A novel D-ring modified taxoid: Synthesis and biological evaluation of a γ-lactone analogue of docetaxel

Article abstract of DOI:10.1039/c1ob06535a

The synthesis of a novel D-ring modified docetaxel analogue, in which the oxetane ring is replaced with a γ-lactone, was achieved from 10-deacetylbaccatin III. The key steps of the synthesis include the direct acetylation of the secondary hydroxyl group a

Full text of DOI:10.1039/c1ob06535a

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PAPER
A novel D-ring modified taxoid: synthesis and biological evaluation of a
c-lactone analogue of docetaxel†
Feng Gao,a,b Zhan-Kun Yang,a Qiao-Hong Chen,a Xiao-Guang Chenc and Feng-Peng Wang*a
Received 7th September 2011, Accepted 3rd October 2011
DOI: 10.1039/c1ob06535a
The synthesis of a novel D-ring modified docetaxel analogue, in which the oxetane ring is replaced with
a g-lactone, was achieved from 10-deacetylbaccatin III. The key steps of the synthesis include the direct
acetylation of the secondary hydroxyl group at C-5 and D-ring opening and intramolecular aldol
reaction to form the g-lactone. In MTT assays, this analogue proved to have equipotent cytotoxicity
relative to paclitaxel towards HCT8, HePG2 and BGC23 cancer cell lines, and be more potent than
paclitaxel against A549 and A375. It represents the first example of D-ring modified taxoids with
significant cytotoxicity.
Introduction
The anticancer drug paclitaxel (Taxol, 1) and its semisynthetic
analogue docetaxel (Taxotere, 2) (Fig. 1), being clinically used
in the treatment of ovarian cancer, breast cancer, and non-
small-cell lung cancer, have contributed significantly to human
health.1
Fig. 1 Structures of paclitaxel and docetaxel.
The early studies of their structure–activity relationships estab-
lished that the oxetane D-ring is essential for biological activity.
This was concluded from the fact that all of the synthesized D-seco
analogues at that time, such as 3 (Fig. 2),2 did not show activity
in both cytotoxicity and tubulin assembly assays. It was also
proposed that the oxetane oxygen is very important for biological
activity, according to the observations that the replacement of the
Fig. 2 Representatives of D-ring modified analogues of paclitaxel and
docetaxel.
aDepartment of Chemistry of Medicinal Natural Products, Sichuan Univer-
sity, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, P. R. China.
E-mail: wfp@scu.edu.cn; Fax: +86-28-85501368; Tel: +86-28-85501368
oxetane oxygen with other atoms, such as analogues 4,3 5,4 and 6,5
bDepartment of Chinese Traditional Medicine, Agronomy College, Sichuan
diminishes greatly the cytotoxic activity and the interaction with
microtubules. With respect to microtubule binding, the oxetane
D-ring in paclitaxel was proposed to serve two functions by the
NMR study6 and the Taxol-epothilone minireceptor model:7 (i)
the oxetane oxygen might serve as a hydrogen-bond acceptor; and
(ii) the four-membered ring might operate to rigidify the paclitaxel
Agricultural University, No. 221, Huiming Road, Wenjing Region, Chengdu
611130, P. R. China
cDepartment of Pharmacology, Institute of Materia Medica, Chinese
Academy of Medical Sciences, Beijing 100050, P. R. China
† Electronic supplementary information (ESI) available: Key NOE re-
lationships of compounds 15 and 16, and NMR spectra for all new
compounds. See DOI: 10.1039/c1ob06535a
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core and thereby enforce a favorable conformational bias on the
side chains at C-2, C-4, and C-13.
Later, the fact that cyclopropane analogue 78 and D-seco
analogues 8–109–11 showed significant activities at microtubule
stabilization demonstrated that neither the intact oxetane ring
nor an oxygen on C-5 are necessary for efficient binding of
taxanes to tubulin. For example, the analogues 78 and 89 are
equipotent with paclitaxel in promoting the polymerization of
tubulin to microtubules. The ratios IC50(7)/IC50(paclitaxel) and
IC50(8)/IC50(paclitaxel) are 1.2 and 1.0, respectively. However,
all of the reported D-ring modified analogues of paclitaxel and
docetaxel have very low or no activity in cytotoxicity assays. It is
envisioned that the oxetane oxygen might play an important role
in cytotoxicity based on the lack of cytotoxicity of the microtubule
stabilizer 5(20)-deoxydocetaxel. Consequently, we designed a new
lactone analogue of docetaxel (20), in which an additional oxygen
atom was incorporated into the D-ring and the g-lactone was
expected to play a similar role to the oxetane ring.
