7-Deaza-2,8-diazaadenine containing oligonucleotides: synthesis, ring opening and base pairing of 7-halogenated nucleosides

Oligonucleotides containing 7-bromo-7-deaza-2,8-diaza-2′-deoxyadenosine (3) and 5-amino-3-bromo-4-carbamoyl-1-(2′-deoxy-β-d-erythro-pentofuranosyl)pyrazole (4) were synthesized. Compound 3 was prepared from 7-bromo-8-aza-7-deaza-2′-deoxyadenosine (5) via the 1,N⁶-etheno derivative 6 and was converted into the phosphoramidite 11. The 7-bromo substituent of 3 increases oligonucleotide duplex stability compared to the non-halogenated nucleoside. Oligonucleotides incorporating 3 are transformed to those containing 4 during long time deprotection at elevated temperature (25% aq ammonia, 60 °C, 30 h). Compound 3 forms a strong base pair with dG. The base pair stability decreases in the order dG>dT>dA>dC. Similar recognition selectivity is observed for the pyrazole nucleoside 4, however, due to decreased stacking and higher flexibility of the pyrazole moiety, duplexes are less stable than those containing 3.     

Hydrogelation and spontaneous fiber formation of 8-aza-7-deazaadenine nucleoside ‘click’ conjugates

Nucleoside hydrogels based on benzyl azide ‘click’ conjugates of 8-aza-7-deaza-2′-deoxyadenosine bearing 7-ethynyl, 7-octa-(1,7-diynyl), and 7-tri-prop-2-ynyl-amine side chains were synthesized (1, 3, 4). The cycloaddition adduct with the shortest linker (1) yields the most powerful hydrogelator forming stable gels at a concentration of 0.3 wt % of 1 in water. One molecule of 1 catches 7500 water molecules. Cycloaddition of the 8-aza-7-deaza-7-azido-2′-deoxyadenosine (9) and 3-phenyl-1-propyne (10) leads to the isomeric conjugate 2, with a C-N connectivity between the nucleobase and triazole moiety. This gel is less stable than that of the adduct 1. Both gels show a similar stability over a wide pH range (4.0-10.0). Xerogels of 1 and 2 studied by scanning electron microscopy (SEM) reveal that both click adducts (1 and 2) form long fibers spontaneously.     

Synthesis and ‘double click’ density functionalization of 8-aza-7-deazaguanine DNA bearing branched side chains with terminal triple bonds

The 7-[di(prop-2-ynl)amino]prop-1-ynyl derivative of 8-aza-7-deaza-2′-deoxyguanosine (1) was synthesized from 7-iodo-8-aza-7-deaza-2′-deoxyguanosine (7) by Sonogashira cross-coupling and converted into the phosphoramidite building block 10. Oligonucleotides bearing branched side chains with terminal triple bonds were prepared by solid-phase synthesis containing single or multiple residues of 1 as 2′-deoxyguanosine surrogates. T_m measurements demonstrate that compound 1 has a positive effect on duplex stability, which is comparable to the stabilizing effect of the octa-1,7-diynylated non-branched nucleoside 2. Nucleoside 1 and corresponding oligonucleotides were functionalized by the Cu(I)-mediated 1,3-dipolar cycloaddition ‘double click’ reaction with diverse ligands (AZT 3, benzyl azide 4, 11-azidoundecanol 5 and m-dPEG™₄-azide 6). The conjugation reactions were carried out in solution and on solid support. Nucleoside 1 allowed ‘double’ functionalization of a single residue with two reporter groups. The ‘double click’ reaction proceeded smoothly even when two residues of nucleoside 1 were arranged in proximal positions. Hybridization with complementary strands led to a stable oligonucleotide duplex. Molecular modeling indicates that inspite of the crowded steric situation with four AZT ligands within closest proximal positions, all ligands are well accommodated in the major groove not disturbing the DNA helix.     

