Convenient synthesis and evaluation of a heptadentate bifunctional ligand for radioimmunotherapy applications

Article abstract of DOI:10.1002/ejoc.201101063

An efficient synthetic route to a bifunctional chelating agent C-NE3TA-NCS for antibody-targeted radioimmunotherapy (RIT) applications was developed. Various synthetic methods centered on key reaction steps, including bimolecular cyclization, ring-opening

Full text of DOI:10.1002/ejoc.201101063

FULL PAPER
DOI: 10.1002/ejoc.201101063
Convenient Synthesis and Evaluation of a Heptadentate Bifunctional Ligand
for Radioimmunotherapy Applications
Hyun-Soon Chong,*[a] Xiang Sun,[a] Pengfei Dong,[a] and Chi Soo Kang[a]
Keywords: Medicinal chemistry / Immunochemistry / Radiochemistry / Radioimmunotherapy / Yttrium / Macrocyclic
ligands / Chelates
An efficient synthetic route to a bifunctional chelating agent
C-NE3TA-NCS for antibody-targeted radioimmunotherapy
(RIT) applications was developed. Various synthetic methods
centered on key reaction steps, including bimolecular cycli-
zation, ring-opening reactions of aziridine and aziridinium
cations, and reductive amination, were explored to optimize
the preparation of a tetraaza-based chelate TANPA and C-
NE3TA analogues. Heptadentate C-NE3TA-NCS was conju-
gated to a tumor-targeting antibody and compared with
hexadentate C-NOTA-NCS for radiolabeling reaction kinet-
ics with lanthanides for RIT. The C-NE3TA–antibody conju-
gate displayed significantly enhanced complexation kinetics
with 90Y compared with the C-NOTA–antibody conjugate.
The synthetic methods for TANPA and C-NE3TA-NCS re-
ported herein have broad applications for the preparation of
bifunctional and macrocyclic chelating agents.
Introduction
carboxylate donor group appended by an ethylene bridge
Bifunctional ligands are essential components for bio- to the macrocyclic ring. C-NOTA is known to be an inade-
medical and radiopharmaceutical applications of biolo- quate chelate for lanthanides by virtue of its cavity size, in-
gically active metals as cytotoxic agents or imaging sufficient number of donors to saturate the coordination
probes.[1–3] In particular, receptor-targeted cancer therapy sphere of a lanthanide cation, and inability to assume an
and imaging techniques, including radioimmunotherapy optimal geometry for the six donor groups.[14,15] It was pro-
(RIT), radioimmunoimaging (RII), and magnetic resonance posed that the acyclic pendant donor groups in C-NE3TA
imaging (MRI), require a bifunctional ligand that can could rapidly initiate coordination to the metal, and there-
tightly and/or rapidly complex a radioactive or nonradioac- after, the macrocyclic component in the chelate could wrap
tive lanthanide, such as 90Y (Emax = 2.3 MeV, t1/2 = 64.1 h), around the metal ion trapped in the acyclic pendant arm to
177Lu (Emax = 0.5 MeV, t1/2 = 6.7 d), and GdIII [2,4,5]
Our achieve maximum complex stability by saturating the metal
.
continued research on chelation chemistry[6–12] led to the coordination sphere, as in the case of C-NOTA. The cavity
design and preparation of heptadentate ligand C-NE3TA[8] size becomes irrelevant because the metal sits above the
(Figure 1). C-NE3TA contains the macrocyclic component plane of the ring nitrogen atoms. Thus, C-NE3TA is pro-
of C-NOTA[13] (Figure 1) and an acyclic pendant amino- posed to bind the metal ion more rapidly than C-NOTA,
Figure 1. Structures of C-NOTA, C-NE3TA, 7, tert-butyl C-NE3TA, and C-NE3TA-NCS.
while maintaining high complex stability of C-NOTA
Chemical, and Physical Science Department, Illinois Institute through dynamic and three-dimensional binding.
[a] 3101 S. Dearborn St, LS 182, Chemistry Division, Biological,
of Technology,
C-NE3TA can also form a neutral complex with various
Chicago, IL 60616, USA
lanthanides. Neutral metal complexes do not require coun-
terions and are known to have lower osmotic activity and
reduced protein binding and toxicity than ionic com-
plexes.[16,17] Neutral metal complexes are also known to be
Fax: 1-312-567-3494
E-mail: Chong@iit.edu
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101063 or from
the author.
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H.-S. Chong, X. Sun, P. Dong, C. S. Kang
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less sensitive to acid-/cation-promoted dissociation than an- C-NOTA–hercpetin conjugate was prepared and evaluated
ionic complexes. For example, the neutral GaIII complex of for the same radiolabeling reaction kinetics study.
NOTA was stable at pH 3.[15]
We previously evaluated C-NE3TA as a chelator of YIII
,
LuIII, and GdIII for RIT and MRI applications, and 90Y,
177Lu, and 153Gd complexes of C-NE3TA produced excel-
lent in vitro and in vivo complex stability profiles.[8] With
Results and Discussion
Compound 7 contains four reactive amines and can be
the potential of using C-NE3TA for targeted cancer therapy further substituted with potential donor groups. With po-
and imaging, we wanted to develop an efficient synthetic tential applications of 7 for the preparation of various che-
route suitable for scaled up production of C-NE3TA-NCS lating systems, we wanted to develop a practical synthetic
containing a functional group for conjugation to a tumor process for 7. The retrosynthetic routes to 7[10] are centered
targeting antibody or peptide (Figure 1). We herein report on the following key reaction steps: ring opening of azirid-
an efficient synthesis and evaluation of C-NE3TA-NCS. ine, reductive amination, and bimolecular cyclization
Various synthetic routes to the key intermediate macro- (Scheme 1).
cyclic chelating backbone 7 (Figure 1) for the preparation
The first synthetic route to 7 includes the preparation
of C-NE3TA-NCS are also presented. C-NE3TA conju- of the precursor molecule 6 through ring opening of N-Ts-
gated to a tumor-targeting antibody, trastuzumab, was protected aziridine 3 by N-Ts-protected 5[18] (Scheme 2). In
evaluated for its radiolabeling reaction kinetics with the β- our initial approach to prepare 6, we wanted to synthesize
emitting radioisotopes 90Y and 177Lu. For comparison, a compound 2 by the reaction of 1[10] with TsCl in the pres-
Scheme 1. Retrosynthetic routes to the key precursor molecule 7 (Ts = tosyl, BOC = tert-butyloxycarbonyl).
