Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging

Article abstract of DOI:10.1016/j.jinorgbio.2015.11.021

A novel pyridyl functionalized analog of the promising hexadentate ⁶⁸Ga^{3 +} chelate H₂dedpa (N₄O₂, 1,2-[[6-carboxy-pyridin-2-yl]-methylamine]ethane) was successfully synthesized and characterized. This

Full text of DOI:10.1016/j.jinorgbio.2015.11.021

JIB-09852; No of Pages 10
Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging
Caterina F. Ramogida a,b, Lisa Murphy a, Jacqueline F. Cawthray a, James D. Ross a,
Michael J. Adam b, Chris Orvig a,
⁎
a
Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada
TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia V6T 2A3, Canada
b
a r t i c l e i n f o
a b s t r a c t
A novel pyridyl functionalized analog of the promising hexadentate 68Ga3+ chelate H2dedpa (N4O2, 1,2-[[6-carboxy-
pyridin-2-yl]-methylamine]ethane) was successfully synthesized and characterized. This new bifunctional chelate
(BFC) was used to prepare the first proof-of-principle bi-modal H2dedpa derivative for fluorescence and nuclear im-
aging. Two bi-modal H2dedpa derivatives were prepared: H2dedpa-propylpyr-FITC and H2dedpa-propylpyr-FITC-
(N,N′-propyl-2-NI) (FITC = fluorescein, pyr = pyridyl functionalized, NI = nitroimidazole). The ligands possess
the strong gallium-coordinating atoms contained within dedpa2− that are ideal for radiolabeling with 68Ga3+ for
positron-emission tomography (PET) imaging, and two fluorophores for optical imaging. In addition, one analog
contains two NI moieties for specific entrapment of the tracer in hypoxic cells. These new bi-modal analogs were
compared to the native unfunctionalized H2dedpa scaffold to determine the extent to which the addition of pyridyl
functionalization would affect metal coordination, and complex stability. The non-radioactive gallium complexes
were tested in a 3D tumor spheroid model. The novel pyridyl bis-functionalized H2dedpa ligand, H2dedpa-
propylpyr-NH2, was quantitatively radiolabeled with 67Ga (RCY N 99%) under reaction conditions commensurate
with unfunctionalized H2dedpa (10 min at room temperature) at ligand concentrations as low as 10−5 M. The
resultant 67Ga-complex withstood transchelation to the in vivo metal-binding competitor apo-transferrin (2 h
at 37 °C, 93% intact), signifying that [Ga(dedpa-propylpyr-NH2)]+ is a kinetically inert complex suitable for in vivo
use, but exhibited slightly reduced stability compared to the native [67Ga(dedpa)] scaffold (N99% intact).
Finally, bi-model fluorescent Ga-dedpa compounds were successfully imaged in a 3D tumor spheroid model. The
Ga-dedpa-FITC-NI derivative was specifically localized in the central hypoxic core of the spheroid.
Article history:
Received 3 September 2015
Received in revised form 12 November 2015
Accepted 17 November 2015
Available online xxxx
Keywords:
Gallium-68
Bifunctional chelates
Positron-emission tomography
Fluorescence imaging
Nitroimidazole
Spheroids
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
imaging, whereas PET and SPECT allow for resolution only in the mm
range and relatively long acquisition times are required (several mi-
nutes); the use of optical imaging is limited by its minimal penetration
in tissue (mm – cm), by contrast nuclear modalities have unlimited pen-
1.1. Molecular imaging techniques
Medical molecular imaging is defined by the ability to visualize the
function of the body providing detail at the molecular and cellular
level. Such techniques have become invaluable to the clinician and
patient in elucidating (diagnosis) disease (especially in oncology) in
early stages and providing essential information of the disease [1].
Current molecular imaging modalities available to the researcher or
clinician include nuclear techniques such as single-photon emission
computed tomography (SPECT) or positron emission tomography
(PET), ultrasound, computed tomography (CT), magnetic resonance
imaging (MRI), and optical imaging. Each modality possesses specific
advantages as far as depth penetration, resolution, and image acquisi-
tion time (Table 1). Fluorescence and nuclear imaging techniques
possess characteristics on opposite sides of the spectrum — resolution
down to nm scale and real-time imaging can be achieved with optical
etration allowing whole body imaging [2,3]. The fusion of fluorescent
and nuclear modalities into one imaging agent would take advantage
of the strengths of each technique. For example, whole-body imaging
using radio-imaging via PET to determine localization of the tumor
can be supplemented by fluorescence techniques to inform clinicians
(through endoscopy or surgical excision) of tumor boundaries by
illuminating the cancerous cells. The synergistic blend of imaging
modalities to produce bi-modal and/or multi-modal agents has been
acknowledged as a promising new facet of molecular imaging with
broadened applicability, and has been extensively reviewed [1–3].
Specifically, the development of bimodal radio- and fluorescent imaging
probes is emerging as a potentially powerful new technique [4,5].
1.2. Radiometals in radiopharmaceuticals: Gallium-68
Metals continue to play an important role in the field of nuclear
⁎
Corresponding author.
E-mail address: orvig@chem.ubc.ca (C. Orvig).
medicine. Radioactive metals possess a range of half-lives, and decay
http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021
0162-0134/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

2
C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Table 1
complexed to c(RGDyK) as a proof-of-principle [15]; however, the
lengthy and challenging synthesis resulted in extremely poor yields.
Therefore, a synthetic route to a novel bifunctional H2dedpa derivative
would add value to this Ga(III) ligand as a viable alternative to NOTA
for PET imaging agent elaboration. Herein, a novel bifunctional
H2dedpa analog possessing two reactive primary amines (H2dedpa-
propylpyr-NH2, Fig. 1; pyr = pyridyl) attached to the pyridyl rings was
synthesized for the first time.
The novel bifunctional H2dedpa analog was used to prepare two bi-
modal H2dedpa derivatives. These novel compounds possess two
attached fluorophores suitable for fluorescence microscopy imaging,
and retain the N4O2 binding sphere provided by the H2dedpa ligand
for coordination to 68Ga3+ for PET imaging. The second analog contains
two 2-nitroimidazole (2-NI) moieties to investigate the specific uptake
of the probe in hypoxic tissue, given that 2-NI can be reduced and
retained exclusively in hypoxic cells via direct competition with intra-
cellular oxygen concentration [19–22]. We recently reported 2-NI
analogs of H2dedpa which do not possess fluorophores, and exhibited
superb hypoxic-to-normoxic uptake ratios in three cell lines [23].
Much effort has been made by others to make a fluorescent probe of
hypoxia [24–28].
Comparison of imaging modalities with corresponding penetration through tissue, resolution
of technique, and acquisition timescale [2].
Timescale for
acquisition/
observation
Modality
Penetration
Resolution
CT
MRI
PET
SPECT
Fluorescence
microscopy (optical)
Ultrasound
Whole body
Whole body
Whole body
Whole body
mm–cm
μm
μm
mm
mm
nm
Min
Min–hour
Min–hour
Min–hour
Sec
Whole body except lungs
μm
Sec
emissions which make them ideal for incorporation into radiopharma-
ceuticals for both imaging and/or therapy.
