1,N2-ethenodeoxyguanosine: properties and formation in chloroacetaldehyde-treated polynucleotides and DNA.

1,N2-Etheno-2'-deoxyguanosine (1,N2-epsilon dGuo), not previously reported as a product of chloroacetaldehyde (CAA) reaction, has been synthesized and characterized. Reaction of deoxyguanosine with CAA in dimethylformamide in the presence of K2CO3 led to preparation of pure 1,N2-epsilon dGuo with 55% yield. pKa values are 2.2 and 9.2. The anionic form of the compound exhibits weak but defined fluorescence; the intensity is similar to that of N2,3-etheno-2'-deoxyguanosine (N2,3-epsilon dGuo) at neutrality. The stability of the glycosyl bond of 1,N2-epsilon dGuo (t1/2 = 2.3 h at 37 degrees C, pH 1) is 10-fold greater than of unmodified deoxyguanosine and at least one thousand-fold greater than of isomeric N2,3-epsilon dGuo. Reaction of CAA with model polynucleotides indicates that hydrogen bonding of guanine residues in the double-stranded structures is, as expected, an important factor in the formation of 1,N2-ethenoguanine. In contrast, the formation of isomeric N2,3-ethenoguanine is relatively independent of whether the DNA is single- or double-stranded. In salmon sperm DNA, reacted with CAA at neutrality, the formation of 1,N2-ethenoguanine could be demonstrated. However, we find the efficiency of formation of this adduct in double-stranded DNA to be lower than that of all other etheno derivatives.     

Development of an on-line liquid chromatography-electrospray tandem mass spectrometry assay to quantitatively determine 1,N(2)-etheno-2'-deoxyguanosine in DNA

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysate. Using the newly developed technique, basal levels of 1,N(2)-etheno-2'-deoxyguanosine were determined in commercial calf thymus DNA (1.70 +/- 0.09 adducts/10(7) dGuo), in cultured mammalian cells (CV1-P) DNA (4.5 +/- 0.4 adducts/10(7) dGuo), and in untreated female rat liver DNA (5.22 +/- 1.37 adduct/10(7) dGuo). The mutagenicity of 1,N(2)-etheno-2'-deoxyguanosine had already been demonstrated by in vitro and in vivo systems. The method described here provides the first evidence of the occurrence of 1,N(2)-etheno-2'-deoxyguanosine as a basal endogenous lesion and may be usefully employed to assess the biological consequences of etheno DNA damage under normal and pathological conditions.     

Structure of the 1,N2-ethenodeoxyguanosine adduct opposite cytosine in duplex DNA: Hoogsteen base pairing at pH 5.2

The exocyclic 1, N(2)-ethenodeoxyguanosine (1,N(2)-is an element of dG) adduct, arising from the reaction of vinyl halides and other vinyl monomers, including chloroacetaldehyde, and lipid peroxidation products with dG, was examined at pH 5.2 in the oligodeoxynucleotide duplex 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCCATGCG)-3' (X = 1,N(2)-is an element of dG). Previously, X(anti).C(anti) pairing was established in this duplex, containing the 5'-TXG-3' sequence context, at pH 8.6 [Shanmugam, G., Goodenough, A. K., Kozekov, I. D., Harris, T. M., Guengerich, F. P., Rizzo, C. J., and Stone, M. P. (2007) Chem. Res. Toxicol. 20, 1601- 1611]. At pH 5.2, the 1,N(2)-is an element of dG adduct decreased the thermal stability of the duplex by approximately 13 degrees C. The 1,N(2)-is an element of dG adduct rotated about the glycosyl bond from the anti to the syn conformation. This resulted in the observation of a strong nuclear Overhauser effect (NOE) between the imidazole proton of 1,N(2)-is an element of dG and the anomeric proton of the attached deoxyribose, accompanied by an NOE to the minor groove A(20) H2 proton from the complementary strand. The syn conformation of the glycosyl bond at 1,N(2)-is an element of dG placed the exocyclic etheno moiety into the major groove. This resulted in the observation of NOEs between the etheno protons and the major groove protons of the 5'-neighboring thymine. The 1,N(2)-is an element of dG adduct formed a Hoogsteen pair with the complementary cytosine, characterized by downfield shifts of the amino protons of the cytosine complementary to the exocyclic adduct. The pattern of chemical shift perturbations indicated that the lesion introduced a localized structural perturbation involving the modified base pair and its 3'- and 5'-neighbor base pairs. A second conformational equilibrium was observed, in which both the modified base pair and its 3'-neighboring G.C base pair formed tandem Hoogsteen pairs. The results support the conclusion that at neutral pH, in the 5'-TXG-3' sequence, the 1,N(2)-is an element of dG adduct exists as a blend of conformations in duplex DNA. These involve the interconversion of the glycosyl torsion angle between the anti and the syn conformations, occurring at an intermediate rate on the NMR time scale.     

