Synthesis, isolation, stereostructure and cytotoxicity of paclitaxel analogs from cephalomannine

Article abstract of DOI:10.1016/j.fitote.2013.07.011

Four paclitaxel derivatives were afforded by preparative HPLC separation of two pairs of diastereoisomers, which were obtained by catalytic hydrogenation and epoxidation of the C-13 side-chain double bond of cephalomannine, a naturally occurring paclitaxel analog. The four paclitaxel derivatives were analyzed using NMR, CD spectroscopy, and side-chain hydrolysis in order to measure their optical rotations and GC characteristics. In this way, the stereoconfigurations of the products were determined. Evaluation of the compounds' activity indicated that they had differing cytotoxic activities: compound 5 had superior activity in BCG-823 tumor cells compared to paclitaxel, while compound 7 had superior activity in HCT-8 and A549 tumor cells compared to paclitaxel. These results indicate that the stereoconfiguration of the paclitaxel N-acyl side chain has a significant impact on its activity.

Full text of DOI:10.1016/j.fitote.2013.07.011

Fitoterapia 90 (2013) 79–84
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Synthesis, isolation, stereostructure and cytotoxicity of
paclitaxel analogs from cephalomannine
⁎
Feng Gao , Dan Wang, Xing Huang
Department of Chinese traditional herbal, Agronomy College, Sichuan Agricultural University, No.211, Huiming Road, Wenjiang Region, Chengdu 611130, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2013
Received in revised form 9 July 2013
Accepted 13 July 2013
Available online 20 July 2013
Four paclitaxel derivatives were afforded by preparative HPLC separation of two pairs of
diastereoisomers, which were obtained by catalytic hydrogenation and epoxidation of the
C-13 side-chain double bond of cephalomannine, a naturally occurring paclitaxel analog. The
four paclitaxel derivatives were analyzed using NMR, CD spectroscopy, and side-chain
hydrolysis in order to measure their optical rotations and GC characteristics. In this way, the
stereoconfigurations of the products were determined. Evaluation of the compounds' activity
indicated that they had differing cytotoxic activities: compound 5 had superior activity in
BCG-823 tumor cells compared to paclitaxel, while compound 7 had superior activity in HCT-8
and A549 tumor cells compared to paclitaxel. These results indicate that the stereoconfiguration
of the paclitaxel N-acyl side chain has a significant impact on its activity.
Keywords:
Paclitaxel
Cephalomannine
Structural modification
Side chain
© 2013 Elsevier B.V. All rights reserved.
Cytotoxic activity
1. Introduction
N-benzoyl group in its C-13 side chain, while cephalomannine
has an N-tigloyl group. Because of these structural similar-
Paclitaxel, or Taxol® (1, Fig. 1), is a structurally peculiar
diterpenoid compound that was isolated from the bark of the
North American Taxus brevifolia by Wani et al. in 1971 [1].
Because of its significant anticancer activity, unique mecha-
nism of action, complex molecular structure, and multiple
chiral centers, research on its chemical synthesis, structural
modification, and structure–activity relationship has long
been a focus in the field of natural product chemistry [2]. At
present, paclitaxel and its semisynthetic derivative, docetaxel
(2, Fig. 1), are mainly used as clinical treatments for breast,
ovarian, and non-small-cell lung cancer [3].
As can be seen from its name, cephalomannine (3, Fig. 1) was
initially considered to have been isolated from Cephalotaxus
mannii, but it was later discovered that there was an error in the
plant classification and that cephalomannine was actually from
Taxus wallichiana, a species of yew [4]. In later phytochemical
studies, cephalomannine was found in several other yew species
[5–7]. The structure of cephalomannine is very similar to that of
paclitaxel, differing only in its C-13 side chain: paclitaxel has an
ities, the separation of cephalomannine from paclitaxel is
very difficult, and several methods for effectively separating
the two compounds have been developed [8–10]. Since the
amount of cephalomannine might even be higher than that
of paclitaxel in certain Taxus plants, it can also be a useful
semi-synthetic raw material that can be converted into paclitaxel
or docetaxel [11,12]. Furthermore, there have been reports
of interesting structural modifications and biotransformations
of cephalomannine [13–15]. Therefore, structural modification
of readily available cephalomannine can contribute to a better
understanding of the structure–activity relationship in the
anticancer effects of taxanes.
