Inverse 1,2,3-Triazole-1-yl-ethyl Substituted Hydroxamates as Highly

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Inverse 1,2,3-Triazole-1-yl-ethyl Substituted Hydroxamates as Highly Potent Matrix Metalloproteinase Inhibitors: (Radio)synthesis, in Vitro and First in Vivo Evaluation Verena Hugenberg,*,† Burkhard Riemann,† Sven Hermann,‡ Otmar Schober,† Michael Schaf̈ ers,‡,§ Katrin Szardenings,∥ Artem Lebedev,∥ Umesh Gangadharmath,∥ Hartmuth Kolb,∥ Joseph Walsh,∥ Wei Zhang,∥ Klaus Kopka,†,⊥ and Stefan Wagner† †

Department of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1, D-48149 Münster, Germany ‡ European Institute for Molecular Imaging, University of Münster, Mendelstrasse 11, D-48149 Münster, Germany § Interdisciplinary Centre of Clinical Research (IZKF), Albert-Schweitzer-Campus 1, Building D3, D-48149 Münster, Germany ∥ Siemens Medical Solutions USA, Inc., 6100 Bristol Parkway, Culver City, California 90230, United States S Supporting Information *

ABSTRACT: 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 18F-labeled candidate in mice were investigated.



INTRODUCTION Matrix metalloproteinases (MMPs) are a family of structurally and functionally related zinc- and calcium-dependent secreted or membrane bound endopeptidases.1 They are involved in many physiological processes such as the degradation of extracellular matrix components and tissue remodeling but also play a critical role in many pathophysiological mechanisms promoting a wide range of diseases. Activated MMPs are tightly controlled by endogenous tissue inhibitors of metalloproteinases (TIMPs). In addition to other direct TIMP/MMP interactions, the TIMPs also bind to the zinc atom of the active site of the MMPs and as a result block their activity. Dysregulated MMP expression and activation can be associated with cancer,2 atherosclerosis,3 stroke, arthritis,4 periodontal disease,5 multiple sclerosis,6 and cardiovascular disease.7 Therefore, noninvasive imaging of MMP activity in vivo is of enormous interest in basic research as well as in clinical © XXXX American Chemical Society

applications. This can be realized through radiolabeled MMP inhibitors (MMPIs), which can be used as radiotracers for the detection of activated MMPs using modern molecular imaging techniques, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET). An alternative approach is to attach a fluorescent-dye to the MMPI and use an optical imaging technique, such as fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT), for MMP detection. A number of different synthetic MMPIs have been developed and extensively explored in recent years.8 Examples of nonpeptidic small molecule broad spectrum MMPIs are Nsulfonyl amino acid hydroxamates CGS 25966 and CGS 27023A, which chelate the Zn2+ ion in the active site of the Received: May 7, 2013

A

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Figure 1. Lead structures 1a and 1b as well as a selection of radiolabeled analogs and a fluorescent photoprobe.

Figure 2. Novel class of 1,2,3-triazol-4-yl-methyl-substituted hydroxamate MMPIs.26

enzyme in a bidentate manner via the hydroxamate moiety.9,10 These hydroxamate lead structures with their ability to inhibit a broad spectrum of MMPs were initially chosen in order to check the possibility of MMP visualization with the aforementioned imaging modalities. After a successful proof of this concept, the development of labeled MMP-specific tracers might be worthwhile. Fluorescent photoprobes and radiolabeled derivatives of the CGS lead structures have already been developed by our group. A Cy5.5-labeled photoprobe of CGS 25966 enabled the specific imaging of MMPs in vitro, ex vivo, and in vivo.11,12 The SPECT-compatible 123I-labeled CGS 27023A derivative (HO[123I]I-CGS 27023A) was successfully used to image activated MMPs in vivo in vascular lesions, which develop after carotid artery ligation in apolipoprotein E-deficient (ApoE−/−) mice.13,14 Next, we focused on the development of 18F-labeled PET-compatible radiotracers. Radiosyntheses, in vitro and in vivo evaluations of three different analogs ([18F]FEtO-CGS 27023A, [18F]FEtO-CGS 25966, and [18F]F-CGS 27023A) have been described.15−18 The radiosynthesis of the most promising candidate [18F]FEtO-CGS 25966 ([18F]1e in Figure 1) under GMP conditions has been enabled for first-in-man studies.19 Initial human PET-scans with [18F]1e indicated the potency of this compound to image activated MMPs in vivo but also showed an extensive tracer accumulation in the intestine and liver complicating for example imaging of atherosclerosis. Therefore, we aimed at the improvement of the pharmacokinetics of these hydroxamate-based radiotracers and recently developed a new class of triazole-substituted CGS derivatives,

exhibiting excellent MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13 (IC50 = 0.006−58 nM) as well as significantly increased hydrophilicities as compared to the lead compounds. The compounds were tested against these specific MMPs because of their distinguished impact in cardiovascular diseases, one main focus of our research so far.20−24 In this MMPI class, the benzyl- and the picolylmoieties of the CGS lead structures were systematically substituted with a 1,2,3-triazol-4-yl-methyl unit (Figure 2). Normally, these substituents reach into the S2′ enzyme pocket, which is solvent-exposed and should tolerate some limited structural changes.25 We propose that the triazole ring itself as well as its orientation play an important role for MMP inhibition. In particular, we discovered that MMPIs bearing the 1,2,3-triazole ring close to the hydroxamate moiety were considerably more potent as compared to analogs with a benzyl polyethylene glycol spacer between the backbone of the molecule with a triazole unit attached at the end of the spacer. Obviously the MMPIs with the 1,2,3-triazole ring close to the hydroxamate moiety form additional attractive interaction(s) and/or hydrogen bonds with the residues of the S2′ enzyme pocket and/or the zinc ion of the active site mediated by the Natoms of the triazole unit. A radiolabeled representative member of this new generation of triazole-substituted hydroxamate-based MMPIs ([18F]3a) revealed increased metabolic stability (63% parent in plasma after 30 min) in mice in comparison to the original compound [18F]FEtO-CGS 25966 ([18F]1e, 37% parent after 20 min in plasma). No nonspecific binding of the radiotracer in nonexcretory organs and tissues nor defluorination was observed in vivo. B

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Scheme 1. Synthesis of the Azidoethyl Substituted Hydroxamic Acids 9a−ca

a Reaction conditions: (a) 4-methoxybenzene-1-sulfonyl chloride (a), 4-methylbenzene-1-sulfonyl chloride (b), 4-(2-fluoroethoxy)benzene-1sulfonyl chloride16 (c), pyridine (5a: 82% 5b: 82%, 5c: 50%); (b) 2-azidoethyl 4-methylbenzenesulfonate,27 K2CO3, DMF, 60 °C (6a: 82%, 6b: 73%, 6c: 84%); (c) KSF clay, CH3CN, 84 °C (7a: 46%, 7b: 30%, 7c: 84%); (d) THPONH2, EDC, NMM, HOBT, DMF (8a: 97%, 8b: 86%, 8c: 88%); (e) 4N HCl in dioxane, MeOH, dioxane (9a: 46%, 9b: 35%, 9c: 72%).

