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The pyrimidine nucleoside beta-d-2‘-deoxy-2‘-fluoro-2‘-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N4-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-β-d-arabinofuranosyl]cytosine (6) to provide N4-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-β-d-ribofuranosyl]cytosine (7a). The protected 2‘-C-methylcytidine (7c) was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2‘-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2‘-C-methylcytidine and low cellular toxicity.

A series of N3, 5-Anhydro-4-(beta-D-ribofuranosyl)-8-azapurin-2-ones were prepared in multistep reactions from uridine as potential anti-hepatitis C virus (HCV) agents. The synthetic details as well as biological evaluations are discussed.

Based on the discovery of beta-D-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of beta-D- and L-2'-deoxy-2'-fluoroibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2'-fluoro group was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely beta-D-2'-deoxy-2',5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As beta-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2'-fluoro were combined into one molecule, yielding beta-D-2'-deoxy-2'-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.

Based on the discovery of (2'R)-d-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2'-deoxy-2'-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2'-fluoro group, was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2'R)-d-2'-deoxy-2',5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N(4)-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N(4)-hydroxyl and the 2'-fluoro into one molecule, resulting (2'R)-d-2'-deoxy-2'-fluoro-N(4)-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.

The synthesis and anti-HBV and anti-HIV activity of a number of 2′,3′-dideoxy-2′-fluoro-3′-C-hydroxymethyl-β-D-arabinofuranosyl pyrimidine nucleosides are reported.

A base-modified nucleoside analogue, β- d -N 4 -hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC 90 ) of 5.4 μM, an observation that was confirmed by virus yield assays (EC 90 = 2 μM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC 90 of 5 μM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2′-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 μM) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 μM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4–800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 μM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 μM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 μM].

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

A detailed investigation on fluorination of 1,3,5-tri-O-benzoyl-α- d -ribofuranose-2-sulphonate esters is reported. Various combinations of sulfonate esters, fluorinating agents and solvents were evaluated in this study. Organic ammonium fluoride, in particular n-Bu4NF, was found to be better fluorinating agent than inorganic fluoride, and 1,3,5-tri-O-benzoyl-α- d -ribofuranose-2-trifluoromethylsulphonate ester appeared to be the best substrate. The developed method is suitable for stereospecific (arabino) incorporation of radiofluorine ( 18 F ) into the sugar moiety.

A synthesis of the C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (9-deaza-2'-deoxyguanosine) was achieved starting from 2-amino-6-methyl-3H-pyrimidin-4-one (5) and methyl 2-deoxy-3,5-di-O-(p-nitrobenzoyl)-D-erythro-pento-furanoside (11). The anomeric configuration of the C-nucleoside was established by 1H NMR, NOEDS and ROESY. This C-nucleoside did not inhibit the growth of T-cell lymphoma cells.

Synthesis of P1, P2-disubstituted methylenebis(phosphonate)s as inhibitors of inosine monophosphate dehydrogenase is presented.

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a Ubeta or Ualpha nucleotide is a 10(7)-fold slower substrate for UDG (t1/2 approximately 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage. When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil. Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (KDns = 1.5 microM) and binds approximately 25-fold more tightly to Ubeta containing dsDNA (KDapp approximately 50 nM). Thus, base flipping contributes less than approximately 2 kcal/mol to the free energy of binding and is not a major component of the10(6)-fold catalytic specificity of UDG. Kinetic studies at 25 degrees C show that site-specific binding occurs by a two-step mechanism. The first step (E + S left and right arrow ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (KD approximately 1.5-3 microM). The second step (ES left and right arrow E'F) involves a rapid step leading to reversible uracil flipping (kmax approximately 1200 s-1). This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence. The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate. The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil. Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism.

