Developing an adult stem cell derived microphysiological intestinal system for predicting oral prodrug bioconversion and permeability in humans.

Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration-time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines.

Integrity and efficiency: AbbVie's journey of building an integrated nonregulated bioanalytical laboratory.

While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost-effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.

Replacement of a Naphthalene Scaffold in Kelch-like ECH-Associated Protein 1 (KEAP1)/Nuclear Factor (Erythroid-derived 2)-like 2 (NRF2) Inhibitors.

Activators of nuclear factor-erythroid 2-related factor 2 (NRF2) could lead to promising therapeutics for prevention and treatment of oxidative stress and inflammatory disorders. Ubiquitination and subsequent degradation of the transcription factor NRF2 is mediated by Kelch-like ECH-associated protein-1 (KEAP1). Inhibition of the KEAP1/NRF2 interaction with small molecules leads to NRF2 activation. Previously, we and others described naphthalene-based NRF2 activators, but the 1,4-diaminonaphthalene scaffold may not represent a drug-like scaffold. Paying particular attention to aqueous solubility, metabolic stability, potency, and mutagenicity, we modified a previously known, naphthalene-based nonelectrophilic NRF2 activator to give a series of non-naphthalene and heterocyclic scaffolds. We found that, compared to previously reported naphthalene-based compounds, a 1,4-isoquinoline scaffold provides a better mutagenic profile without sacrificing potency, stability, or solubility.

Design, Synthesis, Molecular Modeling, and Biological Evaluation of Novel Amine-based Histone Deacetylase Inhibitors.

Histone deacetylases (HDACs) are promising drug targets for a variety of therapeutic applications. Herein we describe the design, synthesis, biological evaluation in cellular models of cancer, and preliminary drug metabolism and pharmacokinetic studies (DMPK) of a series of secondary and tertiary N-substituted 7-aminoheptanohydroxamic acid-based HDAC inhibitors. Introduction of an amino group with one or two surface binding groups (SBGs) yielded a successful strategy to develop novel and potent HDAC inhibitors. The secondary amines were found to be generally more potent than the corresponding tertiary amines. Docking studies suggested that the SBGs of tertiary amines cannot be favorably accommodated at the gorge region of the binding site. The secondary amines with naphthalen-2-ylmethyl, 5-phenylthiophen-2-ylmethyl, and 1H-indol-2-ylmethyl (2 j) substituents exhibited the highest potency against class I HDACs: HDAC1 IC50 39-61 nm, HDAC2 IC50 260-690 nm, HDAC3 IC50 25-68 nm, and HDAC8 IC50 320-620 nm. The cytotoxicity of a representative set of secondary and tertiary N-substituted 7-aminoheptanoic acid hydroxyamide-based inhibitors against HT-29, SH-SY5Y, and MCF-7 cancer cells correlated with their inhibition of HDAC1, 2, and 3 and was found to be similar to or better than that of suberoylanilide hydroxamic acid (SAHA). Compounds in this series increased the acetylation of histones H3 and H4 in a time-dependent manner. DMPK studies indicated that secondary amine 2 j is metabolically stable and has plasma and brain concentrations >23- and >1.6-fold higher than the IC50 value for class I HDACs, respectively. Overall, the secondary and tertiary N-substituted 7-aminoheptanoic acid hydroxyamide-based inhibitors exhibit excellent lead- and drug-like properties and therapeutic capacity for cancer applications.

Hyperoxia-induced p47phox activation and ROS generation is mediated through S1P transporter Spns2, and S1P/S1P1&2 signaling axis in lung endothelium.

