Chem.-Biol. Interactions, 38 (1982) 135--144 135 Elsevier/North-Holland Scientific Publishers Ltd.

SURFACE ACTIVITY AND HUMAN BLOOD PLATELET AGGREGATION-INHIBITORY POTENCY

R.P. QUINTANA, A. LASSLO* and G.S. QUEEN**

Department of Medicinal Chemistry, College of Pharmacy, .University of Tennessee Center for the Health Sciences, Memphis, TN 38163 (U.S.A.)

(Received March 23rd, 1981 ) (Revision received June 12th, 1981) (Accepted June 19th, 1981)

SUMMARY

Surface and interfacial activity is correlated with molecular constitution and inhibitory potency of mono- and bis(carbamoylpiperidino)alkanes and aralkanes, and of some corresponding quaternary pyridinium congeners, in ADP-induced human blood platelet aggregation. The measurements of surface and interracial tension were carried out at concentrations and pH-values approximating those employed in the hemodynamic study. The effect of changes in chemical structure, ranging from relatively minor variations in a specific functional group to the alteration of major com- ponents in molecular constitution, was examined and interpreted in terms of contemporary theoretical chemistry.

INTRODUCTION

In preceding papers [1,3; cf. 2], we reported on relationships between the molecular constitution of carbamoylpiperidines and related compounds, and their inhibition of ADP-induced human blood platelet aggregation; interpretation of the data in terms of hydrophobicity, planarity and inter- atomic distances between aggregation-inhibitory specific functions distinctly suggested characteristically spaced target sites on the platelet membrane. While we and others have associated surface action with chemically induced effects on membrane structures {e.g. Refs. 4--14), to the best of our knowledge, relationships between platelet aggregation-inhibitory potencies

*To whom correspondence should be addressed. **Work reported in this paper comti tutes a segment of the dissertation to be submitted by Galen S. Queen to the Graduate School--Medical Sciences o f the University of Tennessee in partial fulfdlment of the requirements for the Ph.D. degree in medicinal chemistry.

0009--2797/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers Ltd.

1 3 6

and actually measured surface and interfacial tensions have not been meaningfully explored. The surface and interracial activity of carbamoyl- piperidine derivatives yielded, therefore, unique insights on the influence surface-chemical factors exert in chemically induced inhibition of human platelet aggregation.

MATERIALS AND METHODS

The chemistry and properties of the carbamoylpiperidines and related compounds employed in this study have been described elsewhere [15--18]. An of the compounds were analytically pure. They include: 1,4-xylylenebis- [3-(N,N-diethylcarbamoyl)pyridinium bromide] (I); 1-(p-methylbenzyl)-3- (N,N-diethylcarbamoyl)piperidine hydrobromide (II); 1,4-xylylenebis[3- (N,N-diethylcarbamoyl)piperidine hydrobromide] (III); 1,3-xylylenebis [3- (N,N-diethylcarbamoyl)piperidine hydrochloride] (IV); l~lecyl-3-(N,N~li- ethylcarbamoyl)piperidine hydrobromide (V); l~iecyl-3-(N-ethylcarbamoyl)- piperidine hydrobromide (VI); 1-decyl-3-(carbamoyl)piperidine hydrobro- mide (VII); 1,10-bis[3-(N,N-diethylcarb~moyl)piperidino]decane dihydro- bromide (VIII); 1,2-bis[3-(N,N-diethylcarbamoyl)piperidino]ethane dihydro- bromide (IX); 1-hexyl-3-(N,N~liethylcarbamoyl)piperidine hydrobromide (X); l<lecyl-3-(N,N-diethylcarbamoyl)pyridinium bromide (XI).

Surface and interfacial tensions were determined at 25°C by the ring method, employing previously described instrumentation and methodologies [4,19--21]. Solutions of the compounds (1 • 10 -s M to 2 • 10 -4 M) were freshly prepared in pH 7.6 phosphate buffer (surface tension determinations), or in pH 7.6 phosphate buffer saturated with n-hexane (interracial tension determinations) [4]. In the former instance, measurements were made exactly 15 rain after pipetting the solution into the sample dish; in the latter one 25 ml of n-hexane saturated with buffer was layered gently on a 25-ml aliquot of the aqueous solution and the interface was allowed to age for exactly 15 rain prior to measurements. Data from at least two independent experiments were used in obtaining average surface or interracial tension values. Appropriate correction factors [22,23] were applied and surface and interfacial pressures (~) were computed by subtracting surface or inter- facial tensions of solutions (7) from that of the respective controls (70). Values of individual determinations usually did not differ by more than 1.0% from the reported mean values and most were below 0.5%.

