6′-Chloro-7- or 9-(2,3-dihydro-5H-4,1-benzoxathiepin-3-yl)-7H- or 9H-purines and their corresponding sulfones as a new family of cytotoxic drugs

A series of 1-(2,3-dihydro-5H-4,1-benzoxathiepin-3-yl)pyrimidine derivatives were synthesized and two of them (8 and 9) showed a modest antiproliferative activity against the MCF-7 breast cancer cell line. We then decided to change the pyrimidine base for the more lipophilic 6′-chloropurine, and the N-9′ purine (15) and N-7′ purine (17) were obtained. The sulfone N-7′-alkylated-6-chloropurine 18 was the most active derivative. Compound 17 was found to be slightly more active than its regioisomer 15, with an activity similar to that of 5-fluorouracil as a reference drug. Encouraged by these values, we tested these compounds against both the HT-29 human colon cancer cell line and the IEC-6 normal rat intestinal epithelial cell line, and 15 was found to be 12.7-fold more active against HT-29 than versus IEC-6.     

Reactions of trimethylsilyl fluorosulfonyldifluoroacetate with purine and pyrimidine nucleosides

Difluorocarbene, generated from trimethylsilyl fluorosulfonyldifluoroacetate (TFDA), reacts with the uridine and adenosine substrates preferentially at the enolizable amide moiety of the uracil ring and the 6-amino group of the purine ring. 2′,3′-Di-O-benzoyl-3′-deoxy-3′-methyleneuridine reacts with TFDA to produce 4-O-difluoromethyl product derived from an insertion of difluorocarbene into the 4-hydroxyl group of the enolizable uracil ring. Reaction of the difluorocarbene with the adenosine substrates having the unprotected 6-amino group in the purine ring produced the 6-N-difluoromethyl derivative, while reaction with 6-N-benzoyl protected adenosine analogues gave the difluoromethyl ether product derived from the insertion of difluorocarbene into the enol form of the 6-benzamido group. Treatment of the 6-N-phthaloyl protected adenosine analogues with TFDA resulted in the unexpected one-pot conversion of the imidazole ring of the purine into the corresponding N-difluoromethylthiourea derivatives. Treatment of the suitably protected pyrimidine and purine nucleosides bearing an exomethylene group at carbons 2′, 3′ or 4′ of the sugar rings with TFDA afforded the corresponding spirodifluorocyclopropyl analogues but in low yields.     

Quantitation of Purines and Pyrimidines in Human Serum by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatography assay was developed for the purpose of examining xanthine, hypoxanthine, uridine, thymidine, allopurinol, and oxypurinol in serum of patients with advanced carcinoma treated with methorexate and 5-fluorouracil. After the addition of an internal standard, serum samples were extracted of purines and pyrimidines with trichloroacetic acid and neutralized with tert-amine. Aliquots of the extracted serum were injected onto a C18 reverse-phase column and purines and pyrimidines were eluted with a gradient of MeOH/H₂O and KH₂PO₄ solutions. Absorbance was detected with a variable-wavelength UV spectrophotometer at 254 nm and 280 nm. This assay can be readily applied to quantitate baseline and treatment-induced variations in serum purine and pyrimidine levels which may correlate with clinical response and/or toxicity in patients with cancer.     

An original way to use a β-cyclodextrin-bonded silica stationary phase in electrochromatography. Application to the achiral separation of nucleobases and nucleosides

Summary Although β-cyclodextrin-bonded silica stationary phases are usually dedicated to the separation of enantiomers, they can also be used for achiral purposes, taking advantage of the complex interactions between the solutes and the cyclodextrins. The work described in this paper was intended to show how such stationary phases can be used in short-end injection capillary electrochromatography (CEC) for rapid (<6 min) resolution of a mixture of purines and pyrimidines. Several experimental conditions (mobile-phase composition, voltage and temperature) were investigated to determine the optimum operating conditions for the method, which can also be applied to the analysis of nucleosides. Quantitative analysis was also performed; theophylline was used as internal standard to assess the linearity and the repeatability of the method. Limits of detection ranged from 4 to 13 μm and repeatability was good (RSD never exceeded 2.6%).     

