A high-yielding five-step synthesis of the title compound, methyl 7,9-dimethyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepine-1-carboxylate, starting from 2,4-dimethylaniline was developed. This synthesis involved N-alkylation of 2,4-dimethylaniline with ethyl 4-bromobutyrate to obtain ethyl 4-[(2,4-dimethylphenyl)amino] butanoate. Carbamoylation of the latter followed by hydrolysis of the resulting ester provided 4-[(2,4-dimethylphenyl)(methoxycarbonyl) amino] butanoic acid. Activation of the carboxylic acid using thionyl chloride followed by intramolecular cyclization via a Friedel-Crafts reaction using aluminum trichloride provided the title compound in good yield. Analogues of the title compound were also prepared similarly.

Vinorelbine is a semisynthetic vinca alkaloid used in the treatment of advanced breast and non-small cell lung cancers. Vincristine, a related vinca alkaloid, is 9-fold more efficiently metabolized by CYP3A5 than by CYP3A4 in vitro. This study quantified the relative contribution of CYP3A4 and CYP3A5 to the metabolism of vinorelbine in vitro using cDNA-expressed human cytochrome P450s (P450s) and human liver microsomes (HLMs). CYP3A4 and CYP3A5 were identified as the P450s capable of oxidizing vinorelbine using a panel of human enzymes and selective P450 inhibitors in HLMs. For CYP3A4 coexpressed with cytochrome b5 (CYP3A4+b5) and CYP3A5+b5, the Michaelis-Menten constants for vinorelbine were 2.6 and 3.6 μM, respectively, but the Vmax of 1.4 pmol/min/pmol was common to both enzymes. In HLMs, the intrinsic clearance of vinorelbine metabolism was highly correlated with CYP3A4 activity, and there was no significant difference in intrinsic clearance between CYP3A5 high and low expressers. When radiolabeled vinorelbine substrate was used, there were clear qualitative differences in metabolite formation fingerprints between CYP3A4+b5 and CYP3A5+b5 as determined by NMR and mass spectrometry analysis. One major metabolite (M2), a didehydro-vinorelbine, was present in both recombinant and microsomal systems but was more abundant in CYP3A4+b5 incubations. We conclude that despite the equivalent efficiency of recombinant CYP3A4 and CYP3A5 in vinorelbine metabolism the polymorphic expression of CYP3A5, as shown by the kinetics with HLMs, may have a minimal effect on systemic clearance of vinorelbine.

The availability of high performance liquid chromatography (HPLC) columns capable of operation at pH values up to 12 has allowed a greater selectivity space to be explored for method development in pharmaceutical analysis. Ammonium hydroxide is of particular value in the mobile phase because it is compatible with direct interfacing to electrospray mass spectrometers. This paper reports an unexpected N-nitrosation reaction that occurs with analytes containing primary and secondary amines when ammonium hydroxide is used to achieve the high pH and acetonitrile is used as the organic modifier. The nitrosation reaction has generality. It has been observed on multiple columns from different vendors and with multiple amine-containing analytes. Ammonia was established to be the source of the nitroso nitrogen. The stainless steel column frit and metal ablated from the frit have been shown to be the sites of the reactions. The process is initiated by removal of the chromium oxide protective film from the stainless steel by acetonitrile. It is hypothesized that the highly active, freshly exposed metals catalyze room temperature oxidation of ammonia to NO but that the actual nitrosating agent is likely N(2)O(3).

Naveglitazar [LY519818; benzenepropanoic acid, α-methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (α-S)-] is a nonthiozolidinedione peroxisome proliferator-activated receptor α-γ dual, γ-dominant agonist that has shown glucose-lowering potential in animal models and in the clinic. Studies have been conducted to characterize the disposition, metabolism, and excretion of naveglitazar in mice, rats, and monkeys after oral and/or i.v. bolus administration. After oral administration of [14C]naveglitazar, naveglitazar was well absorbed and moderately metabolized in all species evaluated, with total recoveries of radioactivity ranging from 90 to 96%. Naveglitazar was the most abundant peak observed in circulation at Cmax, representing 68 to 81% of the total radioactivity in plasma. The most prominent metabolite observed in circulation was the R-enantiomer of naveglitazar, LY591026, which is formed via enzymatic chiral inversion. para-Hydroxy naveglitazar and the sulfate conjugate of para-hydroxy naveglitazar were also observed in circulation in most species, especially in the monkey. The metabolic pathways observed include enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways. Naveglitazar was highly bound to plasma proteins among the species examined (>99%), and binding was independent of concentration. Biliary excretion was recognized as the most prominent excretion pathway in bile duct-cannulated rats (79 of the 96% recovered), producing an acyl glucuronide conjugate of naveglitazar and a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar, which were shown to be reversible. The primary excretory pathway observed in mice and monkeys was via the feces. In summary, naveglitazar was well absorbed, moderately metabolized, and excreted via the feces in mice, rats, and monkeys.

