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Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in many kinds of life-threatening diseases. Therefore, MMP imaging-for example, with radiolabeled MMP inhibitors (MMPIs)-potentially represents a valuable tool for clinical diagnostics using noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Despite numerous preclinical imaging approaches, translation to a clinical setting has not yet been successful. We introduce and oppose three potential radiofluorinated MMP-targeted imaging probes, modified by the introduction of pentamethine cyanine (Cy5) dyes and therefore containing both radio- as well as fluorescent label with respect to their capability to assess MMP activity in vivo by means of scintigraphic (PET) and/or fluorescent (NIRF) imaging. New hybrid MMPI tracer candidates, structurally based on radiofluorinated pyrimidine-2,4,6-triones (barbiturates) from previous approaches, were synthesized by convenient two-step syntheses. In the first step, Cy5 dyes, varying in the number of sulfonate groups ( n SO 3 - = 1, 2, or 4) and bearing an additional "clickable" alkyne moiety, were coupled to the barbiturate MMPI by amide formation. In the second step, the [ 18 F]fluoride radiolabel was introduced into the resulting Cy5 dye conjugates by "radio-click" chemistry. Biodistribution studies of these hybrid tracer candidates were assessed and compared in C57BL/6 mice by PET as well as fluorescence imaging. MMP activity was imaged in a MMP-positive mouse model of irritant contact dermatitis (ICD) by PET and sequential fluorescence reflectance imaging (FRI), respectively. In vivo data were validated by scintillation counting, gelatin zymography, and MMP-histology. Three new potential hybrid MMP imaging probes were prepared, differing essentially in the number of sulfonate groups, introduced by Cy5 dye components. Although the hydrophilicity of these compounds was substantially increased, 10a ( n SO 3 - = 1) and 10b ( n SO 3 - = 2) were still rapidly eliminated via unfavorable hepatobiliary pathways, as observed in earlier approaches. Only 11 ( n SO 3 - = 4) showed delayed in vivo clearance and a shift towards higher renal elimination. In the chosen mouse model of ICD, only 11 ( n SO 3 - = 4) significantly accumulated in the inflamed mouse ear, which could be precisely visualized by means of PET and FRI.

Integrins are heterodimeric transmembrane protein receptors consisting of different α and β subunits. α(v)β(3) integrins are overexpressed on many tumor cells and tumor-associated angiogenic vessels, whereas α(IIb)β(3) is a receptor for, e.g., fibrinogen and mediates platelet aggregation. In this study, a near-infrared fluorescent imaging probe has been designed and synthesized by conjugating fluorescent dyes to a non-peptidic, pharmacophore-based ligand, based on a molecular modeling design approach. Affinity values were determined, and in vitro cell binding assays and preliminary in vivo xenograft studies in nude mice were performed to evaluate target binding. Competition assays revealed excellent binding and selectivity to α(v)β(3) compared to that for α(IIb)β(3). In vitro, the probe showed high target binding on α(v)β(3)-positive M-21 cells and negligible binding to α(v)β(3)-negative MCF-7 cells. In vivo, the tracer is able to image target expression in U-87 xenografts with a maximum signal-to-noise ratio (SNR) of 2.5:1 at 24 h after injection.

N-Alkylated (S)-7-halogen-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatins were developed as a new group of nonradioactive reference compounds for future radiotracers. Inhibitor potency studies of these compounds suggest that the binding pockets readily accommodate both the 7-halogen substituents and aliphatic side chains (methyl to n-butyl) as well as some ω-fluorinated analogues (3-fluoropropyl and 4-fluorobutyl) at the isatin nitrogen. Indeed, compared to the halogen free parent compounds, some 7-halogenated derivatives exhibited slightly improved inhibitory potencies with IC50 values up to 2.6 nM (caspase-3) and 3.3 nM (caspase-7), respectively. Moreover, the 7-position of isatin, a potential cytochrome P450 hydroxylation site, was substituted by I, Br, Cl, and F to potentially enhance the metabolic stability of isatin sulfonamides. As an example, the radiotracer [(18)F]39 that was produced by (19)F/(18)F isotope exchange was shown to be stable in human blood serum after incubation at 37 °C for at least 90 min.

