468 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
Kroeger Smith et al.
and CBGA) at room temperature. Water (20 mL) was added,
and the reaction mixture was acidified to pH 3-4 with 1 N HCl
and extracted with ethyl ether (2 × 100 mL). The organic phase
was washed with brine and dried over Na2SO4. After evapora-
tion of the solvent, a crude, oily product was obtained. The
products were either recrystallized from ethyl acetate (CMSA)
or chomatographed (CMGA, CBGA) on SiO2 (eluent: hexane/
diethyl ether ) 2:1). Attempts to prepare 1-(2-chloroethyl)-3-
methyl-3-(5-carboxypentanoyl)triazene (CMADA) by this pro-
cedure were unsuccessful.
1-(2-Ch lor oeth yl)-3-m eth yl-3-(3-ca r boxyp r op a n oyl)tr ia -
zen e (CMSA). Yield: 2.3 g (34%), mp 75-83 °C dec. 1H-NMR
(CDCl3/200 MHz): δ 4.127 (ClCH2, AA′), 3.90 (CH2Nd, BB′),
3.246 (CH3, s), 3.147 (CH2, t), 2.779 (CH2, t). 13C-NMR (CDCl3/
200 MHz): δ 178.41, 173.81, 62.75, 41.72, 28.90, 28.62, 27.33.
Exact mass calcd for C7H13ClN3O3 (M + H)+ ) 222.064544,
found 222.0646.
1-(2-Ch lor oet h yl)-3-m et h yl-3-(4-ca r b oxyb u t a n oyl)t r ia -
zen e (CMGA). Yield: 4.52 g (64%). 1H-NMR (CDCl3/200
MHz): δ 4.118 (ClCH2, AA′, ∼6.0 Hz), 3.90 (CH2Nd, BB′, ∼6.0
Hz), 3.235 (CH3, s), 2.91 (CH2, t, 7.3 Hz), 2.49 (CH2, t, 7.3 Hz),
2.05 (CH2, qi, 7.3 Hz). Exact mass calcd for C8H15ClN3O3 (M +
H)+ ) 236.080194, found 236.0798.
1-(2-Ch lor oeth yl)-3-ben zyl-3-(4-ca r boxybu ta n oyl)tr ia z-
en e (CBGA). Yield: 5.7 g (61%). 1H-NMR (CDCl3/200 MHz):
δ 7.23 (Ph, s), 5.13 (PhCH2, s), 4.06 (ClCH2, AA′, ∼6.0 Hz), 3.81
(CH2Nd, BB′, ∼6.0 Hz), 2.94 (CH2, t, 7.3 Hz), 2.49 (CH2, t, 7.3
Hz), 2.07 (CH2, qi, 7.3 Hz). Exact mass calcd for C14H19ClN3O3
(M + H)+ ) 312.111494, found 312.1112.
Gen er a l Ester ifica tion P r oced u r e for CMSA, CBSA,
CMGA, a n d CBGA. Carboxylic acid (10 mmol), methanol (0.5
mL), and a catalytic amount of 4-(dimethylamino)pyridine
(DMAP) were dissolved in 20 mL of CH2Cl2. A solution of
dicyclohexylcarbodiimide (2.06 g, 10 mmol) in methylene chlo-
ride (15 mL) was added at 0 °C. The reaction mixture was
stirred for 5 h at room temperature. The precipitated dicyclo-
hexylurea was filtered off, and the filtrate was concentrated to
give a colorless oil. This oil was purified by column chomatog-
raphy on SiO2 (eluent: hexane/diethyl ether ) 3/1).
1-(2-Ch lor oet h yl)-3-m et h yl-3-(3-ca r b om et h oxyp r op a -
n oyl)t r ia zen e (CMS). Yield: 34%. 1H-NMR (CDCl3/200
MHz): δ 4.12 (ClCH2, AA′, ∼6.5 Hz), 3.89 (CH2Nd, BB′, ∼6.5
Hz), 3.71 (OCH3, s), 3.23 (NCH3, s), 3.14 (CH2, t, 6.75 Hz), 2.72
(CH2, t, 6.75 Hz). Exact mass calcd for C8H14ClN3O3 (M+) )
235.072369, found 235.0712.