With a half-life of 68 min and predominant positron emission (β+,
89%), 68Ga is an attractive radiometal for incorporation into a positron-
emission tomography (PET) imaging agent. Moreover, the isotope can
be supplied via a commercially available 68Ge/68Ga generator system,
thus greatly enhancing the isotope's accessibility to hospitals.
Like other radiometal-based radiopharmaceuticals, the utility of
68Ga-based PET imaging agents is strongly dependent on a bifunctional
chelate (BFC) that possesses the ability to efficiently and securely com-
plex the metal under radiochemical reaction conditions, such as room
temperature and low concentrations. The BFC must also possess a han-
dle for attachment to biologically relevant molecules which will act as
the vehicle to enforce site-specific delivery of the pharmaceutical to
the diseased state in vivo. The current state-of-the-art ligands for 68Ga
include the two macrocycles, 1,4,7-triazacyclononane-1,4,7-triacetic
acid (NOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA) [6–9]. NOTA has been universally recognized as the best
chelating ligand for gallium — the binding constants are the highest
reported, and complexation occurs within minutes at room tempera-
ture. Bifunctional variants of these macrocyclic (closed-chain) ligands
have been prepared (e.g. NODASA and NODAGA, Fig. 1) [10–12]
and conjugated to a variety of bio-targeting groups [13,14]. However,
the synthesis of these ligand systems is far from trivial, and groups
wishing to use these macrocycles for in vitro studies often purchase
the bifunctional derivatives from commercial sources.
Our group recently reported a new acyclic (open-chain) ligand,
H2dedpa (N4O2, 1,2-[[6-carboxy-pyridin-2-yl]-methylamine]ethane),
whose properties rival those of the gold standard NOTA for Ga(III)
chelation [17,18]. This hexadentate chelating system exhibits several
properties of merit for use in Ga(III) PET imaging agent elaboration:
quantitative radiolabeling (radiochemical yield, RCY N 99%) in only
10 min at room temperature at ligand concentrations as low as
10−7 M, and metal-complexes of high thermodynamic stability and
kinetic inertness. A bifunctional analog (derivative with a chemically re-
active/compatible attachment for coupling to biomolecules/targeting
vectors) of H2dedpa was synthesized (H2dedpa-p-Bn-NCS, Fig. 1) and
The ability of the new H2dedpa BFCs to label gallium isotopes was
investigated, followed by assessment of their kinetic inertness via an
apo-transferrin stability assay. Finally, the optical properties of
the novel “bi-modal” probes were evaluated in a 3D tumor spheroid
in vitro model visualized using confocal fluorescence microscopy.
2. Experimental
2.1. Materials and methods
All solvents and reagents were purchased from commercial suppliers
(Sigma Aldrich, TCI America, Fisher Scientific) and were used as received.
Fluorescein isothiocyanate (FITC) was purchased from Sigma-Aldrich
and used as received. Human apo-transferrin was purchased from
Sigma Aldrich. The analytical thin-layer chromatography (TLC) plates
were aluminum-backed ultrapure silica gel 60 Å, 250 μm thickness; the
flash column silica gel (standard grade, 60 Å, 40–63 μm) was provided
by Silicycle. 1H and 13C NMR spectra were recorded at 25 °C unless oth-
erwise noted on Bruker AV300, AV400, or AV600 instruments; NMR
spectra are expressed on the δ scale and referenced to residual solvent
peaks. Low-resolution mass spectrometry was performed using a Waters
ZG spectrometer with an ESCI electrospray/chemical-ionization source,
and high-resolution electrospray–ionization mass spectrometry (HR–
ESI–MS) was performed on a Micromass LCT time-of-flight instrument
at the Department of Chemistry, University of British Columbia. 67Ga-
(chelate) apo-transferrin stability experiments were analyzed using GE
Healthcare Life Sciences PD-10 desalting columns (size exclusion for
MW b 5000 Da) and counted with a Capintec CRC 15R well counter.
The HPLC system used for analysis and purification of nonradioactive
compounds consisted of a Waters 600 controller, Waters 2487 dual
Fig. 1. Left: Bifunctional NOTA analogs used in bioconjugation reactions to targeting vectors; middle: previously synthesized bifunctional H2dedpa derivative H2dedpa-p-Bn-NCS [15,16],
right: novel pyridyl bifunctional H2dedpa derivative (8) synthesized in this work.
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3
wavelength absorbance detector, and a Waters delta 600 pump.
Phenomenex Synergi Hydro-RP 80 Å columns (250 mm × 4.6 mm
analytical or 250 mm × 21.2 mm semipreparative) were used for purifi-
cation of several of the deprotected ligands. Analysis of radiolabelled
complexes was carried out using a Phenomenex Synergi 4 μ Hydro-RP
80 Å analytical column (250 × 4.60 mm 4 μm) using a Waters Alliance
HT 2795 separation module equipped with a Raytest Gabi Star NaI (Tl)
detector and a Waters 996 photodiode array (PDA). 67GaCl3 was
cyclotron-produced and provided by Nordion (Vancouver, BC, Canada)
as a ~0.1 M HCl solution.
7.65 (m, 8H), 4.73 (s, 4H), 3.95 (d, J = 11.8 Hz, 6H), 3.60 (s, 4H). 13
NMR (101 MHz, DMSO) δ 163.6, 157.9, 147.9, 147.5, 134.7, 133.6,
C
132.5, 131.4, 130.0, 128.5, 126.5, 124.4, 52.8, 52.1, 47.4. HR–ESI–MS
79
m/z for C
H
Br2N6NaO12S2 (M + Na+) calcd. (found) 906.9315
30 26
(906.9296).
2.6. Dimethyl 6,6′-((ethane-1,2-diylbis(((2-nitrophenyl)sulfonyl)
azanediyl))bis(meth-ylene))bis(4-(3-((tert-butoxycarbonyl)
amino)prop-1-yn-1-yl)picolinate) (5)
Compound 4 (420 mg, 0.47 mmol) was suspended in freshly
distilled THF (20 mL) and triethylamine (8 mL) in a Schlenk flask
under N2. The vessel underwent three freeze–pump–thaw cycles to
eliminate any oxygen, then tert-butyl prop-2-yn-1-ylcarbamate
(273 mg, 1.76 mmol, 3.7 equiv), bis(triphenylphospine)palladium(II)
dichloride (33 mg, 0.047 mmol, 10 mol%), and copper iodide (18 mg,
0.095 mmol, 20 mol%) were all added quickly. The flask was heated to
60 °C and stirred overnight. The resultant murky brown solution
was cooled to room temperature, filtered, washed with CH2Cl2 and
concentrated in vacuo. The crude product was purified by column
chromatography (CombiFlash Rf automated column system; 24 g HP
silica; A: hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield
5 as a brown fluffy solid (382 mg, 78%). 1H NMR (400 MHz, CDCl3) δ
8.03–7.98 (m, 2H), 7.82 (s, 2H), 7.68–7.56 (m, 6H), 7.37 (s, 2H), 4.63
(s, 4H), 4.13 (d, J = 4.2 Hz, 4H), 3.87 (s, 6H), 3.49 (s, 4H), 1.42
(s, 18H). 13C NMR (101 MHz, CDCl3) δ 164.7, 156.6, 155.4, 147.9,
147.6, 133.9, 133.3, 132.3, 132.1, 131.2, 127.3, 126.2, 124.3, 92.9, 80.2,
79.4, 53.5, 53.1, 52.9, 47.1, 28.3. HR–ESI–MS m/z for C46H51N8O16S2
(M + H+) calcd. (found) 1035.2864 (1035.2865).