Hyperoxia-induced lung injury adversely affects ICU patients and neonates on ventilator assisted breathing. The underlying culprit appears to be reactive oxygen species (ROS)-induced lung damage. The major contributor of hyperoxia-induced ROS is activation of the multiprotein enzyme complex NADPH oxidase. Sphingosine-1-phosphate (S1P) signaling is known to be involved in hyperoxia-mediated ROS generation; however, the mechanism(s) of S1P-induced NADPH oxidase activation is unclear. Here, we investigated various steps in the S1P signaling pathway mediating ROS production in response to hyperoxia in lung endothelium. Of the two closely related sphingosine kinases (SphKs)1 and 2, which synthesize S1P from sphingosine, only Sphk1(-/-) mice conferred protection against hyperoxia-induced lung injury. S1P is metabolized predominantly by S1P lyase and partial deletion of Sgpl1 (Sgpl1(+/-)) in mice accentuated lung injury. Hyperoxia stimulated S1P accumulation in human lung microvascular endothelial cells (HLMVECs), and downregulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1&2, but not S1P3, using specific siRNA attenuated hyperoxia-induced p47(phox) translocation to cell periphery and ROS generation in HLMVECs. These results suggest a role for Spns2 and S1P1&2 in hyperoxia-mediated ROS generation. In addition, p47(phox) (phox:phagocyte oxidase) activation and ROS generation was also reduced by PF543, a specific SphK1 inhibitor in HLMVECs. Our data indicate a novel role for Spns2 and S1P1&2 in the activation of p47(phox) and production of ROS involved in hyperoxia-mediated lung injury in neonatal and adult mice.

Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome.

Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemopreventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the "bare-bones", Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.

Amyloid-ß pathology and APOE genotype modulate retinoid X receptor agonist activity in vivo.

Previous data demonstrate that bexarotene (Bex), retinoid X receptor (RXR) agonist, reduces soluble and insoluble amyloid-ß (Aß) in Alzheimer disease (AD)-transgenic mice either by increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. However, although the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aß, likely the proximal AD neurotoxin. If the APOE4 loss-of-function is lipidation of apoE4, then induction of ABCA1/ABCG1 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aß42 with h-APOE), levels of soluble Aß (Aß42 and oligomeric Aß) are highest in E4FAD hippocampus (HP) > E3FAD-HP > E4FAD cortex (CX) > E3FAD-CX, whereas levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6 months). In E4FAD-HP, short-term RXR agonist treatment (Bex or LG100268; 5.75-6 months) increased ABCA1, apoE4 lipoprotein-association/lipidation, and apoE4/Aß complex, decreased soluble Aß, and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and LG100268. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5-6 months) and actually increased soluble Aß levels in E3FAD-CX and E4FAD-CX with the short-term protocol, possibly the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aß pathology, i.e. low levels of apoE4 lipoprotein association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.

Proteomic profiling of nitrosative stress: protein S-oxidation accompanies S-nitrosylation.

Reversible chemical modifications of protein cysteine residues by S-nitrosylation and S-oxidation are increasingly recognized as important regulatory mechanisms for many protein classes associated with cellular signaling and stress response. Both modifications may theoretically occur under cellular nitrosative or nitroxidative stress. Therefore, a proteomic isotope-coded approach to parallel, quantitative analysis of cysteome S-nitrosylation and S-oxidation was developed. Modifications of cysteine residues of (i) human glutathione-S-transferase P1-1 (GSTP1) and (ii) the schistosomiasis drug target thioredoxin glutathione reductase (TGR) were studied. Both S-nitrosylation (SNO) and S-oxidation to disulfide (SS) were observed for reactive cysteines, dependent on concentration of added S-nitrosocysteine (CysNO) and independent of oxygen. SNO and SS modifications of GSTP1 were quantified and compared for therapeutically relevant NO and HNO donors from different chemical classes, revealing oxidative modification for all donors. Observations on GSTP1 were extended to cell cultures, analyzed after lysis and in-gel digestion. Treatment of living neuronal cells with CysNO, to induce nitrosative stress, caused levels of S-nitrosylation and S-oxidation of GSTP1 comparable to those of cell-free studies. Cysteine modifications of PARK7/DJ-1, peroxiredoxin-2, and other proteins were identified, quantified, and compared to overall levels of protein S-nitrosylation. The new methodology has allowed identification and quantitation of specific cysteome modifications, demonstrating that nitroxidation to protein disulfides occurs concurrently with S-nitrosylation to protein-SNO in recombinant proteins and living cells under nitrosative stress.

