Syntheses of radioiodinated pyrimidine-2,4,6-triones as potential agents for non-invasive imaging of matrix metalloproteinases

Article abstract of DOI:10.3390/ph10020049

Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in many kinds of live-threatening diseases. Therefore, MMP imaging for example with radiolabelled MMP inhibitors (MMPIs) potentially represents a valuable tool for clinical diagnostics using non-invasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. This work includes the organic chemical syntheses and in vitro evaluation of five iodinated barbiturate based MMPIs and the selection of derivative 9 for radiosyntheses of isotopologues [¹²³I]9 potentially useful for MMP SPECT imaging and [¹²⁴I]9 for MMP PET imaging.

Full text of DOI:10.3390/ph10020049

pharmaceuticals
Article
Syntheses of Radioiodinated Pyrimidine-2,4,6-Triones
as Potential Agents for Non-Invasive Imaging of
Matrix Metalloproteinases
Hans-Jörg Breyholz 1, Klaus Kopka 2, Michael Schäfers 3 and Stefan Wagner 1,
*
1
Department of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1,
D-48149 Münster, Germany; hans-joerg.breyholz@ukmuenster.de
Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ),
Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany; k.kopka@dkfz-heidelberg.de
European Institute for Molecular Imaging (EIMI), University of Münster, Waldeyerstraße 15,
D-48149 Münster, Germany; michael.schaefers@ukmuenster.de
2
3
*
Correspondence: stefan.wagner@ukmuenster.de; Tel.: +49-251-83-44713
Academic Editor: Jean Jacques Vanden Eynde
Received: 8 May 2017; Accepted: 28 May 2017; Published: 30 May 2017
Abstract: Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in
many kinds of live-threatening diseases. Therefore, MMP imaging for example with radiolabelled
MMP inhibitors (MMPIs) potentially represents a valuable tool for clinical diagnostics using
non-invasive single photon emission computed tomography (SPECT) or positron emission
tomography (PET) imaging. This work includes the organic chemical syntheses and in vitro
evaluation of five iodinated barbiturate based MMPIs and the selection of derivative
9
for
9
radiosyntheses of isotopologues [123I]
for MMP PET imaging.
9
potentially useful for MMP SPECT imaging and [124I]
Keywords: pyrimidine-2,4,6-triones; radioiodination; matrix metalloproteinase inhibitor; fluorometric
in vitro inhibition assays
1. Introduction
The in vivo molecular imaging of locally upregulated and activated matrix metalloproteinases
(MMPs) that are observed in pathologies such as cardiovascular diseases, inflammation or cancer
remains a substantive clinical issue [
should not only account for high binding affinity and specificity towards the enzyme but also for
drug-target residence time [ ], subgroup selectivity, sensitivity, target-to-background ratio as well
as in vivo stability [ ]. Maybe insufficient consideration of these parameters caused that most of the
preclinical studies of the past 20 years with radiolabeled MMPIs were either disappointing or remained
at a preliminary stage [ ]. In addition an inadequate validation of the animal models regarding their
1]. Current targeting strategies for noninvasive imaging of MMPs
2
3
4,5
level of MMP expression leads to challenging data [6].
Our own approaches towards the development of radiolabelled and fluorescent-dye conjugated
MMP tracers focused on two different classes of non-peptidic MMPIs, on the one hand hydroxamate-based
inhibitors (i.e., derivatives of CGS 27023A and CGS 25966 [
and, on the other hand, pyrimidine-2,4,6-trione-based inhibitors (i.e., barbiturates, derivatives of
RO-2653 [ 11] with sub-group selectivity for the gelatinases A (MMP-2) and B (MMP-9), neutrophil
7] with a broad-spectrum inhibitory profile)
8–
collagenase (MMP-8) and the membrane-bound MMPs MT-1-MMP (MMP-14) and MT-3-MMP
(MMP-16)). Initially, in 2005 we suggested in the latter project a first radiolabelled barbiturate-based
MMPI, compound 12 (see Table 2) labelled with the radionuclide iodine-125 (125I), for first in vitro and
Pharmaceuticals 2017, 10, 49; doi:10.3390/ph10020049
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ex vivo applications [12]. In 2008 we developed for the first time the barbiturate-based near-infrared
fluorescent photo probe Cy5.5-AF443 that was suitable for in vitro and in vivo imaging of the
gelatinases MMP-2 and MMP-9 [13,14]. In 2010 we published the radiosynthesis and evaluation
of the first fluorine-18 (18F) labelled barbiturate-based MMPI [15] and two years later, of several
more hydrophilic radiofluorinated analogues with improved biodistribution behavior [16]. Moreover,
a gallium-68 (68Ga) labelled version was introduced by our group in 2012 [17]. Favorable MMP
binding affinities for our barbiturate-based tracers were indeed measured by in vitro assays and in vivo
biodistribution studies using wt-mice. However, in animal models with increased MMP expression
mentioned barbiturate-based tracers did not meet the expectations. Anyhow in vivo MMP imaging was
feasible and specific with our hitherto most encouraging optical imaging probe Cy5.5-AF443 suggesting
the assumption that improved imaging performance of the photoprobe Cy5.5-AF443 compared to the
barbiturate radiotracers is caused by the cyanine dye substituent with the four hydrophilic sulfonic
acid moieties. Actually, these structural characteristics change the physicochemical properties and
accordingly the essential biodistribution pattern influenced i.a. by the excretion routes (renal or
hepatobiliary), plasma-protein binding, binding to non-target-organs and/or off-target interactions
with other proteins. In summary, adopting or transferring features from optical tracers to radiotracers
could support their development, an aspect that was recently reviewed by Faust et al. [18]. Therefore,
the aim of this work was the synthesis of radioiodinated barbiturate-based MMPI tracers with increased
hydrophilicity for potential SPECT/PET imaging. Radionuclides iodine-123 and iodine-124 were used
for the radiosyntheses and applied on our target molecule 9, which is ca. 3 log units more hydrophilic
as compared to our initial preclinical research tracer [125I]12 (see Table 2) [12]. To achieve increased
hydrophilicity two different chemical modifications of the C5 phenoxyphenyl moiety in 12 that
occupies the S1’ enzyme pocket were realized. Moreover the commercially available radionuclides
t½
β
iodine-123 (for SPECT) and iodine-124 (for PET) exhibit prolonged half-lives
compared to the
most common
γ
-emitter for SPECT technetium-99m ( 13.2 h vs. 6.0 h) and
+-emitter for PET
t½
fluorine-18 ( 4.2 d vs. 110 min) allowing long-term studies with the corresponding 123/124I-labelled
t½
barbiturate-based tracer in the next steps.
