A laccase-catalysed one-pot synthesis of aminonaphthoquinones and their anticancer activity

Article abstract of DOI:10.1016/j.bmc.2012.05.028

Nuclear monoamination of a 1,4-naphthohydroquinone with primary aromatic amines was catalysed by the commercial laccase, Novozym 51003, from Novozymes to afford aminonaphthoquinones. The synthesis was accomplished by reacting a mixture of the primary amine and 1,4-naphthohydroquinone in succinate-lactate buffer and a co-solvent, dimethylformamide, under mild reaction conditions in a vessel open to air at pH 4.5 and pH 6.0. Anticancer screening showed that the aminonaphthoquinones exhibited potent cytostatic effects particularly against the UACC62 (melanoma) cancer cell line (GI₅₀ = 3.98-7.54 μM). One compound exhibited potent cytostatic effects against both the TK10 (renal) and the UACC62 (melanoma) cancer cell line. The cytostatic effects of this compound (GI₅₀ = 8.38 μM) against the TK10 cell line was almost as good as that of the anticancer agent, etoposide (GI₅₀ = 7.19 μM). Two compounds exhibited potent cytostatic effects against both the UACC62 (melanoma) and the MCF7 (breast) cancer cell lines. The total growth inhibition (TGI) of most of the compounds was better than that of etoposide against the UACC62 cell line. Three compounds (TGI = 7.17-7.94 μM) exhibited potent cytostatic effects against the UACC62 cell line which was 7 to 8-fold better than that of etoposide (TGI = 52.71 μM). The results are encouraging for further study of the aminonaphthoquinones for potential application in anticancer therapy.

Full text of DOI:10.1016/j.bmc.2012.05.028

Bioorganic & Medicinal Chemistry 20 (2012) 4472–4481
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A laccase-catalysed one-pot synthesis of aminonaphthoquinones and their
anticancer activity
⇑
Kevin W. Wellington , Natasha I. Kolesnikova
CSIR Biosciences, PO Box 395, Pretoria, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Nuclear monoamination of a 1,4-naphthohydroquinone with primary aromatic amines was catalysed by
the commercial laccase, Novozym 51003, from Novozymes to afford aminonaphthoquinones. The synthe-
sis was accomplished by reacting a mixture of the primary amine and 1,4-naphthohydroquinone in suc-
cinate-lactate buffer and a co-solvent, dimethylformamide, under mild reaction conditions in a vessel
open to air at pH 4.5 and pH 6.0.
Received 7 March 2012
Revised 3 May 2012
Accepted 12 May 2012
Available online 23 May 2012
Anticancer screening showed that the aminonaphthoquinones exhibited potent cytostatic effects par-
Keywords:
Biocatalysis
Laccase
ticularly against the UACC62 (melanoma) cancer cell line (GI50 = 3.98–7.54
ited potent cytostatic effects against both the TK10 (renal) and the UACC62 (melanoma) cancer cell
line. The cytostatic effects of this compound (GI50 = 8.38 M) against the TK10 cell line was almost as
good as that of the anticancer agent, etoposide (GI50 = 7.19 M). Two compounds exhibited potent cyto-
lM). One compound exhib-
l
l
Oxidative enzymes
1,4-Naphthohydroquinones
Aminonaphthoquinones
C–N bond formation
Primary amines
Monoamination
Anticancer agents
Cytostatic effects
Cytotoxic effects
GI50
static effects against both the UACC62 (melanoma) and the MCF7 (breast) cancer cell lines. The total
growth inhibition (TGI) of most of the compounds was better than that of etoposide against the UACC62
cell line. Three compounds (TGI = 7.17–7.94
cell line which was 7 to 8-fold better than that of etoposide (TGI = 52.71
l
M) exhibited potent cytostatic effects against the UACC62
M).
l
The results are encouraging for further study of the aminonaphthoquinones for potential application in
anticancer therapy.
Ó 2012 Elsevier Ltd. All rights reserved.
TGI
LC50
1. Introduction
It must be noted that the amino group is in the ortho position to
the ketone in mitomycin C, streptonigrin,29 actinomycin32 (and the
1,4-Naphthoquinone derivatives have exhibited an interesting
variety of biological responses such as antiallergic,1–3 antibacterial,4,5
antifungal,4–7 anti-inflammatory,1–4,7 antithrombotic,8,9 antiplate-
let,1–3,10–13 antiviral,4,14,10,15 apoptosis,16–18 lipoxygenase,19,20 radical
scavenging21 and anti-ringworm4 activities. There have also been re-
ports on their anticancer activity.5,6,10–12 Human DNA topoisomerase
I and II are known to be inhibited by 1,4-naphthoquinone deriva-
tives.12,21–26 These 1,4-naphthoquinone derivatives can also produce
reactive oxygen species (ROS) such as semiquinone and hydroxyl
radicals by enzymatic reduction (i.e. NADPH-cytochrome P 450
reductase).26–29 It is the hydroxyl radical that is the cause of DNA
strand breaks.30
structurally related aurantins33) as well as in the ansa-antibiotics
rifamycin32 and geldanamycin.34 The position of the amine group
may be a requirement for the biological activity exhibited by these
compounds which has inspired research into new routes to the
synthesis of aminoquinones.
In 2008 7.6 million deaths (13% of all deaths) were attributed to
cancer which is the leading cause of death worldwide. Cancer in
the breast, colon, lung, stomach, and liver cause the most deaths
each year. Deaths from cancer are projected to continue to rise
worldwide with an estimated 13.1 million deaths predicted for
2030.35
Chemotherapy is the primary treatment for cancer and has been
hampered by the development of drug resistant and multidrug
resistant tumours. Multidrug resistance is the principal mechanism
by which many cancers develop resistance to chemotherapy drugs
and is a major factor in the failure of many forms of chemother-
apy.36 One of the largest protein families in biology is the ATP-
binding cassette (ABC), a superfamily of proteins.37 Their role in
resistance to chemotherapy drugs has been known for more than
three decades.38 Fifteen family members of these ABC proteins
The aminoquinone moiety is prevalent in several drugs that are
in use such as the commercial anti-neoplastic agents actinomy-
cin29 and streptonigrin29 ( Fig. 1). The antibiotics, mitomycin31
and rifamycin,32 are also based on an aminoquinone (Fig. 1).