The epoxidation of cephalomannine has been reported in
a patent on the preparation of cephalomannine analogs, but
the diastereomers were not separated in this report [16].
There is no stereoselectivity of hydrogenation and epoxida-
tion reactions on the double bond of cephalomannine's side-
chain and the afforded compounds in the patent were a pair
of diastereoisomers. Therefore, in the current study, catalytic
hydrogenation and epoxidation of cephalomannine were
carried out separately on the side-chain N-tigloyl double bond
to obtain two different diastereoisomers, which were then
⁎
Corresponding author. Tel.: +86 28 8629 0870; fax: +86 28 8629 0780.
E-mail address: gaofeng@sicau.edu.cn (F. Gao).
0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2013.07.011

80
F. Gao et al. / Fitoterapia 90 (2013) 79–84
R2
R1O
11
further subjected to preparative reversed-phase HPLC eluting
with CH3CN–H2O (60/40, v/v) to give compounds 4 (85 mg,
tR = 16.7 min) and 5 (82 mg, tR = 18.6 min).
O
18
12
OH
7
NH
19
O
1'
10
9
16
2'
8
6
Ph 3'
O
13 15
2.3.1. N-debenzoyl-N-(S-2-methylbutanoyl)paclitaxel (4)
White powder, [a]D25 − 49 ° (c 0.10, MeOH); IR νmax 3438,
5
3
17
4
O
OH
2961, 1730, 1714, 1638 cm−1 1H NMR (400 MHz, CDCl3)
;
14
1
2
H
OBz
and 13C NMR (125 MHz, CDCl3): see Table 1. MS (ESI, MeOH)
m/z 834 [M + H]+
HO
20
OAc
.
2.3.2. N-debenzoyl-N-(R-2-methylbutanoyl)paclitaxel (5)
White powder, [a]D25 − 25 ° (c 0.10, MeOH); IR νmax 3438,
1 Paclitaxel
2 Docetaxel
R1 = Ac, R2 = Bz
R1 = H, R2 = Boc
2962, 1730, 1716, 1638 cm−1 1H NMR (400 MHz, CDCl3)
;
and 13C NMR (125 MHz, CDCl3): see Table 1; MS (ESI, MeOH)
m/z 834 [M + H]+. HR-ESI-MS: 834.3632 [M + H]+ (calcd.
for C45H56NO14 834.3622).
R1 = Ac, R2 =1''
CO
3 Cephalomannine
2''
3''
4''
2.4. Preparation of compounds 6 and 7
Fig. 1. Structures of paclitaxel (1), docetaxel (2) and cephalomannine (3).
The solution of cephalomannine (250 mg, 0.30 mmol) in
CH3Cl3 (50 mL) was added in mCPBA (3-chloroperbenzoic
acid, 62 mg, 0.36 mol, 1.2 equiv.). The reaction mixture was
stirred for 8 h under argon prior to being quenched with
aqueous solution of Na2S2O3. The organic phase was separated,
washed with brine and dried over anhydrous Na2SO4. Then
the organic solvent was removed under reduced pressure
to give the mixture of compounds 6 and 7. The mixture
was further subjected to preparative reversed-phase HPLC
eluting with CH3CN–H2O (60/40, v/v) to give compounds 6
(76 mg, tR = 12.7 min) and 7 (72 mg, tR = 15.2 min).
separated using preparative high-performance liquid chroma-
tography (HPLC). In addition, the three-dimensional structures
as well as anticancer activities of the obtained diastereoisomers
were studied.
2. Experimental
2.1. General
1H and 13C NMR spectra were obtained on a Varian Unity
INOVA 400/54 and Bruker ARX-500 spectrometer in CDCl3
with TMS as the internal standard. Mass spectra were
obtained on a VG Auto spec 3000 or on a Finnigan MAT90
instrument. Optical rotations were measured on a Perkin-Elmer
341, and CD spectra were measured on a JASCO J-810
spectropolarimeter. Preparative HPLC was performed on a
Shimadzu LC-8A liquid chromatograph equipped with a
Zorbax PrepHT GF (21.2 mm × 25 cm, 7 μm) or Venusil MP
C18 (20 mm × 25 cm, 5 μm) column. GC–MS analysis was
performed on a GC (Agilent, 6890N) interfaced with a mass
selective detector (Agilent, 5973B). Silica gel H (Qingdao
Sea Chemical Factory, Qingdao, People's Republic of China)
was used for column chromatography.
