Synthesis and structure-activity relationships of thioflavone derivatives as specific inhibitors of the ERK-MAP kinase signaling pathway

Article abstract of DOI:10.1016/j.bmc.2004.02.002

Condensation of nitrobenzaldehydes 3 and α-[o-(p-methoxybenzylthio) benzoyl] sulfoxide 4 gave α-sulfinyl enones 5. Treatment of 5 with formic acid caused cyclization followed by debenzylation to afford 3-(methylsulfinyl) thioflavanones 6. Double-bond formation with elimination of methanesulfenic acid was performed by refluxing 6 in benzene, and, finally, the nitro group of 2-phenyl-4H-1-benzothiopyran-4-one (thioflavones) 7 was reduced with tin in tetrafluoroboric acid. Various 2^′-aminothioflavones 8 thus prepared were evaluated for their inhibitory effects on the ERK-MAP kinase pathway. In a cell-based assay, 2-(2^′-amino-3 ^′-methoxyphenyl)-4H-1-benzothiopyran-4-one (8b) showed a more potent inhibitory effect than the corresponding oxygen compound (PD98059, 1) on the Raf-induced activation of the ERK-MAP kinase pathway as well as cell proliferation. Furthermore, compound 8b selectively and potently inhibited the proliferation of tumor cells in which the ERK-MAP kinase pathway is constitutively activated.

Full text of DOI:10.1016/j.bmc.2004.02.002

Bioorganic & Medicinal Chemistry 12 (2004) 2397–2407
Synthesis and structure–activity relationships of thioflavone
derivatives as specific inhibitors of the ERK-MAP kinase
signaling pathway
Tadashi Kataoka,a,* Shin-ichi Watanabe,a Eiji Mori,a Ryoji Kadomoto,b
Susumu Tanimurab and Michiaki Kohnob,*
aLaboratory of Pharmaceutical Chemistry, Gifu Pharmaceutical University, 6-1, Mitahora-higashi 5-chome, Gifu 502-8585, Japan
bLaboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences,
Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan
Received 13 January 2004; revised 2 February 2004; accepted 2 February 2004
Abstract—Condensation of nitrobenzaldehydes 3 and a-[o-(p-methoxybenzylthio)benzoyl] sulfoxide 4 gave a-sulfinyl enones 5.
Treatment of 5 with formic acid caused cyclization followed by debenzylation to afford 3-(methylsulfinyl)thioflavanones 6. Double-
bond formation with elimination of methanesulfenic acid was performed by refluxing 6 in benzene, and, finally, the nitro group of 2-
phenyl-4H-1-benzothiopyran-4-one (thioflavones) 7 was reduced with tin in tetrafluoroboric acid. Various 20-aminothioflavones 8
thus prepared were evaluated for their inhibitory effects on the ERK-MAP kinase pathway. In a cell-based assay, 2-(20-amino-30-
methoxyphenyl)-4H-1-benzothiopyran-4-one (8b) showed a more potent inhibitory effect than the corresponding oxygen compound
(PD98059, 1) on the Raf-induced activation of the ERK-MAP kinase pathway as well as cell proliferation. Furthermore, compound
8b selectively and potently inhibited the proliferation of tumor cells in which the ERK-MAP kinase pathway is constitutively
activated.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
common to many types of human tumors.4–7 Thus, there
has been an explosion of interest in the components of
the ERK-MAP kinase pathway as attractive targets for
the development of anticancer drugs.8;9
The 41- and 43-kDa mitogen-activated protein (MAP)
kinases (ERK2 and ERK1, respectively) play a pivotal
role in the intracellular signaling pathway and are
essential components of the ERK-MAP kinase signaling
pathway, which includes MAP kinase/ERK kinase
(MEK)1/2 and Raf-1.1–3 The ERK-MAP kinase pathway
is activated by a wide variety of mitogenic stimuli that
interact with structurally distinct receptors, and it rep-
resents a convergence point for most, if not all, mitogenic
signaling pathways. Very precise spatio-temporal control
mechanisms for the ERK-MAP kinase activity have
evolved to ensure homeostasis in multi-cellular organ-
isms. Accordingly, aberrant activation of the ERK-MAP
kinase pathway has been shown to be an essential feature
It has been shown that a flavone derivative, 2-(2-amino-
3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059)
(1), inhibits the MEK activity specifically and
potently.10;11 However, the structure–activity relation-
ship of flavone derivatives related to 1 has not been
reported. The isosteric replacement of an oxygen atom
by a sulfur atom is useful for the design and synthesis of
analogues, which are expected to display modified
chemical and biological behavior.12 In this respect,
although differences in the physical and chemical
behaviors between the flavone derivatives and the thi-
oflavones have been well studied,13;14 the difference in
their biological activity has not been examined.
Keywords: Anti-tumor agents; Inhibitors; Signal transduction; Sulfur
heterocycles.
In this paper, we report the synthesis and structure–
activity relationship of thioflavones bearing the poly-
substituted phenyl group at the 2-position as inhibitors
against the ERK-MAP kinase pathway.
* Corresponding authors. Tel.: +81-58-237-3931; fax: +81-58-237-5979
(T.K.), fax: +81-95-819-2472 (M.K.); e-mail addresses: kataoka@
gifu-pu.ac.jp; kohnom@net.nagasaki-u.ac.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.002

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T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
R2
2. Chemistry
R3
R1
S
PMP
piperidine
The synthetic procedure used for the preparation of 1,15;16
which has the flavone skeleton, cannot be applied to the
synthesis of thioflavones because thioflavones have a
structure in which a sulfur atom is replaced with an
oxygen atom. Although many thioflavones have been
prepared, a limited number of 20-substituted ones are
known. Two methods were selected from several reported
procedures17–23 for the synthesis of the 20-substituted
thioflavones: the direct condensation of a benzoylacetate
with a benzenethiol24–30 and the condensation of an a-[o-
(alkylthio)benzoyl] sulfoxide with a benzenethiol fol-
lowed by cyclization and elimination of a sulfenic acid.31
+
benzene
S
NO2
reflux, 0.5 - 1 h
O
O
CHO
3a-h
4
PMP= p-MeOC6H4
R1
R1
PMP O2N
S
R2
R3
O2N
S
R2
R3
HCOOH
5˚C, 15 - 30 min
S
S
O
O
O
O
5a-h
6a-h
We first attempted to synthesize 20-nitrothioflavone (7a)
by condensation of ethyl o-nitrobenzoylacetate32 (2)
with benzenethiol using an acid, such as polyphosphoric
acid, p-toluenesulfonic acid, or BF3–Et2O; all these tri-
als, however, were unsuccessful. Therefore, a second
approach was adopted.
R3
R2
S
Sn, HBF4
R1
benzene
NO2
reflux, 30 min
80˚C, 0.5 h
O
7a-h
R3
OH
R2
R1
R2
R1
NO2
O
O
OMe
S
S
NH2
COOEt
+
NH2
NH2
O
2
1 (PD98059)
O
O
9c: R1=H, R2=OMe
9e: R1=Cl, R2=H
9g: R1=Me, R2=H
8a-h
The 20-nitrothioflavone derivatives were synthesized as
shown in Scheme 1. o-Nitrobenzaldehydes 3 were pre-
pared from o-nitroanilines or o-nitrobenzoic acids.
Condensation of benzaldehydes 3 with a-benzoyl sulf-
oxide31 4 gave a-sulfinyl enones 5 in good yields. The 1H
NMR spectra of 5 showed the olefinic protons at d 7.5–
8.05 as singlets, but the geometrical structures of the
double bonds could not be determined. Cyclization of 5
followed by debenzylation was performed by the treat-
ment with formic acid at 5 ꢁC to give 3-(methylsulfinyl)-
2,3-dihydro-4H-1-benzothiopyran-4-ones 6 as a mixture
of diastereoisomers. Refluxing the diastereomer mix-
tures of 6 in benzene caused the elimination of meth-
anesulfenic acid to form thioflavones 7.
3, 5-8:
e: R1=Cl, R2=R3=H
b: R1=MeO, R2=R3=H f: R1=R3=H, R2=Cl
c: R1=R3=H, R2=MeO g: R1=Me, R2=R3=H
d: R1=R2=H, R3=MeO h: R1=H, R2=R3=OMe
a: R1=R2=R3=H
Scheme 1.
reoisomers of 6g in chloroform was allowed to stand at
room temperature, 6g was gradually transformed into
thioflavone 7g.
