Molecular modeling of a library of piperazine-based MMP inhibitors The crystal structure of MMP-9 was downloaded from the Protein Data Bank

(PDB) (PDB code 2OW1). All molecules were drawn using ChemaxonMarvinSketch (www.chemaxon.com) and prepared (structure recognition and protonation) using SPORES (www.tcd.uni-konstanz.de/research/spores.php). Molecular docking

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simulations were performed using PLANTS v1.6.140,141. The docking site center was determined by applying a constraint for the hydroxamic group to be able to form a coordination with the zinc in the active site. Fifteen poses were generated for each compound and the docking results were analyzed using Molegro Vir-tual Docker (www.molegro.com). Docking solutions were selected based on the MOLDOCKSCORE and the docking solutions were evaluated manually, followed by energy minimization of the ligand.

5.3 4-(Tert-butoxycarbonyl)-1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxylic acid (6)

An aqueous solution of sodium hydroxide (4.6 mL, 50% w/w, 19.4 N, 88.6 mmol) was slowly added to a solution of dl-piperazine carboxylic acid dihydrochloride 4 (5.99 g, 29.5 mmol) in 60 mL of p-dioxane and 30 mL of water at 0⁰C. Addition of di-tert-butyl dicarbonate (7.09 g, 32.5 mmol) was followed and the mixture was stirred overnight at room temperature yielding 5. Freshly distilled triethylamine (8.23 mL, 59.0 mmol), 4-dimethylaminopyridine (721 mg, 5.90 mmol) and 4-me-thoxyphenylsulfonyl chloride (6.09 g, 29.5 mmol) were added to the mixture at 0⁰C and the reaction was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure and partitioned between EtOAc and 1 N HCl. The EtOAc layer (2 x 100 mL) was washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo to give the title compound 6 (10.2 g, 86%):

1H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 7.83 – 7.74 (m, 2H), 7.10 – 7.01 (m, 2H), 4.32 (t, J = 7.3 Hz, 1H), 4.06 (dd, J = 12.4, 7.3 Hz, 1H), 3.83 – 3.67 (m, 5H), 3.56 – 3.40 (m, 2H), 3.32 (dd, J = 12.5, 7.3 Hz, 1H), 1.39 (s, 9H).

HRMS-ESI: calc for C₁₇H₂₅N₂O₇S ([M + H]⁺), 401.5, found 401.2; calc for C₁₇H₂₈N₃O₇S ([M + NH₄]⁺), 418.5, found 418.3; calc for C₃₄H₄₉N₄O₁₄S₂ ([2M + H]⁺), 801.9, found 801.5.

5.4 Methyl 1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxylate hydrochloride (7)

Thionyl chloride (8.72 mL, 120.0 mmol) was added dropwise to a solution of 4-(tert-butoxycarbonyl)-1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxylic acid 6 (9.60 g, 24.0 mmol) in 50 mL of methanol at 0⁰C. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated under

re-duced pressure to a solid residue, which was triturated with 5% methanol/hexane to give the title compound 7 (6.90 g, 82%):

1H NMR (400 MHz, DMSO-d₆) δ 7.83 – 7.75 (m, 2H), 7.08 – 7.02 (m, 2H), 3.87 (t, J = 8.1 Hz, 1H), 3.65 (s, 3H), 3.41 – 3.13 (m, 3H), 2.95 – 2.71 (m, 3H), 1.91 (s, 1H).

HRMS-ESI: calc for C₁₃H₁₉N₂O₅S ([M + H]⁺ without HCl), 315.4, found 315.2; calc for C₂₆H₃₇N₄O₁₀S₂ ([2M + H]⁺ without HCl), 629.7, found 629.3.

5.5 4-((Trimethylsilyl)ethynyl)benzoyl chloride (9)

Thionyl chloride (6.25 mL, 86.0 mmol) was added dropwise to a solution of 4-((tri-methylsilyl)ethynyl)benzoic acid 8 (4.69 g, 21.5 mmol) in 50 mL of chloroform at 0⁰C. The mixture was kept under reflux for 2 h. The reaction mixture was con-centrated under reduced pressure and coevaporated three times with chloroform to give the corresponding acyl chloride 9, which was immediately used without further purification.

