Thrombin receptor (PAR-1) antagonists. Solid-phase synthesis of indole-based peptide mimetics by anchoring to a secondary amide.

A novel, 10-step, solid-phase method, based on a secondary amide linker, was developed to construct a diverse library of indole-based SFLLR peptide mimetics as thrombin receptor (protease-activated receptor 1, PAR-1) antagonists. The key steps include stepwise reductive alkylation, urea formation, and Mannich reaction. Screening of the library led to a quick development of the SAR and the significant improvement of PAR-1 activity.

Administration of a potent antagonist of protease-activated receptor-1 (PAR-1) attenuates vascular restenosis following balloon angioplasty in rats.

Human platelets possess two distinct thrombin-activated receptors, PAR-1 (protease-activated receptor-1) and PAR-4, whereas human vascular smooth muscle cells possess only PAR-1. Although such thrombin receptors have been studied extensively in vitro, their physiological roles are still rather ill-defined. We have now employed a potent, selective PAR-1 antagonist, RWJ-58259, to probe the in vivo significance of PAR-1 in thrombosis and vascular injury. RWJ-58259 was examined in two thrombosis models in guinea pigs: the arteriovenous (A-V) shunt assay (monitoring thrombus weight) and the Rose Bengal intravascular photoactivation assay (monitoring time to occlusion). Administration of RWJ-58259 (10 mg/kg, total i.v. dose) did not inhibit thrombus formation in either thrombosis model, although local, intrashunt delivery in the A-V shunt model did elicit a modest antithrombotic effect (thrombus weight reduction from 35 +/- 2 to 24 +/- 4 mg). These results are consistent with the presence of more than one thrombin-sensitive receptor on guinea pig platelets, in analogy with human platelets. Indeed, we were able to establish that guinea pig platelets express three thrombin receptors, PAR-1, PAR-3, and PAR-4. We also examined RWJ-58259 in a vascular restenosis model involving balloon angioplasty in rats. Perivascular administration of RWJ-58259 (10 mg) significantly reduced neointimal thickness (77 +/- 5 microm to 45 +/- 5 microm, P < 0.05), clearly demonstrating an important role for PAR-1 in vascular injury. From these results, it is evident that a PAR-1 antagonist is not especially effective for treating platelet-dependent thrombosis; however, it could well be beneficial for treating restenosis attendant to arterial injury.

Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes.

Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.

Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significantly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor.

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Potential anxiolytic agents. 3. Novel A-ring modified pyrido[1,2-a]benzimidazoles.

A variety of pyrido[1,2-a]benzimidazoles (PBIs) modified on the A-ring were prepared and evaluated for affinity to the benzodiazepine binding site on the GABA-A receptor and in animal models predictive of anxiolytic activity in humans. A-ring benzo-fused derivative 7 exhibited potent activity, as did the 6- and 7-pyrido compounds 3 and 4.

Heterocycle-peptide hybrid compounds. Aminotriazole-containing agonists of the thrombin receptor (PAR-1).

The thrombin receptor PAR-1 is activated by alpha-thrombin to stimulate cells, including platelets, through the tethered-ligand sequence SFLLRN. We have discovered a novel series of heterocycle-peptide hybrids comprised of a tripeptide segment, such as Cha-Arg-Phe, and an N-terminal heterocyclic group, many of which behave as full PAR-1 agonists. Certain compounds with an aminotriazole group, such as 4 and 16, are nearly as potent as SFLLRN-NH2 in inducing platelet aggregation. Also, some arylethenoyl "N-capped" compounds, such as 52 and 57, exhibit mixed PAR-1 agonist-antagonist activity.

Macrocyclic hexapeptide analogues of the thrombin receptor (PAR-1) activation motif SFLLRN.

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. Macrocyclic peptide analogues of SFLLRN were synthesized and evaluated in vitro. In general, the compounds were much less potent in inducing platelet aggregation relative to SFLLRN-NH2 and did not act as antagonists of alpha-thrombin. Derivative 3c was the most potent macrocycle in activating PAR-1, with an EC50 of 24 microM.

Thrombin receptor-activating peptides (TRAPs): investigation of bioactive conformations via structure-activity, spectroscopic, and computational studies.

