Development of a novel method for operating magnetic particles, Magtration Technology, and its use for automating nucleic acid purification.

Magnetic particles are useful for simple and efficient nucleic acid extraction. To achieve fully automated nucleic acid extraction and purification using magnetic particles, a new method for operating magnetic particles, Magtration Technology, was developed. In this method, magnetic separation is performed in a specially designed disposable tip. This enables high recovery of magnetic particles with high reproducibility. The features of this technology are (i) a simple mechanism for process control and (ii) flexible software to enable adaptation to commercially available reagents. Automated instruments based on Magtration Technology were developed and used for nucleic acid extraction. Total DNA, total RNA and plasmids were purified by Magtration Technology at an efficiency comparable to that of manual methods.

N-[1-(2-Phenylethyl)pyrrolidin-3-yl]-1-adamantanecarboxamides as novel 5-HT2 receptor antagonists.

A series of 1-adamantanecarboxamides was synthesized and examined for their potency as a selective 5-HT2 receptor antagonist. We found (S)-N-[1-[2-(4-fluorophenyl)ethyl]pyrrolidin-3-yl]-1-adamantane carboxamide hydrochloride hydrate (10-(S), Y-39241) to have a high affinity and selectivity for 5-HT2 receptors, and this potent anti-platelet effect of Y-39241 was confirmed both in vitro and in vivo.

N-[[1-(2-phenylethyl)pyrrolidin-2-yl]methyl]cyclohexanecarboxamides as selective 5-HT1A receptor agonists.

A series of benzamides was synthesized as selective agonists for the 5-HT1A receptor. It was found that (S)-N-[[1-(2-phenylethyl)pyrrolidin-2-yl]methyl]cyclohexanecarb oxamide(7-(S)) has potent and selective agonistic activity for the 5-HT1A receptor (5-HT1A; Ki 0.49 nmol/L, D2; IC50 = >1000 nmol/L, 5-HT2; Ki = 240 nmol/L).

Synthesis and pharmacological evaluation of carboxamide derivatives as selective serotoninergic 5-HT(4) receptor agonists.

A number of new carboxamide derivatives were synthesized. The affinity of these compounds for the serotoninergic 5-HT(4) receptor was evaluated by use of radioligand-binding techniques. The agonistic activity was evaluated as the contractile effect of the ascending colon isolated from guinea-pigs. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[1-[2-[(methylsulfonyl)amino]ethly]-4-piperidinylmethyl]benzamide (24) showed a high affinity for the 5-HT(4) receptor (Ki = 9.6 nM). Compound 24 displayed a higher affinity for 5-HT(4) receptors than the other receptors, including, 5-HT(3) and dopamine D(2) receptors. In addition, compound 24 was confirmed to be a potent 5-HT(4) receptor agonist (ED(50) = 7.0 nM). An interaction model between compound 24 and 5-HT(4) receptor was proposed.

Rapid prediction of rifampin susceptibility of Mycobacterium tuberculosis.

We evaluated the relationship between rifampin (RIF) susceptiblility and amino acid substitution in rpoB gene of Mycobacterium tuberculosis and the usefulness of rpoB gene sequencing in the rapid prediction of RIF susceptibility of M. tuberculosis in clinical specimens. A total of 76 genetic alterations in the 69 bp core region of rpoB gene were detected in 74 of 130 M. tuberculosis strains. Examination of the correlation between the minimum inhibitory concentrations (MICs) of RIF and amino acid substitutions in the 69 bp core region of rpoB gene revealed that all 43 strains containing amino acid substitution with Leu or Trp in codon 531 showed RIF-resistant phenotypes, with MICs > or = 64 microg/ml. In contrast, a variable level of RIF susceptibility was observed among strains containing amino acid substitutions in either codon 516 or codon 526. In the clinical study, we tested 26 sputum samples, two gastric lavages, and one synovial fluid sample obtained from patients with tuberculosis. The RIF susceptibility predicted by direct rpoB sequencing was satisfactorily compatible with the results of the RIF-susceptibility test and the MICs of RIF against isolated organisms. Our results suggest that rpoB gene sequencing is useful for the detection of M. tuberculosis in clinical samples as well as the rapid prediction of RIF susceptibility of these strains.

Benzoxazines. II. Synthesis, conformational analysis, and structure--activity relationships of 3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide derivatives as potent and long-acting serotonin-3 (5-HT3) receptor antagonists.

A series of 3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide derivatives was synthesized and evaluated for serotonin-3 (5HT3) receptor antagonistic activities by means of assays of 5-HT3 receptor binding and the ability to antagonize the von Bezold-Jarisch reflex in rats. Replacement of the 1,4-benzoxazine ring with a 1,4-benzthiepine ring or seven-membered ring (i.e., 1,5-benzoxepine or 1,5-benzthiepine) resulted in decreased affinity for 5-HT3 receptor. Introduction of substituents at the 2 position of the 1,4-benzoxazine ring increased the antagonistic activities (dimethyl > methyl > dihydro > phenyl). The compounds bearing a 9-methyl-9-azabicyclo[3.3.1]non-3-yl moiety as the basic part of 3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide derivatives were equipotent to those bearing 1-azabicyclo[2.2.2]oct-3-yl moiety. The 9-methyl-9-azabicyclo[3.3.1]non-3-yl moiety was confirmed to adopt a boat-chair conformation on the basis of both NMR studies and X-ray analysis. In this series, endo-6-chloro-3,4-dihydro-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-2,2, 4-trimethyl-2H-1,4-benzoxazine-8-carboxamide showed the highest affinity for 5-HT3 receptors (Ki = 0.019 nM), and a long-lasting 5-HT3 receptor antagonistic activity as evidenced by antagonism to the von Bezold--Jarisch reflex in rats. Such a long-lasting 5-HT3 receptor antagonism would be attributed to the introduction of both two methyl groups at the 2 position of the benzoxazine ring and the 9-methyl-9-azabicyclo[3.3.1]non-3-yl moiety, which adopts the boat-chair conformation.

