3,4-Dihydroquinolin-2(1H)-ones as combined inhibitors of thromboxane A2 synthase and cAMP phosphodiesterase.

A series of 1H-imidazol-1-yl- and 3-pyridyl-substituted 3,4-dihydroquinolin-2(1H)-ones was designed and synthesized as combined inhibitors of thromboxane (TXA2) synthase and cAMP phosphodiesterase (PDE) in human blood platelets. A number of structures, e.g. 4b, 7a, 7e, 13a, and 21-25, were superior to dazoxiben 26 as inhibitors of TXA2 synthase in in vitro ADP-induced aggregation experiments with human blood platelets. The TXA2 synthase inhibitory activity was confirmed by measurement of the prostanoid metabolites derived from 14C-labeled arachidonic acid. Three compounds (7a, 7e, and 25) demonstrated in vitro inhibition of human platelet cAMP PDE at micromolar concentrations in conjunction with their TXA2 synthase inhibitory activity. Synergistic enhancement of antiaggregatory and antithrombotic actions was expected when simultaneous stimulation of adenylate cyclase (through increased PGI2 production) and inhibition of platelet cAMP PDE were possible from the same compound. Ex vivo and in vivo experiments were conducted in rats and mice, respectively, to evaluate the effects of compounds 7e and 23 on platelet aggregation and thrombotic events within these animals. Compound 7e, which has a comparable level of TXA2 synthase (IC50 1.2 microM) and human platelet cAMP PDE (IC50 6.4 microM) inhibitory activities, was found to be orally bioavailable with a long duration of action and offered effective protection against mortality in a collagen-epinephrine-induced pulmonary thromboembolism model in mice. Significant blood pressure and heart rate effects were observed for several compounds, e.g. 7e, 9e, 13a, 13d, 18, 20, 21, and 23, when dosed orally in conscious spontaneously hypertensive rats.

Stimulation of prostaglandin D2 receptors on human platelets by analogs of prostacyclin.

RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.

Effects of enprostil on cardiovascular and respiratory parameters in the dog, and on hematologic parameters in the rat, monkey, and human.

The effects of enprostil (+/-)-7-[(1R*,2R*,3R*)-3-hydroxy-2-[(E)-(3R*)-3-hydroxy-4-phenoxy-1- butenyl]-5-oxocyclopentyl]-4,5-heptadienoate) were studied on cardiovascular and respiratory parameters in the dog and on hematologic parameters in the rat, monkey, and human. Anesthetized dogs were instrumented to allow measurement of heart rate, systemic blood pressure, respiratory rate or tracheal pressure, and ventricular contractile force after intragastric (i.g.) or intravenous (i.v.) administration of enprostil in the presence or absence of autonomic challenges. The effects of intraduodenal (i.d.) enprostil on arterial and venous PO2, PCO2, and pH; respiratory rate, flow rate, and volume; blood pressure (b.p.); and heart rate were also examined. Enprostil i.v. (0.3-10 micrograms/kg) significantly increased tracheal pressure in a dose-dependent manner, but minimally altered b.p., heart rate, and ventricular contractile force. Enprostil i.v. (1-10 micrograms/kg) significantly inhibited pressor and depressor responses to several autonomic challenge agents at the highest dose level, indicative of a nonspecific inhibitory effect on b.p. responses. Cardiovascular effects of enprostil (1-100 micrograms/kg i.g.) were absent. Enprostil (10-3,000 micrograms/kg i.d.) had no noteworthy effects on respiratory parameters in the dog. Platelet aggregation effects of enprostil were studied in vitro using platelet rich plasma (PRP) from the rat, monkey, and human; whole blood clotting time and prothrombin time after oral enprostil were studied in the rat; and ex vivo effects on platelet activation were studied in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of cyclic AMP phosphodiesterase. 3. Synthesis and biological evaluation of pyrido and imidazolyl analogues of 1,2,3,5-tetrahydro-2-oxoimidazo[2,1-b]quinazoline.

Hybridization of structural elements of 1,2,3,5-tetrahydro-2-oxoimidazo[2,1-b]quinazoline ring system common to the cyclic AMP (cAMP) phosphodiesterase (PDE) inhibitors lixazinone (RS-82856, 1) and anagrelide (3) with complementary features of other PDE inhibitor cardiotonic agents prompted the design and synthesis of the title compounds 7a-d, 11, 12, and 13a,b. The necessary features of these compounds were determined within the framework of the proposed active-site models for the high affinity form of cAMP PDE inhibited by cGMP (type IV). Evaluation of these targets, both in vitro as inhibitors of platelet or cardiac type IV PDE or in vivo as inotropic agents in the pentobarbital-anesthetized dog model of congestive heart failure, showed that these structure possessed negligibly enhanced activities over the parent heterocyclic system, and remained significantly inferior to 1 in all respects. This difference is ascribed to the absence of the N-cyclohexyl-N-methylbutyramidyl-4-oxy side chain of 1. The proposal that the acidic lactam-type functionality, common to the type IV PDE inhibitor inotropic agents such as 4-6 and 8-10, mimics the polarizable cyclic phosphate moiety of cAMP suggested that the side chain of 1 may function as an effective surrogate for selected characteristics of the adenine portion of cAMP. However, the results of this study show that incorporation of adenine-like hydrogen-bonding functionalities common to other type IV PDE inhibitors into the 1,2,3,5-tetrahydro-2-oxoimidazo[2,1-b]quinazoline system did not enhance activity to the levels observed for 1 and analogues. These observations, coupled with the kinetic pattern of inhibition of type IV PDE observed for 1 and analogues, suggest that access to a secondary, lipophilic-tolerant binding site, possibly coincident with the adenine binding domain, and adjacent to the catalytic ribose-phosphate binding site of platelet and cardiac type IV PDE, is responsible for the increased potency of these compounds.

