New pyrazolylpyrazoline derivatives as dual acting antimalarial-antileishamanial agents: synthesis, biological evaluation and molecular modelling simulations

Figures & data

Figure 1. Some previously reported pyrazoline derivatives (I and II) and pyrazole hybrids with other heterocyclic moieties (III) with dual antimalarial and antileishmanial activity. Compounds (IV) represent our target compounds.

Scheme 1. General synthetic route of the target compounds.

Table 1. Antiplasmodial activities of the synthesised compounds at 20 mg/kg.

Test substance	Dose (mg/kg)	% Parasitaemia*	% Suppression
3a	20	54.3 ± 1.4	−3.8
3b	20	53.1 ± 1.3	−1.9
5b	20	18.8 ± 2.6	66.7
6a	20	30.3 ± 2.8	42.3
7a	20	33.0 ± 1.9	36.5
Chloroquine P.	20	0.0	100
NC**	1 ml/100 g	52.2 ± 3.1	0.0

*Values are M ± SD, P < 0.05, ** Negative control.

Table 2. Antiplasmodial activities of the synthesised compounds at 30 mg/kg.

Test substance	Dose (mg/kg)	% Parasitaemia*	% Suppression
5b	30	17.4 ± 1.2	71.2
6a	30	28.3 ± 2.8	52.4
7a	30	41.1 ± 3.1	30.3
NC**	1 ml/100 g	59.3 ± 3.1	0.0

*Values are M ± SD, P < 0.05, **Negative control.

Table 3. In vitro anti-plasmodial activity against chloroquine-resistant (RKL9) strain of P. falciparum.

Comp. No.	IC50, µM ± SD*
5b	0.0368 ± 0.006
6a	0.0946 ± 0.002
Chloroquine	0.1920 ± 0.003
Pyrimethamine	0.01246 ± 0.002

*Results of two separate determinations.

Table 4. Antileishmanial activity is expressed as antipromastigote and antiamastigote activities of the test compounds and reference standards.

	Antileishmanial activity (IC50*)
Comp. No.	Antipromastigote		Antiamastigote
	μg/ml	μM	μg/ml	μM
3a	3.06 ± 0.12	9.13 ± 0.30	1.10 ± 0.18	2.76 ± 0.45
3b	4.11 ± 0.22	10.86 ± 0.58	2.82 ± 0.36	7.45 ± 0.95
4a	3.84 ± 0.14	9.02 ± 0.33	2.64 ± 0.12	6.18 ± 0.28
5a	3.64 ± 0.18	8.28 ± 0.41	1.89 ± 0.04	4.29 ± 0.09
5b	0.03 ± 0.24	0.05 ± 0.57	0.42 ± 0.32	1.00 ± 0.76
6a	0.42 ± 0.28	0.89 ± 0.60	0.68 ± 0.14	1.45 ± 0.30
7a	0.04 ± 0.24	0.08 ± 0.48	0.44 ± 0.22	0.87 ± 0.44
Miltefosine	3.19 ± 14	7.83 ± 0.34	0.30 ± 0.04	0.74 ± 0.10
Amphotericin B deoxycholate	0.05 ± 0.002	0.04 ± 0.001	0.20 ± 0.02	0.15 ± 0.01

*IC50: values indicate the effective concentration of a compound required to achieve 50% growth inhibition in μg/ml.

Table 5. Data from the acute toxicity studies.

Test substances	Dose (mg/Kg)	Wt. before test* (Day 0)	Wt. after test* (Day 1)
5b	125	33.8 ± 1.2	33.3 ± 2.6
	250	32.2 ± 2.5	32.4 ± 2.0
	500	32.8 ± 2.8	32.5 ± 1.4
6a	125	31.3 ± 2.5	31.5 ± 1.8
	250	32.1 ± 1.7	31.8 ± 1.7
	500	32.2 ± 2.1	32.6 ± 1.3
NC	1 ml/100g	32.1 ± 1.9	32.6 ± 2.1

*Values are M ± SD.

Table 6. AutoDock Vina docking scores (kcal mol⁻¹) of the most active compounds against Pf DHFR-TS (PDB: 1j3k) and PTR1 (PDB: 2bfm).

		Docking score
	Pf-DHFR-TS^a	Pf-DHFR-TS^b	Leishmanial PTR1
5b	−11.2	−9.8	−9.3
7a	−10.9	−10.4	−10.4
6a	−8.5	−7.6	−8.5
Pyrimethamine^c	−7.7	−8.5	–
Dihydropterine^d	–	–	−8.4
Trimethoprim^d	–	–	−7.4

^aQuadruple mutant (N51I, C59R, S108N and I164L) Pf DHFR-TS structure. ^bWild-type Pf DHFR-TS structure. ^cPyrimethamine is a binder to Pf DHFR-TS. ^dDihydropterine and Trimethoprim are binders to PTR1.

Figure 2. The docking pose of the most active compound (5b) as cyan sticks in the binding site of the quadruple mutant Pf-DHFR-TS (PDB code: 1j3k). The hydrophilic and hydrophobic regions are in red and green coloured molecular surfaces, respectively. Non-polar hydrogen atoms were omitted for clarity. The label “NDP-610” represents the NADPH co-factor.

Figure 3. The docking pose of the most active compound (7a) as cyan sticks in the binding site of PTR1 (PDB code: 7pxx). The hydrophilic and hydrophobic regions are in red and green coloured molecular surfaces, respectively. Non-polar hydrogen atoms were omitted for clarity. The label “NDP-302” represents the NADPH co-factor.

Figure 4. MD simulations for the three systems, the apo leishmanial PTR1, 7a-PTR1 complex and co-crystal ligand – PTR1 complex systems. (A) Root mean square deviation (RMSD) of the protein alpha carbon atoms across the 50 ns simulation. (B) The radius of gyration (Rg) for the PTR1 protein across the 50 ns simulation time. The frame number (x-axis) 5000 indicates 50 ns simulation time. (C) Per residue, root means square fluctuation (RMSF). (D) Hydrogen bond counts during the MD simulation for 7a in the binding site during the 50 ns simulation. (E) RMSD of the ligand heavy atoms during the 50 ns simulation of the ligand-complexed systems.

Figure 5. The free energy landscape (FEL) of the simulated PTR1 systems is based on the principal component analysis. (A) Leishmanial PTR1. (B) 7a-PTR1 complex. (C) Co-crystal ligand – PTR1 complex. The colour bar represents the free energy value in kcal mol − 1. The colour ranges from red to yellow to blue spots indicate the energy minima and energetically favoured protein conformations to more unfavourable high-energy conformations.