Design and synthesis of 2-phenyl-1,4-dioxa-spiro[4.5]deca-6,9-dien-8-onesas potential anticancer agents starting from cytotoxic spiromamakone A

Article abstract of DOI:10.1016/j.ejmech.2013.05.030

The spirocycle is a key structure found in many bioactive compounds. From the cytotoxic and spirocyclic natural product, spiromamakone A (1) and its analogues, a more synthetically accessible spiroacetal template 4 was designed based on structural similarity analysis. A total of 50 compounds were rapidly synthesized in only one or two synthetic steps from the starting compound, and their cytotoxicity was evaluated. As a result, (±)-(2R*,5R*)- 2-(4-iodophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-6,9-dien-8-one (7d-II) was discovered and found to be fifteen-fold more cytotoxic than 1. The easily accessible spiroacetal 7d-II appeared to act in a manner similar to the highly oxidized natural product, spiromamakone A (1).

Full text of DOI:10.1016/j.ejmech.2013.05.030

European Journal of Medicinal Chemistry 66 (2013) 180e184
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of 2-phenyl-1,4-dioxa-spiro[4.5]deca-6,9-dien-
8-ones as potential anticancer agents starting from cytotoxic
spiromamakone A
Shinichiro Fuse a, Kennichi Inaba b, Motoki Takagi b, Masahiro Tanaka b,
Takatsugu Hirokawa c, Kohei Johmoto d, Hidehiro Uekusa d, Kazuo Shin-ya e,
,
f,
*
Takashi Takahashi a , Takayuki Doi
*
a Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8552, Japan
b Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), 2-4-7, Aomi, Koto-ku, Tokyo 135-0064, Japan
c Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7, Aomi, Koto-ku, Tokyo
135-0064, Japan
d Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8552, Japan
e Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7, Aomi, Koto-ku, Tokyo 135-0064, Japan
f Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aza-Aoba, Aramaki, Aoba, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The spirocycle is a key structure found in many bioactive compounds. From the cytotoxic and spirocyclic
natural product, spiromamakone A (1) and its analogues, a more synthetically accessible spiroacetal
template 4 was designed based on structural similarity analysis. A total of 50 compounds were rapidly
synthesized in only one or two synthetic steps from the starting compound, and their cytotoxicity was
evaluated. As a result, (ꢀ)-(2R*,5R*)-2-(4-iodophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-6,9-dien-8-one
(7d-II) was discovered and found to be fifteen-fold more cytotoxic than 1. The easily accessible spi-
roacetal 7d-II appeared to act in a manner similar to the highly oxidized natural product, spi-
romamakone A (1).
Received 26 February 2013
Received in revised form
21 May 2013
Accepted 23 May 2013
Available online 3 June 2013
Keywords:
Spiromamakone A
Spiropreussione B
Spirocycle
Ó 2013 Elsevier Masson SAS. All rights reserved.
Spiroacetal
Anticancer agent
1. Introduction
human ovarian carcinoma cell line A2780, 3.0 mM against the
human liver carcinoma cell line BEL-7404) [4,5]. Although spi-
romamakone A and its analogues are attractive candidates for
anticancer agents, their total synthesis is yet to be reported, and it is
difficult to establish a short-step synthetic method for 1e3 [6].
Moreover, the structures of spiromamakone A and its analogues are
not easy to modify, which is needed in order to tune its properties
for drug development. The development of more synthetically
accessible molecules would be very valuable, particularly if they
could retain the same, or higher, biological activity as that of spi-
romamakone A and its analogues [7e9].
Starting from spiromamakone A and its analogues, 2-phenyl-
1,4-dioxa-spiro[4.5]deca-6,9-dien-8-ones 4, the spiroacetal tem-
plate for a rapid synthesis of analogues was designed based on
structural similarity analysis. A total of 50 compounds were pre-
pared in one or two synthetic steps from the starting compound
5, and their cytotoxicity was evaluated. As a result, an easily
Spirocyclic compounds have garnered considerable recent
attention due to their unique three-dimensional orientation and
proven biological activities [1e3]. Spiromamakone A (1) is a potent
cytotoxic natural product (IC50 value, 0.33 mM against the murine
leukemia cell line P388) that was first isolated in 2006 by Munro
and co-workers [4]. It has an unprecedented spiro-nonadiene
skeleton with a high degree of oxidation and unsaturation. Two
analogues, 4-oxo-spiromamakone A (2) and spiropreussione (3),
are reportedly also cytotoxic (2: IC50 value, 1.13
murine leukemia cell line P388, 3: IC50 values, 2.4
m
m
M against the
M against the
* Corresponding authors.