Results and discussion
Our synthetic strategy was to reconstruct the D-ring as a g-
lactone starting from commercially available 10-deacetylbaccatin
III (10-DBA). The construction of the g-lactone was envisioned
with an intramolecular aldol reaction (Scheme 1) by analogy with
Wender’s successful construction of the oxetane D-ring through
the photolysis of a-methoxy ketone.12 For the aldol reaction,
an acetoxy group at C-5a and a carbonyl group at C-4 are
prerequisites. This functionality could be introduced through an
intramolecular transacylation between C-4 and C-5, while the
oxetane D-ring opening could be mediated by Lewis acid.
Starting from 10-DBA, the hydroxyl groups at C-7, C-10, and
C-13 were protected with Boc2O using DMAP as base. These
hydroxyl groups in 10-DBA were mostly protected as TES ethers
in the literature. However, we observed that the TES ethers are
sensitive to Lewis acid during the opening of the oxetane ring.
After screening the various protecting groups, Boc was selected
as the optimized one. Selectively reductive deacylation of the
benzoate at C-2 was completed with Red-Al at low temperature
according to the procedure described in the literature.13 Formation
of the C-1, C-2 cyclic carbonate was easily achieved by addition
of triphosgene to yield compound 11 (Scheme 1).
Scheme 1 Synthesis of lactone analogue of docetaxel: Reagents and
conditions: (a) (Boc)2O, DMAP, CH2Cl2–THF (1 : 1), (95%); (b) Red-Al
(4 equiv), THF, -15 ◦C (85%); (c) triphosgene, CH2Cl2–pyridine (80 : 20),
-10 ◦C (92%); (d) BF3·OEt2, CH2Cl2, -5 ◦C; (12, 64%; 13, 10%); (e) TiCl4
(1 equiv), CH2Cl2 (2 mL for each mg of substrate) (13: 93%); (f) LTA,
CH3CN (85%); (g) LDA, HMPA, THF, -78 ◦C (15: 23%; 16: 42%); (h)
KOtBu, HMPA, THF, -78 ◦C (16: 65%); (i) Ac2O, DMAP (65%); (j) PhLi,
THF, -78 ◦C (35%); (k) NaH (30 equiv), THF, rt (74%); (l) 1 M HCl, THF,
40 ◦C (60%).
And then, Lewis acid-promoted opening of the oxetane ring
was explored on compound 11. The mechanism of the oxetane
ring opening catalyzed by Lewis acid involves the formation of
an orthoester between C-4, C-5, and C-20. The hydrolysis of this
orthoester afforded the C-5 and C-20 acetoxy derivatives,5 with
C-20 acetoxy derivative as the major product. Unsurprisingly,
treatment of 11 with BF3·OEt3 at 0 ◦C generated 12 with a C-
20 acetoxy group in 85% yield, as well as trace amounts of our
desired product 13 (10%). To increase the yield of expected product
13, we examined various Lewis acids, such as AlCl3, SnCl4, TiCl4
etc., in varied solvents, concentrations, and reaction temperature.
Gratifyingly, it was found that treatment of a highly diluted
solution of 11 (50 mg) in DCM (100 mL) with 1 equivalent of TiCl4
With the critical intermediate 14 in hand, we started to explore
the possibility of its intramolecular aldol reaction. Initially, a-
acetoxy ketone◦14 was subjected to aldol reaction with LDA and
HMPA at -78 C to generate a pair of diastereoisomers 15 and
16. The structures of 15 and 16 were established by careful inter-
pretation of their 1D and 2D NMR spectra. The configurations
at C-4 and C-5 in 15 were determined based on the critical NOE
correlations between H-5b and H-2b, and between H2-20 and H-3
(Fig. 3). Similarly, the key NOE correlations between H-5a/H-3a,
and H-5a/H-7a could suggest the b-orientation of the g-lactone
(Fig. 4). The distinguishable configurations at C-4 and C-5 in 15
◦
at 25 C for 10 min could produce the expected product 13 with
an acetoxy group at C5 in 93% yield. The subsequent oxidation of
the vicinal glycol at C-4 and C-20 with lead tetraacetate yielded
the a-acetoxy ketone 14 in excellent yield.
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Table 1 Cytotoxic data of compound 20 and paclitaxel
IC50, (mM)
Cmpd
HCT8
HePG2
BGC823
A549
A375
Paclitaxel 4.49 0.83 0.12 0.47 2.42 0.76 2.76 1.53 2.6 2.0
20 3.74 1.90 0.11 0.006 3.6 2.26 0.13 0.04 0.15 0.04
Note: HCT8: human colon cancer cell line; HePG2: human liver cancer
cell line; BGC-823: human stomach cancer cell line; A549: human lung
cancer cell line; A375: human melanoma cell line.
