Diamination by N-coupling using a commercial laccase

Article abstract of DOI:10.1016/j.bmc.2010.01.025

Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed by an immobilised commercial laccase, Denilite II Base, from Novozymes. The amine and the p-hydrobenzoquinone was reacted under mild conditions (at room t

Full text of DOI:10.1016/j.bmc.2010.01.025

Bioorganic & Medicinal Chemistry 18 (2010) 1406–1414
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Diamination by N-coupling using a commercial laccase
*
Kevin W. Wellington , Paul Steenkamp, Dean Brady
CSIR Biosciences, Modderfontein 1645, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed
by an immobilised commercial laccase, DeniliteÒ II Base, from Novozymes. The amine and the p-hydro-
benzoquinone was reacted under mild conditions (at room temperature and at 35 °C) in a reaction vessel
open to air in the presence of laccase and a co-solvent to afford, exclusively, the diaminated p-benzoqui-
none. These compounds may have potential antiallergic, antibiotic, anticancer, antifungal, antiviral and/
or 5-lipoxygenase inhibiting activity.
Received 8 January 2010
Accepted 11 January 2010
Available online 18 January 2010
Keywords:
Biocatalysis
Ó 2010 Elsevier Ltd. All rights reserved.
Green chemistry
p-Hydrobenzoquinones
p-Benzoquinones
Diaminobenzoquinones
Laccase
DeniliteÒ II Base
Oxidative enzymes
1. Introduction
plastic drugs that are in use contain an aminoquinone moiety. The
common antibiotic, Mitomycin, is an example of such a drug. Nakij-
Laccases are a group of oxidative enzymes that are able to ab-
stract hydrogen from phenolic hydroxyl groups by using molecular
oxygen as an electron acceptor, resulting in phenoxy radicals that
undergo a broad range of reactions.1–3 Laccases catalyse the oxida-
tion of organic compounds such as methoxyphenols, phenols,
o- and p-diphenols, aminophenols, polyphenols, polyamines, and
lignin-related molecules.1–3 Laccase applications span from the
textile to the pulp and paper industries, and from food applications
to bioremediation processes.2
iquinone-derivatives22 andherbimycin-derivatives23 areothersthat
are under development. Reports of simple aminoquinones possess-
ing activity against a number of cancer cell-lines,24–26 antiallergic
or 5-lipoxygenase inhibiting activity27 has prompted research into
new routes to the synthesis of aminoquinones.
In this paper we report on the synthesis of diaminobenzoquinon-
esusingthecommerciallaccase, DeniliteII Base(onaninertsupport)
from Novozymes. This laccase is from the Aspergillus sp. and is used
for bleaching denim in the textile industry. The synthesis of diami-
nated benzoquinones, catalysed by a fungal laccase, has been re-
ported previously, but on a smaller range of substrates.28 This
paper is, to the best of our knowledge, the first on the synthesis of
diaminobenzoquinones using a commercial laccase on an inert
support.
The high oxidative selectivity exhibited by laccases in aqueous
solutionhas made themattractive for a variety of oxidationsin green
chemistry. As a result of these properties, their application as bio-
catalysts in organic chemistry has been investigated. Several exam-
ples have been reported in the literature such as the synthesis of
actinocin4 and cinnabarinic acid,5 the synthesis of substituted tria-
zolobenzothiadiazinones,6 dimerization of various compounds such
as estradiol,7 penicillin X,8 bisphenol A9 salicyclic acids10 synthesis
of polymers,11–13 oxidative coupling of hydroquinone and mithram-
icine14 or (+)-catechin,15 oxidation of substituted imidazoles.16
2. Results and discussion
The p-hydrobenzoquinones (1a–d) and primary amines (2a–f)
used in this study are depicted in Figure 1.
It was anticipated that the reaction of a hydroquinone 1a–d
with a primary amine 2a–f would afford a monoaminated benzo-
quinone 3 or a diaminated benzoquinone 4 or, both 3 and 4
(Scheme 1).
derivatization of dihydrocaffeic acid,17 -tryptophan18 and para-
L
dihydroxylated compounds,19 oxidative domino reactions of dib-
enzofuranones20 and naphthoquinone synthesis.21 Several antineo-
In our first approach, 1 equiv of primary amine was reacted with
1 equiv of p-hydroquinone in an attempt to obtain the monoaminat-
ed product 3. The reactions were conducted at room temperature
* Corresponding author. Tel.: +27 11 605 2749; fax: +27 11 608 3020.
E-mail addresses: kwellington@csir.co.za, kwwellington@gmail.com (K.W. Well
ington).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.025

K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
1407
OH
O
OH
O
OH
O
Table 1
OH
Synthesised diaminated p-benzoquinones (yield in parentheses) at room temperature
in 10% aqueous MeOH
O
O
Entry Hydroquinone Primary
amine
Reaction
time (h)
Methoda Diaminated
product
OH
OH
OH
OH
1d
1a
1b
1c
1
2
1a
1a
1b
1b
1b
1c
1c
1c
1c
1d
1d
2e
2b
2a
2b
2c
2a
2b
2f
116
95
171
95
A
A
A
A
A
A
A
A
A
A
A
5 (2%)
6 (32%)
7 (11%)
8 (14%)
9 (4%)
10 (26%)
11 (20%)
12 (16%)
13 (11%)
14 (33%)
15 (19%)
3
4
5
6
7
8
OH
90
172
144
142
149
172
148
N
9
10
11
2c
2a
2c
NH2
NH2
NH2
NH2
NH2
NH2
2a
2b
2f
2d
2e
2c
a
Method A—0.0750 g laccase, 1 equiv amine, MeOH (1.0 mL).
Figure 1. The p-hydrobenzoquinones and primary amines used in this study.
using a mixture of water, 10% methanol and succinate-lactate buffer
(pH 4.5). No monoaminated products were obtained. The results for
these reactions are summarised in Table 1.
