Synthesis and evaluation of novel macrocyclic and acyclic ligands as contrast enhancement agents for magnetic resonance imaging

Article abstract of DOI:10.1021/jm051009k

Novel chelates PIP-DTPA, AZEP-DTPA, NETA, NPTA, and PIP-DOTA were synthesized and evaluated as potential magnetic resonance imaging (MRI) contrast enhancement agents. The T₁ and T₂ relaxivities of their corresponding Gd(III) complexe

Full text of DOI:10.1021/jm051009k

J. Med. Chem. 2006, 49, 2055-2062
2055
Synthesis and Evaluation of Novel Macrocyclic and Acyclic Ligands as Contrast Enhancement
Agents for Magnetic Resonance Imaging
Hyun-Soon Chong,*,†,‡ Kayhan Garmestani,‡ L. Henry Bryant, Jr.,§ Diane E. Milenic,‡ Terrish Overstreet,‡ Noah Birch,†
Thien Le,† Erik D. Brady,‡ and Martin W. Brechbiel‡
Biological, Chemical, and Physical Sciences Department, Chemistry DiVision, Illinois Institute of Technology, Chicago, Illinois 60616,
Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute,
Bethesda, Maryland 20892, and Diagnostic Radiology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892
ReceiVed October 7, 2005
Novel chelates PIP-DTPA, AZEP-DTPA, NETA, NPTA, and PIP-DOTA were synthesized and evaluated
as potential magnetic resonance imaging (MRI) contrast enhancement agents. The T1 and T2 relaxivities of
their corresponding Gd(III) complexes are reported. At clinically relevant field strengths, the relaxivities of
the complexes are comparable to that of the clinically used contrast agents Gd(DTPA) and Gd(DOTA). The
serum stability of the 153Gd-labeled complexes, Gd(PIP-DTPA), Gd(AZEP-DTPA), Gd(PIP-DOTA), Gd-
(NETA), and Gd(NPTA), was assessed by measuring the release of 153Gd from the complexes. 153Gd-
(NETA), 153Gd(PIP-DTPA), and 153Gd(PIP-DOTA) were found to be stable in human serum for up to 14
days without any measurable loss of radioactivity. Significant release of 153Gd was observed with the 153Gd-
(III) radiolabled NPTA. In vivo biodistribution of the153Gd-labeled complexes was performed to evaluate
their in vivo stability. While Gd(AZEP-DTPA) and Gd(NPTA) were found to be unstable in vivo,
Gd(NETA), Gd(PIP-DTPA), and Gd(PIP-DOTA) were excreted without dissociation. These results suggest
that the Gd(III) complexes of the novel chelates NETA, PIP-DTPA, and PIP-DOTA possess potential as
MRI contrast enhancement agents. In particular, the piperidine backboned chelates Gd(PIP-DTPA) and
Gd(PIP-DOTA) displayed reduced kidney retention as compared to the nonspecific MRI contrast agent
Gd(DOTA) at all time points, although the observed effects were relatively small at 0.5 h postinjection.
Incorporation of the lipophilic piperidine ring appears to confer a moderate effect on the liver uptake of
these two chelates.
Introduction
cellular distribution and the disadvantages of low relaxivity, low
tissue specificity, and rapid clearance. Considerable research
efforts have been directed toward developing safe Gd(III)-based
MR contrast agents with high tissue specificity and sensitivity.
Magnetic resonance imaging (MRI), a noninvasive and high-
resolution imaging technique, has become a powerful tool in
diagnostic medicine.1 MRI gives distribution of the MR signal
intensity resulting from the longitudinal (T1) and the transverse
(T2) magnetic relaxation time of tissue water protons.2 The
images with a clear contrast between tissues with different
relaxation times of water protons T1 and T2 can be obtained
with MRI. However, the innate tissue contrast in images is not
sufficient to distinguish between normal and pathogenic tissues
when they produce similar signal intensity. To enhance contrast
between tissues, paramagnetic metal complexes are injected into
the body and lead to significant improvement in the contrast of
T1-weighted or T2-weighted MR image by increasing the
difference in the signal intensity from two different types of
tissues.3
The lanthanide Gd(III) is known to be an optimal paramag-
netic metal for MRI because of high electronic spin (7/2) and
slow electronic relaxation rate.4 Free Gd(III), however, is toxic
with severe side effects.5 Thus, stable Gd(III) complexes are
required for in vivo use with efficient excretion postadminis-
tration. A number of Gd(III) complexes such as Gd(DOTA)
and Gd(DTPA) (Figure 1) are clinically approved for use in
MRI.2 However, most contrast agents have nonspecific extra-
MRI is proven to be more sensitive and specific than other
medical tests for detecting liver malignancies and for distin-
guishing them from benign lesions.7 The gadolinium complexes
of BOP-DTPA and EOB-DTPA (Figure 1) are the clinically
approved hepatobiliary agents.6 These agents are partially taken
up by normal hepatocytes, probably due to the binding of the
lipophilic aromatic moiety to cytosolic protein in the hepato-
cytes,2 and this selective uptake of the agents into the normal
liver cells yields enhanced contrast between normal and
tumorous liver cells on T1-weighted images.7 The utility of these
agents is in the detection and the characterization of hepato-
celluar lesions.8 However, the Gd(III) complexes provide low
detection and characterization of metastatic lesions.7
We have recently prepared stereochemically pure piperidine
(PIP-DTPA) and azepane (AZEP-DTPA) backboned DTPA
derivatives as potential hepatocellular agents (Figure 2).9a Our
strategy in the design of the chelates was to increase lipophilicity
and rigidity of the chelate system by incorporating either a
piperidine or an azepane ring into the DTPA system. This was
anticipated to result in both enhanced hepatobiliary clearance
and in vivo complex stability. In general, conformational
constraint of chelating agents, i.e., inclusion of a rigid structure
to control the geometry of the metal binding donor groups, has
a significant effect on the stability of the formed metal
complexes.10 High lipophilicity is known to result in greater
hepatobiliary clearance.11 We previously reported that the
Gd(III) complexes of the new chelates PIP-DTPA and AZEP-
* To whom correspondence should be addressed. Address: Biological,
Chemical, and Physical Science Department, Chemistry Division, Illinois
Institute of Technology, 3101 S. Dearborn St, LS 182, Chicago, IL 60616.
Phone: 312-567-3235. Fax: 312-567-3494. E-mail: Chong@iit.edu.
† Illinois Institute of Technology.
