Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

From β‐N‐tert‐butyloxycarbonyl N‐carboxyanhydrides to β‐aminoacid derivatives

Abstract: β‐N‐tert‐butyloxycarbonyl‐N‐carboxyanhydrides are very reactive β‐amino acid derivatives. They react cleanly and smoothly with different nucleophiles like aminoesters, enolates, N‐methyl‐d ‐glucamine, amidoximes to afford in good to excellent yields peptides, β‐amino ketocompounds, β‐aminosugars and functionalized disubstituted 1,2,4‐oxadiazoles.     

Synthesis of 3,7,8‐15N3‐N1‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin

3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was synthesized from the oxidation of 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine with 2 equivalents of Ir(IV) in pH 4.5 potassium phosphate buffer. The synthesis of 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine started with bromination of 1,7,NH₂‐¹⁵N₃‐2′‐deoxyguanosine. The resulting 1,7,NH₂‐¹⁵N₃‐8‐bromo‐7,8‐dihydro‐2′‐deoxyguanosine reacted with sodium benzyloxide to afford 1,7,NH₂‐¹⁵N₃‐8‐benzyloxy‐7,8‐dihydro‐2′‐deoxyguanosine. Subsequent catalytic transfer hydrogenation of 1,7,NH₂‐¹⁵N₃‐8‐benzyloxy‐7,8‐dihydro‐2′‐deoxyguanosine with cyclohexene and 10% Pd/C yielded 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Purification of 3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was first carried out on a C18 column and the product was further purified on a graphite column. ESI‐MS was used to confirm the identity and to determine the isotopic purity of all the labeled compounds. The isotopic purity of 3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was 99.4 atom% based on LC‐MS measurements. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis and Anticonvulsant Properties of New Mannich Bases Derived from 3‐Aryl‐pyrrolidine‐2,5‐diones. Part 1

A series of new Mannich bases of N‐[(4‐arylpiperazin‐1‐yl)‐methyl]‐3‐(chlorophenyl)‐pyrrolidine‐2,5‐diones 10–23 have been synthesized and evaluated for their anticonvulsant activity in maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure threshold tests. Their neurotoxicity was determined using a rotorod screen. Several molecules showed a promising anticonvulsant profile especially in the MES‐test. In this model of seizures, the most active were N‐[{4‐(4‐chlorophenyl)‐piperazin‐1‐yl}‐methyl]‐3‐(3‐chlorophenyl)‐pyrrolidine‐2,5‐dione 16 and N‐[{4‐(3‐trifluoromethylphenyl)‐piperazin‐1‐yl}‐methyl]‐3‐(3‐chlorophenyl)‐pyrrolidine‐2,5‐dione 17 with ED₅₀ values of 21.4 mg/kg and 28.83 mg/kg, respectively. Selected derivatives 10 , 14 , and 16 were tested in the psychomotor seizure 6‐Hz test from which N‐[{4‐(2‐chlorophenyl)‐piperazin‐1‐yl}‐methyl]‐3‐(2‐chlorophenyl)‐pyrrolidine‐2,5‐dione 10 revealed the highest protection with an ED₅₀ of 78 mg/kg. Compounds 10 , 12 , and 17 were also tested in the pilocarpine‐induced status PIPS test. Furthermore, 17 was examined in the hippocampal kindling screen after i. p. administration to rats.     

Transport characteristics of peptides and peptidomimetics: I. N‐methylated peptides as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa

Abstract: Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N‐Methylation has been used successfully in peptide‐based drug design in an attempt to improve the metabolic stability of a peptide‐based lead compound. However, the effect of N‐methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P‐glycoprotein (P‐gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N‐acetylated and C‐amidated) analogs of H‐Ala‐Phe‐Ala‐OH with N‐methyl groups on either the Ala‐Phe or Phe‐Ala peptide bond were synthesized. Transport studies using Caco‐2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N‐methylation of the Ala‐Phe peptide bond of H‐Ala‐Phe‐Ala‐OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N‐methylation of the Phe‐Ala peptide bond of H‐Ala‐Phe‐Ala‐OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N‐methylation of the Phe‐Ala peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ decreased the substrate activity of the molecule for the efflux transporter P‐gp. In contrast, N‐methylation of the Ala‐Phe peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ increased the substrate activity of the molecule for P‐gp.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

