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O -Alkyl and O -aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O -alkyl and O -aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O -aryl carbamates being ca. 10 6 –10 10 more reactive than their O -alkyl counterparts. In human plasma O -alkyl carbamates were stable, whereas in contrast their O -aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O -aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O -aryl and O -alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei , Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n -hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O -alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.

S-Alkylation of an N-protected cysteine ester with a range of ω-iodoalkyl heterocycles affords 11 novel non-proteinogenic heterocyclic amino acids having a sulfur atom in the backboneheterocycle linker.

Carbamate and ester derivatives of the 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) scaffold react readily with thiols via a Michael addition at rates not significantly affected by the nature of the carboxylic or carbamic acid leaving group. These Michael acceptors are irreversible inhibitors of the cysteine proteases papain and human liver cathepsin B, displaying first-order kinetics with respect to inhibitor concentration. In contrast, none of the Bsmoc derivatives inhibited porcine pancreatic elastase, a serine protease. (C) 2006 Elsevier Ltd. All rights reserved.

Human leukocyte elastase (HLE) is a serine protease that very efficiently degrades various tissue matrix proteins such as elastin. The imbalance between HLE and its endogenous inhibitors leads to excessive elastin proteolysis and is considered to be responsible for the onset of chronic obstructive pulmonary disease (COPD). A novel series of C-3-, C-4-, and N-1-substituted azetidin-2-ones were prepared as potential mechanism-based inhibitors of HLE to restore the protease/antiprotease imbalance. N-Acyloxyalkylazetidin-2-ones, 4, and their carbamate counterparts, 5, are weak HLE inhibitors, being 5 times less active than their bicyclic oxazolidin-2,4-dione-substituted analogues, 6, containing an electron-withdrawing substituent at C-4. Compounds 6 containing a C-4 substituent exist as two diastereomeric pairs of enantiomers, each pair presenting similar inhibitory activity against HLE. Comparative docking experiments with the C-4-substituted oxazolidin-2,4-dione inhibitors 6 suggest that only the 4R,5‘S and...

The synthesis of imidazolidin-4-one derivatives of primaquine as potential antimalarial agents is described. The target compounds were synthesized in three steps: (i) condensation of (±)-primaquine with N α -protected amino acids, (ii) removal of the N α -protecting group, and (iii) reaction of the N-acylprimaquine with a carbonyl compound: acetone, three cyclic ketones and veratraldehyde. Using 2-formylbenzoic acid in the third step afforded 1 H -imidazo[2,1- a ]isoindole-2,5(3 H ,9b H )-diones. All products were isolated in good to excellent yields. Whereas imidazolidin-4-ones were formed as mixtures of all possible diastereomers in equal amounts, 1 H -imidazo[2,1- a ]isoindole-2,5(3 H ,9b H )-diones were produced in a stereoselective fashion. The compounds hydrolyse very slowly ( t 1/2 5–30 d) in pH 7.4 buffer to release primaquine. These primaquine derivatives are being submitted to biological assays, and preliminary results of their antimalarial activity are quite encouraging.

Tertiary benzamides are oxidized by the 5,10,15,20-tetraphenylporphyrinatoiron(III) chloride-(BuOOH)-O-t system at the alpha-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-N-methylbenzamides 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 alpha-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.

N -(2-iodoethyl) and N -(3-iodopropyl)pyrimidines and purines undergo stereoselective conjugate radical addition with an optically active oxazolidinone acceptor to give syn -adducts that can be converted into amino acids carrying pyrimidine and purine (nucleobase) side chains.

An orthogonally protected 3-(1-aminoalkyl)isoxazole-4-carboxylic acid has been prepared by 1,3-dipolar cycloaddition of a suitably protected α-aminonitrile oxide with an enaminoester dipolarophile; this protected amino acid has been deprotected and coupled independently at either the C- or N-terminus to produce pseudopeptide segments as peptide mimetics that contain a cis -amide bond replacement.

