A furantaxane with an unusual 6/8/6/5 ring system and potent tumor MDR reversal activity obtained via microbial transformation

Article abstract of DOI:10.1021/ol301755n

A furantaxane (4) with an unusual 6/8/6/5 ring system and two hydroxylated products (2, 3) were isolated following the biotransformation of a taxane (1) by Streptomyces griseus. The structures of the isolates were elucidated by spectroscopic analysis. The absolute configuration of 4, which exhibited potent reversal activity in the A549/taxol MDR tumor cell line, was unambiguously deduced by single-crystal X-ray diffraction.

Full text of DOI:10.1021/ol301755n

ORGANIC
LETTERS
2012
Vol. 14, No. 16
4106–4109
A Furantaxane with an Unusual 6/8/6/5
Ring System and Potent Tumor MDR
Reversal Activity Obtained via Microbial
Transformation
Xiao Liu, Dan Xie, Ridao Chen, Mei Mei, Jianhua Zou, Xiaoguang Chen, and 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
jgdai@imm.ac.cn
Received June 26, 2012
ABSTRACT
A furantaxane (4) with an unusual 6/8/6/5 ring system and two hydroxylated products (2, 3) were isolated following the biotransformation of a
taxane (1) by Streptomyces griseus. The structures of the isolates were elucidated by spectroscopic analysis. The absolute configuration of 4,
which exhibited potent reversal activity in the A549/taxol MDR tumor cell line, was unambiguously deduced by single-crystal X-ray diffraction.
The clinical treatment of cancer with chemotherapeutic
drugs is frequently hindered by either intrinsic or acquired
resistance of the tumor cells. When tumor cells acquire
resistance against a single chemotherapeutic drug, they often
show cross resistance to a variety of antitumor drugs, a state
termed multidrug resistance (MDR).1 According to statis-
tical data, the failure of treatment in over 90% of patients
with metastatic cancer is caused by MDR.2 Although MDR
can develop by several mechanisms, a common cause is
believed to be overexpression of an energy-dependent drug
efflux pump, P-glycoprotein (P-gp). P-gp lowers the intra-
cellular concentration of cytotoxic agents by pumping them
outside of the tumor cells.3 Although several chemical
compounds, including verapamil, quinidine, and cyclosporin
A have been reported to reverse MDR in vivo, these agents
were not developed further due to their unacceptable
toxicity profiles.4 Identifying novel MDR reversal agents
that can act as competitive inhibitors to the binding of
antitumor drugs to P-gp would be an effective strategy
to overcome this clinical problem.5
In a previous investigation, we obtained in high yield the
natural taxane sinenxan A, a type of 4(20),11(12)-taxa-
diene with a C-14 oxygenated substituent (Figure 1), along
with several analogs, from cell cultures of Taxus chinensis.
These compounds possessed potent reversal activity
against several MDR tumor cells6 (see Supporting
Information). In an effort to identify more potent deri-
vatives, these compounds were subjected to structural mod-
ification by chemical and/or enzymatic methods and the
derivatives were evaluated for pharmacological activity.7
(5) (a) Riordan, J. R.; Ling, V. Pharmacol. Therapeut. 1985, 28, 51–75.
(b) Tsuruo, T.; Saito, H. I.; Kawabata, H.; Ohhara, T.; Hamada, H.;
Utakoji, T. Jpn. J. Cancer Res. 1986, 77, 682–692. (c) Naito, M.; Hamada,
H.; Tsuruo, T. J. Biol. Chem. 1988, 263, 11887–11891.
(6) Cheng K. D., Chen W. M., Zhu W. H., Fang Q. C. WO 9,406,740,
1994.
(1) Hosoyama, H.; Shigemori, H.; Tomida, A.; Tsuruo, T.; Kobayashi,
J. Bioorg. Med. Chem. Lett. 1999, 9, 389–394.
(2) Longley, D. B.; Johnston, P. G. J. Pathol. 2005, 205, 275–292.
