Synthesis and Evaluation of an Enantiomerically Enriched Bifunctional Chelator for 64Cu-Based Positron Emission Tomography (PET) Imaging

Article abstract of DOI:10.1002/ejoc.201301499

We report the synthesis and evaluation of an enantiomerically enriched bifunctional chelator, (S)-C-NE3TA. The bifunctional chelator was efficiently prepared by regioselective and stereoselective ring opening of an aziridinium ion. The new chiral chelator instantly and almost completely bound to ⁶⁴Cu at room temperature. The corresponding ⁶⁴Cu-radiolabeled complex remained intact in human serum for 48 h without any measurable transchelation and was tolerant to a rigorous EDTA challenge for 24 h. The ⁶⁴Cu-radiolabeled (S)-C-NE3TA complex was stable in mice and produced an excellent biodistribution profile. The results of the in vitro and in vivo evaluations indicate that the new optically active chelator is a promising candidate for PET imaging applications.

Full text of DOI:10.1002/ejoc.201301499

FULL PAPER
DOI: 10.1002/ejoc.201301499
Synthesis and Evaluation of an Enantiomerically Enriched Bifunctional
64
Chelator for Cu-Based Positron Emission Tomography (PET) Imaging
Hyun-Soon Chong,*[a] Xiang Sun,[a] Yongliang Zhong,[a] Kamil Bober,[a]
Michael R. Lewis,[b,c] Dijie Liu,[c] Varyanna C. Ruthengael,[c] Inseok Sin,[a] and
Chi Soo Kang[a]
Keywords: Chelating agents / Copper / Macrocyclic ligands / PET imaging / Imaging agents
We report the synthesis and evaluation of an enantiomer-
ically enriched bifunctional chelator, (S)-C-NE3TA. The bi-
functional chelator was efficiently prepared by regioselective
and stereoselective ring opening of an aziridinium ion. The
new chiral chelator instantly and almost completely bound to
64Cu at room temperature. The corresponding 64Cu-radio-
labeled complex remained intact in human serum for 48 h
without any measurable transchelation and was tolerant to a
rigorous EDTA challenge for 24 h. The 64Cu-radiolabeled (S)-
C-NE3TA complex was stable in mice and produced an ex-
cellent biodistribution profile. The results of the in vitro and
in vivo evaluations indicate that the new optically active che-
lator is a promising candidate for PET imaging applications.
imaging.[1–3] However, the currently available 64Cu chelators
have limitations related to the harsh radiolabeling condi-
tions or have less favorable in vitro and in vivo complex
stability for their practical applications to PET im-
aging.[2,5–8] We previously reported a heptadentate (2,2Ј-{7-
[(carboxymethyl)amino]ethyl}-1,4,7-triazonanane-1,4-diyl)-
diacetic acid (NE3TA) as a promising 64Cu chelator.[9] The
bifunctional version of NE3TA (C-NE3TA) was efficiently
radiolabeled with 64Cu under mild conditions and produced
a superior in vivo biodistribution profile compared to that
of C-DOTA.[10]
To develop a highly effective 64Cu chelator, we were in-
terested in the synthesis and evaluation of optically active
NE3TA derivatives. Chirality in acyclic and macrocyclic
chelators such as DTPA, DOTA, and 1,4,7-triazacyclo-
nonane-1,4,7-triacetic acid (NOTA) has a significant im-
pact on metal complexation, and metal complexes of
stereoisomeric analogues displayed different in vitro and in
vivo complex stability.[11–14] An optically active chelator is
expected to generate a limited number of possible dia-
stereomeric metal complexes. We previously reported the
application of a facile ring opening of aziridinium ions for
the practical synthesis of bifunctional chelators in the
NETA and NE3TA series.[15–17] We envisioned that stereo-
selective ring opening of aziridinium ions would provide
optically active NE3TA compounds in high enantio-
selectivity. We herein report the synthesis of the first op-
tically active chelator, (S)-C-NE3TA, in the NETA and
NE3TA series. The new chelator (S)-C-NE3TA was evalu-
ated for complexation kinetics and stability with 64Cu in
vitro and in vivo.
Introduction
Positron emission tomography (PET) is a highly sensitive
imaging tool and has been applied for the diagnosis and
staging of cancers.[1–3] 64Cu (t1/2 = 12.7 h) is a positron- and
β-emitting radionuclide and one of the most useful and
readily available radioisotopes for radiotherapy and PET
imaging applications.[1–4] Antibodies and peptides have
been employed as targeting vectors for the delivery of the
short-lived 64Cu nuclide in tumor-specific PET imaging.
Targeted PET imaging requires a bifunctional chelator that
possesses a functional group for conjugation to a sensitive
biomolecule target in tumor cells and can rapidly sequester
64Cu to form a stable complex under mild conditions.[2,3]
Various chelating agents including 1,4,8,11-tetraaza-
cyclotetradecane-N,NЈ,NЈЈ,NЈЈЈ-tetraacetic acid (TETA),
1,4,7,10-tetraazacyclododecane-N,NЈ,NЈЈ,NЈЈЈ-tetraacetic
acid (DOTA), diethylenetriaminepentaacetic acid (DTPA),
and 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic
acid (CB-TE2TA) have been explored for 64Cu-based PET
[a] Chemistry Division, Biological and Chemical Sciences Depart-
ment, Illinois Institute of Technology,
3101 S. Dearborn St, LS 182, Chicago, IL, USA
E-mail: Chong@iit.edu
http://www.iit.edu
[b] Research Service, Harry S. Truman Memorial Veterans’ Hospi-
tal,
Columbia, MO, USA
[c] Department of Veterinary Medicine and Surgery, University of
Missouri,
Columbia, MO, USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301499
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Results and Discussion
Synthesis
excellent isolated yield (96%) from the reaction of 9, which
contains a DMB group, with 10. The DMB and tert-butyl
protecting groups in 12 were readily removed by treatment
of 12 with 6 m HCl (aq.) under reflux to provide the desired
chelator (S)-C-NE3TA (13) in quantitative yield.
