Search

During the course of our study to develop analytical methodology for quantitating the investigative antitumor agent 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran -4-one (DAF; NSC 686288) in plasma, a significant concentration of a metabolite was observed in a post-dosed rat. The results of electron-ionization (EI) mass spectrometric analysis of the metabolite suggested that N-acetylation had occurred, but, interestingly, that only one of the compound's two primary amino groups had been transformed. Comparing the mass spectra and gas chromatographic retention times of a mono-acetylated sample of DAF and that of the metabolite showed both to be the same. A retro-Diels-Alder (RDA) fragmentation of the B ring of DAF results in formation of two abundant product ions, each retaining one of the amino groups. The EI mass spectrum of mono-N-acetamido-d3 DAF shows loss of ketene-d2, leading to formation of an -NHD group. The ensuing RDA fragmentation easily identifies which of the two product ions contains the deuterium, thereby allowing us to assign the site of N-acetylation as the amino group on ring C (the 4' position) of DAF.

Certain naturally occurring isoflavonoids have been shown to inhibit protein-tyrosine kinases, and this has led to investigations of ring-modified structural analogs. Most recently, 2-(3-methyl-4-aminophenyl)-benzothiazole (MAB: NSC 674495) was shown to possess significant activity against certain breast cell cancer lines in vitro and in vivo. Our efforts thus focussed on developing a simple and sensitive method for quantitating MAB in plasma using GC-MS. The GC-MS assay was found to be linear over the range of 0.050 to 5.0 microg/ml, and was applied to monitor the plasma concentration of MAB in a rat dosed with 25 mg/kg as a 1 min intravenous infusion. Plasma was collected at intervals from 3 through 180 min, and concentrations of MAB were determined. Non-linear regression analysis of the plasma concentration-time data revealed that levels declined from a maximum at 3 min of 18 microg/ml to 1 microg/ml at 3 h in a biphasic manner. In another investigation, significant plasma concentrations of a major metabolite was detected and determined to be mono-N-acetylated MAB.

Reversed-phase HPLC conditions for the separation of 4-(2-pyridyl)-1-piperazinethiocarboxylic acid 2-[1-(pyridyl)-ethylidene]hydrazide dihydrochloride (NSC 348977, I), a synthetic thiosemicarbazone with antitumor activity, from mouse plasma have been investigated. Following denaturization and precipitation of the spiked plasma with acetonitrile, an aliquot of the supernatant was diluted with aqueous buffer and subjected to analysis on a Nova-Pak C18 column (150 x 3.9 mm I.D.) by isocratic elution with 50 mM aqueous potassium phosphate buffer (pH 6.8, containing 1 mM EDTA)-acetonitrile (60:40, v/v). The column effluent was monitored for UV absorption at 310 nm. Problems identified in the sample preparation and separation of I include sensitivity to oxygen, light, non-neutral pH and the presence of metal ions. These factors were seen to adversely influence sample recovery, and attempts were made to find conditions which minimize their effects.

A simple and sensitive isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the quantitation of perillic acid, a major circulating metabolite of perillyl alcohol and d-limonene, in plasma is described. Sample preparation involved protein precipitation and subsequent transfer and dilution with 10 mM NaHCO3. The mobile phase consisted of acetonitrile (36%) and 0.05 M ammonium acetate buffer pH 5.0 (64%). Separations were achieved on a C18 column and the effluent monitored for UV absorption at the analytes' respective UVmax. Separation was excellent with no interference from endogenous plasma constituents. This method was found suitable for quantifying drug concentrations in the range of 0.25 to 200.0 micrograms/ml using a 0.05-ml plasma sample, and was used to study the plasma pharmacokinetics of perillic acid in mice.