We examined the effects of 1–(2-deoxy -2-fluoro-β-D-arabinofuranosyl)-thymine (or FMAU, a potent antiviral nucleoside) on the stability of duplex and triplexes. When compared the stability of the self-complementary 5′-A5T5 duplex with 5′-A5X5 (X = FMAU), duplex containing FMAU has much higher melting temperature (Tm). 5′-A6T5T3X3T5F3X3 and T3X3T5A6T5F3X3 form the parallel and antiparallel triplexes T3X3: A6:X3X3, respectively. The former exhibited the typical T:A:T triplex behavior with only one melting temperature at 70 °C and 45 °c in 1.0 M and 0.2 M NaCl solution, respectively, whereas the latter has two Tm values at 56 °C and 28 °C in 1.0 M solution. FMAU clearly stabilize the triplex structure as A6T22 which forms the parallel triplex T6:A6:T6 has also only one Tm at 54 °C and 37 °C in high and iow salt concentration solutions, respectively. A 31mer 5′-TCCTCCTTTTTTAGGAGGATTTTTTGGTGGT and 5′-TCCTCCTTTTTTAGGAGGATTTTTTX'X'TX'X'T (X' = 2′-deoxy-5-methylcytidine) were prepared to study their trip...

2-(4-Nitrophenylethyl) methylenebis(phosphonate) (1) has been prepared by reaction of 2-(4-nitrophenyl)ethyl alcohol with methylenebis(phosphonyl) tetrachloride. Compound 1 was treated with diisopropylcarbodiimide (DIC) to give bicyclic intermediate 2, which in reaction with suitably protected 2′-deoxynucleosides 3 gave P1,P2-disubstituted methylenebis(phosphonate)s 4. Removal of the nitrophenylethyl group by β-elimination with DBU afforded the corresponding 2′-deoxynucleoside 5′-methylenebis(phosphonate) analogues 5. This paper is dedicated to the 60th birthday of Professor Jacques H. van Boom.

Several years ago, we published an hypothesis concerning conformation of the glycone moiety of different substrates in active centers of several DNA metabolizing enzymes (Nucleosides & Nucleotides 1993, 12, 649–670). This hypothesis prompted us to further study the subtle conformational changes on substrates of DNA polymerases. Data collected in our, as well as other laboratories, have been analyzed, and models of active centers of different DNA polymerases are discussed below. Based on the model of substrate requirements, we now can divide DNA polymerases into two distinguished classes.

We have found that several aliphatic and alicyclic diols induce melanogenesis in cultured S91 mouse melanoma cells and normal human epidermal melanocytes (NHEM). In addition, these compounds induce melanogenesis when applied to guinea pig skin, with transfer of melanin to keratinocytes and formation of “supranuclear caps,” as occurs in naturally pigmented skin. The relative order of potency of some of these diols in NHEM is 5-norbornene-2,2-dimethanol bg 3,3-dimethyl-1,2-butanediol bg cis-1,2-cyclopentanediol bg 2,3-dimethyl-2,3-butanediol bg 1,2-propanediol. Following treatment with these diols or 3-isobutyl-1-methylxanthine, melanin and tyrosinase activity are increased within S91 cells and NHEM; however, for cultured NHEM, the largest increases of melanin and tyrosinase occur in an extracellular particulate fraction, shown by electron microscopy to consist almost entirely of stage III and IV melanosomes. These results indicate that cultured NHEM treated with diols export melanosomes in a fashion that is commensurate with natural melanogenic processes. In contrast, S91 mouse melanoma cells exhibit aberrant melanosomal trafficking, in accordance with the known defect in myosin-V mediated melanosomal transport. Both S91 cells and NHEM exhibit morphologic changes and growth arrest indicative of differentiation following treatment with diols. The diols described in this report are candidates for use as cosmeceutical tanning agents.

2-(4-Nitrophenyl)ethyl methylenebis(phosphonate) (6) was prepared by reaction of equimolar amounts of 2-(4-nitrophenyl)ethyl alcohol and methylenebis(phosphonyl) tetrachloride in the presence of tetrazole. Compound 6 was further converted into the corresponding 4-nitrophenylethyl trisanhydride intermediate 7 by dehydration with diisopropylcarbodiimide (DIC). Reaction of 7 with either 2‘,3‘-O-isopropylideneadenosine (8a) or 2‘,3‘-O-isopropylideneguanosine (8b) afforded, after hydrolysis, the desired P1-[2-(4-nitrophenyl)ethyl]-P2-(2‘,3‘-O-isopropylideneadenosin-5‘-yl) methylenebis(phosphonate) (9a) and guanosine analogue 9b, respectively. A similar treatment of intermediate 7 with 3‘-O-acetylthymidine (12a), 3‘-O-acetyl-2‘-deoxy-N4-benzoylcytidine (12b), 3‘-O-acetyl-2‘-deoxy-N6-benzoyladenosine (12c), and 3‘-O-acetyl-2‘-deoxy-N2-isobutyrylguanosine (12d) gave the corresponding 2-(4-nitrophenyl)ethyl methylenebis(phosphonate)s 13a−d. These compounds as well as 9a,b were treated with 1,8-diazabicyclo[5.4.0]u...

