Characterization of a rare triterpenoid and minor phenolic compounds in the root bark of Anisophyllea dichostyla R. Br.

Anisophyllea dichostyla R. Br. (Anisophylleaceae), is a small shrub which grows widely in regions of the Democratic Republic of Congo (DRC) where its root barks are used in folk medicine for the treatment of many debilitating diseases. In a previous work [Khallouki, F., Haubner, R., Hull, W.E., Erben, G., Spiegelhalder, B., Bartsch, H., Owen, R.W., 2007. Isolation, purification and identification of ellagic acid derivatives, catechins and procyanidins from the root barks of Anisophyllea dichostyla R. Br. Food and Chemical Toxicology 45, 472-485] on this species, an appreciable number (16) of phenolic antioxidants (3.32 g/kg) such as ellagitannins (27%) and polyhydroxyflavan-3-ols (catechins and procyanidins; 73%) were isolated and identified. Two fractions, as well as containing minor phenolic compounds also showed evidence of a secondary plant substance similar to a triterpenoid. Following purification of the triterpenoid by semi-preparative HPLC, and recrystallization, the structure was elucidated as bryonolic acid as evinced by comprehensive spectroscopic analyses including (1)H and (13)C NMR, DEPT, COSY, ROESY, HMQC, HMBC, HPLC-ESI-MS and GC-MS experiments. Bryonolic acid, which is extremely rare in nature, is therefore reported in the family Anisophylleaceae for the first time. Furthermore, the following minor phenolic compounds namely tyrosol, 2-(3-methoxy, 4-hydroxyphenyl)-ethanol, vanillin, syringaldehyde, vanillic acid, syringic acid, gallic acid and ferulic acid were also identified by GC-MS in this species for the first time.

Isolation, purification and identification of ellagic acid derivatives, catechins, and procyanidins from the root bark of Anisophyllea dichostyla R. Br.

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120g). The polyphenol content (3.32g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside and (-)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (-)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3'-di-O-methyl ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid, 3'-O-methyl-3,4-methylenedioxo ellagic acid 4'-O-beta-d-glucopyranoside, and 3'-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC-ESI-MS and nano-ESI-MS-MS: (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), epicatechin-(4beta-->8)-epicatechin (procyanidin B(2)), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-beta-d-glucopyranoside, (-)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4'-O- beta-d-glucopyranoside, and 3,3'-di-O-methyl ellagic acid 4-O-beta-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (-)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC(50)=52 micro g/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3'-O-methyl ellagic acid 4'-O-beta-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-beta-d-glucopyranoside, epicatechin-(4beta-->8)-catechin (procyanidin B(1)), and (-)-epicatechin 3-O-gallate, proved to be the most potent (IC(50)=36 micro g/mL).

Analysis of enterolignan glucuronides in serum and urine by HPLC-ESI-MS.

A method involving the coupling of high-performance liquid chromatography with electrospray ionisation mass spectrometry (HPLC-ESI-MS) for the quantitative determination of the mammalian lignans enterolactone and enterodiol in human blood and urine has been developed. In contrast to techniques previously published, the method allows direct measurement of free enterolignans as well as their monoglucuronide conjugates in human biofluids with minimal sample preparation. Thereby the method is suitable for large-scale intervention, case-control and epidemiologic studies. Comprehensive, high-precision (1)H and (13)C nuclear magnetic resonance data (CD3OD as solvent) obtained at 11.7 T in combination with polarimetric data show that the major form of lignan precursor in the linseeds used is (-)-secoisolariciresinol diglucoside ((2R,3R)-2,3-bis(4'-hydroxy-3'-methoxy-benzyl)-1,4-butanediyl-bis-beta-d-glucopyranoside) which is transformed by human intestinal bacteria into (+)-enterodiol and (+)-enterolactone. However, these metabolites are mono-glucuronidated after absorption and are detected as (-)-enterodiol 3'-beta-d-glucuronide=(2R,3R)-2-(3'-O-(beta-d-glucopyranosyluronic acid)benzyl)-3-(3''-hydroxybenzyl)-butane-1,4,diol and (-)-enterolactone 3'-beta-d-glucuronide=(2R,3R)-2-(3'-O-(beta-d-glucopyranosyluronic acid)benzyl)-3-(3''-hydroxybenzyl)-beta-butyrolactone in blood and urine.

