Fluorogenic reagents for amines and thiols which are of value for their determination in high performance liquid chromatography (HPLC) are reviewed. Special attention was made on the development and application of 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F) and ammonium 7-fluorobenzo-2-oxa-1, 3-diazole-4-sulfonate (SBD-F) which are respective reagents for amines and thiols. Finally, usage of a chemiluminescence reaction (TCPO-H2O2-fluorescent compounds) for the detection of fluorescent compounds separated by HPLC column was proposed. The proposed method allowed us to detect 2 fmol of dansylated amino acids.
The reaction of indan-1, 3-dion with carbon disulfide followed by methylation with dimethyl sulfate or methyl iodide in the presence of sodium hydroxide gave 2-bis (methylthio)-methyleneindan-1, 3-dione (2), 2-(1-mercapto-1-methylthio) methylene-1, 3-dione (3), and 2, 5-bis (1, 3-dioxo-2-indanylidene)-1, 3, 5-trithiolane (4). Compound 3 reacted with ammonia to give methyl 3-amino-1-oxoindan-2-dithiocarboxylate (6) which was converted to 8-methylthioindeno [2, 1-c] isothiazol-3 (3H)-one (9) by the treatment with iodine in dimethyl sulfoxide. Compound 9 reacted with amines and active methylene compounds giving the corresponding products substituted on one methylthio group.
Twenty kinds of 4-acyl-3, 4-dihydro-2H-pyrido [3, 2-b]-1, 4-oxazine (12a-21b) were synthesized. The following results were obtained by the preliminary investigation on the local anesthetic and the analgesic (mouse writhing test) activities of the synthesized compounds. There was little local anesthetic activity in all these compounds ; there was an analgesic activity approximately equivalent to aspirin's.
Reduction of the acetoacetates (2, 9, and 14) containing chiral alkyl moieties, derived respectively from three chiral alcohols, (1R)-8-phenylmenthol (1), (1R)-3-diphenylmethylnopinol (8), and (1R)-cis-3-hydroxyisobornyl neopentyl ether (11), has been examined using hydride reagents. Although high asymmetric induction could not be achieved in each case, it is found that the reduction occurs in predictable fashion from the less hindered face of the transition state with an acetoacetate conformation where all alkoxy-carbon-hydrogen bond, the estercarbonyl, and ketone-carbonyl are arranged syn-planar (2A).
The results of screening of biologically active principles in Chinese medicinal prescriptions used for the treatment of hepatic jaundice suggested the presence of strong inhibitors of β-glucuronidase in "Inchinko-to"extracts. Non-dialyzable portion (A-6) of water-soluble fraction of"Inchinko-to"extracts was found to be potently inhibitory against β-glucuronidase from bovine liver. The inhibitor of β-glucuronidase was partially purified by gel-filtration on Toyopearl HW-60S and column chromatography on DEAE-cellulose. The partially purified β-glucuronidase inhibitor (A-8) was supposed to be a glycoprotein or a glycopeptide. Its activity was not affected by acid, pepsin and trypsin. It also inhibited β-glucuronidase obtained from human intestinal bacteria.
The antithrombic activities of 70% methanol extract (I) obtained from Korean red ginseng (root of Panax ginseng C.A. MEYER) were evaluated in the models of experimental disseminated intravascular coagulation (DIC) induced by endotoxin or thrombin in rats, and in the in vitro models of blood platelet aggregation, agglutination of fibrinogen and activation of fibrinolytic system. In the coagulative system, I prevented the exchanges of values of clinical examination (blood platelet, fibrinogen and prothrombin time) at a dose of 500 mg/kg in rats on DIC induced by endotoxin or thrombin. I also inhibited the formation of fibrin thrombi in the renal glomeruli on the thrombin-induced DIC in rats. In the fibrinolytic system, I was found to show fibrinolytic activity in rats as determined by the euglobulin lysis time (ELT) assay. In the in vitro experiments, incubation of I in the plasminogen-containing fibrin plate promoted activation of urokinase action.
Fluorescent characteristics of 3-aminoisocarbostyril derivatives were examined from the viewpoint of the substituent effect in comparison with 3-aminoisocarbostyril (Ia) as a parent compound. 3-Amino-(Ib-f, IIIa, b) and 3-substituted aminoisocarbostyril derivatives (IIa-e) gave larger fluorescence quantum yields in ethanol than that of Ia. The fluorescence quantum yield of Ia was increased by the replacement of methoxy and alkylamino groups at C7 and C3, respectively. It was suggested that the basicity of C3-amino group may play an important part for the enhancement of their fluorescence. On the other hand, the fluorescence quantum yields of 2-amino-3-hydrazinoisocarbostyril derivatives (IVa-f) were negligibly small. On the basis of the consideration of the fluorescence and phosphorescence spectral data, it was assumed that the decrease in the fluorescence quantum yields of IVa-f was attributed to the intersystem crossing from 1(ππ*) to 3(ππ*).
Emission characteristics of hydrazones (Ia-g) and cyclization products (IIa-c, IIIa-c, IVa, b) derived from the reactions of 2-amino-3-hydrazino-(AH) and 2, 3-diaminoisocarbostyril (DA) with carbonyl compounds were examined. The significant enhancement in the phosphorescence was caused by the formation of hydrazones of AH. The large Stokes shifts for Ia-g observed in various solvents were explained in terms of the exciplex formation of charge transfer and hydrogen bonding types between a solute and a solvent molecule in an singlet excited state. The intense fluorescence for IIa-c and IIIa-c was shown in ethanol but no emission for IVa, b. It was suggested by the comparison of the molecular structures between IIa-c-IIIa-c and IVa, b that the bond chacacter of N1-atom plays an important role for the emission of cyclization products.
