Evaluation of mass spectrometric methods applicable to the direct analysis of non-peptide bead-bound combinatorial libraries.

Electrospray, matrix-assisted laser desorption, and time-of-flight secondary ion mass spectrometry have been explored as possible methods for the identification of active members of molecular combinatorial libraries. All three methods are found to yield accurate molecular weight information about a target molecule angiotensin II antagonist synthesized on a 40-microns polystyrene bead. Structural identification is also possible by accurate mass measurements to eliminate candidate structures with the same nominal mass and by analysis of the fragmentation patterns. In addition, the secondary ion mass spectrometry measurements yield spatially resolved spectra from a single bead after exposure to a suitable gas which clips the covalent bond at the linking position. All three approaches appear to offer a viable screening strategy of non-peptide libraries without the use of additional molecular tags.

Potent nonpeptide angiotensin II receptor antagonists. 2. 1-(Carboxybenzyl)imidazole-5-acrylic acids.

The further evolution of the imidazole-5-acrylic acid series of nonpeptide angiotensin II receptor antagonists is detailed (for Part 1, see: J. Med. Chem. 1992, 35, 3858). Modifications of the N-benzyl ring substitution were undertaken in an effort to mimic the Tyr4 residue of angiotensin II. Introduction of a p-carboxylic acid on the N-benzyl ring resulted in the discovery of compounds with nanomolar affinity for the receptor and good oral activity. SAR studies of these potent antagonists revealed that the thienyl ring, the (E)-acrylic acid, and the imidazole ring in addition to the two acid groups were important for high potency. Also, overlay comparisons of the parent diacid with both angiotensin II and a representative biphenylyltetrazole nonpeptide angiotensin II receptor antagonist are presented. The parent diacid analog, SK&F 108566 or (E)-3-[2-butyl-1-(4-carboxybenzyl)-1H-imidazole-5-yl]-2-[(2- thienyl)methyl]propenoic acid, is currently in clinical development for the treatment of hypertension.

Imidazole-5-acrylic acids: potent nonpeptide angiotensin II receptor antagonists designed using a novel peptide pharmacophore model.

A series of novel nonpeptide angiotensin II receptor antagonists containing a substituted (E)-acrylic acid has been developed. The overlay of 1, an imidazole-5-acetic acid found in the patent literature, on a novel pharmacophore model of AII suggested that extension of the acid side chain and attachment of a second aryl residue to mimic the C-terminal phenylalanine region of AII would lead to increased activity. A study of extended acid side chains at C-5 of the imidazole nucleus led to the discovery of the (E)-acrylic acid 5 as a promising starting point for further exploration. As predicted by the modeling, substitution of a benzyl group on the acrylic acid side chain to mimic the phenylalanine gave increased potency. An extensive study of the SAR of the newly introduced aromatic ring revealed that electron-rich heteroaryl rings provided improved activity, most notably in the in vivo rat models. Compound 40, (E)-3-[2-butyl-1- [(2-chlorophenyl)methyl]imidazol-5-yl]-2-[(2-thienyl)methyl]-2- propenoic acid, has been shown to be a potent, competitive, and orally active small molecule AT-1 receptor antagonist. It exhibits a 2 orders of magnitude increase in binding affinity and a 10-fold improvement in in vivo potency as compared to compound 1 and represents an important milestone in the development of even more potent nonpeptide angiotensin II receptor antagonists.

Identification of fenoldopam prodrugs with prolonged renal vasodilator activity.

Fenoldopam (SK&F 82526) is a short-acting selective dopamine-1 agonist in clinical trials for the treatment of hypertension, congestive heart failure and renal failure. In the present study, we tested various N-ethyl carbamate esters of fenoldopam in the conscious dog instrumented with a femoral arterial Vascular-Access-Port and a renal artery flow probe. Oral administration of SK&F R-82526 at 1 and 3 mumol/kg resulted in transient (30-60 min) dose-dependent increases in plasma fenoldopam levels and renal blood flow. Administration of the 7,8-bis-N-ethyl carbamate ester of R-fenoldopam (SK&F R-106114) and the 4',7,8-tris-N-ethyl carbamate ester of R-fenoldopam (SK&F R-105058) at 1, 3 and 10 mumol/kg p.o. also resulted in dose-dependent increases in plasma fenoldopam levels and renal blood flow; however, both parameters remained elevated for at least 4 hr. Intravenous administration of SK&F R-105058 also resulted in sustained plasma fenoldopam levels and increases in renal blood flow, indicating that slow absorption was not the cause of the sustained effect. The present study indicates that N-ethyl carbamate esters of fenoldopam are fenoldopam prodrugs which result in sustained increases in renal blood flow and plasma fenoldopam levels.

