Symmetric benzidine derivatives as anti-HCV agents: Insight into the nature, stereochemistry of the capping amino acid and the size of the terminal capping carbamates.

Novel symmetric molecules, bearing a benzidine prolinamide core, two terminal carbamate caps of variable sizes and nature, including natural and unnatural amino acids were developed. Several terminal N-carbamate substituents of the core structure, ranging from linear methyl, ethyl and butyl groups to branching isobutyl group; and an aromatic substituent were also synthesized. Series 1 has hydrophobic AA residues, namely S and R phenylglycine and a terminal carbamate capping group, whereas Series 2 bears sulphur containing amino acids, specifically S and R methionine and the natural R methylcysteine. The novel compounds were tested for their inhibitory activity (EC50) and their cytotoxicity (CC50), using an HCV 1b (Con1) reporter replicon cell line. Compound 4 with the unnatural capping residue, bearing d-Phenylglycine amino acid residue and N-isobutyloxycarbonyl capping group, was the most active within the two series, with EC50 = 0.0067 nM. Moreover, it showed high SI50 > 14788524 and was not cytotoxic at the highest tested concentration (100 µΜ), indicating its safety profile. Compound 4 also inhibited HCV genotypes 2a, 3a and 4a. Compared to the clinically approved NS5A inhibitor Daclatasvir, compound 4 shows higher activity against genotypes 1b and 3a, as well as improved safety profile.

Expanding the chemical space of anti-HCV NS5A inhibitors by stereochemical exchange and peptidomimetic approaches.

Here we report a series of potent anti-HCV agents bearing a symmetrical benzidine l-prolinamide backbone with different capping groups including alkyl/aryl carbamates of natural and unnatural valine and leucine amino acids. All compounds were investigated for their inhibitory activity in an HCV replicon assay on genotype 1b. The novel compounds share some chemical and clinical attributes of commercially available NS5A inhibitors. Compounds 5 and 6 with unnatural capping residue and ethyl and isobutyl carbamates showed EC50 values in the picomolar range with a low toxicity profile and selectivity indices of several orders of magnitude. These findings enlarge the chemical space from which NS5A inhibitors may be discovered by adopting unnatural amino acids, amino acids other than valine and carbamates other than methyl as the capping groups.

Synthesis of benzidine derivatives via FeCl3·6H2O-promoted oxidative coupling of anilines.

Under open-flask conditions in the presence of commercially available FeCl3·6H2O, N,N-disubstituted anilines can be converted into diversely functionalized benzidines with yields of up to 99%. Oxidative coupling was extended to N-monosubstituted anilines, and the method was applied to the efficient preparation of 6,6'-biquinoline. Mechanistic investigations have also been performed to explain the observed reactivities.

A dual-cell device designed as an oxidase mimic and its use for the study of oxidase-like nanozymes.

A dual-cell device has been designed as an oxidase-like mimic with the oxidation of 3,3',5,5'-tetramethylbenzidine as a model reaction. This dual-cell device could be also used to study oxidase-like nanozymes. It was found that only the catalytic sites for oxygen reduction are essential and necessary for oxidase-like nanozymes.

Synthesis and mutagenicity of 3-halogenated and 3,3',5,5'-tetrahalogenated benzidines.

3-Fluorobenzidine (FBz), 3-chlorobenzidine (ClBz), 3-bromobenzidine (BrBz), 3,3',5,5'-tetrafluorobenzidine (F4Bz), and 3,3',5,5'-tetrachlorobenzidine (Cl4Bz) were synthesized and tested for their ability to revert Salmonella typhimurium. F4Bz was the only compound to show direct-acting mutagenicity and was equally potent in strain TA98 and the acetylase-deficient strain TA98/1,8-DNP6. In the presence of hamster liver S9, all compounds except Cl4Bz were mutagenic. The relative mutagenicities in TA98 were FBz greater than ClBz greater than BrBz greater than F4Bz greater than Bz greater than Cl4Bz = 0. In TA98/1,8-DNP6 the trend was F4Bz approximately BrBz approximately ClBz greater than FBz greater than Bz greater than Cl4Bz = 0.

N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism.

N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-post-labeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared with that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-post-labeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and were shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product that eluted at the same time from the HPLC (32P-dpG-ABZ). Additional experiments demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced approximately 40-fold more dGp-ABZ than horseradish peroxidase (1943 +/- 339 versus 49 +/- 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e. arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different.

Unambiguous synthesis of asymmetrically substituted chlorinated benzidines, and a study of their mutagenicity in the Ames test: potent activity of 3,5,3'-trichlorobenzidine.

3,5-Dichlorobenzidine and 3,5,3'-trichlorobenzidine were prepared by the reaction of 2,6-dichloronitrosobenzene with aniline and 2-chloroaniline, respectively, to give the appropriate substituted azobenzene; reduction and rearrangement gave the desired benzidine derivatives. The products were purified and characterized by mass spectrometry and [1H] nuclear magnetic resonance spectroscopy. The mutagenic activities of the chlorinated benzidines were determined, using the Ames tester strain TA98. 3,5,3'-Trichlorobenzidine is the most potent benzidine derivative yet reported. The activities of five chlorinated benzidines are compared, and rules for the effect of substitution on activity are discussed.

Synthesis and mutagenicity of 3,3'-dihalogenated benzidines.

3,3'-Difluorobenzidine (F2Bz), and 3,3'-dibromobenzidine (Br2Bz) were synthesized and compared with 3,3'-dichlorobenzidine (Cl2Bz) for ability to revert Salmonella typhimurium. The relative mutagenicities in all systems are Cl2Bz approximately equal to Br2Bz greater than F2Bz greater than Bz. F2Bz, Cl2Bz, and Br2Bz are direct-acting mutagens towards S. typhimurium strain TA98. The acetylase-deficient derivative TA98/1,8-DNP6 displays no resistance to induction of mutagenesis by these compounds, in the absence of mammalian activation. With addition of hamster hepatic S-9 activation the mutagenicity of these compounds increases greatly. TA98/1,8-DNP6 shows some resistance to this mutagenicity. Multiple mechanisms must exist for the genotoxicity of 3,3'-dihalogenated benzidines.

Role of N-glucuronidation in benzidine-induced bladder cancer in dog.

The mechanism by which benzidine induces bladder cancer in dog was evaluated by assessing metabolism of [3H]benzidine by dog liver slices and microsomes. Slices incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min peak, which was also produced when microsomal incubations were supplemented with UDP-glucuronic acid. In contrast to microsomes, very little of the 32.5 min peak was produced with the 100,000 g supernatant fraction. Microsomal metabolism was increased 5-fold by pretreatment with Triton X-100. Very little activity was observed with rat microsomes in either the presence or absence of Triton X-100. This metabolite was also generated by incubating benzidine with glucuronic acid at 4 degrees C for 3 days. Thermospray MS identified this metabolite as benzidine N-glucuronide. At 37 degrees C, the t1/2 stability of purified N-glucuronide was 99, 25 and 3 min in dog urine adjusted to pH 7.3, 6.3 and 5.3 respectively. The N-glucuronide was quite stable at pH 9.3, in dog plasma, and in aprotic solvents for 4 h at 37 degrees C. Relative to benzidine, its N-glucuronide is weakly bound to plasma proteins but not more reactive with DNA. Thus, detoxification by liver provides a mechanism for accumulation of benzidine in acidic urine, uptake of benzidine into bladder epithelium, and activation of benzidine in bladder. The liver and N-glucuronidation play a potentially important role in the species specificity of benzidine carcinogenesis.