Discovery and initial structure-activity relationships of trisubstituted ureas as thrombin receptor (PAR-1) antagonists.

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.

Non-peptide GPIIb/IIIa inhibitors. 20. Centrally constrained thienothiophene alpha-sulfonamides are potent, long acting in vivo inhibitors of platelet aggregation.

The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon = 3.3 x 10(6) M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 microg/kg [corrected], and an oral dose of 50-90 microg/kg [corrected] followed by low daily doses of 10 microg/kg [corrected] was sufficient to maintain approximately 80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 +/- 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.

Identification of low molecular weight GP IIb/IIIa antagonists that bind preferentially to activated platelets.

A critical function of fibrinogen in hemostasis and thrombosis is to mediate platelet aggregation by binding selectively to an activated form of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpeptide fibrinogen receptor antagonists have been described, their binding selectivity for resting and activated platelets has not been explored. Therefore, dissociation constants of GP IIb/IIIa antagonists for two biochemically separated forms of purified GP IIb/IIIa and for resting and activated platelets were determined by competitive displacement of the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium silicate scintillation proximity assay fluomicrospheres produced an activated form of the receptor, whose binding affinity for GP IIb/IIIa antagonists was measured conveniently by competition with the arginine-glycine-aspartic acid (RGD) containing heptapeptide [125I]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa antagonists also were performed on resting and activated platelets. We identified two classes of compounds. One class binds to both forms of GP IIb/IIIa, as well as resting and activated platelets, with similar Kd values (e.g., L-736,622 and Echistatin). The other class of compounds binds with much higher affinity to the activated form of GP IIb/IIIa (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, like L-734,217 (KdActivated = 5 nM and KdResting = 620 nM), can effectively inhibit ex vivo platelet aggregation at concentrations of drug that produce low levels of occupancy of the circulating platelet receptors. The potential clinical advantages of selective versus nonselective GP IIb/IIIa antagonists remain to be explored in clinical trials.

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.

The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.

Identification and characterization of endothelin converting activity from EAHY 926 cells: evidence for the physiologically relevant human enzyme.

A neutral proteolytic activity that converts human Big endothelin-1 (Big Et-1) to endothelin-1 has been identified from a human endothelial hybrid cell line, EAHY 926. This enzyme is an integral membrane protein and cofractionates with other enzymes typically found in the plasma membrane. The activity has been solubilized with nonionic detergents and purified 1000-fold by a combination of lectin affinity chromatography, ion-exchange chromatography, and chromatography on red-dye agarose. The partially purified activity is a metalloenzyme based upon its sensitivity to chelating agents, competitive inhibition by phosphoramidon, and reconstitution with ZnCl2 or CoCl2 following EDTA inactivation. The enzyme appears to be unique, however, as it is not inhibited by specific inhibitors of known metalloproteases. It correctly processes Big Et-1 to Et-1 and the complementary C-terminal fragment with a sharp pH optimum near 7.0. Both the Km for Big Et-1 and the Ki for phosphoramidon are pH-dependent, with values of 5-7 and 3.5 microM, respectively, at pH 7.0. The enzyme also cleaves Big Et-2 with a Km of 27.9 microM and a Vmax one-third that for Big Et-1 but has no appreciable activity toward Big Et-3. An s20,w of 9.5 S was determined by sucrose density ultracentrifugation in H2O and D2O. When combined with a Stokes radius of 56 A determined by gel filtration, the enzyme had a calculated apparent molecular weight of 250,000. Conditions have been established to renature the activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions activity was detected in a protein band at 280 kDa, in agreement with the aforementioned molecular weight determination. From these results, a kcat/Km of 1 x 10(6) M-1 s-1 was estimated for the purified enzyme with Big Et-1 as a substrate, which is a reasonable value for a protease acting upon its physiologic substrate. Several criteria indicate that the activity isolated from EAHY cells is the physiologically relevant endothelial-derived endothelin converting enzyme. On the basis of our results, this enzyme is present in low abundance in endothelial cells and at least a 100,000-fold purification will be required to obtain a homogeneous preparation. However, because EAHY cells can be grown in large numbers, they can supply the quantities of enzyme required both for biochemical studies and for the development of specific inhibitors.

Pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity.