Scheme 2. Synthetic route 1 for the formation of 7: Regiospecific ring opening of N-Ts aziridine 3. DIPEA = diisopropylethylamine,
DMAP = 4-(dimethylamino)pyridine.
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A Heptadentate Bifunctional Ligand for Radioimmunotherapy
ence of triethylamine. However, the reaction afforded com- dium triacetoxyborohydride to provide 15, which was fur-
pound 4 through the formation of compound 5 and subse- ther treated with trifluoroacetic acid (TFA) to remove the
quent regiospecific ring opening of compound 3 by nucleo- BOC groups in 15.[10] Of the synthetic routes for the prepa-
philic chloride present in the reaction mixture. Compound ration of 7 explored, this chromatography-free route was
4 was further reacted with 5, which led to the isolation of the most efficient process for the scaled up production of 7.
6 in a yield of 84%. It was reasonably speculated that 3 was Macrocyclic backbone 7 was obtained in two steps with an
initially formed from intramolecular substitution of 4 and overall yield of 87%.
then regiospecific ring opening of 3 at the less hindered car-
A practical and reproducible synthetic route toward C-
bon by the nucleophilic amine in compound 5 provided 6. NE3TA-NCS for RIT applications is shown in Scheme 5.
The regioisomer of 6, which can be formed from the attack The advantages of the synthetic route include (i) formation
of 5 at the more hindered methine carbon in 3, was not of the key intermediate compound 21 by an efficient ring
obtained from the reaction. When the reaction was repeated opening of the aziridinium ion, (ii) formation of the desired
in THF, aziridine analogue 3 was obtained as the major C-NE3TA-NCS in reasonable yields with fewer reaction
product (46% yield). To optimize the synthesis of 6, a mix- steps than the original synthetic route for the chelator,[8]
ture of 3 and 4 isolated from the reaction of 1 with TsCl and (iii) reproducible and relatively convenient chromato-
was directly reacted with 5. Compound 6 was obtained in graphic purification of 21. The original synthetic route[8] to
an improved isolated yield (76%). The tosyl groups in 6 C-NE3TA-NCS includes the preparation of tert-butyl C-
were removed by treatment of 6 with concentrated sulfuric NE3TA by selective base-promoted alkylation of 7 by using
acid to provide 7 in a yield of 68%.
tert-butyl bromoacetate. Although the yield (Ͻ 65%) of
The second synthetic route for the formation of 7 in- tert-butyl C-NE3TA (Figure 1) was reasonably good, flash
cluded base-promoted bimolecular cyclization between chromatographic purification of the macrocyclic compound
compounds 11 and 12 as the key reaction step (Scheme 3). on silica gel was challenging due to its high polarity and
Ester 8 was converted into 9[19] and isolated in 78% yield. unwanted polyalkylated or partially alkylated byproducts
The reaction of compound 9 with TsCl provided N-Ts-pro- present in the reaction mixture. TLC is not a very useful
tected amide 10, which was subsequently reduced to the analytical tool to distinguish between the desired product
primary amine 11. The base-promoted reaction of 11 with and byproducts because both of the compounds appear on
ditosylate 12[20] provided compound 6. Identical 1H and 13
C
TLC as concentration-dependent tailing spots. Protection
NMR spectroscopic data were obtained for 6 prepared by of the secondary amine of tert-butyl C-NE3TA by using a
the two synthetic routes, which further confirmed the regi- benzyl group to enhance the lipophilicity of the product
ochemistry observed in the ring opening of 3.
was expected to allow a relatively straightforward chroma-
The third synthetic approach was based on reductive am- tographic purification process. The ring-opening reaction of
ination of 13 with 14 followed by removal of the BOC aziridinium ion 19 by 20 was expected to avoid the forma-
groups in 15 (Scheme 4). N-BOC-protected macrocyclic tion of byproducts produced in the reaction of 7 with tert-
compound 14[10] was treated with 13 in the presence of so- butyl bromoacetate.[8] Compound 16 was prepared by re-
Scheme 3. Synthetic route 2 for the formation of 7: Bimolecular cyclization of 11 and 12.
Scheme 4. Short and chromatography-free synthetic route 3 for the formation of 7: reductive amination of 13 with 14 (DCE = 1,2-
dichloroethane).
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H.-S. Chong, X. Sun, P. Dong, C. S. Kang
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Scheme 5. Efficient synthesis of C-NE3TA-NCS for conjugation to an antibody (NBS = N-bromosuccinimide, Bn = benzyl, NCS = N-
chlorosuccinimide).
ductive amination of 1 and benzaldehyde followed by re- by using SE-HPLC or instant thin-layer chromatography
duction of the intermediate imine analogue. The amino (ITLC) after challenging the reaction mixture with 10 mm
alcohol 16 was further treated with tert-butylbromoacetate diethylenetriaminepentaacetic acid (DTPA), and the radio-
to provide 17, which was converted to N,N-bisubstituted labeling efficiency was determined. The data given in
β-amino bromide 18 by bromination with NBS and PPh3. Table 1 indicate that the C-NE3TA–trastuzumab conjugate
Aziridinium ion 19 was produced by the reaction of com- displayed relatively rapid radiolabeling with 90Y (5 min,
pound 18 with silver perchlorate and further treated with 47%; 1 h, 89%). Complexation of C-NE3TA–trastuzumab
20 to provide key intermediate 21, which was isolated in a with 177Lu was slow with a radiolabeling efficiency of 38%
yield of 51%; tert-butyl groups in compound 21 were re- after 1 h. C-NE3TA appears to have an insufficient number
moved through the treatment of 21 with 4 m HCl in 1,4- of donors to rapidly complex the larger metal, 177Lu. As
dioxane. Compound 22 was subjected to hydrogenolysis to expected,[14,15] C-NOTA was completely ineffective at bind-
provide compound 23 in quantitative yield. The amino ing both 90Y and 177Lu, which have ionic radii of 104 and
group in compound 23[8] was converted into the isothiocya- 100 pm, respectively. The macrocyclic cavity of NOTA ap-
nate group to produce the desired ligand C-NE3TA-NCS.[8] pears to be too small to bind lanthanides with high kinetic
C-NE3TA-NCS and C-NOTA-NCS were conjugated with stability. The results of complexation kinetics indicate that
a tumor-targeting antibody, trastuzumab. The antibody was the additional acyclic bidentate donor system was essential
approved by the Food and Drug Administration (FDA) for for the improved complexation kinetics of C-NE3TA rela-
the treatment of metastatic breast cancer and is known to tive to C-NOTA.
selectively target the HER2 protein overproduced in various
Table 1. Evaluation of C-NE3TA–trastuzumab[a] and C-NOTA–
tumors, including those found in colon and breast can-
trastuzumab[b] conjugates for radiolabling efficiency [%] with 90Y
cers.[21,22] The corresponding conjugates were evaluated for
and 177Lu (0.25 m NH4OAc, pH 5.5, r.t.) using ITLC or HPLC
(data given in parentheses).
radiolabeling with β-emitting radionuclides 90Y and 177Lu
at room temperature. 90Y and 177Lu have been extensively
90Y
177Lu
investigated as potent cancer therapeutic radionuclides.