Naveglitazar [LY519818; benzenepropanoic acid, alpha-methoxy- 4-[3-(4-phenoxyphenoxy)propoxy], (alpha-S)-] is a nonthiozolidinedione peroxisome proliferator-activated receptor alpha-gamma dual, gamma-dominant agonist that has shown glucose-lowering potential in animal models and in the clinic. Studies have been conducted to characterize the disposition, metabolism, and excretion of naveglitazar in mice, rats, and monkeys after oral and/or i.v. bolus administration. After oral administration of [C-14] naveglitazar, naveglitazar was well absorbed and moderately metabolized in all species evaluated, with total recoveries of radioactivity ranging from 90 to 96%. Naveglitazar was the most abundant peak observed in circulation at C-max, representing 68 to 81% of the total radioactivity in plasma. The most prominent metabolite observed in circulation was the Renantiomer of naveglitazar, LY591026, which is formed via enzymatic chiral inversion. para-Hydroxy naveglitazar and the sulfate conjugate of para-hydroxy naveglitazar were also observed in circulation in most species, especially in the monkey. The metabolic pathways observed include enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways. Naveglitazar was highly bound to plasma proteins among the species examined (> 99%), and binding was independent of concentration. Biliary excretion was recognized as the most prominent excretion pathway in bile duct-cannulated rats (79 of the 96% recovered), producing an acyl glucuronide conjugate of naveglitazar and a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar, which were shown to be reversible. The primary excretory pathway observed in mice and monkeys was via the feces. In summary, naveglitazar was well absorbed, moderately metabolized, and excreted via the feces in mice, rats, and monkeys.

Ruboxistaurin (LY333531), a potent and isoform-selective protein kinase C beta inhibitor, is currently undergoing clinical trials as a therapeutic agent for the treatment of diabetic microvascular complications. The present study describes the disposition and metabolism of [14C]ruboxistaurin following administration of an oral dose to dogs, mice, and rats. The study revealed that ruboxistaurin was highly metabolized in all species. Furthermore, the results from the bile duct-cannulated study revealed that ruboxistaurin was well absorbed in rats. The primary route of excretion of ruboxistaurin and its metabolites was through feces in all species. The major metabolite detected consistently in all matrices for all species was the N-desmethyl metabolite 1, with the exception of rat bile, in which hydroxy N-desmethyl metabolite 5 was detected as the major metabolite. Other significant metabolites detected in dog plasma were 2, 3, 5, and 6 and in mouse plasma 2, 5, and 19. The structures of the metabolites were proposed by tandem mass spectrometry with the exception of 1, 2, 3, 5, and 6, which were additionally confirmed either by direct comparison with authentic standards or by nuclear magnetic resonance spectroscopy. To assist identification by nuclear magnetic resonance spectroscopy, metabolites 3 and 5 were produced via biotransformation using recombinant human CYP2D6 and, likewise, metabolite 6 and compound 4 (regioisomer of 3 which did not correlate to metabolites found in vivo) were produced using a microbe, Mortierella zonata. The unambiguous identification of metabolites enabled the proposal of clear metabolic pathways of ruboxistaurin in dogs, mice, and rats.

A binding constant was determined for the complexation reaction between alprostadil (PGE1) and α-cyclodextrin (α-CD). This constant was used to calculate the fraction PGE1 free upon reconstitution of Caverject dual chamber syringe, indicated for the treatment of erectile dysfunction. The determination was based on the measurement of the chemical shift of the C20 methyl protons of PGE1. The observed chemical shift varies as a linear function of the amount of PGE1 bound. The binding constant was obtained from the binding isotherm, a curve of the observed chemical shift versus free ligand (α-CD) concentration, through the application of non-linear regression analysis. A value K11=966M−1±130M−1 (2s), measured at 27°C, was obtained. This value is in good agreement with those reported in the literature. The percent PGE1 free was subsequently calculated for the reconstituted solution and in the corpora cavernosum after injection. The latter showed PGE1 to be delivered essentially quantitatively to the targeted site.

The application of adiabatic inversion pulses to the detection of (1)H-(15)N heteronuclear correlations is described. The pulse sequences studied were gHSQC, CRISIS-gHSQC, gHMBC and CRISIS-gHMBC. The poor inversion quality of rectangular 180 degrees X pulses can lead to a loss of signal at the peripheries of the spectrum. Replacing these pulses with adiabatic sweeps significantly improves sensitivity across the potentially large (15)N spectral window. Satellite spectrum profiles are shown to demonstrate the increase in sensitivity when employing adiabatic pulses on wide spectral widths. Additionally, the active pharmaceutical nizatidine was used as a model compound to demonstrate the improvements in the long-range correlation data.

