Synthesis of 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester as a novel potential PET gene reporter probe for HBV and HSV-tk in cancers

Acyclic nucleoside 2-amino-6-(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (ABE, 1) is a new hepatitis B virus (HBV) specific antiviral reagent and shows high anti-HBV activity. Carbon-11 labeled ABE may serve as a novel reporter probe for positron emission tomography (PET) to image HBV and herpes simplex virus thymidine kinase (HSV-tk) in cancers. The radiolabeling precursors 2-amino-6-(4-hydroxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (10) and 2-N-Boc protected analogue 2-N-bis(Boc)amino-6-(4-hydroxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (12), and the reference standard ABE were synthesized from bis(trifluoroethyl) (2-iodoethoxy)methylphosphonate (5), guanine (6), and 2-amino-6-chloropurine (8). The target radiotracer 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester ([11C]ABE, [11C]1) was prepared by O-[11C]methylation of the unprotected HO-precursor 10, or 2-N-Boc protected HO-precursor 12 with [11C]methyl triflate followed by a quick deprotection reaction, and isolated by solid-phase extraction (SPE) purification in 40-55% radiochemical yields.     

Kinetics of binding of multisubstrate analogue inhibitor (2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine) with trimeric purine nucleoside phosphorylase

Complex formation of multisubstrate analogue inhibitor--2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) with trimeric purine nucleoside phosphorylase from Cellulomonas sp. was investigated using a stopped-flow spectrofluorimetric approach. Results obtained indicate that, in contrast to binding of guanine, i.e., the transition-state conformation trapping ligand, for which binding at each active site is followed by the enzyme conformational change, association of the ground-state analogue PME-6-thio-Gua is a one-step process.     

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.     

Synthesis, cytostatic and anti-HCV activity of 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides (55 examples of both ribo- and 2'-deoxyribonucleosides), aimed at identifying novel homologues of natural nucleosides, was developed. The key transformation involved nucleophilic substitutions of Tol-protected 6-(mesyloxymethyl)purine nucleosides by primary or secondary amines, alcoholates or thiolates. While the 2'-deoxyribonucleosides were inactive, the ribonucleosides exerted considerable cytostatic effects and some anti-HCV activity with low selectivity.     

A facile synthesis of cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine and its derivative

The synthesis of carbocyclic nucleosides, cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (3) and cis-2-amino-6-cyclopropylamino-9-[4-(1,2-dihydroxyethyl)- cyclopent-2- enyl]purine (4), was achieved from cyclopentadiene (5) in five and six steps, respectively. This route involves a hetero Diels-Alder reaction and a Pd(0)-catalyzed coupling reaction.     

Utilization of [11C]phosgene for radiosynthesis of N-(2-{3-[3,5-bis(trifluoromethyl)]phenyl[11C]ureido}ethyl)glycyrrhetinamide, an inhibitory agent for proteasome and kinase in tumors

N-(2-{3-[3,5-Bis(trifluoromethyl)]phenylureido}ethyl)glycyrrhetinamide (2), an ureido-substituted derivative of glycyrrhetinic acid (1), has been reported to display potent inhibitory activity for proteasome and kinase, which are overexpressed in tumors. In this study, we labeled this unsymmetrical urea 2 using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]2 could become a positron emission tomography ligand for the imaging of proteasome and kinase in tumors. The strategy for the radiosynthesis of [(11)C]2 was to react hydrochloride of 3,5-bis(trifluoromethyl)aniline (4·HCl) with [(11)C]COCl(2) to possibly give isocyanate [(11)C]6, followed by the reaction of [(11)C]6 with N-(2-aminoethyl)glycyrrhetinamide (3).     

Synthesis of 9-[(Phosphonomethoxy)methyl]guanine and 9-[2-Hydroxy-1-(phosphonomethoxy)ethyl]guanine

Abstract

The synthesis of 9-[(phosphonomethoxy)methyl]guanine (3) and 9-[2-hydroxy-1-(phosphonomethoxy)ethyl]guanine (4) is described.     