A facile, sensitive and highly specific HPLC method for assaying 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in plasma has been developed. The drug was efficiently isolated from plasma by extraction with tert.-butyl methyl ether. A structurally related compound with similar physicochemical properties served as the internal standard (I.S.). Following evaporation of the organic solvent, the extract was reconstituted with 0.05 M ammonium acetate buffer, pH 5.0, and loaded onto a 4 micron Nova-Pak C18 column (15 cm x 3.9 mm), which was preceded by a 7 micron Brownlee RP-18 precolumn (1.5 cm x 3.2 mm). Chromatography was performed at ambient temperature using a mobile phase of methanol-0.1 M ammonium formate buffer, pH 3.7 (25:75, v/v). UV absorbance of the effluent was monitored at 240 nm. A flow-rate of 1.0 ml/ min was used for analyzing mouse and dog plasma extracts. Under these conditions, the drug eluted at 4.0 min and was followed by the I.S. at 6.1 min. An automatic switching valve was employed to allow the precolumn to be flushed 1.5 min into the run, without interrupting the flow of the mobile phase to the analytical column, thereby preventing the apparent build-up of extractable, strongly retained, UV-absorbing components present in mouse and dog plasma. Operating in this manner, more than 100 samples could be analyzed during a day using a refrigerated autosampler for overnight injection. The method was readily adapted to the determination of SarCNU in human plasma by simply decreasing the eluent flow-rate to 0.6 ml/min, whereby SarCNU and the I.S. eluted at approximately 5.8 and 9.1 min, respectively. Furthermore, the switching valve was not necessary for the analysis of human plasma samples. With a 50-microliter sample volume, the lowest concentration of SarCNU included in the plasma standard curves, 0.10 micrograms/ml, was quantified with a 7.8% R.S.D. (n = 27) over a 2 month period. Plasma standards, with concentrations of 0.26 to 5.1 micrograms/ml, exhibited R.S.D. values ranging from 1.3 to 4.7%. Thermospray-ionization MS detection was used to definitively establish the specificity of the method. The sensitivity of the assay was shown by application to be more than adequate for characterizing the plasma pharmacokinetics of SarCNU in mice.

A specific, sensitive and technically convenient HPLC method for assaying genistein in biological fluids has been developed. The compound and 4-hydroxybenzophenone, added as an internal standard, were efficiently isolated from both plasma and urine by extraction with tert-butyl methyl ether. Following evaporation of the organic solvent, the extract was reconstituted with methanol-0.05 M ammonium acetate buffer, pH 4.7 (30:70, v/v), and loaded onto a 4 microns Nova-Pak C8 column (15 cm x 3.9 mm I.D.). Chromatography was performed at ambient temperature using a mobile phase of acetonitrile-0.05 M ammonium formate buffer, pH 4.0 (27:73, v/v), at a flow-rate of 1.0 ml/min, with UV detection at 260 nm. Mean values of the tR for the drug and internal standard, determined from chromatograms of the 1 microgram/ml plasma standard during a 6 month period, were 8.27 +/- 0.55 and 11.92 +/- 0.71 min, respectively (S.D., n = 29). With a sample volume of 50 microliters, the lowest concentration of genistein included in the plasma standard curve, 0.020 microgram/ml, was quantified with a 10.7% R.S.D. over a 5 month period. Plasma standards having concentrations of 0.050 to 1.02 micrograms/ml exhibited R.S.D. values ranging from 2.3 to 6.1%. The drug was quantified in urine with similar reproducibility. The sensitivity of the assay was adequate for characterizing the plasma pharmacokinetics of genistein in the mouse and dog. However, a 10-fold improvement in sensitivity was afforded by increasing the sample size to 250 microliters, without otherwise modifying the method. Thus, this procedure may prove suitable for determining plasma and urine levels of genistein in humans consuming dietary isoflavonoids in a much more convenient manner than permitted by existing methodology.

The synthesis and anti-HIV activity of selected (acyloxy)alkyl esters of trisodium phosphonoformate (foscarnet sodium) are described. The conversion of bis(trimethylsilyl) (alkoxycarbonyl)phosphonates 11a-d to the corresponding disilver salts 12a-d and their subsequent reaction with iodoalkyl acrylates 4a-c gave the desired bis(acyloxyalkyl) phosphonates 6-9(a-c). Of the analogs tested, only the dichlorophenyl analog 9a showed a dose-dependent inhibition of HIV activity in H9 cells. Using 31P-NMR, bioreversibility has been investigated in an attempt to rationalize these results.

The preparation of 2-[N-(ethyl)-(N-beta-hydroxyethyl)]amino-ethyl 2,2-diphenylpropionate (1), a metabolite of aprophen [2-diethylaminoethyl 2,2-diphenylpropionate], is described. Hydrolysis of [2-(2-chloroethyl)ethylamino]ethyl acetate hydrochloride (2) in a basic solution, followed by acidic pH adjustment, gave the ethylcholineaziridinium ion (3) that upon treatment with 2,2-diphenylpropionic acid produced 1 in a 56% yield. Synthetic 1 was found to possess antimuscarinic activities, but was approximately 10-fold less potent than the parent compound aprophen.

