Structure-activity relationships of the 7-substituents of 5,4-'diamino- 6,8,3'-trifluoroflavone, a potent antitumor agent

Article abstract of DOI:10.1021/jm970728z

Recently, we reported that 5,4'-diamino-6,8,3'-trifluoroflavone (1b) exhibits potent antitumor activity against certain types of human cancer cell lines both in vitro and in vivo. Since the antiproliferative activity of 5,4'-diaminoflavon (1a), the lead c

Full text of DOI:10.1021/jm970728z

2056
J . Med. Chem. 1998, 41, 2056-2067
Str u ctu r e-Activity Rela tion sh ip s of th e 7-Su bstitu en ts of
5,4′-Dia m in o-6,8,3′-tr iflu or ofla von e, a P oten t An titu m or Agen t
Tsutomu Akama,* Hiroyuki Ishida, Uichiro Kimura, Katsushige Gomi,‡ and Hiromitsu Saito
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Company, Ltd., 1188 Shimotogari, Nagaizumi-cho,
Sunto-gun, Shizuoka-ken 411-8731, J apan
Received October 27, 1997
Recently, we reported that 5,4′-diamino-6,8,3′-trifluoroflavone (1b) exhibits potent antitumor
activity against certain types of human cancer cell lines both in vitro and in vivo. Since the
antiproliferative activity of 5,4′-diaminoflavone (1a ), the lead compound of 1b, was modulated
by the addition of apigenin, we hypothesized that the 7-position is important for the interaction
with a putative target molecule. On the basis of this hypothesis, the structure-activity
relationships of the substituents at the 7-position of 1b were explored. As a result, 7-methyl
(7a ), 7-hydroxymethyl (7l), 7-(acyloxy)methyl (9a ,c,e,g,j), and 7-aminomethyl (12f) derivatives
were found to exhibit comparable or superior antitumor activity to compound 1b against MCF-7
cells both in vitro and in vivo (po administration). In particular, compounds 9e,g,j, and 12f
were sufficiently water-soluble as compared with 1b which hardly solubilizes in water. A
lipophilic 7-(hexanoyloxy)methyl derivative (9c) was also found to exhibit strong antitumor
activity especially in vivo. Since the modes of action and the target molecule(s) are unknown,
a mechanistic study will be important in the future.
Ch a r t 1
Flavonoids, either natural or synthetic, are known to
exhibit various biological activities.1 In particular, there
are many compounds exhibiting antitumor2 or related
activities, such as antimitotic activity3 or inhibition of
aromatase,4 topoisomerase,5 protein kinase C,6 several
protein-tyrosine kinases,7 or cyclin-dependent kinase.8
Previously, we reported that 5,4′-diaminoflavone (1a )
(Chart 1) and some of its congeners exhibit potent cyto-
toxicity against human breast cancer cell line MCF-7.9
In the course of the search for the pharmaceutical
mechanism of compound 1a , we found that the antipro-
liferative activity of compound 1a against MCF-7 cells
was modulated by the addition of apigenin which itself
exhibits both growth-inhibiting and -stimulating activity
to certain types of cancer cell lines.10 The isomeric
isoflavone derivative genistein exhibited no effect on the
activity of 1a . Both the mechanism of action and the
target molecule of compound 1a are still unclear. How-
ever, we hypothesized that the 7-hydroxy group of api-
genin might participate in the interaction with a puta-
tive target molecule and that the introduction of a
functional group to the 7-position of compound 1a might
affect the biological activity, because apigenin possesses
three polar substituents at the 5-, 7-, and 4′-positions
in contrast to compound 1a which has two at the 5- and
4′-positions. In addition, substituents at the 7-position
might improve the physical properties (e.g., water solu-
bility) of compound 1a which exhibits poor solubility
both in water and in organic solvents. The hypothesis
prompted us to explore the structure-activity relation-
ships (SAR) of the substituents at the 7-position of 5,4′-
diaminoflavone derivatives.
In direct contrast, compound 1a was suggested to be
metabolically deactivated in vivo.11 However, the in
vivo potency was enhanced in compound 1b11 in which
fluorine atoms were introduced into potential metabolic
positions, i.e., 6-, 8-, and 3′-positions, probably due to
blocking metabolic deactivation. Therefore, we chose
compound 1b as a parent skeleton and synthesized vari-
ous 7-substituted-5,4′-diamino-6,8,3′-trifluoroflavone de-
rivatives. Herein, we describe the synthesis of these
compounds and their antitumor activity against MCF-7
cells both in vitro and in vivo and agaist HeLa S3 in
vitro.
Ch em istr y
We have already developed a practical synthetic
pathway to compound 2, a key intermediate of com-
pound 1b, using sequential directed ortho metalations
as the key step.12 In the process, we found that the two
fluorine atoms corresponding to the 6- and 8-positions
can be regarded as a good directing group for the
directed ortho metalation.13 Therefore, we planned to
introduce various functional groups into the 7-position
or the corresponding position of an intermediate by
directed ortho metalation.
* Address for correspondence: Tokyo Research Laboratories, Kyowa
Hakko Kogyo Co., Ltd., 6-6, Asahi-machi 3-chome, Machida-shi, Tokyo
194-8533, J apan.
‡ Present address: Kyowa Hakko Kogyo Co., Ltd., 6-1, Ohtemachi
1-chome, Chiyoda-ku, Tokyo 100-8185, J apan.
S0022-2623(97)00728-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/08/1998

SAR of 5,4′-Diamino-6,8,3′-trifluoroflavone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2057
Sch em e 1a
a
(a) TBAF, THF, 0 °C; (b) LDA, electrophile, THF, -78 °C; (c) chloromethyl methyl ether, i-Pr2NEt, CH2Cl2, rt; (d) NaBH4, MeOH, 0
°C; (e) TBDMSCl, imidazole, DMF, rt; (f) NaH, 1,4-dioxane (toluene), reflux; (g) HCl, EtOH, rt; (h) m-CPBA (1 eq), CH2Cl2, 0 °C; (i)
m-CPBA (excess), CH2Cl2, rt; (j) K2CO3, MeI, acetone, reflux; (k) NaH, Cl(CH2)2NMe2‚HCl, DMF, 80 °C; (l) HCl, 1,4-dioxane, reflux or
H2SO4, 50 °C.
Sch em e 2a
Sch em e 3a
a
(a) LDA, electrophile, THF, HMPA, -78 °C; (b) PPh3, THF, 1
N HCl, rt; (c) NaH, MeI, DMF, 0 °C; (d) H2SO4, 50-100 °C; (e) aq
NaOH, EtOH, rt.
a
(a) RCO2H, H2SO4, 100 °C; (b) Me2NH‚HCl, i-Pr2NEt, DMF,
Compounds 7 were synthesized as shown in Scheme
1. The trimethylsilyl group of compound 2, which was
prepared in eight steps from 2,4-difluorophenol,12 was
removed with tetrabutylammonium fluoride to afford
3. The 4-position and the amide group of compound 3
were lithiated by LDA, and the dianion generated was
trapped with various electrophiles to afford 4. Com-
pounds 4 and 511,12 were condensed with sodium hydride
followed by cyclization with HCl to afford 6. Target
compounds 7 were obtained after removal of the two
pivaloyl groups by heating with HCl or H2SO4.
Some of the compounds 7 were obtained by the direct
lithiation of compound 811,12 followed by the deprotection
shown in Scheme 2. In this case, HMPA was needed
as a cosolvent to dissolve the starting material 8 in THF.
Lower fatty acid ester derivatives 9 were synthesized
from 7-hydroxymethyl derivative 6l as shown in Scheme
50 °C.
3. When compound 6l was heated in the corresponding
carboxylic acid in the presence of H2SO4, simultaneous
esterification of the 7-hydroxymethyl group and hy-
drolysis of the pivaloyl groups afforded 9a -d ,f. N,N-
Dimethylglycine ester (9e) and N,N-dimethyl-â-alanine
ester (9g) were obtained by the reactions of 9d ,f with
dimethylamine hydrochloride, respectively.
Long-chain fatty acid ester derivatives 9h ,i and
4-(dimethylamino)butyric acid ester derivative 9j were
synthesized as shown in Scheme 4. Since the reaction
of compound 7l and allyloxycarbonyl (Aloc) chloride in
pyridine afforded a mixture of 10 (major) and 7-(allyl-
oxycarbonyl)oxymethyl derivative of 10 (minor), the
crude mixture was treated with aqueous NaOH to
remove the O-Aloc group to give 10 as a sole product.

2058 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Akama et al.
Sch em e 4a
(Figure 1B). Although both compounds are known to
bind the estrogen receptor (ER) and possess growth-
stimulating activity to MCF-7 cells,9,10b,14 apigenin and
genistein behaved quite differently against the cytotox-
icity of compound 1a , suggesting that the effect of
apigenin was mediated by an alternative pathway to
the ER. Taken together with the fact that the target
molecule of compound 1a is not the ER,9 compound 1a
and apigenin might be anticipated to interact with the
same unknown molecule.
Because compounds 1a ,b have unique antiprolifera-
tive profiles against various human cancer cell lines,9,11
e.g., cytotoxic against breast, ovarian, endometrial, and
liver cancer cell lines but not to uterus, colon, and
stomach cell lines, two cell lines considered to be sen-
sitive and nonsensitive to 1a ,b were selected for in vitro
evaluation of the compounds synthesized. The cytotox-
icity of compounds 7a -r , 9a -j (except 9d ,f), and 12a -l
against HeLa S3 as a nonsensitive cancer cell line and
MCF-7 as a sensitive one was tested. The results are
presented in Table 2.
a
(a) Ally chloroformate, pyridine, rt; (b) 1 N NaOH, THF, 0 °C;
(c) RCOCl, Et3N, CH2Cl2 or RCO2H, CDI, DMF, rt; (d) Pd(PPh3)4,
HCO2H, Et3N, THF, rt.
