Epothilone D and its 9-Methyl analogues: Combinatorial syntheses, conformation, and biological activities

Article abstract of DOI:10.1016/j.ejmech.2013.08.003

Epothilone D (Epo D) and its 9-Methyl conformational analogues were synthesized through a highly efficient combinatorial approach. The fragment E was synthesized in 11 total steps with 6 longest linear steps, and each aldehyde B was prepared via a 3-step sequence. Starting from the common precursor E and a suitable aldehydes B, each target molecule were obtained in only 4 steps. The 9-(S)-epo D and 9-(R)-epo D demonstrated significant difference in inhibition activities against cancer cell lines and in conformational analysis.

Full text of DOI:10.1016/j.ejmech.2013.08.003

European Journal of Medicinal Chemistry 68 (2013) 321e332
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Epothilone D and its 9-Methyl analogues: Combinatorial syntheses,
conformation, and biological activities
Feng Sang a, Peng Feng c, Jie Chen a, Yahui Ding c, Xiyan Duan b, Jiadai Zhai a, Xiaoyan Ma c,
,
a,
*
Bin Zhang a, Quan Zhang a, Jianping Lin a , Yue Chen
*
a State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, PR China
b Chemical Engineering and Pharmaceutics School, Henan University of Science and Technology, Luoyang, Henan 471023, PR China
c Accendatech, Tianjin 300384, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Epothilone D (Epo D) and its 9-Methyl conformational analogues were synthesized through a highly
efficient combinatorial approach. The fragment E was synthesized in 11 total steps with 6 longest linear
steps, and each aldehyde B was prepared via a 3-step sequence. Starting from the common precursor E
and a suitable aldehydes B, each target molecule were obtained in only 4 steps. The 9-(S)-epo D and
9-(R)-epo D demonstrated significant difference in inhibition activities against cancer cell lines and in
conformational analysis.
Received 3 April 2013
Received in revised form
25 July 2013
Accepted 2 August 2013
Available online 11 August 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Epothilone
Analogues
Total synthesis
Structure activity relationship
Bioactivity
1. Introduction
and 14-(S)-MeO epo D [8]. Herein we report the total syntheses
of Epo D and its two conformationally restricted 9-Me analogues,
Compared to palitaxel, epothilones share similar mechanism of
tubulin depolymerization, while show enhanced inhibition activ-
ities against various human cancer cells, especially against multi-
drug-resistant cancer cell lines. The promising bioactivities of
epothilones have attracted vast research on syntheses, conforma-
tional analysis and SAR (structureeactivity relationships) studies of
epothilones [1e6]. However, the bioactive conformation of the
flexible C8eC11 region of epothilones remains to be fully recog-
nized. For example, the 9-oxy epo D (Fig. 1) is thirty times less
active than Epo D [4,5], while the synthetic 9,10-dehydroepothilone
D (Fig. 1) prepared by Danishefsky et al. is more potent than Epo D
[6], and hence entered clinical trial as anti-cancer drug candidate.
Taylor applied the strategy of conformationally restricted ana-
logues to explore the bioactive conformation in region of C10eC14,
which not only provided insights into the biologically active
conformation, but also resulted in the discovery of a few highly
active rational designed analogues including 14-(R)-Me epo D [7]
and the analysis their conformational changes and biological
activities.
2. Results and discussion
2.1. Chemistry
According to the retrosynthetic analysis (Scheme 1), all the three
target molecules can be disconnected into three fragments: frag-
ment A, B and D. And two approaches to synthesize these three
compounds were proposed. Fragment C (R ¼ H) [14] in approach 1,
a known compound, was used to test our new synthetic strategy in
establishing the C6, C7 and C8 chiral centers.
We started the synthesis with compound 1a (Scheme 2) [9]. The
1,4 conjugate addition of Grignard reagent to compound 1a pro-
vided compound 2a with good stereoselectivity (dr >10:1) [10].
Aldehyde 3a was obtained by reduction with DIBAL-H [11]. In this
step, aldehyde 3a is not only highly volatile but also prone to epi-
merize. Fortunately, through aqueous quenching of the reaction,
followed by simple separation of organic phase and precipitation of
the sultam with hexane, the aldehyde 3a was obtained in a hexane
* Corresponding authors. Tel.: þ86 22 2350 8090.
E-mail addresses: jianpinglin@nankai.edu.cn (J. Lin), yuechen@nankai.edu.cn
(Y. Chen).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.003

322
F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
O
OH
11
O
O
OH
O
The keto acid 8 was prepared from sultam 4 via hydrolysis with
LiOH/H2O2 with 72.5% yield (Scheme 3) [16]. Coupling of fragments
8 and 9 with EDCI led to ester 10 in 85.5% yield [14,17], which is the
common precursor for the synthesis of Epo D and its 9-Me ana-
logues. Then, aldol reaction with Ti enolate of 10 and aldehyde 3a in
hexane resulted in the formation of compound 11 with 74% yield
and over 15:1 diastereoselectivity [18], and the next TBS protection
provided known compound 12, which was converted to Epo D
according to the methods in literature: RCM reaction and depro-
tection of TBS groups [14,17,19], and 33.7% yield was obtained when
Zhan catalyst-1B was applied in the RCM step [20].
O
O
N
N
OH
OH
8
8
S
S
O
11
epothilone D
9-oxy epo D
S
O
OH
11
O
O
O
N
OH
O
With the successful synthesis of Epo D, similar strategy was
applied to prepare 9-Me analogues, while with different C7eC12
aldehydes. The various conditions to supply conjugation adducts
2b/2c were listed in Table 1.
N
OH
8
11
S
OH
8
The conjugate addition of Grignard reagent followed by direct
methylation of the generated enolate provided sultam 2c in 75%
(entry 1, Table 1) [21], and the conjugate addition of the same
Grignard reagent to alpha substituted amide [13b,21], followed by
protonation also provided compound 2c as the main product (entry
2, Table 1). The replacement of Grignard reagent with copper
lithium reagent failed to provide any product (entry 3, Table 1) [22].
The addition of methyl magnesium bromide to conjugate amide
provided methyl ketone as the main product (entry 4, Table 1);
however, with catalytic amount of cuprous iodide, compound 2c
was obtained in 62% yield (entry 5, Table 1). Fortunately, with
catalytic amount of cuprous chloride, the conjugate addition fol-
lowed by direct methylation provided sultam 2b and 2c in 70% yield
and 1:1 ratio (entry 6, Table 1) [22,23]. Both compound 2b and 2c
were analyzed with X-ray crystallographic techniques to confirm
their absolute stereochemistry (Fig. 2).
14-(R)-Me epo D
9,10-dehydroepothilone D
Fig. 1. Epo D, 9-oxy epo D, 9,10-dehydroepothilone D and Taylor’s C14-Me epo D.
solution, and was used directly in the next aldol reaction. Then, the
aldol reaction between 3a and the Ti enolate of known ketone
fragment 4 [12] resulted in the formation of 5 in high yield (81%)
and 15:1 diastereoselectivity. Compound 5 was converted to known
compound 7 through TBS protection and hydrolysis to remove the
sultam auxiliary [13]. The structural analysis confirmed that car-
boxylic acid 7 is identical in all aspect to the same molecule in
literature [14]. The synthesis of Epo D from compound 7 was also
reported in the literature [14,15]. This sequence showed that the 1,4
conjugate addition, reduction of sultam derivatives with DIBAL-H
to obtain aldehyde in hexane, and consequently aldol reaction
with Ti enolate is a feasible and efficient strategy to establish the
correct C6, C7 and C8 chiral centers, even with the volatile and
unstable chiral aldehyde. With this strategy, the combinatorial
strategy (approach 2) was applied to prepare Epo D and its 9-Me
analogues.
Aldehydes 3b and 3c were then obtained by reduction with
DIBAL-H and precipitation of the by-product sultam (Scheme 4).
Aldehyde 3b in hexane was used directly in the next aldol reaction,
and the aldol adduct 14 was isolated in 75% yield and over 20:1
diastereoselectivity. The TES protection of 14 supplied compound
Scheme 1. Retrosynthetic analysis of Epo D and its C9-modified analogues.

F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
323
Scheme 2. Synthesis of Epo D through approach 1.
15, and the following RCM reaction with Zhan catalyst-1B provided
16 and 17 as Z/E isomer in about 1:1.7 ratio (Scheme 5). Depro-
tection of two protection groups with TFA/CH2Cl2 provided final
products 18 and 19 respectively.
The 9-(S)-Me epo D 24 along with the E isomer 25 were pre-
pared using the similar sequence to prepare compound 18 (Scheme
6), and the synthetic sequence starting from 10 and 3c was also
only 4 steps.
9-Me epo D (compounds 18 and 24) were more potent than their
corresponding E isomers (compounds 19 and 25). Furthermore, we
evaluated tubulin polymerization of Epo D, compounds 9-(R)-Me
epo D 18 and 9-(S)-Me epo D 24 for their tubulin polymerization
inhibitory activity using literature method [24], tubulin polymeri-
zation for Epo D, compounds 18 and 24 at 5 mM were 43.3%, 15.7%
and 8.5% respectively. This result is consistent to their inhibitory
activities against cancer cells growth.
2.2. Biological activity
2.3. Computational analysis
With Epo D analogues 18, 19, 24, 25 in hand, we conducted a
bioassay against sensitive breast cancer cell line MCF and ADR
resistant breast cancer cell line MCF-7/ADR (Table 2). Although all
analogues were less potent than the mother molecule Epo D, the
9-(R)-Me epo D 18 demonstrated significant higher activity (about
35 times) than the 9-(S)-Me epo D 24. In addition, the Z isomers of
The conformations of epothilones were investigated with
electron-crystallography [25], NMR [3,26e34] and computational
analysis [33e39]. The optimized conformations of Epo D, 18, and 24
were subjected to geometry optimization with DFT at the B3LYP/6-
31G (d) level using Gaussian 03 [40]. Fig. 3 shows the superposition
of conformations of Epo D and 18, Epo D and 24. From Fig. 3, the
Scheme 3. Synthesis of Epo D through approach 2.