2.4.1.
paclitaxel (6)
White powder, [a]D25 − 38 ° (c 0.10, MeOH); IR νmax 3422,
2988, 1710, 1632 cm−1 1H NMR (400 MHz, CDCl3) and 13C
NMR (125 MHz, CDCl3): see Table 1; MS (ESI, MeOH) m/z
848 [M + H]+. HR-ESI-MS: 848.3466 [M + H]+ (calcd. for
C45H54NO15 848.3415).
N-debenzoyl-N-[(2R,3S)-2,3-epoxy-2-methylbutanoyl]
;
2.4.2.
paclitaxel (7)
White powder, [a]D25 − 66 ° (c 0.10, MeOH); IR νmax 3422,
2988, 1712, 1632 cm−1 1H NMR (400 MHz, CDCl3) and 13C
N-debenzoyl-N-[(2S,3R)-2,3-epoxy-2-methylbutanoyl]
;
NMR (125 MHz, CDCl3): see Table 1; MS (ESI, MeOH) m/z
848 [M + H]+. HR-ESI-MS: 848.3446 [M + H]+ (calcd. for
C45H54NO15 848.3415).
Spots on TLC (silica gel G) were detected by spraying with
H2SO4–EtOH. Commercially available reagents and solvents
were directly used without further purification.
2.5. Complete alkaline hydrolysis of compounds 4–7
2.2. Substrates
A solution of 4 (or 5, 6, 7) (50 mg) and KOH (100 mg) in
MeOH (15 mL) was stirred overnight at room temperature.
The reaction mixture was neutralized with 1 N HCl and
extracted with EtOAc (10 mL × 3). Then the combined organic
phase was dried over anhydrous Na2SO4 and evaporated to
give the residue. The two residues, from compounds 4 and 5,
were dissolved in MeOH (5 mL) and treated with excess
CH2N2. After evaporation, the residues were dissolved in CHCl3
(5 mL) and filtered. An aliquot of the two filtrates was analyzed
by GC using a chiral column (Astec Chiraldex G-TA G0012-08,
30 m, 50 °C), to give peaks at tR 12.5 or 11.4 min, respectively.
The standard methyl ester of (R)-or (S)-2-methylbutyric
Cephalomannine (3) was purchased from Kunming Wuyi
Biotechnology Co., Ltd., China. The structures were charac-
terized on the basis of 1H NMR and 13C NMR spectra, and the
purities were determined to be N95% by HPLC analyses.
2.3. Preparation of compounds 4 and 5
A solution of cephalomannine (200 mg, 0.24 mmol) and
Pd–C (10 mg, 5%) under H2 atmosphere was strongly stirred
for 24 h. Then the reaction solution was filtered and evaporat-
ed to give a mixture of compounds 4 and 5. The mixture was

Table 1
1H and 13C NMR data for compounds 3, 4, 5, 6, 7 (δ in ppm, in CDCl3).