Isolation and structure elucidation of phenolic antioxidants from Tamarind (Tamarindus indica L.) seeds and pericarp.

Although it is already known that Tamarind (Tamarindus indica L.) seeds contain phenolic substances, the individual components of the seeds have not been fully identified and quantitated, and in the case of Tamarind pericarp not reported. Therefore, major polyphenolic compounds were extracted using organic solvents and the metabolites were isolated by semi-preparative high performance liquid chromatography. Their structures were elucidated by liquid chromatography-electrospray-ionisation-mass spectrometry (LC-ESI-MS), nano-electrospray-ionisation mass spectrometry (ESI-MS), and where possible by gas chromatography-mass spectrometry (GC-MS) and 1H and 13C NMR. Quantitative analysis of polyphenolic compounds in Tamarind seeds and pericarp was conducted by analytical high performance liquid chromatography (HPLC), calculated against standard curves of authentic compounds. The yields of total phenolic compounds after Soxhlet extraction with methanol were 6.54 and 2.82 g/kg (dry weight) in the seeds and pericarp respectively. The profile (%) of polyphenolics in Tamarind pericarp was dominated by proanthcyanidins (73.4) in various forms (+)-catechin (2.0), procyanidin B2 (8.2), (-)-epicatechin (9.4), procyanidin trimer (11.3), procyanidin tetramer (22.2), procyanidin pentamer (11.6), procyanidin hexamer (12.8) along with taxifolin (7.4), apigenin (2.0), eriodictyol (6.9), luteolin (5.0) and naringenin (1.4) of total phenols, respectively. The content of Tamarind seeds comprised only procyanidins, represented (%) mainly by oligomeric procyanidin tetramer (30.2), procyanidin hexamer (23.8), procyanidin trimer (18.1), procyanidin pentamer (17.6) with lower amounts of procyanidin B2 (5.5) and (-)-epicatechin (4.8). Extraction of Tamarind pericarp and seeds using acetone:methanol:acetic acid gave only procyanidin oligomers, but in much higher yield and variety. The antioxidant capacities of the Soxhlet methanolic extracts were determined, and indicates that Tamarind may be an important source of cancer chemopreventive natural products in tropical regions.

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays.

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and low expression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon-alpha (IFN-alpha). Treatment effects were quantitated using the MTT survival assay and 31P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011 mircroM VBL alone (87% vs. 102% survival) or to 0.011 microM BL + 10 microM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic 31P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.

Isolation and structure elucidation of the major individual polyphenols in carob fibre.

Although it is already known that carob fibre contains several classes of polyphenolic substances, a comprehensive analysis of these has not been conducted to date. Therefore, the major polyphenolic compounds were extracted with organic solvents, and, following fractionation by normal-phase column chromatography on silicic acid, their structures were elucidated by liquid-chromatography electrospray-ionisation mass spectrometry (LC-ESI), nano-electrospray-ionisation mass spectrometry (ESI-MS), and gas-chromatography mass spectrometry (GC-MS). In addition, complete 1H and 13C NMR assignments were obtained for the isolated gallotannins 1,6-di-, 1,2,6-tri- and 1,2,3,6-tetra-O-galloyl-beta-D-glucose. Carob fibre was found to contain a rich variety of phenolic antioxidants. A total of 24 polyphenol compounds were identified with a yield of 3.94 g/kg (dry weight). The profile was dominated by gallic acid in various forms: free gallic acid (42% of polyphenols by weight), gallotannins (29%), and methyl gallate (1%), while simple phenols, mainly cinnamic acid, made up about 2% of the total. Flavonoids represented 26% of the polyphenols, and the major components were identified as the glycosides myricetin- and quercetin-3-O-alpha-L-rhamnoside (ca. 9% and 10%, respectively). These data indicate that carob fibre is rich in both amount and variety of phenolic antioxidant substances, and its inclusion in the diet may have chemopreventive properties.