The new chelator (S)-C-NE3TA was prepared by a syn-
thetic method centered on the ring opening of aziridinium
To prove the stereochemistry and regiochemistry of the
ions (Scheme 1). We previously reported the application of ring-opening product, the chelator 13 was independently
a facile ring opening of aziridinium ions for the practical prepared by a different synthetic route as shown in
synthesis of the racemic chelators in the NETA and NE3TA Scheme 2. We first attempted to prepare the macrocyclic
series. The ring opening of the enantiomerically enriched backbone (S)-23 by reductive amination of aldehyde 16
aziridinium ions 8 and 9 with the macrocyclic amine 10[18] with N-tert-butyloxycarbonyl-protected (N-Boc) 1,4,7-tri-
was expected to provide the desired chelating backbone 11 azacyclononane (TACN) 17.[19] However, Swern oxidation
and 12, respectively, as the ring-opening products in high of (S)-15[20] provided (Ϯ)-16[21] (50%). It appears that the
stereoselectivity. The starting material p-nitro-Bn-alaniol (1) proton at the chiral center of 15 was involved in the reac-
was subjected to reductive amination with benzaldehyde tion; it was possibly removed and then added again. The
and 2,4-dimethoxybenzaldehyde (DMB) to provide the N- reductive amination of 16 with 17 afforded 23 (98%) in the
protected amino alcohols 2 and 3 in isolated yields of 70 racemic form. We then used a synthetic method based on
and 88%, respectively. The amino alcohols 2 and 3 were the base-promoted bimolecular cyclization between 20 and
treated with tert-butyl bromoacetate to provide 4 (86%) and 21 to generate the macrocyclic backbone 22. Amide 18 was
5 (93%), respectively. Halogenation of 4 and 5 with N- obtained in 62% isolated yield from the reaction of 14 with
bromosuccinimide (NBS) or N-iodosuccinimide (NIS) and NH4OH, and the subsequent reaction of 18 with 4-tolu-
triphenylphosphine (PPh3) provided the N,N-disubstituted enesulfonyl chloride (TsCl) followed by reduction with BH3
β-amino halides 6 (70%) and 7 (63%), respectively. We ini- provided the N-Ts-protected amine 20 (80%). The reaction
tially performed the ring-opening reaction of the aziridin- of 20 with the N-Ts-protected ditosylate 21[22] provided 22
ium ion 8, which contains an N-benzyl group, with 10. The (54%), which was further subjected to deprotection of the
aziridinium ion 8 was produced in situ by the reaction of 6 tosyl groups under acidic conditions to give the desired
with silver perchlorate. As expected, aziridinium ion 8 re- compound 23 (40%). The base-promoted alkylation of 23
acted with 10 at the less-hindered methylene carbon atom with tert-butyl bromoacetate provided 24 in a poor isolated
to provide the desired ring-opening product 11 in a poor yield (26%) owing to the expected polyalkylation of 23. The
isolated yield (41%). However, N-debenzylation of 11 was removal of the tert-butyl groups of 24 with HCl (aq.) gave
challenging and failed to provide the desired product. As the final chelator 13 in an excellent isolated yield (91%).
1
expected, the ring-opening product 12 was prepared in an The H and 13C NMR spectroscopic data of 13 prepared
Scheme 1. Synthesis of the enantiomerically enriched bifunctional chelator (S)-C-NE3TA (13).
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An Enantiomerically Enriched Chelator for Positron Emission Tomography
Scheme 2. Synthesis of (S)-C-NE3TA (13) for confirmation of regiochemistry and stereochemistry.
by the two synthetic routes were essentially identical, and measurable radioactivity was released from the complex
the optical rotation data of (S)-13 confirmed that the intra- over 2 d as evidenced by TLC and HPLC analysis (Figure 1,
molecular rearrangement of (S)-7 to form of aziridinium a).
ion (R)-9 and subsequent ring opening of (R)-9 proceeded
with double inversion of stereochemistry at the chiral center by analytical HPLC for comparison (Figure 1, b). The
Cold CuII-(S)-C-NE3TA was prepared and characterized
to provide (S)-12, which was further converted to (S)-13.
64Cu-radiolabeled complex was further evaluated for com-
plex stability on the basis of an ethylenediamine (EDTA)
challenge test.[24] The 64Cu-radiolabeled complex was
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 different time points (0, 1, 4,
and 24 h) and analyzed by instant TLC (ITLC; see Sup-
porting Information). The 64Cu-radiolabeled complex was
very tolerant of the harsh EDTA challenge and remained
intact after 1 h (Figure 1, c). After 24 h, a small portion of
the 64Cu (ca. 9%) was transferred from the complex to
EDTA (Figure 1, d).
In Vitro and In Vivo Evaluation of (S)-C-NE3TA
The new chiral chelator was evaluated for radiolabeling
reaction kinetics with 64Cu (Supporting Information). The
chelator (0.25 m NH4OAc, pH 5.5) instantly bound to 64Cu
with excellent radiolabeling efficiency at room temperature,
and radiolabeling of the chelator with 64Cu was nearly com-
plete (Ͼ99% radiolabeling efficiency) after 1 min as deter-
mined by TLC (Supporting Information). The in vitro se-
rum stability of the 64Cu-radiolabeled chelator (pH 7,
37 °C) was assessed to determine if the complex remained
stable without loss of radioactivity in human serum. This
was achieved by measuring the transfer of 64Cu from the
complex to human serum proteins by TLC and HPLC
(Supporting Information). The 64Cu-radiolabeled complex
of (S)-C-NE3TA remained stable in human serum, and no
The in vivo stability of 64Cu-radiolabeled (S)-C-NE3TA
was evaluated by a biodistribution study with non-tumor-
bearing CF-1 mice (intravenous injection, n = 3). The 64Cu-
radiolabeled complexes were independently prepared by re-
action of the chelator with 64Cu at room temp. for the in
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Figure 1. (a) HPLC chromatogram of 64Cu-(S)-C-NE3TA in human serum at 48 h. (b) HPLC chromatogram of CuII-(S)-C-NE3TA. (c)
ITLC chromatogram of 64Cu-(S)-C-NE3TA in a solution of EDTA at 100-fold molar excess after 1 h. (d) TLC chromatogram of 64Cu-
(S)-C-NE3TA in a solution of EDTA at 100-fold molar excess after 24 h.
vivo biodistribution study. The 64Cu-radiolabeled complex cleared at 4 h. The radioactivity uptake in the muscle and
was evaluated for in vivo stability by measuring the radioac- bone was very low at the 1 h time point (0.09Ϯ0.04 and
tivity that accumulated in selected organs and cleared from 0.10Ϯ0.04%, respectively). The in vivo biodistribution data
the blood of mice at three time points (1, 4, and 24 h post- indicate that the 64Cu-radiolabeled complex remained es-
injection; Figure 2). The radioactivity accumulation in the sentially inert with no dissociation of 64Cu in mice and dis-
blood and organs at 24 h post-injection was negligible played rapid blood clearance and low retention in the or-
[Ͻ0.1% injected dose per gram (ID/g)]. The highest uptake gans.
of the 64Cu-radiolabeled complex was observed in the kid-
ney (1.70Ϯ0.34% ID/g) at 1 h post-injection; this value
rapidly declined over time (0.42Ϯ0.09% ID/g at 4 h and
0.07Ϯ0.01% ID/g at 24 h). The radioactivity uptake in the
Conclusions
A new optically active NE3TA compound was prepared
and evaluated for potential use in 64Cu-based PET imaging.
The chelator instantly and almost completely bound to
64Cu at room temperature. The corresponding 64Cu-radio-
labeled complex remained intact in human serum for 2 d
without any loss of radioactivity, and the chelator was toler-
ant against a rigorous EDTA challenge for 24 h. The 64Cu-
radiolabeled complex of (S)-C-NE3TA was stable in mice
as demonstrated by the extremely low radioactivity level in
the blood and normal organs. The results of the in vitro
and in vivo evaluations indicate that the new optically
active chelator is an excellent candidate for use in PET im-
aging applications.
blood, liver, muscle, and bone was very low at all time
points (Ͻ0.4% ID/g). The radioactivity level in the blood
at 1 h was low (0.31Ϯ0.10%) and rapidly and completely
Experimental Section
Instruments and Methods: 1H and 13C NMR spectra were obtained
with a Bruker 300 instrument, and chemical shifts (δ) are reported
in parts per million relative to tetramethylsilane (TMS). Fast atom
bombardment (FAB) high-resolution mass spectra (HRMS) were
Figure 2. Biodistribution of 64Cu-radiolabeled (S)-C-NE3TA in
non-tumor-bearing CF-1 mice.