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyguanosine

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1) was allowed to react in the presence of 2'-deoxyguanosine (16 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC/MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 24% of 1 reacted with different heteroatoms of the nucleoside. As expected, the principal site of 2'-deoxyguanosine alkylation was N7. Alkylation of N7 caused spontaneous depurination, and N-(7-guaninylethyl)-N-hydroxyethyl-p-aminophenylbutyric acid (5) and the corresponding N7,N7-bis-adduct (6) were the major stable dGuo derivatives. Also several other adducts were detected and tentatively identified by means of MS/MS and UV. From them, the O(6-), N1-, N(2-), and O5'-derivatives can be biologically significant. Our results shed new light on DNA modifications caused by chlorambucil, which is an important chemotherapeutic drug and a known carcinogen.     

Mechanism of 1,N2-etheno-2'-deoxyguanosine formation from epoxyaldehydes

Background levels of etheno adducts have been attributed to the reaction of DNA with 2,3-epoxyaldehydes, a proposed product of lipid peroxidation. We have examined the reaction of (2R,3S)-epoxyhexanal with dGuo to give 7-(1S-hydroxybutyl)-1,N(2)-etheno-dGuo. We observed that the stereochemistry of the side chain scrambled over time. This process provided insight into the mechanism for the formation of 1,N(2)-etheno-dGuo from 4,5-epoxy-2-decenal [Lee, S. H., et al.(2002) Chem. Res. Toxicol. 15, 300-304]. The mechanistic proposal predicts that 2-octenal is a by-product of the reaction. The reaction of 4,5-epoxy-2-decenal was reinvestigated, and the 2-octenal adduct of dGuo was identified as a product of this reaction in support of the mechanistic proposal. Also observed are products that appear to be derived from 2,3-epoxyoctanal, which can be formed through Schiff base formation of 4,5-epoxy-2-decenal with the dGuo followed by hydration of the double bond and retro-aldol reaction.     

Synthesis and cytotoxicity of 4-amino-5-oxopyrido[2,3-d]pyrimidine nucleosides

A number of nucleoside analogues have been either used clinically as anticancer drugs or evaluated in clinical studies, while new nucleoside analogues continue to show promise. In this article, we report synthesis and cytotoxicity of a series of new pyrido[2, 3-d]pyrimidine nucleosides. 2-Amino-3-cyano-4-methoxypyridine was converted, in two steps, to 4-amino-5-oxopyrido[2,3-d]pyrimidine. A variety of 1-O-acetylated pentose sugar derivatives were condensed with silylated 4-amino-5-oxopyrido[2,3-d]pyrimidine, followed by protection, to afford a series of 4-amino-5-oxopyrido[2, 3-d]pyrimidine nucleosides. Further derivatizations provided an additional group of pyrido[2,3-d]pyrimidine nucleosides. These nucleosides were evaluated for in vitro cytotoxicity to human prostate cancer (HTB-81) and mouse melanoma (B16) cells as well as normal human fibroblasts (NHF). A number of compounds (1a,b, 2a-c,f, 3f+4d) showed significant cytotoxicity to cancer cells, with 4-amino-5-oxo-8-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine (1b) being the most potent proliferation inhibitor (EC(50): 0.06-0.08 microM) to all types of cells tested. However, a selective inhibition to the cancer cells was observed for 4-amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (2b), which is a potent inhibitor of HTB-81 (EC(50): 0.73 microM) and has a favorable in vitro selectivity index (28).     

