Novel heterocyclic hybrids of pyrazole targeting dihydrofolate reductase: design, biological evaluation and insilico studies

Figures & data

Figure 1. Some reported pyrazole hybrid molecules as antimicrobial agents.

Figure 2. Potent DHFR inhibitors owning pyrazole scaffold as antimicrobial and antimalarial agents.

Figure 3. Reported antimicrobial leads containing pyridazine, 1,2,4-triazine and 1,2,3-triazole moieties.

Scheme 1. Synthesis of pyrazolopyridazine derivatives.

Scheme 2. Synthesis of pyrazolotriazine and pyrazolotriazole derivatives.

Figure 4. Antimicrobial activity of the most active compounds against different bacterial and fungal strains compared with the reference drugs, ciprofloxacin and amphotericin B, respectively.

Table 1. Minimal inhibitory concentrations (MICs) of the synthesised compounds against the tested pathogenic bacteria and fungi.^a

Compound No.	MIC (Mean ± SEM) (μg/mL)
	Gram-positive Bacteria		Gram-negative Bacteria		Fungi
	S. pneumonia RCMB 010010	B. subtilis RCMB 010067	P. aeruginosa RCMB 010043	E. coli RCMB 010052	A. fumigatus RCMB 002568	C. albicans RCMB 005036
3a	1.95 ± 0.3	0.98 ± 0.1	7.81 ± 0.3	0.98 ± 0.6	1.95 ± 0.1	3.9 ± 0.3
3b	7.81 ± 0.1	15.63 ± 0.5	125 ± 0.3	250 ± 0.5	125 ± 0.2	62.5 ± 0.1
6a	0.98 ± 0.1	0.49 ± 0.2	3.9 ± 0.1	0.49 ± 0.4	0.98 ± 0.2	3.9 ± 0.03
6b	1.95 ± 0.4	0.98 ± 0.1	62.5 ± 0.3	0.98 ± 0.1	1.95 ± 0.3	7.81 ± 0.05
8a	15.63 ± 0.5	125 ± 0.4	125 ± 0.3	62.5 ± 0.3	3.9 ± 0.1	62.5 ± 0.5
9a	3.9 ± 0.3	1.95 ± 0.08	15.63 ± 0.5	1.95 ± 0.3	250 ± 0.2	7.81 ± 0.2
Ciprofloxacin	0.98 ± 0.2	0.1 ± 0.3	0.5 ± 0.05	0.01 ± 0.1	–	–
AmphotricinB	–	–	–	–	1.95 ± 0.3	3.9 ± 0.1

–: Not tested; SEM: standard error mean; each value is the mean of three values.

^aAntibacterial and antifungal activities were expressed as MIC in µg/mL.

Table 2. In vitro inhibitory activities of the screened compounds 3a, 3b, 6a, 6b, 8a and 9a against DHFR enzyme.

Compound No.		IC50 (Mean ± SEM) (µM)
		DHFR
3a		0.11 ± 1.05
3b		18.30 ± 0.81
6a		0.09 ± 0.91
6b		5.24 ± 0.37
8a		1.36 ± 0.12
9a		1.45 ± 0.23
Methotrexate		0.14 ± 1.25

IC₅₀: Compound concentration required to inhibit DHFR enzyme activity by 50%; SEM: standard error mean; each value is the mean of three values.

Table 3. Calculated molecular properties of the synthesised compounds 3, 6, 8 and 9 for assessment of the drug likeness.

Compound No. Rule	miLogP^a <5	% ABS^b	TPSA^c	Natoms^d	MW^e <500	M.Vol.^f	nON^g <10	nOHNH^h <5	nviol.^i	nrotb.^j (<10)
3a	0.07	64.04	130.31	19	281.30	222.79	8	4	0	3
3b	2.54	84.80	70.14	19	252.28	224.06	5	2	0	2
6a	−0.04	56.01	153.59	23	329.35	259.77	9	4	0	2
6b	−0.20	58.09	147.54	23	330.33	256.61	9	3	0	2
6c	2.63	76.77	93.42	23	300.32	261.04	6	2	0	1
6d	2.48	78.85	87.37	23	301.31	257.88	6	1	0	1
8a	1.27	71.49	108.71	27	398.47	316.26	8	2	0	3
8b	3.94	92.25	48.54	27	369.45	317.53	5	0	0	2
9a	0.03	67.74	119.57	19	278.30	216.61	8	3	0	2
9b	2.70	88.50	59.40	19	249.28	217.88	5	1	0	1
Ciprofloxacin	−0.70	83.27	74.57	24	331.35	285.46	6	2	0	3
Amphotericin B	−2.49	1.27	319.61	65	924.09	865.48	18	13	3	3

^aOctanol–water partition coefficient, calculated by the methodology developed by Molinspiration.

^b% ABS: percentage of absorption.

^cTPSA: topological polar surface area.

^dNumber of non-hydrogen atoms.

^eMolecular weight.

^fMolecular volume.

^gNumber of hydrogen-bond acceptors (O and N atoms).

^hNumber of hydrogen-bond donors (OH and NH groups).s

ⁱNumber of “Rule of five” violations.

^jNumber of rotatable bonds.

Table 4. Toxicity risks, solubility, drug-likeness, and drug score of the synthesised compounds.

	Toxicity risks				Solubility	Drug likeness	Drug Score
Comp. no.	Mutagenicity	Tumorigenicity	Irritancy	Reproductive effect
3a	green	green	green	red	−2.09	6.32	0.56
3b	red	red	green	red	−3.8	4.81	0.17
6a	green	green	green	red	−4.15	0.33	0.38
6b	green	green	green	red	−2.79	1.59	0.49
6c	Red	red	green	red	−5.86	−1.42	0.07
6d	Red	red	green	red	−4.5	−0.01	0.12
8a	Red	green	green	red	−4.4	4.87	0.24
8b	Red	red	green	red	−6.11	3.16	0.08
9a	Green	green	green	red	−2.22	4.26	0.56
9b	Red	red	green	red	−3.93	2.52	0.17
Ciprofloxacin	Green	green	green	green	−3.32	2.07	0.82
Amphotericin B	Green	green	green	green	−5.08	−0.14	0.27

Red: high risk; green: low risk.

Figure 5. 2D and 3D Views (A, B) of the original ligand, methotrexate re-docked in the active site of DHFR (PDB ID: 1DLS) using MOE software. 3D representation (C) of the superimposition of the docking pose (yellow) and the co-crystallised (red) of methotrexate with an RMSD of 0.88 Å.

Figure 6. 2D and 3D Views (A, B) of the compound 3a docked in the active site of DHFR (PDB ID: 1DLS) using MOE software. Dotted lines and arrows represent hydrogen bonds.

Figure 7. 2D and 3D Views (A, B) of the compound 6a docked in the active site of DHFR (PDB ID: 1DLS) using MOE software. Dotted lines and arrows represent hydrogen bonds.