Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

Article abstract of DOI:10.1016/j.bmc.2014.09.014

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustar

Full text of DOI:10.1016/j.bmc.2014.09.014

Bioorganic & Medicinal Chemistry 22 (2014) 5935–5949
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based design, synthesis and biological testing of etoposide
analog epipodophyllotoxin–N-mustard hybrid compounds designed
to covalently bind to topoisomerase II and DNA
Arun A. Yadav a, Xing Wu a, Daywin Patel a, Jack C. Yalowich b, Brian B. Hasinoff a,
⇑
a Faculty of Pharmacy, Apotex Centre, University of Manitoba, 750 McDermot Ave, Winnipeg, Manitoba R3E 0T5, Canada
b College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2014
Revised 27 August 2014
Accepted 8 September 2014
Available online 16 September 2014
Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct
and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series
of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds were designed, synthesized and
biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topo-
isomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid
had a mean GI50 in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and
cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis
indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the
N-mustard compounds from which they were derived. These results supported the conclusion that the
hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and
DNA.
Keywords:
Epipodophyllotoxin
Topoisomerase II
Etoposide
Hybrid
Structure-based design
Anticancer
Ó 2014 Elsevier Ltd. All rights reserved.
DNA
Alkylator
COMPARE
K562 cells
Molecular modeling
Docking
Nitrogen mustard
1. Introduction
as an epipodophyllotoxin–lexitropsin hybrids; enediyne–lexitrop-
sin hybrids;5 an epipodophyllotoxin–amsacrine hybrid;6 various
Hybrid drug molecules that contain two distinct pharmaco-
phores and that can simultaneously act at two pharmacological
targets have the potential to exceed the potency, spectrum of activ-
ity, and efficacy of either of the constituent moieties from which
they were derived. Several recent reviews have described the
various approaches taken in designing hybrid drugs.1–5 In the anti-
cancer drug development area these have included such molecules
N-mustard- and cisplatin–steroid hybrids;4 epipodophyllotoxin–
camptothecin hybrids;7 and chlorambucil–distamycin A hybrids.3
The X-ray structure of two molecules of etoposide complexed in
a ternary complex with topoisomerase IIb (PDB ID: 3QX3) has
recently been determined.8,9 In this structure the glycosidic moiety
of etoposide occupies a spacious binding pocket in the protein. The
bound epipodophyllotoxin moiety of etoposide, unlike planar DNA
intercalators that stack with two bases, stacks only with a single
+5 guanine base in a DNA deformed structure.9 Etoposide is an
effective anticancer agent10 and interfacial topoisomerase II inhibi-
tor11,12 which stabilizes a covalent topoisomerase II–DNA cleavable
complex intermediate10–14 resulting in single- and double-strand
DNA breaks that are cytotoxic. Compounds in which the glycosidic
moiety of etoposide is modified (e.g., teniposide) or even replaced
retain excellent activity.15–18 In previous studies we described the
Abbreviations: Boc2O, di-tert-butyl dicarbonate; CHO, Chinese hamster ovary;
Et3N, triethylamine; EtOAc, ethyl acetate; HOBt, (1-hydroxybenzotriazole); ICE,
immunodetection of complexes of enzyme-to-DNA; K562 cells, human erythroleu-
kemic cells; kDNA, catenated DNA; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-
oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
NaBH(OAc)3,
sodium
triacetoxyborohydride; Pd-C, palladium on carbon;
aMEM, Minimal Essential
Medium Alpha.
⇑
Corresponding author. Tel.: +1 204 474 8325; fax: +1 204 474 7617.
E-mail address: B_Hasinoff@UManitoba.ca (B.B. Hasinoff).
synthesis and biological and topoisomerase IIa inhibitory activities
http://dx.doi.org/10.1016/j.bmc.2014.09.014
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
of photoaffinity etoposide probes with a substitution on the glyco-
sidic moiety18 or with the glycoside moiety replaced with a substi-
tuted phenyl group.17 Here, guided by molecular modeling and
docking into the etoposide binding site in the X-ray structure of
topoisomerase IIb, we designed and synthesized a series of epipod-
ophyllotoxin–N-mustard hybrid compounds. These hybrids were
designed to contain a bifunctional N-mustard moiety that could
alkylate protein residues on topoisomerase II or alkylate DNA bases
when DNA and topoisomerase II are in a ternary complex with
inhibitor. These N-mustard hybrids could also alkylate uncom-
plexed DNA.19 It was hypothesized that binding of an epipodo-
phyllotoxin–N-mustard hybrid in the cleaved DNA–topoisomerase
II complex would result in an N-mustard-derived covalent adduct
being produced, and that this covalent adduct would irreversibly
inactivate topoisomerase II. Hence, the hybrid molecules would
have an advantage over etoposide which is bound in a thermody-
namic equilibrium within an established complex between topoiso-
merase II and DNA and is free to dissociate. In addition, since these
hybrid compounds may exert their antitumor effects by two inde-
pendent mechanisms (targeting topoisomerase II and alkylating
DNA), cancer cells should be much less able to develop resistance.
Due to the known clinical utility of the N-mustards19 and of etopo-
side,10 a series of epipodophyllotoxin–N-mustard hybrids based on
chlorambucil, melphalan and bendamustine moieties were
designed, synthesized and tested for their biological activity
in vitro and in cell-based assays.
that was 17-fold more potent than etoposide and was 41-fold more
potent than the most potent N-mustard melphalan. Using a variety
of assays, all of the hybrids showed strong evidence for targeting
topoisomerase II. In addition, the hybrids showed evidence consis-
tent with the ability to function as alkylating agents as expected.
2. Chemistry
Two different strategies were used for the synthesis of the hybrid
molecules containing both the epipodophyllotoxin and the N-mus-
tard anticancer drug motifs. In the first approach the N-mustard
compounds were directly linked to the epipodophyllotoxin as
depicted in Schemes 1 and 2. In the second approach the N-mustard
compounds were linked to the epipodophyllotoxin analog via a
piperazine linker as shown in Schemes 3 and 4. In the first step
(Scheme 1), the 4b-OH group in 1 (40-O-demethylepipodophyllo-
toxin) (AvaChem, San Antonio, TX) was efficiently converted to
the 4b-azide (compound 2) via nucleophilic substitution with
NaN3 and TFA at room temperature as previously reported.20,21
The corresponding 4b-amino compound 3 ((10R,11R,15S,16S)-16-
amino-10-(4-hydroxy-3,5-dimethoxyphenyl)-4,6,13-trioxatetracy-
clo[7.7.0.03,7.011 15
, ]hexadeca-1,3(7),8-trien-12-one) was prepared
by reducing the azide 2 with catalytic hydrogenation.20,21 The
advanced intermediate 3 was then subjected to a reductive amina-
tion reaction with 4-(bis(2-chloroethyl)amino)benzaldehyde 4 to
furnish 5. Similarly the DCC coupling reactions of 3 with chloram-
bucil 6 and bendamustine 8 (OChem, Des Plaines, Il) gave com-
Several of the newly synthesized hybrids incorporated a piper-
azine linker to extend the chain and to improve pharmacological
properties. The most potent of the series had a National Cancer
Institute (http://dtp.nci.nih.gov) mean NCI-60 cell line screen GI50
pounds
7
and 9, respectively (Schemes
1
and 2). The
epipodophyllotoxin–melphalan hybrid was synthesized in three
steps. Melphalan 10 (OChem, Des Plaines, Il) was protected with
Scheme 1. Synthesis of the epipodophyllotoxin advanced intermediate 3 and the N-mustard hybrid 5 and the chlorambucil-derived 7.

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5937
Scheme 2. Synthesis of the epipodophyllotoxin–N-mustard bendamustine-derived hybrid 9 and the melphalan-derived 13.
Scheme 3. Synthesis of the epipodophyllotoxin piperazine-linked advanced intermediates 15 and 16.
the Boc group using Boc2O (di-tert-butyl dicarbonate) to give 11
((2R)-3-{4-[bis(2-chloroethyl)amino]phenyl}-2-{[(tert-butoxy)car-
The Ritter reaction of chloroacetonitrile with 40-O-demethyle-
pipodophyllotoxin 1 was used23 to make the previously described24
4-chloroacetamido-4-deoxy-40-demethylepipodophyllotoxin inter-
mediate 14 in excellent yield (Scheme 3). In the next step 14 was
subjected to nucleophilic substitution with 1-Boc-piperazine to give
the intermediate 15, which on deprotection with TFA furnished the
bonyl]amino}propanoic acid) which was prepared using
a
described procedure.22 Compound 11 on DCC coupling with 3
yielded 12 (Scheme 2). Finally deprotection with 25% (v/v) HCl gave
the desired compound 13.

