Synthesis and evaluation of 3D templates based on a taxane skeleton to circumvent P-glycoprotein-associated multidrug resistance of cancer

Article abstract of DOI:10.1016/j.bmcl.2005.03.038

We have developed a practical synthetic route to construct C-aromatic taxane derivatives as three-dimension templates by way of intramolecular alkylation. The usefulness of synthesized compounds as the core template was evaluated by using a newly developed screening system for P-glycoprotein.

Full text of DOI:10.1016/j.bmcl.2005.03.038

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2601–2605
Synthesis and evaluation of 3D templates based on a
taxane skeleton to circumvent P-glycoprotein-associated
multidrug resistance of cancer
Takashi Takahashi,a,* Kazuoki Nakai,a Takayuki Doi,a Masa Yasunaga,b
Hiroshi Nakagawab and Toshihisa Ishikawab,*
aDepartment of Applied Chemistry, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
bDepartment of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan
Received 5 February 2005; revised 1 March 2005; accepted 10 March 2005
Available online 14 April 2005
Abstract—We have developed a practical synthetic route to construct C-aromatic taxane derivatives as three-dimension templates
by way of intramolecular alkylation. The usefulness of synthesized compounds as the core template was evaluated by using a newly
developed screening system for P-glycoprotein.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Multidrug resistance (MDR) in human cancer is the ma-
jor obstacle to long-term, sustained patient response to
chemotherapy. P-glycoprotein (ABCB1 or MDR1)
causes MDR in cancer cells by actively extruding the
clinically administered chemotherapeutic drugs, such
as doxorubicin, vincristine, and paclitaxel.1 To circum-
vent P-glycoprotein-associated MDR, a new approach
of drug molecular design is needed.2 The nontaxol-type
taxoids have been reported as MDR reversal agents.3–6
Since several functional groups are attached on the tax-
ane skeleton that has a very tight endo form of a tricy-
clic system, the skeleton is regarded as a synthetic core
template.7 We have previously reported that a conform-
ationally restricted b-strand mimetic library based on a
bicyclic template provided potent and selective inhibi-
tors of serine proteases through combinatorial introduc-
tion of substituents.8 Therefore, the 3D core template
could be a useful probe for chemical genomics, when
variable substituents can be attached on the template
at the various positions.9 We design core templates 1,
2, and 3, that have different sizes of the B-ring as well
as different functional groups attached to the B-ring
(Fig. 1).10 These structures may allow fine tuning by
attaching variable substituents on the templates. We
herein report the synthesis of new templates of taxane
derivatives and evaluation of their MDR reversal
activity.
O
AcO
OH
BzHN
Ph
O
4
O
O
H
OBz
OAc
HO
OH
paclitaxel
Conformational analysis based on MM2 calculation
using Monte Carlo method suggested that all mimetics
possess the endo conformation as paclitaxel (Fig. 2).11
An O-functional group at the C4 or C5 position of 1–3
is located at the same position as the C4 acetoxy group
in paclitaxel. The tricyclic compounds 1–3 could be con-
structed by way of intramolecular alkylation of 4–6,
respectively. The precursors of the alkylation can be pre-
pared by coupling between the A-ring anion 7 and the
C-ring aromatic aldehydes 8 or 9 (Fig. 1).
The synthesis of the eight-membered ether skeleton 1 is
shown in Scheme 1. The coupling reaction of TBS-pro-
tected salicylic aldehyde derivatives 8a–c with a vinyl an-
ion 7 generated from the corresponding hydrazone
Keywords: Multidrug resistance reversal agents.
*
Corresponding authors. Tel.: +81
57342884; e-mail: ttak@apc.titech.ac.jp
3 57342120; fax: +81 3
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.038

2602
T. Takahashi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2601–2605
O
opening of the resulting oxirane ring with LiAlH4 pro-
vided the triol 12.13 Selective protection of the phenolic
alcohol with TBSCl, followed by carbonate formation
through exposure to carbonyldiimidazole afforded di-
TBS ether, which was then desilylated to give diol 4.
The construction of the eight-membered ether was car-
ried out by way of intramolecular Mitsunobu reaction.
