Confocal Raman Spectroscopic Characterization of Dermatopharmacokinetics Ex Vivo.

Confocal Raman spectroscopy is being assessed as a tool with which to quantify the rate and extent of drug uptake to and its clearance from target sites of action within the viable epidermis below the skin's stratum corneum (SC) barrier. The objective of this research was to confirm that Raman can interrogate drug disposition within the living layers of the skin (where many topical drugs elicit their pharmacological effects) and to identify procedures by which Raman signal attenuation with increasing skin depth may be corrected and normalized so that metrics descriptive of topical bioavailability may be identified. It was first shown in experiments on skin cross-sections parallel to the skin surface that the amide I signal, originating primarily from keratin, was quite constant with depth into the skin and could be used to correct for signal attenuation when confocal Raman data were acquired in a "top-down" fashion. Then, using 4-cyanophenol (CP) as a model skin penetrant with a strong Raman-active C≡N functionality, a series of uptake and clearance experiments, performed as a function of time, demonstrated clearly that normalized spectroscopic data were able to detect the penetrant to at least 40-80 µm into the skin and to distinguish the disposition of CP from different vehicles. Metrics related to local bioavailability (and potentially bioequivalence) included areas under the normalized C≡N signal versus depth profiles and elimination rate constants deduced post-removal of the formulations. Finally, Raman measurements were made with an approved dermatological drug, crisaborole, for which delivery from a fully saturated formulation into the skin layers just below the SC was detectable.

Quantification of Chemical Uptake into the Skin by Vibrational Spectroscopies and Stratum Corneum Sampling.

Evaluation of the bioavailability of drugs intended to act within the skin following the application of complex topical products requires the application of multiple experimental tools, which must be quantitative, validated, and, ideally and ultimately, sufficiently minimally invasive to permit use in vivo. The objective here is to show that both infrared (IR) and Raman spectroscopies can assess the uptake of a chemical into the stratum corneum (SC) that correlates directly with its quantification by the adhesive tape-stripping method. Experiments were performed ex vivo using excised porcine skin and measured chemical disposition in the SC as functions of application time and formulation composition. The quantity of chemicals in the SC removed on each tape-strip was determined from the individually measured IR and Raman signal intensities of a specific molecular vibration at a frequency where the skin is spectroscopically silent and by a subsequent conventional extraction and chromatographic analysis. Correlations between the spectroscopic results and the chemical quantification on the tape-strips were good, and the effects of longer application times and the use of different vehicles were clearly delineated by the different measurement techniques. Based on this initial investigation, it is now possible to explore the extent to which the spectroscopic approach (and Raman in particular) may be used to interrogate chemical disposition deeper in the skin and beyond the SC.

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition.

Racemases and epimerases catalyse changes in the stereochemical configurations of chiral centres and are of interest as model enzymes and as biotechnological tools. They also occupy pivotal positions within metabolic pathways and, hence, many of them are important drug targets. This review summarises the catalytic mechanisms of PLP-dependent, enolase family and cofactor-independent racemases and epimerases operating by a deprotonation/reprotonation (1,1-proton transfer) mechanism and methods for measuring their catalytic activity. Strategies for inhibiting these enzymes are reviewed, as are specific examples of inhibitors. Rational design of inhibitors based on substrates has been extensively explored but there is considerable scope for development of transition-state mimics and covalent inhibitors and for the identification of inhibitors by high-throughput, fragment and virtual screening approaches. The increasing availability of enzyme structures obtained using X-ray crystallography will facilitate development of inhibitors by rational design and fragment screening, whilst protein models will facilitate development of transition-state mimics.

Novel 2-arylthiopropanoyl-CoA inhibitors of α-methylacyl-CoA racemase 1A (AMACR; P504S) as potential anti-prostate cancer agents.