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A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
Scheme 4. Synthesis of the epipodophyllotoxin–N-mustard piperazine-linked hybrids 17, 18 and 19.
deprotected piperazine 16. The reductive amination reaction of 16
with 4 resulted in formation of 17 in good yield (Scheme 4). Simi-
larly, DCC coupling of 16 with chlorambucil 6 and bendamustine 8
gave 18 and 19, respectively, in moderate to good yield (Scheme 4).
camptothecin and other non-topoisomerase IIa
targeted drugs.25
Decreased cellular topoisomerase II translates to fewer DNA strand
breaks and reduced cytotoxicity. As shown in Table 1, while etopo-
side and the piperazine advanced intermediates 15 and 16 were
highly cross-resistant (10- to 17-fold), the hybrids generally dis-
played low cross resistance (1.3- to 4.8-fold), or even collateral
sensitivity in the case of 13. These results are consistent with most
of the hybrids acting, at least in part, as topoisomerase II poisons in
cells. However, because the cross-resistance of the hybrids is low
compared to etoposide and the piperazine intermediates 15 and
16 that are essentially etoposide analogs, these results suggest that
the hybrids are acting through an additional cytotoxic alkylation
mechanism. In contrast, the N-mustards chlorambucil, melphalan
and bendamustine all displayed collateral sensitivity towards the
K/VP.5 cell line consistent with reports that DNA topoisomerase
II contributes to enhanced alkylating agent adduct repair.30,31
Reduction of DNA topoisomerase II in K/VP.5 cells would result
in compromised DNA repair and increased sensitivity.
3. Results and discussion
3.1. Cell growth inhibitory effects of the hybrids and piperazine
intermediates on human leukemia K562 cells and K/VP.5 cells
with decreased levels of topoisomerase IIa and topoisomerase
IIb
The cell growth inhibitory effects of the synthesized hybrids
and the piperazine advanced intermediates on human leukemia
K562 cells and K/VP.5 cells25,26 were compared to etoposide and
the N-mustards chlorambucil, melphalan and bendamustine
(Table 1). Most of the hybrids displayed sub-micromolar IC50 val-
ues comparable to that of etoposide. All of the hybrids were much
more growth inhibitory than the three N-mustards tested. Cancer
cells can acquire resistance to topoisomerase II poisons by lower-
ing their level and/or activity of topoisomerase II.27 We have previ-
ously used28,29 a clonal K562 cell line selected for resistance to
etoposide as a screen to determine the ability of compounds to
act as topoisomerase II poisons. These K/VP.5 cells were deter-
mined to be 26-fold resistant to etoposide and to contain reduced
3.2. Evaluation of cell growth inhibitory effects of the hybrids in
the NCI-60 human tumor cell line screen
Compounds 5, 7, 9, 13, 17 and 19 were tested by the National
Cancer Institute (http://dtp.nci.nih.gov) to identify which tumor
cell types were most sensitive to these compounds and to be able
to carry out NCI COMPARE analyses (http://dtp.nci.nih.gov/com-
pare) to determine what other anticancer and cytotoxic drugs the
hybrids shared tumor cell growth inhibition profiles with, and to
levels of both topoisomerase II
a
(ꢀ6-fold) and topoisomerase IIß
(ꢀ3-fold).25,26 In addition, these cells are cross-resistant to other
known topoisomerase II poisons, but are not cross-resistant to

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5939
Table 1
Comparison of the cell growth inhibitory effects of the epipodophyllotoxin–N-mustard hybrids 5, 7, 9, 13, 17, 18, and 19, with the epipodophyllotoxin piperazine advanced
intermediates 15 and 16 and some N-mustards and etoposide
Compound
K562 IC50
(l
M)
K/VP.5a IC50
(lM)
RRb
Mean NCI-60 cell GI50 (lM)
5
7
9
13
15
16
17
18
0.46
0.72
2.0
0.77
1.8
2.5
0.66
3.4
8.4
0.85
4.3
26
10
34
5.3
4.9
1.7
2.5
1.3
0.4
16
10
3.1
12
4.8
0.7
<0.3
0.5
17
0.98
1.3
4.2
6.9
NDc
ND
0.71
ND
NDd
60
1.5
0.21
0.83
0.27
0.37
5.4
13
>110
12
19
6, Chlorambucil
8, Bendamustine
10, Melphalan
Etoposide
70
29
12
0.29
a
b
c
The etoposide resistant K/VP.5 cells that were derived from K562 cells have decreased levels of topoisomerase II
RR is relative resistance (K/VP.5 IC50 ꢁ K562 IC50).
a
and topoisomerase IIb.25,26
ND is not determined.
d
1-dose NCI-60 data for this compound in given in Supplemental Figure 6.
identify putative mechanisms by which the hybrids exerted their
activity (see below Fig. 9 results and discussion of hierarchical
cluster analysis). The NCI-60 cell line GI50 5-dose testing results
for compounds 5, 7, 9, 13 and 17 are given in Table 1 and Supple-
mental Figures 1–5, respectively (pages S5, S9, S13, S17 and S21,
advanced intermediate 16 and the hybrid 18 were about as inhib-
itory as etoposide, while the rest of the hybrids were less inhibitory
(compare at 10 and 100 lM).
The topoisomerase II interfacial inhibitors/poisons are cytotoxic
through their ability to stabilize a covalent topoisomerase II–DNA
cleavable complex intermediate which can lead to frank double-
strand DNA breaks.10,13,14 Thus, DNA cleavage assay experi-
ments28,29,34 were carried out to see whether the hybrids stabilized
the cleavable complex. Etoposide was used as a positive control. As
shown in Figure 2, the addition of etoposide to the reaction mix-
respectively). The 1-dose (10 lM) testing results for 19 are given
in Supplemental Figure 6 (page S22). All of the hybrids tested
had lower mean GI50 values compared to etoposide. It can also
be seen from these results that the leukemia cell lines were as a
group the most sensitive to the hybrids tested, with GI50 values
generally in the sub-micromolar range. The hybrids also generally
displayed very good growth inhibitory effects towards non-small
cell lung cancer lines and renal cancer lines. Of the hybrids sub-
jected to 5-dose testing, 17 was the most growth inhibitory with
ture containing topoisomerase II
a and supercoiled pBR322 DNA
induced formation of linear pBR322 DNA. Migration of linear
DNA (LIN) was identified by treating closed circular pBR322 with
the restriction enzyme HindIII to generate authentic linearized
PBR322 (not shown). Figure 2 shows that all of the hybrids tested
induced concentration dependent formation of linear DNA consis-
a mean GI50 over all cell lines tested of 0.71
least growth inhibitory with a mean GI50 of 6.9
l
M, and 13 was the
M (Table 1). The
l
hybrids were the least growth inhibitory towards colon and ovar-
ian cancers with GI50 values in the low micromolar range. The most
potent hybrid (compound 17) tested had a mean NCI-60 cell line
GI50 value that was 17-fold more potent than etoposide and 41-
fold more potent than the most potent N-mustard melphalan
(Table 1).
tent with activity as topoisomerase IIa poisons. Based on the
amount of linear DNA produced, hybrid 5 and the piperazine
advanced intermediate 16 were more potent than etoposide, while
the other hybrids exhibited similar or were less active compared to
etoposide.
Phosphorylated H2AX (cH2AX), which is a variant of an H2A
core histone, rapidly localizes at the site of double-strand DNA
3.3. The hybrids inhibited topoisomerase II
a
decatenation
breaks upon treatment of cells with drugs or ionizing radiation.35
activity, acted as a topoisomerase II
double-strand breaks in cells
a
poisons and induced DNA
The thousands of cH2AX molecules that are localized at the site
of DNA double-strand breaks are thought to amplify the DNA dam-
age signal and are a widely accepted marker of double-strand
breaks.35 Thus, in order to determine if the hybrids could induce
The torsional stress that occurs in DNA during replication and
transcription can be relieved by topoisomerase II. In addition, nor-
mal chromosomal separation of daughter DNA strands during
mitosis is facilitated by topoisomerase II. Topoisomerase II alters
DNA topology by catalyzing the passing of an intact DNA double
helix through a transient double-strand break made in a second
helix.10,13,14 As shown in Figure 1, all of the hybrids inhibited the
double-strand breaks in intact K562 cells, the level of
tein was determined by Western blotting with etoposide as the
positive control (all agents used at 20
M).34 Experiments carried
out as we previously described,29 and shown in Figure 3, indicate
that the compounds 5, 17, 18, 19 and 13 increased levels of H2AX
cH2AX pro-
l
c
in K562 cells while 7 and 9, whose topoisomerase cleavage activity
was low (Fig. 2), were relatively inactive. Compounds 17, 19 and 13
were even more potent than the etoposide positive control. These
results suggest that the hybrids acted as topoisomerase II poisons
in a cellular context to produce damaging DNA double-strand
breaks.
A cellular ICE (immunodetection of complexes of enzyme-
to-DNA) assay was also used to determine if the hybrids could
produce topoisomerase II-covalent complexes in K562 cells.
Experiments were carried out as we previously described29 and
decatenation of concatenated kDNA by topoisomerase II
razoxane (ICRF-187), a known inhibitor of topoisomerase II
a. Dex-
a
decatenation,32 and etoposide were included as positive controls.
The decatenation assay measures the ability of compounds to inhi-
bit the catalytic activity only, and is not a measure of ‘interfacial’
inhibitory or ‘poisoning’ activity which can induce a stabilized
topoisomerase II-DNA covalent complex.11,33 Based on the reduc-
tion in the amount of open circular (OC) DNA produced, compound
13, which inhibited the decatenation activity in the low micromo-
lar range (compare 1 and 10
lM), was the most inhibitory, and
are shown in Figure 4. At a concentration of 30
lM, all of the
even exceeded that of etoposide and dexrazoxane. The piperazine
hybrids tested and the etoposide positive control increased the