324
F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
Table 1
Synthesis of the C7eC12 segment.
Entry
Xc
R1
R2
R3
R4M
2b/2cg
% yieldh
1a
2b
3c
4d
5e
6f
L-camphorsultam
L-camphorsultam
D-camphorsultam
L-camphorsultam
L-camphorsultam
L-camphorsultam
H
H
H
H
H
H
H
Me
Me
Me
CH2]C(CH3)CH2CH2MgBr/HMPA/MeI
CH2]C(CH3)CH2CH2MgBr
[CH2]C(CH3)CH2CH2]2CuLi
CH3MgBr/HPMA/MeI
CuI/CH3MgBr/HPMA/MeI
CH2]C(CH3)CH2CH2MgBr/CuCl/HMPA/MeI
2c
75
67
e
Me
Me
H
H
H
2c
NRi
CH2]C(CH3)CH2CH2
CH2]C(CH3)CH2CH2
Me
Methyl ketone
2c
2b/2c (1:1)
e
62
70j
a
THF, ꢀ78 ꢁC to r.t., 7 h.
b
c
d
e
f
THF, ꢀ78 ꢁC, 3 h.
THF, ꢀ40 ꢁC, 24 h.
THF, ꢀ78 ꢁC to ꢀ40 ꢁC, 3 h.
THF, ꢀ78 ꢁC to r.t., 10 h.
THF, ꢀ78 ꢁC to r.t., 10 h.
The major product after purification.
g
h
i
Yield of the major diastereomer after purification.
No reaction.
The total yield of the 2b and 2c.
j
ring of 18 matches better with Epo D than 24. The RMSD between
the ring atoms of Epo D and 18 is 0.09 Å, whereas the RMSD be-
tween the ring atoms of Epo D and 24 is 0.14 Å. The (S)-methyl
modification changes the shape of the ring of Epo D more than (R)-
methyl. The best docking poses of Epo D, 18 and 24 to the structure
total steps for the syntheses of common precursor E were 6 steps
and 11 steps respectively, and additional 4 linear steps may supply
epothilone D or one of its 9-Me analogues. Therefore, this combi-
natorial approach is highly efficient for the preparation of epothi-
lone analogues with modification at C9 position.
of
a
,
b
-tubulin (PDB ID: 1TVK) using AutoDock v.4.2 (http://
On the other hand, the biological activities of 9-Me analogues
indicated that conformation adopted by 18 (Fig. 3, purple) is
probably closer to the bioactive confirmation than the conforma-
tion adopted by 24 (Fig. 3, yellow), but modification at C9 position
still reduces the biological activity; this phenomena may be
explained by the computer modeling.
autodock.scripps.edu) were further used to calculate the binding
free energy using MM-PBSA from AMBER 11 [41]. The binding en-
ergy of 18 and 24 to the structure of a, b-tubulin are 5.50 kcal/mol
and 8.14 kcal/mol higher than Epo D respectively. This information
is helpful to explain that the biological activities of compounds Epo
D, 18, 24 against MCF-7 cancer cell lines are in the descending order
(IC50 of Epo D, 18, 24 against MCF-7 is 0.014, 0.99, 35.4
mM
4. Experimental section
respectively). Our results indicate that the C9 site is probably not
well tolerated to modification to maintain the bioactivities.
4.1. Chemistry
3. Conclusion
All reactions were carried out under an argon atmosphere with
dry, freshly distilled solvents under anhydrous conditions, unless
otherwise noted. Yields refer to chromatographically and spectro-
scopically (1H NMR) homogeneous materials, unless otherwise
stated. The used solvents were purified and dried according to
common procedures. Other chemicals and solvents were
commercially available. The phosphate buffer solution (pH 7.2,
0.1 M) was prepared by dissolving disodium hydrogen phosphate
(Na2HPO4$12H2O, 25.79 g) and sodium dihydrogen phosphate
(NaH2PO4$2H2O, 4.37 g) in distilled, deionized water (1000 mL).
In summary, we completed the total synthesis of epothilone D
and its 9-Me analogues via a combinatorial strategy. This strategy
involved the common precursor E and varies of aldehydes (com-
pounds 3a, 3b and 3c). The synthesis of each aldehyde was only 3
steps, and the syntheses shared the one-pot strategy of conjugate
addition followed by alkylation. The next reduction with DIBAL-H
provides the volatile and unstable aldehydes in hexane, which
were used directly in the next aldol reactions. The linear steps and
Fig. 2. X-ray of sultam 2c and sultam 2b.

F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
325
Scheme 4. Preparation of fragment B.
High-resolution mass spectra (HRMS) were obtained with an
FTICR-MS (Ion spec 7.0T) spectrometer. 1H NMR spectra were ob-
tained by using a Bruker AC-P 300, AV 400, AV 600. Chemical shifts
are reported in parts per million (ppm) relative to either a tetra-
methylsilane internal standard or solvent signals. Data are reported
as follows: chemical shift, multiplicity (s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, br ¼ broad, m ¼ multiplet), coupling con-
stants and integration. 13C NMR spectra were recorded using a
Bruker AC-P 300 (75 MHz) or AV 400 spectrometer (100 MHz) using
reduced pressure, and the crude product was purified by column
chromatography (5% EtOAc in hexane) to obtain compound 2a as a
white solid (1.63 g, 67.7%). Rf ¼ 0.75 (hexane/EtOAc ¼ 4:1); 1H NMR
(400 MHz, CDCl3)
d
4.66 (m, 2H), 3.89 (t, J ¼ 8.4 Hz, 1H), 3.46 (m,
2H), 3.06 (m, 1H), 1.97e2.05 (m, 4H), 1.74e1.91 (m, 4H), 1.68 (s, 3H),
1.34e1.45 (m, 5H), 1.19 (m, 3H), 1.15 (s, 3H), 0.96 (s, 3H).
4.2.2. Synthesis of compound 3a
To a solution of compound 2a (1.06 g, 3.0 mmol) in CH2Cl2
(6 mL) was added DIBAL-H (4.8 mL, 4.8 mmol, 1 M in CH2Cl2) under
argon atmosphere at ꢀ78 ꢁC. After stirred at ꢀ78 ꢁC for 4 h, the
reaction was quenched by addition of NaHSO4 (1.15 g) in water
(20 mL), and was diluted with hexane (4 mL). The organic phase
was removed by needle to a 25 mL flask which contained 10 g of 4 Å
molecular sieves, and the aqueous phase was extracted with hex-
ane (2 ꢂ 3 mL). The organic solution was concentrated under
reduced pressure carefully until a white crystal appeared. The clear
hexane layer (about 10 mL) was store in ꢀ20 ꢁC overnight, and the
upper clear solution was used for the next step directly.
CDCl3 or CH3CD as the solvent. Chemical shifts (
d) are reported in
parts per million measured relative to the solvent peak. IR spectra
were recorded with
spectrometer.
a Bio-Rad FTS 6000 Fourier infrared
4.2. Synthesis of compounds
4.2.1. Synthesis of compound 2a
To a solution of compound 1 (1.98 g, 7.0 mmol) in THF (30 mL)
was added Grignard reagents (18.9 mmol) under argon atmosphere
at ꢀ78 ꢁC, and the resulting solution was stirred at same temper-
ature for 2 h. Then HMPA (7.4 mL, 49.0 mmol) and CH3I (4.6 mL,
70.0 mmol) was added at ꢀ78 ꢁC and the reaction mixture was
warmed up slowly to 25 ꢁC in 5 h. The reaction was quenched by
addition of sat. NH4Cl (15 mL), and was stirred for 10 min. After the
solvents were removed under reduced pressure, the residue was
diluted with water (20 mL) and EtOAc (50 mL), and the aqueous
phase was extracted with EtOAc (2 ꢂ 30 mL) again. Then the
combined organic phase was washed with H2O (10 mL) and brine
(10 mL), dried over MgSO4. The solvent was removed under
4.2.3. Synthesis of compound 5
To a solution of compound 4 (775 mg, 1.55 mmol) in CH2Cl2
(5 mL) was added TiCl4 (1.7 mL, 1.7 mmol, 1 M in DCM) under argon
atmosphere at ꢀ78 ꢁC, followed by addition of DIEA (0.29 mL,
1.7 mmol) to obtain a dark red solution. The reaction mixture was
stirred at ꢀ78 ꢁC for 1 h. Then compound 3a in hexane (obtained
from above reaction) was added, and the resulting solution was
warmed up slowly to ꢀ12 ꢁC in 10 h. The reaction was quenched
by addition of aqueous of phosphate solution (8 mL, pH ¼ 7.2).
Scheme 5. Preparation of 18 from 10 and 3b.