beta-Methylene-BAD (8), a nonhydrolyzable analogue of benzamide adenine dinucleotide (BAD), was synthesized as potential inhibitor of human inosine monophosphate dehydrogenase (IMPDH). Treatment of 2',3'-O-isopropylideneadenosine 5'-methylenebisphosphonate (15) with DCC afforded P1,P4-bis(2',3'-O-isopropylideneadenosine) 5'-P1,P2:P3,P4-dimethylenetetrakisphosphonate (17). This compound was further converted with DCC to an active intermediate 18 which upon reaction with 3-(2',3'-O-isopropylidene-beta-D-ribofuranosyl)benzamide (19) gave, after hydrolysis and deisopropylidenation, the desired beta-methylene-BAD (8) in 95% yield. In a similar manner, treatment of 18 with 2',3'-O-isopropylidenetiazofurin (21) followed by hydrolysis and deprotection afforded beta-methylene-TAD (5) in 91% yield. Compound 8 (IC50 = 0.665 microM) was found to be a 6-8 times less potent inhibitor of IMPDH than 5 (IC50 = 0.107 microM) and was almost equally potent against IMPDH type I and type II. Although TAD and beta-methylene-TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (Ki = 333 microM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (Ki = 6.3 microM).

5′,9-Anhydro-3-(β- d -ribofuranosyl)xanthine and 3,5′-anhydro-xanthosine were prepared as potential anti-hepatitis C virus (HCV) agents from uridine and xanthosine, respectively.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.

Cofactor type inhibitors (NAD-analogues) of IMP-dehydrogenase (IMPDH) were synthesized and their application as potential anticancer agents are discussed. C-nucleoside isosteres of NAD, C-NAD and C-PAD, showed an effective competitive inhibition of IMPDH, C-NAD but not C-PAD caused extremely potent inhibition of alcohol dehydrogenase. We also synthesized compounds in which nicotinamide riboside was replaced with tiazofurin (TAD-analogues) and the 2' and 3'-positions of adenosine part were fluorinated. The ribose ring of 2'-deoxy-2'-fluoroadenosine is in the C3'-endo conformation whereas 3'-deoxy-3'-fluoroadenosine favors the C2'-endo sugar pucker. These derivatives are good inhibitors of IMPDH type II, the isoenzyme dominant in neoplastic cells. In contrast, all these analogues showed rather week inhibitory activity against alcohol dehydrogenase. Nicotinamide riboside derivatives in which the base and the sugar are linked through an oxygen or a methylene bridge were synthesized. NAD-analogues containing such conformationally restricted nicotinamide nucleoside moiety (syn or anti) are expected to be selective inhibitors of B-specific (IMPDH) or A-specific dehydrogenases, respectively.

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were synthesized via Diels-Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating into double-stranded DNA and bind covalently. Structure-activity relationships were studied. Of these compounds, 2,3-aziridino-4-[[(methylamino)carbonyl]methyl] cyclopent[alpha]anthracene-6,11-dione (4) was shown to have inhibitory activity against several leukemic and solid tumor cell lines. Mice (BDF1) bearing Lewis lung adenocarcinoma were treated with 4 and MMC (i.p., QD x 5). At a dose of 30.0 mg/kg, compound 4 was as effective as MMC (0.8 mg/kg). Compound 4 appears to be less toxic than MMC. DNA unwinding assay indicated that 4 is able to intercalate into DNA double strands and is also a topoisomerase II inhibitor.

