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.

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.

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.

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.

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.

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.

Pyrimidine and nucleoside gamma-esters of L-Glu-Sar: synthesis, stability and interaction with hPEPT1.

The aim of the present study was to improve the synthetic pathway of bioreversible dipeptide derivatives as well as evaluate the potential of using l-Glu-Sar as a pro-moiety for delivering three newly synthesised nucleoside and pyrimidine l-Glu-Sar derivatives. l-Glu(trans-2-thymine-1-yl-tetrahydrofuran-3-yl ester)-Sar (I), l-Glu(thymine-1-yl-methyl ester)-Sar (II) and l-Glu(acyclothymidine)-Sar (III) were synthesised and in vitro stability was studied in various aqueous and biological media. Affinity to and translocation via hPEPT1 was investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay, using [14C] labelled Gly-Sar (glycylsarcosine). Translocation was measured as pHi-changes induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All dipeptide derivatives released the model drugs quantitatively by specific base-catalysed hydrolysis at pH>6.0. II was labile in aqueous buffer solution, whereas I and III showed appropriate stability for oral administration. In 10% porcine intestinal homogenate, the half-lives of the dipeptide derivatives indicated limited enzyme catalyzed degradation. All compounds showed good affinity to hPEPT1, but the Compounds I and III showed not to be translocated by hPEPT1. The translocation of the l-Glu-Sar derivative of acyclovir, l-Glu(acyclovir)-Sar was also investigated and showed not to take place. Consequently, l-Glu-Sar seems to be a poor pro-moiety for hPEPT1-mediated transport.

Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs.

An enzymatically activated liposome-based drug-delivery concept involving masked antitumor ether lipids (AELs) has been investigated. This concept takes advantage of the cytotoxic properties of AEL drugs as well as the membrane permeability enhancing properties of these molecules, which can lead to enhanced drug diffusion into cells. Three prodrugs of AELs (proAELs) have been synthesized and four liposome systems, consisting of these proAELs, were investigated for enzymatic degradation by secretory phospholipase A(2) (sPLA(2)), resulting in the release of AELs. The three synthesized proAELs were (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (1-O-DPPC), (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol)(350) (1-O-DPPE-PEG(350)), and 1-O-DPPE-PEG(2000) of which 1-O-DPPC was the main liposome component. All three phospholipids were synthesized from the versatile starting material (R)-O-benzyl glycidol. A phosphorylation method, employing methyl dichlorophosphate, was developed and applied in the synthesis of two analogues of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine poly(ethylene glycol). Differential scanning calorimetry has been used to investigate the phase behavior of the lipid bilayers. A release study, employing calcein encapsulated in non-hydrolyzable 1,2-bis-O-octadecyl-sn-glycero-3-phosphocholine (D-O-SPC) liposomes, showed that proAELs, activated by sPLA(2), perturb membranes because of the detergent-like properties of the released hydrolysis products. A hemolysis investigation was conducted on human red blood cells, and the results demonstrate that proAEL liposomes display a very low hemotoxicity, which has been a major obstacle for using AELs in cancer therapy. The results suggest a possible way of combining a drug-delivery and prodrug concept in a single liposome system. Our investigation of the permeability-enhancing properties of the AEL molecules imply that by encapsulating conventional chemotherapeutic drugs, such as doxorubicin, in liposomes consisting of proAELs, an increased effect of the encapsulated drug might be achievable due to an enhanced transmembrane drug diffusion.

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.

A one-pot access to 6-substituted phenanthridines from fluoroarenes and nitriles via 1,2-arynes.

[reaction: see text] A one-pot, t-BuLi-induced synthesis of 6-substituted phenanthridines from fluoroarenes and nitriles via 1,2-arynes is reported. Aryl- and hetaryl nitriles, cyanamides, and trimethylacetonitrile gave phenanthridine products. The method was extended to provide bisphenanthridine 10 by a one-pot bis-cyclization, using 1,3-dicyanobenzene and PhF in 1:5 ratio. Reaction of 1-fluoronaphthalene and 4-chlorofluorobenzene with benzonitrile afforded the regioisomerically pure products 11 and 12, respectively.

