Ring substitution influences oxidative cyclisation and reactive metabolite formation of nordihydroguaiaretic acid analogues

Article abstract of DOI:10.1016/j.bmc.2015.09.039

Nordihydroguaiaretic acid (NDGA) is a natural polyphenol with a broad spectrum of pharmacological properties. However, its usefulness is hindered by the lack of understanding of its pharmacological and toxicological pathways. Previously we showed that oxidative cyclisation of NDGA at physiological pH forms a dibenzocyclooctadiene that may have therapeutic benefits whilst oxidation to an ortho-quinone likely mediates toxicological properties. NDGA analogues with higher propensity to cyclise under physiologically relevant conditions might have pharmacological implications, which motivated this study. We synthesized a series of NDGA analogues which were designed to investigate the structural features which influence the intramolecular cyclisation process and help to understand the mechanism of NDGA's autoxidative conversion to a dibenzocyclooctadiene lignan. We determined the ability of the NDGA analogues investigated to form dibenzocyclooctadienes and evaluated the oxidative stability at pH 7.4 of the analogues and the stability of any dibenzocyclooctadienes formed from the NDGA analogues. We found among our group of analogues the catechols were less stable than phenols, a single catechol-substituted ring is insufficient to form a dibenzocyclooctadiene lignan, and only compounds possessing a di-catechol could form dibenzocyclooctadienes. This suggests that quinone formation may not be necessary for cyclisation to occur and the intramolecular cyclisation likely involves a radical-mediated rather than an electrophilic substitution process. We also determined that the catechol dibenzocyclooctadienes autoxidised at comparable rates to the parent catechol. This suggests that assigning in vitro biological activity to the NDGA dibenzocyclooctadiene is premature and requires additional study.

Full text of DOI:10.1016/j.bmc.2015.09.039

Bioorganic & Medicinal Chemistry 23 (2015) 7007–7014
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ring substitution influences oxidative cyclisation and reactive
metabolite formation of nordihydroguaiaretic acid analogues
⇑
Isaac Asiamah, Heather L. Hodgson, Katherine Maloney, Kevin J. H. Allen, Ed S. Krol
Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, SK S7N 5E5, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2015
Revised 10 September 2015
Accepted 22 September 2015
Available online 25 September 2015
Nordihydroguaiaretic acid (NDGA) is a natural polyphenol with a broad spectrum of pharmacological
properties. However, its usefulness is hindered by the lack of understanding of its pharmacological and
toxicological pathways. Previously we showed that oxidative cyclisation of NDGA at physiological pH
forms a dibenzocyclooctadiene that may have therapeutic benefits whilst oxidation to an ortho-quinone
likely mediates toxicological properties. NDGA analogues with higher propensity to cyclise under phys-
iologically relevant conditions might have pharmacological implications, which motivated this study. We
synthesized a series of NDGA analogues which were designed to investigate the structural features which
influence the intramolecular cyclisation process and help to understand the mechanism of NDGA’s autox-
idative conversion to a dibenzocyclooctadiene lignan. We determined the ability of the NDGA analogues
investigated to form dibenzocyclooctadienes and evaluated the oxidative stability at pH 7.4 of the ana-
logues and the stability of any dibenzocyclooctadienes formed from the NDGA analogues. We found
among our group of analogues the catechols were less stable than phenols, a single catechol-substituted
ring is insufficient to form a dibenzocyclooctadiene lignan, and only compounds possessing a di-catechol
could form dibenzocyclooctadienes. This suggests that quinone formation may not be necessary for cycli-
sation to occur and the intramolecular cyclisation likely involves a radical-mediated rather than an elec-
trophilic substitution process. We also determined that the catechol dibenzocyclooctadienes autoxidised
at comparable rates to the parent catechol. This suggests that assigning in vitro biological activity to the
NDGA dibenzocyclooctadiene is premature and requires additional study.
Keywords:
Lignans
Natural products
Nordihydroguaiaretic acid
Quinone
Dibenzocyclooctadiene
Stability
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
nordihydroguaiaretic acid (NDGA) (up to 15% d.w.).2 NDGA is gen-
erally accepted as responsible for both beneficial and adverse
The leaves of the plant creosote bush were used commonly in
traditional medicines among the Native Americans for diverse ben-
eficial effects.1–3 The aqueous extract of this shrub, commonly
referred to as Chaparral tea, was listed in the American pharma-
copeia as an ethnobotanical used to treat tuberculosis, arthritis
and cancer.1 Documented traditional applications of the plant
extract include treatment for infertility, rheumatism, arthritis, dia-
betes, gallbladder and kidney stones, pain and inflammation
among many others.2,3 Creosote bush is rich in lignans, particularly
effects associated with this shrub.2–6 NDGA shows promise in the
treatment of multiple diseases, including cardiovascular dis-
eases,7,8 neurological disorders9–13 and cancers.3,14–20 It also
potently inhibits viruses such as human immunodeficiency virus
(HIV-1), herpes simplex virus (HSV), human papilloma virus
(HPV) and influenza virus.2,21 The radical scavenging22,23 and
antioxidant effects24 as well anti-inflammatory25,26 and anti-prolif-
erative properties may be of relevance in different diseases.
Despite its broad pharmacological activities, NDGA use is associ-
ated with toxicity especially when ingested at higher doses.
Hepato- and nephrotoxicity has been associated with NDGA
use24,27–30 and is likely linked to NDGA bioactivation to a reactive
ortho-quinone.31–33
Abbreviations: NDGA, nordihydroguaiaretic acid; cNDGA, NDGA cyclolignan;
DMPA, 3,4-dimethoxyphenylacetone; ESI-MS, electrospray ionization-mass spec-
trometry; GSH, glutathione; GSSG, oxidized glutathione; HPLC, high performance
liquid chromatography; HIV-1, human immunodeficiency virus; HSV, herpes
simplex virus; HPV, human papilloma virus; NADP(H), reduced nicotine adenine
dinucleotide phosphate; TFA, trifluoroacetic acid.
The origin of the dichotomous biological activity observed for
NDGA remains unknown. Traditionally creosote bush-based
products are prepared by boiling the leaves in water.3 We previ-
ously demonstrated that incubation of NDGA at pH 7.4 gave a
⇑
Corresponding author. Tel.: +1 306 966 2011; fax: +1 306 966 6377.
