Design and synthesis of potent antitumor 5,4'-diaminoflavone derivatives based on metabolic considerations

Article abstract of DOI:10.1021/jm9700326

Recently, we reported that 5,4'-diaminoflavone (1) exhibits potent and specific growth-inhibitory activity against the estrogen receptor (ER)- positive human breast cancer cell line MCF-7. However, when compound 1 was incubated with S-9 mix, its metabolit

Full text of DOI:10.1021/jm9700326

1894
J . Med. Chem. 1997, 40, 1894-1900
Design a n d Syn th esis of P oten t An titu m or 5,4′-Dia m in ofla von e Der iva tives
Ba sed on Meta bolic Con sid er a tion s
Tsutomu Akama,*,† Hiroyuki Ishida, Yasushi Shida, Uichiro Kimura, Katsushige Gomi,‡ Hiromitsu Saito,
Eiichi Fuse, Satoshi Kobayashi, Nobuyuki Yoda, and Masaji Kasai*,§
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun,
Shizuoka-ken 411, J apan
Received J anuary 15, 1997X
Recently, we reported that 5,4′-diaminoflavone (1) exhibits potent and specific growth-inhibitory
activity against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7.
However, when compound 1 was incubated with S-9 mix, its metabolites were observed.
Moreover, addition of S-9 mix to the medium caused the drastic decrease in activity of compound
1. Since the 6-, 8-, and 3′-positions were considered to be metabolized oxidatively in vivo from
MO calculations, a series of 5,4′-diaminoflavone derivatives substituted at such putative
metabolic positions with various functional groups were synthesized aiming at the metabolically
stable derivatives. Among them, 5,4′-diamino-6,8,3′-trifluoroflavone (14d ) exhibited strong
growth-inhibitory activity against MCF-7 cells even in the presence of S-9 mix. Moreover,
orally administered compound 14d completely suppressed the growth of MCF-7 inoculated
into nude mice, and the effect was more potent than that of compound 1. In addition to ER-
positive breast cancer cells, compound 14d exhibited growth-inhibitory activity against a panel
of human cancer cell lines including a part of ER-negative breast, endometrial, ovarian, and
liver cancers. From these results, fluorine introduction to the putative metabolic positions of
compound 1 was elucidated to be effective in the enhancement of the in vivo antitumor activity,
probably due to the block of the metabolic deactivation.
Recently, we reported that 5,4′-diaminoflavone (1) and
some of its congeners exhibit potent and specific growth-
inhibitory activity against the estrogen receptor (ER)-
positive human breast cancer cell line MCF-7.1 Since
they are expected to be a new type of chemotherapeutic
agent, we were then interested in their in vivo antitu-
mor activity. However, flavonoids are liable to be
metabolized oxidatively in vivo.2,3 In particular, the two
benzene rings of compound 1 seemed to be suitable
targets of electrophilic oxidation because of the electron-
donating two amino groups. S-9 mix, the 9000 G
supernatant of the enzyme-induced rat liver homoge-
nate, is often used for the model system of metabolic
activation, e.g., the Ames test.4 When compound 1 was
incubated with S-9 mix, formation of metabolites with
a 16 mass increase was observed by LC-MS analysis.5
Moreover, when S-9 mix was added to the medium, the
drastic decrease in activity of compound 1 against
MCF-7 cells was observed. Thus, it was suggested that
the compound might be metabolically deactivated when
administered in vivo. In order to estimate the position-
(s) that is (are) most susceptible to being metabolized,
the superdelocalizability (Sr)6 values of compound 1
were calculated. From the point of view of drug design,
we hypothesized that introduction of substituents would
be effective in blocking the metabolic deactivation.
Especially, the fluorine atom was considered to be one
of the most suitable substituents for the block of
metabolism.7,8 Therefore, we introduced a fluorine atom
or some other substituent to compound 1 in order to
block the presumed metabolism, retaining potent anti-
tumor activity.
As a result, we found that 5,4′-diamino-6,8,3′-trifluo-
roflavone (14d ) exhibited superior antitumor activity
against MCF-7 in vivo as well as in vitro. To confirm
the specificity of the growth-inhibitory activity of com-
pound 14d , the in vitro antitumor spectrum of 14d was
examined. Compound 14d exhibited growth-inhibitory
activity against not only ER-positive breast cancer cells
but also certain ER-negative breast, endometrial, ova-
rian, and liver cancer cell lines. Herein we describe the
synthesis of 6- or 8-substituted, 6,8-disubstituted, and
polyfluoro-substituted 5,4′-diaminoflavone derivatives
and their antitumor activity both in vitro and in vivo.
Ch em istr y
The AM19 calculation of the Sr values of compound 1
concerning electrophilic reaction was performed using
MOPAC version 6.01.10 The Sr value is known as a
parameter of the reactivity of each position in the
compound.6 From the results, we expected that the
metabolism would occur mainly at the 6- or 8-position
and that the 3′-position would be the next candidate.11
To confirm a suitable substituent for the block of
metabolism, various 6- or 8-substituted derivatives of
compound 1 were synthesized. 6-Chloro (2a) and 8-chlo-
ro (2b) derivatives were obtained by direct chlorination
of compound 1 with N-chlorosuccinimide (Scheme 1).
The chlorinated position was determined by NMR
analysis. At first, the detection of the NOE between
5-NH2 and 6-H had failed. We successfully assigned all
carbons and hydrogens by HMBC, HSQC, and, finally,
deuterium shift effect, i.e., the detection of the changes
in peak shapes of 4a-, 5-, and 6-C in the complete
† Present address: Tokyo Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 6-6 Asahi-machi 3-chome, Machida-shi, Tokyo 194,
J apan.
‡ Present address: Kyowa Hakko Kogyo Co., Ltd., 6-1 Ohtemachi
1-chome, Chiyoda-ku, Tokyo 100, J apan.
§ Present address: Sakai Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 1-53 Takasu-cho 1-chome, Sakai-shi, Osaka 590,
J apan.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(97)00032-0 CCC: $14.00 © 1997 American Chemical Society

Potent Antitumor 5,4′-Diaminoflavone Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1895
Sch em e 1a
Sch em e 3a
a
(a) N-Chlorosuccinimide, 1,4-dioxane, reflux (for 2a ,b) or Cl2,
AcOH, rt (for 2c) or Br2, AcOH, rt (for 2d ).