‡ National Cancer Institute.
§ National Institutes of Health.
10.1021/jm051009k CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/25/2006

2056 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Chong et al.
Figure 1. Magnetic resonance imaging contrast agents in clinical use.
Figure 2. New potential Gd(III)-based magnetic resonance imaging contrast enhancement agents.
DTPA display comparable relaxivity and significantly improved
in vitro stability to Gd(DTPA).9a
labeled complexes of the new chelates were investigated in
human serum and mice, respectively.
In our continuing effort to develop liver-specific contrast
enhancement agents, Gd(PIP-DTPA) and Gd(AZEP-DTPA)
are further evaluated for in vivo stability. Novel Gd(III)
complexes of PIP-DOTA, NETA, and NPTA (Figure 2) have
been prepared and evaluated. The structurally new chelates
NETA and NPTA possess both macrocyclic and acyclic pendent
sidearms. The dynamic participation of the pendent sidearm in
NETA and NPTA in binding to Gd(III) is expected to potentially
impact access and relaxivity of water protons and complex
stability. A novel chelate, PIP-DOTA, possesses a liphophilic
piperidine ring. The liphophilicity and rigidity of PIP-DOTA
is greater than those of the parent DOTA. Like PIP-DTPA,
PIP-DOTA is expected to provide enhanced hepatobiliary
clearance while maintaining the complex stability of DOTA.
We herein report the synthesis and evaluation of PIP-DOTA
as MRI contrast agents. We also evaluated a new series of
Gd(III) chelates, PIP-DTPA, AZEP-DTPA, NETA, and
NPTA. The relaxivities of the corresponding Gd(III) complexes
were measured. The in vitro and in vivo stabilities of the 153Gd-
Results and Discussion
Synthesis of Novel Chelates. The chemical structures of the
chelates PIP-DTPA, AZEP-DTPA, PIP-DOTA, NETA, and
NPTA for the present study are shown in Figure 2. PIP-
DTPA,9a AZEP-DTPA,9a NETA,9b and NPTA9b were prepared
as described previously. The synthesis of PIP-DOTA contain-
ing a piperidine backbone is shown in Scheme 1. The key step
in the synthesis of PIP-DOTA is the coupling reaction between
two precursor molecules 3 and 4 to provide macrocycle 5. This
cyclization was very efficiently achieved under a high dilution
condition. To accomplish this coupling route, precursor molecule
4 was prepared by reaction of ethyl glycine with excess
ethylenediamine.12 Precursor molecule 3 was prepared from the
readily available 2,6-pyridinedicarboxylic acid 1. Thus, catalytic
hydrogenation of pyridine 2,6-dicarboxylic acid 1 over rhodium
on alumina provided enantiomerically pure cis-2,6-piperidine
dicarboxylic acid, which subsequently was reacted with CBZ-
Cl to provide N-CBZ-cis-2,6-dicarboxylic acid 2.13 The diacid
Scheme 1. Synthesis of PIP-DOTA

NoVel Agents for Magnetic Resonance Imaging
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2057
Figure 3. Plots of T1 relaxivity for Gd(NETA) (O), Gd(NPTA) (]), Gd(AZEP-DTPA) (b), Gd(PIP-DTPA) ([), Gd(PIP-DOTA) (9), Gd(DTPA)
(2), and Gd(DOTA) (4) at pH 7 and 23 °C. The data for Gd(AZEP-DTPA) and Gd(PIP-DTPA) were previous results from ref 9a.
Figure 4. Plots of T2 relaxivity for Gd(NETA) (O), Gd(NPTA) (]), Gd(AZEP-DTPA) (b), Gd(PIP-DTPA) ([), Gd(PIP-DOTA) (9), Gd(DTPA)
(2), and Gd(DOTA) (4) at pH 7 and 23 °C. The data for Gd(AZEP-DTPA) and Gd(PIP-DTPA) were previous results from ref 9a.
2 was converted into the activated ester 3 in 78% yield. Coupling
of 3 with 4 under high dilution conditions provided piperidine-
backboned macrocycle 5 in good yield (53%). The CBZ group
in 5 was removed by catalytic hydrogenation over palladium
to provide bicyclic triamide 6. The reduction of carbonyl groups
in 6 was attempted with 1 M BH3-THF at 50 °C, initially
adapting previously reported conditions.14 However, these
reaction conditions provided only a trace of 7 along with
partially reduced byproducts containing a mixture of unreduced
carbonyl groups adjacent to the piperidine ring as confirmed
by mass and NMR spectra. A successful reduction of 6 was
performed under a somewhat harsh reflux condition while
changing the borane reagent. Thus, the bicyclic triamide 6 was
treated with BH3-SMe2 complex and refluxed for 5 days to
cleanly provide the cyclic tetraamine 7 (55%). For the synthesis
of 8, 7 was reacted with tert-butyl bromoacetate in the presence
of NaCO3 at 65 °C. However, these reaction conditions provided
an undesired penta-alkylated quaternary byproduct as confirmed
by mass (m/z ) 783.4 [M + H+]) and 13C NMR (four carbonyl
peaks of tert-butyl ester, 163.7, 169.4, 170.3, and 170.6 at a
2:1:1:1 ratio) analysis. Alkylation of the HCl salt of 7 was then
carried out in DMF in the presence of DIPEA and KI at 90 °C
to provide 8 as the major product in 37% yield. The tert-butyl
groups in 8 were removed by treating with HCl(g) to provide
the desired chelate, PIP-DOTA (9) in 87% yield.