Treatment of diol 4 with PPh3 and DEAD provided tri-
cyclic ethers 1a–c in 65–96% yields.14
O
O
O
5
5
5
Z
Z
4
4
Z
4
O
O
O
O
O
O
O
O
1
2
3
a: Z=H
Intramolecular
Alkylation
b: Z=5-OMOM
c: Z=4-OMOM
The synthesis of the nine-membered ether skeleton 2 was
carried out as summarized in Scheme 2. Aldehyde deriva-
tives 9a–c were coupled to the vinyl anion 7 to give
allylic alcohols 13a–c. Subsequent epoxidation, followed
by reduction induced partial desilylation and then the
primary alcohol was selectively protected with a TBS
ether provided diols 14a,b, while 14c was not obtained
since the reductive opening of epoxide had failed. Treat-
ment of 14a,b with carbonyldiimidazole furnished the
carbonate, which was desilylated, chlorinated. Then, re-
moval of PMB with DDQ afforded alkylation precur-
sors 5a,b. The 15c, prepared from 12c by a similar way
to the preparation of 4 except selective deprotection of
the TBS group, was converted to triflate 16c, which
underwent carbonylation in methanol using Pd(OAc)2-
bis(diphenylphosphono)propane (DPPP) under a CO
atmosphere (15 atm) leading to methyl ester 17c. Then,
the desired 5c was prepared in six steps (66%).
TBSO
X
Shapiro
W
O
Coupling
Y
+
5
5
4
Z
4
Z
O
Li
O
7
8 W=OTBS
9 W=CH2OPMB
O
4 X=Y=OH
5 X=Cl, Y=CH2OH
6 X=Cl, Y=C(OEE)(CN)
Figure 1. Synthetic strategy for the 3D templates 1, 2, and 3.
TBSO
OPMB
Figure 2. 3D structures of 1a, 2a, and 3a based on MM2 calculation.
b
a
+
7
9a–9c
5
4
Z
TBSO
R1O
OH
TBSO
13
OPMB
a
c
+
7
8a–8c
c
5
Z
g
5a,b
5c
4
5
OR2
2a–2c
4
Z
HO
10 R11=TBS, R2=H
OH
b
11 R =H, R2=TBS
14a, 14b
TBSO
TBSO
HO
e
d
R
4a–4c
a: Z=H
1a–1c
f
5
Z
4
2
HO
OH
O
O
OMOM
b: Z=5-OMOM
c: Z=4-OMOM
12a–12c
a: Z=H
O
b: Z=5-OMOM
c: Z=4-OMOM
15c R=OH
16c R=OTf
17c R=CO2Me
d
e
Scheme 1. The synthesis of eight-membered ethers 1. Reagents and
conditions: (a) THF, ꢀ78 ꢁC; (b) (i) K2CO3, MeOH; (ii) TBSCl,
imidazole, CH2Cl2, 10a (89%); 10b (68%); 10c (61%); (c) (i) VO(acac)2,
t-BuOOH, benzene; (ii) LiAlH4, ether, 12a (91%); 12b (93%); 12c
(58%); (d) (i) TBSCl, imidazole, CH2Cl2; (ii) carbonyldiimidazole,
CH3CN, reflux; (iii) TBAF, THF, 4a (52%); 4b (94%); 4c (61%); (viii)
PPh3, DEAD, THF, RT, 1a (85%); 1b (96%); 1c (65%).
Scheme 2. The synthesis of nine-membered ethers 2. Reagents and
conditions: (a) THF, ꢀ78 ꢁC, 13a (83%); 13b (53%); 13c (64%); (b) (i)
VO(acac)2, t-BuOOH, benzene; (ii) LiAlH4, ether, reflux; (iii) TBSCl,
imidazole, CH2Cl2, 14a (88%); 14b (72%); (c) (i) carbonyldiimidazole,
CH3CN, reflux; (ii) TBAF, THF; (iii) MsCl, LiCl, NEt3, THF,
CH2Cl2; (iv) DDQ, CH2Cl2, H2O, 5a (69%); 5b (66%); (d) Tf2O, NEt3,
CH2Cl2, 85%; (e) CO (15 atm), Pd(OAc)2, DPPP, NEt3, MeOH, THF,
80 ꢁC, quant.; (f) (i) LiAlH4, ether; (ii) Ac2O, NEt3, CH2Cl2; (iii)
carbonyldiimidazole, CH3CN, reflux (iv) TBAF, AcOH, THF; (v)
MsCl, LiCl, NEt3, CH2Cl2, THF; (vi) K2CO3, MeOH, 5c (66%); (g)
NaH, PhH, DC-18-crown-6, RT, 2a (66%); 2b (92%); 2c (64%).
