Chemoenzymatic Total Synthesis of (+)-10-Keto-Oxycodone from Phenethyl Acetate.

The total synthesis of (+)-10-keto-oxycodone was attained from phenethyl acetate in a stereoselective manner. Absolute stereochemistry was established via enzymatic dihydroxylation of phenethyl acetate with the recombinant strain JM109 (pDTG601A) that furnished the corresponding cis-cyclohexadienediol whose configuration corresponds to the absolute stereochemistry of the ring C of (+)-10-keto-oxycodone. Intramolecular Heck reaction was utilized to establish the quaternary carbon at C-13, along with the dibenzodihydrofuran functionality. The C-14 hydroxyl and C-10 ketone were installed via SmI2-mediated radical cyclization, and oxidation of a benzylic alcohol (obtained from an intermediate nitrate azide), respectively. The synthesis of (+)-10-keto-oxycodone was completed in a total of 14 operations (21 steps) and an overall yield of ~2%. Experimental and spectral data are provided for key intermediates and new compounds.

Modified in vivo comet assay detects the genotoxic potential of 14-hydroxycodeinone, an α,ß-unsaturated ketone in oxycodone.

14-Hydroxycodeinone (14-HC) is an α,ß-unsaturated ketone impurity found in oxycodone drug substance and has a structural alert for genotoxicity. 14-HC was tested in a combined Modified and Standard Comet Assay to determine if the slight decrease in % Tail DNA noted in a previously conducted Standard Comet Assay with 14-HC could be magnified to clarify if the response was due to cross-linking activity. One limitation of the Standard Comet Assay is that DNA cross-links cannot be reliably detected. However, under certain modified testing conditions, DNA cross-links and chemical moieties that elicit such cross-links can be elucidated. One such modification involves the induction of additional breakages of DNA strands by gamma or X-ray irradiation. To determine if 14-HC is a DNA crosslinker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, 14-HC was administered orally to mice up to 320 mg/kg/day. Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a nonstatistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, 14-HC was identified as a cross-linking agent in the duodenum in the Modified Comet Assay.

The abuse potential of Remoxy(®), an extended-release formulation of oxycodone, compared with immediate- and extended-release oxycodone.

OBJECTIVES: Remoxy(®) is a water-insoluble, highly viscous oral formulation of oxycodone extended release (ER) currently in development. The primary objective was to determine the abuse potential of Remoxy under fed conditions relative to oxycodone ER and immediate release (IR) under fasted conditions and compared with placebo (treatment group X). A secondary objective was to evaluate abuse potential under reversed fed/fasted conditions (treatment group Y). DESIGN: Phase I randomized double-blind triple-dummy placebo- and active-controlled 6-way crossover study. Setting. A single US site. PATIENTS. Healthy men and women aged 18-50 years who were nondependent, recreational opioid users. Interventions. Remoxy 40 mg whole and chewed, oxycodone ER 40 mg whole and crushed, oxycodone IR 40 mg crushed, and placebo. OUTCOME MEASURES: The primary endpoint was the drug liking subscale of the drug effects questionnaire assessed by various pharmacodynamic parameters. Secondary endpoints included additional pharmacodynamic measures, chewing duration, and safety measures. RESULTS: In treatment group X, Remoxy whole (fed) and chewed (fed) had a significantly lower abuse potential compared with oxycodone ER (crushed, fasted) and IR (fasted) based on the majority of pharmacodynamic parameters of interest for the primary endpoint (drug liking subscale) as well as secondary endpoints. Treatment group Y showed generally similar results. CONCLUSIONS: The abuse potential of Remoxy when taken whole or chewed was significantly lower than two comparators with known abuse potential, including oxycodone IR and crushed oxycodone ER, under the fed/fasted conditions tested. Remoxy may be associated with a reduced risk potential for abuse.

Importance of using highly pure internal standards for successful liquid chromatography/tandem mass spectrometric bioanalytical assays.

Internal standards (IS) with similar physicochemical properties to the analyte provide multiple advantages in liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalytical methods such as: reduction of the analysis run time, improvement in the intra-injection reproducibility, impact reduction of matrix and ionization effects. However, it is important to evaluate the purity of the IS prior to their use. Indeed, a minor impurity in the IS may lead to an important issue during bioanalytical method development. Stable labelled internal standards are usually appropriate IS for bioanalysis. The use of oxycodone-D3, ursodiol-D5 and atovaquone-D4 as internal standards in three different bioanalytical methods was evaluated. During oxycodone, oxymorphone and noroxycodone simultaneous quantification method development, oxymorphone was identified as a contaminant in oxycodone-D3. Since the limit of quantification for oxymorphone was very low (10 pg/mL), the presence of an even low percentage of oxymorphone in oxycodone-D3 leads to the change of the stable labelled IS for an analogue, ethylmorphine. 23-Nordeoxycholic acid was preferred to ursodiol-D5 as internal standard for the ursodiol, tauroursodiol and glycoursodiol simultaneous quantification method. Indeed, more than 7% of ursodiol was identified in the ursodiol-D5 which could not be bypassed by decreasing the IS concentration without compromising the linearity. An atovaquone-D4 reference standard revealed the non-negligible presence of atovaquone-D5 to atovaquone-D8 that has a large impact on the method validation. Therefore, atovaquone-D4 was sent for recertification since its isotopic purity was found to be much less than the isotopic purity mentioned on its certificate of analysis. Consequently, during bioanalytical method development, the purity of the IS should be scrutinized.

