Synthesis and biological evaluation of novel macrocyclic ligands with pendent donor groups as potential yttrium chelators for radioimmunotherapy with improved complex formation kinetics

Article abstract of DOI:10.1021/jm0200759

Novel 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) based octadentate ligands [2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1- ylethyl)carbonylmethylamino] acetic acid tetrahydrochloride (1) and [3-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-

Full text of DOI:10.1021/jm0200759

3458
J . Med. Chem. 2002, 45, 3458-3464
Syn th esis a n d Biologica l Eva lu a tion of Novel Ma cr ocyclic Liga n d s w ith
P en d en t Don or Gr ou p s a s P oten tia l Yttr iu m Ch ela tor s for
Ra d ioim m u n oth er a p y w ith Im p r oved Com p lex F or m a tion Kin etics
Hyun-soon Chong, Kayhan Garmestani, Dangshe Ma, Diane E. Milenic, Terrish Overstreet, and
Martin W. Brechbiel*
Chemistry Section, Radiation Oncology Branch, Building 10, Room B3B69, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892
Received February 13, 2002
Novel 1,4,7-triazacyclononane-N,N′,N′′-triacetic acid (NOTA) based octadentate ligands [2-(4,7-
biscarboxymethyl[1,4,7]triazacyclononan-1-ylethyl)carbonylmethylamino]acetic acid tetrahy-
drochloride (1) and [3-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-propyl)carbonyl-
methylamino]acetic acid tetrahydrochloride (2) with pendent donor groups as potential yttrium
chelators for radioimmunotherapy (RIT) have been prepared via a convenient and high-yield
cyclization route. The complexation kinetics of the novel chelates with Y(III) was investigated
and compared to that of 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA),
a macrocyclic chelating agent well recognized as forming very stable complexes with yttrium
but also limited in usage because of slow Y(III) complex formation rates. The in vitro stability
of the corresponding 88Y-labeled complexes in human serum was assessed by measuring the
release of 88Y from the complexes over 14 days. The in vivo biodistribution of 86Y-labeled 1 in
mice was evaluated and compared to that of the 86Y-DOTA complex. Formation of the Y
complex of 1 was significantly more rapid than that of either 2 or DOTA. Serum stability of
the 88Y complex formed with 1 was equivalent to the DOTA complex, while the complex formed
with 2 proved to be significantly unstable. The results obtained from a biodistribution study
indicate that the 86Y-1 complex possesses in vivo stability comparable to the analogous DOTA
complex.
In tr od u ction
that complicates and limits the routine use of these
ligands in RIT applications. In general, DOTA conju-
gated to mAb’s displays relatively slow and inefficient
radiolabeling with Y(III) isotopes under mild condi-
tions.8 This is contrary to the rapid and high-yield
radiolabeling (>90%) of mAb’s conjugated with bifunc-
tional derivatives of the acyclic chelating agent dieth-
ylenetriaminepentaacetic acid (DTPA).3c,8 Low radio-
labeling yields require a chromatographic purification
of the product to separate unchelated Y(III), which is
not always practical for RIT applications. With this
background in mind, this laboratory has endeavored to
prepare a suitable Y(III) chelating agent that would
possess comparable complex stability of DOTA and the
superior, practical complexation kinetics of DTPA. Such
efforts have resulted in the synthesis and evaluation of
several chelates including the CHX-DTPA9 and 1B4M-
DTPA (Scheme 1),10 which display significantly im-
proved complexation kinetics with Y(III) compared to
DOTA.9c However, the corresponding radio-yttrium
complexes remain somewhat less stable in serum and
in vivo.9c Despite this shortcoming, significant clinical
use of these ligands has been made because of their
practical and reproducible high specific activity radio-
labeling chemistry.11
Monoclonal antibodies (mAb’s) have been employed
as a targeting biomolecule for the delivery of radionu-
clide into tumor cells in radioimmunotherapy (RIT), and
numerous clinical trials have been performed to vali-
date this modality of cancer therapy.1 Several useful
â--emitting radionuclides including 131I, 90Y, 177Lu, and
153Sm have been employed for labeling mAb’s for RIT
applications.2 A variety of macrocyclic ligands have been
prepared and evaluated as suitable chelates to bind
specific radionuclides.3 Since the release of the radio-
metals from the chelate is a potential source of ra-
diotoxic effects for nontumor cells and normal tissue,4
the requirement of having a chelate that forms a
kinetically inert complex with the radiometal is a critical
aspect for performing successful targeted radiotherapy.
The pure â--emitting radionuclide, 90Y (Emax
)
2.28 MeV; T1/2 ) 64.1 h) has been extensively studied
in RIT because of its physical properties.5 The macro-
cyclic chelating agent, 1,4,7,10-tetraazacyclododecane-
N,N′,N′′,N′′′-tetraacetic acid (DOTA) is well-known to
be the most effective chelator of Y(III) and the lan-
thanides,6 and numerous bifunctional analogues suit-
able for protein conjugation have been reported in the
literature.7 Although the Y(III)-DOTA complex shows
ideal stability both in vivo3c and in vitro,3b the extremely
slow formation rate of the complex6 is a major obstacle
Our continued interest in finding a suitable chelator
to complex Y(III) isotopes efficiently prompted the
design of the structurally novel chelators 1 and 2
(Scheme 1). The chelators 1 and 2 possess the same
octadentate coordinating groups as both DOTA and
* To whom correspondence should be addressed. Phone: (301) 496-
0591. Fax: (301) 402-1923. E-mail: martinwb@mail.nih.gov.
10.1021/jm0200759 This article not subject to U.S. Copyright. Published 2002 by the American Chemical Society
Published on Web 06/18/2002

Yttrium Chelators for Radioimmunotherapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3459
Sch em e 1
Sch em e 2a
a
Reaction conditions: (a) NaN3, DMSO, 90 °C, 4 h; (b) H2, 10% Pd/C, EtOH, 3 h, 20 psi; (c) HCl(g)
.