Effects of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) on pentobarbital (PB)-induced sleeping time and on hepatic microsomal drug metabolizing enzyme systems were studied in male and female mice. 1) In all i.p. doses used (10, 50 and 100 mg/kg), Δ9-THC and CBD significantly prolonged PB-induced sleeping time in both sexes of mice. CBD was more active than Δ9-THC in this route of administration. 2) Δ9-THC prolonged PB-induced sleeping time by i.c.v. administration (5, 10 and 20 μg/mouse) in both sexes of mice. On the other hand, CBD did not show any significant prolongation by i.c.v. administration in male mice, although it prolonged the sleeping time in female mice by the same treatment. The result indicates that female mice are more sensitive to the sleep prolonging effect of the cannabinoids. 3) CBD showed an inhibitory effect on p-nitroanisole O-demethylase and aniline hydroxylase in vivo and in vitro. Δ9-THC also showed the inhibitory effect, but to lesser extents. 4) Kinetic analysis showed that CBD has lower K1 values for p-nitroanisole O-demethylase and aniline hydroxylase than Δ9-THC in both sexes of mice. The inhibitory effect of the cannabinoids was more remarkable in male than in female. 5) The in vivo inhibitory effect of the cannabinoids was paralleled to the reduction of cytochrome P-450 content in hepatic microsomes. It is concluded that CBD has greater inhibitory effect on hepatic microsomal drug metabolizing enzymes of male and female mice than Δ9-THC.
Some plant lectins have been reported to agglutinate tumor cells. In addition to this function, we report here that some plant lectins can lyse tumor cells in cooperation with polymorphonuclear leukocytes (PMNs). PMNs lysed MM46, MM48, MH134 and EL-4 tumor cells in the presence of wheat germ agglutinin. MM48, MH134, EL-4 and YAC-1 were lysed by PMNs in cooperation with concanavalin A. Neither lectin lysed normal spleen cells. Peanut lectin induced cytolysis of both YAC-1 and normal cells in coopertion with PMNs. In these lectin-dependent PMN-mediated cytolysis, carbohydrate moiety on cell membranes was recognized by lectin. These results show that PMN can lyse tumor cells in the presence of appropriate mediators and that some lectins can participate in tumor lysis.
Methylation of hinokitiol and 7-iodohinokitiol yielded two isomeric methyl ethers by the ratio of about 1 : 1, respectively. The proton and 18C nucleo magnetic resonance spectra of methyl ethers of 7-iodohinokitiol were discussed to elucidate the structures. Isomers of hydrazino-isopropyl-tropone and hydrazino-iodoisopropyl-tropone were obtained by the reaction of the corresponding methyl ethers with hydrazine hydrate. Series of Schiff bases were synthesized by condensation of aromatic aldehydes with these hydrazinotropones and 5-aminohinokitiol, respectively.
The effect of free fatty acids in plasma of rabbits on plasma elimination and renal clearance of chlorpropamide (CPA) was studied. Drip infusion of 0.7 w/v% fat emulsion which elevated the free fatty acids concentration in plasma was made. When 50 mg/kg CPA was intravenously injected, the plasma elimination rate constant of CPA decreased. For the purpose of elucidating the cause, fat emulsion was intravenously injected and renal tubular secretion inhibitory test was conducted in accordance with the standard renal clearance method. The ratios of renal tubular secretion volume (S), renal tubular reabsorption volume (A), and urinary excretion volume (UV) of CPA to CPA glomerular filtration volume (GFR·Pf) were obtained, respectively. It was found that the ratio of renal tubular secretion volume to glomerular filtration volume (S/GFR·Pf) significantly decreased. These findings suggest that renal tubular secretion of CPA is inhibited by increasing free fatty acids concentration in plasma.
A simple, sensitive and accurate method for the determination of tiquizium ion, a quaternary ammonium ion, in the human serum and urine is described. The method is based on the extraction of biological fluids with chloroform under alkaline conditions and on reversed-phase high-performance liquid chromatography. Quantitation is possible down to 0.2 ng/ml of tiquizium ion using 2 ml of serum and down to 2 ng/ml using 2 ml of urine. Serum levels and urinary excretion data are obtained with this method for twelve healthy volunteers who had received a 5 mg, 10 mg and 20 mg of oral dose of tiquizium bromide.
A high-performance liquid chromatographic method for the determination of adenosine-5'-triphosphate (ATP) in the biological materials, blood, heart, liver and kidney is described. The organs were frozen in liquid nitrogen immediately after the removal in situ with precooled tongs and homogenized with cold 10% trichloroacetic acid and 0.1 N HCl. The chromatographic analysis utilized an anion exchange (Polygosil N (CH3)2) column with a mobile phase of 0.35 M KH2PO4 buffer (pH3.35) and ultraviolet monitoring at 260 nm. This method was applied to the determination of ATP in the blood, heart, liver and kidney. The minimum detectable quantity of ATP was 10 pmol (about 55 ng) and the coefficient of variation was about 1%.