Synthesis and topoisomerase inhibitory activities of novel aza-analogues of flavones

Article abstract of DOI:10.1016/S0223-5234(99)80087-7

A series of aza-flavones (3-hydroxy-2-phenyl-4-quinolones) were designed and synthesized as inhibitors of bacterial DNA-gyrase and mammalian topoisomerase II. Structure activity relationships of the compounds against each of the enzymes are discussed.

Full text of DOI:10.1016/S0223-5234(99)80087-7

Eur. J. Med. Chem. 34 (1999) 381−387
© Elsevier, Paris
381
Original article
Synthesis and topoisomerase inhibitory activities
of novel aza-analogues of flavones1
Zhihua Sui*, Van N. Nguyen#, Jason Altom, Jeffrey Fernandez,
Jamese J. Hilliard, Jeffrey I. Bernstein, John F. Barrett#, Kwasi A Ohemeng*#
The R.W. Johnson Pharmaceutical Research Institute, 1000 Route 202 South, Raritan, New Jersey 08869, USA
(Received 20 July 1998; accepted 29 October 1998)
Abstract – A series of aza-flavones (3-hydroxy-2-phenyl-4-quinolones) were designed and synthesized as inhibitors of bacterial DNA-gyrase
and mammalian topoisomerase II. Structure activity relationships of the compounds against each of the enzymes are discussed. © Elsevier,
Paris
aza-flavones / 3-hydroxy-2-phenyl-4-quinolones / DNA-gyrase / topoisomerase II
1. Introduction
DNA topoisomerases catalyse the topological intercon-
versions of DNA molecules which are required for
several essential processes in DNA metabolism, includ-
ing replication, recombination, transcription, and chro-
mosome separation at mitosis [1]. These enzymes have
therefore served as targets for several useful antitu-
mour [2] and antibacterial agents [3]. Both prokaryotic
and eukaryotic topoisomerase II inhibitors, developed as
either antibacterial or antitumour agents, respectively,
share a similar mechanism of action. The formation of a
ternary complex (DNA::topoisomerase II::drug) leading
to the “cleavable-complex” is required for the activity of
the inhibitors [4–8]. Specific binding sites and strand
specificity for covalent catalysis leading to formation of
the ternary drug complex are unique, even though both
enzymes share a common tyrosine for the covalent
catalysis [9–13]. These differences afford the possibility
of finding agents that inhibit only the prokaryotic topoi-
Figure 1. Structures of flavones 1 and aza-analogues of flavo-
nes 2.
somerase
II,
such
as
the
antibacterial
4-quinolones [14–16], or inhibitors of the mammalian
topoisomerase II, such as the antitumour quinolones [15,
17–21].
In the process of searching for prokaryotic topoi-
somerase II (DNA gyrase) inhibitors, we recently identi-
fied a series of flavones 1 as bona fide DNA gyrase
(bacterial type II topoisomerase) inhibitors by the super-
coiling and cleavable complex assays. Some of the
compounds (e.g. quercetin: IC50 = 3.3 µg/mL) were as
potent as some of the currently marketed 4-quinolone
antibacterials (e.g. ofloxacin: IC50 = 1.75 µg/mL) against
the target enzyme [22]. This led us to synthesize a series
of the hitherto unknown aza-analogues of flavones 2
(figure 1). Since literature evidence indicated that some
*Correspondence and reprints
1
Part of the work was presented at the XIVth International
Symposium on Medicinal Chemistry, Maastricht, NL, 1996
(Abst. P-10.17)
#
Current address: Bristol-Myers Squibb Pharmaceutical Re-
search Institute, 5 Research Parkway, Wallingford, CT 06492-
7660

