pharmaceuticals Article

Syntheses of Radioiodinated Pyrimidine-2,4,6-Triones as Potential Agents for Non-Invasive Imaging of Matrix Metalloproteinases Hans-Jörg Breyholz 1 , Klaus Kopka 2 , Michael Schäfers 3 and Stefan Wagner 1, * 1 2 3

*

Department of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1, D-48149 Münster, Germany; [email protected] Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany; [email protected] European Institute for Molecular Imaging (EIMI), University of Münster, Waldeyerstraße 15, D-48149 Münster, Germany; [email protected] Correspondence: [email protected]; Tel.: +49-251-83-44713

Academic Editor: Jean Jacques Vanden Eynde Received: 8 May 2017; Accepted: 28 May 2017; Published: 30 May 2017

Abstract: Dysregulated expression or activation of matrix metalloproteinases (MMPs) is observed in many kinds of live-threatening diseases. Therefore, MMP imaging for example with radiolabelled MMP inhibitors (MMPIs) potentially represents a valuable tool for clinical diagnostics using non-invasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. This work includes the organic chemical syntheses and in vitro evaluation of five iodinated barbiturate based MMPIs and the selection of derivative 9 for radiosyntheses of isotopologues [123 I]9 potentially useful for MMP SPECT imaging and [124 I]9 for MMP PET imaging. Keywords: pyrimidine-2,4,6-triones; radioiodination; matrix metalloproteinase inhibitor; fluorometric in vitro inhibition assays

1. Introduction The in vivo molecular imaging of locally upregulated and activated matrix metalloproteinases (MMPs) that are observed in pathologies such as cardiovascular diseases, inflammation or cancer remains a substantive clinical issue [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 [8–11] 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

Pharmaceuticals 2017, 10, 49; doi:10.3390/ph10020049

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the first time the barbiturate-based near-infrared fluorescent photo probe Cy5.5-A 10, 49 2 of 11 suitable for in vitro and influorescent vivo imaging of the gelatinases MMP-2 andwas MMP-9 [13 the first time the barbiturate-based near-infrared photo probe Cy5.5-AF443 that ex near-infrared vivo applications In 2008 we developed time the evaluation barbiturate-based near-infrared published the for radiosynthesis and of MMP-9 the first fluorine-18 (18F)we labelled ba suitable fluorescent for [12]. in vitro and in probe vivo imaging ofthe thefirst gelatinases MMP-2 and [13,14]. In 2010 e barbiturate-based photo Cy5.5-AF443 that was fluorescent probe Cy5.5-AF443 that was suitable forfirst in vitro in(18vivo imaging of theradiofluorinated MMPI [15] and twoIn later, of and several more hydrophilic the radiosynthesis and evaluation ofyears the fluorine-18 F) labelled barbiturate-based tro and in vivo imaging ofpublished thephoto gelatinases MMP-2 and MMP-9 [13,14]. 2010 we gelatinases MMP-2 and MMP-9 [13,14]. In of 2010 we published the radiosynthesis and evaluation improved biodistribution [16].radiofluorinated Moreover, a gallium-68 (68Ga) labelle MMPI and two years several more behavior hydrophilic analogues with diosynthesis and evaluation of the[15] first fluorine-18 (18F)later, labelled barbiturate-based 18 first fluorine-18 ( F) labelled barbiturate-based MMPI [15] and Favorable two years later, of several introduced by[16]. our group inwith 2012 [17]. MMP binding affinities improved biodistribution behavior Moreover, a gallium-68 (68Ga) labelled version wasfor our ba two years later, ofofthe several more hydrophilic radiofluorinated analogues 68 more hydrophilic radiofluorinated analogues with improved biodistribution behavior [16]. Moreover, indeed measured in vitro assays and in vivo biodistribution studies grouptracers in( 2012 [17]. Favorable MMP binding affinities for our barbiturate-based stribution behavior [16]. introduced Moreover, by a our gallium-68 Ga)were labelled version wasby a gallium-68 (68MMP Ga) labelled version was by our inbiodistribution 2012 [17]. MMP However, invitro animal models with increased MMPFavorable expression mentioned tracers were indeed by in assays and group in vivo studies using wt-mice.barbitura ur group in 2012 [17]. Favorable bindingmeasured affinities forintroduced our barbiturate-based Pharmaceuticals 2017, 10, 49 2 of 11 binding affinities forinour barbiturate-based tracers were indeed measured by in and in vivo did not the expectations. Anyhow invitro vivoassays MMP imaging wastracers feasible and sp However, animal models withmeet increased MMP expression mentioned barbiturate-based eed measured by in vitro assays and vivo biodistribution studies using wt-mice. studies using wt-mice. However, in animal models with increased MMP expression hitherto most encouraging optical imaging probe Cy5.5-AF443 suggesting the a meet the expectations. Anyhow influorescent vivo MMP imaging was feasible and specific with our mal models with biodistribution increaseddid MMP expression mentioned barbiturate-based tracers thenot first time the barbiturate-based near-infrared photo probe Cy5.5-AF443 that was suitable for inimaging vitro and in vivo imaging of the specific gelatinases MMP-2 and MMP-9 [13,14]. 2010 we mentioned barbiturate-based tracers did not meet the expectations. Anyhow vivoInMMP imaging was compared improved imaging performance of thein photoprobe Cy5.5-AF443 to most encouraging optical imaging probe Cy5.5-AF443 suggesting the assumption that expectations. Anyhow inhitherto vivo MMP was feasible and with our published the radiosynthesis and evaluation of the first fluorine-18 (18F) labelled barbiturate-based and specificCy5.5-AF443 with our hitherto most encouraging optical imaging probe Cy5.5-AF443 suggesting radiotracers caused by the cyanine dye substituent four hydrophil improved imaging performance oftheis the photoprobe Cy5.5-AF443 compared towith the the barbiturate ncouraging opticalfeasible imaging probe suggesting assumption that MMPI [15] and two years later, of several more hydrophilic radiofluorinated analogues with assumption that biodistribution improved the photoprobe compared the physicochemical moieties. Actually, structural characteristics changetosulfonic the radiotracers is causedimaging by theperformance cyanine the four hydrophilic acid ng performance the of the photoprobe Cy5.5-AF443 compared to dye theofthese barbiturate improved behavior [16]. Moreover, asubstituent gallium-68 (68with Ga) Cy5.5-AF443 labelled version was introduced by our in accordingly 2012 [17]. Favorable MMP binding affinities forthe our four barbiturate-based barbiturate radiotracers isgroup caused bystructural the cyanine dye substituent with hydrophilic sulfonic the essential biodistribution pattern influenced i.a. by the excretion moieties. Actually, these characteristics change physicochemical properties and aused by the cyanine dye substituent with the four hydrophilic sulfonic acid tracers were indeed measured by in vitro assaysplasma-protein and in vivo biodistribution studies acidcharacteristics moieties. Actually, characteristics change the physicochemical and(renal hepatobiliary), binding, binding to properties non-target-organs and/or accordingly thethese essential biodistribution pattern influenced i.a. by using the wt-mice. excretion routes or off-tar ly, these structural change thestructural physicochemical properties and However, in animal models with increased MMP expression mentioned barbiturate-based tracers accordingly the essential biodistribution pattern influenced i.a. by the excretion (renalinteractions orfeatures from op other proteins. Into summary, adopting orroutes transferring hepatobiliary), binding, binding non-target-organs and/or off-target essential biodistribution pattern i.a. bywith the excretion routes (renal orfeasible did notinfluenced meet theplasma-protein expectations. Anyhow in vivo MMP imaging was and specific with our radiotracers could support their development, an from aspect that was recently hepatobiliary), plasma-protein binding, binding to non-target-organs and/or off-target with other Inoptical summary, adopting or transferring optical tracers to reviewed lasma-protein binding, binding to non-target-organs and/or off-target interactions hitherto mostproteins. encouraging imaging probe Cy5.5-AF443 suggesting thefeatures assumption thatinteractions improved imaging performance of the photoprobe Cy5.5-AF443 compared to the barbiturate [18]. Therefore, the aim ofaspect thistowork was recently thetracers synthesis of radioiodinated radiotracers support their development, an that was reviewed by Faust et al.barbitura withadopting other proteins. Incould summary, adopting or transferring features from optical to radiotracers eins. In summary, or transferring features from optical tracers radiotracers is caused by the cyanine dye substituent with the four hydrophilic sulfonic acid tracers with increased hydrophilicity forFaust potential SPECT/PET imaging. [18].an Therefore, thewas aim recently of work the synthesis MMPIRadionuc d support their development, aspect that reviewed Faust et al.of radioiodinated could support their development, anthis aspect thatwas wasby recently reviewed by et al.barbiturate-based [18]. Therefore, moieties. Actually, these structural characteristics change the physicochemical properties and and iodine-124 were used for the radiosyntheses and applied on our target molecule tracers with increased hydrophilicity for potential SPECT/PET imaging. Radionuclides iodine-123 he aim of this work the synthesis of radioiodinated barbiturate-based MMPI the was aim of this work was the synthesis of radioiodinated barbiturate-based MMPI tracers with increased 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 interactions log units more hydrophilic compared to our initial preclinical research [125I and iodine-124 were used for the radiosyntheses andas applied onoff-target our target molecule 9, used which is ca. tracer 3 eased hydrophilicity for potential imaging. Radionuclides iodine-123 hydrophilicity forSPECT/PET potential SPECT/PET imaging. Radionuclides iodine-123 and iodine-124 were with other proteins. In summary, adopting or transferring features from optical tracers to 125 log units hydrophilic compared to initial research tracer [ I]12 (see Table 2) were used for the radiosyntheses andmore applied on our[12]. target molecule 9, our which ca.preclinical 3 is ca. 3 log To achieve increased hydrophilicity two different chemical modificatio for the radiosyntheses and applied onasour target molecule 9,iswhich units more hydrophilic radiotracers could support their development, an aspect that was recently reviewed by Faust et al. 125 125 phenoxyphenyl moiety in 12 that occupies the S1’ enzyme pocket were realized ydrophilic as compared to our initial preclinical research tracer [ I]12 (see Table 2) [12]. To achieve increased hydrophilicity two different chemical modifications of the C5 as compared to our initial preclinical research tracer [ I]12 (see Table 2) [12]. To achieve increased [18]. Therefore, the aim of this work was the synthesis of radioiodinated barbiturate-based MMPI phenoxyphenyl moiety in 12 modifications that occupies the S1’imaging. enzyme pocket were realized. the commercially available radionuclides iodine-123 (for in SPECT) (fo e increased hydrophilicity twotwo different chemical of the C5phenoxyphenyl hydrophilicity different chemical modifications of the C5 moiety 12Moreover thatand iodine-124 tracers with increased hydrophilicity for potential SPECT/PET Radionuclides iodine-123 and iodine-124 were used for thewere radiosyntheses andMoreover applied on(for our 9, which is ca. 3γ-emitter prolonged half-lives t½ compared to molecule the most common for exhibit SPECT techneti moiety in 12 thatoccupies occupies the S1’ enzyme pocket realized. thetarget commercially available radionuclides iodine-123 SPECT) and iodine-124 (for PET) the S1’ enzyme pocket were realized. Moreover the commercially available radionuclides log units more hydrophilic as compared to our initial preclinical research tracer [125I]12 (see Table 2) h vs. 6,0(for h) β+exhibit -emitter for PET fluorine-18 ( t½ compared 4,2 d vs. 110 prolonged t½and compared to and the most common γ-emitter for SPECT technetium-99m (t½ 13,2 long-te vailable radionuclides iodine-123 (forhalf-lives SPECT) iodine-124 (for PET) exhibit iodine-123 (for SPECT) and iodine-124 PET) prolonged half-lives to min) the allowing [12]. To achieve increased hydrophilicity two different chemical modifications of the C5 + -emitter 123/124 +for the corresponding I-labelled barbiturate-based tracer in the next steps. h vs. 6,0 h) and β -emitter for PET fluorine-18 ( t ½ 4,2 d vs. 110 min) allowing long-term studies with ves t½ compared to the most common γ-emitter for SPECT technetium-99m (t ½ 13,2 most common γ-emitter SPECT technetium-99m ( 13.2 h vs. 6.0 h) and β for PET phenoxyphenyl moiety in 12 that occupies the S1’ enzyme pocket were realized. Moreover the 123/124 I-labelled 123/124 +-emitter for PET fluorine-18 available radionuclides iodine-123 (for SPECT)tracer and the iodine-124 (forsteps. PET) exhibit the(commercially I-labelled in the next ½ 4.2 4,2ddvs. vs. 110min) min) allowingbarbiturate-based long-term studies with fluorine-18 (tcorresponding 110 allowing long-term studies with corresponding prolonged half-lives t½ compared to the most common γ-emitter for SPECT technetium-99m (t½ 13,2 2.steps. Results and Discussion g 123/124I-labelled barbiturate-based tracer ininthe next barbiturate-based tracer the next steps. h vs. 6,0 h) and β+-emitter for PET fluorine-18 (t½ 4,2 d vs. 110 min) allowing long-term studies with 2. Results and Discussion the corresponding 123/124I-labelled barbiturate-based tracer in the next steps. 2. Results and Discussion 2.1. Chemistry iscussion 2.1.2. Chemistry Results and Discussion Phenyl barbiturates 5a–d and 6 were prepared as outlined in Scheme 1 by fou 2.1. Chemistry five-6(compound 5d) or (compound respectively. 5a–d and were prepared assix-step outlined in Scheme6)1 sequences, by four- (compounds 2.1.Phenyl Chemistrybarbiturates 5a–c), Phenyl barbiturates 5a–d in and 6orwere prepared as outlined in Scheme 1 by four- (compounds 5a–c), five(compound 5d) six-step (compound 6) sequences, respectively. iturates 5a–d and 6 were prepared as outlined Scheme 1 by four(compounds Phenyl barbiturates 5a–d and 6 were prepared as outlined in Scheme 1 by four- (compounds 5a–c), five- 5a–c), (compound 5d) or5d) six-step (compound sequences, respectively. five(compound or six-step (compound 6) 6) sequences, respectively. pound 5d) or six-step (compound 6) sequences, respectively.