Position
Cephalomannine (3)
Η (J in Hz)
Compounds 4/5a
Η (J in Hz)
Compounds 6/7b
δΗ (J in Hz)
δ
δC
δ
δC
δC
1
2
3
4
5
6
–
79.0 s
74.9 d
45.5 d
81.1 s
84.4 d
35.5 t
–
78.7 s
74.9 d
45.5 d
81.0 s
84.3 d
35.5 t
–
79.0/78.9 s
74.9/74.8 d
45.5/45.6 d
81.0/81.1 s
84.3 d
5.67 d (7.2)
3.78 d (7.2)
–
5.68 d (7.2)
3.78 d (7.2)
–
5.67 d (6.8)
3.77 t (6.8)
–
4.94 d (8.0)
1.88 m
2.54 m
4.40 m
–
4.94 d (8.0)
1.86 m
2.53 m
4.40 m
–
4.93 d (10.0)
1.81 m
2.50 m
4.39 m
–
35.4/35.5 t
7
8
9
10
11
12
13
14
72.2 d
58.5 s
203.8 s
75.6 d
133.1 s
142.2 s
72.2 d
35.5 t
72.0 d
58.4 s
203.6 s
75.5 d
133.5 s
141.9 s
72.4 d
35.5 t
72.1 d
58.5 s
–
–
–
203.5 s
75.6/75.5 s
133.7 s
141.9 s
72.2/72.3 d
35.5 t
6.27 s
–
6.28 s
–
6.28 d (3.6)
–
–
–
–
6.21 t (8.4)
2.34 m
2.28 m
–
6.18 t (8.8)
2.33 m
2.29 m
–
6.18 q (8.8)
2.36 m
2.24 m
–
15
16
17
18
19
20
43.1 s
21.8 q
26.8 q
14.7 q
9.5 q
43.1 s
21.8 q
26.6 q
14.7 q
9.5 1q
76.4 d
43.1 s
21.8/21.7 q
26.8 q
14.7/14.8 q
9.5 q
76.4 t
1.15 s
1.26 s
1.80 s
1.68 s
4.29 d (8.4)
4.19 d (8.4)
–
1.15 s
1.26 s
1.82 s
1.68 s
4.29 d (8.4)
4.18 d (8.4)
–
1.15 s
1.26 s
1.79 s
1.67 s
4.28 d (8.4)
4.17 d (8.4)
–
76.5 t
1′
2′
3′
4-OAc
172.9 s
73.3 d
54.8 d
170.4 s
22.5 q
171.4 s
20.8 q
167.1 s
129.2 s
130.3 d
128.3 d
133.8 d
131.3 s
127.0 d
128.8 d
129.0 d
–
172.8 s
73.0 d
54.1 d
171.2 s
22.5 q
172.8 s
20.8 q
166.8 s
129.1 s
130.1 d
128.8 d
133.0 d
138.0 s
128.4 d
126.7 d
129.1 d
–
172.7 s
73.0/73.3 d
54.1/54.3 d
170.1 s
22.5 q
171.2 s
20.8 q
166.9 s
129.2 s
130.3 d
128.3 d
133.8 d
131.3 s
127.0 d
128.8 d
129.0 d
–
–
–
–
–
–
–
2.36 s
–
2.24 s
–
2.23 s
–
10-OAc
2.25 s
–
2.17 s
–
2.16 s
–
2-OBz
Ph-1
Ph-2,6
Ph-3,5
Ph-4
3′-Ph-1
3′-Ph-2,6
3′-Ph-3,5
3′-Ph-4
NH
NH-CO
1″
2″
3″
4″
–
–
–
8.13 d (7.2)
7.51 m
7.62 m
–
8.10 d (7.2)
7.50 m
7.61 m
–
8.10 d (7.2)
7.50 m
7.60 m
–
7.40 br. s
7.42 br. s
7.34 m
6.51 d (8.8)
–
7.39 m
7.40 m
7.33 m
6.28 d (8.8)
–
7.38 br. s
7.31 m
7.21 m
–
169.1 s
12.2 q
138.2 s
132.0 d
13.9 q
176.5 s
17.5 q
43.1 q
27.1 q
11.8 q
–
171.5 s
1.62 s
–
1.38 m
2.34 m
1.08 m
0.86 t (7.2)
1.80 s
–
13.5/13.4 q
60.0/59.9 s
59.6/59.3 s
12.6/12.7 q
6.43 q (6.8)
1.72 d (6.8)
2.98/3.00 q (4.4)
1.42 d (4.4)
a
The 1H and 13C NMR data of the diastereoisomers 4 and 5 were completely identical.
The diastereoisomers 6 and 7 had a weak difference in 13C NMR data.
b

82
F. Gao et al. / Fitoterapia 90 (2013) 79–84
Table 2
carcinoma cell line and the A2780 human ovarian were accessed
using MTT method.
Cytotoxicity of paclitaxel and compounds 4, 5, 6 and 7.
Cells were plated in the RPMI 1640 with 10% fetal calf
serum media on 96-well plates in a total volume of
100 μL with a density of 1 × 10−4 cells mL−1. Triplicate
wells were treated with media and tested compounds.
