A dramatic enhancing effect of InBr3 towards the oxidative Sonogashira cross-coupling reaction of 2-ethynylanilines.

The addition of InBr3 to the oxidative Sonogashira cross-coupling reaction of 2-ethynylaniline with (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane led to a dramatic increase in the reactivity to afford the corresponding 1,3-enynes bearing a trifluoromethyl group on their terminal sp(2) carbon. The subsequent cyclization of these 1,3-enynes under palladium catalysis provides access to the corresponding indoles bearing a 3,3,3-trifluoroprop-1-enyl group at their 2-position.

Immature platelet fraction for prediction of platelet engraftment after allogeneic stem cell transplantation.

Platelet regeneration represents an important and separate element in the engraftment process for allogeneic stem cell transplantation. Fully automated flow cytometry using blood cell counters now allows reliable quantification of reticulated platelets, expressed as the immature platelet fraction (IPF). We studied the kinetics of IPF in six patients grafted with allogeneic peripheral blood stem cell transplantation (PBSCT), 12 patients with bone marrow transplantation (BMT) and seven patients with cord blood transplantation (CBT). Preconditioning therapy caused an immediate and rapid fall in tri-lineage hematopoiesis. IPF rose transiently above 3% after a mean duration of 11 days post-PBSCT, 18 days post-BMT and 19 days post-CBT. This was 1, 4 and 13 days earlier than platelet engraftment, respectively. A linear correlation model showed a close association between the rise of IPF and tri-lineage engraftment after transplantation. IPF counting may thus provide an accessible measure of thrombopoietic activity, leading to early evaluation of marrow function and allowing monitoring of platelet regeneration.

Alamethicin-leucine zipper hybrid peptide: a prototype for the design of artificial receptors and ion channels.

In this report, we describe a novel concept of extramembrane control of channel peptide assembly and the eventual channel current modulation. Alamethicin is a peptide antibiotic, which usually forms ion channels in various association states. By introducing an extramembrane leucine zipper segment (Alm-LeuZ), the association number of alamethicin was effectively controlled to produce a single predominant channel open state. The assembly was estimated to be a tetramer, by comparison of the channel conductance with that of the template-assembled Alm-LeuZ tetramer, which was prepared by the conjugation of a maleimide-functionalized peptide template with cysteine-derivatized Alm-LeuZ segments. Employment of an extramembrane segment of a random conformation provided higher levels of channel conductance. The result exemplified the possibility of channel current control by a conformational switch of the extramembrane segments.

Enhanced and prolonged production of plantlets regenerated from carrot callus in a viscous additive-supplemented medium.

To reduce the hydrodynamic stress in plant cell culture and enhance the production of regenerated plantlets, a liquid medium containing a viscous additive was newly designed and plantlet production from embryogenic carrot callus cultivated in the medium was examined. Na-alginate or carboxymethyl cellulose (CMC) was used as the viscous additive. The viscosity of the medium increased with increasing additive content and the number of regenerated plantlets also increased. When carrot calli were cultivated in the medium containing 0.4% CMC, designated as N medium (viscosity, 3 mPa.s), the maximum enhancement of plantlet regeneration, approximately 2.5 times higher than that in the control medium, was obtained. Enlargement of callus size observed in N medium is considered to be the main reason for the enhanced plantlet regeneration. Regeneration enhancement was sufficiently induced after calli were cultivated once in N medium, but this regeneration ability rapidly disappeared after once cultivation in the conventional medium. In repeated batch culture using N medium, plantlet production continued at a high level for 18 batches (250 d) with no significant decrease, while in the control culture without CMC the number of plantlets produced dropped to almost zero by the sixth batch (84 d).

Michael-type reaction of ethyl bromodifluoroacetate with alpha,beta- unsaturated carbonyl compounds in the presence of copper powder.

