From dynamic combinatorial chemistry to in vivo evaluation of reversible and irreversible myeloperoxidase inhibitors

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1 1 Supporting Information From dynamic combinatorial chemistry to in vivo evaluation of reversible and irreversible myeloperoxidase inhibitors Jalal Soubhye,*,a Michel Gelbcke, a Pierre Van Antwerpen, a François Dufrasne, a Mokhtaria Yasmina Boufadi, a,b Jean Nève, a Paul G. Furtmüller, c Christian Obinger, c Karim Zouaoui Boudjeltia d and Franck Meyer*,e a. Chimie Pharmaceutique Organique, Faculty of pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, 1050 Bruxelles, Belgium. b. Laboratory of Beneficial Microorganisms, Functional Food and Health (LMBAFS). Faculty of Natural Sciences and Life. Université de Abdelhamid Ibn Badis, Mostaganem, Algeria. c. Department of Chemistry, BOKU University of Natural Resources and Life Sciences,1190 Vienna, Austria. d. Laboratoire de Médicine Expérimentale, CHU Charleroi, A. Vesale Hospital, Université Libre de Bruxelles (ULB), 6110 Montigny-le-Tilleul, Belgium. e. Laboratory of biopolymers and supramolecular nanomaterials, Faculty of pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, 1050 Bruxelles, Belgium. * Correspondence jal.sub@hotmail.com, franck.meyer@ulb.ac.be Table of content Structure and Activity of Hydralazine and Isoniazid... 2 Names of the compounds of groups A, B and C... 2 Combinatorial Procedure... 3 Preparation of hydrazone compounds... 4 Close view of the best score positions of the best 12 compounds (up: 3D view, bottom: 2D view) Transient-State Kinetics In Vivo test References... 29

2 2 Structure and Activity of Hydralazine and Isoniazid Names of the compounds of groups A, B and C Group A Vanilline 1A; 3,4,5-Trimethoxybenzaldehyde 2A; 4-Aminobenzaldehyde 3A; 3- Aminobenzaldehyde 4A; 2-Methoxybenzaldehyde 5A; 3-Hydroxy-4-methoxybenzaldehyde 6A; 2,5-dihydroxybenzaldehyde 7A; 3,4-Dihydroxybenzaldehyde 8A; Nitrobenzaldehyde 9A; 3-Nitrobenzaldehyde 10A; 4-Hydroxybenzaldehyde 11A; 3-Hydroxybenzaldehyde 12A; 4-Dimethylaminobenzaldehyde 13A; Benzaldehyde 14A; 4-Methoxybenzaldehyde 15A; o- Anisaldehyde 16A; Salicylaldehyde 17A; 2-Chlorobenzaldehyde 18A; Thiophene-2- aldehyde 19A; 2-Nitrobenzaldehyde 20A; 2,6-Dichlorobenzaldehyde 21A; p-anisaldehyde 22A; 3-Fluoro benzaldhyde 23A; Furfural 24A. Group B Paraformaldehyde 1B; Hydrate de chloral 2B; Crotonaldehyde 3B; Formaldehyde 4B; Acetoacetaldehyde dimethylacetal 5B; Isobutyraldehyde 6B; 2-Methyl-n-valeraldehyde 7B; Propionaldehyde 8B; Malondialdehyde 9B; Acetaldehyde 10B; Glutardialdehyde 11B; 1- Chloroacetaldehyde 12B; Glycoladehyde 13B; Glyoxal 14B. Group C Hydralazine 1C; 4-Fluorophenylhydrazine 2C; Isoniazide 3C; 1-Napthylhydrazine 4C; 2,4- Dinitrophenylhydrazine 5C; Methylphenylhydrazine 6C.

