PAF-acether antagonistic pyrrolo[1,2-c]thiazoles: from RP 48740 to RP 59227

The optimisation of the PAF antagonistic activity of RP 48740 led to the discovery of still more potent molecules of the pyrrolo[1,2-c]thiazole family: RP 52629, RP 52770 and RP 59227. The latter compound, for which a stereo-specific synthesis was developed, exhibits a very high potency as a PAF receptor antagonist.     

Temperature dependence of [3H]PAF binding to washed human platelets.

Our results indicate that the binding of [3H]PAF to a single class of high affinity sites on human platelets is endothermic and largely entropy-driven. The increase in entropy can be attributed to the disorganization of water molecules bound to both receptor and ligand during the binding process.     

Human platelets possess multiple [3H]imipramine binding sites

Scatchard analysis of [3H]imipramine binding to human platelets over the concentration range of 0.1-250 nM revealed a biphasic concave upward curve. Computer-assisted analysis indicated the best fit of the data by a two-site model with apparent Kd values of 0.68 and 293 nM and apparent Bmax values of 802 fmol/mg protein and 12.72 pmol/mg protein for the high- and low-affinity components, respectively. These results demonstrate the complex manner in which [3H]imipramine binding to human platelets, suggesting a reason for inconsistencies in Bmax values reported in the literature. Further studies of these low- as well as high-affinity sites may help elucidate the functional role of [3H]imipramine binding sites in affective disorders.     

Characterization of binding sites for [3H]spiroperidol

Experiments were designed to investigate the biochemical properties of binding sites for [3H]spiroperidol ([3H]SPD) solubilized from canine caudate and to define the effect of detergent on the binding of the radioligand. Extraction of canine caudate with 0.75-1.0% digitonin was found to generate the maximum yield of binding sites for [3H]SPD while minimizing extraction of membrane proteins. Although binding sites were solubilized with 1.0% digitonin, a 10-fold reduction in detergent concentration was necessary to achieve maximal binding of [3H]SPD. The rank order of affinity for agonists and antagonists was consistent with the pharmacologic properties of the D2 subtype of the dopamine receptor. However, the binding of antagonists was found to be complex. Studies with some preparations of pooled canine caudate resulted in competition curves for the D2-selective antagonists domperidone and sulpiride that best fit a single-site model. Other preparations exhibited biphasic inhibition curves with these antagonists. The class of binding sites for [3H]SPD with low affinity for D2-selective antagonists constituted as much as 30-40% of the binding sites. Enrichment of solubilized binding sites for [3H]SPD was achieved by size exclusion HPLC followed by adsorption to DEAE-Sephadex and elution with buffer of increasing ionic strength. Enrichment of binding sites was accompanied by a decrease in the affinity of solubilized sites for [3H]SPD.     

Characterization of [3H]tryptamine binding sites in brain

[3H]Tryptamine binds with high affinity to sites on rat brain membranes. The sites have the characteristics of tryptamine receptor recognition sites. These sites are widely distributed among rat brain regions with the highest density occurring in the cerebral cortex, striatum and hippocampus. The site is also found in human cerebral cortex. The binding site is localized mainly to the synaptosomal fraction. Drug competition studies indicate that the [3H]tryptamine binding site is distinct from serotonin receptors. Drugs that are potent inhibitors of [3H]tryptamine binding include tetrahydro-beta-carboline, quipazine, phenylethylamine, amphetamine, p-chloroamphetamine and methamphetamine.     

Efficient one-pot, three-component synthesis of a library of pyrrolo[1,2-c]pyrimidine derivatives

Herein is reported a simple and efficient one-pot three-component synthesis of pyrrolo[1,2-c]pyrimidine derivatives starting from various substituted pyrimidines, 2-bromoacetophenones, and electron deficient alkynes in epoxides acting both as reaction medium and HBr scavanger. This method proved to be very lucrative and avoids formation of ylide inactivation products. The synthesis represents an environmentally benign alternative to classical methods. The new library of compounds was briefly characterized regarding the improved Lipinski rule to asses the potential drug-likeness of the compounds. The majority of compounds are statisfing the Lipinski rule.     