We have reported that the reaction of ethyl bromodifluoroacetate (1) with alkenyl iodides in the presence of copper powder gives ethyl alkenyldifluoroacetates. As an extension of this reaction, reaction of 1 with Michael acceptors in the presence of copper powder was examined and found to give 1,4-addition products selectively, unless the acceptor has a group stabilizing a radical intermediate, such as a phenyl group.

Synthesis of new synthons for organofluorine compounds from halothane containing sulfur functional groups.

To develop new synthons for the syntheses of organofluorine compounds, the treatment of Halothane, 2-bromo-2-chloro-1,1,1-trifluoroethane, (1) with 4-methylbenzenethiol (2) in the presence of sodium hydride gave 1-chloro-2,2,2-trifluoroethyl 4-methylphenyl sulfide (3), which was oxidized with m-chloroperbenzoic acid (m-CPBA) to the corresponding sulfoxide (4) and sulfone (5). Reaction of 3 and 5 with allyltributyltin in the presence of 2,2'-azobis(isobutyronitrile) (AIBN) gave 1-(trifluoromethyl)-3-butenyl compounds (9, 11). Sulfoxide 4 was decomposed in this condition. The treatment of 3 with allyltrimethylsilane in the presence of Lewis acids gave 1-(trifluoromethyl)-3-butenyl compounds (9) in good yield. This result suggests that 4-methylphenylthio substituent stabilizes the alpha-carbocation effectively, though the trifluoromethyl group destabilizes it strongly. Aromatic compounds similarly reacted with 3 in the presence of titanium(IV) chloride to give 2-aryl-1,1,1-trifluoro-2-(4-methylphenylthio)ethanes. Thus, sulfur compounds derived from Halothane were found to be useful new synthons for organofluorine compounds.

[Pharmacological profiles of sildenafil (VIAGRA) in the treatment of erectile dysfunction: efficacy and drug interaction with nitrate].

Penile erection follows relaxation of the corpus cavernosum in which nitric oxide (NO) released during sexual stimulation from non-adrenergic non-cholinergic nerve endings and from endothelial cells of the corpus cavernosum plays a crucial role. Sildenafil (VIAGRA) selectively inhibited phosphodiesterase type 5 (PDE5) activity in the human corpus cavernosum and increased cGMP concentrations in the rabbit cavernosum in the presence of NO. Sildenafil enhanced the NO-dependent relaxation of the isolated human corpus cavernosum and the intracavernosal pressure in the anesthetized dog without affecting systemic blood pressure and heart rate. In the patients with erectile dysfunction, an orally administered sildenafil enhanced the penile rigidity during visual sexual stimulation. Sildenafil did not affect the phenylephrine-induced contraction of the isolated rabbit aorta, but enhanced the relaxant effect of glyceryl trinitrate. The pharmacodynamic interaction with glyceryl trinitrate was also observed in human studies where sildenafil potentiated the hypotensive effect of the nitrate. These results indicate that sildenafil, which enhances the physiological process of penile erection during sexual arousal, is a novel orally effective treatment for erectile dysfunction. It should be noted, however, that sildenafil enhanced the hypotensive effect of glyceryl trinitrate, as a result of inhibition of PDE5 in vascular smooth muscle. Therefore, administration of sildenafil to patients who are using nitrates and NO donors is contraindicated.

Synthesis of fluorine analogs of protoporphyrin potentially useful for diagnosis and therapy of cancer. IV. Synthesis of (trifluorovinyl)vinyl- and (1-chloro-2,2-difluorovinyl)vinyldeuteroporphyrins.

