Novel hexadentate and pentadentate chelators for 64Cu-based targeted PET imaging

Article abstract of DOI:10.1016/j.bmc.2014.02.041

A series of new hexadentate and pentadentate chelators were designed and synthesized as chelators of ⁶⁴Cu. The new pentadentate and hexadentate chelators contain different types of donor groups and are expected to form neutral complexes with Cu(II). The new chelators were evaluated for complex kinetics and stability with ⁶⁴Cu. The new chelators instantly bound to ⁶⁴Cu with high labeling efficiency and maximum specific activity. All ⁶⁴Cu-radiolabeled complexes in human serum remained intact for 2 days. The ⁶⁴Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Among the ⁶⁴Cu- radiolabeled complexes evaluated, ⁶⁴Cu-complex of the new chelator E was well tolerated with a minimal transfer of ⁶⁴Cu to EDTA. ⁶⁴Cu-radiolabeled complex of the new chelator E was further evaluated for biodistribution studies using mice and displayed rapid blood clearance and low organ uptake. ⁶⁴Cu-chelator E produced a favorable in vitro and in vivo complex stability profiles comparable to ⁶⁴Cu complex of the known hexadentate NOTA chelator. The in vitro and in vivo data highlight strong potential of the new chelator E for targeted PET imaging application.

Full text of DOI:10.1016/j.bmc.2014.02.041

Bioorganic & Medicinal Chemistry 22 (2014) 2553–2562
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel hexadentate and pentadentate chelators for 64Cu-based
targeted PET imaging
Inseok Sin a, Chi Soo Kang a, Nilantha Bandara b, Xiang Sun a, Yongliang Zhong a, Buck E. Rogers b,c
,
Hyun-Soon Chong a,
⇑
a Chemistry Division, Department of Biological and Chemical Sciences, Illinois Institute of Technology, 3101 S. Dearborn St., Chicago, IL 60616, United States
b Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, United States
c Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63110, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new hexadentate and pentadentate chelators were designed and synthesized as chelators of
64Cu. The new pentadentate and hexadentate chelators contain different types of donor groups and are
expected to form neutral complexes with Cu(II). The new chelators were evaluated for complex kinetics
and stability with 64Cu. The new chelators instantly bound to 64Cu with high labeling efficiency and max-
imum specific activity. All 64Cu-radiolabeled complexes in human serum remained intact for 2 days. The
64Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Among the
64Cu-radiolabeled complexes evaluated, 64Cu-complex of the new chelator E was well tolerated with a
minimal transfer of 64Cu to EDTA. 64Cu-radiolabeled complex of the new chelator E was further evaluated
for biodistribution studies using mice and displayed rapid blood clearance and low organ uptake.
64Cu-chelator E produced a favorable in vitro and in vivo complex stability profiles comparable to 64Cu
complex of the known hexadentate NOTA chelator. The in vitro and in vivo data highlight strong potential
of the new chelator E for targeted PET imaging application.
Received 21 November 2013
Revised 10 February 2014
Accepted 19 February 2014
Available online 1 March 2014
Keywords:
64Cu
Chelating agents
PET imaging
NOTA
Ó 2014 Published by Elsevier Ltd.
1. Introduction
acyclic DTPA is known to rapidly complex with Cu(II), 64Cu-DTPA
was reported be kinetically labile and dissociated in serum.2 The
64Cu (t1/2 = 12.7 h, Ebþ ¼656 keV;Ebꢀ ¼573 keV;Ecmax ¼511 keV)
macrocyclic chelators, DOTA (logK = 22.3) and TETA (logK = 21.7)
form fairly stable complexes with Cu(II) using the four nitrogens in
the macrocyclic ring and two pendant carboxylate oxygens.3 In par-
ticular, TETA conjugated to an antibody was found to display high
binding selectivity to Cu(II) in the presence of other metals with an
oxidation state of +2 including Zn(II), Mg(II), and Ca(II).4 The 64Cu
complexes of DOTA and TETA remained intact with a minimal loss
of 64Cu in serum.2 However, in vivo biodistribution data suggested
that 64Cu-DOTA and 64Cu-TETA analogues conjugated to a biomole-
cule including octreotide were dissociated in mice, and high liver
uptake was observed with the 64Cu-radiolabeled complexes.5–7
64Cu complexes of cross-bridged cyclam (CB-TE2A) analogues were
shown to possess improved kinetic in vivo stability compared to
64Cu-DOTA and 64Cu-TETA complexes.7,8 However, radiolabeling of
CB-TE2A with 64Cu required a complicated and harsh condition
due to slow complexation kinetics of the chelator with 64Cu.8 NOTA
max
max
is one of the most useful radioisotopes for positron emission
tomography (PET) imaging.1,2 Various 64Cu-radiolabeled antibody
or peptide conjugates have been employed for PET imaging of can-
cers in preclinical and clinical settings.1,2 A bifunctional chelator
that can rapidly complex Cu(II) with high kinetic inertness and
thermodynamic stability is a critical component of clinically viable
64Cu-based radiopharmaceuticals for targeted PET imaging. Many
antibodies and proteins are not tolerant of heating and radiolysis,
and effective radiolabeling of a bifunctional chelator with a
short-lived 64Cu under mild condition is required to minimize
radiolytic damage of an antibody conjugated with the chelator.
Bifunctional DTPA (diethylene triamine pentaacetic acid), TETA
(1,4,8,11-tetraazacyclotetradecane-N,N0,N00,N000-tetraacetic acid),
DOTA(1,4,7,10-tetraazacyclododecane-N,N0,N00,N000-tetraaceticacid),
and CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-
diacetic acid) have been investigated as chelators of 64Cu.2 Although
(1,4,7-triazacyclononane-1,4,7-triacetic acid) is
a hexadentate
macrocyclic chelator containing aminocarboxylate donors and
binds to Cu(II) with high thermodynamic stability (log K = 21.6).3
64Cu-radiolabeled NOTA complexes have produced encouraging
⇑
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.2014.02.041
0968-0896/Ó 2014 Published by Elsevier Ltd.

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I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
in vitro and in vivo profiles.6,9,10 We previously reported that 64Cu-
NOTA complex remained stable in both serum and mice and dis-
played fast blood clearance and low uptake in normal organs.10
In our continued effort on improved chelation chemistry for
development of radiopharmaceuticals,10–14 we synthesized and
evaluated new hexadentate and pentadentate NOTA analogues
(Fig. 1) as potential chelators of 64Cu. The new chelators were
evaluated for radiolabeling with 64Cu to determine the maximum
specific activity and radiolabeling kinetics with 64Cu under mild
condition. 64Cu-radiolabeled complexes of the promising new
chelator were evaluated for complex stability in human serum
and EDTA (ethylenediaminetetraacetic acid) challenge and
in vivo biodistribution using mice.
(d, J = 8.5 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d 28.2 (q), 31.8 (t),
32.7 (t), 51.2 (q), 53.5 (t), 55.5 (t), 56.0 (t), 59.4 (t), 66.4 (d), 80.8
(s), 123.7 (d), 129.4 (d), 146.4 (s), 149.7 (s), 171.4 (s), 173.7 (s).
HRMS (positive ion ESI) Calcd for
C
29H47N4O8 [M+H]+ m/z
521.3388. Found: [M+H]+ m/z 521.3391.
2.3. 2-[4,7-Bis(carboxymethyl)-1,4,7-triazanonan-1-yl]-4-(4-
nitrophenyl)butanoic acid (A)16
Compound 3 (20 mg, 0.032 mmol) was treated with 6 M HCl
solution (2 mL), and the resulting solution was refluxed for 3 h.
