Eduarda Rosa

The microsomal demethylation of N,N-dimethylbenzamides: Substituent and kinetic deuterium isotope effects

The metabolism of N,N-dimethylbenzamides by phenobarbital-induced rat liver microsomes results in the formation of N-methylbenzamides and formaldehyde. The reaction proceeds via the formation of an intermediate N-hydroxymethyl-N-methylbenzamide, which, for the microsomal oxidation of N,N-dimethylbenzamide, was isolated and characterized. Confirmation of the N-hydroxymethyl-N-methylbenzamide was obtained by its independent synthesis from N-methylbenzamide and formaldehyde. The intermolecular kinetic deuterium isotope effects for the reaction are 0.9 (+/- 0.1) for Vmax and 1.4 (+/- 0.1) for Vmax/Km. The intramolecular kinetic deuterium isotope effect, determined from the relative amounts of N-methylbenzamide and N-trideuteriomethylbenzamide formed in the microsomal demethylation of N-trideuteriomethyl-N-methylbenzamide, is 6.0 +/- 0.3. There is no correlation of Vmax or Vmax/Km with the substituent in the aromatic ring, nor with the calculated ionization potentials of the benzamides. The results are interpreted in terms of a mechanism in which the benzamide undergoes direct hydrogen atom abstraction to form a carbon centred radical. This carbon centred radical subsequently forms an N-hydroxymethyl-N-methylbenzamide that decomposes to formaldehyde and an N-methylbenzamide. Semi-empirical AM1 self consistent field molecular orbital calculations identify that loss of a hydrogen atom from the E-methyl group is thermodynamically more favourable than from the Z-methyl group by ca. 5 kJ/mol.

Oxidation of the methyl groups of N,N-dimethylbenzamides by a cytochrome P450 mono-oxygenase model system

Abstract Oxidation of N,N -dimethylbenzamides to the corresponding N -formyl- N -methylbenzamides using tetraphenylporphyrinato-iron(III)-Bu t OOH is independent of the substituent in the aryl ring of the benzamide group and subject to a kinetic deuterium isotope effect of 5.6. These results are consistent with a mechanism involving direct hydrogen atom abstraction from the substrate.

Triazene drug metabolites. Part 4. Kinetics and mechanism of the decomposition of 1-aryl-3-benzoyloxymethyl-3-methyltriazenes in mixed aqueous–organic solvents

Kinetic studies for the hydrolysis of 1-aryl-3-benzoyloxymethyl-3-methyltriazenes to 1-aryl-3-hydroxymethyl-3-methyltriazenes in mixed aqueous–organic media are reported. Reactions are first-order in the benzoyloxymethyltriazene, and are independent of pH above pH 8. Below pH8, specific acid catalysis is observed. No nucleophilic catalysis is detected at any pH. The observed first-order rate constant, kobs, vary with the substituent in both the 1-aryl and benzoyl rings. Hammett σ values of 1.28 and 1.41 are obtained for substituents in the benzoyl group in 50% MeCN–H2O and 60% dioxane–H2O respectively. A Hammett ρ value of –1.84 is obtained in 50% MeCN–H2O for substituents in the 1-aryl ring. Observed first-order rate constants also vary with the composition of aqueous dioxane mixtures and a linear correlation between logkobs and the Grunwald–Winstein Y parameter is found to give a slope of 0.99. The solvent deuterium isotope effect, kH2O/kD2O, is 1.26 for the 4-methoxybenzoyl derivative. Values of the activation parameters are ΔH‡ca. 80 kJ mol–1 and ΔS‡ca.–5 J K–1 mol–1. The data are best interpreted in terms of a unimolecular ionisation of the benzoyloxymethyltriazene to form a iminium cation and a benzoate anion. Hydroxymethyltriazene formation results from the capture of the intermediate iminium ion by water. Consistent with this mechanism, a common ion effect of the benzoate anion is observed, and the benzoate ion is ca. 75 times more effective than water at trapping the iminium ion.