Nucleoside and oligonucleotide pyrene conjugates with 1,2,3-triazolyl or ethynyl linkers: synthesis,duplex stability,and fluorescence changes generated by the DNA-dye connector

Fluorescent nucleosides and oligonucleotides functionalized with pyrene were synthesized using ‘click’ chemistry or the Sonogashira cross-coupling reaction. The dye was connected to position-7 of 7-deaza-2′-deoxyguanosine or to the 2′-deoxyribofuranose moiety. Four different DNA-dye connectors with 1,2,3-triazolyl residues or triple bonds were constructed. Phosphoramidites of the pyrene conjugates (9, 14, 25) were prepared and used in solid-phase synthesis. Short linkers (2, 4) destabilize DNA, while long linkers (1) increased duplex stability. Nucleosides and oligonucleotides with single dye incorporations show linker dependent fluorescence. Linker dependent excimer emission with pyrenes in proximal positions was also observed. A ‘superchromophore’ formed by the 7-deaza-2′-deoxyguanosine ethynylpyrene conjugate shows strong red shifted fluorescence emission at 495 nm.     

Oligonucleotides containing 7-propynyl-7-deazaguanine: synthesis and base pair stability

Oligonucleotides incorporating the propynyl derivative of 7-deaza-2′-deoxyguanosine (1) were synthesized by solid-phase oligonucleotide synthesis. As building blocks the phosphoramidites 7a,b were prepared. The incorporation of 1 into oligonucleotides exerts a positive effect on the DNA duplex stability. The duplex stabilization by 1 was higher than that of 7-iodo-7-deaza-2′-deoxyguanosine (2b). The stabilizing effect of the 7-propynyl group introduced in the 7-deazapurines is similar to that reported for 8-aza-7-deazapurines. From CD spectra it was deduced that the B-DNA structure is not significantly altered by compound 1.     

Regioselective synthesis of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones by the cyclization of 3-acyl-4-methoxy-1-methylquinolinones with hydrazines

Reaction of 3-acyl-4-methoxy-1-methylquinolinones 2 and 5 with hydrazines has been investigated under different experimental conditions. Compound 2 always gave rise selectively and exclusively to the regioisomeric 1,3-disubstituted- or 2,3-disubstituted-pyrazolo[4,3-c]quinolin-4(5H)one (compounds 3a,b or 4a,b, respectively), while reaction of 5 with N-methylhydrazine led to a mixture of pyrazoles 7a and 8a. With N-phenylhydrazine, compounds 7b or 8b were regioselectively obtained. Compound 8a could be selectively synthesized working in solventless conditions. Structural elucidation of all products was independently achieved by NMR spectroscopy.     

Synthesis of Methylenecyclopropane Analogues of Antiviral Nucleoside Phosphonates

Synthesis of methylenecyclopropane analogues of nucleoside phosphonates 6a, 6b, 7a and 7b is described. Cyclopropyl phosphonate 8 was transformed in four steps to methylenecyclopropane phosphonate 16. The latter intermediate was converted in seven steps to the key Z- and E-methylenecyclopropane alcohols 23 and 24 separated by chromatography. Selenoxide eliminations (15→16 and 22→23+24) were instrumental in the synthesis. The Z- and E-isomers 23 and 24 were transformed to bromides 25a and 25b, which were used for alkylation of adenine and 2-amino-6-chloropurine to give intermediates 26a, 26b, 26c and 26d. Acid hydrolysis provided the adenine and guanine analogues 6a, 6b, 7a and 7b. Phosphonates 6b and 7b are potent inhibitors of replication of Epstein-Barr virus (EBV).     