⇑
Corresponding author.
E-mail
addresses:
kwellington@csir.co.za,
kwwellington@gmail.com
(K.W. Wellington).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.028

K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
4473
O
H3CO
H2N
peptide-CO
O
N
N
OH
CH3
N
CH3
O
peptide-CO
O
H2N
HO
H2N
CH3
O
H3CO
OCH3
MeO
Actinomycin
Streptonigrin
CH3
CH3
O
O
HO
O
O
NH2
NH
H3C
OH
O
CH3
O
O
O
O
O
OH
O
O
CH3
N
H
H2N
H3C
OCH3
H3C
NH
H3C
H3C
H3C
MeO
H3C
MeO
CH3
OH
H3CO
N
O
O
O
Mitomycin C
CH3
OCONH2
Geldanamycin
Rifamycin
Figure 1. Drugs having an aminoquinone moiety.
can function as drug-efflux pumps and these have been implicated
as potentially conferring resistance.39,40
Amongst these there have also been reports on amination of p-
hydroquinones with primary aromatic amines.47
Only three of these account for the most observed multidrug
resistance (MDR) in humans. These are P-glycoprotein (Pgp/
MDR1/ANCB1) and the MDR–associated protein (MRP)1 (ABCC1),
and the breast cancer resistance protein (ABCG2).41 The drug efflux
pumps, located in the tumour-cell plasma membrane, actively ex-
pel chemotherapy drugs from the interior thus allowing tumour
cells to avoid the toxic effects of the drug.36 There is thus a need
for new compounds that are effective in treating drug resistant
and multidrug resistant tumours.
Laccase (p-diphenol: oxygen oxidoreductase, EC 1.10.3.2) be-
longs to the group of copper-containing oxidases called oxidore-
ductases.42,43 It catalyses the reduction of molecular oxygen to
water and by-pass hydrogen peroxide formation.42,43 This blue
multi-copper oxidase is able to catalyse the oxidation of various
low-molecular weight compounds, including: benzenediols, ami-
nophenols, polyphenols, polyamines, and lignin-related mole-
cules.44 The demand for green chemical processes has inspired
interest in the application of enzymes to address modern synthetic
organic chemistry challenges. The successful application of laccase
in organic synthesis has resulted in several literature reports.45,46,49
Our goal was to synthesise aminonaphthoquinones having the
amine moiety in the ortho position to the ketone of the quinone
ring. We report here on the synthesis of aminonaphthoquinones
using a commercial laccase (Novozym 51003) from Novozymes
and also on the anticancer screening results of the synthesised
compounds. This report is, to the best of our knowledge, the first
on amination of 1,4-dihydroxy-2-naphthoic acid using laccase
and also the first report on the anticancer activity of the synthes-
ised compounds. We have previously reported on the synthesis
of diaminobenzoquinones from the reaction between p-hydroqui-
nones and primary aryl and alkyl amines using a commercial
laccase.48
2. Results and discussion
2.1. Synthesis
Novozymes has a few laccases available on the market in differ-
ent preparations. Novozym 51003 is a robust, stable, fungal laccase
O
O
OH
OH
OH
O
R3
NH
R4
R2
R1
Laccase
OH
O
R1
R2
3
+
pH 4.5
heat
R4
1
NH2
R3
2a-2k
2b
2e
2f
2k
2a
2c
R1 = H
2g
2h
R1 = H
2j
R1 = F
2d
2i
R1 = H
R1 = F
R1 = H
R1 = H
R1 = H R1 = H R1 = Cl
R1 = H
R
R
R
2 = H
3 = H
4 = H
R
R
R
2 = H
R
R
R
2 = H
3 = Cl
4 = H
R
R
R
2 = Cl
3 = H
4 = H
R
R
R
2 = H
3 = H
4 = H
R
R
R
2 = F
3 = H
4 = H
2 = H
R
R
R
2 = H
R
R
R
2 = H
3 = F
4 = H
R
R
R
2 = H
3 = H
4 = H
R
R
R
2 = H
3 = Cl
4 = H
R
R
R
3 = CH(CH3)2
4 = H
3 = EtOH
4 = H
3 = H
4 = CN
Scheme 1. Laccase-catalysed synthesis of aminonaphthoquinones.

4474
K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
Table 1
The synthesised aminonaphthoquinones (yield in parentheses) at pH 4.5 using Methods A–D
Entry
Hydroquinone
Amine (equiv)
Reaction Time (h)
Method
Product
1
2
3
4
5
6
7
1
1
1
1
1
1
1
2a (2)
2b (2)
2b (2)
2c (2)
2c (2)
2d (2)
2f (2)
48
26
48
27
72
24
24
D
A
D
A
B
C
C
4 (77%)
5 (70%)
5 (47%)
6 (51%)
6 (64%)
7 (32%)
8 (52%)
Method A–2.0 mL (2 220 U) Novozym 51003, 2 equiv amine (1.2 mmol), 1,4-dihydroxy-2-naphthoic acid (0.6 mmol), 2.0 mL water, 2.0 mL succinate-lactate buffer (1.0 M, pH
4.5), 1.0 mL DMF, 35 °C.
Method B–4.0 mL (4 440 U) Novozym 51003, 2 equiv amine (1.2 mmol), 1,4-dihydroxy-2-naphthoic acid (0.6 mmol), 2.0 mL water, 2.0 mL succinate-lactate buffer (1.0 M, pH
4.5), 1.0 mL DMF, 35 °C.
