Structure-based design, synthesis and biological testing of piperazine-linked bis-epipodophyllotoxin etoposide analogs

Article abstract of DOI:10.1016/j.bmc.2015.04.022

Drugs that target DNA topoisomerase II, such as the epipodophyllotoxin etoposide, are a clinically important class of anticancer agents. A recently published X-ray structure of a ternary complex of etoposide, cleaved DNA and topoisomerase IIβ showed that

Full text of DOI:10.1016/j.bmc.2015.04.022

Accepted Manuscript
Structure-based design, synthesis and biological testing of piperazine-linked
bis-epipodophyllotoxin etoposide analogs
Arun A. Yadav, Gaik-Lean Chee, Xing Wu, Daywin Patel, Jack C. Yalowich,
Brian B. Hasinoff
PII:
S0968-0896(15)00320-X
http://dx.doi.org/10.1016/j.bmc.2015.04.022
BMC 12234
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
9 February 2015
1 April 2015
9 April 2015
Please cite this article as: Yadav, A.A., Chee, G-L., Wu, X., Patel, D., Yalowich, J.C., Hasinoff, B.B., Structure-
based design, synthesis and biological testing of piperazine-linked bis-epipodophyllotoxin etoposide analogs,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.022
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Structure-based design, synthesis and biological testing of piperazine-linked bis-
epipodophyllotoxin etoposide analogs
Arun A. Yadava, Gaik-Lean Cheea, Xing Wua, Daywin Patela, Jack C. Yalowichb and Brian B.
Hasinoffa,*
a College 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, Ohio
43210, USA
*Corresponding author. Tel: +1 204 474 8325; fax: +1 204 474 7617. E-mail address:
B_Hasinoff@UManitoba.ca (B. B. Hasinoff)
Keywords: epipodophyllotoxin; topoisomerase II; etoposide; structure-based design; anticancer;
DNA; COMPARE; K562 cells; molecular modeling; docking
.1.

ABSTRACT
Drugs that target DNA topoisomerase II, such as the epipodophyllotoxin etoposide, are a
clinically important class of anticancer agents. A recently published X-ray structure of a ternary
complex of etoposide, cleaved DNA and topoisomerase IIβ showed that the two intercalated
etoposide molecules in the complex were separated by four DNA base pairs. Thus, using a
structure-based design approach, a series of bis-epipodophyllotoxin etoposide analogs with
piperazine-containing linkers was designed to simultaneously bind to these two sites. It was
hypothesized that two-site binding would produce a more stable cleavage complex, and a more
potent anticancer drug. The most potent bis-epipodophyllotoxin, which was 10-fold more growth
inhibitory toward human erythroleukemic K562 cells than etoposide, contained a linker with
eight methylene groups. All of the mono- and bis-epipodophyllotoxins, in a variety of assays,
showed strong evidence that they targeted topoisomerase II. COMPARE analysis of NCI 60-cell
GI50 endpoint data was also consistent with these compounds targeting topoisomerase II.
.2.

1. Introduction1
Etoposide is a widely used anticancer drug and is a topoisomerase II poison/interfacial
inhibitor that exerts its cytotoxicity through its ability to stabilize a covalent topoisomerase II-
DNA cleavable complex intermediate which leads to double-strand DNA breaks that are toxic to
the cell.1-4 The X-ray structure of two molecules of etoposide complexed to a cleaved DNA-
topoisomerase IIβ ternary complex (PDB ID: 3QX3) has recently been determined.5-7 In this
structure two etoposide molecules that intercalate into the cleaved DNA are separated by four
base pairs. Unlike planar DNA intercalators that stack with two bases, the aglycone moiety of
etoposide stacks only with a single +5 guanine base in a deformed DNA structure.6 It has been
shown that etoposide is itself a weak DNA intercalator (dissociation constant 11 µM).8,9 The
glycosidic moieties of the two bound etoposide molecules occupy spacious binding pockets in
the protein. Consequently, compounds in which the glycosidic moiety of etoposide is modified
(e.g., teniposide), or even replaced, retain excellent activity.10-14 This provided us an opportunity
to substitute the glycosides with linkers of various sizes in order to optimize epipodophyllotoxin
two-site binding. We recently used the DNA-topoisomerase IIβ ternary complex X-ray structure
to carry out structure-based design of a series of potent epipodophyllotoxin-nitrogen-mustard
1Abbreviations: ACN, acetonitrile; Boc, di-tert-butyl dicarbonate; CHO, Chinese
hamster ovary; DCM, dichloromethane; DMF, dimelthylformamide; ESI, electrospray ionization;
Et3N, triethylamine; ICE, immunodetection of complexes of enzyme-to-DNA; kDNA, catenated
DNA; K562 cells, human erythroleukemic cells; HRMS, high resolution mass spectrometry;
MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
r.m.s., root-mean-square; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl
sulfate; TFA, trifluoroacetic acid; αMEM, Minimal Essential Medium Alpha.
.3.

hybrid compounds that were designed to covalently bind to topoisomerase II and DNA.14 The
nitrogen-mustard functionalities were based on the clinically efficacious nitrogen mustards
chlorambucil, bendamustine and melphalan. In previous studies we also described the synthesis
and activity of photoaffinity etoposide probes.12,13
We also synthesized and tested a series of DNA-binding bisanthrapyrazole compounds15-
17 that were designed to achieve bisintercalation separated by four DNA base pairs. These
compounds displayed excellent cell growth inhibition, and were also strong topoisomerase IIα
inhibitors. In principle, molecules that can interact with two binding sites on DNA, as opposed to
a single binding site, should have a much stronger DNA interaction and a slower rate of
dissociation. These effects are due to the bisintercalator occupying twice the number of DNA
intercalation sites on the DNA. This potential for stronger DNA binding is based on a simple
model in which the different intercalation sites contribute equally and independently to the free
energy.18 Thus, by analogy, an etoposide analog that contains two epipodophyllotoxin moieties,
separated by a linker of a length that allows bisintercalation into the two etoposide binding sites,
should, in principle, result in a more stable and longer-lived cleavage complex, and consequently
a more selective and cytotoxic etoposide analog.
Bis-epipodophyllotoxins with various sizes and types of linkers that link the two
epipodophyllotoxin moieties through attachment to the E-ring pendant phenolic groups have
been described and tested in vivo for antitumor activity.19 Epipodophyllotoxin-acridine hybrids
with six and eight methylene linkers were shown to target topoisomerase II, and to have good cell
growth inhibitory activity.20 The synthesis and testing of bis-epipodophyllotoxins with linkers of
various sizes and types that had triplex-forming oligonucleotides attached, in order to increase
.4.