Sch em e 5a
With regard to the activity against MCF-7 cells, the
7-methyl derivative (7a ) showed potent cytotoxicity
comparable to that of compound 1b among lower alkyl
derivatives, and the activity was decreased when the
alkyl chains became longer (7b-d ). 7-Chloro (7m ),
7-bromo (7e), 7-methoxy (7j), and 7-methylthio (7f)
derivatives were less active than 1b. In contrast to the
7-hydroxy derivative (7i) which lost activity, the 7-amino
derivative (7o) exhibited the most potent activity against
MCF-7. Although the introduction of ethoxycarbonyl
(7q) and carboxy (7r ) groups resulted in a large decrease
in activity, the 7-hydroxymethyl derivative (7l), its ace-
tate (9a ), and hydrophilic esters possessing a dimeth-
ylamino group (9e,g,j) retained the activity. With
increasing the length of the alkyl chain, decreasing
activity occurred in fatty acid ester derivatives (9b,c,h ,i).
7-Methoxymethyl (12a ), 7-aminomethyl (12f), and 7-(di-
methylamino)methyl (12h ) derivatives retained activity
against MCF-7 cells; however, the activity tended to
decrease in derivatives possessing bulkier substituents
with more than three carbons (12b-e,g). It is interest-
ing that the quaternary amine derivative (12i) lost its
potency in contrast with the tertiary amine derivative
(12h ) which showed strong cytotoxicity. 7-(Hexanoyl-
amino)methyl derivative (12g) showed comparable ac-
tivity to the 7-(hexanoyloxy)methyl derivative (9c).
a
(a) MsCl, Et3N, DMF, 0 °C; (b) RH (base), DMF; (c) H2SO4,
50-100 °C; (d) CH3(CH2)4COCl, Et3N, DMF; rt; (e) MeI, THF, rt.
The 7-hydroxymethyl group of 10 was acylated followed
by deprotection of the Aloc group to afford 9h -j.
Various 7-alkoxymethyl and 7-aminomethyl deriva-
tives 12 were synthesized as shown in Scheme 5. The
hydroxymethyl group of compound 6l was converted into
mesylate (11) by treating with methanesulfonyl chloride
in pyridine. Compounds 12 were obtained by a substi-
tution reaction at the 7-methyl position of 11 with
various nucleophiles, such as alcohols, alkoxides, and
amines, followed by the removal of the pivaloyl groups.
All target compounds synthesized are listed in
Table 1.
With regard to the activity against HeLa S3 cells, half
of the compounds did not show cytotoxicity (IC50 > 100
µM). Among the compounds showing IC50 values below
0.1 µM against MCF-7, 7-methyl (7a ), 7-(dimethylami-
no)methyl (7p ), and 7-methoxymethyl (12a ) showed no
cytotoxicity against HeLa S3 cells. These compounds
were considered to have the same profiles as compound
1b. Against HeLa S3 cells compounds 7l,o, 9a ,e,g,j, and
12f,h showed cytotoxicity with IC50 values of 4.5-24
µM, which is more than 100 times greater than the IC50
values against MCF-7 cells.
Biologica l Resu lts
In Vitr o Activity. The growth of MCF-7 cells in the
absence of estradiol was completely inhibited by 0.05
µM compound 1a .9 When various concentrations of
apigenin were added to the medium in the presence of
compound 1a (0.05 µM), the cells grew in a concentra-
tion-dependent manner up to 6.3 µM apigenin (Figure
1A). In contrast, the addition of genistein had no effect
In Vivo Activity. The in vivo antitumor activity of
the derivatives showing strong cytotoxicity in vitro was
tested. MCF-7 cells were inoculated into female nude
mice and were growth-stimulated by estradiol. Com-
pounds were orally administered on 5 consecutive days/
week for 2 weeks (days 0-4 and 7-11). The results are

SAR of 5,4′-Diamino-6,8,3′-trifluoroflavone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2059
Ta ble 1. Synthesized 7-Substituted-5,4′-diamino-6,8,3′-trifluoroflavone Derivatives
compd
X
empirical formulaa
mp (°C)
1H NMR (δ, ppm)b of C-7 substituents
2.33 (t, J ) 2.0 Hz, 3H)
1.21 (t, J ) 7.6 Hz, 3H), 2.74 (q, J ) 7.6 Hz, 2H)
0.95 (t, J ) 7.4 Hz, 3H), 1.40 (sextet, J ) 7.4 Hz, 2H),
1.63 (quint, J ) 7.4 Hz, 2H), 2.79 (t, J ) 7.4 Hz, 2H)
0.89 (t, J ) 6.9 Hz, 3H), 1.2-1.5 (m, 6H), 1.64 (quint,
J ) 7.4 Hz, 2H), 2.78 (t, J ) 7.4 Hz, 2H)
7a
7b
7c
Me
Et
n-Bu
C
C
C
16H11F3N2O2
17H13F3N2O2
19H17F3N2O2
239
204-205
178-180
7d
n-Hex
C
21H21F3N2O2
179-180
7m
7e
7i
7j
7k
Cl
Br
OH
OCH3
C
C
C
15H8ClF3N2O2
15H8BrF3N2O2
243-244
256-258
>300
215-217
250 dec
c
15H9F3N2O3
C16H11F3N2O3
C19H18F3N3O3‚HCl
11.3 (br s, 1H)
4.09 (t, J ) 1.5 Hz, 3H)
2.89 (s, 6H), 3.56 (t, J ) 5.0 Hz, 2H), 4.67 (t,
J ) 5.0 Hz, 2H)
2.59 (s, 3H)
3.19 (s, 3H)
O(CH2)2NMe2
7f
SMe
C
C
16H11F3N2O2S
16H11F3N2O3S
C16H11F3N2O4S•0.6H2O
205-207
280 dec
300 dec
182 dec
290-291
210-212
237-239
254-255
277 dec
220 dec
214-215
7g
7h
7n
7o
7p
7q
7r
7l
9a
9b
SOMe
SO2Me
N3
NH2
NMe2
3.50 (s, 3H)
C15H8F3N5O2
C15H10F3N3O2
d
5.99 (br s, 2H)
3.00 (t, J ) 2.5 Hz, 6H)
1.34 (t, J ) 7.2 Hz, 3H), 4.43 (q, J ) 7.2 Hz, 2H)
C17H14F3N3O2
C18H13F3N2O4
C16H9F3N2O4‚0.3H2O
C16H11F3N2O3
C18H13F3N2O4
C19H15F3N2O4
CO2Et
CO2H
CH2OH
CH2OCOMe
CH2OCOEt
4.5-4.6 (m, 2H), 5.44 (t, J ) 4.9 Hz, 1H)
2.11 (s, 3H, COCH3), 5.29 (t, J ) 1.5 Hz, 2H)
1.04 (t, J ) 7.6 Hz, 3H), 2.36 (q, J ) 7.6 Hz, 2H),
5.24 (br s, 2H)
9c
9h
9i
CH2OCO(CH2)4CH3
CH2OCO(CH2)8CH3
CH2OCO(CH2)16CH3
C22H21F3N2O4
C26H29F3N2O4
C34H45F3N2O4
166-167
149-150
139-140
0.88 (t, J ) 6.4 Hz, 3H), 1.2-1.3 (m, 4H), 1.64 (quint,
J ) 7.4 Hz, 2H), 2.35 (t, J ) 7.4 Hz, 2H), 5.29 (s, 2H)
0.86 (t, J ) 6.7 Hz, 3H), 1.1-1.4 (m, 12H), 1.64 (quint,
J ) 7.4 Hz, 2H), 2.35 (t, J ) 7.4 Hz, 2H), 5.29 (s, 2H)
0.87 (t, J ) 6.7 Hz, 3H), 1.1-1.4 (m, 28H), 1.63 (quint,
J ) 6.9 Hz, 2H), 2.34 (t, J ) 7.7 Hz, 2H), 5.29 (s, 2H)
2.84 (s, 6H), 4.27 (s, 2H), 5.42 (s, 2H)
9e
9g
CH2OCOCH2NMe2
CH2OCO(CH2)2NMe2
C20H18F3N3O4‚HCl‚0.4H2O
C21H20F3N3O4‚HCl‚0.3H2O
220 dec
227-228
2.75 (d, J ) 4.5 Hz, 6H), 2.93 (t, J ) 7.4 Hz, 2H), 3.31 (t,
J ) 7.4 Hz, 2H), 5.31 (s, 2H)
9j
CH2OCO(CH2)3NMe2
C22H22F3N3O4‚HCl
233 dec
1.94 (quint, J ) 7.9 Hz, 2H), 2.48 (t, J ) 7.9 Hz, 2H),
2.73 (s, 6H), 3.04 (br s, 2H), 5.27 (s, 2H)
3.31 (s, 3H), 4.57 (br s, 2H)
0.93 (t, J ) 7.4 Hz, 3H), 1.63 (sextet, J ) 6.9 Hz, 2H),
3.50 (t, J ) 6.9 Hz, 2H), 4.69 (t, J ) 2.0 Hz, 2H)
0.88 (t, J ) 6.9 Hz, 3H), 1.2-1.4 (m, 6H), 1.60 (q, J )
6.9 Hz, 2H), 3.52 (t, J ) 6.9 Hz, 2H), 4.68 (t,
J ) 2.0 Hz, 2H)
2.76 (d, J ) 5.0 Hz, 6H), 3.29 (q, J ) 5.0 Hz, 2H),
3.83 (t, J ) 5.0 Hz, 2H), 4.72 (br s, 2H)
1.8-2.0 (m, 2H), 2.74 (d, J ) 4.9 Hz, 6H), 3.0-3.2
(m, 2H), 3.55 (t, J ) 5.9 Hz, 2H), 4.64 (br s, 2H)
3.80 (s, 2H)
12a
12b
CH2OMe
CH2O(CH2)2CH3
C17H13F3N2O3
C19H17F3N2O3
221
170-171
12c
CH2O(CH2)5CH3
C22H23F3N2O3
162-163
12d
12e
CH2O(CH2)2NMe2
CH2O(CH2)3NMe2
C20H20F3N3O3‚HCl‚0.6H2O
C21H22F3N3O3‚HCl‚1.6H2O
265
155 dec
12f
12h
12i
12g
CH2NH2
C16H12F3N3O2‚2HCl
C18H16F3N3O2‚HCl‚0.5H2O
C19H19F3IN3O2
>300
CH2NMe2
269-270
244 dec
239-240
2.84 (s, 6H), 4.47 (br s, 2H)
3.19 (s, 9H), 4.73 (s, 2H)
0.83 (t, J ) 6.9 Hz, 3H), 1.1-1.3 (m, 4H), 1.48 (quint,
J ) 7.3 Hz, 2H), 2.07 (t, J ) 7.3 Hz, 2H), 4.39 (d,
J ) 5.0 Hz, 2H)
CH2N+Me3I-
CH2NHCO(CH2)4CH3
C22H22F3N3O3‚0.2H2O
a
All compounds were analyzed for C, H, and N; analytical results were within (0.4% of the theoretical values unless otherwise noted.
