Alkyne-substituted diminazene as G-quadruplex binders with anticancer activities

Article abstract of DOI:10.1016/j.ejmech.2016.04.030

G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to

Full text of DOI:10.1016/j.ejmech.2016.04.030

European Journal of Medicinal Chemistry 118 (2016) 266e275
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Alkyne-substituted diminazene as G-quadruplex binders with
anticancer activities
, c, d
Changhao Wang a, Brandon Carter-Cooper b, Yixuan Du a, Jie Zhou a
,
Musabbir A. Saeed a, Jinbing Liu a, Min Guo a, Benjamin Roembke a, Clinton Mikek e,
, c, d, *
Edwin A. Lewis e, Rena G. Lapidus b, Herman O. Sintim a
a Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
b Translational Core Laboratory, University of Maryland Greenebaum Cancer Center, 655 W. Baltimore Street, Baltimore, MD 21201, USA
c Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA
d Center for Drug Discovery, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907, USA
e Department of Chemistry, Mississippi State University, Mississippi State, MS 39762, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-
quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed
that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes,
with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities.
In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition
assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to
G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both
in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit
micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231)
cancer cell lines and represent interesting new leads to develop anticancer agents.
Received 6 January 2016
Received in revised form
11 April 2016
Accepted 11 April 2016
Available online 20 April 2016
Keywords:
Anticancer therapeutics
G-quadruplex
Western blot
© 2016 Elsevier Masson SAS. All rights reserved.
Telomerase activity
c-MyC
1. Introduction
function of G-quadruplexes [11] could exacerbate neurodegenera-
tive diseases [10]. Due to the expanding role of G-quadruplexes in
Cancers of the human reproductive organs, such as ovarian,
prostate and breast, kill millions of people annually worldwide.
Although there are several therapeutics against these cancers [1],
aggressive forms remain problematic to treat. For example,
although there are myriad hormone-based therapeutics against
breast cancer [2], hormone insensitive breast cancer such as triple
negative breast cancer remains difficult to treat [3].
G-quadruplex structures in both DNA and RNA are emerging as
important regulatory elements that control diverse processes in the
cell, ranging from telomere maintenance [4], gene expression [5],
translation [6], alternative splicing [7], RNA metabolism [8] and
protein sequestration [9]. Additionally G-quadruplex elements
(both DNA and RNA) in the cell have been postulated to bind to and
activate heme to promote oxidative damage [10]. Such enzymatic
biology [12], there has been an explosion of research activities
dedicated to the discovery of G-quadruplex ligands [13]. Small
molecules that stabilize the G-quadruplex structure have been
shown to inhibit telomere extension [14] and transcription of
oncogenic genes (such as c-MYC, [15] c-kit, [16] KRAS5b) and hence
these molecules have the potential to be used as antineoplastics
[4,17]. Beyond potential use as anticancer agents, G-quadruplex li-
gands have also been shown to inhibit HIV replication [18].
We have been interested in repurposing drugs that are used in
both human and veterinary medicine for other indications for
anticancer therapy because we believe that the translational po-
tential for such drugs or analogues thereof is high, since the mol-
ecules are already in clinical use. Along this line, we recently
reported that diminazene (DMZ in Fig. 1), a drug used to treat
sleeping sickness (animal trypanosomiasis), and which was
formerly considered as selective AT-rich duplex binder (via minor
groove) is indeed a potent G-quadruplex binder with nanomolar
dissociation constant (Kd) [19]. DMZ binds to DNA minor groove
* Corresponding author. Department of Chemistry, Purdue University, 560 Oval
Drive, West Lafayette, IN 47907, USA.
E-mail address: hsintim@purdue.edu (H.O. Sintim).
http://dx.doi.org/10.1016/j.ejmech.2016.04.030
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
267
Fig. 1. The structures of diminazene (DMZ) analogues.
with a Kd of 1
m
M so it appears that DMZ is more selective for G-
nitrogen in the pyridine ring could interact with the DNA nucleo-
bases via hydrogen bonding. Fluorine and the methoxy groups
could also accept hydrogen bonds from water and/or the DNA
target. A second criteria that we used for selecting the analogs was
synthetic tractability and the commercial availability of that start-
ing materials.
quadruplex than duplex DNA [19a]. Nonetheless a Kd of 1
mM for
duplex DNA would limit the targeting of G-quadruplexes by DMZ in
the complex cellular environment, where the concentration of
duplex DNA is several orders of magnitude greater than G-quad-
ruplexes. We had earlier observed that the monoamidine analogue
of DMZ (DMZ1 in Fig. 1) could also bind to G-quadruplexes, albeit
not as potent as DMZ [19a]. Importantly DMZ1 only had a weak
Initial attempts to characterize the binding of the analogues
using isothermal titration calorimetry (ITC) was not very successful
due to the low solubility of the compounds in buffer (see SI,
Table S2, Figs. S1 and S2). Others have used FRET melting of
fluorophore-labeled G-quadruplexes to determine the binding of
ligands to G-quadruplexes and also selectivity between G-quad-
ruplexes and duplexes, so we decided to adopt this protocol [20].
Thus, we investigated the change in melting temperature (T1/2) by
fluorescence resonance energy transfer (FRET) assay (Figs. 2 and 3).