their DNA sequence specificity, have been shown to induce topoisomerase IIα DNA
cleavage.21,22 Several tubulin-targeted bis-podophyllotoxin compounds with linkers of various
sizes and types displayed only moderate cell growth inhibitory activity, but were not tested for
their ability to target topoisomerase II.23
Guided by molecular modeling and docking into the two etoposide binding sites in the
DNA-topoisomerase IIβ ternary complex X-ray structure,5-7 we designed and synthesized a series
of mono- and bis-epipodophyllotoxin compounds by substituting the glycosidic moieties with
linkers of varying length in order to determine the minimum and the optimum linker chain length
to optimize topoisomerase II poisoning and cell growth inhibition. Two piperazines were
included in the linker, both to increase solubility, and to exploit the polyamine transport system,
as has been done with the polyamine-linked etoposide analog F14512.10 All of the mono- and
bis-epipodophyllotoxins, in a variety of assays, showed strong evidence of targeting
topoisomerase II. The most potent bis-epipodophyllotoxin of the series 7c (Scheme 1) had an
IC50 value towards K562 cells that was 10-fold more potent than etoposide. A second bis-
epipodophyllotoxin compound 7d tested in the NCI-60 cell screen (http://dtp.nci.nih.gov) had a
mean GI50 value over all cell lines that was 25-fold more potent than etoposide.
2. Chemistry
The piperazine-linked mono- and bis-epipodophyllotoxins were synthesized as shown in
Scheme 1. The Ritter reaction of chloroacetonitrile with 4'-O-demethylepipodophyllotoxin
(AvaChem, San Antonio, TX) was carried out24 to make the 4-chloroacetamido-4-deoxy-4'-
.5.

demethylepipodophyllotoxin (5) in excellent yield as we and others have reported.14,25 The bis-
piperazine intermediates 4a - 4e were prepared in 80 - 90% yields via the reaction of 1-Boc-
piperazine 1 with dibromoalkanes 2a - 2e followed by deprotection of the t-Boc group under
acidic conditions as we and others have reported.14,15,26 Bispiperazines 4a - 4e were then reacted
with 5 (1 or 2 equivalents) in the presence of triethylamine to afford mono- (6a, 6c, 6d) and bis-
epipodophyllotoxins (7a - 7e) in moderate to good yields. The reaction of an equimolar amount
of 5 and 4 gave both mono- and bis-epipodophyllotoxins, but when two equivalents of 5 and one
equivalent of 4 were reacted together, the reaction yielded essentially bis-epipodophyllotoxins.
3. Results and Discussion
3.1 Cell growth inhibitory effects of mono- and bis-epipodophyllotoxins on human
leukemia K562 cells and K/VP.5 cells with a decreased level of topoisomerase IIα
The cell growth inhibitory effects of the mono- and bis-epipodophyllotoxins on human
leukemia K562 cells and etoposide-resistant K/VP.5 cells27,28 are compared to etoposide in Table
1. All the bis-epipodophyllotoxins displayed sub-micromolar IC50 values, with the most potent 7c
being 10-fold more potent than etoposide. The IC50 values systematically decreased with an
increase in methylene linker length and reached a minimum with compound 7c which contained
eight methylenes. The IC50 values then coordinately increased when the linker length was
increased to ten and twelve methylenes (compounds 7d and 7e, respectively). These results
suggested that an optimum chain length was in the order of eight methylene groups, and thus may
.6.

be the optimum chain length needed for the bis-epipodophyllotoxins to achieve bisintercalation
in the DNA within the topoisomerase IIβ-DNA ternary structure. Increasing linker length also
enhanced the cell growth inhibitory potency of the mono-epipodophyllotoxins that contained two
piperazine moieties separated by either three, eight or ten methylene linkers (6a, 6c and 6d,
respectively). The most potent 6d was 6-fold more potent than etoposide. The fact that the mono
compounds were so active may be because they are polyamines that were taken up by the
polyamine transport system as has been proposed for the polyamine-linked etoposide analog
F14512.10
Cancer cells can acquire resistance to topoisomerase II poisons by lowering their level
and/or activity of topoisomerase II.29 We have previously used14,30 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. The etoposide-selected K/VP.5 cells were previously determined to be
26-fold resistant to etoposide, and to contain reduced levels of both topoisomerase IIα (6-fold)
and topoisomerase IIß (3-fold).27,28 In addition, these cells are cross-resistant to other known
topoisomerase II poisons, but are not cross-resistant to camptothecin and other non-
topoisomerase IIα targeted drugs.28 Reduced topoisomerase II in the cell translates to fewer DNA
strand breaks produced by topoisomerase II poisons and less cytotoxicity. As shown in Table 1,
etoposide and compounds 6a, 7a and 7b were similar in their cross-resistance to K/VP.5 cells
(10.5- to 21.5-fold cross-resistant). The other mono- and bis-epipodophyllotoxins were generally
less cross-resistant (2.6- to 8.2-fold). The universal cross-resistant pattern for these newly
synthesized compounds strongly suggests targeting of topoisomerase II.
.7.

