Studies on antitumor agents, 2. Syntheses and antitumor activities of 1-(tetrahydro-2-furanyl)-5-fluorouracil and 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracil.

Convenient and efficient methods were developed for preparing 1-(tetrahydro-2-furanyl)-5-fluorouracil (Thf-FU, 3) [trade name, Futraful (Ftorafur) or FT-207], which is used clinically as an antitumor agent, and 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracil (Thf2-FU, 4). For the syntheses, 2,4-bis(trimethylsily)-5-fluorouracil (Me3Si-FU, 1) and 2-acetoxytetrahydrofuran (Thf-OAc, 2) were condensed in the presence of Friedel-Crafts catalysts, such as SnCl4 and BF3-Et2O in dichloromethane, or in the presence of NaI in acetonitrile to give Thf-Fu or Thf2-FU depending on the reaction conditions and workup procedure. A trace of 3-(tetrahydro-2-furanyl)-5-fluorouracil (3-Thf-FU, 5) was formed in these reactions. Thf2-FU was easily hydrolyzed to Thf-FU. 2-Methoxytetrahydrofuran can be used instead of Thf-OAc for preparation of Thf-FU under similar conditions. The optimal ratios of Me3Si-FU, Thf-OAc, and SnCl4 or NaI for preparation of Thf-FU and Thf2-FU were determined. In all cases, 2-2.5 equiv of Thf-OAc with respect to Me3Si-FU gave the best results. The yields of Thf-FU and more especially of Thf2-FU were greatly dependent on the relative amount of SnCl4, and 0.01-0.1 equiv of the catalyst with respect to Me3Si-FU gave the best results. Thf2-FU was found to be effective against murine solid tumors and it was less toxic than Thf-FU when given orally. The antitumor activity of 3-Thf-FU is also reported.

GLC-mass fragmentographic determination of mannitol and sorbitol in plasma.

A GLC-mass fragmentographic method was developed for the simultaneous determination of mannitol and sorbitol as their n-butyldiboronate derivatives in plasma. The plasma sample was deproteinized, and the subsequent supernatant was concentrated to dryness; the resulting residue was then dissolved in pyridine containing n-butylboronic acid to allow derivation. An aliquot of this solution was injected into the gas chromatograph-mass spectrometer and analyzed by a selected-ion monitoring method using galactitol as the internal standard. Detection was limited to 20 ng/0.1 ml of plasma for both mannitol and sorbitol. A rapid, precise, and sensitive assay for the determination of mannitol and sorbitol in plasma was established.

High-performance liquid chromatographic determination of proglumide in plasma.

A rapid and sensitive method for determining the anticholinergic agent, proglumide, in plasma by high-performance liquid chromatography is described. Samples were acidified with hydrochloric acid and extracted with chloroform. The dried extract was resolved in chloroform and chromatographed on an adsorption chromatographic column using a mobile phase of chloroform-methanol (24:1) on a high-performance liquid chromatograph equipped with a UV absorbance detector (240 nm). The detection limit for proglumide was 0.05 microgram/ml.

Simultaneous high-performance liquid chromatographic determination of suxibuzone and its metabolites in plasma and urine.

A high-performance liquid chromatographic method is described for the simultaneous determination of the anti-inflammatory agent suxibuzone and its metabolites, 4-hydroxymethylphenylbutazone, phenylbutazone, oxyphenbutazone, and gamma-hydroxyphenylbutazone, in plasma and urine. Acidified plasma or urine is extracted with benzenecyclohexane (1:1). The organic extract is reduced to dryness and the resulting residue is redissolved in methanol. Aliquots of this solution are chromatographed on a reversed-phase column using a mobile phase of methanol--0.5 M KH2PO4 (linear gradient from 0 to 100% methanol at 8% min with a flow rate of 2.0 ml/min) on a high-performance liquid chromatograph equipped with a UV absorbance detector (254 nm). Detection is limited to 0.10 microgram/ml for suxibuzone and 4-hydroxymethylphenylbutazone and to 0.05 microgram/ml for the other metabolites.

High-pressure liquid chromatographic determination of ftorafur [1-(tetrahydro-2-furanyl)-5-fluorouracil] and GLC-mass spectrometric determination of 5-fluorouracil and uracil in biological materials after oral administration of uracil plus ftorafur.