The solvent was DMSO-d6 except for 7a ,c,d , 9c,h ,i, and 12b,c (CDCl3). c N: calcd, 7.27; found, 6.74. N: calcd, 13.08; found, 12.62.
b
d
listed in Table 3 by the T/C values which is the ratio of
was diminished when the 7-amino group of 7o was
dimethylated (7p ). The 7-hydroxymethyl derivative (7l)
showed activity comparable to that of compound 1b at
25 mg/kg with slight body weight decrease, and further,
the dose of 50 mg/kg was also tolerable. The acetylated
derivative of 7l (9a ) showed strong activity, but dose
dependency was not observed, probably due to poor
solubility. Compound 9b’s lack of effect seemed to be
due to the same reason. Although compounds 9a ,b have
poor solubility both in water and in organic solvents,
compound 9b is especially insoluble. Interestingly, the
tumor growth rate of the drug-treated group relative
to that of the control group. Representative results
(compounds 9c,e and 12f) are graphed in Figure 2.
The 7-methyl derivative (7a ) showed 89% reduction
of the growth rate at 12.5 mg/kg with some body weight
decrease (-11%), which was equal to the efficacy of 1b
at 25 mg/kg dosage. The 7-amino derivative (7o) which
was the most potent in vitro also showed strong
antitumor activity; however, the body weight decrease
was more serious (-22% at 12.5 mg/kg). The efficacy

2060 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Akama et al.
Ta ble 2. In Vitro Cytotoxicity of
7-Substituted-5,4′-diamino-6,8,3′-trifluoroflavone Derivatives
against HeLa S3 and MCF-7 Cell Lines
IC50 (µM)a
compd
X
HeLa S3
MCF-7
1b
7a
7b
7c
7d
7m
7e
7i
7j
7k
7f
7g
7h
7n
7o
7p
7q
7r
7l
9a
9b
9c
9h
9i
H
Me
Et
n-Bu
n-Hex
Cl
Br
OH
OCH3
O(CH2)2NMe2
SMe
SOMe
SO2Me
N3
NH2
NMe2
CO2Et
CO2H
>100
>100
>100
>100
21
>100
>100
>100
34
0.040
0.013
0.13
8.8
>10
0.21
0.16
>10
0.10
1.4
0.19
2.5
1.5
>100
>100
>100
1.2
>10
0.46
19
0.0077
0.039
1.4
>100
>100
>100
24
>10
CH2OH
CH2OCOMe
CH2OCOEt
0.062
0.026
0.13
0.16
0.10
4.9
69
34
CH2OCO(CH2)4CH3
CH2OCO(CH2)8CH3
CH2OCO(CH2)16CH3
CH2OCOCH2NMe2
CH2OCO(CH2)2NMe2
CH2OCO(CH2)3NMe2
CH2OMe
CH2O(CH2)2CH3
CH2O(CH2)5CH3
CH2O(CH2)2NMe2
CH2O(CH2)3NMe2
CH2NH2
F igu r e 1. Interaction of apigenin with compound 1a on the
growth of MCF-7 cells. MCF-7 cells were incubated with
various concentrations of apigenin (Api) (A) or genistein (Gen)
(B) with or without 0.05 µM compound 1a for 6 days and
counted by a microcell counter (ref 9).
>100
>100
9.2
>10
9e
9g
9j
0.026
0.019
0.029
0.046
1.2
11
4.5
12a
12b
12c
12d
12e
12f
12h
12i
12g
>100
78
(hexanoyloxy)methyl derivative (9c) exhibited intensive
antitumor activity from 6.3 to 25 mg/kg without serious
body weight decrease (also see Figure 2A). The long-
chain fatty acid ester derivatives (9h ,i) exhibited 80-
87% reduction of the growth rate at 25 mg/kg. These
long-chain ester derivatives would have high lipid
solubility which might improve the oral absorption. The
ester derivatives possessing a dimethylamino group
(9e,g,j) showed strong antitumor activity; especially,
compound 9e showed excellent activity (also see Figure
2B). 7-Aminomethyl derivative 12f showed activity
comparable to 7-hydroxymethyl derivative 7l at 25 and
50 mg/kg (also see Figure 2C). As observed in the case
of compounds 7o,p , dimethylation of the amino group
at the 7-methyl position of compound 12f (compound
12h ) did not improve activity.
>100
4.6
7.1
>10
>10
0.026
12
8.8
8.9
32
CH2NMe2
0.072
3.1
0.34
CH2N+Me3I-
CH2NHCO(CH2)4CH3
>100
a
Cells were treated with each compound for 3 days. The uptake
of neutral red dye was measured (ref 9).
Various 7-(acyloxy)methyl derivatives were synthe-
sized, and their antitumor activity was tested to find
derivatives with improved solubility because the 7-hy-
droxymethyl derivative (7l) had a potency comparable
to that of compound 1b despite its poor physical proper-
ties. Long-chain fatty acid ester derivatives (9c,h ,i)
were inactive or less active than the corresponding
alcohol (7l) or its acetate (9a ) in vitro; nevertheless all
the ester derivatives except compound 9b were active
in vivo. Therefore the ester derivatives were considered
to act as prodrugs and to be hydrolyzed to generate
compound 7l in vivo. With respect to the acetoxymethyl
derivative 9a , it is not evident whether the compound
is hydrolyzed in vivo because this compound showed
cytotoxicity as potent as that of compound 7l in vitro.
Compound 9b and the methoxymethyl derivative 12a
also showed potent activity in vitro but no effect in vivo.
A potential cause of these results can be traced to the
poor solubility of the compounds in both water and
organic solvents. Hydrophilic ester derivatives (9e,g,j),
showed potent antitumor activity both in vitro and in
Discu ssion
The SAR of the 7-position of 5,4′-diamino-6,8,3′-
trifluoroflavone (1b) was explored. Interesting findings
were obtained from in vitro experiments: (1) although
both an amino group and a hydroxy group are capable
of being a hydrogen bond donor/acceptor, the 7-amino
derivative (7o) showed excellent cytotoxicity against
MCF-7 cells in contrast that the 7-hydroxy derivative
(7i), which showed no activity; (2) although the 7-methyl
(7a ) and 7-hydroxymethyl (7l) derivatives showed activ-
ity comparable to that of 1b against MCF-7 cells, the
more oxygenated 7-ethoxycarbonyl (7q) and 7-carboxyl
(7r ) derivatives showed weak or no activity.

SAR of 5,4′-Diamino-6,8,3′-trifluoroflavone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2061
Ta ble 3. In Vivo Antitumor Activity of
7-Substituted-5,4′-diamino-6,8,3′-trifluoroflavone Derivatives
against MCF-7 Cells Implanted into Nude Micea
dose
T/C min body weight
compd
X
(mg/kg)
(%)
change (%)
1b
7a
H
Me
25
11
55
11
55
26
18
39
17
1.6
6.1
11
79
27
7.9
0.8
13
20
68
4.0
1.0
4.1
15
89
57
60
11
1.1
16
76
-7.4
-8.7
-11
-5.7
-13
-22
-19
-4
-6.7
-5.9
-4.5
-2.4
-5.9
-8.4
-7.9
-5.0
-4.8
-4.8
-9.0
-11
-12
-8.4
0
6.3
12.5
3.1
6.3
12.5
12.5
25
50
25
50
25
6.3
12.5
25
25
25
6.3
25
50
25
25
7o
NH2
7p
7l
NMe2
CH2OH
9a
CH2OCOMe
9b
9c
CH2OCOEt
CH2OCO(CH2)4CH3
9h
9i
9e
CH2OCO(CH2)8CH3
CH2OCO(CH2)16CH3
CH2OCOCH2NMe2
9g
9j
12a
12d
12f
CH2OCO(CH2)2NMe2
CH2OCO(CH2)3NMe2
CH2OMe
CH2O(CH2)2NMe2
CH2NH2
50
50
6.3
25
50
-7.9
-5.8
-8.5
-11
-17
0
12h
12g
CH2NMe2
CH2NHCO(CH2)4CH3
25
50
a
Tumor fragments were implanted sc into BALB/c-nu/nu female
mice on day -20. Compounds were administered po 5 consecutive
days/week for 2 weeks.
vivo. However, the ether derivatives (12d ,e), which
correspond to decarbonylated derivatives of compounds
9e,g and cannot be hydrolyzed, were inactive both in
vitro and in vivo. Thus, it was suggested that com-
pounds 9e,g,j also act as prodrugs of compound 7l. On
the other hand, the amide type derivative 12g which
exhibited activity comparable to that of the ester
derivative 9c in vitro showed no effect in vivo despite
the fact that the aminomethyl derivative 12f exhibited
strong antitumor activity in vivo, suggesting that the
amide bond was not cleaved in vivo.
F igu r e 2. In vivo antitumor activity of compounds 9c,e, and
12f.
exhibits remarkable antitumor activity in vivo, does not
exhibit water solubility, but it is more soluble in organic
solvents such as methanol (ca. 2 mg/mL) than compound
1b (ca. 1 mg/mL). The increased lipophilicity might
enhance the membrane permeability, which could im-
prove the oral absorption compared to that of compound
1b. We cannot conclude that the prodrugs are prefer-
able to the intact drugs from the present data because
the intact compounds, such as 7a ,l, are sufficiently
potent in vivo despite the poor water solubility. In fact,
the formulation and administration route of the intact
compounds are restricted; however, other problems such
as species-specific activation make the development of
the prodrugs difficult.
