Pyridine-containing octadentate ligand NE3TA-PY for formation of neutral complex with 177Lu(III) and 90Y(III) for radiopharmaceutical applications: Synthesis, DFT calculation, radiolabeling, and in vitro complex stability

Article abstract of DOI:10.1016/j.jinorgbio.2021.111436

Targeted radionuclide therapy is a developing therapeutic modality for cancer and employs a cytotoxic radionuclide bound to a chelating agent and a bioactive molecule with high binding affinity for a specific biomarker in tumors. An optimal chelator is one of the critical components to control therapeutic efficacy and toxicity of targeted radionuclide therapy. We designed a new octadentate ligand NE3TA-PY (7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) for β-particle-emitting ¹⁷⁷Lu and ⁹⁰Y with targeted radionuclide therapy applications. The pyridine-containing polyaminocarboxylate ligand was proposed to form a neutral complex with Lu(III) and Y(III). The new chelator NE3TA-PY was synthesized and experimentally and theorectically studied for complexation with ¹⁷⁷Lu(III) and ⁹⁰Y(III). DFT-optimized structures of Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY complexes were predicted. NE3TA-PY displayed excellent radiolabeling efficiency with both ¹⁷⁷Lu and ⁹⁰Y. The new chelator (NE3TA-PY) bound to ¹⁷⁷Lu was more stable in human serum and better tolerated when challenged by EDTA than ⁹⁰Y-labeled NE3TA-PY. Our findings suggest that the new chelator (NE3TA-PY) produced excellent Lu-177 radiolabeling and in vitro complex stability profiles.

Full text of DOI:10.1016/j.jinorgbio.2021.111436

Journal of Inorganic Biochemistry 221 (2021) 111436
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Pyridine-containing octadentate ligand NE3TA-PY for formation of neutral
complex with 177Lu(III) and 90Y(III) for radiopharmaceutical applications:
Synthesis, DFT calculation, radiolabeling, and in vitro complex stability
Hyun-Soon Chong*, Yunwei Chen, Chi Soo Kang, Inseok Sin, Shuyuan Zhang, Haixing Wang
Department of Chemistry, Lewis College of Science and Letters, Illinois Institute of Technology, Chicago, IL, United States of America
A R T I C L E I N F O
A B S T R A C T
Keywords:
Targeted radionuclide therapy is a developing therapeutic modality for cancer and employs a cytotoxic radio-
nuclide bound to a chelating agent and a bioactive molecule with high binding affinity for a specific biomarker in
tumors. An optimal chelator is one of the critical components to control therapeutic efficacy and toxicity of
targeted radionuclide therapy. We designed a new octadentate ligand NE3TA-PY (7-[2-[(carboxymethyl)(2-
pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) for β-particle-emitting 177Lu and 90Y
with targeted radionuclide therapy applications. The pyridine-containing polyaminocarboxylate ligand was
proposed to form a neutral complex with Lu(III) and Y(III). The new chelator NE3TA-PY was synthesized and
experimentally and theorectically studied for complexation with 177Lu(III) and 90Y(III). DFT-optimized structures
of Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY complexes were predicted. NE3TA-PY displayed excellent radio-
labeling efficiency with both 177Lu and 90Y. The new chelator (NE3TA-PY) bound to 177Lu was more stable in
human serum and better tolerated when challenged by EDTA than 90Y-labeled NE3TA-PY. Our findings suggest
that the new chelator (NE3TA-PY) produced excellent Lu-177 radiolabeling and in vitro complex stability profiles.
Chelating agent
NE3TA-PY
Lu-177
90Y
Radiolabeling
DFT calculation
1. Introduction
is a highly energetic and pure βꢀ -emitting radionuclide with the
advantage of a longer range of penetration and homogeneous dose
Molecularly targeted radionuclide therapy using an alpha- or beta-
particle emitter has been developed as a therapeutic modality for cancer
[1]. Numerous targeted radiotherapeutics built on various radionuclide-
biomolecule combinations have been evaluated for treatment of cancer
in preclinical and clinical settings [1–9]. Successful radionuclide therapy
requires a well-coordinated therapeutic platform consisting of a potent
therapeutic radionuclide, a tumor-targeting biomolecule, and a bifunc-
tional chelator for labeling of the biomolecule with radionuclide [1,3,4].
90Y (t1/2 = 64 h) and 177Lu (t1/2 = 6.7 d) are potent radionuclides
suitable for targeted cancer therapy. Y-90 (t1/2 = 2.7 d, Emax = 2.3 MeV)
distribution at optimal therapeutic range [1,2,7]. 177Lu is a βꢀ -particle
emitter with relatively long half-life (t1/2 = 6.7 d, Emax = 0.5 MeV) and
less energetic and shorter tissue-penetration range (1.6 mm) relative to
90Y (11.3 mm) [5,8,9]. Therapeutic efficacy of the β-particle emitting
radionuclides 177Lu and 90Y has been highlighted in FDA-approvals of
two clinically available radiopharmaceuticals, Zevalin® (90Y-labeled
anti-CD20 antibody) for non-Hodgkin’s lymphoma (NHL) therapy and
Lutathera® (177Lu-labeled tyrosin3-octreotate peptide) for treatment of
neuroendocrine tumors (NETs) [1,7–9]. Zevalin® is structured on 90Y-
1B4M-DTPA (2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriamine
Abbreviations: 1B4M-DTPA, 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriamine pentaacetic acid; B3LYP, Becke, 3-parameter, Lee–Yang–Parr; DFT, Density
functional theory; DEPT, distortionless enhancement by polarization transfer; DIPEA, Diisopropylethylamine; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′
-
tetraacetic acid; DOTA-TATE, 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid-octreotate; DTPA, diethylenetriaminepentaacetic acid; EDTA, ethyl-
enediaminetetraacetic acid; ECP, Effective core potential; LANL2DZ, Los Alamos National Laboratory 2 double Z; MMFF, molecular mechanics force field; NETA, 7-
[2-[bis(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid; NE3TA, 7-[2-(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic
acid; NE3TA-PY, 7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid; NODA, 1,4,7-triazacyclononane-1,4-diacetic acid;
PY, Pyridine; TATE, Octreotate; TFA, Trifluoroacetic acid; Thin layer chromatography, TLC; TMS, tetramethylsilane.
