Synthesis and evaluation of water-soluble docetaxel prodrugs-docetaxel esters of malic acid

Article abstract of DOI:10.1016/j.bmc.2007.04.002

The synthesis of docetaxel esters of malic acid is described. These compounds were found to have greatly improved water solubility and are stable in solution at neutral pH. The C2′ modified compounds 2a-c and 3a-c behave as prodrugs, that is, docetaxel is

Full text of DOI:10.1016/j.bmc.2007.04.002

Bioorganic & Medicinal Chemistry 15 (2007) 6323–6330
Synthesis and evaluation of water-soluble docetaxel
prodrugs-docetaxel esters of malic acid
Wenting Du,a Lan Hong,b Tongwei Yao,b Xiaochun Yang,c
Qiaojun He,c Bo Yangc and Yongzhou Hua,*
aZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
bDepartment of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
cInstitute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Received 5 March 2007; revised 31 March 2007; accepted 3 April 2007
Available online 6 April 2007
Abstract—The synthesis of docetaxel esters of malic acid is described. These compounds were found to have greatly improved water
solubility and are stable in solution at neutral pH. The C20 modified compounds 2a–c and 3a–c behave as prodrugs, that is, docet-
axel is generated upon exposure to human plasma, whereas the C7 and C20,7,10- l modified derivatives do not. 20-DL-Malyl docet-
axel sodium salt demonstrated enhanced antitumor activity in vitro when compared to docetaxel and showed the inhibitory effect on
tumor growth in vivo.
ꢀ 2007 Published by Elsevier Ltd.
1. Introduction
ness because of its haemolyticus and viscidity, espe-
cially for the patients who are hypersensitive to
Tween 80.2 Patients receiving these drugs should be
premedicated.3 To solve the problem of low water sol-
ubility, several research groups have synthesized and
evaluated water-soluble taxoids through the introduc-
tion of solubilizing moieties to C20-, C7- or/and
C10-positions. The solubilizing moieties can be (salts
of) carboxylic acids, phosphates, sulfonates, amines,
sugar derivatives or polyethylene glycol. In most cases
these moieties are coupled via an ester or carbonate
linkage.4
The diterpenoid natural product paclitaxel (Taxolꢁ) 1a
and its semi-synthetic analogue docetaxel (Taxotereꢁ)
1b are two important chemotherapeutic drugs widely
used today for the treatment of various types of cancer.
They are known to exert their therapeutic effect, at least
in part, by promoting the assembly of tubulin into
microtubules and stabilizing the resulting microtubules.
They thereby inhibit microtubule disassembly and nor-
mal dynamic reorganization of the microtubular net-
work required for mitosis.1
However, the clinical usefulness of these drugs is partic-
ularly hampered by their poor solubility. So they should
be co-injected with Cremophor EL or Tween 80.
As we know, docetaxel is a little more water-soluble
than paclitaxel, but just 6–7 lg/mL in aqueous solution
(paclitaxel: 0.25 lg/mL).5–8 Until now, just a few at-
tempts have been undertaken to overcome poor solubil-
ity of docetaxel, though numerous researches on
analogues, prodrugs,6 and derivatives of paclitaxel have
been carried out. Previously, some glycosyl derivatives
of docetaxel were prepared and evaluated.9,10 A direct
7-O-glucosyl docetaxel analogue was reported to be
twice as water-soluble as paclitaxel and with similar
activity.11 Amino acid conjugates linked to the 20-hydro-
xyl of a 30-cyclopropyl docetaxel analogue were also pre-
pared and tested in vivo.12 Some showed better activity
Cremophor EL in paclitaxel for injection frequently
causes untoward hypersensitivity reactions (hypoten-
sion, bronchospasm, urticaria, etc.). And Tween 80
in docetaxel for injection hampers the clinical useful-
Keywords: Docetaxel; Prodrug; Malic acid; Water-soluble; Paclitaxel.
*
Corresponding author. Tel./fax: +86 571 88205460; e-mail:
huyz@zjuem.zju.edu.cn
0968-0896/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.04.002

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W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
than docetaxel in ILS (Increase of Life Span) on B16
melanoma-bearing mice.
pound 6a was prepared by condensing docetaxel with
protected malic acid in the presence of dicyclohexylcar-
bodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) at ꢀ10 ꢂC. Then deprotection of compound
6a was effected in a mixture of HOAc–THF–H2O to pro-
vide compound 2a. Exposure of this compound to aque-
ous NaHCO3 afforded its sodium salt 3a (Scheme 1). In
the same manner, compounds 2b and 3b, 2c and 3c were
synthesized. The significant downfield shift of the C-20H
(ꢁd 5.28) and no great change of C-7H (ꢁd 4.24) and
C-10H (ꢁd 5.28) in 3a–c relative to that in docetaxel in
1H NMR spectrum demonstrated that the malyl was
linked to 20-OH of docetaxel.
Since our aim was to increase water solubility of docet-
axel, we considered converting docetaxel into water-sol-
uble prodrugs, which, after enzymatic cleavage in vivo,
will release the active compound. We chose malic acid,
a non-cytotoxic acid of the Krebs cycle which could be
metabolized easily, as the solubilizing moiety.
In the present paper, we report the synthesis of eight
docetaxel prodrugs, in which the malyl moiety is linked
to different hydroxyl groups: C20-malyl docetaxel 2 and
their sodium salts 3 (including C20-DL-malyl docetaxel
2a, C20-L-malyl docetaxel 2b, C20-D-malyl docetaxel
2c, and their sodium salts 3a–c), 20,7,10-tri-(malyl)
docetaxel 4, and C7-malyl docetaxel 5. All the target
The reaction, carried out with an excess of protected
malic acid in refluxing dichloromethane replacing
DCC by diisopropylcarbodiimide (DIC), afforded
20,7,10- tri-(malyl) docetaxel 4 instead compared with
1
compounds were determined by H NMR, 13C NMR,
1
1
1H–1H COSY, H–13C COSY, and DEPT135. The sta-
the result of the literature3 (Scheme 2). In H NMR
bility of synthetic compounds in PBS buffer and plasma
and the antitumor activity (in vitro and in vivo) were
tested and are described in this paper.
spectrum, the significant downfield shift of the C-20H
(d 5.28), C-7H (d 5.62), and C-10H (d 6.32) in 4 relative
to that in docetaxel and two groups of malyl (d 4.48, 3H,
CH-malyl; d 2.86, 6H, CH2-malyl) demonstrated that
three malyl groups were linked to 20-OH, 7-OH, and
10-OH of docetaxel, respectively, since the reacting
power of 1-OH is lower than the others of docetaxel.
