N‐heterocyclic carbenes as ligands in palladium‐mediated [11C]radiolabelling of [11C]amides for positron emission tomography

A model palladium‐mediated carbonylation reaction synthesizing N‐benzylbenzamide from iodobenzene and benzylamine was used to investigate the potential of four N‐heterocyclic carbenes (N,N′‐bis(diisopropylphenyl)‐4,5‐dihydroimidazolinium chloride ( I ), N,N′‐bis(1‐mesityl)‐4,5‐dihydroimidazolinium chloride ( II ), N,N′‐bis(1‐mesityl)imidazolium chloride ( III ) and N,N′‐bis(1‐adamantyl)imidazolium chloride ( IV )) to act as supporting ligands in combination with Pd₂(dba)₃. Their activities were compared with other Pd‐diphosphine complexes after reaction times of 10 and 120 min. Pd₂(dba)₃ and III were the best performing after 10 min reaction (20%) and was used to synthesize radiolabelled [¹¹C]N‐benzylbenzamide in good radiochemical yield (55%) and excellent radiochemical purity (99%). A Cu(Tp*) complex was used to trap the typically unreactive and insoluble [¹¹C]CO which was then released and reacted via the Pd‐mediated carbonylation process. Potentially useful side products [¹¹C]N,N′‐dibenzylurea and [¹¹C]benzoic acid were also observed. Increased amounts of [¹¹C]N,N′‐dibenzylurea were yielded when PdCl₂ was the Pd precursor. Reduced yields of [¹¹C]benzoic acid and therefore improved RCP were seen for III /Pd₂(dba)₃ over commonly used dppp/Pd₂(dba)₃ making it more favourable in this case. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of stable-isotope-labeled N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-(3-dimethylaminopropyl)-N′-ethylurea

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) is a carbodiimide coupling reagent commonly used for the preparation of amides from carboxylic acids and amines. Because of initial concerns regarding the genotoxicity of EDC and its use in GMP syntheses at Bristol Myers Squibb, the quantitation of residual EDC and its by-product N-(3-dimethylaminopropyl)-N′-ethylurea (EDU) by liquid chromatography–mass spectrometry (LCMS) impurity analysis was required. These analyses required the use of stable-isotope-labeled EDC and EDU to serve as internal standards. To meet this need, stable-isotope-labeled EDC 9 and EDU 10 were prepared from [1,2-¹³C₂] ethylene glycol and [¹³C,¹⁵N] potassium cyanide in overall yields of 6% and 8%, respectively.     

The preparation of tritiated E- and Z-4-aminobut-2-enoic acids,conformationally restricted analogues of the inhibitory neurotransmitter 4-aminobutanoic acid (GABA)

A synthesis of [³H]E- and [³H]Z-4-aminobut-2-enoic acids from methyl 4-N-phthalimidobut-2-ynoate by catalytic hydrogenation using tritium gas in the presence of a homogeneous catalyst, tris(triphenylphosphine)rhodium(I) chloride, is reported. HPLC separation of the E- and Z-isomers, and the saturated analogue, 4-aminobutanoic acid (GABA), is also described.     

