Multitarget drugs as potential therapeutic agents for alzheimer’s disease. A new family of 5-substituted indazole derivatives as cholinergic and BACE1 inhibitors

Abstract Multitarget drugs are a promising therapeutic approach against Alzheimer’s disease. In this work, a new family of 5-substituted indazole derivatives with a multitarget profile including cholinesterase and BACE1 inhibition is described. Thus, the synthesis and evaluation of a new class of 5-substituted indazoles has been performed. Pharmacological evaluation includes in vitro inhibitory assays on AChE/BuChE and BACE1 enzymes. Also, the corresponding competition studies on BuChE were carried out. Additionally, antioxidant properties have been calculated from ORAC assays. Furthermore, studies of anti-inflammatory properties on Raw 264.7 cells and neuroprotective effects in human neuroblastoma SH-SY5Y cells have been performed. The results of pharmacological tests have shown that some of these 5-substituted indazole derivatives 1–4 and 6 behave as AChE/BuChE and BACE1 inhibitors, simultaneously. In addition, some indazole derivatives showed anti-inflammatory (3, 6) and neuroprotective (1–4 and 6) effects against Aβ-induced cell death in human neuroblastoma SH-SY5Y cells with antioxidant properties.


General procedure for the synthesis of piperidinopropylaminoindazole derivatives 1-6.
To a solution of the appropriate 1,3-disubstitiuted 5-aminoindazole derivative in butanone or acetonitrile, K2CO3, catalytic amount of KI and 3-piperidinopropyl chloride hydrochloride were added. The mixture reaction was heated at 140ºC and stirred in microwave reactor. Then, the reaction mixture was cooled and filtered to remove existing inorganic salts. The solvent was evaporated under reduced pressure and purified.

General procedure for the synthesis of derivatives 7-12.
To a solution of the appropriate 5aminoindazole derivative in pyridine, was added, the corresponding acyl chloride at room temperature and the mixture was stirred at room temperature. Amounts of reagents, time reaction, conditions, specific procedures and purification methods are specified in each case.
Reaction time: 20 h. The crude was poured over 50 ml of H2O and was extracted with CH2Cl2 (3x15 mL).

General procedure for the synthesis of 5-nitroindazole ethers 1-substituted 21-26.
To a suspension of the appropriate 1-substituted derivative of 5-nitro-3-indazolol in 2-butanone, K2CO3, and KI, the corresponding bromide or chloride was added and stirred at room temperature until complete elimination of N1-H indazole ether. Then, the reaction mixture was cooled and filtered to remove existing inorganic salts. The solvent was evaporated under reduced pressure and purified. The resulting crude product was processed in each case. Amounts of reagents, time reaction, conditions, specific procedures and purification methods are specified in each case. (21). From 18 (1.00 g, 2.96 mmol), benzyl bromide (0.42 mL 3.55 mmol) and K2CO3 (0.90 g, 6.50 mmol) in butanone (60 mL). Reaction time: 20 h.

3-(benzyloxy)-1-(2,3-dichlorobenzyl)-5-nitroindazole
The dried crude was solved in 10 mL of CH2Cl2. After addition of 50 mL of hexane, the resulting suspension was filtered to obtain a yellow solid. The final product was purified by recrystallisation from isopropanol.

Docking studies
The docking studies as well as the preparation of protein and ligands were performed with Schrödinger Sofware. The BACE1 structure (PDB ID 5tol, (Wu et al. 2016, Bioorg Med Chem Lett, 26, 5729-5731) was prepared using Protein Preparation Wizard into the graphical user interface Maestro. This includes a preprocess steps such as hydrogen atoms addition, modelling of missing side chains using and removal of water molecules. Also refine for hydrogen bonds using PROPKA at pH = 7.0 (Olsson et al. 2011, J Chem Theory Comput, 7, 525-537) and restrain minimization using OPLS3e force field were performed.
Ligand preparation was carried out by LigPrep panel into Maestro. The force field selected was OPLS3e. For generation of possible tautomers and ionization states was used Epik at pH = 7.0.
The docking simulation was performed using Glide.
In order to explain the activity of this family of derivatives and specifically the influence of the piperidinopropyl chain, the docking study of the compound pair 31 and 4 was carried out with the GLIDE program (Fig. S26). The docking simulation revealed the interactions gathered in Table S3. Analyzing Fig. S26 and Table S3, we can see the influence of the piperidinopropyl chain. As can be seen, in the derivative 31, the amino group, form a hydrogen bond with Asn85, but does not establish interactions with the catalytics Asp80 and Asp276 while the piperidinopropyl chain in the compound 4 favours the relocation of the compound to allow to form two ionic bridges between the amino group of piperidinopropyl chain and the catalytic aspartic acids Asp80 and Asp276. In addition, to the interaction with the catalytic aspartic acids, the compound 4 establishes interaction with different subsites (SS) defined according to Hu et al. (ACS Chem Neurosci, 2019, 10, 880-889). The group of Hu performed a new dissection of the binding pocket of BACE1 from a systematic analysis of the results of 354 ligand-protein complexes. Thus, eight subsites, including the catalytic site were identified. Well, our compound 4 establishes interactions with three subsites (SSI, SS3 and SS4) by aromatic H-bond and halogen bond interactions thus explaining its biological activity.