1. Syntheses of phenyl barbiturates5a–d 5a–d and and yields: (a) NaH,(a) dimethyl Scheme 1. Scheme Syntheses of phenyl barbiturates and6.6.Reagents Reagents and yields: NaH, dimethyl 82% (2a), 73% (2b); (b) urea, NaOEt, EtOH, 29% (3a), 91% (3b); (c) NBS, carbonate, carbonate, dioxane,dioxane, 82% (2a), 73% (2b); (b) urea, NaOEt, EtOH, 29% (3a), 91% (3b); (c) NBS, dibenzoyl peroxide, CCl4, 51% (4a); Br2, HBr, H2O, 89% (4b); (d) N-(2-hydroxyethyl)-piperazine or Scheme 1. Syntheses of phenyl barbiturates 5a–d and 6. Reagents and yields: (a) Na dibenzoyl peroxide, CCl4 , 51% (4a); Br 2 , HBr, H2 O, 89% (4b); (d) N-(2-hydroxyethyl)-piperazine or carbonate, dioxane, 82% (2a), (b) MeOH, urea, NaOEt, EtOH, 29% (3a), 91% (3 Scheme 1. Syntheses phenyl barbiturates 5a–d and 6. 73% Reagents and yields: (a) dimethyl 3-(piperazin-1-yl)-propionic acid,of MeOH, 53% (5a), 18% (5b), 28% (5c); (e) H(2b); 82%NaH, (5d); 2 , Pd/C, 4 , 51% (4a); Br 2 , HBr, H 2 O, 89% (4b); (d) N-(2-hydroxyethyl)-p dibenzoyl peroxide, CCl carbonate, dioxane, 82% (2a), 73% (2b); (b) urea, NaOEt, EtOH, 29% (3a), 91% (3b); (c) NBS, yntheses of phenyl barbiturates 5a–d and 6. Reagents (f) NaI, NaOCl, NaOH, MeOH, and 12% yields: (6). (a) NaH, dimethyl 2, HBr, 2O, 89% (4b); (d) N-(2-hydroxyethyl)-piperazine or dibenzoyl peroxide, oxane, 82% (2a), 73% (2b); (b) urea, NaOEt, EtOH, CCl 29%4, 51% (3a), (4a); 91% Br (3b); (c) H NBS,

roxide, CCl4, 51% (4a); Br2, HBr, H2O, 89% (4b); (d) N-(2-hydroxyethyl)-piperazine or

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3-(piperazin-1-yl)-propionic acid, MeOH, 53% (5a), 18% (5b), 28% (5c); (e) H2, Pd/C, MeOH, 82% (5d); (f) NaI, NaOCl, MeOH, 12% (6). Pharmaceuticals 2017, 10, NaOH, 49 3 of 11

In detail, 4-iodophenyl acetic acid methylester (1a), synthesized by the esterification of In detail,4-iodophenyl 4-iodophenyl acetic acid (1a), synthesized by the esterification of to commercial commercial acidmethylester using MeOH/H 2SO4 was methoxycarbonylated give the 4-iodophenyl acid ester using 2a. MeOH/H SO was methoxycarbonylated to give the corresponding correspondingacetic malonic 4-Benzyloxyphenyl malonic acid dimethyl ester (2b) [19] was 2 4 malonic 4-Benzyloxyphenyl malonic acid dimethyl [19] wastoobtained from methyl obtainedester from2a.methyl 2-((4-benzyloxy)phenyl)acetate (1b)ester [20] (2b) analogous 2a by the literature 2-((4-benzyloxy)phenyl)acetate (1b)2b[20] analogous 2a by the using literature procedure. Malonic esters procedure. Malonic esters 2a and were cyclizedtowith urea sodium ethoxide as a base to 2a andthe 2b5-phenylbarbituric were cyclized withacid ureaderivatives using sodium ethoxide as a subsequently base to yield the 5-phenylbarbituric yield 3a and 3b, which were brominated with acid derivatives 3a and(NBS) 3b, which subsequently brominated(compound with N-bromosuccinimide (NBS) N-bromosuccinimide (compound 3a) orwere bromine/HBr 3b), resulting in the (compound 3a) orbarbituric bromine/HBr 3b), resulting in the 5-bromo-5-phenyl barbituric 5-bromo-5-phenyl acids (compound 4a and 4b. These were reacted with either of the commercially acids 4a piperazines and 4b. These were reacted with eitherorof3-(piperazin-1-yl)-propionic the commercially available piperazines available N-(2-hydroxyethyl)-piperazine acid in MeOH N-(2-hydroxyethyl)-piperazine or 3-(piperazin-1-yl)-propionic in MeOH to yield the barbituric to yield the barbituric acid derivatives 5a–c in overall yieldsacid of 6% (5a), 2% (5b) and 17% (5c). acid derivatives 5a–c in overall yields 6%achieved (5a), 2% by (5b)catalytic and 17% (5c). Cleavage(Hof2, the benzyloxy Cleavage of the benzyloxy group in 5cof was hydrogenation Pd/C, MeOH) group in 5c achievedcompound by catalytic5d hydrogenation MeOH)ofresulting the hydroxyl resulting in was the hydroxyl in 14% overall(H yield. Iodination 5d usingin sodium iodide, 2 , Pd/C, sodium hypochlorite sodium yielded iodide, the ortho iodination product and 6 in compound 5d in 14% and overall yield.hydroxide Iodinationinofmethanol 5d using sodium sodium hypochlorite sodium hydroxide 2% overall yield. in methanol yielded the ortho iodination product 6 in 2% overall yield. To obtain obtain carboxy carboxy derivative 9 and fluoro derivative 10 (Scheme 2) the bromo intermediate To intermediate 7, whose synthesis was described previously [12], was reacted either either with with commercially commercially available available 3-(piperazin-1-yl)-propionic acid 3-(piperazin-1-yl)-propionic acid or piperazine 8 [15]. The The yields yields were were 83% 83% for for 99 and 8% for 10 (Scheme 2).

Scheme 2. Syntheses of compounds 9 and 10. Reagents and yields: (a) MeOH, 83% (9), 8% (10).

