Studies on the synthesis of antiulcer agents. VI. Synthesis and antiulcer activity of dihydrobenzofuranone derivatives

The derivatives (2) of 3-(2,3-dihydro-2,2-dimethyl-3-oxo-5-benzofuranyl) acrylic acid (2b) were synthesized. The compounds (3a-g) in which bromo, methoxy, nitro, amino or acetamido group was introduced on the benzene ring of the derivatives (2) and the compounds (3h-k) in which acryloyl moiety was introduced on the 6- or 7-position of the benzofuranone skeleton also synthesized. Furthermore, propionic acid derivatives (4a-c), acetic acid derivatives (4d-g), formic acid derivatives (4h-k) and oxyacetic acid derivatives (5) were prepared by converting the acryloyl moiety of the derivatives (2) into propionyl, acetyl, formyl and oxyacetyl groups. These compounds were tested for antiulcer activities. Among these compounds, 1-[3-(2,3-dihydro-2,2-dimethyl-3-oxo-5-benzofuranyl)acryloyl]piperidine (2d) and 4-[3-(2,3-dihydro-2,2-dimethyl-3-oxo-5-benzofuranyl)acryloyl] morpholine (2g) were found to have stronger antiulcer activities.     

Antihypertensive effect of synthetic atrial natriuretic factor in vasopressin-infused rats

To assess the pathophysiological role of atrial natriuretic factors in the regulation of blood pressure, we studied the effect of chronic infusion of a synthetic atrial natriuretic factor of 25 amino-acid residues on blood pressure and sodium-water excretion. Experimental subjects were rats with hypertension made by chronic infusion of vasopressin on regular intakes of sodium or on sodium loading with 1% NaCl as drinking water. When a subdepressor dose (150 micrograms/kg/day) of synthetic atrial natriuretic factor was delivered via an osmotic minipump into the jugular vein simultaneously with 7.2 U/kg/day of vasopressin infused intraperitoneally by another osmotic minipump, the expected elevation of systolic blood pressure was completely inhibited. This was not accompanied by any changes in urine volume and urinary sodium excretion. The antihypertensive effect was sustained throughout the experimental period lasting 3 days in rats on regular sodium intake (p less than 0.01) or on sodium loading with 1% NaCl as drinking water (p less than 0.01). These results indicate that a subdepressor dose of synthetic atrial natriuretic factor can modulate the vasopressor effect of vasopressin. Therefore it is suggested that an atrial natriuretic factor may be involved in the regulation of blood pressure via its antagonizing effect to vasopressin.     

Cellular sensitization to cis-diamminedichloroplatinum(II) by novel analogues of the protein kinase C activator lyngbyatoxin A

Protein kinase C (PKC) has been implicated in enhancing cellular sensitivity to cis-diamminedichloroplatinum(II) (CP). We have synthesized a series of novel analogues of lyngbyatoxin A (7-linalylindolactam V), a natural tumor promoter and a potent activator of PKC, and investigated the effects of these synthetic compounds on PKC activity and the antiproliferative activity of CP. Lyngbyatoxin A was as effective as phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate, in enhancing the sensitivity of HeLa cells to CP. A 24-h pretreatment of HeLa cells with 1 to 100 nM lyngbyatoxin A caused an approximately 9-fold sensitization to CP. All analogues of lyngbyatoxin A that retained the lactam ring portion of the molecule but contained different hydrophobic substituents at C-7 including indolactam V (ILV), tert-butyl-ILV, or n-hexyl-ILV increased cellular sensitivity to CP in a concentration-dependent manner. Maximum cellular sensitization to CP (9-fold) was seen with 10 nM n-hexyl or tert-butyl compounds, and ILV devoid of any C-7 substitution required higher concentrations (1 microM) for equivalent sensitization. The ability of lyngbyatoxin A analogues to sensitize cells to CP correlated directly with their ability to activate PKC in vitro. Synthetic analogues that lacked the lactam ring structure neither activated PKC nor sensitized cells to CP. The C-9 epi analogue of n-hexyl-ILV was less effective than the corresponding natural stereoisomer in activating PKC as well as sensitizing cells to CP. Exposure of HeLa cells to 100 nM lyngbyatoxin A for 24 h caused a substantial decrease in cellular PKC activity to 20% of the untreated control value, but a similar treatment of cells with n-hexyl- or tert-butyl-ILV led to only a 25% reduction in PKC activity. Concentrations of ILV (e.g. 1 microM) that sensitized HeLa cells to CP caused no down-regulation of PKC. Thus, on the basis of results with these novel lyngbyatoxin A analogues, we conclude that activation but not down-regulation of PKC is necessary for sensitization of HeLa cells to CP.     