Sch em e 2a
a
(a) ClCO2Et, Et3N, CH2Cl2, rt; (b) fuming HNO3, H2SO4, rt;
(c) H2, Pd/C, EtOAc, rt; (d) pivaloyl chloride, pyridine, rt; (e) aq
NaOH, EtOH, rt; (f) Br2, (CH2Cl)2, 0 °C; (g) chloromethyl methyl
ether, i-Pr2NEt, CH2Cl2, 0 °C; (h) n-BuLi, ClCO2Et, THF, -78 °C;
(i) NaH, toluene, 1,4-dioxane, reflux; (j) HCl, EtOH, rt; (k) HCl,
EtOH, reflux.
lithium to afford 8. Compound 9 was obtained from 8
and 4b in a similar manner as shown in Scheme 2.
Fluorinated derivatives were synthesized as shown
in Scheme 4. Compound 1012 was hydrolyzed with HCl
to afford compound 11. Fluorination of the compound
11 with N-fluoro-3,5-dichloropyridinium triflate14 in 1,2-
dichloroethane afforded monofluoro- and difluorophenol
derivatives 12a -c. Each exchangeable OH and NH
proton of compounds 12a -c was protected as a MOM
ether and a pivaloyl amide, respectively. Compounds
13a -c thus obtained led to target compounds 14a -e
via the condensation with acetophenones 4a -c15,16 in
the same way as shown in Scheme 3.
a
(a) NaH, 1,4-dioxane, reflux; (b) HCl, EtOH, rt; (c) HCl, EtOH,
reflux; (d) BBr3, CH2Cl2, -78 °C to rt.
decoupled 13C-NMR spectrum by the addition of CD3OH
and CD3OD in DMSO-d6. When chlorine or bromine
was used in acetic acid, 6,8-dichloro derivative 2c or 6,8-
dibromo derivative 2d was obtained as a major product,
respectively.
8-Methyl (5a ), 8-methoxy (5b), and 8-hydroxy (5c)
derivatives were synthesized as shown in Scheme 2 in
a similar manner as the synthesis of compound 1.1 The
starting materials 3a ,b were prepared according to the
synthesis of compound 1012 from 5-amino-2-methylphe-
nol and 5-amino-2-methoxyphenol, respectively. The
condensation of compounds 3a ,b and acetophenones
4a ,b13 afforded 1,3-diketones. The N-ethoxycarbonyl
group of compound 3 was removed during the reaction
because of the strongly basic conditions. The 1,3-
diketones thus obtained were treated with HCl in EtOH
at room temperature to afford cyclized products. Com-
pounds 5a ,b were obtained after the removal of the
N-pivaloyl groups under more strongly acidic conditions.
Compound 5c was obtained by the demethylation of 5b
using boron tribromide in dichloromethane.
6,8-Dimethyl derivative 9 was synthesized as shown
in Scheme 3. 2,4-Dimethylphenol (6) was converted to
ethyl carbonate followed by nitration in mixed acid to
give the 5-nitro product. Catalytic hydrogenation of the
nitro group and amidation of the amino group formed
with pivaloyl chloride afforded compound 7. The ethox-
ycarbonyl group of 7 was removed by hydrolysis, and
the phenol obtained was ortho brominated. Then the
phenolic hydroxy group was reprotected as a meth-
oxymethyl (MOM) ether, and the ethoxycarbonyl group
was introduced to the position between the amide and
the ether via a halogen-metal exchange by n-butyl-
Biologica l Activity a n d Discu ssion
The growth-inhibitory activities of various 6- or
8-substituted and polyfluoro-substituted 5,4′-diaminofla-
vone derivatives in both the absence and the presence
of S-9 mix are shown in Table 1. In the absence of S-9
mix, introduction of methoxy (5b) and hydroxy (5c)
groups resulted in a drastic decrease in activity com-
pared to compound 1. It was suggested that if these
compounds were formed in vivo as metabolites of
compound 1, compound 1 would not exhibit expected
antitumor activity enough in vivo. It seems probable
because metabolites considered to be hydroxylated
products were formed in vitro by the addition of S-9
mix.5 Methylation (5a ) and 6,8-dimethylation (9) re-
sulted in a somewhat decreased activity. Chlorination
(2a ,b) and fluorination (14a ,b) seemed to retain the
activity. However, 6,8-dichlorination (2c) resulted in a
decrease in activity, despite that 6,8-difluorination (14c)
retained the activity. 6,8-Dibromination (2d ) resulted
in a further decrease in activity. Thus the most suitable
substituent was considered to be fluorine. Fluorine is
the only element which can replace hydrogen without
notable steric consequences.7 Bulkiness of the substit-
uents seemed to be important for decreasing the growth-
inhibitory activity; however, the electron-withdrawing
property did not seem to be important. Then fluorine

1896 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Akama et al.
Sch em e 4a
a
(a) HCl, EtOH, reflux; (b) N-fluoro-3,5-dichloropyridinium triflate, (CH2Cl)2, reflux; (c) chloromethyl methyl ether, i-Pr2NEt, CH2Cl2,
rt; (d) pivaloyl chloride, NaH, THF, 0 °C; (e) NaH, 1,4-dioxane, toluene, reflux; (f) HCl, EtOH, rt; (g) HCl, EtOH, reflux.
Ta ble 1. Growth-Inhibitory Activity of 5,4′-Diaminoflavone
of compounds 1 and 14d was tested. MCF-7 cells were
Derivatives against MCF-7 Cells in the Presence or Absence of
S-9 Mix
inoculated into female nude mice and were growth-
stimulated by estradiol. Compounds were administered
25 mg/kg po on 5 consecutive days in 1 week for 2 weeks
IC50 (µM)a
compd
substituents
S-9 mix (-)
S-9 mix (+)
(days 0-4 and 7-11). As shown in Figure 1 A, while
compound 1 suppressed the tumor growth from day 7
to day 18, compound 14d , as expected, completely
suppressed the tumor growth during the experiment.
It is noteworthy that the growth inhibition by compound
14d was maintained for 2 weeks after the drug admin-
istration. In addition, no significant body weight change
was observed in each group compared to the control
group during the experiment (Figure 1B).
1
2a
H
6-Cl
8-Cl
6,8-diCl
6,8-diBr
8-OMe
0.0073
0.0039
0.0098
0.053
6.3
0.21
>10
0.25
0.61
NTb
19
2b
2c
2d
5b
5c
21
5.3
8-OH
16
5a
9
8-Me, 3′-F
6,8-diMe, 3′-F
6-F
0.015
0.036
0.0044
0.0069
0.0060
0.0012
0.0040
0.18
0.11
0.13
0.17
0.046
0.025
0.089
14a
14b
14c
14d
14e
Then the in vitro antitumor spectrum of compound
14d was examined to confirm the effects against other
cell lines, and the results are shown in Table 2. In
addition to ER-positive breast cancer cell lines, com-
pound 14d exhibited growth-inhibitory activity against
one of two ER-negative breast cancer cell lines (SK-BR-
3) and endometrial, ovarian, and liver cancer cell lines
with IC50 values in the range of 10-2-10-3 µM order.
