Discovery of O6-benzyl glaziovianin A, a potent cytotoxic substance and a potent inhibitor of α,β-tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2016.09.026

We have discovered O⁶-benzyl glaziovianin A, which showed stronger inhibition of microtubule polymerization (IC₅₀?=?2.1?μM) than known α,β-tubulin inhibitors, such as colchicine and glaziovianin A. Also, we performed competition binding experiments of O⁶-benzyl glaziovianin A and revealed that O⁶-benzyl glaziovianin A binds to the colchicine binding site with high affinity. It is interesting that glaziovianin A derivatives change their mode of action in benzylation at the O⁶(α,β-tubulin inhibitor) or O⁷(γ-tubulin-specific inhibitor) position.

Full text of DOI:10.1016/j.bmc.2016.09.026

Accepted Manuscript
Discovery of O 6-benzyl glaziovianin A, a potent cytotoxic substance and a
potent inhibitor of α,β-tubulin polymerization
Ichiro Hayakawa, Shuya Shioda, Takumi Chinen, Taisei Hatanaka, Haruna
Ebisu, Akira Sakakura, Takeo Usui, Hideo Kigoshi
PII:
S0968-0896(16)30711-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.026
BMC 13279
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
3 August 2016
9 September 2016
10 September 2016
Please cite this article as: Hayakawa, I., Shioda, S., Chinen, T., Hatanaka, T., Ebisu, H., Sakakura, A., Usui, T.,
Kigoshi, H., Discovery of O 6-benzyl glaziovianin A, a potent cytotoxic substance and a potent inhibitor of α,β-
tubulin polymerization, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.09.026
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Discovery of O6-benzyl glaziovianin A, a
potent cytotoxic substance and a potent
inhibitor of ,-tubulin polymerization
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Ichiro Hayakawa,* Shuya Shioda, Takumi Chinen, Taisei Hatanaka, Haruna Ebisu, Akira Sakakura, Takeo
Usui,* Hideo Kigoshi*

Bioorganic & Medicinal Chemistry
Discovery of O6-benzyl glaziovianin A, a potent cytotoxic substance and a potent
inhibitor of ,-tubulin polymerization
Ichiro Hayakawaa*, Shuya Shiodab, Takumi Chinenc, Taisei Hatanakaa, Haruna Ebisuc, Akira Sakakuraa,
Takeo Usuic*, Hideo Kigoshib*
aDivision of Applied Chemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530,
Japan
bDepartment of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
cGraduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We have discovered O6-benzyl glaziovianin A, which showed stronger inhibition of microtubule
polymerization (IC50 = 2.1 M) than known ,-tubulin inhibitors, such as colchicine and
glaziovianin A. Also, we performed competition binding experiments of O6-benzyl glaziovianin
A and revealed that O6-benzyl glaziovianin A binds to the colchicine binding site with high
affinity. It is interesting that glaziovianin A derivatives change their mode of action in
benzylation at the O6 (-tubulin inhibitor) or O7 (-tubulin-specific inhibitor) position.
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
O6-Benzyl glaziovianin A
Cytotoxicity
,-Tubulin inhibitor
Structure–activity relationship study
1. Introduction
In a series of structure–activity relationship studies of
glaziovianin A (1), we reported the synthesis of O7-benzyl
Inhibitors of microtubule polymerization are some of the most
common and effective treatment for breast cancer.1 Colchicine,
vinca alkaloids, and taxol are known as typical microtubule
inhibitors of natural product origin. Many kinds of inhibitors of
microtubules bind to either the colchicine binding site,2 the
vinblastine binding site,3 or the taxoid binding site4 in ,-
tubulin. Especially, taxanes and vinca alkaloids, such as taxol and
vinblastine, are used in cancer chemotherapy. In contrast,
inhibitors binding to the colchicine site has not been used in
clinic at the present, although these compounds are currently
undergoing clinical development and evaluation in patients with
a wide array of cancers.
glaziovianin A (3), which showed strong cytotoxicity against
HeLa cells (IC50 = 0.19 M).8 On the other hand, we recently
developed a modified route to the synthesis of O7-modified
glaziovianin A.7b However, the resultant O7-benzyl glaziovianin
A (3) showed much weaker cytotoxicity (IC50 = 12.81 M). We
supposed that the previous lot of O7-benzyl glaziovianin A (3)
contained an impurity that exhibits strong cytotoxicity. In this
paper, we described the discovery of a new glaziovianin A
derivative: O6-benzyl glaziovianin A (4), which showed more
potent cytotoxicity against HeLa cells and inhibitory activity of
polymerization of ,-tubulin than known ,-tubulin inhibitors,
such as colchicine and glaziovianin A (1).