2.2. Enzyme and clogD clogD Values Values 2.2. Enzyme Assays Assays and The MMP MMPinhibition inhibitionpotencies potencies of the barbituric acid derivatives andmeasured 10 were The of the barbituric acid derivatives 5a, 5b, 5a, 6, 9 5b, and 6, 10 9were measured in fluorometric in vitroassays inhibition assays as described[21]. previously TheofIC5a, 50-values of in fluorometric in vitro inhibition as described previously The IC50[21]. -values 5b and 6 5a, 5b and 6 were determined for gelatinases MMP-2 and MMP-9 (Table 1), the IC 50-values of 9 and were determined for gelatinases MMP-2 and MMP-9 (Table 1), the IC50 -values of 9 and 10 for MMP-2, 10 for MMP-2, MMP-8, MMP-9, MMP-13(only and MMP-14 9)results (Table are 2). The results depicted MMP-8, MMP-9, MMP-13 and MMP-14 9) (Table (only 2). The depicted inare Tables 1 andin 2. Tables 1 and The tables also contain the calculated logP/logD values (clogP/clogD) of the The tables also 2. contain the calculated logP/logD values (clogP/clogD) of the synthesized barbituric synthesized barbituric acidsthe derivatives indicate thecaused changes caused by the acids derivatives to indicate changes oftolipophilicities by of thelipophilicities structural modifications. structural modifications. Replacement of the phenoxyphenyl moiety in 12 (see Table 1) by a phenyl group resulted in Replacement of the in 12 (see 1) by5a–b a phenyl resulted in compounds 5a–d and 6. phenoxyphenyl As expected themoiety lipophilicities ofTable iodinated and 6group are significantly 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 reduced, with clogD values between −1.34 and 1.26 compared to 12 with a clogD value of 3.53. On the other hand the ICranging 50 values for MMP-2 and -9 of these compounds are generally increased 3.53. On the other hand the IC 50 values for MMP-2 and -9 of these compounds are generally compared to the derivatives with a phenoxyphenyl residue (Table 2). This is also expected because the increased compared tooptimized the derivatives phenoxyphenyl residue 2). This is also expected phenoxyphenyl core is for thewith deepa and narrow S1 ’ pocket of(Table the target MMPs [8]. While 5b because the phenoxyphenyl core is optimized for the deep and narrow S 1’ pocket of the target MMPs and 6 possess IC50 values for MMP-2 and -9 in the micromolar range (0.67–1.6 µM, Table 1), 5a is at [8]. While 5b MMP-2 and 6 possess ICwith 50 values for MMP-2 and -9 in the micromolar range (0.67–1.6 µ M, least a potent inhibitor an IC50 value of 10 nM. Table 1), 5a is at least a potent MMP-2 inhibitor with an IC50 value of 10 nM.

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Pharmaceuticals 2017, 2017, 10, 10, 49 49 Pharmaceuticals Pharmaceuticals 2017,Table 10, 49 1. IC50 values of phenyl barbiturates 5a, 5b and 6. Pharmaceuticals Table1.1.IC IC5050values valuesofofphenyl phenylbarbiturates barbiturates5a, 5a,5b 5band and6.6. Table Pharmaceuticals 2017, 2017, 10, 10, 49 49 Pharmaceuticals Pharmaceuticals 2017, 2017, 10, 10, 49 49

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Table 1. 1. IC50 50 values of phenyl barbiturates 5a, 5b and 6. Table Table 1. IC IC50 values values of of phenyl phenyl barbiturates barbiturates 5a, 5a, 5b 5b and and 6. 6. Table 1. IC 50 values of phenyl barbiturates 5a, 5b and 6. Table 1. IC50 values of phenyl barbiturates 5a, 5b and 6. Table values of phenyl barbiturates 5a, 5b and 6. Table 1. 1. IC IC50 50 values of phenyl barbiturates 5a, 5b and 6.

Cpd. R1 R2 R2R2 Cpd.Cpd. R1R1 Cpd. R R11 R R22 Cpd. Cpd. 5a −I −H R1 R2 Cpd. R 1 R 2 Cpd. R−I1 R−H 2 5a Cpd. R1 R2

R3 R3 R3 R R33 R R33

R33 R R3

IC50 [nM] IC50 [nM] a clogD a IC50 [nM] MMP-2 MMP-9 clogP IC50 50 [nM] a clogD a IC [nM] MMP-2 MMP-9 clogP a a ICMMP-9 50 [nM] MMP-2 MMP-9 a clogD a clogD clogPclogP a 1.26 a IC 50±[nM] 10 ± MMP-2 3MMP-2 550 180 1.42 MMP-9 clogP clogD a a 50 [nM] MMP-2IC MMP-9 clogP clogD IC [nM] a 10 ± 3 MMP-9 550 ± clogP 180 a 1.42 1.26 IC50 50 [nM] MMP-2 MMP-2 MMP-9 clogP a clogD clogD a

5a −I 1010± ±± 550550 ± 180 180 R 2−H −H 5a Cpd. −5a I 1 R−I ± 180 1.42 1.26 a 1.42 a 1.26 333 3 550 MMP-2 clogD MMP-210 MMP-9 clogP clogD a 1.26 5a −I −H −H 10 ±MMP-9 550 ±±clogP 180 a 1.42 1.42 1.26 5b 5a −I −H 1200 ± 340 1600 ± 600 −0.85 −1.34 −I −H 10 ± 3 550 ± 180 1.42 1.26 5a −I −H 10 ± 3 550 ± 180 1.42 1.26 5a −I 10 ±± 1200 33 ± 340 ±±1600 180 1.42 1.26 5b −I −H −H ±±600 −0.85 −1.34 5a −I −H 101200 550 180 1.42 −0.85 1.26 −1.34 5b −I−H −H −H ±550 340 1600 600 −I 1200 ± 340 1600 ± 600 −0.85 −1.34 5b −5b I5b 1200 ± 340 1600 ± 600 −I −H 1200 ± 340 1600 ± 600 −0.85−0.85 −1.34 −1.34 1200 ±± 600 6 5b 670 ±±± 340 210 1600 1300 ± 320−0.85 0.75 −1.34 5b−OH−I −I −I −H −H 1200 340 1600 600 −0.85 −1.340.53 5b −I −H 1200 ±± 340 600 −1.34 5b −I −H 1200670 340 1600 600 ±±−0.85 −0.85 −1.34 0.53 6 −OH −I 670 ±1600 210 ±±1300 1300 320 0.75 0.75 6 −OH −I −I ± 210 320 0.75 0.53 6 −OH 670 ± 210 1300 ± 320 670± ± 210 1300 1300 ± 3200.75 0.750.53 0.53 6 −OH −I −I 6 −OH 670 210 ± 320 0.53 a clogP/clogD values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP at 66 −OH −I 670 ±± 320 0.75 0.53 a clogP/clogD −OHvalues −I were calculated by ACD/Chemsketch 670 ±± 210 210 1300 1300 320 ACD/Labs 0.75 0.53 version 6.00 (clogD = clogP at at a clogP/clogD a clogP/clogD were calculated by ACD/Chemsketch version 6.00 (clogD =clogP clogP 66 values −OH −I 670 1300 ±±Values 320 0.75 by version ACD/Labs 6.00 (clogD == clogP at a clogP/clogD −OHvalues −I were 670 ±± 210 210 1300 320 ACD/Labs 0.75 0.53 physiological pH (7.4) with consideration of charged species). are the0.53 mean  standard values were calculated calculated by ACD/Chemsketch ACD/Chemsketch version ACD/Labs 6.00 (clogD at a clogP/clogD values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP at physiological physiological pH (7.4) with consideration of charged species). Values are the mean  standard a clogP/clogD values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD == clogP at a clogP/clogD physiological pH (7.4) with consideration charged species). Values are the mean  standard physiological pH (7.4) with consideration of of charged species). Values are the (SD) mean values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD clogP atstandard deviation (SD) of three assays. physiological pH (7.4) with consideration of charged species). Values are the mean standard a clogP/clogD pH (7.4) with consideration of charged species). Values are the mean ± standard deviation of three assays. values were calculated version 6.00 clogP a clogP/clogD deviation (SD) of with three assays. by were calculated by ACD/Chemsketch ACD/Chemsketch version ACD/Labs ACD/Labs the 6.00 (clogD (clogD ==standard clogP at at physiological pH (7.4) consideration of deviation of deviation (SD)values of(SD) three assays. physiological pH (7.4) with consideration of charged charged species). species). Values Values are are the mean mean   standard deviation (SD) of three three assays. assays. physiological pH (7.4) with consideration of charged species). Values are the mean  standard physiological pH (7.4) with consideration of charged species). Values are the mean  standard deviation of assays. deviation (SD) (SD) of three three Table 2. Structures and and IC50and values of phenoxyphenyl barbiturates 9–12. Table 2.assays. Structures IC50 values ofofphenoxyphenyl barbiturates 9–12. deviation three assays. Table 2. Structures Structures IC50 50 values phenoxyphenyl barbiturates 9–12. deviation (SD) (SD) of of three assays. Table 2. values of barbiturates Table 2. Structures and IC 50 values of phenoxyphenyl phenoxyphenylbarbiturates barbiturates 9–12. 9–12. Table 2. Structures and and IC50IC values of phenoxyphenyl 9–12. Table values of phenoxyphenyl barbiturates 9–12. Table 2. 2. Structures Structures and and IC IC50 50 values of phenoxyphenyl barbiturates 9–12. Table values of phenoxyphenyl barbiturates 9–12. Table 2. 2. Structures Structures and and IC IC50 50 values of phenoxyphenyl barbiturates 9–12.

