A Novel Conjugate of Bis[((4-bromophenyl)amino)quinazoline], a EGFR-TK Ligand, with a Fluorescent Ru(II)-Bipyridine Complex Exhibits Specific Subcellular Localization in Mitochondria

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00608

The epidermal growth factor receptor (EGFR) is a key target in anticancer research, whose aberrant function in malignancies has been linked to severe irregularities in critical cellular processes, including cell cycle progression, proliferation, differentiation, and survival. EGFR mutant variants, either transmembrane or translocated to the mitochondria and/or the nucleus, often exhibit resistance to EGFR inhibitors. The ability to noninvasively image and quantify EGFR provides novel approaches in the detection, monitoring, and treatment of EGFR-related malignancies. The current study aimed to deliver a new theranostic agent that combines fluorescence imaging properties with EGFR inhibition. This was achieved via conjugation of an in-house-developed ((4-bromophenyl)amino)quinazoline inhibitor of mutant EGFR-TK, selected from a focused aminoquinazoline library, with a [Ru(bipyridine)₃]²⁺ fluorophore. A triethyleneglycol-derived diamino linker featuring (+)-ionizable sites was employed to link the two functional moieties, affording two unprecedented Ru conjugates with 1:1 and 2:1 stoichiometry of aminoquinazoline to the Ru complex (mono-quinazoline-Ru-conjugate and bis-quinazoline-Ru-conjugate, respectively). The bis-quinazoline-Ru-conjugate, which retains an essential inhibitory activity, was found by fluorescence imaging to be effectively uptaken by Uppsala 87 malignant glioma (grade IV malignant glioma) cells. The fluorescence imaging study and a time-resolved fluorescence resonance energy transfer study indicated a specific subcellular distribution of the conjugate that coincides with that of a mitochondria-targeted dye, suggesting mitochondrial localization of the conjugate and potential association with mitochondria-translocated forms of EGFR. Mitochondrial localization was further documented by the specific concentration of the bis-quinazoline-Ru-conjugate in a mitochondrial isolation assay.

Full text of DOI:10.1021/acs.molpharmaceut.9b00608

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Article
A Novel Conjugate of Bis[((4-Bromophenyl)amino)quinazoline], a
EGFR-TK Ligand, with a Fluorescent Ru(II)-Bipyridine Complex,
Exhibits Specific Subcellular Localization in Mitochondria
Rashid Ilmi, Eleni Tseriotou, Panayiota Stylianou, Yiota A. Christou, Iakovia Ttofi,
Nikolas Dietis, Costas Pitris, Andreani D. Odysseos, and Savvas N Georgiades
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.9b00608 • Publication Date (Web): 11 Sep 2019
Downloaded from pubs.acs.org on September 13, 2019
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A Novel Conjugate of Bis[((4-Bromophenyl)amino)quinazoline], a EGFR-TK
Ligand, with a Fluorescent Ru(II)-Bipyridine Complex, Exhibits Specific
Subcellular Localization in Mitochondria
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Rashid Ilmi,†,ǂ Eleni Tseriotou,‡ Panayiota Stylianou,† Yiota A. Christou,† Iakovia Ttofi,‡ Nikolas
Dietis, Costas Pitris, Andreani D. Odysseos, * and Savvas N. Georgiades *
║
†,├,
‡,
├
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†
. EPOS-Iasis, R&D, 5 Karyatidon Street, Nicosia 2028, Cyprus
‡
. Department of Chemistry, University of Cyprus, 1 Panepistimiou Avenue, Nicosia 2109, Cyprus
║
. Medical School, University of Cyprus, 1 Panepistimiou Avenue, Nicosia 2109, Cyprus
├. Department of Electrical and Computer Engineering, 1 Panepistimiou Avenue, Nicosia 2109, Cyprus
ǂ. Current address: Department of Chemistry, College of Science, Sultan Qaboos University, Al Koudh, Muscat 123, Oman
*. These authors have contributed equally to the manuscript. Corresponding author, E-mail: georgiades.savvas@ucy.ac.cy,
Phone: +357 22892779, Fax: +357 22895456
ABSTRACT
The Epidermal Growth Factor Receptor (EGFR) is a key target in anticancer research, whose aberrant
function in malignancies has been linked to severe irregularities in critical cellular processes, including
cell cycle progression, proliferation, differentiation and survival. EGFR mutant variants, either
transmembrane or translocated to the mitochondria and/or the nucleus, often exhibit resistance to
EGFR inhibitors. The ability to non-invasively image and quantify EGFR provides novel approaches in
the detection, monitoring and treatment of EGFR-related malignancies. The current study aimed to
deliver a new theranostic agent that combines fluorescence imaging properties with EGFR inhibition.
This was achieved via conjugation of a in house-developed ((4-bromophenyl)amino)quinazoline
inhibitor of mutant EGFR-TK, selected from a focused aminoquinazoline library, with a
[Ru(bipyridine)3]2+ fluorophore. A triethyleneglycol-derived diamino linker featuring (+)-ionizable
sites was employed to link the two functional moieties, affording two unprecedented Ru-conjugates
with 1:1 and 2:1 stoichiometry of aminoquinazoline to Ru-complex (mono-quinazoline-Ru-conjugate
and bis-quinazoline-Ru-conjugate, respectively). The bis-quinazoline-Ru-conjugate, which retains an
essential inhibitory activity, was found by fluorescence imaging to be effectively uptaken by U87MG
(grade IV malignant glioma) cells. The fluorescence imaging study and a time-resolved fluorescence
resonance energy transfer study (TR-FRET) indicated a specific subcellular distribution of the
conjugate that coincides with that of a mitochondria-targeted dye, suggesting mitochondrial
localization of the conjugate, and potential association with mitochondria-translocated forms of EGFR.
Mitochondrial localization was further documented by the specific concentration of the bis-
quinazoline-Ru-conjugate in a mitochondrial isolation assay.
KEYWORDS
Ruthenium Conjugate; Aminoquinazoline; Epidermal Growth Factor Receptor Tyrosine Kinase;
Mitochondrial Localization, Mitocan; TEG Linker.
INTRODUCTION
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The human epidermal growth factor receptor family of receptor tyrosine kinases (ErbB1-4) controls a
complex network of ligand-receptor interactions and cellular responses known as the ErbB-axis.1
Alterations and disruptions in the function of the ErbB axis can impact processes such as cell cycle
progression, proliferation, differentiation and survival,2,3 thus leading to common malignancies,4,5
which has rendered EGFR (ErbB1) a prime target for anticancer research. Coincident with this interest
in targeting EGFR was the identification of intrinsic and acquired resistance to EGFR inhibiting agents,
with the first uniform clinical definition of acquired resistance to EGFR inhibitors reported in January
2010.6 The capacity of cancer cells to adapt to treatment suggests that additional mechanisms of
resistance to EGFR inhibitors may have a key role in regulating tumor response, such as (i)
translocation of surface receptors to the mitochondria and/or the nucleus and constitutive activation of
EGFR,7-11 (ii) altered DNA damage response epitomised by genetic and epigenetic variations of genes
in the EGFR oncogenic cascade,12 (iii) expression of the EGFRvIII variant, deprived of the
extracellular ligand binding domain,13 and (iv) as-yet undiscovered mutations.