Kinetics of Binding of Multisubstrate Analogue Inhibitor (2-Amino-9-[2-(Phosphonomethoxy)Ethyl]-6-Sulfanylpurine) with Trimeric Purine Nucleoside Phosphorylase

Complex formation of multisubstrate analogue inhibitor—2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) with trimeric purine nucleoside phosphorylase from Cellulomonas sp. was investigated using a stopped-flow spectrofluorimetric approach. Results obtained indicate that, in contrast to binding of guanine, i.e., the transition-state conformation trapping ligand, for which binding at each active site is followed by the enzyme conformational change, association of the ground-state analogue PME-6-thio-Gua is a one-step process.     

Synthesis and biological evaluation of some alpha-[6-(1'-carbamoylalkylthio)-1 H-pyrazolo[3,4-D]pyrimidin-4-yl]thioalkylcarboxamide acyclonucleosides

The reaction of 1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 11 with compounds 12a-c produces ethyl alpha-[6-(1'-carboethoxyalkylthio)-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylates 13a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 14, 15, and 16 to afford, predominately, the N(1)-acyclic nucleosides (17-19)a-c, which were deprotected to give the desired products (20-22)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The acyclic nucleosides (20-22)a-c were evaluated for their inhibitory effects against the replication of varicella-zoster virus, human cytomegalovirus and M. tuberculosis. No marked biological activity was found.     

Utilization of 1,3-Dioxolanes in the Synthesis of α-branched Alkyl and Aryl 9-[2-(Phosphonomethoxy)Ethyl]Purines and Study of the Influence of α-branched Substitution for Potential Biological Activity

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.     

Rapid C-carboxylation of nitro[(11)C]methane for the synthesis of ethyl nitro[2-(11)C]acetate

The labelling synthesis of ethyl nitro[2-(11)C]acetate, a synthetic intermediate feasible for (11)C-labelled PET tracers, by C-carboxylation of [(11)C]MeNO(2) with 1-ethoxycarbonylbenzotriazole, and its simple application are presented.     

Antiviral potential of a new generation of acyclic nucleoside phosphonates, the 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines

Three acyclic nucleoside phosphonates (ANPs) have been formally approved for clinical use in the treatment of 1) cytomegalovirus retinitis in AIDS patients (cidofovir, by the intravenous route), 2) chronic hepatitis B virus (HBV) infections (adefovir dipivoxil, by the oral route), and 3) human immunodeficiency virus (HIV) infections (tenofovir disoproxil fumarate, by the oral route). The activity spectrum of cidofovir {(S)- 1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC)]}, like that of (S)-HPMPA [(S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine) and (S)-HPMPDAP [(S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2, 6-diaminopurine), encompasses a broad spectrum of DNA viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. Adefovir {9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)} and tenofovir [(R)-9-[2-(phosphonomethoxy) propyl]adenine [(R)-PMPA)]} are particularly active against retroviruses (ie., HIV) and hepadnaviruses (ie., HBV); additionally, PMEA also shows activity against herpes- and poxviruses. We have recently identified a new class of ANPs, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines, named, in analogy with their alkylpurine counterparts, HPMPO-DAPy, PMEO-DAPy, and (R)-PMPO-DAPy. These compounds exhibit an antiviral activity spectrum and potency that is similar to that of (S)-HPMPDAP, PMEA, and (R)-PMPA, respectively. Thus, PMEO-DAPy and (R)-PMPO-DAPy, akin to PMEA and (R)-PMPA, proved particularly active against HIV- 1, HIV-2, and the murine retrovirus Moloney sarcoma virus (MSV). PMEO-DAPy and (R)-PMPO-DAPy also showed potent activity against both wild-type and lamivudine-resistant strains of HBV. HPMPO-DAPy was found to inhibit different poxviruses (ie., vaccinia, cowpox, and orf) at a similar potency as cidofovir. HPMPO-DAPy also proved active against adenoviruses. In vivo, HPMPO-DAPy proved equipotent to cidofovir in suppressing vaccinia virus infection (tail lesion formation) in immunocompetent mice and promoting healing of disseminated vaccinia lesions in athymic-nude mice. The 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines offer substantial potential for the treatment of a broad range of retro-, hepadna-, herpes-, adeno-, and poxvirus infections.     

