Supporting Information

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1 Supporting Information Monoamine oxidase isoform-dependent tautomeric influence in the recognition of 3,5 diaryl pyrazole inhibitors. Franco Chimenti, a Rossella Fioravanti,* a Adriana Bolasco, a Fedele Manna, a Paola Chimenti, a Daniela Secci, a Olivia Befani, b Paola Turini, b Francesco Ortuso c and Stefano Alcaro c Contents Title page S1 Chemistry Biochemistry S2 Molecular modelling S3 S1

2 Chemistry. Melting points were determined on a Büchi 510 apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1605 FT spectrophotometer on potassium bromide disk. 1 H NMR spectra were performed on a Bruker AM-400 (400 MHz) FT spectrometer in DMSO, CDCl 3 or aceton as solvents, and the chemical shifts were reported in ppm referring to the solvent peak. Electron ionization (EI) mass spectra were obtained by a Fisons QMD 1000 mass spectrometer (70 ev, 200 ía, ion source temperature of 200 C). The samples were introduced directly into the ion source. Elemental analyses for C, H, and N were performed on a Carlo Erba model 1106 elemental analyzer, and the analytical results were within (±0.4% of the theoretical values). General Procedure for the synthesis of 3,5-diaryl pyrazoles 3a-k. To a solution of appropriate chalcones 3a-k (0.05 mol) in dry ethanol (50 ml), was added powered K 2 CO 3 (0.15 mol), followed by excess of aqueous hydrogen peroxide (35%, 10 mol), added over 10 min. The mixture was stirred at room temperature for 3 h and reaction progress was monitored by TLC (30:70 v/v EtOAc/hexanes). Upon completation, the MeOH was removed under reduced pressure and resulting residue dissolved in CH 2 Cl 2 (30 ml) and washed with H 2 O (20 ml). The organic phase was separated, dried on Na 2 SO 4, and then the solvent was removed under reduced pressure to yield the corresponding epoxide. The solid was crystallized from suitable solvent. The suitable epoxide (0.1 mol) was dissolved in xylenes (4 ml). CH 2 Cl 2 (2 ml) was added, if required, to achieve solution. p- Toluensulfonic acid monohydrate and hydrazine hydrate (0.3 mol) were then added to the solution. The reaction mixture was stirred under refluxed for 3 h. After the xylenes were removed, under reduced pressure, the obtained solid was washed with hexanes and crystallized from acetonitrile. The chemical and physical data are reported in Table 1. NMR data of derivatives 3a-3k Compounds 3a 3b 3c 3d 3e 3f 3g 3h 3j 3k 1 H-NMR (δ)ppm 3.25 (s,1h,ch); (m,10h,ar); (s,1h, NH) 3.50 (s,1h,ch); (m,9h,ar); (s,1h, NH) 3.60 (s,1h,ch); (m,9h,ar); (s,1h, NH) 3.80 (s,1h,ch); (m,8h,ar); (s,1h, NH) 3.80 (s,1h,ch); (m,8h,ar); (s,1h, NH) 3.30 (s,1h,ch); (m,8h,ar); (s,1h, NH) 2.35 (s,3h,ch 3 );3.35 (s,1h,ch); (m,9h,ar); (s,1h, NH) 2.35 (s,3h,ch 3 );3.35 (s,1h,ch); (m,8h,ar); (s,1h, NH) 2.05 (s,3h,ch 3 );3.60 (s,1h,ch); (m,8h,ar); (s,1h, NH) 2.15 (s,3h,ch 3 ); 3.20 (s,3h,ch 3 ); 3.35 (s,1h,ch); (m,8h,ar); (s,1h, NH) Biochemistry. All chemicals were commercial reagents of analytical grade and were used without purification. Bovine brain mitochondria were used as the enzyme source and were isolated according to the Basford method. 1 Activities of MAO-A and MAO-B were determined by a fluorimetric method with kynuramine as substrate. 2 Briefly, the incubation mixtures contained: 0.1 ml of 0.25 M potassium phosphate buffer (ph 7.4), mitochondria (6 mg/ml), and drug solutions with final concentration ranging from 0 to 10-3 M. The solutions were incubated at 38 C for 30 min. Addition of perchloric acid ended the reaction. The samples were centrifuged at g for 5 min, and the supernatant was added to 2.7 ml of 0.1 N NaOH. The pyrazole derivatives were dissolved in dimethyl sulfoxide (DMSO) and added to the reaction mixture from 0 to 10-3 mm. To study the inhibition of pyrazole derivatives on the activities of both MAO-A and MAO-B separately, the mitochondrial fractions were preincubated at 38 C for 30 min with the appropriate specific irreversible inhibitor (L-deprenyl 0.5 µm to eliminate MAO-B from the assay of MAO-A activity, and clorgyline 0.05 µm to eliminate MAO-A from the assay for the isoform B). The data are the means of three experiments performed in duplicate. References 1. Stahl, W. L.; Smith, J. C.; Napolitano, L. M.; Basford, R. E. Brain mitochondria. I. Brain Mithocondria: Isolation of Bovine Brain Mitochondria. J. Cell Biol. 1963, 19, Matsumoto, T.; Suzuki, O.; Furuta, T.; Asai, M.; Kurokawa, Y.; Nimura, Y.; Katsumata, Y.; Takahashi, I. A sensitive fluorometric assay for serum monoamine oxidase with kynuramine as substrate. Clin. Biochem. 1985, 18, S2