The noninvasive imaging of matrix metalloproteinases (MMPs) activity in postischemic myocardial tissue holds great promise to predict cardiac function post-myocardial infarction. Consequently, development of MMP specific molecular imaging probes for noninvasive visualization and quantification of MMP activity is of great interest. A novel MMP imaging strategy is based on activatable cell-penetrating peptide probes (ACPP) that are sensitive to the proteolytic activity of MMP-2 and -9. The MMP-mediated activation of these ACPPs drives probe accumulation at the target site. The aim of this study was the development and characterization of radiolabeled MMP-2/9 sensitive ACPPs to assess MMP activity in myocardial remodeling in vivo. Specifically, a short and long-circulating MMP activatable cell-penetrating imaging probe (ACPP and Alb-ACPP, respectively; the latter is an ACPP modified with an albumin binding ligand that prolongs blood clearance) were successfully synthesized and radiolabeled. Subsequently, their biodistributions were determined in vivo in a Swiss mouse model of myocardial infarction. Both peptide probes showed a significantly higher uptake in infarcted myocardium compared to remote myocardium. The biodistribution for dual-isotope radiolabeled probes, which allowed us to discriminate between uncleaved ACPP and activated ACPP, showed increased retention of activated ACPP and activated Alb-ACPP in infarcted myocardium compared to remote myocardium. The enhanced retention correlated to gelatinase levels determined by gelatin zymography, whereas no correlation was found for the negative control: an MMP-2/9 insensitive non-ACPP. In conclusion, radiolabeled MMP sensitive ACPP probes enable to assess MMP activity in the course of remodeling post-myocardial infarction in vivo. Future research should evaluate the feasibility and the predictive value of the ACPP strategy for assessing MMP activity as a main player in postinfarction myocardial remodeling in vivo.

Noninvasive imaging and quantification of matrix metalloproteinase (MMP) activity in vivo are of great (pre)clinical interest. This can potentially be realized by using radiolabeled MMP inhibitors (MMPIs) as positron emission tomography (PET) imaging agents. Triazole-substituted MMPIs, discovered by our group, are highly potent inhibitors of MMP-2, -8, -9, and -13. The triazole ring and its position contribute significantly to the potency of the MMP inhibitor. To evaluate structure-activity relationships (SARs) of the initially discovered triazole-substituted MMPIs, an additional CH2-group between the backbone of the molecule and the triazole core was inserted, and the triazole ring was "inversed" by switching the alkyne and azide groups. Similar to the original triazole-substituted hydroxamates, the inverse triazole MMPIs are excellent inhibitors with promising in vivo properties. Pharmacokinetic properties and metabolic stability of an (18)F-labeled candidate in mice were investigated.

We report here the synthesis of a nonpeptide, small-molecule fluorescent imaging agent with high affinity to aminopeptidase N (APN/CD13), a key player in a variety of pathophysiological angiogenic processes. On the basis of a recently described lead structure, we synthesized three putative precursor compounds by introducing polyethylene glycol (PEG) spacers comprising amino groups for dye labeling. Different attachment sites resulted in substantial differences in target affinity, cell toxicity, and target imaging performance. In comparison to bestatin, a natural inhibitor of many aminopeptidases, two of our compounds (22, 23) exhibit comparable inhibition potency, while a third (21) does not show any inhibiting effect. Cell binding assays with APN-positive BT-549 and APN-negative BT-20 cells and the final fluorescent probes Cy 5.5-21 and Cy 5.5-23 confirm these findings. The favorable characteristics of Cy 5.5-23 will now be proven in in vivo experiments with murine models of high APN expression and may serve as a tool to better understand APN pathophysiology.