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The ability to quantitatively image the ErbB kinase axis in a non-invasive manner can aid in lesion
detection, patient stratification, new drug development and validation, dose optimization and treatment
monitoring.14-18 Imaging requires development of sophisticated optical probes, such as far-red and near
infra-red (NIR) emitting probes. These are preferred for biological applications, due to: (i) lack of
native NIR fluorescence in biological systems leading to highly sensitive detection, delineating
molecular structures in pM concentrations;19 (ii) low scattering of NIR photons allowing for better
image resolution;20 and (iii) deep-in tissue penetration permitting non-invasive imaging.21
Optical imaging with anti-EGFR antibodies22 presents limitations in introducing a therapeutic
functionality, while radiolabelled tyrosine kinase inhibitors (TKIs) exhibit non-specific binding and
sub-optimal pharmacokinetic properties23 due to low hydrophilicity. Metal-based fluorophores
(carrying either transition or lanthanide metals), however, exhibit complementary properties over
organic fluorophores, including resistance to photobleaching, long luminescence lifetimes, minimal or
no re-absorption and sharp emission bands.24,25 While a metal/pyridine-based platform has provided a
high synthetic versatility with potential for coupling to biological ligands,26 other recent efforts to
conjugate pyridine-based systems to aminoquinazoline TKIs have fallen short of achieving effective
uptake or quantifiable anti-cancer biological responses.27,28
The present study aims to address some of the aforementioned challenges by delivering a new
theranostic agent intended for both EGFR therapeutic targeting and EGFR diagnostic imaging. This
successfully combines the optical properties of a metal-based fluorophore with the anti-EGFR efficacy
of a small-molecule TKI. The current design is appropriate for creating bias towards mitochondria-
translocated forms of EGFR, since it exploits the ability of the positively-charged metal-complex and
ionizable linker to achieve specific subcellular localization in mitochondria, hence demonstrating
behaviour of a mitocan, in analogy to previous reports on all-organic conjugates.29-31
EXPERIMENTAL SECTION
Chemical synthesis, purification and characterization
Chemicals, organic solvents, silicas, aluminas and TLC plates were purchased from Sigma-Aldrich or
Merck. All reactions were performed under an argon atmosphere and anhydrous solvents were used,
unless otherwise stated. In flash liquid chromatography purifications, silica gel 60 (0.06–0.2 mm) was
used for organic compounds and neutral alumina (0.06-0.2 mm) was used for Ru-containing
compounds. Final purification of compounds 18 and 19 was carried out in XBridge BEH C18 OBD
column, on a Waters 1525EF semi-preparative HPLC system, equipped with autosampler, diode array
detector and fraction collector. NMR spectra were obtained on a Bruker Avance III Ultrashield Plus
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spectrometer (at 500 MHz for H NMR and 125 MHz for 13C NMR, at 25 °C, chemical shifts relative
to tetramethylsilane). MS data were collected on a Bruker Autoflex III Smartbeam MALDI-TOF/TOF
instrument, using a MTP384 ground steel plate. UV spectra were recorded on a Jenway 6715 UV-Vis
spectrophotometer. Fluorescence spectra were recorded on a JASCO FP-6300 spectrofluorometer.
Detailed synthetic procedures for all compounds synthesized in this study, as well as characterization
data and original NMR spectra are provided in the Supporting Information.
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Biological evaluation of compounds
Cell Culture: SW480 (grade II colon cancer cells) and U87MG (grade IV glioblastoma cells), either
parental or stably transfected with Green Fluorescent Protein and Luciferase (SW480-GFP/Luc;
U87MG-GFP/Luc) were a generous gift from Prof. Andreas Evdokiou, University of Adelaide. Both
SW480 and U87MG cell lines constitutively express ∆EGFR Tyr-1068 mutation. Cells were cultured
at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium containing L-glutamine (DMEM;
Biosera), supplemented with 10% v/v heat-inactivated FBS (Gibco ThermoFisher) and 1% v/v
Penicillin/Streptomycin (Gibco ThermoFisher). The cells were sub-cultured twice a week using 0.25%
Trypsin-EDTA (Gibco ThermoFisher) as a detaching agent.
Viability assay: SW480 or U87MG cells were seeded as indicated in 96-well-plates in DMEM
(Biosera), 10% v/v heat-inactivated FBS (Gibco ThermoFisher), 1% v/v Penicillin/Streptomycin
(Gibco ThermoFisher) and allowed to attach overnight. The cells were treated with medium containing
the compounds of interest at five or six increasing concentrations, based on previously reported
efficacies of Gefitinib (positive control) on either cell line and incubated for 48 h at 37 °C, 5% CO2.
Incubation time was based on the cell growth kinetics. The final concentration of the solvent DMSO in
the medium was adjusted to 1% v/v for all treatment concentrations, including controls, to correct for
the concentration effect of DMSO. Following the treatment, 0.5 mg/mL methylthiazolyldiphenyl-
tetrazolium bromide (MTT; Sigma) in DMEM was added and the cells were incubated for 3 h at 37 °C,
5% CO2. MTT solution was removed and the formed crystals were solubilised by addition of DMSO.
Absorbance was measured at 570 nm on a VictorX4 automated plate reader (PerkinElmer). Three
experiments were performed with triplicates for each concentration per experiment.
ELISA-based kinase activity assay: Tyrosine kinase enzymatic activity of the EGFR was assessed with
standard ELISA technique on anti-phosphotyrosine-coated plates (PathScan Phospho-EGF receptor
(Tyr-1068) Sandwich ELISA kit 7240, Cell Signaling). Three experiments for each compound were
performed, each done in triplicate. Compounds 9a-d were assessed against Gefitinib for the ∆EGFR
Tyr-1068 binding and inactivating efficacy in the presence and absence of EGF. Treatment of SW480
cells with EGF stimulates phosphorylation of EGF Receptor at Tyr-1068. The fluorescence signal
detection and quantification was performed on the Multimodal Multilabel Plate Reader Victor X4
(PerkinElmer) as absorbance at 450 nm within 30 min after adding the STOP solution of the kit. Cells
were plated in petri dishes (10 cm) and treated for 2 h with the compounds. One petri dish of each
treatment was incubated for 30 min with EGF. The cells were lysed using the lysis buffer of the kit and
following the provider’s protocol. Detection of microwell strips was performed using the Plate Reader
at 450 nm absorbance. Blue color indicates positive reaction (i.e. active phosphorylation) which
changes to yellow upon addition of STOP solution.
Mathematical and statistical analyses: Inhibitory concentration (IC50) for each sample was calculated
by linear regression analysis of mean normalized absorbance (%) values, using Matlab (The
MathWorks, Inc.). Curves with positive slopes were excluded from the calculation of the IC50. Welch's
t-test (or unequal variances t-test), an adaptation of Student's two-tailed t-test32 has been employed to
compare the IC50 values of different compound pairs due to its higher reliability when the two samples
have unequal variances and unequal sample sizes.33
Evaluation of Intracellular Uptake and Subcellular Biodistribution
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Intracellular trafficking of free Ru complex (10): In order to initially assess the potential of free Ru
complex (10) to be uptaken and internalized by target cells and serve as a potential imaging agent,
U87MG cells, stably transfected with GFP and Luciferase (passage 3), were trypsinized and seeded on
2.5 cm2 cover slips at 20x103 cells/cm2 in 300 µL of complete DMEM and allowed to attach overnight.
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Sustained uptake and intracellular distribution was assessed after 24 h of incubation at 37 oC, 5% CO2
and 95% humidity. In brief, medium was removed from coverslip cultures and complete DMEM
containing the compound (10) at concentrations equivalent to the IC50, as determined in the cell
viability assays, was added. DMSO concentrations in the medium were adjusted to 1% v/v for all
treatments. At the indicated time, cells were subsequently rinsed with PBS twice and incubated with
Hoechst nuclear stain (1:10x103 dilution in PBS) for 5 min. Excess dye was removed by rinsing with
PBS and cells were subsequently subjected to Live Imaging Solution (1X) until imaging under a Z1
Fluorescence Microscope (Zeiss). Imaging data generation was performed using the AxioVision 4.8
software. A freely tunable white light laser was used for excitation under the following stably
maintained settings: Green Fluorescent Protein (Zeiss GFP, excitation 485 ± 50 nm; emission: 510 ± 25
nm); 4',6-Diamidino-2-phenylindole (Zeiss DAPI, excitation: 355 ± 50 nm; emission: 460 ± 50 nm);
and Quantum Dot fluorochrome 615 (Zeiss QD, excitation 350LP nm; emission 615 ± 20 nm).