Relaxivity. The Gd(III) complexes of the new chelates were
prepared using a described method.9a T1 and T2 NMRD profiles
for the Gd(III) complexes are depicted in Figures 3 and 4,
respectively. For comparison purposes, the T1 and T2 relaxivities
of the commercially available Gd(DTPA) (Magnevist) and
Gd(DOTA) (Dotarem) were measured. It appears that the T1
NMRD profiles parallel the T2 NMRD profiles. Qualitatively
and quantitatively, the T1 and T2 NMRD profiles for the newly
synthesized Gd(III) complexes are similar to those of the
clinically used MR contrast agent GdDTPA (Magnevist) and
to those of other previously reported Gd acyclic and macrocyclic
complexes where the Gd(III) is coordinated to the eight donor
atoms of the ligands with one vacant coordination site available
to bind a water molecule.2 In NMRD, the T1 relaxivities of the
evaluated new Gd(III) complexes at 20 MHz were comparable
to that of Gd(DOTA), and the difference of relaxivity between
the Gd(III) complexes are in the range of (1.16 MHz. This
supports that the number of water molecules coordinated to the
Gd(III), q, is equal to 1.2,15 The relaxation rates for the Gd(III)
chelates are dominated by the inner-sphere dipolar coupling

2058 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Chong et al.
between the coordinated water molecule and the paramagnetic
Gd(III). The measured relaxivities arise from the exchange
between the coordinated water molecule and the surrounding
water molecules in solution. An increase in the number of
coordinated water molecules to the Gd(III), which could have
occurred with dissociation of the Gd(III) from the chelate in
solution, would have resulted in a significant increase in the
measured relaxivities. No indication of Gd(III) dissociation is
evident from the NMRD profiles, which is in agreement with
the serum stability studies. In a previous study, we reported
that Gd(PIP-DTPA) and Gd(AZEP-DTPA) display compa-
rable relaxivity to Gd(DTPA) at the clinically relevant field
strength, 61 MHz. Examination of the NMRD profiles at around
15 MHz provides evidence that the T1 and T2 relaxivities are
dominated by the rotational correlation time (tumbling time)
of these low molecular weight compounds.16 Inspection of T1
and T2 relaxivities at 60 MHz in Figures 3 and 4 indicates that
among the evaluated Gd(III) complexes, Gd(NETA) gives the
highest T1 and T2 relaxivity at 61 MHz, while PIP-DOTA gives
the lowest T1 and T2 relaxivity.
Figure 5. Serum stability of 153Gd(NETA) (4), 153Gd(NPTA) (9), 153
-
Gd(AZEP-DTPA) (]), 153Gd(PIP-DTPA) (0), 153Gd(DTPA) (2), and
153Gd(PIP-DOTA) (O) at pH 7 and 37 °C. The data for 153Gd(AZEP-
DTPA), 153Gd(PIP-DTPA), and 153Gd(DTPA) were previous results
from ref 9a.
It is noteworthy that Gd(NETA) and Gd(NPTA) having both
a macrocyclic and acyclic binding moiety display considerably
enhanced T1 relaxivity as compared to Gd(DTPA) and Gd-
(DOTA) and the other new agents evaluated in the present study.
For example, the T1 relaxivities of Gd(NETA), Gd(NPTA),
Gd(DTPA), and Gd(DOTA) at 61 MHz are 4.83, 4.66, 4.32,
and 3.84, respectively. Like DOTA and DTPA, NETA and
NPTA possess eight potential coordinating groups. Unlike
DOTA and DTPA, NETA and NPTA possess both macrocyclic
and acyclic binding moieties. Since the number of coordinated
water molecules (q ) 1) is the same for all complexes reported,15
enhanced relaxivities of Gd(NETA) and Gd(NPTA) compared
to DOTA and DTPA may be ascribed to the presence of both
the macrocyclic and acyclic moieties. All of the flexible acyclic
pendant binders may or may not participate in binding Gd(III).
Increase in q values of the Gd(III) complexes of bimodal ligands,
NETA and NPTA, could occur from a decrease in the ligand
denticity from eight- to seven-coordinate by one of the pendent
coordinating groups not being coordinated at all or from an
“on”-“off” mechanism of one of the pendent coordinating
groups. These types of binding to Gd(III) of one of the pendant
coordinates would have resulted in enhanced relaxivity of
Gd(NETA) and Gd(NPTA), providing values that are intermedi-
ate between one coordinated water molecule (q ) 1) and two
coordinated water molecules (q ) 2).
Gd(DTPA) displayed slightly higher T1 and T2 relaxivities
at all of the field strengths as compared to the Gd(III) complexes
of azepane and piperidine-backboned DTPA. The T1 and T2
relaxivities of Gd(DOTA) have been significantly reduced by
the addition of a piperidine ring into the system (PIP-DOTA).
The addition of a piperidine or azepane ring into DTPA or
DOTA resulted in a decrease in both T1 and T2 relaxivities. In
particular, a high degree of rigidity present in Gd(PIP-DOTA)
might lead to significantly reduced relaxivity as compared to
that in Gd(DOTA). It is interesting to note that at high field
strengths (greater than 10 MHz in units of the proton Larmor
frequency), T1 relaxivities of Gd(PIP-DTPA) are very similar
to those of Gd(DOTA).
measurable loss of radioactivity over 14 days. A number of
reports have shown that Gd(DOTA) is exceptionally stable in
human serum.2 In contrast, there is a rapid loss of 153Gd from
Gd(DTPA) with ∼15% released after 5 days. The behavior of
the Gd(NPTA) complex mirrors that of Gd(DTPA), i.e., a
pronounced loss (>20%) over the first 6 days. The Gd(AZEP-
DTPA) also shows evidence of steady metal transfer to serum
proteins, although the loss is much more gradual than noted
with DTPA or NPTA. The 153Gd-labeled complexes of the
piperidine-backboned chelates are exceptionally stable in serum.
Incorporation of the rigid piperidine ring into the DTPA system
seems to confer a significant effect on the complex stability in
serum. Somewhat surprisingly, the structurally similar chelates
NETA and NPTA have markedly different serum stabilities. A
considerable release of radioactivity was observed from the
153Gd(III) complex formed with NPTA which has a propylene
chain linking the pendent donor groups to the macrocycle.
However, no measurable loss of radioactivity was observed from
153Gd(NETA) which has an ethylene chain versus the propylene
of NPTA. The stability of the Gd(III) radiolabeled complex in
serum appears to be dependent on the carbon chain length
between the pendent donor groups and the macrocyclic ring in
NETA or NPTA. The observed release of radioactivity from
NPTA might be a consequence of the formation of a six-
membered chelate ring as opposed to what is generally
considered to be the more favorable arrangement of donors, i.e.,
a five-membered chelate ring for complexation of Gd(III).
In Vivo Biodistribution of 153Gd-Labeled Complexes. A
biodistribution study of the chelates was performed in athymic
mice to evaluate the in vivo stability of the 153Gd-labeled
chelates. For comparison purposes, a biodistribution of 153Gd-
labeled DOTA was also performed. The results of the biodis-
tribution studies for the new 153Gd-labeled chelates and 153Gd-
(DOTA) are shown in Table 1. If 153Gd is released from the
chelate, radioactivity would be expected to accumulate in the
bone, i.e., the femur, primarily. As a point of interest, the blood
and kidney data are included, which are representative of whole
body clearance. Radioactivity accumulated in selected organs
or cleared from the blood compartment was measured at five
time points, 0.5, 1, 4, 8, and 24 h postinjection of the 153Gd-
labeled complexes. The data are presented as the percent injected
dose per gram (% ID/g) calculated for blood and selected organs.
