The synthesis of novel taxoids for oral administration

Article abstract of DOI:10.1016/j.bmc.2013.11.037

A group of novel taxoids, with modifications at C-7, C-10, C-3′ and C-14 positions of paclitaxel, was synthesized in order to improve their biological profile by decreasing their affinity with P-glycoprotein (P-gp) and increasing cellular permeability. Most of the new taxoids demonstrated the similar potent cytotoxic activities in MCF-7 human tumor cell line as paclitaxel in vitro. In the permeability assay with monolayers of Caco-2 cells, most of the compounds demonstrated an increased trans-cellular transport in A-to-B direction in comparison with paclitaxel. Among them the compounds T-13, T-15 and T-26 showed the highest permeability, and with efflux ratios better than that of ortataxel. The interaction of the compounds T-13 and T-26 with P-gp was evaluated using Madin-Darby canine kidney (MDCK)-multidrug resistance-1(MDR1) and MDCK-wild-type (WT). The results indicated that T-13 and T-26 were poor substrates for P-gp and possessed inhibiting effects of P-gp mediated efflux. It was thus clear that simultaneous modifications at the C-7, C-10 and C-3′ positions of paclitaxel significantly impaired its interactions with P-gp and interfered with P-gp mediated efflux.

Full text of DOI:10.1016/j.bmc.2013.11.037

Bioorganic & Medicinal Chemistry 22 (2014) 194–203
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The synthesis of novel taxoids for oral administration
b,c,
Yun-rong Jing a, Wei Zhou b, Wan-liang Li b, Lin-xiang Zhao a, , Yong-feng Wang
⇑
⇑
a Key Laboratory of Structure-Based Drug Design & Discovery, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China
b Chem-Pharm R&D Institute, Tianjin Tasly Group Co., Ltd, Tasly TCM Garden, No. 2, Pujihe East Road, Beichen District, Tianjin 300402, PR China
c Zhuhai Oxforston PharmTech Co., Ltd, Zhuhai, Guangdong 519085, China
a r t i c l e i n f o
a b s t r a c t
A group of novel taxoids, with modifications at C-7, C-10, C-30 and C-14 positions of paclitaxel, was syn-
thesized in order to improve their biological profile by decreasing their affinity with P-glycoprotein (P-
gp) and increasing cellular permeability. Most of the new taxoids demonstrated the similar potent cyto-
toxic activities in MCF-7 human tumor cell line as paclitaxel in vitro. In the permeability assay with mon-
olayers of Caco-2 cells, most of the compounds demonstrated an increased trans-cellular transport in A-
to-B direction in comparison with paclitaxel. Among them the compounds T-13, T-15 and T-26 showed
the highest permeability, and with efflux ratios better than that of ortataxel. The interaction of the com-
pounds T-13 and T-26 with P-gp was evaluated using Madin-Darby canine kidney (MDCK)-multidrug
resistance-1(MDR1) and MDCK-wild-type (WT). The results indicated that T-13 and T-26 were poor sub-
strates for P-gp and possessed inhibiting effects of P-gp mediated efflux. It was thus clear that simulta-
neous modifications at the C-7, C-10 and C-30 positions of paclitaxel significantly impaired its interactions
with P-gp and interfered with P-gp mediated efflux.
Article history:
Received 19 July 2013
Revised 17 November 2013
Accepted 18 November 2013
Available online 27 November 2013
Keywords:
Paclitaxel
1,14-Carbonate baccatin III
Oral bioavailability
P-gp substrate
Bi-directional Caco-2 cells permeability
MDCK-MDR1 monolayers
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
suffered efflux mediated by P-gp in the gastrointestinal tract. It was
observed that modifications of paclitaxel at the C-7, C-10 and C-30
Taxoids have become one of the most potent and successful
anticancer drugs derived from nature.1 Paclitaxel, in particular,
alone and combined with other drugs has been widely applied in
chemotherapy for treatments of a range of tumors, such as ovarian,
lung, bladder, cervix, and metastatic breast cancer (MBC),2 as well
as melanomas and glioma.3 However its application has been im-
peded by its intrinsic physicochemical properties and enzymatic
affinities, such as poor aqueous solubility,4 and being a substrate
of P-glycoprotein.5 As a result, its clinically available dosage forms
are only injections, some of which contain a excipient6 with the
potented for causing allergic reactions. Development of a viable
injection formulation for taxoids and an oral taxoid have been a
challenging task for pharmaceutical researchers.
Paclitaxel is a highly substituted polyoxygenated diterpenoid. In
recent years, a number of taxoids derived from 14b-hydroxy-10-
deacetylbaccatin III have been showed good oral bioavailability while
maintaining anticancer activity.7 Ortataxel (Fig. 1), in particular, has
entered phase II clinical trials with oral administration. It was sug-
gested that the better oral absorption of these new taxoids resulted
from their impaired affinity with P-gp; therefore, they no longer
positions were crucial in weakening the interactions between taxane
with P-gp,8 for example, BMS-184476,9 milataxel10 and tesetaxel
(Fig. 1)11 in which modifications occurred at C-7 and C-30; while in
the case of simotaxel the modification of docetaxel was at the C-10
and C-30 positions. In summary, modifications at the C-7, C-10, C-
14 and C-30 positions of paclitaxel appear to disturb its interactions
with P-gp, as a result, its poor oral bioavailability and hydrophobic-
ity-related drug resistance were improved.
In order to explore the effects of modified C-7, C-10, C-14, and
C-30 substituents on the affinity of paclitaxel analogs with P-gp
and to find potent oral taxoid candidates, we decided to synthesize
a group of new taxoids with modifications at the northern hemi-
sphere of paclitaxel at C-7 (ester, amino ester, deoxy or methyl
ether), C-10 (ester, amino ester, or methyl ether) and C-30
(30N-Boc and 30-aromatic ring or alkyl group) positions, as well
as a group of taxoids with modifications at the northern and the
southern hemisphere of paclitaxel at C-7, C-10, C-14 (1,14-carbon-
ate moiety) and C-30 positions. Herein, we report the design, syn-
thesis, and biological evaluation of these novel taxoids.
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. at: Zhuhai Oxforston PharmTech Co., Ltd, Zhuhai,
Guangdong 519085, China. Tel.: +86 24 23986420; fax: +86 24 23986420 (L.-x.Z.);
tel.: +86 22 26736391; fax: +86 22 86342716 (Y.-f.W.).
E-mail addresses: linxiang.zhao@vip.sina.com (L.-X. Zhao), yonkenafil@yahoo.
co.uk (Y.-F. Wang).
Following literature methods, a group of oxazolidine carboxylic
acid derivatives 7a–d were prepared, ready for acylation with the
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.037

Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
195
N
R1
O
R2
R6
R5
O
O
NH
O
O
O
O
N
O
NH
O
H
OH
O
O
R4
O
O
O
R3
O
H
O
O
HO
O
OH
O
F
Ortataxel:
R1=AcO, R2=OH, R3,R4=1,14-carbonate
R5=CH2CH(CH3)2, R6=t-butyloxycarbonyl;
Tesetaxel
BMS-184476: R1=AcO, R2=OCH2SCH3, R3=H,
R4=OH, R5=C(CH3)3, R6=t-butyloxycarbonyl;
R1=OH, R2=OCOCH2CH3, R3=H,
R4=OH, R5=2-furanyl, R6=t-butyloxycarbonyl;
Milataxel:
Simotaxel: R1=cyclopentylcarbonyloxy, R2=OH,
R3=H, R4=OH, R5=2-thiophene
R6=isopropyloxycarbonyl
Figure 1. Taxanes bearing modifications at the key positions C-7, C-10, C-14 and C-30 endowed with cytotoxic activity against MDR cell lines.
designed baccatins. First, O-benzyl, Boc-
ester 1 was converted to its enolate with lithium, followed by addi-
tion of (SR)-tert-butanesulfinamide 2 to give enantiopure 3-substi-
a
-hydroxyacetate benzyl
group in 9 with carbonyldiimidazole, followed by acylation at C-
10 and methylation at C-7. Their reverse counterparts B-05 and
B-06 were synthesized by selective acylation of the 7-hydroxyl in
10 (Scheme 2).
tuted isoserine benzyl esters
3 in excellent yield and high
diastereoselectivity.12 After exchange of the amine protective
group and condensation with 1-dimethoxymethyl-4-methoxy-
benzene, the oxazolidine carboxylic acid derivatives 7a–d were ob-
tained in high yields (Scheme 1).
