Organocatalytic conjugate addition promoted by multi-hydrogen-bond cooperation: access to chiral 2-amino-3-nitrile-chromenes.

A new efficient enantioselective conjugate addition strategy has been disclosed to rapidly construct 2-amino-3-nitrile-chromene complexes via a multi-hydrogen-bond cooperative activation model.

Tetrahydrobenzo- and benzofurobenzopyrones as a new class of potential photoreagents toward DNA.

The synthesis and the photobiological activity of new tetrahydrobenzo- and benzofurobenzopyrone derivatives carrying at position 4 of benzopyrone ring of furobenzopyrone moiety a phenyl, or a methyl group with a linear structure or with various angular arrangements, are reported. The new compounds are characterized by having an additional cyclohexene or phenyl ring condensed at the 2, 3 double bond of the furan ring of furobenzopyrone nucleus. The syntheses were performed starting from the appropriate hydroxybenzopyrones on which the tetrahydrobenzofuran or benzofuran moiety was built, which look most promising for enhancement of photoreactivity of compounds toward DNA. All the synthesized compounds were screened for photosensitizing activity and some of them exhibited good activity also a certain effect was observed in the dark.

Synthesis, resolution, and VCD analysis of an enantiopure diazaoxatricornan derivative.

Using simple organic synthetic transformations, a novel diazaoxatricornan derivative, the 12 c-methyl-12-phenyl-8-propyl-12,12 c-dihydro-8 H-4-oxa-8,12-diazadibenzo[ cd, mn]pyrene ( 6a), was prepared. This novel chiral cup-shaped molecule was isolated in racemic form and in excellent yield after the addition of methyl lithium to the BF 4 salt of a novel unsymmetrical diazaoxatriangulenium cation. Compound 6a was found to be stable under classical laboratory conditions-something not obvious considering the extreme stability of the carbenium ion precursor, the electron-rich nature of the core, and the strain induced by the pyramidalization of the central carbon. The enantiomers were readily separated by chiral stationary phase chromatography, and the absolute configuration of (-)-( S)- 6a was determined by a comparison of the experimental and theoretical vibrational circular dichroism (VCD) spectra. This isolation of (-)-( S)- 6a and (+)-( R)- 6a constitutes thus the first report of a nonracemic closed-capped chiral bowl molecule for which the chirality is due to the intrinsic dissymmetry of the central core of the structure only.

Synthesis of dibenzo[def,p]chrysene, its active metabolites, and their 13C-labeled analogues.

Dibenzo[def,p]chrysene (DBC) is a highly carcinogenic polycyclic aromatic hydrocarbon suspected to be involved in initiation of lung cancer in smokers. Efficient new syntheses of DBC, its active metabolites [DBC diol (1), DBC dione (2), DBC diol epoxide (3)], and their previously unknown 13C2-labeled analogues are reported. The 13C2-labeled analogues are required as standards for sensitive methods of analysis of their DNA adducts in human cells using stable isotope dilution liquid chromatography/tandem mass spectrometry.

Quantification of benzo[a]pyrene diol epoxide DNA-adducts by stable isotope dilution liquid chromatography/tandem mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants found in car exhausts, charbroiled food, and tobacco smoke. Three pathways for the metabolic activation of B[a]P to ultimate carcinogens have been proposed. The most widely accepted pathway involves cytochrome-P450 (CYP) 1A1- and/or 1B1-mediated formation of B[a]P-7,8-oxide, which undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further CYP1A1- and/or CYP1B1-mediated activation of the dihydrodiol results in the formation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. In previous studies, it was demonstrated that (+)-anti-B[a]PDE was the most potent tumorigen of the CYP-derived B[a]PDE diastereomers. We have developed a stable isotope dilution, liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay for all eight (+/-)-anti-B[a]PDE-derived dGuo and dAdo DNA-adducts. The LC-MRM/MS assay was rigorously validated and used to show that (+)-anti-trans-B[a]PDE-dGuo was the major adduct formed when naked DNA and human bronchoalveolar adenocarcinoma H358 cells were treated with (+/-)-anti-B[a]PDE. The preference for DNA-adducts derived from (+)-anti-B[a]PDE was even more apparent in cellular DNA. Thus, the increased potency of (+)-anti-B[a]PDE as a tumorigen is most likely due its ability to preferentially form DNA-adducts when compared with (-)-anti-B[a]PDE. Also, the adduct profile suggests that this occurs by binding of (+)-anti-B[a]PDE to DNA in a manner that facilitates covalent binding to dGuo rather than dAdo residues.