The thrombin receptor (PAR-1) is an unusual transmembrane G-protein coupled receptor in that it is activated by serine protease cleavage of its extracellular N-terminus to expose an agonist peptide ligand, which is tethered to the receptor itself. Synthetic peptides containing the agonist motif, such as SFLLRN for human PAR-1, are capable of causing full receptor activation. We have probed the possible bioactive conformations of thrombin receptor-activating peptides (TRAPs) by systematic introduction of certain conformational perturbations, involving alpha-methyl, ester psi(COO), and reduced-amide psi(CH2N) scans, into the minimum-essential agonist sequence (SFLLR) to probe the importance of the backbone conformation and amide NH hydrogen bonding. We performed extensive conformational searches of representative pentapeptides to derive families of putative bioactive structures. In addition, we employed 1H NMR and circular dichroism (CD) to characterize the conformational disposition of certain pentapeptide analogues experimentally. Activation of platelet aggregation by our pentapeptide analogues afforded a structure-function correlation for PAR-1 agonist activity. This correlation was assisted by PAR-1 receptor binding data, which gauged the affinity of peptide ligands for the thrombin receptor independent of a functional cellular response derived from receptor activation (i.e. a pure molecular recognition event). Series of alanine-, proline-, and N-methyl-scan peptides were also evaluated for comparison. Along with the known structural features for PAR-1 agonist peptides, our work adds to the understanding of peptide topography relative to platelet functional activity and PAR-1 binding. The absolute requirement of a positively charged N-terminus for strong agonist activity was contradicted by the N-terminal hydroxyl peptide psi(HO)S-FLLR-NH2. The amide nitrogen between residues 1 and 2 was found to be a determinant of receptor recognition and the carbonyl groups along the backbone may be involved in hydrogen bonding with the receptor. Position 3 (P3) of TRAP-5 is known to tolerate a wide variety of side chains, but we also found that the amide nitrogen at this position can be substituted by an oxygen, as in SF-psi(COO)-LLR-NH2, without diminishing activity. However, this peptide bond is sensitive to conformational changes in that SFPLR-NH2 was active, whereas SF-NMeL-LR-NH2 was not. Additionally, we found that position 3 does not tolerate rigid spacers, such as 3-aminocyclohexane-1-carboxylic acid and 2-aminocycloalkane-1-carboxylic acid, as analogues 1A, 1B, 2A, 2B, 3, 4, 5A and 5B lack agonist activity. On the basis of our results, we suggest that an extended structure of the agonist peptide is principally responsible for receptor recognition (i.e. binding) and that hydrophobic contact may occur between the side chains of the second (Phe) and fourth (Leu) residues (i.e. P2-P4 interaction).

Thrombin receptor (PAR-1) antagonists. Heterocycle-based peptidomimetics of the SFLLR agonist motif.

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. A series of azole-based carboxamides, designed after SFLLR, were synthesized and evaluated in vitro. The compounds inhibited platelet aggregation induced by SFLLRN-NH2 or alpha-thrombin, and blocked the binding of [3H]-S-(p-F-Phe)-Har-L-Har-KY-NH2 to a CHRF membrane preparation of PAR-1. Oxazole 30 bound to PAR-1 with an IC50 of 1.6 microM, and gave IC50 values of 25 microM and 6.6 microM against alpha-thrombin- and SFLLRN-NH2-induced platelet aggregation, respectively.

Azepinoindole derivatives with high affinity for brain dopamine and serotonin receptors.

We synthesized 20 and 21 as conformationally constrained analogues of the dopamine receptor antagonist SKF-83742, as well as analogues 6-9, 16, and 18-22. Although 20 and 21 were inactive, 7, 9, and 19 showed strong binding to D-1, D-2, S-2, and alpha-1 receptors, as well as antipsychotic activity in vivo.

Pyrroloisoquinoline antidepressants. 3. A focus on serotonin.

A collection of hexahydropyrroloisoquinoline derivatives (1-22), which represent a class of compounds that inhibit the neuronal uptake of dopamine (DA), norepinephrine (NE), and serotonin (5-HT), was investigated in vivo for serotonin-potentiating properties in the mouse head-twitch and rat serotonin syndrome assays. The p-methylthio compound 3b (McN-5652-Z) was found to possess exceptional activity in these assays, and the activity was attributable almost exclusively to the (+)-6S,10bR enantiomer. Ten closely related analogues were synthesized, tested, and compared among themselves and with some previously prepared compounds, both in vivo and in vitro. Several trans diastereomers exhibited strong inhibition of 5-HT uptake and substantial potentiation of 5-HT, while the cis diastereomers (3a, 4a, and 10a) tested were virtually devoid of such activity. Although 3b was only moderately selective in inhibiting the uptake of 5-HT vs NE, its 10-substituted analogues 4b, 7b-9b had improved 5-HT selectivity relative to NE, to the extent of 20-25 times (150-200 times relative to DA). Of these more selective compounds (in vitro), only 4b and 7b had substantial activity in vivo. Sulfoxide 11b appeared to function as a prodrug of 3b in vivo.