E-mail address: ed.krol@usask.ca (E.S. Krol).
http://dx.doi.org/10.1016/j.bmc.2015.09.039
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

7008
I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
schisandrin-like dibenzocyclooctadiene lignan,34 however in the
presence of glutathione (GSH) a ring adduct is observed, indicating
that NDGA can form an ortho-quinone.31 Biological evaluations of
NDGA are commonly conducted under aerobic conditions at pH
7.4 or higher, which may confound the results as biological activity
could be the result of NDGA, its ortho-quinone, dibenzocycloocta-
diene or a combination.5,35 Traditional conditions of preparation
favour oxidation and we suggested that the NDGA dibenzocyclooc-
tadiene may make a contribution to the beneficial effects of NDGA,
and that NDGA analogues with a higher propensity to cyclise under
physiologically relevant conditions might have pharmacological
implications. There are numerous reports of biological activity for
naturally-occurring and synthetic dibenzocyclooctadiene lig-
nans,36–39 including a report that for a series of dibenzylbutanedi-
ols the dibenzocyclooctadiene structure enhanced anti-tumor
activity and inhibition of MDA-MB-435 breast cancer cells.40 Phar-
macological activity from the uncyclised lignan cannot be ruled out
however, as anti-viral properties have been reported for tetram-
ethyl and tetraacetyl NDGA which would not be anticipated to
cyclise under these conditions.21
2
7
9
HO
HO
HO
HO
H3CO
HO
3
1
8
4
6
8'
7'
5
9'
1'
6'
2'
3'
NDGA
2
1
5'
OH
4'
OH
H3CO
HO
HO
HO
HO
HO
3
4
5
OH
OCH3
OCH3
OH
OCH3
OH
HO
HO
H3CO
6
7
OH
OH
The precise mechanism of the intramolecular cyclisation is
unknown although it possibly follows one of two pathways
(Scheme 1).34,41–43 We34 and others22 have hypothesized that
cyclisation occurs through a di-radical mechanism, whereas there
are numerous examples where intermolecular coupling of cate-
chols is proposed to occur via electrophilic substitution through
an ortho-quinone intermediate.42,44 We therefore propose to iden-
tify the structural features which control oxidative metabolism of
NDGA-like lignans, including cyclisation. In order to accomplish
these goals we plan to: investigate the oxidative stability of NDGA
lignan and dibenzocyclooctadiene analogues at pH 7.4 and deter-
mine what structural features are necessary for formation of
dibenzocyclooctadienes from the NDGA lignan analogues. We syn-
thesized seven NDGA analogues (Fig. 1) and evaluated their stabil-
ity including lignan half-life at pH 7.4, and product studies to
determine their ability to form dibenzocyclooctadienes using our
previously established conditions.34 We were also interested in
establishing the oxidative stability of the prepared analogues as
stability of a compound in a test medium is an important parame-
ter when investigating biological activity.45,46
OCH3
Figure 1. Structures for NDGA and its structural analogues synthesised for this
study.
process. This will be useful in understanding the mechanisms of
NDGA autoxidative conversion to dibenzocyclooctadiene lignans
and will allow us to control intramolecular cyclisation in the
preparation of additional lignan analogues. We anticipate that
these studies will provide information on the contribution that
dibenzocyclooctadienes play in the in vitro pharmacological activ-
ity of NDGA.
2. Materials and methods
2.1. Materials
Caution: The following chemicals are hazardous and should be han-
dled carefully: meso-Nordihydroguiairetic acid (NDGA, 97%) from
Larrea tridentata, reduced glutathione (GSH), 3,4-dimethoxypheny-
lacetone (DMPA), Tyrosinase from mushroom (EC1.14.18.1),
The analogues were designed to help us understand the
structural features which influence the intramolecular cyclisation
O
OH
O.
OH
O
.
.O
.
HO
HO
A
y
IIa
a
w
HO
HO
HO
IIb
h
t
a
P
OH
- e
- H
OH
OH
OH
- H
- e
.O
HO
HO
I
HO
HO
cNDGA
OH
NDGA
OH
P
-H
- e
OH
a
t
h
OH
w
O
a
y
B
.O
.O
O
IIIa
IIIb
Scheme 1. Intramolecular conversion of NDGA to dibenzocyclooctadiene lignan (cNDGA) via radical-mediated process (pathway A) or electrophilic substitution mechanism
(pathway B). Both pathways involve a 2 proton, 2 electron loss followed by isomerisation of a di-radical intermediate IIa–IIb or an ortho-quinone IIIa–IIIb and subsequent
radical coupling or electrophilic substitution, respectively.

I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
7009
dimethylsulfoxide (Me2SO), NADP(H), AgNO3, K2HPO4 and MgSO4
were purchased from Sigma–Aldrich Co. (Madison, WI). Citric acid
and HCl were purchased from Fisher Scientific (Ottawa, ON). For-
mic acid was purchased from BDH. All solvents were HPLC grade.
Water was purified via a Millipore (Mississauga, ON) Milli-Q sys-
tem with a Quantum EX Cartridge. NDGA analogues were prepared
by modifications of literature procedures.
2H). 13C NMR (500 MHz, CDCl3): d (ppm) 13.95, 16.11, 37.81,
38.94, 41.09, 41.45, 55.84, 111.34, 113.97, 121.67, 125.64, 128.15,
129.08, 133.57, 141.73, 143.55, 146.29. ESI-MS (m/z) = 283.2
[MꢁH]ꢁ found; 283.17 Da calcd.
2.3.4. 4,40-(2,3-Dimethylbutane-1,4-diyl)bis(2-methoxyphenol)
(3)
Compound (3) was obtained as off-white solid (yield 20–30%).
>99% pure by HPLC; 1H NMR (500 MHz, CDCl3): d (ppm) 0.83–
0.86 (6H, 2 overlapping d, J = 6.4, H9, 90), 1.72–1.76 (2H, m, H8,
2.2. Instrumentation
2.2.1. HPLC-UV diode array analysis
80), 2.26–2.32 (1H, dd, J = ,H7
a), 2.37–2.41 (1H, dd, J = 7.4, 13.4,
The HPLC system consisted of a Waters 2690 separation module
equipped with a Waters 996 photodiode array detector and Mass-
lynx V4.1 software (Waters Corp., Milford, MA). An Allsphere ODS-
H7a0), 2.51–2.55 (1H, dd, J = 7.4, 13.7, H7b), 2.72–2.76 (1H, dd,
J = 4.1, 12.9, H7b0), 3.82 (3H, s, p-OCH3), 3.87 (3H, s, o-OCH3), 5.58
(2H, bs, p-ArOH), 6.55 (1H, s, H2), 6.60 (1H d, J = 7.8, H6), 6.64
(1H, s, H20), 6.68 (1H, d, J = 7.8, H60), 6.83 (1H, d, J = 8.0, H5), 6.85
(1H, d, J = 8.0, H50). 13C NMR (500 MHz, CDCl3) d (ppm) 14.04,
16.40, 21.25, 37.62, 39.07, 39.36, 41.26, 55.95, 56.03, 60.62,
111.46, 111.61, 114.04, 114.15, 121.81, 121.89, 133.77, 133.97,
143.67, 143.73, 146.42, 146.50. ESI-MS m/z: 329.1 [MꢁH]ꢁ found;
329.18 Da calcd.
2 microbore column (3
l
m, 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 iso-
cratic phase of 90% A for 2 min, decreased to 60% A over 8 min, then
10% A over 12 min, held isocratic for 10 min, and finally increased
to 90% A over 1 min and held isocratic at 90% A for 6 min to equi-
librate the column.
