Synthesis and comparative biological evaluation of bifunctional ligands for radiotherapy applications of 90Y and 177Lu

Article abstract of DOI:10.1016/j.bmc.2014.12.035

Zevalin is an antibody-drug conjugate radiolabeled with a cytotoxic radioisotope (⁹⁰Y) that was approved for radioimmunotherapy (RIT) of B-cell non-Hodgkin's lymphoma. A bifunctional ligand that displays favorable complexation kinetics and in vivo stability is required for effective RIT. New bifunctional ligands 3p-C-DE4TA and 3p-C-NE3TA for potential use in RIT were efficiently prepared by the synthetic route based on regiospecific ring opening of aziridinium ions with prealkylated triaza- or tetraaza-backboned macrocycles. The new bifunctional ligands 3p-C-DE4TA and 3p-C-NE3TA along with the known bimodal ligands 3p-C-NETA and 3p-C-DEPA were comparatively evaluated for potential use in targeted radiotherapy using β-emitting radionuclides ⁹⁰Y and ¹⁷⁷Lu. The bifunctional ligands were evaluated for radiolabeling kinetics with ⁹⁰Y and ¹⁷⁷Lu, and the corresponding ⁹⁰Y or ¹⁷⁷Lu-radiolabeled complexes were studied for in vitro stability in human serum and in vivo biodistribution in mice. The results of the comparative complexation kinetic and stability studies indicate that size of macrocyclic cavity, ligand denticity, and bimodality of donor groups have a substantial impact on complexation of the bifunctional ligands with the radiolanthanides. The new promising bifunctional chelates in the DE4TA and NE3TA series were rapid in binding ⁹⁰Y and ¹⁷⁷Lu, and the corresponding ⁹⁰Y- and ¹⁷⁷Lu-radiolabeled complexes remained inert in human serum or in mice. The in vitro and in vivo data show that 3p-C-DE4TA and 3p-C-NE3TA are promising bifunctional ligands for targeted radiotherapy applications of ⁹⁰Y and ¹⁷⁷Lu.

Full text of DOI:10.1016/j.bmc.2014.12.035

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and comparative biological evaluation of bifunctional
ligands for radiotherapy applications of 90Y and 177Lu
a
a
a
a
b,c
Hyun-Soon Chong a, , Xiang Sun , Yunwei Chen , Inseok Sin , Chi Soo Kang , Michael R. Lewis
,
⇑
Dijie Liu c, Varyanna C. Ruthengael c, Yongliang Zhong a, Ningjie Wu a, Hyun A. Song a
a Chemistry Division, Department of Biological and Chemical Sciences, Illinois Institute of Technology, 3101 S. Dearborn St, LS 182, Chicago, IL 60616, United States
b Research Service, Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO, United States
c Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO, United States
a r t i c l e i n f o
a b s t r a c t
ZevalinÒ is an antibody-drug conjugate radiolabeled with a cytotoxic radioisotope (90Y) that was approved
for radioimmunotherapy (RIT) of B-cell non-Hodgkin’s lymphoma. A bifunctional ligand that displays
favorable complexation kinetics and in vivo stability is required for effective RIT. New bifunctional ligands
3p-C-DE4TA and 3p-C-NE3TA for potential use in RIT were efficiently prepared by the synthetic route based
on regiospecific ring opening of aziridinium ions with prealkylated triaza- or tetraaza-backboned macrocy-
cles. The new bifunctional ligands 3p-C-DE4TA and 3p-C-NE3TA along with the known bimodal ligands
3p-C-NETA and 3p-C-DEPA were comparatively evaluated for potential use in targeted radiotherapy using
b-emitting radionuclides 90Y and 177Lu. The bifunctional ligands were evaluated for radiolabeling kinetics
with 90Y and 177Lu, and the corresponding 90Y or 177Lu-radiolabeled complexes were studied for in vitro
stability in human serum and in vivo biodistribution in mice. The results of the comparative complexation
kinetic and stability studies indicate that size of macrocyclic cavity, ligand denticity, and bimodality of
donor groups have a substantial impact on complexation of the bifunctional ligands with the radiolantha-
nides. The new promising bifunctional chelates in the DE4TA and NE3TA series were rapid in binding 90Y
and 177Lu, and the corresponding 90Y- and 177Lu-radiolabeled complexes remained inert in human serum or
in mice. The in vitro and in vivo data show that 3p-C-DE4TA and 3p-C-NE3TA are promising bifunctional
ligands for targeted radiotherapy applications of 90Y and 177Lu.
Article history:
Received 11 October 2014
Revised 9 December 2014
Accepted 16 December 2014
Available online xxxx
Keywords:
Bifunctional ligands
Y-90
Lu-177
Radioimmunotherapy
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
thermodynamic stability and rapid radiolabeling kinetics under
mild conditions.5,6 The radiolabeled complexes for RIT also must
90Y (t1/2 = 2.7 days, Emax = 2.3 MeV) and 177Lu (t1/2 = 6.7 days,
Emax = 0.5 MeV) are b-emitting cytotoxic radionuclides for use in
targeted radiation therapy of cancer.1–3 90Y is a pure b-emitter with
a high energy and long range of tissue penetration (ꢀ12 mm) that
may be suitable for treatment of large solid tumors.1 A 90Y-radiola-
beled antibody conjugate (ZevalinÒ) is clinically available for radio-
immunotherapy (RIT) of B-cell non-Hodgkin’s lymphoma.1,4 177Lu
possesses a shorter penetration range (ꢀ2 mm) and lower maximal
energy relative to 90Y and was proposed to selectively target to
small tumors while minimizing tissue damage.1,2 An imageable
kinetically inert to transchelation by metal cations and natural
chelators present in vivo.5,6 Although efficacy and potency of RIT
have been demonstrated in numerous clinical trials, lack of an opti-
mal bifunctional ligands remains one of limitations for practical
applications of RIT.1–4 Research effort has been made to improve
chelation chemistry for RIT applications of 90Y and 177Lu. Better
understanding of chelation chemistry of Y(III) and Lu(III) can lead
to a rational design and development of bifunctional chelators
for potent and safe RIT applications using the radiolanthanides.
Y(III) and Lu(III) with the prevalent oxidation state (+3) have the
respective ionic radius (pm)7 of 116 and 98 for coordination num-
ber 8. An effective chelate of Y(III) and Lu(III) should possesses the
adequate denticity to completely saturate the coordination sphere
of Y(III) or Lu(III) with high complex stability. Y(III) and Lu(III)
with a large ionic radius were shown to be coordinated to the che-
c
-ray of a low abundance emitted from 177Lu can be applied for a
gamma scintigraphy during radiation therapy.1
An effective RIT using a radiolanthanide requires the use of a
bifunctional ligand that can form a metal complex with high
lators of high denticity such as octadentate DOTA and DTPA.6
size-fit between ionic radius of a metal and a macrocyclic cavity
A
⇑
Corresponding author. Tel.: +1 312 567 3235; fax: +1 312 567 3494.
