New insights on repellent recognition by <i>Anopheles gambiae</i> odorant-binding protein 1

It is generally recognized that insect odorant binding proteins (OBPs) mediate the solubilisation and transport of hydrophobic odorant molecules and contribute to the sensitivity of the insect olfactory system. However, the exact mechanism by which OBPs deliver odorants to olfactory receptors and their role, if any, as selectivity filters for specific odorants, are still a matter of debate. In the case of Anopheles gambiae recent studies indicate that ligand discrimination is effected through the formation of heterodimers such as AgamOBP1 and AgamOBP4 (odorant binding proteins 1 and 4 from Anopheles gambiae). Furthermore, AgamOBPs have been reported to be promiscuous in binding more than one ligand simultaneously and repellents such as DEET (N,N-diethyl-3-toluamide) and 6-MH (6-methyl-5-hepten-2-one) interact directly with mosquito OBPs and/or compete for the binding of attractive odorants thus disrupting OBP heterodimerisation. In this paper, we propose mechanisms of action of DEET and 6-MH. We also predict that ligand binding can occur in several locations of AgamOBP1 with partial occupancies and propose structural features appropriate for repellent pharmacophores

Towards an efficient prodrug of the alkylating metabolite monomethyltriazene: Synthesis and stability of N-acylamino acid derivatives of triazenes

A series of 3-[a-(acylamino)acyl]-1-aryl-3-methyltriazenes 6ael, potential cytotoxic triazene prodrugs, were synthesised by coupling 1-aryl- 3-methyltriazenes to N-acylamino acids. Their hydrolysis was studied in isotonic pH 7.4 phosphate buffer and in human plasma, while hydrolysis of the derivative 6a was studied in more depth across a range of pH values. Prodrugs 6ael hydrolyse by cleavage of the triazene acyl group to afford the corresponding monomethyltriazenes. Studies in human plasma demonstrate that acylation of the a-amino group of the amino acid carrier is an effective means of reducing the chemical reactivity of the a-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis. These derivatives displayed log P values that suggest they should be well absorbed through biological membranes

Dopamine- and tyramine-based derivatives of triazenes. Activation by tyrosinase and implications for prodrug design

We report the synthesis of triazene derivatives, and their evaluation as substrates for tyrosinase. The conversion of prodrugs to the ortho-quinone was confirmed by LC-MS

The efficiency of C-4 substituents in activating the -lactam scaffold towards serine proteases and hydroxide ion

The presence of a leaving group at C-4 of monobactams is usually considered to be a requirement for mechanism-based inhibition of human leukocyte elastase by these acylating agents. We report that second-order rate constants for the alkaline hydrolysis and elastase inactivation by N-carbamoyl monobactams are independent of the pKa of the leaving group at C-4. Indeed, the effect exerted by these substituents is purely inductive: electron-withdrawing substituents at C-4 of N-carbamoyl-3,3-diethylmonobactams increase the rate of alkaline hydrolysis and elastase inactivation, with Hammett rho-I values of 3.4 and 2.5, respectively, which indicate the development of a negative charge in the transition-states. The difference in magnitude between these rho-I values is consistent with an earlier transition-state for the enzymatic reaction when compared with that for the chemical process. These results suggest that rate limiting step in elastase inactivation is the formation of the tetrahedral intermediate, and that beta-lactam ring-opening is not concerted with the departure of a leaving group from C-4. Monobactam sulfones emerged as potent elastase inhibitors even when the ethyl groups at C-3, required for interaction with the primary recognition site, are absent. For one such compound, a 1:1 enzyme-inhibitor complex involving porcine pancreatic elastase has been examined by X-ray crystallography and shown to result from serine acylation and sulfinate departure from the β-lactam C-4

Oxidation of tertiary benzamides by 5,10,15,20-tetraphenylporphyrinatoiron (III) chloride – tert-butylhydroperoxide