(3) Gottesman, M. M.; Pastan, I. Annu. Rev. Biochem. 1993, 62, 385–
427.
(7) (a) Zhao, X.; Gu, J.; Yin, D.; Chen, X. Bioorg. Med. Chem. Lett.
2004, 14, 4767–4770. (b) Dai, J.; Qu, R.; Zou, J.-H.; Chen, X. Tetra-
hedron 2008, 64, 8102–8116. (c) Dai, J. Curr. Top. Med. Chem. 2009, 9,
1625–1635. (d) Huang, M.; Zhao, X.; Zhang, M.; Gu, J.; Chen, X.; Yin,
D. Bioorg. Med. Chem. Lett. 2010, 20, 5418–5420. (e) Zhang, Y.; Li, H.;
Wang, H.; Su, F.; Qu, R.; Yin, D.; Dai, J.; Li, Y.; Chen, X. Cancer
Chemother. Pharmacol. 2010, 66, 999–1003.
(4) Tan, B.; Piwnica-Worms, D.; Ratner, L. Curr. Opin. Oncol. 2000,
12, 45–48.
r
10.1021/ol301755n
Published on Web 08/01/2012
2012 American Chemical Society

One derivative of sinenxan A, 10-oxo-2R,5R,14β-triacetox-
ytaxa-4(20),11(12)-diene (1, Figure 1), which was obtained
by chemical synthesis (Scheme S1) and possessed a C-10
carbonyl group in place of an acetoxy group, was found to
have highly potent MDR reversing activity in the A549/
taxol MDR cell line.
To further investigate the structureꢀactivity relationships
of these taxanes and to obtain more potent derivatives,
structural modification of 1 by biological and/or chemical
transformations was performed. Herein, we report three
new products (2ꢀ4) derived from the microbial transforma-
tion of 1 by Streptomyces griseus CACC200300. Of them, 4
is the first taxane reported having an unusual 6/8/6/5 ring
system including a 10R,18-epoxy moiety (a furan ring) in the
skeleton. The absolute structure was determined by HR-MS,
1D-NMR, 2D-NMR, NOE, and single crystal X-ray dif-
fraction analysis. The bioassay results showed that 4 ex-
hibited more potent reversing activity toward MDR tumor
cells A549/taxol than 1.
Compound 2 was obtained as a white amorphous
powder.9 Its molecular formula, C26H36O8, was estab-
lished by positive HR-ESI-MS, which showed a quasimo-
lecular ion peak at m/z 499.2310 [MþNa]þ (calcd for
C26H36O8Na, 499.2308). Its molecular weight was 16
amu more than that of 1, suggesting the introduction of
one hydroxyl group, which was further supported by an IR
absorption at 3447 cmꢀ1. The 1H NMR spectroscopic data
of 2 were similar to those of 1 except for the disappearance
of the methylene signals corresponding to H-9 (δH2.34, d,
15.2 Hz; δH2.84, d, 15.2 Hz; Table 1) and the appearance of
an oxygen-bearing methine signal at δH5.36 (br s). The 13
C
NMR spectrum showed an oxygentaed carbon resonance
at δC77.1 (d) in place of the 1’s C-9 signal at δC58.1 (t).
These dataindicate the introduction ofa hydroxyl groupat
the C-9 position. This result was further supported by the
shift of H-3 of 1 at δH3.04 (d, 6.4 Hz) downfield to δH3.58
(d, 7.5 Hz) in 2 and the shift of C-8/C-10 of 1 at δC40.9 (s)/
204.1 (s) downfield to δC 55.7 (s)/214.6 (s) in 2. The
β-configuration of the 9-OH group was determined by
the NOE difference spectrum, in which the integration
value of H-9 was enhanced when H-3 was irradiated,
whereas the enhancement of H-9 was not observed when
H3-19 was irradiated. Therefore, the structure of 2 was
determined as 9β-hydroxy-10-oxo-2R,5R,14β-triacetoxy-
taxa-4(20),11(12)-diene. This is the first report of a stereo-
selective hydroxylation of a taxane at the 9β position by
biotransformation.
