One-pot laccase-catalysed synthesis of 5,6-dihydroxylated benzo[b]furans and catechol derivatives, and their anticancer activity

Article abstract of DOI:10.1002/ardp.201200413

A commercial laccase, Suberase from Novozymes, was used to catalyse the synthesis of 5,6-dihydroxylated benzo[b]furans and catechol derivatives. The yields were, in some cases, similar to or better than that obtained by other enzymatic, chemical or electr

Full text of DOI:10.1002/ardp.201200413

266
Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
Full Paper
One-Pot Laccase-Catalysed Synthesis of 5,6-Dihydroxylated
Benzo[b]furans and Catechol Derivatives, and Their
Anticancer Activity
Kevin W. Wellington1, Tozama Qwebani-Ogunleye1, Natasha I. Kolesnikova1, Dean Brady1,
and Charles B. de Koning2
1 CSIR Biosciences, Pretoria, South Africa
2 Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Wits, South Africa
A commercial laccase, Suberase1 from Novozymes, was used to catalyse the synthesis of 5,6-
dihydroxylated benzo[b]furans and catechol derivatives. The yields were, in some cases, similar to
or better than that obtained by other enzymatic, chemical or electrochemical syntheses. The
synthesised derivatives were screened against renal (TK10), melanoma (UACC62), breast (MCF7)
and cervical (HeLa) cancer cell lines. GI50, TGI and LC50 are reported for the first time. Anticancer
screening showed that the cytostatic effects of the 5,6-dihydroxylated benzo[b]furans were most
effective against the melanoma (UACC62) cancer cell line with several compounds exhibiting
potent growth inhibitory activities (GI50 ¼ 0.77–9.76 mM), of which two compounds had better
activity than the anticancer agent etoposide (GI50 ¼ 0.89 mM). One compound exhibited potent
activity (GI50 ¼ 9.73 mM) against the renal (TK10) cancer cell line and two exhibited potent
activity (GI50 ¼ 8.79 and 9.30 mM) against the breast (MCF7) cancer cell line. These results
encourage further studies of the 5,6-dihydroxylated benzo[b]furans for their potential application
in anticancer therapy.
Keywords: 1,3-Dicarbonyls / Anticancer / Benzo[b]furans / Biocatalysis / Catechols / C–C coupling / Cytostatic effects /
Cytotoxic effects / GI50 / LC50 / Laccase / Michael addition / TGI
Received: October 30, 2012; Revised: January 4, 2013; Accepted: January 18, 2013
DOI 10.1002/ardp.201200413
Introduction
cassette (ABC) transporters that actively pump drugs out of
tumour cells is one of the best known mechanisms of multi-
drug resistance [1, 2].
Cancer is a leading cause of death worldwide and accounted
for 7.6 million deaths (13% of all deaths) in 2008 (http://
www.who.int/mediacentre/factsheets/fs297/en/). Amongst the
most deadly cancers are lung, stomach, liver, colon and breast
cancer. It is projected that worldwide deaths from cancer will
continue to rise to an estimated 13.1 million in 2030 (http://
www.who.int/mediacentre/factsheets/fs297/en/).
The number of effective drugs available for treating malig-
nant tumours has been reduced by the development of
chemoresistance and this has led to a search for therapeutic
alternatives through the discovery of new classes of anti-
cancer compounds.
The hydroxylated benzo[b]furan moiety has attracted much
attention due to its wide range of biological activities [3–7]. This
group of compounds act as antifungal agents, antioxidant
agents, 5-lipoxygenase inhibitors, cyclooxygenase-2 inhibitors,
Naþ and Kþ-ATPase inhibitors and modulators of the estrogen
receptor [8–12]. Examples of benzofuran scaffolds that exhibit
anticancer activity are shown in Fig. 1.
The primary treatment for many cancers is chemotherapy.
The development of multidrug resistance to chemotherapeu-
tic drugs is a main obstacle for the successful treatment
of malignant tumours. Overexpression of the ATP-binding
Correspondence: Kevin W. Wellington, CSIR Biosciences, P.O. Box 395,
Pretoria, South Africa.
E-mail: kwellington@csir.co.za; kwwellington@gmail.com
Fax: þ2712 841 3388
Usnic acid 1, a common and abundant lichen metabolite,
showed activity against the wild-type p53 breast cancer cell
line MCF7, the breast cancer cell line MDA-MB-231 and the
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Anticancer Activity of 5,6-Dihydroxylated Benzo[b]furans
267
MeO
MeO
H3C
OH
OH
H3C
O
O
OH
H3C
EtO
O
H3C
HO
O
CH3
R
O
O
O
O
OH
CH3
3
2 R = H, F
1 Usnic acid
R
R
OH
OH
O
O
O
R1
R1
R1
R1
Oxidation
O
O
O
5
4
Figure 1. Benzofuran scaffolds.
lung cancer cell line H1299 [13]. Benzofuran derivatives 2 and
3 (Fig. 1) were identified as anticancer agents of which
derivatives displayed selective cytotoxicity against a tumouri-
genic cell line [14]. Laccases (EC 1.10.3.1) are enzymes that
are widely distributed in plants and fungi. They are
characterised by a multinuclear copper-containing active
This study is, to the best of our knowledge, the first on the
synthesis of a variety of 5,6-dihydroxylated benzo[b]furans 4
and catechol derivatives using the commercial laccase,
Suberase1, as well as the first report on the anticancer
activity of the synthesised compounds.
site and have been classified as oxidoreductases. Laccases Results and discussion
catalyse the oxidation of a broad range of substrates
such as phenols, o- and p-diphenols, aminophenols, methoxy-
phenols, aryl thiols, anilines, polyphenols, polyamines and
lignin-derivatives [15–17]. In the monoelectronic oxidation of
substrates molecular oxygen is used and water is produced
as the only by-product (http://www.who.int/mediacentre/
factsheets/fs297/en/) [1, 2, 15–17]. Laccases have been success-
fully applied in organic synthesis which has culminated in
several reports in the field of green chemistry [18].
Synthesis
The catechols (6a–c) and the 1,3-dicarbonyls (7a–f) used in
this study are depicted in Fig. 2.
The method used for the synthesis entailed reacting
one equivalent of the catechol with one equivalent of the
1,3-dicarbonyl at room temperature using Suberase1 in a
vessel open to air at pH 7.15 (Scheme 1). A pH of 7.15
was chosen because it would make the reaction medium
sufficiently basic to deprotonate the alpha-proton from the
1,3-dicarbonyls and thus facilitate the Michael addition
reaction with the in situ-generated o-quinone.