Tertiary benzamides are oxidized by the 5,10,15,20-tetraphenylporphyrinatoiron(III) chloride–ButOOH system at the α-position of the N-alkyl groups. The major products are N-acylamides, although small amounts of secondary amides, the products of dealkylation, are also formed. Plots of initial rate versus initial substrate concentration for these reactions are curved, suggesting formation of an oxidant–substrate complex. The reaction rates are almost insensitive to the substituent in the benzamide moiety, but there is a kinetic deuterium isotope effect of 5.6 for the reaction of the N,N-(CH3)2 and N,N-(CD3)2 compounds. Comparison of the reaction products from N-alkyl-Nmethylbenzamides reveals that, for all compounds studied except N-cyclopropyl-N-methylbenzamide, oxidation of the alkyl group is preferred, strongly so (by a factor of ca. 8) for N-allyl-N-methylbenzamide. In contrast to microsomal oxidation, there is no steric hindrance to oxidation of an isopropyl group. Thus, we propose that these reactions proceed via hydrogen atom abstraction to form an α-carbon-centred radical and we attribute the observed diminished reactivity of the N-cyclopropyl group to its known reluctance to form a cyclopropyl radical. Oxidation of N-methyl-N-(2,2,3,3-tetramethylcyclopropyl)methylbenzamide provides preliminary evidence for rearrangement of an intermediate radical. While it remains unclear how these reactions proceed directly to the N-acyl products, we have established that N-hydroxymethyl, N-alkoxymethyl and N-alkylperoxymethyl intermediates are not involved

Chemical and microsomal oxidation of tertiary amides: regio-and stereoselective aspects

The conformationally restricted tertiary amides N-methyl-2-pyrrolidone 6, N-methyl-2-piperidone 7 and N-methyl-ε-caprolactam 8 were oxidised by 5,10,15,20-tetraphenylporphyrinatoiron() chloride/tert-butyl hydroperoxide (TPPFe/ButOOH) and by phenobarbital-induced rat liver microsomes. The products were the N-demethylated lactams together with the analogous N-methylimides and norimides. For the TPPFe/ButOOH reaction ring oxidation is preferred to N-demethylation, paralleling the relative stabilities of the corresponding intermediate carbon-centred radicals as calculated by the AM1 semi-empirical method. In contrast, the microsomal reaction of the N-methyllactams strongly favours N-demethylation, demonstrating that hydrogen atom abstraction from the alkyl group Z to the amide carbonyl oxygen atom is preferred. The chiral tertiary amides N-methyl-N- (1-phenylethyl)benzamide 9 and N-methyl-5-phenyl-2-pyrrolidone 10 were also oxidised by TPPFe/ButOOH and by phenobarbital-induced rat liver microsomes. Using TPPFe/ButOOH, loss of the secondary alkyl group of 9 is preferred by a factor of ca. 6. Similarly, ring oxidation of 10 is favoured over demethylation by a factor of 9. For the microsomal reaction of (R)-9 dealkylation is preferred over demethylation by a factor of 1.7, whereas for (S)-9 demethylation is favoured by a factor of 1.25. For the microsomal reaction of (R)-10 and (S)-10 ring oxidation at the 5-position of the pyrrolidone ring is preferred over demethylation by factors of ca. 4 and 9 for the two isomers, respectively, and the (S)-enantiomer undergoes ring oxidation 2–3 times more readily than the (R)-enantiomer. For both 9 and 10 there is negligible stereochemical influence of the chiral centre upon the N-demethylation reaction. The results show that the stereochemical preference of the microsomal N-dealkylation reaction is modest

Structure-activity studies of the binding of the flavonoid scaffold to DNA

Background: Flavonoids have been shown to have a wide variety of biological activities and proven to be good scaffolds for the design of DNA-binding agents as anticancer therapeutics. Materials and Methods: In structure-activity relationship studies, flavonoid derivatives were designed and synthesised through various organic synthesis protocols, resulting in novel or previously described molecules. These were studied by UV-Vis absorbance and fluorescence spectroscopy as well as competition dialysis for their binding to DNA isoforms. Their cytotoxic potential was assessed using MTS assays on MCF-7 breast cancer and CCRFCEM leukaemia cell lines. Results and Conclusion: Introduction of moieties such as chloride, nitrogen, acetoxy and methoxy groups did not help to improve binding affinity, but introduction of tertiary amines improved the binding 1,000-fold due to an improved interaction of the compound with the nucleic acid; replacement of oxygen by sulphur increased the binding 7-fold, possibly because sulphur being less electronegative than oxygen would allow the electrons of the molecule to interact more strongly with the nucleic acid. Inhibition of growth by 50% (IG50) values were moderate in breast and leukaemia cancer cell lines possibly due to the flavonoids interacting with other cellular components besides the nucleic acids

