Spiro heterocycles bearing piperidine moiety as potential scaffold for antileishmanial activity: synthesis, biological evaluation, and in silico studies

Abstract New spiro-piperidine derivatives were synthesised via the eco-friendly ionic liquids in a one-pot fashion. The in vitro antileishmanial activity against Leishmania major promastigote and amastigote forms highlighted promising antileishmanial activity for most of the derivatives, with superior activity compared to miltefosine. The most active compounds 8a and 9a exhibited sub-micromolar range of activity, with IC50 values of 0.89 µM and 0.50 µM, respectively, compared to 8.08 µM of miltefosine. Furthermore, the antileishmanial activity reversal of these compounds via folic and folinic acids displayed comparable results to the positive control trimethoprim. This emphasises that their antileishmanial activity is through the antifolate mechanism via targeting DHFR and PTR1. The most active compounds showed superior selectivity and safety profile compared to miltefosine against VERO cells. Moreover, the docking experiments of 8a and 9a against Lm-PTR1 rationalised the observed in vitro activities. Molecular dynamics simulations confirmed a stable and high potential binding to Lm-PTR1.


S1: General consideration:
All melting points were determined on a Koffler melting point apparatus and are uncorrected. 1H-NMR and 13 C NMR spectra were recorded on a Bruker avance 400 MHz spectrometer using TMS as internal reference (chemical shifts in , ppm), and IR spectra were obtained on a Nicolet 710 FT-IR spectrometer (KBr, max in cm -1 ).Mass spectra were recorded on a GC-MSQP 1000EX Schimadzu at the Microanalytical laboratory, Cairo University, Cairo, Egypt.Elemental analyses were recorded on Vario El Fab-Nr elemental analyzer (Cairo University).

General procedure for preparation of ionic liquid Piperidinium acetate-IL:
Piperidine (0.9 mL, 0.1 mol) was cooled in an ice bath with continuous stirring then was treated with acetic acid (0.6 g, 0.1 mol) drop-wise through a separating funnel.

General procedure for synthesis of 1-benzyl-2,6-diarylpiperidin-4-one 1a-d:
A mixture of acetone (0.1 mol), aromatic aldehyde (2.0 mmol) in ethanol (4 mL) was treated with benzylamine (0.1 mol) and Piperidinium acetate-IL (30 mg) were added to a round-bottom flask equipped with a magnetic stir bar and condenser.The mixture was heated at 70 C for the time specified in Table 1.The reaction progress was monitored by TLC (EtOAc/hexane = 2:8).After completion of the reaction, the mixture was cooled to room temperature then poured onto crushed ice.The formed solid was filtered, dried, and purified by crystallization using ethanol as a solvent.

Method B:
The acidified kaolin (2% w/w) (150 mg) was added to a solution of 4-Phenylamino piperidine-4-carbonitrile 2a-d (4 mmol) in water (10 mL) and refluxed for 24 h.After completion of the reaction (as indicated by TLC), the reaction mixture was cooled to room temperature and neutralized with sodium hydroxide solution (4 N) to pH=7 carefully.The reaction mixture was filtered and extracted with ethyl acetate (2×20 mL).The organic layer dried over sodium sulfate and evaporated.The obtained amides were crystallized in H2O-EtOH.

Synthesis of spiro heterocycles 5a-10a:
A Piperidinium acetate-IL (30 mg) were added to a round-bottom flask equipped with a magnetic stir bar and condenser.The mixture was heated at 70 C for 5 h and the reaction progress was monitored by TLC (EtOAc/hexane = 4:8).After completion of the reaction, the mixture was cooled to room temperature for 45 minutes and poured on crushed ice.Thus, acquired solid was filtered, dried, and purified by crystallization using ethanol as a solvent.The remaining ionic liquid was further washed with diethyl ether and dried at 80 C under reduced pressure, for its reuse.