In our laboratories, we have been interested in the use of
enzymes for the development of green methods of synthesis,
and for accessing compounds that have pharmaceutical
value. We have previously reported on the synthesis of di-
aminobenzoquinones [19a] and aminonaphthoquinones
[19b] via C–N bond formation as well as 1,4-naphthoqui-
none-2,3-bis-sulfides [19c] via C–S bond formation using com-
mercial laccases (Denilite II Base on an inert support, and
Novozyme 51003) from Novozymes [19]. In this article, we
report on the synthesis of 5,6-dihydroxylated benzo[b]furan
derivatives using another commercial laccase (Suberase1
from Novozymes) and on the anticancer activity of the
synthesised compounds. We anticipated that the 5,6-di-
hydroxylated benzo[b]furans 4 possessing ortho phenolic sub-
stituents could be metabolised to the o-quinonoid structure 5
in cancer cells (Fig. 1). Since quinonoid compounds display
potent biological properties such as antibacterial, anticancer,
antifungal and antimalarial activity [20], we postulated that
the benzo[b]furan 5 could have similar biological activities.
The first approach, Method A, entailed reacting the cate-
chols 6a–c with the 1,3-dicarbonyls 7a–f at room temperature
(rt) at pH 7.15 for 24 h. The second approach, Method B,
entailed conducting the reaction under the same conditions
but for a longer time (44 h) to determine whether a longer
reaction time would improve the yield of the product. In the
third approach, Method C, a co-solvent, DMF, was added to
the reaction mixture to improve the solubility of the organic
substrates. The number of equivalents of the 1,3-dicarbonyl was
also increased so that a ratio of 1,3-dicarbonyl to catechol was
4:1. These reactions were conducted for 42 h. The results of the
investigations with Methods A to C are depicted in Table 1.
From these results it can be seen that 5,6-dihydroxylated
benzo[b]furans can be accessed using all three synthesis
methods. For Method A the highest yield that was obtained
is 98% for 17 (Entry 15, Table 1) and the lowest is 37% for 15
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268
K. W. Wellington et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
CH3
O
CH3
OH
OH
OH
OH
OH
OH
6a
6b
6c
O
O
O
O
O
O
O
O
O
O
O
O
H3C
CH3
H3C
CH3
CH3
7b
H3C CH3
7a
CH3
Ph
7f
7d
7c
7e
Figure 2. The catechols 6a–c and 1,3-dicarbonyls 7a–f used in this study.
(Entry 12, Table 1). For Method B the highest yield was 77% for
18 (Entry 18, Table 1) and the lowest 49% for 8 (Entry 2,
Table 1). In the case of Method C, the highest yield was
71% for 14 (Entry 11, Table 1) and the lowest 15% for 21
(Entry 23, Table 1).
was obtained in 60% yield using Candida rugosa lipase and
laccase from T. villosa at 238C. Compound 9 was obtained in
62% yield using Candida antarctica (CALB) lipase and laccase
from T. villosa at 238C [23]. We have synthesised compounds 8
and 9 without the use of scandiumtris(trifluoromethane-
sulfonate) or lipase and achieved a 49% yield of compound
8 (Entry 2, Table 1) and a 50% yield of compound 9 (Entry 3,
Table 1).
When comparing the yield of product using Method A to
that obtained using Method B, it can be seen that there is not
a significant increase in yield even though the reaction time
has almost doubled (Entries 1 and 2, 5 and 6, 17 and 18,
Table 1). It is therefore concluded that there is a limit to the
quantity of product that can be formed in the reaction.
The presence of DMF (Method C) may have deactivated the
laccase, Suberase1, resulting in a lower yield of the product.
A higher volume of organic solvent increases the solubility of
a substrate but significantly decreases the overall reaction
rate by deactivating the enzyme [21].
Hajdok et al. [24] was the first to report on the synthesis of
compounds 10–21 using laccase initiated oxidative domino
reactions. One method entailed using a commercial laccase
from Trametes versicolor in an acetate buffer (pH 4.38) at room
temperature while the other method used laccase from
Agaricus bisporus in phosphate buffer (pH 5.96) also at rt.
The latter method was found to be better since it gave the
product in higher yield and purity with yields ranging from
71 to 97%. The yields of compounds 10–21 using our methods
are 37–98%. The commercial fungal laccase, Suberase1 from
Myceliophthora thermophila, was overall less effective than the
laccase from A. bisporus which was used by Hajdok et al. [24].
Compounds 13 and 19 have also been synthesised by
employing tyrosinase and laccase from A. bisporus and were
obtained in 39 and 44% yields, respectively [25]. Our method
was higher yielding since compound 13 was obtained in 59%
yield and compound 19 in 76% yield (Entries 8 and 19,
respectively, Table 1; Fig. 3).
The optimum conditions for synthesising these 5,6-dihy-
droxylated benzo[b]furans 4 using Suberase1 is thus that
used under Method A.
The enzymatic synthesis of 5,6-dihydroxylated benzo[b]fur-
ans has been reported previously. The first enzymatic syn-
thesis of compound 9 was reported in 2007 by Witayakran
et al. [22] using Trametes villosa laccase in phosphate buffer
(pH 7.0) with a Lewis acid, scandiumtris(trifluoromethane-
sulfonate), and sodium lauryl sulfate at 208C and afforded it
in 76% yield. In 2009, Witayakran et al. also reported on a
laccase–lipase co-catalytic system for the synthesis of com-
pounds 8 and 9 using phosphate buffer (pH 7.0) with lipase
[23]. In this reaction, the Michael addition step was enhanced
by lipase addition providing improved yields. Compound 8
There have also been literature reports on the chemical
syntheses of 5,6-dihydroxylated benzo[b]furans. The first
report of compound 8 was a chemical synthesis which
afforded 8 in 47% yield using pyridine and sodium metaio-
date in ethanol at 208C [26]. Our methods afforded similar
yields for compound 8 (48 and 49%, Entries 1 and 2, respect-
ively, Table 1). Duthaler and Scherrer [27] reported on the
chemical synthesis of compound 10 which was obtained in
22% yield using sodium acetate in water. We were able to
obtain compound 10 in 65% yield (Entry 4, Table 1).
Electrochemical syntheses have also been reported for
the synthesis of 5,6-dihydroxylated benzo[b]furans. The first
R
O
O
R
R
OH
OH
O
R
R
1
1
OH
OH
Suberase
O
O
_
7a e
R1
R1
phosphate
buffer, rt,
pH 7.15
_
6a c
O
_
8 21
Scheme 1. Synthesis of the 5,6-dihydroxylated benzo[b]furans
8–21.
´
report on the synthesis of 16 was by Grujic et al. [28] in 1976
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Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
Anticancer Activity of 5,6-Dihydroxylated Benzo[b]furans
269
Table 1. Synthesised benzo[b]furans 8–21 (yield in parentheses) at rt in phosphate buffer at pH 7.15.