The effector caspases-3 and -7 play a central role in programmed type I cell death (apoptosis). Molecular imaging using positron emission tomography (PET) by tracking the activity of executing caspases might allow the detection of the early onset as well as therapy monitoring of various diseases induced by dysregulated apoptosis. Herein, four new fluorinated diastereo- and enantiopure isatin sulfonamide-based potent and selective caspase-3 and -7 inhibitors were prepared by cyclic sulfate ring-opening with fluoride. All fluorohydrins exhibited excellent in vitro affinities (up to IC50 = 11.8 and 0.951 nM for caspase-3 and -7, respectively), which makes them appropriate PET radiotracer candidates. Therefore, N-(4-[(18)F]fluoro-3(R)-hydroxybutyl)- and N-(3(S)-[(18)F]fluoro-4-hydroxybutyl)-5-[1-(2(S)-(methoxymethyl)pyrrolidinyl)sulfonyl]isatin were synthesized in 140 min with 24% and 10% overall radiochemical yields and specific activities of 10-127 GBq/μmol using [(18)F]fluoride in the presence of Kryptofix and subsequent acidic hydrolysis. In vivo biodistribution studies in wild-type mice using PET/computed tomography imaging proved fast clearance of the tracer after tail vein injection.

In vivo imaging of MMPs is of great (pre)clinical interest and can potentially be realized with modern three-dimensional and noninvasive in vivo molecular imaging techniques such as positron emission tomography (PET). Consequently, MMP inhibitors (MMPIs) radiolabeled with positron emitting nuclides (e.g., (18)F) represent a suitable tool for the visualization of activated MMPs with PET. On the basis of our previous work and results regarding radiolabeled and unlabeled derivatives of the nonselective MMPIs, we discovered a new class of fluorinated MMPIs with a triazole-substituted hydroxamate substructure. These novel MMPIs are characterized by an increased hydrophilicity compared with the lead structures and excellent MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13 (IC(50) = 0.006-107 nM). Therefore, one promising fluorinated triazole-substituted hydroxamate (30b) was selected and resynthesised as its (18)F-labeled version to yield the potential PET radioligand [(18)F]30b. The biodistribution behavior of this novel compound was investigated with small animal PET.

Radiolabeled C-5-disubstituted pyrimidine-2,4,6-triones have recently been suggested by our group as a class of potent matrix metalloproteinase (MMP) targeted radiotracers that can noninvasively visualize activated MMPs by means of positron emission tomography (PET). MMPs belong to the zinc- and calcium-dependent endopeptidases which are involved in the proteolytic degradation of components of the extracellular matrix (ECM) but also are capable of processing and releasing bioactive molecules such as growth factors, proteinase inhibitors, and cytokines. Locally increased levels of activated MMPs modulate and contribute to the progression of various diseases, such as cancer, atherosclerosis, stroke, arthritis, and others. Therefore, activated MMPs are suitable biological targets for the specific and noninvasive visualization of aforementioned pathologies in vivo. On the basis of our recent results, we here describe a series of new fluorinated pyrimidine-2,4,6-triones of the second generation with maintained MMP inhibition potencies (IC(50) = 4-605 nM), which are fine-tuned toward more hydrophilic versions, and show the improved biodistribution behavior of one selected radiofluorinated pyrimidine-2,4,6-trione by means of small-animal PET.

The expression and function of endothelin (ET) receptors are abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. A previously reported promising radioligand for positron emission tomography (PET) based on the non-peptide ET(A) receptor antagonist PD 156707 showed specific binding to target receptors in the myocardium but high accumulation in bile and intestine, probably because of its high lipophilicity. In this study we describe the synthesis of a series of fluorinated derivatives with hydrophilic building blocks. All compounds were evaluated as high affinity ET(A) receptor ligands (16, 17, 23-26, K(i) = 1.4-7.9 nM) with high subtype selectivity over the ET(B) receptor. [(18)F]3-Benzo[1,3]dioxol-5-yl-4-{3-[1-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethyl)-1H-[1,2,3]triazol-4-ylmethoxy]-4,5-dimethoxybenzyl}-5-hydroxy-5-(4-methoxyphenyl)-5H-furan-2-one ([(18)F]17) was synthesized as one of the radioligands of this series that possesses a higher hydrophilicity and an excellent stability in human serum. Improved clearance properties and specific uptake in target organs have been confirmed by biodistribution studies and small animal PET imaging.

