6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6,5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

A novel series of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives was discovered as potent dual-acting agents to block the TXA2 receptor and to inhibit the thromboxane synthase (TRA/TSI). Synthesis, structure-activity relationship (SAR), and in vitro and in vivo pharmacology of this series of compounds are described. Modification of the series revolved around the oxazole moiety to increase the hydrophilicity of the compounds and to correlate the biological activity with lipophilicity of the compounds. The most potent in the series was (E)-7-[4-[4-[[(4-cyclohexylbutyl)amino]carbonyl]-2-oxazolyl] phenyl]-7 -(3-pyridyl)hept-6-enoic acid (14) with Kd = 9.9 +/- 0.4 nM for the thromboxane receptor antagonism and IC50 = 55.0 +/- 17.9 nM for thromboxane synthase inhibition. The compound 14 was a selective TRA/TSI which exhibited desirable characteristics for oral activity, "shunt" effect to elevate PGI2 level, and absence of agonist activity.

ADVERTISEMENT RETURN TO ISSUELetterNEXTDibasic Benzo[b]thiophene Derivatives as a Novel Class of Active Site-Directed Thrombin Inhibitors. 1. Determination of the Serine Protease Selectivity, Structure−Activity Relationships, and Binding OrientationDaniel J. Sall, Jolie A. Bastian, Stephen L. Briggs, John A. Buben, Nickolay Y. Chirgadze, David K. Clawson, Michael L. Denney, Deborah D. Giera, Donetta S. Gifford-Moore, Richard W. Harper, Kenneth L. Hauser, Valentine J. Klimkowski, Todd J. Kohn, Ho-Shen Lin, Jefferson R. McCowan, Alan D. Palkowitz, Gerald F. Smith, Kumiko Takeuchi, Kenneth J. Thrasher, Jennifer M. Tinsley, Barbara G. Utterback, Sau-Chi B. Yan, and Minsheng ZhangView Author Information Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 Cite this: J. Med. Chem. 1997, 40, 22, 3489–3493Publication Date (Web):October 24, 1997Publication History Received9 July 1997Published online24 October 1997Published inissue 1 October 1997https://pubs.acs.org/doi/10.1021/jm9704107https://doi.org/10.1021/jm9704107rapid-communicationACS PublicationsCopyright © 1997 American Chemical SocietyRequest reuse permissionsArticle Views439Altmetric-Citations50LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Inhibition,Inhibitors,Peptides and proteins,Reaction products,Thiophenes Get e-Alerts

Cyclic heptapeptide 1, which contains an Arg-Gly-Asp sequence, has good affinity for the platelet receptor GPIIb-IIIa and was chosen for study by 1H NMR techniques. The key RGD sequence of this molecule was found to reside in a conformationally defined type II' Gly-Asp beta-turn, and this information was used in the design of simple non-peptide RGD mimics. Disubstituted isoquinolones, bearing an acidic side chain at position 2 and a basic side chain at position 6, were prepared and were found to have modest affinity for GPIIb-IIIa. Systematic modification of the basic residue contained in these molecules yielded compounds with high affinity for GPIIb-IIIa.

Disubstituted 3,4-dihydroisoquinolones that contain an ether-linked benzamidine at C6 and a β-substituted aspartate mimic at C2 offer enhanced affinity for GPIIb–IIIa relative to the non-substituted isoquinolone propionate. Alkyl substituents afforded a 10-fold increase in intrinsic activity while aryl substituents yielded a 40-fold improvement.

The use of 5,6-bicyclic amidines as arginine surrogates in the design of a novel class of potent platelet glycoprotein IIb-IIIa receptor (GPIIb-IIIa) antagonists is described. The additional conformational restriction offered by the bicyclic nucleus results in 20-400-fold increases in potency compared to the freely flexible, acyclic benzamidine counterpart. The design, synthesis, structure-activity relationships (SAR), and in vitro activity of this novel class of GPIIb-IIIa antagonists are presented.

The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A(2) by human platelets, We found that while SFLLRN evokes an initial transient increase in cytosolic free calcium concentration ([Ca2+](i)) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca2+](i) declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca2+](i) may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the ''activating'' phosphorylation of cytosolic phospholipase A(2) (cPLA(2)). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr(669) derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA(2) and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca2+](i) and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA(2) in the absence of ERK1/2 stimulation suggests that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA(2).

Human platelets have been employed as an assay system to evaluate the pharmacological activity of a group of stable, arene-fused prostacyclin analogs. Prostacyclin (PGI2) is a highly active member of the eicosanoid family and is relatively unstable under physiological conditions. Prostacyclin's best characterized activities are those of inhibition of platelet aggregation and relaxation of vascular smooth muscle. These activities are mediated in large part via elevation of intracellular levels of cyclic AMP subsequent to receptor occupation and activation of adenylate cyclase. We previously described the synthesis of a series of arene-fused prostacyclin analogs with stability in aqueous media at physiological pH. Several of these compounds have prostacyclin-like activities, i.e., competitive binding at the platelet prostacyclin receptor, elevation of intraplatelet cyclic AMP levels and inhibition of human platelet aggregation. One compound in particular (11a) demonstrated these activities with potency similar to PGI2, i.e., Kd at platelet receptor of 3.7 nM and IC50 for inhibition of collagen-induced human platelet aggregation in plasma of 2.9 nM.

