Synthesis of the N2-deoxyguanosine adduct of the potent dietary mutagen IQ

Article abstract of DOI:10.1021/ol047851m

(Chemical Equation Presented) After bioactivation, the potent dietary mutagen 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) reacts with DNA to give two regioisomeric adducts of deoxyguanosine. The synthesis of the minor N ²-adduct has been achi

Full text of DOI:10.1021/ol047851m

ORGANIC
LETTERS
Synthesis of the N2-Deoxyguanosine
Adduct of the Potent Dietary Mutagen
IQ
2004
Vol. 6, No. 26
4985-4988
1
James S. Stover and Carmelo J. Rizzo*
Department of Chemistry, Center in Molecular Toxicology, and
Vanderbilt Institute of Chemical Biology, Vanderbilt UniVersity,
VU Station B 351822, NashVille, Tennessee 37235-1822
c.j.rizzo@Vanderbilt.edu
Received October 18, 2004
ABSTRACT
After bioactivation, the potent dietary mutagen 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) reacts with DNA to give two regioisomeric adducts
of deoxyguanosine. The synthesis of the minor N2-adduct has been achieved utilizing the Buchwald
reaction as the key step.
−Hartwig palladium-catalyzed N-arylation
In the early 1940s, the simple aromatic amines 2-amino-
fluorene (AF) and its N-acetyl analogue (AAF) were
developed as pesticides. They were subsequently found to
be potent animal carcinogens and never used as intended.2
Although there has never been significant environmental
exposure to AF or AAF, exposure to other aromatic amines
is of concern. For example, 2,6-dimethylaniline, a widely
used industrial chemical and a constituent of cigarette smoke,
has been identified as a rodent carcinogen.3 Recently, a strong
link has been demonstrated between bladder cancer and
exposure to 4-aminobiphenyl, a common component of
permanent hair dyes.4,5 The process of cooking meats
produces a wide array of highly mutagenic heterocyclic
amines, including 2-amino-3-methyl-imidazo[4,5-f]quinoline
(IQ, 1).6 Because of their source, a wide cross-section of
the population is exposed to these dietary mutagens and there
is significant interest in their chemistry and biology. Some
members of this family are among the most potent agents
tested in the Ames assay.7
Aromatic amines are procarcinogens and require metabolic
activation in order to covalently modify DNA.8,9 This
involves the initial cytochrome P450 N-oxidation to the
corresponding hydroxylamine followed by O-esterification
by N-acetyl transferase (NAT) or a related enzyme. Sol-
volysis then gives the arylnitrenium ion, which is the ultimate
carcinogenic species. Arylnitrenium ions are ambident elec-
trophiles and react with nucleophiles at both nitrogen and
carbon. Their reaction with DNA is highly selective for
deoxyguanosine. Furthermore, reaction of the arylnitrenium
ion at nitrogen gives the C8-adduct, while reaction at carbon
gives the N2-modified deoxyguanosine. For IQ, both the C8-
and N2-deoxyguanosine adducts have been characterized
(Scheme 1).10,11
(1) This manuscript is dedicated to Professor Amos B. Smith, III, in
celebration of his 60th birthday.
(2) Heflich, R. H.; Neft, R. E. Mutat. Res. 1994, 318, 73-174.
(3) Gonc¸alves, L. L.; Beland, F. A.; Marques, M. M. Chem. Res. Toxicol.
2001, 14, 165-174.
(4) Yu, M. C.; Skipper, P. L.; Tannenbaum, S. R.; Chan, K. K.; Ross,
R. K. Mutat. Res. 2002, 506-507, 21-28.
(6) Turesky, R. J. Drug Metab. ReV. 2002, 34, 625-650.
(7) Sugimura, T. Mutat. Res. 1997, 376, 211-219.
(8) Guengerich, F. P. Drug Metab. ReV. 2002, 34, 607-623.
(9) Novak, M.; Rajagopal, S.; Xu, L. L.; Kazerani, S.; Toth, K.; Brooks,
M.; Nguyen, T. M. J. Phys. Org. Chem. 2004, 17, 615-624.
(5) Turesky, R. J.; Freeman, J. P.; Holland, R. D.; Nestorick, D. M.;
Miller, D. W.; Ratnasinghe, R. D.; Kadlubar, F. F. Chem. Res. Toxicol.
2003, 16, 1162-1173.
10.1021/ol047851m CCC: $27.50
© 2004 American Chemical Society
Published on Web 11/26/2004