Zevalin, containing 90Y, is the first RIT drug approved for
the treatment of non-Hodgkin lymphoma.[1] C-NE3TA-
NCS and C-NOTA-NCS were conjugated to trastuzumab
and the concentration of trastuzumab in the corresponding
conjugates was quantified by the spectroscopic method de-
Time
[min]
C-NE3TA–
trastuzumab
C-NOTA–
C-NE3TA–
C-NOTA–
trastuzumab trastuzumab trastuzumab
23.3Ϯ2.7
(22.9Ϯ2.0)
46.6Ϯ3.5
(39.3Ϯ3.4)
60.1Ϯ0.7
(49.9Ϯ1.7)
73.9Ϯ2.6
(61.8Ϯ1.6)
81.6Ϯ1.6
(66.1Ϯ1.1)
88.9Ϯ1.7
(71.9Ϯ1.3)
13.5Ϯ0.7
1
5
2.9Ϯ0.2
13.0Ϯ8.2
10.1Ϯ4.8
8.6Ϯ0.0
8.7Ϯ2.8
16.1Ϯ5.4
1.1Ϯ0.2
3.7Ϯ1.6
5.0Ϯ1.8
9.8Ϯ2.1
13.3Ϯ1.1
22.9Ϯ4.5
(10.5Ϯ0.8)
21.8Ϯ0.6
(18.0Ϯ0.2)
26.8Ϯ0.5
(21.7Ϯ0.2)
32.1Ϯ1.3
(24.7Ϯ0.7)
33.6Ϯ1.7
(26.0Ϯ0.4)
37.7Ϯ1.6
(29.0Ϯ0.2)
scribed previously.[9] The CuII–arsenazo III (AAIII)-based 10
UV/Vis spectrophotometric assay was used for the determi-
20
nation of the number of C-NE3TA or C-NOTA ligands
linked to trastuzumab (L/P ratio). The respective ligand to
30
protein (L/P) ratios for C-NE3TA–trastuzumab[8] and C-
60
NOTA–trastuzumab were 3.9 and 4.3.
The purified C-NE3TA–trastuzumab and C-NOTA–tra-
stuzumab conjugates (0.25 m NH4OAc, pH 5.5) were lab-
eled with 90Y or 177Lu (60 μCi) at room temperature (r.t.).
[a] Radiolabeling efficiency (mean Ϯ standard deviation, %) was
measured in triplicate by using HPLC and ITLC. [b] Radiolabeling
efficiency (mean Ϯ standard deviation, %) was measured in dupli-
During the reaction (1 h), the components were analyzed cate by using ITLC.
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A Heptadentate Bifunctional Ligand for Radioimmunotherapy
solution of 1 (50 mg, 0.255 mmol), Et3N (77.2 mg, 1.28 mmol), and
DMAP (3.1 mg, 0.0255 mmol) in CH2Cl2 (1.0 mL) at 0 °C . The
resulting mixture was stirred at room temperature for 6 d before
the solvent was evaporated. The residue was purified by column
chromatography on silica gel (60–230 mesh) eluting with 18% ethyl
acetate/hexane to provide pure 4 (76 mg, 81%) as a light yellow
Conclusions
We have presented an efficient synthetic route for the bi-
functional ligand C-NE3TA-NCS for targeted RIT applica-
tions. Bimolecular cyclization and ring-opening reactions of
aziridine and aziridinium ions were explored for improved
and efficient synthesis of 7, which was a useful macrocyclic
chelating backbone and key precursor for the C-NE3TA
analogue. The synthetic methods reported herein can be ap-
plied to the preparation of various macrocyclic ligand sys-
tems. The C-NE3TA–trastuzumab conjugate exhibited sig-
nificantly enhanced complexation kinetics with 90Y relative
to the C-NOTA–trastuzumab conjugate. C-NE3TA can be
further evaluated for RIT application using 90Y and is a
potential bifunctional chelator of other small divalent and
trivalent cations, such as 64Cu and 68Ga, for RII.
1
solid. H NMR (CDCl3, 300 MHz): δ = 2.39 (s, 3 H), 2.83 (dd, J
= 13.8, J = 4.2 Hz, 1 H), 3.04 (dd, J = 13.8; J = 3.0 Hz, 1 H), 3.55–
3.58 (m, 2 H), 3.68–3.78 (m, 1 H), 5.41–5.55 (d, J = 8.9 Hz, 1 H)
7.12–7.22 (m, 4 H), 7.53 (d, J = 8.2 Hz, 2 H), 7.96 (d, J = 8.2 Hz,
2 H) ppm. 13C NMR (CDCl3, 300 MHz): δ = 21.4 (q), 38.2 (t),
47.6 (t), 55.2 (d), 123.7 (d), 126.8 (d), 129.7 (d), 130.1 (d), 136.9
(s), 144.0 (s), 144.2 (s), 146.8 (s) ppm. HRMS (ESI+): calcd. for
C16H17N2O4SClNa [M + H + Na]+ 391.0490, found 391.0506.
N-(1-{4,7-Bis[(4-methylphenyl)sulfonyl]-1,4,7-triazonan-1-yl}-3-(4-
nitrophenyl)propan-2-yl)-4-methylbenzene-1-sulfonamide (6): Prepa-
ration from the reaction of compound 4 with 5. DIPEA (19.4 mg,
0.150 mmol) was added to a solution of 4 (50 mg, 0.136 mmol) and
5[18] (59.4 mg, 0.136 mmol) in CH3CN (3 mL). The resulting mix-
ture was heated at reflux for 4.5 d before the reaction mixture was
cooled to room temperature and the solvent was evaporated. The
residue was purified by column chromatography on silica gel (60–
230 mesh) eluting with 3% ethyl acetate/CH2Cl2 to provide pure 6
(87 mg, 84%) as a light yellow solid. 1H 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, 300 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. HRMS (ESI+):
calcd. for C36H44N5O8S3 [M + H]+ 770.2347, found 770.2311.
