Design and synthesis of 4-substituted benzamides as potent, selective, and orally bioavailable I(Ks) blockers.

Multiple delayed rectifier potassium currents, including I(Ks), are responsible for the repolarization and termination of the cardiac action potential, and blockers of these currents may be useful as antiarrhythmic agents. Modification of compound 5 produced 19(S) that is the most potent I(Ks) blocker reported to date with >5000-fold selectivity over other cardiac ion channels. Further modification produced 24A with 23% oral bioavailability.

Arylcyclopropanecarboxyl guanidines as novel, potent, and selective inhibitors of the sodium hydrogen exchanger isoform-1.

A novel series of arylcyclopropanecarboxyl guanidines was synthesized and evaluated for activity against the sodium hydrogen exchanger isoform-1 (NHE-1). In biological assays conducted in an AP1 cell line expressing the human NHE-1 isoform, the starting cyclopropane 3a (IC(50) = 3.5 microM) shows inhibitory activity comparable to cariporide (IC(50) = 3.4 microM). Structure-activity relationships are used to optimize the affinity of various acyl guanidines for NHE-1 by screening the effect of substituents at both aryl and cyclopropyl rings. It is demonstrated that introduction of appropriate hydrophobic groups at the phenyl ring and a gem-dimethyl group at the cyclopropane ring enhances the NHE-1 inhibitory activity by up to 3 orders of magnitude (compound 7f, IC(50) = 0.003 microM). In addition, the gem-dimethyl series of analogues seem to display improved oral bioavailability and longer plasma half-life in rats. Furthermore, the lead benzodihydrofuranyl analogue 1 (BMS-284640) shows over 380-fold increased NHE-1 inhibitory activity as well as improved selectivity for NHE-1 over NHE-2 compared to cariporide.

Pharmacologic characterization of BMS-191095, a mitochondrial K(ATP) opener with no peripheral vasodilator or cardiac action potential shortening activity.

Previous work described ATP-sensitive K(+) channel (K(ATP)) openers (e.g., BMS-180448), which retain the cardioprotective activity of agents such as cromakalim while being significantly less potent as vasodilators. In this study, we describe the pharmacologic profile of BMS-191095, which is devoid of peripheral vasodilating activity while retaining glyburide-reversible cardioprotective activity. In isolated rat hearts subjected to 25 min of global ischemia and 30 min of reperfusion, BMS-191095 increased the time to onset of ischemic contracture with an EC(25) of 1.5 microM, which is comparable to 4.7 microM and 3.0 microM for cromakalim and BMS-180448, respectively. Comparisons of cardioprotective and vasorelaxant potencies in vitro and in vivo showed BMS-191095 to be significantly more selective for cardioprotection with virtually no effect on peripheral smooth muscle, whereas cromakalim showed little selectivity. In addition to increasing the time to the onset of contracture, BMS-191095 improved postischemic recovery of function and reduced lactate dehydrogenase release in the isolated rat hearts. The cardioprotective effects of BMS-191095 were abolished by glyburide and sodium 5-hydroxydecanoate (5-HD). BMS-191095 did not shorten action potential duration in normal or hypoxic myocardium within its cardioprotective concentration range nor did it activate sarcolemmal K(ATP) current (< or =30 microM). BMS-191095 opened cardiac mitochondrial K(ATP) with a K(1/2) of 83 nM, and this was abolished by glyburide and 5-HD. These results show that the cardioprotective effects of BMS-191095 are dissociated from peripheral vasodilator and cardiac sarcolemmal K(ATP) activation. Agents like BMS-191095 may owe their cardioprotective selectivity to selective mitochondrial K(ATP) activation.

Cardioselective antiischemic ATP-sensitive potassium channel (K(ATP)) openers. 6. Effect of modifications at C6 of benzopyranyl cyanoguanidines.

The effect on potency and selectivity of modifications at the C6 position of the cardioprotective K(ATP) opener BMS-180448 (2) is described. Structure-activity studies show that a variety of electron-withdrawing groups (ketone, sulfone, sulfonamide, etc.) are tolerated for cardioprotective activity as measured by EC(25) values for an increase in time to the onset of contracture in globally ischemic rat hearts. Changes made to the sulfonamido substituent indicate that compounds derived from secondary lipophilic amines are preferred for good cardioprotective potency and selectivity. The diisobutyl analogue 27 (EC(25) = 0.04 microM) is the most potent compound of this series. The cardiac selectivity of 27 results from a combination of reduced vasorelaxant potency and enhanced cardioprotective potency relative to the potent vasodilating K(ATP) openers (e.g., cromakalim). The diisobutylsulfonamide analogue 27 is over 4 orders of magnitude more cardiac selective than cromakalim (1). These results support the hypothesis that the cardioprotective and vasorelaxant properties of K(ATP) openers follow distinct structure-activity relationships. The mechanism of action of 27 appears to involve opening of the cardiac K(ATP) as its cardioprotective effects are abolished by the K(ATP) blocker glyburide.

