Aristoxazole analogues. Conversion of 8-Nitro-1-naphthoic acid to 2-Methylnaphtho[1,2- d ]oxazole-9-carboxylic acid: Comments on the chemical mechanism of formation of DNA adducts by the aristolochic acids

Article abstract of DOI:10.1021/np300137f

2-Methylnaphtho[1,2-d]oxazole-9-carboxylic acid was obtained by reduction of 8-nitro-1-naphthoic acid with zinc-acetic acid. This naphthoxazole is a condensation product between an 8-nitro-1-naphthoic acid reduction intermediate and acetic acid and is a lower homologue of aristoxazole, a similar condensation product of aristolochic acid I with acetic acid that was previously reported. Both oxazoles are believed to arise via a common nitrenium/carbocation ion mechanism that is likely related to that which leads to aristolochic acid-DNA-adducts.

Full text of DOI:10.1021/np300137f

Note
pubs.acs.org/jnp
Aristoxazole Analogues. Conversion of 8-Nitro-1-naphthoic Acid to
2-Methylnaphtho[1,2-d]oxazole-9-carboxylic Acid: Comments on the
Chemical Mechanism of Formation of DNA Adducts by the
Aristolochic Acids
Horacio A. Priestap,*,† Manuel A. Barbieri,† and Francis Johnson‡
†Department of Biological Sciences, Florida International University, 11200 Southwest 8th Street, Miami, Florida 33199, United
States
‡Department of Chemistry, Stony Brook University, Nichols Road, Stony Brook, New York 11794, United States
S
* Supporting Information
ABSTRACT: 2-Methylnaphtho[1,2-d]oxazole-9-carboxylic acid was obtained by
reduction of 8-nitro-1-naphthoic acid with zinc−acetic acid. This naphthoxazole is
a condensation product between an 8-nitro-1-naphthoic acid reduction
intermediate and acetic acid and is a lower homologue of aristoxazole, a similar
condensation product of aristolochic acid I with acetic acid that was previously
reported. Both oxazoles are believed to arise via a common nitrenium/carbocation
ion mechanism that is likely related to that which leads to aristolochic acid−
DNA−adducts.
ristoxazole (1) is a phenanthroxazole that arises from a
condensation of aristolochic acid I (2) (AA-I; 8-methoxy-
A
6-nitrophenanthro[3,4-d][1,3]dioxole-5-carboxylic acid) with
acetic acid and is formed when AA-I (2) is reduced with Zn
in hot acetic acid.1 In this Note we report that the same
reaction can be demonstrated with the simpler 8-nitro-1-
naphthoic acid (NNA; 3) under the same reaction conditions,
and this leads to the corresponding naphthoxazole 4 (2-
methylnaphtho[1,2-d]oxazole-9-carboxylic acid). The forma-
tion of this compound shows that aristolochic acid I (2) and
NNA (3) display the same behavior when reduced with Zn/
acetic acid. Consequently, the only requirement for the reaction
to proceed seems to be the presence of the carboxylic and nitro
groups located in a peri relationship. The present study
indicates that this reaction can be extended to other polycyclic
aromatic hydrocarbons bearing a nitro and a carboxyl group in
the same positional relationship. The present finding is also
important in relation to DNA−adduct formation by the AAs,
because AA−adducts and the oxazoles seem to be generated by
the same mechanistic pathway (see discussion below).
are formed, namely, the naphthoxazole 4 and the acetoxy
derivative 6. The major reaction product is the lactam 5 (ca.
46%), which is accompanied by minor amounts of the 5-
acetoxy derivative 6 (ca. 27%) and the oxazole 4 (ca. 12%), as
determined by HPLC analysis. Unidentified products (ca. 15%)
were also formed.
Compound 4 was isolated from the reaction mixture by
extraction with aqueous NaHCO3 solution, whereas com-
pounds 5 and 6 were separated by preparative TLC.
