Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Structure-Activity Relationships of Acyclic Selenopurine Nucleosides as Antiviral Agents.

A series of acyclic selenopurine nucleosides 3a-f and 4a-g were synthesized based on the bioisosteric rationale between oxygen and selenium, and then evaluated for antiviral activity. Among the compounds tested, seleno-acyclovir (4a) exhibited the most potent anti-herpes simplex virus (HSV)-1 (EC50 = 1.47 µM) and HSV-2 (EC50 = 6.34 µM) activities without cytotoxicity up to 100 µM, while 2,6-diaminopurine derivatives 4e-g exhibited significant anti-human cytomegalovirus (HCMV) activity, which is slightly more potent than the guanine derivative 4d, indicating that they might act as prodrugs of seleno-ganciclovir (4d).

Formation of the N(2)-acetyl-2,6-diaminopurine oligonucleotide impurity caused by acetyl capping.

The acetyl 'capping' reaction routinely employed during phosphorothioate oligonucleotide synthesis has been implicated in the formation of an impurity species with a mass 41 amu greater than the expected oligonucleotide molecule. The impurity has been found to arise by conversion of a protected guanine nucleobase to N(2)-acetyl-2,6-diaminopurine. A two-part mechanism is proposed consisting of transamidation of the protecting group on guanine and substitution of guanine's O(6) atom.

Synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, an impurity of acyclovir.

The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, the process uses an acid catalysed phase transfer catalysis (PTC) process to produce the selective alkylation at the 9 position of the guanine ring.

Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of 2,6-diaminopurine, 2-aminopurine and thymine.

A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into a PNA and was found to selectively bind to T (ΔT(m)≥ +6 °C) in a complementary DNA strand. The 2AP monomer showed excellent discrimination of T (ΔT(m)≥ +12 °C) over the other nucleobases. 2AP also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching dependent on the opposite base.

Synthesis and antiviral activity of N9-[3-fluoro-2-(phosphonomethoxy)propyl] analogues derived from N6-substituted adenines and 2,6-diaminopurines.

An efficient method for the synthesis of N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases has been developed. Both (R)- and (S)-enantiomers of the N(6)-substituted FPMP derivatives of adenine and 2,6-diaminopurine were prepared and their anti-human immunodeficiency virus (HIV) and anti-Moloney murine sarcoma virus (MSV) activity was evaluated. Whereas none of the 6-substituted FPMPA derivatives showed any antiviral activity, several FPMPDAP derivatives had a moderate antiretroviral activity. Moreover, the data obtained from the study of the substrate activity of the active derivatives towards N(6)-methyl-AMP aminohydrolase support the notion that the studied N(6)-substituted FPMPDAP derivatives act as prodrugs of the antiretroviral FPMPG analogues.

Preparation of the 2'-deoxynucleosides of 2,6-diaminopurine and isoguanine by direct glycosylation.

The purine nucleoside 2,6-diaminopurine-2'-deoxyriboside is prepared by the direct glycosylation of the 2,6-bis(tetramethylsuccinimide) derivative of the parent purine heterocycle 4 with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride 5 using the sodium salt method. 2'-Deoxyisoguanosine is prepared from 2,6-diaminopurine by a five-step procedure. The purine heterocycle isoguanine is prepared by selective diazotization of 2,6-diaminopurine and then converted to the N9-trityl derivative to increase solubility. After silylation of the O(2)-carbonyl with TMSCl, the N(6)-amino group is protected as the tetramethylsuccinimide (M(4)SI). The O(2)-carbonyl is protected as the DPC derivative, and the trityl group is removed. The resulting product is glycosylated in good yield to generate fully protected 2'-deoxyisoguanosine.

Additive Pummerer reaction of 3,5-O-(di-tert-butyl)silylene-4-thiofuranoid glycal: a high-yield and beta-selective entry to 4'-thioribonucleosides.

Upon reacting 3,5-O-(di-tert-butyl)silylene-4-thiofuranoid glycal S-oxide (6) with Ac(2)O/TMSOAc/BF(3) x OEt(2) in CH(2)Cl(2), the additive Pummerer reaction proceeded to furnish the corresponding 1,2-di-O-acetyl-4-thioribofuranose 7. Compound 7 serves as a highly beta-selective glycosyl donor in the Vorbruggen condensation carried out in the presence of TMSOTf. Thus, the 4-thio-beta-D-ribofuranosyl derivatives of uracil, thymine, N (4) -acetylcytosine, 6-chloropurine, and 2-amino-6-chloropurine were synthesized. The use of 7 can be extended to the beta-selective synthesis of 4'-thio-C-ribonucleosides.

