Microbial transformation of sesquiterpenes, (-)-Ambrox and (+)-sclareolide

Article abstract of DOI:10.1002/hlca.200490238

The microbial transformation of (-)-Ambrox (1), a perfumery sesquiterpene, by a number of fungi, by means of standard two-stage-fermentation technique, afforded ambrox-1α-ol (2), ambrox-1α,11α-diol (3), ambrox-1α,6α-diol (4), ambrox-1α,6α,11α-triol (5), ambrox-3-one (6), ambrox-3β-ol (7), ambrox-3β,6β-diol (8), 13,14,15,16-tetranorlabdane-3,8,12-triol (9), and sclareolide (10) (Schemes 1 and 2). Further incubation of compound 10 with Cunninghamella elegans afforded 3-oxosclareolide (11), 3β-hydroxysclareolide (12), 2α- hydroxysclareolide (13), 2α,3β-dihydroxysclareolide (14), 1α,3β-dihydroxysclareolide (15), and 3β-hydroxy-8-episclareolide (16) (Scheme 3). Metabolites 2-5, 12, 13, and 16 were found to be new compounds. The major transformations include a reaction path involving hydroxylation, ether-bond cleavage and inversion of configuration. Metabolites 11-16 of sclareolide showed significant phytotoxicity (Table I). The structures of the metabolites were characterized on the basis of spectroscopic techniques.

Full text of DOI:10.1002/hlca.200490238

Helvetica Chimica Acta Vol. 87 (2004)
2685
¾
Microbial Transformation of Sesquiterpenes, (À)-Ambrox and
(‡)-Sclareolide
by M. Iqbal Choudhary*, Syed Ghulam Musharraf, Amtul Sami, and Atta-ur-Rahman
H. E. J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi,
Karachi-75270, Pakistan
(phone: ‡92-21-9243224, 9243211; fax: ‡92-21-9243190-9; e-mail: hej@cyber.net.pk)
¾
The microbial transformation of (À)-Ambrox (1), a perfumery sesquiterpene, by a number of fungi, by
means of standard two-stage-fermentation technique, afforded ambrox-1a-ol (2), ambrox-1a,11a-diol (3),
ambrox-1a,6a-diol (4), ambrox-1a,6a,11a-triol (5), ambrox-3-one (6), ambrox-3b-ol (7), ambrox-3b,6b-diol
(8), 13,14,15,16-tetranorlabdane-3,8,12-triol (9), and sclareolide (10) (Schemes 1 and 2). Further incubation of
compound 10 with Cunninghamella elegans afforded 3-oxosclareolide (11), 3b-hydroxysclareolide (12), 2a-
hydroxysclareolide (13), 2a,3b-dihydroxysclareolide (14), 1a,3b-dihydroxysclareolide (15), and 3b-hydroxy-8-
episclareolide (16) (Scheme 3). Metabolites 2 5, 12, 13, and 16 were found to be new compounds. The major
transformations include a reaction path involving hydroxylation, ether-bond cleavage and inversion of
configuration. Metabolites 11 16 of sclareolide showed significant phytotoxicity (Table 1). The structures of
the metabolites were characterized on the basis of spectroscopic techniques.
Introduction. In continuation of our studies on biotransformation of bioactive
natural and synthetic compounds [1 11], we have recently investigated the bio-
¾
transformation of two sesquiterpenes, (À)-Ambrox (1) and (‡)-sclareolide (10), with
various fungal strains. Ambergris, a metabolite of the sperm whale, is one of the most-
valuable animal perfumes, ranking with civet and musk [12][13]. Ambrein is the major
constituent of the ambergris. During exposure in the sea for many years, ambrein is
oxidatively decomposed by the action of sea water, air, and sunlight to yield some other
¾
odorous compounds [14]. In these compounds, (À)-Ambrox (1) has a very strong
amber-like odor.