* Corresponding author at: Department of Chemistry, Lewis College of Science and Letters, Illinois Institute of Technology, 3101 S. Dearborn St, LS 182, Chicago, IL
60616, United States of America.
E-mail address: chong@iit.edu (H.-S. Chong).
https://doi.org/10.1016/j.jinorgbio.2021.111436
Received 29 December 2020; Received in revised form 20 March 2021; Accepted 20 March 2021
Available online 24 April 2021
0162-0134/© 2021 Elsevier Inc. All rights reserved.

H.-S. Chong et al.
Journal of Inorganic Biochemistry 221 (2021) 111436
Fig. 1. Structure of chelating agents DOTA, DTPA, NETA, NE3TA, and NE3TA-PY.
pentaacetic acid), while Lutathera® contains 177Lu-DOTA-TATE (1,4,7,
10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid-octreotate). An
effective bifunctional chelator is expected to complex a cytotoxic or
imaging radionuclide with a limited half-life rapidly and tightly and
possess a high kinetic inertness to trans-chelation by metal cations and
natural chelators present in vivo. Thus, it is critical to use an optimal
chelator that can tightly and rapidly sequester the radionuclide and
minimize radiotoxicity and enhance therapeutic potency. Various che-
lators including DOTA and DTPA (diethylenetriamine pentaacetic acid)
analogues (Fig. 1) have been employed for radiolabeling of tumor-
targeting peptides and antibodies with 90Y or 177Lu [1–3,7–9]. In gen-
eral, the macrocyclic DOTA can form a stable complex with a metal,
while the acyclic DTPA rapidly bound to a metal.
2.2. Reagents
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) and
used as received unless otherwise noted. 90Y (0.05 M HCl) and 177Lu
(0.05 M HCl) were purchased from Perkin-Elmer. 90Y (t1/2 = 64.1 h) is a
β-emitting radionuclide. 177Lu (t1/2 = 6.7 days) is a β/γ-emitting radio-
nuclide. Appropriate shielding and handling protocols should be in place
when using the radioisotopes.
2.3. 4-[2-(Benzyl-tert-butoxycarbonylmethyl-amino)-ethyl]-7-tert-butox-
ycarbonyl-methyl-[1,4,7]triazonan-1-yl)-acetic acid tert-butyl ester (3)
Compound 3 was prepared via a modification of the method as
previously reported [18]. To a solution of 1 [19] (881.4 mg, 2.46 mmol)
in CH3CN (25 mL) was added diisopropylethylamine (DIPEA, 954.1 mg,
7.40 mmol) and 2 [18] (807.4 mg, 2.46 mmol). The reaction mixture
was stirred under reflux for 14.5 h. The resulting solution was evapo-
rated in vacuo, and the residue is purified via column chromatography
(silica gel, 60 mesh) eluting with 3% methanol in dichloromethane to
afford pure 3 as a light yellow oil (1.36 g, 91%). 1H and 13C NMR spectra
of compound 3 is identical to those reported in the literature [18]. 1H
NMR (CDCl3, 300 MHz) δ 1.33 (m, 27H), 2.53–2.79 (m, 4H), 2.81–3.02
(m, 2H), 3.04–3.09 (m, 4H), 3.16 (s, 2H), 3.21–3.30 (m, 4H), 3.38–3.49
(m, 2H), 3.52–3.65 (m, 4H), 3.73 (s, 2H), 7.26–7.29 (m, 5H); 13C NMR
(CDCl3, 300 MHz) δ 28.1 (q), 49.1 (t), 49.3 (t), 52.2 (t), 52.6 (t), 53.7 (t),
55.3 (t), 57.7 (t), 58.3 (t), 81.5 (s), 81.6 (s), 127.6 (d), 128.5 (d), 129.1
(d), 137.5 (s), 170.3 (s), 170.4 (s).
We have synthesized and evaluated structurally novel chelating
agents in the series of NETA ((7-[2-[bis(carboxymethyl)amino]ethyl]-
1,4,7-triazacyclononane-1,4-diacetic acid) and NE3TA (7-[2-(carbox-
ymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) con-
taining both macrocyclic and acyclic binding moieties for rapid
formation of a stable complex with therapeutic or diagnostic radionu-
clides including 90Y, 177Lu, 64Cu, and 205/6Bi [10–15]. We were inter-
ested in development of chelation chemistry for formation of a neutral
complex with Lu(III) and Y(III). Neutral metal complexes were reported
to be more inert to acid/cation-promoted dissociation when compared
with ionic complexes [16,17].