The positive HR-ESI-MS spectrum of 3 displayed a
quasimolecular ion peak at m/z 515.2262 [MþNa]þ
(calcd for C26H36O9Na, 515.2257),10 consistent with the
molecular formula C26H36O9 and an MW 16 amu more
than that of 2, indicating the presence of an additional
hydroxyl group in the molecule. The 1H NMR spectrum of
3 was very similar to that of 2, except that the signal of the
H3-18 at δH2.01 (3H, s) in 2 was absent, while an additional
coupling of the oxygenated methylene proton signals at
δH4.60 (d, 12.6 Hz) and 4.42 (d, 12.6 Hz) was observed,
suggesting hydroxylation at C-18. This was further sup-
ported by the observation that the signal of C-18 of 3 shifted
downfield to δC63.4 (t) from δC21.3 (q) in 2. Thus, 3 was
identified as 9β,18-dihydroxy-10-oxo-2R,5R,14β-triacetox-
ytaxa-4(20),11(12)-diene, which might be biosynthesized
from 2 by a subsequent 18-hydroxylation.
Figure 1. Structures of sinenxan A and 1.
Fourteen species of filamentous fungi distributed in 10
genera (Absidia, Alternaria, Aspergillus, Botrytis, Cunning-
hamella, Fusarium, Mucor, Penicillium, Rhizopus, and
Gibberella) and four species of actinomyces belonging to
the Nocardia and streptomyces genera were employed as
biocatalysts for screening transformations of 1. Based on
the TLC and HPLC analyses, the strain S. griseus
CACC200300 was selected for preparative scale biotrans-
formation due to the diversity and relatively high yields
of products. After the standard two-stage fermentation
protocol,8 three new products (2ꢀ4, Scheme 1) were obtained
by a combination of open silica gel column chromatography
and semipreparative HPLC. On the basis of IR, HR-MS,
1D-NMR, 2D-NMR, and/or single crystal X-ray diffraction
analysis, their structures were established as 9β-hydroxy-
10-oxo-2R,5R,14β-triacetoxytaxa-4(20),11(12)-diene (2,
∼1.5%), 9β,18-dihydroxy-10-oxo-2R,5R,14β-triacetox-
ytaxa-4(20),11(12)-diene (3, ∼0.5%), and 10R,18-epoxy-
10β-hydroxy-9-oxo-2R,5R,14β-triacetoxytaxa-4(20),11(12)-
diene (4, ∼8.5%). These products evidence several enzymatic
reactions including hydroxylation, oxidation, and intramo-
lecular acetalization.
Compound 4 was obtained as colorless crystals from
MeOH after purification by semipreparative HPLC.11 Its
molecular formula was determined to be C26H34O9 with 10
degrees of unsaturation by HR-ESI-MS, in which an ion
peak [MþNa]þ at m/z 513.2118 (calcd for C26H34O9Na,
513.2101) was observed. The 1H NMR spectrum of 4 was
(10) 9β,18-Dihydroxy-10-oxo-2R,5R,14β-triacetoxytaxa-4(20),11(12) -
diene (3): White powder; mp 147ꢀ148 °C, [R]20D ꢀ61.7 (c 0.06, CH3OH);
IR (νmax): 3427, 2936, 1734, 1715, 1433, 1372, 1261, 1231, 1015, 988, 958
cmꢀ1; for 1H and 13C NMR data, see Table 1; HR-ESI-MS (positive) m/z
[MþNa]þ 515.2262 (calcd 515.2257 for C26H36O9Na).
(8) Betts, R. E.; Walters, D. E.; Rosazza, J. P. J. Med. Chem. 1974, 17,
599–602.
