New insight for fluoroquinophenoxazine derivatives as possibly new potent topoisomerase I inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.12.053

Fluoroquinolones, represented by ciproxacin and norfloxacin, are well-known clinical antimicrobial agents, and their phenyl ring expanded quinophenoxazines are reported as possible antitumor active compounds. These quinophenoxazines are known to inhibit DNA topoisomerase II essential for cell replication cycle. But there were no reports for topoisomerase I inhibition study for these compounds. In this report, we have prepared a few quinophenoxazine analogues and tested their topoisomerases I and II inhibitory activities and cytotoxicity. From the result, we found that quinophenoxazine analogues possessed strong topoisomerase I inhibitory capacity as well as topoisomerase II inhibition. Among the compounds prepared, A-62176 analogues showed strong topoisomerases I and II inhibitory activities. Interestingly, compound 8 missing the 3-aminopyrrolidine moiety at C2 position has similar potent inhibitory capacity against topoisomerases I and II at higher concentrations (20 and 10 μM, respectively). But compound 8 inhibited topoisomerase I function more selectively at lower concentration, 2 μM. Our observation might strongly implicate that fluoroquinophenoxazines can be developed as efficient topoisomerase I inhibitor with the elaborate modification.

Full text of DOI:10.1016/j.bmcl.2007.12.053

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1520–1524
New insight for fluoroquinophenoxazine derivatives as possibly
new potent topoisomerase I inhibitor
Da-Hye Kang,a Jung-Sook Kim,b Mi-Ja Jung,a Eung-Seok Lee,c Yurngdong Jahng,c
Youngjoo Kwona,* and Younghwa Nab,*
aCollege of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
bCollege of Pharmacy, Catholic University of Daegu, Gyeongsan, Gyeongbuk 712-702, Republic of Korea
cCollege of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Republic of Korea
Received 8 October 2007; revised 3 December 2007; accepted 20 December 2007
Available online 25 December 2007
Abstract—Fluoroquinolones, represented by ciproxacin and norfloxacin, are well-known clinical antimicrobial agents, and their
phenyl ring expanded quinophenoxazines are reported as possible antitumor active compounds. These quinophenoxazines are
known to inhibit DNA topoisomerase II essential for cell replication cycle. But there were no reports for topoisomerase I inhibition
study for these compounds. In this report, we have prepared a few quinophenoxazine analogues and tested their topoisomerases I
and II inhibitory activities and cytotoxicity. From the result, we found that quinophenoxazine analogues possessed strong topoiso-
merase I inhibitory capacity as well as topoisomerase II inhibition. Among the compounds prepared, A-62176 analogues showed
strong topoisomerases I and II inhibitory activities. Interestingly, compound 8 missing the 3-aminopyrrolidine moiety at C2 position
has similar potent inhibitory capacity against topoisomerases I and II at higher concentrations (20 and 10 lM, respectively). But
compound 8 inhibited topoisomerase I function more selectively at lower concentration, 2 lM. Our observation might strongly
implicate that fluoroquinophenoxazines can be developed as efficient topoisomerase I inhibitor with the elaborate modification.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Fluoroquinolones, represented by ciproxacin (1a) and
norfloxacin (1b),1 are well-known clinical antimicrobial
agents. The pharmacological activities of this class of
compounds are known to be originated from DNA gyr-
ase and topoisomerase IV inhibition.2 The similarity be-
tween bacterial and eukaryotic DNA topoisomerase II
mechanistic aspects had stimulated many researchers
to prepare and test phenyl ring expanded fluoroquin-
ophenoxazines as possible new antitumor agents.3
Among the compounds, A-62176 (2)3a reported by Ab-
bott researchers showed good inhibitory activity against
human and murine cancer cell lines. Based on this infor-
mation, several types of phenyl ring expanded fluoro-
quinophenoxazines have been synthesized and tested
for the development of prospective anticancer agents.4
Since it has been known that the fluoroquinophenoxa-
zines are topoisomerase II inhibitor,5 most of the biolog-
ical mechanistic tests are devoted to topoisomerase II
inhibition pathways. However, there are no reports
against topoisomerase I for this class of compounds.
O
O
O
N
O
F
F
OH
OH
N
N
R
N
HN
O
H2N
HCl
1a (Ciproxacin) R = Cyclopropyl
1b (Norfloxacin) R = Ethyl
2 (A-62176)
In this report, we have prepared six fluoroquinophenox-
azine analogues with (S)-A-62176. And we have tested
their topoisomerase I inhibitory activities to determine
the possibility if fluoroquinophenoxazines can be
exploitable as new topoisomerase I inhibitors. We also
tested topoisomerase II inhibitory activity to compare
the selectivity between two topoisomerases.
