Ester prodrugs of cyclic 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine: synthesis and antiviral activity

Reaction of 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (1) with dicyclohexylcarbodiimide and N,N,-dicyclohexyl-4-morpholinocarboxamidine in dimethylformamide at elevated temperature afforded the corresponding cyclic phosphonate 2, that is, 1-{[(5S)-2-hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-5-azacytosine. Compound 2 exerts strong in vitro activity against DNA viruses, comparable with activity of parent compound 1. Transformation of 2 to its tetrabutylammonium salt followed by reaction with alkyl or acyloxyalkyl halogenides enabled us to prepare a series of structurally diverse ester prodrugs: alkyl (octadecyl), alkenyl (erucyl), alkoxyalkyl (hexadecyloxyethyl), and acyloxyalkyl (pivaloyloxymethyl) (3-6). The introduction of an alkyl, alkoxyalkyl, or acyloxyalkyl ester group to the molecule resulted in an increase of antiviral activity; the most active compound was found to be the hexadecyloxyethyl ester 5. The relative configuration of the diastereoisomer trans-6 was determined using H,H-NOESY NMR.     

6-[2-(Phosphonomethoxy)alkoxy]pyrimidines with antiviral activity

6-Hydroxypyrimidines substituted at positions 2 and 4 by hydrogen, methyl, amino, cyclopropylamino, dimethylamino, methylsulfanyl, or hydroxyl group afford by the reaction with diisopropyl 2-(chloroethoxy)methylphosphonate in the presence of NaH, Cs(2)CO(3), or DBU a mixture of N(1)- and O(6)-[2-(diisopropylphosphorylmethoxy)ethyl] isomers which were converted to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis. Analogously, 2,4-diamino-6-hydroxypyrimidine gave on reaction with [(R)- and (S)-2-(diisopropylphosphorylmethoxy)propyl] tosylate, followed by deprotection, the enantiomeric 6-[2-(phosphonomethoxy)propoxy]pyrimidines. 2,4-Diamino-6-sulfanylpyrimidine gave, on treatment with diisopropyl 2-(chloroethoxy)methylphosphonate in the presence of NaH and subsequent deprotection, 2,4-diamino-6-[[2-(phosphonomethoxy)ethyl]sulfanyl]pyrimidine. 2-Amino-4-hydroxy-6-[2-(phosphonomethoxy)ethyl]pyrimidine was obtained from the appropriate 2-amino-4-chloropyrimidine derivative by alkaline hydrolysis and ester cleavage. Direct alkylation of 2-amino-4,6-dihydroxypyrimidine afforded a mixture of 2-amino-4,6-bis[2-(phosphonomethoxy)ethyl]- and 2-amino-1,4-bis[2-(phosphonomethoxy)ethyl]pyrimidine. None of the N(1)-[2-(phosphonomethoxy)ethyl] isomers exhibited any antiviral activity against DNA viruses or RNA viruses tested in vitro. On the contrary, the O(6)-isomers, namely the compounds derived from 2,4-diamino-, 2-amino-4-hydroxy-, or 2-amino-4-[2-(phosphonomethoxy)ethoxy]-6-hydroxypyrimidine, inhibited the replication of herpes viruses [herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV), and cytomegalovirus (CMV)] and retroviruses [Moloney sarcoma virus (MSV) and human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)], their activity being most pronounced against the latter. The antiviral activity was lower if the oxygen at the position 6 was replaced by a sulfur atom, as in 2,4-diamino-6-[2-(phosphonomethoxy)ethylsulfanyl]pyrimidine. In analogy to N(9)-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP), solely the (R)-2,4-diamino-6-[2-(phosphonomethoxy)propoxy]pyrimidine exerted antiviral activity, whereas its (S)-enantiomer was essentially inactive.     