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be potent inhibitors of human cytosolic phospholipase A2α (cPLA2α). In the course of structure-activity relationship studies, we investigated the effect of a substitution of indole 3 position with acyl, alkyl, and oxadiazole residues. The highest increase of inhibitory potency could be achieved by a 3-methyl-1,2,4-oxadiazol-5-yl-moiety. Appropriate compound 40 revealed an IC50 of 0.0021 μM against isolated cPLA2α. In a cellular assay applying human platelets 40 blocked cPLA2α activity even with an IC50 of 0.0006 μM. Metabolic stability and aqueous solubility of the target compounds were also determined. Furthermore, one selected compound was tested for peroral bioavailability in mice.

Various ω-substituted 1,1-difluoroalkanes are synthesized in good yields from alkyl aryl thioethers by a new oxidative desulfurization-difluorination protocol with the reagents combination of 1,3-dibromo-5,5-dimethylhydantoin (DBH) as an oxidizer and pyridine·9HF (Py·9HF) as a fluoride source. The reaction proceeds via a fluoro-Pummerer-type rearrangement followed by an oxidative desulfurization-fluorination step. Starting from α-fluorinated thioethers, this reaction is promising for (18)F-labeling (τ(1/2) = 110 min) of ligands applicable for positron emission tomography (PET). Using the combination of DBH and carrier-added Py·9H[(18)F]F, an (18)F-labeled difluoride was synthesized from the corresponding α-fluoro thioether with a radiochemical yield of 9%.

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent inhibitors of human cytosolic phospholipase A(2)alpha (cPLA(2)alpha). In continuation of our attempts to develop clinical active cPLA(2)alpha inhibitors, a series of structurally related indole-5-carboxylic acids with reduced lipophilicity was synthesized and tested for cPLA(2)alpha-inhibitory potency. Furthermore, the thermodynamic solubility of these compounds and their metabolic stability in rat liver microsomes were evaluated. With an IC(50) of 0.012 microM against the isolated enzyme, compound 36 was one of the most potent cPLA(2)alpha inhibitors that emerged during the structure-activity relationship study. Concomitantly, 36 possessed the highest water solubility (212 microg/mL at pH 7.4) of all new target compounds. Despite these favorable properties, peroral application of 36 (100 mg/kg) in mice only led to low concentrations of the substance in blood plasma. A very high plasma clearance was observed after intravenous administration of 36 (10 mg/kg). However, in a topical murine model of contact dermatitis, 36 showed a pronounced anti-inflammatory in vivo activity.

A new strategy for the radiolabeling of porous nanocontainers has been developed, and the first experiments in vivo are reported. Our approach consists of the use of nanometer-sized zeolites whose channels have been filled with the positively charged gamma-emitter (111)In(3+) via simple ion exchange. To avoid leaching of the isotope under physiological conditions, the entrances of the channels have been closed using a specifically designed molecular stopcock. This stopcock has a positively charged group that enters the channels and entraps the loaded radionuclides via electrostatic and steric repulsion. The other side of the stopcock is a bulky triethoxysilane group that can covalently bind to the walls of the zeolite entrances, thereby irreversibly closing the channels. The surface of the zeolites has been functionalized with different chemical groups in order to investigate the different biodistributions depending of the nature of the functionalizations. Preliminary in vivo experiments with Wistar rats have been performed and showed the potential of the approach. This strategy leads to a nanoimaging probe with a very high density of radioisotopes in a confined space, which is highly stable in physiological solution and could allow a large variety of functionalities on its external surface.

Caspases are responsible for the execution of the cell death program and are potentially suitable targets for the specific imaging of apoptosis in vivo. A series of N-1-substituted analogues of the small molecule nonpeptide caspase inhibitor (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (1), which may be useful for the development of caspase-targeted radioligands, were synthesized and their inhibition potencies were evaluated in vitro. Two of the most powerful techniques to introduce fluorine into organic compounds, viz, bromofluorination of olefins and fluorohydrin synthesis by ring-opening of epoxides, were used. Most of the target compounds are potent inhibitors of the two effector caspases-3 and -7. Furthermore, the (18)F-radiolabeled model compound (S)-1-[4-(1-[(18)F]fluoro-2-hydroxyethyl)benzyl]-5-[1-(2-methoxymethyl-pyrrolidinyl)sulfonyl]isatin ([(18)F]37), a putative tracer for the noninvasive imaging of apoptosis by positron emission tomography (PET) was synthesized by nucleophilic epoxide ring-opening of its precursor 36. The radiochemistry utilized in the (18)F-fluorination reverted to carrier-added [(18)F]Et(3)N.3HF, a new fluorine-18 source for radiolabeling.