O
4
O
H
N
3
2
From the analysis of the data for the isolated products of these
reactions reported in Table 1 it was found that only the diaminated
product was observed for each of these reactions despite the use of
only 1 equiv of primary amine. The formation of the diaminated
product accounts for the generally low yields (between 2% and
33%) observed for these reactions. A characteristic red or brown
colour of the products was indicative of the formation of quinon-
amines and simplified product isolation. From the 1H NMR spec-
trum of product 14 two aniline residues and one proton, H-6, for
the quinone ring was observed (Fig. 2).
O
5
6
N
H
1
O
Figure 2. The structure of compound 14 with atom numbering.
products as discussed by Chakraborty et al.29 The results for these
reactions are summarised in Table 2.
From the data in Table 2 it can be seen that the yields range
from moderate (entries 4 and 15) to poor (entries 1, 3 and 8) when
the reactions are done at 35 °C. For 5 (entry 1) only a 4% increase in
yield was observed upon heating using Method C for which previ-
ously only a 2% yield was observed at room temperature using
Method A. The use of Method E, however, had increased the yield
by almost fourfold (22% from 6%) and 11-fold (22% from 2%, Meth-
od A, Table 1) for 5. For 17 a yield of only 5% was obtained (entry 3)
upon heating. For 11 the yield only increased by 5% using Method
G (entry 7). In the case of 15 the yield was increased by almost
twofold using Method G (entry 10). For 13 the yield increased by
almost fivefold (52% from 11%) using Method D (entry 15). Both
methods B and D proved successful for synthesising 7, but only
afforded the product in 16% and 25% yield (entry 14), respectively.
From the results in Table 2 it was concluded that the reactions did
not go to completion and this may be attributed to the hydroqui-
none not being completely converted to the quinone. The struc-
tures of the diaminated products synthesised at 35 °C are shown
in Figure 4.
The amine proton is also observed, but no phenolic hydroxy
groups. For the 13C NMR spectrum, signals characteristic for qui-
nones are observed in the 175–180 ppm range. The molecular
ion peak of 361.1187 in the HRMS spectrum of 14 correlated with
the calculated mass of 361.1188. This mass could be attributed to
the amination of 1d with two molecules of 2a with the loss of six
hydrogen atoms. The structures of the diaminated products from
the reactions in Table 1 are shown in Figure 3.
From the results it was evident that at least 2 equiv of primary
amine would be required in order to increase the yield of the dia-
minated product from these reactions. The conditions for these
reactions also had to be optimised in order to speed up the forma-
tion of the product. For this purpose certain reactions were re-
peated and conducted at 35 °C using at least 2 equiv (Method B)
and 3 equiv (Method C) and the total volume (5 mL) of these reac-
tions was half of that used in Method A (10 mL). The solvent was
also changed from 10% methanol to 20% dimethylformamide, a
more polar solvent, to increase the solubility of the substrates.
Since the hydroquinone was observed by TLC for these reactions,
it was concluded that the concentration of laccase was not sufficient
to catalyse the conversion of all the hydroquinone to the quinone. In
order to increase the yield, it was decided to conductreactions with a
threefold higher concentration of laccase (Methods D, E, F and G). It
was also decided to increase the equivalents of the amine (Method F,
4 equiv) since this would promote the formation of the diaminated
As a consequence it was decided to investigate a set of reactions
in which the quantity of the Denilite II Base was at least doubled
(from 0.23 g to 0.48 g, Methods H and I). It was also decided to
add the enzyme at different time intervals to ensure fresh enzyme
and to circumvent the possibility of denaturing all at once. The re-
sults of this investigation are reported in Table 3 from which it is
apparent that doubling the quantity of enzyme does increase the
OH
O
O
Denilite
II Base
R
R
RHN
R
RNH2
and/or
+
NHR
NHR
2a-f
OH
O
O
4
1a-d
3
R = H, COCH3, CO2CH3, CO2CH2CH3,
Scheme 1.

1408
K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
O
H
N
O
H
N
6
8
5
N
H
N
H
O
O
O
O
O
O
H
N
H
N
7
N
H
N
H
O
O
O
O
O
O
H
N
9
11
13
H
N
10
12
O
OH
HO
N
H
N
H
O
O
O
O
O
O
H
N
H
N
N
O
O
O
N
H
N
N
H
O
O
O
O
O
O
H
N
14
H
N
O
OH
HO
N
H
N
H
O
O
O
O
15
H
N
O
OH
HO
N
H
O
Figure 3. Structures of diaminated products 5–15 synthesised at room temperature.
Table 2
Synthesised diaminated p-benzoquinones (yield in parentheses) at 35 °C in 20%
aqueous DMF
For 7 there has only been a slight increase in yield, 7% (entry 2),
when compared to Method D. A twofold increase (16%) for 7 (entry
2) is observed when compared to Method B. Similarly for 13 and
21, a 6% and 9% yield increase was observed when compared to
using Method D. A sevenfold increase in yield (28%, previously
4%) is observed for 9 when compared to using Method A.
Entry Hydroquinone Primary
amine
Reaction
time (h)
Methoda Diaminated
product
1
1a
2e
72
72
96
68
96
96
72
72
96
72
72
92
96
96
96
72
72
C
E
B
C
B
F
E
G
E
G
G
C
C
C
B
D
D
5 (6%)
5 (22%)
16 (16%)
17 (5%)
8 (52%)
18 (20%)
19 (19%)
11 (25%)
20 (12%)
21 (21%)
15 (36%)
22 (20%)
12 (21%)
23 (32%)
7 (16%)
7 (25%)
13 (52%)
In accordance with the literature,30,31 the second substitution of
monoaminated substances usually take place para to the first ami-
nation site. In these compounds, C2 is believed to be the first ami-
nation site and C5 the second. The diaminated products have a
characteristic aromatic proton peak in the 5.7–6.6 ppm region for
the H6/H3 proton in the 1H NMR spectrum and a characteristic
peak in the region 90–100 ppm in the 13C spectrum. This downfield
shift is attributed to the deshielding of the H5/H3 nucleus caused
by the electron withdrawing nitrogen and oxygen substituents
on the ring. A proposed mechanism for nuclear diamination is
shown in Figure 5.