N-(But-3-enyl)-N-methylbenzamide 14a undergoes microsomal oxidation by rat liver microsomes to yield both N-methyl- and N-(but-3-enyl)benzamides 18a and 19, the products of N-dealkylation. Cyclic products, that could be derived from a carbon-centred radical formed by hydrogen atom abstraction from the N-methyl group, were not observed. When generated independently, this carbon-centred radical underwent cyclisation, the 5-exo-trig mode being preferred to 6-endo-trig by a factor of 5. In contrast, N-(but-3-ynyl)-N-methylbenzamide 15 undergoes microsomal oxidation to yield the products of dealkylation 18a and 23 and also N-benzoylpiperidone 24. Dealkylation is preferred by factor of 3 and the piperidone accounts for ca. 45% of the reaction at the N-methyl group. Piperidone formation is consistent with the generation of a carbon-centred radical α- to the amide nitrogen atom during dealkylation and implies that cyclisation proceeds preferentially via the 6-endo-dig mode. Generated independently the radical undergoes cyclisation by both 5-exo-dig and 6-endo-dig modes, the former being favoured by a factor of 10. Similarly, N,N-dimethylacrylamide 26 and N-methyl-N-(3-pyridyl)acrylamide 27 undergo microsomal oxidation to form, via the 5-endo-trig cyclisation mode, 3-hydroxy-N-methyl-2-pyrrolidone 33 and 3-hydroxycotinine34, respectively, confirming the intermediacy of a carbon-centred radical in the dealkylation process.Attempts to trap N-acyliminium ions during microsomal dealkylation failed. Thus, although N,N-dimethylaniline 35 reacts in the presence of NaCN to form N-cyanomethyl-N-methylaniline 37 (NuCN), N,N-dimethylbenzamide undergoes dealkylation without forming N-cyanomethyl-N-methylbenzamide. Similarly, microsomal reaction of N,N-dimethylaniline in the presence of NaBD4 gives rise to multiple incorporation of deuterium atoms into the methyl groups of the starting material, whereas N,N-dimethylbenzamide undergoes dealkylation but with no such deuterium incorporation into the starting material. Further, microsomal oxidation of N,N-dimethylsalicylamide 38 yields N-methylsalicylamide 40 with no evidence for the formation of N-methyl-2,3-dihydro-4H-1,3-benzoxazin-4-one 39, the potential product of intramolecular cyclisation of the phenolic oxygen atom onto the putative N-aroylmethylene iminium ion. p

Purpose. Dipeptide derivatives of primaquine (PQ) with reduced oxidative deamination to the inactive metabolite carboxypnmaquine were synthesized and evaluated as a novel class of transmission-blocking antimalarials.

Tertiary acyloxymethylsulfonamides undergo hydrolysis via pH-independent and acid- and base-catalysed processes. Reactions are also buffer catalysed for buffer species with pKa values > ca. 10.5. For the pH-independent pathway, hydrolysis takes place via formation of an N-sulfonyl iminium ion. There is no general-base or nucleophilic catalysis in this region. The mechanism of the acid-catalysed process involves pre-equilibrium protonation of the substrate followed by iminium ion formation. General-acid catalysis is not observed. The base-catalysed pathway involves the normal BAc2 mechanism of ester hydrolysis. The buffer-catalysed reaction gives rise to a curved Bronsted plot, with β values of 1.6 and 0.25 for nucleophiles with pKa values 13, respectively. This is indicative of nucleophilic catalysis associated with a change in rate-limiting step from formation of the tetrahedral intermediate for buffer species with pKa > 13 to decomposition of the tetrahedral intermediate for buffer species with pKa