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Intracellular trafficking and subcellular distribution of aminoquinazoline-Ru-conjugates (21 and
22): To further assess the subcellular distribution of Ru-containing compounds and define their
potential role as theranostics for translocated-EGFR-associated malignancies, U87MG cells (passage 3)
were trypsinized and seeded on 2.5 cm2 cover slips at 20x103 cells/cm2 in 300 L of complete DMEM
and allowed to attach overnight. Intracellular distribution of mono-quinazoline- and bis-quinazoline-
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Ru-conjugates was assessed at 30 min, 60 min and 6 h of incubation at 37 C, 5% CO2 and 95%
humidity. In brief, medium was removed from coverslip cultures and complete DMEM containing the
indicated compound (21 or 22) at concentrations equivalent to their IC50, as determined in the cell
viability assays, was added. DMSO concentrations in the medium were adjusted to 1% v/v for all
treatments. At the indicated time, cells were washed twice with PBS and subjected to incubation with
MitoTracker Green FM (excitation 490 nm; emission 516 nm), diluted in plain DMEM (without FBS
o
or Penicillin/Streptomycin) at final concentration 20 nM and incubated at 37 C, 5% CO2 and 95%
humidity for 45 min. Cells were subsequently rinsed with PBS twice and incubated with Hoechst
nuclear stain (1:10x103 dilution in PBS) for 5 min. Excess dye was removed by rinsing with PBS and
cells were subsequently subjected to Live Imaging Solution (1X) until imaging. Localization and
intensity of fluorescence was assessed with a Fluorescent Microscope (Olympus BX51) equipped with
a 40x objective. A freely tunable white light laser was used for excitation under the following stably
maintained settings: Tetramethylrhodamine isothiocyanate (Thorlabs MDF-TRITC, excitation: 542 ±
10 nm; emission: 620 ± 26 nm); Fluorescein isothiocyanate (FITC, Olympus U-MNIB2, excitation:
480 ± 10 nm; emission = 510LP nm); and 4',6-diamidino-2-phenylindole (DAPI Olympus U-MNU2,
excitation = 365 ± 5 nm; emission = 420LP nm).
Time-resolved FRET detection: U87MG cells near confluence were seeded in a black 96-well plate
with clear optical-bottom (Nunc™ MicroWell™, Thermo Fisher Scientific Ltd) at 7000 cells/well
density and allowed to attach overnight. The wells were then randomly assigned in one of the following
four groups (n=14 wells/group): Negative control group (no treatment; NC), Ru group (compound 22-
only treatment; R), MitoTracker group (MitoTracker-only treatment; M), co-administration group
(compound 22 and MitoTracker treatment; RM). For the wells in the R-group, the cell media was
removed and substituted with 200 μL of 0.05 mM compound 22 solution in cell media, followed by
incubation at 37 °C for 1 h. For the wells in the M-group, the cell media was removed and substituted
with 200 μL of 20 nM MitoTracker solution in serum-free and antibiotic-free cell media, followed by
incubation at 37 °C for 45 min. For the wells in the RM-group, the wells were sequentially treated with
compound 22 and the MitoTracker probe as described above, with an intermediate step of three
thorough cell washes with PBS. Every group was treated at an appropriate timing schedule so that all
wells were read at the same final time. At the endpoint of treatment, all wells were emptied of their
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media, washed thoroughly three times with PBS and re-filled with 200 μL of PBS prior to the
application of the fluorescent excitation/detection protocol. Mean data were collected from three
independent identical experiments. An ultra-sensitive multimodal microplate reader with bottom-
reading fluorescent capabilities was used for the fluorescence excitation and detection (Spark 20M,
Tecan Group Ltd, Switzerland). The 96-well plate was subjected to a specific time-resolved
excitation/emission protocol (TR-FRET protocol, see Supplementary data) that combined the optimum
fluorescence excitation/emission wavelengths of compound 22 (excitation 460 nm; emission 635 nm)
with those of the MitoTracker (excitation 490 nm; emission 516 nm). During the TR-FRET
experiment, the microplate was incubated at 37 °C and 5% CO2/humidified air within the reader’s
humidity cassette, in order to maintain the optimum cellular conditions and avoid well content
evaporation. Seventyfive flashes were used for each measurement in order to maximize detection
sensitivity and accuracy from each well (integration time 400 μs; lag time 100 μs; settle time 500 ms).
The optimum parameters for gain (detection potency) and Z-position (3D lens position during flashes)
were calculated by the reader using a plate scan and automatically applied in all measurements. Due to
overlap of the emission spectra of the donor and the acceptor, the calculation of direct excitation (DE)
and spectral bleed-through (SBT) corrections were required in order to quantify the sensitized emission
(SEM) and the percentage apparent FRET efficiency (% Eapp) (TR-FRET formulae, see Supplementary
data). The mean  SE values from each treatment group were calculated after subtracting the emission
values of the NC-group at the same conditions (background fluorescence).
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Mitochondrial isolation assay: U87MG cells were seeded in two T25 flasks (A and B) with 5 x 107
cells/flask and allowed to attach overnight. Cells in flask A were treated with 5 mL of 20 nM of
MitoTracker Green, diluted in cell media and incubated at 37 °C for 45 min, whereas cells in flask B
were treated with 5 mL of 50 μM of compound 22, diluted in serum-free cell media and incubated at 37
°C for 1 h. After the end of the treatment period, cells in both flasks were treated for mitochondrial
isolation using a commercial kit (Amsbio KC010100), following the manufacturer’s instructions. Cells
were washed with cold PBS three times prior to trypsinization and centrifugation, followed by
homogenization in mitochondria isolation buffer. After centrifugation the pellet (containing cell
membranes) was discarded and a sample of the supernatant (containing cell contents) was kept for later
measurements. A further centrifugation of the supernatant (at 12,000 g for 15 min at 4 °C) was applied
to collect the mitochondrial pellet which was resuspended with Gibco® FluoroBrite™ DMEM media
(ThermoFisher A1896701) for minimal background fluorescence, then kept on ice covered by
aluminum foil. A 200 μL sample (n=6) of cellular-content isolates and 200 μL sample of mitochondrial
isolates (n=6) were transferred in a black 96-well microplate, similar to the one used in FRET
measurements as described above. The measurement plate entered a Spark 20M microplate reader for
fluorescent emission measurements at the specified excitation and emission wavelengths described in
FRET measurements (i.e. ex/em: MitoTracker at 490/516 nm; compound 22 at 460/645 nm).
Measurements from wells with media-only but no cells were used as “blank” and were subtracted from
all measurements in order to remove any potential background fluorescence. Three independent
experiments were performed and the mean values were used for statistical analysis using GraphPad
Prism software.
RESULTS AND DISCUSSION
Synthesis of a focused aminoquinazoline library
A well-investigated inhibitor class targeting the EGFR tyrosine kinase (ATP binding site) is the
aminoquinazoline family, which includes the first EGFR-TK inhibitor (Gefitinib) on the market and
several analogues. Most notably, Gefitinib is employed as a second-line therapy in the treatment of
glioblastoma,34 the most malignant brain tumor type with poor prognosis.35
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As part of a screening effort to identify new aminoquinazoline analogues with comparable or improved
activity relative to the clinically available drug Gefitinib, for developing them further into theranostic
agents, a focused library incorporating the validated aminoquinazoline pharmacophore was
synthesized. The library synthesis was achieved via a 5-step route as shown in Scheme 1. This route
initiated with 2-amino-4-nitrobenzoic acid (1), which underwent condensation with formamidine
acetate (2), to afford quinazolinone 3, in 93% yield.36 Intermediate 3 was converted to 4-
chloroquinazoline derivative 4 (94% yield) via treatment with SOCl2 in the presence of catalytic DMF
at high temperature, followed by azeotrope distillation with toluene.37 This treatment proved necessary
for obtaining high conversion in a multi-gram scale reaction, since 4 showed propensity for hydrolysis
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back to 3 in certain solvents such as ‘wet’ DMSO, as suggested by its H and 13C NMR spectra. The
observed reversibility can be attributed to the strong electron-withdrawing effect of the nitro-group.
