Structural studies on bioactive compounds. 40.1 synthesis and biological properties of fluoro-, methoxyl-, and amino-substituted 3-phenyl-4H-1-benzopyran-4-ones and a comparison of their antitumor activities with the activities of related 2-phenylbenzothiazoles

Article abstract of DOI:10.1021/jm060359j

A new series of fluoro-, methoxyl-, and amino-substituted isoflavones have been synthesized as potential antitumor agents based on structural similarities to known flavones and isoflavones (quercetin and genistein respectively) and antitumor 2-phenylbenzothiazoles. Target compounds were synthesized using palladium-catalyzed coupling methodologies to construct the central aryl carbon-carbon single bond. The new isoflavone derivatives were tested for in vitro activity in human breast (MDA-MB-468 and MCF-7) and colon (HT29 and HCT-116) cancer cell lines. Low micromolar GI₅₀ values were obtained in a number of cases, with the MDA-MB-468 cell line being the most sensitive overall. Notably, significant potentiation of growth inhibitory activity (GI₅₀ < 1 μM for 12d, 12f, 12h, 12k, 121, 12o but not the methylene-bridged derivative 12i) was observed when MDA-MB-468 cells were co-incubated with TBDD, a powerful inducer of cytochrome P450 (CYP)-1A1 activity, suggesting that isoflavone derivatives can act as substrates for CYP1A1 bioactivation.

Full text of DOI:10.1021/jm060359j

J. Med. Chem. 2006, 49, 3973-3981
3973
Structural Studies on Bioactive Compounds. 40.1 Synthesis and Biological Properties of Fluoro-,
Methoxyl-, and Amino-Substituted 3-Phenyl-4H-1-benzopyran-4-ones and a Comparison of
Their Antitumor Activities with the Activities of Related 2-Phenylbenzothiazoles
David A. Vasselin,† Andrew D. Westwell,‡ Charles S. Matthews,† Tracey D. Bradshaw,† and Malcolm F. G. Stevens*,†
Cancer Research UK Experimental Cancer Chemotherapy Research Group, Centre for Biomolecular Sciences, School of Pharmacy, UniVersity
of Nottingham, Nottingham NG7 2RD, U.K., and Welsh School of Pharmacy, Cardiff UniVersity, Redwood Building, King Edward VII AVenue,
Cardiff CF10 3XF, U.K.
ReceiVed March 28, 2006
A new series of fluoro-, methoxyl-, and amino-substituted isoflavones have been synthesized as potential
antitumor agents based on structural similarities to known flavones and isoflavones (quercetin and genistein
respectively) and antitumor 2-phenylbenzothiazoles. Target compounds were synthesized using palladium-
catalyzed coupling methodologies to construct the central aryl carbon-carbon single bond. The new isoflavone
derivatives were tested for in vitro activity in human breast (MDA-MB-468 and MCF-7) and colon (HT29
and HCT-116) cancer cell lines. Low micromolar GI50 values were obtained in a number of cases, with the
MDA-MB-468 cell line being the most sensitive overall. Notably, significant potentiation of growth inhibitory
activity (GI50 < 1 µM for 12d, 12f, 12h, 12k, 12l, 12o but not the methylene-bridged derivative 12i) was
observed when MDA-MB-468 cells were co-incubated with TBDD, a powerful inducer of cytochrome P450
(CYP)-1A1 activity, suggesting that isoflavone derivatives can act as substrates for CYP1A1 bioactivation.
Chart 1. Chemical Structures of Bioactive Flavones,
Isoflavones, and Benzothiazoles
Introduction
Flavanoids are plant polyphenols (e.g., quercetin, 1; Chart
1) that are widely represented in nature2 and exert promiscuous
biological effects on mammalian systems3 that might be
exploitable in therapeutic or chemopreventive stategies.4 Re-
cently, an SAR study of a library of natural and synthetic
flavonoids related to quercetin has shown that antiproliferative
activity against the human HT29 cell line is associated with
caspase activation.5 Less abundant in nature are the isoflavones,
represented by the soy constituent genistein (2), which perturb
a number of cancer-relevant molecular targets and have attracted
recent interest particularly in its potential role in preventing
mammary tumor progression.4,6
In an earlier paper we reported the synthesis of polyhydroxy-
lated 2-phenylbenzothiazoles (3) and compared their cytotox-
icities and pharmacological properties with those of quercetin
and genistein but were unable to identify any compounds with
exploitable antitumor activities.7 However, providentially, we
identified two related series of 2-arylbenzothiazoles with
uniquely selective properties. Thus, 2-(4-amino-3-methylphe-
nyl)-5-fluorobenzothiazole 4a (5F 203, NSC 703786), like other
agents of this planar arylamine class, exploits the arylhydro-
carbon receptor (AhR) to translocate the drug to cell nuclei8
wherein cytochrome P450 CYP1A1 is induced. Generation of
a reactive chemical intermediate(s) then forges DNA adducts
but only in sensitive tumor types (e.g., mammary, ovarian, and
lung tumor cell lines).9 Phortress, an L-lysylamide prodrug
modification of amine 4a, was equiactive with doxorubicin
against a panel of human breast tumor xenografts10 and is
currently in phase 1 clinical studies in the U.K.11 A related
structure, 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole 5 (GW
610, NSC 721648), has an extended spectrum of action
compared to 4a, notably in being active against some colon
cell lines (GI50 < 10 nM) that do not have inducible CYP1A1.12
In this paper we sought to examine whether 3-phenyl-4H-1-
benzopyran-4-ones (3-phenylchromones), decorated with ap-
propriate fluoro, methoxy, or amino substituents in the A and
C rings, possessed biological properties comparable to those of
their benzothiazole counterparts 4 and 5. Previously published
work has indicated that palladium(0) coupling chemistry can
be applied to forge the link between alkyl- or alkyloxy-
substituted phenylboronic acids and unsubstituted 3-haloben-
zopyranones,13 and we have explored this route to secure greater
structural variety in the A and C rings of 3-benzopyranones
(Figure 1).
* To whom correspondence should be addressed. Phone: +44 115
9513419. Fax: +44 115 9513412. E-mail: malcolm.stevens@
nottingham.ac.uk.
