Glucolipsin A and B, two new glucokinase activators produced by Streptomyces purpurogeniscleroticus and Nocardia vaccinii.

During the screening of the natural products for their ability to increase the activity of glucokinase by relieving inhibition by long chain fatty acyl CoA esters (FAC), two novel compounds, glucolipsin A (1) and B (2) were isolated from the butanol extracts of Streptomyces purpurogeniscleroticus WC71634 and Nocardia vaccinii WC65712, respectively. The structures of these two compounds were established by spectroscopic methods and chemical degradation. Glucolipsin A (1) and B (2) relieved the inhibition of glucokinase by FAC with RC50 values of 5.4 and 4.6 microM.

Monitoring in vitro experiments using microdialysis sampling on-line with mass spectrometry.

A method has been developed for the real-time analysis of components in in vitro reactions by the on-line combination of microdialysis sampling (MD) with tandem mass spectrometry (MS/MS) and single stage mass spectrometry (MS). Apparatus and parameters associated with the integration have been studied. Analytical figures of merit for the drug gepirone have been determined. The qualitative 'limit of identification' was found to be 100 ng/ml and 200 ng/ml for methods using thermospray and electrospray MS interfaces, respectively. Using this approach, monitoring of in vitro experiments involving drug metabolites, enzymatic reactions, and ligand-protein binding interactions were performed.

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis.

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.

Fusaricide, a new cytotoxic N-hydroxypyridone from Fusarium sp.

A new cytotoxic N-hydroxypyridone, fusaricide (1), was isolated from a Fusarium sp. Its structure was solved by X-ray diffraction and spectroscopic analyses.

Harziphilone and fleephilone, two new HIV REV/RRE binding inhibitors produced by Trichoderma harzianum.

During the screening of the natural products for their ability to inhibit the binding of REV (regulation of virion expression) protein to [33P] labeled RRE (REV responsive element) RNA, two novel fungal metabolites, harziphilone and fleephilone, were isolated from the butanol-methanol (1:1) extract of the fermentation broth of Trichoderma harzianum by bioassay guided fractionation. The structures of these two new compounds were established by spectroscopic methods. Harziphilone and fleephilone showed inhibitory activity against the binding of REV-protein to RRE RNA with IC50 values of 2.0 microM and 7.6 microM, respectively. However both compounds did not protect CEM-SS cells from acute HIV infection at concentration levels up to 200 micrograms/ml using an XTT dye reduction assay. In addition, harziphilone demonstrated cytotoxicity at 38 microM against the murine tumor cell line M-109.

Pyrrolosporin A, a new antitumor antibiotic from Micromonospora sp. C39217-R109-7. II. Isolation, physico-chemical properties, spectroscopic study and X-ray analysis.

Pyrrolosporin A (1) is a new macrolide antitumor antibiotic possessing an unusual spiro-alpha-acyltetronic acid moiety. The antibiotic was isolated from the fermentation broth of Micromonospora sp. by vacuum liquid chromatography, crystallization and reversed phase HPLC (C18). The structure was determined by a combination of NMR, MS, IR, UV, X-ray analysis and degradation studies.

Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques.

A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structures of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy-and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and delta 1, 10 a-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnostic tool for new degradation products.

Predictive strategy for the rapid structure elucidation of drug degradants.

Structural information on drug degradants and impurities can serve to accelerate the drug discovery and development cycle. Traditional structure elucidation methodologies for obtaining this information are often slow and resource-consuming; therefore, LC/MS profiling and LC/MS/MS substructural analysis methodologies have been developed to rapidly and accurately elucidate structures of impurities and degradants. This work is a further development of methodologies used for the elucidation of degradation products of paclitaxel [K.J. Volk et al., Proc. 9th AAPS Ann. Meeting, 1994, p.29]. In this study cefadroxil was used as a model compound for the evaluation of a predictive strategy for the production and elucidation of impurities and degradants induced by acid, base, and heat, using LC/MS and LC/MS/MS profiling methodology, resulting in an LC/MS degradant database which includes information on molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. Furthermore, libraries such as this can provide a predictive foundation for pre-clinical development work involving drug stability, synthesis, and monitoring.

Ascosteroside, a new antifungal agent from Ascotricha amphitricha. II. Isolation and structure elucidation.