E-mail
addresses:
ttak@apc.titech.ac.jp
(T.
Takahashi),
doi_taka@
mail.pharm.tohoku.ac.jp (T. Doi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.030

S. Fuse et al. / European Journal of Medicinal Chemistry 66 (2013) 180e184
181
accessible spiroacetal, (ꢀ)-(2R*,5R*)-2-(4-iodophenyl)-7-chloro-
1,4-dioxa-spiro[4.5]deca-6,9-dien-8-one (7d-II) was discovered
and found to be a 15-fold more cytotoxic agent than 1.
2. Results and discussion
From the structural similarity analysis of spiromamakone A (1)
and its analogues 2 and 3 (Fig. 1bed) [10], we speculated that a
common structure (emphasized in Fig.1a) is pivotal for inducing the
cytotoxicityof these compounds. Syntheticallyaccessible, 2-phenyl-
1,4-dioxa-spiro[4.5]deca-6,9-dien-8-one 4 was designed as a tem-
plate for the rapid synthesis of analogues. From a comparison of the
molecular shapes between 1 and the template 4, the structure of
template 4 overlapped well with the pivotal structure in 1 (Fig. 1d).
Forty-three compounds retaining various substituents were
rapidly synthesized in only one or two synthetic steps from
the starting compound 5, as shown in Fig. 2 (see Supporting
information). Aryl halides
7 were prepared from the acetal
Fig. 2. Rapid synthesis of 43 spiroacetals 7 and 9.
exchange reaction between diols 6 and dimethyl acetal 5 in the
existence of pyridinium p-toluenesulfonate (PPTS). The dimethyl
acetal 5 was derived from the corresponding 4-methoxy phenols in
one step [11]. Two diastereomers, I and II, were obtained from the
compounds of 7c and 7d. Although the relative stereochemistries of
the obtained diastereomers were not determined at this point,
these diastereomers were isolated by silica gel column chroma-
tography. 7a, 7b and a diastereomer of 7d-II, were used for further
modification based on a SuzukieMiyaura coupling reaction [12,13]
with various aryl building blocks 8 [14,15].
The cytotoxicity of synthesized compounds 7 and 9 against
cervical carcinoma HeLa cells was evaluated (Table 1). Twelve
compounds, 7d-II, 9c, 9eeg, 9j, 9sev, 9y, and 9ag (Table 1, entries 7,
10, 12e14, 17, 26e29, 32 and 40) exhibited higher cytotoxicity than
that of spiromamakone A (1). In particular, 7d-II showed a 15-fold
more potent cytotoxicity than 1 (Table 1, entry 7). Interestingly,
both diastereomers 7c-I and 7c-II exhibited a comparable degree of
cytotoxicity (Table 1, entries 4 and 5). On the other hand, the dia-
stereomer, 7d-II was 15-fold more cytotoxic than its diastereomer
7d-I (Table 1, entries 6 and 7). This result clearly indicates that the
relative stereochemistry of 7d-II is important to its cytotoxicity.
From the comparison of the cytotoxicity between 7b and 7d-II, and
between 7c and 7d, it was indicated that the chloro and 4-iodo
groups in 7d were also important for the potency of its
Fig. 1. (a) Structures of spiromamakone
A (1), 4-oxo-spiromamakone A (2), spi-
ropreussione B (3), and the designed spiroacetal template 4. The common structure is
emphasized. (b) Structural similarity analysis of spiromamakone A (1) and 2. (c) Struc-
tural similarity analysis of 1 and 3. (d) Structural similarity analysis of 1 and the designed
template 4. Carbon atoms for 1, 2, 3, and 4 are shown in green, cyan, yellow and purple,
respectively. Construction of 3D models and energy minimization was performed using
the OPLS-AA force field in the Conformational Search algorithm in the MacroModel
program (Schrödinger, LLC). All compounds are aligned by a shape similarity score with
1, which was calculated using the ROCS (OpenEye Scientific Software, Inc).