NO-1886 ameliorates glycogen metabolism in insulin-resistant HepG2 cells by GSK-3ß signalling.

OBJECTIVES: The aim of the study was to elucidate the possible role and mechanism of NO-1886 (ibrolipim, a lipoprotein lipase activator) in ameliorating insulin resistance induced by high palmitate. METHODS: HepG2 cells were cultured in RPMI 1640 medium and were treated with palmitate to induce insulin resistance. Free fatty acids (FFAs), glucose, glycogen, cell viability and mRNA and protein levels were analysed separately. KEY FINDINGS: We found that HepG2 cells treated with 0.5 mm palmitate for 48 h led to a significant decrease of insulin-induced glucose consumption (from 2.89 ± 0.85 mm in the control to 0.57 ± 0.44 mm in palmitate). Insulin resistance (IR) of HepG2 cells was induced by 0.5 mm palmitate for 48 h. NO-1886 stimulated glucose consumption, glycogen synthesis and FFA absorption in insulin-resistant HepG2 cells. Maximum stimulation effects were observed with 10 µm NO-1886 for 24 h. Compared with the dimethyl sulfoxide-treated group, 2.5 µm NO-1886 or higher could induce the mRNA expression of lipoprotein lipase. Meanwhile, NO-1886 increased the protein content of P-GSK-3ßser(9) and decreased the protein level of GSK-3ß in insulin-resistant HepG2 cells, but NO-1886 didn't change the protein levels of PI3-Kp85 and Akt2. CONCLUSION: Lipoprotein lipase activator NO-1886 could increase glycogen synthesis in HepG2 cells and could ameliorate the insulin resistance, which was associated with GSK-3 signalling.

Effects of aromatase inhibition versus gonadectomy on hippocampal complex amyloid pathology in triple transgenic mice.

Alzheimer's disease (AD) is the most common form of dementia among the elderly with women exhibiting a higher risk than men for the disease. Due to these gender differences, there is great interest in the role that estrogens play in cognitive impairment and the onset of the classic amyloid and tau lesions in AD. Human and rodent studies indicate a strong association between low brain aromatase, sex hormone levels, and beta amyloid deposition. Therefore, the effects of depleting both circulating and brain estrogen levels, through gonadectomy and/or treatment with the aromatase inhibitor, anastrozole, upon hippocampal AD-like pathology in male and female 3xTgAD mice were evaluated. Liquid chromatography-mass spectrometry revealed anastrozole serum levels of 10.19 ng/mL and for the first time brain levels were detected at 4.7 pg/mL. Densitometric analysis of the hippocampus revealed that anastrozole significantly increased Aß- but not APP/Aß-immunoreactivity in intact 3xTgAD females compared to controls (p<0.001). Moreover, anastrozole significantly increased the number of Aß- compared to APP/Aß-positive hippocampal CA1 neurons in intact and gonadectomized female mice. Concurrently, anastrozole significantly reduced the APP/Aß plaque load in 9 month old female 3xTgAD mice. These data suggest that anastrozole treatment differentially affects select amyloid species which in turn may play a role in the extraneuronal to intraneuronal deposition of this peptide.

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.

NO-SSRIs: Nitric Oxide Chimera Drugs Incorporating a Selective Serotonin Reuptake Inhibitor.

Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 hours. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter, however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer's disease (GT-1061), was made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide co-therapy of neuropathologies concomitant with depression.

Unexpected hormonal activity of a catechol equine estrogen metabolite reveals reversible glutathione conjugation.