A nitro group of 7 was reduced with tin and tetrafluo-
roboric acid to obtain 20-aminothioflavones 8. We used
tetrafluoroboric acid instead of hydrochloric acid in
order to prevent thioflavones 7 from chlorination of the
aryl group at the 2-position. Reduction of 30- or 40-
substituted 20-nitrothioflavones 7c,e,g gave 50-hydroxyl-
ated products 9c,e,g together with 8c,e,g. The exact mass
data (molecular formula: C16H13NO2S) indicated that
Cyclized product 6g as an example was separated into a
trans-diastereoisomer and a mixture of two cis-isomers
to examine the structures of 6. The coupling constants
between protons of the 2-position (Ha) and the 3-posi-
tion (Hb) of three isomers were small and had similar
values (J ¼ 3–5 Hz) as shown in Figure 1. Therefore,
their stereochemistry was determined by the NOE dif-
ference spectra: a compound showing a small NOE
enhancement (5%) was a trans-isomer (6g-1 or 6g-2),
and the others with a big enhancement (13% and 15%)
were cis-isomers (6g-3 and 6g-4). The two cis-isomers
would be the diastereoisomers due to the difference in
stereochemistry between the sulfinyl group and the
carbon atom at the 3-position, but their stereostructures
have not been assigned yet. When a mixture of diaste-
1
product 9c contained one more oxygen than 8c. The H
NMR spectrum of 9c showed two singlets due to 30- and
60-protons at d 6.34 and 6.90, respectively, and a new
broad singlet due to a hydroxyl group that appeared at d
5.20. An aromatic carbon without a substituent was
reduced, and an aromatic carbon with a substituent was
increased in the 13C NMR spectrum. The IR spectrum
exhibited an absorption at 3400 cmꢀ1 due to the hydroxy
group. The hydroxylation would proceed via the rear-

T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
2399
PD98059 (1) is a unique inhibitor of MEK1/2; it does
not inhibit MEK1/2 activity directly but acts alike as an
allosteric inhibitor, that is, it binds outside the ATP- and
ERK1/2-binding sites on MEK1/2 and the resultant
modification of the three-dimensional structures renders
MEK1/2 not phosphorylatable (activatable) by Raf-1.11
Likewise, 8b did not inhibit the MEK1 activity directly
as determined by the phosphorylation of ERK1/2 in an
in vitro kinase assay, but it inhibited the phosphoryl-
ation of MEK1 by Raf-1 (Table 1). As in the case of the
in vivo assay, 8b exhibited a more potent inhibitory
activity on the phosphorylation of MEK1 by Raf-1 than
1 (IC50 of 1.33 lM vs 7.55 lM). An assessment of other
kinases, which include ERK1/2, p38 MAP kinase, c-Jun
N-terminal kinase, MKK3, MAPKAP kinase 2, protein
kinase Ca, EGF receptor kinase, and cyclin-dependent
kinase 2, showed that none of these enzymes was sig-
nificantly inhibited by 8b or 8c at concentrations as high
as 100 lM. Furthermore, 8b did not inhibit the phos-
phorylation of the myelin basic protein by Raf-1 in an in
vitro assay (data not shown). These results indicate that
substitution by sulfur of oxygen at the position 1 of the
flavone structure, that is, conversion of flavone to thio-
flavone by itself, does not alter its specificity as an MEK
inhibitor.
Trans
Me
Me
Me
Me
O2N
O2N
Ha
Ha
S
S
S
S
Hb
Hb
O
O
O
O
6g-1
6g-2
Cis
Me
Me
Me
Me
O2N
O2N
Ha
Ha
S
S
S
Hb
S
Hb
O
O
O
O
6g-3
6g-4
1H-NMR (400MHz)
Isomers Ha ( ppm) Hb ( ppm) SOMe ( ppm) J ( Hz)
Finally, compound 8b was tested for its ability to inhibit
the proliferation of several tumor cell lines in culture.
Compound 8b inhibited the proliferation of tumor cells
in which the ERK-MAP kinase pathway is constitu-
tively activated (WiDr colon carcinoma cells and
HT1080 fibrosarcoma cells),6 but it did not inhibit sig-
nificantly the proliferation of those in which constitutive
activation of the ERK-MAP kinase pathway is not
detected (PC3 prostate carcinoma cells and Colo320
colon carcinoma cells).6 Furthermore, 8b showed better
inhibitory activity than 1 on the proliferation of tumor
cells (IC50 of 8.6 lM vs 29.5 lM for HT1080 cells and
IC50 of 10.0 lM vs 30.5 lM for WiDr cells), and totally
inhibited their proliferation at 30–50 lM concentration.
In contrast, the corresponding oxygen compound 1 at
50 lM, which is almost the highest concentration
available in the culture medium, inhibited partly (70–
80%) but not completely the proliferation of these tumor
cells. One weak point of 1, for all its high specificity, is
the low solubility in aqueous medium. Conversion of
flavone to thioflavone by itself did not resolve this point
significantly: compound 8b, for example, gave the
maximum concentration of ꢁ100 lM in the culture
medium. However, more potent inhibitory activity
conferred on thioflavone derivatives made 8b readily
possible to totally abolish the proliferation of tumor
cells.
Trans
Cis-1
Cis-2
5.14
4.96
5.18
4.17
4.20
4.49
2.79
2.85
2.97
3
4
5
NOE measurement
Ha-Hb (%) Hb-SOMe (%)
Trans
Cis-1
Cis-2
5
15
13
5
5
6
Figure 1.
rangement of an N-arylhydroxylamine formed in situ
under acidic conditions (Bamberger rearrangement).33;34
3. Biological activity
Compounds 7a–g, 8a–g, and 9c,e,g were examined for
their ability to inhibit the Raf-1-induced activation of
the ERK-MAP kinase pathway in a cell-based assay
with the use of 3T3-DRaf-1:ER cells.35;36 Among those
studies, 8b was found to have the most potent inhibitory
activity on the activation of the ERK-MAP kinase
pathway, which was more potent than that of 1,10;11 the
corresponding oxygen compound of 8b (IC50 of 1.88 lM
vs 4.35 lM as determined by the phosphorylation of
MEK1, and 2.95 lM vs 13.8 lM as determined by the
phosphorylation of ERK1/2). Furthermore, 8b inhibited
the Raf-1-induced proliferation of the cells more
potently than 1 (IC50 of 7.00 M vs 17.5 lM). Although
8c also exhibited inhibitory activity, other compounds
did not significantly inhibit the Raf-1-induced activation
of ERK-MAP kinase pathway or cell proliferation at
concentrations up to 100 lM (Table 1).
4. Structure–activity relationship
All the 20-nitrothioflavones 7a–h totally abolished the
inhibitory activity, indicating that the amino group at
the 20-position is essential to exhibit the inhibitory effect
on the activation of the ERK-MAP kinase pathway.

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T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
Table 1. Inhibitory activity of thioflavone derivatives
R4
R1
R3
R2
O
O
S
OMe
NH2
O
1 (PD98059)
7 - 9
Compd
R1
R2
R3
R4
Cell-based assay [ IC50 (lM)]
In vitro kinase assay
[IC50 (lM)]
Phosphoryl-
ation of
MEK1
Phosphoryl-
ation of
ERK1/2
Cell Prolif-
eration
Raf-1 fi
MEK1
MEK1 fi
ERK1/2
1
NH2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
OCH3
H
H
H
4.35 0.78
13.75 3.18 17.50 1.27
7.55 1.48 >100
7a
7b
7c
7d
7e
7f
H
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
ND
ND
ND
ND
ND
ND
ND
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
OCH3
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
OCH3
H
H
H
Cl
OCH3
H
H
Cl
H
H
7g
7h
8a
8b
8c
8d
8e
8f
CH3
H
H
H
OCH3
H
OCH3
H
H
OCH3
H
H
H
1.88 0.11
21.50 2.12
>100
2.95 1.06 7.00 0.57
26.25 1.06 22.85 3.89
1.33 0.04 >100
8.25 3.18 >100
OCH3
H
H
H
Cl
OCH3
H
>100
ND
ND
ND
ND
ND
ND
ND
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
H
Cl
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H
H
8g
8h
9c
9e
9g
CH3
H
H
H
OCH3
OCH3
H
OCH3
OH
OH
OH
H
Cl
CH3
H
Values are means SEM of three separate assays, each performed in duplicate.
The derivative bearing a methoxy group 8b but not a
methyl group 8g at the 30-position showed inhibitory
activity, suggesting that an electron-donating group
with lone-pair electrons at the 30-position is necessary:
the interaction, such as the hydrogen bond between the
30-substituent and the binding site on the enzyme, would
be important for revealing the inhibitory activity. The
40-methoxy group decreased the activity, and com-
pounds 8d and 9c,e,g bearing the 50-methoxy or hydroxy
group and the 40,50-dimethoxy derivative 8h did not
show the inhibitory activity. All these findings indicate
that the methoxy group of the 2-phenyl group should be
situated at the ortho position of the amino group.
thioflavone with the 30-methoxy group showed the
strongest activity.
