Synthesis, characterization, and comparative 32P-postlabeling efficiencies of 2,6-dimethylaniline-DNA adducts

2,6-Dimethylaniline (2,6-diMeA) is a ubiquitous environmental pollutant that is used in industry as a synthetic intermediate. It is also found in tobacco smoke and as a major metabolite of lidocaine. Although the potential carcinogenicity of 2,6-diMeA in humans is presently uncertain, this aromatic amine has been classified as a rodent carcinogen. In addition, it is known to form hemoglobin adducts in humans, which indicates a profile of metabolic activation similar to that of typical arylamine carcinogens. Like other aromatic amines, 2,6-diMeA has been shown to yield N-(deoxyguanosin-8-yl)-2,6-dimethylaniline (dG-C8-2,6-diMeA) as a major DNA adduct in vitro. In this study, we show that 2,6-diMeA yields an unusual pattern of DNA adducts. In addition to dG-C8-2,6-diMeA, we have isolated two new adducts, 4-(deoxyguanosin-N(2)-yl)-2,6-dimethylaniline (dG-N(2)-2,6-diMeA) and 4-(deoxyguanosin-O(6)-yl)-2,6-dimethylaniline (dG-O(6)-2,6-diMeA), from the reaction of N-acetoxy-2,6-dimethylaniline with deoxyguanosine. A similar reaction conducted with deoxyadenosine yielded 4-(deoxyadenosin-N(6)-yl)-2,6-dimethylaniline (dA-N(6)-2,6-diMeA). All four adducts were detected in DNA reacted with N-acetoxy-2,6-dimethylaniline, with the relative yields being 46% for dA-N(6)-2,6-diMeA, 22% for dG-N(2)-2,6-diMeA, 20% for dG-O(6)-2,6-diMeA, and 12% for dG-C8-2,6-diMeA. This product profile contrasts markedly with the usual pattern of adducts obtained with aromatic amines, where C8-substituted deoxyguanosine products typically predominate. We further analyzed the kinetics of the T(4) polynucleotide kinase (PNK)-catalyzed phosphorylation of the C8 and N(2) deoxyguanosine 3'-phosphate adducts from 2,6-diMeA. The kinetic parameters obtained with these two structurally different adducts are compared to those determined with the parent nucleotide (dG3'p), and with (+/-)-anti-10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene 3'-phosphate, the major adduct derived from the environmental pollutant benzo[a]pyrene. The results indicate that all the adducts were labeled with lower efficiencies than dG3'p, stressing the likely underestimation of adduct levels in typical 32P-postlabeling protocols. Nonetheless, the N(2) adducts derived from 2,6-diMeA and benzo[a]pyrene were both labeled with higher efficiencies than the C8 adduct derived from 2,6-diMeA, with the benzo[a]pyrene adduct being the best substrate for PNK. Thus, the data suggest that N(2) adducts from dG3'p are intrinsically better substrates than their C8 analogues for PNK, and that bulkier aromatic fragments may favor the enzyme-substrate interaction during the labeling step.     

Detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human pancreas tissue using capillary liquid chromatography-microelectrospray mass spectrometry

Cigarette smoking has been associated with various cancers including bladder and pancreas. 4-Aminobiphenyl has been isolated as a constituent of cigarette smoke and has been established as a carcinogen in various animal models and humans. In rodents and humans, 4-aminobiphenyl is N-hydroxylated and forms adducts to DNA, the predominant one being N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). In this study, we report a micro-electrospray mass spectrometric (muESI-MS) isotope dilution method for the detection and quantification of dG-C8-ABP in human pancreatic tissue. A reverse phase capillary column (320 microm ID) was connected to a triple quadrupole mass spectrometer via a commercially available micro-ESI source. The system was operated in the selected reaction monitoring mode transmitting the [M + H]+ --> [M + H - 116]+ transitions for both the analyte and the isotopically labeled internal standard. Twelve human pancreas samples were analyzed, where six were current smokers (three male and three female) and six were considered nonsmokers (three female and three male). Of the samples analyzed, six showed dG-C8-ABP levels above the limit of quantification for the method, five were considered to have levels that were undetectable, and one was discarded due to inconsistent internal standard signal. The age of the human subjects ranged from 17 to 63, and, in samples where adduct was present, levels ranged anywhere from 1 to 60/10(8) nucleotides. Although no correlation between smoking preference, age, or gender was proven with this particular sample pool, this report demonstrates that capillary LC-muESI-MS can provide a sensitive and definitive method for DNA adduct analysis in human tissue.     

