Microbial transformations of taxadienes and the multi-drug resistant tumor reversal activities of the metabolites

Article abstract of DOI:10.1016/j.tet.2012.09.091

Microbial transformation of two taxadienes (1, 2) was individually investigated with two filamentous fungi, Cunninghamella echinulata CGMCC 3.3400 and Aspergillus niger CGMCC 3.1858, and two actinomycete stains, Streptomyces griseus CACC 200300 and Nocard

Full text of DOI:10.1016/j.tet.2012.09.091

Tetrahedron 68 (2012) 9539e9549
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Microbial transformations of taxadienes and the multi-drug resistant tumor
reversal activities of the metabolites
*
Xiao Liu, Ridao Chen, Dan Xie, Mei Mei, Jianhua Zou, Xiaoguang Chen, Jungui Dai
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences,
1 Xian Nong Tan Street, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2012
Received in revised form 18 September 2012
Accepted 21 September 2012
Available online 26 September 2012
Microbial transformation of two taxadienes (1, 2) was individually investigated with two filamentous
fungi, Cunninghamella echinulata CGMCC 3.3400 and Aspergillus niger CGMCC 3.1858, and two actino-
mycete stains, Streptomyces griseus CACC 200300 and Nocardia purpurea CGMCC 4.1182. A total of 21
products were obtained, 13 of which were new compounds. The reactions that occurred exhibited di-
versity, including selective hydroxylation, epoxidation, oxidation, demethylation, acetylation, deacety-
lation, and O-alkylation, and we have proposed plausible bioconversion routes. The results of
a pharmacological evaluation showed that compound 15 displayed significant reversal of activity toward
multi-drug resistant (MDR) tumor cell line A549/taxol when co-administered with paclitaxel at 10 mM.
This investigation provided a useful approach to prepare new active taxane derivatives that were difficult
to access by chemical means.
Keywords:
Taxadiene
Microbial transformation
Tumor MDR reversal activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
oxygenated substituents (Fig.1) from cell cultures of Taxus chinensis,6
possessed potent reversal activity against several MDR tumor cells. In
Clinical treatment of cancer with chemotherapeutic drugs is fre-
quently hindered by either an intrinsic or acquired resistance of the
tumor cells. In both cases, tumors can be refractory to a variety of
antineoplastic drugs that have different structures and mechanisms of
action. This process is termed multi-drug resistance (MDR).1 Accord-
ing to statistical data, the failure of treatment in over 90% of patients
with metastatic cancer is caused by MDR.2 Although MDR can be de-
veloped by several different mechanisms, a common cause is believed
tobeoverexpressionofanMr170,000plasmamembraneglycoprotein
(P-gp), which acts as an energy-dependent drug efflux pump to lower
the intracellular concentration of cytotoxic agents by pumping them
outside of the tumor cells.3 A broad range of chemical compounds,
including verapamil, quinidine, and cyclosporine A, have been re-
ported to reverse MDR in vivo. These reversing agents competitively
inhibit the binding of antitumor drugs to P-gp in MDR tumor cells and
increase the intracellular accumulation of antitumor drugs to suffi-
cient amounts to be cytotoxic to the MDR tumor cells.4 However, these
agents have not been developed for further clinical trials due to their
unacceptable toxicities.5 Therefore, identifying novel MDR reversal
agents is an effective strategy to overcome this clinical problem.
In our previous investigation, we found that our patented natural
taxanes, sinenxan A and other 4(20),11(12)-taxadienes with C-14
an effort to find more potent derivatives, a systematic structural di-
versification and modification of these compounds was performed by
chemical and/or enzymatic approaches.7 Two derivatives, 10-oxo-
2a,5
a
,14
b
-triacetoxytaxa-4(20),11(12)-diene (1, Fig. 1) and 5
a-hy-
droxy-10
b
-methoxy-2 ,14 -diacetoxytaxa-4(20),11(12)-diene
a
b
(2,
Fig. 1) were obtained from sinenxan A by chemical synthesis (see
Experimental section 4.2). The change of an acetoxy group at C-10 to
a carbonyl group (1)oramethoxygroup(2)ledtoaremarkablypotent
MDR reversing activity toward the A549/taxol MDR cell line. There-
fore, in an attempt to obtain more potent derivatives and further in-
vestigatethestructureeactivityrelationshipofthistypeofcompound
with different substituents at C-10 position, the structural modifica-
tion of 1 and 2 by biological transformation was performed. Twenty-
one derivatives were obtained via bioconversion of 1 and 2 through
diverse reactions with two species of filamentous fungi, Cunning-
hamella echinulata CGMCC 3.3400 and Aspergillus niger CGMCC
AcO
R2
18
12
14
9
7
4
5
OAc
R1
H
H
2
OAc
Sinenxan A
OAc
1 R1=OAc, R2=O
H
H
AcO
AcO
20
2 R1=OH, R2=OMe
* Corresponding author. Tel.: þ86 10 63165195; fax: þ86 10 63017757; e-mail
address: jgdai@imm.ac.cn (J. Dai).
Fig. 1. The structures of sinenxan A, substrates 1 and 2.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.091

9540
X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
3.1858, and two actinomycete stains, Streptomyces griseus CACC
200300 and Nocardia purpurea CGMCC 4.1182. Thirteen of the prod-
ucts are new compounds, of which the structures were elucidated via
extensive spectroscopic data analysis. Furthermore, the bioassays
showed thatproduct15 exhibitedmoreactivereversalefficiencythan
verapamil toward taxol-resistant A549 tumor cells.
74.8 (d) from dC 76.0 (d) in 1. These observations suggest the pres-
ence of a hydroxy group rather than an acetoxy group at C-5. This
conclusion was further confirmed by the loss of the acetyl group
signals at [dH 2.16 (s, eCOCH3), dC 169.6 (s, eCOCH3)] and the ob-
servation of IR absorption at 3478 cmꢀ1 for a hydroxyl group. Thus, 3
was elucidated as
5a-hydroxy-10-oxo-2a,14b-diacetoxytaxa-
4(20),11(12)-diene, which is a selectively deacetylated product of 1.
Compound 4 was obtained as a white amorphous powder. The
positive HR-ESI-MSspectrum of 4 showed a quasi-molecular ionpeak
at m/z 457.2215 [MþNa]þ (calcd 457.2203 for C24H34O7Na), which is
consistent with the molecular formula C24H34O7. The molecular
weight of compound 4 was 16 amu higher than that of 3, which
suggested the introduction of one hydroxyl group. The 1H NMR
spectroscopic data of 4 were similar to those of 3, except that the
signalsofH2-6dH 1.63(m)disappeared, andanadditionaloxymethine
signal at dH 3.67 (m,1H) was observed. These data suggested that the
hydroxyl group may be introduced at the C-6 position. This deduction
was further supported by the 13C NMR spectrum of 4, which had an
oxygenated carbonresonanceatdC 70.3(d) inplaceof theC-6 signal at
dC 31.0 (t) in 3. The stereochemistry of the introduced hydroxyl group
2. Results and discussion
In this investigation, 14 species of filamentous fungi distributed
in 10 genera (Absidia, Alternaria, Aspergillus, Botrytis, Cunning-
hamella, Fusarium, Mucor, Penicillium, Rhizopus, and Gibberella) and
4 species of actinomycetes (Nocardia and Streptomyces genera)
were employed as biocatalysts for the biotransformation of 1 and 2.
After analysis by TLC and HPLCeUV, four strains, C. echinulata
CGMCC 3.3400, A. niger CGMCC 3.1858, S. griseus CACC 200300, and
N. purpurea CGMCC 4.1182, were selected for the further scale-up
biotransformations.
2.1. Biotransformations of 1
was determined by NOE difference spectra to be in the
tion; the integration values of H-5, H-7 , and H-19 were enhanced
when H-6 was irradiated. Therefore, the structure of 4 was de-
termined to be ,6 -dihydroxy-10-oxo-2 ,14 -diacetoxytaxa-
4(20),11(12)-diene, a hydroxylated derivative of 3.
a-configura-
b
2.1.1. Biotransformation of 1 by C. echinulata CGMCC 3.3400.
Following a standard two-stage fermentation protocol,8 four new
metabolites (3e6, Fig. 2) were obtained after incubation of 1 with
cultures of C. echinulata for 7 days. The compounds were purified by
silica gel chromatography and semi-preparative HPLC. The
5a
a
a
b
Compound 5 was obtained as a white amorphous powder. The
molecular formula of C26H37O8 was established by positive HR-
ESI-MS for the observation of a quasi-molecular ion peak at m/z
477.2460 [MþH]þ (calcd 477.2489 for C26H37O8). The 1H NMR
spectrum of 5 was similar to that of 4, except that the signal of H-6
at dH 3.67 (m) was down-shifted to dH 4.78 (m). Moreover, the
carbon resonance of C-6 in the 13C NMR spectrum of 5 shifted
downfield to dC 72.4 (d) from dC 70.3 (d) in 4, suggesting the
compounds were identified as 5
ytaxa-4(20),11(12)-diene (3, w52.1%),
,14 -diacetoxytaxa-4(20),11(12)-diene (4, w1.8%), 5
10-oxo-2 ,6 ,14 -triacetoxytaxa-4(20),11(12)-diene (5, w2.2%), and
-hydroxy-10-oxo-2 ,5 ,14 -triacetoxytaxa-4(20),11(12)-diene (6,
a
-hydroxy-10-oxo-2
,6 -dihydroxy-10-oxo-
-hydroxy-
a,14b-diacetox-
5
a a
2a
b
a
a
a
b
7b
a
a
b
w6.6%). A plausible biotransformation pathway for all four metabo-
lites (Fig. 2) was proposed starting from the initial structure 1.
O
OH
O
9
OAc
OH
H
H
14
HO
OAc
OAc
H
H
AcO
AcO
AcO
9
6
4
8
hydroxylation
deacetylation
O
B
C
O
O
OH
7
OH
7
hydroxylation
deacetylation
OAc
OAc
OH
5
A/B
B
H
H
H
OAc
OAc
OAc
H
1
H
H
7
AcO
AcO
AcO
deacetylation
A
O
O
O
OH
OH
OAc
OH
6
6
hydroxylation
acetylation
5
OH
5
A
A
H
H
H
OAc
OAc
OAc
H
H
H
5
AcO
3
Fig. 2. The plausible bioconversion route for metabolites from 1 (A: by Cunninghamella echinulata CGMCC 3.3400; B: by Streptomyces griseus CACC 200300; C: by Nocardia purpurea
CGMCC 4.1182).