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A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
Figure 1. Effect of the hybrids, the piperazine advanced intermediates 15 and 16, and the etoposide and dexrazoxane positive controls on the inhibition of the topoisomerase
II
a
-mediated decatenation activity of kDNA. These fluorescent images of the ethidium bromide-containing gels show that in the absence of added drug (lanes marked +Topo
) topoisomerase II decatenated kDNA to its open circular (OC) and nicked circular (NC) forms. Topoisomerase II was present in the reaction mixture for all lanes but lanes
was shown for all agents tested. The results were typical of
IIa
a
a
marked kDNA. ORI is the gel origin. Concentration dependent inhibition of decatenation of topoisomerase II
a
experiments carried out on 2 different days.
Figure 2. Concentration dependent effect of the hybrids, the piperazine advanced intermediates 15 and 16, and etoposide on the topoisomerase II
cleavage of supercoiled pBR322 plasmid DNA to produce linear DNA. These fluorescent images of ethidium bromide-stained gels show that topoisomerase II
converted supercoiled (SC) pBR322 DNA to relaxed (RLX) DNA. In this assay the relaxed DNA runs slightly ahead of the supercoiled DNA because the gel was run in the
presence of ethidium bromide. Topoisomerase II was present in the reaction mixture in all but the lanes marked pBR322. The etoposide positive controls produced
a-mediated relaxation and
a
(+Topo II
a)
a
significant amounts of linear DNA (LIN). A small amount of nicked circular (NC), which may arise from strand breakage during isolation, is normally present in pBR322 DNA.
The results were typical of experiments carried out on 2 different days.

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5941
links. Thus, a DNA cross-link assay was performed as we previously
described36,37 using 10
M of the agents indicated. Chlorambucil
l
and cisplatin were used as positive controls (Fig. 5). Of the hybrids
tested only compound 9 produced a comparable amount of
cross-linked DNA as did chlorambucil, and both of these agents
produced much less cross-linked DNA compared to cisplatin.
Detectable DNA cross-linking in the presence of 9 suggests that
this mechanism may contribute, in part, to cellular growth inhibi-
tion and cytotoxicity.
Figure 3. The hybrids 7, 9, 5, 17, 18, 19, 13 and the etoposide positive control
induced double-strand DNA breaks in K562 cells as indicated by formation of
c
H2AX. K562 cells were treated with 20
medium, lysed and subjected to SDS–PAGE electrophoresis and Western blotting.
The blots were probed with antibodies to H2AX and with GAPDH (glyceraldehyde
lM of the drugs indicated for 4 h in growth
3.5. Cell cycle analysis and two-color flow cytometry
c
3-phosphate dehydrogenase) as a loading control and a chemiluminescent-induc-
ing horseradish peroxidase-conjugated secondary antibody. The results were
typical of experiments carried out on 2 different days.
Cell cycle analysis was carried out on synchronized CHO cells as
we previously described28,37 on the hybrids indicated (Fig. 6) in
order to determine the possible mechanism(s) by which these
compounds might be acting. Drugs that act on topoisomerase II
a
typically cause a block in G2/M, while N-mustard type DNA
cross-linking drugs such as melphalan typically cause a cell cycle
delay from G1.38 CHO cells (normal doubling time of 12 h) that
were synchronized to G0/G1 through serum starvation were treated
with 20 lM of the hybrid indicated, or etoposide (20 lM) for com-
parison, in order to determine whether these compounds induced
a G2/M cell cycle block and/or a delay from G0/G1. CHO cells were
chosen for these experiments as they are easily and effectively syn-
chronized by serum starvation. Subsequent serum repletion
resulted in the control (DMSO vehicle) cells advancing to G2/M
by 17 h (Fig. 6B). The control cells (Fig. 6B, all panels) then went
through several complete cell cycles as evidenced by the 12 h peri-
odicity for peaks in the various cell cycle stages. Of the compounds
tested 5, 7 and 13 displayed a significant percentage of sub-G0/G1
cells comparable to that observed for etoposide, which was indic-
ative of apoptosis. The percentage of sub-G0/G1 cell was small in
control- and 9-treated cells (<2%). Treatment with etoposide
Figure 4. Chemiluminescent images of
a Western slot blot determination of
cellular covalent topoisomerase II –DNA (upper image) and topoisomerase IIb–
a
DNA cleavage complexes (lower image) produced in K562 cells determined using
an ICE (immunodetection of complexes of enzyme-to-DNA) assay. In these
experiments K562 cells were either treated with DMSO vehicle or with 30
the etoposide positive control or the drugs indicated for 1 h.
lM of
caused
a large block in G2/M by 25 h, which subsequently
decreased at later times as the sub-G1 population, indicative of
apoptosis, increased to about 25%. It can be seen from Figure 6B
that the etoposide-treated and the control cells exited G0/G1 at
almost identical rates as shown by the overlap of the their respec-
tive curves between 9 and 14 h. Importantly, compounds 5, 7 and
13 all displayed a delayed exit of about 5 h from G0/G1 compared to
etoposide and the vehicle-treated control. This delayed progression
from G0/G1 may be indicative of a mechanism typical of a DNA
cross-linking agent such as melphalan.38 However, the large G2/M
blocks observed with these compounds were also typical of a agent
such as etoposide that targets topoisomerase II. Thus, these results
are consistent with some of the hybrids acting both as DNA cross-
linking agents and topoisomerase II poisons. However, it should
also be noted that other agents that do not target topoisomerase
II can also cause G2/M blocks.
Early in the apoptotic process translocation of the phospholipid
phosphatidylserine occurs from the inner to the external plasma
membrane of cells. Fluorescent annexin V-FITC, which strongly
binds phosphatidylserine on cells in this early stage of apoptosis,39
was used to identify apoptotic and apoptotic/necrotic cells by two-
color flow cytometry as we previously described.37 Thus, annexin
V-FITC stained cells in both the lower and upper right quadrants
of Figure 7A were considered to be apoptotic. When used in com-
bination with propidium iodide, which binds DNA, a measure of
cells that are necrotic may be obtained since necrotic cell mem-
branes are permeable to propidium iodide. Thus, cells in the lower
right quadrant were apoptotic-only and those in the upper right
quadrant were apoptotic/necrotic. As shown in Figure 7A and B,
all treatments of K562 cells with hybrids 5, 7, 9, 13 or with etopo-
Figure 5. The DNA cross-linking effects of the hybrids, cisplatin and chlorambucil
on linearized pBR322 DNA. This fluorescent image of the ethidium bromide-stained
gel shows that the bifunctional alkylating agent chlorambucil control and cisplatin
cross-linked DNA while the hybrids only slightly increased cross linking. All agents
were tested at a concentration of 10 lM. Control 1 was not heated and thus
consisted of only double stranded DNA. Control 2 and the other drug-treated
samples were heated to 90 °C for 5 min to cause strand separation and then rapidly
cooled. The strands that were cross-linked by drug treatment recombined to form
double-strand DNA, whereas those that were not cross-linked, did not recombine.
The amount of DNA in each well was the same. The band intensity of the single
stranded DNA is less than that of double strand DNA because of reduced binding of
ethidium bromide.
amount of topoisomerase II
to the untreated control in (Fig. 4, upper image). The hybrids were
about as potent as etoposide in producing topoisomerase II -cova-
a covalently bound to DNA compared
a
lent complexes. Likewise the hybrids and etoposide were about
equipotent in producing increases in the amount of the topoiso-
merase IIb-covalent complexes (Fig. 4, lower image).
3.4. Comparison of the DNA cross-linking activity of the hybrids
to that of chlorambucil and cisplatin
side (24 h, 10 lM) reduced the proportion of viable cells and
increased the proportion of both apoptotic and apoptotic/necrotic
cells.
The hybrids contain an N-mustard moiety that is potentially
capable of damaging DNA through the production of DNA cross

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A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
Figure 6. Cell cycle effects of hybrid treatment of synchronized CHO cells. CHO cells that had been synchronized in G0/G1 through serum starvation were repleted with serum
and were treated with the DMSO vehicle or with 20 M of the hybrid indicated directly after repletion and allowed to grow for the times indicated, after which they were
l
subjected to cell cycle analysis of their propidium iodide-stained DNA. In (A) the cell counts are displayed on the vertical axis and the DNA content is plotted on the horizontal
axis. In (B) the percentage of the cells in the sub-G0/G1, G0/G1, S and G2/M phases is plotted as a function of time for each of the hybrids indicated. The solid lines are a least-
squares calculated spline fit to the data. As shown in the top plots a high percentage of the serum starved cells were initially present in G0/G1. After serum repletion the
percentage of control cells in each phase varied periodically as the cells progressed through several cell cycles.