2.2. Dimethyl 4-bromopyridine-2,6-dicarboxylate (1)
Compound 1 was prepared via a slightly modified version from
previously published [29]. Chelidamic acid monohydrate (2.20 g,
10.9 mmol) and PBr5 (23.4 g, 54.3 mmol, 5 equiv) were heated neat to
90 °C under N2 for 2 h. The deep red melt was then cooled to room tem-
perature, and CHCl3 (25 mL) was added. The solution was filtered, and
filtrate was cooled on ice (0 °C), while methanol (90 mL) was slowly
added. Crystallization was induced with scratching, and the crystalline
solid was collected by vacuum filtration and further dried under
vacuum to yield 1 as a crystalline white solid (2.82 g, 86%). 1H NMR
(300 MHz, CDCl3) δ 8.45 (s, 2H), 4.02 (s, 6H).
2.3. Methyl 4-bromo-6-(hydroxymethyl)picolinate (2)
Compound 1 (5.61 g, 20.5 mmol) was stirred in methanol/CH2Cl2
(200 mL: 50 mL) on ice (0 °C), and NaBH4 (1.17 g, 30,8 mmol, 1.5
equiv) was added in small portions. The reaction mixture was stirred
at 0 °C for 1.5 h, until complete by TLC (Rf (product) = 0.5, Rf (starting
material) = 0.63 in 100% ethyl acetate), subsequently quenched with
sat. NaHCO3 (100 mL), and phases separated. The aqueous phase was
extracted further with CH2Cl2 (4 × 50 mL); all organics were collected,
dried over MgSO4 and concentrated in vacuo to yield the crude product
2 as a white solid (4.79 g, 95%). The product was used in the next step
without purification.
2.7. Dimethyl 6,6′-((ethane-1,2-diylbis(((2-nitrophenyl)sulfonyl)
azanediyl))bis(meth-ylene))bis(4-(3-((tert-butoxycarbonyl)
amino)propyl)picolinate) (6)
Compound 5 (382 mg, 0.37 mmol) was dissolved in ethyl acetate
(40 mL), and palladium on activated carbon (10% Pd, 46 mg) was
added. The system was sealed and charged with hydrogen gas several
times. The solution was stirred under hydrogen at room temperature
for 24 h, after which time the catalyst was filtered off and replaced
anew, with further stirring for an additional 24 h. The solid catalyst
was filtered off, and filtrate was concentrated in vacuo to obtain 6 as a
brown solid (325 mg, 84%). 1H NMR (400 MHz, CDCl3) δ 8.22 (m, 2H),
7.81 (s, 2H), 7.60–7.70 (m, 6H), 7.39 (s, 2H), 4.73 (s, 4H), 3.94 (s, 6H),
3.50 (s, 4H), 3.14 (quart, J = 8 Hz, 4H), 2.67 (t, J = 8 Hz, 4H), 1.79
(quint, J = 8 Hz, 4H), 1.45 (s, 18H). 13C NMR (101 MHz, CDCl3) δ
165.3, 156.3, 156.0, 153.5, 148.1, 147.6, 133.6, 132.6, 132.0, 131.5,
126.0, 124.6, 124.0, 79.4, 53.2, 52.8, 46.4, 40.0, 32.4, 30.5, 28.4. HR–
ESI–MS m/z for C46H59N8O16S2 (M + H+) calcd. (found) 1043.3490
(1043.3472).
2.4. Methyl 4-bromo-6-(bromomethyl)picolinate (3)
Compound 2 (4.79 g, 19.5 mmol) was suspended with stirring in
CHCl3 (220 mL) at 0 °C. To this solution, PBr3 (2.94 mL, 31.1 mmol, 1.6
equiv) was added, and the mixture was stirred at room temperature
for 3.5 h. The yellow solution was subsequently cooled to 0 °C, and
quenched with aq. K2CO3 (200 mL). The organic phase was separated,
and the aqueous layer was further extracted with CH2Cl2
(2 × 120 mL). The organics were collected, dried over MgSO4 and
concentrated in vacuo. The crude solid was purified by column chroma-
tography (CombiFlash Rf automated column system; 80 g HP silica; A:
hexanes, B: ethyl acetate, 100% A to 100% B gradient) to yield 3 as a
white solid (4.27 g, 67%). 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J =
1.6 Hz, 1H), 7.86 (d, J = 1.6 Hz, 1H), 4.59 (s, 2H), 4.02 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 164.4, 158.7, 148.7, 134.8, 130.3, 128.0, 53.5,
32.2. MS (ES+) m/z = 310.1 [M + H]+.
2.8. Dimethyl 6,6′-((ethane-1,2-diylbis(azanediyl))bis(methylene))
bis(4-(3-((tert-butoxy-carbonyl)amino)propyl)picolinate) (7)
Compound 6 (324 mg, 0.31 mmol) was dissolved in THF (10 mL),
and thiophenol (65 μL, 0.64 mmol, 2.05 equiv), followed by K2CO3
(257 mg, 1.86 mmol, 6 equiv) was added. The reaction mixture was
stirred at room temperature for 4 days; subsequently the excess salts
were removed by centrifugation (10 min, 4000 rpm) and the filtrate
was concentrated in vacuo. The crude product was purified by column
chromatography (CombiFlash Rf automated column system; 12 g HP sil-
ica; A: dichloromethane, B: methanol with 2% triethylamine, 100% A to
25% B gradient) to obtain 7 as a light yellow-brown oil (132 mg, 63%).
1H NMR (400 MHz, CDCl3) δ 7.83 (s, 2H), 7.43 (s, 2H), 4.01 (s, 4H),
3.96 (s, 6H), 3.15 (quart, J = 8 Hz, 4H), 2.85 (s, 4H), 2.70 (t, J = 8 Hz,
4H), 1.84 (quint, J = 8 Hz, 4H, quint), 1.43 (s, 18H). 13C NMR
(101 MHz, CDCl3) δ 165.9, 160.2, 155.9, 152.6, 147.5, 125.7, 123.8,
2.5. Dimethyl 6,6′-((ethane-1,2-diylbis(((2-nitrophenyl)sulfonyl)
azanediyl))bis(methylene))bis(4-bromopicolinate) (4)
Compound 3 (889 mg, 2.88 mmol, 2 equiv) and N,N′-(ethane-1,2-
diyl)bis(2-nitrobenzenesulfonamide) [16] (619 mg, 1.44 mmol) were
dissolved in dimethylformamide (7 mL), and Na2CO3 (915 mg,
8.64 mmol, 6 equiv) was added. The reaction mixture was stirred at
room temperature for 3 days; subsequently diethyl ether (20 mL) and
water (20 mL) were added. The white precipitate that formed upon
addition was collected by vacuum filtration, washed with excess
water and diethyl ether, and further dried under vacuum to yield 4 as
a white solid (1.13 g, 89%). 1H NMR (300 MHz, CDCl3) δ 8.06 (m, 4H),
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

4
C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
79.3, 54.7, 52.8, 48.7, 39.9, 32.3, 30.5, 28.4. HR–ESI–MS m/z for
34H53N6O8 (M + H+) calcd. (found) 673.3925 (673.3928).