Interestingly, despite being structurally quite different, [18F]3a and [18F]1e have very similar clearance characteristics. [18F]3a represents a promising new radiotracer for noninvasive PET imaging of activated MMPs in vivo, due to its high inhibition potencies for MMP-2, -8, -9, and -13, metabolic stability and adequate biodistribution properties.26 To further evaluate the structure−activity relationships (SARs), particularly the optimal position of the triazole unit, the initially discovered triazole-substituted MMPIs were chemically modified by the introduction of an additional CH2-group between the backbone of the molecule and the triazole core and by an inverse arrangement of the nitrogens in the triazole ring. These modifications resulted in a 1,2,3triazole-1-yl-ethyl substitution pattern at the sulfonamide nitrogen with increased conformational flexibility. To investigate the relevance of the p-methoxy substituent at the benzenesulfonyl moiety, which occupies the S1′ pocket of the enzyme, a series of p-methyl and p-fluoroethoxy substituted derivatives were prepared. Here, we describe the (radio)synthesis, in vitro and first in vivo evaluation of this new group of hydroxamate-based MMPIs with an inversely attached triazole moiety.

in DMF. Cleavage of the THP protecting groups was performed in hydrochloric acid containing dioxane/methanol mixtures, yielding the azide key intermediates 9a−c with different p-substituents at the benzenesulfonyl moiety (9a: OCH3, 9b: CH3, 9c: OCH2CH2F). 4-(2-Fluoroethoxy)benzene-1-sulfonyl chloride, required for the synthesis of MMPI precursor 9c, was prepared according to literature procedures.16 5-Fluoropent-1-yne, used as the alkyne building block for the 1,3-dipolar cycloaddition, was prepared starting from 4pentyne-1-ol via tosylation reaction and subsequent fluorination with TBAF (Scheme 2). Scheme 2. Synthesis of 5-Fluoropent-1-yne (11)a

a

Reaction conditions: (a) 4-methylbenzene-1-sulfonyl chloride (82%); (b) TBAF, water (46%).



Copper(I)-catalyzed click reactions of the azido key intermediates 9a−c with different alkyne building blocks were accomplished with varying yields (11−71%), resulting in a series of inverse 1,2,3-triazole-substituted hydroxamic acid derivatives 12a−e, 13a−c, and 14a−e (Table 1). Applying this strategy, the synthesis of three groups of fluorinated inverse 1,2,3-triazole-substituted MMPIs, bearing a p-methoxy (12a− e), a p-methyl (13a−c), or a p-fluoroethoxy (14a−e) moiety at the benzenesulfonyl ring, was successfully realized. In contrast to the p-methoxy (12a−e) and p-methyl series (13a−c), the pfluoroethoxy analogs (14a−e) represent reference compounds of radiofluorinated isotopomers with two different potential positions for 18F. Additionally, our previous studies have shown that the p-fluoroethoxy motif at this position should be well tolerated without affecting the MMP inhibition.15,16 Furthermore, the set of the initially discovered highly potent triazole-substituted MMPIs26 was further explored with inhibitors carrying a p-fluoroethoxy moiety at the benzenesulfonyl ring (20a−c) to estimate their inhibition potencies compared to the inverse triazoles. Therefore, a hydroxamate intermediate containing a p-(2-fluoroethoxy)benzenesulfonyl and a propargyl moiety was synthesized according to Scheme 3 (19), and click reactions with different azide building blocks

RESULTS AND DISCUSSION Chemistry. To evaluate the SARs of these new triazolesubstituted hydroxamates, initially a small set of nonradioactive fluorinated standard compounds was synthesized and tested in enzymatic fluorogenic MMP inhibition assays. The key step to prepare these inverse 1,2,3-triazole-1-yl-ethyl substituted MMPIs was a copper(I) catalyzed Huisgen 1,3dipolar cycloaddition of azidoethyl substituted hydroxamic acid derivatives with different fluorinated alkyne building blocks. The preparation of the azidoethyl substituted key intermediates, possessing different p-substituents at the benzenesulfonyl moiety, is summarized in Scheme 1. Nucleophilic substitution of the commercially available tert-butyl ester of Dvaline hydrochloride 4 with different p-substituted benzene-1sulfonyl chlorides in pyridine resulted in the sulfonamides 5a− c. N-Alkylation with 2-azidoethyl 4-methylbenzenesulfonate27 under basic conditions yielded the carboxylic acid esters 6a−c, which were converted to the carboxylic acids 7a−c in acetonitrile with KSF clay under reflux. Preparation of 7a−c into the corresponding hydroxamic acid esters 8a−c was achieved by O-THP hydroxylamine, EDC, HOBT, and NMM C

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Table 1. Synthesis of MMPIs via Copper(I)-Catalyzed Click Reaction

cpd

R1

R2

yield (%)

12a 12b 12c 12d 12e 13a 13b 13c 14a 14b 14c 14d 14e

OCH3 OCH3 OCH3 OCH3 OCH3 CH3 CH3 CH3 OCH2CH2F OCH2CH2F OCH2CH2F OCH2CH2F OCH2CH2F

CH2CH2CH2F CH2(OCH2CH2)4OCH2CH2F CH2CH2CH2CH2O-(3-(2-fluoro-pyridinyl)) N-(4-fluoro-benzenesulfonamidyl) N-methyl-N-(4-fluoro-benzenesulfonamidyl) CH2CH2CH2F CH2(OCH2CH2)4OCH2CH2F CH2CH2CH2CH2O-(3-(2-fluoro-pyridinyl)) CH2CH2CH2F CH2(OCH2CH2)4OCH2CH2F CH2CH2CH2CH2O-(3-(2-fluoro-pyridinyl)) N-(4-fluoro-benzenesulfonamidyl) N-methyl-N-(4-fluorobenzenesulfonamidyl)

38 71 42 53 30 11 30 58 38 40 48 20 17

Scheme 3. Synthesis of MMPI 20a−ca

a

Reaction conditions: (a) propargyl bromide, K2CO3, DMF (78%); (b) KSF clay, CH3CN (78%); (c) THPONH2, EDC, NMM, HOBT, DMF (92%); (d) 4 N HCl in dioxane, MeOH, dioxane (59%); (e) CuSO4·5H2O, sodium ascorbate, DMF, H2O, 1-azido-2-fluoroethane,28 1-azido-2-(2(2-(2-fluoroethoxy)ethoxy)ethoxy)ethane,29 3-(2-(2-(2-(2-azidoethoxy)ethoxy)-ethoxy)ethoxy)-2-fluoropyridine30 (20a: 63%, 20b: 36%, 20c: 63%).