The reaction mixture was cooled to room temperature, and the
resulting solution was filtered, and the filtrate was concentrated
in vacuo to provide chelator A (16 mg, 89%) as a yellow solid. 1H
NMR (D2O, 300 MHz) d 1.90–1.92 (m, 1H), 2.06–2.10 (m, 1H),
2.59–2.84 (m, 2H), 2.95–3.18 (m, 12H), 3.49–3.53 (m, 1H), 3.79
(s, 4H), 7.30 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H); 13C NMR
(D2O, 75 MHz) d 29.7 (t), 32.1 (t), 45.7 (t), 49.2 (t), 50.6 (t), 55.3
(t), 63.7 (d), 123.7 (d), 129.4 (d), 146.4 (s), 149.7 (s), 172.2 (s),
175.6 (s). The data of 1H and 13C NMR data were essentially iden-
tical to those previously reported.16 Analytical HPLC (tR = 7.6 min,
method 1).
2. Materials and methods
2.1. Instruments and reagents
1H, 13C, and DEPT NMR spectra were obtained using a Bruker
300 NMR instrument, and chemical shifts are reported in ppm on
the d scale relative to TMS. Electro spray ionization (ESI) high res-
olution mass spectra (HRMS) were obtained on JEOL double sector
JMS-AX505HA mass spectrometer (University of Notre Dame, IN).
64Cu was prepared on CS-15 cyclotron at Washington University
Medical School, St. Louis, MO according to the previous reported
method.10 Radioactivity was counted with a Beckman Gamma
8000 counter containing a NaI crystal (Beckman Instruments,
Inc., Irvine, CA). Analytical and semi-prep HPLC were performed
on Agilent 1200 (Agilent, Santa Clara, CA) equipped with a diode
array detector (k = 254 and 280 nm), thermostat set at 35 °C and
a Zorbax Eclipse XDB-C18 column (4.6 ꢁ 150 mm, 80 Å, Agilent,
Santa Clara, CA). The mobile phase of a binary gradient (0–100%
B/15 min; solvent A = 0.1% TFA in water; solvent B = 0.1% TFA in
acetonitrile for method 1). The mobile phase of a binary gradient
(0-60% B/40 min, solvent A: 0.1% TFA in H2O, solvent B: 0.1% TFA
in CH3CN, flow rate: 3 mL/min) was used for semi-prep HPLC
(method 2).
2.4. tert-Butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-[3-(4-
nitrophenyl)propyl]-1,4,7-triazanonan-1-yl}acetate (5)
To a solution of 112 (72 mg, 0.29 mmol) in CH3CN (2 mL) at 0 °C
was added dropwise 411 (105 mg, 0.29 mmol) in CH3CN (1 mL) and
DIPEA (112 mg, 0.87 mmol). The resulting mixture was stirred for
61 h at room temperature. The resulting mixture was concentrated
to dryness in vacuo. Water (10 mL) and 0.1 M HCl aqueous solution
(1 mL) were added to the residue, and the resulting mixture was
extracted with CHCl3 (3 ꢁ 10 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated to the dryness
in vacuo. The residue was purified via column chromatography
on silica gel (60–220 mesh) eluting with 20% MeOH in CH2Cl2 to
afford 5 (81 mg, 54%) as an oil. 1H NMR (CDCl3, 300 MHz) d 1.41
(s, 18H), 2.21 (m, 2H), 2.65–2.90 (m, 8H), 3.01–3.18 (m, 5H),
3.37–3.70 (m, 8H), 7.41 (d, J = 6.0 Hz, 2H), 8.12 (d, J = 6.0 Hz, 2H);
13C NMR (CDCl3, 75 MHz) d 25.9 (t), 28.1 (q), 32.8 (t), 49.8 (t),
52.3 (t), 53.5 (t), 55.3 (t), 58.2 (t), 81.7 (s), 123.9 (d), 129.4 (d),
146.7 (s), 147.9 (s), 170.7 (s). HRMS (positive ion ESI) Calcd for
2.2. tert-Butyl 2-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-
triazanonan-1-yl}-4-(4-nitrophenyl)-butanoate (3)
To a solution of 112 (272 mg, 0.761 mmol) in CH3CN (1 mL) was
added portionwise compound 215 (230 mg, 0.761 mmol) and DI-
PEA (295 mg, 2.284 mmol) in CH3CN (1 mL). The resulting mixture
was stirred for 7 days at room temperature. The reaction mixture
was concentrated to dryness in vacuo. 0.1 M HCl aqueous solution
(20 mL) was added to the residue, and the resulting mixture was
extracted with CH2Cl2 (2 ꢁ 20 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated to the dryness
in vacuo. The residue was purified via column chromatography
on silica gel (220–440 mesh) eluting with 15% CH3OH in CH2Cl2
C
27H45N4O6 [M+H]+ m/z 521.3334. Found: [M+H]+ m/z 521.3309.
2.5. 2-[4-(Carboxymethyl)-7-[3-(4-nitrophenyl)propyl]-1,4,7-
triazanonan-1-yl]acetic acid (B)
Compound 5 (20 mg, 0.038 mmol) at 0–5 °C was treated drop-
wise with 4 M HCl (g) in 1,4-dioxane (2 mL) over 10 min. The
resulting mixture was gradually warmed to room temperature
and stirred for 24 h. Diethyl ether (20 mL) was added to the reac-
tion mixture which was stirred for 10 min. The resulting mixture
was capped and placed in the freezer for 1 h. The solid formed
was filtered, washed with ether, and quickly dissolved in deionized
to afford pure product
3
(240 mg, 51%). 1H NMR (CDCl3,
300 MHz) d 1.42 (s, 18H), 1.96–1.99 (m, 2H), 2.67–2.98 (m, 15H),
3.23–3.29 (m, 5H), 3.65 (s, 3H), 7.36 (d, J = 8.5 Hz, 2H), 8.12
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
OH
CO2H
CO2H
N
N
N
N
CO2H
N
CO2H
CO2H
N
CO2H
N
N
N
N
N
N
N
N
O
N
N
N
N
D
A
C
B
N
S
E
F
HO2C
NO2
NO2
NO2
NO2
Figure 1. Potential chelators for PET imaging using 64Cu.

I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
2555
water. The resulting aqueous solution was concentrated in vacuo
to provide chelator B (14 mg, 78%). 1H NMR (D2O, 300 MHz) d
2.02–2.17 (m, 2H), 2.76 (t, J = 7.2 Hz, 2H), 3.18–3.60 (m, 14H),
3.79 (s, 4H), 7.37 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.7 Hz, 2H); 13C
NMR (D2O, 75 MHz) d 24.9 (t), 31.7 (t), 49.6 (t), 50.7 (t), 51.0 (t),
56.9 (t), 57.3 (t), 123.9 (d), 129.5 (d), 146.4 (s), 148.7 (s), 172.9
(s). HRMS (positive ion ESI) Calcd for C19H29N4O6 [M+H]+ m/z
409.2082. Found: [M+H]+ m/z 409.2087. Analytical HPLC (tR =
7.3 min, method 1).