Table 2 Comparison of key coupling constants (in Hz) of 20 with those
of paclitaxel, docetaxel and representatives of D-ring modified analogues
Comp.
J13-14a
J13-14b
J2-3
Fig. 3 Key NOE correlations of compound 15.
Paclitaxel
9.0
8.0
4.4
7.3
6.0
8.0
9.6
9.0
8.0
10.3
9.4
10.0
8.0
9.6
7.0
7.0
5.3
7.4
5.5
7.0
7.2
Docetaxel
313
89
910
1011
20
towards HCT8, HePG2 and BGC23 cancer cell lines; it is more
potent than paclitaxel against A549 and A375 cancer cell lines.
These results suggested that the incorporation of an additional
oxygen atom on D-ring could improve the cytotoxicity towards
certain kinds of cancer cell lines. It is important to note that
lactone 20 is the first D-ring modified analogue of paclitaxel and
docetaxel that showed superior cytotoxic activity. After careful
analysis of the coupling constants between H-13 and H-14a/H-
14b, and between H-2 and H-3 in the D-ring modified analogues
of paclitaxel (Table 2), it was found that these coupling constants
of compound 20 are very close to those of paclitaxel. Compounds
8 and 10, possessing microtubule disassembly activity comparable
to paclitaxel but lacking cytotoxicity, are the compounds next
to compound 20 according to the similarity of their coupling
constants at these positions to paclitaxel. The coupling constants
might reflect, at least in part, the conformation of the whole
molecule.9,16 Accordingly, the above-mentioned research results
might suggest: (i) that the oxetane D-ring is not necessary for the
cytotoxicity of paclitaxel; (ii) that the oxygen of the D-ring seems
to be important to the cytotoxicity; and (iii) the conformation
of the paclitaxel core structure remains after replacement of the
oxetane D-ring with a g-lactone ring.
Fig. 4 Key NOE correlations of compound 16.
and 16 could be explained by the enolization of C-4 ketone during
the aldol reaction. We also observed that the acetoxy group at
C-5a in 14 could be epimerized to the C-5b position during the
chromatography process through a silica gel column. Encouraged
by the successful synthesis of the g-lactone D-ring, we began our
optimization by testing the effect of several bases, such as KOtBu,
LiHMDS, LDA etc. Finally, it was found that employing KOtBu
as base could lead to the exclusive generation of the expected
product.
The docetaxel analogue 20 was synthesized from the key
intermediate 16 employing the Holton–Ojima method.14 The
hydroxyl group at C-4 in 16 was acetylated by reacting with acetic
anhydride in the presence of DMAP. The regioselective opening
of the C-1, C-2 carbonate and the benzoylation at C-2 in 17 were
achieved simultaneously by reacting with phenyllithium. Luckily,
the t-butyloxycarbonyl group at C-13 in the key intermediate 18
obtained in the above-mentioned step was selectively removed. The
docetaxel side chain was then introduced to C-13 of compound
18 through coupling with commercially available b-lactam 19
in the presence of excess sodium hydride. Finally, the global
deprotection of the product obtained from the above-mentioned
step with 1 M hydrochloric acid furnished the expected docetaxel
analogue 20.
Conclusions
In summary, we have successfully synthesized a novel D-ring
modified docetaxel analogue, in which the oxetane ring is replaced
with a g-lactone, in 10 steps and 6% overall yield from the
commercially available 10-deacetylbaccatin III (10-DBA). The
critical reactions include the direct regioselective acetylation of
the secondary hydroxyl group at C-5 and D-ring opening and an
intramolecular aldol reaction to form the g-lactone. Compound
20 represents the first example of the D-ring modified taxoids with
significant cytotoxicity.
The cytotoxicity of docetaxel analogue 20 was evaluated against
a small panel of human cancer cell lines15 As shown in Table 1,
compound 20 showed equipotent cytotoxicity relative to paclitaxel
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0.080 mmol), and the reaction mixture was stirred at -15 ◦C
for 30 min prior to being quenched with saturated K2CO3
solution. After standard workup, the residue was subjected to
column chromatography on silica gel (cyclohexane–acetone 6 : 1)
to generate compound 11 (46 mg, 92%) as an amorphous solid:
Experimental
General procedures
NMR spectra were obtained on a Varian Unity INOVA 400/54
NMR spectrometer in CDCl3 with TMS as the internal standard.
Mass spectra were obtained on a VG Auto spec 3000 or on a
Finnigan MAT 90 instrument. Optical rotations were measured on
a Perkin–Elmer 341. Silica gel H (Qingdao Sea Chemical Factory,
Qingdao, People’s Republic of China) was used for column
chromatography. Spots on TLC (silica gel G) were detected by
spraying with H2SO4–EtOH. Commercially available reagents and
solvents were directly used without further purification. All anhy-
drous reactions were performed under argon. Dichloromethane
was distilled from calcium hydride, and THF was distilled from
sodium–benzophenone. Cytotoxicity were carried out according
to the protocols described in the literature. The “standard workup”
means: the reaction mixture was extracted with EtOAc, the
combined extracts were washed with water or brine and dried
over anhydrous Na2SO4, and the organic solvent was removed
under reduced pressure.
