Synthesis of enantiopure Delta2-isoxazoline derivatives and evaluation of their affinity and efficacy profiles at human beta-adrenergic receptor subtypes

The new enantiomerically pure 3-substituted-Delta(2)-isoxazolin-5-yl-ethanolamines (+)-6a/(-)-6b, (-)-6a/(+)-6b, and (+)-7a/(-)-7b, prepared via a 1,3-dipolar cycloaddition-based approach, were tested for their affinity at human beta(1)-, beta(2)-, and beta(3)-adrenergic receptor (beta-AR) subtypes stably expressed in CHO cells. The corresponding 3-isopropenyl derivatives (+)-5a/(-)-5b, (-)-5a/(+)-5b, and some isoxazole analogs were also tested. The binding affinities at the beta-ARs of the isoxazolinyl amino alcohols were significantly lower than those of the corresponding isoxazole derivatives. A stereochemical effect was observed, since the process of molecular recognition is predominantly controlled by the (S)-configuration of the stereogenic center located at the 5 position of the heterocycle rather than by that of the stereocenter carrying the secondary alcohol group. On the contrary, the stereochemical features marginally affected the efficacy response; as a matter of fact, functional tests carried out on Delta(2)-isoxazoline derivatives provided with a detectable binding affinity showed the overall profile of neutral antagonists at all three beta-AR subtypes.     

Facile synthetic route to enantiopure unsymmetric cis-2,5-disubstituted pyrrolidines

The (+/-)-cis-5-arylcarbamoyl-2-ethoxycarbonylpyrrolidines 6a-g were firstly synthesized in 53-64% yields by using meso-diethyl-2,5-dibromoadipate 3 and (S)-(-)-1-phenylethylamine in three steps. The diastereomeric mixture (S;2S,5R)-(-)-7 and (S;2R,5S)-(+)-8 were prepared by the Grignard reaction and separated by a flash column chromatography in 29 and 52% yields. The absolute configurations of (+)-8 was confirmed by X-ray crystallographic analysis and the enantiopure pyrrolidines (2S,5R)-(-)-9/(2R,5S)-(+)-9 and (2S,5R)-(-)-10/(2R,5S)-(+)-10 were obtained in good yields.     

A new sigma ligand, (+/-)-PPCC, antagonizes kappa opioid receptor-mediated antinociceptive effect

The compound (1R,2S/1S,2R)-2-[4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(+/-)-PPCC] is a ligand with high affinity for sigma (sigma) sites of which the selectivity towards several other receptor systems has been demonstrated. Given the existence of a relationship between the sigma system and the kappa opioid (KOP)-mediated analgesia, to characterize the pharmacological properties of (+/-)-PPCC we analyzed its influence on the analgesic effect of the systemic injected kappa agonist (-)-U-50,488H comparing the effects with those shown by (+)-pentazocine and BD1047. The results demonstrate that the systemic administration of (+/-)-PPCC (1 mg/kg s.c.) does not modify basal tail-flick latency. Pre-treatment with (+/-)-PPCC, at the same dose, significantly decreased the antinociceptive effect of (-)-U-50,488H, analogously to the sigma compounds used. This study confirms that (+/-)-PPCC plays the role of sigma agonist in this model and strengthens the hypothesis of the sigma receptor modulatory role on KOP-mediated analgesia.     

Colorectal cancer metastases affect the biochemical characteristics of the human liver beta-adrenoceptor-G-protein-adenylate cyclase system

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked beta2-adrenoceptors (beta2-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of beta2-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of beta-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of beta-ARs (B(max)) was significantly reduced, compared to NA-NM liver tissues (40.09+/-2.83 vs. 23.09+/-3.24 fmol/mg protein; P<0.001). A similar decrease in the beta-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6+/-2.2 vs. 23.8+/-3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized beta-ARs were uncoupled from stimulatory G-protein (G(S)), as total density of beta-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition-concentration curves of ICI 118.551 showed the presence of a homogeneous population of the beta2-AR subtypes. Neither the binding patterns nor the inhibition constant (K(i)) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic beta-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence beta-AR-mediated functions in both organs.     