Stereoselective inhibition of the binding of [3H]PK 11195 to peripheral-type benzodiazepine binding sites by a quinolinepropanamide derivative

The specific binding of [3H]PK 11195 to the peripheral-type benzodiazepine binding site is inhibited by the l-enantiomer of N,N-diethyl-alpha-methyl-2-phenyl-4-quinolinepropanamide ((-)Q1) but not by its d-enantiomer ((+)Q1). (-)Q1 inhibited [3H]PK 11195 binding to several rat tissues with an IC50 of less than 10 nM whereas (+)Q1 was at least 500 times less potent. This stereoselectivity was observed in all the tissues tested (brain, heart, kidney and adrenals). The same stereoselectivity was found for the displacement of the binding of [3H]PK 11195 in vivo, where (-)Q1 had an ID50 between 4-15 mg/kg and (+)Q1 was completely inactive at all doses tested (i.e. up to 40 mg/kg). Neither isomer had appreciable affinity for central-type benzodiazepine binding sites ([3H]diazepam) nor for voltage-sensitive calcium channels ([3H]PN 200210 and [3H]verapamil).     

Irreversible loss of [3H]forskolin binding sites in human platelets by alpha-haloacetyl analogs of forskolin

The 7-bromoacetyl-7-desacetyl (BrAcFsk) and 7-chloroacetyl-7-desacetyl (CIAcFsk) analogs of forskolin were synthesized as alkylating agents to study the high affinity binding sites for forskolin. BrAcFsk and CIAcFsk activated adenylate cyclase in human platelet membranes with EC50 values of about 20 and 12 microM, respectively. Both analogs increased cyclic AMP in human platelets; however, they were less potent that forskolin. Forskolin inhibited [3H]forskolin binding to human platelet membranes with an IC50 of 20 nM, whereas BrAcFsk and CIAcFsk inhibited [3H] forskolin binding with IC50 values of 0.1 microM. Pretreatment of intact platelets with 10 microM BrAcFsk caused a 90% irreversible loss in [3H]forskolin binding sites, whereas pretreatment with 10 microM CIAcFsk led to a loss of 55% of the binding sites. The loss of binding sites occurred within 5 min for BrAcFsk and within 30 min for CIAcFsk. The time required for the loss of binding sites produced by either alkylating agent was increased by the inclusion of 200 microM forskolin in the pretreatment buffer. The inactive bromoacetyl analog of forskolin 7-bromoacetyl-7-desacetyl-1.9-dideoxyforskolin (1,9-dideoxy-BrAcFsk) did not activate adenylate cyclase, inhibit [3H]forskolin binding, or cause an irreversible loss of [3H]forskolin binding sites. Adenylate cyclase was assayed in membranes from platelets treated with either 10 microM BrAcFsk or 10 microM 1,9-dideoxy-BrAcFsk. The stimulation of adenylate cyclase by prostaglandin E1, guanosine-5'-O-(3-thio)triphosphate, and AIF4 was inhibited by about 50% in membranes from platelets treated with BrAcFsk. However, the stimulation of adenylate cyclase by forskolin was unaffected by preincubation with BrAcFsk. Pretreatment of human platelets with 1,9-dideoxy-BrAcFsk had no effect on the stimulation of adenylate cyclase by prostaglandin E1, AIF4, or forskolin.     

Characterization of L-[3H]glutamate binding sites in bovine brain coated vesicles.

Clathrin-coated vesicles isolated from bovine brain exhibit an L-[3H]glutamate-specific binding. Coated vesicles were purified from bovine brain by differential centrifugation and gel filtration. High purity of coated vesicles was established previously by several enzyme markers and electron microscopy. The binding activity was performed in the absence of Na+, Ca2+, and Cl- ions to avoid binding and/or uptake to uptake sites. Coated vesicles were frozen, thawed, treated with 0.04% Triton X-100 and washed before incubation with L-[3H]glutamate. Saturation binding experiments revealed a single binding site with a Kd = 439 +/- 87 nM and a Bmax = 11.74 +/- 3.4 pmol/mg protein, consistent with kinetics characteristic for glutamate receptors. The glutamate-specific binding was stereospecific for glutamate and aspartate, showing higher affinity for L-forms than D-forms. Pharmacological characterization indicated that specific binding was sensitive to quisqualate and almost insensitive to kainate and N-methyl-D-aspartate. 200 microM guanosine triphosphate (GTP) produced a decrease of 50% in L-[3H]glutamate binding activity and competition experiments produced an affinity shift to the right of the glutamate dose-response curve. These results support the evidence that glutamate receptors are present in bovine brain coated vesicles and, at least in part, are associated to a G-protein.     

