A dual topoisomerase inhibitor of intense pro-apoptotic and antileukemic nature for cancer treatment

Article abstract of DOI:10.1002/cmdc.201700026

Classic cytotoxic drugs remain indispensable instruments in antitumor therapy due to their effectiveness and a more prevalent insensitivity toward tumor resistance mechanisms. Herein we describe the favorable properties of 6-(N,N-dimethyl-2-aminoethoxy)-11-(3,4,5-trimethoxyphenyl)pyrido[3,4-c][1,9]phenanthroline (P8-D6), a powerful inducer of apoptosis caused by an equipotent inhibition of human topoisomerase I and II activities. A broad-spectrum effect against human tumor cell lines at nanomolar concentrations, as well as strong antileukemic effects, were shown to be superior to those of marketed topoisomerase-targeting drugs and dual topoisomerase inhibitors in clinical trials. The facile four-step synthesis, advantageous drugability properties, and initial in vivo data encourage the application of P8-D6 in appropriate animal tumor models and further drug development.

Full text of DOI:10.1002/cmdc.201700026

Accepted Article
Title: A Dual Topoisomerase Inhibitor of Intense Pro-Apoptotic and
Antileukemic Nature for Cancer Treatment
Authors: Christopher Meier, Tamara N. Steinhauer, Fabian Koczian,
Birte Plitzko, Katharina Jarolim, Ulrich Girreser, Simone Braig,
Doris Marko, Angelika M. Vollmar, and Bernd Clement
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A Dual Topoisomerase Inhibitor of Intense Pro-Apoptotic and
Antileukemic Nature for Cancer Treatment
Christopher Meier[a], Tamara N. Steinhauer[a], Fabian Koczian[b], Birte Plitzko[a], Katharina Jarolim[c],
Ulrich Girreser[a], Simone Braig[b], Doris Marko[c], Angelika M. Vollmar[b] and Bernd Clement*[a]
Abstract: Classic cytotoxic drugs constantly remain an indispen-
sable instrument in antitumor therapy due to their effectiveness and
a more prevalent insensibility against tumor resistance mechanisms.
We describe the favorable properties of P8-D6, a powerful inductor
of apoptosis caused by an equipotent inhibition of human topoiso-
merase I and II activities. A broad spectrum effect against human
tumor cell lines in nanomolar concentrations as well as strong anti-
leukemic effects were shown and proven to be superior to marketed
topo-targeting drugs and dual topoisomerase inhibitors in clinical
trials. The facile four-step synthesis, advantageous drugability
proper-ties, and first in vivo data encourage the application of P8-D6
in appropriate animal tumor models and further drug development.
evaluated in phase I/II clinical trials, pyrazoloacridine (PZA) 1,
the benzopyridoindole intoplicine 2, the phenazine derivatives
XR11576
3
and XR5944 4, batracylin
5
and the
indenoquinolinone TAS-103 6 (Scheme 1), as well as the
etoposide derivative tafluposide and the homocamptothecin
elomotecan.[1,14,21-28] Although pre-clinical studies were very
promising for these derivatives, their effectiveness could not be
transferred to the clinic yet.
