Effects of novel aggregation inhibitors on serotonin uptake by human blood platelets.

Novel aggregation inhibitors blocked serotonin uptake by human blood platelets in concentrations ranging from 0.7 +/- 0.1 microM to 237.5 +/- 35.7 microM; a modified procedure, validated by kinetic analysis, was employed in which pH drift was minimized to 0.03 during the active assay period. Structural features in carbamoylpiperidine and nipecotoylpiperazine derivatives which actually constitute molecular probes, and show remarkable specificity for aggregation-inhibitory target sites, disclosed striking differences between the latter and serotonin receptors or other loci affecting serotonin uptake.

Relationships between chemical structure and inhibition of epinephrine-induced human blood platelet aggregation.

The effect of structural features in a series of carbamoylpiperidine and nipecotoylpiperazine congeners on epinephrine-induced aggregation of human blood platelets is examined. Epinephrine-induced primary aggregation is effectively inhibited by the nipecotoylpiperazine derivatives (culminating at an IPA50 of 11.9 microM). While among nipecotoylpiperazine as well as carbamoylpiperidine congeners there are potent inhibitors of ADP-stimulated platelet function (cresting at an IA50 of 12.4 and 11.4 microM, respectively), the carbamoylpiperidine analogs are much less active (e.g., IPA50 of 298.1), or practically inactive, in impeding epinephrine-induced primary aggregation (PA).

Relationships between chemical structure and inhibition of ADP-stimulated human thrombocyte release of serotonin and platelet factor 4.

The inhibitory potencies of carbamoylpiperidinoalkane and N-alkylnipecotoylpiperazine derivatives on ADP-stimulated human blood platelet aggregation, serotonin (5-HT) release and platelet factor 4 (PF-4) release were evaluated. The procedure was designed to allow concurrent determination of all three sets of values. Most compounds were more than twice as potent in blocking PF-4 (X = 91 +/- 1 (S.E., n = 7)%) compared to their inhibition of 5-HT (X = 38 +/- 1(S.E., n = 6)%) release; the one compound which failed to meet these criteria was still decidedly more powerful in impeding PF-4 than 5-HT release. Since the compounds' platelet aggregation-inhibitory values were within the same range as their 5-HT release-blocking potencies, but had a strikingly greater impact in arresting PF-4 release, it is suggested that the platelet plasma membrane and the lining enveloping the dense bodies may share certain commonalities, while the sheathing encasing the alpha-granules may differ from both in a tangible manner.

Molecular determinants of the platelet aggregation inhibitory activity of carbamoylpiperidines.

A series of alpha,alpha'-bis[3-(N,N-dialkylcarbamoyl)piperidino]-p- xylenes were synthesized and tested for their inhibitory activity on ADP-induced aggregation of human platelets. A parabolic curve was obtained when log 1/C (activity) was plotted against log P (octanol/water partition coefficient). Using this as a model, a new analogue, alpha,alpha'-bis-[3-(N-methyl-N-butylcarbamoyl)piperidino]-p-xylen e (3g), was synthesized with a predicted IC50 of 25 microM. When this compound was subsequently evaluated, the IC50 was 22.1 +/- 5.5 microM, demonstrating the applicability of this model. The amide oxygen of the carbamoyl substituent appeared necessary for activity. Thus, for example, when the amide carbonyl group of 3a (IC50 = 44.5 microM) was reduced to CH2, the resulting compound 4 had a dramatically reduced activity, IC50 = 1565 microM. Compound 3a was resolved into (+) and (-) enantiomers and a meso (0) diastereomer using fractional crystallization, diastereomeric tartrate formation, and chiral HPLC. Compared to (-)-3a, the (+) isomer was 15 times more potent when ADP was the agonist and 19 times more active when collagen was used as the agonist. Molecular modeling of R,R- and S,S-3a using the SYBYL program was used to examine their interactions with phosphatidylinositol (PI). There was a better fit between PI and the R,R-3a with the energy of interaction being 17.6 kcal/mol less than that of the S,S-3a/PI complex. Although the absolute stereochemistry of individual enantiomers is not known, this study shows that R,R-3a interacts more favorably with PI than does S,S-3a and that (+)-3a is a more potent inhibitor of human platelet aggregation than (-)-3a. It is postulated that because of their lipophilicity, these compounds penetrate the platelet membrane and are then protonated at the pH of the cytosol. The protonated N then neutralizes the anionic charge on the membrane phosphoinositides, thereby rendering them less susceptible to hydrolysis by phospholipase C. Thus, the determinant parameters for optimum antiplatelet activity in 3-carbamoylpiperidines are (1) the amide carbonyl, (2) appropriate stereochemistry of the 3-substituent and (3) a log P value of about 4.5.