Glaziovianin A (1), a naturally occurred isoflavone derivative,
is an inhibitor of microtubule dynamics (Fig. 1).5,6 Previously, we
reported the synthesis7 and structure–activity relationship study8
of glaziovianin A (1). In that report, we revealed that O7-
alkylated9 glaziovianin A analogues have strong cytotoxicity
against HeLa cells. Especially, O7-propargyl glaziovianin A (2)
indicated more potent cytotoxic effects8b than glaziovianin A (1)
itself and decreased the polymerization rate of ,-tubulin in
vitro.6 We also revealed, from the results of a competition assay,
that glaziovianin A (1) binds to the colchicine site of ,-
tubulin.6

glaziovianin A (4). Thus, we planned to rigorously synthesize O6-
benzyl glaziovianin A (4) to confirm the structure of the
impurity.
Table 1 1H NMR data of A-ring of O7-benzyl glaziovianin A
(3) and the impurity (600 MHz, CD3OD)
Figure 1. Structures of glaziovianin A (1) and its derivatives.
2. Results and Discussion
1H (ppm)
2.1. Discovery of O6-benzyl glaziovianin A (4)
O7-benzyl
We used HPLC analysis to examine the purities of old and
new lots of O7-benzyl glaziovianin A (3) (Fig. 2). As a result, the
previously synthesized O7-benzyl glaziovianin A (3) contained an
impurity (ca. 10%). In contrast, the new lot of O7-benzyl
glaziovianin A (3) did not contain this impurity.
No.
impurity
glaziovianin A (3)
H1
H2
H3
H4
7.58
7.22
5.27
3.95
7.64
7.18
5.21
4.01
We synthesized O6-benzyl glaziovianin A (4) according to our
established strategy (Scheme 1). To convert commercially
available sesamol (5) to known benzophenone 6, we followed the
procedure reported by Tamura et al.10 Regioselective methylation
of benzophenone 6 gave the desired monomethyl benzophenone
7,11 which was converted into benzyl ether 8. Condensation of 8
with N,N-dimethylformamide dimethyl acetal afforded enamine
9, which was transformed into iodochromone 10. The Suzuki–
Miyaura coupling reaction12 between iodochromone 10 and
pinacol boronate 117b furnished O6-benzyl glaziovianin A (4).
Figure 2. HPLC analysis of O7-benzyl glaziovianin A (3); upper:
preparation by the previous synthetic route; bottom: preparation by the
modified synthetic route (column: Develosil ODS-HG-5 (4.6 × 250 mm),
solvent: 75% MeOH aq., detect: UV 254 nm, flow rate: 1 mL/min).
Next, we isolated the impurity in previously synthesized O7-
benzyl glaziovianin A (3) by using preparative HPLC and
determined its structure. The molecular weight of the impurity
was identical to that of O7-benzyl glaziovianin A (3) by ESIMS
analysis. Also, we compared the 1H NMR data between the
impurity and O7-benzyl glaziovianin A (3) (Table 1). The
chemical shifts of the protons at the A-ring portion (H1–H4) in the
impurity were different from those of O7-benzyl glaziovianin A
(3), and the others corresponded well (within 0.01 ppm). These
results suggested that the impurity was an isomer of O7-benzyl
glaziovianin A (3) concerning the A-ring, such as O6-benzyl

against HeLa cells (Table 2) and the effects on the microtubule
polymerization (Fig. 4). Glaziovianin A (1) showed moderate,
and O7-benzyl glaziovianin A (3) weak cytotoxicity against HeLa
cells. In contrast, O6-benzyl glaziovianin A (4) showed potent
cytotoxicity (IC50 = 0.07 M).
