Synthesis and cytotoxic activity of a new series of topoisomerase I inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.055

A series of structurally simple analogues of natural topopyrone C were synthesized and tested for cytotoxic and topoisomerase I inhibitory activities. The removal of the hydroxyl groups at the 5 and 9 positions resulted in an increased cytotoxic potency a

Full text of DOI:10.1016/j.bmcl.2007.12.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1484–1489
Synthesis and cytotoxic activity of a new series
of topoisomerase I inhibitors
Sabrina Dallavalle,a,* Sonia Gattinoni,a Stefania Mazzini,a Leonardo Scaglioni,a
Lucio Merlini,a Stella Tinelli,b Giovanni L. Berettab and Franco Zuninob
a
`
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, via Celoria 2, 20133 Milano, Italy
bDepartment of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale dei Tumori,
via Venezian 1, 20133 Milan, Italy
Received 20 November 2007; revised 20 December 2007; accepted 21 December 2007
Available online 25 December 2007
Abstract—A series of structurally simple analogues of natural topopyrone C were synthesized and tested for cytotoxic and topoi-
somerase I inhibitory activities. The removal of the hydroxyl groups at the 5 and 9 positions resulted in an increased cytotoxic
potency and ability to stabilize topoisomerase-mediated cleavage. In addition, the results suggest that some structural features, such
as the pyrone ring and a polar group in position 11, are fundamental for topoisomerase I inhibitory effect. These structural require-
ments are also consistent with the cytotoxic activity.
Ó 2007 Elsevier Ltd. All rights reserved.
Topoisomerase I (topo I) is a nuclear enzyme that cata-
lyzes the relaxation of superhelical tension in DNA dur-
ing replication and transcription. This process is
initiated by a reversible covalent attachment of the en-
zyme to the DNA backbone, creating a transient sin-
gle-strand break in the DNA duplex and preventing
buildup of torsional energy. The formed DNA–topo I
covalent binary complex, which normally undergoes
religation, can be stabilized by drugs via formation of
a ternary complex. Persistence of the stabilized DNA
breaks leads to cell death.1
alternative mechanism,4 such as inhibition of catalytic
activity. Camptothecins (CPT) are the only topo I inhib-
itors in clinical use, but there are some disadvantages
inherent to the use of CPT. Although CPT is cytotoxic
in a wide range of animal tumors, its therapeutic efficacy
is limited due to the rapid reversibility of the ternary
DNA–enzyme–CPT complex and hydrolysis of the lac-
tone to a hydroxyacid that binds to plasma proteins.6
Moreover, some cancer cells develop resistance to
CPT. In this context, there is a need for chemically sta-
ble compounds that act as topo I inhibitors and that are
cytotoxic in cancer cell lines.
As cancer cells tend to overexpress this enzyme, which is
required during various DNA functions, topo I inhibi-
tors have emerged as important antineoplastic agents.2
A diverse set of anticancer compounds, including camp-
tothecins,3 indolocarbazoles, and indenoisoquinolines,4
are selective topo I inhibitors. These compounds bind
to the transient topo I–DNA covalent complex and inhi-
bit resealing of the single-strand break.5 Other com-
pounds, however, can inhibit topo I through an
Recently, Kanai et al.7 discovered four new specific
inhibitors of topo I, topopyrones A–D (1a–d). All
four compounds were isolated from the culture broth
of a fungus, Phoma sp. BAUA2861, and two of them
from Penicillium sp. BAUA4206. Structural elucida-
tion showed that the compounds are of the anthraqui-
none type and contain a fused 1,4-pyrone moiety8
(Chart 1).
Topopyrones A, B, C, and D selectively inhibit recombi-
nant yeast growth, depending on the expression of hu-
man topo I. All these compounds showed significant
cytotoxic effects (in the range 0.7–20 lM) against a pa-
nel of tumor cell lines when tested in vitro.7 Structural
Keywords: Topopyrones; Cytotoxic activity; Topoisomerase I;
Antitumor.
*
Corresponding author. Tel.: +39 02 50316818; fax: +39 02
50316801; e-mail: sabrina.dallavalle@unimi.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.055

S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1484–1489
1485
ity to topo I inhibitors. This finding prompted us to
investigate the structural requirements for antitumor
activity of this class of compounds.