Nucleotide regulation and characteristics of potassium channel opener binding to skeletal muscle membranes.

[3H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC50 value for MgATP-supported binding (0.4 mM) was decreased approximately 10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [3H]P1075 bound to skeletal muscle with a Kd value of 37 +/- 3 nM and a Bmax value of 280 +/- 14 fmol/mg of protein. [3H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (Ki = 200-300 nM). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [3H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [3H]P1075 dissociation in a manner consistent with noncompetitive interaction.

Cardioselective antiischemic ATP-sensitive potassium channel (KATP) openers. 5. Identification of 4-(N-aryl)-substituted benzopyran derivatives with high selectivity.

This paper describes our studies aimed at the discovery of structurally distinct analogs of the cardioprotective KATP opener BMS-180448 (2) with improved selectivity for the ischemic myocardium. The starting compound 6, derived from the indole analog 4. showed good cardioprotective potency and excellent selectivity compared to 2 and the first-generation KATP opener cromakalim (1). The structure-activity studies indicate that increasing the size of the alkyl ester leads to diminished potency as does its replacement with a variety of other groups (nitrile, methyl sulfone). Replacement of the ethyl ester of 6 with an imidazole gave the best compound 3 (BMS-191095) of this series which maintains the potency and selectivity of its predecessor 6. The results described in this publication further support that there is no correlation between vasorelaxant and cardioprotective potencies of KATP openers. Compound 3 is over 20- and 4000-fold more selective for the ischemic myocardium than 2 and cromakalim (1), respectively. The selectivity for the ischemic myocardium is achieved by reduction of vasorelaxant potency rather than enhancement in antiischemic potency. As for cromakalim (1) and 2, the cardioprotective effects of compound 3 are inhibited by cotreatment with the KATP blocker glyburide, indicating that the KATP opening is involved in its mechanism of cardioprotection. With its good oral bioavailability (47%) and plasma elimination half-life (3 h) in rats, compound 3 offers an excellent candidate to investigate the role of residual vasorelaxant potency of 2 toward its cardioprotective activity in vivo.

Glyburide-reversible cardioprotective effects of calcium-independent phospholipase A2 inhibition in ischemic rat hearts.

OBJECTIVES: A myocardial calcium-independent PLA2 has been described that is activated during myocardial ischemia and this enzyme may modulate ATP-sensitive potassium channels (KATP). The aim of this study was to determine the effect of an inhibitor of this enzyme, a bromoenol lactone, in isolated globally ischemic rat hearts. METHODS: Isolated rat hearts were treated for 10 min with 0.3-6 microM bromoenol lactone and then subjected to 25 min ischemia and 30 min reperfusion. RESULTS: The bromoenol lactone significantly increased coronary flow in nonischemic myocardium, and slightly reduced cardiac function at 6 microM. During global ischemia, time to contracture was significantly increased from vehicle group values in the presence of the bromoenol lactone (EC50 = 1.2 microM). During reperfusion, a concentration-dependent increase in function and a reduction in LDH release were observed for the PLA2 inhibitor. The concentrations of the PLA2 inhibitor which were significantly cardioprotective, inhibited this enzyme in membrane fractions of rat myocardium (IC50 = 0.87 microM). The KATP blocker sodium 5-hydroxydecanoate (5-HD) inhibited the increase in time to contracture observed for the bromoenol lactone. During reperfusion, 5-HD abolished the protective effects of the bromoenol lactone on cardiac function and LDH release. Glyburide had similar effects on the cardioprotective activity of the bromoenol lactone, although it only partially abolished the LDH reducing effect of this agent. CONCLUSIONS: The bromoenol lactone protects ischemic myocardium at concentrations which also inhibit calcium-independent PLA2. This cardioprotection can be attenuated by blockers of KATP, suggesting a potential mechanism for modulation of myocardial KATP.

Cardioselective antiischemic ATP-sensitive potassium channel openers. 4. Structure-activity studies on benzopyranylcyanoguanidines: replacement of the benzopyran portion.