Compound 3 was a white amorphous powder. Its molecular
formula, C24H34O6, was established by positive HR-ESI-MS; the
spectrum contained a quasi-molecular ion peak at m/z 441.2237
[MþNa]þ (calcd 441.2253 for C24H34O6Na). The 1H NMR spectro-
scopic data of 3 were similar to those of 1, except that the signal of H-
presence of an acetoxy group rather than a hydroxy group at C-6.
This conclusion was further confirmed by the appearance of ad-
ditional signals for an acetoxy group at [dH 2.05 (s, eCOCH3); dC
169.8 (s, eCOCH3) and 21.2 (q, eCOCH3)] in the spectrum of 5. The
stereochemistry of 6-OAc was determined to be in the
uration by an NOE difference spectral experiment, in which the
integration values of H-5, H-7 , and H-19 were enhanced when H-
a-config-
5 (dH 5.31, br s) shifted upfield to dH 4.20 (br s). Moreover, in the 13
C
NMR spectrum of 3, the carbon resonance of C-5 shifted upfield to dC
b

X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
9541
6 was irradiated. Therefore, compound 5 was elucidated as 5
a
-
determined to be
4(20),11(12)-diene.
9
a
-hydroxy-10-oxo-2
a
,5a,14b
-triacetoxytaxa-
hydroxy-10-oxo-2 ,6 ,14 -triacetoxytaxa-4(20),11(12)-diene,
a
a
b
which may be converted from 4 via subsequently enzymatic
acetylation.
2.1.3. Biotransformation of
1 by N. purpurea CGMCC 4.1182.
The positive HR-ESI-MS spectrum of 6 had a quasi-molecular ion
peak at m/z 477.2515 [MþH]þ (calcd 477.2489 for C26H37O8), which
is consistent with the molecular formula of C26H36O8. Its molecular
weight was 16 amu more than that of 1, suggesting the introduction
of a hydroxy group, which was further supported by an IR ab-
sorption band at 3427 cmꢀ1. The 1H NMR spectroscopic data of 6
were similar to those of 1, except that the signals of H2-7 [dH 1.74
(m, 1H), 1.25 (m, 1H)] had disappeared, while a new oxymethine
proton signal at dH 3.54 (dd, 11.5, 5.5) was observed. These data
suggest that the OH group may be introduced at C-7 position. This
hypothesis was further supported by the observation that the
chemical shift of C-7 of 6 was shifted downfield to dC 70.9 (d) from
dC 36.3 (t) in 1. The stereochemistry of 7-OH was determined to be
Compound 1 was efficiently bioconverted to one product (9, Fig. 2)
by N. purpurea after 7 days of incubation. The structure of this new
compound was determined to be 5a,14b-dihydroxy-10-oxo-2a-ace-
toxytaxa-4(20),11(12)-diene (9, in ca. 81.4% yield), which was formed
through deacetylation of 1 at the C-5 and C-14 positions.
Compound 9 was obtained as a white amorphous powder. Its
molecular formula, C22H32O5, was established by positive HR-ESI-
MS (m/z 377.2333 [MþH]þ, calcd 377.2329 for C22H33O5). The 1H
NMR data of 9 were very similar to those of 1, except that the
chemical shifts of H-5 dH 5.31 (br s) and H-14 dH 5.17 (dd, 9.2, 4.8) of
1 shifted upfield to dH 4.20 (br s) and dH 4.28 (dd, 9.0, 4.5), re-
spectively. Moreover, in the 13C NMR spectrum of 9, the carbon
resonances of C-5 and C-14 shifted upfield to dC 74.7 (d) and dC 67.9
(d) from dC 76.0 (d) and dC 70.1 (d) in 1, respectively. These chemical
shifts indicated the presence of hydroxy groups rather than acetoxy
groups at positions C-5 and C-14. This conclusion was further
confirmed by the loss of two acetyl signals at [dH 2.16 (s, eCOCH3),
dC 169.6 (s, eCOCH3)] and [dH 2.04 (s, eCOCH3), dC 170.2 (s,
eCOCH3)] and an IR absorption band at 3478 cmꢀ1. Therefore,
deacetylation occurred at both the C-5 and C-14 positions of 1 to
in the
tegration value of H-3 was enhanced when H-7 was irradiated.
Therefore, the structure of 6 was determined to be 7 -hydroxy-10-
oxo-2 ,5 ,14 -triacetoxytaxa-4(20),11(12)-diene, which is a hy-
b-configuration by the NOE experiment, in which the in-
b
a
a
b
droxylated derivative of 1.
2.1.2. Biotransformation of 1 by S. griseus CACC 200300. After in-
cubation of 1 with cell cultures of S. griseus for 7 days, three new
metabolites (6e8) were obtained by a combination of silica gel
chromatography and semi-preparative HPLC (Fig. 2). Their struc-
form 9, and the structure of 9 was determined to be 5
droxy-10-oxo-2 -acetoxytaxa-4(20),11(12)-diene.
a,14b-dihy-
a
tures were identified as 7
ytaxa-4(20),11(12)-diene (6, w29.0%),
,14 -diacetoxytaxa-4(20),11(12)-diene (7, w0.8%), and 9
droxy-10-oxo-2 ,5 ,14 -triacetoxytaxa-4(20),11(12)-diene
w0.8%). Compound 6 was also metabolite of 1 by C. echinulata. The
hypothetical biotransformation route is illustrated in Fig. 2.
The molecular formula of 7 (C24H34O7) was established by
positive HR-ESI-MS (m/z 435.2388 [MþH]þ, calcd 435.2384 for
C24H35O7), which is 42 amu lower than that of 6. The 1H NMR
spectroscopic data of 7 were similar to those of 6, except that
the signal of H-5 (dH 5.30, t) was shifted upfield to dH 4.25 (t).
Moreover, in the 13C NMR spectrum of 7, the carbon resonance
of C-5 was shifted upfield to dC 74.9 (d) from dC 75.7 (d) in 6.
The above observations suggest the presence of a hydroxy group
instead of an acetoxy group at C-5. This conclusion was further
b
-hydroxy-10-oxo-2
a
,5
a
,14
b
-triacetox-
2.2. Biotransformations of 2
5
a,7b-dihydroxy-10-oxo-
2
a
b
a
-hy-
2.2.1. Biotransformation of 2 by C. echinulata CGMCC 3.3400.
Compound 2 was efficiently converted to eight products (10e14,
16e18, Fig. 3) after incubationwith 2-day-old cultures of C. echinulata
a
a
b
(8,
for 7 days. The compounds were identified as 5
methoxy-2 ,14 -diacetoxytaxa-4(20),11(12)-diene (10, w0.7%),
,9 ,10 -trihydroxy-2 ,14 -diacetoxytaxa-4(20),11(12)-diene (11,
w3.1%), 5 ,10 -dihydroxy-2 ,14
-diacetoxytaxa-4(20),11(12)-diene9
(12, w56.8%), 10 -hydroxy-2 ,5 14 -triacetoxytaxa-4(20),11(12)-
diene9 (13, w0.7%),
,10 -dihydroxy-5 ,14 -diacetoxytaxa-
4(20),11(12)-diene7b (14, w1.9%),
,18-dihydroxy-10 -methoxy-
,14 -diacetoxytaxa-4(20),11(12)-diene (16, w0.8%), ,10 ,18-
trihydroxy-2 ,14 -diacetoxytaxa-4(20),11(12)-diene (17, w1.3%),
and 5 -hydroxy-2 ,10 ,14
-triacetoxytaxa-4(20),11(12)-diene9 (18,
a,9a-dihydroxy-10b-
a
b
5a
a
b
a
b
a
b
a
b
a
b
b
a
b
2a
a
b
5a
b
2a
b
5a
b
a
b
a
a
b
b
confirmed by the absence of acetoxy signals
[
dH 2.16 (s,
w1.0%). Among these products, 10, 11, 16, and 17 were new com-
pounds. Based upon the comparison of the structures of the substrate
and metabolites, a plausible biotransformation pathway was pro-
posed in Fig. 3.
eCOCH3); dC 169.4 (s, eCOCH3), 21.5 (q, eCOCH3)] in the spectra
of 6. Accordingly, it was clear that deacetylation occurred at the
C-5 position of 6 to form 7, and the structure of 7 was de-
termined to be
4(20),11(12)-diene.
5
a
,7
b
-dihydroxy-10-oxo-2
a
,14
b
-diacetoxytaxa-
The positive HR-ESI-MS spectrum of 10 displayed a quasi-
molecular ion peak at m/z 473.2517 [MþNa]þ, which was consis-
tent with the molecular formula C25H38O7 (calcd 473.2516 for
C25H38O7Na). Its molecular weight was 16 amu higher than that of
2, suggesting the introduction of one hydroxyl group. The 1H NMR
data of 10 were similar to those of 2, except for the disappearance of
the methylene signals that corresponded to H-9 (dH 2.23, dd, 14.7,
11.7; dH 1.60, m) and the appearance of an oxygen-bearing methine
signal at dH 4.02 (d, 9.2). The 13C NMR spectrum had a signal for an
oxygenated carbon resonance at dC 76.9 (d) in place of C-9 signal at
dC 45.1 (t) for compound 2. These observations indicate the in-
troduction of a hydroxy group at the C-9 position. This deduction
was further supported by the downfield shift of the H3-19 signal of
2 at dH 0.80 (s) to dH 0.98 (s) in 10 and the shift of the C-8/C-10 signal
The molecular formula of 8, C26H36O8, was established by pos-
itive HR-ESI-MS (m/z 499.2332 [MþNa]þ, calcd 499.2308 for
C26H36O8Na) to be 16 amu higher than that of 1, suggesting the
introduction of one hydroxy group. These data are supported by an
IR absorption band at 3449 cmꢀ1. The 1H NMR data of 8 were
similar to those of 1, except for the disappearance of the methylene
signals corresponding to H-9 (dH 2.34, d, 15.2; dH 2.84, d, 15.2) and
the appearance of an oxygen-bearing methine signal at dH 4.48 (s).