DIPEA (163 mg, 1.259 mmol) in CH3CN (2 mL) was added portion-
wise, and the resulting mixture was allowed to room temperature
and stirred for 24 h. The reaction mixture was concentrated to
dryness in vacuo. The residue was treated with deionized water
(10 mL) and extracted with CHCl3 (2 ꢁ 10 mL). The combined
organic layer was concentrated in vacuo. Then the resulting mix-
ture was dissolved with 0.1 M HCl solution (10 mL) and washed
with CHCl3 (2 ꢁ 10 mL). The aqueous layer was neutralized using
0.1 M NaOH (10 mL) and extracted with CHCl3 (3 ꢁ 20 mL). The
combined organic layers were dried over MgSO4, filtered, and con-
centrated to the dryness in vacuo. The residue was purified via col-
umn chromatography on silica gel (60–220 mesh) eluting with 30%
CH3OH in dichloromethane containing Et3N to provide pure 9
(96.8 mg, 42%). 1H NMR (CDCl3, 300 MHz) d 1.38 (s, 18H), 2.89–
3.57 (m, 20H), 4.47 (s, 2H), 7.31 (d, 2H, J = 7.5 Hz), 8.05 (d, 2H,
J = 7.5 Hz). 13C NMR (CDCl3, 75 MHz) d 28.1 (q), 28.8 (t), 40.8 (t),
48.4 (t), 50.3 (t), 51.5 (t), 55.8 (t), 62.9 (t), 82.0 (s), 123.7 (d),
129.3 (d), 146.5 (s), 148.4 (s), 169.4 (s), 203.4 (s). HRMS (positive
ion ESI) Calcd for C28H44N4O7 [M+H]+ m/z 549.6716. Found:
[M+H]+ m/z 549.3403.
2.6. Ethyl 2-[(4-nitrophenyl)methyl]-3-oxobutanoate (6)17
Ethyl acetoacetate (10 g, 76.84 mmol) was added dropwise to
NaH (1.84 g, 76.84 mmol) in the THF (220 ml). p-Nitro benzyl bro-
mide (16.6 g, 76.84 mmol) was added portionwise over 1 h. The
reaction mixture was stirred for 1.5 h. After evaporation of the sol-
vent, the residue was treated with H2O (100 mL) and extracted
with ethyl acetate (2 ꢁ 100 mL). The combined organic layers were
dried over MgSO4, filtered, and concentrated to the dryness in
vacuo. The residue was recrystallized with ethanol to provide pure
product 6 (10 g, 49%). 1H NMR (CDCl3, 300 MHz) d 1.21 (t, 3H,
J = 7.2 Hz), d 2.23 (s, 3H), d 3.23–3.27 (m, 2H), d 3.79 (t, 3H,
J = 7.5 Hz), d 4.15–4.17 (m, 2H), d 7.36 (d, 2H, J = 8.4 Hz), d 8.13
(d, 2H, J = 8.4 Hz). 13C NMR (CDCl3, 75 MHz) d 14.0 (q), 29.5 (q),
33.4 (t), 60.6 (d), 61.9 (d), 123.8 (d), 129.8 (d), 146.1 (s), 146.9
(s), 168.5 (s), 201.2 (s).
2.10. tert-Butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-[2-hydroxy-
4-(4-nitrophenyl)butyl]-1,4,7-triazanonan-1-yl}acetate (10)
A solution of 9 (33 mg, 0.060 mmol) in anhydrous methanol
(1 mL) at 0 °C was added portionwise NaBH4 (10 mg, 0.264 mmol)
over 1 h. The mixture was then warmed to room temperature and
stirred for 3 h. The reaction mixture was concentrated to dryness
and treated with H2O (10 mL) and extracted with ethyl acetate
(2 ꢁ 15 mL). The combined organic layers were dried over MgSO4,
filtered, and the filtrated was concentrated in vacuo to provide
pure 10 (26.5 mg, 81%). 1H NMR (CDCl3, 300 MHz) d 1.43 (s,
18H), 1.59–1.70 (m, 2H), 2.36 (m, 1H), 2.69–2.83 (m, 15H), 3.29
(s, 4H), 3.51–3.69 (m, 1H), 7.35 (d, 2H, J = 8.4 Hz), 8.11 (d, 2H,
J = 8.4 Hz). 13C NMR (CDCl3, 75 MHz) d 28.2 (q), 32.2 (t), 35.8 (t),
55.8 (t), 56.0 (t), 56.4 (t), 58.9 (t), 63.8 (t), 68.0 (d), 80.8 (s), 123.6
(d), 129.3 (d), 146.3 (s), 150.6 (s), 171.5 (s). HRMS (positive ion
ESI) Calcd for C28H47N4O7 [M+H]+ m/z 551.3439. Found: [M+H]+
m/z 551.3463.
2.7. 4-(4-Nitrophenyl)butan-2-one (7)18
Compound 6 (10 g, 37.7 mmol) was dissolved in the mixture of
acetic acid (85 mL) and concd HCl solution (30 mL), and the result-
ing solution was refluxed for 24 h after which the reaction mixture
was allowed to room temperature and concentrated to dryness in
vacuo. The residue was treated with H2O (100 ml) and extracted
with ethyl acetate (2 ꢁ 100 mL). The combined organic layers were
dried over MgSO4, filtered, and concentrated in vacuo to provide
pure 7 (7.3 g, 100%). 1H NMR (CDCl3, 300 MHz) d 2.09 (s, 3H),
2.77 (t, 2H, J = 9 Hz), 2.92 (t, 2H, J = 9 Hz), 7.29 (d, 2H, J = 9 Hz),
8.01 (d, 2H, J = 9 Hz); 13C NMR (CDCl3, 75 MHz) d 29.3 (t), 30.1
(q), 44.2 (t), 123.7 (d), 129.3 (d), 146.5 (s), 149.0 (s), 206.8 (s).
The data of 1H and 13C NMR data were essentially identical to those
of 7 as previously reported.16
2.11. 2-[4-(Carboxymethyl)-7-[4-(4-nitrophenyl)-2-oxobutyl]-
1,4,7-triazanonan-1-yl]acetic acid (C)
2.8. 1-Bromo-4-(4-nitrophenyl)butan-2-one (8)
TFA (800 lL) was added dropwise to compound 9 (22.6 mg
0.041 mmol) at 0 °C, and the resulting solution was stirred for
2 h at 0 °C. The resulting mixture was allowed to room tempera-
ture and stirred for additional 1 h. The reaction mixture was con-
centrated to dryness in vacuo and treated with ether (2 mL), and
the ether layer was decanted. The residue was dissolved in H2O
(2 mL) and washed by CHCl3 (2 ꢁ 5 mL). The aqueous layer was
concentrated to dryness in vacuo to provide pure product C
(16.4 mg, 91%). 1H NMR (D2O, 300 MHz) d 2.85 (m, 4H), 3.20–
3.45 (m, 12H), 3.82 (s, 4H), 4.20 (s, 2H), 7.30 (d, 2H, J = 9.0 Hz),
8.00 (d, 2H, J = 9.0 Hz); 13C NMR (D2O, 75 MHz) d 28.3 (t), 39.8
(t), 50.0 (t), 50.4 (t), 51.3 (t), 56.3 (t), 64.2 (t), 123.6 (d), 129.3
(d), 146.0 (s), 148.8 (s), 171.8 (s), 205.8 (s). HRMS (positive ion
ESI) Calcd for C20H29N4O7 [M+H]+ m/z 437.2031. Found: [M+H]+
m/z 437.2041. Analytical HPLC (tR = 7.8 min, method 1).
To a solution of 7 (5.0 g, 25.88 mmol) in anhydrous methanol
(10 mL) was added dropwise bromine (4.14 mg, 25.88 mmol) in
anhydrous methanol (10 mL) at 7–9 °C over 1 h. The mixture was
stirred for 34 h at the same temperature. DI water (30 mL) was
added to the mixture which was warmed to room temperature
and continuously stirred for 12 h. The reaction mixture was ex-
tracted with CH2Cl2 (2 ꢁ 30 mL). The combined organic layers were
dried over MgSO4, filtered, and the filtrate was concentrated in va-
cuo. The residue was purified via column chromatography on silica
gel (60–220 mesh) and eluted with 10% ethyl acetate in hexanes to
afford 8 (1.26 g, 18%) as a solid. 1H NMR (CDCl3, 300 MHz) d 3.05 (s,
4H), 3.87 (s, 2H), 7.35 (d, 2H, J = 7.8 Hz), 8.12 (d, 2H, J = 8.1 Hz); 13
C
NMR (CDCl3, 75 MHz) d 29.4 (t), 33.9 (t), 40.4 (t), 123.8 (d), 129.3 (d),
146.6 (s), 148.2 (s), 200.4 (s). HRMS (positive ion ESI) Calcd for
C
10H11BrNO3 [M+H]+ m/z 271.9917. Found: [M+H]+ m/z 271.9924.