2.3.5. 4[4-(3,4-Dimethoxyphenyl)-2,3-dimethylbutyl]benzene-
1,2-diol (4)
2.2.2. ESI-MS analysis
All MS experiments were conducted using AB SCIEX 4000
QTRAP (AB SCIEX instruments) quadrupole linear ion trap mass
spectrometer. Samples were infused directly at 10 ll/min. Data
Compound 4 was obtained as dark purple solid (yield 20–30%).
>98% pure by HPLC; 1H NMR (500 MHz, CDCl3): d (ppm) 0.81–0.83
(6H, 2 overlapping d, J = 4.5, H9, 90), 1.69–1.76 (2H, m, H8, 80), 2.31–
acquisition and analyses were performed using Analyst 1.5.1 soft-
ware from AB SCIEX.
2.33 (2H, dd, J = 7.8, 13.4, H7
7
a), 2.38–2.42 (1H, dd, J = 7.7, 13.4,
a0), 2.46–2.51 (1H, dd, J = 7.0, 13.3, H7b), 2.51–2.55 (1H, dd,
J = 7.3, 13.5, 7b0), 3.82 (3H, s, p-OCH3), 3.86 (3H, s, o-OCH3), 6.50
(1H, d, J = 6.2), 6.56 (1H, s), 6.58 (1H, s), 6.77–6.78 (m, 2H), 13C
NMR (500 MHz, MeOD): d (ppm) 13.82, 13.89, 37.31, 37.44,
40.69, 40.98, 55.89, 55.92, 110.99, 112.28, 115.04, 115.96, 121.16,
121.49, 134.40, 134.69, 141.52, 143.29, 146.96, 148.55. ESI-MS
(m/z) = 329.1 [MꢁH]ꢁ found; 269.18 Da calcd.
2.2.3. NMR analysis
All NMR experiments were performed on a Bruker AVANCE
DPX-500 spectrometer and data processed by X-WIN NMR 3.5 soft-
ware or TopSpin 3.2. All compounds were named using ACD/
ChemSketch.
2.3. Methods
2.3.6. 4,40-Butane-1,4-diyldibenzene-1,2-diol (5)
Compound 5 was obtained as brown solid (yield 40–45%).
>98% pure by HPLC; 1H NMR (500 MHz, CD3OD): d (ppm) 1.52
(4H, s, H8, 80), 2.43 (4H, s, H7, 70), 6.44 (2H, d, J = 8.0, H6, 60),
6.56 (2H, s, H2, 20), 6.63 (2H, d, J = 8.0, H5, 50). 13C NMR
(500 MHz, CD3OD): d (ppm) 32.58 (C8, 80), 36.26 (C7, 70), 116.31
(C6, 60), 116.64 (C5, 50), 120.78 (C2, 20), 135.75 (C1, 10), 144.20
(C4, 40), 146.13 (C3, 30). ESI-MS (m/z) = 273.03 [MꢁH]ꢁ found;
273.11 Da calcd.
2.3.1. Synthesis and characterisation of NDGA analogues
The basic 18-carbon lignan skeleton was constructed using con-
secutive Stobbe condensation47,48 or a Stobbe condensation fol-
lowed by alkylation.40,49 We followed literature procedures to
synthesise NDGA analogues 1–7 and characterised them by the
1H and 13C NMR spectroscopy as well as ESI-MS methods. Yields
reported are overall yields.
2.3.2. 4-(2,3-Dimethyl-4-phenylbutyl)benzene-1,2-diol (1)
Analogue 1 was obtained as dark purple oil (yield 8–15%). 96.7%
pure by HPLC; [1H] NMR (500 MHz, CDCl3): d (ppm) 0.84–0.87 (6H,
2 overlapping d, J = 4.3, H9, 90), 1.74–1.84 (2H, m, H8, 80), 2.32–2.36
2.3.7. 3,30-(2,3-Dimethylbutane-1,4-diyl)diphenol (6)
Analogue 6 was obtained as a brown oil (yield 20%). >96% pure
by HPLC; 1H NMR (500 MHz, CDCl3): d (ppm) 0.87–0.89 (6H, 2
overlapping d, J = 6.5, H9, 90), 1.61–1.69 (2H, m, H8, 80), 2.32–
(1H, dd, J = 8.5, 13.4, H7
a
), 2.43–2.48 (1H, dd, J = 8.6, 13.4, H7a0),
2.36 (2H, dd, J = 8.3, 13.3, H7a
, H7a0), 2.59–2.63 (2H, dd, J = 6.3,
2.55–2.59 (1H, dd, J = 6.4, 13.6, H7b), 2.66–2.70 (1H, dd, J = 6.2,
13.6, H7b0), 5.78 (2H, bs, o, p-ArOH), 6.55–6.57 (1H, dd, J = 1.9,
8.1, H6), 6.64 (1H, d, J = 1.9, H2), 6.79 (1H, d, J = 7.9, H5), 7.14
(2H, d, J = 7.2, H20, 60), 7.21 (1H, t, J = 7.4, H40), 7.30 (2H, t, H30,
50). [13C] NMR (500 MHz, CDCl3): d (ppm) 13.97, 16.24, 38.10,
39.33, 40.67, 41.45, 115.18, 116.08, 121.55, 125.64, 128.17,
128.23, 129.13, 129.17, 134.89, 141.28, 141.78, 143.25. ESI-MS
(m/z) = 269.1 [MꢁH]ꢁ, found; 269.15 Da calcd.
13.4, H7b, H7b0), 5.56 (2H, bs, ArOH), 6.68–6.60 (2H, s), 6.70
(1H, d, J = 1.6), 6.76 (2H, d, J = 7.3), 6.83–6.86 (1H, m), 7.14–
7.17 (2H, m). ESI-MS (m/z) = 269.1 [MꢁH]ꢁ, found; 269.15 Da
calcd.
2.3.8. 3,30-(2,3-Dimethylbutane-1,4-diyl)bis(6-methoxyphenol)
(7)
Compound 7 was obtained as off white solid (yield 20–30%).
