Design and synthesis of silicon-containing tubulin polymerization inhibitors: Replacement of the ethylene moiety of combretastatin A-4 with a silicon linker

Article abstract of DOI:10.1016/j.bmc.2013.09.046

Silicon-containing combretastatin analogs were designed, synthesized and evaluated for stability and biological activities. Among them, compound 31 exhibited strong tubulin polymerization-inhibitory activity and very potent tumor cell growth-inhibitory ac

Full text of DOI:10.1016/j.bmc.2013.09.046

Bioorganic & Medicinal Chemistry 21 (2013) 7381–7391
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of silicon-containing tubulin polymerization
inhibitors: Replacement of the ethylene moiety of combretastatin
A-4 with a silicon linker
⇑
Masaharu Nakamura , Daisuke Kajita, Yotaro Matsumoto, Yuichi Hashimoto
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Silicon-containing combretastatin analogs were designed, synthesized and evaluated for stability and
Received 28 August 2013
Revised 17 September 2013
Accepted 19 September 2013
Available online 1 October 2013
biological activities. Among them, compound 31 exhibited strong tubulin polymerization-inhibitory
activity and very potent tumor cell growth-inhibitory activity (IC50 = 0.007
tion assay. This compound also potently inhibited [ H]colchicine binding (90.7% inhibition at 3 M).
l
M) in MCF-7 cell prolifera-
3
l
These activities were comparable to those of combretastatin A-4 (CA-4) (1). In addition, compound 31
was physico-chemically more stable than 1. These results suggest that a silicon linker can act as a bioiso-
ster of a cis carbon–carbon double bond.
Keywords:
Silicon
Tubulin
Ó 2013 Elsevier Ltd. All rights reserved.
Combretastatin
Colchicine
Antitumor agent
8
1
. Introduction
sequent tumor cell death. The phosphate prodrug of CA-4 (CA-4P)
(
2) is currently undergoing phase II trials for the treatment of solid
9
Microtubules are cytoskeletal components that play central
tumors. On the other hand, it is known that CA-4 (1), which has a
cis double bond, isomerizes to the more stable trans-form during
storage and administration, resulting in a decrease of antitumor
roles in a number of cellular processes, including mitosis, motility,
and control of cell shape.1 They are formed by self-assembly of
alpha- and beta-tubulin heterodimers. Tubulin is the target of anti-
,2
1
0,11
activity.
Therefore, some groups have attempted to modify
3
12
mitotic agents that act by interfering with microtubule dynamics.
ethylene moiety of CA-4 (1). Very recently, Dos Santos Edos
Microtubule-targeting agents can be grouped into two classes:
those that inhibit the polymerization process (microtubule-desta-
bilizing agents), and those that promote the polymerization of
tubulin (microtubule-stabilizing agents). The three characterized
binding sites of tubulin are the taxane site, the vinca site, and
the colchicine site. Many tubulin-binding compounds, such as
paclitaxel, vinblastine and eribulin, are in clinical use for cancer
et al. have reported a series of sulfur and selenium-containing
1
3
tubulin polymerization inhibitors (see Fig. 1).
In recent years, silicon-containing bioactive compounds have
1
4–19
been reported.
The incorporation of silicon into organic
compounds can improve various biological properties, including
selectivity, potency, pharmacokinetics, pharmacodynamics and
cell penetration. Indeed, several organosilicon agents have
4
,5
20,21
therapy.
advanced to clinical studies.
Taking into account the bond
The combretastatins were isolated from the stem wood of the
South African tree Combretum caffrumin the 1980s.6 Some related
compounds have been shown to potently inhibit microtubule
activity by binding to tubulin at the colchicine site, thereby
interfering with cell growth and proliferation. Among them, com-
bretastatin A-4 (CA-4) (1) showed the strongest tumor cell
growth-inhibitory activity.7 CA-4 (1) also acts as a vascular
disrupting agent (VDA) and induces blood flow reduction and sub-
length of silicon, we hypothesized that a silicon linker can act as
a stable alternative structure to a cis carbon–carbon double bond.
Based on this hypothesis, we planned to introduce a silicon linker
as a substitute for the cis double bond of combretastatins, with the
objective of increasing the stability and tumor cell growth-inhibi-
tory activity.
In this paper, we describe the design and synthesis of silicon-
containing analogs of combretastatins, and evaluation of their
stability and biological activities by means of tubulin polymeriza-
tion inhibition (TPI) assay and MCF-7 cell proliferation assay, as
well as colchicine site competitive-binding assay.
⇑
Corresponding author. Tel.: +81 3 5841 7847; fax: +81 3 5841 8495.
E-mail address: nakamura-nk@iam.u-tokyo.ac.jp (M. Nakamura).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.046

7
382
M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
HO
OH
OMe
MeO
MeO
A
B
OR
OMe
O
MeO
MeO
OH
OMe
O
OMe
CA-2
OMe
OMe
combrestatin
CA-4 (1 : R = OH)
CA-4P (2 : R = PO(OH)2
)
Figure 1. Chemical structures of combrestatins.
2
2
. Results and discussion
.1. Molecular design and computational studies
Generally, bonds involving silicon are longer than those involv-
ing carbon. The average length of a C–C bond is 1.53 Å, whereas
that of a Si–C bond is 1.89 Å.22 We focused on this feature of silicon
and designed silicon-containing compound 3 with the aim of
obtaining increased stability and tumor cell growth-inhibitory
activity. First, we calculated the distance between the two benzene
ring (d) in compounds 3 and 4. Structure optimizations were
⁄
performed at the MP2/6-31+G level of theory with the GAUSSIAN
2
3,24
0
9 program package.
The results are illustrated in Figure 2.
As expected, the distance (d) in the silicon compound 3 was close
to that of the cis double bond of CA-4 (1). On the other hand, the
distance (d) of the corresponding carbon compound 4 was shorter
than those of 1 and 3.
Figure 3. Binding models of CA-4 (1) (aqua) and the designed compound 3 (pink) at
the colchicine-binding site of tubulin.
Next, we performed a docking study of the designed compound
3
and CA-4 (1) with the reported crystal structure of the tubulin-
Debenzylation of compounds 7, 10, 13 and 18 gave phenols 3, 8,
11 and 17, respectively. Compounds 15 and 9 were prepared by
methylation of phenols 3 and 8, respectively.
Next, we planned to optimize the A-ring of compound 3. Com-
pounds 21–24 were prepared similarly, as illustrated in Scheme 2.
Finally, we attempted to modify the substituent groups on sili-
con. As illustrated in Scheme 3, compounds 26a–b were prepared
according to the procedure described for the diaryldimethylsilanes
N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA-colchicine) com-
plex (PDB ID: 1SA0),25 using the AutoDock 4.2 docking program.26,27
The results are illustrated in Figure 3, as visualized with PyMOL
software. It is known that the colchicine-binding site is a deep
pocket located at the alpha–beta interface of tubulin heterodi-
mers. As expected, compound 3 and CA-4 (1) occupied the space
corresponding to the colchicine-binding site of tubulin. Both of
them formed hydrogen bonds with Cys241and Val181. Therefore,
we anticipated that the designed compound 3 would bind effi-
ciently to tubulin and show tubulin polymerization-inhibitory
activity.
2
8
2
9
(
7, 10, 12–14 and 18), starting from dichlorodiethylsilane 25a and
dichlorosilacyclobutane 25b, respectively. The diaryldiethylsilane
7 was easily prepared by debenzylation of compound 26a. On
2
the other hand, debenzylation reaction of compound 26b in etha-
nol/ethyl acetate gave the ring-opened compound 28. Therefore,
we performed debenzylation reaction using only ethyl acetate.
This afforded the desired compound 29. The diarylmethylsilane
2
.2. Synthesis
Based on our computational studies, we first planned to prepare
3
6
1 was prepared in 2 steps from methoxymethyl (MOM) ether
g. The corresponding carbon compound 35, generically known
silicon-containing compound 3. We also planned to synthesize its
B-ring modified derivatives, that is compounds 7–18, since the
as isoerianin, was prepared by reduction of the olefin 34, which
was synthesized by reaction of the known compound 3330 with
trimethylaluminium in toluene.
