Synthesis and evaluation of etoposide and podophyllotoxin analogs against topoisomerase IIα and HCT-116 cells

Article abstract of DOI:10.1016/j.bmc.2020.115773

Etoposide is a widely-used anticancer agent that targets human type II topoisomerases. Evidence suggests that metabolism of etoposide in myeloid progenitor cells is associated with translocations involved in leukemia development. Previous studies suggest

Full text of DOI:10.1016/j.bmc.2020.115773

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Synthesis and Evaluation of Etoposide and Podophyllotoxin Analogs against
Topoisomerase IIα and HCT-116 Cells
Matthew B. Murphy, Priyanka Kumar, Amber M. Bradley, Christopher E.
Barton, Joseph E. Deweese, Susan L. Mercer
PII:
S0968-0896(20)30603-9
https://doi.org/10.1016/j.bmc.2020.115773
BMC 115773
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Bioorganic & Medicinal Chemistry
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18 August 2020
9 September 2020
Please cite this article as: M.B. Murphy, P. Kumar, A.M. Bradley, C.E. Barton, J.E. Deweese, S.L. Mercer,
Synthesis and Evaluation of Etoposide and Podophyllotoxin Analogs against Topoisomerase IIα and HCT-116
Cells, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115773
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Synthesis and Evaluation of Etoposide and Podophyllotoxin Analogs against
Topoisomerase IIα and HCT-116 Cells
a
b
a
b
Matthew B. Murphy , Priyanka Kumar , Amber M. Bradley , Christopher E. Barton , Joseph E.
a,c
a,d
Deweese , and Susan L. Mercer
aDepartment of Pharmaceutical Sciences, Lipscomb University College of Pharmacy and Health
Sciences, One University Park Drive, Nashville, TN 37204 USA
bDepartment of Biology, Belmont University, 1900 Belmont Boulevard, Nashville, TN 37212
USA
c
d
Departments of Biochemistry and Pharmacology, Vanderbilt University School of Medicine,
Nashville, TN 37235 USA
Corresponding Author:
Susan L. Mercer, Ph.D.
Susan.Mercer@Lipscomb.edu
6
15-966-7178

Abstract
Etoposide is a widely-used anticancer agent that targets human type II topoisomerases.
Evidence suggests that metabolism of etoposide in myeloid progenitor cells is associated with
translocations involved in leukemia development. Previous studies suggest halogenation at the
C-2’ position of etoposide reduces metabolism. Halogens were introduced into the C-2’ position
by electrophilic aromatic halogenation onto etoposide (ETOP, 1), podophyllotoxin (PPT, 2), and
4
-dimethylepipodophyllotoxin (DMEP, 3), and to bridge the gap of knowledge regarding the
activity of these metabolically stable analogs. Five halogenated analogs (6-10) were synthesized.
Analogs 8-10 displayed variable ability to inhibit DNA relaxation. Analog 9 was the only analog
to show concentration-dependent enhancement of Top2-mediated DNA cleavage. Dose response
assay results indicated that 8 and 10 were most effective at decreasing the viability of HCT-116
and A549 cancer cell lines in culture. Flow cytometry with 8 and 10 in HCT-116 cells provide
evidence of sub-G1 cell populations indicative of apoptosis. Taken together, these results
indicate C-2’ halogenation of etoposide and its precursors, although metabolically stable,
decreases overall activity relative to etoposide.
Keywords
Etoposide, podophyllotoxin, topoisomerase, cancer, HCT-116, A549

Highlights
Etoposide E-ring halogenation decreases cellular activity.
Introduction
Traditional anticancer therapeutics primarily utilize mechanisms that target processes
related to replication and cell proliferation. These mechanisms of action bring with them
inherent risks to the patients as they often involve the induction of DNA damage. Etoposide
(ETOP, 1) treats cancer by disrupting the mechanism of topoisomerase II. Etoposide originates
from its natural product, podophyllotoxin (PPT, 2), and synthetic precursor, 4’-
demethylepipodophyllotoxin (DMEP, 3) (Figure 1). Though a widely-used anticancer agent,
etoposide poses a risk to patients through treatment-related secondary leukemias at a rate of 2-
1
2%.1-3 These secondary leukemias result from balanced chromosomal translocations within
several defined loci.4
Figure 1: Chemical structure of etoposide (ETOP, 1), etoposide’s natural product,
podophyllotoxin (PPT, 2), and synthetic precursor, 4’-demethylepipodophyllotoxin (DMEP, 3).
The C-2’ and C-6’ positions are designated locations of targeted synthetic modifications for this
work.

