Design, synthesis, and evaluation of 1,4,7,10-tetraazacyclododecane-1,4,7- triacetic acid derived, redox-sensitive contrast agents for magnetic resonance imaging

Article abstract of DOI:10.1021/jm100592u

The design and synthesis of three 1,4,7,10-tetraazacyclododecane-1,4,7- triacetic acid (DO3A) derivatives bearing linkers with terminal thiol groups and a preliminary evaluation of their potential for use in assembling redox-sensitive magnetic resonance imaging contrast agents are reported. The linkers were selected on the basis of computational docking with a crystal structure of human serum albumin (HSA). Gd(III)-DO3A and Eu(III)-DO3A complexes were synthesized, and the structure of one complex was established by X-ray crystallographic analysis. The binding to HSA of a Gd(III)-DO3A complex bearing a thiol-terminated 3,6-dioxanonyl chain was competitively inhibited by homocysteine and by the corresponding Eu chelate. Binding to HSA was abolished when the terminal thiol group of this complex was absent. The longitudinal water-proton relaxivities (r₁) of the three Gd(III)-DO3A complexes and of two Gd(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexes were measured in saline at 7 T. The DO3A complexes exhibited smaller r₁ values, in both bound and free states, than the DOTA complexes.

Full text of DOI:10.1021/jm100592u

J. Med. Chem. 2010, 53, 6747–6757 6747
DOI: 10.1021/jm100592u
Design, Synthesis, and Evaluation of 1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic Acid Derived,
Redox-Sensitive Contrast Agents for Magnetic Resonance Imaging
Natarajan Raghunand,† Gerald P. Guntle,‡ Vijay Gokhale,§ Gary S. Nichol, Eugene A. Mash, and
Bhumasamudram Jagadish*,
†Department of Radiology, University of Arizona, Tucson, Arizona 85724-5024, ‡Cancer Center Division, University of Arizona,
Tucson, Arizona 85724-5024, §Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721-0240, and
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721-0041
Received May 14, 2010
The design and synthesis of three 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) derivatives
bearing linkers with terminal thiol groups and a preliminary evaluation of their potential for use in assembling
redox-sensitive magnetic resonance imaging contrast agents are reported. The linkers were selected on the
basis of computational docking with a crystal structure of human serum albumin (HSA). Gd(III)-DO3A and
Eu(III)-DO3A complexes were synthesized, and the structure of one complex was established by X-ray
crystallographic analysis. The binding to HSA of a Gd(III)-DO3A complex bearing a thiol-terminated 3,6-
dioxanonyl chain was competitively inhibited by homocysteine and by the corresponding Eu chelate. Binding
to HSA was abolished when the terminal thiol group of this complex was absent. The longitudinal water-
proton relaxivities (r1) of the three Gd(III)-DO3A complexes and of two Gd(III)-1,4,7,10-tetraazacyclodo-
decane-1,4,7,10-tetraacetic acid (DOTA) complexes were measured in saline at 7 T. The DO3A complexes
exhibited smaller r1 values, in both bound and free states, than the DOTA complexes.
Introduction
that are sensitive to physiochemical changes and biochem-
ical changes in their microenvironment, such as pH, pO2,
metal ion concentration, enzymatic activity, and redox
state.7-21 Perturbations of tissue redox are linked to the
development of hyperglycemia-induced vascular and renal
complications in diabetes mellitus.22,23 Evidence also suggests
that altered redox homeostasis in cancer cells plays a role in
tumor progression and metastasis,24-26 chemoresistance,25
and radioresistance.27 The Nrf2 transcription factor, which
is up-regulated in many cancers,26 transcriptionally up-reg-
ulates antioxidant response element (ARE) containing genes.
ARE-containing genes include many that impinge on tissue
levels of reduced glutathione, thioredoxin, and cysteine and
code for drug transporters such as the multidrug resistance-
associated protein (MRP).25 Reduced thiols such as glutathione
and cysteine can directly effect the chemical repair of DNA