1
[a]2D5 +12.1 (CHCl3, c 0.8); H NMR (CDCl3, 400 MHz) d 6.30
(1H, s), 5.92 (1H, t, J = 8.4 Hz), 5.37 (1H, dd, J = 10.0, 7.6 Hz),
5.00 (1H, br.d, J = 8.4 Hz), 4.66 (1H, d, J = 6.0 Hz), 4.64, 4.56
(each 1H, ABq, J = 8.8 Hz), 3.55 (1H, d, J = 6.8 Hz), 1.29 (3H, s),
2.23 (3H, s), 1.83 (3H, s), 1.50, 1.47, 1.45 (each 9H, s), 1.28 (3H, s),
2.12, (3H, s); MS (ESI, MeOH) m/z 767 [M+H]+; HR-ESI-MS:
767.3462 [M+H]+, calc. for C38H55O16 766.3490.
Preparation of compounds 12 and 13. To a solution of co◦m-
pound 11 (100 mg, 0.13 mmol) in anhydrous CH2Cl2 at -15 C
was added 3 drops of BF3·Et2O, and the reaction mixture was
stirred at -15 ◦C for 10 min under argon prior to being quenched
with ammonium hydroxide. After standard workup, purification
of the residue via column chromatography over silica gel (ether–
acetone 6 : 1) yielded compounds 12 (65 mg, 64%) and 13 (10 mg,
10%) as amorphous powders. Compound 12: [a]2D5 -7.5 (CHCl3,
c 2.3); 1H NMR (CDCl3, 400 MHz) d 6.48 (1H, s), 5.72 (1H, dd,
J = 10.0, 2.4 Hz), 5.44 (1H, dd, J = 11.2, 4.4 Hz), 4.52, 4.41 (each
1H, ABq, J = 12.0 Hz), 4.23 (1H, d, J = 4.4 Hz), 3.79 (1H, t, J =
2.8 Hz), 3.74 (1H, t, J = 4.4 Hz), 2.29 (3H, s), 2.13 (3H, s), 1.50,
Preparation of 7,10,13-triBoc-10-DAB (a). To a stirred solu-
tion of 10-DAB (100 mg, 0.18 mmol) in anhydrous CH2Cl2–THF
(1 : 1) at room temperature were added (Boc)2O (240 mg, 1.1 mmol)
and DMAP (30 mg). The reaction mixture was stirred under argon
for 12 h (monitored by TLC, cyclohexane–acetone 4 : 1) prior to
the addition of water. After standard workup, the residue was
subjected to column chromatography over silica gel (cyclohexane–
acetone 8 : 1) to yield 7,10,13-triBoc-10-DAB (147 mg, 95%) as an
amorphous solid: [a]D25 +9.6 (CHCl3, c 1.2); 1H NMR (CDCl3, 400
MHz) d 7.31–8.11 (10H, m), 6.27 (1H, s), 5.86 (1H, t, J = 8.0 Hz),
5.64 (1H, d, J = 6.8 Hz), 5.40 (1H, dd, J1 = 6.8 Hz, J2 = 10.4 Hz),
4.97 (1H, br.d, J = 8.0 Hz), 4.13, 4.32 (each 1H, ABq, J = 8.8 Hz),
3.96 (1H, d, J = 6.8 Hz), 1.50, 1.49, 1.48 (each 9H, s); MS (ESI,
MeOH) m/z 845 [M+H]+.
