Identification of a reduction product of aristolochic acid: Implications for the metabolic activation of carcinogenic aristolochic acid

Article abstract of DOI:10.1021/np100296y

Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated both in endemic nephropathy in the Balkan region and in ailments caused by ingestion of herbal remedies. Aristolochic acids are metabolized to active intermediates that bind to DNA. In this study, reduction of aristolochic acid I with zinc in acetic acid afforded a new product that was characterized as 9-methoxy-7-methyl-2H-1,3-oxazolo[5′,4′-10,9] phenanthro[3,4-d]-1,3-dioxolane-5-carboxylic acid, designated as aristoxazole, along with the expected aristolactam I. This new compound is a condensation product of aristolochic acid and acetic acid that may be related to the aristolochic acid-DNA adducts. The proposed mechanism of formation of aristoxazole involves nucleophilic attack of acetic acid on the nitrenium ion of aristolochic acid I. On the basis of these studies, a route to the metabolic activation of aristolochic acids and formation of adducts with DNA in in vitro systems is proposed and discussed.

Full text of DOI:10.1021/np100296y

J. Nat. Prod. 2010, 73, 1979–1986
1979
Identification of a Reduction Product of Aristolochic Acid: Implications for the Metabolic
Activation of Carcinogenic Aristolochic Acid
Horacio A. Priestap,*,† Carlos de los Santos,‡ and J. Martin E. Quirke§
Department of Biological Sciences, Florida International UniVersity, 11200 Southwest 8th Street, Miami, Florida 33199, United States,
Department of Pharmacological Sciences, Stony Brook UniVersity, School of Medicine, 100 Nicolls Road, Stony Brook,
New York 11794, United States, and Department of Chemistry and Biochemistry, Florida International UniVersity,
11200 Southwest 8th Street, Miami, Florida 33199, United States
ReceiVed May 4, 2010
Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated both in endemic
nephropathy in the Balkan region and in ailments caused by ingestion of herbal remedies. Aristolochic acids are
metabolized to active intermediates that bind to DNA. In this study, reduction of aristolochic acid I with zinc in acetic
acid afforded a new product that was characterized as 9-methoxy-7-methyl-2H-1,3-oxazolo[5′,4′-10,9]phenanthro-
[3,4-d]-1,3-dioxolane-5-carboxylic acid, designated as aristoxazole, along with the expected aristolactam I. This new
compound is a condensation product of aristolochic acid and acetic acid that may be related to the aristolochic acid-DNA
adducts. The proposed mechanism of formation of aristoxazole involves nucleophilic attack of acetic acid on the nitrenium
ion of aristolochic acid I. On the basis of these studies, a route to the metabolic activation of aristolochic acids and
formation of adducts with DNA in in vitro systems is proposed and discussed.
Aristolochic acids are carcinogenic and nephrotoxic nitroarenes
that occur in Aristolochia species, with aristolochic acids I and II
(AAI and AAII, 1 and 2, respectively; for numbering, see structure
1) usually being the most abundant of these compounds. Ingestion
of AAs is implicated in the type of renal fibrosis known as “Chinese
herbs nephropathy” or “Balkan endemic nephropathy”.1-9 A report
on the carcinogen aristolochic acid was recently issued by the U.S.
Department of Health and Human Services.10
conjugates of glucuronic or sulfuric acids. Furthermore, covalent
adducts of DNA such as (deoxyguanosin-N2-yl)aristolactam I (dG-
AAI) (7), (deoxyadenosin-N6-yl)aristolactam I (dA-AAI) (8), (deox-
yguanosin-N2-yl)aristolactam II (dG-AAII) (7a), and (deoxyadenosin-
N6-yl)aristolactam II (dA-AAII) (8a) (Scheme 1) were identified
in the renal tissue of patients that ingested herbal preparations
containing AAI and AAII.5-7 Interestingly, only adducts formed
at C-9 (C-7 in an alternative numbering)5,7,11 of the aristolochic
acids are known. There are no reports of adduct formation at the
C-10 nitrogen that usually occurs with nitroaromatic compounds.
The accepted mechanism for the reductive transformations of
AAs into DNA adducts is shown in Scheme 1.5,7,11 It was proposed
that the aristolactam-nitrenium ion 4 is the ultimate carcinogenic
species that binds to the amine group of purine nucleotides (7, 7a,
8, and 8a) or is hydrolyzed to the corresponding 9-hydroxyaristo-
lactam I (6). Note that in previous reports a different numbering
system was used in which this compound was named 7-hy-
droxyaristolactam.11
In view of the importance of the reduction of AAs with respect
to genotoxicity, we reinvestigated the reduction of AAI using zinc
in acetic acid. As a result, we report the identification of a new
reduction product from AAI, namely 9-methoxy-7-methyl-2H-1,3-
oxazolo[5′,4′-10,9]phenanthro[3,4-d]-1,3-dioxolane-5-carboxylic acid,
which is named aristoxazole (15). We discuss its mechanism of
formation and the possibility that there is a nitrenium ion-based
pathway leading to adduct formation, which is complementary to
that proposed by Pfau et al.11
The toxicity of AAs is due to the formation of active intermedi-
ates during the detoxification process. The crucial step in the
generation of DNA-reactive and mutagenic metabolites is the
reduction of the nitro group of AAs. In rats and other mammals,
the major metabolic pathway involves reduction of AAI (1) and
AAII (2) to aristolactams [5 and 5a (Scheme 1)]. In addition,
O-demethylation of AAI (1) and aristolactam I (5), as well as
oxidation at C-8 of AAII (2), is observed. All of these metabolites
are detected in urine and feces either in their original form or as
Results and Discussion
Identification of Aristoxazole. Previous studies have estab-
lished that AAs underwent reduction to either the corresponding
aristolactam or a complex mixture of uncharacterized products.12
In this study, AAI was reduced with zinc powder because this is a
well-established route for the reduction of nitro groups. The zinc
reduction of AAI (1) in boiling acetic acid yielded the expected
aristolactam I (5) as the major product. However, high-performance
liquid chromatography (HPLC) analyses revealed the presence of
a minor reduction product, subsequently identified as aristoxazole
(15) [for the sake of simplicity, the numbering of the atoms matches
that of AAI (1)]. When AAI was reduced with Zn in acetic acid
between 60 and 118 °C, aristolactam I (5) and aristoxazole (15)
* To whom correspondence should be addressed. E-mail: Priestap@
fiu.edu. Phone: (305) 348-0375.
† Department of Biological Sciences, Florida International University.
‡ Stony Brook University.