E-mail address: Chong@iit.edu (H.-S. Chong).
http://dx.doi.org/10.1016/j.bmc.2014.12.035
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chong, H.-S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.035

2
H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
is important for an optimal complexation of a chelate with a
metal.8,9 Hexadentate NOTA was known to be ineffective in bind-
ing the lanthanides with high complex stability due to inadequate
size of the macrocyclic cavity and insufficient number of donor
groups in the chelator.10–12 Octadentate DOTA, with a larger mac-
rocyclic platform, forms stable complexes with Y(III) and Lu(III),
although slow complexation kinetics of DOTA with the lanthanides
remains a major drawback for its practical RIT applications.13,14 In
general, acyclic chelators such as DTPA analogues rapidly bind to a
metal. However, acyclic DTPA are known to form less stable
complexes than macrocyclic DOTA. The currently available RIT
drug, ZevalinÒ was generated using a DTPA analogue (1B4M-DTPA)
that displays favorable radiolabeling kinetics with 90Y.1
with the structural variations is expected to advance our
understanding on effect of the coordination factors including
bimodality, denticity, and macrocyclic cavity on complexation of
the bimodal chelates with the b-emitting radiolanthanides.
2. Materials and methods
2.1. Instruments and reagents
1H, 13C, and DEPT NMR spectra were obtained using a Bruker
300 MHz instrument and chemical shifts are reported in parts
per million (ppm) on the d scale relative to TMS. Electrospray
(ESI) high-resolution mass spectra (HRMS) were obtained on JEOL
double sector JMS-AX505HA mass spectrometer (University of
Notre Dame, South Bend, IN). 90Y (0.05 M HCl) and 177Lu (0.05 M
HCl) were purchased from Perkin Elmer.
Recently, we reported that bimodal ligands in the NETA and
DEPA series (Fig. 1), containing both macrocyclic and acyclic bind-
ing moieties, are highly effective in binding
a-, b-, or c-emitting
radionuclides.12,14–18 The design of the bimodal chelates is based
on hypothesis that the chelators sequester a metal with enhanced
complexation kinetics and a high level of complex stability via
cooperative and bimodal coordination of macrocyclic and acyclic
binding moieties. We proposed that the acyclic pendant donor
groups in the chelates can rapidly capture and initiate coordination
of the metal, as in the case of the acyclic chelate DTPA, and the
macrocyclic component tightly wraps up the cation trapped in
the acyclic donor groups achieving maximum complex stability,
as in the case of a macrocyclic chelate NOTA or DOTA.12 The
octadentate NETA conjugated to trastuzumab, a tumor-targeting
antibody, was found to instantly complex 90Y and 177Lu, and the
corresponding 90Y or 177Lu-radiolabeled complexes were stable in
human serum and tumor-bearing mice.14
In our continued effort in development of superior chelation
chemistry for 90Y and 177Lu, we sought comparative evaluation of
bimodal chelates for understanding on the effect of denticity,
the size-match between cavity and metal, and bimodality on
complexation. The new bifunctional chelates 3p-C-DE4TA (1) and
3p-C-NE3TA (2) and 3p-C-NOTA (3) were designed for evaluation
of complexation with 90Y and 177Lu (Fig. 1). The known bimodal
chelates decadentate 3p-C-DEPA (4)17 and octadentate 3p-C-NETA
(5)16 were also evaluated for complexation with 90Y and 177Lu for
comparison. The comparative evaluation of the bimodal chelates
2.2. 2-{[(2,4-Dimethoxyphenyl)methyl]amino}-5-(4-
nitrophenyl)pentan-1-ol (6)
To a stirred solution of 516 (1.60 g, 7.13 mmol) in 1,2-dichloro-
ethane (40 mL) was added 2,4-dimethoxy-benzaldehyde (1.19 g,
7.13 mmol). The resulting solution was stirred for 10 min, and
sodium triacetoxyborohydride (2.12 g, 9.99 mmol) was added por-
tionwise to the solution over 10 min. The mixture was stirred at
room temperature for 1 d. The reaction mixture was quenched by
saturated NaHCO3 (100 mL), and the resulting solution was
extracted with ethyl acetate (3 Â 60 mL). The combined organic
layer was concentrated in vacuo. The residue was purified by silica
gel (60–230 mesh) column chromatography eluted with 10%
CH3OH in CH2Cl2 to afford 6 (1.65 g, 62%). 1H NMR (CDCl3,
300 MHz) d 1.41–1.53 (m, 2H), 1.55–1.73 (m, 2H), 2.45 (s, 2H),
2.59–2.76 (m, 3H), 3.32 (dd, J = 10.8, 5.4 Hz, 1H), 3.61–3.74 (m,
3H), 3.78 (s, 6H), 6.34–6.49 (m, 2H), 7.07 (d, J = 7.8 Hz, 1H), 7.27
(d, J = 8.7 Hz, 2H), 8.10 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3,
75 MHz) d 27.3 (t), 31.3 (t), 35.8 (t), 46.1 (t), 55.3 (q), 55.4 (q),
57.4 (d), 62.7 (t), 98.7 (d), 103.9 (d), 120.5 (s), 123.6 (d), 129.1
(d), 130.5 (d), 146.4 (s), 150.1 (s), 158.6 (s), 160.4 (s). HRMS
(positive ion ESI) calcd for C20H27N2O5 [M+H]+ m/z 375.1914.
Found: [M+H]+ m/z 375.1886.
HO2C
N
CO2H
CO2H
CO2H
CO2H
CO2H
N
N
N
N
N
N
N
CO2H
CO2H
N
N
HO2C
N
H
HO2C
N
H
3p-C-NOTA (3)
3p-C-DE4TA (1)
NO2
NO2
3p-C-NE3TA (2)
O2N
HO2C
N
CO2H
CO2H
N
N
CO2H
CO2H
CO2H
CO2H
N
N
CO2H
N
N
N
N
CO2H
N
HO2C
HO2C
N
N
N
NO2
CO2H
HO2C
3p-C-NETA (5)
C-DOTA
3p-C-DEPA (4)
NO2
O2N
Figure 1. Bifunctional chelators for targeted radioimmunotherapy using 90Y and 177Lu in preclinical evaluation.
Please cite this article in press as: Chong, H.-S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.035

H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
2.3. tert-Butyl 2-{[(2,4-dimethoxyphenyl)methyl][1-hydroxy-5-
(4-nitrophenyl)pentan-2-yl] amino}acetate (7)
(s), 170.6 (s), 171.1 (s). HRMS (positive ion ESI) calcd for
C52H85N6O12 [M+H]+ m/z 985.6220. Found: [M+H]+ m/z 985.6224.
To a stirred solution of 6 (1.60 g, 4.27 mmol) in CH3CN (30 mL)
at 0 °C was added K2CO3 (0.62 g, 4.49 mmol). A solution of t-butyl
bromoacetate (0.88 g, 4.49 mmol) in CH3CN (10 mL) was added
dropwise to the resulting mixture over 10 min. The reaction mix-
ture was stirred for at room temperature for 2 days, while the reac-
tion progress was continuously monitored using TLC. The reaction
mixture was filtered and evaporated in vacuo to provide 7 (1.21 g,
58%) as a light yellow oil. The product was directly used for the
next step without further purification. 1H NMR (CDCl3, 300 MHz)
d 1.33 (s, 9H), 1.55–1.73 (m, 4H), 2.62–2.73 (m, 2H), 2.80–2.94
(m, 1H), 3.17 (dd, J = 30.9, 17.1 Hz, 2H), 3.33 (dd, J = 10.2 Hz,
10.2 Hz, 1H), 3.41–3.50 (m, 1H), 3.65 (d, J = 13.8 Hz, 1H), 3.79 (s,
6 H), 4.05–4.16 (m, 1H), 6.35–6.46 (m, 2H), 7.13 (d, J = 8.7 Hz,
1H), 7.28 (d, J = 8.1 Hz, 2H), 8.10 (d, J = 8.1 Hz, 2H); 13C NMR (CDCl3,
75 MHz) d 27.0 (t), 27.9 (q), 28.4 (t), 35.9 (t), 49.5 (t), 52.2 (t), 55.2
(q), 55.3 (q), 62.1 (t), 63.7 (d), 80.9 (s), 98.4 (d), 103.9 (d), 119.1 (s),
123.6 (d), 129.1 (d), 131.4 (d), 146.4 (s), 150.0 (s), 158.9 (s), 160.4
(s), 172.3 (s). HRMS (positive ion ESI) calcd for C26H37N2O7 [M+H]+
m/z 489.2595. Found: [M+H]+ m/z 489.2577.
