Synthesis and characterization of thymidine adducts of arylamines

Article abstract of DOI:10.1081/SCC-120015711

The synthesis of adducts of arylamines with thymidine are reported.

Full text of DOI:10.1081/SCC-120015711

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Synthesis and Characterization of Thymidine Adducts
of Arylamines
N. V. Anil Kumar a & K. S. Rangappa a b
a Department of Studies in Chemistry , Manasa Gangotri , University of Mysore , Mysore,
India
b Department of Studies in Chemistry , Manasa Gangotri , University of Mysore , Mysore, 570
006, India
Published online: 21 Aug 2006.
To cite this article: N. V. Anil Kumar & K. S. Rangappa (2003) Synthesis and Characterization of Thymidine Adducts of
Arylamines, Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
33:2, 259-264, DOI: 10.1081/SCC-120015711
To link to this article: http://dx.doi.org/10.1081/SCC-120015711
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SYNTHETIC COMMUNICATIONSÕ
Vol. 33, No. 2, pp. 259–264, 2003
Synthesis and Characterization of Thymidine
Adducts of Arylamines
*
N. V. Anil Kumar and K. S. Rangappa
Department of Studies in Chemistry, Manasa Gangotri,
University of Mysore, Mysore, India
ABSTRACT
The synthesis of adducts of arylamines with thymidine are reported.
Key Words: Thymidine adducts; Synthesis; Characterization.
Aromatic amines and nitroarenes are very important indus-
trial intermediates. Several scientists[1,2] have extensively studied
the covalent binding of a number of aromatic amines to DNA
and identified various products by studying the in vivo and
in vitro reactions of esters of aryl hydroxylamine.[3] N-Oxidation is
*Correspondence: K. S. Rangappa, Department of Studies in Chemistry, Manasa
Gangotri, University of Mysore, Mysore 570 006, India. Fax: þ91-821-518835 or
421263; E-mail: rangappaks@yahoo.com.
259
DOI: 10.1081/SCC-120015711
Copyright & 2003 by Marcel Dekker, Inc.
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260
Anil Kumar and Rangappa
a key step in the metabolism of aromatic amines and amides to toxic
products. Aromatic amines can be metabolized to highly reactive
N-hydroxy aromatic amines. These are highly reactive inter-
mediates, which are responsible for the genotoxic effects of this
class of compounds. DNA adducts of arylamines have been found
in several organs of exposed experimental animals. With the advance-
ment in analytical technique, scientists have quantified the DNA
adducts of arylamines in human tissue. Therefore, the reference
standards of DNA adducts have been synthesized. The deoxyguano-
sine adducts or arylamines from various animal sources by
synthesis are known[4–6] but not the thymidine adducts and
therefore, this study of the reaction of arylamines with thymidine is
undertaken.
In the present study, the thymidine adducts of the monocyclic aryl-
amines are synthesized, which can be used to develop 32P-postlabeling
and mass spectrometric methods for low-level detection in biological
material.
This synthetic route was based on observations by Lobo et al.[7,8]
on the reaction of N-benzoyloxy-N-arylamines with nucleophiles under
basic conditions. The DNA adducts of arylamines are synthesized from
the corresponding N-benzoyloxy arylamines and nucleoside. These N-
benzoyloxy compounds are obtained after the reaction of the corre-
sponding N-hydroxy arylamines treated with benzoyl chloride at
below 0ꢀC. The freshly prepared solution of the N-benzoyloxy-N-aryla-
mine was added to the aqueous solution of thymidine. The resulting
mixture was purified by solvent extraction (Sch. 1). N-Benzoyloxy
compounds have also been isolated and characterized by NMR and
IR spectroscopy.
All NMR experiments were performed with d6-DMSO as solvent.
The peaks were assigned according to the spectra of thymidine. In the
1H NMR spectra of the thymidine adducts, the peak of 6H disappears
and the NH proton of the arylamine shifted downfield by 3 ppm. The
C-6 peak for unmodified thymidine is 124 ppm where as the C-6 peak
shifted to 137–140 ppm for the adducts. In the 13C NMR spectra,
the C-20 peak of the sugar in the adducts is shifted slightly
upfield by 1 ppm and the resonance of C-5 is shifted by 3 ppm. It is
important to note that C-1 peak of arylamine is shifted upfield by
about8 ppm.
The UV spectra (methanol/water) of thymidine adducts were shifted
to longer wavelengths by 10–45 nm compared to thymidine
alone, depending on the adduct.

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Thymidine Adducts of Arylamines
261
Scheme 1.
EXPERIMENTAL
Synthesis and Characterization of N-(thymidin-6-yl)-aniline (4a)
To a stirred solution of nitro benzene (1a, 8 mmol, 0.985 g), ammo-
nium chloride (0.5 g) and ammonium hydroxide (2 mL) were added
in water (40 mL). The solution was heated to 50ꢀC. Zinc (17.6 mmol,
1.1509 g) was added in such a way that the temperature does not
rise above 60ꢀC. Reaction was monitored by thin layer chromatog-
raphy (TLC). After the reduction of nitro benzene, reaction
mixture was cooled to room temperature, and filtered. The
filtrate was saturated with sodium chloride and cooled in ice bath.
This was extracted with ether:benzene (1:1 v/v, 4 Â 35 mL). The

