Total synthesis of the aristolochic acids, their major metabolites, and related compounds

Article abstract of DOI:10.1021/tx500122x

Plants from the Aristolochia genus have been recommended for the treatment of a variety of human ailments since the time of Hippocrates. However, many species produce the highly toxic aristolochic acids (AAs), which are both nephrotoxic and carcinogenic. For the purposes of extensive biological studies, a versatile approach to the synthesis of the AAs and their major metabolites was devised based primarily on a Suzuki-Miyaura coupling reaction. The key to success lies in the preparation of a common ring-A precursor, namely, the tetrahydropyranyl ether of 2-nitromethyl-3-iodo-4,5-methylendioxybenzyl alcohol (27), which was generated in excellent yield by oxidation of the aldoxime precursor 26. Suzuki-Miyaura coupling of 27 with a variety of benzaldehyde 2-boronates was accompanied by an aldol condensation/elimination reaction to give the desired phenanthrene intermediate directly. Deprotection of the benzyl alcohol followed by two sequential oxidation steps gave the desired phenanthrene nitrocarboxylic acids. This approach was used to synthesize AAs I-IV and several other related compounds, including AA I and AA II bearing an aminopropyloxy group at position-6, which were required for further conversion to fluorescent biological probes. Further successful application of the Suzuki-Miyaura coupling reaction to the synthesis of the N-hydroxyaristolactams of AA I and AA II then allowed the synthesis of the putative, but until now elusive, N-acetoxy- and N-sulfonyloxy-aristolactam metabolites.

Full text of DOI:10.1021/tx500122x

Article
pubs.acs.org/crt
Total Synthesis of the Aristolochic Acids, Their Major Metabolites,
and Related Compounds
Sivaprasad Attaluri,† Charles R. Iden,† Radha R. Bonala,† and Francis Johnson*,†,‡
‡
†Department of Pharmacological Sciences, and Department of Chemistry, Stony Brook University, Stony Brook, New York
11794-3400, United States
S
* Supporting Information
ABSTRACT: Plants from the Aristolochia genus have been
recommended for the treatment of a variety of human ailments
since the time of Hippocrates. However, many species produce
the highly toxic aristolochic acids (AAs), which are both
nephrotoxic and carcinogenic. For the purposes of extensive
biological studies, a versatile approach to the synthesis of the
AAs and their major metabolites was devised based primarily
on a Suzuki−Miyaura coupling reaction. The key to success
lies in the preparation of a common ring-A precursor, namely,
the tetrahydropyranyl ether of 2-nitromethyl-3-iodo-4,5-
methylendioxybenzyl alcohol (27), which was generated in
excellent yield by oxidation of the aldoxime precursor 26.
Suzuki−Miyaura coupling of 27 with a variety of benzaldehyde 2-boronates was accompanied by an aldol condensation/
elimination reaction to give the desired phenanthrene intermediate directly. Deprotection of the benzyl alcohol followed by two
sequential oxidation steps gave the desired phenanthrene nitrocarboxylic acids. This approach was used to synthesize AAs I−IV
and several other related compounds, including AA I and AA II bearing an aminopropyloxy group at position-6, which were
required for further conversion to fluorescent biological probes. Further successful application of the Suzuki−Miyaura coupling
reaction to the synthesis of the N-hydroxyaristolactams of AA I and AA II then allowed the synthesis of the putative, but until
now elusive, N-acetoxy- and N-sulfonyloxy-aristolactam metabolites.
villages there, a substantial number of the inhabitants suffer
from a debilitating ailment10 characterized by a renal fibrosis
that is followed by a urothelial malignancy in approximately
50% of the cases.8,11 The diseased state was termed Balkan
endemic nephropathy (BEN), but its close identity in both
morphological and clinical patterns to the outbreak in Belgium,
known at the time as Chinese herbs nephropathy (CHN), led
Cosyns12 to suggest that they likely had a similar origin. Both
diseased states are now termed aristolochic acid nephropathy
(AAN).