The plates were incubated at 37 °C in 5% CO2 for 72 h. Cell
viability was determined based on mitochondrial conversion of
3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT, Sigma) to formazan. The amount of MTT converted to
formazan is a sign of the number of viable cells. Each well was
supplemented with 50 μL of a 1 mg mL−1 solution of MTT
in uncompleted media. The plates were incubated in 37 °C, 5%
CO2 for an additional 4 h. The media was carefully removed
from each well and then 200 μL of DMSO was added. The plates
were gently agitated until the reaction color was uniform
and the OD570 was determined using a micro-plate reader
(Wellscan MK3, Labsystems Dragon). Microsoft® Excel 2000
was used to analyze data. Media-only treated cells served
as the indicator of 100% cell viability. The 50% inhibitory
concentration (IC50) was defined as the concentration that
reduced the absorbance of the untreated wells by 50% of
the control in the MTT assay.
Compd.
IC50 (nM)
HCT-8
Bel-7402
BGC-823
A549
A2780
Paclitaxel
51
460
680
220
25
6.0
12.5
7.2
10.5
5.2
3.29
16.5
2.14
9.45
2.66
16
240
22
86
5.0
7.86
18.6
9.45
12.8
7.05
4
5
6
7
acid gave a peak at tR 11.4 and 12.5 min. The other two
residues, from compounds 6 and 7, were separated via
column chromatography over silica gel eluting with EtOAc
followed by CHCl3–MeOH (1:2) to yield (2R, 3S)- or (2S, 3R)-2,3-
epoxy-2-methylbutanoic acid, respectively, for measuring
optical rotations.
2.6. Cytotoxicity bioassays
The cytotoxicity assays against the HCT-8 human colorectal
adenocarcinoma cell line, Bel-7402 human liver cancer cell line,
BGC-823 human gastric cancer cell line, A549 human lung
O
1''
AcO
O
OH
NH
O
2''
3''
Ph
O
4''
O
OH
H
OBz
HO
1)H2 / Pd-C / CH3OH
OAc
OH
''
4
2) HPLC Isolation
O
+
AcO
O
NH
O
O
1''
Ph
O
AcO
O
H
OH
O
NH
OH
O
2''
3''
H
HO
OAc
OH
OBz
O
Ph
O
5
4''
O
OH
HO
O
OAc
OBz
Cephalomannine (3)
1''
AcO
NH
O
2''
3''
O
Ph
O
4''
O
OH
H
HO
1) mCPBA / CH2Cl2
2) HPLC Isolation
OAc
OH
OBz
6
O
+
AcO
O
NH
O
O
Ph
O
O
OH
H
OBz
HO
OAc
7
Fig. 2. Synthesis of paclitaxel analogs from cephalomannine.

F. Gao et al. / Fitoterapia 90 (2013) 79–84
83
3. Results and discussion
In order to determine the stereo configuration of the newly
formed epoxy group in compounds 6 and 7, complete hydrolysis
was carried out. A pair of 2,3-epoxy-2-methylbutanoic acid
enantiomers with rotation values of [a]2D5 − 5 ° and +5°(c 0.10,
CHCl3) was obtained, respectively, which is consistent with the
optical rotation values of (2R,3S)- and (2S,3R)-2,3-epoxy-2-
methylbutanoic acid obtained from an asymmetric synthe-
sis [18]. Thus, it can be deduced that the structures of
compounds 6 and 7 were N-debenzoyl-N-[(2R,3S)-2,3-epoxy-
2-methylbutanoyl]paclitaxel and N-debenzoyl-N-[(2S,3R)-2,3-
epoxy-2-methylbutanoyl]paclitaxel, respectively. All 1H and
13C NMR data for compounds 4 and 5 were assigned by
comparison with cephalomannine, paclitaxel, and 2,3-epoxy-2-
methylbutanoic acid NMR data (Table 1).
With paclitaxel and as a positive control, the cytotoxic
activities of compounds 4, 5, 6, and 7 were evaluated in five
types of tumor cells, and the results are shown in Table 2. The
activities of compounds 4 and 6 are weaker than that of
paclitaxel, while the activities of compounds 5 and 7 are
comparable to those of paclitaxel. Furthermore, the activity of
compound 5 in the BGC-823 tumor cell line was superior to
those of paclitaxel, and the activities of compound 7 in HCT-8
and A549 cells were 2 and 3 times higher than those of paclitaxel,
respectively.