2. Chemistry
For preparing our target compounds, we protected one
of the carboxyl groups of malic acid with acetone to ob-
tain 1,2-O-isopropylidene-malic acid,13 then the latter
was reacted with docetaxel in different conditions. Com-
In the preparation of C7-malyl docetaxel 5, 20- and
10-OH were protected first by treatment with 2,2,2-Tri-
chloroethylchloroformate (Troc-Cl) at ꢀ23 ꢂC under
O
O
RO
OH
R' NH O
Ph
O
O
OAc
OH
H
OBz
HO
Paclitaxel 1a (R = Ac; R' = Ph)
Docetaxel 1b (R = H; R' = tBuO)
O
R3O
OR2
O
O
NH O
Ph
O
O
OAc
OR1
H
OBz
HO
2a R1 = DL-malyl, R2 = H, R3 = H
2b R1 = L-malyl, R2 = H, R3 = H
2c R1 = D-malyl, R2 = H, R3 = H
3a sodium salt of 2a
3b sodium salt of 2b
3c sodium salt of 2c
4
5
R1 = DL-malyl, R2 = DL-malyl, R3 = DL-malyl
R1 = H, R2 = DL-malyl, R3 = H
O
O
HO
O
OH
O
HO
O
OH
O
NH O
O
O
HO
OH
O
NH O
O
a
O
NH O
OH
b
Ph
O
O
Ph
O
O
H
OBz
Ph
O
O
OAc
H
OBz
HO
O
OAc
H
OBz
HO
O
OAc
HO
O
HO
RO
O
O
O
2a R = H
3a R = Na
6a
1b
c
Scheme 1. Reagents and conditions: (a) 1,2-O-isopropylidene-malic acid, DCC, DMAP, CH2Cl2, ꢀ10 ꢂC, 7 h, 70%; (b) HOAc:THF:H2O (1:1:1), rt,
20 h, 86%; (c) NaHCO3, acetone, H2O, rt, 1 h, 100%.

W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
6325
O
OH
O
O
OH
O
O
O
O
O
O
O
O
OH
O
O
O
HO
OH
O
O
O
O
O
OH
O
NH O
OH
O
O
O
O
NH O
O
O
a
b
O
NH O
O
Ph
O
O
Ph
O
O
H
OBz
Ph
O
OAc
H
HO
OAc
H
HO
O
OBz
OAc
HO
O
OBz
O
HO
HO
O
O
O
7
1b
4
Scheme 2. Reagents and conditions: (a) 1,2-O-isopropylidene-malic acid, DIC, DMAP, CH2Cl2, reflux, 5 h, 85%; (b) HOAc:THF:H2O (4:1:2), 45 ꢂC,
6 h, 68%.
Ar2 to give compound 8, which was treated with pro-
tected malic acid as described in Scheme 1. Contrarily
the protected malic acid was linked at 7-OH. Removal
of the Troc groups with zinc dust resulted in the desired
compound 10 which was followed by deprotection to
graded to docetaxel in 4 and 3 h, respectively, and 70–
80% of docetaxel was generated in 6 h. In contrast, com-
pounds 4 and 5 were too stable to generate docetaxel.
Therefore, 20-malyl docetaxel and their sodium salts
act as prodrugs but the 7-malyl and 20,7,10-tri-malyl
derivatives do not. Hydrolysis rate of sodium salts in hu-
man plasma is particularly encouraging (Table 1).
1
provide C7-malyl docetaxel 5 (Scheme 3). In H NMR
spectrum, the significant downfield shift of C-7H
(d 5.57) and no great change of C-20H (d 4.51) and
C-10H (d 5.34) in 5 relative to that in docetaxel demon-
strated that the malyl was linked to 7-OH of docetaxel.
3.2. Cytotoxicity
In order to investigate the influence of malyl- on docet-
axel in vitro cytotoxicity, all compounds were screened
for their cytotoxicity in vitro against human cancer cell
lines (A549, K562, KB, and MCF-7). The results are
summarized in Table 1. Derivatives 4 and 5 were signif-
icantly less active against most tested cancer cell lines.
Compound 2a showed similar activity and compound 3a
showed more activity when compared to docetaxel. On
the other hand, 20-DL-malyl docetaxel 2a showed more
activity than 20-L-malyl docetaxel 2b and 20-D-malyl
docetaxel 2c on the whole, which was consistent with
their sodium salts 3a–c. The result was strange and dif-
ferent from forecast. We repeated three times and got
similar results. In order to explain this phenomenon,
the test of the concentration of compounds 2a–c, 3a–c
and docetaxel on cancer cells in vitro with HPLC is in
process.
3. Biological results
3.1. Solubility and stability
Solubility of synthesized compounds 2–5 was measured
by HPLC, the results are listed in Table 1. Compounds
2–5 demonstrated greatly improved water solubility
compared to docetaxel (65–90 times), especially paclit-
axel (1720–2480 times).5–8 According to their water sol-
ubility, we considered that it is possible to prepare them
in the form of lyophilized powder for injection clinically
without any surfactant such as Tween 80.
The test of stability in PBS-buffer showed that all target
compounds were proved to be stable when incubated in
PBS buffer (pH 7.4) at 37 ꢂC for 48 h, since few liberated
docetaxel was detected (HPLC). Otherwise, derivatives
2a–c and 3a–c mostly generated docetaxel when incu-
bated in human plasma in 6 h: 50% of them was de-
Since compound 3a has highly anticancer activity in vitro
and generated docetaxel when incubated in human
plasma in 6 h, we tested its activities in vivo against the
Cl3C
Cl3C
O
O
O
O
O
O
O
O
O
O
O
O
HO
OH
O
O
OH
O
O
O
NH O
OH
O
NH O
O
O
NH O
O
O
a
b
Ph
O
Ph
O
Ph
O
O
O
O
H
OBz
H
OBz
H
OBz
OAc
HO
OAc
OAc
HO
O
HO
O
O
O
CCl3
CCl3
8
9
1b
O
O
O
O
O
OH
O
O
O
HO
O
OH
c
d
O
O
NH O
HO
O
O
O
NH O
Ph
O
O
Ph
O
OH
H
OBz
O
OAc
HO
OH
H
OBz
OAc
HO
10
5
Scheme 3. Reagents and conditions: (a) Troc-Cl, pyridine, CH2Cl2, Ar2, ꢀ23 ꢂC, 1.5 h, 65%; (b) 1,2-O-isopropylidene-malic acid, DCC, DMAP,
CH2Cl2, ꢀ10 ꢂC, 3 h, 82%; (c) zinc dust, CH3OH: HOAc (9: 1), rt, 20 min, 88%; (d) HOAc:THF:H2O (4: 1: 2), 45 ꢂC, 6 h, 86%.