The real role of the 7-substituents is still unclear. To
clarify it, the target molecule of aminoflavone deriva-
tives and how they interact with the molecule must be
identified. Neither the simple alkylation nor the cleav-
age of the DNA is considered to be the mode of action,
because the sensitivities of the two cell lines are quite
different. Therefore, the potential candidates of the
target molecule include various protein kinases, nuclear
receptors, transcriptional factors, and other key en-
Since the lead compound 1b possesses poor solubility
in both water and organic solvents,15 the improvement
of a physical property such as solubility may be crucial.
In addition to the potent antitumor activity, compounds
9e,g,j and 12f which are hydrochlorides exhibited
improved water solubility. Preliminary data of water
solubility are >10 mg/mL for compounds 9e,j and >5
mg/mL for compounds 9g and 12f. Water solubility is
an advantage for a drug with respect to the formulation
and the oral absorption. The prodrug formation is one
potential approach for improving the physical properties
and pharmacokinetics.16 In this study, it is suggested
from the biological data that 7-(acyloxy)methyl deriva-
tives act as prodrugs of the 7-hydroxymethyl derivative
7l. On the other hand, compound 9c, which also

2062 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Akama et al.
Ta ble 4. 1H NMR of the C-4 (C-7 of Target Compounds)
zymes or cofactors in certain types of tumor cell growth.
It will be important to determine the mechanism of the
antitumor activity of the aminoflavone derivatives in
the future. At least, we found that a physical property
of the parent compound 1b can be improved by modi-
fication at the 7-position, while retaining the antitumor
activity.
In conclusion, the structure-activity relationships of
the substituents at the 7-position of 5,4′-diamino-6,8,3′-
trifluoroflavone (1b) were explored based on the hy-
pothesis that the 7-substituents would affect the bio-
logical activity of 5,4′-diaminoflavone derivatives. As
a result, some derivatives, such as compounds 7a ,l,
9a ,c,e,g,j and 12f, were found to exhibit comparable or
superior antitumor activity to the parent compound 1b
against MCF-7 cells both in vitro and in vivo. In
addition, compounds 9e,g,j and 12f were sufficiently
water-soluble compared to 1b which barely solubilizes
in water. A lipophilic derivative (9c) was also found to
exhibit strong antitumor activity in vivo.
Substituents of Compounds 4
compd
1H NMR (δ, ppm) in CDCl3
4b
4c
4d
4f
4h
4i
1.20 (t, J ) 7.7 Hz, 3H), 2.72 (q, J ) 7.7 Hz, 2H)
0.92 (t, J ) 5.9 Hz, 3H), 1.4-2.0 (m, 4H), 2.69 (br t, 2H)
0.7-1.0 (m, 3H), 1.4-2.0 (m, 8H), 2.5-2.9 (m, 2H)
2.50 (br s, 3H)
3.57 (s, 3H), 5.21 (s, 2H)
10.3 (s, 1H)
4j
4k
4.7-4.8 (m, 2H)
0.03 (s, 6H), 0.83 (s, 9H), 4.70 (t, J ) 1.7 Hz, 2H)
E t h yl 4-(1-Bu t yl)-3,5-d iflu or o-6-(p iva loyla m in o)-2-(2-
tetr a h yd r op yr a n yloxy)ben zoa te (4c). 1-Iodobutane was
used as an electrophile, and chromatography (4:1 hexane/
EtOAc) of the crude product gave 4c (29%): FABMS (negative)
m/z 440 (M - H-).
Eth yl 3,5-Diflu or o-4-(1-h exyl)-6-(p iva loyla m in o)-2-(2-
tetr a h yd r op yr a n yloxy)ben zoa te (4d ). 1-Iodohexane was
used as an electrophile, and chromatography (4:1 hexane/
EtOAc) of the crude product gave 4d (71%): FABMS (negative)
m/z 468 (M - H-).
Eth yl 4-Br om o-3,5-d iflu or o-6-(p iva loyla m in o)-2-(2-tet-
r ah ydr opyr an yloxy)ben zoate (4e). 1,2-Dibromoethane was
used as an electrophile, and chromatography (4:1 hexane/
EtOAc) of the crude product gave 4e (49%): FABMS (negative)
m/z 464, 462 (M - H-).
Eth yl 3,5-Diflu or o-4-(m eth ylth io)-6-(p iva loyla m in o)-2-
(2-tetr a h yd r op yr a n yloxy)ben zoa te (4f). Dimethyl disul-
fide was used as an electrophile (89%): FABMS (negative) m/z
430 (M - H-).
Eth yl 3,5-Diflu or o-4-for m yl-6-(p iva loyla m in o)-2-(2-tet-
r a h yd r op yr a n yloxy)b en zoa t e (4i). N,N-Dimethylform-
amide was used as an electrophile, and chromatography (4:1
hexane/EtOAc) of the crude product gave 4i (54%): FABMS
m/z 414 (M + H+).
Eth yl 3,5-Diflu or o-4-(h yd r oxym eth yl)-6-(p iva loyla m i-
n o)-2-(2-tetr a h yd r op yr a n yloxy)ben zoa te (4j). To a solu-
tion of 4i (22.3 g, 54.0 mmol) in MeOH (260 mL) was added
NaBH4 (1.02 g, 27.0 mmol) at 0 °C. The reaction mixture was
stirred for 1 h, and water was added. The mixture was
extracted with EtOAc twice, and the organic layer was washed
with brine to afford 4j (22.0 g, 98%): FABMS m/z 416 (M +
H+).
E t h yl 4-[(ter t-Bu t yld im et h ylsilyloxy)m et h yl]-3,5-d i-
flu or o-6-(pivaloylam in o)-2-(2-tetr ah ydr opyr an yloxy)ben -
zoa te (4k ). To a solution of 4j (213 mg, 0.500 mmol) in CH2-
Cl2 (5 mL) were added imidazole (68 mg, 1.0 mmol) and tert-
butyldimethylsilyl chloride (302 mg, 2.0 mml). The reaction
mixture was refluxed for 30 min. Water was added, and the
mixture was extracted with EtOAc. The organic layer was
washed with brine, and the residue was purified by chroma-
tography (4:1 hexane/EtOAc) to afford 4k (256 mg, 98%):
FABMS m/z 528 (M + H+).
Eth yl 3,5-Diflu or o-4-h yd r oxy-6-(p iva loyla m in o)-2-(2-
tetr a h yd r op yr a n yloxy)ben zoa te (4g). To a solution of
diisopropylamine (7.00 mL, 50.0 mmol) in THF (14 mL) was
added n-butyllithium (1.6 M in hexane, 30 mL) at -10 to 0 °C
under argon atmosphere. The reaction mixture was cooled to
below -60 °C, and a solution of compound 3 (7.70 g, 20.0 mmol)
in THF (60 mL) was added dropwise. After 2 h of stirring,
trimethylborate (2.7 mL, 24 mmol) was added, and the reaction
mixture was warmed to 0 °C during 20 min. Acetic acid (4.0
mL) and 30% H2O2 (8.0 mL) were added, and the mixture was
stirred for 20 h at room temperature; 10% NaHSO3 was added,
and the mixture was washed with EtOAc. The mixture was
acidified with 2 N HCl and extracted with EtOAc. The organic
layer was washed with brine, and the residue was triturated
with hexane to afford 4g (4.33 g, 54%): FABMS (negative) m/z
400 (M - H-).
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. EIMS and FABMS 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.
Eth yl 3,5-Diflu or o-2-(2-tetr a h yd r op yr a n yloxy)-6-(p iv-
a loyla m in o)ben zoa te (3). To a solution of compound 2 (44.0
g, 96.3 mmol) in THF (400 mL) was added tetrabutylammo-
nium fluoride (1.0 M in THF, 28 mL) at 0 °C, and the mixture
was stirred for 30 min. Brine was added, and the mixture
was extracted with Et2O. The organic layer was washed with
brine. The residue was triturated with hexane to afford 3 (37.1
g, 100%): 1H NMR (90 MHz, CDCl3) δ 1.28 (s, 9H, C(CH3)3),
1.38 (t, J ) 7.0 Hz, CH2CH3), 1.4-2.0 (m, 6H, 3′,4′,5′-CH2),
3.3-4.1 (m, 2H, 6′-CH2), 4.36 (q, J ) 7.0 Hz, CH2CH3), 5.32
(br s, 1H, 2′-H), 7.00 (dd, J ) 10.7, 9.6 Hz, 1H, 4-H), 7.57 (br
s, 1H, NH); FABMS m/z 386 (M + H+).
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 s
4: Eth yl 3,5-Diflu or o-4-m eth yl-6-(p iva loyla m in o)-2-(2-
tetr a h yd r op yr a n yloxy)ben zoa te (4a ). To a solution of
diisopropylamine (70 mL, 500 mmol) in THF (140 mL) was
added n-butyllithium (1.6 M in hexane, 288 mL) at -10 to 0
°C under argon atmosphere. The reaction mixture was cooled
to below -60 °C, and a solution of compound 3 (77.0 g, 200
mmol) in THF (600 mL) was added dropwise. After 2 h of
stirring, iodomethane (19.0 mL, 300 mmol) was added, and
the reaction mixture was stirred for 30 min. Water was added,
and the mixture was warmed to room temperature. The
mixture was extracted with EtOAc, and the organic layer was
washed with brine. The residue was triturated with hexane
to afford 4a (75.9 g, 95%): 1H NMR (90 MHz, CDCl3) δ 1.19
(s, 9H, C(CH3)3), 1.37 (t, J ) 7.0 Hz, CH2CH3), 1.4-2.0 (m,
6H, 3′,4′,5′-CH2), 2.23 (t, J ) 2.2 Hz, 3H, 4-CH3), 3.4-4.2 (m,
2H, 6′-CH2), 4.34 (q, J ) 7.0 Hz, CH2CH3), 5.28 (br s, 1H, 2′-
H), 7.69 (dd, J ) 10.7, 9.6 Hz, 1H, 4-H), 7.57 (br s, 1H, NH);
FABMS m/z 400 (M + H+). NMR data of compounds 4 are
listed in Table 4.
Eth yl 3,5-Diflu or o-4-eth yl-6-(p iva loyla m in o)-2-(2-tet-
r a h yd r op yr a n yloxy)ben zoa te (4b). Iodoethane was used
as an electrophile, and chromatography (3:1 hexane/
EtOAc) of the crude product gave 4b (67%): FABMS m/z 414
(M + H+).