proceeded at ꢀ78 ꢁC.10c,d,12 Concomitant migration of
the TBS group in the product 10 provided phenol 11,
which was easily converted to the desired 10. Hydr-
oxy-directed epoxidation of 10, followed by reductive

T. Takahashi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2601–2605
2603
Table 1. P-glycoprotein assay for the synthesized templates
Entry Compound Km Vmax Vmax/Km IC50
[lM] [lM]
b, c
b
6a,b
6c
3a,b
a
5a–5c
[lM]
O
1
1a
1b
40
11
28
20
4
23
35
40
23
41
45
48
47
39
35
65
0.56
3.2
1.4
1.2
>50 (>50)
>50 (>50)
>50 (>50)
>50 (>50)
>50 (>50)
45 (>40)
2
1
3
1c
2a
2
O
a: Z=H
b: Z=5-OMOM
c: Z=4-OMOM
O
OMOM
4
5
2b
2c
10
6
O
18
6
8
7
3a
3b
50
12
35
0.96
3.9
1.1
>50 (>50)
>50 (>50)
>50 (>50)
Scheme 3. The synthesis of eight-membered ketones 3. Reagents and
conditions: (a) (i) TPAP, NMO, CH2Cl2, a (67%); b (91%); c (47%); (ii)
TMSCN then 0.5 M HCl aq, THF; (iii) EVE, CSA, CH2Cl2, two steps
6a (75%); 6b (84%); 6c (79%); (b) (i) LiN(TMS)2, THF, 50 ꢁC; (ii) CSA,
MeOH; (iii) NaOH aq, ether, three steps a (56%); b (79%); 18 (35%);
(c) carbonyldiimidazole, CH3CN, reflux, 3a (65%); 3b (52%).
8
9
18
Paclitaxel
Verapamil
10
11
0.7
2
50
30
0.60 (0.0021)
—
IC50 was measured against KB-G2 cells that have resistance with
paclitaxel and vinblastin. The value against KB-3-1 cells is given in
parentheses.
Intramolecular O-alkylation of 5a–c was performed at
room temperature using NaH in the presence of 2 equiv
of 18-crown-615 to afford nine-membered ethers 2a–c in
good yields.16,17
Sf9 cells and drug-induced ATPase activity of P-glyco-
protein was measured by using the plasma membrane
fraction of Sf9 cells. Figure 3 depicts the relationship be-
tween the ATPase activity of P-glycoprotein and the
concentration of compound 1c, demonstrating a typical
Michaelis–Menten-type kinetics with a Km value of
28 lM and a Vmax of 40 nmol/min/mg protein. As indi-
cated by the Km value, the affinity of this compound 1c
to P-glycoprotein is one order of magnitude lower than
that of paclitaxel (Km = 0.7 lM).
The construction of the eight-membered ketones 3 by
way of an intramolecular alkylation of the protected
cyanohydrin ethers 6 is outlined in Scheme 3.10a,18
Oxidation of the alcohols 5a–c to the aldehyde, followed
by three-step formation of the protected cyanohydrin
provided 6a–c. Alkylation of 6a,b was accomplished in
a refluxing THF solution of lithium hexamethyldisilaz-
ide, and the resulting cyclized products were sequentially
treated with acid and base. As a carbonate group was
deprotected under the cyclization conditions using ex-
cess amount of base, the resulting diols were reprotected
as carbonate to afford 3a,b.19 Alkylation of 6c, however,
did not give a desired 3c but 18 via SN20 alkylation with-
out losing a carbonate group.13,20
The results of the P-glycoprotein assay for the synthe-
sized templates are summarized in Table 1. The Km val-
ues of all of the synthesized compounds were greater
than those of paclitaxel and verapamil, typical sub-
strates for P-glycoprotein. A significant difference was
noticed between the OMOM substituent at the C5 posi-
tion, at the C4 position, and non-substituent with re-
spect to Vmax/Km (1b > 1c > 1a, 2b > 2c > 2a, 3b > 3a).