1-(2-Ch lor oeth yl)-3-ben zyl-3-(3-car bom eth oxypr opan oyl)-
tr ia zen e (CBS). Yield: 74%. 1H-NMR (CDCl3/200 MHz): δ
7.23 (Ph, s), 5.14 (PhCH2, s), 4.07 (ClCH2, AA′, ∼6.0 Hz), 3.81
(CH2Nd, BB′, ∼6.0 MHz), 3.71 (CH3O, s), 3.18 (CH2, t, 6.8 Hz),
2.76 (CH2, t, 6.8 Hz). Exact mass calcd for C14H18ClN3O3 (M+)
) 311.103669, found 311.1066.
1-(2-Ch lor oe t h yl)-3-m e t h yl-3-(4-ca r b om e t h oxyb u t a -
n oyl)tr ia zen e (CMG). Yield: 87%. 1H-NMR (CDCl3/200
MHz): δ 4.12 (ClCH2, AA′, ∼6.0 Hz), 3.895 (CH2Nd, BB′, ∼6.0
Hz), 3.68 (CH3O, s), 3.23 (CH3, s), 2.88 (CH2, t, 7.3 Hz), 2.44
(CH2, t, 7.3 Hz), 2.04 (CH2, t, 7.3 Hz). Exact mass calcd for
C9H17ClN3O3 (M + H) ) 250.0958, found 250.0928.
Chloroethyl azide (2.1 g, 20 mmol) was dissolved in 30 mL of
dry THF and cooled to 0 °C under nitrogen. Methylmagnesium
chloride (7 mL of a 3 N solution in THF) was added dropwise.
The reaction was allowed to warm to 0 °C over a 45 min time
period. After recooling to -30 °C, the above-described THF
solution of the activated adipic acid monomethyl ester was added
dropwise. The cooling bath was removed and the reaction
mixture was stirred at room temperature for 1 h. Diethyl ether
(60 mL) and saturated NaHCO3 solution (10 mL) were added.
The reaction mixture was filtered, and the filtrate was washed
with (10%) NaHCO3 solution (2 × 20 mL) and saturated brine.
The organic phase was dried over Na2SO4. After evaporation
of the solvent, a colorless oil was obtained. The product was
purified by column chromatography on silica gel (eluent: pen-
tane/diethyl ether ) 2/1 v/v). Yield: 750 mg (15%). 1H-NMR
(CDCl3/200 MHz): δ 4.11 (ClCH2, AA′, ∼6.5 Hz), 3.89 (CH2Nd,
BB′, ∼6.5 Hz), 3.67 (NCH3, s), 3.23 (OCH3,s), 2.83 ((O)CCH2),
2.36 ((O)CCH2), 1.72 ((CH2)4, m). Exact mass calcd for C10H18
-
ClN3O3 (M+) ) 263.103669, found 263.1048.
Rea ction s of Tr ia zen es w ith Ca lf Th ym u s DNA. A 10
µL aliquot of a 0.5 M dimethyl sulfoxide solution of a particular
triazene was added to 990 µL of a solution of calf thymus DNA
(1 mg/mL) in 0.02 M sodium phosphate/0.01 M sodium chloride
buffer (pH 7.0). The reactions (5 mM final triazene concentra-
tion) were incubated at 37 °C either alone (48 h) or in the
presence of porcine liver esterase (2 h) (EC 3.1.1.1) (10 µL/1 mL
reaction solution, 5.35 mg/mL). In addition, CMS was also
incubated for 48 h in the presence of esterase. The DNA was
precipitated with 3 mL of absolute ethanol and centrifuged for
15 min at 5000g. The DNA pellet was washed with 2% sodium
acetate in ethanol and then allowed to sit in this solution for
15 min. This solution was decanted and was replaced by
anhydrous ether for 5 min. The ether was decanted off, and
the DNA was dried in a vacuum desiccator overnight and then
stored at -20 °C until hydrolysis.
