Development of GPIIb/IIIa antagonists as antithrombotic drugs.

Thrombosis represents a major target for development of drugs to prevent and treat a variety of cardiovascular and cerebrovascular diseases, which are the leading cause of morbidity and mortality in the Western world. This review by Andy Nichols and colleagues focuses on a central process in thrombosis, namely platelet aggregation, and how it can be inhibited by antagonists of the adhesion molecule GPIIb/IIIa. Successful and future therapeutic applications of GPIIb/IIIa antagonists, and their pharmacology, are considered in detail.

Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat.

An orally active, nonpeptide Arg-Gly-Asp (RGD) mimetic alpha(v)beta3 antagonist, (S)-3-Oxo-8-[2-[6-(methylamino)-pyridin-2-yl]-1-ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepine-4-acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds alpha(v)beta3 and the closely related integrin alpha(v)beta5 with low nanomolar affinity but binds only weakly to the related integrins alpha(IIb)beta3, and alpha5beta1. Compound 1 inhibited alpha(v)beta3-mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast-mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose-dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 approximately 20 microM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)-induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX-induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX-induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (microCT) and static histomorphometry. Furthermore, both the 10-mg/kg and 30-mg/kg doses of compound prevented the OVX-induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the alpha(v)beta3 antagonist compound 1 inhibits OVX-induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast-mediated bone resorption, ultimately preventing cancellous bone turnover.

A novel constrained reduced-amide inhibitor of HIV-1 protease derived from the sequential incorporation of gamma-turn mimetics into a model substrate.

C7 mimetics, designed to lock three amino acid residues of a peptide chain into a gamma-turn conformation, were introduced sequentially between the P3 to P2' positions of a model HIV-1 protease substrate I (resulting in compounds II-IV) to probe its conformational requirements in binding to HIV-1 protease. Of these, compound IIIa with the C7 mimetic replacing Asn-Tyr-Pro, corresponding to the P2 through P1' positions of substrate, was found to be an inhibitor with a Ki of 147 microM. Reduction of the amide bond in the C7 mimetic of IIIa resulted in a novel constrained reduced-amide mimetic VIa with a Ki of 430 nM. This corresponds to over a 300-fold improvement in inhibitory activity over the original C7 mimetic. The inhibitory activity of mimetic VIa was in addition found to be 44-fold better than a similar linear reduced-amide containing inhibitor V. The synthesis of these mimetics are described.

Cyclic enkephalin analogues containing alpha-amino-beta-mercapto-beta,beta-pentamethylenepropionic acid at positions 2 or 5.

Analogues of the highly potent and delta-receptor-selective enkephalins 1-4 were prepared with alpha-amino-beta-mercapto-beta,beta-pentamethylenepropionic acid (Apmp) replacing the beta,beta-dimethylcysteine (Pen) at positions 2 or 5. The peptides 5-8 were prepared by employing D,L-Apmp and, following oxidative cyclization, the resulting diastereomeric peptides were separated and purified by preparative high performance liquid chromatography. Compounds 7 and 8, with D- or L-Apmp substituted at position 5 are approximately 5 orders of magnitude more potent in the MVD assay than analogues 5 or 6 with D- or L-Apmp at position 2. While displaying less delta-receptor selectivity than the corresponding Pen-containing compounds, 7 and 8 are an order of magnitude more potent. All the analogues showed diminished delta-receptor selectivity in the rat brain binding assay. Compounds 7 and 8 displayed delta-receptor affinity comparable to the corresponding Pen-containing analogues.

Potent vasopressin antagonists lacking the proline residue at position 7.

As part of a program to design potent antidiuretic vasopressin antagonists and to define the minimum effective pharmacophore requirements for vasopressin (VP) antagonist activity, we studied the importance of the C-terminal tripeptide of a previously reported peptide antagonist of arginine-vasopressin (AVP,1). The proline residue at position 7 in AVP is proposed to impart a conformational constraint to the peptide backbone that is essential for V2-receptor agonist activity. Since the structure-activity relationships for VP agonists and antagonists are different, we investigated the effect of proline on antagonist activity, by synthesizing analogue 3 lacking this residue. This analogue was found to retain a high degree of antidiuretic antagonist activity. Since deletion of the Gly residue at position 9 of the antagonist did not adversely affect VP antagonist potency, several vasopressin antagonist analogues (4-7 and 9) that lacked both the Pro and Gly residues were also studied. These, too, were found to block vasopressin V2-receptor activity. Our results indicate that neither the proline nor glycine residues are essential for antagonism of the V2 receptor.

A minor modification of residue 1 in potent vasopressin antagonists dramatically reduces agonist activity.

