Design, synthesis, and biological evaluation of the analogues of glaziovianin A, a potent antitumor isoflavone

Article abstract of DOI:10.1016/j.bmc.2012.08.005

Various analogues of glaziovianin A, an antitumor isoflavone, were synthesized, and their biological activities were evaluated. O ⁷-modified glaziovianin A showed strong cytotoxicity against HeLa S₃ cells. Compared to glaziovianin A,

Full text of DOI:10.1016/j.bmc.2012.08.005

Bioorganic & Medicinal Chemistry 20 (2012) 5745–5756
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of the analogues of glaziovianin A,
a potent antitumor isoflavone
Ichiro Hayakawa a, , Akiyuki Ikedo a, Takumi Chinen b, Takeo Usui b, Hideo Kigoshi a,
⇑
⇑
a Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennodai, Tsukuba 305-8571, Japan
b Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba 305-8572, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Various analogues of glaziovianin A, an antitumor isoflavone, were synthesized, and their biological activ-
ities were evaluated. O7-modified glaziovianin A showed strong cytotoxicity against HeLa S3 cells. Com-
pared to glaziovianin A, the O7-benzyl and O7-propargyl analogues were more cytotoxic against HeLa S3
cells and more potent M-phase inhibitors. Furthermore, O7-modified molecular probes of glaziovianin A
were synthesized for biological studies.
Received 20 July 2012
Revised 7 August 2012
Accepted 8 August 2012
Available online 16 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Structure–activity relationship study
Glaziovianin A
Isoflavone
Antitumor activity
Molecular probes
1. Introduction
on cell cycle progression and spindle structure. We also describe
the synthesis of molecular probes of glaziovianin A for biological
Glaziovianin A (1), isolated from the leaves of the Brazilian tree
Ateleia glazioviana, exhibited a broad spectrum of cytotoxicity
against a panel of 39 human cancer cell lines (termed JFCR39) at
the Japanese Foundation for Cancer Research (Fig. 1).1 On the basis
of COMPARE analysis, the pattern of the differential cytotoxicities
of glaziovianin A (1) suggested that glaziovianin A (1) inhibits can-
cer cell proliferation by inhibiting tubulin polymerization.2 Indeed,
we previously reported that glaziovianin A (1) arrested the cell cy-
cle progression in M phase as do tubulin inhibitors.1 Interestingly,
glaziovianin A (1) induced abnormal spindle structures with una-
ligned chromosomes in mitotic cells, but did not influence on the
microtubule network in interphase cells. Although the target mol-
ecule(s) or inhibitory mechanism remained to be revealed, these
results suggest that glaziovianin A (1) is a novel mitotic inhibitor.
Mitotic inhibitors including tubulin polymerization inhibitors have
become clinically important drugs, especially against breast can-
cer.3 Also, glaziovianin A (1) showed antitumor activities in a
mouse xenograft model (unpublished data). These results encour-
aged us to develop novel anticancer drugs based on glaziovianin A
(1). A structure–cytotoxicity relationship study of glaziovianin A
(1) was preliminarily reported by our group.4 In the present paper,
we describe in detail the structure–activity relationships of glazi-
ovianin A, specifically concerning its cytotoxicity and its effects
studies and target identification.
We previously synthesized glaziovianin A (1) by using Suzuki–
Miyaura coupling as a key step (Scheme 1).5 We used a similar
strategy to synthesize glaziovianin A analogues. To develop these
analogues, the structure of glaziovianin A (1) can be divided into
two structural moieties: an A-ring and a B-ring (Fig. 2). Therefore,
we synthesized 3-iodochromone analogues as an A-ring and boron
compounds as a B-ring.
2. Results and discussion
2.1. Synthesis of B-ring analogues of glaziovianin A
First, we prepared B-ring analogues of glaziovianin A (Scheme
2). The diol group in 3,6-dimethoxybenzene-1,2-diol (6)6 was
protected as an acetonide group to give compound 7. The
OMe
5'
4'
O
O
6'
O
B
5
A
8
4
3
MeO
MeO
6
10
9
3'
1'
2'
C
O
OMe
2
7
glaziovianin A (1)
Figure 1. Structure of glaziovianin A (1).
⇑
Corresponding authors.
E-mail address: hayakawa@chem.tsukuba.ac.jp (I. Hayakawa).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.005

5746
I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
OMe
OMe
OMe
OMe
O
O
O
O
OH
O
O
a
b,c
MeO
MeO
O
O
B
O
OH
OH
OMe
4
OMe
8
OMe
6
OMe
7
2
OMe
OMe
OMe
OMe
OMe
OMe
d
OMe
O
O
(HO)2B
MeO
MeO
I
OMe
9
OMe
10
O
O
B
O
O
OMe
5
R2
3
R2
R2
R1
R1
R1
R1
O
e
MeO
MeO
Suzuki–Miyaura
R3
coupling
R2
O
O
O
O
OMe
8 R1, R1 =
1
1
12
R , R
=
, R2 = OMe
O
O
O
O
R2 = OMe, R3 = Bpin
MeO
MeO
13 R1 = R2 = OMe
O
1 = R2 = OMe
10
R
OMe
R3 = B(OH)2
O
14 R1, R1
=
, R2 = H
O
O
O
glaziovianin A (1)
11 R1, R1
=
Scheme 1. Total synthesis of glaziovianin A (1) by our group.
R2 = H, R3 = B(OH)2
OMe
OMe
Br
6'
O
O
O
O
B-ring part
6'
O
O
MeO
MeO
MeO
MeO
OMe
5'
f
A-ring part
4'
O
O
OMe
OMe
O
O
O
O
5
MeO
3'
glaziovianin A (1)
15
2'
OMe
MeO
7
Scheme 2. Synthesis of B-ring analogues of glaziovianin A. Reagents and condi-
tions: (a) 2-methoxypropene, PPTS, benzene, reflux, 72%; (b) NBS, DMF, rt, 57%; (c)
bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMF, 150 °C, 28%; (d) n-BuLi, B(OMe)3,
THF, rt; (e) 3, PdCl2(dppf), 1 M Na2CO3 aq, 1,4-dioxane, rt {64% for 12, 11% for 13
(from 9), 41% for 14}; (f) NBS, DMF, rt, 31%.
glaziovianin A (1)
O
R1
R2
2.2. Synthesis of A-ring analogues of glaziovianin A
O
glaziovianin A analogues
Next, we tried to modify the A-ring part of glaziovianin A
(Scheme 3). The hydroxy group at the C7 position of 1610 was pro-
tected as a THP group to afford compound 17. Condensation of 17
with N,N-dimethylformamide dimethyl acetal gave an enamine in
quantitative yield. Iodinative cyclization of the enamine afforded
iodochromone 18.11 The cross coupling reaction of arylboronate
55 and iodochromone compounds 18 and 19,12 gave glaziovianin
A analogues 20 and 21, respectively.
O
R1
I
R3O
R2
+
B
R3O
boron compounds
O
3-iodochlomone derivatives
Figure 2. Key structural moieties of glaziovianin A.
Next, we attempted to synthesize glaziovianin A analogues that
have various functional groups at the C7 position (Scheme 4).
Treatment of compound 20 with p-TsOHꢀH2O provided O7-demeth-
yl analogue 22, which is a suitable precursor for the synthesis of
O7-modified glaziovianin A. Thus, we tried to synthesize glycoside
25 and diol 27 in order to improve water solubility. Glycosylation
of the O7 position in 22 with tetraacetyl glucopyranosyl trichloro-
acetimidate 23 by using Schmidt glycosylation13 gave tetraacetyl
glycosylisoflavone 24. Removal of the acetyl groups of 24 by using
NaOMe afforded glycoside 25. Next, allylation of O7-demethyl ana-
logue 22 with allyl bromide gave allyl ether 26. Dihydroxylation of
the terminal olefin of 26 afforded diol 27. On the other hand, the
bromination of 7 gave a monobromo compound, which was con-
verted into arylboronate 8 by using PdCl2(dppf), bis(pinacola-
to)diboron, and KOAc in DMF at 150 °C.7 The Suzuki–Miyaura
coupling8 between 3-iodo-6,7-dimethoxy-4H-chromen-4-one (3)5
and boron compounds, such as arylboronate 8, 2,3,4,5-tetrame-
thoxyphenylboronic acid (10),9 or commercially available 3,4-
(methylenedioxy)phenylboronic acid (11), gave glaziovianin A
analogues 12–14, respectively. On the other hand, bromide 15
was synthesized from selective bromination at the C6’ position of
glaziovianin A (1).

I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
5747
Table 1
O
O
Cytotoxicity of glaziovianin A (1) and its analogues against HeLa S3 cells
MeO
MeO
I
b,c
Compound
cLogP
Cytotoxicity
IC50 (lM) Relative value
7
RO
a
OH
THPO
O
16
17
18
R = H
R = THP
Glaziovianin A (1)
2.19
2.79
2.15
2.44
3.02
3.03
2.06
1.92
1.00
0.08
2.88
1.13
3.92
2.40
0.59
1
—
0.027
0.010
0.009
12
13
14
15
20
21
22
24
25
26
27
28
29
>100
22.0
56.2
67.8
>100
>100
>100
>100
>100
0.46
>100
0.19
0.17
OMe
OMe
O
O
R1
O
O
R1
O
O
—
—
—
—
—
1.3
—
MeO
R2O
MeO
R2O
I
d
= H, R2 = THP
R = H, R2 = THP
1
1
18
20
R
19 R1 = OMe, R2 = Me
21 R1 = OMe, R2 = Me
3.1
3.5
Scheme 3. Synthesis of glaziovianin
A analogues 20 and 21. Reagents and
conditions: (a) DHP, PPTS, CH2Cl2, rt, 80%; (b) Me2NCH(OMe)2, 90 °C, quant; (c) I2,
py, CHCl3, rt, 75%; (d) 5, PdCl2(dppf), 1 M Na2CO3 aq, 1,4-dioxane, rt (66% for 20, 16%
for 21).
extra methoxy group at C5 of the A-ring, showed no cytotoxicity
even at 100 lM, indicating that the steric hindrance and electron
density of the A-ring extinguished cytotoxicity. O7-Demethyl ana-
logue 22 exhibited no cytotoxicity, perhaps because of its instabil-
ity. On the other hand, compounds 24, 25, and 27, which have O7-
OMe
O
O
hydrophilic functional groups, showed no cytotoxicity at 100 lM,
O
and the cLogP values of these compounds, 1.00, 0.08, and 1.13,
are much smaller than that of glaziovianin A (1). These results
showed that the improvement of water solubility diminishes cyto-
toxicity. In contrast, compounds 26, 28, and 29, which have,
respectively, an allyl, a benzyl, and a propargyl group at O7 instead
of the methyl group, showed cytotoxicity with IC50 values of 0.46,
MeO
OMe
7
RO
AcO
O
20
R = THP
a
22 R = H
OAc
OAc
OAc
AcO
OAc
OAc
OAc
HO
OH
OH
OH
OH
c
0.19, and 0.17 l
M, respectively.15 Thus, some alkyl groups at O7
b
O
24 R =
25R =
27 R =
had enhanced cytotoxicity against HeLa S3 cells. In particular, O7-
propargyl analogue 29 is more active than glaziovianin A (1) itself.
These results indicated that the hydrophobicity of the O7-alkyl
group in glaziovianin A analogues improves cytotoxicity. However,
compound 20, which has a THP group at O7, showed no cytotoxic-
HN
O
O
O
CCl3
23
e
d
26 R =
OH
f
28
29
R = Bn
R =
ity even at 100 l
M. This indicated that steric hindrance of the O7-
g
substituent reduces cytotoxicity to a large extent.
2.4. Effects of glaziovianin A (1) and O7-alkylated analogues 26,
28, and 29 on cell cycle progression and spindle structures in
HeLa S3 cells
Scheme 4. Synthesis of O7-modified analogues of glaziovianin A. Reagents and
conditions: (a) p-TsOHꢀH2O, MeOH, CHCl3, rt, 85%; (b) 23, BF3ꢀEt2O, MS3A, CH2Cl2, rt,
27% (
a : b = 1:5.6); (c) NaOMe, MeOH, rt, 91%; (d) allyl bromide, K2CO3, MeCN, rt,
81%; (e) OsO4, py, rt, 84%; (f) benzyl bromide, K2CO3, MeCN, rt, 80%; (g) propargyl
bromide, K2CO3, MeCN, rt, 67%.
We previously reported that glaziovianin A (1) inhibited the cell
cycle progression in the M-phase with abnormal spindle struc-
tures.1 Here, we investigated the effects of the cytotoxic O7-alkyl-
ated analogues 26, 28, and 29 on both cell cycle progression and
spindle structures.16,17 As shown in Figure 3A–E, all the analogues
inhibited the cell cycle progression in the G2/M phase at a concen-
hydroxy group at the C7 position in 22 was alkylated into benzyl
ether 28 and propargyl ether 29 in order to increase lipid solubility.
2.3. Cytotoxicity of glaziovianin A (1) and its analogues against
HeLa S3 cells
tration of 1 lM for 24 h treatment. To confirm whether or not
these compounds also induce abnormal spindle structures with
Table 1 summarizes the cytotoxicity of glaziovianin A (1) and its
analogues against HeLa S3 cells and their calculated LogP (cLogP)
values.14 Compound 12, which has an acetonide group instead of
the methylene acetal group of glaziovianin A (1), showed no cyto-
unaligned chromosomes, as does 1, microtubules and chromo-
somes in mitotic cells treated with 1 l
M O7-alkylated analogues
for 6 h were observed (Fig. 3F–J). Normal bipolar spindles were ob-
served in DMSO-treated cells, but abnormal multipolar spindles
were formed in most of the cells treated with 1 (Fig. 3F and G,
respectively). As expected, analogues 26, 28, and 29 also induced
abnormal multipolar spindles, and these structures were resem-
bled with the spindles induced by 1 (Fig. 3H–J). Because it is well
known that the disruption of a mitotic spindle inhibits mitosis by
activating the spindle checkpoint, these results strongly suggested
that O7-alkylated analogues inhibited cell cycle progression in the
M phase by inducing abnormal spindle formation. Especially, ben-
zyl (28) and propargyl (29) analogues completely arrested cell cy-
cle progression, indicating that these compounds are more potent
cell cycle inhibitors than 1 and 26.
toxicity even at 100 lM. Also, compound 13, which has methoxy
groups at C30 and C40 instead of the methylene acetal group, was
much less cytotoxic than glaziovianin A (1). These results indicated
that the steric hindrance of C30 and C40 at the B-ring part reduced
the cytotoxicity of glaziovianin A (1) to a large extent. Compound
14, which lacks methoxy groups at C20 and C50, was about 100-fold
less cytotoxic than glaziovianin A (1). Also, compound 15, which
has a bromo group at C60 of the B-ring, was less cytotoxic than
glaziovianin A (1). These results suggested that the electron den-
sity and/or steric hindrance of the B-ring might be responsible
for cytotoxicity. On the other hand, compound 21, which has an