Phase I oxidative metabolism of nitrogen-containing drug molecules to their corresponding N-oxides is a common occurrence. There are instances where liquid chromatography/tandem mass spectometry techniques are inadequate to distinguish this pathway from other oxidation processes, including C-hydroxylations and other heteroatom oxidations, such as sulfur to sulfoxide. Therefore, the purpose of the present study was to develop and optimize an efficient and practical chemical method to selectively convert N-oxides to their corresponding amines suitable for drug metabolism applications. Our results indicated that efficient conversion of N-oxides to amines could be achieved with TiCl(3) and poly(methylhydrosiloxane). Among them, we found TiCl(3) to be a facile and easy-to-use reagent, specifically applicable to drug metabolism. There are a few reports describing the use of TiCl(3) to reduce N-O bonds in drug metabolism studies, but this methodology has not been widely used. Our results indicated that TiCl(3) is nearly as efficient when the reductions were carried out in the presence of biological matrices, including plasma and urine. Finally, we have shown a number of examples where TiCl(3) can be successfully used to selectively reduce N-oxides in the presence of sulfoxides and other labile groups.

Numerous indoloquinoline alkaloid structures have been identified from extracts of the West African plant Cryptolepis sanguinolenta . Recently, through the use of 2D NMR methods and cryogenic NMR probe technology in conjunction with computer‐assisted structure elucidation (CASE) methods, the structures of some chemical degradation products of this family of alkaloids have also been reported. We now report the characterization of a novel indoloquinoline dimeric alkaloid, quindolinocryptotackieine, through the extensive utilization of CASE methods. The NMR data presented here were collected over a decade earlier before the elucidation of the structure was possible, since manual analysis did not present a conclusive structure, whereas CASE produced a series of structures from which the structure could be verified. The original mass spectrometric (MS) data collected for the sample were problematic. Contemporary MS data were instead recollected from remaining small quantities of this alkaloid using modern instrumentation. The re‐collected data gave a usable molecular ion and several key fragment ions that were diagnostically useful. Copyright © 2003 John Wiley & Sons, Ltd.

During the terminal heat sterilization of the lipid emulsion final dose formulation of the photodynamic therapeutic (PDT) agent tin ethyl etiopurpurin (SnET2), a new degradant was observed at very low levels. The degradant, which was prone to photo-instability, was isolated by preparative chromatography and subsequently characterized by mass spectrometry and NMR methods. Reproducible parent ion clusters were only observable via negative ion APCI methods. Because of the limited isolate sample, NMR characterization was done using 1.7 min SMIDG (SubMicro Inverse-Detection Gradient) NMR probe technology in conjunction with the accordion-optimized IMPEACH-MBC long-range heteronuclear shift correlation experiment. The "static" 8 Hz optimization of the GHMBC experiment failed to allow the observation of a number of long-range correlations that were of critical importance to the determination of the structure of the impurity. In contrast, all of the correlations required to assemble the structure were obtained from an IMPEACH-MBC experiment optimized for long-range heteronuclear couplings in the range from 2-10 Hz.

Synthetic efforts towards the indole alkaloid natural product roquefortine C resulted in the formation of an unknown intermediate. Elucidation of the structure of this molecule relied on the use of long-range H-1-N-15 2D-NMR. Computational predictions were used to facilitate the location of weak responses in long-range H-1-C-13 HMBC spectra. These methods provided conclusive evidence that this compound possessed a novel tetracycle. The complete H-1, C-13, and N-15 chemical shift assignments of this unique fused imidazolidinone tetrahydropyrroloindole derivative are reported.

The application of the randomly optimized RDSQC (Randomly optimized Direct correlation Single Quantum Coherence) experiment for the detection of direct correlations facilitated the characterization of an unknown compound. The expected structure consisted of purely aliphatic moieties. The actual, identified compound contained the desired structure plus an adenosine functionality with two protons whose direct proton-carbon couplings were over 200 Hz. Application of a 130 Hz optimized direct heteronuclear GHSQC experiment afforded no correlations for the adenine responses. The RDSQC experiment allowed for the simultaneous optimization of multiple couplings in a range of 130 to 220 Hz producing a direct correlation spectrum with all the expected responses.

