Tadeusz Kulikowski

Infrared spectra of 2-thiocytosine and 5-fluoro-2-thiocytosine; experimental and ab initio studies

Abstract IR absorption spectra of 2-thiocytosine and 5-fluoro-2-thiocytosine isolated in low-temperature, inert gas matrices and in thin polycrystalline films are reported. The spectra of amino-thiol tautomeric forms of the compounds were studied for matrix isolated species, whereas the spectra of amino-thione tautomeric forms were observed for polycrystalline films. The experimental spectra were compared with the spectra predicted with the ab initio SCF/6-31G** method. The observed IR absorption bands in the spectra of monomers (amino-thiol forms) were assigned to the theoretically calculated normal modes.

Synthesis and biological activity of 1H-benzotriazole and 1H-benzimidazole analogues - inhibitors of the NTPase/helicase of HCV and of some related Flaviviridae

To improve anti-helical activity of analogues of 1H-benzotriazole and 1H-benzimidazole their N-alkyl derivatives were synthesized and tested for anti-helicase activity against enzymes of selected Flaviviridae including hepatitis C virus (HCV), West Nile virus (WNV), Dengue virus (DENV) and Japanese encephalitis virus (JEV). 1- and 2-alkyl derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole were obtained by direct alkylation of 4,5,6,7-tetrabromo-1H-benzotriazole with the use of respective alkyl halides in the presence of KOH in methanol, to give a mixture of 1- and 2- isomers, which was separated by flash column chromatography in good yield. The proportion of isomers strongly depended on the reaction time and temperature. 1- and 2-hydroxyethyl and 1- and 2-chloroethyl derivatives of the tetrabromobenzo-triazole were synthesized with the use of 2-bromoethanol and 1-bromo-2-chloroethane respectively as alkylating agents. N-alkylation of this benzotriazole compound enhanced inhibitory activity and selectivity towards the helicase activity of HCV NTPase/helicase. The most active were the 2-methyl, 2-ethyl and 2-propyl derivatives (IC50∼6.5 μM in the presence of DNA as a substrate). Derivatives of the benzotriazole in which hydroxyethyl or chloroethyl replaced the alkyl substituents lost their inhibitory activity. Brominated or methylated benzotriazole N(1) ribosides also did not exert helicase inhibitory activity. Although a number of N(1) and N(2) alkyl derivatives exerted good HCV and WNV helicase inhibitory activity when DNA was used as substrate, the activity was strongly decreased or even disappeared when RNA was used as substrate. The cytotoxicity tests in Vero and HeLa Tat cells showed a substantial decrease of cytotoxicity of N-alkyl derivatives as compared to the parent benzotriazole.

High-Yield Regioselective Thiation of Biologically Important Pyrimidinones, Dihydropyrimidinones and Their Ribo, 2′-Deoxyribo and 2′, 3′-Dideoxyribo Nucleosides

Abstract Convenient and high-yield regioselective thiation procedures based on the use of the Lawesson reagent in different solvents, are described for conversion of the 2- and 4-keto, and 2, 4-diketo pyrimidines to the corresponding 2(4)-thio, and 2, 4-dithio, derivatives. This method is applicable to thiation of the 4-keto groups of 5, 6-dihydropyrimidinones and pyrimidine nucleosides. The mild reaction conditions employed are such that it is the method of choice for compounds with labile glycosidic bonds, such as 5, 6-dihydropyrimidine nucleosides and the 2′, 3′-dideoxynucleosides currently of interest as antiretroviral, including anti-HIV, agents.

COMPARATIVE STUDY OF THE POTENCY AND SELECTIVITY OF ANTI-HERPES COMPOUNDS

Several anti-herpes compounds, including phosphonoacetic acid, 9-(2-hydroxyethoxymethyl) guanine, 9-β-D-arabinofuranosyladenine, 1-β-D-arabinofuranosylthymine, 5-iodo-5′-amino-2;5′-dideoxyuridine, 5-iodo-2′-deoxyuridine, 5-bromo-2′-deoxycytidine and various other 5-substituted 2′-deoxyuridines and 2′-deoxycytidines were compared in vitro (primary rabbit kidney cells) for both antiviral activity (based on the ID50 required to inhibit the cytopathogenicity of the KOS strain of type 1 herpes simplex virus) and antimetabolic activity (based on the ID50 required to inhibit the incorporation of 2′-deoxyuridine into host cell DNA). Of the whole set of compounds tested, E-5-(2-bromovinyl)- and E-5-(2-iodovinyl)-2′-deoxyuridine emerged as both the most potent and the most selective anti-herpes agents.

Interaction of 5-fluoro-4-thio-2′-deoxyuridine 5′-phosphate with mammalian tumour thymidylate synthase: Role of the pyrimidine N(3)-H dissociation

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.

Synthesis and evaluation of ATP-binding site directed potential inhibitors of nucleoside triphosphatases/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses.

