DNA oxidative damage by terpene catechols as analogues of natural terpene quinone methide precursors in the presence of Cu(II) and/or NADH

Article abstract of DOI:10.1021/tx060021s

Natural terpene quinone methides (QM) and their derivatives have been investigated as therapeutics due to their broad antifungal, antibacterial, and antitumor activities. Recently, we reported that a terpene QM was formed from the catechol precursor through the disproportionation of Cu(II)/(I) redox cycle, and extensive DNA damage was observed throughout the oxidation process. In this paper, we investigate DNA damage with a series of terpene catechols as analogues of natural QM precursors and suggest that reactive oxygen species (ROS) are responsible for the observed DNA damage in the Cu²⁺-induced oxidation despite the stereo- and structural difference of these catechol or subsequent oxidation products. In addition, the presence of NADH significantly enhanced the extent of DNA damage by oxidation of these catechols. Especially with alkene catechols 6-7, the extent of DNA damage was independent of the concentration of catechols, implying that NADH enables the continuous production of ROS through the redox cycle of catechols/quinones.

Full text of DOI:10.1021/tx060021s

828
Chem. Res. Toxicol. 2006, 19, 828-836
DNA Oxidative Damage by Terpene Catechols as Analogues of
Natural Terpene Quinone Methide Precursors in the Presence of
Cu(II) and/or NADH
Miguel A. Zuniga, Jifeng Dai, Mark P. Wehunt, and Qibing Zhou*
Department of Chemistry, Virginia Commonwealth UniVersity, 1001 West Main Street,
Richmond, Virginia 23284-2006
ReceiVed January 31, 2006
Natural terpene quinone methides (QM) and their derivatives have been investigated as therapeutics
due to their broad antifungal, antibacterial, and antitumor activities. Recently, we reported that a terpene
QM was formed from the catechol precursor through the disproportionation of Cu(II)/(I) redox cycle,
and extensive DNA damage was observed throughout the oxidation process. In this paper, we investigate
DNA damage with a series of terpene catechols as analogues of natural QM precursors and suggest that
reactive oxygen species (ROS) are responsible for the observed DNA damage in the Cu2+-induced oxidation
despite the stereo- and structural difference of these catechol or subsequent oxidation products. In addition,
the presence of NADH significantly enhanced the extent of DNA damage by oxidation of these catechols.
Especially with alkene catechols 6-7, the extent of DNA damage was independent of the concentration
of catechols, implying that NADH enables the continuous production of ROS through the redox cycle of
catechols/quinones.
Scheme 1. Diterpenone Catechol 2 as a Precursor for
Terpene Quinone Methides Resulted in Extensive DNA
Damage in the Presence of Cu2+
Introduction
Natural terpene quinone methides (QM)1 are a category of
closely related compounds, and their biological activity attracts
numerous research efforts to develop them as effective thera-
peutics against fungi, bacteria, and tumor growth (1-9). While
many focus on derivatives and analogues of terpene QM (7-
9), we are interested in the biological potential of catechol
precursors and their conversion to terpene QMs through an
oxidation process. Oxidation of catechol has been well-studied
and has significant impacts on biological systems (10-16).
Recently, we reported that terpene QM 1 as an analogue of
tingenone (a triterpene QM) was formed from a catechol
precursor 2 through the disproportionation of Cu(II)/(I) redox
cycle (Scheme 1), and the high reactivity of QM 1 toward
nucleophiles was confirmed (17). As a result, extensive DNA
damage was observed during the oxidation process. Two
possible mechanisms may contribute to this observed DNA
damage: (1) DNA nucleobase alkylation by in situ generated
QM (18-20) and (2) DNA oxidative damage by reactive oxygen
species (ROS) such as superoxide or hydroxyl radical (13-
16). The extent of nucleobase alkylation depends on the stability
and reactivity of QM, while ROS are generated from the
disproportionation of Cu(II)/(I) redox cycle in the oxidation of
catechols (Scheme 1). For catechol oxidation, it has also been
reported that NADH (NADPH) can considerably enhance the
production of ROS by reducing quinones to catechols, thus,
forming a redox cycle with Cu2+-induced oxidation (13-16,
21). In this paper, we investigated DNA damage with a series
of terpene catechols as analogues of natural QM precursors and
suggested that production of ROS was the dominant mechanism
for the observed DNA damage in the Cu (II)-induced oxidation
despite the stereo- and structural differences of catechols or
subsequent oxidation products. In addition, the extent of DNA
damage with alkene analogues increased substantially in the
presence of NADH and independent of the concentration,
implying that NADH enhances the production of ROS through
the redox cycle of catechols/quinones.
To elucidate the Cu2+-induced DNA damage mechanism, a
series of catechol analogues of natural terpene QM precursors
were designed to investigate potential effects of stereochemistry,
substitutional and functional groups on nucleobase alkylation,
and production of ROS (Scheme 2). For example, analogue 3
is a diastereoisomer of 2 in a cis-conformation, while all of the
reported natural terpene QMs are in the trans-conformation.
Interestingly, molecular modeling (Spartan, Wavefunction, Inc.,
Irvine, CA) indicates that analogue 3 adopts a unique bent
conformation as compared to that of trans-analogue 2 (Figure
1), predicting that the QM of analogue 3 will have a different
reactivity toward DNA than 1. The stability of QM 1 can be
improved by incorporating a methyl group on the phenyl ring
to prevent potential polymerization (22, 23), which was
investigated with analogues 4-5. Also, the QM formation from
* To whom correspondence should be addressed. Phone: (804) 828-
3520. Fax: (804) 828-8599. E-mail: qzhou@vcu.edu.
1 Abbreviations: QM, quinone methide; ROS, reactive oxygen species;
DCC, N,N-dicyclohexylcarbodiimide; NOESY, nuclear Overhauser and
exchange spectroscopy; ESI-MS, electrospray ionization mass spectroscopy.
10.1021/tx060021s CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/03/2006

DNA OxidatiVe Damage by Terpene Catechols
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 829
(1.03 g, 1.05 equiv) and 18-crown-6 (5.04 g, 1.2 equiv). The
reaction was stirred under N2 for 16 h. The reaction solution was
then extracted with ether (250 mL × 2). The organic layers were
collected, dried with MgSO4, and concentrated. Flash chromato-
graphic separation (5-10% EtOAc in hexanes) afforded the cyanide
1
product as an oil (2.81 g) in 92% yield. H NMR (CDCl3, 300
MHz): δ 7.02 (d, J ) 8.4 Hz, 1H), 6.74 (d, J ) 8.4 Hz, 1H), 3.83
(s, 3H), 3.76 (s, 3H), 3.58 (s, 2H), 2.23 (s, 3H). 13C NMR (CDCl3,
75 MHz): δ 152.8, 147.8, 130.6, 124.3, 121.6, 118.1, 109.9, 60.6,
55.9, 21.8, 12.1. ESI-MS calcd for C11H15NO2 (M + H+), 192.10;
found, 192.07.