Table 2 Cytotoxicity of colchicine and glaziovianin A
derivatives against HeLa cells
compound
IC50 (M)
0.02 ± 0.01
0.61 ± 0.07
12.81 ± 6.26
0.07 ± 0.01
colchicine
glaziovianin A (1)
O7-benzyl glaziovianin A (3)
O6-benzyl glaziovianin A (4)
Previously we reported that glaziovianin A (1) and O7-
propargyl glaziovianin (2) decreased the in vitro
A
polymerization rate of ,-tubulin.6 In the present study, we used
microtubule polymerization to examine whether O6-benzyl
glaziovianin A (4) also inhibits microtubule dynamics. As shown
in Fig. 4A, pure O7-benzyl glaziovianin A (3) showed no
apparent effects, but O6-benzyl glaziovianin A (4) completely
inhibited microtubule polymerization at the concentration of 3
M. In the same condition, colchicine also inhibited microtubule
polymerization, but less potently compared with O6-benzyl
glaziovianin A (4) (Fig. 4B). The IC50 values for O6-benzyl
glaziovianin A (4) and colchicine were 2.1 ± 0.7 and 9.1 ± 1.3
M, respectively (Fig. 4B), suggesting that O6-benzyl
glaziovianin A (4) is a more potent microtubule inhibitor than
colchicine in vitro. Furthermore, the previous lot of O7-benzyl
glaziovianin A (3) (before HPLC separation) still showed an
intermediate inhibitory effect on microtubule polymerization
(Fig. 4A). Therefore, we concluded that contaminating O6-benzyl
glaziovianin A (4) in the previous lot of O7-benzyl glaziovianin
A (3) caused considerable inhibitory activity against microtubule
polymerization and cell proliferation.14
Scheme 1. Synthesis of O6-benzyl glaziovianin A (4).
Reagents and conditions: (a) MeI, K2CO3, acetone, 60 °C; (b)
BnCl, K2CO3, n-Bu4NI, MeCN, rt, 35% in two steps; (c)
(MeO)2CHNMe2, 95 °C; (d) I2, py, CHCl3, rt, 86% in two
steps; (e) 11, PdCl2(dppf)·CH2Cl2, 1 M Na2CO3 aq, 1,4-
dioxane, rt, 76%.
1
The H NMR data of the impurity were in good agreement
with those of synthetic O6-benzyl glaziovianin A (4) (Fig. 3).
Therefore, the structure of the impurity was confirmed to be O6-
benzyl glaziovianin A (4).13
Figure. 3 1H NMR (600 MHz, CD3OD) spectra of the impurity and of
synthetic O6-benzyl glaziovianin A (4).
2.2. Biological activity of O6-benzyl glaziovianin A (4)
We next evaluated the cytotoxicity of O6-benzyl glaziovianin
A (4), O7-benzyl glaziovianin A (3), and glaziovianin A (1)

Table 3 Inhibitory effects of O6-benzyl glaziovianin A (4) on
the binding of colchicine and vinblastine to tubulin
radiolabelled drug bound (% of control)
compound
[3H]-colchicine
[3H]-vinblastine
151.9 ± 6.8
5.3 ± 0.5
colchicine
18.2 ± 1.1
vinblastine
127. 1 ± 9.7
67.8 ± 7.1
glaziovianin A (1)
112.4 ± 5.5
O6-benzyl
glaziovianin A (4)
20.3 ± 1.5
117.7 ± 4.4
The competition assay of O6-benzyl glaziovianin A (4)
binding to tubulin with radiolabelled vinblastine or colchicine
was monitored by the centrifugal gel filtration method. Values
are the means ± s.d.
Table 4 Drug binding analysis based on tryptophan
fluorescence spectrum changes
compound
Kd (M)
26.6 ± 13.4
80.0 ± 45.7
1.0 ± 0.1
colchicine
glaziovianin A (1)
O6-benzyl glaziovianin A (4)
The Kd value of each drug was calculated from the tryptophan
fluorescence decrease (∆FL) from three independent experiments
using GraphPad Prizm software. Values are the means ± s.d.
Figure 4. Effects of glaziovianin
A derivatives on microtubule
polymerization. Purified porcine tubulin was incubated with or without each
compound at 37 °C and then polymerized by the addition of 10 M taxol (A)
or 1 M glutamate (B). A. O6-Benzyl glaziovianin A (4) inhibited microtubule
polymerization at 3 M, but the O7-derivative did not. ○: DMSO control; ●:
O7-benzyl glaziovianin A (3), which was prepared by the previous synthetic
route (before HPLC separation); ■: HPLC-separated O7-benzyl glaziovianin
A (3); and □: HPLC-separated O6-benzy glaziovianin A (4). B. O6-Benzyl
glaziovianin A (4) inhibited microtubule polymerization in a dose-dependent
manner. Effects of O6-benzyl glaziovianin A (4), prepared by the modified
synthetic route, on microtubule polymerization in vitro. The final
concentrations were 0 M (○), 0.5 M (▲), 1.5 M (△), and 3.0 M (♦) of
O6-benzyl glaziovianin A (4), 5 M (■) and 30 M (□) of colchicine, or 10
M of glaziovianin A (1) (●).