2.12. H2dedpa-propylpyr-FITC (11)
C
To a solution of 8 (3.5 mg, 0.008 mmol) in water (0.3 mL), a solution of
fluorescein isothiocyanate (FITC) (6.1 mg, 0.016 mmol, 2.1 equiv) in DMF/
water (3:1, 1.2 mL) was added. To this yellow mixture, triethylamine
(5.5 μL, 5 equiv) was added at which time the solution turned much
brighter orange in color. The reaction mixture was stirred at ambient tem-
perature under darkness for 18 h. The reaction mixture was subsequently
concentrated in vacuo, redissolved in CH3CN and purified by RP-HPLC
(gradient: A: 0.1% TFA (trifluoroacetic acid) in water, B: CH3CN; 5 to
100% B linear gradient over 25 min, 1 mL/min, tR (mono-FITC) =
16.7 min, tR (product) = 18.7 min, tR (FITC) = 23.4 min). Product frac-
tions were pooled and lyophilized to yield the product as a yellow solid
(3.2 mg, 33%). Purity was confirmed by HPLC re-injection of an aliquot
of final collected product. HR–ESI–MS m/z for C64H55N8O14S2 (M + H+)
calcd. (found) 1223.3279 (1223.3286) (0.6 PPM).
2.9. 6,6′-((Ethane-1,2-diylbis(azanediyl))bis(methylene))
bis(4-(3-aminopropyl) picolinic acid) (8)
Compound 7 (73 mg, 0.11 mmol) was dissolved in THF/water (3:1,
4 mL), and lithium hydroxide (11 mg, 0.43 mmol, 4 equiv) was added.
The reaction mixture was stirred at room temperature for 1 h, and sub-
sequently concentrated in vacuo. The resulting solids were redissolved
in 4 M HCl/dioxane/THF (3:2:1, 8 mL), and stirred at room temperature
overnight. The reaction mixture was then concentrated in vacuo
and purified by semi-preparative RP-HPLC (gradient: A: 0.1% TFA
(trifluoroacetic acid) in water, B: CH3CN, 5 to 100% B linear gradient
over 25 min, 10 mL/min, tR = 8.6 min). Product fractions were pooled,
and lyophilized to yield 8 as an off-white solid (26 mg, 55%). 1H NMR
(400 MHz, D2O) δ 8.03 (s, 2H), 7.57 (s, 2H), 4.62 (s, 4H), 3.69 (s, 4H),
3.01 (t, J = 8 Hz, 4H), 2.83 (t, J = 8 Hz, 4H), 2.02 (quint, J = 8 Hz,
4H). 13C NMR (101 MHz, D2O) δ 168.5, 154.1, 150.9, 148.0, 126.3,
125.3, 50.2, 44.0, 38.9, 31.3, 27.1. HR–ESI–MS m/z for C22H33N6O4
(M + H+) calcd. (found) 445.2563 (445.2570).
2.13. H2dedpa-propylpyr-FITC-(N,N′-propyl-2-NI) (12)
Compound 10 (4.0 mg, 0.005 mmol) was dissolved in DMF/water
(2:1, 0.3 mL) and FITC (4.6 mg, 0.012 mmol, 2.2 equiv) in DMF
(0.1 mL) was added. To this murky yellow solution, triethylamine
(3 μL, 4 equiv) was added at which time the solution turned clear and
became much brighter orange in color. The reaction mixture was stirred
at ambient temperature excluded from light for 20 h. The reaction mix-
ture was subsequently concentrated in vacuo, redissolved in CH3CN and
purified by RP-HPLC (gradient: A: 0.1% TFA (trifluoroacetic acid) in
water, B: CH3CN; 5 to 100% B linear gradient over 25 min, 1 mL/min,
tR (product) = 18.4 min). Product fractions were pooled and lyophilized
to yield the product as a bright yellow solid (2.9 mg, 36%). Purity was
confirmed by HPLC re-injection of an aliquot of final collected product.
HR–ESI–MS m/z for C76H69N14O18S2 (M + H+) calcd. (found)
1529.4356 (1529.4352) (−0.3 PPM).
2.10. Dimethyl 6,6′-((ethane-1,2-diylbis((3-(2-nitro-1H-imidazol-1-yl)
propyl)azane-diyl))bis(methylene))bis(4-(3-((tert-butoxycarbonyl)
amino)propyl)picolinate) (9)
Compound 7 (60 mg, 0.089 mmol) and 1-(3-bromopropyl)-2-nitro-
1H-imidazole [23] (46 mg, 0.20 mmol, 2.2 equiv) were dissolved in
acetonitrile (4 mL), and K2CO3 (49 mg, 0.36 mmol, 4 equiv) was
added. The reaction mixture was stirred at 55 °C for 4 days, subsequent-
ly cooled to room temperature, filtered to remove excess salts and
concentrated in vacuo. The crude product was purified by column chro-
matography (CombiFlash Rf automated column system; 4 g HP silica; A:
dichloromethane, B: methanol, 100% A to 30% B gradient) to yield 9 as a
yellow oil (30 mg, 34%). 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 2H), 7.38
(s, 2H), 7.19 (d, J = 9.4 Hz, 2H), 7.07 (s, 2H), 4.42 (t, J = 7.2 Hz, 4H),
3.93 (s, 6H), 3.77 (s, 4H), 3.13 (dd, J = 13.2, 6.7 Hz, 4H), 2.71–2.66
(m, 4H), 2.63 (s, 4H), 2.54 (s, 4H), 2.02–1.92 (m, 4H), 1.81 (dt, J =
14.6, 7.2 Hz, 4H), 1.40 (s, 18H). 13C NMR (101 MHz, CDCl3) δ 165.9,
156.1, 152.8, 147.6, 144.9, 128.5, 126.6, 126.3, 124.2, 79.4, 60.3, 53.0,
51.8, 51.4, 48.4, 40.1, 32.5, 30.8, 28.5, 28.3. MS (ES+) m/z = 979.6
[M + H]+.