5748
I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
Figure 3. Effects of glaziovianin A (1) and O7-alkylated analogues 26, 28, and 29 on cell cycle progression and spindle structures in HeLa S3 cells. (A–E) O7-alkylated analogues
inhibited cell cycle progression in G2/M phase. HeLa S3 cells were treated with DMSO (A), 1 M of glaziovianin A (1) (B), compound 26 (C), 28 (D), or 29 (E) for 24 h, and DNA
contents were determined with flow cytometric analysis. (F–J) O7-alkylated analogues induced abnormal spindle formation. HeLa S3 cells were treated with DMSO (F), 1
l
lM
of glaziovianin A (1) (G), compound 26 (H), 28 (I), or 29 (J) for 6 h, and microtubules (green) and chromosomes (blue) were stained with anti-a-tubulin antibody (DM1A, Santa
Cruz Biotechnology) and DAPI, respectively.
2.5. Synthesis of biotin, fluorescence, and photoaffinity probes
of glaziovianin A
into bromide 33. The coupling reaction between bromide 33 and
O7-demethyl analogue 22, followed by removal of the Boc group
gave amine 34 as a TFA salt. Amidation of amine 34 with (+)-biotin
N-hydroxysuccinimide ester 3520 afforded O7-biotinyl glaziovianin
A (36).
In Section 2.3, we described that O7-alkylated glaziovianin A
analogues showed more potent cytotoxicity against HeLa S3 cells
than glaziovianin A itself. So, we next examined the synthesis of
O7-modified molecular probes of glaziovianin A for biological stud-
ies and target identification. First, we synthesized a biotin probe of
glaziovianin A (1) to confirm the target biomolecule, tubulin
(Scheme 5). Condensation of mono-Boc-1,6-diaminohexane 3018
with 6-benzyloxyhexanoic acid 3119 gave amide 32. Removal of
the benzyl group of 32 afforded an alcohol, which was converted
Next, we synthesized a fluorescent probe for a dynamic analysis
of glaziovianin A in living cells, as well as a photoaffinity probe for
an analysis of the binding site between glaziovianin A and the tar-
get protein. In Scheme 5, the coupling reaction between bromide
33 and O7-demethyl analogue 22 was in low yield, perhaps because
of the low reactivity of bromide 33. Therefore, we next tried to em-
ploy allylic bromide 42 as a linker (Scheme 6). Reduction of known
aldehyde 3721 gave allylic alcohol 38, which was protected by a
TBS group, thus affording TBS ether 39. Condensation of TBS ether
39 and amine 30 gave amide 40. Removal of the TBS group in 40
afforded allylic alcohol 41, which was converted into allylic bro-
mide 42. The coupling reaction between O7-demethyl analogue
22 and allylic bromide 42 gave the coupling compound 43 in good
(72%) yield. Removal of the Boc group in coupling compound 43
gave amine 44 as a TFA salt. Amidation of compound 44 with N-
hydroxysuccinimide esters, such as (+)-biotin analogue 35,20 trif-
luoromethyldiazirine analogue 48,22 and BODIPY analogue 49,23
afforded O7-modified biotin probe 45, photoaffinity probe 46, and
fluorescent probe 47, respectively.
O
a
OBn
5
+
NH2
BocHN
N
H
HOOC
O
OBn
BocHN
b,c
6
5
31
6
30
32
d,e
O
Br
5
BocHN
N
OMe
6
H
33
O
O
MeO
HO
OMe
O
22
7
The cytotoxicities of biotin probes 36 and 45, as well as that of
fluorescent probe 47, against HeLa S3 cells are shown in Table 2.
Each of these probes was about 30-fold less cytotoxic than glazi-
ovianin A (1). However, they all maintained sufficient cytotoxici-
ties to be used as probes.
OMe
OMe
O
O
O
MeO
O
O
S
N
O
7
RO
O
O
H
HN
H
NH
O
3. Conclusion
34
36
35
R =
O
N
6NH2
5
H
In conclusion, we have investigated the structure–cytotoxicity
relationships of glaziovianin A (1). The results revealed that O7-
modified glaziovianin A analogues have strong cytotoxicity against
HeLa S3 cells. Among them, O7-benzyl and O7-propargyl analogues
28 and 29 completely arrested cell cycle progression, indicating
that these compounds are more potent cell cycle inhibitors than
glaziovianin A (1). Furthermore, we have synthesized O7-modified
molecular probes of glaziovianin A for biological studies. Further
searches for target biomolecules of glaziovianin A (1) by using
these probes are in progress.
TFA
O
f
O
N
R =
S
6 N
5
H
H
H
H
HN
NH
O
Scheme 5. Synthesis of biotin probe of glaziovianin A. Reagents and conditions: (a)
DCC, HOBt, DMF, rt, 81%; (b) H2, 5% Pd/C, EtOH, rt, 89%; (c) NBS, PPh3, CH2Cl2, rt,
40%; (d) 22, K2CO3, MeCN, reflux, 34%, recovery of 22: 46%; (e) TFA, CH2Cl2, rt, 62%;
(f) 35, Et3N, DMF, rt, 41%.

I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
5749
Table 2
O
O
Cytotoxicities of glaziovianin A (1) and the molecular probes of glaziovianin A against
HeLa S3 cells
OR
a
6
MeO
MeO
CHO
37
38
R = H
Compound
Cytotoxicity
b
39 R = TBS
IC50
0.59
15.2
15.5
17.4
(
lM)
Relative value
O
Glaziovianin A (1)
Biotin probe 36
Biotin probe 45
BODIPY probe 47
1
c
R
0.039
0.038
0.034
BocHN
N
H
BocHN
NH2
40 R = OTBS
6
d
e
30
41 R = OH
in ppm, relative to the central line of a triplet at 77.0 ppm for deu-
teriochloroform. IR spectra were recorded on a JASCO FT/IR-300
instrument and are reported in wavenumbers (cmꢁ1). High resolu-
tion ESI mass spectra were recorded on an Applied Biosystems
QStar/Pulsar i spectrometer. Fuji Silysia silica gel BW-820MH was
used for column chromatography. Anhydrous benzene, CH2Cl2,
MeOH, THF, toluene, and MeCN were used as obtained from com-
mercial supplies for moisture-sensitive reaction. Other organic sol-
vents for moisture-sensitive reactions were distilled by standard
procedure. MS3A was heated in a microwave oven for 2 min.
42 R = Br
OMe
O
O
OMe
O
O
MeO
HO
O
OMe
O
MeO
RO
7
O
22
OMe
f
7
O
O
O
O
43 R =
N
H
NHBoc
6
g
4.1.2. 4,7-Dimethoxy-2,2-dimethylbenzo[d][1,3]dioxole (7)
To a stirred solution of catechol 6 (54.3 mg, 319
zene (2.5 mL) were added PPTS (5.5 mg, 21.9
l
l
mol) in ben-
mol) and 2-
44 R =
N
H
NH2 TFA
6
methoxypropene (0.10 mL, 1.04 mmol) at room temperature. After
being stirred at reflux for 48 h, the mixture was cooled to room
temperature, diluted with EtOAc (5 mL), and washed with 1 M
aqueous NaOH (3 mL). The EtOAc solution was washed with brine
(3 mL), dried (Na2SO4), and concentrated. The residual solid was
purified by column chromatography on silica gel (0.8 g, hexane–
EtOAc = 40:1) to give acetonide 7 (48.6 mg, 72%) as a white solid:
O
h
i
45 R =
S
N
H
N
6 H
H
HN
H
NH
O
O
O
46
R =
N
H
N
IR (CHCl3) 3024, 2987, 2941, 1612, 1450, 1423, 1055 cmꢁ1 1H
;
6 H
CF3
NMR (270 MHz, CDCl3) d 6.40 (s, 2H), 3.84 (s, 6H), 1.73 (s, 6H);
13C NMR (67.8 MHz, CDCl3) d 120.5 (2C), 118.6 (2C), 117.0 (2C),
106.5, 60.1 (2C), 20.9 (2C); HRMS (ESI) m/z 233.0772, calcd for
N
N
11H14NaO4 [M+Na]+ 233.0784.
F
C
N
j
O
O
B
N
F
F
47 R =
N
N
4.1.3. 5-Bromo-4,7-dimethoxy-2,2-dimethylbenzo[d][1,3]
dioxole (7a)
To a stirred solution of acetonide 7 (48.6 mg, 231
(2.6 mL) was added NBS (34.9 mg, 196 mol) at 0 °C. After being
H
6 H
lmol) in DMF
F
l
O
O
O
O
N
B
N
stirred at room temperature for 18 h in dark, the mixture was di-
luted with water (3 mL) and extracted with CH2Cl2 (2 mL ꢂ 3).
The combined extracts were washed with brine (5 mL), dried
(Na2SO4), and concentrated. The residual solid was purified by col-
umn chromatography on silica gel (2.0 g, hexane–CH2Cl2 = 8:1) to
give bromide 7a (38.2 mg, 57%) as a white solid: IR (CHCl3) 3007,
N
N
O
O
CF3
O
O
N
N
48
49
Scheme 6. Synthesis of biotin, photoaffinity, and fluorescent probes of glaziovianin
A. Reagents and conditions: (a) NaBH4, MeOH, 0 °C, 70%; (b) TBSCl, imidazole, DMF,
rt, 91%; (c) 30, toluene, reflux, 69%; (d) TBAF, THF, rt, 86%; (e) NBS, PPh3, CH2Cl2, rt,
64%; (f) 22, K2CO3, MeCN, reflux, 72%, recovery of 22: 14%; (g) TFA, CH2Cl2, rt, 55%;
(h) 35, Et3N, DMF, rt, 47% (i) 48, Et3N, MeCN, rt, 49%; (j) 49, Et3N, DMF, rt, 65%.
2992, 2941, 1610, 1453, 1430, 1412, 1045, 1026 cmꢁ1 1H NMR
;
(270 MHz, CDCl3) d 6.64 (s, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 1.72 (s,
6H); 13C NMR (67.8 MHz, CDCl3) d 131.2, 130.7, 128.1, 126.9,
126.4, 115.2, 105.8, 61.5, 59.7, 21.3, 21.2; HRMS (ESI) m/z
310.9899, calcd for C11H13BrNaO4 [M+Na]+ 310.9889.
4. Experimental section
4.1. Chemistry
4.1.4. 2-(4,7-Dimethoxy-2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane (8)
To
207
(54.5 mg, 556
mide 7a (39.7 mg, 138
ture. After being stirred at 150 °C under a stream of N2 for 3 h,
the mixture was cooled to room temperature, diluted with EtOAc
(4 mL), and washed with brine (2 mL). The EtOAc solution was
dried (Na2SO4), filtered through a pad of Florisil, and concentrated.
The residual oil was purified by column chromatography on silica
gel (0.8 g, hexane–EtOAc = 100:1 ? 20:1) to give arylboronate 8
(12.9 mg, 28%) as a white solid: IR (CHCl3) 3010, 2987, 2941,
a
stirred solution of bis(pinacolato)diboron (52.5 mg,
mol), PdCl2(dppf)ꢀCH2Cl2 (11.3 mg, 13.8 mol), and KOAc
mol) in DMF (0.60 mL) was added a solution of bro-
mol) in DMF (0.50 mL) at room tempera-
4.1.1. General method
l
l
1H NMR spectra were recorded on a JEOL JNM-EX270 (270 MHz)
or a Bruker AVANCE 500 (500 MHz) spectrometer. Chemical shifts
for 1H NMR are reported in parts per million (ppm) downfield from
tetramethylsilane as the internal standard, 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, and br = broad. 13C NMR spectra were recorded on
l
l
a
JEOL JNM-EX270 (67.8 MHz) or
a Bruker AVANCE 500
(125 MHz) spectrometer. Chemical shifts for 13C NMR are reported