The first synthesis of nitro-multideoxy-sugar containing nucleosides was achieved. 1-(4,6-O-Benzylidene-3-deoxy-3-nitro-β-D-glucopyranosyl)uracil (3) was converted in 75% yield into 1-(4,6-O-benzylidene-2,3-dideoxy-3-nitro-arabinohexopyranosyl)uracil (7) by acetylation followed by NaBH4 reduction in methanol. De-O-benzylidenation with CF3CO2H afforded crystalline 1-(2,3-dideoxy-3-nitro-β-D-arabinohexopyranosyl)uracil (S) was obtained in 87% yield. Raney Ni reduction of 8 afforded the corresponding 3′-amino-nucleoside 9. Acetylation of 8 followed by NaBH4 treatment afforded an 8:1 mixture from which 1-(2,3,4-trideoxy-3-nitro-β-D-threohexopyranosyl)-uracil (14) was obtained in pure crystalline form. After Raney Ni reduction of the mixture, 1-(3-amino-2,3,4-trideoxy-β-d-threo-hexopyranosyl)uracil (16) and its erythro epimer 21 were isolated. 1-(4,6-O-Benzylidene-2,3-dideoxy-3-nitro-β-d-lyxohexopyranosyl)uracil (24) was prepared in 72% yield from 1-(4,6-O-benzylidene-3-deoxy-3-nitro-β-d-galactopyrano...

A series of DNA-intercalating 9-anilinoacridines, namely 9-phenoxyacridines, 9-(phenylthio)-acridines, and 9-(3',5'-disubstituted anilino)acridines, were synthesized as potential antitumor agents with inhibitory effects on DNA topoisomerase II. Unlike amsacrine (m-AMSA), these agents were designed to avoid the oxidative metabolic pathway. These acridine derivatives were, therefore, expected to have long half-life in plasma. Both 9-phenoxyacridines and 9-(phenylthio)acridines were found to have moderate cytotoxicity against mouse leukemia L1210 and human leukemic HL-60 cell growth in culture. Among 9-(3',5'-disubstituted anilino)-acridines, 3-(9-acridinylamino)-5-(hydroxymethyl)aniline (AHMA) was found to be a potent topoisomerase II inhibitor and exhibited significant antitumor efficacy both in vitro and in vivo. Chemotherapy of solid-tumor-bearing mice with 10, 10, and 5 mg/kg (QD x 4, ip) AHMA, VP-16, and m-AMSA, respectively, resulted in more tumor volume reduction by AHMA than by VP-16 or m-AMSA for E0771 mammary adenocarcinoma and B-16 melanoma. For Lewis lung carcinoma, AHMA was as potent as VP-16 but more active than m-AMSA. Structure-activity relationships of AHMA derivatives are discussed

A number of 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil and -cytosine nucleosides substituted at the 5 position with a nitrophenyl or nitrobenzyl group were synthesized from 5-phenyl- and 5-benzyluracil via condensation of the fluorinated sugar, followed by nitration. The corresponding amino analogues were also prepared by reduction of the nitro nucleosides. The uracil nucleosides were converted into the corresponding cytosine nucleosides by way of the triazole intermediates. None of these nucleosides exhibited significant activity against herpes simplex virus type 1 in Vero cells. However, cytosine nucleosides containing the o-nitrophenyl, p-nitrophenyl, p-nitrobenzyl or p-aminobenzyl substituent were found to be toxic (even at 1 μM) to uninfected Vero cells, although they were essentially nontoxic in HL-60 cells. The 5′-monophosphates of the uracil nucleosides were inhibitors of the reaction catalyzed by purified Ehrlich ascites carcinoma thymidylate synthase, the 5-phenyluracil nucleotides...

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric and isomeric analogue of NAD, in which the nicotinamide ring is linked to the sugar via a C-glycosyl (C5-C1') bond. CNAD acts as a general dehydrogenase inhibitor but shows unusual specificity and affinity for liver alcohol dehydrogenase (ADH, EC 1.1.1.1). The pattern of inhibition is congruent to 4 nM, with NAD as the variable substrate. These values are 3-5 orders of magnitude smaller than those obtained for CNAD in other dehydrogenases and are comparable to values observed for the tightest binding ADH inhibitors known. The specificity and affinity of CNAD for ADH are likely due to coordination of the zinc cation at the ADH catalytic site by the CNAD pyridine nitrogen. This is supported by kinetic and computational studies of ADH-CNAD complexes. These results are compared with those for a related analogue, CPAD. In this analogue, displacement of the pyridine nitrogen to the opposite side of the ring removes the specificity for ADH.

