Evaluation of Nitrosamide Formation in the Cytochrome P450-Mediated Metabolism of Tobacco-Specific Nitrosamines

Article abstract of DOI:10.1021/acs.chemrestox.6b00384

N′-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are carcinogenic tobacco-specific nitrosamines believed to play a vital role in the initiation of tobacco-related cancers. For their carcinogenicities to be exhibited, both NNN and NNK must be metabolically activated by cytochrome P450s, specifically P450 2A6 and P450 2A13, respectively. Prior research has focused on α-hydroxylation, which leads to the formation of several DNA adducts that have been identified and quantified in vivo. However, some studies indicate that P450s can retain substrates within their active sites and perform processive oxidation. For nitrosamines, this would oxidize the highly unstable α-hydroxynitrosamines to potentially more stable nitrosamides, which could also alkylate DNA. Thus, we hypothesized that both NNN and NNK are processively oxidized in vitro to nitrosamides by P450 2A6 and P450 2A13, respectively. To test this hypothesis, we synthesized the NNN- and NNK-derived nitrosamides, determined their half-lives at pH 7.4 and 37 °C, and monitored for nitrosamide formation in an in vitro P450 system with product analysis by LC/NSI⁺-HRMS/MS. Half-lives of the nitrosamides were determined by HPLC-UV and ranged from 7-35 min, which is more than 40 times longer than the corresponding α-hydroxynitrosamines. Incubation of NNN in the P450 2A6 system resulted in the formation of the nitrosamide N′-nitrosonorcotinine (NNC) at low levels. Similarly, the nitrosamide 4-(methylnitrosamino)-1-(3-pyridyl)-1,4-butanedione (CH₂-oxo-NNK) was detected in low amounts in the incubation of NNK with the P450 2A13 system. The other possible NNK-derived nitrosamide, 4-(nitrosoformamido)-1-(3-pyridyl)-1-butanone (CH₃-oxo-NNK), was not observed in the P450 2A13 reactions. CH₂-oxo-NNK readily formed O⁶meGua in reactions with dGuo and calf thymus DNA. These results demonstrate that NNC and CH₂-oxo-NNK are novel metabolites of NNN and NNK, respectively. Though low-forming, their increased stability may allow for mutagenic DNA damage in vivo. More broadly, this study provides the first account of a cytochrome P450-mediated conversion of nitrosamines to nitrosamides, which warrants further studies to determine how general this phenomenon is in nitrosamine metabolism.

Full text of DOI:10.1021/acs.chemrestox.6b00384

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
Evaluation of Nitrosamide Formation in the Cytochrome
P450 Mediated Metabolism of Tobacco-Specific Nitrosamines
Erik S. Carlson, Pramod Upadhyaya, and Stephen S. Hecht
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00384 • Publication Date (Web): 16 Nov 2016
Downloaded from http://pubs.acs.org on November 20, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Chemical Research in Toxicology is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 46
Chemical Research in Toxicology
1
2
3
1
4
Evaluation of Nitrosamide Formation in the Cytochrome P450 Mediated Metabolism of
Tobacco-Specific Nitrosamines
5
6
7
8
2
3
4
5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
,‡
*
Erik S. Carlson,† Pramod Upadhyaya,† and Stephen S. Hecht†,
†
6
7
Masonic Cancer Center, University of Minnesota, 2231 6th Street SE, 2ꢀ210 CCRB, Minneapolis, MN
55455, United States
‡
8
9
Department of Pharmacology, University of Minnesota Medical School, 321 Church Street SE, 6ꢀ120
Jackson Hall, Minneapolis, MN 55455
10
11
12
13
14
15
16
17
18
19
*To whom correspondence should be addressed:
20
21
Masonic Cancer Center, University of Minnesota, 2231 6th Street SE - 2-148 CCRB, Minneapolis, MN 55455, USA.
phone: (612) 624-7604 fax: (612) 624-3869 e-mail: hecht002@umn.edu
1
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 2 of 46
1
2
3
4
5
6
1
2
TOC Graphic:
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
4
2
ACS Paragon Plus Environment

Page 3 of 46
Chemical Research in Toxicology
1
2
3
4
5
1
Abstract:
6
2
7
N′ꢀNitrosonornicotine (NNN) and 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (NNK) are
8
9
3
4
carcinogenic tobaccoꢀspecific nitrosamines believed to play a vital role in the initiation of
tobaccoꢀrelated cancers. To exhibit their carcinogenicity, both NNN and NNK must be
metabolically activated by cytochrome P450s, specifically P450 2A6 and P450 2A13,
respectively. Prior research has focused on αꢀhydroxylation, which leads to the formation of
several DNA adducts that have been identified and quantified in vivo. However, some studies
indicate that P450s can retain substrates within their active sites and perform processive
oxidation. For nitrosamines, this would oxidize the highly unstable αꢀhydroxynitrosamines to
potentially more stable nitrosamides, which could also alkylate DNA. Thus, we hypothesized
that both NNN and NNK are processively oxidized in vitro to nitrosamides by P450 2A6 and
P450 2A13, respectively. To test this hypothesis, we synthesized the NNNꢀ and NNKꢀderived
nitrosamides, determined their halfꢀlives at pH 7.4 and 37 °C, and monitored for nitrosamide
formation in an in vitro P450 system with product analysis by LCꢀNSI+ꢀHRMS/MS. Halfꢀlives
of the nitrosamides were determined by HPLCꢀUV and ranged from 7–35 min, which is more
than 40 times longer than the corresponding αꢀhydroxynitrosamines. Incubation of NNN in the
P450 2A6 system resulted in the formation of the nitrosamide, N'ꢀnitrosonorcotinine (NNC) at
low levels. Similarly, the nitrosamide 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanedione (CH2ꢀ
oxoꢀNNK), was detected in low amounts in the incubation of NNK with the P450 2A13 system.
The other possible NNKꢀderived nitrosamide, 4ꢀ(nitrosoformamido)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone
(CH3ꢀoxoꢀNNK), was not observed in the P450 2A13 reactions. CH2ꢀoxoꢀNNK readily formed
O6meGua in reactions with dGuo and calf thymus DNA. These results demonstrate that NNC
and CH2ꢀoxoꢀNNK are novel metabolites of NNN and NNK, respectively. Though lowꢀforming,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
3
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 4 of 46
1
2
3
4
5
6
7
8
9
1
2
3
4
their increased stability may allow for mutagenic DNA damage in vivo. More broadly, this study
provides the first account of a cytochromeꢀP450 mediated conversion of nitrosamines to
nitrosamides which warrants further studies to determine how general this phenomenon is in
nitrosamine metabolism.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
ACS Paragon Plus Environment