Entry Catechol
Dicarbonyl
Reaction
time (h)
Method
Product
(%Yield)
Yields of other
enzymatic
syntheses
Yields of
other syntheses
1
2
3
4
5
6
7
8
6a
6a
6b
6a
6b
6b
6c
6a
6a
6b
6b
6c
6a
6a
6b
6b
6c
6c
6a
6a
6b
6c
6c
7a
7a
7a
7b
7b
7b
7b
7c
7c
7c
7c
7c
7d
7d
7d
7d
7d
7d
7e
7e
7e
7e
7e
24
44
24
24
24
44
24
24
42
24
42
24
24
42
24
42
24
44
24
42
24
24
42
A
B
A
A
A
B
8 (48)
8 (49)
9 (50)
60 [23]
47 [26]
22 [27]
76 [22], 62 [23]
87 [24]
70 [24]
10 (65)
11 (62)
11 (67)
12 (70)
13 (59)
13 (30)
14 (78)
14 (71)
15 (37)
16 (58)
16 (40)
17 (98)
17 (59)
18 (73)
18 (77)
19 (76)
19 (50)
20 (80)
21 (43)
21 (15)
A
A
C
A
C
A
A
C
A
C
A
B
A
C
A
A
C
89 [24]
85 [24], 39 [25]
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
71 [24]
97 [24]
91 [24]
90 [28], 90 [29], 82 [30], 66 [31]
87 [30], 81 [31]
92 [24]
95 [24]
93 [30], 80 [31]
96 [24], 44 [25]
91 [24]
97 [24]
Method A – Suberase1 (8.0 mL), catechol (2.0 mmol), 1,3-dicarbonyl (2.0 mmol, 1 eq), phosphate buffer (20.0 mL, 0.10 M, pH 7.15),
stirring time at rt ¼ 24 h.
Method B – Suberase1 (8.0 mL), catechol (2.0 mmol), 1,3-dicarbonyl (2.0 mmol, 1 eq), phosphate buffer (20 mL, 0.10 M, pH 7.15),
stirring time at rt ¼ 44 h.
Method C – Suberase1 (4.5 mL), catechol (0.60 mmol), 1,3-dicarbonyl (2.4 mmol, 4 eq), phosphate buffer (4.0 mL, 0.10 M, pH 7.15) and
DMF (2.0 mL), stirring time at rt ¼ 42 h.
´
and afforded it in 90% yield. Later Tabakovic et al. [29] also
furans 8–21. In this case, the aim was to achieve only C–C
bond formation using the 1,3-dicarbonyl 7f. The purpose was
to determine whether anticancer activity would still be
observed without the formation of the furan ring. The results
of the investigation are shown in Table 2.
reported on the electrochemical synthesis of 16 in 90% yield
in water. Another electrochemical synthesis of 16 using
sodium acetate in water by Nematollahi et al. [30] afforded
it in 82% yield. The electrochemical synthesis of 16 by
Davarani et al. [31] only afforded a 66% yield. We could only
obtain a 58% yield for 16 (Entry 13, Table 1). Nematollahi et al.
[30] also reported on the electrochemical synthesis of 17 and
18 which were obtained in 87 and 93% yields, respectively,
also using sodium acetate in water as a reaction medium.
The electrochemical synthesis of 17 and 18 in a sodium
acetate solution by Davarani et al. [31] afforded these com-
pounds in slightly lower yields, 81 and 80%, respectively.
Compound 17 was obtained in 98% yield (Entry 15,
Table 1) which is higher than the yields obtained by electro-
chemical synthesis, while compound 18 was obtained in 77%
yield (Entry 18, Table 1) which was lower than that obtained
by electrochemical syntheses.
Both reactions proceeded in mediocre yield to afford 22
and 23 (Entries 1 and 2, respectively). A proposed mechanism
involving an o-quinone intermediate of 22 and 23 is shown
in Fig. 4 below.
Anticancer evaluation
Screening was conducted against renal (TK10), melanoma
(UACC62), breast (MCF7) and cervical (HeLa) cancer cell lines
using the Sulforhodamine B (SRB) assay to determine the
growth inhibitory effects of the compounds [32]. These cell
lines have been used routinely at the U.S. National Cancer
Institute for screening for new anticancer agents and were
derived from tumours that have different sensitivities to
chemotherapeutic drugs [33]. Etoposide, an anticancer agent,
was used as a positive control. It is known to be an inhibitor of
topoisomerase, particularly topoisomerase II, and aids in
DNA unwinding which causes the DNA strands to break
The synthesis of two novel catechol derivatives 22 and 23
was also investigated (Scheme 2).
These reactions were conducted using Method A which
was used for the synthesis of the 5,6-dihydroxylated benzo[b]-
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270
K. W. Wellington et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
CH3
OH
CH3
OH
OH
OH
OH
OH
O
O
O
O
H3C
H3C
OH
OH
H3C
H3C
O
O
8
O
O
10
11
15
19
9
CH3
O
CH3
CH3
O
OH
OH
OH
OH
OH
OH
O
O
O
O
OH
OH
H3C
H3C
H3C
O
O
O
O
13
12
14
CH3
CH3
O
OH
OH
OH
OH
OH
O
O
O
O
H3C
H3C
OH
OH
OH
H3C
H3C
H3C
H3C
Ph
O
O 17
O
16
O
18
CH3
O
CH3
OH
OH
OH
OH
O
O
Ph
Ph
O
21
O
20
Figure 3. Structures of the 5,6-dihydroxylated benzo[b]furans 8–21 synthesised at rt.
[34]. Three parameters were determined during the screening
process: 50% cell growth inhibition (GI50), total cell growth
inhibition (TGI) and the lethal concentration that kills 50% of
cells (LC50). The results of this investigation are shown in
Table 3 from which it can be seen that several compounds
exhibited potent cytostatic effects.
The GI50 values of the catechols 6a–c, the catechol deriva-
tive 22 and selected benzo[b]furans were compared to that of
etoposide. No selection criteria were used for the compounds
that were screened. Screening against the TK10 cell line
R
R
OH
OH
O
O
O
HO
HO
O
O
Suberase
H3C
H3C
H3C
CH3
+
H3C
CH3
7f
phosphate
buffer, pH
7.15, rt, 24h
CH3
CH3
CH3
O
7f
6b R = Me
22 R = Me
R
OH
O
6c R = OMe
23 R = OMe
R
R
O
H3C
CH3
O
HO
HO
O
O
Scheme 2. Synthesis of C-C coupled products 22 and 23.
CH3
Suberase
HO
CH3
CH3
CH3
H
O
+
6b and 6c
H
R
Table 2. Synthesised catechol derivatives 23 and 24 (yield in
parentheses) at rt in phosphate buffer at pH 7.15.