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the antitumor drug tiazofurin. Beta-methylene TAD (beta-TAD) is a phosphodiesterase-resistant analogue of TAD, active in tiazofurin-resistant cells. Beta-methylene SAD (beta-SAD) is the active selenium derivative of beta-TAD. Both agents are analogues of the cofactor NAD and are capable of acting as general dehydrogenase inhibitors. Crystal structures of beta-TAD and beta-SAD bound to horse liver alcohol dehydrogenase (LADH) are presented at 2.9 and 2.7 A, respectively. Both complexes crystallize in the orthorhombic space group C222(1) and are isomorphous to apo-LADH. Complexes containing beta-TAD and beta-SAD were refined to crystallographic R values of 15% and 16%, respectively, for reflections between 8 A and the minimum d spacing. Conformations of both inhibitors are similar. beta-TAD and beta-SAD bind to the "open" form of LADH in the normal cofactor-binding cleft between the coenzyme and catalytic domains of each monomer. Binding at the adenosine end of each inhibitor resembles that of NAD. However, the positions of the thiazole and selenazole heterocycles are displaced away from the catalytic Zn cation by approximately 4 A. Close intramolecular S-O and Se-O contacts observed in the parent nucleoside analogues are maintained in both LADH-bound beta-TAD and beta-SAD, respectively. These conformational constraints may influence the binding specificity of the inhibitors.

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.

Recent research activities directed toward the synthesis of 2′-β-fluoro-substitutednucleosides by nucleophilic displacement of an activated 2′-hydroxy group of preformedribonucleosides are reviewed. In the pyrimidine- C -nucleoside area, direct fluorination ofa 4,5′-anhydro- C -nucleoside triflate with TASF afforded the desired 2′-β-fluoro-1-methyl-ψ-uridine (C-FMAU). A similar strategy in regular pyrimidine series by using the corresponding2,5′- O -, 2,3′- O - and 5′,6-anhydro nucleoside derivatives did not give thedesired fluoro-containing products. An interesting triflyl migration was discovered whena triflate nucleoside was treated with LiCl in HMPA. The carbocyclic analog of theadenosine containing a fluorine in the 2′-β configuration was prepared from O 5′ , O 3′ , N 6 -tribenzoylaristeromycin in reaction with DAST. A similar treatment of purine nucleosidesled to decomposition. When, however, inosine, adenosine and guanosine were tritylatedat the 3′,5′ positions of the sugar moiety and suitably protected at the base, thecorresponding 2′-β-fluoro-substituted arabino -nucleosides were obtained by treatmentwith DAST in moderate to good yields. Deprotection afforded the desired F- ara -H,F- ara -A and F- ara -G. The role of the bulky trityl groups at the 3′,5′ positions of thesugar and the effect of C-3′- endo to C-2′- endo conformational shift on the reaction courseof 2′-hydroxyl group with DAST is discussed.

Many different modified nucleosides and nucleotides with conformationally restricted partly flattened sugar residues are analyzed as substrates or inhibitors of several groups of enzymes of nucleic acid metabolism. A detailed examination of the sugar moiety of large group of modified nucleosides showed that there is a striking conformational similarity, i.e., they are flattened. We propose herein a hypothesis which can represent a general conformational elements in the structure of the active sites of several different groups of enzymes. This proposal envisions that during the enzymatic process natural substrates should reflect these flattened conformations. This hypothesis allows computation of conformational analyses of the enzyme actives centers as well as the design of new actively metabolized modified nucleosides.