Method C–1.0 mL (1 110 U) Novozym 51003, 2 equiv amine (1.2 mmol), 1,4-dihydroxy-2-naphthoic acid (0.6 mmol), 2.0 mL water, 2.0 mL succinate-lactate buffer (1.0 M, pH
4.5), 1.0 mL DMF, 35 °C.
Method D–3.5 mL (3 885 U) Novozym 51003, 2 equiv amine (1.2 mmol), 1,4-dihydroxy-2-naphthoic acid (0.6 mmol), 2.0 mL water, 2.0 mL succinate-lactate buffer (1.0 M, pH
4.5), 1.0 mL DMF, 35 °C.
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
NH
NH
NH
NH
NH
6
8
4
5
7
CN
Cl
CH3
H3C
OH
Figure 2. Aminonaphthoquinones synthesised at pH 4.5.
from the thermophilic ascomycete Myceliophthora thermophila
used for lignin modification within pulps and effluents. It is pro-
duced by submerged fermentation of genetically modified Aspergil-
lus sp. with molecular weight of 56,000 Da. The goal of our
investigation was to determine whether this commercial laccase
could be used to catalyse C–N bond formation to afford amino-
naphthoquinones. The reaction that was investigated was that be-
tween the hydroquinone 1 and the primary amines 2a–k to form 3
as shown in Scheme 1.
B compared to that obtained using Method A (51%, entry 4), a long-
er reaction time was in this case better for product formation. A
characteristic dark-purple, red or brown color of the product was
indicative of the formation of the aminonapthoquinone and simpli-
fied product isolation. The phenolic hydroxyl groups were not ob-
served and the amine proton was not observed for all of the
synthesised compounds. Signals characteristic of the carbonyl car-
bons of the quinones are observed in the 178–186 ppm range of
the 13C NMR spectrum. The structures of the synthesised amino-
naphthoquinones are shown in Figure 2 below.
The 1,4-naphthoquinone with a carboxyl group was chosen be-
cause the carbon at the 3-position would be a good electrophile
due to the electron withdrawing effect of the carboxyl group in
the 2-position. This would promote C–N bond formation and thus
allow access to the aminonaphthoquinones. The carboxyl group
would also increase the solubility of the 1,4-naphthohydroquinone
in an aqueous medium in addition to providing another point
where structural modification can be done, for example, by amida-
tion and esterification for the synthesis of derivatives. Only pri-
mary amines were used in this investigation and no reactions
were attempted with secondary, tertiary or cyclic amines.
The reactions were conducted using the laccase, Novozym
51003, in succinate-lactate buffer (pH 4.5) and a co-solvent, DMF.
The latter was added to aid the dissolution of the substrates. For
these reactions 2 equiv of the amine was used since this would
promote the formation of the aminonaphthoquinone. The enzyme
was added at different time intervals to the reaction mixture to en-
sure fresh enzyme and to circumvent the possibility of denaturing
all at once. The results of the investigation are shown in Table 1
below.
Since the yields of some of the products were low, it was
decided to investigate whether the aminonaphthoquinones could
be synthesised at pH 6.0 and also whether the yields could be im-
proved. The results of the investigation using Method E are shown
in Table 2 below.
From the results it can be seen that the aminonaphthoquinones
can also be synthesised at pH 6.0. The highest yield was obtained
for 4 (85%, entry 1) and the lowest for 9 (25%, entry 3). The yield
for 4 is 8% more than that obtained at pH 4.5 using Method E
(77%, Table 1, entry 1). The yield for 7 (33%, Table 2, entry 2) is sim-
Table 2
The synthesised aminonaphthoquinones (yield in parentheses) using Novozym 51003
in aqueous DMF at pH 6.0
Entry
Hydroquinone
Amine (2 equiv)
Product time (h)
Reaction
1
2
3
4
5
6
7
8
1
1
1
1
1
1
1
1
2a
2d
2e
2g
2h
2i
48
48
48
48
48
48
48
48
4 (85%)
7 (33%)
9 (25%)
10 (29%)
11 (49%)
12 (28%)
13 (40%)
14 (31%)
The highest yield was obtained for 4 (77%, entry 1) and the low-
est for 7 (32%, entry 6). Method A is better for the synthesis of 5
because a higher yield (70%, entry 2) was obtained which was
23% higher than that obtained using Method D (47%, entry 3). A
longer reaction time in Method D did not increase the yield for 5.
A 13% higher yield for 6 (64%, entry 5) was obtained using Method
2j
2k
Method E–2.5 mL (2 775 U) Novozym 51003, 2 equiv amine, 1,4-dihydroxy-2-
naphthoic acid (0.6 mmol), 1.0 mL DMF, 3.0 mL sodium phosphate buffer (0.01 M,
pH 6.0).

K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
4475
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
OH
NH
NH
NH
NH
NH
NH
F
Cl
F
O
O
O
13
14
11
9
10
12
F
Cl
Cl
F
Figure 3. Aminonaphthoquinones synthesised at pH 6.0.
ilar to that obtained using Method A (32%, Table 1, entry 6). The
additional aminonaphthoquinones are shown in Figure 3.
Cyanide and halides are known to inhibit laccase.45,50 The low
yield for 7 could be due to the nitrile moiety, in both 2d and 7,
inhibiting laccase. This can occur by coordination of the nitrogen
atom of the nitrile moiety to the copper atoms within the laccase
active site. The possibility exists that the presence of halides such
as chloro and fluoro may also inhibit laccase and may account for
the low yields of the halogenated aminonaphthoquinones.
