Synthesis and characterization of adenosine adducts of arylamines

Article abstract of DOI:10.1081/NCN-120014818

The synthesis of adducts of arylamines with adenosine are reported.

Full text of DOI:10.1081/NCN-120014818

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SYNTHESIS AND CHARACTERIZATION OF ADENOSINE
ADDUCTS OF ARYLAMINES
a
a
b
N. V. Anil Kumar , K. Mantelingu & K. S. Rangappa
a
Department of Studies in Chemistry , University of Mysore , Manasagangotri, Mysore,
70006, India
5
b
Department of Studies in Chemistry , University of Mysore , Manasagangotri, Mysore,
70006, India
5
Published online: 31 Aug 2006.
To cite this article: N. V. Anil Kumar , K. Mantelingu & K. S. Rangappa (2002) SYNTHESIS AND CHARACTERIZATION OF
ADENOSINE ADDUCTS OF ARYLAMINES, Nucleosides, Nucleotides and Nucleic Acids, 21:6-7, 463-467
To link to this article: http://dx.doi.org/10.1081/NCN-120014818
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, Nos. 6 & 7, pp. 463–467, 2002
SYNTHESIS AND CHARACTERIZATION OF
ADENOSINE ADDUCTS OF ARYLAMINES
N. V. Anil Kumar, K. Mantelingu, and K. S. Rangappa*
Department of Studies in Chemistry, Manasagangotri,
University of Mysore, Mysore – 570006, India
ABSTRACT
The synthesis of adducts of arylamines with adenosine are reported.
Key Words: Adenosine adducts; Synthesis; Characterization
Aromatic amines and nitroarenes are very important industrial inter-
[
1,2]
mediates. Several scientists
of a number of aromatic amines to DNA and identified various products by
have extensively studied the covalent binding
[3]
studying in vivo and in vitro reactions of esters of aryl hydroxylamine.
Aromatic amines can be metabolised to highly reactive N-hydroxy aromatic
amines. These are highly reactive intermediates 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
advancement 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 deoxyguanosine adducts of aryl-
[4–7]
amines by synthesis are known but not the adenosine adducts. Hence, this
study of the reaction of arylamines with adenosine is undertaken.
*
Corresponding author. Fax: 91-821-518835 or 421263; E-mail: rangappaks@yahoo.com
63
4
DOI: 10.1081=NCN-120014818
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
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464
ANIL KUMAR, MANTELINGU, AND RANGAPPA
Scheme 1.
[8,9]
The synthetic route was based on the observations by Lobo et al.
on
the reaction of N-benzoyloxy-N-arylamines with nucleophiles under basic
conditions. The DNA adducts of arylamines are synthesized from the cor-
responding N-benzoyloxy arylamines and nucleoside. These N-benzoyloxy
compounds are obtained by treating the corresponding N-hydroxy aryla-
ꢀ
mines with benzoyl chloride below 0 C. The freshly prepared solution of the
N-benzoyloxy-N-arylamine was added to the aqueous solution of adenosine.
The resulting mixture was purified by solvent extraction (Sch. 1) and are
characterized by NMR and IR spectral data. NMR characterization was
performed with d -DMSO as solvent. The peaks were assigned according to
6
1
the spectra of adenosine. In the H NMR spectra of the adenosine adducts,
the peak of 8H disappears and the NH proton of the arylamine shifted
downfield by 4 ppm. The C-8 peak for unmodified adenosine is 150 ppm
where as the C-8 peak shifted to 160–164 ppm for the adducts.
1
3
0
C NMR spectra reveals that the C-2 peak of the sugar in the adducts
is shifted slightly upfield by 1 ppm and the resonance of C-5 is shifted by
ppm. It is important to note that, the C-1 peak of arylamine is shifted
3
upfield by about 8 ppm. The UV spectra (methanol=water) of adenosine
adducts were shifted to longer wavelength by 10–45 nm compared to ade-
nosine alone.

ADENOSINE ADDUCTS OF ARYLAMINES
465
The adducts formation of aniline and 3-methyl aniline with adenosine
were carried out to know whether the adduct can be synthesized or not,
so that, the reaction pathway of forming the adduct can be deducted. The
0
possibility of synthesizing the 2 -deoxyadenosine adducts would be similar
3
2
and these can be used to develop P post-labeling and mass spectrometric
methods for low level detection in biological materials. The synthetic route
for the synthesis of 2 -deoxyadenosine adducts with aniline and 3-methyl
0
aniline (5) is similar to that of 4. However, we are interested in the synthesis
of 4 as reference standard adducts. The characterization of 5 is also under
progress. It is important that the mechanism of formation of these adducts
will be known so that, the required reference adducts can be synthesized for
quantification.
EXPERIMENTAL
Synthesis and Characterization of N-(Adenosin-8-yl)-aniline (4a)
To a stirred solution of nitro benzene (1a, 8 mmol, 0.985 g), ammonium
chloride (0.5 g) and ammonium hydroxide (2 mL) were added in water
ꢀ
(
40 mL). The solution was heated to 50 C. Zinc (17.6 mmol, 1.1509 g) was
ꢀ
added in such a way that the temperature does not rise above 60 C. Reaction
was monitored by thin layer chromatography (TLC). After the reduction of
nitro benzene, reaction mixture was cooled to room temperature, and fil-
tered. The filtrate was saturated with sodium chloride and cooled in ice bath.
This was extracted with ether:benzene (1:1 v=v, 4635 mL). The organic layer
was dried over anhydrous magnesium sulphate and the solvent was evapo-
rated. The product, N-phenyl hydroxylamine (2a), so obtained was further
purified by washing with n-hexane followed by cyclohexane. The pure
hydroxylamine (2a) was characterized by m.p., NMR and IR, which is in
[10]
confirmation with the reported data.
To a solution of aryl hydroxylamine (2a, 2 mmol, 0.2183 g) in dry ether
cooled in ice bath), benzoyl chloride (2.4 mmol, 0.3374 g) in dry ether (ice
(
cold) was added. The rate of addition of benzoyl chloride was such that the
ꢀ
temperature does not rise above 0 C. 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 characterised by mp, NMR and IR. Data obtained are in good
[10]
agreement with the reported data.
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
(