Inhibitors of cyclic AMP phosphodiesterase. 4. Synthesis and evaluation of potential prodrugs of lixazinone (N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro-2- oxoimidazo[2,1-b]quinazolin-7-yl)-oxy]butyramide, RS-82856).

The cyclic AMP phosphodiesterase (cAMP PDE) inhibitor and cardiotonic agent lixazinone (N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro-2- oxoimidazo[2,1-b]quinazolin-7-yl)oxy]butyramide, RS-82856, 1) and its acid and base addition salts were found to be insufficiently soluble in formulations suitable for intravenous administration. These results prompted an investigation into potential prodrugs with enhanced aqueous solubility designed to deliver 1 by three distinct mechanisms: (1) decarboxylation of alpha-carboxamides; (2) hydrolytic loss of a solubilizing N-1-(acyloxy)methyl or (N,N-dialkylamino)methyl moiety; or (3) intramolecular closure of a guanidino ester or amide. The target compounds were evaluated as delivery systems for 1 by three criteria: (1) chemical conversion rate to 1 under physiological conditions; (2) inhibition of type IV cAMP PDE at a fixed time point; and (3) in vivo inotropic activity in anesthetized dogs by both intravenous and oral administration. Release of 1 from 4a (series 1) was found to be too slow to be of value as a prodrug of 1, since decarboxylation could be induced only by strong acid, conditions under which hydrolytic ring opening was found to severely compete. Conversely, 1 was released too readily on exposure of (N,N-dialkylamino)methyl derivatives such as 8d (series 2) to physiological conditions, although no large increase in aqueous solubility was realized. Finally, both the physicochemical and in vitro studies indicated that ring closure of the guanidinium esters and amides 17a-k (series 3) to 1 was quantitative and pH- and time-dependent, suggesting the possibility of delivery of the open, water-soluble prodrug form, followed by closure to 1 in plasma. Detailed examination of these agents in vivo, however, demonstrated that only those compounds that rapidly cyclized to 1, as measured by plasma levels of 1, exhibited inotropic activity, indicating that the open prodrug form was not efficiently absorbed upon oral administration.

Inhibitors of cyclic AMP phosphodiesterase. 1. Analogues of cilostamide and anagrelide.

Evaluation of a series of lactam heterocyclic analogues of cilostamide (2) as inhibitors of cyclic AMP phosphodiesterase derived from both human platelets and rat heart in comparison with their corresponding methoxy-substituted heterocycles has revealed that the N-cyclohexyl-N-methyl-4-oxybutyramide side chain of 2 is an important lipophilic and/or steric pharmacophore. Attachment of this side chain to the parent heterocycle of the potent cyclic AMP phosphodiesterase inhibitor anagrelide (3) afforded the hybrid structure RS-82856 (1), shown to be more potent than either of its progenitors as an inhibitor of cyclic AMP phosphodiesterase or of ADP-induced platelet aggregation. The available in vitro data suggest that 1 possesses potentially useful antithrombotic and cardiotonic properties.

Inhibitors of cyclic AMP phosphodiesterase. 2. Structural variations of N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro- 2-oxoimidazo[2,1-b]quinazolin-7-yl)-oxy]butyramide (RS-82856).

A series of analogues of the cyclic AMP phosphodiesterase (PDE) inhibitor N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro- 2-oxoimidazo[2,1-b]quinazolin-7-yl)oxy]butyramide (RS-82856, 1) was prepared by systematic variation of the side-chain substituent, length, position, connecting atom, and the parent heterocycle itself. The compounds were evaluated as inhibitors of cyclic AMP phosphodiesterase from both human platelets and rat or dog heart tissue and as inhibitors of ADP-induced platelet aggregation. Structure-activity correlations for the analogue series revealed significant limitations on the steric bulk of substituents on the 1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one heterocycle and the position and length of the side chain. As inhibitors of cyclic AMP phosphodiesterase (PDE), potency steadily increased with increasingly lipophilic side chains. In platelet aggregation inhibition studies, however, a maximum in activity was reached with 1, while more lipophilic compounds were significantly less active. Major changes in the heterocycle itself, represented by isomeric and other carbonyl variations, also decreased activity. The molecular features defined by this series of analogues of 1 correlate to a high degree with current understanding of the chemical and topographical requirements of the active site of the FIII (type IV) form of cyclic AMP PDE. Selective inhibition of this enzyme has been proposed as the principal component of the positive inotropic action of a number of cardiotonic agents.