A-ring of combrestatins has been established to be crucial to elicit
cytotoxic and antitubulin responses.12 These compounds were
prepared by usual organic-synthetic methods, as illustrated in
Scheme 1. Substituted bromobenzenes, that is compounds 6a, 6b
and 6f, were prepared by benzylation of the corresponding phenol.
Substituted diaryldimethylsilanes (7, 10, 12–14 and 18) were
synthesized by reaction of 3,4,5-tribromobenzene (5) and substi-
tuted bromobenzenes (6a–f) with dichlorodimethylsilane in the
presence of n-butyllithium (n-BuLi) in tetrahydrofuran (THF).
2
.3. Tubulin polymerization-inhibitory activity and tumor cell
growth-inhibitory activity
All of the prepared compounds were initially screened at 30 lM
3
1,32
concentration by means of TPI assay,
which has been widely
Si
d
OH
MeO
MeO
C
d
OH
MeO
d
MeO
MeO
OH
OMe
OMe
OMe
MeO
OMe
OMe
OMe
4
CA-4 (1)
3
d = 3.03 Å
d = 2.47 Å
d = 3.00 Å
Figure 2. The distance between the two benzene rings (d) of CA-4 (1) and the designed compounds.

M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
7383
MeO
MeO
Br
Si
R1
Si
OMe
OMe
MeO
MeO
MeO
MeO
Br
R1
a
A
B
+
+
R
2
R
2
OMe
5
OMe
OMe
OMe
6
6
6
6
6
6
a (R = OBn, R = OMe)
7 (R1 = OBn, R = OMe)
16
1
2
2
b (R1 = OBn, R = H)
10 (R = OBn, R = H)
2
1
2
c (R
d (R
e (R
f (R = OMe, R = OBn)
1
= H, R
= H, R
, R
2
= OMe)
12 (R = H, R = OMe)
1 2
1
2
= OBn)
1 2
13 (R = H, R = OBn)
1
2
= -OCH
2
O-)
1 2 2
14 (R , R = -OCH O-)
1
2
18 (R = OMe, R = OBn)
1
2
c
3 (R
8 (R
11(R
17 (R
= OH, R
= OH, R
= H, R
= OMe)
= H)
= OH)
1
9
5 (R = OMe, R = OMe)
1 2
7
1
1
1
(R1 = OBn, R = OMe)
1
1
2
2
b
(R1 = OMe R = H)
0 (R = OBn, R = H)
2
2
1
2
3 (R = H, R = OBn)
1
2
1
1
2
8 (R
= OMe, R
2
= OBn)
1 2
= OMe, R = OH)
Scheme 1. Synthesis of silicon-containing compound 3 and its B-ring-modified derivatives 7–18. Reagents and conditions: (a) dichlorodimethylsilane n-BuLi, THF, ꢀ70 °C to
rt, 12–42%; (b) H , Pd/C, ethanol, rt, 33–88%; (c) iodomethane, K CO , acetone, reflux/sodium hydride, N,N-dimethylformamide, 41–66%.
2
2
3
R
4
Br
Si
OBn
OMe
Br
OBn
OMe
a
R4
+
R
5
R7
R7
R
5
R
6
R6
= H, R = OMe, R
, R = -OCH
= H, R
1
1
1
9a (R = H, R = OMe, R = OMe, R = OMe) 6a
20a (R
20b (R
20c (R
4
5
6
= OMe, R
= OMe, R
= OBn, R = H)
7
7
= OMe)
= H)
7
4
5
6
7
9b (R , R = -OCH O-, R = OMe, R = H)
4
5
2
O-, R
6
4
5
2
6
7
9c (R = H, R = OMe, R = OBn, R = H)
4
5
= OMe, R
6
4
5
6
7
Si
OH
R
4
b
A
B
R
7
OMe
R
5
R6
2
2
2
2
1 (R4 = H, R5 = OMe, R = OMe, R = OMe)
6 7
3 (R , R = -OCH O-, R = OMe, R = H)
4
4
4
5
2
6
6
6
7
4 (R
2 (R
= H, R
= H, R
5
= OMe, R
= OH, R
7
= H)
c
5
= OMe, R
= OMe, R = H)
7
Scheme 2. Synthesis of the A-ring-modified derivatives 21–24. Reagents and conditions: (a) dichlorodimethylsilane n-BuLi, THF, ꢀ70 °C to rt; (b) H
2
, Pd/C, ethanol, rt, 5–36%
(
2 steps); (c) iodomethane, K CO , acetone, reflux, 15%.
2
3
used in identifying tubulin polymerization inhibitors. Secondly, the
tumor cell growth-inhibitory activity of the synthesized com-
pounds towards human breast cancer cell line (MCF-7) was evalu-
resulted in a large decrease of activity (1% inhibition). The benzy-
lated compound 18 showed weak activity (4% inhibition).
In the MCF-7 cell proliferation assay, CA-4 (1) showed very po-
tent tumor cell growth-inhibitory activity with an IC50 value of
3
3,34
ated.
In this paper, the tumor cell growth-inhibitory activity is
given as the IC50 value, which is defined as the test chemical con-
centration required to elicit 50% cell growth inhibition.
The results for CA-4 (1), compound 3 and its B-ring-modified
derivatives 7–18 are shown in Table 1. In TPI assay, CA-4 (1)
0.004
strong activity (IC50 = 0.043
tion of the methoxy group of 3, that is compound 8, resulted in loss
of activity (IC50 >30 M), as was the case in TPI assay. Deletion of
the hydroxyl group at the R position, that is compound 12, re-
sulted in a decrease of activity (IC50 = 0.15 M). Compounds 14
and 15 showed moderate activity with the IC50 values of 1.12
and 0.49 M, respectively. The trimethoxy compound 16 was inac-
tive (IC50 >30 M). Inversion of the methoxy and hydroxyl groups
of 3, that is compound 17, resulted in loss of tumor cell growth-
inhibitory activity (IC50 >30 M). Broadly speaking, these results
show that tubulin polymerization-inhibitory activity and tumor
cell growth-inhibitory activity were well correlated among the
compounds evaluated in our experiments.
The A-ring modified derivatives 21–24 were next evaluated
(Table 2). Transfer of the methoxy group from R to R in the A-ring
4 7
l
M. The silicon-containing compound 3 also exhibited
lM), but was less potent than 1. Dele-
l
showed 66% inhibition at 30
l
M. As expected, the designed sili-
1
con-containing compound 3 exhibited similar inhibition (62%) at
the same concentration. In other words, compound 3 showed
activity comparable to that of CA-4 (1). The benzylated compound,
that is compound 7, was inactive. Deletion of the methoxy group at
l
l
l
the R
loss of activity. Compounds 9 and 10, which lack the methoxy
group at the R position, were also inactive. The methoxy group
at this position seems to be essential for tubulin polymerization-
inhibitory activity. Deletion of the hydroxyl group at the R posi-
tion, that is compound 12, resulted in a decrease of activity (30%
inhibition at 30 M). Compounds 11, 13 and 15, which lack the hy-
droxyl group at the R position, showed weak activities (2–11%
inhibition). The methylenedioxy derivative 14 also showed weak
activity (14% inhibition). Thus, modifications of the R position
diminished the activity. Addition of a methoxy group at the R
2
position of the B-ring of 3, that is compound 8, resulted in
l
2
1
l
1
of 3, that is compound 21, resulted in a decrease of tubulin poly-
merization-inhibitory activity and cell growth-inhibitory activity
1
(25% inhibition at 30
dimethoxy compound 22 showed weak activities (5% inhibition
at 30 M and IC50 = 5.52 M, respectively). The combrestatin A-2
(CA-2) type compound 23 exhibited moderate tubulin polymeriza-
lM and IC50 = 0.74 lM, respectively). The
3
position, that is compound 16, resulted in loss of activity. Inversion
of the methoxy and hydroxyl groups of 3, that is compound 17,
l
l

7
384
M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
R
R'
Si
OBn
OMe
O
a
MeO
MeO
c
OH
MeO
MeO
Si
R
Cl
R'
Cl
5
+ 6a +
Si
26b
OMe
OMe
d
OMe
OMe
2
2
5a (R = Et, R' = Et)
5b (R, R' = -CH
26a (R = Et, R' = Et)
26b (R, R' = -CH CH CH -)
28
2
CH
2
CH
2
-)
2
2
2
OH
MeO
MeO
Si
b
Si
OH
OMe
MeO
MeO
2
6a
OMe
2
9
OMe
2
7
H
H
Br
OMOM
OMe
OMOM
OMe
Si
OH
OMe
H
Cl
e
MeO
MeO
Si
MeO
MeO
f
Si
Cl
5
+
+
6
g
25c
OMe
OMe
3
1
3
0
O
O
Cl
H
OH
2 steps
MeO
MeO
O
g
MeO
MeO
OH
h
MeO
MeO
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
isoerianin(35)
3
2
33
34
Scheme 3. Conversion of substituent groups on silicon. Synthesis of compounds 27–29, 31 and isoerianin (35). Reagents and conditions: (a) n-BuLi, THF, ꢀ70 °C to rt, 10–33%;
b) H , Pd/C, ethanol, rt, 94%; (c) H , Pd/C, ethanol/ethyl acetate, rt, 30%; (d) H
(
2
2
2
, Pd/C, ethyl acetate, rt, 30%; (e) n-BuLi, THF, ꢀ70 °C to rt; (f) concd HCl, methanol, rt. 18% (2
steps); (g) trimethylaluminium in toluene, reflux, 11%; (h) H
2
, Pd/C, ethanol, rt, 83%.
