Studies with a PNMT inhibitor.

DCTQ (SK&F 64139) is a potent inhibitor of both adrenal and central nervous system (CNS) phenylethanolamine N-methyltransferase (PNMT). In animal studies, a plasma level of 0.35 microgram/ml was associated with 50% inhibition of both adrenal and central PNMT. We performed single-dose phase I studies with DCTQ in man. Plasma drug levels up to 6.26 microgram/ml were readily obtained. There were few subjective and no objective clinical changes. DCTQ did not alter blood pressure or cause CNS symptoms in man. Furthermore, resting plasma and urinary catecholamines did not change after DCTQ. The study suggests that acute inhibition of PNMT under resting conditions is without significant clinical effect.

Inhibitors of phenylethanolamine N-methyltransferase and epinephrine biosynthesis. 2. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonanilides.

1,2,3,4-Tetrahydroisoquinoline-7-sulfonanilides (1-14) related to 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (21,SK&F 29661) were prepared and studied for their ability to inhibit phenylethanolamine N-methyltransferase (PNMT) in vitro. The choice of substituents on the 7-phenyl group of the sulfonanilides was based on the Topliss approach to structure-activity relationship studies. Information about the importance of an acidic hydrogen atom on the sulfonamide nitrogen atom was obtained from the preparation and testing of a tertiary N-methylsulfonanilide (15). Other THIQ's (1,2,3,4-tetrahydroisoquinolines) containing sulfur substituents in the 7 position were prepared and tested and consisted of 7-N-benzyl and 7-N-phenethyl derivatives of SK&F 29661 (16-18) and 7-(phenacylthio)-and 7-(phenacylsulfonyl)-THIQ (19 and 20). The two most potent inhibitors were the 7-p-bromo- and -chlorosulfonanilides, 2 and 6. However, neither was an effective inhibitor of norepinephrine to epinephrine conversion when tested in an in vivo mouse assay at unit doses of 25 or 100 mg/kg.

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.

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.

Tetrahydrothiadiazoloisoquinolines: synthesis and inhibition of phenylethanolamine-N-methyltransferase.

A series of 7,8-fused heterocyclic tetrahydroisoquinolines were synthesized and tested as inhibitors of rabbit adrenal phenylethanolamine-N-methyltransferase (PNMT). 6,7,8,9-Tetrahydro[1,2,3]thiadiazolo[5,4-h]isoquinoline 5 (SK&F 86607) was found to be a potent inhibitor of PNMT with an IC50 similar to that of 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (1, SK&F 64139). The isomeric tetrahydro[1,2,3]thiadiazolo[4,5-h]- and tetrahydro[1,2,5]thiadiazolo[3,4-h]isoquinolines, 13 and 20, were also potent PNMT inhibitors. However, substitution of Cl at position 5 (17) resulted in loss of potency similar to the loss observed in the 5-chloro analogue of 1. The 1,2,5 isomer 20 showed only a small drop in activity at 10(-6) M. All of the thiadiazoles were more potent than the 7,8-benzo-fused analogue 36. Fusion of other five-membered heterocyclic ring systems at the 7,8-position, e.g. triazole 22 and imidazoles 24 and 26, resulted in a decrease of PNMT inhibition. The alpha-adrenoreceptor affinities of 1 and 5 were also compared.

Inhibitors of phenylethanolamine N-methyltransferase and epinephrine biosynthesis. 1. Chloro-substituted 1,2,3,4-tetrahydroisoquinolines.

In a search for inhibitors of epinephrine biosynthesis as potential therapeutic agents, a series of 13 ring-chlorinated 1,2,3,4-tetrahydroisoquinolines was prepared. These compounds were tested initially for their ability to inhibit rabbit adrenal phenylethanolamine N-methyltransferase (PNMT) in vitro. Enzyme-inhibitor dissociation constants, determined for the six most potent members of the series, indicated the following order of decreasing potency: 7,8-Cl2 greater than 6,7,8-Cl3 greater than 7-Cl approximately 5,6,7,8-Cl4 greater than 5,7,8-Cl3. These compounds were subsequently examined for PNMT-inhibiting activity in intact rats and mice. 7,8-Dichloro-1,2,3,4-tetrahydroisoquinoline (13, SK&F 64139) was the most potent member of the series both in vitro and in vivo and is currently undergoing clinical investigation.

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.

Evidence for differing leukotriene receptors in gastric mucosa.

Exogenously administered LTD4 was found to increase pepsin secretion from and decrease the transgastric electrical potential difference across the stomachs of anesthetized cats. The former response was completely blocked by prior administration of a new and selective LTD4 antagonist, L-649,923, while the potential difference response was unaffected. This result, coupled with previous evidence of a difference in leukotriene potency order for the two responses, suggests the presence of functionally different leukotriene receptor subtypes in the gastric mucosa.

Comparison of the effects of SK & F 29661 and 64139 upon adrenal and cardiac catecholamines.