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An Enantiomerically Enriched Chelator for Positron Emission Tomography
layer was adjusted to 12 with 1 m NaOH (aq.), and the solution
obtained with a JEOL double sector JMS-AX505HA mass spec-
trometer (University of Notre Dame, South Bend, IN). Optical ro-
tation data was obtained with a JASCO P-2000 polarimeter. The
analytical HPLC was performed with an Agilent 1200 instrument
equipped with a dioarray detector (λ = 254 and 280 nm), a thermo-
stat set at 35 °C, and a Zorbax Eclipse XDB-C18 column
(4.6ϫ150 mm, 80 Å). A mobile phase with a binary gradient [0–
100% B/40 min; solvent A, 0.05 m AcOH/Et3N, pH 6.0; solvent B,
CH3OH for method 1; 0–100% B/15 min; solvent A, 0.1% tri-
fluoroacetic acid (TFA) in H2O; solvent B, 0.1% TFA in CH3CN
for method 2] at a flow rate of 1 mL/min was used. Semipreparative
HPLC was performed with an Agilent 1200 instrument equipped
with a dioarray detector (λ = 254 and 280 nm), a thermostat set at
35 °C, and a Zorbax Eclipse XDB-C18 column (9.4ϫ250 mm,
80 Å). A mobile phase with a binary gradient (0–40% B/40 min;
solvent A, 0.1% TFA in H2O; solvent B, 0.1% TFA in CH3CN for
method 3; 0–100% B/40 min; solvent A, 0.05 m AcOH/Et3N, pH
6.0; solvent B, CH3OH for method 4) at a flow rate of 3 mL/min
was used. All reagents were purchased from Aldrich (Milwaukee,
WI). All solvents for workup and chromatographic purification
were purchased from VWR international (Radnor, PA). 64Cu in
the chloride form was obtained from Washington University (Saint
Louis, MO).
was extracted with ethyl acetate (3ϫ 20 mL). The organic layer was
dried with MgSO4, filtered, and concentrated in vacuo to afford
1
pure 3 (77.7 mg, 88%). H NMR (CDCl3, 300 MHz): δ = 2.63 (s,
1 H), 2.84–2.89 (m, 3 H), 3.33–3.37 (m, 1 H), 3.58–3.64 (m, 5 H),
3.73–3.80 (m, 4 H), 6.36–6.40 (m, 2 H), 7.0 (d, J = 7.8 Hz, 1 H),
7.26 (d, J = 8.1 Hz, 2 H), 8.10 (d, J = 8.4 Hz, 2 H) ppm. 13C NMR
(CDCl3, 75 MHz) δ = 37.9 (t), 46.3 (t), 55.1 (q), 55.4 (q), 58.3 (d),
62.1 (t), 98.5 (d), 103.8 (d), 119.8 (s), 123.6 (d), 130.0 (d), 130.5 (d),
146.6 (s), 146.9 (2C, s), 158.5 (s), 160.4 (s) ppm. [α]2D6 = –28.8 (c =
1.0, CHCl3). HRMS (ESI+): calcd for C18H23N2O5 [M + H]+
347.1601; found 347.1613.
tert-Butyl 2-{Benzyl[(2S)-1-hydroxy-3-(4-nitrophenyl)propan-2-yl]-
amino}acetate (4): To a solution of 2 (150 mg, 0.52 mmol) in
CH3CN (3 mL) was added K2CO3 (72.4 mg, 0.52 mmol) at 0 °C.
A solution of tert-butyl bromoacetate (102 mg, 0.52 mmol) in
CH3CN (2 mL) was added dropwise to the resulting mixture over
10 min. The reaction mixture was heated to reflux and stirred for
14 h; the reaction progress was continuously monitored by TLC.
The reaction mixture was cooled to room temperature and filtered,
and the solvents were evaporated to dryness. The residue was puri-
fied by column chromatography on silica gel (60–220 mesh) eluted
with 20% ethyl acetate in hexanes to afford pure 4 (180 mg, 86%).
1H NMR (CDCl3, 300 MHz): δ = 1.37 (s, 9 H), 2.59 (dd, J = 12.9,
7.8 Hz, 1 H), 2.97 (dd, J = 13.2, 6.0 Hz, 1 H), 3.14–3.20 (m, 2 H),
3.35–3.50 (m, 3 H), 3.82 (dd, J = 43.8, 13.5 Hz, 2 H), 4.10 (s, 1 H),
7.22–7.29 (m, 7 H), 8.10 (d, J = 7.8 Hz, 2 H) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 28.0 (q), 33.9 (t), 51.5 (t), 56.1 (t), 61.8 (t),
64.9 (d), 81.7 (s), 123.8 (d), 127.4 (d), 128.4 (d), 128.9 (d), 129.9
Caution: 64Cu (t1/2 = 12.7 h) is a positron-emitting radionuclide. Ap-
propriate shielding and handling protocols should be in place when
using this isotope.
(2S)-2-(Benzylamino)-3-(4-nitrophenyl)propan-1-ol (2): To
1
(200 mg, 1.02 mmol) were added benzaldehyde (140.63 mg,
1.33 mmol) and benzene (12 mL). The solution was stirred for 14 h
under reflux with a Dean–Stark trap. The solvent was evaporated
in vacuo, and MeOH (5 mL) was added to the crude imine residue.
The resulting mixture was cooled to 0 °C, and NaBH4 (84.8 mg,
2.2 mmol) was added portionwise over 15 min. The reaction mix-
ture was stirred for 24 h and then concentrated to dryness in vacuo.
The residue was quenched with H2O (20 mL) and extracted with
ethyl acetate (3ϫ 20 mL). The organic layer was dried with MgSO4,
filtered, and concentrated in vacuo. The crude product was dis-
solved in 1 m HCl (aq., 20 mL) and washed with ethyl acetate (3ϫ
20 mL). The pH of the aqueous layer was adjusted to 12 with 1 m
NaOH (aq.), and the solution was extracted with ethyl acetate (3ϫ
20 mL). The organic layer was dried with MgSO4, filtered, and con-
1
(d), 138.3 (s), 146.5 (s), 147.4 (s), 172.3 (s) ppm. The H and 13C
NMR spectroscopic data of (S)-4 are essentially identical to those
of rac-4.[16] [α]2D6 = –36.3 (c = 1.0, CHCl3).
tert-Butyl 2-{[(2,4-Dimethoxyphenyl)methyl][(2S)-1-hydroxy-3-(4-
nitrophenyl)propan-2-yl]amino}acetate (5): To a stirred solution of
3 (70 mg, 0.20 mmol) in CH3CN (3 mL) at 0 °C was added K2CO3
(30.4 mg, 0.22 mmol).
A solution of tert-butyl bromoacetate
(43.4 mg, 0.22 mmol) in CH3CN (2 mL) was added dropwise to the
resulting mixture over 10 min. The reaction mixture was heated to
reflux; the reaction progress was continuously monitored by TLC.
