The effect of GABA on in vitro binding of two novel non-benzodiazepines, PK 8165 and CGS 8216, to benzodiazepine receptors in the rat brain

The effect of gamma-aminobutyric acid (GABA) on the binding of PK 8165, a quinoline derivative, and CGS 8216, a pyrazoloquinoline, was assessed in two different regions of the rat brain. PK 8165, a compound with reported anxiolytic properties, inhibited [3H]-propyl beta-carboline-3-carboxylate labeled receptors in the cerebellum with an IC50 of 844 nM and 370 nM in the absence and presence of micro M GABA, respectively. GABA (100 micro M) was less effective in the cerebral cortex, decreasing the IC50 value from 280 to 197 nM. In saturation isotherm studies with [3H]-CGS 8216, a benzodiazepine receptor antagonist, GABA (100 micro M) induced a small but significant reduction in the apparent affinity of [3H]-CGS 8216 for benzodiazepine receptors in the cerebral cortex but the Bmax was unchanged.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Synthesis of an affinity ligand ('UPHIT') for in vivo acylation of the kappa-opioid receptor

The isothiocyanate analog (1S,2S-trans-2-isothiocyanato-4,5-dichloro-N- methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective kappa-opioid receptor agonist, U50,488, was prepared as a potential site-directed affinity ligand for acylation of kappa-opioid receptors in vivo. The isothiocyanate (3a) which we have designated UPHIT and its enantiomer (3b) were synthesized in 3 steps starting from optically pure (1S,2S)-(+)-trans-2-pyrrolidinyl-N-methyl-cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to kappa receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 +/- 0.36 nM, whereas 827.42 +/- 5.88 and 115.10 +/- 1.23 nM were obtained for the IC50 value of the 1R,2R enantiomer (3b) and (+/-)-3 respectively. Intracerebroventricular (ICV) injection of 100 micrograms of (+/-)-3 into guinea-pig brain followed by analysis of remaining kappa-binding sites 24 h later revealed that (+/-)-3 depleted 98% of the kappa receptors that bind [3H]U69,593 and 40% of those that bind [3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the kappa-opioid receptor.     

Structure-activity relationship for a series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles: potent subtype-selective inhibitors of N-methyl-D-aspartate (NMDA) receptors.

A series of 2-substituted 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles was synthesized as potential antagonists for the NR1A/2B subtype of N-methyl-D-aspartate (NMDA) receptors. Assayed by electrical recording under steady-state conditions, 7-hydroxy-2-(4-phenylbutyl)- 1,2,3,4-tetrahydropyrido-[3,4-b]indole (30) was the most potent compound in the series having an IC50 value of 50 nM at the NR1A/2B receptors.     

Synthesis and biological evaluation of halogenated naphthyridone carboxamides as potential ligands for in vivo imaging studies of substance P receptors

With the aim of developing new radioligands for in vivo studies of substance P receptors using positron emission tomography or single photon emission computed tomography, 2- and 3-halo naphthyridone-6-carboxamide derivatives were synthesized. Their affinities toward the target receptors were evaluated on CHO cells and compared to the unsubstituted analogue EP 00652218 (IC(50) = 100 nM +/- 20). The IC(50) value was not altered in the case of 2-chloro compound 1 (IC(50) = 100 nM +/- 15) and only slightly reduced for the 2-fluoro and -iodo analogues 6 and 8 (IC(50) = 500 nM +/- 80). A drastic reduction in binding (IC(50) > 1000 nM) was observed for the halogenated compounds 2-5, 7, and 9.     

Agonist-Dependent Internalization of γ-Aminobutyric AcidA/Benzodiazepine Receptors in Chick Cortical Neurons

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.     

Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors

A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [125I]CGP 23,996 (des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3, 14-somatostatin), [3H]naloxone or [3H]DPDPE ([D-Pen2-D-Pen5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the synthesis of our most potent and selective mu opioid receptor compound D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, which we refer to as Cys2Tyr3Orn5Pen7-amide. While this octapeptide exhibited high affinity (IC50 = 2.80 nM) for an apparently single population of binding sites (nH = 0.89 +/- 0.1) and exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM). Thus, Cys2Tyr3Orn5Pen7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors.     

Pharmacology of the leukotriene antagonist verlukast: the (R)-enantiomer of MK-571.