>98% pure by HPLC; 1H NMR (500 MHz, CDCl3): d (ppm) 0.80–
0.84 (6H, 2 overlapping d, J = 6.7, H9, 90), 1.74–1.78 (2H, m, H8,
2.3.3. 4-(2,3-Dimethyl-4-phenylbutyl)-2-methoxyphenol (2)
Compound 2 was obtained as yellow oil (yield 20–30%). >99%
pure by HPLC; 1H NMR (500 MHz, CDCl3): d (ppm) 0.86–0.88 (6H,
2 overlapping d, H9, 90), 1.77–1.85 (2H, m, H8, 80), 2.38–2.49 (2H,
80), 2.22–2.27 (1H, dd, J = 9.6, 13.3, H7
a), 2.31–2.35 (1H, dd,
J = 7.9, 12.8, H7a0), 2.54–2.58 (1H, dd, J = 5.8, 13.5, H7b), 2.70–
2.74 (1H, dd, J = 4.5, 13.4, H7b0), 3.89 (6H, s, 3,30 OCH3), 5.58 (2H,
bs, ArOH), 6.58–6.60 (1H, dd, J = 1.6, 8.1), 6.63–6.65 (1H, dd,
J = 1.6, 8.1), 6.71 (1H, d, J = 1.6), 6.75–6.79 (3H, m). ESI-MS (m/z)
= 329.13 [MꢁH+]ꢁ obsd; 269.18 Da calcd.
m, H7a
, 7a0), 2.57–2.68 (2H, m, H7b, H7b0), 3.84 (3H, s, OCH3),
5.51 (1H, bs, p-ArOH), 6.58 (s, 1H), 6.62–6.65 (1H, m,), 6.84–6.88
(1H, m,), 7.13 (d, J = 7.2, 1H), 7.18–7.23 (m, 2H), 7.23–7.33 (m,

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I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
2.3.9. Autoxidation study
2.3.11. Assessment of the effect of nucleophilic trapping agent
on the intramolecular reaction
The prepared analogues were evaluated for their potential to
undergo intramolecular conversion to corresponding dibenzocy-
clooctadiene lignans using a method previously developed by our
group.34 Briefly, the substrate in CH3CN (20 mM) is added to phos-
phate–citric acid buffer (0.5 M, pH 7.4) pre-equilibrated to 37 °C to
give a final substrate concentration of 0.1 mM. The reaction mix-
ture is stirred at the same temperature for 90 min and monitored
by HPLC. The reaction was stopped by acidification to pH 1.5 with
HCl. The reaction mixture is extracted with ethyl acetate, dried
over MgSO4 and solvent evaporated in vacuo. The oxidation pro-
duct was purified over a C-18 column (RedSepÒRf GOLD, Teledyne
Isco) on a Tris Pump connected to UA-6 UV/Vis Detector (Teledyne
Isco) separation system using 70:30 H2O/CH3CN (v/v) containing
0.1% FA. The product was analysed by HPLC, MS and NMR
techniques. Standard NDGA was used as positive control.
The test compound and internal standard (DMPA) at a final con-
centration of 0.5 mM in phosphate–citric acid buffer (0.5 M, pH
7.4) were incubated at 37 °C. In the first experiment, the incuba-
tion was carried out in either the absence or presence (2 fold
excess) of glutathione (GSH) Aliquots were taken at various time
intervals and acidified to pH 1.5. In the second experiment, GSH
was added at various time intervals and similarly acidified to pH
1.5. A third incubation was conducted in the presence of a 20-fold
excess of GSH at 37 °C for 6 h to further investigate the observed
delay in the intramolecular cyclisation process. All samples were
analysed directly by HPLC and further by ESI-MS.
3. Results and discussion
Autoxidation of 5 afforded compound 8 (Scheme 2) as a brown
solid following purification by solid phase extraction. 1H NMR
(500 MHz, CD3OD): d (ppm) 1.37 (2H, m), 1.94 (2H, m), 2.00 (2H,
m), 2.47 (2H, m), 6.54 (2H, s, H2, 20), 6.60 (2H, s, H5, 50). 13C
NMR (500 MHz, CD3OD): d (ppm) 31.33 (C8, 80), 33.36 (C7, 70),
117.02 (C2, 5, 20, 50), 133.76 (C1, 10), 135.63 (C6, 60), 143.87 (C4,
40), 145.71 (C3, 30). The loss of C6, 60 protons and the coupling of
C7, 70 as well as C8, 80 protons are consistent with cyclisation
and are also in agreement with our previous findings for the
NDGA-derived dibenzocyclooctadiene lignan.34 High-resolution
mass spectroscopic data (see Supporting information) further con-
firmed cyclisation.
The main objectives of this study were to determine how func-
tional groups on the aromatic rings of NDGA lignan analogues
influence their stability and the formation of dibenzocyclooctadi-
enes in phosphate buffer (pH 7.4) at 37 °C. From this we hoped
to develop an understanding of the mechanism of autoxidative
intramolecular cyclisation of NDGA and NDGA analogues, with
the goal of controlling dibenzocyclooctadiene formation in the
design of pharmacologically active lignans. We prepared seven
NDGA analogues and investigated their oxidative stability at pH
7.4, their ability to form dibenzocyclooctadienes, and the stability
of any dibenzocyclooctadienes using HPLC-UV, MS and NMR.
The dibenzocyclooctadiene family of lignans exhibit numerous
pharmacological activities36–38 including antiviral,50–52 anti-
cancer,51 anti-inflammatory52 and hepatoprotective effects.53 Our
group previously reported a unique schisandrin-like dibenzocy-
2.3.10. Chemical stability and reaction kinetics
The test compound in CH3CN (20 mM) and internal standard
(DMPA) were added to a phosphate–citric acid buffer (0.5 M, pH
7.4) pre-equilibrated to 37 °C to give final concentrations of
0.5 mM each. The mixture was incubated at 37 °C in incubating
Orbital Shaker (VWR). Aliquots were taken at time intervals and
the reactions quenched by acidification to pH 1.5 with HCl. The
samples were analysed directly by HPLC. Test analogues were ver-
ified by the elution time of standards and quantitated by their cor-
responding standard curves (R2 = 0.97–1.0). All calibration curves
were developed by plotting peak area ratios of substrate versus
internal standard as a function of concentration. The kinetics of
the loss of test analogues were established from the concentration
that remained in solution over time46 in pH 7.4 buffer at 37 °C. The
loss of NDGA in buffer at pH 7.4 follows apparent first-order kinet-
ics.31 Thus, the disappearance rate of the analogues can be
described by:
clooctadiene lignan derived from NDGA via autoxidation under
54
physiologically relevant conditions.34 Wagner and Lewis
had
reported earlier that NDGA converts to ‘activated’ NDGA in the
presence of molecular oxygen, although the structure of the oxida-
tion product was not elucidated. The authors suggested that any
biological action by NDGA treatment could result from either
NDGA itself or its oxidation products54 since many antimicrobial
and antineoplastic agents are known to function by interacting
with DNA and subsequently affecting nucleic acid metabolism.55,56
Given the important biological activities of dibenzocyclooctadiene
lignans,38,39 and the fact that many bioassay conditions for
NDGA5,35 as well as methods of Chaparral tea preparation24,34
favour autoxidation, we speculated that the intramolecular cyclisa-
tion product contributes to the broad spectrum of beneficial prop-
erties reported for NDGA. There is growing evidence in the
literature that toxicity of NDGA can be reduced through structural
modification, combined with enhanced therapeutic effects, indicat-
ing that derivatives of NDGA are potential drug targets.6
C
ln ¼ ꢁkt
Co
where Co and C are initial concentration and concentration at time t,
respectively; k is the reaction rate constant and t is time. k was
3.1. Oxidative stability of NDGA lignan analogues
ꢀ
ꢁ
C
obtained as the gradient from a plot of ln
as a function of time
Co
We evaluated the stability of the NDGA analogues in 0.5 M
phosphate buffer (pH 7.4) at 37 °C. These conditions were used
previously by us34 as they are representative of conditions compa-
rable to those used in in vitro biological activity studies. We fol-
lowed the loss of starting material (initial concentration 0.5 mM)
via HPLC-UV and determined that the lignans followed pseudo-
first order kinetics. The phenolic analogues were the most stable,
displaying half-lives of 5.49 h (2), 6.70 h (3) and 4.68 h (7); a
half-life was not determined for phenol analogue 6 as we observed
less than 5% loss of starting material over 48 h. Conversely, NDGA
and the catechol analogues were all determined to be less stable
with half-lives of 3.94 h (NDGA), 4.22 h (1), 4.62 h (4) and 1.47 h
(5) (Table 1) (see Figs. 2 and 3).