5. Conclusion
We have prepared various derivatives of thioflavones 7,
8, and 9 and evaluated their inhibitory activity on the
activation of the ERK-MAP kinase pathway. The 2-(20-
amino-30-methoxyphenyl) derivative 8b is the most
potent compound among them and inhibits the Raf-1-
induced activation of MEK more efficiently than the
corresponding oxygen compound 1. Furthermore, 8b
potently and selectively inhibits the proliferation of
several tumor cells in which constitutive activation of
the ERK-MAP kinase pathway is detected. These thio-
flavone derivatives would provide new leads for the
development of potent and useful inhibitors against the
ERK-MAP kinase pathway.
The inhibitory activity of thioflavones against the acti-
vation of the ERK-MAP kinase pathway may depend
largely on the polarizability of the chromone skeleton.
The prominent activity observed for compound 8b
suggests that the replacement of 1-oxygen atom of the
chromone skeleton with a sulfur atom promotes the
polarization of the 4-carbonyl group,13;14 which makes
the binding of thioflavone 8b to MEK easier and/or
stronger than that of the flavone derivative 1. This
hypothesis would be supported by the fact that
6. Experimental
6.1. Chemistry
þ
þ 37
MeSCH2 is more stable than MeOCH2 . In contrast,
a through-space effect of the methoxy group seems to be
more important than its electronic effect because the
6.1.1. 4-Methoxy-2-nitrobenzaldehyde (3c). This com-
pound was prepared by the procedure for the synthesis

T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
2401
of 2-bromo-4-methylbenzaldehyde.38 (a) A mixture of
paraformaldehyde (3.67 g, 45 mmol) and hydroxylamine
hydrochloride (3.16 g, 45 mmol) in water (5 mL) was
warmed until the mixture became transparent. Then
sodium acetate (3.71 g, 45 mmol) was added to the
mixture. The whole mixture was gently heated under
reflux for 15 min to give a 10% formaldoxime solution.
(b) Concentrated hydrochloric acid (5.15 mL) was
slowly added to a suspension of 4-methoxy-2-nitroani-
line (5.0 g, 30 mmol) in water (5 mL), and then ice was
added (9.0 g). The mixture was cooled and maintained at
)5 to 5 ꢁC in an ice–salt bath. A solution of sodium
nitrite (2.05 g, 30 mmol) in water (3 mL) was added to
the mixture with vigorous stirring, and the whole mix-
ture was stirred for 15 min. The stirred solution of the
diazonium salt was made neutral with a solution of
sodium acetate (1.66 g, 20 mmol) in water (3 mL). (c) To
the solution of formaldoxime prepared above (step a)
were added copper(II) sulfate (712 mg, 4.5 mmol),
sodium sulfite (120 mg, 0.95 mmol), and a solution of
sodium acetate (19 g) in water (21 mL). The mixture was
vigorously stirred at 10–15 ꢁC, and the solution of the
diazonium salt prepared in step b was gradually added
to it. The whole mixture was stirred for 2 h at room
temperature and then concentrated hydrochloric acid
(27 mL) was added to it. The resulting mixture was
gently refluxed for 2 h, cooled to room temperature, and
then extracted with ether. The extracts were washed with
brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by column
chromatography on silica gel eluting with CH2Cl2–
AcOEt (30:1) to give red powder (1.43 g, 27%). The
spectral data were identical with those of an authentic
sample prepared by an alternative method.39
6.1.3. 3-Chloro-2-nitrobenzaldehyde (3e).
This sample
(0.85 g) was similarly prepared starting from 3-chloro-2-
nitrobenzoic acid (1.75 g, 9 mmol) by the method for 3d.
The spectral data were identical with those of an
authentic sample prepared by an alternative method.41
4-Chloro-2-nitrobenzaldehyde41 (3f) and 3-methyl-2-
nitrobenzaldehyde42 (3g) were prepared by the known
methods.
6.1.4. Methyl 2-(4-methoxybenylthio)benzoate.31 Sodium
hydride (60% pure in mineral oil) (7.36 g, 0.184 mol) was
gradually added to a solution of methyl thiosalicylate43
(31 g, 0.184 mol) in dry THF (300 mL). p-Methoxyben-
zyl chloride (25 mL, 0.184 mol) was added to the mixture
of the thiosalicylate. The mixture was heated at 60 ꢁC for
1 h and concentrated under reduced pressure. To the
residue was added 3 N hydrochloric acid (100 mL), and
the mixture was extracted with chloroform. The extracts
were washed with brine, dried (MgSO4), and concen-
trated under reduced pressure. The oily product (47 g,
89%) was used for the next step without further purifi-
1
cation. H NMR (400 MHz, CDCl3): d ¼ 3:78 (3H, s,
OMe), 3.89 (3H, s, OMe), 4.11 (2H, s, CH2), 6.84 (2H, d,
J ¼ 8 Hz, Ar-H), 7.14 (1H, t, J ¼ 7 Hz, Ar-H), 7.25–
7.45 (4H, m, Ar-H), 7.95 (1H, d, J ¼ 8 Hz, Ar-H).
6.1.5. 1-[2-(4-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)ethanone (4).31 Sodium methylsulfinylmethylide (dim-
sylsodium) was prepared from dry DMSO (162 mL) and
sodium hydride (60% pure in mineral oil) (10.8 g,
0.27 mol) at 65–70 ꢁC for 45 min under argon. Dry THF
(80 mL) was added to the solution of dimsylsodium, and
the mixture was cooled at 0 ꢁC. A solution of methyl
2-(4-methoxybenylthio) benzoate (39 g, 0.135 mol) in
dry THF (40 mL) was gradually added to the dimsyl-
sodium solution at 0 ꢁC under argon, and then the
mixture was stirred for 2 h at room temperature. THF
was evaporated under reduced pressure, and the residue
was acidified by adding 3 N hydrochloric acid. The
mixture was extracted with chloroform and the extracts
were washed with brine, dried (MgSO4), and concen-
trated under reduced pressure. The residue was washed
with ether, and then ether was removed in vacuo. The
product (25.6 g, 57%) was used for the reaction with
aldehydes without further purification. 1H NMR
(400 MHz, CDCl3): d ¼ 2:74 (3H, s, SOMe), 3.78 (3H, s,
OMe), 4.10 (2H, s, SCH2Ar), 4.27 (1H, d, J ¼ 14 Hz,
COCH2SO), 4.44 (1H, d, J ¼ 14 Hz, COCH2SO), 6.83
(2H, d, J ¼ 9 Hz, Ar-H), 7.26 (2H, d, J ¼ 8 Hz, Ar-H),
7.23–7.30 (1H, m, Ar-H), 7.42–7.51 (2H, m, Ar-H), 7.81
(1H, d, J ¼ 8 Hz, Ar-H).
6.1.2. 5-Methoxy-2-nitrobenzaldehyde (3d).
Thionyl
chloride (4.53 g, 38 mmol) and DMF (2 drops) were
added to a solution of 5-methoxy-2-nitrobenzoic acid
(5 g, 25 mmol) in THF (25 mL), and the mixture was
refluxed for 1 h and then cooled. The solution of 5-
methoxy-2-nitrobenzoyl chloride thus prepared was
added dropwise to a suspension of sodium borohydride
(0.96 g, 25 mmol) in a mixture of DMF (12 mL) and
THF (25 mL) at 0 ꢁC. After the mixture was stirred for
3 h at room temperature, 3 N hydrochloric acid (25 mL)
was added to it, and then the mixture was diluted with
water (25 mL). The whole mixture was extracted with
ether and the extracts were washed with brine and water,
dried (MgSO4) and concentrated under reduced pres-
sure. The residue was purified by column chromato-
graphy on silica gel eluting with hexane–AcOEt (2:1) to
give 5-methoxy-2-nitrobenzyl alcohol (3.05 g, 78%). A
solution of the benzylalcohol (3.05 g, 17 mmol) in
CH2Cl2 (12 mL) was added to a solution of pyridinium
chlorochromate (PCC) (5.33 g, 24 mmol) in CH2Cl2
(23 mL). The mixture was stirred for 2 h at room tem-
perature and diluted with ether (75 mL). The organic
layer was washed with brine, dried (MgSO4), and con-
centrated under reduced pressure to give yellow powder
(2.65 g, 88%). The spectral data were identical with those
of an authentic sample prepared by an alternative
method.40
6.1.6. 1-[2-(4-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(2-nitrophenyl)-2-propen-1-ones (5a). A mixture of
a-sulfinyl ketone 4 (2 g, 6 mmol), o-nitrobenzaldehyde
(0.90 g, 6 mmol), and piperidine (2 drops) in benzene
(10 mL) was heated under reflux for 30 min and dehy-
drated using a Dean–Stark apparatus. Benzene was
removed under reduced pressure, and the residue was

2402
T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
dissolved in chloroform. The solution was washed with
3 N hydrochloric acid and brine, dried (MgSO4), and
concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel eluting
with CHCl3–AcOEt (10:1) to give yellow powder (1.84 g,
66%), mp 148 ꢁC. IR (film): m ¼ 1063 (SOMe), 1340 and
6.1.9. 1-[2-(4-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(5-methoxy-2-nitrophenyl)-2-propen-1-ones (5d).