Synthesis and biological activity of nucleosides and nucleotides related to the antitumor agent 2-beta-D-ribofuranosylthiazole-4-carboxamide

Phosphorylation of 2-beta-D-ribofuranosylthiazole-4-carboxamide (1) provided the 5'-phosphate 2, which was converted to the corresponding 5'-triphosphate 4 and the cyclic 3',5'-phosphate 5. Treatment of 2-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiazole-4-carbonitrile (6) with NH3-NH4Cl provided 2-beta-D-ribofuranosylthiazole-4-carboxamidine hydrochloride (7), and treatment with H2S-pyridine provided the corresponding 4-thiocarboxamide 9. Compound 9 was treated with ethyl bromopyruvate, followed by treatment with methanolic ammonia, to yield 2'-(2-beta-D-ribofuranosylthiazol-4-yl)thiazole-4'-carboxamide (11). 5'-Phosphate 2 was cytotoxic to L1210 cells in culture and significantly effective against the intraperitoneally implanted murine leukemias in mice. Amidine 7 was slightly toxic to L1210 in culture and inhibitory to purine nucleoside phosphorolysis. The cyclic 3',5'-phosphate 5 was less effective than the corresponding 5'-phosphate 2 or the parent nucleoside 1 as an antitumor agent.     

Identification of products formed by reaction of 3',5'-di-O-acetyl-2'-deoxyguanosine with hypochlorous acid or a myeloperoxidase-H2O2-Cl- system

Hypochlorous acid (HOCl), generated by myeloperoxidase from H(2)O(2) and Cl(-), plays an important role in host defense and inflammatory tissue injury. We have studied the reaction of 3',5'-di-O-acetyl-2'-deoxyguanosine with reagent HOCl and with a human myeloperoxidase-H(2)O(2)-Cl(-) system in order to characterize polar reaction products. When 100 microM 3',5'-di-O-acetyl-2'-deoxyguanosine was reacted with 100 microM HOCl at pH 7.4 and 37 degrees C and the reaction was terminated by N-acetylcysteine, 3',5'-di-O-acetyl derivatives of previously reported products, such as diastereomers of spiroiminodihydantoin nucleoside, a diimino-imidazole nucleoside, an amino-imidazolone nucleoside, and 8-chloro-2'-deoxyguanosine were formed. In addition, we report the formation of 3',5'-di-O-acetyl derivatives of a guanidinohydantoin nucleoside, an iminoallantoin nucleoside, and a diamino-oxazolone nucleoside in this system. The identification of the products was based on their identical ESI-MS and UV spectra and HPLC retention times with authentic compounds synthesized with other oxidation systems. All of these products were also formed in the reaction of 3',5'-di-O-acetyl-2'-deoxyguanosine with the myeloperoxidase-H(2)O(2)-Cl(-) system under mildly acidic conditions. The yields of the products were greatly affected by the pH of the reaction mixture. The total yields of these products formed by HOCl at pH 7.4 and by the myeloperoxidase-H(2)O(2)-Cl(-) system at pH 4.5 were 72 and 43% of the consumed 3',5'-di-O-acetyl-2'-deoxyguanosine, respectively, indicating that nearly half of the consumption of 3',5'-di-O-acetyl-2'-deoxyguanosine by HOCl and the myeloperoxidase-H(2)O(2)-Cl(-) system can be accounted for by the formation of these products.     

4-Hydroperoxy-2-nonenal-induced formation of 1,N2-etheno-2'-deoxyguanosine adducts

Analysis of the reaction between 4-hydroperoxy-2-nonenal (HPNE) and 2'-deoxyguanosine (dGuo) by liquid chromatography/mass spectrometry (LC/MS) revealed the formation of 1,N2-etheno-dGuo as well as heptanone-etheno-dGuo and trace amounts of dihydroxyheptane-etheno-dGuo. Identities of the dGuo adducts were confirmed by comparison with authentic standards. The minor dihydroxyheptane-etheno-dGuo adducts could be generated from 2,3-epoxy-4-hydroxynonanal (EHN), the epoxidation product of 4-hydroxy-2-nonenal (HNE). An LC/MS method was developed for the analysis of EHN. No EHN was detected by LC/MS during the decomposition of HPNE. Therefore, the dihydroxyheptane-etheno-dGuo adducts are either generated from a direct reaction between HPNE and dGuo or from another intermediate that cannot be detected by LC/MS. In addition, no HNE-derived hydroxypropano-dGuo adducts were observed. On the basis of these findings, we conclude that HPNE, a primary product of lipid peroxidation, is a major precursor to the formation of 1,N2-etheno-dGuo. We propose that it arises from the reaction of dGuo and HPNE through the intermediate formation of a cyclic hydroxy-ethano-epoxide derivative. The minor amounts of heptanone-ethano-dGuo adducts that were formed from HPNE in the absence of vitamin C suggest that heptanone-etheno-dGuo can be generated directly from HPNE without the intermediate formation of ONE. Therefore, HPNE can be considered as another lipid hydroperoxide-derived bifunctional electrophile with the potential for biological activities that are similar to HNE and ONE.     

