Stimulation of the catecholaminergic and serotoninergic neurons in the rat brain by R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane, (-)-BPAP

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane HCl, (-)-BPAP, the recently developed selective and much more potent catecholaminergic/serotoninergic enhancer (CAE/SAE) substance than (-)-deprenyl enhances the performance of midbrain neurons, both in vivo and ex vivo, in a characteristic complex manner, presenting one bell shape dose/concentration effect curve in the low nanomolar range and another at higher micromolar range. For example, 4.7 +/- 0.10 nmol/g wet weight noradrenaline was released within 20 min from the quickly removed locus coeruleus of saline treated rats. This amount was increased 30 min after the subcutaneous administration of 0.0005 mg/kg (-)-BPAP to 15.4 +/- 0.55 nmol/g (P < 0.001). However, following the injection of a hundred times higher, 0.05 mg/kg, dose of (-)-BPAP, the amount of noradrenaline (4.3 +/- 0.25 nmol/g) released from the locus coeruleus did not differ from the control value. In ex vivo experiments, when the isolated locus coeruleus was soaked in an organ bath containing (-)-BPAP, the release of noradrenaline was significantly enhanced from 10(-16) M concentration, reached a peak effect at 10(-13) M concentration, but 10(-10) M (-)-BPAP was ineffective. A significant enhancer effect was detected also in the high concentration range from 10(-8) M, the peak effect was reached at 10(-6) M concentration and 10(-5) M (-)-BPAP was ineffective. (-)-BPAP enhanced in the low concentration range the performance of dopaminergic and serotoninergic neurons with a peak effect at 10(-13) and 10(-12) M concentration, respectively. The results with (-)-BPAP, the highly specific artificial enhancer substance, suggest that (i) high and low affinity "enhancer" receptors may exist in the brain, and (ii) that they may be identified with the recently cloned family of the "trace amine" receptors, activated by beta-phenylethylamine and tryptamine, the prototypes of the endogenous enhancer substances.     

Neuroprotective function of R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane, [R-(-)-BPAP], against apoptosis induced by N-methyl(R)salsolinol, an endogenous dopaminergic neurotoxin, in human dopaminergic neuroblastoma SH-SY5Y cells

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane HCl [R-(-)-BPAP] is one of "catecholaminergic and serotonergic enhancers", which were proposed to improve symptoms through increase in impulse-evoked release of monoamine neurotransmitters for Parkinson's disease. It was reported that (-)-BPAP up-regulated the synthesis of neurotrophic factors in mouse astrocytes, suggesting the neuroprotective potency of (-)-BPAP. In this paper, the neuroprotective function of (-)-BPAP and the related compounds was examined against apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], a possible pathogenic toxin in Parkinson's disease, in human dopaminergic neuroblastoma SH-SY5Y cells. The anti-apoptotic activity was confirmed with some of (-)-BPAP analogues, and the mechanism was found to be due to the direct stabilization of mitochondrial membrane potential and the induction of anti-apoptotic Bcl-2. The studies on structure-activity relationship demonstrated that the potency to stabilize the mitochondrial membrane potential depended on the absolute stereo-chemical structure of BPAP derivatives. The compounds with dextrorotation prevented the mitochondrial permeability transition, whereas those with levorotation did not. The presence of a propargyl or propyl group at the amino residue of R-(-)-1-(benzofuran-2-yl)-2-propylamine increased potency to stabilize the membrane potential and prevent apoptosis. R-FPFS-1169 and R-FPFS-1180 had more potent to induce Bcl-2 and prevent apoptosis than the corresponding S-enantiomers. These results are discussed with the possible application of BPAP derivatives as neuroprotective agents in Parkinson's disease and other neurodegenerative disorders.     

A pharmacological analysis elucidating why,in contrast to (-)-deprenyl (selegiline), alpha-tocopherol was ineffective in the DATATOP study