Circadian rhythm of the B max of [3H]-imipramine binding in rabbit platelets

Summary [³H]-imipramine binding was measured in rabbit blood platelet membranes on a 24 h cycle. Animals were kept on a 14 h light (L) 10 h dark (D) schedule, and blood samples were collected at L + 2, L + 8, D + 2, D + 8 and L – 2 h on a following cycle. Significant differences were found for B_max values of [³H]-imipramine binding, with highest values during the dark phase and lowest during the light phase. No significant differences were found in K _d values. These results suggest the existence of a circadian rhythm for the B_max of [³H]-imipramine binding in blood platelets. Send offprint requests to S. Z. Langer     

Characterization of platelet-activating factor (PAF) receptor by specific binding of [3H]L-659,989, a PAF receptor antagonist, to rabbit platelet membranes: possible multiple conformational states of a single type of PAF receptors

(trans)-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989) is a potent and orally active platelet-activating factor (PAF)-specific and competitive receptor antagonist. The 2,5-tritium-labeled L-659,989 ([3H]L-659,989) specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (KD) of 1.60 +/- 0.20 nM in 10 mM MgCl2. Several selected PAF analogs and PAF receptor antagonists show equilibrium inhibition constants roughly similar to those found in the specific [3H]PAF binding assay. Other pharmacological agents with no PAF antagonistic activities do not inhibit the specific binding of [3H]L-659,989. K+ and divalent cations such as Mg2+, Ca2+, and Mn2+ potentiate the specific [3H]L-659,989 binding. Na+ and Li+ also enhance but GTP shows no effect on the specific binding of [3H]L-659,989. However, Ni2+ inhibits the specific binding. Scatchard analysis demonstrates that the potentiating effect of these cations is due to an increase in the detectable receptor number for L-659,989. In 10 mM MgCl2 [3H]L-659,989 shows higher receptor number than [3H]PAF. Under various ionic conditions with or without GTP, in which [3H] L-659,989 binding remains approximately the same, C16-PAF shows different potencies in competing against the specific [3H] L-659,989 binding. These results demonstrate the existence of multiple conformational states of the PAF-specific receptor. The variation in the detectable receptor number under different conditions is due to the coexistence of the high and low affinity states and the fact that the low affinity state(s) of the receptor with KD value(s) possibly in the micromolar range cannot be detected in the Scatchard analysis with the radioligand at nanomolar concentrations. In the presence of 150 mM NaCl and 1 mM GTP, receptors exist in a single conformational state with an equilibrium dissociation constant (KB) of 0.931 microM for PAF.     

Characterization of [3H]thiocolchicoside binding sites in rat spinal cord and cerebral cortex.

Thiocolchicoside, a semi-synthetic derivative of the naturally occurring compound colchicoside with a relaxant effect on skeletal muscle, has been found to displace both [3H]gamma-aminobutyric acid ([3H]GABA) and [3H]strychnine binding, suggesting an interaction with both GABA and strychnine-sensitive glycine receptors. In order to gain further insight into the interaction of thiocolchicoside with these receptors, the binding of [3H]thiocolchicoside in rat spinal cord-brainstem and cortical synaptic membranes was characterized. [3H]Thiocolchicoside binding was saturable in both tissues examined. In spinal cord-brainstem membranes, we found a K(D) of 254 +/- 47 nM and a Bmax of 2.39 +/- 0.36 pmol/mg protein, whereas in cortical membranes, a K(D) of 176 nM and a Bmax of 4.20 pmol/mg protein was observed. A similar K(D) value was found in kinetic experiments performed in spinal cord-brainstem membranes. Heterologous displacement experiments showed that GABA and strychnine displaced the binding in a dose-dependent manner, whereas glycine was ineffective. [3H]Thiocolchicoside binding was also displaced by several GABA(A) receptor agonists and antagonists, but not by baclofen, flunitrazepam, guvacine, picrotoxin or by other drugs unrelated to GABA transmission. In spinal cord-brainstem, and to a lower extent, in cortical membranes, GABA and its analogs were not able to completely displace [3H]thiocolchicoside specific binding indicating that, besides GABA(A) receptors, thiocolchicoside can bind to another unidentified site. Unlabelled thiocolchicoside, however, completely displaced [3H]muscimol binding both in cortical and in spinal cord-brainstem synaptic membranes with an IC50 in the low microM range. Neurosteroids were found to modulate the binding in cortical but not in spinal cord-brainstem synaptic membranes. We conclude that [3H]thiocolchicoside binding shows a pharmacological profile indicating an interaction with the GABA(A) receptor. The different affinities for the GABA(A) receptor agonists and antagonists and sensitivity to neurosteroids obtained in the cerebral cortex and in the spinal cord may indicate a preferential interaction of the compound with a subtype of the GABA(A) receptor. The data also indicate that [3H]thiocolchicoside binds to another site(s), whose nature remains to be elucidated.     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

Affinity modulation of [3H]imipramine, [3H]paroxetine and [3H]citalopram binding to the 5-HT transporter from brain and platelets.