α-Methylacyl-CoA racemase (AMACR; P504S) catalyses an essential step in the degradation of branched-chain fatty acids and the activation of ibuprofen and related drugs. AMACR has gained much attention as a drug target and biomarker, since it is found at elevated levels in prostate cancer and several other cancers. Herein, we report the synthesis of 2-(phenylthio)propanoyl-CoA derivatives which provided potent AMACR inhibitory activity (IC50 = 22-100 nM), as measured by the AMACR colorimetric activity assay. Inhibitor potency positively correlates with calculated logP, although 2-(3-benzyloxyphenylthio)propanoyl-CoA and 2-(4-(2-methylpropoxy)phenylthio)propanoyl-CoA were more potent than predicted by this parameter. Subsequently, carboxylic acid precursors were evaluated against androgen-dependent LnCaP prostate cancer cells and androgen-independent Du145 and PC3 prostate cancer cells using the MTS assay. All tested precursor acids showed inhibitory activity against LnCaP, Du145 and PC3 cells at 500 µM, but lacked activity at 100 µM. This is the first extensive structure-activity relationship study on the influence of side-chain interactions on the potency of novel rationally designed AMACR inhibitors.

Identification of novel small-molecule inhibitors of α-methylacyl-CoA racemase (AMACR; P504S) and structure-activity relationships.

α-Methylacyl-CoA racemase (AMACR; P504S; EC 5.1.99.4) catalyses epimerization of 2-methylacyl-CoAs and is important for the degradation of branched-chain fatty acids and the pharmacological activation of ibuprofen and related drugs. It is also a novel drug target for prostate and other cancers. However, development of AMACR as a drug target has been hampered by the difficulties in assaying enzyme activity. Consequently, reported inhibitors have been rationally designed acyl-CoA esters, which are delivered as their carboxylate prodrugs. The novel colorimetric assay for AMACR based on the elimination of 2,4-dinitrophenolate was developed for high-throughput screening and 20,387 'drug-like compounds' were screened, with a throughput of 768 compounds assayed per day. Pyrazoloquinolines and pyrazolopyrimidines were identified as novel scaffolds and investigated as AMACR inhibitors. The most potent inhibitors have IC50 values of ~2 µM. The pyrazoloquinoline inhibitor 10a displayed uncompetitive inhibition, whilst 10j displayed mixed competitive inhibition. The pyrazolopyrimidine inhibitor 11k displayed uncompetitive inhibition. This is the first report of the identification of specific drug-like small-molecule AMACR inhibitors by high-throughput screening. Pyrazoloquinolines and pyrazolopyrimidines may also be useful as inhibitors of other CoA-utilizing enzymes.

A simple 1 H NMR based assay of total capsaicinoid levels in Capsicum using signal suppression in non-deuterated solvent.

BACKGROUND: The heat of Capsicum fruits is routinely assayed using high-performance liquid chromatography (HPLC) to determine capsaicin (CA) and dihydrocapsaicin (DHC) levels. The assay can be time consuming, with each HPLC run typically lasting 10 min. Nuclear magnetic resonance (NMR) is eminently suitable for quantification of fruit extracts, although it has been largely ignored for quantitative chilli analysis. The present study describes a novel approach using solvent suppression in protic solvent (i.e. non-deuterated) to quantify total capsaicinoid levels in chilli extracts. RESULTS: Using solvent suppression techniques and maleic acid as an internal standard, capsaicinoid content in a series of accurately weighed standard samples was determined over a range between 40 and 720 ppm (0.13-2.35 mmolar) with high accuracy and precision. The measurement was linear over the entire range. This method was subsequently used with ten authentic Capsicum samples (seven chinense, two annuum and one baccatum) and showed an excellent correlation with the HPLC data. CONCLUSION: The results of the present study confirm that NMR in non-deuterated solvent can provide a rapid and robust assessment of the pungency of capsicum fruits. © 2018 Society of Chemical Industry.

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC50 = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.

A study on the AMACR catalysed elimination reaction and its application to inhibitor testing.