E t h yl 3,5-Diflu or o-4-(m et h oxym et h oxy)-6-(p iva loyl-
a m in o)-2-(2-tetr a h yd r op yr a n yloxy)ben zoa te (4h ). To a
solution of 4g (4.23 g, 10.5 mmol) in CH2Cl2 (50 mL) were
added diisopropylethylamine (2.4 mL, 13.7 mmol) and chlo-

SAR of 5,4′-Diamino-6,8,3′-trifluoroflavone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2063
Ta ble 5. 1H NMR of the C-7 Substituents of Compounds 6
NaHCO3, H2O, and brine. Chromatography (100:1 CHCl3/
MeOH) gave 6g (200 mg, 95%): EIMS m/z 536 (M+).
compd
1H NMR (δ, ppm) in CDCl3
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
(m eth ylsu lfon yl)-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-
on e (6h ). To a solution of 6f (203 mg, 0.391 mmol) in CH2Cl2
(5 mL) was added m-chloroperbenzoic acid (850 mg, 3.91 mmol)
at 0 °C, and the reaction mixture was stirred at room
temperature for 2 h; 10% aqueous NaHSO3 was added, and
the mixture was extracted with CHCl3. The organic layer was
washed with aqueous NaHCO3, H2O, and brine. Chromatog-
raphy (100:1 CHCl3/MeOH) gave 6h (204 mg, 94%): EIMS m/z
552 (M+).
6b
6c
6d
6f
6g
6h
6i
6j
6k
6l
1.35 (t, J ) 7.5 Hz, 3H), 2.7-3.1 (m, 2H)
0.96 (t, J ) 6.3 Hz, 3H), 1.2-1.9 (m, 4H), 2.7-3.0 (m, 2H)
0.8-1.0 (m, 3H), 1.1-1.8 (m, 8H), 2.7-3.0 (m, 2H)
2.66 (t, J ) 1.3 Hz, 3H)
3.23 (s, 3H)
3.43 (s, 3H)
10.9 (br s, 1H)
4.22 (t, J ) 1.8 Hz, 3H)
2.36 (s, 6H), 2.79 (t, J ) 5.5 Hz, 2H), 4.48 (t, J ) 5.5 Hz, 2H)
4.6-4.7 (m, 2H)a
6o
6p
6q
6.58 (br s, 2H)a
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
h yd r oxy-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6i).
This compound was obtained from 4h and 5 in a similar
manner as described for 6a (26%): EIMS m/z 490 (M+).
3.12 (t, J ) 2.6 Hz, 6H)
1.43 (t, J ) 7.0 Hz, 3H), 4.49 (q, J ) 7.0 Hz, 2H)
a
The solvent was DMSO-d6.
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
m eth oxy-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6j).
To a solution of 6i (348 mg, 0.710 mmol) in acetone (35 mL)
were added K2CO3 (166 mg, 1.20 mmol) and iodomethane (0.45
mL, 7.1 mmol), and the reaction mixture was refluxed for 40
min. The mixture was filtered, and to the filtrate were added
water and EtOAc. The organic layer was washed with 1 N
NaOH, H2O, and brine. Chromatography (CHCl3) gave 6j (280
mg, 78%): EIMS m/z 504 (M+).
6,8-Diflu or o-7-[2-(d im eth yla m in o)eth oxy]-2-[3-flu or o-
4-(p iva loyla m in o)p h en yl]-5-(p iva loyla m in o)-4H -1-b en -
zop yr a n -4-on e (6k ). To a solution of 6i (490 mg, 1.00 mmol)
in DMF (40 mL) were added K2CO3 (3.60 g, 26.0 mmol) and
2-(dimethylamino)ethyl chloride hydrochloride (2.88 g, 20.0
mmol), and the reaction mixture was stirred at 50 °C for 12
h. The mixture was filtered, and the filtrate was concentrated
under reduced pressure. To the residue were added water and
EtOAc. The organic layer was washed with H2O and brine.
Chromatography (30:1 CHCl3/MeOH) gave 6k (230 mg, 41%):
EIMS m/z 561 (M+).
romethyl methyl ether (0.96 mL, 12.6 mmol) at 0 °C, and the
reaction mixture was stirred for 30 min. Water was added,
and the mixture was extracted with CHCl3. The organic layer
was washed with brine, and the residue was triturated with
hexane to afford 4h (4.33 g, 93%): FABMS (negative) m/z 444
(M - H-).
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 s
6a -f,i,l: 6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en -
yl]-7-m et h yl-5-(p iva loyla m in o)-4H -1-b en zop yr a n -4-on e
(6a ). To a refluxing suspension of NaH (27.7 g, 693 mmol)
which was washed with hexane three times in 1,4-dioxane/
toluene (1:1, 300 mL) under argon atmosphere was added
dropwise a solution of 4a (99.8 g, 250 mmol) and 5 (50.0 g,
210 mmol) in the above solvent (800 mL). The reaction
mixture was refluxed for 2 h and was cooled on an ice bath.
Water was added, the mixture was extracted twice with
EtOAc, and the organic layer was washed with brine. The
combined extracts were concentrated and dissolved in EtOH
(800 mL). Concentrated HCl (200 mL) was added, and the
reaction mixture was stirred for 14 h at room temperature.
Water was added, and the precipitated product was collected
by filtration. Recrystallization from CHCl3 (1250 mL)/hexane
(500 mL) afforded 6a (50.3 g, 49%). The mother liquid was
concentrated and recrystallized from CHCl3 (300 mL)/hexane
(200 mL) to afford additional 6a (12.0 g, 21%): 1H NMR (90
MHz, CDCl3) δ 1.36 (s, 9H, C(CH3)3), 1.38 (s, 9H, C(CH3)3),
2.41 (t, J ) 2.2 Hz, 3H, 7-CH3), 6.64 (s, 1H, 3-H), 7.5-7.9 (m,
3H, 2′,6′-H and 4′-NH), 8.59 (t, J ) 8.4 Hz, 1H, 5′-H), 10.5 (br
s, 1H, 5-NH); EIMS m/z 488 (M+). NMR data of compounds 6
are listed in Table 5.
6,8-Diflu or o-7-e t h yl-2-[3-flu or o-4-(p iva loyla m in o)-
p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6b).
This compound was obtained from 4b and 5 in a similar
manner as described for 6a (41%): EIMS m/z 502 (M+).
7-(1-Bu tyl)-6,8-d iflu or o-2-[3-flu or o-4-(p iva loyla m in o)-
p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6c).
This compound was obtained from 4c and 5 in a similar
manner as described for 6a (36%): EIMS m/z 530 (M+).
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-(1-
h exyl)-5-(pivaloylam in o)-4H-1-ben zopyr an -4-on e (6d). This
compound was obtained from 4d and 5 in a similar manner
as described for 6a (43%): EIMS m/z 558 (M+).
7-Br om o-6,8-d iflu or o-2-[3-flu or o-4-(p iva loyla m in o)-
p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6e).
This compound was obtained from 4e and 5 in a similar
manner as described for 6a (57%): FABMS m/z 555, 553 (M
+ H+).
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
(m eth ylth io)-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e
(6f). This compound was obtained from 4f and 5 in a similar
manner as described for 6a (58%): EIMS m/z 520 (M+).
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
(m eth ylsu lfin yl)-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-
on e (6g). To a solution of 6f (204 mg, 0.392 mmol) in CH2Cl2
(5 mL) was added m-chloroperbenzoic acid (86 mg, 0.39 mmol)
at 0 °C, and the reaction mixture was stirred for 2 h; 10%
aqueous NaHSO3 was added, and the mixture was extracted
with CHCl3. The organic layer was washed with aqueous
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
(h yd r oxym eth yl)-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-
on e (6l). This compound was obtained from 4k and 5 in a
similar manner as described for 6a (40%): EIMS m/z 504 (M+).
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 s
6m ,n ,q: 7-Ch lor o-6,8-d iflu or o-2-[3-flu or o-4-(p iva loyla m i-
n o)p h en yl]-5-(p iva loyla m in o)-4H -1-b en zop yr a n -4-on e
(6m ). To a solution of diisopropylamine (1.54 mL, 11.0 mmol)
was added n-butyllithium (1.6 M in hexane, 6.3 mL) at -10
to 0 °C under argon atmosphere. The reaction mixture was
cooled to below -60 °C, and a solution of compound 8 (1.19 g,
2.50 mmol) in THF (50 mL) and HMPA (10 mL) was added
dropwise. After 2 h of stirring, N-chlorosuccinimide (802 mg,
6.00 mmol) was added, and the reaction mixture was stirred
for 10 min. Water was added, and the mixture was warmed
to room temperature. The mixture was extracted with EtOAc,
and the organic layer was washed with brine. Chromatogra-
phy (60:1 CHCl3/acetone) gave 6m (384 mg, 30%): EIMS m/z
511, 509 (M+).
7-Azid o-6,8-d iflu or o-2-[3-flu or o-4-(p iva loyla m in o)-
p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6n ).
p-Toluenesulfonylazide17 was used as an electrophile, and the
precipitated product after adding water to the reaction mixture
was collected by filtration to afford 6n (81%): IR (KBr) 2140
cm-1; FABMS (negative) m/z 514 (M - H-).
6,8-Diflu or o-7-(e t h oxyca r b on yl)-2-[3-flu or o-4-(p iv-
a loyla m in o)p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -
4-on e (6q). Ethyl chloroformate was used as an electrophile,
and chromatography (40:1 CHCl3/EtOAc) gave 6q (66%):
EIMS m/z 546 (M+).
7-Am in o-6,8-d iflu or o-2-[3-flu or o-4-(p iva loyla m in o)-
p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -4-on e (6o).
To a solution of 6n (3.50 g, 6.80 mmol) in THF (120 mL) was
added PPh3 (1.96 g, 7.48 mmol), and the reaction mixture was
stirred for 2.5 h at room temperature; 1 N HCl (50 mL) was
added, and the mixture was further stirred for 10 h. The pH
was adjusted to 9 with 10 N NaOH, and the precipitated

2064 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Akama et al.
product was collected by filtration. Chromatography (40:1
CHCl3/MeOH) gave 6o (2.83 g, 85%): EIMS m/z 489 (M+).
