Rationalizing the activities of diverse cholecystokinin 2 receptor antagonists using molecular field points.

Cholecystokinin 2 receptor antagonists encompass a wide range of structures. This makes them unsuitable candidates for existing 3D-QSAR methods and has led us to develop an alternative approach to account for their observed biological activities. A diverse set of 21 antagonists was subjected to a novel molecular field-based similarity analysis. The hypothesis is that compounds with similar field patterns will bind at the same target site regardless of their underlying structure. This initial report demonstrates a linear correlation between ligand similarity and biological activity for this challenging data set. A model generated with three molecules was used to predict the activity of 18 test compounds, with different chemotypes, with a root-mean-square error of 0.68 pKB units. The ability to automatically derive a molecular alignment without knowledge of the protein structure represents an improvement over existing pharmacophore methods and makes the method particularly suitable for scaffold-hopping.

Scaffold hopping with molecular field points: identification of a cholecystokinin-2 (CCK2) receptor pharmacophore and its use in the design of a prototypical series of pyrrole- and imidazole-based CCK2 antagonists.

A new molecular modeling approach has been used to derive a pharmacophore of the potent and selective cholecystokinin-2 (CCK(2)) receptor antagonist 5 (JB93182), based on features shared with two related series. The technique uses "field points" as simple and effective descriptions of the electrostatic and van der Waals maxima and minima surrounding a molecule equipped with XED (extended electron distribution) charges. Problems associated with the high levels of biliary elimination of 5 in vivo required us to design a compound with significantly lower molecular weight without sacrificing its nanomolar levels of in vitro activity. Two new series of compounds were designed to mimic the arrangement of field points present in the pharmacophore rather than its structural elements. In a formal sense, two of the three amides in 5 were replaced with either a simple pyrrole or imidazole, while some features thought to be essential for the high levels of in vitro activity of the parent compounds were retained and others deleted. These compounds maintained activity and selectivity for this receptor over CCK(1). In addition, the reduction in molecular weight coupled with lower polarities greatly reduced levels of biliary elimination associated with 5. This makes them good lead compounds for development of drug candidates whose structures are not obviously related to those of the parents and represents the first example of scaffold hopping using molecular field points.

Quantitative determination of intermolecular interactions with fluorinated aromatic rings.

The chemical double mutant cycle approach has been used to investigate substituent effects on intermolecular interactions between aromatic rings and pentafluorophenyl pi-systems. The complexes have been characterised using 1H and 19F NMR titrations, X-ray crystal structures of model compounds and molecular mechanics calculations. In the molecular zipper system used for these experiments, H-bonds and the geometries of the interacting surfaces favour the approach of the edge of the aromatic ring with the face of the pentafluorophenyl pi-system. The interactions are generally repulsive and this repulsion increases with more electron-withdrawing substituents up to a limit of +2.2 kJ mol(-1), when the complex distorts to minimise the unfavourable interaction. Strongly electron-donating groups cause a change in the geometry of the aromatic interaction and attractive stacking interactions are found (-1.6 kJ mol(-1) for NMe2). These results are generally consistent with an electrostatic model: the polarisation of the pentafluorophenyl ring leads to a partial positive charge located at the centre and this leads to repulsive interactions with the positive charges on the protons on the edge of the aromatic ring; when the aromatic ring has a high pi-electron density there is a large electrostatic driving force in favour of the stacked geometry which places this pi-electron density over the centre of the positive charge on the pentafluorophenyl group.

2,7-Dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1,4]diazonine as a new template for the design of CCK(2) receptor antagonists.