Worldwide population exposure to aristolochic acids (AAs)
is extensive. In China, where the use of herbal remedies is
pervasive, the number of AAN cases is suspected to be very
large despite a governmental ban on the use of Aristolochia
species. Because of the widespread use of herbal products in
Taiwan, a very large number of cases of AAN have been
reported there recently,13 but patients having AAN-like
symptoms have been seen in many countries, best documented
in Spain,14 the United Kingdom,15 and Japan.16 The
epidemiology, diagnosis, and management of AAN have
INTRODUCTION
■
Various species of Aristolochia have been used as herbal
remedies since the time of Hippocrates to treat a variety of
human conditions and diseases.1 A popular use has been in
childbirth,2 and until around 1980, these herbs were
recommended by physicians, especially in the United Kingdom,
to women at term. In searching for antitumor agents, Kupchan
and Doskovich3 examined aristolochic acid I (AA I, 1), the
principal chemical constituent of Aristolochia indica, but found
that it was ineffective as an anticancer agent in mice. Jackson
and his associates4 found AA I to be nephrotoxic in human
clinical trials, and Pezzuto et al.5 subsequently confirmed these
results but also observed the substance to be mutagenic. In
1993, a report from Vanherweghem and his associates6,7
described an unfortunate event at a Belgian clinic where more
than 1500 women were given pills as part of a slimming
regimen that contained a Chinese herbal product derived from
Aristolochia fangchi. More than one hundred developed a
serious nephropathy characterized as a rapidly progressive,
renal, interstitial fibrosis. Shortly thereafter, Cosyns and his
colleagues8,9 reported that patients suffering from aristolochia-
induced nephropathy were also at risk for urothelial cancer.
This drew attention to a similar nephropathic condition that
was known to be endemic along the banks and tributaries of the
Danube River but particularly in Croatia. In many of the
recently been reviewed by Gokman and Lord in association
̈
with other eminent researchers in the field.17
Received: April 1, 2014
Published: May 30, 2014
© 2014 American Chemical Society
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Aristolochia fangchi contains a mixture of AAs, the suspected
AAN-etiologic agents, whose structures were first identified by
Pailer and his associates.18,19 Using a mixture of AA I (1) and
AA II (2), it proved possible to reproduce the main features of
the disease in rodents,20,21 thus removing any doubts that the
aristolochic acids are responsible for AAN in humans. Evidence
that tumor development also could be ascribed to these acids
rests on the isolation of DNA adducts from renal tissue.22,23
These adducts have been identified24 as 3 and 4 (Figure 1),
of derivatives containing a biological marker, either biotin or a
fluorescent probe.
Figure 2. Chemical structures for synthetic intermediates.
RESULTS AND DISCUSSION
■
Synthetic Chemistry of AA I and AA II, Their
Congeners, Related Metabolic Products, and Protein
Probe Substances. AA I and AA II occur together naturally in
many Aristolochia species and are available commercially as a
mixture. However, they cannot be separated by crystallization,
and their separation by column chromatography is a tedious
procedure. By contrast, the corresponding methyl esters can be
separated chromatographically, but attempts to hydrolyze the
esters returned very poor yields of the parent acids. This
unusual result is a reflection of the steric strain (the aromatic
equivalent of 1,3-allylic strain28) that exists between the nitro
and carboxyl functions and parallels identically the group
compression that exists in 8-nitro-1-naphthoic acid (NPA).
