Design, synthesis and cytotoxicity of novel 3′-N-alkoxycarbonyl docetaxel analogs

Article abstract of DOI:10.1016/j.bmcl.2013.10.007

By-product 9a exhibited potent cytotoxicity against both SK-OV-3 and A549 cell lines. The structure of 9a was characterized using 1D and 2D NMR experiments and confirmed by synthesis to afford a diastereomeric mixture (16a) that was identical to 9a, as well as a pair of diastereomers (R)-16b and (S)-16c. The preliminary SAR study demonstrated that analogs with an (R)-configuration were slightly more potent than analogs with an (S)-configuration. In addition, α,α-gem-dimethyl analogs 16g-i were the most potent analogs in this series, exhibiting similar potency to docetaxel and greater potency than Taxol against the SK-OV-3 cell line. For the A549 cell line, analogs 16g-i were more potent (>65-fold) than both docetaxel and Taxol.

Full text of DOI:10.1016/j.bmcl.2013.10.007

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6834–6837
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and cytotoxicity of novel 30-N-alkoxycarbonyl
docetaxel analogs
⇑
Jun Chang, Xiao-Dong Hao, Yun-Peng Hao, Hong-Fu Lu, Jian-Ming Yu, Xun Sun
Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
By-product 9a exhibited potent cytotoxicity against both SK-OV-3 and A549 cell lines. The structure of 9a
was characterized using 1D and 2D NMR experiments and confirmed by synthesis to afford a diastereo-
meric mixture (16a) that was identical to 9a, as well as a pair of diastereomers (R)-16b and (S)-16c. The
preliminary SAR study demonstrated that analogs with an (R)-configuration were slightly more potent
Received 9 August 2013
Revised 19 September 2013
Accepted 4 October 2013
Available online 11 October 2013
than analogs with an (S)-configuration. In addition,
a,a-gem-dimethyl analogs 16g–i were the most
potent analogs in this series, exhibiting similar potency to docetaxel and greater potency than Taxol
against the SK-OV-3 cell line. For the A549 cell line, analogs 16g–i were more potent (>65-fold) than both
docetaxel and Taxol.
Keywords:
30-N-Alkoxycarbonyl docetaxel analogs
Synthesis
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
R2O10
O
Paclitaxel (Taxol, 1) (Fig. 1) is a diterpenoid originally isolated
from the bark of Taxus brevifolia in 19711 and is a promising anti-
cancer agent for the treatment of ovarian and breast cancer, as well
as lung, skin, neck and head cancers.2 Paclitaxel is an antimitotic
agent with a unique mode of action; by binding tubulin and stabi-
lizing microtubule formation, it ultimately disrupts mitosis and
causes cell death.3 Docetaxel (Taxotere, 2)4 is a semi-synthetic
derivative of paclitaxel with a similar mechanism of action. Doce-
taxel was approved by the FDA to treat breast cancer in 1996.
Although both paclitaxel and docetaxel are potent antitumor
agents, they have some drawbacks, such as poor aqueous solubil-
ity, multi-drug resistance (MDR) and low oral bioavailability.5
Therefore, developing new paclitaxel-based anticancer agents
retaining superior clinical antitumor activity while lacking these
side effects remains a goal in drug discovery.
Extensive structure–activity relationship (SAR) studies of paclit-
axel and docetaxel have been undertaken for decades and mainly
focused on modifying the C-13 side chain, B and C ring.2,6 The
C-13 phenylisoserine side chain on paclitaxel is essential for the
activity; analogs without a 30-N-acyl group exhibited significantly
decreased activity compared with paclitaxel.7 Georg et al. reported
that acylating N-debenzoyltaxol generated the tert-butylacetyl
analog; this compound displayed comparable biological activity
in the microtubule assembly assay, but demonstrated slightly
diminished activity in the B16 melanoma cell line assay relative
to Taxol.8 Song and co-workers reported the synthesis of 30-N-
acyl-N-debenzoylpaclitaxel analogs that were highly cytotoxic.
The compound bearing a N-cycloalkenoyl moiety was 20 times
OH
O
9
7
11
6
12
13
O
R1
8
3
C
B
NH
O
5 O
A
2'
H4
1'
3'
14HO1 2
OBz OAc
OH
1
2
paclitaxel R1 = Ph, R2 = Ac
docetaxel R1 = t-BuO, R2 = H
Figure 1.
more potent than paclitaxel against the A549 and SK-OV-3 tumor
cell lines in vitro and caused superior tumor growth inhibition
(T/C = 212%) relative to paclitaxel (T/C = 200%) during in vivo
experiments against ip implanted B16 melanoma.9 Further modifi-
cations demonstrated that some analogs bearing N-alkylcarbonyl
groups, such as trans-crotonyl, 2-butynoyl and trans-2-hexenoyl
substituents, exhibited more potent inhibitory activity than Paclit-
axel against five tumor cell lines.10 Interestingly, except for 30-N-
ethoxycarbonyl11 and a few small 30-N-alkoxycarbonyl analogs,
30-N-chiral alkoxycarbonyl substituted docetaxel analogs have
not been reported so far.
