Amidine Nitrosation

The acidic nitrosation chemistry of nine acyclic secondary and tertiary amidines (Ph-NC(R1)NR2R3; R1 = H, CH3, Ph; R2, R3 = H, Ph or (CH3)2 or C(CH2)4) and several N-acylamidines was investigated. The principal nitrosation products were amides derived from the amino moiety and compounds derived from the benzenediazonium ion, which was independently trapped for quantitation in several cases. Tertiary amidines also produce nitrosamines in minor, but significant, yields. The benzamidines did not react, and the N-acylamidines hydrolyzed much more rapidly than they nitrosated. The data support the hypothesis that the reaction occurs by nitrosation on the imino nitrogen, followed by the addition of H2O to give a tetrahedral intermediate (α-hydroxynitrosamine) for which the main decomposition pathway generates an amide and a diazonium ion. In the case of the pyrrolidine-derived amidines, about 25% of the decomposition results in cleavage of the amine moiety, which nitrosates to give N-nitrosopyrrolidine. Pseudo-first-order rate constants for amidine nitrosation in aqueous acetic acid with excess nitrite at 25 °C ranged from (3 to 106) × 10-5 s-1, while the amidine basicity ranged over 5 pKa units. Rate constants corrected for amidine basicity showed the pyrrolidine derived amidines to be most reactive. The lack of benzamidine nitrosative reactivity is attributed to a very slow rate of H2O additon to the N-nitrosoamidinium ion and reversible nitrosation

<i>N</i>-Nitrosotolazoline: Decomposition Studies of a Typical <i>N</i>-Nitrosoimidazoline

N-Nitrosotolazoline (N-nitroso-2-benzylimidazoline), a N-nitrosated drug typical of N-nitrosoimidazolines, reacts readily with aqueous acid, nitrous acid, or N-acetylcysteine to produce highly electrophilic diazonium ions capable of alkylating cellular nucleophiles. The kinetics and mechanism of the acidic hydrolytic decomposition of N-nitrosotolazoline have been determined in mineral acids and buffers. The mechanism of decomposition in acidic buffer is proposed to involve the rapid reversible protonation of the imino nitrogen atom followed by slow general base-catalyzed addition of H2O to the 2-carbon of the imidazoline ring to give a tetrahedral intermediate, which is also a α-hydroxynitrosamine. Rapid decomposition of this species gives rise to the diazonium from which the products are derived by nucleophilic attack, elimination, and rearrangement. The proposed mechanism is supported by the observations of general acid catalysis, a negligible deuterium solvent kinetic isotope effect (kH/kD = 1.15) and ΔS⧧ = −34 eu. In phosphate buffer at 30 °C, the half-lives of N-nitrosotolazoline range from 5 min at pH 3.5 to 4 h at pH 6. The main reaction product of the hydrolytic decomposition is N-(2-hydroxyethyl)phenylacetamide. This and other products are consistent with the formation of a reactive diazonium ion intermediate. N-Nitrosotolazoline nitrosates 50 times more rapidly than tolazoline and results in a set of products derived from reactive diazonium ions but different from those produced from the hydrolytic decomposition of the substrate. N-Acetylcysteine increases the decomposition rate of N-nitrosotolazoline by 25 times at pH 7 and results in both N-denitrosation and induced decomposition to produce electrophiles. These data suggest that N-nitrosotolazoline shares the chemical properties of many known direct-acting mutagens and carcinogens

<i>N</i>-Nitrosotolazoline: Decomposition Studies of a Typical <i>N</i>-Nitrosoimidazoline

N-Nitrosotolazoline (N-nitroso-2-benzylimidazoline), a N-nitrosated drug typical of N-nitrosoimidazolines, reacts readily with aqueous acid, nitrous acid, or N-acetylcysteine to produce highly electrophilic diazonium ions capable of alkylating cellular nucleophiles. The kinetics and mechanism of the acidic hydrolytic decomposition of N-nitrosotolazoline have been determined in mineral acids and buffers. The mechanism of decomposition in acidic buffer is proposed to involve the rapid reversible protonation of the imino nitrogen atom followed by slow general base-catalyzed addition of H2O to the 2-carbon of the imidazoline ring to give a tetrahedral intermediate, which is also a α-hydroxynitrosamine. Rapid decomposition of this species gives rise to the diazonium from which the products are derived by nucleophilic attack, elimination, and rearrangement. The proposed mechanism is supported by the observations of general acid catalysis, a negligible deuterium solvent kinetic isotope effect (kH/kD = 1.15) and ΔS⧧ = −34 eu. In phosphate buffer at 30 °C, the half-lives of N-nitrosotolazoline range from 5 min at pH 3.5 to 4 h at pH 6. The main reaction product of the hydrolytic decomposition is N-(2-hydroxyethyl)phenylacetamide. This and other products are consistent with the formation of a reactive diazonium ion intermediate. N-Nitrosotolazoline nitrosates 50 times more rapidly than tolazoline and results in a set of products derived from reactive diazonium ions but different from those produced from the hydrolytic decomposition of the substrate. N-Acetylcysteine increases the decomposition rate of N-nitrosotolazoline by 25 times at pH 7 and results in both N-denitrosation and induced decomposition to produce electrophiles. These data suggest that N-nitrosotolazoline shares the chemical properties of many known direct-acting mutagens and carcinogens