Synthesis and biological evaluation of 2-methoxy- and 2-methylthio-6-[2'-alkylamino)ethyl]-4(3H)-pyrimidinones with anti-rubella virus activity.

The synthesis of a new family of antiviral compounds, 2-methoxy-, and 2-methylthio-6-[(2'-alkylamino)ethyl]-4(3H)-pyrimidinones, has been accomplished. The activity of these agents against positive strand (rubella virus and sindbis virus) and negative strand (vesicular stomatitis virus) RNA viruses is reported. Some of these compounds are efficient and selective inhibitors of rubella virus.     

Tissue distribution, autoradiography, and metabolism of 4-(2'-methoxyphenyl)-1-[2' -[N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethyl]piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist for positron emission tomography: An In vivo study in rats

The in vivo behavior of 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-[(18)F]fluorobenzamido ]ethyl]-piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist, was studied in awake, freely moving rats. Biodistribution studies showed that the carbon-fluorine bond was stable in vivo, that this compound was able to cross the blood-brain barrier, and that a general diffusion equilibrium could account for the availability of the tracer. The great quantity of highly polar metabolites found in plasma did not contribute to the small amounts of metabolites found in hippocampus, frontal cortex, and cerebellum. Exvivo p-[(18)F]MPPF and in vitro 8-hydroxy-2-(di-n-[(3)H]propylamino)tetralin autoradiography were compared both qualitatively and quantitatively. Qualitative evaluation proved that the same brain regions were labeled and that the p-[(18)F]MPPF labeling is (a) in total agreement with the known distribution of 5-HT(1A) receptors in rats and (b) characterized by very low nonspecific binding. Quantitative comparison demonstrated that the in vivo labeling pattern obtained with p-[(18)F]MPPF cannot be explained by differences in regional blood flow, capillary density, or permeability. The 5-HT(1A) specificity of p-[(18)F]MPPF and binding reversibility were confirmed in vivo with displacement experiments. Thus, this compound can be used to evaluate parameters characterizing 5-HT(1A) binding sites in the brain.     

Synthesis and anticonvulsant activity of new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-2,5-diones

Twenty-two new 1-[2-oxo-2-(4-phenylpiperazin-1-yl)ethyl]pyrrolidine-2,5-diones were synthesized and tested for anticonvulsant activity. Initial anticonvulsant screening was performed using standard maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens in mice. Several compounds were tested additionally in the 6-Hz psychomotor seizure model. The neurotoxicity was determined applying the rotarod test. Excluding one compound, all other molecules were found to be effective in at least one seizure model. The most active were 1-(2-oxo-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)pyrrolidine-2,5-dione (14), 1-{2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}-3-methylpyrrolidine-2,5-dione (17), 1-{2-[4-(4-chlorophenyl)piperazin-1-yl]-2-oxoethyl}-3,3-dimethylpyrrolidine-2,5-dione (23) and 3,3-dimethyl-1-(2-oxo-2-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}ethyl)pyrrolidine-2,5-dione (26). These compounds showed high activity in the 6-Hz psychomotor seizure test as well as were active in the maximal electroshock and subcutaneous pentylenetetrazole (14 and 23) screens. Initial SAR studies for anticonvulsant activity have been discussed.     

Further exploration of 1-[2-[Bis-(4-fluorophenyl)methoxy]ethyl]piperazine (GBR 12909): role of N-aromatic,N-heteroaromatic,and 3-oxygenated N-phenylpropyl substituents on affinity for the dopamine and serotonin transporter

A series of N-aromatic, N-heteroaromatic, and oxygenated N-phenylpropyl derivatives of 1-(2-benzhydryloxyethyl)-piperazine and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]piperazine, analogues of GBR 12909 (1a) and 12935 (1b), was synthesized and examined for their dopamine (DAT) and serotonin (SERT) transporter binding properties. One of these compounds, racemic 3-[4-(2-benzhydryloxyethyl)piperazin-1-yl]-1-(3-fluorophenyl)-propan-1-ol (33), had DAT affinity as good as, or better than, GBR 12909 and 12935, and was more selective for DAT over SERT than the GBR compounds. Both trans- (43) and cis- (47) (+/-)-2-(4-[2-[bis-(4-fluorophenyl)-methoxy]ethyl]piperazin-1-ylmethyl)-6-methoxy-1,2,3,4-tetrahydronaphthalen-1-ol had relatively good SERT selectivity and, as well, showed high affinity for SERT.     