1,14-Carbonate baccatins III B-07–B-14 also were prepared
(Scheme 3). MnO2 oxidation of the C13-hydroxy group in B-01–
B-06 and 10 gave 13, which underwent an enolation with N-(sulfo-
nyl) oxaziridines followed by a diastereoselective 14b-hydroxyl-
ation to give 14.15 Treatment of 14 with carbonyldiimidazole
afforded the 1,14-carbonate derivatives 15. Diastereoselective
reduction of the C13-oxo group of 15 with BH3ꢀTHF in the presence
The designed baccatins were synthesized starting with 10-
deacetylbaccatin III (10-DAB, 8), an abundant taxoid from renew-
able parts of Taxus spp. plants.13 The first baccatin 7,10-di-
methyl-baccatin III B-01 was prepared by methylation of 8 with
methyl trifluorosulfonate (TfOMe) in quantitative yield (Scheme 2).
The 7-hydroxyl group of 8 was selectively protected by using
triethylchlorosilane in quantitative yield.14 In the presence of
LHMDS, 9 was treated with TfOMe to introduce the methyl group
selectively at the 10-hydroxyl group. After the silyl group was re-
moved, the 7-hydroxyl group of 10 underwent a selective methyl-
of (R)-2-methyl–CBS-oxazaborolidine afforded the 13a-OH epimer
only, yield 85–90% with 99% ee.
The target taxoids T-01–T-35 were synthesized by acylations of
B-01–14 with 7a–d,16 followed by deprotection with acetyl chlo-
ride in MeOH (Scheme 4 and Table 1).
2.2. Biological evaluation
thiocarbonylation with thiocarbonyldiimidazole, ready for
a
radical deoxygenation. Treatment of the resulting compound with
tributyltin hydride in the presence of AIBN at 80 °C14 gave the 7-
deoxy derivative B-02 (Scheme 2).
2.2.1. In vitro growth-inhibition assay
The synthesized target compounds T-01–T-35 (Table 1) demon-
strated almost the same inhibitory activity as paclitaxel (Table 2)
10-Carbonate-7-methyl-baccatin B-03 and 10-carbonate deri-
vate B-04 were prepared through activation of the 10-hydroxyl
in a test against eight selected human tumor cell lines at 1
concentration. In order to substantiate the potency of the new
lM
O
O
S
O
NH2 O
N
a
R1
S
NH
O
c
b
2
OBn
R1
OBn
R1
OBn
OBoc
OBoc
OH
2a: R1=Ph
2b: R1=Py
3a: R1=Ph
4a: R1=Ph
4b: R1=Py
1
3b: R1=Py
2c: R1=CH2CH(CH3)2 3c: R1=CH2CH(CH3)2
4c: R1=CH2CH(CH3)2
R1
R1
R2
R2
R2
O
OBn
O
N
N
O
NH
O
e
d
O
OH
R1
OBn
OH
O
O
5a: R1=Ph, R2=Bz
6a: R1=Ph, R2=Bz
7a: R1=Ph, R2=Bz
7b: R1=Ph, R2=Boc
7c: R1=Py, R2=Boc
5b: R1=Ph, R2=Boc
6b: R1=Ph, R2=Boc
6c: R1=Py, R2=Boc
5c: R1=Py, R2=Boc
5d: R1=CH2CH(CH3)2, R2=Boc
6d: R1=CH2CH(CH3)2, R2=Boc
7d: R1=(CH3)2CHCH2, R2=Boc
Scheme 1. Reagents and conditions: (a) LHMDS, THF, ꢁ70 °C, 70%; (b) HCl/EtOAc, 83%; (c) Boc2O or BzCl, DMAP, CH2Cl2, 90%; (d) 1-dimethoxymethyl-4-methoxy-benzene,
PPTS, toluene, 100 °C, 80%; (e) H2, Pd(OH)2, MeOH, 87%.

196
Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
HO
O
OH
O
O
O
a
O
O
HO
HO
HO
H
H
OAc
OAc
HO
HO
OBz
8
HO
OBz
B-01
b
O
O
O
O
O
OTES
OH
a, c
d, e
f, g
O
O
O
HO
HO
H
H
H
OAc
OAc
O
OAc
HO
HO
HO
OBz
OBz
10
OBz
B-02
9
f, g
O
R2
O
R1
O
R1
O
O
O
OTES
O
c, a
O
O
O
HO
HO
HO
H
H
H
OAc
OAc
OAc
HO
HO
OBz
OBz
HO
OBz
11: R1=CON(Me)2
12: R1=COOMe
B-03: R1=CON(Me)2
B-04: R1=COOMe
B-05: R2=CON(Me)2
B-06: R2=COOMe
Scheme 2. Reagents and conditions: (a) LHMDS, THF, TfOMe, ꢁ72 °C, 75–87%; (b) TESCl, imidazole, DMF, 0 °C, 90%; (c) TBAF, THF, 90–93%; (d) TCDI, DBU, THF, 83%; (e)
Bu3HSn, AIBN, dioxane, 80 °C, 63%; (f) CDI, THF, 85–90%; (g) CH3OH or (CH3)2NH, THF, 76% or 83%.
R
1
R
1
O
O
R
R
R
O
R
1
2
2
2
b
a
c
O
O
O
O
O
HO
H
H
H
OAc
OAc
OAc
HO
HO
HO
OBz
13
OBz
14
HO
OBz
B-01~B-06, and 10
R
R
1
O
O
R
R
1
B-07: R =R =MeO;
2
2
1
1
2
B-08: R =MeO, R =H;
2
B-09: R =MeO, R =OTES;
1
2
B-10: R =OCON(Me) , R =MeO;
d
1
2
2
O
O
O
HO
B-11: R =OCOOMe, R =MeO;
1
2
H
H
OAc
OAc
OBz
B-12: R =MeO, R =OCON(Me) ;
O
O
OBz
1
2
2
O
O
B-13: R =MeO, R =OCOOMe;
1
1
2
2
B-14: R =AcO, R =OTES
O
O
15
Scheme 3. Reagents and conditions: (a) MnO2, acetone, >80%; (b) t-BuOK, THF/DMPU, ꢁ72 °C, N-(sulfonyl) oxaziridines, 80%; (c) CDI, THF, >90%; (d) BH3/(R)-2-methyl-CBS-
oxazaborolidine, THF, rt, 83–87%.
R1
O
R2
R1
O
R2
R5
O
O
R5
a
+
R6
N
O
OH
O
O
R6
HO
N
H
O
H
O
OAc
R4
R3
OBz
OAc
R4
OBz
Ar
R3
Ar
Ar=4-MeO-C6H4
16
B-01~B-14
R1
O
R2
R6
NH
O
b
O
R5
O
H
OH
OAc
R4
OBz
R3
T-01~T-35
Scheme 4. Reagents and conditions: (a) DCC/DMAP/PTSA, CH2Cl2, >90%; (b) AcCl/CH3OH, >90%.

Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
197
Table 1
Syntheses of T-01–T-35
dimethylaminocarbonyl and T-30–T-32 bearing 1,14-carbonate-
10-methyl-7-dimethylaminocarbonyl groups. These compounds
showed a decreased potency against the MCF-7 cell line, however,
they are still cytotoxic agents with IC50 values under 65 nM. There-
fore, it can be concluded that modifications at the C-7, C-10, C-14
or C-30 position of taxane at one or two, three, or four positions
did not reduce cytotoxicity significantly as compared to paclitaxel.
Taxoid
R1
R2
R3
R4
R5
Re
T-01
T-02
T-03
T-04
T-05
T-06
T-07
T-08
T-09
T-10
T-ll
T-12
T-13
T-14
T-15
T-16
T-17
T-18
T-19
MeO
MeO
MeO
MeO
Me2NCOO
Me2NCOO
Me2NCOO
Me2NCOO
MeOCOO
MeOCOO
MeOCOO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
H
H
MeO
MeO
MeO
MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Ph
Ph
Ph
Bz
t-Boc
t-Boc
t-Boc
Bz
t-Boc
t-Boc
t-Boc
Bz
t-Boc
t-Boc
Bz
t-Boc
t-Boc
t-Boc
Bz
Me2CHCH2
Ph
Ph
2-Py
Me2CHCH2
Ph
Ph
Me2CHCH2
Ph
Ph
2-Py
Me2CHCH2
Ph
Ph
2-Py
Me2CHCH2
2.2.2. Caco-2 monolayers permeability assay
Caco-2 monolayers have been widely used as an in vitro model
for prediction of intestinal permeability and hence oral absorption
of therapeutic agents,17 especially during the early drug discovery
process for compounds screening and prioritizing. One of the char-
acteristics affecting intestinal permeability of a compound is its
substrate activity for P-glycoprotein (P-gp), an efflux transporter
responsible for multiple drug resistance expressed in many tissues
including epithelia cells of the intestinal mucosa.18 Therefore, mit-
igating substrate activity for P-gp became one of the strategies in
drug discovery for optimization of oral bioavailability. The trans-
cellular permeability of the new taxoids T-01–T-34 were assessed
and the results obtained are summarized in Table 4. Apparent per-
meability coefficients (Papp) from A-to-B (apical to basolater of the
cell monolayers) and B-to-A (basolateral to apical of the cell mon-
olayers) were obtained by measuring the amount of the compound
MeO
MeO
MeO
Me2NCOO
Me2NCOO
Me2NCOO
Me2NCOO
MeOCOO
MeOCOO
MeOCOO
MeOCOO
t-Boc
t-Boc
t-Boc
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
T-20
MeO
MeO
Ph
Bz
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
T-21
MeO
MeO
Ph
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
t-Boc
transported from the donor compartment at 10 lM to the receiver
T-22
MeO
MeO
2-Py
compartment after 90 min incubation. Quantification was done by
LC-MS/MS. P-gp substrate activity was assessed by the efflux ratio
(ER) (the ratio of Papp in the B-to-A direction over that in the A-to-
B direction). Erythromycin, paclitaxel and ortataxel (T-35) were in-
cluded as the parallel references in the same experiments. The
trans-cellular transport of most of the new taxoids in the A-to-B
direction was increased compared with paclitaxel (Papp A–
B = 0.97), and over half the new taxoids demonstrated a similar
trans-cellular efficacy to ortataxel (Papp A–B = 2.80). It appeared
the compounds T-13, T-15 and T-26 were the most able com-
pounds in the group to undergo the trans-cellular transport (Papp
>10.0 ꢂ 10ꢁ6 cm/s).