6,8-Diflu or o-7-(d im e t h yla m in o)-2-[3-flu or o-4-(p iv-
a loyla m in o)p h en yl]-5-(p iva loyla m in o)-4H-1-ben zop yr a n -
4-on e (6p ). To a solution of 6o (510 mg, 1.04 mmol) in DMF
(15 mL) were added NaH (212 mg, 5.30 mmol) and io-
domethane (0.17 mL, 2.6 mmol) at 0 °C, and the reaction
mixture was stirred for 2.5 h. Water was added, and the
mixture was extracted with CHCl3. The organic layer was
washed with water and brine. The residue was purified by
chromatography (100:1 CHCl3/acetone) to afford 6p (290 mg,
54%): EIMS m/z 517 (M+).
(78%): IR (KBr) 1647 cm-1; EIMS m/z 336 (M+). Anal.
(C16H11F3N2O3) 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-7-[2-
(d im eth yla m in o)eth oxy]-4H-1-ben zop yr a n -4-on e Hyd r o-
ch lor id e (7k ). This compound was obtained from 6k in a
similar manner as described for 7f (97%). To a solution of the
free base of 7k (170 mg) in 2-PrOH (10 mL) were added HCl
(1 N in 2-PrOH, 0.5 mL) and 2-Pr2O (5 mL). The precipitated
hydrochloride was collected: IR (KBr) 1651 cm-1; EIMS m/z
393 (M+). Anal. (C19H18F3N3O3‚HCl) 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-7-h y-
d r oxy-4H-1-ben zop yr a n -4-on e (7l). This compound was
obtained from 6l in a similar manner as described for 7f
(60%): IR (KBr) 3500, 3350, 1657 cm-1; EIMS m/z 336 (M+).
Anal. (C16H11F3N2O3) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-7-ch lor o-6,8-d i-
flu or o-4H-1-ben zop yr a n -4-on e (7m ). This compound was
obtained from 6m in a similar manner as described for 7f
(73%): IR (KBr) 1653 cm-1; EIMS m/z 342, 340 (M+). Anal.
(C15H8ClF3N2O2) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-7-a zid o-6,8-d i-
flu or o-4H-1-ben zop yr a n -4-on e (7n ). This compound was
obtained from 6n in a similar manner as described for 7f
(84%): IR (KBr) 2135, 1653 cm-1; EIMS m/z 347 (M+). Anal.
(C15H8F3N5O2) C, H, N.
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 s
7a -e: 5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-
7-m eth yl-4H-1-ben zop yr a n -4-on e (7a ). A mixture of 6a
(800 mg, 1.64 mmol), 1,4-dioxane (50 mL), and HCl (25 mL)
was refluxed for 2 h. The mixture was poured into ice-water
and was basified with 10 N NaOH. The mixture was extracted
with CHCl3, and the organic layer was washed with brine.
Chromatography (40:1 CHCl3/MeOH) followed by recrystalli-
zation from EtOAc/hexane gave 7a (183 mg, 35%): IR (KBr)
1
1654 cm-1; H NMR (270 MHz, CDCl3) δ 2.33 (t, J ) 2.0 Hz,
3H, 7-CH3), 4.17 (br s, 2H, 4′-NH2), 6.12 (br s, 2H, 5-NH2),
6.45 (s, 1H, 3-H), 6.84 (t, J ) 8.4 Hz, 1H, 5′H), 7.5-7.6 (m,
2H, 2′,6′H); EIMS m/z 320 (M+). Anal. (C16H11F3N2O2) C, H,
N.
2-(4-Am in o-3-flu or op h en yl)-5,7-d ia m in o-6,8-d iflu or o-
4H-1-ben zop yr a n -4-on e (7o). This compound was obtained
from 6o in a similar manner as described for 7f (69%): IR
5-Am in o-2-(4-am in o-3-flu or oph en yl)-6,8-diflu or o-7-eth yl-
4H-1-ben zop yr a n -4-on e (7b). This compound was obtained
from 6b in a similar manner as described for 7a (44%): IR
(KBr) 1658 cm-1; EIMS m/z 334 (M+). Anal. (C17H13F3N2O2)
C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-7-(1-bu tyl)-6,8-d i-
flu or o-4H-1-ben zop yr a n -4-on e (7c). This compound was
obtained from 6c in a similar manner as described for 7a
(47%): IR (KBr) 1653 cm-1; EIMS m/z 362 (M+). Anal.
(C19H17F3N2O2) 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-7-(1-
h exyl)-4H-1-ben zop yr a n -4-on e (7d ). This compound was
obtained from 6d in a similar manner as described for 7a
(53%): IR (KBr) 1655 cm-1; EIMS m/z 390 (M+). Anal.
(C21H21F3N2O2) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-7-b r om o-6,8-d i-
flu or o-4H-1-ben zop yr a n -4-on e (7e). This compound was
obtained from 6e in a similar manner as described for 7a
(38%): IR (KBr) 1649 cm-1; EIMS m/z 386, 384 (M+). Anal.
(C15H8BrF3N2O2) C, H; N: calcd, 7.27; found, 6.74.
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 s
7f-q: 5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-
7-(m eth ylth io)-4H-1-ben zop yr a n -4-on e (7f). 6f (511 mg,
0.983 mmol) in H2SO4 (20 mL) was stirred at 50 °C for 10 min.
The reaction mixture was poured into ice-water and neutral-
ized with 10 N NaOH. The precipitated product was collected
by filtration. Chromatography (CHCl3) followed by recrystal-
lization from EtOAc/hexane gave 7f (309 mg, 89%): IR
(KBr) 1641 cm-1; EIMS m/z 352 (M+). Anal. (C16H11F3N2O2S)
C, H, N.
(KBr) 1662 cm-1
(C15H10F3N3O2) C, H; N: calcd, 13.08; found, 12.62.
; FABMS m/z 322 (M +
H+). Anal.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
(d im eth yla m in o)-4H-1-ben zop yr a n -4-on e (7p ). This com-
pound was obtained from 6p in a similar manner as described
for 7f (97%): IR (KBr) 1653 cm-1; EIMS m/z 349 (M+). Anal.
(C17H14F3N3O2) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
(eth oxyca r bon yl)-4H-1-ben zop yr a n -4-on e (7q). This com-
pound was obtained from 6q in a similar manner as described
for 7f (50%): IR (KBr) 1726, 1637 cm-1; EIMS m/z 378 (M+).
Anal. (C18H13F3N2O4) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-7-ca r boxy-6,8-d i-
flu or o-4H-1-ben zop yr a n -4-on e (7r ). To a suspension of 7q
(121 mg, 0.320 mmol) in EtOH (10 mL) and MeOH (5 mL)
was added 5 N NaOH (0.4 mL), and the reaction mixture was
stirred for 2.5 h at 50 °C. The mixture was cooled on an ice
bath, and the pH was adjusted to 4 with HCl. The precipitated
product was collected by filtration to afford 7r (101 mg, 90%):
IR (KBr) 1734, 1624 cm-1; FABMS m/z 351 (M + H+). Anal.
(C16H9F3N2O4‚0.3H2O) C, H, N.
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 s
9a-d,f: [5-Am in o-2-(4-am in o-3-flu or oph en yl)-6,8-diflu or o-
4H-4-oxo-1-ben zop yr a n -7-yl]m eth yl Aceta te (9a ). A mix-
ture of 6l (6.02 g, 11.9 mmol), AcOH (160 mL), and H2SO4 (40
mL) was stirred at 100 °C for 35 min. The reaction mixture
was cooled on an ice bath and poured into ice-water. The
mixture was extracted with EtOAc, and the organic layer was
washed with 1 N NaOH twice, water, and brine. The residue
was purified by chromatography (100:1 CHCl3/MeOH) followed
by recrystallization from CHCl3/MeOH/hexane to afford 9a
(2.43 g, 54%): IR (KBr) 1738, 1622 cm-1; FABMS m/z 379 (M
+ H+). Anal. (C18H13F3N2O4) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
(m eth ylsu lfin yl)-4H-1-ben zop yr a n -4-on e (7g). This com-
pound was obtained from 6g in a similar manner as described
for 7f (89%): IR (KBr) 1635 cm-1; FABMS m/z 369 (M + H+).
Anal. (C16H11F3N2O3S) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
(m eth ylsu lfon yl)-4H-1-ben zop yr a n -4-on e (7h ). This com-
pound was obtained from 6h in a similar manner as described
for 7f (64%): IR (KBr) 1637 cm-1; FABMS m/z 385 (M + H+).
Anal. (C16H11F3N2O4S‚0.6H2O) 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-7-h y-
d r oxy-4H-1-ben zop yr a n -4-on e (7i). This compound was
obtained from 6i in a similar manner as described for 7f
(52%): IR (KBr) 1651 cm-1; EIMS m/z 322 (M+). Anal.
(C15H9F3N2O3) 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-
4-oxo-1-ben zop yr a n -7-yl]m eth yl P r op ion a te (9b). This
compound was obtained in a similar manner as described for
9a except that propionic acid was used instead of AcOH and
the product was purified by chromatography (20:1 CHCl3/
MeCN) (32%): IR (KBr) 1728, 1651 cm-1; EIMS m/z 392 (M+).
Anal. (C19H15F3N2O4) 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-
4-oxo-1-ben zop yr a n -7-yl]m eth yl Hexa n oa te (9c). This
compound was obtained in a similar manner as described for
9a except that hexanoic acid was used instead of AcOH and
the product was purified by chromatography (20:1 CHCl3/
MeCN) followed by recrystallization from EtOAc/hexane
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
m eth oxy-4H-1-ben zop yr a n -4-on e (7j). This compound was
obtained from 6j in a similar manner as described for 7f

SAR of 5,4′-Diamino-6,8,3′-trifluoroflavone
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2065
(55%): IR (KBr) 1728, 1651 cm-1; EIMS m/z 392 (M+). Anal.