However, 14d was inactive up to 1 µM against another
ER-negative cell line (MDA-MB-453) and stomach,
colon, kidney, pancreas, and vulva cell lines.
8-F
6,8-diF
6,8,3′-triF
6,8,3′,5′-tetraF
a
MCF-7 cells were treated with each compound for 6 days and
b
counted by a microcell counter. Not tested.
was introduced to the 3′- or 3′,5′-positions, which are
the next candidates for metabolism. 6,8,3′-Trifluoro
derivative 14d and 6,8,3′,5′-tetrafluoro derivative 14e
exhibited comparable or somewhat enhanced activity to
compound 1.
The pharmacological mechanism which explains the
selectivity of antitumor activity of 14d has not, as yet,
been clear. Compound 14d did not compete with
estradiol for the ER up to 100 µM, and it did not cause
topoisomerase (I and II) dependent DNA cleavage (data
not shown). Recently, a series of 2-(4-aminophenyl)-
benzothiazoles were reported to exhibit selective growth-
inhibitory activity to breast cancer cells.17 The antitu-
mor profile of 14d seems to resemble that of these
compounds; however, 14d did not show biphasic dose-
response against MCF-7 (data not shown) compared to
these compounds which showed biphasic dose-re-
sponse. Moreover, the antitumor spectrum of 14d
seems to be somewhat different as compared to the
spectra of these compounds. 6,4′-Diaminoflavone and
6-hydroxy-5,7,3′-triaminoflavone were reported as in-
hibitors of protein-tyrosine kinases including the EGF
On the other hand, the addition of S-9 mix decreased
the growth-inhibitory activity in all cases except for
compound 5b which have already lost the potency in
the absence of the S-9 mix. The growth-inhibitory
activity of compound 1 was decreased about 30 times
by S-9 mix addition. Hence it was suggested that these
compounds might be metabolically deactivated in the
liver, and the deactivation would not be blocked by the
introduction of a substituent to at least one position.
While the degree of the deactivation (the ratio of S-9
mix (+) and (-)) was not improved, 6,8-difluoro deriva-
tives 14c-e exhibited more potent growth-inhibitory
activity than other compounds in the presence of S-9
mix. Compound 14d exhibited the most potent activity
(IC50 0.025 µM).
To confirm the effect of fluorine introduction against
metabolic deactivation, the in vivo antitumor activity

Potent Antitumor 5,4′-Diaminoflavone Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1897
binding were reported.19,20 One possible mechanism of
their growth-inhibitory activity was thought to be the
depletion of the ER or the ER-ERE complex, which
probably resulted in the inhibition of the ER responsive
gene expression. In preliminary results, compound 14d
slightly suppressed the estradiol-stimulated ER trans-
activation in the (ERE)2-luciferase reporter gene assay
(data not shown). However, even if compound 14d
possesses such activity, it is not enough to explain its
strong antitumor effect and the activities against ER-
negative cell lines.
The pharmacological mechanism is still unclear;
however, our hypothesis that metabolic deactivation of
compound 1 in vivo would be overcome by introducing
substituents such as fluorine atom to the putative
metabolic positions was supported by the in vivo experi-
ment. Although further investigation is required to
disclose the real effect of fluorine introduction, it might
be one methodology for enhancing the in vivo potency
of a drug which is susceptible to being metabolized.
Con clu sion
We prepared a series of 5,4′-diaminoflavone deriva-
tives substituted at putative metabolic positions with
various functional groups aiming at the metabolically
stable derivatives. Among them, we found that 5,4′-
diamino-6,8,3′-trifluoroflavone (14d ) exhibited strong
growth-inhibitory activity in vivo as well as in vitro.
Since the compound was considered to be a new type of
antitumor agent, further evaluation of the compound
and research on structure-activity relationships of
related derivatives are in progress.
F igu r e 1. Antitumor activity of compounds 1 and 14d against
MCF-7. Tumor fragments were implanted sc into BALB/c-nu/
nu female mice on day -20. Compounds were administered
po 5 consecutive days in 1 week for 2 weeks: (A) tumor growth
rate of the control group (0), compound 1-treated group (4),
and compound 14d -treated group (O); (B) body weight change
of each group (symbols are the same as in A). *P < 0.05 vs
control group.
Exp er im en ta l Section
All melting points were determined on a Yanako micromelt-
ing point apparatus and are uncorrected. IR spectra were
recorded on J ASCO IR-400 and J ASCO IR-810 spectrometers.
1H-NMR spectra were recorded on HITACHI R-90H (90 MHz),
J EOL J NM-GX-270 (270 MHz), and J EOL J NM-EX-270 (270
MHz) spectrometers. Electron impact mass spectra (EIMS)
were recorded on a J EOL J MS-D-300 spectrometer. 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 (NaH) was a 60% oil dispersion. THF was
freshly distilled from sodium and benzophenone.
Ta ble 2. Growth-Inhibitory Activity of Compound 14d against
Various Human Cancer Cell Lines
cell lines
type
breast
breast
breast
ER
IC50 (µM)a
MCF-7
T-47D
SK-BR-3
MD- 453
Ishikawa
A2780
OVCAR-3
Hep-G2
MKN-28
WiDr
+
+
-
-
+
-
-
0.0012
0.031
0.0025
>1.0
breast
5-Am in o-2-(4-am in oph en yl)-6-ch lor o-4H-1-ben zopyr an -
4-on e (2a ) a n d 5-Am in o-2-(4-a m in op h en yl)-8-ch lor o-4H-
1-ben zop yr a n -4-on e (2b). To a solution of compound 11 (2.00
g, 7.93 mmol) in 1,4-dioxane (50 mL) was added N-chlorosuc-
cinimide (1.06 g, 7.93 mmol). The mixture was refluxed for
19 h, and the solvent was evaporated. The mixture was
extracted with EtOAc, and the organic layer was washed with
brine. The residue was chromatographed (4:1 CHCl3/acetone)
and recrystallized from CHCl3/MeOH to afford 2a (415 mg,
18%) and 2b (266 mg, 12%). 2a : mp 242-243 °C; IR (KBr)
1642 cm-1; 1H NMR (270 MHz, DMSO-d6) δ 6.58 (s, 1H, 3-H),
6.67 (d, J ) 8.9 Hz, 2H, 3′,5′-H), 6.75 (d, J ) 8.9 Hz, 1H, 8-H),
7.54 (d, J ) 8.4 Hz, 1H, 7-H), 7.73 (d, J ) 8.9 Hz, 2H, 2′,6′-H);
EIMS m/ z 286, 288 (M+). Anal. (C15H11ClN2O2) C, H, N. 2b:
endometrial
ovarian
ovarian
liver
stomach
colon
kidney
pancreas
vulva
0.013
0.0013
0.014
0.0028
>1.0
>1.0
ACHN
PSN-1
A431
>1.0
>1.0
>1.0
a
Cells were treated with compound 14d for 6 days and counted
by a microcell counter.
receptor.18 Although the direct inhibition of such ki-
nases concerning 14d has not been tested, the inhibition
of [3H]thymidine uptake by the EGF receptor expressing
NRK-49F cells stimulated by EGF (10 ng/mL) in the
serum free medium was examined. The IC50 value of
14d was >10 µM, suggesting that the EGF dependent
pathway is not the major target of the compound.
