Novel 5-aminoflavone derivatives as specific antitumor agents in breast cancer

Article abstract of DOI:10.1021/jm950938g

In the course of our search for new antitumor agents in breast cancer, novel amino-substituted flavone derivatives were synthesized and examined for antitumor activities. Among them, 5,4'-diaminoflavone and some of its congeners showed remarkable antiprol

Full text of DOI:10.1021/jm950938g

J . Med. Chem. 1996, 39, 3461-3469
3461
Novel 5-Am in ofla von e Der iva tives a s Sp ecific An titu m or Agen ts in Br ea st
Ca n cer
Tsutomu Akama,*,† Yasushi Shida, Toru Sugaya,‡ Hiroyuki Ishida, Katsushige Gomi,§ and Masaji Kasai*,‡
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari,
Nagaizumi-cho, Sunto-gun, Shizuoka-ken 411, J apan
Received December 27, 1995X
In the course of our search for new antitumor agents in breast cancer, novel amino-substituted
flavone derivatives were synthesized and examined for antitumor activities. Among them,
5,4′-diaminoflavone and some of its congeners showed remarkable antiproliferative activity
against the estrogen receptor (ER)-positive and estrogen-responsive human breast cancer cell
line MCF-7. The activity was observed irrespective of the presence or absence of estrogen.
The 5-aminoflavone derivatives (5-AFs) are not classical anti-estrogens because they did not
compete with [3H]estradiol to bind the estrogen receptor. Moreover, 5-AFs showed antitumor
activity highly selective to the ER-positive breast cancer cell line, and they showed no effects
against the ER-negative human cancer cell lines HeLa S3, WiDr, and MDA-MB-453. Although
the mechanism of their selective antitumor activity to ER-positive breast cancer cells is unclear,
5-AFs are expected to be a new type of antitumor agents in breast cancer.
Ch a r t 1
The blockade of estrogen action is a major approach
for the treatment of hormone-dependent breast cancer.1-4
Triarylethylene anti-estrogens, such as tamoxifen (TAM)
(Chart 1), are representative of this strategy.5 However,
because these anti-estrogens generally possess partial
estrogenic activity, considerable efforts by many groups
have been devoted to the search for pure anti-estrogens
without estrogenic activity.6-10 Indeed, pure anti-
estrogens might be more effective than partial agonists
in reducing the mitogenic action of estrogen on the
growth of breast cancer cells, but they would exhibit the
antiproliferative effect mostly in the case when the
growth is estrogen-dependent. In addition, there is
another problem that many breast tumors become
resistant to TAM during treatment. Tumors that lose
hormone dependency usually become resistant to TAM.
Among estrogen receptor (ER)-positive breast cancer
tissues, approximately 60% of them respond to anti-
estrogens, whereas the remaining 40% are nonrespon-
sive.1,2,11-13 On the other hand, it is known that MCF-
7, the estrogen receptor-positive and estrogen-respon-
sive human breast cancer cell line, can proliferate in
the absence of estrogen.14 Recently, a new breast cancer
cell line which expresses ER but grows estrogen inde-
pendently was reported.15 It seems that the growth of
breast cancer cells, which are not responsive to anti-
estrogens but are ER-positive, is due to estrogen-
independent growth, although it is poorly understood
how estrogen-independent growth in an ER-positive
tumor is mediated.14 This circumstance prompted us
to search for novel anti-breast-cancer agents which are
effective against both estrogen-dependent and -inde-
pendent growth.
example, antioxidant,17 anti-inflammatry,18 gastropro-
tective,19 antiviral,20 antimutagenic,21 topoisomerase II
inhibitory,22 protein kinase C inhibitory,23 and cy-
totoxic24-26 activities, etc., have been reported. Par-
ticularly, we have been interested in the relationship
between flavonoids and anti-breast-cancer activities
because large numbers of flavonoids are known to
exhibit antiproliferative effects against breast cancer
cells or binding affinities for the ER. For example,
apigenin (1) and some of its congeners were reported to
possess antiproliferative activity against the human
breast cancer cell line ZR-75-1.27 6,4′-Dihydroxyflavone
(2) has a binding affinity for the ER,28 and flavanone
derivative 3 (BE-14348B29) exhibits strong estrogenic
activity. The ER binding affinity and the estrogenic
Flavonoids, either natural or synthetic, are well
known to exhibit various biological activities.16 For
† Present address: Tokyo Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 6-6 Asahi-machi 3-chome, Machida-shi, Tokyo 194,
J apan.
‡ Present address: Sakai Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 1-53 Takatsu-cho 1-chome, Sakai, Osaka 590, J apan.
§ Present address: Kyowa Hakko Kogyo Co., Ltd., 6-1 Ohtemachi
1-chome, Chiyoda-ku, Tokyo 100, J apan.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(95)00938-1 CCC: $12.00 © 1996 American Chemical Society

3462 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Akama et al.
Ta ble 1. Antiproliferative Activity and ER Binding of
Compounds 10 against MCF-7
strongly basic conditions. The 1,3-diketones thus ob-
tained were used for the next step without purification.
They were treated with HCl in EtOH to afford cyclized
products 9. Various compounds 10 were obtained after
the removal of the N-pivaloyl group and other protecting
groups of 9 under more strongly acidic conditions. Some
derivatives were prepared from the obtained compounds
10. The 4′-carboxy derivative 10f was obtained by
hydrolysis of the corresponding cyano derivative 10e.
The 4′-acetamido derivative 10h was obtained by selec-
tive acetylation of 10g. The 4′-diethylamino derivative
10i was obtained as a major product of the mixture of
mono- and trialkylated products by alkylation of 10g.
Compounds 10p ,q were synthesized by alkylation of 9p
followed by deprotection.
6-Amino-4′-(dimethylamino)flavone (13) was synthe-
sized as shown in Scheme 2. Ethyl 5-nitrosalicylate (11)
was converted to its methoxymethyl ether. The nitro
group was then reduced to the amino group with
hydrazine and palladium on charcoal and protected as
a pivaloyl amide to afford compound 12. Compound 13
was obtained from compounds 12 and 8n in the same
way as shown in Scheme 1.
IC50 (µM)a
estradiolb
ER binding
compounds
X
-
+
competition
10a
10b
10c
10d
10e
10f
10g
10h
10j
10k
10l
10m
10p
10q
10n
10i
H
4′-OH
4′-OMe
4′-Br
4′-CN
4′-CO2H
4′-NH2
4′-NHAc
4′-NHMe
4′-NHEt
4′-NHPrn
4′-NHBun
4′-NHHexn
4′-NHCH2Ph
4′-NMe2
4′-NEt2
>100
13
1.5
12
1.0
>100
>10
0.0072
13
0.0023
>100
18
>100
>100
>100
NTc
>100
>100
>100
>100
>100
>100
NTc
>100
>100
NTc
>100
>100
9.5
94
>10
0.0098
18
0.0054
0.0017
0.0087
0.0087
0.44
4.3
0.0050
0.010
0.0013
0.0067
0.0051
0.075
4.1
0.0040
0.0020
apigenin (1)
genistein (5)
tamoxifen
(TAM)
estradiol
95
77
19
>100
11
0.14
14
0.99
3.9
0.0016
a
IC50 values were measured by cell count method described in
6-Amino-3′-(dimethylamino)flavone (16a) and 7-amino-
4′-(dimethylamino)flavone (16b) were synthesized as
shown in Scheme 3. Compounds 14a ,39b were treated
with the derivatives of 3- and 4-(dimethylamino)benzoic
acid under basic condition to afford the corresponding
1,3-diketones, which were successively cyclized and
deprotected under acidic conditions to afford compounds
16a ,b.
the Experimental Section. 10-4 µM. c Not tested.
b
activity were also reported concerning the isoflavone
derivatives daizein (4) and genistein (5).30 Recently,
compound 6 (L86-8275) was reported to exhibit antitu-
mor activities against several types of human breast
cancer cell lines.31
As mentioned above, most flavonoids exhibiting bio-
logical activities associated with breast cancer or estro-
genic action possess at least one hydroxyl group in their
skeletons. On the other hand, some flavonoids possess-
ing amino groups are known, but their previously
mentioned biological activities are scarcely known.32-35
Moreover, to the best of our knowledge, 5-aminoflavones
are not known except for only a few examples.36,37
Therefore, we hypothesized that flavone derivatives
substituted with amino groups, which can function as
hydrogen bond donors or acceptors as hydroxyl groups,
might exhibit antitumor activity in breast cancer.
