Differential Targeting of Human Topoisomerase II Isoforms with Small Molecules

Article abstract of DOI:10.1021/acs.jmedchem.5b00473

(Chemical Equation Presented). The TOP2 poison etoposide has been implicated in the generation of secondary malignancies during cancer treatment. Structural similarities between TOP2 isoforms challenge the rational design of isoform-specific poisons to further delineate these processes. Herein, we describe the synthesis and biological evaluation of a focused library of etoposide analogues, with the identification of two novel small molecules exhibiting TOP2B-dependent toxicity. Our findings pave the way toward studying isoform-specific cellular processes by means of small molecule intervention.

Full text of DOI:10.1021/acs.jmedchem.5b00473

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Brief Article
Differential targeting of human topoisomerase
II isoforms with small molecules
Angelica Mariani, Alexandra Bartoli, Mandeep Atwal, Ka C. Lee, Caroline A. Austin, and Raphaël Rodriguez
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 May 2015
Downloaded from http://pubs.acs.org on May 7, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Differential targeting of human topoisomerase II isoforms
with small molecules
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Angelica Mariani1, Alexandra Bartoli1, Mandeep Atwal2, Ka C. Lee2, Caroline A. Austin2,*, and
Raphaël Rodriguez1,3,*
1Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, Avenue de la Terrasse, 91198
Gif-sur-Yvette, France. 2Institute for Cellular and Molecular Biosciences, Newcastle University, Newcastle upon Tyne,
3
NE2 4HH, United Kingdom. Institut Curie Research Centre, Organic Synthesis and Cell Biology Group, 26 rue
d’Ulm, 75248 Paris Cedex 05, France.
ABSTRACT: The TOP2 poison etoposide has been implicated in the generation of secondary malignancies during cancer
treatment. Structural similarities between TOP2 isoforms challenge the rational design of isoform-specific poisons to further
delineate these processes. Herein, we describe the synthesis and biological evaluation of a focused library of etoposide
analogues, with the identification of two novel small molecules exhibiting TOP2B-dependent toxicity. Our findings pave the
way towards studying isoform-specific cellular processes by means of small molecule intervention.
DNA-breaks and carcinogenesis, whereas the desired
INTRODUCTION
toxicity against cancer cells has been proposed to rely
on the targeting of TOP2A.5,10 While isoform-selective
Small molecules can interfere with DNA-templated
processes with valuable spatiotemporal resolution,
TOP2 poisons can provide valuable mechanistic in-
sights,5b,11 the structural homology between TOP2
providing the means to trigger and modulate disease-
relevant mechanisms.1 TOP2 are nuclear enzymes re-
isoforms has so far challenged the development of po-
quired to unfold and release DNA topological strain
tent isoform-specific VP-16-derived drugs. The high-
through the cleavage, strand passage and re-ligation of
resolution crystal structure of a ternary VP-16-TOP2B
double-stranded DNA.2 This molecular event involves the
catalytic core-DNA complex has revealed unprecedented
information on the binding mode of VP-16.12 Remarka-
formation of reversible enzyme-DNA covalent complex-
es, representing an intrinsic threat that can lead to cell
bly, the glycosidic moiety of VP-16 is in close spatial
death. Clinically approved anti-neoplastic agents that
proximity to a polar glutamine residue (Q778) of TOP2B,
target TOP2 operate by stabilizing the normally transient
a position that is occupied by an apolar methionine
covalent protein-DNA complexes, thereby preventing re-
(M762) in TOP2A. Hence, we sought to take advantage
ligation.3 The proteolytic degradation of such complexes
of this structural variation to explore the ability of synthet-
reveals DNA double-strand breaks (DSBs), which in turn
ic analogues of VP-16 to differentially poison either iso-
activate the DNA-damage response (DDR) machinery to
form in human cancer cells. Interestingly, TOP2B778Q
repair DNA lesions.4 Unrepaired DSBs can either trigger
and TOP2A762M align with residue serine 83 in gyrase
cell death or, alternatively promote genome rearrange-
ments associated with therapy related leukemia.5 Etopo-
side (VP-16)6 targets both TOP2 isoforms in mammalian
cells.7 The α isoform (TOP2A) is required for replication,8
whereas both isoforms are involved in transcription.9
Recent findings implicate TOP2B in VP-16-induced
A, a bacterial type II topoisomerase and it has been
shown that mutations in serine 83 confer resistance to 4-
quinolone antibiotics. This finding functionally demon-
strates that a single residue alteration in the active site
can alter drug targeting, thus lending strong support to
our approach.13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 9
Scheme 1. Synthetic strategy and molecular structures of VP-16 analogues.