§ Department of Chemistry and Biochemistry, Florida International
University.
10.1021/np100296y  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/08/2010

1980 Journal of Natural Products, 2010, Vol. 73, No. 12
Priestap et al.
Scheme 1. Metabolic Activation and DNA Adduct Formation of AAI (1) and AAII (2)a
a For 1 and 3-8, R ) OCH3; for 2 and 3a-8a, R ) H.
Table 1. 1H NMR and 13C NMR Data for Aristoxazole (15) and
Aristoxazole Methyl Ester in DMSO-d6 (δ in parts per million,
J in parentheses in hertz)
were obtained in a ca. 3:1 ratio. The reaction proceeds at room
temperature (25 °C), but the aristolactam I:aristoxazole ratio changes
to 6:1. Aristoxazole (15, MW 351, C19H13NO6) is a stable
compound. It cannot be reduced further when treated under the
same conditions.
aristoxazole (15)
15 methyl ester
a
position
δH
δC
δH
The molecular formula indicates that two carbon atoms were
added to AAI during the formation of aristoxazole. These were
supplied by acetic acid because the reduction was conducted in
this solvent. The UV spectrum shows a strong absorption at 251
nm typical of the phenanthrene nucleus. The possibility that
aristoxazole was an N-acetyl derivative related to aristolactam I
was discounted for several reasons. Evidence of an intact carboxylic
acid group was provided by chemical behavior, the detection of a
fragment ion at m/z 306 (M - 44) under electron impact, the IR
spectrum (carbonyl stretch at 1668 cm-1), and formation of a methyl
ester with diazomethane. The 1H NMR spectrum (Table 1) provided
limited data because the compound contains only a small number
of hydrogens. The presence of a broad singlet at ca. δH 13, which
exchanged with D2O, gave further confirmation of the carboxylic
acid moiety. The doublet at δH 8.74, the triplet at δH 7.62, the singlet
at δH 7.49, and the doublet at δH 7.34 were assigned as the aryl
hydrogens at C-5, C-6, C-2, and C-7, respectively, on the basis of
the splitting patterns and comparison of the chemical shifts with
those of AAI.13 The expected singlet for H-9 (ca. δH 7.20) was
absent, as well as the signal for a probable NH lactam proton (ca.
δH 10.70). The singlets at δH 6.38 and 4.04 were assigned to the
methylenedioxy and methoxy groups, respectively. An extra methyl
group was observed at δH 2.69. These results and structural
1
2
3
4
4a
4b
5
6
7
8
8a
9
10
10a
127.7
109.1
144.4
144.3
114.1
125.0
119.4
127.1
108.9
153.5
111.8
142.6
133.2
117.3
161.6
170.1
101.8
56.0
7.50 s (6.5)
7.57 s
8.74 d (8.4)
7.62 t (8.4)
7.34 d (8.0)
8.80 d (8.4)
7.70 t (8.4)
7.42 d (8.0)
12
1-COOH
3,4-CH2O2
8-OMe
12-Me
1-COOMe
13.00 s (broad)
6.38 s
4.04 s
6.43 s
4.08 s
2.73 s
3.94 s
2.69 s
14.3
a Assignments of signals with similar chemical shifts may be
reversed.
considerations suggested the phenanthroxazole structure 15 for the
unknown compound, the methyl group being attributed to the C-12
methyl substituent.

Identification of a Reduction Product of Aristolochic Acid
Journal of Natural Products, 2010, Vol. 73, No. 12 1981
Figure 1. Calculated ball-and-wire structures of aristolic acid and aristoxazole.
The proton-decoupled 13C NMR spectrum (Table 1) supported
structure 15. The spectrum was composed of 19 signals, seven of
which appeared above δC 130. Four of these signals were assigned
by comparison with those of AAI14 as follows: δC 170.1 (carboxyl
group), δC 153.4 (C-8), and δC 144.3 and 144.2 (C-3 and C-4 or
vice versa). The remaining three downfield signals at δC 161.6,
142.6, and 133.2 were assigned as C-12, C-9, and C-10 of the
benzoxazole unit, respectively, based on a comparison with the
corresponding carbons of isosalviamine C (16) that have chemical
shifts of δC 162.3, 143.0, and 133.0, respectively.15 Further support
for the oxazole ring is provided by the signal at δC 14.3, whose
chemical shift matches very well with that of the corresponding
methyl substituent in 2-methylbenzoxazole (δC 14.4) and natural
products containing this moiety (δC 14.9-15.1).15 Equally impor-
tant, this signal is much further upfield than the methyl carbons of
acetyl groups on aryl substrates such as acetanilide (δC 24.1), phenyl
acetate (δC 21.1), and acetophenone (δC 25.7).
will generate the oxazole unit after several steps. Thus, aristoxazole
is a derivative of an intermediate reduction product of AAI. In
contrast, aristolactam I (5) represents the final product from the
complete reduction of the nitro group of AAI.
Reduction of nitro arenes to amines using Zn occurs via the
nitroso and hydroxylamine intermediates. Each reduction step occurs
by a series of single-electron transfers (SETs) in which an electron
from the Zn and a proton are transferred to the species being
reduced.18 Thus, the formation of the N-hydroxyarylamine 9, and
the aryl amine 10, which are probable intermediates in the formation
of aristoxazole (15) and aristolactam I (5), is thought to occur via
SET reactions (Scheme 2). Both amines 9 and 10 can readily cyclize
to form the more stable aristolactam, 5, either directly or via the
postulated N-hydroxylactam, 3. Thus, formation of aristolactam I
from the reduction process is entirely expected. Furthermore, the
stability of the lactam makes it an unlikely precursor to aristoxazole,
indicating that the pathway must diverge prior to the reduction that
leads to aristolactam.
The UV spectrum of aristoxazole shows a remarkable resem-
blance to that of aristolic acid [19 (Figure 1)] (λmax values of 254.5,
296.2, 319sh, 327.1, 355.0, and 374.5 nm), which indicates that
these compounds have similar chromophores. The two minor bands
of aristoxazole at 363 and 382 nm are ∼8 nm bathochromically
shifted from those of the desnitro AAI, a shift that can be attributed,
in part, to the presence of the oxazole ring at C-9 and C-10. On
the basis of the comparison of the λmax values for benzene (254
nm) and 2-methylbenzoxazole (271 and 277 nm),16 a bathochromic
shift of ∼20 nm would have been expected. The smaller batho-
chromic shift observed with aristoxazole is attributed to steric
compression by crowding of the substituents at the peri 8, 9, 10,
and 1 positions.17 Molecular modeling of the aristoxazole revealed
distortion within the phenanthrene ring (Figure 1), presumably as
a means of reducing steric compression. Support for this proposal
was obtained from the modeling of aristolic acid, which lacks the
oxazole ring. The data revealed that the phenanthrene ring of
aristolic acid was essentially planar (Figure 1). In summary,
chemical and spectroscopic data support the proposed structure 15
for aristoxazole in which the oxazole unit is fused to the C-9-C-
10 bond of the phenanthrene moiety.
