Nordihydroguaiaretic acid autoxidation produces a schisandrin-like dibenzocyclooctadiene lignan

Article abstract of DOI:10.1021/np8001354

The lignan meso-nordihydroguaiaretic acid is known to undergo spontaneous oxidation in alkaline solution. In the presence of the trapping agent glutathione, the major oxidation products are consistent with the formation of a meso-nordihydroguaiaretic acid ortho-quinone. In the absence of a trapping agent however, the major oxidation product of meso-nordihydroguaiaretic acid in aqueous solution is a unique, stable schisandrin-like dibenzocyclooctadiene lignan that may be responsible for some of the biological effects of nordihydroguaiaretic acid.

Full text of DOI:10.1021/np8001354

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J. Nat. Prod. 2008, 71, 1612–1615
Notes
Nordihydroguaiaretic Acid Autoxidation Produces a Schisandrin-like Dibenzocyclooctadiene
Lignan
Jennifer L. Billinsky† and Ed S. Krol*,‡
Graduate Toxicology Program, UniVersity of Saskatchewan, Saskatoon, SK Canada, and College of Pharmacy and Nutrition, UniVersity of
Saskatchewan, Saskatoon, SK Canada
ReceiVed March 4, 2008
The lignan meso-nordihydroguaiaretic acid is known to undergo spontaneous oxidation in alkaline solution. In the presence
of the trapping agent glutathione, the major oxidation products are consistent with the formation of a meso-
nordihydroguaiaretic acid ortho-quinone. In the absence of a trapping agent however, the major oxidation product of
meso-nordihydroguaiaretic acid in aqueous solution is a unique, stable schisandrin-like dibenzocyclooctadiene lignan
that may be responsible for some of the biological effects of nordihydroguaiaretic acid.
Nordihydroguaiaretic acid (NDGA, masoprocol, 1) (Figure 1)
is a naturally occurring lignan from the creosote bush (Larrea
tridentata). NDGA has been utilized in traditional healing practices
for a wide range of ailments1,2 and was licensed for use as a topical
treatment for actinic keratosis (Actinex, Chemex Pharmaceuticals,
Denver, CO). Use of NDGA for therapeutic purposes is currently
limited due to reports of contact hypersensitivity,3 nephrotoxicity,4
hepatotoxicity,5,6 and cytotoxicity.5,7,8 It has been suggested that
the toxicity is the result of oxidation to a reactive ortho-quinone
species,4 the presence of which we recently confirmed via trapping
of NDGA ortho-quinones as glutathione (GSH) adducts.9 NDGA
has been shown to increase oxidized glutathione (GSSG) and lipid
peroxidation levels in murine hematopoietic cells,10 suggesting an
increase in oxidative stress consistent with the formation of an
ortho-quinone. There are numerous reports that NDGA can be
unstable in aqueous media,4,11-13 especially at elevated pH,11,13
with a half-life of 3.1 h at pH 7.4.9 NDGA, in the presence of O2,
has been reported to undergo conversion to an ortho-quinone,12 or
to an unspecified “activated NDGA”, with a λmax 286nm.11 In this
study we have isolated and identified the major non-GSH reactive
product of NDGA autoxidation in aqueous solution and determined
that NDGA forms a unique, stable nonquinone compound with a
Figure 1. Nordihydroguaiaretic acid (NDGA).
species. The peak at 34 min was confirmed as being NDGA (1) by
MS (ES-MS m/z 301 (100%) [M - H]-) and comparison with an
authentic standard.