2.4. Effects of O6-benzyl glaziovianin A (4) on the cell cycle
progression and spindle structure in mitosis
We next examined the effects of O6-benzyl glaziovianin A (4)
on the cell cycle progression and spindle structure in mitosis
(Fig. 5). O6-Benzyl glaziovianin A (4) completely inhibited the
cell cycle progression in mitosis at 10 times lower concentrations
than glaziovianin A (1) did (Fig. 5, Left). Under this condition,
O6-benzyl glaziovianin A (4) inhibited the normal bipolar spindle
formation (Fig. 5, Right). Most of the cells treated with 100 nM
O6-benzyl glaziovianin A (4) showed a multipolar spindle as the
glaziovianin A (1)-treated cells did. At higher concentrations, O6-
benzyl glaziovianin A (4) completely disrupted the microtubule
and only short fragments were observed. These results also
indicate that O6-benzyl glaziovianin A (4) is a potent microtubule
polymerization inhibitor.
2.3. Competition binding experiments of O6-benzyl glaziovianin
A (4)
We previously reported that glaziovianin A (1) and O7-
propargyl glaziovianin
A
(2) decreased the in vitro
polymerization rate of ,-tubulin by binding to the colchicine
binding site.6 To determine whether the binding site of O6-benzyl
glaziovianin A (4) is the same as that of glaziovianin A (1), we
performed competition binding experiments using [3H]-labeled
vinblastine and colchicine (Table 3). Like colchicine and
glaziovianin A (1), O6-benzyl glaziovianin A (4) inhibited the
binding of [3H]-colchicine but enhanced the binding of [3H]-
vinblastine, suggesting that the binding site of O6-benzyl
glaziovianin A (4) is identical or close to those of colchicine and
glaziovianin A (1). Furthermore, the changes in tryptophan
fluorescence showed that O6-benzyl glaziovianin A (4) bound to
,-tubulin with a markedly higher affinity than glaziovianin A
(1) and colchicine (Table 4). These results strongly suggest that
O6-benzyl glaziovianin A (4) binds to the colchicine binding site
with high affinity and inhibits microtubule polymerization more
potently than colchicine and glaziovianin A (1).

We recently reported glaziovianin A (1) and O7-propargyl
glaziovianin A (2) as microtubule dynamics inhibitors,6 and O7-
benzyl glaziovianin A (3) as a -tubulin-specific inhibitor,
gatastatin.14 Our preliminary data suggested that O6-benzyl
glaziovianin A (4) also inhibits GTP binding on -tubulin (89.6 ±
1.7% inhibition at 60 M). This inhibitory activity was weaker
than that of O7-benzyl glaziovianin A (3) (93.3 ± 3.7% inhibition
at 60 M). Thus, it is interesting that glaziovianin A derivatives
change their mode of action in benzylation at the O6 (-tubulin
and -tubulin inhibitor) or O7 (-tubulin-specific inhibitor)
position (Fig. 6).14b Here we showed that glaziovianin A (1) is
good template with which to develop tubulin superfamily
inhibitors. The synthesis of O6-modified glaziovianin
A
derivatives is currently underway in our group.
Figure 6 Summary of the structure–activity relationships of glaziovianin
A derivative.
4. Experimental
4.1. Chemistry
General method
Figure 5. Effects of O6-benzyl glaziovianin
A (4) on cell cycle
progression (Left) and spindle morphology (Right). Left: Effects of
colchicine, glaziovianin A (1) and O6-benzyl glaziovianin A (4) on cell cycle
progression in HeLa cells. HeLa cells were treated with each compound for
18 h. Right: Effects of colchicine, glaziovianin A (1) and O6-benzyl
glaziovianin A (4) on spindle morphology in HeLa cells. HeLa cells were
treated with glaziovianin A (1) and O6-benzyl glaziovianin A (4) for 6 h.
Scale bar, 20 m. Green and blue represent -tubulin and DNA, respectively.
All moisture-sensitive reactions were performed under an
atmosphere of argon or nitrogen, and the starting materials were
azeotropically dried with benzene or toluene before use.