Keywords: Fluoroquinophenoxazines; Topoisomerases I and II inhi-
bition; Anti-cancer agents.
*
Corresponding authors. Tel.: +82 2 3277 4653; fax: +82 2 3277 2851
(Y.K.); tel.: +82 53 850 3616; fax: +82 53 850 3602 (Y.N.); e-mail
addresses: ykwon@ewha.ac.kr; yna7315@cu.ac.kr
The structures of prepared compounds are depicted in
Figure 1. In order to synthesize these compounds we
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.053

D.-H. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1520–1524
1521
O
O
O
N
O
O
N
O
F
F
F
OH
OH
OH
N
N
N
N
S
O
O
HCl
H2N
H2N
H2N
HCl
HCl
NO2
O
Cl
7a
7c
7b
O
O
O
N
O
O
F
F
OH
OH
F
OH
N
N
N
F
N
O
O
O
H2N
H2N
HCl
N
N
HCl
SO3H
OH
7e
7d
8
Figure 1. Structures prepared.
employed methods previously reported3a,6 (see Scheme
1). The key starting compounds, O-aminoaromatic alco-
hol or thiol, were used with commercially available ones.
The in situ coupling of O-aminothiophenol and product
of ethyl 2,3,4,5-tetrafluorobenzoylacetate (3) and tri-
ethyl orthoformate in acetic anhydride afforded 4a.
Double ring cyclization under NaHCO3 basic condition
and subsequent regioselective nucleophilic substitution
reaction at C-2 position with 3(S)-(tert-butoxycarbon-
ylamino)pyrrolidine produced 6a. Final hydrolysis of
ethyl carboxylate and removal of protected group from
amino group produced 7a. With this procedure, six flu-
oroquinophenoxazine analogues including A-62176
were prepared. Among these compounds, five new quin-
ophenoxazines 7a–e and two known ones, 86 and A-
62176,4c were prepared. The spectral data for the com-
pounds, 7a–e, are indicated in the reference section.7
erature methods.8 First, in the topoisomerase II inhibi-
tion results showed that five compounds except 7d and
7e have inhibited the enzyme activity potently compared
Table 1. Topoisomerase I and II inhibitory activities of prepared
compounds
Compounds
Topo I (%
Inhibition)
Topo II (%
Inhibition)
2 lM
20 lM
2 lM
10 lM
Camptothecin
Etoposide
63.3
ND
45.1
87.9
65.7
39.1
32.4
88.9
53.5
84.1
ND
97.2
86.1
95.3
25.0
0.0
ND
0.7
ND
40.9
91.7
88.6
91.6
24.7
28.8
89.4
82.4
7a
7b
57.0
89.0
26.0
1.9
7c
7d
7e
A-62176
8
0.0
90.2
96.6
89.1
0.5
Topoisomerases I and II tests were conducted with
supercoiled pBR322 plasmid DNA according to the lit-
The values of
experiments.
%
inhibition are means from three independent
O
F
O
O
F
O
O
N
O
F
F
F
F
F
F
a
b
OEt
OEt
OEt
39%
70%
N
H
F
F
S
HS
3
4a
5a
O
O
O
O
F
F
OH
c
OEt
d
52%
N
N
N
N
73%
S
S
H2N
BocHN
6a
7a
Scheme 1. General synthetic method for the target compounds. (a) 1. CH(OEt)3, Ac2O; 2. 2-Aminothiophenol; (b) NaHCO3; (c) 3-t-Boc-(S)-
aminopyrrolidine; (d) 1. NaOH; 2. HCl.

1522
D.-H. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1520–1524
activity. Next, we have conducted the topoisomerase I
inhibition test for the compounds. and the results are
surprising. Basically, topoisomerase I inhibitory activity
pattern was quite similar to that of topoisomerase II.
Five compounds except 7d and 7e showed comparable
activity with camptothecin at the range of tested concen-
trations, 2 and 20 lM (Table 1 and Fig. 3). The test
compounds possessing structural similarity with A-
62176 exhibited potent inhibitory activities but others,
7d and 7e, did not. These two topoisomerases inhibition
results are quite interesting and impressive since the
quinophenoxazine core can play the role as a dual
repressor against topoisomerases I and II action. Com-
pound 8 also exhibited enzyme inhibition activity com-
parable to A-62176 at lower concentration, 2 lM. But
compound 8 had not shown inhibitory action against
topoisomerase II at the same concentration. This infor-
mation might suggest the possibility that modification of
3(S)-aminopyrrolidine group attached to quinophenox-
azine core controls the selectivity between the topoi-
D
D
T
T
E
E
1
1
2
2
3
3
4
4
5
6
7
(2 uM)
5
6
7
(10 uM)
Figure 2. Topoisomerase II inhibitory activities of prepared com-
pounds. Compounds were examined in a final concentration of 2 and
10 lM, respectively. Lane D: pBR322 only, Lane T: pBR322 + Topo
II, Lane E: pBR322 + Topo II + etoposide, Lanes 1–7: pBR322 +
Topo II + compounds in designated concentrations (compounds 7a–e,
A-62176, and 8).