Scheme 1
Scheme 2
Lakshman, Johnson, Hopkins and Sigurdsson, and others
demonstrated the utility of the Buchwald-Hartwig reaction
for the synthesis of nucleoside adducts of carcinogens.13,18-32
We have previously reported the synthesis of the C8-
deoxyguanosine adduct of IQ and its subsequent incorpora-
tion into oligonucleotides via phosphoramidite-based solid-
phase synthesis.28,33 The key step was a Buchwald-Hartwig
reaction between a suitably protected 8-bromo-2′-deoxygua-
nosine derivative with IQ. We report here the synthesis of
the N2-deoxyguanosine adduct of IQ, which also uses the
Buchwald-Hartwig reaction as the key step.
Two strategies have been previously employed for the
synthesis of N2-modified deoxyguanosine derivatives utilizing
the Buchwald-Hartwig reaction. The first involved the
reaction of a suitably protected 2-bromopurine riboside with
an arylamine,23-27 while the second strategy utilized the
coupling of the N2-amino group of deoxyguanosine with
haloarenes.19,26,27 To examine both strategies, we synthesized
the protected 5-bromo- and 5-amino-IQ derivatives as shown
in Scheme 2. The electrophilic bromination of IQ was
previously reported to give the desired 5-bromo-IQ (7).34
Protection of the amine as the corresponding benzamide gave
There are multiple lines of evidence suggesting that the
C8- and N2-deoxyguanosine adducts of IQ are repaired at
different efficiencies.6 In feeding studies with rats and
monkeys, it was found that the major DNA lesion 24 h after
a single dose of IQ was the C8-adduct.12 The relative ratio
of the C8- to N2-deoxyguanosine adducts was in the range
of 6:1, which roughly parallels the natural reactivity of the
IQ nitrenium ion toward DNA. However, in chronically
treated monkeys undergoing long-term carcinogen bioassays,
a sharp increase in the proportion of N2-adduct was observed
after 3.6 years. This increase was found in slowly dividing
tissue such as the liver, pancreas, and kidney. There was no
increased persistence of the N2-adduct in colon tissue, which
divides at a high rate. These observations suggest that the
C8-adduct (5) is repaired more efficiently in slow growing
tissue. This has led to speculation that the minor N2-
deoxyguanosine adduct (6) plays a more significant role in
the tumorigenic properties of IQ and other aromatic amines.
The Buchwald-Hartwig palladium-catalyzed N-arylation
reaction has been widely used for the synthesis of modified
nucleosides.13-15 Chida and co-workers used this reaction
as the key step in the total synthesis of the nucleoside
antibiotic spicamycin.16,17 Shortly thereafter, reports by
(18) Lakshman, M. K.; Keeler, J. C.; Hilmer, J. H.; Martin, J. Q. J. Am.
Chem. Soc. 1999, 121, 6090-6091.
(19) De Riccardis, F.; Bonala, R. R.; Johnson, F. J. Am. Chem. Soc.
1999, 121, 10453-10460.
(20) Harwood, E. A.; Sigurdsson, S. T.; Edfeldt, N. B. F.; Reid, B. R.;
Hopkins, P. B. J. Am. Chem. Soc. 1999, 121, 5081-5082.
(21) Harwood, E. A.; Hopkins, P. B.; Sigurdsson, S. T. J. Org. Chem.
2000, 65, 2959-2964.
(22) De Riccardis, F.; Johnson, F. Org. Lett. 2000, 2, 293-295.
(23) Bonala, R. R.; Shishkina, I. G.; Johnson, F. Tetrahedron Lett. 2000,
41, 7281-7284.
(24) Lakshman, M. K.; Gunda, P. Org. Lett. 2003, 5, 39-42.
(25) Johnson, F.; Bonala, R.; Tawde, D.; Torres, M. C.; Iden, C. R. Chem.
Res. Toxicol. 2002, 15, 1489-1494.
(10) Turesky, R. J.; Markovic, J. Chem. Res. Toxicol. 1994, 7, 752-
761.
(26) Chakraborti, D.; Colis, L.; Schneider, R.; Basu, A. K. Org. Lett.
2003, 5, 2861-2864.
(11) Turesky, R. J.; Rossi, S. C.; Welti, D. H.; Lay, J. O.; Kadlubar, F.
F. Chem. Res. Toxicol. 1992, 5, 479-490.
(27) Lakshman, M. K.; Ngassa, F. N.; Bae, S.; Buchanan, D. G.; Hahn,
H.-G.; Mah, H. J. Org. Chem. 2003, 68, 6020-6030.
(28) Wang, Z.; Rizzo, C. J. Org. Lett. 2001, 3, 565-568.
(29) Meier, C.; Gra¨sl, S. Synlett 2002, 802-804.
(30) Gillet, L. C. J.; Scha¨rer, O. D. Org. Lett. 2002, 4, 4205-4208.
(31) Schoffers, E.; Olsen, P. D.; Means, J. C. Org. Lett. 2001, 3, 4221-
4223.
(12) Turesky, R. J.; Box, R. M.; Markovic, J.; Gremaud, E.; Snyderwine,
E. G. Mutat. Res. 1997, 376, 235-241.
(13) Lakshman, M. K. J. Organomet. Chem. 2002, 653, 234-251.
(14) Muci, A. R.; Buchwald, S. L. Top. Curr. Chem. 2002, 219, 131-
209.
(15) Hartwig, J. F. Angew. Chem., Int. Ed. 1998, 37, 2046-2067.
(16) Chida, N.; Suzuki, T.; Tanaka, S.; Yamada, I. Tetrahedron Lett.
1999, 40, 2573-2576.
(17) Suzuki, T.; Suzuki, S. T.; Yamada, I.; Koashi, Y.; Yamada, K.;
Chida, N. J. Org. Chem. 2002, 67, 2874-2880.
(32) Takamura-Enya, T.; Ishikawa, S.; Mochizuki, M.; Wakabayashi,
K. Tetrahedron Lett. 2003, 44, 5969-5973.
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Soc. 2004, 126, 11189-11201.
4986
Org. Lett., Vol. 6, No. 26, 2004