Etoposide blocks the ability of topoisomerase II to ligate cleaved DNA, which is a
5
mechanism known as interfacial poisoning. In this sense, etoposide and compounds that use the
5
same mechanism are referred to as topoisomerase II poisons. Topoisomerase II is responsible
5
for untangling DNA during replication, transcription, and cell division. Humans have two
6
isoforms of topoisomerase II: topoisomerase IIα (TOP2A) and IIβ (TOP2B). TOP2A appears to
be more involved in replication and mitosis, while TOP2B has roles in chromatin remodeling
6
and transcription. Therefore, we focused on TOP2A for the present study.
Previously, it has been proposed that metabolism of etoposide may be involved in
contributing to the strand breaks that result in translocations.7-10 Etoposide is metabolized into a
highly reactive metabolite, etoposide quinone (EQ, 5) (Figure 2), with 5-fold greater activity
compared to the parent compound.7, 11-13 Additionally, etoposide quinone appears to have a
covalent mechanism that is not seen with etoposide or etoposide catechol (EC, 4) (Figure 2).7, 8,
1
2
While etoposide and etoposide catechol lead to increased DNA strand breaks and inhibition of
relaxation in the presence of TOP2A, these compounds are not able to adduct to the enzyme as
the quinone appears to do.7, 12 Etoposide catechol can be chemically oxidized to the more active
quinone form.12 Adduction of etoposide quinone to the enzyme is very likely a covalent
interaction and locks the enzyme halves together causing the enzyme to be trapped on DNA.7, 8
Further support of the etoposide quinone role in leukemogenesis can be seen within
myeloid progenitor cells which possess an enzyme, myeloperoxidase, capable of transforming
etoposide into its quinone metabolite (Figure 2).9, 14 Interestingly, prior studies suggest that the
quinone forming potential of etoposide can be drastically reduced by the placement of a halogen
onto its E-ring.15 This work focused on the synthesis of metabolically stable halogenated

analogs of etoposide and its parent compounds, followed by biological analysis for retention of
anticancer activity.
Figure 2: Metabolism of etoposide. The metabolism of etoposide (ETOP, 1) can involve
hydrolysis, sulfate modification, and demethylation (CYP3A4). While hydrolysis of etoposide
yields hydroxy acid, the major metabolite, CYP3A4 metabolizes etoposide to the catechol (EC,
4
). The parent compound or the catechol can be oxidized to produce the quinone (EQ, 5).
Myeloperoxidase may catalyze this oxidation in bone marrow progenitor cells.
Methods
Chemical Synthesis. Halogens were introduced into the C-2’ position by electrophilic aromatic
halogenation onto etoposide (ETOP, 1), podophyllotoxin (PPT, 2), or 4-
dimethylepipodophyllotoxin (DMEP, 3) as previously published.15, 16 The reagents N-