The 13C NMR spectrum had an oxygenated carbon resonance at dC
81.0 (d) in place of the C-9 signal at dC 58.1 (t) in 1. These obser-
vations indicate that a hydroxy group was introduced at the C-9
position. This deduction was further supported by a downfield
chemical shift of the H3-19 signal of 1 at dH 0.96 (s) to dH 1.10 (s) in 8,
and the chemical shift of the C-8/C-10 signal of 1 at dC 40.9 (s)/204.1
of 2 at dC 39.6 (s)/76.1 (d) to dC 44.2 (s)/81.4 (d) in 10. The a-con-
figuration of the 9-OH group was determined by an NOE difference
spectrum, in which the integration values of H-2 and H3-19 were
enhanced when H-9 was irradiated. Therefore, the structure of 10
(s) to dC 47.1 (s)/210.0 (s) in 8. The a-configuration of the 9-OH
group was determined by an NOE difference spectrum experi-
ment, in which the integration values of H-2 and H3-19 were en-
was determined to be
5a,9a-dihydroxy-10b-methoxy-2a,14b-
hanced when H-9 was irradiated. The structure of
8 was
diacetoxytaxa-4(20),11(12)-diene.

9542
X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
MeO
OH
H
MeO
MeO
OH
HO
OH
18
demethylation
9
18
10
hydroxylation
hydroxylation
OH
OH
OH
OH
A
A
A
H
H
H
OAc
OAc
OAc
OAc
H
H
H
H
17
AcO
AcO
AcO
AcO
AcO
AcO
16
10
2
demethylation
OH
A
demethylation
A/D
HO
HO
HO
HO
10
10
hydroxylation
acetylation
deacetylation
OH
H
OH
OH
OAc
OAc
OAc
5
A/D
A/D
A
H
H
H
2
OAc
H
OAc
OAc
OH
H
H
H
14
AcO
AcO
AcO
11
12
13
3'
HO
acetylation
acetylation
acetylation
A/D
4'
D
D
2'
1'
D
O-alkylation
O
AcO
AcO
AcO
10
10
10
10
OH
OAc
H
H
H
H
OAc
OAc
OAc
OH
15
H
H
H
H
AcO
AcO
AcO
19
18
Sinenxan A
Fig. 3. The plausible bioconversion route for metabolites from 2 (A: by Cunninghamella echinulata CGMCC 3.3400; D: by Aspergillus niger CGMCC 3.1858).
The molecular formula of 11, C24H36O7, was established by positive
a combination of open silica gel chromatography and semi-
HR-ESI-MS ([MþNa]þ 459.2317 (calcd 459.2359 for C24H36O7Na)).
The 1H NMR spectrum of 11 was very similar to that of 10, except that
the signal for H-10 shifted downfield from dH 4.39 (d, 9.2) for 10 to dH
4.84 (d, 9.0) for 11. Moreover, in the 13C NMR spectrum of 11, the
carbonresonanceofC-10wasshiftedupfieldto dC 72.2 (d) from dC 81.4
(d) for 10, which suggested the presence of a hydroxy group rather
than a methoxy group at C-10. This conclusionwas further confirmed
by the disappearance of methoxygroup signals [dH 3.33 (s, eOCH3), dC
preparative HPLC purification (Fig. 3). The compounds were iden-
tified as 5
ene (12, w54.5%), 10
11(12)-diene (13, w0.8%), 2
4(20),11(12)-diene (14, w3.8%), 2
ytaxa-4(20),11(12)-diene (15, w1.4%), 5
acetoxytaxa-4(20),11(12)-diene (18, w7.2%), 5
diacetoxytaxa-4(20),11(12)-diene-10 -O-(butan-2-ol)-ether
w1.8%), and ,5 ,10 ,14 -tetraacetoxytaxa-4(20),11(12)-diene
a
,10
b
-dihydroxy-2
-hydroxy-2
,10 -dihydroxy-5
-hydroxy-5
-hydroxy-2
-hydroxy-2
a
,14
b
-diacetoxytaxa-4(20),11(12)-di-
,5 14 -triacetoxytaxa-4(20),
,14 -diacetoxytaxa-
,10 ,14 -triacetox-
,10 ,14 -tri-
,14
(19,
b
a
a
b
a
b
a
a
b
b
a
b
a
a
b
b
a
a
b-
b
58.9 (q, eOCH3)]. Thus, 11 was elucidated as 5
,14 -diacetoxytaxa-4(20),11(12)-diene.
The positive HR-ESI-MS spectrum of 16 displayed a quasi-
a,9a,10b-trihydroxy-
2a
a
b
b
2a
b
(sinenxan A, w3.13%). Compounds 12e14, and 18 were also me-
tabolites of 2 by C. echinulata. Compound 15 was previously re-
ported, and the spectroscopic data were in agreement with the
reference.10 Compound 19 was new compound. Based upon
a comparison of the structures of the substrate and metabolites,
a plausible biotransformation pathway was proposed (Fig. 3).
Compound 19 was obtained as a white amorphous powder. Its
molecular formula, C28H44O7, was established by positive HR-ESI-
MS, which showed a quasi-molecular ion peak at m/z 515.2947
[MþNa]þ (calcd 515.2985 for C28H44O7Na). The 1H NMR spectrum
was very similar to that of 12, except that a series of additional
protons [dH 3.42 (dq, 3.0, 6.3, H1-10), dH 3.90 (dq, 3.0, 6.3, H1-20), dH
1.12 (d, 6.3, H3-30), dH 1.10 (d, 6.3, H3-40)] were observed. Signals
for carbons connected to the new proton signals were also ob-
served in the 13C NMR spectrum. Thus, a butan-2,3-ol moiety was
deduced to be attached at the C-10 position through an ether
molecular ion peak at m/z 473.2482 [MþNa]þ (calcd 473.2516 for
C25H38O7Na) that was consistent with the molecular formula
C25H38O7. The 16 amu increase of molecular mass indicated the
presence of an additional hydroxy group in the molecule. The 1H
NMR spectrum of 16 was very similar to that of 2, except that the
signal of H3-18 at dH 1.99 (3H, s) in 2 was absent, while an additional
coupling of the oxygenated methylene proton signals at dH 4.71 (d,
12.6 Hz) and 4.00 (d, 12.6 Hz) was observed, suggesting hydroxyl-
ation at C-18. In addition, the signal of C-18 of 16 shifted downfield to
dC 77.2 (t) from dC 24.9 (q) in 2. Thus, 16 was identified as 5
dihydroxy-10 -methoxy-2 ,14 -diacetoxytaxa-4(20),11(12)-diene,
which may be biosynthesized from 2 by hydroxylation at C-18.
a,18-
b
a
b
Compound 17 was obtained as a white amorphous powder. Its
molecular formula, C24H36O7, was established by positive HR-ESI-MS,
which showed a quasi-molecular ion peak at m/z 459.2320 [MþNa]þ
(calcd 459.2359 for C24H36O7Na). The 1H NMR spectroscopic data of
17 were similar to those of 16, except that the signal of H-10 shifted
downfield from dH 4.72 (dd,11.4, 5.4) for 16 to dH 5.22 (dd,11.4, 5.4) for
17. Moreover, in the 13C NMR spectrum of 17, the carbon resonance of
C-10 shifted upfield to dC 70.6 (d) from dC 75.3 (d) for 16, which sug-
gested the presence of a hydroxy group rather than a methoxy group
at C-10. This conclusion was further confirmed by the loss of the
methoxygroupsignalat[dH 3.25(s, eOCH3), dC 59.3(q, eOCH3)]inthe
bond. According to the above evidence, 19 was identified as 5
hydroxy-2 ,14 -diacetoxytaxa-4(20),11(12)-diene-10 -O-(butan-
2-ol)-ether.
a-
a
b
b
2.2.3. Biotransformation of 2 by N. purpurea CGMCC 4.1182. After
incubation with cell cultures of N. purpurea for 7 days, four metab-
olites (13, 20e22) were obtained from 2 by a combination of open
silica gel chromatography and semi-preparative HPLC purification
(Fig. 4). The compounds were identified as 10
,5 ,14 -triacetoxytaxa-4(20),11(12)-diene (13, w30.5%), 10
methoxy-2 ,5 ,14 -triacetoxytaxa-4(20),11(12)-diene (20, w5.5%),
10 -methoxy-2 ,5 ,14 -trihydroxytaxa-4(20),11(12)-diene (21,
w22.6%), and 5 ,10 ,14 -trihydroxy-2 -acetoxytaxa-4(20),11(12)-
b-hydroxy-
spectrum of 16. Thus, 17 was identified as 5a,10b,18-trihydroxy-
2a
a
b
b-
2a
,14 -diacetoxytaxa-4(20),11(12)-diene, which might be bio-
b
a
a
b
synthesized from 16 by a subsequent demethylation at C-10 position.
b
a
a
b
a
b
b
a
2.2.2. Biotransformation of 2 by A. niger CGMCC 3.1858. After in-
cubation of 2 with cell cultures of the fungus A. niger for 7 days,
seven metabolites (12e15, 18, 19 and sinenxan A) were obtained by
diene (22, 7.46%). Of these compounds, 13, 20, and 22 were known,
and the spectroscopic data were in good agreement with the re-
ported data.7b,12 Compound 13 was also metabolite of 2 by C.