Effects of Isoquinolinesulphonamide Compounds on Multidrug-resistant P388 Cells

Abstract— The effects of eight isoquinolinesulphonamide compounds on resistance to vinblastine in adriamycin-resistant mouse leukaemia cells (P388/ADR) which overexpress the relative molecular weight (M_r) 140 kDa P-glycoprotein in the plasma membrane were investigated. N-[2-(Methylamino)ethyl]-5-isoquinolinesulphonamide (H-8) and N-(2-aminoethyl)-5-isoquinolinesulphonamide (H-9) did not reverse vinblastine resistance. N-[2-[N-[3-(4-Chlorophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulphonamide (H-86) and N-[2-[N-[3-(4-chlorophenyl)-1-methyl-2-propenyl]amino]ethyl]-5-isoquinolinesulphonamide (H-87) caused accumulation of intracellular vinblastine and inhibition of vinblastine efflux from the cells and reversed the resistance. Addition of an aminoethyl group to the nitrogen atom of the sulphonamide group (W-66) or a formyl group at the terminal amino group (H-85) of H-86 reduced those activities. Conversion of the chlorophenyl group of H-87 to pyridinyl (H-31) or furanyl (H-34) markedly decreased activities against the drug resistance. The activity against vinblastine accumulation closely correlated with the apparent partition coefficient of compounds. These compounds dose-dependently inhibited photoaffinity labelling of a photosensitive analogue of vinblastine, N-(p-azido-(3-[¹²⁵I])salicyl)-N′-β-aminoethylvindesine ([¹²⁵I]NASV), and there was a good correlation between inhibition of [¹²⁵I]NASV-photolabelling and hydrophobicity. Although these isoquinolinesulphonamides inhibited protein kinase A with different magnitudes, this activity did not correlate with the effect on the drug resistance. These results indicate that isoquinolinesulphonamide compounds with a hydrophobic group interact with antitumour drugs on P-glycoprotein and reverse multidrug resistance without involvement of their activity on protein kinase A.     

Effects of benserazide on tryptophan metabolism in the mouse

Benserazide [N-(seryl)-N′-(2,3,4-trihydroxybenzyl)-hydrazine, Ro4-4602] is known to inhibit aromatic amino acid decarboxylase. David [1] has shown that administration of [1-¹⁴C]tryptophan to mice leads to a considerable evolution of ¹⁴CO₂, and that this is inhibited by prior administration of benserazide. This was interpreted as indicating that decarboxylation to tryptamine may be a major pathway of tryptophan metabolism in the mouse. In the present work, an alternative explanation is advanced. Evolution of ¹⁴CO₂ from [1-¹⁴C]tryptophan could be due to metabolism of the alanine released by the action of kynureninase. In this case there would also be evolution of ¹⁴CO₂ following administration of [2-¹⁴C]tryptophan. This has been demonstrated. It has further been shown that benserazide is a potent inhibitor of kynureninase and kynurenine aminotransferase in the mouse. The result of this inhibition is both a decrease in oxidative metabolism of tryptophan and an increase in the concentration of kynurenine in the liver. These effects are seen at levels of the drug similar to doses used clinically in treatment of Parkinson's disease. One effect of reduced oxidative metabolism of tryptophan would be reduced synthesis of nicotinamide, and it is possible that patients treated with benserazide may show some signs of niacin deficiency.     

Radiosynthesis of N‐[4‐(4‐fluorobenzyl)piperidin‐1‐yl]‐N′‐(2‐[11C]oxo‐1,3‐dihydrobenzimidazol‐5‐yl)oxamide,a NR2B‐selective NMDA receptor antagonist

In order to perform in vivo imaging of the NR2B NMDA receptor system by positron emission tomography, a NR2B selective NMDA receptor antagonist has been labelled with carbon‐11 (half‐life: 20 min). N‐[4‐(4‐fluorobenzyl)piperidin‐1‐yl]‐N′‐(2‐oxo‐1,3‐dihydrobenzimidazol‐5‐yl)oxamide has been described demonstrating high affinity and selectivity for the NR2B receptors (IC₅₀ of 5 nM in [³H]Ro‐25,6981 binding assay). The labelling precursor and the reference compound were synthesized by coupling the 4‐(4‐fluorobenzyl)piperidine with the corresponding oxalamic acid. The reaction of [¹¹C]phosgene with phenylenediamine precursor led the formation of the [¹¹C]benzimidazolone ring present on the ligand. The labelling occurred in THF or acetonitrile and the decay corrected radiochemical yield was 30–40% from the produced [¹¹C]methane. HPLC purification and formulation led to 2.6–3.7 GBq (70–100 mCi) of radioligand within 30–35 min. The specific radioactivity was 72–127 GBq/µmol (2–3.4 Ci/µmol) at the end of synthesis. Copyright © 2009 John Wiley & Sons, Ltd.     