1.47, 1.44 (each 9H, s), 1.35 (3H, s), 1.22 (3H, s), 1.15 (3H, s); 13
C
NMR (CDCl3, 100 MHz) d 202.0 s, 170.5 s, 151.8 s, 144.2 s, 132.0
s, 90.2 s, 82.8 s, 81.1 d, 78.3 d, 75.4 s, 72.1 d, 71.4 d, 69.8 d, 64.7 t,
59.1 s, 43.4 d, 40.6 s, 30.6 t, 32.5 t, 27.7 q, 27.1 q, 20.7 q, 18.8 q,
16.6 q, 12.7 q. MS (ESI, MeOH) m/z 785 [M+H]+; HR-ESI-MS:
785.3567 [M+H]+, calcd. for C38H57O17 785.3596. Compound 13:
1
[a]2D5 -13.2 (CHCl3, c 2.0); H NMR (CDCl3, 400 MHz) d 6.44
(1H, s), 5.82 (1H, dd, J = 8.4, 5.6 Hz), 5.32 (1H, t, J = 2.8 Hz),
5.20 (1H, dd, J = 11.6, 4.8 Hz), 4.24 (1H, d, J = 4.8 Hz), 4.08, 3.57
(each 1H, ABq, J = 9.2 Hz), 3.50 (1H, t, J = 4.4 Hz), 2.31, (3H, s),
2.34 (3H, s), 1.49, 1.47, 1.44 (each 9H, s), 1.29 (3H, s), 1.28 (3H, s),
1.15 (3H, s); 13C NMR (CDCl3, 100 MHz) d 201.9 s, 171.0 s, 151.9
s, 143.8 s, 132.0 s, 90.1 s, 83.0 s, 81.7 d, 78.0 d, 74.0 s, 72.1 d, 71.6
d, 71.3 d, 62.9 t, 59.0 s, 45.3 d, 41.1 s, 32.7 t, 30.0 t, 27.6 q, 26.0 q,
21.3 q, 19.5 q, 16.1 q, 12.7 q; MS (ESI, MeOH) m/z 785 [M+H]+;
HR-ESI-MS: 785.3532 [M+H]+, calcd. for C38H57O17 785.3596.
Preparation of 7,10,13-TriBoc-2-debenzoate-10-DAB (b). To
a stirred solution of 7,10,13-TriBoc-10-DAB (147 mg, 0.175
mmol) in anhydrous THF (10 mL) at -15 ◦C was added
NaAlH2(OCH2CH2OCH3)2 (Red-Al, 3.5 M in toluene, 0.5 mL),
and the reaction mixture was stirred at -15 ◦C for 24 h prior
to addition of water. 1% Hydrochloric acid was added to adjust
pH to 3. After standard workup, the residue was purified on
column chromatography over silica gel (cyclohexane–acetone 6 : 1)
to obtained 7,10,13-triBoc-2-debenzoate-10-DAB (110 mg, 85%)
as an amorphous solid: [a]2D5 +15.2 (CHCl3, c 1.1); 1H NMR
(CDCl3, 400 MHz) d 6.19 (1H, s), 5.83 (1H, t, J = 8.0 Hz), 5.36
(1H, dd, J = 10.4, 6.8 Hz), 4.97 (1H, br.d, J = 8.0 Hz), 4.63, 3.59
(each 1H, ABq, J = 8.8 Hz), 3.90 (1H, d, J = 6.8 Hz), 3.59 (1H,
d, J = 6.8 Hz), 2.22 (3H, s), 2.02, (3H, s), 1.75 (3H, s), 1.50, 1.49,
1.46 (each 9H, s), 1.16 (3H, s), 1.05 (3H, s); 13C NMR (CDCl3,
100 MHz) d 204.9 s, 170.3 s, 139.4 s, 137.3 s, 85.1 d, 83.8 s, 82.8
s, 78.4 d, 75.9 s, 73.5 t, 73.1 d, 73.1 d, 72.3 d, 54.5 s, 53.6 d, 42.2
s, 35.5 t, 33.7 t, 27.6 q, 25.3 q, 21.5 q, 18.8 q, 15.0 q, 14.3 q; MS
(ESI, MeOH) m/z 741 [M+H]+; HR-ESI-MS: 741.3612 [M+H]+,
calc. for C37H57O15 741.3698.
Regioselective preparation of compound 13. To a solution of
compound 11 (100 mg, 0.13 mmol) in anhydrous CH2Cl2 (200 mL)
at 25 ◦C was added TiCl4 (0.13 mL, 1 M in CH2Cl2) and the
◦
reaction was allowed to proceed with stirring at 25 C for 5 min
under argon before quenching with ammonium hydroxide. After
standard workup, the residue was separated on silica gel (ether–
acetone 6 : 1) to give compound 13 (94 mg, 93%) as an amorphous
powder.