The results of our efforts aimed at the replacement of the benzopyran ring of the lead cardiac selective antiischemic ATP-sensitive potassium channel (KATP) opener (4) are described. Systematic modification of the benzopyran ring of 4 resulted in the discovery of a structurally simpler acyclic analog (8) with slightly lower antiischemic potency than the lead compound 4. Further structure-activity studies on the acyclic analog 8 provided the 2-phenoxy-3-pyridylurea analog 18 with improved antiischemic potency and selectivity compared to the benzopyran-based compound 4. These data demonstrate that the benzopyran ring of 4 and its congeners is not mandatory for antiischemic activity and cardiac selectivity. The results described in this paper also show that, as for the benzopyran class of compounds, the structure-activity relationships for the antiischemic and vasorelaxant activities of KATP openers are distinct. The mechanism of action of the acyclic analogs (e.g., 18) still appears to involve KATP opening as their cardioprotective effects are abolished by pretreatment with the KATP blocker glyburide.

Cardioselective anti-ischemic ATP-sensitive potassium channel openers. 3. Structure-activity studies on benzopyranyl cyanoguanidines: modification of the cyanoguanidine portion.

Structure-activity relationships for the cyanoguanidine portion of the lead cardiac selective ATP-sensitive potassium channel (KATP) opener (3) are described. The cyanoguanidine moity appears to be optimal since increasing or decreasing the distance between the aniline nitrogen and the pendant aromatic ring attenuates anti-ischemic potency/selectivity. Similarly, unfavorable results are obtained by replacement of the aniline nitrogen with other linkers (CH2, S, O). Replacement of the phenyl ring with a methyl group diminishes cardiac selectivity. Constraining the urea moiety into a benzimidazolone or imidazolone ring retains anti-ischemic potency with significant improvement in cardiac selectivity. As shown by the ratio of vasorelaxant and anti-ischemic potencies, the cardiac selectivity in vitro varies over 3 orders of magnitude. These data are in agreement with previous results indicating that distinct structure-activity relationships exist for the anti-ischemic and vasorelaxant activities. Since the anti-ischemic effects of this series of compounds are abolished by pretreatment with structurally different KATP blockers (glyburide, sodium 5-hydroxydecanoate, meclofenamic acid), the mechanism for the anti-ischemic actions of these compounds still appears to involve the opening of KATP.

Cardioselective antiischemic ATP-sensitive potassium channel openers. 2. Structure-activity studies on benzopyranylcyanoguanidines: modification of the benzopyran ring.

The ATP-sensitive potassium channel (KATP) openers are of considerable interest as myocardial protecting agents. However, there exists a narrow window of safety for the use of first-generation compounds as antiischemic agents due to their powerful peripheral vasodilating effects, which can result in underperfusion of the area already at risk. We have recently disclosed the discovery of benzopyranylcyanoguanidine type KATP openers (BMS-180448) which are more selective for the ischemic myocardium compared to the first-generation compounds. This publication deals with structure-activity relationships for the antiischemic activity of the lead compound 8. The presence of an electron-withdrawing group at C6, an sp3 center at C4, and a gem-dimethyl group at C2 appears to be essential for antiischemic activity. Cyanoguanidine can be replaced with a urea moiety. The results reported here support the hypothesis that distinct structure-activity relationships exist for antiischemic and vasorelaxant activities of compounds related to 8 and cromakalim. The trifluoromethyl analog 10 is 550-fold more selective in vitro for the ischemic myocardium compared to the first-generation agent cromakalim. The reasons for the selectivity of these compounds for the ischemic myocardium are not clear at the present time. They may be related to the existence of receptor subtypes in smooth muscle and the myocardium.

Molecular Responses to Abscisic Acid and Stress Are Conserved between Moss and Cereals.

Promoter elements from the wheat Em gene have been characterized. These elements are inducible by abscisic acid (ABA) and by osmotic stress. In this study, we demonstrated that the same promoter elements function in a distantly related plant species, the moss Physcomitrella patens. Transient and stable expression of the [beta]-glucuronidase reporter gene was used to determine that the heterologous wheat promoter also responds to osmotic stress and ABA in moss. Mutational analysis of the promoter indicated that the mechanism of gene regulation is conserved in both species. Gel retardation and DNase I footprint analyses were conducted to characterize further the interaction of moss transcription factors with the Em promoter. In addition, the synthesis of stress-related polypeptides in moss was observed. The evolutionary significance of these data and the potential for studying the entire ABA perception-response pathway in moss are discussed.

Cardioprotective profile of the cardiac-selective ATP-sensitive potassium channel opener BMS-180448.