Anhydrous acetone, MeCN, CHCl3, and 1,4-dioxane were
purchased from Kanto Chemical Co., Inc., or Wako Pure
Chemical Industries Ltd., and used without further drying. TLC
analyses were conducted on E. Merck precoated silica gel 60 F254
(0.25 mm layer thickness). Merck silica gel 60 (0.040–0.063 mm
230–400 mesh) and Fuji Silysia silica gel BW-820MH (75–200
m) were used for column chromatography. Melting points were
measured on Yanaco MP-J3 micro melting point apparatus and
are uncorrected. Infrared (IR) spectra were recorded on a JASCO
FT/IR-4100 instrument and only selected peaks are reported in
3. Conclusions
In conclusion, we have discovered O6-benzyl glaziovianin A
(4), which showed stronger inhibition of microtubule
polymerization than known ,-tubulin inhibitors, such as
colchicine and glaziovianin A (1).15 O6-Benzyl glaziovianin A (4)
potently inhibited microtubule polymerization in vitro (IC50 = 2.1
M) and arrested cell cycle progression in mitosis by forming
abnormal spindles, suggesting that the introduction of a benzyl
group at the O6 position potentiates the affinity to the colchicine
binding site of ,-tubulin. Although O6-benzyl glaziovianin A
(3) inhibited microtubule polymerization in vitro more potently
than colchicine, the cytotoxicity of O6-benzyl glaziovianin A (3)
was weaker than that of colchicine, suggesting that cell
permeability of O6-benzyl glaziovianin A (3) is lower than that of
colchicine.
wavenumbers (cm−1). H and 13C NMR spectra were recorded on
1
a Varian INOVA-600 spectrometer, a Varian Mercury-400
spectrometer, a Bruker AVANCE 600 spectrometer, a Bruker
AVANCE 400 spectrometer or a Bruker DPX 400 spectrometer.
1
The H and 13C chemical shifts were referenced to the solvent
peaks, H 7.26 (residual CHCl3) and C 77.0 ppm (CDCl3), or H
3.31 (residual CHD2OD) and C 49.0 ppm (CD3OD),
respectively.
J values are given in Hz. The following
abbreviations are used for spin multiplicity: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, and br = broad.

High resolution ESI/TOF mass spectra were recorded on a JEOL
AccuTOFCS JMS-T100CS spectrometer.
mol) in 1,4-dioxane (450 L) was added aqueous 1 M Na2CO3
(150 L, 150 mol) at room temperature in a glove box. The
mixture was stirred at room temperature under a stream of N2 for
20 h and diluted with H2O (1.0 mL) and CHCl3 (3.0 mL) at room
temperature. The layers were separated, and the aqueous layer
was extracted with CHCl3 (3 × 3.0 mL). The organic layer and
extracts were combined, washed with brine, dried (Na2SO4),
filtered, and concentrated. The residual solid was purified by
column chromatography on silica gel (700 mg, hexane–EtOAc
4:1→1:1) to give O6-benzyl glaziovianin A (4) (containing Pd-
metal). The resultant mixture was dissolved in CHCl3 (3 mL),
and SiliaBond (SILICYCLE, SiliaMetS® Thiourea, 10 mg) was
added. The resulting mixture was stirred at room temperature for
12 h, filtered, and concentrated. The resultant mixture was
purified by recycle HPLC [JAIGEL-1H-40 (20 × 600 mm) and
JAIGEL-2H-40 (20 × 600 mm); flow rate 3.8 mL/min;
detection, UV 254 nm; solvent CHCl3] to give O6-benzyl
glaziovianin A (4) (6.3 mg, 68%) as a white solid: mp 185–188
°C; IR (CHCl3) 3026, 3003, 1636, 1606, 1506, 1451, 1296, 1270
4.1.1. 1-(5-(benzyloxy)-2-hydroxy-4-
methoxyphenyl)ethan-1-one (8)
To a stirred solution of benzophenone 6 (103 mg, 0.61 mmol)
in acetone (3.0 mL) were added K2CO3 (172 mg, 1.25 mmol) and
MeI (40 L, 0.64 mmol) at room temperature. After being stirred
at reflux for 4 h, the reaction mixture was concentrated. The
resultant mixture was dissolved in H2O (1.0 mL), diluted with 2
M HCl (0.3 mL) to pH 3, and extracted with EtOAc (3 × 12 mL).
The combined extracts were washed with brine, dried (Na2SO4),
filtered, and concentrated to give
a crude monomethyl
benzophenone 7 (110 mg), which was used for the next reaction
without further purification.