3.2. Evaluation of cell growth inhibitory effects of the compounds in the NCI-60 human
tumor cell line screen
Compounds 7a, 7b and 7d were accepted for testing in the NCI-60 cell screen by the
National Cancer Institute (http://dtp.nci.nih.gov). These results allowed us to identify which
tumor cell types were the most sensitive to these compounds and to carry out NCI COMPARE
analyses (http://dtp.nci.nih.gov/compare) to determine what other anticancer and cytotoxic drugs
the bis-epipodophyllotoxins had similar tumor cell growth inhibition profiles, and thus to identify
mechanisms by which the bis-epipodophyllotoxins exerted their activity. The NCI-60 cell 5-dose
testing mean GI50 values for compounds 7b and 7d are given in Table 1 and the results for the
individual cell lines are given in Supplemental Figures S1 and S2. The 1-dose (10 µM) testing
results for 7a are given in Supplemental Figure S3. The leukemia cell lines were, as a group, the
most sensitive to the two compounds tested, with GI50 values in the sub-micromolar range.
Compounds 7a, 7b and 7d were the least growth inhibitory toward melanoma and ovarian
cancers, with GI50 values in the low micromolar range. Compounds 7d and 7b that were
subjected to 5-dose testing had mean GI50 values over all cell lines tested of 0.47 µM and 3.7
µM, respectively (Table 1). The mean GI50 value for etoposide over all cell lines tested was 12
µM (Table 1), Hence, compound 7d was 25-fold more potent than etoposide.
3.3 The mono- and bis-epipodophyllotoxins inhibited topoisomerase IIα decatenation
activity, acted as topoisomerase IIα poisons and induced DNA double-strand breaks in cells
.8.

The torsional stress that occurs in DNA during replication and transcription and daughter
strand separation during mitosis can be relieved 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.1-4 As shown in Figure 1 all of the compounds
shown, including etoposide, required concentrations above 10 µM to inhibit the decatenation of
kDNA by topoisomerase IIα. It should be noted that compounds 7c, 7d, and 7e showed some
residual open circular DNA at 100 µM compared to that of etoposide, suggesting that these
compounds were slightly less active than etoposide in the decatenation assay. Compounds 6a and
7a also showed comparable decatenation activity to that of etoposide (data not shown). The
decatenation assay is a measure of the ability of compounds to inhibit the catalytic activity only,
and is not a measure of whether they acted as topoisomerase II poisons.
Stabilization of the covalent complex by a topoisomerase II-targeted drug can lead to
double-strand DNA breaks that are toxic to the cell. Thus, DNA cleavage assay
experiments14,30,31 were carried out to see whether the compounds stabilized the cleavable
complex. As shown in Figure 2 the addition of the etoposide positive control to the reaction
mixture containing topoisomerase IIα and supercoiled pBR322 DNA induced a concentration-
dependent formation of linear pBR322 DNA. Linear DNA was identified by comparison with
linear pBR322 DNA produced by action of the restriction enzyme HindIII acting on a single site
on pBR322 DNA (not shown). As shown in Figure 2, all of the mono- and bis-
epipodophyllotoxins that were tested also induced a concentration-dependent formation of linear
DNA, which indicated that they acted as topoisomerase IIα poisons. Based on the amount of
linear DNA produced, all of the compounds, except mono-epipodophyllotoxin 6a, were as potent
.9.

or more potent than etoposide. The three most cell growth inhibitory bis-epipodophyllotoxin
compounds 7b, 7c and 7d induced topoisomerase II-mediated DNA cleavage to linear DNA at a
concentration as low as 0.1 µM (Figure 2). By comparison, etoposide at this concentration
induced formation of linear DNA in amounts comparable to that observed for the control
conditions (+Topo IIα alone).
H2AX is a variant of an H2A core histone which becomes phosphorylated to produce
γH2AX in the vicinity of double-strand DNA breaks upon treatment of cells with drugs or
ionizing radiation.32 This phosphorylation becomes amplified over megabases of DNA
surrounding the break, thus acting as a scaffold for the recruitment of key signaling and repair
proteins, and, thus, is an accepted marker of double-strand breaks.32 Thus, in order to determine
if the mono- and bis-epipodophyllotoxins could induce double-strand breaks in intact K562 cells,
the level of γH2AX was determined by Western blotting with etoposide as the positive control.31
Experiments carried out as we previously described,30,33 and shown in Figure 3, indicate that
etoposide and all of the mono- and bis-epipodophyllotoxin compounds increased the levels of
γH2AX in K562 cells. Compounds 7d, 6d, 7c, 6c and 7b induced even greater levels of γH2AX
compared to etoposide. Compound 6a was less potent in the γH2AX assay and also in its ability
to inhibit cell growth (Table 1). These results suggest that the compounds acted as topoisomerase
II poisons in a cellular context to produce DNA double-strand breaks.
3.4 Cell cycle analysis and two-color flow cytometry and caspase 3/7 induction as markers
of apoptosis
.10.

Cell cycle analysis was carried out on synchronized CHO cells as we previously
described14,33 on the compounds indicated (Figure 4) in order to determine the mechanism by
which these compounds might be acting. Drugs that act on topoisomerase IIα typically cause a
block in G2/M.34 CHO cells (normal doubling time of 12 h) that were synchronized to G0/G1
through serum starvation were treated with 1 µM of the compounds indicated, or 10 µM
etoposide for comparison. The cells were treated with a 10-fold lower concentration of the mono-
and bis-epipodophyllotoxins (1 µM) than for etoposide (10 µM) to compensate for the higher
potency of these compounds compared to etoposide (Table 1). CHO cells were chosen for these
experiments as they are easily and effectively synchronized by serum starvation. Subsequent to
this starvation, serum repletion resulted in the DMSO vehicle-treated control cells advancing to
G2/M by 14 h (Figures 4A and 4B). The control cells then went through several complete cell
cycles as evidenced by the 12 h periodicity for peaks in the various cell cycle stages. By 49 h
both 7d and 7c displayed a significant percentage of sub-G0/G1 cells comparable to that observed
for etoposide, which was indicative of apoptosis, whereas mono-epipodophyllotoxin 6d had a
sub-G0/G1 level about one-third that of etoposide. The percentage of sub-G0/G1 cells that was
characteristic of induction of apoptosis was small in control-treated cells (< 2%). Treatment with
etoposide caused a large block in G2/M by 21 h, which subsequently decreased at later times as
the sub-G0/G1 population, indicative of late-stage apoptosis, increased to about 39% at 49 h. Both
7d and 7c displayed effects with a similar time-course compared to etoposide; a prolonged G2/M
block followed by increased sub-G0/G1 levels. Compound 6d exhibited a more rapid escape from
the G2/M block compared to 7c and 7d. The large G2/M blocks observed were typical of agents
such as etoposide that target topoisomerase II.34 Hence, these results again are consistent with the
.11.