Preparation of compound 14. LTA (62 mg, 0.14 mmol) was
added to a solution of the compound 13 (98 mg, 0.125 mmol)
in CH3CN (20 mL). The reaction mixture was stirred at room
temperature for 12 h and then quenched with water (10 mL). After
standard workup, the residue was separated on silica gel column
(petroleum ether–acetone 6 : 1) to give compound 14 (80 mg) as an
amorphous powder: [a]D25 +23.6 (CHCl3, c 1.0); 1H NMR (CDCl3,
400 MHz) d 6.39 (1H, s), 5.96 (1H, t, J = 8.0 Hz), 5.50 (1H, dd,
J = 11.2, 5.2 Hz); 5.02 (1H, t, J = 2.8 Hz), 4.29 (1H, d, J = 6.0 Hz),
Preparation of compound 11. To a stirred solution of 7,10,13-
triBoc-2-debenzoate-10-DAB (50 mg, 0.067 mmol) in anhydrous
CH2Cl2–pyridine (5 : 1) at -15 ◦C was added triphosgene (24 mg,
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4.11 (1H, d, J = 6.4 Hz), 1.18 (3H, s), 2.34 (3H, s), 2.32, (3H, s),
1.48, 1.48, 1.46 (each 9H, s), 1.32 (3H, s), 1.26 (3H, s); 13C NMR
(CDCl3, 100 MHz) d 199.8 s, 195.1 s, 169.5 s, 151.9 s, 143.3 s, 131.3
s, 89.3 s, 82.6 s, 78.9 d, 77.7d, 75.3 d, 71.2 d, 70.9 d, 59.8 s, 49.8 d,
40.8 s, 33.9 t, 33.0 t, 27.6 q, 25.4 q, 21.1 q, 19.7 q, 16.0 q, 12.4 q; MS
(ESI, MeOH) m/z 753 [M+H]+; HR-ESI-MS 753.3345 [M+H]+,
calcd. for C37H53O16 753.3333.
was allowed to stir overnight at room temperature before being
quenched with aqueous Na2CO3 solution. After standard workup,
the residue was purified by column chromatography over silica gel
(cyclohexane–acetone 8 : 1) to yield compound 17 (34 mg, 65%) as
an amorphous powder: [a]2D5 +11.2 (CHCl3, c 1.0); 1H NMR (400
MHz) d 6.45 (1H, s), 5.40 (1H, dd, J = 11.2, 4.8 Hz), 4.82 (1H, d,
J = 7.6 Hz), 4.24 (1H, d, J = 4.8 Hz), 3.89 (1H, br.s), 3.77 (1H, d, J =
4.8 Hz), 3.24, 2.96 (each 1H, ABq, J = 11.6 Hz), 2.36 (3H, s), 2.12
(3H, s), 1.36, 1.18, 1.14 (each 3H, s); 13C NMR (CDCl3, 100 MHz)
d 200.5, 198.1, 167.1, 152.2, 152.1, 151.1, 141.8, 132.5, 89.3, 83.2,
82.5, 78.1, 77.4, 77.0, 74.8, 72.2, 68.8, 57.9, 51.1, 40.0, 35.4, 33.7,
30.6, 29.4, 28.1, 27.5, 27.4, 26.6, 18.1, 18.0, 14.5; MS (ESI, MeOH)
m/z 817 [M+Na]+; HR-ESI-MS: 817.3250 [M+Na]+, calcd. for
C39H54NaO17 817.3258.
Preparation of compounds 15 and 16. To a stirred solution of
compound 14 (125 mg, 0.17 mmol) in anhydrous THF at -78 ◦C
was added HMPA (0.05 mL), and the reaction mixture was stirred
under argon at -78 ◦C for 15 min prior to addition of fresh LDA
(0.2 mL, 1 M in THF). The reaction was allowed to proceed for an
additional 2 h prior to being quenched with water. The mixture was
extracted with CH2Cl2, and the combined extracts were washed
successively with water, 1% HCl, and brine. The organic phase
was dried over anhydrous Na2SO4 and evaporated to dryness.
The crude product was purified by silica gel chromatography
(cyclohexane–acetone 5 : 1) to give compounds 15 (28 mg, 23%)
and 16 (52 mg, 42%). Their structures were established by 1D and
2D NMR including 1H–1H COSY, HMQC, HMBC and NOE (see
ESI†).
Preparation of compound 18. To a solution of compound 17
(85 mg, 0.107 mmol) in dry THF at -78 ◦C under argon was
added a solution of phenyllithium (1 M, 0.4 mL, 4 equiv.) in
hexane. The solution was stirred for 1 h at -78 ◦C and then poured
onto a mixture of CH2Cl2 and saturated ammonium chloride, and
the subsequent mixture was stirred vigorously for 5 min at room
temperature. After standard workup, the residue was purified on
silica gel chromatography (petroleum ether–acetone 10 : 1) to give
compound 18 (29 mg, 35%) as an amorphous solid: [a]2D5 +23.6
1
Compound 15. [a]2D5 -13.5 (CHCl3, c 0.8); H NMR (CDCl3,
400 MHz) d 6.37 (1H, s), 5.72 (1H, dd, J = 7.2, 3.6 Hz), 5.15 (1H,
dd, J = 8.0, 6.4 Hz), 4.46 (1H, d, J = 6.8 Hz), 4.61 (1H, t, J =
5.2 Hz), 3.03, 2.79 (each 1H, ABq, J = 18.0 Hz), 2.70 (1H, t, J =
6.8 Hz), 2.19, (3H, s), 1.53 (3H, s), 1.48 (each 27H, s), 1.23 (3H,
s), 1.20 (3H, s); 13C NMR (100 MHz) d 202.2, 170.6, 151.8, 142.9,
131.2, 90.6, 83.4, 81.8, 80.5, 77.8, 77.1, 71.5, 70.7, 57.8, 46.4, 46.1,
40.4, 34.1, 29.2, 27.6, 26.7, 19.0, 16.4, 13.5; MS (ESI, MeOH) m/z
753 [M+H]+; HR-ESI-MS: 753.3351 [M+H]+, calcd. for C37H53O16
753.3333.