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5943
Figure 7. Treatment of K562 cells with the hybrids indicated or the etoposide positive control induced apoptosis as determined by annexin V-FITC/propidium iodide two-
color fluorescence flow cytometry. (A) Two-color flow cytometry scatter plots of untreated control K562 cells, and 10 M of the hybrid or the etoposide positive control
l
treated for 24 h. Annexin V-FITC binds to the exterior surface of cells that are in the early stages of apoptosis and membrane-impermeant propidium iodide only binds the
DNA of necrotic cells that have leaky membranes. Thus, the lower left quadrant contained viable cells; the upper left quadrant contained cells that were necrotic-only; the
lower right quadrant contains cells that were apoptotic-only but were not necrotic; and the upper right quadrant contained cells that were both apoptotic and necrotic. (B)
Changes in relative number of K562 cells that were classified as necrotic, apoptotic/necrotic, apoptotic or viable 24 h after no treatment, or after treatment with 10
hybrid or the etoposide positive control as indicated.
lM of the
3.6. Docking of 7 into the etoposide binding site of etoposide
retain good topoisomerase II-targeted activity. The glycosidic moi-
complexed to cleaved DNA and topoisomerase IIb
ety may even be replaced with a variety of other moieties such as
the polyamines found in F1451240 and in TOP-53 or a p-nitro-
phenyl moiety found in GL-311.16 It has been shown in the PDB
ID: 3QX3 X-ray structure of two molecules of etoposide complexed
to cleaved DNA and topoisomerase IIb8,9 that the glycosidic moiety
of etoposide occupies a spacious binding pocket in the protein with
It is well known that the glycosidic moiety of etoposide (Fig. 8A)
can undergo substitution such as that found in teniposide16 or be
substituted or replaced as in the two photoaffinity etoposide
analogs we synthesized and biologically caracterized,17,18 and still

5944
A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
Figure 8. Docking of the epipodophyllotoxin–chlorambucil hybrid 7 into the etoposide binding site of the PDB ID: 3QX3 X-ray structure of etoposide complexed to cleaved
DNA and topoisomerase IIb yields two equally high scoring poses. (A) Structure of etoposide. (B) Docking pose of hybrid 7 into the X-ray structure in which the N-mustard
moiety extends into the spacious protein cavity is shown in a stick representation in atom colors. (C) Docking pose of hybrid 7 into the X-ray structure that more closely
interacts with DNA is shown in a ball-and-stick representation in atom colors. The X-ray structure of etoposide bound to the 3QX3 structure is shown in a yellow ball-and-
stick structure in both (B) and (C). For clarity DNA is shown in a ladder representation and the protein is shown as a gray semi-transparent surface. The poses shown in (B) and
(C) are the nearly energetically equivalent highest scoring poses of 7 docked into the etoposide binding site of the PDB ID: 3QX3 X-ray structure. The H-atoms of the bound
etoposide or the poses of 7 in (B) and (C) displayed are not shown for clarity. The etoposide was removed from the structure and 7 was docked into its place with the genetic
algorithm docking program GOLD.
few protein interactions. This result explains why etoposide ana-
logs are relatively insensitive to substituents that replace or substi-
tute on the glycosidic moiety.
the glycosidic binding pocket in the two topoisomerase II isoforms
is the replacement of Ala816 in topoisomerase IIb by the more
nucleophilic Ser800 in topoisomerase IIa 9
.
If this Ser800 group in
were to form a covalent bond with the N-mus-
tard hybrids, this would result in a desirable preferential selectivity
of the hybrids for topoisomerase II over topoisomerase IIb. It was
In order to determine if the hybrids synthesized could also fit
into this spacious binding pocket, docking was carried out into
the PDB ID: 3QX3 X-ray structure of etoposide complexed to
cleaved DNA and topoisomerase IIb in which etoposide had been
removed. The results of the docking of 7 are shown in Figure 8B
and C. The X-ray-determined pose of etoposide is shown in each
panel in a yellow ball-and-stick representation. Docking of 7
resulted in two poses (Fig. 8B and C) that gave nearly equal GOLD
fitness scores. In one pose (shown in stick representation in atom
colors Fig. 8B) the N-mustard moiety is fully extended into the pro-
tein pocket normally occupied by the glycosidic moiety. In the
other pose (shown in ball-and-stick representation in atom colors
in Fig. 8C) the N-mustard moiety is in close proximity to the DNA.
The polycyclic and pendant phenol moieties of both of these poses
show a high degree of overlap with that of the X-ray structure of
the bound etoposide (yellow ball-and-stick representation). The
bifunctional N-mustard anticancer drugs such as chlorambucil,
melphalan and bendamustine, which are capable of alkylating
DNA bases or nucleophilic protein residues, act through a highly
reactive aziridinium ion mechanism19 produced by loss of a chlo-
ride from one of the N-mustard arms. The two docking poses of
7 shown in Figure 8 indicate that alkylation of either nucleophilic
amino acids on topoisomerase IIb (Fig. 8B) or on nucleophilic oxy-
gen or nitrogen groups on DNA (Fig. 8C) in the ternary complex
could result in a covalent adduct with either protein or with
DNA. In the case of alkylation of topoisomerase II, this adduct
would be expected to result in irreversible inactivation of the
enzyme. In contrast, the etoposide in the ternary etoposide–topo-
isomerase II–DNA complex is free to dissociate from the complex.
If alkylation of DNA occurs it would be expected to result in perma-
nent double- or single-strand DNA damage that would be difficult
for the cell to repair.
topoisomerase IIa
a
shown in a recent study of 19 topoisomerase II inhibitors, which
included etoposide and teniposide, that most had little or no pref-
erence for inhibition of topoisomerase IIa
over topoisomerase IIb.42
Additionally, it has been proposed that topoisomerase IIb may be
responsible for mediating chromosome translocations in therapy-
related leukemias.43 Thus, a topoisomerase II
a specific agent may
be less leukemogenic and hence more efficacious than the rela-
tively non-selective topoisomerase II-targeted anticancer agents
currently in use.
3.7. Cross correlation and hierarchical cluster analysis obtained
from a COMPARE analysis of NCI 60-cell data
An NCI COMPARE analysis (http://dtp.nci.nih.gov/compare) of
the NCI GI50 endpoint 60-cell line data was carried out on the epip-
odophyllotoxin–N-mustard hybrid compounds, and for compari-
son etoposide and teniposide, and the N-mustard compounds
bendamustine, uramustine, chlorambucil, melphalan and mechlor-
ethamine. These results were further subjected to hierarchical
cluster analysis44 which identifies which groups of compounds
had a similar tumor cell growth inhibition profile in the NCI 60-cell
line data. COMPARE analysis on the NCI 60-cell data has been used
to identify compounds that act through common mechanisms45
and is based on the assumption that compounds with a common
mechanism of action will show a similar profile of log(GI50) values
as identified by significant cross-correlation coefficients r. The
results of the COMPARE analysis are shown in Figure 9. As might
be expected all the N-mustards (except bendamustine) correlated
very well with each other with r values in the 0.82–0.98 range
and also clustered together very well. The second most highly clus-
tered group consisted of the hybrid compounds 7, 9, 13, 17 and
teniposide, all of which contain an epipodophyllotoxin moiety
and had r values in the range of 0.77–0.92. The next most highly
clustered group consisted of compound 19 and etoposide. These
latter two results show that the hybrid compounds which contain
an epipodophyllotoxin moiety all share an NCI GI50 endpoint 60-
cell profile, and presumably a common mechanism of action, with
the epipodophyllotoxins etoposide and teniposide. The hybrids and
There is a high degree of homology (ꢀ78%) in the catalytic
domains of topoisomerase II
a
and topoisomerase IIb.41 While the
X-ray structure of an etoposide–topoisomerase II
a–DNA complex
has not yet been reported, the topoisomerase II
a–DNA complex
with cleaved DNA has been reported.41 It was previously sug-
gested8,9 that one of the two key glycosidic-interacting residues,
Gln778, in the topoisomerase IIb structure, which is replaced by
methionine (Met762) in topoisomerase II
a, may be useful in devel-
oping isoform-specific anticancer drugs. An additional difference in