The optimal pH of most laccases is known to be between 3.5
and 5.0.51 By conducting the reactions at pH 6.0 the catalytic activ-
ity of the commercial laccase could have been reduced resulting in
lower conversion of 1,4-dihydroxy-2-naphthoic acid 1 to the corre-
sponding naphthoquinone intermediate. Lower concentrations of
the intermediate could also account for the generally lower yields
of the aminonaphthoquinones at pH 6.0.
alkylamines was achieved using a Lewis acid catalyst, CeCl3Á7H2O
in ethanol at room temperature.54
Similar synthetic approaches to that reported by Chakraborty52
and Benites et al.54 would have to be attempted to access the
aminonaphthoquinones. Our method allows access to aminonaph-
thoquinones in a one-pot synthesis directly from 1,4-dihydroxy-2-
naphthoic acid 1. We have thus eliminated the use of a chemical
oxidant, an iodinated intermediate, a palladium catalyst and a
phosphine ligand in the one approach, and in the other the use
of a Lewis acid catalyst (CeCl3.7H2O) and a chlorinated inter-
mediate.
2.2. Anticancer evaluation
Screening was conducted against TK10 (renal), UACC62 (mela-
noma), MCF7 (breast) and HeLa (Human Negroid Cervix Epitheloid
Adenocarcinoma, ECACC no. 93021013) cancer cell lines using the
sulforhodamine B (SRB) assay to determine the growth inhibitory
effects of the compounds.55 The TK10 (renal), UACC62 (melanoma)
and MCF7 (breast) 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.56 Etoposide, an anticancer agent, was
used as a positive control. It is known to be an inhibitor of topoiso-
merase, particularly topoisomerase II and aids in DNA unwinding
which causes the DNA strands to break.57 Three parameters were
determined during the screening process such as 50% cell growth
inhibition (GI50), total cell growth inhibition (TGI) and the lethal
concentration that kills 50% of cells (LC50). The results are shown
in Table 3.
A proposed mechanism for the formation of the aminonaphtho-
quinones is shown in Figure 4.
The role of laccase is simply that of an oxidant, two laccase oxi-
dations occur before the aminonaphthoquinone is formed.
Literature reports on nuclear amination by chemical methods
are limited. This is due to the susceptibility of the amino group
to oxidation and hydrolysis. A chemical method for achieving nu-
clear amination on a 1,4-benzohydroquinone was reported by
Chakraborty.52 This was accomplished by first halogenating (iodin-
ating) the 1,4-benzohydroquinone followed by coupling to the pri-
mary amine using a palladium catalyst and a triphenylphosphine
ligand while refluxing under argon.52 Some well-known chemical
oxidants such as cupric acetate, silver (I) oxide and sodium iodate
have also been used to achieve nuclear amination of 1,4-hydro-
benzoquinones with primary aromatic amines.53 Amongst these
sodium iodate can be used to accomplish amination more
conveniently.53
Several compounds exhibited potent growth inhibitory activity
as seen from the results in Table 3. The GI50 concentrations of the
compounds were compared to that of etoposide.
A chemical method which leads to aminonaphthoquinones has
been reported by Benites et al.54 Amination of 1,4-naphthoquinone
and 2,3-dichloro-1,4-naphthoquinone with a variety of aryl- and
Only 5 exhibited potent activity (GI50 = 8.38
lM, entry 2)
against the TK10 cell line which was almost as good as that of
O
O
O
O
OH
+
NHR
OH
NH2R
+
H2NR
Laccase
-H
OH
OH
O
OH
-
-
O
O
O
O
OH
O
hydroquinone 1
+
H
O
O
O
O
O
H
NHR
OH
NHR
OH
NHR
OH
+
-
H
Laccase
OH
O
O
O
aminoketophenol
aminonaphthoquinones
Figure 4. A proposed mechanism for the formation of the aminonaphthoquinones.

4476
K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
etoposide (GI50 = 7.19 lM, entry 12). The other compounds had
medium to weak activity.
Potent growth inhibitory activity (GI50 = 3.98–7.54 lM) was
observed for almost all the compounds against the UACC62 cell
line. The best activity was for 9 (GI50 = 3.98 M, entry 6) and 7
l
(GI50 = 3.99
(GI50 = 0.89
l
M, entry 4) but it was not as good as that of etoposide
l
M, entry 12).
Screening against the MCF7 cell line showed that only the two
fluorinated compounds, 12 (GI50 = 9.08 M, entry 9) and 13
(GI50 = 9.84 M, entry 10) exhibited potent growth inhibitory
activity which was not as good as that of etoposide (GI50 = 0.56 M,
l
l
l
entry 12). The activities of the other compounds were moderate to
weak.
The aminonaphthoquinones exhibited moderate to weak
activity against the HeLa cell line.
A comparison of the TGI concentrations of the compounds with
etoposide was also done. Most of the compounds were inactive
and a few exhibited weak activity against the TK10 cell line.
Compounds 7 (TGI = 7.17
and 14 (TGI = 7.94 M, entry 11) all exhibited potent activity
against the UACC62 cell line. The activities of these compounds
were 7- to 8-fold better than that of etoposide (TGI = 52.71 M,
lM, entry 4), 9 (TGI = 7.83 lM, entry 6)
l
l
entry 12) and the best activity was exhibited by 7. Most of the
other compounds exhibited moderate to weak activities (TGI =
10.81–49.08 lM) which was better than etoposide.
Screening against the MCF7 cell line showed that about half the
number of compounds showed weak activity (TGI = 48.43–
98.93
l
M) while other compounds were inactive like etoposide.
M, Entry 9). Be-
The best activity was exhibited by 12 (TGI = 48.43
l
nites et al. also reported on the anticancer activity of a series of
aminonaphthoquinones.54 Potent activity was observed against
the MCF7 cell line for some of the aminonaphthoquinones.54
The TGIs of the aminonaphthoquinones were weaker than that
of etoposide against the HeLa cancer cell line.
The LC50 concentrations of the compounds were compared to
that of etoposide to get an indication of the cytotoxic effects of
these compounds against the three cell lines. The compounds were
inactive like etoposide against the TK10 cell line.
Almost all of the compounds were more lethal (TGI =
19.13–81.18
which was inactive (LC50 >100
Five compounds, 7, 9, 12, 13 and 14, exhibited more lethal cyto-
toxic effects (TGI = 88.46–93.01 M) against the MCF7 cell line
than that of etoposide which was inactive (LC50 >100 M).
