Structure Elucidation of Macrolide Antibiotics Using MSn Analysis and Deuterium Labelling.

The 14- and 16-membered macrolide antibiotics are an important structural class. Ubiquitously produced by a number of bacterial strains, namely actinomycetes, purification and structure elucidation of the wide array of analogs is challenging, both for discovery efforts and methodologies to monitor for byproducts, metabolites, and contaminants. Collision-induced dissociation mass spectrometry offers an attractive solution, enabling characterization of mixtures, and providing a wealth of structural information. However, interpretation of these spectra can be difficult. We present a study of 14- and 16-membered macrolide antibiotics, including MSn analysis for unprecedented depth of coverage, and complimentary analysis with D2O and H218O labeling to elucidate fragmentation mechanisms. These analyses contrast the behaviors of varying classes of macrolides and highlight how analogues can be identified in relation to similar structures, which will provide utility for future studies of novel macrolides, as well as impurities, metabolites, and degradation products of pharmaceuticals. Graphical Abstract.

Synthesis and antibacterial activities of some novel 17, 18-unsaturated carbonyl compounds derivated from josamycin.

Some novel josamycin derivatives bearing an arylalkyl-type side chain were designed and synthesized. By HWE or Wittig reaction, 16-aldehyde group of josamycin analogs were converted into unsaturated carbonyl compounds. They were evaluated for their in vitro antibacterial activities against a panel of respiratory pathogens. 8b and 8e exhibited comparable activities against a panel of respiratory pathogens, especially to resistant ones in the series of desmycarosyl josamycin analogs. Among of all the target molecules, 21 showed the best antibacterial activities.

Structure elucidation, complete NMR assignment and PM5 theoretical studies of new hydroxy-aminoalkyl-alpha,beta-unsaturated derivatives of the macrolide antibiotic josamycin.

Four new hydroxy-aminoalkyl derivatives of alpha,beta-unsaturated macrolide-josamycin (2-5) have been synthesised and their structures have been studied by means of (1)H and (13)C NMR and FT-IR methods. Complete assignment of resonances in the (1)H and (13)C NMR spectra has been made on the basis of (1)H-(13)C HSQC, (1)H-(13)C HMBC, (1)H-(1)H COSY, (1)H-(1)H NOESY 2D experiments. Spectroscopic data indicated that for the derivatives 3 and 4 some equilibrium between two different structures exists in contrast to derivatives 2 and 5. The lowest-energy structures of the new derivatives of josamycin have been calculated and visualised by PM5 method at semi-empirical level of theory, taking into account the NMR and FT-IR data. The most significant differences between the structures of josamycin and its newly synthesised derivatives' were found in the conformation of the macrolide aglycone part and in the mutual orientation of the 4-O-isovalerylmycarosylmycaminose moiety relative to the aglycone part. PM5 semi-empirical calculations indicated that the structures of the new macrolide derivatives are stabilised by rather weak intramolecular hydrogen bonds in agreement with spectroscopic data. Antimicrobial properties of the new derivatives 2-5 as well as those having an acetate group at C-3 (6 and 7) were determined and compared to that of the parent macrolide antibiotic josamycin (1).

Preparation of floating microspheres for fish farming.

The aim of this study was to develop floating microspheres with practical applications to fish farming. Each microsphere with a central hollow cavity was prepared using a solvent diffusion and evaporation method with Eudragit E100. Various manufacturing parameters were investigated by single factor method. The macrolide antibiotic josamycin was selected as a model drug. The loading efficiency of the drug in the microspheres was 64.7%. In the release study, virtually none of the drug was released into the fresh water whereas the entire drug was released from the josamycin-loaded microspheres into the simulated gastric fluid of rainbow trout (pH 2.7). The buoyancy was excellent with approximately 90% of the microspheres still floating after 24h.

[Identification of the components and products of hydrolysis in acetylleucomycin by LC-MS].

AIM: To identify the components of acetylleucomycin and its hydrolytic products by LC-MS. METHODS: Acetylleucomycin was separated on a Diamonsil C18 column with 0.1 mol x L(-1) ammonium acetate-acetontrile (35 : 65) as mobile phase. The LC-MS was equipped with an electorspray ion source (ESI), which was set at the positive ion mode, and the mass spectra of each component in chromatogram were obtained with difference cone voltage. RESULTS: The components of acetylleucomycin and its hydrolytic products can be separated by HPLC. The components were identified according to the molecular weight and its major mass fragment ions. The major components identified in domastic acetylleucomycin were acetylleucomycin A4, A5; acetylleucomycin A1, A3; acetylleucomycin A6, A7, and acetylleucomycin A13. The hydrolytic products of acetylleucomycin were not kitasamycin, but some non-complete hydrolytic product. CONCLUSION: The method is rapid, sensitive and specific. It' s suitable to application in the fields of multi-components antibiotics analysis.

Kinetics of macrolide action: the josamycin and erythromycin cases.

Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of full-length proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacterio-toxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyl-tRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.

Anodic polarographic determination of josamycin in formulations and spiked human urine.

