Non-prostanoid thromboxane A2 receptor antagonists with a dibenzoxepin ring system. 2.

A series of 11-[2-(1-benzimidazolyl)ethylidene]-6,11-dihydrodibenz[b,e]oxep in-2- carboxylic acid derivatives and related compounds were synthesized and found to be potent TXA2/PGH2 receptor antagonists. Each compound synthesized was tested for its ability to displace [3H]U-46619 binding from guinea pig platelet TXA2/PGH2 receptors. Structure-activity relationship studies revealed that the following key elements were required for enhanced activities: (1) an (E)-2-(1-benzimidazolyl)ethylidene side chain in the 11-position of the dibenzoxepin ring system and (2) a carboxyl group in the 2-position of the dibenzoxepin ring system. The studies also indicated that the TXA2/PGH2 receptor binding affinities of this series of compounds in guinea pig platelet were poorly correlated with those in human platelet. Introduction of substituent(s) to the benzimidazole moiety was effective and sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]- 6,11-dihydrodibenz[b,e]oxepin-2-carboxylate monohydrate (57) recorded the highest affinity for human platelet TXA2/PGH2 receptor with a K(i) value of 1.2 +/- 0.14 nM. It demonstrated potent inhibitory effects on U-46619-induced guinea pig platelet aggregation (in vitro and ex vivo) and human platelet aggregation (in vitro). Compound 57, now designated as KW-3635, is a novel, orally active, and specific TXA2/PGH2 receptor antagonist with neither TXA2/PGH2 receptor agonistic nor TXA2 synthase inhibitory effects. It is now under clinical evaluation.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 1. Synthesis and hypocholesterolemic activity of dibenz[b,e]oxepin-11-carboxanilides.

A series of N-phenyl-6,11-dihydrodibenz[b,e]oxepin-11-carboxamides and related derivatives were prepared on the basis of structures of the reported inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). These compounds were tested for their ability to inhibit ACAT (liver microsomes from cholesterol-fed rabbits) in vitro and to decrease serum total cholesterol in cholesterol-fed golden hamsters in vivo. The structure-activity relationships in vitro were as follows. Substitution at positions 2 and 6 in the anilide resulted in potent inhibitory activity, and the potency increased with increasing size of the substituents, with maximum potency being obtained with a 2,6-diisopropyl substitution. The position of the substituent on the dibenz[b,e]oxepin ring system influenced the activity, and substitution at position 2 was critical for potent activity. The electronic effect of the substituent at position 2 does not influence activity, but bulkiness seems to be a significant factor. The lipophilicity of the compounds also plays an important role in determining ACAT inhibitory activity. Among the compounds tested, 2-bromo-N-(2,6-diisopropylphenyl)-6,11-dihydrodibenz-[b,e]++ +oxepin-11- carboxamide (33, KF17828) showed significant in vitro activity (rabbit liver microsomes IC50 = 23 nM) and the most potent in vivo activity (complete reduction in elevated serum total cholesterol levels at a dose of 10 mg/kg in hamsters).

Dibenzoxepin derivatives: thromboxane A2 synthase inhibition and thromboxane A2 receptor antagonism combined in one molecule.

A new series of 6,11-dihydrodibenz[b,e]oxepin derivatives exerting both thromboxane synthase inhibitory (TXS-I) and thromboxane receptor antagonist (TXRA) activities is described. (-)-11-[(3-Pyridylmethyl)thio]-6,11-dihydrodibenz[b,e]oxepin -2- carboxylic acid [(-)-3] and (E)-11-[2-(3-pyridyl)ethylidene]-6,11-dihydrodibenz[b,e]oxepin+ ++-2- carboxylic acid methanesulfonate (11E) exhibited potent inhibitory effects on bovine platelet thromboxane synthase with IC50 values of 4.0 and 14 nM, respectively, and these derivatives also antagonized guinea pig platelet TXA2/PGH2 receptors with Ki values of 85 and 180 nM, respectively. Compound 11E exhibited the dual inhibitory activity in ex vivo experiments and demonstrated a significant protective effect in a rat acute renal failure model.