A novel series of nonpeptide CCK(2) receptor antagonists has been prepared, in which 2,7-dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1, 4]diazonine (5) was used as a chemical template. This uncommon ring system was obtained in a highly substituted form and in high yield by ozonolysis of the enamine bond of 1,2,3,4-tetrahydro-9H-pyrido[3, 4-b]indole derivatives (4), in which the configuration of the substituents was established stereoselectively via the Pictet-Spengler reaction. Further structural manipulation was guided by molecular modeling through comparison of fieldpoint-based structures of candidate compounds with a selected low-energy conformation of the representative CCK(2) receptor antagonist 5-[[[(1S)-[[(3, 5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyl]amino]carbonyl]-6- [[( 1-adamantylmethyl)amino]carbonyl]indole (JB93182 (3)). By this approach compounds such as (3R, 5S)-4-acetyl-3-(1-adamantyl)methyl-1-(2-chlorobenzyl)-5-carboxymet hyl aminocarbonyl-2,7-dioxo-2,3,4,5,6,7-hexahydro-1H-benzo[h][1, 4]diazonine (32) were prepared. Compound 32 behaved as a competitive CCK(2) receptor antagonist in vitro as judged by its inhibition of pentagastrin-stimulated acid secretion in an isolated, lumen-perfused, immature rat stomach assay (pK(B) = 6.74 +/- 0.27) and by its displacement of [(125)I]CCK-8S from CCK(2) sites in mouse cortical homogenates (pK(i) = 6.99 +/- 0.05). Compound 32 was 100-fold selective for CCK(2) over CCK(1) receptors based on the affinity estimate obtained in a guinea pig pancreas radioligand binding assay (pK(i) = 5.0).

Extended electron distributions applied to the molecular mechanics of some intermolecular interactions. II. Organic complexes.

Extended electron distributions (XEDs) have been used to simulate the formation of complexes by intermolecular interaction via: (i) aromatic stacking; and (ii) hydrogen bonding. The results qualitatively reproduce experimental observations. In contrast, atom-centred partial charges fail to reproduce highly hydrogen-bonded systems, but make little difference in cases where interactions are driven largely by van der Waals forces. The dielectric constant used in the Coulombic term has been shown to be significant in defining the type and properties of these interactions when XEDs are employed. Some consideration has been given to solvation and entropy effects.

Non-peptide cholecystokinin-B/gastrin receptor antagonists based on bicyclic, heteroaromatic skeletons.

A series of potent and selective cholecystokinin-B/gastrin receptor antagonists based on the dibenzobicyclo[2.2.2]octane (BCO) skeleton which have recently been described were found to show species-dependent behavior when examined in rat and dog models. We now report the discovery of compounds in which the BCO skeleton has been replaced with bicyclic, heteroaromatic frameworks, such as a 5,6-disubstituted indole or benzimidazole. These new ligands maintain the affinity and selectivity profile of the previous compounds in vitro but show a much more consistent behavior pattern in vivo. Representative examples of this class of compound have been shown to inhibit pentagastrin-stimulated acid secretion when administered intravenously at doses of 0.1 mumol kg-1 or less.

Multiconformational composite molecular potential fields in the analysis of drug action. I. Methodology and first evaluation using 5-HT and histamine action as examples.

The quality of molecular electrostatic maps generated by non-quantum mechanical methods has been improved using extended electron distributions. Further simplification has been achieved by distilling these maps down to their energy extrema. A new means of defining surface interaction has been added and the resulting composite map has been plotted for a limited number of low-lying conformers of a series of agonists and antagonists of the H2 and H3 receptors and 5-HT1A and 5-HT1D receptors. The results from the cross-comparison of these maps indicate their ability to distinguish the specific receptor. Interesting consequences of the method are that structural overlay is irrelevant, that several conformations may contribute to the overall binding pattern and that lesser pharmacological activities may be deduced from the results.

The matching of electrostatic extrema: a useful method in drug design? A study of phosphodiesterase III inhibitors.

Ligands which bind to a specific protein binding site are often expected to have a similar electrostatic environment which complements that of the binding site. One method of assessing molecular electrostatic similarity is to examine the possible overlay of the maxima and minima in the electrostatic potential outside the molecules and thereby match the regions where strong electrostatic interactions, including hydrogen bonds, with the residues of the binding site may be possible. This approach is validated with accurate calculations of the electrostatic potential, derived from a distributed multiple analysis of an ab initio charge density of the molecule, so that the effects of lone pair and pi-electron density are correctly included. We have applied this method to the phosphodiesterase (PDE) III substrate adenosine-3',5'-cyclic monophosphate (cAMP) and a range of nonspecific and specific PDE III inhibitors. Despite the structural variation between cAMP and the inhibitors, it is possible to match three or four extrema to produce relative orientations in which the inhibitors are sufficiently sterically and electrostatically similar to the natural substrate to account for their affinity for PDE III. This matching of extrema is more apparent using the accurate electrostatic models than it was when this approach was first applied, using semiempirical point charge models. These results reinforce the hypothesis of electrostatic similarity and give weight to the technique of extrema matching as a useful tool in drug design.

Extended electron distributions applied to the molecular mechanics of some intermolecular interactions.