N-羟乙基-2-(3''''-硝基联苯基-4-亚甲磺酰基)乙酰胺的合成

目的：合成磷酸二酯酶3B（PDE3B）选择性抑制剂N-羟乙基-2-（3＇-硝基联苯基-4-亚甲磺酰基）乙酰胺.方法：以对溴苄溴为初始原料经Williamson成醚反应、氧化、酰化和Suzuki偶联4步反应合成了目标化合物.结果：目标产物的结构经核磁共振氢谱、碳谱、质谱及红外光谱等确证,4步反应的总收率为24.8%.结论：该合成路线原料易得,操作简便,可用于放大制备.     

Comparison of (32)P-postlabeling and high-resolution GC/MS in quantifying N7-(2-Hydroxyethyl)guanine adducts.

This study compares (32)P-postlabeling and high-resolution gas chromatography/mass spectrometry (GC/MS) in the quantification of N7-(2-hydroxyethyl)guanine adducts (7-HEG) in DNA obtained from the same tissue samples of control rats and rats exposed to ethene. The samples were obtained from two independent studies. In one study, male Sprague-Dawley rats were exposed to 300 ppm ethene for 12 h/day for 3 days ("Euro samples"). In the other study, male F-344 rats were exposed to 3000 ppm ethene for 6 h/day for 5 days ("U.S. samples"). DNA from liver and kidney from the European study was isolated in the European laboratory, and DNA from liver and spleen from the U.S. study was isolated in the U.S. laboratory. The DNA samples were coded, divided into two portions, and exchanged between the two laboratories. All DNA samples from both laboratories were analyzed with respect to 7-HEG adducts by (32)P-postlabeling and high-resolution GC/MS in the European and U.S. laboratories, respectively. However, the U.S. samples were repurified in the European laboratory before the postlabeling analysis. The data from the Euro and the U.S. samples were therefore treated separately in the regression analysis of the (32)P-postlabeling versus GC/MS data. The slope of the regression line for the Euro samples was 1.19 (r = 0.97), implying that the GC/MS data were slightly lower than the postlabeling data (one possible outlier was excluded). The slope of the regression line for the U.S. samples was 0.61 (r = 0.94), implying that the GC/MS data were somewhat higher than the postlabeling data. The main conclusion from this study is that there is very good agreement between the (32)P-postlabeling and high-resolution GC/MS methods in quantifying 7-HEG adducts to DNA, particularly when identical DNA samples are analyzed and the RNA content is <2%. The paper also discusses the background levels of adducts, the interorgan distribution, comparison between different strains, and exposure conditions.     

Synthesis and antifungal activity of new N-(1-phenyl-4-carbetoxypyrazol-5-yl)-, N-(indazol-3-yl)- and N-(indazol-5-yl)-2-iodobenzamides

N-(1-Phenyl-4-carbetoxypyrazol-5-yl)-, N-(indazol-3-yl)- and N-(indazol-5-yl)-2-iodobenzamides 6, with a Benodanil-like structure, were synthesized by refluxing in acetic acid the corresponding benzotriazinones 5 with potassium iodide for 1 h in order to study the role on the antifungal activity of the N-substitution with an aromatic heterocyclic system on benzamide moiety. Among the tested iododerivatives, compounds 6d,f,g,h possess interesting activities toward some phytopathogenic fungal strains.     

Separation of (+)-syn- and (-)-anti-benzo[a]pyrene dihydrodiol epoxide-DNA adducts in 32P-postlabeling analysis.

The (+)-enantiomer of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is a diagnostic probe for cytochrome P-450 and non-cytochrome P-450 pathways of dihydrodiol epoxidation. The principle products of epoxidation are the (+)-syn-dihydrodiol epoxide [(+)-syn-BPDE] and the (-)-anti-dihydrodiol epoxide [(-)-anti-BPDE]. Chromatographic conditions are described that separate the major deoxynucleoside 3',5'-bisphosphate adducts derived from these dihydrodiol epoxides on commercial poly(ethylenimine) thin-layer plates. Inclusion of boric acid and magnesium chloride in the D4 solvent is a key feature of the separation. Reasonable separation of these bisphosphate adducts from the major deoxynucleoside 3',5'-bisphosphate adduct derived from (+)-anti-BPDE is also observed. 32P-Postlabeling analysis of DNA adducts produced following topical administration of benzo[a]pyrene to mouse skin suggests that cytochrome P-450 plays a major role in its metabolism to DNA binding derivatives.     

