Phenotypic evaluation and in silico ADMET properties of novel arylimidamides in acute mouse models of Trypanosoma cruzi infection

Figures & data

Figure 1 Structures of compounds.

Table 1 The trypanocidal effect (µM) and SI of AIAs and of Bz against T. cruzi (Y strain)

Compound	BTs (24 h/37°C)		Intracellular amastigotes (48 h/37°C)
	EC50 (µM)	SI	EC50 (µM)	SI
DB1957	0.068±0.0014*	17	0.19±0.12#	6
DB1959	0.078±0.01**	45	0.72±0.28#	1.6
DB1890B	0.014±0.007***	760	1.09±0.03#	9.7
Bz	12.94±1.93	>77	3.7±1.72	>360

Notes: EC₅₀ values, compound concentration that reduces the number of parasites by 50%; SI, that corresponds to the ratio of LC₅₀/EC₅₀ – for BT and intracellular parasites calculated on LC₅₀ values of 24 and 48 h of incubation at 37°C, respectively. Statistical analysis (AIA vs Bz):

* P=0.003,

** P=0.003,

*** P=0.0003, and

# P>0.05. All assays were run at least two times in duplicate.

Abbreviations: AIA, arylimidamide; Bz, benznidazole; T. cruzi, Trypanosoma cruzi; BT, bloodstream trypomastigote; SI, selectivity index.

Figure 2 Treatment of T. cruzi-infected mice (inoculum of 10⁴ per animal using BT from Y strain) with DB1957, DB1959, and DB1890B.

Notes: The activities of 25 mg/kg of compounds and 100 mg/kg of the reference drug Bz (by per oral) were also evaluated using similar therapeutic schemes at the 5–9 dpi. Parasitemia curve (A) and survival rates (B) are shown. All assays were run at least two times. Statistical analysis: all groups with P≤0.05.

Abbreviations: T. cruzi, Trypanosoma cruzi; BT, bloodstream trypomastigote; Bz, benznidazole.

Table 2 Biochemical analysis (mean ± SD values) at the 56 dpi of mice blood samples from T. cruzi-infected groups treated with 25 mg/kg/day of each AIA

Compound	BUN	ALT	CK
DB1957	61.2±6.51	36.88±6.08	414.67±295.16
DB1959	44.8±11.17	37.5±3.54	151.5±67.18*
DB1890B	47.05±19.87	141±147.08	1,051.5±212.84*

Notes: Reference values for healthy mice (CECAL/Fiocruz): BUN: 18–29, ALT: 28–132, and CK: 68–1,070. Statistical analysis:

* P=0.03 (DB1959/DB1980B).

Abbreviations: SD, standard deviation; T. cruzi, Trypanosoma cruzi; AIA, arylimidamide; BUN, blood urea nitrogen; ALT, alanine aminotransferase; CK, creatine kinase.

Scheme S1 Reagents and conditions: (a) H₂/Pd (C), EtOH, and EtAOc or SnCl₂-2H₂O, EtOH, and DMSO, 80°C and (b) (i) S-(2-naphthylmethyl)-2-pyridylthioimidate-HBr, EtOH, and CH₃CN and (ii) EtOH/HCL or CHCl₂/CH₃SO₃H.

Abbreviation: DMSO, dimethyl sulfoxide.

Table S1 Physicochemical parameters and Lipinski’s rule of five

Descriptor	DB1890B	DB1957	DB1959
Molecular weight (≤500)	602.74	590.75	654.57
LogP (≤5)	8.34	8.32	8.16
No of acceptors (≤10)	7	7	7
No of donors (≤5)	4	4	4
No of rotatable bonds (≤10)	14	12	14
Surface area	263.244	255.36	262.77

Table S2 In silico ADME

ADME predictors	DB1890B	DB1957	DB1959	Bz
Absorption
Caco-2 permeability (log Papp in 10^6 cm/s)	0.385	0.742	0.133	0.479
Intestinal absorption (human; %)	94.957	98.939	98.031	68.885
Skin permeability (log Kp)	−2.749	−2.859	−2.744	−2.893
Distribution
VDss (human; log L/kg)	−0.586	0.791	0.253	−0.104
Fraction unbound (human; Fu)	0	0.127	0.145	0.503
BBB permeability (log BB)	−1.424	−0.979	−1.929	−0.619
CNS permeability (log PS)	−1.906	−2.044	−2.439	−2.995
Metabolism
CYP2D6 substrate	No	No	No	No
CYP3A4 substrate	Yes	Yes	Yes	No
CYP1A2 inhibitor	No	No	No	No
CYP2C19 inhibitor	No	No	No	No
CYP2C9 inhibitor	Yes	No	No	No
CYP2D6 inhibitor	No	No	No	No
CYP3A4 inhibitor	Yes	Yes	Yes	No
Excretion
Total clearance (log mL/min/kg)	0.586	0.731	0.188	0.625

Abbreviations: ADME, absorption, distribution, metabolism, and excretion; Bz, benznidazole; Papp, apparent permeability coefficient; AMES, assay of the ability of a chemical compound to induce mutations in DNA; Kp, skin permeability constant; Fu, Fraction Unbound; BBB, blood–brain barrier; BB, blood-brain; CNS, central nervous system; PS, permeability-surface area.

Table S3 In silico toxicity

Toxicity predictors	DB1890B	DB1957	DB1959	Bz
AMES toxicity	No	No	No	Yes
Maximum tolerated dose (human; log mg/kg/day)	0.437	−0.64	−0.481	0.984
hERG I inhibitor	No	No	No	No
hERG II inhibitor	Yes	Yes	Yes	No
Oral rat acute toxicity (LD50; mol/kg)	2.777	3.046	2.931	2.454
Oral rat chronic toxicity (LO AEL; log mg/kg_bw/day)	1.307	0.004	−0.152	1.649
Hepatotoxicity	No	Yes	No	Yes
Skin sensitization	No	No	No	No
T. pyriformis toxicity (log µg/L)	0.29	0.348	0.302	1.227
Minnow toxicity (log mM)	−0.455	−0.714	−0.247	1.649

Abbreviations: Bz, benznidazole; T. pyriformis, Tetrahymena pyriformis; AMES, assay of the ability of a chemical compound to induce mutations in DNA; LD, lethal dose; LOAEL, lowest-observed-adverse-effect level.