-
217 °C (decomposition). MS (ESI-EM) m/e: 402.1166 (M)+
Calcd for C23H18N2O5 402.1195.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(2-hydroxy-ethyl)-
piperazin-1-yl]-2-thioxo-dihydro-pyrimidine-4,6-dione 10d.
Yield: 52% (acetone/ethanol/water (1:1:1)). Mp: 116-118 °C.
Anal. (C22H23BrN4O5S‚0.9H2O) C, H, N.
[5-(4-(4-Bromo-phenoxy)-phenyl]-2-thioxo-dihydro-py-
rimidine-4,6-dione 8e. Yield: 37% of a mustard yellow solid.
Mp 195-205 °C. MS (EI): m/e (intensity %): 393 (M + H+,
13), 392 (M+, 70), 391 (M + H+, 13), 390 (M+, 68), 291 (14),
290 (61), 289 (11), 288 (63), 261 (9), 181 (6),152 (8), 89 (100).
Anal. (C16H11N2BrN2O5S‚H2O) C, H, N.
General Procedure for the Preparation of the 5-Bromo-
5-(4-phenoxy-phenyl)-pyrimidine-2,4,6-triones 9a-d and
5-Bromo-5-(4-phenoxy-phenyl)-2-thioxo-dihydro-pyrimi-
dine-4,6-dione 9e.
Method A: A suspension of the corresponding pyrimidine-
2,4,6-trione, 1.2 equiv of N-bromosuccinimide, and a catalytic
amount of dibenzoyl peroxide in carbon tetrachloride was
heated to reflux for a period of 3 h. After being cooled to
ambient temperature, the mixture was concentrated, and the
residue was treated with water and extracted with ethyl
acetate (3×). The combined extracts were washed with brine
and dried (Na2SO4), and the solvent was evaporated.
Method B: A suspension of the pyrimidine-2,4,6-trione in
water (ca. 3 mL/mmol) was cooled to 0-5 °C, and a mixture of
1.76 equiv of 48% HBr and 1.6 equiv of bromine was added
dropwise. After stirring for 4-5 h at 0-10 °C the precipitate
was collected by filtration and dried in vacuo.
5-Bromo-5-[4-(4-bromo-phenoxy)-phenyl]-pyrimidine-
2,4,6-trione 9a. Yield: 52% (Method A) or 56% (Method B) of
a colorless solid (chloroform). Mp: 153-155 °C. MS (MALDI-
TOF) m/e: 375, 377 (M - Br + H)+. Anal. (C16H10Br2N2O4) C,
H, N.
5-Bromo-5-[4-(4-iodo-phenoxy)-phenyl]-pyrimidine-
2,4,6-trione 9b. Yield: 96% (Method A) of an off-white solid.
Mp: 246-249 °C (decomposition). MS (EI): m/e (intensity
%): 502 (M+, 9), 500 (M+, 9), 456 (14), 422 (100), 350 (8), 336
(23), 296 (4), 220 (3), 168 (8), 89 (20), 76 (8).
5-Bromo-5-[4-(4-methoxy-phenoxy)-phenyl]-pyrimidine-
2,4,6-trione 9c. Yield: 96% (Method A) of a yellow solid. MS
(MALDI-TOF) m/e: 405 (M+).
5-[4-(4-Benzyloxy-phenoxy)-phenyl]-5-bromo-pyrimi-
dine-2,4,6-trione 9d. Yield: 78% (Method B) of an off-white
solid.
Synthesis of 5-[4-(4-Bromo-phenoxy)-phenyl]-pyrimi-
dine-2,4,6-trione Derivatives 10e-j. 5-[4-(2-Bromo-ethyl)-
piperazin-1-yl]-5-[4-(4-bromo-phenoxy)-phenyl]-pyrimi-
dine-2,4,6-trione 10e. Triphenylphosphine (1.46 g, 5.57
mmol) and carbon tetrabromide (1.84 g, 5.55 mmol) were added
to a suspension of 1.40 g (2.78 mmol) 10a in 80 mL of
acetonitrile. The mixture was heated to reflux for 18 h, cooled
to ambient temperature, and stored at -30 °C overnight. The
precipitate was collected by suction filtration to give 920 mg
(1.62 mmol) of an off-white solid. Yield: 58%. MS (ESI-EM)
m/e: 565.0046 (M + H)+ Calcd for C22H23Br2N4O4 565.0086.