5750
I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
1608, 1452, 1435, 1302, 1058 cmꢁ1
;
1H NMR (270 MHz, CDCl3) d
1H), 4.00 (s, 3H), 3.98 (s, 3H), 3.93 (s, 3H), 3.91 (s, 3H), 3.87 (s,
3H), 3.86 (s, 3H); 13C NMR (67.8 MHz, CDCl3) d 175.0, 154.4,
153.3, 152.0, 147.4, 139.9, 139.3, 137.8, 137.4, 121.5, 119.0,
117.7, 110.6, 104.8, 99.7, 60.8, 58.7, 57.4, 57.1, 56.4, 56.2; HRMS
(ESI) m/z 425.1201, calcd for C21H22NaO8 [M+Na]+ 425.1207.
6.81 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 1.70 (s, 6H), 1.33 (s, 12H);
13C NMR (67.8 MHz, CDCl3) d 142.9, 142.5, 139.1, 138.8, 137.7,
114.0, 106.6, 83.1 (2C), 61.4, 57.2, 25.0 (4C), 21.6, 21.4; HRMS
(ESI) m/z 359.1646, calcd for C17H25BNaO6 [M+Na]+ 359.1636.
4.1.5. 3-(4,7-Dimethoxy-2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-
6,7-dimethoxy-4H-chromen-4-one (12)
4.1.8. 3-(Benzo[d][1,3]dioxol-5-yl)-6,7-dimethoxy-4H-chromen-
4-one (14)
All solvents were degassed by freeze–thawing. To a stirred solu-
To a stirred solution of boronic acid 11 (50.4 mg, 152
PdCl2(dppf)ꢀCH2Cl2 (17.1 mg, 14.8 mol) in 1,4-dioxane (1.0 mL)
were added 1 M Na2CO3 aq (0.75 mL, 750 mol) and iodochromone
3 (108 mg, 323 mol) in 1,4-dioxane (1.0 mL) at room tempera-
lmol) and
tion of arylboronate 8 (12.9 mg, 38.4
(3.1 mg, 3.79 mol) in 1,4-dioxane (0.25 mL) were added aqueous
1 M Na2CO3 (0.19 mL, 190 mol) and iodochromone 3 (19.1 mg,
57.5 mol) in 1,4-dioxane (0.30 mL) at room temperature. After
lmol) and PdCl2(dppf)ꢀCH2Cl2
l
l
l
l
l
l
ture. After being stirred at room temperature under a stream of
N2 for 12 h, the reaction mixture was diluted with water (1 mL)
and extracted with CH2Cl2 (2 mL ꢂ 3). The combined extracts were
washed with brine (3 mL), dried (Na2SO4), filtered through a pad of
Florisil, and concentrated. The residual oil was purified by column
chromatography on silica gel (0.7 g, hexane–CHCl3 = 1:1 ? 1:2) to
give a brown solid (containing Pd-metal) (21.0 mg). The brown so-
lid (21.0 mg) was dissolved in CHCl3 (3 mL), and SiliaBond (SILICY-
CLE, SiliaBond Thiourea, 200 mg) was added. The resulting mixture
was stirred at room temperature for 30 min, filtered, and concen-
trated to give a glaziovianin A analogue 14 (20.2 mg, 41%) as a
white solid: IR (CHCl3) 3005, 2940, 1635, 1608, 1439, 1274,
being stirred at room temperature under a stream of N2 for 36 h,
the mixture was diluted with EtOAc (2 mL) and filtered through
a pad of Florisil. The filtrate was washed with brine (4 mL), dried
(Na2SO4), and concentrated. The residual solid was purified by col-
umn chromatography on silica gel (0.6 g, hexane–EtOAc = 10:1 ?
1:1) to give a brown solid (containing Pd-metal) (10.1 mg, 64%).
The brown solid (10.1 mg) was dissolved in CHCl3 (1 mL), and Sil-
iaBond (SILICYCLE, SiliaBond Thiourea, 100 mg) was added. The
resulting mixture was stirred at room temperature for 30 min, fil-
tered, and concentrated to give glaziovianin
(10.1 mg, 64%) as a white solid: IR (CHCl3) 3016, 2937, 1634,
1605, 1444, 1278, 1160, 1037 cmꢁ1 1H NMR (270 MHz, CDCl3) d
A analogue 12
;
1154 cmꢁ1 1H NMR (270 MHz, CDCl3) d 7.92 (s, 1H), 7.62 (s, 1H),
;
7.92 (s, 1H), 7.62 (s, 1H), 6.87 (s, 1H), 6.49 (s, 1H), 3.99 (s, 3H),
3.98 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 1.67 (s, 3H), 1.65 (s, 3H);
13C NMR (67.8 MHz, CDCl3) d 175.5, 154.0, 153.2, 152.1, 147.4,
139.0, 138.5, 137.0, 136.5, 121.5, 117.9, 117.7, 110.3, 106.4,
104.7, 99.4, 60.2, 56.9, 56.2, 56.0, 20.9, 20.7; HRMS (ESI) m/z
437.1213, calcd for C22H22NaO8 [M+Na]+ 437.1207.
7.11 (d, J = 1.9 Hz, 1H), 6.98 (dd, J = 8.1, 1.9 Hz, 1H), 6.88 (s, 1H),
6.87 (d, J = 8.1 Hz, 1H), 5.99 (s, 2H), 3.99 (s, 3H), 3.99 (s, 3H); 13C
NMR (67.8 MHz, CDCl3) d 175.2, 154.1, 153.1, 152.2, 147.6, 143.0,
142.2, 139.9, 139.0, 121.8, 121.6, 117.6, 113.8, 104.9, 101.8, 99.7,
56.5, 56.3; HRMS (ESI) m/z 349.0683, calcd for
C18H14NaO6
[M+Na]+ 349.0683.
4.1.6. 2,3,4,5-Tetramethoxyphenylboronic acid (10)
4.1.9. 3-(6-Bromo-4,7-dimethoxybenzo[d][1,3]dioxol-5-yl)-6,7-
dimethoxy-4H-chromen-4-one (15)
To
510
hexane, 0.34 mL, 554
a
stirred solution of tetramethoxybenzene
mol) in THF (1.0 mL) was added n-BuLi (1.63 M solution in
mol) slowly at room temperature. After
9 (101 mg,
l
To a stirred solution of glaziovianin A (1) (10.2 mg, 26.4
lmol)
l
in DMF (0.25 mL) was added NBS (4.7 mg, 26.6 mol) at room tem-
l
the mixture was stirred at room temperature for 15 min,
B(OMe)3 (0.34 mL, 2.99 mmol) was added into the mixture at
ꢁ78 °C. The reaction mixture was stirred at room temperature
for 3 h, diluted with aqueous 1 M HCl (2.5 mL), stirred at room
temperature for 3 h, and extracted with ether (2 mL ꢂ 3). The com-
bined extracts were washed with brine (5 mL) and dried (MgSO4).
Removal of the solvent afforded boronic acid 10 (138 mg) as a
white solid, which was used for the next reaction without further
purification.
perature. After being stirred at room temperature for 6 h, the reac-
tion mixture was diluted with water (0.5 mL) and extracted with
CH2Cl2 (1 mL ꢂ 3). The combined extracts were washed with brine
(2 mL), dried (Na2SO4), and concentrated. The residual oil was puri-
fied by column chromatography on silica gel (0.7 g, hexane–
EtOAc = 5:1 ? 2:1) to give a bromide 15 (3.8 mg, 31%) as a white
solid: IR (CHCl3) 3004, 2937, 1640, 1612, 1505, 1428, 1264, 1155,
1047 cmꢁ1 1H NMR (270 MHz, CDCl3) d 7.75 (s, 1H), 7.62 (s, 1H),
;
6.92 (s, 1H), 6.03 (s, 2H), 4.00 (s, 3H), 3.99 (s, 3H), 3.85 (s, 3H),
3.83 (s, 3H); 13C NMR (67.8 MHz, CDCl3) d 175.2, 154.2, 153.3,
152.0, 147.2, 139.1, 138.7, 137.2, 136.5, 121.6, 118.3, 117.6,
104.9, 101.8, 99.5, 86.4, 60.5, 57.2, 56.4, 56.2; HRMS (ESI) m/z
486.9985, calcd for C20H17BrNaO8 [M+Na]+ 486.9999.
4.1.7. 6,7-Dimethoxy-3-(2,3,4,5-tetramethoxyphenyl)-4H-chro-
men-4-one (13)
To
152
dioxane (1.0 mL) were added 1 M Na2CO3 aq (0.75 mL, 750
and iodochromone 3 (103 mg, 310 mol) in 1,4-dioxane (1.0 mL)
a
stirred solution of crude boronic acid 10 (41.0 mg,
mol) and PdCl2(dppf)ꢀCH2Cl2 (17.8 mg, 15.4 mol) in 1,4-
mol)
l
l
l
4.1.10. 1-(2-Hydroxy-5-methoxy-4-(tetrahydro-2H-pyran-2-
yloxy)phenyl)ethanone (17)
l
at room temperature. After being stirred at room temperature un-
der a stream of N2 for 17 h, the reaction mixture was diluted with
water (1 mL) and extracted with CH2Cl2 (2 mL ꢂ 3). The combined
extracts were washed with brine (3 mL), dried (Na2SO4), filtered
through a pad of Florisil, and concentrated. The residual oil was
purified by column chromatography on silica gel (3.0 g, hexane–
EtOAc = 4:1 ? 2:1) to give a brown solid (containing Pd-metal)
(7.0 mg). The brown solid (7.0 mg) was dissolved in CHCl3 (1 mL),
and SiliaBond (SILICYCLE, SiliaBond Thiourea, 70 mg) was added.
The resulting mixture was stirred at room temperature for
30 min, filtered, and concentrated to give glaziovianin A analogue
13 (7.0 mg, 11% in 2 steps) as a white solid: IR (CHCl3) 3010,
To a stirred solution of phenol 16 (16.5 mg, 90.7
CH2Cl2 (0.90 mL) were added DHP (102 L, 1.12 mol) and PPTS
(3.2 mg, 12.7 mol) at 0 °C. After being stirred at room tempera-
lmol) in
l
l
l
ture for 18 h, the reaction mixture was diluted with saturated
aqueous NaHCO3 (1 mL) and extracted with CH2Cl2 (2 mL ꢂ 3).
The combined extracts were washed with brine (2 mL), dried
(Na2SO4), and concentrated. The residual solid was purified by col-
umn chromatography on silica gel (1.5 g, hexane–EtOAc = 20:1) to
give THP ether 17 (19.3 mg, 80%) as a white solid: IR (CHCl3) 3518,
3014, 2950, 1632, 1504, 1372, 1330, 1261, 1228, 1216, 1208 cmꢁ1
;
1H NMR (270 MHz, CDCl3) d 12.5 (s, 1H), 7.12 (s, 1H), 6.74 (s, 1H),
5.50 (t, J = 3.0 Hz, 1H), 3.89–3.82 (m, 1H), 3.85 (s, 3H), 3.66–3.62
(m, 1H), 2.56 (s, 3H), 2.01–1.89 (m, 3H), 1.74–1.64 (m, 3H); 13C
NMR (67.8 MHz, CDCl3) d 201.8, 159.3, 154.2, 142.1, 113.3, 112.3,
2946, 1637, 1606, 1448, 1297, 1271, 1072 cmꢁ1
;
1H NMR
(270 MHz, CDCl3) d 7.92 (s, 1H), 7.62 (s, 1H), 6.88 (s, 1H), 6.67 (s,