For the purpose to circumvent P-glycoprotein-associ-
ated drug resistance, an O-functional group can be
introduced at the C5 position better than at the C4 posi-
tion on the aromatic ring. Interestingly, the nine-mem-
bered cyclic ether 2b shows better affinity to P-
glycoprotein than taxane skeleton 3b and eight-mem-
bered cyclic ether 1b. All template compounds, except
The synthesized compounds with the 3D core template
were evaluated by using a newly developed screening
system for P-glycoprotein.21,22 For the evaluation, the
cDNA of human P-glycoprotein was overexpressed in
60
40
0.5
20
0
1
2
1/[S]
0
0
20
40
60
80
100
Compound 1c (µM)
Figure 4. The effect of 2b as a MDR reversal agent on the cytotoxicity
of paclitaxel against MDR KB-G2 cells overexpressing P-glycoprotein
(Val-185). The concentration-dependent effects are compared with the
sensitivity of KB-3-1 (parental control) cells.
Figure 3. Relationship between the ATPase activity of P-glycoprotein
(Val-185) and the concentration of compound 1c. The inset demon-
strates the Lineweaver–Burk plot of the experimental data.

2604
T. Takahashi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2601–2605
Takahashi, T. Tetrahedron Lett. 2001, 42, 7855–7857; (d)
Nakai, K.; Miyamoto, S.; Sasuga, D.; Doi, T.; Takahashi,
T. Tetrahedron Lett. 2001, 42, 7859–7862; (e) Miyamoto,
S.; Doi, T.; Takahashi, T. Synlett 2002, 97–99.
2c, did not exhibit detectable cytotoxicity in either MDR
KB-G2 cells or KB-3-1 cells at concentrations of up to
50 lM.
11. The calculation was carried out with the MacroModel 6.0.
Mohamadi, F.; Richards, N. G. J.; Guida, W. C.;
Liskamp, R.; Lipton, M.; Caulfield, C.; Chang, G.;
Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11,
440–467.
12. (a) Nicolaou, K. C.; Yang, Z.; Sorensen, E. J.; Nakada,
M. J. Chem. Soc., Chem. Commun. 1993, 1024–1026; (b)
Di Grandi, M. J.; Jung, D. K.; Krol, W. J.; Danishefsky,
S. J. J. Org. Chem. 1993, 58, 4989–4992.
13. (a) Nicolaou, K. C.; Claiborne, C. F.; Nantermet, P. G.;
Couladouros, E. A.; Sorensen, E. J. J. Am. Chem. Soc.
1994, 116, 1591–1592; (b) Nicolaou, K. C.; Claiborne, C.
F.; Paulvannan, K.; Postema, M. H. D.; Guy, R. K.
Chem. Eur. J. 1997, 3, 399–409.
The reversal activity of compound 2b was further evalu-
ated in MDR KB-G2 cells. Figure 4 demonstrates that
compound 2b sensitized MDR KB-G2 cells to paclitaxel
in a dose-dependent manner. In the presence of com-
pound 2b at the concentration of 50 lM, the IC50 value
of MDR KB-G2 cells was 0.018 · 103 nM, which is
about 30 times lower than the value observed with pac-
litaxel per se (IC50 = 0.60 · 103 nM).
In summary, we have herein demonstrated that the
synthesis and P-glycoprotein assay for the novel 3D
templates based on the taxane skeleton. The affinity to
P-glycoprotein of the synthetic templates suggested that
introduction of the O-functional group would enhance
the affinity to P-glycoprotein. Further study for MDR
reversal agents by optimization of those template com-
pounds is underway in our laboratory.
