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A series of pyrimidyl-5-hydroxamic acids was prepared for evaluation as inhibitors of histone deacetylase (HDAC). Amino-2-pyrimidinyl can be used as a linker to provide HDAC inhibitors of good enzymatic potency.

In this paper we present and discuss a novel, simple and easy to implement parametric modeling approach to assess synergy. An extended three parameter log-logistic model is used to analyse the data and calculate confidence intervals of the interaction indices. In addition the model corrects for the bias due to plate-location effects. The analysis is performed with PROC NLMIXED and SAS-code is provided. The approach is illustrated using data coming from an oncology study in which the inhibition effect of a combination of two compounds is studied using 96-well plates and a fixed-ratio design.

A series of (4-chlorophenyl)-alpha-(1-methyl-1H-imidazol-5-yl)azoloquinolines and -quinazolines was prepared. These compounds displayed potent Farnesyl Protein Transferase inhibitory activity and tetrazolo[1,5-a]quinazolines are promising agents for oral in vivo inhibition.

Replacement of the 1-methylimidazol-5-yl moiety in the farnesyltransferase inhibitor ZARNESTRA series by a 4-methyl-1,2,4-triazol-3-yl group gave us compounds with similar structure-activity relationship profiles showing that this triazole is potentially a good surrogate to imidazole for farnesyltransferase inhibition.

The evaluation of structure-activity relationships associated with the modification of the R115777 quinolinone ring moiety displaying potent in vitro inhibiting activity is described.

Biological assay guided fractionation of a dichloromethane extract of Synaptolepis kirkii led to the isolation of four new and five known daphnane-type diterpene orthoesters, whose structure was established by spectroscopic data. Full spectroscopic data of the new and known natural products are reported here for the first time. Pronounced neurotrophic and substantial antileukaemia activities of these compounds were found in in vitro assays.

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H-benzotriazole) is a selective, non-steroidal aromatase inhibitor containing an asymmetric carbon atom. In this paper, we compare the effects of R76713 (racemate) with its enantiomers R83839 (the levo-isomer) and R83842 (the dextro-isomer) on steroid biosynthesis in rat cells in vitro and in the rat in vivo . In rat granulosa cells, aromatase activity was inhibited by 50% at concentrations of 0.93 nM of R76713, 240 nM of R83839 and 0.44 nM of R83842, revealing a 545-fold difference in activity between both enantiomers. Up to 1 μM, none of the compounds had any effect on steroid production in primary cultures of rat testicular cells. Above this concentration all three compounds showed a similar slight inhibition of androgen synthesis with a concomitant increase in the precursor progestins, indicative for some effect on the 17-hydroxylase/17,20-lyase enzyme. In rat adrenal cells none of the compounds showed any effect on corticosterone synthesis. At concentrations above 1 μM there was an increase in the levels of 11-deoxycorticosterone pointing towards an inhibition of the 11-hydroxylase enzyme. This increase was more pronounced for R83839 than for R76713 and R83842. In vivo , in PMSG-primed rats, R83842 reduced plasma estradiol by 50%, 2 h after oral administration of 0.0034 mg/kg, whereas 0.011 mg/kg of R76713 and 0.25 mg/kg of R83839 were needed to obtain the same result. Oral administration of up to 20 mg/kg of the compounds did not significantly affect plasma levels of adrenal steroids in LHRH/ACTH-injected rats. Plasma testosterone was lowered at 10 and 20 mg/kg of R83842 and at the highest dose (20 mg/kg) of R76713 and R83839. In conclusion, the present study shows that the aromatase inhibitory activity of R76713 resides almost exclusively in its dextro-isomer R83842. R83842 exhibits a specificity for aromatase as compared to other enzymes involved in steroid biosynthesis of at least a 1000-fold in vitro as well as in vivo . This confirms the extreme selectivity previously found for the racemate.

During the course of a screening program intended to identify new antiproliferative agents, a new bafilolide metabolite was discovered. R176502 (1) was isolated from the liquid fermentation cultures of a novel Micromonospora species found in African river bottom sediment. It was purified from ethyl acetate extracts using a series of countercurrent chromatographic steps. The structure was determined using 1- and 2-D NMR experiments. Three previously described bafilomycins (bafilomycins A1 (2), B1 (3), and B2 (4)) were also isolated (from other microbial strains). R176502 exhibited potency for inhibition of tumor cell proliferation in the nM range of concentrations.

Chemical compounds synthesized at Janssen Pharmaceutica are commonly used to identify and characterize neurotransmitter receptors in the brain and in the peripheral system through in vitro or in vivo binding techniques. In this regard, they have played a prominent role in advancing our knowledge about dopamine and serotonin receptors; indeed the identification in vitro or in vivo of these receptors, their solubilization, and their purification were made possible by the availability of 3H-haloperidol, 3H-spiperone, and 3H-ketanserin. Other ligands such as 3H-dexetimide and 3H-lofentanil were used to demonstrate, for the first time, axonal transport of muscarinic and opiate receptors. Finally some of these ligands (spiperone, carfentanil, ketanserin) allowed human brain receptors to be visualized through PET scanner.