This latter acid cannot be esterified by the standard Fischer−
Speier method, and its methyl ester undergoes substantial
substitution of the nitro group when basic hydrolysis is
attempted. Similarly, attempts to form the acid chloride of NPA
with SOCl2 leads mainly to 8-chloro-1-naphthoic acid after
hydrolysis and lesser amounts of 5,7-dichloronaphthostyril.29
Besides these problems, the positions of the electron-donating
groups already present in the AAs restrict their versatility in
further desirable aromatic substitution reactions. On the basis
of such aberrant chemistry, the possibility of using the naturally
occurring AAs in further synthetic manipulations seemed
limited. We elected to attempt to develop a general de novo
total synthesis of the AAs and their principal metabolites (a) to
allow a broader structure−activity study than would be possible
with the natural substances, (b) to permit the metabolic fate of
these substances to be examined under controlled conditions,
and (c) to probe for proteins involved in the nephrotoxicity of
AA I.
Figure 1. Chemical structures for the aristolochic acids, their DNA
adducts, and selected metabolites.
derived from metabolites of AA I,25 and the corresponding
adducts, 5 and 6, derived from AA II. In both cases, the
adenosine adducts are dominant. All of these adducts appear to
arise by the same metabolic pathways that are followed by most
carcinogenic amines and nitro compounds,26,27 in which the
intermediate N-hydroxyamine metabolites (in the cases under
discussion, the N-hydroxylactams, 7 and 8) are the purported
pro-carcinogens. Surprisingly, in the case of the AAs, the
expected C-8 purine metabolic adducts, which are normally
found with many of the known nitro- and amino-polyaromatic
hydrocarbons, are not observed, and only the products of
adduction at the exocyclic amino groups of these nucleobases
are found.
In this current article, we record the total syntheses of the
naturally occurring AAs I−IV together with a series of
analogues intended to support advanced etiologic studies of
AAN. We discuss some of the difficulties associated with their
chemistry and define why we adopted a “total synthesis” route
rather than employing the natural products. In addition, we
have synthesized the proposed penultimate AA metabolites
(Figure 1), namely, the N-sulfonyloxy AA-lactams as their
pyridinium salts, respectively, 9 and 10, and the corresponding
putative metabolites, the N-acetoxy AA-lactams 39 and 40
(Scheme 6), together with two AAs having a 3-aminopropyloxy
linker at the 7-position, namely 11 and 12 (Figure 2). The
latter are precursors for the preparation (to be reported later)
The synthesis of AA I has previously been reported by
Kupchan and Wormser,30 while the synthesis of AA IV was
achieved by Pailer and his associates.31 Both methods are
similar and rely on a photochemical coupling step that is
impracticable for larger-scale development. In addition, too
many steps are involved, yields at some stages are low (in
particular, the final basic hydrolysis of AA I methyl ester gives
AA I in only 6% yield30), and the route lacks versatility and
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would not be easily adaptable to the synthesis of the 7-
bromoaristolactams 13 and 14 that we saw as the critical
intermediates in our synthetic approach to the DNA adducts
3−6. The work of Castedo and his associates,32 which uses an
internal benzyne reaction to construct the aristolactam
framework, did not seem applicable to the synthesis of the
AAs themselves. Similarly, the methodology, principally evolved
by Couture et al.33,34 for the synthesis of a series of the related,
naturally occurring aristolactams, was not easily adaptable to
the synthesis of the AA metabolites 7−10. Although their
methods are elegant, they are relatively inefficient because (a)
the typical isoindolenone intermediate 15 is frequently
obtained as a mixture of geometric isomers and (b) the
cyclization to give the required penultimate lactam 16 using the
Bu3SnH/AIBN catalyst is problematic on a larger scale. We
envisaged that a more efficient synthetic route (see Scheme 1
dimethylformamide gave 23 in 75% yield.37 The latter was
condensed with cyclohexylamine to give 24 (97% yield), which,
when treated with butyl lithium38 followed by iodine both at
−70 °C, led to the ortho-iodinated compound 25 that was still
contaminated by small amounts of unreacted starting material
24. Whereas treatment of the mixture with hydroxylamine
acetate afforded an impure solid, the pure oxime 26 could be
isolated cleanly by trituration with di-isopropyl ether (54%
overall yield from compound 24). Oxidation of 26 to 27 proved
challenging. Although we tried many of the usual proce-
dures39−44 to achieve this transformation, we were successful
only with the rarely used benzo-peroxo-molybdenum com-
plex,45 which under the neutral conditions described by
Ballisteri et al.46 gave the desired compound 27 in 67% yield.