to etoposide used as positive control. (Table 1 and
Fig. 2) These active quinophenoxazine analogues exhib-
ited much higher inhibition efficiency than etoposide at
2 lM concentration. But, compounds 7d and 7e possess-
ing fused naphthalenyl and pyrimidyl moieties have not
shown good biological activity. Interestingly, compound
8 missing 3(S)-aminopyrrolidine group still revealed
good enzyme inhibitory activity at higher concentration,
20 lM, compared with A-62176. Based on this result, we
suspected that the phenyl ring fusion into benzoxazine
core is important to exert topoisomerase II inhibition
somerases
I and II. But the clear factors and
mechanism of compound 8 for selective inhibition on
the topoisomerase I function over II at lower concentra-
tion should be studied in more detail.
With these results, we pursued the cytotoxicity test9 to
correlate the topoisomerases inhibition ability and cyto-
toxic properties for the tested compounds. Disappoint-
ingly, the compounds were not anticancer active ones
as potent as positive controls and even less than A-
62176 (Table 2).
D
T
C
1
2
3
4
5
6
7 (2 uM)
In conclusion, five compounds except 7d and 7e showed
very potent inhibitory capacity against topoisomerases I
and II. These data, however, were not parallel with the
cytotoxic activities, which are common in biological
activity study in vitro.10 Our findings reflect that fluor-
oquinophenoxazizne analogues are potent inhibitor
against topoisomerase I as well as topoisomerase II. In
addition, more elaborate consideration of the SAR for
these analogues could provide better information for
the development of new class of topoisomerase I inhib-
itors. For the clear SAR paradigm of the quinophenox-
azine analogues as selective topoisomerase I, more
compounds should be tested and analyzed.
D
T
C
1
2
3
4
5
6
7 (20 uM)
Figure 3. Topoisomerase I inhibitory activities of prepared com-
pounds. Compounds were examined in a final concentration of 2 and
20 lM, respectively. Lane D: pBR322 only, Lane T: pBR322 + Topo I,
Lane C: pBR322 + Topo I + camptothecin, Lanes 1–7: pBR322 +
Topo I + compounds in designated concentrations (Compounds 7a–e,
A-62176, and 8).
Table 2. Cytotoxicities of prepared compounds (7a–e, A-62176, and 8) against various cancer cells
a
IC50 (lM)
Compound/cells
DU 145
HCT 116
HeLa
MDA-MB231
HL-60
Adriamycin
Camptothecin
1.57 0.06
3.20 0.40
29.79 1.98
19.30 1.98
17.60 0.97
13.83 1.13
>50
1.10 0.07
1.00 0.02
15.11 0.87
2.41 0.26
2.56 0.03
7.45 0.78
>50
3.09 0.32
1.08 0.67
18.24 1.21
5.08 1.36
2.01 0.16
17.74 1.57
>50
1.39 0.07
4.16 0.33
16.25 1.84
19.17 1.17
19.34 0.88
19.76 0.23
>50
0.72 0.04
0.013 0.002
1.44 0.08
11.51 0.51
1.63 0.13
9.93 0.19
>50
Etoposide
7a
7b
7c
7d
7e
>50
1.82 0.23
>50
>50
0.87 0.20
6.30 0.17
>50
2.33 0.08
>50
>50
4.34 0.10
>50
>50
2.40 0.08
>50
A-62176
8
a Each data point represents the mean SD from three different experiments performed in triplicate. Cell lines used are DU 145, human prostate
tumor; HCT 116, human colon tumor; HeLa, human cervix tumor; MDA-MB231, human breast tumor; HL-60, human myeloid leukemic tumor.

