Synthesis and evaluation of a 18F‐labeled 4‐phenylpiperidine‐4‐carbonitrile radioligand for σ1 receptor imaging

We report the design and synthesis of several 4‐phenylpiperidine‐4‐carbonitrile derivatives as σ₁ receptor ligands. In vitro radioligand competition binding assays showed that all the ligands exhibited low nanomolar affinity for σ₁ receptors (K_i(σ₁) = 1.22–2.14 nM) and extremely high subtype selectivity (K_i(σ₂) = 830–1710 nM; K_i(σ₂)/K_i(σ₁) = 680–887). [¹⁸F]9 was prepared in 42–46% isolated radiochemical yield, with a radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^‐ substitution of the corresponding tosylate precursor. Biodistribution studies in mice demonstrated high initial brain uptakes and high brain‐to‐blood ratios. Administration of SA4503 or haloperidol 5 min prior to injection of [¹⁸F]9 significantly reduced the accumulation of radiotracers in organs known to contain σ₁ receptors. Two radioactive metabolites were observed in the brain at 30 min after radiotracer injection. [¹⁸F]9 may serve as a lead compound to develop suitable radiotracers for σ₁ receptor imaging with positron emission tomography.     

Synthesis and biological evaluation of a radioiodinated spiropiperidine ligand as a potential σ1 receptor imaging agent

We report the synthesis and evaluation of 1′‐(4‐[¹²⁵I]iodobenzyl)‐3H‐spiro[isobenzofuran‐1,4′‐piperidine] ([¹²⁵I]Spiro‐I) as a potential SPECT tracer for imaging of σ₁ receptors. [¹²⁵I]Spiro‐I was prepared in 55–65% isolated radiochemical yield, with radiochemical purity of >99%, via iododestannylation of the corresponding tributyltin precursor. In receptor binding studies, Spiro‐I displayed low nanomolar affinity for σ₁ receptors (σ₁: K_i=2.75±0.12 nM; σ₂: K_i=340 nM) and high subtype selectivity (σ₂/σ₁=124). Biodistribution in mice demonstrated relatively high concentration of radioactivity in organs known to contain σ₁ receptors, including the lung, kidney, heart, spleen, and brain. Administration of haloperidol 5 min prior to injection of [¹²⁵I]Spiro‐I significantly reduced the concentration of radioactivity in the above‐mentioned organs. These findings suggest that the binding of [¹²⁵I]Spiro‐I to σ₁ receptors in vivo is specific. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of aromatic amino acid substitutions in the 3-position of cyclic β-casomorphin analogues on μ-opioid agonist/δ-opioid antagonist properties*

The β-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed μ agonist/δ antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37 , 1136-1144]. The 2-Na1³ residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bz1) (4), Tyr(Bz1) (5), 4′-benzoylphenylalanine (Bpa) (6), 4′-benzylphenylalanine (Bzp) (7), thyrnine (Thy) (8), thyroxine (Thx) (9), 4′-biphenylalanine (Bip) (10), 4′-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in μ and δ receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the μ receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the δ receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective δ antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by δ receptor activation. The Bzp³- and Bip³-containing peptides 7 and 10 turned out to be μ antagonists against the μ selective agonist H-Tyr-D-Ala-Phe-Phe-NH₂, in the GPI assay. The other analogues were weak partial μ agonists which displayed remarkably decreased μ receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be δ antagonists in the MVD assay. Analogues 4 and 9 exhibited δ antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher δ antagonist potency against DPDPE and deltorphin I and, in most cases, increased δ receptor affinity. These results indicate that the & delta; receptor tolerates bulky aromatic side chains in the 3-position of cyclic β-casomorphin analogs with either δ agonist or δ antagonist properties. However, these compounds displayed drastically reduced μ receptor affinity in nearly all cases. © Munksgaard 1996.     