More than 100 years ago, Bamberger reported that N-phenyl-
hydroxylamine rearranged in a sulfuric acid solution to give
4-aminophenol.19 The key features of this reaction are the O-
protonation of the NOH group followed by N-O bond cleavage
and loss of water to form an intermediate nitrenium ion.20,21 The
studies mentioned above suggest that formation of aristoxazole (15)
during reduction of AAI with Zn in acetic acid involves formation
of the corresponding N-hydroxylamine and a nitrenium ion as the
reactive intermediate species.
It was important to consider whether the aristoxazole was formed
by a variation of the reactions of the nitrenium ion, 4, of Scheme
1, which was proposed for the activation of AAs in biological
systems. However, we were unable to account for the formation of
aristoxazole via this pathway. The stumbling block is that hydrolysis
of the lactam structure, 3, in Scheme 1, which is required to generate
the N-hydroxylamine 9, the expected initial precursor of aristox-
azole, would not be possible under the reaction conditions. This
topic is discussed further in Probable Routes of Aristoxazole
Formation.
In light of these difficulties, it was necessary to propose an
alternative pathway, which is shown in Scheme 2. Before discussing
the most reasonable pathway to aristoxazole, we needed to examine
the possibility that aristoxazole formation occurred by direct
heterolysis of the N-O bond of the N-hydroxylamine 9 to give
the nitrenium ion 12 prior to the formation of the lactam 3 or
Considerations about the Formation of Aristoxazole.
Reduction of AAI with Zn in acetic acid produces aristolactam I
(5) accompanied by smaller amounts of aristoxazole. Overall, the
formation of aristoxazole from AAI involves the reduction of the
nitro group and the insertion of an acetoxy group at C-9, which

1982 Journal of Natural Products, 2010, Vol. 73, No. 12
Priestap et al.
Scheme 2. Proposed Mechanism for the Formation of Aristoxazole (15)a
a With the exception of structures 1, 5, and 15, none of the structures in this scheme have been isolated. Thus, it is not possible to determine their bioactivities. The
bioactivities of structures 1 and 5 are well-established.5,7,11,25
Table 2. Density Function Calculationsa of Likely Intermediates
reduction (Scheme 2). This conversion of N-hydroxylamine 9 into
to Aristoxazole and Related Compounds
nitrenium ion 12 seems unlikely because the acetic acid medium
compound energy (arbitrary units) compound energy (arbitrary units)
is insufficiently strong to drive the transformation of 9 into 12.
Even if it occurs, it is probable that it would be very slow, which
would mean that spontaneous formation of lactam 3 or reduction
to 10 would be by far the predominant process. Although the 9-to-
12 conversion cannot be ruled out, an alternative and more
reasonable mechanism is shown in Scheme 2. In this mechanism,
formation of aristoxazole is proposed to occur via the formation of
an oxazinone 11.
1
3
5
9
-1235.9836
-1085.5347
-1010.3526
-1161.9774
-1086.8029
11
13
14
15
-1085.5324
-1314.7125
-1314.7325
-1238.2741
10
a Calculations were conducted at the B3LYP 6-311+G** level.
(Table 2), may occur rapidly and synchronously because formation
of the lactam by reaction of the C-1 carboxyl group and the C-10
amino group in 14 was not observed.
The N-hydroxyaristolactam, 3, was not detected directly in this
synthesis, which is not altogether surprising because this compound
has also not been isolated as a product of other chemical or
enzymatic reductions of AAI. Intriguingly, the analogous N-
hydroxyaristolactam of aristolochic acid II is a stable compound
that has been characterized in plants (see Are N-Hydroxyaristo-
lactams Toxic?).
Support for the formation of 11 is provided by the known
cyclization of the oximes of 2-acylbenzoic acids to form 2,3-
benzoxazin-1-ones.22 A similar reaction also occurs during the
reduction of o-nitrobenzoic acid with tin or zinc, leading to the
formation of 2,1-benzisoxazolin-3-one following cyclization of the
reduction intermediate, o-hydroxylaminobenzoic acid.23 Addition-
ally, the final condensation between the amino and acetoxy groups
in adduct 14 to give the oxazole ring resembles the reaction of
It is assumed that the 9-hydroxylamine, 9, can be stabilized by
spontaneous cyclization. The carboxyl group of 9 can undergo an
addition-elimination reaction with the hydroxylamine unit to form
either the lactam 3 by reaction at the nitrogen or the oxazinone 11
by reaction at the oxygen (pathways a and b in Scheme 2). Also,
the nucleophilic addition-elimination reaction of a hydroxylamino
oxygen atom with a carboxylic acid has been documented.22,23 It
is proposed that part of the N-hydroxylamine 9 is converted into
the oxazinone 11 instead of being transformed into either lactam 3
or amine 10 by further reduction. In contrast to N-hydroxylamine
9, oxazinone 11 could readily ionize to give nitrenium ion 12, which
could be attacked by the available nucleophiles such as acetic acid
and water. The 9-acetoxy adduct 13 could then undergo aromati-
zation and dehydration to give aristoxazole (15). Like the nitroso
derivative of AAI (not shown), N-hydroxyarylamine, 9, arylamine,
10, and oxazinone, 11, would be too reactive to be either isolated
or detected among the reaction products. Similarly, it is proposed
that transformation of 13 into 15 via the more stable isomer, 14

Identification of a Reduction Product of Aristolochic Acid
Journal of Natural Products, 2010, Vol. 73, No. 12 1983
2-aminophenols with carboxylic acids, which is usually employed
to prepare oxazole derivatives.24
Formation of aristoxazole is discussed further in connection with
problems concerning the mechanisms of aristolochic acid activation
and formation of adducts (see Probable Routes of Aristoxazole
Formation).