Photochemical reactivity of sulfamethoxazole and other sulfa compounds with photodiode array detection

The photochemical activities of six sulfa compounds [sulfacetamide (CET), sulfadiazine (DIA), sulfaguanidine (GUA), sulfamerazine (MER), sulfamethoxazole (SMX), and sulfamethizole (MET)] under different experimental conditions such as photolysis time, solvent and buffer pH are investigated by photodiode array (PDA) spectrophotometry. With no photolysis, the sulfa drugs CET and DIA show no absorbance at 332 nm and the other compounds only modest absorbance. Upon photolysis for 4 min, absorbance enhancements at 332 nm of three to four times for GUA and MET and 12-15 times for SMX and MER are observed. For CET and DIA after photolysis, the (absorbance) l/mg is now approximately 0.01-0.02 units. Although two pH optima of approximately 3-4 and 7 are noted, the optimum solvent for photolysis is ethanol without pH adjustment. For flow injection (FI) with on-line photolysis and PDA detection, a mobile phase of 100% ethanol with a step flow rate from 0.1 to 1 ml/min is used providing a 4-min reaction time. The FI detection limit for SMX with photolysis at 330 nm is 1 mg/l. The relative standard deviation data (n=4) of seven individual points in a calibration curve from 5 to 150 mg/l are 0-4%. The recovery of SMX from pharmaceutical tablets is 99.7% indicating no interference from trimethoprim which is not photochemically active.     

The Synthesis of D-Heteroannulated 3β-Hydroxy-13α-androst-5-ene Derivatives via α-Oxoketene Dithioacetal and α-Oxohydroxymethylidene Synthons

Summary.  16-Ketene dithioacetal derivatives of 3β-hydroxy-13α-androst-5-en-17-one react with amidine, benzamidine, or guanidine to yield novel pyrimido-fused D-heteroannulated steroids. The reactions of 3β-hydroxy-16-hydroxymethylidene-13α-androst-5-en-17-one with N,N′-dinucleophiles furnish heterocycles containing a pyrazole ring fused to positions 16,17 of the sterane skeleton.     

Determination of purines and pyrimidines in beer samples by capillary zone electrophoresis

Ten purine and pyrimidine bases were separated using capillary zone electrophoresis (CZE) with direct UV detection at 254 nm as well as mass spectrometric (MS) detection using an electrospray ionization (ESI) interface. For this purpose a carrier electrolyte composition compatible with both methods of detection containing 300 mM diethylamine (DEA) was selected. Limits of detection were in the range between 0.1 and 0.3 mg l⁻¹ and calibration plots were found to be linear over at least two orders of magnitude. The applicability of the developed method for the analysis of real samples was demonstrated for some beer samples. A series of “Lager” beer samples from different breweries in Europe as well as a number of completely different types of beers were investigated with respect to their content in the selected purine and pyrimidine bases using the developed CE method with UV detection at 254 nm.     

Mass spectrometric profiling of saturated fatty acid esters of steroids separated by high-temperature gas chromatography

An efficient analytical method for simultaneous determination of 12 SFEs in serum is described. The method involves solid-phase extraction to isolate of SFEs from interfering species, especially cholesteryl esters, conversion to trimethylsilyl (TMS) ether derivatives for the direct analysis by gas chromatography–mass spectrometry (GC–MS) using a high temperature MXT-1 (Silcosteel-treated stainless steel) capillary column. All SFEs as their TMS derivatives were well separated with excellent peak shapes within 12 min. Overall recoveries ranged from 88% to 119%, with a detection limits for SFEs ranged from 2 to 30 μg L⁻¹. The linearity as correlation coefficient was higher than 0.99 except for pregnenolone-3-arachidate (r² = 0.98) in the concentration range of 5–3000 μg L⁻¹. Ten serum samples obtained from volunteers were also analyzed and quantitatively determined of DHEA-3-palmitate and pregnenolone-3-stearate in 1.8–1195.8 μg L⁻¹ concentration. The devised high temperature GC–MS method could be useful for identification of SFEs in biological specimens including serum.     