The cyanide adduct (2.81 g, 14.7 mmol) was refluxed in a
solution of NaOH (8.5 g) in ethanol (30 mL) for 18 h. The reaction
solution was then acidified with HCl to pH 2 and extracted with
CH2Cl2 (250 mL × 2). The organic layers were collected, dried
with MgSO4, and concentrated. Flash chromatographic separation
(2-7% MeOH in CH2Cl2) afforded compound 10 as an oil (1.95
g) in 63% yield. 1H NMR (CDCl3, 300 MHz): δ 6.91 (d, J ) 8.3
Hz, 1H), 6.72 (d, J ) 8.3 Hz, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 3.60
(s, 2H), 2.21 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 178.4, 152.3,
147.5, 131.6, 126.0, 125.5, 109.7, 60.4, 55.8, 38.9, 12.4. ESI-MS
calcd for C11H15O4 (M + H+), 211.10; found, 211.10.
Figure 1. The cis-isomer of diterpenone catechol 3 adopts a unique
bend structure as compared to that of trans-isomer 2, predicting a
different reactivity of QM of 3 in the DNA damage study. Both
structures were obtained using AM1 calculation as the molecular
modeling method.
Scheme 2. Terpene Catechols 2-7 as Analogues of Natural
Terpene QM Precursors
Methyl (4E)-2-(3,4-Dimethoxy-2-methylphenyl)-5,9-dimeth-
yldeca-4,8-dienoate (11). To a solution of freshly distilled diiso-
propylamine (2.20 mL, 15.7 mmol) in dry THF (20 mL) at -78
°C under N2 was added a solution of n-butyllithium (2.5 M, 6.28
mL). After 10 min at -78 °C, a solution of 3,4-dimethoxyphen-
ylacetic acid (1.40 g, 7.13 mmol) in THF (10 mL) was slowly
added. The resulting solution was stirred for 1 h at -78 °C, and
then geranyl chloride (1.45 mL, 7.85 mmol) was added. The
reaction solution was stirred under N2 for 18 h and allowed to slowly
warm to room temperature. The reaction solution was then quenched
with 1 N HCl (250 mL) and extracted with CH2Cl2 (250 mL × 2).
The organic layers were collected, washed with brine, dried with
MgSO4, and concentrated. Flash chromatographic separation (0.5-
7% MeOH in CH2Cl2) afforded the free acid as a yellow oil (1.65
g) in 70% yield. 1H NMR (CDCl3, 300 MHz): δ 7.06 (d, J ) 8.6
Hz, 1H), 6.75 (d, J ) 8.6 Hz, 1H), 5.04 (m, 2H), 3.83 (s, 3H),
3.76 (s, 3H), 3.76 (m, 1H), 2.77-2.70 (m, 1H), 2.41-2.34 (m,
1H), 2.29 (s, 3H), 2.00-1.94 (m, 4H), 1.65 (s, 3H), 1.57 (s, 6H).
13C NMR (CDCl3, 75 MHz): δ 180.5, 151.9, 147.2, 137.8, 131.6,
131.2, 130.3, 124.3, 122.8, 120.9, 109.8, 60.5, 55.8, 47.0, 39.9,
31.6, 26.7, 25.8, 17.8, 16.3, 12.1. ESI-MS calcd for C21H31O4
(M + H+), 347.22; found, 347.22.
catechol 2 may be unique due to the ketone moiety in the
structure (24-26) and is in contrast to the quinone formation
in the oxidation of a variety of catechols (13-16). Thus, as a
comparison, the homoconjugated ketone moiety of catechol 2
was replaced with a conjugated alkene as analogues 6-7 for
the exclusive formation of quinones (Scheme 2).
Experimental Procedures
All chemicals were purchased from Fisher Scientific (Pittsburgh,
PA) or Sigma-Aldrich (Milwaukee, WI) and used without further
purification. NMR spectra of the synthesized compounds were
obtained by Variant NMR spectrometers. Oligonucleotides were
purchased from Invitrogen (Carlsbad, CA) and purified by gel
electrophoresis according to published protocols. Aqueous solutions
of [γ-32]-ATP (250 µCi) were purchased from MP Biomedicals
(Costa Mesa, CA). Gel images of isotopic 32P were obtained by a
Molecular Dynamics Typhoon 8600 Variable Mode Imager (Sunny-
vale, CA), and water was purified with a Barnstead E-pure
4-Module Deionization System (Dubuque, IA). Electrospray ioniza-
tion mass spectroscopy (ESI-MS) analysis was carried out using
Q-TOF2 from Micromass (Manchester, U.K.). Compounds 2 and
14 and thiol-protected citral were obtained as reported previously
(17, 27).
1-(Chloromethyl)-3,4-dimethoxy-2-methylbenzene (9). To a
solution of 2-chloroethyl methyl ether (6.05 g, 64.0 mmol) in acetic
acid (5.5 mL) was added 2,3-dimethoxytoluene (5.20 mL, 34.7
mmol), and the reaction was initiated by slightly heating. The
reaction solution was stirred under N2 at room temperature for 16
h and then cooled in ice to precipitate the product. The solid was
collected by filtration and washed with water. Flash chromato-
graphic separation (5-10% EtOAc in hexanes) afforded the free
acid as a white solid (3.21 g) in 47% yield. 1H NMR (CDCl3, 300
MHz): δ 7.04 (d, J ) 8.3 Hz, 1H), 6.73 (d, J ) 8.3 Hz, 1H), 4.58
(s, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 2.35 (s, 3H). 13C NMR (CDCl3,
75 MHz): δ 153.4, 147.7, 131.9, 129.0, 125.9, 109.5, 60.5, 55.8,
45.6, 11.6. ESI-MS calcd for C10H13ClO2 (M - Cl), 165.09; found,
165.05.
To a solution of the resulting free acid (1.57 g, 4.72 mmol) in
CH2Cl2 (35 mL) were added 4-(dimethylamino)pyridine (681 mg,
1.2 equiv), DCC (1.16 g, 1.2 equiv), and methanol (210 µL, 1.1
equiv). The resulting reaction mixture was stirred under N2 for 18
h at room temperature. The reaction solution was then quenched
with 1 N HCl (250 mL) and extracted with CH2Cl2 (250 mL × 2).
The organic layers were collected, washed with brine, dried with
MgSO4, and concentrated. Flash chromatographic separation (5-
20% EtOAc in hexanes) afforded diastereoisomeric pure trans-
1
compound 11 as a yellow oil (1.03 g) in 60% yield. H NMR
(CDCl3, 300 MHz): δ 7.04 (d, J ) 8.5 Hz, 1H), 6.74 (d, J ) 8.5
Hz, 1H), 5.05-5.00 (m, 2H), 3.83 (s, 3H), 3.77 (s, 3H), 3.76 (m,
1H), 3.64 (s, 3H), 2.76-2.71 (m, 1H), 2.38-2.34 (m, 1H), 2.27
(s, 3H), 2.00-1.93 (m, 4H), 1.66 (s, 3H), 1.57 (s, 6H). 13C NMR
(CDCl3, 75 MHz): δ 174.9, 151.7, 147.2, 137.6, 131.6, 130.9,
130.8, 124.3, 122.6, 121.2, 109.7, 60.4, 55.8, 52.0, 47.1, 39.9, 32.1,
26.8, 25.9, 17.8, 16.3, 12.0. ESI-MS calcd for C22H33O4 (M + H+),
361.24; found, 361.24.