A Highly Abbreviated Synthesis of Dibenzo[def,p]chrysene and Its 12-Methoxy Derivative, a Key Precursor for the Synthesis of the Proximate and Ultimate Carcinogens of Dibenzo[def,p]chrysene.

Dibenzo[def,p]chrysene (DBC) (1), is by far the most mutagenic and toxic polycyclic aromatic hydrocarbon identified. Its metabolic activation leads to trans-11,12-dihydroxy-11,12-dihydro-DBC (2), which is further metabolized to the ultimate metabolite, anti-trans-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydro-DBC (3), that binds to DNA causing mutations and ultimately tumor induction. We report a facile route for the syntheses of DBC (1) and its 12-methoxy derivative (12-methoxy-DBC) (13), a key intermediate for the synthesis of 2 and 3, using a Suzuki cross-coupling approach.

Quantitative detection of benzo[alpha]pyrene diolepoxide-DNA adducts by cryogenic laser induced fluorescence.

In the present report, we describe a fluorescence-based method capable of measuring benzo[alpha]pyrene diolepoxide (BPDE) adducts in intact genomic DNA, with a sensitivity of a few hundreds copies per cell. The assay is based on cryogenic laser-induced fluorescence technology at liquid nitrogen temperatures, coupled with an intensified charge-coupled device camera, and incorporates several enhancements to existing methodologies. One important modification was the incorporation of terbium(III)nitrate pentahydrate, Tb(NO3)3, as an internal fluorescence standard to correct for differences in light scattering and fluctuations in instrument parameters. Since the fluorescence spectrum of Tb(NO3)3 does not overlap with those of BPDE-DNA adducts, use of this lanthanide salt markedly improved the sensitivity of cryogenic laser-induced fluorescence. The limit of quantification of the assay is 6.4 BPDE-DNA adducts/10(8) nucleotides, or 776 adducts/cell, using 22.5 micrograms of genomic DNA. This assay is rapid, highly sensitive, and economical and has been applied to monitor DNA adduct levels as a function of time after exposure to BPDE in repair-competent human lymphoblastoid AHH-1 and TK6 cells.

Synthesis and cytotoxic activity of benzopyranoxanthone analogues of benz.

Condensation of 3-hydroxy-2-naphthalenecarboxylic acid with phloroglucinol afforded 1,3-dihydroxy-12H-benzo[b]xanthen-12-one. Construction of an additional dimethylpyran ring onto this skeleton, by alkylation with 3-chloro-3-methyl-1-butyne followed by Claisen rearrangement, gave access to a series of benzo[b]pyrano[2,3-i]xanthen-6-ones and benzo[b]pyrano[3,2-h]xanthen-7-ones related to psorospermine and benzo[b]acronycine. In contrast with what is observed in the pyridoacridone and benzopyridoacridone series, the linear benzo[b]-pyrano[2,3-i]xanthen-6-one derivatives were more potent than their angular benzo[b]pyrano[3,2-h]xanthen-7-one isomers. cis-3,4-Diacetoxy-5-methoxy-2,2-dimethyl-3,4-dihydro-2H,6H-benzo[b]pyrano[2,3-i]xanthen-6-one, the most active among the new compounds, was more potent than acronycine in inhibiting the proliferation of L1210 murine leukemia cells.

New synthetic approaches to polycyclic aromatic hydrocarbons and their carcinogenic oxidized metabolites: derivatives of benzo[s]picene, benzo[rst]pentaphene, and dibenzo[b,def]chrysene.