Fast separations with open tubular ion exchange capillary columns

Fused silica columns of 4.6 μm i.d. were coated with 3-sulfopropylsilane as a cation exchanger and 3-(2-aminoethyl-amino)-propylsilane as an anion exchanger. Fast separations of cations (< 25 seconds) and of anions (< 35 seconds) were obtained, using a potentiometric microelectrode as a detector. In proceeding towards smaller i.d.s, a mixture of alkali metal cations could be separated successfully in an uncoated fused silica capillary of 2.3 μm i.d. as a result of retention by the surface silanol groups at pH 9.4.     

Automated screening of reversed‐phase stationary phases for small‐molecule separations using liquid chromatography with mass spectrometry

There are various reversed‐phase stationary phases that offer significant differences in selectivity and retention. To investigate different reversed‐phase stationary phases (aqueous stable C₁₈, biphenyl, pentafluorophenyl propyl, and polar‐embedded alkyl) in an automated fashion, commercial software and associated hardware for mobile phase and column selection were used in conjunction with liquid chromatography and a triple quadrupole mass spectrometer detector. A model analyte mixture was prepared using a combination of standards from varying classes of analytes (including drugs, drugs of abuse, amino acids, nicotine, and nicotine‐like compounds). Chromatographic results revealed diverse variations in selectivity and peak shape. Differences in the elution order of analytes on the polar‐embedded alkyl phase for several analytes showed distinct selectivity differences compared to the aqueous C₁₈ phase. The electron‐rich pentafluorophenyl propyl phase showed unique selectivity toward protonated amines. The biphenyl phase provided further changes in selectivity relative to C₁₈ with a methanolic phase, but it behaved very similarly to a C₁₈ when an acetonitrile‐based mobile phase was evaluated. This study shows the value of rapid column screening as an alternative to excessive mobile phase variation to obtain suitable chromatographic settings for analyte separation.     

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 microm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.     

Reversed-phase separations on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase

Summary The chiral separation of eight racemic compounds has been investigated on a cellulose tris(3,5-dimethylphenylcarbamate) chiral column under reversed-phase conditions. The discrimination mechanism under reversed-phase conditions is discussed. Addition of acid to the mobile phase is necessary for resolution of acidic racemic compounds. The presence of ion-pair reagent in the mobile phase is a key factor in the resolution of basic racemic compounds. Retention of the racemates is also affected by addition of acid or salt. The anion in buffer not only interacts with the racemete, but also with the polysaccharide derivative on the silica gel surface.     

HPLC separations with micro-bore columns using high-temperature water and flame ionization detection

Previous work demonstrates that a flame ionization detector (FID) may be used as a detector for high-temperature water separation. However, the relatively high flow rate of the eluent required by standard high-performance liquid chromatography columns causes instability of the FID. In this work, micro-bore columns are packed with poly(styrene-divinylbenzene) (PRP-1) or Daiso gel octadecylsilane-bonded phase particles. Because micro columns require low volume flow rates, the eluent used in high-temperature water chromatography does not cause instability of the FID. Separation of carbohydrates, amino acids, and other organic acids and bases is performed on two micro-bore columns using a homemade high-temperature water chromatograph with FID. Both isothermal and programmed temperatures are used in this work. The limit of detection and the linear range are also determined for amino acids tested.     

The potential of serially coupled alkyl-bonded silica monolithic columns for high resolution separations of pharmaceutical compounds in biological fluids

Summary In this paper we investigate the potential of alkyl-bonded silica monolithic columns for the isolation and identification of drug-related components in biological fluids. Up to 6 columns have been connected in series to produce a chromatographic system with up to 40,000 plates. This high-resolution chromatography system has been coupled to both MS and NMR to enable efficient detection and characterisation of drug-related components in biological fluids. The use of six coupled columns has been shown to give enhanced resolution over a high quality silica particulate column packed with 3 μm material which exhibits the same back pressure. The effect of volume and mass load on the performance of monolithic columns for semi-preparative chromatography of biological fluids has also been investigated. In these studies it was possible to inject up to 100 mL of neat urine with no loss of chromatographic performance. Furthermore, upon re-testing, the columns showed similar chromatographic performance. Again several columns were serially connected, producing enhanced resolution in the semi-preparative mode.     