Yield 75%, yellow powder, mp 153–155 ꢁC; IR (film):
m ¼ 1071 (SOCH3), 1336 (NO2), 1509 (NO2), 1639 (CO);
1H NMR (400 MHz, CDCl3): d ¼ 2:87 (3H, s, SOCH3),
3.79 (3H, s, Ar-OCH3), 3.80 (3H, s, Ar-OCH3), 3.97
(1H, d, J ¼ 12 Hz, SCH2), 4.07 (1H, d, J ¼ 12 Hz,
SCH2), 6.58 (1H, d, J ¼ 3 Hz, Ar-H), 6.74 (1H, dd,
J ¼ 9, 3 Hz, Ar-H), 6.85 (1H, d, J ¼ 8 Hz, Ar-H), 6.91
(1H, dd, J ¼ 9, 1 Hz, Ar-H), 7.15–7.20 (2H, m, Ar-H),
7.25 (2H, d, J ¼ 8 Hz, Ar-H), 7.49 (1H, dd, J ¼ 8, 1 Hz,
Ar-H), 7.97 (1H, d, J ¼ 9 Hz, Ar-H), 8.09 (1H, s,
C@CH); 13C NMR (100 MHz, CDCl3): d ¼ 37:2 (q),
40.4 (t), 55.1 (q), 56.1 (q), 113.8 (d), 114.0 (d), 115.3 (d),
124.2 (d), 126.9 (d), 127.3 (d), 127.4 (s), 130.0 (d), 130.8
(d), 132.6 (d), 133.2 (s), 135.3 (s), 137.6 (d), 139.3 (s),
140.7 (s), 146.3 (s), 158.9 (s), 163.0 (s), 192.3 (s);
FABMS: m=z (relative intensity) 498 (Mþ+1, 4%), 121
(base); elemental analysis calcd (%) for C25H23NO6S2: C,
60.34; H, 4.66; N, 2.81. Found: C, 59.93; H, 4.52; N,
2.79.
1
1522 (NO2), 1617 (CO); H NMR (400 MHz, CDCl3):
d ¼ 2:86 (3H, s, SOMe), 3.79 (3H, s, OMe), 4.02 (1H, d,
J ¼ 12 Hz, SCH2-Ar), 4.05 (1H, d, J ¼ 12 Hz, SCH2-
Ar), 6.83–6.88 (3H, m, Ar-H), 7.13–7.27 (8H, m, Ar-H),
7.48 (1H, d, J ¼ 8 Hz, Ar-H), 7.96 (1H, d, J ¼ 8 Hz,
Ar-H), 8.05 (1H, s, C@CH); 13C NMR (100 MHz,
CDCl3): d ¼ 37:2 (t), 40.7 (q), 55.3 (q), 114.1 (d), 124.4
(d), 124.8 (d), 127.3 (d), 127.9 (s), 129.7 (d), 130.1 (d),
130.4 (s), 130.5 (d), 131.5 (d), 132.5 (d), 133.3 (d), 135.6
(s), 136.9 (d), 140.5 (s), 146.4 (s), 147.0 (s), 159.0 (s),
192.3 (s); FABMS: m=z 468 (Mþ+1), 121 (base); ele-
mental analysis calcd (%) for C24H21NO5S2: C, 61.65; H,
4.53; N, 3.00. Found: C, 61.60; H, 4.65; N, 3.00. The
spectral data showed that product 5a was a single iso-
mer, but the geometrical structure was not determined.
6.1.7. 1-[2-(4-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(3-methoxy-2-nitrophenyl)-2-propen-1-ones (5b).
Yield 82%, yellow powder, mp 164 ꢁC; IR (film):
6.1.10. 1-[2-(p-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(3-chloro-2-nitrophenyl)-2-propen-1-ones (5e). Yield
73%, yellow powder, mp 134–135 ꢁC; IR (film): m ¼ 1074
(SOCH3), 1248 (NO2), 1534 (NO2), 1636 (CO); 1H
NMR (400 MHz, CDCl3): d ¼ 2:78 (3H, s, SOCH3),
3.80 (3H, s, Ar-OCH3), 4.10 (1H, d, J ¼ 12 Hz, SCH2),
4.12 (1H, d, J ¼ 12 Hz, SCH2), 6.86 (2H, d, J ¼ 9 Hz,
Ar-H), 6.97 (1H, t, J ¼ 8 Hz, Ar-H), 7.00–7.07 (2H, m,
Ar-H), 7.26–7.34 (5H, m, Ar-H), 7.46 (1H, s, C@CH),
7.60 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR (100 MHz,
CDCl3): d ¼ 36:7 (t), 40.5 (q), 55.3 (q), 114.1 (d), 124.3
(d), 125.7 (s), 126.5 (d), 127.7 (s), 128.3 (s), 128.9 (d),
129.4 (d), 130.1 (d), 130.5 (d), 131.2 (d), 132.0 (d), 133.4
(d), 133.5 (s), 142.2 (s), 147.5 (s), 151.6 (s), 159.0 (s),
191.6 (s); FABMS: m=z (relative intensity) 502 (Mþ+1,
35%), 121 (base); elemental analysis calcd for
C24H20ClNO5S2: C, 57.42; H, 4.02; N, 2.79. Found: C,
57.23; H, 3.94; N, 2.87.
1
m ¼ 1064 (SOCH3), 1370 (NO2), 1524 (NO2); H NMR
(400 MHz, CDCl3): d ¼ 2:78 (3H, s, SOCH3), 3.80 (3H,
s, Ar-OCH3), 3.83 (3H, s, Ar-OCH3), 4.09 (1H, d,
J ¼ 14 Hz, SCH2), 4.13 (1H, d, J ¼ 14 Hz, SCH2), 6.68
(1H, d, J ¼ 8 Hz, Ar-H), 6.83–6.88 (3H, m, Ar-H), 7.01–
7.03 (2H, m, Ar-H), 7.29–7.33 (4H, m, Ar-H), 7.51 (1H,
s, C@CH), 7.65 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 36:8 (t), 40.6 (q), 55.3 (q), 56.5
(q), 113.5 (d), 114.1 (d), 121.8 (d), 124.2 (d), 126.5 (d),
127.8 (s), 127.9 (s), 130.1 (d), 130.5 (d), 131.0 (d), 132.1
(d), 133.1 (d), 133.8 (s), 139.5 (s), 142.2 (s), 150.1 (s),
151.1 (s), 159.0 (s), 192.0 (s); FABMS: m=z (relative
intensity) 498 (Mþ+1, 7%), 121 (base); elemental anal-
ysis calcd (%) for C25H23NO6S2: C, 60.35; H, 4.66; N,
2.81. Found: C, 60.21; H, 4.63; N, 2.86.
6.1.8. 1-[2-(4-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(4-methoxy-2-nitrophenyl)-2-propen-1-ones (5c).
Yield 70%, yellow powder, mp 152–155 ꢁC; IR (film):
m ¼ 1075 (SOCH3), 1348 (NO2), 1528 (NO2), 1634 (CO);
