Design of new topoisomerase II inhibitors based upon a quinobenzoxazine self-assembly model

Article abstract of DOI:10.1021/jm980265c

A new class of pyridobenzophenoxazine compounds has been developed as topoisomerase II inhibitors for anticancer chemotherapy. These compounds were designed based on a proposed model of a quinobenzoxazine self-assembly complex on DNA. They showed excellen

Full text of DOI:10.1021/jm980265c

J . Med. Chem. 1998, 41, 4273-4278
4273
Design of New Top oisom er a se II In h ibitor s Ba sed u p on a Qu in oben zoxa zin e
Self-Assem bly Mod el
Qingping Zeng,† Yan Kwok,† Sean M. Kerwin,§ Gina Mangold,‡ and Laurence H. Hurley*,†
Drug Dynamics Institute and Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin,
Austin, Texas 78712-1074, and The Institute for Drug Development, 14960 Omicron Drive, San Antonio, Texas 78245-3217
Received May 1, 1998
A new class of pyridobenzophenoxazine compounds has been developed as topoisomerase II
inhibitors for anticancer chemotherapy. These compounds were designed based on a proposed
model of a quinobenzoxazine self-assembly complex on DNA. They showed excellent inhibitory
effects on several tumor cell lines with nanomolar IC50 values. Their cytotoxic potency correlates
with their ability to unwind DNA and inhibit topoisomerase II.
Ch a r t 1. Structures of Fluoroquinolones and
Quinobenzoxazines
In tr od u ction
Quinobenzoxazines are synthetic analogues of anti-
bacterial fluoroquinolones, exemplified by norfloxacin
and ciprofloxacin.1-3 As shown in Chart 1, A-62176 (1-
(3-aminopyrrolidin-1-yl)-2-fluoro-4-oxo-4H-quino[2,3,4-
ij][1,4]benzoxazine-5-carboxylic acid) and A-85226 (3-
amino-1-(3-aminopyrrolidin-1-yl)-2-fluoro-4-oxo-4H-
quino[2,3,4-ij][1,4]benzoxazine-5-carboxylic acid) are two
examples of the quinobenzoxazines. Studies have shown
that a norvaline prodrug derivative of A-62176 has
curative activity against MX-1 and delays tumor growth
in other xenographs together with significant delays in
solid tumor growth in several murine tumor models.4
Fluoroquinolones function through inhibition of bacte-
rial DNA topoisomerase II (gyrase) or IV. The cytotox-
icity of the fluoroquinolones derives from their ability
to shift the cleavage-religation equilibrium required for
topoisomerase action toward cleavage, thereby effec-
tively trapping the enzyme on DNA to form the “cleaved
complex”.5-8 The quinobenzoxazines, on the other hand,
are potent mammalian DNA topoisomerase II inhibi-
tors. It has been proposed that they inhibit the DNA
topoisomerase II reaction at a step prior to the formation
of the “cleaved complex” intermediate.9 Our experi-
mental results are in accord with this proposal but also
demonstrate that A-62176 stimulates the formation of
topoisomerase II-mediated cleavage at certain DNA
sequence sites at low concentrations and pH.10
through two chelated Mg2+ ions. The two magnesium
cations link the two drug molecules in a head-to-tail
fashion, such that the bidentate ligand of the â-keto acid
moiety and the primary amino group of the aminopyr-
rolidine side chain are the head and tail, respectively.
Each magnesium cation also binds with one phosphate
oxygen of the DNA backbone and two water molecules
to form an octahedral complex. Norfloxacin, an anti-
bacterial fluoroquinolone that does not intercalate into
duplex DNA, shows synergistic effects on the DNA
binding of the quinobenzoxazines, suggesting that nor-
floxacin can replace the externally bound quinobenzox-
azine in a DNA-bound 2:2 drug-Mg2+ complex.10-12
On the basis of this model, it can be proposed that
there are three different structural motifs within the
quinobenzoxazines that determine the ability of these
drugs to self-assemble on DNA and interact with
topoisomerase II. One motif is its polyaromatic ring
that intercalates into DNA base pairs and anchors the
whole assembly on DNA, and the other motifs are the
â-keto acid functionality and the 3-aminopyrrolidine
substituent that chelate Mg2+ through which an exter-
nal molecule is bound in the DNA minor groove. The
external portion of the assembly may be responsible for
interacting with topoisomerase II. To develop more
potent topoisomerase II inhibitors, the three motifs can
be changed systematically by altering either the quino-
benzoxazine’s intercalation ability or its magnesium
binding ability.
DNA binding studies reveal that the antibacterial
fluoroquinolones prefer to bind to single-stranded DNA
rather than to duplex DNA. They also bind to the
DNA-gyrase complex in the presence of Mg2+ 8
In
.
contrast to the antibacterial fluoroquinolones, the
quinobenzoxazines bind to duplex DNA through an
intercalation mechanism. A self-assembly model has
been proposed for the quinobenzoxazines on the basis
of results of biophysical and biochemical studies.11,12 In
this model, a 2:2 drug-Mg2+ dimer binds to DNA, with
one drug molecule intercalating between the DNA base
pairs and the other drug molecule externally bound
* To whom correspondence should be addressed. Tel: (512) 471-
4841. Fax: (512) 471-2746. E-mail: dg-dna@mail.utexas.edu.
† Drug Dynamics Institute.
Since the phenyl ring portion of the quinobenzox-
azines is actually inserted between DNA base pairs, its
intercalation ability can be increased by extending the
§ Division of Medicinal Chemistry.
‡ The Institute for Drug Development.
10.1021/jm980265c CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/07/1998

4274 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Zeng et al.