IC50 50 [nM] [nM] IC50 IC [nM] ICIC 50 50 [nM] [nM] a MMP-2 MMP-8 MMP-9 MMP-13 clogP clogP clogDa aa IC 50MMP-9 [nM] MMP-2 MMP-8 MMP-13 clogD IC 50 [nM] a aa clogD a IC50MMP-9 [nM] MMP-13 MMP-2 MMP-8 MMP-9 clogP MMP-2 MMP-8 MMP-13 clogP clogD a MMP-2 MMP-8 IC MMP-9 MMP-13 clogP clogD a 50 [nM] a IC 50 [nM] a aa clogD a MMP-2 MMP-8 MMP-9 MMP-13 clogP clogD a b Cpd. R 1 R 2 MMP-2 MMP-8 MMP-9 MMP-13 clogP MMP-229 ±MMP-8 clogD a 0.92 −I 1170 ± ±MMP-9 80 1.3 1.3 ± ±MMP-13 0.2 362 ± ±clogP 24 a 1.40 1.40 Cpd. 99 R R2 b 1 −I 29 ±±MMP-8 222 1170 80 0.2 24 MMP-2 clogD a a 0.92 MMP-2 MMP-8 MMP-9 MMP-13 clogP clogD 0.92 −I 80 ± 1.3 0.2362 362 362 24 1.40 1.40 0.92 9 b b b −I 9 b 29 ± 2 29 1170 ±1170 80 ±MMP-9 1.3 0.2 ±MMP-13 ± 24±clogP 9 −I 29 ± 2 1170 ± 80 1.3 ± 0.2 362 ± 24 1.40 0.92 − I 29 ± 2 1170 ± 80 1.3 ± 0.2 362 ± 24 1.40 9 9 9b b −I 29 ± 2 1170 ± 80 1.3 ± 0.2 362 ± 24 1.40 0.92 −I 29 ± 2 1170 ± 80 1.3 ± 0.2 362 ± 24 1.40 0.92 0.92 99 bb −I 29 ± 2 1170 ± 80 1.3 ± 0.2 362 ± 24 1.40 0.92 10 −I 29 ± 2 1170 ± 80 1.3 ± 0.2 362 ± 24 1.40 0.92 −I 23 ± 5 146 ± 48 17 ± 5 28 ± 11 3.13 2.99 10 −I 23 ± 55 146 17 ± 55 28 ±± 11 3.13 2.99 −I 146 ±± 48 48 3.13 2.99 10 10 −I 10−I 23 ±±55 23 ±146 146 ±48 48 17± 5± 517 ± 28 28 ±28 11 11 3.13 2.99 23 17 3.13 2.99 10 10 −I −I 23 ± 5 146 ±± 146 ± 48 17 ± 5±± 11 28 ± 11 3.13 2.99 23 ± 5 48 17 ± 5 28 11 3.13 2.99 1010 −I 23 ± 5 146 ± 48 17 ± 5 28 ± 11 3.13 2.99 23 ± 48 17 ± 5 28 10 −I 23 ±± 55 7 ± 146 146 28 ±± 11 11 n. d.3.13 3.13 3.682.99 2.99 3.53 12−I −I 1 ± 48 n. n. d. d. 17 ± 5 22 ± ± 0.2 0.2 12 −I 7 ± 1 n. d. 3.68 3.53 12 −I 7±1 n. d. 2 ± 0.2 n. d. 3.68 3.53 12 12 777±±±111 n. d. d. 0.2 n. d. 3.68 n. 22 2±± ± 0.2 n. 12 −I −I −I n. d. 0.2 n. d. d. 3.683.68 3.53 3.53 3.53 −I 77 ±±7 22 ±± 0.2 n. 3.68 3.53 121212 I−I 711± n.d.d. d. 3.68 2.883.53 3.53 c d ± 23 11 ± 9n. 2 ±720.2 n. ±n. d.17 3.68 12 −13 −I n. d. d.n. 0.2 n. d. d. 645 3.68 3.53 −H 138 ± 12 ±±22 0.2 3.17 c d 13 −H 23 138 77 ± 645 3.17 2.88 13 c −H 23 ±± 99 138 ±± 12 12 ±2 645 ±± 17 17 3.17 2.88 d c d −H 23 138 77 7±± ± 22 2 645 ±± 17 2.88 d 13 c 13 23 138 12 645 13 cc −H −H 23±±±999 by ACD/Chemsketch 138 ±±±12 12 645ACD/Labs 17± 17 3.17 3.17 2.88=dd2.88 a clogP/clogD values were were calculated calculated version 6.003.17 (clogD clogP at at c 13 d −H 23 ±± 99± 138 ±± 138 12 77 ±± 22version 645 ±± 17 3.17 2.88 d values by ACD/Chemsketch ACD/Labs 6.00 (clogD = a clogP/clogD 1313 H 23 9 ± 12 7 ± 2 645 ± 17 3.17 d 13c c a− −H 23 138 12 645 17 3.17 2.88 clogP/clogD values were calculated by9 ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP clogP at2.88 −H 23 ± 138 ± 12 7 ± 2 645 ± 17 3.17 2.88 physiological pH (7.4) with consideration of charged species). Values are the mean  SD of three a clogP/clogD values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP at a clogP/clogD physiological pH (7.4) with of species). Values are the mean = SD of a clogP/clogD values were calculated byACD/Chemsketch ACD/Chemsketch version ACD/Labs (clogD = clogP at physiological pH (7.4) with consideration consideration of charged charged species). Values 6.00 are6.00 the mean SD of three three values were calculated by version ACD/Labs (clogD clogP at a clogP/clogD b IC50 c IC50 (MMP-1) dexperimental were calculated ACD/Chemsketch version ACD/Labs 6.00 (clogD == clogP at a clogP/clogD assays. values (MMP-14) 49 ± ± 88by nM. 50 µ µValues M, IC50 50 are (MMP-3) 7606.00 µSD M. bvalues c IC d were calculated by ACD/Chemsketch ACD/Labs 6.00 clogP at= clogP at physiological pH (7.4) with consideration charged species). the mean of three a aclogP/clogD assays. 50 (MMP-14) === 49 nM. 50 (MMP-1) >>>version 50 M, IC (MMP-3) === (clogD 760 µ M. clogP/clogD values were calculated byof ACD/Chemsketch version ACD/Labs (clogD b IC c IC d experimental physiological pH (7.4) with consideration of charged species). Values are the mean  SD of three values were calculated by ACD/Chemsketch version ACD/Labs 6.00 (clogD = clogP at assays. IC 50 (MMP-14) 49 ± 8 nM. 50 (MMP-1) 50 µ M, IC 50 (MMP-3) 760 µ M. experimental physiological pH (7.4) with consideration of charged species). Values are the mean  SD of three physiological pH (7.4) with consideration of charged species). Values are the mean  SD of three logD 2.15(7.4) ± 0.02. 0.02. For comparison reasons the IC 50 values of compound compound 12 [12] and 13 [15] from b IC50=(MMP-14) c d experimental physiological pH with of species). Values are SD of13 three physiological pH consideration of charged charged species). Values are themean mean ±and SD of[15] three assays. assays. == 49 ±±consideration 88comparison nM. (MMP-1) >> 50 µµ 50 M, IC (MMP-3) ==the 760 µµ12 M. b IC50=(MMP-14) d experimental logD 2.15 ± For reasons the values of [12] from assays. 49 nM.c cc IC IC50 50 (MMP-1) 50IC M, IC50 50 (MMP-3) 760 M. logD = pH 2.15 (7.4) ± 0.02. comparison reasons the 50species). values of Values compound and 13SD [15]of from physiological of charged the mean  three b d experimental b IC dM. bassays. d assays. 50 (MMP-14) =are 49 ±For 8±±cshown. nM. IC (MMP-1) 50IC M, (MMP-3) =are 760 µ[12] 50 (MMP-14) ==with 49 8consideration nM. (MMP-1) >> 50 µµµM, IC 50 50 (MMP-3) ==12760 µµ 12 M. b IC c IC d experimental previous work also IC50IC (MMP-14) = 49For ± 8comparison nM. > 50IC µM, IC (MMP-3) = 760 µM. experimental logD = 2.15 ± 0.02. assays. 50±(MMP-14) 49 8IC nM. IC5050 50 (MMP-1) 50 M, ICIC 50 (MMP-3) 760 M. experimental 50 (MMP-1) 50 logD = 2.15 0.02. reasons the 50> values of compound [12] and 13 [15] from previous work are also shown. logD =previous ± 0.02. For comparison reasons the IC50 values of compound 12 [12]=and 13 [15]d experimental from b 2.15 c work are shown. assays. IC 50 ±(MMP-14) =also 49 ± values 8 nM. 50 the (MMP-1) > 50and µ M, IC 50 from (MMP-3) 760 µ M. For comparison reasons the IC50 ofIC compound [12] 13compound [15] previous work are logD == 2.15 For comparison reasons values of 12 [12] and 13 from logD = 2.15 ±work 0.02. For comparison reasons the IC 5012 values 12 and 13 also [15] from logD 2.15 ± 0.02. 0.02. For comparison reasons the IC IC50 50 values ofofcompound compound 12 [12][12] and 13 [15] [15] fromshown. previous are also shown. previous work are also shown. The second hydrophilic modification included the substitution of the hydroxy group with aa The second hydrophilic modification included the substitution of the hydroxy group with logD = 2.15 ± 0.02. For comparison reasons the IC 50 values of compound 12 [12] and 13 [15] previous work are also shown. The second hydrophilic modification included the substitution of the hydroxy group from with a previous work are alsoalso shown. previous work are shown. Cpd.

Cpd. Cpd. 1Cpd. Cpd. RR 1 Cpd. R Cpd. R Cpd. R111

R11 R R1

R2R 2 R 2 2 RR 2

R22 R R2

carboxy group in the the piperazine piperazine residue of 12 12 to to yield carboxylicofacid acid resultinggroup in aa clogD clogD shift of of The second hydrophilic modification included the substitution the 99hydroxy with ashift carboxy group in residue of yield carboxylic resulting in