2.14. [Ga(dedpa-propylpry-NH2)][NO3], [Ga(8)][NO3]
To a solution of 8 (5.6 mg, 0.013 mmol) in water/methanol (2:1,
0.5 mL), a solution of Ga(NO3)3·6H2O (5.0 mg, 0.014 mmol, 1.1 equiv)
in water (0.2 mL) was added. The pH of this solution was adjusted to
5 using aq. NaOH (0.1 M), and subsequently stirred at 60 °C for
40 min. The solvent was then removed in vacuo to yield [Ga(8)][NO3]
as a white solid. The product was used in subsequent steps without fur-
ther purification. 1H NMR (400 MHz, D2O) δ 8.28 (s, 2H), 8.00 (s, 2H),
4.70 (d, J = 17.8 Hz, 2H), 4.42 (d, J = 17.8 Hz, 2H), 3.32 (d, J =
9.9 Hz, 2H), 3.12 (dd, J = 14.5, 6.8 Hz, 8H), 2.67 (d, J = 10.0 Hz, 2H),
2.24–2.09 (m, 4H). 13C NMR (150 MHz, D2O) δ 167.5, 164.0, 152.8,
2.11. 6,6′-((Ethane-1,2-diylbis((3-(2-nitro-1H-imidazol-1-yl)propyl)
azanediyl)) bis(methylene))bis(4-(3-aminopropyl)picolinic acid) (10)
144.9, 129.3, 125.9, 50.9, 48.6, 40.7, 34.1, 28.8. HR–ESI–MS m/z for
69
Compound 9 (30 mg, 0.03 mmol) was dissolved in THF/water (3:1,
4 mL), and lithium hydroxide (3 mg, 0.12 mmol, 4 equiv) was added.
The mixture was stirred at room temperature until methyl deprotection
was deemed complete by mass spectrometry (MS (ES+) m/z = 951.7
[M + H]+, about 1 h), and subsequently concentrated in vacuo. The res-
idue was redissolved in 4 M HCl/dioxane/THF (3:2:1, 3 mL) and stirred
at room temperature overnight. The reaction mixture was subsequently
concentrated in vacuo, redissolved in water and purified by semi-
preparative RP-HPLC (gradient: A: 0.1% TFA (trifluoroacetic acid) in
water, B: CH3CN, 5 to 100% B linear gradient over 25 min, 10 mL/min,
tR = 12.7 min). Product fractions were pooled, and lyophilized to
yield 10 as an off-white solid (23 mg, 100%). 1H NMR (400 MHz,
MeOD) δ 7.92 (s, 2H), 7.49 (s, 2H), 7.42 (s, 2H), 7.11 (s, 2H), 4.49–4.44
(m, 4H), 4.42 (s br, 4H), 3.58 (s br, 4H), 3.22 (s br, 4H), 3.03–2.97 (m,
4H), 2.85–2.79 (m, 4H), 2.26 (s br, 4H), 2.03 (dd, J = 14.5, 7.1 Hz, 4H).
13C NMR (101 MHz, MeOD) δ 167.2, 154.7, 149.0, 145.9, 128.8, 128.4,
128.1, 125.8, 119.1, 116.2, 59.3, 58.2, 53.7, 51.7, 40.1, 32.6, 28.8, 27.1.
HR–ESI–MS m/z for C34H47N12O8 (M + H+) calcd. (found) 751.3640
(751.3635) (−0.7 PPM).
C H
22 30
GaN6O4 (M+) calcd. (found) 511.1584 (511.1577).
2.15. [Ga(dedpa-propylpyr-NH2-(N,N′-propyl-2-NI)][NO3], [Ga(10)][NO3]
The gallium complex was prepared as above for [Ga(8)][NO3] using
10 and was isolated as a white solid. 1H NMR (400 MHz, D2O) δ 8.33 (s,
2H), 8.02 (s, 2H), 7.38 (s, 2H), 7.19 (s, 2H), 4.65 (d, J = 17.1 Hz, 2H), 4.47
(t, J = 6.0 Hz, 4H), 4.44 (d, J = 9.7 Hz, 2H), 3.25 (d, J = 11.4 Hz, 2H), 3.17
(dd, J = 11.2, 4.0 Hz, 8H), 2.86 (d, J = 11.1 Hz, 2H), 2.83 (d, J = 9.9 Hz,
2H), 2.49–2.38 (m, 2H), 2.38–2.23 (m, 4H), 2.21 (dt, J = 15.8, 7.9 Hz,
4H). 13C NMR (101 MHz, D2O) δ 163.3, 161.8, 148.8, 141.3, 126.8,
126.7, 126.0, 122.9, 116.3, 113.4, 54.4, 45.3, 37.3, 30.8, 25.3, 21.4. HR–
ESI–MS m/z for C
(817.2659) (−0.2 PPM).
H
GaN12O8 (M+) calcd. (found) 817.2661
69
34 44
2.16. [Ga(dedpa-propylpyr-FITC)][NO3], [Ga(11)][NO3]
To a solution of [Ga(8)][NO3] (7.2 mg, 0.013 mmol) in water (0.5 mL),
a solution of FITC (11.0 mg, 0.028 mmol, 2.2 equiv) in DMF/water
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
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C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
5
(2:1, 0.3 mL) was added. To this murky yellow solution, triethylamine
2.20. Confocal microscopy imaging
(7 μL, 4 equiv) was added at which time the solution turned clear and be-
came bright orange in color. The reaction mixture was stirred at ambient
temperature under darkness for 18 h. The solution volume was reduced
by ~2/3 by evaporation with blowing air, then the crude mixture was
purified by RP-HPLC (gradient: A: 0.1% TFA (trifluoroacetic acid) in
water, B: CH3CN; 5 to 100% B linear gradient over 25 min, 1 mL/min, tR
(product) = 18.0 min). Product fractions were pooled and lyophilized
to yield the product as a bright yellow solid (10.7 mg, 63%). Purity was
Confocal images were taken on an Olympus Fluoview FV1000-
inverted laser scanning confocal microscope located in the Life Sciences
Centre at the University of British Columbia. The microscope was
equipped with an Olympus UPLAPO 10×/0.40 air objective lens,
UPLAPO 40×/1.00 oil objective lens and PLAPO 60×/1.40 water objec-
tive lens. A scan rate of 4.0 μs pixel−1 with Kalman averaging was
used. The argon 488 nm laser had the following excitation and emission
ranges: Ex 488 nm: Em 500–550 nm. Images were processed with
ImageJ software, and displayed images are all at a depth (measurement
along z-axis) of 144.3 μm.
confirmed by HPLC re-injection of an aliquot of final collected product.
69
HR–ESI–MS m/z for C
H
GaN8O14S2 (M+) calcd. (found) 1289.2300
64 52
(1289.2291) (−0.7 PPM).
2.17. [Ga(dedpa-propylpyr-FITC-(N,N′-propyl-2-NI)][NO3], [Ga(12)][NO3]
3. Results and discussion
The final product was prepared as above for [Ga(11)][NO3] using 12
and was isolated as a bright yellow solid (5.2 mg, 45%, tR (product) =
18.5 min). Maldi-TOF MS (ES+) m/z = 1595.6 [M+].
3.1. Synthesis and characterization of pro-ligands and metal complexes
In an attempt to fully harness the potential of H2dedpa for radio-
pharmaceutical elaboration, much effort has been made towards
developing new and facile synthetic routes towards preparation of
bifunctionalized analogs of the native unfunctionalized scaffold. These
bifunctional chelating ligands (BFCs) should possess reactive groups
for bioconjugation to targeting vectors. Herein, a novel BFC based on
the strong Ga(III) chelate H2dedpa has been developed where bis-
functionalization has been introduced through the 4-position of each
pyridyl group (H2dedpa-propylpyr-NH2, 8).