Ch a r t 2. Structures of the Pyridobenzophenoxazine Analogues Synthesized in This Study
Sch em e 1a
a
(a) (1) (EtO)3CH, Ac2O, (2) 1-amino-2-naphthol; (b) NaH; (c) t-Boc-3-aminopyrrolidine; (d) OH-/H+.
phenyl ring to a naphthyl ring. A series of new
pyridobenzophenoxazine analogues were thus designed
and synthesized. These analogues showed strong cy-
totoxic effects on MDA-231 breast, H226 non-small-cell
lung, HT-29 colon, Rali lymphoma, and DU-145 prostate
human cancer cells and B16 murine melanoma cells.
The IC50 values of these inhibitors were in the range of
4 nM to 2 µM. They also showed potent inhibitory
effects against human topoisomerase II, with IC50 values
in the micromolar range. Some of these new analogues
have a greater DNA unwinding ability in a decatenation
assay than A-62176. It appears that for these com-
pounds the higher DNA unwinding ability leads to
higher potency in topoisomerase II inhibition and
increased cytotoxicity against tumor cells.
acid, the amino group of the side chain, and a phosphate
oxygen of the DNA backbone. Better intercalation may
be achieved by extending the phenyl ring of A-62176 to
a naphthalene ring (i.e., A-62176 f 1, 2, or 3). Since
the exact orientation of the benzene ring between DNA
base pairs is uncertain, all three possible directions of
benzo-annulation were explored to achieve the maxi-
mum π-stacking interaction with DNA base pairs.
Compounds 4-6 have a piperazine ring instead of a
3-aminopyrrolidine ring as side chains. Since the
3-amino group of A-62176 is proposed to chelate mag-
nesium, the change may have some effect on its ability
to self-assemble on DNA and, therefore, on topo-
isomerase II inhibition. For A-62176, the stereochem-
istry of the 3-aminopyrrolidine side chain affects topo-
isomerase II inhibition potency; i.e., the S-isomer is
more potent than the R-isomer.10,12 To study the
possible effects of the stereochemistry of the pyrido-
benzophenoxazines, the compounds were made either
racemic or enantiomerically pure.
Resu lts
Design a n d Syn th esis of th e P yr id oben zop h en -
oxa zin es. A series of pyridobenzophenoxazines has
been designed and synthesized as topoisomerase II
inhibitors (Chart 2). The design was based on the 2:2
quinobenzoxazine-Mg2+self-assemblymodelfor A-62176.
In this model, the benzene ring of A-62176 intercalates
into DNA and binds with DNA by π-π stacking with
DNA base pairs. Additional stabilization is achieved
by the chelation of two magnesium ions with the â-keto
The synthetic procedure was based on a published
method for the synthesis of the quinobenzoxazines, with
some modification.1 The synthesis of compound 1 is
shown in Scheme 1. The synthesis was started with
ethyl 2,3,4,5-tetrafluorobenzoylacetate, which was pre-
pared according to the published procedure.1 Treatment

Design of New Topoisomerase II Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4275
Ta ble 1. Decatenation Inhibition, DNA Unwinding, and Cytotoxicity Data for the Pyridobenzophenoxazines
IC50 (nM)
topoisomerase II
inhibition
DNA
unwinding
B16
melanoma
MDA-231
breast
H226 non-small-
cell lung
HT-29
colon
Raji
lymphoma
DU-145
prostate
compd
IC50 (µM)a
concn (µM)b
A-62176 (S)
1a (racemic)
1b (R)
1c (S)
2a (racemic)
2b (R)
2c (S)
3a (racemic)
3b (R)
3c (S)
0.51
0.77
1.22
0.72
0.55
0.24
0.49
0.77
0.42
0.44
9.6
50
170 ( 10
440 ( 60
680 ( 50
130 ( 20
40 ( 4
160 ( 20
150 ( 40
350 ( 60
190 ( 40
27 ( 2
90 ( 3
140 ( 10
2400 ( 100
130 ( 10
22 ( 2
160 ( 10
180 ( 10
360 ( 40
270 ( 40
30 ( 3
50 ( 6
86 ( 6
160 ( 30
200 ( 30
360 ( 120
140 ( 10
17 ( 7
35
19
160 ( 70
82 ( 8
18 ( 1
3
10
20 ( 2
5 ( 1
13 ( 5
26 ( 1
34 ( 2
30 ( 10
64 ( 7
180 ( 20
100 ( 10
140 ( 15
20 ( 6
2100 ( 300 2200 ( 300
650 ( 60
45 ( 48
84 ( 7
27 ( 4
4 ( 2
22 ( 15
14 ( 2
2100 ( 200
200 ( 30
>105
51 ( 6
20 ( 4
23 ( 4
28 ( 5
38 ( 6
25 ( 9
15
13
>100
>100
>100
21 ( 2
22 ( 3
32 ( 5
48 ( 8
10 ( 2
23 ( 3
29 ( 5
30 ( 6
4
5
6
2000 ( 200
360 ( 90
6800 ( 1800
800 ( 50
210 ( 40
7.2 ( 7.6
840 ( 270
140 ( 40
75 ( 32
1.84
0.64
380 ( 50
>106
a
b
Drug concentration that inhibits 50% of the conversion of catenated to decatenated KDNA by human topoisomerase II. Drug
concentration that causes a shift of the topoisomers from the original to the middle position between the relaxed and the supercoiled
bands.
of 2,3,4,5-tetrafluorobenzoylacetate with triethyl ortho-
fomate in acetic anhydride followed by various amino-
naphthols generated a small library of enamino keto
acid intermediates. These intermediates were treated
with sodium hydride at -78 °C and then heated to
reflux to complete the double annulation. The double
annulation allowed us to avoid hydroxyl protection and
deprotection, which was necessary in the synthesis of
the quinobenzoxazines.1 As a result, double annulation
shortened the synthesis by three steps and improved
the overall yields. Regioselective nucleophilic substitu-
tion of fluoride with racemic or optically pure 3-(tert-
butoxycarbonylamino)pyrrolidine followed by hydrolysis
of the carboxylate and deprotection of the amino group
produced the final products (compounds 1-3). Com-
pounds 4-6 were obtained by using tert-butyl-1-piper-
azinecarboxylate for the nucleophilic aromatic substitu-
tion. The yields of nucleophilic substitutions with tert-
butyl-1-piperazinecarboxylate were lower than with
3-(tert-butoxycarbonylamino)pyrrolidine.
