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

Figures & data

Figure 1. Some reported spiro-compounds with antileishmanial activities A–C, and the target compounds D.

Scheme 1. Synthesis of 1-benzyl-2,6-diarylpiperidin-4-one 1a–d.

Table 1. Optimisation of reaction conditions of the reaction of acetone, benzaldehyde, and benzylamine.^a

Entry	Catalyst (mol%)	Solvent	Reaction time (h)	Yield (%)
1	0	EtOH	5	12
2	5	EtOH	5	41
3	10	EtOH	3	92
4	15	EtOH	3	92
5	10	MeOH	3	81
6	10	MeCN	3	72
7	10	CH2Cl2	3	55
8	10	Neat	3	40

^aThe reaction was stopped as no further conversion of starting materials (TLC).

Scheme 2. Synthesis of 1-benzyl-2,6-diaryl-4-(phenylamino)piperidine-4-carbonitrile 2a–d.

Scheme 3. Reduction and acid hydrolysis of compounds 2a–d.

Scheme 4. Synthesis of spiro heterocycles 5a–7a.

Scheme 5. Synthesis of spiro heterocycles 8a–10a.

Scheme 6. Synthesis of spiro heterocycles 11a,b.

Scheme 7. Synthesis of spiro heterocycles 12 and 13.

Table 2. Antipromastigote and antiamastigote activity (IC₅₀) of the test compounds and the reference.

Comp. no.	Antipromastigotes	Antiamastigote IC50, µM ± SD
	IC50, µM ± SD
5a	5.3974 ± 0.24	5.4492 ± 0.22
6a	4.4232 ± 0.22	4.6694 ± 0.16
8a	1.1202 ± 0.12	0.8946 ± 0.18
9a	0.4102 ± 0.04	0.50142 ± 0.16
11a	2.4396 ± 0.22	4.86654 ± 0.32
11b	3.2668 ± 0.32	4.88993 ± 0.34
11c	3.9764 ± 0.12	5.2886 ± 0.38
11d	1.4686 ± 0.16	3.6244 ± 0.26
12	2.2088 ± 0.22	3.4266 ± 0.14
13	1.3402 ± 0.14	3.24076 ± 0.22
Miltefosine	7.8974 ± 0.28	8.08 ± 0.24

Note: IC₅₀: value indicates the effective concentration of a compound required to achieve 50% growth inhibition in µM.

Table 3. In vitro evaluation of folate pathway inhibition expressed as percentage survival.^a

Entry	No Cpd added	Folic acid		Folinic acid
1	–	20µM	100 µM	20 µM	100 µM
8a	20%	72%	81%	76%	87%
9a	26%	76%	84%	80%	92%
Trimethoprim (100 µM)	72%	–	99%	–	–

^aPercentage survival ¼ 100 – % AP; where % AP is the percentage growth inhibition.

Table 4. CC₅₀ values of the most active compounds against normal VERO cells and their selectivity index.

Comp. no.	CC50^aµg/ml (µM)	Antileishmanial
		IC50 µM	SI^b
8a	62.5 (128.27)	0.8946	143.38
9a	125 (249.38)	0.5014	497.37
Miltefosine	(99.7)	7.897	12.6

^aCC₅₀ is the concentration at which 50% of the cells survive.

^bSI is the selectivity index; SI = CC₅₀/IC₅₀.

Table 5. Docking scores of the most active compounds against PTR1.

Cpd #	Docking score
8a	−7.8
9a	−8.0
Trimethoprim^a	−6.5

^aTrimethoprim is a binder to LmPTR1.

Figure 2. The docking poses of 8a and 9a poses as cyan sticks in the binding site of the Lm-PTR1 for (A) and (B), respectively, as 3 D and 2 D depictions. The Simon sticks representation is for NADPH co-factor (annotated as “NDP” in the 2 D depiction). Non-polar hydrogen atoms were omitted for clarity.

Figure 3. MD simulations for the three systems, the unliganded leishmanial PTR1 (black line), 8a-PTR1 (red line - dark grey) and 9a-PTR1 (green line - pale grey) complex systems. (A) Root mean square deviation (RMSD) of the protein alpha carbon atoms across the 50 ns simulation. (B) Radius of gyration (Rg) for the PTR1 protein across the 50 ns simulation time. (C) Per residue root mean square fluctuation (RMSF).