McN-5652: a highly potent inhibitor of serotonin uptake.

McN-5652 is one of a series of substituted pyrrolo-isoquinolines that, as a group, potently inhibit the uptake of one or more of the monoamines, norepinephrine, serotonin and dopamine. McN-5652 is characterized by exceptionally high potency as an inhibitor of the uptake of serotonin by rat brain synaptosomes in vitro (Ki approximately 0.6 nM) and ex vivo (ED50 approximately 2 mg/kg p.o.). The high potency of McN-5652 as a serotonin uptake inhibitor in vivo is indicated further by the low doses required to potentiate L-5-hydroxytryptophan-induced head twitches in mice (ED50 = 0.4 mg/kg 2 hr after p.o. dosing) and the serotonin syndrome in rats (ED50 = 1.5 mg/kg 2 hr after p.o. dosing). McN-5652 also potently inhibited the synaptosomal uptake of norepinephrine (Ki approximately 3 nM) and was a moderately potent inhibitor of the synaptosomal uptake of dopamine (Ki approximately 40 nM). McN-5652 inhibited tetrabenazine-induced ptosis in rats and mice but was much less effective in blocking the sedation caused by tetrabenazine. In rats, McN-5652 did not induce the stereotyped behavior often caused by dopamine agonists and inhibitors of dopamine uptake. Receptor binding experiments indicated that McN-5652 has a weak affinity for serotonin 5-HT2 and alpha-1 adrenergic receptors (apparent Ki approximately 200 nM) and a very low affinity for dopamine D1 and D2 receptors, serotonin 5-HT1, alpha-2 adrenergic, muscarinic and gamma-aminobutyric acid-A receptors. Experiments using the guinea pig ileum indicate that McN-5652 is a weak, noncompetitive antagonist of histamine.

Pyrroloisoquinoline antidepressants. 2. In-depth exploration of structure-activity relationships.

A series of pyrrolo[2,1-a]isoquinolines, and related compounds, were examined for antidepressant-like activity, by virtue of their antagonism of tetrabenazine-induced ptosis and sedation, and inhibition of biogenic amine uptake. Thus, we have identified some of the most potent antagonists of TBZ-induced ptosis and some of the most potent inhibitors of the uptake of dopamine, norepinephrine, and serotonin (in rat brain synaptosomes) ever reported. Compounds of particular note, in this regard, are 52b, 29b, 22b, and 48b, respectively. Biological activity was chiefly manifested by the trans isomeric class. Also, through resolution of four compounds, 7b, 24b, 37b, and 48b, biological activity was found to be associated with the (+) enantiomer subgroup (salts measured at 589 nm in MeOH), corresponding to the 6S, 10bR absolute configuration for 7b, 37b, and 48b, and the 6R,10bR configuration for 24b. An X-ray determination on (+)-24b X HBr established its absolute configuration; configurations for the other compounds were verified by enantiospecific synthesis starting with (+)-(R)-2-phenylpyrrolidine. Regarding the pendant phenyl ring, diverse substitution patterns were investigated. Those substitutions that were particularly unfavorable were 3',4',5'-trimethoxy (20b), 2',3',4',5',6'-pentafluoro (34b), 2'-trifluoromethyl (38b), 3',5'-bis(trifluoromethyl) (42b), 4'-n-butyl (44b), 2'-cyano (47b), 4'-methylsulfonyl (50b), and 2'-carboxy (58b). Exceedingly potent compounds, in one way or another, were 10b-12b, 22b, 23b, 25b, 28b, 29b, 33b, 45b, 48b, 51b-53b. The pattern of aromatic substitution had a strong impact on selectivity in the uptake tests (NE vs. DA vs. 5-HT). Activity was significantly diminished by methyl substitution of 7b at the 5 (65, 66), 6 (61b), or 10b (60b) position, by changing the phenyl group of 7b to cyclohexyl (67b), benzyl (68b), or H (72), by moving the phenyl group of 7b to the 5 (69) or 10b (70) position, by expansion of ring B to an azepine (78b), and by modification of ring C to an azetidine (77b), piperidine (75b), or azepine (74b). The interaction of selected analogues with various CNS receptors is reported. Little affinity was shown for the muscarinic cholinergic receptor, suggesting a lack of anticholinergic side effects. Interestingly, 24b and 33b displayed a high affinity for the serotonin-2 receptor, analogous to mianserin and clomipramine. After the body of data was reviewed, derivatives 24b and 48b were chosen for advanced development.