Table 1
Tubulin polymerization-inhibitory activity and tumor cell growth-inhibitory activity of compound 3 and its B-ring modified derivatives 7–18
MeO
MeO
Si
R1
R
2
OMe
R
3
R
1
R
2
R
3
TPI assaya
Inhibition (%) at 30 lM
MCF-7 assayb IC50
0.004
(lM)
CA-4 (1)
—
—
—
66
3
7
8
9
OH
OBn
OH
OMe
OBn
H
OMe
OMe
H
H
H
OH
OMe
OBn
H
H
H
H
H
H
H
H
H
H
62
NA
NA
NA
NA
2
30
4
14
11
0.043
>30
>30
(52% at 30
(57% at 30
>30
0.15
24.9
1.12
0.49
c
c
c
l
l
M)
M)
c
10
11
12
13
14
15
16
17
18
H
H
–OCH
OMe
OMe
OMe
OMe
2
O–
OMe
OMe
OH
c
OMe
H
H
NA
1
4
>30
>30
25.3
OBn
a
b
c
Tubulin polymerization-inhibitory activity.
Tumor cell growth-inhibitory activity against human breast cancer cell line MCF-7. Assays were performed in triplicate.
No activity was observed in the concentration range examined.
tion-inhibitory activity (27% inhibition), but showed weak growth-
The results of the modification of substituent groups on silicon
inhibitory activity. The symmetric compound 24 also showed weak
in 3 are shown in Table 3. In TPI assay, compound 3 (R = Me,
0
activities (12% inhibition at 30
tively). The results of A-ring modification indicate that the
,4,5-trimethoxy group of 3 is important for these activities.
lM and IC50 = 9.24
lM, respec-
R = Me) exhibited strong activity (62% inhibition at 30
l
M). On
0
the other hand, compounds 27 (R = Et, R = Et) and 28 (R = i-Pr,
0
3
R = OEt) showed weaker activity (16% and 5% inhibition,

M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
7385
Table 2
Tubulin polymerization-inhibitory activity and tumor cell growth-inhibitory activity of the A-ring modified derivatives 21–24
R4
Si
OH
R
5
R7
OMe
R6
R
4
R
5
R
6
R
7
TPI assaya
Inhibition (%) at 30 lM
MCF-7 assayb IC50
0.004
(lM)
CA-4 (1)
—
—
—
—
66
3
OMe
H
H
–OCH
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
H
OMe
H
H
H
62
25
5
27
12
0.043
0.74
5.52
(11% at 1
9.24
21
22
23
24
2
O–
lM)
OMe
a
Tubulin polymerization-inhibitory activity.
Tumor cell growth-inhibitory activity against human breast cancer cell line MCF-7. Assays were performed in triplicate.
b
Table 3
Effects of modification of substituent groups on silicon in 3. Tubulin polymerization-inhibitory activity and tumor cell growth-inhibitory activity of compounds 27–29, 31 and 35.
R
R'
MeO
MeO
X
OH
OMe
OMe
0
TPI assaya
Inhibition (%) at 30 lM
MCF-7 assayb IC50
0.004
X
R
R
(lM)
CA-4 (1)
—
—
—
66
3
Si
Si
Si
Si
Si
C
Me
Et
Pr
–CH
Me
Me
Me
Et
OEt
62
16
5
87
51
54
0.043
1.71
0.58
0.062
0.007
0.075
27
28
29
31
35
c
2
CH
2
2
CH –
c
c
H
H
a
b
c
Tubulin polymerization-inhibitory activity.
Tumor cell growth-inhibitory activity towards human breast cancer cell line MCF-7. Assays were performed in triplicate.
Racemic mixture.
respectively) than compound 3. From these results, we
considered that bulky substituent groups on silicon reduced the
activity owing to steric hindrance. Therefore, we designed and
evaluated the silacyclobutane compound 29 and the methylsi-
lane compound 31. In addition, the corresponding carbon
compound 35, generically known as isoerianin, was synthesized
for comparison with 31. As expected, compound 29 exhibited
potent activity (87% inhibition). The tubulin polymerization-
inhibitory activities of compounds 31 and 35 were retained
MCF-7 : Me, H >> Me, Me > -CH
2
CH
2
CH
2
- >> Pr, EtO > Et, Et
TPI : -CH
2 2
CH CH
2
- > Me, Me > Me, H >> Et, Et > Pr, EtO
Removal
DIMINISHES activity
R
R'
Si
OH
MeO
MeO
Inversion
INACTIVE
OMe
OMe
(
51% and 54% inhibition). In the MCF-7 cell proliferation assay,
compounds 27 and 28 showed weaker activity than compound
(IC50 = 1.71 and 0.58 M, respectively), as in the case of TPI as-
OMe (alkoxy group)
REQUIRED
3
l
3,4,5-trimethoxy
IMPORTANT for activity
say. Although the silacyclobutane compound 29 showed the
strongest activity in TPI screening assay, the cell growth-inhibi-
tory activity of 29 was less than that of 3 (compound 29:
Figure 4. Structure–activity relationships (SARs) of the synthesized compounds.
IC50 = 0.062
tent cell growth-inhibitory activity with an IC50 value of
.007 M. Interestingly, the activity of the corresponding carbon
compound 35 was 10 times weaker than that of the silicon
compound 31. This result lends support to our hypothesis that
the silicon linker can act as an alternative structure to a cis
carbon–carbon double bond. The resulting structure–activity
relationships (SARs) are summarized in Figure 4. The SARs of
silicon-containing compounds were similar to those of parent
olefin-based CA-4 derivatives.12
lM). The methylsilane compound 31 exhibited po-
2.4. Colchicine site competitive binding assay
0
l
3
We performed competitive binding assay using [ H]colchicin-
ein order to identify the binding site. The results of binding
3
5
assay and the IC50 values in TPI assay of selected compounds
are shown in Table 4. In the colchicine-binding studies, all of
the tested compounds inhibited colchicine-binding competi-
3
tively. CA-4 (1) effectively inhibited the binding of [ H]colchicine
to tubulin (93.6% inhibition at 3 lM). The silicon-containing

7
386
M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
Table 4
Results of colchicine site competitive binding assay and IC50 in TPI assay
MeO
MeO
Y
OH
OMe
OMe
TPI assaya
Inhibition of [ H]colchicine bindingb (%)
3
Y
IC50 (lM)
3
l
M
10
—
l
M
30
—
lM
CA4 (1)
4.5
93.6
46.6
3
16.0
79.8
99.0
90.8
98.3
2
9
11.8
88.1
3
3
1c
5c
28.0
21.5
90.7
30.9
98.9
73.5
99.6
83.6
a
b
c
Tubulin polymerization-inhibitory activity.
Assays were performed in triplicate.
Racemic mixture.
Table 5
Stability test of CA-4 (1) (remaining amount of 1)
Time (h)
0
4
6.5
24
CA-4 (1) (%)
Product 1 (%)
Product 2 (%)
100.0
0.0
0.0
90.0
7.9
2.1
87.9
9.6
2.5
84.5
11.3
4.2
synthesized compounds 3, 31 and 35. The tested samples were
dissolved in a solution of PBS (pH 7.4):acetonitrile = 1:1, and kept
under room lighting at room temperature. Stability was esti-
mated by means of HPLC analysis. As shown in Figure 5, two
products appeared in the solution of CA-4 (1) within 4 hours. It
is presumed that one of them was the trans-form of 1. After
2
4 hours, the concentration of CA-4 (1) was decreased to 84.5%
of the initial value (Table 5). On the other hand, 3, 31 and 35 were
completely stable under the same conditions (see Supplementary
data).