DTPA. However, these new chelators are a combination
of both macrocyclic and acyclic character. The macro-
cyclic component chosen is based on 1,4,7-triazacy-
clononane-N,N′,N′′-triacetic acid (NOTA), while the
acyclic component is a pendent bis(carboxymethyl)-
amino donor group that is connected by either an
ethylene or a propylene bridge for 1 and 2, respectively.
The cooperative binding of the pendent donor groups
coupled with the preorganization and macrocyclic effect
of the NOTA substructure is expected to accelerate
complexation with Y(III) isotopes while maintaining a
high level of stability of the complexes.
Herein, we report the synthesis of novel octadentate
ligands 1 and 2 for improved kinetics of complexation
with Y(III) isotopes for RIT. The relative Y(III) com-
plexation kinetics of ligands 1, 2, and DOTA was
investigated using a competition reaction with arsenazo-
(III).6b The serum stability of the 88Y-labeled complexes
of 1 and 2 was evaluated. The in vivo biodistribution of
the 86Y-1 complex and 86Y-DOTA complex was evalu-
ated in mice.
plated cyclization,12b and the Richman-Atkin cycliza-
tion conditions.12d These cyclization methods, when
employed to prepare smaller polyaza macrocyclic struc-
tures, are frequently problematic, resulting in low
yields, inconvenient procedures, and difficult purifica-
tion regimens due to the presence of higher homologue
byproducts. Our present report introduces a convenient
approach to cyclization, which employs primary amines
having a pendent hydroxyl functional group as a pre-
cursor molecule. Thus, primary amines bearing a distal
hydroxyl group are reacted with an N-tosyl-protected
ditosylate to provide the 1,4,7-triazacyclononane mac-
rocyclic ring. After cyclization, the pendent hydroxyl
group is available for further chemistry to introduce
additional pendent donor groups as desired. This cy-
clization method is a convenient, clean, and high-yield
route using readily available starting materials. This
strategy can be directly applied to construction of a wide
range of macrocyclic rings by using modified primary
amines.
The synthetic routes for the NOTA-based macrocyles
1 and 2 are shown in Scheme 2. The parent macrocyclic
rings in 1 and 2 are obtained via the efficient cyclization
of ethanolamine (n ) 1) or propanolamine (n ) 2) with
N,N′-ditosylethylenediamine ditosylate 3. Thus, ditosy-
late 313 was reacted with ethanolamine or propanol-
amine in the presence of Na2CO3 to afford the tosyl-
protected macrocycles 4a and 4b in 86% and 92% yields,
respectively. Interestingly, reaction of ditosylate 3 with
ethanolamine in the presence of K2CO3 as the templat-
Resu lt a n d Discu ssion
Syn th esis. For the preparation of ligands 1 and 2,
efficient cyclization using two precursor molecules is a
crucial step. A variety of cyclization methods in the
synthesis of macrocyclic chelating agents have been
developed.12 Representative synthetic methods for bi-
molecular cyclization include reaction of a diamine and
diester under high dilution conditions,12a metal-tem-

3460 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Chong et al.
F igu r e 2. Serum stability of 88Y-1 ([) and 88Y-2 (9) at pH
7 and 37 °C.
F igu r e 1. Plot of absorbance at 652 nm vs time of Y-1 (c),
Y-2 (a), and Y-DOTA (b) at pH 4 and 25 °C.
surements obviously show that 1 forms a complex with
Y(III) at a much greater rate than DOTA.
ing base failed to provide the desired macrocycle 4a . The
choice of the appropriate base for templating these
reactions appears to be quite important. The subsequent
reaction of 4a and 4b with SOCl2 provided 5a and 5b,
which were reacted with NaN3 in DMSO to afford 6a
and 6b, respectively. The diazides 6a and 6b were
hydrogenated to provide the corresponding amines 7a
and 7b. Deprotection of the tosyl groups in compounds
7a and 7b was effected with concentrated sulfuric acid
at 115 °C for 72 h. After deprotection, alkylation of
triamines 8a and 8b with tert-butyl bromoacetate
provided 9a and 9b, respectively. Tetraacids 1 and 2
were obtained as HCl salts after deprotection of the tert-
butyl esters with anhydrous HCl(g)-dioxane. The syn-
thetic route to ligands 1 and 2 reported herein is
efficient and high-yielding. Beginning with the readily
available starting material 3, the straightforward syn-
thetic method provided ligands 1 and 2 in 86% and 85%
overall yields, respectively.