In summary, four paclitaxel derivatives were obtained by
preparative HPLC separation of two pairs of diastereoisomers,
which were obtained from catalytic hydrogenation and epoxi-
dation of the C-13 side-chain double bond of cephalomannine
and their stereostructures were confirmed by chemical and
spectral methods. Various cytotoxic activities of these two pairs
of diastereoisomers indicated that the stereoconfiguration
of the N-acyl in the paclitaxel side chain has a significant
impact on its activity, and increasing the oxygenation of N-acyl
side chains improves the activity. This result can further enrich
our understanding on the structure–activity relationship in
paclitaxel.
Compounds 4 and 5 were produced by catalytic hydroge-
nation of the N-tigloyl double bond of cephalomannine
(Fig. 2). The 1H-NMR results showed that the product of
the hydrogenation appeared to be a single compound. In
particular, the signal for the vinyl proton on the cephalomannine
side-chain double bond disappeared (δH, 6.43, 1H, q, J = 6.8 Hz,
Table 1) and the signal for the 4′-methyl group appeared as a
triplet signal (δH, 0.86, 3H, t, J = 7.2 Hz, Table 1). However,
HPLC results showed that there was a pair of products
present in a 1:1 ratio, which indicates that there was no
stereoselectivity of the hydrogenation of the cephalomannine
Δ2″,3″ double bonds, and a pair of diastereoisomers was formed.
Therefore, optically pure compounds 4 and 5 were obtained
by separating the hydrogenation products using preparative
HPLC.
Next, analysis was performed to confirm the stereo-
configuration of the newly-formed C-2″ chiral carbon in
compounds 4 and 5. There were no significant differences
between the circular dichroism (CD) spectra of compounds 4
and 5, and hence, the absolute configurations of these two
compounds could not be determined using CD spectroscopy.
Gabetta et al. isolated N-debenzoyl-N-(2-methylbutanoyl)
paclitaxel from Taxus media cv. Hicksii and confirmed that
the stereoconfiguration of its 2-methylbutanoyl structural
unit was S, and the optical rotation of this compound is
[a]2D5 − 48 ° (c 0.10, MeOH) [17]. In this study, the optical
rotations of 4 and 5 are [a]2D5 − 49 ° (c 0.10, MeOH) and
[a]2D5 − 25 ° (c 0.10, MeOH), respectively. Hence, it can
be deduced that compound 4 and the N-debenzoyl-N-
(2-methylbutanoyl) paclitaxel isolated by Gabetta et al. are
the same. Therefore, the 2-methylbutanoyl structural unit in
compound 4 has the S configuration, while C2″ in compound
5 has the R configuration. In order to further confirm the
stereoconfigurations of compounds 4 and 5, gas chroma-
tography (GC) was used to analyze the hydrolyzates of
both compounds in comparison to a standard control. The
tR values of the 2-methylbutyric acids obtained from
complete hydrolysis of compounds 4 and 5 were 12.5 min
and 11.4 min, respectively, which correspond to (S)- and
(R)-2-methylbutyric acid. Thus, the structures of compounds 4
and 5 were N-debenzoyl-N-(S-2-methylbutanoyl)paclitaxel
and N-debenzoyl-N-(R-2-methylbutanoyl)paclitaxel, respec-
tively. All 1H and 13C NMR data for compounds 4 and 5 were
assigned by comparison with cephalomannine and paclitaxel
NMR data (Table 1).
Acknowledgments
This project was supported by the National Natural
Science Foundation of China (No. 81001383) and the
Doctoral Foundation of Ministry of Education of China
(No. 20105103120009).
Appendix A. Supplementary data
Compounds 6 and 7 were produced by the epoxidation of
cephalomannine (Fig. 2). The 1H NMR results showed
that the products were a pair of diastereomers, in which the
signal of the proton on the double bond of the side chain of
cephalomannine (δH, 6.43, 1H, q, J = 6.8 Hz, Table 1) was
replaced with a proton on an epoxy carbon (δH, 2.98/3.00, 1H,
q, J = 4.4 Hz). The HPLC results also showed that the
epoxidation products were a pair of compounds with a
ratio of 1:1, which again indicates that that there was no
stereoselectivity of the epoxidation of the double bonds.