4-Hydroxyequilenin (4-OHEN) is a major phase I metabolite of the equine estrogens present in widely prescribed hormone replacement formulations. 4-OHEN is autoxidized to an electrophilic o-quinone that has been shown to redox cycle, generating ROS, and to covalently modify proteins and DNA and thus potentially to act as a chemical carcinogen. To establish the ability of 4-OHEN to act as a hormonal carcinogen at the estrogen receptor (ER), estrogen responsive gene expression and proliferation were studied in ER(+) breast cancer cells. Recruitment by 4-OHEN of ER to estrogen responsive elements (ERE) of DNA in MCF-7 cells was also studied and observed. 4-OHEN was a potent estrogen, with additional weak activity associated with binding to the arylhydrocarbon receptor (AhR). The potency of 4-OHEN toward classical ERalpha mediated activity was unexpected given the reported rapid autoxidation and trapping of the resultant quinone by GSH. Addition of thiols to cell cultures did not attenuate the estrogenic activity of 4-OHEN, and preformed thiol conjugates added to cell incubations only marginally reduced ERE-luciferase induction. On reaction of the 4OHEN-GSH conjugate with NADPH, 4-OHEN was observed to be regenerated at a rate dependent upon NADPH concentration, indicating that intracellular nonenzymatic and enzymatic regeneration of 4-OHEN accounts for the observed estrogenic activity of 4-OHEN. 4-OHEN is therefore capable of inducing chemical and hormonal pathways that may contribute to estrogen-dependent carcinogenesis, and trapping by cellular thiols does not provide a mechanism of termination of these pathways.

Multiple decomposition pathways for the oxenium ion precursor O-(4-(4'-methylphenyl)phenyl)-N-methanesulfonylhydroxylamine.

Although O-arylhydroxylamine derivatives have been claimed to be sources of oxenium ions in a large number of studies, it is not clear that the products of these reactions are due to oxenium ions. Previously, we had shown through azide trapping studies that the quinol ester 2a and the title compound 3a generate the oxenium ion 1a. The ester 2a exclusively generates 1a in water and is also a photoprecursor of 1a in water. This is not true of 3a. The oxenium ion pathway accounts for a significant fraction of the reaction of 3a under neutral and acidic pH conditions, but there are three other pathways that account for hydrolysis of 3a. Both 3a and its conjugate base 3a(-) are present in aqueous solution under mild pH conditions. In addition to the oxenium ion product 4a, two other significant products are generated: the phenol 6a and the rearrangement product 8a. Both 4a and 8a are generated exclusively from 3a, whereas 6a is generated from both 3a and 3a(-). Azide trapping studies show that 6a and 8a are not generated from the oxenium ion. The phenol 6a is generated by two paths, one involving an apparent radical intermediate 7a and the other through a stepwise alpha-elimination pathway through 3a(-). The rearrangement product 8a is generated either through a concerted rearrangement or via an ion-pair rearrangement. Photolysis of 3a does not generate 1a. The only products of photolysis of 3a in water are 6a (major) and 8a (minor). The weak O-N bond of 3a is susceptible to homolysis under photolysis conditions, and the radical 7a is observed after laser flash photolysis of 3a. The cation 1a that is observed during laser flash photolysis experiments on 2a cannot be detected during similar experiments on 3a. These results suggest that the previous attribution of oxenium ions as the source of the decomposition products of other O-arylhydroxylamine derivatives in aromatic solvents via thermolysis or acid-catalyzed decomposition may not be correct.

Hydrolysis and photolysis of 4-Acetoxy-4-(benzothiazol-2-yl)-2,5-cyclohexadien-1-one, a model anti-tumor quinol ester.