Kin etics Stu d y. The complexation kinetics of novel
chelates 1, 2, and DOTA with Y(III) was qualitatively
investigated using a competing reaction with arsenazo-
(III) according to a modification of a previously reported
procedure.6b The absorbance (A652) for the Y(III)-
arsenazo(III) complex was measured in the absence and
in the presence of the chelates (1, 2, and DOTA) over 1
h at room temperature. The absorbance (A652) was 0.089
for arsenazo(III)-Y(III) in the absence of the ligand. A
plot of the absorbance at 652 nm versus time is shown
in Figure 1. Inspection of Figure 1 indicates that the
complex formation of ligand 1 with Y(III) is quite rapid
and is essentially complete very shortly after the start-
ing point of the measurement. However, ligand 2
displays very slow complexation with Y(III) compared
to 1. Furthermore, the degree of complexation of 2 was
slight and remained unchanged over the period of
measurement. In the presence of ligand 2, the absor-
bance of arsenazo(III)-Y(III) declined to 0.083 at 1 h
(Figure 1). Complexation of Y(III) by 2 is assumed to
occur to some degree, but the formed complex might
undergo decomplexation because of an absence of com-
plex stability, ultimately leading to an unfavorable
equilibrium situation. This interpretation also agrees
with the serum stability result of the 88Y-labeled
complex with 2, which displayed a significant loss of
radio-yttrium in serum. As expected,6 DOTA displayed
sluggish complexation with Y(III). The time scan mea-
Ser u m Sta bility. The stability of the corresponding
88Y-labeled complexes formed with chelators 1 and 2 in
human serum was assessed by measuring the release
of 88Y from the complexes at 37 °C over 14 days. The
serum stability of 88Y-labeled complexes thereby ob-
tained is shown in Figure 2, which indicates that 88Y-
labeled ligand 1 having an ethylene bridge is stable in
serum for up to 14 days with no measurable loss of
radioactivity. However, considerable release of radio-
activity was observed from the 88Y chelate formed with
2, which possesses the longer propylene bridge. The
percentage of 88Y released from this complex at 14 days
was about 25%. The data show that the stability of the
Y(III) radiolabeled complex in serum is dependent on
the length of the carbon chain between the pendent
donor groups and the macrocyclic ring. The observed
release of radioactivity from chelate 2 might be a
consequence of the formation of a six-membered chelate
ring as opposed to what is generally considered to be
the best arrangement of donors, i.e., five-membered
chelate ring for complexation of Y(III). A number of
reports have shown that DOTA possesses exceptional
stability in human serum.3b,c Ligand 1 shows in vitro
stability comparable to that of DOTA. Thus, ligand 1,
after appropriate derivatization to provide a bifunctional
analogue, might serve as a viable alternative to the use
of DOTA in RIT applications.
In Vivo Biod ist r ibu t ion . To evaluate the in vivo
stability of 86Y-labeled ligand 1, which displayed excel-
lent serum stability and kinetics, a biodistribution study
was performed in mice. For the purpose of comparison,
a biodistribution of 86Y-labeled DOTA in mice was also
performed. The results of the biodistribution studies for
86Y-1 and 86Y-DOTA are shown in Figure 3. Radioac-
tivity that accumulated in selected organs and cleared
from the blood of mice was measured at five time points,
0.5, 1, 4, 8, and 24 h postinjection, of the two 86Y-labeled
complexes. The data in Figure 3 show that the ac-
cumulated radioactivity in the organs at 4 h postinjec-
tion is negligible for both 86Y-1 and 86Y-DOTA. Both
86Y-1 and 86Y-DOTA display a slightly higher radio-
activity level in the kidney compared to that in other
organs, no doubt associated with whole-body clearance
and elimination of these complexes. At 24 h, radioactiv-
ity that accumulated in the kidney was 0.54 ( 0.13%
injected dose per gram (% ID/g) and 0.84 ( 0.16% ID/g

Yttrium Chelators for Radioimmunotherapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3461
zation of 1 as a bifunctional-chelating agent for conjuga-
tion with mAb’s or peptides for RIT applications is in
progress and will be reported in due course.
Exp er im en ta l Section
Gen er a l. All solvents and reagents were obtained from
1
Aldrich and used as received unless otherwise noted. H, 13C,
and APT NMR spectra were obtained using a Varian Gemini
300 instrument, and chemical shifts are reported in ppm on
the δ scale relative to TMS, TSP, or solvent. Proton chemical
shifts are annotated as follows: ppm (multiplicity, integral,
coupling constant (Hz)). Elemental microanalyses were per-
formed by Galbraith Laboratories, Knoxville, TN. Fast atom
bombardment (FAB-MS) mass spectra were obtained on an
Extrel 4000 in the positive-ion detection mode. Chromatograms
(SE-HPLC) were obtained on a Dionex isocratic system with
a Waters 717 autosampler, a Gilson 112 UV detector, and an
in-line IN/US γ-Ram model 2 radiodetector. Time scan mea-
surements were obtained using an HP-8452A diode array
spectrophotometer. The 88Y was obtained from Los Alamos
National Laboratory and purified as previously reported.9c The
86Y was produced and purified as previously reported.15
Phosphate-buffered saline (PBS) (0.1 M, pH 7.4) consisted of
0.08 M Na2HPO4, 0.02 M KH2PO4, 0.01 M KCl, and 0.14 M
NaCl.
F igu r e 3. Biodistribution of 86Y-1 (A) and 86Y-DOTA (B) in
Balb/c mice following iv injection.
Caution: 88Y (t1/2 ) 106.6 day) is a γ-emitting radionuclide.
86Y (t1/2 ) 14.7 h) is a â+-emitting radionuclide. Appropriate
shielding and handling protocols should be in place when using
these isotopes.
for 86Y-1 and 86Y-DOTA, respectively. The data in Figure
3 also indicate a rapid blood clearance for 86Y-1 and
86Y-DOTA. The 86Y-1 displayed low bone uptake that
peaked at 1.47 ( 0.28% ID/g at 0.5 h to decline to 0.05
( 0.01% ID/g at 24 h. This trend is a noteworthy result
in that free yttrium is known to be efficiently deposited
at the bone.14 Compared to that of complex 86Y-1, the
bone accumulation of 86Y-DOTA was actually slightly
higher at all time points. The results of the biodistri-
bution studies as a whole indicate that 86Y-1 displays
in vivo stability comparable to that of 86Y-DOTA.
