Simple but powerful: Phenanthroline-based small molecules for cellular imaging and cancer screening

Article abstract of DOI:10.1016/j.bmcl.2011.06.051

A two-step synthetic procedure gives highly fluorescent phenanthroline molecular probes. The compounds localize in the endoplasmic reticulum and their potential as bioactive probes was evaluated. The materials are quickly taken up by living cells within 5

Full text of DOI:10.1016/j.bmcl.2011.06.051

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4385–4388
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Simple but powerful: Phenanthroline-based small molecules for cellular
imaging and cancer screening
Natalia N. Sergeeva a,b, , Marion Donnier-Marechal b, Gisela M. Vaz a, Anthony M. Davies c,
⇑
Mathias O. Senge a,b
a Medicinal Chemistry, Institute of Molecular Medicine, Trinity Centre for Health Sciences, Trinity College Dublin, St. James’s Hospital, Dublin 8, Ireland
b SFI Tetrapyrrole Laboratory, School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
c Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 8, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
A two-step synthetic procedure gives highly fluorescent phenanthroline molecular probes. The com-
pounds localize in the endoplasmic reticulum and their potential as bioactive probes was evaluated.
The materials are quickly taken up by living cells within 5 min. Preliminary in vitro studies have shown
that these compounds are selective to esophageal cancer cells and can be used as selective markers in
intracellular cancer diagnostics. The materials show a remarkable cytotoxicity towards cancer cells vs
normal as 7–1.
Received 28 May 2011
Revised 10 June 2011
Accepted 11 June 2011
Available online 17 June 2011
Keywords:
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Esophageal cancer
Fluorescence
Phenanthroline
Imaging
Fluorescence cell imaging has garnered significant attention in
the scientific community over the past years.1 Due to the clinical
significance of the topic, a vast body of data has been generated
for compounds ranging from simple organic molecules to highly
sophisticated fluorescent proteins and bionanotags.2 High-content
screening3 is a high-throughput technical approach based on fluo-
rescent materials targeting cellular functions and processes, and is
widely used in biomedicine, cancer research and drug develop-
ment. These fully automated systems provide quick analyses and
generate a massive amount of information in a relatively short per-
iod of time. For example, they can deliver 108 individual physiolog-
ical measurements at the cellular level in less than a day.4 This
integrated approach also allows screening under more relevant
physiological and biological conditions in comparison to classical
biochemical scenarios.
Cancer is a plague of the 21st century, increasing the number of
its victims every year due to overall population aging, environmen-
tal conditions and life style choices. Esophageal cancer is the eighth
of most common type and sixth most common cause of death from
cancer worldwide.5,6 Due to late detection, the survival rates in
Europe are about 10%.6,7 It has shown a dramatic increase in Eur-
ope8 and North America in the past few years.9,10 According to
the Esophageal Cancer Research Fund (OCRF), the rates of the
disease in Ireland and UK are amongst the highest in Europe and
increase faster than in other European countries.11 This necessi-
tates the development of better screening techniques for this and
other types of cancer.
Small fluorescent molecules offer significant potential in this
area above.12 We have found that the small molecules such as phe-
nanthroline derivatives can be used as versatile materials for cellu-
lar imaging and cancer screening studies. They show strong
absorption in the 280–400 nm region, exhibit high fluorescence
upon excitation and large Stokes shifts. These optical properties
do not alter drastically with changes of the solvent systems and
the compounds are stable in aqueous media with no decay in fluo-
rescence over time.13a The compounds 2 and 3 are soluble in aqua-
tic phosphate buffered salen solution (PBS) and exhibit a strong
fluorescence in PBS at pH 7.4 upon excitation at 405 nm for our
studies (Fig. 1).
These highly fluorescent compounds can be synthesized easily
and on a large scale making them commercially affordable and
cheap. The compounds 2 and 3 have been prepared via two-step
procedure in the yields of 80% and 66%, respectively.13a Commer-
cially available 1,10-phenanthroline can be easily oxidized with
KBr in H2SO4-HNO3 to 1,10-phenanthrolin-5,6-dione 1 in 95% fol-
lowed by reaction with an appropriate aldehyde and NH4OAc in
glacial acetic acid (Scheme 1).
Studies on localization, incubation times, concentration ranges,
and toxicity were carried out in two cell lines OE21 (human
⇑
Corresponding author.
E-mail address: sengem@tcd.ie (N.N. Sergeeva).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.051

