Keith R.F. Elliott

New imidazo(1,2-a)pyrazine derivatives with bronchodilatory and cyclic nucleotide phosphodiesterase inhibitory activities.

New imidazo[1,2-a]pyrazine derivatives have been synthesized either by direct cyclization from pyrazines or by electrophilic substitutions. The presence of electron donating groups on position 8 greatly enhances the reactivity of the heterocycle towards such reactions on position 3 of the heterocycle. The activities of these derivatives in trachealis muscle relaxation and in inhibiting cyclic nucleotide phosphodiesterase (PDE) isoenzyme types III and IV have been assessed. All compounds demonstrated significantly higher relaxant potency than theophylline. All the derivatives were moderately potent in inhibiting the type IV isoenzyme of PDE but only those with a cyano group on position 2 were potent in inhibiting the type III isoenzyme.

Further analysis of the mechanisms underlying the tracheal relaxant action of SCA40.

1 SCA40 (1nm − 10 μm), isoprenaline (1–300 nm) and levcromakalim (100 nm − 10 μm) each produced concentration‐dependent suppression of the spontaneous tone of guinea‐pig isolated trachea. Propranolol (1 μm) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 μm) or 8‐(p)‐sulphophenyltheophylline (8‐SPT; 140 μm). 2 An isosmolar, K+‐rich (80 mm) Krebs solution increased tracheal tone, antagonized SCA40 (approximately 60 fold), antagonized isoprenaline (approximately 20 fold) and very profoundly depressed the log concentration‐effect curve for levcromakalim. Nifedipine (1 μm) did not itself modify the relaxant actions of SCA40, isoprenaline or levcromakalim. However, nifedipine prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by the K+‐rich medium. In contrast, nifedipine did not prevent the equivalent antagonism of levcromakalim. 3 Charybdotoxin (100 nm) increased tracheal tone, antagonized SCA40 (approximately 4 fold) and antagonized isoprenaline (approximately 3 fold). Nifedipine (1 μm) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4 Quinine (30 μm) caused little or no change in tissue tone and did not modify the relaxant action of isoprenaline. However, quinine antagonized SCA40 (approximately 2 fold). Nifedipine (1 μm) prevented the antagonism of SCA40 induced by quinine. 5 Tested on spontaneously‐beating guinea‐pig isolated atria SCA40 (1 nm − 10 μm) increased the rate of beating in a concentration‐dependent manner. Over the concentration‐range 1 μm − 10 μm, SCA40 also caused an increase in the force of atrial contraction. 6 Intracellular electrophysiological recording from guinea‐pig isolated trachealis showed that the relaxant effects of SCA40 (1 μm) were often accompanied by the suppression of spontaneous electrical slow waves but no change in resting membrane potential. When the concentration of SCA40 was raised to 10 μm, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization. 7 SCA40 (10 nm − 100 μm) more potently inhibited the activity of cyclic AMP phosphodiesterase (PDE) than that of cyclic GMP PDE derived from homogenates of guinea‐pig trachealis. Theophylline (1 μm − 10 mm) also inhibited these enzymes but was less potent than SCA40 in each case and did not exhibit selectivity for inhibition of cyclic AMP hydrolysis. 8 Tested against the activity of the isoenzymes of cyclic nucleotide PDE derived from human blood cells and lung tissue, SCA40 proved highly potent against the type III isoenzyme. It was markedly less potent against the type IV and type V isoenzymes and even less potent against the isoenzymes types I and II. 9 It is concluded that the tracheal relaxant action of SCA40 (1 nm − 1 μm) does not involve the activation of β‐adrenoceptors or P1 or P2 purinoceptors. Furthermore, this action is unlikely to depend upon the opening of BKCa channels with consequent cellular hyperpolarization and voltage‐dependent inhibition of Ca2+ influx. The tracheal relaxant action of SCA40 (up to 1 μm) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 μm, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKCa channels.

Mechanical, biochemical and electrophysiological studies of RP 49356 and cromakalim in guinea-pig and bovine trachealis muscle.

Experiments have been performed using guinea-pig and bovine trachealis in order to determine whether cromakalim and RP 49356 share the same relaxant action and to analyse the mechanisms underlying this action. RP 49356 was approximately 3 times less potent than cromakalim in suppressing the spontaneous tone of guinea-pig trachea and, like cromakalim, was antagonised by glibenclamide and by phentolamine. Biochemical studies showed that relaxant concentrations of cromakalim and RP 49356 did not alter the cAMP or cGMP content of guinea-pig trachealis muscle and did not inhibit cAMP or cGMP hydrolysis by tracheal homogenates. Like cromakalim, RP 49356 caused marked hyperpolarisation of guinea-pig trachealis cells. Patch clamp recording using inside-out membrane patches from bovine trachealis showed that cromakalim, RP 49356, glibenclamide and phentolamine were each without effect on the open state probability (Popen) of large conductance, Ca(2+)-activated K(+)-channels. We conclude that cromakalim and RP 49356 share a similar action in opening K(+)-channels in the trachealis cell membrane. This action probably does not involve the intracellular accumulation of cyclic nucleotides and the channel involved is not the large conductance, Ca(2+)-dependent K(+)-channel.