2. Results and Discussion
2.1. Chemistry
Phenyl barbiturates 5a
–d and 6 were prepared as outlined in Scheme 1 by four- (compounds
5a–c), five- (compound 5d) or six-step (compound 6) sequences, respectively.
Scheme 1. Syntheses of phenyl barbiturates 5a–d and 6. Reagents and yields: (a) NaH, dimethyl
carbonate, dioxane, 82% (2a), 73% (2b); (b) urea, NaOEt, EtOH, 29% (3a), 91% (3b); (c) NBS,
dibenzoyl peroxide, CCl4, 51% (4a); Br2, HBr, H2O, 89% (4b); (d) N-(2-hydroxyethyl)-piperazine or
3-(piperazin-1-yl)-propionic acid, MeOH, 53% (5a), 18% (5b), 28% (5c); (e) H2, Pd/C, MeOH, 82% (5d);
(f) NaI, NaOCl, NaOH, MeOH, 12% (6).

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In detail, 4-iodophenyl acetic acid methylester (1a), synthesized by the esterification of commercial
4-iodophenyl acetic acid using MeOH/H2SO4 was methoxycarbonylated to give the corresponding
malonic ester 2a. 4-Benzyloxyphenyl malonic acid dimethyl ester (2b) [19] was obtained from methyl
2-((4-benzyloxy)phenyl)acetate (1b) [20] analogous to 2a by the literature procedure. Malonic esters
2a and 2b were cyclized with urea using sodium ethoxide as a base to yield the 5-phenylbarbituric
acid derivatives 3a and 3b, which subsequently were brominated with N-bromosuccinimide (NBS)
(compound 3a) or bromine/HBr (compound 3b), resulting in the 5-bromo-5-phenyl barbituric
acids 4a and 4b
N-(2-hydroxyethyl)-piperazine or 3-(piperazin-1-yl)-propionic acid in MeOH to yield the barbituric
acid derivatives 5a in overall yields of 6% (5a), 2% (5b) and 17% (5c). Cleavage of the benzyloxy
.
These were reacted with either of the commercially available piperazines
–
c
group in 5c was achieved by catalytic hydrogenation (H2, Pd/C, MeOH) resulting in the hydroxyl
compound 5d in 14% overall yield. Iodination of 5d using sodium iodide, sodium hypochlorite and
sodium hydroxide in methanol yielded the ortho iodination product 6 in 2% overall yield.
To obtain carboxy derivative
9 and fluoro derivative 10 (Scheme 2) the bromo intermediate 7,
whose synthesis was described previously [12], was reacted either with commercially available
3-(piperazin-1-yl)-propionic acid or piperazine
(Scheme 2).
8 [15]. The yields were 83% for 9 and 8% for 10
Scheme 2. Syntheses of compounds 9 and 10. Reagents and yields: (a) MeOH, 83% (9), 8% (10).
2.2. Enzyme Assays and clogD Values
The MMP inhibition potencies of the barbituric acid derivatives 5a
in fluorometric in vitro inhibition assays as described previously [21]. The IC50-values of 5a
were determined for gelatinases MMP-2 and MMP-9 (Table 1), the IC50-values of and 10 for MMP-2,
MMP-8, MMP-9, MMP-13 and MMP-14 (only ) (Table 2). The results are depicted in Tables 1 and 2.
,
5b
,
6
,
9
and 10 were measured
,
5b and
6
9
9
The tables also contain the calculated logP/logD values (clogP/clogD) of the synthesized barbituric
acids derivatives to indicate the changes of lipophilicities caused by the structural modifications.
Replacement of the phenoxyphenyl moiety in 12 (see Table 1) by a phenyl group resulted in
compounds 5a
–
d
and
6. As expected the lipophilicities of iodinated 5a
–b and 6 are significantly
reduced, with clogD values ranging between
3.53. On the other hand the IC50 values for MMP-2 and -9 of these compounds are generally increased
compared to the derivatives with a phenoxyphenyl residue (Table 2). This is also expected because the
−
1.34 and 1.26 compared to 12 with a clogD value of
phenoxyphenyl core is optimized for the deep and narrow S1’ pocket of the target MMPs [
and possess IC50 values for MMP-2 and -9 in the micromolar range (0.67–1.6 M, Table 1), 5a is at
least a potent MMP-2 inhibitor with an IC50 value of 10 nM.
8]. While 5b
6
µ

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Table 1. IC50 values of phenyl barbiturates 5a, 5b and 6.
IC50 [nM]
Cpd.