Nucleophilic displacement of the chloride of 4 in parallel reactions by a small set of amines that
included 4-substituted anilines (4-bromo (5a); 4-isopropyl (5b); 4-tert-butyl (5c)) and the bulky
i
naphthalene-1-ylmethanamine (5d) in PrOH-DCM readily afforded aminoquinazolines 6a-d.38 Our
selection of amines reflected an established requirement for hydrophobic halogen-substituted anilines
or sterically bulky amino head-groups, serving the purpose of favourably occupying a hydrophobic
binding pocket of EGFR-TK.39 The nitro-group of 6a-d was subsequently reduced by using iron
powder in a mixed solvent, to furnish amino-counterparts 7a-b.36 Finally, derivatization of 7a-d with ɑ-
chloroacetyl chloride (8) under basic conditions in a 1:1 THF-DMF mixture led to amides 9a-d.40 The
presence of a chloride ‘handle’ in amides 9a-d was intended to enable further modifications needed for
fluorophore attachment, during the conjugate formation stages. All reactions in this route were scalable
up to multiple grams, and the solid products were isolated via filtration and washing, without need for
1
chromatography purification. All products were characterized and their purity checked by H and 13C
NMR, and by MS MALDI-TOF.
R
AcO
H2N
NH2
O
N
Cl
N
HN
R-NH2
5a-d
H
HO2C
H2N
2
HN
N
N
(iv)
(ii)
NO2
(iii)
(i)
NO2
NO2
N
NO2
3
4
1
6a-d
R
R
O
8
HN
a:
b:
c:
d:
HN
Cl
CH3
CH3
CH3
Cl
N
O
N
CH3
CH3
Br
(v)
R=
Cl
N
N
N
NH2
H
7a-d
9a-d
o
Scheme 1. Synthesis of a focused aminoquinazoline library. Key: (i) MeOCH2CH2OH, 130 C, 18 h, 93%; (ii) SOCl2, DMF,
155 oC, 2 h, then toluene, 110 oC, 24 h, 94%; (iii) iPrOH-DCM, r.t., 2-5 h, 61-93%; (iv) EtOH, H2O, AcOH, Fe, 60 oC, 4 h, 57-
78%; (v) THF, DMF, Et3N, 30 min, r.t., then addition of 8, 0 oC-r.t., 12 h, 53-65%.
Biological evaluation of aminoquinazoline library
Tumor cell growth inhibitory activity: In order to identify compounds with favorable therapeutic
efficacies for subsequent conversion to theranostic agents/conjugates, the new 4-aminoquinazolines
(series 6a-d, 7a-d and 9a-d) were submitted to screening, in comparison to Gefitinib, a clinically
approved anti-EGFR quinazoline, widely applied in both colon cancer41 and glioblastoma.42 This
screen tested compound ability to inhibit the growth of SW480 ΔEGFR Tyr-1068 mutant cancer cells
(Figure 1). Tyr-1068 represents a well-established driver mutation, prevalent in most EGFR-driven
malignancies, including glioblastoma multiforme (GBM),43 lung44 and colon cancer.45 Among the 12
members of the focused library, aminoquinazoline derivatives 9a-d exhibited superior cell growth
inhibitory efficacies compared to Gefitinib and were selected for further evaluation of their efficacies
and specificity to inhibit phosphorylated EGFR in a binding competition assay.
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Figure 1. Cell growth inhibitory efficacy of 4-aminoquinazolines. Compounds 6a-d (first row), 7a-d (second row) and 9a-d
(third row) were assessed for their cell growth inhibitory efficacy on SW480 ΔEGFR* Tyr-1068 cells against Gefitinib in a
colorimetric assay based on tetrazolium incorporation properties (MTT assay). Graph plotting was performed using the WorkOut
2.5 software. Results shown are representative of three experiments in triplicate points; bars indicate ± SE. (*) ΔEGFR denotes
constitutively active mutant of the Epidermal Growth Factor Receptor.
EGFR tyrosine kinase binding competition/phosphorylation inhibition: The compounds’ ability to
inhibit EGFR-TK phosphorylation was evaluated on SW480 ΔEGFR Tyr-1068 cancer cells. The
constitutively active mutant form of the receptor found in these cells demonstrates baseline activity
even in the absence of EGF. Treatment of the cells with EGF resulted in stimulation of the
phosphorylation of EGFR at Tyr-1068, disclosed by increased absorbance at 450 nm in the treated
samples, in a PathScan Phospho-EGF receptor (Tyr-1068) Sandwitch ELISA kit 7240. Exposure to
compounds 9a-d resulted in statistically significant EGFR binding efficacy and ensuing
downregulation of Tyr-1068 phosphorylation for both amides 9a and 9d over Gefitinib and untreated
control (Figure 2).
Figure 2. Treatment of SW480 ΔEGFR Tyr-1068 cells
with EGF stimulates phosphorylation of the EGF
receptor at Tyr-1068. Phospho-ΔEGFR Tyr-1068 is
detected by PathScan Phospho-EGF receptor (Tyr-
1068) Sandwitch ELISA kit 7240. Compounds 9a-d
exhibit specific anti-ΔEGFR Tyr-1068 activity.
Baseline Phospho-EGFR activity detected in
untreated controls is representative of the
constitutive expression of the malignancy-driving
mutation of EGFR in these cells. Graph plotting was performed using the WorkOut 2.5 software. The distribution of the values
of the optical density (OD) readings at 450 nm were compared for statistically significant difference using Student’s t-test.
When compared against untreated controls compounds 9a-9d disclosed highly significant statistical differences (p<0.0001) in
both the absence or presence of EGF. When compared against Gefitinib in the absence of EGF, the differences of compounds
9a and 9d were highly significant (p=0.005862 and 0.006986 respectively; 0.005<p<0.05) while in the presence of EGF only
compound 9a disclosed statistically significant differences (p=0.023499; p<0.05), thus justifying its selection for conjugation
with compound 10. Results shown are representative of three experiments in duplicate points; bars indicate ± SE.
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Testing for additive or synergistic effects between aminoquinazolines and [Ru(bipyridine)3CO2H]2+:
Due to the wide applications and high promise of quinazoline-based TKIs,46 we envisaged their
covalent conjugation with fluorescent ruthenium complexes as capable to deliver biologically active
theranostic agents, combining EGFR-recognizing and -inhibiting properties. Seemingly, ruthenium(II)-
containing scaffolds, besides their superior optical imaging properties,47 exert a caliber of anticancer
activities strongly related to their unique DNA-binding patterns, migration inhibitory potential and
ability to undergo favorable ligand exchange kinetics upon interaction with tumor cells.48-51 These
reports have inspired the design and synthesis of kinetically stable prototype ruthenium complexes with
potent optical (i.e. far-red spectroscopic) properties as building blocks for drug delivery carriers.52
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The question was then raised whether a ruthenium complex in conjugation with an aminoquinazoline
would add to the quinazoline’s cell growth inhibitory activity. In order to determine this, a viability
assay studying cells exposed to equimolar concentrations of each of 9a-d plus free carboxy-
functionalized [Ru(bipyridine)3]2+ complex 10 -our selected complex for use in conjugate formation- in
both the presence or absence of EGF stimulation, was employed (Figure 3). It was observed that cell
viability was not affected in any case by the presence of Ru-complex 10, hence additive or synergistic
effect between each aminoquinazoline and 10 was ruled out.
Figure 3. (A) Carboxy-functionalized [Ru(bipyridine)3]2+
complex used in this study. (B) EGFR-specific cell growth
inhibitory efficacy of 9a-d is neither enhanced nor
compromised by the free Ru-complex 10. SW480 ΔEGFR
Tyr-1068 cells were treated for 48 h with compounds 9a-d
or Gefitinib, either alone or in combination with free Ru-
complex 10 in the presence or absence of EGF and
subsequently subjected to viability assessment with
tetrazolium incorporation (MTT assay). Graph plotting was
performed using the WorkOut 2.5 software. Results shown
are representative of three experiments in triplicate points;
bars indicate ± SE.
Given the fact that 9a has shown one of the most significant EGFR-specific cell growth inhibitory
effects in the presence of 10, and its synthesis has been more efficient compared to 9d, which also
exhibited low solubility issues, quinazoline 9a was selected for incorporation into a theranostic
conjugate, in combination with Ru-complex 10.