Figure 5. Stability test of CA-4 (1) (HPLC charts).
3
. Conclusion
compounds 3 and 29 showed 46.6% and 88.1% inhibition at 3
l
M,
respectively. These results were roughly correlated with the IC50
Based on computational studies, we designed and synthesized
values in TPI assay. The methylsilane compound 31, which
a series of silicon-containing combretastatin analogs with the aim
of increasing the stability and tumor cell growth-inhibitory activ-
ity, since it is known that CA-4 (1) isomerizes to the inactive
trans-form during storage and administration. In the tubulin
polymerization inhibition (TPI) screening assay, several of the
synthesized compounds showed comparable activity to CA-4
(1). Among them, the methylsilane compound 31 exhibited very
potent tumor cell growth-inhibitory activity with an IC50 value
showed the strongest cell growth-inhibitory activity, potently
3
inhibited [ H]colchicine binding (90.7% inhibition at 3
l
M). The
binding-inhibitory activity of the corresponding carbon com-
pound 35 was weaker than that of the silicon compound 31, as
in the case of cell growth inhibition assay (30.9% inhibition at
3 lM). For confirmation, we calculated the distance between
the two benzene ring (d) in compounds 31 and 35. The distances
were 3.04 Å and 2.49 Å, respectively. The distance in the silicon
compound 35 was similar to that of the cis double bond of CA-4
of 0.007 lM (MCF-7), being similar in potency to 1. The activity
of the corresponding carbon compound 35 was 10 times weaker
than that of the silicon compound 31. In the colchicine competi-
tive binding study, the silicon compound 31 also showed stronger
activity than the carbon compound 35. These results lend support
to our hypothesis that the silicon linker can act as an alternative
structure to a cis carbon–carbon double bond. In addition, CA-4
(
1). These results also lend support to our hypothesis that the sil-
icon linker can act as an alternative structure to a cis carbon–car-
bon double bond.
2
.5. Solution stability of CA-4 and the synthesized compounds
(
1) was decomposed in polar solution, whereas 31 was stable un-
It has been reported that CA-4 (1), which has a cis double bond,
der the same conditions. These results suggest that substitution
isomerizes to the more stable trans-form during storage and
administration, resulting in a decrease of antitumor activity.
Therefore, we tested the solution stability of CA-4 (1) and the
of a cis carbon–carbon double bond with a silicon linker would
1
0,11
be
a useful approach for structure optimization of these
inhibitors.

M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
7387
4
4
4
. Experimental
.1. Chemistry
.1.1. General
150.91, 147.76, 139.16, 137.30, 133.48, 129.39, 128.56, 128.02,
27.90, 127.49, 119.91, 111.64, 110.86, 71.30, 60.86, 56.23, 55.89,
ꢀ1.96. HRMS(FAB+) m/z: calcd for C25 Si 438.1863, found
1
30 5
H O
+
4
38.1871 (M ).
Compound 16 (218.1 mg, yield 27.8%), white solid. Mp:
1
1
H NMR spectra were recorded on JEOL JNM-GX500 and JNME-
39–41 °C, H NMR (500 MHz, CDCl
3
): d 6.72 (4H, s), 3.87 (6H, s),
3.85 (12H, s), 0.54 (6H, s). C NMR (500 MHz, CDCl ): d 153.12,
139.32, 133.09, 110.99, 60.88, 56.30, ꢀ1.86. HRMS(FAB+) m/z:
CA-500 (500 MHz) spectrometers. Chemical shifts for 1H NMR are
reported in parts per million (ppm) downfield from tetramethylsil-
ane (d) and coupling constants are in hertz (Hz). The following
abbreviations are used for spin multiplicity: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, br = broad. Chemical shifts for
13
3
+
29 6
calcd for C20H O Si 393.1733, found 393.1732 (M+H ).
4.1.4. 3-(Dimethyl(3,4,5-trimethoxyphenyl)silyl)phenol (8)
1
3
C NMR are reported in ppm relative to the centerline of the triplet
The title compound was prepared according to the procedure
at 77.0 ppm for deuteriochloroform. Mass spectra were recorded on
a JEOL JMS-HX110 spectrometer. Melting points were determined
on a MP-J3 melting point apparatus (Yanaco, Japan). Routine thin
layer chromatography(TLC) and preparative TLC (PTLC) were per-
formed on silica gel 60 F254 plates (Merck, Germany). Flash column
chromatography was performed on Silica gel 60 (spherical, particle
described for 3, starting from compound 10. Yield: 87.9%. White
1
solid. Mp: 104–106 °C, H NMR (500 MHz, CDCl
3
): d 7.27–7.23
(1H, m), 7.08 (1H, d, J = 7.0 Hz), 6.95 (1H, d, J = 3.0 Hz), 6.85–6.83
(1H, m), 6.70 (2H, s), 4.81 (1H, br s), 3.86 (3H, s), 3.84 (6H, s),
1
3
0.53 (6H, s). C NMR (500 MHz, CDCl
3
): d 155.09, 153.09, 140.33,
139.26, 133.01, 129.38, 126.55, 120.71, 116.25, 110.98, 60.87,
size 40–100
l
m; Kanto).The stability of tested compounds was
56.26, ꢀ2.18. HRMS(FAB+) m/z: calcd for C17
22 4
H O Si 318.1287,
+
determined by high-performance liquid chromatography (HPLC
found 318.1291 (M ).
(
[
MD-2010plus, PU-2080, SSC-2120, MX-2080-32, Jasco, Japan))
Inertsil ODS-4 column (5
m, 4.6 ꢁ 150 mm, GL Science), 40–90%
with 0.1% phosphoric acid for 25 min,
.0 mL/min, 30 °C, UV detection at 254 nm].
l
4.1.5. (3-Methoxyphenyl)dimethyl(3,4,5-
trimethoxyphenyl)silane (9)
acetonitrile in
1
2
H O
Methyl iodide (16
lL, 0.25 mmol) was added to a mixture of
3
0
8
-(dimethyl(3,4,5-trimethoxyphenyl)silyl)phenol
.19 mmol), sodium hydride (60% suspension in mineral oil,
.4 mg, 0.21 mmol) and N,N-dimethylformamide (1 mL) at 0 °C.
(8) (61 mg,
4
.1.2. 5-(Dimethyl(3,4,5-trimethoxyphenyl)silyl)-2-
methoxyphenol (3)
The mixture was stirred at room temperature for 2.5 h. The reac-
tion was quenched with H O, and the whole was extracted with
ethyl acetate. The extract was successively washed with water
and brine, and then dried over MgSO . The solvent was evaporated,
To a solution of (3,4-dimethoxyphenyl)dimethyl(3,4,5-trime-
thoxyphenyl)silane (7) (144.6 mg, 0.33 mmol) in ethanol (4 mL)
was added 5% Pd/C (14.5 mg) at room temperature, and the mix-
2
4
2
ture was stirred under an H atmosphere at the same temperature
and the residue was purified by means of PTLC (hexane/ethyl ace-
for 12 h. The reaction mixture was filtered through a Celite pad and
the solvent was evaporated. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 4/1) to give 3
(
7
tate = 5/1) to give 9 (49.1 mg, 66.4%) as a colorless oil. 1H NMR
(
3
500 MHz, CDCl ): d 7.33–7.29 (1H, m), 7.11 (1H, d, J = 6.9 Hz),
94.7 mg, 82.4%) as a colorless oil. 1H NMR (500 MHz, CDCl
7.06 (1H, d, J = 2.3 Hz), 6.93–6.90 (1H, m), 6.71 (2H, s), 3.89 (3H,
3
): d
.08 (1H, d, J = 1.1 Hz), 7.00 (1H, dd, J = 8.0, 1.1 Hz), 6.87 (1H, d,
1
3
s), 3.84 (6H, s), 3.80 (3H, s), 0.54 (6H, s). C NMR (500 MHz,
CDCl ): d 159.07, 153.10, 139.90, 139.27, 133.09, 129.17, 126.51,
119.93, 114.29, 110.97, 60.87, 56.26, 55.19, ꢀ2.13. HRMS(FAB+)
3
J = 8.0 Hz), 6.71 (2H, s), 5.57 (1H, s), 3.89 (3H, s), 3.86 (3H, s),
.84 (6H, s), 0.51 (6H, s). 1 C NMR (500 MHz, CDCl
3
3
1
1
C
3
): d 153.05,
+
25 4
m/z: calcd for C18H O Si 333.1522, found 333.1524 (M+H ).