Intracellular metabolism of the new antiviral compound 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine

1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine [HPMP-5-azaC], the 5-azacytosine analogue of cidofovir (HPMPC), represents a new acyclic nucleoside phosphonate with pronounced activity against DNA viruses, and a selectivity index superior to that of cidofovir. Here we investigated the intracellular metabolic pathway of [6-³H]-HPMP-5-azaC. By comparing the metabolism in mouse lymphosarcoma S49-wild type (S49-WT) and mutant cells deficient for dCMP deaminase, we identified the mono- and diphosphate metabolites generated from HPMP-5-azaC and its deaminated product HPMP-5-azaU. In human lung carcinoma A549 cells, the relative formation of the deaminated metabolites was only 6%, implying that deamination plays a minor role in the overall metabolism of HPMP-5-azaC. The diphosphorylated metabolite of HPMP-5-azaC accounted for 60% of the total radioactivity, and reached intracellular levels which were 60-fold higher in absolute value than the corresponding diphosphate levels obtained with cidofovir. Consequently to its increased activation, HPMP-5-azaC showed about 45-fold higher incorporation into cellular DNA than cidofovir. Herpes-, pox- or adenovirus infection had no marked influence on the metabolism of HPMP-5-azaC. The HPMP-5-azaC-diphosphate metabolite was shown to have long intracellular stability (half-life: 63 h), suggesting that infrequent administration of HPMP-5-azaC should be possible. HPMP-5-azaC represents a new acyclic nucleoside phosphonate compound with promising anti-DNA virus activity and a favorable metabolic profile that is characterized by low sensitivity to catabolic deamination and a high rate of phosphorylation and DNA incorporation.     

Synthesis and antiviral activity of (S)-9-[4-hydroxy-3-(phosphonomethoxy)butyl]guanine

The synthesis of (S)-9-[4-hydroxy-3-(phosphonomethoxy)butyl]guanine (3), starting from (S)-4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane (4), is described. Alkylation of trityl derivative 7 with (diethylphosphono)methyl triflate provided phosphonate 8, which was readily converted to mesylate 12 in three steps. Nucleophilic substitution of the mesylate group of 12 by 2-amino-6-chloropurine sodium salt led to (S)-2-amino-6-chloro-9-[3-[(diethyl-phosphono)methoxy]-4-(tetrahydro- 2H-pyran-2-yloxy)butyl]purine (13). Sequential treatment of 13 with trimethylsilyl bromide and then with 2 N HCl furnished 3. Preliminary in vitro screening indicated that 3 exhibited a potent activity against human cytomegalovirus (HCMV) but was not active against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The adenine and cytosine derivatives (14 and 15) did not exhibit activity against HSV-1 and -2 and HCMV.     

Structure-antiviral activity relationship in the series of pyrimidine and purine N-[2-(2-phosphonomethoxy)ethyl] nucleotide analogues. 1. Derivatives substituted at the carbon atoms of the base.

A series of dialkyl esters of purine and pyrimidine N-[2-(phosphonomethoxy)ethyl] derivatives substituted at position 2, 6, or 8 of the purine base or position 2, 4, or 5 of the pyrimidine base were prepared by alkylation of the appropriate heterocyclic base with 2-chloroethoxymethylphosphonate diester in the presence of sodium hydride, cesium carbonate, or 1,8-diazabicyclo[5,4, 0]undec-7-ene (DBU) in dimethylformamide. Additional derivatives were obtained by the transformations of the bases in the suitably modified intermediates bearing reactive functions at the base moiety. The diesters were converted to the corresponding monoesters by sodium azide treatment, while the free acids were obtained from the diester by successive treatment with bromotrimethylsilane and hydrolysis. None of the PME derivatives in the pyrimidine series, their 6-aza or 3-deaza analogues, exhibited any activity against DNA viruses or retroviruses tested, except for the 5-bromocytosine derivative. Substitution of the adenine ring in PMEA at position 2 by Cl, F, or OH group decreased the activity against all DNA viruses tested. PMEDAP was highly active against HSV-1, HSV-2, and VZV in the concentration range (EC50) of 0.07-2 microg/mL. Also the 2-amino-6-chloropurine derivative was strongly active (EC50 = 0.1-0. 4 microg/mL) against herpes simplex viruses and (EC50 = 0.006-0.3 microg/mL) against CMV and VZV. PMEG was the most active compound of the whole series against DNA viruses (EC50 approximately 0.01-0.02 microg/mL), though it exhibited significant toxicity against the host cells. The base-modified compounds did not show any appreciable activity against DNA viruses except for 7-deazaPMEA (IC50 approximately 7.5 microg/mL) against HIV-1 and MSV. The neutral (diisopropyl, diisooctyl) diesters of PMEA were active against CMV and VZV, while the corresponding monoesters were inactive. The diisopropyl ester of the 2-chloroadenine analogue of PMEA showed substantially (10-100x) higher activity against CMV and VZV than the parent phosphonate. Also, the diisopropyl and diisooctyl ester of PMEDAP inhibited CMV and VZV, but esterification of the phosphonate residue did not improve the activity against either MSV or HIV.     