T-23
MeO
MeO
Me2CHCH2
Ph
T-24
MeO
H
T-25
MeO
OH
Ph
T-26
Me2NCOO
Me2NCOO
Me2NCOO
MeOCOO
MeO
MeO
Ph
T-27
MeO
2-Py
To further assess substrate activity for the P-gp, a group of com-
pounds with the Papp in A-to-B higher than 5 ꢂ 10ꢁ6 cm/s was
chosen for evaluation of efflux ratios in bi-directional Caco-2 cells
permeability studies and the results are summarized in Table 5.
Paclitaxel exhibited asymmetric transport across Caco-2 monolay-
ers with an efflux ratio higher than 34. Ortataxel19 also showed
asymmetric transport but to a much less extent compared with
paclitaxel. A compound with an efflux ratio (Papp B–A/Papp A–B)
greater than 2.0 is generally considered a substrate of an efflux
transporter.20 The compounds T-13 and T-15 showed efflux ratios
less than 2.0, indicating that they are probably not P-gp substrates.
The compounds T-04, T-06, T-08, T-10, T-11, T-17 and T-19
showed efflux ratios smaller than ortataxel (ER = 6.84) suggesting
that modification at C-7, C-10 and C-30 positions could minimize
substrate activity for P-gp.
In addition to the modifications at the position C-7 and C-10, a
1,14-carbonate moiety as seen in ortataxel was introduced in
compounds T-20–T-34. Most of these compounds exhibited Papp
similar to that of ortataxel. To further investigate the structure-
transporter relationship for the new structure features, the
compounds T-21, T-24, T-25, T-32 and T-34 were examined by per-
meability assessment bidirectionally in Caco-2 cell monolayers and
their efflux ratios were determined (Table 5). Although some of
them showed a decreased efflux ratios compared to that of paclit-
axel, they still retained some substrate activity for P-gp. The results
suggested that attempts to modify the C-7, C-10 and C-14 positions
of taxane simultaneously did not provide a synergistic effect on
weakening its affinity for P-gp. Compound T-26 was an interesting
case which showed substantially reduced substrate activity for
T-28
MeO
Me2CHCH2
Ph
T-29
MeO
T-30
Me2NCOO
Me2NCOO
Me2NCOO
MeOCOO
MeOCOO
OH
Ph
T-31
MeO
2-Py
T-32
MeO
Me2CHCH2
Ph
T-33
MeO
T-34
MeO
Me2CHCH2
Me2CHCH2
T-35 (ortataxel)
AcO
compounds against paclitaxel sensitive cancer cell lines, the IC50
values of the new compounds against the MCF-7 cell line were
determined (Table 3). Most of new compounds demonstrated a
comparable potency to paclitaxel against MCF-7 cells, with IC50
values under 10 nM, except for T-12–T-15 bearing 10-methyl-7-

198
Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
Table 2
In vitro growth-inhibition assay of T-01–T-35
Taxoid
Inhibition % at 1 lM
A2780
(ovarian)
HeLa
(cervix)
MCF-7
(breast)
NCI-H460
(NSCL)
A375
(melanoma)
HT29
(colon)
HL60
(leukemia)
DU145
(prostate)
Paclitaxel
T-01
T-02
T-03
T-04
T-05
T-06
T-07
T-08
T-09
T-10
T-ll
T-12
T-13
T-14
T-15
T-16
T-17
T-18
T-19
T-20
T-21
T-22
T-23
T-24
T-25
T-26
T-27
T-28
T-29
T-30
T-31
T-32
T-33
T-34
Ortataxel
96.68
95.22
93.84
95.79
95.63
96.31
94.82
95.53
94.82
95.80
94.13
95.60
95.57
95.81
94.69
95.01
96.88
96.42
96.55
94.87
96.72
94.69
94.65
94.59
96.00
95.02
92.50
95.63
94.98
94.33
95.86
95.73
96.12
96.60
95.51
96.40
101.04
101.55
98.18
101.12
99.31
101.83
99.76
100.30
100.56
100.93
99.53
94.20
92.10
88.53
95.52
97.84
85.73
93.49
82.35
89.78
95.47
95.28
102.90
90.79
102.13
83.46
101.21
106.85
102.71
106.08
99.96
96.00
93.54
100.73
84.86
86.26
87.12
81.28
89.77
85.90
81.75
84.68
95.53
97.22
93.12
85.40
88.69
87.48
85.05
79.84
85.74
84.31
86.54
83.31
83.56
80.65
86.44
80.43
85.75
87.68
86.59
87.75
86.19
90.32
89.49
88.69
81.60
87.94
82.25
81.84
81.98
84.68
82.65
85.19
85.82
81.01
82.32
86.23
86.03
87.23
89.16
86.79
85.51
96.22
89.73
96.95
91.64
104.36
87.65
87.71
86.80
89.00
94.18
102.87
91.86
88.42
90.43
90.34
104.50
93.96
97.39
88.34
90.21
90.80
89.84
82.17
87.90
94.40
92.54
92.85
96.93
94.18
91.97
93.14
97.86
96.29
94.89
93.38
96.65
88.36
87.54
87.25
88.28
88.18
87.89
87.92
88.10
87.29
88.75
87.32
89.09
89.36
89.81
89.11
89.15
89.73
89.65
89.76
88.41
89.41
88.98
88.28
87.93
87.33
86.58
81.67
87.84
86.21
85.44
87.38
88.67
89.19
88.49
87.25
87.65
96.80
97.22
97.84
97.65
97.58
97.45
97.21
98.11
97.66
96.84
97.40
96.94
97.82
97.77
97.75
98.09
97.79
97.01
97.44
99.11
96.57
95.90
95.81
97.17
97.66
97.46
96.85
98.52
97.49
97.19
97.53
97.83
97.81
97.34
96.92
96.52
90.62
86.42
83.14
87.62
87.04
87.14
84.79
84.69
82.89
86.20
83.30
85.74
85.80
87.45
86.30
86.86
88.35
88.52
89.18
84.92
87.76
84.71
83.87
83.88
89.14
86.78
86.73
90.26
87.98
86.65
90.66
90.76
91.41
90.68
88.62
90.58
99.65
102.82
102.87
102.64
102.32
102.11
101.56
101.75
99.99
100.54
98.20
98.28
99.45
99.89
100.10
97.27
101.39
99.86
100.43
100.48
101.19
101.60
100.94
100.57
99.54
Table 3
In vitro growth-inhibition assay of taxoids against MCF-7 tumor cell line
evaluated in MDCK-MDR1 and MDCK-WT cell monolayers in the
presence and absence of known P-gp inbitor verapamil
(100
M)21 (Table 6). As shown in Table 6, all the test compounds
a
Test compound
MCF-7 (IC50, nM)
Test compound
MCF-7 (IC50, nM)
l
Paclitaxel
T-03
T-04
T-06
T-08
T-10
T-ll
T-12
T-13
T-14
T-15
T-17
T-19
T-20
7.05 0.05
3.33 0.06
3.06 0.21
1.58 0.04
1.70 0.06
1.40 0.02
2.90 0.08
64.67 0.18
9.63 0.12
46.63 0.21
13.26 0.12
4.97 0.05
2.81 0.02
6.93 0.16
T-21
T-22
T-23
T-24
T-26
T-27
T-28
T-29
T-30
T-31
T-32
T-33
T-34
2.10 0.08
3.40 0.08
4.04 0.14
4.49 0.18
7.93 0.08
14.31 0.12
6.44 0.25
3.24 0.12
15.40 0.16
18.21 0.21
21.17 0.24
3.84 0.07
2.94 0.11
exhibited efflux in the MDCK-MDR1 cells either similar or stronger
than those in the MDCK-WT cells. The efflux ratios of T-13 and T-
26 were 6-fold and 30-fold less than that of digoxin and paclitaxel,
respectively. The efflux of both compounds were reduced signifi-
cantly in the presence of 100 lM verapamil suggesting that com-
pounds T-13 and T-26 retained P-gp substrate activity although
weaker than that of paclitaxel.
