From a synthetic point of view, total synthesis of (±)-lythranidine, an alkaloid isolated from Lythrum anceps MAKINO, involves the following four interesting problems ; the construction of the 17-membered ring, the formation of trans-2, 6-disubstituted piperidine ring, the settlement of the correct relative stereochemistry of the four asymmetric centers in the mobile macrocyclic ring system, and the construction of unsymmetrically substituted biphenyl moiety. The details how to solve them are discussed. The last problem was rather easily solved by partial demethylation of a 2, 2'-dimethoxybiphenyl derivative with a reagent system including a hard acid and a soft nucleophile. This reagent system has been shown to be effective for the cleavage of a variety of carbonoxygen bonds and some carbon-carbon double bonds. These bond cleavage reactions by a hard acid and soft nucleophile system are shortly reviewed in the latter part of this article.
2-Thiobarbituric acid (TBA) values obtained by 1% H3PO4 method which is applicable to whole tissue homogenate (Anal. Biochem. 86, 271 (1978)) were further evaluated by using highly peroxidized CCl4-intoxicated rat liver homogenate along with the control one. Both homogenates were fractionated by three manners : Trichloroacetic acid treatment to yield supernatant and acid-insoluble precipitate ; chloroform-methanol extraction to obtain lipid, water soluble and residual fractions ; and 105000×g ultracentrifugation to make supernatant and pellet fractions. Of all fractions were measured conjugated diene and peroxide values besides TBA values and a good similarity of indices was obtained. However, the sum of the TBA value of each fraction was far smaller than that of original whole homogenate in CCl4-intoxicated rat, while the sum was rather bigger than original value in the case of normal rat, irrespective to the method of fractionation. Since the color development of TBA reaction is strongly affected by various cell components, it is likely that 1% H3PO4 method applied to whole homogenate does not give only the existing amount of TBA-reactive substances, but can represent the combined status of tissue peroxidation including several antioxidative and prooxidative factors located in other compartments from extracted lipid.
A dye (Neutral Red) uptake method by using cultured human cancer cells and an application of this method to a sensitivity test for anti-cancer drug are described. The amount of Neutral Red (NR) taken by the cells was proportional to the concentration of NR added. The little additional dye was taken up by the cells after incubation for more than 60 min. In addition, the uptake of dye was linear to the number of viable cells by Trypan Blue exclusion. However, in vitro monolayer aging had an effect on the correlation between the uptake of dye and the number of viable cells. EC50s of mitomycin C, bleomycin, adriamycin, neocarzinostatin by this assay method were 0.11 μg/ml, 6.0 μg/ml, 0.054 μg/ml and 1.9 units/ml, respectively. This assay method is suited for a sensitivity test for anti-cancer drug because of its technical simplicity, convenience, allowing efficient handling of large number of specimens, precision and reproducibility.
After the oral administration of N-(3-chloro-2-methylphenyl) anthranilic acid (GEA-6414), the urinary metabolites were examined in human, dogs, rabbits and rats. Two kinds of metabolites, M-I and M-II, besides GEA 6414 were detected from the human urine by TLC and GLC. M-I and M-II were identified as N-(3-chloro-2-hydroxymethylphenyl) anthranilic acid and N-(3-chloro-4-hydroxy-2-methylphenyl) anthranilic acid, respectively. The glucuronides of M-I and M-II were estimated as major metabolites of GEA6414 in the human urine. M-I was also excreted along with its glucuronide in the urine from rabbits and rats. M-II was detected only in the urine from human and rats, but its glucuronide was excreted in the urine from human, rabbits and rats. M-II sulfate was detected only in the rat urine as a major metabolite. In the rabbit urine 64% of dosed GEA 6414 were excreted as its glycine conjugate. A little amount of GEA 6414 and its glucuronide were detected in the dog urine.