X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
9543
HO
10
MeO
O
10
oxidation
demethylation
B
OH
OH
OH
B
H
H
H
OAc
OAc
OAc
H
H
H
3
AcO
AcO
AcO
12
2
acetylation
acetylation
C
B
HO
MeO
AcO
10
10
demethylation
acetylation
OAc
OAc
OAc
OAc
5
5
C
B
H
H
H
OAc
OAc
OAc
Sinenxan A
H
H
H
AcO
AcO
AcO
13
20
deacetylation
deacetylation
epoxidation
C
C
B
MeO
HO
AcO
demethylation
acetylation
4
OH
OH
5
5
H
H
H
O
14
HO
14
HO
OH
OAc
H
21
H
22
OAc
H
23
20
AcO
Fig. 4. The plausible bioconversion route for metabolites from 2 (B: by Streptomyces griseus CACC 200300; C: by Nocardia purpurea CGMCC 4.1182).
echinulata, and 21 was a new compound. Based upon the compari-
son of the structures of the substrate and metabolites, a plausible
biotransformation pathway was proposed (Fig. 4).
obtained (3,12,13, 23, and sinenxan A, Fig. 4). The spectroscopic data
of these four metabolites were in accordance with those of known
compounds, 5
diene (12, w4.5%), 10
11(12)-diene (13, w11.3%),
a
,10
b
-dihydroxy-2
a
,14
a
b
-diacetoxytaxa-4(20),11(12)-
,5 ,14 -triacetoxytaxa-4(20),
,5 ,10 ,14 -tetraacetoxytaxa-
4b,20-epoxy-11(12)-ene (23, w1.4%), and 2a,5a,10b,14b-tetraace-
The positive HR-ESI-MS spectrum of 21 displayed a quasi-
molecular ion peak at m/z 351.2537 [MþH]þ (calcd, 351.2536 for
C21H35O4), which was consistent with the molecular formula
C21H34O4. The 1H NMR spectroscopic data of 21 were very similar to
those of 2, except that the signals for H-2 at dH 5.33 (dd, 6.0,1.8) and H-
14 at dH 5.04(dd, 9.3, 4.5) shifted upfield to dH 4.10 (brd, 6.0)and dH 4.01
(dd, 9.0, 4.5), respectively. Moreover, in the 13C NMR spectrum of 21,
the carbon resonance of C-2 shifted upfield to dC 69.7 (d) from dC 71.1
(d) for 2, and the carbon resonance of C-14 shifted upfield to dC 67.7 (d)
from dC 70.8 (d) for 2, which suggested the presence of hydroxy groups
rather than acetoxy groups at C-2 and C-14. This conclusion was fur-
ther confirmed by the absence of acetoxy signals [dH 2.03, 2.04 (s,
eCOCH3), dC 169.9, 170.3 (s, eCOCH3)] that were present in the spec-
b-hydroxy-2
a
a
b
b
2a
b
toxytaxa-4(20),11(12)-diene (sinenxan A, 28.7%).7b,9a,11 Compounds
12, 13, and sinenxan A were also metabolites of 2 by A. niger. The
new compound, 5a-hydroxy-10-oxo-2a,14b-diacetoxytaxa-4(20),
11(12)-diene (3, w6.9%), was formed through oxidation of 2 at the C-
10 position and was also metabolite of 1 by C. echinulata. Based upon
the comparison of the structures of the substrate and metabolites,
a plausible biotransformation pathway was proposed (Fig. 4).
2.3. The MDR reversal potency of compounds 1e23 against
taxol-resistant A549 tumor cells in vitro
trum of 2. Therefore, 21 was identified as 10b-methoxy-2a,5a,14b-
trihydroxytaxa-4(20),11(12)-diene, which might be biosynthesized
from 2 by deacetylation at both the C-2 and C-14 positions.
The multi-drug resistant A549/taxol tumor cell line was estab-
lished by culturing the cells with gradually increasing concentrations
of paclitaxel. For these experiments, the potency of compounds 1e23
in the reversal of the drug resistance of A549/taxol was examined at
2.2.4. Biotransformation of 2 by S. griseus CACC 200300. After 7 days
of incubation of 2 with S. griseus and separation by silica gel chro-
matography and semi-preparative HPLC, five metabolites were
10
mM. Verapamil was used as the positive control. The effects of the
tested compounds are summarized in Table 5. Several of the
Table 1
1H NMR spectral data of compounds 1 and 3e9 in CDCl3
a
No.
1b
3
4
5
6
7
8
9
1
2
3
5
6
7
7
9
9
13
13
14
16
2.04 (br s)
5.44 (d, 6.4)
3.04 (d, 6.4)
5.31 (br s)
1.63 (m, 2H)
1.25 (m)
2.00 (d, 2.0)
2.01 (d, 2.0)
2.01 (br s)
2.07 (br s)
2.01 (br s)
2.10 (br s)
5.53 (dd, 6.4, 2.0) 5.49 (dd, 6.0, 2.1)
2.86 (d, 6.4)
5.32 (br s)
1.75 (m, 2H)
1.90 (d, 2.1)
5.43 (dd, 6.0, 2.0) 5.44 (dd, 6.5, 2.0) 5.45 (dd, 6.5, 2.5) 5.49 (dd, 6.5, 2.5)
5.10 (dd, 6.0, 2.0)
3.14 (d, 6.0)
4.25 (t)
3.29 (d, 6.0)
4.20 (br s)
1.63 (m, 2H)
1.16 (m)
3.24 (d, 6.5)
4.17 (d, 4.0)
3.67 (m)
1.25 (m)
1.80 (m)
2.85 (d, 16.0)
2.28 (d, 16.0)
2.40 (dd, 19.0,
4.5)
2.91 (dd, 19.0,
9.0)
5.17 (dd, 9.0,
4.5)
1.46 (s)
3.31 (d, 6.5)
4.28 (d, 3.5)
4.78 (m)
1.25 (m)
1.70 (m)
2.93 (d, 15.5)
2.25 (d, 15.5)
2.95 (dd, 19.0,
9.0)
2.44 (dd, 19.0,
4.0)
5.20 (dd, 9.0,
4.0)
1.46 (s)
2.87 (d, 6.5)
5.30 (t)
1.71 (m, 2H)
3.23 (d, 6.0)
4.20 (br s)
1.71 (m, 2H)
1.18 (m)
2.01 (m, 2H)
a
b
a
b
3.54 (dd, 11.5, 5.5) 3.65 (dd, 11.0, 5.0) 1.29 (m)
1.70 (m)
2.85 (d, 16.5)
2.02 (m)
2.91 (dd, 19.0,
9.0)
2.47 (dd, 19.0,
4.5)
5.17 (dd, 9.0,
4.5)
1.74 (m)
1.65 (m)
1.74 (m)
2.34 (d, 15.2)
2.84 (d, 15.2)
2.45 (dd, 18.8,
4.8)
2.93 (dd, 18.8,
9.2)
5.17 (dd, 9.2,
4.8)
1.46 (s)
2.85 (d, 15.6)
2.28 (d, 15.6)
2.40 (dd, 19.2,
4.4)
2.90 (dd, 19.2,
8.8)
5.20 (dd, 8.8,
4.4)
1.46 (s)
2.79 (d, 16.5)
2.02 (m)
2.89 (dd, 19.0,
9.5)
2.42 (dd, 19.0,
4.5)
5.15 (dd, 9.5,
4.5)
1.52 (s)
2.85 (d, 15.6)
2.28 (d, 15.6)
2.78 (dd, 19.0,
9.0)
2.48 (dd, 19.0,
4.5)
4.28 (dd, 9.0,
4.5)
1.45 (s)
4.48 (s)
2.98 (dd, 19.2,
8.8)
2.52 (dd, 19.2,
4.4)
5.09 (dd, 8.8,
4.4)
a
b
1.46 (s)
1.48 (s)
(continued on next page)

9544
X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
Table 1 (continued )
No.
1b
3
4
5
6
7
8
9
17
1.18 (s)
1.16 (s)
1.18 (s)
1.18 (s)
1.16 (s)
1.17 (s)
1.21 (s)
1.21 (s)
18
1.91 (s)
1.92 (s)
1.91 (s)
1.91 (s)
1.91 (s)
1.92 (s)
1.97 (s)
1.91 (s)
19
0.96 (s)
0.90 (s)
0.96 (s)
0.96 (s)
0.90 (s)
0.80 (s)
1.10 (s)
0.90 (s)
20a
20b
5.28 (br s)
4.87 (br s)
5.16 (br s)
4.80 (br s)
5.28 (br s)
4.87 (br s)
2.16 (s),
2.06 (s)
5.33 (br s)
4.96 (br s)
2.09 (s), 2.06 (s),
2.05 (s)
5.28 (br s)
4.80 (br s)
2.16 (s), 2.06 (s),
2.03 (s)
5.18 (br s)
4.88 (br s)
2.08 (s),
2.04 (s)
5.36 (br s)
4.93 (br s)
2.17 (s), 2.09 (s),
2.04 (s)
5.16 (br s)
4.78 (br s)
2.09 (s)
eOAc 2.16 (s), 2.06 (s), 2.06 (s),
2.04 (s) 2.03 (s)
a
1H NMR data were recorded at 300 MHz for 9, at 400 MHz for 1, 3, and 8, and at 500 MHz for 4e7, respectively. Peaks were assigned by analyses of the 1D- and 2D-NMR
spectra.
b
Multiplicities and coupling constants (J) in hertz are in parentheses.
Table 2
13C NMR data for compounds 1 and 3e9 in CDCl3
a
No.
1
3
4
5
6
7
8
9
1
2
3
4
5
6
7
57.4 d
70.3 d
43.9 d
144.1 s
76.0 d
57.4 d
70.5 d
41.4 d
147.3 s
74.8 d
31.0 t
56.9 d
69.6 d
39.4 d
144.0 d
74.8 d
70.3 d
31.0 t
56.6 d
70.0 d
39.9 d
144.0 s
76.2 d
51.4 d
70.2 d
42.0 d
144.8 s
75.7 d
51.0 d
70.0 d
39.5 d
145.7 s
74.9 d
57.1 d
69.7 d
43.9 d
148.0 s
78.5 d
28.5 t
57.9 d
71.0 d
41.3 d
143.8 s
74.7 d
30.4 t
30.5 t
36.3 t
72.4 d
31.1 t
35.6 t
70.9 d
35.5 t
70.2 d
30.7 t
26.9 t
35.5 t
8
9
40.9 s
58.1 t
204.1 s
142.4 s
135.4 s
38.6 t
70.1 d
35.6 s
26.2 q
28.6 q
21.6 q
22.1 q
115.8 t
21.3, 21.4,
21.6 q; 169.6,
169.9, 170.2 s
41.5 s
57.8 t
204.1 s
143.7 s
136.5 s
38.5 t
70.4 d
35.5 s
26.2 q
30.7 q
21.4 q
21.7 q
113.2 t
43.9 s
57.1 t
39.6 s
57.0 t
202.6 s
142.6 s
137.2 s
39.1 t
70.2 d
38.6 s
26.3 q
31.1 q
21.5 q
22.3 q
117.9 t
21.1, 21.2,
21.4 q; 169.8,
2ꢁ170.1 s
46.0 s
57.5 t
203.6 s
140.8 s
136.4 s
38.9 t
69.6 d
36.8 s
26.5 q
30.7 q
21.5 q
21.6 q
117.1 t
21.3, 21.4,
21.5 q; 169.4,
169.9, 170.1 s
46.5 s
57.5 t
203.4 s
144.4 s
137.5 s
39.0 t
69.9 d
38.9 s
26.5 q
31.0 q
21.4 q
15.5 q
114.5 t
21.3, 21.4 q;
169.9, 170.0 s
47.1 s
41.9 s
61.6 t
204.4 s
147.7 s
137.2 s
41.3 t
81.0 d
210.0 s
141.2 s
140.0 s
39.5 t
10
11
12
13
14
15
16
17
18
19
20
eOAc
202.9 s
140.0 s
136.8 s
38.6 t
70.1 d
35.3 s
26.2 q
29.4 q
21.4 q
24.5 q
116.8 t
21.3, 21.4 q;
2ꢁ170.1 s
70.3 d
35.5 s
67.9 d
35.9 t
26.6 q
31.2 q
21.8 q
17.1 q
118.1 t
3ꢁ21.3 q;
3ꢁ170.0 s
26.4 q
31.4 q
21.7 q
21.6 q
112.9 t
21.5 q;
169.8 s
21.3, 21.4 q;
2ꢁ170.1 s
a
13C NMR data were recorded at 100 MHz for 1, 8, and 9, at 125 MHz for 3e6, and at 150 MHz for 7, respectively. Peaks were assigned by analyses of the 1D- and 2D-NMR
spectra.