Chemistry
Synthesis of Fluoro- and Alkoxy-Substituted 3-Iodochro-
mones. Many 2-hydroxyacetophenones (6) required as starting
† University of Nottingham.
‡ Cardiff University.
10.1021/jm060359j CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/03/2006

3974 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Vasselin et al.
Table 1. Yields and Melting Points of 3-Iodo-4H-1-benzopyran-4-ones
8 and 3-Aryl-4H-1-benzopyran-4-ones 12
starting
material
general
yield
(%)
mp
(°C)
molecular
formulab
compd
methoda
8b
8c
8d
8e
8f
8g
8h
8i
7b
7c
7d
7e
Cd
Cd
Cd
Cd
Cd
Cd
Ce
Ce
Ce
Ce
D
D
D
D
D
D
D
D
D
78
66
85
63
41
27
59
64
43
29
20
50
95
99
76
93
79
36
96
67
42
27
72
52
102-103
107-109
120-122
135-137
93-95
118-120
103-105
112-113
143-145
157-159
182-184
194-196
78-79
138-139
136-138
181-183
152-154
221-223
171-173
165-167
146-148
169-171
178-180
181-183
C9H4FIO2
C9H4FIO2
C9H4FIO2
C9H3F2IO2
C9H3F2IO2
C9H3F2IO2
C10H7IO3
C10H7IO3
C10H7IO3
C11H8I2O4
C15H9BrO2
C15H9NO4
C16H12O3
Figure 1. Synthetic strategy to substituted 3-aryl-4H-1-benzopyran-
4-ones.
7fc
7gc
7hc
7ic
7jc
7kc
8a
8a
8a
8a
8a
8d
8d
8d
8c
Scheme 1. Synthesis of 3-Iodo-4H-1-benzopyran-4-ones 8a
8j
8k
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
C16H12O3
C17H14O4
C16H11FO3
C17H13FO4
C16H9FO4
C16H11FO3
C17H13FO4
C17H13FO4
C17H12F2O4
C17H12F2O4
C17H12F2O4
8c
D
D
D
D
8b
8g
8f
8e
D
a See Experimental Section. b Microanalytical and spectroscopic data for
new compounds provided in Supporting Information. c Prepared in situ from
the corresponding 2-hydroxyacetophenone (6) and used without further
purification. d CHCl3 as solvent. e MeOH as solvent.
a Reagents and conditions: (a) N,N-dimethylformamide dimethylacetal,
90 °C; (b) I2 in CHCl3 or MeOH.
materials were available commercially, but certain fluoro-
substituted precursors (6b, 6e, 6g) were synthesized via homo-
Fries rearrangement of the corresponding acetates, themselves
derived from fluoro-substituted phenols.14 Heating the acetates
in 1,2-dichlorobenzene and anhydrous AlCl3 afforded mixtures
of isomeric hydroxyacetophenones from which the required
isomers were recovered by column chromatography; attempted
syntheses of the 3,6- and 4,6-difluoro-2-hydroxyacetophenones
by this route were not successful. The di-O-benzylated hy-
droxyacetophenone 6l was synthesized by reacting 2,4,6-
trihydroxyacetophenone with benzyl chloride under basic
conditions (K2CO3) in DMF.15
Scheme 2. Attempted Borylation of
3-Iodo-4H-1-benzopyran-4-one 8a
Condensation of 2-hydroxyacetophenones (6a-l) with dim-
ethylformamide dimethylacetal afforded enamines (7a-l) that
could be cyclized without further purification directly to
3-iodochromones (8) (Scheme 1) with iodine in CHCl3 or
MeOH, a reaction discovered by Gammill.16 In some cases it
was more efficient to purify crude enamines (eg 7b-e)
chromatographically prior to iodinative cyclization. Yields of
iodochromones were moderate (Table 1) with a tendency for
lower yields in the fluorinated substrates. Attempted conversion
of 6l to 5,7-di-O-benzyl-3-iodochromone (8l) gave only an
inseparable mixture of products.
Suzuki-Miyaura and Stille Couplings. Initially a Pd(0)-
catalyzed borylation of 3-iodochromone (8a) was attempted on
the basis of Murata’s pinacol borane/Pd(dppf)Cl2/base meth-
odology in anhydrous dioxane.17 In no case was the required
boronate ester (9) obtained. The major products (from 1H NMR)
were the dehalogenated chromone (10) and the 3,3′-bis-
chromone product of bimolecular coupling (11) (Scheme 2).
Cross-coupling of 3-iodochromones (8) with, optimally, 3-4
equiv of substituted phenylboronic acids in the presence of Pd-
(PPh3)4 and Na2CO3 in benzene gave 3-aryl-4H-benzopyran-
4-ones (12a-o) generally in moderate to high yields (Scheme
3, Table 1). Because highly efficient coupling was limited to
boronic acids bearing electron-donating methoxyl or electron-
a Reagents and conditions: (a) bis(diphenylphosphino)ferrocene pal-
ladium(0), bis(pinacolato)diboron, NEt3, dioxane, 25 °C.
neutral groups, a study was conducted to reverse the “polarity”
of the coupling partners. Rewardingly, several 3-iodochromones
(8) were converted efficiently to air-stable and crystallizable
3-(trimethylstannyl)chromones (13a-g), employing catalytic Pd-
(PPh3)4 and hexamethylditin in dioxane (Scheme 3).