Correlation between 5-fluorouracil metabolism and treatment response in two variants of C26 murine colon carcinoma.

Following an i.p. dose of 150 mg x kg(-1) 5-fluorouracil (5-FU), drug uptake and metabolism over a 2-h period were studied by in vivo (19)F magnetic resonance spectroscopy (MRS) for the murine colon carcinoma lines C26-B (5-FU-insensitive; n=11) and C26-10 (5-FU-sensitive; n=15) implanted s.c. in Balb/C mice. Time courses for tumour growth, intracellular levels of FdUMP, thymidylate synthase (TS) activity, and 5-FU in RNA were also determined, and the effects of a 9.5-min period of carbogen breathing, starting 1 min before drug administration, on MRS-detected 5-FU metabolism and tumour growth curves were examined. Both tumour variants generated MRS-detectable 5-FU nucleotides and showed similar initial growth inhibition after treatment. However, the growth rate of C26-B tumours returned to normal, while the sensitive C26-10 tumours, which produced larger fluoronucleotide pools, still showed moderate growth inhibition. Carbogen breathing did not significantly influence 5-FU uptake or fluoronucleotide production but did significantly enhance growth inhibition in C26-10 tumours. While both tumour variants exhibited incorporation of 5-FU into RNA and inhibition of TS via FdUMP, clearance of 5-FU from RNA and recovery of TS activity were greater for the insensitive C26-B line, indicating that these processes, in addition to 5-FU uptake and metabolism, may be important determinants of drug sensitivity and treatment response.

Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes.

Because olives represent an important component of the Mediterranean diet, it is necessary to establish unequivocal identification and quantitation of the major potential antioxidant phenolic compounds they contain. The major phenolic antioxidants in two types of brined olives were isolated and purified by semi-preparative high performance liquid chromatography. Structural analysis was conducted using UV spectrophotometry, mass spectrometry and nuclear magnetic resonance spectroscopy. In particular, completely assigned 1H and 13C NMR data are presented and errors in literature data are corrected. The data show that tyrosol, hydroxytyrosol, 3-(3, 4-dihydroxyphenyl) propanoic acid (dihydrocaffeic acid), dihydro-p-coumaric acid (phloretic acid), the phenylpropanoid glucosides acteoside (verbascoside) and isoacteoside, along with the flavonoids luteolin and apigenin are major components of the phenolic fraction of brined black olives. Brined green olives contain only hydroxytyrosol and traces of other minor phenolics. Brined olives contain even higher concentrations of phenolic antioxidants than olive oil and may, therefore, be more important modulators of cancer chemopreventive activity.

Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy.

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.

Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization.

Several radioiodinated N-(dialkylaminoalkyl)benzamides have been used for planar scintigraphy and single-photon emission computed tomography (SPECT) of melanoma metastases. In a quest for improved melanoma uptake and tissue selectivity, structure-activity studies for N-(2-diethylaminoethyl)benzamides with variation of phenyl substituents were performed using C57Bl/6 mice bearing B16 melanoma. Compounds 2 (4-amino-5-bromo-N-(2-diethylaminoethyl)-3-[(131)I]iodo-2-methoxybenz amide) and 6 (4-acetamido-N-(2-diethylaminoethyl)-5-[(131)I]iodo-2-methoxybenzamid e) showed at 6 h post iv injection, for example, melanoma uptake of 16.6 and 23.2% ID/g, respectively (mean values, n = 3). Uptake was 3-5 times higher (P < 0.01) than observed with benzamides known from the literature and was probably facilitated by the relatively slow urinary excretion of 2 or 6. In contrast, analogues lacking either the MeO, Ac, AcNH, or Br substituents exhibited reduced tumor uptake and high urinary excretion of radioactivity in various benzamide metabolites. Uptake of radioiodinated benzamides in B16 melanoma is not mediated by a specific mechanism such as sigma-receptor binding. 2 and 6 exhibited similar melanoma uptake values but quite different sigma(1)-receptor affinities of K(i) = 0.278 +/- 0.018 and 5.19 +/- 0.40 microM, respectively. Uptake studies with IMBA (N-(2-diethylaminoethyl)-3-[(131)I]iodo-4-methoxybenzamide) or BZA (N-(2-diethylaminoethyl)-4-[(131)I]iodobenzamide) showed that with increasing dose of unlabeled compound the measured uptake of label was unchanged (IMBA) or even enhanced (BZA) while receptor binding of label decreased. Differential and equilibrium density-gradient centrifugation revealed that most of the radioactivity from labeled IMBA was associated with fractions containing melanin granules. Thus, structure-activity studies indicate that blood clearance rates and metabolic stability are the main determinants for benzamide uptake in melanoma. The high uptake and slow clearance of 6 offer considerable potential for melanoma imaging in patients, and this compound may also prove to be useful for radionuclide therapy.