3 Molecular modelling Pre-treatment The models of MAO-A (2BXR) and MAO-B (1GOS) were downloaded from the Protein Data Bank and submitted to the following pretreatment. Docking 1. The ligands covalently bound to the FAD moiety were removed. 2. The FAD natural bond order was restored. 3. Water molecules were removed, taking into account the solvation effect by the GB/SA method. 4. All residue sidechains within a radius of 15 Å from the N5 isoalloxazine ring were energy minimized by BatchMin ver 7.2 with AMBER* united atoms notation force field and GB/SA water. In order to prevent a too strong modification with respect to crystallographic model conformation, a constant force equal to 48 kcal/mol Å has been applied to backbone atoms and residues located outer than 15 Å from the FAD. This procedure relaxed the active site of the enzyme isoforms, restoring the natural planarity of the FAD cofactor ring. 5. The resulting MAO structures were used as receptor models. In agreement to the GLIDE docking software, for both pretreated receptor models, a box, centred onto the FAD N5 atom, of about 110,000 Å 3 has been adopted for calculating the energetic map at single precision level (GRID step of GLIDE). Resulting two maps has been used for the docking of both a and b tautomers of compounds 3g and 3h with respect to MAO-A and MAO-B. Binding modes of compounds 3g tautomer a, 3g tautomer b, 3h tautomer a and 3h tautomer b have been simulated respectively within both MAO-A and MAO-B receptor models. The best 10 configurations of each docking calculation have been submitted to energy minimisation using the same software, force field and environment previously reported for the enzyme pre-treatment. No constrains have been considered in this optimisation. Molecular Dynamics refinement In order to perform a deeper exploration of the MAOs molecular recognition, all global minimum energy complex configurations, generated by the previously reported optimisation, have been submitted to molecular dynamics simulation. These calculations have been carried out with AMBER* united atom force field as implemented in Macromodel ver. 7.2 for a total time equal to 500 ps and a time step equal to 1.5 fs. Solvent effects have been considered by means of the GB/SA method. No constrains have been adopted and one structure every 10 ps has been collected, then 50 structures have been sampled for each simulation. After molecular dynamics simulation the tautomer configuration ensembles have been unified and submitted to the same energy minimisation procedure reported into the docking section. Such an approach allowed us to obtain a single ensemble for each compound with respect to each enzyme. The optimised complexes have been submitted to Boltzmann population analysis followed by MM-GBSA dg bind calculation. Table S1: global complex energies of 3g and 3h tautomers into the enzymatic clefts. Comp Tau 3g 3h Global Energy MAO-A Relative energy Number of resulting energy minimized configurations Global Energy MAO-B Relative energy Number of resulting energy minimized configurations a b a b S3

4 Figure S1: most probable and stable complex of 3g (tautomer a) and MAO-A. Interacting residues of the Figure S3: most probable and stable complex of 3h (tautomer a) and MAO-A. Interacting residues of the Figure S2: most probable and stable complex of 3g (tautomer b) and MAO-A. Interacting residues of the Figure S4: most probable and stable complex of 3h (tautomer b) and MAO-A. Interacting residues of the S4

5 Figure S5: most probable and stable complex of 3g (tautomer a) and MAO-B. Interacting residues of the active site are shown in labelled polytubes, FAD in CPK rendering, the compound in blue carbon polytube, and other aminoacids in ribbon. Hydrogen bonds are displayed Figure S7: most probable and stable complex of 3h (tautomer b) and MAO-B. Interacting residues of the active site are shown in labelled polytubes, FAD in CPK rendering, the compound in blue carbon polytube, and other aminoacids in ribbon. Hydrogen bonds are displayed Figure S6: most probable and stable complex of 3h (tautomer a) and MAO-B. Interacting residues of the active site are shown in labelled polytubes, FAD in CPK rendering, the compound in blue carbon polytube, and other aminoacids in ribbon. Hydrogen bonds are displayed S5

6 Elemental Analyses Comp C H N Cl F 3a 81.05/ / / b 70.18/ / / / c 74.98/ / / /7.92 3d 61.87/ / / / e 65.58/ / / / /6.93 3f 69.76/ / / / g 81.32/ / / h 70.98/ / / / j 75.57/ / / /7.45 3k 81.56/ / /