New insights into the chemical behavior of S-oxide derivatives of thiocarbonyl-containing antitubercular drugs and the influence on their mechanisms of action and toxicity.

OBJECTIVES: This work aims at getting more insights into the distinct behavior of S-oxide derivatives of thiocarbonyl-containing antitubercular drugs, in order to better understand their mechanism of action and toxicity. METHODS: Computational calculation of relative free energy (ΔΔG) of S-oxide tautomers (sulfine R-C [SO]NH2), sulfenic acid (R-C [S-OH]NH) and sulfoxide (R-C [SHO]NH) derived from thioamide and thiourea antitubercular drugs and an update of the literature data with a new point of view about how the structural features of oxidized primary metabolites (S-oxide) can influence the outcome of the reactions and be determinant for the mechanisms of action and of toxicity of these drugs. RESULTS: The calculated free energy of S-oxide tautomers, derived from thioamide and thiourea-type antitubercular drugs, supported by some experimental results, revealed that S-oxide derivatives could be found under sulfine and sulfenic acid forms depending on their chemical structures. Thiocarbonyl compounds belonging to the thioamide series are firstly oxidized, in the presence of H2O2, into the corresponding S-oxide derivatives that are more stable under the sulfine tautomeric form. Otherwise, S-oxides of thiourea-type (acyclic and cyclic) compounds tend to adopt the sulfenic acid tautomeric form preferentially. While the intermediate ethionamide-SO under sulfine form can be isolated and in the presence of H2O2 can undergo further oxidation by a mechanism yielding radical species that are toxic for Mycobacterium tuberculosis and human, thioacetazone-SO, found mainly into sulfenic acid form, is unstable and sufficiently reactive in biological conditions to intercept different biochemical pathways and manifests thus its toxicity. CONCLUSION: Based on experimental and theoretical data, we propose that S-oxide derivatives of thioamide and thiourea-type antitubercular drugs have preference for distinct tautomeric forms. S-oxide of ethioamide is preferentially under sulfine form whereas S-oxide of thiourea compound as thioacetazone is mainly found under sulfenic acid form. These structural features lead to individual chemical reactivities that might explain the distinct mechanism of action and toxicity observed for the thioamide and thiourea antitubercular drugs.

Antimicrobial and Anti-biofilm Activity of Thiourea Derivatives Bearing 3-amino-1H-1,2,4-triazole Scaffold.

A set of 21 thiourea derivatives were prepared through reacting 3-amino-1H-1,2,4-triazole with the commercial aliphatic and aromatic isothiocyanates. The aliphatic isothiocyanate was used as reagent leading to substitution on NH atom of 3-aminotriazole ring, whereas the triazole amino group was substituted when isothiocyanate group was bonded to the Csp2 hybridized atom, e.g. an aryl or C=O fragment. All compounds were evaluated in vitro for the antimicrobial activity. The derivatives 1, 2, 4, 8, 9, 10 and 12 showed the highest inhibition against Gram-positive cocci (S. aureus and S. epidermidis). The observed MIC values were in the range of 4-32 µg/mL. Compounds were also tested for their in vitro antimicrobial activity against the hospital methicillin-resistant strains of S. aureus. The observed MIC values varied from 4 to 64 µg/mL. The products 4 and 10 effectively inhibited the formation of biofilms of the methicillin-resistant and standard strains of S. epidermidis. The compound 10 was found to be more promising with IC50 values of 2-6 µg/mL as compared to the control. Moreover, the cytotoxicity against the MT-4 cells of all studied thioureas was evaluated. The compound 18 was significantly cytotoxic (CC50 = 8 µM).

New parameter that supports speculation on the possible mechanism of hypothyroidism induced by chemical substances in repeated-dose toxicity studies.