5-[4-(2-Methoxy-ethyl)-piperazin-1-yl]-5-[4-(4-methoxy-
phenoxy)-phenyl]-1,3-dimethyl-pyrimidine-2,4,6-trione
10f. A mixture of 250 mg (550 µmol) 10c, 347 mg (2.75 mmol)
of dimethyl sulfate, 44 mg (1.1 mmol) of sodium hydroxide,
and 2.5 mL of diisopropyl ether was stirred for 70 h at ambient
temperature. The reaction mixture was extracted with chlo-
roform (3 × 50 mL), and the extracts were washed with brine
and dried (Na2SO4). The solvent was evaporated to give 330
mg of a yellow solid. The precipitate which was formed upon
treatment with ether was filtered off and dried to give a
colorless solid (150 mg, 302 µmol). Yield: 55%. MS (ESI-EM)
m/e: 497.2363 (M + H)+ Calcd for C26H32N4O6 497.2395.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-piperazin-1-yl-pyri-
midine-2,4,6-trione 10g. A solution of 200 mg (440 µmol) 9a
in 5 mL of absol methanol was treated with 75.8 mg (880 µmol)
piperazine. The reaction mixture was stirred for 24 h at
ambient temperature. After ca. 10 min a colorless solid (160
mg, 348 µmol) began to precipitate from the reaction mixture,
which was finally collected by suction filtration, stirred for 1
h in methanol, filtered off, and dried in vacuo. An analytical
sample can be prepared by treatment of a DMSO solution
(heating necessary) of the material with water. After 1-2 h
stirring in water, the formed precipitate is filtered off. Yield:
79%. Mp: 265-266 °C (decomposition). MS (EI): m/e (intensity
%): 461 (M + H+, 7), 460 (M+, 28), 459 (M + H+, 7), 458 (M+,
28), 419 (3), 418 (15), 417 (4), 416 (16), 376 (25), 375 (37), 374
(25), 373 (35), 348 (11), 347 (49), 346 (15), 345 (52), 264 (7),
263 (17), 262 (8), 261 (17), 202 (33), 159 (19), 85 (100), 56 (38).
Anal. (C20H19BrN4O4‚1.0 H2O) C, H, N.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(isopropyl)-pi-
perazin-1-yl]-pyrimidine-2,4,6-trione 10h. A solution of 454
mg (1.00 mmol) 9a in 8 mL of absol methanol was treated with
256 mg (2.00 mmol) of 1-(2-propyl)-piperazine. The reaction
mixture was stirred for 24 h at ambient temperature. After
ca. 5 min, a colorless precipitate (360 mg, 718 µmol) was
formed, which was collected by suction filtration and washed
with methanol. Yield: 72%. Mp: 262-263 °C. MS (ESI-EM)
m/e: 501.1095 (M + H)+ Calcd for C23H26N4O4Br 501.1137.
5-[4-(2-Amino-ethyl)-piperazin-1-yl]-5-[4-(4-bromo-phen-
oxy)-phenyl]-pyrimidine-2,4,6-trione 10i. Compound 9a
(200 mg, 440 µmol) was dissolved in 2 mL of absol methanol
and treated with 125 mg (9.67 µmol) of N-(2-aminoethyl)-
piperazine. The reaction mixture was stirred for 16 h at
ambient temperature. After ca. 30 min, a colorless precipitate
(100 mg, 199 µmol) was formed, which was collected by suction
filtration and dried in vacuo. Yield: 45%. Mp: 220-223 °C
(decomposition). MS (ESI-EM) m/e: 502.1063 (M + H)+ Calcd
for C22H25N5O4Br 502.1090.
5-Bromo-5-[4-(4-bromo-phenoxy)-phenyl]-2-thioxo-di-
hydro-pyrimidine-4,6-dione 9e. Yield: 36% (Method A) of
a brown solid (chloroform/petroleum ether).
General Procedure for the Preparation of 5-[4-(2-
Hydroxy-ethyl)-piperazin-1-yl]-5-(4-phenoxy-phenyl)-py-
rimidine-2,4,6-triones 10a-c and 5-[4-(2-Hydroxy-ethyl)-
piperazin-1-yl]-5-(4-phenoxy-phenyl)-2-thioxo-dihydro-
pyrimidine-4,6-dione 10d. A solution of 5-bromopyrimidine-
2,4,6-trione 9a-c or 9e in methanol (ca. 5-10 mL/mmol) was
treated with 2.0 equiv of N-(2-hydroxy-ethyl)-piperazine and
stirred for 24 h at ambient temperature. Compounds 10a-c
precipitated after approximately 30-60 min, whereas com-
pound 10d did not precipitate from the reaction solution. The
precipitated colorless solids were collected by suction filtration
and dried in vacuo. In the case of 10d the solvent was removed,
and the residue was recrystallized from acetone/ethanol/water
(1:1:1).