Regulation of the black bullhead hepatic beta-adrenoceptors

Black bullhead catfish (Ameiurus melas) were exposed to air for 1 h to examine the effect of an acute stress on the distribution and function of the hepatic beta-adrenoceptors (beta-ARs). Air exposure significantly reduced both adrenaline (ADR)- and noradrenaline (NADR)-stimulated glucose production in isolated hepatocytes with no effect on either receptor affinity (K(d)) or number of binding sites (B(max)). A 24 h exposure of isolated hepatocytes to the beta-agonist isoproterenol also had no significant impact on either binding parameter. Competition studies using selective agonists and antagonists suggest that the hepatic beta-AR in this species is pharmacologically beta(2)-like. However in addition to the beta(2)-AR, molecular evidence provides support for the existence of hepatic beta-ARs that phylogenetically group with the beta(3)-ARs and the beta(1)-ARs. Despite the presence of several potential phosphorylation sites in the third intracellular loop and cytoplasmic tail of the bullhead beta(2)-AR, no significant changes were observed in the binding parameters. While physiological data supports the presence of only a single subtype, molecular data supports the existence of multiple beta-AR subtypes in this species. The mechanisms thought to regulate mammalian beta-ARs exist in the bullhead ARs reported here but these mechanisms are not as effective in this fish system as in mammals.     

Expression of human alpha 2-adrenergic receptors in adipose tissue of beta 3-adrenergic receptor-deficient mice promotes diet-induced obesity

Catecholamines play an important role in controlling white adipose tissue function and development. beta- and alpha 2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte alpha 2/beta-AR balance in obesity, and it has been proposed that increased alpha 2-ARs in adipose tissue with or without decreased beta-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte alpha 2/beta-AR balance was genetically manipulated in mice. Human alpha 2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (-/-) or heterozygous (+/-) for a disrupted beta 3-AR allele. Mice expressing alpha 2-ARs in fat, in the absence of beta 3-ARs (beta 3-AR -/- background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of alpha2-ARs, the absence of beta 3-ARs, and a high fat diet. Of note, obese alpha 2-transgenic beta 3 -/- mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased alpha 2/beta-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (alpha 2 and beta 3-AR) and diet interact to influence fat mass.     

Partial resolution of racemic trans-4-[5-(4-alkoxyphenyl)-2,5-dimethylpyrrolidine-1-oxyl-2-yl]benzoic acids by the diastereomer method with (R)- or (S)-1-phenylethylamine

The partial resolution is described of a series of racemic trans-4-[5-(4-alkoxyphenyl)-2,5-dimethylpyrrolidine-1-oxyl-2-yl]benzoic acids (1), which are the key intermediates for the synthesis of chiral organic radical liquid crystalline compounds and are crystallized to give racemic compounds. Racemic acid 1 [(+/-)-1] with a long alkyl chain (C7 to C13) could be resolved by the conventional diastereomeric salt formation using (R)- or (S)-1-phenylethylamine (2) as the resolving agent, whereas resolution of (+/-)-1 with a short alkyl chain (C4 to C6) was unsuccessful. Use of six equiv of (R)- or (S)-2 for the initial diastereomeric salt formation of (+/-)-1 with a C7-C13 alkyl chain, followed by recrystallization of the resulting salts once or twice, gave 2S,5S- or 2R,5R-enriched 1, respectively, in an ee range of 75-92% and with an overall recovery of 11-27%, based on the original quantity of (+/-)-1.     

Stereoselective synthesis of 2,3,7-trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the MalphaNP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).     

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.     

Syntheses and evaluation of pyridazine and pyrimidine containing bioisosteres of (+/-)-pyrido[3.4-b]homotropane and pyrido-[3.4-b]tropane as novel nAChR ligands.

Bioisosteric replacement of the pyridine pharmacophoric element in (+/-)-pyrido[3.4-b]homotropane (PHT) and pyrido[3.4-b]tropane with the pyridazine and pyrimidine nucleus resulted in hitherto unknown nAChR ligands such as 5-8. Inverse type Diels-Alder reactions constitute the key steps in the new routes to the pyridazine- or pyrimidine-annulated bioisosteres. The enantiopure (+)-2-tropinone (11) from the 'chiral pool' is transformed to the ring-expanded silyl enol ether 12 and to the enamine 15. Both proved to be highly dienophilic species in the inverse type [4+2] cycloaddition reactions with the 1,2,4,5-tetrazines 13 and 16a,b or with the 1,3,5-triazine 19 to provide the enantiopure target compounds 5-7. In the same way the racemic pyrimidine-annulated species 8 was obtained from 3-tropanone 21. The new ligands were tested for their in vitro affinity for (alpha4)2(beta2)3 and alpha7* nAChR subtype. In comparison to PHT, well known to exhibit affinity for agonist binding sites in rat brain approximately equivalent to that of (+)-anatoxin-a (1), replacement of the pyridine by the bioisosteric pyridazine resulted in 30-fold lower affinity at the (alpha4)2(beta2)3 subtype. The annulated diazinotropanes 6-8, ligands with ferruginine-like structures more or less retained the affinity of (-)-norferruginine (3) except of compound 7. Remarkably, all of the novel ligands are devoid of affinity at the alpha7* subtype.     