-H+
R
Entry Catechol
Dicarbonyl
Reaction
time (h)
Product
(Yield)
O
HO
HO
O
O
22 R = Me
1
2
6b
6c
7f
7f
24
24
22 (80)
23 (55)
HO
CH3
23 R = OMe
CH3
CH3
CH3
CH3
CH3
H
O
O
Method
dicarbonyl (2.0 mmol,
0.10 M, pH 7.15), stirring time at rt ¼ 24 h.
A
–
Suberase1 (8.0 mL), catechol (2.0 mmol), 1,3-
eq), phosphate buffer (20.0 mL,
Figure 4. A proposed mechanism for C–C bond formation for the
catechol derivatives 22 and 23.
1
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Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
Anticancer Activity of 5,6-Dihydroxylated Benzo[b]furans
271
showed that only 21 had potent activity (GI50 ¼ 9.73 mM,
Entry 12) which was not as good as that of etoposide
(GI50 ¼ 7.19 mM, Entry 14).
against the TK10 cell line showed that most of the com-
pounds were more lethal than etoposide (LC50 > 100 mM)
with 18 (LC50 ¼ 73.59 mM, Entry 10) being the most lethal.
Again, most of the compounds were more lethal than etopo-
side (LC50 > 100 mM) against the UACC62 cell line and 15
(LC50 ¼ 60.32 mM, Entry 9) was the most lethal. Apart from
18, none of the test compounds were lethal for HeLa cells,
indicating a degree of selectivity between cell lines.
Most of the compounds exhibited potent growth inhibi-
tory activity against the UACC62 cell line. The potent
activity exhibited by 15 (GI50 ¼ 0.78 mM, Entry 9) and 21
(GI50 ¼ 0.77 mM, Entry 12) was slightly better activity than
that of etoposide (GI50 ¼ 0.89 mM, Entry 14).
Moreover, the same two compounds, 15 (GI50 ¼ 8.79 mM,
Entry 9) and 21 (GI50 ¼ 9.30 mM, Entry 12), also exhibited potent
growth inhibitory activity against the MCF7 cell line but this was
not as good as that of etoposide (GI50 ¼ 0.56 mM, Entry 14).
Most compounds exhibited weak TGI activity and three
were inactive against the TK10 cell line. The activities of 18,
20 and 21 (Entries 10–12, respectively) were slightly
better (TGI ¼ 46.14–48.25 mM) than that of etoposide
(TGI ¼ 49.74 mM, Entry 14).
The compounds also exhibited more lethal cytotoxic
effects against the MCF7 cell line than that of etoposide
(LC50 > 100 mM, Entry 14) and 21 (LC50 ¼ 86.93 mM, Entry
12) was the most lethal. Apart from 18, none of the test
compounds were lethal for HeLa cells indicating a degree
of selectivity between cell lines.
The catechols 6a–c and the catechol derivative 22 (Entry 13)
did not exhibit any significant anticancer activity. From the
results it can be seen that the GI50 concentrations of benzo[b]-
furan 9 (Entry 4) are almost half of those of 22 (Entry 13)
against the renal (TK10), melanoma (UACC62) and breast
(MCF7) cancer cell lines. It may thus be concluded that the
presence of the furan ring enhances anticancer activity. The
benzo[b]furans were most effective against the melanoma
(UACC62) cell line. Only one benzo[b]furan, 21, exhibited
growth inhibitory activity against all three cancer cell lines
which may be attributed to the presence of the methoxy
group on the benzene ring. When the methoxy group was
replaced with a methyl group, as in 20, growth activity
against the renal (TK10) and breast (MCF7) cell lines was
diminished. The benzo[b]furan 15 exhibited activity against
melanoma (UACC62) and breast (MCF7) cell lines. The replace-
The compounds also exhibited moderate to weak activity
against the UACC62 cell line with most compounds, 11, 12,
14, 15, 18 and 21, exhibiting better activity (TGI ¼ 18.32–
51.06 mM) than that of etoposide (TGI ¼ 52.71 mM, Entry 14).
The best activity was observed for 15 (TGI ¼ 18.32 mM, Entry 9)
which was almost threefold better than that of etoposide.
The test compounds exhibited weak activity against the
MCF7 cell line, but the cytostatic effects of these compounds
were better than that of etoposide which was inactive
(TGI > 100 mM, Entry 14).
The LC50 values of the compounds were also compared to
that of etoposide to get an idea of the cytotoxic effects of
these compounds against the different cell lines. Screening
Table 3. In vitro anticancer screening of the compounds against renal (TK10), melanoma (UACC62), breast (MCF7) and HeLa cancer cells
expressed as GI50, TGI and LC50 values (mM).
Entry Compound
Renal (TK10)
TGI
Melanoma (UACC62)
Breast (MCF7)
TGI
Cervical (HeLa)
GI50
LC50
GI50
TGI
LC50
GI50
LC50
GI50
TGI
LC50
a
a
a
a
a
a
a
1
2
3
4
5
6
7
8
6a
6b
6c
9
10
11
12
14
15
18
20
21
22
68.02 >100
>100
45.00 >100
>100
52.41 >100
>100
64.31 >100
–
–
–
–
–
–
–
a
38.14
51.63
41.05
75.72 >100
96.98 >100
69.94
15.18
23.64
98.82 32.74
46.22
77.26 27.34
–
a
–
50.26
60.84
68.65
51.06
76.87 27.55
88.94 35.10
99.57 35.53
78.49 25.97
15.85
74.35 25.55
60.32 8.79
66.27 28.44
60.68
66.12
64.19
61.87
54.74
93.81
97.14 60.70 >100 >100
92.85 49.09 >100 >100
97.78 44.55 >100 >100
93.63 56.46 >100 >100
25.93 >100 >100
95.11 26.68 69.67 >100
65.00 >100
24.25 54.74
52.47 >100
>100
37.73
85.23 23.63
>100
6.04
6.50
0.78
6.79
33.15 >100
16.56
26.54
18.69
18.60
9.73
55.32
94.09
76.75
73.59
77.90
85.35
33.85
18.32
28.93
68.82 >100
9
51.65
46.14
48.25
47.15
48.79
60.90
60.79 >100
10
11
12
13
14
93.35 34.37 62.15 89.93
9.76 >100
0.77 23.60
51.29 >100
>100
64.58
>100
17.96
9.30
47.28 >100 >100
46.93 86.93 22.32 73.19 >100
a
a
a
86.99 >100 >100
7.19 49.74 >100
68.03 >100
0.56 >100
>100
>100
–
3.56
–
–
40.18 87.54
Etoposide
0.89
52.71 >100
Inactive, i: GI50 or TGI > 100 mM; weak activity, w: > 30 mM GI50 or TGI < 100 mM; moderate activity, m: < 30 mM GI50 or
TGI > 10 mM; potent activity, p: GI50 or TGI < 10 mM.
a
Not screened.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

272
K. W. Wellington et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
column chromatography was performed using Merck Silica Gel 60
(70–230 mesh). Melting points were determined using a Glassco
melting point apparatus and are uncorrected.