previous work arein also The second hydrophilic modification included theyield substitution ofacid the 9hydroxy group with ashiftwith carboxy group theshown. piperazine residue of 12 to in a clogD of a The second hydrophilic modification included thecarboxylic substitution ofresulting the hydroxy group The second hydrophilic modification included the substitution of the hydroxy group with aa 2.6group units (approximately 400 fold increased increased water solubility, Table 2) from 3.53 0.92. Despite The second hydrophilic modification included the substitution of the hydroxy group with The second hydrophilic modification included the substitution of the hydroxy group with a carboxy in the piperazine residue of 12 to yield carboxylic acid 99 2) resulting in atowards clogD shift of 2.6 units (approximately 400 fold water solubility, Table from 3.53 towards 0.92. Despite carboxy group in the piperazine residue of 12 to yield carboxylic acid resulting in a clogD shift of 2.6 units (approximately 400 fold increased water solubility, Table 2) from 3.53 towards 0.92. Despite carboxy group in the piperazine residue of 12 to yield carboxylic acid 9 resulting in a clogD shift of carboxy group in the piperazine residue of 12 to yield carboxylic acid 9 resulting in a clogD The second hydrophilic modification included the substitution of the hydroxy group with a this modification the high MMP-2and -9-inhibition potency of 12 was only marginally influenced 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 carboxy group in the piperazine residue ofand 12 to yield carboxylic resulting in0.92. a clogD shift ofshift this modification the MMP-2-9-inhibition potency of 12 only influenced 2.6 units 400high fold increased water solubility, Table 2)acid from 3.53 towards Despite this(approximately modification the high MMP-2and -9-inhibition potency of 129was was only marginally marginally influenced 2.6 units (approximately 400 fold increased water solubility, Table 2) from 3.53 towards 0.92. Despite of 2.6 units (approximately 400 fold increased water solubility, Table 2) from 3.53 towards 0.92. (Table 2, 12: IC 50 (MMP-2) = 7 nM, IC 50 (MMP-9) = 2 nM. 9: IC 50 (MMP-2) = 29 nM, IC 50 (MMP-9) = 1.3 2.6 units (approximately 400 fold increased water solubility, Table 2) from 3.53 towards 0.92. Despite carboxy group in the piperazine residue of 12 to yield carboxylic acid 9 resulting in a clogD shift of this the high MMP-2-9-inhibition potency of 12 was only marginally influenced (Table IC (MMP-2) == 77 and nM, IC 50 (MMP-9) == 22 nM. 9: (MMP-2) == 29 nM, 50 0.92. (MMP-9) == 1.3 2.6 units (approximately 400 fold increased solubility, 2) from Despite this modification modification high MMP-2and potency of 1250 was only3.53 marginally (Table 2, 2, 12: 12:the IC50 50 (MMP-2) nM, -9-inhibition ICwater 50 (MMP-9) nM.Table 9: IC IC 50 (MMP-2) 29towards nM, IC ICinfluenced 50 (MMP-9) 1.3 this modification the MMP-2and -9-inhibition potency of was only marginally influenced nM). Moreover, in 400 vitro data showed, that 9solubility, was also a12 nanomolar inhibitor of collagenase this modification the high high MMP-2and -9-inhibition potency 12nanomolar was only marginally influenced Despite this modification the high MMP-2and -9-inhibition potency of 12 was only marginally (Table 2, 12: IC 50 (MMP-2) = 7 nM, IC 50showed, (MMP-9) = 2 nM. 9: IC 50of (MMP-2) = 29 nM, IC 50 towards (MMP-9) = 1.3 2.6 units (approximately fold increased water Table 2) from 3.53 0.92. Despite nM). Moreover, in vitro data that 9 was also a inhibitor of collagenase 333 (Table 2, 12: IC 50 (MMP-2) = 7 nM, IC 50 (MMP-9) = 2 nM. 9: IC 50 (MMP-2) = 29 nM, IC 50 (MMP-9) = 1.3 this modification the high MMP-2and -9-inhibition potency of 12 was only marginally influenced nM). Moreover, in vitro data showed, that 9 was also a nanomolar inhibitor of collagenase (Table 2, 12: IC 50 (MMP-2) = 7 nM, IC 50 (MMP-9) = 2 nM. 9: IC 50 (MMP-2) = 29 nM, IC 50 (MMP-9) = 1.3 (MMP-13) and MMP-14 (MT-1 MMP), but only a micromolar inhibitor of neutrophil collagenase (Table 2, 12: IC 50 (MMP-2) = 7 nM, IC 50 (MMP-9) = 2 nM. 9: IC 50 (MMP-2) = 29 nM, IC 50 (MMP-9) = 1.3 nM). Moreover, in vitro data showed, that 99 nM, was also aa(MMP-9) nanomolar of collagenase 33 = 29 nM, influenced 2, 12: IC (MMP-2) = but 7but IC = 2inhibitor nM. 9: neutrophil IC (MMP-13) and MMP-14 MMP), aa509: micromolar inhibitor of collagenase this the high MMP-2and -9-inhibition potency of 12 was marginally influenced 50(MT-1 5050(MMP-2) nM). Moreover, in vitro showed, that was also nanomolar inhibitor of collagenase (MMP-13) and MMP-14 (MT-1 MMP), only micromolar inhibitor of neutrophil collagenase (Table 2,modification 12: IC50(Table (MMP-2) = 7data nM, IC 50 (MMP-9) 2only nM. 50 (MMP-2) = 29=only nM, IC (MMP-9) nM). Moreover, in vitro data showed, that 99 =was also aaIC nanomolar inhibitor of collagenase 33 = 1.3= (MMP-8) (9: (IC 50 (MMP-14) = 49 49 nM nM (Table 2,micromolar footnote b), inhibitor IC50 50 (MMP-8) 1170 nM, IC50 50 (MMP-13) nM).(MMP-9) Moreover, in(IC vitro data showed, that was also nanomolar inhibitor ofnanomolar collagenase (MMP-13) and MMP-14 (MT-1 MMP), but only a of neutrophil collagenase (MMP-8) (9: 50 (MMP-14) = (Table 2, footnote b), IC (MMP-8) = 1170 nM, IC (MMP-13) = IC = 1.3 nM). Moreover, in vitro data showed, that 9 was also a inhibitor (MMP-13) and MMP-14 (MT-1 MMP), but only a micromolar inhibitor of neutrophil collagenase (Table 2, 12: IC 50 (MMP-2) = 7 nM, IC 50 (MMP-9) = 2 nM. 9: IC 50 (MMP-2) = 29 nM, IC 50 (MMP-9) 50 (MMP-8) (9: (IC 50 (MMP-14) = 49 nM (Table 2, footnote b), IC 50 (MMP-8) = 1170 nM, IC 50 (MMP-13)= nM). (MMP-13) Moreover, in vitro data showed, that 9 was also a nanomolar inhibitor of collagenase 3=1.3of and MMP-14 (MT-1 MMP), but only aa the micromolar inhibitor of collagenase 362 (9: nM). In summary, compound 9 confirms confirms results from Grams etneutrophil al. [8] IC that 5,5-disubstituted (MMP-13) and MMP-14 (MT-1 MMP), but only micromolar inhibitor of neutrophil collagenase (MMP-8) (IC 50 (MMP-14) = 49 nM (Table 2, footnote b), IC 50 (MMP-8) = 1170 nM, 50 (MMP-13) = 362 nM). In summary, compound 9 the results from Grams et al. [8] that 5,5-disubstituted (MMP-8) (9: (IC 50 (MMP-14) = 49 nM (Table 2, footnote b), IC 50 (MMP-8) = 1170 nM, IC 50 (MMP-13) = collagenase 3 (MMP-13) MMP-14 (MT-1 but only a micromolar inhibitor of neutrophil nM). Moreover, vitro and data showed, that 9aMMP), wasb), also a nanomolar inhibitor of collagenase collagenase 3 362 nM). Inin summary, 9 confirms the results from Grams etofal. [8] IC that 5,5-disubstituted (MMP-8) (9: (IC ==compound 49 nM (Table 2, footnote IC 50 (MMP-8) == potent 1170 nM, == (MMP-13) and MMP), but only micromolar inhibitor neutrophil barbiturates with(MT-1 para-substituted phenoxyphenyl unit represent inhibitors for MMP-2, MMP-2, -9 (MMP-8) (9:MMP-14 (IC50 50 (MMP-14) (MMP-14) 49 nM (Table 2,the footnote b), IC 50Grams (MMP-8) 1170 nM, IC50 50 (MMP-13) (MMP-13) 362 In summary, compound 99 confirms results from et [8] that 5,5-disubstituted barbiturates with aa(9: para-substituted phenoxyphenyl unit represent inhibitors for -9 362 nM). nM). In summary, compound confirms the results from Grams et al. al.potent [8]b), that 5,5-disubstituted collagenase (MMP-8) (IC (MMP-14) = 49 nM (Table 2, footnote IC (MMP-8) = 1170 nM, barbiturates with a para-substituted phenoxyphenyl unit represent potent inhibitors for MMP-2, -9 (MMP-13) and MMP-14 (MT-1 MMP), but only a micromolar inhibitor of neutrophil collagenase 50 50 362 In summary, compound confirms results from et al. [8] that 5,5-disubstituted (MMP-8) (9: 50 (MMP-14) = 49 nM99 (Table 2, the footnote IC 50 1170 IC 50the (MMP-13) = and(IC -14. Additionally, as shown shown by comparison comparison of b), the ICGrams 50 (MMP-8) values of and 13nM, with 10 elongation 362 nM). nM). In summary, compound confirms the results from Grams etof al.99= [8] that 5,5-disubstituted barbiturates with aa para-substituted phenoxyphenyl unit represent potent inhibitors for MMP-2, -9 and -14. Additionally, as by of IC values and 13 with 10 the barbiturates with para-substituted phenoxyphenyl unit represent potent for MMP-2, -9al. [8] that and -14. Additionally, as shown by comparison of the the IC50 50 values ofresults 9inhibitors and 13from with 10 the50elongation elongation IC 362 nM). In summary, compound 9from confirms the Grams etdecrease (9: (IC 50=(MMP-14) = 49 nM (Table 2, footnote b), IC 50tri-ethylenglycol (MMP-8) = 1170 nM, IC (MMP-13) = 50 (MMP-13) barbiturates with a para-substituted phenoxyphenyl unit represent potent inhibitors for MMP-2, -9 of the substituent of the piperazine residue with fluorinated resulted in a barbiturates with a para-substituted phenoxyphenyl unit represent potent inhibitors for MMP-2, -9 362(MMP-8) nM). In summary, compound 9 confirms the results Grams et al. [8] that 5,5-disubstituted and Additionally, as shown by of the 50 values of 99 and the of substituent the residue fluorinated tri-ethylenglycol in and -14. -14. Additionally, asof by comparison comparison ofwith the IC IC 50 values of and 13 13 with with 10 10resulted the elongation elongation of the thesummary, substituent ofshown the piperazine piperazine residue with fluorinated tri-ethylenglycol resulted in aa decrease decrease 5,5-disubstituted barbiturates with a para-substituted phenoxyphenyl unit represent potent inhibitors and -14. Additionally, as shown by comparison of the IC 50 values of 9 and 13 with 10 the elongation 362 nM). In compound 9 confirms the results from Grams et al. [8] that 5,5-disubstituted and -14. Additionally, as shown by comparison of the IC 50 values of 9 and 13 with 10 the elongation barbiturates with a para-substituted unit represent potent resulted inhibitors MMP-2, -9 of of residue tri-ethylenglycol in decrease of the the substituent substituent of the the piperazine piperazine phenoxyphenyl residue with with fluorinated fluorinated tri-ethylenglycol resulted in aafor decrease of substituent the piperazine residue with tri-ethylenglycol resulted in decrease for MMP-2, -9 andaof Additionally, as shown by comparison ofofthe IC values of 13MMP-2, with 10 the with para-substituted phenoxyphenyl represent potent inhibitors for -9 of the the substituent of-14. the piperazine residue withoffluorinated fluorinated tri-ethylenglycol resulted in9athe aand decrease 5013 andbarbiturates -14. Additionally, as shown by comparison the ICunit 50 values 9 and with 10 elongation elongation of the substituent of the piperazine residue with fluorinated tri-ethylenglycol resulted in andsubstituent -14. Additionally, as shown by comparison of the IC50tri-ethylenglycol values of 9 and 13 with 10inthe elongationa of the of the piperazine residue with fluorinated resulted a decrease of the substituent of the piperazine residue with fluorinated tri-ethylenglycol resulted in a decrease

Pharmaceuticals 2017, 10, 49 Pharmaceuticals 2017, 10, 49

5 of 11 5 of 11

of selectivity for unknown reasons. FromFrom this this selection of of iodinated decrease of selectivity for unknown reasons. selection iodinatedbarbiturate barbiturate derivatives carboxylic acid acid9 was 9 was chosen for radiochemical synthesis of the radioiodinated isotopologues chosen for radiochemical synthesis of the radioiodinated isotopologues [123/124 I]9 123/124I]9 (see Section 2.3) because this compound possesses the most favourable characteristics [(see Section 2.3) because this compound possesses the most favourable characteristics indicated by indicated and IC50-values. clogD andby ICclogD 50 -values. 2.3.Radiochemistry Radiochemistry 2.3 Palladium-catalyzed cross-coupling cross-coupling reaction reaction (Stille (Stille coupling) coupling) of non-radioactive non-radioactive reference Palladium-catalyzed compound 9 with tributyltin hydride (or hexabutylditin) (yield: 28%) 28%) [20] [20] provided provided the stannyl compound precursor 11 (Scheme 3).