α-Methylacyl-CoA racemase (AMACR; P504S) catalyses a key step in the degradation of branched-chain fatty acids and is important for the pharmacological activation of Ibuprofen and related drugs. Levels of AMACR are increased in prostate and other cancers, and it is a drug target. Development of AMACR as a drug target is hampered by lack of a convenient assay. AMACR irreversibly catalyses the elimination of HF from 3-fluoro-2-methylacyl-CoA substrates, and this reaction was investigated for use as an assay. Several known inhibitors and alternative substrates reduced conversion of 3-fluoro-2-methyldecanoyl-CoA by AMACR, as determined by (1)H NMR. The greatest reduction of activity was observed with known potent inhibitors. A series of novel acyl-CoA esters with aromatic side chains were synthesised for testing as chromophoric substrates. These acyl-CoA esters were converted to unsaturated products by AMACR, but their use was limited by non-enzymatic elimination. Fluoride sensors were also investigated as a method of quantifying released fluoride and thus AMACR activity. These sensors generally suffered from high background signal and lacked reproducibility under the assay conditions. In summary, the elimination reaction can be used to characterise inhibitors, but it was not possible to develop a convenient colorimetric or fluorescent assay using 3-fluoro-2-methylacyl-CoA substrates.

A study on the chiral inversion of mandelic acid in humans.

Mandelic acid is a chiral metabolite of the industrial pollutant styrene and is used in chemical skin peels, as a urinary antiseptic and as a component of other medicines. In humans, S-mandelic acid undergoes rapid chiral inversion to R-mandelic acid by an undefined pathway but it has been proposed to proceed via the acyl-CoA esters, S- and R-2-hydroxy-2-phenylacetyl-CoA, in an analogous pathway to that for Ibuprofen. This study investigates chiral inversion of mandelic acid using purified human recombinant enzymes known to be involved in the Ibuprofen chiral inversion pathway. Both S- and R-2-hydroxy-2-phenylacetyl-CoA were hydrolysed to mandelic acid by human acyl-CoA thioesterase-1 and -2 (ACOT1 and ACOT2), consistent with a possible role in the chiral inversion pathway. However, human α-methylacyl-CoA racemase (AMACR; P504S) was not able to catalyse exchange of the α-proton of S- and R-2-hydroxy-2-phenylacetyl-CoA, a requirement for chiral inversion. Both S- and R-2-phenylpropanoyl-CoA were epimerised by AMACR, showing that it is the presence of the hydroxy group that prevents epimerisation of R- and S-2-hydroxy-2-phenylacetyl-CoAs. The results show that it is unlikely that 2-hydroxy-2-phenylacetyl-CoA is an intermediate in the chiral inversion of mandelic acid, and that the chiral inversion of mandelic acid is via a different pathway to that of Ibuprofen and related drugs.

NMR diffusion-ordered spectroscopy can explain differences in skin penetration enhancement between microemulsion formulations.

Changing the formulation variables of microemulsion systems has a significant influence on the resulting transdermal enhancement effect. NMR diffusion-ordered spectroscopy (DOSY) can offer an extremely valuable tool to interpret the differences in the obtained fluxes based on variations in self-diffusions between the drug and its locus domain. From the clinical editor: Microemulsion systems are often used as vehicles for transdermal drug delivery. In this communication authors interpret quantitative differences between microemulsion formulations based on studying the self-diffusion of testosterone relative to the diffusion of its residing oil phase based on DOSY spectroscopy.

α-Methylacyl-CoA Racemase from Mycobacterium tuberculosis-Detailed Kinetic and Structural Characterization of the Active Site.

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium's survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.

Evaluation of chemical disposition in skin by stimulated Raman scattering microscopy.