2.6. 2-{[5-(4-Nitrophenyl)-1-[4,7,10-tris(carboxymethyl)-
1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl]amino}acetic
acid (1)
To compound 11 (54 mg, 0.0548 mmol) was added 6 M HCl
solution (5 mL), and the resulting solution was refluxed for
15 min. The resulting solution was cooled to room temperature
and washed with CHCl3 (10 mL). The aqueous layer was concen-
trated and dried in vacuo to provide compound 1 (41 mg, 95%)
present in a HCl salt as a waxy yellow solid. d 1.40–1.79 (m, 4H),
2.41–4.01 (m, 29H), 7.32 (d, J = 8.1 Hz, 2H), 8.05 (d, J = 8.1 Hz,
2H). 13C NMR (D2O, 75 MHz) d 25.5 (t), 27.8 (t), 34.3 (t), 45.2 (t),
49.1 (t), 49.8 (t), 50.9 (t), 52.4 (t), 52.7 (t), 54.2 (t), 54.8 (d), 55.7
(t), 123.6 (d), 129.4 (d), 145.8 (s), 150.0 (s), 168.9 (s). HRMS
(negative ion ESI) calcd for C27H41N6O10 [MÀH]À m/z 609.2890.
Found: [MÀH]À m/z 609.2926.
2.7. tert-Butyl-2-{[2-bromo-5-(4-nitrophenyl)pentyl][(2,4-
dimethoxyphenyl)methyl]-amino} acetate (12)
To a solution of 7 (300 mg, 0.614 mmol) and PPh3 (193 mg,
0.737 mmol) in CHCl3 (5 mL) at 0 °C was added portionwise NBS
(131 mg, 0.737 mmol) over 5 min. The resulting mixture was
stirred for 4 h at 0 °C. The ice bath was removed, and the reaction
mixture was warmed to room temperature and stirred for 1 h. The
solvent was evaporated, and the residue was purified by silica gel
column chromatography eluted with 10% ethyl acetate in hexanes
to afford 12 (210 mg, 62%) as a yellow oil. 1H NMR (CDCl3,
300 MHz) d 1.46 (s, 9H), 1.62–2.17 (m, 4H), 2.60–2.81 (m, 2H),
2.96 (dd, J = 13.8, 8.7 Hz, 1H), 3.17 (dd, J = 13.8, 5.4 Hz, 1H), 3.24
(s, 2H), 3.66–3.82 (m, 7H), 3.87 (d, J = 7.4 Hz, 1H), 3.94–4.12 (m,
1H), 6.40–6.46 (m, 2H), 7.15 (d, J = 9.0 Hz, 1H), 7.31 (d, J = 8.4 Hz,
2H), 8.12 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d 28.2 (q),
28.5 (t), 35.1 (t), 35.2 (t), 51.9 (t), 55.1 (d), 55.3 (q), 56.1 (t), 61.8
(t), 80.9 (s), 98.4 (d), 103.9 (d), 119.1 (s), 123.6 (d), 129.2 (d),
131.2 (d), 146.3 (s), 150.0 (s), 158.8 (s), 160.2 (s), 170.9 (s). HRMS
(positive ion ESI) calcd for C26H35N2O6 [MÀBr]+ m/z 471.2490.
Found: [MÀBr]+ m/z 471.2474.
2.4. tert-Butyl 2-{[(2,4-dimethoxyphenyl)methyl][2-iodo-5-
(4-nitrophenyl)pentyl]amino}acetate (8)
To a solution of 7 (100 mg, 0.205 mmol) and PPh3 (64.42 mg,
0.246 mmol) in CHCl3 (5 mL) at 0 °C was added portionwise I2
(62.34 mg, 0.246 mmol) and imidazole (16.75 mg, 0.246 mmol)
over 5 min. The resulting mixture was stirred for 5 h at room tem-
perature. The solvent was evaporated, and the residue was purified
by silica gel column chromatography eluted with 5% EtOAc in
hexanes to afford pure 8 (97.4 mg, 79%) as a light yellow oil.
Compound 8 was directly used for the next step. 1H NMR (CDCl3,
300 MHz) d 1.46 (s, 9H), 1.63–1.78 (m, 2H), 1.82–2.06 (m, 2H),
2.59–2.80 (m, 2H), 2.97 (dd, J = 13.2, 9.0 Hz, 1H), 3.19–3.27 (m,
3H), 3.72–3.85 (m, 8H), 4.07–4.19 (m, 1H), 6.43 (s, 2H), 7.16 (d,
J = 8.7 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 8.11 (d, J = 8.1 Hz, 2H); 13C
NMR (CDCl3, 75 MHz) d 28.3 (q), 30.6 (t), 34.9 (t), 36.2 (t), 37.0
(q), 51.7 (d), 55.4 (q), 55.8 (t), 63.5 (t), 80.9 (s), 98.4 (d), 103.9
(d), 119.1 (s), 123.6 (d), 129.2 (d), 131.2 (d), 146.3 (s), 150.0 (s),
158.8 (s), 160.2 (s), 170.9 (s). HRMS (positive ion ESI) calcd for
2.8. tert-Butyl 2-[(1-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,4,
7-triazonan-1-yl}-5-(4-nitrophenyl)pentan-2-yl)
[(2,4-dimethoxyphenyl)methyl]amino]acetate (15)
C
26H37N2O7 [MÀI+HO+H]+ m/z 489.5812. Found: [M+H]+ m/z
489.2587.
2.5. tert-Butyl 2-{[(2,4-dimethoxyphenyl)methyl][5-(4-
nitrophenyl)-1-{4,7,10-tris[2-(tert-butoxy)-2-oxoethyl]-
1,4,7,10-tetraazacyclododecan-1-yl}pentan-2-yl]amino}
acetate (11)
To a solution of 12 (50 mg, 0.0907 mmol) in CH3CN (1 mL) at
À5 °C was added AgClO4 (18.8 mg, 0.0907 mmol). The resulting
mixture was stirred for 10 min at the same temperature.