acyltransferases are involved in these processes.27,28 The N-
hydroxylamines can undergo N-O bond cleavage under mildly
acidic conditions; however, for this reaction to be biologically
relevant, a previous activation step involving formation of reactive
esters is necessary to render the N-O bond labile enough for
cleavage. There is strong evidence that the sulfuric or carboxylic
acid esters of the resulting N-hydroxylamines are among the more
important carcinogenic metabolites because in aqueous solution they
undergo heterolysis of the N-O bond to yield the arylnitrenium
ions.28-32 It is likely that the N-hydroxylamine 3 is activated as a
sulfate or acyl ester prior to the formation of the nitrenium ion 4
because it was demonstrated that expression of the human sul-
fotransferase SULT1A1 in bacterial and mammalian target cells
enhances the mutagenicity of AAs.33
Molecular Mechanics Calculations. In view of the fact that
most of the compounds that are postulated in Scheme 2 were not
isolated, we conducted molecular mechanics density function
calculations at the B3LYP 6311+G** level. The energies of the
calculated structures are summarized in Table 2. The resultant model
of aristoxazole shows a distortion within the phenanthrene skeleton
(Figure 1) that would explain the smaller than expected bathochro-
mic shifts in its UV spectrum (Identification of Aristoxazole). The
calculations indicated that compound 14 was more stable than its
isomer, 13, which would be expected. Calculations also showed
that the N-hydroxyaristolactam, 3, was only slightly more stable
than the isomeric oxazinone, 11. This result was unexpected because
the 8-demethoxy analogue, 3a, of the N-hydroxylactam, 3, is a stable
compound that was isolated from plants (see Are N-Hydroxyaris-
tolactams Toxic?). We repeated the calculations at different levels
of sophistication, but in all cases, the energies of the two compounds
were similar. Nevertheless, we have included the data (Table 2)
because it is the first time that such information has been reported
for aristolochic acid and related compounds. The bond lengths, bond
angles, and molecular dipole moments for the compounds that are
listed in Table 2 are given in the Supporting Information.
Mechanism of Aristolochic Acid Activation via the N-
Hydroxyaristolactam and Formation of DNA Adducts. The
mechanism for metabolic activation of AAs and DNA adduct
formation proposed by Pfau et al. is shown in Scheme 1 in which
the key features are the formation of the N-hydroxyaristolactam 3
and the aristolactam-nitrenium ion 4.5,7,11 The cyclic nitrenium ion
4 with its delocalized positive charge was suggested to be the
ultimate carcinogen. Formation of the AA adduct occurs by reaction
of the electrophilic C-9 of the nitrenium ion with the nucleophilic
exocyclic amino group of purine nucleotides in DNA. While adducts
of AA reduction intermediates with DNA (7, 7a, 8, and 8a) and
lactams 5 and 5a have been characterized, the reactive species that
covalently binds to DNA and their precursors remain unknown.
Attempts to isolate the N-hydroxyaristolactam (3) were unsuccess-
ful.11 However, support for the proposed mechanism was obtained
by the generation of DNA adducts from both aristolactam I (5)
and N-chloroaristolactam II.25,26 Isolation of 9-hydroxyaristolactam
I (6) from enzymatic reduction of AAI also provided indirect
evidence of the existence of the cyclic hydroxamic acid intermediate
3.11
Although the proposed mechanism explains much of what is
observed, some factors in the formation of DNA adducts remain
unclear. For example, in vitro experiments revealed that AAs readily
form DNA adducts under conditions in which it would be
impossible to activate the hydroxylamine 3 as an O-sulfate or O-acyl
ester. Similarly, the regioselectivity of DNA adduct formation at
C-9 is somewhat surprising because nitroarenes usually afford both
N and C adducts. Another intriguing question is why medicinal
and edible plants containing significant concentrations of N-
hydroxyaristolactams are nontoxic. These issues are discussed below
in relation to the reduction product aristoxazole.
Activation of Nitroarenes: Nitroreduction and O-Esteri-
fication of the N-Hydroxylarylamine. Polycyclic nitroaromatic
compounds are known to be metabolized into potent carcinogens
in laboratory animals. They undergo reduction of the nitro group
to give reactive nitrenium ions that bind to DNA with N-
hydroxyarylamine intermediates being the immediate precursors of
the nitrenium ions. Mutagenicity and carcinogenicity are dependent
upon two activation reactions, namely, nitroreduction and O-
esterification of the N-hydroxylamine. Specific nitroreductases and
O-Esterification of N-hydroxylamine intermediates of nitroarene
carcinogens is required for the exertion of biological effects.
However, in the case of AAs, there is also evidence of formation
of DNA adducts under reduction conditions that are not conducive
to such esterification processes.
This subject is discussed below.
“In Vitro” Experiments. Formation of Adducts by Aris-
tolochic Acids without Activation of the N-Hydroxylamine
by O-Sulfation or O-Esterification. AAs can efficiently form
DNA adducts under in vitro conditions where the activation of the
N-hydroxylamine is not possible. For example, DNA adducts were
formed by reduction of AAs with either zinc in potassium phosphate
buffer (pH 5.8) at 37 °C or zinc in aqueous 1% acetic acid at 37 °C
in the presence of DNA.34-36 Likewise, enzymatic reduction of AAI
by xanthine oxidase in the presence of hypoxanthine and calf thymus
DNA also yielded DNA adducts.11 The nitroreduction products of most
nitroarenes are barely mutagenic unless they are activated by
esterification;27-32 however, a few nitroarenes seem to be mutagenic
in bacteria after nitroreduction without esterification.37-40
As was the case in the reactions described above and the
enzymatic generation of the 9-hydroxyaristolactam 6,11 aristoxazole
was obtained under conditions that were not conducive to activation
of the hydroxy group of the N-hydroxylamine by O-sulfation or
O-acylation. Formation of the oxazinone 11 would provide an
explanation for the apparent lack of activation with aristolochic
acids during in vitro reductions, which will be discussed in Proposed
Mechanism for the Formation of Aristoxazole via Oxazinone 11.
Probable Routes of Aristoxazole Formation. In principle,
it could be possible to account for the formation of aristoxazole by
reduction of AAI with Zn in acetic acid using the mechanism of
Scheme 1 in the following way. Under acidic conditions, N-
hydroxyaristolactam 3 could ionize to nitrenium ion 4, which would
then be trapped by acetic acid. The resulting 9-acetoxy adduct could
undergo hydrolysis of the lactam moiety to yield the acyclic form
(10-amino-1-carboxyl derivative). Then, condensation of the 10-
amino group with the 9-acetoxy group would generate aristoxazole.
Thus, nitrenium ion 4 could be a precursor of both the 9-hy-
droxyaristolactam I (6) and aristoxazole (15). However, this
possibility fails to take into account several factors.