1
2
3
4
5
R2
Type 1
N
O
O
N
N3
N
R2
=
NMe2
OMe
OH
OH
O
O
O
O
6, R1 = H
R2
O
O
6
O
O
O
O
7
NMe2
OMe
OH
OH
8
9
MeO
OMe
MeO
OMe
OH
4'
OR1
OR1
O
O
O
2, R1 = H
3, R1 = Me
R1 = H, Me
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
R3
N
Type 2
N
MeO
OMe
O
O
N
OMe
NMe2
2
OH
OMe
9, R1 = H
OH
R3
=
O
4
4
2
1, Podophyllotoxin
O
7, R1 = H
N3
O
O
O
R3
O
O
O
NMe2
NH2
O
OMe
O
O
O
MeO
OMe
OH
MeO
OMe
OH
4'
OR1
OR1
4, R1 = H
5, R1 = Me
R1 = H, Me
8, R1 = H
10, R1 = Me
exhibited a detectable preference for TOP2B over
TOP2A compared to VP-16. The introduction of a 4’-
methoxy substituent completely abolished the activity of
the small molecules, perhaps reflecting the ability of the
4’-OH to interact with a key residue of TOP2B.12 Moreo-
ver, negatively charged derivatives where inactive, in line
with the poor capacity of such functional groups to op-
erate near nucleic acids due to electrostatic repulsion
with the DNA phosphate backbone. Hence, compound
10 (Scheme 1), which contains both a 4’-methoxy
substitution and a negatively charged group, was used
as a negative control in subsequent experiments.
RESULTS AND DISCUSSION
A library of 34 small molecules was prepared based on
the main scaffold of VP-16 (Scheme 1). Each analogue
consisted of the replacement of the sugar moiety with a
triazole-containing fragment conveniently introduced by
means of thermal or copper catalyzed alkyne-azide cy-
cloadditions (Supporting Information). We explored vari-
ous linker length, steric hindrance and polarity. Ana-
logues could be categorized in two main classes based
on the nature of the triazole orientation (Type 1 vs Type
2, Scheme 1), comprising polar substituents expected
to be either positively or negatively charged at physiolog-
ical pH, H-bond acceptor/donor as well as aromatic and
aliphatic fragments. Additionally, this focused library
contained both 4’-demethyl and -methylated counter-
parts to further explore the effect of this substitution on
the TOP2 isoform-specific targeting capacity of the small
molecules.
These results prompted us to investigate whether the
trapping of TOP2-DNA covalent complexes led to the
production of DSBs. Thus, we monitored the occurrence
of a surrogate mark of DSBs, the phosphorylation of
histone protein H2A, X variant on Ser139 (termed
γ−H2AX), one of the earliest cellular events in response
to DNA damage.16 To do so, we independently treated
K562 cells with 100 µM of each analogue and monitored
γ−H2AX by means of immunofluorescence (Figure
1A/C and Figure S6, Supporting Information). Con-
sistent with the TARDIS data, the small molecules that
poisoned TOP2 triggered the production of γ-H2AX
characteristic of the DDR, with the general trend of 6 > 8
> 9 > 10. In contrast to the TARDIS data, we observed
that compound 7 displayed a superior ability to generate
DSBs compared to the other synthetic analogues that
was comparable to VP-16, whilst exhibiting a similar
TOP2 targeting capacity compared to 6. Since the acti-
vation of the DDR requires the enzymatic processing of
the TOP2-DNA-drug complexes,4 this result suggested
that ternary complexes formed with 7 may be detected
and processed differently than complexes formed with
the other analogues. It is conceivable that a more favor-
able kinetics of ternary complex formation with 7 led to a
faster proteolytic processing and a higher rate of DSBs
mark production. However we cannot rule out that com-
To assess the ability of each analogue to poison TOP2
in cellulo, we performed a “trapped in agarose DNA
immunostaining” (TARDIS) assay, which allows for the
quantification of drug-stabilized TOP2-DNA complexes
using isoform-specific immunofluorescence detection.14
The library was first screened in a single-dose experi-
ment in K562 human leukemia cells, known to express
similar levels of both isoforms (Table S1- S2, Support-
ing Information).15 From this initial screen, we identified
compounds 6, 7, 8 and 9 (Scheme 1) as TOP2 poi-
sons, which were subsequently investigated in more
detail including dose-response treatments (Figure
1A/B and Figure S1-S6, Supporting Information). We
found that analogues harboring an aliphatic spacer
and/or a type 2 triazole-linker exhibited a wide range of
activity, presumably due to a high degree of rotational
freedom enabling stabilizing interactions within the bind-
ing pocket of TOP2. While each active small molecule
targeted both isoforms, our analysis revealed that 6
2
ACS Paragon Plus Environment

Page 3 of 9
Journal of Medicinal Chemistry
pound 7 exerts its activity partly as a result of a TOP2- of such functionalities to engage electrostatic interac-
1
2
3
4
5
6
7
8
independent mechanism.
tions with polar amino acid residues such as the one
found at residue 778 of TOP2B. As anticipated, the neu-
tral (9) and negatively charged (10) analogues, which
induced low levels of γ−H2AX, exhibited little anti-
proliferative properties against the cell lines studied.
Strikingly, we found that compound 8 exhibited almost
identical IC50 values against WT and Nalm-6TOP2A+/- cells
(~3.4/3.5 µM), while it was significantly more potent
against Nalm-6TOP2B-/- cells with an IC50 value of 1.92 µM.
This atypical profile suggested a more complex mecha-
nism of action for compound 8 compared to the other
analogues.