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5945
Figure 9. Cross correlation and hierarchical cluster analysis obtained from a COMPARE analysis of NCI 60-cell log(GI50) data for the epipodophyllotoxin–N-mustard hybrid
compounds, etoposide, teniposide and the N-mustard compounds bendamustine, uramustine, chlorambucil, melphalan and mechlorethamine. The values in the table are the
Pearson correlation coefficients r. The individual cells are colored according to the range of r in which they fall.
the N-mustards also clustered together. Thus, the data in Figure 9
was also examined to see how well the hybrid compounds corre-
lated with the N-mustard compounds from which they were
derived. Compound 7, which was derived from (Scheme 1) and
contains the chlorambucil moiety, correlated well (r 0.76, p
10ꢂ11) and highly significantly with chlorambucil. Compound 13,
which contains the melphalan moiety, correlated very well and
highly significantly (r 0.74, p 10ꢂ10) with melphalan. Compound
9, which was derived from (Scheme 2) and contains the bendamus-
tine moiety, correlated well and very significantly (r 0.56, p
7 ꢃ 10ꢂ6) with melphalan. What these latter set of correlations
show is that in the NCI COMPARE analysis the hybrid compounds
that contain an N-mustard moiety correlate well with the corre-
sponding N-mustards from which they were derived, suggesting
they may have a common mechanism of action. These results are
consistent with the low cross-resistance to the etoposide-resistant
K/VP.5 cell line (Table 1) and the delayed cell cycle progression
results the hybrids displayed in Figure 6.
tested (Fig. 7) were comparable to etoposide in inducing apoptosis
in K562 cells.
The COMPARE analysis showed that the hybrid compounds
clustered well (Fig. 9) with teniposide and etoposide with which
they share the epipodophyllotoxin moiety. The NCI 60-cell growth
inhibition profiles of the hybrids were also highly significantly cor-
related with the N-mustard compounds from which they were
derived. These and our other results strongly suggested that the
hybrids inhibited tumor cell growth both by inhibiting topoiso-
merase II and by alkylating DNA.
The molecular modeling and docking results of 7 (Fig. 8) into
the etoposide binding site of the PDB ID: 3QX3 X-ray structure of
etoposide complexed to cleaved DNA and topoisomerase IIb
showed two nearly energetically equivalent poses (Fig. 8B and C).
In Figure 8B the N-mustard moiety of 7 was directed into the pro-
tein cavity. In Figure 8C an energetically equivalent pose of 7 was
found positioned such that it could possibly interact with the DNA.
Alkylation of nucleophilic protein residues on topoisomerase II by
the hybrids would be expected to permanently inactivate the
enzyme and result in cell kill. Likewise, alkylation of nucleophilic
oxygen or nitrogen bases on DNA would likewise be expected to
result in cell kill. Given that these hybrids have two moieties
through which they can independently, and in tandem, exert their
anticancer activity, they would be expected to have a broader
range of therapeutic activities than either one of their constituent
moieties. Because the hybrids can act through two independent
cell killing mechanisms, emergence of resistance should be
delayed/reduced. In conclusion, a series of hybrid epipodophyllo-
toxin N-mustard analogs have been designed, synthesized and bio-
logically characterized that were more potent than the prototype
epipodophyllotoxin etoposide or their N-mustard analogs. Subse-
quent studies on the hybrids will focus on the molecular mecha-
nisms by which the hybrids are cytotoxic, their selectivity for
4. Conclusions
Etoposide and the N-mustards from which our hybrids are
derived are thought to exert their anticancer activity by distinc-
tively different mechanisms. Etoposide acts as a topoisomerase
II
a
interfacial inhibitor/poison10–13 that results in stabilization of
cleaved DNA and frank double strand breaks that lead to cell death.
The N-mustards are thought to act through the formation of cross-
links with DNA that results in DNA damage that is difficult to
repair and also leads to cell death. The hybrids were designed with
the view that combining epipodophyllotoxin and N-mustard moi-
eties in the same molecule would produce a potent anticancer drug
that combined the anticancer activities of both of these classes of
agent. Seven N-mustard–epipodophyllotoxin hybrid compounds
(and the two piperazine advanced intermediates, 15 and 16) were
synthesized and tested for cell growth inhibitory activity with
K562 cells (Table 1). Six of the hybrids were tested in the NCI-60
cell line assay.
targeting topoisomerase IIa over topoisomerase IIb, their pharma-
cokinetics and pharmacodynamics, and the characterization of the
putative covalent adducts with topoisomerase II and DNA.
All of the hybrid compounds tested showed strong evidence
that they targeted topoisomerase II. This was evidenced by their
strong activities in the kDNA decatenation assay (Fig. 1), the DNA
cleavage assay (Fig. 2) and the cellular ICE assay (Fig. 4). Addition-
5. Experimental
5.1. Chemistry
ally, the results of the cellular
cH2AX assays (Fig. 3) and the cell
1H and 13C NMR spectra were recorded on a Bruker Avance NMR
spectrometer operating at 300 MHz or 500 MHz, as indicated, for
1H NMR and 75 MHz (or 125 MHz as indicated) for 13C NMR,
respectively, in CDCl3 or DMSO-d6. The chemical shifts are
expressed as d units with Si(CH3)4 as the internal standard. Melting
points were uncorrected. The high resolution mass spectra were
run on a Bruker microTOF Focus mass spectrometer (Fremont,
CA) using electron spray ionization. TLC was performed on
cycle analyses which displayed G2/M blocks were also characteris-
tic of topoisomerase II-targeted drugs. A DNA cross-link assay
showed that 9 had N-mustard type activity as it formed DNA
cross-links (Fig. 5) about equal to that of chlorambucil. Also, the
hybrids caused a delayed progression from synchronized CHO cells
compared to that observed for etoposide (Fig. 6) consistent with
the hybrids possessing native N-mustard type activity. The hybrids