Experimental Section
Instruments and Methods: H, 13C, and DEPT NMR spectra were
1
obtained by using a Bruker 300 instrument and chemical shifts are
reported in parts per million on the δ scale relative to tetramethyl-
silane (TMS). Elemental microanalyses were performed by Gal-
braith Laboratories, Knoxville, TN. Fast atom bombardment
(FAB) high-resolution mass spectra were obtained on a JEOL
double sector JMS-AX505HA mass spectrometer (University of
Notre Dame, South Bend, IN). Analytical HPLC was performed
on an Agilent 1200 instrument equipped with a diode array detec-
tor (λ = 254 and 280 nm), a thermostat set at 35 °C, and a Zorbax
Eclipse XDB-C18 column (4.6ϫ150 mm, 80 Å). The mobile phase
of a binary gradient (0–100% B/40 min; solvent A, 0.05 m AcOH/
Et3N, pH 6.0; solvent B, CH3CN for method 1 or 0–50% B/30 min,
50–100% B/31 min, 100%B/40 min; solvent A, 0.05 m AcOH/Et3N,
pH 6.0; solvent B, CH3OH for method 2) at a flow rate of
1 mLminϪ1 was used. Size-exclusion HPLC (SE-HPLC) chromato-
grams were obtained on an Agilent 1200 instrument equipped with
an in-line IN/US γ-Ram Model 2 radiodetector (Tampa, FL), fitted
with a BioSep-SEC-S3000 column (Phenomenex, Torrance, CA).
The isocratic mobile phase (0.05 m NaSO4, 0.02 m NaH2PO4,
0.05% NaN3, pH 6.8) at a flow rate of 1 mLminϪ1 was used for
SE-HPLC. 90Y and 177Lu in the chloride form were obtained from
NEN Perkin–Elmer.
Preparation from the One-Pot Reaction Starting from Compound 1:
TsCl (972 mg, 5.10 mmol) was added portionwise to a solution of
1 (200 mg, 1.02 mmol), Et3N (515 mg, 5.10 mmol), and DMAP
(12.5 mg, 0.102 mmol) in CH2Cl2 (3.0 mL) at 0 °C. The resulting
mixture was allowed to stir at room temperature for 1 d before
the solvent was evaporated. The residue was purified by column
chromatography on silica gel (60–230 mesh) eluting with 20% ethyl
acetate/hexane to provide a mixture of 3 and 4 (332 mg) as a light
yellow solid. The mixture was directly used for the next step with-
out further purification. DIPEA (21.7 mg, 0.168 mmol) was added
to a solution of the mixture of 3 and 4 (50 mg) and 5 (66.9 mg,
0.153 mmol) in CH3CN (3 mL). The resulting mixture was heated
at reflux for 30 h before the reaction mixture was cooled to room
temperature, filtered, and the solvent was evaporated. The residue
was purified by column chromatography on silica gel (60–
230 mesh) eluting with 3% ethyl acetate/CH2Cl2 to provide pure 6
Caution: 90Y (t1/2 = 72 h) and 177Lu (t1/2 = 6.7 d) are β- and/or γ-
emitting radionuclides. Appropriate shielding and handling proto-
cols should be in place when using the isotope.
1-[(4-Methylphenyl)sulfonyl]-2-[(4-nitrophenyl)methyl]aziridine (3):
TsCl (2.14 g, 11.2 mmol), Et3N (3.7 g, 36.7 mmol) and DMAP
(61 mg, 0.50 mmol) were added to a solution of 1 (1.0 g, 5.1 mmol)
in THF (15 mL) at 0 °C . The resulting mixture was allowed to stir
at room temperature for 20 h before the solvent was evaporated.
The residue was purified by column chromatography on silica gel
(60–230 mesh) eluting with 20% ethyl acetate/hexane to provide
pure 3 (780 mg, 46%) as a light yellow solid. 1H NMR (CDCl3,
300 MHz): δ = 2.22 (d, J = 4.2 Hz, 1 H), 2.38 (s, 3 H), 2.53 (dd, J
= 13.8, J = 4.0 Hz, 1 H), 2.80 (d, J = 4.2 Hz, 1 H), 2.83–2.95 (m,
1 H), 3.03–3.12 (m, 1 H), 7.12–7.22 (m, 4 H), 7.59 (d, J = 8.1 Hz,
2 H), 7.93 (d, J = 8.1 Hz, 2 H) ppm. 13C NMR (CDCl3, 300 MHz):
δ = 21.5 (q), 32.7 (t), 37.3 (t), 40.7 (d), 123.5 (d), 127.9 (d), 129.5
(d), 134.5 (s), 144.9 (s), 145.0 (s), 146.6 (s) ppm. HRMS (ESI+):
calcd. for C16H17N2O4S [M + H]+ 333.0904, found 333.0901.
1
(89 mg, yield over two steps was 76%) as a light yellow solid. H
and 13C NMR spectroscopic data of 6 obtained from this reaction
was essentially same as those of 6 described above.
1-(4-Nitrophenyl)-3-(1,4,7-triazonan-1-yl)propan-2-amine (7): Con-
centrated H2SO4 (10 mL) was added to compound 6 (705 mg,
0.92 mmol). The resulting mixture was heated to 115 °C and stirred
for 3 d before the reaction mixture was cooled to room tempera-
ture. The reaction mixture was added dropwise into diethyl ether
(65 mL) at –60 °C over 1 h. 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 resulting aqueous
solution was extracted with CHCl3 (30 mL). The aqueous solution
was adjusted to pH 7 using 5 m NaOH (aq.) and extracted with
N-[2-Chloro-3-(4-nitrophenyl)propyl]-4-methylbenzene-1-sulfon- CHCl3 (2ϫ 50 mL). The aqueous solution was further adjusted to
amide (4): TsCl (243 mg, 1.28 mmol) was added portionwise to a pH 10 and then pH 13. At each pH, the aqueous solution was ex-
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H.-S. Chong, X. Sun, P. Dong, C. S. Kang
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tracted with CHCl3 (3ϫ 50 mL). The organic layers extracted at
pH 10 and pH 13 were combined, dried with MgSO4, filtered, and
concentrated to provide pure 7 (191 mg, 68%) as a light yellow oil.
