Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites

Article abstract of DOI:10.1016/j.bmc.2011.01.062

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13- hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α- hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12- trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.

Full text of DOI:10.1016/j.bmc.2011.01.062

Bioorganic & Medicinal Chemistry 19 (2011) 2464–2469
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microbial transformation of (+)-nootkatone and the antiproliferative
activity of its metabolites
a
a
a
b
´
´
Anna Gliszczynska , Agnieszka Łysek , Tomasz Janeczko , Marta Switalska
b
a,
⇑
´
Joanna Wietrzyk , Czesław Wawrzenczyk
a Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Norwida 25, 50-375 Wroclaw, Poland
b Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology, Weigla 12, 53-114 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the
fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were estab-
Received 6 October 2010
Revised 28 October 2010
Accepted 28 January 2011
Available online 4 March 2011
lished as (+)-(4R,5S,7R,9R)-9
(4R,5S,7R,9R,11S)-11,12-epoxy-9
a
-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-
-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone
a
(5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7)
on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature.
The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2–7) of its biotransformation
has been evaluated.
Keywords:
Biotransformation
Sesquiterpenes
(+)-Nootkatone
Ó 2011 Elsevier Ltd. All rights reserved.
Botrytis cinerea
Didymosphaeria igniaria
Aspergillus ochraceus
Aspergillus niger MB
Chaetomium sp.
Fusarium culmorum
Cancer cell lines: A549 (human lung
adenocarcinoma) and HL-60 (human
promyelocytic leukemia)
Antiproliferative activity
1. Introduction
industrially valuable product in the fragrance, food, cosmetics
and pharmaceutical applications.
Bicyclic sesquiterpene (+)-nootkatone is a natural compound
which was first isolated from the heartwood of Alaska yellow cedar
(Chamaecyparis nootkatensis) and was found in trace amount in
grapefruit (Citrus paradise).1,2 This sesquiterpene ketone possesses
pleasant grapefruit flavor and occurs in orange and grapefruit juice
and in peel oils obtained from orange, grapefruit, lemon, mandarin
and lime.3 The literature presents wide range of biological activity
of nootkatone and describes potential fields of its application.
Nootkatone inhibits the gastric ulcer formation in rats as well as
the activity of acetylcholinesterases and of human cytochrome
P450 monooxygenases CYP450.4–7 It is known as the potential
insecticide against larvae of Drosophila melanogaster and a strong
repellent to termites (Coptotermes formosanus).8,9 As the carries
of specific odor with interesting activity, (+)-nootkatone (1), is
The objective of our research project is the synthesis of new ac-
tive derivatives of natural compounds with enhanced biological
activity. Recently we have published the synthesis of oxideriva-
tives of farnesol by fungal strains.10 Here we report results of func-
tionalization of (+)-nootkatone (1) with the fungal cultures:
Botrytis cinerea, Didymosphaeria igniaria, Aspergillus ochraceus,
Aspergillus niger MB, Chaetomium sp. and Fusarium culmorum.
Nootkatone and products of its microbial transformations were
subjected to the biological tests. The antiproliferative activity to-
wards the cancer cell lines: A549 (human lung adenocarcinoma)
and HL-60 (human promyelocytic leukemia) was investigated.
2. Results and discussion
Biotransformation of (+)-nootkatone (1) was carried out by fun-
gal cultures: B. cinerea AM235 and D. igniaria KCh6670, A. ochraceus
AM456, A. niger MB, Chaetomium sp. KCh6651 and F. culmorum
AM10 which were selected in the screening procedure of 20 fungal
strains.
⇑
Corresponding author. Tel.: +48 71 320 52 57.