To a stirred solution of the crude monomethyl benzophenone
7 (110 mg) in MeCN (3.0 mL) were added K2CO3 (165 mg, 1.19
mmol), BnCl (80 L, 0.700 mmol), and n-Bu4NI (329 mg, 0.89
mmol) at room temperature. After being stirred at room
temperature for 8.5 h, the reaction mixture was filtrated with
Celite, and this Celite was rinsed with EtOAc (4 × 10 mL). The
filtrate and rinse were concentrated. The residual solid was
purified by column chromatography on silica gel (10 g, hexane–
EtOAc 16:1→14:1→12:1→2:1) to give acetophenone 8 (57.3
mg, 35% in two steps) as a white solid; mp 147–149 °C; IR
1
cm–1; H NMR (400 MHz, CDCl3)  7.89 (s, 1H), 7.71 (s, 1H),
7.48 (d, J = 7.2 Hz, 2H), 7.40–7.28 (m, 3H), 6.89 (s, 1H), 6.52 (s,
1H), 6.02 (s, 2H), 5.22 (s, 2H), 3.98 (s, 3H), 3.87 (s, 3H), 3.85 (s,
3H); 13C NMR (100 MHz, CDCl3)  175.4, 154.9, 153.5, 152.5,
146.8, 139.2, 139.0, 137.1, 136.8, 136.2, 128.7 (2C), 128.2, 127.7
(2C), 121.7, 118.1, 117.8, 110.2, 107.0, 101.9, 99.8, 71.1, 60.2,
56.9, 56.4; HRMS (ESI) m/z 485.1215, calcd for C26H22NaO8
[M+Na]+ 485.1206.
1
(CHCl3) 3025, 3012, 1633, 1508, 1444, 1374, 1329 cm–1; H
NMR (400 MHz, CDCl3)  12.59 (s, 1H), 7.45–7.32 (m, 5H),
7.09 (s, 1H), 6.46 (s, 1H), 5.08 (s, 2H), 3.92 (s, 3H), 2.44 (s, 3H);
13C NMR (100 MHz, CDCl3)  202.1, 160.4, 157.7, 140.4, 136.8,
128.6 (2C), 128.1, 127.6 (2C), 116.2, 111.7, 100.6, 72.6, 56.1,
26.2; HRMS (ESI) m/z 271.1001, calcd for C16H15O4 [M–H]–
271.0975.
4.2. Biology
4.2.1. Calculation of IC50 value
HeLa cells were grown in DMEM supplemented with 10%
FBS, 100 units/mL penicillin, and 100 g/mL streptomycin in a
humidified atmosphere containing 5% CO2. Various
concentrations of each compound were then added and incubated
for 48 hours. Cell growth was determined using WST-8 cell
counting kit (Dojindo) and IC50 value was calculated.
4.1.2. 6-(benzyloxy)-3-iodo-7-methoxy-4H-chromen-
4-one (10)
Acetphenone 8 (6.5 mg, 24 µmol) was dissolved in N,N-
dimethylformamide dimethyl acetal (0.10 mL, 0.75 mmol) and
stirred at 95 °C for 2 h. The reaction mixture was concentrated in
vacuo to give crude enamine 9 (10 mg), which was used for the
next reaction without further purification.
4.2.2. In vitro tubulin polymerization assay
MAPs free ,-tubulin was purified from porcine brain using
two cycles of polymerization-depolymerization in a high-
molarity buffer as described previously.16 1 mg/mL ,-tubulin,
1 M glutamate and 1 mM GTP were mixed in RB buffer (100
mM MES (pH 6.8), 1 mM EGTA, 0.5 mM MgCl2). One of the
compounds was then added and incubated for 10 min on ice.
Samples were transferred into cuvette and tubulin polymerization
was determined by monitoring the absorbance at 350 nm at 37 °C
using a thermostatic spectrophotometer (Beckman Coulter). The
IC50 values for O6-benzyl glaziovianin A (4) and colchicine were
calculated from final tubulin polymerization rate (control %) at
20 min of each concentration.
To a stirred solution of crude enamine 9 (10 mg) in CHCl3
(0.4 mL) were added pyridine (4.0 L, 50 mol) and I2 (9.5 mg,
37 mol) at 0 °C. After being stirred in dark at room temperature
for 15 h, the mixture was diluted with saturated aqueous Na2S2O3
(1 mL) at 0 °C and extracted with CHCl3 (4 × 3 mL). The
combined extracts were washed with brine, dried (Na2SO4),
filtered, and concentrated. The residual solid was purified by
column chromatography on silica gel (700 mg, CHCl3) to give
iodochromone 10 (8.4 mg, 86% in two steps) as a yellow solid:
mp 129–132 °C; IR (CHCl3) 3012, 1634, 1618, 1505, 1448,
1
1287, 1270 cm–1; H NMR (400 MHz, CDCl3)  8.21 (s, 1H),
7.58 (s, 1H), 7.47 (d, J = 7.2 Hz, 2H), 7.39–7.28 (m, 3H), 6.86 (s,
1H), 5.21 (s, 2H), 3.96 (s, 3H); 13C NMR (100 MHz, CDCl3) 
172.4, 156.9, 155.3, 152.4, 147.2, 136.0, 128.7 (2C), 128.2, 127.7
(2C), 115.1, 107.0, 99.6, 86.6, 71.2, 56.5.; HRMS (ESI) m/z
430.9744, calcd for C17H13NaIO4 [M+Na]+ 430.9750.
