5-[6-1 -(Cyclohexyl-1 H-tetrazol-5-YL)hexyl]-1,8-naphthyridin-2-(1H)-one, SC-44368, a Potent Anti-aggregatory Agent which Selectively Inhibits Platelet Cyclic AMP Phosphodiesterase.

SC-44368 (5-[6-(1-cyclohexyl-1H-tetrazol-5-y)hexyl]-1,8-naphthyridin-2(1H)-one) is a potent and selective competitive inhibitor of platelet cyclic AMP-dependent phosphodiesterase (cAMP-PDE) (Ki: 1.65 µM). For the phosphodiesterase isoenzyms from human platelets SC-44368 shows a 26-fold selectivity (IC50 ratio) for the inhibition of the cAMP-PDE over the cyclic GMP-dependent phosphodiesterase (cGMP-PDE). By comparison, 3-isobutyl-1-methyl-xanthine (IBMX) inhibited the cAMP-PDE and cGMP-PDE from human platelets with approximately equal efficacy. Broad inhibitory activity was evident against human platelet aggregatory responses in vitro. IC50 values of 18.1 ± 5.3 µM (25 nM platelet activating factor, PAF), 17.3 ± 3.0 µM (1.0 µg/ml collagen) and 24.2 ± 10.3 µM (1µM ADP) were obtained against maximum increases in platelet-rich plasma (PRP) light transmission achieved by each agonist. SC-44368 potentiated the prostacyclin-induced increase of intra-platelet cAMP levels but did not potentiate the sodium nitroprusside-induced increase of intraplatelet cGMP levels. In an ex vivo model of platelet aggregation SC-44368 (3 mg/kg, i.v.) produced a potent inhibition of collagen-induced platelet aggregation. SC-44368 produced only weak hypotensive activity in the rat. Thus, SC-44368 is a novel cAMP-PDE inhibitor which possesses potent, broad spectrum anti-aggregatory properties.

A structurally novel inhibitor of cGMP phosphodiesterase with vasodilator activity.

A novel, potent, competitive inhibitor of smooth muscle cGMP phosphodiesterase is described (Compound I, [4-[2-n-butyl-5-chloro-1-(2- chlorobenzyl)imidazolyl]methyl] acetate). The compound is highly selective for inhibiting cGMP phosphodiesterase compared with cAMP phosphodiesterase. Compound I inhibits the contraction of smooth muscle in response to a variety of agonists in the same concentration range to that which inhibits the enzyme. Compound I produced a dose-related reduction in the pressor responses to angiotensin II infusion while not inhibiting the responses to bolus doses of angiotensin II. Two structural analogues of Compound I which did not inhibit cGMP phosphodiesterase failed to inhibit smooth muscle contraction in vitro and did not affect angiotensin II pressor responses in vivo. We propose a mechanism to account for the effects of a cGMP phosphodiesterase inhibitor on smooth muscle contraction in vitro and in vivo.

Effect of SC 38249, a novel substituted imidazole, on platelet aggregation in vitro and in vivo.

SC 38249 [RS)-1-(2,3-bis-[(4-methoxyphenyl)methoxy]propyl)-1H-imidazole) caused dose-related inhibition of collagen-induced thromboxane A2 formation in human platelet rich plasma (IC50: 9.9 +/- 1.0 microM) accompanied by a dose-dependent increase in plasma PGE2. Broad inhibitory activity was evident against human platelet aggregatory and secretory responses in vitro. IC50 values of 11.9 +/- 1.9 microM (0.64 mM arachidonic acid), 18.3 +/- 3.8 microM (0.5 microgram ml-1 collagen) and 37.6 +/- 6.1 microM (25 nM Paf-acether) were obtained against maximum increase in PRP light transmission achieved by each agonist. Although less potent, SC 38249 retained significant inhibitory activity against PRP responses induced by a higher (3.0 micrograms ml-1) concentration of collagen (IC50: 272.5 +/- 24.6 microM), and against Paf-acether-induced responses in PRP pre-treated with 10 microM indomethacin (I.C.50: 192.0 +/- 16.1 microM). Experimental animal studies confirmed the in vitro anti-aggregatory efficacy of SC 38249, since significant inhibitory activity was observed against Paf-acether and ADP-induced responses in dog PRP ex vivo, anti-Forssman antibody-induced thrombocytopenia in anaesthetized guinea pigs, and collagen-induced intravascular aggregation in anaesthetized rabbits. Thus, SC 38249 is a novel thromboxane synthase inhibitor which possesses interesting anti-aggregatory properties which cannot wholly be attributed to prevention of platelet thromboxane A2 formation.

Thromboxane synthase inhibitors. Synthesis and pharmacological activity of (R)-, (S)-, and (+/-)-2,2-dimethyl-6-[2-(1H-imidazol-1-yl)-1-[[(4-methoxyphenyl)- methoxy]methyl]ethoxy]hexanoic acids.

A series of substituted omega-[2-(1H-imidazol-1-yl)ethoxy]alkanoic acid derivatives were synthesized and evaluated for their ability to inhibit thromboxane synthase both in vitro and in vivo. Compound 13 was identified as a potent and selective competitive inhibitor of human platelet thromboxane synthase having a Ki value of 9.6 X 10(-8) M. In collagen-treated human whole blood, 13 potentiated levels of 6-keto PGF1 alpha. Enantiospecific syntheses afforded the R and S enantiomers of 13, of which the S enantiomer 13b was the more potent. Compounds 13 and 13b were potent in vivo inhibitors of thromboxane synthase with good oral activity and duration of action.