The overall procedure allowed for the preparation of 27 in ∼50
g batches.
It is worth noting that the more logical use of a carboxylic
acid or ester in place of the protected benzyl alcohol in 19 did
not prove feasible because of the formation of cyclic
benzoxazinone products at the later oxime stage. Similarly,
when we began the synthesis with a methyl group in place of
CH2OH in the sequence, it was not possible to functionalize
the former either by halogenation or oxidation at the
phenanthrene stage. Thus, we employed the protected benzyl
alcohol as a surrogate for the carboxylic acid with the intent of
generating the acid later in the sequence.
Scheme 1
Ring C: Component 21. The required triflates (20b−f) were
prepared from the corresponding phenols by standard methods
or, in the case of the trifluoromethoxy analogue 20f, by the
method of Choshi et al.47 The aminopropyl derivative 20g was
synthesized as illustrated in Scheme 3 by an adaptation of the
for the retrosynthetic approach) might be possible, first by
linking the two principal precursors, namely, 19 and 21, of the
aromatic rings A and C by means of a Suzuki−Miyaura
reaction, which together with a likely sequential condensation
would lead directly to the OH-protected form of phenanthrene
18, avoiding the problem of geometrical isomerism. In any
event, this procedure proved to be both efficient and versatile
for the synthesis of the aristolochic acids and their lactams and
by similar chemistry for the synthesis of their N-hydroxyar-
istolactam metabolites 7−10. The efficient synthesis of a series
of AA-related lactams employing a similar Suzuki−Miyaura
strategy has also been described more recently by Kim et al.35,36
Comprehensive Methods of Synthesis of the Aristolo-
chic Acids. Ring-A: Component 19. The key to our general
synthetic approach lies in the efficient synthesis of an α-
nitrotolyl compound (ring-A precursor) represented generically
by 19. This was realized in the iodo compound 27 whose
synthesis was accomplished in the five steps outlined in Scheme
2. Lithiation of 22 at −75 °C followed by quenching with
a
Scheme 3
a
Scheme 2
a
Reagents: (a) DHP/H+; (b) Tf2O/Pyr; (c) DIBAL; (d) CH3I/
K2CO3; (e) CF3COOH/MeOH; (f) I(CH2)3NHBoc/K2CO3.
procedures of Bajwa and Jennings48 for the unambiguous
preparation of substituted salicylaldehydes and the correspond-
ing benzyl alcohols. Compound 20h was prepared similarly
starting from 2,4-dihydroxybenzaldehyde. The syntheses of the
substituted phenyl-2-formyl boronates (21b−f), apart from the
simplest analogue (21a: R = R1= H) which is commercially
a
Reagents: (a) BuLi; (b) DMF; (c) cyclohexylamine; (d) I2; (e)
H3O+; (f) HONH2/HOAc; (g) peroxomolybdate.
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available, were all carried out by the known Pd-catalyzed/
pinacolato diborane substitution reaction of the corresponding
triflates (20b−f), again according to a procedure by Choshi et
al.47 (Scheme 4). Some of these boronates proved to be
unstable to chromatography and were used directly in the
succeeding Suzuki−Miyaura coupling reaction without further
purification.
KMnO4, etc.) was used. Mostly, the aldehydes 38a−h or
decomposition products were obtained. The conversion was
therefore accomplished in two steps, initially by oxidation to
the aldehydes 38a−h using either activated MnO2 in acetone or
CrO3 in acetic acid, followed by a second oxidation with
sodium chlorite in aqueous DMSO buffered by sodium
dihydrogen phosphate.50 This afforded all of the desired
aristolochic acids (1, 2, and 1c−h) in acceptable overall yields.