The reaction mixture was filtered, and the solvent was evaporated
in vacuo to provide the residue, which was purified by column
centrated in vacuo to afford pure 2 (202 mg, 70%). 1H NMR chromatography on silica gel (60–230 mesh) eluted with 20% ethyl
(CDCl3, 300 MHz): δ = 2.14 (s, 1 H), 2.82–2.99 (m, 3 H), 3.35 (dd,
J = 10.5, 3.9 Hz, 1 H), 3.65 (dd, J = 14.1, 3.0 Hz, 1 H), 3.75–3.80
acetate/hexane. Pure 5 (85.5 mg, 93%) was obtained as a light yel-
low oil. H NMR (CDCl3, 300 MHz): δ = 1.38 (s, 9 H), 2.56 (dd,
1
(m, 2 H), 7.21–7.38 (m, 7 H), 8.14 (d, J = 8.4 Hz, 2 H) ppm. 13C J = 12.6, 8.7 Hz, 1 H), 3.02–3.48 (m, 6 H), 3.73–3.86 (m, 8 H), 4.04
NMR (CDCl3, 75 MHz): δ = 37.9 (t), 51.1 (t), 59.0 (d), 62.2 (t), (s, 1 H), 6.39–6.41 (m, 2 H), 7.09 (d, J = 8.4 Hz, 1 H), 7.28 (d, J
123.8 (d), 127.3 (d), 128.0 (d), 128.6 (d), 130.1 (d), 139.6 (s), 146.6
(s), 146.7 (s) ppm. The 1H and 13C NMR spectroscopic data of
(S)-2 are essentially identical to those of rac-2.[16]
= 8.1 Hz, 2 H), 8.11 (d, J = 8.4 Hz, 2 H) ppm. 13C NMR (CDCl3,
75 MHz): δ = 28.0 (q), 34.1 (t), 49.4 (t), 52.2 (t), 55.2 (q), 55.3 (q),
61.5 (t), 65.3 (d), 81.3 (s), 98.5 (d), 103.9 (d), 118.6 (s), 123.7 (d),
129.8 (d), 131.6 (d), 146.5 (s), 147.6 (s), 158.8 (s), 160.5 (s), 172.1
(s) ppm. HRMS (ESI+): calcd for C24H33N2O7 [M + H]+ 461.2282;
found 461.2297. [α]2D6 = –41.2 (c = 1.0, CHCl3).
(2S)-2-{[(2,4-Dimethoxyphenyl)methyl]amino}-3-(4-nitrophenyl)-
propan-1-ol (3): A stirred solution of 1 (50 mg, 0.26 mmol) and 2,4-
dimethoxybenzaldehyde (55 mg, 0.33 mmol) in benzene (10 mL)
was heated to reflux for 16 h with a Dean–Stark trap (5 mL). The
solvent was evaporated in vacuo, and MeOH (10 mL) was added
to the crude imine residue. The resulting mixture was cooled to
0 °C, and NaBH4 (29 mg, 0.77 mmol) was added portionwise over
15 min. The reaction mixture was stirred for 24 h and then concen-
trated to dryness in vacuo. The residue was quenched with H2O
(15 mL) and extracted with ethyl acetate (3ϫ 20 mL). The organic
layer was dried with MgSO4, filtered, and concentrated in vacuo.
The crude product was dissolved in 1 m HCl (aq., 20 mL) and
washed with ethyl acetate (3ϫ 20 mL). The pH of the aqueous
tert-Butyl
2-{Benzyl[(2R)-2-iodo-3-(4-nitrophenyl)propyl]amino}-
acetate (6): To 4 (500 mg, 1.25 mmol) was added CH2Cl2 (7 mL).
The solution was cooled to 0 °C, and triphenylphosphine (393 mg,
1.50 mmol) and imidazole (102 mg, 1.50 mmol) were added. To the
resulting solution was added iodine (380 mg, 1.50 mmol) portion-
wise. The mixture was stirred for 4 h at room temperature. The
solvent was evaporated to dryness, and the residue was purified by
column chromatography on silica gel (60–220 mesh) eluted with
5% ethyl acetate in hexanes. The pure product 6 (449 mg, 70%)
was obtained as a yellowish oil and directly used for the next step.
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1H NMR (CDCl3, 300 MHz): δ = 1.48 (s, 9 H), 2.97 (dd, J = 14.4, The reaction mixture was filtered and concentrated to the dryness.
9.9 Hz, 1 H), 3.08 (dd, J = 14.1, 9.0 Hz, 1 H), 3.32–3.39 (m, 3 H), HCl solution (0.1 m, 10 mL) was added to the residue, and the re-
3.67 (dd, J = 14.7, 3.6 Hz, 1 H), 3.85–3.91 (m, 1 H), 4.05–4.08 (m,
1 H), 7.25–7.40 (m, 7 H), 8.13 (d, J = 8.7 Hz, 2 H) ppm. 13C NMR
(CDCl3, 75 MHz) δ = 28.3 (q), 34.8 (d), 43.1 (t), 56.3 (t), 59.0 (t),
63.7 (t), 81.4 (s), 123.6 (d), 127.5 (d), 128.3 (d), 129.0 (d), 129.9 (d),
138.8 (s), 146.8 (s), 147.6 (s), 170.5 (s) ppm. [α]2D6 = –4.9 (c = 1.0,
CHCl3).
sulting mixture was extracted with CH2Cl2 (3ϫ 10 mL). The com-
bined organic layers were dried with MgSO4, filtered, and concen-
trated to the dryness in vacuo. The residue was purified by column
chromatography on silica gel (220–440 mesh) eluted with 2 %
CH3OH in CH2Cl2 to provide pure product 12 (49 mg, 96%) as a
1
yellow oil. Analytical HPLC (method 1, tR = 39.6 min). H NMR
(CDCl3, 300 MHz): δ = 1.36–1.47 (m, 27 H), 2.65–3.82 (m, 30 H),
6.44–6.46 (m, 2 H), 7.10 (d, J = 7.8 Hz, 1 H), 7.52 (d, J = 8.4 Hz,
2 H), 8.15 (d, J = 8.1 Hz, 2 H) ppm. 13C NMR (CDCl3, 75 MHz):
δ = 28.0 (q), 28.1 (q), 34.1 (t), 49.8 (t), 51.2 (t), 52.1 (t), 53.5 (t),
54.7 (t), 55.4 (q), 55.5 (q), 56.0 (t), 58.4 (d), 59.0 (t), 81.5 (s), 82.1
(s), 98.6 (d), 104.5 (d), 117.6 (s), 123.8 (d), 130.5 (d), 132.1 (d),
146.6 (s), 146.7 (s, 2 C), 158.7 (s), 160.8 (s), 170.5 (s), 173.0 (s) ppm.
