Cyclic amidines as benzamide bioisosteres: EPC synthesis and SAR studies leading to the selective dopamine D4 receptor agonist FAUC 312

Investigation of conformationally restricted benzamide bioisosteres led to the chiral phenyltetrahydropyrimidine derivative ent2a (FAUC 312) displaying strong and highly selective dopamine D4 receptor binding (K(i(high))=1.5 nM). Mitogenesis experiments indicated 83% ligand efficacy when compared to the unselective agonist quinpirole. The target compounds of type 2 and 3 were synthesized in enantiopure form starting from asparagine.     

Fancy bioisosteres: synthesis and dopaminergic properties of the endiyne FAUC 88 as a novel non-aromatic D3 agonist

Enlargement of the pi-electronic system of the non-aromatic D3 agonist FAUC 73 led to dopaminergic endiynes of type 1 being synthesized via the bromovinyl triflate 7a as a key intermediate when palladium catalyzed coupling reactions were exploited for the introduction of the (aza)alkyne substituents. As the first neuroreceptor active endiyne, FAUC 88 (1c) displayed high and selective dopamine D3 receptor affinity (K(ihigh) = 3.2 nM) and substantial ligand efficacy (72%, EC(50) = 2.5 nM). Similarities between molecular electrostatic potentials induced by the catechol subunit of the genuine neurotransmitter and those of its non-aromatic endiyne bioisostere are discussed.     

Benzamide bioisosteres incorporating dihydroheteroazole substructures: EPC synthesis and SAR leading to a selective dopamine D4 receptor partial agonist (FAUC 179).

Conformationally restricted benzamide bioisosteres were investigated when the chiral phenyldihydroimidazole derivative 4e (FAUC 179) showed strong and highly selective dopamine D4 receptor binding (K(i)high=0.95nM). Mitogenesis experiments indicated partial agonist properties (42%). EPC syntheses of the target compounds of type 4 were performed starting from alpha-amino acids.     

2,4-Disubstituted pyrroles: synthesis,traceless linking and pharmacological investigations leading to the dopamine D4 receptor partial agonist FAUC 356

Solution-phase synthesis and a solid-phase supported approach to piperazinylmethyl substituted pyrroles are described. Receptor binding studies and the measurement of D4 ligand efficacy led to the ethynylpyrrole 1d (FAUC 356) exerting selective D4 binding and substantial ligand efficacy (66%, EC(50)=1.9nM). This activity profile might be of interest for the treatment of ADHD.     

Dopaminergic 7-aminotetrahydroindolizines: ex-chiral pool synthesis and preferential D3 receptor binding

Starting from both isomers of enantiopure asparagine, heterocyclic bioisosteres of the preferential dopamine D3 receptor agonist (R)-7-OH-DPAT were investigated when SAR studies led to the 3-formyl substituted aminoindolizine (S)-1e (FAUC 54) displaying a K(i) value of 6.0 nM for the high affinity D3 binding site. In contrast, D3 affinity of the enantiomer (R)-1e was 300 fold lower.     

Synthesis and biological investigations of dopaminergic partial agonists preferentially recognizing the D4 receptor subtype

Aminomethyl-substituted biaryls bearing a pyrazole or triazole moiety were synthesized and investigated for dopamine and serotonin receptor binding. The N-arylpyrazoles 3b,f,g and 4 revealed Ki values in the subnanomolar range (0.28-0.70 nM) for the dopamine D4 receptor subtype. Employing both mitogenesis and GTPgammaS assays, ligand efficacy was evaluated indicating partial agonist properties. Interestingly, the tetrahydropyrimidine 4 (FAUC 2020) displayed significant intrinsic selectivity for D2(long) over D2(short).     