5946
A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
plastic-backed plates bearing 200
l
m (thickness) silica gel 60 F254
J = 2.1 Hz, 1H), 6.52 (s, 1H), 6.24 (s, 2H), 6.01 (d, J = 10.14 Hz, 2H),
5.20 (m, 1H), 4.49 (d, J = 4.95 Hz, 1H), 4.29 (t, J = 7.62, 4.35 Hz,
1H), 3.58–3.82 (m, 18H), 3.18 (m, 1H), 2.95 (m, 1H), 2.85 (m,
2H), 2.27 (m, 2H), 2.02 (m, 2H); 13C NMR (CDCl3, 75 MHz): d
174.6, 171.6, 151.8, 147.1, 146.8, 145.5, 134.4, 132.1, 130.1,
124.7, 113.2, 112.3, 109.3, 108.8, 108.2, 101.2, 94.9, 68.3, 55.9,
52.4, 46.6, 42.7, 41.1, 40.8, 38.0, 36.5, 24.3, 21.9; HRMS (ESI) for
(Silicycle, Quebec City, Canada). Where applicable compounds
were visualized by quenching of fluorescence by UV light
(254 nm). Unless otherwise specified chemicals were from Aldrich
(Oakville, Canada) and were used without further purification.
The following general procedure for DCC coupling was used. To
a solution of the appropriate amine (1 mmol) in anhydrous DMF
under an inert atmosphere the appropriate acid (1 mmol), DCC
(1 mmol) and HOBt (1-hydroxybenzotriazole) (1 mmol) were
added. The reaction mixture was stirred at room temperature over-
night. The medium was then taken up with CH2Cl2 and water. The
organic phase was extracted with water (3 ꢃ 50 mL), then dried
(MgSO4) and filtered. This product was further purified by silica
gel chromatography using CH2Cl2/methanol as the eluant to give
the pure compounds in good to moderate yields.
C
37H41Cl2N4O8 m/z (M+H)+: calcd 739.2296, obsd 739.2317.
5.1.4. tert-Butyl N-[(1R)-2-{4-[bis(2-chloroethyl)amino]phenyl}-
1-{[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dimethoxyphenyl)-
14-oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
,
]hexadeca-1,3(7),
8-trien-10-yl]carbamoyl}ethyl]carbamate (12)
The DCC coupling of 11 with 3 yielded 12 (66%); 1H NMR (CDCl3,
300 MHz): d 7.14 (d, J = 8.46, 2H), 6.67 (d, J = 8.58, 2H), 6.49 (s, 1H),
6.31 (s, 1H), 6.28 (s, 1H), 6.19 (d, J = 7.32 Hz, 1H), 5.97 (d,
J = 2.61 Hz, 2H), 5.44 (s, 1H), 5.08 (m, 1H), 4.98 (m, 1H), 4.53 (d,
J = 4.8 Hz, 1H), 4.36 (t, J = 8.6, 7.5 Hz, 1H), 4.18 (t, J = 8.4, 7.1 Hz,
1H), 3.82–3.63 (m, 14H), 3.06–2.85 (m, 3H), 2.80 (m, 1H), 1.42 (s,
9H). This intermediate was used in the next step without further
purification.
5.1.1. (10R,11R,15S,16S)-16-[({4-[Bis(2-chloroethyl)amino]phenyl}-
methyl)amino]-10-(4-hydroxy-3,5-dimethoxyphenyl)-4,6,13-
trioxatetracyclo[7.7.0.03,7.011 15
,
]hexadeca-1,3(7),8-trien-12-one
(5)
Compound
3
(0.1 g, 0.25 mmol) and the benzaldehyde 4
(0.062 g, 0.25 mmol) were mixed in DCE (5 mL) and then treated
with sodium triacetoxyborohydride (0.064 g, 0.3 mmol). The mix-
ture was stirred at room temperature under a N2 atmosphere for
3 h. After completion of the reaction, the mixture was quenched
by adding aqueous saturated NaHCO3, and the product was
extracted with EtOAc. The EtOAc extract was dried (MgSO4) and
the solvent was evaporated to give the crude free base 5 which
was purified by column chromatography using hexane/EtOAc
(80:20) as an eluant (0.1 g, 72%). Mp: 168–172 °C (decomposed);
1H NMR (CDCl3, 300 MHz): d 7.24–7.28 (m, 2H), 6.75 (m, 2H),
6.57 (s, 1H), 6.48 (s, 1H), 6.29 (s, 2H), 5.92 (s, 2H), 5.35 (br s, 1H),
4.53 (d, J = 6.5, 1H), 4.35 (m, 2H), 3.97 (m, 1H), 3.60–3.82 (m,
16H), 3.35 (m, 1H), 2.76 (m, 1H); 13C NMR (CDCl3, 75 MHz): d
175.5, 147.6, 147.2, 146.3, 145.5, 133.9, 132.7, 131.6, 131.2,
129.8, 128.8, 112.2, 110.2, 108.3, 108.0, 101.3, 68.6, 56.4, 55.4,
54.0, 53.5, 43.6, 41.5, 40.4, 38.6; HRMS (ESI) for C32H35Cl2N2O7
m/z (M+H)+: calcd 629.1816, obsd 629.1800. The synthesis of 5
by a different method has been reported previously.46
5.1.5. (2R)-2-Amino-3-{4-[bis(2-chloroethyl)amino]phenyl}-N-
[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dimethoxyphenyl)-14-
oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
, ]hexadeca-1,3(7),
8-trien-10-yl]propanamide (13)
Compound 12 (175 mg, 0.22 mmol) was dissolved in 3 mL of
THF. To this solution 2.0 mL of hydrochloric acid (25%, v/v) was
added and the mixture was stirred for 20 h at room temperature.
The solvent was removed under reduced pressure. The oil remain-
ing was dissolved in methanol and the product was precipitated
with diethyl ether, filtered and dried, to afford 123 mg (90%)
of a colorless solid 13; mp: 282–285 °C; 1H NMR (DMSO-d6,
300 MHz):
d 8.45 (d, J = 9.8 Hz, 1H), 8.33 (s, 3H), 7.10 (d,
J = 8.43 Hz, 2H), 6.72 (d, J = 8.56, 2H), 6.50 (s, 1H), 6.31 (s, 1H),
6.28 (s, 1H), 6.19 (s, 2H), 5.98 (d, J = 2.60 Hz, 2H), 5.05 (m, 1H),
4.99 (m, 1H), 4.47 (d, J = 4.86 Hz, 1H), 4.28 (t, J = 8.4, 7.2 Hz, 1H), 4.18
(t, J = 8.2, 6.9 Hz, 1H), 3.82–3.63 (m, 14H), 3.09 (m, 1H),
3.02–2.89 (m, 3H); 13C NMR (DMSO-d6, 125 MHz): d 174.3, 167.8,
147.1, 146.3, 145.5, 139.7, 134.6, 132.4, 130.5, 130.0, 129.0,
122.7, 111.4, 108.2, 55.8, 52.0, 47.3, 42.8, 40.0, 39.0, 36.6, 35.8;
HRMS (ESI) for C34H38Cl2N3O8 m/z (M+H)+: calcd 686.2030, obsd
686.2038.
5.1.2. 4-{4-[Bis(2-chloroethyl)amino]phenyl}-N-[(10S,11S,15R,
16R)-16-(4-hydroxy-3,5-dimethoxyphenyl)-14-oxo-4,6,13-tri-
oxatetracyclo[7.7.0.03,7.011 15
, ]hexadeca-1,3(7),8-trien-10-yl]-
butanamide (7)
The DCC coupling reaction of 3 with chlorambucil 6 gave 7
(78%); mp: 138–142 °C (decomposed); 1H NMR (CDCl3,
300 MHz): d 7.09 (d, J = 8.52 Hz, 2H), 6.74 (s, 1H), 6.74 (s, 1H),
6.67 (d, J = 8.58 Hz, 2H), 6.53 (s, 1H), 6.30 (s, 2H), 6.00 (d,
J = 2.61 Hz, 2H), 5.58 (d, J = 7.05, 1H), 5.43 (s, 1H), 5.23 (m, 1H),
4.59 (d, J = 4.8 Hz, 1H), 4.43 (m, 1H), 3.83–3.61 (m, 15H), 2.96 (m,
1H), 2.82 (dd, J = 6.12, 4.89 Hz, 1H), 2.60 (t, J = 7.38, 7.29, 2H),
2.25 (t, J = 7.62, 7.53, 2H), 1.97 (m, 2H); 13C NMR (CDCl3,
75 MHz): d 174.4, 172.9, 148.4, 147.6, 146.5, 134.1, 132.6, 130.2,
129.8, 128.9, 113.7, 110.1, 108.9, 107.8, 101.6, 69.1, 56.4, 54.3,
48.0, 43.5, 41.8, 39.8, 37.2, 35.6, 34.2, 27.2; HRMS (ESI) for
5.1.6. tert-Butyl 4-({[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dime
thoxyphenyl)-14-oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
, ]-
hexadeca-1,3(7),8-trien-10-yl]carbamoyl}methyl)piperazine-1-
carboxylate (15)
To a stirred solution of 14 (0.1 g, 0.21 mmol) and 1-Boc-pipera-
zine (0.043 g, 0.23 mmol) in DMF (3 mL) triethylamine (0.042 g,
0.42 mmol) was added at room temperature. The reaction mixture
was stirred overnight; it was then quenched with water and then
extracted with CH2Cl2 (3 ꢃ 20 mL). The combined organic extract
was washed with brine and dried over Na2SO4. Concentration
under reduced pressure gave a crude product, which on column
chromatography gave the title compound as a colorless solid
(0.12 g, 92%); mp: 233 °C; 1H NMR (CDCl3, 300 MHz): d 7.20 (d,
J = 7.29 Hz, 1H), 6.69 (s, 1H), 6.56 (s, 1H), 6.32 (s, 2H), 6.01 (d,
J = 1.2 Hz, 2H), 5.44 (s, 1H), 5.22 (m, 1H), 4.63 (d, J = 4.74 Hz, 1H),
4.46 (t, J = 7.77, 8.79 Hz, 1H), 3.80 (m, 7H), 3.40 (m, 4H), 3.19–
2.96 (m, 3H), 2.80 (dd, J = 4.89, 4.95 Hz, 1H), 2.49 (m, 4H), 1.47
(s, 9H); 13C NMR (CDCl3, 75 MHz): d 174.2, 170.1, 154.6, 148.4,
147.7, 146.5, 134.2, 132.5, 130.1, 128.8, 110.3, 108.3, 107.9,
101.7, 80.1, 69.0, 61.3, 56.4, 53.3, 47.8, 43.6, 41.9, 37.2, 28.3; HRMS
(ESI) C32H40N3O10 for m/z (M+H)+: calcd 626.2708, obsd 626.2682.
C
35H38Cl2N2NaO8 m/z (M+Na)+: calcd 707.1897, obsd 707.1890.
5.1.3. 4-{5-[Bis(2-chloroethyl)amino]-1-methyl-1H-1,3-benzodi-
azol-2-yl}-N-[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dimethoxy-
phenyl)-14-oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
, ]hexa-
deca-1,3(7),8-trien-10-yl]butanamide (9)
Similarly the DCC coupling reaction of 3 with bendamustine 8
gave 9 (65%); mp: 265–268 °C (decomposed); 1H NMR (DMSO-d6,
300 MHz): d 8.31 (d, J = 10.62 Hz, 1H), 8.24 (s, 1H), 7.34 (d,
J = 8.73 Hz, 1H), 6.91 (d, J = 1.77 Hz, 1H), 6.81 (s, 1H), 6.77 (d,