2.12. 2-[4-(Carboxymethyl)-7-[2-hydroxy-4-(4-
nitrophenyl)butyl]-1,4,7-triazanonan-1-yl]acetic acid (D)
2.9. tert-Butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-[4-(4-
nitrophenyl)-2-oxobutyl]-1,4,7-triazanonan-1-yl}acetate (9)
TFA (1 mL) was added dropwise to compound 10 (24 mg
0.045 mmol) at 0 °C, the resulting mixture was stirred for 3 h at
0 °C. The resulting mixture was allowed to room temperature
and stirred for additional 1 h. The reaction mixture was
Compound 8 (114 mg, 0.420 mmol) was added portionwise to a
solution of 112 (150 mg, 0.420 mmol) in CH3CN (5 mL) at 0 °C.

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I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
concentrated to dryness and treated with ether (3 mL), and the
ether layer was decanted. The residue was dissolved in H2O
(2 mL) and washed by CHCl3 (2 ꢁ 5 mL). The aqueous layer was
concentrated to dryness in vacuo, and the residue was purified
by semi-prep HPLC (method 3, tR = ꢂ21 min) to provide pure prod-
uct D (7.2 mg, 37%). 1H NMR (D2O, 300 MHz) d 1.69 (m, 2H), 2.66–
2.78 (m, 2H), 3.01–3.49 (m, 18H), 3.86 (m, 1H), 7.31 (d, 2H,
J = 8.1 Hz), 8.01 (d, 2H, J = 8.4 Hz). 13C NMR (D2O, 75 MHz) d 30.7
(t), 35.1 (t), 49.8 (t), 50.5 (t), 58.5 (t), 62.3 (t), 65.5 (d), 123.7 (d),
129.4 (d), 145.9 (s), 150.0 (s), 175.5 (s). HRMS (positive ion ESI)
Calcd for C10H31N4O7 [M+H]+ m/z 439.2187. Found: [M+H]+ m/z
439.2203. Analytical HPLC (tR = 8.0 min, method 1).
0.1 M HCl solution (10 mL) and extracted with CHCl3 (3 ꢁ 10 mL).
The combined organic layers were dried over MgSO4, filtered,
and the filtrate was concentrated to the dryness in vacuo. The res-
idue was purified via column chromatography on silica gel (60–
220 mesh) eluting with 15% CH3OH in CH2Cl2 to afford pure 12
(46 mg, 73%). 1H NMR (CDCl3, 300 MHz) d 1.45 (s, 18H), 2.82–
2.96 (m, 12H), 3.31 (s, 4H), 3.87 (s, 2H), 6.89–6.93 (m, 2H), 7.19
(dd, J = 1.2 Hz, J = 4.8 Hz, 1H); 13C NMR (CDCl3, 75 MHz) 28.1 (q),
48.3 (t), 50.4 (t), 51.0 (t), 54.2 (t), 56.7 (t), 82.5 (s), 127.8 (d),
128.6 (d), 131. 5 (d), 132.7 (s), 170.1 (s). HRMS (positive ion ESI)
Calcd for C23H40N3O4S [M+H]+ m/z 454.2734. Found: [M+H]+ m/z
454.2723.
2.13. tert-Butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-(pyridin-2-
ylmethyl)-1,4,7-triazanonan-1-yl}acetate (11)
2.16. 2-[4-(Carboxymethyl)-7-(thiophen-2-ylmethyl)-1,4,7-
triazanonan-1-yl]acetic acid (F)
To a solution of 112 (50 mg, 0.140 mmol) in 1,2-dichloroethane
(1 mL) was added 2-pyridinecarboxaldehyde (15 mg, 0.140 mmol).
The resulting solution was stirred for 10 min and then added with
sodium triacetoxyborohydride (44.5 mg, 0.210 mmol) portionwise
over 10 min. The mixture was stirred at room temperature for
overnight. The reaction mixture was quenched by adding saturated
NaHCO3 (15 mL), and the resulting solution was extracted with
ethyl acetate (3 ꢁ 15 mL). The combined organic layers were con-
centrated to dryness in vacuo. The residue was dissolved in 0.1 M
HCl solution (10 mL) and washed with CHCl3 (2 ꢁ 10 mL). The
aqueous layer was treated with saturated NaHCO3 (10 mL) and ex-
tracted with ethyl acetate (3 ꢁ 20 mL). The combined organic lay-
ers were dried over MgSO4, filtered, and concentrated in vacuo to
provide pure 11 (60 mg, 96%) as a yellowish oil. 1H NMR (CDCl3,
300 MHz) d 1.44 (s, 18H), 2.75–2.90 (m, 12H), 3.31–3.36 (m, 4H),
3.87 (s, 2H), 7.13 (t, J = 5.4 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.61–
7.67 (m, 1H), 8.50 (d, J = 3.9 Hz, 1H); 13C NMR (CDCl3, 75 MHz) d
28.2 (q), 55.3 (t), 55.6 (t, 2C), 59.8 (t), 64.1 (t), 80.6 (s), 121.7 (d),
123.1 (d), 136.2 (d), 148.8 (d), 160.6 (s), 171.5 (s). HRMS (positive
ion ESI) Calcd for C24H41N4O4 [M+H]+ m/z 449.3122. Found: [M+H]+
m/z 449.3118.
Compound 12 (17 mg, 0.037 mmol) at 0–5 °C was treaeted
dropwise with 4 M HCl (g) in 1,4-dioxane (2.5 mL) over 10 min.
The resulting mixture was warmed to room temperature and stir-
red for 24 h. Diethyl ether (40 mL) was added to the mixture which
was continuously stirred for 10 min. The solid formed was filtered,
washed with ether, and quickly dissolved in DI water. The aqueous
solution was concentrated in vacuo to provide chelator F (13.5 mg,
80%) as a yellow solid. 1H NMR (D2O, 300 MHz) d 2.90–3.61 (m,
18H), 6.91–7.08 (m, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.49 (d,
J = 4.8 Hz, 1H). 13C NMR (D2O, 75 MHz) d 49.0 (t), 50.2 (t), 50.3
(t), 54.3 (t), 56.9 (t), 128.0 (d), 129.8 (d), 130.1 (s), 132.7 (d),
173.95 (s). HRMS (positive ion ESI) Calcd for C15H24N3O4S [M+H]+
m/z 342.1482. Found: [M+H]+ m/z 342.1469. Analytical HPLC
(tR = 5.8 min, method 1).
2.17. Preparation and characterization of Cu(II) complexes
Cu(II)-complexes of the chelators A–F were prepared by reaction
of each chelator (5 lL, 10 mM) with CuCl2 (5 lL, 10 mM) in 0.25 M
NH4OAc buffer (pH 5.5) for 24 h at room temperature and 300 rpm.
Each of Cu(II)-complex was purified by semi-prep HPLC (solvent A:
0.1% TFA in H2O, solvent B: 0.1% TFA in CH3CN, 0–60% B/40 min,
flow rate: 3 mL/min). The purified Cu(II)-complex was character-
ized by analytical HPLC (method 1, Supporting information).
2.14. 2-[4-(Carboxymethyl)-7-(pyridin-2-ylmethyl)-1,4,7-
triazanonan-1-yl]acetic acid (E)
Compound 11 (16 mg, 0.036 mmol) was treated with 6 M HCl
solution (3 mL), and the resulting solution was refluxed for 5 h.