ATP-sensitive potassium channel (KATP) openers have direct protective effects on ischemic myocardium that are independent of vasorelaxation. Because reference KATP openers (e.g., cromakalim, pinacidil) are potent relaxants of smooth muscle, their utility for treating myocardial ischemia may be limited by hypotension. Efforts aimed at development of a cardioprotective KATP opener with less vasorelaxant activity led to identification of the arylcyanoguanidine analogue BMS-180448. In globally ischemic rat hearts, BMS-180448 was cardioprotective (EC25 for increasing time to contracture = 2.5 microM), with potency equal to that of cromakalim (EC25 = 4.9 microM) despite being significantly less potent as a peripheral smooth muscle relaxant (methoxamine-constricted rat aorta). The cardioprotective effects of BMS-180448 in isolated perfused rat heart were abolished by the KATP blockers glyburide and sodium 5-hydroxydecanoate, indicating KATP involvement in its mechanism of action. Further confirmation was obtained by demonstration of single KATP opening by BMS-180448 in guinea pig cardiac myocytes. In anesthetized dogs, cromakalim was > 100-fold more potent than BMS-180448 in decreasing blood pressure (BP). In anesthetized dogs subjected to 90-min coronary occlusion/reperfusion, BMS-180448 reduced infarct size (IS) by 50% without hemodynamic effects. BMS-180448 provides the opportunity to explore the cardioprotective actions of this class of agents without the possible complications (hypotension, coronary steal) that may be caused by the currently available KATP openers.

Pharmacology and structure-activity relationships for KATP modulators: tissue-selective KATP openers.

KATP openers are recognized as having a therapeutic potential for the treatment of various cardiovascular and noncardiovascular diseases. However, the first-generation agents open KATP in a variety of tissues that limit their potential clinical utility. This review describes our studies aimed at the identification of a common pharmacophore among structurally diverse KATP openers and the discovery of tissue-selective agents that may offer advantages over the first-generation agents for the treatment of cardiovascular diseases. Compounds (e.g., BMS-182264) combining the structural features of KATP openers cromakalim and pinacidil, show smooth-muscle relaxing and antihypertensive activities comparable to their predecessors, indicating cromakalim and pinacidil may express their biologic effects through similar structural requirements. The hypothesis about common features is further supported by radioligand-binding studies showing cromakalim, pinacidil, and the combination compound BMS-182264 bind to a similar receptor site in rat aortic smooth-muscle cells. Regardless of having a pharmacologic profile similar to cromakalim and pinacidil in vascular smooth muscle, BMS-182264 had no effect on action potential duration in guinea pig papillary muscle, indicating smooth-muscle selectivity for BMS-182264. We demonstrate that no correlation exists between anti-ischemic and smooth-muscle relaxing potencies for a variety of structurally different KATP openers. Efforts to find KATP openers selective for the ischemic myocardium led to the identification of BMS-180448 which, despite having similar anti-ischemic potency to cromakalim, was significantly less active as a smooth-muscle relaxant. This compound was shown to be efficacious as an anti-ischemic agent in vivo without affecting hemodynamic variables, a potential liability of first-generation compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Dihydropyrimidine angiotensin II receptor antagonists.

The discovery of the nonpeptide angiotensin II (AII) receptor antagonist losartan, previously called DuP 753, has stimulated considerable interest in the synthesis of novel analogs of this compound. Our efforts in this area have resulted in the discovery of dihydropyrimidines as potent AII receptor antagonists. The chemistry leading to this novel class of AII antagonists and their biological properties are reported in this publication. Structure-activity studies showed that a variety of substituents are tolerated on the dihydropyrimidine ring, indicating that the AII receptor is permissive in accepting this region of the nonpeptide antagonists. As reported for imidazole-based AII antagonists, the tetrazolyl dihydropyrimidine analogs were found to be more potent than the corresponding carboxylic acids. Our studies show that dihydropyrimidine analogs 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5-yl)[1, 1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester (Ki = 8.3 nM), 2-butyl-4-chloro-1,6-dihydro-6-methyl-1- [[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-5- pyrimidinecarboxylic acid (Ki = 1.0 nM), and 2-butyl-6-chloro-1,4-dihydro-4,4-dimethyl-1-[[2'-(1H-tetrazol-5-yl )[1,1'- biphenyl]-4-yl]methyl]-5-pyrimidinecarboxylic acid, ethyl ester (Ki = 1.1 nM), display affinities for the AII receptor which are comparable to or better than losartan (Ki = 9.0 nM). One of these derivatives, 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5- yl)[1,1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester, showed antihypertensive activity on oral administration to spontaneously hypertensive rats. These results demonstrate that the imidazole of losartan can be successfully replaced with a dihydropyrimidine ring.