5.6 Methyl 1-((4-methoxyphenyl)sulfonyl)-4-(4-((trimethylsilyl)ethynyl) benzoyl)piperazine-2-carboxylate (10)

To a solution of amine hydrochloride 7 (5.02 g, 14.3 mmol) in 10 mL of water and 20 mL of p-dioxane at 0⁰C was added subsequently freshly distilled triethylamine (6.18 mL, 44.3 mmol), 4-dimethylaminopyridine (175 mg, 1.43 mmol) and drop-wise 4-((trimethylsilyl)ethynyl)benzoyl chloride 9 freshly prepared as described above. The reaction was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure and partitioned between EtOAc and water. The organic layer was washed with 1 N HCl, aqueous NaHCO₃, water and brine, dried over Na₂SO₄ and concentrated under reduced pressure to give the title compound (6.11 g, 83%):

1H NMR (400 MHz, DMSO-d₆) δ 8.15 – 8.07 (m, 2H), 7.92 – 7.84 (m, 2H), 7.78 – 7.70 (m, 2H), 7.09 – 7.02 (m, 2H), 4.63 (t, J = 6.7 Hz, 1H), 3.79 (s, 3H), 3.59 – 3.37 (m, 3H), 3.32 (dd, J = 12.5, 6.6 Hz, 1H), 3.25 – 3.13 (m, 2H), 0.08 (s, 9H).

HRMS-ESI: calc for C₂₅H₃₁N₂O₆SSi ([M + H]⁺), 515.7, found 515.3; calc for C₂₅H₃₄N₃O₆SSi ([M + NH₄]⁺), 532.7, found 532.3; calc for C₂₅H₃₀N₂O₆SSiNa ([M + Na]⁺), 537.7, found 537.3; calc for C₅₀H₆₁N₄O₁₂S₂Si₂ ([2M + H]⁺), 1030.3, found 1029.6.

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5.7 Methyl 4-(4-ethynylbenzoyl)-1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxylate (11)

To a solution of methyl 1-((4-methoxyphenyl)sulfonyl)-4-(4-((trimethylsilyl)ethy-nyl)benzoyl)piperazine-2-carboxylate 10 (5.82 g, 11.3 mmol) in 60 mL of THF at 0⁰C was added subsequently acetic acid (403 μL) and acetic anhydride (403 μL).

Dropwise addition of tetra-n-butylammonium fluoride (7.10 g, 22.5 mmol) in 60 mL of THF was followed and the mixture was stirred overnight at room temperature.

The reaction mixture was concentrated under reduced pressure and partitioned between EtOAc and H₂O. The EtOAc layer (2 x 100 mL) was washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo to give the title compound 11 (4.75 g, 95%):

Preparation of NH₂OK/NH₂OH solution: NH₂OH.HCl (6.74 g, 97.0 mmol) was dis-solved in MeOH (35 mL) by heating to reflux overnight. The solution was cooled at 0⁰C, and a solution of KOH (8.16 g, 145.5 mmol) in MeOH (20 mL) was added in one portion. The resulting suspension was used without prior removal of precipitated material. A solution of methyl ester 11 (4.29 g, 9.70 mmol) in NH₂OK/NH₂OH solu-tion (as described above) was stirred at room temperature for 2 days. The reacsolu-tion mixture was taken up in dilute aqueous HCl (pH = 3, 100 mL), extracted with EtOAc (2 x 100 mL), dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography (0-5% MeOH in EtOAc) to afford hydroxamic acid 3 (2.24 g, 52%) as a white solid:

1H NMR (400 MHz, DMSO-d₆) δ 10.44 (s, 1H), 8.87 (s, 1H), 8.08 – 8.00 (m, 2H), 7.81 – 7.68 (m, 4H), 7.10 – 7.02 (m, 2H), 4.45 (t, J = 6.8 Hz, 1H), 3.64 (dd, J = 12.3, 6.7 Hz, 1H), 3.55 – 3.37 (m, 3H), 3.26 – 3.15 (m, 2H), 3.04 (s, 1H).

HRMS-ESI: calc for C₂₁H₂₂N₃O₆S ([M + H]⁺), 444.5, found 444.5; calc for C₂₁H₂₅N₄O₆S ([M + NH₄]⁺), 461.5, found 461.4; calc for C₂₁H₂₁N₃O₆SNa ([M + Na]⁺), 466.5, found 466.5; calc for C₄₂H₄₃N₆O₁₂S₂ ([2M + H]⁺), 888.0, found 887.5; calc for C₄₂H₄₆N₇O₁₂S₂ ([2M + NH₄]⁺), 905.0, found 904.5; calc for C₄₂H₄₂N₆O₁₂S₂Na ([2M + Na]⁺), 909.9, found 910.0.

5.9 4-(4-(1-(4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)butyl)-1H-1,2,3-triazol- 4-yl)benzoyl)-N-hydroxy-1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxamide (1B)

1H-NMR (400 MHz, CD₃OD): 8.41 (s, 1H), 7.88 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.4 Hz), 7.43 (d, 2H, J = 8.4 Hz), 7.08 (d, 2H, J = 8.8 Hz), 4.54 (dd, 1H, J = 4.0, 5.2 Hz), 4.52 (t, 2H, J = 7.2 Hz), 4.42 (m, 1H), 3.89 (s, 3H), 3.74 (dd, 1H, J = 2.8, 4.0 Hz), 3.66-3.56 (m, 11H), 3.53 (t, 2H, J = 6.4 Hz), 2.05 (m, 2H), 1.61 (m, 2H).