¾
Fermentation of (À)-Ambrox (1) with Fusarium lini (NRRL 68751) afforded the
new mono-, di-, and trihydroxylated metabolites 2 5, as a result of enantioselective a-
hydroxylations at C(1), C(6), and C(11), while incubation of compound 1 with
Rhizopus stolonifer (ATCC 10404) yielded metabolites 6 8 and ether-cleaved product
9. The ether cleavage through biotransformation is a rare observation. Fermentation of
compound 1 with Curvularia lunata (NRRL 2178) yielded metabolites 6 and 7, while its
biotransformation with Cunninghamella elegans (NRRL 1392) afforded compounds 6,
7, and (‡)-sclareolide (10). Compounds 6 10 have already been reported as
metabolites of 1 by Cephalosporium aphidicola (wild type), Aspergillus niger (IFO
4049), Aspergillus cellulose (IFO 4040), and Botryatis cinerea (AHU 9424) [15 19].
The oxygenated metabolites of compound 1 did not release any effective odor when
compared to 1, except for the ether-cleaved product 9, which exhibited a strong sweet
odor quit different from the amber-like odor.
(‡)-Sclareolide (10), a constituent of Arnica angustifolia [20], Sideritis nutans [21],
and Kyllinga erecta [22], has exhibited phytotoxic activity and cytotoxicity against
¹ 2004 Verlag Helvetica Chimica Acta AG, Z¸rich

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Helvetica Chimica Acta Vol. 87 (2004)
human cancer cell lines [6][23]. Compound 10 differs from 1 only in the presence of an
oxo group at C(12). Due to the presence of the lactone moiety, compound 10, on
incubation with Cunninghamella elegans, yielded an entirely new set of oxidized
metabolites 11 16, resulting from the enantioselective hydroxylations at C(2) and C(1)
and epimerization at C(8). The inversion of configuration is also a rare phenomenon in
biotransformations. Metabolites 11, 12, and 15 have been reported earlier as fermented
products of compound 10 by various fungi [11][15][24] and have shown cytotoxicity
against various human cancer cell lines [17]. During this study, metabolites of
compound 10 were subjected to phytotoxicity assay where metabolites 11, 12, and 15
showed significant phytotoxicity at higher dose against Lemna minor L. (Table 1).
Table 1. Phytotoxicity against Lemna minor L. of (‡)-Sclareolide (10) and Its Transformed Products 11 16
Concentration [mg/ml]
Growth inhibition [%]a)
10
11
12
13
14
15
16
100
10
1
62.5100
29.7
17.4
87.572.3 0
50
2518.7
5
82.7
54.1
18.7
75
43.7
31.2
65.4
31.2
31.2
24.6
32.9
a) Standard drug (paraquat) showed 100% growth inhibition at 7.5 mg/ml.
Results and Discussion. Screening-scale experiments showed that all four fungal
¾
cultures were capable of converting (À)Ambrox (1) to polar metabolites. Preparative-
scale fermentation was thus carried out to produce sufficient quantities of new and
known metabolites for structure elucidation and bioassay studies. Structures of known
metabolites were elucidated through comparison of their reported data [15 19].
¾
Incubation of (À)-Ambrox (1) with Fusarium lini (NRRL 68751) yielded the new
metabolites 2 5, whose structures were elucidated through comparative spectroscopic
spectral studies with substrate 1. Metabolites 3 and 4 might be formed by
monohydroxylation of compound 2 at C(11) and C(6), respectively, which could be
monohydroxylated in turn to yield 5 (see Scheme 1).
‡
The HR-EI-MS of 2 displayed the M at m/z 252.2056, corresponding to the molecular formula C16H28O2
(calc. 252.2089), indicating one more O-atom than 1. Absorption at 3436 cmÀ1 in the IR spectrum of 2 indicated
the presence of an OH group. The 1H-NMR spectrum of 2 (Table 2) further confirmed the formation of a
monohydroxy derivative through the geminal-proton signal at d 3.46 (t, J ˆ 2.6 Hz). The splitting pattern and
coupling constant indicated that the OH group must be axially situated either at C(1), C(3), or C(7). The
13C-NMR spectrum of 2 (Table 3) showed an additional CH signal at d 72.4, along with the g-upfield shifts of
C(3), C(5), and C(9) as compared to 1, suggesting that the location of the new OH group is C(1). HÀC(1) (d
3.46) showed homonuclear couplings with CH2(2) (d 2.10, 1.52), and HMBC interactions with C(3) (d 35.3) and
C(5) (d 48.8). The configuration of the new OH group was deduced from the NOE experiment, which showed
enhancement of the HÀ(1) signal (d 3.46) on irradiation of the Meb(16) signal. These interactions are only
possible with a disposition (axial) of the OHÀC(1).