Moreover, some compounds which exhibit growth-
inhibitory activity against MCF-7 cells without ER
mp 275-276 °C; IR (KBr) 1640 cm-1 1H NMR (270 MHz,
;
DMSO-d6) δ 6.52 (d, J ) 8.9 Hz, 1H, 6-H), 6.58 (d, J ) 8.9 Hz,
2H, 3′,5′-H), 6.68 (s, 1H, 3-H), 7.42 (d, J ) 8.9 Hz, 1H, 7-H),
7.75 (d, J ) 8.9 Hz, 2H, 2′,6′-H); EIMS m/ z 286, 288 (M+).
Anal. (C15H11ClN2O2) C, H, N.
5-Am in o-2-(4-am in oph en yl)-6,8-dich lor o-4H-1-ben zopy-
r a n -4-on e (2c). To a suspension of compound 1 (940 mg, 3.73
mmol) in AcOH (50 mL) was added a solution of chlorine in
AcOH (2.7 wt %, 24 g, 9.2 mmol). The mixture was stirred

1898 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Akama et al.
for 18 h at room temperature and poured into water. The
mixture was extracted with CHCl3/MeOH (9:1), and the
organic layer was washed with saturated aqueous NaHCO3
and brine. The residue was chromatographed (4:1 CHCl3/
acetone) and recrystallized from CHCl3/MeOH to afford 2c (218
mg, 18%): mp 255-259 °C; IR (KBr) 1624 cm-1; 1H NMR (270
MHz, DMSO-d6) δ 6.58 (s, 1H, 3-H), 6.68 (d, J ) 9.4 Hz, 2H,
3′,5′-H), 7.76 (s, 1H, 7-H), 7.76 (d, J ) 8.9 Hz, 2H, 2′,6′-H);
EIMS m/ z 320, 322, 324 (M+). Anal. (C15H10Cl2N2O2) C, H,
N.
Water was added, and the mixture was extracted with CHCl3.
The organic layer was washed twice with brine and concen-
trated. The crude product was dissolved in concentrated
H2SO4 (50 mL) on an ice bath, and fuming HNO3 (4.0 mL) was
added. The mixture was stirred for 40 min and was poured
into 500 mL of ice-water. The mixture was extracted with
EtOAc, and the organic layer was washed with water and
brine. Chromatography (9:1 n-hexane/EtOAc) gave 2,4-di-
methyl-O-(ethoxycarbonyl)-5-nitrophenol (12.8 g, two steps,
36%).
5-Am in o-2-(4-am in oph en yl)-6,8-dibr om o-4H-1-ben zopy-
r a n -4-on e (2d ). To a suspension of compound 1 (1.46 g, 5.80
mmol) in AcOH (100 mL) was added bromine (0.45 mL, 18
mmol). The mixture was stirred for 18 h at room temperature.
The precipitate was collected by filtration and triturated with
MeOH to afford 2d as a hydrobromide (1.23 g, 43%): mp 240
The obtained nitrophenol (12.2 g, 51.0 mmol) and 10%
palladium on charcoal (1.22 g) in EtOAc (150 mL) were stirred
under hydrogen atmosphere for 1 day at room temperature.
Hydrogen was replaced with nitrogen, and the mixture was
filtered through a Celite pad. The solvent was evaporated,
and the residue was dissolved in pyridine (50 mL) on an ice
bath. Pivaloyl chloride (5.2 mL, 42 mmol) was added, and the
mixture was stirred for 1 h and poured into water. The
mixture was extracted with EtOAc, and the organic layer was
washed with 2 N HCl, H2O, and brine. Recrystallization from
n-hexane gave 7 (10.0 g, two steps, 67%): mp 82-83 °C; IR
1
°C dec; IR (KBr) 1621 cm-1; H NMR (270 MHz, DMSO-d6) δ
6.68 (s, 1H, 3-H), 6.69 (d, J ) 8.8 Hz, 2H, 3′,5′-H), 7.79 (d, J
) 8.6 Hz, 2H, 2′,6′-H), 7.97 (s, 1H, 7-H); EIMS m/ z 408, 410,
412 (M+). Anal. (C15H10Br2N2O2‚0.6HBr) C, H, N.
5-Am in o-2-(4-am in o-3-flu or oph en yl)-8-m eth yl-4H-1-ben -
zop yr a n -4-on e (5a ). To a refluxing suspension of NaH (800
mg, 20.0 mmol) in 1,4-dioxane/toluene (1:1, 10 mL) under
argon atmosphere was added dropwise a solution of 3a (2.56
g, 7.05 mmol) and 4b (1.39 g, 5.88 mmol) in the above solvent
(35 mL) over 5 min. The reaction mixture was refluxed for 2
h and cooled on an ice bath. Water was added, and the basic
solution was washed with n-hexane. Then the solution was
extracted with EtOAc, and the organic layer was washed with
brine. The combined extracts were concentrated and dissolved
in EtOH (40 mL). Concentrated HCl (10 mL) was added, and
the reaction mixture was stirred for 16 h at room temperature.
Water (80 mL) was added, and the precipitated product was
collected by filtration to afford 2-[3-fluoro-4-(pivaloylamino)-
phenyl]-8-methyl-5-(pivaloylamino)-4H-1-benzopyran-4-one (2.03
g, 76%).
To a solution of the compound obtained (1.50 g, 3.32 mmol)
in 1,4-dioxane (40 mL) was added concentrated HCl (20 mL),
and the reaction mixture was refluxed for 4 h and cooled on
an ice bath. The mixture was neutralized with 10 N NaOH,
and the precipitated product was collected by filtration.
Chromatography (40:1 CHCl3/MeOH) followed by trituration
with i-Pr2O/EtOAc afforded 5a (528 mg, 58%): mp 249-250
°C; IR (KBr) 1637 cm-1; 1H NMR (270 MHz, DMSO-d6) δ 2.31
(s, 3H, CH3), 5.98 (br, 2H, 4′-NH2), 6.44 (d, J ) 8.3 Hz, 1H,
6-H), 6.60 (s, 1H, 3-H), 6.87 (t, J ) 8.6 Hz, 1H, 5′-H), 7.19 (br,
2H, 5-NH2), 7.21 (d, J ) 8.3 Hz, 1H, 7-H), 7.5-7.7 (m, 2H,
2′,6′-H); EIMS m/ z 284 (M+). Anal. (C16H13FN2O2) C, H, N.