accomplished via click reactions with precursors 9a−c and alkyne based radiosynthons, i.e., 5-[18F]fluoropent-1-yne, 1[18F]fluoro-3,6,9,12,15-pentaoxaoctadec-17-yne, 2-[18F]fluoro3-(hex-5-yn-1-yloxy)pyridine, 4-[18F]fluoro-N-(prop-2-yn-1yl)benzenesulfonamide, and 4-[18F]fluoro-N-methyl-N-(prop2-yn-1-yl)benzenesulfonamide, that are readily available via one 18 F-labeling step. On the other hand, the propargyl key intermediate 19 can be converted with azide based radiosynthons, i.e., 1-azido-2-[18F]fluoroethane, 1-azido-2-(2-(2-(2[18F]fluoroethoxy)ethoxy)ethoxy)ethane and 3-(2-(2-(2-(2azidoethoxy)ethoxy)ethoxy)ethoxy)-2-[18F]fluoropyridine also producible in one step. Additionally, the preparation of a p[18F]fluoroethoxy substitutent at the benzenesulfonyl unit is

were performed yielding the corresponding 1,2,3-triazole-4-ylmethyl-substituted derivatives 20a−c. In summary, three series of inverse triazole-substituted hydroxamate MMPIs with different p-substituents at the benzenesulfonyl moiety were prepared from azidoethyl substituted key intermediates 9a−c by 1,3-dipolar cycloaddition with alkyne building blocks. Additionally, click reactions from the propargylic key intermediate 19 bearing a p-fluoroethoxysubstituent at the benzenesulfonyl unit were performed yielding the 1,2,3-triazole-substituted CGS-derivatives 20a−c. Fluorinated nonradioactive reference compounds of potential MMPtargeted radioligands 12a−e, 13a−c, 14a−e, and 20a−c were obtained to measure their MMP inhibition potencies by enzymatic fluorogenic assays. 18F-labeling can potentially be D

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Table 2. MMP Inhibition Potencies and clog D (log D) Values of Novel, (Inverse) Triazole-Substituted Hydroxamic Acids 12− 14, and 20 IC50 [nM]a

log D values

compound

MMP-2

MMP-8

MMP-9

1b10 1e17 2a26 2b26 2c26 3a26 3b26 3c26 3d26 9a 12a 12b 12c 12d 12e 9b 13a 13b 13c 9c 14a 14b 14c 14d 14e 19 20a 20b 20c

11d 4±3 2±1 4 ± 0.5 30 ± 10 0.13 ± 0.07 0.1 ± 0.03 3 ± 0.5 0.2 ± 0.09 0.9 ± 0.05 0.7 ± 0.02 4 ± 0.9 0.9 ± 0.08 5±1 3±1 4±1 2 ± 0.4 3±2 3 ± 0.4 4±1 2 ± 0.4 5±2 0.4 ± 0.1 4±1 9±2 0.8 ± 0.2 2 ± 0.01 2 ± 0.5 0.5 ± 0.2

23d 2±1 0.2 ± 0.08 5 ± 0.8 58 ± 6 0.02 ± 0.004 0.04 ± 0.006 5 ± 0.7 0.5 ± 0.2 2 ± 0.3 0.4 ± 0.2 1 ± 0.08 0.5 ± 0.06 0.2 ± 0.05 1 ± 0.3 1 ± 0.1 0.9 ± 0.07 2 ± 0.5 4 ± 0.6 1 ± 0.1 0.4 ± 0.07 35 ± 24 0.05 ± 0.02 0.7 ± 0.04 5±1 0.3 ± 0.1 2 ± 0.5 0.2 ± 0.09 0.3 ± 0.04

27d 50 ± 27 0.6 ± 0.2 5±1 43 ± 3 0.03 ± 0.003 0.05 ± 0.007 4 ± 0.9 0.6 ± 0.2 6±2 0.07 ± 0.006 2 ± 0.4 0.3 ± 0.09 0.6 ± 0.3 3 ± 0.6 4 ± 0.3 3 ± 0.1 0.4 ± 0.1 6 ± 0.5 4 ± 0.3 2 ± 0.7 10 ± 10 0.8 ± 0.1 3 ± 0.4 11 ± 1 0.07 ± 0.009 7±1 0.2 ± 0.1 0.2 ± 0.7

b

MMP-13

clog D

11 ± 0.3 6±3 4±1 18 ± 2 0.006 ± 0.003 0.04 ± 0.007 1 ± 0.3 0.5 ± 0.03 9±2 0.05 ± 0.02 6 ± 0.4 0.04 ± 0.004 0.09 ± 0.01 1 ± 0.2 1 ± 0.5 0.3 ± 0.03 0.1 ± 0.02 3 ± 0.2 1 ± 0.5 0.07 ± 0.002 7±1 0.07 ± 0.01 0.1 ± 0.01 1±1 0.04 ± 0.01 0.7 ± 0.04 0.6 ± 0.4 0.3 ± 0.04

3.81 4.03 2.09 1.21 1.15 1.53 0.65 1.37 0.58 2.57 2.25 0.64 3.05 2.81 3.31 2.51 2.84 0.41 3.89 2.79 3.12 0.70 4.18 3.04 3.54 2.83 1.75 0.87 1.59

log D (exp) 2.02 ± 0.03

0.60 ± 0.01c

1.25 ± 0.01c

a Values are the mean ± SD of three experiments. bclog D values were calculated by ACD/Chemsketch version ACD/Laboratories 6.00 (log D = log P at physiological pH (7.4)). clog D (exp) value was determined for compounds [18F]3a and [18F]12a. dKi values, where SDs are not denoted.

inhibition potencies against activated MMP-2, -8, -9, and -13. These inverse triazoles are still more potent as compared to the MMPIs with a benzyl polyethylene glycol spacer between the backbone of the molecule and the triazole unit 2a−c (IC50 values of 0.2−58 nM). Considering the various p-substituents at the benzenesulfonyl moiety obvious differences in the MMP inhibition potencies were not determined. Apparently, variations of small-sized substituents at this position are tolerated with respect to the investigated MMPs. This observation was also made for the fluoroethoxy and 1,2,3triazole-4-yl-methyl containing MMPIs 20a−c. They also showed potent MMP inhibition with IC50 values comparable to those of the inverse triazole-substituted counterparts 14a−e. By the introduction of hydrophilic groups, such as mini-PEG units and triazole cores, in combination with fluorinated building blocks a series of MMPIs with a wide range of hydrophilicities were obtained while maintaining good MMP inhibition. To evaluate the hydrophilic properties of the target compounds, the corresponding calculated log D values (clog D) of the synthesized hydroxamic acids are also highlighted in Table 2. The inverse triazole-substituted hydroxamic acids show clog D values ranging from 0.41 (MMPI 13b) to 4.18 (MMPI 14c). Additionally, the partition coefficient (log D value) of the radiofluorinated analog [18F]12a (see section Radiochemistry) was determined experimentally (log D (exp) = 1.25 ± 0.01).