All chemicals were reagent grade materials. The 1,3-dicarbonyls
were purchased from Sigma–Aldrich, South Africa. Suberase1
(10757.8 PCU/mL) is a fungal laccase from M. thermophila produced
by submerged fermentation of a genetically modified Aspergillus
oryzae strain. The enzymatic preparation is supplied as a brown
liquid which is completely miscible with water. Suberase1 was
obtained from Novozymes in South Africa.
ment of the methoxy group with that of a methyl, as in 14,
also resulted in diminished activity and in this case against
the breast (MCF7) cancer cell line. When comparing the
structure of 21 to that of 15 it appears that the replacement
of the methyl group with that of a phenyl specifically affords
growth inhibitory activity against the renal (TK10) cancer cell
line. It was concluded that the phenyl and methoxy groups
are essential for activity against all three cancer cell lines.
Conclusions
General methods for the synthesis of the
benzo[b]furan derivatives
The fungal laccase from M. thermophila, commercially avail-
able as an inexpensive preparation known as Suberase1,
can be used in the catalytic synthesis of 5,6-dihydroxylated
benzo[b]furan and catechol derivatives under mild and
environmentally friendly reaction conditions. The yields
are, in some cases, similar to or better than that obtained
by other enzymatic, chemical, or electrochemical synthesis.
This method has eliminated the use of the Lewis acid, scan-
diumtris(trifluoromethanesulfonate), and lipase which was
used in previous laccase methods.
Method A
The laccase (Suberase1, 2.0 mL) was added to a mixture of the
catechol (2.0 mmol), 1,3-dicarbonyl (2.0 mmol) and phosphate
buffer (20.0 mL, 0.10 M, pH 7.15) in a 250-mL round-bottom flask
stirred under air at rt. More laccase (2.0 mL) was added after 2, 18
and 20 h. The mixture was vigorously stirred under air until
the substrates were consumed as judged by TLC. After stirring
the reaction mixture was acidified with 32% HCl to pH 4.0. The
mixture was extracted with EtOAc and washed with water
(20.0 mL). The organic phases were then combined and the solvent
evaporated. The residue, a powder, was purified by washing with
EtOAc or recrystallising from a combination of MeOH and EtOAc.
The 5,6-dihydroxylated benzo[b]furans exhibit potent cyto-
static effects against the three cancer cell lines but are most
effective against UACC62 (melanoma) with two compounds
exhibiting better activity than etoposide based on the
GI50 concentrations. The 5,6-dihydroxylated benzo[b]furans
generally have better TGI activity than that of etoposide.
These results warrant further studies of the 5,6-dihydroxy-
lated benzo[b]furans for their application in anticancer
therapy. These studies will entail further synthesis of 5,6-dihy-
droxylated benzo[b]furans and evaluation of their anticancer
activity in addition to structural modification of hits to deter-
mine whether the anticancer activity can be optimised.
Method B
Same as Method B except that more laccase (2.0 mL) was added
after 4, 24 and 28 h.
Method C
The laccase (Suberase1, 1.5 mL) was added to a mixture of
the catechol (0.60 mmol), 1,3-dicarbonyl (2.40 mmol), phosphate
buffer (4.0 mL, 0.10 M, pH 7.15) and DMF (2.0 mL) in a test tube
stirred under air at rt. More laccase (1.5 mL) was added after 2 h
and then again after 4 h. The mixture was vigorously stirred under
air until the substrates had been consumed as judged by TLC. After
stirring the reaction mixture was transferred to a separating funnel
and the mixture extracted with EtOAc and washed with water
(20.0 mL). The organic phases were combined, the solvent evapor-
ated and the residue (a powder) purified by flash chromatography.
Experimental
1-(5,6-Dihydroxy-2-methyl-1-benzofuran-3-yl)ethanone 8
General
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Varian Mercury 400 MHz spectrometer. Carbon-
13 nuclear magnetic resonance (13C NMR) spectra were recorded
on the same instruments at 100 MHz. Chemical shifts are
reported in parts per million (ppm) relative to the solvent peaks
and coupling constants are given in Hertz (Hz). A Waters UPLC
coupled in tandem to a Waters photodiode array (PDA) detector
and a SYNAPT G1 HDMS mass spectrometer was used to generate
accurate mass data. The PDA detector was used for all purity
determinations (Maxplot 200–500 nm). All chemicals for UPLC-MS
work were of ultra-pure LC-MS grade and purchased from Fluka
(Steinheim, Germany) while ultra-pure solvents were purchased
from Honeywell (Burdick & Jackson, Muskegon, USA). Ultra-pure
water was generated using a Millipore Elix 5 RO system and
Millipore Advantage Milli-Q system (Millipore SAS, Molsheim,
France). Reactions were monitored by thin layer chromatography
(TLC) on aluminium-backed Merck silica gel 60 F254 plates. Gravity
OH
O
H3C
OH
H3C
O
Method A
Stirring time 24 h. The product was recrystallised from 5% MeOH
in EtOAc solution at 358C to afford a dark-brown powder
(198 mg, 48%) (Found: M–Hþ, 205.0518. C11H9O4 requires M–H,
205.0501). UPLC 95.5%. Rf 0.40 (EtOAc/hexane, 1:1). mp 200–
2038C. 1H NMR (400 MHz, DMSO-d6): d 2.51 (3H, s, CH3), 2.68
(3H, s, CH3), 6.91 (1H, s, ArH), 7.33 (1H, s, ArH), 8.94 (1H, br s,
OH), 9.06 (2H, br s, OH); 13C NMR (100 MHz, DMSO-d6): d 15.3,
30.8, 97.8, 106.4, 117.2, 117.2, 143.6, 144.2, 147.0, 160.7, 193.9.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
Anticancer Activity of 5,6-Dihydroxylated Benzo[b]furans
273
62%) (Found: M–Hþ, 231.0683. C13H11O4 requires M–H, 231.0657).
UPLC 99.7%. Rf 0.48 (EtOAc/hexane, 1:1). mp 2508C. 1H NMR
(400 MHz, DMSO-d6): d 2.14 (2H, m, CH2), 2.26 (3H, s, CH3),
2.45 (2H, t J 6.1 Hz, CH2), 2.98 (2H, t J 6.1 Hz, CH2), 7.11 (s, 1H,
ArH); 13C NMR (100 MHz, DMSO-d6): d 9.0, 22.2, 23.3, 37.9, 102.4,
107.9, 113.5, 115.9, 142.2, 143.4, 147.6, 169.6, 194.6.
Method B
Stirring time 44 h. The product was washed with EtOAc to afford
a black powder (202.0 mg, 49%).
1-(5,6-Dihydroxy-2,7-dimethyl-1-benzofuran-3-yl)-
ethanone 9
Method B
Stirring time 44 h. The product was washed with EtOAc to afford
a brown powder (311.0 mg, 67%).