Compound 1415 (32.4 mg, 0.0907 mmol) and DIPEA (35.2 mg,
0.272 mmol) was sequentially added to the reaction mixture at
À5 °C. The resulting mixture was gradually warmed to room tem-
perature and stirred for 20 h. The reaction mixture was filtered and
concentrated to the dryness in vacuo. 0.1 M HCl solution (10 mL)
was added to the residue, and the resulting mixture was extracted
with CH2Cl2 (3 Â 10 mL). The combined organic layers were dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified via column chromatography on silica gel (60–220 mesh)
sequentially eluted with 50% ethyl acetate in hexanes and then
with 3% CH3OH in CH2Cl2 to provide pure product 15 (70 mg,
93%) as a yellowish oil. 1H NMR (CDCl3, 300 MHz) d 1.29–1.48
(m, 27H), 1.50–1.94 (m, 4H), 2.61–3.93 (m, 31H), 6.34–6.45 (m,
2H), 7.05 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 7.8 Hz, 2H), 8.09 (d,
J = 7.8 Hz, 2H). 13C NMR (CDCl3, 75 MHz) d 27.3 (t), 28.0 (q), 28.1
(q), 28.4 (t), 35.8 (t), 50.0 (t), 50.8 (t), 51.4 (t), 51.9 (t), 54.0 (t),
54.4 (t), 55.4 (q), 55.5 (q), 56.9 (d), 57.4 (t), 58.5 (t), 58.7 (t), 81.5
To a solution of 8 (54.3 mg, 0.0907 mmol) in CH3CN (5 mL) at
0 °C was added compound 1018 (46.66 mg, 0.0907 mmol) and
DIPEA (23.43 mg, 0.181 mmol). The resulting mixture was stirred
for 4 d at room temperature. The reaction mixture was concen-
trated to dryness in vacuo. The residue was sequentially washed
with 0.1 M HCl (10 mL) and 0.1 M NaOH (10 mL). The resulting res-
idue was concentrated to dryness in vacuo to provide pure 11
(65.4 mg, 73%). 1H NMR (CDCl3, 300 MHz) d 1.39 (s, 36H), 1.50–
2.09 (m, 4H), 2.52–2.95 (m, 14H), 3.05–3.70 (m, 17H), 3.77 (s, 6
H), 6.34–6.52 (m, 2H), 7.21 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.4 Hz,
2H), 8.12 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3, 75 MHz) d 28.1 (q),
28.2 (q), 28.6 (t), 29.6 (t), 35.8 (t), 48.6 (t), 51.0 (t), 51.6 (t), 51.8
(t), 53.3 (t), 55.3 (d), 55.5 (q), 55.6 (q), 56.3 (t), 56.9 (t), 81.3 (s),
81.3 (s), 81.4 (s), 81.6 (s), 98.4 (d), 104.8 (d), 118.1 (s), 123.6 (d),
129.3 (d), 130.6 (d), 146.4 (s), 150.0 (s), 158.8 (s), 160.7 (s), 170.3
Please cite this article in press as: Chong, H.-S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.035

4
H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(s), 81.6 (s), 82.1 (s), 98.6 (d), 104.4 (d), 117.7 (s), 123.6 (d), 129.5
(d), 131.9 (d), 146.3 (s), 149.8 (s), 158.8 (s), 160.8 (s), 170.5 (s),
170.6 (s), 173.1 (s). HRMS (positive ion ESI) calcd for
and MeOH (7 mL) was added with stirring. The excess solvent
was removed and gave a mixture of yellowish oil (60:40). The
residue was purified via column chromatography on silica gel
(60–220 mesh) eluting 50% CH2Cl2 in hexane afford pure 19
(414 mg, 58.4%) as a colorless oil. 1H NMR (CDCl3, 300 MHz) d
1.61–1.89 (m, 2H), 1.96–2.06 (m, 2H), 2.72 (t, J = 7.5 Hz, 2H), 3.72
(s, 3H), 4.22 (dd, J = 7.5, 6.9 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H), 8.07
(d, J = 8.7 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d 28.4 (t), 34.1 (t),
34.8 (t), 45.2 (d), 53.0 (t), 123.7 (d), 129.2 (d), 146.4 (s), 149.3 (s),
170.0 (s).
C
44H70N5O10 [M+H]+ m/z 828.5117. Found: [M+H]+ m/z 828.5161.
2.9. 2-({1-[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]-5-
(4-nitrophenyl)pentan-2-yl} amino)acetic acid (2)
To compound 15 (67 mg, 0.0809 mmol) was added 6 M HCl
solution (3 mL), and the resulting solution was maintained at
reflux for 15 min. The reaction was allowed to room temperature,
and the resulting solution was filtered and dried in vacuo to pro-
vide the desired chelate 2 (51 mg, 96%) as a waxy yellow solid.
1H NMR (D2O, 300 MHz) d 1.38–1.70 (m, 4H), 2.47–3.36 (m,
16H), 3.27–3.40 (m, 1H), 3.67–3.91 (m, 6H), 7.27 (d, J = 7.8 Hz,
2H), 7.96 (d, J = 7.8 Hz, 1H). 13C NMR (D2O, 75 MHz) d 25.6 (t),
27.1 (t), 34.6 (t), 44.5 (t), 49.1 (t), 49.6 (t), 51.9 (t), 55.6 (d), 56.0
(t), 59.0 (t), 123.9 (d), 129.6 (d), 146.1 (s), 150.3 (s), 169.1 (s),
172.0 (s). HRMS (positive ion FAB) calcd for C23H36N5O8: [M+H]+
m/z 510.2558. Found: [M+H]+ m/z 510.2557.
2.13. Methyl 2-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,
4,7-triazonan-1-yl}-5-(4-nitrophenyl) pentanoate (20)
Compound 19 (50 mg, 0.158 mmol) was added dropwise to a
solution of 14 (56.5 mg, 0.158 mmol) in CH3CN (0.5 mL) at 0 °C.
DIPEA (61.1 mg, 0.474 mmol) in CH3CN (0.5 mL) was added drop-
wise. The resulting mixture was allowed to room temperature
and stirred for 5 d, while monitoring the reaction progress using
TLC. After which period of time, the reaction mixture was evapo-
rated to dryness. The residue was dissolved with 0.1 M HCl solu-
tion (10 mL) and washed with CHCl3 (2 Â 10 mL). The combined
organic layers were dried over MgSO4, filtered, and concentrated
to the dryness in vacuo. The residue was purified via column chro-
matography on silica gel (60–220 mesh) and eluted with 10%
MeOH in CH2Cl2 to provide pure 20 (92 mg, 98%) as a yellow oil.
1H NMR (CDCl3, 300 MHz) d 1.43 (s, 9H), 1.60–1.94 (m, 4H),
2.65–3.03 (m, 14H), 3.28 (s, 5H), 3.65 (s, 3H), 7.33 (d, J = 8.4 Hz,
2H), 8.12 (d, J = 8.4 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d 27.9 (t),
28.2 (q), 30.0 (t), 35.6 (t), 51.0 (q), 53.5 (t), 55.2 (t), 55.9 (t), 59.4
(t), 66.8 (d), 80.7 (s), 123.6 (d), 129.2 (d), 146.4 (s), 150.1 (s),
171.4 (s), 174.0 (s). HRMS (positive ion ESI) calcd for C30H49N4O8
[M+H]+ m/z 593.3545. Found: [M+H]+ m/z 593.3529.
2.10. 1,3-Diethyl 2-[3-(4-nitrophenyl)propyl]propanedioate
(17)19
To a round bottom flask containing 60% NaH in mineral oil
(272 mg, 6.80 mmol) in an ice-bath was added THF (10 mL). A solu-
tion of diethyl malonate (1.04 g, 6.47 mmol) in THF (10 mL) was
added dropwise over 10 min at 0 °C, and the reaction mixture
was stirred for 30 min. To the reaction mixture was added drop-
wise a solution of 1616 (1.58 g, 6.47 mmol) in THF (10 mL) over
10 min. The reaction was warmed to room temperature and stirred
for 1 d. The reaction mixture was quenched by H2O (10 mL) and
evaporated to dryness. H2O (30 mL) was added to the mixture,
and the resulting solution was extracted with ethyl acetate
(3 Â 30 mL). The combined organic layers were dried over MgSO4
and concentrated to the dryness in vacuo. The residue was purified
via column chromatography on silica gel (60–220 mesh) eluted
with 8% ethyl acetate in hexanes to afford pure 17 (1.46 g, 70%)
2.14. 2-[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]-5-
(4-nitrophenyl)pentanoic acid (3)
To compound 20 (55 mg, 0.0928 mmol) was added 6 M HCl
solution (6 mL), and the resulting solution was maintained at
reflux for 3 h. The reaction was allowed to room temperature,
as
a d 1.23
light yellow oil. 1H NMR (CDCl3, 300 MHz)
(t, J = 7.2 Hz, 6H), 1.61–1.79 (m, 2H), 1.83–1.98 (m, 2H), 2.74 (t,
J = 7.5 Hz, 2H), 3.32 (t, J = 7.2 Hz, 1H), 4.07–4.25 (m, 4H), 7.31
(d, J = 8.4 Hz, 2H), 8.11 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3,
75 MHz) d 14.1 (q), 28.1 (t), 28.5 (t), 35.4 (t), 51.7 (d), 61.4 (t),
123.7 (d), 129.2 (d), 146.4 (s), 149.5 (s), 169.2 (s).