potentially feasible by direct substitution of the corresponding tosylate precursor with [18F]fluoride. In Vitro Enzyme Assays and clog D Values. The MMP inhibition potencies of the key intermediates 6a−c, the inverse triazole-substituted hydroxamates 12a−e, 13a−c, 14a−e, and the triazole-substituted hydroxamates 20a−c bearing a fluoroethoxy moiety against MMP-2, -8, -9, and -13 were measured by fluorogenic in vitro enzymatic inhibition assays using a previously described protocol.31 The resulting IC50 values of the investigated MMPs were compared to those of the initially discovered trizaole-based MMPIs and the lead compound 1e. As displayed in Table 2, the new class of inverse triazolesubstituted hydroxamic acids revealed excellent MMP inhibition potencies with IC50 values in the nanomolar and picomolar range (0.04−35 nM), similar to the initially discovered triazole-based MMPIs (IC50 values of 0.006−5 nM). Compared to the lead compound 1e (IC50 values of 2−50 nM), most of the inhibitors of the new series were more potent. Inverse triazole-substituted MMPIs bearing an N-methyl-N-(4fluorobenzenesulfonamidyl) group at the triazole unit (12e and 14e) demonstrated slightly higher IC50 values (1−11 nM). In summary, the formal introduction of an additional CH2group between the backbone of the molecule and the triazole core as well as the inverse arrangement of the nitrogen atoms in the triazole ring did not indicate any significant effect on the E

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Scheme 4. Radiosynthesis of [18F]12aa

a Reaction conditions: (a) K(K222)[18F]F, K2CO3, CH3CN, 110 °C, 120 s ([18F]11: rcy: 72%, decay corrected); (b) CuSO4·5H2O, sodium ascorbate, DMF, H2O, rt, 15 min ([18F]12a: overall rcy: 47 ± 1%, decay corrected).

The log D (exp) differs from the clog D value (clog D (12a) = 2.25) in 1 unit. Compared to the radiofluorinated triazole substituted analog [18F]3a (log D (exp) = 0.60 ± 0.01) the inverse triazole-substituted hydroxamic acid [18F]12a is approximately 4.5 times more lipophilic. Similar to the initially discovered 1,2,3-triazole-4-yl-methylsubstituted MMPIs, the inverse 1,2,3-triazole-1-yl-ethyl-substituted MMPIs also revealed excellent MMP inhibition. Neither the introduction of an additional CH2-group between the backbone of the molecule and the triazole core nor the inverse arrangement of the nitrogen atoms in the triazole ring attenuated the in vitro potencies. Furthermore, different psubstituents at the benzenesulfonyl moiety that occupies the principal specificity S1′ enzyme pocket were tolerated without changing the inhibition of MMP-2, -8, -9, and -13. This finding may be explained by the similar shape of the hydrophobic S1′ pocket of the here investigated enzymes (MMP-2, -8, -9: intermediate S1′ pocket; MMP-13: deep S1′ pocket), while the binding behavior of the compounds against MMPs with shallow S1′ pockets (e.g., MMP-1, -7) may differ.25,32 But also important to potency and specificity of the MMPI besides the S1′ pocket is the chelating moiety which interacts with the catalytic zinc ion. The hydroxamate moiety is one of the most potent zinc-binding groups, and its affinity for Zn2+ can overwhelm the specific protease binding contributions of the substitutents reaching the S1′ pocket.32,33 Anyhow, these results encouraged us to radiosynthesize the representative 18F-labeled MMP-targeted tracer [18F]12a for further in vitro and in vivo evaluation. Radiochemistry. To compare the inverse triazole-substituted MMPIs with the initially discovered triazole-substituted MMPIs, compound 12a was chosen for the radiosynthesis of its 18 F-labeled analog [18F]12a, because of the structural similarity to [18F]3a.26 Analogue to the radiosynthesis of [18F]3a, a semiautomated two-step procedure for the radiosynthesis of [18F]12a was developed and optimized (Scheme 4). In the first step, the labeling synthon 5-[18F]fluoropent-1-yne ([18F]11) was prepared by nucleophilic substitution of the tosylate precursor (solution of DMF) with anhydrous [18F]fluoride in the presence of Kryptofix 2.2.2 (K222), heating of the reaction mixture to 110 °C, and direct distillation of the immediately formed [18F]11 into a cooled (−10 °C) flask within 2 min. [18F]11 was isolated after 22 ± 2 min in an average radiochemical yield (rcy) of 72 ± 1% (decay corrected, n = 5). The second step consisting of the Huisgen 1,3-dipolar cycloaddition was performed outside the automated radiosynthesizer in DMF using aqueous solutions of copper(II)

Figure 3. In vitro stability of [18F]12a (tR = 8.67 min) after incubation in mouse blood serum at 37 °C after 30 min (top), 90 min (middle), and 120 min (bottom) measured at analytical HPLC system A, method A2, starting with 35% CH3CN in water (0.1% TFA) for 13 min, followed by a linear gradient from 35% to 90% CH3CN in water (0.1% TFA) over 2 min, followed by a linear gradient from 90% to 35% CH3CN in water (0.1% TFA) over 3 min with a flow rate of 1 mL·min−1.

sulfate pentahydrate and sodium ascorbate in 15 min under stirring at room temperature (rt). After purification by semipreparative HPLC, evaporation, and formulation, the two-step radiosynthesis of the inverse 1,2,3-triazole [18F]12a F

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Defluorination of the radioligand in vivo potentially impairing image interpretation (indicated by bone uptake of [18F]fluoride ions) was not observed in the entire dynamic imaging study. Furthermore, no accumulation of [18F]12a in organs/tissues such as the brain, myocardium, lung, and muscles, indicating unspecific binding, was observed. The excretion of [18F]12a predominantly via the hepatobiliary pathway was found to be similar to the original triazolesubstituted tracer [18F]3a.26 Biostability and Metabolism of [18F]12a. Three 10month-old female ICR (CD1) wild type mice were examined at 30 min p.i. to determine the biostability of [18F]12a. Representative radio HPLC traces are shown in Figure 6. The %ID/g values of the total organs are shown in Figure 7 and listed in Table 3 for 30 min time points. The retention time of unchanged [18F]12a tracer was between 10.3−13.0 min. A total of five more polar metabolites were detected, with retention times (tR) of 1.9 min (metabolite 1), 5.9 min (metabolite 2), 7.5 min (metabolite 3), 8.6 min (metabolite 4), and 9.9 min (metabolite 5). The muscle %ID/g of [18F]12a at 30 min was 0.50% with no other metabolites present. In plasma, the %ID/g was 0.33% at 30 min, and the radioactivity was partitioned between 35% (0.12% ID/g) of the parent tracer and 65% (0.21% ID/g) of four metabolites: 9% metabolite 1 (0.03% ID/g), 25% metabolite 3 (0.08% ID/g), 23% metabolite 4 (0.07% ID/g) and 8% metabolite 5 (0.03% ID/g). In the liver sample at 30 min 12% parent (0.35% ID/g) were present, while the remaining 88% (2.56% ID/g) of activity were distributed between four metabolites (metabolites 2, 3, 4 and 5). The main metabolites in the liver were metabolite 3 with 61% (1.77% ID/ g) and metabolite 4 with 20% (0.58% ID/g). Examination of the small intestine and the gall bladder both showed 14% (8.6% ID/g for small intestine, 12.4% ID/g for gall bladder) parent compound, while 86% (53.1% ID/g for small intestine, 76.3% ID/g for gall bladder) of the remaining activity were portioned between the five mentioned metabolites. Similar to the liver, the main compounds were metabolite 3 with 49% (30.2% ID/g for small intestine, 43.5% ID/g for gall bladder) and metabolite 4 with 18% (11.1% ID/g for small intestine, 16.0% ID/g for gall bladder). In the kidney homogenate, the parent tracer was present at higher amounts compared to other tissues at 92%

