DNA Adducts from N-Nitrosotolazoline
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 321
5′-O-Phenylacetyl-2-deoxyguanosine 12. To a 4 mL N,N-
dimethylformamide (DMF) solution of 2-deoxyguanosine (100 mg,
0.375 mmol), which had been dried by evaporation twice from
anhydrous pyridine, 115 mg of pyridine (1.49 mmol) and phenyl-
acetyl chloride (144 mg, 0.938 mmol) was added. The reaction
mixture was stirred at room temperature for 6 h. The product was
separated by HPLC on a C-18 semiprep column (methanol and
0.025 M formic acid). In the crude reaction mixture, the product
yield was 56%; mp > 250 °C (dec). 1H NMR (DMSO-d6): δ 10.63
(s, 1 H), 7.82 (s, 1 H), 7.22–7.33 (m, 5 H), 6.46 (s, 2 H), 6.13 (t,
J ) 6.6 Hz, 1 H), 5.42 (d, J ) 3.0 Hz, 1 H), 4.37 (m, 1 H), 4.22
(m, 2 H), 3.98 (m, 1 H), 3.66 (s, 2 H), 2.58 (m, 1 H), 2.23 (m, 1
H). 13C NMR (DMSO-d6): δ 171.00, 156.68, 153.63, 150.92,
135.12, 134.17, 129.30, 128.29, 126.79, 116.73, 83.91, 82.33, 70.51,
64.36, 40.05, 38.68. MS/MS (ESI): 385.2, 152.3, 151.3. HRMS
(FAB): m/z calcd for C18H19N5O5 + H, 386.1464; found, 386.1455.
with methanol and formic acid (0.02 M in water) as the mobile
phase. The methanol in the collected eluent was evaporated in
vacuo. The aqueous solution was neutralized with concentrated
ammonia hydroxide to pH 6.5. Upon neutralization, a white powder
[7-(2-phenylacetamido)ethylguanine 5c] precipitated. The powder
was filtered and washed with cold water three times and ethyl ether
three times. A trace amount of water was removed in vacuo at 80
°C for 5 h; mp > 300 °C. 1H NMR (DMSO-d6): δ 10.75 (s, 1 H),
8.11 (t, 1 H, J ) 5.2 Hz), 7.69 (s, 1 H), 7.16–7.29 (m, 5 H), 6.07
(s, 2 H), 4.20 (t, 2 H, J ) 5.3 Hz), 3.45 (t, 2 H, J ) 5.3 Hz), 3.34
(s, 2 H). 13C NMR (DMSO-d6): δ 170.14, 152.34, 151.16, 142.99,
135.90, 132.86, 128.65, 127.84, 125.99, 108.04, 45.36, 42.05, 39.43.
HRMS (FAB): m/z calcd for C15H17N6O2 + H, 313.1413; found,
313.1412.
O6-(2-Phenylacetamidoethyl)guanine 4a. To a solution of N-2-
hydroxyethylphenylacetamide (27, 400 mg, 2.23 mmol) in 5 mL
of anhydrous DMF was added 48 mg of NaH (2 mmol) in one
portion. The reaction mixture was stirred at room temperature for
30 min followed by the addition of 2-amino-6-chloropurine (70
mg, 0.37 mmol). Semipreparative HPLC was used for the purifica-
tion of the product, O6-(2-phenylacetamidoethyl)guanine; mp
2-Amino-6-mesitylsulfonoxy-9-[3′,5′-bis(t-butyldimethylsilyl)-
2′-deoxyribosyl]purine 29. To a stirred solution of 3′,5′-bis(t-
butyldimethylsilyl)-2′-deoxyguanosine (8) (107 mg, 0.216 mmol)
in a mixture of hexamethylphosphoramide (HMPA, 300 µL) and
dichloromethane (1.5 mL) were added triethylamine (120 µL, 80.71
mg, 0.86 mmol), 4-dimethylaminopyridine (DMAP, 5.8 mg, 0.04
mmol), and 2-mesitylenesulfonyl chloride (110 mg, 0.502 mmol).
