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In common with other producers of fine chemicals, the pharmaceutical industry can deploy flow microreactors to provide a more flexible production regime than is achievable with large-scale batch reactors. With monitoring of output flow, microreactors can be adjusted exquisitely to give enhanced and inherently safer protocols. Bulk production can be achieved through long run times or parallel reactors. However, microreactors can also be used advantageously in pharmaceutical R&D in other ways. This paper describes their use in the creation of a tool for rapid discovery and optimisation of leads to new drugs. Here microreactors are used to create an integrated micro-scaled chemical synthesis and bioassay system able to conduct fast-cycling iterative searches of diverse chemical space as an enhanced method for identifying and optimising novel lead chemotypes. The use of a microreactor to provide point-of-use access to PET ligands is also described. This significant down-scaling and acceleration of the synthesis of potent but short-lived biomarkers of disease could provide a means of significantly shortening the time and resources required to achieve a drug approval.

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The stereoselective control of chemical reactions has been achieved by applying electrical fields in a micro reactor generating controlled concentration gradients of the reagent streams. The chemistry based upon well-established Wittig synthesis was carried out in a micro reactor device fabricated in borosilicate glass using photolithographic and wet etching techniques. The selectivity of the cis (Z) to trans (E) isomeric ratio in the product synthesised was controlled by varying the applied voltages to the reagent reservoirs within the micro reactor. This subsequently altered the relative reagent concentrations within the device resulting in Z/E ratios in the range 0.57-5.21. By comparison, a traditional batch method based on the same reaction length, concentration, solvent and stoichiometry (i.e., 1.0:1.5:1.0 reagent ratios) gave a Z/E in the range 2.8-3.0. However, when the stoichiometric ratios were varied up to ten times as much, the Z/E ratios varied in accordance to the micro reactor i.e., when the aldehyde is in excess, the Z isomer predominates whereas when the aldehyde is in low concentrations, the E isomer is the more favourable form. Thus indicating that localised concentration gradients generated by careful flow control due to the diffusion limited non-turbulent mixing regime within a micro reactor, leads to the observed stereo selectivity for the cis and trans isomers.

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1. We have investigated the bronchodilator potential of type V phosphodiesterase (PDE V) inhibitors in anaesthetized ventilated guinea-pigs using the potent and selective PDE V inhibitor, SK&F 96231. We have compared its activity to that of salbutamol, the PDE III inhibitors, siguazodan and SK&F 95654 and to the PDE IV inhibitor rolipram. 2. Administered as an i.v. infusion SK&F 96231 (0.6 and 1 mg kg-1 min-1, i.v.) caused a slowly developing inhibition of histamine (100 nmol kg-1, i.v.)-induced bronchoconstriction and elevated tracheal cyclic GMP levels in the anaesthetized guinea-pig. SK&F 96231 (0.1 and 0.3 mg kg-1 min-1, i.v.) was without effect on histamine-induced bronchoconstriction. In the presence of a sub-threshold infusion of SNP (0.1 mumol kg-1 min-1, i.v.) there was a marked enhancement of SK&F 96231-induced inhibition of histamine responses such that at infusion rates that were ineffective alone, SK&F 96231 caused a > 50% inhibition of histamine responses. The stimulation of tracheal cyclic GMP accumulation by SK&F 96231 was also potentiated. 3. Administered directly into the airway, SK&F 96231 (300 micrograms in 5 mg lactose carrier) was largely without effect on histamine-induced bronchoconstriction (4.9 +/- 1.9% inhibition). In the presence of SNP (0.1 mumol kg-1 min-1, i.v.) or isosorbide dinitrate (200 micrograms administered by insufflation into the trachea) there was a marked potentiation of the inhibitory activity of SK&F 96231 (40 +/- 4% and 62 +/- 1.8% respectively). 4. Salbutamol and rolipram (3-300 microg by insufflation) caused a dose-related inhibition of histamine responses with a maximum of 91 +/- 2% and 59 +/- 10% respectively. The PDE III inhibitor, siguazodan,was without effect on histamine responses but they were reduced (27.7 +/- 4.8% at 300 microg) by SK&F95654. There was a marked enhancement of the inhibitory activity of rolipram in the presence of SK&F 95654.5. We conclude that SK&F 96231 has weak anti-spasmogenic activity in the guinea-pig in vivo, we suggest that this is primarily a consequence of a low endogenous guanylate cyclase activity in the airway. The potentiation of the anti-spasmogenic activity of SK&F 96231 by SNP suggests that a combination of PDE V inhibitor and guanylate cyclase agonist might provide significant bronchodilator activity.6. We have established that PDE IV inhibitors are bronchodilators when administered directly into the airway of anaesthetized guinea-pigs but that PDE III inhibitors are only weakly active. The marked enhancement of the inhibitory activity of rolipram by the PDE III inhibitor, SK&F 95654, indicates that inhibitors of both PDE III and PDE IV might offer greater potential as bronchodilators than inhibitors of either isoenzyme alone.