47.67, 145.30, 139.18, 133.51, 130.49, 126.54, 119.89, 110.91,
10.50, 60.86, 56.25, 55.86, ꢀ2.02. HRMS(FAB+) m/z: calcd for
+
4.1.6. (3-(Benzyloxy)phenyl)dimethyl(3,4,5-trimethoxyphenyl)
silane (10)
18
H
25
O
5
Si 349.1471, found 349.1478 (M+H ).
To a solution of 5-bromo-1,2,3-trimethoxyphenol (494.1 mg,
2 mmol) and 1-(benzyloxy)-3-bromobenzene (6b) (526.3 mg,
2 mmol) in dry tetrahydrofuran (15 mL) was added n-butyl lithium
in n-hexane (2.65 M, 1.51 mL, 4 mmol) under an Ar atmosphere at
ꢀ78 °C. This was stirred at the same temperature for 1 h, and then
dichlorodimethylsilane (0.27 mL, 2.2 mmol) was added. The mix-
ture was stirred at ꢀ78 °C for 2 h, and at room temperature for
4
.1.3. (3,4-Dimethoxyphenyl)dimethyl(3,4,5-trimethoxyphenyl)
silane (7) and diethylbis(3,4,5-trimethoxyphenyl)silane (16)
To a solution of 5-bromo-1,2,3-trimethoxyphenol (543.6 mg,
2
.2 mmol) in dry tetrahydrofuran (5 mL) was added n-butyl lith-
ium in n-hexane (2.65 M, 0.83 mL, 2.2 mmol) under an Ar atmo-
sphere at ꢀ78 °C. The mixture was stirred at same temperature
for 1 h, and then added to a solution of dichlorodimethylsilane
3 h. The reaction was quenched with NH
was extracted with ethyl acetate. The extract was successively
washed with water and brine, and then dried over MgSO . The sol-
4
Cl aq, and the whole
(
0.3 mL, 2.0 mmol) in dry tetrahydrofuran (2.5 mL) at ꢀ78 °C. The
mixture was stirred at room temperature for 1.5 h, and cooled to
78 °C. To this was added a solution of (3-(benzyloxy)-4-methoxy-
4
ꢀ
vent was evaporated, and the residue was purified by means of sil-
ica gel column chromatography (hexane/ethyl acetate = 9/1) to
phenyl)lithium (2.0 mmol) in dry tetrahydrofuran at ꢀ78 °C. The
1
mixture was stirred at the same temperature for 2 h, and at room
give 10 (327.5 mg, 40.1%) as a colorless oil. H NMR (500 MHz,
temperature for 3 h. The reaction was quenched with NH
4
Cl aq,
3
CDCl ): d 7.42–7.29 (6H, m), 7.14–7.11 (2H, m), 7.00–6.97 (1H,
and the whole was extracted with ethyl acetate. The extract was
successively washed with water and brine, and then dried over
m), 6.70 (2H, s), 5.04 (2H, s), 3.86 (3H, s), 3.83 (6H, s), 0.53 (6H,
s). C NMR (500 MHz, CDCl ): d 158.35, 153.10, 140.00, 139.27,
3
1
3
4
MgSO . The solvent was evaporated, and the residue was purified
137.08, 133.06, 129.19, 128.68, 128.08, 127.67, 126.74, 120.86,
by means of silica gel column chromatography (hexane/ethyl ace-
115.24, 110.96, 70.00, 60.87, 56.26, ꢀ2.14. HRMS(FAB+) m/z: calcd
+
tate = 19/1 to 4/1) to give 7 and 16. Compound 7 (187.2 mg, yield
for C24
H
28
O
4
Si 408.1757, found 408.1762 (M ).
1
2
7
7
1.3%), white solid. Mp: 57–59 °C, H NMR (500 MHz, CDCl
3
): d
.38 (2H, d, J = 9.0 Hz), 7.35–7.31 (2H, m), 7.29–7.27 (1H, m),
.09 (1H, dd, J = 8.0, 1.1 Hz), 7.00 (1H, d, J = 1.1 Hz), 6.92 (1H, d,
4.1.7. 4-(Dimethyl(3,4,5-trimethoxyphenyl)silyl)phenol (11)
The title compound was prepared according to the procedure
J = 8.0 Hz), 6.65 (2H, s), 5.11 (2H, s), 3.89 (3H, s), 3.87 (3H, s),
described for 3, starting from compound 11. Yield: 32.7%. White
.81 (6H, s), 0.47 (6H, s). 13C NMR (500 MHz, CDCl
1
3
3
): d 153.05,
3
solid. Mp: 97–99 °C. H NMR (500 MHz, CDCl ): d 7.39 (2H, d,

7
388
M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
J = 8.6 Hz), 6.84 (2H, d, J = 8.6 Hz), 6.70 (2H, s), 5.38 (1H, br s), 3.86
115.72, 114.40, 110.84, 60.78, 56.16, 55.88, -1.90. HRMS (FAB+)
m/z: calcd for C18H O Si 348.1393, found 348.1411 (M ).
24 5
3H, s), 3.83 (6H, s), 0.51 (6H, s). 1 C NMR (500 MHz, CDCl
3
): d
56.87, 153.01, 139.03, 135.91, 133.88, 128.90, 115.16, 110.96,
+
(
3
1
6
3
0.89, 56.25, ꢀ1.91. HRMS(FAB+) m/z: calcd for
C
17
H
23
O
4
Si
4.1.13. (4-(Benzyloxy)-3-methoxyphenyl)dimethyl(3,4,5-
trimethoxyphenyl)silane(18)
+
19.1366, found 319.1373 (M+H ).
The title compound was prepared according to the procedure
4
.1.8. (4-Methoxyphenyl)dimethyl(3,4,5-trimethoxyphenyl)
described for 10, starting from compound 6f. Yield: 12.4%. Color-
1
silane (12)
less oil. H NMR (500 MHz, CDCl
3
): d 7.44 (d, J = 7.4 Hz, 2H), 7.36
The title compound was prepared according to the procedure
(td, J = 1.7, 6.6 Hz, 2H), 7.28–7.31 (m, 1H), 7.01–7.03 (m, 2H),
described for 10, starting from 4-bromoanisole. Yield:11.9%. Color-
6.91 (d, J = 8.0 Hz, 1H), 6.69 (s, 2H), 5.17 (s, 2H), 3.87 (s, 3H), 3.86
(s, 3H), 3.83 (s, 6H), 0.52 (s, 6H). C NMR (500 MHz, CDCl ): d
3
1
13
3
less oil. H NMR (500 MHz, CDCl ): d 7.45 (1H, dd, J = 6.6, 2.0 Hz),
6
.91 (1H, dd, J = 6.6, 2.0 Hz), 6.70 (2H, s), 3.86 (3H, s), 3.83 (6H,
152.95, 149.30, 149.11, 137.07, 133.45, 129.82, 128.54, 127.81,
127.42, 127.17, 117.13, 113.43, 110.80, 70.67, 60.77, 56.13, 56.08,
13
s), 3.82 (6H, s), 0.52 (6H, s). C NMR (500 MHz, CDCl
3
): d 160.64,
1
5
3
53.06, 139.16, 135.70, 133.73, 128.93, 113.71, 110.93, 60.84,
30.93, ꢀ1.98. HRMS(FAB+) m/z: calcd for C25
30 5
H O Si 438.1863,
+
6.25, 55.12, ꢀ1.91. HRMS(FAB+) m/z: calcd for
C
18
H
25
O
4
Si
found 438.1867 (M) .
+
33.1522, found 333.1522 (M+H ).