2.2.4. Effects of T-13 and T-26 on digoxin uptake
The inhibitory interactions of compounds T-13 and T-26 on P-
gp activity were investigated by assessing their effect on digoxin
efflux in MDCK-MDR1 cells. As shown in Table 7, in the presence
All assays consisted of at least six replicate wells and were performed on three
separate occasions. IC50 values are presented as the mean standard deviation.
Cells were exposed to each agent for 72 h.
of 10 lM of T-13 and T-26, the efflux ratio of digoxin reduced from
27 down to 8 and 6, respectively. In contrast under the same con-
dition, paclitaxel had no effect on P-gp mediated digoxin efflux.
The results suggested that the T-13 and T-26 could inhibit P-gp
activity.
P-gp as indicated by the efflux ratios (ER = 2.04) compared to that
of ortataxel (ER = 6.84).
3. Conclusions
2.2.3. P-gp-mediated BL-AP transport in MDCK-MDR1 and
MDCK-WT monolayers
The further investigating experiment of the interaction of the
new compounds T-13 and T-26 with P-gp were conducted. The
AP-BL and BL-AP Papp of paclitaxel, T-13, T-26, and digoxin were
In summary, a group of novel taxoids was synthesized with
modifications at the C-7, C-10, C-14 and C-30 positions. Their
anticancer activities were evaluated against MCF-7 human breast
tumor cell line in vitro by MTT assay and their affinities with
P-gp were measured in the bi-directional Caco-2 cell permeability

Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
199
Table 4
Apparent permeability coefficients (10ꢁ6 cm/s) of test compounds in A-to-B direction
Test compound
Pappa A–B (10ꢁ6cm/s)
Sample-2
Test compound
Pappa A–B (10ꢁ6 cm/s)
Sample-2
Sample-1
Mean
Sample-1
Mean
Erythromycin
Paclitaxel
Ortataxel
T-26
T-15
T-13
T-ll
T-10
T-06
T-08
T-19
T-04
T-17
T-27
T-18
T-14
T-09
T-21
0.80
1.02
2.76
19.53
11.82
11.19
8.58
8.45
9.00
6.41
6.13
7.85
6.66
5.78
4.71
4.27
4.44
4.47
0.63
0.93
2.84
17.33
9.54
9.91
9.46
9.06
8.38
9.92
8.81
6.49
7.29
3.59
4.35
4.54
4.31
3.87
0.71
0.97
2.80
18.43
10.68
10.55
9.02
8.75
8.69
8.17
7.47
7.17
6.97
4.68
4.53
4.41
4.38
4.17
T-33
T-02
T-22
T-03
T-16
T-12
T-07
T-05
T-24
T-23
T-31
T-32
T-20
T-28
T-34
T-25
T-30
T-29
4.70
4.32
4.46
3.80
3.23
3.16
2.93
3.21
3.24
2.59
2.14
2.76
1.50
1.25
1.08
0.78
<1.30b
<1.36b
3.16
3.15
2.86
3.49
3.41
3.38
3.10
2.68
2.05
2.56
2.78
2.03
1.47
0.89
0.75
0.97
<1.67
<1.43
3.93
3.73
3.66
3.64
3.32
3.27
3.02
2.94
2.64
2.57
2.46
2.40
1.48
1.07
0.92
0.88
<1.67
<1.43
a
Apparent permeability coefficient.
b
Papp values were expressed as ‘<’ than the values that were calculated using the minimum concentration of the standards for receiver sides due to the fact that real
concentration in the receivers were below quantitation limit (BQL).
Table 5
The Efflux ratios of test compounds with Papp A-to-B higher than 5.0 ꢂ 10ꢁ6 cm/sec and some representative compounds which introduced 1, 14-carbonate moiety
Test compound
Caco-2 cell lines (21 days)/HBSS buffer, pH 7.4
Papp (10ꢁ6 cm/s)
Efflux ratioa
A–B
0.58
RSD
B–A
9.89
RSD
Erythromycin
Paclitaxel
Ortataxel
T-15
T-13
T-26
T-ll
T-08
T-06
T-10
T-19
T-04
T-17
T-32
T-21
T-34
T-24
T-25
0.08
0.07
0.02
0.28
0.09
0.04
0.06
0.14
0.10
0.02
0.16
0.08
0.08
0.21
0.19
0.25
0.32
0.15
0.04
0.01
0.22
0.13
0.11
0.07
0.12
0.06
0.02
0.08
0.22
0.32
0.04
0.24
0.18
0.10
0.20
0.09
16.92
34.38
6.84
1.59
1.73
2.04
3.75
3.95
4.08
4.22
5.08
5.12
5.23
2.39
6.42
7.81
9.56
51.04
0.97
2.80
19.43
15.04
16.40
8.87
8.25
8.11
6.91
7.16
7.54
7.42
2.40
2.83
0.92
2.64
0.88
33.39
19.16
30.93
25.99
33.48
33.26
32.58
33.11
29.18
36.37
38.57
38.81
5.74
18.20
7.16
25.26
44.70
a
Efflux Ratio = Papp (B-A)/Papp (A-B) across monolayers of Caco-2.
Table 6
A–B and B–A Papp of digoxin, paclitaxel, T-13 and T-26 across MDCK-WT and MDCK-MDR1 monolayers in the presence and absence of verapamil (100
l
M)
Test compounda
Treatmentb
MDCK-WT
Papp (10ꢁ6 cm/s)
B–A
8.06
MDCK-MDR1
Papp (10ꢁ6 cm/s)
B–A/A–B
B–A/A–B
A–B
A–B
B–A
Digoxin
Paclitaxel
T-13
Control
Verapamil
Control
Verapamil
Control
Verapamil
Control
0.35
0.48
0.09
0.46
1.39
1.56
1.40
3.00
22.74
2.69
344.23
7.06
2.28
1.96
0.19
2.55
0.13
2.02
0.52
1.02
0.63
1.58
12.45
3.89
44.09
24.36
5.26
4.25
6.38
1.92
65.53
1.53
333.79
12.08
10.10
4.16
1.30
29.81
3.24
3.17
3.06
2.99
3.00
T-26
2.13
1.00
10.11
1.21
Verapamil
a
Digoxin, paclitaxel, T-13 and T-26 were added to donor compartments at 5 lM.
Verapamil was added to both AP and BL compartments at 100 lM.
b

200
Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
Table 7
Permeability of digoxin in the presence and absence of test compounds
Test compounds
Treatmenta
Pappb
Efflux ratioc
A–B
B–A
Sample-1
Sample-2
Mean
Sample-1
Sample-2
Mean
Alone
Paclitaxel
T-13
0.63
0.60
1.70
1.37
0.68
0.69
1.62
2.21
0.65
0.64
1.66
1.79
17.22
14.73
13.27
11.34
18.02
19.76
13.78
11.21
17.62
17.25
13.53
11.27
26.92
26.88
8.15
Digoxin
(5 lM)
T-26
6.30
a
b
c
Paclitaxel, T-13 and T-26 were added to donor compartments at 10 lM.
Apparent permeability coefficient.
Effux ratio = Papp (B–A)/Papp (A–B) across monolayers of MDCK-MDR1.
assay in comparison with paclitaxel and ortataxel. T-13, T-15 and
T-26 were found could not to be substrates for P-gp, and demon-
strated significant enhancement of transcellular transport in the
A-to-B direction. Compounds T-04, T-06, T-08, T-10, T-11, T-17
and T-19 appear to be poor substrates for P-gp. Furthermore T-
13 and T-26 were found not only poor substrates for P-gp but also
possessed inhibiting effects of P-gp mediated efflux in MDCK-
MDR1 and MDCK-WT monolayers.
The results indicated simultaneous modifications at C-7 (ester,
amino ester, deoxy or methyl ether), C-10 (ester, amino ester or
methyl ether) and C-30 positions (30N-Boc and 30-aromatic ring or
alkyl group) seem to play an important role in modulating P-gp
mediated efflux. Moreover, the results from the new taxoids T-
20–T-34 indicated the presence of the 1,14-carbonate moiety on
the taxanes seems not be a necessary prerequisite in weakening
the affinity with P-gp. However, the role of the 1,14-carbonate
moiety on weakening taxoid affinity with P-gp was seen in the case
of T-26 and T-06, with efflux ratio improved to 4.08 from 2.04. The
improved physicochemical and biological profiles of the com-
pounds T-13, T-15 and T-26 warrant a continuing study to assess
whether or not they are qualified as oral administration drug can-
didates of taxoids. The results will be reported in due time.