4.2.3. Competition assay
The competition assay of O6-benzyl glaziovianin A (4)
binding to tubulin with radiolabelled vinblastine or colchicine
was monitored by the centrifugal gel filtration method. The
reaction mixture (50 μL) containing 0.5 mg/mL (approx. 5 μM)
tubulin, 50 nM [3H]-labeled vinblastine or colchicine, 10% (v/v)
DMSO and 100 μM mitotic inhibitor was incubated for 5 min at
room temperature and processed by centrifugal gel filtration
using Centri-Spin 20 (Princeton Separations).
4.1.3. O6 -benzyl glaziovianin A (4)
All solvents were degassed by freeze–thawing. To a stirred
solution of pinacol boronate 11 (8.4 mg, 24 mol), iodochromone
10 (8.0 mg, 20 mol), and PdCl2(dppf)·CH2Cl2 (3.5 mg, 4.3

4.2.4. Kd value calculation
References and notes
Tryptophan fluorescence-based drug binding was performed
as described in ref. 17 with minor modification. In briefly, 0.1
mg/mL ,-tubulin and 1 mM GTP were mixed in RB buffer
(100 mM MES (pH 6.8), 1 mM EGTA, 0.5 mM MgCl2). Various
concentrations of test compounds were then added and incubated
for 30 min at room temperature. Samples were transferred into
cuvette and tryptophan fluorescence of tubulin (excitation: 295
nm, emission: 310–450 nm) was monitored using RF-5300
(SHIMADZU). Dissociation constants were calculated from
fitting curves of decreasing fluorescence using GraphPad Prizm.
1.
2.
Review: Kingston, D. G. I. J. Nat. Prod. 2009, 72, 507.
Uppuluri, S.; Knipling, L.; Sackett, D. L.; Wolff, J. Proc. Natl.
Acad. Sci. USA 1993, 90, 11598.
3.
4.
5.
6.
Gigant, B.; Wang, C.; Ravelli, R. B. G.; Roussi, F.; Steinmetz, M.
O.; Curmi, P. A.; Sobel, A.; Knossow, M. Nature 2005, 435, 519.
Rao, S.; He, L.; Chakravarty, S.; Ojima, I.; Orr, G. A.; Horwitz, S.
B. J. Biol. Chem. 1999, 274, 37990.
Yokosuka, A.; Haraguchi, M.; Usui, T.; Kazami, S.; Osada, H.;
Yamori, T.; Mimaki, Y. Bioorg. Med. Chem. Lett. 2007, 17, 3091.
Chinen, T.; Kazami, S.; Nagumo, Y.; Hayakawa, I.; Ikedo, A.;
Takagi, M.; Yokosuka, A.; Imamoto, N.; Mimaki, Y.; Kigoshi, H.;
Osada, H.; Usui, T. ACS Chem. Biol. 2013, 8, 884.
7.
8.
(a) Hayakawa, I.; Ikedo, A.; Kigoshi, H. Chem. Lett. 2007, 36,
1382; (b) Hayakawa, I.; Shioda, S.; Ikedo, A.; Kigoshi, H. Bull.
Chem. Soc. Jpn. 2014, 87, 544.
4.2.5. Analysis of cell cycle progression
HeLa cells were grown in DMEM supplemented with 10%
FBS, 100 units/mL penicillin and 100 g/mL streptomycin in a
humidified atmosphere containing 5% CO2. Various
concentrations of test compounds were then added and incubated
for 18 hours. Cells were collected and fixed with 70% EtOH at –
20 °C. After wash with PBS, cells were stained with PI solution
(0.2% NP-40, 0.1% trisodium citrate dehydrate, 71.1 M
propidium iodide and 10 g/mL RNaseA) at 4 C. DNA contents
were analysed by cytometric analysis (PARTEC CyFlow PA;
Partec GmbH, Munster, Germany). Cell populations of each cell
cycle phase were calculated using Multi Cycle AV (PhoenixFlow
Systems).
(a) Ikedo, A.; Hayakawa, I.; Usui, T.; Kazami, S.; Osada, H.;
Kigoshi, H. Bioorg. Med. Chem. Lett. 2010, 20, 5402; (b)
Hayakawa, I.; Ikedo, A.; Chinen, T.; Usui, T.; Kigoshi, H. Bioorg.