4
.1.14. (3-(Benzyloxy)-4-methoxyphenyl)dimethyl(2,3,4-
4
.1.9. (4-(Benzyloxy)phenyl)dimethyl(3,4,5-trimethoxyphenyl)
trimethoxyphenyl)silane (20a)
silane (13)
The title compound was prepared according to the procedure de-
scribed for 10, starting from 1-(benzyloxy)-4-bromobenzene. Yield:
The title compound was prepared according to the procedure
described for 10, starting from compound 19a. Colorless oil
(crude). This was used in the next step without further purification.
1
3
41.5%. Colorless oil. H NMR (500 MHz, CDCl ): d 7.46–7.24 (1H, m),
6
(
.99 (2H, d, J = 8.6 Hz), 6.70 (2H, s), 5.07 (2H, s), 3.86 (3H, s), 3.83
4.1.15. (3-(Benzyloxy)-4-methoxyphenyl)(7-methoxybenzo[d]
[1,3]dioxol-5-yl)-dimethylsilane (20b)
6H, s), 0.52 (6H, s). 1 C NMR (500 MHz, CDCl
3
3
): d 159.87, 153.07,
1
37.02, 135.74, 133.70, 129.30, 128.70, 128.09, 127.58, 114.60,
The title compound was prepared according to the procedure
described for 10, starting from compound 19b. Colorless oil
(crude). This was used in the next step without further purification.
110.92, 105.30, 69.85, 60.87, 56.25, 56.17, ꢀ 1.90. HRMS(FAB+) m/
+
z: calcd for C24
H O
28 4
Si 408.1757, found 408.1765 (M ).
4
.1.16. Bis(3-(benzyloxy)-4-methoxyphenyl)dimethylsilane (20c)
The title compound was prepared according to the procedure
4
.1.10. Benzo[d][1,3]dioxol-5-yldimethyl(3,4,5-
trimethoxyphenyl)silane (14)
The title compound was prepared according to the procedure
described for 10, starting from 5-bromobenzo[d][1,3]dioxole.
Yield: 35.6%. White solid. Mp: 75–76 °C. H NMR (500 MHz,
CDCl
described for 10, starting from compound 6a. Yield: 38.5%. White
solid. Mp: 82–84 °C. H NMR (500 MHz, CDCl
J = 6.9 Hz, 4H), 7.33 (t, J = 7.4 Hz, 4H), 7.28 (d, J = 7.4 Hz, 2H), 7.01
1
3
): d 7.39 (d,
1
(
dd, J = 1.1, 8.0 Hz, 2H), 6.95 (d, J = 1.7 Hz, 2H), 6.88 (d, J = 7.4 Hz,
3
): d 7.00 (2H, dd, J = 7.6, 1.1 Hz), 6.96 (1H, d, J = 1.1 Hz),
1
3
2
H), 5.08 (s, 4H), 3.89 (s, 6H), 0.39 (s, 6H). C NMR (500 MHz,
6
.85 (1H, d, J = 7.6 Hz), 5.94 (2H, s), 3.86 (3H, s), 3.84 (6H, s), 0.51
1
3
3
CDCl ):d 150.65, 147.59, 137.24, 129.63, 128.45, 127.81, 127.74,
(
6H, s). C NMR (500 MHz, CDCl
3
): d 153.10, 148.64, 147.50,
1
27.45, 119.69, 111.48, 71.10, 55.79, ꢀ2.13. HRMS (FAB+) m/z:
1
6
3
39.27, 133.33, 131.18, 128.31, 113.52, 110.91, 108.71, 100.67,
+
32 4
calcd for C30H O Si 484.2070, found 484.2034 (M ).
0.87, 56.27, ꢀ1.92. HRMS(FAB+) m/z: calcd for
C
18
H
23
O
5
Si
+
47.1315, found 333.1524 (M+H ).
4
.1.17. 5-(Dimethyl(2,3,4-trimethoxyphenyl)silyl)-2-methoxyphenol
21)
The title compound was prepared according to the procedure
described for 7, starting from compound 20a. Yield: 4.8% (2 steps).
(
4
.1.11. (3,4-Dimethoxyphenyl)dimethyl(3,4,5-trimethoxyphenyl)
silane (15)
Methyl iodide (21
methyl(3,4,5-trimethoxyphenyl)silyl)-2-methoxyphenol
l
L, excess) was added to a mixture of 5-(di-
1
Colorless oil. H NMR (500 MHz, CDCl
3
): d 7.09 (d, J = 1.1 Hz, 1H),
.02 (dd, J = 1.7, 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.86 (s, 1H),
.63 (s, 1H), 5.54 (s, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H),
.69 (s, 3H), 0.51 (s, 6H). C NMR (500 MHz, CDCl ): d 158.42,
3
55.39, 147.15, 144.98, 141.37, 131.59, 130.23, 126.35, 123.15,
(3)
7
6
3
1
1
(
30 mg, 0.086 mmol), potassium carbonate (11.9 mg, 0.172 mmol)
and acetone (2 mL) at room temperature. The mixture was stirred
under reflux for 11 h and at room temperature for 12 h. The reac-
tion mixture was filtered and the solvent was evaporated. The res-
13
19.87, 110.23, 107.13, 60.47, 60.36, 55.89, 55.71, -1.78. HRMS
idue was purified by PTLC (hexane/ethyl acetate = 4/1) to give 15
+
(
FAB+) m/z: calcd for C18
H
24
O
5
Si 348.1393, found 348.1411 (M ).
1
(
(
79.1 mg, 40.8%) as
500 MHz, CDCl
a
white solid. Mp: 73–74 °C.
H NMR
3
): d 7.10 (1H, dd, J = 8.0, 1.1 Hz), 7.00 (1H, d,
4
.1.18. 5-((3,4-Dimethoxyphenyl)dimethylsilyl)-2-methoxyphenol
J = 1.1 Hz), 6.91 (1H, d, J = 8.0 Hz), 6.71 (2H, s), 3.89 (3H, s), 3.86
6H, s), 3.84 (6H, s), 0.53 (6H, s). 13C NMR (500 MHz, CDCl
): d
53.08, 150.19, 148.66, 139.20, 133.58, 129.35, 127.60, 116.51,
11.08, 110.94, 60.87, 56.26, 55.98, 55.80, ꢀ1.83. HRMS(FAB+) m/
(
22)
The title compound was prepared according to the procedure
described for 15, starting from compound 24. Yield: 15.0%. Color-
(
1
1
3
1
less oil. H NMR (500 MHz, CDCl
3
): d 7.07–7.09 (m, 2H), 6.99–7.00
+
26 5
z: calcd for C19H O Si 362.1550, found 362.1540 (M ).
(
m, 2H), 6.85–6.89 (m, 2H), 5.56 (s, 1H), 3.89 (s, 3H), 3.88 (s, 3H),
1
3
3
1
1
3
.86 (s, 3H), 0.50 (s, 6H). C NMR (500 MHz, CDCl ): d 149.95,
48.51, 147.37, 145.15, 130.80, 129.68, 127.40, 126.41, 119.80,
16.39, 110.93, 110.37, 55.83, 55.73, 55.67, ꢀ2.08. HRMS (FAB+)
4
.1.12. 4-(Dimethyl(3,4,5-trimethoxyphenyl)silyl)-2-methoxyphenol
17)
The title compound was prepared according to the procedure de-
scribed for 7, starting from compound 18. Yield: 74.1%. White solid.
(
+
m/z: calcd for C17
H
22
O
4
Si 318.1287, found 318.1288 (M ).
1
Mp: 123–125 °C. H NMR (500 MHz, CDCl
3
): d 7.06 (dd, J = 1.4,
.7 Hz, 1H), 6.94–6.96 (m, 2H), 6.70 (s, 2H), 5.73 (s, 1H), 3.87 (s,
H), 3.86 (s, 3H), 3.84 (s, 6H), 0.52 (s, 6H). 13C NMR (500 MHz,
): d 152.97, 146.83, 146.33, 139.09, 133.56, 128.69, 127.96,
4
.1.19. 2-Methoxy-5-((7-methoxybenzo[d][1,3]dioxol-5-yl)
7
3
CDCl
dimethylsilyl)phenol (23)
The title compound was prepared according to the procedure
described for 3, starting from compound 20b. Yield: 8.4% (2 steps).