4b,5,6,7,8,8a-trans-Hexahydro-2,3-dimethoxy-1,4b,8,8-tetra-
methylphenanthrenene- (12). To a solution of 11 (992 mg, 2.73
mmol) in dry CH3NO2 (20 mL) at -15 °C was added BF3‚Et2O
(2.40 mL, 7 equiv), and the resulting reaction solution was stirred
under N2 at -15 °C for 4 h. The reaction solution was diluted with
saturated NaHCO3 (150 mL) and extracted with CH2Cl2 (150 mL
× 3). The organic layers were collected, washed with brine, dried
with MgSO4, and concentrated. Flash chromatographic separation
(5-10% EtOAc in hexanes) afforded the diastereoisomeric mixture
3,4-Dimethoxy-2-methylphenylacetic Acid (10). To a solution
of 9 (3.21 g, 16.0 mmol) in dry DMF (35 mL) was added KCN

830 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Zuniga et al.
of isopropyl ester as a colorless oil (619 mg) in 62% yield. These
diastereoisomers were not further separated since one of the chiral
centers was removed during the decarboxylation step. H NMR
at room temperature for 2 h. The reaction solution was quenched
with brine (100 mL) and extracted with degassed EtOAc (100 mL
× 2). The organic layers were collected, dried with MgSO4, and
concentrated. Flash chromatographic separation (25-35% EtOAc
in hexanes) under N2 afforded product 4 as a brown oil (5.4 mg)
1
(CDCl3, 300 MHz) for the diastereomeric mixture: δ 6.73 (s, 1H),
3.83 (s, 3H), 3.73 (s, 3H), 3.69 (s, 3H), 3.66 (m, 1H), 2.33-2.23
(m, 1H), 2.02 (s, 3H), 1.79-1.17 (m, 11H), 0.94-0.88 (m, 6H).
13C NMR (CDCl3, 75 MHz) observed: δ 177.1, 151.3, 147.1, 145.3,
131.0.3, 123.9, 106.6, 105.9, 60.4, 55.7, 52.3, 48.6, 46.6, 45.2, 44.2,
41.7, 41.3, 39.7, 39.0, 38.0, 33.6, 33.2, 33.0, 25.2, 25.1, 24.7, 24.0,
21.7, 19.5, 19.4, 12.3, 12.1. ESI-MS calcd for C22H33O4 (M + H+),
361.24; found, 361.24.
1
in 54% yield. H NMR (CDCl3 and CD3OD, 300 MHz): δ 6.64
(s, 1H), 3.53 (br s, 2H), 3.30 (d, J ) 21.3 Hz, 1H), 3.21 (d, J )
21.3 Hz, 1H), 2.39 (s, 1H), 2.17-2.14 (m, 1H), 2.01 (s, 3H), 1.63-
1.53 (m, 4H), 1.35-1.30 (m, 1H), 1.24 (s, 3H), 1.02 (s, 3H), 0.97
(s, 3H). 13C NMR (CDCl3 and CD3OD, 75 MHz): δ 207.5, 138.9,
137.2, 136.8, 118.8, 118.2, 104.0, 58.7, 39.6, 38.8, 36.7, 35.1, 28.9,
28.6, 21.2, 17.7, 15.0, 7.6. ESI-MS calcd for C18H25O3 (M+),
288.17; found, 288.09.
To a solution of the resulting ester (617 mg, 1.70 mmol) in
ethanol (20 mL) was added 3 g crushed NaOH pellets, and the
resulting mixture was refluxed for 3 h. The reaction solution was
acidified with 2.5 N HCl and extracted with CH2Cl2 (150 mL ×
3). The organic layers were collected, washed with brine, dried
with MgSO4, and concentrated to afford a diastereoisomeric mixture
4b,5,6,7,8,8a-trans-Hexahydro-4b,8,8-trimethylphenanthre-
nene-2,3-diol (6). To a solution of 14 (100 mg, 0.35 mmol) in
CH2Cl2 (3.0 mL) under N2 was added a solution of BBr3 (1.0 M in
heptane, 1.5 mL). The resulting solution was stirred at room
temperature for 2 h. The reaction solution was quenched with brine
(100 mL) and extracted with EtOAc (100 mL × 2). The organic
layers were collected, dried with MgSO4, and concentrated. Flash
chromatographic separation (25-35% EtOAc in hexanes) under
1
as a viscous oil (432 mg) in 73% yield. H NMR (CDCl3, 300
MHz) for the diastereomeric mixture: δ 6.75 (s, 1H), 3.84 (s, 3H),
3.84 (m, 1H), 3.75 (s, 3H), 2.45-2.17 (m, 2H), 2.11 (s, 3H), 1.89-
1.18 (m, 10H), 0.96-0.94 (m, 6H). 13C NMR (CDCl3, 75 MHz)
observed: δ 182.9, 181.9, 152.1, 151.5, 147.3, 146.9, 145.3, 140.3,
131.1, 125.2, 123.3, 107.2, 106.5, 106.0, 60.5, 60.4, 55.8, 50.3,
48.6, 46.6, 44.8, 44.0, 42.1, 41.8, 41.3, 39.7, 39.0, 38.9, 38.7, 37.9,
37.7, 34.9, 34.4, 33.7, 33.2, 33.0, 32.7, 25.2, 24.6, 24.0, 21.7, 21.7,
19.5, 12.4, 12.0. ESI-MS calcd for C21H31O4 (M + H+), 347.22;
found, 347.22.
To the resulting mixture (398 mg, 1.25 mmol), lead(IV) acetate
(1.20 g, 2.2 equiv) and copper(II) acetate (248 mg, 1.1 equiv), was
added quinoline (15 mL). The resulting dark solution was degassed
under vacuum and then heated under N2 at 140 °C for 3 h. After
cooling to room temperature, quinoline from the resulting residue
was removed by vacuum distillation. The reaction solution was
diluted with 1 N HCl (200 mL) and extracted with CH2Cl2 (150
mL × 3). The organic layers were collected, washed with brine,
dried with MgSO4, and concentrated. Flash chromatographic
separation (5-20% EtOAc in hexanes) afforded product 12 as a
colorless oil (200 mg) in 53% yield. 1H NMR (CDCl3, 300 MHz):
δ 6.68 (d, J ) 2.8 Hz, 1H), 6.64 (s, 1H), 5.95 (d, J ) 2.8 Hz, 1H),
3.86 (s, 3H), 3.75 (s, 3H) 2.26 (s, 3H), 2.19-2.07 (m, 2H), 1.75-
1.50 (m, 4H), 1.24-1.20 (m, 1H), 1.05 (s, 3H), 1.02 (s, 3H), 0.97
(s, 3H). 13C NMR (CDCl3, 75 MHz): δ 151.9, 145.2, 145.0, 128.4,
127.9, 125.2, 124.2, 104.4, 60.5, 55.9, 50.7, 41.2, 38.5, 36.6, 33.0,
32.7, 22.7, 20.1, 19.3, 11.8. ESI-MS calcd for C20H29O2 (M+),
300.21; found, 300.09.