We chose dual-labeled F21T, c-MYC, c-kit2 and k-RAS21R because c-
MYC G-quadruplex affects the c-MYC transcriptional activity [21]
while c-kit and k-RAS G-quadruplexes control the expression of
oncogenic c-kit [16] and KRAS [5] proteins and F21T is a good model
for telomere [5]. 26-mer DNA (hairpin) is a good model to deter-
mine if the ligands could also bind to duplexes [22]. The FRET-
labeled oligonucleotides used in this study have also been used
by others and shown to be well-behaved in determining the po-
tency of G-quadruplex ligands [20].
affinity for duplex DNA (Kd ¼ 34
mM for AT-rich duplex DNA) [19a],
suggesting that perhaps DMZ1 was a better starting point for
developing G-quadruplex interactive anti-cancer agents. A focused
library of DMZ1 analogues (Fig. 1), whereby a
p-moiety was
appended to the parent DMZ1, were easily prepared via Sonoga-
shira coupling and then triazene formation. Herein, we reveal that
DMZ and monoamidine analogue, DMZ1, do not have anticancer
properties but alkyne analogues of DMZ have respectable to good
anticancer properties against ovarian, prostate and triple negative
breast cancers.
2. Results and discussion
2.1. Design and synthesis of alkyne-substituted DMZ analogues as
G-quadruplex binders
TMPyP4, a prototypical G-quadruplex ligand was the most sta-
bilizing ligand (note that a lower concentration of TMPyP4 was
used in the FRET melting assay, compared to the amidine ligands).
Of the tested triazenes, DMZ, which has two amidine groups, was
the best stabilizer of the tested G-quadruplexes but showed low
selectivity between G-quadruplexes and duplex DNA. For example,
DMZ could stabilize k-RAS21R but could also stabilize double-
stranded DNA, see Fig. 3. Removing one of the amidine groups
from DMZ to afford compound DMZ1 reduced affinity for duplex
DNA (△T1/2 < 1 ꢀC for double-stranded DNA). Contrary to our initial
To aid future optimizations of the alkyne DMZ analogues as
anticancer agents, we sought to verify that like the parent DMZ,
these compounds also bind to G-quadruplexes. Also, we asked if the
compounds were selective for G-quadruplexes over DNA duplexes.
Since DMZ requires both amidine groups to bind to duplex DNA,
removing one amidine group will reduce duplex DNA binding af-
finity. However, the G-quadruplex surface has more room to
accommodate on extended pi-surface. We therefore extended the
parent DMZ with additional aromatic surface while maintaining
one amidine for solubility concern. Sonogashira coupling strategy
allowed us to generate alkyne-substituted DMZ analogues in a
timely and economic manner. Details of synthetic pathway are
illustrated in experimental section. We also explored the effects of
substitutions on the aromatic rings by incorporating fragments
such as pyridine, fluorine and methoxy groups. These groups are
commonly found in drugs. Pyridine is a mimic of phenyl group, but
has an enhanced aqueous solubility arising from the nitrogen in the
pyridine forming hydrogen bonds with water. Additionally the
hypothesis that adding a
p-moiety to DMZ1 would increase G-
quadruplex binding, the addition of an alkyne, cyano or phenyl
groups to compound DMZ1 did not afford an analogue with a more
superior G-quadruplex stabilization property than DMZ1 (see Fig. 3
and Table S1 in SI). Encouraging, however, was the observation that
DMZ1 and alkyne analogues (but not the control compound
TMPyP4 or DMZ) were more selective for G-quadruplex over
duplex DNA (see Fig. 4 and Table S1 in SI.). We determined the

268
C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
Fig. 2. FRET model of study.
melting temperature of fluorescently-labeled c-MYC in the pres-
ence of fixed ligand concentration and increasing concentrations of
non-fluorescent double-stranded DNA. When compound DMZ1 or
alkyne derivatives were used as ligands, adding up to 500 equiva-
lence of double-stranded DNA did not affect the T1/2 of c-MYC, in
agreement with the FRET melting data (Fig. 3), which showed that
the compounds stabilized G-quadruplexes but not duplex DNA. For
DMZ or TMPyP4 however, increasing the amount of duplex DNA
marrow (NBM) and normal fibroblast cell (MCR5A) for cytotoxicity.
Amongst the first set of DMZ analogues (compounds DMZ1 to 6,
and DMZ11), bearing different substituents (cyano, amidine, alkyne
and aryl groups), only the alkyne derivatives (DMZ3, DMZ5 and
DMZ11) were active (see Table 1, entries iv, vi and xii), albeit not
very potent. Interestingly DMZ, which potently binds to G-quad-
ruplexes, and the monoamidine analogue DMZ1 were also not
effective at inhibiting cancer proliferation. Encouraged by the initial
positive results with the alkynes analogues we sought to optimize
the anticancer potency and made further alkyne analogues by
appending various aromatic moieties to the alkyne to give com-
pounds DMZ7 to 10 and DMZ12 to 14 (see Fig. 1). We initially
rationalized that by adding aromatic appendages to the initial
alkyne compounds, stacking interactions between the G-quad-
ruplex tetrad and the compounds would increase.