Requirements for triggering of adipocyte differentiation by glucocorticoids and indomethacin.

The stable adipogenic cell line TA1, spontaneously differentiates into mature adipocytes after several days at confluence. Glucocorticoids (e.g. dexamethasone) accelerate the onset of differentiation by precociously activating the transcription of genes that are expressed in the mature adipocyte but not in the preadipocyte. Thus the hormone may induce a critical regulatory factor required for activating the entire set of differentiation-dependent genes. We have found that the nonsteroidal antiinflammatory drug indomethacin also stimulates differentiation of TA1 cells but even more rapidly and completely than does dexamethasone. Contrary to previous suggestions we find that this activity of indomethacin's cannot be ascribed to inhibition of cyclo-oxygenase, the critical enzyme in prostaglandin biosynthesis. Finally, indomethacin's ability to stimulate TA1 cell differentiation synchronously and rapidly has allowed us to document that cell confluence is required for efficient differentiation and that the drug needs only to trigger rather than maintain the differentiation process.

A potent and selective inhibitor of cyclic AMP phosphodiesterase with potential cardiotonic and antithrombotic properties.

Some biochemical and pharmacological properties of a novel, potent inhibitor of cyclic AMP phosphodiesterase, N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimidazo[2,1-b] quinazolin-2-one) butyramide (RS-82856), were investigated. RS-82856 selectively inhibits the high affinity form of cyclic AMP phosphodiesterase (type IV) isolated from human platelets (Ki = 0.5 nM) with only weak effects on both the nonspecific and cyclic GMP-sensitive phosphodiesterases. The inhibitor reduces both maximum velocity and substrate affinity of the type IV enzyme. This mixed pattern of partial competitive and noncompetitive inhibition was also obtained with one of the two high affinity forms of phosphodiesterase found in dog heart (Ki = 0.75 nM). Of several human and dog tissues examined, RS-82856 exhibits marked selectively for the platelet high affinity enzyme. It also has significant inhibitory effects on cardiac membrane-bound phosphodiesterase. RS-82856 inhibits the aggregation of human platelets in response to adenosine 5'-diphosphate (IC50 = 0.11 microM) in vitro and is active ex vivo for at least 2 hr following oral administration (10 mg/kg) to rhesus monkeys. Administration of RS-82856 to instrumented, anesthetized dogs by either intravenous or intraduodenal routes increases cardiac contractile force and reduces afterload. These data suggest that RS-82856 may be useful as an agent to increase cardiac output in the treatment of congestive heart failure.

Synthesis and platelet aggregation inhibition activity of a series of enantiomeric bicyclo[3.2.0]heptane-6-oximinoacetic acids (1).

Opening of racemic epoxide (3) with (3S)- or (3R)-dimethyl-3-(dimethyl-t-butylsilyloxy)oct-1-ynyl aluminum gave two regioisomers, which were separated chromatographically. The separated regioisomers, themselves mixtures of chromatographically inseparable diastereoisomers, were converted into their dicobalthexacarbonyl complexes, which were easily resolved and isolated by chromatography. The individual diastereoisomers were deprotected to give bicyclo[3.2.0]heptan-3-ones, whose absolute stereochemistry was assigned using circular dichroism. One of these compounds, (1R,2R,3S,5R,3'S)-3-(3'-hydroxyoct-1'-ynyl)-bicyclo[3.2.0]++ +heptan-2-ol-6- oximinoacetic acid (11a) was 4.5 times more potent than PGE1 in inhibiting the ADP-induced aggregation of human platelets. The next most potent compound in this series was the "ent-15-epi" compound (11b), which was 0.034 times the potency of PGE1 in the platelet aggregation assay.

Activation of cardiac adenylate cyclase: horminal modification of the magnesium ion requirement.

Histamine and epinephrine stimulate the activity of guinea pig heart adenylate cyclase [ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1], in part, by decreasing the requirement for Mg2+ as an activator. This effect may represent an increase in affinity for Mg2+ and/or a decrease in sensitivity of the enzyme towards inhibition by free ATP. Both of these inotropic hormones also increase maximum velocity. Pretreatment of the membrane-bound enzyme with EDTA, to remove available divalent cations, almost eliminates persistent stimulation by guanyl-5'-yl imidodiphosphate [Gpp(NH)p]. Addition of Mg2+ to the preincubation medium restores the capacity of Gpp(NH)p to acutely activate the enzyme. These results indicate that Mg2+ interacts with the nucleotide (GTP) regulatory site. Persistent stimulation of the enzyme by either Gpp(NH)p or fluoride ion also involves a decrease in the requirement for Mg2+ and an increase in maximum velocity.