Scheme 3
Scheme 4
8 in good overall yield. Likewise, nitration of IQ gave
5-nitro-IQ (9). Protection of the primary amine and reduction
of the nitro group gave 10.
gave the desired product 15 in 30% yield. When Pd(OAc)2/
BINAP was used as the catalyst and Cs2CO3 as the base,
complete consumption of 11 and a 73% isolated yield of 15
was observed. Catalytic hydrogenation removed the O6-
benzyl group and simultaneously reduced the nitro group to
give 16 in 80% yield. Desilylation of 16 gave the N2-
The Buchwald-Hartwig cross-couplings of protected
5-bromo-IQ (8) with protected guanosine derivative 11 as
well as protected 5-amino-IQ (10) with protected 2-bro-
mopurine (12) were attempted under a number of conditions
(Scheme 3). Unfortunately, we were never able to achieve a
successful cross-coupling to give 13. Other protecting groups
for the primary amine of 7 and 9 were examined but were
also unsuccessful.
Previous synthetic studies that utilized the Buchwald-
Hartwig reaction for the synthesis of N2-arylamine deriva-
tives of deoxyguanosine revealed two important reactivity
trends.13,19,26 The cross-coupling of a haloarene with the N2-
amino group of a suitably protected deoxyguanosine deriva-
tive gave the highest yields when electron-deficient halo-
arenes were used. When a 2-halopurine was used as the
reactant, electron-rich arylamines gave the best yield. With
this in mind, we decided to attempt the cross-coupling of
5-bromo-2-nitro-IQ (14) with protected deoxyguanosine 11.
The nitro group would significantly increase the electron-
deficient nature of the halide (14) and also serve as a masked
amino group.19
1
deoxyguanosine adduct of IQ (6). The H NMR spectrum
of 6 was identical to that previously published.10 In Johnson’s
report on the synthesis of N2-deoxyguanosine adducts derived
from simple arylamines, it was noted that the products were
sensitive to air oxidation and characterized as their N-acetyl
Scheme 5
Oxidation of 5-bromo-IQ with sodium nitrite gave 14 in
71% yield.35 Buchwald-Hartwig cross-coupling of 14 and
11 using Pd2(dba)3/BINAP and NaOtBu in toluene at 80 °C
Figure 1. N2-Deoxygunaosine adduct of a simple arylamine.
Org. Lett., Vol. 6, No. 26, 2004
4987

derivatives (Figure 1).19 We have observed no such sensitiv-
ity for the N2-IQ adduct (6). The products obtained by the
Johnson group were o-phenylenediamine derivatives and are
likely to be more sensitive to oxidation than our example
(6).
The amino group of the IQ moiety must be protected for
incorporation into oligonucleotides by solid-phase synthesis.
This amino group was protected as its p-tert-butylphenoxy-
acetyl derivitive by the reaction of 16 with the proper acid
chloride (Scheme 5). Removal of the silyl protecting group
with fluoride gave N-protected nucleoside 18. This interme-
diate is ideal for the preparation of the corresponding
phosphoramidite.
methods. The synthesis proceeds in five linear steps from
IQ in 29% overall yield. The synthesis of the phosphor-
amidite reagent and the incorporation of the N2-IQ adducts
into oligonucleotides is currently underway in our laboratory.
This will allow for conformational studies of the adducted
oligonucleotide and assessment of differential mutagenicity
and repair efficiency of the C8- and N2-adducts.
Acknowledgment. Partial support of this work was
provided by a center grant from the National Institute of
Environmental Health Sciences (ES000267). J.S.S. acknowl-
edges support from an NIEHS predoctoral traineeship
(ES007028).
In conclusion, we have developed an efficient strategy for
the synthesis of the N2-deoxyguanosine adduct of the dietary
mutagen IQ by employing palladium-catalyzed N-arylation
Supporting Information Available: Experimental pro-
cedures for the preparation of 6 and 18 and copies of all 1H
and 13C NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
(34) Waterhouse, A. L.; Rapoport, H. J. Label. Compds. Radiopharm.
1985, 22, 201-216.
(35) Brooks, M. E., Ph.D. Dissertation, Miami University, Oxford, OH,
2002.
OL047851M
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Org. Lett., Vol. 6, No. 26, 2004