Compound 4 was isolated and purified via reversed-phase flash
column chromatography and was found to have an identical UV
absorbance maximum (286 nm) to the previously unidentified
autoxidation product of NDGA.11 NDGA possesses a plane of
symmetry, so that only half of the protons and carbons appear in
the NMR spectrum. 1H and 13C NMR spectroscopic data for
compound 4 indicate that distinct resonances are observed for all
of the alkyl and aromatic protons. There are several pieces of NMR
evidence to suggest that 4 is an intramolecular cyclization product
of NDGA (Figure 3). Four singlets in the aromatic region
corresponding to one proton each were observed (HMQC), sug-
gesting that two of the aromatic protons of NDGA have been
substituted due to the formation of a new carbon-carbon bond. A
coupling reaction involving C-2′, C-2′′, C-5′, or C-5′′ of NDGA
would result in splitting of the aromatic 1H NMR signals. Our results
suggest that coupling has occurred between the C-6′ and C-6′′
positions of NDGA, which have previously been shown to be highly
reactive.9 A second piece of evidence comes from a COSY NMR
experiment for 4, where it was determined that only one of the
CH2 protons at C-7 is coupled to the adjacent CH proton at C-8
(this is also the case for the protons on C-7′ and C-8′), suggesting
that one of the CH2 protons is held at a 90° dihedral angle to the
CH, a conformation that may be attained in an eight-membered
ring. A compound that has cyclized between the aromatic C-6′ and
C-6′′ carbons of NDGA would result in the dibenzocyclooctadiene
lignan 4, as represented in Figure 4. As a result of the nonplanar
eight-membered ring that is formed, compound 4 has no plane of
symmetry and the NMR chemical shifts for all of the protons and
carbon atoms of 4 are distinguishable. The aromatic protons at C-1
and C-9 of compound 4 (the numbering in NDGA 1 differs from
that for the NDGA-derived dibenzocyclooctadiene lignan 4; refer
λ
max of 286 nm, suggesting it is the “activated NDGA” previously
reported.11
NDGA was incubated at pH 7.4 in the absence of GSH, and
HPLC was used to monitor the reaction. A mixture of products
was observed and the reaction mixture was further analyzed by
LC-MS (Figure 2). One of the LC-MS peaks was consistent with
an ortho-quinone of NDGA that has undergone hydroxylation
(tR ) 29 min, 3 ES-MS m/z 315 (100%) [M - H]-). The ES-MS
results for the 22 and 25 min peaks were consistent with either an
ortho-quinone or para-quinone methide (ES-MS m/z 299 (100%)
[M - H]-), although previous studies suggested that para-quinone
methide formation is unlikely.9 Addition of GSH to the incubation
mixture resulted in disappearance of both the 25 min and the 29
min peaks and the appearance of NDGA-SG adducts (data not
shown). The major product, the 22 min peak (4), however was
unaffected by the addition of GSH, suggesting it was not a quinone
* To whom correspondence should be addressed. Tel: (306) 966-2011.
Fax: (306) 966-6377. E-mail: ed.krol@usask.ca.
† Graduate Toxicology Program.
‡ College of Pharmacy and Nutrition.
10.1021/np8001354 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/02/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 9 1613
NDGA would undergo radical coupling of the carbon atoms para
to the 3′- and 3′′-OH of NDGA via the carbon-centered resonance
forms (Figure 5).
A group of dibenzocyclooctadiene lignans known as schisandrins,
isolated from Schisandra chinensis, have been studied for their
potential therapeutic properties including the ability to protect
against oxidative stress as antioxidants21 or via enhancement of
GSH synthesis;22,23 the ability to inhibit P-glycoprotein24 and
multidrug resistance-associated protein 1;25 and enhancement of
apoptosis through caspase-9 activation.26 In addition, schisandrin
B is a substrate for murine CYP2E1,27 and several schisandrins
have been found to be CYP3A4 inhibitors.28 The NDGA-derived
dibenzocyclooctadiene lignan 4 is an analogue of the schisandrins
and thus may also display similar biological activity. It is conceiv-
able that the NDGA-derived dibenzocyclooctadiene lignan is also
responsible for a portion of the biological activity of NDGA,
especially if NDGA undergoing biological study is maintained under
aerobic conditions for extended periods of time at pH 7.4 or higher.
We have observed that in the absence of GSH at pH 8.0, 37 °C,
50% of the NDGA is mainly converted to 4 after a 1 h incubation,
with smaller amounts present as the ortho-quinone and hydroxylated
ortho-quinone. In our microsomal incubations (pH 7.4, 37 °C)
NDGA is preincubated for 5 min followed by a 60 min incubation,
which is sufficient for measurable NDGA autoxidation to occur.