compounds tested acting as topoisomerase II poisons.
Early in the apoptotic process translocation of the phospholipid phosphatidylserine occurs
from the inner to the external plasma membrane of cells.35 Fluorescent annexin V-FITC, which
strongly binds phosphatidylserine on cells in the early stage of apoptosis, was used to identify
apoptotic and apoptotic/necrotic cells by two-color flow cytometry, as we previously
described.14,33 Annexin V-FITC stained K562 cells in both the lower and upper right quadrants of
Figure 5A were considered to be apoptotic. When used in combination with propidium iodide,
which binds DNA, a measure of cells that are necrotic may be obtained since necrotic cell
membranes 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
5B and 5C treatment of K562 cells for 24 h and 48 h with 1 µM of compounds 6d, 7c, 7d, and 10
µM etoposide all progressively reduced the proportion of viable cells, and increased the
proportion of both apoptotic and apoptotic/necrotic cells. All the compounds tested were
similarly, or more effective, than etoposide in inducing apoptosis, even though the compounds
were tested at a 10-fold lower concentration.
The cell cycle results (Figure 4) and the annexin V results (Figure 5) both indicated that
mono- and bis-epipodophyllotoxins were able to induce apoptosis. Further confirmation of
apoptosis induction was established by observation of caspase 3/7 activity, as we previously
described,36 in the presence of 1 ΦM 6d, 7c and 7d (Figure 6). Treatment with these compounds
resulted in a time dependent increase in caspase 3/7 activity compared to vehicle-treated controls,
which was highly statistically significant (unpaired t-test) for all these agents (p < 0.001). These
results are consistent with both the cell cycle results (Figure 4) and the annexin V results of
.12.

Figure 5.
3.5 Docking of bis-epipodophyllotoxins into the two etoposide binding sites of the
etoposide-DNA-topoisomerase IIβ ternary complex
It has been shown in the topoisomerase IIβ-DNA ternary X-ray structure, in which two
molecules of etoposide are complexed to cleaved DNA and topoisomerase IIβ, that the glycosidic
moiety of etoposide faces the DNA major groove, and occupies a spacious binding pocket in the
protein with few protein interactions. The X-ray structure of the etoposide-topoisomerase IIβ-
DNA complex was used in the docking studies because the corresponding topoisomerase IIα
structure has not yet been published. There is, however, a high degree of homology (~78%) in the
catalytic domains of topoisomerase IIα and topoisomerase IIβ.37 Thus, docking into the ternary
topoisomerase IIβ structure5-7 should be a good proxy for the ternary topoisomerase IIα structure.
An examination of the topoisomerase IIβ ternary complex5-7 showed that the pendant E-
ring phenolic group of etoposide interacted significantly with the protein, and thus would not be
a suitable attachment point for constructing a bis-epipodophyllotoxin. In fact, bis-
epipodophyllotoxin compounds that linked two epipodophyllotoxin moieties through attachment
to the pendant phenolic hydroxyl groups exhibited poor antitumor activity.19 However, the
presence of the two spacious glycosidic binding pockets in the X-ray structure suggested that two
epipodophyllotoxins could be linked together through a suitable linker that traversed the DNA
major groove at the glycosidic attachment end of the epipodophyllotoxin. Thus, docking of
proposed bis-epipodophyllotoxins was carried out into the X-ray structure. The results of the
.13.

docking of 7d are shown in Figure 7. The X-ray-determined pose of the two etoposide molecules
are shown in a yellow ball-and-stick representation in Figure 7B. In the highest scoring pose
(shown in stick representation in atom colors), the piperazine linker is fully extended into the
protein pocket normally occupied by the two glycosidic moieties, and the methylene linker
occupies the major groove. The polycyclic and pendant phenolic moieties of this pose show a
high degree of overlap with that of the X-ray structures of the bound etoposide molecules. For
bis-epipodophyllotoxins with six or fewer methylene linkers (Scheme 1), no reasonable docking
poses were found in which both epipodophyllotoxin moieties were simultaneously docked into
both etoposide binding sites. These results suggested that the linkers in these bis-
epipodophyllotoxins were not long enough to enable simultaneous docking into the two
etoposide binding sites. It is, however, notable that 7b, which has a linker number of six, was
more potent than etoposide; a result suggesting that this compound may form a more flexible
complex in a cellular context, compared to that indicated in the fixed X-ray structure.
3.6 COMPARE analysis of NCI-60 cell data
With 7b and 7d as seeds, both public and our private data (Supplementary Figures S1 -
S3) were used in an NCI COMPARE analysis (http://dtp.nci.nih.gov/compare) of the NCI-60 cell
GI50 endpoint data. We previously used COMPARE analysis of the NCI-60 cell data to identify
compounds that act through common mechanisms.14,33 COMPARE analysis 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 correlation coefficients r. Thus, compounds 7b and
.14.

