[Glycol and Phosphate Depot Forms of 4- and/or 5-Modified Nucleosides Exhibiting Antibacterial Activity].

Resistance developed to the majority of drugs used to treat infectious diseases warrants the design of new compounds effective against drug-resistant strains of pathogens. Recently, several groups of modified nucleosides have been synthesized and showed significant antibacterial activity in vitro, but their further studies were difficult to undertake because of their low solubility in aqueous solutions. Nevertheless, new compounds, well soluble in water-organic solutions, were synthesized and found to be more effective in inhibiting the growth of Gram-positive bacteria and mycobacteria. The water-soluble forms of modified nucleosides under study were assumed to be their depot forms. To check the assumption, the compounds were tested for hydrolysis in various media and their molecular docking was performed into the active center of the putative target, Mycobacterium tuberculosis flavin-dependent thymidylate synthase ThyX. Computer modelling showed that the water-soluble analogs do not act as ThyX inhibitors, supporting the assumption of their depot nature. The compounds were resistant to chemical hydrolysis but were hydrolyzed when incubated with porcine liver carboxylesterase, human serum, or Staphylococcus aureus 209P. The results demonstrate that the compounds are most likely depot forms of modified nucleosides.

Development of Liposomal Forms of Modified Pyrimidine Nucleosides and Investigation of Their Antibacterial Properties.

Different phosphocholine-cardiolipin-2'-deoxyuridine inclusion complexes were developed, that allowed to compose a water-soluble form of nucleoside analogues with previously defined antituberculosis activity. It was found that the resulting liposomes effectively penetrated to the cells. The increase of cytotoxicity was undoubtedly indicative of accumulation of the nucleoside in the cell culture. The result proved the ability of the liposomes for delivery of the low-soluble compounds to the cells for further investigation of their efficacy. It was shown that treatment of the bacterial cells with the llposomes of the modified nucleosides did not affect the bacterial growth.

Content of total homocysteine and major aminothiols in rats with experimental renal ischemia.

The contents of total homocysteine, cysteine, and glutathione in blood plasma and tissue of rats with renal ischemia were measured by HPLC. Our study was performed on the "two-kidney, one-clip (0.13 mm)" model. The concentrations of homocysteine and cysteine in blood plasma from treated rats were higher than in sham-operated animals (control; by 36 and 14%, respectively). Homocysteine level in the intact and clipped kidneys of treated rats was 40% higher than in the control. However, no differences were found in homocysteine level in the ischemic and intact kidneys of treated animals. Cysteine concentration in the clipped kidney was lower than in the kidneys of intact and sham-operated animals (by 1.6 and 1.5 times, respectively). Glutathione concentration in the ischemic kidney did not differ from the control. No differences were revealed in the content of aminothiols in liver samples from rats of the treatment and control groups. Our results suggest that functional inactivation of one kidney is accompanied by impairment of homocysteine catabolism (trans-sulfonation).

New 5-modified pyrimidine nucleoside inhibitors of mycobacterial growth.

The WHO has declared tuberculosis (TB) a global health emergency. Therefore, there is an urgent need to discover and develop new anti-TB drugs. Here we report on a new category of 5-substituted pyrimidine nucleosides as potent inhibitors of Myco-bacterium tuberculosis growth in vitro. A series of 2'-deoxy-, 3'-azido-2',3'-dideoxy-, and 3'-amino-2',3'-dideoxypyrimidine nucleoside analogues bearing lengthy flexible alkyloxymethyl substituents exhibited marked inhibitory activity against M. tuberculosis in vitro. 5-Dodecyloxymethyl-2'-deoxyuridine was found to be a potent inhibitor of M. tuberculosis propagation in vitro. In contrast, monophosphates of the tested nucleosides were devoid of antimycobacterial activity. This new class of inhibitors seems to be a promising chemotherapeutic agent against TB and merits further studies.

[Structure-functional analysis of interactions of terminal deoxynucleotidyl transferase with new non-nucleoside substrates].

