Synthesis of partially deuterated N-nitrosamines - New standards in tobacco-smoke analysis

Article abstract of DOI:10.1007/s00706-003-0152-8

N-Nitrosamines of tobacco alkaloids contribute to the cause of tobacco related cancers and, therefore, they are important analytes. Herein the preparation of four partially deuterated N-nitrosamines from simple, commercially available, deuterated educts is described, which can serve as useful standards in tobacco-smoke analysis. Springer-Verlag 2004.

Full text of DOI:10.1007/s00706-003-0152-8

Monatshefte f€ur Chemie 135, 549–555 (2004)
DOI 10.1007/s00706-003-0152-8
Synthesis of Partially Deuterated
N-Nitrosamines – New Standards
in Tobacco-smoke Analysis
ꢀ
¨
Peter Gartner , Katharina Bica, and Christian Einzinger
Institute of Applied Synthetic Chemistry, Vienna University of Technology,
A-1060 Vienna, Austria
Received December 9, 2003; accepted December 16, 2003
Published online February 16, 2004 # Springer-Verlag 2004
Summary. N-Nitrosamines of tobacco alkaloids contribute to the cause of tobacco related cancers and,
therefore, they are important analytes. Herein the preparation of four partially deuterated N-nitros-
amines from simple, commercially available, deuterated educts is described, which can serve as useful
standards in tobacco-smoke analysis.
Keywords. Isotopic labeling; Heterocycles; Tobacco nitrosamines; Analytical standards; Alkaloids.
Introduction
The effect of tobacco smoke from cigarettes and cigars has been excessively
investigated in the last decades. Since then a great number of different illnesses
have been directly related to the abuse of tobacco products.
A class of major interest are the tobacco specific N-nitrosamines formed during
smoking from nicotine and other tobacco alkaloids. Due to their carcinogenic and
mutagenic activity, which has been tested positive in different animals, they con-
ceivably contribute to the incidence of tobacco-related cancers [1].
For accurate measurement in tobacco-smoke analysis the use of standards and
reference materials is inevitable. Applying partially deuterated analogues is a well
established technique and they are highly useful tools in GC=MS and HPLC=MS
analysis.
We report herein the synthesis of four partially deuterated N-nitrosamines
(NNK-d4 (4), NNN-d4 (8), NAT-d10 (11), NAB-d10 (13)), starting from commercially
available deuterated d4-nicotinic acid ethyl ester and d5-pyridine.
ꢀ
Corresponding author. E-mail: peter.gaertner@tuwien.ac.at

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P. Gartner et al.
550
Fig. 1. Major deuterated tobacco-smoke N-nitrosamines
Results and Discussions
Synthesis of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK-d4, 4)
d4-Nicotinic acid ethyl ester (1) was treated with deprotonated N-methylpyrroli-
din-2-one and the following decarboxylation of the formed ꢀ-ketolactam 2 with
6 N hydrochloric acid provided the desired amino dihydrochloride 3 [2]. Finally 3
was dissolved in water, the pH was adjusted to 4 and after treatment with sodium
nitrite the desired d4-N-nitrosamine 4 was obtained. The degree of deuteration
was determined via HPLC=MS analysis using a protocol which has been already
described earlier [3] and proved to be >99%. 4 was obtained in an overall yield
of 30% as a mixture of stereoisomers regarding the N-nitrosamine moiety
(Z:E ¼ 4.1:1).
Synthesis of d4-N-Nitrosonornicotine (NNN-d4, 8)
In case of 8 we also started with 4, but used the TMS-protected pyrrolidin-2-one
instead. Decarboxylation of 5 and in situ ring closure of the formed intermediate
gave myosmine (6). Reduction with sodium borohydride afforded nornicotine (7)
[4], which was finally treated with sodium nitrite=hydrochloric acid [5] to provide
the desired N-nitrosonornicotine (8) in 45% yield and a Z:E ratio of 1.8:1. The
degree of deuteration was determined via HPLC=MS analysis and proved to be
>99%.
Synthesis of d10-N-Nitrosoanatabine (NAT-d10, 11)
and d10-N-Nitrosoanabasine (NAB-d10, 13)
In order to obtain almost completely deuterated products we decided to start with
d5-pyridine(9)e, introducing the second heterocyclic ring in one step as described