bromosuccinimide and N-chlorosuccinimide afforded a series of halogenated analogs (6-10, see
Table 1). Refer to the supplemental materials for additional synthetic details.
Table 1: Scaffold and substituent and resulting synthetic analogs 6-10. NT = not synthesized or
tested.
Scaffold and
Substituent
PPT
ETOP
DMEP
2
2
’-chloro
’-bromo
’,6’-dichloro
6
7
8
9
NT
NT
10
NT
NT
2
Enzymes and materials. Wild-type human TOP2A was expressed in Saccharomyces cerevisiae
1
7
JEL1top1 cells and purified as described previously. The enzyme was stored at -80ºC as a 1.0
mg/mL (~3 μM) stock in 50 mM Tris-HCl, pH 7.7, 0.1 mM EDTA, 750 mM KCl, and 5%
glycerol. Negatively supercoiled pBR322 DNA was prepared using a Plasmid Mega Kit
(Qiagen). Etoposide was obtained from Sigma. Etoposide, podophyllotoxin, and analogs were
stored at 4˚C as 20 mM stock solutions in 100% DMSO.
Topoisomerase II-Mediated Plasmid DNA Relaxation. Reaction mixtures contained ~7 nM
wild-type human TOP2A, 5 nM negatively supercoiled pBR322 DNA, and 1 mM ATP in 20 μL
of 10 mM Tris-HCl, pH 7.9, 175 mM KCl, 0.1 mM Na2EDTA, 5 mM MgCl2, and 2.5%
glycerol. Assays were started by the addition of enzyme, and DNA relaxation mixtures were
incubated for 15 min at 37°C. DNA relaxation reactions were carried out in the presence of
etoposide, podophyllotoxin, or analogs at concentrations denoted in the experiments. Reactions

were stopped by the addition of 3 μL of stop solution (77.5 mM Na EDTA, 0.77% SDS).
2
Samples were mixed with 2 μL of agarose gel loading buffer, heated for 2 min at 45°C, and
subjected to gel electrophoresis in 1% agarose gels. The agarose gel was then stained in
ethidium bromide for 30 min. DNA bands were visualized by UV light and imaged using a Bio-
Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). DNA relaxation
was monitored by the conversion of supercoiled plasmid DNA to relaxed topoisomers.
Topoisomerase II-Mediated Plasmid DNA Cleavage. Plasmid DNA cleavage reactions were
performed using the procedure of Fortune and Osheroff.18 Reaction mixtures contained ~150
nM of human TOP2A and 5 nM negatively supercoiled pBR322 DNA in 20 μL of 10 mM Tris-
HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5% glycerol. Unless stated
otherwise, assays were started by the addition of enzyme, and DNA cleavage mixtures were
incubated for 6 min at 37°C. DNA cleavage reactions were carried out in the absence or
presence of etoposide, podophyllotoxin, or the analogs at concentrations denoted in the
experiments. DNA cleavage complexes were trapped by the addition of 2 μL of 2.5% SDS
followed by 2 μL of 250 mM Na EDTA, pH 8.0. Proteinase K was added (2 μL of a 0.8 mg/mL
2
solution), and reaction mixtures were incubated for 30 min at 37°C to digest TOP2A. Samples
were mixed with 2 μL of agarose gel loading buffer (60% sucrose in 10 mM Tris-HCl, pH 7.9),
heated for 2 min at 45°C, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-
acetate, pH 8.3, and 2 mM EDTA containing 0.5 μg/mL ethidium bromide. Double-stranded
DNA cleavage was monitored by the conversion of negatively supercoiled plasmid DNA to
linear molecules. DNA bands were visualized by UV light and quantified using a Bio-Rad
ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). The DNA cleavage

levels were analyzed using Graphpad Prism 8 with an ANOVA followed by a post-hoc Tukey
multiple comparisons test.
Cell Culture Conditions. HCT116 colorectal cancer cells (ATCC, CCL-247) and A549 lung
carcinoma cells (ATCC, CCL-185) were cultured in Dulbecco's modified Eagle's medium
(DMEM, Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 1% penicillin-
streptomycin (Invitrogen). All cells were cultured at 37°C with 5% CO2.
4
Cell Viability Assay. For HCT-116 and A549 cell viability assays, 1x10 cells were seeded in
triplicate in 96-well plates and grown for 24 h to approximately 20% confluency. Medium was
then replaced with either control medium or medium containing 2-fold dilutions of the indicated
etoposide analog (maximum concentration 400 μM, minimum concentration 390 nM) and
incubated for 72 h. Viability was determined by the addition of alamarBlue for 4 h and reading
at 570 nm. 600 nm readings were used to detect background signal and data were normalized by
determine the ratio of 570 nm/600 nm. Resulting data were used to determine LD values for
5
0
each analog.
HCT-116 Flow Cytometry. HCT116 cells were treated with DMSO (control), or the LD50
concentrations of the indicated analogs for 48 h. Cells were harvested, processed and stained
with propidium iodide using the BD Cycletest Plus DNA Reagent Kit (BD Biosciences) per the
manufacturer’s instructions.19 Cell cycle profiles were generated using a BD Accuri C6 flow