7d were submitted for COMPARE analysis using the NCI Marketed Drugs GI50 target set. For
compound 7b, the five most highly correlated compounds were all well known topoisomerase II
targeted drugs, and included, in order, daunorubicin, doxorubicin, the epipodophyllotoxin
derivative teniposide and epirubicin, with r values ranging from 0.76 to 0.67, respectively. For
compound 7d the most highly correlated compounds in this target set were also all well known
topoisomerase II targeted drugs and included, in order, valrubicin, daunorubicin, teniposide,
mitoxantrone and doxorubicin with r values ranging from 0.81 to 0.73, respectively. When
etoposide itself was used as a seed, compounds 7b and 7d gave COMPARE correlation r values
of 0.62 and 0.68, respectively. A COMPARE cross-correlation r value of 0.76 was found
between 7b and 7d, which is expected since they are close analogs of one other. The 1-dose (at
10 µM) percent growth inhibition data for 7a was also highly correlated with 7b and 7d with r
values of 0.80 and 0.70, respectively, which indicated that these compounds all shared a common
target.
4. Conclusions
Five bis-epipodophyllotoxin compounds were designed using molecular modeling and
docking into an X-ray structure of the topoisomerase IIβ-DNA-etoposide ternary complex.5-7
Results are also reported for three structurally similar bis-piperazine mono-epipodophyllotoxin
analogs with varying methylene linker domains. The bis-epipodophyllotoxins that were designed
through docking into the X-ray structure, and tested for their ability to inhibit cell growth and
target topoisomerase II, contained two piperazine moieties separated by differing numbers of
.15.

methylene groups. This was done in order to determine both the minimum and the optimum
linker chain length in order to optimize cell growth inhibition and topoisomerase II poisoning.
The most potent compound 7c, which contained a linker with eight methylene groups, had an
IC50 toward K562 cells 10-fold lower than etoposide.
The mono- and bis-epipodophyllotoxin compounds showed strong evidence that they
targeted topoisomerase II. This was evidenced not only by their strong activities in the DNA
cleavage assay (Figure 2), but also by the fact that they were cross-resistant toward etoposide-
resistant K/VP.5 cells containing reduced levels of topoisomerase II (Table 1). Moreover, the
ability of these compounds to induce DNA double strand breaks in the cellular γH2AX assays
(Figure 3) and induce G2/M blocks in the cell cycle analyses were also characteristics of
topoisomerase II-targeted drugs (Figure 4). The mono- and bis-epipodophyllotoxins tested were
either as effective, or more effective, than etoposide in inducing apoptosis in cells (Figure 5).
NCI COMPARE analyses of NCI 60-cell GI50 endpoint data showed that the bis-
epipodophyllotoxin compounds correlated most highly with other well known topoisomerase II
poisons. These and our other results confirmed that the mono- and bis-epipodophyllotoxins
inhibited tumor cell growth by targeting topoisomerase II.
There is no direct experimental evidence that the bis-epipodophyllotoxins simultaneously
bound to both etoposide binding sites in the topoisomerase II ternary complex. The docking
results suggested that only bis-epipodophyllotoxins with eight or more methylene linkers (7c, 7d
and 7e) are capable of achieving a bisintercalation binding mode. Continuing studies will focus
on characterizing the relative selectivity of these compounds toward topoisomerase IIα and
topoisomerase IIβ, and their cellular pharmacokinetic and pharmacodynamic properties.
.16.

5. Experimental
5.1 Chemistry
1H and 13C nuclear magnetic resonance (NMR) spectra were recorded at 300 K in 5 mm
NMR tubes on a Bruker Avance 300 or 500 MHz NMR spectrometer, as indicated, operating at
300.13 MHz for 1H NMR and 75.5 MHz (or 125 MHz as indicated) for 13C NMR, respectively,
in CDCl3. Chemical shifts are given in parts per million (ppm) relative to tetramethylsilane in the
case of the 1H NMR spectra, and to the central line of CDCl3 (δ 77.0) for the 13C NMR spectra.
Melting points were taken on a Gallenkamp (Loughborough, England) melting point apparatus
and were uncorrected. The high resolution mass spectra (HRMS) were run on Bruker microTOF
Focus mass spectrometer (Fremont, CA) using electron spray ionization (ESI). TLC was
performed on plastic-backed plates bearing 200 µm silica gel 60 F254 (Silicycle, Quebec City,
Canada). Compounds were visualized by quenching of fluorescence by UV light (254 nm) where
applicable. The reaction conditions were not optimized for reaction yields. Unless otherwise
indicated, all chemicals were from Aldrich (Oakville, Canada) and were used as received.
Epipodophyllotoxin 5 (1 or 2 equivalents) was reacted overnight with the corresponding
bispiperazines 4a - 4e (1 equivalent) in dimethylformamide in the presence of triethylamine (3
equivalents) at room temperature. After completion of reaction as determined by TLC, the
mixture was quenched with water and extracted into dichloromethane. The combined extract was
dried over Na2SO4 and concentrated under vacuum to give the crude product. Final purification
.17.

was done by column chromatography (silica gel) using dichloromethane/methanol/30%
ammonium hydroxide (85:10:5 v/v/v) as an eluant. All products were colorless solids except for
6a which was an oil.
5.1.1 N-[(10S,11S,15R,16R)-16-(4-hydroxy-3,5-dimethoxyphenyl)-14-oxo-4,6,13-
trioxatetracyclo[7.7.0.03,7.011,15]-1,3(7),8-trien-10-yl]-2-{4-[3-(piperazin-1-
yl)propyl]piperazin-1-yl}acetamide (6a)
1H NMR (CDCl3, 300 MHz): δ 7.26 (d, J = 7.6 Hz, 1H), 6.71 (s, 1H), 6.57 (s, 1H), 6.32
(s, 2H), 6.01 (d, J = 3.8 Hz, 2H), 5.25 (m, 1H), 4.64 (d, J = 4.7 Hz,1H), 4.46 (t, J = 7.9, 0.6 Hz ,
1H), 3.86 (t, J = 7.9,0.7 Hz, 1H), 3.80 (s, 6H), 3.10 (d, J = 9.0 Hz, 2H), 3.02 (m, 1H), 2.64-2.28
(m, 10H), 1.66 (m, 2H); 13C NMR (CDCl3, 75 MHz): δ 174.2, 170.1, 147.9, 147.2, 146.8, 134.4,
132.2, 129.8, 129.1, 109.7, 108.4, 108.0, 101.2, 68.6, 61.1, 56.1, 55.8, 53.1, 52.5, 50.0, 47.2,
43.5, 43.3, 41.4, 36.9, 23.7; HRMS (ESI) for C34H46N5O8 m/z (M+H)+: calcd 652.3341, obsd
652.3329; viscous oil; yield: 28%.
5.1.2 2-(4-{3-[4-({[(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(9),2,7-trien-10-yl]carbamoyl}methyl)piperazin-
1-yl]propyl}piperazin-1-yl)-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]acetamide (7a)
1H NMR (CDCl3, 300 MHz): δ 7.25 (d, J = 7.5 Hz, 1H), 6.70 (s, 1H), 6.57 (s, 1H), 6.32
(s, 2H), 6.01 (d, J = 3.3 Hz, 2H), 5.47 (bs, 1H), 5.25 (m, 1H), 4.64 (d, J = 4.7 Hz, 1H), 4.46 (t, J
= 7.8, 8.7 Hz , 1H), 3.87 (t, J = 8.6 Hz, 1H), 3.80 (s, 6H), 3.10 (d, J = 9.0 Hz, 2H), 3.02 (m, 1H),
.18.