[1-(beta,beta-Pentamethylene-beta-mercaptopropionic acid),2-(O-ethyl)-D- tyrosine,4-valine,9-desglycine]arginine-vasopressin (SK&F 101926, 1), a potent in vivo and in vitro vasopressin V2 receptor antagonist, was recently tested in human volunteers and shown to be a full antidiuretic agonist. A new animal model for vasopressin activity has been developed in dogs that duplicates the clinical agonist findings exhibited with SK&F 101926. In this model we have discovered that substitution of a cis-4'-methyl group on the Pmp moiety at residue 1 of vasopressin antagonists results in substantially reduced agonist activity compared to the unsubstituted molecule (SK&F 101926). The corresponding analogue with a trans-4'-methyl group exhibits more agonist activity than the cis molecule. These findings can be explained by viewing the biological activities of compounds such as 1 as the interaction of the vasopressin receptor with a number of discrete molecular entities, conformers of 1, which present different pharmacophores. Models have been developed to assist in the understanding of these results.

Syntheses and in vitro evaluation of 4-(2-aminoethyl)-2(3H)-indolones and related compounds as peripheral prejunctional dopamine receptor agonists.

A series of (beta-aminoethyl)indolones and related compounds was synthesized and evaluated in vitro as peripheral prejunctional dopaminergic agonists in the field-stimulated isolated perfused rabbit ear artery. 4-[2-(Di-n-propylamino)ethyl]-7-hydroxy-2(3H)-indolone was the most potent compound (ED50 = 2 +/- 0.3 nM) tested, while the related secondary amine 24 and the des-OH derivatives 28 and 34 were only slightly less potent. 4-Methoxybenzeneethanamine and 2-methyl-3-nitrophenylacetic acid were employed as starting materials for for the synthesis of the 4-(beta-aminoethyl)indolones. The ring-opened 3-acylamino analogues 46 and 47 were prepared via nitration of the phenethylamine 43 derived from 4-methoxyphenylacetic acid. The inactive isomeric indolones 38, 39, and 41 were derived from 4-nitrobenzeneethanamine and from indolone-6-acetic acid.

A check on rational drug design: crystal structure of a complex of human immunodeficiency virus type 1 protease with a novel gamma-turn mimetic inhibitor.

We have previously reported (Newlander et al., J. Med. Chem. 1993, 36, 2321-2331) the design of human immunodeficiency virus type 1 (HIV-1) protease inhibitors incorporating C7 mimetics that lock three amino acid residues of a peptide sequence into a gamma-turn. The design of one such compound, SB203238, was based on X-ray structures of reduced amide aspartyl protease inhibitors. It incorporates a gamma-turn mimetic in the P2-P1' position, where the carbonyl of the C7 ring is replaced with an sp3 methylene group yielding a constrained reduced amide. It shows competitive inhibition with Ki = 430 nM at pH 6.0. The three-dimensional structure of SB203238 bound to the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction and refined to a crystallographic R-factor (R = sigma magnitude of Fo magnitude of - magnitude of Fc magnitude of /sigma magnitude of Fo magnitude of, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively) of 0.177. The inhibitor lies in an extended conformation in the active site; however, because of the constrained geometry of the C7 ring, it maintains fewer hydrogen bonds with the protein than in most other HIV-1 protease-inhibitor complexes. More importantly, the inhibitor binds to the enzyme differently than predicted in its design, by binding with the P2-P1' alpha-carbon atoms shifted by approximately one-half a residue toward the N-terminus from their presumed positions. This study illustrates the importance of structural information in an approach to rational drug design.

Design, synthesis, and biological activity of a peptide mimic of vasopressin.

Our molecular modeling studies suggested that the conformational effects of the "cystine-line" residue Pmp1-Cys6 on the cyclohexapeptide ring of the vasopressin antagonist [Pmp1,D-Phe2,Val4]AVP might be mimicked by substitution of D-aminoadipic acid at position 6 and cyclization of its side-chain carboxyl to the alpha-amine of residue 2. The peptide was prepared with DL-aminoadipic acid, and following cyclization, the two diastereomeric peptides were separated and purified by preparative high-performance liquid chromatography. The structure of each was confirmed by amino acid analysis and fast atom bombardment mass spectrometry. The chirality of the aminoadipic acid residue of each peptide was determined by chiral gas chromatography. The circular dichroism spectrum of each peptide was run and compared with the appropriate agonist and antagonist peptide standards. These peptides demonstrated in vitro poor V2-receptor affinity and an inability to inhibit or stimulate vasopressin-induced adenylate cyclase formation, suggesting that they lack one or more key features of the agonist/antagonist pharmacophore.

Potent vasopressin antagonists modified at the carboxy-terminal tripeptide tail.

In a continuing effort to design more potent renal vasopressin (V2 receptor) antagonists, we have focused our attention on the carboxy-terminal tripeptide tail (Pro-Arg-Gly-NH2), a fragment common to both agonists and antagonists. Vasopressin antagonist analogues having a dibasic dipeptide tail, e.g., Arg-Arg-NH2 or Arg-Lys-NH2, attached directly to the cyclic hexapeptide ring are potent V2-receptor antagonists. Similar modification of a representative agonist drastically reduces its potency. We report the synthesis and pharmacological properties of a series of potent V2-receptor antagonists 3-9 where a combination of D or L dibasic dipeptide has been utilized to replace the common tripeptide fragment. Our results suggest a difference in the way agonists and antagonists bind to vasopressin receptor and further support the difference in the structure-activity relationships of agonists and antagonists. These results provide potentially useful insights for the design of novel V2-receptor antagonists.