4386
N. N. Sergeeva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4385–4388
The intracellular localization of the dyes 2 and 3 was studied by
confocal microscopy with a LSM 700 confocal laser scanning
microscope. Living HET-1A cells were incubated with the commer-
cially available ER red-tracker for 20 min at 37 °C and stained with
3 (18 lM). The ER tracker was illuminated at 560 nm without any
interference from the dye 3, and images were collected at 575 nm.
The fluorescence of 3 was detected at 420–480 nm upon excitation
at 405 nm. Superimposition of the intracellular images of the red
ER stain and 3 confirmed that the latter accumulates in the endo-
plasmic reticulum (Fig. 3).
Similar results were obtained for 2 as well.12a The experiments
were carried out in OE21 live-cells and showed the same localiza-
tion of the dyes in both cell lines. Moreover, we carried out the
same in vitro experiments using fixed cells. Noteworthy, ER-track-
er has to be administered to living, incubated, and if an experiment
requires cells can be fixed later. In our case the compounds 2 and 3
can be used to stain living as well as fixed cells, both exhibit the
same intracellular localization.
Figure 1. Emission spectra of 2 and 3 at excitation of 405 nm in PBS at pH 7.4
(10 lM).
Additionally, both dyes show remarkable cytotoxicity in cancer
cells and can be used as potential therapeutics for cancer treat-
ment. Cytotoxicity tests were performed on both cell lines and car-
ried out using an MTT assay.13 The cells were incubated with 2 and
CHO
O
N
3 for 24 h at a concentration range of 0.5–15 l
M in DMSO13 (Table
O
KBr
H2SO4-HNO3
+
1).
N
N
(85-90%)
Overall, the cytotoxicity shown is very high and comparable to
established anti-cancer drugs, for example, cisplatin, carboplatin
and 5-fluorouracil, which are used in combination with radiother-
apy.18 Notably, compounds 2 and 3 are more cytotoxic to cancer
cells than for normal HET-1A cells. Compound 2 is in four times
and 3 is seven times more effective towards the cancer cells com-
pared to the normal cell line.
N
R
1
NH4OAc
AcOH
H
N
N
N
2 (80%) R: Br
3 (66%) R:TMS
R
N
Another remarkable feature of compounds 2 and 3, especially
compound 2, is their potential to be used as a probe for cancer
screening in vitro. A few methods have been established to diag-
nose cancer in vitro, for example, antibody-based immunodiagnos-
tic assays involving monoclonal antibodies. However they are
expensive and difficult to perform.19 We evaluated the different
parameters based on localization and intensity of 2 and 3 in both
OE21 and HET-1A cell lines using cell fluorescence analysis. The
experiments were carried out on ca. 1500 cells for each cell line
at different concentrations and incubation times (20 min to 2 h).
Total area and intensity of the inclusions (distribution of the mate-
rials within cytoplasm), cell intensity and cell intensity CV20
(excluding nuclei) were chosen for use in high-content screening
as the key elements. The analysis can already be performed after
20 min of incubation in living cells. The intensity of the inclusions
for both cell lines remains the same and it can be used as a quality
control to check an even probe distribution. The analysis shows a
significant difference between the two cell lines using cell intensity
CV, which is a normalized parameter, describing the co-efficient of
cell intensity variation (Fig. 4).
Scheme 1. Synthesis of compounds 2 and 3.
esophageal squamous carcinoma) and HET-1A (human normal
esophageal epithelium).13a An analysis and evaluation of the
HET-1A and OE21 live-cells stained with the novel compounds at
different time points (5 min to 24 h) and concentration ranges
(2–27 lM) was performed with high-content screening techniques
using the IN Cell 1000 and IN Cell Investigator Image Analysis
Software.
Remarkably, cellular images could be collected after 5 min of
incubation for both compounds in contrast to some metal com-
plexes14 for which longer times are required (Fig. 2).
The dyes 2 and 3 accumulate in the endoplasmic reticulum (ER).
The ER is vital cellular organelle15 which is involved in the regula-
tion of crucial cell events such as intracellular signaling (ER per-
forms a calcium storage function involving release and uptake of
Ca2+) and participates in the initiation of apoptotic cell death
pathways.16,17
Figure 2. IN Cell images of the OE21 live-cells at 405 nm irradiation and 535 nm emission: (a) 2, 5 min, 9 lM; (b) 3, 20 min, 27 lM.

N. N. Sergeeva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4385–4388
4387
Figure 3. Confocal fluorescence images of HET-1A live-cells incubated with: (a) 3; (b) red ER tracker; (c) superimposition of 3 and red ER tracker.
Table 1
cancer treatment. Additionally, intracellular multiparametric anal-
ysis using high-content screening technique indicates a high pros-
pect to employ 2 and 3 as cancer diagnostics. Currently we are
investigating the relative cytotoxicity of these compounds in rela-
tion to established cancer drugs, are expanding the tests to other
cell lines and focus on the intracellular uptake mechanism.
Cytotoxicity of 2 and 3. IC50 values for OE21 and HET-1A cell lines
Cell line
Compound
IC50 value, lM (lg/ml) SE
OE21
Het-1a
OE21
2
2
3
3
0.77 (0.29) 0.13
3.15 (1.18) 0.08
0.44 (0.16) 0.005
3.02 (1.11) 0.32
Het-1a
IC50, inhibitory concentration at 50% survival; SE, standard error.13
Acknowledgments
This work was supported by funding from the Health Research
Board (translational research award 2007 TRA/2007/11), Science
Foundation Ireland (SFI P.I. 09/IN.1/B2650) and Fundação da Ciên-
cia e Tecnologia.
Thus, the assay described here can be used as a primary evalu-
ation analysis of abnormal vs normal cells in vitro.
In conclusion, compounds 2 and 3 can be used in bioassays as
fluorescent markers in living cells. The incubation times are very
short and initial images can be acquired after 5–20 min. Localiza-
tion studies showed that the dyes accumulate in the endoplasmic
reticulum and can be used as facile ER-markers. The materials
proved to be cytotoxic with a remarkable preference for cancer
cells and therefore can be considered as potential candidates for
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.051.
Figure 4. IN Cell analysis of the HET-1A and OE21 live-cells incubated for 20 min and 2 h with 2.

4388
N. N. Sergeeva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4385–4388
11. (a) Beddy, P.; Howard, J.; McMahon, C.; Knox, M.; de Blacam, C.; Ravi, N.;
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