Antiplatelet effects of R,S-(meso)-alpha, alpha'-bis[3-(N,N-diethylcarbamoyl) piperidino]-p-xylene ex vivo in the dog and in vivo in the mouse.

1. The nipecotamide alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene (A-1) is a platelet aggregation inhibitor. The meso diastereomer A-1c is superior in potency and duration to the synthetic diastereomeric mixture consisting of the R,R-, S,S-, and R,S- (meso) isomers in inhibiting collagen-induced platelet aggregation ex vivo in the dog. 2. A-1c also is more potent and longer acting than A-1 in protecting mice from collagen+epinephrine-induced thromboembolic death. 3. The mechanism of antiplatelet action of this compound appears to be related to its ability to prevent agonist-induced inhibition of platelet cyclic adenosine monophosphate (cAMP) levels.

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.

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.

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.

Synergistic antiplatelet effects of a nipecotamide A-1C and low dose aspirin.

1. The effects of an antithrombotic nipecotamide, A-1, and aspirin were examined separately and in combination, on human platelet aggregation in vitro and on collagen+epinephrine-induced thromboembolic death of mice in vivo. 2. Concurrent addition of the two agents to a platelet suspension resulted in a supraadditive inhibition. Racemic A-1 and its meso diastereomer A-1C behaved similarly in this respect. The IC50 value of rac. A-1 declined from 46.25 to 18.4 microM in the presence of aspirin. 3. In vivo, concurrent administration of A-1C and aspirin produced significant potentiation of antithrombotic activity. A 2-fold reduction in the ED50 of A-1C occurred when it was coadministered with aspirin to mice; also, the toxicity reduced slightly, increasing the therapeutic index by a factor of 2.2. 4. The design and synthesis of new compounds possessing the structural features of the two molecules appears to provide superior antithrombotic agents.

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.

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.

In vitro and in vivo activities of the novel antiplatelet agent alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide.

In an effort to develop compounds with high antithrombotic activity and minimal toxicity, our laboratory has synthesized a number of nipecotamides. The effectiveness of one of these compounds, alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide (A-1), in inhibiting both in vitro and in vivo platelet aggregation is reported here, along with its acute toxicity. The IC50 of A-1 in in vitro ADP- and PAF-induced platelet aggregation was 44.5 microM and 21.2 microM, respectively. Suppression of intraplatelet [Ca2+] is suggested as a likely mediator of the aggregation-inhibitory properties of A-1, since both the release of cytosolic Ca2+ and the influx of extracellular Ca2+ were decreased. The ED50 of A-1 in protecting mice against thromboembolism induced by a collagen-epinephrine challenge was 164 mumol/kg. The measurement of the acute toxicity of this compound as the LD50 was 691 mumol/kg, with the therapeutic index being 4.2. These data indicate that compounds in this family hold promise as clinically effective antithrombotic agents.

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.

Cytosolic ionized calcium in human platelets: the influence of collagen and a novel antiplatelet agent.

Two phases of calcium mobilization were observed when aequorin-loaded human platelets, suspended in a nominally calcium-free medium containing 0.1 mM EGTA, were stimulated with collagen. The first phase coincided with platelet shape change, and the second phase corresponded to aggregation. On the other hand, only one [Ca2+]i peak was found in systems containing 1.0 mM Ca, or 1.0 or 2.0 mM EGTA. A novel antiplatelet compound alpha,alpha'-bis [3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, inhibited both [Ca2+]i peaks. It is suggested that inhibition of the mobilization of intraplatelet calcium stores as well as the blocking of transmembrane calcium flux may be responsible for the platelet aggregation-inhibitory action of this compound.

The effect of carbamoylpiperidine and nipecotoylpiperazine analogs on ADP-stimulated human thrombocyte aggregation and platelet factor 3 availability.

There is a striking congruence between the inhibitory effects of three synthetic entities on ADP-induced (i) human blood platelet aggregation and (ii) platelet factor 3 availability as evidenced by prolonged 'Stypven time'. The pronounced parallel between each compound's potency in inhibiting aggregation (e.g. IA 48.9 +/- 1.3, S.E., %; n = 16) and in impeding platelet factor 3 availability (e.g. IPF-3av 42.3 +/- 2.5, S.E., %; n = 12), determined concurrently in platelet-rich plasma of four different donors, further substantiates that the antiplatelet activity of our carbamoylpiperidine and nipecotoylpiperazine congeners is exerted through their interaction with anionic phospholipids.

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.

Some novel inhibitors of platelet aggregation: acute toxicity in mice and its relationship to in vitro activity and toxicity.