We next investigated the contribution of each isoform
to the inhibition of Nalm-6 leukemia cell proliferation
upon treatment with VP-16 analogues. While TOP2B
double knockout cells are viable, TOP2A is essential for
cell survival. Thus, we employed Nalm-6TOP2A+/- express-
ing ~50% of TOP2A compared to wild-type (WT) and
Nalm-6TOP2B-/- lacking TOP2B.5b,11
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
TOP2A
TOP2B
γ-H2AX
DAPI
A
Hoechst
Hoechst
Alexa 488
Alexa 594
Alexa 488
B
TOP2A
Table 1. IC50 values and selectivity against
TOP2A and TOP2B
150
100
50
0
IC50 (µM)
IC50 ratio
TOP2A+/-
TOP2B-/-
WT
TOP2A+/-
TOP2B-/-
/WT
/WT
-ve VP-16
6
7
8
9
10
0.134 ±
0.007
0.234 ±
0.003
0.182 ±
0.004
VP-16
1.7
1.4
2.1
2.0
0.6
TOP2B
150
100
50
0.62 ±
0.01
1.32 ±
0.01
6
7
8
0.85 ± 0.02
1.90 ± 0.08
3.5 ± 0.1
1.4
1.6
1.0
2.36 ±
0.07
1.2 ± 0.1
3.4 ± 0.2
0
-ve VP-16
6
7
8
9
10
1.92 ±
0.07
γ−H2AX
C
3.90 ±
0.06
9
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
100
50
0
10
> 10
Cell lines: Nalm-6 (WT), Nalm-6TOP2A+/- (TOP2A+/-) and
Nalm-6TOP2B-/- (TOP2B-/-). Data are means ±SD from 3
replicate experiments. N.D, not determined.
-ve VP-16
6
7
8
9
10
CONCLUSIONS
Figure 1. A) Microscope images showing the nucleus
(blue), trapped TOP2 (green) and γ-H2AX (red). B) Quantifi-
cation of fluorescence in the TARDIS assay normalized
against VP-16-treated cells. C) Quantification of fluores-
cence in γ-H2AX positive cells normalized against VP-16-
treated cells. Cells were treated with 100 µM VP-16, 6, 7,
8, 9, 10 or DMSO (-ve). Scale bars, 100 µm.
In conclusion, we have described a series of VP-16
analogues where the sugar moiety was strategically
replaced by chemically diverse substituents. Our data
demonstrates that structural reprogramming of the scaf-
fold of VP-16 enables the fine-tuning of TOP2 isoform-
specific targeting in K562 and Nalm-6 human leukemia
cells, thereby providing proof-of-principle for the devel-
opment of isoform-specific TOP2 drugs derived from VP-
16. For instance, we have found that positively charged
substrates such as 6 and 7 showed a propensity to
affect cell viability in a manner that was reliant on TOP2B
to a greater extent than VP-16, thus enabling the study
of TOP2B-mediated genomic processes. Additional
structural investigations are certainly required to interro-
gate the very nature of chemical interactions involved
between small molecules and TOP2 isoforms with atom-
ic resolution.12 Since the introduction of a triazole ring
was not found to be detrimental to the activity of the
most potent derivatives, a future aim is to employ unbi-
ased methodologies such as in situ click chemistry and
dynamic combinatorial processes to select suitable
fragments catalyzed by a specific TOP2 isoform and
We found that compounds 6 and 7 were the most po-
tent analogues of the series with IC50 values of 0.62 µM
and 1.2 µM against WT cells (Table 1), respectively,
consistent with the data obtained from TARDIS and
γ−H2AX assays. Interestingly, Nalm-6TOP2B-/- cells were
less susceptible to growth inhibition by 6 and 7 than WT
and Nalm-6TOP2A+/- cells, with IC50 values of 1.32 µM and
2.36 µM, respectively. In contrast, VP-16 was more
potent against the Nalm-6TOP2B-/- compared to Nalm-
6TOP2A+/- cells. Altogether, these results indicated that cell
death inducted by 6 and 7 involved TOP2B targeting to
a greater extent than for growth inhibition by VP-16. It is
noteworthy that 6 and 7, which contain positively
charged substituents, displayed a higher potency com-
pared to the other analogues, consistent with the ability
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 9
identify small molecules with enhanced potency and
The product was extracted with ethyl acetate and the
selectivity.17
1
2
3
4
5
6
7
8
collected organic phases were dried over MgSO4. The
crude product was purified by flash chromatography
(DCM: ethyl acetate = 9:1). Compound 5 was obtained
EXPERIMENTAL SECTION
1
as a white solid (309 mg, 0.68 mmol, 62%). H NMR
Chemistry
(500 MHz, CDCl3) d 6.94 (1H, s), 6.56 (1H, s), 6.25 (2H,
s), 6.00 (1H, d, J = 1.0 Hz), 5.97 (1H, d, J = 1.0 Hz),
4.79 (1H, d, J = 3.0 Hz), 4.61 (1H, d, J = 5.0 Hz), 4.40-
4.33 (2H, m), 4.27 (2H, d, J = 2.5 Hz), 3.80 (3H, s), 3.74
(6H, s), 3.38 (1H, dd, J = 5.0, 13.5 Hz), 2.95-2.88 (1H,
Podophyllotoxin (1) was purchased from Santa Cruz
Biotechnology and used without further purifications. 4β-
azido-4-deoxy-4’-demethylepipodophyllotoxin (2) and
4β-azido-4-deoxyepipodophyllotoxin (3) were synthe-
tised as previously described.18 NMR spectroscopy was
performed on Bruker 600 MHz, 500 MHz and 300 MHz
apparatus. High-resolution mass spectrometry analysis
(HRMS) was performed on a Waters LCT Premier XE,
under electron spray ionization (ESI). For final com-
pounds the purity was determined by HPLC (Waters,
Alliance 2695, PDA 996 and MS detector) to be >95%.