Page 5 of 46
Chemical Research in Toxicology
1
2
3
4
5
1
Introduction:
6
7
8
9
10
2
Tobacco use is the leading preventable cause of cancer death in the United States resulting in an
estimated 160,000 deaths annually,1 or 30% of all cancer deaths nationwide.2 4ꢀ(Methylnitrosamino)ꢀ1ꢀ
(3ꢀpyridyl)ꢀ1ꢀbutanone (NNK, 1) and N′ꢀnitrosonornicotine (NNN, 2) are tobaccoꢀspecific nitrosamines
thought to play a critical role in tobacco carcinogenesis. Both compounds cause tumors in animal models
and have been classified as carcinogenic to humans.1,3 To exhibit their carcinogenicity, NNK and NNN
must first be metabolically activated by cytochrome P450s.4–6 In the case of NNK, P450 2A13ꢀmediated
hydroxylation at the methylene or methyl carbon adjacent to the nitrosamino group results in highly
unstable αꢀhydroxy species 3 and 43,4 (Scheme 1A). Prior work indicates that αꢀhydroxynitrosamines have
halfꢀlives of ~5 sec7,8, decomposing to diazohydroxides such as 7 and 8,8 which lose H2O producing
diazonium ions that either hydrolyze to urinary products 10 and 11 or react with DNA and proteins to
form adducts4 (Scheme 1A). If left unrepaired, DNA adducts can cause mutations in critical oncogenes or
tumor suppressor genes, initiating tumor development. Methyl adducts such as O6ꢀmethylguanine
(O6meG) are especially tumorigenic in animal models.9 For NNN, αꢀhydroxylation at the 2′ or 5′ position
by P450 2A6 initiates a similar cascade leading to a variety of DNA adducts and urinary products
(Scheme 1A). Both NNKꢀ and NNNꢀderived DNA adducts have been identified and quantified in several
animal models and show promise as biomarkers for carcinogen activation in humans.10–14
3
4
5
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
While the αꢀhydroxynitrosamine hypothesis outlined above accounts nicely for many of the
metabolic products and DNA adducts produced in the metabolism of NNK, NNN, and other carcinogenic
nitrosamines, there are also inconsistencies in this hypothesis.15,16 Important among these is the short
lifetime of αꢀhydroxynitrosamines which raises questions about their ability to alkylate nuclear DNA after
having been formed in the endoplasmic reticulum. A few studies have shown these intermediates exist
long enough for glucuronidation, but measured levels were minor. In addition, it is unclear if this pathway
mediates detoxification or intracellular transportation to the nucleus17–19. Elespuru et al and Guttenplan
have explored the hypothesis that the αꢀhydroxynitrosamines are further oxidized to nitrosamides, which
5
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 6 of 46
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
are also direct DNA alkylating agents, but this alternate hypothesis lacks compelling supportive data15,16
In the study reported here, we explore this hypothesis with respect to the metabolic activation of NNK
and NNN. We hypothesize that the αꢀhydroxynitrosamines could be retained in the active site of P450
.
2A13 or P450 2A6 and further oxidized to the corresponding nitrosamides: 4ꢀ(methylnitrosamino)ꢀ1ꢀ(3ꢀ
pyridyl)ꢀ1,4ꢀbutanedione (CH2ꢀoxoꢀNNK, 13), 4ꢀ(nitrosoformamido)ꢀ1ꢀ(3ꢀpyridyl)ꢀ1ꢀbutanone (CH3ꢀoxoꢀ
NNK, 14) from NNK and N′ꢀnitrosonorcotinine (NNC, 15) from NNN.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
8
There is precedent for retention of substrates in P450s leading to processive oxidation.
Metabolism of nicotine by P450 2A6 proceeds through a retained iminium ion or hemiaminal
intermediate before releasing cotinine as the major metabolite.20 The Guengerich group has shown that
P450 2E1 oxidizes ethanol directly to acetic acid with limited substrate dissociation.21 Likewise, they
showed that formaldehyde and acetaldehyde formed in the metabolism of dimethylnitrosamine and
diethylnitrosamine by P450 2A6 are directly oxidized to formic acid and acetic acid, respectively, without
release of the intermediate aldehydes.22 They later proposed an alternate route for acid formation via a
nitrosamide intermediate; however, it was undetectable.23
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Nitrosamides are recognized as directꢀacting carcinogens with common halfꢀlives being on the
scale of minutes.24 Extensive research shows that their stability and reactivity is dependent on
temperature, steric and electronic factors, and solvent composition.25–29 In nucleophilic environments,30
the major products are the corresponding carboxylic acid derivatives and the diazonium species discussed
earlier (Scheme 1). Thus, if released into a cell, nitrosamides should not only have better stability for
traversing the hydrolytic environment of the cytosol, but also the ability to alkylate DNA. If nitrosamines
are oxidized to nitrosamides, this could lead to the identification of new classes of DNA adducts and a
better understanding of the mechanisms of nitrosamine carcinogenesis.
23
24
In the present study, we examine the hypothesis that NNK and NNN are metabolized to their
corresponding nitrosamides. We synthesized these three nitrosamides and evaluated their halfꢀlives and
6
ACS Paragon Plus Environment

Page 7 of 46
Chemical Research in Toxicology
1
2
3
4
1
major degradation products in vitro. We describe our finding that CH2ꢀoxoꢀNNK and NNC are minor
metabolites of NNK and NNN in in vitro assays with human cytochrome P450s. Further evaluation of
CH2ꢀoxoꢀNNK demonstrated that it methylates dGuo in DNA and is thus potentially mutagenic.
Together, this work provides the first account of a nitrosamine being converted metabolically to a
nitrosamide and furthers our understanding of the metabolism of NNK and NNN.
5
2
6
7
8
9
3
4
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 8 of 46
1
2
3
4
1
Experimental Procedures:
5
6
7
8
9
2
3
4
Caution: NNN and NNK are carcinogenic in animal models and are IARC Group 1 carcinogens. All
nitrosamides are presumed to be carcinogens based on their structure and reactivity. Handle these in a
wellꢀventilated fume hood with personal protective equipment and extreme care.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
Chemicals and Enzymes:
7
8
NNK, NNN, 4ꢀoxoꢀ4ꢀ(3ꢀpyridyl)ꢀbutanol (keto alcohol, 11), 5ꢀ(3ꢀpyridyl)ꢀ2ꢀhydroxytetrahydrofuran
(lactol, 12), O6ꢀmethylguanine (O6meGua), and [CD3]O6meGua were synthesized as previously
described.31–34 5ꢀ(3ꢀPyridyl)ꢀ2ꢀpyrrolidinone (norcotinine, 28) was obtained from AKos GmbH (Steinen,
Germany). P450 2A6 Baculosomes, regeneration system and reaction buffer were available as a Vivid
CYP450 Screening Kit from Life Technologies (Carlsbad, CA). Purified P450 2A13 and P450 reductase
were a generous gift from Dr. Sharon Murphy (University of Minnesota). All other chemicals and
solvents used were obtained from either Sigma Aldrich (Milwaukee, WI) or Thermo Scientific (Waltham,
MA) in reagent grade and used without further purification.
9
10
11
12
13
14
15
16
General Synthetic Procedures:
17
18
19
20
21
22
23
NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are reported as parts per
million (ppm). Residual solvent peaks were used as an internal reference for 1HꢀNMR (7.26 ppm CDCl3;
2.50 ppm D6ꢀDMSO) and 13CꢀNMR (77.2 ppm CDCl3; 39.5 ppm D6ꢀDMSO). Peak splitting used the
following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt =
doublet of triplets, ddd = doublet of doublet of doublets, bs = broad singlet, and m = multiplet. High
resolution mass spectrometry (HRMS) for selected compounds was performed on an LTQ Orbitrap Velos
(Thermo Scientific, Carlsbad, CA) and reported as m/z. Thinꢀlayer chromatography (TLC) utilized
8
ACS Paragon Plus Environment