1
(CHCl3, c 1.0); H NMR (400 MHz) d 8.11–7.46 (5H, m), 6.39
(1H, s), 5.01 (1H, dd, J = 11.2, 4.4 Hz), 5.76 (1H, d, J = 7.2 Hz),
4.89 (1H, t, J = 8.4 Hz), 2.98 (2H, m), 2.90, 2.52 (each 1H, ABq, J =
11.6 Hz), 2.27 (3H, s), 2.25 (3H, s), 1.67, 1.22, 1.18 (each 3H, s),
1.50, 1.47 (each 9H, s); MS (ESI, MeOH) m/z 795 [M+Na]+;
HR-ESI-MS: 795.3201 [M+Na]+, calcd. for C40H52NaO15
795.3204.
Preparation of compound 20. (1) The link of side chain. To a
solution of compound 18 (120 mg, 0.155 mmol) in dry THF were
added the docetaxel side chain (compound 19, 160 mg, 3 equiv.)
and NaH (40 mg, 10 equiv.). The reaction mixture was stirred
at room temperature for 2 h, and then quenched by addition of
water (10 mL). After standard workup, the residue was purified by
preparative TLC (cyclohexane–acetone 2 : 1) to give the side chain
linked product (128 mg, 74%) as an amorphous powder: [a]2D5 -3.5
1
Compound 16. [a]2D5 +5.2 (CHCl3, c 1.0); H NMR (CDCl3,
400 MHz) d 6.30 (1H, s), 5.73 (1H, t, J = 7.2 Hz), 5.00 (1H, dd,
J = 10.0, 4.8 Hz), 4.61 (1H, t, J = 3.6 Hz), 4.05 (1H, d, J = 6.4 Hz),
3.20, 2.73 (each 1H, ABq, J = 18.4 Hz), 2.84 (1H, t, J = 6.4 Hz),
2.05, (3H, s), 1.56 (3H, s), 1.49 (each 27H, s), 1.15 (3H, s), 1.08
(3H, s); 13C NMR (100 MHz) d 202.5 s, 172.4 s, 141.0 s, 132.4 s,
82.9 s, 81.6 d, 78.6 s, 77.1d, 76.9 s, 74.1 d, 72.3d, 71.5 s, 57.0 s, 48.3
t, 48.0 d, 42.2 s, 35.6 t, 27.4 q, 27.3 t, 26.3 q, 19.4 q, 15.2 q, 12.1
q; MS (ESI, MeOH) m/z 753 [M+H]+; HR-ESI-MS: 753.3346
[M+H]+, calcd. for C37H53O16, 753.3333.
1
(CHCl3, c 1.0); H NMR (400 MHz) d 7.98–7.36 (10H, m), 6.39
(1H, s), 5.76 (1H, d, J = 7.2 Hz), 5.69 (1H, d, J = 9.2 Hz), 5.59
(1H, t, J = 8.4 Hz), 5.01 (1H, dd, J = 11.2, 4.4 Hz), 3.90 (1H, s),
2.90, 2.31 (each 1H, ABq, J = 11.6 Hz), 2.27 (3H, s), 2.25 (3H,
s), 1.67 (3H, s), 1.51, 1.48 (each 9H, s), 1.22, 1.18 (each 3H, s);
13C NMR (100 MHz) d 202.8, 173.9, 170.5, 169.8, 167.5, 166.3,
152.2, 146.3, 132.4, 132.0, 129.2, 128.9, 128.2, 126.4, 88.7, 84.0,
81.2, 77.3, 77.1, 76.0, 76.0, 72.8, 72.4, 71.3, 71.3, 70.5, 70.3, 69.9,
69.1, 64.7, 64.5, 62.5, 57.2, 52.5, 43.0, 38.6, 32.9, 32.0, 29.6, 29.2,
21.3, 20.8, 20.3, 20.0, 13.9, 12.5, 11.7, 10.8; MS (ESI, MeOH)
m/z 1130, [M+Na]+; HR-ESI-MS: 1130.4914 [M+Na]+, calcd. for
C58H77NO20Na 1130.4937.