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W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
Table 1. Water solubility, stability, and cytotoxicity of malic acid docetaxel derivatives
Cell linec (IC50 lg/mL)
a
T1/2 (h)
Compound
Water solubility (lg/mL)
pH 7.4
Plasma
A549
K562
KB
MCF-7
2a
2b
>50
>50
9.5 (4.5–12.0)
3.9 (1.2–7.0)
3.5 (1.7–6.2)
>50
39.2 (10.2–66.3)
>50
>50
470–480
620–630
>48
>24
4
3
2c
3a
>50
0.85 (0.27–3.56)
1.5 (1.0–2.1)
>50
0.45 (0.17–1.06)
3.5 (1.1–6.2)
>50
>50
3.6 (0.7–7.5)
>50
>50
3b
3c
38.1 (12. 5–73.2)
18.8 (8.429.1)
17.0 (10.4–24.6)
0.069 (0.025–0.146)
6.9 (3.7–10.9)
>50
>50
4
550
430
6–7
>48
>48
—
NDb
NDb
—
>50
>50
>50
>50
>50
>50
5
Docetaxel
1.4 (0.5–2.5)
11.6 (3.2–20.3)
3.3 (1.1–6.4)
a Half-life values (T1/2), time in which 50% of the derivative is degraded to docetaxel.
b No docetaxel detected (HPLC).
c Values are means of three experiments, standard deviation is given in parentheses.
HPLC: Rheodyne injection valve (20 lL loop); Lichro-
spher 5RP18 column (250 · 4.6 mM, Phenomenex);
UV-detector (Model G1314A, Applied Agilent); eluent:
methanol: acetonitrile: H2O: 15:40:45 in 5 mM
NH4OAc(pH 5.0) and acetonitrile: H2O: 50:50 in
5 mM KH2PO4 (pH 3.0). The detection of the (pro)-
drugs was performed at 227 nM, where it is supposed
that the extinction coefficients of docetaxel and docet-
axel prodrugs are equal. The concentrations were deter-
mined by measuring the relative area of docetaxel or the
docetaxel prodrugs.
Table 2. Effects of 3a against the murine H22 tumor
Groups
Dose
(mg/kg)
No. of
animals
Tumor weight
(g)
Inhibition
rate (%)
Control
3a
0
5
20
10
10
10
10
1.20 0.40
0.97 0.40
0.85 0.22*
0.78 0.17**
0.64 0.16**
—
19.0
28.9
35.0
46.3
3a
3a
10
20
10
Docetaxel
* P < 0.05.
** P < 0.01 versus control.
5.1.1. 20-(1,2-O-Isopropylidene-DL-malyl) docetaxel (6a).
A solution of docetaxel (80.8 mg, 0.10 mmol)and 1,2-O-
isopropylidene-malic acid (19.1 mg, 0.11 mmol) in
CH2Cl2 (2.5 mL) was stirred at ꢀ10 ꢂC. Next, DCC
(30.9 mg, 0.15 mmol) and DMAP (18.3 mg, 0.15 mmol)
were added. After stirring for 7 h at ꢀ10 ꢂC, the mixture
was filtered, diluted with EtOAc (7.5 mL), and washed
with water and brine. The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was
purified via column chromatography (EtOAc–petro-
leum ether, 1:2), yielding 6a (67.4 mg, 70%) as a color-
less sticky oil: IR (KBr, cmꢀ1) 3500, 2995.37, 2925.25,
1790.56, 1724.99, 1389.74, 1275.15, 1221.15, 1128.27,
murine H22 tumor. The results are depicted in Table 2. It
is showed that compound 3a exhibited inhibitory effect
on H22 tumor growth.
4. Conclusion
In conclusion, we have synthesized some novel water
soluble docetaxel derivatives in good yields and have
demonstrated that 20-malyl docetaxel 2a–c and their so-
dium salts 3a–c act as prodrugs. Among them, 20-DL-
malyl docetaxel sodium salt 3a was found to be more
active than docetaxel in vitro and showed the inhibitory
effect on tumor growth in vivo, in addition to the excel-
lent water solubility.
1
1019.46, 924.58, 623.53; H NMR (400 MHz, CDCl3)
d 8.10 (d, J = 7.2 Hz, 2H, H-Ph), 7.59 (m, 1H, H-Ph),
7.50 (m, 2H, H-Ph), 7.37 (m, 2H, H-Ph), 7.28 (m, 3H,
H-Ph), 6.23 (m, 1H, H-13), 5.68 (m, 1H, H-2), 5.35
(m, 3H, H-10, H-20, H-30), 5.21 (s, 1H, NH), 4.95 (d,
J = 9.2 Hz, 1H, H-5), 4.64 (m, 1H, CH-malyl), 4.21
(m, 3H, H-7,H-20), 3.93 (d, J = 7.20 Hz, 1H, H-3),
2.89 (m, 2H, CH2-malyl) 2.58 (m, 1H, H-6a), 2.41 (s,
3H, OCOCH3), 2.30 (m, 1H, H-14a), 2.18 (m, 1H,
H-14b), 2.03 (s, 3H, H-18), 1.84 (m, 1H, H-6b), 1.75
(s, 3H, H-19), 1.53 (m, 9H, t-Bu-H), 1.23 (m, 12H,
H-16, H-17,CH3-malyl); 13C NMR (100 MHz, CDCl3)
d 211.26, 171.68, 169.67, 168.65, 167.76, 166.90,
155.15, 138.81, 135.57, 133.55, 130.12, 129.20, 128.84,
128.62, 128.17, 126.29, 111.38, 84.24, 80.91, 80.31,
78.72, 76.46, 75.06, 74.90, 74.32, 72.10, 71.70, 70.26,
60.40, 57.50, 46.33, 43.03, 41.85, 36.62, 35.92, 29.60,
28.05, 26.60, 22.55, 20.97, 14.10, 9.86.
5. Experimental
5.1. General procedure
¨
Melting points were determined on BUCHI B-540.