Synthesis of certain nucleoside methylenediphosphonate sugars as potential inhibitors of glycosyltransferases

The synthesis of alpha-D-glucopyranosyl 1-(methylenediphosphonate) (11), alpha-D-galactopyranosyl 1-(methylenediphosphonate) (14), and alpha-D-mannopyranosyl 1-(methylenediphosphonate) (17) has been accomplished. [(Di-phenoxyphosphinyl)methyl]phosphonic acid (diphenyl-MDP) (5), synthesized by two different procedures, was fused with beta-D-glucopyranose pentaacetate followed by catalytic hydrogenation to give 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl methylenediphosphonate (glucose-MDP) (10). The anomeric configuration of 10 was assigned on the basis of NMR spectral studies. Condensation of 10 with 2',3'-di-O-acetyladenosine was accomplished by using 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) as coupling agent, and removal of the blocking groups gave adenosine 5'-[(alpha-D-glucopyranosylhydroxyphosphinyl)methyl]phosphonate (20). Uridine 5'-[(alpha-D-galactopyranosylhydroxyphosphinyl)methyl] phosphonate (23) and guanosine 5'-[(alpha-D-mannopyranosylhydroxyphosphinyl)methyl]phosphonate (26) were similarly prepared. Using a specific glycoprotein galactosyltransferase (EC 2.4.1.38) assay, uridine 5'-[(alpha-D-galactopyranosylhydroxyphosphinyl)methyl]phosphonate (23) demonstrated competitive inhibition with an apparent Ki of 97 microM. The adenosine analogue did not inhibit the enzyme. None of the above compounds show any in vitro antitumor or antiviral activity. Such specific inhibitors of glycosyltransferases may serve as specific probes to study various glycosyltransferases that might be involved in the process of metastasis.     

Synthesis and antiviral activity of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine.

A number of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and tested in vitro for anti-herpes and anti-human immunodeficiency virus (HIV) activity. Among these analogues, (R)-2'-methyl-PMEG [(R)-3] and 2',2'-dimethyl-PMEG (7) demonstrated potent anti-HIV activity in the XTT assay with EC50 values of 1.0 and 2.6 microM, respectively. The corresponding (S)-2'-methyl-PMEG [(S)-3] was found to be less potent against HIV. In addition, the (R) and (S) enantiomers of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG, 8) were prepared for comparison of biological activity, and shown to be active and equipotent against herpesviruses, but inactive against HIV.     

5-Substituted-2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines-acyclic nucleoside phosphonate analogues with antiviral activity

