Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.     

Synthesis of new terminator substrates for DNA-polymerases-- nucleoside-5'-triphosphates with modified carbohydrate residue

Synthesis of 3'-chloro- and 3'-cyanothio-2',3'-dideoxythymidine by the reaction of 2,3'-anhydro-2'-deoxythymidine with ammonium chloride and lithium thiocyanate, respectively, has been developed. In addition, 3'-methanesulphonylamido- and 3'-sulphonylamido-2',3'-dideoxythymidines were synthesized starting from 3'-amino-2',3'-dideoxythymidine. All these compounds along with 2',3'-anhydroriboadenosine,2',3'-anhydrolyxoadenosine, 2',3'-O-isopropylidenecytidine, and 2,3'-anhydro-2'-deoxythymidine were transformed into 5'-triphosphates by treatment with phosphoryl tris-1,2,4-triazolide and then with bis(tri-n-butylammonium)pyrophosphate. All 5'-triphosphates of nucleoside analogues were tested as termination substrates in cell-free systems with various DNA polymerases.     

Synthesis of nucleoside 5'-fluoroalkylphosphonate--compounds with potential antiviral properties]

Several 5'-fluoroalkylphosphonates of 3'-azido-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine were synthesized as potential antiviral compounds. Fluoromethyl, difluoromethyl, fluorochloromethyl and vinyl phosphonates were used as phosphonylating components, with dicyclohexylcarbodiimide or triisopropylbenzenesulphonyl chloride as condensing reagents.     

AZT 5'-Cholinephosphate as an anti-HIV agent: the study of biochemical properties and metabolic transformations using its 32P-labelled counterpart

Biochemical and metabolic transformations of 3'-azido-3'-deoxythymidine 5'-choline phosphate (1) were studied using its 32P-labelled counterpart for the evaluation of possible reasons for its enhanced anti-HIV activity. An effective synthesis of 32P-labelled 1 with a specific activity >1,000 Ci/mmol was developed by esterification of 32P-phosphoric acid with choline in the presence of BrCN followed by the coupling of the resulting choline phosphate with 3'-azido-3'-deoxythymidine (AZT). Chemical and enzymatic stabilities of 1 as well as the dynamics of penetration through HL-60 cell membranes were studied at the concentrations comparable to its antiviral concentrations. The products of intracellular transformations of the studied nucleotide were identified.     

Chemical structure of 3-carbethoxypsoralen-DNA photoadducts

The enzymatic digest from salmon sperm DNA photochemically modified by the monofunctional 3-carbethoxypsoralen was analyzed by high-performance liquid chromatography. The modified nucleosides extracted from DNA were compared with model compounds obtained from irradiation in the dry state of mixtures of 3-carbethoxypsoralen with 2'-deoxyribonucleosides whose chemical structures had previously been characterized. The main photoadducts formed in DNA are two cis-syn diastereoisomers formed via a C4-cycloaddition reaction involving the 4', 5' double bond of 3-carbethoxypsoralen and the 5,6 double bond of 2'-deoxythymidine. Among them, the most polar one accounts for 72%. Under the same conditions, photoadducts formed between 3-carbethoxypsoralen and 2'deoxycytidine account for less than 1%.     

Synthesis of a highly active new anti-HIV agent 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine

Compounds having methyl, vinyl, and ethynyl groups at the 4'-position of stavudine (d4T: 2',3'-didehydro-3'-deoxythymidine) were synthesized. The compounds were assayed for their ability to inhibit the replication of HIV in cell culture. The 4'-ethynyl analogue (15) was found to be more potent and less toxic than the parent compound stavudine.     

New phosphonoformic acid derivatives of 3'-azido-3'-deoxythymidine

New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Conformational studies of novel antiretroviral analogs of zidovudine

Conformational properties of three novel zidovudine analogs, namely 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso, 2), (-)-trans-(5S,6S)-5-bromo-6, 5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (3) and (+)-trans-(5R,6R)-5-bromo-6,5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (4), have been investigated by AM1 calculations and NMR studies, and compared with those of the parent nucleoside (AZT, 1). Based on the results obtained the following correlation may be established, a) AZT and AZT-Iso exhibit a conformational behavior analog to other pyrimidinic nucleosides, displaying a dynamic equilibrium in solution where the two conformers (North and South) undergo a constant transformation. b) Compounds 3 and 4 show a different conformational profile. The estimate of the pseudorotation phase angle reveals the rigid structures of the latter compounds, which do not evidence conformational equilibrium in solution; the azide group being the only group free to rotate. c) Diastereoisomers 3 and 4 exhibit an extra conformational parameter compared with other pyrimidinic nucleosides: the chair or boat conformation in the third ring formed between the sugar and the base. In all cases, a reasonable correlation was obtained between theoretical and NMR spectroscopic data.     