D.-H. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1520–1524
1523
3.87 (m, 2H), 3.93–3.97 (m, 1H), 7.41 (d, J = 14.0 Hz, 1H),
7.55 (dd, J = 7.6, 7.2 Hz, 1H), 7.61 (dd, J = 8.0, 7.2 Hz,
1H), 7.83 (s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 8.08 (d,
J = 7.6 Hz, 1H), 8.09 (br s, 3H), 8.90 (d, J = 8.0 Hz, 1H),
9.12 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 30.0, 49.9,
50.0, 55.2, 105.5, 107.6,116.2, 117.1 (JC-F = 8.2 Hz), 119.7,
120.7, 125.2, 126.2, 128.5, 128.6, 129.3, 129.5, 129.6, 134.8
(JC-F = 8.9 Hz), 144.4, 144.6, 146.6, 153.4 (JC-F = 246.2
Hz), 166.2, 176.5.
Acknowledgment
This work was financially supported by Catholic Uni-
versity of Daegu, Korea.
References and notes
Compound 7e: 1H NMR (400 MHz, CD3OD) d 2.06–2.15
(m, 1H), 2.34–2.42 (m, 1H), 3.70–3.77 (m, 2H), 3.91–4.02
(m, 3H), 7.88 (d, J = 14.0 Hz, 1H), 8.05 (s, 1H), 8.56 (s,
1H); 13C NMR (100 MHz, DMSO-d6) 30.4, 50.2, 51.2,
55.8, 108.3, 108.6, 109.0, 114.1, 118.5 (JC-F = 8.1 Hz),
121.2, 141.3, 152.3, 153.7, 162.7, 168.3, 177.9. Other peaks
were not observed due to low concentration of this
compound.
1. (a) Wise, R.; Andrew, J. M.; Edwards, J. M. Antimicrob.
Agents Chemother. 1983, 23, 559; (b) Koga, H.; Itoh, A.;
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Topoisomerase I. Experiments were performed as
described previously. The test compounds were dissolved
in DMSO at 20 mM as stock solution. The activity of
DNA topoisomerase I was determined by assessing the
relaxation of supercoiled DNA pBR322. The mixture of
100 ng of plasmid pBR322 DNA and 0.2 units of calf
thymus DNA topoisomerase I (Fermentas, USA) was
incubated without and with the prepared compounds at
37ꢁC for 30 min in the relaxation buffer (35 mM Tris-HCl
(pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol,
2 mM spermidine, and 0.01% bovine serum albumin). The
reaction in the final volume of 10 lL was terminated by
adding 2.5 lL of the stop solution containing 10% SDS,
0.2% bromophenol blue, 0.2% xylene cyanol, and 30%
glycerol. DNA samples were then electrophoresesed on a
1% agarose gel at 15 V for 7 h with a running buffer of
TAE. Gels were stained for 30 min in an aqueous solution
of ethidium bromide (0.5 lg/mL). DNA bands were
visualized by transillumination with UV light and were
quantitated using AlphaImagerTM (Alpha Innotech Cor-
poration).; (b) Woo, S.; Jung, J.; Lee, C.; Kwon, Y.; Na,
Y. Bioorg. Med. Chem. Lett. 2007, 17, 1163, DNA
relaxation assay of Topoisomerase II. DNA topoisomerase
II inhibitory activity of compounds was measured as
follows. The mixture of 200 ng of supercoiled pBR322
plasmid DNA and 2 units of human DNA topoisomerase
IIa (Amersham, USA) was incubated without and with
the prepared compounds in the assay buffer (10 mM Tris-
HCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl2,
1 mM EDTA, 1 mM ATP, and 15 lg/mL bovine serum
albumin) for 30 min at 30 ꢁC. The reaction in a final
volume of 20 lL was terminated by the addition of 3lL of
7 mM EDTA. Reaction products were analyzed on a 1%
agarose gel at 25 V for 4 h with a running buffer of TAE.
Gels were stained for 30 min in an aqueous solution of
ethidium bromide (0.5 lg/mL). DNA bands were visual-
ized by transillumination with UV light and supercoiled
DNA was quantitated using AlphaImagerTM (Alpha
Innotech Corporation).
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Hurley, L. H. J. Med. Chem. 1998, 41, 4273; (b) Duan, W.;
Rangan, A.; Vankayalapati, H.; Kim, M.-Y.; Zeng, Q.;
Sun, D.; Han, H.; Federoff, O. Y.; Nishioka, D.; Rha, S.
Y.; Izbicka, D.; Von Hoff, D. D.; Hurley, L. H. Mol.
Cancer Ther. 2001, 1, 103; (c) Kim, M.-Y.; Duan, W.;
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6. Whitten, J.P.; Schwaebe, M.; Siddiqui-Jain, A.; Moran, T.
US pat. Appl. Publ. 2006. 8pp, U.S. Ser. No. 903, 975.