The dissociations of [3H]imipramine, [3H]paroxetine and [3H]citalopram from the 5-HT (serotonin 5-hydroxytryptamine) transporter were found to be markedly influenced by several drugs, although concentrations in the microM range were needed. Most of these drugs attenuated the dissociation rate, i.e. increased the affinity between the ligand and the binding site. A few increased the dissociation rate however. The binding of drugs to the affinity-modulating site was specific, although of low affinity and probably changing the conformation of the high-affinity binding site, thereby changing the fit between the ligand and the interacting amino acid side-chains. Although the drugs usually affected the dissociation rates of the three ligands in the same manner, there were some which had different effects on [3H]imipramine, [3H]paroxetine and [3H]citalopram. For example, 5-HT markedly attenuated the dissociation of [3H]imipramine, had a moderate effect on [3H]paroxetine and very little effect on [3H]citalopram dissociation. This indicates that the three ligands are bound to different domains on the 5-HT transporter. [3H]Citalopram dissociation from human brain and rat brain were differently affected by several drugs. Indalpine augmented the dissociation rate of the [3H]citalopram 5-HT transport complex in human brain but attenuated it in rat brain, thus revealing a species difference of the 5-HT transporter.     

Synthesis and oral antiallergic activity of carboxylic acids derived from imidazo[2,1-c][1,4]benzoxazines, imidazo[1,2-a]quinolines, imidazo[1,2-a]quinoxalines, imidazo[1,2-a]quinoxalinones, pyrrolo[1,2-a]quinoxalinones, pyrrolo[2,3-a]quinoxalinones, and imidazo[2,1-b]benzothiazoles

4H-Imidazo[2,1-c][1,4]benzoxazine-2-carboxylic acid (3) was found to possess potent activity in the IgE-induced rat passive cutaneous anaphylaxis model which may be predictive of clinical antiallergic activity. Compared to disodium cromoglycate (DSCG, 1), 3 was less active following iv administration but unlike DSCG showed very significant oral activity. To explore the structural requirements for this activity, a range of tricyclic compounds was prepared and their activities were measured. Individual 2-carboxylic acids derived from imidazo[1,2-a]quinolines, imidazo[1,2-a]quinoxalines, imidazo[1,2-a]quinoxalinones, pyrrolo[1,2-a]quinoxalinones, pyrrolo[2,3-a]quinoxalinones, and imidazo[2,1-b]benzothiazoles showed iv activities up to 10(3) times as potent as DSCG and many of them showed significant oral activity. From these, imidazo[1,2-a]quinoxaline-2-carboxylic acid 114 has been chosen for further development.     

[3H]-5-Methoxytryptoline is not actively accumulated by rabbit platelets

Summary 5-Methoxytryptoline (5-MeO-TLN, 6-methoxytetrahydro--carboline) inhibits with high affinity [³H]-imipramine binding to the serotonin transporter in platelets. To evaluate whether 5-MeO-TLN is a substrate for the serotonin transporter, the accumulation of [³H]-5-MeO-TLN into rabbit platelets was studied in vitro. At short incubation times (5 min), [³H]-5-MeO-TLN accumulation was temperature-sensitive, but not saturable over a concentration range from 0.06 mol/l to 10 mol/l Moreover, [³H]-5-MeO-TLN uptake was not affected by 100 mol/1 ouabain, its structural analogs tryptoline and 5-hydroxytryptoline, nor by the serotonin uptake inhibitors imipramine and citalopram. After longer incubation times (60 min), [³H]-5-MeO-TLN accumulation at O°C approached that seen at 37°C and temperature-sensitive [³H]-5-MeO-TLN uptake could no longer be observed. It is concluded that temperature-sensitive accumulation of [³H]-5-MeO-TLN is not mediated by the serotonin transporter and most likely represents a passive, diffusional process, the rate of which is temperature-dependent. The present studies thus confirm the hypothesis that 5-MeO-TLN affects [³H]-imipramine binding in platelets through a competitive mechanism and not via an allosteric interaction mediated through the substrate recognition site of the macromolecular complex of the serotonin transporter. Send offprint requests to S. Z. Langer at the above address     