Synthesis of Partially Deuterated N-Nitrosamines
551
Scheme 1
by Yang and Tenner [6, 7] for nondeuterated anatabine. This was formed by treat-
ing pyridine with lithium aluminum hydride. The initially formed N-dihydropyr-
idyl complex 10a is hydrolyzed and subsequently oxidized. Therefore, using d5-
pyridine it was envisaged to obtain the desired d10-anatabine (10).
After treatment of lithium aluminum deuteride with an excess of d5-pyridine,
storage for 3 days at ꢁ7ꢂC and filtration, complex 10a was hydrolyzed with H2O
and afterwards oxidized. Although we tried different oxidation procedures, the
formation of bipyridine by overoxidation was a major problem in this step. In
the end a reaction time of 3 days at room temperature furnished the best results.
Furthermore, it was impossible to isolate a pure product at this stage, because
bipyridine exhibited a similar chromatographic behavior.
Nevertheless, after final nitrosation pure d10-N-nitrosoanatabine (11) was
obtained after chromatography. Although the product was obtained only in low
yield, the procedure proved to be simple giving the target compound in two steps
only.
In the case of d10-N-nitrosoanabasine (13) crude 10 was hydrogenated in the
presence of Pd=C. Nitrosation and a final chromatographic purification yielded the
desired d10-N-nitrosoanabasine (13).
For both products a degree of deuteration of >92% was determined.

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P. Gartner et al.
552
Scheme 2
Experimental
The 1H and 13C NMR spectra were recorded on a Bruker AC 200 using TMS as internal standard. GC
Analysis was performed with a CE Instruments TRACE GC using a J & W Scientific-DB-5 column.
Liquid chromatography was carried out on a Shimadzu LC-10ADþ SPD ꢁ 10AV using a Nucleosil 5
C18, 250ꢃ2 mm column. Degree of deuteration was determined by HPLC=APCI-MS on an Agilent
1100 series with a C8 Zorbax column. Thin layer chromatography (TLC) was carried out on precoated
E. Merck silica gel plates (60F-254) using UV light or molybdato phosphoric acid (5% in ethanol)
for visualization. d5-Pyridine was purchased from Euroiso-top, d4-nicotinic acid ethyl ester from
Cambridge Isotope Laboratories, Inc.
3-(d4-Nicotinoyl)-1-methyl-pyrrolidin-2-one (2, C11H8N2O2D4)
A solution of 2.8 cm3 of diisopropylamine (20.3 mmol) in 40cm3 of anhydrous diethyl ether under Ar
was cooled to ꢁ78ꢂC and 7.6 cm3 of n-butyl lithium (19 mmol) were added via a syringe. The solution
was warmed up to ꢁ15ꢂC for 20 min. After cooling again to ꢁ78ꢂC 1.6 cm3 of N-methylpyrrolidin-2-
one (16 mmol) were added and stirred for 20min. Compound 1 (2.3cm3, 16mmol) was added drop-
wise at ꢁ78ꢂC. The yellow solution was warmed up to room temperature and stirred overnight. The
solution was poured in 70 cm3 of 2 N hydrochloric acid and the aqueous layer was extracted 3 times
with ether, in order to remove N-methylpyrrolidin-2-one. The aqueous layer was treated with a 10N