382
Figure 2. Synthesis of compounds 2a–z.
flavones also had inhibitory activity against the mamma-
lian DNA topoisomerase II [23] we decided to study our
compounds against both enzymes. The synthesis and
biological activities of these compounds were reported
herein.
derivatives 2. Compounds 2 are often hydroscopic and
contain hydrobromic acid. This could be due to the
presence of a- and b-hydroxy carbonyl as well as
catechol moieties which can form chelates or intermo-
lecular complexes with water and hydrobromic acid.
2. Chemistry
3. Results and discussion
Literature searches indicated that the synthesis of
3-hydroxy-4-quinolones has been poorly investigated.
The synthesis employed to these compounds are shown in
figure 2. The ketones 4 were synthesized from the appro-
priately substituted anilines 3, by regioselective acylation
of the ortho positions with the corresponding acetoni-
triles [24, 25]. In most cases, titanium tetrachloride was
used as a catalyst. Aluminium chloride and zirconium
chloride can also be used in place of titanium chloride.
For the synthesis of the non-symmetrical compounds 2w,
2x, 2y and 2z, 3-methoxy-aniline was converted to a
mixture of the two regioisomers the 2- and 6-substituted
derivatives. The ratio of the substitution of position 2 to
that of position 6 was 5:7. The mixture was then
separated by chromatography. N-acylation of 4 with
substituted benzoylchlorides under standard conditions
gave the amides 5. The acylated derivatives 5 were then
cyclized under pressure in the presence of sodium ethox-
ide to give the 3-methoxy-4-quinolones 6. Demethylation
of the various methoxy groups, where necessary, was
accomplished in a single step either by refluxing with
hydrobromic acid or heating with excess pyridine hydro-
chloride at 180–200 °C, which provided the hydroxy
The biological activities of compounds 2a–z, ellipti-
cine and nalidixic acid against the prokaryotic topoi-
somerase II (DNA-gyrase) and the eukaryotic topoi-
somerase II are shown in tables I–V. In general, the
synthesized compounds possess activities against both
bacterial DNA-gyrase and mammalian topoisomerase II.
The compounds are usually more potent against topoi-
somerase II. The potencies of the compounds against the
bacterial DNA-gyrase are in the range of the first genera-
tion quinolones, such as nalidixic acid (IC50 = 52 µg/mL).
Compound 2a (IC50 = 81 µg/mL), the aza analogue of
quercetin is about 25-times less potent than its flavone
counterpart (quercetin, IC50 = 3.3 µg/mL). The most
potent compound 2b (IC50 = 0.01 µg/mL) against topoi-
somerase II is 400-times more potent than the standard
ellipticine (IC50 = 4.68 µg/mL).
The aza-flavones display distinct SAR against the two
enzymes. When ring C is substituted with hydroxy groups
(table I), the compounds showed activities against both
enzymes. While eliminating the hydroxy group does not
abolish the activity against topoisomerase II (IC50
=
0.27 µg/mL), the compound (2c) was inactive against
DNA-gyrase (IC50 > 500 µg/mL). Interestingly, the tri-

383
Table I. Importance of the hydroxyl groups on the C-ring.
Compound
R1
R2
R3
DNA-gyrase
IC50 µg/mL
Topoisomerase II
IC50 µg/mL
Ellipticine
Nalidixic Acid
2a
2b
2c
2d
> 500
52.0
81.0
472
> 500
200
4.68
> 500
5.03
0.01
0.27
1.94
OH
OH
H
OH
OH
H
H
OH
H
H
OH
H
hydroxy derivative 2b showed high potency against
topoisomerase II (IC50 = 0.01 µg/mL) but marginal ac-
tivity against DNA-gyrase (IC50 = 472 µg/mL). This
indicates that the binding sites of ring C for the two
enzymes are different. Furthermore, the activity against
topoisomerase II seems to be more sensitive to the
modifications of ring C as illustrated in table II. Replac-
ing the 4-hydroxy group with an n-butyl did not change
the activity against DNA-gyrase but decreased the activ-
ity against topoiomerase II. Replacing the n-butyl with a
t-butyl increased the gyrase activity slightly, but de-
creased the topoisomerase activity dramatically. We
found that the phenolic protons on ring C are not required
for the activities since the hydroxy groups of ring C can
be replaced by halogens in most cases (table III). These
results suggest that the hydroxy groups do not serve as
hydrogen-bonding donors in the binding to the enzymes.
There is no clear evidence according to our data that the
oxygen atoms of the hydroxy groups or the halogens may
act as hydrogen-bonding acceptors though trifluoro-
methyl and bromine decrease or abolish the activities.
The hydroxy group on ring B is essential for the
DNA-gyrase activity but not the topoisomerase activity
as shown in table IV. This further indicates that the
structure requirements for the two enzymes are different.
Table II. Replacement of the 4≠-hydroxyl groups with other substituents.
Compound
R
DNA-gyrase
IC50 µg/mL
Topoisomerase II
IC50 µg/mL
2d
2e
2f
2g
2h
2i
OH
200
185
67.0
314
137
1.94
5.21
45.9
27.0
1.41
27.3
27.5
n-butyl
t-butyl
CO2Et
CO2H
NH2
> 500
> 500
2j
CH3