Structure-activity relationships of novel hematoregulatory peptides.

Hematopoiesis is a lifelong cell renewal process regulated by a family of lineage specific hematopoietic growth factors. Several hematopoietic growth factors such as G-CSF, GM-CSF, and M-CSF have been clinically evaluated for enhancement of host defense in normal and immunocompromised patients and for the treatment of infectious diseases. This paper reports the structure-activity relationships of low molecular weight hematoregulatory peptides based on a nonapeptide (1, SK&F 107647). Like the macromolecular growth factors, these peptides modulate host defense. A molecular target for this class of compounds has not yet been identified. However, the structure-activity relationships established by this study implicate a very specific molecular recognition event that is pivotal for the biological activities of 1 and its analogues.

Conformational preferences in a benzodiazepine series of potent nonpeptide fibrinogen receptor antagonists.

Previously, we reported the direct design of highly potent nonpeptide 3-oxo-1,4-benzodiazepine fibrinogen receptor antagonists from a constrained, RGD-containing cyclic semipeptide. The critical features incorporated into the design of these nonpeptides were the exocyclic amide at the 8-position which overlaid the Arg carbonyl, the phenyl ring which maintained an extended Gly conformation, and the diazepine ring which mimicked the gamma-turn at Asp. In this paper, we investigate conformational preferences of the 8-substituted benzodiazepine analogues by examining structural modifications to both the exocyclic amide and the seven-membered diazepine ring and by studying the conformation of the benzodiazepine ring using molecular modeling, X-ray crystallography, and NMR. We found that the directionality of the amide at the 8-position had little effect on activity and the (E)-olefin analogue retained significant potency, indicating that the trans orientation of the amide, and not the carbonyl or NH groups, made the largest contribution to the observed activity. For the diazepine ring, with the exception of the closely analogous 3-oxo-2-benzazepine ring system described previously, all of the modifications led to a significant reduction in activity compared to the potent 3-oxo-1, 4-benzodiazepine parent ring system, implicating this particular type of ring system as a desirable structural feature for high potency. Energy minimizations of a number of the modified analogues revealed that none could adopt the same low-energy conformation as the one shared by the active (S)-isomer of the 3-oxo-1, 4-benzodiazepines and 3-oxo-2-benzazepines. The overall data suggest that the features contributing to the observed high potency in this series are the orientation of the 3-4 amide and the conformational constraint imposed by the seven-membered ring, both of which position the key acidic and basic groups in the proper spatial relationship.

Flushing and haemodynamic responses to vasopressin peptides in the rhesus monkey.

1. The mechanism of the flushing, hypotension and tachycardia associated with i.v. administration of desGlyd(CH2)5D-Tyr(Et)VAVP (SK&F 101926; 25 micrograms kg-1) and the selective V2 antidiuretic agonist, desamino-8-D-arginine vasopressin (dDAVP; 3 micrograms kg-1) was studied in ketamine-anaesthetized rhesus monkeys. 2. The flushing associated with SK&F 101926 was reduced by pretreatment with a mast cell stabilizer and by repeated administration of peptide (within 2-4 weeks). A similar desensitization to dDAVP-associated flushing was observed on repeated administration. 3. Treatment with dDAVP also resulted in reduced SK&F 101926-associated flushing. 4. The hypotension associated with SK&F 101926 was not affected by pretreatment with a mast cell stabilizer. A similar degree of hypotension was observed with repeated administration of either SK&F 101926 or dDAVP. 5. The tachycardia associated with SK&F 101926 was reduced by pretreatment with a mast cell stabilizer or repeated administration of SK&F 101926. Repeated administration of dDAVP, however, resulted in an enhanced tachycardia. 6. Indomethacin (5 mg kg-1 i.v.) did not alter the flushing or the hypotension associated with the administration of either SK&F 101926 or dDAVP, but resulted in an enhanced tachycardia to SK&F 101926. 7. Administration of a selective V1 vasopressor antagonist did not result in flushing, hypotension or tachycardia. 8. It was concluded that the flushing response to vasopressin-like peptides in rhesus monkeys may be due to an action on mast cells, whereas the haemodynamic responses are not, but probably involve direct vasodilator actions.

Vasopressin receptor antagonism in rhesus monkey and man: stereochemical requirements.

The vasopressin antidiuretic (V2) antagonist activity of the position 6 stereoisomers of four vasopressin analogs were tested for water diuretic activity in the rhesus monkey and for activity to inhibit vasopressin-stimulated adenylate cyclase activity in rhesus monkey and human renomedullary tissue in vitro. Replacement of the mercapto groups of the cysteine residues with methylene groups resulted in compounds having similar in vitro potencies to their disulfide analogs; however, these 'dicarba' compounds demonstrated more potent aquaretic activity. Position 6 D enantiomers were associated with less vasopressin antagonist activity in vitro in both species. Based upon these studies, the most potent aquaretic structure identified was the dicarba analog SK & F 105494.