3 Combinatorial Procedure 3

4 4 Preparation of hydrazone compounds 1 H- and 13 C NMR spectra were taken on a Bruker Avance 300 M Hz spectrometer (Wissemburg, France) at 293 K. Chemical shifts (δ) are given in parts per million (ppm) relative to DMSO-d 6, and the coupling constants are expressed in hertz. Starting materials vanilline, 3-hydroxy-4-methoxybenzaldehyde, 4-(dimethylamino)benzaldehyde, hydralazine and isoniazid compounds were purchased from TCI (Japan) and Sigma Aldrich (Bornem, Belgium). The purity was 95% for all compounds. All the synthesized hydrazones were in E configuration (E configuration 95% and Z configuration 5%). The hydrazine compound (0.1 mol) was added to a solution of the corresponding aldehyde (0.1 mol) in ethanol (50 ml). The reaction mixture was refluxed for 3 hours. After cooling, the precipitated residues were filtered and dried to give the desired hydrazone with a yield of 90% to 100%. (E)-2-methoxy-4-((2-(phthalazin-1-yl)hydrazono)methyl)phenol hydrochloride (1A-1C) [1] 1 HNMR (DMSO-d 6 ): δ (s, 1H), 9.94 (br, s, 1H), 9.26 (m, 1H), 9.04 (s, 2H), 8.20 (m, 3H), 7.84 (s, 1H), 7.28 (dd, J= 9 Hz, 1H), 6.91 (d, J= 6 Hz, 1H), 3.91 (s, 3H). 13 CNMR (DMSO-d 6 ): δ 154.2, 151.5, 149.1, 148.3, 145.4, 136.7, 134.5, 129.0, 128.7, 126.3, 125.5, 125.1, 119.9, 116.2, 110.9, (E)-2-methoxy-5-((2-(phthalazin-1-yl)hydrazono)methyl)phenol (6A-1C) 1 HNMR (DMSO-d 6 ): δ (s, 1H), 9.94 (br, s, 1H), 9.26 (m, 1H), 9.04 (s, 2H), 8.20 (m, 3H), 7.84 (s, 1H), 7.13 (dd, J= 9, 3 Hz, 1H), 6.48 (d, J= 6 Hz, 1H), 3.37 (s, 3H). 13 CNMR (DMSO-d 6 ): δ 154.2, 151.5, 149.1, 148.3, 145.4, 136.7, 134.5, 129.0, 128.7, 127.3, 125.5, 125.1, 122.9, 113.2, 112.9, 56.8.

5 5 (E)-N,N-dimethyl-4-((2-(phthalazin-1-yl)hydrazono)methyl)aniline (13A-1C) HN N N N N 1 HNMR (DMSO-d 6 ): δ (s, 1H), 9.67 (s, 1H), 8.31 (s, 1H), 8.24, (m, 1H), 8.00 (s, 1H), 7.82 (d, J= 6 Hz, 2H), 7.70 (m, 2H), 6.78 (d, J= 9 Hz, 2H), 2.98 (s, 6H). (E)-2-(2-(phthalazin-1-yl)hydrazono)ethanol (13B-1C) 1 HNMR (DMSO-d 6 ): δ (s, 1H), 9.94 (br, s, 1H), 9.26 (m, 1H), 9.04 (s, 2H), 8.20 (m, 3H), 7.84 (s, 1H), 4.52 (d, J= 9 Hz, 2H). 13 CNMR (DMSO-d 6 ): δ 154.2, 148.3, 145.4, 136.7, 134.5, 129.0, 128.7, 125.1, 110.9, (E)-4-((2-(4-fluorophenyl)hydrazono)methyl)-2-methoxyphenol (1A-2C) 1 HNMR (DMSO-d 6 ): δ (s, 1H), 7.28, (s, 1H), 7.25 (d, J= 3 Hz, 1H), 7.16 (d, J= 6 Hz, 1 H), (m, 5H), 3.81 (s, 3H). (E)-5-((2-(4-fluorophenyl)hydrazono)methyl)-2-methoxyphenol (6A-2C) 1 HNMR (DMSO-d 6 ): δ (s, 1H), 7.28, (s, 1H), 7.30 (d, J= 3 Hz, 1H), (m, 6H), 3.81 (s, 3H). (E)-4-((2-(4-fluorophenyl)hydrazono)methyl)-N,N-dimethylaniline (13A-2C)