The mechanism of hydrolysis of aryl ether derivatives of 3-hydroxymethyltriazenes

1-Aryl-3-aryloxymethyl-3-methyltriazenes hydrolyse to the corresponding anilines and phenols by specific-acid-catalysed, general-acid-catalysed and pH-independent mechanisms. All compounds studied exhibit specific- and general acid catalysis, though for 5a general acid catalysis was not observed below a pH of approximately 4, while for compounds 5e,f, such catalysis was absent above a pH of approximately 5. The pH-independent pathway is observed only for those compounds, 5d–f, that contain good aryloxy nucleofugic groups. The specific-acid-catalysed pathway is supported by a solvent deuterium isotope effect (SDIE) of 0.64, consistent with a mechanism involving protonation of the substrate followed by rate-determining unimolecular decomposition of the protonated species. The kH+ values gave rise to a Hammett ρ value of –0.93, reflecting the competing effect of the substituents on the protonation of the substrate and the cleavage of the aryl ether. Correlation of kH+ with the pKa of the phenol leaving group affords a βlg of 0.3. Decomposition of the protonated intermediate proceeds via a triazenyliminium ion that can be trapped by methanol. The general acid- catalysed process exhibits an SDIE of 1.43 and Hammett ρ values of 0.49, 0.84 and 1.0 for reactions catalysed by chloroacetic, formic and acetic acids, respectively. Correlation of kA with the pKa of the acid gave Brønsted α values that diminish from 0.6 for O-aryl systems that are poor nucleofuges (5a,b) to 0.2 for the best nucleofuge (5f), reflecting the different extents of proton transfer required to expel each phenol. Compounds containing powerful nucleofuges exhibit a pH-independent reaction that has an SDIE of 1.1, a Hammett ρ value of 3.4 and a Brønsted βlg value of 1.4. These imply a mechanism involving displacement of the aryloxide leaving group to form a triazenyliminium ion intermediate that again was trapped as a methyl ether

Kinetics and mechanism of hydrolysis of N-acyloxymethyl derivatives of azetidin-2-one

The pH-independent, acid-catalyzed and base-catalyzed hydrolyses of N-acyloxymethylazetidin-2-ones all occur at the ester function. The pH-independent hydrolysis involves rate-limiting alkyl C-O fission and formation of an exocyclic â-lactam iminum ion. This iminium ion is then trapped by water at the exocyclic iminium carbon atom, rather than at the â-lactam carbonyl carbon atom, to form the corresponding N-hydroxymethylazetidin-2-ones. Calculations carried out at the B3LYP/6-31+G(d) level of theory also support that nucleophilic attack by water takes place at the exocyclic carbon rather than at the â-lactam carbonyl carbon of the iminium ion. The mechanism for the acid-catalyzed pathway involves a preequilibrium protonation, probably at the beta-lactam nitrogen, followed by rate-limiting alkyl C-O fission with formation of an exocyclic iminum ion. The basecatalyzed hydrolysis involves rate-limiting hydroxide attack at the ester carbonyl carbon. These results imply formation of a â-lactam system containing a positively charged amide nitrogen atom that hydrolyzes via a pathway that preserves the â-lactam structure in the product and provide further evidence that cleavage of the â-lactam C-N bond is not as facile as is commonly imagined

Hydrolysis of aryl N-methyl-N-arenesulfonylcarbamates

Tertiary sulfonylcarbamates 1 were prepared by reaction of a sulfonamide anion with aryl chloroformates. These previously unreported compounds hydrolyse in aqueous media to the parent sulfonamide and phenol. The pH–rate profile shows both spontaneous and base-catalysed processes. The reaction is also catalysed by buffers. Kinetic data for the hydrolysis of these compounds by HO are best interpreted in terms of a mechanism involving rate-limiting formation of a tetrahedral intermediate from nucleophilic attack of hydroxide ion at the carbamate carbonyl carbon atom. For the 4-nitrophenylsulfonyl compound 1h decomposition of the tetrahedral intermediate appears to be rate-limiting with the sulfonamide anion, rather than the phenoxide, functioning as the leaving group. The buffer catalysed process is consistent with general base-catalysed attack of water at the carbamate carbonyl carbon atom