R1
R2
R3
MMP-2
MMP-9
clogP a
clogD a
5a
5b
6
−I
−I
−H
−H
−I
10 ± 3
550 ± 180
1.42
1.26
1200
±
340 1600
±
±
600
320
−0.85
−1.34
−OH
670 ± 210
1300
0.75
0.53
a
clogP/clogD values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP at physiological
pH (7.4) with consideration of charged species). Values are the mean ± standard deviation (SD) of three assays.
Table 2. Structures and IC50 values of phenoxyphenyl barbiturates 9–12.
IC50 [nM]
Cpd.
R1
R2
MMP-2
MMP-8
MMP-9
MMP-13
clogP a
clogD a
9 b
10
−I
−I
29 ± 2
1170 ± 80
1.3 ± 0.2
362 ± 24
1.40
0.92
23 ± 5
146 ± 48
17 ± 5
28 ± 11
3.13
2.99
12
−I
7 ± 1
n. d.
2 ± 0.2
7 ± 2
n. d.
3.68
3.17
3.53
13 c
−H
23 ± 9
138 ± 12
645 ± 17
2.88 d
a
clogP/clogD values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP at
physiological pH (7.4) with consideration of charged species). Values are the mean
±
SD of three assays.
b
IC50 (MMP-14) = 49 ± 8 nM. c IC50 (MMP-1) > 50
µM, IC50 (MMP-3) = 760
µ
M. d experimental logD = 2.15 ± 0.02.
For comparison reasons the IC50 values of compound 12 [12] and 13 [15] from previous work are also shown.
The second hydrophilic modification included the substitution of the hydroxy group with a
carboxy group in the piperazine residue of 12 to yield carboxylic acid
9 resulting in a clogD shift
of 2.6 units (approximately 400 fold increased water solubility, Table 2) from 3.53 towards 0.92.
Despite this modification the high MMP-2- and -9-inhibition potency of 12 was only marginally
influenced (Table 2, 12: IC50 (MMP-2) = 7 nM, IC50 (MMP-9) = 2 nM.
IC50 (MMP-9) = 1.3 nM). Moreover, in vitro data showed, that was also a nanomolar inhibitor of
collagenase 3 (MMP-13) and MMP-14 (MT-1 MMP), but only a micromolar inhibitor of neutrophil
collagenase (MMP-8) ( : (IC50 (MMP-14) = 49 nM (Table 2, footnote b), IC50 (MMP-8) = 1170 nM,
IC50 (MMP-13) = 362 nM). In summary, compound confirms the results from Grams et al. [ ] that
5,5-disubstituted barbiturates with a para-substituted phenoxyphenyl unit represent potent inhibitors
for MMP-2, -9 and -14. Additionally, as shown by comparison of the IC50 values of and 13 with 10 the
elongation of the substituent of the piperazine residue with fluorinated tri-ethylenglycol resulted in a
9: IC50 (MMP-2) = 29 nM,
9
9
9
8
9

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decrease of selectivity for unknown reasons. From this selection of iodinated barbiturate derivatives
carboxylic acid
was chosen for radiochemical synthesis of the radioiodinated isotopologues [123/124I]
9
9
(see Section 2.3) because this compound possesses the most favourable characteristics indicated by
clogD and IC50-values.
2.3. Radiochemistry
Palladium-catalyzed cross-coupling reaction (Stille coupling) of non-radioactive reference
compound
9 with tributyltin hydride (or hexabutylditin) (yield: 28%) [20] provided the stannyl
precursor 11 (Scheme 3).
Scheme 3. Precursor synthesis of 11 and radiosynthesis of the barbituric acid-based MMP-targeted
model tracer [123/124I]
9. Reagents and yields: (a) Bu3SnH or Bu6Sn2 (2.0 eq.), PdCl2(PMePh2)2 or
PdCl2 (3 mol%), KOAc (3.0 eq.), N-methyl-pyrrolidone, 28%; (b) [123/124I]NaI, chloroamine-T hydrate,
0.1 M K2HPO4.
Subsequent radioiododestannylation of 11 with no-carrier-added (n. c. a.) [123/124I]NaI and
chloroamine-T led to the radioligands [123/124I]
9
. The decay-corrected radiochemical yields were
6% (n = 3) for [124I]
at the end of synthesis (EOS) and the
3% for [123I] 5% for [124I]
and 93 (as determined by radio-HPLC).
The molar activities are 0.2–6.3 GBq/ mol and 0.4–14.0 GBq/ mol, respectively. The identities of
123/124I]9 were proven by HPLC (coinjection and coelution with reference compound 9).
28
±
7% (n = 6) for [123I]
9
and 44
±
9
radiochemical purities were 95
±
9
±
9
µ
µ
[
3. Materials and Methods
3.1. General Methods. Chemistry
All chemicals, reagents and solvents for the synthesis of the compounds were of analytical grade,
purchased from commercial sources and used without further purification, unless otherwise specified.