ꢁ70 °C dropwise over 30 min. The reaction was stirred at ꢁ70 °C
for 4 h. The resulted mixture was poured into sat. NH4Cl solution
(2 L) and extracted with EtOAc (1.20 L ꢂ 3). The combined organic
layer was dried over Na2SO4, filtered and concentrated. The residue
was purified by column chromatography (Petroleum ether, Petro-
leum ether/EtOAc = 10:1) to give a mixture of compound 3c (solid)
with a small amount of compound 2c (oil), which was filtered and
washed with Petroleum ether to give compound 3c (30 g, yield
62%) as a white solid. 1H NMR (400 MHz, CDCl3) d 7.41 (dd,
J = 7.9, 1.3 Hz, 2H), 7.38–7.27 (m, 3H), 5.30 (d, J = 12.2 Hz, 1H),
5.25 (s, 1H), 5.05 (d, J = 12.2 Hz, 1H), 4.32 (dd, J = 11.2, 5.6 Hz,
1H), 3.97 (ddd, J = 10.9, 5.3, 4.0 Hz, 1H), 2.55 (t, J = 10.8 Hz, 1H),
1.72–1.60 (m, 1H), 1.49 (s, 9H), 1.39–1.30 (m, 1H), 1.22 (s, 9H),
0.90 (dd, J = 11.2, 6.6 Hz, 6H).
4.1.2. (2R,3S)-3-Substituted isoserine benzyl ester 4
A typical procedure is given for the preparation of (2R,3S)-3-
isobutyl isoserine benzyl ester:
A mixture of compound 3c
(18.00 g, 39.13 mmol) in 4 N HCl/EtOAc (500 mL) was stirred at
room temperature for 10 h. The resulted mixture was concen-
trated, and the residue was partitioned between DCM (250 mL)
and water (200 mL). The separated aqueous layer was washed with
DCM (150 mL ꢂ 2). The aqueous layer was adjusted to pH 9–10
with NH3ꢀH2O (28%), and then extracted with DCM (200 mL ꢂ 3).
The combined organic layers were dried over Na2SO4, filtered
and concentrated to give compound 4c (9.50 g, yield 95.7%) as a
white solid. 1H NMR (400 MHz, CDCl3) d 7.49–7.30 (m, 5H), 5.24
(s, 2H), 4.06 (d, J = 2.3 Hz, 1H), 3.21–3.09 (m, 1H), 1.77–1.59 (m,
1H), 1.43–1.25 (m, 2H), 0.92 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.6 Hz,
3H).
4. Experimental
4.1. Syntheses
All anhydrous reactions were performed in oven-dried glass-
ware under argon. Tetrahydrofuran was distilled over sodium/ben-
zophenone. Chemicals were obtained from J&K Chemical Co. and
were used without further purification. Reactions were monitored
by thin-layer chromatography on 0.25 mm E. Merck silica gel
plates (60 F254) using UV light as a visualizing agent. For silica
gel chromatography, the flash chromatography technique (Biotage
Isolera I) was used, with silica gel (300–400 mesh) and p.a. grade
solvents. Melting points were determined on a YRT-3 Thomas-
Hoover capillary apparatus and were not corrected. 1H and 13C
NMR and 2D NMR spectra were recorded on a Bruker Avance III
400 spectrometer with Me4Si or CHCl3 (in CDCl3) as internal refer-
ence. Mass spectra were obtained on an Agilent G6300 Trap MSD
equipped with an ESI ionization source.
4.1.3. (2R,3S)-N-Substituted-3-substituted isoserine benzyl ester
5
A typical procedure is given for the preparation of (2R,3S)-N-
tert-butyloxycarboryl-3-isobutyl isoserine benzyl ester: A mixture
of compound 4c (9.03 g, 35.8 mmol), (Boc)2O (9.38 g, 43.00 mmol)
and TEA (9.04 g, 89.50 mol) in DCM (380 mL) was stirred at room
temperature overnight. The resulted mixture was concentrated,
and the residue was purified by column chromatography (Petro-
leum ether, Petroleum ether/EtOAc = 10:1, 5:1) to give compound
5d (11.82 g, yield 94.4%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.41 (m, 5H), 5.30 (d, J = 12.0 Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H),
4.63 (d, J = 10.0 Hz, 1H), 4.22–4.14 (m, 2H), 3.11 (d, J = 5.1 Hz,
1H), 1.67 (m, 1H), 1.56–1.46 (m, 1H), 1.44 (s, 9H), 1.42–1.35 (m,
1H), 0.96 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H).
4.1.1. (SR,2R,3S)-N-tert-Butanesulfinyl-O-methoxycarbonyl-3-
substituted isoserine benzyl ester 3
A typical procedure is given for the preparation of (SR, 2R, 3S)-N-
tert-Butane-sulfinyl-O-methoxy
carbonyl-3-isobutyl isoserine benzyl ester: To a solution of
compound 1 (141.00 g, 0.53 mol) in THF (1.5 L) was added LiHMDS
(530 mL, 0.53 mol) at ꢁ70 °C dropwise over 1 h. The reaction mix-
ture was stirred at ꢁ70 °C for 30 min. Subsequently, a solution of
compound 2c (20.00 g, 0.11 mol) in THF (250 mL) was added at
4.1.4. (4S,5R)-N-Substituted-2-(4-anisyl)-4-substituted-5-
oxazolidinecarboxylic benzyl ester 6
A typical procedure is given for the preparation of (4S,5R)-N-
butyloxycarboryl-2-(4-anisyl)-4-isobutyl-5-oxazolidinecarboxylic
benzyl ester: To a solution of compound 5d (11.00 g, 31.32 mmol)

Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
201
and PPTS (0.74 g, 2.96 mmol) in toluene (160 mL) was added a solu-
tion 1-dimethoxymethyl-4-methoxy-benzene (6.84 g, 37.54 mmol)
dropwise at 90 °C over 20 min. The reaction was stirred at 90–
100 °C for 2 h. 1-Dimethoxymethyl-4-methoxy-benzene (2.45 g)
added to the reaction mixture additionally. The reaction was stirred
for another 2 h. The resulting mixture was concentrated, and the
residue was purified by column chromatography (Petroleum
ether/EtOAc = 30:1, 20:1, 10:1) to give compound 6d (13.83 g, yield
92%) as a yellow oil mixture with 4-methoxybenzaldehyde. 1H NMR
(400 MHz, CDCl3) d 7.42–7.23 (m, 7H), 6.89–6.76 (m, 2H), 6.22 (s,
1H), 5.16 (s, 2H), 4.43 (d, J = 1.3 Hz, 1H), 4.24 (s, 1H), 3.73 (s, 3H),
1.73–1.56 (m, 2H), 1.51 (d, J = 6.1 Hz, 1H), 1.24 (s, 9H), 0.91 (d,
J = 5.4 Hz, 3H), 0.86 (d, J = 5.4 Hz, 3H).
(d, J = 8.5 Hz, 1H, H-20), 3.87 (d, J = 7.3 Hz, 1H, H-3), 3.45 (s, 3H),
2.32 (d, J = 7.0 Hz, 1H, H-14), 2.29 (s, 4H, H-14), 2.26–2.22 (m,
2H, H-6) 2.08 (t, J = 5.2 Hz, 3H), 1.97–1.91 (m, 1H, H-7), 1.74 (s,
3H), 1.60–1.51 (m, 1H, H-7), 1.13 (s, 3H), 1.08 (s, 3H). 13C NMR
(101 MHz, CDCl3)
d 209.67, 170.62, 167.21, 142.99, 133.54,
130.11, 129.61 , 128.64, 128.57, 86.21, 84.43, 82.65, 82.00, 79.07,
75.85, 67.84, 57.77 , 56.44, 53.18, 45.61, 42.55, 42.15 , 38.78),
35.55, 29.69, 27.24, 26.35, 22.65, 19.99, 19.75, 16.56 , 15.37,
14.84, 14.75.
10-Methyl-7-dimethylaminocarbonyl-baccatin III B-05.
To a solution of compound 10 (0.73 g, 1.31 mmol) in THF
(10 mL) was added carbonyldiimidazole (0.44 g, 2.62 mmol) at
room temperature. The reaction was stirred for 4 h. The resulting
mixture was poured into water (20 mL) and extracted with EtOAc
(10 mL ꢂ 3). The combined organic layer was dried over Na2SO4,
filtered and concentrated. The crude product was dissolved in
THF (10 mL), and then 33% aqueous dimethylamine (0.50 mL) solu-
tion was added, stirred for 2 h. The reaction solution was concen-
trated and then purified by column chromatography (n-hexane/
EtOAc = 3:4) gave B-05 (0.46 g, 0.96 mmol, 73%) as a white solid.