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). 13C NMR (CDCl3, 300 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 (s).
acetate/CH2Cl2 to provide pure 6 (176 mg, 55%) as a white solid.
1H and 13C NMR spectroscopic data of 6 obtained in this reaction
is essentially same as those of 6 described above.
1,4-Di-tert-butyl 7-(2-{[(tert-Butoxy)carbonyl]amino}-3-(4-ni-
trophenyl)propyl)-1,4,7-triazonane-1,4-dicarboxylate (15):[10] Com-
pound 14 (2.24 g, 6.80 mmol) was added to a solution of 13 (2.00 g,
6.80 mmol) in DCE (50 mL) at 0 °C . The resulting solution was
stirred for 10 min and sodium triacetoxyborohydride (2.02 g,
9.52 mmol) was added portionwise to the solution over 30 min. The
mixture was stirred at room temperature overnight. The reaction
2-Amino-3-(4-nitrophenyl)propanamide (9):[19] NH4OH (50 mL) was
added to a solution of 8 (7.15 g, 27.8 mmol) in toluene (50 mL)
at room temperature. The resulting mixture was stirred at room mixture was concentrated, treated with a saturated aqueous solu-
temperature overnight. The reaction mixture was filtered and
tion of NaHCO3 (50 mL), and extracted with ethyl acetate (3ϫ
50 mL). The combined organic layers were dried with MgSO4, fil-
tered, and concentrated in vacuo to provide 15 (4.08 g, 99%) as a
yellowish solid. 1H and 13C NMR spectroscopic data of compound
washed with toluene to provide pure 9 (4.46 g, 78%) as a light
1
yellow solid. 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, 15 were the same as those previously reported. Compound 15 was
300 MHz): δ = 41.1 (t), 56.2 (d), 123.5 (d), 131.1 (d), 146.5 (s),
directly used for the next step without further purification.
148.1 (s), 176.6 (s) ppm.
1-(4-Nitrophenyl)-3-(1,4,7-triazonan-1-yl)propan-2-amine (7): TFA
(3 mL) was added dropwise over 10 min to a solution of 15 (70 mg,
0.115 mmol) in CHCl3 (3 mL) at 0–5 °C. The resulting mixture was
warmed to room temperature. After 6 h, the reaction mixture was
concentrated and deionized water (5 mL) was added to the residue.
The aqueous layer was adjusted to pH 7 by using 2 m NaOH (aq.)
and then extracted with CHCl3 (3ϫ 5 mL). The aqueous layer was
2-[(4-Methylphenyl)sulfonamido]-3-(4-nitrophenyl)propanamide (10):
A solution of TsCl (1.09 g, 5.74 mmol) in DMF (5 mL) was added
dropwise over 30 min to a solution of 9 (500 mg, 2.39 mmol) and
Et3N (869 mg, 8.61 mmol) in DMF (10 mL) at 0 °C. The resulting
mixture was warmed to room temperature and stirred for 3 d before
the solvent was evaporated. The residue was washed with CH2Cl2
to provide pure 10 (526 mg, 61%) as a colorless solid. 1H NMR further adjusted to pH 10 and then pH 13. At each step, the aque-
([D6]DMSO, 300 MHz): δ = 2.25 (s, 3 H), 2.64–2.79 (m, 1 H), 2.92– ous layer was extracted with CHCl3 (3ϫ 5 mL). The organic layers
3.04 (m, 1 H), 3.90 (m, 1 H), 7.02–7.13 (m, 3 H), 7.30–7.36 (m, 4 obtained from extraction of the aqueous solution (pH 10 and
H), 7.44 (s, 1 H), 7.94 (d, J = 8.2 Hz, 2 H), 8.06 (d, J = 8.5 Hz, 1 pH 13) were combined and dried with MgSO4, filtered, and con-
H) ppm. 13C NMR ([D6]DMSO, 300 MHz): δ = 21.2 (q), 38.5 (t), centrated in vacuo to provide free amine 7 (31 mg, 87%) as a yellow
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.
oil. 1H and 13C NMR spectroscopic data of 7 obtained in this reac-
tion is essentially the same as those of 7 described above.
N-[1-Amino-3-(4-nitrophenyl)propan-2-yl]-4-methylbenzene-1-
sulfonamide (11): 1 m BH3 in THF (9.5 mL) was added dropwise
over 30 min to a solution of 10 (573.2 mg, 1.58 mmol) in THF
(10 mL) at 0 °C. The resulting mixture was warmed to room tem-
perature and stirred for 2 h and then heated to reflux overnight.
The reaction mixture was concentrated and the residue was treated
with MeOH (10 mL) and concentrated to the dryness. This quench-
ing process was repeated twice. The residue was dried in vacuo and
treated with 6 m HCl (10 mL). The resulting mixture was stirred at
room temperature for 1 h and then heated to reflux for 2 h. The
aqueous solution was cooled to room temperature and was ad-
justed to pH 7 by using 2 m NaOH and then extracted with CHCl3
(2ϫ 30 mL). The aqueous layer was further adjusted to pH 10 and
then pH 13. At each pH, the aqueous solution was extracted with
CHCl3 (2 ϫ 30 mL). The organic layers extracted at pH 7 and
2-(Benzylamino)-3(4-nitrophenyl)propan-1-ol (16): A stirred solution
of 1 (1.0 g, 5.10 mmol) and benzaldehyde (568 mg, 5.35 mmol) in
benzene (20 mL) was heated at reflux for 40 h by using a Dean–
Stark trap (5 mL). The solvent was evaporated in vacuo and MeOH
(20 mL) was added to the crude imine residue. The resulting mix-
ture was cooled to 0 °C and NaBH4 (482 mg, 12.7 mmol) was
added portionwise over 15 min. The reaction mixture was stirred
for 24 h and 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 con-
centrated in vacuo. The crude product was dissolved in saturated
citric acid (20 mL) and extracted with ethyl acetate (3ϫ 20 mL).