We herein report synthesis and evaluation of a NE3TA analogue
containing a pyridine (PY) ring (Fig. 1) for 177Lu(III) and 90Y(III). The
new chelator NE3TA-PY has different acyclic donor groups structured on
1,4,7-triazacyclononane (TACN) and can form 8-coordinate complexes
with metal cations using four tertiary nitrogen atoms, two acetate
groups, and a nitrogen donor in the pyridine ring. The new chelator is
hypothesized to rapidly form a stable neutral complex with the β-par-
ticle emitting radionuclides 177Lu(III) or 90Y(III) with a relatively large
ionic radius. The new chelator NE3TA-PY was synthesized and evalu-
ated for radiolabeling kinetics and in vitro complex stability with 90Y and
177Lu. The structures of the neutral metal complexes Lu(III)-NE3TA-PY
and Y(III)-NE3TA-PY were predicted using DFT methods.
2.4. di-tert-Butyl 2,2′-(7-(2-((3,3-dimethyl-2-oxobutyl)amino)ethyl)-
1,4,7-triazonane-1,4-diyl)diacetate (4)
To a solution of 3 (1 g, 1.65 mmol) in EtOH (50 ml) was added 10%
wet Pd/C (300 mg). The resulting mixture was subjected to hydro-
genolysis at room temperature for 40 h by agitation with excess H2 (g) at
60 psi in a Parr hydrogenator apparatus. The reaction mixture was
filtered through Celite®, and the solvent was evaporated in vacuo to
provide compound 4 (830 mg, 97.6%) as a colorless oil. 1H NMR (CDCl3,
300 MHz) δ 1.26 (m, 27H), 2.74–3.59 (m, 23H). 13C NMR (CDCl3,
75 MHz) δ 27.9 (q), 44.3 (t), 49.1 (t), 49.9 (t), 50.9 (t), 53.2 (t), 56.9 (t),
57.5 (t), 82.2 (s), 83.2 (s), 167.2 (s), 169.8 (s). HRMS (positive ion ESI)
Calcd for C26H50N4O6 [M + H]+ m/z 515.3803. Found: [M + H]+ m/z
515.3798.
2. Experimental
2.1. Instruments and methods
1H, 13C, and DEPT (distortionless enhancement by polarization
transfer) NMR spectra of the new compounds were obtained using a
Bruker 300 NMR instrument, and chemical shifts are reported in ppm on
the d scale relative to TMS (tetramethylsilane). Electrospray ionization
(ESI) high resolution mass spectra (HRMS) of the new compounds were
obtained on JEOL double sector JMS-AX505HA mass spectrometer
(University of Notre Dame, IN). Analytical HPLC was performed on an
Agilent 1200 equipped with diode array detector (λ = 254 and 280 nm),
with the thermostat set at 35 ◦C and with a Zorbax Eclipse XDB-C18
column (4.6 × 150 mm, 80 Å). A combination of a binary gradient
(0%–100% B/15 min; solvent A = 0.1% trifluoroacetic acid (TFA) in
H2O; solvent B = 0.1% TFA in CH3CN) at a flow rate of 1 mL/min was
used for analytical HPLC.
2.5. di-tert-Butyl 2,2′-(7-(2-((3,3-dimethyl-2-oxobutyl)(pyridin-2-
ylmethyl)amino)ethyl)-1,4,7-triazonane-1,4-diyl)diacetate (5)
To a solution of 4 (100 mg, 0.19 mmol) and potassium carbonate
(31.5 mg, 0.22 mmol) and sodium iodide (34.2 mg, 0.22 mmol) in
anhydrous CH3CN (2 ml) at 0 ◦C was dropwise added 2-(chloromethyl)
pyridine (24.2 mg, 0.19 mmol). The reaction mixture was filtered and
concentrated to dryness to provide compound 5 (103.8 mg, 90.2%) as a
colorless oil. 1H NMR (CDCl3, 300 MHz) δ 1.41 (m, 27H), 2.70–2.80 (m,
4H), 3.00–3.75 (m, 18H), 3.93 (s, 2H), 7.23 (t, J = 6.0 Hz, 1H), 7.31 (d,
J = 6.0 Hz, 1H), 7.65 (t, J = 9.0 Hz, 1H), 8.51 (d, J = 6.0 Hz, 1H). 13C
NMR (CDCl3, 75 MHz) δ 28.2 (q), 49.7 (t, 2C), 52.8 (t), 53.0 (t), 54.0 (t),
2

H.-S. Chong et al.
Journal of Inorganic Biochemistry 221 (2021) 111436
56.0 (t), 57.9 (t), 59.4 (t), 81.6 (s), 122.6 (d), 123.7 (d), 136.9 (d), 149.3
(d), 157.7 (s), 170.4 (s), 170.7 (s). HRMS (positive ion ESI) Calcd for
C32H55N5O6 [M + H]+ m/z 606.4225. Found: [M + H]+ m/z 606.4217.
2.8. Radiolabeling of NE3TA-PY with 177Lu or 90
Y
All HCl solutions were prepared from the commercially available
ultra-pure HCl solution (JT baker, #6900–05). For metal-free radio-
labeling, plasticware including pipette tips, tubes, and caps was soaked
in 0.1 M HCl overnight and washed thoroughly with Milli-Q (18.2MΩ)
water and air-dried overnight. Ultra-pure ammonium acetate (Aldrich,
#372331) was purchased from Aldrich and used to prepare 0.25 M
NH4OAc buffer solutions (pH 5.5 or pH 7.0). After adjusting pH using
0.1 M/1 M HCl or NaOH solution, 0.25 M NH4OAc buffer solutions were
treated with Chelex-100 resin (Biorad, #142–2842, 1 g/100 ml buffer
solution), shaken overnight at room temperature, and filtered through
2.6. 7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-
triazacyclononane-1,4-diacetic acid (6)
To Compound 5 (16.6 mg, 0.027 mmol) was added 6 M HCl (3 ml).