13-Oxo-7,10-substituted-1,14-carbonate-baccatin III 15.
Synthesis of compound 15 following the procedures of Ref. 15
7,10-Substituted-1,14-carbonate-baccatin III B-07–B-14.
A typical procedure is given for the preparation of 10-methyl-7-
dimethylaminocarbonyl-1,14-carbonate baccatin III B-12: A solu-
tion of 13-oxo-10-methyl-7-dimethylaminocarbonyl-1,14-carbon-
ate baccatin III (0.78 g, 1.17 mmol) in anhydrous THF (5 mL) was
added to (R)-2-methyl–CBS-oxazaborolidine (0.07 mL, 0.23 mmol)
and 1.0 M BH3/THF (5.85 mL, 5.85 mmol) at room temperature, un-
der nitrogen and stirring. After 5 h, the reaction was quenched by
addition of water. The reaction mixture was extracted with EtOAc
and the organic phase was dried, filtered and evaporated under re-
duced pressure. Chromatography of the residue (SiO2, n-hexane/
EtOAc = 1:1) gave B-12 (0.68 g, 1.02 mmol, 87.3%) as a white solid.
MS (m/z) ESI: 694.2 (M+Na)+; 1H NMR (400 MHz, CDCl3) d 8.03 (d,
J = 7.3 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.49 (t, J = 7.7 Hz, 2H), 6.08
(d, J = 7.2 Hz, 1H, H-2), 5.49 (dd, J = 10.7, 7.2 Hz, 1H, H-7), 5.23 (s,
1H, H-10), 5.04 (t, 1H, H-13), 4.97 (d, J = 8.3 Hz, 1H, H-5), 4.80 (d,
J = 5.8 Hz, 1H, H-14), 4.32 (d, J = 8.4 Hz, 1H, H-20), 4.22 (d,
J = 8.4 Hz, 1H, H-20), 3.87 (d, J = 7.2 Hz, 1H, H-3), 3.67 (d,
J = 5.3 Hz, 1H, OH-13), 3.40 (s, 3H), 2.87 (s, 6H), 2.54 (m, J = 14.5,
9.5, 7.3 Hz, 1H, H-6), 2.31 (s, 3H), 2.17 (s, 3H), 1.92 (ddd, J = 14.2,
8.1, 2.6 Hz, 1H, H-6), 1.81 (s, 3H), 1.24 (s, 3H), 1.14 (s, 3H). 13C
NMR (101 MHz, CDCl3) d 205.73 , 171.28, 170.21, 164.84, 155.01 ,
152.98 , 141.50 , 134.17, 133.99 , 129.83 , 128.91, 128.14 , 88.54 ,
84.21, 83.86, 82.88, 80.06, 75.99, 72.28, 71.79 , 69.45, 57.11, 57.00,
46.25, 41.32 , 36.53, 35.89 , 33.73 , 25.62, 22.21, 21.33, 14.91, 10.85.
General procedure for the syntheses of protected taxoids 16
through the coupling of side-chain precursor 7aꢃd with baccatin
derivatives B-01–B-14.
4.1.5. (4S,5R)-N-Substituted-2-(4-anisyl)-4-substituted-
5-oxazolidinecarboxylic acid 7a–d
A typical procedure is given for the preparation of (4S,5R)-N-
butyloxycarboryl-2-(4-anisyl)-4-isobutyl-5-oxazolidinecarboxylic
acid 7d: To a mixture of compound 6 (15.60 g, 33.20 mmol) in
MeOH (400 mL) was added Pd(OH)2 (7.80 g). The reaction was stir-
red at room temperature under H2 atmosphere (20 psi) for 1 h. TLC
showed the reaction was complete. The resulting mixture was fil-
tered, and the filtrated was concentrated. The residue was purified
by column chromatography (Petroleum ether/EtOAc = 10:1, 5:1) to
give 7d (10.98 g, yield 87.2%) as a white solid.
7a: mp: 127ꢃ128 °C; MS (m/z) ESI: 402.0 (M-H)-; 1H NMR
(400 MHz, CDCl3) d 7.53–7.19 (m, 12H), 6.89 (s, 1H), 6.85 (d,
J = 8.0 Hz, 2H), 5.48 (s, 1H), 4.91 (s, 1H), 3.82 (s, 3H). 13C NMR
(101 MHz, CDCl3)
d 172.74, 159.99, 135.30, 130.73, 129.53,
128.78, 128.70, 128.30, 128.16, 127.06, 113.56, 55.30.
7b: mp: 129ꢃ130 °C; MS (m/z) ESI: 398.0 (M-H)-; 1H NMR
(400 MHz, DMSO) d 7.48–7.23 (m, 7H), 6.96 (d, J = 8.3 Hz, 2H),
6.28 (s, 1H), 5.15 (s, 1H), 4.53 (s, 1H), 3.78 (s, 3H), 0.95 (s, 9H).
13C NMR (101 MHz, DMSO) d 170.83, 160.12, 129.17, 128.92,
128.08, 127.19, 113.81, 91.49, 79.80, 55.64, 27.90.
7c: mp: 125ꢃ126 °C; MS (m/z) ESI: 399.0 (M-H)-; 1H NMR
(400 MHz, CDCl3) d 8.59 (d, J = 4.6 Hz, 1H), 7.85 (td, J = 7.8,
1.5 Hz, 1H), 7.45 (m, 3H), 7.34 (dd, J = 7.1, 5.3 Hz, 1H), 6.93 (d,
J = 8.6 Hz, 2H), 6.23 (s, 1H), 5.58 (d, J = 6.6 Hz, 1H), 4.64 (d,
J = 6.6 Hz, 1H), 3.82 (s, 3H), 1.07 (s, 9H). 13C NMR (101 MHz, CDCl3)
d 169.83, 160.50, 151.95, 148.21, 138.29, 130.48, 128.87, 123.34,
121.49, 113.87, 92.39, 81.08, 63.56, 55.34, 27.80.
7d: mp: 137ꢃ138 °C; MS (m/z) ESI: 378.0 (M-H)-; 1H NMR
(400 MHz, CDCl3) d 9.08 (s, 1H), 7.39 (d, J = 8.1 Hz, 2H), 6.92 (d,
J = 8.5 Hz, 2H), 6.31 (s, 1H), 4.54 (s, 1H), 4.41 (s, 1H), 3.84 (s, 3H),
1.69 (m, 3H), 1.36 (s, 9H), 1.04 (d, J = 5.9 Hz, 6H). 13C NMR
(101 MHz, CDCl3) d 175.66, 159.93, 153.59, 128.16, 113.63, 90.64,
81.29, 58.94, 55.30, 43.97, 28.24, 25.57, 23.13, 21.87.
7,10-Dimethyl-baccatin III B-01
To a solution of compound 8 (0.52 g, 0.96 mmol) in THF (6 mL)
was added 1.0 M LiHMDS (3.82 mL, 3.82 mmol) at ꢁ72 °C
dropwise. The reaction mixture was stirred at ꢁ72 °C for 40 min.
Subsequently, a solution of TfOMe (0.33 mL, 2.87 mmol) in THF
(2 mL) was added at ꢁ72 °C dropwise over 5 min. The reaction
was stirred at ꢁ72 °C for 4 h. The resulting mixture was poured
into sat. NH4Cl solution (20 mL) and extracted with EtOAc
(20 mL ꢂ 3). The combined organic layer was dried over Na2SO4,
filtered and concentrated. The residue was purified by column
chromatography (n-hexane/EtOAc = 1:1) gave B-01 (0.48 g,
0.84 mmol, 87%) as a white solid.
A solution of 4 equiv 7a in dry CH2Cl2, was added with 1equiv
baccatin derivative, 2 equiv DCC, 0.5 equiv DMAP and 0.2 equiv
PTSA, at room temperature with stirring overnight. The reaction
was evaporated under reduced pressure. Chromatography of the
crude mixture (n-hexane/EtOAc = 2:1) afforded 16 as a white solid.
A general procedure for the syntheses of the target taxoids T-
01–T-35 through deprotection of the fully protected taxoids 16 is
given below.
The fully protected taxoids 16 were dissolved in THF and added at
0 °C to a 0.2 M solution of acetyl chloride in MeOH. After 2 h the reac-
tion was quenched by addition of saturated aqueous NH4Cl solution.
The organic phase was dried, filtered, and evaporated under reduced
pressure. Chromatography (SiO2, n-hexane/EtOAc = 5:6) gave the tar-
get taxoids as white solids, in greater than 90% yield.
10-Methyl-7-deoxybaccatin III B-02
Compound B-02 was prepared according to Ref. 14, gave a
white solid in 63% yield. MS (m/z) ESI: 565.2 (M+Na)+; 1H NMR
(400 MHz, CDCl3) d 8.15–8.09 (m, 2H), 7.61 (m, 1H), 7.49 (m,
2H), 5.62 (d, J = 7.4 Hz, 1H, H-2), 5.01–4.95 (m, 2H, H-10, H-10,
H-5), 4.95–4.87 (m, 1H, H-13), 4.32 (d, J = 8.4 Hz, 1H, H-20), 4.20
The analyses of T-01–T-35 were listed in the supporting
information.