‡
The metabolite 3 showed an IR spectrum similar to that of 2. Its HR-EI-MS exhibited the M at m/z
268.2064, consistent with the molecular formula C16H28O3 (calc. 268.2038), 32 amu greater than that of 1. This
suggested dihydroxylation of 1. However, the 1H-NMR spectrum of 3 (Table 2) was remarkably different from
that of 1 in several respects: two downfield OH protons appeared at d 3.69 (t, J ˆ 2.8 Hz) and 4.51 (ddd, J ˆ 10.2,
7.1, 3.9 Hz), while the CH2(12) signals were slightly shifted downfield to d 4.15( dd, J ˆ 9.6, 7.1 Hz) and 3.63
(dd, J ˆ 9.6, 4.1 Hz), indicating the presence of an OH group at C(11). The 13C-NMR spectrum of 3 (Table 3)

Helvetica Chimica Acta Vol. 87 (2004)
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Scheme 1. Metabolism of (À)-Ambrox (1) by Fusarium lini
showed two new CH resonances at d 72.0 and 70.1. The DEPTexperiment revealed the presence of five CH2 and
four CH, indicating that two CH2 moieties of 1 were converted to OH-bearing CH groups. The proton at d 3.69
(t, J ˆ 2.8 Hz) could be assigned to HbÀC(1), while the OH-bearing CH(11) appeared at d 4.51. The
configuration of OHÀC(11) was deduced from an NOE experiment, which showed a 7.5% enhancement of the
HÀC(11) signal (d 4.51) when Meb(13) (d 1.12) was irradiated. Similarly, irradiation of the HÀC(11) signal
caused a 3.6 and 2.1% enhancement of the signals at 1.12 (Meb(13)) and 0.82 (Meb(16)), respectively, supporting
the b-position of HÀC(11).
The structure of ambrox-1a,6a-diol (4) was deduced on the basis of the following considerations. HR-EI-
‡
MS showed the molecular ion at m/z 268.2041 (C16H28O3 ; calc. 268.2038), revealing two new O-functions in the
molecule as compared to 1. These O-functions were deduced to be OH groups from the IR absorption at
3329 cmÀ1. The 1H-NMR spectrum of 4 (Table 2) showed additional signals for two protons geminal to the OH
groups at d 3.42 (t, J ˆ 2.7 Hz) and 4.62 (dd, J ˆ 7.3, 3.0 Hz). The splitting pattern and coupling constants
suggested an axial orientation of the OH groups present at C(1) and C(6). The 13C-NMR spectrum of 4

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Helvetica Chimica Acta Vol. 87 (2004)
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Table 3. 13C-NMR Data (125MHz, CDCl 3) of Compounds 1 and 10 and of Their New Metabolites 2 5 and 13,
14, and 16, respectively. Multiplicities were determined by DEPT experiments.
1
2
3
4
5
10
38.9
18.2
42.3
33.2
56.6
20.2
38.5 39.3
86.3
59.2
37.4
28.7
176.1
21.6
33.2
21.0
15.1
13
48.4
64.4
51.5
34.7
56.2
21.4
44.7
86.0
58.9
38.539.5
28.7
176.0
21.8
14
49.9
69.4
84.6
35.1
56.0
16
37.4
26.9
79.4
38.6
56.3
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
40.0
18.4
42.5
33.1
57.3
20.7
39.8
79.9
60.2
36.1
22.6
65.0
21.2
33.6
21.2
15.1
72.4
25.6
35.3
33.0
48.8
20.4
39.539.9
80.3
52.5
40.1
22.3
64.9
21.3
33.3
20.9
15.9
72.0
24.9
35.5
33.1
49.0
20.3
74.7
25.1
38.8
35.3
52.1
73.2
25.0
37.6
34.1
51.0
69.520.6
39.6
81.3
59.1
71.1
21.2
48.5 48.2
80.9
82.3
59.0
40.3
70.1
73.1
21.9
33.3
20.7
16.7
84.573.6
59.1
C(9)
54.0
41.540.6
58.5
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
36.2
27.1
65.8
71.0
73.0
23.6
32.8
27.7
17.7
32.7
179.4
21.8
33.3
21.0
28.6
179.7
23.6
27.7
21.1
23.9a)
25.1
33.2
21.7
16.2
23.6a
18.4
15.7
16.0
a) Assignments are interchangeable.