The anodic polarographic behaviour of josamycin (a macrolide antibiotic) has been studied using direct current (DC(t)) polarography and differential pulse polarography (DPP). In Britton-Robinson buffers (BRb) josamycin exhibited well-defined anodic polarographic waves in the pH range 2-8. In BRb of pH 6, the diffusion-current constant was 1.62 microA mmol(-1) (n = 6). The current-concentration plots are rectilinear over the range 10-50 and 5-40 microg/ml using the DC(t) and DPP modes, respectively, with a detection limit of 4.83 x 10(-6) M adopting the DPP mode. The method was applied to commercial tablets adopting both DC(t) and DPP modes, the recoveries % were 99.13 +/- 2.22 (n = 10) and 99.82 +/- 1.82 (n = 7), respectively. The method was further extended to the in vitro determination of josamycin in human urine, the recovery % (n = 6) was 100.06 +/- 2.84. The number of electrons involved in the electrode process could be accomplished and a proposal of electrode reaction was presented.

Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate.

Coupled liquid chromatography and ion trap mass spectrometry (LC/MS) was used for the characterization of the semi-synthetic 16-membered ring macrolide josamycin propionate. On-line identification of impurities in this antibiotic complex was performed with an ion trap mass spectrometer without recourse to time-consuming isolation and purification procedures. Ion trap mass spectrometry is ideally suited to identification of impurities because it provides MSn capability, enabling multiple stages of mass spectrometry to obtain the maximum amount of structural information for a given molecule. The ion trap was used with an electrospray ionization source operated in the positive ion mode or with an atmospheric pressure chemical ionization source operated in the negative ion mode. The identity of the unknown compounds was deduced using the MS/MS and MSn collision-induced dissociation spectra of reference substances or structural analogs as interpretative templates, combined with knowledge about the nature of functional group fragmentation behavior. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing josamycin propionate. The knowledge of the fragmentation behavior is also of importance in further research on other 16-membered macrolides.

Synthesis of new 14-membered macrolide antibiotics via a novel ring contraction metathesis.

[reaction: see text] A novel ring opening ring closing metathesis (ROM-RCM) was demonstrated for cyclic conjugated dienes, effecting the excision of a C(2)H(2) unit and a net ring contraction. Applying the ring contraction metathesis, new 14-membered ring macrolide antibiotics were synthesized in a single step from existing 16-membered ring macrolides. This new class of macrolide antibiotics will provide access to new therapeutics for the treatment of macrolide-resistant bacterial infections.

Comparative bioavailability of josamycin, a macrolide antibiotic, from a tablet and solution and the influence of dissolution on in vivo release.

The bioavailability of josamycin from a tablet formulation (2 x Josacine 500 mg tablets) was investigated and compared with the bioavailability of a solution (containing 1 g drug and buffered at pH 4.0) following administration to six healthy human volunteers. Bioavailability profiles for the solution indicated that the drug was inherently rapidly absorbed with a mean Cmax of 1.64 +/- 0.67 mg L-1 attained at a mean tmax of 0.39 +/- 0.08 h. The AUC0-last was 1.510 +/- 0.687 mg h L-1. Bioavailability was significantly lower from the tablets than from the solution. Highly variable serum concentration-time profiles were obtained from the tablets and Cmax values ranged from 0.05 to 0.71 mg L-1 with a tmax range of 0.33-2.0 h. AUC0-last values ranged from 0.03 to 0.95 mg h L-1. Dissolution of josamycin from the tablets was generally unaffected at low pH (pH 1.2-5.0), but, rather, limited predominantly by tablet disintegration. However, dissolution was increasingly limited as the pH increased from 5.0 to 9.0. Besides poor disintegration, the particularly low intrinsic dissolution rate and solubility of josamycin at these pH values is likely to further reduce the dissolution rate. Comparison of the solution and tablet serum concentration-time profiles suggests that the absorption of josamycin from the tablets was dissolution rate limited. This is supported by the in vitro dissolution-pH topogram, which suggests that dissolution will be particularly rate limiting if dissolution of whole or parts of tablets occurs in gastro-intestinal fluid above pH 5.0.

[A 13-week subchronic toxicity study of josamycin in F344 rats].

A 13-week subchronic toxicity study of josamycin was performed in male and female F344 rats to determine the maximum tolerable dose (MTD) for subsequent investigation of the carcinogenicity. As animals refused to take diet containing 5.0% josamycin in our preliminary study, dose levels in the present study were determined as 0, 0.16, 0.32, 0.63, 125 and 2.5% in diet. Rats were randomly allocated to 6 groups, each consisting of 10 males and 10 females. No animal died during the administration period and no group showed significant changes in body weight gain. Definite toxicity of josamycin was not noted in hematological and serum biochemical examinations. Histopathological examinations revealed no particular findings related to josamycin administration except cecal enlargement in the 1.25 and 2.5% groups. based on the results of the present study, it was concluded that the MTD of josamycin in 2.5% in diet, because the dietary dose level of 2.5% proved to exert no significant toxicological signs.

Sensitive determination of josamycin and rokitamycin in plasma by high-performance liquid chromatography with fluorescence detection.

Rokitamycin and josamycin were successfully derivatized with dansylhydrazine in 20 min at 60 degrees C. Rokitamycin and josamycin levels were determined in plasma after ion-pair extraction into hexane-isoamyl alcohol with lauryl sulphate and precolumn derivatization. Resolution was obtained by liquid chromatography with fluorescence detection (352/537 nm) in 12 min. The limit of detection was 20 ng/ml macrolide starting from 1 ml of plasma, and linearity was demonstrated between 50 and 400 ng/ml. Inter-run coefficients of variation were 10.2% at 100 ng/ml and 9.1% at 300 ng/ml. The system was reliably used for pharmacokinetic studies in plasma.