Stille couplings, despite their unpredictable outcomes,18
seemed a logical methodology to adapt to synthesize 3-(4-
nitrophenyl)chromones. Attempted Stille coupling between
3-iodochromone (8a) and (4-nitrophenyl)trimethylstannane did
not lead to the formation of compound 14a. We therefore
examined the interaction of 3-(trimethylstannyl)chromone (13a)
and 4-iodonitrobenzene as a reverse process for the formation
of isoflavone 14a. On the basis of similarities between the
coupling partners, reaction conditions favorable for coupling
iodoarenes and vinylstannanes were employed.19 A range of
catalysts (Pd/C, PdCl2, Pd2(dba)3, Pd(PPh3)4, Pd(MeCN)2Cl2,
Pd(PhCN)2Cl2, Pd(PPh3)2Cl2, and Pd(dppf)Cl2‚CH2Cl2), ligands
(Ph3As, LiCl, LiI), and solvents (dioxane, THF, and NMP) were
reacted in a parallel mode, but in all cases 1H NMR analysis of

Benzopyranones and Their ActiVities
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3975
Scheme 3. Suzuki-Miyaura and Stille Couplingsa
a Reagents and conditions: (a) substituted arylboronic acid, Pd(PPh3)4, Na2CO3, benzene reflux; (b) Me6Sn2, Pd(PPh3)4, dioxane, reflux; (c)
4-iodonitrobenzene, Pd2(dba)3, CuI, LiCl, Ph3As, NMP, 80 °C.
Table 2. Yield and Melting Points of
3-(Trimethylstannyl)-4H-1-benzopyran-4-ones 13 and
3-(4-Nitrophenyl)-4H-1-benzopyran-4-ones 14
Scheme 4. Synthesis of
3-(4-Aminophenyl)-4H-benzopyran-4-ones (16)a
starting
material
general
yield
(%)
mp
(°C)
molecular
formulab
compd
methoda
13b
13c
13d
13e
13f
13g
14b
14c
14d
14e
8d
8c
8i
8h
8g
8e
13b
13c
13d
13e
E
E
E
E
E
E
F
F
F
F
90
80
73
38
77
66
53
46
26
23
118-120
106-108
83-85
C12H13FO2Sn
C12H13FO2Sn
C13H16O3Sn
C13H16O3Sn
C12H12F2O2Sn
C12H12F2O2Sn
C15H8FNO4
C15H8FNO4
C16H11NO3
87-89
171-173
176-178
235-237
222-225
229-231
241-243
C16H11NO3
a See Experimental Section. b Microanalytical and spectroscopic data for
new compounds provided in Supporting Information.
a Reagents and conditions: (a) SnCl2‚2H2O, EtOH, reflux, then excess
NaOH; (b) 8a, diphenylphosphinoferrocene palladium(0), K2CO3, dioxane-
ethanol, reflux; (c) benzophenone imine, Pd2(dba)3, NaOtBu, rac-BINAP,
toluene, 80 °C; (d) 2 M HCl in THF, 25 °C.
the reactions revealed a complex mixture of products, including
low yields of the required nitrophenylchromone (14a) contami-
nated with the 3,3′-bis-chromone (11) and starting material.
Eventually, optimal conditions employing the stannylated
substrate (13), Pd2(dba)3 catalyst (1 mol %), Ph3As (8 mol %),
copper(I) iodide (10 mol %), and LiCl (3 mol equiv) in dry
NMP at 80 °C were adopted. Products 14a-e were isolated in
moderate yields, whereas only traces of the difluorinated
products 14f and 14g were detected in the reaction mixtures
(1H NMR) from 13f and 13g, and products (∼25-30%) were
assigned as the 6,7-difluoro- and 7,8-difluoro-4H-1-benzopyran-
4-ones (15a and 15b), respectively, formed following catalytic
reductive destannylation. Yields and physical properties of
3-(trimethylstannyl)chromones (13) and substituted 3-(4-nitro-
phenyl)chromones (14) are recorded in Table 2.
16a was formed in only 14% yield from 3-iodochromone (8a)
and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (17).
No reaction occurred between chlorophenylchromone (12a) and
the surrogate amine (benzophenone imine) using racemic
BINAP as the ligand, but coupling of 4-bromophenylchromone
12b afforded a crude imine 18, which was successfully
hydrolyzed using 2 M HCl in dioxane to give amine 16a in
61% overall yield (Scheme 4).
As expected, the nature of the substituent at C(3) of the 4H-
benzopyran-4-one moiety has a significant bearing on the
1
chemical shift of the proton H-2. In the H NMR spectrum of
the unsubstituted 3-iodobenzopyranone (8a), this proton reso-
nates at δ 8.32 in CDCl3. Fluorination in the phenyl ring has a
minimal effect on H-2, which resonates in the range δ 8.29-
8.34 for 8b-g. An additional aryl residue at C(3) imparts a
small shielding effect (relative to iodo) with chemical shifts in
Synthesis of 3-(4-Aminophenyl)-4H-benzopyran-4-ones.
High yields of amines 16a-c were obtained by tin(II) chloride
reduction of precursor nitro compounds 14a-c. Less efficient
were direct Pd(0)-mediated routes to these amines. For example,

3976 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Vasselin et al.
Table 3. Growth Inhibitory Propertiesa of 3-Aryl-4H-benzopyran-4-ones
with Pearson correlation coefficients less than 0.6.21 Unlike the
benzothiazole compound whose selective antitumor activity
within the NCI panel correlated with inducible CYP1A1
activity,22 a comparable mechanism involving CYP1A1-medi-
ated DNA damage could be argued as unlikely.8,9 However,
Western blot analyses performed using lysates of MDA-MB-
468 cells treated for 24 h with 12h (1, 10, 100 µM) revealed
weak induction of CYP1A1 protein following exposure of cells
to 10 or 100 µM 12h (data not shown).
and Reference Compounds against Human Cancer Cell Lines in Vitrob
GI50 (µM)c
compd
MDA-MB-468
MCF-7
HT29
63
2.6
88.0
74.0
HCT-116
4ad
<0.0001
<0.0001
29.0
27.2
66.5
0.9
<0.0001
0.008
>100
16
5.4
5e
12d
12e
12f
12h
12i
12j
12k
12l
12m
12n
12o
16a
16b
16c
>100
81.4
88.1
93.1
60.0
43.8
90.6
75.5
41.5
46.7
47.2
64.9
8.8
64.0
>100
>100
65.3
88.2
1.5
9.9
Paucity of growth inhibition by, for example, 12h in MCF-7
(GI50 ) 93 µM) contrasting with MDA-MB-468 (GI50 ) 0.9
µM) cells also appears at first glance to be inconsistent with a
mode of action involving CYP1A1-mediated DNA damage.