The pH of the aqueous layer was adjusted to 12 by using 25%
NaOH (aq.) and extracted with ethyl acetate (3ϫ 20 mL). The or-
ganic layer was dried with MgSO4, filtered, and concentrated in
pH 10 were combined, dried with MgSO4, filtered, and concen- vacuo to afford pure 16 (810 mg, 56%). The product was directly
trated in vacuo to provide free amine 11 (408 mg, 74%) as a light
yellow solid. H NMR ([D6]DMSO, 300 MHz): δ = 2.26 (s, 3 H),
used for the next step without further purification. 1H NMR
(CDCl3, 300 MHz): δ = 1.65–2.20 (br. s, 2 H), 2.80–3.06 (m, 3 H),
1
2.40–2.62 (m, 2 H), 3.16–3.80 (m, 5 H), 7.12 (d, J = 8.2 Hz, 2 H), 3.30–3.41 (m, 1 H), 3.59–3.68 (m, 1 H), 3.74–3.85 (m, 1 H), 7.14–
7.28 (d, J = 8.2 Hz, 2 H), 7.37 (d, J = 8.4 Hz, 2 H), 7.92 (d, J = 7.34 (m, 7 H), 8.15 (d, J = 8.4 Hz, 2 H). 13C NMR (CDCl3,
8.4 Hz, 2 H) ppm. 13C NMR ([D6]DMSO, 300 MHz): δ = 21.2 (q), 300 MHz): δ = 38.0 (t), 51.1 (t), 59.0 (d), 62.2 (t), 123.8 (d), 127.3
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.
(d), 128.0 (d), 128.6 (d), 130.1 (d), 139.7 (s), 146.6 (s).
tert-Butyl 2-{Benzyl[1-hydroxy-3-(4-nitrophenyl)propan-2-
N-(1-{4,7-Bis[(4-methylphenyl)sulfonyl]-1,4,7-triazonan-1-yl}-3-(4- yl]amino}acetate (17): K2CO3 (212 mg, 1.54 mmol) was added to a
nitrophenyl)propan-2-yl)-4-methylbenzene-1-sulfonamide (6): stirred solution of 16 (400 mg, 1.40 mmol) in CH3CN (2 mL) at
Na2CO3 (445.2 mg, 4.2 mmol) was added to a solution of 11
(146 mg, 0.42 mmol) and 12[20] (319.8 mg, 0.42 mmol) in CH3CN
(8 mL). The resulting mixture was heated at reflux for 4 d before
the reaction mixture was cooled to room temperature, filtered, and
the solvent was evaporated. The residue was purified by column
chromatography on silica gel (60–230 mesh) eluting with 3% ethyl
0 °C. A solution of tert-butylbromoacetate (301 mg, 1.54 mmol) in
CH3CN (1 mL) was added dropwise to the resulting mixture over
10 min. The reaction mixture was heated to 60 °C and stirred for
2 d as the reaction progress was continuously monitored by means
of TLC. The reaction mixture was cooled to room temperature and
filtered. The solvent was evaporated in vacuo to provide a residue
6646
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 6641–6648

A Heptadentate Bifunctional Ligand for Radioimmunotherapy
that was purified by column chromatography on silica gel (60–
230 mesh) eluted with 20% ethyl acetate/hexane. Pure 17 (451 mg,
81 %) was obtained as a light yellow oil. 1H NMR (CDCl3,
300 MHz): δ = 1.40 (s, 9 H), 2.52–2.66 (m, 1 H), 2.92–3.04 (m, 1
H), 3.11–3.52 (m, 5 H), 3.71–3.98 (m, 2 H), 7.16–7.33 (m, 7 H),
8.15 (d, J = 8.4 Hz, 2 H) ppm. 13C NMR (CDCl3, 300 MHz): δ =
27.9 (q), 33.9 (t), 51.5 (t), 56.1 (t), 61.9 (t), 64.9 (d), 81.8 (s), 123.8
(d), 127.4 (d), 128.4 (d), 128.9 (d), 129.9 (d), 138.3 (s), 146.6 (s),
147.3 (s), 172.3 (s) ppm.
(d, J = 8.4 Hz, 2 H) ppm. 13C NMR (D2O + NaOD, 300 MHz): δ
= 31.6 (t), 51.4 (t), 53.3 (t), 56.2 (t), 56.3 (d), 57.8 (t), 61.0 (t), 123.6
(d), 127.8 (d), 128.7 (d), 130.0 (d), 137.7 (s), 145.8 (s), 149.2 (s),
179.6 (s), 179.8 (s) ppm. HRMS (ESI+): calcd. for C28H38N5O8Na2
[M + 2Na – H]+ 616.2354, found 616.2322.
2-({1-(4-Aminophenyl)-3-[4,7-bis(carboxymethyl)-1,4,7-triazonon-
1-yl]propan-2-yl}amino)acetic Acid (23):[8] Wet 10% Pd/C (8.1 mg)
was added under argon to a solution of 22 (13.2 mg, 0.0184 mmol)
in ethanol (5 mL) at room temperature . The reaction mixture was
placed in a hydrogenation apparatus setup at a pressure of 60 psi
for 40 h. The resulting mixture was filtered through a pad of Celite
and washed thoroughly with ethanol. The filtrate was concentrated
to provide 23 (11.7 mg, 100%) as a yellowish solid. 1H and 13C
NMR spectroscopic data of 23 were essentially identical to those
of 23 previously reported.
tert-Butyl 2-{Benzyl[2-bromo-3-(4-nitrophenyl)propyl]amino}acetate
(18): NBS (211 mg, 1.18 mmol) was added portionwise over 5 min
to a solution of 17 (395 mg, 0.986 mmol) and PPh3 (310 mg,
1.18 mmol) in CHCl3 (6 mL) at 0 °C. The resulting mixture was
stirred for 4 h at 0 °C. The ice bath was removed and the reaction
mixture was warmed to room temperature and stirred for 1 h. The
solvent was evaporated and the residue was purified by silica gel
column chromatography eluting with 5% EtOAc in hexanes to af-
ford 18 (390 mg, 85 %) as a yellow oil. 1H NMR (CDCl3,
300 MHz): δ = 1.48 (s, 9 H), 2.84–2.96 (m, 1 H), 2.99–3.14 (m, 1
H), 3.24–3.34 (m, 1 H), 3.36 (s, 2 H), 3.61–3.73 (m, 1 H), 3.84–3.96
(m, 2 H), 3.96–4.09 (m, 1 H), 7.21–7.42 (m, 7 H), 8.13 (d, J =
8.4 Hz, 2 H) ppm. 13C NMR (CDCl3, 300 MHz): δ = 28.2 (q), 42.0
(t), 53.8 (d), 56.6 (t), 59.3 (t), 61.9 (t), 81.4 (s), 123.5 (d), 127.5 (d),
128.6 (d), 128.9 (d), 130.1 (d), 138.8 (s), 146.6 (s), 146.8 (s), 170.6 (s)
ppm. HRMS (ESI+): calcd. for C22H28N2O4Br [M + H]+ 463.1227,
found 463.1233.