9
m), 2.56 (1H, t, J = 2.5 Hz). 13C NMR (125 MHz, CDCl) δ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
174.8, 152.6, 148.6, 146.9, 137.3, 135.4, 132.8, 128.3,
111.0, 109.6, 108.3, 101.5, 79.5, 75.6, 71.9, 67.5, 60.8,
56.32, 56.29, 44.0, 41.0, 38.0. HRMS (ESI) m/z calcd for
C25H25O8 [M+H]+: 453.1544, found: 453.1588.
General procedure for the synthesis of type 1
compounds. 4β-azido-4-deoxy-4’-demethylepipodo-
phyllotoxin (2) or 4β-azido-4-deoxyepipodophyllotoxin (3)
(1 equiv) was suspended in a water: t-butanol mixture
(1:1) and degassed for 5 min with argon. The appropriate
alkyne (10 equiv) was added followed, in order, by sodi-
um ascorbate and CuSO4•5H2O.The mixture was stirred
at 50°C till completion. The final suspension was diluted
with H2O and extracted with ethyl acetate. The collected
organic phases were dried over MgSO4 and the solvent
was removed under reduced pressure. The mixture was
purified by flash chromatography or by HPLC.
Synthesis of 4β-(prop-2-ynyloxy)-4’-demethyl-
epipodophyllotoxin (4). Podophyllotoxin (1, 0.47 g,
1.13 mmol, 1 equiv) was dissolved in dry DCM (11 ml).
Sodium iodide (0.51 g, 3.40 mmol, 3 equiv) was added
and the resulting suspension was stirred at RT for 5 min.
Methanesulfonic acid (220 µl, 3.40 mmol, 3 equiv) was
added dropwise at 0°C and the mixture was then
warmed to rt and stirred overnight. The solvent was
removed under vacuum and the crude product was
resuspended in dry THF (10 ml). BaCO3 (223 mg, 1.13
mmol, 1 equiv) and propargyl alcohol (10 ml, 173 mmol,
153 equiv) were added and the mixture was additionally
stirred for 3 h at rt. The reaction was quenched with a
10% Na2S2O3 aqueous solution and diluted with H2O.
The product was extracted with ethyl acetate and the
collected organic phases were dried over MgSO4. The
crude product was purified by flash chromatography
(DCM: ethyl acetate = 8:2). Compound 4 was obtained
4β-(4-N,N-dimethylaminomethyl-1,2,3-
triazolyl)-4’-demethylepipodophyllotoxin hydro-
chloride (6). Yield: 5 mg, 0.009 mmol, 38%. White
1
solid. H NMR (500 MHz, CD3OD, 283 K) δ 8.01 (1H, s),
6.73 (1H, s), 6.68 (1H, s), 6.38 (2H, s), 6.34 (1H, br s),
6.00 (1H, s), 5.97 (1H, s), 4.79 (1H, br s), 4.43 (3H, br s),
3.75 (6H, s), 3.37 (2H, br s), 3.22-3.21 (1H, m), 2.91 (6H,
s). 13C NMR (125 MHz, CD3OD, 283 K) δ 175.9, 150.7,
149.3, 148.7, 138.2, 136.0, 135.3, 131.2, 128.6, 126.7,
111.4, 109.8, 109.2, 103.4, 69.0, 60.2, 56.7, 52.5, 44.8,
43.2, 42.6, 38.5. HRMS (ESI) m/z calcd for C26H29N4O7
[M+H]+: 509.2031, found: 509.2008.
1
as a white solid (245 mg, 0.56 mmol, 49%). H NMR
(500 MHz, CDCl3) δ 6.94 (1H, s), 6.56 (1H, s), 6.26 (2H,
s), 6.00 (1H, s), 5.96 (1H, s), 5.40 (1H, s), 4.79 (1H, d, J
= 2.0 Hz), 4.60 (1H, d, J = 4.5 Hz), 4.39-4.31 (2H, m),
4.27 (2H, s), 3.76 (6H, s), 3.37 (1H, dd, J = 4.5, 14.0
Hz), 2.93-2.87 (1H, m), 2.57 (1H, s). 13C NMR (125 MHz,
CDCl3) δ 174.9, 148.5, 146.8, 146.4, 134.0, 132.9,
130.8, 128.3, 111.0, 109.5, 107.9, 101.5, 79.5, 75.6,
71.9, 67.4, 56.4, 56.3, 43.8, 41.0, 37.9. HRMS (ESI) m/z
calcd for C24H23O8 [M+H]+: 439.1387, found: 439.1377.
General procedure for the synthesis of type 2
compounds. 4β-(prop-2-ynyloxy)-4’-demethylepipodo-
phyllotoxin (4) or 4β-(prop-2-ynyloxy) epipodophyllotoxin
(5) (1 equiv) was suspended in a water: t-butanol mixture
(1:1) and degassed for 5 min with argon. The appropriate
azide (10 equiv) was added followed, in order, by sodium
ascorbate and CuSO4•5H2O. The mixture was stirred at
rt or 50°C till completion. The final suspension was dilut-
ed with H2O and extracted with ethyl acetate. The col-
lected organic phases were dried over MgSO4 and the
solvent was removed under reduced pressure. The mix-
ture was purified by flash chromatography or by HPLC.