This hypothesis did not pan out as the new alkyne analogues
(DMZ7 to 10 and DMZ12 to 14) did not display enhanced G-quad-
ruplex stabilization (see Fig. 4) but the analogues had improved
anticancer properties (see Table 1). Of note, the GI50 values of some
of the DMZ analogues (DMZ7, DMZ9, DMZ10, DMZ12 and DMZ13)
are similar to or better than those of cisplatin against the three
tested cancer cell lines (compare entries viii, x, xi, xiii, xiv with xvi
in Table 1). Also the GI50 values of some of these analogues were
better than TMPyP4, a potent G-quadruplex binder, underscoring
the fact that G-quadruplex binding potency alone does not define
the potency of these drugs. Cisplatin is an anticancer drug that is
resulted in a lower DT1/2 (see Fig. 4).
2.2. Viability assay
The alkyne DMZ analogues only have moderate affinity for G-
quadruplexes and so the key question was whether they could
inhibit the proliferation of cancer cells, and if that happens to be the
case whether the proliferation inhibition would be via G-quad-
ruplex stabilization inside cells. We evaluated the compounds
against three human cancer cell lines, ovarian cancer cell (OVCAR-
3), prostate cancer cell (PC-3) and triple negative breast cancer cell
(MDA-MB-231), using WST-1 assay. A select few compounds were
also tested against two human normal cell lines, normal bone
Fig. 3. FRET stabilization temperature (△T1/2) of various G-quadruplex DNA with DMZ
a
analogues and TMPyP4. G-quadruplex DNA: F21T (50eFAM-GGG TTA GGG TTA GGG
TTA GGG-TAMRAe30), c-kit2 (50eFAM-CCC GGG CGG GCG CGA GGG AGG GGA GG-
TAMRAe30), k-RAS21R (50eFAM-AGG GCG GTG TGG GAA GAG GGA-TAMRAe30), c-MYC
(50-FAM-TGA GGG TGG GTA GGG TGG GTA A-TAMRA-30). Duplex 26-mer (50eFAM-TAT
AGC TAT ATT TTT TTA TAG CTA TA-TAMRAe30). Conditions: dual-labeled DNA (0.4
m
M),
T1/2 is calcu-
lated by melting temperature in the presence and absence of DMZ analogues. bFor
TMPyP4, 0.4 M dual-labeled DNA and TMPyP4 were applied to get meaningful
melting temperatures. *△T1/2 is larger than 5 ꢀC.
DMZ analogues (4
mM), potassium cacodylate buffer (60 mM, pH 7.2).
D
Fig. 4. FRET-melting competition results at 4
dual-labeled c-MYC (0.4 M) and competitor unlabeled ds-DNA (0
and 200 M). T1/2 is calculated by melting temperature in the presence and absence
of DMZ analogues. 0.4 M TMPyP4 was added to get meaningful melting temperature.
m
M DMZ analogues in the presence of
m
m
M, 4 M, 40
m
mM
m
m
D
m

C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
269
Table 1
2.3. PCR stop assay
Anticancer activity of DMZ analogues with different cancer cell lines.
PCR stop assay, using templates that contain G-quadruplex se-
quences, has been used by several investigators to demonstrate the
binding of ligands to G-quadruplex [25]. We investigated the effects
of analogues DMZ9, DMZ13, DMZ, DMZ1 and TMPyP4 on c-MYC G-
quadruplex stabilization via the PCR stop assay. Pu27, Pu27-13,14
and Pu-mutant were used as templates [26]. Pu27 is contained in
the nuclear hypersensitivity element III1 (NHE III1), which controls
80e90% transcription level of c-MYC [27]. Pu27-13,14 [26] and Pu-
mutant (see SI) are two mutated Pu27 strands used as control.
Entry Compound Anticancer activities for different cancer cell
lines(GI50 SD,
m
M)a
OVCAR-3 PC-3
MDA-MB-231 NBM MCR5A
i
ii
iii
iv
v
vi
vii
viii
ix
DMZ
>25
NE
NE
NEb
NE
NE
NE
NE
NE
ed
e
e
DMZ1
DMZ2
DMZ3
DMZ4
DMZ5
DMZ6
DMZ7
DMZ8
DMZ9
DMZ10
DMZ11
DMZ12
DMZ13
DMZ14
Cisplatin
15
e
e
e
11.8 1.7 22.5 8.8 30.2 1.8
NE NE NE
16.8 3.1 18.0 5.7 20.5 4.2
NE
6.5 2.1
e
e
e
e
e
e
NE
5.8 0.2
NE
9.5 1.3
e
e
In the PCR reaction system, 5
various concentrations of DMZ1, DMZ9, DMZ13 (1
25 M, 50 M and 100 M), DMZ (1 M, 10 M and 25
TMPyP4 (1 M and 10 M) were added. The polymerization/
extension of oligomers Pu27 and Pu27rev was completely inhibited
in the presence of 100 M DMZ9 and DMZ13 (see Fig. 5). Also, DMZ
and TMPyP4 showed inhibition at 25 M and 10 M, respectively.