Arg-Gly-Asp-Ser (RGDS) 1, Arg-Val-Asp-Ser (RVDS) 5 Arg-dVal-Asp-Ser (R[d]VDS) 3, and a cyclic RGD peptide, cycle S,R [H-Pen-Arg-Gly-Asp-Pen-Gly-OH] 4,were tested for their ability to antagonize GPIIb-IIIa function. The activities were found to fall in the order 4>>1>>3>2. Simulated annealing and molecular dynamics studies were carried out to estimate the most populated conformations of each molecule. The acyclic molecules 1-3 were found to populate a much wider range of conformations than the cyclic molecule 4. The backbones of all four molecules were found to approximate beta-turn structures in the most populated conformations. In 4 the beta-turn intramolecular hydrogen bond between C=O of the i residue (Arg) and NH of the i + 3 residue (Ser) did not appear to be present. The distance between the beta-carbons of the critical Arg and Asp groups was found to be shorter in 4 (average 7.98 Angstrom) than in the less active acyclic molecules (averages of 8.65-9.33 Angstrom).

The antithrombotic activity of the tripeptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), was compared to heparin in a model of canine coronary artery thrombosis. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. Drug administration was started 15 min before initiation of intimal injury. Clotting times and ex vivo platelet aggregation were determined on citrated blood samples. Gingival template bleeding times were determined. Clotting times (thrombin time; activated partial thromboplastin time, APTT; prothrombin time, PT) increased in a dose-dependent manner with both anticoagulants. The two anticoagulants selectively inhibited thrombin-induced platelet aggregation. GYKI-14766 and heparin were found to delay thrombosis significantly when compared to vehicle-treated animals; minimum effective antithrombotic doses were 0.25 mg/kg/h and 80 U/kg + 30 U/kg/h, respectively. GYKI-14766 (0.25 mg/kg/h) had no effect on template bleeding time, APTT or PT. Heparin (80 U/kg + 30 U/kg/h), however, was associated with a 2.5- to 3.0-min increase in template bleeding time, a 1.8-fold and 1.7-fold increase in APTT and PT, respectively. Antithrombotic efficacy was achieved at doses of GYKI-14766 that did not affect APTT, PT or template bleeding time, whereas antithrombotic efficacy observed with heparin was associated with significant increases in APTT, PT and template bleeding time. These data demonstrate that the tripeptide thrombin inhibitor, GYKI-14766, could potentially prove to be a safer and more effective antithrombotic agent than heparin.

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The stereoisomers of S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor ligand, were compared to TXA2/PGH2 receptor antagonists, SQ29548 and BM13505 in guinea pig platelets, aortas and trachea. Equilibrium binding assays in platelets yielded Kd values (nanomolar) for (+)-S-145 (0.57 +/- 0.04), (-)-S-145 (9.2 +/- 1.3), SQ29548 (11.1 +/- 0.70) and BM13505 (118 +/- 16). In aortas, the corresponding Kb values (nanomolar) were (0.014 +/- 0.002), (1.90 +/- 0.31), (16.8 +/- 3.3) and (142 +/- 29), respectively, whereas in trachea, the Kd values (nanomolar) were (0.019 +/- 0.004), (1.12 +/- 0.18), (1.94 +/- 0.30) and (18.99 +/- 2.59), respectively. S-145 stereoisomers elicited platelet shape change stereoselectively that was characterized by EC50 values 8 to 16-fold higher than the EC50 values for these ligands to block aggregation induced by TXA2/PGH2 mimetic, U44069. S-145 (+)- and (-)-isomers stereoselectively induced transient aortic contraction at concentrations 214,000- and 16,000-fold higher, respectively, than the corresponding Kb values in this tissue. S-145-induced platelet shape change and aortic contraction were inhibitable by low concentrations of SQ29548. We postulate that S-145 may elicit partial agonist activity in platelets and aorta via lower affinity for the active than inactive state of the TXA2/PGH2 receptor in those tissues. S-145 had no agonist activity in isolated trachea possibly indicating different TXA2/PGH2 recognition sites in aorta and trachea or a smaller preligand ratio of active to inactive TXA2/PGH2 receptors in trachea than in aorta.

The stereoisomers of S-1 45, a novel thromboxane A2/prostaglandin H-2 (TXA2/PGH2) receptor ligand, were compared to TXA2/PGH2 receptor antagonists, SQ29548 and BM13505 in guinea pig platelets, aortas and trachea. Equilibrium binding assays in platelets yielded K(d) values (nanomolar) for (+)-S-145 (0.57 +/-0.04), (-)-S-145 (9.2 +/- 1.3), SQ29548 (11.1 +/- 0.70) and BM13505 (118 +/- 16). In aortas, the corresponding K(b) values (nanomolar) were (0.014 +/- 0.002), (1.90 +/- 0.31), (16.8 +/- 3.3) and (142 +/- 29), respectively, whereas in trachea, the K(d) values (nanomolar) were (0.019 +/- 0.004), (1.12 +/- 0.18), (1.94 +/-0.30) and (18.99 +/- 2.59), respectively. S-145 stereoisomers elicited platelet shape change stereoselectively that was characterized by EC50 values 8 to 16-fold higher than the EC50 values for these ligands to block aggregation induced by TXA2/PGH2 mimetic, U44069. S-145 (+)- and (-)-isomers stereoselectively induced transient aortic contraction at concentrations 214,000- and 16,000-fold higher, respectively, than the corresponding K(b) values in this tissue. S-145-induced platelet shape change and aortic contraction were inhibitable by low concentrations of SQ29548. We postulate that S-145 may elicit partial agonist activity in platelets and aorta via lower affinity for the active than inactive state of the TXA2/PGH2 receptor in those tissues. S-145 had no agonist activity in isolated trachea possibly indicating different TXA2/PGH2 recognition sites in aorta and trachea or a smaller preligand ratio of active to inactive TXA2/PGH2 receptors in trachea than in aorta.

Several sulfonamide derivatives related to S-145 were synthesized and tested as thromboxane receptor antagonists. The results reveal the importance of the orientation between the sulfonamide moiety and the caboxyclic acid group of the alpha-side chain in order to obtain high affinity interaction with TXA2 receptors.