Preclinical evaluation of McN-5707 as a potential antidepressant.

Based on its activity in a variety of tests in vivo and in vitro McN-5707 [trans-6-(2-chlorophenyl)-1,2,3,5,6,10b-hexahydropyrrolo- (2,1-a)isoquinoline] is a novel potential antidepressant. McN-5707 blocked tetrabenazine-induced sedation and ptosis in mice and rats, and potently inhibited the uptake of norepinephrine by synaptosomes from rat hypothalamus (Ki approximately 2 nM), and the uptake of serotonin by synaptosomes from rat cerebral cortex (Ki approximately 10 nM). McN-5707 also inhibited the uptake of dopamine by synaptosomes from rat striatum (Ki approximately 40 nM); however, the stereotypic behavior often caused by dopamine uptake inhibitors was not evident in rats at doses of 300 mg/kg (p.o.) or less. In receptor binding assays, McN-5707 potently inhibited ketanserin binding to serotonin 5-HT2 receptors in synaptic membranes from rat cerebral cortex (apparent Ki approximately 8 nM). In mice, McN-5707 antagonized 5-hydroxytryptophan-induced head twitches. Spiperone binding to dopamine D2 receptors in synaptic membranes from rat striatum was weakly inhibited by McN-5707 (apparent Ki approximately 400 nM), as was the binding of WB4101 to alpha-1 adrenergic receptors (apparent Ki approximately 150 nM). McN-5707 was essentially inactive as an inhibitor of [3H]clonidine binding to alpha-2 adrenergic receptors and of [3H]quinuclidinyl benzilate binding to muscarinic receptors. In experiments with guinea pig ileum, McN-5707 weakly antagonized histamine-induced contractions and exhibited virtually no cholinergic or anticholinergic activity. Our observations indicate McN-5707 possesses attributes of both tricyclic and newer atypical antidepressants because it inhibits the uptake of both norepinephrine and serotonin, and blocks 5-HT2 receptors, but lacks some of the anticholinergic and behavioral properties often associated with them.

Cardiac-slowing amidines containing the 3-thioindone group. Potential antianginal agents.

A series of 3-thioindolamidines (and 3-indolamidines) related to mixidine (1) was studied for cardiac-slowing properties, following the discovery of activity for prototype thioindole 2. Structure-activity relationships were explored, leading to many potent antitachycardiac agents (6-9, 12, 13, 15-17, 20, 23, 24, 30, 34, 35, 45, and 47-49). Relative to 2, cardiac-slowing activity is enhanced by substitution of the indole nitrogen with small (C1-C3) saturated alkyl groups (6-9), unsaturated alkyl groups (12, 13, and 15-17), or a methoxyethyl group (20); replacement of the N-methyl group with alkyl (23) or phenyl groups (24); and extension of the ethylene bridge by two methylene units (34). Dethio (i.e., 3-indole) analogues of 2 with alkyl substitution on the indole nitrogen (47-49) have greater activity as well. Several potent compounds were also found to have minimal myocardial depression (6-9, 13, 45, and 47). Secondary pharmacological testing is reported for thioindoles 2, 6, 7, 9, and 28.

Synthesis and stereochemistry of 7-phenyl-2-propionanilidobenzo[a]quinolizidine derivatives. Structural probes of fentanyl analgesics.

The for diastereomers of N-(1,3,4,6,7,11b-hexahydro-7-phenyl-2H-benzo[a]quinolizin-2-yl)-N-phenylpropanamide (7c, 7d, 9c, and 9d), which are conformationally restricted analogues of fentanyl, were synthesized and separately tested for analgesic activity and affinity for the opiate receptor of rat brain. Stereochemical assignments for 7c, 7d, 9c, and 9d were deduced from NMR spectral analyses. Conformational analysis revealed that the 2 alpha isomers (7d and 9d) exist in solution as mixtures of cis- and trans-fused conformers with ca. 90 and 45% cis form, respectively. Other compounds (12a, 12b, and 14) related to these propionanilides were also prepared, stereochemically characterized, and tested. Weak analgesic activity was observed for 7d, and both 7d and 9d bound to the opiate receptor with an I50 of ca. 1100 and 1500 nM, respectively (ca. 0.5% of fentanyl and 2% of morphine). The analgesic activity of 7d was abolished by the opiate antagonist naloxone.