Design, synthesis and in vitro antitumor activity of new trans 2-[2-(heteroaryl)vinyl]-1,3-dimethylimidazolium iodides

The design, the synthesis, and the in vitro antitumor activities of trans 2-[2-(heteroaryl)vinyl]-1,3-dimethylimidazolium iodides versus MCF7 (human mammary carcinoma) and LNCap (prostate carcinoma) cell lines are reported. The design indicates trans 2-[2-[5-(2-chlorophenyl)furan-2-yl]vinyl]-1, 3-dimethylimidazolium iodide 5 and trans 2-[2-[5-(4-bromophenyl)furan-2-yl]vinyl]-1, 3-dimethylimidazolium iodide 6 as highly active compounds in the series. The synthesis of the above new derivatives and in vitro antitumor tests, confirm their significant antiproliferative and cytotoxic activities.     

(S,E)-N-[1-(3-heteroarylphenyl)ethyl]-3-(2-fluorophenyl)acrylamides: synthesis and KCNQ2 potassium channel opener activity

Replacement of the morpholinyl moiety in (S,E)-N-[1-(3-morpholinophenyl)ethyl]-3-phenylacrylamide (1) with heteroaryl groups led to the identification of (S,E)-N-1-[3-(6-fluoropyridin-3-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (5) as a potent KCNQ2 potassium channel opener. Among this series of heteroaryl substituted acrylamides, (S,E)-N-1-[3-(1H-pyrazol-1-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (9) exhibits balanced potency and efficacy. The syntheses and the KCNQ2 opener activity of this series of acrylamides are described.     

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

Summary S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.     

Intramolecular stacking interactions in ternary copper(II) complexes formed by a heteroaromatic amine and 9-[2-(2-phosphonoethoxy)ethyl]adenine, a relative of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine

The stability constants of the mixed-ligand complexes formed between Cu(Arm)2+, where Arm=2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), and the dianions of 9-[2-(2-phosphonoethoxy)ethyl]adenine (PEEA2-) and (2-phosphonoethoxy)ethane (PEE2-), also known as [2-(2-ethoxy)ethyl]phosphonate, were determined by potentiometric pH titrations in aqueous solution (25 degrees C; I=0.1 M, NaNO3). The ternary Cu(Arm)(PEEA) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO3(2-) represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of interaction within the complexes. The increased stability is attributed to intramolecular stack formation in the Cu(Arm)(PEEA) complexes and also, to a smaller extent, to the formation of 6-membered chelates involving the ether oxygen atom present in the -CH2-O-CH2-CH2-PO3(2-) residue of PEEA2-. This latter interaction is separately quantified by studying the ternary Cu(Arm)(PEE) complexes which can form the 6-membered chelates but where no intramolecular ligand-ligand stacking is possible. Application of these results allows a quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PEEA) species; e.g., of the Cu(Bpy)(PEEA) system about 11% exist with the metal ion solely coordinated to the phosphonate group, 4% as a 6-membered chelate involving the ether oxygen atom of the -CH2-O-CH2CH2-PO3(2-) residue, and 85% with an intramolecular stack between the adenine moiety of PEEA2- and the aromatic rings of Bpy. In addition, the Cu(Arm)(PEEA) complexes may be protonated, leading to Cu(Arm)(H;PEEA)+ species for which it is concluded that the proton is located at the phosphonate group and that the complexes are mainly formed (50 and 70%) by a stacking adduct between Cu(Arm)2+ and the adenine residue of H(PEEA)-. Finally, the stacking properties of adenosine 5'-monophosphate (AMP2-), of the dianion of 9-[2-(phophonomethoxy)ethyl]adenine (PMEA2-) and of several of its analogues (=PA2-) are compared in their ternary Cu(Arm)(AMP) and Cu(Arm)(PA) systems. Conclusions regarding the antiviral properties of several acyclic nucleoside phosphonates are shortly discussed.