Examination of the data indicates that 153Gd(NETA), 153Gd-
Serum Stability. The stability of the corresponding 153Gd-
labeled complexes in human serum was assessed by measuring
the release of 153Gd from the complexes at 37 °C over 14 days.
The serum stability of 153Gd-labeled complexes thereby obtained
is shown in Figure 5. The chelates PIP-DTPA, PIP-DOTA,
and NETA sequestered 153Gd as the complex in serum with no

NoVel Agents for Magnetic Resonance Imaging
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2059
Table 1. Biodistribution of 153Gd-Labeled Chelates in Balb/c Mice Following Intravenous Injectiona
time points
0.5 h
1 h
4 h
8 h
24 h
ligand
tissue
PIP-DTPA
blood
liver
spleen
kidney
lung
0.79
1.09
0.30
2.96
0.57
0.43
2.31
0.44
0.71
0.18
1.83
0.39
0.18
0.64
0.96
1.37
0.40
4.16
0.89
0.60
0.87
4.05
11.14
2.81
13.41
3.80
2.97
9.00
1.31
0.56
0.35
3.89
0.92
0.51
1.06
1.60
2.78
1.04
14.06
1.32
0.96
3.74
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0.10
0.10
0.10
1.28
0.23
0.08
1.39
0.22
0.21
0.09
0.65
0.31
0.21
0.14
0.32
0.30
0.12
1.17
0.37
0.16
0.40
0.97
1.54
0.69
1.09
0.68
0.55
1.39
0.15
0.05
0.04
0.43
0.11
0.07
0.20
0.37
0.39
0.18
3.86
0.20
0.27
0.79
0.05
0.78
0.09
1.36
0.17
0.09
1.36
0.13
1.16
0.09
1.28
0.20
0.02
0.48
0.37
1.03
0.18
2.87
0.33
0.62
0.42
2.08
13.08
2.37
8.43
10.15
2.25
10.22
0.54
0.37
0.85
1.88
0.46
0.25
0.56
0.45
2.90
2.21
11.63
0.72
0.47
2.80
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0.02
0.11
0.01
0.11
0.01
0.03
1.04
0.03
0.63
0.01
0.33
0.04
0.035
0.081
0.27
0.35
0.04
0.83
0.11
0.70
0.08
0.43
1.17
0.39
6.85
10.86
0.24
0.75
0.04
0.03
0.86
0.41
0.12
0.07
0.33
0.20
0.33
2.44
1.80
0.22
0.11
0.43
0.01
0.50
0.08
1.16
0.60
0.06
0.46
0.01
0.54
0.06
0.80
0.12
0.07
0.30
0.01
0.29
0.10
1.55
0.09
0.07
0.11
0.20
16.99
1.56
10.67
4.04
3.14
15.23
0.00
0.11
0.29
2.12
0.05
0.02
0.11
0.04
3.11
0.69
14.81
0.26
0.41
1.78
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0.01
0.08
0.01
0.20
0.78
0.01
0.25
0.00
0.16
0.01
0.17
0.07
0.04
0.11
0.00
0.09
0.05
0.50
0.06
0.08
0.05
0.02
1.65
0.15
6.88
1.74
2.37
5.98
0.00
0.05
0.35
1.14
0.01
0.00
0.07
0.01
0.78
0.18
3.91
0.04
0.23
0.43
0.00
0.43
0.06
0.88
0.11
0.05
0.29
0.00
0.37
0.04
0.91
0.04
0.00
0.15
0.02
0.35
0.17
3.47
0.08
0.03
0.08
0.15
20.73
1.55
9.66
2.18
1.19
12.87
0.00
0.08
0.27
0.87
0.05
0.03
0.10
0.04
5.09
1.23
12.05
0.45
0.34
2.29
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0.00
0.02
0.01
0.06
0.03
0.02
0.15
0.00
0.07
0.01
0.13
0.01
0.02
0.01
0.02
0.06
0.07
1.91
0.01
0.01
0.02
0.08
1.42
0.29
4.92
0.34
0.08
1.94
0.00
0.01
0.26
0.16
0.02
0.01
0.05
0.02
1.35
0.40
3.22
0.09
0.09
0.64
0.00
0.37
0.05
0.47
0.23
0.02
0.20
0.00
0.42
0.04
0.66
0.03
0.00
0.22
0.00
0.15
0.04
1.12
0.04
0.02
0.08
0.07
19.19
3.25
14.96
1.77
1.45
13.65
0.00
0.03
0.04
0.59
0.03
0.02
0.03
0.02
2.89
0.70
6.80
0.32
0.20
1.84
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(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
0.00
0.04
0.00
0.17
0.28
0.00
0.03
0.00
0.09
0.01
0.15
0.00
0.01
0.03
0.00
0.02
0.01
0.22
0.01
0.01
0.01
0.03
1.57
0.91
8.43
0.33
0.39
1.96
0.00
0.00
0.01
0.07
0.01
0.00
0.00
0.01
0.31
0.20
1.45
0.03
0.05
0.22
heart
femur
blood
liver
spleen
kidney
lung
PIP-DOTA
heart
femur
blood
liver
spleen
kidney
lung
DOTA
heart
femur
blood
liver
spleen
kidney
lung
AZEP-DTPA
heart
femur
blood
liver
spleen
kidney
lung
NETA
heart
femur
blood
liver
spleen
kidney
lung
NPTA
heart
femur
a Values are the percent injected dose per gram (% ID/g) ( standard deviation.
(PIP-DTPA), and 153Gd(PIP-DOTA) display in vivo stability
comparable to that of 153Gd(DOTA), while 153Gd(AZEP-
DTPA) and 153Gd(NPTA) undergo in vivo dissociation with the
former exhibiting the greatest degree of 153Gd transfer. At 4 h
postinjection, the liver % ID/g values are 0.50 ( 0.08, 0.54 (
0.16, 0.29 ( 0.09, and 0.11 ( 0.05 for the 153Gd complexes of
PIP-DTPA, PIP-DOTA, DOTA, and NETA, respectively.