Dihydropyrimidine calcium channel blockers. 4. Basic 3-substituted-4-aryl-1,4-dihydropyrimidine-5-carboxylic acid esters. Potent antihypertensive agents.

We have examined a series of novel dihydropyrimidine calcium channel blockers that contain a basic group attached to either C5 or N3 of the heterocyclic ring. Structure-activity studies show that a 1-(phenylmethyl)-4-piperidinyl carbamate moiety at N3 and sulfur at C2 are optimal for vasorelaxant activity in vitro and impart potent and long-acting antihypertensive activity in vivo. One of these compounds (11) was identified as a lead, and the individual enantiomers 12a (R) and 12b (S) were synthesized. Two key steps of the synthesis were (1) the efficient separation of the diastereomeric ureido derivatives 29a/29b and (2) the high-yield transformation of 2-methoxy intermediates 30a/30b to the (p-methoxybenzyl)thio intermediates 31a/31b. Chirality was demonstrated to be a significant determinant of biological activity, with the dihydropyridine receptor recognizing the enamino ester moiety (12a) but not the carbamate moiety (12b). Dihydropyrimidine 12a is equipotent to nifedipine and amlodipine in vitro. In the spontaneously hypertensive rat, dihydropyrimidine 12a is both more potent and longer acting than nifedipine and compares most favorably with the long-acting dihydropyridine derivative amlodipine. Dihydropyrimidine 12a has the potential advantage of being a single enantiomer.

Cardioprotective effects of the potassium channel opener cromakalim: stereoselectivity and effects on myocardial adenine nucleotides.

We determined if the cardioprotective effects of the potassium channel opener cromakalim are stereoselective and if it can preserve adenine nucleotides in ischemic myocardium. We subjected isolated isovolumically beating rat hearts to 25 min of global ischemia and reperfusion with and without pretreatment by cromakalim or its enantiomers. All of these compounds significantly increased preischemic coronary flow with the (3S,4R)-(-)-enantiomer being more potent (EC25 = 0.52 microM) compared to cromakalim (EC25 = 1.04 microM) and the (3R,4S)-(+)-enantiomer (EC25 greater than 100 microM). The (-)-enantiomer was also significantly more potent in reducing ischemic/reperfusion damage compared to cromakalim and its (+)-enantiomer. Reperfusion contractile function was improved significantly and lactate dehydrogenase release was reduced by these compounds. Time to contracture was also increased significantly by the (-)-enantiomer (EC25 = 2.27 microM), cromakalim (EC25 = 4.89 microM) and the (+)-enantiomer (EC25 greater than 100 microM). We determined if cromakalim, in a concentration which does not depress cardiac function (10 microM), can preserve high energy phosphates during ischemia in isolated rat hearts. Cromakalim significantly preserved ATP at 15 to 25 min of ischemia. Adenylate energy charge was also significantly improved by cromakalim at 20 to 25 min into an ischemic episode. Thus, the cardioprotective effects of cromakalim are stereoselective and may be due partly to preservation of myocardial energy reserves. It is significant that cromakalim can preserve adenine nucleotides despite its lack of negative inotropic effects.

Dihydropyrimidine calcium channel blockers. 3. 3-Carbamoyl-4-aryl-1,2,3,4-tetrahydro-6-methyl-5-pyrimidinecarboxylic acid esters as orally effective antihypertensive agents.

In order to explain the potent antihypertensive activity of the modestly active (IC50 = 3.2 microM) dihydropyrimidine calcium channel blocker 5, we carried out drug metabolism studies in the rat and found 5 is metabolized to compounds 6-10. Two of the metabolites, 6 (IC50 = 16 nM) and 7 (IC50 = 12 nM), were found to be responsible for the antihypertensive activity of compound 5. Potential metabolism of 6 into 7 in vivo precluded our interest in pursuing compounds related to 6. Structure-activity studies aimed at identifying additional aryl-substituted analogues of 7 led to 17g,j,p with comparable potential in vivo, though these compounds were less potent than 7 in vitro. To investigate the effects of absolute stereochemistry on potency, we resolved 7 via diastereomeric ureas 19a,b, prepared from 18 by treatment with (R)-alpha-methylbenzylamine. Our results demonstrate that the active R-(-)-enantiomer 20a of 7 is both more potent and longer acting than nifedipine (1) as an antihypertensive agent in the SHR. The in vivo potency and duration of 20a is comparable to the long-acting dihydropyridine amlodipine. The superior oral antihypertensive activity of 20a compared to that of previously described carbamates 2 (R2 = COOEt) could be explained by its improved oral bioavailability, possibly resulting from increased stability of the urea functionality.