Design and synthesis of piperidine-3-carboxamides as human platelet aggregation inhibitors.

A detailed structure-activity analysis was carried out using eight 1-alkyl(aralkyl)nipecotamides (type 5), 33 bis-nipecotamidoalkanes and aralkanes (type 6), and 7 N,N'-bis(nipecotoyl)-piperazines (type 7) as inhibitors of human platelet aggregation. Steric factors played an important role in determining the activity of type 5 compounds possessing an an appropriate degree of hydrophobic character. Types 6 and 7 compounds were more potent than the corresponding type 5 molecules. Hydrophobic character appeared to influence the activity of type 6 compounds. A 3-substituent on the piperidine ring was necessary for antiplatelet activity; the substituent should be preferably an amide with its C attached directly to the ring. 3,5-Disubstitution and 2-substitution led to a decline in activity. Optimal activity was attained when the two nipecotoyl ring N atoms were connected by an aralkyl group, and separated by approximately 7 A. It is suggested that van der Waals forces and pi interactions may govern the inhibitor-platelet interaction. The most potent type 6 inhibitor was alpha,alpha'-bis[3-(N-ethyl-N-butylcarbamoyl)piperidino]-p-xylene (6i). The most potent type 5 compound was 1-decyl-3-(N,N-diethylcarbamoyl)piperidine (5a). Any substitution on the piperazine ring of type 7 compounds led to a decline in activity, the most active analog being N,N'-bis(1-decylnipecotoyl)piperazine (7a). It is suggested that nipecotamides interact with anionic platelet sites located 7 A from each other and connected by a hydrophobic well.

The influence of dosage form on papaverine bioavailability.

The bioavailability of sustained-release papaverine HCl dosage forms were compared to equivalent doses of the drug administered as an elixir and conventional compressed tablets to 12 healthy human subjects. Papaverine plasma levels were determined using a gas-chromatographic procedure. The drug was absorbed more rapidly and completely from the two nonsustained-release formulations. There was a large intersubject variability, and the plasma half-life of the drug was esstimated to be 1 hour. The area under the plasma level-time curve for the nine sustained-release products ranged from 18 to 64% relative to the area achieved by the papaverine elixir. It was concluded that the sustained-release dosage forms of papaverine included in each study group could be considered bioequivalent, but they exhibited inadequate bioavailability relative to either the elixir or the compressed tablet dosage form.

Inhibition of PAF-induced human platelet aggregation by antithrombotic nipecotomides.

Nipecotamides (piperidine-3-carboxamides) are potent inhibitors of platelet aggregation induced by a variety of agonists in vitro and in vivo. The inhibitory effects of six structural types of nipecotamides on human platelet aggregation induced by platelet-activating factor (PAF) in vitro, are studied. Evaluation of 15 racemates and stereoisomers of two nipecotamides showed that bis-nipecotoyl alkanes were more active than their mono congeners. Mono- and bis-nipecotoyl decanes were more potent than the corresponding hexanes. Lipophilicity was found to play a significant role in the antiplatelet activity of these compounds. The stereoselectivity in the PAF-antagonist potential of nipecotamides was less pronounced than that resulting from their action on ADP- or collagen-induced aggregation. Oxidation of the two benzylic carbon atoms of alpha, alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene.2HBr (A-1) to form 1,4-bis[3-N,N-diethylcarbamoyl) piperidino]benzenedicarboxamide (A-40K), which has a second set of carbonyl oxygens but lacks basic N atoms, resulted in a remarkable loss of ADP-antagonist potency while retaining PAF-antagonist activity. It is suggested that in addition to their membrane effects, nipecotamides act at other sites, including the PAF receptor. Double reciprocal plots of PAF binding to gel-filtered platelets (GFP) in the presence and absence of a typical nipecotamide (A-1C) were indicative of competitive inhibition (Ki = 19.28 microM). Scatchard analysis of 3H-PAF binding to GFP suggested the presence of high, intermediate (I) and low affinity binding sites, of which the I site gave a KD/app of 0.332 nM with an estimated 564 sites/platelet. Key interactions of nipecotamides with the PAF receptor appear to be the following (i) electrostatic interactions of the two amide oxygens with a primary set of electropositive areas spaced at 5-7 A, (ii) in the case of appropriate compounds, electrostatic interactions of the two amide oxygens spaced at 10-12 A with corresponding secondary receptor sites carrying positive electrostatic potential, (iii) a hydrophobic moiety fitting into a hydrophobic pocket in the receptor, and (iv) the cationic piperidine N+ (at pH 7.4) interacting with a counterion, probably aspartic acid.