Synthesis and characterization of aminoquinazoline-Ru-conjugates
A synthetic effort was undertaken to covalently adjoin the selected aminoquinazoline 9a to the
fluorescent scaffold of Ru-complex 10 and generate a conjugate. This problem required the use of a
bifunctional linker. Attachment of the linker to quinazoline 9a from its terminal chloride-substituted
site was anticipated to avoid interference with EGFR docking, since the 7-position of
aminoquinazolines in addition to the 6-position are the only sites tolerating substituents, which
typically protrude towards a cleft that leads to the enzyme surface, as seen in crystal structures of
clinically used TKIs bound to EGFR.53 The absence of a substituent on the 6-position in our
compounds should avoid steric clashes and ensure rotational flexibility of the linker.
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A triethyleneglycol (TEG)-derived linker bearing amino-groups at both termini was selected for
providing a satisfactory length for the current application. This linker can potentially enhance the
conjugate’s aqueous solubility compared to other all-hydrophobic linker options. Moreover,
ethyleneglycol units are expected to increase membrane permeability and uptake, since they are often
used as preferred nanocarrier building components for crossing the blood-brain barrier (BBB).54 The
resulting conjugates of this type, could provide promise for BBB passage in future applications. The
linker’s terminal amino-groups ensure reactivity towards both the chloro-quinazoline 9a and a carboxy-
bipyridine unit, which serves as a chelator for Ru(II) complexation in the last stage of the synthesis.
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The synthetic route for production of a conjugated ligand for Ru(II) (Scheme 2), which notably avoided
use of any protecting groups, initiated with the TEG unit (11). Compound 11 was first converted to the
corresponding dibromide 12, by using tetrabromomethane and triphenylphosphine in THF (73%
yield).55,56 The dibromide was readily converted to diazide 13 in excellent yield (93%), by applying a
modification of a reported method.56 A single-site reduction of the diazide to the azide-amine
intermediate 14 followed, via use of one equivalent of triphenylphosphine in THF.57,58 The 47% yield
and the TLC profile of the reaction suggested that a more polar by-product (possibly the diamine) also
formed, however due to high polarity it was not isolable by chromatography. The terminal amino-
group of 14 was next coupled to bipyridine-4-carboxylic acid (15), under two similar sets of conditions
that employed carbodiimides. In the first case, DCC was the activating agent, together with HOBt in
THF,59 leading to 48% of amide product 16, isolated after chromatography as a 2:1 inseparable
complex with dicyclohexylurea (DCU). To obtain DCU-free amide 16, we also performed the reaction
using EDC in DMF, in the presence of HOBt and Et3N.60 The yield in this case was comparable to the
previous reaction (41%). Compound 16 (with or without DCU) was submitted to a second reduction
with triphenylphosphine in THF57,58 to provide a new terminal amine (17), ready for coupling to the
quinazoline pharmacophore fragment (9a). Purification of intermediates in all stages up to compound
17 (included) required silica column chromatography.
The pairing of the two fragments, pharmacophore 9a and linker-ligand 17, was carried out in CH3CN
under basic conditions (Hünig’s base), in the presence of KI.61 While the 1:1 nucleophilic substitution
product 18 was the intended product, the high reactivity of amine 17 in conjunction with the order of
reactant addition, led to a mixture of 18 with the unprecedented 2:1 (9a-to-17) adduct 19. The ratio of
products could be shifted in favour of one or the other, by controlling which of the two reactants was
added in excess or by slowing down the rate of addition. The two products were separable by semi-
preparative C18 reverse-phase HPLC, which at this stage became necessary due to their high polarity.
Compounds 18 and 19 are both novel ligands for complexation of Ru(II). Both structures exhibit
potentially ionizable nitrogen atoms at the quinazoline-linker junction, which is expected to mimic the
ionizable amino-groups encountered in clinically available quinazolines such as Gefitinib and Afatinib,
known to increase drug solubility. It is also possible that intracellular protonation of the amines would
direct the conjugates derived from these ligands towards negatively-charged organelles (mitochondria,
nucleus), where translocated EGFR resides.7-11 All intermediates in this synthesis were characterized by
1H and 13C NMR, while novel intermediates containing aromatic fragments were also characterized by
MS MALDI-TOF.
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N3
O
HO
Br
O
O
O
O
O
N3
OH
Br
(ii)
(i)
13
12
11
CO2H
6
7
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O
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N
N
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N3
O
N
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NH2
H
(iii)
(v)
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(iv)
N
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9a 17
= 1:1)
(
/
O
18
(major) + 19
(major) + 18
(minor)
(minor)
9a
(vi)
O
H2N
N
H
O
N
19
N
17
9a 17
(
/
= 2:1)
Br
NH
N
O
N
O
H
O
N
N
H
N
H
O
N
N
18
Br
NH
O
N
O
N
N
H
O
N
O
N
N
O
Br
NH
N
H
N
NH
19
N
Scheme 2. Synthetic route for preparation of the conjugated ligands (quinazoline:bipyridine ratio 1:1 or 2:1, compounds 18 and
o
19, respectively), for Ru complexation. Key: (i) THF, CBr4, PPh3, r.t., 2 h, 73%; (ii) DMF, NaN3, 60 C, 12 h, 93%; (iii) THF,
o
o
PPh3, 0 C-r.t., 24 h, 47%; (iv) A. THF, DCC, HOBt, 0 C-r.t., 18 h, 48% (as 2:1 complex 16:DCU) or B. DMF, EDC, HOBt,
Et3N, r.t., 24 h, 41% (DCU-free 16); (v) THF, PPh3, 0 oC-r.t., 24 h, 88%, (vi) CH3CN, iPr2EtN, KI, 80 oC, 24 h, 47% in 18 (after
HPLC) when ratio of 9a:17=1:1; 46% in 19 (after HPLC) when ratio of 9a:17=2:1.
N
21 .
2Cl
18
OR
N
N
Cl
RuII
N
OR
(i)
Cl
19
22
22
. 2PF6
. 2Cl
(ii)
20
Br
NH
N
2
O
O
N
H
O
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N
N
O
N
N
H
N
N
H
RuII
N
21
N
Br
NH
O
N
2
O
N
H
O
N
N
O
N
N
N
O
N
N
Br
NH
N
H
N
N
RuII
N
NH
22
Scheme 3. Complexation of ligands 18 and 19 to produce the aminoquinazoline-Ru-conjugates 21 and 22, respectively. Key: (i)
EtOH-H2O (1:1), 100 oC, 24 h; (ii) NaPF6, r.t., then 0 oC, 24 h. Yields: 44% in 21.2Cl; 53% in 22.2PF6.
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Figure 4. (A-B) UV-Vis spectra of mono-quinazoline-Ru-conjugate 21 and bis-quinazoline-Ru-conjugate 22, respectively, in
DMSO at 25 µM concentration. (C-D) Emission spectra (excitation at 460 nm) of 21 and 22, respectively, in DMSO at 25 µM
concentration. Graphs were plotted in Origin 8.0.
Compounds 18 and 19 were independently submitted to reflux for 24 h with one equivalent of cis-
[Ru(bipyridine)2Cl2] complex (20), in a 1:1 EtOH-H2O solvent system62,63 (Scheme 3). Each reaction
afforded a characteristic deep-orange colored solution, indicating successful formation of the
corresponding [Ru(bipyridine)3]2+ species, compound 21 and 22, respectively. Attempts to isolate the
-
products in solid form by counterion exchange (Cl- to PF6 ) proved successful only for the bulkier
molecule 22, which was obtained in 53% yield. In the case of 21, it was decided to isolate the
conjugate directly out of the reaction mixture by solvent evaporation, followed by column
chromatography in neutral alumina column, which finally afforded solid 21.2Cl in 44% yield. Both
structures were confirmed by NMR and MS MALDI-TOF analysis. Moreover, their UV-Vis and
fluorescence spectra were obtained and found to be similar (shown for 22, Figure 4). The spectra also
closely matched published spectra of the prototype [Ru(bipyridine)3]2+ complex,63,64 which indicates
successful assembly of the desired Ru-fluorophore.