It is believed that the role of laccase is simply that of an oxidant,
three laccase oxidations occur before the diaminoquinones are
formed. It is assumed that the second amination is kinetically fas-
ter than the first due to ring activation by the first amination.
Niedermeyer et al. has reported on the synthesis of monoamino-
quinones and diaminobenzoquinones using fungal laccases (EC
1.10.3.2)fromTrametesspec. and Myceliophthora thermophila.28 Only
four diaminobenzoquinones were synthesized using a molar ratio of
up to 5:1 of primary amine to hydroquinone in the absence of a co-
2
3
4
5
6
7
8
9
10
11
12
13
14
1b
1b
1b
1c
1c
1c
1d
1d
1d
1b
1c
1d
1b
2a
2e
2b
2d
2e
2b
2e
2b
2c
2f
2f
2f
2a
15
1c
2c
a
Method B—0.075 g laccase, 2 equiv amine, DMF (1.0 mL). Method C—0.075 g
laccase, 3 equiv amine, DMF (1.0 mL). Method D—0.23 g laccase, 2 equiv amine,
DMF (1.0 mL). Method E—0.23 g laccase, 3 equiv amine, DMF (1.0 mL). Method F—
0.23 g laccase, 4 equiv amine, DMF (1.5 mL). Method G—0.23 g laccase, 2 equiv
amine, DMF (1.0 mL).
yield of the products 5, 9, 12 and 22 (entries 2–5) except for 5 (en-
try 1) for which the yield has decreased by 7%.

K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
1409
O
O
O
O
16
17
19
H
N
H
N
N
H
N
H
O
O
O
O
O
H
N
O
18
H
N
O
O
OH
HO
N
H
N
H
O
O
O
O
O
O
20
22
21
23
H
N
H
N
O
O
N
H
N
H
O
O
O
O
O
O
H
N
H
N
N
N
O
N
H
N
N
H
N
O
O
Figure 4. Structures of diaminated products 16–23 synthesised at 35 °C.
In summary, the one-pot, three-step biocatalytic method allows
direct access to diaminobenzoquinones from the p-hydrobenzo-
quinones and affords only the diaminated product. This method
has eliminated the use of an iodinated intermediate, a palladium
catalyst and a phosphine ligand. While the laccase, Denilite II Base,
may not be the ideal enzyme for these reactions, there is potential
for the development of other laccase enzymes (preferably with
high enzyme activity) to be used for the synthesis of
diaminobenzoquinones.
Table 3
Synthesised diaminated p-benzoquinones at 35 °C in 20% aqueous DMF
Entry Hydroquinone Primary
amine
Reaction
time (h)
Methoda Diaminated
product
1
2
3
4
5
1a
1b
1b
1c
1d
2e
2a
2c
2c
2b
72
72
72
72
72
H
H
I
I
I
5 (15%)
7 (32%)
9 (28%)
13 (58%)
21 (30%)
a
Method H—0.48 g laccase, 5 equiv amine, DMF (1.0 mL). Method I—0.48 g lac-
case, 3 equiv amine, DMF (1.0 mL).
3. Conclusions
The commercial laccase, Denilite II Base, can be used to access
diaminobenzoquinones starting from p-dihydroxylated benzoic
acid derivatives and aryl and alkyl primary amines. The yields of
products from the reaction of primary aryl amines with p-dihy-
droxylated benzoic acid derivatives are generally higher than those
from the reaction with alkyl primary amines. The formation of
product is affected by factors such as the nucleophilicity of the
amines, the electrophilicity of the hydroquinones, number of
equivalents of amine, solubility and temperature.
solvent in sodium acetate buffer, pH 5 (laccase from T. spec., final
activity 0.15 units mLÀ1) or citrate-phosphate buffer, pH 7 (laccase
from M. thermophila, final activity 1.0 units mLÀ1). For this method
the formation of mono-aminobenzoquinones was favoured as well
as mixtures of the latter and the diaminobenzoquinone.
Chemical methods for nuclear amination have been reported in
the literature, but these reports are few due to the susceptibility of
the amino group to oxidation and hydrolysis. One method involves
first halogenating (iodinating) the p-benzohydroquinone followed
by coupling to the primary amine using a palladium catalyst and
4. Experimental section
4.1. General
a triphenylphosphine ligand while refluxing under argon.29
A
drawback of this method is that it affords a mixture of monoami-
nated and diaminated benzoquinones. Another method involves
the reaction of the iodinated p-benzohydroquinone with the pri-
mary amine at room temperature in a vessel open to air. A draw-
back of this method is that a mixture of iodinated monoaminated
benzoquinone, monoaminated benzoquinone and diaminated
benzoquinone is obtained.29
Well-known chemical oxidants that have been used to achieve
nuclear amination of simple p-hydroquinones with simple primary
aromatic amines are cupric acetate, silver (I) oxide and sodium
iodate.32 Amination can be accomplished more conveniently using
sodium iodate.32
Proton nuclear magnetic resonance (1H NMR) spectra were re-
corded on a Varian Mercury 200 MHz spectrometer. Carbon-13 nu-
clear magnetic resonance (13C NMR) spectra were recorded on the
same instruments at 50 MHz. Chemical shifts are reported in ppm
relative to the solvent peaks. High-resolution mass spectra were
recorded on a Waters HPLC coupled to a Synapt HDMS mass spec-
trometer. Reactions were monitored by thin layer chromatography
(TLC) on aluminium-backed Merck silica gel 60 F254 plates. Gravity
column chromatography was done using Merck Silica Gel 60
(70–230 mesh). Melting points were determined using a Glassco
melting point apparatus and are uncorrected.

1410
K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
O
O
O
OH
OH
R
R
R
R
laccase
NH2R
NHR'
H2NR'
H
O
O
OH
quinone
1a-d
hydroquinone
-
-H
OH
O
O
O
R
R
laccase
R'H2N
R
R'NH 2
+
NHR'
NHR'
-H
NHR'
O
O
aminohydroquinone
aminoquinone
O
O
O
H
R'HN
R
R'H2N
R
R'HN
R
laccase
5-23
+
-H
NHR'
O
NHR'
NHR'
OH
OH
diaminohydroquinone
diaminoquinone
Figure 5. A proposed mechanism for nuclear diamination.