Oral administration of uracil plus ftorafur [1-(tetrahydro-2-furanyl)-5-fluorouracil] has a greater antitumor effect than that of ftorafur alone. A high-pressure liquid chromatographic methods was developed for the determination of ftorafur in plasma and visceral tissues with a sensitivity of 0.025 microgram/ml or g of wet weight. A combination of GLC--mass fragmentography and GLC--mass spectrometry with total-ion monitoring was developed for the specific, simultaneous determination of 5-fluorouracil, and active metabolite, and uracil as their trimethylsilylated derivatives. The detection limits for 5-fluorouracil and uracil in the first method were 0.001 microgram/ml for plasma and 0.001--0.005 microgram/ml for visceral tissues, and those in the second method were 0.2 microgram/ml or g of wet weight. The precision and sensitivity of the assay appear to be satisfactory for determination of the levels of these compounds in plasma and visceral tissues.

A new macromolecular antitumor antibiotic, C-1027. II. Isolation and physico-chemical properties.

A new macromolecular antibiotic C-1027 was obtained from the broth filtrate of Streptomyces globisporus C-1027 by precipitation with ammonium sulfate, DEAE-cellulose column chromatography and gel filtration chromatography on a Sephadex G-75 column. This antibiotic, prepared as a white powder, is an acidic polypeptide having an isoelectric point of pH 3.5-3.7 and a molecular weight of 15,000 as determined by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography. The acid hydrolysate of the purified antibiotic C-1027 contained no methionine or tryptophan. From the physico-chemical data, it may be considered to possess a very labile non-protein chromophore.

New glutarimide antibiotics, S-632-B1 and B2. I. Taxonomy of producing strain, fermentation and biological properties.

Strain S-632 was found to produce new glutarimide antibiotics, S-632-B1 and B2, which were isolated from the culture fluid. A taxonomic study on strain S-632 was carried out, and the taxonomic characterization demonstrated that it belonged to the species Streptomyces hygroscopicus. The strain was given the name S. hygroscopicus S-632. These antibiotics were active against Saccharomyces sp., but inactive against filamentous fungi and bacteria, and had cytotoxic activity against KB tissue culture cells.

New glutarimide antibiotics, S-632-B1 and B2. II. Isolation, physico-chemical properties and chemical structure.

Antifungal antibiotics S-632-A1,A2,B1 and B2 were extracted with ethyl acetate from the filtered broth of Streptomyces hygroscopicus S-632 and isolated through a combination of conventional and reversed-phase silica gel column chromatography. On the basis of the spectral data, S-632-B1 and B2 were found to be new members of the glutarimide family of antibiotics. The chemical structures of these components were elucidated as two stereo-isomers of 3-(5,7-dimethyl-8,9-epoxy-2-hydroxy-4-oxo-6-decenyl)glutarimide.

A new macromolecular antitumor antibiotic, C-1027. I. Discovery, taxonomy of producing organism, fermentation and biological activity.

Strain C-1027, an actinomycete isolated from a soil sample collected in China, was found to produce the new antibiotic, C-1027. From taxonomical studies on its morphological, cultural and physiological characteristics, this antibiotic-producing strain was identified as Streptomyces globisporus C-1027. Antibiotic C-1027 has antimicrobial activity against most Gram-positive bacteria but not against Mycobacterium sp. or Gram-negative bacteria. This antibiotic shows remarkable activity in spermatogonial assay and potent cytotoxicity against KB carcinoma cells in vitro, and exhibits inhibition on transplantable tumors in mice.

New antibiotics 4181-A and B from Streptomyces griseus; taxonomy, fermentation, isolation and characterization.

The new antibiotics 4181-A and B were isolated from the fermentation broth of Streptomyces griseus, a soil isolate. Their molecular formulae were determined as C29H21NO9 and C28H19NO9, respectively. The UV, IR and NMR spectra suggest that they possess a quinone moiety in their structures. They were found to have antibacterial, antifungal and antitumor activity.

Mass spectra of N-substituted derivatives of 5-fluorouracil.

Several N-substituted derivatives of 5-fluorouracil have been synthesized during the development of new antitumor agents. For determination of the position of the substituent on these compounds, electron impact mass spectrometry was investigated. In their electron impact mass spectra, characteristic fragment ions were produced by the retro Diels--Alder decomposition of the 5-fluorouracil skeleton: [R(or R')N=CHC(F)=C=O]+. was produced from N-1 substituted derivatives, while an isocyanate ion, [RN=C=O]+., or an acylurea ion, [RCONHCONH2]+. was formed from N-3 substituted derivatives. The ions observed were useful for structural determination.

High-performance of liquid chromatographic determination of [3,4-di-(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its metabolites in human plasma and urine.