A new synthetic approach to polycyclic aromatic compounds is described that entails in the key steps double Suzuki coupling of PAH bisboronic acid derivatives with o-bromoaryl aldehydes to furnish aryl dialdehydes that are converted to larger polycyclic aromatic ring systems by either (a) conversion to diolefins by Wittig reaction followed by photocyclization or (b) reductive cyclization with triflic acid and 1,3-propanediol. This synthetic method provides convenient access to as many as three different polycyclic aromatic ring systems from a single Suzuki coupled intermediate. It was utilized to synthesize substituted derivatives of benzo[s]picene, benzo[rst]pentaphene, dibenzo[b,def]chrysene, and 13,14-dihydro-benz[g]indeno[2,1-a]fluorene, as well as the putative carcinogenic bisdihydrodiol metabolites of benzo[s]picene, benzo[rst]pentaphene, and dibenzo[b,def]chrysene.

Preparation, isolation, and characterization of Dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence line-narrowing spectroscopy.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3'-dAMP or 3'-dGMP with either (+/-)-anti- or (+/-)-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13, 14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (+/-)-trans and (+/-)-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (+/-)-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (+/-)-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (+/-)-syn-trans-DB[a,l]PDE-dGMP adducts, a pair of (+/-)-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDE-dGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.

How stereochemistry affects mutagenesis by N2-deoxyguanosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: configuration of the adduct bond is more important than those of the hydroxyl groups.

Previous work has shown that the major adduct from the (+)-anti diol epoxide of benzo[a]pyrene (B[a]P), which forms at N2-deoxyguanosine [(+)-trans-anti-B[a]P-N2-dG], is capable of inducing either predominantely G --> T mutations ( approximately 95%) in a 5'-TGC-3 sequence context or predominantly G --> A mutations ( approximately 80%) in a 5'-CGT-3' sequence context. This is likely to be attributable to the major adduct being in a different mutagenic conformation in each case. In the next phase of this work, the questions to be addressed are what conformation is associated with what mutation and why? To help define what aspect of adduct structure is important to mutagenesis, the work herein reports on the mutations induced in a single sequence context by four stereoisomers of B[a]P-N2-dG: (+)-trans-, (+)-cis-, (-)-trans-, and (-)-cis-. The (+)-trans- and (-)-cis-adducts show a remarkably similar mutational pattern with G --> A mutations predominating ( approximately 80%). The (-)-trans- and (+)-cis-adducts also show a similar mutational pattern with a more even mixture of G --> T, G --> A, and G --> C mutations. Each of these adducts has an adduct bond and three hydroxyl groups at four consecutive saturated carbons in the B[a]P moiety of the adduct; the stereochemistry at these four positions differs in each of the adducts. The (+)-trans- and (-)-cis-adducts are a pair sharing the S configuration for the adduct bond, although they are a mirror image vis-a-vis the hydroxyl groups. The (-)-trans- and (+)-cis-adducts share the opposite adduct bond stereochemistry (R) but differ in the stereochemistry of their hydroxyl groups. Thus, there is a correlation suggesting that anti-B[a]P-N2-dG adduct mutagenesis is more dependent on the stereochemistry of the adduct bond than on the stereochemistry of the hydroxyl groups.

