6,7-Dihydroxy-3-chromanamine: synthesis and pharmacological activity of an oxygen isostere of the dopamine agonist 6,7-dihydroxy-2-aminotetralin.

6,7-Dihydroxy-3-chromanamine, the oxygen isostere of 6,7-dihydroxy-2-aminotetralin (6,7-ADTN), has been synthesized and its dopaminergic activity in various test systems determined. Following bilateral injection into the rat nucleus accumbens, a pattern of locomotor activity similar to that produced by 6,7-ADTN was observed. Its ability to displace N-n-propyl[3H]norapomorphine binding to homogenates of rat brain corpus striatum was found to be about 15 times weaker than 6,7-ADTN and apomorphine. Like 6,7-ADTN it failed to influence dopamine metabolism following an intraperitoneal injection. It is suggested that in addition to the 2-aminotetralins, the 3-chromanamines may be a potential source of new dopamine receptor agonists.

Neuropharmacological profile of a new series of dopamine agonists: N-n-propyl-hexahydronaphthoxazines.

It has been demonstrated that within the series of hydroxylated (7-OH, 9-OH) and non-hydroxylated (N-0498) hexahydronaphthoxazines the 9-OH (N-0500) analogue is a very potent centrally acting DA receptor agonist. In in vitro [3H]DP-5,6-ADTN binding experiments, reflecting D-2 dopaminergic activity, N-0500 was equipotent with apomorphine and RU-29717, whereas both the 7-OH (N-0499) and N-0498 were much less effective. In in vivo tests related to DA receptor stimulation N-0500 was found to be the most active compound. In the gamma-butyrolactone model, a test for DA autoreceptor activation, N-0500 was 10 times as potent as apomorphine, but 3 times less active than RU-29717. The locomotor activity of mice was inhibited more strongly by N-0500 than by N-0499. Striatal concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid were rapidly reduced by N-0500 both after intraperitoneal and oral administration, indicating that this compound is well absorbed from the gastrointestinal tract and passes the blood-brain barrier to activate DA autoreceptors. In models for postsynaptic DA receptor stimulation (induction of stereotypy in rats, reversal of reserpine-induced immobility of mice) N-0500 was found to be as effective as RU-29717 in inducing stereotyped behaviors in rats, but was much less effective than RU-29717 in restoring the mobility of reserpinized mice, suggesting a selectivity for D-2 DA receptors by N-0500 in contrast to the mixed D-1/D-2 receptor activity of RU-29717. In in vitro binding experiments for evaluating the affinity towards other receptor types, N-0500 exhibited only a weak affinity towards 5-HT1 and alpha 2 binding sites and possessed a very weak affinity for 5-HT2 and alpha 1 receptor sites. It was concluded from these in vitro binding experiments that N-0500, has not only a very high affinity for D-2 DA receptors, but is more selective than RU-29717 and much more selective than the ergot bromocriptine. On the basis of its very potent in vivo central D-2 dopamine receptor activities and its in vitro selectivity, N-0500, being the most potent compound within the series, is a much more specifically acting drug than many of the dopaminergic ergolines and might therefore be a good candidate for the treatment of Parkinson's disease.

The synthesis of [1,2,3,4-13C] cortisol.

The preparation of [1,2,3,4-13C] cortisol 21 acetate by total synthesis is described. The C labels are introduced in a way analogous to the one described by us previously for the synthesis of testosterone and estradiol. The cortisol dihydroxyacetone side chain was elaborated using known methods. The 11 beta-hydroxyl function was introduced by addition of hypobromous acid to a 9-double bond followed by reductive debromination. 13C-labeled cortisol can be used as a tracer for the determination of cortisol production rates.

[1,2,3,4-13C] testosterone and [1,2,3,4-13C] estradiol.