(Table 3) showed the appearance of two downfield CH signals at d 74.7 and 71.1, as compared to 1. The HÀC(6)
(d 4.62) of 4 showed COSY-458 interactions with CH2(7) (d 2.05, 1.64) and HÀC(5) (d 1.58), and heteronuclear
interactions with C(8) (d 80.9) and C(10) (d 41.5), which further supported the position of the new OH group at
C(6). The configuration at C(1) and C(6) was further investigated by NOE experiments. An NOE between
Meb(16)/HÀC(6) (24.5%) and HÀC(1) (2.6%) was observed.
‡
The metabolite 5 was isolated as a colorless crystalline compound. The HR-EI-MS showed the M at m/z
284.1913 consistent with the molecular formula C16H28O4 (calc. 284.1987), corresponding to a trihydroxy
derivative. The IR spectrum displayed absorption at 3411 cmÀ1, further indicating the presence of OH groups.
The 1H-NMR spectrum of 5 (Table 2) exhibited three additional downfield CH signals at d 3.62 (t, J(1eq,2eq) ˆ
J(1eq,2ax) ˆ 2.2 Hz), 4.63 (dd, J(6eq,5ax) ˆ 7.4 Hz, J(6eq,7eq) ˆ 2.3 Hz), and 4.59 (ddd, J(11,9ax) ˆ 9.9 Hz,
J(11,12a) ˆ 7.1 Hz, J(11,12b) ˆ 4 Hz) that could be assigned to HbÀC(1), HbÀC(6), and HbÀC(11),
respectively. The 13C-NMR spectrum of 5 (Table 3) showed three downfield CH signals at d 73.2, 69.5, and
71.0 corresponding to the OH-bearing C(1), C(6), and C(11), respectively. The positions and configurations of
the newly introduced OH groups were deduced by 2D-NMR techniques. The COSY-458 plot showed couplings
between HÀC(1) (d 3.62)/CH2(2) (d 2.14, 1.59), HÀC(6) (d 4.63)/CH2(7) (d 2.01, 1.77), and HÀC(5) (d 1.47),
HÀ(11) (d 4.59)/CH2(12) (d 4.20, 3.61) and HÀC(9) (d 2.21), while the HMBC plot showed the heteronuclear
interactions between HÀC(6)/C(8) (d 81.3) and C(10) (d 40.6), HÀC(1)/C(3) (d 37.6), and C(5) (d 51.0), and
HÀC(11)/C(12) (d 73.0). The splitting patterns of HÀC(1) and HÀC(6) indicated their equatorial (b) and axial
(b) orientations, respectively. The configuration at the newly created chiral centers was further deduced by the
NOESY technique (Fig. 1), further supporting the structure as ambrox-1a,6a,11a-triol (5).
¾
Fermentation of (À)-Ambrox (1) with Rhizopus stolonifer (ATCC 10404)
furnished the known metabolites 6 9 (Scheme 2), that with Curvularia lunata (NRRL
2178) 6 and 7, and that with Cunninghamella elegans (NRRl 1392) 6, 7, and the known
10 (see Exper. Part).
Incubation of (‡)-sclareolide (10) with Cunninghamella elegans (NRRL 1392)
resulted in the formation of the new metabolites 13, 14, and 16 and the known
metabolites 11, 12, and 15 (Scheme 3). The structures of these metabolites were
elucidated on the basis of spectral data and comparative spectroscopic studies with

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Fig. 1. Selected NOESY correlations in compound 5
Scheme 2. Metabolism of (À)-Ambrox (1) by Rhizopus stolonifer
substrate 10. The metabolite 14 could be formed by monohydroxylation at C(2) of 12 or
at C(3) of 13 (see Scheme 3).