Aminoquinazoline-Ru-conjugates induce cell growth inhibitory effect on glioblastoma cell lines
The cell growth inhibitory effect of five concentrations (0.01-0.1 mM) of the mono-quinazoline- and
bis-quinazoline-Ru-conjugates (21 and 22, respectively) was assessed against those of their constituent
components, [Ru(bipyridine)3CO2H]2+ free complex (10) and free amine 7a, on U87MG cells in an
MTT colorimetric assay. U87MG is an established cell line derived from a human malignant glioma
tumor65 and has been classified as grade IV glioblastoma/astrocytoma since 2007,66 over-expressing
ΔEGFR Tyr-1068. U87MG cells show a highly tumorigenic potential, consistent with glioblastoma
tumors, when transplanted in nude mouse brain. Dose-dependent effects and IC50 values of receptor
TKIs such as Erlotinib and Lapatinib, with structural similarities to 7a/9a, have been studied in a
number of glioma-initiating and glioblastoma cells and have revealed variable sensitivities at the
micromolar dose level, irrespectively of the expression and phosphorylation status of EGFR.67 Our
study shows that the aminoquinazoline-Ru-conjugates require higher doses for significant reduction of
glioma cell viability.
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The recorded absorbance data were normalized by setting the mean absorbance of the untreated
controls (compound concentration=0 mM) as 100%. Mean normalized absorbance (%) vs. compound
concentration was fitted with a linear model. Due to the directly proportional relationship between
absorbance and number of living cells, this allowed determination of mean IC50 values and their
standard deviations (Figure 5, A-B). The half maximal inhibitory concentrations (IC50) - indicating the
concentrations of the substances needed to inhibit cell growth by half - were evaluated after 24, 48 and
72 h of incubation, in order to rule out cell recovery or reversible effect.
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Figure 5. Ru-conjugate formation does not compromise cell growth inhibitory efficacies in U87MG ΔEGFR Tyr-1068
expressing cancer cell lines. U87MG ΔEGFR Tyr-1068 cells were subjected to incubation with serial concentrations of
compounds 7a, 10, 21 and 22 and the half maximum inhibitory concentrations were evaluated after 24, 48 and 72 h of
incubation, in order to rule out cell recovery or reversible effect. The absorbance was measured using a tetrazolium incorporation
assay (MTT assay) (9-12 measurements per compound). In order to normalize the absorbance data, the mean absorbance of the
negative control samples (0 mM of each compound) at 570 nm was set at 100%. Mean normalized absorbance (%) descended
with compound concentration and was fitted with a linear model, allowing for calculation of mean IC values and standard
50
deviations. (A-B). Distribution of IC values for the compounds under investigation. The diamonds indicate the mean value, the
50
solid boxes the standard deviation, the horizontal lines the maximum and minimum values, and the stars the outliers of the data.
The values of the mean and the standard deviation of each compound in A and B can also be seen in the table below the figures
in rows A and B respectively. The distributions of IC values were compared for statistically significant difference using
50
Student’s t-test. The p values resulting for each pair are indicated in the table. The differences between the IC values of
50
compound 10 vs. 7a and 21 are highly statistically significant (p < 0.005). In part B, the difference of the IC value of compound
50
21 vs. 22 is also statistically significant (p < 0.05). The IC value of 7a is not statistically different than 21 or 22.
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In a first series of experiments against U87MG cells, amine 7a demonstrated superior efficacies over
free Ru-complex 10 (p=0.000001) (Figure 5A). Comparable half-maximum inhibition efficacies were
recorded for 7a and the mono-quinazoline-Ru-conjugate 21 (p=0.0678), suggesting that linkage via the
triethyleneglycol-derived linker and attachment through a C-N bond in the theranostic conjugate does
not compromise the cell growth inhibitory effectiveness of the quinazoline free amine moiety. A
follow-up series of experiments (Figure 5B) further confirmed the absence of any statistically
significant differences between the efficacies of 7a and 21 (p=0.3856) and disclosed a significantly
more favorable growth inhibitory efficacy for the bis-quinazoline-Ru-conjugate 22 over the mono-
quinazoline-Ru-conjugate 21 (p=0.0303). This can be attributed to the higher avidity of 22 for EGFR
receptors, due to the presence of two TKI units in its structure. The delayed cellular uptake of 21
compared to that of 22 (data not shown), attributed to lower membrane solubility (due to the presence
of a polar secondary amine), seems to partially compromise its phospho-tyrosine inhibiting and ensuing
growth inhibitory efficacies. The observed difference is associated to the absence of a second
aminoquinazoline unit in 21, which renders it more polar relative to 22.
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Intracellular uptake and mitochondrial co-localization of Ru-containing compounds
Fluorescence imaging: An initial evaluation of exposure of U87MG cells, stably transfected with
Green Fluorescent Protein and Luciferase (U87MG-GFP/Luc), to the free Ru-complex 10, yielded
favorable and sustained intracellular distribution, following 24 h of incubation. Subcellular organelle
uptake, visualized under a fluorescence microscope as red dots, was associated with prominent
perinuclear, possibly mitochondrial, accumulation of the complex (Figure 6).
Figure 6. U87MG-GFP/Luc cells (green) incubated
with free Ru-complex 10 (red) (50 µM) (A-B, a-b)
for 24 h and stained with Hoechst (blue). Arrows
indicate perinuclear and subcellular organelle
sustained uptake, defining a promising approach to
target malignancies mediated by biomarkers localized
in these organelles. Cells were imaged under an
AxioImager Z1 microscope and the data were
analyzed using AxioVision software. The physical
dimensions of the images can be inferred from the
scale bars representing 25 μm (top) and 10 μm
(bottom).
For the assessment of the potential of mono-quinazoline- and bis-quinazoline-Ru-conjugates (21 and
22) to serve as potent molecular imaging probes for detection and quantification of EGFR-tyrosine
kinase it is essential to obtain a preliminary insight of their capacity to dock through cellular
membranes, be internalized and effectively distributed to intracellular target sites in live U87MG cells.
Such target sites include the mitochondria as subcellular organelles or the nucleus, where constitutively
activated and phosphorylated-EGFR is translocated.68 Internalization and subcellular distribution
o
studies were performed under cell culture conditions (37 C, 5% CO2, 95% humidity) and cellular
uptake was assessed at distinct time points in correlation with the localization of mitochondria (specific
fluorescent biomarker MitoTracker FM) and nucleus (specific staining with Hoechst). Paternal cells
were opted over the GFP/Luc-transfected cell line in order to enable the visualization of the
MitoTracker FM green stain. Cell trafficking and temporal distribution disclosed more favorable
uptake for 22 over the free Ru-complex 10, which was not being localized intracellularly at 30 min, as
disclosed by the lack of fluorescence detection under the TRITC filter. Compound 10 was, however,
visualized at 60 min with the same subcellular characteristics, though at lower intensity. Compound 22
was detected intracellularly at 30 min with fluorescence intensity and subcellular concentration
augmented gradually at 60 min and 6 h. The possibility of intracellular protonation of the nitrogen at
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the linker-quinazoline junction could lead to augmented positive charge, expected to enhance the
uptake by negatively charged subcellular organelles, including the mitochondria and the nucleus. The
mitochondria-specific biomarker (MitoTracker FM) and compound 22 appeared to co-localize in
subcellular organelles, visualized with a FITC filter, as early as 60 min post-incubation (Figure 7).
These observations are strongly suggestive of time-dependent mitochondria uptake. This finding
highlights the ability of 22 to act as a subcellularly localized EGFR optical probe and potentially as an
inhibitor of translocated EGFR in malignancies with acquired EGFR resistance.