Synthesis of Partially Deuterated N-Nitrosamines
553
NaOH solution to pH ¼ 6 and extracted with CH2Cl2. The combined organic layers were dried with
Na2SO4, filtered, and the solvent was removed under vacuo. The obtained crude product was purified
by flash chromatography using CH2Cl2:MeOH ¼ 50:1 as eluent to yield 3.01g (89%) of 2 as a brown
oil. Rf ¼ 0.32 (CH2Cl2:MeOH¼ 9:1); 1H NMR (200 MHz, CDCl3): ꢁ ¼ 4.39 (dd, J ¼ 3.7 Hz,
J ¼ 5.3 Hz, H-3), 3.65–1.83 (m, 7H, with 2.80 (s, CH3)) ppm.
4-(Methylamino)-1-(3-(d4-pyridyl)-1-butanone dihydrochloride (3, C10H12N2OCl2D4)
Compound 2 (2.98 g, 14.3mmol) was dissolved in 130 cm3 of 6 N HCl and refluxed for 70h. The
solvent was removed in vacuo and the brown residue was dissolved in 70cm3 of H2O. The solution was
filtered and the solvent was evaporated under reduced pressure. The crude product was crystallized
1
from methanol to yield 2.81 g (78%) of 3 as white crystals. H NMR (200 MHz, D2O): ꢁ ¼ 3.27
(t, J ¼ 6.75Hz, H-2), 3.06 (t, J ¼ 7.73Hz, H-4), 2.66 (s, CH3), 2.05 (quin, J ¼ 7.38Hz, H-3) ppm.
4-(Methylnitrosamino)-1-(3-(d4-pyridyl)-1-butanone (4, C19H9N3O2D4)
Compound 3 (2.73 g, 10.7mmol) was dissolved in 27cm3 of H2O and cooled to 0ꢂC. A 1 N NaOH
solution was added to pH¼ 4. A solution of 1.30 g of NaNO2 (18.9mmol) in 1.6 cm3 of H2O was
added dropwise at 0ꢂC and the reaction mixture was stirred for 20h at room temperature. The reaction
mixture was extracted with CH2Cl2, the combined organic layers were washed three times with 2 N
NaOH, three times with H2O, and dried over Na2SO4. After filtration the solvent was evaporated under
reduced pressure. The residue was purified by flash chromatography using CH2Cl2:MeOH¼ 50:1 as
eluent to yield 0.98 g (43%) of 4 as light brown crystals. Rf ¼ 0.42 (CH2Cl2:MeOH ¼ 10:1); 1H NMR
(200 MHz, CDCl3): ꢁ ¼ 4.22 (t, J ¼ 6.9 Hz, 1.8H, H-4(trans)), 3.76 (s, 0.3H, CH3(cis)), 3.71
(t, J ¼ 7.1 Hz, 0.2H, H-4(cis)), 3.09–3.04 (m, 4.5H, H-2 und CH3(trans)), 2.91 (t, J ¼ 7.1 Hz, 0.2H,
H-2(cis)), 2.22 (quin, 0.2H, H-3(trans)), 1.97 (quin, 1.8H, H-3(cis)) ppm; 13C NMR (50 MHz, CDCl3)
ꢁ ¼ 197.4 (C ¼ O), 52.8 (C-4(trans)), 43.8 (C-4(cis)), 38.9 (CH (cis)), 35.7 (C-2(cis)), 35.0
ꢄ
3
(C-2(trans)), 21.7 (C-3(trans)), 19.9 (C-3(cis)); LC=APCI-MS: Rt ¼ 2.6 min, m=z ¼ 212 [M]þ.
3-(d4-Nicotinoyl)pyrrolidin-2-one (5, C10H6N2O2D4)
Anhydrous triethylamine (9.1cm3, 65 mmol) and 4.5 cm3 of pyrrolidin-2-one (59 mmol) were added
under Ar to 20cm3 of anhydrous benzene. After addition of trimethylsilylchloride at room temperature
the reaction mixture was stirred for 3 h. It was diluted with 20cm3 of anhydrous benzene and filtered
under argon. The residue was washed with anhydrous benzene and the filtrate was evaporated at
reduced pressure. The crude product was purified by distillation in vacuo (93–96ꢂC, 18–22mbar).
A solution of 2.7 cm3 of diisopropylamine (19 mmol) in 20cm3 of anhydrous diethyl ether under Ar
was cooled to ꢁ78ꢂC and 5.9 cm3 of n-butyl lithium (14 mmol) were added via a syringe. The solution
was allowed to warm up to ꢁ15ꢂC for 20min. After cooling again to ꢁ78ꢂC 2.44 g of N-TMS-
pyrrolidin-2-one (15 mmol) were added and it was stirred for 20min. Compound 1 (1.4cm3, 9.7mmol)
was added dropwise at ꢁ78ꢂC. The yellow solution was warmed to room temperature and stirred over
night. The solution was quenched carefully by addition of 20 cm3 of H2O and extracted with 10 cm3 of
diethyl ether. The aqueous layer was treated with 4 N HCl to pH¼ 7, saturated with NaCl and extracted
with CH2Cl2. The combined organic layers were dried with Na2SO4, filtered, and the solvent was
1
removed under reduced pressure to yield 1.47g of crude 5. H NMR (200 MHz, CDCl3): ꢁ ¼ 6.46
(s, NH), 4.24 (m, H-3), 3.34 (m, H-4), 2.23 (m, H-5) ppm.
2,4,5,6-d4-Myosmine (6, C9H6N2D4)
Compound 5 (1.47 g, 7.6mmol) was dissolved in 8.4cm3 of 12 N HCl (16.8 mmol) and refluxed for
24h. A 20% NaOH solution was added to adjust pH¼ 14. The reaction mixture was extracted with