Infrared spectra were recorded on a JASCO FT/IR-
4100 spectrophotometer. Optical rotations were mea-
sured on a JASCO P-1030 digital polarimeter. NMR
spectra were recorded on a Brucker Advance DMX
400 MHz or 500 MHz spectrometer with TMS as the
internal standard. Chemical shifts are expressed in parts
per million (ppm). Elemental analysis was carried out on
Carlo ERBA-1108 analyzer. Mass spectra were recorded
on Finnigan LCQ DECA spectrometer. All compounds
were at least 90% pure, with some small impurities pres-
ent, which were integrated as less than 2%.
5.1.2. 20-DL-Malyl docetaxel (2a). Compound 6a
(96.4 mg, 0.10 mmol) was dissolved in a mixture of

W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
6327
HOAc–THF–H2O (5:5:5 mL). The mixture was stirred
at room temperature for 20 h. Next, the organic solvents
were removed by evaporation in vacuo. The residue was
d 8.12 (d, J = 7.30 Hz, 2H, H-Ph), 7.65 (m, 1H, H-Ph),
7.59 (m, 2H, H-Ph), 7.42 (m, 4H, H-Ph), 7.26 (br s,
1H, H-Ph), 6.10 (m, 1H, H-13), 5.65 (m, 1H, H-2),
5.45 (m, 1H, H-20), 5.33 (m, 1H, H-10), 5.26 (m, 1H,
H-30), 5.00 (d, J = 8.80 Hz, 1H, H-5), 4.48 (m, 1H,
CH-malyl), 4.22 (m, 3H, H-7, H-20), 3.72 (br s, 1H,
H-3), 2.96 (m, 1H, CH2-malyl), 2.83 (m, 1H, CH2-mal-
yl), 2.42 (m, 4H, H-6a, OCOCH3), 2.24 (m, 1H,
H-14a), 1.92 (s, 5H, H-14b, H-18, H-6b), 1.68 (s, 3H,
H-19), 1.41 (s, 9H, t-Bu-H), 1.16 (s, 6H, H-16, H-17);
Anal. Calcd for C47H57NO18 (924): C, 61.10; H, 6.22.
Found: C, 61.29; H, 6.30.
diluted by water (10 mL) and freeze-dried, yielding 2a
25
(79.5 mg, 86%) as a white solid: ½aꢂ ꢀ37.14 (c 0.70,
D
MeOH); mp 163–166 ꢂC; IR (KBr, cmꢀ1) 3433.64,
2979.48, 2944.88, 1720.19, 1495.53, 1453.10, 1369.21,
1248.68, 1165.76, 1066.44, 983.52, 708.71; 1H NMR
(500 MHz, CD3OD) d 8.11 (d, J = 7.30 Hz, 2H, H-Ph),
7.65 (m, 1H, H-Ph), 7.58 (m, 2H, H-Ph), 7.41 (m, 4H,
H-Ph), 7.26 (br s, 1H, H-Ph), 6.10 (m, 1H, H-13), 5.45
(m, 1H, H-2), 5.28 (m, 3H, H-10, H-20, H-30), 4.99 (d,
J = 8.80 Hz, 1H, H-5), 4.36 (m, 1H, CH-malyl), 4.18
(m, 3H, H-7, H-20), 3.72 (br s, 1H, H-3), 2.94 (m, 1H,
CH2-malyl), 2.83 (m, 1H, CH2-malyl), 2.44 (m, 1H, H-
6a), 2.38 (s, 3H, OCOCH3), 2.24 (m, 1H, H-14a), 1.90
(s, 5H, H-14b, H-18, H-6b), 1.68 (s, 3H, H-19), 1.41
(s, 9H, t-Bu-H), 1.15 (s, 3H, H-16), 1.12 (s, 3H, H-17);
13C NMR (125 MHz, CD3OD) d 210.06, 172.31,
172.01, 171.38, 170.02, 166.68, 158.23, 139.26, 138.59,
137.02, 133.87, 131.41, 131.20, 129.88, 129.68, 129.29,
128.22, 85.80, 82.02, 81.05, 79.13, 77.50, 76.44, 76.40,
75.59, 73.15, 72.55, 69.69, 58.78, 56.25, 47.91, 44.40,
40.89, 37.49, 36.58, 28.69, 26.90, 23.19, 21.66, 14.59,
10.45; Anal. Calcd for C47H57NO18 (924): C, 61.10; H,
6.22. Found: C, 61.21; H, 6.32.
25
D
5.1.5. 20-L-Malyl docetaxel sodium salt (3b). ½aꢂ ꢀ48.59
1
(c 0.70, MeOH); mp 211–214 ꢂC; H NMR (500 MHz,
CD3OD) d 7.97 (d, J = 6.89 Hz, 2H, H-Ph), 7.69 (m,
3H, H-Ph), 7.37 (m, 4H, H-Ph), 7.16 (m, 1H, H-Ph),
5.77 (m, 1H, H-13), 5.38 (m, 1H, H-2), 5.08 (m, 3H,
H-10, H-20, H-30), 4.90 (d, J = 9.20 Hz, 1H, H-5), 4.45
(m, 1H, CH-malyl), 4.24 (m, 1H, H-7), 4.02 (s, 2H, H-
20), 3.85 (br s, 1H, H-3), 2.90 (m, 1H, CH2-malyl),
2.60 (m, 1H, CH2-malyl), 2.44 (m, 1H, H-6a), 2.38 (s,
3H, OCOCH3), 2.23 (m, 1H, H-14a), 1.95 (m, 1H, H-
14b), 1.94 (s, 3H, H-18), 1.82 (m, 1H, H-6b), 1.69 (s,
3H, H-19), 1.43 (s, 9H, t-Bu-H), 1.16 (s, 3H, H-16),
1.10 (s, 3H, H-17); Anal. Calcd for C47H56NNaO18
(946): C, 59.68; H, 5.97. Found: C, 59.94; H, 6.12; MS
(ESI): m/z 947 [M+H]+, 969 [M+Na]+.