and the resulting solution was filtered through CeliteÒ using 18
X
H2O and dried in vacuo to provide compound 3 (47.7 mg, 89.3%)
as a yellow solid. 1H NMR (D2O, 300 MHz) d 1.50–1.79 (m, 4H),
2.49–2.62 (m, 2H), 2.99–3.05 (m, 12H), 3.58–3.67 (m, 1H), 3.70–
3.92 (m, 4H), 7.16 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H); 13C
NMR (D2O, 75 MHz) d 27.3 (t), 27.7 (t), 34.7 (t), 45.7 (t), 47.2 (t),
50.1 (t), 51.0 (t), 55.8 (t), 65.3 (d), 123.6 (d), 129.5 (d), 145.9 (s),
150.5 (s), 171.5 (s), 174.4 (s). HRMS (positive ion ESI) calcd for
2.11. 5-(4-Nitrophenyl)pentanoic acid (18)20
Compound 17 (2.5 g, 7.7 mmol) was dissolved in the mixture of
acetic acid (20 mL) and concd HCl (20 mL), and the resulting solu-
tion was refluxed for 24 h. The reaction mixture was cooled to
room temperature and concentrated in vacuo to provide 18
(1.6 g, 93.2%) as a yellow solid that was used for the next step
without further purification. 1H NMR (D2O + NaOD, 300 MHz) d
1.29–1.42 (m, 4H), 1.97–2.07 (m, 2H), 2.38–2.49 (m, 2H), 7.06 (d,
J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H); 13C NMR (D2O + NaOD,
75 MHz) d 25.5 (t), 30.1 (t), 34.8 (t), 37.3 (t), 123.3 (d), 129.1 (d),
145.3 (s), 151.6 (s), 183.4 (s).
C
21H29N4O8 [MÀH]+ m/z 465.1991. Found: [MÀH]+ m/z 465.1999.
2.15. Radiolabeling of the bifunctional chelates with 90Y and
177Lu
All HCl solutions were prepared from ultra pure HCl (JT baker,
#6900-05). For metal-free radiolabeling, plasticware including
pipet tips, tubes, and caps was soaked in 0.1 M HCl (aq) overnight
and washed thoroughly with 18 M
X water, and air-dried
2.12. Methyl 2-bromo-5-(4-nitrophenyl)pentanoate (19)20
overnight. Ultra pure ammonium acetate (Aldrich, #372331) was
purchased from Aldrich and used to prepare buffer solutions
(0.25 M) at various pHs. After adjusting pH using 0.1 M HCl or
0.1 M NaOH solution, 0.25 M NH4OAc buffer solutions were treated
with Chelex-100 resin (Biorad, #142-2842, 1 g/100 mL buffer solu-
tion), shaken overnight at room temperature, and filtered through
Compound 18 (500 mg, 2.24 mmol) was added to a solution of
CCl4 (0.5 mL) and thionyl chloride (1.07 g, 8.97 mmol). The solution
was brought to reflux for 1 h. At this point, NBS (598.1 mg,
3.36 mmol) was added, and 1 drop of 48% aqueous HBr catalyst
was added to the warm solution. The dark solution was refluxed
for an additional 1 h and became clear. The solution was cooled,
0.22
purchased from Perkin Elmer. TLC plates (6.6 Â 1 cm, Silica gel 60
l
M filter (Corning, #430320) prior to use. 90Y and 177Lu were
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H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
F254, EMD Chemicals Inc., #5554-7) with the origin line drawn at
0.6 cm from the bottom were prepared.
(3:2 v/v) or 20 mM EDTA in 0.15 M NH4OAc. A solution of the
radiolabeled complex in serum was withdrawn at the designated
time point, and the percentage of 90Y or 177Lu released from each
of the radiolabeled complexes into serum was assessed by ITLC
as described above.
To a buffer solution (0.25 M NH4OAc, pH 5.5 or pH 7.0) in a
capped microcentrifuge tube (1.5 mL, #05-408-129) was sequen-
tially added a solution of a chelate in water (20
177Lu (60
Ci). The total volume of the resulting solution was
40 L. The reaction mixture was agitated on the thermomixer
l
g) and 90Y or
l
l
2.17. Biodistribution studies
(Eppendorf, #022670549) set at 1000 rpm at room temperature
for 1 h. The labeling efficiency was determined by ITLC eluted with
CH3CN/H2O (3:2 v/v) or 20 mM EDTA in 0.15 M NH4OAc as the
All animal experiments were conducted in accordance with the
guidelines established by the Animal Care and Use Committee of
the University of Missouri and the Harry S. Truman Memorial
Veterans’ Hospital Subcommittee for Animal Studies. Six to eight
week old CF-1 mice were obtained from Charles River Laboratories
and housed one week prior to the studies. An aliquot of each
mobile phase. A solution of radiolabeled complexes (2 lL) was
withdrawn at the designated time points (1 min, 10 min, 20 min,
30 min, and 60 min), 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). Unbound
(Rf = 0.6) and bound (Rf = 0.9) radioisotope appeared around
30 mm and 50 mm from the bottom of the TLC plate eluted with
CH3CN/H2O (3:2 v/v), respectively. For the ITLC eluted with
20 mM EDTA in 0.15 M NH4OAc system, unbound (Rf = 0.9) and
bound (Rf = 0.6) radioisotope appeared around 50 mm and
30 mm, respectively.
177Lu-radiolabeled complex (60
described above were intravenously injected via the tail vein in a
volume of 100 L of phosphate-buffered saline. At 1 h, 4 h and
lCi) that was prepared as
l
24 h post-injection, mice were sacrificed and blood, liver, kidney,
muscle and bone were collected, weighed and counted in a gamma
counter. The radioactivity from each tissue/organ was decay-
corrected by a known aliquot of the injected dose, and the
percent-injected dose per gram of tissue (% ID/g) was calculated.
Values were presented as mean SD for each group of 3 mice.
2.16. In vitro serum stability of 90Y- and 177Lu-radiolabeled
complexes
3. Results and discussion
3.1. Synthesis
Human serum was purchased from Gemini Bioproducts
(#100110). 90Y- or 177Lu-radiolabeled complexes (0.25 M NH4OAc,
pH 5.5) were prepared from the reaction of chelates with 90Y or
177Lu at room temperature or 37 °C. Completion of radiolabeling
was monitored by ITLC eluted, and the freshly prepared radiola-
beled complexes were used for serum stability without further
purification. 90Y-3p-C-DE4TA, 90Y-3p-C-NE3TA and 90Y-3p-C-NOTA
were prepared by a reaction of 3p-C-DE4TA (1), 3p-C-NE3TA (2)
3p-C-DE4TA (1) contains a 12-membered larger macrocyclic
backbone, while heptadentate 3p-C-NE3TA (2) is structured on a
smaller triazacyclononane (TACN) ring. Both 3p-C-DE4TA and
3p-C-NE3TA possess a less hindered bidentate acyclic pendant
arm relative to 3p-C-DEPA (4) and 3p-C-NETA (5), respectively.