After it was stirred at room temperature overnight, the resulting
solution was washed with saturated solution of sodium bicarbonate
(2 mL × 2) and brine (2 mL × 2). The solution was dried with
anhydrous sodium sulfate. After the solvent was removed in vacuo,
the residue was purified by flash chromatography (ethyl acetate/
hexane, 30/70, v/v) to obtain 128 mg of the 6-mesitylsulfonoxy
1
165–166 °C. H NMR (DMSO-d6): δ 12.39 (s, 1 H), 8.34 (b, 1
H), 7.81 (s, 1 H), 7.23 (m, 5 H), 6.21 (s, 2 H), 4.39 (t, J ) 5.6 Hz,
2 H), 3.47 (t, J ) 5.6 Hz, 2 H), 3.34 (s, 2 H). 13C NMR (DMSO-
d6): δ 170.5, 159.8, 159.7, 155.6, 138.0, 136.4, 129.0, 128.2, 126.3,
112.7, 64.2, 42.2, 38.8. HRMS (FAB): m/z calcd for C15H17N6O2
+ H, 313.1413; found, 313.1418.
1-(2-Phenylacetamidoethyl)adenine 7a. Using the literature as
a guide (10, 11), to a solution of 100 mg of adenosine (0.38 mmol)
in 2 mL of anhydrous DMF, 267 mg of N-(2-bromoethyl)pheny-
lacetamide 35 (1.1 mmol) and 80 mg of CaCO3 (0.8 mmol) were
added. The solution was stirred for 8 h at 80 °C under anhydrous
conditions. The solvent was evaporated to dryness, and the residue
was hydrolyzed in 2 mL of acetic acid at 90 °C for 6 h. Purification
on semipreparative HPLC gave 7a (40.5 mg, 36%) as a white solid;
mp 250 °C (dec.). 1H NMR (DMSO-d6): δ 8.29 (br t, J ) 5.7 Hz,
1 H), 7.86 (s, 1 H), 7.83 (s, 1 H), 7.17–7.28 (m, 7 H), 4.19 (t, J )
5.6 Hz, 2 H), 3.40 (t, J ) 5.6 Hz, 2 H), 3.38 (s, 2 H). 13C NMR
(DMSO-d6): 173.3, 165.9, 148.1, 142.1, 141.0, 135.9, 129.0, 128.2,
126.4, 119.4, 52.0, 42.2, 37.1. HRMS (FAB): m/z calcd for
C15H17N6O + H, 297.1464; found, 297.1453.
3-(2-Phenylaceamidoethyl)adenine 8a and 9-(2-Phenylace-
amidoethyl)adenine 11. Using the literature as a guide (12), a
mixture of adenine (135 mg, 1 mmol) with N-(2-bromoethyl)phe-
nylacetamide (35, 362 mg, 1.5 mmol) in 1 mL of DMF was heated
at 80 °C while stirring for 4 h. After the solvent was evaporated in
vacuo, the residue was purified on aluminum oxide (activated, basic)
with CH2Cl2 and MeOH (9:1, v/v) as an eluting solvent. The
collected fraction contained two products, 8a and 11, which were
then separated on silica gel with CH2Cl2 and MeOH (5:1, v/v).
Purification on silica gel alone could not separate 8a from adenine.
For 3-(2-phenylaceamido)ethyladenine 8a: mp 231–234 °C. 1H
NMR (DMSO-d6): δ 8.21 (br, t, J ) 5.5 Hz, 1 H), 8.10 (s, 1 H),
7.95 (b, 1 H), 7.90 (b, 1 H), 7.77 (s, 1 H), 7.15–7.29 (m, 5 H),
4.35 (t, J ) 5.6 Hz, 2 H), 3.40 (t d, J ) 5.6, 5.5 Hz, 2 H), 3.35 (s,
2 H). 13C NMR (DMSO-d6): 171.2, 154.5, 149.0 (2C), 145.6, 136.1,
129.0, 128.2, 126.4, 116.4, 49.0, 42.2, 37.6. HRMS (FAB): m/z
calcd for C15H17N6O + H, 297.1464; found, 297.1470. For 9-(2-
phenylaceamido)ethyladenine 11: mp 212–214 °C. 1H NMR
(DMSO-d6): δ 8.15 (br, 1 H), 8.13 (s, 1 H), 7.95 (s, 1 H), 7.13–7.26
(m, 7 H), 4.19 (t, J ) 5.2 Hz, 2 H), 3.40 (t, J ) 5.2 Hz, 2 H), 3.34
(s, 2 H). 13C NMR (DMSO-d6): 170.6, 156.0, 152.4, 149.6, 141.0,
136.3, 129.0, 128.2, 126.3, 118.7, 55.1, 42.5, 42.3. HRMS (FAB):
m/z calcd for C15H17N6O + H, 297.1464; found, 297.1460.