2,4-Diamino-6-hydroxypyrimidines substituted in position 5 by an allyl, benzyl, cyanomethyl, ethoxycarbonylmethyl, phenyl, cyclopropyl, or methyl group were prepared either by C5-alkylation or by formation of the pyrimidine ring by cyclization. Their alkylation with 2-[(diisopropoxyphosphoryl)methoxy]ethyl tosylate afforded N1- and O6-regioisomers that were separated and converted to the free phosphonic acids by treatment with bromotrimethylsilane followed by hydrolysis. Reaction of 2,4-diamino-6-[[(diisopropoxyphosphoryl)methoxy]ethoxy]pyrimidine with elemental bromine, N-chloro-, or N-iodosuccinimide gave the corresponding phosphorus-protected 5-bromo-, 5-chloro-, and 5-iodo derivatives, respectively. Their deprotection afforded 2,4-diamino-5-bromo- and -5-chloro-6-[2-(phosphonomethoxy)ethoxy]pyrimidines. 2,4-Diamino-5-methyl-6-[2-(phosphonomethoxy)ethoxy]pyrimidine was synthesized also by cross-coupling of the 5-bromo compound with AlMe(3), followed by deprotection. The compounds showed poor, if any, inhibitory activity against DNA viruses such as herpes simplex virus type 1 and type 2, cytomegalovirus, varicella-zoster virus, and vaccinia virus. In contrast, several 5-substituted 2,4-diaminopyrimidine derivatives markedly inhibited retrovirus replication in cell culture. The 5-methyl derivative was exquisitely inhibitory to human immunodeficiency virus and Moloney murine sarcoma virus-induced cytopathicity in cell culture (EC(50) approximately 0.00018 mumol/mL) but also cytostatic to CEM cell cultures. In contrast, the 5-halogen-substituted derivatives showed pronounced antiretroviral activity (EC(50) = 0.0023-0.0110 mumol/mL), comparable to that of the reference drugs adefovir and tenofovir, but were devoid of measurable toxicity at 0.3 mumol/mL.     

Alpha anomer of 5-aza-2'-deoxycytidine down-regulates hTERT mRNA expression in human leukemia HL-60 cells

DNA methylation inhibitors are being extensively studied as potential anticancer agents. In the present study, we compared the capability of alpha anomer of 5-aza-2'-deoxycytidine (alpha-5-azadCyd) to induce down-regulation of hTERT expression in HL-60 cells with other nucleoside analogs that act as DNA methylation inhibitors: beta-5-azadCyd (decitabine), (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], isobutyl ester of (R,S)-3-(adenin-9-yl)-2-hydroxypropanoic acid [(R,S)-AHPA-ibu] and prospective DNA methylation inhibitors (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine [(S)-HPMPazaC] and 5-fluoro-zebularine (F-PymRf). Exposure to alpha-5-azadCyd induced the down-regulation of hTERT expression in low micromolar concentrations (0.05-50 microM). A more cytotoxic beta anomer caused a transient up-regulation of hTERT and a subsequent reduction in hTERT mRNA levels at concentrations more than 10 times below its GIC50 value. In this respect, (S)-DHPA and (R,S)-AHPA-ibu were less efficient, since a similar effect was achieved at concentrations above their GIC(50). In contrast, F-PymRf treatment resulted in up to a three-fold induction of hTERT expression within a broad range of concentrations. In all cases, the down-regulation of hTERT expression was concentration-dependent. The correlation was found between c-myc overexpression and transiently elevated hTERT expression after treatment with all tested compounds except for alpha-5-azadCyd and (S)-HPMPazaC. Although the primary task of hypomethylating agents in anticancer therapy lies in reactivation of silenced tumour-suppressor genes, the inhibition of hTERT expression might also be a fruitful clinical effect of this approach.     

Synthesis and nicotinic acetylcholine receptor in vivo binding properties of 2-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine: a new positron emission tomography ligand for nicotinic receptors.