The reaction mixture was gradually cooled to room temperature,
filtered, and concentrated to dryness in vacuo to provide chelator
E (15 mg, 86%) as a yellow solid. 1H NMR (D2O, 300 MHz) d 2.59–
2.80 (m, 4H), 3.01 (m, 4H), 3.08 (s, 4H), 3.72 (s, 4H), 4.19 (s, 2H),
7.84 (t, J = 7.2 Hz, 2H), 7.98 (d, J = 7.8 Hz, 1H), 8.39 (dt, J = 7.5 Hz,
J = 1.8 Hz 1H), 8.51 (d, J = 5.4 Hz, 1H). 13C NMR (D2O, 75 MHz) d
47.5 (t), 48.9 (t), 50.5 (t), 55.4 (t), 56.4 (t), 126.4 (d), 127.9 (d),
141.4 (d), 147.3 (d), 153.0 (s), 173.1 (s). HRMS (positive ion ESI)
Calcd for C16H25N4O4 [M+H]+ m/z 337.1870. Found: [M+H]+ m/z
337.1863. Analytical HPLC (tR = 4.2 min, method 1).
2.18. Determination of maximum specific activity (MSA)
Whatman C18 silica gel TLC plates (KC18F, 60 Å) were pur-
chased from Fisher Scientific (Pittsburgh, PA). Radio-TLCs were
developed with 10% NH4OAc/MeOH (3:7) and analyzed using a
Bioscan 200 imaging scanner (Bioscan, Inc., Washington, DC).
64CuCl2 was diluted with a 10-fold excess of 0.1 M NH4OAc (pH
5.5) for radiolabeling. The maximum specific activities were deter-
mined experimentally via titrating 64CuCl2 in 0.1 M NH4OAc (pH
5.5) with the chelators. Briefly, for each chelator, six reaction vials
were prepared in 0.1 M NH4OAc (pH 5.5) via dilution to give final
chelator masses in the range 0.001–0.1 lg. 3.7 MBq (100 lCi) of
64Cu in 0.1 M NH4OAc (pH ꢂ5.5) was added to each vial and ad-
2.15. tert-Butyl 2-{4-[2-(tert-butoxy)-2-oxoethyl]-7-(thiophen-
justed the final volume to 100 lL (final pH 5.5) and vortexed for
2-ylmethyl)-1,4,7-triazanonan-1-yl}acetate (12)
10–15 s. The reactions were incubated on a rotator at 37 °C for
1 h. After incubation, 1 L aliquots were withdrawn from reaction
l
To a solution of 112 (50.0 mg, 0.140 mmol) in 1,2-dichloroeth-
ane (1 mL) was added 2-thiophenecarboxaldehyde (15.7 mg,
0.140 mmol). The resulting solution was stirred for 10 min and
treated with sodium triacetoxyborohydride (44.5 mg, 0.210 mmol)
portionwise over 10 min. The reaction mixture was stirred at room
temperature for 3 d. The reaction mixture was quenched by adding
saturated NaHCO3 (15 mL), and the resulting solution was
extracted with ethyl acetate (3 ꢁ 15 mL). The combined organic
layer was concentrated in vacuo. The residue was treated with
vials and analyzed by TLC (C-18) with a mixture of 10% NH4OAc/
MeOH (3:7) as a mobile phase for labeling percentage. All reactions
were done in triplicate. The data were plotted as % labeling versus
amount of chelator used in the reaction and fitted using sigmoidal
dose response equation in GraphPad Prism (La Jolla, CA). Based on
this, the amount of mass required to achieve 50% labeling was
determined, and this mass was multiplied by two to obtain the
minimal mass for 100% labeling to determine the maximum spe-
cific activity.

I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
2557
2.19. Complexation formation kinetics of chelators with 64Cu
evaluated by ITLC as described above. Stability of the complexes
was also evaluated at 25 h time point by HPLC (method 1, solvent
A: 0.1% TFA in H2O, solvent B: 0.1% TFA in CH3CN, 0–100%
B/15 min, flow rate: 1 mL/min). 64Cu-EDTA complex was eluted
early (tR = ꢂ2.5 min), while 64Cu-radiolabeled complexes of
chelators A–D, E, and F have the respective retention time
(tR = 7–8 min, tR = 4.8 min, and tR = 6.2 min).
All HCl solutions were prepared from ultra-pure HCl (Fisher Sci-
entific, #A466-500). For metal-free radiolabeling, plasticware
including pipette tips, tubes, and caps was soaked in 0.1 M HCl
(aq) overnight and washed thoroughly with Milli-Q (18 M O)
water, and air-dried overnight. 0.25 M NH4OAc buffer solution
(pH 5.5) was prepared using ultra-pure ammonium acetate and
pH of the solution was adjusted using 0.1 and 1 M HCl solution.
The resulting buffer solution was treated with Chelex-100 resin
(Biorad, #142-2842), shaken overnight at room temperature, and
2.22. In vivo biodistribution
All animal studies were performed in compliance with the
Guidelines for Care and Use of Research Animals established by
Washington University’s Animal Studies Committee. Biodistribu-
tion studies were carried out in female CD-1 mice (Charles River
Labs). The tissue uptakes of 64Cu-chelator A and 64Cu-chelator E
were evaluated in mice (n = 4) that were injected via the tail vain
filtered through 0.22 lm filter (Corning, #430320) prior to use.
TLC plates (6.6 ꢁ 1 cm or 6.6 ꢁ 2 cm, Silica gel 60 F254, EMD Chem-
icals Inc., #5554-7) with the origin line drawn at 0.6 cm from the
bottom were prepared. To a buffer solution (11–18 lL, 0.25 M
NH4OAc, pH 5.5) in a capped microcentrifuge tube (1.5 mL, Fisher
Scientific, #05-408-129) was sequentially added a solution of the
with 0.74 MBq (20 lCi) per animal (100 lL). At 1, 4, and 24 h
chelator (6.7
(20 Ci, 1–3
20 L, and the concentration of each chelator in the reaction mix-
l
g, 1–7
l
L) in the buffer and 64Cu in 0.05 M HCl
post-injection, mice were anesthetized with 1–2% isoflurane and
sacrificed by cervical dislocation. Subsequently, the tissues of
interest were harvested, weighed, and measured in a gamma coun-
ter. Samples were corrected for radioactive decay to calculate per-
cent injected-dose per gram (%ID/g) of tissue.
l
lL). The total volume of the resulting solution was
l
ture was 0.6 mM. The reaction mixture was agitated on the ther-
momixerset at 1000 rpm at room temperature for 30 min. The
labeling efficiency was determined by ITLC eluted with 20 mM
EDTA in 0.15 M NH4OAc as the mobile phase. A solution of radiola-
3. Results and discussion
beled complexes (2.0 lL) was withdrawn at the designated time
3.1. Design of new chelators
points (1, 10, and 30 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 hot plate maintained
at 35 °C and scanned using TLC scanner (Bioscan). Bound and
unbound radioisotope appeared 30–35 mm (Rf = 0.6) and
50–55 mm (Rf = 1.0) from the bottom of the TLC plate, respectively.