Table 3
1H NMR spectral data of compounds 2, 10, 11, 16, 17, 19, and 21 in CDCl3
a
No.
2b
10
11
16
17
19c
21
1
2
1.83 (br s)
5.33 (dd, 6.0, 1.8)
1.81 (br s)
5.34 (d, 6.0)
1.81 (br s)
5.35 (d, 6.0)
1.90 (d, 2.1)
5.37 (dd, 6.3,
2.1)
1.89 (br s)
5.37 (dd, 6.0,
2.0)
1.82 (br s)
5.34 (d, 6.0)
1.86 (d, br s)
4.10 (d, 6.0)
3
5
6
3.21 (d, 6.0)
4.18 (br s)
1.71 (m, 2H)
1.26 (m)
1.70 (m)
2.23 (dd, 14.7,
11.7)
3.21 (d, 6.0)
4.20 (br s)
1.71 (m, 2H)
1.26 (m)
1.98 (m)
4.02 (d, 9.2)
3.22 (d, 6.0)
4.19 (br s)
1.75 (m, 2H)
1.23 (m)
1.65 (m)
4.05 (d, 9.0)
3.14 (d, 6.3)
4.18 (br s)
1.75 (m, 2H)
1.01 (m)
2.17 (m)
2.28 (dd, 15.0,
11.4)
3.13 (d, 6.0)
4.15 (br s)
1.70 (m, 2H)
1.09 (m)
2.13 (m)
2.24 (m)
3.19 (d, 6.0)
4.18 (br s)
1.73 (m, 2H)
1.25 (m)
2.01 (m)
2.10 (dd, 15.0,
12.0)
3.07 (d, 6.0)
4.19 (br s)
1.71 (m, 2H)
1.07 (m)
2.14 (m)
2.18 (dd, 15.0,
11.4)
7a
7b
9a
9
b
1.60 (m)
4.65 (dd, 11.7,
5.4)
2.77 (dd, 19.2,
9.3)
2.37 (dd, 19.2,
4.5)
5.04 (dd, 9.3,
4.5)
1.61 (m)
4.72 (dd, 11.4,
5.4)
3.36 (dd, 19.2,
9.0)
2.27 (dd, 19.2,
4.5)
1.61 (dd, 15.0, 5.4)
5.22 (dd, 11.4,
5.4)
3.28 (dd, 19.2,
9.3)
1.59 (dd, 15.0, 4.5)
4.82 (dd, 12.0,
4.5)
2.76 (dd, 18.9,
9.0)
2.34 (dd, 18.9,
4.5)
5.03 (dd, 9.0,
4.5)
1.59 (dd, 15.0, 4.8)
4.62 (dd, 11.4,
4.8)
2.66 (dd, 17.4,
9.0)
2.48 (dd, 17.4,
4.5)
4.01 (dd, 9.0,
4.5)
10
13
13
14
4.39 (d, 9.2)
4.84 (d, 9.0)
a
b
2.80 (dd, 19.6,
9.2)
2.39 (dd, 19.6,
4.4)
5.01 (dd, 9.2,
4.4)
2.78 (dd, 18.9,
9.0)
2.36 (dd, 18.9,
4.8)
5.01 (dd, 9.0,
4.8)
2.37 (m)
5.09 (dd, 9.0,
4.5)
5.07 (dd, 9.3,
4.8)
16
17
18
1.62 (s)
1.17 (s)
1.99 (s)
1.26 (s)
1.17 (s)
1.56 (s)
1.64 (s)
1.18 (s)
1.99 (s)
1.63 (s)
1.19 (s)
4.71 (d, 12.6), 4.00
(d, 12.6)
1.75 (s)
1.20 (s)
4.61 (d, 12.6), 4.04
(d, 12.6)
1.71 (s)
1.10 (s)
1.98 (s)
1.54 (s)
1.24 (s)
1.99 (s)
19
0.80 (s)
0.98 (s)
1.00 (s)
0.82 (s)
0.82 (s)
0.79 (s)
0.83 (s)

X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
9545
Table 3 (continued )
No.
2b
10
11
16
17
19c
21
20a
20b
eOCH3
eOAc
5.09 (br s)
4.78 (br s)
3.28 (s)
5.13 (br s)
4.84 (br s)
3.33 (s)
5.13 (br s)
4.82 (br s)
5.12 (br s)
4.78 (br s)
3.25 (s)
5.11 (br s)
4.77 (br s)
5.09 (br s)
4.77 (br s)
5.18 (br s)
3.27 (s)
2.03, 2.04 (s)
2.03, 2.04 (s)
2.03, 2.05 (s)
2.04, 2.05 (s)
2.03, 2.04 (s)
2.02, 2.03 (s)
a
1H NMR data were recorded at 300 MHz for 2, 11, 16, 17 19 and 21, and at 400 MHz for 10, respectively. Peaks were assigned by analyses of the 1D- and 2D-NMR spectra.
Multiplicities and coupling constants (J) in Hz are in parentheses.
Data for protons of 19: dH 3.42 (dq, 3.0, 6.3, H-10), dH 3.90 (dq, 3.0, 6.3, H-20), dH 1.12 (d, 6.3, H-30), dH 1.10 (d, 6.3, H-40).
b
c
Table 4
13C NMR data for compounds 2, 10, 11, 16, 17, 19, and 21 in CDCl3
a
No.
2
10
11
16
17
19b
21
1
2
3
4
5
6
7
8
55.2 d
71.1 d
40.1 d
136.2 s
76.4 d
30.8 t
33.2 t
39.6 s
45.1 t
76.1 d
147.9 s
135.6 s
39.9 t
70.8 d
37.4 s
22.3 q
31.6 q
24.9 q
20.9 q
113.4 t
59.1 q
2ꢁ21.5 q;
169.9 s, 170.3 s
55.4 d
70.7 d
41.9 d
147.4 s
77.1 d
30.5 t
59.0 d
70.7 d
42.0 d
147.5 s
72.2 d
30.4 t
32.0 t
44.7 s
78.6 d
72.2 d
135.6 s
135.6 s
39.4 t
70.5 d
37.5 s
21.2 q
25.2 q
26.0 q
17.4 q
114.1 t
55.5 d
70.4 d
39.5 d
146.7 s
70.9 d
30.6 t
32.3 t
37.9 s
44.5 t
75.3d
141.0 s
138.6 s
44.3 t
61.5 d
34.1 s
25.2 q
31.2 q
77.2 t
22.5 q
113.8 t
59.3 q
2ꢁ21.5 q,
2ꢁ170.1 s
59.3 d
66.6 d
39.6 d
146.8 s
70.9 d
30.7 t
32.4 t
37.9 s
46.2 t
70.6 d
142.5 s
136.2 s
40.0 t
61.7 d
34.1 s
25.5 q
31.7 q
76.4 t
22.4 q
113.7 t
59.1 d
71.0 d
40.0 d
147.8 s
76.4 d
30.8 t
31.7 t
39.9 s
45.7 t
72.6 d
136.9 s
134.4 s
39.4 t
70.8 d
37.4 s
25.1 q
33.3 q
20.9 q
22.3 q
113.5 t
55.1 d
69.7 d
41.9 d
149.8 s
76.1 d
31.6 t
33.5 t
40.2 s
45.4 t
76.0 d
135.9 s
135.7 s
42.3 t
67.7 d
37.6 s
22.6 q
31.8 q
25.5 q
21.1 q
113.8 t
66.4 q
31.5 t
44.2 s
9
76.9 d
81.4 d
138.0 s
134.1 s
39.8 t
70.5 d
37.4 s
21.1 q
25.3 q
25.7 q
17.2 q
114.1 t
58.9 q
10
11
12
13
14
15
16
17
18
19
20
eOCH3
eOAc
2ꢁ21.5 q;
2ꢁ21.5 q;
2ꢁ21.5 q;
2ꢁ21.5 q;
2ꢁ169.9 s
169.9 s, 170.3 s
169.9 s, 170.3 s
169.9 s, 170.1 s
a
13C NMR data were recorded at 125 MHz for 2, and at 100 MHz for 10, 11, 16, 17, 19, and 21, respectively. Peaks were assigned by analyses of the 1D- and 2D-NMR spectra.
b
Data for carbon of 19: dC (75.7 d, C-10), dC (67.8 d, C-20), dC (17.6 q, C-30), dC (14.2 q, C-40).