A Stable and Highly Potent Hexamethonium Derivative Which Modulates Muscarinic Receptors Allosterically in Guinea-pig Hearts

Abstract— W84 (N, N,N′,N′-tetramethyl-N,N′-bis-(3-phthalimidopropyl)-N,N′-hexane-1,6-diyl-bis-ammomum dibromide) is a potent stabilizer of antagonist binding to muscarinic receptors; however, W84 hydrolyses in aqueous buffered medium. The synthesis of the stable derivative CHIN3/6 is presented containing a 2-phenyl-quinazolinone instead of the labile phthalimide substituent. This compound retarded [³H]N-methylscopolamine-dissociation in guinea-pig cardiac membranes with slightly higher potency than W84, the EC50 values amounting to 7·5 × 10^?7and 13× 10^?7 m , respectively. Molecular modelling revealed differences in the electron density of the substituents and in their molecular shape. It is suggested that the derivatives use partially different sites of attachment when occupying the allosteric binding site of the receptor protein.     

Synthesis, antiplatelet aggregation activity, and molecular modeling study of novel substituted-piperazine analogues

Abstract  

New carbamoylpyridine and carbamoylpiperidine analogues containing nipecotic acid scaffold were designed, synthesized, and evaluated for their platelet aggregation inhibitory activity. Molecular modeling investigation was performed and the impact of lipophilicity on activity was also discussed. Structure activity relationship among this series was obtained. N ¹-[1-(4-bromobenzyl)-3-piperidino-carbonyl]-N ⁴-(2-chlorophenyl)-piperazine hydrobromide (20), and 1,4-bis-[3-[N ⁴-(2-chlorophenyl)-N ¹-(piperazino-carbonyl)]-piperidin-1-yl-methyl]-benzene dibromide (30) are the most active antiplatelet aggregating compounds in this study, both at concentration of 0.06 μM.     

Nitrosamine formation from interaction of isosorbide dinitrate and hydroxyzine HCl, a tertiary amine

The interaction of two drugs—isosorbide dinitrate (ISDN) and hydroxyzine hydrochloride (HZ), a tertiary amine—was studied in vitro, under conditions simulating those found in the stomach, to determine if nitrosamines are formed. Gas chromatography-mass spectrometry was used to monitor the latter compounds. We found that, in the presence of sodium nitrite, HZ undergoes oxidative cleavage and nitrosation, forming three nitrosamine compounds, N-(4-chlorophenyl)benzyl]-N′-nitro-sopiperazine (A), N′-[2-(2′-hydroxyethoxy)ethyl]-N′-nitrosopiperazine (B), and N, N′-dinitrosopiperazine (C). However, when ISDN (0.8 g) and HZ (2.0 g) were incubated together for 1 hr, only N-[α-(4-chlorophenyl)benzyl]-N′-nitrosopiperazine (A) was recovered. Although preparations of HZ contain (A) as an impurity, the quantity is trivial (0.5 ng/mg drug), and the bulk of the material detected is formed by interaction of ISDN with HZ. Because some individuals may ingest isosorbide dinitrate and hydroxyzine HCl, or analogous combinations, over a period of years, the risk posed by this type of drug interaction should be determined.     

Radiosynthesis of 2-[6-chloro-2-(4-iodophenyl)imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-[11C]methyl-acetamide, [11C]CLINME,a novel radioligand for imaging the peripheral benzodiazepine receptors with PET