Odour evaluation,fraction collection and preparative scale separations with glass capillary columns

A simple but effective GC approach to odour evaluation and fraction collection with capillary columns is described. An allglass device splits the column effluent, with one splitter arm going to an FID. For odour evaluation, the other splitter arm is led via teflon tubing to the nose. Off-line heart-cutting is carried out by collecting the split effluent on a glass capillary microtrap, the inner walls of which are coated with OV-101. This allows recovery of nanogram amounts of material. Multipass operation or repeated collection leads to preparative capillary GC for further spectroscopic investigation. These techniques do not involve sophisticated equipment or valves. Applications of this type of smell analysis, heart-cutting and enrichment in the analysis of Rhododendron fragrantissimum essential oil, black pepper essential oil and hop essential oil, illustrating the versatility of the systems, are presented.     

Effective enantiomeric separations of racemic primary amines by the isopropyl carbamate-cyclofructan6 chiral stationary phase

A new chiral stationary phase (CSP) was developed by bonding isopropyl-carbamate functionalized cyclofructan6 (IP-CF6) to the silica gel. It was evaluated by injecting 119 racemic primary amine-containing compounds. This CSP showed pronounced enantioselectivity toward all types of primary amines, separating 93% of all tested compounds. Baseline separation was achieved even for some simple aliphatic racemic amines that contained no other functionality. The polar organic mode was shown to be the effective mobile phase owing to higher efficiency. This new chiral stationary phase showed great potential for preparative-scale separations. It is also interesting that the chiral selector, R-naphthylethyl-carbamate functionalized CF6 (RN-CF6), was found to provide complementary selectivity for the relatively few amine analytes that did not separate on IP-CF6. Thus between the two CSPs, 98% of attempted amine compounds were separated.     

Synthesis of Some New Pyrimidothienopyridazines and Related Heterocycles

5‐Amino‐3,4‐diphenylthieno[2,3‐c]pyridazine‐6‐carbonitrile 2a was reacted with propylene diamine to give tetrahydropyrimidinyl derivative 3 . The latter compound ( 3 ) underwent certain cyclocondensation reactions to produce pyrimidothienopyridazines 4–6 . Also, the reactions of 5‐amino‐3,4‐diphenylthieno[2,3‐c]pyridazine‐6‐carboxamide with heterocyclic aldehydes and/or cycloalkanones were carried out and their products were identified. Moreover, some novel pyridazinothienopyrimidobenzimidazoles 14–17 were synthesized.     

Studies on the chromatographic behavior of nucleosides and bases on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

The chromatographic behavior of some nucleosides, pyrimidines and purines on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS) were studied by high performance liquid chromatography. Their retention behavior on CABS were compared with those on ODS. The influence of mobile phase variables, such as methanol content, pH and ionic strength on the retention behavior were studied. Some nucleosides, pyrimidines and purines on CABS were successfully separated. The results show that the calix[8]arene-bonded phase exhibits high selectivities for the above analytes in high aqueous mobile phases. According to the chromatographic data, it is indicated that hydrophobic interaction, hydrogen-bonding interaction, and dipole-dipole interaction are mainly responsible for the retention behavior. In addition, in some extent, the vertical stacking action of the analytes on CABS can also change the retention behavior. CABS was superior to ODS in the routine fast separation of nucleosides and bases.     

Chemistry and Biochemistry of Unsaturated Fatty Acids

Although unsaturated fatty acids have long been known to accompany saturated fatty acids in most lipids, qualitative and quantitative determination of fatty acid patterns only became possible with the advent of modern analytical methods. Present day knowledge of the chemical structure, physical properties, and metabolism of unsaturated fatty acids provides the basis for the development of new concepts of their function. Thus unsaturated fatty acids crucially determine the properties of biological membranes. Moreover, essential fatty acids are precursors of prostaglandins.