202
Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
4.2. MTT assay and transport experiments
resistance of MDCK-MDR1 monolayers from randomly selected
wells was 2870 210
cm2 (Mean SD) higher than 1000
X
4.2.1. Growth-inhibition assay in vitro
X
cm2 before use in transport experiments. MDCK-WT cells were
The inhibitory effects of test compounds on proliferation rates
were determined by a modified MTT assay (Amresco),22 which
indirectly determines cell number by measuring membrane-asso-
ciated proteins. Breast carcinoma cell lines MCF-7 (ATCC),
non-small cell lung carcinoma cell lines NCI-H 460 (ATCC), ovarian
carcinoma cell line A2780 (ECACC), melanoma carcinoma cell lines
A-375 (ATCC), colorectal carcinoma cell lines HT-29 (ATCC), cervi-
cal carcinoma cell line HeLa (ATCC), leukemia carcinoma cell lines
HL-60 (ATCC), and prostate carcinoma cell lines DU145 (ATCC)
were grown in DMEM or IMDM with 10% fetal calf serum. The cells
seeded at a density of 5.0 ꢂ 104 cells/well, and matured exhibiting
a TEER 280 12
X X
cm2 higher than 200 cm2 before use in trans-
port experiments after 6 days. Cell monolayer integrity was evalu-
ated by measurement of Lucifer yellow permeability by monitoring
the change in TEER over the course of the experiment. Transport
experiments of the test compounds were performed at 37 °C in
HBSS at pH 7.4. Separate the apical plate from the basolateral plate
after 90 min incubation. A 100 ll sample was taken from the donor
compartment to determine the concentration of the compound
remaining in the donor chamber at the end of the experiment.
were plated at a density of 2000–5000 cells/well/100 ll on 96-well
microtiter plates in complete growth medium and incubated at
37 °C overnight to allow the cells to attach to the substrate before
drugs exposure. The test compounds were suspended in DMSO at
100 mM and stored at ꢁ20 °C. Serial dilutions in media were pre-
Acknowledgments
We gratefully acknowledge Tianjin Tasly Group Co., Ltd for the
financial support for the project and the scientist of Shanghai
ChemPartner Co., Ltd for performing pharmacological experiments.
We acknowledge Dr. W.Q. Chen for his many helpful suggestions.
pared to make the final desired concentrations (1 lM). Duplicate
wells were exposed to various treatments. After 72 h at 37 °C, 5%
CO2 incubation, the wells were added for 4 h with 10 l of 5 mg/
mL MTT in phosphate-buffered saline and incubated at 37 °C. At
the end of the incubation period, the cells were fixed with 100
l
Supplementary data
ll
modified lysis reagent (10% SDS, 5% isopropanol with 0.012 N HCl
in distilled water), dispersed by pipetting until homogeneous.
Optical density was measured at 580 to 680 nm by microplate
reader, within 15 min.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.037.
References and notes
1. (a) Stephenson, F.; A Tale of Taxol, FSU Research in Review, Vol. XII, No. 3,
Tallahassee, FL, 2002, p. 12.; (b) Sarosy, G.; Kohn, E.; Stone, D. A.; Rothenberg,
M.; Jacob, J.; Adamo, D. O.; Ognibene, F. P.; Cunnion, R. E.; Reed, E. J. Clin. Oncol.
1992, 10, 1165; (c) Holmes, F. A.; Walters, R. S.; Theriault, R. L.; Forman, A. D.;
Newton, L. K.; Raber, M. N.; Buzdar, A. U.; Frye, D. K.; Hortobagyi, G. N. J. Natl.
Cancer Inst. 1991, 83, 1797; (d) Rowinsky, E. K.; Cazenave, L. A.; Donehower, R.
C. J. Natl. Cancer Inst. 1990, 82, 1247; (e) McGuire, W. P.; Rowinsky, E. K.;
Rosenheim, N. B.; Grumbine, F. C.; Ettinger, D. S.; Armstrong, D. K. Ann. Intern.
Med. 1989, 111, 273; (f) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.;
McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325.
2. (a) Chow, L. W.; Xu, B.; Gupta, S.; Freyman, A.; Zhao, Y.; Abbas, R.; Vo Van, M. L.;
Bondarenko, I. Br. J. Cancer. 2013, 108, 1985; (b) Rugo, H. S.; Campone, M.;
Amadori, D.; Aldrighetti, D.; Conte, P.; Wardley, A.; Villanueva, C.; Melisko, M.;
McHenry, M. B.; Liu, D.; Lee, F.; Pivot, X. Breast Cancer Res. Treat. 2013, 139, 411;
(c) Esteva, F. J.; Franco, S. X.; Hagan, M. K.; Brewster, A. M.; Somer, R. A.;
Williams, W.; Florance, A. M.; Turner, S.; Stein, S.; Perez, A. Oncologist 2013; (d)
Henderson, I. C.; Berry, D. A.; Demetri, G. D.; Cirrincione, C. T.; Goldstein, L. J.;
Martino, S.; Ingle, J. N.; Cooper, M. R.; Hayes, D. F.; Tkaczuk, K. H.; Fleming, G.;
Holland, J. F.; Duggan, D. B.; Carpenter, J. T.; Frei, E., III; Schilsky, R. L.; Wood, W.
C.; Muss, H. B.; Norton, L. J. Clin. Oncol. 2003, 21, 976.
4.2.2. Caco-2 cells permeability assay
The Caco-2 cells were harvested and seeded on the apical side of
a Millicell-24 well plate. After 21 days culture, take cell culture
plate out of incubator, pre-incubated with HBSS, pH 7.4 at 37 °C,
and then measure TEER values at room temperature. The trans-
monolayer electrical resistance of Caco-2 monolayers from ran-
domly selected wells was measured by Millicell-ERS
voltohmmeter reading, TEER value was 359 12
(Mean SD) higher than 250
cm2. Prewarm apical and basolat-
eral plates at 37 °C for about 5 min. The assay was initiated by
the addition of the test compounds (10 M) from the apical
X
cm2
X
l
(0.8 mL) incubation at 37 °C with 5% CO2 and 95% air. The apical
plate was separated from the basolateral plate after 90-min incu-
bation, and 100
to an opaque plate. The samples were prepared for LC-MS/MS anal-
ysis: donor samples (1:10 diluted): 6 l of donor sample +54
0.4% DMSO HBSS + 60 l ACN with IS (Osalmid or Imipramine); re-
ceiver sample: 60 l of receiver sample + 60 l ACN with IS (Osal-
ll samples were transferred from basolateral plate
l
ll
3. (a) Jeyapalan, S.; Boxerman, J.; Donahue, J.; Goldman, M.; Kinsella, T.; Dipetrillo,
T.; Evans, D.; Elinzano, H.; Constantinou, M.; Stopa, E.; Puthawala, Y.; Cielo, D.;
Santaniello, A.; Oyelese, A.; Mantripragada, K.; Rosati, K.; Isdale, D.; Safran, H. J.
Clin. Oncol. 2013; (b) Thakur, A.; Joshi, N.; Shanmugam, T.; Banerjee, R. Cancer
Lett. 2013, 334, 274; (c) Giussani, P.; Bassi, R.; Anelli, V.; Brioschi, L.; De Zen, F.;
Riccitelli, E.; Caroli, M.; Campanella, R.; Gaini, S. M.; Viani, P.; Riboni, L. Cancer
Invest. 2012, 30, 27.
l
l
l
mid or Imipramine). Erythromycin, metoprolol, atenolol as
reference compounds, paclitaxel and ortataxel as positive control.
Drug permeability : Papp
4. Wenk, M. R.; Fahr, A.; Reszka, R.; Seelig, J. J. Pharm. Sci. 1996, 85, 228.
5. (a) Bardelmeijer, H. A.; Ouwehand, M.; Beijnen, J. H.; Schellens, J. H.; van
Tellingen, O. Invest. New Drugs 2004, 22, 219; (b) Bardelmeijer, H. A.; Beijnen, J.
H.; Brouwer, K. R.; Rosing, H.; Nooijen, W. J.; Schellens, J. H.; van Tellingen, O.
Clin. Cancer Res. 2000, 6, 4416; (c) Sparreboom, A.; van Asperen, J.; Mayer, U.;
Schinkel, A. H.; Smit, J. W.; Meijer, D. K. F.; Borst, P.; Noojijen, W. J.; Beijnen, J.
H.; van Tellingen, O. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2031.
6. (a) Singla, A. K.; Garg, A.; Aggarwal, D. Int. J. Pharm. 2002, 235, 179; (b) Adams, J.
