A novel type of retinoic acid receptor antagonist: synthesis and structure-activity relationships of heterocyclic ring-containing benzoic acid derivatives.

A new series of heterocyclic ring-containing benzoic acids was prepared, and the binding affinity and antagonism of its members against all-trans-retinoic acid were evaluated by in vitro assay systems using human promyelocytic leukemia (HL-60) cells. Structure-activity relationships indicated that both an N-substituted pyrrole or pyrazole (1-position) and a hydrophobic region, with these linked by a ring system, were indispensable for effective antagonism. Among the compounds evaluated, optimal antagonism was exhibited by 4-[4,5,7,8,9,10-hexahydro-7,7,10,-10-tetramethyl-1-(3- pyridylmethyl)anthra[1,2-b]pyrrol-3-yl]benzoic acid (31), 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)-5- thiaanthral[1,2-b]pyrrol-3-yl]benzoic acid (40), and 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3- pyridylmethyl)anthra[2,1-d]pyrazol-3-yl]benzoic acid (55), all of which possess a 3-pyridylmethyl group at the five-membered ring nitrogen atom.

Protective effects of E3330, a novel quinone derivative, on galactosamine/tumor necrosis factor-alpha-induced hepatitis in mice.

Oral pretreatment with E3330, a novel quinone derivative, attenuated liver injury induced with tumor necrosis factor-alpha in galactosamine-sensitized mice. Tumor necrosis factor-alpha is known to induce inflammatory mediators such as leukotrienes and prostanoids. An in vitro study showed that E3330 inhibited the generation of leukotriene B4 and thromboxane B2, but enhanced prostaglandin E2 generation from rat peritoneal exudate cells stimulated with the Ca(2+)-ionophore, A23187. These findings suggest that the protective effect of E3330 on galactosamine/tumor necrosis factor-alpha hepatitis is due at least in part to its inhibition of the generation of leukotrienes. The inhibition of thromboxane B2 generation or the enhancement of prostaglandin E2 generation by E3330 may also contribute to its hepatoprotective effect.

Alpha-tocopherol and inhibition of cytolysis in glutathione-depleted hepatocytes in primary culture.

Treatment of cultured rat hepatocytes with 10 microM 1-chloro-2,4-dinitrobenzene (CDNB) resulted in an acute loss of cellular glutathione (GSH) within 30 min and a marked increase in spontaneous lactic dehydrogenase (LDH) leakage to the culture medium after 24 h, with obvious cellular degeneration as viewed by phase-contrast microscopy. Simultaneous treatment of the cells with alpha-tocopherol markedly protected the cells not only against LDH leakage but cellular degeneration in a dose-dependent manner. The EC50 of alpha-tocopherol was 0.1 microM, ca. 200 times less than normal plasma levels in the rat. In response to the inhibitory effects of alpha-tocopherol on the cytolysis as measured by LDH leakage, GSH biosynthesis was stimulated by CDNB, and cellular GSH levels returned to control levels. The recovery was inhibited by 0.2 mM buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. However, the stimulation of GSH biosynthesis apparently was not essential for the protection from cytolysis by GSH depletion during the experimental period, because treatment with 0.2 mM BSO and 20 microM tocopherol completely protected the cells against the lysis induced by BSO up to 32 h without cellular GSH recovery. The results suggest that alpha-tocopherol may be a primary natural inhibitor of the cytolysis induced by xenobiotics which consume the cellular GSH in vivo.

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro.

Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na+-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.

Mechanism of growth promotion of mouse lymphoma L1210 cells in vitro by feeder layer or 2-mercaptoethanol.

Mouse lymphoma L1210 cells require some thiol compounds (such as 2-mercaptoethanol) or feeder layer cells for their growth in normal culture media in vitro. We found that feeder layer cells (human diploid fibroblast IMR-90) constantly produce thiol compounds; cysteine was the major thiol compound accumulating in the culture medium. In the culture medium of L1210 cells, added cysteine was rapidly oxidized and was toxic to the cells at high concentrations. However, cysteine promoted growth of L1210 cells when it was added repeatedly to the medium at low concentrations. These results show that the major role of feeder layer cells is to provide cysteine continuously. The glutathione content of L1210 cells depended largely on the cysteine concentration in the medium. In normal culture media containing cystine but not cysteine, the cellular glutathione content decreased notably within a few hours. Cysteine had to be supplied repeatedly to keep the content high. Cystine promoted the cellular glutathione content at unphysiologically high concentrations. These results were attributable to the extremely low uptake rate of cystine by the cells as compared with that of cysteine. In the presence of 2-mercaptoethanol, the uptake of radioactive cystine by the cells was increased and a high cellular glutathione level was maintained. A thiol-independent variant of L1210 took up cystine far more rapidly than L1210. From these results we concluded that the deficiency of the cystine uptake limits the growth of L1210 cells in normal culture media.

Interleukin-1 alpha enhances hepatotoxicity of tumor necrosis factor-alpha in galactosamine-sensitized mice.