m
M of Pu27, 5
m
M of Pu27rev and
M, 10 M,
M) and
e
e
10.7 1.5 10.4 1.4 14.4 0.9
e
e
m
m
x
xi
5.0 0.1
5.2 0.4
>25
8.1 0.5
9.5 2.2
NE
4.1 1.8
NE
NE
5.3 1.6
5.0 0.9
>50
5.3 0.2
7.4 0.2
NE
10.5 1.3 11.7 1.0
NE
NE
5.5 0.1
5.5
23.2
5.1 0.4
6.2 1.0
NE
5.6
e
8.3 4.0
e
m
m
m
m
m
m
m
m
xii
xiii
xiv
xv
Xvic
xvii
e
e
e
e
m
NE
e
NE
e
m
m
>10 0.41 0.27
e
To eliminate the possibility that the inhibition of DNA extension
was caused by the direct inhibition of the polymerase enzyme by
the ligands, Pu27-13,14 (see Fig. 5) and Pu-mutant were extended
in the presence of various concentrations of DMZ9, DMZ13, DMZ
and TMPyP4. In both controls, amplified PCR product was observed
NE
NE
e
xviii 19
e
e
xix
TMPyP4
14.6 5.7 14.0 5.4 14.3 4.0
16.8 15.2 6.6
a
Human cancer cell lines: ovarian cancer cell (OVCAR-3), prostate cancer cell (PC-
3) and breast cancer cell (MDA-MB-231).
b
in the presence of 100
PCR product or reduced product was observed in the presence of
10 M TMPyP4 for Pu27-13,14 and Pu-mutant, respectively. On the
other hand, the amount of PCR products obtained in the presence of
25 M DMZ was lower than in the absence of DMZ for Pu27-
mM DMZ9, DMZ13 (see Fig. 6). However, no
NE means not effective. Cell viability was the same or greater than 75% of the
DMSO control at 20 mM compound concentration so accurate GI50 values for these
compounds were not determined.
m
c
Cisplatin as a control compound.
Not determined.
d
m
13,14(see Fig. 6). No changes were observed in Pu-mutant reac-
tion (see Fig. S7). These results were unexpected because both DMZ
and TMPyP4 had been shown to bind to both G-quadruplexes and
duplex DNA (See Fig. 4) so both compounds would inhibit the
extension of Pu27 (containing a G-quadruplex) and Pu27-13,14
(containing duplex). Based on these results, we conclude that the
DMZ analogues inhibit the extension of a template containing c-
MYC sequences via the stabilization of a G-quadruplex structure in
the template and not via direct polymerase inhibition, as observed
with the TMPyP4 case [28].
The aforementioned experiments were performed to determine
if alkyne-substituted analogues of DMZ, some of which have anti-
cancer properties interacted with G-quadruplex DNAs selectively
(i.e. did not have affinity for duplex DNA). Although our in vitro
studies revealed that these compounds are moderate G-quadruplex
binders, it remained to be shown if the pharmacology of these
compounds were via G-quadruplex stabilization inside cells. We
proceeded to perform two classic experiments, which are tradi-
tionally done to show the in vivo efficacy of compounds via G-
quadruplex stabilization: telomerase activity assay [29] or Western
analysis of c-MYC expression [24].
still used in the clinic to treat various cancers so the GI50 values that
we have obtained for some of the alkyne DMZ derivatives are
encouraging and calls for further development of alkyne DMZ an-
alogues as anticancer agents.
To gain some insights into potential toxicities of these DMZ an-
alogues, we proceeded to evaluate analogues DMZ9, DMZ13
TMPyP4 and cisplatin (a control) against two human normal cell
lines (NBM and MCR5A) (see entries x, xiv and xix in Table 1). For
analogues DMZ9 and TMPyP4, the GI50 values against NBM and
MCR5A cells are similar to the three tested cancer cells (hence these
compounds might have low therapeutic window). However, and
pleasingly, analogue DMZ13 killed the three tested cancer cells with
single digit GI50 but was not effective against NBM and MCR5A cells.
Cisplatin is used to treat several cancers yet it has a higher toxicity
against MCR5A than DMZ9 and DMZ13 (Table 1). To rule out the
possibility that the anti-proliferative properties of the alkyne DMZ
analogues are not derived from their amine metabolism products,
we also tested the anticancer properties of 4-aminobenzamidine
and amino alkyne 15 but none of these were active.
Having demonstrated that G-quadruplex-interactive DMZ ana-
logues, bearing alkyne moieties, have anticancer properties and with
an eye towards the future development of these molecules, we
soughtfurtherexperiments tocementourbeliefthatthesemolecules
do indeed bind to G-quadruplexes and do so selectively. The afore-
mentioned experiments (FRET melting) that investigated the in-
teractions of alkyne analogues of DMZ with G-quadruplexes are
indirect. NMR has been demonstrated to be a direct tool to study the
interaction between DNA and ligands [23]. We have used NMR to
show that both DMZ9 and DMZ13 interacted with c-kit1, c-MYC G-
quadruplex but not duplex (see Figs. S3eS5). Since c-MYC is a key
oncogene we performed additional experiments, PCR stop assay [7]
(in vitro) and Western analysis of c-MYC expression [24] (in vivo) in
the presence and absence of some of our DMZ analogues (DMZ,
DMZ1, DMZ9, DMZ13 and TMPyP4, as control) to determine if the
pharmacology of the analogues is at least derived from c-MYC
inhibition.