4.2. Materials
4.3.4. Method D
The same as Method B, except that three times the amount of
enzyme was used.
All chemicals were reagent grade materials.
4.2.1. Substrates
The p-hydroquinones and the primary amines were obtained
from Sigma–Aldrich South Africa.
4.3.5. Method E
The same as Method B, except that 3 equiv (0.9 mmol) of pri-
mary amine and three times the amount of enzyme was used.
4.2.2. Enzymes
4.3.6. Method F
Myceliophthora thermophilia laccase was obtained from Novo-
zymes as DeniliteÒ II Base (800 U gÀ1) on an inert carrier.
The same as Method E, except that DMF (1.5 mL) and 4 equiv
(1.2 mmol) of primary amine was used.
4.3. Diamination of 2,5-dihydroxybenzoic acid derivatives with
aromatic and aliphatic primary amines
4.3.7. Method G
The primary amine (0.6 mmol, 2 equiv) was added to a mixture
containing the 2,5-dihydroxybenzoic acid derivative (0.3 mmol),
succinate-lactate buffer (1.0 mL, 1.0 M, pH 4.5), water (3.0 mL),
DMF (1.0 mL) and Denilite II Base (0.23 g). The reaction mixture
was heated at 35 °C and monitored by TLC. After stirring, the sol-
vent was removed on the rotary evaporator to afford a dark-brown
residue that was purified by flash chromatography.
The following methods were used for the synthesis of the dia-
minated products.
4.3.1. Method A
The primary amine (0.3 mmol, 1 equiv) was added to a mixture
containing 2,5-dihydroxybenzoic acid derivative (0.3 mmol), succi-
nate-lactate buffer (2.0 mL, 1.0 M, pH 4.5), water (7.0 mL), MeOH
(1.0 mL) and Denilite II Base (0.075 g). The reaction mixture was
stirred at room temperature and monitored by TLC. After stirring,
the solvent was removed on the rotary evaporator to afford a
dark-brown residue that was purified by flash chromatography.
4.3.8. Method H
The primary amine (1.5 mmol, 5 equiv) was added to a mixture
containing 2,5-dihydroxybenzoic acid derivative (0.3 mmol), succi-
nate-lactate buffer (1.0 mL, 1.0 M, pH 4.5), water (3.0 mL), DMF
(1.0 mL) and Denilite II Base (0.08 g). The reaction mixture was
heated and stirred at 35 °C and after 1 h more Denilite II Base
(0.08 g) was added and after an additional hour an additional Den-
ilite II Base (0.08 g) was added. After 15 h Denilite II Base (0.24 g)
was added at 1 h intervals in 0.08 g quantities. The total mass of
Denilite II Base that was added is 0.48 g. After 72 h the solvent
was removed on the rotary evaporator to afford a dark-brown res-
idue that was purified by flash chromatography.
4.3.2. Method B
The primary amine (0.6 mmol, 2 equiv) was added to a mixture
containing 2,5-dihydroxybenzoic acid derivative (0.3 mmol), succi-
nate-lactate buffer (1.0 mL, 1.0 M, pH 4.5), water (3.0 mL), DMF
(1.0 mL) and Denilite II Base (0.075 g). The reaction mixture was
heated at 35 °C and monitored by TLC. After stirring, the solvent
was removed on the rotary evaporator to afford a dark-brown res-
idue that was purified by flash chromatography.
4.3.3. Method C
4.3.9. Method I
The same as Method B, except that 3 equiv (0.9 mmol) of pri-
mary amine was used.
The same as Method H except that 3 equiv (0.9 mmol) of the
primary amine was used.

K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
1411
4.4. 2,5-Bis-hexylamino-[1,4]benzoquinone 5
4.7. 3-Acetyl-2,5-bis-(4-isopropyl-phenylamino)-
[1,4]benzoquinone 8
4.4.1. Method A
Stirring time = 116 h. Purification by flash chromatography (sil-
ica/CHCl3–MeOH, 80:1) to afford a dark-brown solid (0.0017 g,
2.0%). Rf = 0.31 (MeOH/CHCl3, 1:19).
4.7.1. Method A
Stirring time = 95 h. Purification by flash chromatography (sil-
ica/CHCl3–hexane, 3:1) to afford a red-brown solid (0.0180 g,
14%). Rf = 0.28 (CHCl3).
4.4.2. Method B
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:5) to afford an orange solid (0.0052 g, 6%).
Rf = 0.29 (EtOAc/hexane, 1:4).
4.7.2. Method B
Stirring time = 96 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:9) to afford a dark-brown solid (0.0650 g,
52%). (M+H+ Found: 417.2161. C26H26N2O3 requires M+H,
417.2178); Rf = 0.35 (EtOAc/hexane, 1:9). Mp = 139–141 °C. 1H
NMR (200 MHz, CDCl3): d = 1.15–1.4 (12H, m, 4 Â CH3), 2.64 (3H,
br s, CH3), 2.80–3.10 (2H, m, 2 Â CH), 6.01 (1H, s, ArH), 7.00–7.30
(8H, m, ArH) and 8.16 (1H, br s, NH); 13C NMR (100 MHz, CDCl3):
d = 23.9, 32.6, 33,8, 122.7, 124.0, 127.0, 127.6, 134.6, 136.8, 146.6,
147.1, 147.8, 177.8, 178.4, 191.8 and 200.7.
4.4.3. Method E
Stirring time = 94 h. Purification by flash chromatography (silica/
MeOH–CHCl3, 1:100) to afford a dark-brown solid (0.0134 g, 22%).
(M+H+ Found: 307.2390. C18H31N2O2 requires M+H, 307.2386);
Rf = 0.15 (CHCl3). Mp = 114–116 °C. 1H NMR (200 MHz, CDCl3):
d = 1H NMR (200 MHz, CDCl3): d = 0.87 (6H, t J = 1.8, and 5.0 Hz,
2 Â CH3), 1.30 (10H, br s, 5 Â CH2), 1.50–1.80 (6H, m, 3 Â CH2),
3.13 (4H, q J = 6.0, 6.8 and 7.0 Hz, 2 Â CH2), 5.29 (1H, s, ArH) and
6.58 (1H, br s, NH); 13C NMR (100 MHz, CDCl3): d = 14.0, 22.5, 26.7,
28.2, 31.4, 42.6, 92.6, 151.3 and 178.1.