6 6 1 HNMR (DMSO-d 6 ): δ (s, 1H), 7.56 (d, J= 6 Hz, 2H), 7.28, (s, 1H), (m, 4H), 6.73 (d, J= 9 Hz, 2H), 3.1 (s, 6H). (E)-N'-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide (1A-3C) [1] 1 HNMR (DMSO-d 6 ): δ (s, 1H), 9.61 (s, 1H), 8.76 (d, J= 3 Hz, 2H), 8.35 (s, 1H), 7.80 (dd, J= 6, 3 Hz, 2H), 7.33 (s, J= 3 Hz, 1H), 7.12 (dd, J= 6, 3 Hz, 1H), 6.85 (d, J= 9 Hz, 1H), 3.84 (s, 3H). 13 CNMR (DMSO-d 6 ): δ 161.3, 150.3, 149.6, 149.3, 148.1, 140.7, 125.4, 122.5, 121.5, 115.5, 109.1, (E)-N'-(3-hydroxy-4-methoxybenzylidene)isonicotinohydrazide (6A-3C) [1] 1 HNMR (DMSO-d 6 ): δ (s, 1H), 9.43 (s, 1H), 8.76 (dd, J= 6, 3 Hz, 2H), 8.31 (s, 1H), 7.80 (dd, J= 6, 3 Hz, 2H), 7.29 (s, J= 3 Hz, 1H), 7.09 (dd, J= 9, 3 Hz, 1H), 6.98 (d, J= 9 Hz, 1H), 3.84 (s, 3H). 13 CNMR (DMSO-d 6 ): δ 161.3, 150.3, 150.1, 149.2, 146.9, 140.7, 126.8, 121.5, 120.6, 112.4, 111.9, (E)-N'-(4-(dimethylamino)benzylidene)isonicotinohydrazide (13A-3C) [2] 1 HNMR (DMSO-d 6 ): δ (s, 1H), 8.76 (dd, J= 6, 3 Hz, 2H), 8.33 (s, 1H), 7.81 (dd, J= 6, 3 Hz, 2H), 7.57 (d, J= 9 Hz, 2H), 6.74 (d, J= 9 Hz, 2H), 2.98 (s, 6H). 13 CNMR (DMSO-d 6 ): δ 161.1, 151.7, 150.3, 149.9, 140.9, 128.7, 121.5, 121.2, 111.8, 39.7.

7 7 Close view of the best score positions of the best 12 compounds (up: 3D view, bottom: 2D view). The X-ray structure of human myeloperoxidase complexed to thiocyanate (PDB code 1DNU) was used as the target structure for the docking studies [3]. The protein dimer was loaded into LeadIT and chains B and D were selected to form the receptor components. The binding site was defined by choosing thiocyanate as a reference. Amino acids within 11 Å of the ligand were selected as binding site (the residues 81 to 120 and 330 to 337). The X-ray water and other ligand molecules were removed from the active site. The ligand input files were prepared according to the following procedure. The initial 3D structures of the ligands were generated by use of the LigandScout procedure. The prediction of different protonation states of all ligands was achieved with the protonation state option in LeadIT software. For the stereochemistry, the options E and Z were chosen in stereo mode to cover all the possible stereo models of the ligands. Docking was performed with the LeadIT program (version 2.1.8), No additive filters were applied. Hydrogen bond donors and acceptors, phenyl centers, and hydrophobic features are defined similarly to FlexX15 as pharmacophore features. The FlexX15 docking solution and scoring function were used. The ligand binding were driven by hybrid approach (enthalpy and entropy). The remaining parameters were set to their default values. The experiment was validated by comparing the docking pose of HX1 in MPO with the co-crystal of HX1-MPO resolved by X-Ray crystallography [4].