Nucleoside and DNA Adducts from <i>N</i>-Nitrosotolazoline

The reaction of N-nitrosotolazoline, the nitrosation product of a representative imidazoline receptor drug tolazoline, with DNA, deoxyguanosine (dG), or deoxyadenosine (dA) produces adducts containing the 2-phenylacetamidoethyl group. The synthesis and characterization of 2-phenylacetamidoethyl-guanine derivatives (O6-dG, O6-Gua, N2-Gua, and 7-Gua) and 2-phenylacetamidoethyladenine derivatives (1-Ade, 3-Ade, 7-Ade, and N6-Ade) are described. In addition to the use of an established UV spectral method for confirming the structure of the alkyl adenines, a new 13C NMR method for determining the N-alkylation site is presented. In combination with the synthesized standards, HPLC MS/MS methods were used to determine the nature and the quantity of adducts produced. N-Nitrosotolazoline reacted with dG to give 7-(2-phenylacetamidoethyl)deoxyguanosine (major), O6-(2-phenylacetamidoethyl)deoxyguanosine, and 5′-O-phenyacetyldeoxyguanosine. The reaction of N-nitrosotolazoline with dA produced the 1-, 3-, 7-, N6, and 5-O′-2-phenylacetamidoethyl adenine and dA derivatives as well as several phenylacetyl adducts. Reaction of N-nitrosotolazoline with DNA in vitro resulted in the detection of 2-phenylacetamidoethyl adducts (adduct, relative %): 7-Gua, 60%; 3-Ade, 30%; O6-Gua, 8%; and 7-Ade, 2%. Comparison of these data with appropriate literature data, as well as our work on the mechanism of N-nitrosotolazoline hydrolytic decomposition, is consistent with the adducts being produced from a 2-phenylacetamidoethyldiazonium intermediate. The results show that N-nitrosotolazoline, and presumably other N-nitrosoimidazolines, if produced by endogenous nitrosation pathways, are capable of alkylating DNA without additional metabolic transformation and are probable carcinogens

Mass Spectrometric Methodology for the Determination of Glyoxaldeoxyguanosine and O⁶-Hydroxyethyldeoxyguanosine DNA Adducts Produced by Nitrosamine Bident Carcinogens

N-Nitrosodiethanolamine (NDELA) is a bident carcinogen that undergoes both P-450 mediated α-hydroxylation and β-oxidation, leading ultimately to the formation of two prominent DNA adducts, glyoxaldeoxyguanosine (gdG) and O6-2-hydroxyethyldeoxyguanosine (OHEdG), in rat liver. HPLC coupled with electrospray ionization (ESI) and tandem mass spectrometry was used for both detection and quantification of gdG and OHEdG. The method, which is fast, sensitive, and unambiguous, is a significant improvement over the previous 32P-postlabeling methodology. A rapid procedure for the enzymatic hydrolysis of the DNA under acidic conditions preserved the integrity of the pH sensitive gdG adducts. Glyoxal and 3-nitroso-2-oxazolidinone generated gdG and OHEdG adducts, respectively, in calf thymus DNA (ct-DNA) in a concentration (range of 104) dependent manner permitting optimization. Isotopomeric internal standards were prepared from the modified guanine derivatives by enzymatic trans-glycosylation. Quantitative HPLC−ESI−MS/MS analysis employing selective reaction monitoring (SRM) for the loss of the deoxyribose fragment was utilized. Both adducts could be detected in the liver DNA of rats that were administered NDELA in a dose range of 0.4−0.8 mmol/kg. At the highest dose, gdG adducts (4.4−11 adducts/106 nuc.) were more abundant than OHEdG adducts (0.35−0.87 adducts/106 nuc.). Conversely, OHEdG adducts were produced in higher yields in ct-DNA than were gdG adducts at the same reagent concentrations

Mass Spectrometric Methodology for the Determination of Glyoxaldeoxyguanosine and O⁶-Hydroxyethyldeoxyguanosine DNA Adducts Produced by Nitrosamine Bident Carcinogens