Synthesis of morphine‐[N‐methyl‐14C]‐6‐β‐D‐glucuronide

Protected morphine‐6‐glucuronide was converted into morphine‐[N‐methyl‐¹⁴C]‐6‐glucuronide by a three‐step procedure. Methyl (3‐pivaloylmorphin‐6‐yl 2,3,4‐tri‐O‐isobutyryl‐β‐D‐glucopyranosid)uronate was N‐demethylated by treatment with 1‐chloroethyl chloroformate to afford protected normorphine‐6‐glucuronide as its hydrochloride salt. The normorphine‐6‐glucuronide derivative was alkylated with iodomethane‐[¹⁴C] in the presence of potassium carbonate to produce C‐14 labelled protected morphine‐6‐glucuronide. Finally, hydrolysis of the protecting groups using 5% sodium hydroxide solution gave morphine‐[N‐methyl‐¹⁴C]‐6‐β‐D‐glucuronide with a specific activity of 41.8 mCi mmol^?1 and radiochemical purity of 99.2% (HPLC). Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of (R)‐ and (S)‐[O‐methyl‐11C]N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐methoxy‐phenyl)‐urea as candidate high affinity β1‐adrenoceptor PET radioligands

Molecular imaging and quantification of myocardial β₁‐adrenoceptor (AR) rather than total β‐AR density is of great clinical interest since cardiac biopsy studies suggest that myocardial β₁‐AR density is reduced in patients with chronic heart failure whereas cardiac β₂‐AR density may vary. Positron emission tomography (PET), with appropriate radioligands, offers the possibility to assess β‐AR density non‐invasively in humans. However, no PET radioligand for the selective imaging of cardiac β₁‐ARs is clinically available. Here some derivatives of the well characterized β₁‐AR selective antagonist, ICI 89,406, namely the enantiomers of N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐hydroxy‐phenyl)‐urea ( 5a and 5b ) were synthesized and evaluated in vitro. The (R)‐isomer 5a was more β₁‐selective but has lower affinity than its (S)‐enantiomer 5b (β₁‐AR selectivity: 6100 vs 1240; β₁‐affinity: K₁ = 0.288 nM vs K₁ = 0.067 nM). Etherification of the analogous desmethyl precursors, 5e and 5f , respectively, with [¹¹C]iodomethane gave ¹¹C‐labelled versions of 5a and 5b , namely 5g and 5h , in 44 ± 5% radiochemical yield (decay‐corrected) and 97.4 ± 1.3% radiochemical purity with specific radioactivities of 26.4 ± 9.4 GBq/µmol within 41.2 ± 3.4 min from the end of bombardment (n = 14). 5g and 5h are now being evaluated as candidate radioligands for myocardial β₁‐ARs. Copyright © 2005 John Wiley & Sons, Ltd.     

A facile synthesis of 5,5‐dideutero‐4‐dimethyl(phenyl)silyl‐6‐undecyl‐tetrahydropyran‐2‐one as a deuterium labeled synthon for (−)‐tetrahydrolipstatin and (+)‐δ‐hexadecanolide

Deuterium‐labeled biologically active compounds are gaining importance because they can be utilized as tracers or surrogate compounds to understand the mechanism of action, absorption, distribution, metabolism, and excretion. Deuterated drug molecules (heavy drugs) become novel as well as popular because of better stability and bioavailability compared with their hydrogen analogs. Labeling of organic molecules with deuterium at specific positions is thus gaining popularity. In this work, we have exploited a highly regioselective and enantioselective direct Michael addition of methyl‐d₃ alkyl ketones to dimethyl(phenyl)silylmethylene malonate that was catalyzed by (S)‐N‐(2‐pyrrolidinylmethyl)pyrrolidine/trifluoroacetic acid/ D₂O combination with high yield and isotopic purity. The 5,5‐dideutero‐4‐dimethyl(phenyl)silyl‐6‐undecyl‐tetrahydropyran‐2‐one was obtained from the adduct of methyl‐d₃ undecanyl ketone and dimethyl(phenyl)silylmethylene malonate by a silicon controlled diastereoselective ketone reduction, lactonization, and deethoxycarbonylation. The dideuterated silylated tetrahydropyran‐2‐one is the precursor for geminal ²H₂‐labeled (+)‐4‐hydroxy‐6‐undecyl‐tetrahydropyran‐2‐one, an advanced intermediate for gem‐dideutero (–)‐tetrahydrolipstatin and (+)‐δ‐hexadecanolide syntheses.     