8 1A-1C 8

9 6A-1C 9

10 13A-1C 10

11 13B-1C 11

12 1A-2C 12

13 6A-2C 13

14 13A-2C 14

15 1A-3C 15

16 6A-3C 16

17 13A-3C 17

18 18 Transient-State Kinetics Highly purified myeloperoxidase with purity index (A 430 /A 280 ) of at least 0.86 was purchased from Planta Natural Products ( Its concentration was calculated by use of ε 430 = 91 mm 1 cm 1. Hydrogen peroxide obtained from a 30% solution was diluted and the concentration was determined by absorbance measurement at 240 nm, where the extinction coefficient is 39.4 M 1 cm 1. 1A-1C stock solutions were prepared in RO water and stored in dark flasks. Dilution was performed with 100 mm phosphate buffer, ph 7.0 The multimixing stopped-flow measurements were performed with the Applied Photophysics (UK) instrument SX-18MV. When 100 μl was shot into a flow cell having a 1 cm light path, the fastest time for mixing two solutions and recording the first data point was 1.3 ms. Compound I was formed as described in Furtmüller et al, 1998, Biochemistry 37, Typically, 8 μm MPO was premixed with 80 μm H 2 O 2, and after a delay time of 20 ms, Compound I was allowed to react with varying concentrations of C1, 1A-1C, 13A-1C, or 13B-1C in 200 mm phosphate buffer, ph 7.0. Kinetics was followed both at single wavelength (456 nm, maximum absorbance of compound II) and by use of a diode-array detector. At least three determinations ( data points) of pseudo-first-order rate constants (k obs ) were performed for each substrate concentration (ph 7.0, 25 C) and the mean value was used in the calculation of the second-order rate constants, which were calculated from the slope of the plot of k obs versus substrate concentration. To allow calculation of pseudo-first-order rates, the concentrations of substrates were at least 10 times in excess of the enzyme. Reaction of MPO Compound I with 20 and 200 µm 1C

19 19 The upper part of Fig.1 shows the direct transition of MPO Compound I to Compound II after adding 20 µm 1C followed by a slow transition to Compound III (light blue spectra) with Soret peak at 450 nm. Compound III then slowly decays to ferric MPO (or ferric MPO with modified heme (light green spectra). The inset shows the time trace at 456 nm, the maximum wavelength of Compound II. The lower part of Fig.1 shows the spectral transition after adding 200 µm 1C to Compound I. Figure 1. Reaction of MPO Compound I with 20 and 200 µm 1C Compound I reduction followed at 456 nm could be fitted with a single exponential function and from the determined k obs value the apparent bimolecular rate constant was calculated

20 20 to be 7.1 x 10 5 M -1 s -1. The transition of Compound II to Compound III and the transition of Compound III back to the ferric MPO (ferric MPO with modified heme) was independent of the 1C concentration Figure 2. Plot of k obs versus 1C concentration Reaction of MPO Compound I with µm 1A-1C

21 21 Fig. 3 shows the direct transition of MPO Compound I to Compound II after adding 10 µm 1A-1C. The inset of the figure shows the time trace at 456 nm, the maximum wavelength of Compound II, with a single exponential fit. From the determined k obs value the apparent bimolecular rate constant was calculated to be 2.8 x 10 5 M -1 s -1. Figure 3. Reaction of MPO Compound I with 10 µm 1A-1C With 100 µm 1A-1C (Figure 4), a very fast direct transition of Compound I to Compound II (red spectra) could be seen, followed by a slow transition to Compound III (light blue spectra) with Soret peak at 450 nm and one sharp band in the visible range at 625nm.

22 22 Compound III then slowly decayed to ferric MPO (or ferric MPO with modified heme) (light green spectra). Figure 4. Reaction of MPO Compound I with 100 µm 1A-1C Figure 5 show the corresponding time traces at 456 nm (maximum absorbance of Compound II) and 430 nm (maximum of ferric MPO). Red part of the time traces corresponds to Compound II formation, light blue corresponds to Compound III formation and light green to the shift of Compound III back to ferric MPO (or ferric MPO with modified heme). Figure 5. Reaction of MPO Compounds I with 100 µm 1A-1C followed at 456 and 430 nm Compound III formation was not depentent on 1A-1C concentration (Figure 6).