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P. Gartner et al.
554
CH2Cl2 and dried with Na2SO4. After filtration the solvent was removed under reduced pressure to
1
yield 0.78 g (68%) of 6 as yellow crystals. Rf ¼ 0.71 (CH2Cl2:MeOH¼ 5:1); H NMR (200 MHz,
CDCl3): ꢁ ¼ 4.2 (tt, J ¼ 7.37, 2.05 Hz, H-3), 3.1 (tt, J ¼ 8.25, 2.02Hz, H-5), 2.2 (m, H-4) ppm.
2,4,5,6-d4-Nornicotine (7, C9H8N2D4)
Myosmine (6, 0.78g, 5.2 mmol) was dissolved in 10 cm3 of anhydrous methanol and 0.3 cm3 of acetic
acid under N2 and cooled with a NaCl-ice-bath. After addition of 0.77g of NaBH4 (20.3 mmol) the
reaction was warmed to room temperature and stirred overnight. Since there was still myosmine 6 left,
according to TLC, another 0.20 g of NaBH4 (5.2mmol) were added and the reaction was warmed to
50ꢂC for 2 h. The reaction mixture was quenched by addition of 11 cm3 of H2O and extracted with
CH2Cl2. The combined organic layers were dried with Na2SO4, filtered, and the solvent was removed
1
under reduced pressure to yield 0.77g of crude 7. H NMR (200 MHz, CDCl3): ꢁ ¼ 4.9 (s, NH), 4.1
(m, H-2), 3.3–2.9 (m, H-5), 2.3–1.3 (m, H-3, H-4) ppm.
2,4,5,6-d4-N-Nitrosonornicotine (8, C9H7N3OD4)
Compound 7 (0.77 g, 5 mmol) was dissolved in 11cm3 of 10% HCl and cooled to 0ꢂC. A solution of
1.44g of NaNO2 (2.1mmol) in 1.9 cm3 of H2O was added slowly and the reaction mixture was stirred
for 72 h at room temperature. A saturated NaHCO3 solution was added to pH¼ 9. The reaction mixture
was extracted with CH2Cl2 and dried with Na2SO4. After filtration the solvent was evaporated under
1
reduced pressure to obtain 0.79 g (87%) of 8 as a brown oil. Rf ¼ 0.53 (CH2Cl2:MeOH¼ 5:1); H
NMR (200MHz, CDCl3): ꢁ ¼ 5.68 (t, J ¼ 6.26Hz, 0.64H, H-20 (cis)), 5.3–5.2 (m, 0.36H, H-20 (trans)),
4.7–4.6 (m, 0.36H, H-50 (trans)), 4.5–4.3 (m, 0.36H, H-50 (trans)), 3.9–3.6 (m, 1.28H, H-50 (cis)),
2.6–2.3, 2.2–1.9 (2m, 4H, H-30, H-40) ppm; LC=APCI-MS: Rt ¼ 2.6 min, m=z ¼ 182 [M]þ.
2,20,30,4,40,5,50,6,60,60-Decadeutero-1,2,3,6-tetrahydro-2,30-bipyridine
(d10-Anatabine) (10, C10H2N2D10)
Lithium aluminum deuteride (4.05 g, 97mmol) was added under Ar in small portions to 100 cm3 of
stirred d5-pyridine at ꢁ35ꢂC. After stirring for 1 h at room temperature the orange reaction mixture was
allowed to rest at ꢁ7ꢂC for 72h. Unreacted lithium aluminum deuteride was filtered off and washed
with another 25 cm3 of d5-pyridine. The yellow solution was cooled with an ice bath and carefully
hydrolyzed with 8 cm3 of H2O. After warming to room temperature a slow stream of dried air (KOH,
H2SO4) was passed through the reaction mixture via an inlet tube while the reaction was followed by
GC analyses. After 72h chloroform was added to dilute the slurry and insoluble salts were filtered off
using silica. The solvent was evaporated and the obtained brown oil was purified via vacuum flash
chromatography using CH2Cl2:MeOH:NH3 ¼ 20:1:0.1 as eluent. Though it was not possible to sepa-
rate d10-anatabine 10 from the byproduct d10-3,30-bipyridine the yield of d10-anatabine 10 (0.79 g
corresponding to 4.8%) was determined by GC analyzes. GC: Rt ¼ 14.3min (d10-anatabine),
Rt ¼ 8.3 min (d10-3,30-bipyridine).
1-Nitroso-2,20,30,4,40,5,50,6,60,60-decadeutero-1,2,3,6-tetrahydro-2,30-bipyridine
(d10-N-Nitrosoanatabine) (11, C10HN3OD10)
Compound 10 (1.86 g, 11.0mmol) contaminated with d10-bipyridine was dissolved in 16.4cm3 of 4 N
HCl (65.6mmol) and cooled to 0ꢂC. A solution of 1.40g (20.3 mmol) of NaNO2 in 9 cm3 of H2O was
added dropwise and the reaction mixture was stirred for 20h at room temperature. Since there was still
d10-anatabine 10 left, according to TLC, another 0.65g of NaNO2 (9.4mmol) in 4.5 cm3 of H2O were
added at 0ꢂC and the reaction was stirred for 3 h. A 2 N NaOH solution was added to adjust pH ¼ 14