Recently, a new 2-(iodophenyl)imidazo[1,2-a]pyridineacetamide series has been developed as iodine-123-labelled radioligands for imaging the peripheral benzodiazepine receptors using single photon emission tomography. Within this series, 2-[6-chloro-2-(4-iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl-acetamide (CLINME) was considered as an appropriate candidate for positron emission tomography imaging and was isotopically labelled with carbon-11 (T_1/2: 20.38 min) at the methylacetamide side chain from the corresponding nor-analogue using [¹¹C]methyl iodide and the following experimental conditions: (1) trapping at −10°C of [¹¹C]methyl iodide in a 1/2 (v:v) mixture of DMSO/DMF (300 µl) containing 0.7–1.0 mg of the precursor for labelling and 3–5 mg of powdered potassium hydroxide (excess); (2) heating the reaction mixture at 110°C for 3 min under a nitrogen stream; (3) diluting the residue with 0.6 ml of the HPLC mobile phase; and (4) purification using semi-preparative HPLC (Zorbax^® SB18, Hewlett Packard, 250 × 9.4 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [¹¹C]CO₂ production batch, 120−150 mCi (4.44–5.55 GBq) of [¹¹C]CLINME were obtained (16–23% decay-corrected radiochemical yield, n=12) within a total synthesis time of 24–27 min (Sep-pak^®Plus-based formulation included). Specific radioactivities ranged from 0.9 to 2.7 Ci/µmol (33.3–99.9 GBq/µmol) at the end of radiosynthesis. Copyright © 2007 John Wiley & Sons, Ltd.     

Radiosynthesis of the 11C-methyl derivative of LBQ657 for PET investigation of the neprilysin inhibitor sacubitril

Neprilysin, also known as neutral endopeptidase, is a cell surface membrane metalo-endopeptidase that cleaves various peptides. Altered neprilysin expression has been correlated with various cancers and cardiovascular diseases. In this work, we present the radiosynthesis of the novel O-¹¹C-methylated derivative of LBQ657 (a potent neprilysin inhibitor). (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [¹¹C]methyl ester ([¹¹C]MeOLBQ) is an analog of sacubitril where the alkyl ester is a ¹¹C-methyl instead of an ethyl. [¹¹C]MeOLBQ was produced in a one-pot two-step synthesis. The O-¹¹C-methylation of the pentanoic acid part was done with [¹¹C]methyl triflate followed by the deprotection of the tert-butyl ester precursor in acidic conditions. [¹¹C]MeOLBQ ([¹¹C] 7 ) was produced in 9.5 ± 2.5% RCY (25 ± 6% decay-corrected from [¹¹C]CO₂, n = 3) high molar activity 348 ± 100 GBq/μmol (9425 ± 2720 mCi/μmol) at EOS, in high chemical (>95%) and radiochemical (>99%) purities. The total synthesis time including HPLC purification and reformulation was 29 minutes. To our knowledge, this is the first PET-labeled analog of the clinically used NEP inhibitor sacubitril.     

Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

Synthesis and characterization of site-specific biotinylated probes for the motilin receptor*

The solid-phase synthesis of two porcine motilin derivatives, specifically biotinylated on the side chain of Lys²⁰), was accomplished by preactivation of the protected amino acids N^x-(9-fluorenylmethoxycarbonyl)-N^ε-biotinyl-L-lysine and N^x-(9-fluorenylmethoxycarbonyl)-N^ε-[N-(biotinyl)-6-aminohexanoyl]-L-lysine with BOP/ HOBt/DIEA (1:1:2.5) followed by coupling to the support-bound peptide substrate. The biotin moiety was stable to TFA cleavage and repetitive cycles of acylation, as evidenced by the high level of purity (>80%) of the crude peptides. This direct synthetic approach complements existing orthogonal protection strategies for the site-specific biotinylation of peptides. The derivatized peptides were purified by RP-HPLC and characterized by mass spectral and amino acid analysis. In binding studies using a rabbit antral smooth muscle homogenate, both [Leu¹³, Lys²⁰ (N^ε-biotinyl)]porcine motilin (3) and [Leu¹³, Lys²⁰ (N^ε-[N-(biotinyl)-6-aminohexanoyl])]porcine motilin (4) possessed nearly equal affinities for the motilin receptor (IC₅₀= 0.89 and 1.2 nM, respectively) as native porcine motilin (1) (IC₅₀= 0.76 nM). The biotinylated peptides were also highly potent in tissue bath assays employing rabbit duodenal smooth muscle segments. In contrast, commercially available [N^x-biotinylPhe¹]porcine motilin (5) had markedly lower affinity in the binding assay (IC₅₀= 30 nM). The relative bioactivities of these receptor probes are in accord with previous synthetic studies on motilin which demonstrated the importance of the amino-terminal segment in the high affinity interaction between the peptide and its receptor. Analog 3 retained high affinity for the motilin receptor in the presence of avidin. Therefore, this peptide is expected to be a valuable tool for the isolation and identification of motilin receptors. © Munksgaard 1994.     