Hypothyroidism induced by xenobiotic treatment was analyzed for possible underlying mechanism(s) on the basis of different responses of the thyroid gland and the liver, using a newly-created database of repeated-dose toxicity of 500 chemicals. Two mechanisms are proposed: direct inhibition of thyroid hormone biosynthesis in the thyroid gland, and stimulated degradation of thyroid hormone by induction of hepatic drug-metabolizing enzymes. In the database there were 10 chemicals inducing hypertrophy/hyperplasia of follicular cells in the thyroid gland and having data on thyroid glands. On the basis of the chemical structure and information available in the literature, we judged three chemicals to be typical thioamide derivatives that act directly on the thyroid gland, and the others as non-thioamide derivatives that were unlikely to have any direct action on the thyroid gland. All these chemicals were classified into two groups using the ratios of relative weight increase rate of thyroid gland versus that of the liver. These values were at least 1.7, but 3.2 or more in the most of the cases for thioamide derivatives, and 1.2 or less for non-thioamide derivatives. This background analysis suggests the feasibility of parameter-supported speculation on the possible underlying mechanism when new repeated-dose toxicity data on hypothyroidism becomes available.

Design, synthesis and anticonvulsant evaluation of novel N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothio amides.

Thirty six new N-(4-substituted phenyl)-2-[4-(substituted) benzylidene]-hydrazinecarbothioamides were synthesized and evaluated for anticonvulsant activity and neurotoxicity. The anticonvulsant activity was established in three seizure models i.e. MES, scMET and 6 Hz model. The most active compound was 2-[4-(4-chlorophenoxy)benzylidene]-N-(4-fluorophenyl)hydrazinecarbothioamide PC 31 which showed 100% protection at 0.5 h in the 6 Hz test. Compound 2-[4-(4-bromophenoxy) benzylidene]-N-(4-bromophenyl) hydrazinecarbothioamide PC 23 was found to be active in both the MES and 6 Hz test. A computational study was carried out from calculation of a pharmacophore pattern and the prediction of pharmacokinetic properties. Titled compounds have also exhibited good binding properties with epilepsy molecular targets such as glutamate, GABA (A) delta and GABA (A) alpha-1 receptors, in the Lamarckian genetic algorithm based on flexible docking studies.

N-benzylsalicylthioamides: highly active potential antituberculotics.

A gseries of 29 new derivatives of N-benzylsalicylthioamides was synthesized and the compounds were tested for in-vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium avium. The activity was analyzed by quantitative structure-activity relationship (QSAR). Activity increased with increasing lipophilicity and electron donating effect of the substituents in the acyl moiety and decreased with the electrophilic superdelocalizability of the molecules. The most active compounds are more active than isoniazid (INH) and are active against INH-resistant potential pathogenic strains of mycobacterium.

Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo.

Thiobenzamide (TB) is a potent hepatotoxin in rats, causing dose-dependent hyperbilirubinemia, steatosis, and centrolobular necrosis. These effects arise subsequent to and appear to result from the covalent binding of the iminosulfinic acid metabolite of TB to cellular proteins and phosphatidylethanolamine lipids [ Ji et al. ( 2007) Chem. Res. Toxicol. 20, 701- 708 ]. To better understand the relationship between the protein covalent binding and the toxicity of TB, we investigated the chemistry of the adduction process and the identity of the target proteins. Cytosolic and microsomal proteins isolated from the livers of rats treated with a hepatotoxic dose of [ carboxyl- (14)C]TB contained high levels of covalently bound radioactivity (25.6 and 36.8 nmol equiv/mg protein, respectively). These proteins were fractionated by two-dimensional gel electrophoresis, and radioactive spots (154 cytosolic and 118 microsomal) were located by phosphorimaging. Corresponding spots from animals treated with a 1:1 mixture of TB and TB- d 5 were similarly separated, the spots were excised, and the proteins were digested in gel with trypsin. Peptide mass mapping identified 42 cytosolic and 24 microsomal proteins, many of which appeared in more than one spot on the gel; however, only a few spots contained more than one identifiable protein. Eighty-six peptides carrying either a benzoyl or a benzimidoyl adduct on a lysine side chain were clearly recognized by their d 0/ d 5 isotopic signature (sometimes both in the same digest). Because model studies showed that benzoyl adducts do not arise by hydrolysis of benzimidoyl adducts, it was proposed that TB undergoes S-oxidation twice to form iminosulfinic acid 4 [PhC(NH)SO 2H], which either benzimidoylates a lysine side chain or undergoes hydrolysis to 9 [PhC(O)SO 2H] and then benzoylates a lysine side chain. The proteins modified by TB metabolites serve a range of biological functions and form a set that overlaps partly with the sets of proteins known to be modified by several other metabolically activated hepatotoxins. The relationship of the adduction of these target proteins to the cytotoxicity of reactive metabolites is discussed in terms of three currently popular mechanisms of toxicity: inhibition of enzymes important to the maintenance of cellular energy and homeostasis, the unfolded protein response, and interference with kinase-based signaling pathways that affect cell survival.