Transport properties of 3′-azido-3′-deoxythymidine and 2′,3′-dideoxyinosine in the rat choroid plexus

Transport properties of 3′-azido-3′-deoxythymidine (AZT) and 2′, 3′-dideoxyinosine (DDI) were characterized in the isolated rat choroid plexus. AZT and DDI competitively inhibited the active transport of [³H]benzylpenicillin, a prototypic organic anion, with K_i values of 85·4±13·1 and 155±22 μM, respectively. Accumulation of [³H]DDI was against an electrochemical potential via a saturable process (K_m=29·7±4·9 μM, V_max=13·5±2·4 pmol min⁻¹/μL tissue) that was inhibited by metabolic inhibitors (carbonylcyanide p -trifluoromethoxyphenylhydrazone, 10 μM, and rotenone, 30 μM) and sulphydryl reagents (p -chloromercuribenzoic acid, 100 μM, and p -chloromercuribenzenesulphonic acid, 100 μM), but did not require an inwardly directed Na⁺ gradient. Accumulation of [³H]DDI was inhibited by benzylpenicillin and AZT in a dose-dependent manner, with IC₅₀ values of 91·6±28·9 and 294±84 μM, respectively. In contrast, no significant accumulation of [³H]AZT was observed. These results suggest that DDI is transported, at least in part, by the transport system for organic anions located on the rat choroid plexus, whereas AZT is recognized, but not transported by this system. © 1997 John Wiley & Sons, Ltd.     

18F‐Labeled benzylpiperazine derivatives as highly selective ligands for imaging σ1 receptor with positron emission tomography

We report the design, synthesis, and evaluation of a new series of benzylpiperazine derivatives as selective σ₁ receptor ligands. All seven ligands possessed low nanomolar affinity for σ₁ receptors (K_i(σ₁) = 0.31‐4.19 nM) and high subtype selectivity (K_i(σ₂)/K_i(σ₁) = 50‐2448). The fluoroethoxy analogues also exhibited high selectivity toward the vesicular acetylcholine transporter (K_i(VAChT)/K_i(σ₁) = 99‐18252). The corresponding radiotracers [¹⁸F] 13 , [¹⁸F] 14 , and [¹⁸F] 16 with high selectivity (K_i(σ₂)/K_i(σ₁) > 100, K_i(VAChT)/K_i(σ₁) > 1000) were prepared in 42% to 55% radiochemical yields (corrected for decay), greater than 99% radiochemical purity (RCP), and molar activity of about 120 GBq/μmol at the end of synthesis (EOS). All three radiotracers showed high initial brain uptake in mouse (8.37‐11.48% ID/g at 2 min), which was not affected by pretreatment with cyclosporine A, suggesting that they are not substrates for permeability‐glycoprotein (P‐gp). Pretreatment with SA4503 or haloperidol resulted in significantly reduced brain uptake (35%‐62% decrease at 30 min). In particular, [¹⁸F] 16 displayed high brain‐to‐blood ratios and high in vivo metabolic stability. Although it may not be an optimal neuroimaging agent because of its slow kinetics in the mouse brain, [¹⁸F] 16 can serve as a lead compound for further structural modifications to explore new potential radiotracers for σ₁ receptors.     

3-(ω-Aminoalkyl)-5,5-dialkyl(or spirocycloalkyl-1′,5-)hydantoins as New 5-HT1A and 5-HT2A Receptor Ligands

A series of new 3-(ω-aminoalkyl)-5,5-disubstituted hydantoins, containing 1-phenylpiperazine, 1-(o-methoxyphenyl)piperazine or 1,2,3,4-tetrahydroisoquinoline fragments, were synthesized by standard alkylation procedures and their 5-HT_1A and 5-HT_2A receptor affinities were determined. It has been shown that the investigated derivatives are recognized by 5-HT_1A and 5-HT_2A receptors due to the presence of a 1-arylpiperazine fragment; however, the terminal hydantoin moiety plays an important role in stabilization of the receptor-ligand complex. It has also been found that the two 1-phenylpiperazine derivatives 32 and 36 are new, selective 5-HT_2A receptor ligands (K_i = 34 and 37 nM, respectively), whereas the derivative of 1-(o-methoxyphenyl)piperazine ( 38 ) is a new, highly potent 5-HT_1A receptor ligand (K_i = 0.51 nM) with a moderate affinity for 5-HT_2A receptors (K_i = 213 nM).     