The new chelators (Fig. 1) were designed based on various fac-
tors including donor number (denticity), donor type, charge of the
complex, compatibility between metal ion and donor group that
can influence coordination chemistry of a metal. Cu(II) has a rela-
tively small ionic radius of 73 ppm for coordination number six
and is known to display a high affinity for nitrogen, oxygen, and
sulfur donors.1 A bifunctional version of the known parent NOTA
chelator (A)16 was prepared and evaluated for comparison to the
new NOTA analogues. A new bifunctional chelator B contains five
donor groups and the p-NO2–Bn group that is linked to the macro-
cyclic backbone by a relatively long propyl chain. Hexadentate che-
lators C and D contain a carbonyl donor group and a hydroxyl
group in addition to the donor groups attached to the macrocyclic
backbone, respectively. It would be interesting to evaluate if
b-carbonyl or less flexible secondary hydroxyl group can efficiently
cooperate with the other donor for rapid and tight complexation
with Cu(II). The new hexadentate chelators E and F differ from
NOTA wherein one of the aminocarboxylate donors is replaced
with a pyridyl (E) or a thiophenyl (F) group. The hexadentate NOTA
chelator A can form an anionic complex with Cu(II), while all new
pentadentate and hexadentate chelators B–F are expected to form
a neutral complex that would have an advantage of less protein
2.20. In vitro serum stability of 64Cu-radiolabeled complexes
Human serum was purchased from Gemini Bioproducts
(#100110). 64Cu-radiolabeled complexes were prepared by reac-
tion of the bifunctional chelators (30 l lCi) in
g) with 64Cu (100
0.25 M NH4OAc buffer (pH 5.5) for 1 h at room temperature, and
labeling efficiency of the radiolabeled complexes were found to
be ꢂ100% as determined by ITLC (20 mM EDTA in 0.15 M NH4OAc).
The freshly prepared radiolabeled complexes of chelators were di-
rectly used for serum stability studies without further purification.
64Cu-radiolabeled complex (85
serum (90
l
Ci, 10
lL) was added to human
l
L) in a microcentrifuge tube, and concentration of
chelator in the resulting mixture was 0.5 mM. The stability of
64Cu-radiolabeled complexes in human serum was evaluated at
37 °C for 2 days. A solution of the radiolabeled complex in serum
was withdrawn at the designated time points and evaluated by
ITLC as described above.
interaction and
distribution over a charged complex.
a potentially more favorable in vivo tissue
2.21. EDTA challenge19
64Cu-radiolabeled complexes were prepared by reaction of each
3.2. Synthesis of new chelators and their Cu(II) complexes
chelator (20
(40
l lCi) in 0.25 M NH4OAc buffer
g) with 64Cu (60
Synthesis of chelators A and B is shown in Scheme 1. Bisubsti-
tuted 1,4,7-triazacyclononane (TACN) analogue 112 was reacted
with an alkylating agent 215 and 411 to provide the key precursor
molecules 3 and 5, respectively. Removal of tert-butyl groups in
3 and 5 was accomplished by treatment of 3 and 5 with HCl (aq)
to afford the respective bifunctional chelators A16 and B. Synthesis
of chelators C and D is outlined in Scheme 2. Hydrolysis followed
by decarboxylation of 617 under acidic condition provided com-
l
L, pH 5.5) for 2 h at room temperature. The labeling efficiency
of the 64Cu-radiolabeled complexes was found to be ꢂ100% as
determined by ITLC (20 mM EDTA in 0.15 M NH4OAc). The 64Cu-
radiolabeled complexes were directly used for EDTA challenge
experiments. A solution of the 64Cu-radiolabeled complex contain-
ing each chelator (ꢂ1 mM) in 0.25 M NH4OAc buffer (40
lL) was
L, H2O, pH 5.0) at
mixed with a solution of EDTA (ꢂ100 mM, 40
l
a 100-fold molar excess. The resulting mixture was incubated for
24 h at 37 °C. The stability of 64Cu-radiolabeled complexes in the
solution was evaluated using ITLC (20 mM EDTA in 0.15 M
NH4OAc). A solution of the radiolabeled complex in EDTA solution
pound 718 which was subjected to
a-bromination using Br2 to pro-
duce 8. Base-promoted reaction of 8 with 112 at room temperature
for 24 h provided substitution product 9 which was further treated
with TFA to provide chelator C. Compound 9 containing the
(3–20 lL) was withdrawn at the designated time points and

2558
I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
Br
(2)
CO2H
CO2tBu
CO2tBu
MeO2C
CO2H
CO2tBu
N
N
N
N
CO2tBu
N
N
6M HCl (aq)
NO2
N
N
N
A
reflux, 3 h
(89%)
HO2C
DIPEA, CH3CN
RT, 7 d (51%)
MeO2C
H
1
3
NO2
NO2
Br
DIPEA
CH3CN
RT, 61 h
(54%)
NO2
(4)
CO2H
CO2tBu
4M HCl (g)/
1,4-dioxane
0 oC to RT, 24 h
(78%)
CO2H
CO2tBu
N
N
N
N
N
N
B
5
NO2
NO2
Scheme 1. Synthesis of TACN-based chalators A and B.
carbonyl group was reduced to alcohol 10 using NaBH4. tert-Butyl
groups in 10 was removed by treatment of 10 with TFA in CHCl3 to
furnish chelator D. The chelators E and F containing the heteroar-
omatic rings were synthesized as outlined in Scheme 3. Reductive
amination of 2-pyridyl aldehyde and 2-thiophenyl aldehyde with
bisubstituted TACN analogue 112 provided compounds 11 and 12,
respectively. Compounds 11 and 12 were treated with 6 M HCl
(aq) and heated to reflux for 5 h to afford the desired chelators E
and F, respectively.
determined by titrating chelators with 64Cu, and the result is
shown in Figure 2. The chelators in different concentrations
(0.0001–0.1 l
g) were labeled with 64Cu (0.1 M NH4OAc, pH 5.5,
37 °C). All chelators studied bound to 64Cu with high labeling effi-
ciency (>98%, 1 h). The respective maximum specific activity (Ci/
lmol) of 4.87, 51.73, 27.45, 1.18, 14.69, and 1.82 was determined
for chelators A–F. It is noteworthy that the pentadentate chelator
B bound to 64Cu with the highest maximum specific activity
(51.73 Ci/lmol). A TACN analogue substituted with two N-car-
Cold Cu(II) complexes of the chelators A–F were prepared and
characterized by HPLC. A solution of each chelator was reacted
with CuCl2 in an equal molar concentration at room temperature
for 24 h to provide the corresponding Cu(II) complexes. The Cu(II)
complexes were purified using semi-prep HPLC and characterized
by analytical HPLC (Supporting information). The Cu(II) complexes
of chelator E and F containing pyridyl and thiophenyl ring were
eluted earlier with 2 min window (tR = 4 min and tR = 6 min,
respectively) as compared to Cu(II) complexes of chelators A–D
(tR = 7–8 min).
boxymethyl groups were known to effectively complex with
Cu(II).3 Introduction of a functional linker to the TACN backbone
appears to have little impact on complexation of the donor groups
with Cu(II). The relatively lower maximum specific activity was ob-
served with the hexadentate chelators D (1.18 Ci/lmol) and F
(1.82 Ci/ mol) containing a hydroxyl group and a thiophenyl
l
group. As compared to the known NOTA bifunctional chelator A,
significantly higher specific activity was observed with chelators
B, C, and E.
3.4. Radiolabeling reaction kinetics
3.3. Maximum specific activity
The new chelators were evaluated for radiolabeling reaction
kinetics with 64Cu at room temperature (Table 1 and Supporting
information). Each chelator (6.7 lg) in 0.25 M NH4OAc (pH 5.5)
The new chelators were evaluated for radiolabeling with 64Cu to
determine the maximum specific activity. The specific activity was
O
O
O
O
Br2, MeOH
7~10 oC, 34 h
RT, 12 h (18%)
Br
HCl, AcOH
H3C
OEt
H3C
reflux, 24 h
8
NO2
quantitative
NO2
7
6
CO2tBu
NO2
DIPEA
CH3CN
RT, 24 h
(42%)
CO2tBu
N
N
N
H
(1)
CO2H
CO2tBu
CO2tBu
CO2H
CO2tBu
CO2H
OH
CO2H
CO2tBu
N
N
N
N
N
N
N
N
TFA
TFA
CHCl3
N
NaBH4
N
CHCl3
N
N
0 oC to RT
3h (91%)
OH
MeOH
0 oC to RT
4 h (37%)
O
O
0
oC to RT
9
4h (81%)
10
D
C
NO2
NO2
Scheme 2. Synthesis of chelators C and D.