182
S. Fuse et al. / European Journal of Medicinal Chemistry 66 (2013) 180e184
Table 1
Cytotoxicity of the synthesized compounds 7, 9, and spiromamakone A (1) against
cervical carcinoma HeLa cells.
Entry
Substrate 7
Substrate 8
Evaluated compounds
IC50 (m
M)a
1
e
e
1
2.2
2
e
e
7a
1.6
3
e
e
7b
1.1
4
5
6
7
8
9
e
e
7c-I
7c-II
7d-I
7d-II
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
9u
9v
9w
9x
9y
1.5
1.6
2.4
0.14
1.4
1.6
0.67
1.8
0.85
0.79
0.88
1.7
3.2
0.91
1.3
1.3
2.4
1.4
3.1
1.7
1.5
e
e
e
e
e
e
7a
7a
7a
7a
7a
7a
7a
7a
7a
7a
7a
7a
7a
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7b
7d-II
7d-II
7d-II
7d-II
8a
8b
8c
8d
8e
8o
8p
8s
8v
8x
8y
8z
8aa
8d
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8w
8x
8aa
8r
8t
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Fig. 3. Synthesis of seven analogues of 7d.
cytotoxic than 4-bromo analogues 7h (Table 2, entries 6e9) in
contrast with 4-aryl iodides 7c and 7d (Table 1, entries 4e7).
At this point, we embarked on the structural determination of
7d-II. Fortunately, a single crystal of the less cytotoxic diastereomer
7d-I was obtained, and the X-ray crystal structure revealed its
relative stereochemistry, as shown in Fig. 4. Thus, the relative ste-
reochemistry of 7d-II was unambiguously determined, as shown
in Fig. 4.
2.6
0.80
0.82
0.80
0.96
3.5
1.7
0.90
1.8
1.5
1.3
4.5
3.2
2.5
2.5
0.79
9.4
2.3
Finally, the panel assay of 39 human cancer cell lines for spi-
romamakone A (1) and 7d-II was performed (see Supporting
information), because there reportedly is a significant correlation
between a pattern of differential cytotoxicity against the cell lines
by a drug and its mode of action [16]. The obtained results indicated
that 7d-II exerts higher cytotoxicity against most cell lines than 1,
but the cytotoxic patterns between 1 and 7d-II show high simi-
larity. In addition, these patterns were weak similar (correlation
coefficient ¼ 0.339e0.461) to those of purine antagonists such as 6-
mercaptopurine and 6-thioguanine and were predicted that 1 and
7d-II possess a unique mechanism of action. These results indicate
that our originally developed, synthetically accessible spiroacetal
7d-II acts in a manner similar to the highly oxidized spirocyclic
natural product, spiromamakone A (1). It is conceivable that both 1
and 7d-II act as a nucleoside mimetic to exert their cytotoxicity.
9z
9aa
9ab
9ac
9ad
9ae
9af
9ag
9ah
9ai
9aj
9ak
8u
8aa
4.6
2.7
DMSO was employed as a negative control and no cytotoxicity was observed.
The IC50 value was defined as the concentration at which 50% survival of cells
was observed. The results are listed in the table.
a
cytotoxicity (Table 1, entries 3e7). In the case of 9aem, which were
derived from 7a (Table 1, entries 8e20), the top 4 cytotoxic com-
pounds 9c and 9eeg retained p-substituted phenyl rings at the
ends of their structures (Table 1, entries 10, 12, 13 and 14). On
the other hand, in the case of compounds 9neag, which were
derived from 7b (Table 1, entries 21e40), highly cytotoxic com-
3. Conclusion
In summary, starting from the cytotoxic, spirocyclic natural
product, spiromamakone A (1) and its analogues, 2-phenyl-1,4-
Table 2
pounds 9sev and 9y (IC50 < 1
mM) contained o-, m-, or p-
Cytotoxicity of the synthesized seven analogues of 7d, spiromamakone A (1) and
7d-II against cervical carcinoma HeLa cells.
substituted phenyl rings at the ends of their structures (Table 1,
entries 26e29 and 32). Generally, it seemed that the basic aromatic
ring decreased the cytotoxicity because all less cytotoxic com-
pounds 9ac, 9ah, 9aj (IC50 > 4 mM) contain a pyridine or pyrimidine
ring (Table 1, entries 36, 41 and 43).