I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
5751
104.3, 96.6, 62.1, 57.1, 29.9, 26.2, 24.8, 18.5; HRMS (ESI) m/z
4.1.14. 3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-5,6,7-tri-
methoxy-4H-chromen-4-one (21)
289.1051, calcd for C14H18NaO5 [M+Na]+ 289.1046.
All solvents were degassed by freeze–thawing. To a stirred solu-
tion of arylboronate 5 (20.6 mg, 66.9
mol) and PdCl2(dppf)ꢀCH2Cl2
(5.8 mg, 7.10 mol) in 1,4-dioxane (0.45 mL) were added aqueous
1 M Na2CO3 (0.32 mL, 320 mol) and iodochromone 19 (36.5 mg,
101 mol) in 1,4-dioxane (0.48 mL) at room temperature. After
4.1.11. (E)-3-(Dimethylamino)-1-(2-hydroxy-5-methoxy-4-(tet-
rahydro-2H-pyran-2-yloxy)phenyl)prop-2-en-1-one (17a)
l
l
THP ether 17 (19.3 mg, 72.6
lmol) was dissolved in N,N-
l
dimethylformamide dimethyl acetal (0.10 mL, 754
l
mol). The
l
reaction mixture was stirred at 90 °C for 12 h and concentrated
to afford enamine 17a (23.3 mg, quant.) as a yellow solid. Enamine
17a was used for next reaction without further purification: IR
(CHCl3) 3689, 2949, 1631, 1541, 1507, 1372, 1284, 1222, 1209,
being stirred at room temperature under a stream of N2 for 48 h,
the reaction mixture was diluted with EtOAc (2 mL) and filtered
through a pad of Florisil. The filtrate was washed with brine
(4 mL), dried (Na2SO4), and concentrated. The residual solid was
purified by column chromatography on silica gel (1.5 g, hexane–
EtOAc = 5:1 ? 2:1) to give glaziovianin A analogue 21 (4.5 mg,
16%) as a white solid: IR (CHCl3) 3013, 2998, 2933, 1646, 1611,
1108 cmꢁ1 1H NMR (270 MHz, CDCl3) d 14.0 (s, 1H), 7.84 (d,
;
J = 12.4 Hz, 1H), 7.18 (s, 1H), 6.72 (s, 1H), 5.62 (d, J = 12.4 Hz, 1H),
5.46 (t, J = 3.2 Hz, 1 H), 3.89 (m, 1H), 3.85 (s, 3H), 3.63 (m, 1H),
3.22 (br s, 3H), 3.17 (br s, 3H), 2.01–1.88 (m, 3H), 1.72–1.61 (m,
3H); 13C NMR (67.8 MHz, CDCl3) d 190.2, 160.0, 154.0, 152.7,
141.7, 113.6, 112.9, 105.0, 96.8, 89.8, 62.4, 58.2, 40.3 (2C), 30.2,
25.1, 19.0; HRMS (ESI) m/z 344.1471, calcd for C17H23NNaO5
[M+Na]+ 344.1474.
1508, 1428, 1231, 1157, 1068 cmꢁ1 1H NMR (270 MHz, CDCl3)
;
d7.88 (s, 1H), 6.71 (s, 1H), 6.52 (s, 1H), 6.01 (s, 2H), 3.96 (s, 3H),
3.95 (s, 3H), 3.92 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H); 13C NMR
(67.8 MHz, CDCl3) d 175.2, 154.7, 153.3, 151.6, 148.9, 142.8,
139.0, 138.7, 136.9, 136.6, 121.4, 117.9, 113.1, 109.8, 101.7, 92.5,
60.3, 56.9, 56.6, 56.4, 56.3; HRMS (ESI) m/z 439.0999, calcd for
4.1.12. 3-Iodo-6-methoxy-7-(tetrahydro-2H-pyran-2-yloxy)-4H-
C
21H20NaO9 [M+Na]+ 439.1000.
chromen-4-one (18)
To a stirred solution of enamine 17a (150 mg, 467
CHCl3 (4.7 mL) were added pyridine (0.19 mL, 2.36 mmol) and I2
(250 mg, 984 mol) at 0 °C. After being stirred at room tempera-
lmol) in
4.1.15. 3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-7-hydroxy-
6-methoxy-4H-chromen-4-one (22)
l
To a stirred solution of glaziovianin A analogue 20 (24.0 mg,
ture for 12 h in dark, the mixture was diluted with saturated aque-
ous Na2S2O3 (5 mL), stirred at room temperature for 10 min, and
extracted with EtOAc (5 mL ꢂ 3). The combined extracts were
washed with brine (10 mL), dried (Na2SO4), and concentrated.
The residual solid was purified by column chromatography on sil-
ica gel (8 g, hexane–EtOAc = 10:1 ? 4:1) to give iodochromone 18
(140 mg, 75%) as a white solid: IR (CHCl3) 3024, 2999, 1640, 1605,
52.6
l
mol) in CHCl3 (0.50 mL) and MeOH (0.10 mL) was added p-
mol) at room temperature. After the
TsOHꢀH2O (1.1 mg, 5.79
l
mixture was stirred at room temperature for 3 h, Et3N (0.01 mL)
was added. Removal of solvent gave a solid, which was purified
by column chromatography on silica gel (0.6 g, hexane–
EtOAc = 1:1 ? 1:3) to give O7-demethyl analogue 22 (16.7 mg,
85%) as a white solid: IR (CHCl3) 3610, 3015, 2945, 1635, 1599,
1541, 1503, 1350, 1219, 1203 cmꢁ1 1H NMR (270 MHz, CDCl3) d
;
1502, 1461, 1288 cmꢁ1 1H NMR (270 MHz, CDCl3) d 7.89 (s, 1H),
;
8.22 (s, 1H), 7.55 (s, 1H), 7.20 (s, 1H), 5.55 (t, J = 2.7 Hz, 1H), 3.95
(s, 3H), 3.91–3.82 (m, 1H), 3.68–3.63 (m, 1H), 2.10–1.87 (m, 3H),
1.77–1.62 (m, 3H); 13C NMR (67.8 MHz, CDCl3) d 172.2, 156.8,
156.7, 151.7, 148.5, 115.6, 105.2, 103.7, 97.1, 86.2, 62.0, 56.3,
29.9, 24.9, 18.3; HRMS (ESI) m/z 424.9867, calcd for C15H15INaO5
[M+Na]+ 424.9862.
7.64 (s, 1H), 6.98 (s, 1H), 6.52 (s, 1H), 6.02 (s, 2H), 4.02 (s, 3H),
3.87 (s, 3H), 3.85 (s, 3H), (OH proton was not observed); 13C
NMR (67.8 MHz, CDCl3) d 175.3, 153.8, 151.6, 151.8, 148.2, 139.1,
138.8, 137.1, 136.6, 121.2, 118.5, 117.6, 109.7, 105.3, 104.0,
102.0, 60.1, 56.9, 56.6; HRMS (ESI) m/z 395.0745, calcd for
C
19H16NaO8 [M+Na]+ 395.0737.
4.1.13. 3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-6-methoxy-
7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (20)
All solvents were degassed by freeze–thawing. To a stirred solu-
4.1.16. (3R,4S,5R)-2-(Acetoxymethyl)-6-(3-(4,7-dimethoxybenzo
[d][1,3]dioxol-5-yl)-6-methoxy-4-oxo-4H-chromen-7-yloxy)tetr
ahydro-2H-pyran-3,4,5-triyl triacetate (24)
tion of arylboronate 5 (10.1 mg, 32.8
(2.2 mg, 2.69 mol) in 1,4-dioxane (0.10 mL) were added aqueous
1 M Na2CO3 (0.13 mL) and iodochromone 18 (10.4 mg, 25.9 mol)
l
mol) and PdCl2(dppf)ꢀCH2Cl2
To a stirred solution of O7-demethyl analogue 22 (24.7 mg,
l
66.4
solution of imidate 23 (65.1 mg, 133
(0.50 mL) and BF3ꢀEt2O (0.03 mL, 243 mol) at 0 °C. After being
l
mol) and MS3A (244 mg) in CH2Cl2 (0.50 mL) were added
l
a
l
mol) in CH2Cl2
in 1,4-dioxane (0.22 mL) at room temperature. After being stirred
at room temperature under a stream of N2 for 24 h, the reaction
mixture was diluted with water (1 mL) and extracted with CH2Cl2
(1 mL ꢂ 3). The combined extracts were washed with brine (2 mL),
dried (Na2SO4), filtered through a pad of Florisil, and concentrated.
The residual oil was purified by column chromatography on silica
gel (0.7 g, hexane–EtOAc = 4:1 ? 2:1) to give a brown oil (7.9 mg).
The brown oil (7.9 mg) was dissolved in CHCl3 (1 mL), and Silia-
Bond (SILICYCLE, SiliaBond Thiourea, 80 mg) was added. The
resulting mixture was stirred at room temperature for 30 min, fil-
tered, and concentrated to give a glaziovianin A analogue 20
(7.8 mg, 66%) as a white solid: IR (CHCl3) 3007, 2948, 1639, 1607,
l
stirred at room temperature for 12 h, the mixture was diluted
with water (2 mL) and extracted with CH2Cl2 (2 mL ꢂ 3). The
combined extracts were washed with brine (5 mL), dried
(Na2SO4), and concentrated. The residual solid was purified by
column chromatography on silica gel (0.7 g, hexane–EtOAc = 4:1
? 2:1) to give tetraacetyl glycosylisoflavone 24 (mixture of ano-
meric isomers,
as white solids: 24 (b-isomer): IR (CHCl3) 3009, 2940, 1672,
1667, 1646, 1522, 1434, 1245 cmꢁ1 1H NMR (270 MHz, pyri-
a-isomer: 1.9 mg, 4.1%; b-isomer: 10.7 mg, 23%)
;
dine-d5) d 8.19 (s, 1H), 7.84 (s, 1H), 7.57 (s, 1H), 6.77 (s, 1H),
5.99 (s, 2H), 5.92 (d, J = 8.4 Hz, 1H), 4.67–4.45 (m, 6H), 4.00 (s,
3H), 3.80 (s, 3H), 3.70 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.05 (s,
3H), 2.04 (s, 3H); 13C NMR (67.8 MHz, pyridine-d5) d 173.8,
170.9, 170.4, 169.8, 169.3, 157.8, 153.9, 151.8, 151.4, 140.0,
138.9, 137.8, 136.7, 126.5, 120.7, 118.9, 109.4, 107.1, 106.9,
103.4, 101.5, 72.9, 71.8, 71.3, 68.5, 64.2, 62.0, 58.4, 58.2, 21.8,
21.3, 20.8, 20.5; HRMS (ESI) m/z 725.1694, calcd for C33H34NaO17
[M+Na]+ 725.1688.
1500, 1468, 1430, 1352, 1297 cmꢁ1 1H NMR (270 MHz, CDCl3) d
;
7.90 (s, 1H), 7.62 (s, 1H), 7.22 (s, 1H), 6.52 (s, 1H), 6.01 (s, 2H),
5.56 (s, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 2.06–1.97 (m,
4H), 1.72–1.68 (m, 4H); 13C NMR (67.8 MHz, CDCl3) d 175.4,
153.5, 151.8, 151.4, 148.2, 139.0, 138.8, 136.9, 136.6, 121.3,
118.4, 118.0, 109.9, 105.3, 104.2, 101.9, 97.1, 62.1, 60.2, 56.9,
56.4, 30.1, 25.1, 18.5; HRMS (ESI) m/z 479.1310, calcd for
C
24H24NaO9 [M+Na]+ 479.1313.

