The chemistry of compounds containing a carbon atom bearing three or four different labile functional groups has received little attention. These compounds should have considerable significance in theoretical and synthetic organic chemistry. Among the compounds with multifunctional structures, those having both carbonyl and halogen groups in addition to other heteroatom groups seem especially valuable from a synthetic viewpoint. Their potential as probes in pure and applied synthetic chemistry has not been exploited, because of structural instability and a paucity of synthetic approaches. Keeping with the background mentioned above in mind, we focused on the synthesis of a new class of multifunctional carbon compounds (structure A in Fig.1), in which ester carbonyl, halogen (Br or F), and other non-carbon (N, O, and/or S) functional groups are directly attached to the central carbon atom. Fluorine was chosen as the halogen because of the inherent stability of the C-F bond and because of the fundamental chemical and biological interest in fluorine-containing compounds. Synthesis, reactions, spectral data, optical resolution, and absolute configuration determination of various multifunctional carbon compounds are described. Some applications of fluorine-containing multifunctional compounds are also discussed.
It has been clarified that basic mechanism of deoxyribonucleic acid (DNA) replication is conserved from bacteria to higher cells. What distinguishes prokaryotic and eukaryotic modes of DNA replication most clearly is that bacterial chromosomes form a single replicon copied from a single initiation point and eukaryotic chromosomes consist of multiple replicons that initiate at multiple points. Thus, eukaryotes have to coordinate orderly replication of the genome. In order to understand this complex problem as a whole, three approaches were chosen. First approach is a genetic one. Certain number of temperature-sensitive (ts) mutants were isolated from mouse FM3A cells. One of the ts mutants, designated as tsFT20, was shown to contain heat-labile DNA polymerase α (pol. α). By the use of this mutant strain, it was proved that pol. α is essential for mammalian DNA replication. In addition, the human gene for pol. α on the X chromosome was assigned. Second approach is an enzymological one. FM3A cells were used for the identification and characterization of enzymes and proteins supposed to be involved in DNA replication. Four DNA-dependent ATPases, three pol. α stimulation factors, DNA topoisomerases I and II have been identified, as well as a stimulation factor for the assembly of nucleosome. DNA helicase activity was detected in two of the DNA-dependent ATPase (B and C1). Third approach is the reconstitution of DNA replication in cell-free system. By use of polyoma virus DNA as a template, cell-free extract from FM3A cells supported DNA replication in the presence of polyoma virus large T-antigen. This cell-free system will be useful for the analysis of the function of replication enzymes and proteins as well as the characterization of ts mutants.
Reduction of N-ethoxycarbonyl-ε-caprolactam with sodium borohydride-H+ in ethanol afforded 2-ethoxy-1-ethoxycarbonyl-hexahydro-1H-azepine in good yield, which was converted to various hexahydro-1H-azepine derivatives having O-, N-, S-, and C-functional groups at the α-position of the azepine ring.
The pro-drugs of α, 2-dimethyl-5H- benzopyrano [2, 3-b] pyridine-7-acetic acid (I) with a potent anti-inflammatory activity were synthesized in order to reduce its gastrointestinal side effects. Various esters synthesized were evaluated for their anti-inflammatory activity and ulcerogenicity. Among the compounds maintaining a potent activity of I, N, N-dimethylcarbamoyl-methyl α, 2-dimethyl-5H- benzopyrano [2, 3-b] pyridine-7-acetate (II-18) showed excellent biopharmaceutical characteristics. The ulcerogenic effect of II-18 showed excellent biopharmaceutical characteristics. The ulcerogenic effect of II-18 on the rat gastric mucosa was about 3 times less than that of I. It was suggested that II-18 may be an useful biolabile pro-drug for I among the compounds tested.
A study was carried out to examme the effect of 50% ethanolic extract from green fruit of Citrus unshiu MARKOVICH on type I, II and IV allergic reactions. Forty eight-hour homologous passive cutaneous anaphylaxis (PCA) in rats as a typical model of the type I reaction was significantly inhibited by the oral administration of the extract. Complement-dependent cytolysis of sheep red blood cell as a typical model of the type II reaction was significantly inhibited by incubation with it. Contact dermatitis in mice as a model of the type IV reaction caused by picryl chloride or sheep red blood cell was significantly inhibited by the oral application of it. The anti-allergic actions decreased with the growth of fruit of Citrus unshiu. These results suggested that the 50% ethanolic extract from green fruit of Citrus unshiu have anti-allergic actions against the type I, II and IV reactions.
The Kampo-prescription, Shi-un-kou, and its constituent crude drugs [Lithospermum erythrorhizon (1), Macrotomia euchroma (2) and Angelica acutiloba (3)] were assayed for their inhibitory effects on Epstein-Barr virus activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). The crude drugs exhibited inhibitory activity singly and in combinations. In particular, the combination of 2 and 3 yielded enhanced inhibition and lower cytotoxicity. The anti-tumor promoter activity suggested by these results was further investigated in an in vivo study, which demonstrated that Shi-un-kou markedly inhibited TPA-induced skin tumor formation in mice.
A simple and rapid technique for the fractional determination of bile acids bound with protein in the serum was developed by using high performance liquid chromatography with a dual-column switching system. The serum samples were directly injected onto a first column (hydroxyapatite), which was initially flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on a column of hydroxyapatite, but free bile acids were not retained. The bile acids were adsorbed on a second column (Serumout-25[○!R]) and eluted onto a column of immobilized 3α-hydroxysteroid dehydrogenase (3α-HSD) with an elution solvent (CH3CN-MeOH-30 mM ammonium acetate, 30 : 30 : 40, v/v/v). Reduced nicotinamideadenine dinucleotide was produced on the immobilized 3α-HSD column and then determined fluorometrically. Subsequently, the hydroxyapatite column was flushed with 20 mM phosphate buffer. Bile acids bound with albumin were eluted and condenced on Serumout-25. The phosphate buffer (400 mM) was finally used for the elution of bile acids bound with globulin from the hydroxyapatite column. Each condenced bile acid was eluted onto the immobilized 3α-HSD column as described above.