CH3
1
3
OH
O
OH
O
O
OH
O
O
O
11
R
10
9
R
O
A series of simple analogues were synthesized following
our previously developed synthetic strategy. Although
the number of compounds tested was limited, some
structural features important to the topo I inhibitory ef-
fect can be inferred. We report here the synthesis, the
assessment of the cytotoxic activity, and the stabilizing
activity of the DNA–topo I complex by the derivatives
of this series.
HO
CH3
HO
OH
5
8
6
O
1a R = Cl Topopyrone A
1b R = H Topopyrone C
1c R = Cl Topopyrone B
1d R = H Topopyrone D
Chart 1.
and biological features of topopyrones make them
attractive synthetic targets.
Topopyrone C (1b) was synthesized as previously de-
scribed.9 The synthetic approach was based on the use
of the Marschalk alkylation reaction of 1-hydroxy-
3,6,8-trimethoxyanthraquinone10 followed by a Baker–
Venkataraman chain elongation11 and an acid-catalyzed
cyclization for the construction of the pyrone
framework.
As a part of our ongoing project centered on the study
of novel topo I inhibitors, we recently developed a meth-
od for total synthesis of topopyrone C.
However, in our hands, the compound showed moder-
ate cytotoxic activity (IC50 = 29.50 lM) compared to
the reference CPT topotecan (IC50 = 1.18 lM) when
tested on the human non-small cell lung cancer
NCI-H460 cell line, a cell model selected for its sensitiv-
In an effort to define the structural elements in topopy-
rone C essential for binding to the topo I–DNA covalent
binary complex, we have been altering topopyrone C at
OH
O
OH
OMe O
OH OH
OMe O
OH
a
b
R
R
R
R
R
R
O
O
O
4
2
3
OMe O
OH
O
R
d
c
R
2-5a R = H
2-5b R = OMe
5
O
O
O
R'
O
OMe O
OH
O
R
O
OMe O
g
e
R'
R
R
R
7
O
6
O
f
R'
O
R'
h
OH
O
OMe O
O
O
O
R
R
R
R
O
O
8
9a R = H R' = -Me
1b R = OH R' = -Me
9c R = H R' = -Ph
6-8a R = H R' = -Me
6-8b R = OMe R' = -Me
6-8c R = H R' = -Ph
Scheme 1. Reagents and conditions: (a) TsOCH3, Na2CO3, tetraglyme, 120 °C, 2 h, 81%, or TsOCH3, Na2CO3, MW, 210 W, 15 min, 81–88%; (b)
1—NaOH, Na2S2O4, CH3OH, N2, 0 °C, 2—CH3CHO, N2, 20 °C, 3 h, 3—H2O2, 0 °C, 30–41%; (c) PCC, H5IO6, CH3CN, 0 °C, 30 min, then rt, 3 h,
60–100%; (d) Ac2O, Py, reflux, 10 h, 88–92% or PhCOCl, K2CO3, CH3COC2H5, reflux, 4 h, 93%; (e) LiH, THF, N2, reflux, 20 h, 55–82%; (f) HBr,
AcOH, 8 h, reflux, 53–76%; (g) TFA, 76–94%; (h) BBr3, CH2Cl2, ꢀ60 °C, 90 min, 25–69% or HBr, AcOH, 6 h, reflux, 83%.

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S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1484–1489
single sites deemed to be critical for inhibitory activity.
The first goal of the present investigation was to define
the role of the hydroxyl groups in positions 5, 9, and
11 and to determine whether they could be deleted with
retention of activity. Thus, the synthetic strategy previ-
ously described was followed to obtain the simplified
derivatives 9a and 9c, starting from commercially avail-
able 1,8-dihydroxyanthraquinone (Scheme 1). The com-
pounds were tested for growth inhibition ability against
the human non-small cell lung cancer NCI-H460 cell
line.12 Cytotoxicity was assessed by growth inhibition
assay after a 1-h drug exposure.13 Interestingly, the cyto-
toxic activity of 9a and 9c was higher than that of top-
opyrone C, revealing that the presence of the two
hydroxyl groups in positions 5 and 9 was detrimental
for activity (Table 1). In contrast, the hydroxyl group
in position 11 seemed to play an important role: in fact,
methylation of the OH in position 11 led to a drop in
activity (8a vs 9a, 8c vs 9c, see also 8b).14
and acetamide in acetic acid. Compound 12 was synthe-
sized to improve the water solubility of this very lipo-
philic class of compounds. Conversion of 9a–12 was
achieved using dimethylaminoethyl chloride hydrochlo-
ride in tetraglyme.