Five carbamoylpiperidine congeners and one nipecotoylpiperazine derivative, which inhibit ADP-induced aggregation of human blood platelets in vitro, were evaluated in vivo for acute toxicity to male and female ICR mice. Although the in vitro platelet aggregation inhibiting potency of these compounds covered about a 1150-fold range, the acute ip LD50s (microM/kg) in mice showed only a fourfold range of values. An increase in platelet aggregation inhibiting potency (in vitro) was not paralleled by an increase in acute toxicity in vivo for these compounds; in fact, the most potent aggregation inhibitor (microM/liter) was the least toxic (microM/kg) to mice. A comparison of acute LD50s (microM/kg) to concentrations which produce 50% inhibition of mouse fibroblast cell growth in culture (microM/liter) did not yield a consistent value, nor was the rank order of toxicity the same from these two tests. In hematoxylin and eosin stained slides of major organs from treated mice, no histopathologic lesions were observed which were attributable to administration of these compounds.

Serotonin efflux-inducing effects on human blood platelets by carbamoylpiperidine and nipecotoylpiperazine aggregation inhibitors.

The serotonin efflux-inducing potency of carbamoylpiperidine and nipecotoylpiperazine aggregation inhibitors was evaluated in human blood platelets. Findings were interpreted in terms of structure-activity relationships, and related to actions exerted by the compounds on other human blood platelet functions. Termination of the active efflux period in the presence and absence of acetylsalicylic acid allowed assessment of the influence of aspirin on the quenching process.

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.

Interrelationships between the chemical structure of synthetic entities, their platelet aggregation inhibitory potency and their cellular toxicity, based on in vitro experiments.

Striking relationships were observed, in vitro, between the molecular constitution of synthetic entities, their aggregation-inhibitory potency (as determined in ADP-induced human blood platelet aggregation), and their cellular toxicity (as assessed by their inhibition of cultured mouse fibroblast L-cell growth). Effects exerted on platelets tended to reflect interactions between the molecules' aggregation-inhibitory specific functions and the platelets' corresponding target sites, while fibroblasts were generally more susceptible to the molecular constitution's hydrophobic character. The hyperbolic relationships between concentrations effecting 50% inhibition and slopes of concentration-response curves reflect net activity from both specific and nonspecific receptor site interactions, with the latter being dominant, and indicated that the assays approximated equilibrium systems. Plotting logarithm values of concentrations effecting 50% inhibition against logarithms of reciprocal slopes for concentration response curves yielded multiple regression coefficients of R = 0.97 (platelet aggregation-inhibitory potency) and R = 0.98 (fibroblast growth-inhibitory potency).

In vivo biocompatibility studies. VII. Inflammatory response to polyethylene and to a cytotoxic polyvinylchloride.

The cellular biocompatibility of low-density polyethylene and a cytotoxic polyvinylchloride were investigated using an in vivo cage implant system. Components of the inflammatory response (white cells, extra-cellular alkaline and acid phosphatase, the complement component C3, and total protein content) were monitored over a 21-day implantation period. Scanning electron microscopy was used to evaluate the morphologic condition of leukocytes adherent to the implanted polymers. Prior to implantation, each polymer was evaluated using an established primary acute toxicity screen. The results showed that the cytotoxic polyvinylchloride stimulated an intense acute phase inflammatory response, and at later observation periods, an intense and increasing chronic inflammatory response. In contrast, the polyethylene promoted relatively small increases in the acute and chronic phases of inflammation; the overall cellular response being essentially resolved by the third week after implantation. The initial toxicity screen of each polymer suggested that the observed differences in inflammation were primarily caused by the release from the polyvinylchloride of the added cytotoxic agent (dioctyltinbisoctylmercaptoacetate).

Acrylate and methacrylate esters: relationship of hemolytic activity and in vivo toxicity.

Quantitative hemolysis assays of acrylate and methacrylate esters provided estimates of the intrinsic hemolytic activity (Hi, the slope of the concentration-response curve) and the concentrations effecting 5% (H5) and 50% (H50) hemolysis. The dependence of hemolytic activity and LD50 (mice) on physical properties (lipophilicity, molar refraction, and molecular volume) of the esters was determined by multiple regression analysis. The observed correlations were: Hi, R2 = 0.94; H5, R2 = 0.95; H50, R2 = 0.94; and LD50, R2 (all compounds) = 0.80, R2 (all compounds less the methyl esters) = 0.94. The difference of the methyl esters was associated with the smaller steric volume of the methyl ester substituent and the presence (methacrylates) or absence (acrylates) of the branched methyl group. Associative steric contributions of the branched methyl group and the ester substituents were probably responsible for greater variability in the methyacrylate series. The results were consistent with the conclusion that the mechanism of the action of the esters is membrane mediated and relatively nonspecific and that in vivo biotransformation was not a significant factor. Also, long-term toxic liability of the esters may be more closely related to intrinsic toxicity than acute toxicity.