Trifluoro or chlorodifluoro analogs of protoporphyrin, the compounds in the title, were synthesized for use in the diagnosis and therapy of cancer. 3- Or 8-acetyldeuteroporphyrin dimethyl esters (2 and 3) were iodinated with iodine in the presence of potassium carbonate to the corresponding iodo compounds (5 and 6). The iodo compounds (5 and 6) were treated with bis(trifluorovinyl)zinc in the presence of tetrakis(triphenylphosphine)-palladium to give trifluorovinyl derivatives (7 and 8) in good yields. Reduction of the acetyl group of 7 and 8 with sodium borohydride afforded the corresponding hydroxyethyl derivatives (9 and 10). Compounds (9 and 10) were dehydrated with methanesulfonyl chloride and triethylamine to give (trifluorovinyl)vinyldeuteroporphyrin dimethyl esters (11 and 12). Treatment of 5 and 6 with bis(1-chloro-2,2-difluorovinyl)zinc in the presence of tetrakis(triphenylphosphine)palladium, followed by similar reactions as above gave (1-chloro-2,2-difluorovinyl)-vinyldeuteroporphyrin dimethyl esters (17 and 18).

Interaction between the L-type Ca2+ channel and the Ca(2+)-activated K+ channel in the fluctuating myogenic contraction in the rabbit basilar artery.

Ring preparations of the rabbit basilar artery spontaneously developed a fluctuating contraction that was not inhibited by the removal of the endothelium or by blockade of the synthesis or action of neurotransmitters and prostanoids. Nifedipine greatly relaxed the spontaneously-developed contraction. Charybdotoxin, an antagonist of the Ca(2+)-activated K+ channel, converted the fluctuating contraction to a tonic contraction, whereas the ATP-sensitive K+ channel blocker, glibenclamide, had no effect. These results indicate that the increased Ca2+ influx via the L-type Ca2+ channel in the rabbit basilar artery may induce myogenic contraction and consequently activate the Ca(2+)-activated K+ channel, which might lead to the fluctuation of the contraction by feedback regulation of the Ca2+ channel.

Rhythmic relaxations of active tension in the rabbit large arteries induced by a combination of cyclopiazonic acid and Bay K 8644.

1. We previously demonstrated that cyclopiazonic acid (CPA), an inhibitor of Ca(2+)-ATPase in the sarcoplasmic reticulum, induced rhythmic relaxations of active tension in the endothelium-denuded small arteries of the mesentery and the ear of the rabbit, but that this agent failed to induce rhythmic responses in the endothelium-denuded rabbit femoral artery. 2. In the present study, an attempt was made to induce rhythmic relaxations of active tension in the endothelium-denuded rabbit femoral artery and the thoracic aorta, both of which were suspended in organ chambers for isometric tension recordings, by using CPA plus Bay K 8644, an L-type Ca2+ channel agonist, to induce an excessive increase in cytosolic Ca2+. 3. CPA or Bay K 8644 alone failed to produce rhythmic relaxations in the femoral artery that had been contracted with phenylephrine. In contrast, rhythmic responses were induced by the sequential treatment of the femoral artery with CPA and Bay K 8644. 4. The rhythmic relaxations of active tension in the femoral artery induced by CPA plus Bay K 8644 were inhibited by charybdotoxin and by iberiotoxin, both of which are antagonists of the Ca(2+)-activated K+ channel, but not by glibenclamide, a blocker of the ATP-sensitive K+ channel. 5. The endothelium-denuded rabbit aorta also exhibited rhythmic responses by the sequential addition of CPA and Bay K 8644. These responses were sensitive to charybdotoxin. 6. These findings indicate that, like small arteries, the large femoral and aortic arteries of the rabbit are also capable of displaying rhythmic relaxations of active tension; these relaxations may be in part attributed to the activation of the Ca(2+)-activated K+ channel as a result of the Ca2+ overload caused by CPA and Bay K 8644.

In vitro effects of the novel anti-epileptic agent vigabatrin on alanine aminotransferase and aspartate aminotransferase activities in rat serum.