Melting points were determined in capillary tubes on a SMP3 capillary melting point apparatus
1
(Stuart Scientific, Staffordshire, UK) and are uncorrected. H-NMR, 13C-NMR and 19F-NMR spectra
were recorded on ARX 300 and/or AMX 400 spectrometers (Bruker, Karlsruhe, Germany). CDCl3
contained tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on a MAT 212
(EI = 70 eV) spectrometer (Varian Medical Systems, Palo Alto, CA, USA) and a Bruker MALDI-TOF-MS
Reflex IV instrument (matrix: DHB). Exact mass analyses were conducted on a Quattro LC (Waters,
Milford, MA, USA) and a Bruker MicroTof apparatus. Elemental analyses were realized by a Vario EL
III analyzer (Elementar Analysensysteme Comp., Hanau, Germany). All aforementioned spectroscopic
and analytical investigations were done by staff members of the Institute of Organic Chemistry,
University of Münster, Germany. All purifications of compounds and determinations of purity by
HPLC were performed by using a gradient RP-HPLC system (Knauer, Berlin, Germany) equipped
with two K-1800 pumps, an S-2500 UV detector and RP-HPLC Nucleosil Eurosphere 100-10 C-18
columns for analytical (250 mm
otherwise): eluent A: water (0.1% TFA), eluent B: acetonitrile (0.1% TFA). The following conditions
were used (unless specified otherwise): Gradient from 90% A to 20% A over 30 min, constant 20% A
×
4.6 mm) purposes. The following eluents were used (unless specified
over 5 min and from 20% A to 90% A over 5 min, at a flow rate of 1.5 mL/min, detection at
λ = 254 nm.

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3.2. General Procedure for 4-Iodophenyl Malonic Acid Dimethyl Ester (2a) and 4-Benzyloxyphenyl Malonic
Acid Dimethyl Ester (2b)
A suspension of 4.00 g NaH (166 mmol, 6.66 g of a 60% suspension in paraffin; the paraffin
was removed by repeated washings with petroleum benzene) and 48.0 g (533 mmol) dimethyl
carbonate in 80 mL absolute dioxane was heated to 100–120 ◦C and a solution of 23.00 g (83.3 mmol)
4-Iodophenyl acetic acid methylester (1a, prepared by the esterification of 4-iodophenyl acetic acid
using MeOH/H2SO4) in absolute dioxane (125 mL) was added dropwise over a period of 1 h.
Refluxing was continued for 3 h and the reaction mixture was allowed to come to room temperature
overnight. The mixture was poured onto ice water and subsequently extracted with methylene chloride
(3
×
). The combined organic layers were washed with water (1
×
), brine (1
×
), dried (Na2SO4) and
concentrated. The crude 2a was used in the next step without further purification. Yield: 22.8 g
(68.2 mmol, 82%). 1H-NMR (300 MHz, DMSO-d6):
δ
[ppm]: 7.90 (d, J = 8.3 Hz, 2 H, HAryl), 7.35
3
(d, J = 8.3 Hz, 2 H, HAryl), 5.18 (s, 1 H, CH), 3.83 (s, 6 H, CH3). 13C-NMR (75.5 MHz, DMSO-d6):
3
δ
[ppm]: 168.46, 138.14, 133.10, 131.88, 94.86, 55.99, 53.11. 4-Benzyloxyphenyl malonic acid dimethyl
ester (2b) [19] was prepared from methyl 2-((4-benzyloxy)phenyl)acetate 1b [20] analogous to 2a.
3.3. General Procedure for 5-(4-Iodophenyl)-pyrimidine-2,4,6-trione (3a) and
5-(4-Benzyloxyphenyl)-pyrimidine-2,4,6-trione (3b)
Under an argon atmosphere 2 eq. of sodium were dissolved in ethanol (0.35 mL/mmol Na)
and 1.7 eq. of urea were added. A solution of malonic ester 2a or 2b in ethanol (2.2 mL/mmol)
was added dropwise and the reaction mixture was heated to reflux for 6 h. After cooling to room
temperature, the mixture was poured onto ice water and adjusted to pH 2 using dilute hydrochloric
1
acid. The precipitate was collected by suction and dried in vacuo. 3a: Yield: 29%. H-NMR (300 MHz,
DMSO-d6):
δ
[ppm]: 11.34 (broad, s), 7.68 (broad, d, 2 H, HAryl), 7.09 (broad, d, 2 H, HAryl), 4.92 (s, 1 H,
[ppm]: 187.77 (C-4/6 enol form), 169.73 (C-4/6 keto form),
CH). 13C-NMR (75.5 MHz, DMSO-d6):
δ
151.31 (C-2), 137.41, 132.46, 128 .89, 93.48, 92.09 (C-5 enol form), 54.60 (C-5 keto form). Anal. Calcd for
C10H7IN2O4: C 36.39, H 2.14, N 8.49. Found: C 36.61, H 2.29, N 8.23. 3b: Yield: 91%. Mp 233–236 ◦C.
1H-NMR (300 MHz, DMSO-d6):
δ
[ppm]: 11.34 (broad, s, OH), 7.4–6.94 (m, 9 H, HAryl), 5.10 (s, 1 H,
CH). 13C-NMR (75.5 MHz, DMSO-d6):
[ppm]: 187.75 (C-4/6 enol form), 169.71 (C-4/6 keto form),
158.25, 151.29 (C-2), 137.39, 132.44, 130.64, 128.14, 115.31, 114.36, 92.06 (C-5 enol form), 69.58, 54.59 (C-5
keto form). Anal. Calcd for C10H14IN2O4 H2O: C 62.19, H 4.91, N 8.53. Found: C 62.38, H 4.56, N 8.14.
δ
·
3.4. 5-Bromo-5-(4-iodophenyl)-pryrimidine-2,4,6-trione (4a)
A suspension of 3a (6.51 g, 19.7 mmol), N-bromosuccinimide (4.20 g, 23.6 mmol, 1.2 eq.) and
a catalytic amount of dibenzoylperoxide in carbon tetrachloride (400 mL) was heated to reflux for
a period of 3 h. After cooling to room temperature the mixture was concentrated, the residue was
treated with water and extracted with ethyl acetate (3 ). The combined extracts were washed with
×
brine, dried (Na2SO4) and the solvent was evaporated. The residue was stirred in CHCl3 for 2 h to
give a colorless solid. Yield: 4.10 g (10.0 mmol, 51%). Mp 183–186 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ
[ppm]: 11.47 (broad, s), 10.94 (broad, s), 8.64 (broad, s), 7.71 (d, 2 H, HAryl), 7.15 (d, 2 H, HAryl).
