C-5-Disubstituted Barbiturates as Potential Molecular Probes for

membrane associated.1 The MMPs are secreted as inactive zymogens .... phenylphosphine)-palladium(0) in refluxing toluene. Structure-Activity .... cal ...
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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 subgroupselective 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 The zinc- and calcium-dependent matrix metalloproteinases (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 characterized. On the basis of their specificity, the MMPs are classified into collagenases, gelatinases, stromelysins, and matrilysins. Another subclass of MMPs is represented by the membrane-type MMPs (MT-MMPs) that additionally contain a transmembrane and intracellular domain, a membrane linker domain, or are membrane associated.1 The MMPs are secreted as inactive zymogens (pro-MMPs). Once activated via the ‘cysteine switch’ mechanism, a zinc active site is provided that initiates its proteolytic activity.2 MMP activity is tightly controlled by several endogenous inhibitors, e.g. the plasma inhibitor R2-macroglobulin, four tissue inhibitors of metalloproteinases (TIMPs),3 or the recently described plasma-membrane-anchored cell surface receptor RECK (reversion-inducing cysteine-rich protein with kazal motifs).4 In pathophysiological processes an increased cytokine and growth-factor-stimulated MMP gene transcription, followed by a pro-MMP secretion and zymogen activation, 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: +49-25183-47362. Fax: +49-251-83-47363. E-mail: [email protected]. † University Hospital of the Westfa ¨ lische Wilhelms-Universita¨t. ‡ Westfa ¨ lische Wilhelms-Universita¨t. § Center of Internal Medicine, University Hospital.

also known as ‘deep pocket’ MMPIs. Nonpeptidyl MMPI classes with a zinc chelating functionality (e.g. carboxylic or hydroxamic acids) have previously been described.9,10 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 hydroxamates 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).

10.1021/jm049145x CCC: $30.25 © 2005 American Chemical Society Published on Web 04/12/2005

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Scheme 1. Synthesis of Barbiturate Derivatives 8a-ea

a Reagents: (a) K CO , DMF; (b) S , morpholine; (c) CH CO H, H O, H SO ; (d) benzyl bromide, KOH, EtOH, H O; (e) SOCl , MeOH; 2 3 8 3 2 2 2 4 2 2 (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-trione, was chosen as the lead compound which is a potent non-hydroxamate MMPI with the phenoxyphenyl residue (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 pyrimidine 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 candidates 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 associated 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 compound 10a as well as the structurally modified barbiturate 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 MT1-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 corresponding radiolabeled analogues, or as the nonradioactive 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

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Scheme 2. Synthesis of Barbiturate Derivatives 10a-pa

a Reagents: (k) NBS, dibenzoylperoxide, CCl ; (l) HBr, Br , H O; (m) R′H, MeOH; (n) [125I]NaI, 2,5-dihydroxybenzoic acid, ascorbinic 4 2 2 acid, CuSO4‚5H2O, EtOH, H2O; (o) CBr4, PPh3, CH3CN; (p) (CH3O)2SO2, NaOH, H2O, ((CH3)2CH)2O (q) N-succinimidyl-3-(tri-nbutylstannyl)-benzoate, DMF; (r) N-succinimidyl-3-iodobenzoate, DMF; (s) ICl, MeOH.

8a-d as well as 2-thioxo-dihydro-pyrimidine-4,6-dione 8e were brominated either with N-bromosuccinimide/ dibenzoyl peroxide or with bromine/hydrobromic acid to yield the bromo compounds 9a-e, that were subsequently 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 converted into 10f with an excess of dimethyl sulfate. Compound 10e was prepared from 10a by heating with carbon tetrabromide and triphenylphosphine in acetonitrile. 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)-benzoate or with N-succinimidyl 3-iodobenzoate28 in refluxing DMF. N-Succinimidyl-3-(tri-n-butylstannyl)-benzoate was synthesized by treatment of N-succinimidyl 3-iodobenzoate with hexabutylditin and tetrakis(triphenylphosphine)-palladium(0) in refluxing toluene. Structure-Activity Relationships (SAR). The potencies of the modified barbiturate analogues 10b-p were measured with MMP-2 and MMP-9 using fluorogenic 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. MMP Inhibition of Barbiturate Derivatives 8a, 10a-p compd

IC50 (MMP-2) [nM]

IC50 (MMP-9) [nM]

8a 10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k 10l 10m 10n 10o 10p

3700 9 7 9 721 30 inactive 5 26 10 85 1500 12 16 86 n. d. 15

n.d. 4 2 6 n.d. 26 inactive 2 1 14 26 550 19 57 10 n. d. 20

a

MMP-9 selectivitya 2.3 3.5 1.5 1.2 2.5 26 0.71 3.3 2.7 0.63 0.28 8.6 0.75

log Db 0.41 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

b

IC50 (MMP-2)/IC50(MMP-9) ratio. log D values calculated by ACD/LogD Suite (log D ) log P at physiological pH 7.4 with consideration of charged species).