Forodesine (BCX-1777, Immucillin H)--a new purine nucleoside analogue: mechanism of action and potential clinical application

Recently a few new purine nucleoside analogues (PNA) have been synthesized and introduced into preclinical and clinical trials. The transition-state theory has led to the design of 9-deazanucleotide analogues that are purine nucleoside phosphorylase (PNP) inhibitors, termed immucillins. Among them the most promising results have been obtained with forodesine. Forodesine (BCX-1777, Immucillin H, 1-(9-deazahypoxanthin)-1,4-dideoxy-1,4-imino-D-ribitol) has carbon-carbon linkage between a cyclic amine moiety that replaces ribose and 9-deaza-hypixanthine. The drug is a novel T-cell selective immunosuppressive agent which in the presence of 2'-deoxyguanosine (dGuo) inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction and phytohemagglutinin. In the mechanism of forodesine action two enzymes are involved: PNP and deoxycytidine kinase (dCK). PNP catalyzes the phosphorolysis of dGuo to guanine (Gu) and 2'-deoxyribose-1-phosphate, whereas dCK converts dGuo to deoxyguanosino-5'-monophosphate (dGMP) and finally to deoxyguanosino-5'-triphosphate (dGTP). The affinity of dGuo is higher for PNP than for dCK. Nevertheless, if PNP is blocked by forodesine, plasma dGuo is not cleaved to Gu, but instead it is intracellularly converted to dGTP by high dCK activity, which leads to inhibition of ribonucleotide reductase (RR), an enzyme required for DNA synthesis and cell replication, which eventually results in apoptosis. Forodesine is active in some experimental tumors in mice, however it could be used for the treatment of human T-cell proliferative disorders and it is undergoing phase II clinical trials for the treatment of T-cell non-Hodgkin's lymphoma, which includes cutaneous T-cell lymphoma (CTCL). Moreover, recent preclinical and clinical data showed activity of forodesine in B-cell acute lympholastic leukemia (ALL).     

Metabolic fate of N6-benzyladenosine and N6-benzyladenosine-5'-phosphate in rats.

The radiolabeled antitumor nucleoside (14C-8)-N6-benzyladenosine and its (14C-8)-5'-phosphate were administered to rats intravenously, and their metabolic fate was studied. Twenty-nine percent of the radioactivity was recovered in the 48-hr urine collection after (14C-8)-N6-benzyladenosine administration. The following metabolites were isolated: unchanged N6-benzyladenosine (20%), adenine (12%), uric acid (5%), and N6-benzyladenine (0.3%). In the case of (14C-8)-N6-benzyladenosine-5'-phosphate, a total of 28% of the radioactivity was recovered in the 48-hr urine collection and the following metabolites were isolated: N6-benzyladenosine (40%), uric acid (12%), adenine (trace), and unidentified urea derivatives (30%). Metabolism of N6-benzyladenosine appears to involve N-debenzylation to some extent, followed by conversion to adenine and uric acid. N6-Benzyladenosine and its 5'-phosphate differ from other adenosine analogs in being retained in significant amounts by the animals.     

Type II guanine oxidation photoinduced by the antibacterial fluoroquinolone Rufloxacin in isolated DNA and in 2'-deoxyguanosine

The role played by type I (radical) and type II (singlet oxygen) mechanisms in the Rufloxacin (RFX)-photoinduced production of 8-hydroxy-2'-deoxyguanosine in DNA has been evaluated. This fluoroquinolone drug has been shown to be able to photoinduce increased levels of some DNA base oxidation products, such as 8-OH-dGuo, that are indicative of mutagenic and carcinogenic events, with probable implications in aging processes. The relative weight of the two photosensitization mechanisms was obtained via determination of two different photoproducts of 2'-deoxyguanosine (dGuo), which are diagnostic of the two different pathways, namely, (4R)- and (4S)-4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine and 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2,5-dihydrooxazol-5-one. The observed predominance of type II reaction is in agreement with the fact that the triplet state of RFX is able to transfer with high efficiency its energy to molecular oxygen, giving rise to singlet oxygen. Photophysical measurements suggest that hydrated electrons produced by Rufloxacin photoionization react with dGuo, Thd, and DNA, whereas these biomolecules quench the RFX triplet state with low efficiency. Static quenching of Rufloxacin fluorescence indicates an interaction of this drug both with DNA and with dGuo. On the basis of these experimental data, Rufloxacin photosensitization of DNA is proposed to occur by a type II mechanism.     