326
F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
Scheme 6. Preparation of 24 from 10 and 3c.
The resulting solution was stirred for 10 min and was diluted with
Et2O (20 mL). The aqueous phase was extracted with Et2O
(2 ꢂ 10 mL) again, and the combined organic phase was dried over
MgSO4. The solvent was concentrated under reduced pressure. The
crude product was purified by column chromatography (5% EtOAc
addition of TBSOTf (0.95 mL, 4.13 mmol). After stirred at ꢀ45 ꢁC for
6 h, the reaction mixture was warmed to 20 ꢁC. Then the reaction
was quenched by addition of H2O (20 mL), and was diluted with
CH2Cl2 (40 mL) and H2O (20 mL). The aqueous phase was extracted
with CH2Cl2 (2 ꢂ 20 mL), and the combined organic phase was
dried over MgSO4. The solvent was concentrated under reduced
pressure. The resulting residue was purified by column chroma-
in hexane) to obtain alcohol 5 as a colorless oil (404 mg, 81.3%).
20
Rf ¼ 0.50 (hexane/EtOAc ¼ 4:1); [
a
]
D
¼ ꢀ75.0 (c ¼ 10 mg/mL,
CHCl3); IR (KBr/cmꢀ1): 3507, 3073, 2957, 2953, 2886, 2857.6, 1691,
tography (3% EtOAc in hexane) to obtain compound 6 as a colorless
20
1650, 1461,1388,1331,1168,1078, 991, 843, 775; 1H NMR (400 MHz,
oil (1.41 g, 91.0%). Rf ¼ 0.70 (hexane/EtOAc ¼ 4:1); [
a]
¼ ꢀ51.0
D
CDCl3)
d
4.66 (s, 1H), 4.64 (s, 1H), 4.56 (t, J ¼ 4.0 Hz, 1H), 3.85 (m,
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3384, 3374, 2955, 2931,
1H), 3.47 (m, 1H), 3.43 (m, 2H), 3.27 (d, J ¼ 9.2 Hz, 1H), 3.20 (m, 1H),
2.93 (dd, J ¼ 17.6, 5.2 Hz, 1H), 2.60 (dd, J ¼ 17.6, 3.6 Hz, 1H), 2.19e
2.23 (m, 1H), 1.96e2.10 (m, 3H), 1.85e1.90 (m, 3H), 1.69 (s, 3H),
1.24e1.54 (m, 6H), 1.19 (s, 3H), 1.15 (s, 3H), 1.14 (s, 3H), 1.01 (d,
J ¼ 6.8 Hz, 3H), 0.94 (s, 3H), 0.86 (s, 9H), 0.82 (d, J ¼ 6.8 Hz, 4H), 0.10
2886, 2857, 1695, 1464, 1388, 1332, 1252, 1136, 1082, 987, 939, 833,
773; 1H NMR (400 MHz, CDCl3)
d 4.68 (s, 1H), 4.65 (s, 1H), 4.57 (t,
J ¼ 4.4 Hz), 3.86 (m, 1H), 3.73 (m, 1H), 3.42 (m, 2H), 3.10 (m, 1H),
2.90 (dd, J ¼ 17.6, 4.8 Hz, 1H), 2.60 (dd, J ¼ 17.2, 3.6 Hz, 1H), 2.25e
2.29 (m, 1H), 1.95e2.12 (m, 3H), 1.88e1.90 (m, 3H), 1.69 (s, 3H),
1.26e1.58 (m, 7H), 1.22 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 1.03 (d,
J ¼ 6.8 Hz, 3H), 0.96 (s, 3H), 0.89 (s, 9H), 0.87 (s, 9H), 0.85 (m, 4H),
0.11 (s, 3H), 0.07 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H); 13C NMR
(s, 3H), 0.07 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 170.1, 146.4, 109.8,
75.1, 72.1, 65.4, 54.6, 53.2, 48.6, 47.9, 44.8, 41.8, 41.0, 38.5, 38.4, 35.5,
33.0, 32.7, 26.6, 26.1, 24.9, 22.7, 22.6, 21.0, 20.1, 18.6, 18.2, 15.6,
9.8, ꢀ4.1, ꢀ4.9; HRMS (ESI) (M þ Hþ) calculated for C34H61NO6SSi:
640.4062, found 640.4063.
(100 MHz, CDCl3)
d 218.1, 170.2, 146.0, 110.0, 78.3, 72.2, 65.3,
54.3, 53.1, 48.5, 47.9, 45.3, 44.9, 42.2, 38.5, 38.4, 33.0, 30.2,
26.6, 26.4, 26.1, 25.7, 23.4, 22.5, 21.0, 20.1, 18.7, 18.3, 18.2,
16.2, ꢀ3.4, ꢀ3.5, ꢀ4.1, ꢀ4.8; HRMS (ESI) (M þ Hþ) calculated for
C40H75NO6SSi2: 754.4926, found 754.4935.
4.2.4. Synthesis of compound 6
To a solution of alcohol 5 (1.32 g, 2.06 mmol) in CH2Cl2 (15 mL)
was added 2,6-lutidine (0.72 mL, 6.20 mmol) at ꢀ45 ꢁC, followed by
4.2.5. Synthesis of compound 7
To a solution of compound 6 (613 mg, 0.81 mmol) in THF (35 mL)
and H2O (8.7 mL) was added LiOH$H2O (509 mg, 12.1 mmol) and
H2O2 (10.8 mL, 30%, 131.2 mmol) at 0 ꢁC. The reaction mixture was
stirred at the same temperature for 30 min, and was stirred at 25 ꢁC
for 24 h. The reaction mixture was quenched by addition of satu-
rated NaHSO4 (50 mL) at 0 ꢁC, and was stirred for 10 min. The
solvent was removed under reduced pressure, and NaHSO4 solid
was added to adjust the pH ¼ 4e5. The resulting solution was
extracted with EtOAc (3 ꢂ 30 mL), and the combined organic phase
was dried over MgSO4. The solvent was concentrated under
reduced pressure. The resulting residue was purified by column
chromatography (0.5% AcOH and 6% EtOAc in hexane) to obtain acid
Table 2
Cytotoxocity and tubulin polymerization inhibitory activity of Epo D and its new
analogues.
Compound
MCF-7
(IC50
MCF-7/ADR
(IC50
M)a
% tubulin polymerization
at 5 mM
,
m
M)a
,
m
Epo D
18
19
24
25
0.014
0.99
7.7
35.4
93.4
0.010
0.51
7.3
43.3
15.7
NDb
8.5
NDb
>100
NDb
a
Cells were exposed to compounds for 72 h.
Not determined.
b

F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
327
Fig. 3. Left: Superposition of geometry optimized conformations of Epo D (cyan) and 18 (purple). Right: Superposition of geometry optimized conformations of Epo D (cyan) and 24
(yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
7 as a colorless oil (252 g, 56.0%). Rf ¼ 0.50 (1% AcOH and 20% EtOAc
Et2O (2 ꢂ 120 mL). The combined organic phase was dried over
MgSO4, and the solvent was concentrated under reduced pressure.
The crude product was purified by column chromatograph (10%
in hexane); IR (KBr/cmꢀ1): 3074, 2954, 2930, 2857, 1711, 1650, 1469,
1253,1086, 987, 941, 876, 832, 773; 1H NMR (100 MHz, CDCl3)
d 4.69
(s, 1H), 4.66 (s, 1H), 4.39 (dd, J ¼ 6.8, 3.2 Hz, 1H), 3.78 (d, J ¼ 6.4 Hz,
1H), 3.14 (dq, J ¼ 7.2, 7.2 Hz, 1H), 2.49 (dd, J ¼ 16.4, 3.2 Hz, 1H), 2.30
(dd, J ¼ 16.4, 6.4 Hz, 1H), 2.04e1.96 (m, 2H), 1.70 (s, 3H), 1.63e1.36
(m, 5H), 1.24 (s, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 1.05 (d, J ¼ 6.8 Hz, 3H),
0.92 (d, J ¼ 7.2 Hz, 3H), 0.90 (s, 9H), 0.89 (s, 9H), 0.10 (s, 3H), 0.06
EtOAc in hexane) to obtain ester 10 as a colorless oil (1.60 g, 85.5%).
20
Rf ¼ 0.65 (0.5% AcOH and 7% EtOAc in hexane); [
a
]
¼ ꢀ28.2
D
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 2954, 2930, 2856,1737, 1705,
1643, 1505, 1471, 1374, 1293, 1248, 1177, 1088, 1047, 825, 769; 1H
NMR (400 MHz, CDCl3) d 6.94 (s, 1H), 6.48 (s, 1H), 5.71 (m, 1H), 5.28
(3s, 9H); 13C NMR (400 MHz, CDCl3)
d
218.5, 177.6, 146.2, 110.0, 77.9,
(t, J ¼ 6.8 Hz, 1H), 5.11e5.03 (m, 2H), 4.46 (dd, J ¼ 6.4, 4.0 Hz, 1H),
2.69 (s, 3H), 2.60e2.42 (band, 5H), 2.31 (dd, J ¼ 16.8, 6.4 Hz, 1H),
2.06 (s, 3H), 1.09 (s, 3H), 1.05 (s, 3H), 0.97 (t, J ¼ 7.2 Hz, 3H), 0.84 (s,
73.6, 53.7, 45.4, 40.3, 38.8, 38.5, 31.2, 30.5, 29.9, 26.4, 26.2, 25.8,
25.7, 23.8, 22.6, 19.1, 18.7, 18.4, 18.0, 16.0, ꢀ3.4, ꢀ3.5, ꢀ4.1, ꢀ4.4.
9H), 0.06 (s, 3H), 0.02 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 215.2,
4.2.6. Synthesis of compound 8
171.3, 164.8, 152.6, 136.9, 133.5, 121.4, 118.1, 116.6, 79.0, 73.7, 52.8,
39.7, 37.7, 31.9, 26.1, 21.1, 20.9,19.4,18.3,14.8,17.9, ꢀ4.0, ꢀ4.8; HRMS
(ESI) (M þ Hþ) calculated for C26H43NO4SSi: 494.2755, found
494.2759.
To a solution of compound 4 (7.5 g, 15.0 mmol) in THF (620 mL)
and H2O (156 mL) was added LiOH$H2O (9.45 g, 22.5 mmol) and
H2O2 (200 mL, 30%) at 0 ꢁC. The reaction mixture was stirred at the
same temperature for 30 min, and was stirred at 25 ꢁC for 24 h.
Then the reaction was quenched by addition of NaHSO3 (66.8 mg)
in water (800 mL) at 0 ꢁC, and was stirred for 10 min. The solution
was concentrated under reduced pressure, and NaHSO4 was added
to adjust the pH ¼ 4e5. The solution was extracted with EtOAc
(3 ꢂ 100 mL), and the organic phase was dried over MgSO4. The
solvent was concentrated under reduced pressure. The resulting
residue was purified by column chromatography (0.5% AcOH and
7% EtOAc in hexane) to obtain acid 8 as a colorless oil (3.29 g, 72.5%).
Rf ¼ 0.30 (1% AcOH and 20% EtOAc in hexane); 1H NMR (400 MHz,
4.2.8. Synthesis of compound 11
To a solution of ester 10 (253 mg, 0.51 mmol) in CH2Cl2 (2 mL)
was added TiCl4 (0.56 mL, 1 M in CH2Cl2) at ꢀ78 ꢁC, followed by
addition of DIEA (0.09 mL, 0.56 mmol) to obtain a dark red solution.
After stirred at ꢀ78 ꢁC for 1 h, compound 3a (1.5 mmol) was added
to the reaction solution. The reaction mixture was warmed up
slowly to ꢀ12 ꢁC, and was stirred for 15 h. Then the reaction was
quenched by addition of aqueous of phosphate solution (3 mL,
pH ¼ 7.2), and was stirred for 10 min. The resulting mixture was
diluted with Et2O (20 mL), the aqueous layer was extracted with
Et2O (20 mL) again. The combined organic phase was dried over
MgSO4, and the solvent was concentrated under reduced pressure.
The crude product was purified by column chromatograph (5%
CDCl3)
1.13 (s, 3H), 1.07 (s, 3H), 0.99 (t, J ¼ 7.2 Hz, 3H), 0.83 (s, 9H), 0.04 (s,
3H), 0.02 (s, 3H); 13C NMR (100 MHz, CDCl3)
215.4, 178.3, 73.7,
d
4.47 (m, 1H), 2.51 (m, 3H), 2.31 (dd, J ¼ 16.4, 6.8 Hz, 1H),
d
52.8, 39.5, 32.0, 26.1, 21.2, 20.8, 18.3, 7.9, ꢀ4.2, ꢀ4.7.
EtOAc in hexane) to obtain alcohol 11 as a colorless oil (239 mg,
20
4.2.7. Synthesis of compound 10
74.0%). Rf ¼ 0.35 (hexane/EtOAc ¼ 9:1); [
a]
D
¼ ꢀ51.4 (c ¼ 10 mg/
To a solution of compound 8 (1.1 g, 3.62 mmol) and alcohol 9
(1.51 g, 7.22 mmol) in CH2Cl2 (20 mL) was added DMAP (0.885 g,
7.22 mmol) and EDCI (2.77 g, 14.44 mmol) at room temperature,
and the reaction mixture was stirred at 20 ꢁC for 2 h. Then the
reaction was diluted with water (60 mL), and was extracted with
mL, CHCl3); IR (KBr/cmꢀ1): 2929, 2856, 1735, 1684, 1506, 1471, 1376,
1292, 1252, 1178, 1079, 976, 283, 832, 776; 1H NMR (400 MHz,
CDCl3)
d
6.94 (s, 1H), 6.48 (s, 1H), 5.70 (m, 1H), 5.28 (t, J ¼ 6.8 Hz,
1H), 5.07 (d, J ¼ 17.2 Hz, 1H), 5.04 (d, J ¼ 10.0 Hz, 1H), 4.66 (s, 1H),
4.64 (s, 1H), 4.40 (m, 1H), 3.44 (s, 1H), 3.26 (m, 2H), 2.69 (s, 3H),