To elucidate the detailed structureeactivity relationship, 7 an-
alogues of 7d with different halogen atoms at different positions
were prepared (Fig. 3). The cytotoxicity of synthesized analogues
against the HeLa cells was evaluated, and the results are shown in
Table 2. From a comparison of the cytotoxicities of 7d-II, 7f and 7h,
the importance of the 4-iodo group for potent cytotoxicity was
confirmed (Table 2, entries 2, 4, 5, 8 and 9). Interestingly, in the case
of aryl bromides 7g and 7h, 3-bromo analogues 7g were more
Entry
Evaluated compounds
IC50 (m
M)a
1
2
3
4
5
6
7
8
9
1
7d-II
7e
7f-I
7f-II
7g-I
7g-II
7h-I
7h-II
2.2
0.14
2.4
2.9
2.7
0.64
0.61
4.5
2.1
DMSO was employed as a negative control and no cytotoxicity was observed.
a
The IC50 value was defined as the concentration at which 50% survival of cells
was observed. The results are listed in the table.

S. Fuse et al. / European Journal of Medicinal Chemistry 66 (2013) 180e184
183
anhydrous magnesium sulphate, filtered, and concentrated under
reduced pressure. The residue was purified by preparative TLC to
give the title compound.
4.2.1. (ꢀ)-(2R*,5S*)-2-(4-Iodophenyl)-7-chloro-1,4-dioxa-spiro[4.5]
deca-6,9-dien-8-one (7d-I)
Mp. 101e103 ꢂC; yield: 19%; 1H NMR (270 MHz, CDCl3):
d 7.75 (d,
J ¼ 8.1 Hz, 2H), 7.13 (d, J ¼ 8.1 Hz, 2H), 6.88 (d, J ¼ 2.7 Hz, 1H), 6.78
(dd, J ¼ 2.7, 8.1 Hz, 1H), 6.31 (d, J ¼ 8.1 Hz, 1H), 5.23 (dd, J ¼ 5.4,
8.1 Hz, 1H), 4.52 (dd, J ¼ 5.4, 8.1 Hz, 1H), 3.86 (t, J ¼ 8.1 Hz, 1H); 13
C
NMR (67.5 MHz, CDCl3):
d 177.9, 143.3, 139.3, 138.0, 136.2, 133.8,
127.9, 127.8, 100.7, 94.4, 78.7, 71.9; IR (neat) 3060, 2889, 1685, 1653,
1615, 1487, 1333, 1132, 1020, 1006, 976, 822; HRMS (ESI-TOF): calcd.
for [C14H10ClIO3 þ H]þ 388.9441, found 388.9463.
Fig. 4. X-ray single crystal structure of 7d-I, and the determined relative stereo-
chemistries of 7d-I and 7d-II.
dioxa-spiro[4.5]deca-6,9-dien-8-ones 4, the syntheticallyaccessible
spiroacetal template was designed for a synthesis of analogues
based on structural similarity analysis. A total of 50 compounds
were rapidly synthesized in only one or two synthetic steps from the
starting compound 5, and its cytotoxicity was evaluated. As a result,
4.2.2. (ꢀ)-(2R*,5R*)-2-(4-Iodophenyl)-7-chloro-1,4-dioxa-spiro
[4.5]deca-6,9-dien-8-one (7d-II)
Yield: 13%; 1H NMR (270 MHz, CDCl3):
d
7.75 (d, J ¼ 8.1 Hz, 2H),
7.12 (d, J ¼ 8.1 Hz, 2H), 6.93 (d, J ¼ 2.7 Hz, 1H), 6.73 (dd, J ¼ 2.7,
8.1 Hz, 1H), 6.31 (d, J ¼ 8.1 Hz, 1H), 5.23 (t, J ¼ 8.1 Hz, 1H), 4.51 (t,
J ¼ 8.1 Hz, 1H), 3.88 (t, J ¼ 8.1 Hz, 1H); 13C NMR (67.5 MHz, CDCl3):
a total of 12 compounds exerted higher cytotoxicity (IC50 < 1 mM)
than 1. In particular, (ꢀ)-(2R*,5R*)-2-(4-iodophenyl)-7-chloro-1,4-
dioxa-spiro[4.5]deca-6,9-dien-8-one (7d-II) was discovered as a
15-fold more cytotoxic agent than 1. The panel assay for various
cancer cell lines suggested that the spiroacetal 7d-II which can be
prepared in only one synthetic step from 5b acts in a manner similar
to the highly oxidized natural product, spiromamakone A (1). It is
conceivable that both 1 and 7d-II act as a nucleoside mimetic to
exert their cytotoxicity. The synthetically accessible, cytotoxic spi-
roacetal 7d-II would be a valuable aid in the drug discovery process.