CH3
OH
O
H3C
7,8-Dihydroxy-6-methoxy-3,4-dihydrodibenzo[b,d]furan-
1(2H)-one 12
OH
H3C
O
CH3
O
Method A
OH
O
Stirring time 24 h. The product was washed with EtOAc to afford a
red-brown powder (220 mg, 50%) (Found: M–Hþ, 219.0615. C12H11O4
requires M–H, 219.0657). UPLC 97.3%. Rf 0.46 (EtOAc/hexane, 1:1).
mp 2348C. 1H NMR (400 MHz, DMSO-d6): d 2.19 (3H, s, CH3), 2.45
(3H, s, CH3), 2.64 (3H, s, CH3), 7.17 (1H, s, ArH), 8.38 (1H, br s, OH),
9.25 (1H, br s, OH); 13C NMR (100 MHz, DMSO-d6): d 8.9,
15.4, 30.7, 103.2, 107.1, 116.2, 117.4, 141.8, 143.1, 146.5,
160.5, 193.9.
OH
O
Method A
Stirring time 24 h. The product was recrystallised from 5% MeoH
in EtOAc solution at 358C to afford a brown powder (348 mg,
70%) (Found: M–Hþ, 247.0557. C13H11O5 requires M–H, 247.0606).
UPLC 97.0%. Rf 0.38 (EtOAc/hexane, 2:1). mp 2328C. 1H NMR
(400 MHz, DMSO-d6): d 2.14 (2H, m, CH2), 2.46 (2H, t J 6.4 Hz,
CH2), 3.00 (2H, t J 6.4 Hz, CH2), 3.94 (3H, s, CH3), 7.00 (1H, s, ArH),
8.71 (1H, br s, OH), 9.24 (1H, br s, OH); 13C NMR (100 MHz, DMSO-
d6): d 22.1, 23.2, 37.4, 60.4, 99.9, 114.8, 115.6, 133.3, 136.2, 140.3,
145.0, 169.9, 194.5.
7,8-Dihydroxy-3,4-dihydrodibenzo[b,d]furan-1(2H)-one 10
OH
O
OH
O
7,8-Dihydroxy-3-methyl-3,4-dihydrodibenzo[b,d]furan-
1(2H)-one 13
Method A
Stirring time 24 h. The product was recrystallised from 5% MeOH
in EtOAc solution at 358C to afford a brown powder (284 mg,
65%) (Found: M–Hþ, 217.0458. C12H9O4 requires M–H, 217.0501).
UPLC 96.2%. Rf 0.48 (EtOAc/hexane, 1:1). 1H NMR (400 MHz,
DMSO-d6): d 2.21 (2H, m, CH2), 2.47 (2H, t J 6.4 Hz, CH2)
2.96 (2H, t J 6.4 Hz, CH2), 7.00 (1H, s, ArH), 7.22 (1H, s, ArH),
9.18 (2H, br s, OH); 13C NMR (100 MHz, DMSO-d6): d 22.2,
23.2, 37.3, 98.4, 105.4, 114.4, 115.6, 143.7, 144.4, 147.9, 169.7,
194.4.
OH
O
OH
H3C
O
Method A
Stirring time 24 h. The product was washed with EtOAc to
7,8-Dihydroxy-6-methyl-3,4-dihydrodibenzo[b,d]furan-
1(2H)-one 11
afford
a
brown powder (274 mg, 59%) (Found: M–Hþ,
231.0610. C13H11O4 requires M–H, 231.0657). UPLC 95.3%.
Rf 0.38 (EtOAc/hexane, 1:1). mp 2638C. 1H NMR (400 MHz,
DMSO-d6): d 1.11 (3H, d, J 6.0 Hz, CH3), 2.30 (1H, dd, J 4.6,
12.8 Hz, CH), 2.43 (2H, dd J 3.6, 12.8 Hz, CH2), 2.68 (1H,
m, CH), 3.01 (1H, dd J 4.6, 12.8 Hz, CH), 6.98 (1H, s, ArH), 7.21
(1H, s, ArH) and 9.15 (2H, br s, OH); 13C NMR (100 MHz, DMSO-d6):
d 20.7, 30.3, 40.0, 45.6, 98.5, 105.3, 114.3, 115.3, 143.7, 144.4,
148.2, 169.4 and 194.0.
CH3
OH
O
OH
O
Method C
Method A
Stirring time 24 h. The product was recrystallised from 5% MeOH
in EtOAc solution at 358C to afford a brown powder (288 mg,
Stirring time 42 h. The product was purified by flash chroma-
tography (silica: EtOAc/hexane, 0.5:9.5, 1:9, 2.5:7.5, 1:1) to afford
a brown powder (84 mg, 30%).
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274
K. W. Wellington et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
245.0867. C14H14O4 requires M–H, 245.0814). UPLC 99.8%.
Rf 0.64 (EtOAc/hexane, 1:1). mp 278–2808C [lit. [28] 2808C].
7,8-Dihydroxy-3,6-dimethyl-3,4-dihydrodibenzo[b,d]furan-
1(2H)-one 14
1H NMR (400 MHz, DMSO-d6):
d
1.09 (6H, s, 2ꢀ CH3),
2.38 (2H, s, CH2), 2.88 (2H, s, CH2), 6.98 (1H, s, ArH), 7.20
(1H, s, ArH), 9.15 (2H, br s, OH); 13C NMR (100 MHz, DMSO-d6):
d 28.0 (2ꢀ CH3), 34.9, 36.7, 51.5, 98.5, 105.3, 114.2, 114.4, 143.7,
144.3, 148.3, 168.6, 193.7.
CH3
OH
O
OH
H3C
Method C
O
Stirring time 42 h. The product was purified by flash chroma-
tography (silica: EtOAc/hexane, 0.5:9.5, 1:9, 2.5:7.5, 1:1) to afford
a yellow powder (118 mg, 40%).
Method A
Stirring time 24 h. The product was washed with EtOAc to afford a
light-brown powder (384 mg, 78%) (Found: M–Hþ, 245.0817.
C14H13O4 requires M–H, 245.0814). UPLC 96.0%. Rf 0.74
7,8-Dihydroxy-3,3,6-trimethyl-3,4-dihydrodibenzo-
[b,d]furan-1(2H)-one 17
1
(EtOAc/hexane, 1:1). mp 165–1688C. H NMR (400 MHz, DMSO-d6):
d 1.11 (3H, d J 6.0 Hz, CH3), 2.25 (3H, s, CH3), 2.29 (1H, dd J 4.7,
12.4 Hz, CH2), 2.43 (2H, dd J 3.4, 12.8 Hz, CH2), 2.68 (1H, m, CH), 3.03
(1H, dd J 4.7, 12.4 Hz, CH), 7.11 (1H, s, ArH), 8.47 (1H, br s, OH), 9.38
(1H, br s, OH); 13C NMR (100 MHz, DMSO-d6): d 9.0, 20.7, 30.3, 31.0,
45.6, 102.3, 107.9, 113.4, 115.5, 142.1, 143.3, 147.8, 169.2, 194.1.