The lead compound of a new series of 3-pyridyl ethers, the azetidine derivative A-85380 (3-[(S)-2-azetidinylmethoxy]pyridine), is a potent and selective ligand for the human alpha4beta2 nicotinic acetylcholine receptor (nAChR) subtype. In vitro, the fluoro derivative of A-85380 (2-fluoro-3-[(S)-2-azetidinylmethoxy]pyridine or F-A-85380) competitively displaced [3H]cytisine or [3H]epibatidine with Ki values of 48 and 46 pM, respectively. F-A-85380 has been labeled with the positron emitter fluorine-18 (t1/2 (half-life) = 110 min) by no-carrier-added nucleophilic aromatic substitution by K[18F]F-K222 complex with (3-[2(S)-N-(tert-butoxycarbonyl)-2-azetidinylmethoxy]pyridin-2-yl) tri methylammonium trifluoromethanesulfonate as a highly efficient labeling precursor, followed by TFA removal of the Boc protective group. The total synthesis time was 50-53 min from the end of cyclotron fluorine-18 production (EOB). Radiochemical yields, with respect to initial [18F]fluoride ion radioactivity, were 68-72% (decay-corrected) and 49-52% (non-decay-corrected), and the specific radioactivities at EOB were 4-7 Ci/micromol (148-259 GBq/micromol). In vivo characterization of [18F]F-A-85380 showed promising properties for PET imaging of central nAChRs. This compound does not bind in vivo to alpha7 nicotinic or 5HT3 receptors. Moreover, its cerebral uptake can be modulated by the synaptic concentration of the endogenous ligand acetylcholine. The preliminary PET experiments in baboons with [18F]F-A-85380 show an accumulation of the radiotracer in the brain within 60 min. In the thalamus, a nAChR-rich area, uptake of radioactivity reached a maximum at 60 min (4% I.D./100 mL of tissue). [18F]F-A-85380 appears to be a suitable radioligand for brain imaging nAChRs with PET.     

2-Amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters as novel HBV-specific antiviral reagents

Novel 2-amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters were synthesized and evaluated for antihepatitis B virus (HBV) activity in vitro using HB611, HuH-6 cell line, stably transfected with the HBV genome. Among the compounds synthesized, 2-amino-6-phenylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (8), 2-amino-6-(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (16), 2-amino-6-(3-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (17), and 2-amino-6-(2-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (18) showed considerably high anti-HBV activity, as represented by IC(50) values of 0.05, 0.03, 0.04, and 0.08 microM, respectively, and exhibited low cytotoxicity, as represented by CC(50) values of more than 1000 microM. It was suggested that these compounds did not have anti-HIV activity, and compound 8 showed only weak anti-HSV-1 activity. An antiviral agent, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used as a control in the present study, showed moderate anti-HBV activity, as represented by an IC(50) value of 0.2 microM. Furthermore, compound 16 was administered orally to mice at a dose of 100 mg/kg in order to examine its gastrointestinal absorbability. Consequently, the main active metabolite was observed in mouse plasma, with especially high concentrations in the liver.     

Development, synthesis, and biological evaluation of (-)-trans-(2S,5S)-2-[3-[(2-oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)-phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran, a potent orally active platelet-activating factor (PAF) antagonist and its water-soluble prodrug phosphate ester.

(-)-trans-(2S,5S)-2-[3-[(2-Oxopropyl)sulfonyl]-4-n-propoxy-5-(3- hydroxypropoxy)phenyl]-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran (10) is one of the most potent platelet-activating factor (PAF) antagonists in vitro and in vivo developed to date. This diaryltetrahydrofuran derivative evolved from modifications of MK 0287 which has been evaluated in clinical studies for asthma. Two structural modifications of MK 0287 were made: (1) elaboration of the 3'-[(hydroxyethyl)sulfonyl] group to a beta-keto propylsulfonyl, and (2) replacement of the 5'-methyl ether by a 3-hydroxypropyl ether. Compound 10 potently and specifically inhibits the binding of [3H]-C18-PAF to human platelet membranes (Ki 1.85 nM) and PMN membranes (Ki 2.89 nM). In vivo, 10 inhibits PAF-induced plasma extravasation and elevated N-acetyl-beta-D-glucosaminidase (NAGA) levels in male rats with ED50 values of 60 micrograms/kg, po and 4 micrograms/kg, iv respectively, and inhibits PAF-induced bronchoconstriction in guinea pigs with an ED50 value of 15 micrograms/kg after intraduodenal administration. Compound 15, a water-soluble phosphate ester prodrug derivative of 10 is at least equipotent to 10 in the in vivo models. Compound 19S, the primary and major metabolite of 10 and 15, is equipotent in in vitro and in vivo models.     

