Synthesis and stability of strongly acidic benzamide derivatives.

Reactivity studies of strong organic acids based on the replacement of one or both of the oxygens in benzoic acids with the trifluoromethanesulfonamide group are reported. Novel derivatives of these types of acids were synthesized in good yields. The generated N-triflylbenzamides were further functionalized through cross-coupling and nucleophilic aromatic substitution reactions. All compounds were stable in dilute aqueous solutions. Studies of stability under acidic and basic conditions are also reported.

Sequential direct SNAr reactions of pentafluorobenzenes with azole or indole derivatives.

Sequential regioselective N-arylations through high-yielding catalyst-free direct SNAr reactions of pentafluorobenzene derivatives with azole or indole derivatives are described. The N-arylated derivatives were further functionalized through a microwave-assisted cross-coupling reaction via C-H bond activation or Suzuki conditions. The order of the reactions could be reversed, proving full orthogonality between the reactions which led to well-defined fully substituted benzene derivatives.

Synthesis and structure-activity relationships of N-benzyl phenethylamines as 5-HT2A/2C agonists.

N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor.

The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1.

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH(2)]-Ser(Bz)-X(aa)-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived ß-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K(i)-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [(14)C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.

Affinity and translocation relationships via hPEPT1 of H-X aa-Ser-OH dipeptides: evaluation of H-Phe-Ser-OH as a pro-moiety for ibuprofen and benzoic acid prodrugs.

The intestinal di/tri-peptide transporter 1 (hPEPT1) has been suggested as a drug delivery target for peptide-based prodrugs. The aim of the study was to synthesize a series of 11 serine-containing dipeptides (H-X(aa)-Ser-OH) and to investigate the relationship between binding to and transport via hPEPT1. An additional aim was to design a dipeptide which could serve as a pro-moiety for prodrugs targeted to hPEPT1. X(aa) was chosen from the 20 proteogenic amino acids. The dipeptides were synthesized using solid phase peptide synthesis. The K(i)-values of H-X(aa)-Ser-OH dipeptides for hPEPT1 in MDCK/hPEPT1 cells ranged from 0.14 mM (logIC(50)=-0.85 ± 0.06) for H-Tyr-Ser-OH to 0.89 mM (logIC(50)=-0.09 ± 0.02) for H-Gly-Ser-OH, as measured in a competition assay with [(14)C]Gly-Sar. The dipeptides were translocated via hPEPT1 with K(m)-values in the range of 0.20 (logIC(50)=-0.69 ± 0.04) for H-Met-Ser-OH to 1.04 (logIC(50)=0.02 ± 0.04) mM for H-Gly-Ser-OH. The relationship between ligand and transportate properties indicated that the initial binding of the ligand to hPEPT1 is the major determinant for translocation of the investigated dipeptides. H-Phe-Ser-OH was selected as a pro-moiety, and two prodrugs were synthesized, i.e. H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH. Both H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH had high affinity for hPEPT1 with K(i)-values of 0.07 mM (logIC(50)=-0.92 ± 0.12) and 0.12 mM (logIC(50)=-1.17 ± 0.40), respectively. However, none of the prodrugs were translocated via hPEPT1. This indicated that the coupling of the drug compounds to the peptide backbone did not decrease transporter binding, but abolished translocation, and that high affinity of prodrugs does not necessarily translate into favourable permeation properties.

Radiosynthesis and in vivo evaluation of a series of substituted 11C-phenethylamines as 5-HT (2A) agonist PET tracers.

