Synthesis and anti-HIV activities of bis-(cycloSaligenyl) pronucleotides derivatives of 3′-fluoro-3′-deoxythymidine and 3′-azido-3′-deoxythymidine

Anti-HIV nucleoside monophosphates have limited cellular uptake due to the presence of negatively-charged phosphate group. Bis-(cycloSaligenyl) derivatives containing two anti-HIV nucleosides, 3′-fluoro-3′-deoxythymidine (FLT) and 3′-azido-3′-deoxythymidine (AZT) were synthesized to increase intracellular delivery of nucleoside monophosphates. 2,5-Bis(hydroxymethylene)benzene-1,4-diol was selected as a monocyclic bidentate scaffold and synthesized by three different methods from bis(hydroxymethylene)cyclohexan-1,4-diene-1,4-diol, or diethyl 2,5-dihydroxyterephthalate. The reaction of the tetraol with diisopropylphosphoramidous dichloride in the presence of 2,6-lutidine, followed by conjugation reactions with nucleosides (i.e., FLT and AZT) and oxidation afforded symmetrical and unsymmetrical bis-(cycloSaligenyl) diphosphate triester products, AZT-AZT, FLT-FLT, and FLT-AZT conjugates, in 63-74% overall yields and modest anti-HIV activities (IC₅₀ = 2.8-69.6 μM).     

On the design of polymeric 5′-O-ester prodrugs of 3′-azido-2′,3′-dideoxythymidine (AZT)

A new 5′-O-AZT prodrug was synthesized by conjugating 3′-azido-2′,3′-dideoxythymidine (AZT) with poly(oxyethylene H-phosphonate) at room temperature under Atherton-Todd reaction conditions. The acute toxicity of poly(5′-O-AZT-oxyethylene phosphate) was reduced significantly in comparison with non-immobilized AZT.     

Dinucleotides containing two allyl groups by combinations of allyl phosphotriesters, 5-allyl-, 2′-O-allyl- and 2′-arabino-O-allyl uridine derivatives as substrates for ring-closing metathesis

Five different dinucleotides, each containing two allyl groups in various positions, were prepared and studied as substrates for ring-closing metathesis reactions. These dinucleotides were designed from appropriate nucleoside building blocks combining four different positions for the allyl group; the allyl phosphotriester linkage, 5-allyl-2′-deoxyuridine, and ribo- as well as arabino-configured 2′-O-allyluridine. Thus, convenient procedures for these building blocks were developed. From the dinucleotides, two new cyclic nucleotide structures were obtained; one connecting two adjacent nucleobase moieties and the other forming an unsaturated four-carbon linkage between the phosphate moiety and the adjacent pyrimidine nucleobase. The latter cyclic dinucleotide was also prepared with a saturated four-carbon linkage using a tandem ring-closing metathesis-hydrogenation procedure. This compound was found to be significantly more stable towards a nucleophilic ring-opening than its unsaturated counterpart.     

Synthesis of new acyclic nucleoside phosphonates (ANPs) substituted on the 1′ and/or 2′ positions

A series of 2′ functionalized acyclic nucleoside phosphonate derivatives of 1-[3′-(phosphonomethoxy)propyl]uracil (1-4) have been synthesized together with the 1′ and 2′-ethynyl derivatives of 9/1-[2′-(phosphonomethoxy)ethyl]adenine/thymine (5-7). Key intermediates leading to the latter series are (±)-[2-{diethyl(phosphonomethoxy)}-1-hydroxy]-but-3-yne (25) and (±)-diisopropyl{[2-hydroxy-4-(trimethylsilyl)but-3-yn-1-yl]oxy}methylphosphonate (30). Compounds 25 and 30 are easily obtained starting from (±)-solketal.     

Synthesis and biological evaluation of andrographolide derivatives as potent anti-HIV agents

A series of Andro derivatives were described and evaluated for their anti-HIV activity in vitro.Compound 10 and 16b,of which TI were>10,had some anti-HTV-l activity in vitro.Therein,compound 10 which was the best potent compound,could serve as a new lead for further development of anti-AIDS agents.     

Convenient synthesis of (E)-5-aminoallyl-2′-deoxycytidine and some related derivatives

Preparation via the Heck reaction of 5-(E)-(3-trifluoroacetamidoallyl)-2′-deoxycytidine (3a) and some N4-formamidine protected derivatives is reported. Difficulties with coupling N-allyl-trifluoroacetamide to 5-iodo-2′-deoxycytidine were overcome by using Pd₂(dba)₃ as catalyst and temporary dimethylformamidine protection of the N4-amine of the nucleoside. Compound 3a was isolated with 60% overall yield by crystallization.     