In h ibition of Top oisom er a se II by th e P yr id o-
ben zop h en oxa zin es. The inhibitory effect of these
compounds against human topoisomerase II was meas-
ured by kinetoplast DNA (KDNA) decatenation assays
in which a catenated network of DNA rings was decat-
enated by topoisomerase II to generate the monomeric
DNA. Decatenation is a highly specific assay for topo-
isomerase II activity13 and can be used to screen for
inhibitors that block the catalytic activity of topo-
isomerase II. The IC50 values for decatenation are
shown in Table 1 and are based on three parallel
experiments. Compounds 2, 3, and 6 show comparable
or slightly higher activities than A-62176, compounds
1 and 5 are slightly less active than A-62176, and
compound 4 was about 20-fold less active than A-62176.
Un w in d in g of DNA by th e P yr id oben zop h en ox-
a zin es. Unwinding of duplex DNA is a hallmark
feature of intercalating agents such as chloroquine,
ethidium bromide, and m-AMSA.14 The antineoplastic
activity of the quinobenzoxazines was shown to correlate
with their ability to unwind DNA in a decatenation
assay.9,11 The DNA unwinding effect of the pyridoben-
zophenoxazines was investigated, and the concentra-
tions of compounds required to produce a 50% conver-
sion of catenated to decatenated KDNA are shown in
Table 1. Compounds 2 and 3 showed the highest ability
to unwind DNA, with unwinding concentrations in the
3-15 µM range. The unwinding ability of compound 1
(19-35 µM) was lower than that of 2 and 3 but higher
than that of A-62176 (50 µM). Compounds 4-6 showed
no significant unwinding effect, even at concentrations
higher than 100 µM.
Cytotoxic P oten cy of th e P yr id oben zop h en ox-
a zin es. The cytotoxicity of each of these compounds
against MDA-231 breast, H226 non-small-cell lung, HT-
29 colon, Raji lymphoma, and DU-145 prostate human
cancer cells and B16 murine melanoma cells was
measured and is shown in Table 1. In general, com-
pounds 2 and 3 are the most potent derivatives, with
IC50 values in the 10-50 nM range. The R-enantiomer
of compound 2 shows selectivity against MDA-231
breast cancer cell lines, with an IC50 value of 5 nM.
Compounds 1 and 5 and A-62176 are in the same range
of potency against all tested cell lines. They are about
10-100-fold less potent than compounds 2 and 3.
Compound 6 shows some selective cytotoxicity, with IC50
values of 7 nM against lymphoma, 75 nM against DU-
145 prostate, and 50 nM against B16 murine melanoma
cells. Compound 4 is the least potent derivative, with
IC50 values in the 0.8-2 µM range.
Discu ssion
The pyridobenzophenoxazines were designed and
synthesized based on a quinobenzoxazine self-assembly
model11 with the premise that increasing the intercala-
tion ability of this class of molecules will increase their
potency against topoisomerase II and, therefore, their
antineoplastic activity. Our experimental results are
consistent with this premise. Compounds 2 and 3
showed a stronger DNA unwinding ability than A-62176.
They also showed higher potency than A-62176 in both
the cell-free topoisomerase II inhibition assay and the
in vitro cytotoxicity assay using a range of cell lines.
Because of the steric hindrance between the hydrogens
on the pyrido and benzo rings, the naphthyl and
quinolone portions of compound 1 are not coplanar. This
may have a negative effect on its DNA intercalation
ability. Therefore, compound 1 was less potent than
compounds 2 and 3 and A-62176. Compounds 4-6 have
a piperazine ring as side chains instead of an amino-
pyrrolidine ring, as found in A-62176 and compounds
1-3. Compounds 4-6 showed the least activity in the

4276 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Zeng et al.
Hz, 2H), 7.30-7.40 (m, 3H), 7.51 (m, 1H), 7.71 (d, J ) 7.8 Hz,
cell-free and in vitro assays. This may be because the
secondary amino nitrogen of the piperazine side chain
is in a less optimal position to chelate Mg2+ and to form
a 2:2 self-assembly ternary complex in the DNA minor
groove. The stereochemistry of the 3-aminopyrrolidine
side chain of compounds 1-3 may play some role in
their potency, and the 2:2 model would allow racemic
mixtures to have different potencies than an average
of the R- and S-isomers. However, the relationship
between the stereochemistry of these compounds and
their biological activity remains to be determined. In
summary, it appears that there is a correlation among
DNA unwinding, topoisomerase II inhibition, and cyto-
toxicity with these pyridobenzophenoxazine derivatives.
In conclusion, a novel group of pyridobenzophenox-
azine compounds has been designed and synthesized
based upon the quinobenzoxazine self-assembly model
on DNA. These compounds produce potent unwinding
of duplex DNA, inhibit human topoisomerase II catalytic
activity, and show potent inhibitory effects on several
tumor cell lines with IC50 values ranging from sub-
nanomolar to micromolar.
1H), 7.85 (d, 7.0 Hz, 1H), 8.20 (br, 1H), 8.87 (br, 1H).