These Gd(III) complexes display uniformly low bone-uptake
which is promising considering that Gd(III) is a bone-seeking
metal.17 In addition, the data indicate a rapid blood clearance
for the new Gd(III) complexes. The control species 153Gd-
(DOTA) displays higher radioactivity levels in the kidney than
in other organs, although the values (<5% ID/g) are still low
on an overall scale. Data obtained with 153Gd(NETA) parallel
that noted with the DOTA analogue. 153Gd(PIP-DOTA)
exhibits lower radioactivity levels in blood and the organs over
all time points compared to 153Gd(PIP-DTPA), 153Gd(NETA),
and 153Gd(DOTA). It is noteworthy that 153Gd(PIP-DOTA) and
153Gd(PIP-DTPA) display a decreased kidney-to-liver ratio (up
to 5.7 times lower) as compared to Gd(DOTA) and Gd(NETA)
at all time points. At 0.5 h postinjection, the ratios for 153Gd-
(PIP-DOTA), 153Gd(PIP-DTPA), Gd(DOTA), and Gd(NETA)
are 2.58, 2.72, 3.04, and 6.95, respectively (Table 2), and kidney
retention of piperdine-backboned DOTA and DTPA 153Gd(PIP-
DOTA), 153Gd(PIP-DTPA), and 153Gd(DOTA) was 1.83, 2.96,
and 4.16, respectively. However, the difference in kidney
retention between these chelates was gradually larger over time
and maximized at 24 h, wherein the kidney-to-liver ratios for
153Gd(PIP-DTPA), Gd(PIP-DOTA), and Gd(DOTA) are 1.27,
1.57, and 7.45, respectively (Table 2). Incorporation of the
liphophilic piperidine ring into the chelate may account for a
modest increase in liver uptake of 153Gd(PIP-DOTA) and
153Gd(PIP-DTPA).
As predicted from the in vitro data, Gd(NPTA) was confirmed
to be unstable in vivo as well, and high radioactivity levels in
the kidney and liver at 24 h were observed. 153Gd-AZEP-
DTPA also clearly undergoes dissociation in vivo resulting in
significant radioactivity accumulation in the liver and bone.
These chelates have unacceptable stabilities and do not warrant
further evaluation. The results of the biodistribution studies as
a whole indicate that 153Gd-NETA, 153Gd-PIP-DTPA, and
153Gd-PIP-DOTA display comparable in vivo stability to
153Gd-DOTA and that these chelates might be further exploited
for use as MRI contrast agents. In particular, the in vivo
biodistribution results of 153Gd-PIP-DTPA and 153Gd-PIP-
DOTA indicate that incorporation of the lipophilic piperidine
ring provides a measurable effect on liver uptake of these two
chelates as evidenced by reduced kidney-to-liver ratio as

2060 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Chong et al.
Table 2. Biodistribution of 153Gd-Labeled Chelates in the Liver and the Kidneya
time points
4 h
complexes
tissue
0.5 h
1 h
8 h
24 h
153Gd(DOTA)
liver
kidney
liver
kidney
liver
kidney
liver
1.37 ( 0.30
4.16 ( 1.17
0.71 ( 0.21
1.83 ( 0.65
1.09 ( 0.10
2.96 ( 1.28
0.56 ( 0.18
3.89 ( 0.43
1.03 ( 0.35
2.87 ( 0.83
1.16 ( 0.63
1.28 ( 0.33
0.78 ( 0.11
1.36 ( 0.11
0.37 ( 2.44
1.88 ( 0.41
0.29 ( 0.09
1.55 ( 0.50
0.54 ( 0.16
0.80 ( 0.17
0.50 ( 0.08
1.16 ( 0.20
0.00 ( 0.18
2.12 ( 1.14
0.35 ( 0.06
3.47 ( 1.91
0.37 ( 0.07
0.91 ( 0.13
0.43 ( 0.02
0.88 ( 0.06
0.08 ( 0.40
0.87 ( 0.16
0.15 ( 0.02
1.12 ( 0.22
0.42 ( 0.09
0.66 ( 0.15
0.37 ( 0.04
0.47 ( 0.17
0.03 ( 0.20
0.59 ( 0.07
153Gd(PIP-DOTA)
153Gd(PIP-DTPA)
153Gd(NETA)
kidney
a Values are the percent injected dose per gram (% ID/g) ( standard deviation.
compared to the results of 153Gd-DOTA. The strategy of
backbone modification, in this case adding a lipophilic piperidine
ring into the system, appears to provide an entry point for
modification of the biological uptake properties of macrocyclic
MRI contrast agents.
chemical shifts are annotated as follows: ppm (multiplicity, integral,
coupling constant (Hz)). Fast atom bombardment mass spectra
(FAB-MS) were obtained on an Extrel 4000 in the positive ion
detection mode. Melting points were obtained on Mel-Temp
(Laboratory Device Inc., Dubuque, IA). Chromatograms (SE-
HPLC) were obtained on a Lab Alliance Qgrad isocratic system
with a Waters 717 plus autosampler, a Gilson 112 UV detector,
and an in-line IN/US γ-Ram model 2 radiodetector. Elemental
microanalyses were performed by Galbraith Laboratories, Knoxville,
TN. HRMS analyses of compound 7 was performed by the
Laboratory of Analytical Chemistry (Dr. Sonja Hess, NIDDK,
Bethesda, MD). HRMS analyses of compound 8 and 9 were
performed by the mass spectrometry laboratory at the University
of Notre Dame, IN. PIP-DTPA,9a AZEP-DTPA,9a NETA,9b and
NPTA9b were synthesized as described previously. Gd(DTPA) was
used as its N-methylglucamine salt, also known as gadopentate
dimeglumine (Magnevist), and was obtained from Berlex Labora-
tories, Wayne, NJ. Gd(DOTA) (Dotarem) was obtained from
Guerbet LLC (Bloomington, IN). The 153Gd was obtained from
Isotope Products, Valencia, CA. Phosphate buffered saline (PBS),
1X, pH 7.4 consisted of 0.08 M Na2HPO4, 0.02 M KH2PO4, 0.01
M KCl, and 0.14 M NaCl. Caution: 153Gd (t1/2 ) 241.6 days) is a
γ-emitting radionuclide. Appropriate shielding and handling pro-
tocols should be in place when using this isotope.