Synthesis of 4β-(prop-2-ynyloxy) epipodo-
phyllotoxin (5). Podophyllotoxin (1, 0.46 g, 1.11
mmol, 1 equiv) was dissolved in dry acetonitrile (11 ml).
Sodium iodide (333 mg, 2.22 mmol, 2 equiv) was added
and the resulting suspension was stirred at RT for 5 min.
Methanesulfonic acid (144 ml, 2.22 mmol, 2 equiv) was
added dropwise at 0°C and the mixture was then
warmed to rt and stirred for 15 min. The solvent was
removed under vacuum and the crude product was
resuspended in dry THF (10 ml). BaCO3 (219 mg, 1.11
mmol, 1 equiv) and propargyl alcohol (10 ml, 173 mmol,
156 equiv) were added and the mixture was additionally
stirred for 30 min at rt. The reaction was quenched with
a 10% Na2S2O3 aqueous solution and diluted with H2O.
4β-(1-N,N-dimethylaminopropyl-4-methyl-
1,2,3-triazolyl)-4’-demethylepipodophyllotoxin
hydroformate (7). Yield: 5.5 mg, 0.009 mmol, 26%.
White solid. 1H NMR (500 MHz, CD3OD, 283 K) δ 8.46
(1H, s), 8.06 (1H, s), 6.93 (1H, s), 6.53 (1H, s), 6.27 (2H,
s), 5.98 (1H, s), 5.97 (1H, s), 4.82-4.73 (3H, m), 4.58
(1H, d, J = 5.0 Hz), 4.54-4.52 (2H, m), 4.40-4.37 (1H,
4
ACS Paragon Plus Environment

Page 5 of 9
Journal of Medicinal Chemistry
Corresponding Author
m), 4.33-4.29 (1H, m), 3.70 (6H, s), 3.40 (1H, dd, J =
1
2
3
4
5
6
7
8
5.0, 14.0 Hz), 3.08-3.04 (2H, m), 3.01-2.94 (1H, m), 2.80
(6H, s), 2.32 (2H, br s). 13C NMR (125 MHz, CD3OD, 283
K) δ 177.5, 169.4, 149.8, 148.5, 148.1, 146.3, 135.6,
134.2, 132.0, 130.7, 125.4, 111.5, 110.8, 109.1, 102.9,
75.2, 69.2, 63.8, 56.6, 56.2, 48.3, 44.9, 43.7, 42.4,
39.7, 26.7. HRMS (ESI) m/z calcd for C29H35N4O8
[M+H]+: 567.2449, found: 567.2457.
*raphael.rodriguez@curie.fr; *caroline.austin@ncl.ac.uk
Funding Sources
This research was generously supported by la Fondation
pour la Recherche Médicale, La Ligue contre le Cancer,
LLR specialist programme 12031 and BCC studentship.
R.R. thanks the CNRS for funding.
4β-(1-(4-hydroxyphenyl)-4-methyl-1,2,3-
ACKNOWLEDGMENT
9
triazolyl)-4’-demethylepipodophyllotoxin
(8).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
We thank Rodriguez’s and Austin’s laboratory members for
fruitful discussions.
Yield: 8.7 mg, 0.015 mmol, 45%. Grey solid. H NMR
(600 MHz, (CD3)2SO, 313 K) δ 8.68 (1H, s), 8.21 (1H, br
s), 7.66 (2H, d, J = 9.0 Hz), 7.06 (1H, s), 6.94 (2H, d, J =
9.0 Hz), 6.53 (1H, s), 6.19 (2H, s), 6.03 (1H, s), 6.00 (1H,
s), 4.83-4.75 (3H, m), 4.51 (1H, d, J = 5.0 Hz), 4.39-4.37
(1H, m), 4.24-4.21 (1H, m), 3.62 (6H, s), 3.26 (1H, br s),
2.92-2.87 (1H, m). 13C NMR (150 MHz, (CD3)2SO, 313 K)
δ 174.4, 157.7, 147.5, 147.1, 146.0, 144.8, 134.7,
132.4, 130.2, 129.5, 128.7, 122.1, 121.8, 115.9, 109.9,
109.6, 108.4, 101.2, 72.9, 67.3, 62.7, 56.0, 42.8, 40.4,
37.7. HRMS (ESI) m/z calcd for C30H28N3O9 [M+H]+:
574.1820, found: 574.1838.
ABBREVIATIONS
TOP2, Topoisomerase II; TOP2A, Topoisomerase IIα;
TOP2B, Topoisomerase IIβ; DSBs, double stranded breaks;
DDR, DNA-damage response; TARDIS, trapped in agarose
DNA immunostaining.
REFERENCES
1. a) Dervan, P. B. Design of sequence-specific DNA-
binding molecules. Science 1986, 232, 464 – 471; b)
Gottesfeld, J. M.; Neely, L.; Trauger, J. W.; Baird, E.
E.; Dervan, P. B. Regulation of gene expression by
small molecules. Nature 1997, 387, 202 – 205; c)
Sun, D.; Thompson, B.; Cathers, B. E. Salazar, M.;
Kerwin S. M.; Trent, J. O.; Jenkins, T. C.; Neidle, S.
Hurley, L. H. Inhibition of human telomerase by a G-
quadruplex-interactive compound. J. Med. Chem.
1997, 40, 2113 – 2116 ; d) Siddiqui-Jain, A.; Grand,
C. L.; Bearss, D. J.; Hurley, L. H. Direct evidence for a
G-quadruplex in a promoter region and its targeting
with a small molecule to repress c-MYC transcription.