‡
The HR-EI-MS of metabolite 13 showed the M at m/z 266.1739, corresponding to the molecular formula
C16H26O3 (calc. 266.1884). The IR spectrum exhibited absorption at 3421 cmÀ1, indicating the presence of an OH
group. The 1H-NMR spectrum of 13 (Table 2) showed the characteristic signals of a sclareolide derivative with
an additional secondary OH group at a CH resonating at d 3.90 (m, w1/2 ˆ 17.1 Hz). The splitting pattern of this
latter signal indicated that an axially oriented OH group could be present at HÀC(2). The position of this OH
group at C(2) was further ascertained by downfield shifts of the C(1) (d 48.4) and C(3) (d 51.5) signals in the
13C-NMR spectrum of 13 (Table 3) when compared with 10. HÀC(2) of 13 showed homonuclear interactions

Helvetica Chimica Acta Vol. 87 (2004)
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Scheme 3. Metabolism of (‡)-Sclareolide (10) by Cunninghamella elegans
Fig. 2. Selected NOE interactions in compound 16
with CH2(1) (d 1.81, 1.42) and CH2(3) (d 1.88, 1.15) in the COSY-458 plot. The configuration of OHÀC(2) was
further investigated by a NOESY experiment, which showed cross-peaks between HÀC(2) (d 3.90)/Meb(15) (d
0.93) and Meb(16) (d 0.87).
Compound 14 (C16H26O4) has two additional O-atoms as compared to 10. The IR spectrum indicated the
presence of OH groups at 3415cm À1. The 1H-NMR data of 14 (Table 2) exhibited two additional downfield
signals at d 3.79 (m, w1/2 ˆ 18.6 Hz) and 3.02 (d, J ˆ 9.1 Hz). The multiplicity of these resonances indicated that
OH groups could be present at C(2) (a position), C(3) (b position), or C(1) (b position). The 13C-NMR
(Table 3) also showed the appearance of two new CH signals at d 84.6 (C(3)) and 69.4 (C(2)). The new CH

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Helvetica Chimica Acta Vol. 87 (2004)
protons at d 3.79 (HÀC(2)) and 3.02 (HÀC(3)) exhibited mutual coupling in the COSY-458 plot. Furthermore,
HÀC(5) (d 1.21) also interacted with HÀC(3), indicating the presence of OH groups at C(2) and C(3).
HÀC(2) showed heteronuclear interactions with C(1) (d 49.9) and C(4) (d 35.1), while HÀC(3) interacted with
C(5) (d 56.0) and C(2) (d 69.4). The configuration of the newly introduced OH groups was deduced to be a and
b, respectively, based on the NOE interaction between HÀC(2)/Meb(15) and Meb(16) and HÀC(3)/Mea(14).
The IR, mass and 1H-NMR spectra (Table 2) of 16 were found to be completely identical with that of the
known 3b-hydroxysclareolide (12) [24]. However analysis of the 13C-NMR spectrum (Table 3) did not support
the conclusion that compound 16 had the same structure as 12. It showed an upfield shifted C(8) signal at d 73.6.
Furthermore, HÀC(9) (d 1.93) and 2 HÀC(7) (d 1.81, 1.52) exhibited heteronuclear interactions with C(8). The
NOESY plot of 16 did not show any correlation between Meb(16) and Me(13), which supported the structure of
an 8-episclareolide. The inversion at the stereogenic center C(8) as compared to 12 was confirmed by a NOE
differential experiment (see Fig. 2), which further supported the structure of 3b-hydroxy-8-episclareolide (16).
We thank Mr. Abdul Qayyum of Medics Laboratories, Karachi, Pakistan, for providing financial assistance
to S. G. M.
