A new example of 1α-hydroxylation of drimanic terpenes through combined microbial and chemical processes

Article abstract of DOI:10.1016/S0040-4020(01)00595-6

Among various filamentous fungi, Aspergillus niger ATCC 9142 was the most efficient strain to convert confertifolin in high yield into its 3β-hydroxy derivative, which was then chemically converted to 1α-hydroxy-, 1α,11α-dihydroxy- and 1α-acetoxy-11α-hydroxyconfertifolin, new compounds structurally related to bioactive natural products isolated from plants or marine sponges.

Full text of DOI:10.1016/S0040-4020(01)00595-6

TETRAHEDRON
Pergamon
Tetrahedron 57 82001) 6051±6056
A new example of 1a-hydroxylation of drimanic terpenes through
combined microbial and chemical processes
a
Gerard Aranda, Luis Moreno, Manuel Cortes, Thierry Prange, Michele Maurs
b
b
c
d
Â
Â
Â
Á
and Robert Azeradd,p
a
Á
Laboratoire de Synthese Organique, UMR 7652, Ecole Polytechnique, 91128 Palaiseau Cedex, France
 Â
Facultad de Quõmica, Ponti®cia Universidad Catolica de Chile, Casilla 306, Correo 22-Santiago, Chile
b
c
 Â
Chimie Structurale Biomoleculaire, UPRES A 7031, UFR Biomedicale, 93017 Bobigny Cedex, France
Â
d
Â
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, Universite Rene Descartes-Paris V,
45 rue des Saints-Peres, 75270 Paris 06, France
Received 20 February 2001; revised 14 May 2001; accepted 30 May 2001
AbstractÐAmong various ®lamentous fungi, Aspergillus niger ATCC 9142 was the most ef®cient strain to convert confertifolin in high
yield into its 3b-hydroxy derivative, which was then chemically converted to 1a-hydroxy-, 1a,11a-dihydroxy- and 1a-acetoxy-11a-
hydroxyconfertifolin, new compounds structurally related to bioactive natural products isolated from plants or marine sponges. q 2001
Elsevier Science Ltd. All rights reserved.
1. Introduction
3b-hydroxylated derivatives, sometimes naturally found as
minor compounds. Hydroxylation at the 1-position, a distinc-
tive feature of several bioactive terpenes, cannot be achieved
by this method. However, starting from 3b-hydroxy deriva-
tives, we have previously shown that a simple sequence of
reactions can result in a functionalization transfer, allowing
the preparation in good yields of the corresponding
1a-hydroxylated compounds.7,9,10
Microbial hydroxylations of terpenoid compounds1 have
been repeatedly reported and employed for the regioselec-
tive functionalization of a number of commonly found
sesqui- and diterpenes, representing a unique and inexpen-
sive source of structural diversity. Much attention has been
paid to the synthesis of hydroxylated drimanic compounds2±8
due to their wide range of biological activities and possible
industrial applications. The microbial hydroxylation of this
type of 4,4-dimethyl sesquiterpenes invariably leads to
In this report, we describe the extension of these reactions
to the preparation of 1a-hydroxylated derivatives of
Table 1. 3b-Hydroxylation of confertifolin 1 80.5 g L21) upon incubation with some 65 h-grown fungal cultures 8278C, 250 rpm)
Microorganisms
Incubation time
8days)
3b-hydroxyconfertifolin 2a,b
Absidia blakesleeana ATCC 6811
A. niger ATCC 9142
8
5
3
3
3
2
8
5
5
11
1111
111
111
1
1
11
Chaetomium indicum LCP 98-4200
Cunninghamella echinulata NRRL 3655
Cunninghamella elegans ATCC 36112
Curvularia lunata NRRL 2380
Mortierella isabellina NRRL 1757
Mucor plumbeus ATCC 4740
111
111
Rhizopus arrhizus ATCC 11145
a
GC/MS measurements.
1: 5±15%; 11: 16±40%; 111: 41±65%; 1111: 66±90%
b
Keywords: confertifolin; biotransformation; microbial hydroxylation; functionalization transfer.