The in vitro effects of vigabatrin (CAS 60643-86-9, MDL 71,754) on alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were examined in rat serum. Initially, vigabatrin (30-100 micrograms/ml) produced a concentration-dependent decrease in ALT activity at 100 micrograms/ml or more after 15 min incubation with the rat serum. AST activity, in contrast, was unaffected by concentrations up to 1,000 micrograms/ml. Next, treating the rat serum with vigabatrin (30-300 micrograms/ml) for up to 5 h produced a concentration- and time-dependent decrease in ALT activity. On the other hand, a decrease in AST activity was observed only after incubation with the highest concentration of vigabatrin for 3 h or more. Finally, an investigation was made on the antagonistic effect of L-alanine, a natural substrate for ALT, on the vigabatrin-induced decrease in ALT activity to elucidate possible mechanisms underlying the inhibitory effect of vigabatrin on ALT activity. L-alanine, depending on its concentration, countered the effects of vigabatrin (100 micrograms/ml) at a 10- or 100-fold higher molar ratio than vigabatrin. These findings suggest that vigabatrin favorably decreases ALT activity by blocking the L-alanine binding site of enzymes.

Endothelium-dependent rhythmic contractions induced by cyclopiazonic acid, a sarcoplasmic reticulum Ca(2+)-pump inhibitor, in the rabbit femoral artery.

The vascular responses to cyclopiazonic acid (CPA), an inhibitor of the Ca(2+)-ATPase in the sarcoplasmic reticulum, were investigated in the rabbit femoral artery, suspended in an organ chamber for isometric tension recordings. CPA produced rhythmic contractions in the femoral artery which had been contracted with phenylephrine. CPA, however, did not induce the rhythmic responses in endothelium-denuded arteries. NG-nitro-L-arginine methyl ester and methylene blue, inhibitors of the formation and the action of nitric oxide, respectively, failed to antagonize the CPA-induced rhythmic contractions in the phenylephrine-contracted artery. In contrast, the CPA-induced rhythmic contractions were abolished by charybdotoxin, a Ca(2+)-activated K+ channel antagonist, but not by glibenclamide, a blocker of the ATP-sensitive K+ channel. Nifedipine also inhibited the CPA-induced rhythmic contractions in the endothelium-intact artery and relaxed the endothelium-denuded artery treated with CPA. These results indicate that the CPA-induced rhythmic contractions in the phenylephrine-contracted rabbit femoral artery may be attributed to the periodic inactivation of the voltage-dependent Ca2+ channel, presumably regulated by the Ca(2+)-activated K+ channel. The activation of the K+ channel by CPA might occur only when the endothelium is present.

Acute effects of deflazacort and its metabolite 21-desacetyl-deflazacort on allergic reactions.

The acute effects of deflazacort (MDL 458, CAS 14484-47-0) and its metabolite, 21-desacetyl-deflazacort, on allergic reactions in animal models were investigated and compared with those of prednisolone. Deflazacort, 21-desacetyl-deflazacort and prednisolone all inhibited 48-h homologous passive cutaneous anaphylaxis in rats, but had no significant effects on active systemic anaphylaxis in mice, on the Schultz-Dale reaction in the isolated guinea-pig trachea or on compound 48/80-induced histamine release in rat peritoneal mast cells. All three agents inhibited reversed cutaneous anaphylaxis in rats and the Arthus reaction in mice. The inhibitory effects of deflazacort on the passive cutaneous anaphylaxis, the reversed cutaneous anaphylaxis and the Arthus reaction were similar to those of 21-desacetyl-deflazacort and were stronger than those of prednisolone. Delayed type hypersensitivity in mice was also inhibited by deflazacort and 21-desacetyl-deflazacort, but prednisolone, at the doses used in the present study, had little effect on this immune response. These findings indicate that while deflazacort and 21-desacetyl-deflazacort have stronger anti-allergic effects than prednisolone, they seem to have little acute effect on mast cell degranulation or on chemical mediators at the receptor site.

Endothelium-derived nitric oxide protection of spontaneous coronary contractions in the rabbit by countering Ca2+ influx via the voltage-sensitive Ca2+ channel.