Agonist and antagonist properties of benzazepine and thienopyridine derivatives at the D1 dopamine receptor.

Nine structurally related 1-phenyl-1H-3-benzazepine derivatives and two thienopyridines were tested for agonist and antagonist properties at the adenylate cyclase-coupled D1 dopamine receptor in homogenates of the striatum of the rat. The benzazepines SK&F 77434 and SK&F 82958, both of which contain a catechol ring, were agonists; the intrinsic activity of SK&F 77434 was similar to that of SK&F 38393, whereas SK&F 82958 was a full agonist. The remaining benzazepines inhibited the stimulation of adenylate cyclase by dopamine. Antagonist potency depended on the nature of the substituent at position 7 of the benzazepine molecule, 7-halogen compounds being the most potent. The Ki values, obtained from analysis of the antagonism of dopamine-stimulated adenylate cyclase, were significantly correlated with the Ki values for displacement of D1 ligands in binding experiments. Furthermore, antagonist activity of the resolved racemic benzazepine SK&F 83566 resided almost exclusively in the R-enantiomer. The thienopyridine derivatives SK&F 89641 and SK&F 89145 were partial agonists with greater efficacies than SK&F 38393.

Synthesis and evaluation of non-catechol D-1 and D-2 dopamine receptor agonists: benzimidazol-2-one, benzoxazol-2-one, and the highly potent benzothiazol-2-one 7-ethylamines.

Our interest in identifying D-1 and D-2 dopamine receptor agonists that are not catechols led us to extend previous studies with oxindoles by investigating analogues of dopamine, N,N-dipropyldopamine, m-tyramine, N,N-dipropyl-m-tyramine, and epinine in which the m-hydroxyl is replaced by the NH portion of a thiazol-2-one, oxazol-2-one, or imidazol-2-one group fused to the 2,3-position. These compounds were evaluated for their affinity and agonist activity at D-1 and D-2 receptors by using in vitro assays. Replacement of the m-hydroxy in N,N-dipropyldopamine with the thiazol-2-one group resulted in a dramatic increase in D-2 receptor affinity and activity compared to that of N,N-dipropyldopamine itself or that of the corresponding oxindole, 1. The resulting compound, 7-hydroxy-4-[2-(di-n-propylamino)ethyl]benzothiazol-2(3H)-one (4), is the most potent D-2 receptor agonist reported to date in the field-stimulated rabbit ear artery (ED50 = 0.028 nM). The benzoxazol-2-one (6), benzimidazol-2-one (5), and isatin (51) analogues showed D-2 receptor agonist potency similar to that of 1. The des-7-hydroxyl analogue of 4 (21) also has enhanced D-2 receptor activity compared to that of the corresponding oxindole, 8. 7-Hydroxy-4-(2-aminoethyl)benzothiazol-2(3H)-one, 27, a non-catechol, has enhanced D-1 and D-2 receptor activity in vitro compared to that of the corresponding oxindole, 7. In vivo, 27 increased renal blood flow and decreased blood pressure in the dog. However, these effects were mediated primarily by D-2 receptor agonist activity. This may be a result of the D-1 partial agonist activity of 27 coupled with its potent D-2 receptor activity.

Synthesis and dopaminergic activity of some halogenated mono- and dihydroxylated 2-aminotetralins.

