l-1,2,3,4-Tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine: a potent and selective agonist at alpha 1-adrenoceptors.

1,2,3,4-Tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine (SK&F-89748) has been resolved into d and l enantiomers and characterized pharmacologically. The more active isomer is the l, which has the S configuration as established by single-crystal X-ray diffraction studies. This compound is a potent and selective alpha 1-agonist with an EC50 in the isolated perfused rabbit ear artery of 9 +/- 2 nM. In several preparations, it has shown an alpha 1/alpha 2 selectivity ratio of over 100 and will be a useful tool for characterizing receptor subtypes.

Alpha-adrenergic agents. 1. Direct-acting alpha 1 agonists related to methoxamine.

A series of phenylethylamines related to methoxamine has been prepared and evaluated for direct alpha 1-receptor agonist activity. It has been observed that for open-chain compounds such as methoxamine, in which the amine-containing portion is free to adopt numerous conformations, an hydroxyl group is necessary for direct alpha 1-adrenergic activity. When the hydroxyl is removed, however, the direct component of activity is greatly reduced unless the amine is incorporated into a more sterically defined structure. From our studies we have concluded that in order for a phenylethylamine to be active as a direct alpha 1-receptor agonist it should have a beta nitrogen in a fully extended conformation relative to a substituted phenyl ring. For optimum potency, the nitrogen should be exocyclic to a saturated six-membered ring. It may be further incorporated exocyclic or endocyclic into an additional ring as long as the amine occupies a well-defined region of space relative to the aromatic portion of a molecule. The ED50 values of some of the more potent compounds as alpha 1-receptor agonists are on the order of 1 X 10(-7) M.

N-[2-hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide. A potent agonist which releases intracellular calcium by activation of alpha 1-adrenoceptors.

N-[2-Hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide (SK&F 102652) has been prepared and characterized pharmacologically. It is a potent agonist with an EC50 of 25 nM at alpha 1-adrenoceptors as determined in the isolated perfused rabbit ear artery. On the presynaptic alpha 2-adrenoceptors of the guinea pig atrium it was considerably weaker, demonstrating an EC50 for inhibition of neurotransmission of 1200 nM and thus an overall alpha 1/alpha 2 selectivity ratio of greater than or equal to 48. In the vascular smooth muscle of the canine saphenous vein an EC100 concentration of this agonist in the presence of zero external Ca2+ induced 37.9 +/- 1.4% of the maximal contractile response due to this agent while the endogenous ligand norepinephrine evoked only 14.5 +/- 0.4% of the maximum. In the presence of low (1 microM) external calcium, this agent produced 78.3 +/- 5.3% of maximum while norepinephrine gave 45.3 +/- 7.4%. This agent produces alpha 1-adrenoceptor-mediated contraction primarily by release of intracellular Ca2+ and should provide a useful tool for characterizing alpha 1-receptor subtypes.

alpha-Adrenergic agents. 2. Synthesis and alpha 1-agonist activity of 2-aminotetralins.

Substituted 2-aminotetralins are potent, selective, direct-acting agonists at postjunctional alpha 1 receptors. Within this series, substituent alterations on the ring, as well as on the nitrogen, change the potency of compounds by over three orders of magnitude (EC50 = 12 to greater than 10 000 nM). It has been demonstrated experimentally that substitution at both the 5 and 8 positions of the aromatic ring produces optimum agonist potency. Removal of either substituent results in a loss of potency and efficacy relative to norepinephrine. Substitution at positions 6 and/or 7 is generally detrimental to activity. Methyl, ethyl, or dimethyl substitution on nitrogen is compatible with high agonist potency, while substitution with larger groups is not. The most potent agonist in this series is 5-(thiomethyl)-8-methoxy-2-aminotetralin, which has an EC50 of 12 nM.

Inhibitors of phenylethanolamine N-methyltransferase and epinephrine biosynthesis. 3. Bis[tetrahydroisoquinoline]s.