Corresponding author. Tel.: 133-1-42-86-21-71; fax: 133-1-42-86-83-87; e-mail: robert.azerad@biomedicale.univ-paris5.fr
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020801)00595-6

6052
G. Aranda et al. / Tetrahedron 57 =2001) 6051±6056
Scheme 1. 8i) A. niger ATCC 9142. 8ii) Ph3P, DEAD in re¯uxing THF. 8iii) SeO2, NPO in dioxane, 1008C. 8iv) AC2O, DMAP, 48C.
confertifolin 1, a D8-9-drimenic lactone from a South
American tree commonly found in Chile and Argentina,
Drimys winteri Forst.11
Application to 3b-hydroxyconfertifolin 2 of the previously
10
described dehydration/oxidation reaction sequence lead-
ing to a functionalization transfer to position 1a- afforded
8Scheme 1) two hydroxylated derivatives which were more
easily separated as their acetate esters. After mild alkaline
hydrolysis 8K2CO3, MeOH), the new alcohols were respec-
tively identi®ed as the 1a-hydroxy-2,3-dehydroconfertifolin
4a and the 1a,11a-dihydroxy-2,3-dehydroconfertifolin 5a
by high ®eld NMR and mass spectrometry.
2. Results and discussion
As a complement to previous studies,8 a number of fungal or
bacterial microorganisms 820 strains) generally employed
for terpene metabolism1 were investigated for the bio-
transformation of confertifolin. The most signi®cant results
are summarized in Table 1. Aspergillus niger ATCC 9142
remained the most ef®cient strain and afforded the corre-
sponding 3b-hydroxylated derivative in 80±90% yield after
simple crystallisation from the crude incubation extract.
The expected 1a-hydroxy derivative 4a 820±35%, see Table
2) exhibited a broad singlet at 3.87 ppm by 1H NMR, with a
characteristic pattern similar to that previously described for
1a-derivatives 8an ABX system between 2-, 3- and 1-hydro-
gens, with a H-1/H-2 coupling constant of 5.7 Hz).10
Table 2. Selenium dioxide oxidation of 2,3-dehydroconfertifolin 83) 8pyridine-N-oxide: 7±8 equiv.; dioxane; 1008C)
SeO2 equivalents
Hours
Conversion %
Total yield 84a15a) %
4a/5a ratio
1.5
2
48
67
65
93
80
80
43:57
26:74
Table 3. 13C Chemical shifts 8ppm) of confertifolin derivatives. Unless otherwise indicated, NMR spectra were run in CDCl3 at 50 MHz. Bracketted data
indicating the number of hydrogens attached to each carbon atom were determined by distorsionless enhancement by polarization transfer 8DEPT) using a ¯ip
angle of 1358
Carbon no.
2a
3
4a
4b
5ab
5b
5c
6
7a
7b
7c
1
2
3
4
5
6
7
8
34.282)
27.182)
78.381)
39.080)
50.681)
18.182)
21.682)
123.880)
170.080)
36.480)
68.282)
174.780)
15.683)
28.283)
20.983)
±
36.282)
119.881)
138.781)
34.680)
48.281)
18.982)
21.582)
124.180)
168.580)
35.580)
68.382)
174.180)
22.283)
31.783)
20.683)
±
69.481)
122.681)
143.081)
35.180)
41.381)
18.882)
21.282)
125.980)
167.680)
41.380)
69.282)
174.780)
21.883)
31.483)
20.983)
±
71.581)
118.981)
144.681)
35.080)
42.281)
18.882)
21.382)
126.780)
165.480)
39.780)
68.582)
174.080)
22.083)
31.283)
21.183)
170.580)
21.683)
70.081)
123.681)
143.081)
35.980)
42.581)
20.082)
22.082)
131.080)
166.980)
43.080)
99.281)
173.680)
22.483)
31.583)
19.683)
71.881)
119.281)
144.081)
35.280)
42.781)
18.682)
21.382)
130.680)
163.380)
40.080)
97.381)
171.580)
22.283)
31.383)
19.583)
171.680)
21.383)
70.981)
119.581)
144.081)
35.280)
42.781)
18.782)
21.782)
132.680)
161.280)
40.580)
91.081)
170.680)
21.983)
31.483)
20.683)
169.580)
169.880)
21.383)
71.481)
25.882)
34.582)
33.080)
43.981)
17.882)
21.782)
124.980)
169.880)
41.980)
68.982)
175.080)
21.783)
33.183)
21.383)
71.381)
26.282)
34.582)
33.280)
44.281)
17.782)
21.482)
130.680)
166.380)
43.480)
99.981)
171.780)
21.483)
33.183)
21.483)
74.781)
22.382)
35.182)
33.480)
45.281)
17.782)
21.682)
129.780)
164.980)
40.880)
97.281)
171.480)
21.583)
32.983)
21.083)
171.380)
21.783)
73.681)
22.082)
35.082)
32.980)
45.481)
17.982)
22.082)
132.080)
162.980)
42.080)
90.881)
170.580)
21.783)
33.583)
20.683)
169.680)
169.980)
21.583)
9
10
11
12
13
14
15
COCH3
±
a
See Ref. 8.