NO2
NO2

I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
2559
CO2tBu
CO2H
CO2tBu
CO2tBu
N
N
CO2H
N
N
CO2tBu
N
6M HCl (aq)
N
CHO
N
N
N
N
N
reflux, 5 h
(86%)
NaBH(OAc)3
DCE, RT, 14 h
(96%)
N
H
E
1
11
NaBH(OAc)3
DCE
RT, 72 h
(73%)
S
CHO
CO2H
CO2tBu
4M HCl (g)
1,4-dioxane
0 oC to RT, 24 h
CO2H
S
CO2tBu
N
N
N
N
N
N
S
(80%)
F
12
Scheme 3. Synthesis of TACN-based chalators E and F containing a heteroaromatic ring.
3.5. In vitro serum stability
In vitro serum stability of the radiolabeled complexes was
performed to determine if the chelators radiolabeled with 64Cu
remained stable without loss of the radioactivity in human serum.
This was assessed by measuring the transfer of 64Cu from the com-
plex to human serum proteins using ITLC (20 mM EDTA in 0.15 M
NH4OAc, Table 2 and Supporting information). 64Cu-radioabeled
chelators were readily prepared from the reactions of the chelators
with 64Cu at room temperature. Essentially no unbound 64Cu was
detected in the reaction mixture at 2 h time point after the reaction
at room temperature as determined by ITLC. The 64Cu-radioabeled
chelators were directly used for serum stability studies (pH 7, 37
°C) without further purification. All 64Cu-radiolabeled complexes
of chelator A–F remained intact in human serum for 2 days as evi-
denced by ITLC analysis (Supporting information). 64Cu-chelator C
was found to be least stable in serum, and ꢂ3% of 64Cu was
dissociated from the complex containing the carbonyl group over
2 days. No measurable radioactivity was released from 64Cu-chela-
tor E over 2 days. A tiny amount of 64Cu (<0.3%) was detected from
other 64Cu-complexes of chelators A, B, D, and F.
Figure 2. Radiolabeling (%) of chelators in different concentration (0.0001–0.1
chelator) with 64Cu (100
Ci).
lg
l
was radiolabeled with 64Cu (20
lCi) at room temperature. During
the reaction time (30 min), the components were withdrawn at
the designated time points (1, 10, and 30 min), and the radiolabel-
ing efficiency (%) was determined using ITLC (20 mM EDTA in
0.15 M NH4OAc). The bifunctional NOTA chelator (A) was em-
ployed for comparison and displayed rapid complexation with
64Cu as expected.10 All new chelators instantly bound to 64Cu with
excellent radiolabeling efficiency (>99%) at room temperature.
Radiolabeling of the chelators with 64Cu was nearly complete with-
in 1 min as determined by ITLC. 64Cu-EDTA migrated with the sol-
vent front on TLC (Rf = 1.0), while 64Cu-radiolabeled chelator
complexes travel slower on the TLC (Rf = 0.6). The 64Cu-radiola-
beled complexes of the chelators and 64Cu-EDTA were well sepa-
rated on the ITLC. All 64Cu-radiolabeled complexes were shown
to be stable against EDTA present in the eluent of TLC.
3.6. Stability of 64Cu-radiolabeled complexes in EDTA solution
64Cu-radiolabeled complexes were further evaluated for com-
plex stability based on EDTA challenge. 64Cu-radiolabeled com-
plexes were freshly prepared and treated with a solution of EDTA
at a 100-fold molar excess, and the resulting solution (pH 5.5)
was incubated at 37 °C for 24 h. A sample was withdrawn at differ-
ent time points (0, 1, 4, 19, and 24 h) and analyzed using both ITLC
and HPLC (Table 3 and Supporting information). 64Cu-radiolabeled
complexes of chelators A and E remained intact against EDTA
challenge, and a small portion of the activity (ꢂ5%) was transferred
from the complexes to EDTA at 24 h time point (ITLC). Among the
Table 1
Radiolabeling kinetics of chelators with 64Cu (0.25 M NH4OAC, pH 5.5, RT)#
Time (min)
Bound complex (%)
A
B
C
D
E
F
1
10
30
99.8 0.1
99.8 0.2
99.6 0.3
99.7 0.1
99.6 0.4
100 0.0
99.3 0.3
99.2 0.9
99.5 0.5
99.0 0.0
99.6 0.2
99.5 0.4
99.9 0.1
99.9 0.1
99.9 0.1
99.8 0.2
99.9 0.2
99.8 0.2
#
Radiolabeling efficiency (mean standard deviation %) was measured in triplicate using ITLC (eluent: 20 mM EDTA in 0.15 M NH4OAc).

2560
I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
Table 2
Complex stability of 64Cu-radiolabeled complexes in human serum (pH 7, 37 °C)#
Time (day)
Bound complex (%)
A
B
C
D
E
F
0
1
2
99.9 0.0
100.0 0.0
99.9 0.1
99.9 0.1
100 0.1
99.7 0.4
99.9 0.1
98.7 0.5
97.8 1.1
100 0.1
100 0.0
99.9 0.1
99.9 0.0
100.0 0.0
100.0 0.0
99.9 0.1
99.9 0.1
99.9 0.0
#
Bound complex (mean standard deviation %) was measured in triplicate using ITLC.
complexes tested, 64Cu-radiolabeled pentadentate chelator B was
least tolerant of EDTA treated, and most of 64Cu was dissociated
from the complex (ꢂ80%) at 24 h time point. 64Cu-radiolabeled
chelator C containing the carbonyl donor group was found dissoci-
ated rapidly in the presence of the excess EDTA, and 50% of the
activity was transchelated by EDTA at 1 h time point. 64Cu-radiola-
beled chelators D and F with the respective hydroxyl and thio-
phenyl donor group were slower in dissociation than 64Cu-
radiolabeled chelators B and C and released >45% of 64Cu at 24 h
time point. Dissociation of the activity from the 64Cu-radiolabeled
chelators was also measured using radio-HPLC at 25 h time point
(Fig. 3). The peak related to 64Cu-EDTA (tR = 2.5 min) was clearly
separated from bound 64Cu complex of the chelators. 64Cu-radiola-
beled complex of polar chelators E and F have the respective reten-
tion time at 4.8 min and 6.2 min, while other 64Cu-radiolabeled
complexes have similar retention time (tR = 7–8 min). The 64Cu-
radiolabeled chelators C and D gave a small peak (tR = ꢂ3 min
and tR = ꢂ7 min, respectively). It is speculated that the less stable
complexes interact with mobile phase during HPLC to give the
minor unbound peaks.
In summary, the in vitro complexation kinetic and stability data
indicate that substitution of the N-carboxymethyl group in the
NOTA chelating backbone with a different donor group including
heteroaromatic ring, carbonyl group, or hydroxyl group gave no
measurable effect on radiolabeling efficiency of the chelators and
stability of the corresponding complexes in serum. It appears that
the donor type in the chelators has little impact on complexation
with Cu(II) since the chelators possess the adequate macrocyclic
cavity and denticity for effective complexation of Cu(II). All new
chelators were found to be highly effective in binding 64Cu. It is
noteworthy that pentadentate chelator B rapidly bound to 64Cu
and the complex remained intact in serum. However, when rigor-
ously challenged by EDTA at a 100-fold molar excess, 64Cu-radioa-
beled complexes produced different complex stability profiles. The
bifunctional NOTA chelator (A) and pyridyl-containing chelator (E)
were well tolerant of EDTA challenge, and only a small amount of
the activity was transferred to EDTA. Other chelators B–D and F
radiolabeled with 64Cu were slowly or rapidly dissociated to
produce 64Cu transchelated to EDTA, although the complexes
remained stable in human serum at 37 °C over 2 days. The results
of EDTA challenge experiment indicate that among the new
chelators B–F tested, the chelator E containing the pyridyl group
was the best in forming a stable complex with 64Cu that was com-
parable to the 64Cu complex of the known chelator A. The penta-
dentate chelator B was less effective in tightly binding 64Cu than
other hexadentate chelators C–F.