328
F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
2.48e2.43 (band, 3H), 2.31 (dd, J ¼ 17.2, 6.0 Hz, 1H), 2.05 (s, 3H),
1.96 (m, 2H), 1.69 (s, 3H), 1.69e1.31 (band, 4H), 1.18 (s, 3H), 1.09 (s,
3H), 1.02 (d, J ¼ 6.8 Hz, 3H), 0.87 (s, 9H), 0.82 (d, J ¼ 6.8 Hz, 3H), 0.10
the resulting mixture was stirred at ꢀ78 ꢁC for 2 h. Then HMPA
(31.7 mL, 220.5 mmol) and CH3I (19.7 mL, 315 mmol) was added by
syringe at ꢀ78 ꢁC, and the reaction mixture was warmed up slowly
to 20 ꢁC in 5 h. The reaction was quenched by addition of sat NH4Cl
(50 mL), and was stirred for 10 min. The organic solvent was
removed by reduced pressure. The residue diluted with water
(100 mL), and was extracted with EtOAc (3 ꢂ 150 mL). The com-
bined organic phase was washed with H2O (50 mL) and brine
(50 mL), dried over MgSO4. The solvent was concentrated under
reduced pressure. The crude product purified by column chro-
(s, 3H), 0.05 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 171.1, 164.8, 152.6,
146.4, 136.8, 133.5, 121.3, 118.0, 116.6, 109.8, 79.0, 74.8, 73.7, 54.1,
41.5, 40.4, 38.4, 37.7, 35.7, 32.8, 26.2, 25.0, 22.6, 22.2, 20.2, 19.4, 18.3,
15.5, 14.8, 9.9, ꢀ4.1, ꢀ4.6; HRMS (ESI) (M þ Hþ) calculated for
C35H59NO5SSi: 634.3956, found 634.3954.
4.2.9. Synthesis of compound 12
To a solution of alcohol 11 (200 mg, 0.32 mmol) in CH2Cl2 (5 mL)
was added 2,6-lutidine (0.11 mL, 0.95 mmol) at ꢀ45 ꢁC, followed by
addition of TBSOTf (0.14 mL, 0.63 mmol). The reaction mixture was
warmed up to 20 ꢁC after stirred at ꢀ45 ꢁC for 5 h. Then the reaction
was quenched by addition of H2O (5 mL), and was diluted with
CH2Cl2 (20 mL) and H2O (20 mL). The aqueous layer was extracted
with CH2Cl2 (2 ꢂ 20 mL), and the combined organic phase was
dried over MgSO4. The solvent was concentrated under reduced
pressure. The crude product was purified by column chromato-
graph (3% EtOAc in hexane) to obtain compound 12 as a colorless oil
(184 mg, 78.2%). Rf ¼ 0.75 (hexane/EtOAc ¼ 9:1); 1H NMR
matograph (5% EtOAc in hexane) to obtain compound 2c as a white
20
solid (8.65 g, 75.0%). Rf ¼ 0.70 (hexane/EtOAc ¼ 4:1); [
a
]
¼ þ82.0
D
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3073, 2965,1683, 1649,1453,
1411, 1391, 1323, 1271, 1219, 1162, 1135, 1064, 1038, 879, 773, 723,
640, 620, 501, 447; 1H NMR (400 MHz, CDCl3)
d 4.66 (s, 2H), 3.89 (t,
J ¼ 6.4 Hz,1H), 3.46 (q, J ¼ 26.8,14.0 Hz, 2H), 2.88 (m,1H), 2.10e2.04
(band, 2H), 1.97e1.81 (band, 5H), 1.70 (s, 3H), 1.68 (m, 1H), 1.42e
1.30 (band, 2H), 1.18 (d, J ¼ 7.2 Hz, 3H), 1.14 (s, 3H), 0.95 (s, 3H), 0.93
(d, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 176.0, 145.9, 109.7,
65.0, 53.2, 48.1, 47.6, 45.7, 44.5, 38.4, 34.6, 34.4, 32.7,30.6 26.4,
22.4, 20.8, 19.8, 17.6, 16.5; HRMS (ESI) (M þ Hþ) calculated for
C20H34NO3S: 368.2254, found 368.2251.
(400 MHz, CDCl3)
d 6.94 (s, 1H), 6.49 (s, 1H), 5.70 (m, 1H), 5.29 (t,
J ¼ 6.8 Hz, 1H), 5.05 (m, 2H), 4.68 (s, 1H), 4.65 (s, 1H), 4.34 (dd,
J ¼ 6.0, 3.6 Hz, 1H), 3.72 (d, J ¼ 4.8 Hz, 1H), 3.15 (m, 1H), 2.69 (s, 3H),
2.54e2.44 (band, 3H), 2.28 (dd, J ¼ 16.8, 6.0 Hz, 1H), 2.06 (s, 3H),
1.97 (m, 2H), 1.69 (s, 3H), 1.50 (m, 1H), 1.33e1.29 (band, 3H), 1.25e
1.21 (band, 4H), 1.04e1.03 (band, 6H), 0.91e0.88 (band, 21H), 0.10
(s, 3H), 0.06 (s, 3H), 0.04 (s, 6H); 13C NMR (100 MHz, CDCl3)
4.2.13. Synthesis of compound 2b
To a solution of Grignard reagents (41.0 mmol) and CuCl (1 g,
cat) was added compound 1b (4.40 g, 15.55 mmol, entry 6, Table 1)
in THF (200 mL) at ꢀ78 ꢁC, and the resulting mixture was stirred
at ꢀ78 ꢁC for 2 h. Then HMPA (14.8 mL, 108.9 mmol) and CH3I
(9.2 mL, 155.5 mmol) was added by syringe at ꢀ78 ꢁC, and the re-
action mixture was warmed up slowly to 20 ꢁC in 5 h. The reaction
was quenched by addition of sat NH4Cl (30 mL), and was stirred for
10 min. The organic solvent was removed under reduced pressure.
The residue was diluted with water (40 mL), and was extracted
with EtOAc (3 ꢂ 100 mL). The combined organic phase was washed
with H2O (30 mL) and brine (30 mL), dried over MgSO4. The solvent
was concentrated under reduced pressure. The crude product was
purified by column chromatograph (5% EtOAc in hexane) to obtain
d
217.9, 171.4, 164.7, 152.7, 146.2, 137.0, 133.6, 121.3, 118.0, 116.6,
110.0, 78.9, 77.8, 74.2, 53.5, 45.4, 40.5, 38.9, 38.5, 37.7, 31.1, 30.7,
26.4, 26.2, 25.9, 23.4, 22.6, 20.5, 19.4, 18.7, 18.4, 17.9, 15.6, 14.8,
1.2, ꢀ3.4, ꢀ3.6, ꢀ4.1, ꢀ4.5.
4.2.10. Synthesis of compound 13
To a solution of Zhan Catalyst-1B (20 mg, 0.027 mmol) in 1,2-
dichoroethane (80 mL) was added compound 12 (55 mg,
0.074 mmol) in 1,2-dichoroethane (3 mL) at 80 ꢁC. After stirred for
4 h, the reaction mixture was cooled to 20 ꢁC, and was stirred at
20 ꢁC for overnight. The reaction solution was concentrated under
reduced pressure. The crude product was purified by column
chromatograph (2% EtOAc in hexane) to obtain compound 13 and
its isomer as a colorless oil (18 mg, 33.7%). Rf ¼ 0.70 (hexane/
EtOAc ¼ 9:1).
compound 2b as a white solid (2.02 g, 35.2%). Rf ¼ 0.70 (hexane/
20
EtOAc ¼ 4:1); [
a]
¼ þ46.8 (c ¼ 10 mg/mL, CHCl3). IR (KBr/cmꢀ1):
D
3339, 3080, 2979, 1774, 1678, 1649, 1456, 1418, 1383, 1336, 1270,
1218, 1194, 1136, 1063, 1039, 878, 684, 588; 1H NMR (100 MHz,
CDCl3)
d
4.66 (s, 2H), 3.90 (t, J ¼ 6.2 Hz,1H), 3.46 (q, J ¼ 25.2, 13.6 Hz,
2H), 2.88 (m, 1H), 2.11e2.04 (band, 3H), 1.99e1.84 (band, 5H), 1.70
(s, 3H), 1.52 (m, 1H), 1.40e1.23 (band, 2H), 1.17 (d, J ¼ 7.2 Hz, 3H),
1.15 (s, 3H), 0.96 (s, 3H), 0.92 (d, J ¼ 6.8 Hz, 3H); 13C NMR (400 MHz,
4.2.11. Synthesis of epothilone D
To a solution of 13 (5 mg, 0.0069 mmol) was added a freshly
prepared 20% CF3COOH solution in CH2Cl2 (0.5 mL) at ꢀ20 ꢁC. The
reaction mixture was stirred at ꢀ20 ꢁC for 1 h, and warmed to 0 ꢁC
in 2 h. Sodium Bicarbonate was added to adjust the pH to 7. The
resulting solution was extracted with EtOAc (3 ꢂ 2 mL), and the
combined organic phase was concentrated under reduced pressure.
The crude product was purified by column chromatograph (10%
EtOAc in hexane) to obtain Epo D as a colorless oil (3 mg, 87.9%).
CDCl3) d 176.1, 146.0, 109.7, 65.0, 53.2, 48.1, 47.7, 46.0, 44.5, 38.4,
35.3, 33.9, 33.2,32.7, 26.4, 22.4, 20.8, 19.8, 16.3, 15.5; HRMS (ESI)
(M þ Hþ) calculated for C20H34NO3S: 368.2254, found 368.2256.
4.2.14. Synthesis of compound 3c
To a solution of compound 2c (682 mg, 1.86 mmol) in CH2Cl2
(5 mL) was added DIBAL-H (3.2 mL, 3.2 mmol, 1 M in CH2Cl2)
at ꢀ78 ꢁC. The reaction mixture was stirred at ꢀ78 ꢁC for 4 h. Then
the reaction was quenched by addition of NaHSO4 (800 mg) in
water (8 mL), and was diluted with hexane (4 mL). The organic
phase was removed by syringe to a 25 mL flask which contained 8 g
4 Ao molecular sieve. The aqueous phase was extracted with hex-
ane (3 mL). The combined organic phase was concentrated under
reduced pressure carefully until a white crystal appeared. The clear
hexane layer (about 7 mL) was store in ꢀ20 ꢁC overnight, and the
upper clear solution was used for the next step directly.
Rf ¼ 0.75 (hexane/EtOAc ¼ 1:1); 1H NMR (400 MHz, CDCl3)
d 6.95 (s,
1H), 6.58 (s, 1H), 5.21 (d, J ¼ 8.8 Hz, 1H), 5.14 (dd, J ¼ 10.0, 4.8 Hz,
1H), 4.29 (d, J ¼ 7.2 Hz,1H), 3.72 (m,1H), 3.54 (br s,1H), 3.16 (m,1H),
3.05 (br s, 1H), 2.69 (s, 3H), 2.65e2.59 (band, 1H), 2.46 (dd, J ¼ 14.8,
11.2 Hz,1H), 2.35e2.21 (band, 3H), 2.06 (d, J ¼ 0.8 Hz, 3H),1.91e1.85
(m, 1H), 1.76e1.68 (band, 2H), 1.66 (s, 3H), 1.34 (s, 3H), 1.31e1.25
(band, 4H), 1.19 (d, J ¼ 6.8 Hz, 3H), 1.07 (s, 3H), 1.01 (d, J ¼ 6.8 Hz,
3H).
4.2.12. Synthesis of compound 2c
4.2.15. Synthesis of compound 3b
To the solution of Grignard reagents (63.0 mmol) was added 1b
To a solution of compound 2b (1.39 g, 3.94 mmol) in CH2Cl2
(8.9 g, 31.5 mmol, Entry 1, Table 1) in THF (130 mL) at ꢀ78 ꢁC, and
(8 mL) was added DIBAL-H (6 mL, 6 mmol, 1 M in CH2Cl2) at ꢀ78 ꢁC.