Conjugation of C-NE3TA-NCS and C-NOTA-NCS to Trastu-
zumab: C-NOTA and C-NOTA-NCS were purchased from Macro-
cylics (Dallas, TX). Trastuzumab was provided as a gift from Dr.
Martin Brechbiel (NCI, NIH). All absorbance measurements were
obtained on an Agilent 8453 diode array spectrophotometer
equipped with an 8-cell transport system (designed for 1 cm cells).
Metal-free stock solutions of all buffers were prepared by using
Chelex-100 resin (200–400 mesh, Bio-Rad Lab, Hercules, CA, Cat#
142–2842). Chelex resin (1 g/100 mL) was added to the buffer solu-
tion and the mixture was shaken overnight in a shaker and filtered
through a Corning filter system (Cat# 430513, pore size 0.2 μm).
Disposable PD-10 columns (Sephadex, G-25M, GE Healthcare,
#17-0851-01) were rinsed with the appropriate buffer solution
(25 mL) prior to the addition of antibody or its ligand conjugate.
Amicon centricon C-50 (50,000 MWCO) centrifugal filter devices
(Millipore, Cat# UFC805008) were used for purification of the tra-
stuzumab conjugate. The initial concentration of trastuzumab was
determined by UV spectroscopic methods. Phosphate buffered sa-
line (PBS; 1ϫ, 11.9 mm Phosphates, 137 mm NaCl, and 2.7 mm
KCl, pH 7.4) was purchased from Fisher and used as received.
Conjugation buffer (50 mm HEPES, 150 mm NaCl, pH 8.6) was
prepared as 1ϫ solution, chelexed, and filtered through a Corning
filter. C-NE3TA-NCS was conjugated to trastuzumab as previously
reported.[8] The ratio of ligand to protein (L/P) for the C-NE3TA–
trastuzumab conjugate was measured by using a CuII–AAIII-based
spectrometric method and found to be 3.9 to 1. Trastuzumab
(6.7 mg for C-NOTA) was diluted to 2.5 mL using conjugation
buffer and the resulting solution was added to a PD-10 column.
Conjugation buffer (6.0 mL) was added to the PD-10 column to
exchange the buffer solution of the antibody and was collected in
a sterile test tube and checked for the presence of trastuzumab by
analysis of the UV spectrum at 280 nm. A 10-fold excess of C-
NOTA-NCS (39.6 μL, 10 mm) was added to a sterile test tube con-
taining the recovered trastuzumab (6.1 mg for C-NOTA). The re-
sulting solution was gently agitated for 16 h at room temperature
and placed on a Centricon C-50 membrane and spun down to re-
tert-Butyl 2-[Benzyl(1-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-tri-
azonan-1-yl}-3-(4-nitrophenyl)propan-2-yl)amino] Acetate (21):
AgClO4 (44.8 mg, 0.216 mmol) was added to a solution of 18
(100 mg, 0.216 mmol) in CH3CN (1 mL) at –5 °C. The resulting
mixture was stirred for 10 min at the same temperature. Compound
20[7] (77.2 mg, 0.216 mmol) and DIPEA (83.7 mg, 0.648 mmol) in
CH3CN (1 mL) were sequentially added to the reaction mixture at
–5 °C. The resulting mixture was gradually warmed to room tem-
perature and stirred for 4.5 d. The reaction mixture was filtered
and concentrated to the dryness. 0.1 m HCl (10 mL) was added to
the residue and the resulting mixture was extracted with CH2Cl2
(3ϫ 10 mL). The combined organic 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)
eluting with 2% CH3OH in CH2Cl2 to provide pure product 21
(81 mg, 51%) as a yellowish oil. 1H NMR (CDCl3, 300 MHz): δ =
1.31–1.45 (m, 27 H), 2.68 (s, 4 H), 2.76–3.95 (m, 21 H), 7.18–7.34
(m, 5 H), 7.52 (d, J = 8.6 Hz, 2 H), 8.14 (d, J = 8.6 Hz, 2 H) ppm.
13C NMR (CDCl3, 300 MHz): δ = 28.0 (q), 28.0 (q), 28.1 (q), 33.3
(t), 49.8 (t), 51.1 (t), 51.6 (t), 53.5 (t), 53.6 (t), 56.3 (t), 57.9 (d),
58.2 (t), 58.4 (t), 81.6 (s), 81.7 (s), 82.6 (s), 124.1 (d), 128.0 (d),
128.8 (d), 129.4 (d), 129.9 (d), 130.5 (d), 137.2 (s), 146.0 (s), 146.8
(s), 170.4 (s), 172.7 (s) ppm. HRMS (ESI+): calcd. for C40H62N5O8
[M + H]+ 740.4593, found 740.4579.
2-[Benzyl({1-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]-3-(4- duce the volume. PBS (3ϫ2 mL) was added to the remaining solu-
nitrophenyl)propan-2-yl})amino]acetic Acid (22): 4 m HCl (g) in 1,4-
dioxane (3 mL) was added dropwise over 10 min to a round-bot-
tomed flask containing 21 (33.0 mg, 0.0446 mmol) at 0–5 °C . The
tion of C-NOTA–trastuzumab conjugate, followed by centrifuga-
tion to remove unreacted ligand. The volume of the purified anti-
body conjugate solution was brought to 1.0 mL. To measure [Tras-
resulting mixture was warmed to room temperature. After 18 h, tuzumab] in the conjugate, a UV/Vis spectrometer was zeroed
diethyl ether (20 mL) was added and the resulting mixture was
stirred for 10 min and capped and placed in the freezer for 1 h. The
resulting precipitate was filtered, washed with diethyl ether, and
quickly dissolved in deionized water. The aqueous solution was
concentrated in vacuo to provide 22 (26.5 mg, 83%) as a light yel-
against a cuvette filled with 2.0 mL of PBS with a window open
from 190 to 1100 nm. A 50 μL portion of PBS was removed and
discarded, 50 μL of C-NOTA–trastuzumab conjugate solution was
added and the absorbance at 280 nm was noted. Beer’s law was
used to calculate [Trastuzumab] in the conjugate with a molar ab-
sorptivity of 1.42. After centrifugation, 2.7 mg (1.83ϫ10–5 m,
44.0%) of Trastuzumab remained.