Fused azaindole derivatives: molecular design,synthesis and in vitro pharmacology leading to the preferential dopamine D3 receptor agonist FAUC 725

Computational studies based on the similarity of molecular electrostatic potential maps initiated the synthesis of the tricyclic target compounds 1 (FAUC 725) and 2. Receptor binding studies at the dopamine receptor subtypes D1, D2(long), D2(short), D3 and D4 showed that the azaindole 1 revealed D3 affinity (K(i)=0.54 nM) comparable to the lead pramipexole and enhanced selectivity over D2 and D4. Mitogenesis experiments indicated substantial intrinsic activity for the D3 selective dipropylamine 1. Based on the structure of (S)-3-PPP, bioisosteric replacement and conformational restriction leading to the test compound 2 was not fruitful.     

Analogues of FAUC 73 revealing new insights into the structural requirements of nonaromatic dopamine D3 receptor agonists

Employing the nonaromatic D3 agonist FAUC 73 as a lead compound, the dopaminergic enynes 1a,b and the diene 2 (FAUC 206) were synthesized via palladium-catalyzed cross-coupling. FAUC 206 showed remarkable D3 affinity and enhanced selectivity over D4 when compared to the lead compound. To learn more about the bioactive structure of the diene moiety, computational studies including DFT-based conformational analysis and calculations of the magnetic shielding properties were performed. The electrostatic properties of the pharmacophoric pi-systems were visualized by diagnostic MEP maps.     

Fancy bioisosteres: synthesis, SAR, and pharmacological investigations of novel nonaromatic dopamine D3 receptor ligands

Structural variations of the lead compound FAUC 88 led to dopaminergic enynes with an extended pi-system when Pd-catalyzed cross coupling reactions were employed for the key reaction steps. The dienyne 9b displayed substantial affinity for the dopamine receptor subtype D3 and remarkable selectivity over D4. Compared to FAUC 88, the novel fancy bioisostere 9b displayed reduced ligand efficacy. DFT-based conformational analysis of the test compound 9b, including the calculation of diagnostic magnetic shielding properties and their comparison with experimentally derived NMR data, indicated a clear energetic preference for the s-trans geometry of the diene substructure.     

Novel azulene derivatives for the treatment of erectile dysfunction

Based on the dopamine D₄ receptor partial agonist FAUC 3019, a series of azulenylmethylpiperazines was synthesized and affinities for the monoaminergic GPCRs including dopamine, serotonin, histamine and α-adrenergic receptor subtypes were determined. Ligand efficacies of the most promising test compounds revealed the N,N-dimethylaminomethyl substituted azulene 11 to be the most potent D₄ partial agonist (EC₅₀ = 0.41 nM). This candidate was investigated for its ability to promote penile erection. Applying an in vivo animal model, test compound 11 turned out to stimulate penile erection in male rats with superior potency in low concentrations when compared to apomorphine.     

Synthesis and radioiodination of selective ligands for the dopamine D3 receptor subtype

Starting from FAUC 365, a series of iodine substituted heteroaryl carboxamides has been synthesized revealing high affinity and selectivity for the dopamine D3 receptor. Binding data showed a 15-560-fold selectivity for the dopamine D3 over D2. A 2,3-dichloro substitution pattern on the phenylpiperazine moiety led to the highest subtype selectivity, whereas the 2-methoxy substituted compounds showed superior D3 affinity. Suitable precursors were radioiodinated with high radiochemical yields (53-85%) leading to potential imaging agents for the D3 receptor by SPET.     

Epidermal growth factor in plasma, serum and urine before and after prolonged exercise

The substance concentration of epidermal growth factor immunoreactivity (EGF IR) and certain other components were studied in plasma, serum and urine from 25 individuals before and after a 2 h cross-country run. The substance concentration of plasma EGF IR increased from a median of 0.10 nM (range 0.04-0.26 nM) to a median of 0.16 nM (range 0.10-0.36 nM) after 2 h of exercise, while serum EGF showed no change. The values obtained for B-platelets were a median of 192 x 10(9)/litre (range 109-282 x 10(9)/litre) before the run, and a median of 265 x 10(9)/litre (range 216-387 x 10(9)/litre) after the run. No correlation was observed between the values obtained for B-platelets and the values for plasma or serum EGF IR. The substance concentration of EGF IR in urine increased from a median of 3.2 nM (range 0.5-7.7 nM) to a median of 7.0 nM (range 1.5-15.7 nM) after the run. Expressed relative to the output of carbamide the output of urinary EGF IR increased with a median factor of 2 following the run. Expressed relative to the output of creatinine no increase was observed.     