[α]2D6 = 7.4 (c = 1.0, CHCl3). HRMS (ESI+): calcd for C42H66N5O10
[M + H]+ 800.4804; found 800.4818.
tert-Butyl 2-{[(2R)-2-Bromo-3-(4-nitrophenyl)propyl][(2,4-dimeth-
oxyphenyl)methyl]amino}acetate (7): To a solution of 5 (600 mg,
1.303 mmol) and PPh3 (513 mg, 1.954 mmol) in CHCl3 (8 mL) at
0 °C was added NBS (348 mg, 1.954 mmol) portionwise 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 tempera-
ture and stirred for 1 h. The solvent was evaporated, and the resi-
due was purified by silica gel column chromatography eluted with
10% ethyl acetate in hexanes to afford 7 (430 mg, 63%) as a yellow
1
oil. H NMR (CDCl3, 300 MHz): δ = 1.48 (s, 9 H), 2.87–3.03 (m,
2 H), 3.28–3.32 (m, 3 H), 3.78–3.84 (m, 9 H), 4.09–4.12 (m, 1 H),
2-{(2S)-1-[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]-3-(4-nitro-
phenylpropan-2-yl)amino}acetic Acid (13): To 12 (22 mg,
6.46–6.48 (m, 2 H), 7.20 (d, J = 9.0 Hz, 1 H), 7.33 (d, J = 8.7 Hz, 0.028 mmol) was added 6 m HCl solution (aq., 3 mL), and the re-
2 H), 8.13 (d, J = 8.7 Hz, 2 H) ppm. 13C NMR (CDCl3, 75 MHz) sulting solution was maintained at reflux for 15 min. The reaction
δ = 28.3 (q), 41.8 (t), 52.4 (t), 54.0 (q), 55.4 (q), 56.5 (t), 62.0 (t),
was warmed to room temperature, and the resulting solution was
filtered and concentrated in vacuo to provide 13 (19 mg, 100%) as a
yellow solid. Compound 13 was further purified by semipreparative
HPLC (method 3, 21.5–22.5 min). Analytical HPLC (method 2, tR
= 7.8 min). 1H NMR (D2O, 300 MHz): δ = 2.49–2.52 (m, 2 H),
2.87–3.05 (m, 6 H), 3.23 (m, 7 H), 3.71–3.82 (m, 7 H), 7.45 (d, J
= 8.1 Hz, 2 H), 8.13 (d, J = 8.4 Hz, 2 H) ppm. 13C NMR (D2O,
75 MHz): δ = 34.0 (t), 45.2 (t), 48.5 (t), 49.3 (t), 56.0 (d), 56.1 (t),
58.5 (t), 124.2 (d), 130.2 (d), 143.2 (s), 147.0 (s), 169.4 (s), 171.9
(s). [α]2D6 = +2.12 (c = 0.19, H2O). HRMS (ESI–): calcd for
C21H30N5O8 [M – H]– 480.2100; found 480.2121.
81.1 (s), 98.6 (d), 103.9 (d), 119.0 (s), 123.4 (d), 130.2 (d), 131.5
(d), 146.7 (s), 147.0 (s), 158.9 (s), 160.5 (s), 170.9 (s) ppm. [α]2D6
=
+4.0 (c = 1.0, CHCl3). HRMS (ESI+): calcd for C24H32BrN2O6 [M
+ H]+ 523.1438; found 523.1446.
tert-Butyl
2-{Benzyl[(2S)-1-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-
1,4,7-triazonan-1-yl}-3-(4-nitrophenyl)propan-2-yl]amino}acetate (11):
To a solution of 6 (74.0 mg, 0.145 mmol) in CH3CN (0.5 mL) at
–5 °C was added AgClO4 (30 mg, 0.145 mmol). The resulting mix-
ture was stirred for 20 min at the same temperature. Compound
10 (50.0 mg, 0.140 mmol) and N,N-diisopropylethylamine (DIPEA;
56 mg, 0.435 mmol) in CH3CN (0.5 mL) were sequentially added to
the reaction mixture at –5 °C. The resulting mixture was gradually
warmed to room temperature and stirred for 24 h. The reaction
mixture was filtered and concentrated to dryness. HCl solution
(0.1 m, 20 mL) was added to the residue, and the resulting mixture
tert-Butyl N-[(2S)-1-Hydroxy-3-(4-nitrophenyl)propan-2-yl]carb-
amate (15):[20] To a solution of 14 (849 mg, 3.79 mmol) in CH3OH
(20 mL) was added NaBH4 (716 mg, 18.93 mmol) portionwise over
10 min at 0 °C. The solvent was removed in vacuo, and the residue
was treated with deionized H2O (30 mL) and extracted with ethyl
was extracted with CH2Cl2 (3ϫ 20 mL). The combined organic acetate (3ϫ 20 mL). The combined organic layers were dried with
layers were dried with MgSO4, filtered, and concentrated in vacuo
to dryness. The residue was purified by column chromatography
on silica gel (220–440 mesh), first eluted with 25% ethyl acetate in
hexanes and then 50% ethyl acetate in hexanes. The column was
then dried and eluted with 2% CH3OH in CH2Cl2 to provide pure
11 (43.6 mg, 41%) as yellowish oil. Analytical HPLC (method 1,
tR = 38.8 min). 1H NMR (CDCl3, 300 MHz): δ = 1.36–1.44 (m, 27
MgSO4 and filtered, and the filtrate was concentrated in vacuo to
provide 1, which was directly used for the next step. To a solution of
1 in CH3CN (15 mL) was added di-tert-butyl dicarbonate (556 mg,
2.55 mmol) dropwise. The reaction mixture was stirred for 5 h at
room temp. Then, the reaction mixture was concentrated under
reduced pressure, and the residue was purified by column
chromatography on silica gel (60–230 mesh) eluted with 30% ethyl
H), 2.68–3.73 (m, 25 H), 7.18–7.34 (m, 5 H), 7.52 (d, J = 8.6 Hz, acetate/hexanes to provide pure 15 (658 mg, 88 %). 1H NMR
2 H), 8.14 (d, J = 8.6 Hz, 2 H) ppm. 13C NMR (CDCl3, 75 MHz): (CDCl3): δ = 1.39 (s, 9 H), 2.98 (d, J = 6.0 Hz, 2 H), 3.57 (dd, J =
δ = 27.9 (q), 28.0 (q), 28.1 (q), 33.3 (t), 49.8 (t), 51.1 (t), 51.6 (t),
3.1 Hz, 1 H), 3.68 (dd, J = 3.1 MHz, 1 H), 3.90 (s, 1 H), 4.84 (d,
J = 6.8 Hz, 1 H), 7.40 (d, J = 8.8 Hz, 2 H), 8.15 (d, J = 8.8 Hz, 2
53.6 (t), 55.6 (t), 57.9 (d), 58.2 (t), 81.6 (s), 81.7 (s), 82.6 (s), 124.1
(d), 128.0 (d), 128.8 (d), 129.4 (d), 130.4 (d), 137.2 (s), 146.0 (s), H) ppm. 13C NMR (CDCl3, 75 MHz): δ = 28.06 (q), 37.22 (t), 53.0
146.8 (s), 170.4 (s), 172.7 (s) ppm. [α]2D6 = +3.3 (c = 1.0, CHCl3).
HRMS (ESI+): calcd for C40H62N5O8 [M + H]+ 740.4593; found
740.4621.