14. Selected spectral data of 1a: IR (neat) 2927, 1801, 1605,
1485, 1458, 1289, 1179, 1036, 978, 750 cmꢀ1 1H NMR
;
(270 MHz, CDCl3) d 0.92 (s, 3H), 1.22 (s, 3H), 1.42–1.60
(m, 1H), 1.55 (s, 3H), 1.91–2.04 (m, 1H), 2.20–2.31 (m,
1H), 2.40–2.55 (m, 1H), 4.66 (d, 1H, J = 11.2 Hz), 4.73 (d,
1H, J = 11.2 Hz), 5.77 (s, 1H), 7.08–7.42 (m, 4H); 13C
NMR (67.8 MHz, CDCl3) d 20.0 (CH3), 20.7 (CH3), 23.2
(CH2), 24.1 (CH3), 28.5 (CH2), 39.1 (C), 72.0 (CH2), 78.3
(CH), 92.3 (C), 124.0 (CH), 124.3 (CH), 124.6 (CH), 124.6
(C), 127.2 (C), 130.2 (CH), 132.0 (C), 143.1 (C), 153.7 (C).
15. Takahashi, T.; Nemoto, H.; Kanda, Y.; Tsuji, J.; Fukaz-
awa, Y.; Okajima, T.; Fujise, Y. Tetrahedron 1987, 43,
5499–5520.
16. Selected spectral data of 2a: IR (neat) 2923, 1801, 1464,
1376, 1292, 1205, 1179, 1044, 757 (cmꢀ1); 1H NMR
(270 MHz, CDCl3) d 1.11 (s, 3H), 1.14 (s, 3H), 1.54 (s,
3H), 1.20–1.45 (m, 2H), 1.95–2.25 (m, 2H), 4.21 (d, 1H,
J = 12.5 Hz), 4.30 (d, 1H, J = 12.5 Hz), 4.50 (d, 1H,
J = 15.2 Hz), 4.95 (d, 1H, J = 15.2 Hz), 6.32 (s, 1H), 7.17
(dd, 1H, J = 3.96, 4.95 Hz), 7.22–7.32 (m, 2H), 7.59 (ddd,
1H, J = 1.98, 3.96, 4.95 Hz); 13C NMR (99.6 MHz,
CDCl3, d) 20.2 (CH3), 20.3 (CH3), 23.9 (CH3), 27.2
(CH2), 28.1 (CH2), 39.8 (C), 66.5 (CH2), 72.3 (CH2), 77.8
(CH), 91.6 (C), 126.1 (CH), 126.9 (CH), 128.5 (CH), 128.7
(CH), 130.7 (C), 132.4 (C), 138.5 (C), 139.5 (C), 154.1 (C).
17. Under reflux conditions, deprotection of the carbonate,
followed by intramolecular ether formation of the hydroxy
group at the C2 position was observed.
Acknowledgements
The authors cordially thank Prof. Kazumitsu Ueda
(Kyoto Univ.) for generously providing us with MDR
KB-G2 and parental KB-3-1 cells. This work was sup-
ported by a Grant-in-Aid from the Ministry of Educa-
tion, Culture, Sports, Science, and Technology, Japan
(No. 14103013 to T.T.) as well as a research grant
(No. 13NP0401 to T.I.) for Creative Scientific Research
from the Japan Society for the Promotion of Science.
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1
1599, 1462, 1377, 1281, 1204, 1039, 748 (cmꢀ1); H NMR
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T. Takahashi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2601–2605
2605
22. The cDNA of P-glycoprotein was expressed in Sf9 cells
using the pFASTBAC1 vector and recombinant baculo-
viruses. The plasma membrane fraction of Sf9 cells was
prepared by sucrose-density gradient ultracentrifugation.
The ATPase activity of the isolated Sf9 cell membranes
was determined by measuring inorganic phosphate liber-
ation. The Sf9 cell membranes (2 lg of protein) were
suspended in 10 ll of the incubation medium containing
50 mM Tris-Mes (pH 6.8), 2 mM EGTA, 2 mM dithio-
threitol, 50 mM potassium chloride, 5 mM sodium azide,
2 mM ouabain. This medium was mixed with 10 ll of a
test compound solution (0–1.2 mM) and then pre-incu-
bated at 37 ꢁC for 3 min. The ATPase reaction was
started by adding 10 ll of 4 mM ATP solution to the
reaction mixture (30 ll) and the incubation was main-
tained at 37 ꢁC for 30 min. The reaction was then stopped
by the addition of 20 ll of 5% trichloroacetic acid, and
liberated inorganic phosphate was measured in a Multis-
kan JX system (Dainippon Pharmaceuticals Co., Osaka,
Japan).