Gen er a l P r oced u r e for Syn th esis of Ma cr ocyclics 4a
a n d 4b. To a solution of 313 (1 mmol) and Na2CO3 (10 mmol)
in CH3CN (10 mL) under argon was added either ethanolamine
or propanolamine (1 mmol), and the resulting mixture was
heated to reflux for 24 h. The reaction mixture was allowed to
cool gradually to ambient temperature and was filtered, and
the filtrate was concentrated in vacuo.
2-[4,7-Bis(p-tolu en e-4-su lfon yl)[1,4,7]tr iazacyclon on an -
1-yl]eth a n ol (4a ). The residue was purified via column
chromatography on silica gel eluting with EtOAc. Pure 4a (414
mg, 86%) was thereby obtained as a colorless oil: 1H NMR
(CDCl3) δ 2.03 (s, 1 H), 2.40 (s, 6 H), 2,79 (t, J ) 3.2 Hz, 2 H),
3.01 (t, J ) 3.8 Hz, 4 H), 3.26 (t, J ) 3.6 Hz, 4 H), 3.44 (t, J )
1.8 Hz, 4 H), 3.61 (t, J ) 2.7 Hz, 2 H), 7.31 (d, J ) 9.1 Hz, 4
H), 7.67 (d, J ) 9.1 Hz, 4 H); 13C NMR (CDCl3) δ 21.2 (q), 52.3
(t), 52.7 (t), 54.9 (t), 59.3 (t), 59.9 (t), 126.8 (d), 129.5 (d), 134.8
(s), 143.2 (s). Anal. Calcd for C22H31N3S2O5: C, 54.86; H, 6.49.
Found: C, 54.51; H, 6.64.
3-[4,7-Bis(p-tolu en e-4-su lfon yl)[1,4,7]tr iazacyclon on an -
1-yl]p r op a n -1-ol (4b). The residue was purified via column
chromatography on silica gel, eluting with EtOAc. Pure 4b
(454 mg, 92%) was thereby obtained as a colorless oil: 1H NMR
(CDCl3) δ 1.78 (t, J ) 3.5 Hz, 2 H), 2.13 (s, 1 H), 2.50 (s, 6 H),
2.87 (t, J ) 3.3 Hz, 2 H), 3.04-3.12 (m, 4 H), 3.26-3.41 (m, 4
H), 3.50 (s, 2 H), 3.87 (t, J ) 2.9 Hz, 4 H), 7.40 (d, J ) 8.7 Hz,
4 H), 7.41 (d, J ) 8.7 Hz, 4 H); 13C NMR (CDCl3) δ 21.4 (q),
29.5 (t), 51.5 (t), 52.8 (t), 54.4 (t), 54.7 (t), 61.5 (t), 127.0 (d),
129.7 (d), 135.0 (s), 143.5 (s). Anal. Calcd for C23H33N3S2O5:
C, 55.74; H, 6.71. Found: C, 55.87; H, 6.84.
Gen er a l P r oced u r e for Ch lor in a tion of 5a a n d 5b. A
solution of either 4a or 4b (2 mmol) in dry benzene (20 mL)
was saturated with HCl(g) at 0 °C. After addition of thionyl
chloride (40 mmol), the mixture was heated at 60 °C for 3 h.
The cooled reaction mixture was concentrated and neutralized
with 5% Na2CO3 solution (5 mL). The resulting mixture was
extracted with CH2Cl2 (3 × 50 mL), the combined organic
layers were dried (MgSO4) and filtered, and the filtrate was
concentrated in vacuo.
Con clu sion s
Novel NOTA-based chelators (1 and 2) with pendent
donor groups are prepared to provide stable complexes
while also providing improved Y(III) complexation
kinetics as an alternative to the use of DOTA derivatives
in RIT. The syntheses of 1 and 2 are performed via a
convenient and high-yield cyclization route. The relative
complexation kinetics of these new chelates (1, 2) and
DOTA with Y(III) indicates that 1 forms a Y(III)
complex most rapidly among those studied and displays
a dramatically enhanced formation rate compared to
DOTA. Serum stability studies indicate that the ethyl-
ene-bridged ligand 1 in serum forms a more stable
complex with 88Y(III) than the analogous propylene-
bridged ligand 2. There is no measurable loss of
88Y(III) from the 88Y-labeled chelate 1 for up to 14
days, indicating serum stability comparable to that of
88Y-DOTA. However, 88Y-2, having a longer sidearm,
exhibits a substantial release of 88Y from the ligand.
Thus, the strategy of providing a partial NOTA platform
for the metal ion to set in to increase the complex
formation rate and then to stabilize the complex by
introducing pendent donor groups appears to be suc-
cessful. The biodistribution studies show that 86Y-1
is stable in vivo and is compared favorably with
86Y-DOTA. The results of serum stability, in vivo
biodistribution, and the kinetics study suggest that the
novel chelator 1 reported herein might be a potential
yttrium chelating agent for RIT applications. Derivati-
1-(2-C h lo r o e t h y l)-b is (p -t o lu e n e -4-s u lfo n y l)[1,4,7]-
tr ia za cyclon on a n e (5a ). The residue was purified via column
chromatography on silica gel, eluting with 20% EtOAc-
hexane. Pure 5a was obtained as a colorless oil (930 m g, 93%):
1H NMR (CDCl3) δ 2.53 (s, 6 H), 3.05-3.14 (m, 8 H), 3.62-
3.75 (m, 8 H), 7.42 (d, J ) 9.2 Hz, 4 H), 7.67 (d, J ) 9.2 Hz, 4
H); 13C NMR (CDCl3) δ 21.4 (q), 42.4 (t), 51.4 (t), 52.6 (t), 56.3

3462 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Chong et al.