D.; Flora, K. P.; Goldspiel, B. R.; Wilson, J. W.; Arbuck, S. G.; Finley, R. J. Natl.
Cancer Inst. Monogr. 1993, 15, 141; (c) Weiss, R. B.; Donehower, R. C.; Wiernik,
P. H.; Ohnuma, T.; Gralla, R. J.; Trump, D. L.; Bakerjr, J. R.; Van Echo, D. A.; Von
Hoff, D. D.; Leyland-Jones, B. J. Clin. Oncol. 1990, 8, 1263.
7. (a) Vredenburg, M. R.; Ojima, I.; Veith, J.; Pera, P.; Kee, F. C.; Sharma, A.; Kanter,
P.; Greco, W. R.; Bernacki, R. J. J. Natl. Cancer Inst. 2001, 93, 1234; (b) Polizzi, D.;
Pratesi, G.; Monestiroli, S.; Tortoreto, M.; Zunino, F.; Bombardelli, E.; Riva, A.;
Morazzoni, P.; Colombo, T.; D’Incalci, M.; Zucchetti, M. Clin. Cancer Res. 2000, 6,
2070; (c) Nicoletti, M. I.; Colombo, T.; Rossi, C.; Monardo, C.; Stura, S.;
Zucchetti, M.; Riva, A.; Morazzoni, P.; Donati, M. B.; Bombardelli, E.; D’Incalci,
M.; Giavazzi, R. Cancer Res. 2000, 60, 842; (d) Ferlini, C.; Distefano, M.;
Pignatelli, F.; Lin, S.; Riva, A.; Bombardelli, E.; Mancuso, S.; Ojima, I.; Scambia, G.
Br. J. Cancer 2000, 83, 1762; (e) Ojima, I.; Kuduk, S. D.; Pera, P.; Veith, J. M.;
Bernacki, R. J. J. Med. Chem. 1997, 40, 279; (f) Ojima, I.; Slater, J. C.; Kuduk, S. D.;
ꢀ
ꢁꢀ
ꢁ
VA
½drugꢄacceptor; 90 ꢁ min
¼
Area ꢂ Time ð½drugꢄdonor; 0 ꢁ minÞDilutionFactor
Where VA is the volume in the acceptor well, the area is the surface
area of the membrane and time is the total transport time in sec-
onds. [drug]donor, 0-min is the donor drug concentration count mea-
sured at 0-min; [drug]acceptor,
is the acceptor drug
90-min
concentration count measured at 90-min; The dilution factor is
the fold of dilution of the donor sample before loaded to LC-MS/
MS. For Millicell-24 cell culture plates: surface area of the mem-
brane = 0.7 cm2, VA = 0.8 mL (A-to-B) or 0.4 mL (B-to-A).
4.2.3. MDCK-MDR1 cells and MDCK-WT cells transport23
MDCK-MDR1 cells were seeded onto polycarbonate filter Trans-
wells, and confluent MDCK-MDR1 monolayers expressing P-gp
were obtained 5 days postseeding. The transmonolayer electrical

Y. Jing et al. / Bioorg. Med. Chem. 22 (2014) 194–203
203
Takeuchi, C. S.; Gimi, R. H.; Sun, C.-M.; Park, Y. H.; Pera, P.; Bernacki, R. J. J. Med.
Chem. 1996, 39, 3889; (g) Ojima, I.; Duclos, O.; Kuduk, S. D.; Sun, C.-M.; Slater, J.
C.; Lavelle, F.; Veith, J. M.; Bernacki, R. J. Bioorg. Med. Chem. Lett. 1994, 4, 2631.
8. (a) Wu, Q.; Bounaud, P.-Y.; Kuduk, S. D.; Yang, C.-P. H.; Ojima, I.; Horwitz, S. B.;
Orr, G. A. Biochemistry 1998, 37, 11272; (b) Ojima, I.; Slater, J. C.; Michaud, E.;
Kuduk, S. D.; Bounaud, P.-Y.; Vrignaud, P.; Bissery, M.-C.; Veith, J. M.; Pera, P.;
Bernacki, R. J. J. Med. Chem. 1996, 39, 3889; (c) Ojima, I.; Duclos, O.; Dorman, G.;
Simonot, B.; Prestwich, G. D.; Rao, S.; Lerro, K. A.; Horwitz, S. B. J. Med. Chem.
1995, 38, 3891.
16. (a) Battaglia, A.; Baldelli, E.; Bombardelli, E.; Carenzi, G., et al Tetrahedron 2005,
61, 7727; (b) Laccabue, D.; Pratesi, G. Drugs Future 2001, 26, 533.
17. (a) Hubatsch, I.; Ragnarsson, E. G.; Artursson, P. Nat. Protoc. 2007, 2, 2111; (b)
Artursson, P.; Palm, K.; Luthman, K. Adv. Drug Deliv. Rev. 2001, 46, 27; (c)
Hilgers, A. R.; Conradi, R. A.; Burton, P. S. Pharm Res 1990, 7, 902; (d) Hidalgo, I.
J.; Raub, T. J.; Borchardt, R. T. Gastroenterology 1989, 96, 736.
18. (a) Sugano, K.; Kansy, M.; Artursson, P.; Avdeef, A.; Bendels, S.; Di, L.; Ecker, G.
F.; Faller, B.; Fischer, H.; Gerebtzoff, G.; Lennernaes, H.; Senner, F. Nat. Rev. Drug
Disc. 2010, 9, 597; (b) Ballatore, C.; Hyde, E.; Deiches, R. F.; Lee Virginia, M.-Y.;
Trojanowski, J. Q.; Huryn, D.; Smith, A. B., III Bioorg. Med. Chem. Lett. 2007, 17,
3642; (c) Rice, A.; Liu, Y.; Michaelis, M. L.; Himes, R. H.; Georg, G. I.; Audus, K. L.
J. Med. Chem. 2005, 48, 832.
9. Altstadt, T. J.; Fairchild, C. R.; Golik, J.; Johnston, K. A.; Kadow, J. F.; Lee, F. Y.;
Long, B. H.; Rose, W. C.; Vyas, D. M.; Wong, H.; Wu, M.-J.; Wittman, M. D. J. Med.
Chem. 2001, 44, 4577.
10. Sampath, D.; Discafani, C. M.; Loganzo, F.; Beyer, C.; Liu, H.; Tan, X.; Musto, S.;
Annable, T.; Gallagher, P.; Rios, C.; Greenberger, L. M. Mol. Cancer Ther. 2003, 2,
873.
11. Shionoya, M.; Jimbo, T.; Kitagawa, M.; Soga, T.; Tohgo, A. Cancer Sci. 2003, 94,
459.
19. Vredenburg, M. R.; Ojima, I.; Veith, J.; Pera, P.; Kee, K.; Cabral, F.; Sharma, A.;
Kanter, P.; Greco, W. R.; Bernacki, R. J. J. Natl. Cancer Inst. 2001, 93, 1234.
20. FDA, 2006. Drug Interaction Studies-Study Design, Data Analysis, and
Implications for Dosing and Labeling. http://www.fda.gov/OHRMS/DOCKETS/
98fr/06d-0344-gdl0001.pdf.
12. Wang, Y.; He, Q. F.; Wang, H. W.; Qin, Y. J. Org. Chem. 2006, 71, 1588.
13. Blechert, S.; Guenard, D. The Alkaloids In Brossi, A., Ed.; Academic Press: San
Diego, 1990; p 39, 195.
21. (a) Kim, R. B. Drug Metab. Rev. 2002, 34, 47; (b) Litman, T.; Druley, T. E.; Stein,
W. D.; Bates, S. E. Cell. Mol. Life Sci. 2001, 58, 931; (c) Wacher, V. J.; Wu, C.-Y.;
Benet, L. Z. Mol. Carcinog. 1995, 13, 129.
14. (a) Ishiyama, T.; Iimura, S.; Yoshino, T.; Chiba, J.; Uoto, K.; Terasawa, H.; Soga, T.
Bioorg. Med. Chem. Lett. 2002, 12, 2815; (b) Chen, S.-H.; Huang, S.; Gao, Q.;
Golik, J.; Farina, V. J. Org. Chem. 1994, 59, 1475; (c) Chaudhary, A. G.; Rimoldi, J.
M.; Kingston, D. G. I. J. Org. Chem. 1993, 58, 3798.
22. Sakehan, P.; Streng, R.; Scudierok, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
23. Taub, M. E.; Podila, L.; Ely, D.; Almeida, L. Drug Metab. Dispos. 2005, 33, 1679.
15. Baldelli, E.; Battaglia, A.; Bombardelli, E.; Carenzi, G.; Fontana, G.; Gambini, A.;
Gelmi, M. L.; Guerrini, A.; Pocar, D. J. Org. Chem. 2003, 68, 9773.