Synthesis of novel pyrimidine nucleoside analogues owning multiple bases/sugars and their glycosidase inhibitory activity

A series of novel nucleoside analogues having dual bases (pyrimidine and triazole) and sugars have been synthesized by CuAAC reaction of azido sugars with propynylated pyrimidines. These compounds were evaluated for their in vitro α-glucosidase inhibitory activity. In this series, compounds 4b, 4d, 8a, 8b, 8c, 8e, 8g, 8h, and 8i exhibited very good inhibition of α-glucosidase enzyme. Nucleoside analogues 8a, 4b, 8h, and 8c displayed 47.4%, 41.8%, 39.4%, and 34.6% inhibition, respectively, comparable to the standard drug acarbose (53.4%).     

Silylative N-hydroxyalkylation of amide compounds: application to the synthesis of acyclic alditol-based nucleoside analogues

A rare silylative hydroxyalkylation of amide compounds with chiral aldehydes has been developed utilizing a Lewis acid-Lewis base promoter system consisting of an equimolecular mixture of tert-butyldimethylsilyl trifluoromethanesulfonate and N-diisopropylethylamine. This approach culminated in the synthesis of several enantiopure acyclic nucleoside representatives comprising thymidine analogues 6, 7, 9, 10, 12 and 13, uridine analogues 15 and 16, and 6-chloropurine derivatives 18 and 19.     

Structural and stereochemical investigations into bromotyrosine-derived metabolites from southern Australian marine sponges, Pseudoceratina spp.

Chemical investigation of a southern Australian sponge, Pseudoceratina sp., resulted in the isolation of twelve bromotyrosine-derived alkaloids, comprising four new metabolites, aplysamine-7 (1), (−)-purealin B (2), purealin C (3) and purealin D (4); two new spiroisoxazole enantiomers, (−)-purealidin R (5) and (−)-aerophobin-2 (6); five known metabolites (−)-pseudoceratinine A (7), (−)-aeroplysinin-1 (8), aplysamine-2 (9), purpuramine G (10) and purpuramine J (11); and an artifact 12 derived from ethanolysis of 5. Structures for 1-12 were assigned on the basis of detailed spectroscopic analysis. A second southern Australian Pseudoceratina sp. afforded the first recorded account of a racemic bromotyrosine-derived spiroisoxazole, (±)-purealin (13b), together with the known achiral precursor purealidin A (15). A literature review of marine bromotyrosine-derived spiroisoxazoles reaffirmed the published dominance of (+)-spiroisoxazoles, acknowledging several accounts of (−)-spiroisoxazoles, while also revealing a wide range of chiroptical measurements suggestive of variable optical purity. The Pseudoceratina sp. metabolites 1-12, 13b and 15 were assessed for antibiotic properties, with the new metabolites 3 and 13b exhibiting broad spectrum activity against several Gram-positive bacteria.     

(R)-2,3-Cyclohexylideneglyceraldehyde: a novel template for simple entry into both cis- and trans-2,5-disubstituted tetrahydrofurans

Sharpless asymmetric dihydroxylation at the terminal olefin of benzoates 3a and 3b, using both AD-mix α and AD-mix β afforded only one diastereomer of diols 5a and 5b, respectively. Diols 5a and 5b were easily transformed into cis- and trans-2,5-disubstituted tetrahydrofurans 7 and 14, respectively, which were subsequently converted into known compounds 12 and 19.     

Hydrogen bonding-mediated self-assembly of square and triangular metallocyclophanes

Intramolecular three-centered hydrogen bonding has been used to induce dipyridyl anthranilamide 5 and diphenylacetylene anthranilamide 11 to adopt rigid straight conformation. Compound 5 reacted with Pd(dppp)(OTf)₂ (12a) or Pt(dppp)(OTf)₂ (12b) in dichloromethane to afford square metallocyclophanes 13a and 13b in 70% and 40% yield, respectively. In contrast, the reaction of 11 with 12b in dichloromethane gave triangular metallocyclophane 14 in 15% yield.     