5-Am in o-2-(4-a m in op h en yl)-8-m eth oxy-4H-1-ben zop y-
r a n -4-on e (5b). This compound was obtained from 3b and
4a in a similar manner as described for 5a (overall 22%): mp
216-217 °C; IR (KBr) 1641 cm-1; 1H NMR (270 MHz, DMSO-
d6) δ 3.84 (s, 3H, OCH3), 5.92 (br, 2H, 4′-NH2), 6.43 (d, J ) 8.8
Hz, 1H, 6-H), 6.50 (s, 1H, 3-H), 6.68 (d, J ) 8.8 Hz, 2H, 3′,5′-
H), 6.89 (br, 2H, 5-NH2), 7.18 (d, J ) 8.8 Hz, 1H, 7-H), 7.71
(d, J ) 8.8 Hz, 2H, 2′,6′-H); EIMS m/ z 282 (M+). Anal.
(C16H14N2O3) C, H, N.
5-Am in o-2-(4-a m in op h en yl)-8-h yd r oxy-4H-1-ben zop y-
r a n -4-on e (5c). To a suspension of 5b (286 mg, 1.01 mmol)
in CH2Cl2 (25 mL) was added BBr3 (1.0 M in CH2Cl2, 5.6 mL,
5.6 mmol) at -78 °C under argon atmosphere. The cold bath
was removed, and the mixture was stirred for 9.5 h. Water
was added, and the aqueous layer was neutralized with 2 N
NaOH. Insoluble precipitate was collected by filtration and
recrystallized from EtOH/n-hexane to afford 5c (166 mg,
61%): mp 250 °C dec; IR (KBr) 3330, 1641 cm-1; 1H NMR (270
MHz, DMSO-d6) δ 5.88 (br, 2H, 4′-NH2), 6.35 (d, J ) 8.6 Hz,
1H, 6-H), 6.45 (s, 1H, 3-H), 6.66 (d, J ) 8.8 Hz, 2H, 3′,5′-H),
6.71 (br, 2H, 5-NH2), 6.99 (d, J ) 8.8 Hz, 1H, 7-H), 7.78 (d, J
) 8.8 Hz, 2H, 2′,6′-H), 8.72 (br, 1H, OH); EIMS m/ z 268 (M+).
Anal. (C15H12N2O3‚0.3H2O) C, H; N: calcd, 10.24; found, 9.81.
2,4-Dim et h yl-O-(et h oxyca r b on yl)-5-(p iva loyla m in o)-
p h en ol (7). To a solution of 2,4-dimethylphenol (17.8 mL, 150
mmol) were added Et3N (25.0 mL, 180 mmol) and a solution
of ethyl chloroformate (17.2 mL, 180 mmol) in CH2Cl2 (20 mL),
and the mixture was stirred for 15 min at room temperature.
1
(KBr) 1760, 1650 cm-1; H NMR (90 MHz, CDCl3) δ 1.32 (s,
9H, C(CH3)3), 1.37 (t, J ) 7.0 Hz, 3H, CH2CH3), 2.16 (s, 3H,
CH3), 2.19 (s, 3H, CH3), 4.29 (q, J ) 7.0 Hz, 2H, CH2), 7.01 (s,
1H, 3-H), 7.20 (br, 1H, NH), 7.79 (s, 1H, 6-H); EIMS m/ z 293
(M+). Anal. (C16H23NO4) C, H, N.
E t h yl 3,5-Dim et h yl-2-(m et h oxym et h oxy)-6-(p iva loy-
la m in o)ben zoa te (8). To a solution of 7 (6.28 g, 21.4 mmol)
in EtOH (30 mL) was added 10 N NaOH (6 mL, 60 mmol) at
0 °C. The mixture was stirred for 30 min and acidified with
2 N HCl. The precipitated product was collected by filtration.
To the solution of the crude product in dichloroethane (100
mL) was added dropwise bromine (1.0 mL, 18 mmol) at 0 °C.
The mixture was stirred for 30 min and poured into water.
The mixture was extracted with CHCl3, and the organic layer
was washed with brine. Chromatography (3:1 n-hexane/
EtOAc) gave 2-bromo-4,6-dimethyl-3-(pivaloylamino)phenol
(4.09 g, two steps, 64%): 1H NMR (90 MHz, CDCl3) δ 1.36 (s,
9H, C(CH3)3), 2.13 (s, 3H, CH3), 2.24 (s, 3H, CH3), 5.48 (br,
1H, OH), 6.93 (s, 1H, 5-H), 6.98 (br, 1H, NH); EIMS m/ z 299,
301 (M+).
To a solution of the bromophenol (3.98 g, 13.3 mmol) in
CH2Cl2 (100 mL) were added diisopropylethylamine (2.7 mL,
16 mmol) and chloromethyl methyl ether (1.2 mL, 16 mmol)
at 0 °C. The mixture was stirred for 3 h, and 1 N NaOH was
added. The mixture was extracted with CHCl3, and the
organic layer was washed with water and brine. Trituration
with n-hexane gave a MOM ether (4.09 g, 89%): 1H NMR (90
MHz, CDCl3) δ 1.36 (s, 9H, C(CH3)3), 2.17 (s, 3H, ArCH3), 2.32
(s, 3H, ArCH3), 3.62 (s, 3H, OCH3), 5.03 (s, 2H, OCH2O), 7.00
(s, 1H, 5-H), 7.05 (br, 1H, NH); EIMS m/ z 343, 345 (M+).
To a solution of n-butyllithium (1.6 M in n-hexane, 16 mL,
25 mmol) in THF (20 mL) were added a solution of the MOM
ether (3.78 g, 11.0 mmol) in THF (50 mL) and ethyl chloro-
formate (2.1 mL, 22 mmol) under argon atmosphere below -60
°C. The mixture was stirred for 10 min, and water was added.