Page 9 of 46
Chemical Research in Toxicology
1
2
3
4
1
Polygram preꢀcoated silica gel TLC plate (40 x 80 mm, 0.2 mm thick) with 254 nm fluorescent indicator.
TLC plates were visualized with permanganate stain when necessary, otherwise UV lamp irradiation
sufficed. Flash chromatography was performed on SiliCycle 60 (70ꢀ150) mesh silica gel. Reactions were
performed under an atmosphere of N2 unless specified otherwise.
5
2
6
7
8
9
3
4
5
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methyl 4-oxo-4-(3-pyridyl)-1-butanoate (18):
7
8
Sodium cyanide (0.104 g, 2.12 mmol) was suspended in anhydrous N,Nꢀdimethylformamide (DMF, 10
mL) and brought to 35 °C. 3ꢀPyridinecarboxaldehyde (2.27 g, 21.2 mmol, 2 mL) was added dropwise to
the suspension. After 10 min of stirring, the resulting red solution was treated dropwise with methyl
acrylate (1.90 g, 22.1 mmol, 2 mL). Over 4 h, the solution became increasingly yellow and slightly
viscous. The reaction was quenched with acetic acid (100 ꢀL). The resulting yellow solution was diluted
in CH2Cl2 and washed with H2O and brine. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo to a crude, yellow solid. Purification by column chromatography (50 to 100%
EtOAc in hexanes) yielded pure product as a white, crystalline solid (3.02 g, 73.8%). 1HꢀNMR (500 MHz;
CDCl3): δ 9.21 (dd, J = 2.2, 0.8 Hz, 1H, 2ꢀPy), 8.79 (dd, J = 4.8, 1.7 Hz, 1H, 6ꢀPy), 8.25 (ddd, J = 8.0,
2.2, 1.8 Hz, 1H, 4ꢀPy), 7.43 (ddd, J = 8.0, 4.8, 0.9 Hz, 1H, 5ꢀPy), 3.72 (s, 3H, CH3), 3.33 (t, J = 6.5 Hz,
2H, COCH2CH2), 2.80 (t, J = 6.5 Hz, 2H, COCH2CH2).; 13CꢀNMR (126 MHz; CDCl3): δ 197.1 (CO),
173.2 (CO2CH3), 153.8 (2ꢀPy), 149.8 (5ꢀPy), 135.5 (4ꢀPy), 131.9 (3ꢀPy), 123.8 (5ꢀPy), 52.1 (CH3), 33.8
(COCH2CH2CO2), 27.9 (COCH2CH2CO2) ppm.
9
10
11
12
13
14
15
16
17
18
19
20
21
4-Oxo-4-(3-pyridyl)-butanoic acid (19):
22
23
Compound 18 (393 mg, 2.03 mmol) was dissolved in 1N NaOH (4 mL) and stirred for 3 h at room
temperature. The solution went from colorless to yellow. The pH was adjusted to ~5ꢀ6 with an equal
9
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 10 of 46
1
2
3
4
5
6
1
2
volume of 1N HCl and a precipitate formed. The solid was filtered and dried under vacuum to give a
white, crystalline solid (251 mg, 69%).
7
8
9
3
4
5
6
7
1HꢀNMR (500 MHz; DMSOꢀd6): δ 12.18 (s, 1H, COOH), 9.14 (d, J = 2.1 Hz, 1H, 2ꢀPy), 8.80 (dd, J = 4.8,
1.6 Hz, 1H, 6ꢀPy), 8.31 (dd, J = 8.0, 1.8 Hz, 1H, 4ꢀPy), 7.57 (dd, J = 8.0, 4.8 Hz, 1H, 5ꢀPy), 3.30 (t, J =
6.3 Hz, 2H, COCH2), 2.60 (t, J = 6.3 Hz, 2H, CH2COOH). 13CꢀNMR (126 MHz; DMSO): δ 198.1 (CO),
173.7 (COOH), 153.5 (2ꢀPy), 149.1 (6ꢀPy), 135.4 (4ꢀPy), 131.7 (3ꢀPy), 123.9 (5ꢀPy), 33.4 (COCH2), 27.7
(CH2COOH) ppm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
9
Methyl 4-oxo-4-(3-pyridyl)-butanamide (20a):
10
11
12
13
14
15
16
A solution of 19 (43.57 mg, 0.243 mmol), methylamine hydrochloride (25.41 mg, 0.376 mmol), and Nꢀ
hydroxysuccinimide (NHS, 45.2mg, 0.393 mmol) in anhydrous dimethylsulfoxide (DMSO, 3 mL) was
treated with N,Nꢀdiisopropylethylamine (DIPEA, 75.7 mg, 0.586 mmol, 102 ꢀL) and 1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide (EDAC, 196 mg, 1.03 mmol). The reaction was stirred for 22 h at
room temperature before diluting with EtOAc. The mixture was washed with H2O and brine, dried over
MgSO4, filtered, and concentrated in vacuo to yield a crude, pink oil. Purification by column
chromatography (5:100 MeOH/CH2Cl2) yielded pure product as an offꢀwhite solid (32.9 mg, 70%).
17
18
19
20
21
1HꢀNMR (500 MHz; DMSOꢀd6): δ 9.13 (d, J = 1.2 Hz, 1H, 2ꢀPy), 8.79 (dd, J = 4.7, 1.3 Hz, 1H, 6ꢀPy),
8.30 (d, J = 7.9 Hz, 1H, 4ꢀPy), 7.84 (s, 1H, NH), 7.57 (dd, J = 7.9, 4.7 Hz, 1H, 5ꢀPy), 3.27 (t, J = 6.6 Hz,
2H, COCH2), 2.57 (d, J = 4.6 Hz, 3H, CH2CONH), 2.48 (t, J = 6.6 Hz, 2H, NHCH 3). 13C NMR (126
MHz; DMSO): δ 198.6 (CO), 171.3 (CONH), 153.3 (6ꢀPy), 149.1 (2ꢀPy), 135.4 (4ꢀPy), 131.8 (3ꢀPy),
123.9 (5ꢀPy), 33.7 (COCH2), 29.0 (CH2CONH), 25.5 (NHCH3) ppm.
22
23
10
ACS Paragon Plus Environment

Page 11 of 46
Chemical Research in Toxicology
1
2
3
4
5
1
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanedione (CH2-oxo-NNK, 13):
6
7
8
9
10
2
A solution of 20a (31.4mg, 0.163 mmol) in a 5:1 mixture of acetic anhydride and acetic acid (6 mL) was
brought to 0 °C. To this was added NaNO2 (30.7 mg, 0.445 mmol) all at once. After 4 h, the mixture was
poured onto iceꢀcold H2O. The aqueous mixture was extracted with CH2Cl2. The pooled organics were
dried over MgSO4, filtered, and concentrated in vacuo to yield a crude yellow oil. Purification by column
chromatography on silica gel (100% EtOAc) yielded pure product as a bright, yellow oil (28.8 mg, 65%).
1HꢀNMR (500 MHz; CDCl3): δ 9.27 (s, 1H, 2ꢀPy), 8.83 (d, J = 4.1 Hz, 1H, 6ꢀPy), 8.31 (d, J = 7.9 Hz, 1H,
4ꢀPy), 7.48 (dd, J = 7.8, 4.9 Hz, 1H, 5ꢀPy), 3.67 (t, J = 6.0 Hz, 2H, COCH2), 3.55 (t, J = 6.0 Hz, 2H,
CH2CON), 3.14 (s, 3H, CH3). 13C NMR (126 MHz; CDCl3): δ 196.7 (CO), 175.9 (CONNO), 153.5 (6ꢀ
Py), 149.4 (2ꢀPy), 135.6 (4ꢀPy), 131.9 (3ꢀPy), 123.8 (5ꢀPy), 33.0 (CH2CONNO), 28.7 (COCH2), 25.9
(NCH3) ppm. HRMS Calc: 222.08732, Found: 222.08719
3
4
5
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
8
9
10
11
12
13
CH2-oxo-NNK from 5′-Hydroxycotinine:
14
15
16
17
18
19
20
21
5'ꢀHydroxycotinine (54.23 mg, 0.282 mmol) in CH2Cl2 (25 mL) was brought to 0 °C and treated with pꢀ
toluenesulfonic acid (111 mg, 0.584 mmol). After 5 min of stirring, NaNO2 (151.2 mg, 2.2 mmol) was
added. Stirring was continued for 3 h before pouring onto iceꢀcold H2O. The organic layer was separated
and the aqueous extracted with CH2Cl2. The pooled organics were dried over MgSO4, filtered, and
concentrated in vacuo to give a crude orange oil. The product was purified by HPLC using a 150 x 4.6
mm, 5 ꢀm, Kinetix HILIC column (Phenomenex) with 1:1 hexanes/CHCl3 and isopropanol as mobile
phases. The gradient was 5% to 20% isopropanol over 10 min at 1 mL/min. The product eluted at ~4 min.
The purified product was a bright, yellow oil (4.98 mg, 8%).
22
23
11
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 12 of 46
1
2
3
4
1
N′-Nitrosonorcotinine (NNC, 15):
5
6
7
8
9
2
3
A solution of norcotinine (31.4 mg, 0.163 mmol) in a 5:1 mixture of acetic anhydride and acetic acid (6
mL) was brought to 0 °C. To this was added NaNO2 (30.7 mg, 0.445 mmol) all at once. After 4 h, the
mixture was poured onto iceꢀcold H2O. The aqueous mixture was extracted with CH2Cl2. The pooled
organics were dried over MgSO4, filtered, and concentrated in vacuo to yield a crude yellow oil.
Purification by column chromatography (100% EtOAc) yielded pure product as a bright, yellow oil
(92%). 1HꢀNMR (500 MHz; CDCl3): δ 8.56 (d, J = 4.3 Hz, 1H, 6ꢀPy), 8.46 (d, J = 1.8 Hz, 1H, 2ꢀPy), 7.36
(d, J = 8.0 Hz, 1H, 4ꢀPy), 7.30ꢀ7.28 (m, 1H, 5ꢀPy), 5.29 (dd, J = 9.1, 3.1 Hz, 1H, NCH), 2.97 (dt, J =
18.6, 9.4 Hz, 1H, COCH2), 2.87 (ddd, J = 18.5, 9.4, 4.0 Hz, 1H, COCH2'), 2.66 (dq, J = 13.4, 9.4 Hz, 1H,
CHCH2), 2.15ꢀ2.09 (m, 2H, CHCH2') ppm; 13CꢀNMR (126 MHz; CDCl3): δ 172.7 (CO), 149.4 (6ꢀPy),
147.1 (2ꢀPy), 134.1 (3ꢀPy), 132.7 (4ꢀPy), 123.8 (5ꢀPy), 55.7 (NCH), 26.2 (COCH2), 22.2 (CHCH2) ppm.
HRMS Calc: 192.07675, Found: 192.07670
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
11
12
13
14
2-(3-Pyridyl)-1,3-dithiane (21):
15
16
A solution of 3ꢀpyridylcarboxaldehyde (114 mg, 1.06 mmol, 100 ꢀL) and 1,3ꢀpropanedithiol (162 mg,
1.50 mmol, 150 ꢀL) in anhydrous tetrahydrofuran (THF, 5 mL) was treated with BF3•Et2O (173 mg, 1.22
17
18
19
20
21
mmol 300 ꢀL) dropwise at room temperature. The mixture was then heated to reflux at 80 °C and stirred
for 24 h before quenching with sat'd NaHCO3 solution. The aqueous phase was extracted several times
with CH2Cl2. The pooled organic layers were washed with brine, dried over MgSO4, filtered, and
evaporated in vacuo to give crude yellow crystals. Purification by column chromatography
(hexanes/EtOAc 1:1) yielded the product as a fine, white powder (199.4 mg, >95%).
22
23
1HꢀNMR (500 MHz; CDCl3): δ 8.72 (d, J = 2.0 Hz, 1H, 2ꢀPy), 8.57 (dd, J = 4.9, 1.5 Hz, 1H, 6ꢀPy), 7.85
(dt, J = 7.9, 2.0 Hz, 1H, 4ꢀPy), 7.31 (dd, J = 7.9, 4.9 Hz, 1H, 5ꢀPy), 5.21 (s, 1H, S2CH), 3.10 (ddd, J =
12
ACS Paragon Plus Environment