Compound 6, C13H9NO3, was found to be 2-oxo-1,2-
It is known that reduction of NNA (3) produces the lactam 5
{benz[c,d]indol-2(1H)-one} by spontaneous cyclization of the
reduction product 8-aminonaphthalene-1-carboxylic acid (re-
duction is instantly achieved by Fe2+ ions in aqueous solution at
room temperature).2,3 In this study, reduction of NNA with
Zn/HOAc at room temperature also yielded the expected
lactam 5. However, when the reaction is carried out in boiling
acetic acid, in addition to lactam 5, two new reduction products
Received: February 21, 2012
Published: July 2, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Note
dihydrobenzo[c,d]indol-6-yl acetate. The possible formation of
the corresponding isomer with the acetoxy group at position 7
of the lactam 5 can be eliminated on the basis of the NMR data
(Table 1).
δC 134.8, 148.3, and 162.3, which are coherent with those of
other oxazole derivatives.1 The position of CH3-10 at δC 14.2
matches that of the corresponding methyl group in 2-
methylbenzoxazole (δC 14.4), thus eliminating the possibility
that the methyl is part of an an acetyl group. The above
considerations together with evidence gathered from other
oxazole derivatives1 support structure 4 for this reduction
product of NNA.
The biological activity of NNA (3) has been previously
studied by Pfau et al.4 NNA (3) displays a mutagenic activity
similar to AA-I (2) in Salmonella typhimurium strain TA 100 in
the Ames assay.4 Since compounds such as 3-nitro-2-naphthoic
acid are devoid of mutagenic activity, Pfau et al. concluded that
the mutagenicity is associated with compounds in which the
carboxyl and nitro groups are peri positioned as in 2 and 3.
Molecular orbital calculations of AA-I (2) and NNA (3)
showed that the nitro and carboxyl groups are in a similar
compressed situation (the aromatic version of A1,3-strain)5 in
these molecules.4 Pfau et al. proposed that the observed
mutagenic activity of peri-substituted nitro aromatic carboxylic
acids (2, 3) is due to the reductive formation of a cyclic
hydroxamic acid (= N-hydroxylactam; 7, 9), which by solvolysis
generates a cyclic nitrenium ion (e.g., 10) with delocalized
positive charge. The nitrenium ion reacts, through the carbon
in ortho position to the N-substitution, with nucleophiles such
as the exocyclic amino groups of purine nucleotides in DNA to
form adducts.4,6
On the basis of the mechanism of metabolic activation of
peri-substituted nitro aromatic carboxylic acids proposed by
Pfau et al.,4 the formation of naphthoxazole 4 could be
explained as arising from the 3 → 7 → nitrenium ion → 4
pathway. However, generation of 4 via this mechanism appears
to be quite unlikely in view of the considerations discussed in a
previous paper.1 An alternative mechanism for the formation of
the oxazole 4 is proposed in Scheme 1. It may be assumed that
the NOH group of the N-hydroxy intermediate 11 can react
with the COOH either to give the lactam 7 or through the OH
group to yield the dihydronaphthoxazinone 12. Compound 7
would be further reduced to the lactam 5. On the other hand,
the dihydronaphthoxazinone 12 can either be reduced to give 5
or undergo a solvolysis to a nitrenium/carbocation ion, which
then acquires a nucleophile such as acetate to generate the
1
Table 1. H NMR and 13C NMR Data for 2-
Methylnaphtho[1,2-d]oxazole-9-carboxylic Acid (4),
Benz[c,d]indol-2(1H)-one (5), and 2-Oxo-1,2-
dihydrobenzo[c,d]indol-6-yl Acetate (6) in DMSO-d6 (δ in
parts per million, J in parentheses in herz)
4
5
6
a
a
a
position
δH
δC
130.4
δH
δC
127.1
δH
δC
1
127.1
126.2
2
3
4
7.69 dd
125.8 8.01 d
(6.8)
123.6 8.08 d
(6.8)
(7.2, 1.2)
7.58 dd
(8.4, 7.2)
8.16 dd
(8.4, 1.2)
125.5 7.77 dd
128.8 7.85 dd
129.3
124.3
(8.0, 6.8)
130.1 8.13 d
(8.0)
(8.2, 6.8)
130.8 8.10 d
(8.2)
4a
5
130.8
128.2
119.4
123.4
141.2
7.97 d
(8.8)
124.4 7.56 d
(8.4)
6
7
7.93 d
(8.8)
111.6 7.48 dd
129.0 7.24 d
(7.6)
120.8
105.9
(8.4, 6.8)
148.3 6.99 d
(6.8)
106.1 6.98 d
(7.6)
8
134.8
121.1
162.3
170.4
138.1
125.6
136.0
126.1
8a
CN
1-COOH 13.25 s
(broad)
CH3
2.68 s
14.2
8-NH
10.80 s
10.86 s
169.0
1-CO
CH3CO
CH3CO
169.8
168.8
20.6
a
Assigments of signals with similar chemical shifts may be reversed.