(t), 59.0 (t), 127.1 (d), 129.7 (d), 135.2 (s), 143.8 (s). Anal. Calcd
for C22H30N3S2O4Cl: C, 52.84; H, 6.05. Found: C, 52.78; H,
6.35.
55.4 (t), 126.9 (d), 129.6 (d), 135.0 (s), 143.3 (s). HRMS
(positive-ion FAB) Calcd for C23H34N4S2O4: [M + H]+ m/z
495.2100. Found: [M + H]+ m/z 495.2094. Anal. Calcd for
23H34N4S2O4(H2O)0.5: C, 54.85; H, 7.00. Found: C, 54.47; H,
6.91.
Gen er a l P r oced u r e for Det osyla t ion of 7a a n d 7b .
C
1-(3-Ch lor op r op yl)-b is(p -t olu e n e -4-su lfon yl)[1,4,7]-
tr ia za cyclon on a n e (5b). The residue was purified via column
chromatography on silica gel, eluting with 15% EtOAc-
hexane. Pure 5b was obtained as a colorless oil (966 mg,
94%): 1H NMR (CDCl3) δ 2.00 (t, J ) 6.1 Hz, 2 H), 2.51 (s, 6
H), 2.82 (t, J ) 6.0 Hz, 2 H), 2.94-2.94 (m, 4 H), 3.33 (s, 4 H),
3.58 (s, 2 H), 3.80 (t, J ) 6.0 Hz, 4 H), 7.41 (d, J ) 8.7 Hz, 4
H), 7.75 (d, J ) 8.7 Hz, 4 H); 13C NMR (CDCl3) δ 21.3 (q), 30.1
(t), 43.1 (t), 51.4 (t), 52.7 (t), 53.5 (t), 55.7 (t), 127.0 (d), 129.7
(d), 135.0 (s), 143.4 (s). HRMS (positive-ion FAB) Calcd for
Compound 7a or 7b (1 mmol) was dissolved in concentrated
H2SO4 (5 mL) and heated to 115 °C for 72 h under argon. The
resulting solution was cooled to ambient temperature and
added in portions to Et2O (150 mL) at -60 °C. The resulting
precipitate was collected, washed with Et2O (20 mL), and
immediately dissolved in H2O (25 mL). The aqueous solution
was extracted with Et2O (10 mL), concentrated to 5 mL, and
neutralized with 50% NaOH. The resulting mixture was
extracted with CH2Cl2 (3 × 50 mL), and the combined organic
layers were dried (MgSO4) and filtered, and the filtrate was
concentrated in vacuo.
2-[1,4,7]Tr iazacyclon on an -1-yleth ylam in e (8a). The crude
8a was thereby obtained as a pale-yellow oil (159 mg, 92%):
1H NMR (CDCl3) δ 2.27-2.41 (m, 16 H), 3.72 (4 H); 13C NMR
(CDCl3) δ 39.4 (t), 45.8 (t), 46.0 (t), 52.4 (t), 59.7 (t). This
material was observed to be unstable; accordingly, it was used
immediately as obtained in the next step.
C
23H32N3S2O4Cl: [M + H]+ m/z 514.1601. Found: [M + H]+
m/z 514.1600. Anal. Calcd for C23H32N3S2O4Cl(H2O)0.5
: C,
52.81; H, 6.36. Found: C, 52.72; H, 6.49.
Gen er a l P r oced u r es for th e Rea ction of 5a a n d 5b
w ith Azid e. A mixture of compound 5a or 5b (3.3 mmol) and
NaN3 (6.93 mmol) in DMSO (20 mL) was heated to 90 °C for
4 h. The resulting mixture was poured into ice-water and
extracted with ethyl ether (2 × 50 mL). The combined organic
layers was washed with H2O (3 × 50 mL), dried (MgSO4),
filtered, and concentrated in vacuo.
3-[1,4,7]Tr ia za cyclon on a n -1-ylp r op yla m in e (8b ). The
crude 8b was thereby obtained as a colorless, viscous oil (164
mg, 89%): 1H NMR (CDCl3) δ 0.94 (t, J ) 3.2 Hz, 2 H), 1.48
(s, 4 H), 1.89-2.09 (m, 16 H); 13C NMR (CDCl3) δ 30.1 (t), 39.0
(t), 45.56 (t), 45.58 (t), 51.9 (t), 54.0 (t). HRMS (positive ion
FAB) Calcd for C9H22N4: [M + H]+ m/z 187.1923. Found: [M
+ H]+ m/z 187.1929.
Gen er a l P r oced u r e for Alk yla tion of Tetr a a m in es 8a
a n d 8b. To a suspension of 8a or 8b and K2CO3 in CH3CN
under argon was added dropwise tert-butyl bromoacetate, and
the resulting mixture was heated at 65 °C for 24 h. The
reaction mixture was allowed to cool gradually to ambient
temperature and was filtered, and the filtrate was concen-
trated in vacuo.