DNA adducts from N-nitrosodiethanolamine and related beta-oxidized nitrosamines in vivo: (32)P-postlabeling methods for glyoxal- and O(6)-hydroxyethyldeoxyguanosine adducts

The mechanism by which environmentally prevalent N-nitrosodiethanolamine (NDELA) and related 2-hydroxyethyl- or other beta-oxidized nitrosamines initiate the carcinogenic process has remained obscure. (32)P-Postlabeling assays for the pH sensitive glyoxal-deoxyguanosine (gdG) and the O(6)-2-hydroxyethyldeoxyguanosine (OHEdG) DNA adducts have been developed as probes in this mechanistic investigation and used in both in vitro and in vivo experiments. The ready cleavage of the glyoxal fragment from gdG at pH 7 and greater has required methods of optimization in order to achieve a detection limit of 0.05 micromol/mol of DNA. Nuclease P1 treatment enhances the detection of gdG adducts but does not increase the detection limit for OHEdG. For OHEdG, best results were achieved using fraction collection from HPLC (0.3 micromol/mol of DNA). Using radiochemical methods, both adducts could be detected either by HPLC or 2D TLC. NDELA, N-nitrosomorpholine (NMOR), N-nitrosomethyethanolamine (NMELA), and N-nitrosoethylethanolamine (NEELA) all produce both gdG and OHEdG adducts in rat liver DNA in vivo and are called bident carcinogens because fragments from both chains of the nitrosamine are incorporated into DNA. N-Nitroso-2-hydroxymorpholine (NHMOR), a metabolite of NDELA and NMOR, generates gdG in DNA in vitro and in vivo. gdG DNA adducts were found in the range 1.1-6.5 micromol/mol of DNA. OHEdG DNA adducts were produced from equimolar amounts of nitrosamines in rat liver in vivo over the range 4-25 micromol/mol of DNA and in the order NMELA > NEELA > NDELA > NMOR. Deuterated isotopomers of NDELA showed a marked isotope effect on DNA OHEdG adduct formation. alpha-Deuteration markedly decreased OHEdG adduct formation while beta-deuteration had the opposite effect. These data support the hypothesis that NDELA and related nitrosamines are activated by both enzyme mediated alpha-hydroxylation and beta-oxidation. The formation of OHEdG adducts from NDELA requires alpha-hydroxylation of the 2-hydroxyethyl chain, and formation of gdG necessitates a beta-oxidation as well. The bident nature of these carcinogens may explain why they are relatively potent carcinogens despite the fact that major proportions of doses are excreted unchanged.     

Synthesis, characterization, and 32p-postlabeling analysis of DNA adducts derived from the environmental contaminant 3-nitrobenzanthrone

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. 3-NBA forms DNA adducts in rodent tissues that arise principally through reduction to N-hydroxy-3-aminobenzanthrone (N-OH-ABA), esterification to its acetate or sulfate ester, and reaction of this activated ester with DNA. We detected 3-NBA-derived DNA adducts in rodent tissues by (32)P-postlabeling and generated them chemically by acid-catalyzed reaction of N-OH-ABA with DNA, but their structural identification has not yet been reported. We have now prepared 3-NBA-derived adducts by reaction of a possible reactive metabolite, N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA), with purine nucleosides and nucleotides, characterized them, and have shown that they are present in DNA treated with this 3-NBA derivative. Three of these adducts have been characterized as the C-C adduct N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone, the C-N adduct N-acetyl-N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone, and an unusual 3-acetylaminobenzanthrone adduct of deoxyadenosine, which involves a double linkage between adenine and benzanthrone (N1 to C1, N(6) to C11b), creating a five-membered imidazo type ring system. According to IUPAC fused ring conventions, we propose the following systematic name for this adduct: (9'-(2' '-deoxyribofuranosyl))purino[6',1':2,3]imidazo[5,4-p](1,11b-dihydro-(N-acetyl-3-amino))benzanthrone. The 3'-phosphates of these novel adducts could be 5'-postlabeled using [gamma-(32)P]ATP, although the efficiency of labeling was found to be low (less than 20%). However, none of these adducts could be detected in DNA from 3-NBA-treated rats by (32)P-postlabeling. Two of these synthetic adducts were treated with alkali to generate nonacetylated adducts, and these were also shown by HPLC to differ from those adducts found in rat DNA. Therefore, a different approach to the synthesis of authentic standards is needed for the structural characterization of 3-NBA-derived DNA adducts formed in vivo.     