384
Table III. Replacement of C-ring hydroxyl groups with halogens.
Compound
R1
R2
R3
DNA-gyrase
IC50 µg/mL
Topoisomerase II
IC50 µg/mL
2k
2l
H
F
F
H
Cl
Cl
CF3
H
F
H
F
Br
H
Cl
H
CF3
H
F
H
H
Cl
H
CF3
H
141
131
123
277
159
82.0
103
> 500
29.3
0.12
0.85
> 500
5.07
4.06
30.0
51.3
2m
2n
2o
2p
2q
2r
Unlike ring B, the existence of the hydroxy groups on
ring A is not critical to the activities against both
enzymes, though the structure-activity relationships are
once again different for each of the two enzymes
(table V). While the replacement of the hydroxy groups
with fluorine did not affect the activities against both
enzymes, the methyl substituted compound 2v showed
malian topoisomerase II. The compounds displayed di-
vergent structure-activity relationships against each of the
enzymes. While some compounds are more potent and
selective against mammalian topoisomerase II than ellip-
ticine, others showed comparable potency and selectivity
against DNA gyrase as nalidixic acid.
much higher potency against topoisomerase II (IC50
0.07 µg/mL) and no significant change against DNA-
gyrase activity (IC50 = 113 µg/mL).
=
5. Experimental protocols
Melting points were determined on a Meltemp II
apparatus and are uncorrected. Mass spectral data (chemi-
cal ionization technique) were performed by the analyti-
cal group at the R.W. Johnson Pharmaceutical Research
Institute. All proton NMR spectra were recorded on a
GE-300 spectrometer, and values are reported in ppm
4. Conclusion
We have identified a series of novel aza analogues of
flavones as inhibitors of bacterial DNA-gyrase and mam-
Table IV. Importance of the 3-hydroxyl groups.
Compound
R1
DNA-gyrase
IC50 µg/mL
Topoisomerase II
IC50 µg/mL
2a
2s
2t
OH
H
C2H5
81.0
> 500
> 500
5.03
0.29
21.0