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(2-hydroxy-ethyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione 10a. Yield: 70%.

3408 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Breyholz et al.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(2-carboxy-ethyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione 10j. Compound 9a
(200 mg, 440 µmol) was dissolved in 2 mL of absol methanol
and treated with 83.5 mg (5.28 mmol) of 3-(piperazin-1-yl)-
propionic acid. The reaction mixture was heated to reflux for
6 h and then concentrated to give 170 mg (320 µmol) of a
colorless amorphous solid (water). Yield: 73%. Mp: 208-210
°C (decomposition). MS (ESI-EM) m/e: 553.0644 (M + Na)+
Calcd for C23H23BrN4O6Na 553.0693.
242 °C (decomposition). MS (ESI-EM) m/e: 710.9710 (M +
Na)+ Calcd for C27H22BrIN4O5Na 710.9710.
Radiosynthesis of 5-[4-(2-Hydroxy-ethyl)-piperazin-1-
yl]-5-[4-(4-[125I]iodo-phenoxy)-phenyl]-pyrimidine-2,4,6-
trione [125I]10b. 2,5-Dihydroxybenzoic acid (0.6 mg), ascor-
binic acid (0.8 mg), water for injection (20 µL), and CuSO4‚
5H2O (5 µL, c ) 3.26 g/L water for injection) were added to a
conical vial containing 50 µL (209 nmol) of a solution of 10a
in ethanol (c ) 2.10 g/L).
The ice-cooled mixture was degassed for 10 min under a
helium flow. [125I]NaI (10.39 MBq, 4 µL in 0.05 N NaOH) was
added. The mixture was vortexed and heated to 113 °C for 60
min. After the mixture was cooled to ambient temperature,
water for injection (50 µL) was added. The solution was
injected onto the HPLC (see Synthetic Methods for HPLC
conditions), and the radioactive product fraction was sepa-
rated. The decay-corrected radiochemical yield was 49% (n )
4). Each product fraction was evaporated to dryness and
redissolved in phosphate-buffered saline (PBS, 200 µL). An
aliquot (200 µL) was taken for analytical HPLC (see Synthetic
Methods for HPLC conditions). The radiochemical purity was
>90%. Retention times tR were: tR(10a) ) 31.62 min; tR([125I]-
10b) ) 33.96 min. The identity of the radioactive product [125I]-
10b was verified by HPLC using the nonradioactive reference
10b.
MMP-2 and MMP-9 Fluorogenic Assay. The synthetic
broad-spectrum 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, Minneapolis, MN)
was used to assay MMP-2 and MMP-9 activity as described
previously.41 The inhibition of human active MMP-2 and
MMP-9 by pyrimidine-2,4,6-triones 8a, 10a-p was assayed
by preincubating MMP-2 (1 nM) or MMP-9 (2 nM) and
inhibitor compounds at varying concentrations (10 pM to 1
mM) in 50 mM Tris‚HCl, pH 7.5, containing 0.2 M NaCl, 5
mM CaCl2, 20 µM ZnSO4, and 0.05% Brij 35 at 37 °C for 30
min. An aliquot of substrate (10 µL of a 50 µM solution) was
then added to 90 µL of the preincubated MMP/inhibitor
mixture, and the activity was determined at 37 °C by following
product release with time. The fluorescence changes were
monitored using a Fusion Universal Microplate Analyzer
(Packard Bioscience, Meriden, CT) with excitation and emis-
sion wavelengths set to 330 and 390 nm, respectively. Reaction
rates were measured from the initial 10 min of the reaction
profile where product release was linear with time and plotted
as a function of inhibitor dose. From the resulting inhibition
curves, the IC50 value for each inhibitor was calculated by
nonlinear regression analysis using Grace 5.1.8 software
(Linux).