E-mail addresses: anna.gliszczynska@wp.pl, czeslaw.wawrzenczyk@up.wroc.pl
(C. Wawrzen´ czyk).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.062

´
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A. Gliszczynska et al. / Bioorg. Med. Chem. 19 (2011) 2464–2469
In the culture of B. cinerea AM235 two products: (+)-(4R,5S,
7R,9R)-9 -hydroxynootkatone (2) and (+)-(4R,5S,7R)-13-hydrox-
out with P450 mutants.12 9b-Isomer of product (2), was obtained
from the microbial conversion of (+)-nootkatone (1) byF. culmorum
(14.9%).13
a
ynootkatone (3) were formed ( Fig. 1). After eight days of
incubation they were isolated in 32% (61 mg) and 28% (54 mg)
yields, respectively. The reaction course was monitored by means
of TLC and GC. The GC analysis afforded the information about
changes in quantitative ratio of products in the course of the process
(Table 1). In the first 24 h only substrate was observedin the reaction
Hydroxylation at C-13 in the isopropenyl group of
(+)-(4R,5S,7R)-13-hydroxynootkatone (3) was confirmed by both
1H NMR and 13C NMR data. In the 1H NMR spectrum of this product
two multiplets at d = 4.90 ppm and d = 5.06 ppm from protons of
double bond C11–C12 and characteristic singlet from two protons
of isolated methylene group was observed. The chemical shift of
this singlet (d = 4.14 ppm) confirmed the introduction the hydroxy
group at the position C-13. The presence of hydroxy group in prod-
uct 3 is confirmed by the signal of carbon atom C-13 at 65.2 ppm in
the 13C NMR spectrum (Table 3). In the IR spectrum absorption
bands at 3399 cmꢀ1 and 1047 cmꢀ1 characteristic for stretching
vibrations O–H and C–O in alcohols, respectively are visible.
(+)-Nootkatone (1) transformed in the culture of Chaetomium
sp. KCh6651 gave after two days (+)-(4R,5S,7R,11S)-11,12-
epoxynootkatone (5), which was isolated in 44% yield (51 mg) as
the mixture of epimers with 50% diastereoisomeric excess (de)
with predominance of isomer with configuration 11S. The configu-
ration at C-11 was determined by comparison of spectral (1H NMR)
data of product with the literature 1H NMR data.12 The biotransfor-
mation of 1 in the culture Chaetomium sp. KCh6651 proceeded very
fast. After 24 h of incubation the conversion of substrate was on
the level 43% and after 48 h nootkatone (1) was completely trans-
formed to the product 5. The structure of 5 was confirmed by spec-
troscopic data. In the 1H NMR spectrum of 5 two doublets
(J = 4.7 Hz) at d = 2.59 and d = 2.66 ppm of protons CH2-12. The sig-
nals at d = 53.7 and d = 58.9 ppm in the 13C NMR spectrum indicate
the presence of oxirane ring. The characteristic absorption bands at
1288 cmꢀ1 and 840 cmꢀ1 in the IR spectrum also confirm the epox-
idation of nootkatone.
mixture. In the second day (+)-(4R,5S,7R,9R)-9a-hydroxynootk-
atone (2) was detected and since then its content increased propor-
tionally. The second product (+)-(4R,5S,7R)-13-hydroxynootkatone
(3) was formed in fourth day of biotransformation, independently
from product (2). Both products are the result of hydroxylation at
allylic positions C-9 (2) and C-13 (3) in the nootkatone (1).
The product 2 was obtained as a single isomer. Its structure was
established on the basis of spectral data. The complete 1H and 13C
NMR analyzes with COSY and HMQC let to confirm the structure of
(+)-(4R,5S,7R,9R)-9a
-hydroxynootkatone (2). In the 1H NMR spec-
trum of product (2) triplet at d = 4.45 ppm (J = 2.9 Hz) of the proton
H-9 was observed. Hydroxylation at C-9 was confirmed by signal of
carbon atom C-9 in the 13C NMR spectrum shifted downfield to
73.3 ppm as well as by the absorption band at 3405 cmꢀ1 in IR
spectrum. The relative stereochemistry at C-9 was confirmed by
the analyzes of the coupling constants of related proton signals
and by comparison with the literature spectral data of this com-
pound.11 The coupling constants of 2.9 Hz between H-9 (d = 4.45)
and both of H-8 indicated that H-9 is equatorial orientation. The
orientation of hydroxy group at C-9 is then axial and it is trans sit-
uated to the propenyl group at C-7.