Synthesis and intermembrane transfer of pyrene-labelled liponucleotides: ceramide phosphothymidines.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against human immunodeficiency virus (HIV) in vitro. Here we report on the synthesis and characterization of two pyrene containing conjugates: 2-N-(4-(pyren-1-yl)butanoyl)ceramide 5'-phosphothymidine (Pbs-Cer-P-T) (XII) and 2-N-(10-(pyren-1-yl)decanoyl)ceramide 5'-phosphothymidine (Pds-Cer-P-T) (XIII). These fluorescent labelled conjugates served as model compounds to study incorporation of sphingoliponucleotides into membranes. The complex compounds were prepared by condensation of 3'-acetylthymidine and labelled ceramides using the phosphite triester coupling procedure. UV absorption, fluorimetry as well as 1H-, 31P-, 13C-NMR analyses were used for structure confirmation of the synthesized substances. When incorporated into small unilamellar 1-palmitoyl-2-oleoyl-glycerophosphatidyl-choline (POPC) vesicles and incubated with unlabelled acceptor POPC vesicles, the compounds (XII) and (XIII) exhibited spontaneous transfer. Kinetic data suggest that transfer from donor to acceptor vesicles occurred via the intervening aqueous phase. The non-specific lipid transfer protein from bovine liver stimulated the transfer of Pds-Cer-P-T between phospholipid vesicles in a concentration dependent manner.     

Degradation of phosphatidylnucleosides induced by phospholipases

Hydrolysis of phosphatidylnucleosides, 5'-(rac-1-hexadecyl-2-palmitoyl-sn-glycero-3-phosphoryl)-3'-azido- 3'-deoxythymidine and -2',3'-didehydro-3'-deoxythymidine, effected by phospholipases (PL) A2, C, and D was studied to reveal the metabolism of these derivatives. It was shown that PLA2 deacetylates the glycerol residue at position 2, PLC is inactive, and PLD hydrolyzes the phosphatidylnucleosides to give free nucleosides.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.     

New derivatives of alkyl- and aminocarbonylphosphonic acids containing 3'-azido-3'-deoxythymidine

New 5'-phosphonates of 3'-azido-3'-deoxythymidine were synthesized and shown to be low-toxic and markedly active in MT-4 cell cultures infected with HIV-1.     

AIDS dementia: synthesis and properties of a derivative of 3'-azido-3'-deoxythymidine (AZT) that may become 'locked' in the central nervous system

In an attempt to provide a derivative of 3'-azido-3'-deoxythymidine (AZT) which might be sequestered in the central nervous system and release AZT, the dihydropyridine ester 5'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-3'-deoxythymidine, was synthesized in a three step sequence. This material showed potent anti-HIV-1 activity in MT-4 cells most probably by hydrolysis to the parent nucleoside, AZT. This dihydropyridine derivative of AZT could be easily oxidized to a positively charged pyridinium derivative of AZT in rat brain cytosol. In turn the pyridinium form could be hydrolyzed, non-enzymatically, to AZT.     

Syntheses, crystal structures, and hydrogen bonding patterns of 3'-C-methylenecarboxylic-3'-deoxythymidine and 3'-C-methyleneamidilylic-3'-deoxythymidine

Thymidine derivatives containing carboxylic acid and amide groups have been synthesized and the hydrogen-bonding patterns of 3'-C-methylenecarboxylic-3'-deoxythymidine 6 and 3'-C-methyleneamidilylic-3'-deoxythymidine 9 have been characterized by using X-ray crystallography.     

A simple procedure for constructing 5'-amino-terminated oligodeoxynucleotides in aqueous solution.

A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.     

Mechanisms of inhibitions of DNA polymerase gamma by nucleotide analogues having anti-HIV activities.

Inhibition mechanisms of 5'-triphosphates of 3'-azido-3'-deoxythymidine (AZT-TP) and 3'-deoxythymidine (ddTTP) on extensively purified DNA polymerase gamma from bovine testes were examined by analysis of the products synthesized on singly primed M13mp18 single-stranded DNA or synthetic oligonucleotide template-primer in the presence of analogues. The results indicate that AZT-TP inhibits DNA polymerase gamma in competition with dTTP but is not incorporated into DNA, whereas ddTTP is incorporated into DNA and causes chain termination.