The in vitro metabolism of N,N-dialkylamides by phenobarbital-induced rat liver microsomes yields an N-alkylamide and the corresponding aldehyde. Although, N-hydroxymethyl-N-alkylamide intermediates can be detected from N-methyl-N-alkylamides, no N-hydroxyalkyl-N-alkylamide inter- mediates are detected from the N,N-dialkylamide substrates. Vmax values were independent of amide structure, whereas V max K m values were dependent on the lipophilidty of the N,N-dialkylbenazamide studied. These results suggest that diffusion of substrate into the membrane-bound enzyme active site limits the rate of the microsomal oxidation of the amides. Metabolism of N-alkyl-N-methylamides reveals identical values of Vmax for demethylation and dealkylation. Values of V max K m for demethylation depend upon the lipophilicity of the N-alkyl group, whereas V max K m values for dealkylation appear to be dependent upon the steric bulk of the alkyl group, particularly around the α-carbon. Moreover, V max K m values for demethylation are larger than for dealkylation, implying the reactions are under kinetic control. Comparison of the kinetic data with theoretical AMI semi-empirical molecular orbital calculations suggests a mechanism involving formation of a carbon-centred radical. Use of an N-cyclo-propylmethylbenzamide substrate to trap such a radical failed, presumably because oxygen rebound is faster than radical rearrangement. An N-cyclopropylamide substrate did not undergo metabolism of the cyclopropyl ring, consistent with carbon-centred radical, but not nitrogen radical cation, formation.

The synthesis, inhibitory activity and mode of action of oxazolidine-2,4-diones against porcine pancreatic elastase, here used as a model for human neutrophil elastase, are reported. The nature of N-substitution at the oxazolidine-2,4-dione scaffold has large effect on the inhibitory potency against elastase. N-Acyl and N-sulfonyloxazolidine-2,4-diones emerged as potent pseudo-irreversible inhibitors, displaying high second-order rate constants for PPE inactivation. The title compounds were also shown to be potent inhibitors of human neutrophil elastase (HNE) and proteinase-3, and weak inhibitors of human cathepsin G. The results herein presented show that the oxazolidine-2,4-diones represent a new promising class of serine protease inhibitors.

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.

A series of N-acyloxymethyl- and N-aminocarbonyloxymethyl derivatives of 2-azetidinones, 3, with different substituent patterns at the beta-lactam C-3 and C-4 positions, were designed as potential mechanism-based inhibitors for human leukocyte elastase and found to exhibit inhibitory potency and selectivity for the enzyme.

N -(2-iodoethyl) and N -(3-iodopropyl)pyrimidines and purines undergo stereoselective conjugate radical addition with an optically active oxazolidinone acceptor to give syn -adducts that can be converted into amino acids carrying pyrimidine and purine (nucleobase) side chains.

Molecular modeling studies carried out with experimental DNA models with the sequence d[AG(3)(T(2)AG(3))(3)] suggest that the introduction of a net positive charge onto the side chain of a series of fluorenone carboxamides can improve G-quadruplex binding. The terminal morpholino moiety was replaced with a novel N-methylmorpholinium cation starting from two 4-carboxamide compounds. A different substitution on the fluorenone ring was also investigated and submitted to the same quaternarization process. All compounds were analyzed for their DNA binding properties by competition dialysis methods. In vitro antiproliferative tests were carried out against two different tumor cell lines. Docking experiments were conducted by including all four known human repeat unit G-quadruplex DNA sequences (27 experimentally determined conformations) against the most active fluorenone derivatives. The results of theoretical, biophysical, and in vitro experiments indicate two novel derivatives as lead compounds for the development of a new generation of G-quadruplex ligands with greater potency and selectivity.

We report here a simple entry into naphtho[2,3-d]isoxazole-4,9-dione system containing a EWG in position 3 using the readily available 2,3-dichloro-1,4-naphthoquinone and nitromethyl derivatives in the presence of base. Antifungal activity of synthesised naphthoquinones was evaluated against ATCC and PYCC reference strains of Candida. The results suggest that the naphtho[2,3-d]isoxazole-4,9-dione scaffold has the potential to be developed into novel and safe therapeutic antifungal agents.

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 V max and 1.4 (±0.1) for V max / K m . 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 V max or V max / K m 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 AMI 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.

