Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide.

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5'-O-monophosphate, 7-methylinosine 5'-O-diphosphate, and 7-methylinosine 5'-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).

C6-O-alkylated 7-deazainosine nucleoside analogues: Discovery of potent and selective anti-sleeping sickness agents.

African trypanosomiasis, a deadly infectious disease caused by the protozoan Trypanosoma brucei spp., is spread to new hosts by bites of infected tsetse flies. Currently approved therapies all have their specific drawbacks, prompting a search for novel therapeutic agents. T. brucei lacks the enzymes necessary to forge the purine ring from amino acid precursors, rendering them dependent on the uptake and interconversion of host purines. This dependency renders analogues of purines and corresponding nucleosides an interesting source of potential anti-T. brucei agents. In this study, we synthesized and evaluated a series of 7-substituted 7-deazainosine derivatives and found that 6-O-alkylated analogues in particular showed highly promising in vitro activity with EC50 values in the mid-nanomolar range. SAR investigation of the O-alkyl chain showed that antitrypanosomal activity increased, and also cytotoxicity, with alkyl chain length, at least in the linear alkyl chain series. However, this could be attenuated by introducing a terminal branch point, resulting in the highly potent and selective analogues, 36, 37 and 38. No resistance related to transporter-mediated uptake could be identified, earmarking several of these analogues for further in vivo follow-up studies.

Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase.

8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy.

Synthesis and biological properties of C-2 triazolylinosine derivatives.

O(6)-(Benzotriazol-1H-yl)guanosine and its 2'-deoxy analogue are readily converted to the O(6)-allyl derivatives that upon diazotization with t-BuONO and TMS-N(3) yield the C-2 azido derivatives. We have previously analyzed the solvent-dependent azide·tetrazole equilibrium of C-6 azidopurine nucleosides, and in contrast to these, the O(6)-allyl C-2 azido nucleosides appear to exist predominantly in the azido form, relatively independent of solvent polarity. In the presently described cases, the tetrazole appears to be very minor. Consistent with the presence of the azido functionality, each neat C-2 azide displayed a prominent IR band at 2126-2130 cm(-1). A screen of conditions for the ligation of the azido nucleosides with alkynes showed that CuCl in t-BuOH/H(2)O is optimal, yielding C-2 1,2,3-triazolyl nucleosides in 70-82% yields. Removal of the silyl groups with Et(3)N·3HF followed by deallylation with PhSO(2)Na/Pd(PPh(3))(4) gave the C-2 triazolylinosine nucleosides. In a continued demonstration of the versatility of O(6)-(benzotriazol-1H-yl)purine nucleosides, one C-2 triazolylinosine derivative was converted to two adenosine analogues via these intermediates, under mild conditions. Products were desilylated for biological assays. The two C-2 triazolyl adenosine analogues demonstrated pronounced antiproliferative activity in human ovarian and colorectal carcinoma cell cultures. When evaluated for antiviral activity against a broad spectrum of DNA and RNA viruses, some of the C-2 triazolylinosine derivatives showed modest inhibitory activity against cytomegalovirus.

Two-step, one-pot synthesis of inosine, guanosine, and 2'-deoxyguanosine O6-ethers via intermediate O6-(benzotriazol-1-yl) derivatives.

A simple method for the etherification at the O(6)-position of silyl-protected inosine, guanosine, and 2'-deoxyguanosine is described. Typically, a THF solution of the silylated nucleoside is treated with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and Cs(2)CO(3) under a nitrogen atmosphere. Conversion to the O(6)-(benzotriazol-1-yl) ethers occurs within about 10 min for inosine, and within about 60 min for guanosine and 2'-deoxyguanosine. Then, for reaction with alcohols, the reaction mixture is evaporated and the O(6)-(benzotriazol-1-yl) ether is treated with Cs(2)CO(3) and an appropriate alcohol, at room temperature. On the other hand, for reaction with phenols, Cs(2)CO(3) and the appropriate phenol are added to the reaction mixture without evaporation, and the reaction is carried out at 70°C. Subsequently, workup, isolation, and purification lead to the requisite O(6)-alkyl or O(6)-aryl ethers in good to excellent yields.

Synthesis of 2'-substituted inosine analogs via unusual masking of the 6-hydroxyl group.

2'-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylamine transfer from dimethylformamide. Graphical Abstract:

Towards the synthesis of inosine building blocks for the preparation of oligonucleotides with hydrophobic alkyl chains between the nucleotide units.

