Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.     

Design of novel antimicrobial peptide dimer analogues with enhanced antimicrobial activity in vitro and in vivo by intermolecular triazole bridge strategy

Currently, antimicrobial peptides have attracted considerable attention because of their broad-sprectum activity and low prognostic to induce antibiotic resistance. In our study, for the first time, a series of side-chain hybrid dimer peptides J-AA (Anoplin-Anoplin), J-RR (RW-RW), and J-AR (Anoplin-RW) based on the wasp peptide Anoplin and the arginine- and tryptophan-rich hexapeptide RW were designed and synthesized by click chemistry, with the intent to improve the antimicrobial efficacy of peptides against bacterial pathogens. The results showed that all dimer analogues exhibited up to a 4–16 fold increase in antimicrobial activity compared to the parental peptides against bacterial strains. Furthermore, the antimicrobial activity was confirmed by time-killing kinetics assay with two strains which showed that these dimer analogues at 1, 2 × MIC were rapidly bactericidal and reduced the initial inoculum significantly during the first 2–6 h. Notably, dimer peptides showed synergy and additivity effects when used in combination with conventional antibiotics rifampin or penicillin respectively against the multidrug-resistant strains. In the Escherichia coli-infected mouse model, all of hybrid dimer analogues had significantly lower degree of bacterial load than the untreated control group when injected once i.p. at 5 mg/kg. In addition, the infected mice by methicillin-resistant (MRSA) strain could be effectively treated with J-RR. All of dimer analogues had membrane-active action mode. And the membrane-dependent mode of action signifies that peptides functions freely and without regard to conventional resistant mechanisms. Circular dichroism analyses of all dimer analogues showed a general predominance of α-helix conformation in 50% trifluoroethanol (TFE). Additionally, the acute toxicities study indicated that J-RR or J-AR did not show the signs of toxicity when adult mice exposed to concentration up to 120 mg/kg. The 50% lethal dose (LD₅₀) of J-AA was 53.6 mg/kg. In conclusion, to design and synthesize side chain-hybrid dimer analogues via click chemistry may offer a new strategy for antibacterial therapeutic option.     

Synthesis and Anti-HIV Activity of Novel 2′-Deoxy-2′-β-Fluoro-Threosyl Nucleoside Phosphonic Acid Analogues

Novel 2′-deoxy-2′-β-fluoro-threose purine phosphonic acid analogues were designed and racemically synthesized from 2-propanone-1,3-diacetate. Condensation successfully proceeded from a glycosyl donor 9 under Vorbrüggen conditions. Cross-metathesis of vinyl analogues 13 and 23 with diethyl vinylphosphonate yielded the desired nucleoside phosphonate analogues 14 and 24, respectively. Ammonolysis and hydrolysis of phosphonates yielded the nucleoside phosphonic acid analogues 16, 19, 26, and 29. The synthesized nucleoside analogues were subjected to antiviral screening against human immunodeficiency virus (HIV)-1. Adenine analogue 18 exhibited weak in vitro activities against human immunodeficiency virus (HIV)-1.     

SYNTHESIS OF HALOGEN-SUBSTITUTED 3-DEAZAADENOSINE AND 3-DEAZAGUANOSINE ANALOGUES AS POTENTIAL ANTITUMOR/ANTIVIRAL AGENTS

Various 2-halogen-substituted analogues (38, 39, 43 and 44), 3-halogen- substituted analogues (51 and 52), and 2′, 3′-dihalogen-substituted analogues (57–60) of 3-deazaadenosine and 3-halogen-substituted analogues (61 and 62) of 3-deazaguanosine have been synthesized as potential anticancer and/or antiviral agents. Among these compounds, 3-deaza-3-bromoguanosine (62) showed significant cytotoxicity against L1210, P388, CCRF-CEM and B₁₆F₁₀ cell lines in vitro, producing IC₅₀ values of 3, 7, 9 and 7,μM, respectively. Several 3-deazaadenosine analogues (38, 51, 57 and 59) showed moderate to weak activity against hepatitis B virus.

     

Synthesis of Novel 2′-Spirocyclopropyl-5′- Deoxyphosphonic Acid Furanosyl Nucleoside Analogues as Potent Antiviral Agents

Novel 5′-deoxyfuranosyl purine phosphonic acid analogues with 2 ′-electropositive moiety, such as spirocyclopropanoid, were designed and synthesized from commercially available diethyl malonate. Condensation reaction successfully proceeded from a glycosyl donor 15 at low reaction temperature in Vorbruggen conditions to give desired phosphonate analogues 16b and 23b. The synthesized nucleotide analogues 19, 22, 26, and 29 were subjected to antiviral screening against HIV-1. Adenine phosphonic acid analogue 22 shows significant anti-HIV activity (EC₅₀ = 7.9 μM).     