Eth yl 1,2-Diflu or o-4-oxo-4H-p yr id o[3,2,1-kl]ben zo[h ]-
p h en oxa zin e-5-ca r boxyla te (11). Ethyl 3-((2-hydroxynaph-
thyl)amino)-2-((2,3,4,5-tetrafluorophenyl)carbonyl)prop-2-
enoate (110 mg, 0.25 mmol) and NaH (60% in mineral oil) (23.2
mg, 0.58 mmol) were mixed with freshly distilled THF and
stirred at -78 °C for 15 min. The mixture was gradually
warmed to room temperature and then heated at 65 °C for 30
min. The excess NaH was quenched by addition of methanol
(30 mL). The solution was evaporated to dryness, and the
product was purified by silica gel chromatography (hexanes/
ethyl acetate ) 3:1), yielding a yellow powder (94 mg, 93%):
1H NMR (CDCl3) δ 1.42 (t, J ) 7.2 Hz, 3H), 4.42 (q, J ) 7.2
Hz, 2H), 7.35 (d, J ) 8.7 Hz, 1H), 7.53 (t, J ) 7.2 Hz, 1H),
7.64 (t, J ) 9.0 Hz, 1H), 7.76 (dd, J F-H ) 7.5, 9.9 Hz, 1H), 7.80
(d, J ) 8.7 Hz, 1H), 7.89 (d, J ) 7.5 Hz, 1H), 8.20 (d, J ) 8.7
Hz, 1H), 8.36 (s, 1H).
Eth yl 1,2-Diflu or o-4-oxo-4H-p yr id o[3,2,1-kl]ben zo[f]-
p h en oxa zin e-5-ca r boxyla te (12). The synthesis of com-
pound 12 was accomplished in a manner similar to compound
11 and yielded a yellow amorphous solid (60%): 1H NMR
(CDCl3) δ 1.46 (t, J ) 7.2 Hz, 3H), 4.45 (q, J ) 7.2 Hz, 1H),
7.50 (d, J ) 9.1 Hz, 1H), 7.54 (t, J ) 7.5 Hz, 1H), 7.60 (t, J )
7.5 Hz, 1H), 7.66 (d, J ) 9.1 Hz, 1H), 7.74 (dd, J F-H ) 7.5,
10.8 Hz, 1H), 7.77 (d, J ) 8.4 Hz, 1H), 8.18 (d, J ) 8.4 Hz,
1H), 8.93 (s, 1H).
Exp er im en ta l Section
Eth yl 1,2-Diflu or o-4-oxo-4H-p yr id o[3,2,1-kl]ben zo[g]-
p h en oxa zin e-5-ca r boxyla te (13). The synthesis of com-
pound 13 was accomplished in a manner similar to compound
11 and yielded a light-yellow amorphous solid (52%): 1H NMR
(CDCl3) δ 1.48 (t, J ) 7.2 Hz, 3H), 4.49 (q, J ) 7.2 Hz, 2H),
7.51 (m, 2H), 7.62 (s, 1H), 7.75 (m, 1H), 7.79-7.85 (m, 2H),
7.89 (s, 1H), 9.19 (s, 1H).
E t h yl 1-(3-(ter t-Bu t oxyca r b on yla m in o)p yr r olid in -1-
yl)-2-flu or o-4-oxo-4H -p yr id o[3,2,1-k l]b en zo[h ]p h en ox-
a zin e-5-ca r boxyla te (14a ). Ethyl 1, 2-difluoro-4-oxo-4H-
pyrido[3,2,1-kl]benzo[h]phenoxazine-5-carboxylate (11) (100
mg, 0.25 mmol) and racemic 3-(tert-butoxycarbonylamino)-
pyrrolidine (140 mg, 0.77 mmol) were dissolved in pyridine
(15 mL), and the mixture was stirred under argon at 110 °C
for 36 h. After the pyridine was removed, the residue was
purified by silica gel chromatography (dichloromethane/ethyl
acetate ) 2:3), yielding a yellow powder (120 mg, 91%): 1H
NMR (CDCl3) δ 1.41 (t, J ) 7.2 Hz, 3H), 1.46 (s, 9H), 1.94 (m,
1H), 2.20 (m, 1H), 3.52 (br, 1H), 3.65 (m, 1H), 3.81 (m, 1H),
3.92 (m, 1H), 4.33 (br, 1H), 4.40 (q, J ) 7.2 Hz, 2H), 5.05 (br,
1H), 7.18 (d, J ) 8.7 Hz, 1H), 7.45 (t, J ) 7.8 Hz, 1H), 7.54 (t,
J ) 7.8 Hz, 1H), 7.56 (d, J F-H ) 13.5 Hz, 1H), 7.69 (d, J ) 8.7
Hz, 1H), 7.83 (d, J ) 8.7 Hz, 1H), 8.09 (d, J ) 8.7 Hz, 1H),
9.12 (s, 1H).
E t h yl 1-(3-(ter t-Bu t oxyca r b on yla m in o)p yr r olid in -1-
yl)-2-flu or o-4-oxo-4H-pyr ido[3,2,1-kl]ben zo[f]ph en oxazin e-
5-ca r boxyla te (15). The synthesis of compound 15 was
accomplished in a manner similar to compound 14a and
yielded a yellow amorphous solid (18 mg, 90%): 1H NMR
(CDCl3) δ 1.42 (t, J ) 7.2 Hz, 3H), 1.48 (s, 9H), 2.02 (m, 1H),
2.28 (m, 1H), 3.55 (br, 1H), 3.65 (m, 1H), 3.87 (br, 1H), 3.92
(m, 1H), 4.33 (m, 1H), 4.39 (q, J ) 7.2 Hz, 2H), 5.24 (m, 1H),
7.37 (d, J ) 9.0 Hz, 1H), 7.51-7.61 (m, 3H), 7.78 (d, J ) 7.8
Hz, 1H), 7.99 (d, J ) 7.8 Hz, 1H), 8.71 (s, 1H).