(+)-(4R,5S,7R,9R)-9a-Hydroxynootkatone (2) was obtained pre-
viously in very small yield (8%) as the product of biotransformation
of (+)-nootkatone (1) with cultured plant cells of Marchantia poly-
morpha.11
9a-Hydroxynootkatone (2) was also isolated from
In the culture of D. igniaria KCh6670 the process of biotransfor-
mation of (+)-nootkatone (1) was enantioselective and led to the
whole-cell biotransformation reaction of (+)-valencene carried
OH
1
O
O
O
9
9
6
10
2
3
8
a b
a
12
12
5
7
11
4
11
15
14
13
13
OH
1
3
2
f
b
c d
OH
O
O
O
9
OH
11
O
7
O
OH
11
11
OH
4
7
5
c e
O
12
OH
11
OH
6
Figure 1. Metabolic pathway of (+)-nootkatone (1) in the culture: B. cinerea AM235 (a), D. igniaria KCh6670 (b), A. ochraceus AM456 (c), Chaetomium sp. KCh6651 (d),
F. culmorum AM10 (e), A. niger MB (f).

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A. Gliszczynska et al. / Bioorg. Med. Chem. 19 (2011) 2464–2469
2466
Table 1
The compositions (according to GC) of products mixture in the biotransformations of (+)-nootkatone by fungal strains
Microorganism
Time of incubations (days)
Biotransformation products (%)
1
2
3
4
5
6
7
B. cinerea AM235
1
2
4
6
8
0.5
1
2
1
2
3
2
4
6
8
10
1
2
3
100
89
63
48
0
74
3
0
53
15
0
100
83
71
18
0
—
—
—
7
—
—
—
—
—
—
—
—
—
41
33
36
—
—
—
—
—
43
—
—
—
—
—
—
—
—
6
52
64
0
17
29
82
100
—
—
—
—
—
—
—
11
30
36
60
26
76
68
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
21
32
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
18
37
96
16
40
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
D. igniaria KCh6670
A. ochraceus AM456
F. culmorum AM10
Chaetomium sp. KCh6651
A. niger MB
57
0
0
72
63
4
100
100
—
—
—
2
3
4
—
mixture of two products 2 and 4 (Fig. 1). The biotransformation of
(+)-nootkatone (1) with D. igniaria KCh6670 proceeded very fast.
The progress of the reaction monitored by TLC and GC showed that
after 12 h 26% of (+)-9a-hydroxynootkatone (2) in the reaction
mixture was observed. After one day of incubation the mixture
conversion of substrate 1. The products 5 and 6 were isolated in
the 20% (26 mg) and 12% (17 mg) yields, respectively. In the 1H
NMR spectrum of 6 the replacement of two doublets of the protons
of double bond in 1 by the new signals at d = 3.44 and d = 3.59 ppm
was observed. There are two doublets (J = 10.9 Hz) from the diaste-
reotopic protons of the methylene group at C-12 and they indicate
the presence of hydroxy groups at the C-11 and C-12. The shift of
of products contained only 3% of unreacted substrate, 76% of
(+)-9
11,12-epoxy-9
the biotransformation the amount of
a
-hydroxynootkatone (2) and 21% of (+)-(4R,5S,7R,9R,11S)-
-hydroxy-nootkatone (4). In the second day of
gradually decreased,
a
the singlet of methyl group at C-11 from d = 1.73 in 1 to
2
d = 1.07 ppm in 6 also confirms the transformation of double bond
into 11,12-dihydroxymoiety. Two signals at d = 68.5 ppm and
74.4 ppm (13C NMR of 6) instead of the signals from the vinyl car-
bons in the 13C NMR spectrum of 1 also confirm this transforma-
tions. The characteristic strong absorption band for stretching
vibrations of O–H in the IR spectrum at 3412 cmꢀ1 also indicates
the hydroxy groups in the product 6.
whereas the amount of 4 increased proportionally. This observa-
tion led to the conclusion that product 4 is formed in two step of
biotransformation: first the hydroxylation at C-9 takes place and
then double bond C11–C12 is epoxydized. Products 2 and 4 were
isolated in high yields: 70% (82 mg) and 19% (24 mg), respectively.
