3,5-Dichloro-2,2'-di-O-methylanziaic acid

Vinclozolin (V) is a fungicide with anti-androgenic properties whose metabolism is not fully understood, and data on urinary elimination of either V or its metabolites are limited. Therefore the kinetics of urinary elimination of V and its metabolites, after an oral dose in adult male rats were investigated. A single oral dose of V (100 mg/kg) suspended in corn oil was administered to male adult Wistar rats, and urine was collected at different times after dosing. V and its metabolites were extracted from urine, then enzymatically hydrolyzed using β-glucuronidase/sulfatase of H. pomatia, and analyzed by HPLC/DAD. Urinary pharmacokinetic parameters were calculated using the analyte concentrations adjusted by creatinine levels. V and its metabolites 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA, formerly denoted as M5), 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3,5-dichloroaniline (M3), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) were efficiently detected. The mean urine concentrations of V and M1 metabolite were fitted to a two-compartmental model for pharmacokinetic analysis. DTMBA approximately represented 88% of the total excreted metabolites, it was easily detected up to 168 h after dosing and its half-lives were 21.5 and 74.1 h, respectively. M1 was the second most abundant metabolite and was detected up to 144 h after being void. V and M3 were detected before 48 h, and M2 exhibited the lowest levels during the first 8 h after dosing. DTMBA, the most abundant V metabolite is quickly eliminated by urine, it is chemically stable, specific and could represent a useful alternative to be used as a biomarker of exposure to V.

The title compounds, bis(μ-3,5-dichloro-2-oxidobenzoato)-κ(3)O(1),O(2):O(2);κ(3)O(2):O(1),O(2)-bis[(3,5-dichloro-2-hydroxybenzoic acid-κO(1))(1,10-phenanthroline-κ(2)N,N')copper(II)], [Cu(2)(C(7)H(2)Cl(2)O(3))(2)(C(7)H(4)Cl(2)O(3))(2)(C(12)H(8)N(2))(2)], (I), and bis(μ-5-chloro-2-oxidobenzoato)-κ(3)O(1),O(2):O(1);κ(3)O(1):O(1),O(2)-bis[(5-chloro-2-hydroxybenzoic acid-κO(1))(1,10-phenanthroline-κ(2)N,N')copper(II)] ethanol monosolvate, [Cu(2)(C(7)H(3)ClO(3))(2)(C(7)H(5)ClO(3))(2)(C(12)H(8)N(2))(2)]·C(2)H(6)O, (II), contain centrosymmetric dinuclear complex molecules in which Cu(2+) cations are surrounded by a chelating 1,10-phenanthroline ligand, a chelating 3,5-dichloro-2-oxidobenzoate or 5-chloro-2-oxidobenzoate anionic ligand and a monodentate 3,5-dichloro-2-hydroxybenzoic acid or 5-chloro-2-hydroxybenzoic acid ligand. The chelating benzoate ligand also bridges to the other Cu(2+) ion in the molecule, but the O atom involved in the bridge is different in the two complexes, being the phenolate O atom in (I) and a carboxylate O atom in (II). The bridge completes a 4+1+1 axially elongated tetragonal-bipyramidal arrangement about each Cu(2+) cation. The complex molecules of both compounds are linked into one-dimensional supramolecular chains through O-H...O hydrogen bonds.

1. Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites. There is no information about Vin biotransformation in humans, therefore, the aim of this study was to characterize its in vitro metabolism using human liver microsomes. 2. Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually converted to 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate inhibition kinetics. The KM app for co-eluted M4/DTMBA and M7 was 24.2 ± 5.6 and 116.0 ± 52.6 μM, the VMax app was 0.280 ± 0.015 and 0.180 ± 0.060 nmoles/min/mg protein, and the CLint app was 11.5 and 1.5 mL/min/g protein, respectively. The Ki for M7 was 133.2 ± 63.9 μM. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2), ketoconazole (CYP3A4), pilocarpine (CYP2A6) and sulfaphenazole (CYP2C9) inhibited M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP. 3. DTMBA is a stable metabolite and specific of Vin, therefore, it could be used as a biomarker of Vin exposure in humans to perform epidemiological studies.