Stimulation of spreading and aggregation of platelets on immobilized type V collagen: mechanisms depending on Ca2+ and protein kinase C

The effects of phorbol ester (PMA) and stable prostaglandin endoperoxide analog (U46619) on platelet interaction with a surface coated with monomeric type V collagen (CV substrate) and free Ca2+ concentration in platelet cytoplasm ([Ca2+]in) have been studied. In the absence of PMA and U46619, the discoid and spherical platelets from suspension are attached to CV substrate but are incapable of spreading and aggregation on the substrate. An addition of PMA (0.15-1.5 nM) or U46619 (1.5 microM) to the reaction mixture stimulates platelet spreading and the formation of multilayer (thrombi-like) aggregates on CV substrate. Using the fluorescent probe Quin 2, it was found that U46619 (0.1 microM) increases [Ca2+]in from the basal level (100-120 mM) to 600 nM. PMA (0.75-15 nM) exerts only a slight effect increasing [Ca2+]in by 30-40 nM. The data obtained suggest that the PMA-induced spreading and aggregation of platelets on CV substrate can occur via activation of protein kinase C at relatively low [Ca2+]in values. These results also testify to the existence of a substrate-independent mechanism of spreading of platelets activated in suspensions by soluble inducers.     

Soybean-derived phosphatidylinositol inhibits in vivo low concentrations of amyloid beta protein-induced degeneration of hippocampal neurons in V337M human tau-expressing mice

In our previous reports using primary cultured rat hippocampal neurons, pathophysiological concentrations (< or =10 nM) of amyloid beta proteins (Abetas) showed neurotoxicity via a phosphatidylinositol metabolism disorder, and soybean-derived phosphatidylinositol protected the neurons against the Abeta's neurotoxicity. In the present study, such a neurotoxic effect of Abeta and a neuroprotective effect of phosphatidylinositol were examined in vivo using transgenic mice expressing V337 M human tau. Intrahippocampal CA1 injection of 1.5 mul of 100 nM or 1 microM Abeta25-35 increased the number of degenerating neurons with an apoptotic feature in bilateral hippocampal CA1, CA2, CA3 and dentate gyrus regions in 1 month, demonstrating an in vivo neurotoxic effect of Abeta at lower concentrations after diffusion. Intrahippocampal co-injection or intracerebroventricular administration of 1.5 microl of 500 nM phosphatidylinositol prevented the Abeta25-35-induced neuronal degeneration in all the hippocampal regions, while co-injection of another acidic phospholipid, phosphatidylserine (1.5 microl, 500 nM) with Abeta25-35 showed no protective effects. Thus, exogenously applied phosphatidylinositol appeared to minimize the toxic effects of Abeta in vivo. These results suggest that soybean-derived phosphatidylinositol may be effective in the treatment of Alzheimer's disease.     

Novel generic UPLC/MS/MS method for high throughput analysis applied to permeability assessment in early Drug Discovery

A novel generic ultra performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method for the high throughput quantification of samples generated during permeability assessment (PAMPA) has been developed and validated. The novel UPLC/MS/MS methodology consists of two stages. Firstly, running a 1.5min isocratic method, compound-specific multiple reaction monitoring (MRM) methods were automatically prepared. In a second stage, samples were analyzed by a 1.5min generic gradient UPLC method on a BEH C18 column (50mmx2.1mm). Compounds were detected with a Waters Micromass Quattro Premier mass spectrometer operating in positive electrospray ionization using the compound-specific MRM methods. The linearity for the validation compounds (caffeine, propranolol, ampicillin, atenolol, griseofulvin and carbamazepine) typically ranges from 3.05nM to 12,500nM and the limits of detection for all generically developed methods are in the range between 0.61nM and 12nM in an aqueous buffer. The novel generic methodology was successfully introduced within early Drug Discovery and resulted in a four-fold increase of throughput as well as a significant increase in sensitivity compared to other in-house generic LC/MS methods.     