In CD3OD 8125 MHz).
b

G. Aranda et al. / Tetrahedron 57 =2001) 6051±6056
6053
Figure 1. Energy-minimized models for 1a,11a-dihydroxy 8left) and 1a,11b-dihydroxy-2,3-dehydroconfertifolin 8right).
13C NMR data 8Table 3) and heteronuclear 2D studies
entirely con®rmed this assignement. Acetate 4b was easily
prepared by acetylation. Catalytic hydrogenation 8PtO2 in
EtOAc) of 4a selectively afforded the corresponding
2,3-reduced derivative 81a-hydroxyconfertifolin) 6. Other
catalysts, such as palladium on charcoal or Crabtree's
catalyst were ineffective for reducing the D2,3 double bond.
vs CH3-15/H-11aˆ4.22 A and CH3-15/H-1bˆ3.03 A
measured for an 11b-OH group.
Ê
Ê
The additional allylic oxidation by SeO2 in the lactone ring
is strictly dependent on the presence of the 2,3-unsaturation
and/or the 1a-hydroxy structure because, in identical reac-
tion conditions, confertifolin remained unchanged. Such
11a-hydroxylated derivatives have been already found as
minor natural products such as valdiviolide 8 in various
Drimys species or fuegine 9 in Polygonum hydropiper L.,
but their stereochemistry at C-11 remains to be clari®ed.12±14
Oxidation of 2,3-dehydroconfertifolin 3 with excess
selenium dioxide 8see Table 2) afforded mainly the unex-
pected 1a,11a-hydroxy derivative 5a. The 2,3-dehydro-11-
hydroxy derivative was not detected. The assignment of
1
an additional 11a-hydroxylation was based on H and 13C
Mild acetylation of the dihydroxy derivative 5a provided
regioselectively the monoacetate 5b and the diacetate 5c
while catalytic hydrogenation of 5a 8PtO2 in EtOAc,
15 psi) afforded the corresponding 2,3-reduced derivative
81a,11a-dihydroxy-confertifolin) 7a. Catalytic hydrogena-
tion of the monoacetate derivative 5b was not successful,
because the 1a-acetyl group was transferred in part to the
11a-position and no pure compound 7b could be isolated.
Hydrogenation of the diacetate 5c afforded a diacetate 7c,
but this one did not present convincing NMR data for
an 11a-8acet)oxy derivative, as previously observed with
5a. However, an X-ray determination15 unambiguously
demonstrated a 1a,11a-diacetoxy structure 8Fig. 2).
NMR data, and particularly on NOESY experiments which
showed very similar nuclear Overhauser effects between
CH3-15 and H-11 or CH3-15 and H-1b, indicating very
similar distances. Molecular modelling of 11a- and 11b-
hydroxylated derivatives 8Fig. 1) showed that this pattern
was only compatible with an 11a-hydroxyl group: in mini-
Ê
mized models a distance of 3.15^0.1 A was measured
between CH3-15/H-11b or H-1b for an 11a-OH group,
3. Conclusions
For the ®rst time, an 11a-hydroxy hemiketal structure in the
drimenic lactone series is unambiguously demonstrated and
should be compared to previously known natural products,
such as valdiviolide, fuegin12,13 and cinnamolide deriva-
tives.14
Figure 2. ORTEP representation of 7c with ellipsoids at 50% probability
level. Oxygen atoms are dashed.

6054
G. Aranda et al. / Tetrahedron 57 =2001) 6051±6056
Moreover, it is of some interest that some of the prepared
derivatives and particularly the 2,3-reduced monoacetate
7b present marked similarities with part of the unusual
structures of several cytotoxic sesterpenes of marine
sponges,16±18 such as scalarin 10 for example.
compound was prepared from 3 by oxidation with SeO2 in
dry dioxane at 1008C, as previously described.10 After two
crystallizations from pentane±CH2Cl2, mp 188±1908C.