Enantioselective antiplatelet actions of nipecotamides.

Two synthetic racemic nipecotamides, 1-decyl-3-(N,N- diethylcarbamoyl)piperidine hydrobromide (1) and alpha, alpha'-bis[3-(N-benzyl-N-methylcarbamoyl)piperidino]-p-xylene dihydrobromide (2) were resolved on a chiral alpha 1-acid glycoprotein semipreparative HPLC column. Thus, rac.1 was resolved into two enantiomers 1A-(+) and 1B-(-); rac.2 was separated into the optical antipodes 2A-(-) and 2C-(+), and the meso diastereomer 2B-(0). Also on a preparative scale, 97% pure 2C was obtained via diastereomeric salt formation using dibenzoyl-L-(-)-tartaric acid. The individual isomers were evaluated for their platelet aggregation inhibitory potency. In inhibiting ADP-induced aggregation of human platelets in vitro, 1B-(-) was 4 times more potent than its optical antipode 1A-(+), and 2C-(+) was 6 times as active as 2A-(-); the meso diastereomer 2B-(0) had intermediate activity. With collagen as the 1B-(-) was twice as active as 1A-(+), and 2C-(+), the most active compound in this series (IC50 = 0.96 microM), was 10 times more potent than its antipode 2A-(-). Again, the meso diastereomer 2B-(0) had intermediate activity. These results demonstrate the enantioselective antiplatelet actions of mono- and bis- nipecotamide derivatives.

Antithrombotic nipecotamide increases cyclic AMP levels and inhibits protein phosphorylation in human platelets.

alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide (A-1), a typical antithrombotic nipecotamide, elevated the levels of cyclic adenosine monophosphate (cAMP) in human platelets in vitro, without inhibiting cAMP-phosphodiesterase (PDE). The compound elevated the basal cAMP levels, enhanced the prostaglandin (PG)E1-stimulated platelet adenylyl cyclase (AC) activity, and prevented the ADP-induced decline of the latter. Collagen-induced phosphorylation of 20 and 47 kDa proteins was inhibited by IC50 and 0.5 x IC50 concentrations. In light of the known actions of A-1, it is suggested that stimulation of AC and inhibition of agonist-induced rise in cytosolic ionized calcium ([Ca2+]i) may constitute an aspect of its mechanism of action.

Influence of inducers and inhibitors on the metabolism in vitro and neurochemical effects in vivo of MDMA.

(S)-(+)- and (R)-(-)-3,4-methylenedioxymethamphetamine (MDMA) were metabolized in vitro by rat liver microsomes via N-demethylation to 3,4-methylenedioxyamphetamine (MDA). Whereas no difference was found in the biotransformation of the two enantiomers in the male rat or in the phenobarbital (PB) treated animals of either sex, more than twice as much MDA was formed from (S)-(+)- than from (R)-(-)-MDMA in the untreated female rat. Although 3-methylcholanthrene (3MC) pretreated rat liver microsomes were less active than those from the untreated rats of the same sex, they formed more MDA from (+)- than from (-)-MDMA. The enantioselective metabolism thus appears to be associated with the relative abundance of individual cytochrome P-450 isozymes. (S)-(+)- and (R)-(-)-MDMA.HCl (20 mg/kg) were about equipotent in depleting serotonin (5-HT) levels in the frontal cortex at 3 hrs and 1 wk following oral administration to female rats. Pretreatment of rats with SKF-525A attenuated and that with PB enhanced the 5-HT depleting potential of either isomer at 3 hrs. The 5-HT depleting potency of (+)-MDMA was significantly greater than that of its (-)-antipode at 3 hr in PB pretreated, but not in SKF-525A pretreated animals. The results suggest that the neurochemical effects of MDMA are caused by the formation of an active metabolite in vivo, and since both enantiomers were N-demethylated in vitro to approximately the same extent by PB pretreated rat liver microsomes, the active metabolite may be other than MDA.

Inhibition of agonist-induced rise in platelet Ca2+ by antithrombotic nipecotamides.