A comparison of norepinephrine- and ACTH-stimulated lipolysis in white and brown adipocytes of female rats

1. Isolated white and brown adipocytes (WFA and BFA) from the rat were compared with respect to their lipolytic responsiveness towards norepinephrine (NE) and adrenocorticotrophic hormone (ACTH). 2. NE yielded a Km value of 702.7 +/- 30.6 nM for WFA and 142.5 +/- 7.2 nM for BFA. The maximum lipolytic response (Vm) was 145.7 +/- 1.2 nmol glycerol/micrograms DNA/90 min for WFA and 23.7 +/- 0.2 nmol glycerol/micrograms DNA/90 min for BFA. 3. ACTH yield Km values of 31.6 +/- 1.5 and 31.9 +/- 3.1 nM for WFA and BFA, respectively. Vm values of 141.9 +/- 1.0 and 34.2 +/- 0.5 nmol glycerol/micrograms DNA/90 min were observed for WFA and BFA, respectively.     

A fluorescein-methotrexate-based flow cytometric bioassay for measurement of plasma methotrexate and trimetrexate levels

We describe a bioassay for the quantitation of plasma methotrexate and trimetrexate levels employing intact cells. This assay is based on the intracellular saturation of dihydrofolate reductase with fluorescein-methotrexate (F-MTX) and its dose-dependent displacement by methotrexate or trimetrexate as monitored by flow cytometry. Serially diluted methotrexate-containing plasma, representing a wide chemotherapeutic range, produces F-MTX displacement curves similar to those of standard methotrexate solutions. There is no interference by normal plasma components such as folate and its reduced forms. Plasma methotrexate or trimetrexate concentration is the product of the 50% displacing concentration of standard antifolate (IC50) and the reciprocal of the plasma dilution which yields the same displacement. F-MTX competition with standard methotrexate displayed linear displacement from 18.0 +/- 3.1 to 33.7 +/- 1.5 nM (n = 10). The standard trimetrexate calibration curve was linear from 0.28 +/- 0.03 to 1.5 +/- 0.33 nM (n = 8). Thus, the bioassay sensitivities for methotrexate and trimetrexate are at least 18 and 0.3 nM, respectively. Comparison of methotrexate levels in 10 plasma specimens from cancer patients determined by the clinical enzyme inhibition assay and by our bioassay showed a high degree of correlation (r = 0.987).     

Synthesis and anti-HIV activities of symmetrical dicarboxylate esters of dinucleoside reverse transcriptase inhibitors

Three nucleoside analogues, 3'-fluoro-2',3'-dideoxythymidine (FLT), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-dideoxy-3'-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC(50) values of 0.8-1.0nM and 3-4nM against HIV-1(US/92/727) and HIV-1(IIIB) cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC(50)=3-60nM) was improved by 1.5-66 fold when compared to 3TC (EC(50)=90-200nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile.     

Effects of tumor promoters (mezerein, teleocidin and palytoxin) on growth hormone secretion from rat anterior pituitary cells cultured in monolayer

The effects of compounds with tumor promoting activity (mezerein, teleocidin and palytoxin) on rat growth hormone (rGH) release was compared to that of TPA (12-O-tetradecanoyl phorbol-13-acetate). Mezerein and teleocidin both of which are activators of protein kinase C (TPA type tumor promoter), elicited rGH release about 3.5 to 4 fold above control values. The ED 50 was 16 nM for mezerein, 1.1 nM for teleocidin and 1.5 nM for TPA. In contrast to mezerein or teleocidin, a non-TPA type tumor promoter (palytoxin) which does not activate protein kinase C failed to stimulate rGH release. These observations suggest that the activation of protein kinase C is essential in the release of rGH induced by the tumor promoters.     

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.