The National Cancer Institute (NCI) is pursuing preclinical development of Brefeldin A (BFA), a macrolide isolated from Penicillium brefeldianum, as an antitumor agent. BFA exhibits a unique spectrum of activity against the human tumor cell line panel that composes the NCI's recently established in vitro antitumor screen. A specific method to assay the compound in biological fluids has been developed in which BFA and 1-eicosanol, added as the internal standard, are extracted from plasma specimens with diethyl ether. The residue afforded by evaporation of the organic solvent is dried in vacuo and derivatized with heptafluorobutyrylimidazole prior to analysis by capillary gas chromatography with electron capture detection. Derivatization of both secondary hydroxyl groups of BFA is rapid and quantitative, as confirmed by mass spectrometry. The lowest concentration of BFA quantified with acceptable reproducibility in 50 microliters of plasma is 0.1 microgram/ml, for which a 5.6% coefficient of variation has been determined from six standard curves assayed on separate days. Detector response exhibits a positive deviation from linearity for samples with BFA concentrations greater than 2.5 micrograms/ml. The assay is shown by application to be suitable for preliminary investigations of BFA plasma pharmacokinetics in mice. These studies reveal that BFA is subject to rapid elimination from the mouse, with plasma levels declining in an apparent biphasic manner from an initial concentration of 33 to 0.2 micrograms/ml at 60 min after intravenous treatment with a 26.3 mg/kg dose.

The metabolism of the anticholinergic drug aprophen was studied in rats after oral administration via stomach intubation. beta-Hydroxyethylaprophen, a major urinary metabolite of aprophen, was isolated and identified by normal-phase high-performance liquid chromatography and electron ionization mass spectrometry. More than 22% of the parent drug was recovered and quantified over a 72-h collection period. Results show that 2,2-diphenylpropionic acid, another major metabolite of aprophen which lacks anticholinergic properties, was also isolated and identified in this study. Experiments are currently underway to synthesize and test the anticholinergic properties of beta-hydroxyethylaprophen in mammals.

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.

N-Acetylmuramoyl-L-alanyl-D-isoglutamine (MDP) was synthesized by a series of condensations of appropriate reagents, followed by hydrogenolysis. Each intermediate step resulted in a stable, crystalline product. D-Isoglutamine 4-benzyl ester was condensed with N-(tert-butoxycarbonyl)-L-alanine N-hydroxysuccinimide ester, to give N-(tert-butoxycarbonyl)-L-alanyl-D-isoglutamine benzyl ester. Condensation of L-alanyl-D-isoglutamine benzyl ester with N-acetyl-1-O-benzyl-4,6-O-benzylidenemuramic acid, followed by hydrogenolysis, gave MDP. The synthetic scheme was shown to be capable of producing gram quantities of highly pure MDP, as well as a few of its analogs. The synthetic MDP was characterized by analytical and biological methods, and it was found that the use of fast-atom-bombardment-mass spectrometry may greatly simplify the characterization process.

31P- and 1H-NMR spectroscopy were used to demonstrate that the primary metabolites of the anticancer drug cyclophosphamide (4-hydroxycyclophosphamide and its acyclic tautomer, aldophosphamide) are quantitatively converted by O-methylhydroxylamine, at pH 7.4 and 37 degrees, into the E and Z isomers of aldophosphamide O-methyl oxime. These trapping products are readily extracted from aqueous media with either chloroform or ethyl acetate, are stable at pH 6-8 toward oxime hydrolysis and elimination of phosphoramide mustard (a secondary metabolite of cyclophosphamide), and showed no evidence for transoximination with either ketone or aldehyde acceptors. All of these features support the use of aldophosphamide O-methyl oxime in quantitative studies related to cyclophosphamide metabolism.

N- and O-Triorganosilylated compounds related to various anticancer agents were synthesized for evaluation as potential anticancer prodrugs. 1H-NMR and UV kinetic measurements of hydrolytic desilylation were used to correlate relative rates of structural unmasking with steric bulk about the silicon reaction center. The tert-butyldimethylsilyl ester of chlorambucil and a number of O- triorganosilylated carbamate derivatives of nor-nitrogen mustard showed significant activity against P-388 lymphocytic leukemia in mice.

The structure of the repeating oligosaccharide of the pneumococcal capsular polysaccharide type 18C has been investigated. The repeating oligosaccharide, isolated from an aqueous hydrofluoric acid hydrolyzate of the polysaccharide, was shown, by fast atom bombardment-mass spectrometry, to have a molecular weight of 928, and to contain an O-acetyl group and a glycerol residue. Information about the sequence in the per-O-methylated oligosaccharide was derived from electron-impact mass spectrometry. Supporting data were obtained from methylation analysis, periodate and chromium trioxide oxidations, and enzymic and acid hydrolyses of the oligosaccharide. These studies indicated that the polysaccharide consists of the following pentasaccharide repeating unit. (formula see text)