PURPOSE: Positron emission tomography (PET) imaging of serotonin 2A (5-HT(2A)) receptors with agonist tracers holds promise for the selective labelling of 5-HT(2A) receptors in their high-affinity state. We have previously validated [(11)C]Cimbi-5 and found that it is a 5-HT(2A) receptor agonist PET tracer. In an attempt to further optimize the target-to-background binding ratio, we modified the chemical structure of the phenethylamine backbone and carbon-11 labelling site of [(11)C]Cimbi-5 in different ways. Here, we present the in vivo validation of nine novel 5-HT(2A) receptor agonist PET tracers in the pig brain. METHODS: Each radiotracer was injected intravenously into anaesthetized Danish Landrace pigs, and the pigs were subsequently scanned for 90 min in a high-resolution research tomography scanner. To evaluate 5-HT(2A) receptor binding, cortical nondisplaceable binding potentials (BP(ND)) were calculated using the simplified reference tissue model with the cerebellum as a reference region. RESULTS: After intravenous injection, all compounds entered the brain and distributed preferentially into the cortical areas, in accordance with the known 5-HT(2A) receptor distribution. The largest target-to-background binding ratio was found for [(11)C]Cimbi-36 which also had a high brain uptake compared to its analogues. The cortical binding of [(11)C]Cimbi-36 was decreased by pretreatment with ketanserin, supporting 5-HT(2A) receptor selectivity in vivo. [(11)C]Cimbi-82 and [(11)C]Cimbi-21 showed lower cortical BP(ND), while [(11)C]Cimbi-27, [(11)C]Cimbi-29, [(11)C]Cimbi-31 and [(11)C]Cimbi-88 gave rise to cortical BP(ND) similar to that of [(11)C]Cimbi-5. CONCLUSION: [(11)C]Cimbi-36 is currently the most promising candidate for investigation of 5-HT(2A) receptor agonist binding in the living human brain with PET.

Radiosynthesis and evaluation of 11C-CIMBI-5 as a 5-HT2A receptor agonist radioligand for PET.

UNLABELLED: PET brain imaging of the serotonin 2A (5-hydroxytryptamine 2A, or 5-HT(2A)) receptor has been widely used in clinical studies, and currently, several well-validated radiolabeled antagonist tracers are used for in vivo imaging of the cerebral 5-HT(2A) receptor. Access to 5-HT(2A) receptor agonist PET tracers would, however, enable imaging of the active, high-affinity state of receptors, which may provide a more meaningful assessment of membrane-bound receptors. In this study, we radiolabel the high-affinity 5-HT(2A) receptor agonist 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2-[(11)C-OCH(3)]methoxybenzyl)ethanamine ((11)C-CIMBI-5) and investigate its potential as a PET tracer. METHODS: The in vitro binding and activation at 5-HT(2A) receptors by CIMBI-5 was measured with binding and phosphoinositide hydrolysis assays. Ex vivo brain distribution of (11)C-CIMBI-5 was investigated in rats, and PET with (11)C-CIMBI-5 was conducted in pigs. RESULTS: In vitro assays showed that CIMBI-5 was a high-affinity agonist at the 5-HT(2A) receptor. After intravenous injections of (11)C-CIMBI-5, ex vivo rat studies showed a specific binding ratio of 0.77 ± 0.07 in the frontal cortex, which was reduced to cerebellar levels after ketanserin treatment, thus indicating that (11)C-CIMBI-5 binds selectively to the 5-HT(2A) receptor in the rat brain. The PET studies showed that the binding pattern of (11)C-CIMBI-5 in the pig brain was in accordance with the expected 5-HT(2A) receptor distribution. (11)C-CIMBI-5 gave rise to a cortical binding potential of 0.46 ± 0.12, and the target-to-background ratio was similar to that of the widely used 5-HT(2A) receptor antagonist PET tracer (18)F-altanserin. Ketanserin treatment reduced the cortical binding potentials to cerebellar levels, indicating that in vivo (11)C-CIMBI-5 binds selectively to the 5-HT(2A) receptor in the pig brain. CONCLUSION: (11)C-CIMBI-5 showed a cortex-to-cerebellum binding ratio equal to the widely used 5-HT(2A) antagonist PET tracer (18)F-altanserin, indicating that (11)C-CIMBI-5 has a sufficient target-to-background ratio for future clinical use and is displaceable by ketanserin in both rats and pigs. Thus, (11)C-CIMBI-5 is a promising tool for investigation of 5-HT(2A) agonist binding in the living human brain.