Table 5
Reversal activities of 1e23 against MDR A549/taxol cellsa
Samples
IC50 of paclitaxelb (nM)
RFc (A549/T)
Samples
IC50 of paclitaxelb (nM)
RFc (A549/T)
Paclitaxel (blank control)
Verapamil (positive control)
1
2
3
4
5
6
7
8
9
10
11
26.0
5.9
d
12
13
14
15
16
17
18
19
20
21
22
23
23.6
13.0
17.3
2.5
17.3
15.3
13.7
13.0
11.3
12.4
11.8
6.0
1.1
2.0
1.5
10.6
1.5
1.7
1.9
2.0
2.3
2.1
2.2
4.3
4.4
1.8
2.0
1.6
1.0
1.3
0.7
2.8
1.8
1.9
0.8
1.5
14.4
13.0
16.4
26.0
19.4
36.2
9.3
14.4
13.7
32.5
17.3
a
All the samples at 10
m
M were co-administered with paclitaxel, and paclitaxel concentrations were designated to 10ꢀ10, 10ꢀ9, 10ꢀ8, 10ꢀ7, 10ꢀ6, and 10ꢀ5 M. Paclitaxel
M verapamil with paclitaxel was used as positive control.
without reversal agent was used as blank control, and 10
m
b
A549/taxol, an MDR subline of human lung adenocarcinoma cell line A549.
RF (reversal fold)¼IC50 (antitumor agent)/IC50 (reversal agentþantitumor agent).
c
metabolites exhibited higher reversal activities than the substrates 1
and 2. Compound 15 had approximately two-fold higher activity than
verapamil and five times higher activity than substrate 2. Compound
23hadcomparableactivitytoverapamilandtwotimeshigheractivity
than2. All of the metabolites exhibited weak or nocytotoxicity, which
is a good property for a reversing agent.13 Thus, the results showed
that compounds 15 and 23 might be promising lead compounds for
reversal agents against A549/taxol tumor MDR cells.
3. Conclusion
In summary, we report the successful structural diversification of
two 4(20),11(12)-taxadienes (1 and 2) by microbial transformation.
Seven transformation systems were established in two fungal and
two actinomycete stains. In total, 21 derivatives were obtained,13 of
which were new compounds. Plausible bioconversion routes were
predicted, and the reactions exhibited diversity, including selective

9546
X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
hydroxylation, epoxidation, oxidation, demethylation, acetylation,
deacetylation, and O-alkylation. Most of these transformations are
not easily accessible by a chemical approach. Moreover, bioassays
showed that product 15 exhibited more active reversal efficiency
than its parent substrateand the positive control (verapamil) toward
taxol-resistant A549 tumor cells. These results suggest that bio-
transformation is a powerful approach to the structural di-
versification of bioactive natural products, perhaps even providing
bioactive derivatives with increased potency.
eluent to give 1 (600 mg, 89.3%). The same procedure was repeated
additional three times, and totally obtain ca. 2.4 g of 1.
4.2.1.1. 10-Oxo-2
a a,14b-triacetoxytaxa-4(20),11(12)-diene
,5
(1). White powder; [a20
]D þ3.6 (c 0.11, CH3OH); IR (nmax): 3001, 2955,
2936, 1738, 1679, 1436, 1372, 1241, 1103, 1030, 978, 928 cmꢀ1 1H
;
and 13C NMR data see Tables 1 and 2; HR-ESI-MS (positive) m/z:
[MþNa]þ 483.2314 (calcd 483.2359 for C26H36O7Na).
4.2.2. Preparation of
5a-hydroxy-10b-methoxy-2a,14b-diacetox-
ytaxa-4(20),11(12)-diene (2). To a solution of sinenxan A (5.0 g,
10 mmol) in methanol (150 mL) was added 10 M aqueous KOH
(14 mL). The reaction mixture was stirred at ꢀ10 ꢂC for 20 min. The
reaction was quenched with 1 M aqueous HCl, and the resulting
mixture was concentrated and extracted with EtOAc (300 mLꢁ3).
The organic layer was washed with saturated aqueous NaHCO3,
washed with brine, dried over anhydrous Na2SO4, and concentrated
in vacuo. The residue was purified by flash chromatography to give
4. Experimental section
4.1. General experimental procedures
Optical rotations in CH3OH were recorded on a PerkineElmer
Model-343 digital polarimeter. IR spectra were obtained on
a Thermo Nicolet 5700 FT-IR microscope spectrometer. HR-ESI-MS
were measured on a Q-trap ESI mass spectrometer (Applied Bio-
systems/MDS Sciex, Carlsbad, CA, USA). NMR spectra were recorded
on Mercury-300, Mercury-400, and Bruker-500 spectrometers us-
2
a a,10b,14b-tetrahydroxytaxa-4(20),11-diene (2.85 g, 85%). Ac2O
,5
(3 ml, 30 mmol) was added to a stirred pyridine (50 mL) solution of
the compound ,5 ,10 ,14 -tetrahydroxytaxa-4(20),11-diene
2a
a
b
b
ing the solvent (CDCl3, 1H
d
7.26, 13
C
d
77.0) as an internal reference.
(2.85 g, 8.5 mmol). After the mixture was stirred at room temper-
ature for 16 h, methanol (5 mL) was added to quench the reaction.
The resulting mixture was neutralized with 1 M aqueous HCl and
extracted with EtOAc (200 mLꢁ3). The organic layer was succes-
sively washed with 1 M aqueous HCl (100 mL), saturated aqueous
NaHCO3 (100 mL), and brine (100 mL) before it was dried over
anhydrous Na2SO4, concentrated, and purified by column chro-
matography (petroleum ether/EtOAc¼5:1, v/v as elution) to afford 2
(1.1 g, in a total yield of 30%) as a white powder. The same pro-
cedure was repeated an additional time, and totally obtained ca.
2.2 g of 2.
The chemical shifts ( ) are given in parts per million (ppm), and the
d
coupling constants (J) are given in hertz (Hz). Analytical HPLC was
performed on an Agilent series 1200 HPLC instrument equipped
with a quaternary pump, a diode-array detector (DAD), an auto-
sampler, a column compartment and a GraceSmart C18 column
(250 mmꢁ4.6 mm, i.d., 5
mm). The mobile phase consisted of ul-
trapure water and acetonitrile (CH3CN) with a two-pump gradient
program at the flow rate of 1.0 mL/min at 30 ꢂC. Semi-preparative
HPLC was performed on a Shiseido HPLC instrument equipped
with a YRU-883A RI/UV detector and a Grace Allsphere silica col-
umn (250 mmꢁ10 mm, i.d., 5
m
m) by elution with mixtures of n-
Grace Adsorbosphere C18 column
m) by eluting with the mixtures of
hexane and acetone or
a
4.2.2.1. 5
a
-Hydroxy-10
b
-methoxy-2
a,14b-diacetoxytaxa-
(250 mmꢁ10 mm, i.d., 5
m
4(20),11(12)-diene (2). White powder; [a20
]D þ34.4 (c 0.125, CH3OH);
CH3OH and H2O. Silica gel (200e300 mesh, 300e400 mesh, Qing-
dao Haiyang Chemical Co. Ltd., Qingdao, PR China) and Sephadex
LH-20 gel (Pharmacia Fine Chemical Co., Ltd., Sweden) were used
for column chromatography (CC). Analytical TLC was performed
using pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical
Co. Ltd., Qingdao, PR China). The visualization of TLC plates was
performed by illuminating at UV 254 nm, followed by spraying
with 5% H2SO4 in EtOH and heating at 105 ꢂC. All solvents used for
CC were of analytical grade (Beijing Chemical Works., Beijing, PR
China), and the solvents used for HPLC were HPLC grade (Mal-
linckrodt Baker Co. Ltd., Phillipsburg, USA).
IR (nmax): 3432, 3020, 2924, 2816, 1733, 1639, 1434, 1369, 1249,
1090, 1019 cmꢀ1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-
MS (positive) m/z: [MþNa]þ 457.2566 (calcd 457.2568 for
C25H38O6Na).
4.3. Screening experiment
Fourteen species of filamentous fungi that were distributed in
10 genera (Absidia, Alternaria, Aspergillus, Botrytis, Cunninghamella,
Fusarium, Mucor, Penicillium, Rhizopus, and Gibberella) and four
species of actinomycetes (Nocardia and Streptomyces genera) were
employed as biocatalysts for the biotransformations. Analytical
scale fermentation was performed using 250 mL flasks containing
70 mL of culture medium. After 2 days of cultivation, 2 mg of the
substrate was added. The culture controls consisted of fermenta-
tion blanks, in which the organisms were grown under identical
conditions without addition of the substrate. After one week of
incubation, the cultures were filtered, and the filtrate was extracted
with EtOAc. The solvent was removed under vacuum, and the
resulting residue was dissolved in CH3OH before analysis via TLC
and HPLC.
4.2. Substrate preparation
Sinenxan A [2
a,5a,10b,14b-tetraacetoxytaxa-4(20),11(12)-diene]
was isolated from callus cultures (Ts-19 strain) of T. chinensis.6
4.2.1. Preparation of 10-oxo-2a,5a,14b-triacetoxytaxa-4(20),11(12)-
diene (1). 10-Deacetyl sinenxan A was prepared as reported by
Yin.14 To a solution of 675 mg (1.46 mmol) of 10-deacetyl sinenxan
A in 15 mL of CH2Cl2 was added 855.8 mg (7.30 mmol) of N-
methylmorpholine N-oxide (NMO). After the mixture was stirred
for 10 min, 50.0 mg (0.15 mmol) of tetrapropylammonium per-
ruthenate (TPAP) was added, and the mixture was allowed to stir
for 10 h at 26 ꢂC. The reaction mixture was filtered and then con-
centrated in vacuo. The residue was diluted with 20 mL of ddH2O
before extraction with EtOAc (60 mLꢁ3). The pooled EtOAc extract
was dried over anhydrous Na2SO4 and concentrated in vacuo at
40 ꢂC to afford 1.5 g of residue, which was subjected to silica gel CC.