Verlukast (MK-679) (3-[(3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl)[3-(dimethylamino)- 3- oxopropyl)thio)methyl)-thio)propionic acid) is a potent and selective inhibitor of [3H]leukotriene D4 binding in guinea-pig (IC50 = 3.1 +/- 0.5 nM) and human (IC50 = 8.0 +/- 3.0 nM) lung homogenates and dimethyl sulfoxide differentiated U937 cell membrane preparations (IC50 = 10.7 +/- 1.6 nM) but is essentially inactive versus [3H]leukotriene C4 binding in guinea-pig lung homogenates (IC50 values of 19 and 33 microM). Functionally, when tested at 60 nM, it antagonized contractions of guinea-pig trachea (GPT) induced by leukotriene C4, leukotriene D4, and leukotriene E4 (respective-log KB values of 8.6, 8.8, and 8.9) and contractions of human trachea (HT) induced by leukotriene D4 (-log KB value 8.3 +/- 0.2). In contrast, verlukast (20-200 nM) failed to antagonize contractions of GPT induced by leukotriene C4 in the presence of 45 mM L-serine borate. Intravenous (i.v.) and aerosol verlukast antagonized bronchoconstriction (BC) induced in anaesthetized guinea pigs by i.v. leukotriene D4 but did not block BC to arachidonic acid or histamine. Intraduodenal verlukast (0.25 mg/kg) antagonized leukotriene D4 (0.2 micrograms/kg) induced BC in guinea pigs. Oral and aerosol administration blocked leukotriene D4-induced BC in conscious squirrel monkeys. Orally administered compound also blocked ovalbumin-induced BC in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile for verlukast is similar to that of the racemic compound, MK-571. Verlukast is currently in clinical development for the treatment of asthma and related diseases.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 3: Orally active anti-inflammatory agents

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as I kappaB kinase beta (IKK-beta) inhibitors. Modification of a novel IKK-beta inhibitor 1 (IKK-beta IC(50)=1500 nM, Cell IC(50)=8000 nM) at the 4-phenyl ring and 6-phenol group on the pyridine core ring resulted in a marked increased in biological activities. An optimized compound, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile, exhibited excellent in vitro profiles (IKK-beta IC(50)=8.5 nM, Cell IC(50)=60 nM) and a strong oral efficacy in in vivo anti-inflammatory assays (significant effects at 1mg/kg, po in arachidonic acid-induced ear edema model in mice).     

Identification and synthesis of [1,2,4]triazolo[3,4-a]phthalazine derivatives as high-affinity ligands to the alpha 2 delta-1 subunit of voltage gated calcium channel

We have identified and synthesized a series of [1,2,4]triazolo[3,4-a]phthalazine derivatives as high-affinity ligands to alpha 2 delta-1 subunit of voltage gated calcium channels. Structure-activity relationship studies directed toward improving the potency and physical properties of 2 lead to the discovery of 20 (IC(50)=15 nM) and (S)-22 (IC(50)=30 nM). A potent and selective radioligand, [(3)H]-(S)-22 was also synthesized to demonstrate that this ligand binds to the same site as gabapentin.     

Organic phenyl arsonic acid compounds with potent antileukemic activity

A series of 12 organic arsonic acid compounds has been synthesized and evaluated against human B-lineage (NALM-6) and T-lineage (MOLT-3) acute lymphoblastic leukemia (ALL) cell lines. The lead compounds 2-trichloromethyl-4-[4'-(4"-phenylazo)phenylarsonic acid]aminoquinazoline (compound 19, PHI-P518; IC(50)=1.1+/-0.5 microM against NALM-6 and 2.0+/-0.8 microM against MOLT-3) and 2-methylthio-4-(2'-phenylarsonic acid)aminopyrimidine (compound 15, PHI-P381; IC(50)=1.5+/-0.3 microM against NALM-6 and 2.3+/-0.5 microM against MOLT-3) exhibited potent antileukemic activity at low micromolar concentrations.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

Inhibition of progesterone synthesis in normal and transformed human placental cells by tight binding inhibitors of 3 beta-hydroxysteroid dehydrogenase

The biosynthesis of progesterone from [3H]pregnenolone was curvilinear over a 6 h time course in human placenta cytotrophoblasts and in human placenta choriocarcinoma cells (JEG-3 cells). Mass measurements determined independently by radioimmunoassay indicate that the progesterone synthesized by cytotrophoblasts (21.0 +/- 5.20 ng/6 h/mg protein) is substantially higher than that synthesized by the JEG-3 cells (4.48 +/- 0.56 ng/6 h/mg protein). Two tight binding inhibitors of 3 beta-hydroxysteroid dehydrogenase (2 alpha-cyanoprogesterone I and cyanoketone II), and a potent inhibitor of the microsomal conversion of pregnenolone to progesterone (2 alpha-bromo-5 alpha-androstan-3-one-17 beta-acetate III) were compared as inhibitors of progesterone synthesis in the two cell-types. Compounds I and II were very potent inhibitors yielding IC50 values of between 10 and 20 nM. At higher concentrations (100 nM - 1,000 nM) compound I promoted a complete cessation of progesterone synthesis which could be reversed by washing the cells free of inhibitor. By contrast compound III was ineffectual as an inhibitor yielding an IC50 value greater than 10 microM. This 1,000-fold difference in inhibitory potency suggests that 2 alpha-cyano-substituted steroids display an unusual capacity to inhibit progesterone biosynthesis and secretion in normal and transformed human cells.     