F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
329
The reaction mixture was stirred at ꢀ78 ꢁC for 4 h. Then the reac-
tion was quenched by addition of NaHSO4 (1.44 mg) in water
(11 mL), and was diluted with hexane (8 mL). The organic phase
was removed by syringe to a 50 mL flask which contained 15 g 4 Ao
molecular sieve. And the aqueous phase extracted with hexane
(5 mL). The combined organic phase was concentrated under
reduced pressure carefully until a white crystal appeared. The clear
hexane layer (about 13 mL) was store in ꢀ20 ꢁC overnight, and the
upper clear solution was used for the next step directly.
4.2.18. Synthesis of compounds 16 and 17
To a solution of Zhan Catalyst-1B (25 mg, 0.034 mmol) in 1,2-
dichoroethane (125 mL) was added compound 15 (95 mg,
0.125 mmol) in 1,2-dichoroethane (5 mL) slowly at 80 ꢁC. After
stirred for 4 h, the reaction mixture was cooled to 20 ꢁC, and was
stirred for overnight. Then the reaction solution was concentrated
under reduced pressure. The crude product was purified by column
chromatograph (2% EtOAc in hexane) to obtain compound 16 and
compound 17 as a colorless oil (55 mg, 60.0%). Rf ¼ 0.70 (hexane/
20
EtOAc ¼ 9:1); Compound 16: [
a]
¼ ꢀ7.2 (c ¼ 10 mg/mL, CHCl3); IR
D
4.2.16. Synthesis of compound 14
(KBr/cmꢀ1): 3151, 2972, 2902, 2841, 2723, 1739, 1696, 1505, 1461,
To a solution of ester 10 (705 mg, 1.43 mmol) in CH2Cl2 (7 mL)
was added TiCl4 (1.6 mL, 1 M in CH2Cl2) at ꢀ78 ꢁC, followed by
addition of DIEA (0.27 mL, 1.6 mmol). After stirred at ꢀ78 ꢁC for 1 h,
a solution of compound 3b (1.7 mmol) in hexane (6 mL) was added
to the reaction solution. The resulting reaction mixture was
warmed up slowly to ꢀ12 ꢁC in 10 h. Then the reaction was
quenched by addition of aqueous of phosphate solution (3 mL,
pH ¼ 7.2). The resulting solution was stirred for 10 min and was
diluted with Et2O (30 mL). The aqueous layer was extracted with
Et2O (2 ꢂ 10 mL), and the combined organic phase was dried over
MgSO4. The solvent was concentrated under reduced pressure. The
crude product was purified by column chromatography (5% EtOAc
1410, 1379, 1299, 1249, 1200, 1161, 983, 939, 775; 1H NMR
(400 MHz, CDCl3)
d 6.96 (s, 1H), 6.56 (s, 1H), 5.13 (m, 1H), 5.04 (dd,
J ¼ 9.2, 2.4 Hz, 1H), 4.10e4.08 (band, 2H), 3.07 (m, 1H), 2.71 (s, 3H),
2.76e2.67 (band, 1H), 2.59 (dd, J ¼ 16.0, 8.4 Hz, 1H), 2.34 (m, 1H),
2.11 (s, 3H), 1.81e1.71 (band, 2H), 1.65 (s, 3H), 1.49e1.39 (band, 2H),
1.25 (s, 3H), 1.19 (s, 3H), 1.15 (s, 3H), 1.10 (d, J ¼ 6.8 Hz, 3H), 0.99 (t,
J ¼ 8.0 Hz, 9H), 0.93e0.91 (band, 6H), 0.85 (s, 9H), 0.67 (q, J ¼ 16.0,
8.0 Hz, 6H), 0.10 (s, 3H), ꢀ0.08 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
213.8, 169.9, 163.5, 151.7, 139.5, 137.4, 118.9, 118.1, 115.1, 78.9,
74.7, 52.4, 46.5, 40.5, 38.8, 34.0, 31.9, 31.2, 28.8, 28.7, 25.1, 25.0,
23.5, 23.3, 18.2, 17.5, 16.3, 14.1, 13.9, 13.2, 6.2, 4.5, ꢀ4.7, ꢀ6.5; HRMS
(ESI) (M þ Hþ) calculated for C40H72NO5SSi2: 734.4664, found
734.4664.
in hexane) to obtain alcohol 14 as a colorless oil (701 mg, 75.5%).
20
Rf ¼ 0.35 (hexane/EtOAc ¼ 9:1); [
a
]
D
¼ ꢀ37.8 (c ¼ 10 mg/mL,
CHCl3); IR (KBr/cmꢀ1): 3519, 3069, 2953, 2856, 1730, 1681, 1645,
4.2.19. Synthesis of compound 18
1506, 1464, 1379, 1290, 1253, 1183, 1095, 997, 968, 875, 835, 777; 1H
To a solution of 16 (30 mg, 0.041 mmol) was added a freshly
prepared 20% CF3COOH solution in CH2Cl2 (0.7 mL) at ꢀ20 ꢁC. The
reaction mixture was stirred at ꢀ20 ꢁC for 1 h, and warmed to 0 ꢁC
in 30 min. Sodium bicarbonate was added to adjust the pH to 7. The
resulting solution was extracted with EtOAc (3 ꢂ 3 mL), and the
combined organic mixture was concentrated under reduced pres-
sure. The crude product was purified by column chromatograph
NMR (400 MHz, CDCl3) d 6.94 (s, 1H), 6.48 (s, 1H), 5.71 (m, 1H), 5.29
(t, J ¼ 6.8 Hz, 1H), 5.07 (m, 2H), 4.66 (br s, 2H), 4.43 (t, J ¼ 4.8 Hz,
1H), 3.46 (s, 1H), 3.39 (d, J ¼ 9.6 Hz, 1H), 3.23 (m, 1H), 2.69 (s, 3H),
2.50e2.42 (band, 3H), 2.33 (dd, J ¼ 17.2, 6.0 Hz, 1H), 2.06 (s, 3H),
2.03e1.96 (band, 3H), 1.71 (s, 3H), 1.60 (m, 1H), 1.35e1.25 (band,
2H), 1.17 (s, 3H), 1.12 (s, 3H), 1.04e1.03 (band, 3H), 0.88 (s, 9H),
0.69e0.67 (band, 6H), 0.10 (s, 3H), 0.06 (s, 3H). 13C NMR (100 MHz,
(10% EtOAc in hexane) to obtain target molecule 18 as a colorless oil
20
CDCl3)
d
170.9, 164.5, 152.4, 146.6, 136.6, 133.3, 121.2, 117.8, 116.4,
(15 mg, 72.4%). Rf ¼ 0.25 (hexane/EtOAc ¼ 3:1); [
a
]
D
¼ ꢀ58.6
109.3, 78.9, 73.3, 71.7, 54.0, 41.1, 40.1, 38.4, 37.5, 36.0, 34.1, 31.1, 26.0,
22.5, 21.9, 19.9, 19.2, 18.1, 14.6, 12.6, 9.4, 9.3, ꢀ4.3, ꢀ4.9; HRMS (ESI)
(M þ Hþ) calculated for C36H62NO5SSi: 648.4112, found 648.4112.
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3496, 2958, 2931, 1737, 1685,
1507, 1376, 1249, 1155, 1083, 1002, 977, 858, 849, 765, 704; 1H NMR
(400 MHz, CDCl3)
d
6.94 (s, 1H), 6.57 (s, 1H), 5.22 (d, J ¼ 10.2 Hz, 1H),
5.07 (dd, J ¼ 9.2, 2.4 Hz 1H), 4.30 (dd, J ¼ 10.8, 2.0 Hz, 1H), 3.81 (d,
J ¼ 6.4 Hz, 1H), 3.60 (br s, 1H), 3.23 (q, J ¼ 6.8 Hz, 1H), 3.18 (s, 1H),
2.68 (s, 3H), 2.60 (m, 1H), 2.45 (m, 1H), 2.33e2.19 (band, 3H), 2.06
(s, 3H), 1.78 (m, 1H), 1.68 (s, 3H), 1.66e1.58 (band, 1H), 1.44e1.36
(band, 1H), 1.33 (s, 3H), 1.28e1.21 (band, 2H), 1.12 (t, J ¼ 8.0 Hz, 6H),
1.06 (s, 3H), 1.00 (d, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3)
4.2.17. Synthesis of compound 15
To a solution of alcohol 14 (183 mg, 0.283 mmol) in CH2Cl2
(5 mL) was added 2, 6-lutidine (0.14 mL, 1.13 mmol) at ꢀ78 ꢁC,
followed by addition of TESOTf (0.20 mL, 0.849 mmol). After stirred
1.5 h at ꢀ78 ꢁC, the reaction was quenched by addition of sat
NaHCO3 (7 mL), diluted with CH2Cl2 (20 mL) and H2O (20 mL). The
aqueous layer was extracted with CH2Cl2 (2 ꢂ 20 mL). The com-
bined organic phase was dried over MgSO4, and the solvent was
concentrated under reduced pressure. The crude product was pu-
rified by column chromatograph (3% EtOAc in hexane) to obtain
d
221.0, 170.6, 165.1, 152.0, 139.5, 139.4, 119.2, 119.2, 115.7, 79.1, 72.2,
72.1, 53.8, 41.8, 41.8, 39.8, 38.0, 33.0, 32.8, 30.9, 23.9, 22.2, 19.7, 19.1,
17.7, 16.2, 15.7, 11.6; HRMS (ESI) (M
C28H44NO5S: 506.2935, found 506.2934.
þ
Hþ) calculated for
compound 15 as a colorless oil (195 mg, 90.1%). Rf ¼ 0.75 (hexane/
4.2.20. Synthesis of compound 19
20
EtOAc ¼ 9:1); [
a
]
¼ ꢀ48.8 (c ¼ 10 mg/mL, CHCl3). IR (KBr/cmꢀ1):
To a solution of 17 (35 mg, 0.047 mmol) was added a freshly
prepared 20% CF3COOH solution in CH2Cl2 (0.7 mL) at ꢀ20 ꢁC. The
reaction mixture was stirred at ꢀ20 ꢁC for 1.5 h, and warmed to 0 ꢁC
in 30 min. Sodium Bicarbonate was added to adjust the pH to 7. The
resulting solution extracted with EtOAc (3 ꢂ 3 mL), and the com-
bined organic mixture was concentrated under reduced pressure.
The crude product was purified by column chromatograph (10%
D
3077, 2957, 2878, 2858, 1737, 1699, 1646, 1506, 1471, 1382, 1293,
1253, 1181, 1081, 988, 885, 817, 812; 1H NMR (400 MHz, CDCl3)
d
6.94 (s, 1H), 6.49 (s, 1H), 5.71 (m, 1H), 5.28 (t, J ¼ 6.8 Hz, 1H), 5.06
(m, 2H), 4.67 (d, J ¼ 4.4 Hz, 2H), 4.22 (m, 1H), 3.69 (dd, J ¼ 5.7,
2.0 Hz, 1H), 3.22 (m, 1H), 2.69 (s, 3H), 2.62 (dd, J ¼ 17.2, 3.2 Hz, 1H),
2.47 (m, 2H), 2.21 (m, 1H), 2.06 (s, 3H), 1.99 (m, 1H), 1.71 (s, 3H), 1.48
(m, 1H), 1.36e1.25 (band, 7H), 1.04 (s, 3H), 0.99 (d, J ¼ 6.4 Hz, 3H),
0.94 (t, J ¼ 8.0 Hz, 9H), 0.87 (s, 9H), 0.75 (t, J ¼ 6.6 Hz, 6H), 0.59 (m,
EtOAc in hexane) to obtain target molecule 19 as a colorless oil
20
(21 mg, 85.5%). Rf ¼ 0.25 (hexane/EtOAc ¼ 3:1); [
a]
¼ ꢀ85.2
D
6H), 0.11 (s, 3H), 0.05 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
171.2,
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3493, 2969, 2930,1776,1736,
164.5, 152.5, 146.4, 136.7, 133.4, 121.2, 117.8, 116.4, 109.5, 78.8, 75.4,
74.4, 52.9, 44.7, 41.7, 39.9, 37.5, 36.0, 34.6, 31.4, 26.0, 22.7, 22.5,
21.4, 19.2, 18.1, 14.5, 13.8, 11.1, 11.0, 7.2, 5.5, ꢀ4.5, ꢀ4.6; HRMS
(ESI) (M þ Hþ) calculated for C42H76NO5SSi2: 762.4977, found
762.4976.
1689, 1508, 1376, 1181, 1026, 960, 852, 713; 1H NMR (400 MHz,
CDCl3)
d
6.96 (s, 1H), 6.59 (s, 1H), 5.26 (t, J ¼ 5.2 Hz, 1H), 5.10
(t, J ¼ 7.2 Hz, 1H), 4.32 (d, J ¼ 10.0 Hz, 1H), 4.07 (br s, 1H), 3.71
(d, J ¼ 4.8 Hz, 1H), 3.48e3.43 (band, 2H), 2.69 (s, 3H), 2.56 (m,
1H), 2.43e2.29 (band, 3H), 2.12 (m, 1H), 2.04 (s, 3H), 1.91 (m, 1H),