Compound 4, C13H9NO3, is a carboxylic acid that was
obtained as a white, crystalline solid. ESIMS displayed a
1
protonated molecular ion at m/z 228. The H and 13C NMR
data of this compound are shown in Table 1. The oxazole
structure in this molecule was corroborated by carbon signals at
Scheme 1. Proposed Mechanism for the Formation of 2-Methylnaphtho[1,2-d]oxazole-9-carboxylic Acid (4) and 2-Oxo-1,2-
dihydrobenzo[c,d]indol-6-yl Acetate (6) from 8-Nitro-1-naphthoic Acid (3)
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Journal of Natural Products
Note
addition compounds at C-5 or C-7 of the naphthalene nucleus.
This is in contrast to the AAs, in which only C-9, ortho to the
nitrogen, is involved. HPLC profiles and amounts of the
purified reduction products 4 and 6 recovered from the
reaction mixture indicate that the initial adduct 15 was formed
in higher proportion than the isomer 13. After formation of the
initial adduct 13, aromatization to 14 and condensation would
then produce the naphthoxazole 4. The 5-acetoxy intermediate
16 is converted and isolated as the lactam 6. Among the
reaction products, the assumed 7-acetoxy derivative of the
lactam 5 could not be isolated. This suggests that the 13 → 14
→ 4 pathway may proceed rapidly; that is, addition,
aromatization, and condensation may occur in quick succession,
so that lactamization by reaction of the 8-amino group with the
1-carboxyl group does not take place. This may especially be
true if the carboxyl group is present in the unreactive anionic
form of the zinc salt. Thus, a significant reduction product of
NNA (3) is the oxazole 4. There is no evidence for the
formation of the assumed intermediate 12. However,
compounds such as o-nitrobenzoic acid (17), under reduction
conditions, can give the benzo[c]isoxazolin-3-one (19) by
cyclization of the reduction intermediate 2-(hydroxylamino)-
benzoic acid (18) (Scheme 2).7 This reaction supports the
same type of cyclization for the formation of 12 from 11.
formation of DNA adducts derived from the aristolochic acids
(Scheme 3).
Biological Implication. The equal mutagenic potency of
NNA (3) and AA-I (2) in Ames assays with Salmonella
thyphimurium TA100 shows that both compounds are
metabolized in a similar way and to the same extent by this
microorganism. The properties of NNA (3) and AA-I (2) as
mutagens are likely due to DNA binding. These results also
suggest that they exert their effect via a structurally related
reactive intermediate, possibly the corresponding dihydroox-
azinones described above. NNA (3) and AA-I (2) also show
similar behavior by reduction with Zn/HOAc to give oxazole
derivatives by addition of a molecule of HOAc. Thus, both
compounds exhibit the same biological activity and the same
chemical reactivity. Mutagenicity and chemical behavior are
associated with an aromatic substructure in which a carboxyl
group and a nitro group are located in a peri position. The
above considerations suggest that NNA may also form adducts
with DNA in a similar way to the AAs. Assays will be performed
to explore the potential of NNA to generate DNA adducts in
vitro. One drawback when studying the metabolism of AA-I, in
in vivo and in vitro experiments, is the scarcity of this
compound. Chemical modifications of the AA-I molecule are
also precluded for this reason and the limited positional
reactivity of the phenanthrene core. If NNA forms adducts like
the AAs, it could be used as a simpler analogue of AA-I that is
readily available and could be conveniently modified and
labeled. Consequently, NNA could help to elucidate the
intricate mechanisms leading to formation of DNA adducts by
aristolochic acids in biological systems. Investigations are also
under way to explore the mechanisms underlying this reductive
conversion of NNA (3) into naphthoxazole 4.