Bioisosteres of 9-carboxymethyl-4-oxo-imidazo[1,2-a]indeno-[1,2-e]pyrazin-2-carboxylic acid derivatives. Progress towards selective, potent in vivo AMPA antagonists with longer durations of action

A novel series of 2- and 9-disubstituted heterocyclic-fused 4-oxo-indeno[1,2-e]pyrazin derivatives was synthesized. One of them, the 9-(1H-tetrazol-5-ylmethyl)-4-oxo-5,10-dihydroimidazo[1,2-a]indeno[1,2-e]pyrazin-2-yl phosphonic acid 4i exhibited a strong and a selective binding affinity for the AMPA receptor (IC50 = 13 nM) and demonstrated potent antagonist activity (IC50 = 6nM) at the ionotropic AMPA receptor. This compound also displayed good anticonvulsant properties against electrically-induced convulsions after ip and iv administration with ED50 values between 0.8 and 1 mg/kg. Furthermore, a strong increase in potency was observed when given iv 3 h before test (ED50 = 3.5 instead of 25.6 mg/kg for the corresponding 9-carboxymethyl-2-carboxylic acid analogue). These data confirmed that there is an advantage in replacing the classical carboxy substituents by their bioisosteres such as tetrazole or phosphonic acid groups.     

Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain

Rimcazole (BW 234U) is a potential antipsychotic agent which in open-clinical trials appears to be effective in acute schizophrenic patients. In the present study, rimcazole was found to block the specific binding of [3H]-(+)-SKF 10,047 to sigma sites in rat and guinea pig brain (IC50 = 5.0 X 10(-7) M). The compound was 100 times weaker as a blocker of phencyclidine sites (IC50 = 4.3 X 10(-5) M). At 1 X 10(-5) M, rimcazole had only weak effects on mu, delta, kappa and epsilon opioid receptors. Scatchard analysis of the binding data from guinea pig brain revealed an apparent KD for [3H]-(+)-SKF 10,047 of 85 +/- 5 nM and a Bmax of 824 +/- 27 fmole/mg protein. In the presence of 5 X 10(-7) M BW 234U, the apparent KD was 165 +/- 35 nM, but the Bmax (892 +/- 146 fmoles/mg protein) was not affected. This suggests that rimcazole is a competitive inhibitor of sigma sites. The agent was also capable of blocking sigma sites in vivo (ID50 = 6 mg/kg i.p., mice) as judged by an in vivo sigma receptor binding assay. Thus, if the antipsychotic activity of rimcazole is confirmed in double-blind, placebo-controlled trials, it would be the first compound whose mechanism of antipsychotic activity may best be explained by a direct blockade of sigma sites and not by a direct blockade of dopamine (D2) receptors in brain.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 mu g/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd=3.34 +/- 0.36 nM without tagetone and Kd,t=5.86 +/- 0.86 nM with tagetone; p&lt;0.05, two tailed Student's t-test) without affecting maximal binding (Bmax=488 +/- 24 fmoles/mg protein, and Bmax,t=500 +/- 25 fmoles/ mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 mu M with and without tagetone respectively). GABA was able to decrease merocyanine Delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 mu M. Tagetone, at a concentration of 50 mu g/ml and in the presence of GABA 30 mu M or 60 mu M, enhanced the ability of GABA alone on decreasing Delta A570-610. Tagetone alone did not change Delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine Delta A value are mainly exerted at the membrane potential level (Delta Psim). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding; on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

Synthesis and biological evaluation of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidines as dual EGFR/ErbB-2 kinase inhibitors

A series of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidine derivatives were elaborately designed based on the skeleton of Lapatinib, and evaluated for their potential to inhibit epidermal growth factor receptor (EGFR) and ErbB-2 tyrosine kinase activities and antiproliferative activities against A431 and SKOV-3 cell lines. Among these synthesized pyrimidine derivatives, 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-acrylamidophenoxy)pyrimidine (6), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-cyanoacetamidophenoxy)pyrimidine (9), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-{3-[6-(4-amino)pyrimidinyl]amino) phenoxy}pyrimidine (11) and 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-phenoxyacetamidophenoxy)pyrimidine (14) could significantly inhibit dual EGFR/ErbB-2 kinase activities (IC(50)=37/29 nM, 48/38 nM, 61/42 nM, 65/79 nM, respectively). And compounds 6 and 11 also showed the most potent antiproliferative activities in vitro, with the IC(50) value of 6 being 3.25 μM for A431 and 0.89 μM for SKOV-3, as for 11, 4.24 μM for A431 and 0.71 μM for SKOV-3, respectively. Docking study was also performed to determine the possible binding model.