A facile automated synthesis of N‐succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) for 18F‐labeled cell‐penetrating peptide as PET tracer

A fully automated synthesis of N‐succinimidyl 4‐[¹⁸F]fluorobenzoate ([¹⁸F]SFB) was carried out by a convenient three‐step, one‐pot procedure on the modified TRACERlab FX_FN synthesizer, including [¹⁸F]fluorination of ethyl 4‐(trimethylammonium triflate)benzoate as the precursor, saponification of the ethyl 4‐[¹⁸F]fluorobenzoate with aqueous tetrapropylammonium hydroxide instead of sodium hydroxide, and conversion of 4‐[¹⁸F]fluorobenzoate salt ([¹⁸F]FBA) to [¹⁸F]SFB treated with N,N,N′,N′‐tetramethyl‐O‐(N‐succinimidyl)uranium tetrafluoroborate (TSTU). The purified [¹⁸F]SFB was used for the labeling of Tat membrane‐penetrating peptide (containing the Arg‐Lys‐Lys‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Pro‐Leu‐Gly‐Leu‐Ala‐Gly‐Glu‐Glu‐Glu‐Glu‐Glu‐Glu‐Glu sequence, [¹⁸F]CPP) through radiofluorination of lysine amino groups. The uncorrected radiochemical yields of [¹⁸F]SFB were as high as 25–35% (based on [¹⁸F]fluoride) (n=10) with a synthesis time of～40 min. [¹⁸F]CPP was produced in an uncorrected radiochemical yields of 10–20% (n=5) within 30 min (based on [¹⁸F]SFB). The radiochemical purities of [¹⁸F]SFB and [¹⁸F]CPP were greater than 95%. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and Anticancer Activity of 2-(Alkyl-, Alkaryl-, Aryl-, Hetaryl-)-[1,2,4]triazolo[1,5-c]quinazolines

The combinatorial library of novel potential anticancer agents, namely, 2-(alkyl-, alkaryl-, aryl-, hetaryl-)[1,2,4]triazolo[1,5-c]quinazolines, was synthesized by the heterocyclization of the alkyl-, alkaryl-, aryl-, hetarylcarboxylic acid (3H-quinazoline-4-ylidene)hydrazides by oxidative heterocyclization of the 4-(arylidenehydrazino)quinazolines using bromine, and by the heterocyclization of N-(2-cyanophenyl)formimidic acid ethyl ester. The optimal method for synthesis of the s-triazolo[1,5-c]quinazolines appeared to be cyclocondensation of the corresponding carboxylic acid (3H-quinazoline-4-ylidene)hydrazides. The compounds’ structures were established by ¹H, ¹³C NMR, LC- and EI-MS analysis. The in vitro screening of anticancer activity determined the most active compound to be 3,4,5-trimethoxy-N′-[quinazolin-4(3H)-ylidene]benzohydrazide (3.20) in micromolar concentrations with the GI₅₀ level (MG_MID, GI₅₀ is 2.29). Thus, the cancer cell lines whose growth is greatly inhibited by compound 3.20 are: non-small cell lung cancer (NCI-H522, GI₅₀=0.34), CNS (SF-295, GI₅₀=0.95), ovarian (OVCAR-3, GI₅₀=0.33), prostate (PC-3, GI₅₀=0.56), and breast cancer (MCF7, GI₅₀=0.52), leukemia (K-562, GI₅₀=0.41; SR, GI₅₀=0.29), and melanoma (MDA-MB-435, GI₅₀=0.31; SK-MEL-5, GI₅₀=0.74; UACC-62, GI₅₀=0.32). SAR-analysis is also discussed.     