Derivatives of pyridinones were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevent the spread of HIV-1 infection in cell culture without an appreciable effect on other retroviral or cellular polymerases. 3-[( (4,7-Dimethyl-1,3-benzoxazol-2-yl) methyl]amino ]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,639) and 3-[[ (4,7-dichloro-1,3-benzoxazol-2-yl) methyl]amino]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,661), two compounds within this series, had HIV-1 RT IC50 values in the range of 20-800 nM, depending upon the template-primer used. The most potent inhibition was obtained with rC.dG and dA.dT as template--primers. With rC.dG, reversible slow-binding non-competitive inhibition was observed. [3H]L-697,639 bound preferentially to enzyme-template-primer complexes. This binding was magnesium-dependent and saturable with a stoichiometry of 1 mol of [3H]L-697,639 per mol of RT heterodimer. Displacement of [3H]L-697,639 was seen with phosphonoformate. In human T-lymphoid-cell culture, L-697,639 and L-697,661 inhibited the spread of HIV-1 infection by at least 95% at concentrations of 12-200 nM. Synergism between 3'-azido-3'-deoxythymidine or dideoxyinosine and either of these compounds was also demonstrated in cell culture. Based upon their specificity for HIV-1 RT activity, template-primer dependence on potency and ability to displace [3H]L-697,639; a tetrahydroimidazo [4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione derivative R82150 and the dipyridodiazepinone BI-RG-587 appear to inhibit RT activity by the same mechanism as the pyridinones.

Comparative effects of verapamil, diltiazem and felodipine during experimental digitalis-induced arrhythmias.

We compared the abilities of three different calcium (Ca2+) entry blockers, verapamil, diltiazem and felodipine to abolish ouabain-induced ventricular ectopy (100 X ectopic/total beats, VE) in anesthetized, closed-chest dogs. Ventricular tachycardia (VT) was produced in anesthetized, bilaterally vagotomized, closed-chest dogs by an average dose of 65 +/- 19 micrograms/kg ouabain. 30 min after establishing VT, either verapamil (25-50 micrograms/kg + 5-10 micrograms/kg/min), diltiazem (50-100 micrograms/kg + 20-50 micrograms/kg/min), felodipine (3 micrograms/kg + 0.3 micrograms/kg/min) or saline was administered for another 30 min. Verapamil, at the higher dose utilized, practically abolished ouabain-induced VT (97 +/- 3 to 8 +/- 19% VE); diltiazem was moderately effective (96 +/- 4 to 50 +/- 8% ectopy) at 100 micrograms/kg, and felodipine exerted no antiarrhythmic effects in this model. All three Ca2+ entry blockers lowered mean aortic pressure, felodipine lowering this parameter most prominently. Thus, these structurally and electrophysiologically dissimilar Ca2+ entry blockers differed in their abilities to abolish the digitalis glycoside-induced arrhythmias in vivo. The superiority of verapamil may be related to its multiple, additional electrophysiologic effects.

3-Hydroxy-alpha-methyltyrosine progenitors: synthesis and evaluation of some (2-oxo-1,3-dioxol-4-yl)methyl esters.

The (5-methyl-2-oxo-1,3- dioxol -4-yl)methyl and (5-tert-butyl-2-oxo-1, 3- dioxol -4-yl)methyl esters of 3-hydroxy-alpha-methyltyrosine (methyldopa) were prepared and evaluated as progenitors of the amino acid. 1H NMR experiments reveal that the esters are converted cleanly to methyldopa and the corresponding alpha-diketone at pH 7.4, with the 5-methyl derivative undergoing hydrolysis faster than the 5-tert-butyl analogue. Bioavailability studies in dogs show that the esters, particularly the 5-methyl derivative, yield significant plasma levels of methyldopa. Both esters are orally effective antihypertensive agents in spontaneously hypertensive (SH) rats. These studies indicate that (2-oxo-1,3- dioxol -4-yl)methyl esters are viable prodrugs for the latentiation of methyldopa.

Comparative effects of enalapril, enalaprilic acid and captopril in blocking angiotensin I-induced pressor and dipsogenic responses in spontaneously hypertensive rats.

The role of central angiotensin converting enzyme (ACE), in the maintenance of high blood pressure, was examined in unanesthetized spontaneously hypertensive rats (SHR). Pressor and dipsogenic responses induced by intracerebroventricular (ICV) injections of angiotensin I (AI) were elicited before and 30 min after either captopril (120-800 nanomoles ICV), enalapril (66-460 nanomoles ICV) and enalaprilic acid (70-280 nanomoles ICV). Enalapril was 1.6 (0.7-3.9) and 1.7 (0.9-2.9) times more potent than captopril in inhibiting AI-induced pressor and dipsogenic responses, respectively. Enalaprilic acid was 2.7 (1.1-7.1) and 2.9 (1.9-4.8) times more potent than captopril in inhibiting AI- (ICV administration) induced pressor and dipsogenic responses, respectively. None of the ACE inhibitors, in contrast, reduced the central actions of AII. Basal mean arterial pressure was not reduced by these ACE inhibitors after ICV administration. Administered orally at doses which produced similar hypotensive responses, neither captopril (30 mg/kg) nor enalapril (3 mg/kg) blocked the responses induced by AI given ICV (10 ng). These findings indicate that ACE inhibitors given acutely do not penetrate into the central nervous system sufficiently to block the dipsogenic and pressor responses induced by AI given ICV, and suggest that inhibition of central ACE may not be important to the acute antihypertensive activity of the ACE inhibitors tested.