7,8-Dichloro-1,2,3,4-tetrahydroisoquinoline (SK&F 64139) is a potent inhibitor of phenylethanolamine N-methyltransferase (IC50 = 10 muM) that may have therapeutic utility in man. A series of related compounds in which two 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline molecules have been bridged from nitrogen to nitrogen by an unbranched alkyl chain have been prepared and have demonstrated potent inhibitory properties (0.08 to 2 muM). In contrast simple substitution on the nitrogen of 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline with a variety of substituents gives compounds with greatly diminished inhibitory potencies (IC50 = 2 to greater than 100 muM) relative to SK&F 64139. Kinetic studies with a C6 analogue have shown that it is competitive with respect to phenylethanolamine and uncompetitive with respect to S-adenosylmethionine. The increased potency of some of the bis analogues relative to that seen with the tetrahydroisoquinolines having larger alkyl groups on nitrogen suggests that several of the bis compounds show supplemented or cooperative binding to the enzyme, presumably as a result of the second tetrahydroisoquinoline moiety.

Dopamine agonists related to 3-allyl-6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzaz epi ne-7, 8-diol. 6-Position modifications.

The N-allyl derivative (SK&F 85174) of 6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol (SK&F 82526) retains the DA-1 agonist potency of the latter compound but unlike the parent also shows substantial DA-2 agonist activity. In a previous study of N-substituted benzazepines these combined agonist effects were shown to be uniquely associated with the N-allyl group. A continuation of this research has examined dependency of combined DA-2/DA-1 agonist activities on 6-position modification with the specific objective of developing an agonist with maximum effectiveness and potency at the DA-2 receptor subtype. DA-2 agonist activity was measured in a rabbit ear artery assay, and DA-1 agonist activity was determined in an adenylate cyclase assay. Replacing chloro with bromo retains the activity pattern and the potency of the chloro compound; replacement with a hydrogen causes a decrease of both DA-1 and DA-2 receptor activating potency. Introduction of a 6-methyl group causes loss of DA-2 agonist activity and reduction in DA-1 agonist potency. Substitution with a 6-fluoro provides the best balance of DA-2 and DA-1 agonist activities; this compound was moderately potent in both assays.

Dopamine receptor agonists: 3-allyl-6-chloro-2,3,4,5-tetrahydro- 1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol and a series of related 3-benzazepines.

The N-allyl derivative (SK&F 85174) of 6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-dio l (SK&F 82526) not only retains the exceptional D-1 agonist potency of its parent but also displays reasonably potent D-2 agonist activity, as measured by a dopamine-sensitive adenylate cyclase test and a rabbit ear artery assay, respectively. Several additional N-substituted compounds were prepared to explore the D-2/D-1 agonist relationship. The N-methyl analogue retained good D-2 agonist potency, but this substitution converted D-1 agonist activity into antagonist activity. Most other N-substituents sharply decreased D-2 agonist potency including the N-n-propyl group. This observation was surprising since the introduction of mono- or di-N-n-propyl substituent(s) is commonly linked with retention or enhancement of D-2 agonist potency in other series of dopamine agonists. The N-(2-hydroxyethyl) analogue retains about one-fourth the D-2 potency of SK&F 85174. Several synthetic methods were used to prepare these compounds. N-Allylation of a trimethoxybenzazepine followed by cleavage of the methyl ethers with boron tribromide was the preferred method. Other methods used were direct alkylation of the trihydroxy secondary amine, i.e., SK&F 82526, and an acylation-amide reduction-cleavage method.

Semisynthetic cephalosporins. Synthesis and structure-activity relationships of 7-sulfonylacetamido-3-cephem-4-carboxylic acids.

The synthesis and in vitro and in vivo activities of a series of 7-sulfonylacetamido-3-cephem-4-carboxylic acids with acetoxymethyl or heterocyclic thiomethyl substituents at the 3 position are described. Lengthening the alkyl chain attached to the sulfonyl group increased gram-positive activity but the effect on gram-negative activity was variable. Other structural changes on the 7-acyl side chain resulted in only minor changes in vitro activity. the protective effectiveness in infected mice generally paralleled the in vitro activity, except that the butylsulfonyl derivatives were less protective than predicted by in vitro activity. replacement of the 3-acetoxymethyl by a 3-heterocyclic thiomethyl group resulted in an overall improvement of activity both in vitro and in vivo.