The human topoisomerase (topo) enzyme family plays a crucial
role in a plenty of cellular actions involving movement and the
solution of torsional strains of the genetic material. Its detailed
intra-nuclear functions have been studied extensively and are
well described in literature.[1-5] As these operations are required
for vital processes such as DNA-replication, transcription,
chromatin assembly and DNA-methylation, topoisomerase types
I and II emerged as fundamental targets for the chemothera-
peutic treatment of malignant tumor diseases.[6-10] Approved
topo-targeting drugs like camptothecin derivatives, acting as
topo I poisons, and primarily epipodophyllotoxins and anthra-
cyclins that interfere with topo II are highly effective front-line
cytostatics for the treatment of systemic cancers and solid tumor
types.[5,8] The efficacy and experience in clinical use on the one
hand, but also dose- and/or therapy-limiting side effects and
upcoming resistance mechanisms against these agents on the
other demonstrate a high medical need of novel topo-targeting
molecules.[1,5,6,11-13] Many efforts were made designing dual topo
I/II inhibitors that exhibit a high cytotoxicity and a strong affinity
to both enzyme classes. In this regard, the complete abolish-
ment of topological processes in the tumor cell was shown to be
extremely effective in vitro and in vivo in a broad spectrum of
tumor types.[1,14-20] Consequently, dual topo I/II inhibitors were
Scheme 1. Dual topoisomerase I/II inhibitors in clinical development.[1,14,21-27]
Naturally occurring benzo[c]phenanthridine alkaloids offer a
broad variety of pharmacological activities.[29-33] Among them the
quarternary ammonium salts fagaronine and nitidine 7 were
found to possess moderate dual topo I/II poisoning activity.[34-36]
In the mid-90’s our laboratories evaluated
straightforward synthetic route to 11-substituted
a
facile and
6-
aminobenzo[c]-phenanthridines.[37] Antitumoral activity of
derivatives 8 was observed to be high in vitro and in vivo, but
unfortunately the dissatisfactory physicochemical properties
(solubility: lower µM range, logD(7.4) ~5) limited further
development of this substance class (Scheme 2).[38,39]
[a]
Dr. C. Meier, Dr. T. N. Steinhauer, Dr. B. Plitzko, Dr. U. Girreser and
Prof. Dr. B. Clement
Department of Pharmaceutical and Medicinal Chemistry
Pharmaceutical Institute of the Christian-Albrechts-Universität Kiel;
Gutenbergstraße 76, 24118 Kiel (Germany)
E-mail: bclement@pharmazie.uni-kiel.de
[b]
[c]
F. Koczian, Dr. S. Braig and Prof. Dr. A. M. Vollmar
Department of Pharmacy – Center for Drug Research
Pharmaceutical Biology; University of Munich
Butenandtstraße 5-13, 81377 Munich (Germany)
K. Jarolim and Prof. Dr. D. Marko
Department of Food Chemistry and Toxicology
University of Vienna
Währinger Straße 38, 1090 Vienna (Austria)
Supporting information for this article is available at:
Scheme 2. Natural product-inspired development of aza-analogous benzo[c]-
phenanthridines.[37-42]
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To overcome these disadvantages we synthesized aza-analogs
of benzo[c]phenanthridines (9-14) using different o-methyl-
hetarenecarbonitriles and the core system was systematically
adapted and optimized regarding both physicochemical
properties and cytotoxicity (Scheme 2).[39-42] Applying the
concept of an amino-alkyl side chain linked to a planar core
system to achieve DNA-intercalation as well as anti-
topoisomerase activity,[18] the pyrido-[3,4-c][1,9]phenanthroline
P8-D6 19 was found to be the most potent derivative of our
compound library by far. 19 was accessible via a simple four-
step synthetic protocol commencing with commercially available
starting materials to form the 6-amino-11,12-dihydro derivative
17 in a two-fold ring closure including one-pot synthesis
(Scheme 3).[39]
independent DNA-attraction as it has been reported for structu-
rally related substances before is postulated likewise.[18, 46]
Table 1. Cytotoxicity (µM) of topoisomerase targeting agents in comparison.
∑
ꢀꢁ
ꢂꢃ
[a]
Compound
Leukemia[b]
NSCLC[b]
Colon cancer[b]
ꢄꢃ
Topoisomerase I inhibitors
CPT[c]
0.04
0.84
0.04
0.02
0.20
0.01
0.02
0.08
1.70
0.09
Irinotecan
Topotecan
0.07
0.02
Topoisomerase II inhibitors
Etoposide
Teniposide
Doxorubicin
1.32
0.40
0.14
0.21
0.05
0.07
0.79
2.51
0.71
0.26
0.27
0.07
Dual topoisomerase I/II inhibitors
P8-D6
0.05
0.20
0.53
0.02
0.17
0.47
0.03
0.17
0.23
0.06
0.12
0.50
PZA[d]
Intoplicine
Data taken from DTP-NCI databank, NCI-60 screening data set from
09/2014.[45] [a] Mean growth inhibition 50% over all 60 cell lines. [b] Average
GI50 for all comparable cell lines of corresponding tumor type. [c] Campto-
thecin. [d] Pyrazoloacridine.