Synthesis and tumor-initiating activity in mouse skin of dibenzo[a,l]pyrene syn- and anti-fjord-region diolepoxides.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen among polycyclic aromatic hydrocarbons. Because the fjord-region diolepoxide (DE) pathway is one of the mechanisms of activation, (+/)-trans-DB[a,l]P-11,12-dihydrodiol, (+/-)-anti-DB[a,l]PDE and (+/-)-syn-DB[a,l]PDE were synthesized. The key intermediate for these syntheses, 12-methoxy-DB[a,l]P, was successfully obtained by cyclization of 6-(3-methoxybenzyl)benzanthrone with methanesulfonic acid, which in turn was prepared by 1,4 conjugate addition of 3-methoxybenzyl magnesium bromide to benzanthrone. The presence of the DB[a,l]P nucleus in the dihydrodiolepoxides and diolepoxides was proven by conversion of 12-methoxyDB[a,l]P into the parent compound in several steps. The tumor-initiating activity of the two diolepoxides in mouse skin was compared to that of DB[a,l]P-11,12-dihydrodiol and the parent DB[a,l]P. Groups of 24 8 week old female SENCAR mice were topically initiated with 12, 4 or 1.33 nmol of compound in 100 microliters of acetone. Starting 1 week later, promotion with 12-O-tetradecanoylphorbol-13-acetate (1.62 nmol in 100 microliters acetone) was begun and continued twice weekly for 30 weeks. At the 12, 4 and 1.33 nmol doses, anti-DB[a,l]PDE induced 2.0, 0.7 and 0.7 tumors per mouse (t/m) respectively, whereas syn-DB[a,l]PDE induced 1.8, 1.5 and 1.8 t/m. At the same three doses, DB[a,l]P-11,12-dihydrodiol induced 4.6, 4.3 and 2.8 t/m, and DB[a,l]P resulted in 9.3, 7.1 and 5.2 t/m. These results confirm that DB[a,l]P is more potent than its 11,12-dihydrodiol and show that the two diolepoxides are less tumorigenic than their precursors. At the medium and low doses, syn-DB[a,l]PDE is more tumorigenic than its congener anti-DB[a,l]PDE.

Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo[a,l]pyrene.

Extensive tumorigenicity studies in rodents revealed that dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen among all polycyclic aromatic hydrocarbons (PAHs) tested so far. The structure of the genotoxic metabolite(s) responsible for this exceptional carcinogenicity is unknown. The fjord-region syn- and anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (syn- and anti-DB[a,l]PDE) were synthesized to clarify their role as possible ultimate mutagenic and carcinogenic metabolites of DB[a,l]P.9-Formyl-11,12-dimethoxybenzo[g] chrysene was prepared from 9-phenanthrylacetic acid by a photochemical route. After reaction of the aldehyde with trimethylsulfonium iodide to generate an oxiranyl side-chain, treatment with boron trifluoride produced the key intermediate 11,12-dimethoxy-DB[a,l]P in 14% overall yield. From 11,12-dimethoxy-DB[a,l]P the syn- and anti-DB[a,l]PDE were stereoselectively prepared via the trans-11,12-dihydrodiol. The mutagenicity of the syn- and anti-DB[a,l]PDE was examined in four his- strains of Salmonella typhimurium and in Chinese hamster V79 cells. In all five test systems, the new dihydrodiolepoxides were more potent than any of the previously investigated dihydrodiolepoxides. The specific mutagenicity observed with anti-DB[a,l]PDE in strain TA104 exhibited the highest value ever found with any compound in any his- strains of S.typhimurium. The same appears to be true for the activity observed with this compound in V79 cells. In all five systems, syn-DB[a,l]PDE was only moderately less active than its anti-diastereomer (approximately 2-fold). The exceptional mutagenic activities of these dihydrodiolepoxides may be one of the reasons for the exceptional carcinogenic activity of DB[a,l]P.

Synthesis of Fjord region diol epoxides as potential ultimate carcinogens of dibenzo[a,l]pyrene.

Dibenzo[a,l]pyrene (DB[a,l]P) is one of the strongest polynuclear aromatic hydrocarbon carcinogens known. This paper describes the synthesis of potential ultimate carcinogens of DB[a,l]P: anti- and syn-11,12-dihydroxy-13,14-epoxy-11,12,13,14- tetrahydroDB[a,l]P (DB-[a,l]P-11,12-diol-13,14-epoxides). The method employed is also useful for the preparation of key intermediates for the synthesis of 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrobenzo-[g]chrysene (BgC-11,12-diol-13,14-epoxide). Photochemical cyclization of the appropriately substituted phenanthrylphenylethylenes provided 9-carbomethoxy-11-methoxyBgC (4) and 11-methoxyBgC (3). The former was converted by reduction, oxidation, one-carbon chain extension, and cyclization to 11-methoxy DB[a,l]P (7). Compounds 3 and 7 were converted by hydrolysis and oxidation to BgC-11,12-dione (10) and DB[a,l]P-11,12-dione (11), respectively. The diones are the precursors for the synthesis of the corresponding diol epoxides. anti- and syn-DB-[a,l]P-11,12-diol-13,14-epoxides 13 and 14 were prepared in 38% and 55% yields, respectively, from 11. Both diol epoxides had predominantly pseudodiequatorial hydroxyl groups, as seen in other sterically hindered diol epoxides.