The pharmacokinetics of sodium 4-[α-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3, 5-dimethylbenzoate (I-Na) in the beagle dog was investigated after p.o. and i.v. administration. After the administration of I-Na at 3 mg/kg (p.o.), 70.9% of the dose was absorbed, and the maximum plasma concentration of free acid (I) was observed at 0.33 h. After p.o. administration, the area under the plasma concentration-time curve of I increased almost linearly in proportion to the dose. The metabolite, 4-[α-hydroxy-2-hydroxymethyl-5-(1-imidazolyl) benzyl]-3, 5-dimethylbenzoic acid (II) was also detected in the plasma, but the concentration of II was lower than that of I. After the administration at 3 mg/kg (p.o.), 27.7% and 3.8% of the dose were recovered as I and II, respectively, in the urine, and 32.2% and 30.1% recovered as I and II in the feces. Therefore, 93.8% of the dose was totally recovered within five days. The inhibitory effect of II on the aggregation of rabbit platelets was studied in vitro. This metabolite showed only one sixth activity of I-Na. Thus, the inhibitory effect on the platelet aggregation of II is considered to be almost negligible in the beagle dog administered with I-Na.
The sequence dependency of the antitumor effect of etoposide and ara-C was investigated against the L1210 ascites tumor in BDF1 mice. Etoposide (7.5 mg/kg or 15 mg/kg) and ara-C (25 mg/kg or 500 mg/kg) were administered intraperitoneally on days 1, 4 and 7 after inoculation of L1210 with or without time interval of 3 or 6 h. Antitumor effect was decided for cure rate and post inoculation survival time of the mice died of tumor. Six hours pretreatment with 15 mg/kg of etoposide followed by 500 mg/kg of ara-C yielded 100% of cure rate, but only 20% of cure rate was obtained with the reverse sequence. Simultaneous administration of etoposide and ara-C produced 70% of cure rate. At every dosage examined, pretreatment with etoposide given 6 h before ara-C was the most effective antitumor schedule in L1210 leukemia.
The inhibitory mechanism of 6 traditional Chinese medicines on rabbit platelet aggregation in vitro, and the suppressive effect of oral administration of Toki-syakuyakusan on hyper-aggregability of the platelet from rabbit fed high cholesterol diet for 2 months, were investigated. Collagen-induced aggregation was inhibited by Keisi-bukuryogan, Kami-syoyo-san, Dai-saiko-to, Toki-syakuyaku-san, Hatimi-zio-gan and Syo-saiko-to in their lower concentrations than those inhibiting arachidonic acid- and thrombin-induced aggregation. These traditional Chinese medicines inhibited the release of [3H] arachidonic acid from membrane phospholipids by phospholipase A2, in [3H] arachidonic acid-labelled platelets under stimulation with collagen and thrombin in the concentration ranges that inhibited each aggregation. In their higher concentrations to inhibit arachidonic acid-induced aggregation, they suppressed the conversion of arachidonic acid to thromboxane A2 by about 50%. However, they had no effect on diacylglycerol formation induced by thrombin. The oral administration of Toki-syakuyaku-san depressed the increased aggregability of platelets from rabbit fed high cholesterol diet by 20-40% at the period of 1-2 months of feeding, without affecting plasma and platelet cholesterol level. These results indicate that the traditional Chinese medicines used here have an inhibitory effect on platelet phospholipase A2 activation, rather than on cyclooxygenase, and therefore inhibit platelet activation in vitro and ex vivo.
Some chemical and serological properties of water-soluble and undialysable fraction (WSF) and water-insoluble and undialysable fraction (WISF) of human seminal plasmas were studied. Both of the fractions contained proteins as the main components and also carbohydrates as minor components, and each fraction gave 9 bands which were stained with the Periodate-Schiff and/or Coomassie brilliant blue reagents on sodium dodecyl sulfate-polyacrylamide slub gel electrophoresis. WSF reacted with 17 of 19 hemagglutinins, whereas WISF reacted with 12 of them. Although WSF and WISF did not react with anti-M and -N sera respectively, both of the fractions reacted with the anti-N lectins of Vicia graminea and V. unijuga and also with Arachis hypogaea anti-T (Thomsen-Friedenreich) lectin. Perchloric acid-soluble fractions separated from a large number of human intact organ tissues and serum did not react with the above-mentioned 5 hemagglutinins. In double immuno-diffusion, both of the fractions interacted with rabbit anti-WSF serum to form 4 precipitation lines.
A simple and high sensitive method for the determination of disaccharidase in various biological samples of rats by high-performance liquid chromatography (HPLC) with a differential refractometric detection was developed. This method, as compared with the Dahlqvist method, has the following advantages ; 1) There is no necessity for the elimination of biological components which have an influence on the determination of glucose. 2) Only a little enzyme sample is used. 3) The detection limit of a reaction product, glucose, by the HPLC method is about 10 times higher than that by the Dahlqvist method. 4) The maltase activity in the small intestinal mucosa estimated by HPLC method is positively correlated with that estimated by the Dahlqvist method (r=0.9661, p<0.001, n=8). 5) This method developed in the present study is applicable to not only the small intestinal mucosa, but also serum, bile, urine and various tissues.