Page 13 of 46
Chemical Research in Toxicology
1
2
3
4
1
14.6, 12.3, 2.4 Hz, 2H, SCH2CH2 ꢀax), 2.96 (dt, J = 14.0, 3.8 Hz, 2H, SCH2CH2 ꢀeq), 2.22 (dtt, J = 14.2,
4.6, 2.4 Hz, 1H, SCH2CH2 ꢀax), 2.02ꢀ1.93 (m, 1H, SCH2CH2 ꢀeq) ppm; 13C NMR (126 MHz; CDCl3): δ
150.1 6ꢀPy, 149.5 (2ꢀPy), 135.7 (4ꢀPy), 135.4 (3ꢀPy), 124.0 (5ꢀPy), 48.8 (S2CH), 32.3 (SCH2CH2), 25.2
(SCH2CH2) ppm.
5
2
6
7
8
9
3
4
5
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tert-Butyl 3-(2-(3-pyridyl)-1,3-dithianyl)-1-propylcarbamate (22):
7
8
A solution of 21 (222 mg, 1.13 mmol) and tetramethylethylenediamine (TMEDA, 131.8 mg, 1.13 mmol,
170 ꢀL) in anhydrous THF (6 mL) was cooled to ꢀ78 °C and treated with nꢀBuLi in hexanes dropwise
(1.28 mmol, 800 ꢁL). The resulting dark red solution was stirred at ꢀ78 °C for 30 min before dropwise
addition of 27 in THF (360 mg, 1.26 mmol, 3 mL). The mixture was stirred at ꢀ78 °C for 2 h before
allowing the bath to come to room temperature. After 14 h of stirring, the reaction was quenched with
H2O. The aqueous phase was extracted several times with EtOAc. The pooled organics were dried over
MgSO4, filtered, and concentrated in vacuo to give a crude, yellow oil. Purification by column
chromatography (hexanes/EtOAc 1:1) yielded the product as yellow crystals (290 mg, 72.5%).
9
10
11
12
13
14
15
16
17
18
19
20
21
1HꢀNMR (500 MHz; CDCl3): δ 9.13 (d, J = 2.1 Hz, 1H, 2ꢀPy), 8.52 (dd, J = 4.7, 1.6 Hz, 1H, 6ꢀPy), 8.20
(ddd, J = 8.1, 2.4, 1.6 Hz, 1H, 4ꢀPy), 7.32 (ddd, J = 8.1, 4.7, 0.6 Hz, 1H, 5ꢀPy), 4.41 (bs, 1H, NH), 3.03ꢀ
3.00 (m, 2H, NHCH2), 2.72ꢀ2.61 (m, 4H, SCH2CH2), 2.03ꢀ2.00 (m, 2H, CCH2), 1.99ꢀ1.91 (m, 2H,
SCH2CH2), 1.48ꢀ1.42 (m, 2H, CH2CH2CH2), 1.40 (s, 9H, C(CH3)3) ppm; 13C NMR (126 MHz; CDCl3): δ
155.9 (NHCO), 150.8 (2ꢀPy), 148.4 (6ꢀPy), 137.6 (3ꢀPy), 136.8 (4ꢀPy), 123.4 (5ꢀPy), 79.4 (C(CH3)3),
56.5 (SCS), 42.5 (CCH2), 40.4 (NHCH2), 28.5 (CH3), 27.6 (SCH2CH2), 25.1 (SCH2CH2), 24.7
(CH2CH2CH2) ppm.
22
23
13
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 14 of 46
1
2
3
4
5
6
1
2
3-(2-(3-Pyridyl)-1,3-dithianyl)-1-propylformamide (23):
7
8
9
3
4
A solution of 22 (87.6 mg, 0.247 mmol) in CH2Cl2 (3 mL) was treated with trifluoroacetic acid (TFA,
1.49 g, 13.1 mmol, 1 mL), which resulted in gas evolution. After 3 h, the solvent was evaporated in vacuo
to remove excess TFA. The resulting oil was reconstituted in CH2Cl2 and washed with sat'd. NaHCO3
solution and brine. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give a
yellow oil. This was dissolved in MeOH (5 mL) and treated with triethylamine (29.04 mg, 0.288 mmol,
40 ꢀL) and methyl formate (95.7 mg, 1.60 mmol, 110 ꢀL). The flask was sealed and heated to 55 °C with
stirring. After 4 h, the mixture was concentrated in vacuo to yield a yellow oil. 1HꢀNMR indicated the
product was a 9:1 mixture of cisꢀ and transꢀformamide isomers. Purity was sufficient to carry forward
without column purification.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1HꢀNMR (500 MHz; CDCl3): δ 9.09 (d, J = 2.5 Hz, transꢀ2ꢀPy), 9.08 (d, J = 2.1 Hz, 1H, cisꢀ2ꢀPy), 8.49
(dd, J = 4.8, 1.6 Hz, transꢀ6ꢀPy), 8.48 (dd, J = 4.7, 1.5 Hz, 1H, cisꢀ6ꢀPy), 8.19ꢀ8.16 (m, 1H, 4ꢀPy), 8.07
(d, J = 1.1 Hz, 1H, cisꢀNHCHO), 7.92 (d, J = 11.9 Hz, transꢀNHCHO ), 7.32ꢀ7.29 (m, 1H, 5ꢀPy), 5.94 (s,
1H, cisꢀNHCHO), 5.84 (s, transꢀNHCHO ), 3.18 (q, J = 6.7 Hz, 2H, cisꢀCH2NH), 3.10 (q, J = 6.8 Hz,
transꢀCH2NH), 2.63 (m, 4H, SCH2), 2.02ꢀ1.99 (m, 2H, CCH2), 1.98ꢀ1.85 (m, 2H, SCH2CH2), 1.52ꢀ1.46
(m, 2H, CH2CH2NH). 13CꢀNMR (126 MHz; CDCl3): δ 164.4 (transꢀCHO), 161.2 (cisꢀCHO), 150.5 (2ꢀ
Py), 148.37 (transꢀ6ꢀPy), 148.25 (cisꢀ6ꢀPy), 137.45 (cisꢀ3ꢀPy), 137.35 (transꢀ3ꢀPy), 136.73 (cisꢀ4ꢀPy),
136.67 (transꢀ4ꢀPy), 123.33 (transꢀ5ꢀPy), 123.29 (cisꢀ5ꢀPy), 56.28 (cisꢀSCS), 56.17 (transꢀSCS), 42.3
(cisꢀCCH2), 42.0 (transꢀCCH2), 41.4 (transꢀNHCH2), 37.6 (cisꢀNHCH2), 27.5 (SCH2), 25.7 (transꢀ
CH2CH2CH2), 24.85 (cisꢀSCH2CH2), 24.79 (transꢀSCH2CH2), 24.1 (cisꢀCH2CH2CH2) ppm.
22
23
4-(Formamido)-1-(3-pyridyl)-1-butanone (24):
14
ACS Paragon Plus Environment