Mechanism of action of enalapril in experimental hypertension and acute left ventricular failure.

The mechanism of action of angiotensin converting enzyme (ACE) inhibitors to lower blood pressure remains unclear, but the weight of available data favour peripheral blockade of the formation of angiotensin II (AII). Previous work in rats has shown that the prodrug ACE inhibitor, enalapril (MK-421), lowered blood pressure most effectively when PRA was elevated [sodium deficiency, two-kidney, one figure 8 hypertension, diuretic-treated spontaneously hypertensive rats (SHR)]. In sodium-deficient rats, the enalapril-sensitive component of the blood pressure was greatly reduced after salt loading, and nephrectomy blocked the antihypertensive response to enalapril in SHR. In the present study, further support that the mechanism of action of enalapril involves a reduction in AII has been obtained from rats made hypertensive by continuous intravenous (i.v.) AII infusion for 10 days. Enalapril administered for seven days did not significantly lower blood pressure, suggesting that there were no important non-angiotensin mechanisms (such as bradykinin potentiation) involved in its action. From earlier studies in SHR, the time course for blockade of angiotensin I (AI) pressor responses and the blood pressure reduction did not correspond, suggesting a tissue site of action. In the present studies in adult SHR, a central site of action was ruled out since the parent inhibitor, enalaprilic acid (MK-422), injected into the brain ventricles did not acutely reduce blood pressure. An interaction of enalaprilic acid with the sympathetic nervous system was evaluated in dogs in which adrenergic activity was enhanced as a result of diuretic-induced renin release. Enlaprilic acid did not alter the enhanced hindquarter vasoconstrictor responses to sympathetic nerve stimulation. Enalapril increased renal blood flow, glomerular filtration rate and sodium excretion. The mechanism of the natriuresis in dogs probably involves several mechanisms including a decrease in aldosterone biosynthesis, changes in renal function (glomerular filtration rate and renal blood flow) and possibly blockade of a direct tubular effect of AII on sodium reabsorption. Enalaprlic acid was also studied in a closed chest dog model of acute left ventricular (LV) failure caused by embolization via the left main coronary artery with 50 microns plastic microspheres. Enalaprilic acid at 100 micrograms/kg i.v. reduced preload, afterload and improved LV performance without changing the heart rate. In conclusion, enalapril the prodrug, and enalaprilic acid the active inhibitor, are potentially useful in the treatment of hypertension and LV failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between angiotensin I blockade and antihypertensive properties of single doses of MK-421 and captopril in spontaneous and renal hypertensive rats.

The exact mechanism of action of angiotensin converting enzyme (ACE) inhibitors in reducing blood pressure is not known, although inhibition of angiotensin II formation is the generally accepted mechanism. Experiments were performed in two models of experimental hypertension to determine whether or not inhibition of the pressor response to angiotensin I, 300 ng/kg i.v., would correlate with the antihypertensive response to single oral doses of N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]-L-Ala-L-Pro (MK-421), a new ACE inhibitor. Captopril, given as a single oral dose, was studied in spontaneously hypertensive rats (SHR) for comparative purposes. In SHR, MK-421 at 0.1-3 mg/kg p.o. and captopril at 0.1-3 mg/kg p.o. were approximately equipotent with regard to inhibiting the pressor response to angiotensin I (relative potency=1.7; 95% C.I.=0.7-4.5). The magnitude of ACE inhibition and onset of action were similar with both agents, but MK-421 had a longer duration of action. The decrement in systolic pressure following each ACE inhibitor consisted of an initial decrease in blood pressure corresponding to the maximal inhibition of angiotensin I pressor response and a secondary fall in blood pressure which was evident 5-6 h after treatment. At this time, the inhibition of the pressor response to angiotensin I was minimal. Thus, the time course for blockade of angiotensin I and the blood pressure reduction did not correspond. The dose-response regression lines for the antihypertensive effect of each inhibitor, unlike those for ACE inhibition, were flat. The potency ratio computed on the basis of the maximum fall in blood pressure over 6 h revealed that MK-421 was 11.5 times (P less than 0.05) more potent thant captopril. In 2-kidney Grollman renal hypertensive rats (RHR), MK-421 at 0.3-10 mg/kg p.o. inhibited the pressor response to angiotensin I by 65-95%, but produced significant decrements in blood pressure only at 10 mg/kg p.o. The finding that MK-421 was more potent than captopril in lowering blood pressure in SHR, yet equally active in its ability to block angiotensin I pressor responses, suggests that a mechanism(s) other than inhibition of plasma ACE is involved in the decrease in blood pressure was not reduced. However, a higher dose which produced a similar degree of blockade was associated with a significant decrease in blood pressure.