(C19H15F3N2O4) C, H, N.
added, and the mixture was extracted with EtOAc. The
organic layer was washed with water and brine, and the
residue was purified by preparative TLC (20:1 CHCl3/MeOH)
followed by recrystallization from EtOAc/hexane to afford 9h
(125 mg, 59%): IR (KBr) 1743, 1659 cm-1; FABMS m/z 491
(M + H+). Anal. (C26H29F3N2O4) 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-
4-oxo-1-ben zop yr a n -7-yl]m eth yl Stea r oa te (9i). This com-
pound was obtained in a similar manner as described for 9h
except that stearoyl chloride was used instead of decanoyl
chloride (two steps, 29%): IR (KBr) 1738, 1657 cm-1; FABMS
m/z 603 (M + H+). Anal. (C34H45F3N2O4) 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-
4-oxo-1-b en zop yr a n -7-yl]m et h yl 4-(Dim et h ya m in o)b u -
tyr a te (9j). To a solution of 4-(dimethylamino)butyric acid
hydrochloride (3.99 g, 23.8 mmol) in DMF (50 mL) was added
1,1′-carbonyldiimidazole (3.86 g, 23.8 mmol), and the mixture
was stirred at 80 °C for 2.5 h. Compound 10 (1.00 g, 2.38
mmol) was added, and the mixture was further stirred for 2
h. Water was added, and the mixture was extracted with
EtOAc. The organic layer was washed with brine, and the
residue was purified by chromatography (9:1 CHCl3/MeOH)
to afford the 4-(dimethylamino)butyrate of 10 (1.27 g, 100%):
1H NMR (90 MHz, CDCl3) δ 1.6-2.0 (m, 2H, COCH2CH2), 2.1-
2.5 (m, 4H, COCH2CH2CH2), 2.23 (s, 6H, N(CH3)2), 4.72 (d, J
) 5.7 Hz, 2H, OCH2), 5.2-5.5 (m, 2H, CHdCH2), 5.30 (s, 2H,
7-CH2), 5.7-6.2 (m, 1H, CHdCH2), 6.23 (br s, 2H, 5-NH2), 6.56
(s, 1H, 3-H), 7.10 (br s, 1H, 4′-NH), 7.5-7.7 (m, 2H, 2′,6′-H),
8.31 (t, J ) 7.9 Hz, 5′-H); FABMS m/z 534 (M + H+). To a
solution of the ester (1.27 g, 2.38 mmol) in THF (20 mL) were
added HCO2H-Et3N (1.6 mL, 12 mmol) and Pd(PPh3)4 (275
mg, 0.24 mmol), and the mixture was stirred under argon
atmosphere at room temperature for 19 h. Water was added,
and the mixture was extracted with EtOAc. The organic layer
was washed with water and brine, and the residue was
purified by chromatography (9:1 CHCl3/MeOH) followed by
recrystallization from EtOAc/hexane to afford the free base of
9h (567 mg, 53%). This was converted into the hydrochloride
in a similar manner as described for 9e: IR (KBr) 1732, 1624
cm-1; FABMS m/z 450 (M + H+). Anal. (C22H22F3N3O4‚HCl)
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-
4-oxo-1-ben zopyr an -7-yl]m eth yl Ch lor oacetate (9d). This
compound was obtained in a similar manner as described for
9a except that chloroacetic acid was used instead of AcOH and
the crude product was triturated with isopropyl ether (97%):
1H NMR (90 MHz, DMSO-d3) δ 4.40 (s, 2H, ClCH2), 5.36 (s,
2H, 7-CH2), 6.00 (br s, 2H, 4′-NH2), 6.66 (s, 1H, 3-H), 6.87 (t,
J ) 8.9 Hz, 1H, 5′-H), 7.06 (br s, 2H, 5-NH2), 7.5-7.7 (m, 2H,
2′,6′-H); FABMS m/z 415, 413 (M + H+).
[5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-4H-
4-oxo-1-ben zop yr a n -7-yl]m eth yl 3-Br om op r op ion a te (9f).
This compound was obtained in a similar manner as described
for 9a except that 3-bromopropionic acid was used instead of
AcOH and the crude product was purified by chromatography
(20:1 CHCl3/MeCN) (35%): 1H NMR (90 MHz, CDCl3) δ 2.97
(t, J ) 6.8 Hz, 2H, COCH2), 3.58 (t, J ) 6.8 Hz, 2H, BrCH2),
5.36 (t, J ) 1.5 Hz, 2H, 7-CH2), 6.49 (s, 1H, 3-H), 6.83 (t, J )
8.7 Hz, 1H, 5′-H), 7.4-7.7 (m, 2H, 2′,6′-H); FABMS m/z 473,
471 (M + H+).
[5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-4H-
4-oxo-1-b en zop yr a n -7-yl]m et h yl (Dim et h yla m in o)a ce-
ta te (9e). To a solution of 9d (1.50 g, 3.63 mmol) in DMF (30
mL) were added dimethylamine hydrochloride (1.48 g, 18.2
mmol) and K2CO3 (2.50 g, 18.2 mmol), and the reaction
mixture was stirred at 50 °C for 30 min. Water was added,
and the mixture was extracted with EtOAc. The organic layer
was washed with water and brine. The residue was recrystal-
lized from EtOAc/hexane to afford the free base of 9e (1.18 g,
77%). To a solution of the free base in EtOAc was added 1 N
HCl in 2-PrOH, and the precipitated hydrochloride was
collected by filtration: IR (KBr) 1753, 1653 cm-1; FABMS m/z
422 (M+H+). Anal. (C20H18F3N3O4‚HCl‚0.4H2O) 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-
4-oxo-1-ben zop yr a n -7-yl]m eth yl 3-(Dim eth yla m in o)p r o-
p ion a te (9g). This compound was obtained in a similar
manner as described for 9e (86%) and converted into the
hydrochloride: IR (KBr) 1738, 1622 cm-1; FABMS m/z 436 (M
+ H+). Anal. (C21H20F3N3O4‚HCl‚0.3H2O) C, H, N.
2-[4-[(Allyloxyca r b on yl)a m in o]-3-flu or op h e n yl]-5-
a m in o-6,8-d iflu or o-7-(h yd r oxym eth yl)-4H-1-ben zop yr a n -
4-on e (10). To a solution of 7l (6.72 g, 20.0 mmol) in pyridine
(200 mL) was added allyl chloroformate (10.6 mL, 100 mmol),
and the reaction mixture was stirred at room temperature for
1.5 h. Water was added, and the precipitated product was
collected by filtration. This was dissolved in EtOH (500 mL),
and 2 N NaOH (18 mL) was added. The mixture was stirred
at room temperature for 1.5 h. Water was added, and the
precipitated product was collected by filtration to afford 10
(6.92 g, 82%): 1H NMR (90 MHz, DMSO-d6) δ 4.5-4.7 (m, 4H,
7-CH2 and OCH2), 5.1-5.5 (m, 2H, CHdCH2), 5.7-6.2 (m, 1H,
CHdCH2), 6.90 (s, 1H, 3-H), 7.01 (br s, 2H, 5-NH2), 7.7-8.0
(m, 3H, 2′,5′,6′-H), 9.79 (br s, 1H, 4′-NH); FABMS m/z 421 (M
+ H+).
[5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-4H-
4-oxo-1-ben zop yr a n -7-yl]m eth yl Deca n oa te (9h ). To a
solution of 10 (420 mg, 1.00 mmol) in DMF (20 mL) were added
Et3N (1.4 mL, 10 mmol) and decanoyl chloride (1.3 mL, 6.0
mmol), and the reaction mixture was stirred at room temper-
ature for 7 h. Water was added, and the mixture was
extracted with EtOAc. The organic layer was washed with
water and brine, and the residue was purified by chromatog-
raphy (4:1 CHCl3/hexane) to afford a decanoate of 10 (258 mg,
45%): 1H NMR (90 MHz, CDCl3) δ 0.7-1.0 (m, 3H, CH3), 1.0-
1.8 (m, 14H, (CH2)7CH3), 2.35 (t, J ) 6.4 Hz, 2H, COCH2), 4.72
(d, J ) 5.5 Hz, 2H, OCH2), 5.2-5.5 (m, 2H, CHdCH2), 5.30 (s,
2H, 7-CH2), 5.7-6.2 (m, 1H, CHdCH2), 6.58 (s, 1H, 3-H), 7.09
(br d, J ) 2.6 Hz, 1H, 4′-NH), 7.5-7.7 (m, 2H, 2′,6′-H), 8.31 (t,
J ) 8.6 Hz, 1H, 5′-H); FABMS m/z 575 (M + H+). To a solution
of the decanoate (249 mg, 0.434 mmol) in THF (20 mL) were
added HCO2H-Et3N (0.285 mL, 2.17 mmol) and Pd(PPh3)4 (51
mg, 0.044 mmol), and the mixture was stirred under argon
atmosphere at room temperature for 50 min. Water was
6,8-Diflu or o-2-[3-flu or o-4-(p iva loyla m in o)p h en yl]-7-
[(m eth ylsu lfon yloxy)m eth yl]-5-(pivaloylam in o)-4H-1-ben -
zop yr a n -4-on e (11). To a solution of 6l (3.30 g, 6.55 mmol)
in DMF (70 mL) were added Et3N (4.6 mL) and methanesulfo-
nyl chloride (1.0 mL, 13 mmol), and the reaction mixture was
stirred at room temperature for 10 min. Water was added,
and the mixture was extracted with EtOAc twice. The organic
layer was washed with water and brine. Evaporation of the
solvent gave 11 (3.57 g, 94%): 1H NMR (90 MHz, CDCl3) δ
1.36 (s, 9H, C(CH3)3), 1.38 (s, 9H, C(CH3)3), 3.10 (s, 3H, CH3S),
5.48 (br s, 2H, 7-CH2), 6.69 (s, 1H, 3-H), 7.5-7.9 (m, 3H, 2′,6′-H
and 4′-NH), 8.61 (t, J ) 8.4 Hz, 1H, 5′-H), 10.4 (br s, 1H, 5-NH);
FABMS m/z 583 (M + H+).
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
(m eth oxym eth yl)-4H-1-ben zop yr a n -4-on e (12a ). A mix-
ture of 11 (800 mg, 1.37 mmol) and MeOH (200 mL) was
refluxed for 24 h, and the solvent was removed under reduced
pressure. To the residue was added H2SO4 (15 mL), and the
mixture was stirred at 50 °C for 15 min. The reaction mixture
was poured into ice-water, and the pH was adjusted to 9 with
10 N NaOH. The mixture was extracted with EtOAc, and the
organic layer was washed with water and brine. The residue
was purified by chromatography (9:1 CHCl3/MeCN) and
recrystallized from EtOAc to afford 12a (267 mg, two steps,
56%): IR (KBr) 1655 cm-1; EIMS m/z 350 (M+). Anal.