4.8. 3-Acetyl-2,5-bis-(4-(-2-hydroxy-ethyl-phenylamino)-
[1,4]benzoquinone 9
4.4.4. Method H
4.8.1. Method A
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:8, 1:6, 1:4) to afford an orange-red crystalline
solid (0.0147 g, 15%). Rf = 0.37 (EtOAc/hexane, 1:4).
Stirring time = 90 h. Purification by flash chromatography (sil-
ica/CHCl3–hexane, 3:1) to afford a black solid (0.0050 g, 4%).
Rf = 0.43 (CHCl3).
4.5. 2,5-Bis-(4-isopropyl-phenylamino)-[1,4]benzoquinone 6
4.8.2. Method I
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:1; EtOAc; EtOAc/MeOH, 9:1) to afford a black
solid (0.0377 g, 28%). (M+H+ Found: 419.1601. C23H19N2O6 requires
M+H 419.1243); Rf = 0.29 (EtOAc). Mp = 160–162 °C. 1H NMR
(200 MHz, CDCl3): d = 2.65 (3H, s, CH3), 2.88 (4H, t J = 6.0 and
12.2 Hz, 2 Â CH2), (4H, t J = 6.4 and 6.6 Hz, 2 Â CH2), 6.00 (1H, s,
ArH), 7.00–7.40 (8H, m, ArH) and 8.18 (1H, s, NH); 13C NMR
(100 MHz, CDCl3): d = 32.7, 38.6, 38.8, 63.4, 63.5, 122.9, 124.4,
126.4, 129.7, 129.3, 130.3, 135.4, 136.9, 137.6, 139.4, 146.4, 177.8
and 178.4.
4.5.1. Method A
Stirring time = 95 h. Purification by flash chromatography (silica/
CHCl3) to afford a dark-brown solid (0.0360 g, 32%). (M+H+ Found:
375.2088. C24H24N2O2 requires M+H 375.2073. Rf = 0.34 (CHCl3).
Mp = 241–242 °C [lit.33 189.5–190 °C]. 1H NMR (200 MHz, CDCl3):
d = 1.28 (12H, m, 4 Â CH3), 2.95 (2H, 2 Â CH), 6.05 (1H, s, 2 Â ArH),
7.15–7.40 (8H, m, ArH) and 8.11 (2H, s, 2 Â NH); 13C NMR
(100 MHz, CDCl3): d = 24.2, 34.0, 95.7, 123.0, 127.8, 135.0, 147.3
and 180.3.
4.6. 3-Acetyl-2,5-bis-phenylamino-[1,4]benzoquinone 7
4.9. 3,6-Dioxo-2,5-bis-phenylamino-cyclohexa-1,4-
dienecarboxylic acid methyl ester 10
4.6.1. Method A
Stirring time = 171 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:4) to afford an orange-brown solid (0.0110 g,
11%). Rf = 0.32 (CHCl3).
4.9.1. Method A
Stirring time = 172 h. Purification by flash chromatography (sil-
ica/CHCl3) to afford a dark-brown solid (0.0270 g, 26%). (Found:
MÀH+ 347.1027. C20H16N2O6 requires MÀH 347.1032); Rf = 0.13
(CHCl3). Mp = 185–187 °C [lit.33 202–203 °C]. 1H NMR (200 MHz,
CDCl3): d = 3.16 (3H, s CH3), 6.11 (1H, s, ArH), 7.15–7.60 (10H, m,
ArH), 8.19 (1H, br s, NH) and 8.72 (1H, br s, NH); 13C NMR
(100 MHz, CDCl3): d = 51.4, 95.9, 123.0, 123.8, 126.5, 127.14,
129.34, 129.7, 136.7, 137.3, 146.7, 177.0 and 178.6.
4.6.2. Method B
Stirring time = 96 h. Purification by flash chromatography (sil-
ica/CHCl3; MeOH/CHCl3, 1:19, 3:7, 2:3, 1:1) to afford an orange–
brown solid (0.0144 g, 16%). (M+H+ Found: 333.1228. C20H16N2O3
requires M+H 333.1229); Rf = 0.15 (MeOH/CHCl3, 1:9). Mp = 169–
171 °C. 1H NMR (200 MHz, CDCl3): d = 2.68 (3H, s, CH3), 6.07 (1H,
s, ArH) 7.00–7.60 (10H, m, ArH) and 8.19 (2H, s, 2 Â NH); 13C
NMR (100 MHz, CDCl3): d = 32.9, 99.3, 123.0, 124.5, 126.4, 127.3,
129.3, 130.0, 137.3, 139.4, 146.6, 178.1 and 178.8.
4.10. 2,5-Bis-(4-isopropyl-phenylamino-3,6-dioxo-cyclohexa-
1,4-dienecarboxylic acid methylester 11
4.6.3. Method D
4.10.1. Method A
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/CHCl3; MeOH/CHCl3, 1:100, 3:100; 5:100) to afford a dark-
brown solid (0.0252 g, 25%). (M+H+ Found: 333.1234. C20H16N2O3
requires M+H, 333.1239); Rf = 0.09 (CHCl3).
Stirring time = 144 h. Purification by flash chromatography (sil-
ica/CHCl3–hexane, 1:1; MeOH/CHCl3, 1:20) to afford a brown solid
(0.0180 g, 20%). Rf = 0.64 (MeOH/CHCl3, 1:19).
4.10.2. Method G
4.6.4. Method H
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:6; 1:3; 1:1) to afford a dark-brown powder
(0.0325 g, 25%). (M+H+ Found: 433.2112. C26H28N2O4 requires
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:9, 1:8) to afford an orange-red crystalline so-
lid (0.0329 g, 32%).