The assessment of matrix metalloproteinase (MMP) activity in vivo is highly desirable in various human diseases such as cancers. Hydroxamic acids based on CGS27023A or CGS25966 are nonpeptidyl lead structures that specifically target activated MMPs in vivo. The aim of this study was the modification and fluorescent labeling of these lead structures to develop a highly affine, nonpeptide MMP inhibitor (MMPI)-ligand for molecular optical imaging of activated MMPs. An 11 step synthesis was developed involving a PEGylated benzyl derivative as a spacer to minimize the interactions between the activated MMP and the dye of conjugate 11 with an azide as a protected amino function. After reducing the azide (Staudinger reaction) and labeling with Cy5.5, we obtained a CGS-based MMP inhibitor 11 with a fluorescent signaling flag. To evaluate the biological properties of this photoprobe, three human cancer cell lines (A-673, HT-1080 and BT-20) were characterized with respect to their MMP-2 and -9 (gelatinases) expression levels (real-time PCR) and protein levels (Western blotting). Initially, fluorogenic inhibition assays were used to assess the MMP inhibition potential. The PEGylated CGS 10 showed complete inhibition of MMP-2 and MMP-9 activities in vitro both for purified MMP-2/-9 (active and pro-forms) and MMP-2/-9 containing cell culture supernatants. To test the imaging potential in biological tissues, gelatinase activity was measured on tumor cryostat sections of the above-mentioned tumor cells using FITC-labeled dye-quenched gelatin. Gelatinase positive tumors revealed strong binding of CGS-Cy5.5 11, while gelatinase negative tumors were not targeted. In conclusion, this new CGS-based MMP photoprobe has a high affinity for MMP-2 and -9 and is thus a promising candidate for sensitive imaging of MMP activity in various diseases in patients.

The measurement of matrix metalloproteinase (MMP) activity in diseases like inflammation, oncogenesis, or atherosclerosis in vivo is highly desirable. Fine-tuned pyrimidine-2,4,6-triones (barbiturates) offer nonpeptidyl lead structures for developing imaging agents for specifically visualization of activated MMPs in vivo. The aim of this study was to modify a C-5-disubstituted barbiturate and thus design a highly affine, nonpeptidic, optical MMP inhibitor (MMPI)-ligand for imaging of activated MMPs in vivo. A convergent 10 step synthesis was developed, starting with a malonic ester and (4-bromophenoxy)benzene to generate 5-bromo-pyrimidine-2,4,6-trione as the key intermediate. To minimize the interactions between activated MMPs and the dye of the conjugate 6, a PEGylated piperazine derivative was used as a spacer and an azide as a protected amino function. After linking both building blocks, reducing the azide ( Staudinger reaction) and labeling with Cy 5.5, we obtained the nonhydroxamate MMP inhibitor 6 with high affinity (IC 50-value: 48 nM for MMP-2) measured in a fluorogenic assay using commercially available MMP-substrates and the purified enzyme. Zymography revealed an efficient blocking of enzyme activity of purified MMP-2 and MMP-9 and of MMP-containing cell supernatants (HT-1080), (A-673) using the PEGylated barbiturate 5. Fluorescence microscopy studies using a highly (A-673) and a moderate (HT-1080) MMP-2 secreting cell line showed efficient binding of the Cy 5.5 labeled tracer 6 to the MMP-2 positive cells while MMP-2 negative cells (MCF-7) did not bind. Therefore, this new barbiturate-based MMP-probe has a high affinity and specificity toward MMP-2 and -9 and is thus a promising candidate for sensitive MMP detection in vivo.