(C17H13F3N2O3) C, H, N
5-Am in o-2-(4-am in o-3-flu or oph en yl)-6,8-diflu or o-7-[(pr o-
p yloxy)m eth yl]-4H-1-ben zop yr a n -4-on e (12b). A mixture
of 11 (582 mg, 1.00 mmol) and 1-propanol (100 mL) was stirred
at 100 °C for 2 h, and the solvent was removed under reduced
pressure. To the residue was added H2SO4 (30 mL), and the
mixture was stirred at 100 °C for 15 min. The reaction
mixture was poured into ice-water, and the pH was adjusted

2066 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Akama et al.
to 7 with 10 N NaOH. The precipitated product was collected
by filtration, purified by chromatography (100:1 CHCl3/MeOH),
and recrystallized from EtOAc/hexane to afford 12b (286 mg,
two steps, 76%): IR (KBr) 1626 cm-1; FABMS m/z 379 (M +
H+). Anal. (C19H17F3N2O3) C, H, N.
afford 12g (106 mg, 29%): IR (KBr) 1657, 1647 cm-1; FABMS
m/z 434 (M + H+). Anal. (C22H22F3N3O3‚0.2 H2O) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
[(d im eth yla m in o)m eth yl]-4H-1-ben zop yr a n -4-on e (12h ).
To a solution of 11 (800 mg, 1.38 mmol) in DMF (10 mL) were
added dimethylamine hydrochloride (558 mg, 6.85 mmol) and
K2CO3 (945 mg, 6.85 mmol), and the reaction mixture was
stirred at room temperature for 45 min. Water was added,
and the mixture was extracted with EtOAc. The organic layer
was washed with water and brine. The solvent was removed
under reduced pressure to afford the 7-(dimethylamino)methyl
derivative (727 mg, 100%): 1H NMR (90 MHz, CDCl3) δ 1.36
(s, 9H, C(CH3)3), 1.38 (s, 9H, C(CH3)3), 2.36 (s, 6H, N(CH3)2),
3.81 (br s, 2H, 7-CH2), 6.67 (s, 1H, 3-H), 7.5-7.8 (m, 3H, 2′,6′-
H, and 4′-NH), 8.59 (t, J ) 8.4 Hz, 1H, 5′-H), 10.4 (br s, 1H,
5-NH); FABMS m/z 532 (M + H+). This compound (715 mg,
1.35 mmol) was treated with H2SO4 as in the case of 7f and
purified by chromatography (9:1 CHCl3/MeOH) followed by
recrystallization from MeOH/EtOH to afford the free base of
12h (360 mg, 73%), which was converted into the hydrochloride
in the similar manner as described for 12f: IR (KBr) 1654
cm-1; FABMS m/z 364 (M + H+). Anal. (C18H16F3N3O2‚-
HCl‚0.5H2O) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
[(h exyloxy)m eth yl-4H-1-ben zop yr a n -4-on e (12c). This
compound was obtained in a similar manner as described for
12b except that 1-hexanol was used instead of 1-propanol (two
steps, 79%): IR (KBr) 1655 cm-1; FABMS m/z 421 (M + H+).
Anal. (C22H23F3N2O3) 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-7-[[2-
(d im e t h yla m in o)e t h oxy]m e t h yl]-4H -1-b e n zop yr a n -4-
on e (12d ). To a solution of 11 (800 mg, 1.38 mmol) in DMF
(20 mL) were added 2-(dimethylamino)ethanol (0.28 mL, 2.8
mmol) and NaH (220 mg, 5.50 mmol) at 0 °C under argon
atmosphere. The reaction mixture was stirred for 2.5 h at
room temperature. Water was added, and the mixture was
extracted with EtOAc. The organic layer was washed with
water and brine. The residue was purified by chromatography
(9:1 CHCl3/MeOH) to afford a (dimethylamino)ethyl ether of
11 (477 mg, 60%): 1H NMR (90 MHz, CDCl3) δ 1.36 (s, 9H,
C(CH3)3), 1.38 (s, 9H, C(CH3)3), 2.35 (s, 6H, N(CH3)2), 2.63 (t,
J ) 5.7 Hz, 2H, CH2N), 3.71 (t, J ) 5.7 Hz, 2H, CH2O), 4.79
(br s, 2H, 7-CH2), 6.67 (s, 1H, 3-H), 7.6-7.9 (m, 3H, 2′,6′-H
and 4′-NH), 8.62 (t, J ) 8.4 Hz, 1H, 5′-H), 10.5 (s, 1H, 5-NH);
FABMS m/z 576 (M + H+). A mixture of the above ether (454
mg, 0.790 mmol) and H2SO4 (10 mL) was stirred at 50 °C for
15 min. The reaction mixture was poured into ice-water, and
the pH was adjusted to 7 with 10 N NaOH. The precipitated
product was collected by filtration and purified by chromatog-
raphy (20:1:1 CHCl3/MeOH/NH3)18 to afford the free base of
12d (266 mg, 83%). This was converted into the hydrochloride
5-Am in o-2-(4-a m in o-3-flu or op h en yl)-6,8-d iflu or o-7-(tr i-
m eth yla m m on iu m yl)-4H-1-ben zop yr a n -4-on e (12i). To a
solution of 12h (349 mg, 0.961 mmol) in THF (20 mL) was
added iodomethane (0.30 mL, 4.8 mmol), and the reaction
mixture was stirred at 40 °C for 30 min. The mixture was
cooled on an ice bath, and the precipitated product was
collected by filtration to afford 12i (468 mg, 96%): IR (KBr)
1657 cm-1; FABMS m/z 378 (M + H+). Anal. (C19H19F3IN3O2)
C, H, N.
Cell Gr ow th -In h ibitor y Activity. Assays were conducted
according to published method.9
in a similar manner as described for 9e: IR (KBr) 1655 cm-1
;
In Vivo An titu m or Activity. Tumor fragments (8 mm3)
were transplanted subcutaneously in the flank of 7-9-week
old 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/week for 2 weeks (n ) 5). Estradiol
propionate was administered on the day of initial administra-
tion of test compounds. Length and width of the tumor were
determined on days 0, 4, 7, 11, 14, and 18, and the tumor
volume were calculated according to the following equation:
FABMS m/z 408 (M + H+). Anal. (C20H20F3N3O3‚HCl‚0.6H2O)
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-7-[[3-
(d im eth yla m in o)p r op yloxy]m eth yl]-4H-1-ben zop yr a n -4-
on e (12e). This compound was obtained in a similar manner
as described for 12d except that 3-(dimethylamino)-1-propanol
was used instead of 2-(dimethylamino)ethanol (two steps,
4.6%): IR (KBr) 1653 cm-1; FABMS m/z 422 (M + H+). Anal.
(C21H22F3N3O3‚HCl‚1.6H2O) C, H, N.
5-Am in o-2-(4-a m in o-3-flu or op h en yl]-7-(a m in om eth yl)-
6,8-d iflu or o-4H-1-ben zop yr a n -4-on e (12f). To a solution
of 11 (1.12 g, 1.91 mmol) in DMF (20 mL) was added sodium
diformylamide (219 mg, 2.31 mmol),19 and the reaction mixture
was stirred at room temperature for 5 h. Water was added,
and the precipitated product was collected to afford the
7-(diformylamino)methyl derivative (1.06 g, 99%): 1H NMR
(270 MHz, CDCl3) δ 1.37 (s, 18H, C(CH3)3 × 2), 5.08 (br s, 2H,
7-CH2), 6.67 (s, 1H, 3-H), 7.6-7.8 (m, 2H, 2′,6′-H), 7.83 (d, J
) 4.0 Hz, 1H, 4′-NH), 8.62 (t, J ) 8.4 Hz, 1H, 5′-H), 8.94 (s,
2H, CHO × 2), 10.5 (s, 1H, 5-NH); FABMS m/z 560 (M + H+).
A mixture of the above compound (1.01 g, 1.81 mmol), EtOH
(20 mL), and HCl (20 mL) was refluxed for 2.5 h and poured
into ice-water. The precipitated product was collected by
filtration and recrystallized from DMF/H2O (9:1) to afford the
free base of 12f (404 mg, 67%). The free base was suspended
in MeOH (15 mL). To the suspension were added 4 N HCl in
EtOAc (0.67 mL) and isopropyl ether. The precipitated
hydrochloride was collected by filtration: IR (KBr) 1660
cm-1; FABMS m/z 336 (M + H+). Anal. (C16H12F3N3O2‚2HCl)
C, H, N.
tumor volume (mm3) ) {length (mm) × [width (mm)]2}/2
The tumor volumes at initial administration (V0) and on the
day of judgment (V) were calculated, and the tumor growth
rate (V/V0) was calculated. The T/C values were obtained from
the following equation:
T/C (%) )
(V/Vo of treated group)/(V/Vo of control group) × 100
Ack n ow led gm en t. We are grateful to Ms. Miki
Haibara, Akiko Mimura, Taimi Sano, and Miyoko Asano
for their excellent technical assistance.
Su p p or t in g In for m a t ion Ava ila b le: Completely as-
signed 1H NMR data of compounds 4, 6, 7, 9, and 12 and 1H
NMR spectra of compounds 7h ,r , 9e,g, and 12d ,e,g,h (18
pages). Ordering information is given on any current mast-
head page.
5-Am in o-2-(4-a m in o-3-flu or op h e n yl)-6,8-d iflu or o-7-
[(h exa n oyla m in o)m eth yl]-4H-1-ben zop yr a n -4-on e (12g).
To a solution of 12f (285 mg, 0.851 mmol) in DMF (20 mL)
were added Et3N (0.24 mL, 1.7 mmol) and hexanoyl chloride
(0.20 mL, 1.4 mmol) at -50 °C. The reaction mixture was
gradually warmed to 0 °C. Water was added, and the mixture
was extracted with EtOAc. The organic layer was washed with
water and brine. The residue was purified by chromatography
(100:1 CHCl3/MeOH) and trituration with isopropyl ether to
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J M970728Z