Only compounds 7, 9 and 14 (TGI = 82.33, 86.14 and 85.85
respectively) were slightly more lethal than etoposide (TGI =
87.54 M, Entry 12) against the HeLa cell line.
lM) against the UACC62 cell line than etoposide
lM).
l
l
lM,
l
Compound 5 having the isopropyl substituent on the phenyl ring
exhibited potent activity against both the TK10 and UACC62 cell
lines. The two fluorinated compounds, 12 and 13, exhibited potent
growth inhibitory activity against both the UACC62 and MCF7 cell
lines. Compound 7, having a nitrile substituent in the meta position
on the phenyl ring, and 9 having a chloro substituent in the para
position on the phenyl ring, were the most potent against the UACC62
cell line. The compounds exhibited better cytostatic (lower TGI con-
centrations) and cytotoxic effects (lower LC50 concentrations) than
etoposide against the UACC62 cell line. Overall, the aminonaphtho-
quinones were most effective against the UACC62 cell line in spite
of the HeLa cell line being a more sensitive one.
The aminonaphthoquinones were most effective against the
UACC62 (melanoma) cancer cell line exhibiting potent activity
against it. Only compound 5 exhibited potent cytostatic effects
against both the TK10 (renal) and the UACC62 (melanoma) cancer
cell lines. The activity of 5 (GI50 = 8.38
line was almost as good as that of the known drug, etoposide
(GI50 = 7.19 M). Two compounds, 12 and 13, exhibited potent
lM) against the TK10 cell
l

K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
4477
cytostatic effects against both the UACC62 (melanoma) and the
4.3.1. Method A
MCF7 (breast) cancer cell lines. The aminonaphthoquinones also
exhibited better TGI than that of etoposide against the UACC62 cell
Novozym 51003 (1.0 mL) was added to a mixture of 1,4-dihy-
droxy-2-naphthoic acid (0.6 mmol), amine (1.2 mmol, 2 equiv),
succinate-lactate buffer (2.0 mL, 1.0 M, pH 4.5), water (2.0 mL)
and DMF (1.0 mL) at 35 °C. More enzyme (1.0 mL) was added after
6 h. After heating the reaction mixture was transferred to a sepa-
rating funnel to which water (25 mL) was added. The mixture
was extracted with EtOAc, the solvent evaporated and the residue
purified by flash chromatography. The product was then washed
with a 25% Et2O/hexane solution.
line of which
7 (TGI = 7.17 lM), 9 (TGI = 7.83 lM) and 14
(TGI = 7.94 M) all exhibited potent cytostatic effects 7- to 8-fold
l
better than that of etoposide (TGI = 52.71 lM).
3. Conclusions
A new biocatalytic method was developed for the synthesis of
aminonaphthoquinones by using laccase, a non-hazardous oxidis-
ing agent. The commercial laccase, Novozym 51003, can be used
to access aminonaphthoquinones at both pH 4.5 and at pH 6.0
in a one-pot synthesis from the reaction of 1,4-dihydroxy-2-naph-
thoic acid and a primary amine. The formation of the product is
affected by factors such as the solubility of the substrates, pH of
the reaction mixture, reaction temperature in addition to the
nucleophillicity and number of equivalents of the amine. There
is potential for other laccases, particularly those with a higher
activity and better substrate specificity, to be employed for the
synthesis of aminonaphthoquinones. The aminonaphthoquinones
were most effective against the UACC62 (melanoma) cancer cell
line exhibiting potent cytostatic effects. These results are encour-
aging for further studies of the aminonaphthoquinones for their
application in anticancer therapy. Future work will entail further
synthesis of aminonaphthoquinones and structural modification
of compounds exhibiting potent activity to enhance their antican-
cer activity.
4.3.2. Method B
Novozym 51003 (1.0 mL) was added to a mixture of 1,4-dihy-
droxy-2-naphthoic acid (0.6 mmol), amine (1.2 mmol, 2 equiv),
succinate-lactate buffer (2.0 mL, 1.0 M, pH 4.5), water (2.0 mL)
and DMF (1.0 mL) at 35 °C. More enzyme (1.0 mL) was added after
2, 4 and 24 h. After heating the reaction mixture was transferred to
a separating funnel to which water (25 mL) was added. The mix-
ture was extracted with EtOAc, the solvent evaporated and the res-
idue purified by flash chromatography. The product was then
washed with a 25% Et2O/hexane solution.
4.3.3. Method C
Novozym 51003 (1.0 mL) was added to a mixture of 1,4-dihy-
droxy-2-naphthoic acid (0.6 mmol), amine (1.2 mmol, 2 equiv),
succinate-lactate buffer (2.0 mL, 1.0 M, pH 4.5), water (2.0 mL)
and DMF (1.0 mL) at 35 °C. After heating the reaction mixture
was transferred to a separating funnel to which water (25 mL)
was added. The mixture was extracted with EtOAc, the solvent
evaporated and the residue purified by flash chromatography.
The product was then washed with a 25% Et2O/hexane solution.
4. Experimental
4.1. General
4.3.4. Method D
Novozym 51003 (1.5 mL) was added to a mixture of 1,4-dihy-
droxy-2-naphthoic acid (0.6 mmol), amine (1.2 mmol, 2 equiv),
succinate-lactate buffer (2.0 mL, 1.0 M, pH 4.5), water (2.0 mL)
and DMF (1.0 mL) at 35 °C. More enzyme (1.0 mL) was added after
heating for 1.5 h and 3 h. After heating the reaction mixture was
transferred to a separating funnel to which water (25 mL) was
added. The mixture was extracted with EtOAc, the solvent evapo-
rated and the residue purified by flash chromatography. The prod-
uct was then washed with a 25% Et2O/hexane solution.