The scientific objective of the research reported in this manuscript was the synthesis of novel phosphoramidite building blocks for the preparation of lipophilic oligonucleotides. Reaction of inosine (4) with 4-oxopentyl-4-methylbenzoate (2c) in the presence of triethyl orthoformate and 4M HCl in 1,4-dioxane gave a diastereoisomeric mixture of the ketals 5. Subsequent 4,4'-dimethoxytritylation at the 5'-hydroxyl afforded (R)-6 + (S)-6 which could be separated chromatographically. Detoluoylation gave compounds (R)-7 and (S)-7. Phosphitylation of a diastereoisomeric mixture of 7 led to a mixture of four diastereoisomers of the corresponding 2-cyanoethylphosphoramidites 8.

Synthesis and anti-Hantaan virus activity of N(1)-3-fluorophenyl-inosine.

As part of an ongoing effort to develop new antiviral nucleoside analogs, our interest was drawn to N(1)-aryl purines as a novel structural class and potential scaffold for drug discovery. Herein, we describe the synthesis of N(1)-3-fluorophenyl-inosine (FPI) and N(1)-3-fluorophenyl-hypoxanthine (FP-Hx) and their antiviral activity against hantaviruses. The EC(50) for FPI and FP-Hx were 94 and 234microM, respectively, against Hantaan virus. FPI was not toxic to mammalian cells at concentrations that exhibited antiviral activity. Analysis of its metabolism revealed a low conversion of FPI in Vero E6 or human cells to a 5'-triphosphate, and it was a poor substrate for human purine nucleoside phosphorylase. Further, the compound did not alter GTP levels indicating FPI does not inhibit inosine monophosphate dehydrogenase. With respect to the virus, FPI did not decrease viral RNA levels or increase the mutation frequency of the viral RNA. This suggests that the antiviral activity of FPI might be solely due to the interaction of FPI or its metabolites with viral or host proteins involved in post-replication events that would affect the levels of infectious virus released. Synthesis of other compounds structurally similar to FPI is warranted to identify more potent agents that selectively abrogate production of infectious virus.

The application of the phosphoramidate ProTide approach confers micromolar potency against hepatitis C virus on inactive agent 4'-azidoinosine: kinase bypass on a dual base/sugar modified nucleoside.

Novel phosphoramidate ProTides derived from 4'-azidoinosine have been prepared and evaluated in the replicon assay against hepatitis C Virus (HCV). The parent nucleoside analogue is inactive in this assay, while the ProTides are active at low microM levels in some cases. This is a rare example of an inosine nucleoside analogue with potent antiviral activity and further supports the notion of ProTides as a drug discovery motif.

Studies on the glycosylation of pyrrolo[2,3-d] pyrimidines with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose: the formation of regioisomers during toyocamycin and 7-deazainosine syntheses.

Glycosylation of silylated 4-amino-6-bromo-5-cyano-7H-pyrrolo[2,3-d]pyrimidine (9) with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (10) under "one-pot" glycosylation conditions (MeCN, TMSOTf) yielded the N-7 isomer 11 together with the N-1 compound 13 (ratio = 2:1). When the same conditions were applied to 4-hydroxy-7H-pyrrolo[2,3-d]pyrimidine (21) the N-3 isomer 22 was the only glycosylation product formed in almost quantitative yield.

7-deazainosine derivatives: synthesis and characterization of 7- and 7,8-substituted pyrrolo [2,3-d]pyrimidine ribonucleosides.

The synthesis of model 7 deazapurine derivatives related to tubercidin and toyocamycin has been performed. Tubercidin derivatives were obtained by simple conversion of the amino group of the heterocyclic moiety of the starting 7-deazadenosine compounds, into a hydroxyl group. Preparation of toyocamycin derivatives was accomplished by treatment of the silylated 6-bromo-5-cyanopyrrolo[2,3-d]pyrimidin-4-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-d-ribofuranose. The glycosylation reaction afforded a mixture of 8-bromo 7-cyano 2',3',5' tri-O-benzoyl 7-deazainosine and 6-bromo-5-cyano-3-(2',3',5'-tri-O-benzoyl-beta-d-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4-one isomers: The structures were assigned on the basis of NMR spectroscopy studies. Next deprotection treatment gave the novel 7-deazainosine ribonucleosides.

Inhibition of adenosine deaminase by analogues of adenosine and inosine, incorporating a common heterocyclic base, 4(7)-amino-6(5)H-imidazo[4,5-d]pyridazin-7(4)one.