In this study, we synthesized various amino-substi-
tuted flavones and evaluated their antitumor activity
against MCF-7 in both the presence and absence of
estrogen. In addition, to characterize their physiological
properties, their binding affinity for the ER was studied
by binding competition with [3H]estradiol. Further-
more, we evaluated the antiproliferative activity against
hormone-independent human cancer cell lines HeLa S3
(uterus), WiDr (colon), and MDA-MB-453 (breast, with-
out the ER). We wish to describe the synthesis of
amino-substituted flavones and their attractive antitu-
mor activity.
8-Amino-4′-(dimethylamino)flavone (19) was prepared
in another way (Scheme 4). Although the coupling of
17 with 15b or some of its derivatives was unsuccessful,
the aldol condensation of compound 17 and 4-(dimeth-
ylamino)benzaldehyde (18) successfully afforded a chal-
cone derivative. Compound 19 was obtained by oxida-
tive cyclization40 of the chalcone followed by acidic
deprotection.
Biologica l Activity a n d Discu ssion
5-AFs 10a -q and compounds 13, 16a ,b, and 19 were
evaluated for their antiproliferative activity against
MCF-7, the ER-positive and estrogen-responsive human
breast cancer cell line, in both the presence and absence
of estrogen. Although the growth rate was very slow,
estrogen-independent growth of MCF-7 was observed
in the absence of estrogen.14 Under this condition, we
could evaluate not only antiproliferative activity of test
compounds against the estrogen-independent growth of
MCF-7 but also estrogenic activity (growth-stimulating
activity). In addition, the binding affinity of 5-AFs for
the ER was studied by the binding competition with [3H]-
estradiol. These results are listed in Table 1.
TAM exhibited antiproliferative activity against MCF-7
in the presence of estrogen (IC50 0.14 µM). Apigenin
was almost inactive. Genistein exhibited weak anti-
proliferative activity in the presence of estrogen (IC50
11 µM) and had an affinity to the ER 4 times stronger
than TAM. 5-Aminoflavone 10a exhibited weak estro-
genic activity at 6.3 µM in the absence of estradiol (data
not shown) and weak antiproliferative activity (IC50 13
µM) in the presence of estradiol. In the course of the
chemical modification of 10a , 5-amino-4′-hydroxyflavone
(10b) showed a similar property to 10a with enhanced
Ch em istr y
The 5-aminoflavone derivatives (5-AFs) listed in Table
1 were synthesized as shown in Scheme 1. The con-
densation of the benzoate 738 and various acetophenone
derivatives 8 with sodium hydride afforded 1,3-dike-
tones. If compound 8 was substituted with a functional
group, which is unstable or reactive under basic condi-
tions, suitable protecting groups (THP, MOM, pivaloyl,
etc.) were used. During the reaction, the N-ethoxycar-
bonyl group of compound 7 was removed because of the

5-Aminoflavones as Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3463
Sch em e 1a
a
(a) NaH, 1,4-dioxane, reflux; (b) HCl, EtOH, rt; (c) HCl, EtOH or 1,4-dioxane, reflux; (d) H2SO4, AcOH, H2O, 100 °C; (e) Ac2O, pyridine,
0 °C; (f) NaH, EtI, DMF, 0 °C; (g) NaH, DMF, rt, n-HexI (for 10p ) or PhCH2Br (for 10q).
Sch em e 2a
Sch em e 4a
a
(a) MeOCH2Cl, NaH, THF, reflux; (b) H2NNH2‚H2O, Pd/C,
EtOH, rt; (c) pivaloyl chloride, pyridine, rt; (d) NaH, 1,4-dioxane,
reflux; (e) HCl, EtOH, rt; (f) HCl, EtOH, reflux.
a
(a) NaH, DMF, rt; (b) SeO2, i-AmOH, 150 °C; (c) HCl, 1,4-
Sch em e 3a
dioxane, reflux.
electron-withdrawing groups such as cyano (compound
10e) and carboxy (compound 10f) groups resulted in a
decrease in activity. Only the 4′-bromo derivative 10d
showed comparable activity to TAM. From a further
investigation of the substituents at the 4′-position, we
found that the 4′-amino derivative 10g exhibited an
intensive antiproliferative effect against MCF-7 ir-
respective of the presence or absence of estradiol (IC50
0.0072 and 0.0098 µM, respectively). Acetylation of the
4′-amino group of 10g resulted in a decrease in activity
(compound 10h ). Activity was somewhat enhanced
when the 4′-amino group was alkylated (compounds
10j-m ), whereas with increasing bulkiness, decreasing
activity occurred with compounds 10p ,q. Dialkylated
derivatives 10n ,i also seemed to enhance antitumor
activity; hence, the 4′-amino hydrogen was not impor-
tant as a hydrogen bond donor. Further, the basicity
of the 4′-amino group is important because secondary
39
a
(a) NaH, THF, rt; (b) HCl, EtOH, rt; (c) HCl, EtOH, reflux.
potency (IC50 1.5 µM in the presence of estradiol). In
addition, 10b bound to the ER with an affinity 5 times
less than TAM. Substitution of the 4′-position with

3464 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Akama et al.
Ta ble 2. Antiproliferative Activity of Various Diaminoflavones
against MCF-7
IC50 (µM),a estradiolb
compounds
X
Y
-
+
10g
10o
13
10n
16a
16b
19
5-NH2
5-NH2
6-NH2
5-NH2
6-NH2
7-NH2
8-NH2
4′-NH2
3′-NH2
0.0098
>100
0.0072
27
0.013
0.0040
13
5.0
0.021
0.14
4′-NMe2
4′-NMe2
3′-NMe2
4′-NMe2
4′-NMe2
0.035
0.0050
13
6.9
2.0
tamoxifen
19
a
IC50 values were measured by cell count method described in
the Experimental Section. 10-4 µM.
b
or tertiary amines seemed to enhance the activity.
These data indicated that 4′-amino groups with some
basicity and without bulkiness are essential for exhibit-
ing strong antitumor activity. However, the alkylation
of the 4′-amino group is not essential for exhibiting the
antiproliferative activity because compound 10g showed
sufficiently strong activity. In addition, when the
compounds are used in in vivo, the lower alkyl side
chain might be metabolically removed. So we consid-
ered compound 10g as a prototype. Interestingly, all
the compounds exhibiting strong antitumor activity did
not compete with [3H]estradiol in terms of receptor
binding even at a concentration of 100 µM.
F igu r e 1. Response of MCF-7 cells to compound 10g (0.5 µM)
and tamoxifen (TAM; 0.5 µM) in both the presence and absence
of estradiol (E; 10-4 µM). MCF-7 cells were preincubated in
phenol red free MEM containing 5% calf serum for 5 days.
The cells (2.5 × 103) were treated with each compound in both
the presence and absence of estradiol and counted 6 days later.
The results presented are means ( SD of three independent
replications, and the vertical bars represent the standard
deviations. *P < 0.05 vs lane 1. **P < 0.05 vs lane 4.
