Formation of 2'-deoxy-2-nitroadenosines by reaction of 2'-deoxyadenosines with copper(II) nitrate/acetic anhydride.

Article abstract of DOI:10.1081/NCN-120014815

Nitration of 9-substituted [ethyl, (Ac)2-2'-deoxyribosyl, (Ac)3-ribosyl] N6-acetyladenine derivatives with Cu(NO3)2.3H2O/Ac2O was examined. Nitration proceeded at the 2-position, although the yield was low. Removal of the acetyl groups gave 2'-deoxy-2-nitroadenosine derivatives.

Full text of DOI:10.1081/NCN-120014815

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FORMATION OF 2′-DEOXY-2-NITROADENOSINES BY
REACTION OF 2′-DEOXYADENOSINES WITH COPPER(II)
NITRATE/ACETIC ANHYDRIDE
Toyo Kaiya a , Haruko Tanaka a & Kohfuku Kohda b
a Graduate School of Pharmaceutical Sciences , Nagoya City University , Tanabedori, Mizuho-
ku, Nagoya, 467-8603, Japan
b Graduate School of Pharmaceutical Sciences , Nagoya City University , Tanabedori, Mizuho-
ku, Nagoya, 467-8603, Japan
Published online: 31 Aug 2006.
To cite this article: Toyo Kaiya , Haruko Tanaka & Kohfuku Kohda (2002) FORMATION OF 2′-DEOXY-2-NITROADENOSINES BY
REACTION OF 2′-DEOXYADENOSINES WITH COPPER(II) NITRATE/ACETIC ANHYDRIDE, Nucleosides, Nucleotides and Nucleic
Acids, 21:6-7, 427-433
To link to this article: http://dx.doi.org/10.1081/NCN-120014815
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, Nos. 6 & 7, pp. 427–433, 2002
FORMATION OF 20-DEOXY-2-
NITROADENOSINES BY REACTION OF
20-DEOXYADENOSINES WITH COPPER(II)
NITRATE/ACETIC ANHYDRIDE
Toyo Kaiya, Haruko Tanaka, and Kohfuku Kohda*
Graduate School of Pharmaceutical Sciences, Nagoya City
University, Tanabedori, Mizuho-ku, Nagoya, 467-8603, Japan
ABSTRACT
Nitration of 9-substituted [ethyl, (Ac)2-20-deoxyribosyl, (Ac)3-ribosyl]
N6-acetyladenine derivatives with Cu(NO3)2ꢀ3H2O=Ac2O was examined.
Nitration proceeded at the 2-position, although the yield was low.
Removal of the acetyl groups gave 20-deoxy-2-nitroadenosine derivatives.
INTRODUCTION
Nitric oxide (NO) is involved in important physiological functions such
as vasodilation[1] and neurotransmission.[2] However, it can have adverse
effects: NO forms harmful peroxynitrite (ONOOꢁ ) by reaction with super-
oxide, and it has been reported that peroxynitrite reacts with the guanine
moiety of DNA to form 8-nitroguanine and 8-oxoguanine,[3] the latter of
which is considered to be a DNA modification responsible for mutation=
cancer induction.[4] The adenine moiety of DNA also forms 8-oxoadenine
and 2-hydroxyadenine by treatment with oxidizing agents.[5–6] Nitrated
adenine derivatives, however, had not been described until a recent report by
*Corresponding author. Fax: +81-52-834-9309; E-mail: kohda@phar.nagoya-cu.ac.jp
427
DOI: 10.1081=NCN-120014815
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