After addition, the reaction mixture was refluxed for 5 min. The result-
ing solution was cooled to the room temperature and washed with
CH2Cl2. The aqueous layer was evaporated in vacuo to afford compound
6 (15.1 mg, 90.2%) as a light yellowish solid. Analytical HPLC
(tR = 4.2 min). 1H NMR (D2O, 300 MHz) δ 3.13–3.30 (m,6H), 3.36–3.52
(m, 8H), 3.55–3.64 (m,4H), 3.82(s,4H), 4.25 (s, 2H), 7.85 (m, 2H), 8.40
(t, J = 9.0 Hz, 1H), 8.60 (d, J = 6.0 Hz, 1H). 13C NMR (D2O, 75 MHz) δ
49.9 (t, 2C), 50.8 (t), 51.2 (t), 54.6 (t), 55.4 (t), 55.5 (t), 57.0 (t), 126.3
(d), 126.8 (d), 141.3 (d), 147.1 (d), 152.4 (s), 172.7 (s), 174.6 (s). HRMS
(Negative ion ESI) Calcd for C20H31N5O6 [M - H]ꢀ m/z 436.2202. Found:
[M - H]ꢀ m/z 436.2232.
0.22
μ
M filter (Corning, #430320) prior to use. 90YCl3 and 177LuCl3
were purchased from Perkin Elmer. Thin layer chromatography (TLC)
plates (6.6 × 2 cm, Silica gel 60 F254, EMD Chemicals Inc., #5554–7)
with the origin line drawn at 0.6 cm from the bottom were prepared. To
a buffer solution (0.25 M NH4OAc, pH 5.5 or pH 7.0) in a capped
microcentrifuge tube (1.5 mL) was sequentially added a solution of
NE3TA-PY (10
μ
g/1.6
μ
l H2O). A solution of 90YCl3 or 177LuCl3 (0.05 M
HCl, 1.11 MBq, 30
μ
Ci) was added to the aqueous solution of the
2.7. Computational studies
chelator, and the total volume of the resulting solution was brought up
to 20 μl by adding the buffer solution [12]. The reaction mixture was
All calculations were performed using Spartan’18 Parallel Suite [20].
A structure of Y(III)-NE3TA-PY or Lu(III)-NE3TA-PY complex was
initially built and energy-minimized using the molecular mechanics
force field (MMFF) model. MMFF conformational search for the energy-
minimized Y(III)-NE3TA-PY or Lu(III)-NE3TA-PY complex were per-
formed. Geometry optimizations of the major conformations for Y(III)-
NE3TA-PY and Lu(III)-NE3TA-PY were performed in gas phase using
density functional B3LYP (Becke, 3-parameter, Lee–Yang–Parr) model
[21–23]. Atoms in the chelator (H, C, N, O) were described by using
standard 6-31G* basis set [24,25], while Y(III) and Lu(III) were modeled
using the LANL2DZ (Los Alamos National Laboratory 2 double Z)-
effective core potential (ECP) basis set [26]. The use of the LANL2DZ-
ECP is based on the Dolg, Stoll, and Preuss ECP, where 4f [14] elec-
trons in the lanthanide are placed in the core [27]. The structure of the
DFT-optimized Y(III)-NE3TA-PY or Lu(III)-NE3TA-PY conformer with
energy minimum was calculated and visualized using the UCSF (Uni-
versity of California, San Francisco) molecular analysis program
(Chimera 1.14) [28] or CYLview (Supporting information) [29]. Mo-
lecular surface of Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY complexes
was calculated using molecular surface method (MSMS) included in the
UCSF molecular analysis program (Chimera 1.14).
agitated on the thermomixer (Eppendorf, #022670549) set at 1000 rpm
at room temperature for 1 h. The labeling efficiency was determined by
TLC eluted with a binary mobile phase (20 mM EDTA/0.15 M NH4OAc).
A solution of radiolabeled complexes (2 μl) was withdrawn at the
designated time points, spotted on a TLC plate, and then eluted with the
mobile phase. After completion of elution, the TLC plate was warmed
and dried on the surface of a heater maintained at 35 ◦C and scanned
using TLC scanner (Bioscan, #FC-1000). 90Y-NE3TA-PY or 177Lu-
NE3TA-PY was detected at ~40 mm from the bottom of the TLC plate,
while radionuclide 90Y or 177Lu bound to EDTA moved faster (~60 mm).
2.9. In vitro stability of 177Lu-NE3TA-PY and 90Y-NE3TA-PY
Human serum was purchased from Gemini Bioproducts (#100110).
90Y-NE3TA-PY and 177Lu-NE3TA-PY was prepared by reaction of
NE3TA-PY (50
μ
g/8.0
μ
l H2O) with 90Y (5.55 MBq, 150
μCi, 18.3 μl) or
177Lu (5.55 MBq, 150
μCi, 3.46 μl) in 0.25 M NH4OAc buffer (pH 5.5,
88.54
μ
l for 177Lu or pH 7.0, 73.7
μ
l for 90Y). Completion of radio-
labeling was determined by TLC, and the resulting complexes 90Y-
NE3TA-PY or 177Lu-NE3TA-PY were directly used for serum stability
studies without further purification. 177Lu-NE3TA-PY (5.48 MBq,
148
μ
Ci, 99
μ
l) was added to human serum (500
μ
l) in a microcentrifuge
tube. 90Y-NE3TA-PY (5.03 MBq, 136
μCi, 99 μl) was added to human
Scheme 1. Synthesis of Chelator NE3TA-PY (6).
3

H.-S. Chong et al.