Scheme 2. Synthesis of Benzo[c]isoxazolin-3-one (19) from
o-Nitrobenzoic Acid (17)7
EXPERIMENTAL SECTION
■
General Experimental Procedures. NNA was synthesized by a
literature procedure.2 Zinc dust was purchased from Mallinckrodt
(8681). NMR spectra were recorded on a Bruker 400 MHz FT-NMR
spectrometer in DMSO-d6 with TMS as internal standard. ESI mass
spectra were acquired on a Thermo Scientific LCQ Deca XP MAX
instrument. HPLC/PDA analyses were performed with a P4000
Thermo-Finnigan chromatograph (Thermo Electron Corporation, San
Jose, CA, USA). Column effluent was monitored at 254 nm with a
SpectraSystem UV6000LP variable-wavelength PDA detector. Ana-
lytical separations were carried out with a C18 RP Hypersil GOLD
column (RP5, 250 × 4.6 mm, pore size 5 μm, Thermo Electron
Corporation). The mobile phase consisted of 0.1% TFA in MeCN
(phase A) and 0.1% TFA in H2O (phase B). The linear gradient
program was as follows: 10% to 100% A over 30 min at a flow rate of
1.0 mL/min. GC-MS analyses were carried out in a Hewlett-Packard
model 6890 instrument coupled to a Q-Mass 910 quadrupole selective
detector at 70 eV. A fused capillary column was used (DB-5MS, 30 m
× 0.25 mm i.d.; film thickness 0.25 μm; J & W Scientific); injection
port temperature, 230 °C; split ratio 1:20; detector temperature, 330
°C; carrier gas, helium at 1 mL/min; temperature program: 200 to 300
°C linear increase at 8 °C/min. GC/FID analyses were performed on a
Trace GC Ultra apparatus (Thermo Electron Corporation) equipped
with a flame ionization detector (FID). The output was recorded using
a ChromQuest version 4.1 data system. Analyses were performed on a
DB-5MS capillary column, and the conditions were as indicated above.
2-Methylnaphtho[1,2-d]oxazole-9-carboxylic Acid (4). 8-Nitro-1-
naphthoic acid (3) (300 mg) and Zn (900 mg) were refluxed for 1 h in
glacial HOAc (21 mL) containing less than 1% water (FisherBiotech,
BP1185-500; Alfa Aesar, Johnson Matthey Company, #33252) with
magnetic stirring. The reaction mixture was treated with EtOAc (50
mL) and filtered. The insoluble material was washed with EtOAc and
H2O. The EtOAc phase was washed with H2O and extracted with 5%
This study shows that the carboxyl group may be essential for
the formation of the naphthoxazole 4 and by extension of the
phenanthroxazoles and AA-DNA adducts; otherwise the simple
1-nitronaphthalene, or nitro aromatic compounds in general,
could give the oxazole structure by reduction with Zn/HOAc,
which is not the case. In the present Note it is suggested that
the COOH participates in the activation of the molecule by
forming a dihydro-oxazinone structure such as 12. The 1-
COOH group may also interact with the peri-nitrogenated
function at C-8 in other ways, e.g., by forming a lactam
structure as represented in 5 and 7. As stated above, in order to
rationalize the observed mutagenic activity of peri-substituted
nitro-carboxylic acids and DNA adduct formation, Pfau et al.
suggested4 that ring closure of the uncyclized precursors, such
as the N-hydroxylamine 11, to give compounds 7 and 9, occurs
because of steric stress. This also likely contributes to
stabilization of the nitrenium ion, the ultimate carcinogen, by
delocalization of the positive charge.4,6 This hypothesis is
questionable because (a) solvolysis of the N-hydroxylactam 7
would be less likely than 12 to give a nitrenium ion and (b) the
conversion of 7 into oxazole 4 is, mechanistically, extremely
difficult to explain otherwise. These and other reasons
discussed in ref 1 point to the more rational formation of an
oxazinone ring intermediate (12) for this reaction to proceed.