However, multiple mechanisms may be engaged and could
include an estrogenic effect that would mask growth inhibitory
effects in ER+ (e.g., MCF-7) cell lines. Indeed, the 6-hydroxy-
lated product 4c of CYP1A1-metabolized metabolism of 2-(4-
amino-3-methylphenyl)benzothiazole (4b)22,23 and apigenin
possess estrogenic mitogenic properties, impeding activity of
4b and 5,4′-diaminoflavone,24 respectively, in MCF-7 cells. In
addition, 4c and 4b have been shown to inhibit induced CYP1A1
activity, crucial for 4b-evoked growth inhibition. It is believed
that these mechanisms combine to generate the observed
biphasic dose response relationship for 4b. Indeed, the dose
response observed following treatment of MDA-MB-468 cells
with 12h is also biphasic, inferring a role for metabolic processes
(Figure 2). Moreover, the CYP1A1 antagonist 4c22 was able to
completely abrogate growth inhibitory activity of 12h, increasing
the GI50 value to greater than 100-fold. It has been established
that of the isoflavone analogues examined (12h, 12k, 12e, and
12g) all inhibit deethylation of ethoxyresorufin25 (CYP1A1
activity, EROD assay) in a dose-dependent manner (IC50 < 100
µM), as shown in Figure 3. One explanation for this is that
these compounds act as substrates for CYP1A1 metabolism but
do not themselves, or only weakly, induce cyp transcription.
As noted, 12h did confer meagre induction of the CYP1A1
protein.
17.6
87.0
88.0
22.6
7.2
31.7
8.1
6.1
8.5
80.2
67.8
39.7
8.1
26.0
17.3
8.5
58.9
41.9
17.2
6.3
21.6
6.0
4.8
6.4
7.9
8.6
7.2
a MTT assays: 72 h of drug exposure. b Cancer cell line origin: MDA-
MB-468 (breast); MCF-7 (breast); HT29 (colon); HCT-116 (colon). c Mean
of two experiments. d Ref 22. e Ref 12.
tight ranges of δ 7.98-8.07 for series 12a,b,d-o and δ 8.11-
8.16 for the nitrophenyl analogues 12c and 14a-e. The
3-(trimethylstannyl) group exerts a stronger shielding influence
with H-2 resonating in the range δ 7.50-7.61 for compounds
13a-g.
Biological Results and Discussion
Evaluation of the series of 3-aryl-4H-1-benzopyran-4-ones
12 and 16 in an in vitro panel of human cancer cell lines (breast
MDA-MB-468, breast MCF-7, colon HCT-116 and colon HT29
carcinoma cells) allowed profiles of activity to be compared
with those of (aminophenyl)benzothiazole (4a,b) of defined
mechanism8,9,11 and the (dimethoxyphenyl)benzothiazole (5) of
unknown mechanism.12 Overall, the MDA-MB-468 (ER-) cell
line was the most chemosensitive line (Table 3) and benzothia-
zoles 4a and 5 were the most potent compounds, particularly
against the breast models. Within the series 12 bearing at least
one methoxy group in ring C, the presence and position of
fluorine atoms in ring A have a significant impact on in vitro
activity. Higher potency against MDA-MB-468 cells is seen in
the 6-fluorobenzopyranones (12h,i) (GI50 g 1.5 µM) and the
6,8-difluoro analogue (12n) (GI50 ) 6.3 µM) than in the
7-fluoro- (12j), 6,7-difluoro- (12m) and 7,8-difluorobenzopy-
ranones (12o) (GI50 < 25 µM) and in unfluorinated agents
(12d-f) (GI50 > 25 µM). Evidently, in the monofluoro series,
a fluoro group in the 6-position in the 3-arylbenzopyranone
series has an enhancing growth inhibitory role, as is the case
for the 5-fluoro group in the two benzothiazole compounds 4a
and 5.
The aminoisoflavonoid 16a yielded GI50 values less than 10
µM in MDA-MB-468 and HT29 carcinoma cells and GI50 values
of 65 and 17 µM in MCF-7 and HCT 116 cell lines, respectively.
Fluorinated congeners 16b and 16c lead to enhanced potency
in all cell lines (GI50 < 10 µM). However, these agents did not
match the exquisite potency of the benzothiazoles (4a or 5) in
the breast cell lines (Table 3), and in contrast to aminophenyl-
benzothiazole analogues, no selectivity between CYP1A1
inducible and noninducible phenotypes could be determined.
Representative compounds in series 12 and 16 were also
evaluated in the NCI 60-cell panel in vitro.20 Mean GI50 values
were in the 50-100 µM range with no cell line selectivity
observed (data not shown). Compounds were not COMPARE
positive with the exemplar (4-aminophenyl)benzothiazole (4a)
Intriguingly, when MDA-MB-468 cells were co-incubated
with 2,3,7,8-tetrabromodioxin (TBDD, 10 nM) and isoflavone
analogues (12d, 12f, 12h, 12i, 12k, 12l, 12o), growth inhibition
was significantly potentiated, giving GI50 values less than 1 µM
(Figure 3). Such observations corroborate a role for CYP1A1
activity in antitumor activity, since TBDD is a potent AhR
ligand and powerful inducer of CYP1A1 activity. Furthermore,
analogues that alone cause negligible growth inhibition (e.g.,
12l and 12k where GI50 > 25 µM) evoke a cytotoxic response
at 3 µM in combination with TBDD and an overall biphasic
profile. TBDD is not, however, a CYP substrate, being resistant
to metabolism, and alone demonstrates no growth inhibitory
properties against MDA-MB-468 cells. The notable exception
within series 12 contains a methylenedioxy bridge (12i). GI50
values of 1.5 and 2.0 µM in the absence and presence of 10
nM TBDD were not potentiated by TBDD-induced CYP1A1.
The methylene bridge of 12i may render this analogue resistant
to metabolic modification. Thus, CYP-mediated production of
(DNA-reactive) putative oxonium ion intermediates of isofla-
vones 12d, 12f, 12h, 12k, 12l, and 12o is suggested.