NDGA is a known antioxidant29 and a lipoxygenase inhibitor2 and
has been shown to inhibit P45030 and suppress growth of breast
cancer cells.31 All of these systems incubate NDGA at or near pH
7.4, suggesting that at least some of the observed activity may be
the result of the NDGA-derived dibenzocyclooctadiene lignan or
an NDGA ortho-quinone. An ortho-quinone of NDGA would be
expected to be involved in redox cycling or adduct formation with
biological macromolecules,9 whereas the dibenzocyclooctadiene
lignan appears to be more stable and may be responsible for
mediating biological processes associated with NDGA that are not
necessarily associated with toxicity.
Figure 2. LC-MS-ES(-) (microbore column, see Experimental
Section) for NDGA autoxidation products, pH 7.4, 37 °C. tR ) 22
min, m/z 299 [M - H]- (4); tR ) 25 min, m/z 299 [M - H]- (2);
tR ) 29 min, m/z 315 [M - H]- (3); tR ) 34 min, m/z 301 [M -
H]- (NDGA 1).
Figure 3. Structure of the NDGA-derived dibenzocyclooctadiene
lignan 4, the major NDGA autoxidation product at pH 7.4, 37 °C.
Extracts from Creosote bush containing NDGA used in traditional
healing practices are typically prepared by boiling in water and
then being either applied topically as a salve or paste or consumed
as a tea (T. D. Johnson, personal communication). It is possible
that the traditional extraction process may result in a significant
conversion of NDGA- to NDGA-derived dibenzocyclooctadiene;
however, this will require further investigation. An NDGA-derived
dibenzocyclooctadiene lignan, in which the stereochemistry was
not specified, has been screened by the National Cancer Institute
(NSC 669349) in the United States and displays LD50 values in
the µM range. In order to determine whether the NDGA-derived
dibenzocyclooctadiene lignan 4 is responsible for the effects in the
NCI study or any other biological activity associated with NDGA,
it will be necessary to test the NDGA-derived dibenzocycloocta-
diene lignan derived from meso-NDGA in a variety of in Vitro
systems. Of perhaps greater issue is that due to the rapid formation
of the NDGA-derived dibenzocyclooctadiene lignan under condi-
tions used for many in Vitro studies, interlaboratory differences in
incubation conditions for NDGA-dependent experiments could lead
to significantly different results depending on the actual amount of
NDGA- or NDGA-derived dibenzocyclooctadiene lignan present.
Figure 4. Representation of NDGA-derived dibenzocyclooctadiene
lignan 4 in the R-biphenyl configuration from meso-NDGA 1. This
representation is based on NMR and CD data, after energy
minimization in the MMFF94 force field using Spartan ’06
(Wavefunction Inc., Irvine, CA). Image generated using VMD
(Urbana, IL).
to Figures 1 and 3) were tentatively assigned from observed NOEs
1
with H-5 and H-8, respectively. The H and 13C NMR results for
the alkyl region were comparable to those of other dibenzocy-
clooctadiene lignans including gomisin J14 and the (R,R)-tet-
ramethoxy analogue of 4.15,16 A crystal structure reported for the
(R,R)-tetramethoxy analogue of 4 indicates the phenyl rings are
twisted about the biphenyl bond by an angle of 117.8°.16 Compound
4, determined by CD to be in the R-biphenyl configuration, displays
a similar out-of-plane twist.
Experimental Section
General Experimental Procedures. Caution: NDGA is hazardous
and should be handled carefully. meso-Nordihydroguiairetic acid
(NDGA) from Larrea tridentata was purchased from Sigma-Aldrich
Co. (Madison, WI). Citric acid and K2HPO4 were purchased from Fisher
Scientific (Ottawa, ON). HCl was purchased from BDH (Toronto, ON).
All solvents were HPLC grade. Water was purified via a Millipore
Milli-Q system (Mississauga, ON) with a Quantum EX cartridge.