Ta ble 3. Specificity of Antiproliferative Activity to Breast
Cancer Cell Line
We then investigated the relationship between the
positions of the amino groups and antitumor activities
(Table 2). The relocation of the 4′-amino group to the
3′-position (compound 10o) resulted in a drastic de-
crease in activity. The 4′-dimethylamino derivative 10n
also exhibited a very strong antiproliferative effect
which is comparable to 10g. The relocation of the
5-amino group of compound 10n to the 6-position
IC50 (µM)a
compounds
MCF-7b
HeLa S3
WiDr
10g
10j
10k
adriamycin
0.052
0.076
0.092
0.25
>10
6.3
>10
>10
>10
0.62
>10
0.030
a
IC50 values were measured by neutral red dye uptake method
b
described in the Experimental Section. In the presence of 10-2
(compound 13) resulted in a decrease in activity.
A
µM estradiol.
further decrease in activity was observed when the 4′-
dimethylamino group of 13 relocated to the 3′-position
(compound 16a ). Although the 7-amino derivative 16b
exhibited only weak effects, the 8-amino derivative 19
exhibited antiproliferative activity at an IC50 of 0.021
µM in the presence of estradiol. Reviewing the results,
the best positions of amino groups seemed to be the 5-
and 4′-positions. Because the decrease in activity was
larger when the 4′-amino group was relocated to the 3′-
position than when the 5-amino group was relocated to
the 6-, 7-, or 8-position, the 4′-amino group evidently
plays a critical role in growth inhibition of MCF-7.
The antiproliferative activity of compound 10g against
MCF-7 in both the presence and absence of estradiol is
shown in Figure 1. In the absence of estradiol, the cell
numbers increased from 2.5 × 103 to 42 × 103 in 6 days,
and this seemed to be the estrogen-independent growth
(lane 1). The growth of MCF-7 cells was stimulated 8.3-
fold by the addition of 10-4 µM estradiol (lane 4 vs lane
1). TAM (0.5 µM) stimulated the growth of MCF-7 cells
in the absence of estradiol (lane 2); hence, the partial
agonistic activity of TAM was confirmed,41,42 and it
inhibited the growth of MCF-7 about 55% in the
presence of estradiol (lane 5). On the other hand,
compound 10g (0.5 µM) inhibited almost completely the
growth of MCF-7 cells in both the absence (lane 3) and
presence (lane 6) of estradiol.
The 5,4′-diaminoflavone derivatives exhibited remark-
able antiproliferative activity against MCF-7 cells with-
out ER competition; thus, we were next interested in
the antiproliferative effects of these compounds against
other human cancer cell lines and the ER-negative
breast cancer cell line. As shown in Table 3, a repre-
sentative cytotoxic agent, adriamycin, exhibited anti-
tumor activity against not only MCF-7 but also HeLa
S3 and WiDr, while compounds 10g,j,k exhibited selec-
tive and strong antiproliferative activity against the
breast cancer cell line. However, compound 10g exhib-
ited no effect against the growth of MDA-MB-453, the
ER-negative human breast cancer cell line, at least up
to 5 µM (Table 4).
Although the mechanism of selective antitumor activ-
ity of 5-AFs to ER-positive MCF-7 breast cancer cells is
unclear, it is noteworthy that they are not competitive
inhibitors of estrogen like TAM and pure anti-estrogens

5-Aminoflavones as Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3465
Ta ble 4. Antiproliferative Activity of Compound 10g against
the ER-Negative Breast Cancer Cell Line MDA-MB-453
in 1,4-dioxane (50 mL) under argon atmosphere was added
dropwise a solution of 7 (20.0 g, 47.5 mmol) and 8g (12.6 g,
57.3 mmol) in 1,4-dioxane (110 mL) over 10 min. The reaction
mixture was refluxed for 3 h and cooled on an ice bath. Water
was added, and the basic solution was washed with n-hexane.
Then the solution was twice extracted with EtOAc, and the
organic layer was washed with brine. The combined extracts
were concentrated and dissolved in EtOH (250 mL). Concen-
trated HCl (30 mL) was added, and the reaction mixture was
stirred for 2 h at room temperature. The precipitated product
was collected by filtration, washed with EtOH, and dried to
afford 9g (15.3 g, 77%): 1H NMR (270 MHz, CDCl3) δ 1.33 (s,
9H, C(CH3)3), 1.35 (s, 9H, C(CH3)3), 6.73 (s, 1H, 3-H), 7.25 (dd,
J ) 8.3, 1.0 Hz, 1H, 8-H), 7.66 (t, J ) 8.4 Hz, 1H, 7-H), 7.77
(d, J ) 9.0 Hz, 2H, 2′,6′-H), 7.91 (d, J ) 9.0 Hz, 2H, 3′,5′-H),
8.68 (dd, J ) 8.3, 1.0 Hz, 1H, 6-H), 12.8 (br s, 1H, 5-NH).
2-P h en yl-5-(pivaloylam in o)-4H-1-ben zopyr an -4-on e (9a).
This compound was obtained from 7 and 8a (19%) and was
used for the next step without purification.
2-(4-Hyd r oxyp h en yl)-5-(p iva loyla m in o)-4H-1-ben zop y-
r a n -4-on e (9b). This compound was obtained from 7 and 8b
(29%): 1H NMR (90 MHz, DMSO-d6) δ 1.30 (s, 9H, C(CH3)3),
6.82 (s, 1H, 3-H), 6.95 (d, J ) 9.0 Hz, 2H, 3′,5′-H), 7.31 (dd, J
) 8.3, 1.1 Hz, 1H, 8-H), 7.69 (t, J ) 8.3 Hz, 1H, 7-H), 7.92 (d,
J ) 8.8 Hz, 2H, 2′,6′-H), 8.57 (dd, J ) 8.3, 1.1 Hz, 1H, 6-H).
2-(4-Meth oxyp h en yl)-5-(p iva loyla m in o)-4H-1-ben zop y-
r a n -4-on e (9c). This compound was obtained from 7 and 8c
(39%): 1H NMR (90 MHz, CDCl3) δ 1.39 (s, 9H, C(CH3)3), 3.89
(s, 3H, OCH3), 6.65 (s, 1H, 3-H), 7.03 (d, J ) 8.8 Hz, 2H, 3′,5′-
H), 7.17 (d, J ) 8.6 Hz, 1H, 8-H), 7.61 (t, J ) 8.4 Hz, 1H, 7-H),
7.96 (d, J ) 9.0 Hz, 2H, 2′,6′-H), 8.70 (d, J ) 8.1 Hz, 1H, 6-H),
12.9 (br, 1H, NH).
IC50 (µM)a
compounds
MCF-7b
MDA-MB-453
10g
tamoxifen
0.0072
0.14
>5.0
>5.0
a
IC50 values were measured by cell count method described in
the Experimental Section. In the presence of 10-4 µM estradiol.
b
because they did not compete with [3H]estradiol even
at a concentration of 100 µM. Recently, some amino-
substituted flavones were reported as inhibitors of
protein-tyrosine kinases.27 Thus, the possibility cannot
be excluded that 5-AFs may affect the signal transduc-
tion downstream of the ER, including the phosphory-
lation of the ER.43-46 However, although some 5-AFs
reported here weakly inhibit a certain type of protein-
tyrosine kinase (data not shown), it is probably not
enough to explain their strong and selective antitumor
activity. On the other hand, compound 6 (L86-8275)
was reported to exhibit antiproliferative activities against
some cancer cell lines, including breast cancer cells, by
inhibiting Cdc2 kinase activity.47 But the antiprolif-
erative activity of compound 6 is not selective to breast
cancer cells.48 Therefore, it is unlikely that 5-AFs
exhibited antitumor activity by the same mechanism as
compound 6.
2-(4-Br om oph en yl)-5-(pivaloylam in o)-4H-1-ben zopyr an -
4-on e (9d ). This compound was obtained from 7 and 8d
(67%): 1H NMR (270 MHz, CDCl3) δ 1.39 (s, 9H, C(CH3)3),
6.71 (s, 1H, 3-H), 7.21 (dd, J ) 8.3, 1.0 Hz, 1H, 8-H), 7.65 (t,
J ) 8.6 Hz, 1H, 7-H), 7.68 (d, J ) 8.8 Hz, 2H, 3′,5′-H), 7.77 (d,
J ) 8.9 Hz, 2H, 2′,6′-H), 8.74 (dd, J ) 8.3, 1.0 Hz, 1H, 6-H),
12.8 (br, 1H, NH).