The possible involvement of interleukin-1 alpha (IL-1 alpha) in the pathogenesis of murine hepatitis model induced with galactosamine and lipopolysaccharide (LPS) was investigated. The injection of 10 ng/mouse of LPS in combination with 10 mg/mouse of galactosamine into mice induced hepatic damage at 24 hours. Treatment with anti-mouse IL-1 alpha antiserum 30 min before galactosamine/LPS injection showed a tendency to reduce the liver injury, while pretreatment with anti-mouse tumor necrosis factor-alpha (TNF) antiserum significantly protected mice from liver injury. The use of recombinant murine TNF, instead of LPS, in combination with galactosamine could elicit hepatic damage, whereas recombinant murine IL-1 alpha could not substitute for LPS. However, recombinant murine IL-1 alpha enhanced the hepatotoxic effect of recombinant murine TNF in galactosamine-sensitized mice. These results suggest that TNF plays a major role in the pathogenesis of galactosamine/LPS hepatitis in mice and that IL-1 alpha acts synergistically with TNF in this hepatitis model.

[Effects of E-3123, a new protease inhibitor, on several protease activities and on experimental acute pancreatitis].

4-(2-Succinimidoethylthio) phenyl 4-guanidinobenzoate (E-3123) potently inhibited trypsin, plasmin and thrombin with IC50 values of 3.9 x 10(-8) M, 9.5 x 10(-7) M and 1.9 x 10(-6) M, respectively. Experimental acute pancreatitis was induced by injection of a mixture of trypsin and taurocholate into the pancreas in rats and rabbits or by an application of a closed duodenal loop in dogs. Intravenous infusion of E-3123 at 0.03-0.3 mg/kg in rats or at 0.3-3.0 mg/kg in rabbits reduced mortality after the induction of pancreatitis in a dose-dependent manner. Light microscopy of the pancreas in the E-3123-treated rabbits revealed marked decrease in cell necrosis and acinar cell vacuolation. Increase in plasma lipase activities associated with the progression of pancreatitis in rabbits was also reduced by the infusion of E-3123. In dogs with pancreatitis, increases in serum trypsin and lipase activities were significantly reduced by infusion of E-3123 at 1.0 and 3.0 mg/kg. The efficacies of E-3123 in the in vivo experiments were higher than those of nafamostat mesilate. These results show that E-3123 may possess suppressing effects on pathogenesis and development of acute pancreatitis.

[Effects of E3123 on pancreatic injury in the isolated, perfused rat pancreas and in a pancreatic slice].

We studied the effect of E3123 on pancreatic injury induced in the isolated, perfused pancreas or in a pancreatic slice. Exposure of the isolated, perfused rat pancreas to trypsin-taurocholate or phospholipase A2 caused the leakage of pancreatic enzyme (lipase) into the perfusate. In trypsin-taurocholate-induced pancreatic injury, E3123 and nafamostat mesilate suppressed the leakage of lipase at concentrations of 0.1-1 microM and 1-10 microM, respectively. In phospholipase A2 induced pancreatic injury, E3123 at 10 microM significantly suppressed the enzyme leakage, and nafamostat mesilate had a weak suppressive effect. Exposure of a rat pancreatic slice to phospholipase A2 also caused the leakage of pancreatic enzyme, while the inhibition of enzyme leakage by E3123 was similar to that observed in the isolated, perfused pancreas; nafamostat mesilate was not effective. This finding suggests the possible involvement of a unique mechanism of action in the protection against pancreatic injury by E3123. Therefore, we studied the effect of E3123 on hemolysis by osmotic shock using rat red blood cells. E3123 demonstrated a potent protective effect against the hemolysis, suggesting that a membrane-stabilizing action may contribute to the protection E3123 affords against pancreatic injury.

Involvement of tumor necrosis factor-alpha in development of hepatic injury in galactosamine-sensitized mice.

Intravenous injection of lipopolysaccharide and D-galactosamine, at doses of 0.2 micrograms/kg and 800 mg/kg, respectively, elicited massive hepatic necrosis within 24 hr in C3H/HeN mice. The plasma L-alanine aminotransferase (ALT, E.C. 2.6.1.2) or L-aspartate aminotransferase (AST, E.C. 2.6.1.1) activities at this point reached more than 2,000 IU/L. However, overt hepatic injury as evaluated by the plasma aminotransferase activities did not develop in mice in which only lipopolysaccharide or only D-galactosamine was injected. No tumor necrosis factor-like activities could be detected in the plasma of galactosamine- and lipopolysaccharide-injected mice as determined by the assay of cytotoxicity to highly tumor necrosis factor-sensitive L-P3 cells through the experimental period of 24 hr. However, passive immunization against mouse tumor necrosis factor-alpha with polyvalent rabbit anti-mouse tumor necrosis factor-alpha antiserum, which was able to neutralize the cytotoxic effects of recombinant mouse tumor necrosis factor-alpha on L-P3 cells, could protect the mice from the development of hepatic injury in a dose-dependent manner. Simultaneous injection of recombinant human tumor necrosis factor-alpha, instead of lipopolysaccharide, with 800 mg/kg of D-galactosamine in lipopolysaccharide-resistant C3H/HeJ mice sensitized the animals more than one thousand-fold to the development of hepatic injury. The livers appeared to be morphologically similar to those of galactosamine- and lipopolysaccharide-injected C3H/HeN mice.