Analysis of the relaxant effects of AH 21-132 in guinea-pig isolated trachealis

1 Experiments have been performed with the dual intent of analysing the mechanism by which AH 21–132 relaxes airways smooth muscle and determining whether the effects of this compound can be distinguished from those of theophylline. 2 AH 21–132 (0.25–8 μm) and theophylline (1–1000 μm) each caused concentration‐dependent suppression of the spontaneous tone of guinea‐pig isolated trachealis. The maximal effect of AH 21–132 was equivalent to that of theophylline. No evidence was obtained that the tissue became sensitized or desensitized to the action of AH 21–132. 3 Propranolol (1 μm) profoundly antagonized the tracheal relaxant action of isoprenaline but not that of AH 21–132. 4 In indomethacin (2.8 μm)‐treated tissues, tone was induced by K+‐rich (120 mm) Krebs solution, acetylcholine (ACh, 1 mm) or histamine (200 μm). Log concentration‐relaxation curves for AH 21–132, isoprenaline and theophylline were all moved to the right in the presence of the spasmogens, the smallest rightward shift being induced by histamine and the greatest by ACh. While maximal effects of AH 21–132 and theophylline were unaffected by the spasmogens, that of isoprenaline was reduced by KCl and ACh. 5 In tissues treated with indomethacin (2.8 μm), AH 21–132 (0.1–100 μm) inhibited the spasmogenic effects of potassium chloride (KCl), ACh and histamine in a concentration‐dependent manner. The inhibition was characterized by rightward shifts in the spasmogen concentration‐effect curves with depression of their maxima. 6 In tissues treated with both indomethacin (2.8 μm) and ACh (1 mm), the removal of tracheal epithelium caused a small but significant leftward shift in the log concentration‐relaxation curve for AH 21–132 but did not alter that for theophylline. 7 In tissues treated with indomethacin (2.8 μm) and maintained at 12°C, theophylline (0.1–3.2 mm) caused concentration‐dependent spasm. This effect was not shared by AH 21–132. 8 AH 21–132 (0.1–1000 μm) more potently inhibited the activity of cyclic AMP‐dependent than of cyclic GMP‐dependent phosphodiesterase derived from homogenates of guinea‐pig trachealis. Theophylline, too, inhibited these enzymes but was less potent in each case than AH 21–132 and did not exhibit selectivity for the cyclic AMP‐dependent enzyme. 9 It is concluded that AH 21–132 exerts a non‐specific (i.e. effective no matter what agent is used to support tone) relaxant effect on the trachealis muscle which does not involve the activation of β‐adrenoceptors. The profile of the relaxant action of AH 21–132 more closely resembles that of theophylline than that of isoprenaline. However, AH 21–132 can be differentiated from theophylline in that: (a) its relaxant potency is increased by epithelial removal; (b) it does not cause tracheal spasm; (c) it exhibits selectivity as an inhibitor of cyclic AMP‐dependent as opposed to cyclic GMP‐dependent phosphodiesterase. It is possible that the relaxant effects of AH 21–132 are related to its ability to inhibit cyclic nucleotide phosphodiesterases.

Interactions of formylmethionyl-leucyl-phenylalanine, adenosine, and phosphodiesterase inhibitors in human monocytes Effects on superoxide release, inositol phosphates and cAMP

Cessation of the fMLF‐induced burst of human monocyte superoxide release was associated with a rise in cAMP. This was not due to inhibition of phophodiesterase (PDE), the major form of which was the PDE IV isozyme. The action of burst inhibitors did not correlate with cAMP levels: Rolipram, a PDe IV inhibitor, increased cAMP 6‐fold, with minimal effects on the burst; whereas theophylline increased cAMP less than 2‐fold but decreased the burst to less than half. Although theophylline and the adenylate cyclase activator, adenosine, inhibited fMLF‐induced superoxide release, they did not inhibit production of inositol phosphates. Thus, these studies on inhibition of superoxide release implicated neither cAMP nor inositol phosphates.

Detergents modify the form of Arrhenius plots of 5'-nucleotidase activity.

5’-Nucleotidase is an intrinsic glycoprotein of rat hepatocyte plasma membranes [l-3] with its active site on the external surface of the membrane [ 1,4-61. The enzyme has been solubilised with non-ionic detergents, and purified to homogeneity as a lipoprotein complex with tightly-associated endogeneous sphingomyelin [3,7] . Solubilized preparations have been reported to have mol. wt 52 000-237 000 [3,8-123 which may be a reflection of the formation of aggregate species. The activity of the enzyme is modulated by the physical properties of the bilayer in that Arrhenius plots ofits activity show a well defined break at 28”C, characteristic of a lipid phase separation occurring in the outer half of the bilayer [13-l 51. Addition of the local anaesthetic, benzyl alcohol, increases the fluidity of the bilayer, and depresses the temperature of the lipid phase separation by about 6”C, achieving activation of 5’-nucleotidase and a shift in the temperature of the break in its Arrhenius plot down to about 22’C [ 161. Many membrane-bound enzymes are solubilized using detergents, although little is known about the interaction of detergents with the protein species or any effect on the activity and properties of the enzyme. In this paper we demonstrate that soluble preparations of 5’-nticleotidase obtained using different detergents exhibit markedly different Arrhenius plots, the form of which appears to be related to the physical properties of the detergent. 2. Materials and methods

Cholera toxin mediated activation of adenylate cyclase in intact rat hepatocytes.