Extended electron distributions (XEDs) have been added to the molecular mechanics Coulombic term and applied to a selection of intermolecular interactions. The results from this approach have been compared with the commonly used atom-centred charges and more rigorous AM1-derived natural atom orbital point densities. The use of XEDs generally improves the simulation of experimental and ab initio results over the other two charge allocations and corrects geometries in those cases for which the others yield wrong results.

Crystal and molecular structures of pyridazinone cardiovascular agents.

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.

The effect of endotoxin-induced fever on thermoregulation in the newborn rabbit.

1. Metabolic responses of endotoxin-injected newborn rabbits were measured in a closed circuit calorimeter at a constant environmental temperature within their thermoneutral range. Oxygen consumption and colonic temperatures were then measured over a range of environmental temperatures from 21.0 to 40.0 degrees C and the responses of endotoxin-injected rabbits compared with non-injected litter mates. 2. To measure their preferred thermal environment, endotoxin-injected and non-injected litter mates were allowed to settle on a thermal gradient and their colonic temperatures measured. 3. In a constant environmental temperature of 37 degrees C, rises in oxygen consumption and colonic temperature following endotoxin injection were found to be biphasic. Oxygen consumption rose from 21 ml kg-1 min-1 to a maximum 35 ml kg-1 min-1 and colonic temperature rose from 39.0 to 39.8 degrees C. 4. The maximal rate of oxygen consumption was the same in both injected and non-injected animals, 53 ml kg-1 min-1, being provoked at the same environmental temperature of 24 degrees C. Minimal rates of oxygen consumption were also similar for the two groups but in the injected animals they were achieved at an environmental temperature of 39 degrees C, 2 degrees C higher than for non-injected animals. 5. We conclude that newborn rabbits challenged with a pyrogen have both behavioural and physiological responses. The thermogenic response is consistent with a change in sensitivity to feedback information rather than a simple shift in the central thermoregulatory set point. We find no evidence to support the view that in the febrile response a higher body temperature necessitates an increase in metabolic rate, the so-called Q10 effect.

Age-related changes in endotoxin sensitivity and the febrile response of newborn rabbits.

Thermoregulatory responses to pyrogens in the immediate newborn period have been reported suppressed or undeveloped. In this study we investigated these responses in the newborn rabbit. Endotoxin (Escherichia coli LPS) was injected intraperitoneally in rabbits at different doses on day 0 (day of delivery), 1 or 3 of life, each animal being used only once. Oxygen consumption (VO2) and colonic temperature (Tc) were measured continuously for up to 5 h following endotoxin administration. On all days, a thermogenic response of a similar pattern was seen. At a low dose of endotoxin, there was a monophasic rise in VO2 that reached a peak at 2 h, followed by a plateau phase lasting a further 2-3 h. At an intermediate dose the response was biphasic with an initial smaller peak after 45-60 min and a second more sustained peak developing by about 2 h. At a higher dose the depression between the two peaks became deeper and longer with the second peak becoming more delayed and weaker. As animals got older, the doses needed to produce this sequence increased. On day 0 the increased VO2 caused by endotoxin was not accompanied by an increase in Tc. By day 3, Tc rose with the increase in VO2. We conclude that rabbits on the day of delivery respond to endotoxin with an increase in VO2 and by day 3 this is accompanied by an increase in Tc. The pattern of VO2, changes from monophasic to biphasic with increasing doses of endotoxin and the sensitivity to endotoxin decreases over the first 3 days of life.

Maternal fever at parturition and its effects on the newborn rabbit.

The febrile response to administration of endotoxin has been reported to be suppressed in both pregnant animals at term and in their newborn. In a previous study we found that newborn rabbits under appropriate conditions to develop a febrile reaction to injected endotoxin. In this investigation we sought to discover whether pregnant rabbits at term had a febrile response to endotoxin, and if so, its effect on thermoregulation in their newborn. Endotoxin (E. Coli LPS) was injected into 19 pregnant rabbits at term. Six delivered spontaneously within an hour. At one hour, 13 were given oxytocin, and a further 8 delivered within five minutes. The colonic temperature (Tc) of the mothers before endotoxin administration and at delivery, and of their young, was measured. The results were compared with those of 10 pregnant rabbits not given endotoxin, and their young. Within 15 min of delivery newborn rabbits from each litter were placed on a thermal gradient to assess their thermoregulatory responses. Pregnant rabbits at term developed an impressive febrile response to injected endotoxin and their young were born with high colonic temperatures. Newborn rabbits from febrile mothers selected higher thermal environments and maintained a higher colonic temperature than the newborn of non-febrile mothers. We conclude that fever is sustained in the first hours of life in the newborn of mothers injected with endotoxin. The possible mechanisms are of considerable interest. None of the pregnant rabbits died after endotoxin administration, but the stillbirth rate was 50% compared with 10% in non-febrile does.