The structure of (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9a-hydrox-
ynootkatone (4) was confirmed by spectroscopic data. In the 1H
NMR spectrum of product (4) the characteristic triplet at
d = 4.45 ppm (J = 2.9 Hz) of the proton H-9 could be detected. The
chemical shift of this signal confirms the presence of the hydroxy
group at the C-9. The presence of oxirane ring in the structure of
product 4 was confirmed by presence two doublets (J = 4.7 Hz) at
d = 2.58 and d = 2.70 ppm of protons CH2-12. The signals from
C-11 and C-12 in the 13C NMR spectrum were shifted from 149.0
and 109.2 ppm in nootkatone to 59.0 and 53.0 ppm, respectively
in compound (4).
The relative stereochemistry at C-9 was the same like in the
product (2). The orientation of hydroxy group at C-9 as axial and
trans for the propenyl group at C-7 was compared with the litera-
ture spectral data of this compound.11 The configuration at C-11
was also determined by comparison of its spectral 1H NMR data
with the literature 1H NMR data.12
The product 6 was formed also in the biotransformation of nootk-
atone (1) in the culture F. culmorum AM10. After 10 days incubation
of nootkatone (1) the (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6)
was isolated in 71% yield (142 mg) as the mixture of epimers at C-11
(55:45). The process of transformation was very effective. The epox-
ynootkatone, which is certain the intermediate compound, was not
observed in the reaction mixture.
Both products (+)-(4R,5S,7R,11S)-11,12-epoxynootkatone (5)
and (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) are known in
the literature. The first one, was obtained in the biotransformation
of 1 with fungal strains: A. niger, Botryosphaeria dothidea and F. cul-
morum.13 Epoxy derivative (5) of nootkatone to that time was ob-
tained as
a racemic mixture in the chemical oxidation of
nootkatone with m-CPBA and in the process of transformation of
valencen using genetically modified cytochrome P450BM-3 of bacte-
ria Bacillus megaterium as the catalyst.12
(+)-(4R,5S,7R,9R,11S)-11,12-Epoxy-9
a
-hydroxynootkatone (4)
A. niger MB oxidized nootkatone (1) at the double bond as well
as at the C-7 to form the triol 7. The product 7 was identified as
(+)-(+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone. The time course
for the biotransformation of (+)-nootkatone (1) by A. niger MB
are shown in Table 1. The formation of 7 reached the maximum
(96%) after four days and then was isolated in 33% yield (74 mg)
with 20% de. The transformation of 1 to triol 7 seems to be closely
related to the formation of 6. However the intermediate products 5
and 6 which was not detected in the reaction mixture. The
structure of 7 was determined by spectroscopic (1H and 13C
was not reported previously. In the literature epoxynootkatone with
oxirane ring at C11–C12 is known as well as the product of hydrox-
ylation reaction of nootkatone at C-9 but it is the first report about
the formation of (4) as the results both of these reactions.11–13
In the reaction catalyzed by the strain A. ochraceus AM456 two
products were formed: (+)-(4R,5S,7R,11S)-11,12-epoxynootkatone
(5) with 78% de and the 11,12-dihydroxynootkatone (6). The latter
was obtained as the mixture of epimers (54:46) at C-11. The pro-
cess of biotransformation was carried out three days to the final

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A. Gliszczynska et al. / Bioorg. Med. Chem. 19 (2011) 2464–2469