Synthesis and biological activity of some broad-spectrum N-acylphenyglycine cephalosporins.

The synthesis and the in vitro and in vivo antibacterial activities of a series of N-acylated phenylglycine cephalosporins are described. These compounds exhibit activity against a broad spectrum of gram-positive and gram-negative bacteria including some strains of Pseudomonas aeruginosa, a bacterial species normally insensitive to the cephalosporin antibiotics. The cephalosporins were prepared by acylation of cephaloglycin or its 3-tetrazolylthiomethyl analogue. In several cases, the acylations produced mixtures of diastereomeric cephalosporins, the components of which, when separated, showed different levels of antibiotic activity. Optimum activity was obtained when the acyl moiety on the phenylglycine nitrogen contained an oxygen atom centrally located between the amide carbonyl and a carboxyl substituent, preferably in a three- or five-membered ring. Replacement of acetoxymethyl by (1-methyl-1H-tetrazol-5-yl)thiomethyl at the 3 position resulted in overall improvement in activity both in vitro and in vivo. Against a group of P. aeruginosa strains, the best compounds of this series showed activity on the order of carbenicillin.

Semisynthetic cephalosporins. Synthesis and structure-activity relationships of analogues with 7-acyl groups derived from 2-(cyanomethylthio)acetic acid or 2-[(2,2,2-trifluoroethyl)thio]acetic acid and their sulfoxides and sulfones.

The synthesis and in vitro and in vivo activities of a series of cephalosporins having side chains derived from 2-[(2,2,2-trifluoroethyl)thio]acetic acid or 2-(cyanomethylthio)acetic acid and with acetoxymethyl or 3-heterocyclic thiomethyl substituents at the 3 position are described. In both series, increasing the oxidation state of the side-chain sulfur atom from sulfide to sulfoxide/sulfone decreased the in vitro gram-positive activity, but the effect on gram-negative activity was variable and less pronounced. The protective effectiveness in mice infected with Escherichia coli increased as the oxidation level of the side-chain sulfur was raised from sulfied to sulfoxide/sulfone. Replacement of the 3-acetoxymethyl by a 3-heterocyclic thiomethyl group resulted in overall improvement of activity both in vitro and in vivo for all oxidation states.

4-[2-(Di-n-propylamino)ethyl]-2(3H)-indolone: a prejunctional dopamine receptor agonist.

4-[2-(Di-n-propylamino)ethyl]-2(3H)-indolone (1c) (SK&F 101468) is a potent and selective prejunctional dopamine receptor agonist. It caused a dose-related inhibition of the constrictor response to electrical stimulation in the isolated perfused rabbit ear artery (EC50 = 100 nM), and this response was antagonized by (S)-sulpiride (KB = 7 nM). Compound 1c did not stimulate or block dopamine-sensitive adenylate cyclase and did not produce stimulation of the central nervous system in rats. It was prepared from (2-methyl-3-nitrophenyl)acetic acid in a multistep sequence based on the Reissert indole synthesis.

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.

Development of an affinity ligand for purification of alpha 2-adrenoceptors from human platelet membranes.

Human platelets contain alpha 2-adrenoceptors which are negatively coupled to the enzyme adenylate cyclase. In order to better understand the interaction of this subtype of alpha receptor with this key enzyme, we have initiated a program to isolate and characterize the alpha 2-adrenoceptor. This report describes the synthesis and biological characterization of a series of molecules that were prepared as affinity ligands for this purpose. The best of these is 9-(allyloxy)-6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 101253). This compound is an alpha 2-adrenoceptor antagonist, which was obtained by synthetic modification of 6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 86466), a novel antagonist with high affinity for the alpha 2-receptor.