Thus, nitrenium ion formation via dihydrooxazinone (12) is
likely the mechanistic pathway that leads to 4 and 6 through
interaction with HOAc. This also suggests that dihydroox-
azinone formation may represent an alternative pathway for the
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Note
a
Scheme 3. Proposed Mechanism of Activation and Adduct Formation of Aristolochic Acid I (2)
a
It was postulated that a dihydrooxazinone (20) is the active intermediate formed by reduction of AAs.1 The dihydrooxazinone (20), or its
nitrenium ion (21), may react with nucleophiles (H2O, the amino group of adenine and guanine, HOAc) to give the corresponding adducts 22, 23,
and 24. These adducts undergo intramolecular cyclization to give the more stable lactam (25, 26) or oxazole (1) derivatives. Compound 25 was
obtained by in vitro reduction of AA-I with xanthine oxidase.6 AA-DNA adducts (26) are formed by in vitro reduction of AAs with xanthine oxidase
in presence of DNA, in rats administered with AAs or in humans exposed to AAs.6 Compound 1 may be obtained by reduction of AA-I with Zn/
HOAc.1
aqueous NaHCO3. The NaHCO3 extract was acidified to pH 3 with
diluted HCl and extracted with EtOAc. The organic phase was washed
with water and evaporated to dryness. In order to remove dark
impurities, the residue was dissolved in 20% EtOAc in CHCl3 and the
solution passed through a Si gel column (1 g, 70−230 mesh, 60A,
Aldrich, catalog no. 28,862-4). The column was washed with 30%
EtOAc in CHCl3. Percolate and washes were combined and
evaporated to dryness to give a white residue (35.9 mg) of 4:
[M]+ (100), 141 [M − CO]+ (23), 140 [M − CHO]+ (35), 114 [M −
CO − CNH]+ (23), 113 [M − CHO − CNH]+ (17).
2-Oxo-1,2-dihydrobenzo[c,d]indol-6-yl Acetate (6): HPLC, tR
12.66 min; GC, tR 10.22 min; UV/PAD λmax (nm), 248, 271 (sh),
1
322, 338, 368; H NMR and 13C NMR data, see Table 1; GC-MS, tR
21.25 min; m/z (rel int) 227 [M]+ (7), 185 [M − H2CCO]+
(100), 156 [185 − HCO]+ (4), 129 [185 − HCO − CNH]+ (17), 101
(11), 75 (8); DART-TOF-MS 228.0651 [M + H]+ (calcd for
C13H10NO3, 228.0655), 245.0918 [M + NH4]+ (calcd for
C13H13N2O3, 245.0921).
1
HPLC, tR 13.96 min; UV/PAD λmax (nm) 248, 290, 311, 324; H
NMR and 13C NMR data, see Table 1; ESIMS (MeOH) positive
mode, 228.2 [M + H]+, 244.9 [M + NH4]+; HPLC-ESIHRMS
250.0471 [M + Na]+ (calcd for C13H9NO3Na, 250.0475), 272.0294
[M − H + 2Na]+ (calcd for C13H8NO3Na2, 272.0294).
ASSOCIATED CONTENT
■
S
* Supporting Information
Separation of Compounds 5 and 6. After separation of the
oxazole 4, a portion of the crude reaction mixture (112 mg) was
subjected to preparative TLC (silica gel G, Uniplate, 250 μm, 20 × 20
cm, Analtech, Inc.; 20% EtOAc in CHCl3). After development, two
main yellow bands (Rf 0.50 and Rf 0.30) were scraped from the plates
and eluted with 20% MeOH in CHCl3. The upper band afforded a
yellow residue of the lactam 5 (37 mg), whereas the lower band
yielded a yellow residue of the acetoxy derivative 6 (26 mg).
1H and 13C NMR spectra of compounds 4 and 6. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 305-348-0375. Fax: 305-348-1986. E-mail: priestap@
fiu.edu.
■
Benz[c,d]indol-2(1H)-one (5): HPLC, tR 12.99 min; GC, tR 6.92
min; UV/PAD λmax (nm) 245, 273, 325 (sh), 337, 359; 1H NMR and
13C NMR data, see Table 1; GC-MS, tR 17.82 min; m/z (rel int) 169
Notes
The authors declare no competing financial interest.
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Journal of Natural Products
Note
ACKNOWLEDGMENTS
■
We thank R. Bonala (Stony Brook University, NY) for the
preparation of 8-nitro-1-naphthoic acid. We are also grateful to
Y. L. Hsu and Y. Song (Florida International University) for
recording NMR and GC-MS spectra and J. V. Johnson and M.
C. Dancel (University of Florida, Gainesville, FL) for HPLC-
ESIHRMS measurements. H.A.P. thanks Florida International
University for financial support.
REFERENCES
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