Conclusion
A series of chelates, PIP-DOTA, PIP-DTPA, AZEP-
DTPA, NETA, and NPTA, were prepared and their potential
as MRI contrast enhancement agents was assessed. A novel
piperidine-backboned DOTA derivative, PIP-DOTA, was
synthesized based on a high dilution cyclization method. The
serum stability, relaxivity, and in vivo biodistribution of the
Gd(III) complexes of the new chelates were investigated and
compared to the clinically used MRI contrast agents Gd(DOTA)
and Gd(DTPA).
At clinically relevant field strengths, the T1 and T2 relaxivities
of the new Gd(III) complexes compared favorably to the contrast
agents, Gd(DTPA) and Gd(DOTA). It is noteworthy that among
the evaluated Gd(III) complexes, Gd(NETA) and Gd(NPTA)
having both macrocyclic and acyclic binding moiety displayed
the highest T1 relaxivities at 61 MHz indicating considerably
enhanced relaxivity compared to Gd(DTPA) and Gd(DOTA).
Serum stability studies show that the novel Gd(III)-labeled
complexes, 153Gd(NETA), 153Gd(PIP-DTPA), and 153Gd(PIP-
DOTA), were stable in serum for up to 14 days without any
measurable loss of radioactivity, a very promising result as the
clinically used Gd(DTPA) released 20% of 153Gd after 5 days.
However, 153Gd(NPTA) was found to be unstable in serum as
indicated by a ∼15% loss of 153Gd from NPTA over 5 days.
The results obtained from the biodistribution studies indicate
that 153Gd(NETA), 153Gd(PIP-DTPA), and 153Gd(PIP-DOTA)
possess in vivo stability similar to the analoguous 153Gd(DOTA),
while 153Gd(AZEP-DTPA) and Gd(NPTA) dissociated in vivo
as evidenced by high accumulation of radioactivity in the organs.
The relaxivity and in vitro and in vivo stability data suggest
that PIP-DOTA, NETA, and PIP-DTPA may be effective Gd-
(III) chelates for use in MRI. The Gd(III) complexes of the
piperidine backboned PIP-DOTA and PIP-DTPA display
reduced kidney accumulation with respect to the nonspecific
Gd(DOTA). The strategy to increase lipophilicity and rigidity
of the chelate system and thus enhance hepatobiliary clearance
and complex stability by incorporating either a piperidine or
an azepane ring into the DTPA system appears promising for
the design of liver specific MRI contrast agents. Further
evaluations of these two complexes, Gd(PIP-DTPA) and Gd-
(PIP-DOTA), along with Gd(NETA) for use as MRI liver
agents or nonspecific agents are planned.
N-Carbobenzoxy-piperidine-cis-2,6-dicarboxylic Acid 2. To
a solution of 2,6-pyridinedicarboxylic acid (25 g, 0.15 mmol) in 2
M NaOH (160 mL) and water (40 mL) was added 5% Rh/alumina
catalyst (2.5 g). The resulting mixture was subjected to hydrogena-
tion by agitation with excess H2(g) at 45 psi in a Parr hydrogenation
apparatus at ambient temperature for 24 h. The reaction mixture
was filtered through Celite, and the Celite was washed with water
(100 mL). The filtrate was cooled to 0 °C, and 2 M NaOH (63
mL) and CBZ-Cl (35.2 mL, 0.21 mmol) were added into the
filtrate. The resulting mixture was allowed to warm to room
temperature and stirred for 18 h. The aqueous layer was washed
with ethyl ether (200 mL), adjusted to pH 2.5-3 with 3 M HCl,
and extracted with EtOAc (2 × 300 mL). The combined EtOAc
layers were washed with water (2 × 200 mL) and saturated NaCl
solution (200 mL), dried (MgSO4), filtered, and evaporated. The
residue was dissolved into EtOAc (50 mL), and hexanes (300 mL)
were added to the solution. The resulting slurry was placed in the
freezer for 4 h. The white precipitate was filtered and dried under
1
vacuum to provide pure 2 as a white solid (36.9 g, 76%). The H
and 13C NMR spectra of the material thereby obtained are essentially
identical to data reported previously for 2:13 mp ) 172 °C;1H NMR
(DMSO) δ 1.51-1.69 (m, 4 H), 2.01 (d, J ) 7.1 Hz, 2 H), 4.65 (d,
J ) 4.3 Hz, 2 H), 5.11 (s, 2 H), 7.30-7.36 (m, 5 H); 13C NMR
(DMSO) δ 16.3 (t), 25.5 (t), 52.9 (d), 66.9 (t), 127.3 (d), 128.0
(d), 128.4 (d), 136.6 (s), 155.5 (s), 173.4 (s).
N-Carbobenzoxy-cis-2,6-bis(succinimidylcarbonyl)piperi-
dine 3. A mixture of 2 (27.6 g, 85 mmol), N-hydroxysuccinimide
(20.7 mmol, 180 mmol), EDC (34.5 g, 180 mmol) in EtOAc (375
mL), and DMF (375 mL) was stirred for 18 h. The active ester 3
precipitated during the reaction was filtered. The filtrate was washed
with water (300 mL), 1 M NaHCO3 (2 × 300 mL), and 1 M HCl
(2 × 300 mL). Additional EtOAc (150 mL) was added to dissolve
the white precipitate formed during extractions. The combined
EtOAc layer was dried (MgSO4), filtered, and evaporated in vacuo.
Experimental Section
General. 1H, 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 tetramethylsilane (TMS). Proton

NoVel Agents for Magnetic Resonance Imaging
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2061
The residue was crystallized from EtOAc/hexanes and placed in
the freezer. The white precipitate was filtered. The combined
precipitate was dried on high vacuum to provide pure 3 (35.2 g,
{(1R,11S)-6,9,15-Tris-tert-butoxycarbonylmethyl-3,6,9,15-tet-
raaza-bicyclo[9.3.1]pentadec-3-yl}-acetic Acid tert-Butyl Ester
8. To a slurry of 7‚HCl (92 mg, 0.26 mmol) in DMF (3 mL) at 0
°C was added DIPEA (445 mg, 3.44 mmol), KI (68 mg, 0.41
mmol), and tert-butyl bromoacetate (220 mg, 11.3 mmol). The
resulting mixture was stirred for 2 h at 0 °C and for 2 h at room
temperature. The reaction mixture was heated to 90 °C and stirred
for 18 h, cooled to room temperature, and concentrated in vacuo.