2-(4-Cyan oph en yl)-5-(pivaloylam in o)-4H-1-ben zopyr an -
4-on e (9e). This compound was obtained from 7 and 8e
(58%): 1H NMR (270 MHz, CDCl3) δ 1.39 (s, 9H, C(CH3)3),
6.79 (s, 1H, 3-H), 7.22 (dd, J ) 8.3, 1.0 Hz, 1H, 8-H), 7.68 (t,
J ) 8.6 Hz, 1H, 7-H), 7.84 (d, J ) 8.8 Hz, 2H, 2′,6′-H), 8.03 (d,
J ) 8.9 Hz, 2H, 3′,5′-H), 8.76 (dd, J ) 8.3, 1.0 Hz, 1H, 6-H),
12.7 (br, 1H, NH).
2-[4-(Meth ylam in o)ph en yl]-5-(pivaloylam in o)-4H-1-ben -
zop yr a n -4-on e (9j). This compound was obtained from 7 and
8j (26%): 1H NMR (270 MHz, CDCl3) δ 1.39 (s, 9H, C(CH3)3),
2.93 (d, J ) 5.1 Hz, 3H, CH3), 4.28 (br, 1H, 4′-NH), 6.60 (s,
1H, 3-H), 6.67 (d, J ) 7.0 Hz, 2H, 3′,5′-H), 7.17 (dd, J ) 8.6,
1.0 Hz, 1H, 8-H), 7.59 (t, J ) 8.4 Hz, 1H, 7-H), 7.77 (d, J ) 7.1
Hz, 2H, 2′,6′-H), 8.69 (dd, J ) 8.3, 1.0 Hz, 1H, 6-H), 13.0 (br,
1H, NH).
2-[4-(N-Eth yla ceta m id o)p h en yl]-5-(p iva loyla m in o)-4H-
1-ben zop yr a n -4-on e (9k ). This compound was obtained from
7 and 8k (78%): 1H NMR (270 MHz, CDCl3) δ 1.15 (t, J ) 7.1
Hz, 3H, CH3), 1.39 (s, 9H, C(CH3)3), 1.93 (s, 3H, COCH3), 3.81
(q, J ) 7.2 Hz, 2H, CH2), 6.76 (s, 1H, 3-H), 7.22 (dd, J ) 8.3,
1.0 Hz, 1H, 8-H), 7.30 (d, J ) 8.5 Hz, 2H, 3′,5′-H), 7.67 (t, J )
8.4 Hz, 1H, 7-H), 7.97 (d, J ) 8.8 Hz, 2H, 2′,6′-H), 8.75 (dd, J
) 8.3, 1.0 Hz, 1H, 6-H), 12.8 (br, 1H, NH).
These results suggest the presence of an alternative
receptor or a signal transduction pathway to the ER in
the ER-positive breast cancer cells because the 5-AFs
only inhibited the growth of MCF-7 cells among the
human cancer cell lines tested, including ER-negative
breast cancer cell line. Further, it would appear that
there is a new therapeutic target for the treatment of
breast cancer.
Con clu sion s
We prepared a series of amino-substituted flavones.
Among them, 5,4′-diaminoflavone (10g) and some of its
congeners exhibited a remarkable antiproliferative ef-
fect against the human breast cancer cell line MCF-7
irrespective of the presence or absence of estrogen.
Although the mechanism of selective antitumor activity
of 5-AFs to the ER-positive MCF-7 breast cancer cells
is unclear, they are expected to be a new type of
chemotherapeutic agent in breast cancer. Further
evaluation of the antitumor activities against other cell
lines and research on the structure-activity relation-
ships of related derivatives are in progress.
Exp er im en ta l Section
All melting points were determined on Yanako micromelting
point apparatus and are uncorrected. IR spectra were re-
corded on a J ASCO IR-400 spectrometer. 1H-NMR spectra
were recorded on HITACHI R-90H (90 MHz) and J EOL J NM-
GX-270 (270 MHz) spectrometers. Electron impact mass
spectra (EIMS) were recorded on a J EOL J MS-D-300 spec-
trometer. Elemental analyses were performed by a Perkin-
Elmer 2400 C, H, N analyzer. Organic extracts were dried
over anhydrous Na2SO4, and the solvents were evaporated
under reduced pressure. Merck Kieselgel 60 was used for
column chromatography. Sodium hydride used was a 60% oil
dispersion.
5-(P iva loyla m in o)-2-[4-(N-p r op yla cet a m id o)p h en yl]-
4H-1-ben zop yr a n -4-on e (9l). This compound was obtained
from 7 and 8l and used for the next step without isolation.
2-[4-(N-Bu tyla ceta m id o)p h en yl]-5-(p iva loyla m in o)-4H-
1-ben zop yr a n -4-on e (9m ). This compound was obtained
from 7 and 8m (59%): 1H NMR (90 MHz, CDCl3) δ 0.90 (t, J
) 6.8 Hz, 3H, CH3), 1.1-1.6 (m, 4H, NCH2CH2CH2), 1.39 (s,
9H, C(CH3)3), 1.93 (s, 3H, COCH3), 3.76 (t, J ) 6.8 Hz, 2H,
NCH2), 6.76 (s, 1H, 3-H), 7.1-7.4 (m, 3H, 8,3′,5′-H), 7.66 (t, J
) 8.4 Hz, 1H, 7-H), 7.99 (d, J ) 8.1 Hz, 2H, 2′,6′-H), 8.75 (d,
J ) 8.4 Hz, 1H, 6-H), 12.8 (br, 1H, NH).
Typ ica l P r oced u r e for P r ep a r a tion of 2-Ar yl-4H-1-
ben zop yr a n -4-on es 9a -e,g,j-p : 5-(P iva loyla m in o)-2-[4-
(p iva loyla m in o)p h en yl]-4H-1-ben zop yr a n -4-on e (9g). To
a refluxing suspension of sodium hydride (4.60 g, 115 mmol)
2-[4-(Dim eth yla m in o)p h en yl]-5-(p iva loyla m in o)-4H-1-
ben zop yr a n -4-on e (9n ). This compound was obtained from
7 and 8n (51%): 1H NMR (270 MHz, CDCl3) δ 1.38 (s, 9H,

3466 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Akama et al.
C(CH3)3), 3.08 (s, 6H, N(CH3)2), 6.60 (s, 1H, 3-H), 6.74 (d, J )
9.0 Hz, 2H, 3′,5′-H), 7.17 (dd, J ) 8.3, 1.0 Hz, 1H, 8-H), 7.59
(t, J ) 8.3 Hz, 1H, 7-H), 7.80 (d, J ) 9.0 Hz, 2H, 2′,6′-H), 8.69
(dd, J ) 8.6, 1.0 Hz, 1H, 6-H), 13.0 (br, 1H, NH).
5-(P iva loyla m in o)-2-[3-(p iva loyla m in o)p h en yl]-4H -1-
ben zop yr a n -4-on e (9o). This compound was obtained from
7 and 8o (49%): 1H NMR (270 MHz, CDCl3) δ 1.37 (s, 9H,
C(CH3)3), 1.39 (s, 9H, C(CH3)3 ), 6.75 (s, 1H, 3-H), 7.25 (dd, J
) 8.1, 1.0 Hz, 1H, 8-H), 7.48 (t, J ) 7.9 Hz, 1H, 5′-H), 7.50
(br, 1H, 3′-NH), 7.63 (dd, J ) 8.0, 1.2 Hz, 1H, 6′-H), 7.64 (t, J
) 8.3 Hz, 1H, 7-H), 7.70 (ddd, J ) 8.0, 2.2, 1.0 Hz, 1H, 4′-H),
8.19 (t, J ) 2.0 Hz, 1H, 2′-H), 8.73 (dd, J ) 8.3, 0.7 Hz, 1H,
6-H), 12.8 (br, 1H, 5-NH).