A final piece of evidence to support mediation of activity
via the AhR/CYP1A1 pathway has emerged following treatment
of MDA-MB-435 cells with 12h, 12j, 12m, and 12n. MDA-
MB-435 cells express a splice variant of ARNT that traps the
AhR constitutively in the nucleus.26 Therefore, cytoplasmic
AhR/ligand bound complexes cannot be formed. No growth

Benzopyranones and Their ActiVities
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3977
Figure 2. Effect of 12d, 12f, 12h, 12k, 12l, 12o, and 12i on the growth of MDA-MB-435 cells. MTT assays determined cellular viability following
72 h of exposure to isoflavone analogues in the absence and presence of 10 nM TBDD.
inhibitory activity was encountered, GI50 values greater than
In conclusion, the evidence presented here indicates that novel
30 µM being consistently observed (data not shown).
substituted isoflavones such as 12h, while being only moderately

3978 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Vasselin et al.
7.53 (1H, m, ArH), 6.73 (1H, m, ArH), 2.64 (3H, s, CH3); MS-CI
(m/z) 173 (M+ + 1). Anal. (C8H6F2O2) C, H.
The di-O-benzylated hydroxyacetophenone (6l) was synthesized
by reacting 2,4,6-trihydroxyacetophenone with benzyl chloride.
General Method B for the Synthesis of Substituted 3-Dim-
ethylamino-1-(2-hydroxyphenyl)propenones.16 A mixture of a
substituted 2-hydroxyacetophenone (1.2 mmol) and N,N-dimeth-
ylformamide dimethylacetal (2.4 mmol, 2 mol equiv) was heated
at 90 °C overnight and allowed to cool. The residual solvent was
removed by vacuum evaporation, and the product was purified by
column chromatography using hexanes-EtOAc as eluent.
3-Dimethylamino-1-(2-hydroxyphenyl)propenone (7a). 7a was
obtained from 2-hydroxyacetophenone (6a) as a brown solid (82%),
1
mp 127-128 °C (lit.,16 132-134 °C); H NMR δH (CDCl3) 7.88
(1H, d, J ) 12.5, olefinic CH), 6.60-6.75 (4H, m, ArH), 5.75 (1H,
d, J ) 12.5, olefinic CH), 3.11 (3H, s, NCH3), 2.89 (3H, s, NCH3).
The following new propenones were synthesized.
Figure 3. Inhibition of CYP1A1 activity by 12h (EROD assay). The
ability of 12h (1, 10, and 100 µM) to inhibit deethylation of
ethoxyresorufin, catalyzed by CYP1A1 microsomes, was determined.
Microsomes were excluded from negative control incubates to ascertain
noncatalyzed production of resorufin. Control incubates contained
DMSO vehicle only. Phortress (100 µM)11 was used as a positive control
for inhibition of catalysis.
3-Dimethylamino-1-(3-fluoro-2-hydroxyphenyl)propenone (7b).
7b was obtained from 3-fluoro-2-hydroxyacetophenone (6b) as a
yellow solid (46%), mp 110-112 °C; IR (KBr) 1640, 1614, 1587,
1
1557, 1518, 1494, 1441, 1363, 1272, 1219 cm-1; H NMR δH
(CDCl3) 14.26 (1H, s, OH), 7.93 (1H, d, J ) 12.0, olefinic CH),
7.47 (1H, d, J ) 8.0, H-6), 7.17 (1H, m, ArH), 6.73 (1H, m, ArH),
5.75 (1H, d, J ) 12.0, olefinic CH), 3.23 (3H, s, NCH3), 3.00 (3H,
s, NCH3). HRMS-EI (m/z): calcd for C11H11FNO2, 208.0774 (M+
- 1); found, 208.079 65.
active in their own right, have potent in vitro antitumor activity
when coadministered with a CYP1A1 inducer such as TBDD.
The isoflavones examined appear to inhibit CYP1A1 activity
(EROD assay) and are therefore likely CYP1A1 substrates,
despite their apparent weak ability to induce CYP1A1. The
development of other CYP-inducing agents that will uncover
the latent antitumor activity associated with these novel isofla-
vones may add further interest to potential therapeutic applica-
tion; for example, the selective AhR modulator 6-alkyl-1,3,8-
trichlorodibenzofuran has been shown to exhibit agonist activity
with respect to induction of CYP1A1 (Fretland et al.27).
3-Dimethylamino-1-(4-fluoro-2-hydroxyphenyl)propenone (7c).
7c was obtained from 4-fluoro-2-hydroxyacetophenone (6c) as light-
brown crystals (65%), mp 113-115 °C; IR (KBr) 1635, 1614, 1545,
1
1511, 1425, 1373, 1279, 1238 cm-1; H NMR δH (CDCl3) 14.43
(1H, s, OH), 7.90 (1H, d, J ) 12.0, olefinic CH), 7.69 (1H, m,
ArH), 6.57 (2H, m, ArH), 5.69 (1H, d, J ) 12.0, olefinic CH),
3.21 (3H, s, NCH3), 2.99 (3H, s, NCH3); MS-CI (m/z) 210 (M+
1). Anal. (C11H12FNO2) C, H, N.
+
3-Dimethylamino-1-(5-fluoro-2-hydroxyphenyl)propenone (7d).
7d was obtained from 5-fluoro-2-hydroxyacetophenone (6d) as
yellow crystals (60%), mp 106-107 °C; IR (KBr) 1634, 1592,
Experimental Section
1540, 1489, 1433, 1422, 1400, 1342, 1287, 1272, 1243, 1226 cm-1
;
1H NMR δH (CDCl3) 13.62 (1H, s, OH), 7.92 (1H, d, J ) 12.5,
olefinic CH), 7.36 (1H, dd, J ) 2.5, 10.0, ArH), 7.10 (1H, td, J )
2.5, 10.0, ArH), 6.90 (1H, m, ArH), 5.68 (1H, d, J ) 12.5, olefinic
CH), 3.23 (3H, s, NCH3), 3.03 (3H, s, NCH3); MS-CI (m/z) 210
(M+ + 1). Anal. (C11H12FNO2) C, H, N.