Convergent synthesis of 6-substituted phenanthridines via anionic ring closure.

[reaction: see text] The addition of organometallic derivatives to the cyano group of 2-(2-fluorophenyl)benzonitrile followed by intramolecular nucleophilic substitution produces 6-substituted phenanthridines. Alkyllithiums, aryllithiums, and sterically nondemanding lithium amides reacted at -78 degrees C to produce the 6-substituted phenanthridines in 82-98% yield upon warming to room temperature. The addition of the corresponding Grignard reagents requires an excess of the organometallic reagent and extented reaction times at elevated temperature.

The respective N-hydroxypyrazole analogues of the classical glutamate receptor ligands ibotenic acid and (RS)-2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid.

We have determined the pharmacological activity of N-hydroxypyrazole analogues (3a and 4a) of the classical glutamate receptor ligands ibotenic acid and (RS)-2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA), as well as substituted derivatives of these two compounds. The pharmacological profile of 3a is closer to that of thioibotenic acid rather than ibotenic acid, while 4a is a selective N-methyl-D-aspartic acid (NMDA) receptor agonist. Ring substitution of 3a and 4a leads to NMDA receptor antagonists. Whereas efficacy of 3a derivatives at mglu2 receptor decreases from agonism via partial agonism to antagonism with increasing substituent size, substitution abolishes affinity for mglu1 and mglu4 receptors. Ligand- and receptor-based modelling approaches assist in explaining these pharmacological trends among the metabotropic receptors and suggest a mechanism of partial agonism at mglu2 receptor similar to that proposed for the GluR2 glutamate receptor.

Prodrugs of purine and pyrimidine analogues for the intestinal di/tri-peptide transporter PepT1: affinity for hPepT1 in Caco-2 cells, drug release in aqueous media and in vitro metabolism.

A general drug delivery approach for increasing oral bioavailability of purine and pyrimidine analogues such as acyclovir may be to link these compounds reversibly to stabilized dipeptide pro-moieties with affinity for the human intestinal di/tri-peptide transporter, hPepT1. In the present study, novel L-Glu-Sar and D-Glu-Ala ester prodrugs of acyclovir and 1-(2-hydroxyethyl)-linked thymine were synthesized and their affinities for hPepT1 in Caco-2 cells were determined. Furthermore, the degradation of the prodrugs was investigated in various aqueous and biological media and compared to the corresponding hydrolysis of the prodrug valaciclovir. Affinity studies showed that the L-Glu-Sar prodrugs had high affinity for hPepT1 (K(i) approximately 0.2-0.3 mM), whereas the D-Glu-Ala prodrugs had poor affinity (K(i) approximately 50 mM). The pH-rate profiles of the prodrugs D-Glu[1-(2-hydroxyethyl)thymine]-Ala and L-Glu[acyclovir]-Sar showed specific base catalyzed degradation at pH above 4.5 and 5.5, respectively. This implicates that the degradation rates at pH approximately 7.4 (t(1/2) approximately 3.5 and 5.5 h) are approximately 25 times faster than at upper small intestinal pH approximately 6.0. In 10% porcine intestinal homogenate and 80% human plasma the half-lives of the L-Glu-Sar prodrugs were approximately between 45 and 90 min indicating a limited enzyme catalyzed degradation. In contrast, valaciclovir underwent extensive enzyme catalyzed hydrolysis in 10% porcine intestinal homogenate (t(1/2) approximately 1 min). In conclusion, L-Glu-Sar may potentially function as pro-moiety for purine and pyrimidine analogues, where release of parent compound primarily is controlled by a specific base catalyzed hydrolysis. Acyclovir is quantitatively released at the relevant pH 7.4, whereas the 1-(2-hydroxyethyl)-linked thymine is released instead of the parent compound thymine.

Regioselective Introduction of Electrophiles in the 4-Position of 1-Hydroxypyrazole via Bromine-Lithium Exchange.