The first demonstrated example of F-19-N-15 long-range heteronuclear shift correlation spectroscopy at natural abundance is reported. Because of the very large variation in the size of (2)J(N,F) vs (3)J(N,F) long-range heteronuclear couplings, the utilization of one of the new accordion-optimized long-range heteronuclear shift correlations experiments is essential if all possible correlations are to be observed in a single experiment. A modified IMPEACH-MBC pulse sequence was used in conjunction with an optimization range from 4 to 50 Hz to demonstrate the technique using a mixture of 2- and 3-fluoropyridine, which had (2)J(N,F) and (3)J(N,F) long-range couplings of -52 and 3.6 Hz, respectively. Because of the size of the (2)J(N,F) long-range coupling constant, a J-modulation of the long-range correlation response is observed in the spectrum resulting in a 'doublet' in F-1 due to amplitude modulation. The size of the 'doublet' is shown to be a function of the parameter selection (t(1max), T-max, T-min and spectral width in F-1). This behavior is similar to F-1 'skew' associated with long-range correlation responses in ACCORD-HMBC spectra which has been analyzed in detail previously. Copyright (C) 2002 John Wiley Sons, Ltd.

Identification of degradants of pharmaceuticals is a necessary challenge of the drug development process following the subjection of candidate molecules to a variety of physico-chemical stresses. It would be desirable to be able to conduct such studies on a minimal amount of material. As a prototypical study, the isolation and identification of degradants of a sample of the complex indoloquinoline alkaloid, cryptospirolepine, was undertaken after prolonged storage in DMSO solution using a combination of cryogenic NMR probe technology and CASE (Computer-Assisted Structure Elucidation) programs. None of the starting alkaloid remained after storage; a chromatogram of the DMSO solution demonstrated the presence of >25 components in the mixture. The two most abundant degradation products were identified as the known alkaloid cryptolepinone (similar to35%) and an unprecedented rearrangement product, DP-2, (similar to16%).

A direct comparison of the spectral data for synthetic 2-methyl-6,7-dimethoxy-3'-methoxy-4'-hydroxyoxobenzylisoquinoline iodide (1) and its positional isomer 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2) with the data obtained for the oxobenzylisoquinoline alkaloid thalprzewalskiinone revealed that the original structural assignment of the alkaloid as 1 was in error. These results mandate the revision of structure of thalprzewalskiinone to 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2).

A novel tetrahydroprotoberberine-aporphine dimeric alkaloid, (-)-thalibealine (1), was isolated from the roots of Thalictrum wangiii, and its structure established via spectroscopic analysis. Three other alkaloids were isolated, including the benzyltetrahydroisoquinoline-aporphine dimer (+)-thalmelatidine, the aporphine (+)-magnoflorine, and the protoberberine berberine. This is the first reported isolation of a tetrahydroprotoberberine-aporphine dimer from nature, as well as the first reported isolation of constituents from Thalictrum wangii.

The development of new NMR probe technologies has been an active area of research effort for the past decade. Recently, cryogenically cooled NMR probes have been the subject of considerable interest in the light of the large gains in sensitivity and hence savings in spectrometer time that can be realized by utilizing this technology. With low gamma nuclides, such as N-15, time savings may be less of an issue than the ability to work with available samples rather than having to isolate additional material for analysis. Results of 5-10 Hz optimized CIGAR-HMBC H-1-N-15 experiments at natural abundance obtained using a 5 mm Varian H-1-N-15 Cryo-Q NMR probe are compared with those from a 3 mm gradient inverse triple resonance probe using a 2 mg sample of the oxazolidinone antibiotic eperezolid in a 3 mm NMR tube. Using the cryogenic NMR probe, a H-1-N-15 CIGAR-HMBC spectrum in which most previously reported long-range couplings are observed was recorded in similar to 10 min; almost all of the expected long-range responses were observed within 26 min. Acquiring the same data set with identical parameters using a conventional 3 mm gradient inverse triple resonance probe gave data that were of substantially lower quality. Acquiring data comparable to the 26 min cryogenic probe data with the conventional 3 mm probe required similar to4 h to observe all of the responses and similar to 18 h to obtain a spectrum with a signal-to-noise ratio comparable to the data set acquired with the cryogenic NMR probe. Copyright (C) 2001 John Wiley & Sons, Ltd.

A novel optimization method is described for the acquisition of direct one-bond heteronuclear correlations. The RDSQC (Randomly optimized Direct correlation Single Quantum Coherence) experiment utilizes an optimization based on the randomly ordered sampling of a range of couplings. The random order of the 1/(2*((1)J(CHmin))) delays removes the signal dependency on a single type of apodization, thus eliminating a significant portion of the F-1 artifacts induced in the accordion-optimized ADSQC experiment. Compared to the statically optimized GHSQC, the randomly optimized data maintains the desired signal intensity in most cases, with a small loss for the weakly coupled proton-carbon pairs and significant gains for the more strongly coupled pairs. Compared to the accordion-optimized ADSQC data, the randomly optimized data afforded similar signal-to-noise without the F-1 modulated artifacts simplifying spectral interpretation.