428
KAIYA, TANAKA, AND KOHDA
Koomen’s group who synthesized 2-nitroadenosine via 6-chloro-2-nitro-
purineriboside derivatives that were prepared by the nitration of 6-chloro-
purine derivative with tetrabutylammonium nitrate=trifluoroacetic anhydride
(TBAN=TFAA).[7] T0hese investigators also obtained 2-nitroadenosine by
0
0
nitration of N6,N6,O2 ,O3 ,O5 -pentaacetyladenosine with TBAN=TFAA and
subsequent deacetylation, however, the yield was low due to a side reaction in
the course of the deacetylation.[7] Except for their study, direct nitration of
adenine derivatives has been unreported, although many studies have been
published on direct nitration of uracil derivatives using a variety of nitrating
agents; NO2 þ BF4
,
ꢁ [8] NO2OCOCF3,[9–10] NO2-O3,[11] and Cu(NO3)2ꢀ3H2O=
Ac2O.[12] Sodum et al. reported quite recently on the reaction products of 20-
deoxyadenosine with peroxynitrite. They showed that the main product was
20-deoxy-8-oxoadenosine, however, the minor one was only suggested to be
some nitrated derivative.[13] In this study, we examined the preparation of
nitrated 20-deoxyadenosine derivatives. Since the glycosidic bond of 20-
deoxyadenosine is less stable than that of adenosine against acid, milder
conditions that cause low yield of nitration product are required for the
30 50
preparation of 20-deoxy-2-nitroadenosine. Reaction of N6,O ,O -triacetyl-20-
deoxyadenosine with Cu(NO3)2ꢀ3H2O=Ac2O afforded 2-nitro derivatives and
subsequent removal of the acetyl groups gave 20-deoxy-2-nitroadenosine.
RESULTS AND DISCUSSION
Nitration of 9-substituted N6-acetyladenine derivative (1) (Sch. 1) was
carried out with Cu(NO3)2ꢀ3H2O=Ac2O. The reaction gave a small yield of
Scheme 1. Syntheses of 20-deoxy-2-nitroadenosine derivatives.

20-DEOXY-2-NITROADENOSINES
429
Figure 1. HMBC correlations of compound 2a.
products (2 and 3) with unreacted starting material. The products were
separated by preparative thin layer chromatography (PLC) (silica gel). The
yield of 2 was slightly larger than that of 3 and the total yield of 2 and 3 was
7 ꢂ 33%. The mass spectrum of 2a showed that it has a mono nitrated
structure. The NMR spectrum of 2a suggested that the position of the nitro
group is C2 or C8. The correct position of the nitro group was finally
determined to be C2 by means of HMBC correlations (Fig. 1). From these
results, the structure of 2a was determined to be N6-acetyl-9-ethyl-2-nitro-
adenine. Similarly, the structures of 2b,c were determined to be 2-nitroade-
nine derivatives. Deacetylation of 2a–c with NH4OH=MeOH gave the
corresponding 9-ethyl-2-nitroadenine (4a), 20-deoxy-2-nitroadenosine (4b)
and 2-nitroadenosine (4c), respectively. Compound 4 has a characteristic UV
absorption spectrum with a lmax at a long wavelength due to the effect of
the nitro group (Fig. 2). In the course of deacetylation, 2-methoxyadenine
derivatives were obtained as a by-product by replacement of the nitro group
of 4 with the methoxy group as reported.[7] The yield of 2-methoxyadenine
derivatives was less than 25%. Structures of 3a–c were determined to be
N6-deacetylated 2a–c from spectral data. Deacetylation of 3b,c with
NH4OH=MeOH also gave 4b,c. When adenine derivatives without the
N6-acetyl group were employed as substrate, no reaction proceeded within
the Cu(NO3)2ꢀ3H2O=Ac2O system. This indicates that radical nitration
mechanisms may be involved.[7] It has been reported that 20-deoxy-8-nitro-
guanosine is very unstable and decomposes to 8-nitroguanine within a few
minutes.[3,13] The stability of 4b was then examined using HPLC. Compound
4b was fairly stable and no decomposition was observed under treatment
with aqueous solution (pH 6.8) at 60ꢃC for 4 h. Acid treatment (0.1 N HCl at
60ꢃC for 40 min) of 4b gave 2-nitroadenine (5). When N6-acetyladenine was
allowed to react with Cu(NO3)2ꢀ3H2O=Ac2O, the reaction did not give N6-
acetyl-2-nitroadenine but gave other products (unstable ring-nitrogen-mono-
nitrated N6-acetyladenine and stable N6-acetyl-2(8)-nitro-8(2)-oxoadenine)
and their structures were deduced from NMR and mass spectroscopic data.
When 9-ethylguanine was allowed to react with Cu(NO3)2ꢀ3H2O=Ac2O,
the reaction did not give a nitro derivative but gave instead N-ethylpara-
banic acid.

