Synthesis of some S-3''-deoxyadenosyl-L-homocysteine analogues.

Condensation of 3'-deoxy-3-deazaadenosine, 3'-deoxy-7-deazaadenosine and 3'-deoxyadenosine with N,N'-bis-trifluoroacetyl-L-homocystine dimethyl ester and subsequent deprotection of the resulting N-trifluoroacetyl-S-3'-deoxyadenosyl-L-homocysteine analogues afforded S-3'-deoxy-3-deazaadenosyl-L-homocysteine, S-3'-deoxy-7-deazaadenosyl-L-homocysteine and S-3'-deoxyadenosyl-L-homocysteine respectively. 3'-Deoxy-3-deazaadenosine and 3'-deoxy-7-deazaadenosine were prepared by transformation of the corresponding ribonucleosides with 2-acetoxyisobutyryl bromide. 3'-Deoxy-7-deazaadenosine and 3'-deoxyadenosine were also converted into their 5'-chloro-3',5'-dideoxy derivatives which in turn were condensed with L-homocysteine sodium salt to give S-3'-deoxy-7-deazaadenosyl-L-homocysteine and S-3'-deoxyadenosyl-L-homocysteine which were identical with those synthesized by condensation of the protected L-homocystine with the 3'-deoxynucleosides.     

7-substituted 8-aza-7-deazapurines and 2,8-diaza-7-deaza-purines: synthesis of nucleosides and oligonucleotides

7-Substituted 8-aza- 7-deazaadenosines 1a-e were synthesized by Sonogashira cross coupling from the corresponding 7-iodo nucleoside in 36-79% yields. Starting from 7-bromo (or 7-iodo)-8-aza-7-deazaadenine, 2a,b were obtained by acid-catalyzed glycosylation followed by deprotection in 53 and 35% yields, repectively. Compounds 2b was applied to cross coupling reaction to give 2c-d in 34-95% yield. Compounds 2a and 4b were further transformed to the phosphoramidites 5 and 6b in 9 and 49% overall yields, which were incorporated into oligonucleotides.     

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.     

Synthesis and inhibitory activity of new benzimidazole derivatives against Burkitt's lymphoma promotion

As a continuation to our previous work concerning antitumor benzimidazoles, we have synthesized series of new derivatives of 2-(1-benzyl-2-methyl-1H-benzimidazol-5-ylimino)-3-(substituted)-thiazolidin-4-one (6a-e), 3-(2-methyl-1H-benzimidazol-5-yl)-2-substituted-thiazolidin-4-one (9a-f) and we have studied their inhibitory activity against the Epstein-Barr virus-early antigen (EBV-EA) activation introduced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Compound 6d was found to be significantly active and compounds 5a and 6e were also active.     

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.     

Synthesis of 2'-methylene-substituted 5-azapyrimidine, 6-azapyrimidine, and 3-deazaguanine nucleoside analogues as potential antitumor/antiviral agents

2'-Deoxy-2'-methylene-6-azauridine (5) and 2'-deoxy-2'-methylene-6-azacytidine (8) have been synthesized via a multi-step procedure from 6-azauridine. 2'-Deoxy-2'-methylene-5-azacytidine (14a) and 2'-deoxy-2'-methylene-3-deazaguanosine (19a) and their corresponding alpha-anomers (14b and 19b) have been synthesized by the transglycosylation of 3',5'-O-(1,1,3,3- tetraisopropyldisiloxane-1,3-diyl)-2'-deoxy-2'-methyleneu ridine (12) with silylated 5-azacytosine and silylated N2-palmitoyl-3-deazaguanine, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by separation of the isomers and deprotection of the blocking groups. These compounds were tested for cytotoxicity against B16F10, L1210, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1, HSV-1, and HSV-2.     