E t h yl 1-(3-(ter t-Bu t oxyca r b on yla m in o)p yr r olid in -1-
yl)-2-flu or o-4-oxo-4H -p yr id o[3,2,1-k l]b en zo[g]p h en ox-
a zin e-5-ca r boxyla te (16). The synthesis of compound 16 was
accomplished in a manner similar to compound 14a and
yielded a yellow amorphous solid (33 mg, 45%): 1H NMR
(CDCl3) δ 1.45 (t, J ) 7.2 Hz, 3H), 1.48 (s, 9H), 1.96 (m, 1H),
2.22 (m, 1H), 3.54-3.70 (m, 2H), 3.86-4.02 (m, 2H), 4.33 (m,
1H), 4.44 (q, J ) 7.2 Hz, 2H), 5.22 (br, 1H), 7.34 (s, 1H), 7.40-
7.48 (m, 2H), 7.55 (d, J F-H ) 13.5 Hz, 1H), 7.65 (d, J ) 7.8 Hz,
1H), 7.67 (s, 1H), 7.72 (d, J ) 7.8 Hz, 1H), 8.93 (s, 1H).
1-(3-Am in op yr r olid in -1-yl)-2-flu or o-4-oxo-4H -p yr id o-
[3,2,1-kl]ben zo[h ]p h en oxa zin e-5-ca r boxylic Acid Hyd r o-
gen Ch lor id e Sa lt (1a ). Ethyl 1-(3-(tert-butoxycarbonyl-
Gen er a l Meth od s for Ch em istr y. All melting points were
recorded on a Thomas-Hoover capillary melting point ap-
paratus and are uncorrected. 1H and 19F NMR data were
obtained on a Varian Unity 300-MHz NMR spectrometer. 13C
NMR data were obtained on a Varian Unity 500-MHz NMR
spectrometer. The chemical shifts are relative to the trace
proton, carbon, or fluorine signals of the deuterated solvent.
Coupling constants, J , are reported in Hz and refer to apparent
peak multiplicity and not true coupling constants. Mass
spectroscopic experiments were performed by the Mass Spec-
troscopy Center at The University of Texas at Austin. Ele-
mental analysis of C, H, N was done by Quantitative Tech-
nologies Inc., White House, NJ . Flash column chromatography
was performed on silica gel 60, 230-400 mesh, purchased from
Spectrum. All starting materials were obtained from com-
mercial sources unless otherwise specified.
Eth yl 3-((2-Hyd r oxyn a p h th yl)a m in o)-2-((2,3,4,5-tetr a -
flu or op h en yl)ca r bon yl)p r op -2-en oa te (8). A solution of
ethyl 2,3,4,5-tetrafluorobenzoylacetate (1100 mg, 4.18 mmol)
in triethyl orthoformate (1 mL, 6.06 mmol) and acetic anhy-
dride (1.8 mL, 19 mmol) was heated to 130 °C and stirred for
4 h. During the reaction, ethyl acetate was formed and then
removed by distillation. The resulting reaction mixture was
distilled under vacuum to yield an orange oil, which was
dissolved in 30 mL of dichloromethane. A solution of 1-ami-
nonaphthalen-2-ol hydrogen chloride (1360 mg, 6.26 mmol),
in 2 equiv of pyridine (1.0 mL, 12.4 mmol), was added to the
dichloromethane solution, and the mixture was stirred over-
night at 22 °C. After the solvent was removed under reduced
pressure, the remaining residue was purified by silica gel
chromatography (gradient ethyl acetate/hexanes ) 8:1 to 4:1),
yielding a bright-yellow powder (1690 mg, 93%): 1H NMR
(CDCl3) δ 1.02 (t, J ) 7.2 Hz, 3H), 4.01 (q, J ) 7.2 Hz, 2H),
4.10 (m, 1H), 7.34 (d, J ) 9.0 Hz, 1H), 7.41 (m, 1H), 7.60 (m,
2H), 7.82 (8, 2H), 7.91 (d, J ) 8.1 Hz, 1H), 8.54 (br, 1H).
Eth yl 3-((1-Hyd r oxy-2-n a p h th yl)a m in o)-2-((2,3,4,5-tet-
r a flu or op h en yl)ca r bon yl)p r op -2-en oa te (9). The synthe-
sis of compound 9 was accomplished in a manner similar to
compound 8 and yielded a yellow amorphous solid (90%): 1H
NMR (CDCl3) δ 1.14 (t, J ) 7.2 Hz, 3H), 4.13 (q, J ) 7.2 Hz,
2H), 6.86 (m, 1H), 7.40-7.64 (m, 4H), 7.80 (m, 1H), 7.93 (d, J
) 8.4 Hz, 1H), 8.69-8.79 (m, 1H).
Eth yl 3-((3-Hyd r oxy-2-n a p h th yl)a m in o)-2-((2,3,4,5-tet-
r a flu or op h en yl)ca r bon yl)p r op -2-en oa te (10). The syn-
thesis of compound 10 was accomplished in a manner similar
to compound 8 and yielded a yellow amorphous solid (90%):
1H NMR (CDCl3) δ 1.08 (t, J ) 7.2 Hz, 3H), 4.07 (q, J ) 7.2

Design of New Topoisomerase II Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4277
amino)pyrrolidin-1-yl)-2-fluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo-
[h]phenoxazine-5-carboxylate (14a ) (77 mg, 0.138 mmol) was
mixed with ethanol (3 mL) and 1 N KOH (1 mL). This mixture
was refluxed for 30 min; 2 N HCl (3 mL) and ethanol (2 mL)
were added to the mixture, and the resulting mixture was
refluxed for 4 h. After the reaction mixture was slowly cooled,
a yellow powder was isolated by filtration. The powder was
washed with water and ethanol and then dried in vacuo (65
Hz, 1H), 8.11 (d, J ) 8.7 Hz, 1H), 8.28 (d, J ) 8.7 Hz, 1H),
10.06 (s, 1H); HRMS calcd for C24H19FN3O4 432.1360, found
432.1366.