Synthesis and antiviral activity of some 7-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine analogues of sangivamycin and toyocamycin

The sodium salt of 4-amino-3-cyanopyrazolo[3,4-d]pyrimidine (1) was condensed with (2-acetoxyethoxy)methyl bromide (2) to provide the corresponding protected acyclic nucleoside, 4-amino-3-cyano-1-[(2-acetoxyethoxy)methyl]-pyrazolo[3,4-d]pyrimid ine (3). Treatment of 3 with sodium methoxide in methanol provided a good yield of methyl 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- formimidate (4). Treatment of the imidate (4) with sodium hydrogen sulfide gave the thiocarboxamide derivative 5. Aqueous base transformed 4 into 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- carboxamide (6) in good yield. Treatment of 5 with mercuric chloride furnished the toyocamycin analogue 7. Evaluation of compounds 1, 3-7 revealed that only the heterocycle (1) and the thiocarboxamide acyclic nucleoside (5) were active. Compound 5 was the more potent with activity against human cytomegalovirus and herpes simplex virus type 1.     

Secretion of antiretroviral chemokines by human cells cultured with acyclic nucleoside phosphonates

Acyclic nucleoside phosphonates are novel class of clinically broadly used antivirotics effective against replication of both DNA viruses and retroviruses including human immunodeficiency virus (HIV). We have investigated their in vitro effects on immune defence mechanisms in human peripheral blood mononuclear cells, with the main emphasis on expression of cytokines which are able to suppress the entry of HIV in cells. Included in the study were prototype acyclic nucleoside phosphonates, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), (R)-and (S)-enantiomers of 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] and [(S)-PMPA], and of 9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine [(R)-PMPDAP] and [(S)-PMPDAP], and their N(6)-substituted derivatives. Some of the compounds were found to substantially enhance secretion of chemokines such as macrophage inflammatory protein-1alpha (MIP-alpha/CCL3), and "regulated on activation of normal T cell expressed and secreted" (RANTES/CCL5). Secretion of MIP-1beta/CCL4 was only marginally increased, whereas production of interleukin-16 (IL-16) and interferon-gamma (IFN-gamma) remained uninfluenced. The most effective proved to be the N(6)-cyclooctyl-PMEDAP, N(6)-isobutyl-PMEDAP, N(6)-pyrrolidino-PMEDAP, N(6)-cyclopropyl-(R)-PMPDAP, and N(6)-cyclopentyl-(R)-PMPDAP derivatives. Remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture, and was observed at concentration of 2-5 microM. It may be suggested that acyclic nucleoside phosphonates represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties.     

Facile radiosynthesis of fluorine-18 labeled beta-blockers. Synthesis, radiolabeling, and ex vivo biodistribution of [18F]-(2S and 2R)-1-(1-fluoropropan-2-ylamino)-3-(m-tolyloxy)propan-2-ol

An efficient and general method has been developed for fluorine-18 labeling of beta-blockers that possess the propanolamine moiety. A new synthetically versatile intermediate, 3-(1-(benzyloxy)propan-2-yl)-2-oxooxazolidin-5-yl)methyl 4-methylbenzenesulfonate (13), was prepared and can be conjugated to any phenoxy core. To demonstrate the synthetic methodology, fluorinated derivatives of toliprolol were prepared, namely, [(18)F]-(2S and 2R)-1-(1-fluoropropan-2-ylamino)-3-(m-tolyloxy)propan-2-ol ((2S and 2R)-[(18)F]1). The radiosyntheses were accomplished in <1 h, with 20-24% (uncorrected for decay, n = 7) radiochemical yields, >96% radiochemical and >99% enantiomeric purities, with specific activities of 0.9-1.1 Ci/micromol (EOS). Ex vivo biodistribution studies with the radiotracers demonstrated excessively rapid washout that may limit their use for cerebral PET imaging.     

Synthesis and antiviral activity of deoxy analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent and selective anti-HIV-1 agents.