Experimental Part
¾
1. General. (À)-Ambrox (1) and (‡)-sclareolide (10) were purchased from Sigma-Aldrich. TLC: silica gel
precoated plates (Merck, PF254; 20 Â 20, 0.25mm); detection by vanillin spray reagent. Column chromatog-
raphy (CC): silica gel (70 230 mesh, Merck). M.p.: B¸chi 535 melting-point apparatus. Optical rotations:
MeOH solns.; Jasco DIP 360 digital polarimeter. IR Spectra: CHCl3 solns.; FTIR-8900 spectrophotometer; in
cm-1. 1H- and 13C-NMR Spectra: CDCl3 solns.; Bruker Avance-500-NMR at 500 and 125 MHz, resp.; 2D
experiments with CDCl3 solns. and the same instrument; chemical shifts d in ppm rel. to SiMe4 as an internal
standard, coupling constants J in Hz. EI-MS and HR-EI-MS: Jeol JMS-600 H mass spectrometer; in m/z (rel.
%).
2. Organisms and Culture Media. The fungal cultures were purchased from different sources such as
International Mycological Institute (IMI), American Type Culture Collection (ATCC), Northern Regional
Research Laboratories (NRRL), and Institute of Fermentation, Osaka, Japan (IFO). Stock cultures were
maintained at 40 on agar slants (Sabouraud dextrose agar). The medium for Fusarium lini (NRRL 68751) was
prepared by adding the following chemicals into dist. H2O (3.0 l): glucose (30.0 g), glycerol (30.0 g), peptone
(15.0 g), yeast extract (15.0 g), KH2PO4 (15.0 g), and NaCl (15.0 g), while the medium for Rhizopus stolonifer
(ATCC 10404) (3.0 l, dist. H2O) included glucose (60.0 g), glycerol (30.0 g), peptone (30.0 g), yeast extract
(8.0 g), and KH2PO4 (15.0 g); the pH of the soln. was adjusted to 5.6 by adding few drops of 0.04n NaOH. The
medium for Cunninghamella elegans (NRRL 1392) and Curvularia lunata (NRRL 2178) were prepared by
mixing the following ingredients into distilled H2O (3.0 l): glucose (30.0 g), peptone (15.0 g), yeast extract
(15.0 g), KH2PO4 (15.0 g), and NaCl (15.0 g).
3. General Fermentation and Extraction Conditions. The fermentation medium was distributed among 30
flasks of 250-ml capacity (100 ml in each) and autoclaved at 1210 for 20 min. The fermentation was carried out
according to a standard, two-stage protocol [25]. The substrate was dissolved in acetone, and the resulting clear
soln. was evenly distributed among the 30 flasks (20 mg/0.5ml in each flask) containing 24-h-old stage-II
cultures, and fermentation was carried out for further days on a rotatory shaker (200 rpm) at 290. During the
fermentation period, aliquots from culture were taken out daily and analyzed by TLC to determine the degree of
transformation of substrate. In all experiments, one control flask without fungus (for checking substrate
stability) and another flask without exogenous substrate (for checking endogenous metabolites) were used. The
culture media and mycelium were separated by filtration. The mycelium was washed with CH2Cl2 (1.5l), and the
filtrate was extracted with CH2Cl2 (3 Â 2 l). The combined org. extract was dried (Na2SO4) and evaporated and
the residue analyzed by TLC. Control flasks were also harvested and compared with the test by TLC to confirm
the presence of biotransformed products.
¾
¾
4. Fermentation of (À)-Ambrox (1) with Fusarium lini (NRRL 68751). (À)-Ambrox (1; 600 mg),
dissolved in acetone (15ml), was evenly distributed in 30 flasks containing 24-h-old stage-II culture.
Fermentation was carried out for 8 days. After filtration, extraction, and evaporation, the obtained brown gum
(1.63 gm) was subjected to repeated CC (petroleum ether/AcOEt gradient): 2 (16.4 mg, 2.7%; with petroleum
ether/AcOEt 62 :28), 3 (8.2 mg, 1.3%; with petroleum ether/AcOEt 61:39), 4 (19.1 mg, 3.2%; with petroleum
ether/AcOEt 56 :44), and 5 (27.8 mg, 4.6%; with petroleum ether/AcOEt 29 :71).