1-(2-Azid oet h yl)-4,7-b is(p -t olu en e-4-su lfon yl)[1,4,7]-
tr ia za cyclon on a n e (6a ). The crude 6a could be used directly
for the next step or purified via column chromatography on
neutral alumina, eluting with 15% CH2Cl2-hexane. Pure 6a
(1.56 g, 96%) was thereby obtained as a colorless viscous oil:
1H NMR (CDCl3) δ 2.44 (s, 6 H), 2.82 (t, J ) 6.2 Hz, 4 H), 2.99
(s, 2 H), 3.18-3.35 (m, 4 H), 3.38 (t, J ) 6.2 Hz, 4 H), 3.54 (s,
2 H), 7.34 (d, J ) 8.7 Hz, 4 H), 7.71 (d, J ) 8.7 Hz, 4 H); 13C
NMR (CDCl3) δ 21.4 (q), 49.5 (t), 51.0 (t), 52.1 (t), 55.4 (t), 55.5
(t), 126.7 (d), 129.4 (d), 134.9 (s), 143.1 (s). Anal. Calcd for
C
22H30N6S2O4: C, 52.16; H, 5.97. Found: C, 52.33; H, 6.10.
1-(3-Azid op r op yl)-4,7-bis(p-tolu en e-4-su lfon yl)[1,4,7]-
tr ia za cyclon on a n e (6b). The crude 6b could be used directly
for the next step or purified via column chromatography on
neutral alumina, eluting with 15% CH2Cl2-hexane. Pure 6b
(1.54 g, 92%) was thereby obtained as a colorless viscous oil:
1H NMR (CDCl3) δ 1.84 (t, J ) 5.1 Hz, 2 H), 2.52 (s, 6 H), 2.75
(t, J ) 6.8 Hz, 2 H), 2.90-3.04 (m, 4 H), 3.30 (s, 4 H), 3.49-
3.64 (s, 6 H), 7.41 (d, J ) 8.6 Hz, 4 H), 7.53 (d, J ) 8.6 Hz, 4
H); 13C NMR (CDCl3) δ 21.4 (q), 27.0 (t), 49.1 (t), 51.5 (t), 52.7
(t), 53.8 (t), 55.5 (t), 127.1 (d), 129.7 (d), 135.0 (s), 143.5 (s).
HRMS (positive-ion FAB) Calcd for C23H32N6S2O4: [M + H]+
m/z 521.2005. Found: [M + H]+ m/z 521.1996. Anal. Calcd for
[3-(4,7-B i s -t er t -b u t o x y c a r b o m e t h y l[1,4,7]t r i a z a -
cyclon on a n -1-yleth yl)-ter t-bu toxyca r bon ylm eth yla m in o]-
a cetic Acid ter t-Bu tyl Ester (9a ). Compound 8a (500 mg,
2.94 mmol), K2CO3 (913 mg, 11.76 mmol), and tert-butyl
bromoacetate (2.86 g, 14.7 mmol) in CH3CN (35 mL) afforded
9a (888 mg, 48%) as a colorless oil after purification by
chromatography on silica gel, eluting with 5% MeOH-CH2-
Cl2: 1H NMR (CDCl3) δ 1.38-1.72 (m, 36 H), 2.8 (s, 4 H), 3.05-
3.73 (m, 20 H); 13C NMR (CDCl3) δ 27.4 (q), 47.9 (t), 49.3 (t),
50.7 (t), 52.3 (t), 53.6 (t), 55.2 (t), 56.2 (t), 81.2 (s), 81.3 (s),
169.5 (s), 169.8 (s). HRMS (positive ion FAB) Calcd for
C
23H32N6S2O4(H2O)0.5: C, 52.16; H, 6.28. Found: C, 52.02; H,
6.26.
Gen er a l P r oced u r e for th e Red u ction of Azid es 6a a n d
C
32H60N4O8: [M + H]+ m/z 629.4489. Found: [M + H]+ m/z
629.4479.
[3-(4,7-B i s -t er t -b u t o x y c a r b o m e t h y l[1,4,7]t r i a z a -
6b. To a solution of the azides (4 mmol) in CH3OH (20 mL)
was added 10% Pd/C catalyst (100 mg). The resulting mixture
was subjected to hydrogenation by agitation with excess H2
(gas) at 25 psi in a Parr hydrogenator apparatus at ambient
temperature for 3 h. The reaction mixture was filtered through
Celite, and the filtrate was concentrated in vacuo.
2-[4,7-Bis(p-tolu en e-4-su lfon yl)[1,4,7]tr iazacyclon on an -
1-yl]eth yla m in e (7a ). The residue was purified via column
chromatography on neutral alumina, eluting with 10%
CH3OH-EtOAC. Pure 7a (1.79 g, 93%) was thereby obtained
as a colorless oil: 1H NMR (CDCl3) δ 2.05-2.43 (m, 6 H),
2.69-2.95 (m, 10 H), 3.25 (s, 4 H), 3.49 (s, 4 H), 7.34 (d, J )
8.4 Hz, 4 H), 7.65 (d, J ) 8.4 Hz, 4 H); 13C NMR (CDCl3) δ
21.0 (q), 39.4 (t), 51.6 (t), 52.5 (t), 55.2 (t), 59.8 (t), 126.7 (d),
129.4 (d), 134.8 (s), 143.1 (s). Anal. Calcd for C22H32N4S2O4:
C, 54.98; H, 6.71. Found: C, 55.00; H, 7.13.
