Synthesis and biological evaluation of biguanide and dihydrotriazine derivatives as potential inhibitors of dihydrofolate reductase of opportunistic microorganisms

Figures & data

Scheme 1.  Scheme for the synthesis of A1 (01–03) and A2 (01–04). (a) K₂CO₃, CH₃CN, reflux; (b) potassium salt of phthalimide, DMSO, MW, 70 W, 120°C; (c) Sn/HCl, ethanol; (d) dicyandiamide, ethanol, reflux; (e) dicyandiamide, acetone, reflux.

Scheme 2.  Scheme for the synthesis of B1 (01–07) and B2 (01–07). (a) K₂CO₃, CH₃CN, reflux; (b) Sn/HCl, ethanol; (c) dicyandiamide, ethanol, reflux; (d) dicyandiamide, acetone, reflux.

Table 1.  Inhibitory concentration (IC₅₀, μM) against rlDHFR, pcDHFR, tgDHFR, and maDHFR and selectivity ratios versus rlDHFR by target compounds.

Mol ID	n	R	Activity, IC50 (μM)				Selectivity index
			pc	tg	ma	rl	rl/pc	rl/tg	rl/ma
A1-01	0	H	51.386 (27)	40.265	51.386 (26)	67.720	ND	1.682	ND
A1-02	1	H	106.031 (18)	135.465	106.031 (7)	106.031 (16)	ND	ND	ND
A1-03	2	H	287.314	145.281	97.241 (7)	100.815	0.351	0.694	ND
A2-01	0	H	15.439	0.424	44.615	1.156	0.075	2.724	0.0259
A2-02	1	H	6.491	0.169	35.170	0.556	0.086	3.287	0.0158
A2-03	2	H	2.605	0.079	8.901	0.033	0.013	0.422	0.0037
A2-04	1	OH	34.593	0.523	153.745	1.944	0.056	3.717	0.0126

Mol ID	R1	R2	R3	Activity, IC50 (μM)				Selectivity index
				pc	tg	ma	rl	rl/pc	rl/tg	rl/ma
B1-01	H	H	H	131.649 (10)	131.649 (25)	131.649 (3)	131.649 (22)	ND	ND	ND
B1-02	H	H	Cl	103.914	58.464	63.771	68.740	0.662	1.176	1.078
B1-03	Cl	H	H	293.027	9.948	152.442 (30)	20.834	0.071	2.094	ND
B1-04	Cl	Cl	H	123.006	111.146 (19)	111.146 (16)	27.941	0.227	ND	ND
B1-05	H	Br	H	163.839	21.430	127.419 (36)	74.946	0.457	3.497	ND
B1-06	H	H	OCH3	15.522 (5)	8.404	15.522 (2)	14.716	ND	1.751	ND
B1-07	H	Ph	H	103.565 (24)	111.371	103.565 (3)	66.964	ND	0.601	ND
B2-01	H	H	H	5.399	0.032	14.411	0.120	0.022	3.801	0.0083
B2-02	H	H	Cl	1.109	0.020	4.081	0.034	0.031	1.738	0.0084
B2-03	Cl	H	H	2.531	0.040	10.348	0.073	0.029	1.841	0.0071
B2-04	Cl	Cl	H	1.076	0.029	11.240	0.018	0.017	0.627	0.0016
B2-05	H	Br	H	0.829	0.033	7.321	0.033	0.040	1.017	0.0045
B2-06	H	H	OCH3	1.689	0.019	3.383	0.048	0.029	2.551	0.0143
B2-07	H	Ph	H	0.403	0.012	10.208	0.026	0.064	2.151	0.0025
Trimethoprim^a	—	—	—	12	2.80	0.30	180	14	65	600
Piritrexim^a	—	—	—	0.013	0.0043	0.00061	0.0033	0.26	0.76	5.3
Trimetrexate^a	—	—	—	0.042	0.01	0.0015	0.003	0.07	0.3	2.0

Note. Triplicate assays were performed as previously described^14,15. Number in parentheses indicates percent inhibition obtained at that concentration. Selectivity index: rl/pc, IC₅₀ rlDHFR/IC₅₀ pcDHFR; rl/tg, IC₅₀ rlDHFR/IC₅₀ tgDHFR; rl/ma, IC₅₀ rlDHFR/IC₅₀ maDHFR.

^aData taken from reference 3.

Figure 1.  Binding mode of B2-07 in the active site of developed tgDHFR homology model. Hydrogen bonding interactions are shown by yellow dotted lines, π–π stacking interaction with Phe91 is noticeable.

Table 2.  Comparison of experimental binding energy with docking score.

Mol ID	tgDHFR acti-vity, IC50 (μM)	ΔGexpt^a	XP GScore^a	Error^a	Evdw^a	Ecoul^a
A1-01	40.265	−6.23	−7.49	−1.26	−46.30	−4.69
A1-02	135.465	−5.49	−7.49	−2.01	−45.47	−11.18
A1-03	145.281	−5.44	−8.52	−3.08	−47.53	−11.34
A2-01	0.424	−9.04	−5.24	3.80	−29.64	−7.69
A2-02	0.169	−9.61	−9.37	0.24	−30.30	−5.57
A2-03	0.079	−10.07	−11.46	−1.38	−45.17	−7.64
A2-04	0.523	−8.91	−11.95	−3.04	−42.65	−10.09
B1-01	131.649 (25)	>–5.50	−7.44	>1.93	−33.00	−7.22
B1-02	58.464	−6.00	−4.44	1.57	−40.30	−2.86
B1-03	9.948	−7.09	−7.39	−0.30	−34.24	−4.33
B1-04	111.146 (19)	>–5.61	−8.45	>2.84	−37.96	−8.41
B1-05	21.430	−6.62	−8.38	−1.76	−40.61	−6.49
B1-06	8.404	−7.20	−7.73	−0.53	−37.95	−5.85
B1-07	111.371	−5.61	−7.78	−2.17	−48.64	0.53
B2-01	0.032	−10.63	−9.01	1.62	−23.82	−5.52
B2-02	0.020	−10.92	−6.54	4.38	−28.92	−7.26
B2-03	0.040	−10.49	−9.19	1.31	−27.50	−3.95
B2-04	0.029	−10.69	−6.09	4.60	−30.10	−9.19
B2-05	0.033	−10.61	−10.73	−0.12	−38.50	−6.99
B2-06	0.019	−10.95	−10.02	0.93	−37.30	−5.87
B2-07	0.012	−11.23	−11.99	−0.75	−36.73	−5.83
Trimethoprim	2.800	−7.88	−10.08	−2.20	−32.29	−4.70
Piritrexim	0.0043	−11.87	−10.34	1.52	−35.69	−5.80
Trimetrexate	0.010	−11.35	−10.42	0.92	−36.08	−5.73

^aExperimental free energies from ΔG_expt = RT ln IC₅₀ in kcal/mol; XP Gscore, predicted binding energy using Glide in kcal/mol; Error (kcal/mol), difference between experimental and predicted binding energy from ΔG_expt – XP GScore; Evdw and Ecoul, van der Waals and electrostatic interaction energies between ligand and receptor calculated using Glide.

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