Two isosteric analogues of nicotinamide adenine dinucleotide, C-NAD (11) and C-PAD (12), in which the nicotinamide riboside portion is replaced by a C-nucleoside, were synthesized from 5-(beta-D-ribofuranosyl)nicotinamide (7) and 6-(beta-D-ribofuranosyl)picolinamide (8), respectively. Nucleoside 7 was prepared from the 2,3-O-isopropylidene-5-O-(tetrahydropyranyl)-D-ribonolactone (13) and 3-cyano-5-lithiopyridine as reported earlier. Nucleoside 8 was obtained by conversion of the bromo function of the 6-(2,3:4,5-di-O-isopropylidene-D-altro-pentitol-1-yl)-2-bromopyrid ine (14) into a carboxamido group followed by mesylation of the anomeric hydroxyl group to give derivative 18. Treatment of 18 with CF3COOH/CHCl3 caused deisopropylidenation with simultaneous cyclization into the desired 6-(beta-D-ribofuranosyl)picolinamide (8). NAD analogues, C-NAD (11) and C-PAD (12), were synthesized by imidazole-catalyzed coupling of the corresponding 5'-monophosphates of 7 and 8 with the adenosine-5'-monophosphate. Dinucleotide 11 was found to inhibit the proliferation of L1210 cells (IC50 = 7 microM) and to be a good competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH, ID50 = 20 microM) as well as bovine glutamate dehydrogenase (GDH, Ki = 15 microM). Interestingly, C-NAD (11) caused extremely potent noncompetitive inhibition of horse liver alcohol dehydrogenase (ADH, Ki = 1.1 nM), whereas C-PAD (12) was found to be a much less potent competitive inhibitor (Ki = 20 microM) of ADH.

As an epimerization resistant synthon in the synthesis of oligo-nucleotides consisting of C-nucleoside analogues, hitherto unknown 5-benzyloxy-methyl-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[3,2-d]pyrimpyrimidine (7-benzyloxymethyl-2′-deoxy-9-deazaadenosine) was prepared in seven steps from the known 3-amino-2-cyano-4-(2,3-O-isopropylidene-5-O-trityl-β-D-ribofuranosyl)-pyrrolpyrrole (1). Treatment of 1 with benzyl chloromethyl ether in the presence of potassium t-butoxide and 18-crown-6 afforded the N-protected pyrrole 2, which was converted into the 9-deazapurine derivative 3 in high yield by heating in EtOH. 7-Benzyloxymethyl-9-deazaadenosine 4 was obtained from 3 by acid hydrolysis in 2.5% methanolic hydrogen chloride. After protection of the hydroxyl groups of 4 with Markievicz's reagent, the product 5 was converted into the 2′-O-phenoxythiocarbonyl derivative 6. Reduction of 6 with butyltin hydride in the presence of 2,2′-azobis(2-methylpropionitrile), followed by desilylation with trie...

2,3-Dihydro-1H-cyclopent[a]anthracene-6,11-dione (cyclopentanthraquinone) derivatives bearing a mustard side chain at C-4 and an aziridine ring at the cyclopentene moiety were synthesized. Such a compound may be viewed as an analogue of mitomycin C (MC) and may exhibit bifunctional DNA-alkylating and DNA-intercalating agent. The key intermediate, 4-hydroxy-2,3-di-O-substituted cyclopentanthraquinone, was synthesized by Diels-Alder reaction of naphthoquinone with various 4,5-dihydroxy-1-vinylcyclopent-1-ene derivatives.Introduction of a mustard side chain to the OH group at C-4 and subsequent construction of an aziridine-ring on C 2 -C 3 of the cyclopentanthroquinone molecule furnished the target compound, 2,3-aziridino-4-[2-[N,N-bis(2-chloroethyl)amino]ethoxy]-2,3-dihydro-1H-cyclopenta[a]anthrcene-6,11-dione.It was found that both the aziridine function and the mustard side chain play a significant role in their cytotoxicity against leukemic L1210 and HL-60 cell growth in culture and the inhibition of topoisomerase II kDNA decatenation

A module type, computer-controlled, multipurpose synthesizer displaying a novel device for the transport of liquids, was constructed and used in the synthesis of oligomers containing some C-nucleosides and 2′-deoxy-2′-fluoro-ara-nucleoside moieties. H-Phosphonate method was applied in terms of a further adjustment of construction features of the synthesizer versus chemistry of the process. Results of preliminary studies on the effects of the modified nucleosides on the stability of duplexes showed a clear tendency of destabilization of duplexes in the case of C-nucleosides while fluorinated nucleosides in most cases stabilize the formed duplexes.