5.1.3. 20-DL-Malyl docetaxel sodium salt (3a). To a solu-
tion of 20-malyl docetaxel (2a) (92.4 mg, 0.10 mmol) in
acetone (4 mL) was added a solution of NaHCO3
(8.4 mg, 0.1 mmol) in demineralized water (8 mL). The
reaction mixture was stirred at room temperature for
1 h. Sodium salt 3a (94.6 mg, 100%) was isolated after
25
5.1.6. 20-D-Malyl docetaxel (2c). ½aꢂ ꢀ29.46 (c 0.70,
D
MeOH); mp 171–174 ꢂC; 1H NMR (500 MHz, CD3OD)
d 8.05 (d, J = 7.30 Hz, 2H, H-Ph), 7.62 (m, 1H, H-Ph),
7.58 (m, 2H, H-Ph), 7.40 (m, 4H, H-Ph), 7.22 (br s,
1H, H-Ph), 6.05 (m, 1H, H-13), 5.41 (m 1H, H-2), 5.23
(m, 2H, H-10, H-20), 5.11 (m, 1H, H-30), 4.91 (d,
J = 8.80 Hz, 1H, H-5), 4.30 (m, 1H, CH-malyl), 4.12
(m, 3H, H-7, H-20), 3.70 (br s, 1H, H-3), 2.90 (m, 1H,
CH2-malyl), 2.83 (m, 1H, CH2-malyl), 2.43 (m, 1H, H-
6a), 2.32 (s, 3H, OCOCH3), 2.24 (m, 1H, H-14a), 1.86
(s, 5H, H-14b, H-18, H-6b), 1.60 (s, 3H, H-19), 1.41
(s, 9H, t-Bu-H), 1.14 (s, 3H, H-16), 1.12 (s, 3H, H-17);
Anal. Calcd for C47H57NO18 (924): C, 61.10; H, 6.22.
Found: C, 61.27; H, 6.35.
removal of the acetone in vacuo and freeze-drying:
25
D
½aꢂ ꢀ37.30 (c 0.70, MeOH); mp 200–203 ꢂC; IR
(KBr, cmꢀ1
) 3405.67, 2977.55, 2942.84, 1712.48,
1
1602.56, 1370.18, 1251.58, 1166.72, 1068.37, 708.71; H
NMR (500 MHz, CD3OD) d 8.12 (d, J = 6.89 Hz, 2H,
H-Ph), 7.65 (m, 1H, H-Ph), 7.58 (t, J = 7.47 Hz, 2H,
H-Ph), 7.40 (m, 4H, H-Ph), 7.25 (br s, 1H, H-Ph), 6.10
(m, 1H, H-13), 5.64 (m, 1H, H-2), 5.28 (m, 3H, H-10,
H-20, H-30), 5.00 (d, J = 9.15 Hz, 1H, H-5), 4.36 (m,
1H, CH-malyl), 4.24 (m, 1H, H-7), 4.19 (s, 2H, H-20),
3.87 (br s, 1H, H-3), 2.93 (m, 1H, CH2-malyl), 2.64
(m, 1H, CH2-malyl), 2.47 (m, 1H, H-6a), 2.38 (s, 3H,
OCOCH3), 2.24 (m, 1H, H-14a), 1.95 (m, 1H, H-14b),
1.90 (s, 3H, H-18), 1.82 (m, 1H, H-6b), 1.66 (s, 3H, H-
19), 1.40 (s, 9H, t-Bu-H), 1.15 (s, 3H, H-16), 1.12 (s,
3H, H-17); 13C NMR (125 MHz, CD3OD) d 211.26,
172.38, 171.49, 170.38, 170.33, 167.76, 158.00, 139.76,
139.11, 137.74, 134.57, 131.42, 131.20, 129.86, 129.71,
129.29, 128.32, 85.97, 82.22, 81.05, 79.13, 77.56, 76.44,
76.40, 75.62, 73.20, 72.55, 69.75, 58.83, 56.25, 47.92,
44.45, 40.92, 37.50, 36.60, 28.69, 26.99, 23.23, 21.66,
14.63, 10.47; Anal. Calcd for C47H56NNaO18 (946): C,
59.68; H, 5.97. Found: C, 59.85; H, 6.09; MS (ESI):
m/z 947 [M+H]+, 969 [M+Na]+.
25
D
5.1.7. 20-D-Malyl docetaxel sodium salt (3c). ½aꢂ ꢀ28.98
1
(c 0.70, MeOH); mp 215–219 ꢂC; H NMR (500 MHz,
CD3OD) d 8.00 (d, J = 6.89 Hz, 2H, H-Ph), 7.72 (m,
3H, H-Ph), 7.38 (m, 4H, H-Ph), 7.20 (m, 1H, H-Ph),
5.80 (m, 1H, H-13), 5.41 (m, 1H, H-2), 5.10 (m, 3H,
H-10, H-20, H-30), 4.93 (d, J = 9.20 Hz, 1H, H-5), 4.48
(m, 1H, CH-malyl), 4.27 (m, 1H, H-7), 4.05 (s, 2H, H-
20), 3.82 (br s, 1H, H-3), 2.92 (m, 1H, CH2-malyl),
2.60 (m, 1H, CH2-malyl), 2.47 (m, 1H, H-6a), 2.38 (s,
3H, OCOCH3), 2.24 (m, 1H, H-14a), 1.97 (m, 1H, H-
14b), 1.92 (s, 3H, H-18), 1.80 (m, 1H, H-6b), 1.66 (s,
3H, H-19), 1.40 (s, 9H, t-Bu-H), 1.15 (m, 6H, H-16,
H-17); Anal. Calcd for C47H56NNaO18 (946): C, 59.68;
H, 5.97. Found: C, 59.82; H, 6.11; MS (ESI): m/z 947
[M+H]+, 969 [M+Na]+.
In the same manner, compounds 2band 3b, 2c and 3c
were synthesized.
5.1.8. 20,7,10-Tri-(1,2-O-isopropylidene-DL-malyl) docet-
axel (7). A solution of docetaxel (80.8 mg, 0.10 mmol) and
1,2-O-isopropylidene-malic acid (19.1 mg, 0.11 mmol) in
25
5.1.4. 20-L-Malyl docetaxel (2b). ½aꢂ ꢀ44.29 (c 0.70,
D
MeOH); mp 165–168 ꢂC; 1H NMR (500 MHz, CD3OD)

6328
W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
CH2Cl2 (8 mL) was stirred at 0 ꢂC. Next, DIC (173 lL,
1.10 mmol) and DMAP (6.1 mg, 0.05 mmol) were added.
After 1 h, the mixture was heated to reflux temperature
and stirred for 5 h. The mixture was filtered, diluted with
EtOAc (7.5 mL), and washed with water and brine. The
organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was purified via column chromatog-
solution was cooled to ꢀ23 ꢂC and pyridine (160 lL)
was added. 2,2,2-Trichloroethylchloroformate (14 lL,
0.1 mmol) was slowly added. Stirring was maintained
at ꢀ23 ꢂC for 45 min, and then more 2,2,2-trichloroeth-
yl- chloroformate (12.6 lL, 0.09 mmol) was added. Stir-
ring at ꢀ23 ꢂC was continued for an additional 45 min.