Further pharmacological characterization of the adenosine receptor subtype mediating inhibition of oxidative burst in human isolated neutrophils

The aims of this study were to characterize the adenosine receptor subtype mediating inhibition of superoxide anion generation induced by N-formyl-methionyl-leucylphenylalanine (fMLP) in human neutrophils and to test the hypothesis that adenosine 3′:5′-cyclic monophosphate (cAMP) is the second messenger mediating such inhibition. Superoxide anion generation induced by a submaximal concentration of fMLP (1 μM) was inhibited in a concentration-dependent manner by adenosine receptor agonists with a rank order of potency ofN-ethylcarboxamidoadenosine (NECA) > 2(4-[(2- carboxyethyl)phenyl]ethylamino)-5′-N-ethylcarboxamido adenosine (CGS 21680) > (R)-N⁶-phenyl-2-propyladenosine ((R)-PIA) > 2-Chloro-N⁶-(3-iodobenzyl)9-[5-methylcarbamoyl)-β-D-ribofuranosyl] adenine (2-Cl-IB-MECA) > N⁶-cyclopentyladenosine (CPA) > (S)-N⁶-phenyl-2-propyladenosine ((S)-PIA) ≥ N⁶-(4-amino-3-iodobenzyl) adenosine-5′-N-methyl-uronamide (AB-MECA); this order of potency is consistent with the activation of A_2A adenosine receptors. The nonselective A₁, A_2A, and A_2B receptor antagonist 8-p-(sulphophenyl) theophylline (8-SPT; 10 μM) produced blockade of each of the agonists (pK_B values 4.79–5.68). The selective A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM) and the selective A₃ adenosine receptor antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX; 200 nM) also produced blockade of all the agonists (PK_B values 7.37–7.61 and 6.75–7.52, respectively); however, the concentrations required were higher than those which are considered selective for the A₁ or A₃ receptors. The selective A_2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl) [1,2,4] triazolo [2,3-a] [1,3,5] triazin-5-yl amino]ethyl) phenol (ZM 241385; 100 nM), powerfully suppressed the inhibition of the oxidative burst induced by each of the agonists. A Schild analysis of the effects of ZM 241385, 1–100 nM, against NECA and CGS 21680 was carried out. ZM 241385 produced concentration-dependent, parallel shifts of the concentration–effect curves to both NECA and CGS 21680, with pA₂ values of 9.62 and 9.59, respectively. Together, these data establish that inhibition of the oxidative burst in human isolated neutrophils, induced by adenosine receptor agonists, is mediated by the A_2A receptor. NECA (0.01–10 μM) induced a concentration-dependent increase in the intracellular cAMP content of neutrophils. This effect was inhibited in a dose-dependent manner by ZM 241385 (0.001–10 μM), consistent with activation of A_2A adenosine receptors. The results clearly demonstrate that in human neutrophils inhibition of the fMLP—induced oxidative burst by adenosine receptor agonists is mediated via activation of A_2A adenosine receptors linked positively to cAMP. No evidence of A₁, A_2B, or A₃ adenosine receptor-mediated modulation of oxidative burst was found. Drug Dev. Res. 43:214–224, 1998. © 1998 Wiley-Liss, Inc.     

Comparative Metabolic Studies of Phenacetin and Structurally-related Compounds in the Rat

1. A comparative study of the metabolism of [acetyl-¹⁴C]phenacetin, [acetyl-¹⁴C]methacetin, [acetyl-¹⁴C]paracetamol and [acetyl-¹⁴C]acetanilide in the rat is reported.

2. The extent of N-deacetylation, evidenced by the measurement of respired ¹⁴CO₂ varied, being greatest with acetanilide (25—31%) and least with paracetamol (6%).

3. The major urinary metabolites in each case were N-acetyl-p-aminophenyl sulphate and N-acetyl-p-aminophenyl glucuronide; the relative proportions varied with the sex of the animals and as a result of extended dosage.