Synthesis and characterisation of novel 3′-O- and 5′-O- modified azobenzene-thymidine phosphoramidites and their oligonucleotide conjugates as colorimeter DNA probes and FRET quenchers

The synthesis of two new ‘first generation’ azobenzene based thymidine phosphoramidites 1 and 2 having the chromophore (DABCYL) covalently incorporated as an ester in the 3′-O- and 5′-O-positions of the deoxyribose, and the incorporation of these molecules into 16-mer Chronic Myeloid Leukaemia (CML) antisense oligonucleotides, giving 7 and 8, respectively, is described. These compounds were designed as highly coloured probes, and to participate in a Fluorescence Resonance Energy Transfer (FRET) mechanism in the design of novel molecular beacons.     

Synthesis and in vitro biological evaluation of nitric oxide-releasing derivatives of hydroxylcinnamic acids as anti-tumor agents

Novel furoxan-based nitric oxide-releasing derivatives 6a-p of hydroxylcinnamic acids were synthesized by coupling the carboxyl group of hydroxylcinnamic acids with furoxan through various alkylol amines. Compounds 6a, e-i and m-p displayed more potent anti-tumor activities superior to control 5-fluorouracil (5-FU) in most cancer cells tested. Furthermore, 6f could selectively inhibit tumor cells, but not non-tumor cell proliferation. This inhibition was attributed to high levels of NO released in cancer cells and potentially synergistic effect of NO donor moieties and the bioactivity of hydroxylcinnamic acids.     

Synthesis and hybridization properties of 2′-O-(tetrazol-5-yl)ethyl-modified oligonucleotides

2′-O-(1H-Tetrazol-5-yl)ethyladenosine was synthesized using 2′-O-cyanoethyladenosine derivative as a key intermediate. The 2′-O-(1H-tetrazol-5-yl)ethyl modifications exhibited intriguing properties such as the change in the structure of the tetrazole residue between a protonated and a deprotonated form. The T_m experiments of various oligodeoxynucleotides having a 2′-O-(1H-tetrazol-5-yl)ethyl-modified adenosine showed reduced hybridization affinity in comparison to the unmodified oligonucleotides toward their complementary oligodeoxynucleotides. The mechanism of the reduced hybridization affinity was discussed on the basis of the structure and the physicochemical properties of the tetrazole moiety.     

Synthesis and biological evaluation of triazepane derivatives as DPP-IV inhibitors

A series of triazepane derivatives such as (R)-3-amino-1-(1,2,5-triazepan-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-ones (7, 13a-p) and (R)-3-amino-1-(1,2,5-triazepan-5-yl)-4-(2,4,5-trifluorophenyl)butan-1-ones (17a-e) was synthesized and evaluated for their ability to inhibit dipeptidyl peptidase IV (DPP-IV) enzyme. Compounds with the acid moiety were found to be potent inhibitors of DPP-IV without inhibiting CYP 3A4. Among them, compound 13p ((R)-4-[1-acetyl-2-{3-amino-4-(2,4,5-trifluorophenyl)butanoyl-1,2,5-triazepan-5-carbonyl}benzoic acid]) showed a good in vitro activity without inhibiting CYP.     

Synthesis of Acyclic Nucleosides with N‐[(Benzyloxy)(aryl)methyl] Substituents as Potential HEPT,EBPU, and TNK‐651 Analogues

The syntheses of the novel acyclic nucleosides 5a – 5m , carrying different N‐[(benzyloxy)(aryl)methyl] substituents, are described (Scheme). These compounds could be prepared in medium‐to‐good yields by either direct or silyl‐assisted coupling of the electrophiles 6 with either purine or pyrimidine nucleobases, or with different imidazole derivatives. The reactivity of the positively charged electrophilic intermediates derived from 6 upon Cl^? abstraction was rationalized by ab initio HF/6‐311G quantum‐mechanic calculations. The positive charge was found to be dispersed differently, depending on the electronic properties of the aryl substituents.     

Synthesis and biological evaluation of novel cyanuric acid-tethered tris-pyridinium derivatives

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Scalable synthesis of substituted 2,7-dimethyl-9-phenylxanthen-9-ol (DMPx-OH): useful for the preparation of crystalline 5′-O-DMPx-protected nucleosides

A convenient single-step synthesis of several 2,7-dimethyl-9-phenylxanthen-9-ol (DMPx-OH) analogs has been accomplished using a Friedel-Crafts reaction. Treatment of various DMPx-OH with unprotected 2′-O-methoxyethyl-ribonucleosides (MOE) in the presence of B(C₆F₅)_3, as a Lewis Acid catalyst, furnished 5′-O-protected derivatives of 2′-MOE-ribonucleosides in good yields. The deprotection of the DMPx groups was accomplished by acid hydrolysis under very mild conditions. Among the five DMPx analogs synthesized, the 2,7-dimethyl-9-(4-nitrophenyl)xanthene-9-yl group furnished crystalline products enabling non-chromatographic isolation of 5′-O-protetced nucleosides.     

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.