Table 3
13C NMR chemical shifts (d) of compounds 1–7
Carbon
Compounds
1
2
3
4
5
6
7
C-1
124.63 127.12 124.7
127.43 124.94 124.50 124.65
C-2
199.63 200.62 199.68 200.18 199.47 199.97 199.47
C-3
C-4
C-5
C-6
C-7
C-8
C-9
43.89
40.28
39.29
42.03
40.42
31.58
32.99
43.62
41.14
38.76
42.27
33.94
37.88
73.27
44.34
40.35
39.39
41.97
35.94
33.08
32.04
42.36
41.28
38.53
40.35
33.00
34.97
73.00
42.09
39.57
39.04
40.41
40.56
28.65
32.50
42.08
40.61
39.67
38.69
39.25
27.39
32.99
42.04
40.77
39.11
39.29
82.85
26.96
32.77
C-10
C-11
C-12
C-13
C-14
C-15
170.53 168.40 170.37 167.49 168.83 171.07 170.31
149.03 148.86 152.51
109.20 109.33 109.03
58.96
53.05
18.65
14.59
18.09
58.94
53.49
17.82
14.94
16.80
74.30
68.36
20.12
15.03
16.86
72.72
73.00
21.65
14.92
16.79
20.77
14.86
16.81
20.91
14.47
18.06
65.17
14.85
16.74
NMR) data. In the 1H NMR spectra of 7 two doublets (J = 8.5 Hz) at
d = 3.67 and 3.88 ppm of protons at C-12 were observed. The sig-
nals for C-11 and C-12 in the spectrum of 13C NMR of 7 were
found at d = 72.7 ppm and 73.0 ppm. In this spectrum the signal
from C-7 at d = 82.85 ppm was also observed. In the literature
microbial hydroxylation at the C-7 position of nootkatone (1)
was described and diol of nootkatone (6) is also known.13 How-
ever the formation of metabolite 7 have not been previously
reported.
The results of biotransformation of (+)-nootkatone (1) pre-
sented here indicate that two metabolic pathways in the cultures
of fungal strains studied could be considered (Fig. 1). The major
pathway is the epoxidation of double bond between C11–C12
in the culture of A. ochraceus AM456 and Chaetomium sp. 6651
and possibly in A. niger MB and F. coulmorum AM10. In the culture
of strains of B. cinerea AM235 and D. igniaria KCha6670 hydroxyl-
ation of allylic positions at C-13 and C-9, respectively took place.
D. igniaria KCha6670 additionally oxidized the double bond of
isopropenyl group in 9-hydroxynootkatone (2).
In the next step of our studies the proliferation of the cancer
cells lines A549 (human lung adenocarcinoma) and HL-60 (hu-
man promyelocytic leukemia) exposed either to (+)-nootkatone
(1) and its derivatives (2–7) was investigated. The compounds
(1–7) were tested in SRB or MTT (for cell lines A549 and HL-60,
respectively) assay. The results of cytotoxic activity in vitro were
expressed as an ID50 (lg/ml) that is the concentration of com-
pound which inhibits the proliferation of 50% of tumor cells.
The results of the cytotoxicity studied are summarized in Table 4.
In the series of compounds 1–7 tested against A549 cells the
most potent activity showed 13-hydroxynootkatone (3) with
the ID50 value (36 lg/ml) lower than that of nootkatone (1). The
presence of hydroxy group at C-13 led to increase of activity to-
wards tested cancer cell line. The other compounds studied in
this series did not showed the interesting activity.
The antiproliferative activity of compounds studied against
HL-60 leukemia cells was higher than this observed for A549
cells. The proliferation of HL-60 cells was strongly inhibited by
substrate 1 and the metabolites 2, 3, 5 and 7 with ID50 values
4.27–27.6 lg/ml (Table 4) while epoxyalcohol (4) and diol (6)
did not show any significant activity against leukemia cells. These
results may suggest that compounds 1–3, 5 and 7 can be consid-
ered as good candidates for further studies.