2.4. Inhibition of telomerase activity in breast cancer cells
Some G-quadruplex interactive ligands have been shown to
achieve cancer cell killing via the inhibition of telomerase expres-
sion and/or inhibition of telomere extension [29]. We therefore
investigated if the anticancer activities of some of our analogues are
derived (at least partially) from telomerase or telomere extension
inhibition. We evaluated the effect of compounds DMZ9 and
DMZ13 at various concentrations (0.5 ꢁ GI50, 1 ꢁ GI50, 2 ꢁ GI50) on
telomerase activity in human breast cancer cell line (MDA-MB-231)
using TRAPeze XL Telomerase Detection Kit (Intergen). TMPyP4 was
evaluated at higher concentrations (1 ꢁ GI50, 2 ꢁ GI50, 4 ꢁ GI50
)
since it was less efficacious at cancer cell killing and so higher
concentrations could be used and yet adequate amounts of cells
would remain for post-treatment analysis. Cells were treated with

270
C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
Fig. 5. Effects of DMZ analogues (DMZ1, DMZ 9, DMZ13), DMZ and TMPyP4 on the PCR stop assay with c-MYC Pu27. Compounds were added to the reaction mixture containing 1x
PCR buffer (New England Biolabs), 5
control.
mM Pu27, 5 mM Pu27rev, 200 mM dNTPs and 5 units of Taq polymerase (New England Biolabs) separately. No compound added was treated as
compounds for 48 h and 72 h respectively. Inhibition of telomerase
was evaluated by comparing the telomerase activity in untreated
cells. The results showed that telomerase activity in MDA-MB-231
could be inhibited by up to 50% after treating the cell lines with
analysis of analogue DMZ9 and TMPyP4 on reducing c-MYC protein
levels in MDA-MB-231 cells (see Fig. 8).
2.5. Western blot analysis of c-MYC protein expression
11
25
m
m
M (2 ꢁ GI50) DMZ9 for 48 h or 72 h (see Fig. 7). For TMPyP4,
M (2 ꢁ GI50) could inhibit telomerase activity in MDA-MB-231
c-MYC protein levels in MDA-MB-231 cells were evaluated by
performing Western blot. MDA-MB-231 breast cancer cells were
by ~ 60 and 80% for 48 h and 72 h respectively. It appears that the
effect of DMZ9 on telomerase expression deceases over time
whereas that of TMPyP4 increases over time. This could probably
due to differential metabolism or degradation. Importantly the ef-
fects of both DMZ9 and TMPyP4 were dose-dependent. This was
treated with 22.3 or 111.5
mM TMPyP4, 5.45 or 27.25 mM DMZ9 for 2,
6 or 24 h and 19.5 M DMZ13 for 6 or 24 h. Here too, higher con-
m
centrations of TMPyP4 and DMZ13 than DMZ9 could be used
because TMPyP4 is not as efficacious as DMZ9 so higher concen-
trations could be used without killing all of the cells. TMPyP4 has
previously been shown to down regulate c-MYC protein expression
level [24] so TMPyP4 was included in this experiment as a positive
however not the case for DMZ13, which at 12.4 mM could inhibit
about 20% and 30% of telomerase activity in MDA-MB-231 after
treating the cells for 48 h and 72 h respectively (see Fig. 7). At 48 h,
the inhibition of telomerase activity by DMZ13 was not dose
dependent. We currently do not have a hypothesis to explain this
phenomenon but the low level of telomerase inhibition by DMZ13
suggests that its anti-cancer properties might be derived from the
control. The results showed that at 19.5
mM DMZ13 had no effect on
c-MYC expression at 6 h while at 27.25
mM DMZ9 and 22.3 mM
TMPyP4 caused an approximately 40% decrease in the c-MYC pro-
tein expression at 6 h. After 24 h, DMZ13, DMZ9 and TMPyP4
caused 100%, 67% and 92% reductions in c-MYC expression
stabilization of other G-quadruplexes or even via
quadruplex stabilization pathway.
a non-G-
respectively. At a higher concentration of 111.5 mM, TMPyP4 caused
The inhibition of telomerase expression could occur via several
pathways. It has been proposed that this inhibition may be caused
by the stabilization of G-quadruplex in the core promoter of hTERT
(catalytic domain of telomerase) [30]. Also, hTERT is activated by c-
MYC protein [31] so the effects of analogue on telomerase inhibi-
tion could be an indirect one. Next, we performed a Western blot
a 100% reduction in c-MYC expression after 24 h (see Fig. 8). We
conclude that both DMZ9 and DMZ13 down regulate c-MYC protein
expression. Whether this is solely due to G-quadruplex stabiliza-
tion or due to a combination of G-quadruplex stabilization and yet
to be identified mechanism remains to be elucidated in future
studies.

C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
271
Fig. 6. Effects of DMZ analogues (DMZ1, DMZ 9, DMZ13), DMZ and TMPyP4 on the PCR stop assay with mutated c-MYC template Pu27-13,14. Compounds were added to the
reaction mixture containing 1x PCR buffer (New England Biolabs), 5
No compound added was treated as control.
m
M Pu27e13.14, 5
mM Pu27rev, 200
m
M dNTPs and 5 units of Taq polymerase (New England Biolabs) separately.