Cholera toxin exerts its action upon target tissues by causing the irreversible activation of the enzyme, adenylate cyclase [l&22]. This activation is apparently dependent upon the A-subunit of the toxin, NAD’ and ATP fl,2]. The mechanism by which this effect is achieved is probably the ADP-~bosylation of a guanine nucleotide regulatory protein associated with the catalytic unit of adenylate cyclase [3]. In intact cells, cholera toxin first binds rapidly and irreversibly to cell surface receptors, which are believed to be G,, gangliosides [2]. Tbis is followed by a characteristic lag phase, the duration of which appears to vary widely from about 20 mm to about 4 h depending upon cell type [ 1,2,4,5] before the onset of the irreversible activation of adenylate cyclase on the inner surface of the membrane. We have shown in isolated plasma memories from rat liver that both the extent of activation of adenylate cyclase and the lag time of onset could be influenced by manipulating either the NAD’ or cholera toxin concentrations [6]. These lag times however were very short compared with those found using various other types of intact cells w.,4,51. This study reports on our attempts to influence the lag time for onset, and the degree of cholera toxin activation of adenylate cyclase in isolated rat hepatocytes to compare with our observations using isolated rat liver plasma membr~es [6].

The isoenzyme selectivity of AH 21-132 as an inhibitor of cyclic nucleotide phosphodiesterase activity.

The smooth muscle relaxant, AH 21-132, was tested for its inhibitory effect on the cyclic nucleotide phosphodiesterase (PDE) activities fractionated from guinea-pig cardiac ventricle and bovine trachealis muscle. Both tissues yielded significant PDE-I and PDE-II activities. The cardiac ventricle also contained a significant amount of PDE-III whilst the trachealis contained PDE-IV. AH 21-132 inhibited PDE-III and PDE-IV selectively (Ki values 0.30-0.55 microM) compared with PDE-I and PDE-II (Ki values 20-140 microM).

Is the receptor-mediated endocytosis of cholera toxin a pre-requisite for its activation of adenylate cyclase in intact rat hepatocytes?

Cholera toxin exerts its effects on target cells by irreversibly activating the enzyme adenylate cyclase [ 1,2]. This toxin consists of two non-identical subunits which are held together by non-covalent bonds. The A subunit consists of two non-identical peptide chains, Ar and Aa, of which Ar is the active peptide responsible for catalysing the NAD-dependent ribosylation of the guanine nucleotide regulatory unit of adenylate cyclase [3]. Associated with the two A peptides are 5 identical B subunits which each can bind toa G ml ganglioside. These glycolipids act as a cell surface receptor for the toxin molecule. In isolated membranes either cholera toxin itself or the isolated A subunit can activate adenylate cyclase directly [4,5]. However in intact cells there is a characteristic lag period before the onset of activation of adenylate cyclase [ 1,2]. The duration of this lag period in intact hepatocytes is dependent upon cholera toxin concentration and upon the fluidity of the cell plasma membrane [6]. The lag time has also been shown to be related to the cell surface redistribution of fluorescent-labelled cholera toxin [7,8]. These observations led to the suggestion [6] that the lateral redistribution of bound toxin may be of importance to the process that allows the A subunit to gain access to the cytosol surface of the plasma membrane where it can act on the guanine nucleotide regulatory unit of adenylate cyclase. Receptor-mediated endocytosis has been shown to be an important mechanism by which cells rapidly bind and internalise specific extracellular ligands [9-121. A number of compounds interfere with aspects of this process [ 1 I], some of which are the lysomotropic agents, which specifically elevate the

Imidazo[1,2-a]quinoxalines: synthesis and cyclic nucleotide phosphodiesterase inhibitory activity.

A group of imidazo[1,2-a]quinoxalines have been synthesised from quinoxaline by condensation of an appropriate haloester or intramolecular cyclisation of a keto moiety on an intracyclic nitrogen atom. The reactivity of the heterocycle was explored through diverse reactions such as electrophilic substitution, lithiation and halogen-metal exchange to give access to a new series of derivatives. Confirmation of their structure was mainly performed by NMR, after careful assignment of the signals in comparison to previous attributions made on the parent imidazo[1,2-a]quinoxaline and discussion of available data in the literature. The cyclic nucleotide phosphodiesterase inhibitor activity of some of these derivatives has been assessed on isoenzymes type III and type lV. Compound 15, 4-(methylamino)imidazo[1,2-a]quinoxaline-2-carbonitrile, exhibited potent relaxant activity on smooth muscle, with a potency similar to the one measured with SCA 40, its structural analogue in the imidazo[1,2-a]pyrazine series.