Considering the overall activities of 1–7 it can be postulated
that the presence of an additional hydroxy groups in the cyclo-
hexane ring increases the activity. However it looks that the
isopropenyl group is the most important moiety in the structure

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2468
Table 4
The antiproliferative activity in vitro against the human cancer cell lines
Cell line
Compounds ID50 (lg/ml)
1
2
3
4
5
6
7
A549
HL-60
58.4 6.6
4.27 1.61
62.7 20
25.7 8.9
36
4.4 2.3
3
288.6 34.4
345 108
167
27.6 11.3
9
325 210
284 143
290 19.6
25.8 10.2
of studied compounds which showed antiproliferative activity. On
the other hand, the leukemia cells (HL-60) appear to be more
sensitive to the cytotoxic effects of compounds studied (1–7).
concentrated in vacuo. Residues were dissolved in 1 ml of acetone
and analyzed by TLC and GC.
Screening procedure was carried out for 20 fungal strains which
were from the Institute of Biology and Botany, Medical Academy of
Wrocław: Rhodotorula rubra AM4, Nigrospora oryzae AM8, F. culmo-
rum AM10, Fusarium avenaceum AM12, Fusarium oxysporum AM13,
Fusarium equiseti AM15, Pezicula cinnamomea AR53, Rhodotorula
marina AM77, Penicillium chermesinum AM113, B. cinerea AM235,
Beauveria bassiana AM278, Acremonium roseum AM346, A. ochrac-
eus AM456, Pleurotus ostreatus AM482, Laetiporus sulphurens
AM525, Trametes versicolor AM536, and A. niger MB, Penicillium dig-
itatum KCh904, Chaetomium sp. KCh6651, D. igniaria KCh6670 were
from Department of Chemistry at Wroclaw University of Environ-
mental and Life Sciences (Poland). The screening of fungal strains
led to the selection of six microorganisms that had ability to bio-
transformation of (+)-nootkatone (1).
3. Experimental
3.1. Analysis
The course of microbial transformations as well as the purity of
isolated products were checked by TLC technique (SiO2, DC-
Alufolien Kieselgel 60 F254, Merck). Chromatograms were devel-
oped using the following developing systems: hexane/acetone
2:1. Visualization was made using a solution of 10 g Ce(SO4)2 and
20 g phosphoromolybdenic acid in 1 dm3 H2SO4, followed by heat-
ing. Column chromatography (SiO2, Kieselgel 60, 230–400 mesh,
40–63 lm, Merck) was performed using the same eluents (hex-
ane/acetone, 2:1) as in TLC technique. Gas chromatography (GC)
analysis was carried out on Agilent Technologies 6890N (Network
GC System) instrument with DB-17 column (cross-linked methyl
3.3.2. Preparative biotransformation
In order to isolate and identify the products, preparative-scale
biotransformation were performed. Portion of 1 ml of the pre-
incubation culture solution were used to inoculate 11–18 flasks
(300-ml Erlenmeyer) containing 100 ml of medium in each flask.
The cultures were incubated at 25 °C for four days on rotary shaker
and then the substrate (10 mg (+)-nootkatone on the 100 ml of med-
ium) dissolved in acetone was added to each flask. After one and se-
ven days of incubation the mixtures were extracted three times with
dichloromethane, dried (MgSO4) and concentrated in vacuo. The
crude product mixtures were separated by column chromatography
(silica gel, hexane/acetone 2:1). Pure biotransformation products
were identified by means of spectral analyzes (TLC, 1H NMR and
13C NMR) and optical rotation measurements.
silicone, 30 m ꢁ 0.25 mm ꢁ 0.25
l
m). 1H NMR and 13C NMR spec-
tra were recorded on a Bruker Avance DRX 300 spectrometer. IR
spectra were determined on a Mattson IR 300 Thermo Nicolet
spectrometer. Optical rotation were measured on an Autopol IV
automatic polarimeter (Rudolph). Elemental analysis were
performed on the Vario EL III CHNS (Elementor).
3.2. Materials
The substrate used for the biotransformation experiments, (+)-
nootkatone (1), was purchased from Fluka.
3.2.1. (+)-Nootkatone (1)
3.3.3. Products of biotransformations
Melting point 32 °C, boiling point 125 °C at 0.5 mm Hg, 1H NMR
see Table 1, 13C NMR see Table 2.