A petroleum ether/acetone mixture (12:1, v/v) was used as the
4.4. Organisms, media, and cultivation conditions
Two fungal (C. echinulata CGMCC 3.3400, A. niger CGMCC
3.1858) and two actinomycete stains (S. griseus CACC 200300, N.
purpurea CGMCC 4.1182), which were purchased from the China
General Microorganism Collection Center (Beijing, for code CGMCC)
and the China Center for Antibiotics Culture Collection (Beijing, for
code CACC), were screened for preparative scale bio-
transformations. The two fungal strains C. echinulata CGMCC

X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
9547
3.3400 and A. niger CGMCC 3.1858 were kept on PDA medium,
containing 200 g of potato, 20 g of dextrose, 3.0 g of KH2PO4, 0.75 g
of MgSO4, and micro amounts of VB1 in 1 L of distilled water. The
culture medium for the actinomycete strains S. griseus CACC
200300 and N. purpurea CGMCC 4.1182 contained 20 g of dextrose,
5 g of yeast extract, 5 g of soy peptone, 5 g of NaCl, and 1.0 g of
K2HPO4$3H2O in 1 L of distilled water. The pH of the media was
adjusted to 6.0/7.0 before autoclaving at 121 ꢂC for 25 min. Micro-
bial cultures were grown according to the standard two-stage fer-
mentation protocol. The seed cultures were prepared in 250 mL
flasks with 70 mL of liquid medium and incubated for 2 days. The
larger biotransformation cultures (1000 mL flask containing
200 mL of medium) were inoculated with 2 mL of the seed culture
and cultivated on a rotary shaker at 200 rpm at 28ꢃ1 ꢂC in the dark.
among 36 flasks at a final concentration of 56 mg/L after 2 days of
cultivation. The cultures were pooled and centrifuged after in-
cubation for an additional 7 days. The supernatant was saturated
with NaCl and extracted with EtOAc (10 Lꢁ3). The pooled EtOAc
extracts were dried over anhydrous Na2SO4 and concentrated un-
der reduced pressure at 40 ꢂC. The dried cell mass was extracted
three times in an ultrasonic bath with 300 mL EtOAc, and the
resulting extract was concentrated under vacuum at 40 ꢂC. The two
extracts were combined to afford 1.5 g of residue, which was sub-
jected to silica gel CC. The fractions were further separated by
a
combination of normal phase semi-preparative HPLC and
reverse-phase semi-preparative HPLC to afford 6 (120.0 mg, ca.
29.0%), 7 (3.1 mg, ca. 0.8%; tR 19.80 min; mobile phase: n-hexane/
acetone¼2:1, v/v at 4 mL/min), and 8 (3.4 mg, ca. 0.8%; tR 21.87 min;
mobile phase: n-hexane/acetone¼7:1, v/v at 4 mL/min). There was
20.0 mg (5.0%) of recovered substrate (1). Compound 6 was also
a metabolite of 1 by C. echinulata.
4.5. Biotransformations of 1
4.5.1. Biotransformation of 1 by C. echinulata CGMCC 3.3400. After 2
days of cultivation, compound 1 was added to the cultures for a fi-
nal concentration of 60 mg/L (stock solution of 300 mg of 1 in
12.5 mL ethanol). The cultures were pooled and filtered after 7 days
of incubation. The supernatant was saturated with NaCl and
extracted with EtOAc (7 Lꢁ3). The pooled EtOAc extract was dried
over anhydrous Na2SO4 and concentrated under reduced pressure
at 40 ꢂC. The dried mycelia were extracted three times in an ul-
trasonic bath with 300 mL EtOAc, and the resulting extract was
concentrated under vacuum at 40 ꢂC. The two extracts were com-
bined to afford 2.1 g of residue, which was subjected to silica gel CC.
The fractions were further separated by a combination of normal
phase semi-preparative HPLC and reverse-phase semi-preparative
HPLC to afford 3 (142.1 mg, ca. 52.1%; tR 26.05 min; mobile phase:
n-hexane/EtOAc¼3:1, v/v at 4 mL/min), 4 (5.0 mg, ca. 1.8%; tR
44.85 min; mobile phase: CH3OH/H2O¼65:35, v/v at 3.5 mL/min), 5
(6.8 mg, ca. 2.2%; tR 13.83 min; mobile phase: CH3OH/H2O¼70:30,
v/v at 3.5 mL/min), and 6 (20.6 mg, ca. 6.6%; tR 21.40 min; mobile
phase: n-hexane/acetone¼5:1, v/v at 4 mL/min). There was 12.0 mg
(3.0%) of recovered substrate (1). All of the products were new
compounds. Their physical and chemical data are as follows:
4.5.2.1. 5
a
,7
b
-Dihydroxy-10-oxo-2
a,14b-diacetoxytaxa-
4(20),11(12)-diene (7). White powder; [a20
(
]
D þ6.0 (c 0.05, CH3OH); IR
nmax): 3439, 2932, 2897, 1733, 1668, 1440, 1373, 1249, 1100,
1042 cmꢀ1
;
1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS
(positive) m/z: [MþH]þ 435.2388 (calcd 435.2384 for C24H35O7).
4.5.2.2. 9
4(20),11(12)-diene (8). White powder; [a20
IR (nmax): 3449, 2949, 2854, 1737, 1675, 1435, 1372, 1252, 1104,
a
-Hydroxy-10-oxo-2
a
,5
a,14b-triacetoxytaxa-
]D ꢀ33.3 (c 0.045, CH3OH);
1028 cmꢀ1 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS
;
(positive) m/z: [MþNa]þ 499.2332 (calcd 499.2308 for C26H36O8Na).
4.5.3. Biotransformation of 1 by N. purpurea CGMCC 4.1182. Four
hundred milligrams of 1 in 18 mL ethanol was evenly distributed
among 35 flasks at a final concentration of 57 mg/L after 2 days of
cultivation. The cultures were pooled and centrifuged after an ad-
ditional 7 days of incubation. The supernatant was saturated with
NaCl and extracted with EtOAc (10 Lꢁ3). The dried mycelia were
extracted three times in an ultrasonic bath with 300 mL of EtOAc.
All the extracts were pooled, dried over anhydrous Na2SO4, and
concentrated under vacuum at 40 ꢂC to afford 2.2 g of residue,
which was subjected to silica gel CC. A petroleum ether/acetone
gradient (100:0 to 0:100, v/v) was used as the eluent to give 19
fractions based on the TLC analysis. Fraction 10 (210.2 mg) and
fraction 11 (98.6 mg) were further separated via normal phase
semi-preparative HPLC using a mobile phase of n-hexane/EtOAc
(1:1, v/v) at 4 mL/min to yield 9 (266.1 mg, ca. 81.4%; tR 16.34 min).
There was 6.0 mg (1.5%) of recovered substrate (1).
4.5.1.1. 5
a
-Hydroxy-10-oxo-2
a,14b-diacetoxytaxa-4(20),11(12)-
diene (3). White powder; [a20
]
þ16.0 (c 0.225, CH3OH); IR (nmax):
D
3478, 3012, 2963, 2914, 1717, 1677, 1437, 1375, 1258, 1124, 1063,
1032 cmꢀ1 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS
;
(positive) m/z: [MþNa]þ 441.2237 (calcd 441.2253 for C24H34O6Na).
4.5.1.2. 5
a
,6
a
-Dihydroxy-10-oxo-2
a
,14
b-diacetoxytaxa-
4(20),11(12)-diene (4). White powder; [a20
]
D
þ34.3 (c 0.07, CH3OH);
IR (nmax): 3436, 2928, 2858, 1807, 1736, 1679, 1434, 1373, 1247, 1180,
4.5.3.1. 5
a
,14
b
-Dihydroxy-10-oxo-2
a-acetoxytaxa-4(20),11(12)-
1033 cmꢀ1
;
1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS
diene (9). White powder; [a20
]
D
þ0.45 (c 0.22, CH3OH); IR (nmax):
(positive) m/z: [MþNa]þ 457.2215 (calcd 457.2203 for C24H34O7Na).
3420, 2994, 2924, 2860, 1734, 1663, 1616, 1436, 1377, 1236, 1084,
1016 cmꢀ1 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS
;
4.5.1.3. 5
a
-Hydroxy-10-oxo-2
a
,6
]
a
,14
b
-triacetoxytaxa-4(20),11
(positive) m/z: [MþH]þ 377.2333 (calcd 377.2329 for C22H33O5).
(12)-diene (5). White powder; [a20
(
ꢀ7.3 (c 0.165, CH3OH); IR
D
nmax): 3448, 3012, 2923, 2854, 1746, 1710, 1681, 1445, 1378, 1274,
4.6. Biotransformations of 2
1245, 1175, 1097, 1035 cmꢀ1
;
1H and 13C NMR data see Tables 1
and 2; HR-ESI-MS (positive) m/z: [MþH]þ 477.2460 (calcd
4.6.1. Biotransformation of 2 by C. echinulata CGMCC 3.3400. Four
hundred milligrams of 2 in 18 mL acetone was evenly distributed
among 36 flasks at a final concentration of 56 mg/L after 2 days of
cultivation. The cultures were pooled and centrifuged after 7 days
of additional incubation. The supernatant was saturated with NaCl
and extracted with EtOAc (10 Lꢁ3). The dried mycelia were
extracted three times in an ultrasonic bath with 300 mL EtOAc. All
the extracts were pooled, dried over anhydrous Na2SO4, and con-
centrated under vacuum at 40 ꢂC to afford 3.1 g of residue, which
was subjected to silica gel CC. The fractions were further separated
by a combination of normal phase semi-preparative HPLC and
477.2489 for C26H37O8).
4.5.1.4. 7
b
-Hydroxy-10-oxo-2
a
,5
]
a
,14
b-triacetoxytaxa-
4(20),11(12)-diene (6). White powder; [a20
þ20.6 (c 0.16, CH3OH);
D
IR (nmax): 3427, 3019, 2941, 2936, 1738, 1691, 1436, 1374, 1236, 1092,
1019 cmꢀ1 1H and 13C NMR data see Tables 1 and 2; HR-ESI-MS
;
(positive) m/z: [MþH]þ 477.2515 (calcd 477.2489 for C26H37O8).
4.5.2. Biotransformation of 1 by S. griseus CACC 200300. Four hun-
dred milligrams of 1 in 18 mL of ethanol was evenly distributed

9548
X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
reverse-phase semi-preparative HPLC to afford 10 (3.1 mg, ca. 0.7%;
tR 16.32 min; mobile phase: n-hexane/acetone¼8:1, v/v at 4 mL/
min), 11 (12.6 mg, ca. 3.1%; tR 25.81 min; mobile phase: n-hexane/
acetone¼5:1, v/v at 4 mL/min), 12 (220.0 mg, ca. 56.8%; tR
25.86 min; mobile phase: CH3OH/H2O¼55:45, v/v at 3.5 mL/min),
13 (3.0 mg, ca. 0.7%; tR 16.79 min; mobile phase: n-hexane/
acetone¼8:1, v/v at 4 mL/min), 14 (7.2 mg, ca. 1.9%; tR 28.21 min;
mobile phase: n-hexane/acetone¼6:1, v/v at 4 mL/min), 16 (3.4 mg,
ca. 0.8%; tR 30.77 min; mobile phase: n-hexane/acetone¼5:1, v/v at
4 mL/min),17 (5.3 mg, ca.1.3%; tR 48.56 min; mobile phase: CH3OH/
H2O¼47:53, v/v at 2.5 mL/min), and 18 (4.1 mg, ca. 1.0%; tR
21.61 min; mobile phase: n-hexane/acetone¼7:1, v/v at 4 mL/min).