2.84 (dd, J = 4.8, Hz, 1H), 2.64-2.28 (m, 10H), 1.66 (m, 2H); 13C NMR (CDCl3, 75 MHz): δ
174.3, 170.5, 148.3, 147.6, 146.6, 134.2, 132.5, 130.1, 129.0, 110.2, 108.5, 107.9, 101.6, 69.0,
61.3, 56.4, 53.6, 47.6, 43.6, 41.9, 37.2, 24.3; HRMS (ESI) for C57H67N6O16 m/z (M)+: calcd
1091.4608, obsd 1091.4626; mp: 248 - 252 °C; yield: 42%.
5.1.3 2-(4-{6-[4-({[(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(9),2,7-trien-10-yl]carbamoyl}methyl)piperazin-
1-yl]hexyl}piperazin-1-yl)-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]acetamide (7b)
1H NMR (CDCl3, 300 MHz): δ 8.25 (bs, 1H), 8.05 (d, J = 8.2 Hz, 1H), 6.76 (s, 1H), 6.54
(s, 1H), 6.24 (s, 2H), 5.99 (s, 2H), 5.20 (m, 1H), 4.50 (d, J = 5.1 Hz, 1H), 4.31 (t, J = 7.9 Hz,
1H), 3.72- 3.63 (m, 8H), 3.08-2.87 (m, 4H), 2.45-2.28 (m, 10H), 1.40-1.20 (m, 4H); 13C NMR
(DMSO-d6, 125 MHz): δ 174.5, 169.3, 147.1, 146.5, 134.6, 132.3, 130.2, 130.1, 109.4, 108.7,
108.4, 101.1, 68.3, 61.0, 57.6, 55.9, 53.6, 52.4, 46.4, 42.8, 40.7, 36.5, 26.8; HRMS (ESI) for
C60H73N6O16 m/z (M+H)+: calcd 1133.5078, obsd 1133.5076; mp: 210 - 214 °C; yield: 62%.
5.1.4 N-[(10S,11S,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-{4-[8-(piperazin-1-
yl)octyl]piperazin-1-yl}acetamide (6c)
1H NMR (CDCl3, 300 MHz): δ 7.28 (s, 2H), 6.75 (s, 1H), 6.59 (s, 1H), 6.32 (s, 2H), 6.01
(d, J = 1.8 Hz, 2H), 5.26 (m, 1H), 4.64 (d, J = 4.8 Hz, 1H), 4.43 (t, J = 7.8, 8.2 Hz ,1H),
3.89-3.75 (m, 7H), 3.12-2.63 (m, 8H), 2.58-2.14 (m, 16H), 1.44-1.28 (m, 12H); 13C NMR
.19.

(CDCl3, 75 MHz): δ 174.3, 170.6, 148.3, 147.6, 146.7, 134.4, 132.5, 130.0, 129.1, 110.2, 108.5,
107.9, 101.6, 69.0, 61.3, 59.4, 58.5, 56.4, 54.4, 53.7, 52.9, 47.5, 45.9, 43.6, 42.0, 37.2, 29.4, 27.5,
26.8; HRMS (ESI) for C39H56N5O8 m/z (M+H)+: calcd 722.4123, obsd 722.4127; mp: 102 - 105
°C; yield: 32%.
5.1.5 2-(4-{8-[4-({[(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(9),2,7-trien-10-yl]carbamoyl}methyl)piperazin-
1-yl]octyl}piperazin-1-yl)-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]acetamide (7c)
1H NMR (CDCl3, 300 MHz): δ 7.30 (s, 1H), 6.71 (s, 1H), 6.57 (s, 1H), 6.32 (s, 2H), 6.01
(d, J = 1.71 Hz, 2H), 5.49 (bs, 1H), 5.24 (m, 1H), 4.64 (d, J = 4.7 Hz, 1H), 4.46 (t, J = 7.4, 6.8
Hz , 1H), 3.87-3.75 (m, 7H), 3.18-2.95 (m, 3H), 2.87-2.77 (dd, J = 4.6 Hz, 1H) 2.65-2.25 (m,
10H), 1.79-1.30 (m, 6H); 13C NMR (CDCl3, 75 MHz): δ 174.3, 170.5, 148.3, 147.6, 146.5,
134.2, 132.5, 130.1, 129.1, 110.2, 108.6, 107.9, 101.6, 69.0, 61.3, 58.4, 56.4, 53.6, 52.9, 47.5,
43.6, 42.0, 37.2, 29.4, 27.4, 26.8; HRMS (ESI) for C62H77N6O16 m/z (M+H)+: calcd 1161.5391,
obsd 1161.5407; mp: 152-155 °C; yield: 41%.
5.1.6 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-{4-[10-(piperazin-1-
yl)decyl]piperazin-1-yl}acetamide (6d)
1H NMR (CDCl3, 300 MHz): δ 7.31 (s, 2H), 6.73 (s, 1H), 6.59 (s, 1H), 6.29 (s, 2H), 6.04
(d, J = 1.8 Hz, 2H), 5.26 (m, 1H), 4.64 (d, J = 4.6 Hz, 1H), 4.46 (t, J = 7.6, 8.7 Hz ,1H),
.20.