1412
K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
M+H, 447.2284); Rf = 0.31 (Et2O/hexane, 1:1). Mp = 189–191 °C. 1H
NMR (200 MHz, CDCl3): d = 1.15–1.35 (12H, m, 4 Â CH3), 2.85–3.05
(2H, m, 2 Â CH), 3.12 (3H, s, CH3), 6.06 (1H, s, ArH), 7.00–7.35 (8H,
m, ArH), 8.17 (1H, br s, NH) and 8.67 (1H, s, NH); 13C NMR
(100 MHz, CDCl3): d = 23.9, 33.7, 33.8, 51.3, 95.6, 109.7, 123.1,
123.8, 127.3, 127.6, 134.4, 134.9, 147.0, 147.5, 148.2, 177.0 and
178.5.
124.0, 126.7, 127.4, 129.5, 130.0, 137.0, 137.6, 138.5, 146.9,
164.5, 177.4 and 179.0.
4.14. 2,5-Bis-[4-(2-hydroxy-ethyl)-phenylamino]-3,6-dioxo-
cyclohexa-1,4-dienecarboxylic acid ethyl ester 15
4.14.1. Method A
Stirring time = 148 h. Purification by flash chromatography (sil-
ica/CHCl3; MeOH/CHCl3, 1:40, 1:20) to afford a dark-brown solid
(0.0256 g, 19%). Rf = 0.52 (MeOH/CHCl3, 1:9).
4.11. 3,6-Dioxo-2,5-bis-(2-pyridin-2-yl-ethylamino)-cyclohexa-
1,4-dienecarboxylic acid methyl ester 12
4.11.1. Method A
4.14.2. Method G
Stirring time = 142 h. Purification by flash chromatography (sil-
ica/MeOH–CHCl3, 1:19, 3:7, 2:3, 1:1) to afford a dark-brown solid
(0.0200 g, 16%). Rf = 0.48 (MeOH/CHCl3, 1:9).
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/MeOH–CHCl3, 1:100) to afford a dark-brown solid (0.0489 g,
36%). (M+H+ Found: 451.1857. C25H26N2O6 requires M+H,
451.1869); Rf = 0.29 (MeOH/CHCl3, 1:19). Mp = 154–156 °C. 1H
NMR (200 MHz, CDCl3): d = 1.01 (2H, t, J = 7.0 and 8.0 Hz, CH2),
2.87 (4H, m, 2 Â CH2), 3.57 (3H, q, J = 7.0 and 7.2 Hz, CH2), 3.87
(4H, m, 2 Â CH2), (6.07 (1H, s, ArH), 7.00–7.20 (8H, m, ArH), 8.18
(1H, s, NH) and 8.70 (1H, br s, NH); 13C NMR (100 MHz, CDCl3):
d = 13.8, 38.6, 38.7, 61.0, 63.4, 95.7, 123.1, 123.8, 129.8, 130.3,
135.0, 135.6, 137.2, 138.0, 146.7, 177.0 and 178.6.
4.11.2. Method C
Stirring time = 142 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:2; EtOAc; MeOH/EtOAc, 1:19) to afford an or-
ange–brown solid (0.0260 g, 21%). (M+H+ Found: 407.1733.
C22H22N4O4 requires M+H 407.1719); Rf = 0.19 (MeOH/EtOAc,
1:19). Mp = 93–95 °C. 1H NMR (200 MHz, CDCl3): d = 3.10 (4H, t
J = 6.2 and 6.4 Hz, CH2), 3.62 (4H, q J = 6.0, 6.2 and 6.4 Hz, CH2),
3.85 (3H, m, CH3), 4.41 (1H, m, NH), 5.46 (1H, s, ArH), 7.00–7.35
(4H, m, ArH), 7.55–7.70 (2H, m, ArH), 8.08 (1H, br s, NH) and
8.56 (2H, s, ArH); 13C NMR (100 MHz, CDCl3): d = 36.1, 36.6, 42.0,
43.8, 52.5, 93.2, 122.2, 123.4, 123.5, 136.9, 138.5, 148.2, 149.8,
149.9, 158.0, 176.2 and 176.6.
4.15. 3-Acetyl-2,5-bis-phenylamino-[1,4]benzoquinone 16
4.15.1. Method A
Stirring time = 171 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:4) to afford an orange-brown solid (0.0110 g,
11%). Rf = 0.32 (CHCl3).
4.12. 2,5-Bis-(4-(2-hydroxyl-ethyl)-phenylamino]-3,6-dioxo-
cyclohexa-1,4-diene carboxylic acid methylester 13
4.15.2. Method B
Stirring time = 96 h. Purification by flash chromatography (sil-
ica/CHCl3; MeOH/CHCl3, 1:19, 3:7, 2:3, 1:1) to afford an orange–
brown solid (0.0144 g, 16%). (M+H+ Found: 333.1228. C20H16N2O3
requires M+H 333.1229); Rf = 0.15 (MeOH/CHCl3, 1:9). Mp = 169–
171 °C. 1H NMR (200 MHz, CDCl3): d = 2.68 (3H, s, CH3), 6.07 (1H,
s, ArH) 7.00–7.60 (10H, m, ArH) and 8.19 (2H, s, 2 Â NH); 13C
NMR (100 MHz, CDCl3): d = 32.9, 99.3, 123.0, 124.5, 126.4, 127.3,
129.3, 130.0, 137.3, 139.4, 146.6, 178.1 and 178.8.
4.12.1. Method A
Stirring time = 149 h. Purification by flash chromatography (sil-
ica/CHCl3, MeOH/CHCl3, 1:40, 1:20) to afford
a black solid
(0.0146 g, 11%). Rf = 0.18 (MeOH/CHCl3, 1:19).
4.12.2. Method D
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/CHCl3; MeOH/CHCl3, 1:100, 3:100; 5:100) to afford a black solid
(0.0610 g, 52%). (MÀH+ Found: 435.1552. C24H24N2O6 requires
MÀH, 435.1556); Rf = 0.28 (CHCl3). Mp = 183–185 °C. 1H NMR
(200 MHz, CDCl3): d = 2.91 (4H, m, 2 Â CH2), 3.21 (3H, s, CH3),
3.90 (4H, m, 2 Â CH2), 6.10 (1H, s, ArH), 7.05–7.4 (8H, m, ArH)
and 8.18 (1H, s, NH); 13C NMR (100 MHz, CDCl3): d = 38.6, 38.7,
51.4, 63.3, 95.8, 123.2, 124.0, 128.8, 129.9, 130.3, 130.9, 135.1,
135.6, 137.3, 138.1, 146.8, 177.0 and 178.6.