The mixture was extracted with EtOAc, and the organic layer
was washed with brine. Chromatography (4:1 n-hexane/
acetone) gave 8 (2.32 g, 63%): mp 85-86 °C; IR (KBr) 1715,
1650 cm-1; 1H NMR (90 MHz, CDCl3) δ 1.28 (s, 9H, C(CH3)3),
1.37 (t, J ) 7.0 Hz, 3H, CH2CH3), 2.14 (s, 3H, ArCH3), 2.28 (s,
3H, ArCH3), 3.53 (s, 3H, OCH3), 4.34 (q, J ) 7.0 Hz, 2H, CH2-
CH3), 4.96 (s, 2H, OCH2O), 7.12 (s, 1H, 4-H), 7.73 (br, 1H, NH);
EIMS m/ z 337 (M+). Anal. (C18H27NO5) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d im eth yl-4H-
1-ben zop yr a n -4-on e (9). This compound was obtained from
8 and 4b in a similar manner as described for 5a (overall
54%): mp 222-223 °C; IR (KBr) 1647 cm-1 1H NMR (270
;
MHz, DMSO-d6) δ 2.05 (s, 3H, CH3), 2.29 (s, 3H, CH3), 5.98
(br, 2H, 4′-NH2), 6.60 (s, 1H, 3-H), 6.88 (t, J ) 8.6 Hz, 1H,
3′-H), 7.07 (br, 2H, 5-NH2), 7.17 (s, 1H, 7-H), 7.5-7.7 (m, 2H,
2′,6′-H); EIMS m/ z 298 (M+). Anal. (C17H15N2O2‚0.2H2O) C,
H, N.
E t h yl 2-[N-(E t h oxyca r b on yl)a m in o]-6-h yd r oxyb en -
zoa te (11). To a solution of 1010 (200 g, 475 mmol) in EtOH
(900 mL) was added concentrated HCl (300 mL). The mixture

Potent Antitumor 5,4′-Diaminoflavone Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1899
was refluxed for 3.5 h and cooled on an ice bath. Water (500
mL) was added, and the precipitated product was collected by
filtration to afford 11 (89.1 g, 74%): mp 76-77 °C; IR (KBr)
3380, 1735, 1655 cm-1; 1H NMR (90 MHz, CDCl3) δ 1.31 (t, J
) 7.0 Hz, 3H, CH2CH3), 1.50 (t, J ) 7.0 Hz, 3H, CH2CH3),
4.22 (q, J ) 7.0 Hz, 2H, CH2CH3), 4.53 (q, J ) 7.0 Hz, 2H,
CH2CH3), 6.65 (dd, J ) 8.2, 1.2 Hz, 1H, Ar-H), 7.37 (t, J ) 8.4
Hz, 1H, 4-H), 7.86 (dd, J ) 8.4, 1.1 Hz, 1H, Ar-H), 9.48 (br,
1H, NH), 10.7 (s, 1H, OH); EIMS m/ z 253 (M+).
in a similar manner as described for 5a (overall 50%): mp
247-249 °C; IR (KBr) 1641 cm-1; 1H NMR (270 MHz, DMSO-
d6) δ 5.97 (br, 2H, 4′-NH2), 6.51 (s, 1H, 3-H), 6.65 (d, J ) 8.9
Hz, 2H, 3′,5′-H), 6.67 (dd, J ) 9.2, 3.7 Hz, 1H, 8-H), 7.25 (br,
2H, 5-NH2), 7.37 (dd, J ) 11.4, 9.4 Hz, 1H, 7-H), 7.72 (d, J )
8.4 Hz, 2H, 2′,6′-H); EIMS m/ z 270 (M+). Anal. (C15H11FN2O2)
C, N; H: calcd, 4.10; found, 3.64.
5-Am in o-2-(4-a m in op h en yl)-8-flu or o-4H-1-ben zop yr a n -
4-on e (14b). This compound was obtained from 12b and 4a
in a similar manner as described for 5a (overall 66%): mp
248-250 °C; IR (KBr) 1619 cm-1; 1H NMR (270 MHz, DMSO-
d6) δ 6.00 (br, 2H, 4′-NH2), 6.43 (d, J ) 8.9 Hz, 2H, 3′,5′-H),
6.54 (s, 1H, 3-H), 6.67 (dd, J ) 8.9, 3.5 Hz, 1H, 6-H), 7.19 (br,
2H, 5-NH2), 7.34 (t, J ) 9.4 Hz, 1H, 7-H), 7.69 (d, J ) 8.4 Hz,
2H, 2′,6′-H); EIMS m/ z 270 (M+). Anal. (C15H11FN2O2) C, H,
N.
Eth yl 2-[N-(Eth oxycar bon yl)am in o]-3-flu or o-6-h ydr oxy-
ben zoa te (12a ), Eth yl 6-[N-(Eth oxyca r bon yl)a m in o]-3-
flu or o-2-h yd r oxyben zoa te (12b), a n d Eth yl 2-[N-(Eth oxy-
ca r bon yl)a m in o]-3,5-d iflu or o-6-h yd r oxyben zoa te (12c).
A mixture of 11 (12.7 g, 50.0 mmol) and N-fluoro-3,5-dichlo-
ropyridinium triflate (31.6 g, 100 mmol) in CH2Cl2 (100 mL)
was refluxed for 22 h. Et2O (200 mL) was added to remove
the pyridinium salt as the precipitate, and the mixture was
filtered. The filtrate was washed with 1 N HCl, water, and
brine. Chromatography (6:1-5:1 n-hexane/EtOAc) gave 12a
(5.70 g, 42%), 12b (1.29 g, 9.5%), and 12c (1.76 g, 12%). 12a :
5-Am in o-2-(4-a m in oph en yl)-6,8-d iflu or o-4H-1-ben zop y-
r a n -4-on e (14c). This compound was obtained from 12c and
4a in a similar manner as described for 5a except that the
final product was isolated as a hydrochloride by filtration of
the precipitate from the cooled reaction mixture (overall
mp 110-112 °C; IR (KBr) 3280, 1745, 1710 cm-1 1H NMR
;
17%): mp 242-244 °C; IR (KBr) 1653 cm-1 1H NMR (270
;
(90 MHz, CDCl3) δ 1.30 (t, J ) 7.0 Hz, 3H, CH2CH3), 1.42 (t,
J ) 7.0 Hz, 3H, CH2CH3), 4.21 (q, J ) 7.0 Hz, 2H, CH2CH3),
4.44 (q, J ) 7.0 Hz, 2H, CH2CH3), 6.80 (dd, J ) 9.2, 4.4 Hz,
1H, 5-H), 7.07 (br, 1H, NH), 7.22 (t, J ) 9.2 Hz, 1H, 4-H), 10.4
(s, 1H, OH); EIMS m/ z 271 (M+). 12b: mp 97-99 °C; IR (KBr)
3400, 1730, 1660 cm-1; 1H NMR (90 MHz, CDCl3) δ 1.31 (t, J
) 7.0 Hz, 3H, CH2CH3), 1.51 (t, J ) 7.0 Hz, 3H, CH2CH3),
4.22 (q, J ) 7.0 Hz, 2H, CH2CH3), 4.56 (q, J ) 7.0 Hz, 2H,
CH2CH3), 7.22 (t, J ) 9.2 Hz, 1H, 4-H), 7.79 (dd, J ) 9.2, 4.4
Hz, 1H, 5-H), 9.22 (br, 1H, NH), 10.4 (s, 1H, OH); EIMS m/ z
271 (M+). 12c: mp 146-148 °C; IR (KBr) 3270, 1705, 1690
MHz, DMSO-d6) δ 6.58 (s, 1H, 3-H), 6.68 (d, J ) 8.9 Hz, 2H,
3′,5′-H), 7.67 (t, J ) 10.9 Hz, 1H, 7-H), 7.71 (d, J ) 8.9 Hz,
2H, 2′,6′-H); EIMS m/ z 288 (M+). Anal. (C15H10F2N2O2‚
HCl‚0.7H2O) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-4H-1-
ben zop yr a n -4-on e (14d ). This compound was obtained from
12c and 4b in a similar manner as described for 5a (overall
35%): mp 228-229 °C; IR (KBr) 1653 cm-1 1H NMR (270
;
MHz, DMSO-d6) δ 6.10 (br, 2H, 4′-NH2), 6.69 (s, 1H, 3-H), 6.68
(t, J ) 8.7 Hz, 1H, 5′-H), 7.05 (br, 2H, 5-NH2), 7.59 (dd, J )
8.4, 2.0 Hz, 1H, 6′-H), 7.65 (dd, J ) 12.9, 2.0 Hz, 1H, 2′-H),
7.69 (t, J ) 11.1 Hz, 1H, 7-H); EIMS m/ z 306 (M+). Anal.