COSMIC(90): an improved molecular mechanics treatment of hydrocarbons and conjugated systems.

Four modifications to the COSMIC molecular mechanics force field are described, which greatly increase both its versatility and the accuracy of calculated conformational energies. The Hill non-bonded van der Waals potential function has been replaced by a two-parameter Morse curve and a new H-H potential, similar to that in MM3, incorporated. Hydrocarbon energies in particular are much improved. A simple iterative Hückel pi-electron molecular orbital calculation allows modelling of conjugated systems. Calculated bond lengths and rotational barriers for a series of conjugated hydrocarbons and nitrogen heterocycles are shown to be as accurate as those determined by the MM2 SCF method. Explicit hydrogen-bonding potentials for H-bond acceptor-donor atom pairs have been included to give better hydrogen bond energies and lengths. The van der Waals radii of protonic hydrogens are reduced to 0.5 A and the energy well depth is increased to 1.0 kcal mol-1. Two new general atom types, N+sp2 and O-sp3, have been introduced which allow a wide variety of charged conjugated systems to be studied. A minimum of parameterisation is required, as the new types are easily included in the Hückel scheme which automatically adjusts bond and torsional parameters according to the defined bond-order relationships.

Molecular modelling approaches to host-guest complexes.

Host-guest interactions can be modelled as a non-bonding recognition process using long-range electrostatic forces. By using molecular isopotential maps the differences between the methotrexate-dihydrofolate reductase and folate-dihydrofolate reductase complexes can be predicted. By extending the technique to molecule-molecule docking the interaction of formamide with the crown ether 18-crown-6 can be simulated with reasonable accuracy. The closely related problem of predicting the separation of enantiomers of chiral molecules by chromatography has been attempted with encouraging results. A preliminary report is presented on the progress being made towards a better model for simulating stacking arrangement of pi systems by charge distribution.

Forces in molecular recognition: comparison of experimental data and molecular mechanics calculations.

NMR studies of the rotation barrier of the disaccharide of the glycopeptide antibiotic vancomycin have been used to test the performance of computer simulation techniques using molecular mechanics. In the absence of any solvated water, no correlation could be found between experiment and calculation. By introducing solvent water molecules into the binding region of the antibiotic, the NMR results could be simulated both qualitatively and quantitatively within experimental error without using massive computational resources.

The effect of limit feeding on thermogenesis and thermoregulation in genetically obese (ob/ob) mice during cold exposure.

Thermogenesis and thermoregulation in ad-lib-fed and limit-fed lean (+/ob or +/+) and obese (ob/ob) mice during acute cold exposure were studied by measuring oxygen consumption and body temperature. No significant differences in oxygen consumption were found between the lean ad-lib, obese ad-lib- or obese limit-fed groups. The oxygen consumption of the lean-limit-fed group was decreased by 25-30 per cent compared with the other groups. The body temperature of the obese ad-lib-fed group fell at a rate of at least twice that of any other group. The weight, total cytochrome c oxidase activity and protein content of the brown adipose tissue (BAT) of the lean groups was similar, and there appeared to be little difference in cell size or fat content. The BAT of both obese groups showed a several-fold increase in weight, and a 50 per cent increase in total protein, compared with the lean groups. The limit-fed obese group showed a significant increase in cytochrome c oxidase activity compared with all other groups. The BAT cells of both obese groups were much enlarged and contained considerable amounts of fat. These observations indicate that the susceptibility of obese mice to hypothermia is not due to a reduced capacity for thermogenesis, but to a failure to conserve heat. Failure of thermoregulation in obese animals may be due to postural constraints that result in increased heat loss by radiation. The results are discussed in relation to the accredited role of BAT thermogenesis in rodents exposed to the cold.

Strategic approaches to drug design. II. Modelling studies on phosphodiesterase substrates and inhibitors.

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.