CH3
OH
O
OH
H3C
H3C
Method C
Stirring time 42 h. The product was purified by flash chroma-
tography (silica: EtOAc/hexane, 0.5:9.5, 1:9, 1:1; EtOAc) to afford a
light-brown powder (209.0 mg, 71%).
O
Method A
Stirring time 24 h. The product was recrystallised from a
5% MeOH in EtOAc solution at 358C to afford a light-brown
powder (510 mg, 98%). (Found: M–Hþ 259.0931. C15H15O4
requires M–H, 259.0970). UPLC 96.6%. Rf 0.45 (EtOAc/hexane,
1:2). mp 2558C [lit. [33] 260–2628C]. 1H NMR (400 MHz, DMSO-
d6): d 1.09 (6H, s, 2ꢀ CH3), 2.26 (3H, s, CH3) 2.37 (2H, s, CH2),
2.90 (2H, s, CH2), 7.10 (1H, s, ArH), 8.41 (1H, br s, OH), 9.32
(1H, br s, OH); 13C NMR (100 MHz, DMSO-d6): d 9.0, 28.0,
34.9, 36.8, 51.5, 102.4, 107.9, 113.3, 114.7, 142.0, 143.3,
168.5, 193.8.
7,8-Dihydroxy-6-methoxy-3-methyl-3,4-dihydrodibenzo-
[b,d]furan-1(2H)-one 15
CH3
O
OH
O
OH
H3C
O
Method C
Method A
Stirring time 42 h. The product was purified by flash chroma-
tography (silica: EtOAc/hexane, 5:95, 1:9, 1:1) to afford a light-
brown powder (184 mg, 59%).
Stirring time 24 h. The product was washed with EtOAc to afford a
light-brown powder (194 mg, 37%) (Found: M–Hþ, 261.0843. C14H13O4
requires M–H, 261.0763). UPLC 99.6%. Rf 0.50 (EtOAc/hexane, 1:1).
mp 179–1828C. 1H NMR (400 MHz, DMSO-d6): d 1.12 (3H, d J 6.4 Hz,
CH3), 2.32 (1H, dd J 4.0, 12.4 Hz, CH) 2.44 (2H, dd J 3.4, 13.2 Hz, CH2),
2.72 (1H, m, CH), 3.06 (1H, dd J 5.0, 12.4 Hz, CH), 3.94 (3H, s, CH3), 6.97
(1H, s, ArH). 13C NMR (100 MHz, DMSO-d6): d 20.7, 30.3, 31.0, 45.7, 60.4,
99.8, 114.8, 115.3, 133.4, 136.3, 140.6, 145.1, 169.6, 194.1.
7,8-Dihydroxy-3,3,6-trimethyl-3,4-dihydrodibenzo-
[b,d]furan-1(2H)-one 18
CH3
O
OH
O
7,8-Dihydroxy-3,3-dimethyl-3,4-dihydrodibenzo[b,d]furan-
1(2H)-one 16
OH
H3C
H3C
OH
O
O
H3C
H3C
OH
O
Method A
Stirring time 24 h. The product was recrystallised from 5% MeOH
in EtOAc solution at 358C to afford a brown powder (403 mg,
73%). (Found: M–Hþ, 275.0922. C15H15O5 requires M–H,
275.0919). UPLC 94.5%. Rf 0.44 (EtOAc/hexane, 1:1). mp 288–
Method A
Stirring time 24 h. The product was washed with EtOAc
to afford a yellow powder (286 mg, 58%). (Found: M–Hþ,
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Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
Anticancer Activity of 5,6-Dihydroxylated Benzo[b]furans
275
2918C [lit. [31] 289–2918C]. 1H NMR (400 MHz, MeOH-d4): d 1.16
(6H, s, 2ꢀ CH3), 2.43 (2H, s, CH2) 2.90 (2H, s, CH2), 4.06 (3H, s, CH3),
7.01 (1H, s, ArH); 13C NMR (100 MHz, DMSO-d6): d 28.1, 35.0, 36.8,
51.2, 60.4, 99.8, 114.5, 114.7, 113.4, 136.2, 140.8, 145.1, 168.9,
193.9.
8.0 Hz, ArH), 8.45 (1H, br s, OH), 9.36 (1H, br s, OH); 13C NMR
(100 MHz, DMSO-d6): d 9.1, 30.7, 44.7, 102.4, 108.0, 113.4, 115.8,
126.9, 127.0, 127.1, 128.6, 142.3, 143.2, 143.4, 148.0, 168.8,
193.2.
7,8-Dihydroxy-6-methoxy-3-phenyl-3,4-dihydrodibenzo-
[b,d]furan-1(2H)-one 21
Method B
Stirring time 44 h. The product was washed with EtOAc to afford
a brown powder (425 mg, 77%).
CH3
O
7,8-Dihydroxy-3-phenyl-3,4-dihydrodibenzo[b,d]furan-
1(2H)-one 19
OH
O
OH
Ph
OH
O
O
OH
Ph
Method A
O
Stirring time 24 h. The product was washed with EtOAc to
afford a dull grey powder (279 mg, 43%). (Found: M–Hþ,
323.0922. C19H15O5 requires M–H, 323.0919). UPLC 96.8%.
Rf 0.38 (EtOAc/hexane, 1:2). mp 165–1688C. 1H NMR
(400 MHz, DMSO-d6): d 2.57 (1H, dd J 4.0, 16.0 Hz, CH),
2.93 (1H, dd J 12.0 Hz, 12.4 Hz, CH), 3.26 (2H, m, CH2), 3.67
(1H, m, CH), 3.95 (3H, s, CH3), 7.03 (1H, s, ArH), 7.26 (1H, t J
7.2 Hz, ArH), 7.35 (2H, t J 7.2, 8.0 Hz, ArH), 7.42 (2H, d J 8.0 Hz,
ArH), 8.76 (1H, br s, OH) and 9.33 (1H, br s, OH); 13C NMR
(100 MHz, DMSO-d6): d 30.6, 44.6, 60.4, 99.8, 114.6, 115.5,
126.8, 127.0, 128.5, 133.4, 136.2, 140.6, 143.0, 145.0, 169.0,
193.0.