2-(4-Acet a m id op h en yl)-5-(p iva loyla m in o)-4H -1-b en -
zop yr a n -4-on e (9p ). This compound was obtained from 7 and
8p (52%): 1H NMR (270 MHz, CDCl3) δ 1.29 (s, 9H, C(CH3)3),
2.10 (s, 3H, COCH3), 6.96 (s, 1H, 3-H), 7.39 (dd, J ) 8.3, 1.0
Hz, 1H, 8-H), 7.45 (t, J ) 8.3 Hz, 1H, 7-H), 7.79 (d, J ) 9.3
Hz, 2H, 2′,6′-H), 8.06 (d, J ) 8.8 Hz, 2H, 3′,5′-H), 8.57 (dd, J
) 8.3, 0.7 Hz, 1H, 6-H), 10.3 (br, 1H, 4′-NH), 12.9 (br, 1H,
5-NH).
Typ ica l P r oced u r e for P r ep a r a tion of 2-Ar yl-4H-1-ben -
zopyr an -4-on es 10a -e,g,j-o: 5-Am in o-2-(4-am in oph en yl)-
4H-1-ben zop yr a n -4-on e (10g). To a suspension of 9g (14.5
g, 34.5 mmol) in EtOH (200 mL) was added concentrated HCl
(200 mL). The mixture was refluxed for 5 h. During the
reaction, starting material gradually dissolved and the product
precipitated. After cooling on an ice bath, the precipitate was
collected by filtration, washed with 2-propanol, and dried to
afford 10g (9.44 g, 84%) as a hydrochloride: mp 186-188 °C;
IR (KBr) 1624 cm-1; 1H NMR (270 MHz, DMSO-d6) δ 6.52 (dd,
J ) 8.3, 0.9 Hz, 1H, 6-H), 6.59 (s, 1H, 3-H), 6.64 (dd, J ) 8.1,
0.9 Hz, 1H, 8-H), 6.91 (d, J ) 8.6 Hz, 2H, 3′,5′-H), 7.33 (t, J )
8.2 Hz, 1H, 7-H), 7.36 (d, J ) 8.8 Hz, 2H, 2′,6′-H); EIMS m/ z
252 (M+). Anal. (C15H12N2O2‚1.9HCl) C, H, N.
3′,5′-H), 7.99 (d, J ) 8.6 Hz, 2H, 2′,6′-H); EIMS m/z 262 (M+).
Anal. (C16H10N2O2) C, H, N.
5-Am in o-2-[4-(m eth yla m in o)p h en yl]-4H-1-ben zop yr a n -
4-on e (10j). This compound was obtained as a free base in a
similar manner as described for 10c (42%): mp 184-185 °C;
IR (KBr) 1646 cm-1; 1H NMR (270 MHz, CDCl3) δ 2.91 (d, J )
2.0 Hz, 3H, CH3), 6.41 (dd, J ) 8.2, 1.0 Hz, 1H, 6-H), 6.49 (s,
1H, 3-H), 6.65 (d, J ) 8.8 Hz, 2H, 3′,5′-H), 6.66 (dd, J ) 8.4,
1.0 Hz, 1H, 8-H), 7.29 (t, J ) 8.1 Hz, 1H, 7-H), 7.74 (d, J ) 8.8
Hz, 2H, 2′,6′-H); EIMS m/z 266 (M+). Anal. (C16H14N2O2) C,
H; N: calcd, 10.52; found, 9.81.
5-Am in o-2-[4-(eth yla m in o)p h en yl]-4H-1-ben zop yr a n -4-
on e (10k ). This compound was obtained as a free base in a
similar manner as described for 10c (72%): mp 194-195 °C;
IR (KBr) 1640 cm-1; 1H NMR (270 MHz, CDCl3) δ 1.29 (t, J )
7.2 Hz, 3H, CH3), 3.23 (m, 2H, CH2), 4.05 (br, 1H, NH), 6.41
(dd, J ) 8.2, 0.9 Hz, 1H, 6-H), 6.48 (s, 1H, 3-H), 6.50 (br, 2H,
NH2), 6.64 (d, J ) 7.0 Hz, 2H, 3′,5′-H), 6.67 (dd, J ) 8.3, 1.0
Hz, 1H, 8-H), 7.29 (t, J ) 8.2 Hz, 1H, 7-H), 7.72 (d, J ) 7.0
Hz, 2H, 2′,6′-H); EIMS m/z 280 (M+). Anal. (C17H16N2O2) C,
H, N.
5-Am in o-2-[4-(p r op yla m in o)p h en yl]-4H-1-ben zop yr a n -
4-on e (10l). This compound was obtained in a similar manner
as described for 10g (29%): mp 159-162 °C; IR (KBr) 1641
cm-1 1H NMR (270 MHz, DMSO-d6) δ 0.95 (t, J ) 7.3 Hz,
;
3H, CH3), 2.50 (m, 2H, NCH2CH2), 3.07 (t, J ) 7.0 Hz, 2H,
NCH2), 6.50 (s, 1H, 3-H), 6.51 (d, J ) 8.2 Hz, 1H, 6-H), 6.64
(d, J ) 8.2 Hz, 1H, 8-H), 6.72 (d, J ) 8.8 Hz, 2H, 3′,5′-H), 7.31
(t, J ) 8.2 Hz, 1H, 7-H), 7.76 (d, J ) 8.9 Hz, 2H, 2′,6′-H); EIMS
m/ z 294 (M+). Anal. (C18H18N2O2‚1.8HCl) C, H, N.
5-Am in o-2-[4-(b u t yla m in o)p h en yl]-4H-1-b en zop yr a n -
4-on e (10m ). This compound was obtained as a free base in
a similar manner as described for 10c (44%): mp 138-139
°C; IR (KBr) 1639 cm-1; 1H NMR (270 MHz, CDCl3) δ 0.97 (t,
J ) 7.2 Hz, 3H, CH3), 1.4-1.7 (m, 4H, NCH2CH2CH2), 3.18
(q, J ) 6.4 Hz, 2H, NCH2), 4.10 (br, 1H, NH), 6.41 (dd, J )
8.3, 0.9 Hz, 1H, 6-H), 6.48 (s, 1H, 3-H), 6.51 (br, 2H, NH2),
6.64 (d, J ) 9.0 Hz, 2H, 3′,5′-H), 6.67 (dd, J ) 8.2, 0.9 Hz, 1H,
8-H), 7.28 (t, J ) 8.2 Hz, 1H, 7-H), 7.72 (d, J ) 8.8 Hz, 2H,
2′,6′-H); EIMS m/z 308 (M+). Anal. (C19H20N2O2) C, H, N.
5-Am in o-2-[4-(d im eth yla m in o)p h en yl]-4H-1-ben zop y-
r a n -4-on e (10n ). This compound was obtained as a free base
in a similar manner as described for 10c (77%): mp 229-230
°C; IR (KBr) 1626 cm-1; 1H NMR (270 MHz, CDCl3) δ 3.06 (s,
6H, N(CH3)2), 6.50 (dd, J ) 8.0, 0.9 Hz, 1H, 6-H), 6.50 (br,
2H, NH2), 6.51 (s, 1H, 3-H), 6.68 (dd, J ) 8.2, 1.1 Hz, 1H, 8-H),
6.75 (d, J ) 7.0 Hz, 2H, 3′,5′-H), 7.29 (t, J ) 8.0 Hz, 1H, 7-H),
7.77 (d, J ) 7.0 Hz, 2H, 2′,6′-H); EIMS m/z 280 (M+). Anal.
(C17H16N2O2) C, H, N.