The ionization of N-hydroxylamines is moderate under weakly
acidic conditions at pH ∼6. Such cleavage should be even slower
with the N-hydroxyaristolactam 3 because of “intramolecular
acylation”. The N-O bond heterolysis is energetically less favorable
in arylamides than in the parent N-hydroxyarylamines. For example,
studies with model esters of N-arylhydroxylamines established that
the reaction rate of heterolysis of the N-O bond to give the
nitrenium ion would be 106-fold slower when the NH group is
replaced with the N-acetyl group.41 Consistently, computational
studies show that N-acylation is deactivating (as opposed to
O-acylations that are activating).42,43 Consequently, the N-O bond
cleavage in the N-hydroxyaristolactam 3 would be strongly sup-
pressed, and the 3-to-4 ionization without activation via O-
esterification would be extremely slow for reactions such as the in

1984 Journal of Natural Products, 2010, Vol. 73, No. 12
Priestap et al.
vitro reductions described above. The same problem is likely to
occur in the formation of aristoxazole from the N-hydroxyaristo-
lactam 3. To form aristoxazole from 3, the cleavage of the N-O
bond (3 to 4) must be as fast as reduction (of 3 to 5 or of 9 to 10)
which does not seem to be the case. Thus, the participation of the
N-hydroxyaristolactam 3 in the formation of aristoxazole seems
unlikely.
The strongest evidence against the formation of aristoxazole from
either the N-hydroxyaristolactam I (3) or the putative 9-acetoxy
derivative of 5 is that these species would have to undergo
hydrolysis of the stable lactam ring to form the acyclic 10-amino-
1-carboxylic acid. In that way, the 9-acetoxy group can react with
the C-10 amine. Ring opening of the lactam is difficult, at best.
For example, the amide units of aristolactams and related com-
pounds (e.g., benzanilide) are not cleaved when treated with boiling
acetic acid. Furthermore, the opening of the lactam unit of
aristolactams probably generates substantial strain from interactions
of the substituents at C-9, C-10, and C-1. Clearly, opening the
lactam ring is an energetically unfavorable process, which is very
unlikely to occur under the reaction conditions that lead to the
formation of aristoxazole. An alternative pathway for aristoxazole
formation is proposed below.
Proposed Mechanism for the Formation of Aristoxazole
via Oxazinone 11. The 9-hydroxyaristolactam I (6) and aristox-
azole (15) are significant in vitro reduction products of AAI,
accompanying the major product aristolactam I (5). It seems likely
that these compounds were not formed by the simple dissociation
of the N-hydroxyaristolactam 3 (without activation) for the reasons
discussed above. If the reduction products 9-hydroxyaristolactam
I and aristoxazole are congeners to the known AA-DNA adducts,
both of them may be derived from a common nitrenium ion
precursor. The high reactivity of AA intermediates during in vitro
reductions suggests that aristoxazole is formed via the pathway
shown in Scheme 2 in which the oxazinone 11, which would be
the activated form of the N-hydroxylamine 9, forms the nitrenium
ion. This pathway also avoids the need for the unfavorable ring
opening of the lactam ring, which is a key step in the route based
on N-hydroxyaristolactam 3 (Scheme 1).
Considerations about the Pattern of Adducts Formed.
Only One Type of Aristolochic Acid Adduct Is Identified,
whereas Nitroaromatic Compounds Usually Generate Two
or More Types of Adducts. Oxidation of aromatic amines or
amides or reduction of aromatic nitro compounds produces nitre-
nium ions with reactive sites that are targets for nucleophilic
reagents. Although nitroarenes may produce several types of
adducts,44,45 the two that are most often encountered involve the
N atom or the ortho ring carbon that can bind to C-8 or the amino
group of the purine base, respectively.44,45 For example, 6-nitro-
chrysene (17), which has an aromatic skeleton similar to AAs, forms
adducts at the N and at the adjacent carbon (see arrows in formula
17).45,46 The carcinogenic N-acetyl-2-aminofluorene (18) also forms
two adducts after being metabolically activated by N-hydroxylation
followed by O-esterification (see arrows in formula 18). Structure
18 resembles the N-hydroxyaristolactam 3 in that it too is an
N-acylated compound.
bound to either a guanine or adenine amino group (7, 7a, 8, and
8a). The apparently preferred formation of C-9 adducts by AAs,
as opposed to N-6 adducts formed by 6-nitrochrysene, suggests
that the AAI carboxyl group plays a role in controlling the
regiospecificity of the reaction and the pattern of adducts formed.
Possible explanations are given below.
Tentative Explanation for the Formation of C-9 Adducts
by Aristolochic Acids Based on the Oxazinone Pathway.
There are two possible mechanisms that account for the regiospeci-
ficity. The reaction might occur via an SN2′ mechanism in which
the intermediate 11 undergoes a concerted addition-elimination
reaction upon being attacked by the nucleophile (H2O or CH3CO2H).
Alternatively, 11 could generate the nitrenium ion 12 as an “ion
pair” in which the ring carbon will be the more reactive site for
nucleophilic attack because of the influence of the carboxylic acid
group.
(i) SN2′ Mechanism. Concerted Addition and Elimination
without Formation of a Nitrenium Ion. In this mechanism,
there is direct attack of acetic acid, or another nucleophile, on C-9
of the oxazinone 11 (see arrows in structure 11) to give the imine
13. Subsequently, tautomerization and condensation between the
substituents at C-9 and C-10 would afford aristoxazole (15). This
pathway would seem to be the less likely of the two on the basis
of studies with small model molecules, which indicate that
solvolysis of N-O bond cleavage occurs through an ionic pathway
(SN1′ mechanism) involving a nitrenium ion.52
(ii) Regiospecicific Formation of the C-9 Adducts via a
Nitrenium Ion Pair in Which the Nitrogen Is Blocked from
Attack. “Ion pairs” or “electrostatic complexes” are implicated in
the formation of nitrenium ions by carcinogens.43,52,53 The nitre-
nium ions from AAs may have the intriguing ability to form a “tight
ion pair” by intramolecular interaction of the cation with the
carboxylate ion. A tight ion pair consists of a cation and an anion
held strongly together by electrostatic attraction, and further
stabilized by hydrogen bond interaction between the ions, which
prevent the solvent from forming solvent-separated ions.43,52
Consequently, tight ion pairs are insensitive to the presence of
trapping agents (H2O, Cl-, and reducing agents) in the medium.