The physical data for the synthetic aristolochic acids that are
also known natural products were identical to those reported in
the literature.51 In the cases of 1g and 1h, direct treatment with
HCl generated 1i and 1j as their hydrochloride salts, suitable
for further coupling with a dyestuff or a fluorescent probe.
Synthesis of Putative Metabolites of the Aristolochic
Acids. As noted previously, carcinogenic aromatic amines and
nitro compounds generally share a common metabolic pathway
that ultimately leads to DNA adduction followed by mutations
that can lead to tumor induction. In the case of the nitro-
polyaromatic hydrocarbons, this involves phase I enzymatic
reduction to a hydroxylamine, which is subsequently activated
by phase II metabolism either by O-acetylation or O-
sulfonylation. Solvolysis of these derivatives to generate the
“ultimate carcinogens”, namely, the intermediate nitrenium/
carbenium ions, in the presence of DNA then leads to covalent
adduction.26,27 In the case of the aristolochic acids, this
translates into the pathway shown in Scheme 6.
a
Scheme 4
a
Reagents: B(Pin)2/Pd(dppf)2Cl2/KOAc.
Coupling of Ring Components A and C: Aristolochic
Acid Synthesis. The coupling/condensation of the aldehydic
borate esters 21a−h with the phenylnitromethane 27 (Scheme
5) proceeded smoothly in all cases in aqueous dioxane at 95 °C
using tetrakis(triphenylphosphine) palladium as the catalyst in
the presence of cesium carbonate. Yields of products 36a−h
varied from 56 to 77% after chromatographic purification over
silica gel. Removal of the tetrahydropyranyl group to give the
alcohols 37a−h was accomplished almost quantitatively by
heating to boiling in methanol containing 0.1% sulfuric acid,
then cooling immediately to room temperature.49 However, all
attempts to oxidize the very poorly soluble alcohols directly to
the carboxylic acids 1, 2, and 1c−h failed, no matter which
common oxidant (e.g., CrO3, chromyl chloride, NaOCl,
The synthesis of the metabolic derivatives of the AAs follows
a pathway similar to that described above for the AAs, again
using a Suzuki−Miyaura reaction to establish the phenanthrene
framework. This required the synthesis of 46b as the basis of
ring-A and was accomplished as shown in the first three steps of
Scheme 7. The coupling reactions of 46b with either 21a or
21b to obtain 7 or 8 in good yield were accomplished directly
by means of the Pd-based catalyst, [1,1-bis-
(diphenylphosphino)ferrocenyl]dichloro-palladium(II), in the
presence of potassium carbonate in dioxane/water at 100 °C.
a
Scheme 5
a
Reagents: (a) Pd(PPh3)4/Cs2CO3, 95 °C; (b) H3O+; (c) MnO2 or CrO3/Pyr; (d) NaClO2/NaH2PO4/DMSO/H2O.
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a
Scheme 6
a
Reagents: (a) NQOI; (b) NAT; (c) SULT1B1; (d) electrophile, e.g., H3O+; (e) DNA.
a
Scheme 7
a
Reagents: (a) NaClO2/NaH2PO4; (b) (CH3O)2SO2/NaOH; (c) Br2/HOAc; (d) NBS; (e) NH2OH·HCl/NaOH; (f) TBDMSCl/Pyr; (g)
d(dppf)Cl2/K2CO3/dioxane/H2O, 100 °C, 16 h; (h) SO3/Pyr; (i) Ac2O/Pyr.
Surprisingly, not only was the Suzuki−Miyaura coupling
accomplished between the A and C ring components, but it was
accompanied by the condensation of the aldehyde with the
methylene of the lactam to form ring-B. Additionally, the
TBDMS protecting group was lost from the hydroxylactam in a
highly desirable cascade sequence that gave only one major
product in each case. The completion of the syntheses of the
four purported AA metabolites 9, 10, 39, and 40 was
straightforward by treatment of 7 or 8 with either sulfur
trioxide/pyridine or acetic anhydride/pyridine, respectively.