Method A
Stirring time 24 h. The product was washed with EtOAc to
afford
white solid (449 mg, 76%). (Found: M–Hþ,
a
293.0821. C18H13O4 requires M–H, 293.0814). UPLC 99.8%.
Rf 0.50 (EtOAc/hexane, 1:1). mp 240–2438C. 1H NMR
(400 MHz, DMSO-d6): d 2.58 (1H, dd J 3.2, 16.0 Hz, CH), 2.93
(1H, dd J 12.4, 16.0 Hz, CH), 3.24 (2H, m, H-11), 3.66 (1H, m, CH),
7.01 (1H, s, ArH), 7.26 (1H, s, ArH), 7.35 (2H, t J 7.2, 7.6 Hz, ArH),
7.42 (2H, d J 7.6 Hz, ArH), 9.09 (1H, s, CH), 9.13 (1H, s, CH);
13C NMR (100 MHz, DMSO-d6): d 30.7, 44.6, 62.5, 63.4, 68.8, 71.5,
72.3, 73.8, 98.5, 105.4, 114.2, 115.5, 126.9, 128.6, 143.1, 143.9,
144.5, 148.3, 169.0, 193.1.
Method C
Stirring time 42 h. The product was purified by flash chroma-
tography (silica: EtOAc/hexane, 5:95, 1:9, 1:1; EtOAc) to afford a
black powder (58.0 mg, 15%).
Method C
Stirring time 42 h. The product was purified by flash chroma-
tography (silica: EtOAc/hexane, 5:95, 1:9, 1:1; EtOAc) to afford a
white solid (132 mg, 50%).
3-(3,4-Dihydroxy-5-methylphenyl)-3-methylpentane-2,4-
dione 22
7,8-Dihydroxy-6-methyl-3-phenyl-3,4-dihydrodibenzo-
[b,d]furan-1(2H)-one 20
Me
OH
O
CH3
OH
O
H3C
OH
CH3
H3C
OH
Ph
O
O
Method A
Method A
Stirring time 24 h. The product was washed with EtOAc to
afford a dark-brown powder (1.60 g, 80%) (Found: M–Hþ,
235.0926. C13H14O4 requires M–H, 235.0970. UPLC 98.0%.
Rf 0.48 (EtOAc/hexane, 1:1). mp 82–858C. 1H NMR (400 MHz,
DMSO-d6): d 1.60 (3H, s, CH3), 2.03 (6H, s, CH3), 2.08 (3H, s, CH3),
6.43 (1H, d J 2.0 Hz, ArH), 6.46 (1H, d J 2.4 Hz, ArH), 8.36
(1H, br s, OH), 9.29 (1H, br s, OH); 13C NMR (100 MHz,
DMSO-d6): d 16.2, 19.0, 27.2, 30.7, 68.8, 112.4, 120.1, 124.7,
127.7, 143.0, 144.8, 207.8.
Stirring time 24 h. The product was recrystallised from
5% MeOH in EtOAc solution at 358C to afford a light-brown
powder (279 mg, 80%) (Found: M–Hþ, 307.0977.1120. C19H15O4
requires M–Hþ, 307.0970). UPLC 99.0%. Rf 0.45 (EtOAc/hexane,
1:1). mp 250–2538C. 1H NMR (400 MHz, DMSO-d6): d 2.28 (3H, s,
CH3), 2.59 (1H, dd J 4.0, 16.4 Hz, CH), 2.91 (1H, dd J 12.2, 16.2 Hz,
CH), 3.28 (1H, m, CH2), 3.66 (1H, m, H-11 CH), 7.16 (1H, s, ArH),
7.26 (1H, t J 7.0 Hz, ArH), 7.35 (2H, t J 7.6 Hz, ArH), 7.42 (2H, d J
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276
K. W. Wellington et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 266–277
Unbound dye was removed and protein-bound dye was extracted
with 10 mM Tris base for optical density determination at a
wavelength of 540 nm using a multiwell spectrophotometer.
Optical density measurements were used to calculate the net
percentage cell growth.
The optical density of the test well after a 48 h period of
exposure to test drug is Ti, the optical density at time zero is
T0, and the control optical density is C. Percentage cell growth is
calculated as:
3-(3,4-Dihydroxy-5-methoxyphenyl)-3-methylpentane-2,4-
dione 23
OMe
OH
OH
O
H3C
H3C
CH3
O
ꢀ
ꢁ
ꢁ
ðTi ꢁ T0Þ
ðC ꢁ T0Þ
ꢀ 100 for concentrations at which Ti ꢂ T0
ꢀ 100 for concentrations at which Ti < T0:
Method A
Stirring time 24 h. The product was washed with EtOAc to afford
a dark-brown powder (1.10 g, 55%) (Found: M–Hþ, 251.0933.
C13H14O5 requires M–H, 251.0919. Rf 0.48 (EtOAc/hexane, 1:1).
mp 108–1108C. 1H NMR (400 MHz, DMSO-d6): d 1.62 (3H, s, CH3),
2.05 (6H, s, CH3), 3.74 (3H, s, OMe), 6.27 (1H, s, ArH), 6.28 (1H, s,
ArH), 8.44 (1H, br s, OH), 9.03 (1H, br s, OH); 13C NMR (100 MHz,
DMSO-d6): d 19.1, 27.3, 56.0, 69.0, 103.1, 108.7, 127.7, 133.9, 145.8,
148.5, 207.7.
ꢀ
ðTi ꢁ T0Þ
T0
The results of a five dose screening were reported as TGI (total
growth inhibition). The TGI is the concentration of test drug
where 100 ꢀ (T ꢁ T0)/(C ꢁ T0) ¼ 0. The TGI signifies a cytostatic
effect.
The biological activities were separated into four categories:
inactive (GI50 or TGI > 100 mM), weak activity (30 mM < GI50 or
In vitro anticancer activity evaluation
Assay background
TGI < 100 mM),
TGI < 30 mM) and potent activity (GI50 or TGI < 10 mM).
moderate
activity
(10 mM < GI50
or
The growth inhibitory effects of the compounds were tested in a
3-cell line panel consisting of TK10 (renal), UACC62 (melanoma)
and MCF7 (breast) cancer cells using the Sulforhodamine B (SRB)
assay [32]. The SRB assay was developed by Skehan and colleagues
to measure drug-induced cytotoxicity and cell proliferation. Its
principle is based on the ability of the protein dye, sulforhod-
amine B (Acid Red 52), to bind electrostatically in a pH-dependent
manner to protein basic amino acid residues of trichloroacetic
acid-fixed cells. Under mild acidic conditions it binds to the fixed
cellular protein, while under mild basic conditions it can be
extracted from cells and solubilised for measurement. The SRB
assay is performed at CSIR in accordance with the protocol of the
Drug Evaluation Branch, NCI, and the assay has been adopted for
this screen.
For each tested compound, three response parameters, GI50
(50% growth inhibition and signifies the growth inhibitory
power of the test agent), TGI (which is the drug concentration
resulting in total growth inhibition and signifies the cytostatic
effect of the test agent) and LC50 (50% lethal concentration and
signifies the cytotoxic effect of the test agent), were calculated for
each cell line.
We thank Dr Paul Steenkamp for HRMS analysis, the CSIR (Thematic A
grant) for financial support and Novozymes SA (Pty) Ltd for a kind
donation of the enzyme.
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