1-(P ip er a zin -1-yl)-2-flu or o-4-oxo-4H -p yr id o[3,2,1-k l]-
ben zo[f]p h en oxa zin e-5-ca r boxylic Acid Hyd r ogen Ch lo-
r id e Sa lt (5). The synthesis of compound 5 was accomplished
in a manner similar to compound 4 and yielded a yellow
amorphous solid (42%): mp >210 °C dec; 1H NMR (CF3COOD)
δ 3.81 (s, 4H), 4.07 (s, 4H), 7.80 (m, 3H), 7.97 (m, 3H), 8.30
(br, 1H), 9.65 (s, 1H); HRMS calcd for C24H19FN3O4 432.1360,
found 432.1351.
1
mg, 94%): mp >225 °C dec; H NMR (CF3COOD) δ 2.65 (m,
2H), 4.10 (m, 1H), 4.37-4.54 (m, 4H), 7.44 (d, J ) 9.0 Hz, 1H),
7.66 (t, J ) 7.5 Hz, 1H), 7.78 (t, J ) 7.5 Hz, 1H), 7.90 (d, J )
13.0 Hz, 1H), 8.03 (d, J ) 8.5 Hz, 1H), 8.08 (d, J ) 9.0 Hz,
1H), 8.20 (d, J ) 8.5 Hz, 1H), 9.90 (s, 1H); 13C NMR (DMSO-
d6) δ 29.3, 49.0, 49.1, 54.3, 104.8 (d, J C-F ) 24.6 Hz), 106.8,
114.1, 116.6 (d, J C-F ) 9.2 Hz), 117.0, 118.5, 120.1, 123.8,
1-(P ip er a zin -1-yl)-2-flu or o-4-oxo-4H -p yr id o[3,2,1-k l]-
ben zo[g]p h en oxa zin e-5-Ca r boxylic Acid Hyd r ogen Ch lo-
r id e Sa lt (6). The synthesis of compound 6 was accomplished
in a manner similar to compound 4 and yielded a yellow
amorphous solid (37%): mp >200 °C dec; 1H NMR (CF3COOD)
δ 3.79 (s, 4H), 4.03 (s, 4H), 7.60-7.72 (m, 2H), 7.81 (s, 1H),
7.89 (d, J ) 8.7 Hz, 1H), 7.97-8.02 (m, 2H), 8.53 (s, 1H), 9.93
(s, 1H); HRMS calcd for C24H19FN3O4 432.1360, found 432.1348.
125.8, 128.5, 128.8 (d, J C-F ) 14.2 Hz), 129.4, 130.4 (d, J C-F
)
20.1 Hz), 131.9, 134.3 (d, J C-F ) 9.2 Hz), 143.7, 145.4, 153.0
(d, J C-F ) 248 Hz), 165.4, 175.9, 180.2; 19F NMR (CF3COOD)
δ -113.2 (d, J F-H ) 12.7 Hz); HRMS calcd for C24H19FN3O4
432.1360, found 432.1375. 1a : Anal. (C24H18FN3O4‚HCl‚
1/3H2O) C, H, N. 1b: Anal. (C24H18FN3O4‚HCl‚4/3H2O) C, H,
N. 1c: Anal. (C24H18FN3O4‚HCl‚3/2H2O) C, H, N.
Ma t er ia ls for Bioch em ist r y. KDNA, human topo-
isomerase II (170-kDa form), and DNA Unwinding Kit includ-
ing supercoiled pHOT1 DNA, relaxed DNA marker, buffers,
and human topoisomerase I were purchased from TopoGEN,
Inc. Agarose was purchased from Fisher. Quinobenzoxazine
A-62176 (S-enatiomer) was provided by Abbott Laboratories
or prepared according to published procedures.1
1-(3-Am in op yr r olid in -1-yl)-2-flu or o-4-oxo-4H -p yr id o-
[3,2,1-kl]ben zo[f]p h en oxa zin e-5-ca r boxylic Acid Hyd r o-
gen Ch lor id e Sa lt (2). The synthesis of compound 2 was
accomplished in a manner similar to compound 1a and yielded
1
a yellow amorphous solid (18 mg, 99%): mp >207 °C dec; H
Deca ten a tion Assa y. KDNA (0.25 µg) was incubated with
various concentrations of the quinobenzoxazines in a 20-µL
reaction mixture containing 50 mM Tris-HCl (pH 8), 120 mM
KCl, 10 mM MgCl2, and 0.5 mM each of ATP, dithiothreitol,
and 30 mg/mL BSA for 10 min. Two units of human topo-
isomerase II (170-kDa form) was added to the mixture, which
was then incubated at 37 °C for 30 min. The reaction was
terminated with 0.1 volume of stop buffer (5% sarkosyl, 0.025%
bromophenol blue, and 50% glycerol). The decatenation
products were analyzed on 1% agarose gels run with 0.5 µg/
mL ethidium bromide. The gels were scanned by FluorImager
(Molecular Dynamics) and analyzed by ImageQuaNT soft-
ware.