3-[4,7-Bis(p-tolu en e-4-su lfon yl)[1,4,7]tr iazacyclon on an -
1-yl]p r op yla m in e (7b ). The residue was purified via col-
umn chromatography on neutral alumina, eluting with 10%
CH3OH-EtOAc. Pure 7b (1.76 g, 91%) was thereby obtained
as a colorless oil: 1H NMR (CDCl3) δ 1.67 (t, J ) 6.2 Hz, 2 H),
1.95 (s, 2 H), 2.48 (s, 6 H), 2.67 (t, J ) 5.3 Hz, 2 H), 2.82 (t, J
) 5.6 Hz, 2 H), 2.95 (s, 4 H), 3.26 (s, 4 H), 3.53 (s, 4 H), 7.37
(d, J ) 8.2 Hz, 4 H), 7.72 (d, J ) 8.2 Hz, 4 H): 13C NMR
(CDCl3) δ 21.3 (q), 31.2 (t), 40.1 (t), 51.3 (t), 52.4 (t), 54.9 (t),
cyclon on a n -1-ylp r op yl)-ter t-bu toxyca r bon ylm eth yla m i-
n o]a cetic Acid ter t-Bu tyl Ester (9b). Compound 8b (360
mg, 1.95 mmol), K2CO3 (1.08 g, 7.80 mmol), and tert-butyl
bromoacetate (1.52 g, 7.82 mmol) in CH3CN (23 mL) afforded
9b (652 mg, 52%) as a colorless oil after purification by
chromatography on silica gel, eluting with 4% MeOH-CH2-
Cl2: 1H NMR (CDCl3) δ 1.21-1.62 (m, 36 H), 2.00 (s, 2 H),
2.60-2.95 (m, 6 H), 3.03-3.84 (m, 18 H); 13C NMR (CDCl3) δ
22.7 (t), 27.9 (q), 49.2 (t), 51.4 (t), 52.0 (t), 52.7 (t), 54.1 (t),
55.9 (t), 57.7 (t), 81.1 (s), 81.3 (s), 170.2 (s), 170.4 (s). HRMS
(positive-ion FAB) Calcd for C33H62N4O8: [M + H]+ m/z
643.4646. Found: [M + H]+ m/z 642.4641.
Dep r otection of Tetr a -ter t-bu tyl Ester s 9a a n d 9b. A
solution of either 9a or 9b in dry 1,4-dioxane cooled in a ice-
water bath was saturated with HCl(g) for 4 h, after which the
mixture was warmed to ambient temperature and then stirred
for 12 h. The precipitate was collected, washed with ethyl
ether, and dissolved in water. The solution was then lyophi-
lized to give 1 or 2 as a pale-yellow solid.
[2-(4,7-Bisca r boxym eth yl[1,4,7]tr ia za cyclon on a n -1-yl-
eth yl)ca r bon ylm eth yl-a m in o]a cetic Acid Tetr a h yd r o-
ch lor id e (1). Compound 9a (130 mg, 0.21 mmol) in 1,4-
dioxane (20 mL) afforded 1 (78 mg, 92%) as a salt: 1H NMR

Yttrium Chelators for Radioimmunotherapy
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3463
Metal Chelates. Pure Appl. Chem. 1991, 63, 427-463. (c)
Chakrabarti, M. C.; Le, N.; Paik, C. H.; Graff, W. G. De.;
Carrasquillo, J . A. Prevention of Radiolysis of Monoclonal
Antibody During Labeling. J . Nucl. Med. 1996, 37, 1384-1388.
(d) Sharkey, R. M.; Kaltovich, F. A.; Shin, L. B.; Fand, I.;
Govelitz, G.; Goldenberg, D. M. Radioimmunotherapy of Human
Colonic Cancer Xenografts with 90Y-Labeled Monoclonal Anti-
bodies to Carcinoembryonic Antigen. Cancer Res. 1988, 48,
3270-3275. (e) Lee, Y.-C.; Washburn, L. C.; Sun, T. T. H.
Radioimmunotherapy of Human Colorectal Carcinoma Xe-
nografts Using 90Y-Labeled Monoclonal Antibody CO17-1A
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1990, 50, 4546-4551.
(CDCl3) δ 3.21-3.45 (m, 10 H), 3.75-3.93 (m, 8 H), 4.63-4.78
(m, 8 H); 13C NMR (CDCl3) δ 49.6 (t), 50.7 (t), 51.2 (t), 51.6 (t),
55.6 (t), 56.9 (t), 169.7 (s), 170.2 (s). MS (positive-ion FAB) [M
+ H]+ m/e 405. Anal. Calcd for C16H28N4O8(HCl)4(H2O)4: C,
30.88; H, 6.48. Found: C, 30.71; H, 6.38.
[3-(4,7-Bisca r boxym eth yl[1,4,7]tr ia za cyclon on a n -1-yl-
p r op yl)ca r bon ylm eth yla m in o]a cetic Acid Tetr a h yd r o-
ch lor id e (2). Compound 9b (280 mg, 0.44 mmol) in 1,4-
dioxane (30 mL) afforded 2 (162 mg, 88%) as a salt: 1H NMR
(D2O, pD ) 1) δ 3.23-3.62 (m, 8 H), 3.90 (s, 4 H), 4.21 (s, 4 H),
4.81 (s, 10 H); 13C NMR (D2O, pD ) 1) δ 49.3 (t), 50.3 (t), 52.7
(t), 54.0 (t), 54.3 (t), 56.0 (t), 167.4 (s), 171.1 (s). MS (positive-
ion FAB) m/e 419 [M + H]+. Anal. Calcd for C17H30N4O8(HCl)4-
(H2O): C, 35.07; H, 6.23. Found: C, 35.45; H, 6.15.