Scheme 3. Precursor and radiosynthesis radiosynthesis of of the the barbituric barbituric acid-based acid-based MMP-targeted MMP-targeted Scheme 3. Precursor synthesis synthesis of of 11 11 and 123/124 model I]9.I]9. Reagents and and yields: (a) Bu 3SnH or Bu6Sn2 (2.0 eq.), PdCl2(PMePh2)2 or PdCl2 (3 model tracer tracer [[123/124 Reagents yields: (a) Bu3 SnH or Bu6 Sn2 (2.0 eq.), PdCl2 (PMePh2 )2 or mol%), KOAc (3.0 eq.), N-methyl-pyrrolidone, 28%; (b) [123/124 chloroamine-T hydrate,hydrate, 0.1 M PdCl2 (3 mol%), KOAc (3.0 eq.), N-methyl-pyrrolidone, 28%; (b)I]NaI, [123/124 I]NaI, chloroamine-T K 2HPO4. 0.1 M K HPO . 2

4

Subsequent radioiododestannylation of 11 with no-carrier-added (n. c. a.) 123/124 [123/124I]NaI and Subsequent radioiododestannylation of 11 with no-carrier-added (n. c. a.) [ I]NaI and 123/124 chloroamine-T led to the radioligands [ 123/124 I]9. The decay-corrected radiochemical yields were 28 ± chloroamine-T led to the radioligands [ 124 I]9. The decay-corrected radiochemical yields were 7% (n = 6) for [123I]9 and 44 ± 6% (n = 3) for [ I]9 at the end of synthesis (EOS) and the radiochemical 28 ± 7% (n = 6) for [123 I]9123and 44 ± 6% (n = 3) for [124 I]9 at the end of synthesis (EOS) and the 124 purities were 95 ± 3% for [ I]9 and 93 ±1235% for [ I]9 (as determined by radio-HPLC). The molar radiochemical purities were 95 ± 3% for [ I]9 and 93 ± 5% for [124 I]9 (as determined by radio-HPLC). activities are 0.2–6.3 GBq/μmol and 0.4–14.0 GBq/μmol, respectively. The identities of [123/124I]9 were The molar activities are 0.2–6.3 GBq/µmol and 0.4–14.0 GBq/µmol, respectively. The identities of proven by HPLC (coinjection and coelution with reference compound 9). [123/124 I]9 were proven by HPLC (coinjection and coelution with reference compound 9). 3. Materials and Methods 3. Materials and Methods 3.1. 3.1. General General Methods. Methods. Chemistry Chemistry All chemicals, reagents reagentsand andsolvents solvents synthesis of compounds the compounds of analytical All chemicals, forfor thethe synthesis of the were were of analytical grade, grade, purchased from commercial sources and used without further purification, unless purchased from commercial sources and used without further purification, unless otherwiseotherwise specified. specified. Melting points were determined in tubes capillary a SMP3melting capillary melting point Melting points were determined in capillary on atubes SMP3oncapillary point apparatus 1H-NMR, 13C-NMR and 1 13 19 apparatus (Stuart Scientific, Staffordshire, UK) and are uncorrected. (Stuart Scientific, Staffordshire, UK) and are uncorrected. H-NMR, C-NMR and F-NMR spectra 19F-NMR spectra were recorded on ARX 300 and/or AMX 400 spectrometers (Bruker, Karlsruhe, were recorded on ARX 300 and/or AMX 400 spectrometers (Bruker, Karlsruhe, Germany). CDCl3 Germany). CDCl3 contained(TMS) tetramethylsilane as an internal Mass on spectra were contained tetramethylsilane as an internal(TMS) standard. Mass spectrastandard. were obtained a MAT 212 obtained on a MAT 212 (EI = 70 eV) spectrometer (Varian Medical Systems, Palo Alto, CA, USA) and (EI = 70 eV) spectrometer (Varian Medical Systems, Palo Alto, CA, USA) and a Bruker MALDI-TOF-MS aReflex Bruker Reflex IV instrument DHB). mass analyses wereLC conducted IVMALDI-TOF-MS instrument (matrix: DHB). Exact mass(matrix: analyses wereExact conducted on a Quattro (Waters, on a Quattro LC (Waters, Milford, MA, USA) and a Bruker MicroTof apparatus. Elemental analyses Milford, MA, USA) and a Bruker MicroTof apparatus. Elemental analyses were realized by a Vario EL were realized by a Vario EL III analyzer (Elementar Analysensysteme Hanau, Germany). All III analyzer (Elementar Analysensysteme Comp., Hanau, Germany). AllComp., aforementioned spectroscopic aforementioned spectroscopic were and analytical investigations done by of staff members of the and analytical investigations done by staff members ofwere the Institute Organic Chemistry, Institute of Organic Chemistry, University of Münster, Germany. All purifications of compounds University of Münster, Germany. All purifications of compounds and determinations of purity by and determinations of by purity bya HPLC were performed by (Knauer, using a Berlin, gradient RP-HPLC system HPLC were performed using gradient RP-HPLC system Germany) equipped (Knauer, equipped withdetector two K-1800 an Nucleosil S-2500 UVEurosphere detector and RP-HPLC with twoBerlin, K-1800Germany) pumps, an S-2500 UV and pumps, RP-HPLC 100-10 C-18 Nucleosil Eurosphere 100-10 C-18 columns for analytical (250 mm × 4.6 mm) purposes. The columns for analytical (250 mm × 4.6 mm) purposes. The following eluents were used (unless specified following used(0.1% (unless specified otherwise): eluent water TFA),conditions eluent B: otherwise):eluents eluent were A: water TFA), eluent B: acetonitrile (0.1%A: TFA). The(0.1% following acetonitrile (0.1% TFA). The following conditions were used (unless specified otherwise): Gradient were used (unless specified otherwise): Gradient from 90% A to 20% A over 30 min, constant 20% A 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, 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. at a flow rate of 1.5 mL/min, detection at λ = 254 nm.

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3.2. General Procedure for 4-Iodophenyl Malonic Acid Dimethyl Ester (2a) and 4-Benzyloxyphenyl Malonic Acid Dimethyl Ester (2b) A suspension of 4.00 g NaH (166 mmol, 6.66 g of a 60% suspension in paraffin; the paraffin was removed by repeated washings with petroleum benzene) and 48.0 g (533 mmol) dimethyl carbonate in 80 mL absolute dioxane was heated to 100–120 ◦ C and a solution of 23.00 g (83.3 mmol) 4-Iodophenyl acetic acid methylester (1a, prepared by the esterification of 4-iodophenyl acetic acid using MeOH/H2 SO4 ) 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 (Na2 SO4 ) and concentrated. The crude 2a was used in the next step without further purification. Yield: 22.8 g (68.2 mmol, 82%). 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 7.90 (d, 3 J = 8.3 Hz, 2 H, HAryl ), 7.35 (d, 3 J = 8.3 Hz, 2 H, HAryl ), 5.18 (s, 1 H, CH), 3.83 (s, 6 H, CH3 ). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 168.46, 138.14, 133.10, 131.88, 94.86, 55.99, 53.11. 4-Benzyloxyphenyl malonic acid dimethyl ester (2b) [19] was prepared from methyl 2-((4-benzyloxy)phenyl)acetate 1b [20] analogous to 2a. 3.3. General Procedure for 5-(4-Iodophenyl)-pyrimidine-2,4,6-trione (3a) and 5-(4-Benzyloxyphenyl)-pyrimidine-2,4,6-trione (3b) Under an argon atmosphere 2 eq. of sodium were dissolved in ethanol (0.35 mL/mmol Na) and 1.7 eq. of urea were added. A solution of malonic ester 2a or 2b in ethanol (2.2 mL/mmol) was added dropwise and the reaction mixture was heated to reflux for 6 h. After cooling to room temperature, the mixture was poured onto ice water and adjusted to pH 2 using dilute hydrochloric acid. The precipitate was collected by suction and dried in vacuo. 3a: Yield: 29%. 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 11.34 (broad, s), 7.68 (broad, d, 2 H, HAryl ), 7.09 (broad, d, 2 H, HAryl ), 4.92 (s, 1 H, CH). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 187.77 (C-4/6 enol form), 169.73 (C-4/6 keto form), 151.31 (C-2), 137.41, 132.46, 128 .89, 93.48, 92.09 (C-5 enol form), 54.60 (C-5 keto form). Anal. Calcd for C10 H7 IN2 O4 : C 36.39, H 2.14, N 8.49. Found: C 36.61, H 2.29, N 8.23. 3b: Yield: 91%. Mp 233–236 ◦ C. 1 H-NMR (300 MHz, DMSO-d ): δ [ppm]: 11.34 (broad, s, OH), 7.4–6.94 (m, 9 H, H 6 Aryl ), 5.10 (s, 1 H, CH). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 187.75 (C-4/6 enol form), 169.71 (C-4/6 keto form), 158.25, 151.29 (C-2), 137.39, 132.44, 130.64, 128.14, 115.31, 114.36, 92.06 (C-5 enol form), 69.58, 54.59 (C-5 keto form). Anal. Calcd for C10 H14 IN2 O4 ·H2 O: C 62.19, H 4.91, N 8.53. Found: C 62.38, H 4.56, N 8.14. 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 (Na2 SO4 ) and the solvent was evaporated. The residue was stirred in CHCl3 for 2 h to give a colorless solid. Yield: 4.10 g (10.0 mmol, 51%). Mp 183–186 ◦ C. 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 11.47 (broad, s), 10.94 (broad, s), 8.64 (broad, s), 7.71 (d, 2 H, HAryl ), 7.15 (d, 2 H, HAryl ). 13 C-NMR (75.5 MHz, DMSO-d ): δ [ppm]: 179.66, 170.80, 137.81, 133.18, 127.65, 95.76, 75.99. MS (EI): 6 m/e (intensity %): 410 (M+ , 15), 408 (M+ , 15), 329 (100), 282 (45), 244 (45), 196 (62), 129 (29), 89 (38), 43 (39). Anal. Calcd for C10 H6 BrIN2 O3 : C 29.37, H 1.48, N 6.85. Found: C 29.96, H 1.48, N 6.80. 3.5. 5-(4-Benzyloxyphenyl)-5-bromo-pryrimidine-2,4,6-trione (4b) A suspension of the 3b (5.00 g, 16.1 mmol) in water (48 mL) was cooled to 0–5 ◦ C and 48% HBr (3.25 mL, 28.4 mmol) and bromine (1.32 mL, 25.8 mmol) were added dropwise. After stirring for 4–5 h at 0–10 ◦ C the precipitate was collected by filtration and dried in vacuo. Yield: 5.59 g (14.4 mmol, 89%). Mp 145–147 ◦ C. 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 11.44 (broad, s), 7.43–7.01 (m, 9 H, HAryl ), 5.08 (s, 2 H, CH2 ). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 171.34, 159.00, 150.21, 137.18, 130.86,