3

M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
7389
Colorless oil. 1H NMR (500 MHz, CDCl
): d 7.06 (d, J = 1.7 Hz, 1H),
.99 (dd, J = 1.7, 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.66 (s, 1H),
.65 (s, 1H), 5.95 (s, 2H), 5.56 (s, 1H), 3.890 (s, 3H), 3.886 (s, 3H),
raphy (hexane/ethyl acetate = 4/1) and PTLC (hexane/ethyl ace-
3
6
6
0
1
1
C
tate = 4/1) to give 28 (21.8 mg, 30.3%) as a colorless oil. 1H NMR
3
(500 MHz, CDCl ): d 7.14 (1H, d, J = 1.1 Hz), 7.08 (1H, dd, J = 8.0,
1.1 Hz), 6.89 (1H, d, J = 8.0 Hz), 6.78 (2H, s), 5.60 (2H, s), 3.90
(3H, s), 3.87 (3H, s), 3.84 (6H, s), 3.75 (2H, q, J = 7.0 Hz),
1
3
.48 (s, 6H). C NMR (500 MHz, CDCl
3
):d 148.78, 147.46, 145.18,
43.72, 136.19, 132.21, 130.36, 126.39, 119.76, 113.48, 110.39,
07.44, 101.13, 56.66, 55.74, -2.11. HRMS (FAB+) m/z: calcd for
1.48–1.43 (2H, m), 1.21 (3H, t, J = 7.0 Hz), 1.11–1.07 (2H, m), 0.98
+
13
17
H
20
O
5
Si 332.1080, found 332.1076 (M ) .
3
(3H, t, J = 7.4 Hz). C NMR (500 MHz, CDCl ): d 153.04, 148.04,
1
45.29, 139.58, 130.70, 127.58, 127.33, 120.35, 111.31, 110.43,
0
4
.1.20. 5,5 -(Dimethylsilanediyl)bis(2-methoxyphenol) (24)
60.86, 59.28, 56.24, 55.82, 18.54, 18.34, 16.85, 16.74. HRMS(FAB+)
m/z: calcd for C21H O Si 407.890, found 407.1895 (M+H ).
31 6
+
The title compound was prepared according to the procedure de-
scribed for 3, starting from compound 20c. Yield: 85.8%. White solid.
1
Mp: 130–133 °C. H NMR (500 MHz, CDCl
3
): d 7.06 (d, J = 1.7 Hz,
H), 6.99 (dd, J = 1.7, 8.0 Hz, 2H), 6.85 (d, J = 7.4 Hz, 2H), 5.54 (s,
H), 3.88 (s, 6H), 0.48 (s, 6H). 13C NMR (500 MHz, CDCl
): d
47.36, 145.12, 130.74, 126.42, 119.82, 110.37, 55.75, -2.21. HRMS
4.1.25. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)siletan-1-yl)
phenol (29)
To a mixture of 1-(3-(benzyloxy)-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)siletane (26b) (201.9 mg, 0.45 mmol) and ethyl
acetate (5 mL) was added 5% Pd/C (20 mg) at room temperature,
2
2
1
3
+
(
FAB+) m/z: calcd for C16
20 4
H O Si 304.1131, found 304.1155 (M) .
2
and the whole was stirred under an H atmosphere at the same
4
.1.21. (3-(Benzyloxy)-4-methoxyphenyl)diethyl(3,4,5-
temperature for 13 h. The reaction mixture was filtered through
a Celite pad and the solvent was evaporated. The residue was puri-
fied by silica gel column chromatography (hexane/ethyl ace-
trimethoxyphenyl)silane (26a)
The title compound was prepared according to the procedure de-
scribed for 7, starting from dichlorodiethylsilane. Yield: 32.6%,
White solid. Mp: 61–62 °C. H NMR (500 MHz, CDCl
tate = 9/1 to 4/1) to give 29 (48.5 mg, 30.0%) as a colorless oil. 1
H
1
3
): d 7.38 (2H,
3
NMR (500 MHz, CDCl ): d 7.19 (1H, d, J = 1.1 Hz), 7.11 (1H, dd,
d, J = 8.6 Hz), 7.33–7.30 (2H, m), 7.27–7.25 (1H, m), 7.07 (1H, dd,
J = 8.0, 1.1 Hz), 6.98 (1H, d, J = 1.1 Hz), 6.92 (1H, d, J = 8.0 Hz), 6.63
J = 8.0, 1.1 Hz), 6.92 (1H, d, J = 8.0 Hz), 6.82 (2H, s), 5.66 (1H, br
s), 3.91 (3H, s), 3.89 (3H, s), 3.86 (6H, s), 2.26–2.22 (2H, m), 1.47
1
3
(
2H, s), 5.11 (2H, s), 3.90 (3H, s), 3.88 (3H, s), 3.80 (6H, s), 0.99–
3
(4H, t, J = 8.3 Hz). C NMR (500 MHz, CDCl ): d 153.23, 148.08,
13
0
1
1
3
.94 (10H, m). C NMR (500 MHz, CDCl ): d 152.97, 150.83,
145.49, 139.58, 131.60, 128.60, 127.11, 120.20, 111.15, 110.63,
47.61, 139.12, 137.34, 131.43, 128.77, 128.56, 127.86, 127.40,
27.21, 120.60, 111.54, 71.24, 60.87, 56.22, 58.84, 7.54, 4.31.
60.90, 56.29, 55.89, 18.25, 14.34. HRMS(FAB+) m/z: calcd for C19-
+
H
24
O
5
Si 360.1393, found 360.1394 (M ).
HRMS(FAB+) m/z: calcd for
66.2184 (M ).
27 34 5
C H O Si 466.2176, found
+
4
4.1.26. (4-Methoxy-3-(methoxymethoxy)phenyl)(methyl)(3,4,5-
trimethoxyphenyl)-silane (30)
4
.1.22. 1-(3-(Benzyloxy)-4-methoxyphenyl)-1-(3,4,5-
To a solution of 4-bromo-2-(methoxymethoxy)phenol (6b)
(494.18 mg, 2 mmol) and 5-bromo-1,2,3-trimethoxyphenol
(494.1 mg, 2 mmol) in dry tetrahydrofuran (15 mL) was added n-
butyl lithium in n-hexane (1.76 M, 2.5 mL, 4 mmol) under an Ar
atmosphere at ꢀ78 °C. This was stirred at same temperature for
1.5 h, and then methyldichlorosilane (0.23 mL, 2.2 mmol) was
added. Stirring was continued at ꢀ78 °C for 2 h and at room tem-
trimethoxyphenyl)siletane (26b)
The title compound was prepared according to the procedure
described for 7, starting from 1,1-dichlorosilacyclobutane. Yield:
1
1
0.1%, White solid. Mp: 95–97 °C. H NMR (500 MHz, CDCl
3
): d
7
.39–7.37 (2H, m), 7.35–7.32 (2H, m), 7.29–7.25 (1H, m), 7.20
(
1H, dd, J = 8.0, 1.1 Hz), 7.10 (1H, d, J = 1.1 Hz), 7.96 (1H, d,
J = 8.0 Hz), 6.77 (2H, s), 5.13 (2H, s), 3.91 (3H, s), 3.90 (3H, s),
4
perature for 13.5 h. The reaction was quenched with NH Cl aq,
1
3
3
.83 (6H, s), 2.22–2.19 (2H, m), 1.45–1.40 (4H, m). C NMR
and the whole was extracted with ethyl acetate. The extract was
successively washed with water and brine, and then dried over
(
500 MHz, CDCl ): d 153.22, 151.34, 147.92, 129.58, 137.20,
3
1
7
31.50, 128.59, 127.93, 127.56, 127.47, 119.89, 111.71, 111.10,
4
MgSO . The solvent was evaporated, and the residue was purified
1.24, 60.91, 56.27, 55.93, 18.24, 14.42. HRMS(FAB+) m/z: calcd
by means of silica gel column chromatography (hexane/ethyl ace-
tate = 9/1 to 4/1) to give crude (30) (426.7 mg) as a colorless oil,
which was used in the next step without further purification.
+
for C26
H
30
O
5
Si 450.1863, found 450.1863 (M ).