During our initial work to improve the metabolic stability and
pharmacokinetic profile of docetaxel, we synthesized a series of
fluorinated docetaxel analogs. These analogs revealed improved
metabolic stability, as expected.12 When synthesizing fluorinated
docetaxel analog 9 (Scheme 1), precursor 8 was obtained in 23%
yield alongside an unexpected by-product (8a) in 18% yield; 8a
was converted into 9a by removing the 7,10-troc protecting
groups. The preliminary analysis of the NMR data for 9a indicated
⇑
Corresponding author. Tel./fax: +86 21 51980003.
E-mail address: sunxunf@shmu.edu.cn (X. Sun).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.007

J. Chang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6834–6837
6835
O
MeMgBr
BTC
PhCH2OH
OH
O
O
F
F
F
Cl
O
F
O
O
5
6
3
4
TrocO
O
F
F
HO
O
TrocO
O
OTroc
O
OH
OTroc
O
O
O
O
NH
O
NH2
O
NH
O
Zn / HOAc
O
O
H
O
O
O
H
H
OAc
HO
HO
HO
HO
OH
OAc
OH
OH
OAc
OBz
OH
OBz
OBz
8
9
7
O
5''
1''
TrocO
O
5''
1''
OTroc
O
O
3''
2''
3''
2''
O
NH O
O
NH O
Zn / HOAc
O
4''
O
4''
H
O
H
OBz
O
OAc
HO
OAc
HO
OH
OBz
OH
9a
8a
Scheme 1. Synthesis of 9 and 9a.
that it had the same core structure as docetaxel, but the structural
details remained unknown at this stage. Nevertheless, 9a was also
tested for its cytotoxicity with 9. Surprisingly, 9a demonstrated
more potent cytotoxicity (IC50 = 47 nM) than 9 (IC50 = 140 nM)
and docetaxel (IC50 = 850 nM) against the A549 cell line. This result
encouraged us to elucidate the structure of 9a. Herein, we report
the structure of 9a and a follow-up SAR study based on the con-
firmed structure of 9a.
Compound 9a was obtained as a white powder. The molecular
formula of 9a was determined to be C44H55NO14 through analysis
of ESIMS, HRESIMS (m/z 844.3515 [M+Na]+), 1H NMR, 13C NMR,
and DEPT experiments. In the aromatic region of the 1H NMR spec-
trum, ten protons were assigned to the two mono-substituted aro-
matic rings and confirmed by the 13C NMR spectrum. The higher
field portion of the 1H NMR spectrum revealed four oxygen-bear-
ing methine signals at d = 5.67 (1H, d, J = 6.5 Hz, H-2), 4.22 (1H,
m, H-7), 5.21 (1H, s, H-10), and 6.22 (1H, t, J = 8.7 Hz, H-13), as well
as one pair of methylene signals at d = 4.19 and 4.20 (2H, 2d,
J = 8.0 Hz, 2H-20). These data suggested the presence of represen-
tative signals from the 10-deacetylbaccatin III (10-DAB) skeleton.
Further analysis of the 1H–1H COSY, HMBC, and HMQC spectra of
9a revealed the ester linkage between the C-10 on the side chain
and the C-13 from the 10-DAB. According to the molecular formula
and the docetaxel skeleton, there was a 100,200-dimethyl-propoxy-
carbonyl group attached to the side chain. Interestingly, there were
a pair of doublets (1.07 (1.5H, d, J = 6.6 Hz) and 1.00 (1.5H, d, J =
5.7 Hz)) in the high field region of the 1H NMR spectrum. Mean-
while, there were two doublet peaks (0.84 (1.5H, d, J = 2.7 Hz)
and 0.82 (1.5H, d, J = 2.7 Hz)) and one multiple peak (0.77 (3H,
m)). These peaks suggested that 9a might be a mixture of diaste-
reomers at the 100 site with a R/S = 1 to 1 ratio.