4. The metabolism of [ethyl-¹⁴C]phenacetin and [ethyl-¹⁴C]phenetidine was investigated and the extent of O-dealkylation determined by measurement of respired ¹⁴CO₂

5. The metabolic pathways of some related glycolanilides and oxanilic acids included N-deacylation, and in the glycolanilides, oxidation of the glycollic group.     

Gas phase production of [11C]methyl iodide‐d3. Synthesis and biological evaluation of S‐[N‐methyl‐11C]citalopram and deuterated analogues

Three ¹¹C‐labelled tracers for the serotonin reuptake site, S‐[N‐methyl‐¹¹C]citalopram ( [¹¹C]‐4 ), S‐[N‐methyl‐d₃‐¹¹C]citalopram ( [¹¹C]‐12 ), and S‐[N‐methyl‐¹¹C]citalopram‐α,α‐d₂ ( [¹¹C]‐13 ) were synthesized and the distribution of radioactivity after injection of radioligand was examined ex vivo in rats. The deuterated analogue of (S)‐desmethylcitalopram, (S)‐1‐(4‐fluorophenyl)‐1‐(3‐methylamino‐[3‐d₂]‐propyl)‐1,3‐di‐hydro‐isobenzofuran‐5‐carbonitrile ( 11 ), was synthesized in a multi‐step synthesis from escitalopram ( 4 ) and used as precursor in the synthesis of [¹¹C]‐13 . In analogy with the reported gas phase synthesis of [ ¹¹ C]methyl iodide the first gas phase synthesis of [¹¹C]Methyl iodide‐d₃ is reported. The ¹H/²H kinetic isotope effect related to the synthesized compounds were investigated in ex vivo rat studies, where the brain regions of interest to cerebellum ratios of the tracers [¹¹C]‐4 , [¹¹C]‐12 and [¹¹C]‐13 were compared. The ex vivo data indicated no significant differences in binding in any of the investigated brain regions after injection of the three tracers. Copyright © 2004 John Wiley & Sons, Ltd.     

G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A_2A receptor agonist 2-[4-(2-p-carboxyethyl[³H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([³H]CGS 21680) and of the adenosine A₁ receptor agonist [³H]R-phenylisopropyladenosine ([³H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [³H]CGS 21680 (30 nM) binding in the cortex than [³H]R-PIA (2 nM) binding to cortical or striatal membranes or [³H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple G_i/G_o protein-coupled receptors, more effectively inhibited [³H]R-PIA binding to cortical or striatal membranes and [³H]CGS 21680 binding in the cortex than [³H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples G_s protein-coupled receptors, more effectively inhibited [³H]CGS 21680 binding in the striatum than [³H]CGS 21680 binding in the cortex and less effectively inhibited [³H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [³H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with G_i/G_o proteins. The K _i for displacement of [2-³H]-(4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol) ([³H]ZM 241385, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K _i for displacement of [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([³H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A₁ antagonist, [³H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A₁ agonist, [³H]R-PIA (2 nM), in 2 or 10 mM Mg²⁺-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A₁ receptors and that limbic CGS 21680 binding sites differ from striatal-like A_2A receptors since they couple to G_i/G_o proteins, as well as to G_s proteins. Received: 22 July 1998 / Accepted: 18 January 1999     

[125I], [127I]‐and [14C]‐Labelling of the GLP‐1‐(7‐37) derivative NN2211

Arg³⁴Lys²⁶(N^ε‐(γ‐L ‐glutamyl(N^α‐palmitoyl)))‐GLP‐1(7‐37) (NN2211) is currently in development as a diabetes type 2 drug. The fatty acid attached to the GLP‐1(7‐37) ensures a long and controlled duration of action. The synthesis of [¹²⁵I]NN2211, [¹²⁷I]NN2211 and [¹⁴C]NN2211 used for preclinical ADME studies are described. NN2211 was iodinated using the lactoperoxidase/hydrogen peroxide method, and [¹⁴C]NN2211 was synthesized in 4 steps by two routes both starting from an α‐protected [U‐¹⁴C]glutamic acid. Copyright © 2003 John Wiley & Sons, Ltd.