All new compounds were characterized by elemental analysis
(% C, H, and N values within 0.4% of theoretical values). Melting
points were measured on a Gallenkamp apparatus and are reported
uncorrected. IR spectra were recorded on a Mattson 2020 Galaxy
series or Perkin-Elmer Spectrum One FT-IR spectrometer. Mass
spectra were recorded on a Micromass Platform spectrometer, an
AEI MS-902 (nominal mass), a VG Micromass 7070E, or a Finigan
MAT900XLT spectrometer (accurate mass). NMR spectra were
recorded on a Bruker ARX 250 instrument. Coupling constants are
in Hz. TLC systems for routine monitoring of reaction mixtures
and for confirming the homogeneity of analytical samples used
Kieselgel 60F254 (0.25 mm) silica gel TLC aluminum backed sheets.
Sorbsil silica gel C 60-H (40-60 µm) was used for flash
chromatographic separations. All commercially available starting
materials were used without further purification.
General Method A for the Synthesis of Substituted 2-Hy-
droxyacetophenones. Typically, to prepare hydroxyacetophenones
6b, 6e, 6g, which were not available commercially, substituted
phenyl acetates (1.50 mmol) were added to a mixture of anhydrous
aluminum(III) chloride (1.65 mmol, 1.1 mol equiv) in 1,2-
dichlorobenzene (25 mL) and heated to 100 °C for 24 h. To the
cooled mixtures was added dichloromethane (25 mL), and the
solutions were poured into iced 2 M HCl (40 mL). The aqueous
layers were extracted with further portions of DCM (2 × 25 mL),
and the combined organic fractions were dried (MgSO4) and
concentrated under vacuum. Products were purified by flash column
chromatography using cyclohexanes-EtOAc as eluent. The fol-
lowing new 2-hydroxyacetophenone was synthesized by general
method A.
3-Dimethylamino-1-(3,4-difluoro-2-hydroxyphenyl)prope-
none (7e). 7e was obtained from 3,4-difluoro-2-hydroxyacetophe-
none (6e) as yellow-orange crystals (37%), mp 171-173 °C; IR
(KBr) 1631, 1553, 1509, 1492, 1459, 1436, 1420, 1370, 1321, 1258
1
cm-1; H NMR δH (CDCl3) 14.76 (1H, s, OH), 7.92 (1H, d, J )
12.0, olefinic CH), 7.43 (1H, m, ArH), 6.60 (1H, m, ArH), 5.65
(1H, d, J ) 12.0, olefinic CH), 3.23 (3H, s, NCH3), 3.00 (3H, s,
NCH3); MS-CI (m/z) 228 (M+ + 1). Anal. (C11H11F2NO2) C, H,
N.
3-Dimethylamino-1-(4,6-dibenzyloxy-2-hydroxyphenyl)prope-
none (7l). 7l was obtained from 4,6-dibenzyloxy-2-hydroxyac-
etophenone (6l) as a yellow solid (83%), mp 125-127 °C; IR (KBr)
1
1608, 1542, 1478, 1427, 1399, 1357, 1280, 1235, 1208 cm-1; H
NMR δH (CDCl3) 15.97 (1H, s, OH), 7.83 (1H, d, J ) 12.5, olefinic
CH), 7.41 (10H, m, ArH), 6.11-6.25 (3H, m, ArH and olefinic
CH), 5.07 (2H, s, OCH2), 5.00 (2H, s, OCH2), 3.05 (3H, br s,
NCH3), 2.32 (3H, br s, NCH3). HRMS-EI (m/z): calcd for C25H25-
NO4, 403.1783; found, 403.1765.
General Method C for the Synthesis of Substituted 3-Iodo-
4H-1-benzopyran-4-ones (8).16 To a solution of a substituted
3-(dimethylamino)-1-(2-hydroxyphenyl)propenone (7, 0.6 mmol)
in CHCl3 (15 mL) or MeOH (15 mL) was added iodine (1.2 mmol,
1.5 mol equiv), and the mixture was stirred at 25 °C overnight.
The solution (reduced under vacuum if MeOH used) was washed
with saturated Na2S2O3 (15 mL), and the aqueous layer was
extracted with CHCl3 (20 mL). The combined organic fractions
were purified by column chromatography using hexanes-EtOAc
as eluent.
3,4-Difluoro-2-hydroxyacetophenone (6e). From 2,3-difluo-
rophenyl acetate, the difluoroacetophenone 6e was formed (28%)
as a white solid, mp 40-42 °C; IR (KBr) 1659, 1616, 1512, 1447,
1
1358, 1323, 1282 cm-1; H NMR δH (CDCl3) 12.59 (1H, s, OH),

Benzopyranones and Their ActiVities
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3979
3-Iodo-4H-1-benzopyran-4-one (8a). 8a was prepared according
to method C, in chloroform from 3-(dimethylamino)-1-(2-hydrox-
yphenyl)propenone (7a), as a white solid (78%), mp 102-103 °C;
1H NMR δH (CDCl3) 8.32 (1H, s, H-2), 8.25 (1H, dd, J ) 2.0, 8.0,
ArH), 7.72 (1H, m, ArH), 7.45 (2H, m, ArH); 13C NMR δC (CDCl3)
173.0 (CdO), 157.5 (CH), 155.8 (C), 133.9 (CH), 126.2 (CH),
125.7 (CH), 121.4 (CH), 117.8 (C), 86.1 (C-I); MS-CI (m/z) 273
(M+ + 1). Anal. (C9H5IO2) C, H.
Yields and melting points of 3-iodopyranones (8b-k) prepared
by general method C are listed in Table 1. Analytical data and
spectroscopic properties of new compounds are provided in
Supporting Information.
7.64 (1H, td, J ) 1.8, 8.0, H-7), 7.61 (1H, s, H-2), 7.41 (2H, m,
H-6, H-8), 0.35 (9H, s, SnMe3); 13C NMR δC (CDCl3) 181.6 (Cd
O), 157.6 (CH), 157.2 (C), 133.8 (CH), 126.3 (CH), 125.4 (CH),
124.2 (C), 123.3 (C), 118.4 (CH), -8.9 (CH3); MS-CI (m/z) 310
(M+ + 1), 295 (-CH2). Anal. (C12H14O2Sn) C, H.