330
F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
1.68e1.61 (band, 1H), 1.59 (s, 3H), 1.53e1.45 (band, 1H), 1.32 (s, 3H),
1.27e1.21 (band, 2H), 1.16 (d, J ¼ 7.2 Hz, 3H), 1.03 (s, 3H), 0.93 (d,
J ¼ 6.8 Hz, 3H), 0.80 (d, J ¼ 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)
4.2.23. Synthesis of compounds 22 and 23
To a solution of Zhan Catalyst-1B (40 mg, 0.055 mmol) in 1,2-
dichoroethane (180 mL) was added compound 21 (180 mg,
0.236 mmol) in 1,2-dichoroethane (10 mL) slowly at 80 ꢁC. After
stirred at the same temperature for 4 h, the reaction mixture was
cooled to 20 ꢁC, and was stirred for overnight. Then the reaction
solution was concentrated under reduced pressure. The crude
product was purified by column chromatograph (2% EtOAc in
hexane) to obtain compound 22 and compound 23 as a colorless oil
(90 mg, 52.0%). Rf ¼ 0.70 (hexane/EtOAc ¼ 9:1); Compound 22: 1H
d
219.5, 170.3, 163.5, 151.2, 139.1, 135.6, 118.1, 117.2, 114.3, 77.0, 73.8,
70.3, 52.1, 43.4, 41.2, 37.7, 36.1, 32.2, 31.7, 29.6, 19.4, 18.1, 17.7, 15.3,
15.0, 14.4, 14.0, 11.2; HRMS (ESI) (M
C28H44NO5S: 506.2935, found 506.2937.
þ
Hþ) calculated for
4.2.21. Synthesis of compound 20
To a solution of compound 10 (710 mg, 1.44 mmol) in 7 mL
CH2Cl2 was added TiCl4 (1.6 mL, 1.6 mmol, 1 M in CH2Cl2) at ꢀ78 ꢁC,
followed by addition of DIEA (0.27 mL, 1.6 mmol). After being
stirred at ꢀ78 ꢁC for 1 h, a solution of compound 3c (1.5 mmol) in
hexane (6 mL) was added to the reaction solution. The resulting
reaction mixture was warmed up slowly to ꢀ12 ꢁC in 10 h. The
reaction was quenched by addition of aqueous of phosphate solu-
tion (3 mL, pH ¼ 7.2). Then the resulting solution was stirred for
10 min, and was diluted with ether (30 mL). The aqueous layer was
extracted with Et2O (2 ꢂ 10 mL), and the combined organic phase
was dried over MgSO4. The solvent was removed under reduced
pressure. The crude product was purified by column chromatog-
NMR (400 MHz, CDCl3)
d
6.94 (s, 1H), 6.56 (s, 1H), 5.31 (d, J ¼ 9.2 Hz,
1H), 5.15 (d, J ¼ 4.4 Hz,1H), 4.81 (m, 1H), 4.08 (d, J ¼ 7.2 Hz,1H), 3.17
(m, 1H), 2.81e2.74 (band, 1H), 2.71 (s, 3H), 2.62e2.54 (band, 2H),
2.36 (d, J ¼ 16.0 Hz, 1H), 2.15 (s, 3H), 2.12e2.01 (band, 3H), 1.65 (s,
3H), 1.62 (m, 1H), 1.25 (m, 1H), 1.18 (d, J ¼ 6.8 Hz, 1H), 1.07 (s, 3H),
1.07e0.97 (band, 12H), 0.92 (d, J ¼ 6.8 Hz, 3H), 0.88 (m, 1H), 0.85 (s,
9H), 0.74 (d, J ¼ 6.4 Hz, 3H), 0.67 (m, 6H), 0.11 (s, 3H), 0.07 (s, 3H);
HRMS (ESI) (M þ Hþ) calculated for C40H72NO5SSi2: 734.4664,
found 734.4654.
4.2.24. Synthesis of 24
raphy (5% EtOAc in hexane) to obtain alcohol 20 as a colorless oil
To a solution of 22 (35 mg, 0.047 mmol) was added a freshly
prepared 20% CF3COOH solution in CH2Cl2 (0.7 mL) at ꢀ20 ꢁC. The
reaction mixture was stirred at ꢀ20 ꢁC for 30 min, and warmed to
0 ꢁC in 30 min. Sodium bicarbonate was added to adjust the pH to 7.
The resulting solution extracted with EtOAc (3 ꢂ 3 mL), and the
combined organic mixture was concentrated under reduced pres-
sure. The crude product was purified by column chromatograph
20
(700 mg, 74.8%). Rf ¼ 0.35 (hexane/EtOAc ¼ 9:1); [
a
]
¼ ꢀ33.2
D
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3509, 2960, 2858,1737, 1684,
1646, 1506, 1471, 1378, 1295, 1254, 1181, 1094, 995, 975, 883, 866,
778; 1H NMR (400 MHz, CDCl3)
d 6.94 (s, 1H), 6.49 (s, 1H), 5.70 (m,
1H), 5.29 (t, J ¼ 6.6 Hz, 1H), 5.06 (m, 2H), 4.65 (br s, 2H), 4.41 (t,
J ¼ 4.8 Hz, 1H), 3.47 (d, J ¼ 10 Hz, 1H), 3.44 (s, 1H), 3.21 (m, 1H), 2.68
(s, 3H), 2.50e2.45 (band, 3H), 2.31 (dd, J ¼ 17.2, 5.6 Hz, 1H), 2.06 (s,
3H), 1.99e1.85 (band, 3H), 1.68 (s, 3H), 1.52 (band, 4H), 1.22e1.17
(band, 5H), 1.11 (s, 3H), 1.03e1.01 (band, 3H), 0.87 (s, 9H), 0.71e0.69
(band, 3H), 0.10 (s, 3H), 0.05 (s, 3H). 13C NMR (100 MHz, CDCl3)
(10% EtOAc in hexane) to obtain target molecule 24 as a colorless oil
20
(18 mg, 75.8%). Rf ¼ 0.25 (hexane/EtOAc ¼ 3:1); [
a]
¼ ꢀ68.4
D
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3482, 2959, 1775,1733, 1689,
1507, 1455, 1378, 1253, 1159,1035, 1006, 983, 819, 775, 715; 1H NMR
d
222.1, 171.0, 164.6, 152.4, 146.6, 136.6, 133.3, 121.2, 117.9, 116.5,
(400 MHz, CDCl3)
d
6.94 (s, 1H), 6.50 (s, 1H), 5.26 (dd, J ¼ 8.8, 3.2 Hz,
109.5, 78.9, 73.6, 71.7, 53.8, 41.2, 40.4, 40.2, 37.5, 36.1, 31.2, 27.9,
26.0, 22.4, 21.7, 20.4, 19.2, 18.1, 18.1, 14.6, 10.3, 9.4, ꢀ4.3, ꢀ4.8; HRMS
(ESI) (M þ Hþ) calculated for C36H62NO5SSi: 648.4112, found
648.4111.
1H), 5.14 (t, J ¼ 6.4 Hz,1H), 4.08 (m,1H), 3.83 (d, J ¼ 6.8 Hz,1H), 3.44
(br s, 1H), 3.24 (q, J ¼ 6.8 Hz, 1H), 2.85 (br s, 1H), 2.70 (s, 3H), 2.59e
2.51 (band, 2H), 2.41e2.37 (band, 1H), 2.16e2.11 (band, 1H), 2.08 (s,
3H), 1.93 (m, 1H), 1.74 (m, 1H), 1.63 (s, 3H), 1.55e1.48 (band, 2H),
1.33 (s, 3H), 1.25 (m, 1H), 1.19e1.11 (band, 1H), 1.09 (s, 3H), 1.07
(d, J ¼ 6.8 Hz, 3H), 0.92 (d, J ¼ 6.8 Hz, 3H), 0.81 (d, J ¼ 7.2 Hz, 3H);
4.2.22. Synthesis of compound 21
To a solution of alcohol 20 (380 mg, 0.586 mmol) in CH2Cl2
(13 mL) was added 2,6-lutidine (0.28 mL, 2.35 mmol) at ꢀ78 ꢁC,
followed by addition of TESOTf (0.40 mL, 1.76 mmol). After stirred
at ꢀ78 ꢁC for 1.5 h, the reaction mixture was quenched by addi-
tion of sat NaHCO3 (15 mL), diluted with CH2Cl2 (50 mL) and H2O
(30 mL). The aqueous layer was separated and was extracted with
CH2Cl2 (2 ꢂ 20 mL). The combined organic phase was dried over
MgSO4, and the solvent was removed under reduced pressure.
The crude product was purified by column chromatograph (3%
13C NMR (100 MHz, CDCl3)
d 221.1, 170.8, 164.7, 152.3, 140.0, 137.6,
119.5, 118.7, 116.1, 79.2, 73.3, 72.0, 52.2, 42.7, 42.7, 38.3, 37.2,
32.6, 31.7, 30.4, 21.3, 20.5, 19.2, 18.7, 16.9, 15.5, 10.8, 10.0; HRMS
(ESI) (M þ Hþ) calculated for C28H44NO5S: 506.2935, found
506.2936.
4.2.25. Synthesis of 25
To a solution of 23 (18 mg, 0.025 mmol) was added a freshly
prepared 20% CF3COOH solution in CH2Cl2 (0.6 mL) at ꢀ20 ꢁC. The
reaction mixture was stirred at ꢀ20 ꢁC for 50 min, and warmed to
0 ꢁC in 1.5 h. Sodium bicarbonate was added to adjust the pH to 7.
The resulting solution was extracted with EtOAc (3 ꢂ 3 mL), and the
combined organic mixture was concentrated under reduced pres-
sure. The crude product was purified by column chromatography
EtOAc in hexane) to obtain compound 21 as a colorless oil
20
(379 mg, 84.8%). Rf ¼ 0.75 (hexane/EtOAc ¼ 9:1); [
a]
¼ ꢀ77.2
D
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3076, 2958, 2879, 2858,
1738, 1697, 1646, 1506, 1471, 1378, 1293, 1263, 1180, 1087, 988, 917,
885, 813; 1H NMR (400 MHz, CDCl3)
d 6.93 (s, 1H), 6.48 (s, 1H),
5.70 (m, 1H), 5.27 (t, J ¼ 6.8 Hz, 1H), 5.05 (m, 2H), 4.66 (br s, 2H),
4.25 (m, 1H), 3.84 (t, J ¼ 4.8 Hz, 1H), 3.18 (m, 1H), 2.68 (s, 3H), 2.59
(dd, J ¼ 17.2, 3.2 Hz, 1H), 2.46 (m, 2H), 2.23 (dd, J ¼ 17.2, 6.0 Hz,
1H), 2.10 (m, 1H), 2.06 (s, 3H), 1.87 (m, 1H), 1.70 (s, 3H), 1.74e1.63
(band, 2H), 1.34 (band, 1H), 1.26 (s, 3H), 1.05e0.97 (band, 7H), 0.94
(t, J ¼ 8.0 Hz, 9H), 0.86 (s, 9H), 0.82e0.72 (band, 6H), 0.60 (m, 6H),
(10% EtOAc in hexane) to obtain target molecule 25 as a colorless oil
20
(10 mg, 79.2%). Rf ¼ 0.25 (hexane/EtOAc ¼ 3:1); [
a
]
¼ ꢀ10.4
D
(c ¼ 10 mg/mL, CHCl3); IR (KBr/cmꢀ1): 3498, 2962, 2921, 1777, 1731,
1684, 1507, 1456, 1375, 1176,1016, 975, 867, 758; 1H NMR (400 MHz,
CDCl3)
d
6.97 (s, 1H), 6.53 (s, 1H), 5.40 (dd, J ¼ 11.2, 3.2 Hz, 1H), 5.11
(t, J ¼ 11.2 Hz, 1H), 4.31 (m, 1H), 3.34e3.22 (band, 2H), 2.70 (s, 3H),
2.50 (m, 1H), 2.40e2.26 (band, 2H), 2.13 (m, 1H), 2.07 (s, 3H), 2.03
(m, 1H), 1.80 (m, 1H), 1.66 (s, 3H), 1.57e1.49 (band, 2H), 1.35 (s, 3H),
1.25 (s, 3H), 1.22e1.11 (band, 1H), 1.06e1.03 (band, 6H), 0.94 (d,
J ¼ 10.8 Hz, 3H), 0.68 (d, J ¼ 11.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
0.10 (s, 3H), 0.04 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 217.0, 171.3,
164.5, 152.6, 146.6, 136.8, 133.4, 121.1, 117.8, 116.4, 109.6, 78.7, 75.5,
75.1, 53.0, 44.9, 43.8, 40.1, 37.5, 35.9, 31.9, 29.5, 26.0, 23.2, 22.5,
21.1, 19.3, 18.9, 18.2, 14.6, 12.8, 12.1, 7.2, 5.5, ꢀ4.4, ꢀ4.6; HRMS
(ESI) (M þ Hþ) calculated for C42H76NO5SSi2: 762.4977, found
762.4969.
d
221.1, 172.2, 164.8, 152.3, 141.5, 137.8, 120.1, 118.7, 116.4, 79.7, 71.1,
69.7, 54.0, 41.1, 40.2, 38.3, 38.0, 32.9, 31.9, 31.7, 29.7, 22.0, 19.1, 17.0,