HSQC-NMR (100 MHz, CD₃OD): 130.8, 128.5, 126.4, 122.5, 115.4, 84.8, 83.1, 71.6, 71.6,70.9, 70.6, 56.0, 50.9, 48.8, 43.2, 28.0, 27.2.

HRMS-ESI: calc for C₃₁H₄₂FN₆O₉S ([M + H]⁺), 693.8, found 693.6.

5.10 Production of n.c.a. ¹⁸F-fluorine and preparation of dry ¹⁸ F-fluorine-cryptate complex

Aqueous ¹⁸F-fluorine was produced by irradiation of ¹⁸O-water with a Scanditronix MC-17 cyclotron via the ¹⁸O(p,n) ¹⁸F nuclear reaction. The ¹⁸F-fluorine solution was trapped on a SepPak Light Accell plus QMA anion exchange cartridge (precondi-tioned with 5 mL of sodium bicarbonate 1.4% and 100 mL of H₂O and then dried under a flow of argon) to recycle the ¹⁸O-enriched water. The ¹⁸F-fluorine was eluted from the QMA anion exchange cartridge with 1 mg potassium carbonate in 1 mL of water and collected into a vial containing 5 mg kryptofix[2.2.2]. Subsequently, 1 mL of acetonitrile was added and the solvents were removed at 130°C under an argon stream. The [¹⁸F]KF / kryptofix[2.2.2] complex was then dried by azeotropic distillation with 3 times addition 0.5 mL anhydrous acetonitrile at 130°C.

5.11 1-Azido-4-(2-(2-(2-[¹⁸F]fluoroethoxy)ethoxy)ethoxy)butane ([¹⁸F]-18) (two-step reaction (first approach))

A solution of 17 (1.0 mg, 2.49 μmol) in anhydrous DMSO (500 μL) was added to the dry [¹⁸F]KF / kryptofix[2.2.2] complex and was heated for 10 min at 120⁰C.

The mixture was allowed to cool down for 10 min and was then passed through an

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Alumina N Sep-Pak light cartridge. The reaction vial was rinsed with 700 μL H₂O which was passed through the Alumina N cartridge. The cartridge was then dried by airflow. To remove unreacted [¹⁸F]fluorine and unreacted 17, [¹⁸F]-18 was puri-fied by semi-preparative reverse phase HPLC using a Symmetry Prep C18 column (7.8 mm x 300 mm, 7 μm) from Waters, equipped with a 20 x 4.6 mm² precolumn.

The mobile phase used was a mixture of 0.05 M monosodium phosphate buffer NaH₂PO₄/MeOH/THF 550/220/180 at a flow rate of 2.5 mL.min^-1. The column effluent was monitored using an Elite Lachrom VWR Hitachi L-2400 UV detector (λ = 254 nm, AUFS = 0.5) and a Bicron frisk-tech radioactivity detector. Sample injection was carried out using an injector block with a loop of 1 mL. The retention time of [¹⁸F]-18 was 9.4-13.6 min (the retention time of 17 was 14.2 min). The HPLC-collected fraction was diluted with about 100 mL of water for injection and passed through a Sep-Pak Light C18 cartridge. The cartridge was washed with 10 mL of water for injection and eluted with 700 μL of DMSO to yield purified [¹⁸F]-18.

5.12 4-(4-(1-(4-(2-(2-(2-[¹⁸ F]Fluoroethoxy)ethoxy)ethoxy)butyl)-1H-1,2,3- triazol-4-yl)benzoyl)-N-hydroxy-1-((4-methoxyphenyl)sulfonyl)piperazine-2-carboxamide ([¹⁸F]-18) (one pot two-step reaction (second approach))

The copper(I) species was prepared in situ by using copper(II) sulfate / sodium ascorbate 10 min before addition to the crude [¹⁸F]-18. To a solution of copper(II) sulfate pentahydrate (0.4 M, 31.1 μL, 12.4 μmol) was added sodium ascorbate (0.6 M, 31.1 μL, 18.7 μmol) and 62.8 μL H₂O. The mixture was stirred for 10 min.