The Parkinson Study Group who conducted the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) trial designed their study in the belief that the MAO inhibitor (-)-deprenyl (selegiline), the antioxidant alpha-tocopherol, and the combination of the two compounds will slow the clinical progression of the disease to the extent that MAO activity and the formation of oxygen radicals contribute to the pathogenesis of nigral degeneration. In fact, (-)-deprenyl only delayed the onset of disability associated with early, otherwise untreated Parkinson's disease, however, in contrast to the expectation of the authors, alpha-tocopherol proved to be ineffective in the DATATOP study. Enhancer substances, (-)-deprenyl, (-)-1-phenyl-2-propylaminopentane [(-)-PPAP] the (-)-deprenyl analogue free of MAO inhibitory potency, and R-(-)1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] the presently known most potent enhancer substance, are peculiar stimulants. They enhance the impulse propagation mediated release of the catecholamines in the brain. Due to their enhancer effect, the amount of catecholamines released from selected discrete brain areas (striatum, substantia nigra, tuberculum olfactorium, locus coeruleus) is significantly higher in rats treated with an enhancer substance than in saline treated rats. We compared the effect of (-)-deprenyl 0.025 and 0.25 mg/kg, (-)-PPAP 0.1 mg/kg, (-)-BPAP 0.0001 mg/kg, and alpha-tocopherol 25 and 50 mg/kg, in this test. The doses of (-)-deprenyl and alpha-tocopherol were selected to be in compliance with the dose given in the DATATOP study. Compared to saline treated rats, the enhancer substances significantly increased the amount of dopamine released from the striatum, substantia nigra and tuberculum olfactorium and the amount of norepinephrine released from the locus coeruleus; alpha-tocopherol was ineffective. The results indicate that alpha-tocopherol was ineffective, because, unlike (-)-deprenyl it dose not enhance the activity of the nigrostriatal dopaminergic neurons.     

The L-DOPA-sparing effect of R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane hydrochloride [(-)-BPAP] in reserpine-pretreated rats

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane hydrochloride [(-)-BPAP], a highly potent enhancer of impulse propagation-mediated release of catecholamines and serotonin in the brain, and significantly increased the locomotor activity of normal rats at the doses of 0.3 and 1 mg/kg s.c. (P < 0.05), while L-DOPA (200 and 400 mg/kg i.p.) had no significant effect. The locomotor activity of rats simultaneously administered L-DOPA and (-)-BPAP was significantly higher than with (-)-BPAP alone (P < 0.05). In rats pretreated with reserpine (1 mg/kg i.v.), the hypolocomotion was significantly reversed by 400 mg/kg i.p. L-DOPA, or 1 or 3 mg/kg s.c. (-)-BPAP (P < 0.05). Furthermore, the combined administration of subthreshold doses of 200 mg/kg i.p. L-DOPA and 0.3 mg/kg s.c. (-)-BPAP highly potentiated the locomotor activity in the reserpine-pretreated rats. However, (-)-BPAP failed to reverse the hypolocomotion in rats pretreated with reserpine + alpha-methyl-DL-p-tyrosine. Thus, (-)-BPAP was demonstrated to possess the L-DOPA-sparing effect in normal and reserpine-pretreated rats.     

Cytoprotective effect of two synthetic enhancer substances, (-)-BPAP and (-)-deprenyl, on human brain capillary endothelial cells and rat PC12 cells

Enhancer regulation is a new control mechanism in the brain [Knoll, J., 2003. Enhancer regulation/endogenous and synthetic enhancer compounds: a neurochemical concept of the innate and acquired drives. Neurochemical Research 28(8), 1275-1297]. Enhancer substances exert their effect in bi-modal form with a highly characteristic dose-dependency. Two bell-shaped concentration curves have been published. The one in ultra low concentration range (fM) specific form of enhancer regulation and the other at high concentration (100 microM) non-specific form of enhancer regulation. Catecholaminergic neurons proved to be enhancer-sensitive cells. Since rat PC12 cells and human brain endothelial cells (HBEC) work under catecholaminergic influence, it was reasonable to expect that both the specific and non-specific form of the enhancer regulation might be detectable in these cells. We tested this possibility on these cultured cells under normoxia and hypoxia-reoxygenation. After 1 h hypoxia produced by Argon gas and 0, 2, 4, and 20 h reoxygenation the cell loss was calculated by propidiumiodide assay and the cell activity was investigated by Alamar Blue assay colorimetric measurement. The percentages of living and necrotic cells were expressed after propidiumiodide staining. Broad scale concentrations of the two compounds (1 fM-100 microM) were added to the culture strait after the oxygen deprivation. (-)-BPAP and (-)-deprenyl, due to their enhancer effect, exerted a significant cytoprotective effect on both HBECs and PC12 cells. In harmony with Knoll's publications we were able to demonstrate by the aid of (-)-BPAP and (-)-deprenyl that both HBEC and PC12 are enhancer-sensitive cells. We detected the specific form of the enhancer regulation in the ultra low concentration range (fM-pM) and also the non-specific form of the enhancer regulation was visible (mM-microM).     