3.87-3.80 (m, 7H), 3.15-2.90 (m, 7H), 2.84-2.79 (m, 1H), 2.58-2.25 (m, 15H), 1.44-1.28 (m,
16H); 13C NMR (CDCl3, 75 MHz): δ 174.3, 170.6, 148.3, 147.6, 146.6, 134.3, 132.5, 130.0,
129.1, 110.2, 108.5, 107.9, 101.6, 69.0, 61.3, 59.3, 58.5, 56.4, 54.0, 53.7, 52.9, 47.5, 45.7, 43.6,
42.0, 37.2, 29.5, 27.5, 26.9, 26.6; HRMS (ESI) for C41H60N5O8 m/z (M+H)+: calcd 750.4436,
obsd 750.4465; mp: 135 - 140°C; yield: 36%.
5.1.7 2-(4-{10-[4-({[(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(9),2,7-trien-10-yl]carbamoyl}methyl)piperazin-
1-yl]decyl}piperazin-1-yl)-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]acetamide (7d)
1H NMR (CDCl3, 300 MHz): δ 7.25 (s, 1H), 6.71 (s, 1H), 6.57 (s, 1H), 6.32 (s, 2H), 6.01
(d, J = 1.29 Hz, 2H), 5.49 (bs, 1H), 5.26 (m, 1H), 4.64 (d, J = 4.8 Hz, 1H), 4.46 (t, J = 7.8, 8.7
Hz , 1H), 3.87-3.80 (m, 7H), 3.46-3.51 (m, 1H), 3.10 (d, J = 8.04 Hz, 2H), 2.94-3.02 (m, 1H),
2.85 (dd, J = 4.8, Hz, 1H) 2.64-2.25 (m, 10H), 1.64-1.20 (m, 8H); 13C NMR (CDCl3, 75 MHz): δ
174.3, 170.5, 148.3, 147.6, 146.5, 134.2, 132.5, 130.1, 129.1, 110.2, 108.6, 107.9, 101.6, 69.0,
61.3, 58.5, 56.4, 53.6, 52.9, 47.5, 43.6, 42.0, 37.2, 29.5, 27.5, 26.8; HRMS (ESI) for
C64H81N6O16 m/z (M)+: calcd 1189.5704, obsd 1189.5686; mp: 178 - 182 °C; yield: 54%.
5.1.8 2-(4-{12-[4-({[(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(9),2,7-trien-10-
yl]carbamoyl}methyl)piperazin-1-yl]dodecyl}piperazin-1-yl)-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-
.21.

1,3(7),8-trien-10-yl]acetamide (7e)
1H NMR (CDCl3, 300 MHz): δ 7.32 (s, 1H), 6.71 (s, 1H), 6.57 (s, 1H), 6.32 (s, 2H), 6.01
(s, 2H), 5.40 (bs, 1H), 5.22 (m, 1H), 4.64 (d, J = 4.8 Hz, 1H), 4.46 (t, J = 7.8, 8.7 Hz , 1H),
3.87-3.80 (m, 7H), 3.16-2.93 (m, 3H), 2.85-2.79 (dd, J = 4.8 Hz, 1H), 2.65-2.24 (m, 10H),
1.52-1.20 (m, 10H); 13C NMR (CDCl3, 75 MHz): δ 174.3, 170.5, 148.3, 147.6, 146.5, 134.2,
132.5, 130.1, 129.1, 110.2, 108.6, 107.9, 101.6, 69.0, 61.3, 58.5, 56.4, 53.6, 52.8, 47.5, 43.6,
41.9, 37.2, 29.5, 27.5, 26.8; HRMS (ESI) for C66H85N6O16 m/z (M+H)+: calcd 1217.6017, obsd
1217.6039; mp: 142 - 145°C; yield: 68%.
5.2 Materials, cell culture and growth inhibition assays
Unless specified, reagents were obtained from Sigma-Aldrich (Oakville, Canada). The
extraction and purification of recombinant full-length human topoisomerase IIα were described
previously.38 Human leukemia K562 cells, obtained from the American Type Culture Collection,
and K/VP.5 cells (a 26-fold etoposide-resistant K562-derived sub-line with decreased levels of
topoisomerase IIα mRNA and protein)29 were maintained as suspension cultures in αMEM
(Invitrogen, Burlington, Canada) containing 10% fetal calf serum. The spectrophotometric 96-
well plate 72 h cell growth inhibition MTS assay (Promega, Madison, WI) has been described
previously.14,33 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 IC50
values for cell growth inhibition were measured by fitting the absorbance-drug concentration data
to a four-parameter logistic equation as described.14,33 Typically nine different drug
.22.

concentrations, determined in duplicate and measured on two different days, were used to
characterize the growth inhibition curves. The catenated kDNA and the primary anti-
topoisomerase IIα antibody were from TopoGEN (Port Orange, FL) and Santa Cruz (Dallas, TX),
respectively, and the secondary horseradish peroxidase-conjugated antibody was from Cell
Signaling Technology (Danvers, MA).
5.3 Topoisomerase IIα kDNA decatenation, pBR322 DNA relaxation and cleavage assays
A gel assay as previously described14,33 was used to determine if the compounds inhibited
the catalytic decatenation activity of topoisomerase IIα. kDNA, which consists of highly
catenated networks of circular DNA, is decatenated by topoisomerase IIα in an 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 complexed
enzyme with SDS.14,31,33 The drug-induced cleavage of double-strand closed circular plasmid
pBR322 DNA to form linear DNA at 37 °C was followed by separating the SDS-treated reaction
products by ethidium bromide gel electrophoresis, essentially as described, except that all
components of the assay mixture were assembled and mixed on ice prior to addition of the
drug.14,31,33
5.4 γH2AX assay for DNA double-strand breaks in drug-treated K562 cells
The γH2AX assay was carried essentially out as described.14,33 K562 cells in growth
.23.