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Figure 7. Top row: Distribution of compounds at 60 min. (A) Distribution of MitoTracker (green), (B) Distribution of bis-
quinazoline-Ru-conjugate 22 (red), and (C) Co-localization of the two, appearing yellow (red+green). Bottom row: Distribution
of compounds at 6 h. (D) Distribution of MitoTracker (green), (E) Distribution of bis-quinazoline-Ru-conjugate 22 (red), and (F)
Co-localization of the two, appearing orange, implying accumulation of more bis-quinazoline-Ru-conjugate 22 (red). The cell
nuclei are stained blue in all images. The physical dimensions of the images can be inferred from the scale bars representing 25
μm (full images) and 10 μm (zoomed regions).
Time-resolved fluorescence resonance energy transfer (TR-FRET) discloses mitochondrial
localization of bis-quinazoline-Ru conjugate: In order to independently confirm the mitochondrial
localization of bis-quinazoline-Ru-conjugate 22, a time-resolved fluorescence resonance energy
transfer (TR-FRET)-based plate-reader assay was used in live U87MG cells with MitoTracker-stained
mitochondria, treated with compound 22. The following line of data suggests that compound 22 and
MitoTracker behave as a pair of donor and acceptor, respectively, in terms of energy transfer. First,
upon excitation of the donor/acceptor-treated cells at the donor’s specific excitation wavelength (Dex;
460 nm), a significant quenching of the donor’s emission (Dem; 635 nm) was detected, compared to
cells treated with donor alone, as shown in Table 1 (30849 RFU vs. 17635 RFU, p<0.005). At the same
time, excitation of the donor/acceptor-treated cells with Dex produced a significantly higher
fluorescence intensity at the acceptor’s specific emission wavelength (Aem; 516 nm), compared to cells
treated with acceptor alone (12419 RFU vs. 9216 RFU, p<0.05). These results insinuate a transfer of
energy from the donor to the acceptor in donor/acceptor-treated cells, which is characteristic of FRET
and can occur upon close proximity of bis-quinazoline-Ru-conjugate 22 to MitoTracker. Additionally,
since the donor-acceptor pair have overlapping fluorescence spectra, at proximity their emission should
be quenched when together in donor/acceptor-treated cells, compared to when alone after excitation at
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their specific excitation wavelengths. Our data shows that quenching indeed occurs, both for the donor
(30849 RFU vs. 17635 RFU, p<0.005) and the acceptor (20927 RFU vs. 9224 RFU, p<0.005).
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Table 1. Measured fluorescence (mean value, in relative fluorescence units, RFU) in donor-treated, acceptor-treated and mixed
donor/acceptor-treated U87MG cells, using different sets of excitation (ex) and emission (em) wavelengths. Key: Dex: donor’s
excitation (460 nm); Dem: donor’s emission (635 nm); Aex: acceptor’s excitation (490 nm); Aem: acceptor’s emission (516 nm).
All values detected in mixed treated samples were found significantly different (* p<0.005; ** p<0.05) compared to their
respective donor- or acceptor-alone samples (Student’s unpaired two-sided t-test).
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Detected Fluorescence (in relative fluorescence units, RFU)
Excitation-Emission
sets used
Donor alone
Acceptor alone
Donor + Acceptor
Dex Dem
Dex Aem
Aex Aem
30849
4638
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17635 *
12419 **
9224 *
9216
20927
Nevertheless, the raw fluorescence signal obtained from the plate-reader in donor/acceptor-treated cells
was ‘contaminated’ by the direct excitation (DE) of the acceptor by the donor’s excitation light and the
overlap of the donor’s emission spectrum with that of the acceptor (spectral bleed-through, SBT). The
contributions of DE and SBT were quantified and subtracted from the detected fluorescence signal
using the set of equations shown in Table 2, in order to determine the corrected FRET signal (Fc) and
ultimately the percentage apparent efficiency (% Eapp) of the produced FRET signal, which was
calculated to be 24.44%. The magnitude of Eapp is a function of the sixth power of the distance between
donor and acceptor. It is also dependent on the Förster radius (Ro), which corresponds to the critical
distance between two fluorophores at which the energy transfer is half-maximal.69 Ro is usually in the
range of 2–6 nm and depends on various factors, such as the quantum yield of the donor, the spectral
overlap integral between donor and acceptor, the amount of molecules interacting and the molecular
orientation factor of the FRET pair molecules. Typically, resonance energy transfer between two
fluorophores occurs within the range of 0.5-10 nm,70 which translates to FRET efficiency between
100% and 0%, respectively. The efficiency of 24.44%, therefore, insinuates a physical proximity of
approximately 7-8 nm between the MitoTracker and the bis-quinazoline-Ru-conjugate 22, with the
assumption that the factors mentioned above take average values. This estimated distance allows us to
reasonably conclude that compound 22 localizes in the mitochondria of live U87MG cells.
Table 2. FRET parameters calculated in order to determine the FRET apparent efficiency (Eapp) using the detected fluorescence
and factoring in the direct excitation (DE) of the acceptor by the donor’s excitation wavelength and the spectral bleed-through
(SBT) of the donor’s emission to the acceptor’s emission. Key: Dex: donor’s excitation (460 nm); Dem: donor’s emission (635
nm); Aex: acceptor’s excitation (490 nm); Aem: acceptor’s emission (516 nm).
Parameter/Equation
Calculation
• Dex Aem: 9216 RFU
1.
2.
Direct excitation (DE): acceptor’s excitation at donor’s excitation wavelength.
• Aex Aem: 20927 RFU
• δ = 0.44
DE factor: δ (acceptor-only cells) = RFU Dex Aem / RFU Aex Aem
• DE = 4059 RFU
Spectral bleed-through (SBT): contribution of donor’s fluorescence emission to
measured acceptor’s fluorescence emission.
SBT factor: β (donor-only cells) = RFU Dex Aem / RFU Dex Dem
• Dex Aem: 4638 RFU
• Dex Dem: 30849 RFU
• β = 0.15
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• SBT = 2645 RFU
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Corrected FRET signal (Fc): acceptor’s fluorescence emission after excitation of
the donor, corrected with the DE and SBT.
Fc (donor-acceptor cells) = RFU Dex Aem – [(β * RFU Dex Dem) + (δ * RFU Aex Aem)]
• Dex Aem: 12419 RFU
• Dex Dem: 17635 RFU
• Aex Aem: 9224 RFU
• Fc = 5715 RFU
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Sensitized emission (SEM): Fc divided by the acceptor’s fluorescence emission
before DE and SBT corrections.
SEM (donor-acceptor cells) = Fc / RFU Aex Aem
• Aex Aem: 9224 RFU
• SEM = 0.62
FRET apparent efficiency (Eapp): acceptor’s corrected fluorescence emission
divided by the sum of emissions of acceptor and donor (expressed as percentage).
• Dex Dem: 17635 RFU
• Eapp = 24.44%
% Eapp (donor-acceptor cells) = 100 * Fc /(Fc + RFU Dex Dem
)
Evidence of mitochondrial targeting provided by mitochondrial isolation assay: Specific analysis in
mitochondrial isolates from U87MG cells treated with compound 22 produced further evidence of
mitochondrial targeting (Table 3). Mitochondrial isolates extracted from cells previously treated with
compound 22 emitted significantly higher fluorescence than non-mitochondrial cellular isolates from
the same cell samples (2476 ± 139 vs. 225 ± 82 in RFU ± SE from n=6; One-Way ANOVA with
Sidak’s multiple comparison test; p<0.05) which is a similar behavior to that produced by a similar
analysis with Mitotracker Green treated cells (21864 ± 1483 vs. 1810 ± 212 in RFU ± SE from n=6;
One-Way ANOVA with Sidak’s multiple comparison test; p<0.005). Although the mitochondrial-
specific dye produced an expected stronger fluorescent signal than compound 22, there was no
statistical significance between the compartmental ratio (i.e. the ratio of mean emitted fluorescence of
mitochondrial over non-mitochondrial cellular isolates) of the two treatments (8.89 ± 0.82 vs. 10.45 ±
4.08 in RFU ± SE from n=6; Student’s t-test, p=0.72). Finally, the resulting mitochondrial specificity
(i.e. the percentage of mitochondrial-isolate emitted fluorescence over the total emitted fluorescence)
for compound 22 and Mitotracker Green were found to be very similar (90.91% and 92.80%
respectively). This data clearly documents that compound 22 is specifically concentrated in cellular
mitochondria after 1 h of treatment.