t.
HO
3
2
7
HO
HO
2
3
4
1
8
HO
HO
4
1
Phosphate
7
8'
6
buffer, pH 7.4
5
8
7'
6
5
1'
6
'
incubation at 37oC
5'
8
'
6'
2'
3'
7'
1'
5
4'
5'
2
'
OH
4'
3
'
8
HO
OH
Scheme 2. Intramolecular cyclisation of 5 to its dibenzocyclooctadiene derivative
8.

I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
7011
Table 1
We originally hypothesized that NDGA autoxidation occurred
through a radical-mediated process in which the slightly lower
pKa of the meta hydroxyl group results in a preferential deprotona-
tion/oxidation, followed by cyclisation of the carbon-centered res-
onance forms (Scheme 1 pathway A). Our proposed pathway has
subsequently been supported through physical and theoretical
studies.22 The biosynthesis of dibenzocyclooctadiene lignans in
plants is known to involve enzymatic intramolecular oxidative
coupling of phenolic precursors via a radical cation intermediate,57
which provides additional support for this mechanism. This
approach has been exploited in synthetic strategies to construct
the biaryl linkage of the dibenzocyclooctadiene lignan core struc-
ture.38,41,57 Lignans belonging to the dibenzylbutane class have
served as precursors to dibenzocyclooctadiene derivatives via
intramolecular coupling reactions using coupling reagents such
as RuO2 for phenols57 or 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ) for non-phenols.38 No similar report exists in the
literature involving a catechol containing substrate such as NDGA.
Oxidation of polyphenols by molecular oxygen has resulted in
intermolecular reaction products,42,44 and oxidation of polyphe-
nols present in tea have resulted in dimers and other oligomers,
proposed to occur via intermolecular nucleophilic attack of an
un-oxidised ring on an ortho-quinone.42,44 Thus an alternative
mechanism for NDGA dibenzocyclooctadiene formation could be
envisioned proceeding through initial ortho-quinone formation fol-
lowed by nucleophilic attack by the un-oxidised aromatic ring. This
alternative mechanism would imply that only one catechol ring
would be necessary for autoxidative lignan cyclisation to a diben-
zocyclooctadiene, provided that the second aromatic ring was suf-
ficiently nucleophilic. A potentially useful tool to assess relative
aRate of autoxidative degradation of NDGA and its analogues in 0.5 M phosphate–
citric acid buffer (pH 7.4) at 37 °C
Compound
aRate constant k (sꢁ1
)
Half-life t1/2 (h)
R2
NDGA
4.88 ꢀ 10ꢁ5
4.56 ꢀ 10ꢁ5
3.50 ꢀ 10ꢁ5
2.87 ꢀ 10ꢁ5
4.16 ꢀ 10ꢁ5
13.1 ꢀ 10ꢁ5
§
3.94
4.22
5.49
6.70
4.62
1.47
0.9936
0.9116
0.9473
0.9512
0.9413
0.9598
1
2
3
4
5
6
7
4.11 ꢀ 10ꢁ5
4.68
0.888
§ Compound 6 was stable over 48 h under these conditions (less than 5% loss of
starting material).
a
Lignan autoxidation was determined by following the loss of parent lignan
(0.5 mM) via HPLC as described in the Experimental Procedures.
We also incubated NDGA and the seven analogues (0.1 mM) in
0.5 M phosphate buffer (pH 7.4) at 37 °C for 90 min and monitored
the formation of dibenzocyclooctadiene products by HPLC. Diben-
zocyclooctadienes typically elute earlier than the parent lignan41
and HPLC peaks consistent with dibenzocyclooctadiene lignans
were isolated and further characterised by MS and NMR methods.
As shown in Scheme 2, analogue 5 underwent intramolecular cycli-
sation to form a dibenzocyclooctadiene derivative 8. A 2 min
decrease in retention time and a 3.5 nm shift to a higher absor-
bance observed by HPLC is consistent with previous34 results
obtained for NDGA under the same conditions. ESI-MS in negative
ionization mode gave consistent molecular ions at m/z 299.1 and
271.1 Da for the dibenzocyclooctadiene derivatives of NDGA and
analogue 5, respectively, (Fig. 4). Both products were two mass
units less than their acyclic lignan precursors at m/z 301.1 and
273.2, respectively, consistent with intramolecular cyclisation. 1H
and 13C NMR and high-resolution mass spectroscopic data (see
Supporting information S23) for the dibenzocyclooctadiene pro-
duct 8 derived from analogue 5, confirmed that intramolecular
cyclisation occurred under oxidative conditions consistent with
our previous report for NDGA.34 We were unable to identify prod-
ucts consistent with dibenzocyclooctadienes from any of our other
analogues.
nucleophilicity of the un-oxidised aromatic ring is Hammett
r con-
stants for the ring substituents.58 We designed our lignan ana-
logues such that our catechol analogues possessed substituents
on the second ring which rendered the ring less nucleophilic (ana-
logue 1, H = 0.0) or similar (analogue 4, m-OCH3 = 0.12, p-OCH3 = -
ꢁ0.27) compared to NDGA (m-OH = 0.12, p-OH = ꢁ0.37). We
predicted that if ortho-quinone formation was necessary for
dibenzocyclooctadiene formation, both analogues 1 and 4 should
0.00
100.00
0
2
4
6
8
(A)
-0.50
-1.00
-1.50
-2.00
-2.50
-3.00
80.00
(B)
60.00
40.00
20.00
0.00
0
1
2
3
4
5
6
7
8
9
Time (h)
Time (h)
Figure 2. Typical degradation profile (panel A) and first-order regression lines (panel B) for the catechol analogues 1 (blue), 4 (red) and 5 (green) in 0.5 M phosphate–citric
acid buffer (pH 7.4) at 37 °C.