Suppressive effects of E3330, a novel quinone derivative, on tumor necrosis factor-alpha generation from monocytes and macrophages.

E3330 [(2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid], a novel synthesized hepatoprotective compound, has suppressive effects on tumor necrosis factor-alpha (TNF-alpha) generation from monocytes/macrophages in vitro. E3330 (1-100 microM) reduced lipopolysaccharide (LPS, 10 mg/ml or 1 microgram/ml)-induced TNF-alpha generation from rat resident and Propionibacterium acnes (P. acnes)-elicited peritoneal macrophages, rat and human monocytes, rat Kupffer cells, and splenic mononuclear cells in a concentration-dependent manner. E3330 also (1-100 microM) suppressed TNF-alpha generation stimulated with egg-albumin immune complex in rat P. acnes-elicited peritoneal macrophages. Northern blot analysis showed that LPS-induced expression of TNF-alpha messenger RNA (mRNA) in human blood monocytes was suppressed by E3330. These findings indicate that E3330 has a suppressive effect on TNF-alpha generation from monocytes/macrophages, regardless of origin or species, and this effect is based in part on the suppression of TNF-alpha mRNA expression.

Involvement of tumor necrosis factor-alpha in the pathogenesis of activated macrophage-mediated hepatitis in mice.

The possible involvement of tumor necrosis factor-alpha in the pathogenesis of an experimentally induced hepatitis was investigated. Balb/c mice were primed with Propionibacterium acnes to induce the infiltration of mononuclear cells into the liver. Immunohistochemical study showed that most of the accumulated mononuclear cells at 7 days were Mac-2 positive, suggesting that they were activated macrophages. An injection of lipopolysaccharide resulted in massive hepatic necrosis and high mortality in the mice within 24 hours. Plasma tumor necrosis factor-alpha activity initially rose sharply and then declined over 3 hours. The increase in plasma aminotransferase activity correlated well with the elevation of plasma tumor necrosis factor-alpha activity. Pretreatment with dexamethasone or 16,16-dimethyl-prostaglandin E2 attenuated not only the elevation of plasma tumor necrosis factor-alpha activity but also the increase in plasma aminotransferase activity and improved the survival rate. Passive immunization against tumor necrosis factor-alpha showed protective effects. These findings suggest that tumor necrosis factor-alpha released from activated macrophages may play a crucial role in the pathogenesis of this murine hepatitis.

Protective effects of (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4- benzoquinoyl)]-2-nonyl-2-propenoic acid on endotoxin-mediated hepatitis in mice.

E3330 [(2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid] is a newly synthesized hepatoprotective quinone derivative. We examined the protective effects and possible mechanism of action of E3330 in three different endotoxin (lipopolysaccharide)-induced murine hepatitis models, in which tumor necrosis factor is suggested to play a critical role in the pathogenesis. One of these models was induced by i.v. injection of lipopolysaccharide in combination with D-galactosamine to mice. Oral pretreatment with E3330 improved the survival rate and attenuated the increase in plasma aminotransferase activities of the survivors. The other two models were induced by i.v. injection of lipopolysaccharide or a mixture of D-galactosamine and lipopolysaccharide in Propionibacterium acnes-primed mice. In both of these models, tumor necrosis factor was detected in the plasma within 3 hr of the injection. Oral pretreatment with E3330 attenuated the elevation of plasma tumor necrosis factor activity and protected mice from liver injury. Furthermore, E3330 inhibited the production of tumor necrosis factor from cultured Propionibacterium acnes-elicited murine peritoneal macrophages on stimulation with lipopolysaccharide in vitro. These findings suggest that the inhibition by E3330 of tumor necrosis factor production is the major mechanism of the protective effect of E3330 in these endotoxin-mediated hepatitis models in mice.

Protective effect of E3330, a novel quinone derivative, in galactosamine-induced hepatitis in rats.

The effect of E3330 ((2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2-++ +propenoic acid), a novel quinone derivative, was studied in the galactosamine-induced hepatitis model in F344 rats, in which endogenous endotoxin is believed to play a critical pathogenetic role. Subcutaneous injection of 300 mg/kg of galactosamine into rats resulted in liver injury. Oral treatment with E3330 (10-100 mg/kg) 1 hr after galactosamine challenge attenuated the liver injury. E3330 was also effective when administered p.o. 6 or 12 hr after galactosamine challenge. Subcutaneous injection of 1000 mg/kg of galactosamine into rats resulted in more severe liver injury with endotoxemia. The plasma endotoxin was detected 24 to 48 hr after the galactosamine challenge. The time course of increase in plasma endotoxin level was in good agreement with that in plasma aminotransferase activity. E3330 (100 mg/kg) significantly attenuated the liver injury, but did not affect the endotoxin level. Exogenous administration of endotoxin enhanced the hepatotoxicity of galactosamine. Pretreatment with E3330 also protected rats from severe liver injury induced with endotoxin plus galactosamine. These results suggest that E3330 may exert its hepatoprotective effects through inhibition of an effect of endotoxin in galactosamine-induced hepatitis in rats.