The mechanism of spontaneous coronary contractions was investigated in the rabbit. Arteries with intact endothelium did not spontaneously contract. However, spontaneous coronary contractions were generated after the removal of the endothelium. NG-nitro-L-arginine methyl ester and methylene blue, the inhibitors of the formation and the action of nitric oxide, respectively, induced contractions in endothelium-intact arteries. Spontaneously produced contractions in endothelium-denuded arteries were reversibly inhibited by a Ca(2+)-free solution and by nifedipine, but not by inhibitors of neurotransmitters or prostanoids. These findings may indicate that spontaneous coronary contractions in the rabbit may be attributed to Ca2+ influx via the voltage-sensitive Ca2+ channel. The basal release of endothelium-derived nitric oxide may, in part, antagonize this Ca2+ influx.

Effects of cyclopiazonic acid on phenylephrine-induced contractions in the rabbit ear artery.

1. Upon stimulation with phenylephrine, the rabbit ear artery displays endothelium-regulated rhythmic contractions, which may be attributed to the periodical activation of the dihydropyridine-sensitive Ca2+ channel, presumably regulated by the Ca(2+)-activated K+ channel. The effect of cyclopiazonic acid (CPA), an inhibitor of the Ca(2+)-ATPase of the sarcoplasmic reticulum (SR), on phenylephrine-induced contractions was examined in endothelium-denuded rabbit ear arteries suspended in an organ chamber for isometric tension recordings. 2. Phenylephrine-induced tonic contractions were converted to rhythmic ones by the addition of CPA. 3. The CPA-induced rhythmic contractions were abolished by the blockade of the dihydropyridine-sensitive Ca2+ channel and the Ca(2+)-activated K+ channel by nifedipine and charybdotoxin, respectively. In contrast, glibenclamide, an ATP-sensitive K+ channel antagonist, had no effect on the CPA-induced rhythmic responses. 4. CPA attenuated both Ca2+ repletion by the SR and Ca2+ influx across the plasmalemma without having a significant effect on Ca2+ release from the SR, as evaluated by phenylephrine-induced contractions. In contrast, these three parameters were not altered by the presence of the endothelium. 5. These findings indicate that the CPA-induced rhythmic contractions in the endothelium-denuded rabbit ear artery may be induced by the same ionic mechanism as endothelium-regulated rhythmic responses, by which the K+ channel could regulate the probability of the Ca2+ channel being opened. The CPA-induced rhythmic contractions may correlate with the inhibitory effects of CPA on the SR function, although this is not true for the endothelium-regulated rhythmic contractions.

The role of sarcoplasmic reticulum in endothelium-dependent and endothelium-independent rhythmic contractions in the rabbit mesenteric artery.

Upon stimulation with phenylephrine, the rabbit mesenteric artery displays endothelium-dependent and endothelium-independent rhythmic contractions in the absence and the presence of ryanodine, respectively. For examination of the involvement of the sarcoplasmic reticulum (SR) in these two types of rhythmic contractions, the mesenteric ring was suspended in an organ chamber for isometric tension recordings. Phenylephrine induced endothelium-dependent rhythmic contractions (EDRC), which were converted to endothelium-independent rhythmic contractions (EIRC) by the subsequent addition of ryanodine. Cyclopiazonic acid (CPA) also induced EIRC in the artery contracted with phenylephrine. The nifedipine-treated artery displayed neither EDRC upon phenylephrine stimulation nor EIRC by the addition of ryanodine or CPA: however, these agents relaxed the arteries. Phenylephrine induced EDRC in the artery treated with the K+ channel antagonist sparteine, but these rhythmic contractions were converted to a sustained contraction by ryanodine and CPA without producing relaxation of the artery. Ryanodine and CPA inhibited both phenylephrine-induced Ca2+ release from the SR and Ca2+ sequestration, without affecting Ca2+ influx across the plasmalemma, evaluated by monitoring agonist-induced contractions. These findings indicate that: (1) the EDRC may be attributed to Ca2+ release from the SR, which may be charged by Ca2+ influx via the voltage-dependent Ca2+ channel; and (2) the EIRC may arise from functional impairment of the SR and by the subsequent increase in the K+ efflux, presumably via the Ca(2+)-activated K+ channel.