Proton nuclear magnetic resonance (1H NMR) spectra were re-
corded on a 200 MHz Varian Gemini spectrometer and also on a
400 MHz Varian Unity Plus spectrometer. Carbon-13 nuclear
magnetic resonance (13C NMR) spectra were recorded on the
same instruments at 50 MHz and 100 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 Syn-
apt HDMS mass spectrometer. 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.
4.3.5. Method E
Novozym 51003 (1.0 mL) was added to a mixture containing
amine (1.8 mmol, 2 equiv), 1,4-dihydroxy-2-naphthoic acid
(0.9 mmol), sodium phosphate buffer (3.0 mL, 0.01 M, pH 6.0)
and DMF (1.0 mL) while stirring at 40 °C. After heating for 2 h more
enzyme (1.0 mL) was added. More enzyme (0.5 mL) was again
added after 24 h. After heating for 48 h the reaction mixture was
transferred to a separating funnel to which water (25 mL) was
added. The mixture was extracted with EtOAc, the solvent evapo-
rated and the residue purified by flash chromatography. The prod-
uct was then washed with a 25% Et2O/hexane solution.
4.2. Materials
All chemicals were reagent grade materials.
4.2.1. Substrates
The 1,4-naphthohydroquinone-2-carboxylic acid, primary
amines and were obtained from Sigma–Aldrich South Africa.
4.3.6. 1,4-Dioxo-3-(phenylamino)-1,4-dihydronaphthalene-2-
carboxylic acid 4
4.2.2. Enzymes
The laccase, Novozym 51003 (1,110.00 U/g) from the ascomy-
cete M. thermophila was obtained from Novozymes SA.
O
O
OH
4.3. Synthetic methods
NH
O
The following methods were used for the synthesis of the
aminonaphthoquinones.

4478
K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
4.3.6.1. Method D.
Purification by flash chromatography (sil-
4.3.8.2. Method B.
Heating time = 72 h. Purification by flash
ica: EtOAc/hexane, 1:6, 1:4, 1:2 and EtOAc) to afford a brown solid
(0.1394 g, 77%).
chromatography (silica: EtOAc/hexane, 1:2, 1:1; EtOAc; EtOAc/
MeOH, 19.9:0.1 and 19.5:0.5) to afford
a dark-brown solid
(0.1305 g, 64%). (MÀH+ Found: 336.0865. C19H14NO5 requires
MÀH, 336.0872). Rf = 0.24 (EtOAc/hexane, 1:1). Mp = 157–159 °C.
1H NMR (200 MHz, CDCl3): d = 1.68 2H, br s, NH and OH), 2.92
(2H, t J = 6.4 and 6.8 Hz, CH2), 3.91 (2H, t J = 6.4 and 6.6 Hz, CH2),
7.11 (2H, d J = 8.2 Hz, ArH), 7.29 (2H, d J = 7.8 Hz, ArH), 7.64–7.87
(2H, m, ArH), 7.91 (1H, m, ArH), 8.23 (1H, d J = 7.8 Hz, ArH) and
13.10 (1H, br s, CO2H); 13C NMR (100 MHz, CDCl3): d = 38.7, 63.4,
100.2, 124.6, 126.9, 127.0, 130.0, 131.0, 131.9, 133.8, 135.6,
136.8, 138.3, 153.8, 171.3, 180.2 and 184.4.
4.3.6.2. Method E.
Heating time = 48 h. Purification by flash
chromatography (silica: DCM/hexane, 1:2, 1:1; DCM) to afford a
dark brown solid (0.2208 g, 85%). (M+H+ Found: 294.0771.
C17H12NO4 requires M+H, 294.0766). Rf = 0.35 (EtOAc/hexane,
1:2). Mp = 177–178 °C. 1H NMR (200 MHz, CDCl3): d = 7.16 (2H, d
J = 8.0 Hz, ArH), 7.40 (3H, m, ArH), 7.65–7.88 (2H, m, ArH), 7.92
(1H, d J = 7.4 Hz, ArH), 8.24 (1H, d J = 7.6 Hz, ArH) and 13.12; 13C
NMR (100 MHz, CDCl3): d = 100.2, 124.5, 126.9, 127.1, 127.7,
129.3, 131.0, 131.9, 133.8, 135.6, 138.4, 153.9, 171.3, 180.1 and
184.5.
4.3.9. 3-[(3-Cyanophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 7
4.3.7. 1,4-dioxo-3-{[4-(propan-2-yl)phenyl]amino}-1,4-
dihydronaphthalene-2-carboxylic acid 5
O
O
O
OH
NH
O
O
OH
NH
O
CN
H3C
CH3
4.3.9.1. Method C.
Heating time = 24 h. Purification by flash
chromatography (silica: EtOAc/hexane, 1: 5 1:4, 1:3 and 1:2) to af-
4.3.7.1. Method A.
Heating time = 26 h. Purification by flash
ford a dark-brown solid (0.0607 g, 32%). (M-H+ Found: 317.0540.
chromatography (silica: CHCl3; MeOH/CHCl3, 1: 100 and 1:50) to
C18H9N2O4 requires M-H, 317.0562). Rf = 0.18 (EtOAc/hexane,
afford
a
dark-brown solid (0.1394 g, 70%). (MÀH+ Found:
1:2). Mp = 214–216 °C.
334.1082. C20H16NO4 requires MÀH, 334.1082). Rf = 0.56 (DCM).
Mp = 148–150 °C. 1H NMR (200 MHz, CDCl3): d = 1.29 (6H, m,
2 Â CH3), 2.88–3.10 (1H, s, CH), 7.09 (2H, d J 8.2 Hz, ArH), 7.28
(2H, d J 8.40 Hz, ArH), 7.65–7.90 (2H, m, ArH), 7.95 (1H, d J
7.0 Hz, ArH), 8.25 (1H, d J 7.6 Hz, ArH), and 13.1 (1H, br s, CO2H);
13C NMR (50 MHz, CDCl3): d = 24.1, 34.0, 100.5, 124.3, 126.9,
127.0, 127.3, 131.1, 132.1, 133.7, 135.5 and 136.0.