4-Acetoxy-4-(benzothiazol-2-yl)-2,5-cyclohexadien-1-one, 1, a quinol derivative that exhibits significant anti-tumor activity against human breast, colon, and renal cancer cell lines, undergoes hydrolysis in aqueous solution to generate an oxenium ion intermediate, 3, that is selectively trapped by N(3)(-) in an aqueous environment. The 4-(benzothiazol-2-yl) substituent slows the rate of ionization of 1 compared to analogues with 4-phenyl or 4-(p-tolyl) substituents, 4a or 4b. However, once generated, 3 is somewhat more selective than the 4-phenyl-substituted cation 5a. Calculations performed at the B3LYP/6-31G(d) level agree that the 4-(benzothiazol-2-yl) substituent does significantly stabilize 3. The structure of the major isolated azide adduct, 4-(6-azidobenzothiazol-2-yl)phenol, 9, confirms that the positive charge is highly delocalized in 3. The results of hydrolysis of 1 show that the 4-(benzothiazol-2-yl) substituent has a significant inductive electron-withdrawing effect as well as a significant resonance effect that is electron-donating. Photolysis of 1 in aqueous solution generates the quinol 2 as one of several photolysis products. The presence of the quinol suggests that photolysis also leads, in part, to generation of 3, but photoionization of 1 is significantly less efficient than is the case for the esters 4a and 4b. This study proves that 3 is generated by ionization of 1 in an aqueous environment. A significant number of other 2-benzothiazole derivatives that are not quinols, including ring-substituted derivatives of 2-(4-aminophenyl)benzothiazole 15, are under development as anti-tumor agents as well. The possible generation of the reactive intermediate 17 by hydrolysis of the putative metabolite 16 is under investigation.

Characterization of reactive intermediates generated during photolysis of 4-acetoxy-4-aryl-2,5-cyclohexadienones: oxenium ions and aryloxy radicals.

Aryloxenium ions 1 are reactive intermediates that are isoelectronic with the better known arylcarbenium and arylnitrenium ions. They are proposed to be involved in synthetically and industrially useful oxidation reactions of phenols. However, mechanistic studies of these intermediates are limited. Until recently, the lifetimes of these intermediates in solution and their reactivity patterns were unknown. Previously, the quinol esters 2 have been used to generate 1, which were indirectly detected by azide ion trapping to generate azide adducts 4 at the expense of quinols 3, during hydrolysis reactions in the dark. Laser flash photolysis (LFP) of 2b in the presence of O(2) in aqueous solution leads to two reactive intermediates with lambda(max) 360 and 460 nm, respectively, while in pure CH(3)CN only one species with lambda(max) 350 nm is produced. The intermediate with lambda(max) 460 nm was previously identified as 1b based on direct observation of its decomposition kinetics in the presence of N(3)(-), comparison to azide ion trapping results from the hydrolysis reactions, and photolysis reaction products (3b). The agreement between the calculated (B3LYP/6-31G(d)) and observed time-resolved resonance Raman (TR(3)) spectra of 1b further confirms its identity. The second intermediate with lambda(max) 360 nm (350 nm in CH(3)CN) has been characterized as the radical 5b, based on its photolytic generation in the less polar CH(3)CN and on isolated photolysis reaction products (6b and 7b). Only the radical intermediate 5b is generated by photolysis in CH(3)CN, so its UV-vis spectrum, reaction products, and decay kinetics can be investigated in this solvent without interference from 1b. In addition, the radical 5a was generated by LFP of 2a and was identified by comparison to a published UV-vis spectrum of authentic 5a obtained under similar conditions. The similarity of the UV-vis spectra of 5a and 5b, their reaction products, and the kinetics of their decay confirm the assigned structures. The lifetime of 1b in aqueous solution at room temperature is 170 ns. This intermediate decays with first-order kinetics. The radical intermediate 5b decomposes in a biphasic manner, with lifetimes of 12 and 75 mus. The decay processes of 5a and 5b were successfully modeled with a kinetic scheme that included reversible formation of a dimer. The scheme is similar to the kinetic models applied to describe the decay of other aryloxy radicals.