The hydroxylactams and the corresponding O-acetyl derivatives
proved to be very stable in the solid state, in aqueous solution
buffered to pH 6 and in 1 N hydrochloric acid for 18 h with no
loss of integrity. However, the O-sulfonylpyridinium salts
decompose in aqueous media (pH 6) but can be stored in the
solid state almost indefinitely at −20 °C. Biological studies with
these substances will be reported separately.52
CONCLUSIONS
■
In summary, we have developed new, efficient, and total
syntheses of the naturally occurring AAs I−IV together with a
series of metabolites and AA-analogues. These include the
proposed penultimate AA metabolites involved in DNA adduct
formation, namely, the N-sulfonyloxy AA-lactams derived from
AA I and AA II and the corresponding putative metabolites, the
N-acetoxy AA-lactams, together with two AAs having a 3-
aminopropyloxy linker at the 7-position. The latter are
precursors for the preparation of derivatives containing a
biological marker, either biotin or a fluorescent probe. The total
synthesis of these substances in quantity has not only allowed a
broad array of biological studies to be carried out but also has
provided the chemical basis for the site-specific incorporation of
AA metabolites into oligomeric DNA of any designated
sequence (unpublished material).
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(8) Cosyns, J.-P., Jadoul, M., Squifflet, J.-P., Van Cangh, P.-J., and van
Ypersele de Strihou, C. (1994) Urothelial malignancy in nephropathy
due to Chinese herbs. Lancet 344, 188.
ASSOCIATED CONTENT
* Supporting Information
Details of the synthetic methods; NMR and MS data for the
characterization of each intermediate or final product in the
synthesis of AA or related compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
(9) Cosyns, J.-P., Jadoul, M., Squifflet, J.-P., Wese, F.-X., and van
Ypersele de Strihou, C. (1999) Urothelial lesions in Chinese-herb
nephropathy. Am. J. Kidney Dis. 33, 1011−1017.
́
(10) Grollman, A. P., and Jelakovic, B. (2007) Role of environmental
toxins in endemic (Balkan) nephropathy. J. Am. Soc. Nephrol. 18,
2817−2823.
AUTHOR INFORMATION
Corresponding Author
*Tel: 631-632-8866. Fax:631-632-7394. E-mail: francis.
johnson@stonybrook.edu.
Funding
This work was supported by grant P01ES004068 from the
National Institute of Environmental Health Sciences and by a
generous financial gift from Henry and Marsha Laufer.
■
(11) Cosyns, J.-P., Goebbels, R.-M., Liberton, V., Schmeiser, H. H.,
Bieler, C. A., and Bernard, A. M. (1998) Chinese herbs nephropathy-
associated slimming regimen induces tumours in the forestomach but
no interstitial nephropathy in rats. Arch. Toxicol. 72, 738−743.
(12) Cosyns, J.-P., Jadoul, M., Squifflet, J.-P., De Plaen, J. F., Ferluga,
D., and van Ypersele de Strihou, C. (1994) Chinese herbs
nephropathy: a clue to Balkan endemic nephropathy? Kidney Int. 45,
1680−1688.
(13) Grollman, A. P. (2013) Aristolochic acid nephropathy:
Harbinger of a global iatrogenic disease. Environ. Mol. Mutagen. 54,
1−7.
Notes
The authors declare no competing financial interest.
́
(14) Pena, J. M., Borras, M., Ramos, J., and Montoliu, J. (1996)
̃
ACKNOWLEDGMENTS
Rapidly progressive interstitial renal fibrosis due to a chronic intake of
a herb (Aristolochia pistolochia) infusion. Nephrol. Dial. Transplant. 11,
1359−1360.