medium (1.5 mL in a 24-well plate, 3.4 x 105 cells/mL) were incubated with drug or with DMSO
vehicle control for 4 h at 37 °C. The lysis buffer (pH 6.7, 60 mM Tris) also contained 10%
glycine, 2% SDS, 5% mercaptoethanol, and 0.002% bromophenol blue. Cell lysates (50 µg
protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 14% gel. Separated
proteins were transferred to polyvinylidene fluoride (PVDF) membranes, and then treated
overnight with rabbit anti-γH2AX (Upstate, Charlottesville, VA) primary antibody diluted
1:1000. This was followed by incubation for one h with both peroxidase-conjugated goat-anti-
rabbit secondary antibody (Cell Signaling Technology) diluted 1:2000, and anti-GAPDH (Cell
Signaling Technology) primary antibody diluted 1:1000. After incubation with
luminol/enhancer/peroxide solution (Bio-Rad, Mississauga, Canada), chemiluminescence of the
γH2AX and GAPDH bands were imaged on a Cell Biosciences (Santa Clara, CA) FluorChem
FC2 imaging system equipped with a charge-coupled-device camera.
5.5 Cell cycle synchronization, cell cycle analysis and annexin V flow cytometry
The cell cycle synchronization experiments were carried out as previously described for
the determination of the proportion of cells in sub-G0/G1, G0/G1, S, and G2/M phases of the cell
cycle.14,33 For the synchronization experiments CHO cells were grown to confluence in α-MEM
supplemented with 10% FCS. Following serum starvation with α-MEM-0% FCS for 48 h, the
cells were seeded at 2 x l05 cells/mL, repleted with α-MEM-10% FCS. Directly after repletion
they were continuously treated with either 1 µM of the compound indicated or 10 µM etoposide
in 35-mm diameter dishes for different periods of time.
.24.

The fraction of apoptotic cells induced by treatment of K562 cells with the compounds
and the etoposide positive control were quantified by two-color flow cytometry by
simultaneously measuring integrated green (annexin V-FITC) fluorescence, and integrated red
(propidium iodide) fluorescence as we previously described.14,33 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 (1 X 106
cells/ml) were untreated or treated with either 1 µM of the compound indicated or 10 µM
etoposide at 37°C for either 24 h or 48 h.
5.6 Caspase-3/7 apoptosis assay
The caspase-3/7 assay that measures caspase-3 and caspase-7 activities was carried out on
K562 lysates as we previously described for myocytes36 on a BMG (Cary, NC) Fluostar Galaxy
plate reader in luminescence mode according to the manufacturer=s directions (Caspase-Glo 3/7,
Promega, Madison, WI). The assay utilizes a proluminescent caspase-3/7 DEVD-aminoluciferin
substrate to produce a luminescent signal proportional to caspase-3/7 activity. K562 cells were
treated with either the vehicle control or 1 µM of the compounds indicated for either 24 or 48 h at
a cell density of 2 X 105 cells/mL.
5.7 Molecular modeling and docking of the bis-epipodophyllotoxins into an X-ray structure
of etoposide complexed to cleaved DNA and topoisomerase IIβ
.25.

The molecular modeling and the docking were carried out as described14 using the genetic
algorithm docking program GOLD version 3.2 (CCDC Software, Cambridge, UK) with default
GOLD parameters and atom types and with up to 2000 starting runs. The major diprotonated
microspecies at pH 7.4 of the bis-epipodophyllotoxins were docked into both etoposide binding
sites of the X-ray structure (www.rcsb.org/pdb; PDB ID: 3QX3)5 which contains two molecules
of etoposide complexed to cleaved DNA and topoisomerase IIβ. The structure was prepared by
removing the etoposide molecules, the water molecules and Mg2+ to avoid potential interference
with the docking. In order to obtain satisfactory docking solutions of these highly
conformationally flexible molecules into the two etoposide binding sites, three distance
constraints on each epipodophyllotoxin moiety were used. Docked poses in which the
epipodophyllotoxin moiety of the bis-epipodophyllotoxins differed from that of the X-ray
determined etoposide poses were given a penalty score. The three atoms on the
epipodophyllotoxin moiety of etoposide used to set the distance constraints are denoted with
asterisks in Figure 7. The two atoms on the polycyclic moiety and an atom on the pendant
phenolic group were chosen to orient the core epipodophyllotoxin structure into a bound
etoposide-like pose. Their respective distance constraint partners were the closest heavy atoms in
the X-ray structure. Etoposide docked without constraints back into its topoisomerase IIβ-DNA
structure with a heavy atom r.m.s. distance of 0.48 Å,14 compared to the X-ray structure-
determined pose.5 Values of 2.0 Å or less in the extensive GOLD test set are considered to be
good.39 The graphics were prepared with DS Visualizer 2.5 (Accelrys, San Diego, CA).
MarvinSketch and its associated calculator plugins were used for displaying the compounds and
calculating the major protonated microspecies present at pH 7.4 (Marvin version 6, 2013,
.26.

ChemAxon, http://www.chemaxon.com).
Acknowledgments
This research was supported by grants from the Canadian Institutes of Health Research (grant
MOP13748), the Canada Research Chairs Program, and a Canada Research Chair in Drug
Development to Brian Hasinoff and an NIH grant CA090787 to Jack Yalowich. The authors
declare no competing financial interests. The funding sources 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.
Supplementary data
Supplementary data from the National Cancer Institute (http://dtp.nci.nih.gov) NCI-60 cell line
panel testing results for compounds 7a, 7b and 7d and the NMR data) associated with this article
can be found in the online version, at http://xx.
.27.

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