385
Table V. Importance of the A-ring hydroxyl groups.
Compound
R1
R2
R3
R4
DNA-gyrase
IC50 µg/mL
Topoisomerase II
IC50 µg/mL
2a
2u
2v
2w
2x
2y
2z
OH
F
CH3
H
OH
H
OH
OH
F
CH3
OH
H
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
H
81.0
58.0
113
> 500
133
5.03
9.16
0.07
2.80
1.14
75.7
4.76
OH
H
> 500
> 500
H
H
from Me4Si. Physical data of the final products are shown
in table VI. The elemental analyses of all compounds are
within the range of experimental error (± 0.4%).
5.1. 3,5-Dimethoxy-2-(methoxyacetyl)aniline (4a),
general procedure for the preparation of the ketones 4
a
To a cooled solution of 3,5-dimethoxyaniline (46.0 g,
0.30 mol) in benzene (360 mL) under nitrogen was added
BCl3 (1.0 M in CH2Cl2, 300 mL, 0.30 mol). The suspen-
sion was stirred in an ice bath for 45 min. Methoxy-
acetonitrile (23.5 g, 0.33 mol) was added, followed by the
addition of TiCl4, 1 M solution in CH2Cl2 (39 mL,
0.039 mol). The reaction mixture was heated at reflux for
22 h. The mixture was allowed to cool to 25 °C then 2 N
HCl (600 mL) was slowly added. The resulting mixture
was heated at reflux for 1 h and then cooled and basified
with 5 M NaOH (240 mL). The product was extracted
with EtOAc (5 × 500 mL). The organic layers were dried
(MgSO4) and concentrated in vacuo to give 40.2 g of a
tan solid. Column chromatography on silica gel (CH2Cl2)
gave 38.6 g (57%) of the title compound 4a as an
off-white solid, m.p. 129–131 °C, MS(CI, CH4) MH+ at
226. 1H-NMR (CDCl3): δ 6.60 (br s, 2H, NH2), 5.72 (2s,
2H, ArH), 4.53 (s, 2H, COCH2OCH3), 3.82 (s, 3H,
ArOCH3), 3.78 (s, 3H, ArOCH3), 3.47 (s, 3H,
COCH2OCH3). Anal. (C11H15NO4) C, H, N.
Table VI. Physical data of the final products.
Compound
M.p.
Formula
2a
2b
2c
2d
2e
> 300
> 300
C15H11NO6 0.5H2O
C15H11NO6 HBr 0.5H2O
C15H11NO4 HBr H2O
C15H11NO5 HBr 0.5H2O
C15H11NO5 0.7HBr
C19H19NO4 0.9HBr
C18H15NO6 HBr
175–177
> 300
137–139
191–193
185–189
> 300
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
C16H11NO6 HBr
> 300
C15H11N2O4 1.8HBr 0.6H2O
C15H11N2O4 HBr 0.8H2O
C15H10NO4F HBr
C15H9NO4F2 HBr 0.8H2O
C15H9NO4F2 HBr
C15H10BrNO4 HBr 0.7H2O
C15H9Cl2NO4 HBr H2O
C15H9Cl2NO4 HBr 0.8H2O
C17H9F6NO4 HBr 1.7H2O
C19H19NO4 0.9HBr
261–263
232–233
213
> 300
215–217
> 300
> 300
298–299
191–193
283–285
299–300
300–301
255–256
279–280
259–260
150–151
165–166
5.2.
N-(3,5-Dimethoxy-2-methoxyacetylphenyl)-3,4-
C15H11NO5 HBr 1.6H2O
C17H15NO4 HBr
dimethoxybenzamide (5a), a general procedure for the
preparation of the ketone-amide 5
2t
2u
2v
2w
2x
2y
2z
C15H9F2NO4 HBr 0.8H2O
C17H15Cl2NO4 HBr 0.4H2O
C15H11NO5 HBr 0.05H2O
C15H11NO4 HBr 0.4H2O
C15H11NO3 0.8HBr
To a solution of 3,5-dimethoxy-2-(methoxyacetyl)-
aniline (4a), (2.20 g, 9.77 mmol) in pyridine (75 mL) was
added
3,4-dimethoxybenzoyl
chloride
(2.10 g,
10.5 mmol). The mixture was heated at reflux under
nitrogen for 18 h then poured into ice (500 mL). The
C15H11NO3 HBr 0.9H2O