General Procedure for the Synthesis of Phenyl-piper-
azine Derivatives 10k-n. The corresponding phenyl-pi-
perazine (2.0 equiv) was added in portions to a solution of 9a
(1.0 equiv) in absol methanol (ca. 2-3 mL/mmol). The reaction
mixture was stirred at ambient temperature for 20 h. The
precipitate was collected by suction filtration and washed with
methanol.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-trimethylsilyl-
phenyl)-piperazin-1-yl]-pyrimidine-2,4,6-trione 10k. Yield:
82% of a colorless solid. Mp: 205-210 °C. MS (ESI-EM) m/e:
607.1391 (M + H)+ Calcd for C29H32BrN4O4Si 607.1376.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-fluoro-phenyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione 10l. Yield: 60% of
a colorless solid (chloroform). Mp: 247-250 °C. MS (ESI-EM)
m/e: 552.0784 (M + H)+ Calcd for C26H22N4O4BrF 552.0766.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-nitro-phenyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione 10m. Yield: 63%
of a bright yellow solid. Mp: 174-177 °C. MS (ESI-EM) m/e:
579.0711 (M + H)+ Calcd for C26H22 BrN5O6 579.0706.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(4-iodo-phenyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione 10n. To a suspen-
sion of 280 mg (459 µmol) of 10k in 25 mL of methanol was
added a solution of 300 mg (1.85 mmol) of iodine monochloride
in 5 mL of methanol over 40 min at -70 °C under an argon
atmosphere. The orange solution was allowed to warm to
ambient temperature over a period of 1.5 h, diluted with
dichloromethane, and was washed colorless with 10% sodium
thiosulfate. The aqueous layer was extracted with dichlo-
romethane (3 × 50 mL), washed with brine, and dried (Na2-
SO4). The solvent was evaporated and the residue was dried
in vacuo to give 62 mg (94 µmol) of the colorless crystalline
solid (methanol). Yield: 20%. Mp: 210-211 °C. MS (MALDI-
TOF) m/e: 661 (M+). Anal. (C26H22N4O4BrI) C, H, N.
Synthesis of Benzoylpiperazine Derivatives 10o,p.
Synthesis of N-Succinimidyl-3-(tri-n-butylstannyl)-ben-
zoate. Tetrakis(triphenylphosphine)-palladium(0) (109 mg, 94
µmol) and hexabutylditin (1.50 g, 2.59 mmol) were added to
N-succinimidyl 3-iodobenzoate28 (178 mg, 520 µmol) in 13 mL
of absol toluene under an argon atmosphere. The resulting
yellow solution was stirred for 6 h under reflux and subse-
quently 15 h at ambient temperature. The black reaction
mixture was concentrated under reduced pressure, and the
residue was purified by silicagel chromatograhy (petroleum
ether/ethyl acetate 4:1) to give 130 mg (260 mmol) of a colorless
oil. Yield: 50%. Rf (ethyl acetate/petroleum ether (1:4)): 0.49.
Anal. (C23H35NO4Sn) C, H, N.
Acknowledgment. This work was supported in part
by a research grant from Radiopharmaceuticals Re-
search Amersham Health plc., Amersham, U.K. The
authors gratefully acknowledge the technical assistance
of S. Schro¨er, S. Fatum, and M. Trub.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(3-(tri-n-butyl-
stannyl)-benzoyl)-piperazin-1-yl]-pyrimidine-2,4,6-tri-
one 10o. To a suspension of 10g (90.5 mg, 197 µmol) in 5 mL
of dimethylformamide was added N-succinimidyl 3-(tri-n-
butylstannyl)-benzoate (100 mg, 197 µmol). The mixture was
heated to 150 °C for a period of 6 h, stirred at ambient
temperature for further 70 h, and finally purified by silica gel
chromatography (ethyl acetate/petroleum ether (1:1)) to give
75 mg (88.0 µmol) of a colorless to slight yellow solid. Yield:
45%. MS (ESI-EM) m/e: 875.1798 (M + Na)+ Calcd for C39H49-
BrN4O5SnNa 875.1806.
5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(3-iodo-benzoyl)-
piperazin-1-yl]-pyrimidine-2,4,6-trione 10p. To a suspen-
sion of 10g (2.00 g, 4.35 mmol) in 50 mL of dimethylformamide
was added N-succinimidyl 3-iodobenzoate28 (1.50 g, 4.35 mmol).
The mixture was heated to 150 °C for 4.5 h. The solvent was
removed in vacuo, and the residue was purified by silica gel
chromatography (ethyl acetate/petroleum ether (1:1)) to give
1.30 g (1.89 mmol) of a colorless solid. Yield: 43%. Mp: 236-
Supporting Information Available: Elemental analyses,
exact mass analyses, and spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org
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JM049145X