The introduction of a dimethylaminoethyl chain linked
to the oxygen at position 11 led to a more water-soluble
derivative (12), which retained good cytotoxic activity.
The incorporation of a nitro or a bromo group ortho
or para to the 11-OH resulted in the maintenance of
activity. The most active derivative was compound 13,
with an additional bromo group on the pyrone ring.
In order to investigate the ability of the compounds to
stimulate DNA damage, topopyrone C analogues were
submitted to a test of topo I-mediated DNA cleavage.
All the compounds analyzed appeared less potent than
SN38 in producing DNA damage. Compared to SN38,
no change in sequence selectivity was observed. Com-
pound 13 was the most effective of the series, whereas
9a, 11, and 12 revealed a lower intensity of DNA cleav-
age. The activity was markedly reduced in 8a. This trend
is consistent with the changes in the cytotoxic activity.
The only exception seems to be compound 10, which
is not active on topo I, although showing some cytotoxic
activity. This effect could reflect a steric hindrance which
influences the drug interaction in the ternary complex.
The cytotoxic effect of this compound is likely related
to a topo I-independent mechanism.
Topo I-mediated DNA cleavage assay was used to
investigate the ability of the compounds to stimulate
DNA damage.15 Purified human topo I was used with
SN38 as the reference drug (Fig. 1). Compound 9a in-
duced the same sequence selectivity of DNA cleavage
by topo I shown by CPT SN38. This result indicates
that, although less potent than a CPT, compound 9a
stabilizes in a similar way the binary DNA–topo I cleav-
able complex. The formation of a stable ternary com-
plex is deemed to be the reason for the cytotoxic
activity of CPT.
Another goal of the present study was to assess the role
of the pyrone ring. Compounds 3a, 14, and 15 where a
simple methoxy group replaces the pyrone ring (Scheme
3) were tested for their cytotoxic activity and compared
with the corresponding topopyrones (9a, 12, and 10).
The results showed that removal of the pyrone ring led
to complete loss of antiproliferative activity in all three
cases (Table 2). Moreover, analysis of the topo I-medi-
ated DNA cleavage assay showed that these compounds
were substantially not able to induce DNA damage.
In accord with these findings, compound 9a was further
modified by introducing either lipophilic or hydrophilic
moieties on the skeleton of the molecule (Scheme 2).
Treatment of 9a with Cu(NO3)2 in trifluoroacetic acid
gave compound 10 in a 20% yield. The monobromo
derivative 11 was obtained by treatment of 9a with
NBS and p-toluenesulfonic acid, whereas the dibromo-
derivative 13 was obtained in an 83% yield using Br2
Table 1. In vitro cytotoxic activity of topopyrone C and analogues on H460 cell lines
R7
R6
OR1 O
O
R2
R3
O
R5
R4
O
R1
R2
R3
R4
R5
R6
R7
IC50 (lM)
a
Compound
1b
8a
H
Me
H
H
H
H
H
H
H
H
H
Br
OH
H
H
OH
H
H
H
H
H
H
H
H
Br
H
Br
Me
Me
Me
Ph
29.50
>260
>100
>100
5.50
7.06
9.10
6.50
7.15
4.95
1.18
H
8b
8c
Me
Me
OMe
H
H
OMe
H
H
9a
9c
H
H
H
H
Me
Ph
H
H
H
H
10
11
H
H
NO2
H
H
Me
Me
Me
Me
H
H
H
12
13
(CH2)2N(Me)2
H
H
H
H
H
H
H
Topotecan
a The IC50 values are means of three inhibition assays.

S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1484–1489
1487
Figure 1. Topoisomerase I-mediated DNA cleavage by SN38 and different topopyrones. Samples were reacted with 1, 10, and 50 lM drug at 37 °C
for 30 min. Reaction was then stopped by adding 0.5% SDS, 0.3 mg/ml of proteinase K and incubating for 45 min at 42 °C before loading on a
denaturing 8% polyacrylamide gel. Experiments were performed three times and a representative image is reported. C, Control DNA; T, reaction
without drug; M, purine markers.
