The Premarket Tobacco Product Applications (PMTA) guidance issued by the Food and Drug Administration for electronic nicotine delivery systems (ENDSs) recommends that in addition to reporting harmful and potentially harmful constituents (HPHCs), manufacturers should evaluate these products for other chemicals that could form during use and over time. Although e-vapor product aerosols are considerably less complex than mainstream smoke from cigarettes and heated tobacco product (HTP) aerosols, there are challenges with performing a comprehensive chemical characterization. Some of these challenges include the complexity of the e-liquid chemical compositions, the variety of flavors used, and the aerosol collection efficiency of volatile and semi-volatile compounds generated from aerosols. In this study, a non-targeted analysis method was developed using gas chromatography-mass spectrometry (GC-MS) that allows evaluation of volatile and semi-volatile compounds in e-liquids and aerosols of e-vapor products. The method employed an automated data analysis workflow using Agilent MassHunter Unknowns Analysis software for mass spectral deconvolution, peak detection, and library searching and reporting. The automated process ensured data integrity and consistency of compound identification with >99% of known compounds being identified using an in-house custom mass spectral library. The custom library was created to aid in compound identifications and includes over 1,100 unique mass spectral entries, of which 600 have been confirmed from reference standard comparisons. The method validation included accuracy, precision, repeatability, limit of detection (LOD), and selectivity. The validation also demonstrated that this semi-quantitative method provides estimated concentrations with an accuracy ranging between 0.5- and 2.0-fold as compared to the actual values. The LOD threshold of 0.7 ppm was established based on instrument sensitivity and accuracy of the compounds identified. To demonstrate the application of this method, we share results from the comprehensive chemical profile of e-liquids and aerosols collected from a marketed e-vapor product. Applying the data processing workflow developed here, 46 compounds were detected in the e-liquid formulation and 55 compounds in the aerosol sample. More than 50% of compounds reported have been confirmed with reference standards. The profiling approach described in this publication is applicable to evaluating volatile and semi-volatile compounds in e-vapor products.

In our long and broad program to explore structure-activity relationships of the natural product azepinomycin and its analogues for inhibition of guanase, an important enzyme of purine salvage pathway of nucleic acid metabolism, it became necessary to investigate if the nucleoside analogues of the heterocycle azepinomycin, which are likely to be formed in vivo, would be more or less potent than the parent heterocycle. To this end, we have resynthesized both azepinomycin (1) and its two diastereomeric nucleoside analogues (2 and 3), employing a modified, more efficient procedure, and have biochemically screened all three compounds against a mammalian guanase. Our results indicate that the natural product is at least 200 times more potent toward inhibition of guanase as compared with its nucleoside analogues, with the observed Ki of azepinomycin (1) against the rabbit liver guanase = 2.5 (± 0.6) × 10−6 M, while Ki of Compound 2 =1.19 (± 0.02) × 10−4 M and that of Compound 3 = 1.29 (± 0.03) × 10−4 M. It is also to be noted that while IC50 value of azepinomycin against guanase in cell culture has long been reported, no inhibition studies nor Ki against a pure mammalian enzyme have ever been documented. In addition, we have, for the first time, determined the absolute stereochemistry of the 6-OH group of 2 and 3 using conformational analysis coupled with 2-D 1H NMR NOESY

Synthesis and biochemical inhibition studies of a novel transition state analogue inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five steps from a known compound and biochemically screened against the rabbit liver guanase. The compound exhibited competitive inhibition profile with a K i of 16.7 ± 0.5 µM.

1,4-Dioxan-2-one, 1, was synthesized and the equilibrium constant between it and the hydrolysis product 2-(2-hydroxyethoxy) acetic acid, 2, was determined as KO = 70 ± 3.5 in acidic aqueous media, 25 °C, ionic strength 1M, (KCl), 5% by volume acetonitrile. The carboxylic acid dissociation constant of 2 was determined under the same conditions to be pKa = 3.31 ± 0.02. On the basis of these two determinations, the equilibrium constant between 1 and carboxylic acid anion, 3, and the proton was calculated to be KOA = 0.034 ± 0.002 M. The stability of 1 was determined in the range of pH between 1 and 8.5 in buffered aqueous solutions under the conditions above by UV spectrophotometric methods and exhibited simple first order kinetics of decay. On the basis of buffer dilution plots, the values of ko, the rate constant for solvent mediated decomposition, were determined. The plot of log ko against pH is consistent with a three term rate law for solvolysis with a hydrogen ion catalyzed rate constant kH+ = 1.1 (± 0.1) M−1 min−1, a water catalyzed rate constant, kw = 9.9 (± 0.5) × 10−4 min−1, and a hydroxide ion catalyzed rate constant, kOH = 4.1 (± 0.3) × 104 M−1 min−1. The t1/2 for decay at pH = 7.0, at 25 °C, is ~2 h. Treatment of F344 rats with aflatoxin B1 (AFB1) (positive control) elicited the expected preneoplastic foci in the livers of each rat tested, while subsequent administration of 1 (a total of 1.32 g over 12 weeks) failed to statistically increase focal number or focal volume percent. In 8 rats administered 1 (1.32 g, 12 weeks) alone, no increase above background foci was detected. This study does not support compound 1 as a common carcinogen.

Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported transition state analog inhibitor of guanase, does not represent the true transition state of the enzymecatalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O5 oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O5 results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a simultaneous increase in hydrophobicity at O5 considerably diminishes any gain in inhibition made by solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near N3 has also a deleterious effect on inhibition. The most potent compound in the series has a Ki value of 8.0 ±1.5 M against rabbit liver guanase.