A range of triazene derivatives were synthesized and investigated as prodrug candidates for melanocyte-directed enzyme prodrug therapy (MDEPT). The prodrugs contained a tyramine or dopamine promoiety required for tyrosinase activation and this was joined via a urea functional group to the cytotoxic triazene. The stability of each of the prodrugs in phosphate buffer, human plasma and in mushroom tyrosinase is discussed. The identification of the main peak formed after the tyrosinase reaction was attempted by LC-MS and the conversion of prodrug to the quinone was confirmed.

A new class of inhibitors 4-oxo-beta-lactams (azetidine-2,4-diones), containing the required structural elements for molecular recognition, inhibit porcine pancreatic elastase (PPE) but show a dramatically lower reactivity toward hydroxide compared with the analogous inhibitors 3-oxo-beta-sultams. Inhibition is the result of acylation of the active site serine and electron-withdrawing substituents at the N-(4-aryl) position in 3,3-diethyl- N-aryl derivatives increasing the rate of enzyme acylation and generating a Hammett rho-value of 0.65. Compared with a rho-value of 0.96 for the rates of alkaline hydrolysis of the same series, this is indicative of an earlier transition state for the enzyme-catalyzed reaction. Docking studies indicate favorable noncovalent interactions of the inhibitor with the enzyme. Compound 2i, the most potent inhibitor against PPE, emerged as a very potent HLE inhibitor, with a second-order rate for enzyme inactivation of approximately 5 x 10 (5) M (-1) s (-1).

The metabolism of 1-aryl-3,3-dimethyltriazenes by phenobarbital-induced rat liver microsomes results in the formation of 1-aryl-3-methyltriazenes and formaldehyde. Intermolecular kinetic deuterium isotope effects for the reaction are found to be 1.0 (±0.03) in both V max and V max / K m respectively. The intramolecular kinetic deuterium isotope effects in V max and V max / K m are found to be 4.8 (±0.05). There is no correlation of V max or V max / K m with calculated ionization potentials of the triazenes. For 3-ethyl-3-methyltriazene comparison of values of V max and V max / K m for ethyl VS methyl loss give rise to values of 3.68 in V max and 2.02 in V max / K m . Thus, loss of an ethyl group is favoured. The results are discussed in terms of a mechanism in which the triazene undergoes direct hydrogen atom abstraction to form a carbon centred radical. This radical subsequently forms a hydroxymethyltriazene that collapses to formaldehyde and a monomethyltriazene.

A series of 1,4-naphthoquinone derivatives diversely substituted at C-2, C-3, C-5 and C-8, prepared by reaction of amines, amino acids and alcohols with commercial 1,4-naphthoquinones, has been evaluated against papain and bovine spleen cathepsin B. These 1,4-naphthoquinone derivatives were found to be irreversible inhibitors for both cysteine proteases, with second-order rate constants, k(2), ranging from 0.67 to 35.4 M-1 s(-1) for papain, and from 0.54 to 8.03 M-1 s(-1) for cathepsin B. Some derivatives display a hyperbolic dependence of the first-order inactivation rate constant, k(obs) with the inhibitor concentration, indicative of a specific interaction process between enzyme and inhibitor. The chemical reactivity of the compounds towards cysteine as a model thiol is dependent on the naphthoquinone LUMO energy, whereas papain inactivation is not. The 1,4-naphthoquinone derivatives are inactive against the serine protease, porcine pancreatic elastase. (c) 2007 Elsevier Ltd. All rights reserved.

A series of fluorenone-carboxamide compounds was analyzed with regard to DNA binding properties by UV spectroscopy and competition dialysis methods. The morpholino derivative 10 provided interesting results in terms of affinity and specificity toward the DNA G-tetraplex structures. Interactions against this target were evaluated by a comparative molecular modeling study in agreement with the experimental data, proposing a model for the rational design of new agents with potent and selective DNA tetraplex binding properties.

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 pK(a) 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 p(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 p(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 the 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 beta- lactam C-4.

Amino acids carrying aromatic carbo- and heterocycles in the side chain, such as naphthyl-, biphenyl- and pyridylalanines, have been prepared by alkylation of a glycine enolate with a haloalkyl carbocycle or heterocycle, with enantiomeric excess up to 87% using the ephedrine amide protocol.