7-Azido-8-[125I]ketanserin ([125I]AZIK) was characterized as a potent photoaffinity probe for serotonin-S2 receptors. In reversible binding experiments, [125I]AZIK bound with high affinity (Kd = 0.69 nM) to rat frontal cortex membranes. When incubation with [125I]AZIK was followed by UV irradiation, the binding was found to be irreversible. Protection experiments with various drugs demonstrated the serotonin-S2 nature of the photoaffinity labelling. SDS-polyacrylamide gel electrophoresis of the photolabelled membranes allowed one to identify the serotonin-S2 receptor ligand binding site as a single polypeptide with a molecular mass of approx. 67500 Da. [125I]AZIK will be a valuable tool for the elucidation of the serotonin-S2 receptor structure.

7-Azidoketanserin, a potent photoaffinity probe for serotonin-S2 receptors was shown to irreversibly photoinactivate histamine-H1receptors as well. The photolabelling of H1-receptors could be prevented by several selective histamine-H1 antagonists. In guinea pig cerebellum, a brain area that is highly enriched in H1-receptors, photolabelling could be directed exclusively to these receptors by adding a high concentration of pipamperone, which selectively blocks serotonin-S2 receptors. In rat pre-frontal corex, a region that is enriched in serotonin-S2 receptors, pyrilamine was used to block H1-receptors, thereby directing the photolabelling exclusively to S2-receptors.

Serotonin-S2 receptors from rat frontal cortex were solubilized with a mixture of 6.8 mM CHAPS (3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulphonate) and 1.4 M sodium chloride. This new solubilization procedure solubilized about 40% of the membrane-bound serotonin-S2 receptors in an active form. The solubilized receptors were not sedimented after 1 h of centrifugation at 100 000 × g and they passed freely through 0.20 μm filters. The solubilized preparation showed high affinity binding of [3H]ketanserin and revealed a typical serotonin-S2 receptor profile: binding could be displaced by nanomolar concentrations of different serotonin antagonists and by micromolar concentrations of serotonin agonists. Compounds belonging to other pharmacological classes were poorly, or not active. Upon density gradient sedimentation, Svedberg coefficients of approximately 5 S were found on sucrose gradients made with H2O or D2O as the solvent. This was much lower than the value of 11.5 S previously reported from lysophosphatidylcholine-solubilized serotonin-S2 receptors.

A new compound, azapride, i.e. 4-azido-5-chloro-2-methoxy-N-[1-(phenylmethyl)-4-piperidinyl]benzamide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation-induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation-induced inactivation by azapride remained unchanged in the presence of the scavenger p-aminobenzoic acid. These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors. The identification of brain dopamine receptors has been closely associated with the development of neuroleptic drugs: they were for the dopamine receptor what cc-bungarotoxin was for the nicotinic receptor [l]. Indeed, in contrast to hormones and neuropeptides, classical neurotransmitters (acetylcholine, dopamine, 5-hydroxytryptamine, noradrenaline, etc) cannot be used to characterize receptor sites in vitro because of their very low affinity and rapid rate of dissociation 121. Biochemical and pharmacological studies first suggested the existence of dopamine receptors 13, 41; more direct evi

During recent years, a considerable amount of research has been directed towards the characterization and purification of hormone and drug receptors. Neuroleptlc drugs are thought to act by blocking brain dopamine receptors; in vitro binding assays using either [3H] haloperidol [1,2] or [3H] spiperone [3] have provided more direct evidence for this interaction. Although [3H] spiperone was also found to label serotonin-S2 receptors [4], it remains the more appropriate ligand when used with selective displacers since it is capable of labeling dopamine receptors both in vitro and in vivo [5] and even when the receptor has been reduced to a macromolecular form [6]. Dopamine receptors have also been identified in human brain by in vitro binding assays [7]. Photoaffinity labeling has been a valuable tool for the elucidation of the molecular structure of several receptor sites [8,9]. Recently, we developed a photoaffinity compound azapride (4-azido-5-chloro-2-methoxy-N-[l-(phenylmethyl)-4-piperidinyl]benzamide) which is an azide analogue of clebopride, a selective dopamine antagonist [10,11]. The synthesis and the binding characteristics in dog striatum will be described elsewhere [12]. In the present paper, we report on the use of this photoaffinity probe to label dopamine receptors from human brain.

Several [ 3 H] radioligands have been proposed for In vitro biochemical characterization of serotonin-S 2 receptors. Among the most widely used are [ 3 H]spiperone and [ 3 H]ketanserin. Recently, a derivative of ketanserin, [ 3 H]7-aminoketanserin has been Introduced as a high affinity selective serotonin-S 2 receptor ligand with a very low non-specific binding [1]. A major drawback of all these ligands, however. Is the low specific radioactivity especially if one wants to study tissues with low receptor contents. [ 125 I]-Labelled compounds have much greater sensitivity than tritiated probes. The available iodinated serotonin-S 2 receptor ligands [ 125 I]LSD [2] and [ 125 I]MIL (a methylated derivative of [ 125 I]LSD) [3] show, however, the major disadvantage of interacting with other serotonin binding sites at nanomolar ligand concentrations [4]. It therefore seemed desirable to develop a [ 125 I]-labelled compound, based on a selective ligand for the serotonin-S 2 receptor subtype, such as 7-aminoketanserin.