5-Am in o-2-p h en yl-4H-1-ben zop yr a n -4-on e (10a ). This
compound was obtained in a similar manner as described for
10g (70%): mp 191-193 °C; IR (KBr) 1644 cm-1; 1H NMR (270
MHz, DMSO-d6) δ 6.24 (dd, J ) 8.1, 0.9 Hz, 1H, 6-H), 6.62 (s,
1H, 3-H), 6.69 (dd, J ) 8.1, 0.9 Hz, 1H, 8-H), 7.24 (t, J ) 8.1
Hz, 1H, 7-H), 7.4-7.6 (m, 3H, 3′,4′,5′-H), 7.8-8.0 (m, 2H, 2′,6′-
H); EIMS m/z 237 (M+). Anal. (C15H11NO2‚HCl) C, H, N.
5-Am in o-2-(4-h yd r oxyp h e n yl)-4H -1-b e n zop yr a n -4-
on e (10b). This compound was obtained in a similar manner
as described for 10g (77%): mp 244-245 °C; IR (KBr) 1634
1
cm-1; H NMR (270 MHz, DMSO-d6) δ 6.59 (dd, J ) 8.1, 1.1
Hz, 1H, 6-H), 6.62 (s, 1H, 3-H), 6.71 (dd, J ) 8.1, 0.9 Hz, 1H,
8-H), 6.95 (d, J ) 8.9 Hz, 2H, 3′,5′-H), 7.46 (dd, J ) 8.1, 8.3
Hz, 1H, 7-H), 7.86 (d, J ) 8.2 Hz, 2H, 2′,6′-H); EIMS m/z 253
(M+). Anal. (C15H11NO3‚HCl) C, H, N.
5-Am in o-2-(3-a m in op h en yl)-4H -1-b en zop yr a n -4-on e
(10o). This compound was obtained in a similar manner as
described for 10g (88%): mp 238-239 °C; IR (KBr) 1637 cm-1
;
5-Am in o-2-(4-m e t h oxyp h e n yl)-4H -1-b e n zop yr a n -4-
on e (10c). The reaction mixture was concentrated, poured
into water, and extracted with CHCl3. The organic layer was
washed with brine. Chromatogaphy (50:1 CHCl3/MeOH) and
recrystallization from EtOAc/n-hexane afforded 10c as a free
1H NMR (270 MHz, DMSO-d6) δ 6.56 (dd, J ) 8.3, 0.8 Hz, 1H,
6-H), 6.64 (dd, J ) 8.2, 0.8 Hz, 1H, 8-H), 6.77 (s, 1H, 3-H),
7.42 (t, J ) 8.4 Hz, 1H, 5′-H), 7.44 (dd, J ) 8.6, 1.3 Hz, 1H,
4′-H), 7.59 (t, J ) 8.0 Hz, 1H, 7-H), 7.85 (s, 1H, 2′-H), 7.91 (d,
J ) 8.1 Hz, 1H, 6′-H); EIMS m/z 252 (M+). Anal. (C15H12N2O2‚
2HCl) C, H, N.
base (48%): mp 144-145 °C; IR (KBr) 1647 cm-1 1H NMR
;
(270 MHz, CDCl3) δ 3.88 (s, 3H, OCH3), 6.43 (dd, J ) 8.1, 0.9
Hz, 1H, 6-H), 6.51 (br, 2H, NH2), 6.55 (s, 1H, 3-H), 6.69 (dd, J
) 8.3, 0.9 Hz, 1H, 8-H), 7.00 (d, J ) 9.0, 2H, 3′,5′-H), 7.31 (t,
J ) 8.2 Hz, 1H, 7-H), 7.84 (d, J ) 9.0 Hz, 2H, 2′,6′-H); EIMS
m/z 267 (M+). Anal. (C16H13NO3) C, H, N.
5-Am in o -2-(4-c a r b o xyp h e n y l)-4H -1-b e n zo p y r a n -4-
on e (10f). A mixture of 10e (100 mg, 0.375 mmol), AcOH (1
mL), H2SO4 (1 mL), and water (1 mL) was heated at 100 °C
for 16 h. After cooling, the precipitated solid was collected by
filtration, washed with water, and chromatographed (90:10:1
CHCl3/MeOH/Et3N) to afford 10f (92 mg, 86%): mp >300 °C;
5-Am in o-2-(4-b r om op h en yl)-4H -1-b en zop yr a n -4-on e
(10d ). This compound was obtained in a similar manner as
described for 10g (57%): mp 197-199 °C; IR (KBr) 1653 cm-1
;
1
IR (KBr) 1711, 1642 cm-1; H NMR (270 MHz, DMSO-d6) δ
1H NMR (270 MHz, DMSO-d6) δ 6.55 (dd, J ) 8.2, 0.9 Hz, 1H,
6-H), 6.68 (dd, J ) 8.4, 1.0 Hz, 1H, 8-H), 6.85 (s, 1H, 3-H),
7.37 (t, J ) 8.3 Hz, 1H, 7-H), 7.77 (d, J ) 8.6 Hz, 2H, 3′,5′-H),
7.99 (d, J ) 9.0 Hz, 2H, 2′,6′-H); EIMS m/z 315, 317 (M+). Anal.
(C15H10BrNO2‚0.5HCl) C, H, N.
6.56 (dd, J ) 8.3, 0.7 Hz, 1H, 6-H), 6.69 (d, J ) 8.1 Hz, 1H,
8-H), 6.89 (s, 1H, 3-H), 7.38 (t, J ) 8.2 Hz, 1H, 7-H), 7.46 (br
s, 2H, NH2), 8.08 (d, J ) 8.6 Hz, 2H, 2′,6′-H), 8.15 (d, J ) 8.6
Hz, 2H, 3′,5′-H); EIMS m/z 281 (M+). Anal. (C16H11NO4) C,
H, N.
5-Am in o-2-(4-cya n op h e n yl)-4H -1-b e n zop yr a n -4-on e
(10e). This compound was obtained as a free base in a similar
manner as described for 10c (82%): mp 270-273 °C; IR (KBr)
2234, 1640 cm-1; 1H NMR (DMSO-d6) δ 6.48 (dd, J ) 8.2, 0.8
Hz, 1H, 6-H), 6.67 (s, 1H, 3-H), 6.70 (dd, J ) 8.1, 0.9 Hz, 1H,
8-H), 7.36 (t, J ) 8.3 Hz, 1H, 7-H), 7.80 (d, J ) 8.6 Hz, 2H,
2-(4-Acet a m id op h en yl)-5-a m in o-4H -1-b en zop yr a n -4-
on e (10h ). To a solution of 10g (100 mg, 0.397 mmol) in
pyridine (2 mL) was added acetic anhydride (41 µL, 0.40
mmol), and the mixture was stirred at 0 °C for 20 min. The
reaction mixture was dissolved in CHCl3 and washed with 10%
aqueous citric acid, 5% aqueous copper sulfate, and brine.

5-Aminoflavones as Antitumor Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3467
Chromatography (20:1 CHCl3/MeOH) and recrystallization
CH3), 5.20 (s, 2H, OCH2O), 7.14 (d, J ) 9.2 Hz, 1H, 3-H), 7.30
(br, 1H, NH), 7.6-7.8 (m, 2H, 4,6-H).
from CHCl3/MeOH/n-hexane afforded 10h (44 mg, 37%): mp
1
272-274 °C; IR (KBr) 1689, 1633 cm-1; H NMR (270 MHz,
6-Am in o-2-[4-(d im eth yla m in o)p h en yl]-4H-1-ben zop y-
r a n -4-on e (13). This compound was obtained from 12 and
8n in a similar manner as described for compounds 9g and
10g and isolated as a free base in a similar manner as
described for 10c (overall 5.5%): mp 248 °C; IR (KBr) 1681
DMSO-d6) δ 2.09 (s, 3Η, CΗ3), 6.52 (dd, J ) 8.3, 0.9 Hz, 1H,
6-H), 6.65 (dd, J ) 8.1, 0.9 Hz, 1H, 8-H), 6.71 (s, 1H, 3-H),
7.35 (t, J ) 8.2 Hz, 1H, 7-H), 7.44 (br, 2H, NH2), 7.76 (d, J )
8.8 Hz, 2H, 2′,6′-H), 7.98 (d, J ) 8.8 Hz, 2H, 3′,5′-H), 10.3 (br,
1H, NH); EIMS m/z 294 (M+). Anal. (C17H14N2O3) C, H, N.