The nitrenium ion 12 would be an ion pair in which the cation and
the anion (the carboxylate group) occur in the same molecule. The
regiospecificity of DNA base attack at C-9 may be due to the
occurrence of this intramolecular ion pair that fixes the anion at
one side of the reacting centers. The ion pair prevents the
participation of the nitrenium nitrogen atom in reactions with
nucleophiles, leaving the electron-deficient C-9 position as the only
site for nucleophilic attack. In summary, formation of an intramo-
lecular nitrenium ion pair could account for the generation of the
dominant C-9 adducts with DNA. In contrast, other nitroarenes
lacking the carboxyl group can generate adducts at both the nitrogen
and ortho carbon. Although computational studies are needed to
test this hypothesis, the proposed oxazinone 11 intermediate
provides a plausible explanation not only for the unusual pattern
of adducts from AAs but also for the activation processes needed
to generate nitrenium ions.
Are N-Hydroxyaristolactams Toxic? N-Hydroxyaristolactams
such as 3 and 3a seem to be implicated in nitrenium ion formation
and toxicity, but there is evidence that questions the suspected
toxicity of N-hydroxyaristolactams. N-Hydroxyaristolactams have
not been documented in the Aristolochiaceae; however, N-methoxy-
and N-hydroxyaristolactams are stable compounds that have been
isolated from Piper spp. (Piperaceae).54-56 Of particular interest
is Piper umbellatum, a medicinal plant widely used in Cameroon
traditional medicine.56 It contains significant amounts of N-
hydroxyaristolactams (∼100 mg/kg of air-dried branches).56 Leaves,
stems, inflorescences, and fruits of P. umbellatum are also used as
food in many parts of Africa.57 There have been no reports that
this plant is toxic. Because the N-hydroxyaristolactams (3 and 3a)
A priori predictions of the base or carcinogen type of adduct
formed are difficult because multiple factors are involved, which
include, for example, DNA repair mechanisms and the stability of
the adducts during isolation.44,47-49 Nevertheless, the relative
preferences of carcinogenic aromatic nitrenium ions for adduct
formation with different DNA base sites have been predicted by
computational studies.49-51 All these theoretical studies point
toward the specificity of the N-site of the nitrenium ion toward
C-8 of purine bases as well as the specificity of the ortho C-site of
the nitrenium ion toward the adenine and guanine amino group.
In contrast to typical nitroaromatic compounds, only one type
of adduct is commonly encountered with AAs in which C-9 is

Identification of a Reduction Product of Aristolochic Acid
Journal of Natural Products, 2010, Vol. 73, No. 12 1985
[M + C2H5]+ (calcd for C21H18NO6, 380.1134), 352.0824 [M + H]+
(calcd for C19H14NO6, 352.0821), 334 [M + H - H2O]+, 308 [M + H
- CO2]+; ESI/MS (MeOH) positive mode 352.0 [M + H]+, 406.0 [M
+ MeOH + Na]+, 703.0 [2M + H]+, 725.0 [2M + Na]+; EIMS m/z
(relative intensity) 350.9 [M]+ (98), 306.9 [M - CO2]+ (66), 291.9
[M - CO2 - CH3]+ (100), 263.9 [M - CO2 - CH3 - CO]+ (15).
Aristoxazole Methyl Ester. Aristoxazole was treated with ethereal
diazomethane as usual to give aristoxazole methyl ester: tR 57.3 min
(system 1); UV/PDA λmax 254, 300, 324, 363, 384 nm; APCI/MS m/z
394.1298 [M + C2H5]+ (calcd for C22H20NO6, 394.1291), 366.0952
[M + H]+ (calcd for C20H16NO6, 366.0978), 365.0872 [M]+ (calcd for
C20H15NO6, 365.0899), 350.0864 [M - MeO]+.
Reduction at Lower Temperatures. Aristoxazole can also be
produced when the AAI/Zn/HOAc mixture is heated at lower temper-
atures. HPLC analyses of reaction mixtures and yields of products (5
and 15) revealed that the ratio of aristolactam I to aristoxazole on
heating at 60 °C, at 90 °C, and at reflux (118 °C) ranged from 2.2-3
to 1. In contrast, the ratio of aristolactam I to aristoxazole was 6:1
when the reaction was conducted at room temperature (25 °C).
are the immediate precursors of the nitrenium ions (4a and 4b),
they are expected to be as toxic as the AAs themselves because
they can be activated in vivo by sulfotransferases and acyltrans-
ferases. The lack of toxicity of N-hydroxyaristolactam-containing
plants and a number of additional questions concerning AA
activation and toxicity remain unanswered.
The major reduction product of AAI in both chemical and
biological reductions is aristolactam I (5). The AA-DNA adducts
(7, 7a, 8, and 8a) and 9-hydroxyaristolactam I (6) are byproducts
from reactive intermediates generated during reduction of AAI. The
new reduction product, aristoxazole (15), may also belong to the
same family of adducts. The formation of aristoxazole probably
occurs via a nitrenium ion pathway in which an acetic acid molecule
attacks C-9 of the nitrenium ion pair 12. Although the formation
of aristoxazole may proceed via the N-hydroxyaristolactam 3
(Scheme 1), it seems more likely that the compound is formed from
the oxazinone 11 (Scheme 2). The oxazinone 11, which can be
considered an activated form of the N-hydroxylamine, could also
provide a rational explanation for adduct formation in in vitro
systems that lack other means of activating the N-hydroxylamine.
In addition to the aristolochic acid-DNA adducts generated via
the N-hydroxyaristolactam pathway (Scheme 1), the oxazinone 11
and the nitrenium ion pair 12 could also serve as important
electrophiles involved in DNA adduct formation in vivo.
Acknowledgment. We thank Mayron Georgiadis (Florida Interna-
tional University), Maria C. Dancel (University of Florida, Gainesville,
FL), and Charles R. Iden (Stony Brook University) for MS measure-
ments. We are grateful to the Department of Chemistry and Biochem-
1
istry at Florida International University for allowing us to record H
and 13C NMR and FTIR spectra. We are grateful to Mr. Ya Li Hsu for
recording our NMR spectra and Ms. Monica Joshi for recording the
FT IR spectrum. We also thank Drs. Francis Johnson and Arthur P.
Grollman (Stony Brook University) for helpful discussions. C.d.l.S. is
partially supported by National Institute of Environmental Health
Sciences Grant PO1 ES004068. We are indebted to Dr. Alexander
Mebel for his assistance in obtaining the molecular mechanics calcula-
tions. We are also grateful to the referees for their helpful suggestions
that have resulted in substantial improvements to this work.