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262
Anil Kumar and Rangappa
organic layer was dried over anhydrous magnesium sulphate and
the solvent was evaporated. The product, N-phenyl hydroxylamine
(2a), so obtained was further purified by washing with n-hexane
followed by cyclohexane. The pure hydroxylamine (2a) was, character-
ized by m.p., NMR, and IR, which is in confirmation with the
reported data.[9]
To a solution of aryl hydroxylamine (2a, 2 mmol, 0.2183 g) in dry
ether (cooled in the ice bath), ice-cold benzoylchloride (2.4 mmol,
0.3374 g) in dry ether was added. The rate of addition of benzoylchloride
was such that the temperature does not rise above 0ꢀC. The solution
was stirred vigorously for 3 h, and the reaction mixture was stirred for
another 30 min so that it attains room temperature. Finally, this was
neutralized with saturated solution of sodium bicarbonate. The organic
layer was dried over anhydrous sodium acetate and the solvent was
evaporated. The product was purified by recrystallization with water.
Pure N-benzoyloxy-N-phenyl hydroxylamine (3a) was characterized by
m.p., NMR, and IR. Data obtained were in good agreement with the
reported data.[9]
To the ester (3a, 0.206 mmol, 43.98 mg) in chloroform:alcohol:
water (7:3:4 v/v, 35 mL), was added triethylamine (0.2 mL). The
temperature was maintained at 37ꢀC. Thymidine (0.206 mmol, 50.00 mg)
was added to a stirred reaction mixture and the reaction was
monitored by TLC. After 24 h, the reaction mixture was evaporated to
dryness and redissolved in water. This was extracted with diethyl ether
(4 Â 50 mL) and n-butanol (3 Â 25 mL). The n-butanol extract was con-
centrated in vacuo. Yield ¼ 3.2 mg (4.7%). Analysis of the product (4a)
by HPLC [chloroform:methanol (50:50 v/v), ꢀ ¼ 264 nm] showed 98.9%
1
purity. This was characterized by H and 13C NMR.
Elemental analysis of 4a: (Calcd.) C 57.65%, H 5.74%, N 12.61%;
(found) C 57.68%, H 5.71%, N 12.64%.
1H NMR of 4a: (400 MHz, d6-DMSO): ꢁ (ppm): 1.84 (m, 1H, 20H);
2.51 (m, 1H, 20H); 2.12 (s, 3H, CH3); 3.66 (m, 2H, 50H); 3.83 (m,
1H, 40H); 4.24 (m, 1H, 30H); 4.84 (s, 1H, Ar-NH); 5.02 (s, 1H,
30OH); 5.20 (s, 1H, 50OH); 6.24 (q, 1H, 10H); 6.42 (q, 2H, Ar-2H,
Ar-6H); 6.61 (d, 1H, Ar-4H); 6.94 (q, 2H, Ar-3H, Ar-5H); 7.76
(s, 1H, NH).
13C NMR of 4a: (400 MHz, d6-DMSO): ꢁ (ppm): 12.26 (CH3);
39.39 (20C); 61.36 (50C); 70.49 (30C); 83.72 (10C); 87.27 (40C); 110.08
(5C); 116.34 (Ar-2C); 116.42 (Ar-6C); 120.14 (Ar-4C); 127.5
(Ar-5C); 128.14 (Ar-3C); 136.73 (6C); 139.41 (Ar-1C); 150.42 (2C);
153.69 (4C).

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Thymidine Adducts of Arylamines
263
Synthesis and Characterization of
N-(Thymidin-6-yl)-3-methylaniline (4b)
The procedure for the synthesis of 4b is similar to that of 4a. In this
case, 8 mmol of 1b, 2 mmol of 2b, and 0.206 mmol of 3b were used to get
4.9 mg (6.9%) of the product 4b. Analysis of the product (4b) by HPLC
[chloroform:methanol (50:50 v/v), ꢀ ¼ 264 nm] showed 99.2% purity.
1
This was characterized by H and 13C NMR.
Elemental analysis of 4b: (Calcd.) C 58.78%, H 6.09%, N
12.10%; (found) C 58.80%, H 6.07%, N 12.11%.
1H NMR of 4b: (400 MHz, d6-DMSO): ꢁ (ppm): 1.82 (m, 1H, 20H);
2.51 (m, 1H, 20H); 2.13 (s, 3H, CH3); 2.56 (s, 3H, Ar-CH3); 3.68 (m, 2H,
50H); 3.79 (m, 1H, 40H); 4.23 (m, 1H, 30H); 4.92 (s, 1H, Ar-NH); 4.99 (s,
1H, 30OH); 5.22 (s, 1H, 50OH); 6.21 (q, 1H, 10H); 6.47 (q, 1H, Ar-4H);
6.59 (q, 1H, Ar-6H); 6.89 (m, 1H, Ar-5H); 7.02 (q, 1H, Ar-2H); 7.69 (s,
1H, NH).
13C NMR of 4b: (400 MHz, d6-DMSO): ꢁ (ppm): 12.21 (CH3); 17.92
(Ar-CH3); 39.35 (20C); 61.26 (50C); 70.29 (30C); 83.62 (10C); 87.15 (40C);
109.49 (5C); 118.92 (Ar-6C); 120.72 (Ar-4C); 122.47 (Ar-3C); 125.93
(Ar-2C); 128.42 (Ar-5C); 138.27 (Ar-1C); 139.84 (6C); 150.32 (2C);
153.58 (4C).
ACKNOWLEDGMENT
The authors would like to thank the Council of Scientific and
Industrial Research (CSIR), New Delhi for the financial support
(No. 01/1458/97-EMR II).
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264
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Received in the Netherlands December 10, 2001