Simplified determination of serum cholesterol sulfate by gas-liquid chromatography combined with cyclohexylsilane-bonded phase column purification

Summary A new gas-liquid chromatographic (GLC) determination of cholesterol sulfate (CS) and dehydroepiandrosterone sulfate (DHEAS) for a biochemical diagnosis of recessive X-linked ichthyosis (RXLI) is described. Although the GLC method for determination of CS is known to be more sensitive than the thin layer chromatographic (TLC) method, the former method has not been widely employed because of its complicated pre-purification steps. The present method allows us to measure the serum levels of CS and DHEAS without tedious purification steps such as multiple conventional column chromatography and preparative thin layer chromatography. Sulfated steroids are rapidly purified with a commercially available mini disposable cyclohexylsilane-bonded phase (CH) column, CH BOND ELUT, and the purified steroids after desulfation are converted to water-resistant tert-butyldimethylsilyl ether derivatives for the GLC analysis on dual 2 m glass columns packed with 2% XE-60 on Chromosorb W.By the present method, serum CS concentrations in RXLI patients were shown to be about 10 times higher than those in patients with ichthyosis vulgaris, carriers of RXLI, and healthy subjects. This method is more suitable not only for a biochemical diagnosis of RXLI but also for studies on the metabolism of sulfated steroids than the previous time-consuming GLC methods.     

Ketamine Suppresses Platelet Aggregation Possibly by Suppressed Inositol Triphosphate Formation and Subsequent Suppression of Cytosolic Calcium Increase

Background: Ketamine has been shown to suppress platelet aggregation, but its mechanisms of action have not been defined. The purpose of the current study is to clarify the effects of ketamine on human platelet aggregation and to elucidate the underlying mechanisms of its action.

Methods: Platelet aggregation was measured using an eight-channel aggregometer, and cytosolic free calcium concentration was measured in Fura-2/AM-loaded platelets using a fluorometer. Inositol 1,4,5-triphosphate (IP3) was measured with use of a commercially available IP3 assay kit. To estimate thromboxane A2 (TXA2) receptor binding affinity and expression, Scatchard analysis was performed using [3H]S145, a specific TXA2 receptor antagonist. TXA2 agonist binding assay was also performed. The membrane-bound guanosine 5'-triphosphatase activity was determined using [[gamma]-32P]guanosine triphosphate by liquid scintillation analyzer.

Results: Ketamine (500 [mu]m) suppressed aggregation induced by adenosine diphosphate (0.5 [mu]m), epinephrine (1 [mu]m), (+)-9,11-epithia-11,12-methano-TXA2 (STA2) (0.5 [mu]m), and thrombin (0.02 U/ml) to 39.1 +/- 30.9, 46.3 +/- 4.3, -2.0 +/- 16.8, and 86.6 +/- 1.4% of zero-control, respectively. Ketamine (250 [mu]m-1 mm) also suppressed thrombin- and STA2-induced cytosolic free calcium concentration increase dose dependently. Although ketamine (2 mm) had no effect on TXA2 receptor expression and its binding affinity, it (1 mm) suppressed intracellular peak IP3 concentrations induced by thrombin and STA2 from 6.60 +/- 1.82 and 4.39 +/- 2.41 to 2.41 +/- 0.98 and 1.90 +/- 0.86 pmol/109 platelets, respectively, and it suppressed guanosine triphosphate hydrolysis induced by thrombin (0.02 units/ml) and STA2 (0.5 [mu]m) to 50.3 +/- 3.2 and 67.5 +/- 5.5%versus zero-control, respectively.     

Different postnatal ontogenic profiles of neurons containing beta (beta 1, beta 2 and beta 3) subunit mRNAs of GABAA receptor in the rat thalamus.

The postnatal ontogeny of neurons containing different GABAA receptor beta (beta 1, beta 2 and beta 3) subunit mRNAs were examined in the rat thalamus using in situ hybridization histochemistry. Neurons containing beta 1 or beta 2 subunit mRNA developed remarkably postnatally, while most neurons were already strongly labeled with beta 3 probe at birth. However, beta 3 subunit mRNA decreased rapidly after birth, few cells being labeled with this probe at day 35 and thereafter.