An a lysis of DNA Ad d u cts. Hydrolysis and HPLC of the
DNA adducts (7-MeG, 7-EtG, 7-HOEtG, 7-ClEtG, O6-MeG, and
O6-HOEtG) were carried out as previously described (11). The
presence of expected 7-BzG from the 7-benzyl triazenes, identi-
fied by coelution with a standard sample, was also estimated.
All results are reported as millimoles of alkylated base per mole
of guanine on at least duplicate experiments.
Kin etic Stu d ies. Rates of triazene decomposition in aqueous
solution at 70 °C were carried out on a Milton Roy MR3000 UV/
visible spectrophotometer. The disappearance of each triazene
(final concentration 3.0 × 10-5 M) was followed by monitoring
the change in absorbance at its respective λmax, ca. 245 nm. The
experimental procedure for a typical kinetic run in 0.1 M
phosphate buffer, pH 7.0 (ionic strength ) 0.45 M maintained
with added NaCl), and the calculation of the first-order rate
constants have been previously detailed (12). A minimum of
100 absorbance vs time readings were obtained over at least
3.5 half-lives. The values reported are the average of two
separate determinations which agree within (2%.
P r od u ct Stu d ies. The products of the decomposition of the
1
various triazenes were determined by H-NMR following reac-
tion at 37 and 70 °C in 0.05 M sodium phosphate/0.25 M Na2-
SO4 in D2O, adjusted to pH 7.5 with a D2O solution of NaOD.
Buffer was added to a weighed amount of triazene, sealed in
vials, and incubated for at least four half-lives, as determined
by kinetic measurements. At the end of the reaction time, an
aliquot of the reaction solution was removed and analyzed by
1H NMR. Assignment of the NMR peaks arising from the
various products was made by comparison and spiking with
authentic samples. Yields were determined by comparative
integration of the product peaks versus an internal standard,
fumaric acid.
1-(2-Ch lor oe t h yl)-3-b e n zyl-3-(4-ca r b om e t h oxyb u t a -
n oyl)t r ia zen e (CBG). Yield: 87%. 1H-NMR (CDCl3/200
MHz): δ 7.23 (Ph, s), 4.06 (ClCH2, AA′, ∼6.0 Hz), 3.81 (CH2Nd,
BB′, ∼6.0 Hz), 3.67 (CH3O, s), 2.91 (CH2, t, 7.3 Hz), 2.44 (CH2,
t, 7.3 Hz), 2.06 (CH2, qi, 7.3 Hz). Exact mass calcd for C15H24
ClN4O3 (M + NH4)+ ) 343.153694, found 343.1571.
-
1-(2-Ch lor oeth yl)-3-m eth yl-3-(5-car bom eth oxypen tan oyl)-
tr ia zen e (CMAD). Adipic acid monomethyl ester (2.96 mL,
20 mmol) and hydroxysuccinimide (2.416 g, 21 mmol) were
dissolved in 30 mL of dry THF. Dicyclohexylcarbodiimide (4.326
g, 21 mmol) was added, and the reaction mixture was stirred
overnight at room temperature. After filtration of the precipi-
tated dicyclohexylurea, the filtrate was concentrated, taken up
in 10 mL of dry THF, and refiltered. This solution of the crude
hydroxysuccinimide ester of adipic acid monomethyl ester was
used in the next reaction step.
The potential conversion of acyltriazenes esters to their
respective free acid forms was checked by HPLC following
incubation at 37 °C. Reactions with CMC, CBS, CMS, CBG,
CMG, or CMAD in buffer alone, or in the presence of calf thymus
DNA in buffer (1 mg/mL), both in the presence and in the
absence of esterase, were carried out for times up to 48 h. The