Table 3: Fluorescence emission measurements of treated U87MG cells with Mitotracker Green (A) and compound 22 (B) (RFU
± SE). Results are presented from mean measurements of three independent experiments performed in n=6 each. Fluorescence
emitted from mitochondrial isolates from each group was significantly higher than that emitted from the cellular-content isolates
of the same treated group (One-Way ANOVA with Sidak’s multiple comparison test; A: ** p<0.005, B: * p<0.05).
Compartmental ratio (i.e. the ratio of mean emitted fluorescence of mitochondrial over cellular-content isolates) was not
statistically significant between the two treatments (Student’s t-test; p=0.72 two-tailed). Mitochondrial target-specificity (i.e. the
percentage of mitochondrial-isolate emitted fluorescence over the total emitted fluorescence) is also presented in this table for
both treatments.
Fluorescence emission of treated cells (in RFU)
Mitotracker Green treated (A)
21864 ± 1483 **
1810 ± 212
Compound 22 treated (B)
2476 ± 139 *
225 ± 82
Mitochondrial isolates
Cellular-content isolates
Compartment Mean Ratio (A/B)
Mitochondrial target-specificity
8.89 ± 0.82
10.45 ± 4.08
90.91 %
92.80 %
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CONCLUSIONS
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The current study describes a structure-guided development of two novel theranostic conjugates (21
and 22), by applying chemical biology approaches.71 Their design involved four key moieties: a) a
ruthenium fluorophore, [Ru(bipyridine)3]2+, selected for its biological compatibility and favorable
optical properties; b) a quinazoline scaffold with target (EGFR-TK)-recognizing attribute, tethered to
the ruthenium complex; c) a 4-bromoaniline head-group on the quinazoline C4, suitable for recognition
by the gatekeeper residue in order to achieve kinase-inhibiting activity; and d) a ethylene glycol-
derived bridge which keeps the fluorophore farther away from the kinase hinge region.
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The application of a selection process from a focused aminoquinazoline library afforded a TKI (9a)
with promising profile of EGFR-TK inhibition, as reflected by the binding competition assay and the
antiproliferative study results. Its conjugation to the [Ru(bipyridine)3]2+ fluorophore resulted in
generation of conjugates 21 and 22, with the latter in particular retaining a meaningful affinity for
sustained receptor binding and sensing.
The choice of a ethylene-glycol-derived linker aimed at taking advantage of all the beneficial
pharmacokinetic properties offered by this type of linker to carriers for delivery of anticancer drugs to
tumors.72-74 Additionally, opting for a charged metal-based fluorophore and a (+)-ionizable linker,
whose protonation augments the overall positive charge of bis-quinazoline-Ru-conjugate 22, proved
highly successful in enhancing the TKI’s dispersity in biological fluids and facilitating cellular
membrane permeability and uptake by negatively charged subcellular organelles, epitomized by the
mitochondria,7,8 as evidenced by the fluorescence imaging study, TR-FRET and mitochondrial
isolation assay. This renders 22 a potential platform for targeting translocalized mutant forms of EGFR.
Efficient uptake and intracellular delivery of minimally-toxic metal scaffold-based conjugates with
mitocan behavior, such as the one described herein, is expected to reduce dosing and minimize ensuing
side effects if applied within a therapeutic context.
Finally, positively charged conjugates of this type, equipped with TEG units, offer promise for
successful delivery of theranostics across the blood brain barrier. Endothelial cells have strong negative
charge, attributed to the abundance of surface glycocalyx, favoring the uptake of cationic carriers over
anionic or neutral ones.75,76 The transport of charged scaffolds through the hyperplastic endothelium of
glioblastoma and the ensuing repulsive forces among the multiple layers of glycocalyx remains an
outstanding challenge in the delivery of therapeutics. Evaluation of the delivery of bis-quinazoline-Ru-
conjugate (22) across both intact and disrupted BBB is intended to be undertaken in three-dimensional
models of BBB. Therefore, evolution of bis-quinazoline-Ru-conjugate 22 could afford promising
candidates for further evaluation in glioblastoma detection, therapy and monitoring.
ACKNOWLEDGEMENTS
A.D.O. and C.P. thank the Cyprus Research Promotion Foundation for Research Grant
YGEIA/BIOS/0311(BIE)/04. S.N.G. thanks the University of Cyprus for a Faculty Start-up
Grant and Professor Epaminondas Leontides (Department of Chemistry, UCY) for access to a
JASCO FP-6300 spectrofluorometer.
ASSOCIATED CONTENT
Supporting Information
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Detailed chemical synthesis procedures and characterization data for all compounds synthesized in this
study (1H NMR, 13C NMR, MS-MALDI-TOF); original NMR spectra; TR-FRET Protocol and
Formulae.
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ABBREVIATIONS
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Ac: Acetyl
ANOVA: Analysis of Variance
ATP: Adenosine Triphosphate
BEH: Ethylene Bridged Hybrid
Bt: Benzotriazole
DAPI: 4’,6-Diamidino-2-phenylindole
DCC: Dicyclohexylcarbodiimide
DCM: Dichloromethane
DCU: Dicyclohexylurea
DE: Direct Excitation
DMEM: Dulbecco’s Modified Eagle’s Medium
DMF: Dimethylformamide
DMSO: Dimethylsulfoxide
EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDTA: Ethylenediaminetetraacetic Acid
EGF: Epidermal Growth Factor
EGFR: Epidermal Growth Factor Receptor
EGFRvIII: Epidermal Growth Factor Receptor Variant III
ELISA: Enzyme-Linked Immunosorbent Assay
ErbB: Erythroblastic Leukemia Viral Oncogene (homologous to EGFR)
Et: Ethyl
FBS: Fetal Bovine Serum
FITC: Fluorescein Isothiocyanate
GFP: Green Fluorescent Protein
GMB: Glioblastoma Multiforme
HPLC: High Performance Liquid Chromatography
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IC50: Half Maximal Inhibitory Concentration
iPr: Iso-propyl
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Luc: Luciferase
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M-group: Cell group treated with MitoTracker Green only
Me: Methyl
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MS MALDI-TOF: Mass Spectrometry, Matrix-Assisted Laser Desorption/Ionization – Time of Flight
MTT: Methylthiazolyldiphenyl Tetrazolium Bromide
NC-group: Negative Control cell group
NIR: Near Infra-Red
NMR: Nuclear Magnetic Resonance
PBS: Phosphate Buffer Saline
Ph: Phenyl
QD: Quantum Dot
R-group: Cell group treated with Ru-conjugate only
RFU: Relative Fluorescence Units
RM-group: Cell group treated with both Ru-conjugate and MitoTracker Green
SBT: Spectral Bleed-Through
SE: Standard Error
SEM: Sensitized Emission
TEG: Triethyleneglycol
THF: Tetrahydrofuran
TK: Tyrosine Kinase
TKI: Tyrosine Kinase Inhibitor
TLC: Thin Layer Chromatography
TR-FRET: Time-Resolved Förster (Fluorescence) Resonance Energy Transfer
TRITC: Tetramethylrhodamine Isothiocyanate
Tyr: Tyrosine
U87MG: Uppsala 87 Malignant Glioma
UV-Vis: Ultraviolet-Visible
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Mitochondrial
Localization
Br
NH
Fluorophore
O
N
EGFR-TKI
Modalities
2
O
N
N
H
O
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O
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NH
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H
RuII
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TEG-Derived Linker
N
A Novel Conjugate of Bis[((4-Bromophenyl)amino)quinazoline], a EGFR-TK
Ligand, with a Fluorescent Ru(II)-Bipyridine Complex, Exhibits Specific
Subcellular Localization in Mitochondria
Rashid Ilmi, Eleni Tseriotou, Panayiota Stylianou, Yiota A. Christou, Iakovia Ttofi, Nikolas Dietis,
Constantinos Pitris, Andreani D. Odysseos,* and Savvas N. Georgiades*
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