The ionic mechanism of phenylephrine-induced rhythmic contractions in rabbit mesenteric arteries treated with ryanodine.

Phenylephrine induces endothelium-independent rhythmic contractions in ryanodine-treated rabbit mesenteric arteries. To elucidate the ionic mechanism of this rhythmic behaviour, rabbit mesenteric arterial rings were suspended in an organ chamber for isometric tension studies. Yohimbine, propranolol, and atropine had no effect on these contractions, minimizing the possibility that transmitter release from nerve terminals was involved. Additionally, the oscillatory contractions were not altered by diphenhydramine, cimetidine, and indomethacin, thus ruling out the involvement of histamine and prostaglandins. This oscillatory response was completely abolished after the removal of extracellular Ca2+, as well as after Ca2+ channel blockade by diltiazem or nifedipine. Sparteine and quinidine, Ca(2+)-activated K+ channel blockade by diltiazem or nifedipine. Sparteine and quinidine, Ca(2+)-activated K+ channel antagonists, also abolished the oscillation. In contrast, tetraethylammonium and 3,4-diaminopyridine, voltage-dependent K+ channel antagonists, augmented the response. Glibenclamide, an antagonist of the ATP-sensitive K+ channel, had no effect on the rhythmic contractions. These results suggest that the rhythmic contractions observed in rabbit mesenteric arteries after ryanodine treatment were caused by the movement of Ca2+ and K+ across the plasmalemma via the voltage-dependent Ca2+ channel and the Ca(2+)-activated K+ channel, respectively.

Phenylephrine-induced rhythmic activity in the rabbit ear artery.

Isolated rabbit ear arteries displayed rhythmic contractions when stimulated with alpha 1 agonist phenylephrine. These rhythmic responses were greatly attenuated by endothelium removal. However, contractions were sufficiently rhythmic in the arteries treated with NG-monomethyl-L-arginine, NG-nitro-L-arginine and indomethacin, synthetic inhibitors of endothelium-derived nitric oxide and prostanoids. Phenylephrine-induced rhythmic contractions were converted to tonic contractions by the blockade both of the voltage-dependent Ca2+ channel and the Ca(2+)-activated K+ channel by nifedipine and charybdotoxin, respectively. In contrast, glibenclamide, an ATP-sensitive K+ channel antagonist, did not alter the rhythmic contractions. These results suggest that endothelium may in part regulate the phenylephrine-induced rhythmic contractions in the rabbit ear artery, although endothelium-derived nitric oxide or prostanoids may not be involved in these responses. These endothelium-involved rhythmic responses may be attributed to the activation of the voltage-dependent Ca2+ channel and the Ca(2+)-activated K+ channel.

The role of endothelium in the phenylephrine-induced oscillatory responses of rabbit mesenteric arteries.

Phenylephrine-induced oscillatory contractions in rabbit mesenteric arteries were investigated in vitro. Adrenergic, cholinergic, or histamine antagonists as well as cyclooxygenase and lipoxygenase inhibitors had no effect on this phenylephrine-induced oscillation. The removal of extracellular calcium ions or treatment with a calcium antagonist reduced the amplitude and frequency of the oscillation. Removal of the endothelium or treatment with inhibitors of the synthesis or the target enzyme of endothelium-derived relaxing factor (EDRF) also reduced the amplitude and frequency of the oscillation. In a perfusion bioassay, the perfusate from an endothelium-intact arterial segment induced oscillation of an endothelium-denuded arterial ring recipient. These results suggest that phenylephrine-induced oscillation is mediated by an endothelium-derived factor such as EDRF and depends on the influx of extracellular calcium ions.