Effect of targeted deletions of beta1- and beta2-adrenergic-receptor subtypes on heart rate variability

Beta-adrenergic receptors (beta-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (beta1, beta2, and beta3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of beta-ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, beta1-KO, beta2-KO, and beta1/beta2 double KO (DL-KO), to examine the role that beta-AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in beta1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] mice compared with wild-type [WT; 638 (SD 30)] or beta2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking beta1-ARs (beta1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF powers compared with mice with intact beta1-ARs (WT and beta2-KO). These results demonstrate the differential role of beta-AR subtypes in regulating autonomic signaling.     

The enantiomers of epiboxidine and of two related analogs: synthesis and estimation of their binding affinity at α4β2 and α7 neuronal nicotinic acetylcholine receptors

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.     

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

Synthesis of 6-substituted 1-phenylbenzazepines and their dopamine D(1) receptor activities

A series of racemic 6-aryl substituted 1-phenylbenzazepines 7a-e, and 17a,b were prepared. All these compounds showed binding potencies compatible to or much higher than that of the prototypic (+/-)-SKF-38393 ((+/-)-1) and (+/-)-SKF-83959 (3) for the D(1) receptor. Among analogs of (+/-)-SKF-38393, compounds 7b, 7c and 7e possess 10-, 2- and 7-fold enhancement in binding for the D(1) receptor, respectively. Lower but compatible potency to that of (+/-)-1 was observed for compounds 7a and 7d. The optimal 6-substituents (m-tolyl, and 2'-naphthyl) were applied to the skeleton of (+/-)-SKF-83959 (3). The resulting compounds 17a,b displayed high affinity at the D(1) receptor, only slightly lower than that of 3. These two compounds also showed good binding at the D(2) receptor.     

Synthesis of enantiopure 2-aryl-2-methoxypropionic acids and determination of their absolute configurations by X-ray crystallography

Racemic 2-aryl-2-methoxypropionic acids were enantioresolved by the use of (S)-(-)-phenylalaninol 4. For instance, racemic 2-methoxy-2-phenylpropionic acid (+/-)-7 was condensed with phenylalaninol (S)-(-)-4 yielding a diastereomeric mixture of amides, which was easily separated by HPLC on silica gel affording the first-eluted amide (-)-13a and the second-eluted amide (+)-13b: alpha = 3.19, Rs = 3.49. The absolute configuration of amide (-)-13a was determined to be (R;S) by X-ray crystallography by reference to the S configuration of the phenylalaninol moiety. Amide (R;S)-(-)-13a was converted to oxazoline (R;S)-(-)-14a, from which enantiopure 2-methoxy-2-phenylpropionic acid (R)-(-)-7 was recovered. Other 2-aryl-2-methoxypropionic acids, (R)-(-)-8, (R)-(-)-9, (R)-(+)-10, (R)-(-)-11, and (R)-(-)-12, were similarly prepared in enantiopure forms with the use of phenylalaninol (S)-(-)-4, and their absolute configurations were clearly determined by X-ray crystallography or by chemical correlation.     

Differential effects of intravenous R,S-(+/-)-3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its S(+)- and R(-)-enantiomers on dopamine transmission and extracellular signal regulated kinase phosphorylation (pERK) in the rat nucleus accumbens shell and core