7. Compound 7a: 1H NMR (400 MHz, CD3OD + DMSO-
d6) d 2.04–2.15 (m, 1H), 2.48–2.57 (m, 1H), 3.43–3.50 (m,
2H), 3.58 (dd, J = 14.8, 8.8Hz, 1H), 3.71 (dd, J = 10.4,
6.4 Hz, 1H), 3.99–4.04 (m, 1H), 7.38–7.48 (m, 3H), 7.63 (d,
J = 7.6 Hz, 1H), 7.80 (d, J = 12.4 Hz, 1H), 9.26 (s, 1H);
13C NMR (100 MHz, CD3OD + DMSO-d6) 29.9, 48.7,
50.4, 54.1, 108.8 (JC-F = 23.8 Hz), 109.6, 120.6, 123.6,
125.2 (JC-F = 8.1 Hz), 125.8, 128.2, 128.6, 129.4, 134.4,
135.3 (JC-F = 14.9 Hz), 135.4, 143.7, 158.8 (JC-
F = 252.1 Hz), 167.1, 177.6.
Compound 7b: 1H NMR (400 MHz, DMSO-d6) d 1.98–
2.03 (m, 1H), 2.21–2.25 (m, 1H), 3.71–3.74 (m, 2H), 3.81–
3.83 (m, 2H), 3.91–3.94 (m, 1H), 7.29 (d, J = 8.8 Hz, 1H),
7.40 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 14.0 Hz, 1H), 8.20 (s,
1H), 9.10 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 30.1,
49.9, 50.0, 55.1, 105.2 (JC-F = 24.5 Hz), 108.2, 116.8
(JC-F = 8.1 Hz), 117.7, 119.9, 125.3, 126.4, 129.5 (JC-F
=
14.2 Hz), 129.6, 129.8, 134.4 (JC-F = 8.9 Hz), 139.5, 143.2,
154.1 (JC-F = 246.9 Hz), 165.9, 176.5.
Compound 7c: 1H NMR (400 MHz, CD3OD) d 2.12–2.20
(m, 1H), 2.42–2.48 (m, 1H), 3.83–3.88 (m, 2H), 4.01–4.05
(m, 2H), 4.07–4.10 (m, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.44
(d, J = 13.2 Hz, 1H), 8.19 (d, J = 8.8 Hz, 1H), 8.55 (s, 1H),
9.06 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 30.0, 49.9,
50.0, 55.1, 105.7, 108.5, 114.1, 116.4 (JC-F = 9.7 Hz), 119.3,
125.0, 125.3, 126.4, 129.9 (JC-F = 16.4 Hz), 133.6, 134.4,
140.2, 144.5, 149.3, 154.0 (JC-F = 246.1 Hz), 165.8, 176.4.
Compound 7d: 1H NMR (400 MHz, DMSO-d6) d 1.94–
1.98 (m, 1H), 2.19–2.24 (m, 1H), 3.67–3.70 (m, 2H), 3.83–
9. Cytotoxicity assay: Cancer cells were cultured according to
the supplier’s instructions. Cells were seeded in 96-well
plates at a density of 2–4 · 104 cells per well and incubated
overnight in 0.1 mL of media supplied with 10% Fetal
Bovine Serum (Hyclone, USA) in 5% CO2 incubator at
37 ꢁC. On day 2, culture medium in each well was
exchanged with 0.1 mL aliquots of medium containing
graded concentrations of compounds. On day 4, each well
was added with 5 lL of the cell counting kit-8 solution

1524
D.-H. Kang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1520–1524
(Dojindo, Japan) and then incubated for additional 4 h
10. (a) Zhao, L. X.; Kim, T. S.; Ahn, S. H.; Kim, T. H.; Kim,
E. K.; Cho, W. J.; Choi, H.; Lee, C. S.; Kim, J. A.; Jeong,
T. C.; Chang, C.-j.; Lee, E.-S. Bioorg. Med. Chem. Lett.
2001, 11, 2659; (b) Cho, W. J.; Le, Q. M.; Van, H. T. M.;
Lee, K. Y.; Kang, B. Y.; Lee, E.-S.; Lee, S. K.; Kwon, Y.
Bioorg. Med. Chem. Lett. 2007, 17, 3531; (c) Seo, C. S.; Li,
G.; Kim, C. H.; Lee, C. S.; Jahng, Y.; Chang, H. W.; Son,
J. K. Arch. Pharm. Res. 2007, 30, 1404.
under the same condition. The absorbance of each well
was determined by an Automatic Elisa Reader System
(Bio-Rad 3550) at a 450 nm wavelength. For determina-
tion of the IC50 values, the absorbance readings at 450 nm
were fitted to the four-parameter logistic equation. The
compounds of adriamycin, etoposide, and camptothecin
were purchased from Sigma and used as positive controls.