Arylazide photoaffinity probe for alpha 2-adrenoceptors.

An arylazide photoaffinity probe for alpha 2-adrenoceptors has been developed and characterized. The compound, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), had a Ki for the human platelet alpha 2-adrenoceptor of approximately 40 nM. Upon exposure to ultraviolet light, SKF 102229 irreversibly blocked the binding of [3H]yohimbine to both membrane bound and solubilized, partially purified, receptors. The extent of alpha 2-adrenoceptor blockade was dependent upon both the length of exposure to ultraviolet light and the concentration of SKF 102229. Typically, a 60% decrease in alpha 2-adrenoceptor number is obtained following 8 min of photolysis in the presence of 100 nM SKF 102229. The pharmacologic characteristics of the irreversible blockade produced by SKF 102229 were those of an alpha 2-adrenoceptor. Thus, photodependent, irreversible blockade of alpha 2-adrenoceptors by SKF 102229 was prevented by the concomitant presence of phentolamine or p-aminoclonidine but not by prazosin. Given its specificity and efficient blockade of the ligand binding site, SKF 102229 should prove useful for studies of the structure and function of alpha 2-adrenoceptors.

alpha-Adrenergic agents. 3. Behavioral effects of 2-aminotetralins.

Studies with 5-substituted-8-methoxy-2-amino-tetralin compounds suggest that some are alpha 1-adrenoceptor agonists, which readily penetrate the blood-brain barrier. They potentiate the locomotor activity that is induced by apomorphine (AP) in reserpinized mice, an effect that has been suggested to result from activation of central alpha-receptors. This effect is selectively blocked by the preferential alpha 1-antagonist phenoxybenzamine, but not by drugs that block other types of receptors. The effect is also produced by the centrally administered alpha 1-agonists phenylephrine and methoxamine, but not by various types of standard CNS stimulants. When administered in high doses, some of the aminotetralin compounds induce locomotor activity in reserpinized mice without AP, an effect also found with high doses of centrally administered phenylephrine and methoxamine. This effect is blocked by a series of drugs at doses that correspond to their alpha 1-antagonist potencies.

Alpha 1-adrenoceptor-mediated vasoconstriction in vivo to enantiomers of SK & F 89748-A.

The pressor activity of the 1-enantiomer of SK & F 89748-A, 1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine, in pithed normotensive rats was found comparable with that of 1-phenylephrine. The d-enantiomer was half as potent. The log dose-pressor effect curves for d- and 1-SK & F 89748-A were not influenced by reserpine treatment (2 X 5 mg/kg i.p., -48 and -24 h), were virtually unaffected by yohimbine (1 mg/kg i.v., -15 min) but were markedly shifted to the right by prazosin (0.1 mg/kg i.v., -15 min) and phentolamine (1 mg/kg i.v., -15 min). Similar observations were made for the 1-enantiomer in pithed cats. It is concluded that d- and 1-SK & F 89748-A are potent, directly acting highly selective agonists of (vascular) postjunctional alpha 1-adrenoceptors. Potency and selectivity were equally pronounced for both enantiomers. The currently available selective agonists of alpha 1-adrenoceptors, including the optical isomers of SK & F 89748-A, cannot distinguish between alpha 1- and alpha 2-adrenoceptors. This conclusion is based on binding affinity since these affinities are linearly correlated as shown by radioligand displacement experiments.

Assessment of alpha-adrenergic receptor subtypes in isolated rat aortic segments.