Purine nucleoside phosphorylase inhibitor BCX-1777 (Immucillin-H)--a novel potent and orally active immunosuppressive agent.

Patients with purine nucleoside phosphorylase (PNP) deficiency present a selective T-cell immunodeficiency. Inhibitors of PNP are, therefore, of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP from various species including human, mouse, rat, monkey and dog, with IC50 values ranging from 0.48 to 1.57 nM. BCX-1777, in the presence of 2'-deoxyguanosine (dGuo, 3-10 microM), inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction (MLR) and phytohemagglutinin (PHA) (IC50 values < 0.1-0.38 microM). BCX-1777 is a 10-100-fold more potent inhibitor of human lymphocyte proliferation than other known PNP inhibitors like PD141955 and BCX-34. Nucleotide analysis of human lymphocytes indicate that inhibition of proliferation by BCX-1777 correlates with dGTP levels in the cells. BCX-1777 has excellent oral bioavailability (63%) in mice. At a single dose of 10 mg/kg in mice, BCX-1777 elevates dGuo to approximately 5 microM. BCX-1777 was not effective in mouse T-cell models such as delayed type hypersensitivity (DTH) and splenomegaly because mouse T-cells do not accumulate dGTP as do human T-cells. However, in the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mouse model, BCX-1777 was effective in prolonging the life span 2-fold or more. This is the first known example of a PNP inhibitor that elevates dGuo in mice similar to the levels observed in PNP-deficient patients. Furthermore, these dGuo levels are also required for in vitro T-cell inhibition by BCX-1777. Thus, BCX-1777 represents a novel class of selective immunosuppressive agents that could have therapeutic utility in various T-cell disorders.     

The formation of substituted 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine adducts by cis-2-butene-1,4-dial, a reactive metabolite of furan

Furan is an environmental chemical that induces liver toxicity and tumor formation in rodents, leading to its classification as a probable human carcinogen. cis-2-Butene-1,4-dial, the metabolite considered responsible for furan's toxicological effects, is mutagenic in the Ames assay and reacts with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), and 2'-deoxyguanosine (dGuo) to form previously characterized diastereomeric adducts. The initially formed dCyd adducts are stable to rearrangement, while the dAdo and dGuo adducts are unstable and rearrange to form secondary products. On the basis of UV absorbance, fluorescence, 1H NMR, and mass spectral data, the rearrangement product of the dAdo adduct was identified as the substituted etheno-dAdo adduct, 1'-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2,1-i]purin-8-yl]ethane-2'-al. The NMR characterization of the O-methyloxime derivative of the secondary dGuo adduct, along with mass spectral and UV data on the underivatized adduct, allowed for its structural assignment as the substituted etheno-dGuo compound, 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(ethane-2'-al)[1,2-alpha]purine-9-one. The characterization of the primary and secondary products formed in the reaction of cis-2-butene-1,4-dial with nucleosides is important for understanding the mechanism of furan-induced carcinogenesis. These secondary adducts retain a reactive aldehyde with the potential to form cross-links and are likely to contribute significantly to furan's toxic and carcinogenic effects.     

Synthesis and biological studies of 3-(beta-D-ribofuranosyl)-2,3,-dihydro-6H-1,3-oxazine-2,6-dione, a new pyrimidine nucleoside analog related to uridine.

Reaction of the trimethylsilyl derivative of 2,3-dihydro-6H-1,3-oxazine-2,6-dione (2, "uracil anhydride") with protected 1-O-acetylribofuranoses in the presence of stannic chloride gave the corresponding block nucleosides. 3-(2,3-5-Tri-O-2',2',2'-trichloroethoxycarbonyl-beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (4c) thus prepared from the protected sugar 3c, 1-O-acetyl-2,3,5-tri-O-(2,2,2-trichloroethoxycarbonyl)ribofuranose, gave, on removal of the protecting groups with zinc dust,3-(beta-d-ribofuranosyl)-2,3-dihydro-6H-1,3-oxazine-2,6-dione (1). The structure of 1 was confirmed by uv, ir, NMR, and CD spectral data and was shown to be an N nucleoside. Uracil anhydride, 2, and, to a lesser extent, its ribonucleoside 1 exert a moderate growth inhibition of mouse leukemia L5178Y, HeLa, and Novikoff hepatoma cells i- culture. Both compounds produce weak inhibition of vaccinia viral replication in HeLa cells.     