Preparation of the novel BFC (8), began with the conversion of
chelidamic acid to methyl 4-bromo-6-(bromomethyl)picolinate (3) in
three steps (55% overall yield, Scheme 1). This bromo-picolinate was
used in N,N′-alkylation of 2-nitrobenzenesulfonamide (nosyl) protected
ethylenediamine [16] to yield 4 in high yield (89%, Scheme 2). The
di-bromo substituents on the nosyl-protected Me2dedpa precursor 4
were cross-coupled to tert-butyl-prop-2-yn-1-ylcarbamate (Boc-N
propargylamine) using Sonogashira methodology to yield 5 (78%
yield). In order to prevent unwanted nucleophilic addition of
thiophenol to the alkynes, the alkyne bonds were reduced under mild
conditions (10% Pd on activated carbon, H2) to produce the analogous
alkane product 6 (84% yield) before nosyl-deprotection was performed.
The alkyne reduction reaction was monitored closely and quenched be-
fore reduction of the nitro moieties off the nosyl groups began. Both
nosyl groups were removed through the addition of thiophenol under
basic conditions to yield protected pro-ligand 7 (63% yield). Methyl
ester and N-Boc deprotections were performed in one-pot to yield the
novel bifunctional H2dedpa analog H2dedpa-propylpyr-NH2 (8) (26%
overall yield in 5 steps). Compound 8 contains two primary amines as
reactive sites for further conjugation to biomolecules.
2.18. Attempted Ga(III) complexation of derivatives 11 and 12
To a solution of 11 or 12 (0.02 mmol) in water/methanol (2:1,
0.5 mL), a solution of Ga(NO3)3·6H2O (0.022 mmol, 1.1 equiv) in water
(0.2 mL) was added. The pH of this solution was adjusted to 5 using aq.
NaOH (0.1 M), and subsequently stirred at 60 °C for 40 min. The reaction
progress was followed by ES–MS by presence of the [M(L)]+ peak. No
complexation was evident after 40 min, and the reaction was subse-
quently stirred at 90 °C overnight. Again, no complexation was evident.
2.19. 3D tumor spheroids cell culture and compound dosing
DLD-1 human colon carcinoma cells were maintained in Advanced
DMEM (Invitrogen) and supplemented with 2% FBS and 2 mM gluta-
mine in a humidified incubator at 37 °C and 5% CO2. Spheroid culture
procedures followed those of a previously reported protocol. [30,31]
Spheroids were formed by plating 100 μL of a 1.5 × 105 cell mL−1 single
cell suspension of DLD-1 cells onto agarose (0.75%) coated 96 well
plates and they were allowed to aggregate for 5 to 7 days which resulted
in the formation of one spheroid per well. Stock solutions (So) of
[Ga(11)]+, [Ga(12)]+, 11, and 12, and FITC were made up in media
with 6% DMSO to an original concentration of approx. 0.6 mM. Aliquots
of each stock solution (So) were used to make working solutions (S100
,
S20, S5) of 100, 20, and 5 μM for each compound. These were dosed in
triplicate for each independent compound, concentration, and time
point. At the time of dosing, 50 μL of medium from each well containing
a spheroid was replaced with 50 μL of each working solution S100, S20 or
S5 such that the final concentration of compound in the well was 50, 10,
or 2.5 μM, respectively (final concentration of DMSO in each well did not
exceed 1%). Dosed spheroids were then incubated at 37 °C for 1, 2, or
4 h. At the end of each incubation period, spheroids were transferred
to 1.5 mL Eppendorf tubes and washed thrice with phosphate buffered
saline (PBS, pH 7.4, 3 × 200 μL) to remove excess compound. Spheroids
were then transferred to a 96-well imaging plate.
The protected pro-ligand 7 was also further functionalized off the
secondary amines with 1-(3-bromopropyl)-2-nitroimidazole, followed
by complete deprotection to give 10; this scaffold now contains 2-
nitroimidazoles, which are common trapping moieties in hypoxic
tissue, as well as two primary amines situated off the 4-position of the
pyridyl rings which can be used in further bioconjugation reactions.
As a proof-of-principle, fluorescein isothiocyanate (FITC) was conju-
gated to the reactive primary amines of BFCs 8 and 10 (Scheme 3)
Scheme 1. Synthesis of methyl 4-bromo-6-(bromomethyl)picolinate (3). Reagents and conditions: (i) a. PBr5, N2, 90 °C, 2 h; b. CHCl3, methanol, 0 °C (ii) methanol/CH2Cl2, NaBH4, 0 °C,
1.5 h (iii) PBr3, CHCl3, 0 °C – RT, 3.5 h.
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

6
C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Scheme 2. Synthesis of novel pyridyl-functionalized H2dedpa BFCs H2dedpa-propylpyr-NH2 (8) and H2dedpa-propylpyr-NH2-(N,N′-propyl-2-NI) (10). Reagents and conditions: (i) DMF,
Na2CO3, RT, 3 d; (ii) THF, Et3N, Pd(PPh3)2Cl, CuI, N2, 60 °C, 18 h; (iii) EtOAc, Pd/C, H2, RT, 2 × 24 h; (iv) THF, thiophenol, K2CO3, RT, 4 d; (v) THF/water (3:1), LiOH, RT, 1 h, then 4 M
HCl/dioxane/THF (3:2:1), RT, 18 h; (vi) CH3CN, K2CO3, 55 °C, 4 d; (vii) same as (v).
through thiourea bond formation, to generate “bi-modal” imaging
probes. FITC is a commercially available fluorophore (λex/λem = 490/
525 nm) used commonly as a dye in fluorescence microscopy and cell
biology because of its high absorptivity, excellent fluorescence quantum
yield, and good water solubility. The pro-ligand 11 contains both
the promising backbone H2dedpa (N4O2) for chelation to radiometals
67/68Ga, as well as the fluorophore FITC for optical imaging. Similarly,
12 also contains both the chelating backbone (N4O2) and fluorophore,
but also 2-nitroimidazole moieties which will act as hypoxia targeting/
trapping vectors.
revealed excitation and emission peaks at 492 and 527 nm respectively
(Fig. 3), which are typical for wavelengths of the FITC dye.