Hexadentate 3p-C-NOTA (3) does not have the flexible pendant
arm required for bimodal binding by cooperation of acyclic and
macrocyclic binding moieties and was designed to compare the
effect of bimodality on complexation with the radiolanthanides.
The new bifunctional chelates 3p-C-DE4TA (1) and 3p-C-NE3TA
(2) as shown in Schemes 1 and 2 were prepared based on the reg-
iospecific ring opening of labile aziridinium ions (9 and 13) with a
prealkylated macrocycles DO3A (tri-tert-butyl 1,4,7,10-tetraazacy-
clododecane-1,4,7-triacetate 10) and NO2A (di-tert-butyl-1,4,7-tri-
azacyclononane-1,4-diacetate, 14). Synthesis of 3p-C-DE4TA is
outlined in Scheme 1. Reductive amination of amino alcohol 516
with dimethoxybenzaldehyde provided N-dimethoxybenzyl
(DMB) protected compound 6 which was further alkylated to
afford bi-substituted amino alcohol 7. Halogenation of 7 using I2
and PPh3 afforded secondary b-amino iodide 8. Conversion of 8
to aziridinium ion 9 followed by nucleophilic reaction of 9 with tri-
alkylated macrocyclic cyclen 1018 at the less hindered carbon pro-
vided the desired ring opening product 11 as the regiospecific
isomer. Subsequent removal of the tert-butyl and DMB groups in
11 by treatment of 11 with 6 M HCl (aq) under reflux provided
3p-C-DE4TA (1) in quantitative yield. Synthesis of 3p-C-NE3TA
(2) is outlined in Scheme 2. Reaction of 7 with brominating agent
(NBS and PPh3) afforded secondary b-amino bromide 12. Intramo-
lecular rearrangement of b-bromoamine 12 to aziridinium ion 13
was promoted by halogen sequestering agent (AgClO4). Reaction
of NO2A (14)15 with the alkylating agent 13 provided the nucleo-
philic ring opening product 15 as the regiospecific isomer. Com-
pound 15 was subjected to acidic hydrolysis using HCl(aq) for
removal of the protective tert-butyl groups in 15. Synthesis of
3p-C-NOTA is shown in Scheme 3. A base-promoted reaction of
p-nitrophenylpropyl bromide (16)16 with diethyl malonate pro-
vided compound 17 which was subsequently subjected to hydroly-
sis to afford a functionalized carboxylic acid 18. Chlorination of 18
and 3p-C-NOTA (3) (50 l lCi) in 0.25 M NH4OAc
g) with 90Y (150
buffer (pH 7.0). Radiolabeling of 3p-C-DE4TA, 3p-C-NE3TA and
3p-C-NOTA with 90Y were complete in 2 h at 37 °C (600 rpm).
90Y-3p-C-DEPA (4) was prepared by a reaction of 3p-C-DEPA
(100 lg, 100 l lCi) in 0.25 M NH4OAc buffer (pH
L) with 90Y (300
5.5). Radiolabeling of 3p-C-DEPA with 90Y was complete in 4 h at
rt (1000 rpm) and 2 h at 37 °C (300 rpm). The complexes
90Y-3p-C-DE4TA, 90Y-3p-C-NE3TA, 90Y-3-C-NOTA, and 90Y-3p-C-
DEPA prepared from the reactions were directly used for serum
stability studies without further purification. 90Y-3p-C-DE4TA
(144
NOTA (144
microcentrifuge tube. 90Y-3p-C-DEPA (90
l
Ci, 99
l
L), 90Y-3p-C-NE3TA (144
Ci, 99 L) was added to human serum (500
Ci, 63 L) was added
l
Ci, 99
l
L), or 90Y-3-C-
l
l
l
L) in a
l
l
to human serum (330 lL) in a microcentrifuge tube.
177Lu-3p-C-DE4TA, 177Lu-3p-C-NE3TA, 177Lu-3-C-NOTA, and
177Lu-3p-C-DEPA were prepared by a reaction of 3p-C-DE4TA,
3p-C-NE3TA, 3p-C-NOTA, 3p-C-DEPA (50 l lCi)
g) with 177Lu (150
in 0.25 M NH4OAc buffer (pH 5.5), respectively. Radiolabeling of
3p-C-DE4TA and 3p-C-DEPA with 177Lu was complete in 3 h at
room temperature and 1000 rpm. Radiolabeling of 3p-C-NE3TA
and 3p-C-NOTA with 177Lu was complete in 2 h at room tempera-
ture and 1000 rpm. The complexes 177Lu-3p-C-DE4TA, 177Lu-3p-C-
DEPA, 177Lu-3p-C-NE3TA, and 177Lu-3-C-NOTA prepared from
the reactions were directly used for serum stability studies with-
out further purification. 177Lu-3p-C-DE4TA (146
177Lu-3p-C-DEPA (146 L), 177Lu-3p-C-NE3TA (149
Ci, 100
100 Ci, 100 L) was added to human
L), or 177Lu-3-C-NOTA (149
serum (500 L) in a microcentrifuge tube. The stability of the radi-
lCi, 100
l
L),
l
l
l
Ci,
l
l
l
l
olabeled complexes in human serum was evaluated at 37 °C over
14 days. The serum stability of the radiolabeled complexes was
assessed by measuring the transfer of the radionuclide from each
complex to serum proteins using ITLC eluted with CH3CN/H2O
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6
H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
O2N
O2N
OCH3
H3CO
OHC
O2N
6
Br
CO2tBu
7
5
HN OH
OCH3
N
OH
H2N OH
NaBH(OAc)3
DCE, 24 h (62%)
CH3CN, RT
14 h (58%)
ButO2C
OCH3
H3CO
H3CO
CO2tBu
N
ButO2C
CO2tBu
O2N
O2N
N
N
I2, PPh3
N
H
8
imi, CHCl3
9
(10)
I
N
0 oC to RT
5 h (79%)
I
N
CO2tBu
DIPEA, CH3CN
RT, 20 h
H3CO
CO2tBu
OCH3
OCH3
H3CO
(73%)
ButO2C
HO2C
N
N
N
N
N
CO2tBu
CO2tBu
CO2H
CO2H
N
N
N
ButO2C
HO2C
6M HCl(aq)
OCH3
N
N
reflux, 15 min
(95%)
H
1
OCH3
11
O2N
O2N
Scheme 1. Synthesis of 3p-C-DE4TA (1).