18
[a]D ˆ12958 8c 0.56, CHCl3). HRMS 8CI±NH3) calc.
for C15H22O3 8M11) 249.1491, found 249.1495. IR
8CCl4), nmax 8cm21): 3615, 3446, 3020, 2963, 2868, 1763,
1736 8sh.), 1668, 1388, 1153, 1034. H NMR 8200 MHz,
1
4. Experimental
4.1. General
CDCl3) d ppm: 0.96, 1.07, 1.09 89H, 3s, CH3-15, -14 and
-13), 3.87 81H, br.s, d after D2O addition, J1±2ˆ5.2 Hz,
H-1b), 4.72, 4.80, 4.94, 5.02 82H, AB part of an ABXY
system, between H2-11 and H2-7, JABˆ16.5 Hz, JAX
ˆ
General experimental methods have been already
described.19 High resolution mass spectrometry 8HRMS)
was performed on a JEOL MS700 spectrometer. EI- and
CIMS were performed on a Hewlett±Packard 5989B instru-
ment. Incubation course was monitored by GC-MS, using a
25 m£0.2 mm Ultra 2 8Hewlett±Packard) capillary column
8temp. programmed 110±2708 at 88 min21). Column
chromatography was performed on silicagel Merck 60H
870±230 mesh). 1H NMR and 13C NMR spectra were
acquired in CDCl3 or CD3OD solutions at 200 or 500 and
50 or 125 MHz, respectively.
JAYˆ2.7 Hz, JBXˆ1.4 Hz and JBYˆ3.5 Hz, H-11a and
H-11b), 5.64, 5.69, 5.74, 5.77, 5.78, 5.81 82H, AB part of
an ABX system between H-2, H-3 and H-1b, JABˆ10 Hz,
JAXˆ0, JBXˆ5.7 Hz, H-2 and H-3). 13C NMR, see Table 3.
4.2.3. 1a-Hydroxyconfertifolin 6. 62 mg of 4a were hydro-
genated in the presence of PtO2 839 mg) in EtOAc 83.4 mL)
during 8 h. After ®ltration through Celite and two crystal-
21
lizations from pentane±CH2Cl2, mp 185±186.58C. [a]D
ˆ
1968 8c 0.38, CHCl3). HRMS 8CI±NH3) calc. for C15H23O3
8M11) 251.1647, found 251.1648. IR 8CCl4), nmax 8cm21):
3688, 3608, 3464, 3008, 2943, 2872, 1747, 1674, 1459,
1
Acetylations were performed in CH2Cl2 at the ice bath
temperature with acetic anhydride 83±4 equiv.) in the
presence of DMAP 84±5 equiv.). Total conversion was
checked by thin layer chromatography. Hydrogenation of
2,3-dehydro derivatives was performed with PtO2 catalyst
in EtOAc 8kept on K2CO3) at room temperature and atmos-
pheric pressure during 6±8 h.
1086, 1028; H NMR 8200 MHz, CDCl3) d ppm: 0.95,
1.02, 1.19 89H, 3s, CH3-15, -14 and -13), 3.83 81H, br.s,
H-1b), 4.69, 4.77, 4.94, 5.03 82H, AB part of an ABXY
system, between H2-11 and H2-7, JABˆ16.6 Hz, JAX
ˆ
JAYˆ2.7 Hz, JBXˆ3.4 Hz and JBYˆ1.5 Hz, H-11a and
H-11b). 13C NMR, see Table 3.
4.2.4. 1a-Acetoxy-2,3-dehydroconfertifolin 4b. This
compound was obtained by acetylation of 4a, usual work-
up and crystallization from pentane±EtOAc 8yield 80±
4.2. Starting material
20
85%): mp 190±2008C dec. [a]D ˆ13588 8c 0.45,
Pure confertifolin 1 was prepared by vacuum distillation of
the natural extract mixture of D. winteri, puri®ed by
column chromatography 8Silicagel, cyclohexane±ethyl
ether) and crystallized from CH2Cl2±pentane: mp 150±
CHCl3). IR 8CCl4), nmax 8cm21): 3026, 2964, 2943, 1767,
1740, 1680, 1449, 1370, 1334, 1028. HRMS 8CI±NH3) calc.