To confirm the proposed structure of 9a unambiguously, the
diastereomeric mixture (16a) and a separated pair of diastereo-
mers (16b and 16c) were synthesized. The synthesis of compounds
16a–c began from commercially available 10-DAB (10), as illus-
trated in Scheme 2. First, the hydroxyl groups at the C7 and the
C10 of 10 were protected using 2,2,2-trichloroethyl chloroformate
(TrocCl) with pyridine to obtain 7,10-ditroc-10-deacetylbaccatin
(11) in 91% yield.13 Subsequently, 11 was coupled with enantio-
pure oxazolidine 1214 using dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) to generate 13 in 86% yield.
After removing the 30-N-Boc and acetonide protective group from
13 using 98% formic acid at room temperature, amino alcohol
7 was obtained in 55% yield.15 With 7 in hand, intermediates
CO2H
TrocO
O
TrocO
O
OTroc
OTroc
TrocO
O
HO
O
OH
OTroc
BocN
O
O
NH2
O
12
b
O
O
H
OBz
O
H
O
O
O
OAc
a
c
OAc
HO
H
OBz
H
HO
HO
HO
R2
BocN
O
OH
OBz
OAc
OAc
HO
HO
O
OBz
7
13
10
11
16a R1 = H,
16b R1 = H,
16c R1 = H,
16d R1 = H,
16e R1 = H,
16f R1 = H,
R2 = i-propyl
R1
TrocO
O
R2 = i-propyl (1''R)
R2 = i-propyl (1''S)
R2 = ethyl
HO
O
OTroc
R1
O
OH
R1
O
R2
R2
O
14a-i
Cl
O
NH
O
NH
O
O
1''
R2 = ethyl (1''R)
R2 = ethyl (1''S)
O
O
H
OBz
O
H
OBz
e
d
O
OAc
HO
OAc
HO
OH
OH
16g R1 = methyl, R2 = ethyl
16h R1 = methyl, R2 = n-propyl
16i R1 = methyl, R2 = i-propyl
15a-i
16a-i
Scheme 2. Synthesis of 16a–i. Reagents and conditions: (a) TrocCl, pyridine, rt, 30 min; (b) 12, DCC, DMAP, toluene; (c) HCOOH, rt, 2 h; (d) 14a–i, Et3N, CH2Cl2; (e) Zn/HOAc,
MeOH, 50 °C.

6836
J. Chang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6834–6837
Table 1
1
The H NMR data for the 100,200-dimethyl-propoxycarbonyl side chain
9a (300 MHz, CDCl3)
16a (500 MHz, CDCl3)
(100R)-16b (500 MHz, CDCl3)
(100S)-16c (500 MHz, CDCl3)
100
200
300
400
4.51 (1H, m)
1.72 (1H, m)
4.51 (1H, m)
1.69 (1H, m)
4.52 (1H, m)
1.71 (1H, m)
0.76 (6H, br s)
4.50 (1H, m)
1.70 (1H, m)
0.84 (3H, d, J = 4.1 Hz)
0.82 (3H, d, J = 3.2 Hz)
0.84 (1.5H, d, J = 2.7 Hz)
0.82 (1.5H, d, J = 2.7 Hz)
0.77 (3H, m)
1.07 (1.5H, d, J = 6.6 Hz)
1.00 (1.5H, d, J = 5.7 Hz)
0.84 (1.5H, d, J = 4.1 Hz)
0.82 (1.5H, d, J = 3.2 Hz)
0.76 (3H, br s)
1.07 (1.5H, d, J = 6.3 Hz)
0.99 (1.5H, br s)
500
1.07 (3H, d, J = 6.3 Hz)
0.99 (3H, br s)
Table 2
40
30
20
10
0
Cytotoxicities (IC50, nM) of the docetaxel analogs
Compd
R1
R2
SK-OV-3
A549
16a (9a)
16b
16c
16d
16e
16f
16g
16h
16i
H
H
H
H
H
H
i-Propyl
i-Propyl (R)
i-Propyl (S)
Ethyl
Ethyl (R)
Ethyl (S)
Ethyl
n-Propyl
i-Propyl
Methyl
16
13
17
22
13
27
3
2
3
4
2
5
47
35
60
30
18
40
13
10
12
4
3
4
2
2
3
2
2
3
Methyl
Methyl
Methyl
Methyl
5.9 1.2
2.9 0.5
2.7 0.4
5.0 0.8
Docetaxel
Taxol
850 43
930 52
35
5
15a–c were prepared by acylating the amino group using freshly
prepared acyl chlorides 14a–c; the acyl chlorides were synthesized
by combining the corresponding alcohols with triphosgene in ether
at low temperatures. Finally, analogs 16a–c were obtained in desir-
able yields by removing 7,10-troc protecting groups from 15a–c
using zinc in acetic acid.