3.3.3.1. (+)-(4R,5S,7R,9R)-9a
-Hydroxynootkatone (2). 1H NMR
(see Table 2), 13C NMR (see Table 3), IR (film, cmꢀ1): 3405(s),
2966(s), 1663(s),1443(m), 1375(s), 1296(s), 1049(m), 888(s); ½a D20
ꢂ
3.2.2. Microorganisms
+42.4 (c 1.22, CH2Cl2).
The fungal strains: B. cinerea AM235 and D. igniaria KCh6670 A.
ochraceus AM456, A. niger MB, Chaetomium sp. KCh6651 and F. cul-
morum AM10 were cultivated on a Sabouraud agar consisting of:
aminobac 5 g, peptone K 5 g, glucose 40 g and agar 15 g in distilled
water 1 l at 28 °C and pH 5.5 and stored in refrigerator at 4 °C.
3.3.3.2. (+)-(4R,5S,7R)-13-Hydroxynootkatone (3). 1H NMR (see
Table 2), 13C NMR (see Table 3), IR (film, cmꢀ1): 3340(s), 2935(s),
1656(s),1454(m), 1357(s), 1298(s), 1047(s), 902(w); ½a D20
ꢂ
+114.2
(c 0.93, CH2Cl2).
3.3. Biotransformation
3.3.3.3.
(+)-(4R,5S,7R,9R,11S)-11,12-Epoxy-9
a
-hydroxynootk-
atone (4). 1H NMR (see Table 2), 13C NMR (see Table 3), IR (film,
cmꢀ1): 3416(m), 2934(m), 1664(s), 1289(m); ½a 2D0
ꢂ
+48.6 (c 0.90,
3.3.1. Screening procedure
The fungal strains were transferred from the slants to 300-ml
Erlenmeyer flask containing 100 ml of medium (3% glucose, 1%
aminobac in water). Pre-incubation was performed at 25 °C for
48 h. After this time the portions of 1 ml of the culture solution
were transferred to inoculate 300-ml flasks, each containing
100 ml of the medium. After cultivation at 25 °C for four days on
a rotary shaker, 10 mg of substrate, dissolved in 0.5 ml of acetone
was added to the grown cultures. In control experiments, the
substrates were incubated in the medium without fungi. For the
time-course analysis after 0.5, 1, 2, 4, 6, 8 and 10 days 10 ml of
the transformation mixture were taken out and extracted with
dichloromethane. The extracts were dried over MgSO4 and
CH2Cl2). Anal. Calcd for C15H22O3: C, 71.97; H, 8.86. Found: C,
71.86; H, 8.98.
3.3.3.4.
NMR (see Table 2), 13C NMR (see Table 3), IR (film, cmꢀ1):
2932(m), 1667(s), 1456(w), 1288(m), 840(w), 798(w);
(+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5). 1H
½ ꢂ
a 2D0
+157.1 (c 1.30, CH2Cl2).
3.3.3.5. (+)-(4R,5S,7R)-11,12-Dihydroxynootkatone (6). 1H NMR
(see Table 2), 13C NMR (see Table 3), IR (film, cmꢀ1): 3412(s),
2969(s), 1655(s), 1300(s), 1046(s), 942(w), 736(s); ½a D20
ꢂ
+151.9
(c 1.00, CH2Cl2) Lit. ½a D20
ꢂ
+130.5 (c 0.81, CHCl3).13

´
2469
A. Gliszczynska et al. / Bioorg. Med. Chem. 19 (2011) 2464–2469
3.3.3.6. (+)-(4R,5S,7R)-7,11,12-Trihydroxynootkatone (7). 1H NMR
water. The background optical density was measured in the wells
filled with culture medium, without the cells. The cellular material
fixed with TCA was stained with 0.4% sulforhodamine B dissolved
in 1% acetic acid for 30 min. Unbound dye was removed by rinsing
(4ꢁ) with 1% acetic acid. The protein-bound dye was extracted
with 10 mM unbuffered Tris base for determination of optical den-
sity (at 540 nm) in a computer-interfaced, 96-well microtiter plate
reader Multiskan RC photometer.