A
C
B
100
80
60
40
20
0
100
100
80
60
40
20
0
48h
48h
48h
80
60
40
20
0
0
3.1
6.2
12.4
0
12.5
25
50
0
2.7
5.5
11
Concentrition of DMZ13 (μM)
Concentrition of TMPyP4 (μM)
Concentrition of DMZ9 (μM)
E
100
80
60
40
20
0
F
100
80
60
40
20
0
D
100
72h
72h
72h
80
60
40
20
0
0
3.1
6.2
12.4
0
12.5
25
50
0
2.7
5.5
11
Concentrition of DMZ13 (μM)
Concentrition of TMPyP4 (μM)
Concentrition of DMZ9 (μM)
Fig. 7. Telomerase activity in lysates of TMPyP4-, DMZ9-or DMZ13-treated MDA-MB-231 cells for 48 h (upper panel) and 72 h (lower panel). Telomerase activity was determined
using the TRAPeze XL Telomerase Detection Kit (Intergen). Lysate (1000 cell-equivalents) was mixed with TRAPeze XL reaction mix containing Amplifuor primers, and incubated for
30 min at 30 ꢀC. Telomerase products were quantitated using a Fluorescence plate reader. A) percentage inhibition of Telomerase activity in TMPyP4 treated MDA-MB-231 cells
relative to untreated control after 48 h. B) percentage inhibition of telomerase activity in DMZ9-treated MDA-MB-231 cells relative to untreated control after 48 h. C) percentage
inhibition of telomerase activity in DMZ13-treated MDA-MB-231 cells relative to untreated control after 48 h. D) percentage inhibition of Telomerase activity in TMPyP4 treated
MDA-MB-231 cells relative to untreated control after 72 h. E) percentage inhibition of telomerase activity in DMZ9-treated MDA-MB-231 cells relative to untreated control after
72 h. F) percentage inhibition of telomerase activity in DMZ13-treated MDA-MB-231 cells relative to untreated control after 72 h.

272
C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
Fig. 8. Western blot analysis of c-MYC protein expression in MDA-MB-231 after treated with DMZ9, DMZ13 and TMPyP4. MDA-MB-231 cells were treated with DMSO vehicle,
DMZ9 (5.45 M and 27.25 M) and TMPyP4 (22.3 M and 111.5 M) for 2 h, 6 h and 24 h. DMZ13 (19.5 M) for 6 h and 24 h. Total protein was extracted and analyzed by Western
m
m
m
m
m
blot with 1:1000 c-MYC (Cell Signaling) and 1:10,000 HRP-linked anti-rabbit IgG (Cell Signaling). *Observed high level of cytotoxicity and low protein levels. The graphs show the
relative density as compared with the DMSO vehicle. Scanned images were analyzed using image J software.
3. Conclusion
concentrations of DNA stock solutions were determined by
measuring the absorbance at 260 nm using the extinction coefficient
values published in the literature [32]. Nuclear magnetic resonance
(NMR) spectra were recorded on Bruker DRX-400 MHz instrument
(1H, 400 MHz; 13C, 100 MHz) or Bruker DRX-500 MHz instrument
(1H, 500 MHz; 13C, 125 MHz). Data for 1H NMR and 13C spectra were
DMZ analogues are easy to synthesize and monoamidine ana-
logues that bear alkyne moieties are selective G-quadruplex
binders with good anticancer properties. These alkyne derivatives
of DMZ therefore serve as good lead molecules that warrant further
studies. Key insights from this study are, G-quadruplex binding or
selectivity are not sole determinants of anticancer efficacy, a fact
that is increasingly being appreciated by the many workers in the
field. For example TMPyP4 and DMZ stabilized G-quadruplexes far
better than all of the alkyne-substituted DMZ analogues yet they
were not the most efficacious anti-cancer agents. In fact DMZ was
not effective at killing any of the three tested cancer cell lines.
Secondly the majority of the DMZ analogues tested had similar G-
quadruplex binding profile and selectivity yet the anticancer
properties of these DMZ analogues varied greatly. Other factors,
such as cell permeation or metabolism or sub-cellular localization
or even alternative therapeutic target(s) could all come into play to
modulate the efficacy of a particular agent. The synthetic tracta-
bility of the DMZ analogues makes it easy to make diverse libraries
to screen and future efforts will be directed at expanding the DMZ
library members and to increase the solubility profiles of the ana-
logues. G-quadruplexes are emerging as important structural ele-
ments that regulate other disease states and so we anticipate that
the molecules described in this manuscript and variants thereof
could find utility in diverse areas, such as anti-HIV therapy.
recorded as follows: chemical shift (d, ppm), multiplicity (s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet), integration, coupling
constant (Hz). High-resolution mass spectra (HRMS) have been
obtained by a TOF instrument with ESI positive mode as the ioni-
zation method.
4.2. Synthesis of the DMZ analogues
4.2.1. Synthesis of alkyne-substituted aromatic amines via
Sonogashira coupling
The syntheses of alkyne-substituted aromatic amines (15e18)
followed the general Scheme 1. A 50 mL Schlenk flask was charged
with aryl iodide (2 mmol, 1.0 eq.), bis-(triphenylphosphine) palla-
dium dichloride (70 mg, 0.05 eq.), cuprous iodide (10 mg, 0.05 eq.),
triphenylphosphine (13 mg, 0.025 eq.), and a stir bar and sealed
with rubber septum [33]. The flask was evacuated and refilled three
times with Argon. Ethynylaniline (1.1 eq.) was added to 10 mL of
distilled dry iPr2NH and degassed together in a separated round
bottom flask for 15 min and then transferred to the Schlenk flask
through cannula. The mixture was stirred for 16 h at room tem-
perature (65 ꢀC in the case of aryl bromide). After completion of the
reaction, the mixture was diluted with ethyl acetate (50 mL) and
the slurry was filtered through a pad of Celite in a sintered glass
funnel (medium frit). The tan solids were additionally washed with
ethyl acetate until the filtrate was nearly colorless. The filtrate was
washed with H2O and brine and dried over magnesium sulfate. The
combined organic fraction filtrates were concentrated in vacuum,
yielding a black solid. The residue was further purified by flash
column chromatography on silica gel using ethyl acetate/hexane
mixture as eluent.