Insulin-stimulated glucose transport regulated by adenylate cyclase system in rat adipocytes.

1. In rat adipocytes, there was an inverse correlation between insulin-stimulated 2-deoxyglucose (2-DG) uptake and cAMP levels, indicating that cAMP suppressed the 2-DG uptake stimulated by insulin. 2. This inhibitory effect of cAMP was due to suppression of translocation of glucose transporters rather than that of insulin-binding to its receptors. 3. Phosphodiesterase inhibitors (IBMX, Ro 20-1724, and cilostamide) inhibited the 2-DG uptake, which was brought about by direct interaction with glucose transporters in the plasma membranes.

Protective effects of the novel thromboxane A2 receptor antagonist sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11- dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate against 9,11-dideoxy- 9 alpha, 11 alpha-epoxymethano-prostaglandin F2 alpha-induced sudden death in guinea-pigs and rats.

Injection of U-46619 (9,11-dideoxy-9 alpha, 11 alpha-epoxymethano- prostaglandinF2 alpha; 130 micrograms/kg i.v.) produced sudden death in anesthetized guinea-pigs and rats within 10-15 min. This sudden death is typified by a precipitous drop in mean arterial blood pressure (MABP) and a dramatic decrease in the circulating platelet counts. In guinea-pigs, KW-3635 (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0) at doses of 0.1 mg/kg or greater dramatically protected animals against sudden death induced by injection of U-46619. Pretreatment with KW-3635 (0.3, 1.0 mg/kg i.v.) inhibited the decrease in circulating platelet counts and the decline in blood pressure associated with the i.v. injection of U-46619. Oral administration of KW-3635 (10, 30 mg/kg) also protected the animals from the U-46619-induced sudden death. The effect of KW-3635 was almost the same as that of daltroban, and was more potent than that of sulotroban. In rats, intravenous administration of KW-3635 at doses of 0.3 mg/kg or greater protected against sudden death. In contrast, acetylsalicylic acid a cyclooxygenase inhibitor, did not protect against sudden death induced by U-46619, indicating that the formation of endogenous thromboxane does not play a major role in the lethal effect of U-46619, and that the blockade of the lethal effects of U-46619 is specific for thromboxane receptor antagonists. Our data show that KW-3635 protects guinea-pigs and rats against U-46619-induced sudden death. Therefore, KW-3635 may be useful for the investigation of diseases where thromboxane is involved.

Effect of benidipine hydrochloride (KW-3049), on cerebral ischemia induced by bilateral occlusion of the common carotid arteries in rats.

The effect of benidipine on experimental cerebral ischemia was investigated in rats subjected to occlusion of the bilateral common carotid arteries. Benidipine (30 micrograms/kg, i.p.) improved neurological symptoms such as ataxia, convulsion and loss of righting reflex, and prolonged survival time after occlusion of the bilateral common carotid arteries. In the nicardipine (100 micrograms/kg, i.p.)-treated group, a similar effect was observed, whereas nifedipine (100, 300 micrograms/kg, i.p.) and verapamil (300 micrograms/kg, i.p.) did not show any beneficial effect in this model. Furthermore, pretreatment with benidipine (30 micrograms/kg, i.p.) suppressed the increase in cerebral water content 3 h after the occlusion. Nicardipine (100 micrograms/kg, i.p.) showed a tendency to reduce the increase in cerebral water content, though the effect was not statistically significant. Nifedipine (100 micrograms/kg, i.p.) produced no improvement. After occlusion of the bilateral common carotid arteries, depletion of adenosine triphosphate (ATP) and phosphocreatine (CP) and an accumulation of lactate occurred in a time-dependent manner. Prophylactic administration of benidipine (30 micrograms/kg, i.p.), 20 min before occlusion, attenuated the depletion of ATP and CP and the accumulation of lactate 3h after the occlusion. Furthermore, post-treatment with benidipine 30 min after occlusion also suppressed these metabolic disorders. In conclusion, the beneficial effects of benidipine in this severe cerebral ischemia model show that the compound has advantages over nicardipine, nifedipine and verapamil. Thus, these results suggest that benidipine may be useful in the treatment of acute ischemic cerebral damage.