13C-NMR (75.5 MHz, DMSO-d6):
m/e (intensity %): 410 (M+, 15), 408 (M+, 15), 329 (100), 282 (45), 244 (45), 196 (62), 129 (29), 89 (38),
δ [ppm]: 179.66, 170.80, 137.81, 133.18, 127.65, 95.76, 75.99. MS (EI):
43 (39). Anal. Calcd for C10H6BrIN2O3: C 29.37, H 1.48, N 6.85. Found: C 29.96, H 1.48, N 6.80.
3.5. 5-(4-Benzyloxyphenyl)-5-bromo-pryrimidine-2,4,6-trione (4b)
A suspension of the 3b (5.00 g, 16.1 mmol) in water (48 mL) was cooled to 0–5 ◦C and 48% HBr
(3.25 mL, 28.4 mmol) and bromine (1.32 mL, 25.8 mmol) were added dropwise. After stirring for 4–5 h
◦
at 0–10 C the precipitate was collected by filtration and dried in vacuo. Yield: 5.59 g (14.4 mmol, 89%).
Mp 145–147 ◦C. 1H-NMR (300 MHz, DMSO-d6):
δ
[ppm]: 11.44 (broad, s), 7.43–7.01 (m, 9 H, HAryl),
5.08 (s, 2 H, CH2). 13C-NMR (75.5 MHz, DMSO-d6):
δ
[ppm]: 171.34, 159.00, 150.21, 137.18, 130.86,

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128.79, 128.03, 127.99, 126.88, 115.30, 76.28, 69.70. MS (ESI-EM) m/e: 410.9951 (M + Na)+ calcd for
C17H13BrN2O4Na 410.9956. Anal. Calcd for C17H13BrN2O4
C 51.79, H 3.12, N 6.96.
·
0.3 H2O: C 52.10, H 3.42, N 7.15. Found:
3.6. General Procedure for Compounds 5a–5c
A solution of 4a or 4b in methanol (2–4 mL/mmol) was treated with 2 eq. of N-(2-hydroxyethyl)-
piperazine (in case of 5a and 5c) or 3-(piperazin-1-yl)-propionic acid (in case of 5b) and stirred for 2 d
at room temperature. The colorless precipitate was collected by suction and dried in vacuo.
3.6.1. 5-[4-(2-Hydroxyethyl)piperazin-1-yl]-5-(4-iodophenyl)pryrimidine-2,4,6-trione (5a)
Yield: 53%. Mp 168–172 ◦C. 1H-NMR (400 MHz, DMSO-d6):
δ
[ppm]: 7.72 (d, 3J = 8.7 Hz, 2 H,
HAryl), 7.16 (d, 3J = 8.7 Hz, 2 H, HAryl), 3.43–2.31 (m, 12 H, CH2). 13C-NMR (75.5 MHz, DMSO-d6):
δ
[ppm]: 169.36, 151.07, 138.72, 131.17, 130.13, 95.72, 85.71, 59.68, 58.20, 53.59, 47.24. MS (ESI-EM)
m/e: 459.0518 (M + H)+ calcd for C16H20IN4O4 459.0524. Anal. Calcd for C16H19IN4O4
H2O: C 40.35,
H 4.23, N 11.76. Found: C 40.93, H 4.54, N 11.47.
·
3.6.2. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-(4-iodophenyl)pyrimidine-2,4,6-trione (5b)
Yield: 18%. Mp 139–142 ◦C. 1H-NMR (300 MHz, DMSO-d6):
3J = 8.7 Hz, 2 H, HAryl), 7.61 (d, 3J = 8.7 Hz, 2 H, HAryl), 3.30–3.21 (m, 4 H, CH2), 2.85–2.59 (m, 8 H,
δ [ppm]: 9.42 (broad, s), 8.00 (d,
CH2). 13C-NMR (75.5 MHz, DMSO-d6):
δ [ppm]: 173.74, 164.25, 151.49, 138.89, 135.07, 131.70, 86.35,
85.64, 53.23, 49.47, 43.38, 32.01. MS (ESI-EM) m/e: 487.0453 (M + H)+ calcd for C17H20IN4O5 487.0473.
Anal. Calcd for C17H19IN4O5·1.8 H2O: C 39.88, H 4.04, N 10.80. Found: C 39.59, H 4.23, N 10.48.
3.6.3. 5-[4-(2-Hydroxyethyl)piperazin-1-yl]-5-(4-benzyloxyphenyl)pryrimidine-2,4,6-trione (5c)
Yield: 28%. Mp 211–213 ◦C. 1H-NMR (400 MHz, DMSO-d6):
5.09 (s, 2 H, CH2), 3.48–3.44 (m, 2 H, CH2OH), 2.57–2.38 (m, 10 H, CH2). 13C-NMR (100 MHz,
δ [ppm]: 7.48–7.02 (m, 9 H, HAryl),
DMSO-d6):
δ [ppm]: 171.08, 159.63, 150.35, 137.67, 130.00, 129.32, 128.78, 128.28, 128.08, 115.76, 74.99,
70.26, 61.07, 59.28, 48.10, 44.91. MS (MALDI-TOF) m/e: 461 (M + Na)+, 439 (M + H)+. Anal. Calcd for
C23H26N4O5·1 H2O: C 60.52, H 6.18, N 12.27. Found: C 60.35, H 5.71, N 12.27.