N-Nitrosodiethanolamine (NDELA) is a bident carcinogen that undergoes both P-450 mediated α-hydroxylation and β-oxidation, leading ultimately to the formation of two prominent DNA adducts, glyoxaldeoxyguanosine (gdG) and O6-2-hydroxyethyldeoxyguanosine (OHEdG), in rat liver. HPLC coupled with electrospray ionization (ESI) and tandem mass spectrometry was used for both detection and quantification of gdG and OHEdG. The method, which is fast, sensitive, and unambiguous, is a significant improvement over the previous 32P-postlabeling methodology. A rapid procedure for the enzymatic hydrolysis of the DNA under acidic conditions preserved the integrity of the pH sensitive gdG adducts. Glyoxal and 3-nitroso-2-oxazolidinone generated gdG and OHEdG adducts, respectively, in calf thymus DNA (ct-DNA) in a concentration (range of 104) dependent manner permitting optimization. Isotopomeric internal standards were prepared from the modified guanine derivatives by enzymatic trans-glycosylation. Quantitative HPLC−ESI−MS/MS analysis employing selective reaction monitoring (SRM) for the loss of the deoxyribose fragment was utilized. Both adducts could be detected in the liver DNA of rats that were administered NDELA in a dose range of 0.4−0.8 mmol/kg. At the highest dose, gdG adducts (4.4−11 adducts/106 nuc.) were more abundant than OHEdG adducts (0.35−0.87 adducts/106 nuc.). Conversely, OHEdG adducts were produced in higher yields in ct-DNA than were gdG adducts at the same reagent concentrations

Nitrosation Chemistry of Pyrroline, 2-Imidazoline, and 2-Oxazoline: Theoretical Curtin−Hammett Analysis of Retro-Ene and Solvent-Assisted C−X Cleavage Reactions of α-Hydroxy-<i>N</i>-Nitrosamines

The results are presented of a theoretical study of the nitrosation chemistry of pyrroline 1 (X = CH2), imidazoline 2 (X = NH), and 2-oxazoline 3 (X = O). Imines 1−3 are converted to the α-hydroxy-N-nitrosamines 7−9 via the N-nitrosoiminium ions 4−6. The NN-cis isomers of 7−9 may undergo retro-ene reactions to the δ-oxoalkyl diazotic acids 10−12. With the opportunity for microsolvation, C−X cleavage becomes possible for 8 and 9 and leads to the formation of N-(2-aminoethyl)- and N-(2-hydroxyethyl)-N-nitrosoformamides 15 and 16, respectively. The NN-isomerization barriers are comparable to the barriers for the ring-opening reactions, and the consideration of two Curtin−Hammett scenarios is required:  CH-I for the NN-trans-rotamers of 7−9 to undergo C−X cleavage or NN-isomerization and CH-II for the NN-cis-rotamers to undergo C−X cleavage, C−N cleavage, or NN-isomerization. We determined all stereoisomers of the substrates, the products, and of all transition states structures for the retro-ene reactions of 7−9, the C−X cleavages of microsolvated 8 and 9, and the NN-isomerizations of 8 and 9. The potential energy surfaces were explored at the B3LYP/6-31G** level, and the results are discussed with emphasis on the comparison of the kinetics and thermodynamics of C−N versus C−X cleavage. The study shows all decompositions to be very fast with activation barriers below 21 kcal·mol-1, and the comparitive analysis predicts that the chemical toxicologies of 1 and 3 should be similar and remarkably different from that of 2

DNA Adducts from <i>N-</i>Nitrosodiethanolamine and Related β-Oxidized Nitrosamines in Vivo: ³²P-Postlabeling Methods for Glyoxal- and <i>O</i>⁶-Hydroxyethyldeoxyguanosine Adducts

The mechanism by which environmentally prevalent N-nitrosodiethanolamine (NDELA) and related 2-hydroxyethyl- or other β-oxidized nitrosamines initiate the carcinogenic process has remained obscure. 32P-Postlabeling assays for the pH sensitive glyoxal-deoxyguanosine (gdG) and the O6-2-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 μmol/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 μmol/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 μmol/mol of DNA. OHEdG DNA adducts were produced from equimolar amounts of nitrosamines in rat liver in vivo over the range 4−25 μmol/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. α-Deuteration markedly decreased OHEdG adduct formation while β-deuteration had the opposite effect. These data support the hypothesis that NDELA and related nitrosamines are activated by both enzyme mediated α-hydroxylation and β-oxidation. The formation of OHEdG adducts from NDELA requires α-hydroxylation of the 2-hydroxyethyl chain, and formation of gdG necessitates a β-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

