Duplex Stability of Oligonucleotides Containing 7-Substituted 7-Deaza- and 8-Aza-7-Deazapurine Nucleosides

Abstract

The synthesis of 7-substituted 7-deaza- and 8-aza-7-deazapurine 2′-deoxyribonucleosides, their incorporation into oligonucleotides, and the stability of corresponding duplexes is described.     

Propynyl groups in duplex DNA: stability of base pairs incorporating 7-substituted 8-aza-7-deazapurines or 5-substituted pyrimidines

Oligonucleotides incorporating the 7-propynyl derivatives of 8-aza-7-deaza-2′-deoxyguanosine (3b) and 8-aza-7-deaza-2′-deoxyadenosine (4b) were synthesized and their duplex stability was compared with those containing the 5-propynyl derivatives of 2′-deoxycytidine (1) and 2′-deoxyuridine (2). For this purpose phosphoramidites of the 8-aza- 7-deazapurine (pyrazolo[3,4-d]pyrimidine) nucleosides were prepared and employed in solid-phase synthesis. All propynyl nucleosides exert a positive effect on the DNA duplex stability because of the increased polarizability of the nucleobase and the hydrophobic character of the propynyl group. The propynyl residues introduced into the 7-position of the 8-aza-7-deazapurines are generally more stabilizing than those at the 5-position of the pyrimidine bases. The duplex stabilization of the propynyl derivative 4b was higher than for the bromo nucleoside 4c. The extraordinary stability of duplexes containing the 7-propynyl derivative of 8-aza-7- deazapurin-2,6-diamine (5b) is attributed to the formation of a third hydrogen bond, which is apparently not present in the base pair of the purin-2,6-diamine 2′-deoxyribonucleoside with dT.     

The influence of modified purine bases on the stability of parallel DNA

The stability of the parallel-stranded (ps) DNA duplexes is increased when the dA-residues are replaced by the 7-substituted 7-deaza-2'-deoxyadenosine derivatives 3a,b or the dG-residues by the 8-aza-7-deazapurine 2'-deoxynucleosides 6 and 7a,b. Also the N-7-glycosylated adenine 5 forms stable base pairs in ps-DNA while it destabilizes oligonucleotide duplexes with antiparallel chain orientation. The presence of a 2-amino group as in compound 4b is critical for the DNA-structure, leading to a much greater destabilization of the ps-hybrids than of aps-DNA.     

7-SUBSTITUTED 8-AZA-7-DEAZAPURINES AND 2,8-DIAZA-7-DEAZA-PURINES: SYNTHESIS OF NUCLEOSIDES AND OLIGONUCLEOTIDES

7-Substituted 8-aza-7-deazaadenosines 1a–e were synthesized by Sonogashira cross coupling from the corresponding 7-iodo nucleoside in 36–79% yields. Starting from 7-bromo (or 7-iodo)-8-aza-7-deazaadenine, 2a,b were obtained by acid-catalyzed glycosylation followed by deprotection in 53 and 35% yields, repectively. Compounds 2b was applied to cross coupling reaction to give 2c-d in 34–95% yield. Compounds 2a and 4b were further transformed to the phosphoramidites 5 and 6b in 9 and 49% overall yields, which were incorporated into oligonucleotides.     

8-Aza-1-deazapurine Nucleosides as Antiviral Agents

Abstract

2′,3′-Dideoxy-8-aza-1-deazaadenosine (21) and its α-anomer (20) were synthesized via glycosylation of 7-chloro-3H-1,2,3-triazolo[4,5-b]pyridi-ne with 2,3-dideoxy-5-O-[(1, 1)-dimethylethyl)diphenylsilyl]-D-glycero-o-pen-tofuranosyl chloride. The reaction gave a mixture of α- and β-anomers of N³-, N⁴- and N¹-glycosylated regioisorners (12–15). The α- and β-anomers of the N⁴-glycosylated isomer 26 and 27 were also synthesized through the glycosylation of 8-aza-1-deazaadenine with 1-acetoxy-2,3-dideoxy-5-O-f(1,1-di-methylethyl)dimethylsilyl]-D-glycero-pentouranose. These dideoxynucleo-sides and a series of previously synthesized 8-aza-1-deazapurine nucleosidcs were tested for activity against several DNA and RNA viruses, HIV-1 included. The α- and β-anomers of 7-chloro-3-(2-deoxy-D-erythro-pentofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (3a and 4) showed activities against Sb-1 and Coxs viruses. The α- and β-anomers of 2′,3′-dideoxy-8-aza-1-deazaadenosine (20 and 21) were found active as inhibitors of adenosine deaminase.     