Chemistry of 4-alkylaryloxenium ion "precursors": sound and fury signifying something?

Quinol esters 2b, 2c, and 3b and sulfonamide 4c were investigated as possible precursors to 4-alkylaryloxenium ions, reactive intermediates that have not been previously detected. These compounds exhibit a variety of interesting reactions, but with one possible exception, they do not generate oxenium ions. The 4-isopropyl ester 2b predominantly undergoes ordinary acid- and base-catalyzed ester hydrolysis. The 4-tert-butyl ester 2c decomposes under both acidic and neutral conditions to generate tert-butanol and 1-acetyl-1,4-hydroquinone, 8, apparently by an SN1 mechanism. This is also a minor decomposition pathway for 2b, but the mechanism in that case is not likely to be SN1. Decomposition of 2c in the presence of N3- leads to formation of the explosive 2,3,5,6-tetraazido-1,4-benzoquinone, 14, produced by N3--induced hydrolysis of 8, followed by a series of oxidations and nucleophilic additions by N3-. No products suggestive of N3--trapping of an oxenium ion were detected. The 4-isopropyl dichloroacetic acid ester 3b reacts with N3- to generate the two adducts 2-azido-4-isopropylphenol, 5b, and 3-azido-4-isopropylphenol, 11b. Although 5b is the expected product of N3- trapping of the oxenium ion, kinetic analysis shows that it is produced by a kinetically bimolecular reaction of N3- with 3b. No oxenium ion is involved. The sulfonamide 4c predominantly undergoes a rearrangement reaction under acidic and neutral conditions, but a minor component of the reaction yields 4-tert-butylcresol, 17, and 2-azido-4-tert-butylphenol, 5c, in the presence of N3-. These products may indicate that 4c generates the oxenium ion 1c, but they are generated in very low yields (ca. 10%) so it is not possible to definitively conclude that 1c has been produced. If 1c has been generated, the N3--trapping data indicate that it is a very short-lived and reactive species in H2O. Comparisons with similarly reactive nitrenium ions indicate that the lifetime of 1c is ca. 20-200 ps if it is generated, so it must react by a preassociation process. Density functional theory calculations at the B3LYP/6-31G*//HF/6-31G* level coupled with kinetic correlations also indicate that the aqueous solution lifetimes of 1a-c are in the picosecond range.

Direct detection of a transient oxenium ion in water generated by laser flash photolysis.

Laser flash photolysis of the quinol ester 2b in O2-saturated aqueous phosphate buffer at pH 7.1 with excitation at 266 nm generates a transient intermediate with lambda(max) 460 nm that decays in a first-order manner with an aqueous solution lifetime of (170 +/- 10) ns at 22 degrees C. This intermediate is not affected by O2, but reacts rapidly with N3- with an apparently diffusion-limited rate constant of (6.6 +/- 0.2) x 10(9) M-1 s-1. Steady state photolysis of 2b yields the quinol 3b as a major reaction product with a yield of ca. 30-35% after correction for photolytic decomposition of 3b. This is the same product that is quantitatively produced by hydrolysis of 2b in the dark. Photolysis of 2b in the presence of 40 mM N3- completely suppresses the yield of 3b The photolytic intermediate is identified as the aryloxenium ion 1b, that was previously indirectly detected by N3--trapping during the hydrolysis of 2b, based on the chemical behavior of the transient and the quantitative agreement of the N3-/solvent selectivity ratio, kaz/ks, measured directly during the flash photolysis experiment, and indirectly by the azide clock procedure during the hydrolysis reaction. Other, as of yet unidentified, transients are produced during the photolysis reaction. A strong transient absorbance band observed at 360 nm decays in a biphasic manner with two first-order rate constants, neither of which are affected by O2 or N3-. The lifetimes of the two intermediates of ca. 12 and 75 mus are considerably longer than that of 1b. Another very short-lived species can be detected at early reaction times (