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Ambrox-2a-ol (ˆ(3aR,5aS,9s,9aS,9bR)-Dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan-9-ol; 2):
25
M.p. 101 1028. ‰aŠ ˆ ‡1.72 (c ˆ 0.1, MeOH). IR (CHCl3): 3436, 2938, 2870. 1H-NMR (CDCl3, 500 MHz):
D
Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 252 (17, M ), 237 (100, [M À Me] ), 219 (54, [M À Me À
‡
‡
‡
‡
‡
H2O] ), 163 (4), 152 (14), 111 (19), 97 (37), 55 (69). HR-EI-MS: 252.2056 (M , C16H28O2 ; calc. 252.2089).
Ambrox-1a,11a-diol (ˆ(1S,3aR,5aS,9S,9aS,9bS)-Dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan-
25
1,9-diol; 3): M.p. 174 1768. ‰aŠ ˆ À1.53 (c ˆ 0.1, MeOH). IR (CHCl3): 3354, 2941, 2868, 1124. 1H-NMR
D
(CDCl3, 500 MHz): Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 268 (7, M ), 253 (60, [M À Me] ),
‡
‡
‡
235(87, [ M À Me À H2O] ), 163 (4.6), 205 (24), 164 (12), 136 (79.2), 121 (52), 95 (55), 81 (100), 55 (95). HR-
‡
‡
EI-MS: 268.2064 (M , C16H28O3 ; calc. 268.2038).
Ambrox-1a,6a-diol (ˆ(3aR,5S,5aS,9S,9aS,9bR)-Dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan-
25
5,9-diol; 4): M.p. 119 1208. ‰aŠ ˆ À18.7 (c ˆ 0.1, MeOH). IR (CHCl3): 3329, 2931, 2877. 1H-NMR (CDCl3,
D
500 MHz): Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 268 (7, M ), 253 (100, [M À Me] ), 235(66,
‡
‡
‡
[M À Me À H2O] ), 163 (4), 217 (9), 191 (24), 167 (7), 139 (13), 111 (21), 81 (23), 55 (62). HR-EI-MS:
‡
‡
268.2041 (M , C16H28O3 ; calc. 268.2038).
Ambrox-1a,6a,11a-triol (ˆ(1S,3aR,5S,5aS,9s,9aS,9bS)-Dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]-
25
furan-1,5,9-triol; 5): M.p. 191 1928. ‰aŠ ˆ À26 (c ˆ 0.1, MeOH). IR (CHCl3): 3411, 2930, 2874, 1217. 1H-NMR
D
(CDCl3, 500 MHz): Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 284 (2, M ), 269 (32, [M À Me] ),
‡
‡
‡
251 (93, [M À Me À H2O] ), 218 (11), 215(13), 175(9), 139 (23), 109 (99), 81 (69), 55(100). HR-EI-MS:
‡
‡
284.1913 (M , C16H28O4 ; calc. 284.1987).
¾
5. Fermentation of (À)-Ambrox (1) with Rhizopus stolonifer (ATCC 10404). Incubation of (À)-1 (600 mg)
with Rhizopus stolonifer (ATCC 10404) for 12 days yielded a brown oily matter (1.8 gm), which was subjected to
CC (silica gel, AcOEt/petroleum ether gradient): 6 (7.9 mg, 1.3%; with 18% AcOEt), 7 (6.3 mg, 1%; with 31%
AcOEt), 8 (11.4 mg, 1.9%; with 44% AcOEt), and 9 (28.1 mg, 4.7%; with 56% AcOEt).
¾
6. Fermentation of (À)-Ambrox (1) with Cunninghamella elegans (NRRL 1392) and Curvularia lunata
(NRRL 2178). The fermentation of (À)-1 (600 mg) with Curvularia lunata for 10 days afforded 6 (14 mg, 2.3%)
and 7 (41 mg, 6.8%), while fermentation with Cunninghamella elegans for 12 days afforded 6 (3.2 mg, 0.53%), 7
(17.2 mg, 2.8%), and (‡)-10 (11.4 mg, 1.9%).
7. Fermentation of (‡)-Sclareolide (10) with Cunninghamella elegans (NRRL 1392). (‡)-Sclareolide (10;
600 mg), dissolved in acetone (15ml), was evenly distributed in 30 flasks containing stage-II cultures.
