Repurposing strategies on pyridazinone-based series by pharmacophore- and structure-driven screening

Abstract We report here in silico repurposing studies on 52 new pyridazinone-based small-molecules through inverse virtual screening (iVS) methodologies. These analogues were originally designed as formyl peptide receptor (FPR) ligands. As it is sometimes the case in drug discovery programmes, subsequent biological screening demonstrated the inefficacy of the molecules in binding FPRs, failing in the identification of new hits. Through a focussed drug-repurposing approach we have defined a variety of potential targets that are suitable to interact with this library of pyridazinone-based analogues. A two-step approach has been conducted for computational analysis. Specifically, the molecules were initially processed through a pharmacophore-based screening. Secondly, the resulting features of binding were investigated by docking studies and following molecular dynamic simulations, in order to univocally confirm “pyridazinone-based ligand-target protein” interactions. Our findings propose aspartate aminotransferase as the most favourable repurposed target for this small-molecule series, worth of additional medicinal chemistry investigations in the field.

The compounds 1a,b were then obtained in two steps. The first reaction was a nucleophilic substitution leading to the selective displacement of the chlorine at C-5 of the pyridazinone ring of 23a,b, using sodium methoxide in anhydrous methanol. The second step was the coupling of 24a,b with 4-butoxyphenylboronic acid using tetrakis(triphenylphosphine)-palladium(0) catalyst under standard Suzuki conditions, to give the final 4-arylated-5-methoxy-pyridazinones 1a,b in good yields.
To obtain the final compounds 10a-g, 11 and 12 (see Scheme S7), the pyridazinones 32,39 26 were reacted with the appropriate (hetero)arylaldehyde through Knovenagel condensation (intermediates 33 and 40a-g). The subsequent alkylation with bromoethane and potassium carbonate in anhydrous acetonitrile led to the final compounds 10a-g. The dehydration with POCl3 at reflux of products 10a and 10c afford the final compounds 11 and 12. In the Scheme S8 is reported the synthetic pathway to obtain the compounds 13a-c. Starting from 4,6-diphenyl-isoxazol[3,4-d]pyridazin-7(6H)-one (41 27 ), the reaction with the appropriate amine (for products 13a,b) or cyclohexanol (for 13c) in 1,4-dioxane, carried out in closed tube at 90°C, induced an opening of the isoxazole nucleus followed by the formation of the amide, or ester, at position 5 of the pyridazinone scaffold (13a-c).
For the synthesis of compound 14a-c and 15a-l (Scheme S9), the starting materials were the appropriate isoxazolo[4,3-d]pyridazinones 43a-g [27][28][29] . The isoxazolopyridazinone 43e is obtained for alkylation reaction of 42 30 with bromopropane and K2CO3 in acetone at reflux. The formation of the styryl derivatives 44a-m (44a 31 ) was performed by using the appropriate (hetero)arylaldehyde and MeONa in methanol. The opening of the isoxazol ring (for compounds 44a, 44b and 44m) with molybdenumhexacarbonyl in CH3CN at reflux gave the acryloyl derivatives 14a-c and afterwards the products 14a,b were reduced with ammonium formate and Pd/C in ethanol to obtain the final compounds 15a,b, respectively. Indeed, the same reduction (ammonium formate and Pd/C) starting from the other styryl derivatives 44 furnished directly the final compounds 15c-l through a reduced opening.
The Scheme S10 shows the synthetic procedure for pyridazinone derivatives 16a,b and 17a-c, starting from the precursors 43f 27 and 46b, the latter obtained by a cyclization reaction of compound 45 32 with polyphosphoric acid and ethanol under heat. The reduction and opening of isoxazolo [3,4d]pyridazinone nucleus with ammonium formate and Pd/C afforded the compounds 47a,b (47a 27 ).

General remarks
Reagents and starting materials were obtained from commercial sources. Extracts were dried over Na2SO4, and the solvents were removed under reduced pressure. All reactions were monitored by thin layer chromatography (TLC) using commercial plates pre-coated with Merck silica gel 60 F-254. Micro TOF) and reported mass values are within the error limits of ± 5 ppm mass units. All melting points were determined on a microscope hot stage Büchi apparatus and are uncorrected.