Page 15 of 46
Chemical Research in Toxicology
1
2
3
4
1
NꢀChlorosuccinimide (NCS, 101.8 mg, 0.762 mmol) and AgNO3 (168.5 mg, 0.992 mmol) were
suspended in 1:1 MeCN/H2O (1 mL) and cooled to 0 °C. To this was added a solution of 23 in MeCN
(0.247 mmol, 1.5 mL), which resulted in immediate precipitate formation. After 30 min, the reaction was
quenched with sat. Na2SO3, sat'd NaHCO3, and brine solutions in succession (1 mL each). The mixture
was filtered and extracted with CH2Cl2. The pooled organics were dried over MgSO4, filtered, and
concentrated in vacuo to give a crude solid. Purification by column chromatography on silica gel (6%
MeOH in CHCl3) yielded pure product as a white solid (20.5 mg, 43% over three steps). 1HꢀNMR (500
MHz; CDCl3): δ 9.15 (dd, J = 2.2, 0.7 Hz, 1H, 2ꢀPy), 8.78 (dd, J = 5.5, 1.7 Hz, 1H, transꢀ6ꢀPy), 8.77 (dd,
J = 4.8, 1.7 Hz, 1H, cisꢀ6ꢀPy), 8.22 (dt, J = 8.0, 2.1 Hz, 1H, transꢀ4ꢀPy), 8.21 (dt, J = 8.0, 2.0 Hz, 1H, cisꢀ
4ꢀPy), 8.18 (s, 1H, cisꢀCHO), 8.06 (d, J = 11.9 Hz, transꢀCHO ), 7.42 (m, 1H, 5ꢀPy), 5.96 (s, 1H,
NHCHO), 3.41 (q, J = 6.6 Hz, 2H, cisꢀCH2NH), 3.36 (q, J = 6.8 Hz, transꢀCH2NH), 3.08 (t, J = 6.9 Hz,
2H, cisꢀCOCH2), 3.07 (t, J = 6.8 Hz, transꢀCOCH2), 2.01 (quintet, J = 6.9 Hz, 2H, cisꢀCH2CH2CH2), 2.01
(quintet, J = 6.9 Hz, transꢀCH2CH2CH2); 13CꢀNMR (126 MHz; CDCl3): δ 198.6 (CO), 161.5 (CHO),
153.8 (6ꢀPy), 149.7 (2ꢀPy), 135.5 (4ꢀPy), 132.0 (3ꢀPy), 123.8 (5ꢀPy), 37.8 (CH2NH), 36.3 (COCH2), 23.5
(CH2CH2CH2) ppm.
5
2
6
7
8
9
3
4
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
8
9
10
11
12
13
14
15
16
17
tert-Butyl 3-hydroxypropyl-1-carbamate (26):
18
19
20
21
22
23
A solution of 3ꢀaminoꢀ1ꢀpropanol (4.94 g, 65.8 mmol, 25) and triethylamine (7.26 g, 71.7 mmol, 10 mL)
in CH2Cl2 (175 mL) was treated with Boc anhydride (16.15 g, 74.0 mmol,17 mL) dropwise, which
resulted in vigorous gas evolution. Once bubbling ceased, the reaction was quenched with sat'd NH4Cl.
The organic layer was collected and the aqueous layer was further extracted with CH2Cl2. The pooled
organics were washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. NMR indicated
the product was sufficiently pure to be brought directly to the next step.
15
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 16 of 46
1
2
3
4
5
6
7
8
9
1
2
3
4
1HꢀNMR (500 MHz; CDCl3): δ 4.84 (bs, 1H, OH), 3.67 (q, J = 5.5 Hz, 2H, HOCH2), 3.30 (d, J = 5.7 Hz,
2H, CH2NH), 3.07 (bs, 1H, NH), 1.68 (quintet, J = 5.7 Hz, 2H, CH2CH2CH2), 1.46 (s, 9H, tꢀBu) ppm. 13C
NMR (126 MHz; CDCl3): δ 157.2 (CONH2), 79.6 (C(CH3)3, 59.2 (HOCH2), 36.9 (CH2NH), 32.9
(CH2CH2CH2), 28.4 (tꢀBu) ppm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
tert-Butyl 3-iodopropyl-1-carbamate (27):
7
8
Imidazole (Im., 5.42 g, 79.7 mmol) and triphenylphospine (20.72 g, 79.0 mmol) were dissolved in CH2Cl2
(250 mL) and brought to 0 °C. To this was added I2 (20.50 g, 80.8 mmol) scoopwise, resulting in a dark
orange solution. After 20 min of stirring, 26 in CH2Cl2 (11.5 g, 65.7 mmol, 50 mL) was added and the
solution was allowed to come to room temperature. After 22 h of stirring, the mixture was filtered over
Celite and washed with 5% Na2S2O3. The organics were dried over MgSO4, filtered, and concentrated in
vacuo to give a yellow solid. Purification by column chromatography (hexanes/EtOAc 4:1) gave pure
product as a light yellow solid (83.2% over 2 steps).1HꢀNMR (500 MHz; CDCl3): δ 4.63 (bs, 1H, NH),
3.19 (m, 4H, NHCH2/ICH2), 2.00 (quintet, J = 6.6 Hz, 2H, CH2CH2CH2), 1.44 (s, 9H, tꢀBu) ppm. 13C
NMR (126 MHz; CDCl3): δ 155.9 (CONH), 79.4 (C(CH3)3), 41.0 (CH2NH), 33.4 (CH2), 28.4 (C(CH3)3),
3.1 (ICH2) ppm.
9
10
11
12
13
14
15
16
17
18
4-(Nitrosoformamido)-1-(3-pyridyl)-1-butanone (CH3-oxo-NNK, 14)
19
20
21
22
23
A solution of 24 (31.4mg, 0.163 mmol) in a 5:1 mixture of acetic anhydride and acetic acid (6 mL) was
brought to 0 °C. To this was added NaNO2 (30.7mg, 0.445 mmol) all at once. After 4 h, the mixture was
poured onto iceꢀcold H2O. The aqueous mixture was extracted with CH2Cl2. The pooled organics were
dried over MgSO4, filtered, and concentrated in vacuo to yield a crude yellow oil. Purification by column
chromatography (100% EtOAc) yielded pure product as a bright, yellow oil (28.8mg, 80%).1HꢀNMR
16
ACS Paragon Plus Environment