(d), 63.2 (t), 79.7 (s), 123.4 (d), 130.1 (d), 146.3 (s), 146.5 (s), 155.9
(s) ppm. [α]2D6 = –43.4 (c = 1.0, CHCl3) [ref.[20] [α]2D0 = –25.3 (c =
2.0, CH3OH)].
tert-Butyl 2-{[(2S)-1-{4,7-Bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-tri- tert-Butyl N-[(2S)-1-(4-Nitrophenyl)-3-oxopropan-2-yl]carbamate
azonan-1-yl}-3-(4-nitrophenyl)propan-2-yl][(2,4-dimethoxyphenyl)-
methyl]amino}acetate (12): To a solution of 7 (50 mg, 0.096 mmol)
in CH3CN (1 mL) at –5 °C was added AgClO4 (19.8 mg,
0.096 mmol). The resulting mixture was stirred for 10 min at the
same temperature. Compound 10 (34.2 mg, 0.096 mmol) and
(16):[21] Oxalyl chloride (257 mg, 2.03 mmol) was dissolved in
CH2Cl2 (5 mL), and the resulting solution was cooled to –60 °C.
Dimethyl sulfoxide (237 mg, 3.04 mmol) was added over 15 min,
and the mixture was stirred for 10 min. Compound 15[20] (300 mg,
1.01 mmol) in CH2Cl2 (20 mL) was added dropwise over 30 min,
DIPEA (37.0 mg, 0.287 mmol) in CH3CN (0.5 mL) were sequen- and the mixture was stirred for 2 h. Triethylamine (705.4 mg,
tially added to the reaction mixture at –5 °C. The resulting mixture
was gradually warmed to room temperature and stirred for 20 h.
6.97 mmol) was added dropwise, and the mixture was stirred for
1 h. NH4Cl (aq. 50 mL) was then added, and the mixture was ex-
1310
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1305–1313

An Enantiomerically Enriched Chelator for Positron Emission Tomography
tracted with CH2Cl2 (3ϫ 100 mL). The combined organic layers in THF (15 mL) at 0 °C. The resulting mixture was warmed to
were concentrated under reduce pressure, and the residue was puri-
fied by column chromatography on silica gel (60–230 mesh) eluted
with 30% ethyl acetate/hexanes to provide pure 16 (149 mg, 50%).
room temperature and stirred for 2 h and then heated to reflux for
14 h. The reaction mixture was concentrated, and the residue was
treated with CH3OH (2ϫ 20 mL). This quenching process was re-
1H NMR (CDCl3, 300 MHz): δ = 1.40 (s, 9 H), 3.11 (dd, J = 13.8, peated twice. The residue was dried in vacuo and treated with 6 m
7.2 Hz, 1 H), 3.32 (dd, J = 13.8, 5.7 Hz, 1 H), 4.40–4.49 (m, 1 H), HCl (10 mL). The resulting mixture was stirred at room tempera-
5.18 (d, J = 6.8 Hz, 1 H), 7.34 (d, J = 8.6 Hz, 2 H), 8.14 (d, J = ture for 1 h and then heated to reflux for 2 h. The aqueous solution
8.6 Hz, 2 H), 9.64 (s, 1 H) ppm. 13C NMR (CDCl3, 75 MHz): δ =
was cooled to room temperature, adjusted to pH 7 with 2 m NaOH,
28.20 (q), 35.01 (t), 60.46 (d), 80.60 (s), 123.80 (d), 130.29 (d), and then extracted with CHCl3 (2ϫ 30 mL). The aqueous layer
1
144.12 (s), 147.05 (s), 155.26 (s), 198.24 (d) ppm. The H and 13C
NMR spectroscopic data are essentially identical to those pre-
viously reported.[21]
was further adjusted to pH 10 and 13. At each pH, the aqueous
solution was extracted with CHCl3 (2ϫ 30 mL). The organic layers
extracted at pH 7 and 10 were combined, dried with MgSO4, fil-
tered, and concentrated in vacuo to provide free amine 20
(613.2 mg, 80%) as a light yellow solid. 1H NMR ([D6]DMSO,
300 MHz): δ = 2.26 (s, 3 H), 2.40–2.62 (m, 2 H), 3.16–3.80 (m, 5
H), 7.12 (d, J = 8.2 Hz, 2 H), 7.28 (d, J = 8.2 Hz, 2 H), 7.37 (d, J
= 8.4 Hz, 2 H), 7.92 (d, J = 8.4 Hz, 2 H) ppm. 13C NMR ([D6]-
DMSO, 75 MHz): δ = 21.2 (q), 37.9 (t), 46.9 (t), 58.4 (d), 123.4 (d),
126.5 (d), 129.6 (d), 130.8 (d), 139.1 (s), 142.4 (s), 146.2 (s), 147.7
(s) ppm. The 1H and 13C NMR spectroscopic data of (S)-20 are
essentially identical to those of rac-20.[16] [α]2D6 = –17.8 (c = 0.225,
DMSO).
1-(4-Nitrophenyl)-3-(1,4,7-triazonan-1-yl)propan-2-amine (rac-23):
To a solution of 16 (100 mg, 0.34 mmol) in 1,2-dichloroethane
(8 mL) was added 17 (112 mg, 0.34 mmol) at 0 °C. NaBH(OAc)3
(107.3 mg, 0.51 mmol) was added portionwise over 5 min, and the
resulting mixture was warmed to room temperature and stirred for
24 h, after which the solvent was removed in vacuo. The residue
was treated with saturated NaHCO3 solution (20 mL) and ex-
tracted with CH2Cl2 (2ϫ 30 mL). The organic layers were com-
bined and concentrated in vacuo, and the product (190 mg, 92%)
from reductive amination was isolated by column chromatography
on silica gel (60–230 mesh) eluted with 20% ethyl acetate in hex-
anes. The product was treated with 4 m HCl in 1,4-dioxane (8 mL),
and the mixture was stirred for 16 h at room temp. Diethyl ether
(50 mL) was added, and the resulting mixture was stirred for
30 min. The slurry formed was filtered and dissolved in 18 MΩ
H2O, and the resulting solution was concentrated to dryness to
N-{(2S)-1-[4,7-Bis(4-methylphenylsulfonyl)-1,4,7-triazonan-1-yl]-3-
(4-nitrophenyl)propan-2-yl}-4-methylbenzene-1-sulfonamide (22): To
a solution of 20 (600 mg, 1.72 mmol) and 21[22] (1.335 g,
1.72 mmol) in CH3CN (25 mL) was added Na2CO3 (1.82 g,
17.2 mmol). The resulting mixture was heated to reflux for 4 d,
after which the reaction mixture was cooled to room temperature
and filtered, and the solvents were evaporated. The residue was
purified by column chromatography on silica gel (60–230 mesh)
eluted with 3% ethyl acetate/CH2Cl2 to provide pure 22 (710 mg,
afford rac-23 (75 mg, 98%). The H and 13C NMR spectroscopic
1
data are essentially identical to those previously reported.[16]
(2S)-2-Amino-3-(4-nitrophenyl)propanamide (18): NH4OH (20 mL)
was added to a solution of 14 (1.03 g, 4.48 mmol) in toluene
(20 mL) at room temperature. The resulting mixture was stirred for
14 h at room temperature. The reaction mixture was filtered and
1
54%) as a white solid. H NMR (CDCl3, 300 MHz): δ = 2.17 (s, 3
H), 2.43 (s, 6 H), 2.70–3.60 (m, 17 H), 7.10 (d, J = 8.0 Hz, 2 H),
7.28–7.34 (m, 6 H), 7.63–7.69 (m, 6 H), 7.99 (d, J = 8.3 Hz, 2 H)
ppm. 13C NMR (CDCl3, 75 MHz): δ = 21.1 (q), 21.5 (q), 40.0 (t),
52.4 (t), 53.4 (t), 53.7 (d), 55.6 (t), 60.4 (t), 123.4 (d), 127.0 (d),
127.1 (d), 129.4 (d), 129.9 (d), 130.4 (d), 135.0 (s), 137.8 (s), 142.9
(s), 143.8 (s), 145.8 (s), 146.6 (s) ppm. The 1H and 13C NMR spec-
1
washed with toluene to provide pure 18 (572 mg, 62%). H NMR
([D6]DMSO, 300 MHz): δ = 2.42–3.10 (m, 4 H), 3.42 (t, J = 3.0 Hz,
1 H), 7.32–7.04 (s, 1 H), 7.30–7.53 (m, 3 H), 8.12 (d, J = 8.5 Hz, 2
H) ppm. 13C NMR ([D6]DMSO, 75 MHz): δ = 41.1 (t), 56.2 (d),
123.5 (d), 131.1 (d), 146.5 (s), 148.1 (s), 176.6 (s) ppm. The 1H and
13C NMR spectroscopic data of (S)-18 are essentially identical to
those of rac-18.[16] [α]2D6 = +13.2 [c = 0.35, dimethyl sulfoxide
(DMSO)].