430
KAIYA, TANAKA, AND KOHDA
Figure 2. UV absorption spectrum of compound 4b at pH 6.8.
EXPERIMENTAL SECTION
Materials and Methods
1H and 13C NMR spectra were recorded on a JEOL EX 270, GSX 400,
or ALPHA 500 spectrometer, and chemical shifts were expressed in ppm
using Me4Si as the internal standard. EI and FAB mass spectra were
obtained with a JEOL JMS-DX300 spectrometer. ESI mass spectra were
obtained with a Micromass Quattro II spectrometer. HPLC analyses
were carried out using a Shimadzu LC-10AD apparatus equipped with an
SPD-M6A, a photodiode array UV detector. The Merck LiChrosphere 100
RP-18e, 46250 mm column used was eluted at a flow rate of 0.6 mL=min
with a 0.15 M phosphate buffer (pH 7.0)–MeOH system. Melting points were
measured with a Yanagimoto micro melting point apparatus and are
uncorrected. Silica gel 60 PF254 (Merck) was used for preparative thin-layer
chromatography (PLC).
Nitration of N6-Acetyladenine Derivatives (1) with Cu(NO3)2ꢀ3H2O=Ac2O
As a general practice, the reaction of 1c which showed the highest yield
was described. Cu(NO3)2ꢀ3H2O (800 mg, 3.32 mmol) was dissolved in 6 mL of

20-DEOXY-2-NITROADENOSINES
431
Ac2O. After 15 min, 1c (200 mg, 0.46 mmol) was added and the mixture was
left at room temperature for 22 h. MeOH (80 mL) was then added and the
mixture was evaporated to dryness. Water was added to the residue and
the products were extracted with AcOEt. Separation and purification of the
products were carried out by PLC (silica gel, AcOEt). The yields of 2c and 3c
were 41 mg (19%) and 29 mg (14%), respectively, and 1c was recovered in a
20 mg (10%) yield. In the case of 1b, separation of the products was carried
out by PLC (silica gel, AcOEt:MeOH ¼ 40:1), and the yields of 2b and 3b
were 3.4% and 3.3%, respectively, and the recovery of starting material was
11%. A similar procedure was employed for the reaction of 1a and products
2a and 3a were separated by PLC (silica gel, AcOEt) in total yield 19%.
N6-Acetyl-9-ethyl-2-nitroadenine (2a). Colorless needles (MeOH-
1
AcOEt), mp 194–196ꢃC; H NMR (500 MHz, DMSO-d6): d 1.49 (t, 3H,
J ¼ 7.3 Hz, CH2CH3), 2.32 (s, 3H, COCH3), 4.34 (q, 2H, CH2CH3), 8.80 (s,
1H, 8-H), 11.28 (bs, 1H, NH); 13C NMR (DMSO-d6) d 14.8 (CH2CH3), 24.4
(COCH3), 39.1 (CH2CH3), 125.2 (5-C), 147.5 (8-C), 149.6 (6-C), 151.8 (4-C),
153.6 (2-C), 168.9 (COCH3); EI-MS: m=z 250 (Mþ ), 208 (Mþ ꢁ Ac þ H),
162 (Mþ ꢁ Ac þ H ꢁ NO2); HRMS calcd for C9H10N6O3: 250.0814, found
250.0814.
30 50
N6,O ,O -Triacetyl-20-deoxy-2-nitroadenosine (2b). Colorless solid;
1H NMR (CDCl3): d 2.10, 2.17 (each s, each 3H, 30-, 50-OCOCH3), 2.77 (m,
1H, 20-H), 2.78 (s, 3H, N-COCH3), 2.89 (m, 1H, 200-H), 4.42 (m, 3H, 40-H, 50-
H, 500-H), 5.45 (m, 1H, 30-H), 6.55 (dd, 1H, J ¼ 6.1, 7.6 Hz, 10-H), 8.45 (s, 1H,
8-H), 8.94 (bs, 1H, NH); EI-MS: m=z 422 (Mþ ); HRMS calcd for
C16H18N6O8: 422.1185, found 422.1183.
0
0
0
N6,O2 ,O3 ,O5 -Tetraacetyl-2-nitroadenosine (2c). Colorless solid; 1H
NMR (CDCl3): d 2.10, 2.14, 2.20 (each s, each 3H, 20-, 30-, 50-OCOCH3), 2.79
(s, 3H, N-COCH3), 4.47 (m, 2H, 40-H, 50-H), 4.53 (m, 1H, 500-H), 5.62 (dd,
1H, J ¼ 4.3, 5.5 Hz, 30-H), 5.78 (m, 1H, 20-H), 6.30 (d, J ¼ 5.8 Hz, 10-H), 8.41
(s, 1H, 8-H), 8.90 (bs, 1H, NH): EI-MS: m=z 480 (Mþ ); HRMS calcd for
C18H20N6O10: 480.1241, found 480.1241.
9-Ethyl-2-nitroadenine (3a ¼ 4a). Yellow powder (H2O), mp 280ꢃC
(decomp); 1H NMR (DMSO-d6): d 1.44 (t, 3H, J ¼ 7.3 Hz, CH2CH3), 4.23 (q,
2H, CH2CH3), 8.16 (bs, 2H, NH2), 8.44 (s, 1H, 8-H); EI-MS: m=z 208 (Mþ );
HRMS calcd for C7H8N6O2: 208.0708, found 208.0704; UV lmax (nm) (pH
6.8) 245, 268 (sh), 346.
30 50
O ,O -Diacetyl-20-deoxy-2-nitroadenosine
(3b). Colorless
solid;
1H NMR (CDCl3): d 2.10, 2.16 (each s, each 3H, 30-, 50-OCOCH3), 2.74 (m,
1H, 20-H), 2.88 (m, 1H, 200-H), 4.39 (m, 2H, 50-H, 500-H), 4.42 (m, 1H, H-40),