Synthesis of Partially Deuterated N-Nitrosamines
555
and the reaction mixture was extracted with CH2Cl2. The combined organic layers were dried with
Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by
vacuum flash chromatography using CH2Cl2:MeOH:NH3 ¼ 30:1:0.1 as eluent followed by column
chromatography to yield 103 mg (26%) yellow oil. 2H NMR (61 MHz, CCl4 þ 5% d4-MeOH):
ꢁ ¼ 8.53, 7.60, 7.30, 6.43, 6.10, 5.98, 5.84, 5.68, 5.02, 4.75 ppm; GC: Rt ¼ 17.7 min; LC=UV:
Rt ¼ 10.0 min; LC=APCI-MS: Rt ¼ 4.4 min, m=z ¼ 201 [M]þ.
2,20,3,4,40,5,50,6,6060-Decadeutero-3-(2-piperidyl)pyridine (d10-Anabasine) (12, C10H4N2D10)
A solution of 1.90 g of d10-anatabine 10 (0.6mmol) contaminated with d10-bipyridine in 250 cm3 of
anhydrous methanol was hydrogenated over 0.95 g of Pd=C at 3.5 bar for 3 h. The reaction mixture was
filtered over silica and the solvent was evaporated under reduced pressure to yield 1.82g of 12 as a
brown oil. GC: Rt ¼ 13.9min (d10-anabasine), Rt ¼ 8.4 min (d10-3,30-bipyridine).
2,20,3,4,40,5,50,6,60,60-Decadeutero-N-nitroso-3-(2-piperidyl)pyridine
(d10-N-Nitrosoanabasine) (13, C10H3N3OD10)
Compound 12 (1.82 g, 10.6 mmol) contaminated with d10-bipyridine was dissolved in 16cm3 of 4 N
HCl (65.6 mmol) and cooled to 0ꢂC. A solution of 1.39 g (20.2mmol) of NaNO2 in 11cm3 of H2O was
added dropwise and the reaction mixture was stirred for 20h at room temperature. Since there was still
d10-anabasine 12 left, according to TLC, another 0.39g of NaNO2 (5.7mmol) in 3 cm3 of H2O were
added at 0ꢂC and the reaction was stirred for 4 h at room temperature. A 2 N NaOH solution was added
to pH¼ 14 and the reaction mixture was extracted with CH2Cl2. The combined organic layers were
dried with Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The residue was
purified by vacuum flash chromatography using CH2Cl2:MeOH:NH3 ¼ 30:1:0.1 as eluent followed by
column chromatography to yield 117 mg (26%) of a yellow oil. 2H NMR (61 MHz, CCl4 þ 5%
d4-MeOH): ꢁ ¼ 8.58, 8.39, 7.67, 7.40, 6.33, 5.90, 4.8, 4.56, 3.65, 2.92, 2.6–1.5 ppm; LC=UV:
Rt ¼ 11.9 min; LC=APCI-MS: Rt ¼ 5.3 min, m=z ¼ 203 [M]þ.
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