Experimental Section
General Experimental Procedures. UV spectra were recorded with
an Agilent 8453 UV-vis spectrophotometer. IR spectra were recorded
on a Perkin-Elmer Spectrum 2000 instrument with KBr pellets. NMR
spectra were recorded on a Bruker 400 MHz FT-NMR spectrometer
in DMSO with TMS as an internal standard. ESI and APCI mass spectra
were recorded on a Thermo Scientific LCQ Deca XP MAX instrument
and Thermo Scientific DSQ mass spectrometer, respectively. HPLC
analysis was conducted with a Thermo-Finnigan chromatograph
(Thermo Electron Corp., San Jose, CA). The chromatograph consisted
of a SpectraSystem SMC1000 solvent delivery system, a vacuum
membrane degasser, P4000 gradient pumps, and an AS3000 autosam-
pler. Column effluent was monitored at 254 nm with a SpectraSystem
UV6000LP variable-wavelength PDA detector and ChromQuest version
4.1. Analytical separations were performed using a C18 RP Hypersil
GOLD column (RP5, 250 mm × 4.6 mm, pore size of 5 µm, Thermo
Electron Corp.). HPLC solvents were employed without further
purification. They were filtered through a 0.22 µm Millipore membrane.
The water used was deionized and filtered through a nylon membrane
(0.45 µm). The following eluting systems were used: system 1, 0.1%
TFA in MeCN (A), 0.1% TFA in H2O (B), linear gradient from 10 to
100% A over 120 min; system 2, MeCN (A), 0.1 M NH4OAc buffer
(pH 7.5) (B), linear gradient from 10 to 100% A over 120 min; system
3, 0.1% TFA in MeCN (A), 0.1% TFA in H2O (B), linear gradient
from 10 to 100% A over 30 min. The flow rate was 1.0 mL/min at
room temperature. In systems 1-3, aristolactam I exhibited tR values
of 48.4, 47.6, and 19.0 min, respectively.
Supporting Information Available: 1H NMR (400 MHz, DMSO-
d6) spectrum of the new compound aristoxazole (15). This material is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Vanherweghem, J.-L.; Depierreux, M.; Tielemans, C.; Abramowicz,
D.; Dratwa, M.; Jadoul, M.; Richard, C.; Vandervelde, D.; Verbeelen,
D.; Vanhaelen-Fastre, R.; Vanhaelen, M. Lancet 1993, 341, 387–391.
(2) Vanhaelen, M.; Vanhaelen-Fastre, R.; But, P.; Vanherweghem, J.-L.
Lancet 1994, 343, 174.
(3) van Ypersele de Strihou, C.; Vanherweghem, J.-L. Nephrol., Dial.,
Transplant. 1998, 10, 157–160.
(4) Nortier, J. L.; Muniz, M.-C.; Schemeiser, H. H.; Arlt, V. M.; Bieler,
C. A.; Petein, M.; Depierreux, M. F.; De Pauw, L.; Abramowicz, D.;
Vereerstraeten, P.; Vanherweghem, J.-L. N. Engl. J. Med. 2000, 342,
1686–1692.
(5) Arlt, V. M.; Stiborova, M.; Schmeiser, H. H. Mutagenesis 2002, 17,
265–277.
(6) Grollman, A. P.; Jelakovic, B. J. Am. Soc. Nephrol. 2007, 18, 2817–
2823.
(7) Stiborova, M.; Frei, E.; Arlt, V. M.; Schmeiser, H. H. Mutat. Res.
2008, 658, 55–67.
(8) Podzik, A. A.; Salmon, U.; Debelle, F. D.; Decaestecker, C.; van den
Branden, C.; Verbeelen, D.; Deschodt-Lanckman, M. M.; Vanher-
weghem, J.-L.; Nortier, J. L. Kidney Int. 2008, 73, 595–607.
(9) Debelle, F. D.; Vanherweghem, J.-L.; Nortier, J. L. Kidney Int. 2008,
74, 158–159.
(10) Report on Carcinogens, Background Document for Aristolochic Acids,
2008, U.S. Department of Health and Human Services, Public Health
Services, National Toxicology Program, Research Triangle Park, NC.
(11) Pfau, W.; Schmeiser, H. H.; Wiessler, M. Carcinogenesis 1990, 11,
313–319.
Molecular mechanics calculations were conducted using Spartan ’06
for Windows (Wave function Inc.). The calculations were conducted
at the B3LYP 6311+G** level.
Aristolochic acid I was obtained from Aristolochia argentina as
previously described and purified by recrystallization from dioxane.13
9-Methoxy-7-methyl-2H-1,3-oxazolo[5′,4′-10,9]phenanthro[3,4-d]-
1,3-dioxolane-5-carboxylic Acid. Aristoxazole (15). Aristolochic acid
I (1) (15.3 mg) was refluxed for 50 min with zinc powder (70 mg) and
glacial HOAc (1 mL) with magnetic stirring. The reaction mixture was
treated with H2O (5 mL) and EtOAc (5 mL), shaken, and centrifuged.
The upper phase was removed, washed with H2O, and extracted with
aqueous 5% NaHCO3. Evaporation to dryness of the EtOAc phase gave
a yellow residue of aristolactam I (5) (9.1 mg). The NaHCO3 solution
containing aristoxazole was acidified to pH 3 with dilute HCl and
extracted with EtOAc. Removal of the solvent from the organic phase
under vacuum yielded a white residue of aristoxazole (3.3 mg): colorless
needles (isoPrOH); HPLC tR ) 46.60 min (system 1), 24.1 min (system
2), 18.2 min (system 3); UV/PDA λmax 251, 300, 326, 363, 383 nm;
UV (H2O/NaOH) λmax 253.0, 299.5, 325.1, 363.8, 382.0 nm; IR (KBr)
(12) Mukhopadhyay, S.; Funayama, S.; Cordell, G. A.; Fong, H. H. S. J.
Nat. Prod. 1983, 46, 507–509.
(13) Priestap, H. A. Phytochemistry 1987, 26, 519–529.
(14) Priestap, H. A. Magn. Reson. Chem. 1989, 27, 460–469.
(15) Lin, F.-W.; Damu, A. G.; Wu, T.-S. J. Nat. Prod. 2006, 69, 93–96.
(16) Ferris, J. P.; Antonucci, F. R. J. Am. Chem. Soc. 1974, 96, 2010–
2014.
(17) Balasubramaniyan, V. Chem. ReV. 1966, 66, 567–641.
(18) Ung, S.; Falguieres, A.; Guy, A.; Ferroud, C. Tetrahedron Lett. 2005,
46, 5913–5917.
ν
max 1668, 1587, 1459, 1310, 1104, 1018 cm-1; APCI/MS m/z 380.1101
(19) Bamberger, E. Ber. Dtsch. Chem. Ges. 1894, 27, 1347–1350.