Synthesis and application of a new fluorous-tagged ammonia equivalent.

A novel fluorous-tagged ammonia equivalent has been developed. It is based on a nitrogen-oxygen bond, which can be cleaved in a traceless manner by a molybdenum complex or samarium diiodide. The application in the synthesis of ureas, amides, sulfonamides, and carbamates is described. The scope of the fluorous N-O linker is exemplified by the synthesis of itopride, a drug used for the treatment of functional dyspepsia. Itopride was synthesized with the aid of fluorous purification methods and the product was isolated in good overall yield, with high purity.

Radiosynthesis and ex vivo evaluation of (R)-(-)-2-chloro-N-[1-11C-propyl]n-propylnorapomorphine.

INTRODUCTION: Several dopamine D(2) agonist radioligands have been used with positron emission tomography (PET), including [(11)C-]-(-)-MNPA, [(11)C-]-(-)-NPA and [(11)C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET brain images with limited contrast or have affinity for both D(2) and D(3) receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(-)-NPA, a novel PET-tracer candidate with high in vitro D(2)/D(3) selectivity. METHODS: 2-Cl-[(11)C]-(-)-NPA and [(11)C]-(-)-NPA were synthesized by a two step N-acylation-reduction process using [(11)C]-propionyl chloride. Awake rats were injected with either tracer, via the tail vein. The rats were decapitated at various times, the brains were removed and quickly dissected, and plasma metabolites were measured. Radioligand specificity, and P-glycoprotein involvement in brain uptake, was also assessed. RESULTS: 2-Cl-[(11)C]-(-)-NPA and [(11)C]-(-)-NPA were produced in high specific activity and purity. 2-Cl-[(11)C]-(-)-NPA accumulated slower in the striatum than [(11)C]-(-)-NPA, reaching maximum concentrations after 30 min. The maximal striatal uptake of 2-Cl-[(11)C]-(-)-NPA (standard uptake value 0.72+/-0.24) was approximately half that of [(11)C]-(-)-NPA (standard uptake value 1.37+/-0.18). Nonspecific uptake was similar for the two compounds. 2-Cl-[(11)C]-(-)-NPA was metabolized quickly, leaving only 17% of the parent compound in the plasma after 30 min. The specific binding of 2-Cl-[(11)C]-(-)-NPA was completely blocked and inhibition of P-glycoprotein did not alter the brain uptake. CONCLUSION: Ex vivo experiments showed, despite a favorable D(2)/D(3) selectivity, that 2-Cl-[(11)C]-(-)-NPA is inferior to [(11)C]-(-)-NPA as a PET tracer in rat, because of slower brain uptake and lower specific to nonspecific binding ratio.

Effect of synthetic and natural phospholipids on N-acylphosphatidylethanolamine-hydrolyzing phospholipase D activity.

N-Acylethanolamines (NAEs) constitute a family of endogenous bioactive lipids that includes arachidonoylethanolamide (anandamide), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). These lipids are formed from their respective N-acylated ethanolamine phospholipid (NAPE) precursor by the action of a phospholipase D enzyme (NAPE-PLD). Anandamide, OEA, and PEA are all bioactive lipids that may influence, amongst others: neuroinflammation, food intake, and oocyte implantation. Here we have synthesized a number of NAPE analogues with variation in the phosphoester structure. The NAPE analogues as well as selected phospholipids and beta-lactamase substrates were tested as potential modifiers of cloned human NAPE-PLD in an enzyme assay involving a (14)C-labeled diether-NAPE substrate. One hit was identified, namely 1,2-dihexanoyl-glycero-N-(3-(tetradecanoylamino)propyl)phosphoramidate (AHP-71B) which showed inhibitory activity and may serve as template for further structure-activity developments. Furthermore, it was found that NAPE-PLD was activated by phosphatidylethanolamine and inhibited by the beta-lactamase substrate nitrocefin.