1
N2 afforded product 6 as a brown oil (72 mg) in 80% yield. H
NMR (CDCl3, 300 MHz): δ 6.71 (s, 1H), 6.58 (s, 1H), 6.39 (d,
J ) 8.7 Hz, 1H), 5.90 (d, J ) 8.7 Hz, 1H), 5.14 (br s, 1H), 4.94
(br s, 1H), 2.06 (br m, 2H), 1.72-1.49 (m, 3H), 1.27-1.21 (m,
2H), 1.02 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H). 13C NMR (CDCl3, 75
MHz): δ 142.9, 142.6, 140.9, 128.8, 126.9, 126.7, 113.8, 110.1,
51.3, 41.2, 37.9, 36.3, 33.0, 32.8, 22.7, 20.5, 19.2. ESI-MS calcd
for C17H23O2 (M+), 258.16; found, 258.08.
4b,5,6,7,8,8a-trans-Hexahydro-1,4b,8,8-tetramethylphenan-
threnene-2,3-diol (7). To a solution of 13 (32 mg, 0.11 mmol) in
CH2Cl2 (2.0 mL) under N2 was added a solution of BBr3 (1.0 M in
heptane, 1.0 mL). The resulting solution was stirred at room
temperature for 2 h. The reaction solution was quenched with brine
(100 mL) and extracted with EtOAc (100 mL × 2). The organic
layers were collected, dried with MgSO4, and concentrated. Flash
chromatographic separation (25-35% EtOAc in hexanes) under
1
N2 afforded product 7 as a brown oil (20 mg) in 68% yield. H
NMR (CDCl3 and CD3OD, 300 MHz): δ 6.61 (d, J ) 3.0 Hz,
1H), 6.53 (s, 1H), 5.86 (d, J ) 2.8 Hz, 1H), 3.51 (br s, 2H), 2.18
(s, 3H), 2.00-1.97 (m, 2H), 1.67-1.42 (m, 3H), 1.21-1.15 (m,
1H), 0.98 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H). 13C NMR (CDCl3
and CD3OD, 75 MHz): δ 143.2, 141.6, 140.3, 127.9, 124.5, 124.2,
120.9, 106.9, 50.9, 41.2, 38.0, 36.5, 32.9, 32.7, 22.5, 20.2, 19.2,
11.3. ESI-MS calcd for C18H25O2 (M+), 272.17; found, 272.09.
General Synthetic Procedures to cis-Analogues 3 and 5. To
a solution of thiol-protected citral (as a 1:1 cis/trans mixture) in
dry THF (20 mL) at -40 °C (acetonitrile/dry ice bath) was slowly
added a solution of 1.05 equiv n-BuLi (1.6 M in hexanes) under
N2. The resulting reaction solution was stirred at -40 °C for 1 h,
and then a solution of 0.95 equiv 15a or 15b in dry THF (10 mL)
was added. After 4 h, the reaction flask was transferred into a
desiccator and kept in a freezer (-25 °C) for 48 h. The reaction
solution was quenched with brine (100 mL) and extracted with ether
(100 mL × 2). The organic layers were collected, dried with
MgSO4, and concentrated. The desired products 16a,b were purified
as a cis/trans diastereoisomeric mixture by a flash column separa-
tion. Further separation of the isomers was not carried out because
the subsequent cyclization step afforded diastereoisomerically pure
cis-compounds.
4b,5,6,7,8,8a-trans-Hexahydro-2,3-dimethoxy-1,4b,8,8-tetra-
methylphenanthren-9(10H)-one (13). To a solution of 12 (270
mg, 0.899 mmol) in CH2Cl2 (10 mL) at 0 °C was added
m-chloroperoxybenzoic acid (70-75%, 443 mg, 1.5 equiv). The
resulting reaction solution was stirred at 0 °C under N2 for 3 h.
The reaction solution was quenched with a solution of 5% Na2S2O3
and extracted with CH2Cl2 (100 mL × 2). The organic layers were
collected, washed with brine, dried with MgSO4, and concentrated.
The residue was dissolved in CHCl3 (10 mL), and trifluoroacetic
acid (0.2 mL) was added. The resulting reaction solution was stirred
under N2 for 18 h and then diluted in CH2Cl2 (100 mL). The organic
solution was washed with saturated NaHCO3 and brine, dried with
MgSO4, and concentrated. Flash chromatographic separation (10-
15% EtOAc in hexanes) afforded product 13 as a colorless oil (119
1
mg) in 41% yield. H NMR (CDCl3, 300 MHz): δ 6.76 (s, 1H),
3.85 (s, 3H), 3.76 (s, 3H), 3.46 (d, J ) 21.4 Hz, 1H), 3.37 (d, J )
21.4 Hz, 1H), 2.44 (s, 1H), 2.30-2.28 (m, 1H), 2.10 (s, 3H), 1.72-
1.64 (m, 4H), 1.43-1.38 (m, 1H), 1.32 (s, 3H), 1.18 (s, 3H), 1.06
(s, 3H). 13C NMR (CDCl3, 75 MHz): δ 209.7, 151.4, 145.8, 144.7,
129.7, 124.0, 105.8, 62.4, 60.5, 56.0, 43.5, 42.8, 41.1, 39.1, 33.0,
32.7, 25.1, 21.8, 19.0, 12.1. ESI-MS calcd for C20H29O3 (M + H+),
317.21; found, 317.20.
To a solution of 16a,b in MeOH/H2O (9:1, 25 mL) was added
1.1 equiv HgO and HgCl
2. The resulting reaction solution was
stirred at room temperature for 12 h. The reaction solution was
diluted with CH2Cl2, and the precipitation was filtered through
Celite. The solution was washed with brine, collected, dried with
MgSO4, and concentrated. The desired products 17a,b were purified
as a cis/trans diastereomeric mixture by a flash column separation.
To a solution of 17a,b in dry CH3NO2 (10 mL) at room
temperature was added 10 equiv BF3‚Et2O, and the resulting
reaction solution was stirred under N2 for 2 h. The reaction solution
was diluted with saturated NaHCO3 (150 mL) and extracted with
4b,5,6,7,8,8a-trans-Hexahydro-2,3-dihydroxy-1,4b,8,8-tetra-
methylphenanthren-9(10H)-one (4). To a solution of 13 (11 mg,
0.35 mmol) in CH2Cl2 (2.0 mL) under N2 was added a solution of
BBr3 (1.0 M in heptane, 1.5 mL). The resulting solution was stirred

DNA OxidatiVe Damage by Terpene Catechols
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 831
CH2Cl2 (150 mL × 3). The organic layers were collected, dried
with MgSO4, and concentrated. The desired compounds 18a,b were
purified as a diastereoisomeric pure product by a flash column
separation.