1986 Journal of Natural Products, 2010, Vol. 73, No. 12
Priestap et al.
(20) Sone, T.; Tokuda, Y.; Sakai, T.; Shinkai, S.; Manabe, O. J. Chem.
Soc., Perkin Trans. 2 1981, 298–302.
(39) Djuric, Z.; Fifer, E. K.; Beland, F. A. Carcinogenesis 1985, 6, 941–
944.
(21) Fishbein, J. C.; McClelland, R. A. Can. J. Chem. 1996, 74, 1321–
1328.
(22) Helberger, J. H.; von Rebay, A. Liebigs Ann. Chem. 1937, 531, 279.
(23) Wunsch, K. H. In AdVances in Heterocyclic Chemistry; Katritzky,
A. R., and Boulton, A. J., Eds.; Academic Press: New York, 1967;
Vol. 8, p 305.
(24) de Raadt, A.; Griengl, H.; Petsch, M.; Plachota, P.; Schoo, N.; Weber,
H. Tetrahedron: Asymmetry 1996, 7, 473–490.
(25) Stiborova, M.; Frei, E.; Breuer, A.; Bieler, C. A.; Schmeiser, H. H.
Exp. Toxicol. Pathol. 1999, 51, 421–427.
(26) Pfau, W.; Schmeiser, H. H.; Wiessler, M. Chem. Res. Toxicol. 1991,
4, 581–586.
(27) McCoy, E. C.; Anders, M.; Rosenkranz, H. S. Mutat. Res. 1983, 121,
17–23.
(28) Hein, D. W.; Doll, M. A.; Fretland, A. J.; Gray, K.; Deitz, A. C.;
Feng, Y.; Jiang, W.; Rustan, T. D.; Satran, S. L.; Wilkie, T. R., Sr.
Mutat. Res. 1997, 376, 101–106.
(29) Shinohara, A.; Saito, K.; Yamazoe, Y.; Kamataki, T.; Kato, R. Cancer
Res. 1986, 46, 4362–4367.
(30) Turesky, R. J.; Lang, N. P.; Butler, M. A.; Teitel, C. H.; Kadlubar,
F. F. Carcinogenesis 1991, 12, 1839–1845.
(31) Wild, D.; Feser, W.; Michel, S.; Lord, H. L.; Josephy, P. D.
Carcinogenesis 1995, 16, 643–648.
(32) Glatt, H. FASEB J. 1997, 11, 314–321.
(33) Meinl, W.; Pabel, U.; Osterloh-Quiroz, M.; Hengstler, J. G.; Glatt, H.
Int. J. Cancer 2006, 118, 1090–1097.
(34) Schmeiser, H. H.; Frei, E.; Wiessler, M.; Stiborova, M. Carcinogenesis
1997, 18, 1055–1063.
(35) Chan, W.; Zheng, Y.; Cai, Z. J. Am. Soc. Mass Spectrom. 2007, 18,
642–650.
(40) Bieler, C. A.; Wiessler, M.; Erdinger, L.; Suzuki, H.; Enya, T.;
Schmeiser, H. H. Mutat. Res. 1999, 439, 307–311.
(41) Novak, M.; Kaley, M. J.; Eiger, E.; Helmick, J. S.; Peters, H. E. J. Am.
Chem. Soc. 1993, 115, 9453–9460.
(42) Ford, G. P.; Herman, P. S. J. Chem. Soc., Perkin Trans. 2 1991, 607–
616.
(43) Borosky, G. L. Chem. Res. Toxicol. 2007, 20, 171–180.
(44) Dipple, A. Carcinogenesis 1995, 16, 437–441.
(45) Purohit, V.; Basu, A. K. Chem. Res. Toxicol. 2000, 13, 673–692.
(46) El-Bayoumy, K.; Sharma, A. K.; Lin, J.-M.; Krzeminski, J.; Boyiri,
T.; King, L. C.; Lambert, G.; Padgett, W.; Nesnow, S.; Amin, Sh.
Chem. Res. Toxicol. 2004, 17, 1591–1599.
(47) Kennedy, S. A.; Novak, M.; Kolb, B. A. J. Am. Chem. Soc. 2007,
119, 7654–7664.
(48) Novak, M.; Kennedy, S. A. J. Phys. Org. Chem. 1998, 11, 71–76.
(49) Parks, J. M.; Ford, G. P.; Cramer, C. J. J. Org. Chem. 2001, 66, 8997–
9004.
(50) Sengupta, J.; Duncan Lyngdoh, R. H. J. Biol. Phys. 1995, 21, 285–
294.
(51) Ford, G. P.; Thomson, J. W. Chem. Res. Toxicol. 1999, 12, 53–59.
(52) Novak, M.; Pelecanou, M.; Roy, A. K.; Andronico, A. F.; Plourde,
F. M.; Olefirowicz, T. M.; Curtin, T. J. J. Am. Chem. Soc. 1984, 106,
5623–5631.
(53) Novak, M.; Kahley, M. J.; Lin, J.; Kennedy, S. A.; James, T. G. J.
Org. Chem. 1995, 60, 8294–8304.
(54) Wu, Q.-L.; Wang, S.-P.; Tu, G.-Z.; Feng, Y.-X.; Yang, J.-S. Phy-
tochemistry 1997, 44, 727–730.
(55) Kuo, Y. C.; Yang, N. S.; Chou, C. J.; Lin, L. C.; Tsai, W. J. Mol.
Pharmacol. 2000, 58, 1057–1066.
(36) Chan, W.; Yue, H.; Poon, W. T.; Chan, Y.-W.; Schmitz, O. J.; Kwong,
D. W. J.; Wong, R. N. S.; Cai, Z. Mutat. Res. 2008, 646, 17–24.
(37) McCoy, E. C.; McCoy, G. D.; Rosenkranz, H. S. Biochem. Biophys.
Res. Commun. 1982, 108, 1362–1367.
(38) Howard, P. C.; Heflich, R. H.; Evans, F. E.; Beland, F. A. Cancer
Res. 1983, 43, 2052–2058.
(56) Tabopda, T. K.; Ngoupayo, J.; Liu, J.; Mitaine-Ofer, A.-C.; Tanoli,
S. A. K.; Khan, S. N.; Ali, M. S.; Ngadjui, B. T.; Tsamo, E.; Lacaille-
Dubois, M.-A.; Luu, B. Phytochemistry 2008, 69, 1726–1731.
(57) P. umbellatum L., Protabase Record display. www.prota.org.
NP100296Y