4.3.9.2. Method E.
Heating time = 48 h. Purification by flash
chromatography (silica: EtOAc/hexane, 1:2, 1:1; EtOAc; MeOH/
EtOAc, 1:50, 1:30) to afford an orange-brown solid (0.0982 g,
33%). (M+H+ Found: 319.0739. C18H11N2O4 requires M+H,
319.0719). Rf = 0.21 (EtOAc/hexane, 1:1). Mp = 296–297 °C. 1H
NMR (200 MHz, CDCl3): d = 1.62 (1H, br s, NH), 7.39–7.88 (6H, m,
ArH), 7.94 (1H, d J = 7.6 Hz, ArH), 8.25 (1H, d J 7.6 Hz, ArH) and
13.10 (1H, br s, CO2H); 13C NMR (50 MHz, CDCl3): d = 101.0,
113.6, 117.7, 127.1, 127.3, 128.4, 130.3, 130.7, 131.0, 131.6,
134.2, 136.0, 139.6, 153.5, 171.1, 180.0 and 184.9.
4.3.7.2. Method D.
chromatography (silica: EtOAc/hexane, 1: 10, 1:6, 1:4, 1:2; EtOAc)
to afford a dark-brown solid (0.0940 g, 47%).
Heating time = 48 h. Purification by flash
4.3.10. 3-[(3-Chlorophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 8
4.3.8. 3-{[4-(2-Hydroxyethyl)phenyl]amino}-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 6
O
O
O
O
O
OH
OH
NH
NH
O
Cl
OH
4.3.10.1. Method C.
Heating time = 24 h. Purification by flash
4.3.8.1. Method A.
chromatography (silica: EtOAc/hexane, 1:2, 1:1; EtOAc; EtOAc/
MeOH, 19.9:0.1 and 19.5:0.5) to afford
(0.1027 g, 51%).
Heating time = 27 h. Purification by flash
chromatography (silica: MeOH/CHCl3, 1: 49 1:39 and 1:19) to af-
ford a dark-brown solid (0.1046 g, 52%). (MÀH+ Found: 326.0226.
a dark-brown solid
C
17H9NO4Cl requires MÀH, 326.0220). Rf = 0.34 (EtOAc/hexane,
1:3). Mp = 203–205 °C. 1H NMR (200 MHz, CDCl3): d = 1.57 (1H,

K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
4479
br s, NH), 7.07 (1H, m, ArH), 7.19 (1H, s, ArH), 7.35 (2H, d J = 4.4 Hz,
4.3.13.1. Method E.
Heating time = 48 h. Purification by flash
ArH), 7.65–8.00 (3H, m, ArH), 8.24 (1H, d J 7.4 Hz, ArH), and 13.10
(1H, br s, CO2H); 13C NMR (50 MHz, CDCl3): d = 123.0, 124.3, 125.0,
127.0, 127.2, 127.9, 130.3, 130.9, 131.7, 134.0, 123.9, 135.8, 139.6,
153,7, 167.1, 171.2, 179.9 and 184.7.
chromatography (silica: DCM/hexane, 1:2, 1:1.5, 1:1; DCM) to af-
ford a brown solid (0.1358 g, 49%). (M-H+ Found: 312.0682.
C17H11NO4F requires M-H, 312.0672). Rf = 0.43 (EtOAc/hexane,
1:3). Mp = 235–237 °C. 1H NMR (200 MHz, CDCl3): d = 7.05–7.19
(4H, m, ArH), 7.66–7.88 (2H, m, ArH), 7.91 (1H, d J = 7.6 Hz, ArH),
8.23 (1H, d J = 7.2 Hz, ArH) and 13.06 (1H, br s, CO2H); 13C NMR
(50 MHz, CDCl3): d = 100.6, 116.4, 116.9, 126.6, 126.8, 127.3,
127.4, 131.2, 132.1, 134.2, 134.7, 136.0, 154.1, 159.5, 164.4,
171.6, 180.4 and 184.9.
4.3.10.2. 2-Acetyl-3-[(4-chlorophenyl)amino]naphthalene-1,4-
dione 9.
O
O
O
OH
NH
4.3.14. 3-[(3-Fluorophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 12
Cl
O
O
O
4.3.11. Method E
Heating time = 48 h. Purification by flash chromatography (sil-
ica: DCM/hexane, 1:2, 1:1; DCM) to afford brown solid
OH
NH
a
(0.0737 g, 25%). (MÀH+ Found: 328.0386. C17H11NO4Cl requires
MÀH, 328.0377). Rf = 0.23 (DCM). Mp = 259–262 °C. 1H NMR
(200 MHz, DMSO-d6): d = 7.33 (2H, d J = 8.8 Hz, ArH), 7.42 (2H, d
J = 8.8 Hz, ArH), 7.82 (1H, t J = 6.8 and 7.6 Hz, ArH), 7.91 (1H, t
J = 7.20 and 7.6 Hz, ArH), 7.98 (1H, d J = 7.2 Hz, ArH), 8.07 (1H, d J
7.2 Hz, ArH); 13C NMR (50 MHz, CDCl3): d = 125.8, 126.3, 126.4,
128.5, 130.5, 130.6, 131.8, 133.4, 135.2, 137.6, 148.7, 167.7, 180.7
and 182.3.
F
4.3.14.1. Method E.
Heating time = 48 h. Purification by flash
chromatography (silica: DCM/hexane, 1:2, 1:1; DCM) to afford a
dark-brown solid (0.0797 g, 28%). (M-H+ Found: 312.0655.