Synthesis of 7-trifluoromethyl-7-deazapurine ribonucleoside analogs and their monophosphate prodrugs

Abstract

Novel 7-trifluoromethyl-7-deazapurine ribonucleoside analogs (13a-c) and their Protides (15a-c) were successfully synthesized from ribolactol or 1-α-bromo-ribose derivatives using Silyl-Hilbert-Johnson or nucleobase-anion substitution reactions followed by key aromatic trifluoromethyl substitution. Newly prepared compounds were evaluated against a panel of RNA viruses, including HCV, Ebola or Zika viruses.     

Mismatch discrimination of base-modified nucleic acids and their constituents: non-Watson-Crick base pairing induced by tautomerization

The effect of tautomerism on the mismatch discrimination was studied on 7-deazapurine and 8-aza-7-deazapurine analogues of isoguanosine. 7-Halogenated 7-deaza-2'-deoxyisoguanosines show better base pair discrimination than 2'-deoxyisoguanosine due to the more favored keto tautomer formation. 8-Aza-7-deazaisoguanosine and its 7-halogeno derivatives also show higher keto tautomer population than that of isoguanosine, but the 7-halogens do not bias the tautomeric equilibrium significantly as it is observed for the 7-deaza-2'-deoxyisoguanosine derivatives.     

Synthesis of non-purine analogs of 6-aryl-9-benzylpurines,and their antimycobacterial activities. Compounds modified in the imidazole ring

Purine analogs modified in the five-membered ring have been synthesized and examined for antibacterial activity against Mycobacterium tuberculosis H₃₇Rv in vitro employing the microplate alamar blue assay (MABA). The 9-deaza analogs were only found to be weak inhibitors, but the 8-aza-, 7-deaza- and 8-aza-7-deazapurine analogs studied displayed excellent antimycobacterial activities, some even substantially better than the parent purine. In the 7-deazapurine series, MIC values between 0.08 and 0.35 μM, values comparable or better than the reference drugs used in the study (MIC rifampicin 0.09 μM, MIC isoniazid 0.28 μM and MIC PA-824 0.44 μM). The five most active compounds were also examined against a panel of drug-resistant Mtb strain, and they all retained their activity. The compounds examined were significantly less active against M. tuberculosis in a state of non-replicating persistence (NRP). MIC in the low-oxygen-recovery assay (LORA) ?60 μM. The 7-deazapurines were somewhat more toxic towards mammalian cells, but still the selectivity indexes were excellent. The non-purine analogs exhibit a selective antimycobacterial activity. They were essentially inactive against Staphylococcus aureus and Escherichia coli.     

8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE: SYNTHESIS AND PROPERTIES OF NUCLEOSIDES AND OLIGONUCLEOTIDES WITH NUCLEOBASES LINKED AT POSITION-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N⁸-(2′-deoxy-ribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C⁸-(2′-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2′-deoxyisoguanosine.     

The base pairing properties of 8-aza-7-deaza-2'-deoxyisoguanosine and 7-halogenated derivatives in oligonucleotide duplexes with parallel and antiparallel chain orientation

The base pairing properties of oligonucleotide duplexes containing 8-aza-7-deaza-2′-deoxyisoguanosine, its 7-bromo or its 7-iodo derivative are described. The nucleosides were synthesized on a convergent route, protected and converted into phosphoramidites. Oligonucleotides were prepared on a solid-phase and were hybridized to yield duplexes with parallel (ps) or antiparallel (aps) chain orientation. The 8-aza-7-deaza-2′-deoxyisoguanosine-containing duplexes show almost identical base pairing stability as those containing 2′-deoxyisoguanosine, while the 7-substituted derivatives induce a significant duplex stabilization both in ps and aps DNA. Self-complementary duplexes with parallel chain orientation are exceptionally stable due to the presence of 5′-overhangs. The bulky halogen substituents were found to be well accommodated in the grooves both of aps and ps DNA.     