In a series of 7,8-dihydroxy-1-phenyltetrahydro-3-benzazepine dopamine receptor agonists introduction of a chloro or fluoro substituent into the 6-position increases dopaminergic potency. Also, in this series replacement of the 7-hydroxyl group with a halogen results in inversion of activity from dopamine receptor agonist to antagonist. The present study was aimed at exploring the possibility that the structure-activity observations in the 3-benzazepine series of dopaminergic agents might be extrapolated to another class of dopamine receptor agonists, the 2-aminotetralins. Thus, a series of chloro- and fluoro-substituted mono- and dihydroxylated 2-aminotetralins was prepared and evaluated for dopaminergic properties in D-1 and D-2 receptor-related tests. Introduction of a chloro substituent into the 8-position of the prototype of this series, i.e. 2-amino-6,7-dihydroxytetralin (ADTN), resulted in a compound with a high degree of selectivity for the D-1 subpopulation of dopamine receptors; it was equally or more potent than ADTN in the D-1 receptor-related tests with greatly decreased effectiveness in the tests involving D-2 receptors. A similar effect was observed with 8-fluoro-ADTN; however, the N-(4-hydroxyphenethyl)-N-propyl derivative 4g of the 8-chloro-substituted ADTN showed marked D-2 binding affinity. Conversely, introduction of a chloro substituent into the 5-position of ADTN markedly decreased D-1 receptor affinity and efficacy. This effect was not seen with the related 5-fluoro derivative, suggesting D-1 receptors are more sensitive to bulk in the 5-position of ADTN than are the D-2 receptors. Replacement of either the 6- or 7-hydroxyl groups of ADTN with a chloro or fluoro substituent, in contrast, did not parallel the response seen in the benzazepine series (i.e., the compounds uniformly demonstrated less receptor affinity and did not have dopamine receptor antagonist activity); however, the decrease in agonist potency was less marked in the case of 2-amino-6-fluoro-7-hydroxytetralins than in the chlorinated monohydroxyaminotetralins. Thus, a parallelism in structure-activity relationships in the benzazepine and aminotetralin series of dopamine receptor agonists was not observed. The differences may reflect altered modes of receptor binding in the two series.

Synthesis, resolution, absolute stereochemistry, and enantioselectivity of 3',4'-dihydroxynomifensine.

3',4'-Dihydroxynomifensine, 8-amino-1,2,3,4-tetrahydro-4-(3,4-dihydroxyphenyl)-2-methylisoquinoli ne (1a), is an agonist of dopamine receptors in central and peripheral systems. Since this dopamine receptor agonist bears an asymmetric center at position 4, its synthesis and resolution were undertaken as part of a study directed toward determining the mode of interaction of these agents with the receptor(s). The enantiomers of 3',4'-dihydroxynomifensine are of particular interest, as they provide additional probes of present conceptual models of the dopamine receptor(s). Initial attempts to prepare 1a were inefficient or unsuccessful; instead, an isomeric compound, 1,2,4,5-tetra-hydro-2-(3,4-dihydroxyphenyl)-4- methyl-3H-1,4-benzodiazepine (9), was obtained. For this reason, a new route to 3',4'-dihydroxynomifensine was employed. The racemic dimethoxy intermediate 1d, thus obtained, was resolved. Methoxyl cleavage of the isomers of 1d afforded the enantiomers of 1a. Enantiomeric excess of these antipodes or appropriate derivatives was examined by NMR, CD, and HPLC methods. CD analysis suggests an enantiomeric excess greater than 99%. Determination of the absolute configuration of the enantiomers of 1a was determined by single-crystal X-ray diffractometric analysis. Examination of the isomers in several pharmacological test systems revealed a high degree of enantioselectivity. D-1 dopaminergic activity resides almost exclusively in the S enantiomer. The findings of the study have been employed to suggest an accessory binding site on the dopamine receptor(s) that differs from that advanced earlier. This accessory binding site may be specific for the D-1 subpopulation of dopamine receptors.

Racemic diastereoisomers of 1-amino-2-hydroxycyclopentanecarboxylic acid.

The synthesis and characterization of the two diastereoisomeric forms of 1-amino-2-hydroxycyclopentanecarboxylic acid have been accomplished. A previously reported synthesis produced a racemic mixture of the threonine analog trans-2-hydroxy-1-aminocyclopentanecarboxylic acid (trans with respect to the hydroxy and carboxyl group). The alternate allothreonine analog was produced by conversion of cyclopentene oxide to trans-2-methoxycyclopentanol, followed by oxidation to 2-methoxycyclopentanone and conversion to a hydantoin. Fractional crystallization of the hydantoin sample, followed by hydrolysis, produced cis-2-hydroxy-1-aminocyclopentanecarboxylic acid (cis with respect to the hydroxy and carboxyl group) in high purity. Neither of the isomeric forms significantly inhibited the growth of the bacterial strains examined nor were they effective in inhibiting Jensen sarcoma cells in tissue culture.