O2N
O2N
O2N
AgClO4
NBS, PPh3
CHCl3
0 oC to RT
5 h (62%)
CH3CN
7
13
Br
N
OH
ClO4
12
N
− 5 oC
N
ButO2C
CO2tBu
H3CO
CO2tBu
OCH3
H3CO
OCH3
H3CO
OCH3
ButO2C
ButO2C
CO2H
H3CO
ButO2C
N
N
ButO2C
CO2H
CO2H
N
N
N
N
(14)
N
H
OCH3
N
N
6M HCl(aq)
N
N
H
15
reflux, 15 min
CO2tBu
DIPEA, CH3CN
RT, 20 h
(96%)
2
(93%)
NO2
O2N
Scheme 2. Synthesis of 3p-C-NE3TA (2).
followed by
a
-bromination and esterification afforded
a-bromo-
(2), 3p-C-NOTA (3), and 3p-C-DEPA (4)17 were evaluated for
methyl ester 19.20 Subsequent reaction of 19 with bisubstituted
TACN 1415 provided compound 20 which was treated with 6 M
HCl (aq) to produce 3p-C-NOTA (3).
radiolabeling efficiency with 90Y and 177Lu (Tables 1 and 2 and
Supporting information). A chelate (20
l
g) in 0.25 M NH4OAc
buffer solution was radiolabeled with 90Y or 177Lu (60
l
Ci) at room
temperature (rt). During the reaction time (1 h), the radiolabeling
kinetics was determined using ITLC. Radiolabeling of 3p-C-DE4TA
(1) with 90Y or 177Lu was nearly complete at 10 min time point
(>99% radiolabeling efficiency, pH 7). It should be noted that
3p-C-DE4TA (1) was very slow in binding both 90Y and 177Lu at
pH 5.5 (1 min, 7% and 21% radiolabeling efficiency for 90Y and
177Lu, respectively, Supporting information). It is speculated that
the protonated secondary amine in the chelate may not participate
in complexation under acidic condition and lead to slow radiola-
beling of the chelate. A dramatic increase in radiolabeling kinetics
was observed when 3p-C-DE4TA (1) was subjected to radiolabeling
3.2. Radiolabeling kinetics and in vitro serum stability
We previously reported 3p-C-NETA (5) instantly bound to 90Y or
177Lu (1 min, >99% radiolabeling efficiency).16 C-DOTA was slower
than 3p-C-NETA in binding both 90Y and 177Lu.16 Both 90Y- and
177Lu-radiolabeled 3p-C-NETA and C-DOTA complexes remained
stable in human serum for 2 weeks without releasing any measur-
able radioactivity.16 3p-C-NETA was used as a positive control for
radiolabeling kinetics and serum stability studies as previously
reported.16 The bifunctional chelates 3p-C-DE4TA (1), 3p-C-NE3TA
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H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
EtO2C
NaH, THF
O2N
CO2Et O2N
AcOH, HCl O2N
reflux, 24 h
(93%)
CO2Et
CO2H
i) SOCl2, CCl4
Br
18
16
17
0
oC to RT
EtO2C
24 h (70%)
ii) NBS, 48% HBr(aq)
iii) MeOH
CO2tBu
CO2H
CO2tBu
CO2tBu
N
N
(49%)
CO2H
CO2tBu
N
N
N
N
N
(14)
N
N
H
6M HCl (aq)
O2N
HO2C
DIPEA, CH3CN
6 days, RT
(67%)
reflux 3 h H3CO2C
CO2CH3
(91%)
19
Br
3
20
NO2
NO2
Scheme 3. Synthesis of 3p-C-NOTA (3).
Table 1
Radiolabeling efficiency (%) of bifunctional chelates with 90Y (pH 5.5, rt)#
Time (min)
Radiolabeling efficiency (%)
3p-C-DE4TA* (1)
3p-C-NE3TAà (2)
3p-C-NOTAà (3)
3p-C-DEPA (4)
3p-C-NETA+ (5)
C-DOTA+
1
10
20
30
60
88.4 1.9
3.1 0.28
22.5 0.28
37.6 0.64
49.1 1.6
68.7 3.4
10.3 0.6
50.8 1.8
73.9 0.6
82.7 0.4
89.3 0.1
89.3 2.8
96.8 0.5
98.0 0.9
97.9 0.2
98.5 0.2
97.4 0.7
98.7 1.6
98.7 2.2
99.4 0.9
99.5 1.0
77.1 3.7
99.2 0.58
99.3 0.61
99.2 0.72
99.5 0.49
69.4 10.6
71.2 11.2
76.1 9.52
83.5 8.13
#
Radiolabeling efficiency (mean standard deviation%) was measured in triplicate using ITLC.
Radiolabeling efficiency was determined at pH 7 using ITLC.
*
à
Duplicate run.
+
The data were cited for comparison.16
Table 2
Radiolabeling efficiency (%) of bifunctional chelates with 177Lu (pH 5.5, rt)#
Time (min)
Radiolabeling efficiency (%)
3p-C-DE4TA* (1)
3p-C-NE3TA (2)
3p-C-NOTA (3)
3p-C-DEPA (4)
3p-C-NETA+ (5)
C-DOTA+
1
10
20
30
60
85.2 0.7
99.8 0.3
99.8 0.1
99.9 0.1
100.0 0.1
6.7 0.1
36.6 0.4
63.2 0.8
78.1 1.1
95.0 0.1
17.5 0.5
81.3 0.2
95.0 0.2
99.1 0.4
100 0.1
93.9 1.3
98.7 0.2
99.2 0.3
99.4 0.1
99.3 0.2
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
100.0 0.0
94.5 3.9
99.5 0.5
99.9 0.1
99.9 0.1
100.0 0.0
#
*
Radiolabeling efficiency (mean standard deviation%) was measured in triplicate using ITLC.
Radiolabeling efficiency was determined at pH 7 using ITLC.
The data cited for comparison.16
+
at pH 7 (1 min, >85% radiolabeling efficiency for 90Y and 177Lu).
Both 3p-C-NE3TA (2) and 3p-C-NOTA (3) were more sluggish in
binding 90Y than 177Lu. 3p-C-NE3TA and 3p-C-NOTA bound to 90Y
with the respective radiolabeling efficiencies of 69% and 89% at
the 1 h time point, while radiolabeling of 3p-C-NE3TA and
3p-C-NOTA with the smaller metal cation 177Lu was nearly com-
plete at 1 h time point. When compared to 3p-C-NETA (5) with
the same macrocyclic cavity, 3p-C-NE3TA (2) and 3p-C-NOTA (3)
were significantly slower in binding 90Y and 177Lu, although the
chelates were more efficient in binding the metals at pH 7
(Supporting information). This result clearly demonstrates that
the tridentate acyclic moiety is critical in enhancing complexation
kinetics via bimodal binding. It is interesting to note that hexaden-
tate 3p-C-NOTA was more efficient in binding both 90Y and 177Lu
than 3p-C-NE3TA, although 3p-C-NOTA has an insufficient number
of donor groups for complex with the large metal cations.
Decadendate 3p-C-DEPA (4) rapidly sequestered 90Y and 177Lu with
the respective radiolabeling efficiency of 89% and 94% at 1 min
time point. 3p-C-DE4TA (1) and 3p-C-DEPA (4) with the same
macroyclic cavity displayed similar complexation kinetics with
the metals.
90Y- or 177Lu-radiolabeld chelates were further evaluated for
in vitro serum stability (n = 2, Figs. 2 and 3 and Supporting infor-
mation). 90Y-3p-C-DE4TA (1) was found to be stable in serum for
at least for 2 weeks, while a small amount of 177Lu (ꢀ5%) was lost
from 177Lu-3p-C-DE4TA (1) over 2 weeks. A significant amount of
the radioactivity from both 90Y-3p-C-DEPA (4) and 177Lu-3p-C-
DEPA (4) was transchelated to serum (25% and 45% in 72 h, respec-
tively). As expected,10,11 both 90Y and 177Lu complexes of 3p-C-
NOTA (3) were not stable in serum, and ꢀ80% of 90Y and ꢀ36% of
177Lu was released from the complexes on day 7. The result
indicate that NOTA rapidly can form a kinetically labile complex,
that is, instantly dissociated in serum. 177Lu-3p-C-NE3TA (2)
remained quite stable in serum, and only ꢀ4% of the radioactivity
was transferred from the complex to serum. However, 90Y-3p-C-
NE3TA underwent rapid dissociation, losing ꢀ25% of 90Y to serum
in 2 days. It appears that heptadentate chelate 3p-C-NE3TA failed
to tightly hold the larger metal, 90Y due to an insufficient number
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H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 2. In vitro serum stability of 90Y-radiolabeled complexes (pH 7 and 37 °C).