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128.79, 128.03, 127.99, 126.88, 115.30, 76.28, 69.70. MS (ESI-EM) m/e: 410.9951 (M + Na)+ calcd for C17 H13 BrN2 O4 Na 410.9956. Anal. Calcd for C17 H13 BrN2 O4 ·0.3 H2 O: C 52.10, H 3.42, N 7.15. Found: C 51.79, H 3.12, N 6.96. 3.6. General Procedure for Compounds 5a–5c A solution of 4a or 4b in methanol (2–4 mL/mmol) was treated with 2 eq. of N-(2-hydroxyethyl)piperazine (in case of 5a and 5c) or 3-(piperazin-1-yl)-propionic acid (in case of 5b) and stirred for 2 d at room temperature. The colorless precipitate was collected by suction and dried in vacuo. 3.6.1. 5-[4-(2-Hydroxyethyl)piperazin-1-yl]-5-(4-iodophenyl)pryrimidine-2,4,6-trione (5a) Yield: 53%. Mp 168–172 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ [ppm]: 7.72 (d, 3 J = 8.7 Hz, 2 H, HAryl ), 7.16 (d, 3 J = 8.7 Hz, 2 H, HAryl ), 3.43–2.31 (m, 12 H, CH2 ). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 169.36, 151.07, 138.72, 131.17, 130.13, 95.72, 85.71, 59.68, 58.20, 53.59, 47.24. MS (ESI-EM) m/e: 459.0518 (M + H)+ calcd for C16 H20 IN4 O4 459.0524. Anal. Calcd for C16 H19 IN4 O4 ·H2 O: C 40.35, H 4.23, N 11.76. Found: C 40.93, H 4.54, N 11.47. 3.6.2. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-(4-iodophenyl)pyrimidine-2,4,6-trione (5b) Yield: 18%. Mp 139–142 ◦ C. 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 9.42 (broad, s), 8.00 (d, = 8.7 Hz, 2 H, HAryl ), 7.61 (d, 3 J = 8.7 Hz, 2 H, HAryl ), 3.30–3.21 (m, 4 H, CH2 ), 2.85–2.59 (m, 8 H, CH2 ). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 173.74, 164.25, 151.49, 138.89, 135.07, 131.70, 86.35, 85.64, 53.23, 49.47, 43.38, 32.01. MS (ESI-EM) m/e: 487.0453 (M + H)+ calcd for C17 H20 IN4 O5 487.0473. Anal. Calcd for C17 H19 IN4 O5 ·1.8 H2 O: C 39.88, H 4.04, N 10.80. Found: C 39.59, H 4.23, N 10.48. 3J

3.6.3. 5-[4-(2-Hydroxyethyl)piperazin-1-yl]-5-(4-benzyloxyphenyl)pryrimidine-2,4,6-trione (5c) Yield: 28%. Mp 211–213 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ [ppm]: 7.48–7.02 (m, 9 H, HAryl ), 5.09 (s, 2 H, CH2 ), 3.48–3.44 (m, 2 H, CH2 OH), 2.57–2.38 (m, 10 H, CH2 ). 13 C-NMR (100 MHz, DMSO-d6 ): δ [ppm]: 171.08, 159.63, 150.35, 137.67, 130.00, 129.32, 128.78, 128.28, 128.08, 115.76, 74.99, 70.26, 61.07, 59.28, 48.10, 44.91. MS (MALDI-TOF) m/e: 461 (M + Na)+ , 439 (M + H)+ . Anal. Calcd for C23 H26 N4 O5 ·1 H2 O: C 60.52, H 6.18, N 12.27. Found: C 60.35, H 5.71, N 12.27. 3.7. 5-[4-(2-Hydroxyethyl)piperazin-1-yl]-5-(4-hydroxyphenyl)pryrimidine-2,4,6-trione (5d) Compound 5c (1.66 g, 3.79 mmol) was dissolved in absolute methanol (150 mL), treated with Pd/C (10%, 250 mg) and heated to reflux for 12 h under an H2 atmosphere. After cooling to room temperature, the mixture was stirred overnight. The catalyst was filtered off and washed with methanol (80 mL). The solvent was evaporated and the solid residue was dried in vacuo. The crude product was taken up in a CHCl3 /ethylacetate mixture (1/1, v/v) and stirred at room temperature for 2–3 h and finally re-isolated by suction filtration. Yield: 1.08 g (3.11 mmol, 82%). Mp 185 ◦ C. 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 7.45 (d, 3 J = 8.6 Hz, 2 H, HAryl ), 7.03 (d, 3 J = 8.6 Hz, 2 H, HAryl ), 3.79 (m, 2 H, CH2 OH), 2.99–2.69 (m, 10 H, CH2 ). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 170.66, 158.47, 149.82, 131.22, 129.29, 115.94, 74.63, 59.74, 57.66, 53.73, 46.67. MS (ESI-EM) m/e: 349.1521 (M + H)+ calcd for C16 H21 N4 O5 349.1506. Anal. Calcd for C16 H20 N4 O5 ·2.2 H2 O: C 49.56, H 5.77, N 14.44. Found: C 49.08, H 5.54, N 14.02. 3.8. 5-(4-Hydroxy-3-iodophenyl)-5-[4-(2-hydroxyethyl)piperazin-1-yl]pryrimidine-2,4,6-trione (6) Compound 5d (500 mg, 1.44 mmol) was dissolved in methanol (10 mL) and treated with 58 mg (1.44 mmol) sodium hydroxide and 216 mg (1.44 mmol) sodium iodide. The solution was cooled in an ice bath and 824 mg (687 µL, 1.44 mmol) sodium hypochlorite (13% active chlorine) were added dropwise over a period of 60 min. The orange suspension was stirred in the ice bath for further 2 h until a nearly colorless solution had formed. The ice bath was removed and 2–3 crystals sodium