(see Table 2), 13C NMR (see Table 3), IR (film, cmꢀ1): 3318(s), 2977(s),
1668(s), 1378 (s), 1207(s), 1057(s), 733(s); ½a D20
ꢂ
+126.4 (c 1.12, CH2Cl2).
Anal. Calcd for C15H24O4: C, 67.14; H, 9.01. Found: C, 67.03; H, 9.08.
3.4. Antiproliferative assay in vitro
3.4.1. Cells
The following established in vitro cancer cell lines were applied:
A549 (human lung adenocarcinoma) and HL-60 (human promyelo-
cytic leukemia). Both cancer cell lines were obtained from Ameri-
can Type Culture Collection (Rockville, Maryland, USA) and are
being maintained in the Institute of Immunology and Experimental
Therapy, Wroclaw, Poland.
A-549 cells were cultured in RPMI 1640+Opti-MEM (1:1) (both
from Gibco, Scotland, UK), HL-60 cells in RPMI 1640 medium
(Gibco, Scotland, UK) supplemented with 2 mM L-glutamine and
1.0 mM sodium pyruvate, 10% fetal bovine serum (all from Sig-
ma–Aldrich Chemie GmbH, Steinheim, Germany). All culture
media were supplemented with 100 units/ml penicillin, and
100 lg/ml streptomycin (both from Polfa Tarchomin S.A., Warsaw,
Poland). All cell lines were grown at 37 °C with 5% CO2 humidified
atmosphere.
3.4.3. MTT assay
This technique was applied for the cytotoxicity screening
against human promyelocytic leukemia cells growing in suspen-
sion culture. For the last 3–4 h of incubation, 20 ll of MTT solution
was added to each well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide; stock solution: 5 mg/ml). The mito-
chondria of viable cells reduce the pale yellow MTT to a navy blue
formazan: the more viable cells are present in well, the more MTT
will be reduced to formazan. When incubation time was com-
pleted, 80 ll of the lysing mixture was added to each well (lysing
mixture: 225 ml dimethylformamide, 67.5 g sodium dodecyl sul-
fate and 275 ml of distilled water). After 24 h, when formazan
crystals had been dissolved, the optical densities of the samples
were read on an Multiskan RC photometer at 570 nm wavelength.
Twenty-four hours before addition of tested agents, the cells
were plated in 96-well plates (Sarstedt, Germany) at a density of
3.4.4. Statistical evaluation
One-way analysis of variance (ANOVA) followed by
Mann–Whitney U Test was applied. P-values <0.05 were considered
104 cells per well in 100
l
l of culture medium. An assay was per-
formed after 72 h of exposure to varying concentrations of the
tested agents (from 0.1 to 100 g/ml). The results were calculated
a
significant.
l
as the ID50 (inhibitory does 50%), the dose of tested agent which
inhibits 50% of the proliferation of the cancer cell population.
ID50 values were calculated for each experiment separately and
mean values SD are presented in the tables. Each compound at
each concentration was tested in triplicate in a single experiment,
which was repeated 3–5 times.
Acknowledgements
This Project was financed by European Union from the
European Social Fund, the State budget and Silesia Region budget.
Project No. Grant/I/15/2009P.
Ethanol, which was used as a solvent (in a dilution correspond-
ing to its highest concentration applied to the tested compounds),
did not exert any inhibitory effect on cell proliferation. In the anti-
proliferative assays to evaluate cytostatic effect, the SRB or MTT
methods were applied
Human lung adenocarcinoma A549 cells were routinely grown
at 37 °C in RPMI 1640+OptiMEM medium supplemented with 5%
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3.4.2. SRB assay
The details of this technique were described by Skehan.14 The
cells attached to the plastic were fixed by gently layering cold
50% TCA on the top of the culture medium in each well. The plates
were incubated at 4 °C for 1 h and then washed five times with tap