Activation of glucose transport by activatory receptor agonists of adenylate cyclase in rat adipocytes.

1. Catecholamine, glucagon, and adrenocorticotropic hormone stimulated 2-deoxyglucose (2-DG) uptake via an increase in glucose transporters in plasma membranes, similarly to insulin. 2. In contrast to the action of insulin, the stimulating effects of these agonists on 2-DG uptake were abolished when Gi was not activated. 3. The mode of the 2-DG uptake stimulation was partially different among these agonists.

Actions of the novel thromboxane A2 receptor antagonist sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11- dihydrodibenz[b,e]oxepine-1-carboxylate monohydrate on smooth muscle preparations.

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)- ethylidene]-6,11-dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0) on smooth muscle preparations were examined. In isolated guinea-pig aorta, KW-3635 competitively inhibited the U-46619 (a thromboxane mimetic) induced contractions (pA2 = 7.74), the effect being more potent than those of sulotroban and daltroban. In canine saphenous vein, KW-3635 also antagonized the U-46619-induced contraction (pA2 = 8.11). In this preparation, solutroban and daltroban, but not KW-3635, exhibited intrinsic agonistic action. KW-3635, even at a high concentration of 10(-5) mol/l did not affect the norepinephrine- or KCl-induced contractions of guinea-pig or rat aorta, prostaglandin (PG)E2- or PGF2 alpha-induced contractions of guinea-pig ileum nor the PGE2-induced contraction of rat fundus. KW-3635 at concentrations higher than its thromboxane A2- (TxA2-)antagonistic one, non-competitively inhibited the PGF2 alpha-induced contractions of guinea-pig aorta (pD2' = 6.23), as was the case with daltroban. The inhibitory effect of KW-3635 (3 x 10(-6) mol/l) on U-46619-induced contractions of guinea-pig aorta persisted for longer than 2 h following washout of the tissue, whereas that of daltroban (10(-5) mol/l completely disappeared at 1 h after the washout. In anesthetized guinea-pigs, KW-3635 at doses of 10 to 1000 micrograms/kg (i.v.) inhibited U-46619 (1 microgram/kg i.v.)-induced pressor responses in a dose-dependent manner. The effect of KW-3635 (0.1 to 1 mg/kg i.v.) persisted for longer than 3 h. These results demonstrate that KW-3635 is a potent and specific TxA2 antagonist without agonistic action in vascular smooth muscles. KW-3635 is considered to be a promising candidate for the treatment of patients with disorders mediated via TxA2.

Antithrombotic effects of KW-3635, a thromboxane A2-receptor antagonist, in guinea pigs.

Antithrombotic effects of KW-3635, a newly synthesized thromboxane (TX) A2-receptor antagonist, were studied in guinea pigs. In the extracorporeal circulation thrombosis model, the shunt was filled with thrombi, and reduction of platelet count and increase in plasma TXA2 concentration were observed. KW-3635 (30 and 100 mg/kg, p.o.) inhibited the thrombus formation in the shunt and prevented the decrease in platelet count in the circulating blood without affecting the red blood cell count. BM13,505 (30, 100 mg/kg, p.o.), another TXA2-receptor antagonist, and ticlopidine (300 mg/kg, p.o.), an antiplatelet drug, also inhibited the thrombus formation, while aspirin (10, 300 mg/kg, p.o.) did not. Peripheral arterial occlusive disease was induced by injection of sodium laurate into the femoral artery in guinea pigs. Daily oral administration of KW-3635 (3-30 mg/kg) significantly prevented the progression of vascular lesions. BM13,505 (3-30 mg/kg, p.o.) and ticlopidine (100 mg/kg, p.o.) also ameliorated the vascular lesions, whereas aspirin (10, 100 mg/kg, p.o.) did not. KW-3635 at concentrations up to 10(-4) M did not affect coagulation parameters in vitro. These results suggest that TXA2 is involved in the pathogenesis of arterial thrombotic and ischemic disorders. KW-3635 may be useful for the treatment of thrombotic disease and peripheral arterial occlusive diseases.