This paper is dedicated to the memory of Professor Roland K. Robins The synthesis of 4-[(1,3-diaminopyrrolo[3′,4′:4,5]pyrido[2,3-d]pyrimidin-8-yl)benzoyl]-L-glutamic acid (18), a potential antifolate and anticancer agent, has been achieved starting from 1,4-dibromobutan-2-ol with alkyl p-aminobenzoic acids. Condensation of these two agents gave 1-(4-alkoxycarbonylphenyl)pyrrolidin-3-ols 7a,b, which were oxidized to the corresponding pyrrolidin-3-one derivatives 8a,b. Compounds 8a,b were converted into 1,3-diamino-8-(4-alkoxycarbonylphenyl)-7,8-dihydro-9H-pyrrolo[3′,4′:4,5]pyrido[2,3-d]pyrimidines 12a,b in 4 steps. Saponification of 12b the benzoate ester and coupling with di-tert-butyl glutamate afforded a mixture of 7,8-dihydro product 16 and its aromatized derivative 17. Finally hydrolysis of esters 16 or 17 gave only the title compound 18. The 7,8-dihydro tricyclic derivatives were easily air-oxidized to form their fully aromatized compounds. The title compound 18 was one tenth less active than MTX against HL-60 cells in culture.

Many different modified nucleosides and nucleotides with conformationally restricted partly flattened sugar residues are analyzed as substrates or inhibitors of several groups of enzymes of nucleic acid metabolism. A detailed examination of the sugar moiety of large group of modified nucleosides showed that there is a striking conformational similarity, i.e., they are flattened. We propose herein a hypothesis which can represent a general conformational elements in the structure of the active sites of several different groups of enzymes. This proposal envisions that during the enzymatic process natural substrates should reflect these flattened conformations. This hypothesis allows computation of conformational analyses of the enzyme actives centers as well as the design of new actively metabolized modified nucleosides.

Recent research activities directed toward the synthesis of 2′-β-fluoro-substitutednucleosides by nucleophilic displacement of an activated 2′-hydroxy group of preformedribonucleosides are reviewed. In the pyrimidine- C -nucleoside area, direct fluorination ofa 4,5′-anhydro- C -nucleoside triflate with TASF afforded the desired 2′-β-fluoro-1-methyl-ψ-uridine (C-FMAU). A similar strategy in regular pyrimidine series by using the corresponding2,5′- O -, 2,3′- O - and 5′,6-anhydro nucleoside derivatives did not give thedesired fluoro-containing products. An interesting triflyl migration was discovered whena triflate nucleoside was treated with LiCl in HMPA. The carbocyclic analog of theadenosine containing a fluorine in the 2′-β configuration was prepared from O 5′ , O 3′ , N 6 -tribenzoylaristeromycin in reaction with DAST. A similar treatment of purine nucleosidesled to decomposition. When, however, inosine, adenosine and guanosine were tritylatedat the 3′,5′ positions of the sugar moiety and suitably protected at the base, thecorresponding 2′-β-fluoro-substituted arabino -nucleosides were obtained by treatmentwith DAST in moderate to good yields. Deprotection afforded the desired F- ara -H,F- ara -A and F- ara -G. The role of the bulky trityl groups at the 3′,5′ positions of thesugar and the effect of C-3′- endo to C-2′- endo conformational shift on the reaction courseof 2′-hydroxyl group with DAST is discussed.

A series of DNA-intercalating 9-anilinoacridines, namely 9-phenoxyacridines, 9-(phenylthio)-acridines, and 9-(3',5'-disubstituted anilino)acridines, were synthesized as potential antitumor agents with inhibitory effects on DNA topoisomerase II. Unlike amsacrine (m-AMSA), these agents were designed to avoid the oxidative metabolic pathway. These acridine derivatives were, therefore, expected to have long half-life in plasma. Both 9-phenoxyacridines and 9-(phenylthio)acridines were found to have moderate cytotoxicity against mouse leukemia L1210 and human leukemic HL-60 cell growth in culture. Among 9-(3',5'-disubstituted anilino)-acridines, 3-(9-acridinylamino)-5-(hydroxymethyl)aniline (AHMA) was found to be a potent topoisomerase II inhibitor and exhibited significant antitumor efficacy both in vitro and in vivo. Chemotherapy of solid-tumor-bearing mice with 10, 10, and 5 mg/kg (QD x 4, ip) AHMA, VP-16, and m-AMSA, respectively, resulted in more tumor volume reduction by AHMA than by VP-16 or m-AMSA for E0771 mammary adenocarcinoma and B-16 melanoma. For Lewis lung carcinoma, AHMA was as potent as VP-16 but more active than m-AMSA. Structure-activity relationships of AHMA derivatives are discussed