CH3
CH3
N
OH
O
O
O
O
O
O
O
O
O
CH3
a
b
12
OH
O
O
O
10
NO2
O
d
c
9a
CH3
CH3
Br
O
Br
OH
O
O
OH
O
O
Br
O
11
13
O
O
Scheme 2. Reagents and conditions: (a) Cu(NO3)2Æ3H2O, CF3COOH, rt, 5 days, 20%; (b) (Me)2N(CH2)2ClÆHCl, K2CO3, tetraglyme, 120 °C, 3 h,
27%; (c) Br2, CH3CONH2, CH2Cl2, AcOH, reflux, 6 h, 83%; (d) NBS, pTsOHÆH2O, MeOH,reflux, 3 h, 40%.
Thus, it appears evident that addition of the 4-pyrone
moiety to the anthraquinone ring is determinant for
the poisoning activity of topo I. The capability to inhibit
the enzymatic activity of purified topo I was evaluated
using the DNA relaxation assay.16 Compounds 9a and
13, the most active analogues in reducing cell growth,
were analyzed. Neither compound was active in inhibit-
ing the enzymatic activity (Fig. 2).
lular uptake, subcellular distribution, and additional
biological targets may contribute to determine antitu-
mor activity. Such observations suggest that optimal
drug efficacy can be achieved from a balance between
cellular pharmacokinetics and intrinsic ability to induce
DNA cleavage.
In conclusion, the first series of structurally simple ana-
logues of natural topopyrone C were synthesized and
tested for their biological activity. The available results
support that some structural features are fundamental
for the topo I inhibitory effect, such as the pyrone ring
and a polar group in position 11, whereas the presence
of hydroxy groups in positions 5 and 9 is detrimental
for activity. Interestingly, most of the new analogues
showed a stronger cytotoxic activity than the parent
compound. Therefore, a new lead structure was found
and further profiling of selected compounds will be re-
ported in due course.
These data lead us to suggest that the biological activity
of topopyrone C derivatives is similar to that of other
planar polycyclic compounds that show stabilization
of the binary DNA–topo I complex, (e.g., indenoiso-
quinolines, benzo[c][1,7] and [1,8]phenanthrolines,
benzo[i]phenanthridines, and other analogues of protob-
erberine alkaloids), but with cytotoxicity of a lower or-
der of magnitude than CPT. The weak correlation
between cell growth inhibition and topo I inhibition
by the tested compounds suggests that differences in cel-

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S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1484–1489
Supplementary data
N
OH
O
O
OCH3
O
O
O
OCH3
Experimental procedures and characterization data for
all compounds. Supplementary data associated with this
article can be found, in the online version, at
doi:10.1016/j.bmcl.2007.12.055.
b
3a
14
References and notes
a
c
1. (a) Wang, J. C. Annu. Rev. Biochem. 1985, 54, 665; (b)
Chen, A. Y.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol.
1994, 94, 194.
2. DNA Topoisomerases in Cancer; Potmesil, M., Kohn, K.
W., Eds.; Oxford University Press: New York, 1991.
3. Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol.
Chem. 1985, 260, 14873.
OH
O
O
OCH3
OH
O
OH
15
NO2
O
4. Meng, L. H.; Liao, Z. Y.; Pommier, Y. Curr. Top. Med.
Chem. 2003, 3, 305.
5. Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.;
Zembower, D.; Stewart, L.; Burgin, A. B. J. Med. Chem
2005, 48, 2336.
Scheme 3. Reagents and conditions: (a) Na2CO3, TsOCH3, MW,
15 min, 81%; (b) tetraglyme, K2CO3 2-(dimethylamino)ethylchloride
hydrochloride, 5 h, 120 °C, 40%; (c) TFA, CuNO3Æ3H2O, 90 min, rt,
30%.
6. Burke, T. G.; Mi, Z. H. J. Med. Chem. 1994, 37, 40.
7. Kanai, Y.; Ishiyama, D.; Senda, H.; Iwatani, W.; Takah-
ashi, H.; Konno, I.; Tokumasu, S.; Kanazawa, S. J.
Antibiot. 2000, 53, 863.
9a
13
15
8. Ishiyama, D.; Kanai, Y.; Senda, H.; Iwatani, W.; Takah-
ashi, H.; Konno, I.; Kanazawa, S. J. Antibiot. 2000, 53,
873.