Proc. Natl. Acad. Sci. USA 2002, 99, 11593 –
11598; e) Hurley, L. H. DNA and its associated pro-
cesses as targets for cancer therapy. Nat. Rev. Can-
cer 2002, 2, 188 – 200; f) Neidle, S.; Parkinson, G.
Telomere maintenance as a target for anticancer drug
discovery. Nat. Rev. Drug Discov. 2002, 1, 383 –
393; g) Pommier, Y. Topoisomerase I inhibitors:
camptothecins and beyond. Nat. Rev. Cancer 2006,
6, 789 – 802; h) Moorhouse, A. D.; Santos, A. M.;
Gunaratnam, M.; Moore, M.; Neidle S.; Moses, J. E.
Stabilization of G-quadruplex DNA by highly selective
ligands via click chemistry. J. Am. Chem. Soc. 2006,
128, 15972 – 15973; i) Rodriguez, R.; Müller, S.;
Yeoman, J. A.; Trentesaux, C.; Riou, J.-F.; Bal-
asubramanian, S. A novel small molecule that alters
shelterin integrity and triggers a DNA-damage re-
sponse at telomeres. J. Am. Chem. Soc. 2008, 130,
15758 – 15759; j) Rodriguez, R.; Miller, K. M.; For-
ment, J. V.; Bradshaw, C. R.; Nikan, M.; Britton, S.;
Oelschlaegel, T.; Xhemalce, B.; Balasubramanian, S.;
Jackson, S. P. Small-molecule–induced DNA damage
identifies alternative DNA structures in human genes.
Nat. Chem. Biol. 2012, 8, 301 – 310; k) Wei, D.; Wil-
son, W. D.; Neidle, S. Small-molecule binding to the
DNA minor groove is mediated by a conserved water
cluster. J. Am. Chem. Soc. 2013, 135, 1369 – 1377;
l) Rodriguez, R.; Miller, K. M. Unravelling the genomic
targets of small molecules using high-throughput se-
quencing. Nat. Rev. Genet. 2014, 15, 783 – 796; m)
Kang, J. S.; Meier, J. L.; Dervan P. B. Design of se-
quence-specific DNA binding molecules for DNA me-
4β-(1-carbomethoxypentyl-4-methyl-1,2,3-
triazolyl)-4’-demethylepipodophyllotoxin
(9).
Yield: 12.9 mg, 0.021 mmol, 62%. White solid. 1H NMR
(500 MHz, CDCl3, 283 K) δ 7.55 (1H, s), 6.71 (1H, s),
6.53 (1H, s), 6.24 (2H, s), 5.98 (1H, s), 5.95 (1H, s), 5.43
(1H, s), 4.76 (3H, br s), 4.60 (1H, d, J = 5.5 Hz), 4.42-
4.35 (2H, m), 4.32-4.26 (2H, m), 3.76 (6H, s), 3.66 (3H,
s), 3.37 (1H, dd, J = 5.5, 14.0 Hz), 2.92-2.86 (1H, m),
2.32 (2H, t, J = 7.5 Hz), 1.98-1.92 (2H, m), 1.71-1.65
(2H, m), 1.38-1.35 (2H, m). 13C NMR (125 MHz, CDCl3,
283 K) δ 175.1, 173.8, 148.4, 146.7, 146.2, 144.7,
133.7, 132.5, 130.7, 128.9, 122.6, 110.7, 109.5, 107.5,
101.4, 73.4, 67.4, 63.0, 56.3, 51.6, 50.2, 43.7, 41.1,
38.0, 33.6, 30.0, 25.9, 24.1. HRMS (ESI) m/z calcd for
C31H36N3O10 [M+H]+: 610.2395, found: 610.2356.
4β-(1-(4-carboxybenzyl)-4-methyl-1,2,3-
triazolyl) epipodophyllotoxin (10). Yield: 11.6 mg,
0.018 mmol, 83%. White solid. 1H NMR (500 MHz,
CDCl3, 283 K) δ 8.10 (2H, d, J = 6.0 Hz), 7.52 (1H, s),
7.36 (2H, d, J = 6.0 Hz), 6.69 (1H, s), 6.51 (1H, s), 6.21
(2H, s), 5.98 (1H, s), 5.94 (1H, s), 5.68-5.58 (2H, m),
4.76-4.75 (3H, m), 4.58 (1H, d, J = 5.0 Hz), 4.33-4.25
(2H, m), 3.78 (3H, s), 3.71 (6H, s), 3.36 (1H, dd, J = 5.0,
14.0 Hz), 2.92-2.86 (1H, m). 13C NMR (125 MHz, CDCl3,
283 K) δ 175.0, 170.2, 152.4, 148.4, 146.8, 145.5,
139.9, 136.8, 135.2, 132.4, 131.0, 129.9, 128.7, 127.9,
122.8, 110.7, 109.5, 107.8, 101.5, 73.7, 67.4, 63.0,
60.7, 56.1, 53.8, 43.8, 41.0, 38.0. HRMS (ESI) m/z calcd
for C33H32N3O10 [M+H]+: 630.2082, found: 630.2097.
ASSOCIATED CONTENT
Supporting information, including detailed synthetic proce-
dures, compound characterization and bioassays, is availa-
ble free of charge via the Internet at http:// pubs.acs.org.