F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
331
15.4, 14.8, 10.0, 8.9; HRMS (ESI) (M
C28H44NO5S: 506.2935, found 506.2938.
þ
Hþ) calculated for
Acknowledgment
This work was supported by National Natural Science Founda-
tion NSFC (No. 21072106 to Y.C., No. 31070640 to J.L. and No.
81001377 to Q.Z.), Fok Ying Tong Education Foundation (No.
122037), and National Program on Key Basic Research Project (No.
2013CB967200).
4.3. Procedure for testing tubulin inhibitory activity of Epo D,
compounds 18 and 24
Fresh animal brain tissue was isolated, and the meninges and
large blood vessels were removed. The cut tissue was washed with
cold MES buffer (1 mL per gram) for 2 times. Then the mixture was
mashed until become homogenate. The mixture was centrifuged at
105,000 g, 4 ꢁC for 1 h. The supernatant was separated, and the
microtubule polymerization buffer was added. And after incubated
at 37 ꢁC or 30 min, the mixture was centrifuged at 105,000 g, 26 ꢁC
for 1 h. Cold MES buffer with 1/10 volume of homogenate was
added into the supernatant, the mixture was gently vortex until the
precipitate was completely dissolved. The solution was stored in ice
for 0.5 h, and the low and high temperature centrifugals were
repeated for two times. The precipitate was suspended in MES
buffer, and the tubulin was diluted with MES buffer to 2 mg/mL.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.08.003.
References
[1] K.C. Nicolaou, F. Sarabia, S. Ninkovic, M.R.V. Finlay, C.N.C. Boddy, Angew.
Chem. 110 (1998) 85e89.
[2] K.C. Nicolaou, D. Vourloumis, T. Li, J. Pastor, N. Winssinger, Y. He, S. Ninkovic,
F. Sarabia, H. Vallberg, F. Roschangar, N.P. King, M.R.V. Finlay, P. Verdier-
Pinard, E. Hamel, Angew. Chem. 109 (1997) 2181e2187.
[3] R.E. Taylor, Y. Chen, G.M. Galvin, P.K. Pabba, Org. Biomol. Chem. 2 (2004) 127e
132.
[4] L. Tang, R.G. Qiu, Y. Li, L. Katz, J. Antibiot. 56 (2003) 16e23.
[5] R.L. Arslanian, L. Tang, S. Blough, W. Ma, R.G. Qiu, L. Katz, et al., J. Nat. Prod. 65
(2002) 1061.
[6] A. Rivkin, F. Yoshimura, A.E. Gabarda, T.C. Chou, H.J. Dong, W.P. Tong,
S.J. Danishefsky, J. Am. Chem. Soc. 125 (2003) 2899e2901.
[7] R.E. Taylor, Y. Chen, A. Beatty, J. Am. Chem. Soc. 125 (2003) 26e27.
[8] J.D. Frein, R.E. Taylor, D.L. Sackett, Org. Lett. 11 (2005) 3186e3189.
[9] M.C. William, B. Corey, J. Org. Chem. 63 (1998) 6732e6734.
[10] G. Koch, O. Loiseleur, D. Fuentes, A. Jantsch, K.-H. Altmann, Org. Lett. 4 (2002)
3811e3814.
5 mL ATPNa2 (40 mmol/L) and 195 mL tubulin were added into a cold
96-well plates, and then a certain concentration of compound was
added into each well of the cold 96-well plates. The values of
OD350 were detected every 3 min with Ultraviolet spectropho-
tometer at 37 ꢁC for 30 min.
4.4. Computational methods
The structure of Epo D, 9-(R)-Me epo D and 9-(S)-Me epo D were
built in Maestro [42] and cleaned up by performing a short force-
field minimization. Epo D, 9-(R)-Me epo D and 9-(S)-Me epo D
were subjected to full geometry optimization with DFT at the
B3LYP/6-31G (d) level using Gaussian 03.
Autodock 4.2 was used to perform the docking simulation of
fully optimized Epo D, 9-(R)-Me epo D and 9-(S)-Me epo D. Struc-
ture of a, b-tubulin (PDB ID: 1TVK) was used as our initial protein
[11] a) E.B. Watkins, A.G. Chittiboyina, M.A. Avery, Eur. J. Org. Chem. (2006)
4071e4084;
b) T. Gaich, J. Mulzer, Org. Lett. 7 (2005) 1311e1314.
[12] J.K. De Brabander, S. Rosset, G. Bernardinelli, Synlett (1997) 824e826.
[13] a) M. Capet, F. David, L. Bertin, J.C. Hardy, Synth. Commun. 25 (1995)
3323e3327;
b) W. Oppolzer, R. Moretti, S. Thomi, Tetrahedron Lett. 30 (1989) 5603e
5606.
[14] S.C. Sinha, J. Sun, G.P. Miller, M. Wartmann, R.A. Lerner, Chem. e Eur. J. 7
(2001) 1691e1702.
[15] a) D.F. Meng, P. Bertinato, A. Balog, D.-S. Su, T. Kamenecka, E.J. Sorensen,
S.J. Danishefsky, J. Am. Chem. Soc. 119 (1997) 10073e10092;
b) K.C. Nicolaou, Y. He, D. Vourloumis, H. Vallberg, F. Roschangar, F. Sarabia,
S. Ninkovic, Z. Yang, J.I. Trujillo, J. Am. Chem. Soc. 119 (1997) 7960e7973.
[16] a) T. Inukai, R. Yoshizwa, J. Org. Chem. 32 (1967) 404e407;
b) J.-C. Jung, R. Kache, K.K. Vines, Y.-S. Zheng, P. Bijoy, M. Valluri, M.A. Avery,
J. Org. Chem. 69 (2004) 9269e9284.
model for docking (Fig. 4).
The binding energy [43] of 9-(R)-Me epo D and 9-(S)-Me epo D
to the structure of
higher than Epo D respectively.
protein) þ E (ligand in water).
a
,
b
-tubulin are 5.50 kcal/mol and 8.14 kcal/mol
D
G binding ¼ E (ligand in
D
D
[17] S.D. Dong, K. Sundermann, K.M.J. Smith, J. Petryka, F.H. Liu, D.C. Myles, Tet-
rahedron Lett. 45 (2004) 1945e1948.
[18] Peng Feng. 201110038175, published date: 2012-8-15.
[19] K.C. Nicolaou, S. Ninkovic, F. Sarabia, D. Vourloumis, Y. He, H. Vallberg,
M.R.V. Finlay, Z. Yang, J. Am. Chem. Soc. 119 (1997) 7974e7991.
[20] Y. Chen, F. Sang, P. Feng, X.Y. Duan, Q. Zhang, J.D. Zhai, X.X. Li, B. Zhang.
CN101973987B, published date: 2012-8-22.
[21] B.E. Rossiter, N.M. Swingle, Chem. Rev. 92 (1992) 771e806.
[22] W. Oppolzer, A.J. Kingma, G. Poli, Tetrahedron 45 (1989) 479e488.
[23] W. Oppolzer, A.J. Kingma, Helv. Chim. Acta 72 (1989) 1337e1345.
[24] M.L. Shelanski, F. Gaskin, C.R. Cantor, Proc. Natl. Acad. Sci. U S A 70 (1973) 765e768.
[25] J.H. Nettles, H.L. Li, B. Cornett, J.M. Krahn, J.P. Snyder, K.H. Downing, Science
305 (2004) 866e869.
[26] T. Carlomagno, M.J.J. Blommers, J. Meiler, W. Jahnke, T. Schupp, F. Petersen,
D. Schinzer, K.-H. Altmann, C. Griesinger, Angew. Chem. Int. Ed. 42 (2003)
2511e2515.
[27] D.W.Heinz, W.-D. Schubert,G.Höfle,Angew.Chem. Int. Ed. 44(2005)1298e1301.
[28] T. Carlomagno, Nat. Prod. Rep. 29 (2012) 536e554.
[29] A. Kumar, H. Heise, M.J.J. Blommers, P. Krastel, E. Schmitt, F. Petersen,
S. Jeganathan, E.-M. Mandelkow, T. Carlomagno, C. Griesinger, M. Baldus,
Angew. Chem. Int. Ed. 49 (2010) 7504e7507.
[30] V.M. Sánchez-Pedregal, K. Kubicek, J. Meiler, I. Lyothier, L. Paterson,
T. Carlomagno, Angew. Chem. Int. Ed. 45 (2006) 7388e7394.
[31] T. Carlomagno, V.M. Sánchez, M.J.J. Blommers, C. Griesinger, Angew. Chem.
Int. Ed. 42 (2003) 2515e2517.
[32] F. Yoshimura, A. Rivkin, A.E. Gabarda, T.C. Chou, H.J. Dong, G. Sukenick, F.F. Morel,
R.E. Taylor, S.J. Danishefsky, Angew. Chem. Int. Ed. 42 (2003) 2518e2521.
[33] R.E. Taylor, J. Zajicek, J. Org. Chem. 64 (1999) 7224e7228.
[34] M. Erdélyi, B. Pfeiffer, K. Hauenstein, J. Fohrer, J. Gertsch, K.-H. Altmann,
T. Carlomagno, J. Med. Chem. 51 (2008) 1469e1473.
Fig. 4. Superposition of best docking poses of Epo D (cyan), 9-(R)-Me epo D (purple)
and 9-(S)-Me epo D (yellow) to structure of -tubulin (PDB ID: 1TVK). (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
a, b
web version of this article.)
[35] D. Rusinska-Roszak, M. Lozynski, J. Mol. Model. 15 (2009) 859e869.