d 177.9,143.7,138.9,138.0,136.4,133.4,128.3,127.9,100.7, 94.4, 78.6,
71.9; IR (neat) 3058, 2887, 1686, 1652, 1615, 1487, 1334, 1278, 1216,
1132, 1020, 1006, 975, 822; HRMS (ESI-TOF): calcd. for
[C14H10ClIO3 þ H]þ 388.9441, found 388.9461.
4.2.3. (ꢀ)-2-(4-Bromophenyl)-1,4-dioxa-spiro[4.5]deca-6,9-dien-
8-one (7e)
Yield: 46%; 1H NMR (270 MHz, CDCl3)
d
7.54 (d, J ¼ 8.1 Hz, 2H),
7.27 (d, J ¼ 8.1 Hz, 2H), 6.69e6.79 (m, 2H), 6.19e6.26 (m, 2H), 5.25
(dd, J ¼ 5.4, 8.1 Hz, 1H), 4.51 (dd, J ¼ 5.4, 8.1 Hz, 1H), 3.87 (t,
4. Experimental
J ¼ 8.1 Hz, 1H); 13C NMR (67.5 MHz, CDCl3):
d 185.1, 143.2, 142.7,
136.1, 132.0, 129.4, 128.9, 127.8, 122.6, 99.3, 72.0, 78.4.
4.1. Chemistry
4.2.4. (ꢀ)-2-(4-Chlorophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-
6,9-dien-8-one (7f-I)
NMR spectra were recorded on either a JEOL Model ECP-400
(400 MHz for 1H, 100 MHz for 13C) or on a JEOL Model EX-270
(270 MHz for 1H, 67.5 MHz for 13C) instrument for the indicated
solvent. Chemical shifts were reported in units of parts per million
(ppm) relative to the signal for internal tetramethylsilane
(0.00 ppm for 1H) for solutions in CDCl3. The 1H NMR spectral data
were reported as follows: CDCl3 (7.26 ppm). 13C NMR spectral data
were reported as follows: CDCl3 (77.1 ppm). Multiplicities were
reported using the following abbreviations: s; singlet, d; doublet, t;
triplet, m; multiplet, J; coupling constants in Hertz. IR spectra were
recorded on a Perkin Elmer Spectrum One FT-IR spectrophotom-
eter. Only the strongest and/or structurally important absorption is
reported as the IR data given in cmꢁ1. All reactions were monitored
by thin-layer chromatography carried out on 0.2 mm E. Merck silica
gel plates (60F-254) with UV light, visualized by p-anisaldehyde
solution, ceric sulphate or 10% ethanolic phosphomolybdic acid.
Column chromatography was performed on Merck silica gel 60
(0.063e0.200 mm). ESI-TOF Mass spectra were measured using a
Waters LCT PremierÔ XE. The HRMS (ESI-TOF) were calibrated
using leucine enkephalin.
Yield: 29%; 1H NMR (270 MHz, CDCl3):
d
7.39 (d, J ¼ 8.1 Hz, 2H),
7.32 (d, J ¼ 8.1 Hz, 2H), 6.88 (d, J ¼ 2.7 Hz, 1H), 6.74 (dd, J ¼ 2.7,
10.8 Hz, 1H), 6.33 (d, J ¼ 10.8 Hz, 1H), 5.27 (dd, J ¼ 5.4, 8.1 Hz, 1H),
4.52 (dd, J ¼ 5.4, 8.1 Hz, 1H), 3.89 (t, J ¼ 8.1 Hz, 1H); 13C NMR
(100 MHz, CDCl3):
d 178.0, 143.9, 139.0, 135.3, 134.8, 133.5, 129.2,
128.5, 127.6, 100.8, 72.1, 78.6.