Journal of Inorganic Biochemistry 221 (2021) 111436
Table 1
Distance (Å) of selected bonds in DFT-calculated structure of Lu(III)-NE3TA-PY
and Y(III)-NE3TA-PY and their relative binding energy (kcal/mol).
Lu(III)-NE3TA-PY
Y(III)-NE3TA-PY
Lu-N1
2.610
2.586
2.525
2.563
2.475
2.552
2.193
2.145
2.172
2.170
0.000
Y-N1
2.596
2.644
2.614
2.619
2.545
2.604
2.265
2.214
2.250
2.243
8.797
Lu-N2
Y-N2
Lu-N3
Y-N3
Lu-N4
Y-N4
Lu-N5
Y-N5
Lu-N (Mean)
Y-N (Mean)
Lu-O6
Y-O6
Lu-O7
Y-O7
Lu-O8
Y-O8
Lu-O (Mean)
Lu-O (Mean)
Relative Binding Energy (kcal/
mol)
Relative Binding Energy (kcal/
mol)
Fig. 2. DFT-optimized structures for Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY
with energy minimum in gas phase.
All hydrogens are omitted for clarity, and carbon (grey), oxygen (red), nitrogen
(blue), yttrium (grey), and lutetium (green) are highlighted in different color.
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
of compound 4 with 2-pyridylmethyl chloride produced compound 5
that was further treated with 6 M HCl(aq) solution for acid-promoted
deprotection of tert-Butyl groups to afford the desired chelator NE3TA-
PY (6).
serum (500 μl) in a microcentrifuge tube. The stability of the radio-
labeled complexes in human serum was evaluated at 37 ◦C for 7 days.
The serum stability of the radiolabeled complexes was assessed by
measuring the transfer of the radionuclide from each complex to serum
proteins using TLC (eluent: 20 mM EDTA/0.15 M NH4OAc). A solution
3.2. DFT calculations
We conducted theoretical complexation studies of the new chelator
NE3TA-PY with Lu(III) and Y(III) using density functional model. Ge-
ometries and relative energies of Lu(III)-NE3TA-PY and Y(III)-NE3TA-
PY complexes in gas phase were calculated using the hybrid density
functional B3LYP and Pople 6-31G* basis set. The most stable con-
formers with energy minimum for Y(III)-NE3TA-PY and Lu(III)-NE3TA-
PY (Fig. 2 and Supporting information) were obtained from the DFT
calculations.
of the radiolabeled complex in serum (5–16 μl for TLC) was withdrawn
at the designated time point and evaluated by TLC. At each of the time
points, the percentage of 177Lu or 90Y released from each of the radio-
labeled complexes into serum was assessed by TLC. The radiolabeled
complex 90Y-NE3TA-PY or 177Lu-NE3TA-PY was detected at ~40 mm
from the bottom of the TLC plate, while unbound radionuclide 90Y or
177Lu moved faster (~60 mm).
The chelator NE3TA-PY contains four tertiary nitrogens that are
connected via ethylene chains and can form diastereomeric complexes
with a metal cation. The four nitrogens (N1, N2, N3, and N4, Fig. 2) in
the chelator bound to Lu(III) or Y(III) to form four 5-membered chelate
rings in two possible conformations (λ or δ). Each of the two pendant
acetate attached to the TACN ring (N1 and N2) coordinated with the
metal cation can adopt two different orientations (Δ or Λ helicity).
Complexation of the tertiary nitrogen (N4) in NE3TA-PY with the metal
cation leads to formation of non-interconvertible enantiomeric isomers
(R or S configuration).
2.10. EDTA (ethylenediaminetetraacetic acid) challenge of 177Lu-
NE3TA-PY and 90Y-NE3TA-PY
177Lu-NE3TA-PY or 90Y-NE3TA-PY was prepared by reaction of
NE3TA-PY (10
μ
g/1.6
μ
l H2O) with 177Lu (1.11 MBq, 30
μ
Ci, 0.68
μ
l) in
0.25 M NH4OAc buffer (17.72
μ
l, pH 5.5) or 90Y (1.11 MBq, 30
μ
Ci,
3.66
μ
l) in 0.25 M NH4OAc buffer (14.74
μ
l, pH 7.0). Completion of
radiolabeling was determined by TLC, and the resulting complexes 90Y-
NE3TA-PY or 177Lu-NE3TA-PY were directly used for EDTA challenge
The DFT-optimized structures for both Lu(III)-NE3TA-PY and Y(III)-
NE3TA-PY (Fig. 2) were calculated to adopt the nitrogen atoms
(N1ꢀ N3) of the TACN ring in a syn conformation and the pendant donor
groups in Δ helicity and the four five-membered rings in the same
gauche orientations (λλλλ). For the DFT-optimized Lu(III)-NE3TA-PY
and Y(III)-NE3TA-PY complexes, the chiral nitrogen (N4) donor in the
pendant arm is predicted to have R configuration. The DFT calculations
on the relative free energies of the enantiomeric Lu(III)-NE3TA-PY and Y
(III)-NE3TA-PY complexes predicts that Δ(λλλλ)-N4(R) is slightly more
stable than its enantiomeric counterpart Λ(δδδδ)-N4(S) (Supporting in-
formation). The oxygen atoms in the DFT-optimized Lu(III)-NE3TA-PY
studies without further purification. A solution of EDTA (18 μl, 80 mM/
H2O, pH 7.0) at a 100-fold molar excess was added to a solution of 177Lu-
NE3TA-PY (0.99 MBq, 27 μCi/18 μl 0.25 M NH4OAc buffer). A solution
of EDTA (19 μl, 80 mM/H2O, pH 7.0) at a 100-fold molar excess was
added to a solution of 90Y-NE3TA-PY (0.78 MBq, 21
μCi/19 μl 0.25 M
NH4OAc buffer). The resulting mixture was incubated for 24 h at 37 ◦C.