C
T
C
T
C T
9. Gattinoni, S.; Merlini, L.; Dallavalle, S. Tetrahedron Lett.
2007, 48, 1049.
10. (a) Marschalk, C.; Koenig, F.; Ourossoff, N. Bull. Soc.
Chim. Fr. 1936, 3, 1545; (b) Krohn, K.; Hemme, C. Liebigs
Ann. Chem. 1979, 19; (c) Krohn, K.; Vitz, J. Eur. J. Org.
Chem 2004, 1, 209.
11. (a) Bbadhwar, I. C.; Kang, K. S.; Venkataraman, K. J.
Chem. Soc 1932, 1107; (b) Baker, W. J. Chem. Soc. 1933,
1381.
12. Giaccone, G.; Gazdar, A. F.; Beck, H.; Zunino, F.;
Capranico, G. Cancer Res. 1992, 52, 1666.
13. Human non-small cell lung cancer NCI-H460 cells were
cultured in RPMI 1640 containing 10% fetal calf serum.
Cytotoxicity was assessed by growth inhibition assay after
1 h drug exposure. Cells in the logarithmic phase of
growth were harvested and seeded in duplicates into 6-well
plates. Twenty-four hours after seeding, cells were exposed
to the drug, harvested 72 h, and counted with a Coulter
counter. IC50 is defined as the inhibitory drug concentra-
tion causing a 50% decrease of cell growth over that of
untreated control.
Figure 2. Topoisomerase I-mediated DNA plasmid relaxation in
presence of 9a, 13, and 15. Samples were reacted with 1, 10, and
50 lM compound at 37 °C for 30 min. Reaction was then stopped by
adding 0.5% SDS, 0.3 mg/ml of proteinase K and incubating for
45 min at 42 °C before loading on 0.8% agarose gel. C, Control DNA;
T, reaction without drug.
Table 2. In vitro cytotoxic activity of anthraquinones on H460 cell
lines
OR1 O OCH3
14. The cytotoxic activity of compound 8a on human A 431
cells has been recently reported.
R2
O
15. A 30-end labeled gel purified 751-bp BamHI–EcoRI
fragment of SV40 DNA was used for the cleavage assay.
SV40 plasmid was first linearized with BamHI enzyme and
then 30-labeled by using DNA polymerase I large (Kle-
now) fragment (Invitrogen, Paisley, UK) in the presence of
dGTP and a32P ddATP. The labeled DNA was then
restricted with EcoRI enzyme and the corresponding 751-
bp was purified on agarose gel. Topoisomerase I DNA
cleavage reactions (20,000 cpm/sample) were performed in
20 ll of 10 mM Tris–HCl (pH 7.6), 150 mM KCl, 5 mM
MgCl2, 15 lg/ml BSA, 0.1 mM dithiothreitol, and 640 ng
of human recombinant enzyme (full length topoisomerase
I) for 30 min at 37 °C. Reactions were stopped by adding
0.5% SDS and 0.3 mg/ml of proteinase K for 45 min at
42 °C. After precipitation, DNA was resuspended in
denaturing buffer (80% formamide, 10 mM NaOH,
R1
H
R2
IC50 (lM)
a
Compound
Topotecan
1.18
>100
>100
160
3a
14
15
H
(CH2)2N(Me)2
H
H
NO2
a The IC50 values are means of three inhibition assays.
Acknowledgment
The study was supported by the University of Milan
(FIRST funds).

S. Dallavalle et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1484–1489
1489
0.01 M EDTA, and 1 mg/ml dyes) before loading on a
denaturing 8% polyacrylamide gel in TBE buffer.
16. DNA relaxation activity was assayed with 200 ng of
supercoiled pBR322 DNA in 20 mM Tris–HCl (pH
7.6), 0.1 mM dithiothreitol, 5 mM MgCl2, 0.5 lg/ml
BSA, 0.1 mM EDTA, 150 mM KCl in the presence of
purified protein and with 1, 10, and 50 lM of
compound. Samples were incubated for 30 min at
37 °C and then stopped by adding 0.5% SDS and
0.3 mg/ml proteinase K at 42 °C for 45 min. Plasmid
relaxation was analyzed on 0.8% agarose gel followed
by ethidium bromide staining.