Hoogsteen vs. Watson-Crick base pairing: incorporation of 2-substituted adenine- and 7-deazaadenine 2'-deoxy-beta-D-ribonucleosides into oligonucleotides

Various 2-substituted 2'-deoxyadenosines and 7-deazaadenosines have been synthesized. The phosphonate building block 9 of 2-chloro-7-deaza-2'-deoxyadenosine (7-deazacladribine; 2) was prepared by 4,4'-dimethoxytritylation of the parent nucleoside (-->7), followed by protection of the amino function with a formamidine residue (-->8). The latter was reacted with PCl3/N-methylmorpholine/1,2,4-triazole to give compound 9. Moreover, 2-methoxy-2'-deoxyadenosine (2'-deoxyspongosine; 1b) was converted into the fully protected derivative 12, which was then transformed into the 2-cyanoethyl phosphoramidite 14. Also the 2-(trifluoromethyl)-substituted 2'-deoxyadenosines 19-21 were prepared by glycosylation of the chromophore 16 with the halogenose 17, followed by one-pot deprotection and nucleophilic displacement of the 6-Cl substituent. The new DNA building blocks 9 and 14 were used--together with formerly prepared cladribine derivative 4--for solid-phase synthesis of a series of oligodeoxyribonucleotides. These were studied with respect to their thermal stability as well as of the base pairing mode (Watson-Crick vs. Hoogsteen) of modified bases.     

A new synthesis of certain 7-(beta-D-ribofuranosyl) and 7-(2-deoxy-beta-D-ribofuranosyl) derivatives of 3-deazaguanine via the sodium salt glycosylation procedure.

A facile synthesis of 7-beta-D-ribofuranosyl-3-deazaguanine (1) and certain 8-substituted derivatives of 1 via the sodium salt glycosylation method has been developed. Glycosylation of the sodium salt of methyl 2-chloro(or methylthio)-4(5)-cyanomethylimidazole-5(4)-carboxylate (5 and 13b) with 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide (6) gave exclusively methyl 2-chloro(or methylthio)-4-cyanomethyl-1-(2,3, 5-tri-O-benzoyl-beta-D-ribofuranosyl)imidazole-5-carboxylate (7 and 14a), respectively. Ammonolysis of 7 and 14a provided 6-amino-2-chloro(or methylthio)-3-beta-D-ribofuranosylimidazo-[4,5-c]pyridin-4(5H)-one (11 and 17), which on subsequent dehalogenation (or dethiation) gave 1. Similarly, reaction of the sodium salt of 5 and 13b with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose (8), and ammonolysis of the glycosylated imidazole precursors (9 and 16) gave 6-amino-2-chloro(or methylthio)-3-(2-deoxy-beta-D-erythro-pentofuranosyl) imidazo[4,5-c]-pyridin-4(5H)-one (10a and 15), respectively. Dehalogenation of 10a or dethiation of 15 gave 2'-deoxy-7-beta-D-ribofuranosyl-3-deazaguanine (10b). This procedure provided a direct method of obtaining 10b without the contaminating 9-glycosyl isomer 4.     

Novel ofloxacin derivatives: synthesis, antimycobacterial and toxicological evaluation

Thirty novel 9-fluoro-2,3-dihydro-8,10-(mono/di-sub)-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acids were synthesized from 2,3,4,5-tetrafluoro benzoic acid and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC(2)) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from mycobacteria. Among the synthesized compounds, 10-[2-carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active compound in vitro with MIC99 of 0.19 microM and 0.09 microM against MTB and MTR-TB, respectively. In the in vivo animal model also the same compound decreased the bacterial load in lung and spleen tissues with 1.91 and 2.91--log10 protections, respectively, at the dose of 50mg/kg body weight. Compound 10-[(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)]-9-fluoro-2,3-dihydro-3-methyl-8-nitro-7-oxo-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid was found to be the most active in the inhibition of the supercoiling activity of DNA gyrase with an IC(50) of 10.0 microg/mL. The results demonstrate the potential and importance of developing new oxazino quinolone derivatives against mycobacterial infections.     

Effects of the S-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on RNA methylation and synthesis

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 microM 3-deazaaristeromycin or with 10 microM 3-deazaadenosine and 50 microM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 microM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.     

Alumina-supported synthesis of antibacterial quinolines using microwaves

7-(5'-Alkyl-1',3',4'-thiadiazol/oxadiazol-2'-ylthio)-6 -fluoro-2,4-dimethylquinolines and 3-formyl-2-(2'-hydroxy- 1',4'-naphthoquinon-3'-yl)-4-methyl/6-methyl/7-quinolines have been synthesised by the reaction of 5-alkyl-1,3,4-thiadiazol/oxadiazol-2-thiols with 7-chloro-6-fluoro-2,4-dimethylquinoline and by the reaction of 2-hydroxy-1,4-naphthoquinone with 2-chloro-3-formyl-4-methyl/6-methyl/7-methyl/8-methylquinolines respectively on basic alumina using microwaves, the reaction time has been brought down from hours to seconds with improved yield as compared to conventional heating. The compounds were tested for their in vitro antibacterial activity. All compounds showed promising antibacterial activity. The best activity was observed by compounds 3a and 3f.     