Tracking drug disposition in the skin in a non-destructive and at least semi-quantitative fashion is a relevant objective for the assessment of local (cutaneous) bioavailability. Confocal Raman spectroscopy has been shown potentially useful in this regard and, importantly, recent advances have enabled the presence of applied chemicals in the viable epidermis below the stratum corneum (SC) to be determined without ambiguity and having addressed the challenges of (a) background signals from endogenous species and noise and (b) signal attenuation due to absorption and scattering. This study aimed to confirm these observations using a different vibrational spectroscopy approach - specifically, stimulated Raman scattering (SRS) microscopy - and the more conventional in vitro skin penetration test (IVPT). SRS is a nonlinear optical imaging technique which enables more precise location of the skin surface and enhanced skin depth resolution relative to confocal Raman microscopy. The method can also probe larger areas of the sample under investigation and identify the localization of the permeating chemical in specific structural components of the skin. Here, SRS was shown capable of tracking the uptake and distribution of 4-cyanophenol (CP), the same model compound used in the recent confocal Raman investigation, at depths beyond the SC following skin treatment with different vehicles and for different times. The SRS results correlated well with those from the confocal Raman experiments, and both were consistent with independent IVPT measurements. Acquired images clearly delineated CP preference for the intercellular lipid layers of the SC relative to the corneocytes. The stage is now set to apply these and other correlative techniques to examine commercial drug products.

Regioselective opening of myo-inositol orthoesters: mechanism and synthetic utility.

Acid hydrolysis of myo-inositol 1,3,5-orthoesters, apart from orthoformates, exclusively affords the corresponding 2-O-acyl myo-inositol products via a 1,2-bridged five-membered ring dioxolanylium ion intermediate observed by NMR spectroscopy. These C-2-substituted inositol derivatives provide valuable precursors for rapid and highly efficient routes to 2-O-acyl inositol 1,3,4,5,6-pentakisphosphates and myo-inositol 1,3,4,5,6-pentakisphosphate with biologically interesting and anticancer properties. Deuterium incorporation into the α-methylene group of such alkyl ester products (2-O-C(O)CD2R), when the analogous alkyl orthoester is treated with deuterated acid, is established utilizing the novel orthoester myo-inositol 1,3,5-orthobutyrate as an example. Such deuterated ester products provide intermediates for deuterium-labeled synthetic analogues. Investigation into this selective formation of 2-O-ester products and the deuterium incorporation is presented with proposed mechanisms from NMR experiments.

Observation by NMR of cationic Wheland-like intermediates in the deiodination of protected 1-iodonaphthalene-2,4-diamines in acidic media.

1-Iodonaphthalene-2,4-diamines in trifluoroacetic acid/chloroform give stable Wheland-like tetrahedral cationic species observable by NMR, through an initial intramolecular protonation. Dynamic equilibria allow proton-deuterium exchange of aromatic protons and provide a mechanism for deiodination of 1-iodonaphthalene-2,4-diamines.

Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR).

α-Methylacyl-CoA racemase (AMACR; P504S) catalyzes the conversion of R-2-methylacyl-CoA esters into their corresponding S-2-methylacyl-CoA epimers enabling their degradation by ß-oxidation. The enzyme also catalyzes the key epimerization reaction in the pharmacological activation pathway of ibuprofen and related drugs. AMACR protein levels and enzymatic activity are increased in prostate cancer, and the enzyme is a recognized drug target. Key to the development of novel treatments based on AMACR inhibition is the development of functional assays. Synthesis of substrates and purification of recombinant human AMACR are described. Incubation of R- or S-2-methylacyl-CoA esters with AMACR in vitro resulted in formation of epimers (at a near 1-1 ratio at equilibrium) via removal of their α-protons to form an enolate intermediate followed by reprotonation. Conversion can be conveniently followed by incubation in buffer containing 2H2O followed by 1H NMR analysis to monitor conversion of the α-methyl doublet to a single peak upon deuterium incorporation. Incubation of 2-methylacyl-CoA esters containing leaving groups results in an elimination reaction, which was also characterized by 1H NMR. The synthesis of substrates, including a double labeled substrate for mechanistic studies, and subsequent analysis is also described.