5-Am in o-2-[4-(d ieth yla m in o)p h en yl]-4H-1-ben zop yr a n -
4-on e (10i). To a suspension of sodium hydride (1.44 g, 36.0
mmol) in DMF (5 mL) was added a solution of 10g (3.02 g,
12.0 mmol) in DMF (30 mL) at 0 °C. Then EtI (2.1 mL, 26
mmol) was added and the mixture stirred at 0 °C for 50 min.
The reaction mixture was poured into aqueous NH4Cl and
extracted with CHCl3. The organic layer was washed with
water and brine. Chromatography (4:1-1:0 CHCl3/n-hexane)
and recrystallization from EtOAc/n-hexane afforded 10i (0.99
1
cm-1; H NMR (270 MHz, DMSO-d6) δ 3.02 (s, 6H, N(CH3)2),
5.44 (br s, 2H, NH2), 6.64 (s, 1H, 3-H), 6.81 (d, J ) 9.2 Hz,
2H, 3′,5′-H), 7.02 (dd, J ) 8.8, 2.9 Hz, 1H, 7-H), 7.09 (d, J )
2.8 Hz, 1H, 5-H), 7.43 (d, J ) 8.8 Hz, 1H, 8-H), 7.86 (d, J )
9.0 Hz, 2H, 2′,6′-H); EIMS m/z 280 (M+). Anal.
(C17H16N2O2‚0.3H2O) C, H, N.
6-Am in o-2-[3-(d im eth yla m in o)p h en yl]-4H-1-ben zop y-
r a n -4-on e (16a ). To a solution of 15a (1.61 g, 5.16 mmol) in
THF (30 mL) were added 14a (710 mg, 3.68 mmol) and sodium
hydride (475 mg, 11.9 mmol). The reaction mixture was
stirred at room temperature for 5 h. Then 1 N KOH was
added, and the mixture was washed with CHCl3. The aqueous
layer was acidified with 6 N HCl and extracted with CHCl3.
The organic layer was washed with brine and concentrated to
afford 1-(5-acetamido-2-hydroxyphenyl)-3-[3-(dimethylamino)-
phenyl]propane-1,3-dione (400 mg, 44%). The propanedione
obtained (400 mg, 1.18 mmol) was dissolved in EtOH (10 mL),
and concentrated HCl (2 mL) was added. The reaction mixture
was stirred at room temperature for 4 h. The precipitated
product was filtered to afford 6-acetamido-2-[3-(dimethylami-
no)phenyl]-4H-1-benzopyran-4-one hydrochloride (327 mg,
77%). The compound (200 mg, 0.620 mmol) was dissolved in
a mixture of EtOH (10 mL) and concentrated HCl (5 mL)
followed by refluxing for 2.5 h. After cooling, the precipitated
product was collected by filtration and triturated with CHCl3/
MeOH to afford 16a (100 mg, 57%): mp 175 °C; IR (KBr) 1634
1
g, 21%): mp 219-220 °C; IR (KBr) 1626 cm-1; H NMR (270
MHz, CDCl3) δ 1.21 (t, J ) 7.4 Hz, 6H, N(CH2CH3)2), 3.43 (q,
J ) 7.4 Hz, 4H, N(CH2CH3)2), 6.41 (dd, J ) 7.9, 1.0 Hz, 1H,
6-H), 6.48 (s, 1H, 3-H), 6.50 (br, 2H, NH2), 6.67 (dd, J ) 7.4,
1.5 Hz, 1H, 8-H), 6.71 (d, J ) 9.4 Hz, 2H, 3′,5′-H), 7.28 (t, J )
8.2 Hz, 1H, 7-H), 7.75 (d, J ) 8.9 Hz, 2H, 2′,6′-H); EIMS m/z
308 (M+). Anal. (C19H20N2O2) C, H, N.
5-Am in o-2-[4-(h exyla m in o)p h en yl]-4H-1-ben zop yr a n -
4-on e (10p ). To a solution of 9p (3.00 g, 7.93 mmol) in DMF
(30 mL) were added sodium hydride (634 mg, 15.9 mmol) and
1-iodohexane (1.40 mL, 9.52 mmol) at 0 °C. The mixture was
stirred at room temperature for 3 h and poured into ice and
water. The mixture was extracted with CHCl3; the extracts
were washed with water and brine and chromatographed (1:1
EtOAc/n-hexane) to afford 2-[4-(N-hexylacetamido)phenyl]-5-
(pivaloylamino)-4H-1-benzopyran-4-one (2.66 g, 70%). The
compound (2.16 g, 4.67 mmol) was then treated in a similar
manner as described for compound 9g. Recrystallization from
CHCl3/MeOH gave 10p (1.40 g, 73%): mp 162-165 °C; IR
(KBr) 1645 cm-1; 1H NMR (270 MHz, DMSO-d6) δ 0.88 (t, J )
6.7 Hz, 3H, CH3), 1.3-1.4 (m, 6H, (CH2)3CH3), 1.5-1.6 (m, 2H,
NCH2CH2), 3.11 (t, J ) 7.1 Hz, 2H, NCH2), 6.52 (s, 1H, 3-H),
6.53 (d, J ) 8.2 Hz, 2H, 6-H), 6.58 (br, 2H, NH2), 6.66 (dd, J
) 8.3, 0.9 Hz, 1H, 8-H), 6.71 (d, J ) 9.0 Hz, 2H, 3′,5′-H), 7.32
(t, J ) 8.2 Hz, 1H, 7-H), 7.78 (d, J ) 8.8 Hz, 2H, 2′,6′-H); EIMS
m/z 336 (M+). Anal. (C21H24N2O2‚2HCl) C, H, N.
1
cm-1; H NMR (270 MHz, DMSO-d6) δ 3.08 (s, 6H, N(CH3)2),
7.11 (s, 1H, 3-H), 7.29 (br d, J ) 7.9 Hz, 1H, 4′-H), 7.50 (t, J
) 7.9 Hz, 1H, 5′-H), 7.62 (br d, J ) 7.3 Hz, 1H, 6′-H), 7.69 (br,
1H, 2′-H), 7.74 (dd, J ) 8.9, 2.6 Hz, 1H, 7-H), 7.89 (d, J ) 8.9
Hz, 1H, 8-H), 7.93 (br d, J ) 2.6 Hz, 1H, 5-H); EIMS m/z 280
(M+). Anal. (C17H16N2O2‚2HCl‚0.1H2O) C, H, N.
7-Am in o-2-[4-(d im eth yla m in o)p h en yl]-4H-1-ben zop y-
r a n -4-on e (16b). This compound was obtained from 14b and
4-(dimethylamino)benzoyl chloride (15b) in a similar manner
as described for 16a (overall 4%): mp 180-185 °C; IR (KBr)
5-Am in o-2-[4-(ben zyla m in o)p h en yl]-4H-1-ben zop yr a n -
4-on e (10q). This compound was obtained by the same way
as described for compound 10p except that benzyl bromide was
used instead of 1-iodohexane (overall 26%): mp 157-159 °C;
IR (KBr) 1638 cm-1; 1H NMR (270 MHz, DMSO-d6) δ 4.37 (s,
2H, CH2), 6.48 (s, 1H, 3-H), 6.50 (dd, J ) 8.3, 0.9 Hz, 1H, 6-H),
6.61 (dd, J ) 8.1, 0.9 Hz, 1H, 8-H), 6.71 (d, J ) 9.0 Hz, 2H,
3′,5′-H), 7.30 (t, J ) 8.4 Hz, 2H, 2′,6′-H), 7.3-7.4 (m, 5H, Ph);
EIMS m/z 342 (M+). Anal. (C22H18N2O2‚1.9HCl) C, H, N.