(11) 10R,18-Epoxy-10β-hydroxy-9-oxo-2R,5R,14β-triacetoxytaxa-
4(20),11(12)-diene (4): Colorless crystal; mp 170ꢀ172 °C, [R]20D ꢀ90.8
(c 0.26, CH3OH); IR (νmax): 3432, 2986, 2943, 2859, 1730, 1693, 1439,
(9) 9β-Hydroxy-10-oxo-2R,5R,14β-triacetoxytaxa-4(20),11(12)-diene
D
(2): White amorphous powder; [R]20 ꢀ89.6 (c 0.115, CH3OH); IR
(νmax): 3447, 3001, 2931, 1739, 1693, 1435, 1383, 1370, 1253, 1026,
1014, 973, 932 cmꢀ1; for 1H and 13C NMR data, see Table 1; HR-ESI-
MS (positive) m/z [MþNa]þ 499.2310 (calcd 499.2308 for C26H36O8Na).
1382, 1247, 1189, 1051, 1034, 1018, 957, 919, 898 cmꢀ1; for 1H and 13
C
NMR data, see Table 1; HR-ESI-MS (positive) m/z [MþNa]þ 513.2118
(calcd 513.2101 for C26H34O9Na).
Org. Lett., Vol. 14, No. 16, 2012
4107

a
Table 1. 1H NMR and 13C NMR Data for Compounds 1ꢀ4 in CDCl3
a 1H NMRdata were recorded at 400 MHz for 1 and 4, at 500 MHz for 2 and at 300 MHz for 3, respectively. 13C NMR data were recorded at 100 MHz
for 1 and 4 and at 125 MHz for 2 and 3. Peaks were assigned by analyses of the 1D- and 2D-NMR spectra. b Multiplicities and coupling constants (J) in
Hz are in parentheses. c Data for hydroxy protons of 3: δH 5.47 (s, OH-9). d Data for hydroxy protons of 4: δH 5.57 (s, OH-10).
very similar to that of 3 except for the absence of H1-9
(δH4.15, s). The 13C NMR spectrum of 4 was also very
similar to that of 3 except for the disappearance of C-9
(δC76.9, d) and the appearance of a quaternary carbon
resonance at δC107.6(s). The long-range heteronuclear
correlations of 10-OH/C-10 and C-12, as well as H2-18/
C-11 and C-12, were observed in the HMBC spectrum.
Based upon the HR-MS and NMR data, the presence of a
furan hemiacetal moiety composed of C12ꢀC18ꢀOꢀ
C10ꢀC11 in 4 was deduced. To prove the above assignment
and to determine the absolute configuration of 4, a single
crystal X-ray diffraction pattern was obtained by anom-
alous scattering of Cu KR radiation. An ORTEP drawing
with the atom-numbering scheme indicated is shown in
Figure 2 and demonstrates a configuration of 1S,2S,3R,
5S,8R,10S,14S for 4. The structure of 4 was determined
as10R,18-epoxy-10β-hydroxy-9-oxo-2R,5R,14β-triacetoxy-
taxa-4(20),11(12)-diene.
Figure 2. ORTEP diagram of 4.
To the best of our knowledge, this is the first report of a
10R,18-epoxy taxane (a furantaxane). Based on a compar-
intramolecular acetalization. Considering that acetalization
could occur under acidic conditions, the pH kinetics
during the whole incubation was investigated, and the
results displayed that the lowest pH value was ca. 6.3
during the whole process (Figure S3). Thus, to verify
whether this acetalization step was enzymatic or chemical
under acidic conditions, the incubations of 1 and 2 with
ison of the chemical structures of substrate (1) and the
products (2ꢀ4), a plausible bioconversion route is pro-
posed (Scheme 1). Stereoselective hydroxylation at the
C-9β position of 1 yields 2, and 3 is obtained via further
hydroxylation at the C-18 position. The furantaxane 4
could be formed from 3a, which should be formed from 3
by oxidation of the 9-hydroxy to a 9-keto followed by an
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Org. Lett., Vol. 14, No. 16, 2012

resting cells and/or crude enzymes in the phosphate buffer
solutions (PBS, 50 mM), in which the pH was designated
as 6.3 and 7.0 (a neutral and normal pH value for P450
enzymes), were performed, respectively. The production
of 4 was detected by HPLC analysis, while it was not
detected in the controls (incubations of 1 and 2 in PBS
buffer without resting cells or crude enzymes). These
results solidly indicated that 4 was an enzymatic product
(Figures S4ꢀS6).