Identification of lignans as major components in the phenolic fraction of olive oil.

BACKGROUND: Because olive oil is an important component of the Mediterranean diet, it is necessary to establish unequivocal identification of the major potential antioxidant phenolic compounds it contains. METHODS: The major phenolic antioxidants in extra virgin olive oil were isolated and purified. Structural analysis was conducted using several spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). In particular, detailed (1)H and (13)C NMR data are presented, and several assignment errors in the literature are corrected. RESULTS: The data show for the first time that the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol are major components of the phenolic fraction of olive oils. These lignans, which are potent antioxidants, are absent in seed oils and virtually absent in refined virgin oils but are present at concentrations of up to 100 mg/kg (mean +/- SE, 41.53+/-3.93 mg/kg; range, 0.65-99.97 mg/kg) in extra virgin oils. As with the simple phenols and secoiridoids, there is considerable interoil variation in lignan concentrations. Foods containing high amounts of lignan precursors have been found to be protective against breast, colon, and prostate cancer. CONCLUSION: Lignans, as natural components of the diet, may be important modulators of cancer chemopreventive activity.

The antioxidant/anticancer potential of phenolic compounds isolated from olive oil.

In our ongoing studies on the chemoprevention of cancer we have a particular interest in the health benefits of the Mediterranean diet, of which olive oil is a major component. Recent studies have shown that extravirgin olive oil contains an abundance of phenolic antioxidants including simple phenols (hydroxytyrosol, tyrosol), aldehydic secoiridoids, flavonoids and lignans (acetoxypinoresinol, pinoresinol). All of these phenolic substances are potent inhibitors of reactive oxygen species attack on, e.g. salicylic acid, 2-deoxyguanosine. Currently there is growing evidence that reactive oxygen species are involved in the aetiology of fat-related neoplasms such as cancer of the breast and colorectum. A plausible mechanism is a high intake of omega-6 polyunsaturated fatty acids which are especially prone to lipid peroxidation initiated and propagated by reactive oxygen species, leading to the formation (via alpha,beta-unsaturated aldehydes such as trans-4-hydroxy-2-nonenal) of highly pro-mutagenic exocyclic DNA adducts. Previous studies have shown that the colonic mucosa of cancer patients and those suffering from predisposing inflammatory conditions such as ulcerative colitis and Crohn's disease generates appreciably higher quantities of reactive oxygen species compared with normal tissue. We have extended these studies by developing accurate high performance liquid chromatography (HPLC) methods for the quantitation of reactive oxygen species generated by the faecal matrix. The data shows that the faecal matrix supports the generation of reactive oxygen species in abundance. As yet, there is a dearth of evidence linking this capacity to actual components of the diet which may influence the colorectal milieu. However, using the newly developed methodology we can demonstrate that the antioxidant phenolic compounds present in olive oil are potent inhibitors of free radical generation by the faecal matrix. This indicates that the study of the inter-relation between reactive oxygen species and dietary antioxidants is an area of great promise for elucidating mechanisms of colorectal carcinogenesis and possible future chemopreventive strategies.

Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignansand squalene.

The aim of this study was to evaluate the phenolic antioxidant and squalene content in a range of olive and seed oils. A mean of 290 +/- 38 (SEM) mg squalene/100 g was detected. However, while there was a weak significant difference between extra virgin (424 +/- 21 mg/kg) and refined virgin (340 +/- 31 mg/100 g; P<0.05) olive oils, highly significant differences were evident between extra virgin olive oils (P<0.0001) refined virgin olive oils (P<0.0001) and seed oils (24 +/- 5 mg/100 g). While seed oils were devoid, on average, the olive oils contained 196 +/- 19 mg/kg total phenolics as judged by HPLC analysis, but the value for extra virgin (232 +/- 15 mg/kg) was significantly higher than that of refined virgin olive oil (62 +/- 12 mg/kg; P<0.0001). Appreciable quantities of simple phenols (hydroxytyrosol and tyrosol) were detected in olive oils, with significant differences between extravirgin (41.87 +/- 6.17) and refined virgin olive oils (4.72 +/- 215; P<0.01). The major linked phenols were secoiridoids and lignans. Although extra virgin contained higher concentrations of secoiridoids (27.72 +/- 6.84) than refined olive oils (9.30 +/- 3.81) this difference was not significant. On the other hand, the concentration of lignans was significantly higher (P<0.001) in extra virgin (41.53 +/- 3.93) compared to refined virgin olive oils (7.29 +/- 2.56). All classes of phenolics were shown to be potent antioxidants. In future epidemiologic studies, both the nature and source of olive oil consumed should be differentiated in ascertaining cancer risk.

Olive-oil consumption and health: the possible role of antioxidants.

In the Mediterranean basin, olive oil, along with fruits, vegetables, and fish, is an important constituent of the diet, and is considered a major factor in preserving a healthy and relatively disease-free population. Epidemiological data show that the Mediterranean diet has significant protective effects against cancer and coronary heart disease. We present evidence that it is the unique profile of the phenolic fraction, along with high intakes of squalene and the monounsaturated fatty acid, oleic acid, which confer its health-promoting properties. The major phenolic compounds identified and quantified in olive oil belong to three different classes: simple phenols (hydroxytyrosol, tyrosol); secoiridoids (oleuropein, the aglycone of ligstroside, and their respective decarboxylated dialdehyde derivatives); and the lignans [(+)-1-acetoxypinoresinol and pinoresinol]. All three classes have potent antioxidant properties. High consumption of extra-virgin olive oils, which are particularly rich in these phenolic antioxidants (as well as squalene and oleic acid), should afford considerable protection against cancer (colon, breast, skin), coronary heart disease, and ageing by inhibiting oxidative stress.

Assignment and pH dependence of the 19F-NMR resonances from the fluorouracil anabolites involved in fluoropyrimidine chemotherapy.

Fluoropyrimidine chemotherapy relies on the intracellular anabolic conversion of 5-fluorouracil and the corresponding nucleosides to cytotoxic fluorinated nucleotides (F-Nuctd), such as 5-fluorouridine-5'-triphosphate (FUTP) or 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), which can be detected by 19F-NMR spectroscopy. We have made 19F-NMR signal assignments at 11.7 T and 4 degrees C for model solutions containing 5-fluorouracil (FUra), 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorouridine-5'-monophosphate (FUMP), FdUMP, 5-fluorouridine-5'-diphosphate (FUDP), FUTP and 5-fluorouridine-5'-diphospho(1)-alpha-D-glucose (FUDPG), and we have studied the effects of pH over the range 4.5-7.8, of Mg2+ concentration and addition of EDTA. This information provides a basis for the analysis of 19F-NMR spectra obtained from cells, tissues or extracts following fluoropyrimidine treatment.

Structure elucidation and chemical synthesis of stigmolone, a novel type of prokaryotic pheromone.