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were synthesized via Diels−Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating into double-stranded DNA and bind covalently. Structure−activity relationships were studied. Of these compounds, 2,3-aziridino-4-[[(methylamino)carbonyl]methyl]cyclopent[a]anthracene-6,11-dione (4) was shown to have inhibitory activity against several leukemic and solid tumor cell lines. Mice (BDF1) bearing Lewis lung adenocarcinoma were treated with 4 and MMC (ip, QD × 5). At a dose of 30.0 mg/kg, compound 4 was as effective as MMC (0.8 mg/kg). Compound 4 appears to be less toxic than MMC. DNA unwinding assay indicated that 4 is able to intercalate into DNA double strands and is also a topoisomerase II inhibitor.

2-(4-Nitrophenyl)ethyl methylenebis(phosphonate) (6) was prepared by reaction of equimolar amounts of 2-(4-nitrophenyl)ethyl alcohol and methylenebis(phosphonyl) tetrachloride in the presence of tetrazole. Compound 6 was further converted into the corresponding 4-nitrophenylethyl trisanhydride intermediate 7 by dehydration with diisopropylcarbodiimide (DIC). Reaction of 7 with either 2‘,3‘-O-isopropylideneadenosine (8a) or 2‘,3‘-O-isopropylideneguanosine (8b) afforded, after hydrolysis, the desired P1-[2-(4-nitrophenyl)ethyl]-P2-(2‘,3‘-O-isopropylideneadenosin-5‘-yl) methylenebis(phosphonate) (9a) and guanosine analogue 9b, respectively. A similar treatment of intermediate 7 with 3‘-O-acetylthymidine (12a), 3‘-O-acetyl-2‘-deoxy-N4-benzoylcytidine (12b), 3‘-O-acetyl-2‘-deoxy-N6-benzoyladenosine (12c), and 3‘-O-acetyl-2‘-deoxy-N2-isobutyrylguanosine (12d) gave the corresponding 2-(4-nitrophenyl)ethyl methylenebis(phosphonate)s 13a−d. These compounds as well as 9a,b were treated with 1,8-diazabicyclo[5.4.0]u...

2-(4-Nitrophenylethyl) methylenebis(phosphonate) (1) has been prepared by reaction of 2-(4-nitrophenyl)ethyl alcohol with methylenebis(phosphonyl) tetrachloride. Compound 1 was treated with diisopropylcarbodiimide (DIC) to give bicyclic intermediate 2, which in reaction with suitably protected 2′-deoxynucleosides 3 gave P1,P2-disubstituted methylenebis(phosphonate)s 4. Removal of the nitrophenylethyl group by β-elimination with DBU afforded the corresponding 2′-deoxynucleoside 5′-methylenebis(phosphonate) analogues 5. This paper is dedicated to the 60th birthday of Professor Jacques H. van Boom.

Tritylation of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)guanosine (4) with TrCl/DMAP followed by TrCl/AgNO 3 afforded a mixture of isomeric 3',5'-di-O-trityl and 2',5'-di-O-trityl derivatives 6 and 7, which were separated on a silica gel column to give 6 and 7 in 40% and 50% yield, respectively. Upon treatment with DAST, 6 was converted into the corresponding 2'-β-fluoro nucleoside 8 in 43% yield. Deprotection of the 6-N-acetyl group occurred during the reaction. Removal of the 6-O-NPE group from 8 with DBU/ pyridine, followed by detritylation with CF 3 COOH/CHCl 3 , gave F-ara-G (1b) in good yield. The same treatment of 7 with DAST did not lead to nucleophilic substitution with fluoride ion, but only decomposition took place