332
F. Sang et al. / European Journal of Medicinal Chemistry 68 (2013) 321e332
[36] D. Rusinska-Roszak, H. Tatka, R. Pawlak, M. Lozynski, J. Phys. Chem. B 115
(2011) 3698e3707.
[37] K. Kamel, D. Rusinska-Roszak, Inc. Int. J. Quantum Chem. 108 (2008) 967e973.
[38] V.O. Jiménez, J. Chem. Inf. Model. 50 (2010) 2176e2190.
[39] C. Coderch, J. Klett, A. Morreale, J.F. Díaz, F. Gago, ChemMedChem. 7 (2012)
836e843.
[40] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman Jr.,
J.A. Montgomery, T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson,
H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian,
J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann,
O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala,
K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski,
S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck,
K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford,
J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi,
R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara,
M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez,
J.A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford, CT, 2004.
[41] D.A. Case, T.A. Darden, T.E. Cheatham III, C.L. Simmerling, J. Wang, R.E. Duke,
R. Luo, K.M. Merz, B. Wang, D.A. Pearlman, M. Crowley, S. Brozell, V. Tsui,
H. Gohlke, J. Mongan, V. Hornak, G. Cui, P. Beroza, C. Schafmeister,
J.W. Caldwell, W.S. Ross, P.A. Kollman, AMBER 11, University of California, San
Francisco, 2010.
[42] L.L.C. Schrodinger, Maestro, Version 9.1. New York, NY, 2010.
[43] R. Buettner, R. Corzano, R. Rashid, J. Lin, M. Senthil, M. Hedvat, A. Schroeder,
A. Mao, A. Herrmann, J. Yim, H. Li, Y.-C. Yuan, K. Yakushijin, F. Yakushijin,
N. Vaidehi, R. Moore, G. Gugiu, T.D. Lee, R. Yip, Y. Chen, R. Jove, D. Horne,
J.C. Williams, ACS Chem. Biol. 6 (2011) 432e443.