R,S(+/-)-3,4-methylenedioxymethamphetamine (R,S(+/-)-MDMA, 'Ecstasy') is known to stimulate dopamine (DA) transmission in the nucleus accumbens (NAc). In order to investigate the post-synaptic correlates of pre-synaptic changes in DA transmission and their relationship with MDMA enantiomers, we studied the effects of R,S(+/-)-MDMA, S(+)-MDMA, and R(-)-MDMA on extracellular DA and phosphorylated extracellular signal regulated kinase (pERK) in the NAc shell and core. Male Sprague-Dawley rats, implanted with a catheter in the femoral vein and vertical concentric dialysis probes in the NAc shell and core, were administered i.v. saline, R,S(+/-)-MDMA, S(+)-MDMA, or R(-)-MDMA. Extracellular DA was monitored by in vivo microdialysis with HPLC. Intravenous R,S(+/-)-MDMA (0.64, 1, and 2 mg/kg) increased dialysate DA, preferentially in the shell, in a dose-related manner. S(+)-MDMA exerted similar effects but at lower doses than R,S(+/-)-MDMA, while R(-)-MDMA (1 and 2 mg/kg) failed to affect dialysate DA. R,S(+/-)- and S(+)-MDMA but not R(-)-MDMA increased ERK phosphorylation (expressed as density/neuron and number of pERK-positive neurons/area) in both subdivisions of the NAc. The administration of the D1 receptor antagonist, SCH 39166, prevented the increase in pERK elicited by R,S(+/-)-MDMA and S(+)-MDMA, while the D2/3 receptor antagonist, raclopride, increased pERK in the NAc core per se but failed to affect the R,S(+/-)-MDMA-elicited stimulation of pERK. The present results provide evidence that the DA stimulant effects of racemic MDMA are accounted for by the S(+)-enantiomer and that pERK may represent a post-synaptic correlate of the stimulant effect of R,S(+/-)-MDMA on D1-dependent DA transmission.     

Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists

The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[3H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

New route to enantiopure MalphaNP acid, a powerful resolution and chiral 1H NMR anisotropy reagent

MalphaNP acid (+/-)-1, 2-methoxy-2-(1-naphthyl)propionic acid, was enantioresolved by the use of phenylalaninol (S)-(-)-4; a diastereomeric mixture of amides formed from acid (+/-)-1 and amine (S)-(-)-4 was easily separated by fractional recrystallization and/or HPLC on silica gel, yielding amides (R;S)-(-)-5a and (S;S)-(+)-5b. Their absolute configurations were determined by X-ray crystallography by reference to the S configuration of the phenylalaninol moiety. Amide (R;S)-(-)-5a was converted to oxazoline (R;S)-(+)-8a, from which enantiopure MalphaNP acid (R)-(-)-1 was recovered. In a similar way, enantiopure MalphaNP acid (S)-(+)-1 was obtained from amide (S;S)-(+)-5b. These reactions provide a new route for the large-scale preparation of enantiopure MalphaNP acid, a powerful chiral reagent for the enantioresolution of alcohols and simultaneous determination of their absolute configurations by (1)H NMR anisotropy.     

Are developing beta-adrenoceptors able to desensitize? Acute and chronic effects of beta-agonists in neonatal heart and liver

During fetal and neonatal development, beta-adrenergic receptors (beta-ARs) appear to be resistant to desensitization by beta-agonist drugs. To determine the mechanisms underlying the regulatory differences between adults and neonates, we administered isoproterenol, a mixed beta(1)/beta(2)-AR agonist, and terbutaline, a beta(2)-selective agonist. Effects were examined in the ensuing 4 h after a single injection, or after the last of four daily injections. We prepared cell membranes from heart (predominantly beta(1)-ARs) and liver (predominantly beta(2)-ARs) and assessed signal transduction in the adenylyl cyclase (AC) pathway. In the first few hours after a single administration of isoproterenol to adult rats, cardiac beta-ARs showed activation of G proteins (elevated AC response to forskolin) and desensitization of beta-AR-mediated responses; after the fourth injection, heterologous desensitization emerged, characterized by a loss of signaling mediated either through beta-ARs or glucagon receptors. Terbutaline evoked an increase in the forskolin response but no desensitization of receptor-mediated responses. When we gave the same treatments to neonatal rats, we observed cardiac G protein activation, but there was neither homologous nor heterologous desensitization of beta-ARs or glucagon receptors. In the adult liver, isoproterenol and terbutaline both failed to evoke desensitization, regardless of whether the drugs were given once or for 4 days. In neonates, however, acute or chronic treatment elicited homologous desensitization of beta-AR-mediated AC signaling, while sensitizing the response to glucagon. These results show that neonatal beta-ARs are inherently capable of desensitization in some, but not all, cell types; cellular responses can be maintained through heterologous sensitization of signaling proteins downstream from the receptor. Differences from adult patterns of response are highly tissue selective and are likely to depend on ontogenetic differences in subtypes of beta-ARs and AC.