5′-O-(4-fluorosulphonylbenzoyl)-esters of ribavirin (FSBR), adenosine (FSBA), guanosine (FSBG) and inosine (FSBI) were obtained by acylation of the 5′-OH of adenosine, guanosine, inosine, and ribavirin with 4-fluorosulphonylbenzoyl chloride (FSBCl) in HMPA. The above derivatives were tested as inhibitors of nucleoside triphosphatase (NTPase)/helicase activities of Flaviviridae: hepatitis C virus (HCV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and dengue virus (DENV) and polymerase activity of HCV and WNV. When the unwinding activity of viral NTPase/helicases was tested under standard conditions, only weak inhibition was obtained with FSBI (IC50≥120 μM) and in the case of FSBG even an activation was seen. The preincubation of the NTPase/helicases with the 5′-O-FSB derivatives increased the inhibitory effect. Screening of the 5′-O-FSB derivatives on inhibition of the WNV and HCV RNA polymerases employing GTP or UTP substrates revealed rather modest inhibitory effect. FSBI exhibited the highest inhibitory activity against WNV (IC50=70 μM with UTP substrate) and HCV polymerase (IC50=80 μM with GTP substrate). Other 5′-O-FSB derivatives were very weak inhibitors or completely failed to show any activity against HCV and WNV enzymes. In contrast to the NTPase/helicases the preincubation of the polymerases did not influence the inhibition.

Synthesis and Biological Activity of 5-Fluoro-2-thiocytosine Nucleosides

Abstract Two pathways are described for the synthesis of the 2′-deoxynucleosides of 2-thiocytosine and 5-fluoro-2-thiocytosine: (a) by nucleoside condensation, (b) by amination of the corresponding nucleosides of 2,4-dithiouracil. Biological activities vs two cell systems are described. The nucleosides are moderate to weak substrates of deoxycytidine kinase and, partly as a result of this, reasonable good inhibitors of the enzyme

Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1-24 months after infection) and Trichinella pseudospiralis (isolated 5.5-13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.

Crystal structures of 5-fluoro-dUrd and its 2 and/or 4-thio analogues: Models of substituted dUMP pyrimidine ring interacting with thymidylate synthase

In order to understand the influence on thymidylate synthase interactions with dUMP analogues of the pyrimidine ring 2- and/or 4-thio, and 5-fluoro substitutions, X-ray diffractions by crystals of 5-fluoro-dUrd and its 2- and 4-thio, and 2,4-dithio analogues were measured, the four structures solved and refined. The following conclusions were suggested by results of comparative analyses of structural parameters (bond lengths, valence angles), followed by theoretical considerations based on calculated resonance structure distributions and aromaticity indices of the uracil, thiouracil, fluorouracil and fluorothiouracil rings. The effect of 4-thio substitution of FdUMP, altering specificity of inactivation of thymidylate synthases from various sources, is probably due to weaker proton acceptor power of the 4-thio substituent and increasing acidity (enhanced proton-donor power) of the N(3)-H moiety, resulting in an impaired fitness into the network of hydrogen bonds in the enzyme active center cleft. 2,4-Dithio substitution results in (i) impaired pyrimidine ring recognition by the enzyme active center, due to the 4-thio substituent (ii) increased pyrimidine ring aromaticity in dUMP, leading to resistance of C(6) to nucleophilic attack by the enzyme active center cysteine and (iii) altered planarity of the pyrimidine ring and deflections, with respect to the ring plane, of substituents at C(2), C(4) and C(5). 5-Fluoro substitution apparently activates the pyrimidine ring towards the interaction with thymidylate synthase by producing local strain, which results in an increased reactivity as predicted by the Walsh-Bent rule.

Theoretical and matrix-isolation experimental studies on 2-thiocytosine and 5-fluoro-2-thiocytosine

2-Thiocytosine (s2Cyt) and 5-fluoro-2-thiocytosine (f5s2Cyt) were studied by means of IR spectroscopy under different environmental conditions: isolated in low-temperature inert gas matrices, associated in thin amorphous and polycrystalline films. The compounds isolated in matrices were only very slightly influenced by the environment. From the analysis of the IR spectra of both compounds it appears that they exist in inert gas matrices only in the amino-thiol tautomeric form. Strong environmental effects were observed for s2Cyt and f5s2Cyt deposited in the form of thin polycrystalline films. Contrary to matrices, in polycrystalline films the amino-thione form dominates for both s2Cyt and f5s2Cyt. The experimental findings are in agreement with the ab initio quantum mechanical calculations of the relative total energies of the tautomeric forms. Those energies were calculated using the Self Consistent Field method corrected for electron correlation effects with the use of the second-order many-body perturbation theory (SCF+MBPT(2)). The theoretical calculations show that the amino-thiol tautomeric form is more stable than the amino-thione form by 38 kJ mol-1 and 48 kJ mol-1 for s2Cyt and f5s2Cyt, respectively. Both molecules, s2Cyt and f5s2Cyt, may also appear in the uracil-like imino-thione tautomeric form, which is predicted to be only 8 kJ mol-1 less stable than the amino-thione form. A new method of the preparation of f5s2Cyt is reported.