Table 2. Reversal Activities of 1ꢀ4 against MDR A549/Taxol
Cells
samples (10 μM)
IC50 of paclitaxel (nM)a RF (A549/T)b
paclitaxel
26
ꢀ
paclitaxel þ 1
14.4
13.0
11.8
6.3
1.8
2.0
2.2
4.1
4.4
paclitaxel þ 2
paclitaxel þ 3
paclitaxel þ 4
paclitaxel þ Verapamil
5.9
a A549/taxol, an MDR subline of human lung adenocarcinoma cell
line A549. b RF (reversal fold) = IC50 (antitumor agent)/IC50 (reversal
agent þ antitumor agent).
Scheme 1. Plausible Bioconversion Route from 1 to 2ꢀ4
In summary, this communication reports successful
structural diversification by microbial transformation
of a 4(20),11(12)-taxadiene (1). The biotransformations
observed included hydroxylations, oxidation of a hydroxy
group to a ketone, and an intramolecular acetalization.
The transformationsprovided three newcompounds, 2ꢀ4.
The stereoselective hydroxylation at C-9β and intramole-
cular acetalization are the first reported for biotransforma-
tions of taxanes. 4 is an unusual 6/8/6/5 taxane bearing an
additionalfuran ring comprising C12ꢀC18ꢀOꢀC10ꢀC11. 4
exhibited significant reversal activity, comparable with
verapamil, toward MDR A549/taxol cells when coadmi-
nistered with paclitaxel. These results suggest that 4 is a
good lead as an MDR reversal agent and warrants further
study. Also, bioconversion is a versatile means for obtain-
ing diversity from structural leads and can play an important
role in the discovery of drug leads/candidates.
Taxanes 1ꢀ4, coadministered with paclitaxel, were
tested at 10 μM for reversal activity toward the MDR
tumor cell line of taxol-resistant A549 having a P-gp
overexpressing phenotype (Table 2). 4 displayed signifi-
cant reversal activity with a reversal fold (RF) of 4.1, which
is two times higher than that of 1. The RF of verapamil, the
positive control, was 4.4 at 10 μM. 2 and 3 exhibited
comparable reversal activity to 1. All the compounds
showed low cytotoxicity against five human tumor cell
lines, including HCT-8, Bel-7402, BGC-823, A549, and
A2780 (IC50 > 10ꢀ5 M, Table S4). Low cytotoxicity is a
good property for a reversing agent.12
Acknowledgment. This work was supported by 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 Creation and Manufacturing’, China
(2009ZX09103-033).
Supporting Information Available. Experimental pro-
cedure, selected NMR (1D-NMR, 2D-NMR, and NOE),
HR-ESI-MS, and IR spectra of compounds 1ꢀ4. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(12) (a) Kobayashi, J.; Hosoyama, H.; Wang, X.; Shigemori, H.;
Sudo, Y.; Tsuruo, T. Bioorg. Med. Chem. Lett. 1998, 8, 1555–1558.
(b) Ojima, I.; Borella, C. P.; Wu, X.; Bounaud, P. Y.; Oderda, C. F.;
Sturm, M.; Miller, M.; Chakravarty, S.; Chen, J.; Huang, Q.; Pera, P.;
Brooks, T. A.; Baer, M. R.; Bernacki, R. J. J. Med. Chem. 2005, 48,
2218–2228.
The authors declare no competing financial interest.The authors
declare no competing financial interest.
Org. Lett., Vol. 14, No. 16, 2012
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