Investigation was made on the rate and degree of mixing of a micronized lactose powder (containing eosine as tracer ; weight mean diameter, 17 μm) with small-sized lactose granules (177-500 μm) by using a vertical shaking vessel at a frequency of 150 cycle/min and the amplitude of 30 cm. The mixture was compressed in its intact state and the compressed sample was divided into 16 segments to measure individual powder content of these segments. The coefficient of variation with respect to the powder content was then calculated. Analysis of the particle motion has revealed that the granules were separated easily from the bottom of the vessel during mixing to cause a dispersion of the cohesive powder capable of floating only as agglomerates. At the initial stage of mixing, the agglomerates thus formed were random-mixed with the granules ; however the granule-intake by the agglomerate was observed to form agglomerate complexes. Through the special agglomeration process as such, the mixture was found to become homogeneous and the coefficient of variation gave the minimum of 4% (with the sample obtained by mixing for 1-2 min). Such a homogeneous mixture was formed at a powder content of 10%, but not at 5 and 25%. Prolongation of the shaking time over five minutes caused fine particles to be shaken off to sediment onto the bottom. The homogeneous mixture, formed intermediately as has been stated above, was obteined specifically in the system composed of a fine powder and small-sized granules whose cohesive and adhesive forces (0.022 and 0.026 dynes, respectively) were in balance ; the mixture is therefore different from a simple rendomized-mixture or from an ordered one.
The synthetic prostaglandin, 7-oxo-prostaglandin E1 analogs were tested for prevention of ulcer formation induced by indomethacin in rat, and its structure-activity relationship was studied. 7-Oxo-15-methyl prostaglandin E1 methyl ester (TEI-1226) was the most potent compound of the analogs when given orally. The prevention of indomethacin induced gastrointestinal lesion by TEI-1226 was about 2 times more potent than that by PG E2, while the potency of diarrhea induction by TEI-1226 was almost equivalent to that of PG E2. Prostaglandin-like activity of TEI-1226 on smooth muscles (rabbit aorta, guinea-pig trachea, rat stomach, and rat colon) was about 100 times less active than that of PG F2a or PG E2. In conclusion, synthetic 7-oxo-PG E1 analogs appeared to be useful in the treatment for the gastric side effect caused by nonsteroidal anti-inflammatory drugs.
Cathartic effects of Aloe pulv. (J. P. IX) and pulv. of Aloe arborescens MILL. var. natalensis BERGER (Kidachialoe) were examined in mice and rats by oral administration. It was found that rats were more suitable than mice. Additionally, no sex difference in rats was observed. Cathartic activity (ED50) in male rats was 84.3 mg/kg in Aloe pulv., and 900 mg/kg in Kidachialoe pulv. Several experiments to find the mechanism of cathartic effect of Aloe were done. It was considered that Aloe acted on the large intestine mainly, and that process of activation of Aloe by intestinal flora was necessary to act. It was considered that main cathartic component of Aloe was barbaloin by comparision of barbaloin contents in Aloe and cathartic activity. Then, it was concluded that barbaloin represented cathartic activity of Aloe.
As a part of studies on antispasmodics, the structure-activity relationship was examined in N-alkyl diarylmethylenequinolizidinium bromides and N-alkyl diarylmethyleneindolizidinium bromides (6-33), which can be classified in to γ-substituted piperidine, β-substituted piperidine and β-substituted pyrrolidine. Compounds (6-33) exhibited potent anticholinergic activity due to having conformationally rigid bicyclic hetero rings compared to monocyclic antispasmodics such as diphemanil methylsulfate (1), timepidium bromide (2), prifinium bromide (4) and their unmodified compounds (3 and 5). Several compounds among them exhibited potent anticholinergic activity greater than that of atropine.
The metabolites of 1-(3-chlorophenyl)-3-(N, N-dimethylcarbamoyl)-5-methoxypyrazole (I) were investigated in the rabbit urine after oral administration of I. The five metabolites (M-1-M-5) were isolated as crystals from the rabbit urine to which I was administered. M-1, M-2 and M-5 were identified as 1-(3-chlorophenyl)-5-methoxy-3-(N-methylcarbamoyl) pyrazole (II), 3-carbamoyl-1-(3-chlorophenyl)-5-methoxypyrazole (III) and 3-carboxy-1-(3-chlorophenyl)-5-methoxypyrazole (IV), respectively, by thin layer chromatography and infrared spectroscopy. M-3 and M-4 were identified as 3-carbamoyl-1-(3-chloro-4-hydroxyphenyl)-5-methoxypyrazole (VI) and 1-(3-chloro-4-hydroxyphenyl)-5-methoxy-3-(N-methylcarbamoyl)-pyrazole (V), respectively, by physicochemical properties, mechanical analysis and chemical syntheses.