386
precipitate was collected via filtration and air dried to
give 3.61 g (95%) of the title compound 5a as an
off-white solid. The product was used to prepare 3,5,7-
trimethoxy-2-(3,4-dimethoxyphenyl)-4-oxoquinoline
(6a) without further purification.
posed of 1.4 mM ATP, 1.8 mM spermidine, 5 mM DTT,
0.14 mM Na2EDTA, 6.5% glycerol, 24 mM KCl, 4 mM
MgCl2, 0.36 µg/ml bovine serum albumin (mol. biol.
grade), and 35 mM Tris-HCl, pH 7.5. To this reaction,
drug was added from DMSO-solubilized stocks (such
that the final concentration of DMSO is < 3.5%), fol-
lowed by 1 unit of gyrase holoenzyme (reconstituted Gyr
A and Gyr B subunits). Each reaction was incubated for
30 min at 37 °C, and reactions were stopped by the
addition of SDS (to 0.5%), Na2EDTA (to 6 mM), and
5.35% glycerol containing 0.013% bromophenol blue (as
tracking dye). The total reaction mixture was loaded onto
either a 1% TAE or TBE agarose gel and was electro-
phoresed in a horizontal submarine apparatus to separate
different DNA topoisomers. Gels were stained with EtBr,
and visualized by Polaroid film 667 photography of
fluoresced gels. The percent supercoiling was determined
by the densitometric tracing (Collagey, Image Dynamics
Corporation) of supercoiling versus relaxed DNA, nor-
malized against no-drug control lanes.
5.3.
3,5,7-trimethoxy-2-(3,4-dimethoxyphenyl)-4-oxo-
quinoline (6a), a general procedure for the preparation
of the aza-flavone precursors 6a–6z
Sodium ethoxide solution, prepared from sodium
(0.30 g, 13.0 mmol) and ethanol (100 mL), was trans-
fered into a Parr bomb containing a solution of N-(3,5-
dimethoxy-2-methoxyacetylphenyl)-3,4-dimethoxy-ben
zamide (3.60 g, 9.24 mmol) in ethanol (absolute, 50 mL).
The bomb was sealed and heated at 160–165 °C, 180 psi
for 3 h. The reaction vessel was allowed to cool to room
temperature and the solvent was removed under vacuum.
Column chromatography (silica gel, 10% MeOH in
EtOAc) gave 1.23 g (36%) of 6a as a brown solid, m.p.
134–135 °C, MS (CI, CH4): MH+ at 372. 1H-NMR
(DMSO-d6): δ 10.93 (br, s, 1H, NH), 7.22 (s, 1H, ArH),
7.20 (d, J = 8 Hz, 1H, ArH), 7.11 (d, J = 8 Hz, 1H, ArH),
6.67 (s, 1H, ArH), 6.26 (s, 1H, ArH), 3.83 (s, 6H,
ArOCH3), 3.80 (s, 3H, ArOCH3), 3.78 (s, 3H, ArOCH3),
3.60 (s, 3H, ArOCH3). Anal. (C20H21NO6•0.50 H2O) C,
H, N.
-
6.2. Topoisomerase II assay [27, 28]
Each reaction contained an equal volume of Kineto-
plast DNA (kDNA, TopoGen, Inc., Columbus, OH,
1.1–1.8 µg/reaction), 67 mM Tris-HCl, pH 8, 160 mM
KCl, 13 mM MgCl2, 0.7 mM ATP, 0.7 mM dithiothreitol,
0.06 µg BSA, with an equal volume of test compound,
and 10 units of p170 human topoisomerase II enzyme
(TopoGen, Inc.). Each reaction mixture was incubated for
5 min at 37 °C, and the reaction was stopped with 0.5%
Sarkosyl, 0.0025% bromophenol blue, and 25% glycerol.
Reaction samples were electrophoresed in 0.6% TBE-
agarose gels at 30 mA. The gel was stained with EtBr,
and visualized by Polaroid film 667 or 665 (Polaroid
Corporation, Cambridge, MA) photography of fluoresced
gels at 300 nm. The decatenated kDNA band was quan-
tified by densitometric analysis (Collegey, Image Dy-
namics Corporation).
5.4.
3,5,7-Trihydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-
quinoline (2a), a general procedure for the preparation
of the aza-flavones 2a–2z
A
mixture of 3,5,7-trimethoxy-2-(3,4-dimethoxy-
phenyl)-4-oxoquinoline (0.70 g, 1.88 mmol) in aqueous
48% HBr (10 mL) was heated at reflux for 3.5 h. The
reaction mixture was then cooled and the solvent was
removed under vacuum. The residue was recrystallized
from H2O and EtOH and gave 0.35 g (49%) of the title
compound 2a as a dark yellow solid, m.p. > 300 °C,
MS(CI, CH4): MH+ at 302. 1H-NMR (DMSO-d6): δ
11.32 (s, 1H, NH), 10.5–8.50 (br, s, 5H, ArOH), 7.20 (d,
J = 2Hz, 1H, ArH), 7.04 (dd, J = 8, 2Hz, 1H, ArH), 6.87
(d, J = 8Hz, 1H, ArH), 6.44 (d, J = 2Hz, 1H, ArH), 5.97
(d, J = 2Hz, 1H, ArH). Anal. (C15H11NO6•0.50 H2O) C,
H, N.
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