16a 16b 16c 16d 16e 16f 16g 16h 16i DTX Taxol
Cytotoxicities (IC50, nM) of docetaxel analogs against SK-OV-3
1000
The peaks in 1H NMR spectrum of 9a were identical to those of
( )-16a. Further analysis of the high field portion of the 1H NMR
data for (R)-16b and (S)-16c revealed the strange peaks (Table 1).
Therefore, the structure of 9a was assigned as ( ) 30-N-De-t-butox-
ycarbonyl-N-[100,200-dimethyl-propoxycarbonyl]docetaxel, which is
identical to ( )-16a.
The formation of 9a was puzzling. After examining the prepara-
tion of the fluorinated tert-butyl alcohol, GC/MS and 1H NMR data
confirmed that 2-( )-3-methyl-2-butanol was present after the
Grignard reaction (Scheme 1), even though the mechanism for pro-
ducing the 2-( )-3-methyl-2-butanol remained unclear. Because
the boiling points of the fluorinated tert-butyl alcohol and
2-( )-3-methyl-2-butanol were very similar, they co-distilled and
reacted with triphosgene to form the corresponding acyl chlorides,
forming both the desired product 8 and the by-product 8a.
According to the literature, the biological activity of 30-N-eth-
oxycarbonyltaxol was 2.5-fold less potent than Taxol while inhib-
iting microtubule disassembly.11 After accounting for the activity
750
500
250
0
16a 16b 16c 16d 16e 16f 16g 16h 16i DTXTaxol
Cytotoxicities (IC50, nM) of docetaxel analogs against A549
of 9a (Table 2), we reasoned that the
a-methyl group on the
30-N-alkoxycarbonyl portion may be crucial for the increase in
potency against both the SK-OV-3 and A549 cell lines. Therefore,
a series of 30-N-alkoxycarbonyldocetaxel analogs (16d–i) with a
Figure 2.
a
-methyl or
a,a
-gem-dimethyl group were designed, synthesized
molar values (16g–i) were achieved when the mono-methyl sub-
stitution at -position was changed to a gem-dimethyl group.
and evaluated for their cytotoxicity.16
a
The syntheses of 16a–i followed the same procedure as was
used for 16a. Subsequently 16d–i, as well as (R)-16b and (S)-16c,
were tested in vitro for cytotoxicity against the SK-OV-3 and
A549 cell lines (Table 2 and Fig. 2).17 Although there were no sig-
nificant differences between the racemic and optically pure ana-
logs (16a vs (R)-16b and (S)-16c; 16d vs (R)-16e and (S)-16f),
both (R)-16b and (R)-16e were slightly more potent than (S)-16c
and (S)-16f against both cell lines. Interestingly, single digit nano-
Analogs 16g–i exhibited similar potency (IC50 = 2.7 – 5.9 nM) to
docetaxel (IC50 = 5.0 nM) and were more potent than Taxol (IC50
= 35 nM) against the SK-OV-3 cell line. Additionally, 16g–i were
more potent (>65-fold) than both docetaxel and Taxol against the
A549 cell line.
By-product 9a exhibited potent cytotoxicity against both the
SK-OV-3 and A549 cell lines. The structure of 9a was carefully
characterized with 1D and 2D NMR and was confirmed using

J. Chang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6834–6837
6837
12. (a) Lu, H.-F.; Sun, X.; Xu, L.; Lou, L.-G.; Lin, G.-Q. Eur. J. Med. Chem. 2009, 44,
482; (b) Lu, H.-F.; Xie, C.; Chang, J.; Lin, G.-Q.; Sun, X. Eur. J. Med. Chem. 2011,
46, 1743.
13. Guéritte-Voegelein, F.; Sénilh, V.; David, B.; Guénard, D.; Potier, P. Tetrahedron
1986, 42, 4451.
synthesis to afford a diastereomeric mixture (16a) that was identi-
cal to 9a and separate diastereomers (R)-16b and (S)-16c. A preli-
minary SAR study revealed that analogs with an (R)-configuration
were slightly more potent than analogs with an (S)-configuration.
14. (a) Commercon, A.; Bezard, D.; Bernard, F.; Bourzat, J. D. Tetrahedron Lett. 1992,
33, 5185; (b) Bourzat, J. D.; Commerçon, A. Tetrahedron Lett. 1993, 34, 6049.
15. (a) Harada, N.; Hashiyama, T.; Ozaki, K.; Yamaguchi, T.; Ando, A.; Tsujihara, K.
Heterocycles 1997, 44, 305; (b) Mangatal, L.; Adeline, M. T.; Guénard, D.;
Guéritte-Voegelein, F.; Potier, P. Tetrahedron 1989, 45, 4177.