N-Alkylation of 4,5-dihydroimidazoles with alkene-containing bromomethyl ketones and treatment of the so-formed 4,5-dihydroimidazolium ions with DBU gives rise to an intramolecular 1,3-dipolar cycloaddition reaction that affords (via a reaction cascade involving eliminative ring-opening, recyclisation and prototropic tautomerism) unexpected hexahydropyrrolo[1,2,3-de]quinoxaline products. Steric bulk in both the dihydroimidazole and the dipolarophile allows isolation of an imidazo[1,2-a]indole, the initial product of cycloaddition. When the bromomethyl ketone contains no other functionality, or when cycloaddition is inhibited due to steric constraints, the dihydroimidazolium ion undergoes ring-opening hydrolysis followed by recyclization of the exposed amino ketone to afford either 3-alkyl-1-formylpiperazine-2-ones or 3-aryl-1-formyl-1,4,5,6-tetrahydropyrazines.

The synthesis of phosphonate derivatives of N-phenyl- and N-benzyl-gamma- and delta-sultams, and their application in the Wittig-Horner reaction with N-Boc-L-phenylalanine aldehyde to afford E- and Z-isomers, are described. These compounds were further processed to provide five dipeptide vinyl sultams, which were found to be inactive against papain at concentrations up to 50 microM. In contrast, vinyl sultams demonstrated weak activity against recombinant falcipain-2 and Plasmodium falciparum W2.

In contrast to peptide-based imidazolidin-4-ones, those synthesized from N-(alpha-aminoacyl) derivatives of the antimalarial drug, primaquine and ketones are unexpectedly stable in pH 7.4 at 37 degrees C. The kinetics of hydrolysis of primaquine-based imidazolidin-4-ones were investigated in the pH range 0.3-13.5 at 60 degrees C. The hydrolysis to the parent alpha-aminoacylprimaquine is characterized by sigmoidal-shaped pH-rate profiles, reflecting the spontaneous decomposition of both unionized and protonated (at N-1) forms of the imidazolidin-4-one. The kinetically determined pK(a) values are ca. 3.6-4.0, i.e., 4 pKa units lower than those of amino acid amides, thus implying that hydrolysis of imidazolidin-4-ones at pH 7.4 involves the unionized form. Reactivity of this form decreases with the steric crowding of the amino acid alpha-substituent. In contrast, the rate constant for the spontaneous decomposition of the unionized form increases sharply for imidazolidin-4-ones derived from cyclic ketones, an observation that can be explained by the I-strain (internal strain) effect. These results are consistent with a mechanism of hydrolysis involving an S(N)1-type unimolecular cleavage of the imidazolidin-4-one C2-N3 bond with departure of an amide-leaving group. The mechanism for the decomposition of the protonated imidazolidin-4-one is likely to involve an amide-carbonyl oxygen protonated species, followed by the C2-N3 bond scission, as supported by computational studies. The results herein presented suggest that imidazolidin-4-ones derived from simple N-alkyl alpha-aminoamides are too stable and therefore, may be useful as slow drug release prodrugs.

Imidazolidin-4-one derivatives of primaquine were synthesized as potential double prodrugs of the parent drug. The title compounds inhibit the development of the sporogonic cycle of Plasmodium berghei, affecting the appearance of oocysts in the midguts of the mosquitoes. The imidazolidin-4-ones are very stable, both in human plasma and in pH 7.4 buffer, indicating that they are active per se. Thus, imidazolidin-4-ones derived from 8-aminoquinolines represent a new entry in antimalarial structure-activity relationships.