3.2. 67Ga radiolabeling studies
The γ-emitter 67Ga (t1/2 = 3.26 d) was used in initial radiolabeling
studies of the novel bi-modal ligands 11 and 12 to determine their abil-
ities to complex gallium isotopes. Because of its longer half-life, 67Ga
was used as a model for 68Ga in the radiolabeling experiments
performed herein. Unfortunately, neither fluorescent ligand (11 or 12)
displayed any 67Ga labeling (radiochemical yield ~0%) under 10 min
and room temperature reaction conditions. Extended reaction times
and elevated temperatures (1 h, 85 °C) did not result in an increase in
radiolabeling yield. Although disappointing, this result was not surpris-
ing since direct complexation of the ligands 11 and 12 with non-
radioactive gallium was also futile (vide supra). It is hypothesized that
the added structural ‘bulk’ introduced by two fluorescein molecules
(MW = 389.3 Da each) dangling from the 4-position of each pyridyl
ring impedes the movement/reorganization of donor atoms of the
metal binding sphere of the linear H2dedpa chelate, thus obstructing
metal complexation. Moreover, fluorescein contains many functional
groups that can potentially interfere with chelation of gallium. For
example, the highly acidic phenolic acid moieties as well as the acid–
base tautomeric equilibria of lactone to carboxylate are potential func-
tionalities that can compete for metal chelation. Given this possibility,
Complexation of each pro-ligand (11 and 12) with non-radioactive
gallium was also attempted, but was unsuccessful. No evidence of
metal complexation was observed by ESI–MS, NMR, or HPLC analysis,
even when the reaction mixture was heated to reflux overnight. Instead,
the ‘fluorophore-free’ precursors 8 and 10 were allowed to complex with
Ga(III) under the mild conditions previously used for unfunctionalized
H2dedpa (Scheme 3). The Ga-precursors [Ga(8)]+ and [Ga(10)]+ were
isolated and fully characterized by HR–ESI–MS, and NMR spectroscopy.
Diagnostic diastereotopic splitting of hydrogens in the ethylenediamine
bridge was tracked by NMR spectroscopy. These two Ga-complexes
were then successfully conjugated to FITC under basic conditions, and
purified by RP-HPLC to give [Ga(11)]+ and [Ga(12)]+ as bright yellow
solids. Again, the diastereotopic splitting of hydrogens in the metal
complexes was identified in the 1H NMR spectrum (Fig. 2). The absorp-
tion and emission spectra of [Ga(dedpa-propylpyr-FITC)]+, [Ga(11)]+
,
Scheme 3. Synthesis of bi-modal H2dedpa derivatives H2dedpa-propylpyr-FITC (11) and H2dedpa-propylpyr-FITC-(N,N′-propyl-2-NI) (12) and corresponding Ga-complexes.
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
7
Fig. 2. 1H NMR spectra of (top) [Ga(dedpa-propylpyr-FITC)]+ ([Ga(11)]+) and (bottom) [Ga(dedpa-propylpyr-FITC-(N,N′-propyl-2-NI)]+ ([Ga(12)]+) in DMSO-d6 (400 MHz, 25 °C)
highlighting diastereotopic splitting due to gallium complexation.
the lack of metal chelation for fluorescent dedpa ligands 11 and 12 may
be from a combination of steric and functional group interference.
Consequently, precursors without the fluorescent probe (8 and 10)
were labeled with 67Ga as a model. Both displayed quantitative 67Ga
labeling (RCY N 99%) under reaction conditions commensurate with
unfunctionalized H2dedpa [17] and its recently reported 2-NI deriva-
tives [23] (10 min at room temperature, 10−4 to 10−5 M ligand).
pyridyl rings leads to complexes of (somewhat) reduced kinetic inert-
ness. The propyl linker serves as a weak electron donor and introduces
some degree of steric ‘bulk’ around the metal binding pocket of the
ligand. These steric and electronic effects imposed by the addition of
the alkyl linker seemed to have translated to ligands of reduced stability
compared to their unfunctionalized analogs. Furthermore, it is also
possible that intramolecular interaction of the chelated 67Ga(III) with
nucleophilic primary amine moieties of derivatives 8 and 10 may affect
the stability of the resultant metal complex. Further stability studies
with Ga-complexes in which the amines have been acylated with place-
holders (e.g. benzyl groups) prior to complexation are planned in the
future to discern the stability of these derivatives compared to those
which possess free primary amines (e.g. derivatives 8 and 10).
3.3. Human apo-transferrin stability studies
67Ga-labeled precursors 8 and 10 were tested in a human apo-
transferrin stability assay, and were used as a model to assess the kinetic
inertness of the bi-modal H2dedpa derivatives. Radiolabeling of the final
‘bi-modal’ probed 11 and 12 was unsuccessful, and therefore could not
be tested in the apo-transferrin challenge assay. The iron transport
protein in humans, transferrin, possesses a strong binding affinity for
Fe(III) and other similar metal ions, especially Ga(III). Consequently, it
is imperative that any chelate-bound 67/68Ga3+ be sufficiently stable
(both thermodynamically stable and kinetically inert) to prevent
transchelation of the radiometal to the endogenous protein in vivo.
The unaltered H2dedpa ligand previously demonstrated exceptional
stability in a human apo-transferrin stability assay remaining N99%
intact after 2 h, [17] this result is compared to ligand 8 from this work
where propyl linkers in the 4-position of the pyridyl rings have been
added to introduce bifunctionality of the native H2dedpa scaffold
(Table 2). [67Ga(8)]+ exhibited slightly reduced stability after 2 h com-
pared to [67Ga(dedpa)]+ (93 versus N99% intact). A similar trend was
seen when comparing analogs which include 2-NI moieties (10 and
H2dedpa-N,N′-propyl-2-NI): [67Ga(10)]+ was approximately 27% less
stable than the corresponding complex in which there is no pyridyl
functionalization ([67Ga(dedpa-N,N′-propyl-2-NI)]+). These results
suggest that functionalization of H2dedpa at the 4-position of the
Fig. 3. Excitation (blue dashed) and emission (red solid, λex = 490 nm) spectra of [Ga(11)]+
,
[Ga(dedpa-propylpyr-FITC)]+, in PBS (pH 7.4). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

8
C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Table 2
resulting in reduced oxygen diffusion near the centre of the spheroid,
hence older spheroids tend to be more hypoxic [32].
apo-Transferrin stability challenge assay (37 °C, 2 h) of 67Ga-labeled pyridyl-functionalized
dedpa2− ligands 8 and 10, and non-functionalized dedpa2− standards for comparison, with
stability shown as the percentage of intact 67Ga complex.
Spheroids (grown for 5 days) treated with [Ga(dedpa-propylpyr
-
FITC-(N,N′-propyl-2-NI))]+ yielded confocal microscopy images with
slight observable uptake of tracer, but no discernable elevated uptake
of the 2-NI probe in the central hypoxic core of the spheroid over the
course of 4 h (Fig. 4). Fluorescence images of ‘negative control’
[Ga(dedpa-propylpyr-FITC)]+ exhibited some non-specific uptake
evinced by even distribution of fluorescence throughout the spheroid
over the course of 4 h. Due to the qualitative nature of the fluorescent
images, it was impossible to discern real differences in probe uptake be-
tween [Ga(11)]+ and [Ga(12)]+ with 5 day old spheroids. Nonetheless,
the results show that the novel fluorescent probes [Ga(11)]+ and
[Ga(12)]+ were successfully imaged using fluorescence confocal
microscopy in an in vitro 3D spheroid model.