Chemistry
General Procedure for 23a,b. K2CO3 (6.06 mmol) and tetrabutylammonium bromide (0.30 mmol) were added to a stirred solution of 4,5-dichloro-3(2H)-pyridazinone 22 (3.03 mmol) in anhydrous acetonitrile (3 mL). 3-or 4-methoxybenzyl chloride (4.54 mmol) was added to the mixture and the reaction was carried out at reflux for 5 h. The mixture was then allowed to cool down and the solvent was evaporated in vacuo. Ice-cold water was added to the residue. After 1 h stirring in ice-bath, compounds 11a,b were filtered off and recrystallised from ethanol.  General Procedure for 24a,b. Compounds 23a or 23b (0.88 mmol) was added to a stirred solution of Na 0 (1.76 mmol) in 3 mL of anhydrous methanol. The reaction mixture was stirred for 1 h at room temperature. After removal of the solvent in vacuo, ice-cold water was added to the residue and the precipitate was filtered off by suction and purified by crystallisation from ethanol.  Extra 4-butoxyphenylboronic acid (1.07 mmol) was added and the reaction was refluxed for additional 6 h. The solvent was evaporated under vacuum and the suspension was diluted with icecold water. After extraction with CH2Cl2, the organic layer was dried over Na2SO4 and the residue was purified by flash column chromatography using cyclohexane/ethyl acetate 3:1 as eluent.     mL) and extracted with CH2Cl2 (3 x 15 mL). The solvent was evaporated to afford final compounds 3a,b, which were purified by column chromatography using cyclohexane/ethyl acetate 1:1 (for compound 3a) and cyclohexane/ethyl acetate 1:2 (for compound 3b) as eluents.  General procedure for 5a,b. A mixture of 4a 24 or 4b 24 (0.20 mmol), acetone (2 mL) and 6 M HCl (4 mL) was warmed in a sealed tube at 100°C for 5 h. The solvent was removed in vacuo and the residue was treated with cold water. The precipitate was purified by recrystallisation from ethanol to give pure 5a and 5b as colourless crystals or yellowish crystals, respectively.  General procedure for 6a,b. A mixture of 4a 24 or 4b 24 (0.09 mmol) acetone (1 mL) and 47% HBr (1 mL) was warmed in a sealed tube at 90°C for 2-3 h. After concentration in vacuo, ice-cold water was added and the was collected by suction. Recrystallisation from ethanol gave 6a as colourless solid.

6-Methyl-4-(3-(pyrimidin-5-yl)benzyl)pyridazin-3(2H)-one (40g
(1.17 mmol), ethyl bromide (0.88 mmol) and anhydrous CH3CN (8 mL) was stirred at reflux for 4-6 hours about. After cooling, the solvent was evaporated and ice-cold water was added. The formed precipitate was recovered by vacuum filtration and the final compounds 10a-g were purified by crystallization from ethanol. CONH-H + 1H pyridaz.).      in 5 mL of POCl3 was stirred at 60-70 °C for 2h about. After cooling, ice-cold water (20 mL) was slowly added, and the precipitate was filtered under vacuum and washed with abundant cold-water to obtain the desired compounds 11 and 12, which were recrystallized from ethanol.  General procedure for compounds 13a-c. 0.35 mmol of 4,6-diphenyl-isoxazol[3,4-d]pyridazin-7(6H)-one 41 27 was dissolved in 0.5 mL of 1,4-dioxane in a sealed tube. 1.22 mmol of suitable amine or alcohol was added and the reaction was stirred at 90 °C for 2-3 h (for compound 13c some drops of Et3N were added). After cooling, the solvent was evaporated and ice-cold water was added. The formed precipitate was filtered off to obtaine the desired compounds 13a-c which were recrystallized from ethanol. Hz).

4-Cyclohexyl-3-methyl-6-propylisoxazolo[3,4-d]pyridazin-7(6H)-one (43e).
A mixture of compound 42 31 (0.45 mmol), K2CO3 (0.90 mmol), bromopropane (2.25 mmol, added twice) and anhydrous acetone (2 mL) was warmed at 90 °C for 5 h. After cooling, the solvent was evaporated and ice cold-water was added. The suspension was extracted with ethyl acetate (3 x 10 mL), dried on sodium sulfate and evaporated to obtain the desired compound which was purified by column chromatography to remove the excess of bromopropane using cyclohexane/ethyl acetate 3:1 as eluent. General procedure for compounds 44b-m. To a solution of intermediates 43b-g 27-29 (0.60 mmol) in 1.25 mL of anhydrous MeOH, 1.5 mmol of appropriate (hetero)arylaldehyde and a solution of MeONa (0.65 mmol of Na 0 in 1 mL of MeOH) were added. The mixture was stirred at reflux for 2-20 min about. After cooling, the precipitate was filtered off to obtain the styril derivatives 44b-m.
The mixture reaction was warmed at 80 °C for 3 h. After cooling, the solvent was removed in vacuo, the residue was recovered with ethyl acetate and the organic phase was washed with a mixture of H2O/NH4OH 1:1 (3 x 10 mL). After evaporation of the solvent, the final compounds 14a-c were purified by column chromatography using cyclohexane/ethyl acetate 3:1 as eluent. Ar, J = 7.6 Hz).  General procedure for compounds 15a-l. A suspension of intermediates 14a,b or 44c-l (0.15 mmol), 10% Pd/C (0.05 mmol) and ammonium formate (0.4 mmol) in absolute ethanol (1.5 mL) was refluxed for 2 h. After cooling, the ethanol was evaporated and methylene chloride (15 mL) was added. The precipitate was removed by filtration and the solvent was recovered and evaporated to afford the desired compounds which were purified by crystallization from ethanol.        -6-phenyl-2-propyl-5-(3-(thiophen-2-yl)

Experimental Details -Molecular modelling
Uncharacterized oxidoreductase ytbE 2 3BHY Death-associated protein kinase 3 2 3DRA Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha 2 3FFV Protein syd a number of molecules that have the indicated target among the best 10 proteins targets identified by the pharmacophore mapping analysis.