The allylation of 5-position in ethyl 5-hydroxy-1-phenylpyrazole-3-carboxylate (I) was attempted and ethyl 5-allyloxy-1-phenylpyrazole-3-carboxylate (II) and ethyl 4, 4-diallyl-5-oxo-1-phenyl-2-pyrazoline-3-carboxylate (III) were obtained. The chemical structures of II and III were determined by infrared and nuclear magnetic resonance spectroscopies. To clarify the mechanism to form III, I was allylated under appropriate conditions and it was found that at room temperature II was obtained as a sole product and that heating was necessary for the formation of III. 4-Allyl-5-hydroxy-1-phenylpyrazole-3-carboxylate (IV) was obtained by heating II in benzene. The reaction of II or IV with allyl bromide gave III. These results suggest that Claisen rearrangement occurred in the above reaction.
Two steroidal glycosides, SL-1 (1) and SL-0 (2), and an additional steroid-alkaloidal glycoside, SL-2, were isolated from the stem parts of Solanum lyratum THUNB. (Solanaceae). Among them, two steroidal glycosides, 1 (mp 245-249°, [α] D-45.1°) and 2 (mp 214-218°, [α] D-26.5°, Ehrlich reaction +, a bitter principle), were characterized as 3-O-(β-D-glc·pyr-^^(1→2)β-D-glc·pyr-^^(1→4)β-D-gal·pyr)-spirostanol derivative (a mixture of tigogenin, neotigogenin, diosgenin and yamogenin) (1) and the corresponding furostanol 26-O-β-D-glc·pyranoside (2).
Recently several papers have shown that proteins can be separated by using gradient elution method in reversed phase partition high performance liquid chromatography (HPLC) system, but there has been no report on the use of isocratic elution method. The HPLC method, which has been developed in previous report, was examined whether it can be applied to achieve efficient separation and identification of proteins. Under isocratic elution system by use of Nucleosil CN as stationary phase and sodium octanesulfonate as ion-pair reagent in mobile phase, we studied simultaneous spearation of five kinds of proteins ; insulin, ribonuclease, myoglobin, lysozyme chloride and cytochrome C. Under optimal conditions all of the proteins tested could be satisfactorily chromatographed.
Three possible sulfates of pregnanediol (1) were prepared by the following procedure. Partial acetylation of 1 with acetic anhydride in pyridine gave chromatographic separable monoacetates, 3α-acetoxy-5β-pregnan-20α-ol (3) and 20α-acetoxy-5β-pregnan-3α-ol (4), in appropriate amount. Sulfation with pyridine-SO3 complex of 3 and 4, followed by saponification gave desirable pregnanediol monosulfates, 20α-sulfooxy-5β-pregnan-3α-ol (6) and 3α-sulfooxy-5β-pregnan-20α-ol (8), respectively. Pregnanediol 3, 20-disulfate (9) was obtained from 1 with excess reagent.
2, 3-Dihydroxyestra-1, 3, 5 (10)-triene-6, 17-dione (5) and its methylated derivatives (4, 6, and 7) have been prepared for biological investigation. The preparation method and the results of instrumental analyses including nuclear magnetic resonance and ultraviolet spectra of these materials are described. The additivity rule of the λmax observed in benzocyclanone derivatives was also applicable to the present estra-1, 3, 5 (10)-triene-6, 17-dione skeleton.
From the rat liver mitochondria, protoheme ferro-lyase [E. C. 4.99.11], an enzyme localized on its inner membrane, was purified about 44-fold over the specific activity of the homogenate. The purification was carried out by (NH4)2SO4 fractionation, gel filtration on Sepharose-4B, column chromatographies on DEAE-cellulose and hydroxylapatite in the presence of detergents. From the results of DISC and SDS-polyacrylamide gel electrophoresis, this enzyme was estimated to be an oligomer enzyme with an approximate molecular weight of 380000 consisting of NA-H-1, an extrinsic protein on the inner membrane, and D-1 and D-2, intrinsic proteins linked by a hydrophobic bond to the inner membrane, and the approximate molecular weights of these subunit proteins were 120000, dimer of 74000 and 110000, respectively.