Protective effect of benidipine against the development of glomerular sclerosis in experimental nephrotic syndrome.

An experimental focal segmental glomerular sclerosis (FSGS) was induced by the combined administration of puromycin aminonucleoside (PAN) and protamine sulfate (PS). Blood collections were made on days 0, 37, 70 and 94. Urine collections were made on days 0, 24, 80 and 94. Vehicle-treated rats showed severe proteinuria and an increase in serum total cholesterol (sTC). Benidipine (1 or 3 mg/kg, p.o.)-treated rats exhibited less proteinuria and lower sTC than the vehicle-treated rats. On days 70 and 94, both blood urea nitrogen (BUN) and serum creatinine (sCR) values in the vehicle-treated rats were significantly higher than those in normal rats (without treatment with PAN and PS). On the other hand, the treatment with benidipine (1 or 3 mg/kg, p.o.) attenuated the increases in BUN and sCR. On day 94, vehicle-treated rats showed a significant decrease in creatinine clearance as compared with normal rats, but benidipine (1 or 3 mg/kg, p.o.)-treated rats did not. The histology was examined on day 94. Vehicle-treated rats demonstrated a significantly greater percentage of glomeruli with segmental areas of glomerulosclerosis/hyalinosis, mesangial cell proliferation, and mesangial foam cell. Benidipine (3 mg/kg, p.o.) ameliorated the development of renal regeneration as estimated by histological examination. These results suggest that the Ca-channel blocker benidipine is a favorable drug for preventing the progression of glomerular sclerosis.

Protective effects of benidipine on arachidonic acid-induced acute cerebral ischemia in rats.

Acute cerebral ischemia was produced in rats by injection of arachidonic acid (AA) into the internal carotid artery. Evans blue (EB) was intravenously injected and its extravasation into the brain was determined as an indicator of disturbances in the blood-brain barrier and endothelial cells. Control animals showed severe cerebral edema and marked blue staining of the brain. Benidipine (30 micrograms/kg, i.p.) suppressed the increase in cerebral water content and the extravasation of EB. Similarly nicardipine (100 micrograms/kg, i.p.) suppressed the elevation of water content and the extravasation of EB. Furthermore, both benidipine (30 micrograms/kg, i.p.) and nicardipine (100 micrograms/kg, i.p.) improved the neuronal injuries following AA-injection. An antiplatelet agent, ticlopidine (100 mg/kg, i.p.), and a thromboxane A2 synthetase inhibitor, OKY-1581 (3 mg/kg, i.p.), also suppressed the elevation of cerebral water content. A lipoxygenase inhibitor, AA-561 (200 mg/kg, p.o.), and a cyclooxygenase inhibitor, indomethacin (10 mg/kg, i.p.), did not prevent the increase in cerebral water content. Neither benidipine (3-30 micrograms/kg, i.v.) nor nicardipine (100 micrograms/kg, i.v.) inhibited the AgNO3-induced thrombus formation of the abdominal aorta, whereas ticlopidine (100 mg/kg, p.o.) and OKY-1581 (3 mg/kg, i.v.) prevented the thrombus formation. From the present results, it is suggested that benidipine, as well as nicardipine, may protect against AA-induced acute cerebral infarction via a mechanism independent of antithrombotic action.

Beneficial effect of the novel thromboxane A2 receptor antagonist sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate on collagen-induced coronary ischemia in guinea-pigs.

The effect of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl) ethylidene]-6,11-dihydrodibenz[b,e] oxepine-2-carboxylate monohydrate, CAS 127166-41-0), a novel thromboxane A2 (TxA2) receptor antagonist, on collagen-induced coronary ischemia was studied in guinea-pigs. Under pentobarbital anaesthesia, intravenous injection (i.v.) of collagen (1 mg/kg) induced abnormal ECG changes such as ST-T changes, elevation of T-wave arrhythmia and cardiac arrest in severe cases. The changes of ECG (leads I, II and III) were recorded for 10 min following collagen injection. KW-3635 (25-50 mg/kg p.o.) remarkably improved the collagen-induced ischemic ECG changes. The effect of KW-3635 was more potent than those of daltroban, isbogrel and ticlopidine. Neither nifedipine nor propranolol had any effect. The plasma thromboxane B2 level in the KW-3635-treated animals was lower in comparison with those in both the control and daltroban-treated animals. These results suggest that TxA2 may play a role in this model of coronary ischemia and that KW-3635 is effective in the treatment of ischemic heart disease.