Design, synthesis, and structure-activity analysis of isoform-selective retinoic acid receptor beta ligands.

We recently discovered the isoform selective RAR beta 2 ligand 4'-octyl-4-biphenylcarboxylic acid (3, AC-55649). Although 3 is highly potent at RAR beta 2 and displays excellent selectivity, solubility issues make it unsuitable for drug development. Herein we describe the exploration of the SAR in a biphenyl and a phenylthiazole series of analogues of 3. This ultimately led to the design of 28, a novel, orally available ligand with excellent isoform selectivity for the RAR beta 2.

Library of biphenyl privileged substructures using a safety-catch linker approach.

A biphenyl privileged structure library containing three attachment points were synthesized using a catechol-based safety-catch linker strategy. The method requires the attachment of a bromo-acid to the linker, followed by a Pd-catalyzed Suzuki cross-coupling reaction. Further derivatization, activation of the linker with strong acid and aminolysis afforded the respective products in high purity and good overall yield. To show the versatility of the synthesis, a 199-member library was generated. The library samples both conformational and chemical diversity about a well-known privileged substructure.

Involvement of Y(5) receptors in neuropeptide Y agonist-induced analgesic-like effect in the rat hot plate test.

Neuropeptide Y (NPY) induces analgesic-like effects after central administration across diverse pain models in rodents. In spinal pain models, previous studies indicate a prominent role for Y(1) receptors at mediating this effect of NPY. In supraspinal pain models like the hot plate test, the NPY receptors involved have not been thoroughly explored. By intracerebroventricular (i.c.v.) administration of selective NPY receptor ligands, the possible involvement of Y(5) receptors in analgesic-like mechanisms was investigated using the hot plate test in rats. Both NPY and selective Y(5) agonists induced analgesic-like effects as revealed by prolonged hot plate latencies. Further consistent with a role for Y(5) receptors, pretreatment with a selective Y(5) receptor antagonist blocked the Y(5) agonist-induced analgesic-like effect. The present study indicates involvement of Y(5) receptors probably at the supraspinal level in mediation of NPY agonist-induced analgesic-like effects in the hot plate test.

Design, synthesis, and evaluation of tripeptidic promoieties targeting the intestinal peptide transporter hPEPT1.

The human intestinal proton coupled di/tri-peptide transporter hPEPT1 promotes the oral bioavailability of several drug compounds. The strategy behind the present work is that by linking a suitable di- or tripeptidic promoiety to a drug substance, by a hydrolysable ester bond, it may give rise to a prodrug that targets hPEPT1. 29 tripeptides were designed based on known structural requirements for substrates binding hPEPT1. Serine, homoserine, or threonine was incorporated in the tripeptide as hydroxy group donors in order for them to be linked to carboxylic drug substances. Optimisation of the promoiety included a study of 14 unnatural tripeptides whose diversity was expressed by VolSurf descriptors. A total of 29 tripeptides was synthesised by solid phase peptide synthesis and a standard Fmoc protocol. The affinity of the tripeptides to hPEPT1 was determined by measuring the inhibition of [(14)C]Gly-Sar in mature Caco-2 cell monolayers which resulted in K(i) values ranging from 0.22 to 25 mM or above. Translocation through the intestinal membrane, mediated by hPEPT1, was measured by recording the membrane potential relative to that induced by the known substrate Gly-Sar. The change in membrane potential is caused by influx of protons due to the co-transport of substrates and protons by hPEPT1 and is, as such, an indication of translocation. A K(i) value of 0.30 mM combined with efficient translocation indicated that H-Phe-Ser-Ala-OH is a suitable lead promoiety for targeted hPEPT1 prodrug design.