The reaction solution was diluted with CH2Cl2, washed
with water and brine, dried over Na2SO4, concentrated,
and purified via column chromatography (EtOAc–
petroleum ether, 1:2), yielding 8 (75.3 mg, 65%) as a col-
orless sticky oil: IR (KBr, cmꢀ1) 3440.96, 2978.53,
1712.01, 1496.98, 1452.26, 1370.83, 1242.92, 1167.00,
1066.47, 1025.50, 987.01, 737.36, 708.65; 1H NMR
(400 MHz, CDCl3) d 8.11 (d, J = 7.6 Hz, 2H, H-Ph),
7.61 (m, 1H, H-Ph), 7.51 (t, J = 7.6 Hz, 2H, H-Ph),
7.39 (m, 2H, H-Ph), 7.33 (m, 3H, H-Ph), 6.28 (m, 1H,
H-13), 5.65 (d, J = 7.6 Hz, 1H, H-2), 5.51 (m, 2H, H-
20, NH), 5.38 (s, 1H, H-10), 5.22 (br s, 1H, H-30), 4.96
(d, J = 8 Hz, 1H, H-5), 4.74 (m, 2H, H-20), 4.24 (m,
5H, H-7, CH2-Troc),, 3.92 (br s, 1H, H-3), 2.58 (m,
1H, H-6a), 2.46 (s, 3H, OCOCH3), 2.32 (m, 1H, H-
14a), 2.19 (m, 1H, H-14b), 1.91 (m, 4H, H-6b, H-18),
1.74 (s, 3H, H-19), 1.33 (s, 9H, t-Bu-H), 1.23 (s, 3H,
H-16), 1.12 (s, 3H, H-17); 13C NMR (100 MHz, CDCl3)
d 211.14, 171.35, 169.83, 167.35, 166.93, 155.04, 153.24,
138.57, 136.82, 135.69, 133.58, 130.12, 129.16, 128.90,
128.63, 128.33, 126.41, 93.88, 84.28, 80.94, 80.31,
78.73, 78.11, 77.40, 77.07, 76.76, 75.00, 74.32, 71.76,
71.13, 61.19, 57.58, 46.35, 43.04, 36.61, 35.54, 28.04,
26.32, 22.59, 20.94, 14.10, 9.86.
raphy (EtOAc–petroleum ether, 1:2), yielding
7
)
(108.4 mg, 85%) as a colorless sticky oil: IR (KBr, cmꢀ1
3383.16, 2923.89, 2852.79, 1795.61, 1749.66, 1379.31,
1
1270.64, 1171.94, 1127.99, 1065.62, 913.23, 742.14; H
NMR (400 MHz, CDCl3) d 8.09 (d, J = 7.2 Hz, 2H, H-
Ph), 7.61 (m, 1H, H-Ph), 7.51 (m, 2H, H-Ph), 7.42 (m,
2H, H-Ph), 7.32 (br s, 3H, H-Ph), 6.31 (m, 1H, H-10),
6.20 (br s, 1H, H-13), 5.63 (m, 5H, H-7, H-2, H-20, H-30,
NH), 4.31 (d, J = 8 Hz, 1H, H-5), 4.80 (m, 3H, CH-mal-
yl), 4.13 (m, 2H, H-20), 3.92 (br s, 1H, H-3), 3.03 (m,
6H, CH2-malyl), 2.63 (m, 1H, H-6a), 2.44 (s, 3H,
OCOCH3), 2.32 (m, 1H, H-14a), 2.23 (m, 1H, H-14b),
1.97 (s, 3H, H-18), 1.81 (m, 4H, H-6b, H-19), 1.59 (m,
18H, CH3-malyl), 1.35 (s, 9H, t-Bu-H); 1.25 (m, 3H, H-
16), 1.12 (m, 3H, H-17); 13C NMR (100 MHz, CDCl3) d
201.15, 171.90, 171.87, 171.77, 171.54, 171.00, 167.96,
167.65, 166.72, 157.03, 142.04, 137.07, 133.58, 131.85,
130.07, 129.07, 128.80, 128.59, 128.17, 126.35, 111.26,
111.01, 110.72, 83.74, 80.58, 80.18, 78.54, 77.48, 76.12,
75.79, 74.79, 74.04, 71.68, 70.56, 70.46, 70.16, 60.23,
55.75, 43.08, 41.81, 36.72, 36.32, 36.05, 33.16, 32.95,
28.03, 25.63, 25.54, 25.42, 23.32, 22.38, 20.83, 14.07,
10.71.
5.1.9. 20,7,10-tri-(DL-malyl)-docetaxel (4). Compound 7
(127.6 mg, 0.10 mmol) was dissolved in a mixture of
HOAc: THF–H2O (12:3:6 mL). The mixture was stirred
at 45 ꢂC for 6 h. Next, the organic solvents were removed
by evaporation in vacuo. The residue was diluted by
water (20 mL) and freeze-dried, yielding 4 (78.6 mg, 68%)
5.1.11. 20,10-Bis-(2,2,2-trichloroethoxycarbonyl)-7-(1,2-
O- isopropylidene-DL-malyl)-docetaxel (9). A solution of
compound 8 (115.9 mg, 0.10 mmol)and 1,2-O-isopropy-
lidene-malic acid (19.1 mg, 0.11 mmol) in CH2Cl2
(16 mL) was stirred at ꢀ10 ꢂC. Next, DCC (30.9 mg,
0.15 mmol) and DMAP (6.1 mg, 0.05 mmol) were added.