4. Experimental
4.1. General information
Diminazene aceturate (DMZ), 4-aminobenzamidine dihydro-
chloride, cisplatin and most of aromatic amines were purchased
from SigmaeAldrich and used without further purification. DMZ1
was obtained following published procedure [19a].
kis(1-methylpyridinium-4-yl)porphyrin p-Toluenesulfonate
a,b,g,d-Tetra-
(TMPyP4) was purchased from TCI America. 3-(Phenylethynyl)ani-
line and 3-(pyridin-2-ylethynyl)aniline were purchased from
EnamineStore Ltd. c-kit1 22-mer (50-AGG GAG GGC GCT GGG AGG
GAG G-30), F21T 21-mer (50eFAM-GGG TTA GGG TTA GGG TTA GGG-
TAMRAe30), c-kit2 26-mer (50eFAM-CCC GGG CGG GCG CGA GGG
AGG GGA GG-TAMRAe30), k-RAS21R 21-mer (50eFAM-AGG GCG
GTG TGG GAA GAG GGA-TAMRAe30), c-MYC 22-mer (50-FAM-TGA
GGGTGG GTAGGGTGG GTA A-TAMRA-30), duplex 26-mer (50eFAM-
TAT AGC TAT ATT TTT TTA TAG CTA TA-TAMRAe30) and duplex
competitor 26-mer DNA (50-CAATCG GAT CGA ATT CGATCC GAT TG-
30) were purchased from IDT, where FAM is 6-carboxyfluorescein
4.2.2. General procedure for the synthesis of DMZ analogues
The synthesis of DMZ analogues followed previously reported
procedure [19a], see Scheme 2. 4-Aminobenzamidine dihydro-
chloride (212 mg, 1.0 mmol) was added to a stirred solution of 12 N
HCl (0.27 mL) and water (1.5 mL) in a 10 mL flask at 0 ꢀC and stirring
was continued for 15 min. To the mixture was added (dropwise)
cold (~0 ꢀC) NaNO2 solution (76 mg in 0.27 mL water, 1.1 eq.) and
stirring was continued for 15 min before cold (~0 ꢀC) NaOAc solu-
tion (328 mg in 1.5 mL water, 4.0 eq.) was added dropwise over
15 min to adjust the pH to 6.0. Cold (~0 ꢀC) aromatic amine solution
(1.0 mmol in 1.0 mL methanol) was added dropwise to the above
and
TAMRA
is
6-carboxytetramethylrhodamine.
The

C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
273
Scheme 1. Sonogashira coupling to prepare compounds 15e18.
Scheme 2. Facile synthesis of DMZ analogues via diazonium intermediates. Stability test is shown in Fig. S6.
solution and stirring was continued for another 1e12 h at 0 ꢀC. After
Prism Software (Graphpad, La Jolla, CA).
the reaction was completed, the solvent was removed under
reduced pressure. Water (100 mL) was added to the residue and the
aqueous mixture was washed with dichloromethane (2 ꢁ 15 mL).
The aqueous layer was then basified with 2.5% NaOH solution to
make the pH > 10.0. The desired compound was then extracted
from the aqueous layer with ethyl acetate (2 ꢁ 100 mL). The organic
layer was washed with brine and dried with sodium sulfate. Finally,
the solvent was removed under reduced pressure and the final
product was obtained with purity >95%.
4.5. Fluorescence resonance energy transfer (FRET) assay to
determine T1/2 in absence and presence of ligands
All dual-labeled DNAs were diluted from stock (50
mM) to
800 nM in 60 mM potassium cacodylate buffer (pH 7.2) and then
annealed by heating at 85 ꢀC for 10 min followed by cooling to room
temperature in the heating block, following literature precedent
[22]. Compounds were diluted from stock (20 mM in DMSO) to
8
m
M in 60 mM cacodylate buffer (pH 7.2). 25
mL annealed DNA was
4.3. Cell lines and culturing
added to 96-well plate followed by 25 L diluted compounds.
m
Samples were then allowed to incubate for at least 12 h at 4 ꢀC to
come to equilibrium. Measurements were made in triplicate by
using a LightCycler 480 System RT-PCR machine (Roche) and
average values were reported. Fluorescence readings were made
with excitation at 450e495 nm and detection at 515e545 nm,
taken at intervals of 1.2 ꢀC/min in the range 25 ꢀCe95 ꢀC. The se-
quences of labeled oligonucleotides were as follows: F21T 21-mer
(50eFAM-GGG TTA GGG TTA GGG TTA GGG-TAMRAe30), c-kit2 26-
mer (50eFAM-CCC GGG CGG GCG CGA GGG AGG GGA GG-
TAMRAe30), k-RAS21R 21-mer (50eFAM-AGG GCG GTG TGG GAA
GAG GGA-TAMRAe30), c-MYC 22-mer (50-FAM-TGA GGG TGG GTA
GGG TGG GTA A-TAMRA-30) and duplex 26-mer (50eFAM-TAT AGC
TAT ATT TTT TTA TAG CTA TA-TAMRAe30).