(C15H9F3N2O2) C, H, N.
cm-1 1H NMR (90 MHz, CDCl3) δ 1.29 (t, J ) 7.0 Hz, 3H,
;
CH2CH3), 1.43 (t, J ) 7.1 Hz, 3H, CH2CH3), 4.21 (q, J ) 7.2
Hz, 2H, CH2CH3), 4.47 (q, J ) 7.1 Hz, 2H, CH2CH3), 6.83 (br,
1H, NH), 7.13 (t, J ) 9.9 Hz, 1H, 4-H), 10.5 (s, 1H, OH); EIMS
m/ z 289 (M+).
5-Am in o-2-(4-a m in o-3,5-d iflu or op h en yl)-6,8-d iflu or o-
4H-1-ben zop yr a n -4-on e (14e). This compound was obtained
from 12c and 4c in a similar manner as described for 5a
Typ ica l P r oced u r e for th e P r ep a r a tion of Com p ou n d
13: Eth yl 3,5-Diflu or o-2-[N-(eth oxyca r bon yl)-N-p iva loyl-
a m in o]-6-(m eth oxym eth oxy)ben zoa te (13c). To a solution
of 12c (5.72 g, 19.8 mmol) in CH2Cl2 (70 mL) were added
diisopropylethylamine (4.1 mL, 24 mmol) and chloromethyl
methyl ether (1.8 mL, 24 mmol) at 0 °C. The mixture was
stirred for 20 min, and 1 N NaOH was added. The mixture
was extracted with Et2O, and the organic layer was washed
with water and brine. To a solution of the crude product in
THF (35 mL) were added NaH (792 mg, 19.8 mmol) and
pivaloyl chloride (1.7 mL, 20 mmol) at 0 °C. The mixture was
stirred for 1.5 h, and saturated aqueous NH4Cl was added.
The mixture was extracted with ether, and the organic layer
was washed with brine. Chromatography (4:1 n-hexane/
EtOAc) gave 13c (6.63 g, two steps, 80%): 1H NMR (90 MHz,
CDCl3) δ 1.20 (t, J ) 7.0 Hz, 3H, CH2CH3), 1.33 (t, J ) 7.1
Hz, 3H, CH2CH3), 1.39 (s, 9H, C(CH3)3), 3.55 (s, 3H, OCH3),
4.18 (q, J ) 7.2 Hz, 2H, CH2CH3), 4.33 (q, J ) 7.1 Hz, 2H,
CH2CH3), 5.12 (s, 2H, OCH2O), 7.00 (dd, J ) 10.2, 9.1 Hz, 1H,
4-H); EIMS m/ z 417 (M+).
1
(overall 14%): mp 230 °C dec; IR (KBr) 1637 cm-1; H NMR
(270 MHz, DMSO-d6) δ 6.17 (br, 2H, 4′-NH2), 6.80 (s, 1H, 3-H),
7.06 (br, 2H, 5-NH2), 7.60 (dd, J ) 7.7, 2.7 Hz, 2H, 2′,6′-H),
7.71 (t, J ) 11.1 Hz, 1H, 7-H); EIMS m/ z 324 (M+). Anal.
(C15H8F4N2O2) C, H, N.
Cell Gr ow th -In h ibitor y Activity. Assays were conducted
according to the published method.1 T-47D (3 × 103 cells/well),
OVCAR-3 (3 × 103 cells/well), MKN-28 (1.5 × 103 cells/well),
and PSN-1 (1.5 × 103 cells/well) cells were cultured in RPMI
1640 medium supplemented with 10% fetal bovine serum
(FBS; GIBCO, NY); SK-BR-3 (1 × 104 cells/well) cells were
cultured in McCoy’s 5a medium supplemented with 10% FBS;
Ishikawa (3 × 103 cells/well), Hep-G2 (5 × 103 cells/well), WiDr
(2.5 × 103 cells/well), and ACHN (1.5 × 103 cells/well) cells
were cultured in modified Eagle’s medium supplemented with
10% FBS; A2780 (1 × 103 cells/well) cells were cultured in
RPMI 1640 medium supplemented with 5% FBS; and A431
(1.5 × 103 cells/well) cells were cultured in Dulbecco’s modified
Eagle’s medium supplemented with 10% FBS.
Eth yl 3-F lu or o-2-[N-(eth oxyca r bon yl)-N-p iva loyla m i-
n o]-6-(m eth oxym eth oxy)ben zoa te (13a ). This compound
was obtained from 12a in a similar manner as described for
13c (two steps, 82%): 1H NMR (90 MHz, CDCl3) δ 1.20 (t, J )
7.0 Hz, 3H, CH2CH3), 1.32 (t, J ) 7.2 Hz, 3H, CH2CH3), 1.39
(s, 9H, C(CH3)3), 3.48 (s, 3H, OCH3), 4.19 (q, J ) 7.0 Hz, 2H,
CH2CH3), 4.33 (q, J ) 7.2 Hz, 2H, CH2CH3), 5.14 (s, 2H,
OCH2O), 7.0-7.3 (m, 2H, Ar-H); EIMS m/ z 399 (M+).