A novel method for the synthesis of carbon‐14‐labeled N‐[3‐(1‐methyl‐4‐piperidinyl)‐1H‐pyrrolo[3,2‐b]pyridin‐5‐yl]propanamide and its use in quantitative whole‐body autoradiography studies

Sumitriptan ( 1 ), a non‐selective 5‐HT_1B/1D agonist is an effective therapeutic agent for the acute treatment of migraine, but it is contraindicated for use in patients with known heart disease. The first Selective Serotonin One F Receptor Agonist (SSOFRA), 5‐(4′‐fluorobenz‐amido)‐3‐(N‐methyl‐piperidin‐4‐yl)‐1H‐indole ( 2 ) was demonstrated to be clinically useful in the treatment of migraine. Although 2 exhibited high affinity for the 5‐HT_1F receptor as well as high selectivity for the 5‐HT_1F receptor relative to 5‐HT_1B and 5‐HT_1D receptors, it demonstrated appreciable affinity for the 5‐HT_1A receptor. Subsequently, a program was launched to discover SSOFRA's with improved selectivity over other 5‐HT₁ receptor subtypes. As a result of these efforts, N‐[3‐(1‐methyl‐4‐piperidinyl)‐1H‐pyrrolo[3,2‐b]pyridin‐5‐yl]propanamide ( 3 ) was found to possess greater than 100‐fold selectivity over 5‐HT_1A, 5‐HT_1B and 5‐HT_1D receptors. Pursuant to a potential clinical investigation of 3 , its carbon‐14‐labeled isotopomer has been prepared by a circuitous route from unlabeled 3 and used in quantitative whole‐body autoradiography studies in rats. The results of these efforts are reported herein. Copyright © 2005 John Wiley & Sons, Ltd.     

Effects of N‐Methylated Amyloid‐β30–40 Peptides on the Fibrillation of Amyloid‐β1–40

Alzheimer's disease is a neurodegenerative disorder associated with amyloid‐β (Aβ) fibrillation. N‐Methylated amyloid‐β peptides are potent inhibitors of amyloid‐β fibrillation. We investigated the inhibitory effect of N‐Methylated Aβ_30–40 peptides on Aβ_1–40 fibrillation. N‐Methylated Aβ_30–40 peptides affected the fibrillation, and this effect was dependent on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. N‐Methylated Aβ_30–40 peptides were co‐aggregated with Aβ_1–40. Spectroscopic technique was adopted to investigate an origin of the observed dependence. Suppression of thioflavin T (ThT) fluorescence count was correlated with the dissociation constant K_d of monomer–dimer equilibrium of each N‐Methylated Aβ_30–40 peptide. Monomeric N‐Methylated peptides decreased ThT fluorescence count during Aβ_1–40 fibrillation. Secondary structure content was not largely different between Aβ_1–40 fibrils and co‐aggregates. These results suggested that N‐Methylated Aβ_30–40 peptides disrupted the regular β‐sheet structure of Aβ_1–40 fibrils and affected the ThT fluorescence count. The monomer–dimer equilibrium of N‐Methylated peptides was (partly) responsible for the observed dependence of their inhibitory effect on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. Our study provides a hint to design new N‐Methylated inhibitor peptides of fibrillation.     

Synthesis of 1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐[Methyl‐11C]thymine ([11C]FMAU) via a Stille cross‐coupling reaction with [11C]methyl iodide

1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐[methyl‐¹¹C]thymine ([¹¹C]FMAU) [¹¹C]‐ 1 was synthesised via a palladium‐mediated Stille coupling reaction of 1‐(2′‐deoxy‐2′‐fluoro‐β‐D‐arabinofuranosyl)‐5‐(trimethylstannyl)uracil 2 with [¹¹C]methyl iodide in a one‐pot procedure. The reaction conditions were optimized by screening various catalysts and solvents, and by altering concentrations and reaction temperatures. The highest yield was obtained using Pd₂(dba)₃ and P(o‐tolyl)₃ in DMF at 130°C for 5 min. Under these conditions the title compound [¹¹C]‐ 1 was obtained in 28±5% decay‐corrected radiochemical yield calculated from [¹¹C]methyl iodide (number of experiments=7). The radiochemical purity was >99% and the specific radioactivity was 0.1 GBq/μmol at 25 min after end of bombardment. In a typical experiment 700–800 MBq of [¹¹C]FMAU [¹¹C]‐ 1 was obtained starting from 6–7 GBq of [¹¹C]methyl iodide. A mixed ¹¹C/¹³C synthesis to yield [¹¹C]‐ 1 /(¹³C)‐ 1 followed by ¹³C‐NMR analysis was used to confirm the labelling position. The labelling procedure was found to be suitable for automation. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Synthesis and radiolabelling of Re(CO)3‐β‐elemene derivatives as potential therapeutic radiopharmaceuticals