Rapid quantification of urinary oxycodone and oxymorphone using fast gas chromatography-mass spectrometry.

Human urine specimens that were determined to be presumptively positive for oxycodone and its metabolite, oxymorphone, by immunoassay screening were assayed using fast gas chromatography-mass spectrometry to positively identify and quantify the oxycodone and oxymorphone present. Urine specimens were first spiked with deuterated internal standards, oxycodone-d(3) and oxymorphone-d(3), subjected to acid hydrolysis, and then extracted using a positive-pressure manifold and mixed-bed solid-phase cartridge extraction methodology. Extracts were derivatized using methoxylamine and acetic anhydride. The acetylated-oxime derivatives of oxycodone and oxymorphone were identified and quantified using a selective ion monitoring (SIM). The method was found to be linear for both analytes to 1600 ng/mL, and limits of detection for oxycodone and oxymorphone were found to be 40 ng/mL and 20 ng/mL, respectively. Interlaboratory data comparisons (n = 40) showed correlation coefficients of 0.9999 and 0.9997 for oxycodone and oxymorphone, respectively. Twelve semisynthetic, structurally similar compounds at concentrations of 5000 ng/mL were assayed in the presence of oxycodone and oxymorphone and found not to interfere with identification and quantitation by this method. Finally, exact mass and tandem mass spectrometry techniques were employed to elucidate the structures of the SIM ions.

Determination of 6-oxo-morphinans, as the oximes, by difference circular dichroism spectroscopy.

A negative Cotton effect is observed in the circular dichroism (CD) spectra of 6-oxo-morphinans in the wavelength range of n-pi* electron transitions. 6-oxo-Morphinans can be transformed into oxime derivatives with hydroxylamine and after oxime formation the CD spectra are significantly different. Oxime formation was monitored by CD and by HPLC. It was established that under the experimental conditions used oxime formation was complete within 90 min. The method suggested for the determination of 6-oxo-morphinans is based on the considerable differences between the ellipticities before and after the oxime formation. The ellipticity difference varies linearly with concentration in the range 2 x 10(-5)-5 x 10(-4) mol L(-1) for the three 6-oxo-morphinans examined (oxycodone, hydrocodone, and 14-hydroxycodeinone). For hydrocodone the dependence is also linear in a lower concentration range (5 x 10(-6)-10(-4) mol L(-1)). The new difference CD spectroscopic method can be applied to the selective determination of 6-oxo-morphinans in bulk and dosage forms.

The [4+2]] addition of singlet oxygen to thebaine: new access to highly functionalized morphine derivatives via opioid endoperoxides.

The photooxidation of thebaine (3) with a sun lamp affords two main products: hydrodibenzofuran 10 (major) and benzofuran 11 (minor). The latter compound becomes predominant if a Hg lamp is used instead of a sun lamp. The formation of 10 proceeds via an endoperoxide intermediate that undergoes oxidation at the nitrogen atom. Protection of the nitrogen either by protonation or quaternization prevents its oxidation and thus the photooxidation of 3 in acid media constitutes a one-pot procedure for the preparation of 14-hydroxycodeinone 35. Photooxidation of the thebaine ammonium salt 31 allows the isolation in good yields of the corresponding to thebaine endoperoxide 32. The selective protection and reduction of the keto, aldehyde, and olefinic groups of hydrodibenzofuran 10 allowed the preparation of several diol and keto alcohol derivatives. This is the first report on the use of photooxidation to functionalize thebaine at C(6) and C(14) and also the first on the isolation of opioid endoperoxides.

Transformations of codeine to important semisynthetic opiate derivatives by Pseudomonas putida m10.

A biotransformation mixture which contained codeine and washed cells of Pseudomonas putida M10 gave rise to a number of transformation products that are of clinical importance which included hydrocodone, dihydrocodeine and 14beta-hydroxycodeine. Incubations with the same organism and codeinone gave rise to 14beta-hydroxycodeinone and 14beta-hydroxycodeine. Cell-free extracts and membrane fractions of P. putida M10 were shown to catalyse the 14beta-hydroxylation of codeinone. In addition, the potent analgesic oxycodone was shown to be produced from 14beta-hydroxycodeinone.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans.

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.

Affinity profiles of novel delta-receptor selective benzofuran derivatives of non-peptide opioids.

Highly selective heterocyclic opioid ligands with potent delta-antagonist activity have been developed on the basis of the "message-address" concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the mu-opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the delta-opioid receptor, much less potency toward the mu-binding site, and were the least effective at the kappa-site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers delta-receptor selectivity. The Na+ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dose-dependent.

Determination of oxycodone in plasma and identification of a major metabolite.

An electron-capture GLC method is described for oxycodone and its major metabolite, noroxycodone, in plasma and urine. The method involves extraction of the two substances into benzene-isopropranol at pH 10.4, followed by back-extraction into 0.1 N HCl. The acid phase is washed with hexane and made alkaline prior to reextraction into benzene-isopropanol. The solvent is removed by evaporation, and the heptafluorobutyryl derivatives of the test substances are formed. After removal of excess reagent, oxycodone and noroxycodone are quantitated by GLC. The characteristics of both substances, with respect to plasma levels in dogs and analgesic activity in mice, are reported. Isolation of noroxycodone from human urine and its identification by TLC, GLC, and mass spectrometry are described.