1
low solid. H NMR (D2O, pH 14, 300 MHz): δ = 1.50–3.14 (m, 22
H), 3.31–3.50 (m, 2 H), 3.61–3.75 (m, 1 H), 7.07–7.31 (m, 7 H), 7.88
Eur. J. Org. Chem. 2011, 6641–6648
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6647

H.-S. Chong, X. Sun, P. Dong, C. S. Kang
FULL PAPER
The L/P of C-NOTA–trastuzumab conjugate was measured by
using a CuII–AAIII-based spectrometric method. A stock solution
of CuII–AAIII reagent was prepared in 0.15 m NH4OAc, pH 7.0,
by adding an aliquot of copper (1.55ϫ10–2 m) atomic absorption
solution to a 10 μm solution of AAIII to afford a 5 μm solution of
CuII. This solution was stored in the dark to avoid degradation
over time. A UV/Vis spectrometer was zeroed against well-dried
blank 8 cuvettes with a window open from 190 to 1100 nm. One
cuvette was filled with AAIII solution (2 mL) and the other seven
cuvettes were filled with CuII–AAIII solution (2 mL). CuII–AAIII
solution (50 μL) in the seven cuvettes was removed and discarded.
Milli-Q water (50 μL) was added to the second cuvette and one to
five 10 μL additions of C-NOTA (0.1 mm) were added to five cu-
vettes to give a series of five different concentrations (2 mL total
volume). The solutions in the third to seventh cuvettes were diluted
to 2.0 mL by adding an aliquot of Milli-Q water. C-NOTA–trastu-
zumab conjugate (50 μL) was added to the eighth cuvette contain-
ing CuII–AAIII reagent (1950 μL). After addition of the conjugate
to CuII–AAIII solution, the resulting solution was equilibrated for
10 min. The absorbance of the resulting solution at 610 nm (for
CuII–AAIII) was monitored every 30 s over 6 min. The average of
the absorbance of each solution was calculated and the absorbance
data from CuII–AAIII solutions containing six different concentra-
tions were used to construct a calibration plot of A610nm versus [C-
NOTA] by the equation, Y = 0.0596 – (8.125ϫ 103)[C-NOTA] (R2
= 0.9938), wherein Y = A610nm. The concentration of C-NOTA in
the C-NOTA–trastuzumab conjugate was calculated to be
7.79ϫ10–5 m. The L/P ratio of the C-NOTA–trastuzumab conju-
gate ([7.79ϫ10–5 m]/[1.83ϫ10–5 m]) was measured to be 4.3.
and 50 mm from the origin, respectively. Bound and unbound ra-
dioisotope peaks appeared around 9.0 and 12.0 min in SE-HPLC
(see the Supporting Information).
Supporting Information (see footnote on the first page of this arti-
cle): HPLC and ITLC chromatograms for radiolabeling reaction
kinetics of C-NE3TA–trastuzumab and C-NOTA–trastuzumab
conjugates.
Acknowledgments
We acknowledge financial support from the National Institutes of
Health (NIH) (R01CA112503) and thank Dr. Mamta Dadwal for
the preparation of compound 3.
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Radiolabeling Reaction Kinetics of C-NE3TA–Trastuzumab and C-
NOTA–Trastuzumab Conjugates with 90Y and 177Lu: All HCl solu-
tions were prepared from ultra-pure HCl (Fisher, Cat# A466-500).
For metal-free radiolabeling, plasticware, including pipette tips,
tubes, and caps, was soaked in 0.1 m HCl overnight, washed thor-
oughly with Milli-Q (18.2 MΩ) water, and air-dried overnight. Ul-
tra-pure NH4OAc (Aldrich, #372331) was purchased from Aldrich
and used to prepare all NH4OAc buffer solutions (0.25 m, pH 5.5).
The buffer solutions were treated with Chelex-100 resin (Biorad,
#142-2842, 1 g/100 mL buffer solution), shaken overnight at room
temperature, and filtered through a 0.22 μm filter (Corning,
#430320) prior to use. A solution of C-NE3TA–trastuzumab or C-
NOTA–trastuzumab conjugate (30 μg) in PBS (4.5 μL) and 177Lu
(0.05 m HCl, 60 μCi, 2.4 μL) was sequentially added to a buffer
solution (30 μL, pH 5.5, 0.25 m NH4OAc) in a capped microcentri-
fuge tube (1.5 mL, Fisher Scientific #05-408-129). A solution of
0.05 m HCl (1.6–2.9 μL) and 90Y (0.05 m HCl, 60 μCi, 2–3 μL) was
sequentially added to a solution of C-NE3TA–trastuzumab or C-
NOTA–trastuzumab conjugate (30 μg) in PBS (4.5 μL) in a micro-
centrifuge tube containing a buffer solution (30 μL, pH 5.5, 0.25 m
NH4OAc). The final volume of the resulting solution was around
40 μL and the pH of the resulting reaction mixture was 5.5. The
reaction mixture was agitated on a thermomixer (Eppendorf,
#022670549) set at 1000 rpm at room temperature for 1 h. The la-
beling efficiency was determined by SE-HPLC (Phenomenex,
BioSep-SEC S 3000, 7.8ϫ30 cm, eluent: 0.05 m NaSO4/0.02 m
NaH2PO4/0.05% NaN3, pH 6.8) and ITLC (eluent: 20 mm ethyl-
enediaminetetraacetic acid (EDTA)/0.15 m NH4OAc). A solution
of radiolabeled mixture (6 μL) was withdrawn at the designated
time points (1, 5, 10, 20, 30, and 60 min), and DTPA solution (10
or 5 mm; 0.6 μL) was added to the mixture, and the resulting mix-
ture was left for at least 20 min at room temp. In ITLC analysis,
peaks for bound and unbound radioisotope appeared around 30
[22] J. Baselga, Ann. Oncol. 2001, 12, S49–55.
Received: July 20, 2011
Published Online: October 19, 2011
6648
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Eur. J. Org. Chem. 2011, 6641–6648