Kinetics and mechanism of nitrosation of clonidine: a bridge between nitrosation of amines and ureas

Nitrosation of clonidine has been studied kinetically both in acid medium (with nitrous acid) and in basic medium (with 2,2-dichloroethyl nitrite). The reactive form in acid medium was found to be the protonated clonidine (pKa 8.18). The absence of catalysis by halides or thiocyanate, the existence of general base catalysis, and the measured solvent isotope effect all indicate that the reaction mechanism is different from that for the N-nitrosation of amines. Specifically, kinetic results indicate that the attack of the nitrosating agent on the substrate is not the rate determining step of the process, and suggest a mechanism that shows parallels with that found for ureas. However, in slightly basic medium, the reaction of clonidine with the alkyl nitrite occurs through the free base form of clonidine, as shown by the influence of acidity upon the reaction rate. In this case, the kinetic behaviour is similar to that exhibited by amines.

Triazene drug metabolites. Part 13. The decomposition of 3-acyl-3-alkyl-1-aryltriazenes in aqueous sulfuric acid

The hydrolysis of 1-aryl-3-acyl-3-methyltriazenes in aqueous sulfuric acid is described. The 3-formyl derivative undergoes an acid-catalysed deacylation reaction, characterised by a monotonic rise in the pseudo-first-order rate constant, k0 with increasing acidity, solvent deuterium isotope effects, kH2SO4/kD2SO4, of 0.9 (at 0.95 mol dm–3 H2SO4) and 0.8 (at 2.85 mol dm–3 H2SO4) and an entropy of activation of –80 J mol–1 K–1. The 3-alkanoyl derivatives also undergo acid-catalysed decomposition involving cleavage of either the N3–C acyl bond or the N2–N3 triazene bond. Below 3 mol dm–3 H2SO4, only acyl bond cleavage is observed. At higher acidities the extent of N2–N3 bond cleavage increases. The reaction is characterised by (i) solvent deuterium isotope effects of ca. 0.6 at 2 and 5 mol dm–3 H2SO4 and ca. 0.4 at 8 mol dm–3 H2SO4, (ii)ΔS‡ values of –6.7 and –51 J mol–1 K–1 at 2 and 6.1 mol dm–3 H2SO4, respectively, (iii) Hammett ρ values for the substituent in the triazene N-aryl ring of –0.7 and –0.9 at 3 and 9 mol dm–3 H2SO4, respectively, and (iv) an increase in reactivity with electron donating ability of the alkyl substituent of the acyl group. The 3-trifluoroacetyl triazenes are subject to solvolysis of the neutral, as well as the protonated, substrate. The hydrolysis of the neutral substrate involves N–acyl bond cleavage and is characterised by a solvent deuterium isotope effect, kH2O/kD2O, of 2.4, and a Hammett ρ value of +0.8 for the substituent in the N-aryl ring. The reactivity of the neutral substrate diminishes with increasing acidity until 6 mol dm–3 H2SO4, beyond which acid-catalysed N–acyl bond cleavage predominates, for which the solvent isotope effect, kH2SO4/kD2SO4, is 0.8 and the Hammett ρ value –0.5. The 3-aroyl substrates suffer acid-catalysed decomposition, the extent of the N2–N3 bond cleavage process being greater than for the N-alkanoyl counterparts. The reactions are rationalised in terms of a process that involves pre-equilibrium protonation of the substrate either at the N1 triazene atom or the amide oxygen atom, followed by subsequent decomposition of the protonated substrate via either N3–C bond cleavage, involving attack of water at the amide carbonyl, or unimolecular N2–N3 bond cleavage. The relative extents of the N3–C and N2–N3 bond cleavage processes depend on the reactivity of the acyl group and the water activity; the higher the water activity and the more reactive the acyl group, the more deacylation is favoured.

Acyloxymethyl as a drug protecting group. Kinetics and mechanism of the hydrolysis of N-acyloxymethylbenzamides