4
.1.23. 5-(Diethyl(3,4,5-trimethoxyphenyl)silyl)-2-methoxyphenol
27)
The title compound was prepared according to the procedure
described for 3, starting from compound 26a. Yield: 94.0%. White
(
4.1.27. 2-Methoxy-5-(methyl(3,4,5-trimethoxyphenyl)silyl)
phenol (31)
To
a
solution of crude (4-methoxy-3-(methoxyme-
(30)
1
3
solid. Mp: 62–63 °C. H NMR (500 MHz, CDCl ): d 7.07 (1H, d,
thoxy)phenyl)(methyl)(3,4,5-trimethoxyphenyl)silane
J = 1.1 Hz), 6.99 (1H, dd, J = 8.0, 1.1 Hz), 6.87 (1H, d, J = 8.0 Hz),
(31 mg) in methanol (1 mL) was added one drop of concd HCl aq
at room temperature. The mixture was stirred at room tempera-
ture for 1 h and then evaporated, and the residue was purified by
6
1
1
6
.69 (2H, s), 5.57 (2H, s), 3.90 (3H, s), 3.87 (3H, s), 3.83 (6H, s),
.05–0.99 (10H, m). 1 C NMR (500 MHz, CDCl
45.25, 139.14, 131.48, 128.34, 127.34, 120.54, 111.60, 110.43,
0.87, 56.24, 55.80, 7.56, 4.23. HRMS(FAB+) m/z: calcd for C20
3
3
): d 152.98, 147.53,
PTLC (hexane/ethyl acetate = 3/1) to give 31 (8.8 mg, 18.1% (2
1
H
29-
steps)) as a colorless oil. H NMR (500 MHz, CDCl
3
): d 7.10 (1H,
+
O
5
Si 377.1784, found 377.1777 (M+H ).
d, J = 1.1 Hz), 7.05 (1H, dd, J = 8.0, 1.1 Hz), 6.89 (1H, d, J = 8.0 Hz),
.75 (2H, s), 5.60 (1H, s), 4.87 (1H, q, J = 3.6 Hz), 3.90 (3H, s), 3.86
6
1
3
4
.1.24. 5-(Ethoxy(propyl)(3,4,5-trimethoxyphenyl)silyl)-2-meth
(3H, s), 3.85 (3H, s), 0.59 (3H, d, J = 3.6 Hz). C NMR (500 MHz,
CDCl ): d 153.21, 147.94, 145.45, 139.45, 130.62, 127.42, 127.32,
120.36, 110.64, 110.41, 60.89, 56.25, 55.88, ꢀ4.51. HRMS(FAB+)
oxyphenol (28)
3
To a mixture of 1-(3-(benzyloxy)-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)siletane (26b) (79.7 mg, 0.18 mmol), ethanol
+
23 5
m/z: calcd for C17H O Si 335.1315, found 335.1306 (M+H ).
(
5 mL) and ethyl acetate (1 mL) was added 5% Pd/C (14.5 mg) at
room temperature, and the whole was stirred under an H atmo-
2
4.1.28. 2-Methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl 2-chloro-
acetate (33)
sphere at the same temperature for 14 h. The reaction mixture
was filtered through a Celite pad and the solvent was evaporated.
The residue was purified by means of silica gel column chromatog-
The title compound was prepared according to the reported
2
9
method.

7
390
M. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 7381–7391
1
Yield: 71.7%. White solid. Mp: 149–151 °C (lit. 149–151 °C). H
2.0 mg/ml. Tubulin polymerization was followed by means of tur-
NMR (500 MHz, CDCl
3
): d 7.79 (1H, dd, J = 8.6, 2.3 Hz), 7.62 (1H, d,
bidity measurements (absorbance at 400 nm) at 37 °C in microtu-
J = 2.3 Hz), 7.07 (1H, d, J = 8.6 Hz), 7.03 (2H, s), 4.36 (2H, s), 3.94
bule assemble buffer containing 100 mM MES, 1 mM EGTA,
0.5 mM MgCl , 1 mM 2-mercaptoethanol, and 1 mM GTP (pH 6.5).
2
13
(
3H, s), 3.93 (3H, s), 3.89 (6H, s). C NMR (500 MHz, CDCl
3
): d
1
1
4
93.67, 165.49, 154.53, 153.00, 141.88, 138.82, 132.75, 130.47,
30.33, 124.93, 111.79, 107.49, 61.07, 56.42, 56.31,
4.4. MCF-7 cell proliferation assay
+
0.61.MS(FAB+) m/z: 395 (M+H ).
The tumor cell growth-inhibitory activity of the compounds
was evaluated by measurement of inhibition of MCF-7 cell growth,
4
.1.29. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenol
3
3,34
(
34)
To a solution of 2-methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl
-chloroacetate (33) (197.4 mg, 0.5 mmol) in dry toluene (2 mL)
was added a solution of trimethylaluminium in toluene (1.8 M,
.56 mL) under an Ar atmosphere at 0 °C. The mixture was stirred
as described previously.
-MEM (Minimum Essential Medium) supplemented with 10%
FBS in the presence or absence of a test compound for three days
at 37 °C under 5% CO in air. The number of cells with vehicle alone
Briefly, MCF-7 cells were cultured in
D
2
2
0
was defined as 100%. The concentration of test compounds that
inhibited by 50% the increase of the cell number was quantified
(IC50). The assay was performed in triplicate.
at room temperature for 6 h and at 100 °C for 3 h, and then poured
into ice water. The whole was extracted with ethyl acetate. The ex-
tract was successively washed with water and brine, and then dried
over MgSO
4
. The solvent was evaporated, and the residue was puri-
4.5. Colchicine competitive binding assay35
fied by means of silica gel column chromatography (hexane/ethyl
3
acetate = 10/1 to 0/1) to give 34 (17.5 mg, 11.1%) as a colorless oil.
Mixtures of 1.0
l
M (0.1 mg/mL) tubulin, 1.5
l
M [ H]colchicine
1
3
H NMR (500 MHz, CDCl ): d 6.97 (1H, d, J = 1.7 Hz), 6.85 (1H, dd,
(20 pCi/ L) and test compounds at 3, 10, or 30
l
l
M were prepared
J = 8.6, 1.7 Hz), 6.81 (1H, d, J = 8.6 Hz), 6.55 (2H, s), 5.60 (1H, s),
.38 (1H, d, J = 1.1 Hz), 5.31 (1H, d, J = 1.1 Hz), 3.91 (3H, s), 3.87
and incubated for 30 min at 37 °C. After treatment with 1.5% dex-
tran-coated charcoal solution, they were further incubated for
20 min at 4 °C and centrifuged. The amount of radiolabeled colchi-
cine bound to the tubulin was determined with a scintillation coun-
5
1
3
3
(3H, s), 3.81 (6H, s). C NMR (500 MHz, CDCl ): d 152.90, 149.63,
1
1
46.50, 145.28, 137.79, 137.53, 134.78, 120.27, 114.55, 112.96,
10.23, 105.80, 61.01, 56.22, 56.06. MS(FAB+) m/z: 317 (M+H ).
+
ter. The inhibitory activity in the presence of added 10 lM CA-4 (1)
was defined as 100%. The assay was performed in triplicate.
4
.1.30. 2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)ethyl)phenol
(
35)
To
phenyl)vinyl)phenol (34) (17.5 mg, 0.043 mmol) in ethanol
2 mL) was added 5% Pd/C (14.5 mg) at room temperature, and
the mixture was stirred under an H atmosphere at the same tem-
Acknowledgments
a
solution
of
2-methoxy-5-(1-(3,4,5-trimethoxy-
The work described in this paper was partially supported by a
Grant-in-aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Sciences and Technology, Japan, and a Grant
from the Japan Society for the Promotion of Science.
(
2
perature for 13 h. The reaction mixture was filtered through a Cel-
ite pad and the solvent was evaporated. The residue was purified
by PTLC (hexane/ethyl acetate = 2/1) to give 35 (11.7 mg, 83.3%)
Supplementary data
1
as a colorless oil. H NMR (500 MHz, CDCl
3
): d 6.81 (1H, d,
J = 2.3 Hz), 6.78 (1H, d, J = 8.6 Hz), 6.71 (1H, dd, J = 8.6, 2.3 Hz),
.43 (2H, s), 5.58 (1H, s), 3.99 (1H, q, J = 7.2 Hz), 3.86 (3H, s), 3.82
9H, s), 1.58 (3H, d, J = 7.2 Hz). 13C NMR (500 MHz, CDCl
): d
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.09.046.
6
(
3
1
1
3
53.12, 145.47, 144.94, 142.34, 139.79, 136.26, 118.86, 113.84,
References and notes
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+
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