Using flow cytometric analysis of Annexin V-PE and 7-AAD
stained HeLa cells as well as the detection of caspase-mediated
PARP-cleavage it could be demonstrated that P8-D6 is a strong
and rapid inductor of apoptosis (Figure 1, C-G).[47,48] Low micro-
molar concentrations of P8-D6 showed pro-apoptotic effects al-
ready after 8 h of incubation whilst no PARP cleavage was
observed for etoposide (ETO) or the dual topo inhibitor PZA
before reaching a concentration of 100 µM (Figure 1, D+E). After
24 h incubation time, 100-1000 fold lower amounts of P8-D6
were sufficient to induce apoptosis in cells compared to the
cultivation in the presence of etoposide or PZA (Figure 1, D+E).
PZA treatment also led to partial necrosis of the HeLa cells at
these concentrations (Figure 1, F).
P8-D6 was furthermore tested for antileukemic effects on Jurkat
ALL cells. With 30% of all childhood cancers, leukemia is the
most common cancer in children with acute lymphoid leukemia
(ALL) being most prevalent.[49] The Jurkat ALL cell line was
shown to be sensitive towards P8-D6 treatment in accordance to
the NCI-60 results. Using proliferation assays, flow cytometric
analysis (referred to Nicoletti et al.) and Western Blotting, strong
growth-inhibitory and pro-apoptotic effects at low nanomolar
concentrations could be confirmed (Figure 2, A-C).[50,51]
Scheme 3. Four-step synthesis of P8-D6 19 starting from commercially
available building blocks.[39,40,43,44]
Dehydratation with Pd/C followed by diazotation with NaNO2 in
acidic aqueous medium yielded 18.[40,43] The final step gave the
O-alkylated derivative P8-D6 19 via Mitsunobu reaction in an
overall yield of 12% (Scheme 3).[44]
Cytotoxicity was evaluated by incubation of 60 human tumor cell
lines of nine different tumor types with P8-D6 (NCI-60 DTP
human tumor cell line screening, NCI, Bethesda, Maryland).
The substance showed an outstanding overall activity (expres-
sed as average growth inhibition 50% over all 60 cell lines) of
49 nM in the range of the natural alkaloid camptothecin. Its
growth-inhibitory effect was 4-10 fold higher compared with
other dual topo I/II inhibitors recently elaborated in clinical trials
(Table 1). Besides the good response of all tumor types, a
sensitivity of leukemia cells and NSCLC cells (non-small cell
lung cancer) towards P8-D6 was noticed. [45]
P8-D6 is able to inhibit the catalytic activities of both type I and II
topoisomerases in low micromolar concentrations in vitro (Figure
1, A+B). In this connection there is no significant discrimination
between topo IIα, which reaches maximum protein levels in
G2/M-phase in human cells, and topo IIβ that is expressed
independently of proliferative status and cell cycle.[1] A topo-
Propidium iodide staining and FACS analysis was also used to
evaluate the effects of P8-D6 on the cell cycle. As shown in
Figure 2, D the presence of the substance led to an arrest in the
G2-phase as expected for a topo inhibitor.[52]
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Additionally, dose-dependent phosphorylation of histone H2A.X
(γH2A.X) in Jurkat ALL cells clearly indicates DNA double-strand
breaks as main apoptotic trigger of P8-D6 (Figure 2, E).[53]
In order to examine selectivity towards tumor cells, the cytotoxic
effect of P8-D6 was also investigated on healthy human
lymphocytes isolated from whole blood. Results indicate no
response of lymphocytes towards P8-D6 concentrations that are
already intensively cytotoxic and pro-apoptotic to the leukemia
cell line (Figure 2, F).
Recently, the selective protein disulfide isomerase inhibitor
PS89 was found to sensitize cancer cells towards etoposide
treatment (structure shown in Figure 2, G).[54] In this regard, we
analyzed the effects of P8-D6 in combination with the chemo-
sensitizing compound PS89 pointing out a clearly enhanced
growth inhibition and triggering of apoptosis of per se sub/less
toxic P8-D6 concentrations towards Jurkat ALL cells while
incubated with PS89 (Figure 2, H+I).
A
B
C
D
E
F
G
Figure 1. P8-D6 is a highly pro-apoptotic dual topoisomerase inhibitor. A) Inhibition of topoisomerase I activity (relaxation assay). B) Inhibition of topoisomerase
IIα and IIβ (decatenation assay). C) HeLa cells after incubation (24 h) with P8-D6 or etoposide. D+E) PARP apoptosis assay in HeLa cells (ETO/PZA reference).