In vitro evaluation of N-methyl amide tripeptidomimetics as substrates for the human intestinal di-/tri-peptide transporter hPEPT1.

Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degradation in the GI-tract by various peptidases. The aim of the present study was to evaluate the general application of N-methyl amide bioisosteres as peptide bond replacements in tripeptides in order to decrease degradation by peptidases and yet retain affinity for and transport via hPEPT1. Seven structurally diverse N-methyl amide tripeptidomimetics were selected based on a principal component analysis of structural properties of 6859 N-methyl amide tripeptidomimetics. In vitro extracellular degradation of the selected tripeptidomimetics as well as affinity for and transepithelial transport via hPEPT1 were investigated in Caco-2 cells. Decreased apparent degradation was observed for all tripeptidomimetics compared to the corresponding natural tripeptides. However, affinity for and transepithelial transport via hPEPT1 were only seen for Gly-Sar-Sar, AsnPsi[CONCH(3)]PhePsi[CONCH(3)]Trp, and Gly-Sar-Leu. This implies that tripeptidomimetics originating from tripeptides with neutral side chains are more likely to be substrates for hPEPT1 than tripeptidomimetics with charged side chains. The results of the present study indicate that the N-methyl amide peptide bond replacement approach for increasing bioavailability of tripeptidomimetic drug candidates is not generally applicable to all tripeptides. Nevertheless, retained affinity for and transport via hPEPT1 were shown for three of the evaluated N-methyl amide tripeptidomimetics.

Regioselectivity in lithiation of 1-methylpyrazole: experimental, density functional theory and multinuclear NMR study.

Reaction of 1-methylpyrazole with n-BuLi in THF followed by reaction with monodeuteromethanol (CH3OD) under kinetically controlled conditions leads to functionalisation at the methyl group, whereas reaction under thermodynamically controlled conditions leads to functionalisation at the pyrazole 5-position. The observed regioselectivity can be correctly predicted, at least qualitatively, using density functional B3LYP/6-31+G(d,p) calculations only when solvation effects (IEFPCM) are taken into account. The 1H,6Li HOESY and NOESY NMR spectra of the thermodynamic product 5-lithio-1-methylpyrazole (5-Li) in [D8]THF are consistent with an oligomeric structure.

Synthesis of substituted 2-cyanoarylboronic esters.

The synthesis of substituted 2-cyanoarylboronic esters is described via lithiation/in situ trapping of the corresponding methoxy-, trifluoromethyl-, fluoro-, chloro-, and bromobenzonitriles. The crude arylboronic esters were obtained in high yields and purities and with good regioselectivities.

Solid-phase synthesis with resin-bound triarylbismuthanes: traceless and multidirectional cleavage of unsymmetrical biphenyls.

A multistep solid-phase organic synthesis with resin-bound bismuth linker is described. The flexibilities inherent in this system through novel chemoselective cross-coupling reactions, in conjunction with multidirectional and/or traceless cleavage methodologies, are exploited.

Synthesis and binding studies of 2-arylapomorphines.

From codeine, four different 2-aryl substituted apomorphines were synthesised in 6 steps each. Oxidation of codeine with IBX followed by acid catalysed rearrangement gave morphothebaine, which was selectively triflylated at the 2-position and subsequently O-acetylated at the 11-position. The resulting triflate was coupled in a Suzuki-Miyaura type reaction with a series of 4-substituted arylboronic esters which, after deprotection, gave the desired 2-aryl apomorphines. The analogues were tested for affinity towards a range of dopaminergic, serotonergic and adrenergic receptors. 2-(4-Hydroxyphenyl)-apomorphine exhibited high affinity for the dopamine D2 receptor. A putative ligand-receptor interaction was put forward.

Synthesis of tertiary benzamides via Pd-catalyzed coupling of arylboronic esters and carbamoyl chlorides.

Ortho-substituted arylboronic esters are efficiently coupled with carbamoyl chlorides under Pd-catalysis to give tertiary benzamides.