Figure 4. In vivo biodistribution of radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]12a. Maximum intensity projections of selected time frames p.i. demonstrate elimination of radioactivity from the blood primarily via the liver and less pronounced via the kidneys.

was accomplished with an overall radiochemical yield of 47 ± 1% (decay corrected, n = 5) in 97 ± 2 min from the end of radionuclide production. [18F]12a was isolated in radiochemical purities of >98% with specific activities in the range of 9−46 GBq/μmol at the end of the synthesis. The radioligand was formulated in phosphate-buffered saline (PBS) to determine the log D (exp) value and to study its in vitro stability in mouse blood serum at 37 °C. In Vitro Stability. An in vitro stability study was carried out using mouse blood serum. During long-term incubation for up to 120 min at 37 °C, [18F]12a revealed high serum stability. As shown in Figure 3, only the parent compound [18F]12a was detected by radio HPLC. Significant radiometabolites or decomposition products could not be observed. In Vivo Biodistribution Study. Representative coronal whole body images 0−1, 1−5, 5−10, and 90−120 min after injection of [18F]12a in C57/BL6 mice are shown in Figure 4. Overall, [18F]12a is cleared very fast and efficiently from the body through hepatic and renal elimination with no significant tracer remaining in nonexcretion organs 90−120 min p.i. Because of the fast clearance MMP imaging with [18F]12a is realistic within the first minutes p.i. in C57/Bl6 mice but not within several hours p.i. in contrast to the radioiodinated HO[123I]I-CGS 27023A that showed significantly higher lesional uptake in ApoE−/− mice compared to blocked mice at imaging time points ≥80 min p.i.14

Figure 5. In vivo biodistribution of radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]12a. Time-activity curves illustrate tracer dynamics in selected regions of interests (ROI). %ID: percentage injected dose. G

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Figure 6. Representative radio HPLC traces for 30 min p.i. of the metabolism study of [18F]12a. The radiochemical purity of [18F]12a was >98% before injection. The samples were analyzed by radio-HPLC using a γ-detector (Raytest GmbH/Agilent). For the HPLC analysis, a Phenomenex C18 column (250 × 4.6 mm) was used and a gradient method with acetonitrile and water (both modified with 0.05% TFA).

metabolite 3 again as the main metabolite with 21% (13.7% ID/g). The tracer and the metabolites were predominantly cleared through liver, gall bladder, and small intestine. The activity

(10.3% ID/g) along with two metabolites: metabolite 1 (2%, 0.22% ID/g) and metabolite 3 (6%, 0.67% ID/g). In urine, 67% (43.7% ID/g) of the parent compound was observed at 30 min. The five mentioned metabolites were also present, exposing H

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Table 3. % ID/g Values of the Metabolites of [18F]12a in Muscle, Plasma, Kidney, Urine, Liver, Small Intestine, and Gall Bladder percent injected dose per gram (% ID/g) 30 min p.i.

muscle

plasma

kidney

urine

liver

small intestine

gall bladder

[18F]12a: tR ∼ 10.9 min metabolite 1: tR ∼ 1.9 min metabolite 2: tR ∼ 5.9 min metabolite 3: tR ∼ 7.5 min metabolite 4: tR ∼ 8.6 min metabolite 5: tR ∼ 9.9 min

0.50 0.00 0.00 0.00 0.00 0.00

0.12 0.03 0.00 0.08 0.07 0.03

10.28 0.22 0.00 0.67 0.00 0.00

43.74 1.96 3.26 1.30 13.71 1.31

0.35 0.00 0.12 1.77 0.58 0.09

8.65 4.32 4.94 30.27 11.12 2.47

12.42 6.21 7.10 43.48 15.97 3.55

Figure 7. % ID/g values of the total organs at 30 min post injection of [18F]12a.

additional CH2-group and the inverse arrangement of the nitrogen atoms in the triazole ring, nor the different psubstituents at the benzenesulfonyl moiety negatively affected MMP inhibition. In conclusion, these results suggest that the introduction of a heteroaromatic system (e.g., the triazole) provide additional potential hydrogen bond acceptors, increasing the binding potencies of the inhibitors to the enzyme active site. One promising MMPI lead, [18F]12a, with moderate hydrophilicity compared to tracer 3a,26 was successfully radiolabeled with an overall radiochemical yield of 47 ± 1% (decay corrected). The radiofluorinated MMPI [18F]12a showed excellent serum stability in vitro and rapid clearance in vivo in mice with a clear preference for the hepatobiliary over the urinary excretion pathway, similar to previous observations in the noninverted triazole-substituted [18F]3a. Compared to [18F]3a, the inverse triazole-substituted radiotracer [18F]12a appears to be less stable. However, no defluorination or nonspecific binding of the radioligand in nonexcretory organs were observed in vivo.

remaining in the liver at 30 min p.i. was four times lower than in the kidneys. The amount of activity excreted by the liver into the gall bladder and the small intestine was two times higher than the activity in urine excreted by the kidneys. Therefore this tracer appears to be cleared faster through the liver into small intestine and gall bladder than through the kidneys into urine. At 30 min p.i. 35% of the plasma radioactivity in mice was confirmed as nonmetabolized [18F]12a, whereas 65% resulted from metabolites more polar than [18F]12a (Table 3). Compared to [18F]3a, which shows radioactive metabolites in plasma in a fraction of about 37% at 30 min p.i., the inverse triazole substituted radioligand [18F]12a was less stable in vivo. Obviously, the more flexible conformation of the triazole moiety and/or rather the additional CH2-group between the backbone of the molecule and the triazole core cause increased metabolic instability in mice.



CONCLUSION Our previous results with triazole-substituted MMPIs indicate that the triazole ring greatly influences enzyme inhibition. To further evaluate the SAR and the relevance of the position of the triazole unit, we herein present the synthesis and in vitro characterization of a chemically modified series of fluorinated inverse triazole-substituted hydroxamate-based MMPIs. These modifications result in a 1,2,3-triazole-1-yl-ethyl substitution pattern at the sulfonamide nitrogen with an increased conformational flexibility of this moiety and different psubstitutents at the benzenesulfonyl moiety to estimate the relevance of the original p-methoxy substitutent. Similar to the original triazole-substituted MMPI series the inverse triazole hydroxamates are excellent inhibitors of MMP-2, -8, -9, and -13 (IC50 values of 0.04−35 nM) and show calculated log D values ranging from 0.41 to 4.18. Thus, neither the introduction of an