Multiple [3H]-nemonapride binding sites in calf brain

[³H]-Nemonapride has been the ligand of choice to label D₄ dopamine receptors. Its specificity was questioned when it was discovered that sigma (σ) sites were also labeled by [³H]-nemonapride. To further characterize the binding of [³H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [³H]-nemonapride labeled multiple sites. Dopaminergic and σ sites were the most prominent. The σ binding profile was σ-1 like with a K_i binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and σ sites, respectively, revealed additional, not previously characterized binding sites for [³H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [³H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met. Received: 19 September 1996 / Accepted: 12 March 1997     

The binding characteristics of [3H]-dihydroergocryptine on intact human platelets.

1 We have characterized the binding of [3H]-dihydroergocryptine to intact human platelets. 2 The values of the association and dissociation rate constants, affinity and capacity of specific [3H]-dihydroergocryptine binding on intact cells closely resemble those previously reported on the human platelet lysate preparation. 3 The affinity of alpha-adrenoceptor antagonists, determined from inhibition of [3H]-dihydroergocryptine binding, is similar in intact and lysed platelet preparations, but the affinity of agonists is considerably lower in intact cells. 4 The potency of alpha-adrenoceptor antagonists as inhibitors of noradrenaline-induced platelet aggregation and as inhibitors of [3H]-dihydroergocryptine binding on intact platelets demonstrate a significant correlation (r = 0.92, p less than 0.01). 5 The affinity and capacity of [3H]-dihydroergocryptine binding to platelets from a group of healthy, young, male subjects show a high degree of consistency both between subjects (Kd = 2.81 +/- 0.27 nM; Bmax = 63 +/- 3 fmol/10(8) platelet: mean +/- s.e. mean, n = 10) and between sampling occasions in a single subject (Kd = 3.28 nM +/- 13%; Bmax = 70 fmol/10(8) platelet +/- 16%: mean +/- coefficient of variation, n = 5). 6 There is no significant difference in the binding capacity of platelets from a group of elderly male subjects (mean age 73) compared to those from young males (mean age 27) or elderly females (mean age 77). The affinity of binding is slightly but significantly (P less than 0.05) higher in the elderly male group compared to the two other groups. 7 We conclude that [3H]-dihydroergocryptine binds to the alpha 2-adrenoceptor of intact human platelets which is responsible for noradrenaline-induced platelet aggregation. The high consistency of the binding characteristics of [3H]-dihydroergocryptine indicate that this assay may be useful as a monitor of platelet alpha-adrenoceptor sensitivity in clinical investigation.     

Binding of [3H]idazoxan and of its methoxy derivative [3H] RX821002 in human fat cells: [3H]idazoxan but not [3H] RX821002 labels additional non-alpha 2-adrenergic binding sites

Binding studies were carried out in human fat cell membranes with two alpha 2-adrenergic antagonists, [3H]idazoxan and its methoxy derivative [3H]RX821002. Inhibition studies with epinephrine enantiomers indicate that [3H]RX821002 only binds to alpha 2-adrenoceptors, whereas [3H]idazoxan labels alpha 2-adrenoceptors and additional nonadrenergic sites (NAIBS). NAIBS and alpha 2-adrenoceptors display different affinities towards drugs from various chemical families. Imidazoline and some guanidine derivatives exhibit a high affinity for NAIBS. Pharmacological studies of human NAIBS indicate that they are slightly different from those previously reported in the rabbit, suggesting the existence of several subtypes of NAIBS. Furthermore, NAIBS are different from the previously described "imidazoline-preferring sites." [3H]idazoxan and [3H]RX821002 saturation analyses were performed in human adipocytes from different anatomical locations, in order to compare the number of NAIBS and alpha 2-adrenoceptors. Although there was an important variation in NAIBS and alpha 2-adrenoceptor numbers in the studied samples, a very poor correlation was obtained between the Bmax values of the two sites. Moreover, alkylation of alpha 2-adrenoceptors by phenoxybenzamine produces a 90% reduction in accessible [3H]RX821002 binding sites, without modification of [3H]idazoxan binding. These data show that NAIBS are not closely related to the alpha 2-adrenergic molecule. In addition, benextramine appears to be a reversible competitor at NAIBS. [3H]idazoxan binding, but not [3H]RX821002 binding, is sensitive to K+, suggesting that the domains involved in the ligand-NAIBS interaction are different from those involved in the ligand-alpha 2-adrenoceptor interaction.