Synthesis Of 2,2,3-Tris(Hydroxymethyl)Methylenecyclopropane Analogues Of Nucleosides

Synthesis of 2,2,3-tris(hydroxymethyl)methylenecyclopropane analogues 16a, 16b, 17a, and 17b is described. Diethyl ester of Feist's acid 18b was hydroxymethylated via carbanion formation using formaldehyde under simultaneous isomerization to cis diester to give intermediate 19. Reduction followed by acetylation gave triacetate 22. Addition of bromine afforded reagent 23, which was used for alkylation-elimination of adenine and 2-amino-6-chloropurine to provide Z,E-isomeric mixtures of 24a and 24b. Deacetylation and separation furnished the Z-isomers 16a, 16c and E-isomers 17a, 17c. Hydrolytic dechlorination of 16c and 17c gave guanine analogues 16b and 17b. None of the analogues exhibited a significant antiviral activity. Adenosine deaminase is refractory toward adenine analogues 16a and 17a.     

Efficient Electrophilic Fluorination for the Synthesis of Novel 2′-Fluoro-3′-Methyl-5′-Deoxyphosphonic Acid Apiosyl Nucleoside Analogues

Novel 5′-deoxyapiosyl purine phosphonic acid analogues with a 2′-electropositive moiety, such as, a fluorine atom were designed and synthesized from commercially available hydroxylacetone. Condensation of a glycosyl donor 10 with purines under Vorbruggen conditions and cross-metathesis give the desired nucleoside phosphonic acid analogues 14, 17, 21, and 24. The synthesized nucleoside analogues were subjected to antiviral screening against HIV-1, and the adenine analogue 17 exhibited weak in vitro anti-HIV-1 activity (EC₅₀ = 26.6 μM)     

Synthesis of various 5-alkoxymethyluracil analogues and structure-cytotoxic activity relationship study

A number of 5-alkoxymethyluracil analogues were synthesized to evaluate their cytotoxic activity. 5-Alkoxymethyluracil derivatives 1 were prepared via known nucleophilic substitution of 5-chloromethyluracil 5 and subsequently transformed to their corresponding nucleosides 2. All prepared compounds were submitted to cytotoxic activity testing against drug sensitive and drug resistant leukaemia cells and solid tumour derived cell lines. In addition, the cytotoxic activity of 5-alkoxymethyluracil analogues 1 and 2 was compared with the previously published 5-[alkoxy(4-nitrophenyl)methyl]uracil analogues 3 and 4. Extensive structure–cytotoxic activity relationship studies are reported.     

Synthesis and Evaluation of a Series of 2′-Deoxy Analogues of The Antiviral Agent 5,6-Dichloro-2-Isopropylamino-1-(β-L-Ribofuranosyl)-1H-Benzimidazole (1263W94)

Abstract

A series of 2′-deoxy analogues of the antiviral agent 5,6-dichloro-2-isopropylamino-1-(β-L-ribofuranosyl)-1H-benzimidazole (1263W94) were synthesized and evaluated for activity against human cytomegalovirus (HCMV) and for cytotoxicity. The 2-substituents in the benzimidazole moiety correspond to those that were used in the 1263W94 series. In general, as was found in the 1263W94 series, cyclic and branched alkylamino groups were needed for potent activity against HCMV. Three analogues 3a, 3b and 3d were as potent as 1263W94. Further evaluation of two analogues, 3a and 3b, suggested that these 2′-deoxy analogues may act via a novel mechanism of action similar to that of 1263W94. These 2′-deoxy analogues generally lacked cytotoxicity in vitro. Pharmacokinetic parameters in mice and protein binding properties of 3a were quite similar to 1263W94. However, the oral bioavailability of 3a was only half of that observed for 1263W94.     