3.7. 5-[4-(2-Hydroxyethyl)piperazin-1-yl]-5-(4-hydroxyphenyl)pryrimidine-2,4,6-trione (5d)
Compound 5c (1.66 g, 3.79 mmol) was dissolved in absolute methanol (150 mL), treated with
Pd/C (10%, 250 mg) and heated to reflux for 12 h under an H2 atmosphere. After cooling to room
temperature, the mixture was stirred overnight. The catalyst was filtered off and washed with methanol
(80 mL). The solvent was evaporated and the solid residue was dried in vacuo. The crude product was
taken up in a CHCl3/ethylacetate mixture (1/1, v/v) and stirred at room temperature for 2–3 h and
◦
finally re-isolated by suction filtration. Yield: 1.08 g (3.11 mmol, 82%). Mp 185 C. 1H-NMR (300 MHz,
DMSO-d6):
[ppm]: 7.45 (d, 3J = 8.6 Hz, 2 H, HAryl), 7.03 (d, 3J = 8.6 Hz, 2 H, HAryl), 3.79 (m, 2 H,
CH2OH), 2.99–2.69 (m, 10 H, CH2). 13C-NMR (75.5 MHz, DMSO-d6):
[ppm]: 170.66, 158.47, 149.82,
131.22, 129.29, 115.94, 74.63, 59.74, 57.66, 53.73, 46.67. MS (ESI-EM) m/e: 349.1521 (M + H)+ calcd for
δ
δ
C16H21N4O5 349.1506. Anal. Calcd for C16H20N4O5
H 5.54, N 14.02.
·
2.2 H2O: C 49.56, H 5.77, N 14.44. Found: C 49.08,
3.8. 5-(4-Hydroxy-3-iodophenyl)-5-[4-(2-hydroxyethyl)piperazin-1-yl]pryrimidine-2,4,6-trione (6)
Compound 5d (500 mg, 1.44 mmol) was dissolved in methanol (10 mL) and treated with 58 mg
(1.44 mmol) sodium hydroxide and 216 mg (1.44 mmol) sodium iodide. The solution was cooled in
an ice bath and 824 mg (687 µL, 1.44 mmol) sodium hypochlorite (13% active chlorine) were added
dropwise over a period of 60 min. The orange suspension was stirred in the ice bath for further 2 h
until a nearly colorless solution had formed. The ice bath was removed and 2–3 crystals sodium

Pharmaceuticals 2017, 10, 49
8 of 11
thiosulfate were added at room temperature. The solution was acidified to pH 6.8 by adding 1 M
HCl and extracted with ethylacetate (3
×
30 mL). The combined extracts were washed with bri◦ne
(1
×
30 mL), dried (MgSO4) and evaporated to dryness. Yield: 80 mg (0.17 mmol; 12%). Mp 168–170 C
1
(decomposition). H-NMR (300 MHz, DMSO-d6):
δ
[ppm]: 11.50 (s, broad, 2 H), 7.60–6.69 (m, 3 H,
[ppm]: 169.87,
HAryl), 4.34 (s, 1 H, OH) 3.40–2.28 (m, 12 H, CH2). 13C-NMR (75.5 MHz, DMSO-d6):
δ
157.32, 149.35, 137.99, 129.10, 127.04, 115.37, 84.56, 73.25, 59.71, 58.27, 53.70, 47.11. MS (ESI-EM) m/e:
475.0467 (M + H)+ calcd for C16H20IN4O5 475.0473. Anal. Calcd for C16H19IN4O5
N 11.28. Found: C 39.47, H 4.23, N 11.21.
·
H2O: C 39.04, H 4.30,
3.9. General Procedure for Compounds 9 and 10
A solution of 5-bromo-5-[4-(4-iodo-phenoxy)-phenyl]pyrimidine-2,4,6-trione
7 [8,12] in methanol
(ca. 5–10 mL/mmol) was treated with 2.0 eq. of the appropriate piperazine ( or 3-(piperazin-1-yl)-
8
propionic acid) and stirred at room temperature overnight. The colorless solids which precipitated
after ca. 1 h were collected by suction and dried in vacuo. Compound 10 was further purified by silica
gel column chromatography (EtOAc/MeOH (4/1, v/v) + 1% NEt3).
3.9.1. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-[4-(4-iodophenoxy)phenyl]pyrimidine-2,4,6-trione (9)
◦
Yield: 83%. Mp: 236–242 C (decomposition). 1H-NMR (400 MHz, DMSO-d6):
δ [ppm]: 9.31
(s, broad, 2 H), 7.95 (d, 3J = 8.6 Hz, 2 H, HAryl), 7.72 (d, 3J = 9.0 Hz, 2 H, HAryl), 6.92 (d, 2J = 9.0 Hz,
2 H, HAryl), 6.87 (d, 3J = 8.6 Hz, 2 H, HAryl), 3.16–3.09 (m, 2 H, CH2), 2.73–2.58 (m, 8 H, CH2), 2.51–2.44
(m, 2 H, CH2). 13C-NMR (100 MHz, DMSO-d6):
δ
[ppm]: 173.80, 164.31, 158.77, 151.62, 150.29,
138.66, 135.36, 131.05, 120.00, 118.05, 86.65, 85.42, 53.24, 49.46, 43.32, 40.53. MS (ESI-EM) m/e: 420.9687
(M 2 H2O: C 44.96,
C7H13N2O2)+ calcd for C16H10IN2O4 420.9685. Anal. Calcd for C23H23IN4O6
H 4.43, N 9.12. Found: C 45.16, H 4.70, N 9.52.