Structure-activity relationships of dermorphin analogues containing N-substituted amino acids in the 2-position of the peptide sequence

A series of dermorphin analogues containing an N-alkylated amino-acid residue Xaa in the 2-position of the peptide sequence was synthesized (Xaa =N-methylalanine, proline, pipecolic acid, N-methylphenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic]). These peptides have the potential of assuming a cis Tyr^l-Xaa² peptide bond. Their in vitro opioid activity profiles were determined in μ and δ-receptor-representative binding assays and bioassays. Aside from [D-Pro²]dermorphin, all analogues showed high affinity for μ and/or δ-opioid receptors. Whereas most compounds were found to be full μ-agonists in the guinea pig ileum (GPI) assay, [Tic²]dermorphin (compound 7) was a partial μ-agonist. Replacement of Gly⁴in 7 with Phe resulted in an analogue (8) with weak μ-antagonist activity. Furthermore, analogues 7 and 8 both were potent § antagonists (K_c= 3–40 nM) against the §-agonists Leuenkephalin, DPDPE and deltorphin I in the mouse vas deferens (MVD) assay. Compound 3, containing l -Pro in the 2-position, turned out to be one of the most μ receptor-selective linear dermorphin analogues reported to date. Low-temperature HPLC experiments using micropellicular octadecyl silica as stationary phase revealed conformational heterogeneity of the dermorphin analogues which was ascribed to cis-trans isomerization around the Tyr^l-Xaa²-and Tyr⁵-Pro⁶ peptide bonds. In the case of analogue 7 four separate peaks corresponding to the four possible isomers were apparent at -5°C. Since opioid peptide analogues with a non-N-akylated l -amino acid residue in the 2-position are nearly inactive and cannot assume a cis peptide bond at the 1–2 position, these results support the hypothesis that the bioactive conformation of opioid peptides containing an N-alkylated l -amino acid residue in position 2 is characterized by a cis Tyr^l-Xaa² peptide bond.     

Optimization and synthesis of an 18F‐labeled dopamine D3 receptor ligand using [18F]fluorophenylazocarboxylic tert‐butylester

There is still no efficient fluorine‐18‐labeled dopamine D₃ subtype selective receptor ligand for studies with positron emission tomography. We aim at improving the D₃ selectivity and hydrophilicity of a candidate ligand by changing the substitution pattern to a 2,3‐dichlorophenylpiperazine and hydroxylation of the butyl chain. The compound [¹⁸F]3 exhibited D₃ affinity of K_i = 3.6 nM, increased subtype selectivity (K_i(D₂/D₃) = 60), and low affinity to 5‐HT_1A and α₁ receptors (K_i (5‐HT_1A/D₃) = 34; K_i (α₁/D₃) = 100). The two‐step radiosynthesis was optimized for analog [¹⁸F]4 by reducing the necessary concentration of the precursor amine (57 mM), which reacted with [¹⁸F]fluorophenylazocarboxylic tert‐butylester under basic conditions. The optimization of the base (Cs ₂CO₃, 23 mM) and the adjustment of reaction temperature led to the radiochemical yield of 63% after 5 min at 35°C. The optimized reaction conditions were transferred on to the synthesis of [¹⁸F]3 with an overall non‐decay corrected yield of 8‐12% in a specific activity of 32‐102 GBq/µmol after a total synthesis time of 30‐35 min. This provides a D ₃ radioligand candidate with improved attributes concerning selectivity and radiosynthesis for further preclinical studies.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Reduced number of α- and β-adrenergic receptors in the myocardium of rats exposed to tobacco smoke