Review ArticleNaphthalene Combretastatin Analogues: Synthesis,Cytotoxicity and Antitubulin Activity

Synthesis and evaluation of new combretastatin analogues with varied modifications on the bridge and the aromatic rings, have shown that the 2-naphthyl moiety is a good surrogate for the 3-hydroxy-4-methoxyphenyl (B-ring) of combretastatin A-4. Other bicyclic systems, such as 6(7)-quinolyl and 5-indolyl, can replace the B-ring, but they produce less potent analogues in the cytotoxicity and tubulin polymerization inhibition assays. Other modifications are detrimentral for the potency of the studied analogues. The 2-naphthyl combretastatin 53 and the related 6-quinolyl combretastatin 106 analogues are the most potent among the derivatives of this type, whereas 92 and 95 are the most potent among the naphthalene derivatives with a heterocycle in the bridge. Previous and new results in this family of combretastatin analogues are discussed.     

Structure-Activity Relationships Among a New Class of Antiviral Heterosubstituted 2′,3′-Dideoxynucleoside Analogues

Abstract

3′-Oxa-4′-thiocytidine nucleoside analogues 14–17 were prepared from oxathiolanes 10 and 11, and evaluated for activity against HIV-1 and HBV in vitro. The nucleoside analogues were found to possess potent activities against HIV-1 in a panel of cell lines. Compound 16 is moderately active against HBV in 2.2.15 cells.     

Pyrazolopyridines as a novel structural class of potent and selective PDE4 inhibitors

Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase 4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred substituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4 active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhibits LPS induced TNF-α production from isolated human peripheral blood mononuclear cells and has an encouraging rat PK profile suitable for oral dosing.     

Phosphodiester Amidates of Unsaturated Nucleoside Analogues as Anti-HIV Agents

Abstract

Lipophilic phosphodiester L-alaninates of acyclic unsaturated nucleoside analogues 1d, 1e, 2d, 2e, 3d, 3e, 4d and 5d were prepared and their antiretroviral activity was examined in ATH8 cell culture infected with HIV-1. A possible mechanism of action of these analogues is discussed.     

The role of rapid-acting insulin analogues and inhaled insulin in type 2 diabetes mellitus

sBackground:The availability of rapid-acting insulin analogues and inhaled insulin gives clinicians additional treatmentoptions in the management of patients with diabetes mellitus (DM). Combining rapid-acting insulin analogues with basal insulin can more closely mimic physiologic insulin release to maximize glycemic control.Objective:The objective of this article was to discuss the role of rapid-acting insulin analogues and inhaled insulin inthe treatment of patients with type 2 DM.Methods:Materials for this article were obtained through an online search of MEDLINE/PubMed and Google(1996-2006) using the search terms bolus insulin, postprandial, rapid-acting insulin analogues, titration, hypoglycemia, glycemic control, inhaled insulin, and insulins lispro, aspart, and glulisine.Results:Glycosylated hemoglobin (A1C) levels and number of all hypoglycemic episodes were similar in patients withtype 2 DM taking either mealtime rapid-acting insulin analogues or regular human insulin (RHI). Rapid-acting insulins have been successfully used in basal-bolus regimens with a variety of long- and intermediate-acting insulins, as well as with oral hypoglycemic agents. Injectable rapid-acting insulin analogues markedly decreased postprandial glucose (PPG) levels compared with RHI. Better reduction in PPG levels may be key to achieving target A1C levels in some patients, but long-term outcome studies are needed to assess whether lowering PPG levels decreases cardiovascular risk in patients with type 2 DM. Inhaled insulin may be an option for patients who cannot inject insulin, but route of administration and dosing issues limit its use in many patients. The effect of inhaled insulin on PPG is unclear at this time.Conclusions:Although rapid-acting insulin analogues are effective in the management of patients with type 2 DM, the limited numbers of studies have yet to demonstrate that these agents have any significant long-term advantage compared with RHI. In addition, they cost more than RHI. Further studies are needed to compare the efficacy of the rapid-acting insulin analogues, to compare the different dosing regimens used with mealtime insulin administration, and to ascertain if the decrease in PPG levels seen with the use of rapid-acting insulin analogues translates into improved glycemic control and perhaps even a reduction in cardiovascular risk in patients with type 2 DM. (Insulin. 2007;2:61-67) Copyright 2007 Excerpta Medica, Inc.     

Design,Synthesis and Anti-HIV Activity of Homologous PMEA Derivatives

This article describes an efficient route for synthesizing novel cyclopropyl homologous PMEA analogues. The condensation of the bromide 8 with nucleosidic bases (A, U, T, C, 5-FU, G) under standard nucleophilic substitution and deprotection conditions, afforded the target phosphonic acid analogues 14～18 and 21. These compounds were evaluated for their potential antiviral properties against various viruses. Guanine derivative 21 showed significant antiviral activity.     