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The crystal and molecular structures of 11 6-substituted pyridazinone derivatives: 6-phenyl-3(2H)-pyridazinone-acetic acid (1/1) (1), 6-(4-aminophenyl)- 3(2H)-pyridazinone (2), 6-(4-aminophenyl)- 5-methyl-3(2H)-pyridazinone (3), 6-(4-acetamidophenyl)- 3(2H)-pyridazinone (4), 6-(4-acetamido-2- methoxyphenyl)-3(2H)-pyridazinone (5), 6-(2-aminophenyl)-3(2H)- pyridazinone (6), 6-phenyl-3(2H)- pyrazinone (7), 6-(4-aminophenyl)-4,5-dihydro- 3(2H)-pyridazinone (8), (R)-(-)-6[4-(3-bromopropionamido)phenyl]- 4,5-dihydro-5-methyl-3(2H)-pyridazinone (9), (R)-(-)-6-(4-ammoniophenyl)-4,5- dihydro-5-methyl-3(2H)-pyridazinone (-)-tartrate-dichloromethane-methanol (1/1/1) (10), 4,5-dihydro-6-methyl-3(2H)-pyridazinone (11) have been determined as part of a study to determine the relationship between their cardiovascular properties and molecular structure and dimensions. For the two optically resolved chiral derivatives (9) and (10) the absolute configuration has been determined.

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8-(4-Chlorophenyl)thio-cyclic AMP (8-CPT-cAMP), extensively used as selective activator of cyclic AMP-dependent protein kinase, has been found to be a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). Indeed, 8-CPT-cAMP (IC50 = 0.9 microM) inhibited PDE VA with a potency identical to that of zaprinast. 8-CPT-cAMP was also metabolized by PDE VA at a rate half that of cyclic GMP. The cyclic GMP-inhibited phosphodiesterase (PDE III) (IC50 = 24 microM) and the cyclic AMP-specific phosphodiesterase (PDE IV) (IC50 = 25 microM) were also inhibited by 8-CPT-cAMP. In contrast, most of the other cAMP-derivative studies showed little inhibition of any phosphodiesterase isoenzyme. These observations provide further reasons why the mechanism of the physiological effects of 8-CPT-cAMP should be interpreted with caution.

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1,4-Bis(3-oxo-2,3-dihydropyridazin-6-yl)benzene and a series of related bis(azinone) compounds were synthesized. These novel compounds were evaluated for inhibition of the low Km, cAMP-selective, cGMP-inhibited phosphodiesterase (PDE III) derived from cat heart and hemodynamic activity in the ganglion- and beta-blocked anesthetized cat. The most potent PDE III inhibitor of the series was 6-[4-(5-methyl-3-oxo-2,3,4,5-tetrahydropyridazin-6-yl)-phenyl]p yridazin- 3(2H)-one (IC50 = 0.07 microM), which also retained the greatest inotrope and vasodilator (inodilator) potency (ED50 for first derivative of left ventricular pressure (dLVP/dt(max)) = 0.02 mumol/kg, ED15 for 15% fall in perfusion pressure = 0.01 mumol/kg). The structure-activity relationships observed within the bis(azinone) series were consistent with those reported for formally analogous 6-(4-substituted-phenyl)pyridazin-3(2H)-one-based PDE III-inhibiting inodilators with less-extended phenyl substituents (see e.g. Sircar et al. J. Med. Chem. 1987, 30, 1955, Moos et al. J. Med. Chem. 1987, 30, 1963). PDE III inhibitory potency is associated with overall planar topology of the phenylpyridazinone moiety and the presence of two critically separated electronegative centers. A methyl group at the 5-position of a dihydropyridazinone ring leads to enhanced potency. However, the generally higher levels of PDE III inhibitory potency shown by compounds in the bis(azinone) series relative to earlier 6-(4-substituted-phenyl)pyridazin-3(2H)-one derivatives appears to derive from a closer to optimal separation of two interacting points in the inhibitor molecule achieved through the more extended bis(azinone) structure. Correlation between the pharmacological and PDE III inhibitory activities of compounds in the bis(azinone) series provides additional evidence for PDE III being an important mediator of inodilator action.

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Modelling studies have been carried out on the phosphodiesterase (PDE) substrates, adenosine- and guanosine-3'5'-cyclic monophosphates, and on a number of non-specific and type III-specific phosphodiesterase inhibitors. These studies have assisted the understanding of PDE substrate differentiation and the design of potent, selective PDE type III inhibitors.

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