Design and synthesis of hybrid compounds as novel drugs and medicines.

The development of highly effective conjugate chemistry approaches is a way to improve the quality of drugs and of medicines. The aim of this paper is to highlight and review such hybrid compounds and the strategies underpinning their design. A variety of unique hybrid compounds provide an excellent toolkit for novel biological activity, e.g. anticancer and non-viral gene therapy (NVGT), and as templates for killing bacteria and preventing antibiotic drug resistance. First we discuss the anticancer potential of hybrid compounds, containing daunorubicin, benzyl- or tetrahydroisoquinoline-coumarin, and cytotoxic NSAID-pyrrolizidine/indolizine hybrids, then NVGT cationic lipid-based delivery agents, where steroids or long chain fatty acids as the lipid moiety are bound to polyamines as the cationic moiety. These polyamines can be linear as in spermidine or spermine, or on a polycyclic sugar template, aminoglycosides kanamycin and neomycin B, the latter substituted with six amino groups. They are highly efficient for the delivery of both fluorescent DNA and siRNA. Molecular precedents can be found for the design of hybrid compounds in the natural world, e.g., squalamine, the first representative of a previously unknown class of natural antibiotics of animal origin. These polyamine-bile acid (e.g. cholic acid type) conjugates display many exciting biological activities with the bile acids acting as a lipidic region and spermidine as the polycationic region. Analogues of squalamine can act as vectors in NVGT. Their natural role is as antibiotics. Novel antibacterial materials are urgently needed as recalcitrant bacterial infection is a worldwide problem for human health. Ribosome inhibitors founded upon dimers of tobramycin or neomycin, bound as ethers by a 1,6-hexyl linker or a more complex diether-disulfide linker, improved upon the antibiotic activity of aminoglycoside monomers by 20- to 1200-fold. Other hybrids, linked by click chemistry, conjugated ciprofloxacin to neomycin, trimethoprim, or tedizolid, which is now in clinical trials.

Practical Synthesis of Polyamine Succinamides and Branched Polyamines.

Antibiotic resistance is now a growing threat to human health, further exacerbated by the lack of new antibiotics. We describe the practical synthesis of a series of substituted polyamine succinamides and branched polyamines that are potential new antibiotics against both Gram-positive and Gram-negative bacteria, including MRSA and Pseudomonas aeruginosa. They are prepared via 1,4-Michael addition of acrylonitrile and then hydrogenation of the nitrile functional groups to primary amines. They are built upon the framework of the naturally occurring polyamines thermine (3.3.3, norspermine) and spermine (3.4.3), homo- and heterodimeric polyamine succinic amides. Linking two of the same or different polyamines together via amide bonds can be achieved by introducing a carboxylic acid group on the first polyamine, then coupling that released carboxylic acid to a free primary amine in the second polyamine. If the addition of positive charges on the amino groups along the polyamine chains are a key factor in their antimicrobial activity against Gram-negative bacteria, then increasing them will increase the antimicrobial activity. Synthesising polyamine amide dimers will increase the total net positive charge compared to their monomers. The design and practical synthesis of such homo- and hetero-dimers of linear polyamines, spermine and norspermine, are reported. Several of these compounds do not display significant antibacterial activity against Gram-positive or Gram-negative bacteria, including MRSA and Pseudomonas aeruginosa. However, the most charged analogue, a branched polyamine carrying eight positive charges at physiological pH, displays antibiofilm activity with a 50 % reduction in PAO1 at 16-32 µg mL-1 .

Practical Synthesis of Antimicrobial Long Linear Polyamine Succinamides.