The novel antifungal agent ascosteroside (1) was isolated from cultured broth of Ascotricha amphitricha (ATCC 74237). The structure based on spectroscopic data was determined to be an alpha-linked glycoside of a lanostane-type triterpenoid.

Analysis of monoclonal antibody and immunoconjugate digests by capillary electrophoresis and capillary liquid chromatography.

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.

Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri.

During the screening of natural products for their ability to inhibit the binding of HIV-REV protein to [33P]-labeled RRE RNA, one novel compound, niruriside (1), was isolated from the MeOH extract of the dried leaf of Phyllanthus niruri L. by bioassay-guided fractionation. The structure of niruriside was determined by spectroscopic methods. Niruriside showed specific inhibitory activity against the binding of REV protein to RRE RNA with an IC50 value of 3.3 microM; however, niruriside did not protect CEM-SS cells from acute HIV infection at concentrations up to 260 microM using an XTT dye reduction assay.

Rakicidins, new cytotoxic lipopeptides from Micromonospora sp. fermentation, isolation and characterization.

The new cytotoxic agents rakicidins A and B were isolated from cultured broth of a Micromonospora sp. Spectroscopic and amino acid analysis has shown that rakicidin A is a new cyclic lipopeptide, consisting of 4-amino-penta-2,4-dienoic acid, 3-hydroxy-2,4,16-trimethyl-heptadecanoic acid, sarcosine, and 3-hydroxyasparagine. Rakicidin B differs by one methylene group in the lipid side chain. These compounds exhibited cytotoxicity against the M109 cell line.

BMS-192548, a tetracyclic binding inhibitor of neuropeptide Y receptors, from Aspergillus niger WB2346. II. Physico-chemical properties and structural characterization.

The structure of BMS-192548, a tetracyclic binding inhibitor of neuropeptide Y receptors, was established by spectroscopic methods. The compound has an unusual B-C-D ring beta-diketone moiety.

Manumycins E, F and G, new members of manumycin class antibiotics, from Streptomyces sp.

Three new manumycin class antibiotics, namely manumycins E, F and G, were isolated from the culture broth of Streptomyces sp. strain WB-8376. Their structures were established by spectroscopic methods, and the S configuration of C-4 in the epoxycyclohexenone moiety was determined by CD exciton chirality method for each of the three compounds. Manumycins E, F and G are active against Gram-positive bacteria, and have moderate inhibitory effects on the farnesylation of p21 ras protein. They demonstrated weak cytotoxic activity against human colon tumor cell HCT-116.

Eupenifeldin, a novel cytotoxic bistropolone from Eupenicillium brefeldianum.

Eupenifeldin was isolated from cultures of Eupenicillium brefeldianum ATCC 74184 by extraction and crystallization. The compound was identified as a pentacyclic bistropolone on the basis of spectral data and its complete structure was established by single-crystal X-ray analysis. The compound is cytotoxic against the HCT-116 cell line and has in vivo antitumor activity in the P388 leukemia model.

Structural characterization of glycoprotein digests by microcolumn liquid chromatography-ionspray tandem mass spectrometry.

An in-house modified microcolumn liquid chromatography (LC) system has been coupled to a PE-SCIEX API III triple-quadrupole mass spectrometer through an ionspray interface for the structural characterization of model glycoproteins, bovine ribonuclease B and human alpha 1-acid glycoprotein. In conjunction with enzymatic digestion approaches using trypsin and peptide-N-glycosidase F, the feasibility of packed-capillary (250 microns I.D.) LC columns, coupled with ionspray mass spectrometry (MS) in a tandem format, have been assessed for glycopeptide mapping and structural determination. This configuration demonstrates a highly promising approach for the determination of glycosylation sites and the corresponding sequence structures of related tryptic fragments. A glycosylated tetrapeptide, Asn-Leu-Thr-Lys with carbohydrate moieties on Asn-34, was readily located for bovine ribonuclease B. Preliminary results using micro-LC-MS also show the identification of a class A carbohydrate attachment on a tryptic fragment of human alpha 1-acid glycoprotein. The microheterogeneity of carbohydrate moieties can be quickly screened using this approach for either tryptic digests or the intact glycoprotein. These methods demonstrate potential applications for structural characterization of recombinant glycoproteins of pharmaceutical interest.