The effect of substitution in the acyclic structure of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine (HEPT) on anti-HIV-1 activity was investigated by synthesizing a series of deoxy analogs and related compounds. Preparation of 1-[(2-alkyloxyethoxy)methyl]-6- (phenylthio)thymine (2-4) derivatives was carried out based on alkylation of HEPT with primary alkyl halides. Preparation of the 1-[(alkyloxy)methyl]-6-(phenylthio)thymine (26-31) and 1-[(alkyloxy)methyl]-6-(arylthio)-2-thiouracil (32-45) derivatives was carried out on the basis of LDA lithiation of 1-[(alkyloxy)-methyl]thymine (9-14) and 1-[(alkyloxy)methyl]-2-thiouracil (15-25) followed by reaction with diaryl disulfides. The oxidative hydrolysis of the 2-thiouracil derivatives gave 1-[(alkyloxy)methyl]-6-(arylthio)uracil derivatives (46-57). 1-Alkyl-6-(phenylthio)thymine (59-61) derivatives were prepared on the basis of alkylation of 6-(phenylthio)thymine (58). Methylation of the hydroxyl group of HEPT did not affect the anti-HIV-1 activity of HEPT. Substitution of the 1-(2-hydroxyethoxy)methyl group by ethyl, butyl, methoxymethyl, (propyloxy)methyl, and (butyloxy)-methyl groups somewhat improved the original anti-HIV-1 activity of HEPT. Substitution with ethoxymethyl and (benzyloxy)methyl groups further potentiated the activity [EC50: 1-(ethoxy-methyl)-6-(phenylthio)thymine (27), 0.33 microM; 1-[(benzyloxy)methyl]-6-(phenylthio)thymine (31), 0.088 microM]. When the 5-methyl group of 27 and 31 was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity was improved remarkably [EC50: 5-ethyl-1-(ethoxymethyl)-6-(phenylthio)-uracil (46), 0.019 microM; 5-ethyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (52), 0.0059 microM; 5-isopropyl-1-(ethoxymethyl)-6-(phenylthio)uracil (55), 0.012 microM; 5-isopropyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (56), 0.0027 microM]. Introduction of two m-methyl groups into the phenylthio ring also potentiated the activity.     

Structure-activity relationships of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine analogues: effect of substitutions at the C-6 phenyl ring and at the C-5 position on anti-HIV-1 activity.

The effect of substitution on the pyrimidine moiety of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiothymine (HEPT-S) on anti-HIV-1 activity was investigated by synthesizing a series of 5-methyl-6-(arylthio) and 5-substituted-6-(phenylthio) derivatives. Preparation of the 5-methyl-6-(arylthio) derivatives was carried out based on either LDA lithiation of 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]thymine (3) and 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2-thiothymine (4) followed by reaction with diaryl disulfides or an addition-elimination reaction of 1-[[2-(tert-butyldimethylsiloxy)ethoxy]-methyl]-6- (phenylsulfinyl)thymine (31) with aromatic thiols. Preparation of the 5-substituted-6-(phenylthio) derivatives was carried out based on either C-5 lithiation of the 1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-6-(phenylthio)uraci l (41) with LTMP or the LDA lithiation of 5-alkyl-1-[[2-(tert-butyldimethylsiloxy)ethoxy]methyl]-2-thiouraci l derivatives 45-47. Substitution at the meta position of the C-6-(phenylthio) ring by the methyl group improved the original anti-HIV-1 activity of HEPT, and introduction of two m-methyl groups to the phenylthio ring further potentiated the activity [EC50: 6-[(3,5-dimethylphenyl)thio]-1-[(2-hydroxyethoxy)methyl]thymine (28), 0.26 microM; 6-[(3,5-dimethylphenyl)thio]-1-[(2-hydroxyethoxy)methyl]-2-thiothymin e (30), 0.22 microM]. When the 5-methyl group was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity of HEPT was also improved remarkably [EC50: 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (48), 0.11 microM; 5-isopropyl-1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)-2-thiouracil (50), 0.059 microM; 5-ethyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (54), 0.12 microM; 5-isopropyl-1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-2-thiouracil (56), 0.063 microM]. 6-[(3,5-Dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]thymine derivatives 51 and 57 and 6-[(3,5-dimethylphenyl)thio]-5-isopropyl-1-[(2- hydroxyethoxy)methyl]thymine derivatives 52 and 58 inhibited the replication of HIV-1 in the nanomolar concentration range.     