There are many severe bacterial infections notorious for their ability to become resistant to clinically relevant antibiotics. Indeed, antibiotic resistance is a growing threat to human health, further exacerbated by the lack of new antibiotics. We now describe the practical synthesis of a series of substituted long linear polyamines that produce rapid antibacterial activity against both Gram-positive and Gram-negative bacteria, including meticillin-resistant Staphylococcus aureus. These compounds also reduce biofilm formation in Pseudomonas aeruginosa. The most potent analogues are thermine, spermine, and 1,12-diaminododecane homo- and heterodimeric polyamine succinic acid amides. They are of the order of activity of the aminoglycoside antibiotics kanamycin and tobramycin as positive controls. Their low human cell toxicity is demonstrated in ex vivo hemolytic assays where they did not produce even 5% hemolysis of human erythrocytes. These long, linear polyamines are a new class of broad-spectrum antibacterials active against drug-resistant pathogens.

Antibacterial activity of novel linear polyamines against Staphylococcus aureus.

New therapeutic options are urgently required for the treatment of Staphylococcus aureus infections. Accordingly, we sought to exploit the vulnerability of S. aureus to naturally occurring polyamines. We have developed and tested the anti-staphylococcal activity of three novel linear polyamines based on spermine and norspermine. Using a panel of genetically distinct and clinically relevant multidrug resistant S. aureus isolates, including the polyamine resistant USA300 strain LAC, compound AHA-1394 showed a greater than 128-fold increase in inhibition against specific S. aureus strains compared to the most active natural polyamine. Furthermore, we show that AHA-1394 has superior biofilm prevention and biofilm dispersal properties compared to natural polyamines while maintaining minimal toxicity toward human HepG2 cells. We examined the potential of S. aureus to gain resistance to AHA-1394 following in vitro serial passage. Whole genome sequencing of two stable resistant mutants identified a gain of function mutation (S337L) in the phosphatidylglycerol lysyltransferase mprF gene. Inactivation of mutant mprF confirmed the importance of this allele to AHA-1394 resistance. Importantly, AHA-1394 resistant mutants showed a marked decrease in relative fitness and increased generation time. Intriguingly, mprF::S337L contributed to altered surface charge only in the USA300 background whereas increased cell wall thickness was observed in both USA300 and SH1000. Lastly, we show that AHA-1394 displays a particular proclivity for antibiotic potentiation, restoring sensitivity of MRSA and VRSA isolates to daptomycin, oxacillin and vancomycin. Together this study shows that polyamine derivatives are impressive drug candidates that warrant further investigation.

N3-alkylation during formation of quinazolin-4-ones from condensation of anthranilamides and orthoamides.

Dimethylformamide dimethylacetal (DMFDMA) is widely used as a source of electrophilic one-carbon units at the formate oxidation level; however, electrophilic methylation with this reagent is previously unreported. Reaction of anthranilamide with DMFDMA at 150 °C for short periods gives mainly quinazolin-4-one. However, prolonged reaction with dimethylformamide di(primary-alkyl)acetals leads to subsequent alkylation at N(3). 3-Substituted anthranilamides give 8-substituted 3-alkylquinazolin-4-ones. Condensation of anthranilamides with dimethylacetamide dimethylacetal provides 2,3-dimethylquinazolin-4-ones. In these reactions, the source of the N(3)-alkyl group is the O-alkyl group of the orthoamides. By contrast, reaction with the more sterically crowded dimethylformamide di(isopropyl)acetal diverts the alkylation to the oxygen, giving 4-isopropoxyquinazolines, along with N(3)-methylquinazolin-4-ones where the methyl is derived from N-Me of the orthoamides. Reaction of anthranilamide with the highly sterically demanding dimethylformamide di(t-butyl)acetal gives largely quinazolin-4-one, whereas dimethylformamide di(neopentyl)acetal forms a mixture of quinazolin-4-one and N(3)-methylquinazolin-4-one. The observations are rationalised in terms of formation of intermediate cationic electrophiles (alkoxymethylidene-N,N-dimethylammonium) by thermal elimination of the corresponding alkoxide from the orthoamides. These are the first observations of orthoamides as direct alkylating agents.