1H NMR (400 MHz, CDCl3): d ¼ 2:86 (3H, s, SOCH3),
3.77 (3H, s, Ar-OCH3), 3.80 (3H, s, Ar-OCH3), 4.05
(1H, d, J ¼ 12 Hz, SCH2), 4.08 (1H, d, J ¼ 12 Hz,
SCH2), 6.72 (1H, d, J ¼ 8 Hz, Ar-H), 6.85–6.89 (3H, m,
Ar-H), 7.02 (1H, d, J ¼ 8 Hz, Ar-H), 7.20–7.21 (2H, m,
Ar-H), 7.26–7.28 (2H, m, Ar-H), 7.47 (1H, s, Ar-H),
7.52 (1H, d, J ¼ 8 Hz, Ar-H), 7.96 (1H, s, C@CH); 13C
NMR (100 MHz, CDCl3): d ¼ 36:9 (t), 40.8 (q), 55.3 (q),
55.9 (q), 109.4 (d), 114.1 (d), 119.69 (d), 122.2 (s), 124.2
(d), 127.0 (d), 127.9 (s), 130.1 (d), 130.8 (d), 132.6 (d),
132.7 (d), 135.2 (s), 136.5 (d), 140.8 (s), 145.8 (s), 147.3
(s), 159.0 (s), 160.3 (s), 192.5 (s); FABMS: m=z (relative
intensity) 498 (Mþ+1, 7%), 154 (base); elemental anal-
ysis calcd (%) for C25H23NO6S2: C, 60.35; H, 4.66; N,
2.81. Found: C, 59.97; H, 4.61; N, 2.67.
6.1.11. 1-[2-(p-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(4-chloro-2-nitrophenyl)-2-propen-1-ones (5f). Yield
80%, yellow powder, mp 158 ꢁC; IR (film): m ¼ 1073
(SOCH3), 1343 (NO2), 1510 (NO2), 1624 (CO); 1H
NMR (400 MHz, CDCl3): d ¼ 2:85 (3H, s, SOCH3),
3.80 (3H, s, Ar-OCH3), 4.05 (1H, d, J ¼ 12 Hz, SCH2),
4.08 (1H, d, J ¼ 12 Hz, SCH2), 6.86 (2H, d, J ¼ 9 Hz,
Ar-H), 6.92 (1H, td, J ¼ 8, 2 Hz, Ar-H), 7.05 (1H, d,
J ¼ 8 Hz, Ar-H), 7.12 (1H, dd, J ¼ 8, 2 Hz, Ar-H),
7.23–7.28 (3H, m, Ar-H), 7.46 (1H, dd, J ¼ 8, 1 Hz, Ar-
H), 7.94 (1H, s, C@CH); 13C NMR (100 MHz, CDCl3):
d ¼ 36:9 (t), 40.6 (q), 55.3 (q), 114.1 (d), 124.4 (d), 124.9
(d), 127.1 (d), 127.8 (s), 128.7 (s), 130.1 (d), 130.6 (d),
132.5 (d), 132.8 (d), 133.2 (d), 135.2 (s), 135.3 (d), 135.6
(s), 140.8 (s), 146.7 (s), 147.9 (s), 159.0 (s), 191.9 (s);
FABMS m=z (relative intensity) 502 (Mþ+1, 25%), 154
(base); elemental analysis calcd (%) for C24H20ClNO5S2:
C, 57.42; H, 4.02; N, 2.79. Found: C, 57.08; H, 3.95; N,
2.64.

T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
2403
6.1.12. 1-[2-(p-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(3-methyl-2-nitrophenyl)-2-propen-1-ones (5g). Yield
69%, yellow powder, mp 157 ꢁC; IR (film): m ¼ 1068
(SOCH3), 1246 (NO2), 1523 (NO2), 1628 (CO); 1H NMR
(400MHz, CDCl3): d ¼ 2:30 (3H, s, CH3), 2.81 (3H, s,
SOCH3), 3.80 (3H, s, Ar-OCH3), 4.08 (1H, d, J ¼ 12 Hz,
SCH2), 4.11 (1H, d, J ¼ 12 Hz, SCH2), 6.86 (2H, d,
J ¼ 8 Hz, Ar-H), 6.96 (3H, m, Ar-H), 7.07 (1H, dd,
J ¼ 9, 3 Hz, Ar-H), 7.23–7.28 (2H, m, Ar-H), 7.30 (2H, d,
J ¼ 9 Hz, Ar-H), 7.58 (1H, s, C@CH), 7.63 (1H, d,
J ¼ 8 Hz, Ar-H); 13C NMR (100 MHz, CDCl3): d ¼ 18:0
(q), 36.8 (t), 40.7 (q), 55.3 (q), 114.0 (d), 124.1 (d), 126.5
(d), 127.1 (s), 127.8 (s), 128.4 (d), 129.3 (s), 130.1 (d),
131.0 (d), 131.8 (s), 132.1 (d), 132.5 (d), 133.0 (d), 134.0
(d), 141.9 (s), 149.1 (s), 149.2 (s), 159.0 (s), 192.1 (s);
FABMS: m=z (relative intensity) 482 (Mþ+1, 8%), 121
(base); elemental analysis calcd (%) for C25H23NO5S2: C,
62.35, H, 4.81, N, 2.91; found C, 62.06, H, 4.77, N, 2.85.
149.5 (s), 180.0 (s); MS: m=z (relative intensity) 283 (Mþ,
42%), 136 (base); elemental analysis calcd (%) for
C15H9NO3S: C, 63.59; H, 3.29; N, 4.94. Found: C,
63.31; H, 3.21; N, 4.84.
6.1.15. 30-Methoxy-20-nitrothioflavone (7b). Yield 42%,
white powder, mp 157 ꢁC; IR (film): m ¼ 1375 (NO2),
1527 (NO2), 1630 (C@O); 1H NMR (400 MHz, CDCl3):
d ¼ 3:96 (3H, s, OCH3), 6.96 (1H, s, C@CH), 7.11 (1H,
d, J ¼ 8 Hz, Ar-H), 7.19 (1H, d, J ¼ 8 Hz, Ar-H), 7.52–
7.68 (4H, m, Ar-H), 8.49 (1H, d, J ¼ 8 Hz, Ar-H); 13C
NMR (100 MHz, CDCl3): d ¼ 56:7 (q), 114.4 (d), 114.9
(d), 121.3 (d), 126.2 (d), 128.0 (d), 128.5 (d), 129.9 (s),
130.6 (s), 131.5 (d), 131.8 (d), 137.7 (s), 140.0 (s), 146.9
(s), 151.1 (s), 179.7 (s); MS: m=z (relative intensity) 313
(Mþ, base); elemental analysis calcd (%) for
C16H11NO4S: C, 61.33; H, 3.54; N, 4.47. Found: C,
61.12; H, 3.66; N, 4.39.
6.1.13. 1-[2-(4-Methoxybenzylthio)phenyl]-2-(methylsulfin-
yl)-3-(4,5-dimethoxy-2-nitrophenyl)-2-propen-1-ones (5h).
Yield 61%, pale yellow powder, mp 181–182 ꢁC; IR
(film): m ¼ 1064 (SOCH3), 1324 (NO2), 1520 (NO2),
6.1.16. 40-Methoxy-20-nitrothioflavone (7c). Yield 47%,
white powder, mp 158 ꢁC. IR (KBr): m ¼ 1333, 1531
(NO2), 1630 (C@O); 1H NMR (400 MHz, CDCl3):
d ¼ 3:94 (3H, s, OCH3), 6.89 (1H, s, C@CH), 7.23 (1H,
d, J ¼ 9 Hz, Ar-H), 7.46 (1H, d, J ¼ 9 Hz, Ar-H), 7.56–
7.63 (4H, m, Ar-H), 8.54 (1H, d, J ¼ 8 Hz, Ar-H). 13C
NMR (100 MHz, CDCl3): d ¼ 56:1 (q), 114.0 (d), 119.2
(d), 123.2 (s), 125.8 (d), 126.2 (d), 128.0 (d), 128.7 (d),
130.8 (s), 131.8 (d), 132.4 (d), 137.7 (s), 148.8 (s), 149.7
(s), 161.2 (s), 180.2 (s). MS: m=z (relative intensity) 313
(Mþ, 42%), 136 (base); elemental analysis calcd (%) for
C16H11NO4S: C, 61.33; H, 3.54; N, 4.47. Found: C,
61.13; H, 3.77; N, 4.35.
1
1635 (CO); H NMR (400 MHz, CDCl3): d ¼ 2:87 (3H,
s, SOCH3), 3.80 (3H, s, Ar-OCH3), 3.86 (3H, s, Ar-
OCH3), 3.88 (3H, s, Ar-OCH3), 3.95 (1H, d, J ¼ 12 Hz,
SCH2), 4.09 (1H, d, J ¼ 12 Hz, SCH2), 6.50 (1H, s, Ar-
H), 6.86 (2H, d, J ¼ 9 Hz, Ar-H), 6.93 (1H, t, J ¼ 9 Hz,
Ar-H), 7.16–7.25 (4H, m, Ar-H), 7.54 (1H, s, Ar-H),
7.59 (1H, d, J ¼ 9 Hz, Ar-H), 8.03 (1H, s, C@CH); 13C
NMR (100 MHz, CDCl3): d ¼ 36:9 (t), 40.5 (q), 55.3 (q),
56.4 (q), 56.7 (q), 107.6 (d), 112.4 (d), 114.1 (d), 124.1
(d), 125.0 (s), 126.4 (d), 127.1 (d), 130.0 (d), 131.4 (d),
133.1 (d), 134.2 (s), 136.7 (d), 139.1 (s), 141.8 (s), 145.8
(s), 149.0 (s), 153.1 (s), 159.0 (s), 192.6 (s); FABMS: m=z
(relative intensity) 528 (Mþ+1, 4%), 154 (base); ele-
mental analysis calcd (%) for C26H25NO7S2: C, 59.19; H,
2.65; N, 4.78. Found: C, 59.07; H, 2.63; N, 4.69.