3.7. Biodistribution of 64Cu-radiolabeled complexes
64Cu-radiolabeled complexes of chelators A and E which pro-
duced excellent in vitro complexation kinetic and stability data
were further evaluated for a biodistribution study using non-tumor
bearing mice (iv injection, n = 4). The 64Cu-radiolabeled complexes
were independently prepared from reaction of the chelators with
64Cu at 37 °C for 1 h for in vivo biodistribution studies. 64Cu-radio-
labeled complexes were evaluated for in vivo stability by measur-
ing radioactivity that accumulated in selected organs and cleared
from the blood of mice at three time points, 1, 4, and 24 h post-
injection (n = 4). The results of the biodistribution studies for
64Cu-chelator A and 64Cu-chelator E are shown in Figures 4 and
5, respectively. Both 64Cu-chelator A and 64Cu-chelator E displayed
a rapid blood clearance and low uptake in the normal organs. 64Cu-
chelator A and 64Cu-chelator E exhibited the highest radioactivity
level in the blood at 1 h (0.13 0.03% ID/g and 0.25 0.05% ID/g,
respectively) which decreased over the time points. The radioactiv-
ity accumulated in the blood at 24 h was negligible for both 64Cu-
chelator A (0.06 0.00% ID/g) and 64Cu-chelator E (0.10 0.01% ID/
g). The 64Cu-radiolabeled complexes exhibited low uptake in the
lung, muscle, bone, and pancreas (<0.57% ID/g) which peaked at
1 h and decreased at 24 h. A very low radioactivity level in the
spleen over the time points was observed with 64Cu-chelator A
(ꢂ0.2% ID/g) and 64Cu-chelator E (ꢂ0.1% ID/g). 64Cu-chelator E dis-
played higher renal and liver uptake as compared to 64Cu-chelator
A over the course of the study. 64Cu-chelator A exhibited the high-
est radioactivity level in the liver (2.20 0.62% ID/g) and kidney
(1.25 0.35% ID/g) at 1 h that declined to (0.52 0.07% ID/g) and
(0.40 0.04% ID/g) at 24 h. A higher radioactivity level at the liver
and kidney relative to other normal organs was measured with
64Cu-chelator E. Renal and liver uptake of 64Cu-chelator E peaked
at 1 h (3.20 0.21% ID/g and 3.11 0.39% ID/g, respectively) and
decreased over the time points (0.67 0.12% ID/g and
0.98 0.12% ID/g at 24 h, respectively). The in vivo biodistribution
Table 3
Stability of 64Cu-radiolabeld complexes in EDTA solution (37 °C, pH 5.5)#
Time (h)
Bound complex (%)
A
B
C
D
E
F
0
1
4
19
24
25*
99.9 0.1
98.6 0.6
96.8 0.0
95.3 0.5
94.9 0.0
90.3 0.3
99.9 0.1
90.4 0.1
69.2 0.3
22.9 2.1
19.4 1.1
11.9 0.6
97.7 0.2
50.4 0.4
45.8 1.5
41.0 0.4
39.0 0.2
21.7 1.3
99.5 0.3
86.8 0.7
79.1 0.4
59.3 0.0
55.0 0.6
41.0 2.6
99.7 0.1
98.2 0.3
92.6 0.3
93.6 0.1
94.3 0.6
93.1 0.1
100 0.1
95.7 0.5
87.5 0.4
59.5 0.6
53.4 1.7
42.3 0.3
#
Bound complex (mean standard deviation %) was measured in duplicate using ITLC.
Bound complex (mean standard deviation %) was measured in duplicate using radio-HPLC.
*

I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
2561
ADC1 A, Cu-64 (CS071813-1\047-0901.D)
mAU
ADC1 A, Cu-64 (CS071813-1\045-0701.D)
mAU
350
300
250
200
150
100
50
250
200
150
64Cu-A
64Cu-EDTA
64Cu-B
100
50
min
min
min
2
4
6
8
10
12
12
12
14 min
2
4
6
8
10
10
10
12
12
12
14
14
14
ADC1 A, Cu-64 (CS071813-1\041-0301.D)
ADC1 A, Cu-64 (CS071813-1\043-0501.D)
mAU
mAU
180
220
200
180
160
140
120
100
80
64Cu-D
160
140
120
100
80
64Cu-C
60
60
40
40
min
2
4
6
8
10
14
2
4
6
8
ADC1 A, Cu-64 (CS071813-1\033-1101.D)
mAU
ADC1 A, Cu-64 (CS071813-1\035-1301.D)
mAU
64Cu-F
225
200
175
150
125
100
75
64Cu-E
350
300
250
200
150
100
50
50
min
2
4
6
8
10
14
2
4
6
8
Figure 3. HPLC chromatograms of 64Cu-radiolabeled complex against EDTA challenged for 25 h (37 °C, pH 5.5).
Figure 4. Biodistribution of 64Cu-radiolabeled chelator A in female CD-1 mice
following iv injection.
Figure 5. Biodistribution of 64Cu-radiolabeled chelator E in female CD-1 mice
following iv injection.
data suggest that 64Cu-chelator E displayed favorable in vivo sta-
bility as evidenced by rapid blood clearance and low organ uptake
and produced a biodistribution profile comparable to the 64Cu
complex of the known NOTA-based chelator A.
4. Conclusion
The novel pentadentate or hexadentate NOTA analogues with
different donor groups were prepared and evaluated as chelators

2562
I. Sin et al. / Bioorg. Med. Chem. 22 (2014) 2553–2562
of 64Cu. The radiolabeling efficiency data indicate that all new
chelators instantly and almost completely bound to 64Cu at room
temperature. All chelators were efficiently radiolabeled with 64Cu
in a broad range of maximum specific activity. The corresponding
64Cu-radiolabeled complexes remained intact in human serum
for 2 days. No obvious effect of donor atom and denticity on
complexation kinetics and serum stability with 64Cu was observed
with the chelators studied. However, the 64Cu-radiolabeled chela-
tors B–D and F when challenged by EDTA lost a significant amount
of 64Cu, while 64Cu-radiolabeled complexes of two hexadentate
chelators (A and E) were quite inert against the rigorous EDTA
challenge and released a minimal amount of the activity for 24 h.
The results of EDTA challenge experiments indicate that the pyri-
dyl donor (E) in the chelating backbone is more effective in tightly
holding 64Cu than the thiophenyl, carbonyl, and hydroxyl group.
The pentadentate chelator B was shown to form a thermodynam-
ically less stable complex with 64Cu and not tolerant of the EDTA
challenge. The 64Cu-chelator E containing a pyridyl group pro-
duced a biodistribution profile comparable to the known NOTA
chelator A and remained stable in mice and displayed low radioac-
tivity level in the blood and the normal organs. The results of the
in vitro and in vivo evaluations indicate that the new chelator E
is effective in binding 64Cu and will be further modified for use
in targeted PET imaging.
EDTA challenge 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.2014.02.041.
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The authors thank for the financial support from the National
Institutes of Health (R01 CA136695). Dr. Nilantha Bandara was
supported by a DOE Integrated Research Training Program of
Excellence in Radiochemistry (DE-SC0002032).
Supplementary data
Supplementary data (HPLC and ITLC chromatograms for assess-
ment of radiolabeling reaction kinetics and serum stability and