4.15.3. Method D
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/CHCl3; MeOH/CHCl3, 1:100, 3:100; 5:100) to afford a dark-
brown solid (0.0252 g, 25%). (M+H+ Found: 333.1234. C20H16N2O3
requires M+H, 333.1239); Rf = 0.09 (CHCl3).
4.15.4. Method H
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:9, 1:8) to afford an orange-red crystalline so-
lid (0.0329 g, 32%).
4.12.3. Method I
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:1; EtOAc; EtOAc/MeOH, 9:1) to afford a black
solid (0.0767 g, 58%). Rf = 0.23 (EtOAc).
4.16. 3-Acetyl-2,5-bis-hexylamino-[1,4]benzoquinone 17
4.13. 3,6-Dioxo-2,5-bis-phenylamino-cyclohexa-1,4-
dienecarboxylic acid ethyl ester 14
4.16.1. Method B
Stirring time = 68 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:4) to afford a brown solid (0.0049 g, 5%).
Rf = 0.17 (EtOAc/hexane, 1:4). Mp = 33–35 °C. (Found: MH+
349.2485. C20H32N2O3 requires MH 349.2491). d = 1H NMR
(200 MHz, CDCl3): d = 0.89 (6H, m, 2 Â CH3), 1.32 (10H, m,
5 Â CH2), 1.67 (6H, m, 3 Â CH2), 2.60 (3H, s, CH3, 3.13 (2H, q
J = 6.0 and 6.8 Hz, 2 Â CH2), 3.99 (2H, q J = 5.4, 6.8 and 7.2 Hz,
2 Â CH2), 5.45 (1H, s, ArH), 6.62 (1H, br s, NH) and 13.34 (1H, br
s, NH); 13C NMR (100 MHz, CDCl3): d = 14.0, 22.5, 26.6, 26.7, 28.2,
30.0, 31.4, 32.8, 42.7, 47.5, 97.1, 150.5, 157.4, 176.4, 178.7 and
200.5.
4.13.1. Method A
Stirring time = 90 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:4) to afford a dark-brown solid (0.0360 g,
33%). (MÀH+ Found: 361.1177. C21H18N2O4 requires MÀH
361.1188); Rf = 0.64 (MeOH/CHCl3; 1:49). Mp = 162–163 °C [lit.34
178–179 °C]. 1H NMR (200 MHz, CDCl3): d = 1.02 (3H, t, J = 7.0
and 7.2 Hz, CH3), 3.57 (2H, q J = 7.0 and 7.4 Hz, CH2), 6.13 (1H, s,
ArH), 7.20–7.55 (10H, m, ArH), 8.21 (1H, s, NH) and 8.72 (1H, s,
NH); 13C NMR (100 MHz, CDCl3): d = 14.1, 61.2, 96.1, 123.3,

K. W. Wellington et al. / Bioorg. Med. Chem. 18 (2010) 1406–1414
1413
4.17. 2,5-Bis-(naphthalen-2-ylamino- 3,6-dioxo-cyclohexa-1,4-
4.21. 3-Acetyl-2,5-bis(2-pyidin-2-yl-ethylamino)-
dienecarboxylic acid methyl ester 18
[1,4]benzoquinone 22
4.17.1. Method F
4.21.1. Method B
Stirring time = 96 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:6; 1.5:5.5; 2:5) to afford a light-brown pow-
der (0.0267 g, 20%). (M+H+ Found: 449.1488. C28H20N2O4 requires
M+H, 449.1501); Rf = 0.22 (EtOAc/hexane, 5:12). Mp = 201–
203 °C. 1H NMR (200 MHz, CDCl3): d = 2.97 (3H, s, CH3), 6.07 (1H,
s, ArH), 7.40–8.00 (14H, m, ArH), 8.39 (1H, br s, NH) and 8.70
(1H, br s, NH); 13C NMR (100 MHz, CDCl3): d = 51.4, 96.3, 120.3,
121.6, 122.0, 126.4, 126.6, 127.2, 127.6, 2 Â 127.8, 129.4, 129.9,
133.2, 134.3, 146.5, 164.0 and 178.8.
Stirring time = 92 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:2; EtOAc; MeOH/EtOAc, 1:19) to afford a
brown solid (0.0235 g, 20%). (M+H+ Found: 391.1789. C22H22N4O3
requires M+H 391.1770); Rf = 0.5 (MeOH/CHCl3). Mp = 82–84 °C.
1H NMR (200 MHz, CDCl3): d = 2.55 (3H, s, CH3), 3.15 (4H, m,
2 Â CH2), 3.59 (2H, q J = 6.2 and 6.4 Hz), 3.42 (2H, q J = 6.2, 6.4
and 6.8 Hz), 5.52 (1H, s, ArH), 7.12–7.28 (4H, m, ArH), 7.63 (2H, t
J = 7.6 Hz, ArH), 8.58 (2H, s, ArH) and 13.36 (1H, s, NH); 13C NMR
(100 MHz, CDCl3): d = 32.8, 35.9, 38.3, 41.7, 46.6, 97.1, 106.3,
121.7, 121.9, 123.2, 123.4, 136.5, 136.7, 149.5, 149.6, 150.6,
157.7, 158.2, 176.7, 178.2 and 200.3.