AUTHOR INFORMATION
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 9
thyltransferase inhibition. J. Am. Chem. Soc. 2014,
136, 3687 – 3694.
Jung, J.; Schübeler, D.; Burger, L.; Tiwali, V. K. Gene
1
2
3
4
5
6
7
8
regulation and priming by topoisomerase IIα in em-
bryonic stem cells. Nat. Commun. 2013, 4, 2478.
10. Errington, F.; Willmore, E.; Tilby, M. J.; Li. L.; Li, G.;
Li. W.; Baguley, B. C.; Austin, C. A. Murine transgenic
cells lacking DNA topoisomerase IIbeta are resistant
to acridines and mitoxantrone: analysis of cytotoxicity
and cleavable complex formation. Mol. Pharmacol.
1999, 56, 1309 – 1316.
11. Toyoda, E.; Kagaya, S.; Cowell, I. G.; Kurosawa, A.;
Kamoshita, K.; Nishikawa, K.; Iiizumi, S.; Koyama, H.;
Austin, C. A.; Adachi, N. NK314, a topoisomerase II
inhibitor that specifically targets the α isoform. J. Biol.
Chem. 2008, 283, 23711 – 23720.
12. Wu, C.-C.; Li, T.-K.; Farh, L.; Lin, L.-Y.; Lin, T.-S.; Yu,
Y.-J.; Yen, T.-J.; Chiang, C.-W.; Chan, N.-L. Struc-
tural basis of type II topoisomerase inhibition by the
anticancer drug etoposide. Science 2011, 333, 459–
462.
13. Cullen, M. E.; Wyke, A. W.; Kuroda, R.; Fisher, L. M.
Cloning and characterization of a DNA gyrase A gene
from Escherichia coli that confers clinical resistance to
4-quinolones. Antimicrob. Agents Chemother. 1989,
33, 886 – 894.
14. Cowell, I. G.; Tilby, M. J.; Austin, C. A. An overview of
the visualisation and quantitation of low and high MW
DNA adducts using the trapped in agarose DNA im-
munostaining (TARDIS) assay. Mutagenesis 2011,
26, 253 – 260.
15. Padget, K.; Pearson, A. D. J.; Austin, C. A. Quantita-
tion of DNA topoisomerase IIα and β in human leu-
kaemia cells by immunoblottin. Leukemia 2000, 14,
1997 – 2005.
16. Bonner, W. M.; Redon, C. E.; Dickey, J. S.; Nakamu-
ra, A. J.; Sedelnikova, O. A.; Solier, S.; Pommier, Y.
GammaH2AX and cancer. Nat. Rev. Cancer 2008,
8, 957 – 967.
17. a) Huc, I.; Lehn, J.-M. Virtual combinatorial libraries:
Dynamic generation of molecular and supramolecular
diversityꢀbyꢀself-assembly. Proc. Natl. Acad. Sci. USA
1997, 94, 2106 – 2110; b) Krasiński, A.; Radić, Z.;
Manetsch, R.; Raushel, J.; Taylor, P.; Sharpless, K.
B.; Kolb, H. C. In situ selection of lead compounds by
click chemistry:ꢀ target-guided optimization of acetyl-
cholinesterase inhibitors. J. Am. Chem. Soc. 2005,
127, 6686 – 6692; c) Moses, J. E.; Moorhouse, A. D.
The growing applications of click chemistry. Chem.
Rev. Soc., 2007, 36, 1249 – 1262; d) Di Antonio, M.;
Biffi, G.; Mariani, A.; Raiber, E.-A.; Rodriguez, R.; Bal-
asubramanian, S. Selective RNA versus DNA G-
quadruplex targeting by in situ click chemistry. An-
gew. Chem. Int. Ed. 2012, 51, 11073 – 11078.
18. a) Zhou, X.-M.; Wang, Z.-Q.; Chang, J.-Y.; Chen, H.-
X.; Cheng, Y.-C.; Lee, K.-H. Antitumor agents. 120.
New 4-substituted benzylamine and benzyl ether de-
rivatives of 4'-O-demethylepipodophyllotoxin as po-
tent inhibitors of human DNA topoisomerase II. J.
Med. Chem. 1991, 34, 3346 – 3350; b) Kamal, A.;
Laxman, N.; Ramesh, G. Facile and efficient one-pot
synthesis of 4β-arylaminopodophyllotoxins: synthesis
of DNA topoisomerase II inhibitors (NPF and W-68).
Bioorg. Med. Chem. Lett. 2000, 10, 2059 – 2062.
2. a) Vos, S. M.; Tretter, E. M.; Schmidt, B. H.; Berger,
J. M. All tangled up: how cells direct, manage and
exploit topoisomerase function. Nat. Rev. Mol. Cell
Bio. 2011, 12, 827 – 841; b) Chen, S. H.; Chan, N.-
L.; Hsieh T. S. New mechanistic and functional in-
sights into DNA topoisomerases. Annu. Rev. Bio-
chem. 2013, 82, 139 – 170.
3. Bailly, C. Contemporary challenges in the design of
topoisomerase II inhibitors for cancer chemotherapy.
Chem. Rev. 2012, 112, 3611 – 3640.