Yields and melting points of 3-stannylpyranones (13b-g)
prepared by general method E are listed in Table 2. Analytical data
and spectroscopic properties of new compounds are provided in
Supporting Information.
General Method F for the Stille Coupling of 3-(Trimethyl-
stannyl)-4H-1-benzopyran-4-ones (13) and 4-Iodonitrobenzene.
A Schlenk tube was charged with 4-iodonitrobenzene (0.3 mmol),
a substituted 3-(trimethylstannyl)-4H-1-benzopyran-4-one (0.33
mmol, 1.1 mol equiv), triphenylarsine (0.024 mmol, 8 mol %), Pd2-
(dba)3(0) (0.003 mmol, 1 mol %), copper(I) iodide (0.03 mmol, 10
mol %), and LiCl (0.9 mmol, 3 mol equiv). Dry NMP (15 mL)
was added to give a solution of 0.13 M with respect to the iodide.
The tube was evacuated, sealed under nitrogen, and heated at 80
°C for 48 h. Products were partitioned between EtOAc (20 mL)
and saturated brine (20 mL), and the dried (MgSO4) organic layer
was concentrated in vacuo. Products were purified by column
chromatography using hexanes-EtOAc as eluent.
Attempted Pd(0)-Mediated Borylations of 3-Iodo-4H-1-ben-
zopyran-4-one (8a). A mixture of bis(diphenylphosphino)ferrocene
palladium(0) (0.026 g, 0.03 mmol), triethylamine (0.43 mL), bis-
(pinacolato)diboron (0.46 mL, 3.20 mmol), 8a (0.2272 g, 1.0 mmol),
and dry dioxan (4.0 mL) were stirred in a sealable tube at 25 °C
until hydrogen liberation ceased. The tube and contents were flushed
with nitrogen, and the sealed tube was heated at 80 °C for 24 h.
Chromatographic fractionation of the reaction products afforded
small samples of 4H-1-benzopyran-4-one (10) (1H NMR δH
(CDCl3) 8.22 (1H, m, ArH), 7.86 (1H, d, J ) 6.0, CdCH), 7.67
(1H, m, ArH), 7.45 (2H, m, ArH), 6.35 (1H, d, J ) 6.0, CdCH);
MS-CI (m/z) 147 (M+ + 1)) and the bis-chromone (11) (mp 251-
3-(4-Nitrophenyl)-4H-1-benzopyran-4-one (14a). 14a was
prepared (68%) from 4-iodonitrobenzene (0.11 g, 0.44 mmol), 13a
(0.15 g, 0.49 mmol), triphenylarsine (0.011 g, 0.035 mmol), Pd2-
(dba)3(0) (0.004 g, 0.0044 mmol), copper(I) iodide (0.0084 g, 0.044
mmol), and LiCl (0.056 g, 1.32 mmol) in NMP (3.4 mL). The
nitrophenylbenzopyranone was isolated as an orange solid, mp
203-204 °C (lit., 202-204 °C28); IR (KBr) 1632, 1594, 1514, 1463,
1
254 °C; IR (KBr) 1644, 1615, 1463, 1369, 1317, 1260 cm-1; H
NMR δH (CDCl3) 8.91 (2H, s, H-2,22), 8.30 (2H, dd, J ) 1.8, 8.0,
ArH), 7.70 (2H, td, J ) 1.8, 8.0, ArH), 7.54 (2H, dd, J ) 1.8, 8.0,
ArH), 7.46 (2H, td, J ) 1.8, 8.0, ArH); MS-CI (m/z) 291 (M+
1)]. H NMR analysis of reaction mixtures employing a range of
different bases (K2CO3, Cs2CO3, KH2PO4, BaOH2) similarly showed
the presence of 10 and 11.
+
1
1
1369, 1317, 1289, 1260 cm-1; H NMR δH (CDCl3) 8.13 (1H, s,
H-2), 7.38-8.46 (8H, m, ArH); MS-CI (m/z) 268 (M+ + 1).
General Method D for the Synthesis of 3-Aryl-4H-1-ben-
zopyran-4-ones (12) by Suzuki-Miyaura Coupling. To a solution
of a substituted 3-iodo-4H-1-benzopyran-4-one (8, 0.35 mmol), Pd-
(PPh3)4 (0.035 mmol, 0.1 mol equiv), and 2M Na2CO3 (0.7 mmol,
2 mol equiv) in benzene (15 mL) was added phenylboronic acid
(1.4 mmol, 4 mol equiv) in EtOH (15 mL). The mixture was heated
under reflux under nitrogen (20 h). After addition of water (20 mL),
the aqueous phase was extracted with dichloromethane (2 × 30
mL) and the combined organic fractions were dried (MgSO4) and
concentrated under vacuum. Products were purified by column
chromatography using hexanes-EtOAc as eluent.
3-(4-Chlorophenyl)-4H-1-benzopyran-4-one (12a). 12a was
prepared (57%), from 8a and 4-chlorophenylboronic acid by general
method D, as a white solid, mp 186-188 °C (lit. 182 °C27); IR
(KBr) 1638, 1617, 1605, 1597, 1574, 1566, 1492, 1466, 1373, 1356,
1289, 1229 cm-1; 1H NMR δH (CDCl3) 8.22 (1H, dd, J ) 2.0, 8.0,
ArH), 7.93 (1H, s, H-2), 7.58 (2H, m, ArH), 7.41 (4H, m, ArH),
7.32 (1H, m, ArH); MS-CI (m/z) 257/259 (M+ + 1).
Yields and melting points of 3-(4-nitrophenyl)-4H-1-benzopyran-
4-ones (14b-e) prepared by general method F are listed in Table
2. Analytical data and spectroscopic properties of new compounds
are provided in Supporting Information.