Eth yl 2-(Meth oxym eth oxy)-5-(p iva loyla m in o)ben zoa te
(12). To a solution of ethyl 5-nitrosalicylate (5.00 g, 23.4 mmol)
in THF (50 mL) were added sodium hydride (1.23 g, 30.8
mmol) and chloromethyl methyl ether (2.3 mL, 31 mmol) at 0
°C. The reaction mixture was refluxed for 30 min. The
mixture was then poured into ice and water and extracted with
CHCl3. The organic layer was washed with brine and con-
centrated. The residue was dissolved in EtOH (30 mL), and
10% palladium on charcoal (610 mg) and a solution of hydra-
zine monohydrate (2.38 g, 47.5 mmol) in EtOH (3 mL) were
added. The reaction mixture was stirred at room temperature
for 30 min. The mixture was then filtered, concentrated, and
extracted with CHCl3. The organic layer was washed with
brine and chromatographed (3:2 n-hexane/EtOAc) to afford
ethyl 5-amino-2-(methoxymethoxy)benzoate (3.75 g, 71%). To
a solution of this amino MOM ether (1.91 g, 8.48 mmol) in
pyridine (20 mL) was added pivaloyl chloride (1.2 mL, 9.3
mmol), and the reaction mixture was stirred at room temper-
ature for 30 min. The mixture was then filtered, concentrated,
and extracted with CHCl3. The organic layer was washed with
5% aqueous copper sulfate and brine and chromatographed
(7:3 n-hexane/EtOAc) to afford 12 (2.01 g, 77%): 1H NMR (90
MHz, CDCl3) δ 1.31 (s, 9H, C(CH3)3), 1.38 (t, J ) 7.0 Hz, 3H,
CH2CH3), 3.51 (s, 3H, OCH3), 4.35 (q, J ) 7.0 Hz, 2H, CH2-
1648 cm-1 1H NMR (270 MHz, DMSO-d6) δ 3.03 (s, 6H,
;
N(CH3)2), 6.62 (s, 1H, 3-H), 6.67 (d, J ) 2.2 Hz, 1H, 8-H), 6.71
(dd, J ) 8.3, 2.1 Hz, 1H, 6-H), 6.87 (d, J ) 9.2 Hz, 2H, 3′,5′-
H), 7.70 (d, J ) 8.6 Hz, 1H, 5-H), 7.86 (d, J ) 9.0 Hz, 2H,
2′,6′-H); EIMS m/z 280 (M+). Anal. (C17H16N2O2‚2HCl) C, H;
N: calcd, 7.93; found, 7.40.
8-Am in o-2-[4-(d im eth yla m in o)p h en yl]-4H-1-ben zop y-
r a n -4-on e (19). To a mixture of 17 (1.10 g, 5.69 mmol) and
18 (790 mg, 5.30 mmol) in DMF (30 mL) was added sodium
hydride (640 mg, 16.0 mmol) at 0 °C. The reaction mixture
was stirred at room temperature for 3.5 h and then neutralized
with concentrated HCl and extracted with CHCl3. The organic
layer was washed with water and brine and chromatographed
(40:1 CHCl3/MeOH) to afford 3-(3-acetamido-2-hydroxyphenyl)-
1-[4-(dimethylamino)phenyl]propen-3-one (1.34 g, 73%).
A
mixture of the propenone (500 mg, 1.54 mmol) and selenium
dioxide (500 mg, 4.51 mmol) in isoamyl alcohol (25 mL) was
heated at 150 °C for 2 days. The solution was filtered to
remove the precipitated solid and extracted with CHCl3. The
organic layer was washed with water and brine. The crude
product was chromatographed (20:1 CHCl3/MeOH) to afford
8-acetamido-2-[4-(dimethylamino)phenyl]-4H-1-benzopyran-4-
one (140 mg, 28%). The compound (64 mg, 0.19 mmol) was
dissolved in 1,4-dioxane (4 mL), and concentrated HCl (4 mL)
was added followed by refluxing for 2.5 h. After cooling, the
reaction mixture was neutralized with 10% aqueous NaOH
and extracted with CH2Cl2. The organic layer was washed
with water and brine and chromatographed (20:1 CHCl3/
MeOH) to afford 19 (15 mg, 28%) as a free base. The
compound (15 mg) was converted into the hydrochloride with
hydrogen chloride in a mixture of CHCl3 and 2-propanol. The
precipitate was collected by filtration and dried to give the
hydrochloride (15 mg): mp 230 °C dec; IR (KBr) 1605 cm-1
;

3468 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Akama et al.
1H NMR (270 MHz, DMSO-d6) δ 3.04 (s, 6H, N(CH3)2), 6.75
(s, 1H, 3-H), 6.81 (d, J ) 9.2 Hz, 2H, 3′,5′-H), 7.17 (t, J ) 7.3
Hz, 1H, 6-H), 7.27 (dd, J ) 6.9, 2.7 Hz, 1H, 7-H), 7.17 (m, 1H,
5-H), 8.05 (d, J ) 9.0 Hz, 2H, 2′,6′-H); EIMS m/z 280 (M+).
Anal. (C17H16N2O2‚HCl‚0.9H2O) C, H, N.
Ack n ow led gm en t. We thank Mr. Hiromitsu Saito
and Mr. Shun-ichi Ikeda for reviewing the manuscript
and Hiroe Tanaka for the evaluation of anticellular
activity by the neutral red dye uptake method. We are
also grateful to Akiko Mimura and Taimi Sano for their
excellent technical assistance.
Biologica l Assa y. An tip r olifer a tive Activity a ga in st
MCF -7 (Cell Cou n t Meth od for Ta bles 1 a n d 2). MCF-7
cells were suspended in a medium comprised of phenol red
free Eagle’s MEM (Nissui Pharmaceutical Co., Ltd.) and 5%
calf serum (Hyclone) with or without 10-4 µM estradiol (Sigma
Fine Chemical Inc.) (here after referred to as medium A) to a
concentration of 3.3 × 103 cells/mL. The cell suspension thus
prepared was put into wells of 24-well multidishes (NUNC)
at 0.75 mL/well. The cells on the plate were incubated in a
CO2 incubator at 37 °C for 24 h, and 0.25 mL of a sample
containing a test compound and appropriately diluted with
medium A was added to each well (n ) 3). The cells were
further incubated in the CO2 incubator at 37 °C for 144 h. The
growth-inhibitory activity of the compounds was evaluated by
counting cell numbers using a microcell counter (Toa Medical
Electronics Co.).
An tip r olifer a tive Activity a ga in st MDA-MB-453 (Cell
Cou n t Meth od for Ta ble 4). MDA-MB-453 cells (3.3 × 103
cells/mL) suspended in medium A were put into wells of 24-
well multidishes (NUNC) in the amount of 0.75 mL/well. The
subsequent procedures were carried out in the same manner
as described above.
Estr ogen Recep tor Bin d in g Assa y. MCF-7 cells (2.5 ×
105/well) were preincubated in 24-well multidishes containing
0.75 mL of medium A in each well at 37 °C for 24 h. The cells
were then treated with [3H]17â-estradiol (Amersham; final
concentration, 10-2 µM) with various concentrations of test
compounds or 17â-estradiol and incubated at 37 °C for 1 h.
The cells were treated with PBS containing 10% (v/v) glycerol
and 0.5% (w/v) bovine serum albumin at 4 °C for 0.5 h and
washed twice with the above buffer solution. The cells were
lysed with 1 N NaOH at 37 °C overnight, and their radioactiv-
ity was measured with a liquid scintillation counter. Non-
specific binding was calculated using 20 µM 17â-estradiol as
a competing ligand.
1
Su p p or tin g In for m a tion Ava ila ble: Preparation and H
1
NMR data of compounds 8b,g,j-p , 14b, and 17 and H NMR
spectra of target compounds 10g,o, 13, 16a ,b, and 19 (9 pages).
Ordering information is given on any current masthead page.
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