The concentration of α- and β-adrenergic receptors-as measured by specific [³H]WB-4101 and (−)-[³H]-dihydroalprenolol binding-was diminished by 60% below control values in the hearts of hearts of rats exposed to tobacco smoke. These changes in receptor numbers took place almost immediately after tobacco smoke exposure and were rapidly reversible after termination of the exposure. The dissociation constant, K , for [³H]WB-4101 was identical in exposed (K_D = 0.34 ± 0.09 nM) and control (K_D = 0.35 ± 0.07 nM) hearts but was significantly different i in the case of (-)-[³H] dihydroalprenolol binding (exposed, (K_D = 2.83 ± 0.30 nM vs control K_D = 5.22 ± 0.61 nM). For β-receptor binding there was no significant difference between exposed and control animals in the K_i values (−)-epinephrine, (−)-norepinephrine, (−)-alprenolol, (±)-propranolol or timolol. (−)-Isoproterenol, however, was found to bind with lower affinity in exposed compared with control hearts. For α-receptor binding there was no significant difference between control and ‘smoked’ animals in the K_i values for (−)-epinephrine, (−)-norepinephrine or phentolamine. The decrease in α- and β-adrenergic receptor concentration may be related to the phenomenon of receptor desensitization resulting from a release of catecholamines in rats exposed to tobacco smoke.     

Labelling of I2B-imidazoline receptors by [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) in rat brain and liver: characterization, regulation and relation to monoamine oxidase enzymes

The novel selective imidazoline radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) was used to characterize and assess further the nature of I₂-imidazoline receptors in rat brain and liver. In the cerebral cortex, 2-BFI displayed high affinity (K _i= 9.8 nM) for a single class of [³H]2-BFI binding sites. Other imidazoline/guanidine compounds (e.g. aganodine, cirazoline and idazoxan) displayed biphasic competition curves, indicating the existence of high (K _iH= 2.9-78 nM; R _H= 61-83%) and low (K _iL= 4.7-158 μM) affinity sites. The pharmacological profile for [³H]2-BFI binding (aganodine > cirazoline > 2-BFI >> clonidine > amiloride >> efaroxan) was typical of that for I₂-sites. This profile was almost identical to that obtained against [³H]idazoxan (correlation between pK _i values, r = 0.97) which indicated that the sites characterized with [³H]2-BFI in brain corresponded to I₂-imidazoline receptors. The low affinity of amiloride against [³H]2-BFI (K _i= 900 nM) further indicated that these brain I₂-sites belong to the I_2B-subtype. [³H]2-BFI binding sites (B _max= 72 fmol/mg protein) in brain were differentially modulated by treatment (7 days) with cirazoline (up-regulation: 25%) and the MAO inhibitor phenelzine (down-regulation: 31%), indicating that these I₂-sites are regulated in vivo, as is the case for those labelled by [³H]idazoxan. Chronic treatment with 2-phenylethylamine, a phenelzine metabolite and endogenous amine, did not alter the density of brain of I₂-imidazoline receptors labelled by [³H]idazoxan. Preincubation of liver membranes with the MAO inhibitor clorgyline (10^-7 M) abolished the binding of [³H]Ro 41-1049 (N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide) to MAO-A, but it did not alter the binding of [³H]Ro 19-6327 (N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide) to MAO-B or that of [³H]2-BFI to I₂-sites. At 10^-4 M it also abolished MAO-B sites, but a substantial proportion of I₂-sites (40%) remained intact. Preincubation of liver membranes at 60 °C also abolished MAO-A/B sites, whereas still 22% of I₂-sites remained. The results indicate that [³H]2-BFI is a good tool for the identification of I₂-imidazoline receptors and suggest further that certain I₂-sites and MAO are different proteins. Received: 10 January 1997 / Accepted: 6 March 1997     

Novel 2(3H)-Benzothiazolones as Highly Potent and Selective Sigma-1 Receptor Ligands

In an effort to produce a new pharmacological probe with high affinity and selectivity for the sigma-1 receptor, we have synthesized a series of original 2(3H)-benzothiazolones utilizing compound 4 [3-(1-piperidinoethyl)-6-propylbenzothiazolin-2-one] as a lead. Receptor binding affinities were determined at sigma-1 and sigma-2 receptors. The best ligand (9, sigma-1 K_i = 0.56 nM, selectivity ratio >1000) was obtained with an azepine side-chain. When tested on a wide battery of receptors, including 5HT_2A(h), 5HT₃(h), α₁, α ₂, β₁, β₂, H₁, H₂, opioids, D₁(h), D₂(h), 5HT uptake, and DOPA uptake, compound 9 showed submicromolar affinity only for α₂ (K_i = 205 nM) and H₁ (K_i = 311 nM).     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic α‐hydroxymethylamino acids