The stability of 90Y-NE3TA-PY or 177Lu-NE3TA-PY complex in the
presence of EDTA at a 100-fold molar excess was determined using TLC
(eluent: 20 mM EDTA/0.15 M NH4OAc) [14]. The radiolabeled complex
90Y-NE3TA-PY or 177Lu-NE3TA-PY was detected at ~40 mm from the
bottom of the TLC plate, while unbound radionuclide 90Y or 177Lu
moved faster (~60 mm).
Table 2
Radiolabeling efficiency of NE3TA-PY with 177Lu or 90Y (pH 5.5 and 7.0, RT)a.
3. Results and discussion
Time
Radiolabeling efficiency (%)
177Lu-NE3TA-PY
90Y-NE3TA-PY
pH 5.5
3.1. Synthesis
pH 5.5
pH 7.0
pH 7.0
Synthesis of the new chelator NE3TA-PY (6) is centered on reaction
of N-tert-Butyl protected NE3TA (4) with 2-pyridylmethyl chloride
(Scheme 1). Compound 3 was prepared from reaction of tert-Butyl pro-
tected NODA (1,4,7-triazacyclononane-1,4-diacetic acid) 1 using a
modified synthetic method as we previously reported [18]. Removal of
the N-Benzyl group in compound 3 by hydrogenolysis was achieved to
provide compound 4 in a nearly quantitative yield. Substitution reaction
1 min
38.4 ± 2.4
94.8 ± 0.4
99.5 ± 0.4
99.6 ± 0.3
93.0 ± 1.3
97.7 ± 0.6
99.0 ± 0.4
99.4 ± 0.0
11.3 ± 0.5
53.4 ± 0.2
82.1 ± 0.1
92.6 ± 0.8
58.8 ± 1.8
87.1 ± 0.6
93.2 ± 0.1
96.1 ± 0.1
10 min
30 min
60 min
a
Radiolabeling efficiency (mean ± standard deviation%) was measured in
duplicate using TLC eluted with a binary mobile phase (20 mM EDTA/0.15 M
NH4OAc).
4

H.-S. Chong et al.
Journal of Inorganic Biochemistry 221 (2021) 111436
177Lu-NE3TA-PY or 90Y-NE3TA-PY was incubated with EDTA at a 100-
fold molar excess and monitored for release of radioactivity using TLC
analysis (Fig. 4 and Supporting Information). When challenged by
EDTA, 177Lu-NE3TA-PY remained stable and lost a minimum amount of
177Lu in 48 h (~2.5%). However, 90Y-NE3TA-PY was shown to undergo
transchelation with EDTA and released a significant amount of 90Y
bound to EDTA (~30% at 24 h post-incubation time).
100
80
60
Y-90-NE3TA-PY
40
Lu-177-NE3TA-PY
20
The radiolabeling and in vitro complex stability data indicate that
NE3TA-PY with potential 8 donor groups displayed excellent radio-
labeling kinetics and stability with 177Lu. The chelator was slower in
binding to 90Y under mild radiolabeling conditions to form a less stable
complex with 90Y. While both 177Lu and 90Y have the prevalent oxida-
tion state (+3), 177Lu has a smaller ionic radius than 90Y (0.977 Å vs
1.019 Å for coordination number 8) [30]. As predicted by DFT calcu-
lations on Lu(III)-NE3TA-PY and Y(III)-NE3TA-PY complexes, the small
macrocyclic cavity (TACN) is more tightly coordinated with Lu(III) than
Y(III) (Table 1). Lu(III) is more closely bound to the aromatic amine in
0
0
24
48
72
96
120
144
168
Time (hr)
Fig. 3. In vitro complex stability of 177Lu-NE3TA-PY and 90Y-NE3TA-PY in
human serum at pH 7 and 37 ◦C.
and Y(III)-NE3TA-PY complexes are more tightly coordinated to Lu(III)
or Y(III) than the nitrogen atoms (mean distance of M-O and M-N,
~2.2 Å vs 2.6 Å, Table 1). The pyridyl ring in the chelator NE3TA-PY is
predicted to adopt a favorable conformation for coordination to Lu(III)
or Y(III), and the nitrogen atom in the pyridyl ring (N5) was estimated to
make a tighter bond (distance of M-N5 bond, 2.475 Å for Lu3+ and
2.545 Å for Y3+) with Lu(III) or Y(III) than the nitrogen atoms in the
tertiary amines (N1-N4, mean distance of M-N bond, 2.571 Å for Lu3+
and 2.618 Å for Y3+). Calculations on relative binding energies of the
complexes predicts that Lu(III)-NE3TA-PY is more stable than Y(III)-
NE3TA-PY by 8.797 kcal/mol (Table 1 and Supporting Information).
–
the pyridyl ring (Lu N5 distance: 2.475 Å vs 2.545 Å) than Y(III). The
pyridyl ring in Y(III)-NE3TA-PY complex is postulated to cause a
considerable ligand strain on Y(III)-coordination sphere, leading to the
formation of less stable Y(III)-NE3TA-PY complex.