Kin etics. Formation kinetics of Y(III) complexes of 1, 2, and
DOTA (Macrocyclics) was measured by monitoring the absorp-
tion at 652 nm. To a solution of YCl3 (2 mL, 1.6 µM, atomic
absorption standard solution, Aldrich) was added arsenazo-
(III) (5 µM, Sigma) in NH4OAc (0.15 M, pH 4.0, metal-free).
The resulting Y(III)-arsenazo(III) mixture was stirred for at
least 2 h for complete equilibration and added to a solution of
ligand (20 µL, 10 mM) in a cell. After mixing (<5 s), absorption
of the solution at 652 nm (absorption wavelength of Y(III)-
arsenazo(III))6b was immediately followed over time at room
temperature using a HP 8452A Diode Array Spectrophotom-
eter which was calibrated with a 5 µM arsenazo(III) solution.
Without any ligand added, the absorption was 0.089 for the
YCl3-arsenazo(III) complex at 652 nm. The competition
between arsenazo(III) (5 µM) and ligand (100 µM) for Y(III)
(1.6 µM) was followed by the decrease in the absorbance at
652 nm.
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and Model Absorbed Dose Calculations for Radiolabeled Tumor
Associated Antibodies. Med. Phys. 1984, 11, 638-645. (c) Chinol,
M.; Hnatowich, D. J . Generator-Produced Yttrium-90 for Ra-
dioimmunotherapy. J . Nucl. Med. 1987, 28, 1465-1470. (d)
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B.; Kimura. Thermodynamic and Kinetic Studies of Lantha-
nide Complexes of 1,4,7,10,13-Pentaazacyclopentadecane-
N,N′,N′′,N′′′,N′′′′-Pentaacetic Acid and 1,4,7,10,13,16-Hexaaza-
cyclooctadecane-N,N′,N′′,N′′′,N′′′′,N′′′′′-Hexaacetic Acid. Inorg.
Chem. 1991, 30, 1270-1273. (c) Kasprzyk, S. P.; Wilkins, R. G.
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(7) (a) Cox, J . P. L.; Craig, A. S.; Helps, I. M.; J ankowski, K. J .;
Parker, D.; Eaton, M. A. W.; Millican, A. T.; Millar, K.; Beeley,
N. R. A.; Boyce, B. A. Synthesis of C- and N-Functionalized
Derivatives of 1,4,7-Triazacyclononane-1,4,7-Triyltriacetic Acid
(NOTA), 1,4,7,10-Tetra-Azacyclododecane-1,4,7,10-Tetrayltetra-
Acetic Acid (DOTA), and Diethylenetriaminepenta-acetic Acid
(DTPA): Bifunctional Complexing Agents for the Derivatisation
of Antibodies. J . Chem. Soc., Perkin Trans. 1 1990, 2567-2576.
(b) Kline, S. J .; Betebenner, D. A.; J ohnson, D. K. Carboxym-
ethyl-Substituted Bifunctional Chelators: Preparation of Aryl
Isothiocyanate Derivatives of 3-(Carboxymethyl)-3-Azapen-
tanedioic Acid, 3,12-Bis(carboxymethyl)-6,9-Dioxa-3,12-Diaza-
Ser u m Sta bility. The 88Y complexes of 1 and 2 were
prepared by the addition of 350 µCi of 88Y (0.1 M HCl,
adjusting the pH to 4.5 with 5 M NH4OAc) to 20 µL of 0.2 M
ligand solution in 0.15 M NH4OAc of pH 4.5. The reactions
were forced to completion by heating the reaction mixture at
80 °C for 18 h, after which they were loaded onto a column of
Chelex-100 resin (1 mL volume bed, equilibrated with 0.15 M
NH4OAc). The complexes were eluted from the resin with 0.15
M NH4OAc, while the resin retained the free 88Y. The pH of
the 88Y complex solutions was adjusted to 7.0 with PBS buffer,
and 250 µCi of either complex was added to 1 mL of human
serum incubated at 37 °C. An aliquot of the serum (5-10
µL) was taken at selected times (Figure 1) and analyzed by
SE-HPLC. The serum stability of the 88Y complexes was
assessed by measuring the release of 88Y radionuclide from
the complexes to serum proteins using SE-HPLC with a TSK-
3000 column. The column was eluted with PBS at a 1 mL/
min flow rate.
In Vivo Biod istr ibu tion Stu d ies. Female Balb/c mice
were obtained from Charles River Laboratories (Wilmington,
MA) at 4-6 weeks of age. The pH of the 86Y-labeled ligands
were adjusted to pH ∼7.0 with 0.5 M sodium bicarbonate (pH
10.5) and diluted in phosphate-buffered saline. The radio-
labeled ligands (5.9 µCi of 86Y-DOTA, 5.0 µCi of 86Y-1) were
administered to the mice in 200 µL via tail vein injection. The
mice (five per data point) were sacrificed by exsanguination
at 0.5, 1, 4, 8, and 24 h. Blood and the major organs were
harvested and wet-weighed, and the radioactivity was mea-
sured in a γ-scintillation counter (Minaxi-γ; Packard, Downers
Grove, IL). The % ID/g was determined for each tissue. The
values presented are the mean and standard error of the mean
for each tissue. All animal experiments were performed in
compliance with current regulations and guidelines of the U.S.
Department of Agriculture and the NIH Animal Research
Advisory Committee.
Ack n ow led gm en t. We thank the structural Mass
Spectra Group (Dr. L. Pannell, NIDDK, Bethesda, MD)
for obtaining the mass spectra.
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