N,N-dimethylformamide (DMF), an organic solvent widely used in industry, is bioactivated by cytochrome P450 (P450) to reactive metabolites which are believed to be responsible for the hepatotoxicity observed in animals and humans. A decrease of the activating enzyme has been reported in rats treated with DMF, although the specific P450 isoform(s) involved and the nature of the reactive species responsible for this and the other toxic effects are still being investigated. In the present work, the effect of DMF and of the structurally related N,N-dimethylacetamide (DMAc) on the activating enzyme and the nature of the reactive species involved in the mechanism of P450 inactivation by the two chemicals were investigated in vitro. Incubation of liver microsomes from pyridine-induced rats with either substrate resulted in a dose-dependent (0-20 mM) loss of P450 (up to 28 and 24% for DMF and DMAc, respectively), microsomal haem (up to 24 and 20% for DMF and DMAc, respectively), but not protoporphyrin IX content. Moreover, bubbling of CO through the incubation mixture gave almost complete protection against substrate-dependent P450 inactivation, and the spin trapping agent N-tert-butyl-alpha-phenylnitrone, but neither glutathione nor vitamin C, provided a significant protection against DMF- or DMAc-dependent haem loss. Finally, electron spin resonance analysis of microsomal incubations in presence of DMF or DMAc showed spectral evidence for a carbon centered radical intermediate. The results indicate, overall, that both compounds are metabolized in vitro by P450, probably CYP2E1, to free radical metabolites which attack the haem prosthetic group, leading to suicidal enzyme inactivation.

A series of N-acyloxymethyl- and N-aminocarbonyloxymethyl derivatives of 2-azetidinones. 3, with different substituent patterns at the beta -lactam C-3 and C-4 positions, were designed as potential mechanism-based inhibitors for human leukocyte elastase and found to exhibit inhibitory potency and selectivity for the enzyme, (C) 2001 Elsevier Science Ltd. All rights reserved.

The hydrolysis of tertiary amidomethyl ester prodrugs of carboxylic acids by rat liver homogenates is reported. Amidomethyl esters are rapidly and quantitatively converted to the corresponding acid and secondary amide. Reactivity is inversely dependent upon the molar refractivity and lipophilicity of the ester, as well as with steric bulk in the carboxylic acid moiety. In contrast to chemical and plasma hydrolyses, no dependence upon the pKa of the carboxylate leaving group was observed, nor was there any dependence upon the amide N-substituent. The rate of decomposition was inhibited by the carboxylesterase inhibitor eserine but not by the cytochrome P450 inhibitor SKF-525A, indicating the involvement of esterases in the hydrolysis reaction. These results indicate that amidomethyl esters may be expected to be readily cleaved in vivo.

1. The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identified by HPLC retention times, UV spectroscopy, mass spectrometry and by comparison with authentic standards. 2. DEET (1a) produces three major metabolites, N-ethyl-m-toluamide (1b), N,N-diethyl-m-(hydroxymethyl)benzamide (2a) and N-ethyl-m-(hydroxymethyl)benzamide (2b), and, at low substrate concentrations or extended reaction times, two minor metabolites, toluamide (1c) and N,N-diethyl-m-formylbenzamide (3a). 1b and 2a are primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low DEET concentrations, ring methyl group oxidation is favoured; at saturation concentrations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of formation of 2b decreases with increasing DEET concentration; 2b is therefore a secondary metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of 1b and 2a. 3. Except for the primary amides, where N-dealkylation is impossible, metabolism of all subsequent compounds, 1b,c, 2a-c, 3a-c and 4a,b, involves an N-deethylation (NEt2 --NHEt or NHEt --NH2) competitive with a ring substituent oxidation (CH3 --CH2OH, CH2OH --CHO or CHO --CO2H). Surprisingly, the aldehydes 3a-c are also reduced to the corresponding alcohols 2a-c (CHO --CH2OH); CO inhibits the oxidative metabolism of 3a-c, but reduction to 2a-c continues uninhibited. 4. The outcomes of this work are that (1) previously unreported aldehydes 3b and 3c form part of the DEET network of metabolites, (2) the reduction of the aldehydes 3a-c has the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3) amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for N-(1-hydroxyethyl)amide intermediates.

Phthalimidomethyl derivatives 1, encompassing a wide range of leaving group abilities, are rapidly hydrolysed to the corresponding phthalamic acid via rate-determining attack at the phthalimide carbonyl group.