432
KAIYA, TANAKA, AND KOHDA
5.44 (m, 1H, 30-H), 6.48 (bs, 2H, NH2), 6.49 (dd, 1H, J ¼ 5.9, 7.6 Hz, 10-H),
8.24 (s, 1H, 8-H); EI-MS: m=z 380 (Mþ ); HRMS calcd for C14H16N6O7:
380.1082, found 380.1082.
0
0
0
1
O2 ,O3 ,O5 -Triacetyl-2-nitroadenosine (3c). Colorless solid; H NMR
(CDCl3): d 2.10, 2.13, 2.18 (each s, each 3H, 20-, 30-, 50-OCOCH3), 4.47 (m,
2H, 40-H, 50-H), 4.50 (m, 1H, 500-H), 5.64 (dd, 1H, J ¼ 4.6, 5.5 Hz, 30-H), 5.77
(m, 1H, 20-H), 6.24 (d, J ¼ 5.2 Hz, 10-H), 6.27 (bs, 2H, NH2), 8.17 (s, 1H, 8-
H); EI-MS: m=z 438 (Mþ ); HRMS calcd for C16H18N6O9: 438.1134, found
438.1137.
Preparation of 2-Nitroadenine Derivatives (4)
Compound 2a, 2b, 2c, 3b or 3c was treated with conc. NH4OH=MeOH
(1:3) at 50ꢃC for 30 min and the product was separated and purified by PLC.
The yield of 4 was about 70%.
1
20-Deoxy-2-nitroadenosine (4b). Pale yellow powder; H NMR (D2O):
d 2.60 (ddd, 1H, J ¼ 3.7, 6.4, 14.0 Hz, 20-H), 2.87 (m, 1H, 200-H), 3.83 (dd, 1H,
J ¼ 4.6, 12.5 Hz, 50-H), 3.88 (dd, 1H, J ¼ 3.4, 12.5 Hz, 500-H), 4.19 (m, 1H, 40-
H), 4.69 (dd, J ¼ 3.4, 6.1 Hz, 30-H), 6.50 (t, J ¼ 6.7 Hz, 10-H), 8.49 (s, 1H, 8-
H); ESI-MS: positive ion mode, m=z 296.95 (M þ H)þ , negative ion mode,
m=z 294.98 (M ꢁ H)ꢁ ; UV: lmax (nm) (pH 6.8) 236, 262 (sh), 334.
1
2-Nitroadenosine (4c).[7] Pale yellow powder; H NMR (CD3OD): d
3.79, 3.90 (each m, each 1H, 50-H, 500-H), 4.15 (m, 1H, 40-H), 4.36 (dd, 1H,
J ¼ 3.7, 4.9 Hz, 30-H), 4.69 (m, 1H, 20-H), 6.03 (d, 1H, J ¼ 5.5 Hz, 10-H), 8.53
(s, 1H, 8-H); ESI-MS: positive ion mode, m=z 312.91 (M þ H)þ , negative ion
mode, m=z 310.98 (M ꢁ H)ꢁ; UV: lmax (nm) (pH 6.8) 236, 261 (sh), 334.
1
2-Methoxyadenosine.[7] Yield <25%; H NMR (DMSO-d6): d 3.53.
3.64 (each m, each 1H, 50-H, 500-H), 3.82 (s, 3H, CH3), 3.93 (m, 1H, 40-H),
4.15 (dd, 1H, J ¼ 3.4, 4.9 Hz, 30-H), 4.62 (m, 1H, 20-H), 5.78 (d, J ¼ 6.1 Hz, 10-
H), 7.31 (bs, 2H, NH2), 8.14 (s, 1H, 8-H); FAB-MS m=z 298 (M þ H)þ .
Preparation of 2-Nitroadenine (5)
Acid hydrolysis of 4b and 4c was carried out in 0.1N HCl at 60ꢃC for
40 min and in 1N HCl at 90ꢃC for 1h, respectively. Yellow precipitates of 5
which appeared after cooling the reaction mixture in almost quantitative
yield were collected.
2-Nitroadenine (5). mp >300ꢃC; 1H NMR (DMSO-d6): d 8.05 (bs, 2H,
NH2), 8.40 (s, 1H, 8-H); EI-MS m=z 180 (Mþ ), 134 (Mþ ꢁ NO2); HRMS