The solvent was evaporated, and the residue was purified via
column chromatography on silica gel eluting with 15% CH3OH-
CH2Cl2. Pure 8 (65 mg, 37%) was thereby obtained as a colorless
oil: 1H NMR (CDCl3) δ 1.32-1.56 (m, 36 H), 1.75-1.98 (m, 4
H), 2.30-2.42 (m, 2 H), 2.60-3.04 (m, 8 H), 3.21-3.60 (m, 10
H), 4.02-4.09 (m, 2 H); 13C NMR (CDCl3) δ 20.2 (t), 26.0 (t),
28.1 (q), 51.4 (t), 52.2 (t), 52.7 (t), 53.2 (t), 54.3 (t), 56.0 (t), 56.2
(t), 57.7 (t), 80.5 (s), 80.6 (t), 80.7 (t), 170.5 (s), 170.7 (s), 171.1
(s). HRMS (positive ion FAB): calcd for C35H64N4O8Na, [M +
1
78%): mp ) 196 °C; H NMR (DMSO) δ 1.63-1.74 (m, 2 H),
2.09 (d, J ) 8.3 Hz, 4 H), 3.36 (s, 1 H), 5.02 (t, J ) 7.2 Hz, 2 H),
5.15 (s, 2 H), 7.31-7.42 (m, 5 H); 13C NMR (DMSO) δ 15.8 (t),
25.5 (t), 51.8 (d), 67.4 (t), 127.6 (d), 127.9 (d), 128.4 (d), 136.0
(s), 155.2 (d), 167.2 (s), 170.0 (s). Anal. Calcd for C23H23N3O10:
C, 55.09; H, 4.62. Found: C, 54.76; H, 4.78.
(1S,11R)-2,5,10-Trioxo-3,6,9,15-tetraaza-bicyclo[9.3.1]-
pentadecane-15-carboxylic Acid Benzyl Ester 5. To dioxane (1.5
L) at 90 °C was added a solution of active succinimide ester 3 (2.5
g, 5 mmol) in DMSO (30 mL) and a solution of diamine 4 (585
mg, 5 mmol) in DMF (30 mL), each taken up into 100 mL in a
gastight syringe over 24 h. Four more additions of each 5 mmol
solutions were added over 4 days. After the fifth addition, the
reaction was heated for 24 h, cooled to room temperature, and
concentrated in vacuo. The thick brown residue was dissolved in
CH2Cl2 (300 mL). The CH2Cl2 layer was washed with H2O (2 ×
100 mL), 1 M HCl (2 × 100 mL), brine (2 × 100 mL), 1 M
NaHCO3 (2 × 100 mL), and water (2 × 100 mL). The combined
CH2Cl2 layers were dried (MgSO4), filtered, and evaporated to
dryness. The residue was purified via column chromatography on
silica gel by eluting with 3% CH3OH-CH2Cl2. Pure 5 (1.03 g,
+
Na]+ m/z 691.4635; found, [M + Na] m/z 691.4622.
[(1R,11S)-6,9,15-Tris-carboxymethyl-3,6,9,15-tetraaza-bicyclo-
[9.3.1]pentadec-3-yl]-acetic Acid 9. A solution of 8 (110 mg, 0.17
mmol) in ice-water bath was treated with 4 M HCl in 1,4-dioxane
(5 mL). The mixture was allowed to warm to ambient temperature
and then was stirred for 18 h. The precipitate was collected and
washed with ethyl ether. The collected solid was dissolved in water
immediately and lyophilized to provide pure 9 (PIP-DOTA) as a
white solid (66 mg, 87%): 1H NMR (D2O) δ 1.43-1.54 (m, 2 H),
1.62-2.07 (m, 4 H), 3.16 (s, 6 H), 3.24-3.56 (m, 4 H), 3.74 (s, 2
H), 3.90 (s, 4 H), 4.06 (s, 2 H); 13C NMR (D2O) δ 18.78 (t), 32.14
(t), 53.0 (t), 53.4 (t), 53.5 (t), 54.2 (t), 56.8 (t, 2C), 57.0 (t), 57.7
(t), 57.9 (t), 170.0 (s), 172.5 (s), 175.0 (s). HRMS (positive ion
FAB): calcd for C19H31N4O8, m/z 443.2153; found, m/z 443.2142.
Measurements of Relaxivity. NMR dispersion measurements
were made on a custom-designed variable field T1-T2 analyzer
(Southwest Research Institute, San Antonio, TX) at 23 °C. The
magnetic field was varied from 0.02 to 1.5 T (corresponding to a
proton Larmor frequency of 1-64 MHz). T1 was measured by using
a saturation recovery pulse sequence with 32 incremental recovery
times. The relaxivities (relaxation rates per mM Gd(III) concentra-
tion) were obtained after subtracting the water contribution. The
commercial Gd(DTPA) (Magnevist) and Gd(DOTA) (Dotarem)
were used without further modifications. The other Gd chelates
were prepared by dissolving the appropriate ligand and GdCl3 at a
1:0.9 mole ratio in water with stirring and by adjusting the pH of
the resulting solution to 6.5 with 1 M NaOH. The total Gd
concentration for each sample was determined by ICP-AAS (Desert
Analytics, Tuscon, AZ).
Serum Stability. The 153Gd-labeled complexes of the new
chelates, PIP-DOTA, NETA, and NPTA, were prepared by the
addition of 200 µL/350 µCi of 153Gd (0.1 M HCl with the pH
adjusted to 4.5 with 5 M NH4OAc) to a small tube containing 200
µL of a 7 µmol ligand solution in 0.15 M NH4OAc of pH 4.5. The
reaction tube was capped, and the mixture was heated at 80 °C for
12 h. The mixture was loaded onto a column of Chelex-100 resin
(1 mL volume bed, equilibrated with phosphate-buffered saline
(PBS), pH 7.4). The complexes were eluted from the resin with
PBS, pH 7.4, while the resin retained free 153Gd. Radiolabeled
complexes were diluted to an appropriate volume that allowed for
preparation of multiple samples containing 5-10 µCi and were
filter-sterilized using a Millex-GV 0.22 µm filter. This stock solution
was then mixed with 1400 µL of human serum. Aliquots (200 µL)
were drawn and separated into individual tubes for subsequent
analysis using aseptic technique. All samples awaiting analysis were
1
53%) was thereby obtained as a white solid: mp ) 182 °C; H
NMR (CDCl3) δ 1.48-1.62 (m, 3 H), 1.95-2.4 (m, 4 H), 3.03-
3.27 (m, 3H), 3.44-3.82 (m, 2 H), 4.23-4.42 (m, 1 H), 4.69-
4.86 (m, 2 H), 5.11 (d, J ) 9.8 Hz, 1 H), 5.42 (d, J ) 9.8 Hz, 1
H), 5.63 (s, 0.6 H), 6.29 (s, 0.4 H), 6.56 (s, 0.4 H), 6.71 (s, 0.6 H),
7.30-7.56 (m, 5 H); 13C NMR (CDCl3) δ 16.0 (t), 17.9 (t), 23.9
(t), 24.0 (t), 24.2 (t), 36.9 (t), 37.1 (t), 37.6 (t), 37.7 (t), 44.2 (t),
51.2 (d), 51.7 (d), 52.4 (d), 53.0 (d), 57.4 (t), 68.2 (t), 68.5 (t),
127.8 (d), 127.9 (d), 128,2 (d), 128.4 (d), 135.2 (s), 156.2 (s), 156.4
(s), 170.2 (s), 170.9 (s), 171.3 (s), 171.4 (s), 171.5 (s), 171.9 (s).