Enhancer Regulation/Endogenous and Synthetic Enhancer Compounds: A Neurochemical Concept of the Innate and Acquired Drives

This review is to summarize experimental evidence and theoretical consideration in support of the concept that a mesencephalic enhancer regulation is the basis of the limited number of innate drives indispensable for the survival of the individual and the species, while a specifically organized telencephalic enhancer regulation is the basis of the acquired drives to reach an unlimited number of dispensable goals. The study is also an overview of the experimental and clinical data supporting the proposal that, due to the progressive decay of the mesencephalic enhancer regulation with the passing of time, the prophylactic administration of a synthetic enhancer substance [(—)-deprenyl, (—)-BPAP] during postdevelopmental life could significantly slow the age-related decay of behavioral performances, prolong life, and prevent the precipitation or delay the onset of Parkinson's disease and Alzheimer's disease.     

Enantioselective synthesis and absolute configuration of (-)-1-(benzofuran-2-yl)-2-propylaminopentane, ((-)-BPAP), a highly potent and selective catecholaminergic activity enhancer

Enantioselective synthesis and absolute configuration of (-)-1-(benzofuran-2-yl)-2-propylaminopentane ((-)-BPAP), which is a highly potent and selective catecholaminergic activity enhancer (CAE) substance, are described. The synthetic approach consists of the coupling reaction of benzofuran with (R)-N-tosyl-2-propylazirizine or (R)-N-methoxy-N-methylnorvaliamide, followed by appropriate modifications of the resulting coupling products. As the results, (-)-BPAP turned out to have the R configuration, which was finally confirmed by X-ray crystallographic analysis.     

Enhancer substances: selegiline and R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] enhance the neurotrophic factor synthesis on cultured mouse astrocytes

R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP] is a potent "catecholaminergic and serotonergic activity enhancer (CAE/SAE)", which enhances the impulse-evoked catecholamines and serotonin release, e.g. (-)-BPAP enhances in vitro norepinephrine efflux from the slices of locus coeruleus in a bipolar manner with the two effective ranges of low (fM-pM level) and high (nM-microM level) concentrations. Here, the effects of (-)-BPAP and selegiline on the cultured mouse astrocytes were studied. The protein levels of the neurotrophic factors (NGF, BDNF and GDNF) in the conditioned medium of cultured astrocytes were determined by using ELISA. In the cultured astrocytes incubated for 24 h with selegiline, the synthesis of NGF and BDNF was significantly enhanced in the concentration dependent manner, with minimum effective concentrations of 4 x 10(-4) and 5 x 10(-4) M, respectively. (-)-BPAP also enhanced the NGF, BDNF and GDNF synthesis, with minimum effective concentrations of 5 x 10(-5), 1 x 10(-5), and 1 x 10(-6) M, respectively. Although the effects of (-)-BPAP on the NGF synthesis was tested in the range of 1 x 10(-15)-5 x 10(-4) M, the concentration response curve of (-)-BPAP was a single bell shape with the peak effect at 1 x 10(-4) M, and did not show any effects in low concentrations such as fM-pM level. Each concentration response curve of (-)-BPAP on BDNF and GDNF synthesis was a single bell shape with peak effects at 1 x 10(-3) M and 1 x 10(-4) M, respectively.     

Analysis of the effect of (-)-BPAP,a selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

Endogenous and synthetic enhancer substances enhance in low concentration the impulse propagation mediated release of transmitters from the catecholaminergic and serotonergic neurons in the brain. The purpose of this study was to see whether uptake or MAO inhibition or agonists have similar enhancing prospectives as the enhancer substances. We measured the electrical stimulation induced release of [3H]-norepinephrine or [3H]-dopamine or [3H]-serotonin from the isolated brain stem of rats. (-)-1-Benzofuran-2-yl)-2-propylaminopentane HCl [(-)-BPAP] was used as a prototype of the enhancer compounds. 50 ng/ml (-)-BPAP was the most effective concentration in enhancing the nerve stimulation induced release of [3H]-norepinephrine and [3H]-dopamine, 10 ng/ml (-)-BPAP was highly effective in enhancing the release of [3H]-serotonin. In contrast, 250 ng/ml desmethylimipramine (DMI), a selective inhibitor of the uptake of norepinephrine, did not change significantly the nerve stimulation induced release of [3H]-norepinephrine and 50 ng/ml fluoxetine, a selective inhibitor of the uptake of serotonin, did not change the release of [3H]-serotonin. Neither 250 ng/ml clorgyline, a selective inhibitor of MAO-A, nor 250 ng/ml lazabemide, a selective inhibitor MAO-B, was capable to significantly increase the nerve stimulation induced release of either [3H]-serotonin or [3H]-norepinephrine. The potent dopamine receptor agonists, pergolide and bromocriptine did not change significantly the release of [3H]-dopamine in 50 ng/ml concentration, which is sufficient to stimulate the dopamine receptors. The results prove that stimulation of catecholaminergic and serotonergic neurons in the brain via the enhancing mechanism is clearly different from influencing uptake or MAO.     