23 23 Figure 6. k obs of the transition of Compound II to Compound III with 100 µm 1A-1C followed at 456 nm Reaction of MPO Compound I with 13B-1C

24 24 The reaction of 13B-1C with MPO Compound I was similar to 1C and 1A-1C. First there was a fast Compound I reduction (Fig. 7, red spectra, 20 µm 13B-1C) followed by a slow Compound II to Compound III transition. Inset of Fig.7 shows the time trace at 456 nm, the maximum wavelength of Compound II. From the determined k obs value (Fig. 8) the apparent bimolecular rate constant was calculated to be 3.3 x 10 5 M -1 s -1. Figure 7. Reaction of MPO Compound I with 20 µm 13B-1C Figure 8. k obs versus 13B-1C followed at 456 nm Figure 9 shows the transition of MPO Compound I after adding 500 µm 13B-1C. A direct transition of MPO Compound I to Compound II occurred followed by a slow transition to Compound III (light blue spectra). Compound III then slowly decays to ferric MPO (or ferric MPO with modified heme) (light green spectra).

25 25 Figure 9. Reaction of MPO Compound I with 500 µm 13B-1C Reaction of MPO Compound I with 13A-1C

26 26 Fig.10 shows the direct transition of MPO Compound I to Compound II after adding 20 µm 13A-1C. The inset shows the time trace at 456 nm, the maximum wavelength of Compound II. Compound I reduction can be fitted with a single-exponential function and the apparent bimolecular rate constant was calculated to be 7.1 x 10 5 M -1 s -1 (Fig. 11). Figure 10. Reaction of MPO Compound I with 20 µm 13A-1C Figure 11. k obs versus 13A-1C followed at 456 nm At higher concentration (Fig.12) after Compound II formation (red spectra), it is directly reduced to ferric MPO (violet spectra, isosbestic point at 442 nm) with a bimolecular rate constant of 83 M -1 s -1.

27 Figure 12. Reaction of MPO Compound I with 500 µm 13A-1C 27

28 28 In Vivo test Animals and accommodation conditions 20 male Wistar albino rats weighting between 110 and 150 g were used in this experiment. The rats were obtained from the Pasteur Institute (Dely Ibrahim, Algeria). The protocol is consistent with the guidelines of the National Institutes of Health (NIH). The rats were randomly placed in metabolic cages for a period of acclimatization (2 weeks) in the animal house of the Faculty of Science (University Abdelhamid Ibn Badis, Mostaganem) with the temperature room and a natural cycle of light and darkness. The rats had free access to food and water. Induction of acute inflammation by carrageenan The Acute carrageenan-induced inflammatory reaction in the peritoneal cavity of rats was performed according to Paulino et al. [5] with slight modifications. After two weeks of acclimatization, the animals were divided into 5 groups (4 rats for each group). The first and second groups were used (water and food ad libitum) as control negative and positive, respectively. With the exception of the first group (negative control ref), the rats received an intraperitoneal (i.p) injection of 100 µl of carrageenan (1 µm). 4h after induction of inflammation, the rats were maintained under light anesthesia with chloroform just before being sacrificed in order to avoid change of biochemical parameters before blood collection. 10 mg/kg of the tested hydrazones were administrated by intravenous injection [6]. The peritoneal liquid was collected after 24h of the drug administration and the cell debris were removed by centrifugation at 5000 t/min for 5 min. Then concentration and the activity of MPO were measured. Inhibition of Myeloperoxydase (MPO) Assay The reaction mixture contains the following reagents in a 96 well plates final volume of 200 µl for quantifying the activity of myeloperoxidase (MPO) using the H 2 O 2 -dependent oxidation of 3,3,5,5 -tetramethylbenzidine (TMB). The final concentrations are between the brackets. After addition of 20 μl peritoneal exudate, 50 μl TMB (1.35 mm), 10 μl H 2 O 2 (100 µm) and 10 µl catalase (8 U/µL) were added. Then, the absorbance of the solutions was measured at 450 nm with a microplate reader. The quantity of MPO was determined according to the kit MPO, Rat, ELISA kit, (Hyclut.Biotec, Germany).

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