To a solution of 18a,b in CH2Cl2 (2.0 mL) under N2 was added
a solution of BBr3 (1.0 M in heptane, 1.0 mL). The resulting solution
was stirred at room temperature for 2 h. The reaction solution was
quenched with brine (100 mL) and extracted with EtOAc (100 mL
× 2). The organic layers were collected, dried with MgSO4, and
concentrated. The desired products 3 and 5 were purified by a flash
column separation.
in 62% yield (5-10% EtOAc in hexanes). 1H NMR (CD3CN, 400
MHz): 1H NMR (CDCl3, 300 MHz): δ 6.76 (s, 1H), 3.87 (s, 3H),
3.77 (s, 3H), 3.38 (s, 2H), 2.47-2.43 (m, 1H), 2.10 (s, 3H), 2.08
(s, 1H), 1.58-1.52 (m, 2H), 1.34-1.28 (m, 3H), 1.03 (s, 3H), 0.93
(s, 3H), 0.36 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 212.6, 151.5,
145.8, 137.1, 130.0, 125.5, 106.0, 66.8, 60.7, 56.1, 42.2, 42.0, 39.1,
36.7, 34.2, 33.9, 32.2, 22.6, 19.1, 12.2. ESI-MS calcd for C20H29O3
(M + H+), 317.21; found, 317.21.
4b,5,6,7,8,8a-cis-Hexahydro-2,3-dihydroxy-4b,8,8-trimeth-
ylphenanthren-9(10H)-one (3). A colorless oil: 16 mg in 72%
yield (5-20% EtOAc in hexanes). 1H NMR (CDCl3 and CD3OD,
300M Hz): δ 6.86 (s, 1H), 6.58 (s, 1H), 5.35 (br, s), 5.33 (br, s),
3.59 (d, J ) 23.0 Hz, 1H), 3.39 (d, J ) 23.0 Hz, 1H), 2.39-2.35
(d, J ) 13.9 Hz, 1H), 2.06 (s, 1H), 1.63-1.48 (m, 2H), 1.33-1.22
(m, 3H), 1.02 (s, 3H), 0.92 (s, 3H), 0.36 (s, 3H). 13C NMR (CDCl3
and CD3OD, 75 MHz): δ 214.1, 143.3, 142.4, 133.8, 125.7, 115.2,
111.3, 66.7, 43.6, 42.3, 38.4, 36.5, 34.4, 33.6, 32.3, 22.6, 18.9. ESI-
MS calcd for C17H23O3 (M + H+), 275.16; found, 275.07.
4b,5,6,7,8,8a-cis-Hexahydro-2,3-dihydroxy-1,4b,8,8-tetrameth-
ylphenanthren-9(10H)-one (5). A colorless oil: 18 mg in 82%
yield (25-35% EtOAc in hexanes). 1
2-(3,4-Dimethoxybenzyl)-2-(2,6-dimethylhepta-1,5-dienyl)-1,3-
dithiane (16a). A colorless oil: 1.29 g in 63% yield (10-25%
1
EtOAc in hexanes). H NMR (CDCl3, 300 MHz) for the cis/trans
diastereomeric mixture: δ 6.80-6.77 (m, 3H), 5.40 (s, 1H), 5.15-
5.07 (2 sets of triplet, J ) 6.7 Hz, 1H), 3.83 (s, 6H), 3.27-3.21 (2
sets of singlet, 2H), 2.92-2.78 (m, 4H), 2.49-2.44 (m, 1H), 2.10-
2.05 (m, 5H), 1.80 (2 sets of singlet, 3H), 1.66 (s, 3H), 1.59 (2 sets
of singlet, 3H). 13C NMR (CDCl3, 75 MHz) observed: δ 148.2,
142.8, 142.2, 132.0, 131.9, 128.6, 128.4, 128.2, 127.2, 124.4, 124.2,
123.3, 114.4, 110.5, 55.9, 54.8, 54.0, 46.8, 46.5, 41.8, 32.6, 28.1,
27.9, 26.8, 26.3, 25.9, 25.7, 25.6, 24.7, 17.9, 17.2. ESI-MS calcd
for C22H33O2S2 (M + H+), 393.19; found, 393.19.
2-(3,4-Dimethoxy-2-methylbenzyl)-2-(2,6-dimethylhepta-1,5-
dienyl)-1,3-dithiane (16b). A colorless oil: 1.18 g in 54% yield
(5-25% EtOAc in hexanes). 1H NMR (CDCl3, 300 MHz) for the
cis/trans diastereomeric mixture: δ 7.05 (d, J ) 8.5 Hz, 1H), 6.67
(d, J ) 8.5 Hz, 1H), 5.42 (s, 1H), 5.05 (t, J ) 5.4 Hz, 1H), 3.80
(s, 3H), 3.72 (s, 3H), 3.27-3.21 (2 sets of singlet, 2H), 2.97-2.87
(m, 2H), 2.74-2.66 (m, 2H), 2.25 (s, 3H), 2.10-1.96 (m, 6H),
1.66 (s, 3H), 1.62 (s, 3H), 1.58 (s, 3H). 13C NMR (CDCl3, 75 MHz)
observed: δ 151.7, 147.2, 142.6, 141.9, 132.4, 131.8, 131.5, 128.5,
127.9, 127.7, 127.6, 127.5, 124.6, 124.4, 108.7, 60.2, 56.0, 55.7,
55.2, 43.7, 43.4, 42.1, 32.0, 28.2, 27.9, 26.6, 26.2, 26.0, 25.9, 25.8,
25.6, 24.9, 17.9, 16.5, 13.4. ESI-MS calcd for C23H35O2S2 (M +
H+), 407.21; found, 407.21.
1-(3,4-Dimethoxyphenyl)-4,8-dimethylnona-3,7-dienyl-2-
one (17a). A colorless oil: 125 mg in 62% yield (5-20% EtOAc
in hexanes). 1H NMR (CDCl3, 300 MHz) for the cis/trans
diastereomeric mixture: δ 6.80-6.71 (m, 3H), 6.07 (s, 1H), 5.10-
5.00 (2 sets of multiplet, 1H), 3.84 (s, 6H), 3.62-3.60 (2 sets of
multiplet, 2H), 2.57 (t, J ) 7.8 Hz, 1H), 2.12-2.09 (5H), 1.83 (s,
1H), 1.64 (s, 3H), 1.56 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ
198.6, 197.9, 160.8, 160.2, 149.1, 148.1, 132.7, 132.3, 127.7, 123.9,
123.1, 122.5, 121.8, 112.7, 111.5, 56.0, 51.3, 41.5, 34.2, 31.8, 26.9,
26.2, 26.0, 25.9, 22.8, 19.7, 17.9, 14.3. ESI-MS calcd for C19H27O3
(M + H+), 303.20; found, 303.08.
H NMR (CDCl3, 300 MHz):
δ 6.74 (s, 1H), 5.28 (s, 1H), 5.19 (s, 1H), 3.38 (s, 2H), 2.38-2.34
(d, J ) 15.0 Hz, 1H), 2.09 (s, 3H), 2.08 (s, 1H), 1.60-1.48 (m,
2H), 1.33-1.22 (m, 3H), 0.99 (s, 3H), 0.92 (s, 3H), 0.37 (s, 3H).
13C NMR (CDCl3, 75 MHz): δ 213.1, 142.1, 140.5, 133.7, 125.4,
122.7, 108.6, 66.7, 42.2, 42.0, 38.6, 36.6, 34.1, 32.2, 22.6, 22.5,
19.0, 11.8. ESI-MS calcd for C18H25O3 (M + H+), 289.18; found,
289.09.