for C17H23O4 8M11) 291.1596, found 291.1594. H NMR
1
22
8200 MHz, CDCl3) d ppm: 0.99, 1.10, 1.18 89H, 3s, CH3-15,
-14 and -13), 1.99 83H, s, CH3CO), 4.44, 4.53, 4.72, 4.80
82H, AB part of an ABXY system, between H2-11 and H2-7,
1518C, [a]D ˆ171.78 8c 1.83, CHCl3). IR and NMR data
were in full agreement with earlier literature.11,20,21
JABˆ16.7 Hz, JAXˆ1.3 Hz, JAYˆ3.7 Hz, and JBYˆJBX
ˆ
Cultivation and incubation of fungal strains with conferti-
folin 1, and isolation of 3b-hydroxyconfertifolin 2 have
been previously described.8
2.8 Hz, H-11a and H-11b, 4.96 81H, d, J1±2ˆ4.6 Hz,
H-1b), 5.74, 5.79, 5.80, 5.82, 5.87 82H, complex m, H-2
and H-3). 13C NMR, see Table 3.
4.2.1. 2,3-Dehydroconfertifolin 3. This compound was
prepared, following the earlier described protocol,9 from
3b-hydroxyconfertifolin 2 8539 mg), triphenylphosphine
82.26 g) and diethyldiazodicarboxylate 81.35 mL) in re¯ux-
ing anhydrous THF821.6 mL). After usual work-up,
chromatographic puri®cation and two crystallizations from
pentane±ethyl ether, dehydroconfertifolin 3 8438 mg, 86%)
From a mixture of 4a15a 8470 mg, 26:74), obtained after
oxidation with excess SeO2 8see Table 2) and acetylated
overnight, a chromatographic separation afforded the mono-
acetates 4b 8203 mg) and 5b 8128 mg) and the diacetate 5c
8245 mg).
20
4.2.5. 1a,11a-Dihydroxy-2,3-dehydroconfertifolin 5a.
This compound was obtained by mild hydrolysis of 5b 8or
5c) with K2CO3 in aqueous MeOH. After two crystalliza-
was obtained: mp 1318C. [a]D ˆ11558 8c 0.865, CHCl3).
HRMS 8CI±NH3) calc. for C15H22O2 8M11) 233.1542,
found 233.1546. IR 8CCl4), nmax 8cm21): 3017, 2963,
2868, 1765, 1676, 1449, 1374, 1344, 1028. 1H NMR
8200 MHz, CDCl3) d ppm: 0.96, 1.03, 1.17 89H, 3s,
CH3-15, -14 and -13), 4.70 82H, AB part of an ABXY
21
tions from EtOAc±pentane, mp 179±1808C; [a]D ˆ13218
8c 0.35, CH3OH). HRMS 8CI±NH3) calc. for C15H24O4N
8M118) 282.1704, found 282.1705. H NMR 8500 MHz,
1
CD3OD) d ppm: 0.99, 1.09, 1.19 89H, 3s, CH3-15, -14 and
-13), 1.93 82H, s1dd, Jˆ6 and 12 Hz, H-5 and H-6a), 1.65
81H, m, H-6b), 2.13 81H, m, H-7a), 4.08 81H, br.s, H-1b),
2.39 81H, dd, J7b±7aˆ18 Hz, J7b ±6aˆJ7b ±6bˆ5 Hz, H-7b),
5.66, 5.68, 5.72, 5.73, 5.74, 5.75 82H, AB part of an ABX
system between H-2, H-3 and H-1b, JABˆ10 Hz, JAXˆ0,
system between H2-11 and H2-7, JABˆ16.9 Hz, JAX
ˆ
3.6 Hz, JAYˆ1.5 Hz and JBXˆJBYˆ2.7 Hz, H-11a and
H-11b), 5.50±5.52 8br.m, 2H, H-2 and H-3). 13C NMR,
see Table 3.
4.2.2. 1a-Hydroxy-2,3-dehydroconfertifolin 4a. This

G. Aranda et al. / Tetrahedron 57 =2001) 6051±6056
6055
JBXˆ5 Hz, H-2 and H-3), 6.30 81H, s, H-11b). 13C NMR,
found 351.1802. 1H NMR 8200 MHz, CDCl3) d ppm:
0.93, 1.00, 1.24 89H, 3s, CH3-15, -14 and -13), 2.05 and
2.09 86H, 2s, COCH3), 4.77 81H, t, Jˆ2.7 Hz, H-1b), 6.94
81H, t, Jˆ1.0 Hz, H-11b). 13C NMR, see Table 3.
see Table 3.