F) Flow cytometric analysis of pro-apoptotic effects on HeLa cells (after 24 h). G) Quantitative determination of apoptotic cells using flow cytometry (after 24 h). ***
p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 significant difference towards DMSO control.
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Figure 2. P8-D6’s strong antileukemic effects and activity on healthy human lymphocytes. A) Dose-dependent inhibition of Jurkat ALL cell proliferation by P8-
D6 treatment (Cell TiterBlue® assay, 72 h). B) Dose-dependent induction of apoptosis in Jurkat ALL cells by P8-D6 treatment (Nicoletti assay, 24 h). C)
Decrease of Pro-Caspase-3 level and PARP-cleavage in Jurkat ALL cells as hallmarks of apoptosis (P8-D6 treatment, 24 h); Actin = loading control. D) Cell
cycle arrest in the G2-phase by P8-D6 treatment (Propidium iodide staining, 24 h). E) Phosphorylation of Histone H2A.X as sensitive proof of DNA double-
strand breaks in Jurkat ALL cells (P8-D6 treatment, 24 h). F) No pro-apoptotic effects of P8-D6 on healthy human lymphocytes (FACS analysis, 24 h). G) The
chemosensitizing protein disulfide isomerase inhibitor PS89. H) Growth inhibition of Jurkat ALL cells by P8-D6 in combination with PS89 (Cell TiterBlue® assay,
72 h). I) Induction of apoptosis in Jurkat ALL cells by P8-D6 in combination with PS89 (Nicoletti assay, 24 h).
In addition to its pharmacological advantages, P8-D6 exhibits a
physicochemical profile that encourages its development as
promising drug candidate in antineoplastic chemotherapy. A
solubility of approximately 1 mM in phosphate buffer pH 7.4 as
well as the accordance of Lipinski Ro5-parameters for the pre-
diction of oral bioavailability including a logD of 3.7 would allow
even a peroral administration (parameters determined using
HPLC analytics).[55]
This MTD (maximum tolerated dose) study revealed a good
tolerability of 1 mg/kg per body weight (bw) of P8-D6 with regard
to further in vivo efficacy studies (Figure 3). In higher concentra-
tions the substance showed dose-dependent toxicity (para-
meters: body weight, clinical signs), while loss of body weight
and apathy of study animals in the 5 mg/kg group were rever-
sible over the period of study (Figure 3).
Contrary to macroscopic alterations that had been reported for
i.v. etoposide low and high dose treated mice in comparable
studies,[56] no significant changes in organs could be observed
for study animals that received P8-D6 injection up to 5 mg/kg bw
(necropsy findings).
To evaluate first in vivo tolerability of P8-D6, female athymic
nude mice (n = 9 vs. 9 mice receiving PBS-control) were treated
intravenously with different unique doses of P8-D6 in PBS.
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Since tumor cells require an enormous machinery of compo-
nents involved in DNA-related growth and replication processes,
the human topoisomerase family continuatively represents a
pivotal target in antitumor therapy. In summary, our drug candi-
date P8-D6 introduced herein exhibits a plenty of advantages.
The strong and equipotent inhibition of topo I, IIα and IIβ with a
for a dual topoisomerase inhibitor hitherto undescribed excellent
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10.1002/cmdc.201700026
ChemMedChem
COMMUNICATION
Entry for the Table of Contents
Layout 1:
COMMUNICATION
P8-D6 is a novel designed drug can-
didate for antineoplastic chemothera-
py which acts as highly pro-apoptotic
dual topoisomerase I and II inhibitor.
A broad spectrum effect against
human tumor cell lines, its strong anti-
leukemic activity, the facile synthetic
access, advantageous drugability
properties as well as first in vivo data
encourage the application of P8-D6 in
appropriate in vivo efficacy studies
and further drug development.
Christopher Meier, Tamara N.
Steinhauer, Fabian Koczian, Birte
Plitzko, Katharina Jarolim, Ulrich
Girreser, Simone Braig, Doris Marko,
Angelika M. Vollmar and Bernd
Clement*
Page No. – Page No.
A Dual Topoisomerase Inhibitor of
Intense Pro-Apopotic and Anti-
leukemic Nature for Cancer
Treatment
This article is protected by copyright. All rights reserved.