Syntheses of Radioiodinated Pyrimidine-2,4,6-Triones as Potential Agents for Non-Invasive Imaging of Matrix Metalloproteinases

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.


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 [1]. Current targeting strategies for noninvasive imaging of MMPs should not only account for high binding affinity and specificity towards the enzyme but also for drug-target residence time [2], subgroup selectivity, sensitivity, target-to-background ratio as well as in vivo stability [3]. 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 [4,5]. In addition an inadequate validation of the animal models regarding their 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 [7] with a broad-spectrum inhibitory profile) and, on the other hand, pyrimidine-2,4,6-trione-based inhibitors (i.e., barbiturates, derivatives of RO-2653 [8][9][10][11] with sub-group selectivity for the gelatinases A (MMP-2) and B (MMP-9), neutrophil collagenase (MMP-8) and the membrane-bound MMPs MT-1-MMP (MMP-14) and MT-3-MMP (MMP-16)). 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 ( 125 I), for first in vitro and 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 ( 18 F) labelled barbiturate-based MMPI [15] and two years later, of several more hydrophilic radiofluorinated analogues with improved biodistribution behavior [16]. Moreover, a gallium-68 ( 68 Ga) 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 [ 125 I]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 iodine-123 (for SPECT) and iodine-124 (for PET) exhibit prolonged half-lives t½ compared to the most common γ-emitter for SPECT technetium-99m (t½ 13,2 h vs. 6,0 h) and β + -emitter for PET fluorine-18 ( t½ 4,2 d vs. 110 min) allowing long-term studies with the corresponding 123/124 I-labelled barbiturate-based tracer in the next steps.
al models with increased MMP expression mentioned barbiturate-based tracers expectations. Anyhow in vivo MMP imaging was feasible and specific with our couraging optical imaging probe Cy5.5-AF443 suggesting the assumption that g performance of the photoprobe Cy5.5-AF443 compared to the barbiturate used by the cyanine dye substituent with the four hydrophilic sulfonic acid y, these structural characteristics change the physicochemical properties and ssential biodistribution pattern influenced i.a. by the excretion routes (renal or asma-protein binding, binding to non-target-organs and/or off-target interactions ins. In summary, adopting or transferring features from optical tracers to support their development, an aspect that was recently reviewed by Faust et al. e aim of this work was the synthesis of radioiodinated barbiturate-based MMPI ased hydrophilicity for potential SPECT/PET imaging. Radionuclides iodine-123 ere used for the radiosyntheses and applied on our target molecule 9, which is ca. 3 drophilic as compared to our initial preclinical research tracer [ 125 I]12 (see Table 2) increased hydrophilicity two different chemical modifications of the C5 oiety in 12 that occupies the S1' enzyme pocket were realized. Moreover the ailable radionuclides iodine-123 (for SPECT) and iodine-124 (for PET) exhibit es t½ compared to the most common γ-emitter for SPECT

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. 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 ( 18 F) labelled barbiturate-based MMPI [15] and two years later, of several more hydrophilic radiofluorinated analogues with improved biodistribution behavior [16]. Moreover, a gallium-68 ( 68 Ga) 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 [ 125 I]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 iodine-123 (for SPECT) and iodine-124 (for PET) exhibit prolonged half-lives t½ compared to the most common γ-emitter for SPECT technetium-99m (t½ 13,2 h vs. 6,0 h) and β + -emitter for PET fluorine-18 (t½ 4,2 d vs. 110 min) allowing long-term studies with the corresponding 123/124 I-labelled barbiturate-based tracer in the next steps.
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 −1.34 and 1.26 compared to 12 with a clogD value of 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 phenoxyphenyl core is optimized for the deep and narrow S1' pocket of the target MMPs [8]. While 5b and 6 possess IC50 values for MMP-2 and -9 in the micromolar range (0.67-1.6 µ M, Table 1), 5a is at least a potent MMP-2 inhibitor with an IC50 value of 10 nM. Scheme 2. Syntheses of compounds 9 and 10. Reagents and yields: (a) MeOH, 83% (9), 8% (10).

Enzyme Assays and clogD Values
The MMP inhibition potencies of the barbituric acid derivatives 5a, 5b, 6, 9 and 10 were measured in fluorometric in vitro inhibition assays as described previously [21]. The IC 50 -values of 5a, 5b and 6 were determined for gelatinases MMP-2 and MMP-9 (Table 1), the IC 50 -values of 9 and 10 for MMP-2, MMP-8, MMP-9, MMP-13 and MMP-14 (only 9) ( Table 2). The results are depicted in Tables 1 and 2. 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 −1.34 and 1.26 compared to 12 with a clogD value of 3.53. On the other hand the IC 50 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 phenoxyphenyl core is optimized for the deep and narrow S 1 ' pocket of the target MMPs [8]. While 5b and 6 possess IC 50 values for MMP-2 and -9 in the micromolar range (0.67-1.6 µM, Table 1), 5a is at least a potent MMP-2 inhibitor with an IC 50 value of 10 nM.   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,     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,    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,    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,     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,    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,    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,    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,    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] 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,

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 (Stuart Scientific, Staffordshire, UK) and are uncorrected. 1 H-NMR, 13 C-NMR and 19 F-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 × 4.6 mm) purposes. The following eluents were used (unless specified 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 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.

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 (Stuart Scientific, Staffordshire, UK) and are uncorrected. 1 H-NMR, 13 C-NMR and 19 F-NMR spectra were recorded on ARX 300 and/or AMX 400 spectrometers (Bruker, Karlsruhe, Germany). CDCl 3 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 × 4.6 mm) purposes. The following eluents were used (unless specified 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 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. 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/H 2 SO 4 ) 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 (Na 2 SO 4 ) and concentrated. The crude 2a was used in the next step without further purification.  [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 acid. The precipitate was collected by suction and dried in vacuo. 3a: Yield: 29%. 1  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 (Na 2 SO 4 ) and the solvent was evaporated. The residue was stirred in CHCl 3 for 2 h to give a colorless solid. Yield 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 9) by the barbituric acid derivatives 9 and 10, of human active MMP-2 and MMP-9 by 5a, 5b and 6 were assayed by preincubating MMP-2, MMP-3, MMP-8, MMP-9, MMP-13 or MMP-14 (each at 2 nM) and inhibitor compounds at varying concentrations (10 pM-1 mM) in 50 mM Tris·HCl, pH 7.5, containing 0.2 M NaCl, 5 mM CaCl 2 , 20 µM ZnSO 4 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 IC 50 values for each inhibitor were calculated by non-linear regression analysis, performed using the Grace 5.1.8 software (Linux).

Conclusions
Starting from the lipophilic preclincal research tracer [ 125 I]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 9 with a hydrophilic shift compared to 12 (clogD 0.92 vs. 3.53). Similar to 12 carboxylic acid 9 represents a very potent inhibitor of MMP-2 and -9 (IC 50 (MMP-2) = 29 nM, IC 50 (MMP-9) = 1.3 nM). Therefore radioiodinated analogues [ 123/124 I]9 were successfully synthesized for further in vivo evaluations with SPECT and PET.