4.2.6. 1a-Acetoxy-11a-hydroxy-2,3-dehydroconfertifolin
5b. After crystallization from pentane±EtOAc, mp 183±
22
1868C. [a]D ˆ13078 8c 0.305, CHCl3). HRMS 8CI±
NH3) calc. for C17H26O5 8M11) 324.1811, found
324.1804. IR 8CCl4), nmax 8cm21): 3027, 3013, 2986,
2931, 1760, 1728, 1602, 1465, 1422, 1370, 1264, 1083,
Acknowledgements
L. Moreno gratefully acknowledges CONICYT, Chile
8Research Grant 2990102) for ®nancial support. We want
also to warmly thank Nicole Morin for all HRMS measure-
ments.
1
1019. H NMR 8200 MHz, CD3OD) d ppm: 0.96, 1.04,
1.26 89H, 3s, CH3-15, -14 and -13), 1.98 83H, s, CH3CO),
5.11 81H, d, J1-2ˆ5.3 Hz, H-1b), 5.68, 5.73, 5.76, 5.79, 5.81,
5.84, 5.91 82H, AB part of an ABX system, between H-2,
H-3 and H-1b, JABˆ10 Hz, JAXˆ0, and JBXˆ5.3 Hz, H-2
and H-3), 6.0 81H, br.s, H-11b). 13C NMR, see Table 3.
References
4.2.7. 1a,11a-Diacetoxy-2,3-dehydroconfertifolin 5c.
After two crystallizations from pentane±CH2Cl2, mp 142±
1. Azerad, R. In Stereoselective Biocatalysis; Patel, R., Ed.;
Marcel Dekker: New York, 2000; pp. 153±180.
2. Hollinshead, D. M.; Howell, S. C.; Ley, S. V.; Mahon, M.;
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Trans. 1 1983, 1579±1589.
20
1458C. [a]D ˆ12388 8c 0.5, CHCl3). IR 8CCl4), nmax
8cm21): 2979, 2927, 2854, 1786, 1736, 1371, 1242, 1204,
1075, 1018, 985. HRMS 8CI±NH3) calc. for C19H24O6 8M1)
348.1573, found 348.1577. H NMR 8200 MHz, CDCl3) d
1
3. Aranda, G.; Facon, I.; Lallemand, J. Y.; Leclaire, M.; Azerad,
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ppm: 0.96, 1.08, 1.18 89H, 3s, CH3-15, -14 and -13), 1.98
and 2.09 86H, 2s, COCH3), 4.85 81H, d, J1-2ˆ5.4 Hz, H-1b),
5.70, 5.75, 5.79, 5.81, 5.83, 5.86 82H, AB part of an ABX
system, between H-2, H-3 and H-1b, JABˆ9.8 Hz, JAXˆ0,
and JBXˆ5.4 Hz, H-2 and H-3), 6.97 81H, t, Jˆ0.75 Hz,
H-11b). 13C NMR, see Table 3.
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Prod. 1994, 57, 1251±1255.
4.2.8. 1a,11a-Dihydroxyconfertifolin 7a. 68 mg of 5a
were hydrogenated in the presence of PtO2 849 mg) in
EtOAc 84.5 mL) during 6.5 h. After ®ltration through Celite
and two crystallizations from EtOAc±pentane, mp 152±
6. Herlem, D.; Ouazzani, J.; Khuong-Huu, F. Tetrahedron Lett.
1996, 37, 1241±1243.
7. Aranda, G.; Bertranne-Delahaye, M.; Lallemand, J.-Y.;
Azerad, R.; Maurs, M.; Cortes, M.; Lopez, J. J. Mol. Catal:
B.Enzymatic =Special Issue: Biotrans'97) 1998, 5, 255±259.
8. Maurs, M.; Azerad, R.; Cortes, M.; Aranda, G.; Bertranne-
Delahaye, M.; Ricard, L. Phytochemistry 1999, 52, 291±296.
9. Aranda, G.; Lallemand, J.-Y.; Azerad, R.; Maurs, M.; Cortes,
M.; Ramirez, H.; Vernal, G. Synth. Commun. 1994, 24, 2525±
2535.
20
1548C. [a]D ˆ11008 8c 0.95, CH3OH). HRMS 8CI±NH3)
1
calc. for C15H23O4 8M11) 267.1596, found 267.1593. H
NMR 8500 MHz, CD3OD) d ppm: 0.95, 0.98, 1.25 89H,
3s, CH3-15, -14 and -13), 1.21 and 1.74 83H, 2m, H2-3
and H-5), 1.61 and 2.04 82H, 2m, H2-3), 1.66 81H, m,
H-6), 1.92 81H, m, H-6), 2.12 81H, m, H-7a), 2.33 81H,
dd, J7b±7aˆ18 Hz, J7b±6aˆJ7b±6bˆ5.8 Hz, H-7b), 4.05
81H, br.s, H-1b), 6.30 81H, br.s, H-11b). 13C NMR, see
Table 3.