Acyloxymethyl derivatives of secondary and tertiary amides undergo hydrolysis via. acid-catalysed, base-catalysed and pH-independent processes. The pH-independent pathway involves rate-limiting iminium ion formation and is characterised by the following: a Hammett ρ value for the substituent in the benzamide moiety of ca.–1.2 for both types of substrate; the absence of general-base or nucleophilic catalysis; a common benzoate ion effect; a solvent deuterium isotope effect, kobsH2O/kobsD2O, of ca. 1.6; ΔS‡ values of –4 and –12 J K–1 mol–1 for secondary and tertiary substrates respectively; and higher reactivity of the tertiary amides over their secondary counterparts. The acid-catalysed process involves protonation of the substrate followed by iminium ion formation, and is characterised by the following: a Hammett ρ value of ca.–1.5 for the substituent effect of the benzamide moiety; a solvent deuterium isotope effect of ca. 0.4; a monotonic rise in the pseudo-first-order rate constant kobs with increasing [H2SO4]; ΔS‡ values > 0 J K–1 mol–1; higher reactivity of the tertiary substrates over their secondary counterparts; and a value of 0.85 for the Bronsted coefficient, βIg for the carboxylate nucleofuge. The base-catalysed hydrolysis of tertiary substrates involves normal ester hydrolysis via. acyl–oxygen bond cleavage, and is characterised by a Hammett ρ value of +0.38, a solvent deuterium isotope effect, kOH–/kOD–, of 0.85, and a ΔS‡ value of –96 J K–1 mol–1. The corresponding base-catalysed process for the secondary substrates involves imine formation via an E2 elimination reaction. The secondary acyloxymethylamides are some 7 × 104 times more reactive than their tertiary counterparts in the base-catalysed region. Hammett ρ values of +1.1 and +0.6 are obtained for the substituents in the ester and amide moieties, respectively. Buffer catalysis is observed, and the value of ca. 0.5 for the Bronsted β coefficient identifies the amide proton as approximately 50% transferred to the buffer species in the transition state.Heats of formation, ΔHf, calculated using the AM1 SCF MO package reveal that iminium ion formation is thermodynamically equi-energetic for cyclic and acyclic systems. Iminium ion formation from tertiary substrates is favoured by ca. 25 kJ mol–1 over the corresponding secondary analogues.

Synthesis of S-cysteinyl, S(N-acetylcysteinyl) and S-glutathionyl conjugates op N-hydroxymethyltriazenes

Abstract The title compounds are made by direct condensation of N -hydroxymethyltriazenes with cysteine, N -acetylcysteine or glutathione in trifluoroacetic acid which acts as both acid catalyst and solvent.

Triazene drug metabolites: base catalysed formation of N-alkyltriazenes from N-hydroxymethyl-N-alkyltriazenes

Abstract Decomposition of the title compounds is base catalysed and requires the presence of a heteroatom - hydrogen bond in the catalyst and depends on the pKa of the base; aminomethylation of the base is not observed.

The carbohydrate-catalysed hydrolysis of cephalosporins

The decomposition of cephaloridine, cephtazidime, cephaclor, cephalothin, cephaloglycin, cephalexin and cephradine catalysed by glucose, galactose, maltose, sucrose, mannitol and α-methylglucoside in aqueous solutions of pH 9–11 is reported. The rate of decomposition depends upon the structure of the cephalosporin, the more important feature being the electron withdrawing nature of the substituent attached to the exocyclic C-3 methylene carbon atom. At pH 9.5 a Hammett ρ1 value of 2.9 is obtained from a plot of log k2, the apparent second-order rate constant, versusσ1 for the glucose catalysed reaction. At pH ca. 9 plots of k∘versus catalyst concentration appear linear, but above pH ca. 10 such plots are curved. The extent of catalysis increases at higher pH values. Carbohydrates with a hemiacetal OH group are significantly better catalysts than those that do not contain this functionality, though catalysis by non-hemiacetal groups is evident. The difference in reactivity between the two types of OH at pH 9.5 lies between 10 and 15-fold. The results are interpreted in terms of a mechanism that involves nucleophilic catalysis via the hemiacetal anion of the carbohydrate. Curvature of the koversus[catalyst] plots is explained by the formation of non-catalytic dimer between the anionic and neutral forms of the catalyst.

Triazene drug metabolites. Part 11. Synthesis of S-cysteinyl and related derivatives of N-hydroxymethyltriazenes

S-Cysteinyl-, S-(N-acetyl)cysteinyl-, S-glutathionyl and some related thioether derivatives of anticancer triazenes have been synthesised in high yield. The synthesis involves reaction between N-alkyl-N-hydroxymethyltriazenes with cysteine, N-acetylcysteine, glutathione or appropriate thiol in trifluoroacetic acid. The cysteinyl derivative is not a substrate for a mammalian β-lyase. Consequently the cysteinyl, N-acetylcysteinyl and glutathionyl derivatives lack anticancer activity against the Walker, L1210 and V79 tumour cell lines, mutagenic activity against Salmonella TA 100 and TA 2638 cell lines and cytotoxicity against renal proximal tubule cells.