(A)
0.00
-0.20
-0.40
-0.60
-0.80
-1.00
-1.20
-1.40
-1.60
100.00
80.00
60.00
40.00
20.00
0.00
0
2
4
6
8
(B)
0
1
2
3
4
5
6
7
8
9
Time (h)
Time (h)
Figure 3. Typical degradation profile (panel A) and first-order regression lines (panel B) for the phenol analogues 2 (blue), 3 (red) and 7 (green) in 0.5 M phosphate–citric acid
buffer (pH 7.4) at 37 °C.

7012
I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
Figure 4. Enhanced resolution ESI-MS spectrum for NDGA and compound 5: Panels (A) and (B) show NDGA before and after incubation; Panels (C) and (D) show compound 5
before and after incubation in a phosphate buffer (0.5 M pH 7.4) at 37 °C for 90 min. The decrease of 2 mass units after incubation is consistent with intramolecular
cyclisation.
Figure 5. Formation of dibenzocyclooctadiene derivatives for NDGA (panel A) and 5 (panel B) over time in the absence of GSH (blue), GSH added at time t (red) and GSH
added at time t = 0 h (green). The graphs show that the presence of GSH in the incubation mixture significantly affected the amount of dibenzocyclooctadiene formed over
time.
form dibenzocyclooctadienes, and 1 would cyclise at a greatly
reduced rate in comparison to NDGA while the cyclisation rate
for 4 would be comparable to NDGA. We observed no evidence
of oxidative cyclisation for the mono-catechol analogues 1 or 4,
although both compounds autoxidised at comparable rates to
NDGA (t1/2 NDGA: 3.94 h; 1: 4.22 h; 4: 4.62 h) forming ortho-qui-
nones (manuscript in preparation). As predicted, the phenolic
analogues 2 and 3 did not cyclise at pH 7.4. The absence of diben-
zocyclooctadiene cyclisation products for mono-catechols 1 and 4
suggests that ortho-quinone formation on one lignan ring followed
by nucleophilic attack by the unoxidised ring may be insufficient
for dibenzocyclooctadiene formation, and implies that a radical-
mediated process more accurately describes NDGA oxidative
cyclisation.

I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
7013
Table 2
Relative composition of
following 6 h incubation in phosphate–citrate buffer (pH 7.4) at 37 °C in the absence
or presence of a 20 fold excess of glutathione
a similar trend for NDGA with no intramolecular reaction occurring
in the presence of 20 fold excess GSH compared with 33% conver-
sion in the absence of GSH. We did not assay for oxidised glu-
tathione (GSSG), although it is likely that GSH acts as a radical
scavenger in this reaction. This result is consistent with a radical-
mediated cyclisation pathway.
5 or NDGA and their dibenzocyclooctadiene derivatives
Compound
Incubation for 6 h at 37 °C in
Incubation for 6 h at 37 °C in
the presence of GSH (%)
the absence of GSH (%)
SM
CycloL
Adduct
SM
8.52
66.90
CycloL
Adduct
5
95.46
87.08
4.54
—
—
12.91
91.47
33.09
—
—
3.3. meta-Phenol lignan analogues as probes of radical-
NDGA
mediated cyclisation
SM = starting material; CycloL = dibenzocyclooctadiene derivative.
In order to further test our hypothesis that NDGA cyclisation
proceeds through a di-radical intermediate, we synthesized ana-
logue 6. We incubated analogue 6 at pH 7.4 as this meta-phenol lig-
nan can form the comparable di-radical species as predicted for
NDGA, but cannot form an ortho-quinone. However, we were
unable to detect the formation of a dibenzocyclooctadiene from 6
and speculated that the stability of a radical or di-radical interme-
diate might be important for cyclisation.57 We based this assump-
tion on the observation that enterolactone, a dibenzylbutanolide
lignan, bearing meta-substituted phenols treated under oxidative
coupling conditions, did not undergo intramolecular cyclisation
although other phenolic natural dibenzylbutanolide lignans
yielded dibenzocyclooctadiene derivatives under the same condi-
tions.57 In addition, our observation was similar to a result
reported by Robin and Landais, where RuO2 was employed for
cyclisation of an ortho-phenol substituted lignan.57 Robin observed
that additional methoxy substituents on the aromatic ring facili-
tated cyclisation, suggesting that the electron-donating methoxy
substituents may be critical to cyclisation, possibly due to
enhanced stability of a radical intermediate. We investigated this
hypothesis with analogue 7 where the para-methoxy substituents
were expected to stabilize the intermediate radical formed at the
meta-hydroxy groups to allow for cyclisation. In spite of the pres-
ence of these methoxy groups on both aromatic rings, incubation
of 7 at pH 7.4 gave no intramolecular cyclisation product. It is pos-
sible that formation of the intermediate radical of 7 is inhibited by
the electron-donating effect of the adjacent methoxy group,
Table 3
aThe rate of formation of dibenzocyclooctadiene derivatives cNDGA and
8 in
phosphate buffer (pH 7.4) at 37 °C. bThe rate of degradation of dibenzocyclooctadiene
derivatives cNDGA and 8 in phosphate buffer (pH 7.4) at 37 °C
Compound
ak ꢀ 10ꢁ5 sꢁ1
aRel rate
bk ꢀ 10ꢁ5 sꢁ1
bRel rate
cNDGA
8
2.78
8.70
1
3.1
0.47
1.75
1
3.7
3.2. Effect of a nucleophilic trapping agent on the rate of
intramolecular cyclisation
The use of GSH as a nucleophilic trapping agent is common
when investigating quinone formation from catechols and phe-
nols.31 When we added GSH to pH 7.4 incubations of NDGA or ana-
logue 5 at various time points, we observed a combination of GSH
adducts and dibenzocyclooctadiene products (manuscript in
preparation). If GSH was present at the beginning of the reaction
however, we instead observed that the intramolecular reaction of
NDGA or analogue 5 was inhibited (Fig. 5). The dibenzocycloocta-
diene derivatives were formed within 30 min for both NDGA and
5 in the absence of GSH or when GSH was added at various time
points. When GSH was present in the incubation mixture however,
no intramolecular cyclisation occurred until after 3 h (NDGA) or
2 h (5). We saw no significant difference between the amounts of
dibenzocyclooctadiene formed in the absence of or with GSH
added at different time points, but product formation for both
NDGA and 5 were significantly different when GSH was incubated
together with the substrates. These observations triggered further
investigations into whether the intramolecular reaction will be
inhibited completely at a higher concentration of GSH. In the pres-
ence of 20 fold excess GSH, barely 5% of 5 converted to its dibenzo-
cyclooctadiene derivative within a 6 h incubation compared with
over 91% conversion in the absence of GSH (Table 2). We observed
although the difference between the
r
values for OH (ꢁ0.37) and
OCH3 (ꢁ0.27) is small58 and we would anticipate this to slow,
but not prevent, radical formation. Alternatively, any radical
formed may be too stable to react, although given the observations
of others this seems unlikely.44 It is more likely that the
intramolecular cyclisation reaction we observe under these condi-
tions follows a di-radical coupling mechanism as proposed by us34
and others,22 but requires a di-catechol system.