Evaluation of the toxicity of substituted benzthioanilides by using in vitro tests.

The cytotoxicity of 12 benzthioanilides substituted in the N-aromatic ring, and of two commercial preparations (imaverol and thiuram) for comparison, was studied with clone 81 cat cells, by determining the highest tolerated dose, and by using the neutral red uptake assay and the kenacid blue assay for total protein. The concentrations that induced 20%, 50% and 80% (IC20, IC50 and IC80) inhibition relative to controls were calculated from dose-response curves. For some compounds, rat LD50 values were also determined. All the benzthioanilide preparations showed in vitro toxicities lower than those of the fungicides imaverol and thiuram. It was confirmed that the cytotoxicities of the compounds depend on the type of substituent. The least toxic compound contained a CONHCH(2)CO(2)H substituent in the para position of the N-aromatic ring, and the most toxic compounds contained chloro and fluoro, or three chloro substituents in the anilide moiety. All the benzthioanilides tested showed fungistatic activity for dermatophytes; two of the compounds (compound 5 and compound 12) also inhibited the development of yeasts at concentrations lower than those which caused toxicity in vitro. The LD50 values and the cytotoxic concentrations in vitro were linearly related.

Synthesis and in vitro anti-mycobacterium activity of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides. Preliminary toxicity and pharmacokinetic evaluation.

Disseminated infections with Mycobacterium tuberculosis (MT) and Mycobacterium avium complex (MAC) are increasingly opportunistic diseases in patients with advanced acquired human immunodeficiency syndrome (AIDS). A series of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides has been synthesized, and MICs for MT and MAC strains, either standard or isolated from infected patients, have been determined. Preliminary tests show a good activity and a very low toxicity for some derivatives. Pharmacokinetic studies in the rat show a very rapid elimination from the body after intravenous administration and a poor absorption after oral administration.

Relative hepatotoxicity of 2-(substituted phenyl)thiazoles and substituted thiobenzamides in mice: evidence for the involvement of thiobenzamides as ring cleavage metabolites in the hepatotoxicity of 2-phenylthiazoles.

The hepatotoxicity of the 3 isomers of para-substituted thiobenzamides and the 3 isomers of 2-(para-substituted phenyl)-4-methylthiazoles was evaluated in mice depleted of glutathione (GSH) by pretreatment with buthionine sulfoximine (BSO). In accordance with previous studies with the rat, p-methoxythiobenzamide was more toxic than thiobenzamide, and conversely p-chlorothiobenzamide was markedly less toxic as assessed by serum alanine aminotransferase (ALT) activity. The hepatotoxicity of 2-phenyl-4-methylthiazole was also altered by the addition of para-substituents to the phenyl ring in the same way as observed for thiobenzamide derivatives: the rank order of toxicity was 4-methylthiazoles having p-methoxyphenyl > phenyl >> p-chlorophenyl at the 2-position. This good correlation of the rank order of hepatotoxicity between series of 2-(para-substituted phenyl)-4-methylthiazoles and para-substituted thiobenzamides supports the concept that thiobenzamides as ring cleavage metabolites play a role in the hepatotoxicity of 2-phenylthiazole derivatives.

Effects of the herbicide chlorthiamid on the olfactory mucosa.