The inhibitory potency of our compounds in ADP-induced aggregation of human blood platelets, as previously reported [1,3], was ranked as follows: 0 = up to 10%; 1+ = 11--19%; 2+ = 20--29%; 3+ = 30-39%; 4+ = 4 0 - 49%; 5+ -- 50-59%; 6+ = 60% or greater.

RESULTS AND DISCUSSION

Relationships between surface and interfacial pressure and human blood platelet aggregation-inhibitory potency are highlighted in Tables I--III.

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TABLE III

RELATIONSHIP BETWEEN SURFACE ACTIVITY, STRUCTURAL CHARACTERISTICS AND HUMAN BLOOD PLATELET AGGREGATION-INHIBITORY POTENCY

HsC 2 ~, ~ 0[[ /C2H $

N--C A C - - N

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Corn- n Inhibitory Surface presstwe (it, mN m - l ) , p o u n d p o t e n c y a a i r - p h o s p h a t e b u f f e r (DH 7.6),

at c o n c . (M)

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2 • 1 0 -4 1 * 1 0 -4 5 " 1 0 - s 2 " 1 0 -4 1 " 1 0 -4 5 " 1 0 - s 1 " 1 0 - s

VIII I0 5+ at 5 • I 0 "s M 4.8 3.2 3.2 . b 13,3 12.0 8.2 IX 2 I + at 1 . I 0 -4 M 2.5 0.9 --b 8.5 6.5 5.4 _ b

a Summarized f rom Quin tana et al. [ 1,3]. b Not determined.

Compound III was the most potent one among those hitherto designed and synthesized in our laboratories; comparing it with its congeners reflects parallel trends between surface, interfacial and platelet aggregation-inhibitory activity. Undoubtedly, molecules of III adsorbed at the air-buffer and n-hexane-buffer interfaces assume a horizontal orientation, 'anchored' to the aqueous phase by the piperidine nitrogens and amide functions on both ends of the predominantly planar xylylene moiety (Fig. 1A); this conclusion is corroborated by the findings o f Vasil'kevich et al. [27]. The substantial drop in interfacial pressure from III to II is obviously reflecting the loss of one of the two diethylcarbamoylpiperidino substituents; a corresponding difference is reflected in the latter's capacity to inhibit platelet aggregation. Consistent with its lack of antiplatelet activity, compound I did not generate any significant surface or interfacial pressure. This can be attributed to the compound's enhanced hydrophilic character bestowed upon it by its t w o

totally ionized quaternary functions with an unequivocal predisposition for the aqueous bulk phase [ 28].

Examining the influence of the amide moiety in otherwise identically constituted compounds {Table II), one of the most interesting observations of our study was the inverse relationship registered for interfacial with respect to surface pressure. In this sub,series, it is evident that platelet aggregation inhibitory potency and surface pressure values follow a similar trend, and the effect is clearly related to the relative hydrophobic character evidenced in the corresponding amide group polarization and partition coefficient values. As delineated by Kier et al. [26], the connectivity index is computed by encoding the nature of connection and/or branching of atoms in molecules. Since it correlates well with physical properties relating to molecular surface area (e.g., partition coefficient) and volume (e.g., polarizability), it is not surprising that the connectivity index values follow the same trend [26]. While in Table II we were able to glean and correlate

140

A.

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Fig. 1. Projected molecular orientations of surface- or interface-adsorbed carbamoylpipe- ridine derivati~s: A: compound HI; B: compound IX; C: compound VIII; D: com- pound V. See text for discussion. From the pK a (7.91) determined for compound II in water [18], and its ionization computed for pH 7.6 (67.1%), it is reasonable to assume that the molecular/ionic ratio of the compounds would not affect significantly the depicted orientations.

highly specific data elicited by variations in the minutiae o f a particular functional group in a series o f otherwise unaltered molecules, the values in Table I reflect variances in several major molecular components which did no t lend themselves to an equally penetrat ing analysis of the otherwise systematic and gradual changes in chemical const i tut ion.