OH
OH
OH
OH
OH
OH
O
2 H
2e
O
O
HO
5b
O
5a
1
NDGA
- H+
HO
O
O
OH
HO
HO
OH
O
O
O
O
IIa
IIb
HO
cNDGA 2
Scheme 3. Modification to the proposed mechanism of intramolecular cyclisation of dibenzylbutane lignans to dibenzocyclooctadiene derivatives.

7014
I. Asiamah et al. / Bioorg. Med. Chem. 23 (2015) 7007–7014
4. Lambert, J. D.; Zhao, D.; Meyers, R. O.; Kuester, R. K.; Timmermann, B. N.; Dorr,
R. T. Toxicon 2002, 40, 1701.
5. Lambert, J. D.; Sang, S.; Dougherty, A.; Caldwell, C. G.; Meyers, R. O.; Dorr, R. T.;
Timmermann, B. N. Phytochemistry 2005, 66, 811.
3.4.
Rate
of
formation
and
degradation
of
the
dibenzocyclooctadiene derivatives
6. Lu, J. M.; Nurko, J.; Weakley, S. M.; Jiang, J.; Kougias, P.; Lin, P. H.; Yao, Q.; Chen,
C. Med. Sci. Monit. 2010, 16, RA93.
7. Kumar, S.; Wedgwood, S.; Black, S. M. DNA Cell Biol. 2007, 26, 853.
8. Ramasamy, S.; Drummond, G. R.; Ahn, J.; Storek, M.; Pohl, J.; Parthasarathy, S.;
Harrison, D. G. Mol. Pharmacol. 1999, 56, 116.
9. Aktan, S.; Aykut, C.; Yegen, B. C.; Okar, I.; Ozkutlu, U.; Ercan, S. Prostaglandins
Leukot. Essent. Fatty Acids 1993, 49, 633.
10. Goodman, Y.; Steiner, M. R.; Steiner, S. M.; Mattson, M. P. Brain Res. 1994, 654,
171.
11. Guzman-Beltran, S.; Pedraza-Chaverri, J.; Gonzalez-Reyes, S.; Hernandez-
Sanchez, F.; Juarez-Figueroa, U. E.; Gonzalez, Y.; Bobadilla, K.; Torres, M.
Oxid. Med. Cell. Longevity 2013, 2013, 375893.
12. Rothman, S. M.; Yamada, K. A.; Lancaster, N. Neuropharmacology 1993, 32,
1279.
We had previously hypothesized that the NDGA dibenzocy-
clooctadiene lignan may contribute to the biological activities
reported for NDGA and therefore stability in commonly used con-
ditions of biological evaluation may be relevant for inferring activ-
ity. We determined that the dibenzocyclooctadiene derivatives
cNDGA and 8 (derived from NDGA and analogue 5, respectively)
are unstable in pH 7.4 phosphate buffer. The rate of loss of starting
material was determined using the same conditions as for ana-
logues 1–7 and we found that compound 8 (0.5 mM) degraded at
a rate of 1.75 ꢀ 10ꢁ5 sꢁ1 in comparison with 4.70 ꢀ 10ꢁ6 sꢁ1 for
cNDGA (Table 3). Given that the dibenzocyclooctadiene derivatives
were unstable in phosphate buffer at pH 7.4, we inferred the rates
of formation from the loss of their respective substrates since they
were the only product peak by HPLC. Although the rate of cyclisa-
tion was faster for analogue 5 than NDGA (Fig. 5), the resulting
dibenzocyclooctadiene lignan 8 also degraded faster than cNDGA.
The steady rise in concentration of the dibenzocyclooctadiene
derivatives fell precipitously after 6 or 9 h for 8 and cNDGA,
respectively.
13. Shishido, Y.; Furushiro, M.; Hashimoto, S.; Yokokura, T. Pharmacol. Biochem.
Behav. 2001, 69, 469.
14. Ansar, S.; Iqbal, M.; Athar, M. Carcinogenesis 1999, 20, 599.
15. Hofmanova, J.; Soucek, K.; Pachernik, J.; Kovarikova, M.; Hoferova, Z.;
Minksova, K.; Netikova, J.; Kozubik, A. Neoplasma 2002, 49, 362.
16. Huang, J. K.; Chen, W. C.; Huang, C. J.; Hsu, S. S.; Chen, J. S.; Cheng, H. H.; Chang,
H. T.; Jiann, B. P.; Jan, C. R. Life Sci. 2004, 75, 2341.
17. Moody, T. W.; Leyton, J.; Martinez, A.; Hong, S.; Malkinson, A.; Mulshine, J. L.
Exp. Lung Res. 1998, 24, 617.
18. Rowe, D. L.; Ozbay, T.; Bender, L. M.; Nahta, R. Mol. Cancer Ther. 2008, 7, 1900.
19. Soriano, A. F.; Helfrich, B.; Chan, D. C.; Heasley, L. E.; Bunn, P. A., Jr.; Chou, T. C.
Cancer Res. 1999, 59, 6178.
20. Seufferlein, T.; Seckl, M. J.; Schwarz, E.; Beil, M.; v Wichert, G.; Baust, H.; Luhrs,
H.; Schmid, R. M.; Adler, G. Br. J. Cancer 2002, 86, 1188.
4. Conclusions
21. Craigo, J.; Callahan, M.; Huang, R. C.; DeLucia, A. L. Antiviral Res. 2000, 47, 19.
22. Galano, A.; Macias-Ruvalcaba, N. A.; Medina Campos, O. N.; Pedraza-Chaverri, J.
J. Phys. Chem. B 2010, 114, 6625.
23. Floriano-Sanchez, E.; Villanueva, C.; Medina-Campos, O. N.; Rocha, D.;
Sanchez-Gonzalez, D. J.; Cardenas-Rodriguez, N.; Pedraza-Chaverri, J. Free
Radical Res. 2006, 40, 523.