There was 16.0 mg (4.0%) of the recovered substrate (2). Among
these products, 10, 11, 16, and 17 were new compounds, and their
physical and chemical data are detailed as follows:
1103, 1026 cmꢀ1; 1H and 13C NMR data see Tables 3 and 4; HR-ESI-
MS (positive) m/z: [MþNa]þ 515.2947 (calcd 515.2985 for
C28H44O7Na).
4.6.3. Biotransformation of 2 by N. purpurea CGMCC 4.1182. Four
hundred milligrams of 2 in 18 mL ethanol was evenly distributed
among 35 flasks at a final concentration of 57 mg/L after 2 days of
cultivation. The cultures were pooled and centrifuged after 7 days
of additional incubation. The supernatant was saturated with NaCl
and extracted with EtOAc (10 Lꢁ3). The dried mycelia were
extracted three times in an ultrasonic bath with 300 mL of EtOAc.
All the extracts were pooled, dried over anhydrous Na2SO4, and
concentrated under vacuum at 40 ꢂC to afford 2.4 g of residue,
which was subjected to silica gel CC. The fractions were further
separated by a combination of normal phase semi-preparative
HPLC and reverse-phase semi-preparative HPLC to afford 13
(130.0 mg, ca. 30.5%), 20 (24.0 mg, ca. 5.5%; tR 14.98 min; mobile
phase: n-hexane/acetone¼15:1, v/v at 4 mL/min), 21 (73.0 mg, ca.
22.6%; tR 18.60 min; mobile phase: n-hexane/acetone¼5:1, v/v at
4 mL/min), and 22 (26.0 mg, ca. 7.46%; tR 21.00 min; mobile phase:
n-hexane/acetone¼5:1, v/v at 4 mL/min). There was 15.0 mg (3.8%)
of recovered substrate (2). Compound 13 was also a metabolite of 2
by C. echinulata. Compound 21 is a new compound, and its physical
and chemical data are shown as follows:
4.6.1.1. 5
a
,9
a
-Dihydroxy-10
b
-methoxy-2
a
,14
b
-diacetoxytaxa-
4(20),11(12)-diene (10). White powder;
[
a20
]
þ25.1 (c 0.215,
D
CH3OH); IR (nmax): 3524, 3014, 2962, 2922, 1734, 1711, 1640, 1450,
1369, 1265, 1106, 1018 cmꢀ1; 1H and 13C NMR data see Tables 3 and
4; HR-ESI-MS (positive) m/z: [MþNa]þ 473.2517 (calcd 473.2516 for
C25H38O7Na).
4.6.1.2. 5
a,9a,10b-Trihydroxy-2a,14b-diacetoxytaxa-4(20),11(12)-
diene (11). White powder; [a20
]
þ42.0 (c 0.2, CH3OH); IR (nmax):
D
3451, 2982, 2936, 1733, 1641, 1436, 1372, 1238, 1107, 1026 cmꢀ1; 1H
and 13C NMR data see Tables 3 and 4; HR-ESI-MS (positive) m/z:
[MþNa]þ 459.2317 (calcd 459.2359 for C24H36O7Na).
4.6.3.1. 10
ene (21). White solid; [
b-Methoxy-2a,5a,14b-trihydroxytaxa-4(20),11(12)-di-
a]
20 þ55.0 (c 0.12, CH3OH); IR (nmax): 3391,
D
3014, 2979, 2921, 1635, 1445, 1381, 1309, 1199, 1072, 1011 cmꢀ1; 1H
and 13C NMR data see Tables 3 and 4; HR-ESI-MS (positive) m/z:
[MþH]þ 351.2537 (calcd 351.2536 for C21H35O4).
4.6.1.3. 5
4(20),11(12)-diene (16). White powder; [a20
IR (nmax): 3420, 3021, 2984, 2927, 1733, 1644, 1435, 1371, 1250, 1091,
a
,18-Dihydroxy-10
b
-methoxy-2
a,14b-diacetoxytaxa-
]
D þ5.0 (c 0.08, CH3OH);
4.6.4. Biotransformation of 2 by S. griseus CACC 200300. Three
hundred milligrams of 2 in 12.5 mL acetone was evenly distributed
among 25 flasks at a final concentration of 60 mg/L after 2 days of
cultivation. The cultures were pooled and centrifuged after 7 days of
additional incubation. The supernatant was saturated with NaCl and
extracted with EtOAc (10 Lꢁ3). The pooled EtOAc extract was dried
over anhydrous Na2SO4 and concentrated in reduced pressure at
40 ꢂC. The dried mycelia were extracted three times in an ultrasonic
bath with 300 mL of EtOAc, and the resulting extract was concen-
trated under vacuum at 40 ꢂC. The two extracts were combined to
afford 2.2 g of residue, which was subjected to silica gel CC. The
resulting fractions were further separated by a combination of
normal phase semi-preparative HPLC and reverse-phase semi-pre-
parative HPLC to afford 3 (20.0 mg, ca. 6.9%),12 (13.0 mg, ca. 4.5%),13
(36.0 mg, ca.11.3%), sinenxan A (100.0 mg, ca. 28.7%), and 23(5.0 mg,
ca. 1.4%; tR 22.31 min; mobile phase: CH3OH/H2O¼70:30, v/v at
3.5 mL/min). There was 8.0 mg (2.7%) of recovered substrate (2).
Compound 3 was also metabolite of 1 by C. echinulata, and 12,13, and
sinenxan A were also metabolites of 2 by A. niger.
1020 cmꢀ1 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS
;
(positive) m/z: [MþNa]þ 473.2482 (calcd 473.2516 for C25H38O7Na).
4.6.1.4. 5
diene (17). White powder; [
3412, 3020, 2985, 2926, 1733, 1647, 1434, 1372, 1252, 1102,
a,10
b
,18-Trihydroxy-2
a
,14
b-diacetoxytaxa-4(20),11(12)-
20
a]
D
þ5.0 (c 0.08, CH3OH); IR (nmax):
1028 cmꢀ1 1H and 13C NMR data see Tables 3 and 4; HR-ESI-MS
;
(positive) m/z: [MþNa]þ 459.2320 (calcd 459.2359 for C24H36O7Na).
4.6.2. Biotransformation of 2 by A. niger CGMCC 3.1858. Five hun-
dred milligrams of 2 in 20 mL acetone was evenly distributed
among 40 flasks at a final concentration of 62.5 mg/L after 2 days of
cultivation. The cultures were pooled and filtered after 7 days of
additional incubation. The supernatant was saturated with NaCl
and extracted with EtOAc (10 Lꢁ3). The dried mycelia were
extracted three times in an ultrasonic bath with 300 mL EtOAc. All
the extracts were pooled, dried over anhydrous Na2SO4, and con-
centrated under vacuum at 40 ꢂC to afford 3.6 g of residue, which
was subjected to silica gel CC. The fractions were further separated
by a combination of normal phase semi-preparative HPLC and re-
verse-phase semi-preparative HPLC to give 12 (263.5 mg, ca.
54.5%), 13 (4.2 mg, ca. 0.8%), 14 (18.5 mg, ca. 3.8%), 15 (7.2 mg, ca.
1.4%; tR 11.58 min; mobile phase: CH3OH/H2O¼72:28, v/v at 3.5 mL/
min), 18 (38.3 mg, ca. 7.2%), 19 (10.1 mg, ca. 1.8%; tR 11.58 min;
mobile phase: CH3OH/H2O¼72:28, v/v at 3.5 mL/min), and sinen-
xan A (18.2 mg, ca. 3.13%; tR 24.21 min; mobile phase: n-hexane/
acetone¼18:1, v/v at 4 mL/min). There was 10.0 mg (2.0%) of re-
covered substrate (1). Compounds 12e14 and 18 were also me-
tabolites of 2 by C. echinulata. Compound 19 is new compound, and
its physical and chemical data are shown as follows:
4.7. Evaluation of MDR reversal activities for metabolites
in vitro
The human non-small cell lung adenocarcinoma cell line A549
was maintained in the Department of Pharmacology, at the In-
stitute of Materia Medica, Chinese Academy of Medical Sciences &
Peking Union Medical College. The drug resistant subline of A549/
taxol was established by culturing the cells with gradually in-
creasing concentrations of paclitaxel, and it was characterized as
a phenotype with P-gp overexpression.7a The MDR tumor cells
were incubated in the medium RPMI 1640 supplemented with 10%
fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL of
4.6.2.1. 5
b
a
-Hydroxy-2
a
,14
b
-diacetoxytaxa-4(20),11(12)-diene-
þ17.3 (c 0.075,
streptomycin at 37 ꢂC in a humidified atmosphere of 5% CO2 at-
mosphere. Cells were subcultured twice every week by digesting
with mixture of 0.025% trypsin and 0.01% EDTA solution. The
10
-O-(butan-2-ol)-ether (19). White powder; [a20
]
D
CH3OH); IR (nmax): 3451, 2980, 2928, 1734, 1643, 1446, 1372, 1250,

X. Liu et al. / Tetrahedron 68 (2012) 9539e9549
9549
procedures were performed as described previously.15 Reversing
References and notes
agents (10
m
M) combined with paclitaxel (1ꢁ10ꢀ9, 1ꢁ10ꢀ8, 1ꢁ10ꢀ7
,
and 1ꢁ10ꢀ6 M) were dissolved in 100
mL of DMSO, and a final DMSO
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (81102469), the Science & Technology Project
of Guangdong Province (2011A080403020), the Program for New
Century Excellent Talents in University (NCET-06-0155), and the
National Science & Technology Major Project ‘Key New Drug Cre-
ation and Manufacturing’, China (2009ZX09103-033).
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Supplementary data
Selected NMR, MS, and IR spectra of substrates (1e11) and new
compounds (3e11, 16, 17, 19, and 21). Supplementary data associ-
ated with this article can be found in the online version, at http://
dx.doi.org/10.1016/j.tet.2012.09.091.