Figure 3. In vitro serum stability of 177Lu-radiolabeled complexes (pH 7 and 37 °C).
of donors, while 3p-C-NE3TA can form a complex with the smaller
metal 177Lu with enhanced complex stability.
balanced with the nine donor groups. It seems that 3p-C-DE4TA
with the larger macrocyclic cavity have too many donors to hold
smaller Lu(III) with high complex stability. The effect of the
size-match was also demonstrated from evaluation of 3p-C-NE3TA
(2) and 3p-C-NOTA (3). 3p-C-NE3TA with the small cavity, was
completely ineffective in complexing the larger metal cation
Y(III) with high stability, while Lu(III) was quite tightly chelated
with 3p-C-NE3TA. It is demonstrated that 3p-C-NOTA (3) was inca-
pable of chelating the lanthanides effectively due to the poor size
match. It is noteworthy that replacement of the bidentate amino-
carboxylate group in the NOTA with a more flexible tridentate
group led to enhanced complex stability with 177Lu as shown in
complexation of the NE3TA. This result clearly demonstrate that
the pendant acyclic donors are essential in effective complexation
with the metals, and the improved complexation kinetics and sta-
bility of other bimodal chelates as compared to the NOTA, predom-
inantly resulted from cooperative and bimodal binding of acyclic
and macrocyclic donors. The comparative complexation kinetic
and stability data indicate that a well-coordinated interplay of
bimodality, cavity size, and ligand denticity is critical for the
dynamic and tight binding of the bimodal chelates with a metal.
Octadentate 3p-C-NETA with the smaller nine-membered ring
was shown to be the most effective chelate in binding the
lanthanides. The nanodentate chelate (3p-C-DE4TA) with the
The radiolabeling and serum stability data indicate that decad-
entate chelate 3p-C-DEPA (4) instantly bound to 90Y and 177Lu, but
failed to hold 90Y or 177Lu in serum. It seems that the DEPA built on
the larger macrocyclic cavity has too many donor groups to form a
stable complex with the relatively smaller metal cations Y(III) and
Lu(III), and this excessive ligand denticity may promote formation
and dissociation of the Lu(III)- or Y(III)-DEPA complex in equilib-
rium. It should be noted that DE4TA and DEPA are structured on
the same cyclen-based ring and display completely different
complex stability with Y(III) and Lu(III). The comparative data of
3p-C-DE4TA (1) and 3p-C-DEPA (4) suggest that denticity in the
chelates with the large macrocyclic cavity plays a critical role in
complexation. 3p-C-DE4TA with 9 donors were significantly more
effective than 3p-C-DEPA with 10 donors in complexing 90Y. No
substantial difference in complexation of 90Y between 3p-C-DE4TA
(1) and 3p-C-NETA (5), the standard gold chelate, was observed. It
appears that the nine donor groups on the large cyclen ring of the
nonadentate DE4TA were well tolerated to complex with 90Y with
high complexation kinetics and stability. The slightly enhanced
complex stability observed with 90Y-3p-C-DE4TA relative to
177Lu-3p-C-DE4TA may be explained by
between the cavity and the larger metal Y(III), that is, well
a better size-match
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H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
larger macrocyclic cavity formed a metal complex with the larger
metal 90Y with fast radiolabeling kinetics and high complex
stability. The heptadentate chelate (3p-C-NE3TA) with the smaller
nine-membered ring bound to the smaller metal 177Lu in a decent
level of complexation kinetics and stability.
were harvested, weighed, and the radioactivity was measured in
-counter (Fig. 4 and Supporting information). As expected,
a
c
3p-C-NETA used as a positive control displayed excellent in vivo
complex stability as demonstrated by the low radioactivity level
in the blood and organs at all time points studied. A very low radio-
activity level in the blood (<0.5% ID/g) was observed with the
177Lu-radiolabeled complexes at all of the time points studied.
Among the organs, the highest accretion of radioactivity was
observed in the liver at 1 h time point (6.7% ID/g for 177Lu-3p-C-
DE4TA, 1.4% ID/g for 177Lu-3p-C-NETA, and 17.2% ID/g for
177Lu-3p-C-NE3TA). 177Lu-3p-C-NE3TA exhibited a 3-fold higher
retention in the liver as compared to 177Lu-3p-C-DE4TA at 1 h
post-injection, although the NE3TA complex exhibited negligible
accumulation of radioactivity in the liver at 24 h. All of the
3.3. In vivo biodistribution
The in vivo stability of 177Lu-radiolabeled complexes of two
promising chelates 3p-C-DE4TA (1) and 3p-C-NE3TA (2) was eval-
uated by performing biodistribution studies in CF-1 normal mice
(iv injection, n = 3). 3p-C-NETA (5) as a positive control was also
evaluated for in vivo biodistribution for comparison. The mice
were euthanized at 1 h, 4 h, and 24 h. Selected organs and blood
Figure 4. Biodistribution of 177Lu-radiolabeled chelates in non-tumor bearing CF-1 Mice.
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10
H.-S. Chong et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
177Lu-radiolabeled complexes displayed low radioactivity
accumulation in bone and muscle, which peaked at 1 h (<0.5%
ID/g) and decreased by 24 h (<0.1% ID/g). The highest renal
retention at the 1 h time point (5.5% ID/g) was observed with
177Lu-3p-C-NE3TA, while 177Lu-3p-C-DE4TA displayed a very low
uptake in the kidney (<0.6% ID/g) at all of the time points studied.
the Department of Veterans Affairs, for providing resources and use
of facilities at the Harry S. Truman Memorial Veterans’ Hospital in
Columbia, MO.
Supplementary data
Supplementary data (ITLC chromatograms for assessment of
radiolabeling reaction kinetics and serum stability and in vivo
biodistribution data) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.12.035.
4. Conclusion
The new bifunctional chelates 3p-C-DE4TA (1), 3p-C-NE3TA (2),
and 3p-C-NOTA (3) were prepared and evaluated for complexation
with 90Y and 177Lu. The known bimodal chelates 3p-C-DEPA (4)
and 3p-C-NETA (5) with different coordination chemistry were
evaluated for comparison. 3p-C-DE4TA (1) was shown to rapidly
produce 90Y- or 177Lu-radiolabeled complexes at room tempera-
ture. The serum stability data indicate that 90Y and 177Lu-radiola-
beled complexes of 3p-C-DE4TA (1) displayed excellent to good
complex stability in serum. 3p-C-NE3TA (2) with a smaller macro-
cyclic cavity and lower denticity than 3p-C-DE4TA (1) was slower
in binding 90Y or 177Lu. 177Lu-3p-C-NE3TA (2) remained quite inert
in serum, while 90Y-3p-C-NE3TA (2) was gradually dissociated in
serum. The bifunctional version of NOTA (3p-C-NOTA) was shown
to be ineffective in binding 90Y and 177Lu. The results of the
comparative complexation kinetic and stability studies indicate
that bimodality, cavity size, and ligand denticity have an impact
on complexation of the bifunctional chelates with the radiolantha-
nides, and a well-coordinated interplay of the factors is critical for
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Acknowledgments
We acknowledge the financial support from the National
Institutes of Health (2R01CA112503 to H.S. Chong). We also thank
Please cite this article in press as: Chong, H.-S.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.035