6.1.17. 50-Methoxy-20-nitrothioflavone (7d). Yield 45%,
white powder, mp 157 ꢁC; IR (KBr): m ¼ 1357 (NO2),
1520 (NO2), 1621 (C@O); 1H NMR (400 MHz, CDCl3):
d ¼ 3:94 (3H, s, OCH3), 6.91 (1H, s, C@CH), 6.97 (1H,
s, Ar-H), 7.08 (1H, d, J ¼ 9 Hz, Ar-H), 7.55–7.64 (3H,
m, Ar-H), 8.17 (1H, d, J ¼ 9 Hz, Ar-H), 8.55 (1H, d,
J ¼ 8 Hz, Ar-H); 13C NMR (100 MHz, CDCl3):
d ¼ 55:7 (q), 114.9 (d), 116.2 (d), 124.6 (d), 125.8 (d),
127.3 (d), 127.4 (d), 128.2 (d), 130.3 (s), 131.3 (d), 133.3
(s), 137.0 (s), 140.0 (s), 149.8 (s), 152.7 (s), 179.5 (s); MS:
m=z (relative intensity) 313 (Mþ, base); elemental anal-
ysis calcd (%) for C16H11NO4S: C, 61.33; H, 3.54; N,
4.47. Found: C, 61.05; H, 3.66; N, 4.45.
6.1.14. 20-Nitrothioflavone (7a). Propenone 5a (1.84 g,
3.9 mmol) was dissolved in 99% formic acid (4 mL) at
5 ꢁC, and the solution was stirred for 30 min at that
temperature. The mixture was made basic by adding
sodium carbonate and then extracted with chloroform.
The extracts were washed with brine, dried (MgSO4),
and concentrated under reduced pressure to give a cy-
clized product 6a. The raw product of 6a was dissolved
in benzene (10 mL), and the solution was refluxed for
30 min. The cooled organic layer was separated, and the
residue was extracted with chloroform. The combined
organic layers were washed with brine, dried (MgSO4),
and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel
eluting with CHCl3–AcOEt (10:1) to give white powder
(602 mg, 54%), mp 142 ꢁC. IR (film): m ¼ 1358, 1528
(NO2), 1622 (C@O); 1H NMR (400 MHz, CDCl3):
d ¼ 6:92 (1H, s, C@CH), 7.55–7.76 (6H, m, Ar-H), 8.08
(1H, d, J ¼ 8 Hz, Ar-H), 8.54 (1H, d, J ¼ 8 Hz, Ar-H);
13C NMR (100 MHz, CDCl3): d ¼ 114:0 (d), 125.0 (d),
125.5 (d), 126.2 (d), 128.0 (d), 128.7 (d), 130.8 (s), 131.0
(d), 131.1 (s), 131.4 (d), 131.8 (d), 137.4 (s), 147.9 (s),
6.1.18. 30-Chloro-20-nitrothioflavone (7e).
Yield 58%,
yellow powder, mp 207 ꢁC; IR (KBr): m ¼ 1360, 1532
(NO2), 1620 (C@O); 1H NMR (400 MHz) (CDCl3):
d ¼ 6:95 (1H, s, C@CH), 7.50 (1H, d, J ¼ 8 Hz, Ar-H),
7.56–7.67 (5H, m, Ar-H), 8.52 (1H, d, J ¼ 8 Hz, Ar-H);
13C NMR (100 MHz) (CDCl3): d ¼ 126:3 (d), 126.8 (d),
127.3 (s), 128.3 (d), 128.7 (d), 128.8 (d), 128.2 (s), 130.6
(s), 130.7 (d), 131.3 (d), 132.1 (d), 132.3 (d), 137.1 (s),
145.9 (s), 148.1 (s), 179.6 (s); MS: m=z (relative intensity)
317 (Mþ, 30%), 136 (base); elemental analysis calcd (%)
for C15H8ClNO3S: C, 56.70; H, 2.54; N, 4.17. Found: C,
56.44; H, 2.58; N, 4.37.

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T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
6.1.19. 40-Chloro-20-nitrothioflavone (7f).
Yield 63%,
236 (base); HRMS: calcd for C15H11NOS: 253.0561,
found: 253.0556.
yellow powder, mp 202 ꢁC; IR (KBr): m ¼ 1334 (NO2),
1533 (NO2), 1624 (C@O); 1H NMR (400 MHz, CDCl3):
d ¼ 6:89 (1H, s, C@CH), 7.52 (1H, d, J ¼ 8 Hz, Ar-H),
7.57–7.67 (3H, m, Ar-H), 7.72 (1H, d, J ¼ 8 Hz, Ar-H),
8.08 (1H, s, Ar-H), 8.54 (1H, d, J ¼ 8 Hz, Ar-H); 13C
NMR (100 MHz), CDCl3): d ¼ 125:3 (d), 125.8 (d),
126.3 (d), 127.1 (s), 128.2 (d), 128.8 (d), 129.5 (d), 130.7
(d), 132.0 (d), 132.5 (d), 133.4 (s), 137.1 (s), 137.2 (s),
148.5 (s), 180.0 (s); MS: m=z (relative intensity) 317 (Mþ,
40%), 136 (base); elemental analysis calcd (%) for
C15H8ClNO3S: C, 56.70; H, 2.54; N, 4.42. Found: C,
56.51; H, 2.58; N, 4.35.
6.1.23. 20-Amino-30-methoxythioflavone (8b). Yield 89%,
yellow powder, mp 194 ꢁC; IR (KBr): m ¼ 1610 (C@O),
1
3260 (NH2), 3420 (NH2); H NMR (400 MHz, CDCl3):
d ¼ 3:90 (3H, s, OCH3), 4.15 (2H, br s, NH2), 6.78–6.91
(3H, m, Ar-H), 7.13 (1H, s, C@CH), 7.55–7.63 (3H, m,
Ar-H), 8.55 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 55:8 (q), 111.3 (d), 117.5 (d),
120.8 (s), 121.4 (d), 125.8 (d), 126.3 (d), 127.8 (d), 128.6
(d), 130.9 (s), 131.5 (d), 134.1 (s), 138.6 (s), 147.3 (s),
151.9 (s), 180.7 (s); MS: m=z (relative intensity) 283 (Mþ,
60%), 266 (base); HRMS: calcd for C15H11NOS:
283.0667, found: 283.0666; elemental analysis calcd (%)
for C16H13NO2S: C, 67.82; H, 4.62; N, 4.94. Found: C,
67.62; H, 4.63; N, 4.80.
6.1.20. 30-Methyl-20-nitrothioflavone (7g).
Yield 45%,
white powder, mp 178 ꢁC; IR (KBr); m ¼ 1330 (NO2),
1520 (NO2), 1624 (C@O); 1H NMR (400 MHz, CDCl3):
d ¼ 2:43 (3H, s, OCH3), 6.94 (1H, s, C@CH), 7.37–7.67
(6H, m, Ar-H), 8.53 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 17:2 (q), 125.7 (s), 125.8 (d),
127.5 (d), 127.6 (d), 128.2 (d), 128.7 (s), 130.0 (d), 130.3
(s), 130.6 (s), 131.3 (d), 132.7 (d), 137.0 (s), 147.2 (s),
149.2 (s), 179.2 (s). MS m=z: 297 (Mþ, 45%), 136 (base);
elemental analysis calcd (%) for C16H11NO3S: C, 64.63;
H, 3.73; N, 4.71. Found: C, 64.40; H, 3.88; N, 4.64.
6.1.24. 20-Amino-40-methoxythioflavone (8c). Yield 64%,
yellow powder, mp 150 ꢁC; IR (KBr): m ¼ 1594 (C@O),
1
3318 (NH2), 3407 (NH2); H NMR (400 MHz, CDCl3):
d ¼ 3:80 (3H, s, OCH3), 4.16 (2H, br s, NH2), 6.30 (1H,
s, Ar-H), 6.41 (1H, d, J ¼ 9 Hz, Ar-H), 7.09 (1H, s,
C@CH), 7.19 (1H, d, J ¼ 9 Hz, Ar-H), 7.51–7.61 (3H,
m, Ar-H), 8.53 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 55:5 (q), 101.4 (d), 105.2 (d),
114.7 (s), 125.8 (d), 126.58 (d), 128.0 (d), 128.8 (d), 131.2
(s), 131.7 (d), 131.8 (d), 138.9 (s), 145.5 (s), 152.4 (s),
162.4 (s), 181.0 (s); MS: m=z (relative intensity) 283 (Mþ,
50%), 266 (base); HRMS: calcd for C16H13NO2S:
283.0667, found: 283.0655.
6.1.21. 40,50-Dimethoxy-20-nitrothioflavone (7h).