The preparation of [1,2,3,4-13C] testosterone and of [1,2,3,4-13C] estradiol by total synthesis is described. The 13C labels are introduced by alkylating intermediate 1 with [1,2,3,4-13C]l-iodo-3,3-ethylenedioxybutane (2) to obtain intermediate 10. Hydrolysis of the ketal function, cyclization, aromatization and removal of protective groups gave [1,2,3,4-13C] estradiol. Labeled testosterone was prepared by methylating intermediate 10 and by subsequent treatment with acid. The labeled steroids can be used as tracers for in vivo metabolic studies and as internal standards for the development of definitive gc-ms quantitative methods.

The hydroxy-hexanydronaphthoxazines: a new group of very potent and selective dopamine agonists.

Replacement of the pyrrole ring system in the dopaminergic oxaergolines (e.g. RU 29717) by a phenolic OH group leads to the hydroxy-hexahydronaphthoxazines, a new group of potent dopamine agonists which exhibit increased selectivity due to their lower affinity for adrenergic and 5-HT receptors.

Synthesis of 1,2-Epoxyoctane by Pseudomonas oleovorans During Growth in a Two-Phase System Containing High Concentrations of 1-Octene.

We have optimized and compared the synthesis of 1,2-epoxyoctane from 1-octene by resting and by growing cells of Pseudomonas oleovorans. The net production of 1,2-epoxyoctane by resting cells never exceeded 0.6 mg/ml of suspension. In contrast, P. oleovorans produced much more epoxide when it was grown on high levels of 1-octene. To raise the total production of epoxide, the octene layer was repeatedly transferred to fresh, growing cultures of P. oleovorans. By using this approach, a maximum of 28 mg of epoxide was synthesized per ml of total culture, resulting in the accumulation of ca. 75 mg of epoxide per ml in the octene phase.

Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role.

The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.

Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane.

The growth of Pseudomonas oleovorans on n-octane was characterized by the formation of intracellular structures. These inclusions were isolated and characterized. Morphologically, they resembled the poly-beta-hydroxybutyrate granules found in Bacillus cereus, as shown by freeze-fracture electron microscopy. The elemental analysis of isolated granules showed, however, that they do not contain poly-beta-hydroxybutyric acid. Instead, the analysis was consistent with a C8 polyester, which interpretation was supported by the fatty acid analysis of hydrolyzed granules. From the evidence presented here, we conclude that P. oleovorans forms poly-beta-hydroxyoctanoate granules when grown on n-octane.

Synthesis of carbon-11 labelled glycine and the dipeptides L-phenylalanylglycine and L-leucylglycine.

Carbon-11 labelled glycine has been prepared in 30 min by carboxylation of alpha-lithiomethylisocyanide with a radiochemical yield of 10-15%. After coupling with L-phenylalanine-N-carboxyanhydride and L-leucine-N-carboxyanhydride followed by HPLC purification, the corresponding dipeptides were obtained in 20 min with a radiochemical yield of 30-40%. Consequently, starting with 11CO2, non carrier added L-phenylalanyl[1-(11)C]glycine and L-leucyl-[1-(11)C]glycine in 0.1 N NaH2PO4 were obtained in 50 min with a radiochemical yield of 3-6%. The radiochemical yield figures are not corrected for decay.

Syntheses of haptens containing dioxaphosphorinan methoxyacetic acid linker arms for the production of antibodies to organophosphate pesticides.

Four generic heterobifunctional reagents, namely 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid methyl ester, p-sulfide, 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)-methoxyacetic acid methyl ester, p-oxide, 2-(2-mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl)-methoxyacetic acid bispotassium salt, p-sulfide-, and (2-methoxy-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid, methyl ester, have been synthesized and used to prepare organophosphate, thiophosphate, and dithiophosphate haptens containing a functional carboxyl group which can be used to conjugate the haptens to proteins. These hapten-protein conjugates have been used as antigens for preparing polyclonal sera against all classes of organophosphate pesticides. The eight examples used protein-hapten conjugates of chlorpyrifos, parathion, diazinon, paraoxon, azinphos, dimethoate, demeton, and dichlorvos. These were all immunogenic and resulted in sera containing antibodies that recognized the corresponding parent pesticide with high specificity.