7-(2-Phenylacetylaminoethyl)adenine 9c. To a solution of 135
mg of adenine (1 mmol) in 2 mL of anhydrous DMF was added
324 mg of 1-phenylacetylaziridine 40 (2 mmol) (13). The mixture
was stirred for 4 h at 70 °C, and the solvent was evaporated in
vacuo. The residue, containing a mixture of all of the ring
N-alkylated isomers (7a, 8a, 9c, and 11) was chromatographed over
silica gel with CH2Cl2 and MeOH as eluting solvents (from 9:1,
1
nucleoside 29 as a white solid (yield: 90%). H NMR (CDCl3):
7.95 (s, 1 H), 6.26 (t, J ) 6.5 Hz, 1 H), 4.82 (b, s, 2 H), 4.55 (m,
1 H), 3.95 (m, 1 H), 3.74 (m, 2 H), 2.73 (s, 6 H), 2.53 (m, 1 H),
2.36 (m, 1 H,), 2.31 (s, 3 H), 0.89 (s, 18 H), 0.08 (s, 6 H), 0.04 (s,
6 H).
3′,5′-Bis(t-butyldimethylsilyl)-O6-(2-phenylacetylamino)ethyl-
2′-deoxyguanosine 31. Using an adaptation of a literature procedure
(9), 1,4-diazabicyclo[2.2.2]octane (DABCO, 2 molar equiv), 50 mg
of 4 Å molecular sieves, and 5 molar equiv of 2-phenylacetami-
doethanol 27 were added to a solution of 29 (96 mg, 0.14 mmol)
in 1.4 mL of 1,2-dimethoxyethane. After the mixture was allowed
to stir at room temperature for 30 min, 1,8-diazabicyclo[5,4,0]undec-
7-ene (DBU, 1.5 molar equiv) was added. The mixture was allowed
to stir at room temperature for 24 h and then diluted with EtOAc.
The organic layer was dried over Na2SO4 and evaporated. The crude
products were chromatographed on silica gel using hexane and
EtOAc (1:1, v/v). The yield of 31 (white solid) was 67%. 1H NMR
(CDCl3): 7.92 (s, 1 H), 7.26 (m, 5 H), 6.34 (t, J ) 5.5 Hz, 1 H),
6.20 (b, 1 H), 4.84 (s, 2 H), 4.57 (m, 3 H), 4.00 (m, 1 H), 3.83 (m,
2 H), 3.72 (m, 2H), 3.55 (s, 2 H), 2.58 (m, 1H), 2.39 (m, 1H), 0.93
(s, 18 H), 0.12 (s, 6 H), 0.10 (s, 6 H).
O6-(2 Phenylacetamidoethyl)deoxyguanosine 4b. To 20 mg of
31 in 0.5 mL of THF (0.031 mmol) was added 1.0 M TBNF in
THF solution (0.15 mL, 0.15 mmol). After 30 min of stirring at
room temperature, TLC showed that the starting material had
disappeared. Purification on a flash column (methanol and CH2Cl2,
1:9 v/v) provided 14 mg of 4b as a white solid in 96% yield; mp
151–153 °C. 1H NMR (DMSO-d6): 8.30 (t, J ) 5.5 Hz, 1 H), 8.09
(s, 1 H), 7.23 (m, 5 H), 6.40 (s, 2H), 6.22 (t, J ) 6.1 Hz, 1 H),
5.24 (b, 1 H), 4.99 (b, 1H), 4.43 (t, J ) 5.5 Hz, 2 H), 4.35 (m,
1H), 3.83 (m, 1 H), 3.48 (t, J ) 5.4 Hz, 2 H), 3.42 (s, 2 H), 2.55
(m, 1 H), 2.22 (m, 1 H). 13C NMR (DMSO-d6): 170.46, 160.22,
159.63, 153.93, 137.72, 136.31, 128.92, 128.10, 136.23, 113.90,
87.60, 82.81, 70.77, 64.33, 61.73, 42.19, 39.50, 38.42. HRMS
(FAB): m/z calcd for C20H24N6O5 + Li, 435.1968; found, 435.1965.
7-(2-Phenylacetamido)ethylguanine 5c. To a solution of 2′-
deoxyguanosine (100 mg, 0.37 mmol) in 5 mL of DMF, N-(2-
bromoethyl)phenylacetamide 35 (200 mg, 0.83 mmol) was added.
The reaction mixture was heated at 80 °C for 6 h and then cooled
to room temperature. Because 7-alkylguanines are sparingly soluble
in any organic solvent, the reaction mixture was diluted with 0.1
N HCl aqueous solution and purified on a preparative HPLC column