Influence of ionizing radiation on the antimicrobial activity of the antibiotics sulfamethoxazole and trimethoprim.

The response of the antimicrobial compounds sulfamethoxazole (SMX) and trimethoprim (TMP) - individually and in mixtures - to ionizing radiation was investigated using laboratory prepared mixtures and a commercial pharmaceutical formulation. The residual antibacterial activity of the solutions was monitored using Staphylococcus aureus and Escherichia coli test strains. Based on antibacterial activity, SMX was more susceptible to ionizing radiation as compared to TMP. The antibacterial activity of SMX and TMP was completely eliminated at 0.2 kGy and 0.8 kGy, respectively. However, when SMX and TMP were in a mixture, the dose required to eliminate the antibacterial activity was 10 kGy, implying a synergistic antibacterial activity when these are present in mixtures. Only when the antibiotic concentration was below the Minimum Inhibitory Concentration of TMP (i.e., 2 µmol dm-3) did the antibacterial activity of the SMX and TMP mixture disappear. These results imply that the synergistic antimicrobial activity of antimicrobial compounds in pharmaceutical waste streams is a strong possibility. Therefore, antimicrobial activity assays should be included when evaluating the use of ionizing radiation technology for the remediation of pharmaceutical or municipal waste streams.

Synthesis and enzymatic activity of some new purine ring system analogues of adenosine 3',5'-cyclic monophosphate.

A series of novel adenosine 3',5'-cyclic monophosphate (cAMP) analogues, as well as their 6-deamino and 6-nitro derivatives, were synthesized where the purine ring was replaced by indazole, benzotriazole, and benzimidazole. The 3',5'-cyclic monophosphates of indazole and benzotriazole ribofuranosides, where the sugar-phosphate moiety is attached to the N-2 nitrogen atoms of the heterocycles, were also prepared. The biological efficiency of the analogues was tested by their ability to activate purified cAMP-dependent protein kinase I (PK-I) from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart. Each cyclic nucleotide is capable of activating both PK isozymes in half-maximum concentrations (Ka) ranging from 2.0 x 10(-8) to 4.8 x 10(-6) M. The cyclic phosphate of N-1-beta-D-ribofuranosylindazole (13) proved to be a very poor activator for both PK-I and PK-II, but when indazole binds by N-2 to ribose or when the hydrogen atom at C-4 is substituted by a nitro or amino group, activities of the analogues increase considerably. The activating potencies of benzotriazole derivatives are similar to that of cAMP, irrespective of the C-4 substituents. The Ka' values of cyclic nucleotides containing benzimidazole were found to be higher for PK-II than for PK-I; e.g. the activity of 4-nitro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphate (32) is nearly 20 times as high for PK-II than for PK-I.

Synthesis and antiviral activities of 8-alkynyl-, 8-alkenyl-, and 8-alkyl-2'-deoxyadenosine analogues.

Palladium-catalyzed cross-coupling of 8-bromo-2'-deoxyadenosine with terminal alkynes in the presence of copper(I) iodide in dimethylformamide resulted in a series of 8-(1-alkyn-1-yl)-2'-deoxyadenosines. Hydrogenation of alkynyl derivatives over 10% Pd/C under atmospheric pressure gave 8-n-alkyl analogues in nearly quantitative yields. On partial saturation of heptynyl, pentynyl, and propynyl derivatives over Lindlar catalyst, the corresponding cis-olefins were obtained along with minor amounts of trans isomers. Of the analogues tested, the following showed some activity, i.e. they were found to be active at concentrations that were at least 3-fold lower than the cytotoxic concentrations: the 8-heptynyl derivative against vaccinia virus (VV), vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and respiratory syncytial virus (RSV); the 8-propyl derivative against varicella-zoster virus (VZV) and CMV; the 8-pentyl derivative against CMV; the 8-heptyl derivative against VV, CMV, RSV, and influenza A; and the 8-heptenyl derivative against VV, RSV, and influenza A. The unsubstituted 2'-deoxyadenosine did not show any antiviral effect, except against RSV. Except for 8-propyl-dA, the antivirally active dA analogues were rather inhibitory to the growth of human embryonic lung cells. The most cytotoxic was the 8-ethynyl derivative.

Synthesis and antitumor and antiviral properties of 5-halo- and 5-(trifluoromethyl)-2'-deoxyuridine 3',5'-cyclic monophosphates and neutral triesters.