Chlorthiamid (2,6-dichlorothiobenzamide) and its major metabolite 2,6-dichlorobenzonitrile are olfactory toxicants with a high in vivo covalent binding in the olfactory mucosa of mice. This study showed that the cytochrome P450 (P450) inhibitors, metyrapone and sodium-diethyldithiocarbamate, abolished the chlorthiamid-induced toxicity (12 mg/kg; 0.06 mmol/kg) in C57B1/6 mice suggesting a P450-dependent toxicity. Incubation of [14C]-labelled chlorthiamid with rat olfactory microsomes showed a low NADPH-dependent oxidative covalent binding which was only 3-fold higher than that in liver microsomes. Thus the results do not support a major in situ metabolic activation of chlorthiamid and it is suggested that metabolic activation of the major chlorthiamid metabolite (2,6-dichlorobenzonitrile) is responsible for most of the covalent binding and toxicity of chlorthiamid at this site in vivo. Thiobenzamide (16 mg/kg; 0.12 mmol/kg), a dechlorinated chlorthiamid-analog, induced no marked morphological changes in the olfactory mucosa demonstrating that chlorines in the 2,6-position are important for the chlorthiamid-induced toxicity at this site.

Formation of toxic metabolites from thiabendazole and other thiazoles in mice. Identification of thioamides as ring cleavage products.

The metabolism of three nephro- or hepatotoxic thiazoles--2-(thiazol-4-yl)benzimidazole (thiabendazole) (1a), 4-tert-butyl-2-methyl-thiazole (1b), and 2-(p-methoxyphenyl)-4-methylthiazole (1c)--was examined in mice with special regard to the formation of ring cleavage products. By GC/MS analyses of derivatized metabolites and comparison with authentic samples, thioformamide and benzimidazol-2-ylglyoxal as the accompanying fragment were identified as urinary metabolites in mice dosed with 1a. Similarly, 1b produced thioacetamide and tert-butylglyoxal, and 1c produced p-methoxy-thiobenzamide (and its S-oxide) and methylglyoxal. These results could be explained by the postulated metabolic pathways where thiazoles would undergo microsomal epoxidation of the C = C double bond and, after being hydrolyzed, the resulting epoxide would then be decomposed to form the corresponding thioamides and alpha-dicarbonyl fragments.

Formation, characterization, and immunoreactivity of lysine thioamide adducts from fluorinated nephrotoxic cysteine conjugates in vitro and in vivo.

Fluorinated nephrotoxic cysteine conjugates undergo bioactivation via the beta-lyase pathway to thionoacetyl fluorides (TAF), the putative reactive intermediates. The TAF derived from S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC) difluorothionoacetylates amine nucleophiles found in proteins and lipids. A specific antisera, raised against (trifluoroacetamido)lysine adducts formed in vivo after halothane treatment, has previously been used to localize TFEC-derived protein adducts immunohistochemically, and a good correlation between adduction and toxicity was demonstrated. Interestingly, thioamide formation is facilitated by acyl-transfer catalysts such as imidazoles and phenols. However, although putative lysine adducts have been reported to be formed from the related TAF derived from S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), protein adducts derived from CTFC metabolism have not been completely characterized. In the present investigation we characterize (chlorofluorothionacetamido)lysine (CFTAL) adduct formation during S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) metabolism, both in vitro and in vivo. Our data indicate that formation of CTFC-derived lysine thioamides was not as dependent on nucleophilic catalysis as observed for TFEC, and this appears to be due to an apparent greater reactivity of the TAF resulting in a higher trapping efficiency in the absence of catalyst. Also, qualitative and quantitative differences in the structures and time course of CTFC versus TFEC adduct breakdown were observed. Antibodies raised against the halothane metabolite protein adduct (trifluoroacetamido)lysine cross-react with specific mitochondrial proteins from the kidneys of TFEC-treated rats. Using this antibody, we have found that the pattern of adducted proteins from TFEC- and CTFC-treated Fischer rats was similar, but the intensity was considerably lower after treatment with equimolar concentrations of CTFC in vivo.

The effect of partial hepatectomy on the metabolism, distribution, and nephrotoxicity of para-methylthiobenzamide in the rat.