Identification of a novel, N7-deoxyguanosine adduct as the major DNA adduct formed by a non-bay-region diol epoxide of benzo[a]pyrene with low mutagenic potential.

A metabolite of benzo[a]pyrene, 9-r,10-t-dihydroxy-7,8-c-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-III), that is not thought to be involved in carcinogenesis has nevertheless been shown to bind extensively to DNA in vitro. The adducts formed by this non-bay-region diol epoxide in Chinese hamster ovary cells are much less mutagenic than those formed by an isomeric diol epoxide that is carcinogenic. We have isolated and characterized three major adducts formed by in vitro reaction of BPDE-III with DNA. The major adduct, accounting for over half of the total is formed by reaction of BPDE-III with the N7 position of dGuo and is recovered after enzymatic digestion as an N7-Gua adduct. A second major adduct involves the N2 position of dGuo, while the third adduct is tentatively identified as a C8-substituted dGuo. Little or no reaction with deoxyadenosine residues is detected. The N7 adduct is unstable in DNA at 37 degrees C and is released as the modified base with a half-life of about 24 h. This adduct lability apparently leads to single-strand breaks and alkali-sensitive sites in the DNA and may account in part for some of the biological properties of BPDE-III adducts. This represents the first description of an N7-dGuo adduct that is formed in DNA as the major adduct by a diol epoxide derived from a carcinogenic polycyclic aromatic hydrocarbon.

Synthesis of 1- and 3-fluorobenzo[a]pyrene.

Fluoro-substituted aromatic hydrocarbons are useful probes for studying mechanistic details of oxygen transfer in metabolism catalyzed by cytochrome P450. Benzo[a]pyrene (BP) is a particularly suitable substrate for investigating this mechanism. Because 3-hydroxybenzo[a]-pyrene is one of the major metabolites of BP, preparation of 3-fluorobenzo[a]pyrene (3-FBP) was undertaken. Synthesis of 3-FBP was achieved in five steps starting from 6-chlorobenzo[a]pyrene (6-ClBP). In this synthesis 1-FBP was also produced. The overall yield was 16% for both 1-FBP and 3-FBP. After nitration of 6-ClBP at C-1 and C-3 with N2O4 and reduction by SnCl2 to the amino group, diazotization with NaNO2 in the presence of NaBF4 followed. The diazonium tetrafluoroborate salts were reacted with (CH3)2NH to produce the dimethyltriazonium tetrafluoroborate salts. By heating in toluene, a mixture of 1-F-6-ClBP and 3-F-6-ClBP was obtained. The two isomers were separated by normal-phase medium-pressure liquid chromatography. The chloro substituent was then selectively removed from both isomers by hydrogenolysis to yield 1-FBP and 3-FBP.

Synthesis of a novel fluorinated benzo[a]pyrene: 4,5-difluorobenzo[a]pyrene.

The synthesis of 4,5-difluorobenzo[a]pyrene, as a fluorinated probe to investigate the involvement of the K-region in the further metabolic activation of benzo[a]pyrene metabolites, is described. Benzo[a]pyrene-4,5-dione obtained from 2,3-dichloro-5,6-dicyano-1,4-benzoquinone oxidation of cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene was fluorinated with dimethylaminosulfur trifluoride to give 4H,5H,4,4,5,5,-tetra-fluorobenzo[a]pyrene. Defluorination using lithium aluminum hydride in tetrahydrofuran gave 4,5,-difluorobenzo[a]pyrene.