Abstract: New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic α‐hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to μ and δ opioid receptors. The analogue with (R)‐HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to δ and a significant increase of affinity to μ receptors. In contrast, an analogue with (S)‐HmPhe in the same position, was very potent and more specific to δ receptors than parent DT I. The analogue with (R)‐HmVal at position 5 expressed higher δ affinity and selectivity than parent DT I. The analogue with other possible isomer (S)‐HmVal was less selective for δ opioid receptors, as a result of decreasing affinity to δ and increasing affinity to μ receptors. The analogues with (R)‐ or (S)‐HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Synthesis of [18F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine ([18F]NicFP): a potential α4β2 nicotinic acetylcholine receptor radioligand for PET

Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the α4β2 subtype of nicotinic receptor, we synthesized [¹⁸F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine (NicFP). The in vitro K_D of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [¹⁸F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [¹⁸F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/μmol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of an 18F‐labelled high affinity β1‐adrenoceptor PET radioligand based on ICI 89,406

To date, some non‐selective β‐adrenoceptor (β‐AR) positron emission tomography (PET) radioligands are in clinical use, but no PET radioligand for the selective imaging of cardiac β₁‐ARs is clinically available. Therefore, the aim of this study was to develop a potential high‐affinity PET radioligand for the β₁‐subtype of ARs. Here, the synthesis and in vitro evaluation of (S)‐ and (R)‐N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐[4‐(2‐fluoro‐ethoxy)‐phenyl]‐urea ( 8a–b ), derivatives of the well‐characterized β₁‐AR selective antagonist, ICI 89,406, are described. The (S)‐isomer 8a shows both higher β₁‐AR selectivity and β₁‐AR affinity than the (R)‐enantiomer 8b (selectivity: 40 800 vs 1580; affinity: K_I1=0.049 nM vs K_I1=0.297 nM). Therefore, the ¹⁸F‐labelled analogue 8e of compound 8a was synthesized. While the direct nucleophilic ¹⁸F‐fluorination of the tosylate precursor 8d produced 8e in low radiochemical yields (?2.9% decay‐corrected) and specific activities (?3.5 GBq/µmol at the end of synthesis (EOS), n=9) the alternative two‐step synthesis of 8e from ethylene glycol di‐p‐tosylate 9 , [¹⁸F]fluoride ion and phenol precursor 8f gave satisfying results (16.4±3.2% radiochemical yield (decay‐corrected), 99.7±0.5% radiochemical purity, 40±8 GBq/µmol specific activity at the EOS within 174±3 min from the end of bombardment (EOB) (n=5)). Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis and receptor binding analysis of dermorphin hepta-, hexa- and pentapeptide analogues

Sixteen dermorphin analogues were synthesized and characterized for μ- and δ-opioid receptor binding properties using [³H]DAGO and [³H]DPDPE, respectively. The analogues included the following: substitutions at position 4 and/or the C-terminal residue; deletions of Gly⁴ or Pro⁶-Ser⁷; inclusion of Z or an acetyl group on the β-amino group of Lys⁷; and the presence of either a C-terminal amide or free acid group. Two peptides, [Lys7-OH]- and [Lys⁷-NH₂]dermorphin, had μ-affinities (K_iμ= 0.15–0.13 nm ) and μ-selectivities (K_iδ/K_iμ= 1158–1482) higher than dermorphin (K_iμ= 0.28 nm ; K_iδ/K_iμ= 295) and best fitted a one-site binding model similar to dermorphin. Significantly better (P <0.0001) fits to a two-site binding model vs. a one-site model were observed with four dermorphin analogues: [Lys(Z)⁷-OH]heptapeptide, [des-Gly⁴(Tyr⁴,Pro⁵,Asn⁶-OH)]hexapeptide and two pentapeptides, [Tyr⁵-NH₂] and [Trp⁴,Asn⁵-OH]. Our data revealed a complex binding pattern for dermorphin analogues to brain μ-receptors in which Hill coefficients less than 0.85 generally suggest heterogeneity of μ-receptors; however, only detailed analyses of the data derived from the non-linear regression fits for one- or two-components gave evidence for the possible existence of two separate [³H]DAGO binding sites. Eight of our dermorphin analogues had significantly better fits for a two-site model (P <0.05), but only four seemed to have two distinct Kⁱ, values (P <0.0001).     