After 7 day dosing with SK & F 64139, an inhibitor of adrenal and CNS PNMT, a stoichiometric relationship is observed between epinephrine decrease and norepinephrine increase in adrenal tissue. However, this relationship is not found with the adrenal PNMT inhibitor, SK & F 29661. SK & F 64139 was found to possess significant adrenal MAO inhibitory activity, but SK & F 29661 does not. Three factors are presented which argue against MAO inhibition as the cause of the catecholamine profile difference. We then performed various studies with SK & F 29661 and 64139 to examine whether enzymes in the norepinephrine biosynthetic pathway are differentially affected by these drugs and found that they were not. However. when rats were dosed with either SK & F 29661 or 64139 over 24 h and the adrenal catecholamine stores were then radioactively labeled with a tracer dose of 3H-norepinephrine, the rate of 3H-norepinephrine disappearance was reduced in the SK & F 64139-treated animals as compared with those given SK & F 29661. A similar result was observed in the heart. These results suggest that SK & F 64139 may reduce norepinephrine turnover in the adrenal gland and heart via an effect not related to peripheral PNMT inhibition. These findings also demonstrate quite clearly that in intact animals all PNMT inhibitors do not produce identical endogenous catecholamine profiles.

Studies on the H2-receptor antagonism of MK-208 in isolated rabbit gastric glands.

Using isolated rabbit gastric glands, the H2-receptor antagonist MK-208 was investigated with respect to its effects on [14C]aminopyrine uptake as an index of gastric acid secretion. In addition to shifting the histamine concentration-response curve to the right in a parallel fashion, the antagonism produced by MK-208 was reversible, contrary to that previously seen in the guinea pig atria. These observations suggest that the H2-receptors responsible for mediating gastric secretion in the rabbit and the chronotropic response in guinea pig atria are different.

Studies on the properties of 3-N-[3-[3-(1-piperidinomethyl)phenoxy]-propyl]amino-4-amino-1,2, 5-thiadiazole-1-oxide (L-643,441) as an H2-receptor antagonist in isolated gastric glands.

The H2-receptor blocking properties of L-643,441 were compared with ranitidine in isolated rabbit gastric glands. [14C]Aminopyrine uptake was used as the indicator of gastric acid secretion. Unlike ranitidine, inhibition by L-643,441 was time-dependent, only partially reversible, and was not surmounted by increasing doses of histamine. These and other findings are similar to those found previously in guinea pig atria, providing further evidence that at the receptor level, the mechanism of action of L-643, 441 is different from standard H2-receptor antagonists such as ranitidine.

The effects of PNMT inhibitors upon cardiovascular changes induced by hemorrhage in the rat.

Rapid bleeding in normotensive Sprague-Dawley rats produces a marked fall in arterial blood pressure and a profound decrease in heart rate. The bradycardia, which is abolished by vagotomy and partially antagonized by atropine, was significantly prolonged by pretreatment with SK&F 64139, a potent in vivo inhibitor of peripheral and central (CNS) phenylethanolamine N-methyltransferase (PNMT). This effect of the drug was accompanied by a prolonged hypotensive period and was not seen with SK&F 29661, a selective inhibitor of peripheral (adrenal) PNMT or with SK&F 72223, a structural analog of SK&F 64139 which has no effect on PNMT. These data suggest that the effects of SK&F 64139 are a result of inhibition of central PNMT and that epinephrine may function as a central neurotransmitter mediating cardiovascular responses to hemorrhage, at least under the conditions of these studies.

Leukotriene effects upon the transgastric potential difference and pepsin secretion.

The effects of leukotrienes on gastric mucosal function in vivo, and on acid and pepsin secretion in vitro were investigated. In cats, treatment with leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) caused significant decreases in the transgastric electrical potential difference (P.D.) and significant increases in pepsin secretion, which returned toward control levels over 30-90 min. No detectable changes were observed in either acid concentration or gastric secretion volume over the entire 210 min experimental period. Leukotriene B4 (LTB4) had no effect upon any of these parameters. Treatment with LTD4 in isolated rabbit gastric glands resulted in significant increases in pepsin secretion, with no changes observed in aminopyrine accumulation (acid secretion). These results indicate that exogeneous LTC4, LTD4 and LTE4 can affect certain gastric mucosal functions.

Studies on renal dopamine receptors with a new agonist.

SK & F 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) is a new dopamine receptor agonist which selectively increased renal blood flow when administered i.v. to dogs at cumulative doses of 3.3-1333 microgram/kg. Consistent changes in arterial blood pressure heart rate and cardiac output were not observed. The renal response, which was mediated locally in the kidney, was not antagonized by adequate blocking doses of atropine, propranolol, metiamide and/or mepyramine nor by reserpinization or treatment with indomethacin. It was inhibited, however, by the selective peripheral dopamine receptor antagonist, bulbocapnine. Perhaps as a result of its action on renal blood flow, SK & F 38393 produced a diuresis in normally hydrated rats which was characterized by an increased excretion of sodium, potassium and chloride and a increased urinary pH. Compounds of this type may be useful in better defining dopaminergic receptors and in the treatment of disease states where renal ischemia is present.