5752
I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
4.1.17. 3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-6-methoxy-
7-((3R,4S,5S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-
2H-pyran-2-yloxy)-4H-chromen-4-one (25)
37.7 lmol) and benzyl bromide (3.2 lL, 26.9 lmol). After being
stirred at room temperature for 5 h, the mixture was diluted with
saturated aqueous NaHCO3 (1 mL) and extracted with CH2Cl2
(2 mL ꢂ 3). The combined extracts were washed with brine
(2 mL), dried (Na2SO4), and concentrated. The residual solid was
purified by column chromatography on silica gel (0.6 g, hexane–
EtOAc = 5:1 ? 3:1) to give benzyl ether 28 (6.8 mg, 80%) as a white
solid: IR (CHCl3) 3007, 2939, 1606, 1470, 1298, 1227, 1153,
To a stirred solution of tetraacetyl glycosylisoflavone 24 (mix-
ture of anomeric isomers, 4.5 mg, 6.41
lmol) in MeOH (0.09 mL)
was added NaOMe (3.5 mg, 69.4 mol) at 0 °C. After being stirred
l
at room temperature for 1 h, the mixture was diluted with water
(1 mL) and extracted with CHCl3 (1 mL ꢂ 6). The combined extracts
were dried (Na2SO4) and concentrated to give mixture of glycoside
1064 cmꢁ1 1H NMR (270 MHz, CDCl3) d 7.87 (s, 1H), 7.63 (s, 1H),
;
25 (
25 (b-isomer): IR (CHCl3) 3540, 3015, 2944, 1646, 1530, 1456,
1435, 1246 cmꢁ1 1H NMR (270 MHz, pyridine-d5) d 8.07 (s, 1H),
a
-isomer: 0.4 mg, 12%; b-isomer 2.7 mg, 79%) as white solids:
7.49–7.19 (m, 5H), 6.90 (s, 1H), 6.51 (s, 1H), 6.02 (s, 1H), 5.27 (s,
2H), 3.99 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H); 13C NMR (67.8 MHz,
CDCl3) d 175.3, 153.3, 153.1, 151.9, 147.9, 139.0, 138.9, 137.0,
136.7, 135.4, 128.7 (2C), 128.3, 127.2 (2C), 121.6, 118.0, 117.9,
110.0, 105.1, 101.8, 101.3, 71.1, 60.2, 56.9, 56.4; HRMS (ESI) m/z
463.1379, calcd for C26H23O8 [M+H]+ 463.1387.
;
7.86 (s, 1H), 7.64 (s, 1H), 6.74 (s, 1H), 5.99 (s, 2H), 5.89 (d,
J = 7.0 Hz, 1H), 4.67–4.61 (m, 1H), 4.44–4.26 (m, 5H), 3.99 (s, 3H),
3.79 (s, 3H), 3.63 (s, 3H), (Signals due to four protons (OH) were
not observed); 13C NMR (67.8 MHz, pyridine-d5) d 173.7, 157.5,
153.4, 150.7, 150.4, 141.1, 139.3, 137.7, 136.6, 127.7, 120.9,
115.6, 109.2, 107.1, 106.3, 103.5, 100.9, 74.8, 71.8, 71.4, 68.5,
64.4, 61.9, 58.4, 58.1; HRMS (ESI) m/z 557.1253, calcd for
4.1.21. 3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-6-methoxy-
7-(prop-2-ynyloxy)-4H-chromen-4-one (29)
To a stirred solution of O7-demethyl analogue 22 (6.1 mg,
C
25H26NaO13 [M+Na]+ 557.1266.
16.4
32.6
l
l
mol) in MeCN (0.16 mL) were added K2CO3 (4.5 mg,
mol) and propargyl bromide (2.1 L, 23.6 mol). After being
l
l
4.1.18. 7-(Allyloxy)-3-(4,7-dimethoxybenzo[d][1,3]dioxol-5-yl)-
6-methoxy-4H-chromen-4-one (26)
stirred at room temperature for 5 h, the mixture was diluted with
saturated aqueous NaHCO3 (1 mL) and extracted with CH2Cl2
(2 mL ꢂ 3). The combined extracts were washed with brine
(2 mL), dried (Na2SO4), and concentrated. The residual solid was
purified by column chromatography on silica gel (0.6 g, hexane–
EtOAc = 5:1 ? 3:1) to give propargyl ether 29 (4.5 mg, 67%) as a
white solid: IR (CHCl3) 3306, 3008, 2934, 1638, 1609, 1502, 1470,
To a stirred solution of O7-demethyl analogue 22 (6.1 mg,
16.4
32.6
l
l
mol) in MeCN (0.16 mL) were added K2CO3 (4.5 mg,
mol) and allyl bromide (2.1 L, 23.6 mol) at room tempera-
l
l
ture. After being stirred at room temperature for 5 h, the mixture
was diluted with saturated aqueous NaHCO3 (1 mL) and extracted
with CH2Cl2 (2 mL ꢂ 3). The combined extracts were washed with
brine (2 mL), dried (Na2SO4), and concentrated. The residual solid
was purified by column chromatography on silica gel (0.6 g, hex-
ane–EtOAc = 5:1 ?3:1) to giveallylether 26(5.5 mg, 81%) as a white
solid: IR (CHCl3) 3008, 2938, 1639, 1607, 1503, 1469, 1430, 1267,
1099, 1063 cmꢁ1; 1H NMR (270 MHz, CDCl3) d 7.89 (s, 1H), 7.62 (s,
1H), 6.89 (s, 1H), 6.52 (s, 1H), 6.11 (ddt, J = 17.6, 10.5, 5.4 Hz , 1H),
6.02 (s, 2H), 5.48 (ddt, J = 17.6, 1.4, 1.4 Hz, 1H), 5.38 (ddt, J = 10.5,
1.4, 1.4 Hz, 1H), 4.72 (dt, J = 5.4, 1.4 Hz, 2H), 3.98 (s, 3H), 3.87 (s,
3H), 3.85 (s, 3H); 13C NMR (67.8 MHz, CDCl3) d 175.2, 154.4, 153.4,
152.0, 147.8, 139.0, 138.9, 137.0, 136.6, 135.8, 121.5, 118.0, 117.7,
116.5, 110.0, 105.2, 101.8, 100.9, 73.3, 62.1, 57.0, 56.4; HRMS (ESI)
m/z 435.1057, calcd for C22H20NaO8 [M+Na]+ 435.1050.
1431, 1398, 1267, 1231, 1209, 1099 cmꢁ1 1H NMR (270 MHz,
;
CDCl3) d 7.92 (s, 1H), 7.64 (s, 1H), 7.08 (s, 1H), 6.52 (s, 1H), 6.03
(s, 2H), 4.90 (d, J = 2.2 Hz, 2H), 3.99 (s, 3H), 3.87 (s, 3H), 3.85 (s,
3H), 2.61 (s, 1H); 13C NMR (67.8 MHz, CDCl3) d 175.2, 153.5,
151.7, 147.8, 139.0, 138.9, 137.0, 136.7, 132.0, 121.7, 118.5,
117.9, 110.0, 105.4, 101.8, 101.6, 84.1, 77.2, 60.2, 60.1, 56.9, 56.4;
HRMS (ESI) m/z 411.1068, calcd for C22H18NaO8 [M+H]+ 411.1074.
4.1.22. tert-Butyl 6-(6-(benzyloxy)hexanamido)hexylcarbamate
(32)
To a stirred solution of 6-benzyloxyhexanoic acid 31 (97.3 mg,
450
and DCC (103 mg, 500
at 0 °C for 15 min, mono-Boc-1,6-diaminohexane 30 (107 mg,
461 mol) was added at 0 °C. After being stirred at room tempera-
l
mol) in DMF (3.5 mL) were added HOBt (67.3 mg, 499
lmol)
lmol) at 0 °C. After the mixture was stirred
4.1.19. 7-(2,3-Dihydroxypropoxy)-3-(4,7-dimethoxybenzo[d] [1,3]
dioxol-5-yl)-6-methoxy-4H-chromen-4-one (27)
l
ture for 12 h, the mixture was filtered and concentrated. The crude
product was diluted with EtOAc (10 mL), washed with saturated
aqueous NaHCO3 (5 mL), water (5 mL), saturated aqueous NH4Cl
(5 mL), and brine (5 mL). The combined extracts were dried
(Na2SO4) and concentrated. The residual oil was purified by col-
umn chromatography on silica gel (3.0 g, CHCl3–MeOH = 1:0 ?
20:1) to give amide 32 (153 mg, 81%) as a white solid: IR (CHCl3)
To a stirred solution of allyl ether 26 (5.0 mg, 12.1
idine (0.12 mL) was added OsO4 (0.4 M solution in THF, 0.04 mL,
16.0 mol) at room temperature. After being stirred at room tem-
lmol) in pyr-
l
perature for 1.5 h, the reaction mixture was diluted with saturated
aqueous NaHSO3 (1 mL), stirred at room temperature for 1 h, and ex-
tracted with CH2Cl2 (1 mL ꢂ 3). The combined extracts were washed
with brine (2 mL), dried (Na2SO4), and concentrated. The residualso-
lid was purified by column chromatography on silica gel (0.6 g, hex-
ane–EtOAc = 1:1 ? 1:4) to give diol 27 (4.5 mg, 84%) as a white solid:
IR (CHCl3) 3508, 2968, 1637, 1608, 1505, 1458, 1267, 1101,
3321, 3031, 2981, 1661, 1562, 1559, 1243 cmꢁ1
;
1H NMR
(270 MHz, CDCl3) d 7.37–7.26 (m, 5H), 5.76 (br s, 1H), 4.61 (br s,
1H), 4.49 (s, 2H), 3.47 (t, J = 6.5 Hz, 2H), 3.21 (dt, J = 6.8, 6.5 Hz,
2H), 3.09 (dt, J = 6.8, 6.5 Hz, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.71–1.58
(m, 6H), 1.48–1.31 (m, 8H), 1.44 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 172.9, 156.1, 138.6, 128.3 (2C), 127.6 (2C), 127.5, 79.0, 72.9, 70.2,
40.2, 39.1, 36.7, 33.9, 30.0, 29.5, 28.4 (3C), 26.2, 26.0, 25.6, 25.5;
1060 cmꢁ1 1H NMR (270 MHz, CDCl3) d 7.91 (s, 1H), 7.62 (s, 1H),
;
6.92 (s, 1H), 6.52 (s, 1H), 6.03 (s, 2H), 4.28–4.19 (m, 5H), 3.96 (s,
3H), 3.87 (s, 3H), 3.86 (s, 3H), (Signals due to two protons (OH)
was not observed); 13C NMR (67.8 MHz, CDCl3) d 175.3, 154.4,
153.4, 151.8, 147.6, 139.1, 138.9, 137.0, 136.6, 135.8, 121.5, 118.0,
117.7, 116.5, 110.0, 105.2, 101.8, 100.9, 73.3, 62.1, 57.0, 56.4; HRMS
(ESI) m/z 435.1089, calcd for C22H22NaO10 [M+Na]+ 469.1105.
HRMS (ESI) m/z 443.2880, calcd for
C
24H40N2NaO4 [M+Na]+
443.2880.
4.1.23. tert-Butyl 6-(6-hydroxyhexanamido)hexylcarbamate (32a)
To a stirred solution of amide 32 (153 mg, 364 mol) in EtOH
l
4.1.20. 7-(Benzyloxy)-3-(4,7-dimethoxybenzo[d][1,3]dioxol-5-
yl)-6-methoxy-4H-chromen-4-one (28)
(4.0 mL) was added 5% Pd/C (50% water wet, 80.0 mg) at room
temperature. After being stirred at room temperature for 12 h
under H2, the mixture was filtered through a pad of Celite and
concentrated. The residual solid was purified by column chroma-
To a stirred solution of O7-demethyl analogue 22 (6.8 mg,
18.3 lmol) in MeCN (0.18 mL) were added K2CO3 (5.2 mg,