EXPERIMENTAL SECTION

General. All chemicals, reagents, and solvents for the synthesis of the compounds were analytical grade, purchased from commercial sources and used without further purification unless otherwise specified. All air and moisture-sensitive reactions were performed under argon atmosphere. Solvents were purified and dried by literature methods where necessary. The melting points (mp) are uncorrected and were determined in capillary tubes on a Stuart Scientific SMP3 capillary melting point apparatus. Column chromatography was performed on Merck silica gel 60 (0.040−0.063 mm). Thin layer chromatography (TLC) was carried out on silica gel-coated polyester backed TLC plates (Polygram, SIL G/UV254, Macherey-Nagel) using solvent mixtures of cyclohexane (CH), ethyl acetate (EA) and methanol (MeOH). Compounds were visualized by UV light (254 I

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heated to reflux for 3 h. After complete conversion, as indicated by TLC, the reaction mixture was filtered and washed with EA (2 × 20 mL). The combined organic layers were dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. Column chromatographic purification (silica gel, CH/EA 2:1) yielded the product as colorless oil (2.05 g, 5.8 mmol, 46%). HRMS-ES-EM: m/z = 379.1045 [(M + Na) + ] calcd for C14H20N4O5SNa+: 379.1047. (2R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-3-methyl-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (8a). To a solution of the carboxylic acid 7a (3.25 g, 9.1 mmol) in DMF (25 mL) HOBT (1.47 g, 10.9 mmol, 1.2 equiv), NMM (3.0 mL, 27.3 mmol, 3.0 equiv), THPONH2 (3.3 g, 28.2 mmol, 3.1 equiv) and EDC (2.45 g, 12.8 mmol, 1.4 equiv) were added. After being stirred at rt for 16 h, the reaction mixture was diluted with water (150 mL) and extracted with EA (3 × 50 mL). The combined organic phases were washed with water, 5% aqueous KHSO4, saturated aqueous NaHCO3, and brine. After being dried over magnesium sulfate, the solvent was removed under reduced pressure. The product was purified by column chromatography (silica gel, CH/EA 4:1) yielding a diastereomeric mixture of THP-protected hydroxamic ester 8a as a colorless wax (4.02 g, 8.8 mmol, 97%). HRMS-ES-EM: m/z = 478.1725 [(M + Na)+] calcd for C19H29N5O6SNa+: 478.1731. HRMS-ES-EM: m/z = 454.1762 [(M − H)−] calcd for C19H28N5O6S−: 454.1766. (R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-N-hydroxy-3-methylbutanamide (9a). (2R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-3-methyl-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (8a, 2.25 g, 4.9 mmol) was dissolved in dry dioxane (3.0 mL). 4 N hydrochloric acid in dioxane (4.9 mL, 19.7 mmol, 4.0 equiv) and dry MeOH (3.0 mL) were added. After being stirred for 1.5 h at rt, the reaction mixture was diluted with EA (20 mL). The organic layer was washed with water and dried over magnesium sulfate, and the solvent was removed under reduced pressure yielding the hydroxamic acid 9a as beige solid (835 mg, 2.3 mmol, 46%); mp 147 °C. HRMS-ES-EM: m/z = 394.1156 [(M + Na)+] calcd for C14H21N5O5SNa+: 394.1146. General Procedure for the Preparation of Triazoles 12a−e, 13a−c, 14a−e, and 20a−c. To a solution of the azide compound (0.032−2.0 mmol, 1.0 equiv) in DMF (8 mL/mmol) and H2O (2 mL/ mmol) were added CuSO4·5H2O (50 mol %), sodium ascorbate (60 mol %), and the corresponding alkyne (0.032−2.0 mmol, 1.0−3.7 equiv) in sequence. After being stirred at rt, the reaction mixture was diluted with H2O (20 mL) and extracted with EA (3 × 15 mL). The combined organic layers were washed with brine and dried (magnesium sulfate). After evaporation of the solvent, the residue was purified by silica gel column chromatography. Experimental and spectroscopic data of triazoles 12b−e, 13a−c, 14a−e, and 20a−c are listed in the Supporting Information. (R)-2-(N-(2-(4-(3-Fluoropropyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-methoxyphenylsulfonamido)-N-hydroxy-3-methylbutanamide (12a). 12a was obtained from 9a (100 mg, 0.27 mmol) and 5-fluoropent1-yne (11, ca. 1.0 mmol) after 2 h of stirring at rt. Column chromatographic purification (silica gel, EA) gave a colorless solid (47 mg, 0.10 mmol, 38%), mp 54 °C. 1H NMR (300 MHz, CDCl3) δ 9.47 (s, OH, 1H), 7.74 (d, ArH, 3JH,H = 8.9 Hz, 2H), 7.46 (s, CCHN, 1H), 6.97 (d, ArH, 3JH,H = 8.9 Hz, 2H), 4.72 (m, CH2CH2N, 1H), 4.58 (m, CH2CH2N, 1H), 4.50 (dt, 2JH,F = 47.2 Hz, 3JH,H = 5.9 Hz, CH2F, 2H), 3.97 (m, CH2CH2N, 1H), 3.86 (s, OCH3, 3H), 3.62 (d, NCH, 3JH,H = 10.9 Hz, 1H), 3.47 (m, CH 2 CH 2 N, 1H), 2.93−3.78 (m, CH2CH2CH2F, 2H), 2.21 (m, CH(CH3)2, 1H), 2.10 − 1.97 (m, CH2CH2F, 2H), 0.84 (d, CH(CH3)2, 3JH,H = 6.6 Hz, 3H), 0.37 (d, CH(CH3)2, 3JH,H = 6.5 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 168.03 (CONH), 163.46 (qArCOCH3), 146.86 (CCHN), 130.51 (qArCSO2), 129.42 (ArCH), 122.11 (CCHN), 114.44 (ArCH), 83.09 (d, CH2F, 1JC,F = 47.2 Hz), 62.97 (NCH), 55.69 (OCH3), 49.35 (CH2CH2N), 44.31 (CH2CH2N), 29.92 (d, CH2CH2F, 2JC,F = 20.0 Hz), 26.58 (CH(CH3)2), 21.24 (d, CH2CH2CH2F 3JC,F = 5.7 Hz), 19.37 (CH(CH3)2), 18.57 (CH(CH3)2). 19F NMR (282 MHz, CDCl3) δ −220.45 (tt, 2JH,F = 47.2, 3JH,F = 25.8 Hz, 1F). HRMSES-EM: m/z = 480.1684 [(M + Na)+] calcd for C19H28FN5O5SNa+:

nm). NMR spectra were recorded in CDCl3, CD3OH, or DMSO-d6 on a Bruker ARX300, a Bruker DPX300 (1H NMR, 300 MHz, 13C NMR, 75 MHz, 19F NMR, 282 MHz), a Bruker AMX 400 (1H NMR, 400 MHz, 13C NMR, 100 MHz) and a Varian Unity plus 600 (1H NMR, 600 MHz, 13C NMR, 151 MHz) spectrometer. TMS (1H), CDCl3, DMSO-d6, CD3OH (13C), and CFCl3 (19F) were used as internal standards and all chemical shift values were recorded in ppm (δ). Exact mass analyses were conducted on a Bruker MicroTof apparatus. The chemical purities of each new nonradioactive compound were ≥95% and assessed by analytical gradient reversed-phase HPLC system A or B (λ = 254 nm). HPLC System A: Two Smartline 1000 pumps and a Smartline UV detector 2500 (Herbert Knauer GmbH), a GabiStar γdetector (Raytest Isotopenmessgeräte GmbH) and a Nucleosil 100−5 C-18 column (250 mm × 4.6 mm). The recorded data were processed by the GINA Star software (Raytest Isotopenmessgeräte GmbH). The HPLC method A1 started with a linear gradient from 10% to 90% CH3CN in water (0.1% TFA) over 9 min, followed by a linear gradient from 90% to 10% CH3CN in water (0.1% TFA) over 6 min, with a flow rate of 1 mL·min−1 (unless otherwise specified). HPLC method A2 started with 35% CH3CN in water (0.1% TFA) for 13 min, followed by a linear gradient from 35% to 90% CH3CN in water (0.1% TFA) over 2 min, followed by a linear gradient from 90% to 35% CH3CN in water (0.1% TFA) over 3 min with a flow rate of 1 mL· min−1. HPLC system B: Two K-1800 pumps and an S-2500 UV detector (Herbert Knauer GmbH), a GabiStar γ-detector (Raytest Isotopenmessgeräte GmbH). The recorded data were processed by the ChromGate HPLC software (Herbert Knauer GmbH). HPLC method B1 using a Nucleosil 100−5 C18 column (250 mm × 4.6 mm) started with a linear gradient from 10% to 80% CH3CN in water (0.1% TFA) over 18 min, holding for 20 min and followed by a linear gradient from 80% to 10% CH3CN in water (0.1% TFA) over 2 min, with a flow rate of 1.5 mL·min−1. HPLC method B2 using a Eurospher column (100 C18, 250 mm × 20 mm) started with a linear gradient from 10% to 80% CH3CN in water (0.1% TFA) over 18 min, holding for 20 min and followed by a linear gradient from 90% to 10% CH3CN in water (0.1% TFA) over 2 min, with a flow rate of 7.0 mL·min−1. N[(Methoxyphenyl)sulfonyl]-D-valine tert-butyl ester (5)9 2-azidoethyl 4-methylbenzenesulfonate,27 1-azido-2-fluoroethane,28 1-azido-2-(2(2-(2-fluoroethoxy)ethoxy)ethoxy)ethane, 2 9 3-(2-(2-(2-(2azidoethoxy)ethoxy)-ethoxy)ethoxy)-2-fluoropyridine,30 2-fluoroethyl 4-methylbenzenesulfonate (10),16 1-fluoro-3,6,9,12,15-pentaoxaoctadec-17-yne,26 2-fluoro-3-(hex-5-yn-1-yloxy)pyridine,30 4-fluoro-N(prop-2-yn-1-yl)benzenesulfonamide34 and 4-fluoro-N-methyl-N(prop-2-yn-1-yl)benzenesulfonamide35 were synthesized following literature procedures. For some long chain compounds 13C NMR signals at δ ∼ 70 ppm do have multiple intensities. All animal experiments were conducted in accordance with local institutional guidelines for the care and use of laboratory animals. Synthesis of MMPI Precursor 9a. (R)-tert-Butyl 2-(4-methoxyphenylsulfonamido)-3-methylbutanoate (5a). A white solid, yield: 75%, mp 120.3 °C, analytical data see ref 9. (R)-tert-Butyl 2-(N-(2-azidoethyl)-4-methoxyphenylsulfonamido)-3-methylbutanoate (6a). To a solution of (R)-tert-butyl 2-(4methoxyphenylsulfonamido)-3-methylbutanoate (5a) (7.96 g, 23.2 mmol) in DMF (ca. 65 μmol/mL, 360 mL) 2-azidoethyl 4methylbenzenesulfonate27 (5.60 g, 23.2 mmol) and potassium carbonate (32.1 g, 232 mmol) were added. The resulting suspension was stirred at 50 °C for 2 days. The mixture was diluted with water (400 mL) and extracted with EA (3 × 100 mL). The combined organic phases were washed with brine and dried over magnesium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica gel, CH/EA 9:1). The product was obtained as gray oil (7.85 g, 19.0 mmol, 82%). HRMS-ES-EM: m/z = 435.1671 [(M + Na)+] calcd for C18H28N4O5SNa+: 435.1673. (R)-2-(N-(2-Azidoethyl)-4-methoxyphenylsulfonamido)-3-methylbutanoic Acid (7a). To a solution of (R)-tert-butyl 2-(N-(2azidoethyl)-4-methoxyphenylsulfonamido)-3-methylbutanoate (6a, 5.16 g, 12.5 mmol) in CH3CN (25 mL) montmorillonite KSF clay (4.17 g, ca. 1.0 g/3 mmol ester) was added, and the suspension was J