UV spectra were recorded on an Agilent 8453 using Chem Station
software. Negative electrospray mass spectra were obtained using an
API Qstar XL with an Agilent 1100 HPLC (Saskatchewan Structural
Similar coupling products have been prepared via oxidation of
4-methylcatechol with phenoloxidase,17 and numerous synthetic
dibenzocyclooctadiene lignans have been prepared from intramo-
lecular cyclization reactions of matairesinol derivatives.18-20 One
possible mechanism for formation of dibenzocyclooctadiene lignan
4 from NDGA via a radical intermediate is suggested in Figure 5,
although other radical and nonradical processes cannot be ruled
out. This mechanism requires initial formation of phenoxy radicals
at the 3′-OH and 3′′-OH of NDGA. This diphenoxy radical of

1614 Journal of Natural Products, 2008, Vol. 71, No. 9
Notes
Figure 5. Possible reaction pathway for the formation of NDGA autoxidation products 3 and 4 in the absence of GSH.
Sciences Centre). NMR data were obtained on a Bruker AVANCE
DPX-500 (Dept of Chemistry, University of Saskatchewan) operating
at 500 and 125 MHz for proton and carbon, respectively, and the data
analyzed using XWIN-NMR version 3.0. Residual signals from CD3OD
[3.30 ppm (1H) and 49.0 ppm (13C)] served as an internal standard.
Programs for 2D experiments were available from the software package
XWINNMR, provided by Bruker. The DEPT experiment together with
2D NMR experiments including COSY, HMQC, and NOESY experi-
ments were performed with gradient pulses. CD data were obtained
on an Applied Photophysics PiStar-180 spectrometer.
Autoxidation Incubation for ES-MS Analysis. A solution of
NDGA in CH3CN (20 mM) was added to 0.5 M pH 7.4 phosphate-
citric acid buffer pre-equilibrated to 37 °C to give a final NDGA
concentration of 0.1 mM. The reaction mixture was placed in a shaking
water bath for 60 min, during which time the colorless solution became
pink. The reaction was stopped by acidification to pH 1.5 with HCl.
Autoxidation was not effected by light, as no difference was observed
for dark control reactions. Aliquots of the supernatant (50 µL) were
analyzed directly by electrospray LC-MS operating in the negative
mode. An Allsphere ODS-2 microbore column (3 µm, 150 × 2.1 mm)
operating at a flow rate of 0.2 mL/min was used to run a gradient
elution. Solvent A: 0.1% formic acid/H2O, solvent B: 0.1% formic acid/
CH3CN. An initial isocratic phase of 80% A for 2 min, decreased to
65% A over 38 min, isocratic for 1 min, and finally increased to 80%
A over 1 min.
Synthesis of the NDGA Autoxidation Product. A solution of
NDGA in CH3CN (20 mM) was added to 0.5 M pH 8.5 phosphate-
citric acid buffer pre-equilibrated to 37 °C to give a final NDGA
concentration of 0.1 mM. The reaction mixture was placed in a shaking
water bath for 90 min, during which time the colorless solution became
pink. The reaction was stopped by acidification to pH 1.5 with HCl.
The reaction mixture was concentrated in Vacuo to yield a purple solid.
The oxidation product was purified by C-18 flash column chromatog-
raphy, using 70:30 H2O/CH3CN (v/v) containing 0.1%TFA, yielding
an off-white solid.
(6R,7S)-2,3,10,11-Tetrahydroxy-6,7-dimethyl-5,6,7,8-tetrahydro-
dibenzo[a,c]cyclooctene (4): CD (c 0.62, CH3OH) [θ]286 +15158.7;
UV (CH3CN) λmax (log ꢀ) 286 nm (3.87); 1H NMR (CD3OD, 500 MHz)
δ 0.78 (3H, d, J ) 6.9 Hz, CH3-6), 0.99 (3H, d, J ) 7.0 Hz, CH3-7),
1.79 (1H,m, CH-7), 1.88 (1H, m, CH-6), 1.93 (1H, d, J ) 13.2 Hz,
CH2-8b), 2.21 (1H, dd, J ) 9.6, 13.2 Hz, CH2-8a), 2.39 (1H,d, J )

Notes
Journal of Natural Products, 2008, Vol. 71, No. 9 1615
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Acknowledgment. This work was funded by HSURC, NSERC, and
CFI. J.L.B. was a recipient of an NSERC PGS award. The authors
wish to thank Dr. D. R. J. Palmer for help with generating the
dibenzocyclooctadiene lignan image.
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NP8001354