Design and synthesis of 6-chloro-3,4-dihydro-4-methyl-2H-1,4-benzoxazine-8-carboxamide derivatives as potent serotonin-3 (5-HT3) receptor antagonists.

Several 3-substituted 5-chloro-2-methoxybenzamides were synthesized and evaluated for serotonin-3 (5-HT3) receptor binding affinity. The 5-HT3 receptor antagonistic activity of zacopride, a representative 5-HT3 receptor antagonist, was unchanged by the replacement of the 4-amino substituent on the aromatic moiety by a 3-dimethyl-amino substituent. This finding prompted a structural modification of azasetron, another 5-HT3 receptor antagonist. Consequently, a new series of 3,4-dihydro-2H-1,4-benzoxazine-8-carboxamides was obtained and these compounds were found to be more potent than 3,4-dihydro-3-oxo-2H-1,4-benzoxazine-8-carboxamides. In particular, (S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-3,4-dihydro-4-methyl-2H-1,4 -benzoxazine-8-carboxamide showed a high affinity for 5-HT3 receptors K(i) = 0.051 nM) and especially potent antagonistic activity against the von Bezold-Jarisch reflex (ED50 = 0.089 micrograms/kg i.v.) in rats.

Synthesis and structure-activity relationships of 2,3-dihydrobenzofuran-7-carboxamide derivatives as potent serotonin-3 (5-HT3) receptor antagonists.

A series of N-(azabicyclo-3-yl)-2,3-dihydrobenzofuran-7-carboxamide derivatives were synthesized and evaluated for serotonin-3 (5-HT3) receptor antagonistic activities assessed by 5-HT3 receptor binding (in vitro) and by the ability to antagonize the von Bezold-Jarisch reflex in rats (in vivo). In these compounds, 1-azabicyclo[2.2.2]oct-3-yl derivatives were more potent than 8-methyl-8-azabicyclo[3.2.1]oct-3-yl derivatives for 5-HT3 receptor antagonistic activities. The introduction of methyl groups at position 2 of the dihydrobenzofuran ring increased the pharmacological activities (dimethyl > monomethyl > dihydro). Furthermore, the stereoisomers of dimethyl-, monomethyl-, and dihydrobenzofuran derivatives were prepared to evaluate the stereoselectivity of their 5-HT3 receptor binding affinities. Concerning the basic part, the compounds bearing (S)-1-azabicyclo[2.2.2]octan-3-yl moiety were more potent than their counterparts. With respect to the methyl substituent at position 2 of the dihydrobenzofuran ring, the rank order of the potency was dimethyl > or = (2S)-methyl > (2R)-methyl > dihydro. These results suggest that the (2S)-methyl group of the dihydrobenzofuran part contributes to the enhancement of the pharmacological activity. Among these compounds, (S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-2,3-dihydro-2,2- dimethylbenzofuran-7-carboxamide hydrochloride (24) showed the highest affinity for 5-HT3 receptors (Ki = 0.055 nM), and the most potent antagonistic activity on the von Bezold-Jarisch reflex (ED50 = 0.18 microgram/kg i.v.).

Synthesis and pharmacology of 3,4-dihydro-3-oxo-1,4-benzoxazine-8-carboxamide derivatives, a new class of potent serotonin-3 (5-HT3) receptor antagonists.

A series of 3,4-dihydro-3-oxo-1,4-benzoxazine-8-carboxamide derivatives was synthesized and evaluated for serotonin-3 (5-HT3) receptor antagonistic activity assessed by their ability to antagonize the von Bezold-Jarish (BJ) effect in rats. Derivatives bearing 1-azabicyclo[2.2.2]oct-3-yl moiety as a basic function attached to the carboxamide at position 8 showed more potent antagonistic activity than those bearing the other three basic moieties. Structure-activity relationships of this series showed that methyl and chloro groups were more effective as substituents at positions 4 and 6, respectively. The representative compound 15 (Y-25130) in this series showed potent antagonistic activity on the BJ effect (ED50 = 1.3 micrograms/kg i.v.), high affinity for 5-HT3 receptor (Ki = 2.9 nM) and complete protection against cisplatin-induced emesis in dogs at a dose of 0.1 mg/kg i.v.

Growth stimulating activity of heat shock protein 90 alpha to lymphoid cell lines in serum-free medium.

A growth stimulating factor was purified from the culture supernatant of human-human hybridoma SH-76 cells in serum-free RPMI 1640 medium by the serial use of DEAE anion and heparin affinity chromatographies. The factor was a 85 kDa protein on SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence (PEETQTQDQPME) of the protein was coincident with that of heat shock protein 90 alpha (HSP90 alpha). The protein reacted with anti-HSP90 monoclonal antibody. These results suggest that the protein was a member of HSP90 alpha family after taking other circumstantial evidence into account. The protein stimulated the growth of some lymphoid cell lines such as human B-lymphoblastoid cell line HO-323-3 hybridomas derived from HO-323, and several other lymphoid cell lines. There were several lymphoid cell lines which did not respond to the protein. Growth stimulating activity of the protein was heat-unstable and significantly decreased at above 60 degrees C. These are the first data that describe growth-stimulating activity of heat shock protein 90 alpha.