Approximately 2 micromol of a novel prokaryotic pheromone, involved in starvation-induced aggregation and formation of fruiting bodies by the myxobacterium Stigmatella aurantiaca, were isolated by a large-scale elution procedure. The pheromone was purified by HPLC, and high-resolution MS, IR, 1H-NMR, and 13C-NMR were used to identify the active substance as the hydroxy ketone 2,5, 8-trimethyl-8-hydroxy-nonan-4-one, which has been named stigmolone. The analysis was complicated by a solvent-dependent equilibrium between stigmolone and the cyclic enol-ether 3,4-dihydro-2,2, 5-trimethyl-6-(2-methylpropyl)-2H-pyran formed by intramolecular nucleophilic attack of the 8-OH group at the ketone C4 followed by loss of H2O. Both compounds were synthesized chemically, and their structures were confirmed by NMR analysis. Natural and synthetic stigmolone have the same biological activity at ca. 1 nM concentration.

In vivo 1H-NMR microimaging with respiratory triggering for monitoring adoptive immunotherapy of metastatic mouse lymphoma.

The metastatic ESb-MP murine lymphoma in DBA/2 mice has been used as a model for investigating metastatic disease and its cure by adoptive immunotherapy (ADI) as monitored by in vivo multislice spin-echo 1H NMR microimaging at 7 T. isoflurane inhalation anesthesia facilitated long measurement sessions, and respiratory gating with a fiber-optic sensor greatly reduced motional artifacts. With T2 weighting (TR = 2 s, TE = 30 ms) mean signal-to-noise ratios of 30 and 15 for kidney and liver, respectively, were achieved in 20 min (100-micron pixels, 1-mm slices, 25-mm field of view). Without the use of contrast agents, metastases with diameters > or = 0.3 mm in the imaged plane could be detected as hyperintense lesions in kidney (contrast ratio ca. 1.4) and liver (contrast ratio ca. 2) with a confidence level of > 98%. For the first time the complete eradication of late-stage macroscopic metastases by ADI could be demonstrated noninvasively by MRI.

Pharmacokinetics of iodine-123-IMBA for melanoma imaging.

UNLABELLED: The development of an effective radiopharmaceutical with affinity for malignant melanoma has been a research goal for some time. The early detection of melanoma metastases would greatly improve the therapy outcome for this disease. This article describes the synthesis of radioiodinated IMBA, N-(2-diethylaminoethyl)-3-[123I/131I]iodo-4-methoxybenzamide 8, its organ distribution, its comparison with BZA and other benzamides, and demonstrates the scintigraphic efficacy of the title compound with three melanoma patients. METHODS: The syntheses and radioiodination of eight benzamide derivatives are described. After intravenous injection into C57B16-mice subcutaneously transplanted with B16 melanoma, the organ distribution of the respective benzamides were investigated at 1 and 6 hr. n-octanol/phosphate buffer partition coefficients. The wholebody retention, erythrocyte and serum protein bound fractions of radioiodinated benzamides were measured. RESULTS: While structural changes in the amide substituents of N-(2-dialkylaminoalkyl)-4-iodobenzamides 2-7 resulted in no improvement in organ distribution compared with BZA, the 3-iodo-4-methoxyphenyl form of IMBA showed high melanoma uptake with significantly higher melanoma/nontarget tissue ratios. Compared with BZA the average ratio improved after 1 hr by a factor of eight and was still four times better after 6 hr. BZA and IMBA exhibit almost identical n-octanol/ phosphate buffer partition coefficients, however, IMBA has a faster urinary excretion facilitated by a lower affinity to erythrocytes and serum proteins; this could explain the improved tissue partinioning observed. Scintigraphy of patients with melanoma metastases confirmed the promising characteristics derived from the animal studies. CONCLUSION: Due to rapid background clearance and high melanoma affinity, IMBA showed high tumor contrast already at 4 hr after injection which makes it a promising new radiopharmaceutical for the scintigraphic detection of melanoma metastases.