β‐Elemene, (1S, 2S, 4R)‐(?)‐(1‐methy‐1‐vinyl‐2,4‐diisopropenyl cyclohexane) is an anticancer agent from the Traditional Chinese Herb Medicinal. Three novel Re(CO)₃‐β‐elemene derivatives including their radioactive conjugates containing N,N,N tridentate ligands and tricarbonyl rhenium (complex 12, 13, 14) were synthesized. Their structures were characterized by infrared (IR), ¹H‐NMR and HRMS. Good radioactive yield (above 90%) and radioactive chemical purity with Re‐188 (above 95%) were obtained for all of the three derivatives (complex 15, 16, 17). The antiproliferative activity of non‐radioactive β‐elemene‐Re(CO)₃ derivatives on Lewis lung cancer cells and HeLa cell lines were evaluated by WST‐1 methods. The result shows substantial decrease in IC₅₀ values compared with the parent compound β‐elemene. The synthesis and radiosynthesis of β‐elemene tricarbonyl rhenium conjugates provide the possibility to find a new kind of potential radiopharmaceuticals on β‐elemene. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and Anticonvulsant Properties of New N‐Mannich Bases Derived from 3,3‐Diphenyl‐ and 3‐Ethyl‐3‐methyl‐pyrrolidine‐2,5‐diones. Part III

Twenty‐four new N‐[(4‐phenylpiperazin‐1‐yl)‐methyl] derivatives of 3,3‐diphenyl‐ ( 7 – 18 ) and 3‐ethyl‐3‐methyl‐pyrrolidine‐2,5‐dione ( 19 – 30 ) were synthesized and evaluated for their anticonvulsant activity in the maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The acute neurological toxicity was determined using the rotorod screen. Eleven compounds were active and revealed protection only in electrically induced seizures (MES). In the whole series the most effective compound was N‐[{4‐(3‐trifluoromethylphenyl)‐piperazin‐1‐yl}‐methyl]‐3,3‐diphenyl‐pyrrolidine‐2,5‐dione ( 14 ) with an ED₅₀ value of 30.3 mg/kg (p.o. rats) in the MES test. To explain the possible mechanism of action, for chosen active derivatives 7 , 8 , 9 , 11 , 14 , 23 , and 26 , their influence on Na_V1.2 sodium channel currents was evaluated in vitro. The crystallographic structures for several molecules ( 8 , 10 , and 11 ) were solved.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Synthesis and characterization of tritium labeled N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide

N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide is a potent C‐C chemokine receptor 1 (CCR1) antagonist. The compound, possessing benzamide functionality, successfully underwent tritium/hydrogen (T/H) exchange with an organoiridium catalyst (Crabtree's catalyst). The labeling pattern in the product was studied with liquid chromatography–mass spectrometry, time‐of‐flight mass spectrometry, and ³H‐NMR. Overall, multiple labeled species were identified. In addition to the anticipated incorporation of tritium in the benzamide moiety, tritium labeling was observed in the valine portion of the molecule including substitution at its chiral carbon. Using authentic standards, liquid chromatography analysis of the labeled compound showed complete retention of stereochemical configuration.     

Radiosynthesis of 7‐chloro‐N,N‐dimethyl‐5‐[11C]methyl‐4‐oxo‐3‐phenyl‐3,5‐dihydro‐4H‐pyridazino[4,5‐b]indole‐1‐acetamide, [11C]SSR180575, a novel radioligand for imaging the TSPO (peripheral benzodiazepine receptor) with PET

SSR180575 (7‐chloro‐N,N,5‐trimethyl‐4‐oxo‐3‐phenyl‐3,5‐dihydro‐4H‐pyridazino[4,5‐b]indole‐1‐acetamide) is the lead compound of an original pyridazinoindole series of potent and highly selective TSPO (peripheral benzodiazepine receptor) ligands. Isotopic labeling of SSR180575 with the short‐lived positron‐emitter carbon‐11 (T_1/2: 20.38 min) at its 5‐methylpyridazino[4,5‐b]indole moiety as well as at its N,N‐dimethylacetamide function by methylation of the corresponding nor‐analogues was investigated. Best results in terms of radiochemical yields and purities were obtained for the preparation of [indole‐N‐methyl‐¹¹C]SSR180575, where routine production batches of 4.5–5.0 GBq of radiochemically pure (>99%) i.v. injectable solutions (specific radioactivities: 50–90 GBq/ µ mol) could be prepared within a total synthesis time of 25 min (HPLC purification included) starting from a 55 GBq [¹¹C]CO₂ cyclotron production batch (non‐decay‐corrected overall radiochemical yields: 8–9%). The process comprises (1) trapping at ?10°C of [¹¹C]methyl triflate in DMF (300 µ l) containing 0.2–0.3 mg of the indole precursor for labeling and 4 mg of K₂CO₃ (excess); (2) heating at 120°C for 3 min; (3) dilution of the residue with 0.5 ml of the HPLC mobile phase and (4) purification using semi‐preparative reversed‐phase HPLC (Zorbax^® SB‐C‐18). In vivo pharmacological properties of [indole‐N‐methyl‐¹¹C]SSR180575 as a candidate for imaging neuroinflammation with positron emission tomography are currently evaluated. Copyright © 2010 John Wiley & Sons, Ltd.