A new concept of cyclic conjugation as a modification of the Huckel rule has proven to be useful as a general method of predicting thermal stabilities and electonic peoperties of unsaturated cyclic organic molecules. We have been applying this concept to the design of reagents such as p-benzoquinone and 1, 4-dithins. Pericyclic syntheses are very valuable method for the high stereo-, regio-, and periselective controls, and they could provide a logical assembling of molecules, especially highly strained cage compounds which are available or not available for natural sources. These pericyclic reactions are discussed in terms of frontier molecular orbital theory.
Esters of p-amidinophenol have been demonstrated to undergo efficient and specific tryptic hydrolysis. These esters are characterized by their linkage, i.e., the site specific groups for the enzyme (charged amidinium) is not involved in their carbonyl groups but in the leaving portion. Thus, these esters were named "inverse substrates" with respect to their structure and kinetic properties. It was also found that these esters were hydrolyzed by various proteases which show trypsin-like specificity. A facile procedure for the preparation of acyl-enzymes carrying non specific residue was described and the potential usefulness of the "inverse substrates" concept was proposed.
In order to investigate the constituents of Osmanthus species, the components of the leaves of Osmanthus ilicifolius (HASSK.) MOUILLEFERT were examined. A new secoiridoid glycoside (named hiiragilide) and a new caffeoyl glycoside (named cis-acteoside) were isolated, and their structures were elucidated on the basis of chemical and physicochemical evidence. Ligustroside, 10-hydroxyligustroside, 10-acetoxyligustroside, oleuropein, 10-hydroxyoleuropein, 10-acetoxyoleuropein, acteoside, 3-O-p-coumaroylquinic acid, lignans, flavonoids, triterpenes, sterols and others were also identified from the methanolic extract.
As a study to develop new drugs from natural source, Ca2+-blocking action which is expected to be effective for hypertension was examined for over 1500 plants including Japanese and Chinese herbal medicines, and the results are described in this report. Among these medicines, Cnidii monnieri, a Chinese harbal medicine, was found to be most effective in the initial screening test. Its active principal was found to be osthole, a coumarin derivative.
A high performance liquid chromatographic procedure for the assay of carteolol in the human plasma and urine was investigated in the present study to simplify the procedure, to increase the assay sensitivity, and to established a more appropriate assay procedure for routine analysis than the fluorescent method. The chromatogram obtained under the following elution conditions : internal standard, 1-methyl-carteolol ; column, μ-Bondapak C18 ; and mobile phase, a mixture of 30% acetonitrile, 0.02 M NH4H2PO4 and 0.02 M (NH4)2HPO4 ; in the human plasma and urine were satisfactory without interfering peak. The coefficients of correlation for the calibration curves in the plasma and urine were 0.9988 and 0.9998, respectively. The plasma and urinary concentrations of unchanged compound for the first 24 h after a single oral administration at 15 mg/body in healthy volunteers by this assay procedure were satisfactory. The correlation between the plasma concentration and urinary excretion rate of carteolol was good. Therefore, it was considered to be possible to evaluate the bioavailability of carteolol by using the urinary excretion rate.
Phosphatase activities, determined mainly by the hydrolysis of p-nitrophenyl phosphate (p-NPP), were found in porcine brain microtubule proteins prepared by successive cycles of assembly and disassembly. The phosphatase activity showed a pH-optimum around 5 with p-NPP as a substrate in the presence of ethylenediaminetetraacetic acid or Mg2+. The addition of Zn2+ suppressed acid and neutral phosphatase activities, but enhanced the activities 5-70 folds over pH 9. The phosphatase activity contained in microtubule proteins showed low substrate specificity. Fructose-1, 6-diphosphate was hydrolyzed more rapidly than p-NPP and fructose-6-phosphate. Furthermore, the enzyme(s) catalyzed the hydrolysis of phospho-D, L-tyrosine, but rarely acted on phospho-D, L-threonine and phospho-L-serine. The addition of ammonium molybdate which considerably inhibits the hydrolysis of p-NPP, showed a slight effect on phosphatase activity toward casein or phosvitin as substrates. The addition of ascorbic acid enhanced the hydrolysis of p-NPP, whereas it rather suppressed the phosphatase activity against casein and phosvitin. Ammonium molybdate and ascorbic acid did not affect microtubule polymerization. When microtubule proteins were separated into tubulin and microtubule-associated proteins (MAPs) by phosphocellulose column chromatography, the activities at pH 5, 7, and 10 were mostly recovered in 0-0.8 M KCl eluted fractions, indicating that total phosphatases are contained in MAPs, not in tubulin. The addition of tubulin, actin, and calmodulin which are capable of binding to MAPs, had little effect on the activity.
The decomposition compound formed by xenon irradiation of nafamstat mesilate in aqueous solution and its kinetics were studied and the following findings were obtained. Nafamstat mesilate was decomposed by xenon irradiation to form 4-guanidinobenzoic acid (PGBA), 6-amino-2-naphthol (AN) and compound II having yellow color. The structure of the compound II was studied in comparison with nafamstat mesilate by chemical and physical methods and shown to be an amide type compound of PGBA and AN. Photodemposition reaction of nafamstat mesilate was found to follow the pseudo-second order reaction.