Development of a (32)P-postlabeling method for the detection of 1,N(2)-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal in vivo

A (32)P-postlabeling method was developed for the sensitive detection of 1,N(2)-propanodeoxyguanosine adducts of the lipid peroxidation product trans-4-hydroxy-2-nonenal in vivo. The method development was based on the chemically synthesized HNE-1, N(2)-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by nuclease P1. They were subsequently reacted with [gamma-(32)P]ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose TLC and quantitated by autoradiography. The medium labeling efficiency for the mixture of the two pairs of diastereomers was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. The method is applicable for the separation and quantitation of HNE-dGp-propano adducts in vivo. It was applied to DNA from colon and brain tissue of untreated Fischer 344 rats and humans. The determination of the limit of quantitation in DNA from rat colon by spiking of adduct standard revealed a sensitivity of <21 adducts/10(9) nucleotides. The analytical quantitation of 4-HNE-dGp-propano adducts resulted in adduct-levels per 10(9) normal nucleotides +/- the standard deviation of 223.32 +/- 79.84 in rat colon tissue, 90.37 +/- 11.94 in rat brain tissue, 378.44 +/- 52.42 in human colon tissue, and 185.15 +/- 6.48 in human brain tissue. The results clearly demonstrate the applicability of this method for the sensitive detection of endogenously formed 1,N(2)-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal, a specific marker for the lipid peroxidation process.     

N-(4-氯-3-三氟甲基苯基)-N'-(4-溴苯基)脲的合成

4-氯-3-三氟甲基苯胺和三光气在乙酸乙酯中反应制得4-氯-3-三氟甲基苯异氰酸酯,不经分离纯化,直接与对溴苯胺反应制得抗肿瘤药索拉非尼的中间体N-(4-氯-3-三氟甲基苯基)-N'-(4-溴苯基)脲,总收率约75%,纯度99.8%.     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Development of potential antiandrogens: N-(4-nitro-3-trifluoromethyl-phenyl)-cyclohexanoylamides and -benzamides, N-(3,4-diochlorophenyl)- and N-(3,4,5-trichlorophenyl)-benzamides

Development of potential nonsteroidal antiandrogens: N-(4-nitro-3-trifluoromethylphenyl)-cyclohexanoylamides and -benzamides and N-(3,4-dichlorophenyl)- and N-(3,4,5-trichlorophenyl)-benzamides For the development of potential nonsteroidal antiandrogens, N-(4-nitro-3-trifluoromethylphenyl)-cyclohexanoylamides and -benzamides, and N-(3,4-dichlorophenyl)- and N-(3,4,5-trichlorophenyl)-benzamides were synthesized. The hydroxycyclohexane analog 3 exerted a higher binding affinity to the androgen receptor (AR) than hydroxyflutamide. Changing the α-hydroxycyclohexane ring of 3 to a hydroxyphenyl moiety led to a strong decrease in AR affinity. Compounds showing AR affinity were further tested for antiandrogenic and androgenic activity.     

4-(4-氨基苯基)-3-吗啉酮的合成

用两种方法制得抗凝剂利伐沙班的中间体4-(4-氨基苯基)-3-吗啉酮(1):①溴苯相继与乙醇胺、氯乙酰氯反应得4-苯基吗啉酮,再经硝化、还原生成1,总收率约32%;②溴苯先硝化,再与乙醇胺反应得到2-(4-硝基苯胺基)乙醇,再经氯乙酰氯闭环、还原反应制得1,总收率约47%.     

(S)-1-(N-烯丙氧羰基)亚胺乙基-3-巯基吡咯烷的合成

目的 合成帕尼培南关键中间体 (S)-1-( N-烯丙氧羰基)亚胺乙基-3-巯基吡咯烷。方法 以氯甲酸烯丙酯为酰化剂,与盐酸乙脒进行 N-酰化反应得到 1-亚胺乙基氨基甲酸烯丙酯,该化合物与 3-R-羟基吡咯烷进行缩合,再经甲磺酰化、S_N2 取代、水解共 5 步反应得到目标化合物。结果与结论 该合成路线中使用新型保护基烯丙氧羰基替代传统的保护基—对硝基苄氧羰基,目标化合物的结构经 ¹H-NMR、MS 谱确证,总收率为 41.6%,各步反应操作简便,条件温和,有利于工业化生产。     

帕尼培南关键中间体的合成

目的: 改进帕尼培南关键中间体(S)-1-[N-(对硝基苄氧羰基)-1-亚胺基乙基]-3-巯基吡咯烷的合成工艺。方法:以对硝基苄醇为起始原料,经酰化、缩合、与3-R-羟基吡咯烷发生N-烃基化反应,再经磺酰化、缩合、水解反应得到目标化合物。结果与结论:以对硝基苄醇为起始原料经6步反应得到目标化合物,结构经IR、1H-NMR、MS谱图数据确证,比旋光度值与文献报道一致。改进后的工艺总收率为37.2%,各步反应操作简便,条件温和,更适合工业化生产。