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5947
5.1.7. N-[(10S,11S,15R,16R)-16-(4-Hydroxy-3,5-dimethoxyphenyl)-
14-oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
]hexadeca-1,3(7),
8-trien-10-yl]-2-(piperazin-1-yl)acetamide (16)
1H), 7.13 (d, J = 7.26 Hz, 1H), 7.08 (d, J = 1.86 Hz, 1H), 6.85 (dd,
J = 1.44, 1.35 Hz, 1H), 6.68 (s, 1H), 6.56 (s, 1H), 6.31 (s, 2H), 6.01
(d, J = 2.91 Hz, 2H), 5.21 (m, 1H), 4.63 (d, J = 4.59 Hz, 1H), 4.43 (t,
J = 7.83, 8.52 Hz, 1H), 3.83–3.40 (m, 22H), 3.20–2.94 (m, 5H), 2.86
(d, J = 4.8, 3.6 Hz, 1H), 2.55–2.40 (m, 6H), 2.25 (m, 2H); 13C NMR
(CDCl3, 75 MHz): d 174.2, 170.6, 169.9, 153.8, 148.5, 147.7, 146.5,
143.6, 134.2, 132.6, 130.1, 128.8, 128.2, 111.4, 110.4, 110.3, 108.3,
107.9, 101.7, 68.9, 61.2, 56.4, 54.5, 53.5, 47.8, 45.0, 43.6, 41.9,
41.2, 40.7, 37.2, 31.6, 30.1, 26.0, 22.4; HRMS (ESI) C43H51Cl2N6O9
for m/z (M)+: calcd 865.3089, obsd 865.3090.
,
Compound 15 (0.1 g, 0.16 mmol) was dissolved in CH2Cl2 (1 mL)
to which TFA (3 mL) was added at room temperature. The reaction
was stirred overnight and was then quenched with a saturated solu-
tion of NaHCO3. The usual work up procedure and purification by
column chromatography gave the desired compound 16 (0.075 g,
90%); mp: 178–182 °C; 1H NMR (CDCl3, 300 MHz): d 8.28 (br s,
1H), 8.02 (d, J = 8.22 Hz, 1H), 6.76 (s, 1H), 6.54 (s, 1H), 6.24 (s, 2H),
6.0 (s, 2H), 5.18 (m, 1H), 4.48 (d, J = 4.98 Hz, 1H), 4.32 (t, J = 7.98,
7.74 Hz, 1H), 3.72 (t, J = 10.44, 8.79 Hz, 1H), 3.63 (m, 6H), 3.30 (m,
5H), 3.09–2.86 (m, 3H), 2.65 (m, 4H); 13C NMR (CDCl3, 75 MHz): d
174.3, 170.5, 148.4, 147.7, 146.6, 134.2, 132.5, 130.1, 128.9, 110.3,
108.4, 107.9, 101.6, 61.8, 56.4, 54.5, 47.7, 45.6, 43.6, 41.9, 37.2.
HRMS (ESI) C27H32N3O8 for m/z (M+H)+: calcd 526.2184, obsd
526.2165. The synthesis of 16, isolated as the dihydrochloride salt,
by a different method has been reported previously.47
5.2. Materials, cell culture and growth inhibition assays
Unless specified, reagents were obtained from Sigma–Aldrich
(Oakville, Canada). The assay conditions and the expression,
extraction and purification of recombinant full-length human
topoisomerase II
a
were described previously.48 Human leukemia
K562 cells, obtained from the American Type Culture Collection
and K/VP.5 cells (a 26-fold etoposide-resistant K562-derived
5.1.8. 2-[4-({4-[Bis(2-chloroethyl)amino]phenyl}methyl)pipera-
sub-line with decreased levels of topoisomerase II
topoisomerase IIb mRNA and protein)25,26 were maintained as
suspension cultures in MEM (Minimal Essential Medium Alpha)
a and
zin-1-yl]-N-[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dimethoxy-
phenyl)-14-oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
, ]hexa-
a
deca-1,3(7),8-trien-10-yl]acetamide (17)
(Invitrogen, Burlington, Canada) containing 10% fetal calf serum.
The spectrophotometric 96-well plate cell growth inhibition
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium) CellTiter 96 AQueous
One Solution Cell Proliferation assay (Promega, Madison, WI),
which measures the ability of the cells to enzymatically reduce
MTS after drug treatment, has been described.29,49 The drugs were
dissolved in DMSO and the final concentration of DMSO did not
exceed an amount (typically 0.4% or less) that had any detectable
effect on cell growth. The cells were incubated with the drugs for
72 h and then assayed with MTS. The IC50 values for cell growth
inhibition were measured by fitting the absorbance-drug concen-
tration data to a four-parameter logistic equation as described.28,29
Typically 9 different drug concentrations determined in duplicate
and measured on two different days were used to characterize
the growth inhibition curves. The catenated kDNA and the primary
Compound 16 (0.1 g, 0.19 mmol) and the benzaldehyde 4
(0.056 g, 0.23 mmol) were mixed in 1,2-dichloroethane (5 mL)
and then treated with sodium triacetoxyborohydride (0.081 g,
0.38 mmol). The mixture was stirred at room temperature under
a N2 atmosphere for 3 h. After completion of reaction, it was
quenched by adding aqueous saturated NaHCO3, and the product
was extracted with EtOAc. The EtOAc extract was dried (MgSO4)
and the solvent was evaporated to give the crude free base 17
which was purified by column chromatography using CH2Cl2/
methanol (95:5) as eluant (0.115 g, 80%); mp: 155–158 °C; 1H
NMR (CDCl3, 300 MHz): d 7.18 (d, J = 8.31 Hz, 2H), 6.70 (s, 1H),
6.66 (d, J = 8.52 Hz, 2H), 6.56 (s, 1H), 6.32 (s, 2H), 6.02 (d,
J = 2.25 Hz, 2H), 5.4 (br s, 1H), 5.21 (m, 1H), 4.67 (d, J = 5.7 Hz,
1H) 4.46 (t, J = 10.8, 8.88 Hz, 1H), 3.83–3.58 (m, 15H), 3.40 (m,
2H), 3.10 (d, J = 6.3 Hz, 2H), 2.95 (m, 1H), 2.85 (dd, J = 4.77,
3.33 Hz, 1H), 2.55 (m, 4H), 2.39 (m, 4H); 13C NMR (CDCl3,
75 MHz): d 174.3, 170.6, 148.4, 147.6, 146.5, 145.3, 134.2, 132.5,
130.6, 130.1, 129.1, 111.8, 110.2, 108.6, 107.9, 101.6, 69.0, 62.0,
61.3, 56.4, 53.6, 52.5, 47.5, 43.6, 42.0, 40.4, 37.2; HRMS (ESI)
anti-topoisomerase IIa and topoisomerase IIb antibodies were
from TopoGEN (Port Orange, FL) and Santa Cruz (Dallas, TX), respec-
tively, and the secondary horseradish peroxidase-conjugated
antibody was from Cell Signaling Technology (Danvers, MA).
C
38H45Cl2N4O8 for m/z (M)+: calcd 755.2609, obsd 755.2589.
5.3. Topoisomerase IIa kDNA decatenation, pBR322 DNA
relaxation and cleavage assays
5.1.9. 2-[4-(4-{4-[Bis(2-chloroethyl)amino]phenyl}butanoyl)pip
erazin-1-yl]-N-[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dime-
thoxyphenyl)-14-oxo-4,6,13-trioxatetracyclo[7.7.0.03,7.011 15
, ]-
A gel assay as previously described28,29 was used to determine if
the hybrids inhibited the catalytic decatenation activity of topoiso-
hexadeca-1,3(7),8-trien-10-yl]acetamide (18)
The DCC coupling of 16 with 6 yielded 18 (76%); mp: 159–
163 °C (decomposed); 1H NMR (CDCl3, 300 MHz): d 7.10 (d,
J = 8.4 Hz, 2H), 6.70 (s, 1H), 6.68 (d, J = 8.58 Hz, 2H), 6.56 (s, 1H),
6.31 (s, 2H), 6.00 (s, 2H), 5.44 (br s, 1H), 5.24 (m, 1H), 4.64 (d,
J = 4.8 Hz, 1H), 4.42 (m, 1H), 3.84–3.64 (m, 15H), 3.55–3.18 (m,
6H), 3.08–2.89 (m, 3H), 2.72–2.52 (m, 5H), 2.38 (m, 2H), 1.95 (m,
2H); 13C NMR (CDCl3, 75 MHz): d 174.3, 171.4, 148.4, 147.7,
146.5, 144.4, 134.2, 132.8, 130.6, 129.6, 128.7, 112.2, 110.3,
108.4, 107.9, 101.6, 68.8, 56.5, 53.6, 47.9, 43.6, 42.0, 40.5, 37.1,
34.1, 32.3, 26.7; HRMS (ESI) C41H49Cl2N4O9 for m/z (M)+: calcd
811.2871, obsd 811.2857.
merase II
a
. kDNA, which consists of highly catenated networks of
in an
circular DNA, is decatenated by topoisomerase II
a
ATP-dependent reaction to yield individual minicircles of DNA.
Topoisomerase II–cleaved DNA covalent complexes produced by
anticancer drugs may be trapped by rapidly denaturing the com-
plexed enzyme with sodium dodecyl sulfate (SDS).28,34 The drug-
induced cleavage of double-strand closed circular plasmid
pBR322 DNA to form linear DNA at 37 °C was followed by separat-
ing the SDS-treated reaction products by ethidium bromide gel
electrophoresis, essentially as described, except that all compo-
nents of the assay mixture were assembled and mixed on ice prior
to addition of the drug.28,29,34
5.1.10. 2-[4-(4-{5-[Bis(2-chloroethyl)amino]-1-methyl-1H-1,3-
benzodiazol-2-yl}butanoyl)piperazin-1-yl]-N-[(10S,11S,16R)-
16-(4-hydroxy-3,5-dimethoxyphenyl)-14-oxo-4,6,13-trioxatetra
5.4.
cH2AX assay for DNA double-strand breaks in drug-treated
cyclo[7.7.0.03,7.011 15
(19)
, ]hexadeca-1,3(7),8-trien-10-yl]acetamide
K562 cells
The c
H2AX assay was carried out essentially as described.29,37
The DCC coupling of 16 with 8 yielded 19 (52%); mp: 198–202 °C
(decomposed); 1H NMR (CDCl3, 300 MHz): d 7.25 (d, J = 8.91 Hz,
K562 cells in growth medium (1.5 mL in a 24-well plate, 4 ꢃ 105