Fermentation was stopped after 12 days, including control flasks. The org. metabolites were extracted from the
medium. The extract was evaporated and the obtained brown gum (2.3 gm) subjected to CC (petroleum ether/
AcOEt gradient): 11 (81 mg, 13.5%; with petroleum ether/AcOEt 76 :24), an impure fraction containing 12/13
(with petroleum ether/AcOEt 63 :37), which was purified by TLC (AcOEt/petroleum ether 3 :7) to give 12
(21.7 mg, 2.1%) and 13 (31.7 mg, 5.3%), 14 (142 mg, 23.6%; with petroleum ether/AcOEt 53 :47), 15 (7.9 mg,
1.3%; with petroleum ether/AcOEt 39 :61), and 16 (16.5mg, 2.7%; petroleum ether/AcOEt 9 :91).
2a-Hydroxysclareolide (ˆ(3aR,5aS,8S,9aS,9bR)-Dodecahydro-8-hydroxy-3a,6,6,9a-tetramethylnaph-
25
tho[2,1-b]furan-2(1H)-one; 13): M.p. 154 1558. ‰aŠ ˆ À100 (c ˆ 0.006, MeOH). IR (CHCl3): 3421, 2927,
D
2869, 1227. H-NMR (CDCl3, 500 MHz): Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 251 (8, [M À
1
‡
‡
Me] ), 233 (19, [M À Me À H2O] ), 221 (24), 204 (49), 165(41), 139 (31), 107 (43), 81 (34), 55(100). HR-EI-
‡
‡
MS: 266.1739, (M , C16H26O3 ; calc. 266.1884).
2a,3b-Dihydroxysclareolide (ˆ(3aR,5aR,7R,8R,9aS,9bR)-Decahydro-7,8-dihydroxy-3a,6,6,9a-tetramethyl-
25
naphtho[2,1-b]furan-2(1H)-one; 14): M.p. 189 1908. ‰aŠ ˆ À136 (c ˆ 0.08, MeOH). IR (CHCl3): 3415, 2933,
D
2873, 1757. H-NMR (CDCl3, 500 MHz): Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 282 (2, M ),
1
‡
‡
266 (3, [M À H2O] ), 238 (4), 195 (15), 135 (19), 123.1 (100), 81 (31), 71 (77), 55 (58). HR-EI-MS: 282.1884,
‡
‡
(M , C16H26O4 ; calc. 282.1831).
3b-Hydroxy-8-episclareolide (ˆ(3aS,5aR,7S,9aS,9bR)-Decahydro-7-hydroxy-3a,6,6,9a-tetramethylnaph-
25
tho[2,1-b]furan-2(1H)-one; 16): M.p. 181 1828. ‰aŠ ˆ À136 (c ˆ 0.08, MeOH). IR (CHCl3): 3330, 2925,
D
2856, 1712. 1H-NMR (CDCl3, 500 MHz): Table 2. 13C-NMR (CDCl3, 125MHz): Table 3. EI-MS: 266 (3, M ),
‡
‡
‡
251 (10, [M À Me] ), 233 (12, [M À Me À H2O] ), 218 (11), 204 (12), 167 (13), 139 (38), 109 (43), 81 (38), 55
‡
‡
(100). HR-EI-MS: 266.1891 (M , C16H26O3 ; calc. 266.1884).
Phytotoxicity Assay. Phytotoxicity of the sesquiterpene lactones 10 16 was determined against Lemna
minor L. according to the modified protocol of McLaughlin and co-workers [26]. The test compounds were
incorporated with E-medium at different concentrations, i.e., 1, 10, and 100 mg/ml in MeOH. Conical flasks were
inoculated with compounds at the desired concentration prepared from the stock soln. and allowed to evaporate
overnight. Each flask was inoculated with 2 ml of E-medium and ten Lemna minor L., each containing a rosette
of three fronds. Other flasks were supplemented with MeOH serving as negative control and reference inhibitor,

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Helvetica Chimica Acta Vol. 87 (2004)
i.e., paraquat serving as positive control. Treatments were replicated three times, and the flasks were incubated
at 378 for 7 days. Growth of Lemna minor L. in a compound-containing flask was determined by counting the
number of fronds per dose, and growth inhibition was calculated with reference to negative control.
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Received June 21, 2004