The effects of three structural types of nipecotamides and their stereoisomers on collagen-induced aggregation and intraplatelet ionized calcium ([Ca2+]i) rise in human platelets were evaluated using aequorin as the [Ca2+]i indicator. The orders of potencies of racemic nipecotamides were different when collagen was the agonist compared with those obtained using ADP. It is suggested that in addition to their earlier hypothesized interactions with platelet anionic phospholipids of the plasma and organelle membranes, nipecotamides may, in addition, act at other receptor sites. In general, the inhibition of collagen-induced aggregation paralleled their inhibitory effects on the rise of [Ca2+]i. The compounds were stereoselective in inhibiting aggregation as well as [Ca2+]i rise. The meso diastereomers of I and II were more potent than the corresponding enantiomeric pairs. A single [Ca2+]i peak was noticed when the incubate contained 1.0 mM extracellular calcium [Ca2+]o. On the other hand a biphasic [Ca2+]i rise was noticed when the nominally Ca(2+)-free buffer contained 75 microM ethylene glycol tetraacetate (EGTA). The first peak corresponded with platelet shape change, suggesting Ca2+ discharge from internal stores, and the second, with aggregation. The second peak may reflect either Ca2+ flux across the plasma membrane or aequorin leak from internal cellular locations or from the canicular system. Inhibition of the rise in intraplatelet Ca2+ appears to be associated with the platelet aggregation-inhibitory actions of nipecotamides.

Urinary excretion of methenamine and formaldehyde: evaluation of 10 methenamine products in humans.

The urinary excretion of both methenamine and formaldehyde was measured for 48 hr after the oral administration of 10 different methenamine products to 10 human subjects in a crossover study. The following dosage forms were evaluated: a tablet of methenamine base, a methenamine hippurate tablet, and eight products containing methenamine mandelate, including six enteric-coated tablets, a suspension, and a granule dosage form. The nonenteric-coated dosage forms were absorbed more rapidly, based on maximum excretion rates that occurred within 3 hr after dosing. The enteric-coated tablets, which were designed not to release methenamine until reaching the intestinal tract, exhibited maximum excretion rates that did not occur until 7-17 hr after dosing. There were no significant differences (p greater than 0.05) among products in terms of total excretion of free formaldehyde in the urine. However, large differences (p less than 0.05) were noted among products for urinary recovery of total methenamine, with the amount of administered dose recovered ranging from 16 to 83%.

Antiplatelet activity of nipecotamides in experimental thrombosis in mice.

A group of nipecotamides (3-carbamoylpiperidines) were designed, synthesized, and evaluated for their ability to protect platelets from induced aggregation. An in vivo mouse thrombosis model was used to determine the protection afforded by these compounds from sudden thrombotic death induced by intravenous collagen plus epinephrine. Enantioselectivity appears to play a pivotal role in determining the activity of these compounds. Lipophilicity, whereas previously found to correlate well with in vitro activity, did not directly influence in vivo activity. The presence of an amide function on the 3-position of the piperidine ring was essential for activity. Of the 10 compounds reported here, alpha,alpha'-bis[3-(N-benzyl-N-methylcarbamoyl)-piperidino]-p-xyle ne dihydrobromide (4) was the most potent in preventing induced intravascular platelet aggregation in mice, with a 50% effective dose of (ED50) of 27.5 mumol (20 mg)/kg.

Synthesis of stereoisomers of antithrombotic nipecotamides.

The stereoisomers of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)-piperidino]-p-xylene (1) were synthesized. Rac ethyl nipecotate was resolved by diastereomeric (-)-D- and (+)-L-tartrate salt formation. The enantiomeric esters were hydrolyzed to the corresponding nipecotic acids, which were then converted into t-BOC derivatives. Treatment of the latter with diethylamine/isobutyl chloroformate and removal of the t-BOC protecting group afforded (R)- and (S)-N,N-diethylnipecotamides. Condensation of the latter with alpha,alpha'-dibromo-p-xylene gave (R,R)- and (S,S)-1. The meso-diastereomer was obtained by stereospecific synthesis in addition to our earlier procedure involving fractional crystallization of the diastereomeric mixture obtained by synthesis. The latter was resolved earlier into 1A, 1B, and 1C using chiral high-performance liquid chromatography (HPLC). Based on the stereospecific synthesis now achieved, 1A and 1B are assigned the configurations, (R,R) and (S,S) respectively, and 1C is assigned the meso configuration. The (R,S) structure of the latter is also confirmed by X-ray crystallography.