After stirring for 3 h at ꢀ10 ꢂC, the mixture was filtered,
diluted with EtOAc (7.5 mL), and washed with water
and brine. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified via col-
umn chromatography (EtOAc–petroleum ether, 1:2),
yielding 9 (107.8 mg, 82%) as a colorless sticky oil: IR
as a white solid: mp 155–158 ꢂC; IR (KBr, cmꢀ1
)
3418.21, 2942.84, 1737.55, 1633.41, 1371.14, 1252.54,
1
1170.58, 1105.98, 1065.48, 708.71; H NMR (500 MHz,
CD3OD) d 8.11 (d, J = 7.41 Hz, 2H, H-Ph), 7.66 (m,
1H, H-Ph), 7.58 (m, 2H, H-Ph), 7.40 (m, 4H, H-Ph),
7.26 (br s, 1H, H-Ph), 6.32 (s, 1H, H-10), 6.07 (br s,
1H, H-13), 5.62 (m, 2H, H-7, H-2), 5.30 (m, 2H, H-20,
H-30), 4.99 (d, J = 9.29 Hz, 1H, H-5), 4.48 (m, 3H, CH-
malyl), 4.19 (m, 2H, H-20), 3.89 (br s, 1H, H-3), 2.86
(m, 6H, CH2-malyl), 2.55 (m, 1H, H-6a), 2.40 (s, 3H,
OCOCH3), 2.23 (m, 1H, H-14a), 1.98 (m, 1H, H-14b),
1.92 (s, 3H, H-18), 1.78 (m, 4H, H-6b, H-19), 1.41 (s,
9H, t-Bu-H), 1.16 (s, 3H, H-16), 1.12 (s, 3H, H-17); 13C
NMR (125 MHz, CD3OD) d 203.83, 172.99, 171.65,
171.40, 171.32, 171.05, 170.42, 170.22, 167.64, 157.84,
143.13, 138.84, 134.64, 134.04, 133.95, 131.21, 129.90,
129.72, 129.39, 128.31, 85.23, 81.90, 80.87, 78.99, 77.28,
76.95, 76.55, 75.94,73.46, 72.98, 68.32, 68.14, 57.20,
56.20, 48.04, 44.51, 40.43, 40.08, 39.81, 36.38, 33.97,
28.68, 26.68, 23.16, 22.15, 14.96, 11.45; Anal. Calcd for
C55H65NO26 (1156): C, 57.14; H, 5.67. Found: C,
57.38; H, 5.90.
(KBr, cmꢀ1
) 3434.25, 2983.56, 1714.86, 1497.19,
1
1452.59, 1383.17, 1243.34, 1174.69, 738.19, 706.56; H
NMR (400 MHz, CDCl3) d 8.12 (d, J = 8 Hz, 2H, H-
Ph), 7.63 (m, 1H, H-Ph), 7.52 (m, 2H, H-Ph), 7.42 (m,
2H, H-Ph), 7.36 (m, 3H, H-Ph), 6.28 (br s, 1H, H-13),
5.70 (d, J = 6.8 Hz, 1H, H-2), 5.52 (m, 3H, H-10, H-7,
H-20), 5.39 (s, 1H, NH), 5.22 (br s, 1H, H-30), 4.96 (m,
1H, H-5), 4.74 (m, 3H, H-20, CH-malyl), 4.24 (m, 4H,
CH2-Troc), 3.92 (dd, J = 16, 1.2 Hz, 1H, H-3), 2.88 (m,
1H, CH2-malyl), 2.73 (m, 1H, CH2-malyl), 2.56 (m, 1H,
H-6a), 2.48 (s, 3H, OCOCH3), 2.36 (m, 1H, H-14a),
2.20 (m, 1H, H-14b), 1.98 (m, 3H, H-18), 1.87 (m, 4H,
H-6b, H-19), 1.65 (m, 3H, CH3-malyl), 1.58 (m, 3H,
CH3-malyl),1.36 (s, 9H, t-Bu-H), 1.23 (s, 3H, H-16),
1.11 (s, 3H, H-17); 13C NMR (100 MHz, CDCl3) d
210.68, 171.75, 169.70, 167.98, 167.72, 167.24, 166.93,
154.97, 153.19, 139.12, 136.86, 135.26, 133.63, 130.12,
129.11, 128.92, 128.67, 128.30, 126.37, 111.31, 93.85,
83.56, 80.54, 80.47, 78.82, 78.12, 77.30, 76.98, 76.67,
74.77, 74.39, 72.33, 70.47, 70.30, 56.33, 56.26, 48.86,
5.1.10. 20,10-Bis-(2,2,2-Trichloroethoxycarbonyl)-docet-
axel (8). To docetaxel (80.8 mg, 0.10 mmol) under argon
was added CH2Cl2 (2 mL) at room temperature. The

W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
6329
42.93, 36.27, 35.47, 33.85, 28.05, 26.83, 26.61, 26.09,
22.51, 20.73, 13.98, 10.89.
5.2. Biological activity
5.2.1. Water solubility. Docetaxel and docetaxel deriva-
tives were suspended in water until a concentration
was reached of 2 mg/mL. The suspensions were soni-
cated for 15 min and centrifuged (13,000 rpm) for
10 min. The above fluid was analyzed, using HPLC.
5.1.12. 7-(1,2-O-Isopropylidene-DL-malyl) docetaxel (10).
Compound 9 (131.5 mg, 0.10 mmol) was dissolved in a
mixture of CH3OH: HOAc (7.3:0.8 mL) and zinc dust
was added to the mixture. The reaction suspension was
stirred at room temperature for 20 min. The zinc dust
was removed by filtration. The filtrate was concentrated
to dryness, treated with CH2Cl2, and washed with water
and brine. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified via col-
umn chromatography (EtOAc–petroleum ether, 1:2),
yielding 10 (84.8 mg, 88%) as a colorless sticky oil: IR
5.2.2. Stability in PBS-buffer and human plasma. The
docetaxel derivatives (2a–c, 3a–c, 4, and 5) were dis-
solved in water, sonicated, and centrifuged. 10 lL of
the above fluid was mixed with 900 lL of plasma
(heparin) or PBS-buffer (pH 7.4). The solutions were
incubated at 37 ꢂC and on different points (for 2a–c
and 3a–c, T = 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6 h; for com-
pounds 4 and5, T = 0, 3, 6, 9, 12, 24, 36, 48 h)
100 lL was precipitated with 900 lL acetonitrile, then
extracted with 2000 lL of EtOAc. After mixing for
3 min (using a vortex), this mixture was centrifuged
(10 min, 13,000 rpm) and evaporated (4 h, in vacuo).
The (pro)drugs were dissolved in 100 lL methanol
and analyzed by HPLC.