Page 17 of 46
Chemical Research in Toxicology
1
2
3
4
1
(500 MHz; CDCl3): δ 10.00 (s, 1H, CHO), 9.13 (d, J = 1.1 Hz, 1H, 2ꢀPy)), 8.80 (dd, J = 4.8, 1.1 Hz, 1H,
6ꢀPy), 8.24 (dd, J = 8.0, 1.6 Hz, 1H, 4ꢀPy), 7.47 (dd, J = 8.0, 4.9 Hz, 1H, 5ꢀPy), 4.11 (q, J = 7.1 Hz, 1H,
NCH2), 2.92 (t, J = 6.9 Hz, 2H, COCH2), 1.91 (quintet, J = 6.9 Hz, 2H, CH2CH2CH2) ppm. 13ꢀC NMR
(126 MHz; CDCl3): δ 196.8 (CO), 168.5 (CHO), 153.1 (6ꢀPy), 149.0 (2ꢀPy), 136.0 (4ꢀPy), 132.2 (3ꢀPy),
124.1 (5ꢀPy), 37.6 (NCH2), 35.8 (COCH2), 20.9 (CH2CH2CH2) ppm; HRMS Calc: 222.08732, Found:
222.08719
5
2
6
7
8
9
3
4
5
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
8
9
Determination of t1/2 of Nitrosamides:
10
11
12
13
14
15
16
HPLCꢀUV analysis was performed using a 250 x 4.6 mm GeminiꢀNX C18 column (Phenomenex,
Torrance, CA) with the following solvent gradients for the analyses indicated below: (1) isocratic for 5
min at 4% B followed sequentially by a linear gradient to 12% B over 15 min, a 10 min hold, a linear
gradient to 30% B over 10 min, and a final linear gradient to 40% B over 2 min; (2) isocratic for 5 min at
12% B followed sequentially by a linear gradient to 30% B over 10 min, a linear gradient to 40% B over
15 min, and a final linear gradient to 70% B over 2 min. In both systems, solvent A was 15 mM NH4OAc
and solvent B was methanol.
17
18
19
20
21
22
23
For t1/2 determination, an aliquot of NNC (180 nmol) in CH2Cl2 was dried and reconstituted in 30
ꢀL of 0.5X P450 2A6 Reaction Buffer (Life Technologies) and incubated for 0 to 30 min at 37 °C. After
the desired incubation time, 5 ꢁL of sample was analyzed by HPLC using gradient 1. A254 was monitored
and the peaks for NNC and its decomposition products were integrated. NNC eluted at 36.0 min. Peak
area for NNC was fit to a singleꢀorder exponential plot while using the 0 min incubation peak area as a
normalizing factor. The analysis was similarly performed for CH2ꢀoxoꢀNNK and CH3ꢀoxoꢀNNK, except
HPLC gradient 2 was used. CH2ꢀoxoꢀNNK and CH3ꢀoxoꢀNNK eluted at 33.0 and 29.5 min, respectively.
17
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 18 of 46
1
2
3
4
5
6
1
2
Decomposition products were identified by retention time comparisons and coꢀinjection with synthetic
standards.
7
8
9
3
4
5
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vitro detection of CH2-oxo-NNK using P450 2A13:
7
8
9
Incubations with P450 2A13 were performed as previously reported.35 Briefly, purified P450 2A13 and
cytochrome P450 reductase were reconstituted with dilauroylphosphatidylcholine (DLPC, Sigma Aldrich)
for 45 min on ice before diluting with Tris buffer to give a final concentration of 1 ꢀM P450 2A13, 2 ꢀM
10
11
12
13
14
15
16
P450 reductase, 0.1 ꢀg/ꢀL DLPC, and 50 mM Tris, pH = 7.4. To initiate the reaction, an aliquot of this
(containing 5 pmol P450) was added to a Trisꢀbuffered solution of NNK (4 ꢀM) and NADPH (0.2 mM).
Final reaction volumes were always 100 ꢀL. The mixture was brought to 37 °C for 1–60 min before
quenching with 10 ꢀL of both Ba(OH)2 and ZnSO4. After centrifuging the sample at 8000 g for 4 min, the
supernatant was collected and 2 ꢀL were immediately analyzed by liquid chromatographyꢀpositive
nanoelectrosprayꢀionization highꢀresolution tandem mass spectrometry (LCꢀNSI+ꢀHRMS/MS) with an
LTQ Orbitrap Velos (Thermo Scientific, Carlsbad, CA). LC employed a handꢀpacked, Luna C18 (5 ꢀm),
17
18
19
20
21
22
23
100 mm x 75 ꢀm, 15 ꢀm orifice capillary column with a multiꢀstep gradient. Initially, 5% B at 1 ꢁL/min
from 0–5 min was used to load the sample. Afterwards, the flow rate was dropped to 0.3 ꢁL/min and a
linear gradient was started from 5% to 20% B over 4 min, followed by a ramp to 55% B over 10 min, and
reꢀequilibration, where solvent A was 5 mM NH4OAc and solvent B was acetonitrile. CH2ꢀoxoꢀNNK and
CH3ꢀoxoꢀNNK were monitored by both full scan and MS2 fragmentation. Full scan was performed at a
resolution of 60,000 and the accurate parent mass of both nitrosamides (m/z = 222.08719) was extracted
at a mass tolerance of 5 ppm. For MS2 fragmentation, parent ions were isolated (2.0 amu isolation width)
18
ACS Paragon Plus Environment

Page 19 of 46
Chemical Research in Toxicology
1
2
3
4
1
and fragmented by collisionꢀinduced dissociation (CID) with a collision energy of 25 eV, resolution of
15,000, and scan time of 30 ms. Accurate product ion masses from characteristic transitions for CH2ꢀoxoꢀ
NNK (m/z 222 → m/z 180.06542, ꢀH3CNNO +OH), CH3ꢀoxoꢀNNK (m/z 222 → m/z 106.02852), NNK
(m/z 208 → m/z 178.11002), and keto alcohol 11 (m/z 166 → m/z 148.07564) were extracted at a mass
tolerance of 5 ppm.
5
2
6
7
8
9
3
4
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
8
In vitro detection of NNC using P450 2A6:
9
10
11
12
13
14
15
16
17
18
19
Incubations with P450 2A6 were performed as described by the manufacturer with modifications.36 After
thawing the P450 2A6 Baculosomes and VividꢀNADPHꢀRegeneration System (Life Technologies) on
ice, aliquots were combined and diluted 1:10 and 1:50, respectively, with 0.5X Vivid Reaction Buffer
(Life Technologies). For each incubation, an aliquot of the combinedꢀenzyme system (containing 5 pmol
P450) was added to a 0.5X ReactionꢀBuffered solution of NNN (4 ꢀM) and this new mixture was preꢀ
incubated for 2 min at 37 °C. To initiate the reaction, an aliquot of NADP+ (containing 3 nmol) was
added. Final reaction volumes were 100 ꢀL. The incubation and workꢀup were as described earlier for
NNKꢀP450 2A13 incubations. NNC detection was performed by adapting the NNKꢀP450 2A13 LCꢀNSI+ꢀ
HRMS/MS method described above to the accurate parent mass of NNC (m/z 192.07670) in full scan.
Likewise, the MS2 analysis was used to monitor for the accurate product ion masses from characteristic
transitions of NNC (m/z 192 → m/z 134.04739, 162.07874), NNN (m/z 178 → m/z 148.09941), and lactol
20
21
22
12 (m/z 166 → m/z 148.07571).
in vitro Methylation of dGuo by CH2-oxo-NNK:
19
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 20 of 46
1
2
3
4
5
6
7
8
9
1
2
A solution of CH2ꢀoxoꢀNNK in CH2Cl2 was dried under a stream of N2 and reconstituted in a phosphateꢀ
buffered solution of dGuo (4.34 mM dGuo, 25 mM NaHPO4, pH = 7.4) so that the molar ratio of CH2–
oxoꢀNNK to dGuo was 1:1. This was brought to 37 °C and incubated for 18 h. To assess methylation, 200
fmol of [CD3]O6meGua was added as internal standard. Samples were brought up to 1 mL with 0.1N HCl
and incubated at 90 °C for 30 min. After cooling on ice, the samples were neutralized with 1.0N NaOH
and purified by solidꢀphase extraction (StrataꢀX polymeric reversed phase, 30 mg, Phenomenex,
Torrance, CA). Before sample addition, the cartridge was activated using 1 mL each of MeOH and H2O.
After sample addition, the cartridge was washed with 1 mL of both H2O and 10% MeOH. The sample
was eluted and collected with 1 mL of MeOH. The collected fraction was evaporated to dryness in a
Speedvac. The residue was reconstituted in 30 ꢁL of H2O and analyzed by LCꢀMS/MS.37
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
7
8
9
10
11
12
13
14
15
16
17
18
We used a wellꢀestablished liquid chromatographyꢀpositive electrospray ionizationꢀtandem mass
spectrometry (LCꢀESI+ꢀMS/MS) method. A 0.5 x 150 mm Zorbax SBꢀC18, 5 ꢁm column (Agilent, Santa
Clara, CA) was eluted with a multiꢀstep gradient and flow rate of 10 ꢁL/min. After a linear gradient from
5% to 10% B over 10 min, the eluant was brought to 40% B over 5 min, followed by a wash at 90% B
and reꢀequilibration, where solvent A was 15mM NH4OAc and solvent B was methanol. MS was
performed on a TSQ Vantage triple quadrupole mass analyzer (Thermo Scientific). The SRM transitions
were m/z 166.1 → m/z 149.1 and m/z 166.1 → m/z 124.1 for O6meGua and m/z 169.1 → m/z 152.1 for
[CD3]O6meGua using a collision energy of 30 eV and a 0.2 amu scan width.
19
20
in vitro Methylation of calf thymus DNA by CH2-oxo-NNK:
21
22
23
24
A solution of CH2ꢀoxoꢀNNK was dried under a stream of N2 and reconstituted in a phosphateꢀbuffered
solution of calf thymus DNA so that the ratio was 3 nmol CH2ꢀoxoꢀNNK :1 ꢁg DNA. This was brought to
37 °C and incubated for 18 h. The aqueous sample was extracted twice with equal volumes of
CHCl3:isoamyl alcohol (24:1). The DNA was precipitated by addition of an equal volume of isopropanol
20
ACS Paragon Plus Environment