4. a) Nitiss, J. L. Targeting DNA topoisomerase II in can-
cer chemotherapy. Nat. Rev. Cancer 2009, 9, 338 –
350; b) Sunter, N. J.; Cowell, I. G.; Willmore, E.; Wat-
ters, G. P.; Austin, C. A. Role of topoisomerase IIβ in
DNA damage response following IR and Etoposide. J.
Nucleic Acids 2010, Article ID 710589, 8 pages.
5. a) Azarova, A. M.; Lyu, Y. L.; Lin, C.-P.; Tsai Y.-C.;
Lau, J. Y-N.; Wang, J. C.; Liu L. F. Roles of DNA
topoisomerase II isozymes in chemotherapy and sec-
ondary malignancies. Proc. Natl. Acad. Sci. USA,
2007, 104, 11014 – 11019; b) Cowell, I. G.; Sondka,
Z.; Smith, K.; Lee, K. C.; Manville, C. M.; Sidorczuk-
Lesthuruge, M.; Rance, H. A.; Padget, K.; Jackson,
G. H.; Adachi, N.; Austin, C. A. Model for MLL trans-
locations in therapy-related leukemia involving topoi-
somerase IIβ-mediated DNA strand breaks and gene
proximity. Proc. Natl. Acad. Sci. USA 2012, 109,
8989 – 8994; c) Ezoe, S. Secondary leukemia asso-
ciated with the anti-cancer agent, etoposide, a topoi-
somerase II inhibitor. Int. J. Environ. Res. Public
Health 2012, 9, 2444 – 2453; d) Pendleton, M.;
Lindsey, R. H.; Felix, C. A.; Grimwade, D.; Osheroff,
N. Topoisomerase II and leukemia. Ann. N. Y. Acad.
Sci. 2014, 1310, 98 – 110.
6. Keller-Juslén, C.; Kuhn, M.; Von Wartburg, A. Syn-
thesis and antimitotic activity of glycosidic lignan de-
rivatives related to podophyllotoxin. J. Med. Chem.
1971, 14, 936 – 940.
7. Willmore, E.; Frank A. J.; Padget, K.; Tilby, M. J.;
Austin, C. A. Etoposide targets topoisomerase IIalpha
and IIbeta in leukemic cells: isoform-specific cleavable
complexes visualized and quantified in situ by a novel
immunofluorescence technique. Mol. Pharmacol.
1998, 54, 78 – 85.
8. Niimi, A.; Suka, N.; Harata, M.; Kikuchi, A.; Mizuno, S.
Co-localization of chicken DNA topoisomerase IIα,
but not β, with sites of DNA replication and possible
involvement of a C-terminal region of α through its
binding to PCNA. Chromosoma 2001, 110, 102–
114.
9. a) Ju, B.-G.; Lunyak, V. V.; Perissi, V.; Garcia-
Bassets, I.; Rose, D. W.; Glass, C. K.; Rosenfeld, M.
G. A topoisomerase IIbeta-mediated dsDNA break
required for regulated transcription. Science 2006,
312, 1798 – 1802; b) Tiwari, V. K.; Burger, L.; Ni-
koletopoulou, V.; Deogracias, R.; Thakurela, S.; Wir-
belauer, c.; Kaut, J.; Terranova, R.; Hoerner, L.; Miel-
ke, C.; Boege, F.; Murr, R.; Peters, A. H.; Barde, Y.
A.; Schübeler, D. Target genes of topoisomerase IIβ
regulate neuronal survival and are defined by their
chromatin state. Proc. Natl. Acad. Sci. USA 2012,
109, E934 - E943; c) Thakurela, S.; Garding, A.;
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
ACS Paragon Plus Environment

Page 7 of 9
Journal of Medicinal Chemistry
Table of Contents graphic:
1
2
3
4
5
6
7
8
9
H
OH
O
N
TOP2 poison
O
OH
O
H
N
N
HCOO-
N
N
O
N
TOP2
Cl-
N
O
O
N
O
O
O
O
O
O
O
O
O
O
O
DNA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
OH
O
O
O
O
Etoposide
OH
OH
TOP2-dependent cytotoxicity
TOP2A-preference!
TOP2B-preference!
7
ACS Paragon Plus Environment

6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
R2
Type 1
N
O
O
N
N3
N
R2 =
NMe2
OMe
OH
OH
O
O
O
O
6, R1 = H
R2
O
O
O
O
O
O
NMe2
OMe
OH
OH
MeO
OMe
MeO
OMe
OH
4'
OR1
OR1
O
O
O
2, R1 = H
3, R1 = Me
R1 = H, Me
O
R3
Type 2
N N
MeO
OMe
O
O
N
OMe
NMe2
2
OH
OMe
OH
R3 =
O
4
4
2
1, Podophyllotoxin
O
7, R1 = H
9, R1 = H
N3
O
O
O
R3
O
O
O
NMe2
NH2
O
OMe
O
O
O
MeO
OMe
OH
MeO
OMe
OH
4'
OR1
OR1
4, R1 = H
5, R1 = Me
R1 = H, Me
8, R1 = H
10, R1 = Me

TOP2A
TOP2B
γ-H2AX
DAPI
A
Hoechst
Hoechst
Alexa 488
Alexa 594
Alexa 488
B
TOP2A
150
100
50
0
-ve VP-16
6
7
8
9
10
TOP2B
150
100
50
0
-ve VP-16
6
7
8
9
10
γ−H2AX
C
100
50
0
-ve VP-16
6
7
8
9
10