4.18. 2,5-Bis[4-(2-hydroxyethyl)-phenylamino]-3,6-dioxo-
cyclohexane-1,4-diene = carboxylic acid methyl ester 19
4.22. 3,6-Dioxo-2,5-bis-(2-pyridin-2-yl-ethylamino)-cyclohexa-
1,4-dienecarboxylic acid ethyl ester 23
4.18.1. Method E
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/Et2O–hexane, 1:6; 1:3; 1:1) to afford a red solid (0.0205 g,
19%). (M+H+ Found: 365.2460. C20H32N2O4 requires M+H,
365.2440); Rf = 0.27 (Et2O/hexane, 1:1). Mp = 62–63 °C. 1H NMR
(200 MHz, CDCl3): d = 0.89 (6H, m, 2 Â CH3), 1.32 (10H, m,
5 Â CH2), 1.64 (6H, m, CH2), 3.1.7 (4H, m, CH2), 3.86 (3H, m, CH3),
5.40 (1H, d, J = 7.8 Hz, NH) and 6.7 (1H, s, ArH); 13C NMR
(100 MHz, CDCl3): d = 14.0, 22.4, 22.5, 26.4, 26.6, 28.1, 29.0, 31.3,
42.7, 44.4, 51.8, 52.2, 92.7, 96.4, 147.9, 150.4, 166.9, 175.8, 176.2
and 178.1.
4.22.1. Method B
Stirring time = 96 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:2; EtOAc; MeOH/EtOAc, 1:19) to afford a
red–brown solid (0.0403 g, 32%). (M+H+ Found: 421.1858.
C23H24N4O4 requires M+H 421.1876); Rf = 0.20). Mp = 90–92 °C.
1H NMR (200 MHz, CDCl3): d = 1.33 (3H, t J = 7.2 Hz, CH3), 1.78
(1H, s, NH), 3.07 (4H, t J = 6.4 and 6.8 Hz, 2 Â CH2), 3.59 (4H, q
J = 6.4 and 6.6 Hz, 2 Â CH2), 4.25 (2H, m, CH2), 5.40 (1H, s, ArH),
7.14 (4H, t J = 5.2 and 7.6 Hz, ArH), 7.54–7.66 (2H, m, ArH), 8.00
(1H, s, NH) and 8.56 (2H, d J = 4.2 Hz, ArH); 13C NMR (100 MHz,
CDCl3): d = 14.2, 30.9, 35.8, 36.4, 41.7, 43.4, 61.3, 92.8, 121.8,
123.1, 123.2, 136.6, 2 Â 138.2, 149.5, 149.6, 150.2, 157.7, 176.0
and 176.6.
4.19. 2,5-Bis-hexylamino]-3,6-dioxo-cyclohexa-1,4-
dienecarboxylic acid ethylester 20
Acknowledgement
4.19.1. Method E
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/Et2O–hexane, 1:6; 1:4; 1:2) to afford a red solid (0.0138 g,
12%). (MÀH+ Found: 379.2591. C21H35N2O4 requires MÀH,
379.2597); Rf = 0.20 (Et2O/hexane, 1:2). Mp = 63–65 °C. d = 0.85
(6H, m, 2 Â CH3), 1.18–1.49. (10H, m, 5 Â CH2), 1.51–1.75 (6H, m,
3 Â CH2), 3.15 (3H, m, CH3), 4.32 (2H, q, J = 7.2 and 7.9 Hz), 5.37
(1H, s, ArH), 6.64 (1H, br s, NH) and 7.23 (1H, br s, NH); 13C NMR
(100 MHz, CDCl3): d = 13.9, 14.2, 22.4, 22.5, 26.4, 26.5, 28.2, 29.1,
42.7, 61.3, 92.6, 147.6, 150.5, 166.6, 176.0 and 176.5.
We thank the CSIR (Parliamentary Grant and the Young
Researchers Establishment Fund) for financial support.
References and notes
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Burgos, C.; Plou, F. J.; Ballesteros, A.; Alcalde, M. Microb. Cell Fact. 2008, 7, 32;
(d) Xu, F.; Damhus, T.; Danielsen, S.; Østergaard, L. H. In Modern Biooxidations.
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VCH: Weinheim, 2007; p 43.
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3. Riva, S. TRENDS Biotechnol. 2006, 24, 219.
4. Osiadacz, J.; Al-Adhami, A.; Bajraszewska, D.; Fischer, P.; Peczynska-Czoch, W. J.
Biotechnol. 1999, 72, 141.
4.20. 2,5-Bis-(4isopropyl)-phenylamino]-3,6-dioxo-cyclohexa-
1,4-dienecarboxylic acid ethylester 21
5. Eggert, C.; Temp, U.; Dean, J.; Eriksson, K. FEBS Lett. 1995, 376, 202.
6. Bhalerao, U.; Muralikrishna, C.; Rani, B. Tetrahedron 1994, 50, 4019.
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Biochem. Biophys. Res. Commun. 2001, 287, 355.
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Schauer, F. Tetrahedron 2005, 61, 4615.
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12. Ikeda, R.; Tanaka, H.; Uyama, H.; Kobayashi, S. Macromol. Rapid Commun. 1998,
19, 423.
4.20.1. Method G
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/Et2O–hexane, 1.5:12; 1:6; 1:4; 1:2) to afford a dark-brown
powder (0.0289 g, 21%). (M+H+ Found: 447.2299. C27H28N2O4 re-
quires M+H, 433.2127); Rf = 0.18 (Et2O/hexane, 1:2). Mp = 118–
120 °C [lit.28 132–133 °C]. 1H NMR (200 MHz, CDCl3): d = 0.96
(3H, t J = 7.0 and 7.2, CH3), 1.3–1.7 (12H, m, 4 Â CH3), 2.80–
3.10 (2H, m, 2 Â CH), 3.51 (2H, q, CH2), 6.06 (1H, s, ArH),
7.05–7.35 (8H, m, ArH), 8.16 (1H, br s, NH) and 8.65 (1H, s,
NH); 13C NMR (100 MHz, CDCl3): d = 13.8, 23.9, 33.8, 33,9, 60.8,
95.6, 123.1, 123.8, 127.2, 127.6, 134.4, 135.0, 147.0, 147.5,
148.1 and 178.6.
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4.20.2. Method I
Stirring time = 72 h. Purification by flash chromatography (sil-
ica/EtOAc–hexane, 1:10, 1:9, 1:8; 1:7; 1:6) to afford a brown-black
powder (0.0390 g, 30%). Rf = 0.20 (EtOAc/hexane, 1:6).
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