An approach to the in vivo imaging of locally upregulated and activated matrix metalloproteinases (MMPs) found in many pathological processes is offered by positron emission tomography (PET). Hence, appropriate PET radioligands for MMP imaging are required. Here, we describe the syntheses of novel fluorinated MMP inhibitors (MMPIs) based on lead structures of the broad-spectrum inhibitors N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](benzyl)-amino]-3-methyl-butanamide (CGS 25966) and N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)-amino]-3-methyl-butanamide (CGS 27023A). Additionally, tailor-made precursor compounds for radiolabeling with the positron-emitter 18F were synthesized. All prepared hydroxamate target compounds showed high in vitro MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13. As a consequence, the promising fluorinated hydroxamic acid derivative 1f was resynthesized in its 18F-labeled version via two different procedures yielding the potential PET radioligand [18F]1f. As expected, the biodistribution behavior of this novel compound and that of the more hydrophilic variant [18F]1j, also developed by our group, indicates that there was no tissue specific accumulation in wild-type (WT) mice.

A novel fluorescent photoprobe for the imaging of endothelin A receptors (ET(A)R) was developed. Based on the nonpeptidyl, high-affinity, and selective ET(A)R antagonist 3-benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one (PD 156707), a modification of the lead structure with a PEG-spacer containing an amino moiety was performed. Labeling of this precursor with the fluorescent marker Cy 5.5 NHS-ester was accomplished by adaption of common peptide labeling procedures. The affinity of the Cy 5.5-labeled receptor antagonist was evaluated using human carcinoma cell lines with different degrees of ET(A)R expression. Fluorescence microscopy revealed that ET(A)R-positive MCF-7 human breast adenocarcinoma and HT-1080 human fibrosarcoma cells effectively bind the photoprobe at very low doses (nM), while ET(A)R-negative MDA-MB-435 human breast cancer cells showed no fluorescence signal. Binding specificity of the probe could be demonstrated by predosing with a specific ET(A)R antibody or the parent antagonist PD 156707 as a competing inhibitor. The results suggest that the modified photoprobe tightly binds to ET(A) receptors and thus may be a possible candidate for the imaging of ET(A)R-overexpressing tissues in vivo.

Caspases are the unique enzymes responsible for the execution of the cell death program and may represent an exclusive target for the specific molecular imaging of apoptosis in vivo. 5-Pyrrolidinylsulfonyl isatins represent potent nonpeptidyl caspase inhibitors that may be suitable for the development of caspase binding radioligands (CBRs). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin (7) served as a lead compound for modification of its N-1-position. Corresponding pairs of N-1-substituted 2-methoxymethyl- and 2-phenoxymethylpyrrolidinyl derivatives were examined in vitro by biochemical caspase inhibition assays. All target compounds possess high in vitro caspase inhibition potencies in the nanomolar to subnanomolar range for caspase-3 (Ki=0.2-56.1 nM). As shown for compound (S)-1-(4-(2-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (35), the class of N-1-substituted 5-pyrrolidinylsulfonyl isatins competitively inhibits caspase-3. All caspase inhibitors show selectivity for the effector caspases-3 and -7 in vitro. The 2-methoxymethylpyrrolidinyl versions of the isatins appear to possess superior caspase inhibition potencies in cellular apoptosis inhibition assays compared with the 2-phenoxymethylpyrrolidinyl inhibitors.

Studies have demonstrated a positive correlation between inflammation, metastasis, or atherosclerosis and the unbalanced or culminated expression of matrix metalloproteinases (MMPs). The molecular imaging of locally upregulated MMP activity in vivo is a clinical challenge. Actually, radioligands based on nonpeptidyl MMP inhibitors (MMPIs) are currently in development as putative radiopharmaceutical agents for the noninvasive in vivo assessment of activated MMPs. Nonpeptidyl MMPIs bind to the zinc active site of the activated enzyme via mono- (e.g. carboxylate) or bidentate (e.g. hydroxamate) complexation thereby exhibiting a broad-spectrum MMP binding potency. Thus, these mentioned endopeptidase inhibitors should be useable lead compounds for the redevelopment as diagnostic MMPI radiotracers. Recently, the non-hydroxamate C-5-disubstituted pyrimidine-2,4,6-triones were disclosed as subgroup-selective MMP inhibitors. We here describe a set of fine-tuned barbiturates as a new class of MMPI radiotracers for the noninvasive in vivo visualization of activated MMPs using scintigraphic techniques such as SPECT or PET.