20-DEOXY-2-NITROADENOSINES
433
calcd for C5H4N6O2: 180.0396, found 180.0397. UV: lmax (nm) (pH 6.8)
235, 262 (sh), 280 (sh), 334.
Reaction of N6-Acetyladenine with Cu(NO3)2ꢀ3H2O=Ac2O
N6-Acetyladenine was allowed to react with Cu(NO3)2ꢀ3H2O=Ac2O as
described for the nitration of N6-acetyladenine derivatives (1). Products were
separated by PLC (CHCl3:MeOH ¼ 9:1).
1
Ring-nitrogen-mono-nitrated adenine. Yield 3.1%; H NMR (DMSO-
d6): d 7.73 (bs, 2H, NH2), 8.33 and 8.97 (each s, each 1H, 2-H and 8-H); EI-
MS: m=z 180 (Mþ ); HRMS calcd for C5H4N6O2: 180.0395, found 180.0401.
The position (1, 3, 7 or 9) of the N-nitro group could not be deduced.
1
N6-Acetyl-2(8)-nitro-8(2)-oxoadenine. Yield 2.1%; H NMR (DMSO-
d6): d 2.15 (s, 3H, CH3), 11.22 (bs, 1H, NH); 13C NMR (DMSO-d6): d 23.0
(CH3), 115.0, 136.5, 152.0, 152.6, 152.8, 169.5; EI-MS: m=z 238 (Mþ ), 196
(Mþ ꢁ C2H2O), 150 (Mþ ꢁ C2H2O ꢁ NO2); HRMS calcd for C7H6N6O4:
238.0450, found 238.0455.
ACKNOWLEDGMENT
We thank Dr. R. Taguchi for ESI mass analyses.
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Received March 11, 2002
Accepted May 28, 2002