The following human cell lines were purchased from ATCC
(ATCC, Manassas, VA): prostate cancer cells (PC-3), triple negative
breast cancer cells (MDA-MB-231), ovarian cancer cells (OVCAR-3),
and normal fibroblast cells (MCR5A). Normal bone marrow (NBM)
was purchased from Lonza (Maryland). MDA-MB-231 cells were
grown at 37 ꢀC with 5% CO2 atmosphere with DMEM (Life tech-
nologies, Carlsbad, CA) supplemented with heat-inactivated 10%
(V/V) fetal bovine serum. NBM was cultured in RPMI and OVCAR-3
and PC-3 cells were grown at 37 ꢀC with 5% CO2 atmosphere with
RPMI (Life technologies, Carlsbad, CA) supplemented with heat-
inactivated 10% (V/V) fetal bovine serum. Cell lines were grown
and maintained according to ATCC recommendations.
4.4. GI50 determination
4.6. FRET DNA melting in competition with duplex DNA
Cell lines were seeded into 96-well plates the afternoon prior to
treatment. Cell seeding number was determined by growing cells in
log phase growth during drug treatment. Approximately 18 h later,
compounds were semi-serially diluted in dimethyl sulfoxide
(DMSO) and then growth medium, and added to cells. Plates were
incubated for 72 h prior to addition of WST-1 (Promega, Madison
WI). Plates were read after 4 additional hours of incubation at 37 ꢀC
using a Bio-Tek Synergy HT plate reader (Bio-Tek, Winooski, VT).
Data was analyzed, graphed and GI50s generated using GraphPad
FRET competition assay was performed by adding 4
and 200
M duplex competitor 26-mer DNA (50-CAA TCG GAT CGA
ATT CGA TCC GAT TG-30) in the presence of 0.4
M c-MYC G-
quadruplex and 4 M DMZ analogues. Both c-MYC G-quadruplex
and duplex competitor 26-mer DNA were diluted in 60 mM po-
tassium cacodylate buffer (pH 7.2) and then annealed by heating at
85 ꢀC for 10 min followed by cooling to room temperature in the
heating block. Experiments were performed in triplicate.
mM, 40 mM
m
m
m

274
C. Wang et al. / European Journal of Medicinal Chemistry 118 (2016) 266e275
Table 2
Sequences of Oligomers used in the PCR Stop Assay.
Name of
Sequence
Description
oligomers
Pu27
50-
Partial sequence of promoter of oncogene c-MYC.
TGGGGAGGGTGGGGAGGGTGGGGAAGG-
30
Pu27-13,14
Pu-mutant
Pu27rev
50-
A mutated Pu27 (a control template) whose guanines at 13th and 14th positions were changed to adenosines.
TGGGGAGGGTGGAAAGGGTGGGGAAGG-
30
50-
Another mutated Pu27 (another control template) whose qunines at 4th, 8th, 13th, 14th and 17th positions
TGGAGAGAGTGGAAAGAGTGGGGAAGG- were changed to adenosines.
30
50-ATCGATCGC TTCTCGTCCTTCCCCA-30 Complementary sequence of Pu27
4.7. PCR stop assay
At each time point the cells were harvested, washed with 1x PBS
and lysed in radioimmunoprecipitation (RIPA) buffer with protease
inhibitors. The protein in the resulting lysates was quantified using
BCA (Pierce) and subjected to SDS PAGE, then subsequently trans-
ferred to Nylon for Western blot analysis with 1:1000 c-MYC (Cell
Signaling) and 1:10,000 HRP-linked anti-rabbit IgG (Cell Signaling).
Scanned images were analyzed for densitometry using ImageJ
This assay was performed by modifying a previously published
protocol [34]. Sequences of the tested oligomers, Pu27, Pu27-13,14,
Pu-mutant and the corresponding complementary oligomer
Pu27rev were presented in Table 2.
To the PCR reaction mixture (25
Biolabs), 5 M Pu27 and Pu27rev oligomers, various concentrations
of ligands (DMZ, TMPyP4, DMZ1, DMZ9 and DMZ13) were added
and incubated at 4 ꢀC for 6 h. After that, 200
M dNTPs, 5 units of
mL),1x PCR buffer (New England
m
software and b-actin as a loading control. Conditions were
compared by percent to vehicle control for each time point.
m
Taq DNA polymerase (New England Biolabs) were added and PCR
reactions were performed in a thermocycler, with the following
conditions: 94 ꢀC for 3 min, followed by 20 repeated cycles each
having 94 ꢀC for 30 s, 58 ꢀC for 30 s, and 72 ꢀC for 30 s. After PCR,
amplified products were analyzed on 12% polyacrylamide gel with
1 ꢁ TBE and SYBR Gold stained. This assay was also performed
under the same conditions with Pu27-13,14 and Pu27rev, and Pu-
mutant and Pu27rev.
Acknowledgment
We thank NSF (Award# CHE 1518006) and UMD Graduate
Dean's Dissertation Fellowship (JZ) for funding.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.030.
4.8. Telomerase activity assay
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