Spheroids grown for 7 days were similarly treated with 10 μM of
both [Ga(dedpa-propylpyr-FITC-(N,N′-propyl-2-NI))]+ and [Ga(dedpa-
propylpyr-FITC)]+ for 2 h. It is evident that the 2-NI-containing probe
([Ga(12)]+) exhibits elevated and concentrated localization near the
central hypoxic core of the spheroid compared to the negative control
[Ga(11)]+ which displays only some residual non-specific uptake
distributed evenly throughout the spheroid (Fig. 5). These results clear-
ly demonstrate the ability of [Ga(dedpa-propylpyr-FITC-(N,N′-propyl-2-
NI))]+ to specifically localize at the hypoxic region within the spheroid
model. The evident difference between probe uptake in 5 or 7 day old
spheroids may suggest that younger spheroids (5 days old) are not
sufficiently hypoxic to warrant 2-NI trapping.
Complex
15 min (%)
1 h (%)
2 h (%)
[
[
[
[
67Ga(8)]+
99.0 0.9
87.8 3.1
N99
92.3 2.3
82.3 2.4
N99
92.7 2.2
72.4 2.0
N99
67Ga(10)]+
67Ga(dedpa)]+ [17]
67Ga(dedpa-N,N-propyl-2-NI)]+ [23]
97.6 1.2
97.2 0.9
N99
3.4. In vitro 3D spheroid imaging
The uptake of the novel fluorescent Ga-dedpa probes was tested in a
multicellular spheroid model and visualized using confocal microscopy.
Spheroids are multicellular 3-dimensional structures of cancerous cells
that act as in vitro tumor models [32]. Multicellular spheroids can
effectively simulate the features of solid tumors in vivo such as tumor
micro-environment, cell–cell interactions, and the extracellular matrix;
accordingly, they have found great utility for screening of potential drug
candidates [30,33–35]. It has been shown that cells on the periphery of
the spheroid actively proliferate and act as well-oxygenated healthy
(normoxic) cells, while diffusion of oxygen near the centre of the spher-
oid is limited and hence cells at the centre are hypoxic [30]. Thus 3D
spheroids are a good model for visualizing the distribution and targeting
ability of fluorescent drugs in hypoxic conditions.
Spheroids were treated with both Ga-complexes, [Ga(11)]+ and
[Ga(12)]+ at concentrations of 50, 10, and 2.5 μM and incubated for 1,
2, or 4 h to track the course of fluorescence uptake over time. The only
difference between these two fluorescent probes is the addition of
two 2-NI moieties on [Ga(12)]+. It was hypothesized that the addition
of hypoxia trapping functionalities onto [Ga(12)]+ would result in
specific uptake of the fluorescent probe in the hypoxic core of the spher-
oid, whilst [Ga(11)]+ would display no such preference in cell uptake.
Pro-ligands 11 and 12, and FITC were also treated as controls. After
removal of excess fluorescent probe and washing of spheroids with
phosphate buffered saline, each was imaged using fluorescence confocal
microscopy. Spheroids used for imaging were grown for either 5 or
7 days. As spheroids mature their cells pack more densely in the core
4. Conclusions
Herein we report the preparation of a novel bifunctional analog of
the promising Ga(III) chelate H2dedpa using Sonogashira cross-
coupling chemistry. This H2dedpa bifunctional chelate (BFC) has been
altered to encompass two propyl-NH2 functionalities at the 4-position
of each pyridyl ring (H2dedpa-propylpyr-NH2). The two primary amines
in the H2dedpa BFC 8 are sufficient for bioconjugation to targeting
vectors bearing compatible reactive groups, such as an isothiocyanate.
This new BFC was used in a conjugation reaction with the commercially
Fig. 4. Overlaid fluorescence (green) and optical (gray) images of 3D tumor spheroids (5 days old) treated with 10 μM [Ga(dedpa-propylpyr-FITC-(N,N′-propyl-2-NI))]+, [Ga(12)]+ (top
row) or [Ga(dedpa-propylpyr-FITC)]+, [Ga(11)]+ (bottom row) incubated for (A,D) 1 h, (B,E) 2 h, or (C,F) 4 h.
Please cite this article as: C.F. Ramogida, et al., Novel “bi-modal” H2dedpa derivatives for radio- and fluorescence imaging, J. Inorg. Biochem.
(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021

C.F. Ramogida et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
9
Fig. 5. Overlaid fluorescence (green) and optical (gray) images of 3D tumor spheroids (7 days old) dosed with (left) [Ga(dedpa-propylpyr-FITC-(N,N′-propyl-2-NI))]+, [Ga(12)]+, (right)
negative control [Ga(dedpa-propylpyr-FITC)]+, [Ga(11)]+ (10 μM complex for 2 h). Highlighting elevated uptake of 2-NI fluorescent probe (left) in the central hypoxic core of the spheroid,
compared to the negative control compound without 2-NI moieties (right).
available fluorophore FITC to generate a fluorescent H2dedpa conjugate,
as proof-of-principle. This “bi-modal” agent was evaluated for its
potential as a combined 68Ga PET and optical fluorescent imaging
agent. A second H2dedpa analog, which also contains two 2-NI moieties
as a hypoxia marker in addition to the FITC fluorophore, was also
successfully synthesized and characterized.
RCY
SPECT
radiochemical yield
single-photon emission computed tomography
Dedication
In memory of Professor Graeme Hanson and his contributions to
The addition of bulky fluorescein units suspended off each pyridyl
ring of H2dedpa precluded metal-complexation, and consequently re-
sulted in unsuccessful 67Ga radiolabeling. This result will ultimately
limit the utility of these novel H2dedpa fluorescent probes in future
studies. Still, the ‘fluorescent-free’ precursors 8 and 10 were successfully
radiolabelled with 67Ga (N99% RCY, 10 min, RT), suggesting the new
BFC, H2dedpa-propylpyr-NH2 (8), may be a promising candidate for
conjugation with other lighter molecular weight targeting vectors.
Human apo-transferrin stability assays of 67Ga-labeled radiotracers 8
and 10 indicated that these BFCs form gallium complexes of good to
moderate stability (93 and 72% intact after 2 h, respectively). Future
studies to evaluate the mono-derivatization of the propylamine groups
of the new bifunctional dedpa analog, H2dedpa-propylpyr-NH2 (8), are
currently underway. Such derivatives can be used to add a protein-
reactive group as well as a fluorophore onto the same chelate.
Bioinorganic Chemistry and Electron Paramagnetic Resonance.
Acknowledgments
We acknowledge Nordion (Canada) and the Natural Sciences and
Engineering Research Council (NSERC) of Canada for grant support
(CR&D, Discovery) and NSERC CGS-M/CGS-D fellowships (to C.F.R.),
the University of British Columbia for 4YF fellowships (to C.F.R.), and
the UBC Centre for Blood Research for a summer student scholarship
(J.D.R.). C.O. acknowledges the Canada Council for the Arts for a Killam
Research Fellowship (2011–2013).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2015.11.021.
For the first time a novel fluorescent H2dedpa derivative was syn-
thesized, characterized, and successfully imaged using confocal fluores-
cence imaging in an in vitro 3D spheroid model. Fluorescence imaging
using spheroids treated with a derivative in which the hypoxia marker
2-nitroimidazole was integrated resulted in specific localization of the
fluorescent tracer in the central hypoxic core of the spheroid.
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(2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.11.021