Yield
51%, pale brown powder, mp 157–159 ꢁC. IR (KBr):
m ¼ 1331, 1520 (NO2), 1621 (C@O); 1H NMR
(400 MHz, CDCl3): d ¼ 4:00 (3H, s, OCH3), 4.03 (3H, s,
OCH3), 6.90 (1H, s, C@CH), 6.93 (1H, s, Ar-H), 7.56–
7.65 (3H, m, Ar-H), 7.70 (1H, s, Ar-H), 8.56 (1H, d,
J ¼ 8 Hz, Ar-H); 13C NMR (100 MHz, CDCl3):
d ¼ 56:7 (q), 56.8 (q), 108.1 (d), 112.9 (d), 125.4 (d),
126.2 (d), 127.9(d), 128.7 (d), 130.9 (s), 131.8 (d), 137.7
(s), 140.2 (s), 150.0 (s), 150.7 (s), 153.0 (s), 180.3 (s). MS:
m=z (relative intensity) 343 (Mþ, 55%), 179 (base); ele-
mental analysis calcd (%) for C17H13NO5S: C, 59.47; H,
3.82; N, 4.08. Found: C, 59.31; H, 4.00; N, 3.90.
6.1.25. 20-Amino-50-hydroxy-40-methoxythioflavone (9c).
Yield 18%, yellow powder, mp 207 ꢁC; IR (KBr):
1
m ¼ 1610 (C@O), 3340 (NH2), 3415 (NH2); H NMR
(400 MHz, CDCl3): d ¼ 1:25 (2H, br s, NH2), 3.95 (3H,
s, OCH3), 5.20 (1H, br s, OH), 6.34 (1H, s, Ar-H), 6.90
(1H, s, Ar-H), 7.10 (1H, s, C@CH), 7.54–7.63 (3H, m,
Ar-H), 8.55 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 55:9 (q), 100.0 (d), 113.6 (s),
115.2 (d), 125.7 (d), 126.3 (d), 127.7 (d), 128.6 (d), 130.9
(s), 131.5 (d), 137.5 (s), 138.6 (s), 138.7 (s), 149.1 (s),
151.9 (s), 180.8 (s); MS: m=z (relative intensity) 299 (Mþ,
75%), 282 (base); HRMS: calcd for C16H13NO2S:
299.0616, found: 299.0620.
6.1.22. 20-Aminothioflavone (8a). 20-Nitrothioflavone (7a)
(592 mg, 2.1 mmol) was dissolved in hot tetrafluoroboric
acid (42%) (5 mL), and tin (1.23 g, 10 mmol) was added
to it. The mixture was stirred for 30 min at 80 ꢁC, then
cooled and made basic with an aqueous NaOH solution.
The precipitate was collected by filtration and washed
with chloroform. The washings were dried (MgSO4) and
concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel eluting
with CHCl3–AcOEt (10:1) to give yellow powder
(200 mg, 38%), mp 131 ꢁC. IR (KBr): m ¼ 1586 (C@O),
6.1.26. 20-Amino-50-methoxythioflavone (8d). Yield 83%,
yellow powder, mp 174 ꢁC; IR (KBr): m ¼ 1620 (C@O),
1
3380 (NH2), 3420 (NH2); H NMR (400 MHz, CDCl3):
d ¼ 3:78 (3H, s, OCH3), 4.25 (2H, br s, NH2), 6.75 (1H,
d, J ¼ 9 Hz, Ar-H), 6.83 (1H, s, Ar-H), 6.88 (1H, d,
J ¼ 9 Hz, Ar-H), 7.11 (1H, s, C@CH), 7.55–7.59 (1H, m,
Ar-H), 7.64 (2H, m, Ar-H), 8.56 (1H, d, J ¼ 8 Hz, Ar-
H); 13C NMR (100 MHz, CDCl3): d ¼ 55:9 (q), 114.4
(d), 117.9 (d), 118.0 (d), 122.0 (s), 126.1 (d), 126.4 (d),
127.9 (d), 128.7 (d), 130.9 (s), 131.7 (d), 137.3 (s), 138.5
(s), 151.7 (s), 152.5 (s), 180.7 (s); MS: m=z (relative
intensity) 283 (Mþ, 48%), 266 (base); HRMS: calcd for
C16H13NO2S: 283.0667, found: 283.0670; elemental
1
3420 (NH2), 3440 (NH2); H NMR (400 MHz, CDCl3):
d ¼ 4:05 (2H, br s, NH2), 6.77–6.85 (2H, m, Ar-H), 7.11
(1H, s, C@CH), 7.23–7.27 (2H, m, Ar-H), 7.54–7.64
(3H, m, Ar-H), 8.56 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 116:3 (d), 118.5 (d), 121.2 (s),
125.9 (d), 126.4 (d), 127.8 (d), 128.6 (d), 130.3 (d), 130.9
(s), 131.2 (d), 131.6 (d), 138.2 (s), 143.6 (s), 151.7 (s),
180.6 (s); MS: m=z (relative intensity) 253 (Mþ, 43%),

T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
2405
analysis calcd (%) for C16H13NO2S: C, 67.82; H, 4.62; N,
4.94. Found: C, 67.56; H, 4.61; N, 4.94.
analysis calcd (%) for C16H13NOS: C, 71.88; H, 4.90; N,
5.24. Found C, 71.68; H, 4.91; N, 5.17.
6.1.27. 20-Amino-30-chlorothioflavone (8e).
Yield 47%,
6.1.31. 20-Amino-50-hydroxy-30-methylthioflavone (9g).
Yield 26%, yellow powder, mp 203 ꢁC; IR (KBr):
m ¼ 1588 (C@O), 3367 (OH). The absorption due to the
amino group was overlapped with that of the hydroxy
white powder, mp 203 ꢁC; IR (KBr): m ¼ 1600 (C@O),
1
3200 (NH2), 3260 (NH2); H NMR (400 MHz, CDCl3):
d ¼ 4:50 (2H, br s, NH2), 6.78 (1H, t, J ¼ 8 Hz, Ar-H),
7.09 (1H, s, C@CH), 7.16 (1H, d, J ¼ 8 Hz, Ar-H), 7.36
(1H, d, J ¼ 8 Hz, Ar-H), 7.54–7.59 (1H, m, Ar-H), 7.61–
7.64 (2H, m, Ar-H), 8.54 (1H, d, J ¼ 8 Hz, Ar-H); 13C
NMR (100 MHz, CDCl3): d ¼ 126:4 (d), 126.8 (d),
128.40 (d), 128.7 (d), 128.8 (d), 130.7 (s), 130.8 (s), 131.3
(d), 131.8 (s), 132.1 (d), 132.3 (d), 137.1 (s), 145.9 (s),
148.1 (s), 179.6 (s); MS: m=z (relative intensity) 287 (Mþ,
85%), 270 (base); HRMS: calcd for C15H10ClNOS:
287.0172, found: 287.0177; elemental analysis calcd (%)
for C15H10ClNOS: C, 62.61; H, 3.50; N, 4.87. Found: C,
62.42; H, 3.55; N, 4.88.
1
group; H NMR (400 MHz, CDCl3): d ¼ 2:20 (3H, s,
CH3), 3.65 (2H, br s, NH2), 5.10 (1H, br s, OH), 6.64
(1H, s, Ar-H), 6.76 (1H, s, Ar-H), 7.07 (1H, s, C@CH),
7.57–7.63 (3H, m, Ar-H), 8.56 (1H, d, J ¼ 8 Hz, Ar-H);
13C NMR (100 MHz, CDCl3): d ¼ 18:1 (q), 113.8 (d),
120.0 (d), 121.9 (s), 125.1 (s), 126.1 (d), 126.4 (d), 127.9
(d), 128.7 (d), 130.9 (s), 131.7 (d), 135.3 (s), 138.6 (s),
147.8 (s), 152.3 (s), 180.8 (s); MS: m=z (relative intensity)
283 (Mþ, 45%), 266 (base); HRMS: calcd for
C16H13NOS: 283.0667, found: 283.0656.
6.1.32. 20-Amino-40,50-dimethoxythioflavone (8h). Yield
39%, pale yellow powder, mp 170–171 ꢁC; IR (KBr):
6.1.28. 20-Amino-30-chloro-50-hydroxythioflavone (9e).
Yield 32%, yellow powder, mp 239 ꢁC; IR (KBr):
m ¼ 1587 (C@O), 3366 (NH2), 3400 (OH), 2450 (NH2);
1H NMR (400 MHz, CD3OD): d ¼ 6:71 (1H, d,
J ¼ 2 Hz, Ar-H), 6.91 (1H, d, J ¼ 2 Hz, Ar-H), 7.06
(1H, s, C@CH), 7.64 (1H, td, J ¼ 8, 1 Hz, Ar-H), 7.74
(1H, td, J ¼ 8, 1 Hz, Ar-H), 7.83 (1H, d, J ¼ 8 Hz, Ar-
H), 8.50 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR (100 MHz,
DMSO-d6): d ¼ 115:1 (d), 117.9 (d), 119.3 (s), 122.1 (s),
125.9 (d), 126.8 (d), 127.6 (d), 127.9 (d), 130.3 (s), 131.8
(d), 134.0 (s), 137.9 (s), 148.2 (s), 150.2 (s), 179.4 (s); MS:
m=z (relative intensity) 304 (Mþ+1, 45%), 154 (base);
HRMS: calcd for C15H10ClNO2S: 304.0199, found:
304.0206.