Optically pure compounds 6 and 7 were obtained using
preparative HPLC, and there were negligible differences
between the 1H and 13C NMR signals of the two compounds
(Table 1).
Supplementary data associated with this article, including
1H and 13C NMR spectra, CD and HPLC spectra, can be found
in the online version, at doi: http://dx.doi.org/10.1016/j.fitote.
2013.07.011.
References
[1] Wang YF, Shi QW, Dong M, Kiyota H, Gu YC, Cong B. Natural taxanes:
developments since 1828. Chem Rev 2011;111:7652–709.
[2] Oberlies NH, Kroll DJ. Camptothecin and taxol: historic achievements in
natural products research. J Nat Prod 2004;67:129–35.
[3] Nicolas A, Christophe M. Taxanes in paediatric oncology and now.
Cancer Treat Rev 2006;32:65–73.
[4] Miller RW, Powell RG, Smith CR, Arnold E, Clardy J. Antileukemic
alkaloids from Taxus wallichiana Zucc. J Org Chem 1981;46:1469–74.

84
F. Gao et al. / Fitoterapia 90 (2013) 79–84
[5] Guo Y, Vanhaelen-Fastré R, Diallo B, Vanhaelen M, Jaziri M, Homes J,
[12] Gunatilaka AAL, Chordia MD, Kingston DGI. Efficient conversion of
cephalomannine to paclitaxel and 3′-N-acyl-3′-N-debenzoylpaclitaxel
analogs. J Org Chem 1997;62:3775–8.
[13] Li J, Dai J, Chen X, Zhu P. Microbial transformation of cephalomannine
by Luteibacter sp. J Nat Prod 2007;70:1846–9.
[14] Wang H, Li H, Zuo M, Zhang Y, Liu H, Fang W, et al. Lx2-32c, a novel
taxane and its antitumor activities in vitro and in vivo. Cancer Lett 2008;268:
89–97.
[15] Yang CG, Barasoain I, Li X, Matesanz R, Liu R, Sharom FJ, et al.
Overcoming tumor drug resistance with high-affinity taxanes: a SAR
study of C2-modified 7-acyl-10-deacetyl cephalomannines. ChemMedChem
2007;2:691–701.
et al. Immunoenzymatic methods applied to the search for bioactive
taxoids from Taxus baccata. J Nat Prod 1995;58:1015–23.
[6] Van Rozendaal EL, Lelyveld GP, van Beek TA. Screening of the needles of
different yew species and cultivars for paclitaxel and related taxoids.
Phytochemistry 2000;53:383–9.
[7] Zhang J, Sauriol F, Mamer O, You XL, Alaoui-Jamali MA, Batist G, et al. New
taxane analogues from the needles of Taxus canadensis. J Nat Prod 2001;64:
450–5.
[8] Beckvermit JT, Anziano DJ, Murray CK. An improved method for
separating paclitaxel and cephalomannine using ozone and Girard
reagents. J Org Chem 1996;69:9038–40.
[9] Kingston DGI, Gunatilaka AA, Ivey CA. Modified taxols, 7. A method for the
separation of taxol and cephalomannine. J Nat Prod 1992;55:259–61.
[10] Sun R, Fu K, Fu Y, Zu Y, Wang Y, Luo M, et al. Preparative separation and
enrichment of four taxoids from Taxus chinensis needles extracts by
[16] Zheng QY, Murray CK, Daughenbaugh RJ. Cephalomannine epoxide, its
analogues and a method for preparing the same. US Patent 1999;
5892063.
[17] Gabetta B, Fuzzati N, Orsini P, Peterlongo F, Appendino G, Vander Velde
DG. Paclitaxel analogues from Taxus media cv. Hicksii. J Nat Prod 1999;62:
219–23.
macroporous resin column chromatography.
J Sep Sci 2009;32:
1284–93.
[11] Ganem B, Franke RR. Paclitaxel from primary taxanes: a perspective on
creative invention in organozirconium chemistry. J Org Chem 2007;72:
3981–7.
[18] Torres-Valencia JM, Cerda-Garcia-Rojas CM, Joseph-Nathan P. Preparation
of (2R,3S)-(−)- and (2S,3R)-(+)-2,3-epoxy-2-methylbutanoic acids and
some of their esters. Tetrahedron-Asymmetry 1995;7:1611–6.