In addition, the
a,a-gem-dimethyl analogs 16g–i were the most
potent analogs in this series, exhibiting similar potency to doce-
taxel and exceeding the potency of Taxol against the SK-OV-3 cell
line. For the A549 cell line, 16g–i were all more potent (>65-fold)
than docetaxel and Taxol. Based on these findings, an additional
SAR study was carried out in our laboratory and will be reported
in due course.
16. Representative experimental procedure for 30-N-de-t-butoxycarbonyl-N-[100,100,200-
trimethyl-propoxycarbonyl]docetaxel (16i): To
a solution of 1,1,2-trimethyl-
propanol (10 mmol) and triphosgene (3.33 mmol) in ether (10 mL) was added
pyridine (11 mmol) in ether (8 mL) dropwise at ꢀ30 °C. The mixture was
stirred for 2 h at this temperature before being slowly warmed to room
temperature to be stirred further for 10 h. The resulting solution (14i) was used
directly for the next step without purification. To a stirred solution of 7
(500 mg, 0.46 mmol) in anhydrous THF (10 mL) was added excess 14i
dropwise at 0 °C and, subsequently, triethylamine (0.13 mL, 0.93 mmol) in
anhydrous THF (5 mL). The resulting mixture was stirred at room temperature
for 1 h and extracted with ethyl acetate (20 mL ꢁ 3). The combined organic
layer was washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The obtained residue was purified by silica gel flash chromatography
column (petroleum ether/ethyl acetate: 2/1) to afford 15i as a white solid. To a
solution of 15i (0.19 mmol) in methanol (10 mL) were added glacial acetic acid
(4.60 mL) and zinc powder (0.46 g, 7.08 mmol). The resulting mixture was
stirred at 50 °C for 1 h. The reaction mixture was filtered to remove the zinc
and a solid formed. The solvent was removed by distillation, providing a white
solid. The obtained solid was dissolved in ethyl acetate (60 mL), washed with
saturated NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo.
The obtained residue was purified by silica gel flash chromatography column
(petroleum ether/acetone: 2/1) to furnish 16i as a white solid. Mp 200–202 °C;
1H NMR (300 MHz, CDCl3): d 0.75–0.87 (6H, m), 1.13 (3H, s), 1.24 (3H, s), 1.27
(3H, s), 1.28 (3H, s), 1.75(3H, s), 1.84 (1H, m), 1.85 (3H, s), 2.25 (1H, m), 2.26
(2H, m), 2.38 (3H, s), 2.58(1H, m), 3.46 (1H, s), 3.91 (1H, d, J = 6.9 Hz), 4.19 (1H,
d, J = 8.7 Hz), 4.26 (1H, s), 4.27 (1H, m), 4.31 (1H, d, J = 8.7 Hz), 4.63 (1H, br s),
4.94 (1H, d, J = 9.3 Hz), 5.21 (1H, s), 5.27 (1H, m), 5.48 (1H, m), 5.67 (1H, d,
J = 7.2 Hz), 6.22 (1H, t, J = 7.8 Hz), 7.38 (5H, m), 7.50 (2H, t, J = 7.5 Hz), 7.62 (1H,
t, J = 7.5 Hz), 8.10 (2H, d, J = 7.5 Hz); 13C NMR (100 MHz, CDCl3): d 9.4, 13.5,
16.7, 20.4, 22.1, 22.3, 22.4, 22.5, 26.1, 35.8, 35.9, 36.7, 43.2, 46.5, 57.0, 57.6,
71.2, 71.5, 74.2, 74.3, 75.1, 75.9, 77.7, 80.8, 83.6, 84.1, 127.1, 127.3, 128.3,
128.5, 129.9, 130.3, 133.1, 136.6, 137.5, 139.7, 155.3, 165.7, 170.0, 172.7,
210.5; ESI-MS m/z 858.6 [M+Na]+; HRMS (MALDI) m/z calcd for C45H57NO14Na+
[M+Na]+: 858.3708, found 858.3671.
Acknowledgments
Financial support was provided by the National Natural Science
Foundation of China (No: 21242003), the Shanghai Municipal Com-
mittee of Science and Technology (No: 10431903100), and the Na-
tional Basic Research Program of China (973 Program, No:
2010CB912603).
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treated with the drug (100
for 48 h, and cells were incubated for further 4 h at 37 °C. The purple formazan
crystals were dissolved in 100 L DMSO. After 5 min, the plates were read at
lM, 10 lM, 1 lM, 0.1 lM, 0.01 lM, 1 nM, 0.1 nM)
l
570 nm by an automated microplate spectrophotometer. The experiments
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