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thiosulfate were added at room temperature. The solution was acidified to pH 6.8 by adding 1 M HCl and extracted with ethylacetate (3 × 30 mL). The combined extracts were washed with brine (1 × 30 mL), dried (MgSO4 ) and evaporated to dryness. Yield: 80 mg (0.17 mmol; 12%). Mp 168–170 ◦ C (decomposition). 1 H-NMR (300 MHz, DMSO-d6 ): δ [ppm]: 11.50 (s, broad, 2 H), 7.60–6.69 (m, 3 H, HAryl ), 4.34 (s, 1 H, OH) 3.40–2.28 (m, 12 H, CH2 ). 13 C-NMR (75.5 MHz, DMSO-d6 ): δ [ppm]: 169.87, 157.32, 149.35, 137.99, 129.10, 127.04, 115.37, 84.56, 73.25, 59.71, 58.27, 53.70, 47.11. MS (ESI-EM) m/e: 475.0467 (M + H)+ calcd for C16 H20 IN4 O5 475.0473. Anal. Calcd for C16 H19 IN4 O5 ·H2 O: C 39.04, H 4.30, N 11.28. Found: C 39.47, H 4.23, N 11.21. 3.9. General Procedure for Compounds 9 and 10 A solution of 5-bromo-5-[4-(4-iodo-phenoxy)-phenyl]pyrimidine-2,4,6-trione 7 [8,12] in methanol (ca. 5–10 mL/mmol) was treated with 2.0 eq. of the appropriate piperazine (8 or 3-(piperazin-1-yl)propionic acid) and stirred at room temperature overnight. The colorless solids which precipitated after ca. 1 h were collected by suction and dried in vacuo. Compound 10 was further purified by silica gel column chromatography (EtOAc/MeOH (4/1, v/v) + 1% NEt3 ). 3.9.1. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-[4-(4-iodophenoxy)phenyl]pyrimidine-2,4,6-trione (9) Yield: 83%. Mp: 236–242 ◦ C (decomposition). 1 H-NMR (400 MHz, DMSO-d6 ): δ [ppm]: 9.31 (s, broad, 2 H), 7.95 (d, 3 J = 8.6 Hz, 2 H, HAryl ), 7.72 (d, 3 J = 9.0 Hz, 2 H, HAryl ), 6.92 (d, 2 J = 9.0 Hz, 2 H, HAryl ), 6.87 (d, 3 J = 8.6 Hz, 2 H, HAryl ), 3.16–3.09 (m, 2 H, CH2 ), 2.73–2.58 (m, 8 H, CH2 ), 2.51–2.44 (m, 2 H, CH2 ). 13 C-NMR (100 MHz, DMSO-d6 ): δ [ppm]: 173.80, 164.31, 158.77, 151.62, 150.29, 138.66, 135.36, 131.05, 120.00, 118.05, 86.65, 85.42, 53.24, 49.46, 43.32, 40.53. MS (ESI-EM) m/e: 420.9687 (M − C7 H13 N2 O2 )+ calcd for C16 H10 IN2 O4 420.9685. Anal. Calcd for C23 H23 IN4 O6 ·2 H2 O: C 44.96, H 4.43, N 9.12. Found: C 45.16, H 4.70, N 9.52. 3.9.2. 5-(4-(2-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)-5-(4-(4-iodophenoxy)phenyl)pyrimidine-2,4,6-trione (10) Yield: 8%. Mp 176-177 ◦ C. 1 H-NMR (300 MHz, DMSO-d6 ) δ [ppm]: 9.28 (s, 2 H), 7.84 (d, 3 J = 7.8 Hz, 2 H, H 3 3 Aryl ), 7.64 (d, J = 7.8 Hz, 2 H, HAryl ), 6.81 (d, J = 6.8 Hz, 2 H, HAryl ), 6.76 (d, 3 J = 6.8 Hz, 2 H, H 2 13 C-NMR Aryl ), 4.51 (dt, J H,F = 48.1 Hz, 2 H, CH2 F), 3.56–2.50 (m, 22 H, CH2 ). (75.5 MHz, DMSO-d6 ): δ [ppm]: 169.93, 158.40, 151.31, 149.89, 138.31, 135.00, 130.71, 119.61 117.75, 86.35, 85.13, 83.11 (d,|1 JC,F | = 165.7 Hz), 70.54, 70.39, 70.12, 69.76, 69.57, 68.15, 56.71, 49.62, 42.98. 19 F-NMR (282 MHz, CDCl3 ): δ [ppm]: −216.61. MS (ESI-EM) m/e: 685.1518 (M + H)+ calcd for C28 H35 FIN4 O7 685.1529. The purity of 10 was determined by analytical gradient HPLC to be > 95% (system and conditions see Section 3.1), tR = 25.92 ± 0.36 min (n = 3). 3.10. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-[4-(4-(tributylstannyl)phenoxy)phenyl]pyrimidine-2,4,6- trione (11) PdCl2 (PMePh2 )2 (18 mg, 30 µmol, 3 mol%) or PdCl2 (18 mg, 100 µmol, 10 mol%), KOAc (294 mg, 3.00 mmol, previously dried for several hours at 100 ◦ C in vacuo before use) and N-methyl-pyrrolidinone (NMP, 15 mL) were mixed under an argon atmosphere [20]. Compound 9 (578 mg, 1.00 mmol) and tributyltin hydride (582 mg, 2.00 mmol) or hexabutylditin (870 mg, 1.50 mmol) were added and the mixture was heated to 110 ◦ C for 10–15 h. The progress of the reaction was monitored by HPLC (system and conditions see Section 3.1). The retention times tR were: tR (9): 34.15 ± 1.38 min (n = 10), tR (11): 40.83 ± 1.17 min (n = 6). When the conversion was complete, the hot black reaction mixture was filtered through a pad of Celite by suction and the filter cake was washed with a small amount of NMP. The orange filtrate was stored at −30 ◦ C overnight. The beige solid which precipitated upon cooling was isolated by suction. When there was no precipitation, the volatile components of the filtrate were removed by short-path distillation. The solid residue was taken up in hot methanol and insoluble components were filtered off. The solvent was removed in vacuo and the residue was treated with acetone. Yield: 208 mg (0.28 mmol, 28%). The purity of the product

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was >95% as determined by analytical gradient HPLC. Mp: 285–286 ◦ C (decomposition). 1 H-NMR (400 MHz, DMSO-d6 ): δ [ppm]: 7.89–7.12 (m, 8 H, HAryl ), 3.56–1.94 (m, 12 H, CH2 ), 1.77–1.67 (m, 6 H, CH2 ), 1.56–1.44 (m, 6 H, CH2 ), 1.25–1.20 (m, 6 H, CH2 ), 1.06 (t, 3 J = 8.3 Hz, 9 H, CH3 ). MS (ESI-EM) m/e: 585.1847 (M − C7 H13 N2 O2 )+ calcd for C28 H37 N2 O4 Sn 585.1775. 3.11. General Methods. Radiochemistry N. c. a [124 I]NaI was provided by Department of Nuclear Medicine, University Hospital Essen, University Duisburg-Essen, Germany. Typical radioactivities used for the radioiodination were 134 ± 48 MBq [124 I]NaI in 50 ± 18 µL in 0.01 N NaOH. N. c. a. [123 I]NaI was purchased from GE Healthcare Buchler GmbH & Co KG (Braunschweig, Germany). Typical radioactivities used for the radioiodination were 136 ± 63 MBq [123 I]NaI in 10 ± 3 µL in 0.05 N NaOH. Separation, purification and analyses of the radiochemical yields and the radiochemical purities of all radioiodinated compounds were performed by a gradient radio-HPLC chromatograph system (HPLC A) using a Knauer K-500 and a Latek P 402 pump, a Knauer K-2000 UV-detector (λ = 254 nm) a Crismatec NaI(Tl) Scintibloc 51 SP51 γ-detector and a RP-HPLC Nucleosil column 100-5 C-18 (250 mm × 4.6 mm), a corresponding precolumn (20 mm × 4.0 mm). Sample injection was carried out using a Rheodyne injector block (type 7125 incl. 200 µL loop). The recorded data were processed by the NINA version 4.9 software (GE Medical Systems-Functional Imaging GmbH, Münster, Germany). Separation and purification of the radiosynthesized compounds were—unless specified otherwise—performed by radio-HPLC (HPLC A, see above) using a Nucleosil 100-10 C18 column (250 mm × 8 mm). The recorded data were processed by the NINA version 4.9 software . The radiochemical purities and the specific activities were determined using a radio-HPLC system (HPLC B) composed of a Sykam S1021 pump, a Knauer K-2501 UV-detector (λ = 254 nm), a Crismatec NaI(Tl) Scintibloc 51 SP51 γ-detector, a Nucleosil 100-3 C18 column (200 mm × 3 mm), a VICI injector block (type C1 incl. 20 µL loop) and the GINA Star version 4.07 radiochromatography software (raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany). Radio-TLC was analyzed using a miniGITA TLC-Scanner (raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany). 3.12. 5-[4-(2-Carboxyethyl)piperazin-1-yl]-5-[4-(4-[123/124 I]-iodophenoxy)phenyl]pyrimidine-2,4,6-trione ([123/124 I]9) In a conical glass vial 89 µg (0.12 µmol) stannyl precursor 11 in 39 µL MeOH was added to n. c. a. [123 I]NaI or [124 I]NaI. The radiosynthesis was started by addition of 34 mg (0.14 µmol) chloroamine-T hydrate in 39 µL 0.1 M K2 HPO4 buffer (pH 7.34). The mixture was vortexed for 10 s and allowed to stand for 5 min at room temperature. To quench the reaction 50 µL of a 10% Na2 S2 O5 solution (in water for injection) was added and the mixture was vortexed again for 10 s. After 10 min the quenched reaction solution was injected onto the HPLC column (see Section 3.11. HPLC A) to isolate the fraction of the radiolabelled product [124 I]9 (HPLC conditions: eluent A: CH3 CN/H2 O/TFA 950/50/1, v/v/v, eluent B: CH3 CN/H2 O/TFA 50/950/1, v/v/v; gradient: from 92% B to 40% B within 45 min, 5 min constant at 40% B, and from 40% B to 92% B within 5 min at a flow rate of 2.0 mL/min, λ = 254 nm; tR (stannyl precursor 11) = 40.80 ± 0.81 min (n = 3), tR ([124 I]9) = 32.75 ± 2.13 min (n = 3)). The product fraction was evaporated to dryness and redissolved in 0.9% NaCl (200 µL). The decay-corrected radiochemical yield (after evaporation) was 28 ± 7% (n = 6) for [123 I]9 and 44 ± 6% (n = 3) for [124 I]9, respectively. An aliquot (50 µL) was taken for analytical radio-HPLC. The radiochemical purities were 95 ± 3% and 93 ± 5% for [123 I]9 and [124 I]9, and the molar activities were 0.2–6.3 GBq/µmol and 0.4–14.0 GBq/µmol, respectively. The identity of [123 I]9 and [124 I]9 was proven by HPLC using the non-radioactive analog 9. 3.13. In Vitro Enzyme Inhibition Assays The synthetic fluorometric substrate (7-methoxycoumarin-4-yl) acetyl pro-Leu-Gly-Leu-(3-(2,4dinitrophenyl)-L-2,3-diamino-propionyl)-Ala-Arg-NH2 (R & D Systems, Minneapolis, MN, USA)

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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 CaCl2 , 20 µM ZnSO4 and 0.05% Brij 35 at 37 ◦ C for 30 min. An aliquot of substrate (10 µL of a 50 µM solution) was then added to 90 µL of the preincubated MMP/inhibitor mixture, and the fluorescence was determined at 37 ◦ C by following product release with time. The fluorescence changes were monitored using a Fusion Universal Microplate Analyzer (Packard Bioscience, Boston, MA, USA) with excitation and emission wavelengths set to 330 and 390 nm, respectively. Reaction rates were measured from the initial 10 min of the reaction profile where product release was linear with time and plotted as a function of inhibitor dose. From the resulting inhibition curves, the IC50 values for each inhibitor were calculated by non-linear regression analysis, performed using the Grace 5.1.8 software (Linux). 4. Conclusions Starting from the lipophilic preclincal research tracer [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 (IC50 (MMP-2) = 29 nM, IC50 (MMP-9) = 1.3 nM). Therefore radioiodinated analogues [123/124 I]9 were successfully synthesized for further in vivo evaluations with SPECT and PET. Acknowledgments: This work was supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft (DFG)), Collaborative Research Centre CRC 656, Münster, Germany (CRC 656 projects A02, B01 and Z05). We thank Prof. Wolfgang Brandau, Department of Nuclear Medicine, University Hospital Essen, Essen, Germany, for providing [124 I]NaI. We thank Sandra Höppner and Sven Fatum for excellent technical assistance, Heinrich Luftmann and Klaus Bergander, Organic Chemistry Institute, WWU Münster for performing mass spectrometry and NMR spectroscopy, respectively. Many thanks also to Werner Henkel for his temporarily work in our laboratories. Author Contributions: H.J.B., K.K. and S.W. conceived and designed the experiments; H.J.B., K.K. and S.W. performed the experiments; H.J.B., K.K., M.S. and S.W. analyzed the data; H.J.B. and S.W. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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