Four nucleoside analogues ( 1- 4) containing a common heterocyclic base, 4(7)-amino-6(5) H-imidazo[4,5- d]pyridazin-7(4)one, were screened against calf-intestine adenosine deaminase. Compounds 1 and 3 with K(i) values of 10-12 microM are more than four times as potent inhibitors of ADA compared with 2 and 4, with K(i) values of 51-52 microM. Also, 3 is not a substrate of ADA. Nucleosides 3 and 4 also exhibit moderate in vitro activity against breast cancer cell lines, while all four are only minimally or nontoxic to the normal cells.

7,5'-O-dibenzylinosines: synthesis and studies on their conformational properties.

Reaction of 2',3'-O-isopropylidene inosine with benzyl bromide (1 h, rt) led to the 1,5'-O-dibenzylderivative 4, but by increasing the reaction time or the temperature, compound 4 is further transformed into the 1,7,5'-O-tribenzylinosine derivative 5. Similarly, the 7-methyl-1,5'-O-dibenzylderivative 6 has been synthesized from 4. The 1H-NMR spectra of 5 and 6 showed peculiar chemical shifts for geminal protons (H5' and H5'' of the ribose, and the CH2 of the benzyl groups). Preliminary NMR studies have been performed, including NOESY experiments that point toward the predominant existence of conformers that are stabilized by an electrostatic interaction between the positively charged imidazole of the base moiety and the high electron density of the 5'-benzyl substituent.

Efficient syntheses of 5'-deoxy-5'-fluoroguanosine and -inosine.

Substitution of oxygen with a weak hydrogen bond acceptor such as fluorine provides a single-atom modification that can have grave effects on the chemical and medicinal properties of nucleoside analogues. To that end, we present a simple and high-yielding method for the novel synthesis of 5'-deoxy-5'-fluoroguanosine and 5'-deoxy-5'-fluoroinosine utilizing an intramolecular electron-withdrawing approach. The properties of the resulting modified nucleosides, as well as the halogenated intermediates, are notable for their similarity to nucleoside analogues used in the treatment of cancer, as well as enzyme inhibitors designed to target parasitic protozoa.

5'-O-tritylinosine and analogues as allosteric inhibitors of human thymidine phosphorylase.

On the basis of our previous findings that 5'-O-tritylinosine (KIN59) behaves as an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase (TPase), we have undertaken the synthesis and enzymatic evaluation of a novel series of nucleoside analogues modified at positions 1, 2, or 6 of the purine ring and at the 5'-position of the ribose moiety of the lead compound KIN59. SAR studies indicate that quite large structural variations can be performed on KIN59 without compromising TPase inhibition. Thus, incorporation of a cyclopropylmethyl or a cyclohexylmethyl group at position N(1) of 5'-O-tritylinosine increases the inhibitory activity against TPase 10-fold compared to KIN59. Moreover, the trityl group at the 5'-position of the ribose seems to be crucial for TPase inhibition. The here reported results further substantiate that 5'-O-trityl nucleosides represent a new class of TPase inhibitors that should be further explored in those biological systems where TPase plays an instrumental role (i.e. angiogenesis).

Markedly enhancing lipase-catalyzed synthesis of nucleoside drugs' ester by using a mixture system containing organic solvents and ionic liquid.

Eightfold higher yields and three times faster reaction rates were achieved by means of using a mixture solvent system composed of 90% acetone and 10% [BMIM]BF4 in the lipase-catalyzed regioselective synthesis of polymerizable ester of nucleoside drugs.

Synthesis and reactions of 2-chloro- and 2-tosyloxy-2'-deoxyinosine derivatives.

Convenient syntheses of 2-chloro- and 2-tosyloxy-2'-deoxyinosine as their tert-butyldimethylsilyl ethers are described. Both compounds can be synthesized via a common route and rely on commercially available 2'-deoxyguanosine. The present method leading to the chloro nucleoside is operationally simpler compared to previously reported glycosylation techniques where isomeric products were obtained. Both electrophilic nucleosides can be used for the preparation of N-substituted 2'-deoxyguanosine analogues via displacement of the leaving groups, and a comparison of their reactivities shows the chloro analogue to be superior. Interestingly, a Pd catalyst-mediated, two-step, one-pot conversion of an allyl-protected chloro nucleoside intermediate to the final modified 2'-deoxyguanosine derivatives is also feasible. On the basis of these observations, initial assessments of Pd-catalyzed aryl amination as well as a C-C cross-coupling have also been performed with the chloro and tosyloxy nucleoside substrates. Results indicate a potentially high synthetic utility of 2-chloro-2'-deoxyinosine and in many instances this derivative can supplant the bromo and fluoro analogues that are more cumbersome to prepare or are not readily available.

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine.

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies.

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2'-deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2'-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2'-deoxyinosine 9 was prepared from 2'-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-15N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-beta-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.