−
·
3.9.2. 5-(4-(2-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)-5-(4-(4-iodophenoxy)phenyl)-
pyrimidine-2,4,6-trione (10)
◦
1
Yield: 8%. Mp 176-177 C. H-NMR (300 MHz, DMSO-d6)
δ [ppm]: 9.28 (s, 2 H), 7.84 (d,
3J = 7.8 Hz, 2 H, HAryl), 7.64 (d, J = 7.8 Hz, 2 H, HAryl), 6.81 (d, J = 6.8 Hz, 2 H, HAryl), 6.76 (d,
3J = 6.8 Hz, 2 H, HAryl), 4.51 (dt, JH,F = 48.1 Hz, 2 H, CH2F), 3.56–2.50 (m, 22 H, CH2). 13C-NMR
3
3
2
(75.5 MHz, DMSO-d6):
85.13, 83.11 (d,|1JC,F| = 165.7 Hz), 70.54, 70.39, 70.12, 69.76, 69.57, 68.15, 56.71, 49.62, 42.98. 19F-NMR
(282 MHz, CDCl3): [ppm]:
216.61. MS (ESI-EM) m/e: 685.1518 (M + H)+ calcd for C28H35FIN4O7
δ [ppm]: 169.93, 158.40, 151.31, 149.89, 138.31, 135.00, 130.71, 119.61 117.75, 86.35,
δ
−
685.1529. The purity of 10 was determined by analytical gradient HPLC to be > 95% (system and
conditions see Section 3.1), tR = 25.92 ± 0.36 min (n = 3).
3.10. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-[4-(4-(tributylstannyl)phenoxy)phenyl]pyrimidine-2,4,6- trione (11
PdCl2 (PMePh2)2 (18 mg, 30 mol, 3 mol%) or PdCl2 (18 mg, 100 mol, 10 mol%), KOAc
(294 mg, 3.00 mmol, previously dried for several hours at 100 ◦C in vacuo before use) and
N-methyl-pyrrolidinone (NMP, 15 mL) were mixed under an argon atmosphere [20]. Compound
)
µ
µ
9
(578 mg, 1.00 mmol) and tributyltin hydride (582 mg, 2.00 mmol) or hexabutylditin (870 mg, 1.50 mmol)
◦
were added and the mixture was heated to 110 C for 10–15 h. The progress of the reaction was
monitored by HPLC (system and conditions see Section 3.1). The retention times tR were: tR (
34.15 1.38 min (n = 10), tR (11): 40.83 1.17 min (n = 6). When the conversion was complete, the hot
black reaction mixture was filtered through a pad of Celite by suction and the filter cake was washed
with a small amount of NMP. The orange filtrate was stored at
30 ◦C overnight. The beige solid
9):
±
±
−
which precipitated upon cooling was isolated by suction. When there was no precipitation, the volatile
components of the filtrate were removed by short-path distillation. The solid residue was taken up
in hot methanol and insoluble components were filtered off. The solvent was removed in vacuo and
the residue was treated with acetone. Yield: 208 mg (0.28 mmol, 28%). The purity of the product

Pharmaceuticals 2017, 10, 49
9 of 11
1
was >95% as determined by analytical gradient HPLC. Mp: 285–286 ◦C (decomposition). H-NMR
(400 MHz, DMSO-d6): δ [ppm]: 7.89–7.12 (m, 8 H, HAryl), 3.56–1.94 (m, 12 H, CH2), 1.77–1.67 (m, 6 H,
CH2), 1.56–1.44 (m, 6 H, CH2), 1.25–1.20 (m, 6 H, CH2), 1.06 (t, 3J = 8.3 Hz, 9 H, CH3). MS (ESI-EM)
m/e: 585.1847 (M − C7H13N2O2)+ calcd for C28H37N2O4Sn 585.1775.
3.11. General Methods. Radiochemistry
N. c. a [124I]NaI was provided by Department of Nuclear Medicine, University Hospital Essen,
University Duisburg-Essen, Germany. Typical radioactivities used for the radioiodination were
134
Healthcare Buchler GmbH & Co KG (Braunschweig, Germany). Typical radioactivities used for the
radioiodination were 136 L in 0.05 N NaOH. Separation, purification and
63 MBq [123I]NaI in 10
±
48 MBq [124I]NaI in 50
±
18 µ
L in 0.01 N NaOH. N. c. a. [123I]NaI was purchased from GE
±
±
3 µ
analyses of the radiochemical yields and the radiochemical purities of all radioiodinated compounds
were performed by a gradient radio-HPLC chromatograph system (HPLC A) using a Knauer K-500
and a Latek P 402 pump, a Knauer K-2000 UV-detector (
SP51 -detector and a RP-HPLC Nucleosil column 100-5 C-18 (250 mm
precolumn (20 mm × 4.0 mm). Sample injection was carried out using a Rheodyne injector block
(type 7125 incl. 200 L loop). The recorded data were processed by the NINA version 4.9 software
λ
= 254 nm) a Crismatec NaI(Tl) Scintibloc 51
γ
×
4.6 mm), a corresponding
µ
(GE Medical Systems-Functional Imaging GmbH, Münster, Germany). Separation and purification
of the radiosynthesized compounds were—unless specified otherwise—performed by radio-HPLC
(HPLC A, see above) using a Nucleosil 100-10 C18 column (250 mm
processed by the NINA version 4.9 software . The radiochemical purities and the specific activities
×
8 mm). The recorded data were
were determined using a radio-HPLC system (HPLC B) composed of a Sykam S1021 pump, a Knauer
K-2501 UV-detector (
100-3 C18 column (200 mm
Star version 4.07 radiochromatography software (raytest Isotopenmeßgeräte GmbH, Straubenhardt,
Germany). Radio-TLC was analyzed using a miniGITA TLC-Scanner (raytest Isotopenmeßgeräte
GmbH, Straubenhardt, Germany).