Formation of Diterpenone Quinone and QM under Organic
Conditions. The oxidation of catechols was achieved using Ag2O
as an oxidant. After vigorous stirring at ambient temperature for
20 min, the solids were removed by filtration with a 0.2 µm filter
(Acrodisc, 13 CR, PTFE). The resulting yellow solution was
confirmed as analytically pure QM or quinone by 1H and 13C NMR
analysis.
Diterpenone QM 19 (5 mg in 0.8 mL CDCl3). 1H NMR (CDCl3,
300 MHz): δ 6.90 (s, 1H), 6.52 (s, 1H), 6.50 (s, 1H), 6.31 (s, 1H),
2.25 (m, 1H), 2.23 (s, 1H), 1.61-1.44 (m, 2H), 1.35-1.23 (m,
3H), 1.18 (s, 3H), 0.95 (s, 3H), 0.61 (s, 3H). 13C NMR (CDCl3, 75
MHz): δ 201.8, 182.1, 155.6, 149.9, 143.2, 132.3, 122.1, 111.0,
66.9, 41.9, 41.5, 36.3, 35.8, 35.5, 31.0, 24.7, 18.5.
Diterpenone quinone 20 (5 mg in 0.8 mL CDCl3). 1H NMR (300
MHz): δ 6.54 (d, J ) 2.2 Hz, 1H), 6.52 (d, J ) 2.2 Hz, 1H), 6.20
(s, 1H), 6.12 (s, 1H), 2.11 (s, 1H), 2.03 (d, J ) 12.1 Hz, 1H),
1.75-1.72 (m, 2H), 1.58-1.54 (m, 1H), 1.26-1.14 (m, 2H), 1.21
(s, 3H), 1.03 (s, 3H), 1.01 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ
181.4, 180.7, 163.8, 145.0, 143.4, 127.3, 123.2, 121.6, 51.0, 41.5,
40.4, 35.7, 33.5, 32.9, 22.5, 22.5, 18.6.
DNA Oxidative Damage of Diterpenone Catechols 2-7 in
the Presence of CuCl2. The 30-mer oligonucleotide of the DNA
target was radiolabeled with 32P-phosphate at the 5′-position by
T4 polynucleotide kinase (New England Biolabs, MA) according
to the manufacturer’s instructions. Hybridization of complementary
strands was achieved by heating a solution of oligonucleotides (0.5
µM each, 0.28 µCi/µL) in a 90 °C water bath and then cooling to
room temperature slowly. A series of reaction solutions containing
compounds 2-7 were prepared, and the DNA lesion was initiated
by the addition of the catechols. The final reaction solutions (10
µL each) contained 0.25 µM duplex DNA (0.07 µCi/µL), 10 mM
phosphate buffer (pH 7.0), 1 mM MgCl2, CuCl2 (40 µM),
compounds 2-7 (0, 10, 20, 30, and 40 µM, respectively), and 10%
acetonitrile. The resulting reaction solutions were incubated at 37
°C for 12 h. A portion of the reaction solutions (0.1 µCi) was mixed
with formamide and directly separated by a 20% denatured PAGE
for the investigation of direct DNA cleavage. The piperidine
treatment was achieved by mixing the reaction solutions with a
10% piperidine in water (100 µL) and then heating at 90 °C for 20
min. The resulting solutions were lyophilized, and the residues were
dissolved in 90% formamide loading buffer. Each reaction solution
(0.15 µCi) was separated by 20% denatured PAGE and analyzed
by gel image analysis software. The percentage of DNA damage
1-(3,4-Dimethoxy-2-methylphenyl)-4,8-dimethylnona-3,7-di-
enyl-2-one (17b). A colorless oil: 156 mg in 50% yield (25-35%
1
EtOAc in hexanes). H NMR (CDCl3, 300 MHz) for the cis/trans
diastereomeric mixture: δ 6.84 (d, J ) 8.3 Hz, 1H), 6.72 (d, J )
8.3 Hz, 1H), δ 6.80-6.71 (m, 3H), 6.07 (s, 1H), 5.10-5.00 (2 sets
of multiplet, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 3.64 (s, 2H), 2.60 (t,
J ) 7.4 Hz, 1H), 2.14-2.11 (m, 8H), 1.84 (s, 1H), 1.66 (s, 3H),
1.57 (s, 3H). 13C NMR (CDCl3, 75 MHz) observed: δ 198.6, 197.8,
160.4, 159.7, 151.8, 147.6, 132.6, 132.2, 131.4, 127.4, 126.0, 123.9,
123.1, 123.0, 122.5, 109.6, 60.4, 55.8, 49.3, 41.4, 34.1, 26.9, 26.2,
26.0, 25.9, 25.8, 19.7, 17.9, 17.8, 12.5. ESI-MS calcd for C20H29O3
(M + H+), 317.21; found, 317.21.
4b,5,6,7,8,8a-cis-Hexahydro-2,3-dimethoxy-4b,8,8-trimeth-
ylphenanthren-9(10H)-one (18a). A colorless oil: 120 mg in 44%
yield (25-35% EtOAc in hexanes). 1H NMR (CDCl3, 300 MHz):
δ 6.84 (s, 1H), 6.58 (s, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.64 (d,
J ) 23.0 Hz, 1H), 3.54 (d, J ) 23.0 Hz, 1H), 2.47-2.42 (m, 1H),
2.09 (s, 1H), 1.58-1.53 (m, 2H), 1.34-1.31 (m, 3H), 1.05 (s, 3H),
0.94 (s, 3H), 0.36 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 212.7,
148.1, 147.7, 133.7, 126.3, 111.6, 107.8, 66.7, 56.4, 56.1, 43.9,
42.3, 38.6, 36.6, 34.4, 33.6, 32.3, 22.6, 19.1. ESI-MS calcd for
C19H27O3 (M + H+), 303.20; found, 303.21.
4b,5,6,7,8,8a-cis-Hexahydro-2,3-dimethoxy-1,4b,8,8-tetra-
methylphenanthren-9(10H)-one (18b). A colorless oil: 131 mg

832 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Zuniga et al.
Scheme 3. Synthesis of trans-Terpene Catechol Analogues
Scheme 4. Synthesis of cis-Terpene Catechol Analogues
also implied a different mechanism in the cyclization of
conjugated polyenes, possibly involving an enolate intermediate.
The synthesis is summarized in Schemes 3 and 4.