New non-nucleoside esters of phosphoric acid containing various hydrophobic groups, namely (1) N-(2-tripticencarbonyl)-4-aminobutyl; (2) 5-phenylsubstituted N-(2,4-dinitrophenyl)-4-aminobutyl; (3) N-(4-phenylbenzoyl)- and N-(4-(N-benzylamino)benzoyl)-2-aminoethyl groups, as well as (4) diphenylmethyl and fluorenyl groups were synthesized and studied as substrates of terminal deoxynucleotidyl transferase. With the exception of the two latter derivatives, all the analogues displayed substrate properties and could incorporate into the deoxyoligonucleotide 3'-end. As it was shown in biochemical experiments and by computer modeling, a linker joining the triphosphate and hydrophobic fragments of the molecule was necessary for these compounds to display substrate properties.

Base-modified ribonucleosides as potential anti-hepatitis C virus agents.

Four novel series of base modified ribonucleoside analogues were synthesized and evaluated as potential anti-HCV agents. For two compounds notable anti-HCV activity was observed The triphosphates of bicyclic pyrimidine ribonucleosides were studied as substrates/inhibitors of HCV RNA-dependent RNA polymerase (RdRp, NS5B protein) and RNA helicase/NTPase (NS3 protein).

Furano- and pyrrolo [2,3-d] pyrimidine nucleosides and their 5'-O-triphospates: synthesis and enzymatic activity.

A series of bicyclic [2,3-d]furano- and pyrrolopyrimidine ribonucleosides were synthesized and converted chemically into corresponding 5'-O-triphosphates. Substrate properties of the triphosphates toward some RNA and DNA polymerases are reported.

Aryl-containing esters of triphosphoric acid as substrates of terminal deoxynucleotidyl transferase.

A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized.

2'-Deoxynucleoside 5'-triphosphates modified at alpha-, beta- and gamma-phosphates as substrates for DNA polymerases.

Replacement of alpha-, beta- and gamma-phosphate groups in 2'-deoxynucleoside 5'-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at alpha-, beta- and gamma-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that gamma-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the gamma-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase alpha. For dUTP analogues substituted at both gamma-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at gamma-phosphonate or C5 position. Meanwhile, C5-substituted beta, gamma-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2'-deoxythymidine 5'-[beta, gamma-(methylphosphinyl)methylphosphonyl]-alpha-phosphate and its 3'-azido-3'-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.

Modified substrates of DNA polymerases and design of antivirals.

The results obtained in our laboratory on investigating of substrate properties of a large number of compounds towards different DNA polymerases have been summarized. On the basis of systematic analysis a directed synthesis of nucleotides with antiviral properties was performed.

Purine arabinonucleoside 5'-triphosphates with substituents at 2' position as substrates for DNA polymerases.

Analogues of araNTPs carrying an azido or aminogroup instead of the 2' hydroxyl exhibited substrate properties towards several mammalian and viral DNA polymerases. At the same time, introduction of a bulky hydrophobic DNP group into the 2' position inactivated the compounds as substrates. HSV-1 and CMV DNA polymerases were an interesting exception: they effectively incorporated the modified nucleotide residues with DNP group into the 3'-termini of the DNA chain. This is a reliable distinction of these enzymes from cellular DNA polymerases.

Oligonucleotide derivatives bearing reactive and stabilizing groups attached to C5 of deoxyuridine.

Oligonucleotides bearing an aliphatic amino group at the C5-position of deoxyuridine (ULNH2TCCCA, TULNH2CCCA, ULNH2CCACTT, where L = -CH2-, -CH2OCH2CH2- or -CH2NHCOCH2CH2-) have been synthesized. The photoactive (p-azidotetrafluorobenzamido, 2-nitro-5-azidobenzamido, or p-azidobenzamido), alkylating [4-[N-(2-chloroethyl)-N-methylamino]benzyl], or intercalating [N-(2-hydroxyethyl)phenazinium] groups were attached to the amino linker of oligonucleotides. The Tm values were determined for the duplexes formed by the above oligonucleotide derivatives. The alkylating group does not change the melting temperature of the corresponding duplex. The duplex stability is increased a little in the case of photoactive groups. The influence of the phenazinium residue on the duplex stability strongly depends on its location in the oligonucleotide. The spacer length between the C5 atom of deoxyuridine and the photoactive or phenazinium group was shown to influence the complementary duplex stability.