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1-4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1-4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6-7) and tributyltin(IV) complexes (8-11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1-3 are also comparable to those of its o-analogs i.e. 4-7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41-53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1-3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8-11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1-7 and tributyltin(IV) complexes 8-11 is also discussed against a panel of human tumor cell lines.     

Structure-activity studies leading to (-)1-(benzofuran-2-yl)-2-propylaminopentane, ((-)BPAP), a highly potent, selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

The catecholaminergic and serotoninergic neurons in the brain change their performance according to the physiological need via a catecholaminergic/serotoninergic activity enhancer (CAE/SAE) mechanism. Phenylethylamine (PEA), tyramine and tryptamine are the presently known endogenous CAE/SAE substances which enhance the impulse propagation mediated release of catecholamines and serotonin in the brain. A PEA derivative, (-)deprenyl (selegiline), known as a selective inhibitor of MAO-B, is for the time being the only CAE/SAE substance in clinical use. Aiming to develop a selective CAE/SAE substance much more potent than (-)deprenyl, a series of new 1-aryl-2-alkylaminoalkanes, structurally unrelated to PEA and the amphetamines, was designed and prepared. Among them, (-)1-(benzofuran-2-yl)-2-propylaminopentane ((-)BPAP) was selected as a promising candidate substance for further studies. (-)BPAP significantly enhanced in rats the impulse propagation mediated release of catecholamines and serotonin in the brain 30min after acute injection of 0.36nmol/kg sc. In the shuttle box, (-)BPAP was in rats about 130 times more potent than (-)deprenyl in antagonizing tetrabenazine induced inhibition of performance. (+/-)BPAP protected cultured hippocampal neurons from the neurotoxic effect of beta-amyloid in 10(-14)-10(-15)M concentration.     

Two-step regio- and stereoselective syntheses of [19F]- and [18F]-2-deoxy-2-(R)-fluoro-beta-D-allose

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.     

Synthesis, crystal structures, cytotoxicity and qualitative structure-activity relationship (QSAR) of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes, (n)Bu2Sn(L)2

A series of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes has been synthesized and characterized by ¹H-, ¹³C-, ¹¹⁹Sn NMR, ESI-MS (electrospray ionization mass spectrometry), IR and ^119mSn Mössbauer spectroscopic techniques in combination with elemental analyses. The structures of four di-n-butyltin(IV) complexes, viz., ⁿBu₂Sn(L³)₂ (3), ⁿBu₂Sn(L⁴)₂ (4), ⁿBu₂Sn(L⁵)₂ (5) and ⁿBu₂Sn(L⁷)₂ · 0.5C₆H₆ (7) (LH = 5-[(E)-2-(aryl)-1-diazenyl)quinolin-8-ol) were determined by single crystal X-ray diffraction. In general, the complexes were found to adopt a distorted cis-octahedral arrangement around the tin atom. These complexes retain their solid-state structure in non-coordinating solvent as evidenced by ¹¹⁹Sn and ¹³C NMR spectroscopic results. The in vitro cytotoxicity of di-n-butyltin(IV) complexes (3-8) is reported against seven well characterized human tumour cell lines. The basicity of the two quinolinolato donor N and O atoms of the ligands are discussed in relation to the cytotoxicity data.     

Production of D-(--)-3-hydroxyalkanoic acid by recombinant Escherichia coli

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.     

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Monoamine Interactions Measured by Microdialysis in the Ventral Tegmental Area of Rats Treated Systemically with (±)-8-Hydroxy-2-(Di-n-Propylamino)tetralin