Synthesis of trans-catechols 2, 4, and 6-7 was accomplished
through the ester-derivative cyclization pathway. Catechol 2 was
obtained as reported previously (17), and the synthesis of 3 was
carried out similarly except starting from a methylated car-
boxylic acid 10 (Scheme 3). For acid 10, 2,3-dimethoxytoluene
was first converted to benzyl chloride 9 (29), followed by
cyanide addition and then hydrolysis in NaOH solutions (30)
to the desired acid in an overall yield of 27%. Subsequent
synthetic steps to 4 included coupling with geranyl chloride,
ester formation, BF3‚OEt2 cyclization, hydrolysis, decarboxyla-
tion, oxidation, and deprotection, which were carried out
similarly as described for catechol 2 (17). We found that all of
the steps afforded reasonable yields except that a methyl ester
11, instead of the isopropyl ester, must be used in the polyene
cyclization, possibly to reduce the steric bulkiness around the
aromatic ring. Catechols 6-7 were obtained directly from the
deprotection of the alkene intermediates 13-14 from the
synthesis of analogues 2 and 4 (Scheme 3).
was calculated based on the amount of the originally radiolabeled
DNA band versus the total amount of radioactive DNA using the
quantitative analysis software provided by the imager manufacturer.
The DNA oxidative damage by free radicals using Fe3+-EDTA and
H2O2 was carried out according to the published protocol (28). The
investigation of the effect by radical scavengers and copper chelators
on the DNA damage with terpenone 2 and Cu2+ was carried out
similarly as described above. The final concentrations for compound
2 and CuCl2 were 20 µM each. The concentrations of radical
scavengers and chelators used were 5% for ethanol, 0.1 M for
mannitol, 0.1 M for sodium formate, 5% for DMSO, 1.5 unit/10
µL for superoxide dismutase, 1.5 unit/10 µL for catalase, 50 µM
for bathacuproine, and 0.1 M for methional, respectively. The
piperidine treatment was carried out after incubating the reaction
mixtures at 37 °C for 10 h. All the experiments were repeated at
least three times independently.
DNA Oxidative Damage of Diterpenone Catechols 2-7 in
the Presence of CuCl2 and NADH. A series of reaction solutions
containing compounds 2-7 were prepared similarly as described
above. The duplex DNA solution was mixed with a solution of
CuCl2 and NADH first, and then followed by the addition of
catechol solutions. The final reaction solutions (20 µL each)
contained 0.25 µM duplex DNA (0.07 µCi/µL), 10 mM phosphate
buffer (pH 7.0), 1 mM MgCl2, 100 µM NADH, CuCl2 (5 µM),
compounds 2-7 (0, 0.5, 1, 2, and 5 µM, respectively), and 5%
acetonitrile. The resulting reaction solutions were incubated at 37
°C for 6 h. The piperidine treatment and subsequent PAGE
separation were carried out similarly as described above. All the
experiments were repeated at least three times independently.
cis-Analogues 3 and 5 were obtained using the conjugated
polyene synthetic approach (Scheme 4). First, dimethoxylbenzyl
chloride 15 was coupled with n-BuLi and 1,3-dithiol-protected
citral (1:1 cis/trans isomers) at -40 °C to compound 16 as a
diastereoisomeric mixture in 54-63% yield (29). The thiol
groups were then removed with HgCl2 and HgO in a 9:1
MeOH-water solution in 55-62% yield (31). The resulting
polyene 17 as a diastereoisomeric mixture was cyclized in the
presence of BF3‚OEt2, affording the cis-isomers diastereoiso-
meric selectively. The cis-conformation of the resulting diter-
penone 18 was confirmed by 2-D NMR NOESY analysis (see
Supporting Information). Finally, methyl ethers of 15 were
removed with BBr3 to the desired cis-catechol analogues (17).
During the synthesis, we noticed that compounds with the
Result and Discussion
Synthesis of Terpene Catechols. The cis- and trans-catechols
2-7 were synthesized diastereoselectively using a Lewis-acid-
catalyzed polyene cyclization. The trans-isomers 2, 4, and 6-7
were formed selectively through cyclization of ester derivatives
of polyenes such as derivative 11 (Scheme 3), as reported
previously (7, 17). Interestingly, we found that if one of the
double bonds of polyenes was conjugated with a carbonyl group
as in compound 17 (Scheme 4), cyclization afforded the cis-
isomer dominantly. This unique diastereoisomeric selectivity
not only enabled us to obtain cis-conformational analogues, but
1
same conformation had similar H NMR chemical shifts of
methyl groups on the C-ring (Scheme 1). For example, the
chemical shifts of three methyl groups of cis-isomers 3, 5, and
18 are at approximately 1.02, 0.93, and 0.36 ppm, respectively,
while those of trans-isomers 6, 7, and 12-14, are between 1.32
and 0.97 ppm. These unique chemical shifts may be helpful to
confirm the stereo-conformation of terpene isomers.
Quinone Methide and o-Quinone Formation upon Oxida-
tion. Both p-QM and o-quinone were obtained upon oxidation

DNA OxidatiVe Damage by Terpene Catechols
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 833
Scheme 5. Formation of QMs or Quinones of Terpene
Catechols upon Oxidation
versus the relative flat trans-conformation of 2 (Figure 1), which
may effectively prevent polymerization upon concentration.
o-Quinone 20 was formed exclusively in the oxidation of
catechol 6 and also confirmed by both 1H NMR and 13C NMR
analysis. The conjugated o-quinone 16 remained unchanged over
3 days by 1H NMR analysis, however, polymerized upon
concentration. Therefore, these results indicated that p-QMs
formations are unique to homo-conjugated catechols as the
oxidation products, while o-quinones are formed preferably for
simple catechols or conjugated catechols.
DNA Damage by Catechols 2-7 in the Presence of Cu2+
.
DNA damage was observed by all of catechol analogues in the
presence of Cu2+ to various extents, possibly due to the stereo-,
substitutional, and functional group effects. More importantly,
analysis of DNA damage after piperidine treatment showed an
identical fragment pattern, which is similar to that of the
oxidative damage by H2O2/Fe(III)-EDTA. These results sug-
gested that production of ROS in the Cu(II)-induced oxidation
of catechols is the predominant cause for the observed DNA
damage.
of catechols 3 and 6, respectively (Scheme 5). This was
consistent with our previously reported QM formation and also
confirmed the exclusive formation of quinones from catechol
6 in our design.
Oxidation of cis-catechol 3 with Ag2O in CDCl3 afforded
1
analytically pure p-QM 19, which was confirmed by both H
and 13C NMR analysis (17, 23). In the 1H NMR spectrum, the
signals of benzylic protons of 3 at 3.5 ppm disappeared
completely upon oxidation, while four singlets were observed
between 6.31 and 6.90 ppm (see Supporting Information). Also,
the 13C spectrum showed 8 carbon signals in the region above
100 ppm and 9 below 70 ppm as compared to 7 and 10 of
catechol 3. All of these spectral analyses are consistent with
the exclusive conversion of catechol 3 to p-QM 19 (17, 23).
In contrast to the high reactive p-QM 1 as we previously
reported, p-QM 19 of the cis-catechol 3 remained unchanged
upon concentration and storage over several weeks at -4 °C.