3-(4-Aminophenyl)-4H-1-benzopyran-4-one (16a). (i) A sus-
pension of 3-(4-nitrophenyl)-4H-1-benzopyran-4-one (14a) (0.117
g, 0.43 mmol) and tin(II) chloride dehydrate (0.395 g, 1.75 mmol)
in EtOH (3.0 mL) was heated under reflux (20 h), cooled, and
concentrated in vacuo, and the products were partitioned between
EtOAc and excess 1 M NaOH (to pH 11). The organic layer was
washed with brine and water, dried (MgSO4), and concentrated in
vacuo to yield the aminophenylpyranone (0.07 g, 67%), mp 194-
196 °C; IR (KBr) 3461, 3352, 1626, 1609, 1566, 1516, 1464, 1379
cm-1; 1H NMR δH (CDCl3) 8.33 (1H, dd, J ) 2.0, 8.0, ArH), 8.00
(1H, s, H-2), 7.68 (1H, m, ArH), 7.45 (4H, m, ArH), 6.77 (2H, m,
ArH), 3.77 (2H, br s, NH2); MS-CI (m/z) 238 (M+ + 1). Anal.
(C15H11NO2) C, H, N.
(ii) 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (17)
(0.29 g, 1.0 mmol) in dioxan (5.0 mL) was mixed with diphe-
nylphosphinoferrocene palladium(0) (0.026 g, 0.03 mmol) and
EtOH (2.0 mL). To the solution was added 3-iodo-4H-1-benzopy-
ran-4-one (8a) and K2CO3, and the mixture was heated under reflux
(72 h) under nitrogen. The products were partitioned between
EtOAc and water, and the organic fraction was concentrated in
vacuo. Chromatographic purification (EtOAc-hexane as eluent)
gave the same (IR and 1H NMR) aminophenylpyranone (16a)
(14%).
(iii) Into an oven-dried Schlenk tube were combined 3-(4-
bromophenyl)-4H-1-benzopyran-4-one (12b) (0.25 g, 0.83 mmol),
Pd2(dba)3(0) (0.0092 g, 0.001 mmol), (()-BINAP (0.0019 g, 1.0
mmol), benzophenone oxime (1.0 g, 1.162 mmol), and sodium tert-
butoxide (0.11 g, 1.16 mmol) in toluene (5.0 mL). The tube was
flushed with nitrogen and heated at 80 °C for 16 h. Diethyl ether
(50 mL) was added. The mixture was filtered, and the solvents
were removed in vacuo. Chromatographic separation of the products
(EtOAc-hexane as eluent) afforded crude imine 18, which was
hydrolyzed with 2 M HCl in THF at 25 °C to give the same (IR
and 1H NMR) aminophenylpyranone 16a (61%). No reaction
occurred between 3-(4-chloro-phenyl)-4H-1-benzopyran-4-one (12a)
Yields and melting points of 3-aryl-4H-1-benzopyran-4-ones
(12b-o) prepared by general method D are listed in Table 1.
Analytical data and spectroscopic properties of new compounds
are provided in Supporting Information.
General Method E for the Palladium(0)-Mediated Stanny-
lation of 3-Iodo-4H-1-benzopyran-4-ones (8). A substituted
3-iodo-4H-1-benzopyran-4-one (0.4 mmol) and Pd(PPh3)4 (0.008
mmol, 0.02 mol equiv) were placed in a two-necked flask fitted
with a reflux condenser, and the system was evacuated and filled
with nitrogen. Dioxane (20 mL), followed by hexamethylditin (4.0
mmol, 10 mol equiv), was added, and the mixture was refluxed
overnight. Products were partitioned between EtOAc (25 mL) and
water (25 mL), and the pooled organic fractions were dried (MgSO4)
and concentrated in vacuo. Products were purified by column
chromatography using hexanes-EtOAc as eluent.
3-(Trimethylstannyl)-4H-1-benzopyran-4-one (13a). 13a was
prepared (1.87 g, 80%) from 8a (2.05 g, 7.60 mmol), Pd(PPh3)4
(0.174 g, 0.152 mmol), and hexamethylditin (5.0 g, 15.2 mmol) in
dioxane (30 mL) by general method E as above. The trimethyl-
stannylbenzopyranone formed white crystals (from EtOH), mp 85-
86 °C; IR (KBr) 1609, 1556, 1463, 1378, 1340, 1333, 1313, 1251,
1209 cm-1; 1H NMR δH (CDCl3) 8.15 (1H, dd, J ) 1.8, 8.0, H-5),

3980 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Vasselin et al.
and the same reagents utilizing sodium tert-butoxide or potassium
phosphate as bases.
Acknowledgment. This work was supported by a Cancer
Research U.K. Programme Grant (A.D.W., C.S.M., T.D.B. and
M.F.G.S.) and by the University of Nottingham (studentship to
D.A.V.). The authors thank Dr. Bob Schultz and colleagues
(NCI Developmental Therapeutics program) for analyses of
agent activity against the 60-cell-line panel.
3-(4-Aminophenyl)-6-fluoro-4H-1-benzopyran-4-one (16b).
16b was prepared (94%) by reduction of 6-fluoro-3-(4-nitrophenyl)-
4H-1-benzopyranone (14b) with tin(II) chloride dihydrate in EtOH.
The amine had mp 136-138 °C; IR (KBr) 3358, 1638, 1579, 1518,
1483, 1449, 1367 cm-1; 1H NMR δH (DMSO-d6) 8.43 (1H, s, H-2),
8.20 (1H, m, ArH), 7.66 (1H, m, ArH), 7.40 (1H, m, ArH), 7.30
(2H, d, J ) 8.5, H-2′, H-6′), 6.65 (2H, d, J ) 8.5, H-3′, H-5′),
3.85 (2H, br m, NH2); MS-EI (m/z) 255 (M+). HRMS-EI (m/z):
calcd for C15H10FNO2, 255.0696 (M+); found, 255.0703.
3-(4-Aminophenyl)-7-fluoro-4H-1-benzopyran-4-one (16c). 16c
was prepared (75%) by reduction of 7-fluoro-3-(4-nitrophenyl)-
4H-1-benzopyranone (14c) with tin(II) chloride dihydrate in EtOH.
Supporting Information Available: Full compound NMR
characterization data for compounds 8b-k, 12b-o, 13b-g, and
14b-e; additional references; microanalytical data for new com-
pounds; and details of the attempted synthesis of compounds 14f-
g. This material is available free of charge via the Internet at http://
pubs.acs.org.
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The amine had mp 153-155 °C; H NMR δH (CDCl3) 7.99 (1H,
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