(KBr, cmꢀ1
) 3434.32, 2962.70, 2882.42, 1576.21,
1
1475.20, 1382.50, 1205.24, 1031.59, 882.68, 706.87; H
NMR (400 MHz, CDCl3) d 8.09 (d, J = 7.6 Hz, 2H, H-
Ph), 7.61 (m, 1H, H-Ph), 7.50 (m, 2H, H-Ph), 7.35 (m,
5H, H-Ph), 6.20 (m, 1H, H-13), 5.67 (d, J = 7.2 Hz, 1H,
H-2), 5.54 (m, 2H, H-10, H-30), 5.29 (m, 2H, NH, H-7),
4.92 (d, J = 9.2 Hz, 1H, H-5), 4.65 (m, 2H, CH-malyl,
H-20), 4.31 (d, J = 8.40 Hz, 1H, H-20a) 4.19 (d,
J = 9.2 Hz, 1H, H-20b), 3.96 (m, 2H, H-3, OH), 3.54 (s,
1H,OH), 2.85(m,1H,CH2-malyl),2.72(m,1H,CH2-mal-
yl), 2.54 (m, 1H, H-6a), 2.39 (s, 3H, OCOCH3), 2.28 (m,
2H, H-14), 1.87 (m, 7H, H-6b, H-18, H-19), 1.34 (m, 9H,
t-Bu-H), 1.64 (m, 3H, CH3-malyl), 1.57 (m, 3H, CH3-mal-
yl),1.21 (s, 3H, H-16), 1.09 (s, 3H, H-17); 13C NMR
(100 MHz, CDCl3) d 210.64, 171.81, 170.25, 168.05,
167.85, 166.86, 155.29, 138.36, 135.41, 133.66, 130.10,
129.03, 129.01, 128.74, 128.67, 127.92, 126.66, 111.36,
83.50, 80.52, 80.05, 78.71, 77.35, 77.03, 76.71, 74.63,
73.60, 72.71, 70.29, 60.37, 56.36, 45.88, 42.84, 36.23,
35.95, 35.66, 28.12, 26.81, 25.75, 22.41, 21.00, 14.13, 10.86.
5.2.3. In vitro cytotoxicity assay. The cytotoxic activity
in vitro was measured using the MTT assay. The MTT
solution (final concentration: 0.5 mg/ml) in RPMI-1640
(Sigma, St. Louis, MO) was added after cells were treated
with the drug for 48 h, and cells were incubated for fur-
ther 4 h at 37 ꢂC. The purple formazan crystals were dis-
solved in 100 lL DMSO. After 5 min, the plates were
read at 570 nm by an automated microplate spectropho-
tometer (Bio-Tek Instruments, Winooski, VT). The con-
centration required for 50% inhibition of cell viability
(IC50) was calculated using the software ‘Dose–Effect
Analysis with Microcomputers’. The cancer cell lines’ pa-
nel consisted of A549, K562, KB, and MCF-7. In all of
these experiments, three replicate wells were used to
determine each point, and all of the experiments were
performed in triplicate.
5.1.13. 7-DL-Malyl-docetaxel (5). Compound 10
(96.4 mg, 0.10 mmol) was dissolved in a mixture of
HOAc: THF–H2O (12:3:6 mL). The mixture was stirred
at 45 ꢂC for 6 h. Next, the organic solvents were removed
by evaporation in vacuo. The residue was diluted by water
(10 mL) and freeze-dried, yielding 5 (79.4 mg, 86%) as a
white solid: mp 164–168 ꢂC; IR (KBr, cmꢀ1) 3439.42,
2979.48, 2942.84, 1731.76, 1496.49, 1452.14, 369.21,
1248.68, 1168.65, 1102.12, 982.55, 708.71; 1H NMR
(500 MHz, CD3OD) d 8.10 (d, J = 7.36 Hz, 2H, H-Ph),
7.66 (m, 1H, H-Ph), 7.57 (t, J = 7.17 Hz, 2H, H-Ph),
7.38 (m, 4H, H-Ph), 7.25 (m, 1H, H-Ph), 6.15 (m, 1H,
H-13), 5.65 (dd, J = 6.45, 1.71 Hz, 1H, H-2), 5.57 (m,
1H, H-7), 5.34 (s, 1H, H-10), 5.11 (br s, 1H, H-30), 4.98
(d, J = 8.99 Hz, 1H, H-5), 4.51 (br s, 1H, H-20), 4.42 (br
s, 1H, CH-malyl), 4.21 (s, 2H, H-20), 3.92 (m, 1H, H-3),
2.76 (m, 1H, CH2-malyl), 2.60 (m, 1H, CH2-malyl), 2.47
(m, 1H, H-6a), 2.35 (s, 3H, OCOCH3), 2.24 (m, 1H, H-
14a), 2.04 (m, 1H, H-14b), 1.91 (m, 4H, H-6b, H-18),
1.80 (s, 3H, H-19), 1.40 (s, 9H, t-Bu-H), 1.15 (s, 3H, H-
16), 1.09 (s, 3H, H-17); 13C NMR (125 MHz, CD3OD)
d 209.66, 173.19, 170.77, 169.77, 169.68, 166.47, 156.53,
139.43, 138.64, 136.36, 133.39, 130.15, 129.96, 128.49,
128.37, 127.56, 127.00, 83.92, 80.68, 79.54, 77.99, 76.30,
75.06, 74.52, 74.17, 72.63, 71.23, 67.34, 57.36, 56.25,
46.31, 43.19, 39.35, 35.63, 32.94, 27.50, 25.59, 21.87,
20.42, 13.28, 10.27; Anal. Calcd for C47H57NO18 (924):
C, 61.10; H, 6.22. Found: C, 61.25; H, 6.37.
5.2.4. Antitumor activity in ICR mice Hepatoma-22
(H22) tumor. Tumor bearing mice models were estab-
lished by s.c. injections of H22 cells (5 · 106 cells per ani-
mal, subcutaneously into the armpit) in male ICR mice
(National rodent laboratory animal resource, Shanghai
branch, China). Treatments were initiated 24 h after
the injections of tumor cells. Mice in different treatment
groups (negative control group containing 20 female
mice, other groups containing 10 female mice) were in-
jected intraperitoneally (ip) with the compound 3a (5,
10, 20 mg/kg) or docetaxel once a day for consecutive
7 days. Docetaxel of 10 mg/kg was used as a positive
control and physiological saline as negative control. Tu-
mors were dissected and weighed at day 8, and inhibi-
tion rates were calculated as follows: (C ꢀ T)/C · 100,
T, average tumor weight of treated group; C, average tu-
mor weight of negative control group.
Acknowledgments
We thank Department of Chemistry and Pharmaceuti-
cal of Informatics Institute of Zhejiang University for

6330
W. Du et al. / Bioorg. Med. Chem. 15 (2007) 6323–6330
1H NMR, 13C NMR, and ESI-MS spectra. We are also
grateful to other staff of ZJU-ENS Joint Medicinal
Chemistry Laboratory.
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