Differential effects of acyclic nucleoside phosphonates on nitric oxide and cytokines in rat hepatocytes and macrophages

Acyclic nucleoside phosphonates (ANP) are virostatics effective against viruses like hepatitis B virus and human immunodeficiency virus. Our previous reports indicated immunomodulatory activities of ANP in mouse and human innate immune cells. Recently, evidence has increased that hepatocytes may play an active role in immune regulation of the liver homeostasis or injury. In this study we investigated possible immunomodulatory effects of ANP on rat hepatocytes and macrophages. Nitric oxide (NO) production and secretion of cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IL-18, IFN-γ, TNF-α and GM-CSF) were analyzed under in vitro conditions. Test compounds included: 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir); 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP); (R)- and (S)-enantiomers of 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] and [(S)-PMPA]; 9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine [(R)-PMPDAP] and [(S)-PMPDAP]. The group of test compounds also included their N(6)-substituted derivatives. Some of ANP which are able to induce NO production and cytokine secretion in cultured macrophages possess the same immunobiological activity in isolated hepatocytes. The extent of responses is in range of LPS/IFN-γ stimulation in both types of cells. The effects of active ANP on NO expression and cytokine secretion are dose- and time-dependent. Interestingly, the spectrum of detected cytokines induced by ANP is broader in hepatocytes. The results also confirm immunomodulatory effects of some ANP on rodent macrophages. Moreover, we demonstrate for the first time immunobiological reactivity of primary rat hepatocytes induced by exogenous ANP compounds. The potential of hepatocytes to synthesize cytokines can contribute to better understanding of liver immune function and can serve for pharmacological intervention in liver diseases.     

Immunobiological activity of N-[2-(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines,and 2,6-diaminopurines

Acyclic nucleoside phosphonates are novel class of virostatics effective against replication of both DNA-viruses and retroviruses. We found recently, that in addition to the antimetabolic mode of action, some acyclic nucleoside phosphonates such as 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir], which is used in treatment of human immunodeficiency virus (HIV) infection, possess immunostimulatory and immunomodulatory activities known to interfere with replication of viruses. The present experiments analyzed immunobiological effects of more than 70 novel derivatives of acyclic nucleoside phosphonates. They comprise substitutions at the N6-amino function of adenine (A) or 2,6-diaminopurine (DAP) by monoalkyl, dialkyl, cycloalkyl, alkenyl, alkynyl or substituted alkyl group, and at the N9-side chain represented by (R)- or (S)-enantiomeric 9-[2-(phosphonomethoxy)ethyl] (PME) and 9-[2-(phosphonomethoxy)propyl] (PMP) moieties. Their biological effects were investigated in vitro using mouse resident peritoneal macrophages. A number of the compounds under scrutiny, mainly the N6-cycloalkyl derivatives of 9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (PMEDAP) and (R)-enantiomeric 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPDAP] stimulate secretion of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10)] and chemokines ["regulated-upon-activation, normal T expressed and secreted" (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha)]. Moreover, they substantially augment production of nitric oxide (NO) triggered by interferon-gamma. The effects are produced in a dose-dependent fashion. The most potent derivatives, i.e. N6-isobutyl-PMEDAP, N6-cyclopentyl-PMEDAP, N6-cyclooctyl-PMEDAP, N6-dimethylaminoethyl-(R)-PMPDAP, N6-cyclopropyl-(R)-PMPDAP, and N6-cyclopentyl-(R)-PMPDAP are more effective than (R)-PMPA (tenofovir) itself. They exhibit immunostimulatory effects at concentrations as low as 1 to 5 microM. It is suggested that these compounds might be prospective candidates for antiviral therapeutic exploitation.