10. Aranda, G.; Bertranne-Delahaye, M.; Azerad, R.; Maurs, M.;
Â
Cortes, M.; Ramirez, H.; Vernal, G.; Prange, T. Synth.
Commun. 1997, 27, 45±60.
11. Appel, H. H.; Connolly, J. D.; Overton, K. H.; Bond, R. P. M.
J. Chem. Soc. 1960, 4685±4692.
4.2.9. 1a-Acetoxy,11a-hydroxyconfertifolin 7b. Hydro-
genation of 5b 850 mg) in EtOAc 83.5 mL) in the presence
of PtO2 845 mg) during 5 h provided quantitatively a 3:1
mixture of 1a-acetoxy,11a-hydroxy and 1a-hydroxy,11a-
acetoxy derivatives which were not separated by TLC.
Despite repeated crystallization 8CH2Cl2±pentane), a pure
sample of the major constituent 87b) could not be obtained.
HRMS 8CI±NH3) calc. for C17H25O5 8M11) 309.1702,
found 309.1696. 1H NMR 8200 MHz, CDCl3) d ppm:
0.94, 0.98, 1.33 89H, 3s, CH3-15, -14 and -13), 2.07 83H,
s, COCH3), 5.19 81H, t, Jˆ2.8 Hz, H-1b), 5.84 81H, d, Jˆ
1.85 Hz, H-11b). 13C NMR, see Table 3.
12. Appel, H. H.; Bond, R. P. M.; Overton, K. H. Tetrahedron
1963, 19, 635.
13. Ley, S. V.; Mahon, M. J. Chem. Soc., Perkin 1 1983, 1379±
1381.
14. Fukuyama, Y.; Sato, T.; Miura, I.; Asakawa, Y. Phyto-
chemistry 1985, 24, 1521±1524.
15. 7c was crystallized by slow evaporation of a methanol solu-
tion. A crystal 0.1£0.3£0.4 mm3 in size was recorded on a
PHILIPS PW1100 diffractometer operating the CuKa
radiation. Space group P21. Parameters are aˆ8.50183);
Ê
bˆ13.97984); cˆ7.84782) A; bˆ90.2687)8 and Zˆ2. The
structure was solved by direct methods and re®ned with aniso-
tropic thermal factors 8except for hydrogens) to a ®nal Rˆ
6.9% for 1325 observed structure factors. The coordinates
have been deposited with the Cambridge Crystallographic
Data Centre 8CCDC 157588).
4.2.10. 1a,11a-Diacetoxyconfertifolin 7c. Hydrogenation
of 5c 866 mg) in EtOAc 84.5 mL) in the presence of PtO2
845 mg) during 4 h provided quantitatively 7c. After crystal-
20
lization from CH2Cl2±pentane, mp 156±1578C. [a]D
ˆ
115.88 8c 0.228, CHCl3). IR 8CCl4), nmax 8cm21): 2957,
2933, 1784, 1735, 1374, 1248, 1203, 1046, 1012, 984.
HRMS 8CI±NH3) calc. for C19H27O6 8M11) 351.1806,
16. Fattorusso, E.; Magno, S.; Santacroce, C.; Sica, D.
Tetrahedron 1972, 28, 5993±5997.
17. Lu, Q.; Faulkner, D. J. J. Nat. Prod. 1997, 60, 195±198.

6056
G. Aranda et al. / Tetrahedron 57 =2001) 6051±6056
18. Rueda, A.; Zubia, E.; Ortega, M. J.; Carballo, J. L.; Salva, J.
J. Org. Chem. 1997, 62, 1481±1485.
20. Nakano, T.; Maillo, M. A. Synth. Commun. 1981, 11, 463.
21. Hueso-Rodriguez, J. A.; Rodriguez, B. Tetrahedron 1989, 45,
1567±1576.
19. Aranda, G.; El Kortbi, M. S.; Lallemand, J.-Y.; Hammoumi,
A.; Facon, I.; Azerad, R. Tetrahedron 1991, 47, 8339±8350.