I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
5753
tography on silica gel (2.5 g, CHCl3–MeOH = 100:1 ? 20:1) to give
alcohol 32a (107 mg, 89%) as a white solid: IR (CHCl3) 3342, 2975,
1655, 1558 cmꢁ1; 1H NMR (270 MHz, CDCl3) d 5.98 (br s, 1H), 4.67
(br s, 1H), 3.60 (t, J = 6.5 Hz, 2H), 3.19 (q, J = 6.5 Hz, 2H), 3.07 (q,
J = 6.5 Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H), 1.65 (quin, J = 7.2 Hz, 2H),
1.57 (quin, J = 6.5 Hz, 2H), 1.50–1.41 (m, 6H), 1.44 (s, 9H), 1.37–
1.29 (m, 4H), (A signal due to one proton (OH) was not observed);
13C NMR (125 MHz, CDCl3) d 173.0, 156.1, 79.1, 62.5, 40.1, 39.1,
36.6, 32.2, 29.9, 29.4, 28.4 (3C), 26.1, 26.0, 25.3, 25.1; HRMS (ESI)
m/z 353.2423, calcd for C17H34N2NaO4 [M+Na]+ 353.2411.
(s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.20–3.13 (m, 2H), 2.96–2.90
(m, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.95–1.89 (m, 2H), 1.74–1.25 (m,
12H), (Signals due to two protons (NH) were not observed); 13C
NMR (67.8 MHz, CDCl3) d 175.2, 171.6, 153.8, 151.8, 151.6, 148.2,
139.1, 138.8, 137.1, 136.6, 121.2, 118.5, 117.6, 109.7, 105.3,
104.0, 102.0, 67.4, 60.1, 56.9, 56.6, 40.5, 39.2, 37.5, 36.7, 36.6,
36.0, 32.0, 29.7, 29.5, 25.5; HRMS (ESI) m/z 607.2638, calcd for
C
31H40N2NaO9 [M+Na]+ 607.2626.
4.1.27. 6-(3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-6-meth-
oxy-4-oxo-4H-chromen-7-yloxy)-N-(6-(5-((3aS,4S,6aR)-2-oxo-
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexyl)
hexanamide (36)
4.1.24. tert-Butyl 6-(6-bromohexanamido)hexylcarbamate (33)
To a stirred solution of alcohol 32a (107 mg, 324
CH2Cl2 (3.2 mL) were added NBS (69.4 mg, 390 mol) and Ph3P
(102 mg, 389 mol) at room temperature. After being stirred at
lmol) in
l
To a stirred solution of amine 34 (2.7 mg, 3.87
biotin N-hydroxysuccinimide ester 35 (2.0 mg, 5.87
DMF (0.08 mL) was added Et3N (3.0 L, 21.6 mol) at room tem-
l
mol) and (+)-
l
lmol) in
room temperature for 14 h, the mixture was diluted with water
(5 mL) and extracted with CH2Cl2 (5 mL ꢂ 3). The combined
extracts were washed with brine (10 mL), dried (Na2SO4), and con-
centrated. The residual oil was purified by column chromatogra-
phy on silica gel (5.0 g, CHCl3–MeOH = 100:1) to give bromide 33
(51.4 mg, 40%) as a white solid: IR (CHCl3) 3327, 2970, 1649,
l
l
perature. After being stirred at room temperature for 14 h, the mix-
ture was diluted with brine (0.5 mL) and extracted with CH2Cl2
(1 mL ꢂ 3). The combined extracts were dried (Na2SO4) and con-
centrated. The residual oil was purified by column chromatogra-
phy on silica gel (0.6 g, CHCl3–MeOH = 50:1 ? 20:1) and by
recycle HPLC [JAIGEL-1H-40 (600 ꢂ 20 mm) and JAIGEL-2H-40
(600 ꢂ 20 mm), flow rate 3.8 mL/min; detection, UV 265 nm; sol-
vent CHCl3] to give O7-biotinyl glaziovianin A (36) (2.7 mg, 41%)
as a white solid: IR (CHCl3) 3340, 3031, 2974, 1719, 1643, 1562,
1553, 676 cmꢁ1 1H NMR (270 MHz, CDCl3) d 5.88 (br s, 1H), 4.61
;
(br s, 1H), 3.39 (t, J = 7.0 Hz, 2H), 3.21 (q, J = 6.5 Hz, 2H), 3.09 (q,
J = 6.5 Hz, 2 H), 2.17 (t, J = 7.3 Hz, 2H), 1.85 (quin, J = 7.0 Hz, 2H),
1.65 (quin, J = 7.3 Hz, 2H), 1.52–1.29 (m, 10H), 1.43 (s, 9H); 13C
NMR (67.8 MHz, CDCl3) d 171.8, 156.2, 79.4, 67.4, 40.5, 39.2,
36.6, 32.0, 29.7, 29.5, 28.9 (3C), 27.5, 26.7, 26.0, 25.5; HRMS (ESI)
m/z 415.1569 calcd for C17H33BrN2NaO3 [M+Na]+ 415.1567.
1510, 1286, 1202 cmꢁ1 1H NMR (270 MHz, CDCl3) d 7.90 (s, 1H),
;
7.56 (s, 1H), 6.85 (s, 1H), 6.52 (s, 1H), 6.31 (br s, 1H), 6.16 (br s,
1H), 6.00 (s, 2H), 5.27 (s, 1H), 5.02 (s, 1H), 4.60–4.53 (m, 1H),
4.39–4.31 (m, 1H), 4.10 (t, J = 5.9 Hz, 2H), 3.95 (s, 3H), 3.87 (s,
3H), 3.85 (s, 3H), 3.25–3.09 (m, 5H), 2.94–2.89 (m, 2H), 2.31 (t,
J = 7.3 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.84–1.33 (m, 20H); 13C
NMR (125 MHz, CDCl3) d 178.8, 175.3, 171.4, 170.0, 153.8, 151.8,
151.5, 148.1, 139.1, 138.6, 137.0, 136.8, 121.2, 118.4, 117.5,
109.7, 105.3, 103.9, 102.1, 67.4, 60.0, 56.9, 56.7, 55.6, 53.9, 45.4,
40.7, 40.5, 39.1, 37.5, 37.0, 36.4, 35.9, 35.5, 32.3, 29.8, 29.5, 27.8,
26.7, 26.4, 25.5; HRMS (ESI) m/z 833.3385, calcd for
4.1.25. tert-Butyl 6-(6-(3-(4,7-dimethoxybenzo[d][1,3]dioxol-5-
yl)-6-methoxy-4-oxo-4H-chromen-7-yloxy)hexanamido)
hexylcarbamate (33a)
To a stirred solution of O7-demethyl analogue 22 (6.9 mg,
18.5
lmol) and bromide 33 (21.1 mg, 53.8
lmol) in CH2Cl2
(0.27 mL) was added K2CO3 (7.4 mg, 53.6
l
mol) at 0 °C. After being
stirred at reflux for 18 h, the mixture was diluted with water
(1 mL) and extracted with CH2Cl2 (1 mL ꢂ 3). The combined ex-
tracts were washed with brine (1 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatography on
silica gel (0.6 g, hexane–EtOAc = 1:4) to give coupling compound
33a (4.3 mg, 34%) as a white solid, and recovery of O7-demethyl
analogue 22 (3.2 mg, 46%) as a white solid: IR (CHCl3) 3344,
C
41H54N4NaO11S [M+Na]+ 833.3402.
4.1.28. (E)-Methyl 6-hydroxyhex-4-enoate (38)
To a stirred solution of aldehyde 37 (535 mg, 3.77 mmol) in
MeOH (15 mL) was added NaBH4 (171 mg, 4.52 mmol) at 0 °C.
After being stirred at 0 °C for 1 h, the mixture was diluted with sat-
urated aqueous NH4Cl (8 mL) and water (30 mL) and extracted
with CH2Cl2 (20 mL ꢂ 3). The combined extracts were dried
(Na2SO4) and concentrated. The residual oil was purified by col-
umn chromatography on silica gel (12 g, EtOAc–hexane = 3:1) to
give allylic alcohol 38 (380 mg, 70%) as a white solid: IR (CHCl3)
3020, 2967, 1657, 1600, 1568, 1502, 1288 cmꢁ1
;
1H NMR
(270 MHz, CDCl3) d 7.89 (s, 1H), 7.60 (s, 1H), 6.86 (s, 1H), 6.52 (s,
1H), 6.02 (s, 2H), 5.64 (br s, 1H), 4.55 (br s, 1H), 4.10 (t,
J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.24 (q,
J = 6.5 Hz, 2H), 3.10 (q, J = 6.5 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.95
(quin, J = 7.0 Hz, 2H), 1.80–1.70 (m, 2H), 1.57–1.23 (m, 10H), 1.44
(s, 9H); 13C NMR (67.8 MHz, CDCl3) d 175.3, 171.6, 156.2, 153.8,
151.8, 151.6, 148.2, 139.1, 138.8, 137.1, 136.6, 121.2, 118.5,
117.6, 109.7, 105.3, 104.0, 102.0, 79.4, 67.4, 60.1, 56.9, 56.6, 40.5,
39.2, 37.5, 36.9, 36.6, 36.0, 32.0, 29.7, 29.5, 28.9 (3C), 25.5; HRMS
(ESI) m/z 707.3139, calcd for C36H48N2NaO11 [M+Na]+ 707.3150.
3340, 2986, 1739, 1670 cmꢁ1 1H NMR (270 MHz, CDCl3) d 5.71–
;
5.68 (m, 2H), 4.34 (dt, J = 5.4, 1.6 Hz, 2H), 3.68 (s, 3H), 2.38 (t,
J = 7.6 Hz, 2H), 2.18 (dt, J = 7.6, 5.1 Hz, 2H), (A signal due to one
proton (OH) was not observed); 13C NMR (125 MHz, CDCl3) d
173.1, 132.5, 129.6, 74.2, 53.0, 33.5, 29.9; HRMS (ESI) m/z
167.0698, calcd for C7H12NaO3 [M+Na]+ 167.0679.
4.1.26. N-(6-Aminohexyl)-6-(3-(4,7-dimethoxybenzo[d][1,3]
dioxol-5-yl)-6-methoxy-4-oxo-4H-chromen-7-yloxy)
hexanamide (34)
4.1.29. (E)-Methyl 6-(tert-butyldimethylsilyloxy)hex-4-enoate
(39)
To a stirred solution of allylic alcohol 38 (370 mg, 2.57 mmol) in
DMF (26 mL) were added imidazole (385 mg, 5.65 mmol) and
TBSCl (427 mg, 2.83 mmol) at room temperature. After being stir-
red at room temperature for 2 h, the mixture was diluted with
water (10 mL) and extracted with EtOAc (15 mL ꢂ 3). The com-
bined extracts were washed with brine (10 mL), dried (Na2SO4),
and concentrated. The residual oil was purified by column chroma-
tography on silica gel (17 g, EtOAc–hexane = 30:1 ? 10:1) to give
TBS ether 39 (603 mg, 91%) as a white solid: IR (CHCl3) 2978,
To a stirred solution of coupling compound 33a (4.3 mg,
6.29
67.5
l
mol) in CH2Cl2 (0.10 mL) was added TFA (0.05 mL,
lmol) at 0 °C. The mixture was stirred at room temperature
for 4 h and concentrated. The residual oil was purified by column
chromatography on silica gel (0.6 g, CHCl3–MeOH = 20:1 ? 10:1)
to give amine 34 (TFA salt; 2.7 mg, 62%) as a white solid: IR (CHCl3)
3326, 3024, 2980, 1648, 1591, 1554, 1510, 1285 cmꢁ1 1H NMR
;
(270 MHz, CDCl3) d 7.89 (s, 1H), 7.57 (s, 1H), 6.85 (s, 1H), 6.50 (s,
1H), 6.20 (br s, 1H), 6.01 (s, 2H), 4.09 (t, J = 5.9 Hz, 2H), 3.94
1743, 1661 cmꢁ1 1H NMR (500 MHz, CDCl3) d 5.72–5.64 (m, 2H),
;