N-(2-Iodoethyl) and N-(3-iodopropyl)pyrimidines and purines undergo stereoselective conjugate radical addition with an optically active oxazolidinone acceptor to give syn-adducts that can be converted into pyrimidine and purine amino acids.

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.

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.

The metal ions Fe2+, Zn2+ and Cu2+ bring about the rapid decomposition of 1-aryl-3-alkyltriazenes to the corresponding anilines. For Fe2+, a linear dependence of the pseudo-first-order rate constant, k0 on [Fe2+] was observed, while for Zn2+ and Cu2+ plots of k0versus[metal ion] were curved and indicative of complex formation. For Fe2+, second-order rate constants k2Fe2+ for substituted 1-aryl-3-methyltriazenes follow a Hammett relationship giving rise to a ρ value of –3.0. For Zn2+ and Cu2+, the data were analysed in terms of an equilibrium constant, KM2+, for the dissociation of a metal-ion-triazene complex and the first-order rate constant for the collapse of this complex to products, k2M2+. Hammett ρ values of 1.0 for both KZn2+ and KCu2+ are found, and the corresponding ρ values for k2Zn2+ and K2Cu2+ are –1.3 and –1.9. There is reasonable correlation between the Taft Es parameter for the alkyl group and KCu2+, giving a δ value of –1.6. The dependence of k2Cu2+ on the alkyl group is not simple: k2Cu2+ decreases in the order Pr > Et ≠ PhCH2ca. 4-MeOC6H4CH2 > CD3ca. Me. The reactions catalysed by Cu2+ are inhibited by added nucleophilies e.g. Br– and N-methylimidazole.A mechanism is proposed in which the triazene complexes to the metal ion via the N(1) nitrogen atom of the E–cis conformer, then undergoes a fast proton transfer to form a complex involving the unconjugated tautomer which subsequently decomposes via unimolecular scission of the N(2)–N(3) bond to form an alkyldiazonium ion and an aniline–metal complex. The observed products then arise from rapid solvolysis of the metal–aniline complex and the alkyl diazomum ion.

Atraves da carbamoilacao do 2-aminobenzimidazole com diferentes cloroformatos substituidos, sintetizaram-se novos N-[1-(2-amino)benzimidazolil]carbamatos. Estudouse a hidrolise em meio aquoso destes carbamatos numa gama de pH compreendida entre 1 e 13, a uma temperatura de 25 oC. Os parâmetros cineticos avaliados levaram a concluir que ate pH 4 a reacao se processa por mecanismo que envolve o ataque bimolecular da agua ao substrato N-protonado. Esta e a primeira vez que este comportamento e observado em carbamatos, sendo muito provavelmente devido a diferenca de basicidade entre um dos azotos do anel de benzimidazolilo e o oxigenio da funcao carbamato. Para valores mais elevados de pH, os resultados sao consistentes com um mecanismo B Ac 2, atuando a agua como nucleofilo entre pH 4 e 7 enquanto que em pH superior o ion hidroxido e o nucleofilo. Synthesis of new 2-aminobenzimidazole-1-carbamates was accomplished by carbamoylation of 2-aminobenzimidazole using different substituted phenyl chloroformates. The aqueous hydrolysis of the new compounds was examined in the pH range 1-13 at 25 o C. The evaluated kinetic parameters led to the conclusion that up to pH 4 reaction proceeds by a bimolecular attack of water to the N-protonated substrate. This is the first time this behavior is described for carbamates, and can be ascribed to the higher basicity of the benzimidazolyl moiety when compared with the carbonyl oxygen. For higher values of pH, the results are consistent with a B Ac 2 mechanism with nucleophilic catalysis, but while between pH 4 and pH 7 water acts as the nucleophile, for pH> 7 the hydroxide ion is the acting species.