Synthesis of no-carrier-added L- and D-[1-11C]-DOPA.

No-carrier-added DL-[1-11C]-DOPA has been synthesized by carboxylation of an alpha-lithioisocyanide with a radiochemical yield of up to 15% without correction for decay. The total synthesis time is 30 min. The resolution of the D- and L-isomers was accomplished within 16 min by HPLC using a chiral stationary phase and a phosphate buffer of pH 4.5 as eluent.

The preparation of carbon-11 labelled proline for positron emission tomography. Preliminary distribution studies in rats with Walker 256 carcinosarcoma.

DL-[1-11C]Proline has been synthesized by carboxylation of alpha-lithiopyrrolidyl-N-tert-butyl-formamidine with a radiochemical yield of up to 18% without correction for decay. The total synthesis time is 45 min. Accumulation of DL-[1-11C]proline has been shown in Walker 256 carcinosarcoma transplanted in rats. A tumor/non-tumor ratio of 5.9 was found at 45 min after i.v. injection.

Syntheses of carbon-11 labelled ornithine and lysine. Preliminary accumulation studies in rats with Walker 256 carcinosarcoma.

DL-[1-11C]ornithine and lysine have been synthesized by carboxylation of the corresponding alpha-lithioisocyanide with a radiochemical yield of up to 14% without correction for decay. The total preparation time is 50 min. Accumulation of both DL-[1-11C]ornithine and lysine in Walker 256 carcinosarcoma is observed, with a tumor/non-tumor ratio of 4.9 and 4.5 respectively, at 45 min after intravenous injection.

Carbon-11 labelled tyrosine to study tumor metabolism by positron emission tomography (PET).

To measure the rate of protein synthesis in human neoplasms by positron emission tomography, we prepared no carrier added DL-(1-11C)-tyrosine by 11C-carboxylation of the appropriate alpha-lithioisocyanide followed by hydrolysis of the isocyanide function and removal of the protecting methoxy group. The purification, resolution and solvent switch to saline was performed by high performance liquid chromatography (HPLC). DL-(1-11C)-Tyrosine in 0.1 N NaH2PO4 buffer was prepared with a radiochemical yield of 8%-16% (EOS, 35 min). The enantiomeric separation and solvent switch to saline were achieved in 5 min and 10 min respectively. Consequently L-(1-11C)-tyrosine in physiological saline was obtained in 2%-4% radiochemical yield. Tumor accumulation in rats with the experimental WALKER 256 carcinosarcoma was observed for both the L- and D-isomer. Using positron emission tomography a tumor/muscle ratio of two was observed for the L-isomer 15 min after injection. The corresponding figure for the D-isomer was 2.5. The first clinical results with DL-(1-11C)-tyrosine show accumulation of radioactivity in meningioma, a primary breast carcinoma and in liver metastases of a colonic carcinoma.

The resolution, isolation, and pharmacological characterization of the enantiomers of a benzamide containing a chiral sulfoxide.

Rac-ML-1035 (MDL 201,035: 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2-(diethylamino)ethyl] benzamide hydrochloride) is a racemic gastroprokinetic with serotonergic (5-hydroxytryptamine, 5-HT) activity and a novel chiral sulfoxide substituent. Chromatographic and chemical methods have been developed to resolve the enantiomers of rac-ML-1035, and the absolute configuration of the (R)-enantiomer has been determined. We also report pharmacological characterization of rac-ML-1035 and its respective isomers. Radioligand binding to rat cortical membranes revealed that (R)-ML-1035 (MDL 201,226) and (S)-ML-1035 (MDL 201,227) had equivalent activity at the 5-HT3 receptor. However, in isolated tissue studies including field-stimulated guinea pig ileum, field-stimulated rat fundic strip, and nonstimulated guinea pig ileum, (S)-ML-1035 was equally potent yet had greater maximal activity than (R)-ML-1035 in eliciting or facilitating cholinergic contractions. Thus, enantiomers of rac-ML-1035 can be resolved, and the relative configuration of these isomers influences their pharmacological activity.