5948
A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
cells/mL) were incubated with drug or with DMSO as a control for
4 h at 37 °C. Cell lysates (60 g protein) were subjected to SDS–
polyacrylamide gel electrophoresis on 14% gel. Separated
proteins were transferred to polyvinylidene fluoride (PVDF) mem-
branes and then treated overnight with rabbit anti- H2AX primary
antibody diluted 1:2000 (Upstate, Charlottesville, VA). This was
followed by incubation for 1 h with peroxidase-conjugated goat-
anti-rabbit secondary antibody (Cell Signaling Technology) diluted
1:2000. After incubation with luminol/enhancer/peroxide solution
Biosciences, Mississauga ON, Canada) and analyzed with FlowJo
software (Tree Star, Ashland OR) for the proportion of cells in
sub-G0/G1, G0/G1, S, and G2/M phases of the cell cycle.
l
a
The fraction of apoptotic cells induced by treatment of K562
cells with the hybrids and the etoposide positive control were
quantified by two-color flow cytometry by simultaneously mea-
suring integrated green (Annexin V-FITC) fluorescence, and inte-
grated red (propidium iodide) fluorescence as we previously
described.37 The Annexin V-FITC binding to phosphatidylserine
present on the outer cell membrane was determined using an
Apoptosis Detection Kit (BD Biosciences, Mississauga, Canada).
Briefly, K562 cells in suspension were untreated or treated with
the compounds indicated at 37 °C for 24 h. The cells were collected
by centrifugation at 1000g for 3 min and the pooled cells were
washed with the manufacturer-supplied binding buffer. Approxi-
c
(Bio-Rad, Mississauga, Canada), chemiluminescence of the
cH2AX
band was imaged on a Cell Biosciences (Santa Clara, CA) FluorChem
FC2 imaging system equipped with
camera.
a charge-coupled-device
5.5. Cellular assays for the detection of covalent DNA–
topoisomerase II
complexes
a
and DNA–topoisomerase IIb protein
mately 2.5 ꢃ 105 cells were resuspended in 500
l
L of manufac-
turer-supplied binding buffer, and mixed with 5
lL of Annexin
V-FITC and 5
lL of propidium iodide at a final concentration of
The cellular ICE (immunodetection of complexes of enzyme-to-
1 lg/mL. After 15 min of incubation in the dark, the cells were ana-
DNA), assays for topoisomerase II
a
or topoisomerase IIb covalently
lyzed using flow cytometry.
bound to DNA were carried out as described.28,29 The ICE assay
used for the detection of covalent complexes of topoisomerase
5.8. Molecular modeling and docking of the hybrids into an X-
ray structure of etoposide complexed to cleaved DNA and
topoisomerase IIb
IIa and topoisomerase IIb bound to DNA was a modification of
the original cesium chloride ultracentrifugation gradient assay
used to isolate DNA.50 The modification of this assay instead
employed the selective precipitation of genomic DNA using DNA-
zol (Invitrogen).
The molecular modeling and the docking were carried out as
described29 using the genetic algorithm docking program GOLD
version 3.2 (CCDC Software, Cambridge, UK) with default GOLD
parameters and atom types and with 50 starting runs as
described.29 The hybrids were docked into one of the etoposide
binding sites of the X-ray structure (www.rcsb.org/pdb; PDB ID:
3QX3)8 which contains two molecules of etoposide complexed to
cleaved DNA and topoisomerase IIb. The structure was prepared
by removing the etoposide molecules, the water molecules and
Mg2+ to avoid potential interference with the docking. The binding
site (4 Å) was defined using etoposide in the PDB ID: 3QX3 struc-
ture. Etoposide docked back into its protein–DNA structure with
a heavy atom rmsd distance of 0.48 Å, compared to the X-ray struc-
ture.8 Values of 2.0 Å or less in the extensive GOLD test set are con-
sidered to be good.51 The graphics were prepared with DS
Visualizer 2.5 (Accelrys, San Diego, CA).
5.6. DNA cross-link assay
The hybrids and the positive controls were tested for their abil-
ity to cross-link linearized pBR322 DNA as previously
described.36,37 In this assay double-stranded linear DNA was
heated which caused the strands to separate. Upon rapid cooling
the strands that were cross-linked by drug treatment recombined
to form double-stranded DNA, whereas those that were not
cross-linked, did not recombine. Briefly, the pBR322 DNA (1.2
was linearized at a single restriction site with HindIII restriction
enzyme (20 units, Invitrogen) in 20 L of buffer (50 mM Tris/
1 mM MgCl2/5 mM NaCl, pH 8.0) and purified by a Wizard SV Gel
and PCR Clean-up System (Promega). The 10 L reaction mixture,
which contained linear DNA (70 ng), was incubated with 10
lg)
l
l
lM
of the agents for 2 h at 37 °C. The bifunctional DNA cross-linking
agents chlorambucil and cisplatin were used as a positive controls.
The mixture was then heated at 90 °C for 5 min and was then
immediately placed in an ice-water bath for 5 min. The double-
stranded DNA was then separated by electrophoresis from single
stranded DNA on an ethidium bromide agarose gel (1.1%, w/v).
The DNA in the gel was imaged by its fluorescence on a Alpha Inno-
tech Fluorochem 8900 imaging system.
5.9. COMPARE and hierarchical clustering analysis of hybrid
compounds
An NCI COMPARE analysis of the NCI GI50 endpoint 60-cell line
data was carried out online (http://dtp.nci.nih.gov/compare) using
both public data and private data (Supplemental Figs. 1–6) for
specific hybrid compounds tested by the NCI (data in Supporting
information) to obtain the Pearson correlation coefficients r. As a
log(GI50) value was not determined for 19 percentage growth val-
5.7. Cell cycle synchronization, cell cycle analysis and annexin V
flow cytometry
ues from a single point (10 lM) NCI 60-cell result were used in the
analysis. The p values were calculated in SigmaPlot (Systat Soft-
ware, San Jose, CA). The hierarchical clustering analysis was carried
out online (http://www.wessa.net/) using Ward’s method.44
The cell cycle synchronization experiments were carried out as
previously described.28,37 For the synchronization experiments
CHO cells were grown to confluence in
a-MEM supplemented with
Acknowledgments
10% fetal calf serum. Following serum starvation with
a-MEM-0%
fetal calf serum for 48 h, the cells were seeded at 2 ꢃ l05 cells/mL
Supported by Grants from the Canadian Institutes of Health
Research, the Canada Research Chairs Program, and a Canada
Research Chair in Drug Development to Brian Hasinoff and a
United States NIH Grant CA090787 to Jack Yalowich. The authors
declare no competing financial interest. The funding source(s)
had no involvement in the study design; in the collection, analysis
and interpretation of data; in the writing of the report; and in the
decision to submit the article for publication.
and repleted with
a-MEM-10% fetal calf serum. Directly after
repletion they were continuously treated with 20
in 35-mm diameter dishes for different periods of time. Cells were
fixed in 75% ethanol and stained with a solution containing 20 g/
mL propidium iodide, 100 g/mL RNase A in 0.1% (v/v) Triton
X-100 at room temperature for 30 min. Flow cytometry was
carried out on a BD FACSCanto II flow cytometry system (BD
lM of the hybrid
l
l

A.A. Yadav et al. / Bioorg. Med. Chem. 22 (2014) 5935–5949
5949
25. Ritke, M. K.; Roberts, D.; Allan, W. P.; Raymond, J.; Bergoltz, V. V.; Yalowich, J. C.
Br. J. Cancer 1994, 69, 687.
Supplementary data
26. Ritke, M. K.; Yalowich, J. C. Biochem. Pharmacol. 1993, 46, 2007.
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28. Yalowich, J. C.; Wu, X.; Zhang, R.; Kanagasabai, R.; Hornbaker, M.; Hasinoff, B. B.
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29. Hasinoff, B. B.; Wu, X.; Nitiss, J. L.; Kanagasabai, R.; Yalowich, J. C. Biochem.
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5, 7, 9, 13, 17 and 19 and NMR data) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2014.09.014.
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