troscopic data are essentially identical to those of rac-22.[16] [α]2D6
–2.4 (c = 0.51, DMSO).
=
(2S)-1-(4-Nitrophenyl)-3-(1,4,7-triazonan-1-yl)propan-2-amine (23):
Concentrated H2 SO4 (10 mL) was added to 22 (650 mg,
0.844 mmol). The resulting mixture was heated to 115 °C and
stirred for 72 h, after which the reaction mixture was cooled to
room temperature. The reaction mixture was added dropwise into
diethyl ether (65 mL) at –60 °C over 40 min. The resulting mixture
was stirred for 20 min, filtered, and washed with cold diethyl ether.
The residue was quickly dissolved in water (30 mL), and the re-
sulting aqueous solution was extracted with CHCl3 (30 mL). The
aqueous solution was adjusted to pH 7 with 5 m NaOH (aq.) and
extracted with CHCl3 (2ϫ 50 mL). The aqueous solution was fur-
ther adjusted to pH 10 and 13. At each pH, the aqueous solution
was extracted with CHCl3 (3 ϫ 50 mL). The organic layers ex-
tracted at pH 10 and 13 were combined, dried with MgSO4, fil-
tered, and concentrated to provide 23 (105 mg, 40%). 1H NMR
(CDCl3, 300 MHz): δ = 2.25–2.42 (m, 5 H), 2.50–2.89 (m, 15 H),
3.10–3.18 (m, 1 H), 7.35 (d, J = 8.5 Hz, 2 H), 8.12 (d, J = 8.5 Hz,
2 H) ppm. 13C NMR (CDCl3, 75 MHz): δ = 42.1 (t), 46.1 (t), 46.5
(t), 51.1 (d), 53.2 (t), 64.8 (t), 123.6 (d), 129.9 (d), 146.4 (s), 147.6
(2S)-2-[(4-Methylphenyl)sulfonamido]-3-(4-nitrophenyl)propanamide
(19): A solution of TsCl (1.137 g, 5.97 mmol) in N,N-dimethylform-
amide (DMF; 5 mL) was added dropwise over 30 min to a solution
of 18 (520 mg, 2.49 mmol) and Et3N (906 mg, 8.95 mmol) in DMF
(5 mL) at 0 °C. The resulting mixture was warmed to room tem-
perature and stirred for 24 h before the solvent was evaporated.
H2O (50 mL) was added to the resulting residue, and a solid
formed. The solid was collected by filtration and washed with
1
CH2Cl2 (2ϫ 20 mL) to provide pure 19 (840 mg, 93%). H NMR
([D6]DMSO, 300 MHz): δ = 2.25 (s, 3 H), 2.64–2.79 (m, 1 H), 2.92–
3.04 (m, 1 H), 3.90 (m, 1 H), 7.02–7.13 (m, 3 H), 7.30–7.36 (m, 4
H), 7.44 (s, 1 H), 7.94 (d, J = 8.2 Hz, 2 H), 8.06 (d, J = 8.5 Hz, 1
H) ppm. 13C NMR ([D6]DMSO, 75 MHz): δ = 21.2 (q), 38.5 (t),
57.7 (d), 123.4 (d), 126.6 (d), 129.5 (d), 131.0 (d), 138.6 (s), 142.5
(s), 146.2 (s), 146.5 (s), 172.5 (s) ppm. The 1H and 13C NMR spec-
troscopic data of (S)-19 are essentially identical to those of rac-
19.[16] [α]2D6 = +15.9 (c = 0.44, DMSO).
1
(s) ppm. The H and 13C NMR spectroscopic data are essentially
identical to those of rac-23.[16] [α]2D6 = +12.2 (c = 0.30, CHCl3).
N-[(2S)-1-Amino-3-(4-nitrophenyl)propan-2-yl]-4-methylbenzene-1-
sulfonamide (20): BH3 in tetrahydrofuran (THF; 1 m, 15 mL) was
added dropwise over 30 min to a solution of 19 (800 mg, 363 mmol)
tert-Butyl 2-{[(2S)-1-{4,7-Bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-tri-
azonan-1-yl}-3-(4-nitrophenyl)propan-2-yl]amino}acetate (24): To a
Eur. J. Org. Chem. 2014, 1305–1313
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1311

H.-S. Chong et al.
FULL PAPER
slurry of 23 (105 mg, 0.23 mmol) as an acidic salt and KI (46 mg,
0.28 mmol) in DMF (5 mL) at 0 °C were added tert-butyl bromo-
acetate (149 mg, 0.77 mmol) and DIPEA (301 mg, 2.33 mmol)
1:1 (v/v) as the mobile phase. A solution of 64Cu-labeled (S)-C-
NE3TA (2.0 μL) was withdrawn at the designated time points (1,
10, and 30 min), spotted on a TLC plate, and then eluted with the
dropwise over 10 min. The resulting mixture was stirred for 2 h at mobile phase. After completion of the elution, the TLC plate was
0 °C and 2 h at room temperature. The reaction mixture was heated
to 50 °C and stirred for 24 h, after which the reaction mixture was
cooled to room temperature and then to 0 °C. HCl (6 m, 5 mL)
and heptane (30 mL) were sequentially added to the solution. The
resulting mixture was vigorously stirred for 5 min, and the heptane
layer was separated. The aqueous layer was further extracted with
heptane (2ϫ 30 mL) and treated with 10% Na2CO3 (45 mL). Ad-
ditional heptane (30 mL) was added into the aqueous solution. The
resulting solution was stirred for 30 min, and the heptane layer was
separated. The combined organic layers were washed with water
(20 mL), dried with MgSO4, filtered, and concentrated to dryness
to give crude 24 (71 mg), which was further purified by semiprepar-
ative HPLC (method 4) to provide pure 24 (39 mg, 26%). Analyti-
warmed and dried on the surface of a hotplate maintained at 35 °C
and scanned with a TLC scanner (Bioscan). Unbound and bound
radioisotopes appeared at 30–35 mm (Rf = 0.54) and 40–45 mm (Rf
= 0.71) from the bottom of the TLC plate, respectively.