On the other hand, flash chromatography with silica gel
completely degraded p-QM 19. The exceptional stability of
p-QM 19 can be attributed to its unique bent cis-conformation
For analysis of DNA damage, a 32P-radiolabeled oligonu-
cleotide duplex target was selected as described previously (17)
and treated with catechols 2-7 (10-40 µM) and 40 µM Cu2+
at 37°C for 12 h (Figure 2). Gel electrophoresis analysis of both
nonpiperidine- and piperidine-treated reactions were carried out
similarly as described previously (17). While the analysis of
nonpiperidine-treated reactions showed direct cleavage of the
DNA strand (see Supporting Information), the analysis of
piperidine-treated ones indicated not only DNA strand cleavage
but also nucleobase depurinations due to oxidation and alky-
lation (13-16, 32). As shown in Figure 2, the extent of DNA
damage after piperidine treatment was concentration-dependent
within each compound yet varied from one another. The
percentage of undamaged DNA was calculated based on the
Figure 2. DNA damage with compounds 2-7 in the presence of Cu2+ after piperidine treatment. The concentration of duplex DNA was 0.25 µM
in 10 mM phosphate buffer (pH 7.40) and 1 mM MgCl2. aThe percentage was calculated as the average from three independent experiments using
the amount of undamaged radiolabeled DNA versus the total amount of radioactive DNA. The average standard deviation was 5%.

834 Chem. Res. Toxicol., Vol. 19, No. 6, 2006
Zuniga et al.
amount of originally labeled DNA versus the total amount of
radioactive fragments in the phosphor-imaging analysis. On the
basis of the percentage of undamaged DNA, trans-catechols 2
and 6 have higher amount of DNA damage than other analogues
at the same concentrations. When comparing catechol 2 to 4,
the additional methyl group on the A-ring decreased the amount
of DNA damage significantly, and moderated decrease was
observed in the case of catechols 6 to 7 (Figure 2). On the other
hand, the methyl effect was not observed on the cis-analogues
3-4, while the amount of DNA damage by cis-catechol 3 was
much lower than that by trans-catechol 2, probably due to its
unique bent cis-conformation. These results implied that the
extent of DNA damage with catechols 2-7 was possibly due
to steric effects on interactions between Cu2+ and catechol (15,
16), although a more thorough computational study is needed
to verify this.
Figure 3. Identical fragment pattern was observed in the DNA damage
by Cu2+-induced oxidation of catechols versus that by hydroxyl radical,
revealing ROS as the DNA damaging mechanism. DNA fragment
patterns were obtained with (a) 2 at 10 µM; (b) Fe3+-EDTA and H2O2.
Despite the different extent of DNA damage, all of the
catechols 2-7 induced a similar fragment pattern as shown in
Figure 2. When compared with the same percentage of DNA
damage, almost identical patterns were observed (Figure 2), that
is, DNA damage with 30 µM of 3, 10 µM of 2, and 20 µM of
6. The similarity of the fragment pattern indicated that DNA
damage was due to a common mechanism in the presence of
Cu2+. On the basis of the Cu2+-induced oxidation mechanism,
the common product from these analogues were ROS generated
through the disproportion of Cu(II)/(I) redox cycle (13-16).
Thus, the fragment pattern from Cu2+-induced oxidation of
catechols was compared to that of DNA damage by H2O2/Fe3+
-
EDTA and was found to be identical (Figure 3). These results
suggested that the observed DNA fragment pattern with
catechols 2-7 was due to the nonselective DNA damage by
radical species (28). In addition, the mechanism of catechol
oxidation with Cu2+ has been well-established over recent years
using radical scavenge studies and EPR analysis (13-16), which
indicated many ROS were involved including O2•-, Cu(I)-OOH,
HO•, and H2O2. The DNA damage mechanism was proposed
Figure 4. The effect of radical scavengers and copper chelators on
DNA damage by diterpenone 2 (20 µM) and Cu2+ (20 µM) after
piperidine treatment. The concentration of duplex DNA was 0.25 µM
in 10 mM phosphate buffer (pH 7.40) and 1 mM MgCl2. The
concentrations for radical scavengers and chelators were 5% for ethanol,
0.1 M for mannitol, 0.1 M for sodium formate, 5% for DMSO, 1.5
unit/10 µL for superoxide dismutase, 1.5 unit/10 µL for catalase, 50
µM for bathacuproine, and 0.1 M for methional, respectively.
Figure 5. Enhanced DNA damage in the presence of 100 µM NADH and 5 µM Cu2+ with compounds 2-7 at concentrations of 0.5-5 µM after
a
piperidine treatment. The concentration of duplex DNA was 0.25 µM in a 10 mM phosphate buffer (pH 7.40) and 1 mM MgCl2. The percentage
was calculated using the amount of undamaged radiolabeled DNA versus the total amount of radioactive DNA, and the average standard deviation
was 5%. All the reactions were repeated three independent times.

DNA OxidatiVe Damage by Terpene Catechols
Chem. Res. Toxicol., Vol. 19, No. 6, 2006 835
to occur via the in situ released HO• from the DNA-Cu(I)-
OOH complex. This Cu(I)-radical complex mechanism was
further verified by the study of the effect of radical scavengers
and copper chelators on the observed DNA damage with
diterpenone 2 and Cu2+ (Figure 4). Consistent to reported studies
(13-16), free HO• radical scavengers including ethanol, man-
nitol, formate, and DMSO had no effects on the extent of DNA
damage. While in contrast, catalase, bathocuproine, and me-
thional completely quenched the observed oxidative damage on
DNA. These results further supported that the oxidation of
catechol derivatives through disproportionation of Cu(II)/Cu(I)
is different from the free radical mechanism of Fe(III)-H2O2,
although similar DNA damaging patterns were observed.
Scheme 6. The Redox Cycle of trans-Terpene Catechol/
Quinone through Cu2+ and NADH
of catechols regardless of structural differences and oxidation
products. The observed substantial DNA damage by alkene
catechols 6-7 in the presence of NADH revealed their potential
detrimental effects in cells upon oxidation. These results
provided a fundamental basis for future biological studies on
the oxidative metabolism of terpene catechols and may further
contribute the understanding of neoplastic development by
natural catechol carcinogens.
Although nucleobase adducts could be formed with reactive
QMs or quinones in the oxidation process, this was not observed
under the conditions studied on the basis of the similar DNA
fragment pattern by catechols 2-7 versus that by H2O2/Fe3+
-
EDTA. An alternative reaction pathway for the formed QMs
and quinones could be competitive water addition, which was
observed in the nucleobase alkylation with QMs (18, 20).
Therefore, these results suggested that production of ROS was
the dominant mechanism for DNA damage in the Cu2+-induced
oxidation, regardless of the structural difference of catechols
and subsequent oxidation products as QM or quinone.
Acknowledgment. We thank the financial support (startup
fund) from Virginia Commonwealth University. We also thank
John B. Mangrum for his help on the mass analysis.
DNA Damage by Catechols 2-7 with NADH and Cu2+
.
In the presence of NADH, DNA damage by the Cu2+-induced
oxidation of catechols 2-7 occurred to a higher extent at lower
concentrations. With alkene catechols 6-7, the extent of DNA
damage was enhanced substantially, implying that NADH
enabled the generation of ROS through the redox cycle of
catechols/quinones.
Supporting Information Available: NMR spectra of com-
pounds 3-20 and the gel image of direct DNA cleavage by
compounds 2-7 (24 pages). This material is available free of charge
via the Internet at http://pubs.acs.org.
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21). After incubation for 6 h at 37 °C, reactions were treated
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