Detection and characterization of DNA adducts of 3-methylindole

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.     

Synergistic inhibition of human leukemia cell growth by deoxyguanosine and 1-beta-D-arabinofuranosylcytosine

We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.     

Aralkylation of 2'-deoxyguanosine: medium effects on sites of reaction with 7-(bromomethyl)benz[a]anthracene

Product distributions were determined for reactions between 2'-deoxyguanosine, or its anion, and 7-(bromomethyl)benz[a]anthracene in either acetone/H2O (1:1) or 2,2,2-trifluoroethanol at 50 or 70 degrees C. The exocyclic amino-substituted product, N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine, was always the major nucleoside product formed in these reactions, although its yield was higher in reactions involving 2'-deoxyguanosine anion than the neutral nucleoside. Reaction with the anion also led to formation of the 1-substituted 2'-deoxyguanosine and a guanidinoimidazole nucleoside resulting from reaction of 2'-deoxyguanosine at carbon 5. Reactions of 2'-deoxyguanosine anion in 2,2,2-trifluoroethanol are shown to produce significant amounts of the N2-substituted product, which is difficult to prepare by other routes.     

Synthesis and biological activity of a novel adenosine analogue, 3-beta-D-ribofuranosylthieno[2,3-d]pyrimidin-4-one

The title nucleoside 5 was prepared by a condensation of the silylated heterocycle thieno[2,3-d]pyrimidin-4-one (1) with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (2a) in the presence of a Lewis acid or with 2,3,5-tri-O-acetyl-D-ribofuranosyl bromide (2b) in the presence of mercuric oxide and mercuric bromide. The site of ribosylation and anomeric configuration of this nucleoside were established by 1H NMR. The synthesis of 3-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidin-4-one (8), 1-phenyl-5-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one (9), 5-methyl-3-beta-D-ribofuranosylthieno[2,3-d]pyrimidin-4-one (10), and 2-methyl-6-beta-D-ribofuranosyltriazolo[5,4-d]pyrimidin-7-one (11) is also described. The title compound inhibited the growth of murine L-1210 leukemic cells in vitro with an ID50 of 3 X 10(-5)M. The growth inhibition could not be prevented by uridine, cytidine, thymidine, deoxycytidine, cytosine, hypoxanthine, or uridine and hypoxanthine together. On the other hand, inhibition of adenosine kinase by 10(-7) M 5-iodotubercidin prevented the cytotoxic effect. Also a subline of L-1210 cells resistant to several cytotoxic adenosine analogues was also resistant to this nucleoside. Thus it appears that this compound 5 may act as an adenosine analogue.     

Syntheses and properties of 1-methyl-3-phenylaminobenzimidazolium salts, models of DNA adducts of N7-arylaminodeoxyguanosinium salt

When arylaminating carcinogens are administered to cells, they mainly generate the C8-arylamino-2'-deoxyguanosine adduct in DNA. A mechanism for this was proposed in which N7-arylaminated 2'-deoxyguanosine acts as an intermediate; however, it remained unclear whether this is actually the case. To elucidate the mechanisms involved in the generation of this adduct, a series of 5-substituted 1-methylbenzimidazole derivatives were used as models of the imidazole moiety of 2'-deoxyguanosine. Syntheses of a series of 5-substituted (CH(3), H, F, CF(3), or NO(2)) 1-methyl-3-phenylaminobenzimidazolium salts (7) and their related compounds were carried out, and the chemical characteristics of these products were examined. Heating compound 7 at 80 degrees C for 48 h in H(2)O/MeOH provided 5-substituted 1-methyl-2-oxo-2, 3-dihydrobenzimidazoles but only when this compound contained a CF(3) or NO(2) substituent. Compound 7 decomposed in alkaline media, and its rate of decomposition increased when this compound had a stronger electron-withdrawing substituent. The product obtained under these conditions was 4-substituted N(1)-methyl-2-phenylazoaniline. On the other hand, when 1-methyl-3-(4-nitrophenylamino)benzimidazolium salt was treated under the same conditions as described above, it generated a demethylated product, 1-(4-nitrophenylamino)benzimidazole, when heated in H(2)O/MeOH and N(1)-formyl-N(1)-methyl-2-phenylazoaniline when treated in alkaline media. When the chemical characteristics of 3-phenylamino and 3-amino groups were compared using 3-substituted 1-methyl-5-(trifluoromethyl)benzimidazoles, the 3-phenylamino derivative was found to be more reactive.