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

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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). 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-(4bromophenoxy-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 derivative 10g were prepared. Compared to 10a, all these variations influenced the inihibitory effectiveness of the respective compounds only moderately (Table 1): substitution 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 compound 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, compounds 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). With the series of barbiturates 10k-p we prepared some derivatives with a para-substituted phenylpiperazine residue (10k-n) or a meta-substituted benzoylpiperazine residue (10o and 10p) serving as the S2′

substituent. All nonradioactive representatives of feasible 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 (MMP2)/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. 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 ∼400fold 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 thiocarbonyl group. This modification resulted in a 80-fold loss in MMP-2 potency. Compound 10f represents the 1,3N,N-dimethylated methoxyethyl piperazine analogue of 10c. This modification resulted in a complete loss of gelatinase potency. Radioiodinated Model Compound. The 125I-labeled model radiotracer [125I]10b was obtained using nocarrier-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 nonradioactive reference compound of [125I]10b to prove its identity. Compound [125I]10b was isolated in 49% average radiochemical yield. The chemical and radiochemical purities were >90%. The calculated specific activity of [125I]10b was 0.146 GBq/µg (2.176 Ci/µmol) at the endof-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 (calculated log D ) 1.60 (pH 7.4)) that specifically accumulates 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 comparison with the parent compound CGS 27023A (experimental 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]benzenesulfonyl]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-methylbutyric acid [18F]SAV0316 (IC50(MMP-2) ) 1.9 µM). The micromolar MMPI radiotracer [18F]SAV03 was signifi-

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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 carboxylate-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 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 pyrimidine-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 5,5disubstituted 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-spectrum MMPIs which additionally are known to show side effects such as musculoskeletal syndrome and maculopapular rash if therapeutically applied (e.g. peptidomimetic batimastat (BB94) and marimastat (BB-2516),9 e.g. nonpeptidomimetic CGS 27023A33). Recently, the structurally related pyrimidine trione derivative Ro-282653, 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 without 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 barbiturates 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(MMP9) 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 selectivity was ascertained for the amino derivative 10i, which obviously shows a slight preference for MMP-2 (IC50

(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 radiofluorinated PET tracer which would result from a substitution 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 radiotracer. In the series of aryl piperazine derivatives 10k-p, the trimethylsilyl derivative 10k and the stannyl derivative 10o were synthesized as precursor compounds of the corresponding radioiodinated analogues of 10n (iododesilylation) and 10p (iododestannylation), respectively. 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 analogue of 10l. As nucleophilic radiofluorination of aromatic systems is restricted only to strong electrondeficient 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 presence 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 relevant 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 400and 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 complete 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 modifications 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

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computed tomography (SPECT) or positron emission tomography (PET). Four complete pairs of precursors with the corresponding 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.

(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-(4Phenoxy-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-ethanethione 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-ethanethione 4b. Yield: 88% of a mustard yellow solid. Mp: 123127 °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). 2-[4-(4-Methoxy-phenoxy)-phenyl]-1-morpholin-4-yl-ethanethione 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-ethanethione 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-Phenoxy-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.

Experimental Section General Methods. Chemistry. All chemicals, reagents, and solvents for the synthesis of the compounds were analytical grade, purchased from commercial sources and used without further purification, unless otherwise specified. Melting 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-dimensional 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 performed by gradient radio-HPLC each with a RP-HPLC Nucleosil 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: CH3CN/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-(4Phenoxy-phenyl)-ethanones 3a-d. 4-Fluoroacetophenone 2 (25.0 g, 181 mmol) was dissolved in 180 mL of dimethylformamide, 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. 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

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[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. Potassium 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 recyristallised 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-Phenoxy-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 concentration 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 Ester 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. [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 Ester 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-Benzyloxyphenoxy)-phenyl]-acetic Acid 5e. A few drops of concentrated 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. Method B: Reaction of [4-(4-Hydroxy-phenoxy)-phenyl]-acetic Acid Methyl Ester 6d with Benzyl Trichloroacetimidate. Methyl ester 6d (600 mg, 2.32 mmol) was dissolved in 10 mL of methylene chloride. To the stirred

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 temperature 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 (Na2SO4). 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-Phenoxy-phenyl)-malonic Acid Dimethyl Esters 7a-d. A suspension of 2.0 equiv of NaH (60% suspension in paraffin oil (paraffin oil was removed by repeated washings with petroleum 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 overnight. 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 Dimethyl Ester 7c. The raw material was purified by silica gel chromatography (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]-malonicAcidDimethyl 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. General Procedure for the Preparation of the 5-(4Phenoxy-phenyl)-pyrimidine-2,4,6-triones 8a-d and 5-(4Phenoxy-phenyl)-2-thioxo-dihydro-pyrimidine-4,6-dione 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, dimethyl 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-trione 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.