466
ANIL KUMAR, MANTELINGU, AND RANGAPPA
ꢀ
maintained at 37 C. Adenosine (0.206 mmol, 55.05 mg) was added to a stir-
red 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 (4650 mL) and n-butanol (3625 mL). The
n-butanol extract was concentrated invacuo. Yield ¼ 4.4 mg (5.9%). Analysis
of the product (4a) by HPLC [Chloroform:Methanol (50:50 v=v),
1
l ¼ 264 nm] showed 98.7% purity. This was characterised by H and
1
3
C NMR.
Elemental analysis of 4a: (Calcd.) C 53.63%, H 5.06%, N 23.45% (found) C
3.66%, H 5.07%, N 23.42%.
5
ꢁ
1
IR of 4a (in cm ) 672.4, 1087.7, 1151.6, 1221.7, 1309.0, 1436.8, 1643.3,
697.5, 3184.2, 3341.4, 3508.9.
1
1
H NMR of 4a (400 MHz, d -DMSO):
d (ppm): 3.72 (m, 2H, 5 H); 3.91 (m, 1H, 4 H); 3.96 (m, 2H, NH ); 4.24
6
0
0
2
0 0 0
(m, 1H, 3 H); 4.51 (m, 1H, 2 H); 4.62 (s, 1H, 2 OH); 4.69 (s, 1H,
Ar-NH); 4.81 (s, 1H, 5 OH); 4.94 (s, 1H, 3 OH); 6.18 (q, 1H,
H); 6.62 (d, 2H, Ar-2H, Ar-6H); 6.79 (t, 1H, Ar-4H); 7.02 (t,
H, Ar-3H, Ar-5H); 8.42 (d, 1H, 2H).
0
0
0
1
2
1
3
C NMR of 4a (75.5 MHz, d -DMSO):
6
0
0
0
0
0
d (ppm): 63.89 (5 C); 72.93 (3 C); 77.86 (2 C); 82.27 (1 C); 92.06 (4 C); 119.34
Ar-2C, Ar-6C); 120.78 (Ar-4C); 122.32 (C5); 128.17 (Ar-3C, Ar-
C); 141.24 (Ar-1C); 147.76 (4C); 151.39 (2C); 153.19 (6C); 161.23
8C).
(
5
(
Synthesis and Characterisation of N-(Adenosin-8-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
6
.7 mg (8.7%) of the product 4b. Analysis of the product (4b) by HPLC
[
was characterised by H and C NMR.
Chloroform:Methanol (50:50 v=v), l ¼ 264 nm] showed 99.4% purity. This
1
13
Elemental analysis of 4b: (Calcd.) C 54.83%, H 5.41%, N 22.57% (found) C
5
4.85%, H 5.44%, N 22.55%.
ꢁ
1
IR of 4b(in cm ) 669.1, 1091.9, 1152.2, 1224.8, 1304.7, 1439.3, 1648.4,
691.5, 3187.6, 3345.3, 3511.7.
H NMR of 4b (400 MHz, d -DMSO):
1
1
6
0
0
d (ppm): 2.28 (s, 3H, Ar-CH ); 3.77 (m, 1H, 5 H); 3.86 (m, 1H, 4 H); 4.01 (m,
3
0 0 0
H, NH ); 4.29 (m, 1H, 3 H); 4.47 (m, 1H, 2 H); 4.66 (s, 1H, 2 OH);
2
2
4
1
4
0
0
.76 (s, 1H, Ar-NH); 4.80 (s, 1H, 5 OH); 4.93 (s, 1H, 3 OH); 6.17 (q,
0
H, 1 H); 6.71 (d, 1H, Ar-2H); 6.74 (d, 1H, Ar-6H); 6.79 (d, 1H, Ar-
H); 7.11 (d, 1H, Ar-5H); 8.44 (d, 1H, NH).
1
3
C NMR of 4b (75.5 MHz, d -DMSO):
6

ADENOSINE ADDUCTS OF ARYLAMINES
467
0
0
0
0
d (ppm): 19.24 (Ar-CH ); 63.92 (5 C); 72.92 (3 C); 77.96 (2 C); 82.57 (1 C);
3
0
9
2.16 (4 C); 118.72 (Ar-6C); 121.38 (Ar-4C); 122.37 (5 C); 123.99
(
(
Ar-3C);125.94 (Ar-5C); 128.41 (Ar-2C); 142.27 (Ar-1C); 147.77
4C); 151.32 (2C); 156.71 (6C); 163.89 (8C).
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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Received January 16, 2002
Accepted April 30, 2002