Mechanism for gastric antisecretory effects of desmethylimipramine in rats.

The mechanism for the gastric antisecretory action of desmethylimipramine (DMI) was studied using the pylorus-ligated rat preparation. DMI was approximately 40 times more potent in decreasing gastric acid secretion when given into the lateral ventricles of the brain than when administered intravenously. The antisecretory effects of DMI could be blocked by the alpha 2-adrenoceptor antagonists yohimbine and SK&F 72223 and mimicked by central administration of an alpha 2-agonist. It could not be blocked by the alpha 1-antagonist prazosin or mimicked by alpha 1-adrenoceptor agonists. SK&F 72223 and yohimbine themselves produced small increases in gastric acid, but the increase output by SK&F 72223 failed to reduce the antisecretory response to atropine. Since DMI is not an alpha 2-adrenoceptor agonist, but is a potent inhibitor of norepinephrine uptake, these data suggest that the effects of DMI on gastric acid secretion are mediated indirectly via inhibition of catecholamine uptake at central synapses containing alpha 2-adrenoceptors.

The blockade of alpha 2-adrenoceptors by the PNMT inhibitaor SK&F 64139.

Three tetrahydroisoquinolines were tested for activity on central alpha 2-adrenoceptors by determining their ability to inhibit [3H]clonidine binding to rat cerebral cortical membrane homogenates. The compounds included the PNMT inhibitors SK&F 64139 (7,8-dichloro-1,2,3,4-tetrahydroisoquinoline) and SK&F 29661 (1,2,3,4-tetrahydroisoquinoline 7-sulfonamide) as well as SK&F 72223 (5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline) a structural analogue inactive as a PNMT inhibitor. SK&F 64139 and SK&F 72223 but not SK&F 29661 inhibited [3H]clonidine binding. Studies in the isolated guinea pig atrium confirmed that SK&F 64139 and SK&F 72223 are alpha 2-adrenoceptor antagonists.

Lidamidine inhibits intrinsic contractile patterns of the rat proximal colon.

Lidamidine is a clinically effective antidiarrheal agent that inhibits intestinal secretion, reduces intestinal transit, and inhibits smooth muscle contraction. Yet, its specific effects upon colonic motility have not been thoroughly examined. The purpose of our studies was to examine lidamidine's inhibitory effects upon colonic contractile patterns in the rat and identify the responsible receptor mechanism. Fasted male rats were anesthetised and equipped with an intraluminal cannula positioned at the proximal end of a 10 cm fluid-filled segment of the ascending colon (basal pressure, 10 cm H2O). Intraluminal pressure was monitored by a transducer attached to a closed fluid-filled system. All drugs were administered intravenously by slow infusion. A regular pattern of distinct contractile complexes was observed over a 70 min period. These contractions increased intraluminal pressure to 39 +/- 1.2 cm H2O (mean +/- S.E.), occurred at a frequency of 0.3 per min and lasted from 1 to 2.5 min. Inhibition of these contractile patterns was observed with either atropine (0.1 mg/kg) or lidamidine (3.0 mg/kg). A 20 min pretreatment with idazoxan (3.0 mg/kg) antagonized lidamidine's but not atropine's effect. Trimazosin (1 mg/kg) or propranolol (0.3 mg/kg) pretreatment did not antagonize the lidamidine-generated inhibition. These results indicate that lidamidine inhibits an intrinsically generated, cholinergically controlled pattern of colonic contractions primarily by an alpha 2-receptor-mediated mechanism.

Effect of metoclopramide, bethanechol and the cholecystokinin receptor antagonist, L-364,718, on gastric emptying in the rat.

The prokinetic effects of metoclopramide, bethanechol and L-364,718 on a semisolid meal and solid pellet gastric emptying were evaluated and compared. Each compound increased the rate of meal emptying as measured 90 min post-dose. L-364,718, a non-peptide CCK antagonist, was the most potent of these three agents with statistically significant activity observed at 0.03, 0.1 and 0.3 mg/kg p.o. Only metoclopramide significantly enhanced pellet emptying in a dose-dependent manner (3-30 mg/kg p.o.). The effects of each test agent and the potential physiologic role of cholecystokinin in regulating gastric emptying are discussed.

Effects of ring substitution on the pre- and postjunctional alpha-adrenergic activity of aryliminoimidazolidines.

The pre- and postjunctional alpha-adrenergic agonist potency of a series of aryliminoimidazolidines was determined in the isolated rabbit ear artery. This series included clonidine, an antihypertensive agent thought to act by stimulating brainstem alpha-receptors and known to be a preferentially prejunctional alpha-adrenergic agonist. Although all of the compounds acted preferentially on the prejunctional alpha-adrenoceptor, ring substitution had a dramatic effect on both potency and the degree of selectivity. 2-(3,4-Dihydroxyphenylimino) imidazolidine was both the most potent and most selective prejunctional alpha-agonist in this series.