Antiplatelet effects of the novel thromboxane A2 receptor antagonist sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11- dihydrodibenz[b,e] oxepine-2-carboxylate monohydrate.

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)- ethylidene]-6,11-dihydrodibenz[b,e] oxepine-2-carboxylate monohydrate, CAS 127166-41-0) on platelet aggregation were examined. In human washed platelets, KW-3635 shifted the concentration-aggregation curves for U-46619, a thromboxane A2 (TxA2) mimetic, to the right. The pA2 value for KW-3635 was 8.8 +/- 0.10, while those for sulotroban and daltroban were 6.31 +/- 0.18 and 7.75 +/- 0.07, respectively. In human platelet rich plasma (PRP), KW-3635 at 10(-8) mol/l to 10(-6) mol/l inhibited the aggregations induced by U-46619 (1 mumol/l) or collagen (1.5 micrograms/ml). However, KW-3635 at up to 10(-5) mol/l did not affect the primary phase of platelet aggregation induced by adenosine diphosphate or epinephrine. KW-3635 at 10(-5) mol/l did not affect the antiaggregatory effects of the prostaglandins PGI2, PGE1 and PGD2. These results indicate that KW-3635 is a potent and selective TxA2 receptor antagonist. The TxA2 antagonistic effects of KW-3635 were compared with that of daltroban in PRP from various animals species. The effects of KW-3635 on platelet aggregation were species-dependent and KW-3635 exhibited the most prominent activity in human platelets. The activities of KW-3635 in mouse and rabbit PRP were much less potent. In PRP from guinea-pigs, dogs, cats and rats, KW-3635 exhibited moderate anti-aggregatory effects. In the guinea-pig PRP, KW-3635 at 10(-7) mol/l to 3 x 10(-6) mol/l inhibited both the platelet aggregation and the concomitant adenosine triphosphate secretion in a concentration-dependent manner, the effect being more potent than those of sulotroban and daltroban. In the experiments on the platelet aggregation ex vivo in guinea-pigs, KW-3635 at oral doses of 3 and 10 mg/kg inhibited the aggregations induced by U-46619 (1, 3 mumol/l), collagen (3, 6, 9 micrograms/ml) and arachidonate (50, 100 mumol/l). The effects lasted for longer than 7 h following oral administration. These results indicate that KW-3635 is a specific and orally active TxA2 receptor antagonist. KW-3635 is expected to be a drug useful for the treatment of patients with thrombotic disorders.

Inhibitors of acyl-CoA: cholesterol acyltransferase. II. Preparation and hypocholesterolemic activity of optically active dibenz[b,e]oxepin-11-carboxanilides.

In a previous paper, we reported a novel inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT), 2-bromo-N-(2,6-diisopropylphenyl)-6,11- dihydrodibenz[b,e]oxepin-11-carboxamide (1). In this work, we prepared both enantiomers and tested them for ability to inhibit ACAT (liver microsomes from cholesterol-fed rabbits) in vitro and to decrease serum total cholesterol in cholesterol-fed golden hamsters in vivo. The precursor carboxylic acid 4 was optically resolved with cinchonidine. The obtained (-)- and (+)-4 were converted to (-)- and (+)-1 without racemization, respectively. The enantiomer (-)-1 showed potent ACAT inhibitory activity in vitro with an IC50 value of 8 nM and was approximately 10-fold more active than (+)-1. Furthermore, (-)-1 showed strong hypocholesterolemic activity in vivo, whereas (+)-1 was inactive. A molecular modeling study showed that the difference of ACAT inhibitory activity between the enantiomers was derived from the spatial alignment of the bromine. Compound (-)-1 was selected for further evaluation as KW-3033.