A high-performance liquid chromatographic method is described for the simultaneous determination of the non-steroidal analgesic and anti-inflammatory agent [3,4-di-(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its three metabolites in plasma and urine. Deproteinized plasma (with acetonitrile) or urine was applied to a Sep-Pak C18 cartridge, washed with distilled water and then eluted with methanol. The methanol eluate was reduced to dryness. The resulting residues from the plasma and urine were redissolved in methanol aqueous solution, respectively. Aliquots of each solution were chromatographed on a reversed-phase column using a mobile phase of methanol-20 mM potassium dihydrogenphosphate (pH 6.4) (linear gradient from 0 to 100% methanol at 3%/min with a flow-rate of 1.5 ml/min) on a liquid chromatograph equipped with an ultraviolet absorbance detector (254 nm). Detection was limited to 10 ng/ml in plasma and 100 ng/ml in urine for each compound. An accurate and sensitive assay for the determination of [3,4-di-(4-methoxyphenyl)-5-isoxazolyl]acetic acid and its metabolites was established.

Quantitative determination of 1,3-bis(tetrahydro-2- furanyl)-5-fluoro-2,4-pyrimidinedione and its metabolites in plasma by high-pressure liquid chromatography and gas chromatography-mass fragmentography.

1,3-Bis(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione has been developed clinically as an antitumor agent. A high-pressure liquid chromatographic method was developed with which it could be measured in plasma with a sensitivity of 0.050 microgram/ml. Two of its metabolites, 1-(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione and 3-(tetrahydro-2-furanyl)-5-fluoro-2,4-pyrimidinedione, could be determined at the same time with a sensitivity of 0.025 microgram/ml. A gas chromatographic-mass fragmentographic method was developed for the specific determination of the third metabolite, 5-fluoro-2,4-pyrimidinedione, as its silylated derivative with a sensitivity of 0.001 microgram/ml. The precision and sensitivity of the assay appear to be satisfactory for determination of the plasma level of the drug.

Determination of cefodizime in biological materials by high-performance liquid chromatography.

Cefodizime (THR-221) is a new semi-synthetic cephalosporin. A high-performance liquid chromatographic method has been developed for the determination of cefodizime in biological materials. A plasma or serum sample was deproteinized with methanol and the resulting methanol eluate was concentrated to a volume of 0.5 ml. Urine and bile samples were diluted with buffer and each diluted sample was filtered. Faeces samples were homogenized and the supernate obtained after centrifugation was filtered. Visceral tissue samples were homogenized, the centrifuged supernate was deproteinized with methanol, and the methanol eluate was concentrated to a volume of 0.5 ml. Aliquots of each preparation were chromatographed on a reversed-phase column with an ion-pair chromatographic technique on a high-performance liquid chromatograph equipped with an UV detector set at 264 nm. The detection limits for cefodizime were 0.1 microgram/ml in plasma or serum, 0.3 microgram/ml in bile, and 0.5 microgram/ml in urine, 0.5 microgram/g in faeces and visceral tissue. This precise and sensitive assay for the determination of cefodizime is described, and its stability in several media is reported.

Analysis of pyrimidine bases in biological materials by gas chromatography-mass spectrometry.

Simultaneous detection by a combination of gas chromatography-mass fragmentography and gas chromatography-mass spectrometry for total ion monitoring was developed for determination of uracil, thymine and cytosine present in biological materials as pyrimidine bases, as their silylated derivatives. The detection limits for uracil, thymine and cytosine in the first method were 0.001 microgram/ml for plasma and 0.001-0.005 microgram/g wet weight for tissues. Those for uracil and thymine in plasma and tissues and for cytosine in plasma in the second method were 0.2 microgram/ml or g wet weight, and that for cytosine in tissues was 2.5 microgram/g wet weight. An accurate and sensitive assay for determination of pyrimidine bases was established.

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine.

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (+/-)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the selected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02-0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.

High-performance liquid chromatographic determination of a new beta-lactamase inhibitor and its metabolite in combination therapy with piperacillin in biological materials.

[2S-(2 alpha,3 beta,5 alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-yl- methyl)-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid 4,4-dioxide (YTR-830H) is a new beta-lactamase inhibitor and the combination therapy of this compound with piperacillin is now under study. For the determination of the beta-lactamase inhibitor and piperacillin in biological materials, plasma and visceral tissue homogenates were deproteinized, whereas diluted urine and filtered faeces homogenates were treated with a Sep-Pak C18 cartridge. In order to assay the inactive metabolite of beta-lactamase inhibitor, each sample was treated with a Sep-Pak C18 cartridge. Aliquots of each preparation were chromatographed using ion-pair and reversed-phase chromatographic techniques on a high-performance liquid chromatograph equipped with a UV detector, set at 220 nm. The detection limits of beta-lactamase inhibitor and piperacillin were 0.2 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 0.2-0.5 microgram/g in visceral tissue and faeces. Those of the metabolite were 1.0 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 1.0 microgram/g in visceral tissue and faeces. A precise and sensitive assay for the determination of the beta-lactamase inhibitor, its metabolite and piperacillin is described, and their stabilities in several media are reported.