Synthesis of the new nucleoside analogue connecting 2-amino-6-vinylpurine to the 2'-deoxyribose skeleton via the methylene linker.

We have previously reported that the 2-amino-6-vinylpurine nucleoside exhibits the highly efficient and selective cross-linking reaction toward the cytosine base at the target site in the duplex DNA. The nucleoside analogues that connect the 2-amino-6-vinylpurine to the 2'-deoxyribose skeleton through the ethylene or the butylene linker formed the cross-link selectively to the adenine base of the TA pair or the cytosine base of the GC pair in the triplex DNA, respectively. They did not form cross-link in the duplex DNA. These results lead us to study in detail the relationship between the linker length and the cross-linking ability. In this study, we describe the synthesis of the new nucleoside analogue that connects 2-amino-6-vinylpurine to the 2'-deoxyribose unit via the methylene linker.

A Pd(0) based cross-coupling approach to the synthesis of 2-amidopurines and their evaluation as CDK inhibitors.

Two new series of 2-amido- and 2-aminocarbonylpurines have been synthesized using a Pd catalyst cross-coupling reaction either with amides or amines in the presence of CO. Moderate in vitro inhibitory activity against CDK1 and CDK5 was observed with IC(50) of 0.9muM for the most active compound (18c).

Synthesis and antiviral evaluation of alkoxyalkyl esters of phosphonopropoxymethyl-guanine and phosphonopropoxymethyl-diaminopurine.

Phosphonopropoxymethyl-guanine is the methylene phosphonate analogue of acyclovir. Although not highly active against HSV, 4-38 microM of phosphonopropoxymethyl-guanine has been reported to be active against human and murine cytomegalovirus. Recently we found that cidofovir, when esterified with alkoxyalkyl moieties, showed greatly increased antiviral activity against cytomegalovirus, herpes simplex virus and orthopoxviruses, in vitro. The alkoxyalkyl esters of cidofovir are orally active in murine models of human and murine cytomegalovirus and orthopoxviruses in vivo. To see if the antiviral activity of phosphonopropoxymethyl-guanine, phosphonopropoxymethyl-diaminopurine and phosphonopropoxymethyl-N6-cyclopropyl-diaminopurine could be increased by this approach, we synthesized their hexadecyloxypropyl- and octadecyloxyethyl- esters and evaluated antiviral activity and cytotoxicity in cells infected with HSV-1 and HCMV, in vitro. Marked increases in antiviral activity were noted in the alkoxyalkyl esters of phosphonopropoxymethyl-guanine. Alkoxyalkyl esters of diaminopurine and N6-cyclopropyl-diaminopurine showed slight increases in activity against HSV-1 and marked increases in activity against HCMV. The results suggest that esterification with alkoxyalkyl moieties may be a generally useful way to increase antiviral activity of nucleoside phosphonates.

Improved industrial syntheses of penciclovir and famciclovir using N2-acetyl-7-benzylguanine and a cyclic side chain precursor.

We have established practical synthetic methods for penciclovir (PCV, 1) and famciclovir (FCV, 2) from N2-acetyl-7-benzylguanine (NAc7BnG, 3) and 6,6-dimethyl-5, 7-dioxaspiro[2.5]octane-4,8-dione (4)--the latter being a more easily prepared cyclic precursor of the diacetate side chain (5) used in the conventional process. The coupling of 4 with 3 proceeded regioselectively at the N9 position of guanine in good yield. The coupling product was then successfully transformed into the known antiviral agents in a short number of steps.

Synthesis, characterisation and theoretical calculations of 2,6-diaminopurine etheno derivatives.

Four derivatives of 2,6-diaminopurine (1) were synthesised and characterised. When 1 was reacted with chloroacetaldehyde, 5-aminoimidazo[2,1-i]purine (2), 9-aminoimidazo[2,1-b]purine (3), 9-aminoimidazo[1,2-a]purine (4) and diimidazo[2,1-b:2',1'-i]purine (5) were formed. The purified products (3-5) were fully characterised by MS, complete NMR assignments as well as fluorescence and UV spectroscopy. The purified, isolated yields of these products (3-5) varied from 2.5 to 30%. The relative stability of different tautomers was investigated by theoretical calculations. Fluorescence characteristics are also discussed and compared to the starting material 1 and a reference molecule 2-aminopurine.

LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides.

LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).

Synthesis and biological activity of 2-aminopurine methylenecyclopropane analogues of nucleosides.

Synthesis and biological activity of 7- and 9-isomers (Z+E) of methylenecyclopropane analogues of 2-aminopurine nucleosides is described. The (S,Z)-9-isomer is a substrate for xanthine oxidase.