1
m ¼ 1586 (C@O), 3348 (NH2), 3404 (NH2); H NMR
(400 MHz, CDCl3): d ¼ 2:95 (2H, br s, NH2), 3.84 (3H,
s, OCH3), 3.89 (3H, s, OCH3), 6.35 (1H, s, Ar-H), 6.81
(1H, s, Ar-H), 7.10 (1H, s, C@CH), 7.52–7.65 (3H, m,
Ar-H), 8.54 (1H, d, J ¼ 8 Hz, Ar-H); 13C NMR
(100 MHz, CDCl3): d ¼ 56:0 (q), 56.6 (q), 100.8 (d),
112.5 (s), 113.2 (d), 125.7 (d), 126.3 (d), 127.8 (d), 128.6
(d), 130.9 (s), 131.6 (d), 138.5 (s), 142.3 (s), 151.8 (s),
152.1 (s), 180.8 (s); MS: m=z (relative intensity) 313 (Mþ,
90%), 296 (base); HRMS: calcd for C17H15NO3S:
313.0773, found: 313.0759.
6.2. Bioassays
6.1.29. 20-Amino-40-chlorothioflavone (8f).
Yield 57%,
Thioflavone derivatives were dissolved in DMSO to give
100 mM solutions and successive 10-fold dilutions were
prepared. The test solution was added to each of culture
media (cell-based assay) or reaction mixtures (in vitro
kinase assay) to give the indicated final concentration:
all the culture media/reaction mixtures received DMSO
at a final concentration of 0.1%.
yellow powder, mp 180 ꢁC; IR (KBr): m ¼ 1600 (C@O),
1
3200 (NH2), 3260 (NH2). H NMR (400 MHz, CDCl3):
d ¼ 4:12 (2H, br s, NH2), 6.79 (1H, d, J ¼ 9 Hz, Ar-H),
6.80 (1H, s, C@CH), 7.17 (1H, d, J ¼ 9 Hz, Ar-H), 7.54–
7.58 (1H, m, Ar-H), 7.61–7.63 (2H, m, Ar-H), 8.54 (1H,
d, J ¼ 8 Hz, Ar-H); 13C NMR (100 MHz, CDCl3):
d ¼ 115:8 (d), 118.5 (d), 119.5 (s), 126.1 (d), 126.3 (d),
128.0 (d), 128.7 (d), 130.9 (s), 131.0 (d), 131.7 (d), 137.0
(s), 138.3 (s), 144.7 (s), 150.7 (s), 180.5 (s). MS: m=z
(relative intensity) 287 (Mþ, 32%), 270 (base); HRMS:
calcd for C15H10ClNOS: 287.0172, found: 287.0169.
6.2.1. Cell-based assays. NIH3T3 cells expressing DRaf-
1:ER (3T3-DRaf-1:ER)35;36 were kindly provided by Dr.
Martin McMahon (DNAX Research Institute) and were
cultured in DulbeccoÕs modified EagleÕs medium
(DMEM) supplemented with 10% fetal bovine serum.
For the experimental use, sub-confluent cultures of 3T3-
DRaf-1:ER cells in DMEM containing 10% fetal bovine
serum were rendered quiescent by incubation for 24 h in
serum-free medium (DMEM containing 2 mg/mL of
bovine serum albumin, 1 lg/mL of insulin, 2 lg/mL of
transferrin, 20 nM Na2SeO3, and 10 mM HEPES,
pH 7.4) and pre-treated with respective thioflavone
derivatives for 1 h. The cells were then stimulated with
1 lM b-estradiol to activate DRaf-1:ER for 2 h (for the
analysis of Raf-1-induced activation of the ERK-MAP
kinase pathway) or 48 h (for the analysis of Raf-1-in-
duced cell proliferation). Raf-1-induced activation of the
6.1.30. 20-Amino-30-methylthioflavone (8g). Yield 26%,
yellow powder, mp 161 ꢁC; IR (KBr): m ¼ 1623 (C@O),
1
3220 (NH2), 3265 (NH2); H NMR (400 MHz, CDCl3):
d ¼ 2:23 (3H, s, CH3), 4.25 (2H, br s, NH2), 6.77 (1H, t,
J ¼ 8 Hz, Ar-H), 7.10 (1H, s, C@CH), 7.12–7.17 (2H, m,
Ar-H), 7.54–7.57 (3H, m, Ar-H), 8.56 (1H, d, J ¼ 8 Hz,
Ar-H); 13C NMR (100 MHz, CDCl3): d ¼ 17:8 (q), 118.0
(d), 121.1 (s), 123.2 (s), 126.1 (d), 126.4 (d), 127.6 (d),
127.8 (d), 128.6 (d), 130.9 (s), 131.6 (d), 132.2 (d), 138.7
(s), 141.7 (s), 152.4 (s), 180.7 (s); MS: m=z (relative
intensity) 267 (Mþ, 28%), 250 (base); HRMS: calcd for
C16H13NOS: 267.0718, found: 267.0708; elemental

2406
T. Kataoka et al. / Bioorg. Med. Chem. 12 (2004) 2397–2407
ERK-MAP kinase pathway was analyzed by immuno-
blotting as described previously.6;15;16 Briefly, the cells
were scrapped off plates in a hypotonic cell lysis buffer
(25 mM Tris–HCl, pH 7.4, 25 mM NaCl, 1 mM sodium
orthovanadate, 10 mM NaF, 10 mM sodium pyrophos-
phate, 25 mM glycerophosphate, 25 mM p-nitrophenyl
phosphate, 20 nM okadaic acid, 0.2 mM sodium
molybdate, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 10 lg/mL leupeptin, and 1% aprotinin) and
lysed by sonication for 60 s. Lysates were cleared by
centrifugation at 12,000g for 30 min, and protein con-
centrations were determined using the BCA protein
assay reagent (Pierce). Cell lysates (20 lg of protein)
were separated by SDS-PAGE, electrophoretically
transferred to an Immobilon-P membrane (Millipore
Corp.), and subjected to immunodetection using the
polyclonal anti-ERK1/2 antibody,44;45 the monoclonal
anti-phospho-ERK1/2 antibody (Sigma), the monoclo-
nal anti-MEK1 antibody (Transduction Laboratories)
or the polyclonal anti-phospho-MEK1/2 antibody (Cell
Signaling) as the primary antibody and horseradish
peroxidase-conjugated secondary antibody (Promega).
Immunoreactive bands were visualized with the
enhanced chemiluminescence system (Amersham
Biosciences)15;16 and were analyzed using a Fuji Lumi-
nescent image analyzer (LAS-1000 plus; Fuji Photo
Film, Tokyo). For the analysis of Raf-1-induced cell
proliferation, b-estradiol-stimulated 3T3-DRaf-1:ER
cells were harvested by trypsinization and viable cells
which excluded trypan blue were counted using a
hemocytometer.
technology) for 3 h at 4 ꢁC. After collecting on protein-A
Sepharose beads, immune complexes were washed twice
with R-cell lysis buffer and three times with Raf-1 kinase
buffer (30 mM Tris–HCl, pH 7.4, 7 mM MnCl2, 5 mM
MgCl2, and 1 mM dithiothreitol), treated for 30 min at
4 ꢁC with respective thioflavone derivatives, and then
incubated for 60 min at 30 ꢁC with 20 lM ATP and 5 lg
of MEK1(FL) (Santa Cruz Biotechnology) in 100 lL of
Raf-1 kinase buffer. After further incubation with 2.5 lg
of GST-ERK2[KR] for 60 min at 30 ꢁC, the extent of
GST-ERK2[KR] phosphorylation was determined as
described above. Assays of ERK1/2, p38 MAP kinase,
c-Jun N-terminal kinase, MKK3, MAPKAP kinase 2,
protein kinase Ca, EGF receptor kinase, and cycline-
dependent kinase 2, and phosphorylation of myelin
basic protein by Raf-1 were performed as described
previously.6;15;16;46;47
Acknowledgements
We thank Dr. Martin McMahon (DNAX Research
Institute) for supplying 3T3-DRaf-1:ER cells and Dr.
Yoshihiro Matsumura (Nagasaki University) for his
participation in helpful discussions during the prepara-
tion of this manuscript. This work was supported in part
by grants-in aid for Scientific Research from the Min-
istry of Education, Culture, Sports, Science and Tech-
nology of Japan.
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