4.2.5. (ꢀ)-2-(4-Chlorophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-
6,9-dien-8-one (7f-II)
Yield: 17%; 1H NMR (270 MHz, CDCl3):
d
7.40 (d, J ¼ 8.1 Hz, 2H),
7.32 (d, J ¼ 8.1 Hz, 2H), 6.89 (d, J ¼ 2.7 Hz, 1H), 6.79 (dd, J ¼ 2.7,
10.8 Hz, 1H), 6.31 (d, J ¼ 10.8 Hz, 1H), 5.26 (dd, J ¼ 5.4, 8.1 Hz, 1H),
4.52 (dd, J ¼ 5.4, 8.1 Hz, 1H), 3.86 (t, J ¼ 8.1 Hz, 1H); 13C NMR
(67.5 MHz, CDCl3):
d 178.0, 143.3, 139.4, 135.0, 134.8, 133.8, 129.1,
127.8, 127.5, 100.7, 78.7, 72.0.
4.2.6. (ꢀ)-2-(3-Bromophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-
6,9-dien-8-one (7g-I)
Yield: 39%; 1H NMR (400 MHz, CDCl3):
d 7.53 (s, 1H), 7.49e7.53
(m, 1H), 7.29e7.31 (m, 2H), 6.89 (d, J ¼ 2.9 Hz, 1H), 6.78 (dd, J ¼ 2.9,
10.2 Hz, 1H), 6.31 (d, J ¼ 10.2 Hz, 1H), 5.24 (dd, J ¼ 6.1, 8.3 Hz, 1H),
4.51 (dd, J ¼ 6.1, 8.3 Hz, 1H), 3.89 (t, J ¼ 8.3 Hz, 1H); 13C NMR
4.2. General procedure for the synthesis of compounds 7deh
A diol 6c or 6d or 6e (2 equiv) and PPTS (0.1 equiv) was added to
a toluene (25.0 mL/1 mmol of substrate) solution of a substituted-
4,4-dimethoxycyclohexa-2,5-dienone 5a or 5b (1 equiv) at room
temperature. After stirring at 40 ꢂC for 18 h, the reaction mixture
was quenched by the addition of saturated aqueous NaHCO3
and the product was extracted with ethyl acetate three times.
The combined organic extracts were washed with brine, dried over
(100 MHz, CDCl3): d 177.9, 143.2, 139.3, 138.8, 133.8, 131.9, 130.4,
129.1, 127.8, 124.6, 123.0, 100.7, 78.4, 72.0.
4.2.7. (ꢀ)-2-(3-Bromophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-
6,9-dien-8-one (7g-II)
Yield: 29%; 1H NMR (400 MHz, CDCl3):
d
7.53 (s, 1H), 7.49e7.53
(m, 1H), 7.26e7.29 (m, 2H), 6.95 (d, J ¼ 2.9 Hz, 1H), 6.75 (dd, J ¼ 2.9,

184
S. Fuse et al. / European Journal of Medicinal Chemistry 66 (2013) 180e184
10.2 Hz, 1H), 6.34 (d, J ¼ 10.2 Hz, 1H), 5.26 (dd, J ¼ 6.1, 8.3 Hz, 1H),
4.53 (dd, J ¼ 6.1, 8.3 Hz, 1H), 3.90 (t, J ¼ 8.3 Hz, 1H); 13C NMR
Dojindo). The 384-well plates were seeded with aliquots of a 20 mL
medium containing 1.0 ꢃ 103 cells per well and were incubated
overnight before being treated with compounds dissolved in DMSO
at various concentrations for 48 h. Plates were incubated for 1 h at
(100 MHz, CDCl3):
d 177.9, 143.7, 139.1, 138.8, 133.4, 131.9, 130.4,
129.1, 128.4, 124.7, 123.0, 100.8, 77.3, 72.0.
37 ꢂC after the addition of 2
mL WST-8 reagent solution per well. The
4.2.8. (ꢀ)-2-(4-Bromophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-
6,9-dien-8-one (7h-I)
absorption of the formazan dye formed was measured at 450 nm.
The vehicle solvent (DMSO) was used as a negative control.
Yield: 20%; 1H NMR (270 MHz, CDCl3):
d
7.55 (d, J ¼ 8.1 Hz, 2H),
7.26 (d, J ¼ 8.1 Hz, 2H), 6.88 (d, J ¼ 2.7 Hz, 1H), 6.79 (dd, J ¼ 2.7,
10.8 Hz, 1H), 6.31 (d, J ¼ 10.8 Hz, 1H), 5.25 (dd, J ¼ 5.4, 8.1 Hz, 1H),
4.52 (dd, J ¼ 5.4, 8.1 Hz, 1H), 3.86 (t, J ¼ 8.1 Hz, 1H); 13C NMR
Acknowledgements
This work was supported by a grant from the New Energy and
Industrial Technology Department Organization (NEDO).