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5-[4-(4-Methoxy-phenoxy)-phenyl]-pyrimidine-2,4,6trione 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 material, 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 (decomposition). MS (MALDI-TOF) m/e: 349 (M + Na)+, 327 (M + H)+. Anal. (C17H14N2O5) C, H, N. 5-[4-(4-Benzyloxy-phenoxy)-phenyl]-pyrimidine-2,4,6trione 8d. Yield: 88% of a luminous yellow solid. Mp: 215217 °C (decomposition). MS (ESI-EM) m/e: 402.1166 (M)+ Calcd for C23H18N2O5 402.1195. [5-(4-(4-Bromo-phenoxy)-phenyl]-2-thioxo-dihydro-pyrimidine-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-Bromo5-(4-phenoxy-phenyl)-pyrimidine-2,4,6-triones 9a-d and 5-Bromo-5-(4-phenoxy-phenyl)-2-thioxo-dihydro-pyrimidine-4,6-dione 9e. Method A: A suspension of the corresponding pyrimidine2,4,6-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]-pyrimidine2,4,6-trione 9a. Yield: 52% (Method A) or 56% (Method B) of a colorless solid (chloroform). Mp: 153-155 °C. MS (MALDITOF) m/e: 375, 377 (M - Br + H)+. Anal. (C16H10Br2N2O4) C, H, N. 5-Bromo-5-[4-(4-iodo-phenoxy)-phenyl]-pyrimidine2,4,6-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]-pyrimidine2,4,6-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-pyrimidine-2,4,6-trione 9d. Yield: 78% (Method B) of an off-white solid. 5-Bromo-5-[4-(4-bromo-phenoxy)-phenyl]-2-thioxo-dihydro-pyrimidine-4,6-dione 9e. Yield: 36% (Method A) of a brown solid (chloroform/petroleum ether). General Procedure for the Preparation of 5-[4-(2Hydroxy-ethyl)-piperazin-1-yl]-5-(4-phenoxy-phenyl)-pyrimidine-2,4,6-triones 10a-c and 5-[4-(2-Hydroxy-ethyl)piperazin-1-yl]-5-(4-phenoxy-phenyl)-2-thioxo-dihydropyrimidine-4,6-dione 10d. A solution of 5-bromopyrimidine2,4,6-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 compound 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%.

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-phenoxy)-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-methoxyphenoxy)-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-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. Synthesis of 5-[4-(4-Bromo-phenoxy)-phenyl]-pyrimidine-2,4,6-trione Derivatives 10e-j. 5-[4-(2-Bromo-ethyl)piperazin-1-yl]-5-[4-(4-bromo-phenoxy)-phenyl]-pyrimidine-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-methoxyphenoxy)-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 chloroform (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-pyrimidine-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)-piperazin-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-phenoxy)-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.

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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. General Procedure for the Synthesis of Phenyl-piperazine Derivatives 10k-n. The corresponding phenyl-piperazine (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-trimethylsilylphenyl)-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 suspension 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 dichloromethane (3 × 50 mL), washed with brine, and dried (Na2SO4). 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 (MALDITOF) m/e: 661 (M+). Anal. (C26H22N4O4BrI) C, H, N. Synthesis of Benzoylpiperazine Derivatives 10o,p. Synthesis of N-Succinimidyl-3-(tri-n-butylstannyl)-benzoate. 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 subsequently 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. 5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(3-(tri-n-butylstannyl)-benzoyl)-piperazin-1-yl]-pyrimidine-2,4,6-trione 10o. To a suspension of 10g (90.5 mg, 197 µmol) in 5 mL of dimethylformamide was added N-succinimidyl 3-(tri-nbutylstannyl)-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 C39H49BrN4O5SnNa 875.1806. 5-[4-(4-Bromo-phenoxy)-phenyl]-5-[4-(3-iodo-benzoyl)piperazin-1-yl]-pyrimidine-2,4,6-trione 10p. To a suspension 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-

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-1yl]-5-[4-(4-[125I]iodo-phenoxy)-phenyl]-pyrimidine-2,4,6trione [125I]10b. 2,5-Dihydroxybenzoic acid (0.6 mg), ascorbinic 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 separated. 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-4yl)-acetyl-pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)-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 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 value for each inhibitor was calculated by nonlinear regression analysis using Grace 5.1.8 software (Linux).

Acknowledgment. This work was supported in part by a research grant from Radiopharmaceuticals Research Amersham Health plc., Amersham, U.K. The authors gratefully acknowledge the technical assistance of S. Schro¨er, S. Fatum, and M. Trub. 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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