24. Sheikh, N. M.; Philen, R. M.; Love, L. A. Arch. Intern. Med. 1997, 157, 913.
25. Bhattacherjee, P.; Boughton-Smith, N. K.; Follenfant, R. L.; Garland, L. G.; Higgs,
G. A.; Hodson, H. F.; Islip, P. J.; Jackson, W. P.; Moncada, S.; Payne, A. N. Ann. N.
Y. Acad. Sci. 1988, 524, 307.
26. Salari, H.; Braquet, P.; Borgeat, P. Prostaglandins Leukot. Med. 1984, 13, 53.
27. Smith, A. Y.; Fau, F. R.; Gardner, K. D. J.; Fau-Davis, C. J. J.; Davis, C. J. J. Urol.
1994, 152, 2089.
28. Stickel, F.; Egerer, G.; Seitz, H. K. Public Health Nutr. 2000, 3, 113.
29. Goodman, T.; Grice, H. C.; Becking, G. C.; Salem, F. A. Lab. Invest. 1970, 23, 93.
30. Gordon, D. W.; Rosenthal, G.; Hart, J.; Sirota, R.; Baker, A. L. JAMA 1995, 273,
489.
31. Billinsky, J. L.; Marcoux, M. R.; Krol, E. S. Chem. Res. Toxicol. 2007, 20, 1352.
32. Cavalieri, E. L.; Li, K.; Balu, N.; Saeed, M.; Devanesan, P.; Higginbotham, S.;
Zhao, J.; Gross, M. L.; Rogan, E. G. Carcinogenesis 2002, 23, 1071.
33. Grice, H. C.; Becking, G.; Goodman, T. Food Cosmet. Toxicol. 1968, 6, 155.
34. Billinsky, J. L.; Krol, E. S. J. Nat. Prod. 2008, 71, 1612.
35. McDonald, R. W.; Bunjobpon, W.; Liu, T.; Fessler, S.; Pardo, O. E.; Freer, I. K.;
Glaser, M.; Seckl, M. J.; Robins, D. J. Anticancer Drug Des. 2001, 16, 261.
36. Choi, Y. W.; Takamatsu, S.; Khan, S. I.; Srinivas, P. V.; Ferreira, D.; Zhao, J.; Khan,
I. A. J. Nat. Prod. 2006, 69, 356.
In conclusion we have determined that cyclisation of NDGA lig-
nan analogues to a dibenzocyclooctadiene does not readily occur
for mono-catechol lignan analogues, suggesting that catechol oxi-
dation to an ortho-quinone followed by nucleophilic attack by
the un-oxidised ring is an unlikely pathway. Our results lend fur-
ther support to a di-radical coupling mechanism as previously sug-
gested.22,34 We were unable to show that meta-substituted phenols
can cyclise in this manner, suggesting that the di-radical pathway
describes the intramolecular cyclisation process for catechols, but
not phenols. An intriguing possibility remains, which we have out-
lined in Scheme 3, involving nucleophilic attack of an ortho-qui-
none by an ionised ring. NDGA has a pKa of 9.3059 and the
intramolecular cyclisation of NDGA is a base-catalysed process,31
suggesting that this mechanism would be favoured at more alka-
line pH. We are currently investigating this pathway using a mixed
catechol–phenol lignan. The loss of the methyl substituents on the
aliphatic side-chain of NDGA enhances the rate of dibenzocyclooc-
tadiene formation, likely as a result of diminished steric interac-
tions during cyclisation. Finally, we determined that the stability
of the dibenzocyclooctadienes is comparable to that of their pre-
cursors, suggesting that any activity associated with the cyclised
lignans may be related to their oxidised species. We are currently
studying the oxidation products of cNDGA and will report on this
in due course.
37. Charlton, J. L. J. Nat. Prod. 1998, 61, 1447.
38. Chang, J.; Reiner, J.; Xie, J. Chem. Rev. 2005, 105, 4581.
39. Ward, R. S. Nat. Prod. Rep. 1995, 12, 183.
40. Xia, Y.; Bi, W.; Zhang, Y. J. Chil. Chem. Soc. 2009, 54, 428.
41. Coleman, R. S.; Gurrala, S. R.; Mitra, S.; Raao, A. J. Org. Chem. 2005, 70, 8932.
42. Krafczyk, N.; Heinrich, T.; Porzel, A.; Glomb, M. A. J. Agric. Food Chem. 2009, 57,
6838.
43. Spanos, G. A.; Wrolstad, R. E. J. Agric. Food Chem. 1992, 40, 1478.
44. Roginsky, V.; Alegria, A. E. J. Agric. Food Chem. 2005, 53, 4529.
45. Maini, S.; Hodgson, H. L.; Krol, E. S. J. Agric. Food Chem. 2012, 60, 6966.
46. Li, N.; Taylor, L. S.; Ferruzzi, M. G.; Mauer, L. J. J. Agric. Food Chem. 2012, 60,
12531.
47. Mizufune, H.; Nakamura, M.; Mitsudera, H. Tetrahedron 2006, 62, 8539.
48. Johnson, W. S.; McCloskey, A. L.; Dunnigan, D. A. J. Am. Chem. Soc. 1950, 72, 514.
49. Xia, Y.; Zhang, Y.; Wang, W.; Ding, Y.; He, R. J. Serb. Chem. Soc. 2010, 75, 1325.
50. Lee, K. H. J. Nat. Prod. 2004, 67, 273.
Acknowledgements
We would like to thank the Natural Sciences and Engineering
Council of Canada (RPGIN 238538) for funding this work.
Supplementary data
51. Kuo, Y.; Huang, H.; Kuo, L. Y.; Chen, C. J. Org. Chem. 1999, 64, 7023.
52. Wang, J. P.; Raung, S. L.; Hsu, M. F.; Chen, C. C. Br. J. Pharmacol. 1994, 113, 945.
53. Ko, K. M.; Ip, S. P.; Poon, M. K.; Wu, S. S.; Che, C. T.; Ng, K. H.; Kong, Y. C. Planta
Med. 1995, 61, 134.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.039.
54. Wagner, P.; Lewis, R. A. Biochem. Pharmacol. 1980, 29, 3299.
55. Wells, R. D.; Larson, J. E. J. Mol. Biol. 1970, 49, 319.
56. Cozzarelli, N. R. Annu. Rev. Biochem. 1977, 641.
References and notes
57. Robin, J. P.; Landais, Y. J. Org. Chem. 1988, 53, 224.
58. Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and
Biology; Wiley-Interscience, 1979.
59. Moridani, M. Y.; Siraki, A.; Chevaldina, T.; Scobie, H.; O’Brien, P. J. Chem. Biol.
Interact. 2004, 147, 297.
1. Tyler, V. E. The Honest Herbal, a Sensible Guide to the use of Herbs and Related
Remedies; Pharmaceutical Products Press: New York, 1994.
2. Lambert, J. D.; Dorr, R. T.; Timmermann, B. N. Pharm. Biol. 2004, 42, 149.
3. Arteaga, S.; Andrade-Cetto, A.; Cardenas, R. J. Ethnopharmacol. 2005, 98, 231.