Parallel-Stranded DNA Formed by New Base Pairs Related to the Isoguanine-Cytosine or Isocytosine-Guanine Motifs

Abstract

Parallel-stranded (ps) oligonucleotide duplexes containing several new base pairs formed between 7-deazaisoguanine and cytosine, 8-aza-7-deaza-isoguanine and cytosine, and 5-aza-7-deaza guanine and guanine are described. The stability of the pshybrids increased if the duplex contains 8-aza-7-deazaisoguanine instead of isoguanine and is decreased by 7-deazaisoguanine incorporation. The purine-purine base pair between 5-aza-7-deazaguanine and guanine was found to be more stable than that of 5-methylisocytosine with guanine.     

PARALLEL DNA CONTAINING PYRAZOLO[3,4-D]PYRIMIDINE ANALOGUES OF ISOGUANINE

The phosphoramidites of 8-aza-7-deaza-2′-deoxyisoguanosine (1a) and its bromo derivative 1b as well as of 6-aza-2′-deoxyisocytidine and its 5-methyl derivative (3a,b) were synthesized. Parallel-stranded duplexes containing the nucleosides 1a,b show a significantly enhanced duplex stability compared to those containing 2′-deoxyisoguanosine.     

7-DEAZAPURIN-2,6-DIAMINE AND 7-DEAZAGUANINE: SYNTHESIS AND PROPERTY OF 7-SUBSTITUTED NUCLEOSIDES AND OLIGONUCLEOTIDES

The synthesis of 7-substituted 7-deazaguanine and 7-deazaadenine ribonucleosides 1–2, the incorporation of 3a–d into oligonucleotides, and the stability of the corresponding duplexes and base discrimination are described. The pK_a values of 3–4 are determined.     

7-Aminobutynyl-8-Aza-7-Deazahypoxanthine as a Quasi-Universal Nucleobase

Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C～X:A～X:T > X:G. The improvement in PCR performance directly correlated with primer's T_m. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3′ end.     

7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7IdTP incorporation during PCR-amplification and protection from endodeoxyribonuclease hydrolysis.

The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c7Gd), -adenosine (c7Ad) and -inosine (c7Id). c7GdTP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c7AdTP) and (c7IdTP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c7GdTP much better than c7AdTP or c7IdTP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phosphodiester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c7Gd is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae III, Rsa I, Hind III, Pvu II or Taq I) do still hydrolyze the modified DNA. c7Ad protects DNA less efficiently, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis.     

Preparation of Oligonucleotides Containing Non-Natural Base Analogs.

Abstract

The preparation of oligonucleotides containing 5-amino-2′-deoxyuridine, 5-Nacetamido- 2′-deoxyuridine, 5-aza-2′-deoxycytidine and N²-substituted guanosine derivatives is described. In each case selection of the appropriate protective group, synthesis and deprotection conditions is discussed.     

Small molecule purine and pseudopurine derivatives: synthesis,cytostatic evaluations and investigation of growth inhibitory effect in non-small cell lung cancer A549

Novel halogenated purines and pseudopurines with diverse aryl-substituted 1,2,3-triazoles were prepared. While p-(trifluoromethyl)-substituted 1,2,3-triazole in N-9 alkylated purine and 3-deazapurine was critical for strong albeit unselective activity on pancreatic adenocarcinoma cells CFPAC-1,1-(p-fluorophenyl)-1,2,3-triazole derivative of 7-deazapurine showed selective cytostatic effect on metastatic colon cancer cells SW620. Importantly, 1-(p-chlorophenyl)-1,2,3-triazole-tagged benzimidazole displayed the most pronounced and highly selective inhibitory effect in nM range on non-small cell lung cancer A549. This compound revealed to target molecular processes at the extracellular side and inside the plasma membrane regulated by GPLD1 and growth factor receptors PDGFR and IGF-1R leading to the inhibition of cell proliferation and induction of apoptosis mediated by p38 MAP kinase and NF-κB, respectively. Further optimisation of this compound as to reduce its toxicity in normal cells may lead to the development of novel agent effective against lung cancer.     

Design and optimisation of orally active TLR7 agonists for the treatment of hepatitis C virus infection

The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.     

Anticancer Activities of 7-, and 9-Substituted 3-Deazaguanine Derivatives

Abstract

Interesting differences in anticancer activities of 7- and 9-substituted derivatives of 3-deazaguanine are described.     

Phosphoramidate pronucleotides of cytostatic 6-aryl-7-deazapurine ribonucleosides

A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2′,3′-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.