4. Conclusion
The novel chelator NE3TA-PY containing three aminocarboxylates
and a pyridyl ring as donor groups was synthesized and evaluated for
complexation with 177Lu and 90Y. The radiolabeling and in vitro complex
stability data confirm that the new chelator produced excellent radio-
labeling kinetics and complex stability profiles with 177Lu. The chelator
NE3TA-PY was relatively slower in binding 90Y, and the corresponding
90Y-NE3TA-PY complex was relatively less stable than 177Lu-NE3TA-PY
in human serum and EDTA solution. The results of the DFT calculations
also suggest that the chelator NE3TA-PY forms more stable complex
with Lu(III) than Y(III). The experimental and computational data sug-
gest that NE3TA-PY is a promising octadentate ligand that can be further
structurally modified and studied for conjugation to a bioactive mole-
cule for targeted radionuclide therapy and imaging.
3.3. Radiolabeling kinetics and in vitro serum stability
The chelator NE3TA-PY was evaluated for radiolabeling efficiency
with 90Y and 177Lu (Table 2). The new chelator in 0.25 M NH4OAc buffer
solution (pH 5.5 or pH 7.0) was radiolabeled with 90Y or 177Lu at room
temperature (RT). During the reaction time (1 h), the radiolabeling ki-
netics was determined by TLC analysis (Table 2 and Supporting Infor-
mation). The data shown in Table 2 indicate that radiolabeling of
NE3TA-PY with 177Lu or 90Y was significantly more efficient at pH 7.0
compared to pH 5.5. The rapid sequestration of the chelator with the
radionuclides under neutral conditions may be explained by enhanced
ligating capability of the basic nitrogen atom in the pyridyl ring. NE3TA-
PY instantly and nearly completely bound to 177Lu at the beginning of
the reaction under neutral conditions (~93% labeling efficiency at
1 min, pH 7.0). NE3TA-PY was relatively slower in binding 90Y (~59%
at 1 min, pH 7.0) but almost completely sequestered 90Y at 1 h time point
(96%, pH 7.0, RT). 177Lu-NE3TA-PY and 90Y-NE3TA-PY were evaluated
for complex stability in human serum. The radiolabeled complexes were
freshly prepared and incubated in human serum (37 ◦C, pH 7) and
determined for release of radioactivity to serum using TLC analysis
(Fig. 3 and Supporting Information). The data in Fig. 3 show that 177Lu-
NE3TA-PY was stable in human serum for at least 7 days without a
measurable loss of 177Lu, while 90Y-NE3TA-PY released a considerable
amount of 90Y (>12%) to serum over 7 days. (See Fig. 3.)
CRediT authorship contribution statement
Hyun-Soon Chong: Conceptualization, Investigation, Visualization,
Writing - original draft. Yunwei Chen: Investigation. Chi Soo Kang:
Investigation. Inseok Sin: Investigation. Shuyuan Zhang: Investiga-
tion. Haixing Wang: Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
The radiolabeled complexes 177Lu-NE3TA-PY and 90Y-NE3TA-PY
were further assessed for complex stability in the presence of EDTA.
Fig. 4. Complex stability of 177Lu-NE3TA-PY and 90Y-NE3TA-PY in EDTA solution (100-fold molar excess) at pH 7 and 37 ◦C.
5

H.-S. Chong et al.
Journal of Inorganic Biochemistry 221 (2021) 111436
[11] H.A. Song, C.S. Kang, K.E. Baidoo, D.E. Milenic, Y. Chen, A. Dai, M.W. Brechbiel,
H.-S. Chong, Efficient Bifunctional Decadentate ligand 3p-C-DEPA for targeted
alpha Radioimmunotherapy applications, Bioconjug. Chem. 22 (2011) 1128–1135.
[12] H.-S. Chong, H.A. Song, C.S. Kang, T. Le, X. Sun, M. Dadwal, H.B. Lee, X. Lan,
Y. Chen, A. Dai, A highly effective bifunctional ligand for radioimmunotherapy of
cancer, Chem. Commun. 47 (2011) 5584–5586.
Acknowledgement
We acknowledge the financial support from the National Institutes of
Health (R01CA112503 to Hyun-Soon Chong). Molecular graphics were
generated using UCSF Chimera as developed by the Resource for Bio-
computing, Visualization, and Informatics at the University of Califor-
nia, San Francisco (NIH P41-GM103311).
[13] H.-S. Chong, X. Sun, Y. Chen, I. Sin, C.S. Kang, M.R. Lewis, D. Liu, V.C. Ruthengael,
Y. Zhong, N. Wu, H.A. Song, Synthesis and comparative biological evaluation of
bifunctional ligands for radiotherapy applications of 90Y and 177Lu, Bioorg. Med.
Chem. 23 (2015) 1169–1178.
[14] N. Wu, C.S. Kang, I. Sin, S. Ren, D. Liu, V.C. Ruthengael, M.R. Lewis, H.-S. Chong,
Promising bifunctional chelators for copper 64-PET imaging: practical 64Cu
radiolabeling and high in vitro and in vivo complex stability, J. Biol. Inorg. Chem.
21 (2016) 177–184.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jinorgbio.2021.111436.
[15] H.-S. Chong, S. Mhaske, M. Lin, S. Bhuniya, H.A. Song, M.W. Brechbiel, X. Sun,
Novel synthetic ligands for targeted PET imaging and radiotherapy of copper,
Bioorg. Med. Chem. Lett. 17 (2007) 6107–6110.
[16] E.T. Clarke, A.E. Martell, Stabilities of the Fe(III), Ga(III) and In(III) chelates of N,
N′,N′′-triazacyclononanetriacetic acid, Inorg. Chim. Acta 181 (1991) 273–280.