Para-Methylthiobenzamide (PMTB) produces injury to the liver and kidney. Toxicity is mediated via its biotransformation to a reactive S,S-dioxide metabolite. The objective of this study was to examine the role of hepatic metabolism in the production of PMTB-induced renal toxicity. Renal injury was assessed in partially hepatectomized and sham-operated rats and the effect of this procedure on the distribution and metabolism of PMTB was examined. The in vitro oxidation of PMTB and [14C]thiobenzamide by rat kidney microsomes was also examined. Plasma urea levels and renal cortical slice uptake of organic ions were used to monitor renal function. Partial hepatectomy alone did not alter renal function nor raise blood urea nitrogen levels. Nephrotoxicity resulted when a nonnephrotoxic dose of PMTB (1.2 mmol/kg) was given to partially hepatectomized rats. An HPLC method was used for measurement of PMTB and its metabolites para-methylthiobenzamide S-oxide (PMTBSO) and para-methylbenzamide (PMBA) in plasma and kidney. Hepatectomy delayed the removal of this dose of PMTB from plasma and allowed greater concentrations of PMTB and PMTBSO to accumulate in plasma and kidney at 6 and 15 hr. The level of PMBA was similar in both groups at 6 hr, but was increased in plasma and kidney of the hepatectomized group at 15 hr. Kidney microsomes rapidly converted PMTB to PMTBSO and small amounts of PMBA. [14C]TB was oxidized by microsomes to thiobenzamide S-oxide, benzamide, and covalently bound metabolites. The results indicate that partial hepatectomy lowered the threshold for the expression of nephrotoxicity by PMTB. This procedure is associated with an increased renal accumulation of PMTB and PMTBSO, which are both sequentially transformed to the toxic metabolite.

Thiobenzamide-induced hepatotoxicity: effects of substituents and route of administration on the nature and extent of liver injury.

Differences in the nature and extent of hepatic injury were examined after administration of para-substituted thiobenzamides to rats. In accordance with previous studies, the extent of hepatotoxicity varied with the electron-donating ability of the substituent. There was also a good correlation between the extent of hepatic necrosis and the amount of substituted thiobenzamide sulfoxide found in the plasma after intraperitoneal dosing. The nature of the hepatic lesion, characterized as a combination of hepatic necrosis, ballooning degeneration, and biliary dysfunction, varied qualitatively with each thiobenzamide analog. When the hepatotoxicity of thiobenzamide was compared after either intraperitoneal or oral dosing, differences in the extent of hepatic necrosis, ballooning degeneration, transaminase elevation, and biliary dysfunction were observed. Intraperitoneal dosing with thiobenzamide gave less severe necrosis and more pronounced elevations in bile acids, while oral dosing led to more severe necrosis along with impaired biliary function. The route of administration was shown to dramatically affect the pharmacokinetics of thiobenzamide and thiobenzamide sulfoxide. Intraperitoneal administration of thiobenzamide gave high plasma and liver levels of both thiobenzamide and thiobenzamide sulfoxide, whereas oral administration gave slightly lower levels of the sulfoxide but much lower levels of thiobenzamide. The reason for greater hepatic necrosis after oral administration may be due to a greater ability to further metabolize the sulfoxide to a reactive metabolite in the absence of high levels of thiobenzamide.

Toxicity of 2,6-dichlorothiobenzamide (chlorthiamid) and 2,6-dichlorobenzamide in the olfactory nasal mucosa of mice.

The toxic effects of the herbicide chlorthiamid (2,6-dichlorothiobenzamide) and its major environmental metabolite 2,6-dichlorobenzamide (DCBA) were examined in the nasal passages of C57Bl mice following single ip injections. Chlorthiamid (12.25, and 50 mg/kg) induced an extensive destruction of the olfactory region, similar to that previously observed with the analogue dichlobenil (2,6-dichlorobenzonitrile). Necrosis of Bowman's glands was evident first, whereas degeneration and necrosis of the olfactory neuroepithelium developed less rapidly. The lesions were most severe in the dorsomedial region of the nasal cavity. At longer post-treatment intervals, the olfactory epithelium was replaced by a respiratory-like epithelium, and there was fibrosis of the lamina propria. DCBA was also toxic to the olfactory region (100 mg/kg), inducing necrosis of the Bowman's glands and the neuroepithelium in the dorsomedial region of the nasal cavity. No lesions were observed in other parts of the nasal cavity or in the liver after administration of chlorthiamid or DCBA. Chlorthiamid (IC50 = 51 microM), but not DCBA, inhibited the covalent binding of 14C-labeled dichlobenil in the olfactory mucosa in vitro. It is proposed that the toxic effects of chlorthiamid and dichlobenil in the olfactory mucosa are mediated by common or closely related metabolites.