Dibenzoquinazoline diones as antihypertensive cyclic guanosine monophosphate phosphodiesterase inhibitors.

The discovery and structure-activity of a new class of renal artery phosphodiesterase inhibitors is reported, some of which are highly selective for the guanosine cyclic 3',5'-monophosphate phosphodiesterase. One of these compounds, 5,6-dihydro-8,9,11,12-tetramethoxy-1,3-dioxo-1H-benz[f]- isoquino [8,1,2- hij]quinazoline-2(3H)-carboxylic acid, ethyl ester (9), is amongst the most potent and selective compounds of this class yet identified. Furthermore, this compound demonstrates an antihypertensive effect in vivo which is presumably mediated through vascular smooth muscle relaxation.

Structure-activity relationships in an imidazole-based series of thromboxane synthase inhibitors.

Analogues of 4-[[2-(1H-imidazol-1-yl)-1-[[(4-methoxyphenyl)methoxy]methyl] ethoxy]methyl]benzoic acid (5m) were prepared and evaluated as thromboxane synthase inhibitors. A series of esters of 5m showed a parabolic relationship between lipophilicity and inhibition of TxB2 generation in intact platelets, with activities up to 50 times greater than that of dazoxiben. However, on administration to rabbits the ethyl ester 5d had a short duration of action, due to rapid metabolism and excretion via deesterification and beta-glucuronidation. Attempts at replacing the carboxylate group with other potential pharmacophores were unsuccessful.

The effect of thromboxane A2 synthesis inhibitors on platelet aggregation in whole blood.

Aggregatory responses to arachidonic acid and collagen in vitro were compared in blood (single platelet counting) and PRP (light aggregometry) from four species. Sensitivity of mouse, rat and rabbit PRP to these agonists was not predictive of respective potency in whole blood, whereas for human platelets, responses in blood did not differ significantly from PRP. Indomethacin (3-300 microM) inhibited arachidonic acid-induced aggregation in each species, and collagen responses in all except mouse. In contrast, the thromboxane synthase inhibitors dazoxiben (3.7-372 microM) and SC 38249 (2.6-260 microM) demonstrated activity only in rabbit and human blood. However, since in many experiments drug efficacy decreased significantly in blood compared to corresponding PRP, the concentrations of each agent necessary to inhibit responses were above those at which selectivity has previously been demonstrated against isolated enzyme preparations or in PRP. A fundamental reappraisal of both the potency and selectivity of these inhibitors in whole blood appears essential before their mechanism of action can be firmly established.

Effect of calcium and calcium antagonists on [3H]-Paf-acether binding to washed human platelets.

[3H]-Platelet activating factor (Paf-acether, 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine) binds to washed human platelets in a specific, dose-dependent, and saturable manner. Scatchard analysis reveals a high affinity site with a KD value of 0.25 +/- 0.033 nM (245 +/- 30 sites per platelet), and a second low affinity site with a KD value of 9.22 +/- 1.17 nM (1616 +/- 165 sites per platelet). Binding to the high affinity site is independent of buffer calcium concentration, inhibited on an equimolar basis by unlabelled 1-O-octadecyl-Paf-acether, but remains unchanged in the presence of 1-O-octadecyl-lyso-Paf-acether. The relative inhibitory effect of four calcium antagonists on [3H]-Paf-acether high affinity binding correlates closely with their respective anti-aggregatory activity against Paf-acether induced responses in human PRP; order of potency being (+)-cis diltiazem greater than (+/-)-verapamil greater than (-)-cis diltiazem greater than nifedipine. In the case of (+)-cis diltiazem, the effect is competitive, stereo-specific and progressively reversed by addition of calcium (1.0 mM and 5.0 mM). A close spatial relationship may thus exist between the Paf-acether receptor and membrane calcium channels in the human platelet.

Specific binding of [3H]-1-O-octadecyl Paf-acether to washed human platelets.

[3H]-Paf-acether binds to washed human platelets in a dose-dependent manner. Scatchard analysis reveals two distinct binding sites; a high affinity site with a KD value of 0.259 +/- 0.33 nM (245 +/- 30 sites per platelet) and a lower affinity site with a KD value of 9.22 +/- 1.17 nM (1616 +/- 165 sites per platelet). Association of 3H-Paf-acether to the high affinity receptor is rapid, being maximal within two minutes and remaining constant for at least twenty minutes. Dissociation from the low affinity receptor is also rapid (t1/2: less than 10s) whereas dissociation from the high affinity site is significantly slower (t1/2 : approximately 70s). [3H]-Paf-acether binding is inhibited by unlabelled (R)-C16-Paf (IC50: 0.08 +/- 0.01 nM) greater than (R)-C18-Paf (0.48 +/- 0.03 nM) greater than (RS)-C18-Paf (1.06 +/- 0.19 nM), but remains unchanged in the presence of lyso-C18-Paf at 3.0-300 nM. [3H]-Paf-acether binding and its inhibition by unlabelled (R)-C18-Paf-acether is independent of buffer Ca2+ within the range 0-5.0 mM.