[17] L.L. Chappell, E. Dadachova, D.E. Milenic, K. Garmestani, C. Wu, M.W. Brechbiel,
Synthesis, characterization, and evaluation of a novel bifunctional chelating agent
for the lead isotopes 203Pb and 212Pb, Nucl. Med. Biol. 27 (2000) 93–100.
[18] H.-S. Chong, X. Ma, H. Lee, P. Bui, H.A. Song, N. Birch, Synthesis and evaluation of
novel polyaminocarboxylate-based antitumor agents, J. Med. Chem. 51 (2008)
2208–2215.
References
[1] D.E. Milenic, E.D. Brady, M.W. Brechbiel, Antibody-targeted radiation cancer
therapy, Nat. Rev. Drug Discov. 3 (2004) 488–498.
[2] S.J. Knox, R.F. Meredith, Clinical radioimmunotherapy, Semin. Radiat. Oncol. 10
(2000) 73–93.
[3] T.I. Kostelnik, C. Orvig, Radioactive main group and rare earth metals for imaging
and therapy, Chem. Rev. 119 (2019) 902–956.
[19] H.-S. Chong, H.A. Song, N. Birch, T. Le, S.Y. Lim, X. Ma, Efficient synthesis and
evaluation of bimodal ligand NETA, Bioorg. Med. Chem. Lett. 18 (2008)
3436–3439.
[4] C.S. Cutler, H.M. Hennkens, N. Sisay, S. Huclier-Markai, S.S. Jurisson, Radiometals
for combined imaging and therapy, Chem. Rev. 113 (2013) 858–883.
[5] S. Banerjee, M.R.A. Pillai, F.F. Knapp, Lutetium-177 therapeutic
radiopharmaceuticals: linking chemistry, radiochemistry, and practical
applications, Chem. Rev. 115 (8) (2015) 2934–2974.
[20] Spartan’18, Wavefunction, Inc. Irvine, CA.
[21] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange,
J. Chem. Phys. 98 (1993) 5648–5652.
[6] S. Hassfiell, M.W. Brechbiel, The development of the α-particle emitting
[22] C. Lee, W. Yang, R.G. Parr, Development of the Colle-Salvetti correlation-energy
formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785–789.
[23] P.J. Stephens, F.J. Devlin, C.F. Chablowski, M. Frisch, J. Phys. Chem. 98 (1994)
11623–11627.
radionuclides 212Bi and 213Bi, and their decay chain related radionuclides, for
therapeutic applications, Chem. Rev. 101 (7) (2001) 2019–2036.
[7] G.A. Wiseman, C.A. White, R.B. Sparks, W.D. Erwin, D.A. Podoloff, D. Lamonica, N.
L. Bartlett, J.A. Parker, W.L. Dunn, S.M. Spies, R. Belanger, T.E. Witzig, B.R. Leigh,
Biodistribution and dosimetry results from a phase III prospectively randomized
controlled trial of Zevalin radioimmunotherapy for low-grade, follicular, or
transformed B-cell non-Hodgkin’s lymphoma, Crit. Rev. Oncol. Hematol. 39 (2001)
181–194.
[24] W.J. Hehre, R. Ditchfield, J.A. Pople, Self-consistent molecular orbital methods.
XII. Further extensions of Gaussian-type basis sets for use in molecule orbital
studies of organic molecules, J. Chem. Phys. 56 (1972) 2257–2261.
[25] M.S. Gordon, J.S. Binkley, J.A. People, W.J. Pietro, W.J. Hehre, Self-consistent
molecular-orbital methods. 22. Small split-valence basis sets for second-row
elements, J. Am. Chem. Soc. 104 (1982) 2797–2803.
[8] R.D. Alvarez, E.E. Partrudge, M.B. Khazaeli, G. Plott, M. Austin, L. Kilgore, C.
D. Russell, T. Liu, W.E. Grizzle, J. Schlom, A.F. LoBuglio, R.F. Meredith,
Intraperitoneal radioimmunotherapy of ovarian cancer with 177Lu-CC49: a phase
I/II study, Gynecol. Oncol. 65 (1997) 94–101.
[26] P.J. Hay, W.R. Wadt, Ab initio effective core potentials for molecular calculations.
Potentials for K to Au including the outermost core orbitals, J. Chem. Phys. 82
(1985) 299–310.
[9] J. Strosberg, G. EI-Haddad, E. Wolin, A. Hendifar, J. Yao, B. Chasen, E. Mittra, P.
L. Kunz, M.H. Kulke, H. Jacene, D. Bushnell, T.M. O’Dorisio, et al., Phase 3 trial of
177Lu-Dotatate for Midgut neuroendocrine tumors, N. Engl. J. Med. 376 (2017)
125–135.
[27] M. Dolg, H. Stoll, H. Preuss, Energy-adjusted ab initio pseudopotentials for the rare
ether elements, J. Chem. Phys. 90 (1989) 1730–1734.
[28] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF chimera–a visualization system for exploratory research and
analysis, J. Comput. Chem. 13 (2004) 1605–1612.
[10] C.S. Kang, X. Sun, F. Jia, H.A. Song, Y. Chen, M. Lewis, H.-S. Chong, Synthesis and
preclinical evaluation of bifunctional ligands for improved chelation chemistry of
90Y and 177Lu for targeted radioimmunotherapy, Bioconjug. Chem. 23 (2012)
1775–1782.
´
[29] C.Y. Legault, CYLview, 1.0b, Universite de Sherbrooke, 2009. http://www.cylview.
org.
[30] R.D. Shannon, Revised effective ionic radii and systematic studies of interatomic
distances in halides and chalcogenides, Acta Crystallogr. A32 (1985) 751–767.
6