Chiral resolution of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene, a novel antiplatelet compound.

alpha,alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, a novel antiplatelet agent, was resolved into three isomers A, B, and C, on a chiral alpha 1-acid glycoprotein analytical column using a mobile phase of 0.025 M phosphate buffer containing 0.025 M tetrabutylammonium hydrogen sulfate, at a pH of 6.5. The effect of molarity, temperature, pH, flow rate, and organic modifiers on the enantioselectivity was examined. Based on circular dichroic spectra at 220 nm, A and C appear to be the (-)- and (+)-enantiomers, respectively, and B the meso diastereomer. Attempts at resolution using Pirkle type columns gave unsatisfactory results. It appears that both hydrophobic and polar interactions between the compound and the stationary phase are important determinants of resolution.

A new substrate for the measurement of N-acetyltransferase activity.

A sensitive colorimetric method for the measurement of N-acetyltransferase (NAT) is described. It is based on the high rate of acetylation of 2-(p-aminobenzamido)pyridine by the liver enzyme and the lack of it by the blood NAT. A linear relationship was found between enzyme concentration and reaction rate. The reaction rate was also proportional to the substrate concentration. Inhibition of the reaction was observed at high substrate concentrations. The NAT levels in the liver and kidney of rat, rabbit, mouse and man were measured using this procedure. The tissues of dog failed to acetylate this substrate. The method is applicable to kinetic studies such as the analysis of inhibition reactions with o-phenanthroline and p-chloromercuribenzoate.

Relative bioavailability of acetazolamide tablets.

Plasma acetazolamide concentrations were determined enzymatically after administration of three tablet dosage forms and a reference solution to 12 human subjects in a crossover study. Two of the tablet products represented different lots from the same manufacturer. There were no significant differences in area under the plasma level-time curves among the four treatments. However, significant differences were found among tablets in terms of peak plasma concentration and time to reach peak concentration. These apparent differences in rate of absorption were correlated with in vitro dissolution data obtained in pH 1.5 dissolution medium.

Antiplatelet agents affecting the interaction of Tissue Factor-Factor VIIa complex with Factor X in a continuous-flow reactor.

The purpose of the present study was to examine the role of antithrombotic agents in the activation of Factor X in the presence of the Tissue Factor-Factor VIIa (TF-VIIa) complex in a continuous-flow reactor. Tissue Factor immobilized in a phospholipid bilayer on the inner surface of a capillary tube (internal diameter = 0.27 mm) was exposed to a perfusate containing Factors VIIa and X flowing at a flow rate of 12.7 microliters/min, corresponding to a wall shear rate of 100 s-1. Factor Xa (the activated form of Factor X) in the effluent was determined by a chromogenic assay. The effectiveness of two platelet aggregation inhibitors, alpha,alpha'-bis-[3-(N,N-diethylcarbamoyl)piperidino-p-xylene dihydrobromide (A-1) and alpha,alpha'-bis-[3-N-benzyl-N-methylcarbamoyl)piperidino]-p-xylen e dihydrobromide (A-4) in inhibiting Factor X activation is reported here. The results suggest that the Tissue Factor pathway, mediated through TF-VIIa complex, produces significantly lower levels of Factor Xa in the presence of compounds A-1 and A-4. On the basis of these findings, it appears that the anticoagulation action of these compounds reinforces their platelet aggregation-inhibitory properties. These carbamoylpiperidines (nipecotamides) therefore appear to be useful antithrombotic agents.

N-Acetyltransferases of rat liver and blood: substrate specificities.

The substrate specificities of N-acetyltransferases (NAT) were investigated by measuring Vmax and Km values with p-aminobenzoic acid (I), p-aminobenzamide (II), p-amino-benzamidopyridine (III), 2-(p-aminobenzamido)-4,6-dimethylpyrimidine (VI), and the corresponding p-aminobenzenesulfonamides (VII, VIII and XI) using at rat liver and blood preparations. With liver NAT, II, III and VI had lower Km and higher Vmax values than did their corresponding sulfonyl analogs (VII, VIII, XI). III was extraordinarily active (Vmax 854 nmol/mg protein/h); in contrast, II gave a Vmax of 22.4. Sulfadiazine (IX) and sulfamerazine (X) were acetylated at a very slow rate. The activities of the blood enzymes on these compounds were very different. The Vmax values obtained with blood NAT for II, VI and VII were sharply decreased. Surprisingly, acetylation of III, VIII, IX and X could not be detected. In contrast to liver, the blood NAT gave lower values of both Vmax and Km for the S analog of II and a much higher Km for I. While p-aminobenzoic acid was the best substrate for blood NAT, substitution of the amido nitrogen of p-aminobenzamide with an aromatic substituent enhanced the substrate potential for liver NAT, III may be useful as a substrate for the rapid classification of slow and fast acetylators.