Beta adrenoceptor blocking properties of MK-761.

MK-761 is a new class of compound which has beta adrenoceptor antagonist and vasodilating properties in a single molecule. The compound has in vitro beta adrenoceptor blocking activity in the isolated cat heart papillary muscle and isolated rat atria. Unlike propranolol, it did not depress contactile force. Intrinsic sympathomimetic activity was not observed in vitro, (isolated cat papillary muscle, isolated atria) but the compound has some intrinsic sympathomimetric activity because it slightly increased heart rate in reserpinized rats. MK-761 was found to be approximately as potent as timolol and pindolol in blocking cardiac and vascular beta adrenergic receptors in anesthetized dogs. The drug was not, however, cardioselective. Oral beta adrenoceptor blocking activity was observed in rats at doses similar to those which decreased blood pressure.

Antagonism of conditioned salivation in conscious dogs by antihypertensive drugs.

Methyldopate (methyldopa (ethyl ester)), carbidopa, clonidine, and ST-91 were evaluated for their effects on conditioned salivation in unanesthetized dogs. Clonidine produced dose-dependent inhibition of salivation 20 min after an intravenous injection. At equivalent and larger doses, ST-91, a clonidine analog which does not penetrate the blood-brain barrier, was ineffective in inhibiting conditioned salivation, suggesting that central rather than peripheral mechanisms are involved in clonidine-induced inhibition of salivation. Methyldopate also produced a dose-dependent inhibition of salivation in dogs. The mechanism involved in methyldopa-induced inhibition of salivation may involve both central and peripheral mechanisms because carbidopa, an inhibitor (like methyldopa) of peripheral aromatic decarboxylase (EC 4.1.1.28), significantly inhibited salivation.

Central antihypertensive effects of inhibitors of the renin-angiotensin system in rats.

The possibility that mean arterial pressure (MA) might be maintained by an effect of angiotensin II or its precursors on the central nervous system in rats made hypertensive by occluding the aorta between the renal arteries was investigated. Aortic coarctation produced severe hypertension (MAP greater than 150 mmHg) and plasma renin activity values (radioimmunoassay) at least 10 times normal within 2-6 days after surgery. [Sar1, IIe8]angiotensin II, an angiotensin II antagonist administered centrally via an intracerebroventricular (icv) injection (10-100 mug), lowered the MAP in a dose-dependent manner. Peripheral administration of [Sar1, IIe8]angiotensin II (bolus injection) at 100 mug intra-arterially was ineffective, but the antagonist did lower arterial pressure when infused intravenously for 1 h at 4 times this dose. Less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, a converting enzyme inhibitor, and pepstatin, a renin inhibitor, were ineffective via an icv injection. These results suggest that angiotensin II is in part responsible for the elevation in blood pressure following aortic coarctation in rats. Both central and peripheral administration of [Sar1, Ile8]-angiotensin II lowered mean arterial pressure but the antagonist lowered arterial pressure at lower doses and produced a more rapid decline in arterial pressure when administered into the central nervous system then when administered intra-arterially or intravenously.

Attenuation of hydrochlorothiazide-induced hypokalemia in dogs by a beta-adrenergic blocking drug, timolol.

Hydrochlorothiazide, administered at 1, 3 and 9 mg/kg/day p.o. for 4 days, produced a dose-dependent lowering of plasma potassium and an elevation in plasma renin activity in unanesthetized dogs. When plasma renin activity was suppressed in diuretic-treated dogs by the potent beta-adrenergic receptor-blocking drug, timolol (0.5, 2 mg/kg/day p.o. for 4 days), the hypokalemia and hyperreninemia were significantly less pronounced. The data suggest that beta-adrenergic blocking drugs can be used to antagonize these side effects of diuretics.