Med. Chem. 2012, 20, 5745; (c) There was a printing error in
references 8a and 8b. The cytotoxicities of glaziovianin A and its
derivatives were evaluated against HeLa cells (not HeLa S3 cells).
In this paper, we name O-demethyl-O-alkyl glaziovianin
derivatives as O-alkyl glaziovianins for simplification.
9.
10. Tamura, S.; Yoshihira, K.; Tokumaru, M.; Zisheng, X.;
Murakami, N. Bioorg. Med. Chem. Lett. 2010, 20, 3872.
11. (a) Monomethyl benzophenone 7 gave spectral data (1H and 13C
NMR) in full agreement with the reported data11b; (b) O’Reilly, E.;
Corbett, M.; Hussain, S.; Kelly, P. P.; Richardson, D.; Flitsch, S.
L.; Turner, N. J. Catal. Sci. Technol. 2013, 3, 1490.
12. Hoshino, Y.; Miyaura, N.; Suzuki, A. Bull. Chem. Soc. Jpn. 1988,
61, 3008.
13. A plausible formation pathway of the O6-benzyl glaziovianin A
(4) is shown in the Supplementary data.
4.2.6. Observation of mitotic spindle
14. (a) We revealed that pure O7-benzyl glaziovianin A (3) does not
inhibit the polymerization of ,-tubulin and shows the specific
inhibition of -tubulin. O7-Benzyl glaziovianin A (3) was named
"gatastatin (gamma tubulin activity)"14b; (b) Chinen, T.; Liu, P.;
Shioda, S.; Pagel, J.; Cerikan, B.; Lin, T.; Gruss, O.; Hayashi, Y.;
Takeno, H.; Shima, T.; Okada, Y.; Hayakawa, I.; Hayashi, Y.;
Kigoshi, H.; Usui, T.; Schiebel, E. Nat. Commun. 2015, 6, 8722.
15. From these results, previously synthesized O7-modified
glaziovianin A derivatives8 had possibility of contaminating by-
products. Hence, we have reevaluated cytotoxicity against HeLa
cells of O7-propargyl glaziovianin A, which was the most
cytotoxic derivative in reference 8. The previous lot of O7-
propargyl glaziovianin A showed the same IC50 value as reported
(IC50 = 0.17 M). In contrast, cytotoxicity of a new lot of O7-
propargyl glaziovianin A (prepared by using modified synthetic
route) is weaker (IC50 = 0.45 M). Therefore, IC50 values of O7-
benzyl and propargyl glaziovianin A should be corrected as
described in this paper, respectively.
HeLa cells were grown in DMEM supplemented with 10%
FBS, 100 units/mL penicillin and 100 g/mL streptomycin in a
humidified atmosphere containing 5% CO2. Various
concentrations of test compounds were then added and incubated
for 6 hours. Cell growth was then fixed with cold MeOH (–20
°C). Cells were blocked with 0.5% bovine serum albumin and
incubated with anti--tubulin antibody (1:500 dilution, Santa
Cruz, sc-32293) for 1 h at 37 °C. After staining with Alexa488
-
conjugated anti-mouse IgG (1:2000 dilution, Invitrogen) for 30
min at 37 °C, coverslips were washed with PBS and mounted
PBS containing 0.1 g/mL DAPI. Microtubule cytoskeleton was
observed under the LAS AF 6000 fluorescence microscope
(Leica Microsystems).
16. Castoldi, M.; Popov, A. V. Protein Expr. Purif. 2003, 32, 83.
17. Sardar, P. S.; Maity, S. S.; Das, L.; Ghosh S. Biochemistry 2007,
46, 14544.
Acknowledgments
This work was supported by Grants-in-Aid for Scientific
Research on Innovative Areas ‘Chemical Biology of Natural
Products’ (Grant Numbers JP23102014, JP23102013) and for
Scientific Research (Grant Number JP16K07710) from the
Ministry of Education, Culture, Sports, Science and Technology
(MEXT)/Japanese Society for the Promotion of Science (JSPS).
I.H. thanks the Okayama Foundation for Science and
Technology, Kurata Grant awarded by the Kurata Memorial
Hitachi Science and Technology Foundation, the NOVARTIS
Foundation (Japan) for the Promotion of Science, the Naito
Foundation, and the Suzuken Memorial Foundation for their
financial support. We would also like to thank Prof. Elmar
Schiebel (Zentrum für Molekulare Biologie der Universität
Heidelberg) for his helpful discussion.