(67.5 MHz, CDCl3):
d 178.4, 143.8, 139.8, 136.6, 134.3, 132.5, 128.2,
128.2, 123.3, 101.2, 79.2, 72.4.
4.2.9. (ꢀ)-2-(4-Bromophenyl)-7-chloro-1,4-dioxa-spiro[4.5]deca-
6,9-dien-8-one (7h-II)
Supplementary data
Yield: 19%; 1H NMR (270 MHz, CDCl3):
d
7.54 (d, J ¼ 8.1 Hz, 2H),
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.05.
030. These data include MOL files and InChiKeys of the most
important compounds described in this article.
7.26 (d, J ¼ 8.1 Hz, 2H), 6.95 (d, J ¼ 2.7 Hz, 1H), 6.74 (dd, J ¼ 2.7,
10.8 Hz, 1H), 6.33 (d, J ¼ 10.8 Hz, 1H), 5.26 (dd, J ¼ 5.4, 8.1 Hz, 1H),
4.52 (dd, J ¼ 5.4, 8.1 Hz, 1H), 3.88 (t, J ¼ 8.1 Hz, 1H); 13C NMR
(67.5 MHz, CDCl3):
d 178.4, 144.2, 139.4, 136.2, 133.9, 132.5, 128.8,
128.2, 123.3, 101.21, 79.00, 72.5.
References
4.3. Crystallographic studies for (ꢀ)-7d-II
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Single X-ray diffraction data were collected on a Rigaku R-AXIS
RAPID imaging plate diffractometer with MoK
a
radiation
ꢀ
(
l
¼ 0.71075 A). Empirical formula: C14H10ClIO3, formula weight:
388.57, temperature: 123(2) K, crystal system: triclinic, space
ꢀ
group: Pꢁ1 (no. 2), unit cell dimensions: a ¼ 4.8452(8) A,
b ¼ 15.210(3)ꢂA, c ¼ 18.506(3) A,
¼ 86.232(4)ꢂ,
b
¼ 88.992(4)ꢂ,
ꢀ
ꢀ
a
3
ꢀ
g
¼ 87.240(4) , volume: 1359.1(4) A , Z ¼ 4, density (calculated):
1.899 Mg/m3, absorption coefficient: 2.552 mmꢁ1, F(000): 752,
crystal size: 0.16 ꢃ 0.06 ꢃ 0.05 mm3, theta range for data collection:
3.31e27.46ꢂ, index ranges: ꢁ6 ꢄ h ꢄ 6, ꢁ19 ꢄ k ꢄ 19, ꢁ23 ꢄ l ꢄ 21,
reflections collected: 20,622, independent reflections: 6167
[R(int) ¼ 0.0772], completeness to theta ¼ 27.46ꢂ, 99.3%, Absorption
correction: semi-empirical from equivalents, max. and min. trans-
mission: 0.9600 and 0.6342, refinement method: full-matrix least-
squares on F2, data/restraints/parameters: 6167/0/343, Goodness-
of-fit on F2: 1.051, final R indices [I > 2sigma(I)]: R1 ¼ 0.0570,
wR2 ¼ 0.1090, R indices (all data): R1 ¼ 0.1244, wR2 ¼ 0.1391, largest
ꢀꢁ3
diff. peak and hole: 1.927 and ꢁ1.699 e A . CCDC 905767 contains
the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
4.4. Cytotoxicity assay
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The human cancer cell line used in this study was cervical car-
cinoma HeLa cells. The cells were maintained in Dulbecco’s modi-
fied Eagle medium (Sigma) supplemented with 10% fetal bovine
serum (Gibco), penicillin (100 U/mL) and streptomycin (100 mg/mL)
at 37 ꢂC in a humidified incubator under a 5% CO2 atmosphere. The
cytotoxicity of HeLa cells was assayed with WST-8 [2-(2-methoxy-
4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetra-
zolium, monosodium salt] colorimetric assay (Cell Counting Kit;