Rate constants for the decomposition of N-nitro-N-methylamides to N-nitro-N-methylamine and the corresponding carboxylic acid in aqueous buffer solutions are reported. For N-nitro-N-methylacetamide (1a) and N-nitro-N-methylbenzamide (1b), the pH-rate profiles indicate that below pH 5 the reaction is independent of [H+]. At pH values > 7 the reactions are strongly HO– catalysed. Moreover, the basic component of the buffer also catalyses the decomposition reaction. Second-order rate constants, kB, for this buffer catalysis are dependent on the structure of the base. Thus Bronsted plots of log kBversus base pKa for (1a) and (1b) yield slopes of 0.64 and 0.60, respectively, for nitrogen bases. The oxygen bases AcO–, HPO42– and HO– appear to fall on another line of slope ca. 0.5. Solvent deuterium kinetic isotope effects for both the AcO– and HO– catalysed reactions are ca. 1, whereas that for the non-catalysed reaction is ca. 2. Catalysis is found to be nucleophilic in nature; thus, for each of the reactions of (1b) with morpholine, piperidine and 4-chlorophenol the corresponding benzoylated base could be isolated. Further, the observed firstorder rate constants for the reaction of either (1a) or (1b) with imidazole reach a limiting value identical to that for N-acetylimidazole itself. For (1a), the ratios of kB for piperidine to 2,2′,6,6′-tetramethylpiperidine and for pyridine to acetate are ca. 300 and 100, respectively. Again, this is consistent with nucleophilic catalysis. The aromatic substituent effect for the HO– catalysed reaction yields a Hammett ρ value +2.8, whereas for the non-catalysed reaction a value of 0.8 is obtained. The data are discussed in terms of a mechanism in which nucleophilic attack of the catalyst at the carbonyl C-atom to form a tetrahedral intermediate is rate-limiting. Lack of 18O-exchange during hydrolysis is consistent with this proposal. This mechanism is unusual for amide hydrolysis and must reflect the enhanced nucleofugacity of the N-nitroamine fragment. The mechanism of the noncatalysed process is less clear. The substituent effects are much smaller than-those for HO–, and therefore unlikely to involve attack of H2O at the carbonyl carbon. N-Methyl cleavage via H2O attack or thermal rearrangement are possible candidates.

The conformationally restricted tertiary amides N-methyl-2-pyrrolidone 6, N-methyl-2-piperidone 7 and N-methyl-e-caprolactam 8 were oxidised by 5,10,15,20-tetraphenylporphyrinatoiron(III) 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.

Imidoyl nitrates, formed by the reaction of imidoyl chlorides with AgNO3, rearrange via a unimolecular, intramolecular mechanism probably involving homolytic fission of the O–N bond to yield N-nitro amides; migration of the nitro group in N-arylimidoyl nitrates to the ortho- and para-positions of the N-aryl ring does not involve a special ortho-directing effect.

Paramagnetic induced chemical shifts were obtained for a series of 1-aryl-3-hydroxymethyl-3-methyltriazenes and related compounds by the addition of Pr(thd)3 or Eu(fod)3. Induced shifts were shown to be pseudo-contact in origin. Qualitative work using Pr(thd)3 inducated that the N–CH2–O proton resonances are shifted the most, followed by the N–Me resonances. Neither 1-aryl-3-methyl-nor 1-aryl-3,3-dimethyl-triazenes exhibit induced shifts of the N–Me resonances, which indicates that binding of the hydroxymethyltriazenes to the lanthanide ion occurs through the hydroxyl function. Quantitative work using Eu(fod)3 allowed values of ΔB, the paramagnetic shift of the bound hydroxymethyltriazene, and KB, the equilibrium binding constant, to be calculated. The data are consistent with 1 : 1 complex formation and values of KB, which lie between 10–60 l mol–1, are little affected by the aromatic substituent. Correlation of the pseudo-contact induced shifts, ΔB, with r3(where r is the distance between the resonating nucleus and the Eu3+ ion) occurs when the hydroxymethyltriazene is assumed to bind to Eu3+ through the hydroxyl oxygen atom. In contrast, 3-hydroxymethyl-3-methyl-1-(3′-pyridyl)triazene binds to both Pr3+ and Eu3+ through the pyridyl nitrogen atom.