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480.1687. HPLC system B, method B1: tR = 18.05 min (>99%). HPLC system A, method A1: tR = 7.88 min (>99%). Radiochemistry. General Methods. Radiofluorinations were carried out on a modified PET tracer radiosynthesiser (TRACERLab FxFDG, GE Healthcare). The recorded data were processed by the TRACERLab Fx software (GE Healthcare). Separation and purification of the radiosynthesised compounds were performed on the following semipreparative radio-HPLC system C (λ = 254 nm): K500 and K-501 pump, K-2000 UV detector (Herbert Knauer GmbH), NaI(TI) Scintibloc 51 SP51 γ-detector (Crismatec) and an ACE 5 AQ column (250 mm × 10 mm). The recorded data were processed by the GINA Star software (Raytest Isotopenmessgeräte GmbH). Radiochemical purities and specific activities were determined using the analytical radio-HPLC system A. No-carrier-added aqueous [18F]fluoride was produced on an RDS 111e cyclotron (CTI-Siemens) by irradiation of a 1.2 mL water target using 10 MeV proton beams on 97% enriched 18O-water by the 18O(p,n)18F nuclear reaction. Extraction and Drying of [18F]fluoride. To recover the [18O]water, the batch of aqueous [18F]fluoride was passed through an anion exchange resin (Sep-Pak Light Waters Accell Plus QMA cartridge, preconditioned with 5 mL 1 M K2CO3 and 10 mL of water). [18F]Fluoride was eluted from the resin with a mixture of 40 μL of 1 M K2CO3, 200 μL of water for injection, and 800 μL of DNA-grade CH3CN containing 18 mg of Kryptofix 2.2.2 (K222). Subsequently, the aqueous [18F]K(K222)F solution was carefully evaporated to dryness in vacuo (2 min vacuum with helium stream, 1 min at 56 °C without helium, then 8 min at 84 °C without helium). Reaction of [18F]fluoride with Pentynyl Tosylate. A solution of the tosylate precursor 10 (20 mg, 75 μmol) dissolved in DMF (0.5 mL) was added to the reaction vessel containing the dried [18F]fluoride. The vessel was heated to approximately 110 °C. The immediately formed [18F]fluoropentyne [18F]11 was distilled directly from the reaction vessel into a 5 mL-flask with 400 μL of DMF (cooled to −10 °C) within 2 min. The click labeling was performed outside the automated radiosynthesiser. The azide precursor 9a (6 mg, dissolved in 100 μL of DMF), CuSO4 (0.4 M, 60 μL) and sodium ascorbate (8 mg, dissolved in 100 μL of H2O) were added to the reaction mixture and the reaction was stirred at rt for 15 min. The crude was passed through a Waters Sep-Pak Light cartridge filled with quartz wool. The cartridge was rinsed with DMF (0.2 mL). The eluate was diluted with 500 μL of water, and the resulting mixture was purified by gradient-radio-HPLC system C (flow = 5.5 mL/min; eluents: A: CH3CN/TFA, 1000/1, B: H2O/TFA, 1000/1; isocratic: A/B 33/67 (v/v). The product fraction of compound [18F]10a (retention time tR([18F]12a) = 10.3−13.0 min) was evaporated to dryness in vacuo and redissolved in 0.5 mL of NaCl/EtOH (9/1 v/v). Product compound [18F]12a was obtained in an overall radiochemical yield of 47 ± 1% (decay-corrected, based on cyclotron-derived [18F]fluoride ions, n = 5) in 97 ± 2 min from the end of radionuclide production. [18F]12a was isolated in radiochemical purities of >98% with specific activities in the range of 9−46 GBq/μmol at the end of the synthesis. Radiochemical purity and specific activity were determined using the analytical radio-HPLC system A (method A2). In Vitro Enzyme Inhibition Assays (Table 3). The inhibition potencies of hydroxamic acid derivatives 12a−e, 13a−c, 14a−e, and 20a−c against activated MMP-2, -8, -9, and -13 were assayed using the synthetic fluorogenic substrate (7-methoxycoumarin-4-yl)acetyl-ProLeu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl)Ala-ArgNH2 (R&D Systems) as described previously.31 Briefly, MMP-2, -8, -9, or -13 (each at 2 nM) and test compounds at varying concentrations (10 pM to 1 mM) in Tris-HCl (50 mM), pH 7.5, containing NaCl (0.2 M), CaCl2 (5 mM), ZnSO4 (20 μM), and 0.05% Brij 35 were preincubated at 37 °C for 30 min. An aliquot of substrate (10 μL of a 50 μM solution) was added to the enzyme−inhibitor mixture (90 μL), and the fluorescence changes were monitored using a Fusion Universal Microplate analyzer (Packard Bioscience) with excitation and emission wavelengths of 330 and 390 nm, respectively. Reaction rates were measured from the initial 10 min and plotted as function of inhibitor concentration. From the resulting inhibition curves, the IC50 values

were calculated by nonlinear regression analysis using the Grace 5.1.8 software (Linux). Determination of the Partition Coefficient (log D (exp)). The lipophilicity of radioligand [18F]12a was assessed by determination of the water−octanol partition coefficient following a published procedure.36 In brief, approximately 20 kBq of [18F]12a was mixed with equal amounts (0.5 mL) of PBS (pH 7.4) and 1-octanol, and the resulting biphasic system was mixed vigorously for 1 min at rt. The tubes were centrifuged (3000 rpm, 2 min), and three samples of 100 μL of each layer were counted in a gamma counter (Wallac Wizard, Perkin-Elmer Life Science). The partition coefficient was determined by calculating the ratio cpm (octanol)/cpm (PBS) and expressed as log D (exp) ± log(cpmoctanol/cpmPBS)). Two independent experiments were performed in triplicate, and data were provided as mean values ± standard deviation. Stability in Mouse Serum. The serum stability of radioligand [18F]12a was evaluated by incubation in mouse serum at 37 °C for up to 120 min. An aliquot of the PBS-formulated [18F]12a (20 μL, 5 MBq) was added to a sample of mouse serum (200 μL), and the mixture was incubated at 37 °C. Samples of 20 μL each were taken after periods of 10, 20, 30, 60, 90, and 120 min and quenched in MeOH/CH2Cl2 (1:1 (v/v), 100 μL) followed by centrifugation for 2 min. The organic layer was analyzed by analytical radio-HPLC A (method A2, tR = 8.67 min). Biostability and Metabolism Study. Approximately 11.1 MBq of [18F]12a (in a maximum volume of 200 μL) was injected into three mice via tail vein injection. The animals were sacrificed at 30 min p.i. Whole blood was obtained, weighed, and centrifuged at 3000 rpm (3 min) to isolate plasma. Urine was also collected. The muscle, kidneys and liver were harvested, weighed, and homogenized in lysis buffer (1% SDS in PBS buffer). An aliquot of each sample (400 μL) was subsequently removed, mixed with 400 μL of acetonitrile and 100 μL of 3% acetic acid in acetonitrile, vigorously mixed, and placed on dry ice for 3 min. After thawing, the samples were centrifuged at 13000 rpm (8 min) to allow for the separation of supernatant from the pellet. To separate the gall bladder the whole liver was first extracted, the bile duct was pinched with a tweezers and cut. The gall bladder was put in a glass vial, 500 μL of lysis buffer and 500 μL of 5% acetic acid in acetonitrile were added, and the sample was vortexed for 5 min followed by centrifugation (13000 rpm, 8 min). Approximately 0.1− 0.2 g of small intestine sample from each mouse (n = 3 mice) was dissected clean from mesenteric fat, the luminal contents was squeezed out with tweezers. 2.00 g of lysis buffer was added to the sample and the sample was weighted. The sample was then homogenized using a mechanical tissue homogenizer, 200−400 mg of the homogenate were added to a preweighted counting tube and counted in the gamma counter. The homogenate was then frozen in the dry ice and subsequently thawed at room temperature. The homogenate was diluted with 2.0 mL of 3% acetic acid in acetonitrile and mixed again using the tissue homogenizer. A 400 μL sample of the resulting slurry was centrifuged (13000 rpm, 8 min). The supernatant was then removed and assayed for radioactivity in a PerkinElmer Wizard γcounter (20 s). A 2 μL aliquot from the dose sample was counted along with the samples and was used to calculate the % ID/g. The samples were analyzed by HPLC, using a γ-detector (Raytest GmbH/ Agilent). The HPLC was done on a Phenomenex C18 column (250 × 4.6 mm) using a gradient method with acetonitrile and water (both having 0.05% TFA). Tebia bone was extracted from the hind leg, cleaned of the muscle, ligaments and parts of the joint placed in a preweighted plastic tube, weighted and counted in a γ-counter. Animals. Adult C57/BL6 mice (male, 21−24 g) were anaesthetised by isoflurane/O2 and one lateral tail vein was cannulated using a 27 G needle connected to 15 cm polyethylene catheter tubing. [18F]12a (250 kBq/g bodyweight) was injected as a bolus (100 μL of compound flushed with 100 μL of saline) via the tail vein and subsequent PET scanning was performed. Experiments were conducted according to German Animal Welfare guidelines. Small Animal PET Scanning. PET experiments were carried out using a submillimeter high resolution (0.7 mm full width at halfmaximum) small animal scanner (32 module quadHIDAC, Oxford K

dx.doi.org/10.1021/jm4006753 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Positron Systems Ltd., Oxford, UK) with uniform spatial resolution (