■
We thank Irina Zaitseva for her assistance in obtaining
analytical data and HPLC purification of selected compounds,
and we are grateful to Dr. Arthur Grollman for his advice and
continued interest in this research.
(15) Cronin, A. J., Maidment, G., Cook, T., Kite, G. C., Simmonds,
M. S. J., Pusey, C. D., and Lord, G. M. (2002) Aristolochic acid as a
causative factor in a case of Chinese herbal nephropathy. Nephrol.,
Dial., Transplant. 17, 524−525.
ABBREVIATIONS
■
́
(16) Arlt, V. M., Stiborova, M., and Schmeiser, H. H. (2002)
Aristolochic acid as a probable human cancer hazard in herbal
AA, aristolochic acid; AAN, aristolochic acid nephropathy;
BEN, Balkan endemic nephropathy; Boc, tert-butoxycarbonyl;
BPin, boron pinacolate; B(Pin)2, bis-pinacolato diboron; BuLi,
butyl lithium; CHN, Chinese herbs nephropathy; DHP,
dihydropyran; DIBAL, di-isobutylaluminum hydride; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; NAT, N-
acetyl transferase; NPA, 8-nitro-1-naphthoic acid; NQO1,
NAD(P)H dehydrogenase, quinone 1; Pyr, pyridine;
SULT1B1, sulfotransferase 1B1; TBDMS, tert-butyldimethyl-
silyl; Tf2O, triflic anhydride; THP, tetrahydropyran
remedies: a review. Mutagenesis 17, 265−277.
́
(17) Gokmen, M. R., Cosyns, J.-P., Arlt, V. M., Stiborova, M.,
̈
Phillips, D. H., Schmeiser, H. H., Simmonds, M. S. J., Cook, H. T.,
Vanherweghem, J.-L., Nortier, J. L., and Lord, G. M. (2013) The
epidemiology, diagnosis, and management of aristolochic acid
nephropathy: a narrative review. Ann. Int. Med. 158, 469−477.
(18) Pailer, M., and Schleppnik, A. (1957) Natural plant substances
with a nitro group. II. Constitution of aristolochic acid II. Monatsh.
Chem. 88, 367−387.
(19) Pailer, M. (1960) Naturally Occurring Nitrogen Compounds, in
Fortschr. Chem. Org. Naturstoffe (Zechmeister, L., Ed.) Vol. 18, pp 55−
82, Wein Springer Verlag.
(20) Mengs, U., Lang, W., and Poch, J.-A. (1982) The carcinogenic
action of aristolochic acid in rats. Arch. Toxicol. 51, 107−119.
(21) Mengs, U. (1988) Tumor induction in mice following exposure
to aristolochic acid. Arch. Toxicol. 61, 504−505.
(22) Schmeiser, H. H., Bieler, C. A., Wiessler, M., van Ypersele de
Strihou, C., and Cosyns, J.-P. (1996) Detection of DNA adducts
formed by aristolochic acid in renal tissue from patients with Chinese
herbs nephropathy. Cancer Res. 56, 2025−2028.
(23) Grollman, A. P., Shibutani, S., Moriya, M., Miller, F., Wu, L.,
Moll, U., Suzuki, N., Fernandes, A., Rosenquist, T., Medverec, Z.,
Jakovina, K., Brdar, B., Slade, N., Turesky, R. J., Goodenough, A. K.,
Rieger, R., Vukelic, M., and Jelakovic, B. (2007) Aristolochic acid and
́ ́
the etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci.
U.S.A. 104, 12129−12134.
(24) Pfau, W., Schmeiser, H. H., and Wiessler, M. (1990)
Aristolochic acid binds covalently to the exocyclic amino group of
purine nucleotides in DNA. Carcinogenesis 11, 313−319.
(25) Krumbiegel, G., Hallensleben, J., Mennicke, W. H., Rittmann,
N., and Roth, H. J. (1987) Studies on the metabolism of aristolochic
acids I and II. Xenobiotica 17, 981−991.
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