Formation of two centre three electron bond by hydroxyl radical induced reaction of thiocoumarin: evidence from experimental and theoretical studies

Radiation chemical studies of thioesculetin (1), a thioketone derivative of coumarin, were performed by both pulse radiolysis technique and DFT calculations. Hydroxyl (^?OH) radical reaction with 1 resulted transients absorbing at 320, 360 and 500?nm. To identify the nature of the transients, the reaction was studied with specific one-electron oxidant (N₃^?) radical, where 360?nm band was absent. The transient absorption at 500?nm was concentration-dependent. The overall impression for ^?OH radical reaction was that the transient absorbing at 320, 360 and 500?nm was due to sulphur centred monomer radical, hydroxysulfuranyl and dimer radical of 1 respectively. The equilibrium constant between the monomer to dimer radical was 3.75?×?10⁴ M^?1. From the transients’ redox nature, it was observed that 57 and 24% of ^?OH radical yielded to oxidising and reducing products respectively. Further, the product analysis by HPLC suggested that the dimer radical disproportionate to esculetin and thioesculetin. DFT energy calculation for all the possible transients revealed that dimer radical has the lowest energy. The HOMO of 1 and its monomer radical suggested that the electron density was localised on the sulphur atom. The bond length between the two sulphur atoms in dimer radical was 2.88 Å which was less than the van der Waals distance. Bond order between the two sulphur atoms was 0.55, suggesting that the bond was two centre three electron (2c–3e). From TD-DFT calculation, the electronic transition of dimer radical was at 479?nm which was in close agreement with the experimental value. The nature of the electronic transition was σ → σ* from a 2c???3e bond.     

Synthesis and Anti-HIV Evaluation of 7-Deaza Analogues of Carbovir

Abstract

Three analogues of Carbovir 1 have been synthesized and evaluated for antiviral activity in vitro. Anti-HIV-1 and anti-HIV-2 activities have been observed with 7-deaza analogues 3 and 5 of 1. Compound 5 was about ten times more potent than 3 against HIV-1 and HIV-2 on different cell lines.     

Synthesis and anti-HCV Evaluation of 4′(α)-ethyl and 2′(β)-methyl-carbodine Analogues

Novel 4′(α)-ethyl-2′(β)-methyl carbocyclic nucleoside analogues have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The construction of cyclopentene intermediate 12β was successfully made via sequential Johnson-Claisen orthoester rearrangement and ring-closing metathesis (RCM) starting from Weinreb amide 5. Selective dihydroxylation and desilylation gave the target carbodine analogues. The synthesized nucleoside analogues mentioned above 18 and 19 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides neither showed any significant antiviral activity nor toxicity up to 50 μM.     

Temperature-jump and voltage-jump experiments at planar lipid membranes support an aggregational (micellar) model of the gramicidin A ion channel

Summary The kinetics of formation and dissociation of channels formed by gramicidin A and two analogues in planar lipid membranes was studied using a laser temperature-jump technique developed earlier [Brock, W., Stark, G., Jordan, P.C. (1981),Biophys. Chem. 13:329–348]. The time course of the electric current was found to agree with a single exponential term plus a linear drift. In case of gramicidin A the relaxation time was identical to that reported for V-jump experiments [Bamberg, E., Läuger, P. (1973),J. Membrane Biol. 11:177–194], which were interpreted on the basis of a dimerization reaction. The same results were obtained for gramicidin A and for chemically dimerized malonyl-bis-desformylgramicidin. It is therefore suggested that the dimerization represents a parallel association of two dimers to a tetramer. There is evidence that the tetramer, contrary to the presently favored dimer hypothesis, is the smallest conductance unit of an active gramicidin channel. An additional V-jump-induced relaxation process of considerably larger time constant is interpreted as a further aggregation of gramicidin dimers.Abbreviations GA gramicidin A - OPG O-pyromellitylgramicidin A - MBDG malonyl-bis-desformylgramicidin     

Synthesis of a Carboxamide Linked UBr∗UBr Dimer–Duplex and Triplex Stabilities of the Corresponding Oligodeoxynucleotides

Abstract

A U^Br?U^Br dimer was synthesized by connecting the appropiate nucleoside monomers through a 5-atom carboxamide linkage. The dimer was incorporated in oligodeoxynucleotides and investigated for hybridization properties toward single and double stranded DNA.