A solution of 17 (1.0 mg, 2.49 μmol) in anhydrous DMSO (400 μL) was added to the dry [¹⁸F]KF / kryptofix[2.2.2] complex and heated for 10 min at 120⁰C. The mixture was allowed to cool down for 10 min. A solution of 3 (1.66 mg, 3.74 μmol) in anhydrous DMSO (100 μL) and a solution of copper(II) sulfate pentahydrate / sodium ascorbate (as prepared above) were added. The mixture was heated at 60⁰C during 15 min. The mixture was allowed to cool down for 5 min then it was diluted with a solution of 0.1 M Na₂EDTA (15 mL) and passed through a Sep-Pak Plus C18 cartridge (preconditioned with 10 mL of ethanol and 10 mL of water). The cartridge was washed with 10 mL of water for injection, followed by elution of crude [¹⁸F]-1B with 2 mL of absolute ethanol. The solvent was evaporated under reduced pressure and a stream of argon at 60⁰C within 10-15 min. The dried crude product was re-suspended in water. Separation of the radiosynthesized and unlabelled compounds was performed by semi-preparative reverse phase HPLC using a Symmetry Prep

C18 column (7.8 mm x 300 mm, 7 μm) from Waters, equipped with a 20 x 4.6 mm² precolumn. The mobile phase used was a mixture of 0.05 M monosodium phosphate buffer NaH₂PO₄/MeOH/THF 700/70/180 at a flow rate of 3.5 mL.min^-1. The column effluent was monitored using an Elite Lachrom VWR Hitachi L-2400 UV detector (λ = 254 nm, AUFS = 0.5) and a Bicron frisk-tech radioactivity detector. Sample injection was carried out using an injector block with a loop of 1 mL. The retention time of [¹⁸F]-1B was 14.8-16.6 min (the retention time of 3 was 36.4 min). The HPLC-collected fraction was diluted with about 100 mL of water for injection and passed through a Sep-Pak Light C18 cartridge preceded of an Alumina N cartridge.

The cartridge was washed with 10 mL of water for injection and eluted with 0.7 mL of absolute EtOH. The obtained product was redissolved in saline to decrease the percentage of EtOH to less than 10% for the subsequent animal experiments.

Quality control was performed by analytical HPLC, using a X-Terra RP 18 column (4.6 mm x 250 mm, 5 μm) from Waters, equipped with a 20 x 4.6 mm² precolumn.

The mobile phase employed was ACN/H₂O 30/70 at a flow rate of 1.5 mL.min^-1. The column effluent was monitored using an Elite Lachrom VWR Hitachi L-2400 UV detector (λ = 254 nm, AUFS = 0.010) and a Bicron frisk-tech radioactivity detector.

Sample injection was carried out using an injector block with a loop of 100 µL. The retention time of [¹⁸F]-1B was 13.0 min.

The cycloaddition of the alkyne 3 with [¹⁸F]-1B was monitored by silica gel TLC analysis using EtOAc/MeOH 9/1 as mobile phase. The reaction was followed from 0 to 30 min and reaction mixture samples (2.5 μL of reaction mixture diluted in 1 mL EtOAc) were taken every 5 min.

5.13 In vitro evaluation of 1B in a fluorogenic inhibition assay

Recombinant ADAM-17 (ectodomain) was purchased from R&D Systems (Min-neapolis, MN, USA). Recombinant catalytic domain (CD) of human MMP-2 was from Biomol International (Butler Pike, PA, USA). Recombinant human MMP-9 CD without fibronectin type II insert (expressed in E. Coli as described [30,31]) was a kind gift from AstraZeneca R&D (Lund & Moelndal, Sweden).

This competitive enzyme activity assay was performed by monitoring the conver-sion of the fluorogenic substrate Mca-PLAQAV-Dpa-RSSSR-NH₂ (R&D systems) by recombinant ADAM-17 in presence of increasing concentrations of 1B. For MMP-2 and -9, the conversion of the fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (Bachem, Bubendorf, Switzerland) was followed. Measurements

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were performed in Costar White 96-well plates (Corning, Schiphol-Rijk, The Netherlands), where each well contained 10 ng ADAM-17 and a final concentration of 10 µM substrate in a final volume of 100 µL ADAM assay buffer (25 mM Tris pH 9.0, 2.5 µM ZnCl₂, 0.005% w/v Brij-35). Inhibition of MMP proteolytic activity was determined with 10 ng of MMP-2 or MMP-9 per well with a final concentration of 2 µM substrate in 100 µL MMP assay buffer (50 mM Tris pH 7.4, 0.2 M NaCl, 10 mM CaCl₂, 2.5 µM ZnCl₂, 0.05% (v/v) Brij-35). Proteolysis rates were followed by measuring fluorescence (λ_ex,em = 320, 440 nm) increase using a Fluostar Optima plate reader (BMG Labtech, Offenburg, Germany), at 20°C for recombinant MMPs and at 37°C for recombinant ADAM-17, for 15 min (conditions of the experiments not in the stationary phase). For MMP-2 and MMP-9, nine-point inhibition curves (0, 0.5, 1, 2, 6, 12.5, 25, 50 and 100 nM) were plotted in GraphPad Prism. For ADAM-17, nine-point inhibition curves (0, 20, 50, 100, 125, 250, 500, 750 and 1000 nM) were plotted. IC₅₀ values were determined by sigmoidal fitting. Each experiment was performed in triplicate.