cytometer and data were analyzed using Accuri C6 Software (BD Biosciences). 20,000 events
were analyzed for each sample.
Results and Discussion
In order to explore the biological activity of the podophyllotoxin and etoposide analogs,
we performed in vitro studies using purified human topoisomerase IIα (TOP2A). The first
assays examined the ability of TOP2A to perform relaxation of supercoiled plasmids in the
presence of the analogs. During this assay, TOP2A converts supercoiled plasmid substrate into a
relaxed, circular plasmid substrate. This is measured by comparing the migration of DNA in the
gel with supercoiled substrate migrating faster through the gel than the relaxed substrate (see
DNA and DMSO lanes of Figure 3). Relaxation is inhibited by increasing concentrations of
etoposide but not of podophyllotoxin. Similar to podophyllotoxin, neither 6 nor 7 inhibited
relaxation, as indicated by the relaxed DNA bands in the corresponding lanes. However, 8-10 all
displayed some ability to inhibit relaxation by TOP2A.
Figure 3: Plasmid DNA relaxation assays in the presence of topoisomerase IIα (TOP2A).
Analogs 8-10 demonstrated inhibition of DNA relaxation in a concentration dependent manner.
Analogs 8 and 9 both inhibited DNA relaxation to a greater extent than etoposide.
Podophyllotoxin (PPT), 6, and 7 did not inhibit DNA relaxation. Gel is representative of three or
more independent experiments. DNA = plasmid DNA substrate only; DM = 1% DMSO control;
Rel = Relaxed plasmid; SC = supercoiled plasmid

Since DNA relaxation alone does not clarify the mechanism of inhibition, we performed
an additional assay to examine DNA cleavage. This assay is performed in the absence of ATP,
5
which is needed for relaxation activity but not for DNA cleavage. The assay enables
comparison of the effects of compounds on the cleavage of DNA by TOP2A through tracking
the supercoiled, linearized (double-stranded break), and nicked (single-stranded break) forms of
the plasmid. Etoposide acts by inhibiting DNA ligation, which leads to an increase in the level
5
of DNA cleavage observed in the gel as the concentration of etoposide increases. As seen in
Figure 4, unlike 8, analog 10 does have limited ability to increase DNA cleavage. The increase
seen with 10 is approaching significance at 200 M (p = 0.09). In contrast, 9 significantly
increases DNA cleavage levels at both 100 and 200 M (p <0.0001), but the analog is less potent
than etoposide.
2
2
1
1
5
0
5
0
5
0
30
20
10
0
Etoposide
9
0
50
100
150
200
ND 100 200 100 200 100 200 M
Drug [M]
8
9
10
Figure 4: Quantified Results from plasmid DNA cleavage assays with topoisomerase IIα
TOP2A). Etoposide and 9 increase DNA cleavage levels in a concentration dependent manner,
(
but etoposide is more potent at increasing DNA cleavage. By comparison, 8 does not increase
DNA cleavage by topoisomerase IIα. Analog 10 does result in a small increase in DNA cleavage
at 200 µM. Results are representative of three or more independent experiments and error bars
represent the standard deviation of the mean.