Page 21 of 46
Chemical Research in Toxicology
1
2
3
4
1
and gentle shaking. Isolated DNA was washed with 500 ꢁL of 70% EtOH and 100% EtOH, and dried
under N2. To assess methylation, isolated DNA was dissolved in 100 ꢁL of sodium phosphate buffer (25
mM, pH = 7.4) and 200 fmol of [CD3]O6meGua was added as internal standard. The samples were then
processed and analyzed as described above for dGuo methylation.
5
2
6
7
8
9
3
4
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 22 of 46
1
2
3
4
5
6
7
8
9
1
2
3
Results:
Synthesis of Nitrosamides:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
Retrosynthetic analysis identified 3ꢀpyridinecarboxaldehyde (17) as a common precursor for both
NNKꢀderived nitrosamides (Scheme 2). The synthesis of CH2ꢀoxoꢀNNK started with the formation of
keto ester 18 by using the Stetter reaction38 to couple aldehyde 17 with methyl acrylate, followed by
hydrolysis of 18 to keto acid 19. This method is a convenient alternative to the more commonly used
routes to this compound.39,40 Keto acid 19 was coupled to methylamine using 1ꢀethylꢀ3ꢀ(3ꢀ
6
7
8
9
dimethylaminopropyl)carbodiimide (EDAC) and Nꢀhydroxysuccinimide (NHS) in dimethylsulfoxide
(DMSO), resulting in the openꢀchain and lactam conformers of 20 in a 2:1 ratio, respectively. Nitrosation
of the openꢀchain conformer produced CH2ꢀoxoꢀNNK (13) as a single rotamer.41 Nitrosation of the lactam
conformer also produced CH2ꢀoxoꢀNNK; however, new conditions using a strong acid catalyst were
required. These conditions also degraded the product which limited the isolatable yield (<10%).
Ultimately, only the openꢀchain route was synthetically useful for CH2ꢀoxoꢀNNK production.
10
11
12
13
14
15
16
17
18
19
20
21
Synthesis of CH3ꢀoxoꢀNNK (Scheme 2) started with protection of 17 with 1,3ꢀpropanedithiol in
nearly quantitative yield to give 21. This was coupled to 27 by classic umpolung chemistry42 to give 22 in
excellent yield. Compound 27 was prepared in two steps from 25 on a multiꢀgram scale by first Bocꢀ
protecting the amine and then converting the alcohol to an iodide using a modified Appel reaction.43 After
Boc removal from 22 and Nꢀformylation to achieve 23, the dithiane group was oxidatively removed44 to
produce 24 in 43% yield over 3 steps. To complete the synthesis, 24 was nitrosated to give CH3ꢀoxoꢀ
NNK (14) in excellent yield.
22
23
NNC (15) was prepared by nitrosation of norcotinine (28) in 92% yield. The three nitrosamides
were stored in CH2Cl2 at 4 °C. They were stable for at least three months under these conditions.
22
ACS Paragon Plus Environment

Page 23 of 46
Chemical Research in Toxicology
1
2
3
4
1
Attempts to store these compounds neat or in H2Oꢀmiscible solvents (MeOH, MeCN, acetone, etc.)
resulted in decomposition.
5
2
6
7
8
9
3
10
11
12
4
Stability of Nitrosamides
13
14
15
5
The stabilities of the nitrosamides were determined at pH 7.4, 37 ⁰C, in buffers to be used in our
P450 assays. Reactions were followed by HPLC and major products were identified (Figure S1,
Supporting Information). Decay curves for each nitrosamide are shown in Figure 1. The halfꢀlives of
CH2ꢀoxoꢀNNK and CH3ꢀoxoꢀNNK were 35.5 min and 6.7 min, respectively. The halfꢀlife of NNC under
these conditions was 12.3 min. The major product in each case was that expected by nitrosamide
hydrolysis, namely keto acid 19 from CH2ꢀoxoꢀNNK, keto alcohol 11 from CH3ꢀoxoꢀNNK, and hydroxy
acid 29 from NNC (Scheme 3).
16
6
7
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
9
10
11
12
13
In vitro cytochrome P450-catalyzed metabolism of NNK to CH2-oxo-NNK:
14
15
16
17
18
19
20
21
22
23
With synthetic nitrosamide standards in hand and an understanding of their stability, we designed
an assay to detect their formation by P450ꢀmediated metabolism. P450 2A13 is the most efficient enzyme
for αꢀhydroxylation of NNK and was chosen for this part of the study.45 The NNK concentration was set
at 4 ꢁM, the Km for production of hydroxylated products. Samples were analyzed immediately after workꢀ
up to minimize nitrosamide decomposition. We used an LTQ HighꢀResolution Orbitrap Velos MS system
to enhance sensitivity and minimize background noise. We monitored for the accurate masses of the most
abundant product ions of CH2ꢀoxoꢀNNK and CH3ꢀoxoꢀNNK resulting from MS2 fragmentation, and also
extracted their accurate parent masses in full scan mode. Similar monitoring was performed for NNK and
keto alcohol 11 to ensure catalytic turnover as 11 is the most abundant product from methyl
hydroxylation.4,45
23
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 24 of 46
1
2
3
4
5
6
7
8
9
1
2
3
4
5
When NNK was incubated with the relevant enzymes and cofactors, we detected a peak that
matched the accurate parent ion mass, accurate product ion mass resulting from MS2 of [M+H]+ = 222,
and the retention time of synthetic CH2ꢀoxoꢀNNK (Figure 2). This peak was detectable as early as the 1ꢀ
min time point, reached its maximum concentration at 5 min, tapered off by 10–30 min, and was nearly
undetectable by 60 min (Figure 2CꢀF). We did not detect CH3ꢀoxoꢀNNK at any time point.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
The signal for NNK decreased 8ꢀfold while the signal for keto alcohol 11 simultaneously
increased 4ꢀfold over the 60ꢀmin period (data not shown). This indicates that NNK metabolism was rapid
over the assay time period. CH2ꢀoxoꢀNNK was a minor metabolite as its signal was >4000ꢀfold less than
that of keto alcohol 11. No metabolites were observed in control incubations lacking enzyme or cofactors
(data not shown).
8
9
10
11
12
13
14
When identical incubations containing CH2ꢀoxoꢀNNK (10 nM) were performed, the peak area
was 10ꢀfold higher than in unspiked samples, indicating that CH2ꢀoxoꢀNNK was recoverable under our
conditions. Based on this, we estimate that CH2ꢀoxoꢀNNK was produced at concentrations less than 1 nM
in our incubations.
15
16
In vitro cytochrome P450-catalyzed metabolism of NNN to NNC:
17
18
19
20
21
22
The NNN incubations were performed in essentially the same way as those with NNK except that
P450 2A6 was used instead of P450 2A13 as it is the most efficient enzyme for NNN metabolism.46
Catalytic turnover was assessed by measuring lactol 12, because it is the major product from 5′ꢀ
hydroxylation of NNN (Scheme 1A).47 We detected NNC as early as the 1ꢀmin time point (Figure 3). The
peak matched the synthetic standard with respect to the accurate parent ion mass in full scan, the accurate
mass of the most abundant product ions in the MS2 of [M + H]+ = 192, and retention time.
24
ACS Paragon Plus Environment

Page 25 of 46
Chemical Research in Toxicology
1
2
3
4
1
The NNC signal was maximal at 5 min and was approximately 1000ꢀfold lower in intensity than
5
2
6
that of lactol 12 (Scheme 1A). The concentration of NNC at the 5ꢀmin time point was estimated to be ~10
7
8
9
3
4
5
nM.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vitro Methylation of dGuo and DNA by CH2-oxo-NNK:
6
7
We tested the ability of CH2ꢀoxoꢀNNK to methylate DNA by incubating it with a molar
equivalent of dGuo and calf thymus DNA in phosphate buffer for 18 h. Levels of O6ꢀmeGua were 61.7
and 802 ꢀmol/mg Gua for the dGuo and DNA reactions, respectively, as determined by LCꢀMS/MS
analysis. (Figures S2, Supporting Information). Formation of 7ꢀmeGua was also noted, but it was not
quantified.
8
9
10
11
25
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 26 of 46
1
2
3
4
1
Discussion:
5
6
7
8
9
2
3
This study presents the first account of nitrosamines being directly converted to nitrosamides by
P450 catalysis. This breaks new ground in our knowledge of nitrosamine metabolism and provides an
impetus to determine if this phenomenon applies to all nitrosamines. Specifically, we found that CH2ꢀoxoꢀ
NNK and NNC are novel metabolites of P450ꢀmediated oxidation of NNK and NNN, respectively. We
did not observe formation of CH3ꢀoxoꢀNNK, perhaps due to its short halfꢀlife (6.7 min). These novel
metabolites also provide a potentially new mechanism for NNKꢀ and NNNꢀDNA adduct formation
(Scheme 1B). It has long been known that the αꢀhydroxynitrosamine intermediates 3–6 (Scheme 1A)
alkylate DNA, but their short lifetimes raise questions regarding their ability to traverse the hydrolytic
cytosol. The detected nitrosamides had halfꢀlives of 12–35 min, 100ꢀfold more than those of the αꢀ
hydroxynitrosamines.7,8 Additionally, we showed that CH2ꢀoxoꢀNNK methylates both dGuo and calf
thymus DNA (Figure S2, Supplementary Information). Thus, in the case of NNK, it is plausible that CH2ꢀ
oxoꢀNNK could be partially responsible for the methyl adducts previously thought to be formed purely by
αꢀhydroxynitrosamine 3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
However, we note that both CH2ꢀoxoꢀNNK and NNC are quite minor metabolites of NNK and
NNN. It was estimated that CH2ꢀoxoꢀNNK and NNC form at concentrations of ~1 nM and 10 nM,
respectively, while keto alcohol 11 and lactol 12, the hydrolysis products of αꢀhydroxyNNK and αꢀ
hydroxyNNN, form at levels ~4000ꢀfold higher. Because P450 2A13 and P450 2A6 are the most efficient
enzymes for NNK and NNN oxidation and the formation of their known products keto alcohol 11 and
lactol 12 (Scheme 1A) was rapid, it is unlikely that the low levels of CH2ꢀoxoꢀNNK and NNC result from
low catalytic turnover. Likewise, the positive controls indicate that analyte recovery was achievable under
our incubation conditions. We noted that formation of both CH2ꢀoxoꢀNNK and NNC started at 1 min,
peaked at 5 min, and that both were nearly undetectable by 30 min. This may indicate that as metabolism
proceeds, newly formed side products and P450ꢀrelated reactiveꢀoxygen species are eliminating the
nitrosamides via secondary reactions at a rate faster than nitrosamide formation.
26
ACS Paragon Plus Environment