Abstract: The effect of (±)-8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT), a selective serotonin 5-HT_1A agonist, on levels of extracellular norepinephrine (NE), dopamine (DA), and 5-HT (measured simultaneously) was investigated by microdialysis in the ventral tegmental area (VTA) of freely moving rats, and their behavioral activity was monitored. At 50 µg/kg s.c., 8-OH-DPAT reduced 5-HT levels but enhanced NE and DA levels in VTA dialysate. These effects were not altered by pretreatment with systemic idazoxan (5 mg/kg i.p.), a selective α₂ antagonist, or local sulpiride (10 µ M ), a selective D₂/D₃ antagonist. At 500 µg/kg s.c., 8-OH-DPAT further enhanced or more persistently reduced dialysate NE or 5-HT content but had little effect on dialysate DA content. Its DA level-increasing effect could be seen dramatically with local infusion of cocaine (30 µ M ) and, to a lesser extent, sulpiride (10 µ M ). Depletion of endogenous 5-HT with p -chlorophenylalanine attenuated both the 5-HT level-reducing and DA level-enhancing effects of 8-OH-DPAT without affecting its maximal NE effect and the locomotor-stimulatory effect. Partial depletion of endogenous NE with N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine failed to change the monoamine response but diminished the locomotion induced by 8-OH-DPAT. These results suggested that (a) the low dose of 8-OH-DPAT may act at presynaptic 5-HT_1A receptors to modulate 5-HT and DA release, while acting at postsynaptic 5-HT_1A receptors to modulate NE release; (b) the high dose of 8-OH-DPAT may activate D₂ receptors to offset its DA level-increasing effect; and (c) the locomotor-stimulatory effect of 8-OH-DPAT might be mediated primarily by postsynaptic 5-HT_1A receptors and the NE system.     

Enhanced production of D-(-)-3-hydroxybutyric acid by recombinant Escherichia coli

Wild-type bacteria including Escherichia coli normally do not produce extracellular D-(-)-3-hydroxybutyric acid (3HB). To produce extracellular chiral 3HB, a new pathway for synthesis of 3HB was constructed by simultaneous expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), phosphor-transbutyrylase (ptb) and butyrate kinase (buk) in E. coli strain DH5alpha. E. coli DH5alpha containing any one of the four plasmids pBHR69, pUCAB, p68CM or pKKAB that harbor the phbA and phbB genes produced small amounts of 3HB, ranging from 75 to 400 mg l(-1), while E. coli DH5alpha harboring p68CMPTK containing genes of phbA, phbB, ptb and buk increased the 3HB concentration to 1.4 g l(-1) in shake flasks supplemented with LB broth and 20 g l(-1) glucose. 3HB production was further improved to over 2 g l(-1) in shake flasks when E. coli DH5alpha hosted two plasmids simultaneously that separately contained phbA and phbB in one plasmid while ptb and buk in the other. A batch fermentation run in a 5-l fermenter produced approximately 5 g l(-1) 3HB after 24 h. A fed-batch process increased 3HB production to 12 g l(-1) after 48 h of fermentation.     

Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione: Tautomery and coordination to copper(II)

(E)-2-(2-(2-hydroxyphenyl)hydrazono)-1-phenylbutane-1,3-dione (H₂L) was synthesized by azocoupling of diazonium salt of 2-hydroxyaniline with 1-phenylbutane-1,3-dione and characterized by IR, ¹H and ¹³C NMR spectroscopies and X-ray diffraction analysis. In solution, H₂L exists as a mixture of the enol-azo and hydrazone tautomeric forms and a decrease of temperature and of solvent polarity shifts the tautomeric balance to the hydrazone form. In the solid state, H₂L crystallizes from ethanol-water in the monohydrate hydrazone form, as shown by X-ray analysis. The dissociation constants of H₂L (pK₁ = 5.98 ± 0.04, pK₂ = 9.72 ± 0.03) and the stability constants of its copper(II) complex (log β₁ = 11.01 ± 0.07, log β₂ = 20.19 ± 0.08) were determined by the potentiometric method in aqueous-ethanol solution. The copper(II) complex [Cu₂(μ-L)₂]_n was isolated in the solid state and found by X-rays to be a coordination polymer of a binuclear core with a distorted square pyramidal metal coordination geometry.     

Biomimetic conversion of (-)-fusoxypyridone and (-)-oxysporidinone to (-)-sambutoxin: further evidence for the structure of the tricyclic pyridone alkaloid, (-)-fusoxypyridone

Biomimetic-type reactions of the tricyclic pyridone alkaloid, (−)-fusoxypyridone [(−)-4,6′-anhydrooxysporidinone] (1), recently encountered in an endophytic strain of Fusarium oxysporum, and (−)-oxysporidinone (2) afforded (−)-sambutoxin (3) and an analogue of 1, identified as (−)-1′(6′)-dehydro-4,6′-anhydrooxysporidinone (4), thus confirming the structure previously proposed for 1 and suggesting that 1-3 bear the same relative stereochemistry. Oxidation of 4 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) yielded a hitherto unknown sambutoxin analogue, (−)-4,2′-anhydrosambutoxin (5).