Independent coupling of the human tachykinin NK2 receptor to phospholipases C and A2 in transfected Chinese hamster ovary cells

The human tachykinin NK₂ receptor stably expressed in Chinese hamster ovary cells (CHO-hNK₂R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [¹²⁵I]NKA showed that CHO-hNK₂R cells express binding sites which have high affinity for NKA (K _i=3.4±0.9 nM), GR 64349 (K _i=12±3 nM) and [βAla⁸]NKA(4–10) (K _i=21±8 nM) and for the antagonists MEN 10627 (K _i=0.55±0.2 nM), and MEN 11420 (K _i=2.4±0.8 nM). In contrast, the tachykinin NK₁ and NK₃ receptor agonists [Sar⁹,Met(O₂)¹¹]SP and senktide, respectively, were recognized with low affinity (K _i>10 μM). NKA (EC₅₀=68±18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP₃). The concentration-response curve to GR 64349 (EC₅₀=155±14 nM) was close to that of NKA, whereas [βAla⁸]NKA(4–10) (EC₅₀=445±78 nM) and SP (EC₅₀=3197±669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E₂ (PGE₂) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC₅₀ values were 3±0.3, 9±3, 7.8±0.9 and 217±37 nM for NKA, GR 64349, [βAla⁸]NKA(4–10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP₃ formation and PGE₂ release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK₂ receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE₂ release by about 35%. The phospholipase C inhibitor U-73122 (10 μM) prevented the NKA-induced formation of IP₃ but did not affect PGE₂ release. Conversely, the phospholipase A₂ inhibitor quinacrine (100 μM) blocked the release of arachidonic acid and PGE₂ without affecting the NKA-stimulated formation of IP₃. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE₂ release by 81% but was without effect on basal and NKA-stimulated IP₃ production. The calcium channel blockers verapamil (10 μM) and ω-conotoxin GVIA (0.1 μM) did not modify the basal PGE₂ production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 μM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP₃ formation. In conclusion, our data demonstrate that the human tachykinin NK₂ receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK₂ receptor. No pharmacological evidence for heterogeneity of the human NK₂ receptor was obtained in this study. Our findings indicate that the human tachykinin NK₂ receptor is independently coupled to both PLC and PLA₂ signaling pathways. Activation of the PLA₂ pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca²⁺-dependent PLA₂. Received: 7 April 1998 / Accepted: 17 July 1998     

Synthesis and preliminary PET study of the 5‐HT7 receptor antagonist [11C]DR4446

DR4446 (1‐methyl‐2a‐[4‐(4,5,6,7‐tetrahydrothieno[3,2‐c]pyridin‐5‐yl)butyl]‐2a,3,4,5‐tetrahydro‐1H‐benz[cd]indole‐2‐one) is a potent 5‐HT₇ receptor antagonist (K_i=9.7 nM) with a high selectivity over other 5‐HT family receptors (K_i for 5‐HT_1A: 770 nM; for other 5‐HT receptors: >1000 nM). As a positron emission tomography (PET) tracer for the 5‐HT₇ receptor, [¹¹C]DR4446 was synthesized at high radiochemical purity ( >98%) with specific activity of 73–120 GBq/μmol at the end of synthesis by the alkylation of the desmethyl precursor (1) with [¹¹C]CH₃I in the presence of NaH. A PET study in monkey demonstrated that [¹¹C]DR4446 had good permeability into the brain, and had a specific binding component in the brain regions including the thalamus, possibly an area in the 5‐HT₇ receptors. Metabolite analysis showed that [¹¹C]DR4446 was relatively stable and low percentages of two radio‐labeled metabolites were detected in the plasma of monkey using HPLC. Copyright © 2002 John Wiley & Sons, Ltd.