In vitro Serum Stability of 64Cu-(S)-C-NE3TA: Human serum was
purchased from Gemini Bioproducts. 64Cu-(S)-C-NE3TA was pre-
pared by reaction of (S)-C-NE3TA (30 μg) with 64Cu (100 μCi) in
0.25 m NH4OAc buffer (pH 5.5) for 2 h at room temperature, and
the labeling efficiency of (S)-C-NE3TA was ca. 100% as deter-
mined by ITLC [10% NH4OAc/MeOH 1:1 (v:v)]. The freshly pre-
pared 64Cu-(S)-C-NE3TA was directly used for the serum stability
study without further purification. 64Cu-(S)-C-NE3TA (85 μCi,
10 μL) was added to human serum (90 μL) in a microcentrifuge
tube. The stability of 64Cu-(S)-C-NE3TA in human serum was
evaluated at 37 °C for 2 d. A solution of the 64Cu-(S)-C-NE3TA in
serum was withdrawn at the designated time points and evaluated
by ITLC (as described above) and HPLC (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 bound to serum eluted earlier (tR = 2.5 min)
than 64Cu-(S)-C-NE3TA (tR = 7 min).
1
cal HPLC (method 1, tR = 39 min). H NMR (CDCl3): δ = 1.41
(s, 27 H), 2.30–2.52 (m, 1 H), 2.63–2.94 (m, 15 H), 3.20–3.36 (m,
8 H), 7.36 (d, J = 8.01 Hz, 2 H), 8.15 (d, J = 8.01 Hz, 2 H) ppm.
13C NMR (CDCl3, 75 MHz): δ = 28.1 (q), 37.5 (t), 53.4 (t), 55.5
(t), 55.7 (t), 56.4 (t), 59.8 (t), 60.3 (t), 62.71 (d), 80.8 (s), 80.9 (s),
123.32 (d), 130.3 (d), 146.30 (s), 149.10 (s), 171.1 (s), 171.4 (s) ppm.
1
The H and 13C NMR spectroscopic data are essentially identical
to those of rac-24.[23] [α]2D6 = +14.2 (c = 0.30, CHCl3).
EDTA Challenge: 64Cu-(S)-C-NE3TA was prepared by reaction of
(S)-C-NE3TA (5 μg) with 64Cu (30 μCi) in 0.25 m NH4OAc buffer
(pH 5.5) for 2 h at room temperature, and the freshly prepared
64Cu-(S)-C-NE3TA was directly used for the experiment. 64Cu-(S)-
C-NE3TA was mixed with EDTA at a 100-fold molar excess.[24]
The resulting mixture was incubated for 24 h at 37 °C. The stability
of 64Cu-(S)-C-NE3TA in the solution was evaluated by ITLC
(20 mm EDTA in 0.15 m NH4OAc). A solution of the 64Cu-(S)-C-
NE3TA in EDTA (3–9 μL) was withdrawn at the designated time
points and evaluated by ITLC as described above.
2-{[(2S)-1-[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]-3-(4-nitro-
phenyl)propan-2-yl]amino}acetic Acid (13): HCl in dioxane (4 m,
3 mL) was added to a flask containing 24 (4.5 mg, 6.9 μmol), and
the resulting solution was stirred for 16 h. Diethyl ether (20 mL)
was added, and the mixture was stirred for 30 min and filtered. The
funnel was moved quickly to another Buchner flask. The filter cake
was washed with 18 MΩ H2O, and the resulting solution was con-
1
centrated to dryness to afford 13 (3.69 mg, 91%). The H and 13C
NMR and analytical HPLC data of (S)-13 are essentially identical
to those reported above. [α]2D6 = +3.40 (c = 0.19, H2O).
In vivo Biodistribution: 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 stud-
ies. An aliquot of the 64Cu-radiolabeled complex (60 μCi) was pre-
pared as described above and intravenously injected into the tail
vein in 100 μL of phosphate-buffered saline. At 1, 4, and 24 h post-
injection, the mice were sacrificed, and blood, liver, kidney, muscle,
and bone were collected, weighed, and counted in a gamma
counter. The radioactivity from each tissue or organ was decay-
corrected with a known aliquot of the injected dose, and the per-
centage of injected dose per gram of tissue (% ID/g) was calculated.
The values are presented as mean Ϯstandard deviation (SD) for
each group of three mice.
Preparation and Characterization of CuII-(S)-C-NE3TA: The CuII
complex of (S)-C-NE3TA was prepared by reaction of (S)-C-
NE3TA (5 μL, 10 mm) with CuCl2 (5 μL, 10 mm) in 0.25 m
NH4OAc buffer (pH 5.5) for 24 h at room temperature and
300 rpm. The prepared CuII-(S)-C-NE3TA was characterized by
analytical HPLC (tR = 6.4 min, method 2).
Radiolabeling Reaction Kinetics of (S)-C-NE3TA with 64Cu: All
HCl solutions were prepared from ultrapure HCl (Fisher Scien-
tific). For metal-free radiolabeling, plasticware including pipette
tips, tubes, and caps were soaked in 0.1 m HCl (aq.) overnight,
washed thoroughly with Milli-Q (18 MΩ) water, and air-dried over-
night. NH4OAc buffer solution (0.25 m, pH 5.5) was prepared with
ultrapure ammonium acetate (Aldrich), and the pH of the solution
was adjusted with 0.1 m and 1 m HCl solutions. The resulting buffer
solution was treated with Chelex-100 resin (Biorad 1 g/100 mL
buffer solution), shaken overnight at room temperature, and fil-
tered through a 0.22 μm filter (Corning) prior to use. TLC plates
(6.6ϫ1 cm, Silica gel 60 F254, EMD Chemicals Inc.) with the origin
line drawn at 0.6 cm from the bottom were prepared. To a buffer
solution (12.41–16.95 μL, 0.25 m NH4OAc, pH 5.5) in a capped
microcentrifuge tube (1.5 mL, Fisher Scientific) was sequentially
added a solution of (S)-C-NE3TA in 0.25 m NH4OAc buffer
(1.59 μL, 7.97ϫ 10–4 m) and 64Cu in 0.05 m HCl (30 μCi, 1.5–
6.0 μL). The total volume of the resulting solution was 20 μL. The
reaction mixture was agitated with a thermomixer (Eppendorf) set
at 1000 rpm at room temperature for 30 min. The labeling effi-
Supporting Information (see footnote on the first page of this arti-
cle): HPLC and ITLC chromatograms for assessment of radiolabel-
ing reaction kinetics and serum stability, EDTA challenge, and in
vivo biodistribution data.
Acknowledgments
This work was in part supported by the Department of Veterans
Affairs through the provision of resources and use of facilities at
the Harry S. Truman Memorial Veterans’ Hospital in Columbia,
ciency was determined by ITLC eluted with 10% NH4OAc/MeOH MO.
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1305–1313

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Received: October 3, 2013
Published Online: January 15, 2014
Eur. J. Org. Chem. 2014, 1305–1313
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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