Efficient synthesis of various acycloalkenyl derivatives of pyrimidine using cross-metathesis and Pd(0) methodologies

Novel acyclonucleosides (9a-d, 10a-d, 18a,b and 19a,b) have been prepared using Pd(0) and cross-metathesis methodologies. The allylic N-alkylation under Tsuji-Trost conditions was used to introduce the nucleobase, while the Suzuki-Miyaura reaction afforded C-5 substituted uracil analogues. The cross-metathesis performed with a ruthenium catalyst was used to provide new acycloalkenyl nucleosides. The antiviral activities of all final compounds have been evaluated.     

A New Alkylation-Elimination Method for Synthesis of Antiviral Fluoromethylenecyclopropane Analogues of Nucleosides

A new method for the synthesis of fluoromethylenecyclopropane nucleosides by alkylation-elimination procedure is described. Fluorination of methylenecyclopropane carboxylate 6 gave fluoroester 7. Treatment of 7 with phenylselenenyl bromide afforded the desired ethyl (E)-2-bromomethyl-1-fluoro-2-phenylselenenylcyclopropane-1-carboxylate 11 in 85% yield. DIBALH reduction of 11 gave 13, which after acetylation to 14 was reacted with 2-amino-6-chloropurine to give the 9-alkylated product 15 in 87% yield. Se-oxidation of 15 with hydrogen peroxide afforded 16, which underwent smooth elimination in a mixture of THF-DMF at 60 °C giving rise to a Z,E mixture of protected nucleosides 17. Deacetylation gave Z-1a and E-1a which were separated on a silica gel column. Both Z-1a and E-1a were converted into the respective guanine analogues Z-1b and E-1b.     

Synthesis of imidazo[5,1-b]thiazoles or spiro-β-lactams by reaction of imines with mesoionic compounds or ketenes generated from N-acyl-thiazolidine-2-carboxylic acids

New mesoionic compounds (2H, 3H-thiazolo[3,2-c]oxazol-7-ones) (β) or ketenes ((3-acyl-1,3-thiazolidin-2-ylidene)methanone) (β′) were generated from N-acetyl and N-benzoyl-thiazolidine-2-carboxylic acids (7a,b) using different methods, and their reactivity towards N-(phenylmethylene)benzenesulfonamide (2) and N-(phenylmethylene)aniline (3) was tested. When (7a,b) were treated with (2) and acetic anhydride in refluxing toluene solution, only imidazo[5,1-b]thiazoles (8a,b) were obtained from the mesoionic compound intermediates (β). When the ketene intermediates (β′) were generated from (7a,b) by means of Mukaiyama's reagent, only spiro-β-lactams (9a,b) were isolated.     

Methylene-2-ethynylcyclopropanes: synthesis and biological activity of (Z)- and (E)-9-{[2-ethynyl-2-(hydroxymethyl)cyclopropylidene]methyl}adenine and -guanine

Synthesis of methylene-2-ethynylcyclopropane analogues of nucleosides 12a, 12b, 13a, and 13b is described. Ethyl methylenecyclopropane carboxylate 14 was hydroxymethylated to give alcohol 15, which was reduced to diol 16. Selective protection with tert-butyldimethylsilyl group gave derivative 17, which was oxidized to aldehyde 18. Wittig reaction with CBr₄ gave dibromoalkene 19. Elimination of both bromine atoms afforded methylene-2-ethynylcyclopropane 20. Bromoselenenylation using N-bromosuccinimide and diphenyldiselenide gave intermediate 21. Alkylation of adenine and 2-amino-6-chloropurine with 21 provided the Z,E-isomeric mixtures 22a and 22c. Oxidation afforded selenoxides 23a and 23c. Mild thermolysis furnished methylenecyclopropanes Z-24a, E-24a, and 24c. Deprotection and separation of Z,E-isomers gave adenine analogues 12a and 13a, and 2-amino-6-chloropurine intermediates 12c and 13c. Hydrolytic dechlorination of 12c and 13c afforded guanine analogues 12b and 13b. Adenine Z-isomer 12a inhibits replication of Epstein-Barr virus through its cytotoxicity. The E-isomer 13a is a substrate for adenosine deaminase.     