Given that analogs 8, 9, and 10 affected the relaxation and/or DNA cleavage of TOP2A
in vitro, we next analyzed whether the same molecules exhibited anti-proliferative properties
against different cancer cells grown in culture. To this end, HCT116 and A549 cells were
exposed to serially-diluted concentrations of DMSO (control), etoposide, and analogs 8-10
(range 400 µM – 390 nM) for 72 hours and then assayed for viability using alamarBlue reagent.
It should be noted that cells were treated at a low confluency (~20%) in order to assure cells
were actively cycling throughout the assay timeframe. Consistent with the in vitro data, all of
the analogs were effective at decreasing the viability of HCT116 cells, with 8 and 10 displaying
significantly lower LD values as compared to 9 (Table 2). Given the high LD for 9 in HCT-
5
0
50
1
16 cells, we chose to further analyze only 8 and 10 in A549 cells. Both analogs decreased the
viability of A549 cells and displayed similar LD values as compared to HCT-116 cells (Table
5
0
2
). These results indicate that activity against TOP2A does not necessarily reflect whether the
compounds will have cellular activity and may indicate that additional target(s) and/or
mechanism(s) of action are involved. For example, podophyllotoxin is known to inhibit
microtubule polymerization, which may explain the antiproliferative activity of 8 and 10.20, 21

Table 2: LD Values for Compounds in the Presence of HCT-116 and A549 Cells. Data are
5
0
a
representative of three independent experiments. NT = not tested. A549 LD etoposide
5
0
reference value.22
Compound
HCT116
A549
LD , µM
LD , µM
5
0
50
(
95% CI)
(95% CI)
11.8a
Etoposide
1.6 (1.3-2.0)
8
9
20 (16-27)
17 (15-19)
NT
4
1
20 (1.4-10 )
1
0
33 (14-78)
35 (14-91)
Given that both 8 and 10 were significantly more effective at decreasing the viability of
cultured cancer cells, we next analyzed the effect of each molecule on cell cycle profiles. To
avoid the high levels of toxicity observed at 72 h of drug exposure, cells were treated with LD50
concentrations of the indicated analogs and collected for cell cycle analysis 48 h after treatment.
Flow cytometric analysis of cells revealed that both 8 and 10 decreased the ratio of S-phase cells
when compared to the control-treated population. Most importantly, we observed a significant
increase in the sub-G1 cell fraction following treatment with both 8 and 10 (Figure 5).
2
3
Consistent with previously-published literature, the increase in sub-G1 cells is indicative of
cells experiencing cell death via apoptosis.

1
00
G /M
2
S
7
5
2
5
0
5
0
G /G
0
1
Sub-G1
DMSO
8
10
Figure 5: HCT116 cells were cultured for 48h in the presence of 8, 10, or DMSO as a control.
Cells were harvested and further processed for flow cytometric analysis as described in the
methods section. DNA content was analyzed and the percentage of total cells in each phase of
the cell cycle was quantified. Sub-G1 fraction indicates sub-diploid cells and is consistent with
cellular death via apoptosis.
Conclusion
Analogs 8-10 were all able to inhibit DNA relaxation with 8 and 9 more potent than
etoposide. Furthermore, 9 was the only analog to increase TOP2A-mediated DNA cleavage,
which indicates that it retained the poisoning mechanism seen with etoposide. However, analogs
8
and 10 were not found to be poisons of TOP2A. Thus, it appears that these two analogs impact
TOP2A in a manner similar to catalytic inhibitors rather than poisons, but the exact mechanism
remains to be demonstrated. Recent docking studies with a PPT analog indicate the ability to
potentially bind to the ATPase domain of TOP2, which would be consistent with catalytic
inhibition.21, 24 Analogs 8-10 all demonstrated cytotoxicity towards HCT116 and A549 cells
albeit less than etoposide. Analogs 8 and 10 were capable of increasing sub-G1 HCT116 cells
indicative of apoptotic cell death. The relatively weak activity of 9 in HCT116 cells may reflect
an inability of the analog to enter cells due to the addition of the 2’-chloro. Although cytotoxic,

the lack of potency compared to etoposide suggests that the metabolically stable analogs are not
practical molecules for further therapeutic exploration.
Acknowledgements
This work was supporting by funding from the American Foundation of Pharmaceutical
Education (AFPE) – Gateway to Research Scholarship (MBM). Additional financial support
was provided by the Lipscomb University College of Pharmacy and Belmont University Biology
Department.

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Graphical abstract