NMR (CF3COOD) δ 2.69 (m, 2H), 4.19 (m, 1H), 4.40-4.67 (m,
4H), 7.73-7.81 (m, 3H), 7.87-7.99 (m, 3H), 8.20 (m, 1H), 9.47
(s, 1H); 13C NMR (DMSO-d6) δ 29.4, 49.0, 49.3, 54.5, 104.6 (d,
J C-F ) 24.8 Hz), 106.8, 114.1, 116.9 (d, J C-F ) 9.2 Hz), 118.8,
121.4, 122.6, 125.2, 126.2, 127.6, 128.0 (d, J C-F ) 10.6 Hz),
128.6 (d, J C-F ) 14.2 Hz), 132.6, 134.7 (d, J C-F ) 8.7 Hz), 137.7,
138.7, 154.0 (d, J C-F ) 248 Hz), 165.4, 175.4, 180.2; 19F NMR
(CF3COOD) δ -111.4 (d, J F-H ) 12.1 Hz); HRMS calcd for
C
24H19FN3O4 432.1360, found 432.1355. 2a : Anal. (C24H18-
FN3O‚HCl‚H2O) C, H, N. 2b : Anal. (C24H18FN3O4‚HCl‚
4/3H2O) C, H, N. 2c: Anal. (C24H18FN3O3‚HCl‚4/3H2O) C, H,
N.
1-(3-Am in op yr r olid in -1-yl)-2-flu or o-4-oxo-4H -p yr id o-
[3,2,1-kl]ben zo[g] p h en oxa zin e-5-ca r boxylic Acid Hyd r o-
gen Ch lor id e Sa lt (3). The synthesis of compound 3 was
accomplished in a manner similar to compound 1a and yielded
DNA Un w in d in g Assa y. A 22-µL mixture of 0.25 µg of
supercoiled pHOT1 DNA and 5 units of topoisomerase I in
topoisomerase I buffer (provided with the kit) was incubated
at 37 °C for 30 min; 2 µL of various concentrations of drug
was added, and the incubation was continued for another 30
min. The reactions were terminated by addition of SDS to
1%. Proteinase K was added to the reaction mixtures to ca.
50 µg/mL, and the reaction mixtures were incubated for 15-
20 min at 56 °C. After adding 0.1 volume of 10X gel loading
buffer, the reaction mixtures were extracted with an equal
volume of CIA (chloroform-isoamyl alcohol, 24:1). The samples
were loaded on a 1% agarose gel that was cast in TPE buffer
(36 mM Tris-HCl, pH 7.8, 1 mM EDTA, 30 mM NaH2PO4) and
0.2 µg/mL chloroquine. The gel was run in TPE buffer at room
temperature at 10 V for 20 h, destained in water for 15 min,
stained in 0.05 µg/mL ethidium bromide for 15 min, and
destained again in water for 15 min. The gel was then
photographed, scanned by FluorImager (Molecular Dynamics),
and analyzed by ImageQuaNT software.
1
a yellow amorphous solid (29 mg, 99%): mp >220 °C dec; H
NMR (CF3COOD) δ 2.67 (m, 2H), 4.17 (m, 1H), 4.48-4.60 (m,
4H), 7.60-7.67 (m, 2H), 7.76 (s, 1H), 7.84 (m, 1H), 7.92-7.96
(m, 2H), 8.40 (s, 1H), 9.78 (s, 1H); 13C NMR (DMSO-d6) δ 29.4,
49.1, 49.2, 54.5, 104.4 (d, J C-F ) 24.6 Hz), 108.0, 113.3, 114.9,
116.2 (d, J C-F ) 9.0 Hz), 123.1, 125.4, 126.3 (d, J C-F ) 15.1
Hz), 127.7, 128.3, 129.0 (d, J C-F ) 14.2 Hz), 130.3, 132.3, 133.6
(d, J C-F ) 8.7 Hz), 138.6, 141.8, 153.0 (d, J C-F ) 248 Hz), 165.4,
175.9, 180.2; 19F NMR (DMSO-d6) δ -119.2 (d, J F-H ) 14.0
Hz); HRMS calcd for C24H19FN3O4 432.1360, found 432.1358.
3a : Anal. (C24H18FN3O4‚HCl‚3/2H2O) C, H, N. 3b: Anal.
(C24H18FN3O4‚HCl‚1/2H2O) C, H, N. 3c: Anal. (C24H18FN3O4‚
HCl‚1/2H2O) C, H, N.
1-(P ip er a zin -1-yl)-2-flu or o-4-oxo-4H -p yr id o[3,2,1-k l]-
ben zo[h ]p h en oxa zin e-5-ca r boxylic Acid Hyd r ogen Ch lo-
r id e Sa lt (4). Ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-
benzo[h]phenoxazine-5-carboxylate (11) (77 mg, 0.2 mmol) was
treated with tert-butyl 1-piperazinecarboxylate (109 mg, 0.6
mmol) in pyridine (5 mL) at 110 °C for 60 h. After removal of
the solvent, the residue was purified by chromatography,
yielding a yellow solid. The solid was refluxed in a 1:3 mixture
of 1 N KOH and ethanol (4 mL) for 30 min. The solution was
then mixed with equal volumes of 1:1 2 N HCl (aq) and ethanol
(3 mL) and refluxed again for 2 h. After allowing the mixture
to cool slowly to room temperature, a yellow amorphous solid
was isolated by filtration. This solid was extensively washed
with water and ethanol and dried in vacuo (55.6 mg, 60%):
Assa y for In h ibition of Cell Gr ow th . Exponentially
multiplying cells in 0.1 mL of medium were seeded on day 0
in 96-well microtiter plates. On day 1, 0.1-mL aliquots of
medium containing graded concentrations of test agent were
added in duplicate to the cell plates. After incubation at 37
°C in a humidified incubator, the plates were centrifuged
briefly (1-2 min at 1000 rpm), and 100 µL of the growth
medium was removed. Cell cultures were incubated with 50
µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide MTT, 1 mg/mL in Dubecco’s PBS, for 4 h at 37 °C. The
resulting purple formazan precipitate was solubilized with 200
µL of 0.4 N HCl in isopropyl alcohol. Absorbance was
quantitated using a Bio-Rad model 3550 microplate reader at
a test wavelength of 570 nm and a reference wavelength of
630 nm. Absorbance values were transferred to a 486 personal
1
mp >230 °C dec; H NMR (CF3COOD) δ 3.73 (s, 4H), 3.97 (s,
4H), 7.44 (d, J ) 9.0 Hz, 1H), 7.68 (t, J ) 7.2 Hz, 1H), 7.80 (t,
J ) 7.2 Hz, 1H), 7.96 (d, J F-H ) 10.8 Hz, 1H), 8.05 (d, J ) 7.5

4278 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Zeng et al.