The postsynaptic alpha-adrenoceptors in the isolated rat aorta have been characterized according to the sensitivity of the tissue to selective alpha 1- and alpha 2-adrenoceptor agonists and antagonists. The potency (-log EC50) order of the non-selective alpha-agonist norepinephrine and relatively selective agonists was as follows: norepinephrine (alpha 1 = alpha 2; 7.30); clonidine (alpha 2 greater than alpha 1; 7.01); phenylephrine (alpha 1 greater than alpha 2; 6.99), SK & F 89748--A (alpha 1 greater than alpha 2; 6.65); BHT-920 (alpha 2 much greater than alpha 1; 5.56) and M-7 (alpha 2 greater than alpha 1; 4.66). The isolated rat aorta was 12-200-fold more sensitive to the alpha 1-adrenoceptor agonists phenylephrine and SK & F 89748-A, than to the alpha 2-agonists, BHT-920 and M-7. Prazosin is 245-1259-fold more potent than rauwolscine as an antagonist of contractions induced by various alpha 1- and alpha 2-agonists in the rat aorta. These data indicate that constriction of the smooth muscle of the rat aorta to alpha-adrenergic agonists is mediated through alpha 1- but not alpha 2-adrenoceptors.

Pharmacologic differentiation between pre- and postjunctional alpha 2-adrenoceptors by SK&F 104078.

Most alpha 2-adrenoceptor antagonists do not discriminate between pre- and postjunctional alpha 2-adrenoceptors, and this has led to the commonly held belief that pre- and postjunctional alpha 2-adrenoceptors may represent one homogeneous population of receptors. SK&F 104078 has been shown to be a potent antagonist at postjunctional alpha 2-adrenoceptors at concentrations that do not block prejunctional alpha 2-adrenoceptors. Thus, SK&F 104078 is a competitive postjunctional alpha 2-adrenoceptor antagonist in canine and rabbit saphenous veins, canine saphenous artery and human platelet with a dissociation constant of approximately 100 nmol/l. Conversely, SK&F 104078 is inactive as a prejunctional alpha 2-adrenoceptor antagonist in atria from dog, guinea pig, rabbit and rat, and in guinea-pig ileum at concentrations up to 10,000 nmol/l. Likewise, SK&F 104078 has the ability to block postjunctional arterial alpha 2-adrenoceptors in vivo in the pithed rat at doses that do not inhibit prejunctional alpha 2-adrenoceptors in the same model. The results suggest that pre- and postjunctional alpha 2-adrenoceptors may not represent one homogeneous class, but rather are discrete subtypes of the alpha 2-adrenoceptor that may be differentiated by SK&F 104078.

Comparison of central and peripheral alpha 1-adrenoceptors.

A series of alpha-adrenergic agonists and antagonists having diverse chemical structure was examined for both central and peripheral alpha 1-adrenoceptor activity. The agonists tested included several novel aminotetralin derivatives which were potent and selective alpha 1-agonists. Peripheral alpha 1-activity was determined in the isolated rabbit ear artery; central alpha 1-receptor affinity was measured as the ability to inhibit 3H-WB 4101 binding to rat brain homogenates. In the agonist series, an excellent correlation between peripheral alpha 1-activity and central alpha 1-affinity was obtained, providing that partial agonists were excluded. Likewise, the receptor dissociation constant for blockade of the peripheral alpha 1-adrenoceptor correlated well with affinity for the central receptor for all of the alpha-antagonists. These data support the conclusion that central and peripheral alpha 1-adrenoceptors are similar or identical.

Evidence for and against heterogeneity of alpha 1-adrenoceptors.

Recent experimental evidence has suggested that the alpha 1 adrenoceptor may need to be further subdivided. It can no longer be stated categorically that alpha 1-adrenoceptors are present only at postjunctional sites, in view of several reports of alpha 1-mediated modulation of adrenergic and cholinergic neurotransmission. Furthermore, comparison of the pharmacologic characteristics of the alpha 1-adrenoceptor in different species and/or tissues can show clear differences in sensitivity to selective agonists and antagonists, and differences in the degree of dependence on extracellular calcium. However, in other cases, alpha 1-adrenoceptors at diverse sites have been found to have identical characteristics. Furthermore, the subcategories identified by the various selective agents do not fall into the same discrete groups, in contrast to division of alpha-adrenoceptors into alpha 1 and alpha 2-adrenoceptors. Therefore, at this time it seems premature to subdivide the alpha 1-adrenoceptor further.