Synthesis,anti-HIV and cytostatic evaluation of 3′-deoxy-3′-fluorothymidine (FLT) pro-nucleotides

A series of pro-nucleotide phosphoramidates and phosphorodiamidates of the antiviral lead compound 3′-deoxy-3′-fluorothymidine (FLT) have been designed and synthesized. In vitro antiretroviral and cytostatic studies revealed potent (sub-micromolar) inhibition of HIV-1 and HIV-2 replication, with retention of activity in thymidine kinase-negative cell models, as predicted by the ProTide concept.     

HPLC and mass spectrometry analysis of the enzymatic hydrolysis of anti-HIV pronucleotide diastereomers

In one current strategy to develop membrane-soluble pronucleotides, the phosphoramidate derivatives of the approved anti-HIV nucleosides 2',3'-didehydro-3'-deoxythymidine (d4T), 3'-azido-3'-deoxythymidine (AZT), (-)-beta-L-2',3'-dideoxy-3'- thiacytidine (3TC), and 2',3'-dideoxyadenosine (ddA) exhibit promising antiviral activity. However, the non-stereoselective synthetic route results in a mixture of diastereoisomers, which differ in the configuration of the phosphorus chiral center. Since it is believed that enzymatic ester hydrolysis is the first step in the intracellular activation of these prodrugs and that this process could be dependent on the stereochemistry at the phosphorus center, analytical methods must be developed. In the present work, in vitro evaluation of the selectivity of pig liver esterase (PLE) towards each diastereomer of d4T, AZT, 3TC, and ddA prodrugs has been investigated, applying our recently published HPLC-MS procedure using a polysaccharide-type chiral stationary phase. This method has been used to analyze the products of the PLE-catalyzed hydrolysis of the pronucleotides. It was found that both diastereomers of the four prodrugs were substrates for PLE.     

31P NMR and Computational Studies on Stereochemistry of Conversion of Phosphoramidate Diesters into the Corresponding Phosphotriesters

³¹P NMR spectroscopy was used to investigate a stereochemical course of a nitrite-promoted conversion of phosphoramidate diesters into the corresponding phosphotriesters. It was found that this reaction occurred with almost complete epimerization at the phosphorus center and at the C1 atom in the amine moiety. On the basis of the ³¹P NMR data, a plausible mechanism for the reaction was proposed. The density functional theory calculation of the key step of the reaction, i.e., breaking of the P–N bond and formation of the P–O bond, suggested a one-step S_N2(P) process with retention of configuration at the phosphorus center.     

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.     

A NEW APPROACH TO OLIGONUCLEOTIDE N3′⇉P5′ PHOSPHORAMIDATE BUILDING BLOCKS

A new synthetic approach to 5′-phosphoramidites of 3′-aminonucleosides was developed. The methodology relies upon the use of 3′-amino-2′,3′-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2–3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3′-aminonucleoside sugar and base moieties.     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.