The plasma protein binding of phenytoin in man is about 90% and the impaired plasma binding may lead to large variations in the unbound fraction of phenytoin in the plasma. In the present study, the percent of unbound phenytoin was accurately determined by equilibrium dialysis by using 14C-labeled phenytoin. When intra- and intersubject differences in the plasma binding were studied in 22 patients during long-term treatment with phenytoin, a strong linear correlation was observed between the unbound fraction of phenytoin, a strong linear correlation was observed between the unbound fraction of phenytoin and albumin concentration. The obtained results of the linear correlation were demonstrated to be of clinical significance in predicting the percentage of unbound phenytoin in patients with moderate hypoalbuminemia (plasma albumin of 2.0 to 3.0 g/100ml).
Seventy seven crystalline organic drugs listed mainly in JPX were investigated by polarizing microscope by using the already reported improved immersion method. Thirty six drugs were classified as group A and 14 drugs as group B, from which two or one key refractive indices were measured, respectively. As for 26 drugs it was difficult to measure definite two refractive indices from their natural positions at the immersion method and they were classified as group C. However, two refractive indices were measured and described from their frequently observable positions. It was found that graphic representation of the correlation between refractive indices (n1, n2) and common logarithms of birefringence, log (n2-n1), was mostly convenient to use the results of polarizing microscopy for the purpose of analysis or identification of crystalline drugs not only in the cases of groups A and B, but also in the case of group C, sometimes by properly observing interference colors connected with birefringence.
Some properties of angiotensin I-converting enzyme existed in rat testis (RT-ACE) were compared with those of angiotensin I-converting enzyme in rat lungs (RP-ACE). In gel filtration through Sephadex G-200 column, distribution coefficient of RT-ACE (0.34) was higher than that of RP-ACE (0.27). Furthermore, RT-ACE migrated faster than RP-ACE on SDS-polyacrylamide gel electrophoresis. The molecular weights of RT-ACE and RP-ACE were estimated to be 104 and 150 kilodaltons, respectively, on the SDS-polyacrylamide gel electrophoresis. On the other hand, enzymo-chemical properties of both ACEs were very similar when they were assayed by using Nα-hippuryl-His-Leu-OH (HHL) as substrate. Namely, 1) HHL hydrolyzing activities of both ACEs were strongly activated by the addition of NaCl. And the effects of NaCl concentrations on the enzyme activities were similar between RT-ACE and RP-ACE. 2) The effects of various pH on the HHL hydrolyzing activities were also very similar between both ACEs under both conditions where NaCl was present or absent. 3) Km values for HHL hydrolysis of RT-ACE and RP-ACE were estimated to be the same (2.5 mM) at pH 8.3, in the presence of 0.8 M NaCl. 4) The HHL hydrolyzing activity of RT-ACE was inhibited by the addition of captopril or MK-422, specific inhibitors of angiotensin I-converting enzyme, to similar extent to RP-ACE. From these results, it was speculated that the portions of RT-ACE contributing to the appearance of enzyme activities would resemble those of RP-ACE although the molecule of RT-ACE is different from that of RP-ACE.
It has become apparent that trypsin activatable inactive elastase was present in the hog blood. 1) After anti-hog pancreatic elastase I antibody-Sepharose 4B immunoaffinity chromatography, about 9 μg of inactive elastase was detected with enzymic activity in 11 of citrated hog plasma. 2) The inactive latent enzyme was activated by the action of little amount of trypsin and then hydrolyzed both substrates of Congo red-elastin and succinyl-L-trialanine p-nitroanilide. 3) The molecular weight of the inactive enzyme which was estimated by Sephadex G-75 gel filtration was a little larger than that of hog pancreatic elastase I. 4) The activated enzyme generated from the inactive elastase by the action of trypsin had remarkable resemblance of enzymochemical properties with hog pancreatic elastase I. It can therefore be presumed that the inactive latent elastase in the hog plasma is a proelastase-like enzyme but not be any complex forms of elastase with α1-antitrypsin and/or α2-macroglobulin, and is transferred from the hog pancreas into the blood.
5β-Pregn-20-en-3α-ol, which is a key compound for the elucidation of reaction mechanism of pregnanediol 20-sulfate to D-homo-and Δ13-steroids by hot mineral acid, was derived from lithocholic acid by the degradation method of side chain. The overall yield of the desired product through these four steps from acetyllithocholic acid was 27%.
Kinetics of nafamstat mesilate in aqueous solution was studied and the following findings were obtained. In aqueous solution, nafamstat mesilate was exclusively hydrolyzed to form its moieties of 4-guanidinobenzoic acid and 6-amidino-2-naphthol. The hydrolysis followed a pseudo-first order reaction and was catalyzed by each hydrogen ion, hydroxyl ion and water depending on the pH conditions in the pH range of 1-7, the following the kinetic law of k0=kHαH+kH2O+kOHαOH. In the pH range of 3-9, the buffer concentration effect was observed. The ionic strength effect at pH 1 and 9 among pH 1, 3 and 9 studies was also observed. Nafamstat mesilate was most stable under acidic conditions, i.e. at pH 3.29 and 3.21 at 50°C and 60°C, respectively, as shown by pH-rate profile.