5754
I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
4.21 (dt, J = 6.0, 1.4 Hz, 2H), 3.60 (s, 3H), 2.30 (t, J = 7.6 Hz, 2H), 2.16
(dt, J = 7.6, 5.2 Hz, 2H), 0.89 (s, 9H), 0.07 (s, 6H); 13C NMR
(125 MHz, CDCl3) d 173.2, 131.9, 128.5, 74.4, 53.2, 33.0, 29.6,
25.2 (3C), 18.4, ꢁ4.8 (2C); HRMS (ESI) m/z 281.1537, calcd for
4.1.33. (E)-tert-Butyl 6-(6-(3-(4,7-dimethoxybenzo[d][1,3]
dioxol-5-yl)-6-methoxy-4-oxo-4H-chromen-7-yloxy)hex-4-
enamido)hexylcarbamate (43)
To a stirred solution of O7-demethyl analogue 22 (10.3 mg,
C
13H26NaO3Si [M+Na]+ 281.1543.
27.7
l
mol) and allylic bromide 42 (19.2 mg, 58.5
lmol) in MeCN
(0.28 mL) was added K2CO3 (9.4 mg, 68.1
l
mol) at room tempera-
4.1.30. (E)-tert-Butyl 6-(6-(tert-butyldimethylsilyloxy)hex-4-
enamido)hexylcarbamate (40)
ture. After being stirred at reflux for 13 h, the mixture was cooled
to room temperature, diluted with saturated NH4Cl (0.5 mL), and
extracted with CH2Cl2 (1 mL ꢂ 3). The combined extracts were
washed with brine (2 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(0.6 g, CHCl3–MeOH = 400:1 ? 100:1) to give coupling compound
43 (11.0 mg, 72%) as a white solid, and recovery of phenol 22
(1.4 mg, 14%) as a white solid: IR (CHCl3) 3356, 3024, 2985,
To a stirred solution of TBS compound 39 (314 mg, 1.22 mmol)
in toluene (4.1 mL) was added amine 30 (394 mg, 1.82 mmol) at
room temperature. After being stirred at reflux for 24 h, the mix-
ture was cooled to room temperature, diluted with water (5 mL),
and extracted with CH2Cl2 (5 mL ꢂ 3). The combined extracts were
washed with brine (5 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(18 g, CHCl3–MeOH = 1:0 ? 100:1) to give amide 40 (371 mg,
1676, 1660, 1611, 1575, 1499, 1282 cmꢁ1 1H NMR (500 MHz,
;
CDCl3) d 7.90 (s, 1H), 7.60 (s, 1H), 6.85 (s, 1H), 6.52 (s, 1H), 6.00
(s, 2H), 5.86–5.80 (m, 3H), 4.62 (br s, 1H), 4.48 (dt, J = 7.0, 1.8 Hz,
2H), 3.96 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.27 (q, J = 6.3 Hz, 2H),
3.19 (q, J = 6.3 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H), 2.02 (dt, J = 7.3,
5.2 Hz, 2H), 1.58–1.50 (m, 4H), 1.44 (s, 9H), 1.35–1.29 (m, 4H);
13C NMR (125 MHz, CDCl3) d 175.5, 171.4, 156.4, 153.8, 151.8,
151.2, 147.7, 138.9, 138.7, 137.1, 136.5, 136.3, 132.5, 121.2,
118.4, 117.1, 109.5, 105.0, 104.0, 102.2, 79.6, 77.6, 60.0, 56.9,
56.7, 39.8, 39.2, 36.9, 36.0, 31.8, 31.4, 29.4, 28.7 (3C), 25.3; HRMS
(ESI) m/z 705.2996, calcd for C36H46N2NaO11 [M+Na]+ 705.2994.
69%) as
a
;
white solid: IR (CHCl3) 3375, 2968, 1661, 1652,
1568 cmꢁ1
1H NMR (500 MHz, CDCl3) d 5.92 (br s, 1H), 5.75–
5.65 (m, 2H), 4.88 (br s, 1H), 4.21 (dt, J = 6.3, 1.5 Hz, 2H), 3.27 (q,
J = 6.0 Hz, 2H), 3.16 (q, J = 6.0 Hz, 2H), 2.22 (t, J = 7.8 Hz, 2H), 2.16
(dt, J = 7.8, 5.8 Hz, 2H), 1.51–1.46 (m, 4H), 1.40 (s, 9H), 1.34–1.30
(m, 4H), 0.89 (s, 9H), 0.07 (s, 6H); 13C NMR (125 MHz, CDCl3) d
173.7, 157.0, 132.0, 128.5, 78.6, 74.6, 41.1, 40.2, 34.0, 29.9, 29.3,
29.1, 28.3 (3C), 25.7, 25.5, 25.2 (3C), 18.4, ꢁ4.8 (2C); HRMS (ESI)
m/z 465.3117, calcd for C23H46N2NaO4Si [M+Na]+ 465.3119.
4.1.31. (E)-tert-Butyl 6-(6-hydroxyhex-4-enamido)hexylcarba
mate (41)
4.1.34. (E)-N-(6-Aminohexyl)-6-(3-(4,7-dimethoxybenzo[d][1,3]
dioxol-5-yl)-6-methoxy-4-oxo-4H-chromen-7-yloxy)hex-4-
enamide (44)
To a stirred solution of TBS ether 40 (360 mg, 814 lmol) in THF
(8.1 mL) was added TBAF (276 mg, 1.06 mmol) at room tempera-
ture. After being stirred at room temperature for 4 h, the mixture
was diluted with saturated NH4Cl (7 mL) and extracted with
CH2Cl2 (8 mL ꢂ 3). The combined extracts were washed with brine
(10 mL), dried (Na2SO4), and concentrated. The residual oil was
purified by column chromatography on silica gel (10 g, CHCl3–
MeOH = 50:1 ? 20:1) to give allylic alcohol 41 (230 mg, 86%) as
To
11.7
135
a stirred solution of coupling compound 43 (8.0 mg,
mol) in CH2Cl2 (0.20 mL) was added TFA (0.010 mL,
mol) at 0 °C. The mixture was stirred at room temperature
l
l
for 3 h and concentrated. The residual solid was purified by column
chromatography on silica gel (0.6 g, CHCl3–MeOH = 20:1 ? 10:1)
to give amine 44 (TFA salt; 4.4 mg, 55%) as a white solid: IR (CHCl3)
3339, 3030, 2978, 1672, 1654, 1607, 1562, 1510, 1293 cmꢁ1 1H
;
a white solid: IR (CHCl3) 3362, 2972, 1669, 1649, 1551 cmꢁ1
;
1H
NMR (500 MHz, CDCl3) d 7.90 (s, 1H), 7.61 (s, 1H), 6.85 (s, 1H),
6.55 (s, 1H), 6.13 (br s, 1H), 6.01 (s, 2H), 5.82–5.76 (m, 2H), 4.47
(dt, J = 7.2, 1.6 Hz, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H),
3.27–3.21 (m, 2H), 2.95–2.90 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 2.07
(dt, J = 7.4, 5.4 Hz, 2H), 1.63–1.55 (m, 4H), 1.36–1.30 (m, 4H), (Sig-
nals due to two protons (NH) were not observed); 13C NMR
(125 MHz, CDCl3) d 175.8, 171.4, 153.8, 152.1, 151.5, 147.3,
138.9, 138.7, 137.1, 136.4, 136.1, 132.7, 121.1, 118.2, 117.1,
109.5, 105.0, 104.0, 102.0, 77.5, 60.3, 56.9, 56.5, 40.1, 39.4, 36.6,
35.8, 31.7, 31.4, 29.4, 25.2; HRMS (ESI) m/z 605.2480, calcd for
NMR (500 MHz, CDCl3) d 5.81–5.70 (m, 3H), 4.79 (br s, 1H), 4.25
(dt, J = 5.2, 1.9 Hz, 2H), 3.27 (q, J = 6.0 Hz, 2H), 3.19 (q, J = 6.0 Hz,
2H), 2.20 (t, J = 7.5 Hz, 2H), 2.15 (dt, J = 7.5, 5.4 Hz, 2H), 1.51–1.47
(m, 4H), 1.43 (s, 9H), 1.36–1.30 (m, 4H), (A signal due to one proton
(OH) was not observed); 13C NMR (125 MHz, CDCl3) d 173.5, 156.9,
132.3, 128.5, 78.4, 74.6, 41.1, 40.0, 33.7, 29.6, 29.3, 29.0, 28.2 (3C),
25.9, 25.6; HRMS (ESI) m/z 351.2281, calcd for C17H32N2NaO4
[M+Na]+ 351.2254.
4.1.32. (E)-tert-Butyl 6-(6-bromohex-4-enamido)hexylcarbamate
C
31H38N2NaO9 [M+Na]+ 605.2470.
(42)
To a stirred solution of allylic alcohol 41 (222 mg, 677
CH2Cl2 (6.8 mL) were added Ph3P (213 mg, 813 mol) and NBS
(145 mg, 815 mol) at room temperature. After being stirred at
l
mol) in
4.1.35. (E)-6-(3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-6-met
hoxy-4-oxo-4H-chromen-7-yloxy)-N-(6-(5-((3aS,6aR)-2-oxo hexa-
hydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexyl) hex-4-
enamide (45)
l
l
room temperature for 13 h, the mixture was diluted with water
(5 mL) and extracted with CH2Cl2 (7 mL ꢂ 3). The combined
extracts were washed with brine (10 mL), dried (Na2SO4), and con-
centrated. The residual oil was purified by column chromatogra-
phy on silica gel (13 g, CHCl3–MeOH = 1:0 ? 100:1) to give
allylic bromide 42 (169 mg, 64%) as a white solid: IR (CHCl3)
To a stirred solution of amine 44 (2.8 mg, 4.02
biotin N-hydroxysuccinimide ester 35 (2.1 mg, 6.16
DMF (0.08 mL) was added Et3N (2.8 L, 20.2 mol) at room
lmol) and (+)-
lmol) in
l
l
temperature. After being stirred at room temperature for 16 h,
the mixture was diluted with brine (0.5 mL) and extracted with
CH2Cl2 (1 mL ꢂ 3). The combined extracts were dried (Na2SO4)
and concentrated. The residual oil was purified by column chroma-
tography on silica gel (0.6 g, CHCl3–MeOH = 50:1 ? 20:1) and by
recycle HPLC [JAIGEL-1H-40 (600 ꢂ 20 mm) and JAIGEL-2H-40
(600 ꢂ 20 mm), flow rate 3.8 mL/min; detection, UV 265 nm;
solvent CHCl3] to give O7-modified biotin probe 45 (1.5 mg, 47%)
as a white solid: IR (CHCl3) 3350, 3020, 2982, 1723, 1673, 1650,
3368, 2976, 1660, 1642, 1539, 691 cmꢁ1 1H NMR (500 MHz,
;
CDCl3) d 5.82 (br s, 1H), 5.69–5.65 (m, 2H), 4.69 (br s, 1H), 4.03
(dt, J = 5.7, 1.6 Hz, 2H), 3.26 (q, J = 5.9 Hz, 2H), 3.20 (q, J = 5.6 Hz,
2H), 2.20 (t, J = 7.7 Hz, 2H), 2.12 (dt, J = 7.7, 5.5 Hz, 2H), 1.55–1.48
(m, 4H), 1.45 (s, 9H), 1.37–1.29 (m, 4H); 13C NMR (125 MHz, CDCl3)
d 173.6, 157.1, 129.7, 126.9, 78.5, 69.2, 41.1, 40.6, 33.8, 29.5, 29.1,
28.7, 28.2 (3C), 26.0, 25.5; HRMS (ESI) m/z 413.1404, calcd for
C
17H31BrN2NaO3 [M+Na]+ 413.1410.
1607, 1574, 1286, 1199 cmꢁ1 1H NMR (500 MHz, CDCl3) d 7.90
;