(8) Willmott, C. J .; Maxwell, A. A Single Point Mutation in the DNA
Gyrase A Protein Greatly Reduces Binding of Fluoroquinolones
to the Gyrase-DNA Complex. Antimicrob. Agents Chemother.
1993, 37, 126-127.
(9) Permana, P. A.; Snapka, R. M.; Shen, L. L.; Chu, D. T. W.;
Clement, J . J .; Plattner, J . J . Quinobenoxazines: A Class of
Novel Antitumor Quinolones and Potent Mammalian DNA
Topoisomerase II Catalytic Inhibitors. Biochemistry 1994, 33,
11333-11339.
(10) Kwok, Y.; Zeng, Q.; Hurley, L. H. Specific Interaction of
Quinobenzoxazine A-62176 at Topoisomerase II Cleavage
Sites: A 2:2 Quinobenzoxazine:Mg2++ Self-Assembly Complex
in the Presence of Topoisomerase II. J . Biol. Chem. 1998,
submitted.
computer, and the IC50 values were determined using the
computer program EZ-ED50.15
Ack n ow led gm en t. This research was supported by
grants from the National Institutes of Health (CA-
49751) and the Welch Foundation. We thank Mark
Olsen who participated in the early part of this research.
We are grateful to David Bishop for proofreading,
editing, and preparing the final version of the manu-
script.
Refer en ces
(11) Fan, J .-Y.; Sun, D.; Yu, H.; Kerwin, S. M.; Hurley, L. H. Self-
(1) Chu, D. T. W.; Maleczka, R. E., J r. Synthesis of 4-Oxo-4H-quino-
[2,3,4-i,j][1, 4]benoxazine-5-carboxylic Acid Derivatives. J . Het-
erocycl. Chem. 1987, 24, 453-456.
(2) Chu, D. T. W.; Hallas, R.; Clement, J . J .; Alder, J .; McDonald,
E.; Plattner, J . J . Synthesis and Antitumour Activities of
Quinolone Antineoplastic Agents. Drugs Exp. Clin. Res. 1992,
18, 275-282.
(3) Chu, D. T. W.; Hallas, R.; Tanaka, S. K.; Alder, J .; Balli, D.;
Plattner, J . J . Synthesis and Antitumour Activities of Tetracyclic
Quinolone Antineoplastic Agents. Drugs Exp. Clin. Res. 1994,
20, 177-183.
(4) Clement, J . J .; Burres, N.; J arvis, K.; Chu, D. T. W.; Swiniarski,
J .; Alder, J . Biological Characterization of a Novel Antitumor
Quinolone. Cancer Res. 1995, 5, 830-835.
(5) Shen, L. L.; Baranowski, J .; Pernet, A. G. Mechanism of
Inhibition of DNA Gyrase by Quinolone Antibacterials. Specific-
ity and Cooperativity of Drug Binding to DNA. Biochemistry
1989, 28, 3879-3885.
(6) Shen, L. L.; Kohlbrenner, W. E.; Weigl, D.; Baranowski, J .
Mechanism of Quinolone Inhibition of DNA Gyrase. Appearance
of Unique Norfloxacin Binding Sites in Enzyme-DNA Complexes.
J . Biol. Chem. 1989, 264, 2973-2978.
(7) Shen, L. L.; Mitscher, L. A.; Sharma, P. N.; O′Donnell, T. J .;
Chu, D. W. T.; Cooper, C. S.; Rosen, T.; Pernet, A. G. Mechanism
Assembly of a Quinobenzoxazine-Mg2+ Complex on DNA:
A
New Paradigm for the Structure of a Drug-DNA Complex and
Implications for the Structure of the Quinolone Bacterial Gyrase-
DNA Complex. J . Med. Chem. 1995, 38, 408-424.
(12) Yu, H.; Hurley, L. H.; Kerwin, S. M. Evidence for the Formation
of 2:2 Drug-Mg2+ Dimers in Solution and for the Formation of
Dimeric Drug Complexes on DNA from the DNA-Accelerated
Photochemical Recation of Antineoplastic Quinobenzoxazines.
J . Am. Chem. Soc. 1996, 118, 7040-7048.
(13) Marini, J . C.; Miller, K. G.; Englund, P. T. Decatenation of
Kinetoplast DNA by Topoisomerases. J . Biol. Chem. 1980, 255,
4976-4979.
(14) Liu, L. F. DNA Topoisomerase Poisons as Antitumor Drugs.
Annu. Rev. Biochem. 1989, 58, 351-375.
(15) Chen, S. F.; Behrens, D. L.; Behrens, C. H.; Czerniak, P. M.;
Dexter, D. L.; Dusak, B. A.; Fredericks, J . R.; Gale, K. C.; Gross,
J . L.; J iang, J . B.; Kirshenbaum, M. R.; McRipley, R. J .; Papp,
L. M.; Patten, A. D.; Perrella, F. W.; Seitz, S. P.; Stafford, M.
P.; Sun, J . H.; Sun, T.; Von Hoff, D. D. XB596, a Promising Bis-
naphthalimide Anticancer Agent. Anti-Cancer Drugs 1993, 4,
447-457.
of Inhibition of DNA Gyrase by Quinolone Antibacterials:
A
Cooperative Drug-DNA Binding Model. Biochemistry 1989, 28,
3886-3894.
J M980265C