Synthesis and anti-HIV-1 activity of 4-substituted-7-(2'-deoxy-2'-fluoro-4'-azido-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine analogues

Three novel 4-subsituted-7-(2'-deoxy-2'-fluoro-4'-azido-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine analogues were designed, synthesized, and tested for their anti-HIV-1 activity. Initial biological studies indicated that among these pyrrolo[2,3-d]pyrimidine ribonucleoside analogues, 4-amino-7-(2'-deoxy-2'-fluoro-4'-azido-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine 10 exhibited the most potent anti-HIV-1 activity (EC(50)=0.5±0.3 μM), while 4-hydroxy-7-(2'-deoxy-2'-fluoro-4'-azido-β-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine 9 and 4-amino-5-fluoro-7-(2'-deoxy-2'-fluoro-4'-azido-β-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine 11 showed moderate activity (EC(50)=13±8 and 5.4±0.3 μM, respectively). The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds with concentrations up to 25 μM.     

Preparation of 2- and 3-substituted gibberellins A9 and A4 for bioassay

To test the effects of preventing enzymatic 2β- and 3β-hydroxylation on the biological activities of gibberellins, the preparation of the following compounds is described: 2β-methyl- and 2,2-dimethyl-gibberellins A₄ and A₉; 2α-fluoro-, 2β-fluoro- and 2β-methoxy-gibberellin A₉; and 3β-chloro-, 3β-fluoro-, 3β-methoxy- and 3-methylene A₉.     

Synthesis,structural characterization and in vitro antitumor activity of novel 6-chloro-1,1-dioxo-1,4,2-benzodithiazie derivatives

A series of nonconventional aminium N-(6-chloro-7-R-1,1-dioxo-1,4,2-benzodithiazin-3-yl)arylsulfonamidates 7-15 have been synthesized by the reactions of 6-chloro-7-R-3-methylthio-1,4,2-benzodithiazine 1,1-dioxides with 4-dimethylaminopyridine or Et(3)N and some arylsulfonamides. The free N-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-3-yl)benzenesulfonamides 16-18 were obtained by treatment of their aminium salts with H(2)SO(4) in boiling acetic acid. The in vitro antitumor activity of the compounds 9, 11-14 and 16-18 has been tested in the antitumor screening of the National Cancer Institute (NCI), and relationships between structure and antitumor activity are discussed. 4-Dimethylaminopyridinium 4-chloro-N-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-3-yl)benzenesulfonamidate 9 is the prominent of the compounds due to its remarkable activity (log GI(50)<-8.00, log TGI=-5.50) and selectivity for the leukemia SR cell line. For that reason experimental and theoretical analysis of the geometric and electronic properties of 9 was carried out.     

Studies on the Chemical Synthesis of Potential Antimetabolites. 35.1 Synthesis of 2-Chloro-1-deazaadenosine and 2-Chloro-1-deazainosine as Candidate Inhibitors for S-Adenosylhomocysteinases and Methyltransferases

Abstract

The syntheses of 2-chloro-1-deazaadenosine (2) and 2-chloro-1-deazainosine (3) are described. Conversion of 7-ribosylated 6-chloro-1-deazapurine 3-oxide to the desired 2,6-disubstituted 9-ribosyl-1-deazapurines was effected by a series of reactions involving “deoxygenative chlorination” and transglycosylation in satisfactory yields.     

The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol.

Syntheses of H-phosphonate salts (4a-e) of N/S-protected alcohols such as 6-aminohexan-1-ol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol are described using 2-chloro-5,6-benzo-1,3,2-phosphorin-4-one (2) as the phosphonylating agent. The H-phosphonate salts (4a-e), in the presence of pivaloyl chloride or adamantoyl chloride as an activator, were coupled to the 5'-end of synthetic oligonucleotides on solid supports to produce amino or thio-linked oligonucleotides. Following deprotection and purification, fluorescent dyes, biotin derivatives and poly-L-lysine-maleimide were separately attached to the functionalised oligonucleotides. Identical derivatized oligomers were obtained with cyanoethyl-N,N-diisopropylamidite chemistry and amidites (5a-e) of the respective alcohols.     