C
17H11NO4F requires M-H, 312.0655). Rf = 0.37 (EtOAc/hexane,
4.3.12. 3-[(2-Chlorophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 10
1:3). Mp = 194–195 °C. 1H NMR (200 MHz, CDCl3): d = 6.94 (2H, t
J = 8.2 and 11.4 Hz, ArH), 7.08 (1H, t J = 8.0, and 6.8 Hz, ArH), 7.39
(2H, q J = 8.0 and 6.2 Hz, ArH), 7.65–7.89 (2H, m, ArH), 7.93 (1H,
d J = 6.6 Hz, ArH), 8.23 (1H, d J = 7.4 Hz, ArH) and 13.06 (1H, br s,
CO2H); 13C NMR (50 MHz, CDCl3): d = 100.6, 112.2, 112.7, 114.6,
115.0, 120.5, 120.6, 127.0, 127.1, 130.5, 130.6, 130.9, 131.8,
134.0, 135.7, 138.2, 139.8, 140.1, 153.8, 160.3, 165.3, 171.2, 180.0
and 184.7.
O
O
OH
NH
Cl
O
4.3.15. 3-[(2-Fluorophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 13
4.3.12.1. Method E.
Heating time = 48 h. Purification by flash
chromatography (silica: DCM/hexane, 1:2, 1:1.5; 1:1; DCM) to af-
ford a brown solid (0.0853 g, 29%). (M+H+ Found: 328.0361.
C
17H11NO4Cl requires M+H, 328.0377). Rf = 0.43 (EtOAc/hexane,
1:3). Mp = 189–191 °C. 1H NMR (200 MHz, CDCl3): d = 7.20–7.55
(4H, m, ArH), 7.65–7.88 (2H, m, ArH), 7.93 (1H, d J = 7.6 Hz, ArH),
8.24 (1H, d J = 7.6 Hz, ArH) and 12.92 (1H, br s, CO2H); 13C NMR
(50 MHz, CDCl3): d = 100.1, 126.4, 127.0, 127.1, 127.8, 128.8,
129.3, 130.2, 130.6, 131.8, 133.9, 135.6, 136.4, 138.2, 154.7,
171.1, 179.9 and 184.9.
O
O
O
OH
NH
F
4.3.13. 3-[(4-Fluorophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 11
4.3.15.1. Method E.
Heating time = 48 h. Purification by flash
chromatography (silica: DCM/hexane, 1:2, 1:1.5, 1:1; DCM) to af-
ford a red-brown solid (0.1119 g, 40%). (MÀH+ Found: 312.0668.
O
O
O
C17H11NO4F requires MÀH, 312.0672). Rf = 0.43 (EtOAc/hexane,
OH
1:3). Mp = 193–195 °C. 1H NMR (200 MHz, CDCl3): d = 7.08 (4H,
m, ArH), 7.66–7.89 (2H, m, ArH), 7.95 (1H, d J = 7.6 Hz, ArH), 8.23
(1H, m, ArH) and 12.86 (1H, br s, CO2H); 13C NMR (50 MHz, CDCl3):
d = 100.4, 116.0, 116.4, 124.8, 124.9, 125.8, 126.9, 127.1, 127.8,
128.9, 129.0, 130.7, 131.8, 133.9, 135.6, 153.3, 154.4, 158.2,
171.1, 180.1 and 184.9.
NH
F

4480
K. W. Wellington, N. I. Kolesnikova / Bioorg. Med. Chem. 20 (2012) 4472–4481
4.3.16. 3-[(4-Chloro-2-fluorophenyl)amino]-1,4-dioxo-1,4-
dihydronaphthalene-2-carboxylic acid 14
with 10 mM Tris base for optical density determination at a wave-
length 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 expo-
sure 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:
O
O
O
OH
NH
[(TiÀT0)/(CÀT0)] Â 100 for concentrations at which Ti P T0
[(TiÀT0)/T0] Â 100 for concentrations at which Ti < T0.
F
The results of a five dose screening were reported as TGI (total
growth inhibition). The TGI is the concentration of test drug where
100 Â (TÀT0)/(CÀT0) = 0. The TGI signifies a cytostatic effect.
The biological activities were separated into 4 categories: inac-
Cl
tive (GI50 or TGI >100
<100 M, moderate activity (10
tent activity (GI50 or TGI <10 M).
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.
l
M), weak activity (30
l
M < GI50 or TGI
4.3.16.1. Method E.
Heating time = 48 h. Purification by flash
l
lM < GI50 or TGI <30 l
M and po-
chromatography (silica: DCM/hexane, 1:6, 1:4; 1:3 and 1:2) to af-
l
ford a red-brown solid (0.0947 g, 31%). (M+H+ Found: 346.0301.
C17H10NO4ClF requires M+H, 346.0282). Rf = 0.29 (EtOAc/hexane,
1:1.5). Mp = 193–195 °C. 1H NMR (200 MHz, CDCl3): d = 7.12–7.30
(3H, m, ArH), 7.68–7.90 (2H, m, ArH), 7.95 (1H, m, ArH), 8.23
(1H, m, ArH) and 12.82 (1H, br s, CO2H); 13C NMR (50 MHz, CDCl3):
d = 101.0, 117.2, 117.7, 125.4, 125.5, 125.8, 126.8, 127.3, 127.4,
130.8, 132.0, 134.4, 136.0, 153.4, 154.4, 158.4, 171.4, 180.4 and
185.3.
Acknowledgments
4.4. In vitro anticancer activity evaluation
We thank the CSIR (Thematic A grant) for financial support and
Novozymes SA for a donation of Novozym 51003.
4.4.1. Assay background
The growth inhibitory effects of the compounds were tested in
duplicate in a 3-cell line panel consisting of TK10 (renal), UACC62
(melanoma) and MCF7 (breast) cancer cells using the Sulforhoda-
mine B (SRB) assay.55 The SRB assay was developed by Skehan
and colleagues to measure drug-induced cytotoxicity and cell pro-
liferation. Its principle is based on the ability of the protein dye,
sulforhodamine B (Acid Red 52), to bind electrostatically in a pH-
dependent manner to protein basic amino acid residues of trichlo-
roacetic 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.
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