Eth yl 3-F lu or o-6-[N-(eth oxyca r bon yl)-N-p iva loyla m i-
n o]-2-(m eth oxym eth oxy)ben zoa te (13b). This compound
was obtained from 12b in a similar manner as described for
13c (two steps, 95%): 1H NMR (90 MHz, CDCl3) δ 1.19 (t, J )
7.0 Hz, 3H, CH2CH3), 1.35 (t, J ) 7.0 Hz, 3H, CH2CH3), 1.35
(s, 9H, C(CH3)3), 3.55 (s, 3H, OCH3), 4.17 (q, J ) 7.0 Hz, 2H,
CH2CH3), 4.32 (q, J ) 7.0 Hz, 2H, CH2CH3), 5.17 (s, 2H,
OCH2O), 6.85 (dd, J ) 8.8, 4.4 Hz, 1H, 5-H), 7.16 (dd, J )
10.3, 9.0 Hz, 1H, 4-H); EIMS m/ z 399 (M+).
S-9 Mix Tr ea tm en t. A 30% S-9 mix solution was prepared
from S-9 (0.3 mL, Oriental yeast; Tokyo, J apan), glucose
6-phosphate (1.3 mg), â-NADPH (3.6 mg), 20 mM HEPES
buffer (0.2 mL), 50 mM MgCl2 (0.1 mL), 330 mM KCl (0.1 mL),
and distilled water (0.3 mL). The solution was added to the
medium at a concentration of 0.125%. No influence against
the cell growth was observed under this condition.
In Vivo An titu m or Activity. Tumor fragments (8 mm3)
were transplanted subcutaneously in the flank of 7-9 weeks
of age female BALB/c-nu/nu mice (Nihon Crea, Tokyo, J apan).
For promoting the growth of the tumor, 12.5 µg of estradiol
propionate was intramuscularly administered in the femora
on the date of transplantation and 2 weeks after; 20 days after
transplantation, mice with a tumor volume of 25-200 mm3
were selected, and the test compounds were orally adminis-
tered on 5 consecutive days in 1 week for 2 weeks (n ) 5).
Estradiol propionate was administered on the day of initial
administration of test compounds. Length and width of the
tumor were determined on days 0, 4, 7, 11, 14, 18, 21, and 25,
5-Am in o-2-(4-a m in op h en yl)-6-flu or o-4H-1-ben zop yr a n -
4-on e (14a ). This compound was obtained from 12a and 4a

1900 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Akama et al.
(9) Dewar, M. J . S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J . J . P.
AM1: A New General Purpose Quantum Mechanical Molecular
Mode. J . Am. Chem. Soc. 1985, 107, 3902-3909.
and the tumor volumes were calculated according to the
following equation:
(10) (a) Stewart, J . J . P. MOPAC: A General Molecular Orbital
Package (Ver. 6.0). QCPE Bull. 1990, 10, 86-87. (b) Hirano, T.
MOPAC Version 6: Revised as Version 6.01 by Hirano for UNIX
Machines. J CPE Newslett. 1991, 3, 28-29.
(11) Actually, the position 3 seemed to be the most reactive to the
electrophilic reaction. However, in the case of flavonoids, hy-
droxylation of an aromatic ring is often observed in vivo.2,3
(12) Sugaya, T.; Mimura, Y.; Kato, N.; Ikuta, M.; Mimura, T.; Kasai,
M.; Tomioka, S. Synthesis of 6H-Pyrazolo[4,5,1-de]acridin-6-one
Derivative: A Useful Intermediate of Antitumor Agents. Syn-
thesis 1994, 73-76.
(13) Compound 4b was prepared from 4-bromo-2-fluoroaniline as
follows: (a) pivaloyl chloride, pyridine, room temperature (99%);
(b) n-butyllithium, MeCONMe2, THF, -78 °C (66%).
(14) Umemoto, T.; Fukami, S.; Tomizawa, G.; Harasawa, K.; Kawada,
K.; Tomita, K. Power and Structure-Variable Fluorinating
Agents. The N-Fluoropyridinium Salt System. J . Am. Chem. Soc.
1990, 112, 8563-8575.
(15) Compound 4c was prepared by the amidation of 4′-amino-3′,5′-
fluoroacetophenone16 with pivaloyl chloride in pyridine (72%).
(16) Asato, G.; Baker, P. K.; Bass, R. T.; Bentley, T. J .; Chari, S.;
Dalrymple, R. H.; France, D. J .; Gingher, P. E.; Lences, B. L.;
Pascavage, J . J .; Pensack, J . M.; Ricks, C. A. Repartitioning
Agents: 5-[1-Hydroxy-2-(isopropylamino)ethyl]anthranilonitrile
and Related Phenethanolamines; Agents for Promoting Growth,
Increasing Muscle Accretion and Reducing Fat Deposition in
Meat-producing Animals. Agric. Biol. Chem. 1984, 48, 2883-
2888.
(17) Shi, D.-F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J .; Lelieveld,
P.; Fichtner, I.; Stevens, M. F. G. Antitumor Benzothiazoles. 3.
Synthesis of 2-(4-Aminophenyl)benzothiazoles and Evaluation
of Their Activities against Breast Cancer Cell Lines in Vitro and
in Vivo. J . Med. Chem. 1996, 39, 3375-3384.
(18) Cushman, M.; Zhu, H.; Geahlen, R. L.; Kraker, A. J . Synthesis
and Biochemical Evaluation of a Series of Aminoflavones as
Potential Inhibitors of Protein-Tyrosine Kinases p56lck, EGFr,
and p60v-src. J . Med. Chem. 1994, 37, 3353-3362.
tumor volume (mm3) ) {length (mm) × [width (mm)]2}/2
The tumor volumes at initial administration (V0) and on the
day of judgement (V) were calculated, and the tumor growth
rate (V/V0) was calculated.
Ack n ow led gm en t. We thank Mr. Shingo Kakita for
the measurement of the NMR spectra and the structure
elucidation of compounds 2a ,b, Mr. Hiroshi Magara for
the LC-MS analysis, and Dr. Taisuke Nakada for the
ERE-luciferase assay. We are also grateful to Mses
Akiko Mimura, Miki Haibara, Taimi Sano, Miyoko
Asano, and Miyoko Suzuki for their excellent technical
assistance.
1
Su p p or tin g In for m a tion Ava ila ble: Preparation and H-
NMR data of compounds 3a ,b and 4b,c (2 pages). Ordering
information is given on any current masthead page.
Refer en ces
(1) Akama, T.; Shida,Y.; Sugaya, T.; Ishida, H.; Gomi, K.; Kasai,
M. Novel 5-Aminoflavone Derivatives as Specific Antitumor
Agents in Breast Cancer. J . Med. Chem. 1996, 39, 3461-3469.
(2) Griffiths, L. A. Mammalian Metabolism of Flavonoids. In
Flavonoids: Advanced Research; Harborne, J . B., Mabry, T. J .,
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