The title diesters (11-15; halo substituents F, Cl, Br, I) were prepared by DCC-induced cyclization of the precursor 5'-monophosphate or direct halogenation of the 2'-deoxyuridine 3',5'-cyclic monophosphate. Antitumor activities of 11-15 in cell systems (L1210 and Raji/0) were compared to those of the corresponding nucleosides and 5'-monophosphates. Thus, the 5-F- and 5-CF3-2'-deoxyuridines proved to be highly active derivatives [ID50 values (microgram/mL) for L1210, 0.002 and 0.06, respectively], with the 5'-monophosphates showing comparable potencies. The corresponding 3',5'-cyclic monophosphate diesters were 20-30 times less potent but nonetheless highly cytostatic. All derivatives including 11-15 had greatly increased ID50 values for the thymidine kinase deficient (TK-) L1210 and Raji cells. The 3',5'-cyclic diesters (11-15) evidently are not efficient prodrug sources of the nucleoside 5'-monophosphates in TK- cells. They also proved to be 100- to 2000-fold less efficient inhibitors of L1210 thymidylate synthetase than were the 5'-monophosphates. The 5-substituted 2'-deoxyuridines and their 5'-monophosphates were potent inhibitors of herpes simplex virus (MIC50 mostly 0.07-10 micrograms/mL) and vaccinia virus (MIC50 0.07-0.2 microgram/mL), with antiviral activity decreasing in the order 5-I, 5-Br greater than 5-CF3 greater than 5-Cl greater than 5-F. The 3',5'-cyclic monophosphates (11-15) were for the most part 10- to 40-fold less active than the 5'-monophosphates in the virus assay systems (e.g., MIC50 for the 5-Br and 5-I derivatives ranged 1-20 micrograms/mL). By contrast 11-15 were considerably more potent inhibitors of vaccinia virus growth (MIC50 0.4-2 micrograms/mL). As the neutral 3',5'-cyclic methyl phosphate triesters (16-18), the 5-I and 5-Br compounds were less potent in antiviral and cytostatic agents than the 3',5'-cyclic diesters, while the 5-iodo benzyl triester was in several cases as active as the 3',5'-cyclic diester. The title compounds (11-15) appear to require extracellular hydrolysis to the nucleoside before functioning as antitumor or antiviral agents.

Stereospecific synthesis and antiviral properties of different enantiomerically pure carbocyclic 2'-deoxyribonucleoside analogues derived from common chiral pools: (+)-(1R,5S)- and (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one.

Enantiomerically pure (+)- and (-)-carbocyclic thymidine, (-)-carbocyclic 3'-epi-thymidine, (+)-carbocyclic 3'-deoxy-3'-azidothymidine, (+)-carbocyclic 2,3'-O-anhydrothymidine, (+)-carbocyclic 3'-O,6'-methylenethymidine, and (+)-(6'S)-carbocyclic 6'-methylthymidine were synthesized in a stereospecific manner from common chiral pools of (+)-(1R,5S)- and (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and evaluated for antiviral activity. (+)-Carbathymidine and, to a lesser extent, (+)-carbocyclic 2'-deoxyadenosine proved to be effective against HSV-1 [minimum inhibitory concentration (MIC): 0.2 and 2 micrograms/mL, respectively] and HSV-2 (MIC: 2 and 20 micrograms/mL, respectively), but virtually inactive against TK- HSV-1 (MIC: 40 and 100 micrograms/mL, respectively). (+)-Carbathymidine was also active against vaccinia virus (2 micrograms/mL). None of the compounds had a specific effect on the replication of HIV or other RNA viruses.

Synthesis and NMR characterization of diastereomeric CPSMeG derivatives.

Synthesis and stereochemical characterization of enantiomerically pure CPSMeG derivatives by NMR methods are reported. The effect of 5'-dimethoxytrityl on the conformational properties is described. It was found that in P-diastereomers the conformational differences about the C3'-O3' bond, as discernible from the delta J values, are enhanced by the presence of this protecting group.

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.

Enzymatic hydrolysis and biological activity of oligonucleotides containing 5-substituted pyrimidine bases.

Two series of alternating ODNs containing 5-n.alkyl-, alkenyl- and alkynyl-dU and -dC units have been prepared in order to study the kinetics of their hydrolysis by SV PDE and human serum, respectively. Both in (r5dUpdA)10 and (r5dCpdG)6 series the rate of hydrolysis decreased with increasing length of side-chain. Replacement of thymidines by 5-hexynyl-dU in different antisense oligomers resulted in considerably higher biological activity relative to that of the thymidine-containing counterparts.

Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions.

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.