141

The quaternary pyridinium analog of V, compound XI, registered con- siderably higher inteffacial (15.7, 12.4 and 11.0 mN m -~) and substantially lower surface (6.7, 4.1 and 3.2 mN m -l) pressure values than the tertiary amino congener at 2- 10 -4 M, 1 . 10 -4 M and 5 . 10 -5 M concentrations, respectively; the platelet aggregation inhibitory activity of XI was similarly lower (2+ at 1 • 10 -4 M). The reduced hydrophobic character effected by the totally ionized quaternary function with a decided affinity for the aqueous bulk phase accounts for this occurrence. While the loss in hydro- phobicity by a compound containing a single quaternary function (XI) is obviously less pronounced than by one which has two such functional groups (I), it is still materially reflected in both the physicochemical data and the hemodynamic response.

In contrast to the relatively high level of surface pressure generated by the n~lecyl derivative (V), the corresponding n-hexyl (C6) compound (X) elicited considerably lesser surface (2.0 and 1.2 mN m -1) and interfacial (4.0 and 1.3 mN m -1) pressures at 2 • 10 -4 M and 1 • 10 -4 M concentrations, respec- tively. This is certainly a reflection of the enhanced hydrophobicity of the longer aliphatic chain, and augmented cohesive interactions in the vertically oriented surface-adsorbed molecules [29]. Interestingly, in terms of hydro- phobic character, the n-hexyl moiety is approximately equivalent to the p-methylbenzyl component of compound II (Table I)*; accordingly, both compounds, X and II, elicited closely matching surface and interfacial pressure values as well as low orders of antiplatelet activity (2+ at 1 • 10 -4 M and 1+ at 1 • 10 -4 M, respectively).

Like the bis-substituted aralkanes (III and IV) depicted in Table I, the bis(dialkylcarbamoylpiperidino)alkanes in Table III are seen to generate greater pressures at the n-hexane-buffer interface than at the air-buffer interface. The substantial influence of the length of the connecting link is evident in the greater surface activity of the bis-substituted~tecane (VIII) compared to that measured for the bis-substituted~thane (IX); and the augmented hydrophobicity provided by the eight additional methylene units seems to be also reflected in the substantially greater platelet aggregation inhibitory potency of VIII. The length of the connecting chain may also affect considerably the orientation of surface- or interface-adsorbed molecules of the respective compounds. While the short two~arbon chain of IX would necessarily constrain the molecule to lie parallel to the interface (Fig. 1B), the 10-carbon chain of VIII has sufficient flexibility to coil vertically resulting, possibly, in a 'wicket-like loop' conformation such as is depicted in Fig. 1C [27,29,31,32]; the frequency of loop-formation by VIII is probably contingent on the population density of molecules situated at the interface at any given time.

A comparison of effects of corresponding mono- and bis-substituted compounds can be made with compounds V (Table II) and VIII (Table III).

*Hydrophobic fragmental constants [30]: CH~(CH2)s_, 3.337; CH~C, H4CH2-, 2.961.

142

The mono-subst i tuted decane (V) generates substantial surface pressure bu t weak interfacial pressure while the converse is true of the otherwise identical but bis-substituted decane (VIII); this finding parallels, for example, that reported by Menger and Wrenn [31]. The effects of the mono~ubs t i tu ted compound presumably reflect embellished hydrophobic i ty and enhanced cohesional interactions in the perpendicularly oriented chains (Fig. 1D) at the air-buffer interface; the bis-substituted compound, on the other hand, would be expected to have lower cohesional interactions even in the loop-like conformat ion depicted in Fig. 1C. Cohesional attraction for both mono- and b i~compounds at the n-hexane-buffer interface would be antici- pated to be negligible [28].

The relevance of surface activity to the biodynamic response exerted by the compounds discussed in this communicat ion is clearly evidenced. It would be inappropriate, however, to assume that surface action is the sole dominant factor in their platelet aggregation-inhibitory potency; our studies with the very compounds pictured in Table I revealed, for example, that the interatomic distance between aggregation-inhibitory~pecific functions is much more essential in eliciting strong hemodynamic effects. By the same token, it is our considered opinion that we are merely beginning to appreciate the significance of influences exerted by surface-active factors in the design of truly effective medicinal agents.

ACKNOWLEDGEMENTS

Support of this research by USPHS Grants HL-22236 and HL-25884, and by the Gustavus and Louise Pfeiffer Research Foundation, is most gratefully acknowledged. We also wish to thank Research Assistant Lynnet te P. Williams for her assistance in the determination of surface and interfacial tensions.

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