Page 27 of 46
Chemical Research in Toxicology
1
2
3
4
1
We were not able to quantify nitrosamide formation in these reactions. We initially attempted
quantification by HPLCꢀradioflow techniques, but this approach was not sensitive enough (LOD = 400
fmol on column, data not shown). After nitrosamide detection was achieved by LCꢀNSI+ꢀHRMS, we
attempted to trap these products with Nꢀacetylꢀlysine and Nꢀacetylꢀcysteine. Though trapping was
achieved with synthetic standards, this method was unsuccessful in our assay due to low trapping
efficiency and low sample recovery after solid phase extraction, which resulted in no analyte detection
even with accurate mass detection (data not shown). Therefore, we settled on estimating formation levels
by comparing peak areas to those of spiked positive controls.
5
2
6
7
8
9
3
4
5
6
7
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
10
11
12
13
14
15
16
17
18
Despite being minor metabolites, the long halfꢀlives and strong DNAꢀbinding properties of the
nitrosamides suggest potential biological relevance. However, determining whether it is more important
to be lowꢀforming and stable versus highꢀforming and unstable would require further study. Additionally,
though CH2ꢀoxoꢀNNK and NNC are formed to low extents, the nitrosamide pathway may be more
efficient for other nitrosamines. For example, studies by Chowdhury et al noted considerable, processive
conversion of dimethylnitrosamine and diethylnitrosamine to acid byproducts by P450 2A6.22 It was
hypothesized that the αꢀhydroxynitrosamine intermediate decomposed within the active site and the
resulting aldehyde was then oxidized to the acid. The nitrosamide hypothesis was also tested, but
detection was unsuccessful. Given our results, it is plausible that nitrosamides were readily produced, but
instability limited their detection.
19
20
21
22
23
24
25
Our synthesis of each nitrosamide proceeded essentially as expected, except for a few key
findings. First, our method for keto acid 19 (Scheme 2), though not entirely novel,48 is considerably more
convenient than previously reported routes.39,40 The twoꢀstep process involves milder conditions, gives
reproducible yields, and simpler product purification; the latter step provides >99% pure product after
only filtration. Next, the conversion of keto acid 19 to keto amide 20 (Scheme 2) was noteworthy because
a previous study49 reported compound 20 to be in a ringꢀchain equilibrium heavily favoring the lactam
(~6:1). In our hands, the compounds were readily separable on silica gel and showed no isomerization
27
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 28 of 46
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
while stored neat at 2–8 °C. They were clearly distinct by NMR (Figure S3, Supporting Information). The
openꢀchain product had two clean triplets integrating to 2H each while these signals collapsed into nonꢀ
distinct multiplets integrating to 4H in the ring product. Additionally, the methyl resonance in the lactam
is a singlet as opposed to a doublet in the openꢀchain product. In support of the results reported by
Nguyen et al,49 when we performed this reaction in solvents other than DMSO, the lactam 5′ꢀ
hydroxycotinine (20b, Scheme 2), predominated. Similarly, 20b was the major product when harsher
amide coupling conditions were used, such as in situ acid chloride formation by oxalyl chloride or AlMe3ꢀ
mediated amide formation50,51 from 18. It is apparent that DMSO and mild coupling conditions favor the
open chain conformer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10
11
12
13
14
Our nitrosamides were each isolated exclusively as one rotamer. This contrasts to nitrosamines
commonly occur as a mixture of both (E)ꢀ and (Z)ꢀisomers. Past studies indicate that the (E)ꢀconformer is
electronically favored for most nitrosamides.24 Furthermore, rotation to the (Z)ꢀconformer is commonly
believed to be the rateꢀlimiting step for nitrosamide decomposition by a pericyclic process24 and thus,
may not be isolatable.
15
16
17
18
19
20
21
22
23
24
The order of compound stability was CH2ꢀoxoꢀNNK > NNC > CH3ꢀoxoꢀNNK. This ranking fits
with known factors contributing to nitrosamide decomposition.24,26,41 In hydrolytic environments,30
nitrosamides with bulkier groups adjacent to the carbonyl group are more stable. This suggests that CH3ꢀ
oxoꢀNNK should be the least stable, consistent with our observations. Likewise, bulky groups adjacent to
the nitrogen decrease stability. This is consistent with CH2ꢀoxoꢀNNK being most stable and NNC being
relatively less stable. The decomposition products suggest that the mechanism is primarily hydrolysis.
The products shown in Scheme 3B were all either the major or only identified product. However, for
NNC and CH3ꢀoxoꢀNNK, we did identify a lactone and ester as minor products, respectively (Figure S1,
Supporting Information). These presumably result from the extensively studied 1,3ꢀsigmatropic
rearrangement mechanism.28 Though this rearrangement is highly favored when nitrosamides are heated
28
ACS Paragon Plus Environment

Page 29 of 46
Chemical Research in Toxicology
1
2
3
4
1
in organic solvents, aqueous conditions seem to favor hydrolysis and are most relevant to the in vivo
5
2
6
situation.
7
8
9
3
With data supporting the formation of CH2ꢀoxoꢀNNK, we were interested in testing one of its
possible modes of DNA damage: methylation. Methylation was expected since keto acid is the major
product of CH2ꢀoxoꢀNNK hydrolysis (Scheme 2). This implies that methane diazohydroxide, a known
methylating agent, is also released. Our results clearly demonstrated the forrmation of O6meGua in these
reactions, indicating that CH2ꢀoxoꢀNNK methylates DNA. In addition to the methyl DNA adducts readily
formed by CH2ꢀoxoꢀNNK, both CH2ꢀoxoꢀNNK and NNC could potentially generate a set of novel DNA
adducts (Scheme 1B). Further studies are needed to establish the structures, level, and importance of
possible adducts derived from the nitrosamide pathway.
10
4
5
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
8
9
10
11
12
13
14
15
16
17
18
19
In summary, we hypothesized that NNK and NNN are metabolized by P450 2A13 and P450 2A6,
respectively, to their corresponding nitrosamides in vitro. We tested this by synthesizing CH2ꢀoxoꢀNNK,
CH3ꢀoxoꢀNNK, and NNC and evaluating their stability at pH = 7.4 and 37 °C. They were quite stable
relative to the corresponding αꢀhydroxynitrosamines. We then showed that CH2ꢀoxoꢀNNK and NNC are
novel, though minor, metabolites of NNK and NNN, respectively, in an in vitro P450 model. With the
knowledge that CH2ꢀoxoꢀNNK has a relatively long halfꢀlife and methylates DNA, it could potentially
play a role in the mechanism of carcinogenesis by NNK. More broadly, this is the first direct evidence for
the conversion of nitrosamines to nitrosamides by P450 catalysis and provides rationale for further studies
to determine whether this is a general transformation in nitrosamine metabolism.
20
29
ACS Paragon Plus Environment