2,6-Diamino-5,8-diaza-7,9-dicarba-purine.

[reaction: see text] The title compound, a constitutional isomer of the natural nucleobase 2,6-diaminopurine, undergoes regioselective electrophilic substitutions at carbon C-9.

An economical synthesis of famciclovir.

An economical synthesis of famciclovir from N-2-acetyl-7-benzylguanine by a novel regioselective alkylation with the diester cyclopropane compound was developed.

Synthesis and in vivo antitumor activity of 2-amino-9H-purine-6-sulfenamide, -sulfinamide, and -sulfonamide and related purine ribonucleosides.

A number of 6-sulfenamide, 6-sulfinamide, and 6-sulfonamide derivatives of 2-aminopurine and certain related purine ribonucleosides have been synthesized and evaluated for antileukemic activity in mice. Amination of 6-mercaptopurine ribonucleoside (7a) and 6-thioguanosine (7b) with chloramine solution gave 9-beta-D-ribofuranosylpurine-6-sulfenamide (8a) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 8b), respectively. Selective oxidation of 8a and 8b with 3-chloroperoxybenzoic acid (MCPBA) gave (R,S)-9-beta-D-ribofuranosylpurine-6-sulfinamide (9a) and (R,S)-2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine, 9b), respectively. However, oxidation of 8a and 8b with excess of MCPBA gave 9-beta-D-ribofuranosylpurine-6-sulfonamide (10a) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 10b), respectively. Similarly, amination of 5'-deoxy-6-thioguanosine (7c) afforded the 6-sulfenamide derivative (8c), which on controlled oxidation gave (R,S)-2-amino-9-(5-deoxy-beta-D-ribofuranosyl)purine-6-sulfinamide (9c) and the corresponding 6-sulfonamide derivative (10c). Treatment of 6-thioguanine (12) with aqueous chloramine solution gave 2-amino-9H-purine-6-sulfenamide (13). Oxidation of 13 with 1 molar equiv of MCPBA afforded (R,S)-2-amino-9H-purine-6-sulfinamide (14), whereas the use of 4 molar equiv of MCPBA furnished 2-amino-9H-purine-6-sulfonamide (15). The resolution of R and S diastereomers of sulfinosine (9b) was accomplished by HPLC techniques. The structures of (R)-9b and 10b were assigned by single-crystal X-ray diffraction studies. (R)-9b exists in the crystal structure in four crystallographically independent conformations. Of the 18 compounds evaluated, 13 exhibited very significant anti-L1210 activity in mice. Sulfenosine (8b) at 22 mg/kg per day X 1 showed a T/C of 170, whereas sulfinosine (9b) at 173 mg/kg per day X 1 showed a T/C of 167 against L1210 leukemia. The 5'-deoxy analogue of sulfinosine (9c) at 104 mg/kg per day also showed a T/C of 172. A single treatment with 8b, 9b, and 9c reduced body burdens of viable L1210 cells by more than 99.8%.

Prodrugs of the selective antiherpesvirus agent 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (BRL 39123) with improved gastrointestinal absorption properties.

Potential oral prodrugs of the antiherpesvirus acyclonucleoside 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (1, BRL 39123) have been synthesized and evaluated for bioavailability of 1 in the blood of mice. Reduction of 9-[4-acetoxy-3-(acetoxymethyl)but-1-yl]-2-amino-6-chloropurine (13) using ammonium formate and 10% palladium on carbon afforded the 2-aminopurine 14, which was hydrolyzed to the monoacetate 15 and to 2-amino-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purine (5). The 2-aminopurine 5 was subsequently converted to additional monoester (17, 21-23) and diester (16, 24) derivatives and to its di-O-isopropylidene derivative 18. Both 5 and its esters (14-17, 21, 22) and also 18 were well absorbed after oral administration and converted efficiently to 1, the diacetyl (14) and dipropionyl (16) esters providing concentrations of 1 in the blood that were more than 15-fold higher than those observed after dosing either 1 or its esters (25-27). Some 6-alkoxy-9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]purines (8-10), the preparation of which has been reported previously, also showed improved absorption properties, but their conversion to 1 was less efficient than for the 2-aminopurine derivatives. On the basis of these results and subsequent experiments involving determinations of rates of conversion to 1 in the presence of rat and human tissue preparations, 9-[4-acetoxy-3-(acetoxymethyl)but-1-yl]-2-aminopurine (14, BRL 42810) was identified as the preferred prodrug of 1. Oral bioavailability studies in healthy human subjects confirmed 14 as an effective prodrug, and this compound is now being evaluated in clinical trials.