Selective preparation of compound 16. To a stirred solution
of compound 14 (100 mg, 0.13 mmol) in anhydrous THF at
-78 ◦C was added HMPA (0.05 mL), and the reaction mixture
was stirred under argon at -78 ◦C for 15 min before addition
of tBuOK (0.15 mL, 1 M in THF). The reaction was allowed
to proceed for an additional 1 h prior to being quenched with
water. The subsequent mixture was extracted with CH2Cl2, and the
combined extracts were washed successively with water, 1% HCl
and brine. The organic phase was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The crude product was
purified by silica gel chromatography (cyclohexane–acetone 5 : 1)
to give compound 16 (65 mg, 65%).
(2): The global deprotection. To a solution of the previously
prepared side chain linked product (100 mg, 0.097 mmol) in THF
was added 1 M HCl (0.5 mL). The reaction mixture was stirred at
40 ◦C for 3 h before being quenched with 5% NaHCO3 solution.
After standard workup, the residue was purified by preparative
TLC (CHCl3–MeOH 10 : 1) to afford compound 20 (48 mg, 60%)
Preparation of compound 17. To a stirred solution of com-
pound 16 (50 mg, 0.066 mmol) in CH2Cl2 was added DMAP
(50 mg) and acetic anhydride (0.4 mL), and the reaction mixture
1
as an amorphous powder: [a]2D5 -29.6 (CHCl3, c 1.0); H NMR
(400 MHz) 8.13–7.31 (10H, m), 6.30 (1H, d, J = 8.0 Hz), 6.22 (1H,
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t, J = 9.2 Hz), 5.78 (1H, t, J = 11.2 Hz), 5.70 (1H, d, J = 7.2 Hz),
5.30 (1H, br.s), 4.97 (1H, d, J = 8.0 Hz), 4.44 (1H, m), 3.80 (1H, d,
J = 7.2 Hz), 2.85, 2.19 (each 1H, ABq, J = 11.2 Hz), 2.57 (1H, m,
H-14), 2.42 (3H, d, J = 3.2 Hz), 2.36 (1H, m, H-14), 2.23 (3H, s,
OAc), 1.95 (3H, s), 1.85 (1H, m, H-6), 1.45 (9H, s, Boc), 1.39 (1H,
m, H-6), 1.26, 1.15 (each 3H, s); 13C NMR (100 MHz) 203.8, 176.2,
176.1, 171.1, 169.8, 168.3, 166.9, 152.3, 142.8, 133.5, 132.7, 130.2,
129.2, 128.9, 128.6, 128.1, 128.1, 126.4, 84.4, 84.0, 81.0, 79.0, 76.4,
75.9, 75.6, 75.1, 71.9, 58.5, 51.9, 51.7, 45.5, 43.2, 43.1, 42.9, 35.5,
27.5, 27.1, 26.9, 26.6, 22.6, 20.7, 17.4, 14.7. MS (ESI, MeOH)
m/z 858 [M+Na]+; HR-ESI-MS: 858.3304 [M+Na]+, calcd. for
C44H53NO15Na 858.3313.
Acknowledgements
We are grateful to the National Natural Science Foundation of
China (No. 30873147 and No. 30630069) for financial support of
this research.
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Determination of cell viability by MTT assay
Cells were plated in the RPMI 1640 with 10% fetal calf serum
media on 96-well plates in a total volume of 100 mL with a
density of 1 ¥ 104 cells mL-1. Triplicate wells were treated with
media and tested compounds. The plates were incubated at
37 ◦C in 5% CO2 for 72 h. Cell viability was determined based
on mitochondrial conversion of 3[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT, Sigma) to formazan. The
amount of MTT converted to formazan is a sign of the number of
viable cells. Each well was supplemented with 50 mL of a 1 mg mL-1
solution of MTT in uncompleted media. The plates were incubated
in 37 ◦C, 5% CO2 for an additional 4 h. The media was carefully
removed from each well and then 200 mL of DMSO was added.
The plates were gently agitated until the reaction color was uniform
and the OD570 was determined using a microplate reader (Wellscan
R
MK3, Labsystems Dragon). Microsoftꢀ Excel 2000 was used to
analyze data. Media-only treated cells served as the indicator
of 100% cell viability. The 50% inhibitory concentration (IC50)
was defined as the concentration that reduced the absorbance
of the untreated wells by 50% of the control in the MTT
assay.
16 L. Barboni, A. Datta, D. Dutta, G. I. Georg, D. V. V. Velde, R. H.
Himes, M. Wang and J. P. Snyder, J. Org. Chem., 2001, 66, 3321.
366 | Org. Biomol. Chem., 2012, 10, 361–366
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The Royal Society of Chemistry 2012
©