Possible role of the acetone-inducible cytochrome P-450IIE1 in the metabolism and hepatotoxicity of thiobenzamide.

The effect of acetone pretreatment (5% in drinking water for 10 days on rat liver metabolism and toxicity of thiobenzamide (TB) was investigated. Hepatic microsomes from acetone-pretreated rats showed a significant increase of TB-S-oxidation rate which, on the basis of selective thermal inactivation of FAD-containing monooxygenase (FADM), appeared dependent only on cytochrome P-450. Furthermore, TB was able to competitively inhibit acetone hydroxylase (AcH), an enzymatic reaction highly specific for the P-450IIE1 isozyme. Acetone pretreatment of rats also produced an exacerbation of liver damage induced by acute administration of TB (150 mg/kg), as judged by the extent of liver necrosis and serum alanine-amino transferase (ALAT) activities. Coadministration of acetone with TB reduced on the other hand the extent of liver damage. The findings suggest that P-450 species induced by acetone, and in particular the P-450IIE1 isozyme, could be involved in the biotransformation of TB.

Nephrotoxicity of para-substituted thiobenzamide derivatives in the rat.

Histological examination, plasma urea nitrogen levels (BUN), and renal cortical slice uptake of paminohippurate (PAH) or tetraethylammonium (TEA) were used to assess the nephrotoxicity of thiobenzamide and its para-substituted derivatives in Sprague-Dawley rats. Intraperitoneal injection of p-methylthiobenzamide (PMTB) to rats resulted in dose-dependent nephrotoxicity as judged by increased BUN levels, decreased TEA uptake and histologic examination of the kidney. Para-methoxythiobenzamide and PMTB were more potent nephrotoxins than thiobenzamide, which was itself minimally nephrotoxic. Para-methylthiobenzamide-S-oxide (PMTBSO) was more nephrotoxic than PMTB. Rats were pretreated with 1-methyl-1-phenylbenzoylthiourea (MPBTU), a non-toxic arylthiourea which inhibits the metabolism and toxicity of thiocarbonyl compounds. The nephrotoxicity and hepatotoxicity of PMTB was reduced by treatment with MPBTU 30 min prior to PMTB. Pretreatment with MPBTU protected against the renal toxicity of PMTBSO. The results indicate that electron donating para-substituted thiobenzamides produce dose-dependent renal injury, dependent upon oxidative biotransformation.

Effect of reserpine on N-methylthiobenzamide-induced pulmonary edema: role of lung norepinephrine and hypothermia.

N-Methylthiobenzamide (NMTB) is a pneumotoxin which causes pulmonary edema and hydrothorax in rodents. Reserpine has been shown to attenuate the pneumotoxicity induced by NMTB. Some of that evidence suggests that the protection afforded by reserpine occurs independently of its capacity to reduce peripheral 5-hydroxytryptamine (5-HT). We therefore investigated 2 other pharmacologic properties of reserpine, namely: (1) its capacity to reduce lung norepinephrine (NE); and (2) its capacity to induce hypothermia, in order to more fully understand its mechanism of protection. Pretreatment of mice or rats with 6-hydroxydopamine at a dose which reduced lung NE by approximately 80% did not affect the pneumotoxic response to NMTB. Thus a decrease in lung NE probably does not account for reserpine's protective effect. An investigation of reserpine's effects on core temperature revealed that mice dosed with a combination of reserpine + NMTB presented with core temperatures lower than mice treated with either compound alone. Mice placed in a cold environment (2 degrees C) and dosed with NMTB presented with hypothermia and an attenuated toxic response to NMTB. Thus a reserpine-induced hypothermia could be allowing for a reduction of NMTB metabolism and consequent diminution of toxicity. These observations suggest that reserpine's capacity to protect animals against NMTB-induced pulmonary edema may in part be due to its capacity to induce hypothermia.