DNA Guanine Adducts from 3-Methyl-1,2,3-oxadiazolinium Ions

The reaction of 3-methyl-1,2,3-oxadiazolinium tosylate 10, a close model for a putative reactive intermediate in the carcinogenic activation of ethanol nitrosamines such as (2-hydroxyethyl)methylnitrosamine 1, with various guanine derivatives, including acycloguanosine 12, deoxyguanosine, deoxyguanosine monophosphate, and cyclic guanosine monophosphate, various DNA oligomers, and calf-thymus DNA has been examined to determine whether this compound methylates and hydroxyethylates guanine residues as proposed. In all of the transformations, 7-(2-(methylnitrosamino)ethyl)guanine (14) is the major product, following acidic hydrolysis, and exceeds the formation of 7-methylguanine by ratios ranging from 4:1 to 48:1, depending upon the guanine bearing substrate. O6-(2-(Methylnitrosamino)ethyl)deoxyguanosine (20) was prepared from the Mitsunobu coupling of 1 and a protected deoxyguanosine derivative. 20 is not produced in the reaction of 10 and deoxyguanosine and decomposes to 1 and guanine upon mild acid treatment, suggesting possible neighboring group participation in its facile hydrolytic cleavage. All of the major products from the reaction of 10 and 12 have been characterized, including the direct alkylation product, 7-(2-(methylnitrosamino)ethyl)acycloguanosine (13), and N2-(2-(methylnitrosamino)ethyl)guanine, which was independently synthesized. Elucidation of the reactions of DNA with 10 and other electrophiles was facilitated by the development of both partial and total enzymatic hydrolysis assays utilizing 32P-5‘-labeled DNA oligotetramers containing one of each base type and HPLC with radiometric detection. The partial hydrolysis assay gives information as to the type of base being modified, and the total hydrolysis assay permits a determination of the number of adducts produced for a given base. The assays permit a comparison between reactions where the same type of base adduct could be expected. Comparisons of the reactions of ethylene oxide and 10 using this methodology showed that 10 does not hydroxyethylate guanine in DNA

The Carcinogenic Significance of Reactive Intermediates Derived from 3-Acetoxy- and 5-Acetoxy-2-hydroxy-<i>N-</i>nitrosomorpholine

N-Nitroso-2-hydroxymorpholine (NHMOR), a relatively reactive metabolite of two potent carcinogens, N-nitrosodiethanolamine (NDELA) and N-nitrosomorpholine (NMOR), has been reported to not be carcinogenic. Two isomeric acetate esters of the α-hydroxynitrosamines expected to be produced from the cytochrome P450-mediated metabolism of NHMOR have been synthesized, and their hydrolytic decomposition products, hydrolysis rates, and deoxyguanosine (dG) reaction adducts have been determined. N-Nitroso-3-acetoxy-2-hydroxymorpholine was prepared in high yield from the reaction of N-nitroso-2,3-dehydromorpholine with dry peracetic acid in glacial acetic acid or by the reaction of its dimethyldioxirane-produced epoxide with glacial acetic acid. The hydrolysis of this α-acetoxynitrosamine gave acetaldehyde (10%), ethylene glycol (55%), glyoxal (95%), and acetic acid. The pH rate profile for the hydrolysis of this nitrosamine was abnormal in that it exhibited pronounced base-catalyzed hydrolysis beginning at pH 5. The mechanism of hydrolytic decomposition is proposed to involve neighboring group participation with the formation of a reactive epoxide intermediate. N-Nitroso-3-acetoxy-2-hydroxymorpholine reacted with dG to give these guanine adducts after acidic deglycosylation:  1,N2-glyoxal (65%), 7-(2-hydroxyethyl)guanine (9%), and O6-hydroxyethylguanine (3%). N-Nitroso-5-acetoxy-2-hydroxymorpholine was synthesized from 2-hydroxyethylvinylnitrosamine by its oxidative conversion to the corresponding aldehyde followed by reaction with dry peracetic acid in glacial acetic. The hydrolytic decomposition products of this nitrosamine were 2-acetoxyacetaldehyde (65%), a rearrangement product, glycol aldehyde (15%), a trace of glyoxal, and acetic acid. The pH rate profile for the hydrolysis of this acetate is similar to other α-acetoxynitrosamines in that it exhibits a pH-independent region which gives way to base-catalyzed ester hydrolysis beginning at pH 7. The lower pH (≈ 7 < 9) onset of base catalysis is proposed to involve base-catalyzed opening of the hemiacetal and intramolecular acyl transfer to give an unstable α-hydroxynitrosamine. N-Nitroso-5-acetoxy-2-hydroxymorpholine was less reactive toward dG and gave the 1,N2-etheno-dG adduct (44%). The products from both of the isomeric α-acetoxy nitrosamines were judged to arise from diazonium ions produced from unstable α-hydroxynitrosamine intermediates. The high yield of the rearrangement product 2-acetoxyacetaldehyde could explain the low carcinogenic potential of NHMOR if it is mainly α-hydroxylated at the 5 carbon. Hydroxylation of NHMOR at carbon 3 is expected to yield a carcinogenic outcome