λ
= 254 nm), a Crismatec NaI(Tl) Scintibloc 51 SP51
3 mm), a VICI injector block (type C1 incl. 20
γ
-detector, a Nucleosil
×
µL loop) and the GINA
3.12. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-[4-(4-[123/124I]-iodophenoxy)phenyl]pyrimidine-2,4,6-trione
([123/124I]9)
In a conical glass vial 89
123I]NaI or [124I]NaI. The radiosynthesis was started by addition of 34 mg (0.14
hydrate in 39 L 0.1 M K2HPO4 buffer (pH 7.34). The mixture was vortexed for 10 s and allowed to
stand for 5 min at room temperature. To quench the reaction 50 L of a 10% Na2S2O5 solution (in water
µg (0.12
µmol) stannyl precursor 11 in 39
µL MeOH was added to n. c. a.
[
µmol) chloroamine-T
µ
µ
for injection) was added and the mixture was vortexed again for 10 s. After 10 min the quenched
reaction solution was injected onto the HPLC column (see Section 3.11. HPLC A) to isolate the fraction
of the radiolabelled product [124I]
9
(HPLC conditions: eluent A: CH3CN/H2O/TFA 950/50/1, v/v/v,
eluent B: CH3CN/H2O/TFA 50/950/1, v/v/v; gradient: from 92% B to 40% B within 45 min, 5 min
constant at 40% B, and from 40% B to 92% B within 5 min at a flow rate of 2.0 mL/min, = 254 nm;
tR (stannyl precursor 11) = 40.80 ) = 32.75 2.13 min (n = 3)). The product
0.81 min (n = 3), tR ([124I]
fraction was evaporated to dryness and redissolved in 0.9% NaCl (200 L). The decay-corrected
radiochemical yield (after evaporation) was 28 and 44
7% (n = 6) for [123I] 6% (n = 3) for [124I]
respectively. An aliquot (50 L) was taken for analytical radio-HPLC. The radiochemical purities were
95 3% and 93 , and the molar activities were 0.2–6.3 GBq/ mol and
5% for [123I] and [124I]
0.4–14.0 GBq/ was proven by HPLC using the
mol, respectively. The identity of [123I] and [124I]
λ
±
9
±
µ
±
9
±
9,
µ
±
±
9
9
µ
µ
9
9
non-radioactive analog 9.
3.13. In Vitro Enzyme Inhibition Assays
The synthetic fluorometric 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, USA)

Pharmaceuticals 2017, 10, 49
10 of 11
was used to assay activated MMP-2, MMP-8, MMP-9 and MMP-13 as described previously [21].
The inhibitions of human active MMP-2, MMP-8, MMP-9 MMP-13 and MMP-14 (only ) by the
barbituric acid derivatives and 10, of human active MMP-2 and MMP-9 by 5a 5b and were assayed
by preincubating MMP-2, MMP-3, MMP-8, MMP-9, MMP-13 or MMP-14 (each at 2 nM) and inhibitor
9
9
,
6
compounds at varying concentrations (10 pM–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 fluorescence was determined at 37 ◦C by following product release with time. The fluorescence
changes were monitored using a Fusion Universal Microplate Analyzer (Packard Bioscience, Boston,
MA, USA) with excitation and emission 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
values for each inhibitor were calculated by non-linear regression analysis, performed using the Grace
5.1.8 software (Linux).
4. Conclusions
Starting from the lipophilic preclincal research tracer [125I]12 (clogD 3.53, Table 2), that was
already developed by our group [12], we intended to develop a more hydrophilic radioiodinated
barbiturate-based MMP-targeted tracer for the potential non-invasive visualization of activated MMPs
in vivo. This was achieved by the substitution of the hydroxy group of 12 by a carboxy group
yielding derivative
carboxylic acid
9 with a hydrophilic shift compared to 12 (clogD 0.92 vs. 3.53). Similar to 12
9
represents a very potent inhibitor of MMP-2 and -9 (IC50 (MMP-2) = 29 nM, IC50
(MMP-9) = 1.3 nM). Therefore radioiodinated analogues [123/124I]
further in vivo evaluations with SPECT and PET.
9 were successfully synthesized for
Acknowledgments: This work was supported by grants from the German Research Foundation (Deutsche
Forschungsgemeinschaft (DFG)), Collaborative Research Centre CRC 656, Münster, Germany (CRC 656 projects
A02, B01 and Z05). We thank Prof. Wolfgang Brandau, Department of Nuclear Medicine, University Hospital
Essen, Essen, Germany, for providing [124I]NaI. We thank Sandra Höppner and Sven Fatum for excellent technical
assistance, Heinrich Luftmann and Klaus Bergander, Organic Chemistry Institute, WWU Münster for performing
mass spectrometry and NMR spectroscopy, respectively. Many thanks also to Werner Henkel for his temporarily
work in our laboratories.
Author Contributions: H.J.B., K.K. and S.W. conceived and designed the experiments; H.J.B., K.K. and S.W.
performed the experiments; H.J.B., K.K., M.S. and S.W. analyzed the data; H.J.B. and S.W. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).