Gas-liquid chromatographic analysis of fluoropyrimidine nucleosides and fluorouracil in plasma and urine

A gas-liquid chromatographic method employing on-column alkylation and a nitrogen-sensitive detector was developed for the analysis of 5-fluoro-2'-deoxyuridine, 5-fluorouridine, and 5-fluorouracil in plasma and urine. Samples (0.72 ml) containing the fluoropyrimidine and internal standard (5-chloro-2'-deoxyuridine for nucleoside analyses and 6-methyluracil for 5-fluorouracil analyses) were prepared for gas-liquid chromatography by sequential cation-exchange and anion-exchange column chromatography. Recoveries of fluoropyrimidines were 71-95% over the concentration ranges studied. The dried eluate from the anion-exchange column was dissolved in p-tolyltrimethylammonium hydroxide in methanol before gas-liquid chromatographic analysis. Columns packed with either 3% SP-2100 on Supelcoport or 3% OV-1 on Gas-Chrom Q were suitable for nucleoside analyses; a column packed with 0.75% Carbowax 20M-5% KOH on Chromsorb G was used for 5-fluorouracil analyses. The fluoropyrimidine nucleosides were well separated from each other and from the potentially interfering endogenous compounds 2'-deoxyuridine and uridine; 5-fluorouracil was well separated from uracil. Linear standard curves (peak area ratio method) were obtained for plasma containing 0.025 to 20 micrograms FdUrd (0.1 to 81 microM) or 0.05 to 1.0 microgram FUrd (0.2 to 3.8 microM), and for urine containing 0.2 to 1.0 microgram (0.8 to about 4 microM) of the nucleosides. Standard curves for 5-fluorouracil (1.5 to 7.9 microM) and 2'-deoxyuridine (0.9 to 4.4 microM) were also linear. A measurable amount of 5-fluorouracil, equivalent to 4 to 7% of the 5-fluoro-2'-deoxyuridine injected, was formed from the nucleoside on the gas-liquid chromatographic column, requiring correction of 5-fluorouracil concentrations measured in the presence of 5-fluoro-2'-deoxyuridine.     

Synthesis and antitumor activity of 1-substituted-2-methyl-5-nitrobenzimidazoles

Different substituents were introduced in position 1 of 2-methyl-5(6)-nitro-1H-benzimidazole (2) in order to obtain different side chains having different heterocyclic compounds, for example, thiadiazoles (5-7), tetrazoles (8, 9a, b), triazoles (11-13), thiazoles (14a-e), triazines (10, 16, 17), and imidazoles (18a-c). The antitumor effect of compounds 1, 2, 2a, 4, 5, 7, 8, 9a, 10, 13, 14a, 15, 16, and 18c was studied against breast cancer (MCF7) and compounds 2 [IC(50)=4.52 microg] and 7 [IC(50)=8.29 microg] were found to be active.     

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of, followed by condensation with and ester cleavage with TFA, yielded. The IC(50) of and against CCRF-CEM human leukemic lymphoblasts was 1.8+/-0.1 and 2.1+/-0.8 microM, respectively.     

Actions of the Klenow fragment of DNA polymerase I and some DNA glycosylases on chemically stable analogues of N7-methyl-2′-deoxyguanosine

N7-methyl-9-deaza-dG was synthesized and incorporated into oligonucleotides. Thermal melting studies showed that replacement of dG by N7-methyl-9-deaza-dG only slightly decreased DNA duplex stability. Replication of DNA templates containing N7-methyl-9-deaza-dG and the related 7-methyl-7-deaza-dG and 7-deaza-dG by the Klenow fragment of Escherichia coli DNA polymerase I was examined. The dNTP misinsertion frequencies on all three templates were comparably low, although the 7-methyl group significantly slowed down the turnover rates of the polymerase when dCTP was incorporated. The stabilities of N7-methyl-9-deaza-dG and 7-methyl-7-deaza-dG against the actions of formamidopyrimidine DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) were also examined. N7-methyl-9-deaza-dG was stable in the presence of both enzymes. In contrast, 7-methyl-7-deaza-dG was cleaved by Fpg, and possibly by hAAG but at an extremely slow rate. This study suggests that N7-alkyl-9-deaza-dG is a better analogue than 7-alkyl-7-deaza-dG for cellular studies.