Synthesis of novel 4′-C-methyl-1′,3′-dioxolane pyrimidine nucleosides and evaluation of its anti-HIV-1 activity

The key glycosyl donor for the target molecule 12 was prepared by two-step sequences; (1) acetalization of tert-butyldimethylsilyloxyacetaldehyde with 3-bromopropanediol, (2) DBN-initiated β-elimination of the resulting 2-(tert-butyldimethylsilyloxy)methyl-4-bromomethyl-1,3-dioxolane 11. Electrophilic glycosidation between 12 and silylated pyrimidine nucleobase proceeded efficiently to provide a mixture of β- and α-anomers of the respective glycosides 14 and 15. Tin radical-mediated reduction of the bromomethyl functional group of 14 and 15 gave protected 4′-C-methyl-dioxorane uracil- 16 and thymine nucleoside 17. The respective cytosine nucleoside 18 was synthesized from 16. De-silylation of 4′-methyl-1′,3′-dioxolane pyrimidine nucleosides 16–18 gave the target molecules. Evaluation of the anti-HIV-1 activity of the β- and α-anomers of the novel 4′-C-methyl-1′,3′-dioxolane nucleosides 22β,α–24β,α revealed that none of the nucleoside derivatives possess anti-viral activity against HIV-1 and show cytotoxicity against MT-4 cells at 100 μM.     

Syntheses of 4′-spirocyclic phosphono-nucleosides as potential inhibitors of hepatitis C virus NS5B polymerase

To discover novel nucleosides as potential antiviral agents, 4′-spirocyclic phosphono-nucleosides were designed to mimic the monophosphate of R-1479, a known nucleoside inhibitor of HCV NS5B. Bypassing the first kinase step to nucleoside monophosphate is viewed as advantageous since this phosphorylation is often observed as the rate-limiting transformation to the active NTP for many nucleosides. Efficient synthetic routes were developed with a triphenylphosphine–iodine cyclization reaction as the key step to form the tetrahydrofuran 4′-spirocycle. The desired 4′-spirocyclic phosphono-cytidine analogs 12a, 12b, and 16 were prepared in 11 steps.     

Synthesis, IR-, NMR- and X-ray investigations on some novel N-hetaryl-dihydro-pyrazolyl ferrocenes. Study on ferrocenes, part 16

Cyclocondensation of 1-aryl-3-ferrocenyl-2-propen-1-ones (1) with hetaryl hydrazines resulted in N-hetaryl-3-aryl-5-ferrocenyl pyrazolines (3, 4). The analogous 3-aryl-1-ferrocenyl-2-propen-1-ones (5) gave the isomeric N-hetaryl-5-aryl-3-ferrocenyl-pyrazolines (6, 10), but in lower yield. The reaction of aryl-chalcones (7) with 4-hydrazino-phthalazinone led to 3,5-bis-aryl-N-hetaryl-pyrazolines (8) or to the corresponding ene-hydrazones (9). The structure of the new compounds was established by IR, ¹H and ¹³C NMR spectroscopy, including DNOE, HMQC, HMBC and DEPT methods. For compounds 1b, 3b and 8b the stereo structure was elucidated also by X-ray diffraction.     

Cycloaddition of methyl 2-(2,6-dichorophenyl)-2H-azirine-3-carboxylate to electron-rich 2-azadienes

tert-Butyldimethylsililoxy-2-aza-1,3-butadienes react with 2H-azirine 3 leading to Diels-Alder cycloadducts in moderate yields. The reactions are endo- and regioselective with the azirine being added by its less hindered face. There is only one product in the case of 1b, 4b. There are two isomers (4 and 5) from 1a, 1c and 1d. A different result was obtained with the diene 1e. Diene 1e formed products 4e and 8. Some of compounds 4 and 5 have been hydrolysed leading to functionalised aziridines 7. Compound 8 gave aziridine 9.