I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
5755
(s, 1H), 7.60 (s, 1H), 6.82 (s, 1H), 6.56 (s, 1H), 6.15 (br s, 1H), 6.04
(br s, 1H), 6.02 (s, 2H), 5.81–5.76 (m, 2H), 5.23 (s, 1H), 4.97 (s,
1H), 4.62–4.57 (m, 1H), 4.44 (dt, J = 7.0, 1.5 Hz, 2H), 4.36–4.32
(m, 1H), 3.96 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.27–3.13 (m, 5H),
2.93–2.88 (m, 2H), 2.33 (t, J = 7.2 Hz, 2H), 2.24 (t, J = 7.6 Hz, 2H),
2.07 (dt, J = 7.6, 5.2 Hz, 2H), 1.87–1.70 (m, 4H), 1.65–1.50 (m,
6H), 1.35–1.29 (m, 4H); 13C NMR (125 MHz, CDCl3) d 179.0,
175.8, 171.2, 169.7, 154.0, 152.1, 151.5, 147.5, 139.0, 138.8,
137.1, 136.1, 135.7, 132.8, 121.0, 118.4, 117.1, 109.5, 105.4,
104.1, 101.8, 77.9, 60.1, 56.9, 56.6, 55.4, 54.0, 45.4, 40.5, 39.4,
37.2, 36.6, 35.8, 35.3, 32.0, 31.4, 29.6, 27.7, 26.7, 26.1, 25.5; HRMS
(ESI) m/z 831.3241, calcd for C41H52N4NaO11S [M+Na]+ 831.3245.
56.5, 40.1, 39.5, 36.4, 35.8, 31.5, 31.2, 30.5, 29.4, 27.4, 26.5, 25.1,
16.3 (2C), 14,4 (2C); HRMS (ESI) m/z 921.4025, calcd for
C
48H57BF2N4NaO10 [M+Na]+ 921.4028.
4.2. Bioassay
Cell survival was determined by a WST-8 assay kit (Dojindo
Laboratories, Kumamoto, Japan). HeLa S3 cells (3 ꢂ 103 cells/well)
in 96 well plates were incubated overnight. Then, cells were trea-
ted with various concentrations of each compound. After 48 h
incubation, 10 lL of WST-8 reagents were added to the culture.
After 2 h incubation, the absorbance at 450 nm was measured with
iMark microplate reader (BioRad Laboratories, Inc.). Absorbance
correlates with the number of living cells. The number of living
cells (% control) was calculated with the following formula: (each
4.1.36. (E)-N-(6-(6-(3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)-
6-methoxy-4-oxo-4H-chromen-7-yloxy)hex-4-enamido)hexyl)-
4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamide (46)
absorbance ꢁ absorbance of blank well)/(absorbance of 0
lM well
To a stirred solution of amine 44 (2.1 mg, 3.02
fluoromethyl-3-phenyldiazirine succinimide ester 48 (1.5 mg,
4.59 mol) in MeCN (0.06 mL) was added Et3N (2.0 L, 14.4 mol)
lmol) and 3-tri-
ꢁ absorbance of blank well) ꢂ 100.
l
l
l
4.3. Cell culture and reagents
at room temperature. After being stirred at room temperature for
24 h, the mixture was diluted with brine (0.5 mL) and extracted
with CH2Cl2 (1 mL ꢂ 3). The combined extracts were dried
(Na2SO4) and concentrated. The residual oil was purified by col-
umn chromatography on silica gel (0.6 g, CHCl3–MeOH = 300:1 ?
100:1) to give O7-modified photoaffinity probe 46 (1.2 mg, 49%)
as a white solid: IR (CHCl3) 3345, 3028, 2980, 1672, 1658, 1602,
Human cervix epidermoid carcinoma HeLa S3 cells were cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal-calf serum (FCS, Cell Culture Bioscience). Cells were
grown at 37 °C in a 5% CO2 atmosphere. Antibodies specific for a-
tubulin (DM1A, #sc-32293) was purchased from Santa Cruz Bio-
technology (Santa Cruz, CA). Alexa488-conjugated anti-mouse
IgG antibody (#A11001) was purchased from Invitrogen. DAPI
solution was purchased from Wako Pure Chemical Industry (Osaka,
Japan).
1578, 1559, 1514, 1288 cmꢁ1 1H NMR (500 MHz, CDCl3) d 7.98
;
(d, J = 8.0 Hz, 2H), 7.89 (s, 1H), 7.63 (s, 1H), 7.19 (d, J = 8.0 Hz,
2H), 6.86 (s, 1H), 6.51 (s, 1H), 6.30 (br s, 1H), 6.13 (br s, 1H), 6.02
(s, 2H), 5.85–5.79 (m, 2H), 4.47 (dt, J = 7.0, 1.8 Hz, 2H), 3.98 (s,
3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.37–3.25 (m, 4H), 2.26 (t,
J = 7.5 Hz, 2H), 2.05 (dt, J = 7.5, 5.0 Hz, 2H), 1.64–1.55 (m, 4H),
1.38–1.30 (m, 4H); 13C NMR (125 MHz, CDCl3) d 176.0, 172.4,
171.1, 153.8, 152.5, 151.7, 147.3, 139.0, 138.6, 137.2, 136.4,
135.9, 134.8, 132.7, 130.2 (2C), 130.0, 126.7 (2C), 122.9 (q,
1JC,F = 275 Hz), 121.1, 118.4, 117.2, 109.7, 104.9, 104.1, 102.0,
77.2, 60.3, 57.0, 56.6, 39.6, 39.3, 36.5, 35.9, 31.7, 31.4, 29.5, 28.4
4.3.1. Analysis of cell cycle progression
3 ꢂ 104 cells/mL HeLa S3 cells were treated with various con-
centrations of each compound for 24 h, then harvested and fixed
with 70% EtOH (ꢁ20 °C). After staining with propidium iodide solu-
tion, DNA contents were measured by flow cytometric analysis (At-
tuneÒ Acoustic Focusing Cytometer; Applied Biosystems).
2
(q, JC,F = 50.0 Hz), 25.2; HRMS (ESI) m/z 817.2675, calcd for
4.3.2. Immunofluorescence procedures
C
40H41F3N4NaO10 [M+Na]+ 817.2667.
3 ꢂ 104 cells/mL HeLa S3 cells were seeded on sterile coverslips.
After treatment of each compound for 6 h, coverslips were fixed
with ꢁ20 °C MeOH for 5 min. After being washed with PBS-B
(PBS containing 0.5% (w/v) BSA), coverslips were overlaid with
4.1.37. (E)-10-(4-(6-(6-(3-(4,7-Dimethoxybenzo[d][1,3]dioxol-5-
yl)-6-methoxy-4-oxo-4H-chromen-7-yloxy)hex-4-enamido)hex
ylamino)-4-oxobutyl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipy
rrolo[1,2-c:1’,2’-f][1,3,2]diazaborinin-4-ium-5-uide (47)
anti-a-tubulin antibody in PBS-B then placed in a humidified con-
tainer at 37 °C and incubated for 1 h. After being washed twice
with PBS-B, coverslips were overlaid with Alexa488-conjugated
anti-mouse IgG antibody in PBS-B, incubated for 1 h, washed with
To a stirred solution of amine 44 (1.4 mg, 2.05
IPY succinimide ester 49 (1.4 mg, 3.25 mol) in MeCN (0.05 mL)
was added Et3N (1.5 L, 10.8 mol) at room temperature. After
lmol) and BOD-
l
l
l
PBS and mounted with 0.1 lg/mL DAPI solution. The morphology
being stirred at room temperature for 24 h, the mixture was
diluted with brine (0.5 mL) and extracted with CH2Cl2 (1 mL ꢂ 3).
The combined extracts were dried (Na2SO4) and concentrated.
The residual oil was purified by column chromatography on silica
gel (0.6 g, CHCl3–MeOH = 100:1 ? 40:1) and by recycle HPLC [JAI-
GEL-1H-40 (600 ꢂ 20 mm) and JAIGEL-2H-40 (600 ꢂ 20 mm), flow
rate 3.8 mL/min; detection, UV 265 nm; solvent CHCl3] to give O7-
modified fluorescent probe 47 (1.2 mg, 65%) as a white solid: IR
(CHCl3) 3358, 3025, 3019, 2982, 2850, 1665, 1653, 1607, 1555,
of microtubule and nuclei were observed under a Leica LAS AF
6000 fluorescent microscope (Leica Microsystems GmbH, Wetzlar,
Germany). Dilutions of antibodies were 1:250 (antibodies specific
for
a-tubulin) and 1:2000 (Alexa488-conjugated anti-mouce IgG
antibody).
Acknowledgments
This work was supported by Grants-in-Aid for Scientific
Research on Innovative Areas ‘Chemical Biology of Natural Prod-
ucts’ and for Scientific Research (B) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT)/Japanese Society
for the Promotion of Science (JSPS); by a grant from the Uehara
Memorial Foundation; and by a grant from the University of
Tsukuba, Strategic Initiatives (A), Center for Creation of Functional
Materials (CCFM). I.H. thanks The Society of Synthetic Organic
Chemistry, Japan (Meiji Seika Award in Synthetic Organic Chemis-
try, Japan) and the Suntory Institute for Bioorganic Research (SUN-
BOR GRANT) for their financial support.
1294 cmꢁ1 1H NMR (500 MHz, CDCl3) d 7.90 (s, 1H), 7.61 (s, 1H),
;
6.84 (s, 1H), 6.55 (s, 1H), 6.22 (br s, 1H), 6.11 (br s, 1H), 6.06 (s,
2H), 6.02 (s, 2H), 5.80–5.74 (m, 2H), 4.45 (dt, J = 7.2, 1.6 Hz, 2H),
3.96 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.28–3.20 (m, 4H), 3.05 (t,
J = 8.4 Hz, 2H), 2.52 (s, 6H), 2.45 (s, 6H), 2.41 (t, J = 7.0 Hz, 2H),
2.28 (t, J = 7.4 Hz, 2H), 2.05–1.98 (m, 4H), 1.65–1.59 (m, 4H),
1.36–1.29 (m, 4H); 13C NMR (125 MHz, CDCl3) d 175.8, 173.9,
171.7, 154.3, 153.9, 152.0, 151.2, 147.3, 144.7 (2C), 140.4 (2C),
139.1, 138.7, 137.4, 136.4, 136.0, 132.6, 131.5 (2C), 121.9 (2C),
120.9, 118.2, 117.1, 109.4, 105.1, 104.3, 101.8, 77.8, 60.0, 56.8,

5756
I. Hayakawa et al. / Bioorg. Med. Chem. 20 (2012) 5745–5756
14. The calculated LogP (cLogP) values of glaziovianin A and its analogues were
Supplementary data
obtained by using ChemBioDraw Ultra 11.0 (CambridgeSoft): Hansch, C.;
Fujita, T. J. Am. Chem. Soc. 1964, 86, 1616.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.005.
15. Although we previously reported the IC50 values of compounds 26, 28, and 29
to be 0.19, 0.75 and 0.74 l
M, respectively,4 we have reevaluated their
cytotoxicities against HeLa S3 cells and found that those IC50 values should
be corrected as described in this paper.
16. Kondoh, M.; Usui, T.; Nishikiori, T.; Mayumi, T.; Osada, H. Biochem. J. 1999, 340,
411.
References and notes
17. Although we previously reported the effects of glaziovianin
A (1) and
1. Yokosuka, A.; Haraguchi, M.; Usui, T.; Kazami, S.; Osada, H.; Yamori, T.; Mimaki,
Y. Bioorg. Med. Chem. Lett. 2007, 17, 3091.
2. Yamori, T.; Matsunaga, A.; Saito, S.; Yamazaki, K.; Komi, A.; Ishizu, K.; Mita, I.;
Edatsugi, H.; Matsuda, Y.; Takezawa, K.; Nakanishi, O.; Kohno, H.; Nakajima, Y.;
Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer Res. 1999, 59, 4042.
3. Review: Kingston, D. G. I. J. Nat. Prod. 2009, 72, 507.
4. Ikedo, A.; Hayakawa, I.; Usui, T.; Kazami, S.; Osada, H.; Kigoshi, H. Bioorg. Med.
Chem. Lett. 2010, 20, 5402.
5. Hayakawa, I.; Ikedo, A.; Kigoshi, H. Chem. Lett. 2007, 36, 1382.
6. Piggott, M. J.; Wege, D. Aust. J. Chem. 2000, 53, 749.
7. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
8. Hoshino, Y.; Miyaura, N.; Suzuki, A. Bull. Chem. Soc. Jpn. 1988, 61, 3008.
9. Tremblay, S. M.; Sames, D. Org. Lett. 2005, 7, 2417.
10. Adityachaudhury, N.; Kirtaniya, C. L.; Mukherjee, B. Tetrahedron 1971, 27, 2111.
11. Gammill, R. B. Synthesis 1979, 901.
12. Igarashi, Y.; Kumazawa, H.; Ohshima, T.; Satomi, H.; Terabayashi, S.; Takeda, S.;
Aburada, M.; Miyamoto, K. Chem. Pharm. Bull. 2005, 1008, 53.
13. Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. 1980, 19, 731.
compound 26 on cell cycle progression and spindle structures,4 we have
reevaluated theirs effects along with compounds 28 and 29 and found that
those effects should be corrected as described in this paper.
18. Callahan, J. F.; Ashton-Shue, D.; Bryan, H. G.; Bryan, W. M.; Heckman, G. D.;
Kinter, L. B.; McDonald, J. E.; Moore, M. L.; Schmidt, D. B.; Silvestri, J. S.; Stassen,
F. L.; Sulat, L.; Yim, N. C. F.; Huffman, W. F. J. Med. Chem. 1989, 32, 391.
19. Lermer, L.; Neeland, E. G.; Ounsworth, J. P.; Sims, R. J.; Tischler, S. A.; Weiler, L.
Can. J. Chem. 1992, 70, 1427.
20. Becker, J. M.; Wilchek, M. Biochim. Biophys. Acta. 1972, 264, 165.
21. Ku, T. W.; McCarthy, M. E.; Weichman, B. M.; Gleason, J. G. J. Med. Chem. 1847,
1985, 28.
22. (a) Stromgaard, K.; Saito, R. D.; Shindou, H.; Ishii, S.; Shimizu, T.; Nakanishi, K. J.
Med. Chem. 2002, 45, 4038; (b) Nassal, M. Liebigs Ann. Chem. 1983, 1510.
23. (a) Li, Z.; Mintzer, E.; Bittman, R. J. Org. Chem. 2006, 71, 1718; (b) Tera, M.; Iida,
K.; Ikebukuro, K.; Seimiya, H.; Shin-ya, K.; Nagasawa, K. Org. Biomol. Chem.
2010, 8, 2749.