Anal. Calcd for C19H24N4O5: C, 58.75; H, 6.23. Found: C, 59.06;
H, 6.51.
(1S,11R)-3,6,9,15-Tetraaza-bicyclo[9.3.1]pentadecane-2,5,10-
trione 6. To a solution of 5 (1.0 g, 2.57 mmol) in EtOH (30 mL)
was added 10% Pd/C catalyst (200 mg). The resulting mixture was
subjected to hydrogenation by agitation with excess H2(g) at 30
psi in a Parr hydrogenation apparatus at ambient temperature for 3
h. The reaction mixture was filtered through Celite, and the filtrate
was concentrated in vacuo. The residue was purified via column
chromatography on neutral alumina and was eluted with 10%
CH3OH-EtOAc. Pure 6 (630 mg, 97%) was thereby obtained as a
white solid: 1H NMR (CDCl3) δ 1.61-1.74 (m, 4 H), 2.00-2.06
(m, 2 H), 3.21-3.41 (m, 3 H), 3.54-3.74 (m, 4 H), 3.90 (d, J )
14.1 Hz, 1 H); 13C NMR (CDCl3) δ 19.4 (t), 26.5 (t), 27.2 (t), 40.1
(t), 40.4 (t), 47.1 (t), 54.9 (d), 55.6 (d), 176.7 (s), 179.3 (s), 179.6
(s). Anal. Calcd for C11H18N4O3(H2O)0.5: C, 51.05; H, 7.21.
Found: C, 50.89; H, 7.21.
(1R,11S)-3,6,9,15-Tetraaza-bicyclo[9.3.1]pentadecane 7. To a
slurry of 6 (540 mg, 2.12 mmol) in THF (20 mL) at 0 °C under
argon was added dropwise 2 M BH3-Me2S (9.6 mL, 9.6 mmol)
over 1 h. The resulting mixture was stirred at 0 °C for 1 h and at
room temperature for 2 h and was refluxed for 5 days. The reaction
was cautiously quenched by dropwise addition of 10% H2O/THF
(1 mL), and the reaction mixture was concentrated. The residue
was treated with CH3OH (30 mL) and evaporated. This was
repeated twice, and the residue was applied to high vacuum for 2
h and was treated with 6 M HCl (5 mL). The resulting mixture
was refluxed for 1 h, cooled to room temperature, and washed with
CHCl3 (2 × 50 mL). The pH of the aqueous solution was adjusted
to 13 with 5 M NaOH, and the solution was extracted with CHCl3
(3 × 100 mL). The combined CHCl3 layers were dried, filtered,
and evaporated to provide pure 7 (248 mg, 55%) as a colorless oil:
stored at 37 °C in a CO
2 incubator. An aliquot of the mixture (30-
50 µL) was taken at selected times (Figure 4) and analyzed by
SE-HPLC. The serum stability of the 153Gd complexes was assessed
by measuring the transfer of the 153Gd from the complexes to serum
proteins by use of a SE-HPLC fitted with a TSK 3000 column.
Samples were eluted with PBS at a flow rate of 1 mL/min.
In Vivo Biodistribution Studies. Female athymic mice were
obtained from Charles River Laboratories (Wilmington, MA) at
4-6 weeks of age. The pH of the 153Gd-labeled ligands was adjusted
1H NMR (D2O) δ 1.12-1.82 (m, 8 H), 2.96-3.42 (m, 16 H); 13
C
NMR (D2O) δ 22.3 (t), 26.6 (t), 44.4 (t), 45.2 (d), 49.7 (t), 52.0 (t).
HRMS (positive ion FAB): calcd for C11H24N4, [M + H]+ m/z
213.2097; found, [M + H]+ m/z 213.2083.

2062 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Chong et al.
(7) Reimer, P.; Schneider, G.; Schima, W. Hepatobiliary contrast agents
for contrast-enhanced MRI of the liver: properties, clinical develop-
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(9) (a) Chong, H. S.; Garmestani, K.; Bryant, L. H.; Brechbiel, M. W.
Synthesis of DTPA analogues derived from piperidine and azepane:
potential contrast enhancement agents for magnetic resonance imag-
ing. J. Org. Chem. 2001, 66, 7745-7750. (b) Chong, H. S.;
Garmestani, K.; Ma, D.; Milenic, D. E.; Overstreet, T.; Brechbiel,
M. W. Synthesis and biological evaluation of novel macrocyclic
ligands with pendent donor groups as potential yttrium chelators for
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to pH ∼7.0 with 0.5 M sodium bicarbonate (pH 10.5) and diluted
in phosphate-buffered saline. The radiolabeled ligands (5-7 µCi)
were administered to the mice in 200 µL of solution 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
measured in a γ-scintillation counter (Minaxi-γ; Packard, Downers
Grove, IL). The percent injected dose per gram (% ID/g) was
determined for each tissue. The values presented are the mean and
standard deviation for each tissue. All animal experiments were
performed in compliance with current regulations and guidelines
of the U.S. Department of Agriculture and approved by the NCI
Animal Care and Use Committee.
(10) (a) Fossheim, R.; Dugstad, H.; Dahl, S. G. Structure-stability
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Acknowledgment. This research was supported in part by
the Intramural Research Program of the National Institutes of
Health, National Cancer Institute, Center for Cancer Research.
We thank the Structural Mass Spectra Group, Laboratory of
Analytical Chemistry, (Dr. Sonja Hess, NIDDK, Bethesda, MD)
for obtaining the mass spectra of 7.
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