Two protocols for introduction of electrophiles at the 4-position of 1-hydroxypyrazoles have been developed. The first is deprotonation of 4-bromo-1-[(tert-butyldiphenylsilyl)oxy]pyrazole (6) with LDA to produce the 5-lithio derivative in which the silyl group migrates spontaneously from oxygen to C-5 affording 4-bromo-5-(tert-butyldiphenylsilyl)-1-lithoxypyrazole (8). Subsequent treatment with t-BuLi causes bromine-lithium exchange to give 5-tert-butyldiphenylsilyl-4-lithio-1-lithoxypyrazole which is trapped with electrophiles affording 4-substituted 5-(tert-butyldiphenylsilyl)-1-hydroxypyrazoles 9a-e in a one-pot sequence. The second is treatment of 4-bromo-1-hydroxypyrazole (5) with excess LDA and trimethylsilyl chloride to produce 3,5-bis(trimethylsilyl)-4-bromo-1-hydroxypyrazole (12), which upon sequential metalation with n-BuLi and reaction with electrophiles affords 4-substituted 3,5-bis(trimethylsilyl)-1-hydroxypyrazoles 13a-e. The tert-butyldiphenylsilyl group of 9a and the trimethylsilyl groups of 12 can be removed selectively by treatment with tetrabutylammonium fluoride in THF in the presence of trifluoroacetic acid.

Selective Synthesis of 2-, 4-, and 5-Cyano Substituted Imidazoles from Imidazole N-Oxides and Trimethylsilyl Cyanide.

1-Substituted 2-, 4-, and 5-cyanoimidazoles were produced by reaction of 3-cyclohexyl or 3-p-tolylimidazole 1-oxide and trimethylsilyl cyanide in the presence of triethylamine. The product composition depended on reaction temperature and solvent polarity. By proper choice of these parameters each isomer could be obtained selectively in reasonable yield.

Directed ortho-Lithiation on Solid Phase.

Resin-bound 1-hydroxyimidazole, obtained by alkylation of the sodium salt of 1-hydroxyimidazole with chloromethyl polystyrene, was lithiated at C-2 with n-butyllithium. Subsequent treatment with carbon, halogen, or sulfur electrophiles followed by detachment from the solid support by heating with trifluoracetic acid at 100 degrees C for 20 h gave 2-substituted 1-hydroxyimidazoles in 52-93% yields.

Synthesis of 2-Substituted 1-Hydroxyimidazoles through Directed Lithiation.

Benzylation of 3-hydroxyimidazole 1-oxide gave 3-(benzyloxy)imidazole 1-oxide, which was deoxygenated with phosphorous trichloride to produce 1-(benzyloxy)imidazole. 1-(Benzyloxy)imidazole was deprotonated selectively at C-2 by n-butyllithium. The anion formed was reacted with electrophiles to give 1-(benzyloxy)imidazoles with carbon, halogen, silicon, or sulfur substituents at the 2-position. Subsequent debenzylation afforded 2-substituted 1-hydroxyimidazoles which in turn could be deoxygenated to give 2-substituted imidazoles.

Synthesis of analogs of L-valacyclovir and determination of their substrate activity for the oligopeptide transporter in Caco-2 cells.

L-Valacyclovir, a prodrug of acyclovir, is a substrate for the peptide transporter (PepT1) in the intestinal mucosa, which accounts for its higher than expected oral bioavailability. The substrate activity of L-valacyclovir for PepT1 is surprising, particularly when one considers that the molecule has the structural features of a nucleoside rather than a peptide. In an attempt to better understand the structure-transport relationships (STR) for the interactions of L-valacyclovir with PepT1, analogs of this molecule with structural changes in the guanine moiety were synthesized and their substrate activity for PepT1 in Caco-2 cell monolayers was determined. The analogs synthesized include those that had the guanine moiety of L-valacyclovir substituted with purine, benzimidazole, and 7-azaindole. All three analogs (purine, benzimidazole, and 7-azaindole) exhibited affinity for PepT1 in binding studies, but only the purine analog (as the L-valine ester) showed PepT1-associated transcellular transport across Caco-2 cell monolayers. The benzimidazole and 7-azaindole analogs (as their L-valine esters) were rapidly metabolized by esterase when applied to the apical surface of Caco-2 cells, which probably explains their low penetration as the intact prodrugs via PepT1.

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.