damage produced by ionizing radiation, thereby providing a
degree of radioprotection.28 Nrf2-regulated γ-glutamyltrans-
ferase (GGT) can modulate redox equilibria in both the intra-
cellular and extracellular compartments.29,30 GGT can confer a
degree of protection to cancer cells from electrophilic drugs such
as cisplatin by increasing the extracellular levels of thiols.29,30
Knowledge of tumor redox by itself and knowledge of tumor
redox as a potential downstream biomarker of tumor Nrf2
status are thus greatly relevant to the management and treat-
ment of cancer. We report the development of thiol-sensitive
MRI contrast agents for potential use in the noninvasive
interrogation of tumor redox.
Advances in our understanding of the molecular pathogen-
esis of cancer and many other diseases have led to the current
push toward the development of novel, targeted drugs. This is
being accompanied by a transformation of diagnostic radi-
ology from a discipline focused on the imaging of anatomy to
one that seeks to interrogate tissue physiology and molecular
phenotypes and to provide noninvasive imaging biomarkers
of drug targets and drug action. Magnetic resonance imaging
(MRIa) is a powerful medical diagnostic tool, and while not
all MRI examinations require the administration of exogenous
contrast material, in clinical practice many MRI examinations
employ gadolinium(III)-based contrast agents (CAs).1-3 The
efficacy of an MRI contrast agent is dependent on its relaxivity,
and in the past 2 decades much work has been devoted to
understanding the parameters that influence the relaxation
properties of these CAs.4-6 Clinically approved MRI CAs
are suited primarily for highlighting anatomical features on
MR images, after intravenous administration and vascular
distribution throughout the body. In recent years, the
focus has shifted toward the development of MRI CAs
*To whomcorrespondence should beaddressed. Address:Department
of Chemistry and Biochemistry, University of Arizona, 1306 East
University, P.O. Box 210041, Tucson, AZ 85721-0041. Phone: (520)
621-2396. Fax: (520) 621-8407. E-mail: bjagadis@email.arizona.edu.
a Abbreviations: ARE, antioxidant response element; CAs, contrast
agents; DCM, dichloromethane; DO3A, 1,4,7,10-tetraazacyclododecane-
1,4,7-triacetic acid; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HSA, human serum albumin; GGT, γ-glutamyltransferase; MRI, magnetic
resonance imaging; MRP, multidrug resistance-associated protein; PBS,
phosphate-buffered saline; PEG, polyethylene glycol; TBAI, tetrabutylam-
monium iodide.
Previously we reported the synthesis and characterization
of compounds 1 and 2, 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA) complexes of gadolinium(III)
bearing alkyl chains that terminate with a thiol group.31
r
2010 American Chemical Society
Published on Web 08/19/2010
pubs.acs.org/jmc

6748 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Raghunand et al.
These compounds bound to human serum albumin (HSA) in a
redox-sensitive manner, as evidenced by increased MRI relax-
ivity and retention in a 30 kDa MWCO filter in the presence of
HSA. Loss of both these properties occurred when 1 and 2 were
competed with excess homocysteine, which is known to form a
disulfide link with cysteine-34 of HSA. More recently, Lacerda,
et al. prepared a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid (DO3A) macrocycle bearing a terminal β-thioethyl appen-
dage for use as a chelating agent for radionuclides.32 The
synthesis of glyconanoparticles capped with a Gd-DO3A che-
late bearing pentyl and undecyl appendages with terminal thiol
groups has also been reported.33 In a continuation of our work,
we have designed and synthesized three DO3A chelators bear-
ing different types of linkers that terminate with thiol groups,
prepared the corresponding Gd and Eu complexes 3-5, ob-
tained a crystal structure of Gd-DO3A complex 4a, studied the
binding of these complexes to HSA, and evaluated the relaxivity
of these complexes in the presence and absence of HSA as
reported herein.
Figure 1. HSA with the putative binding region shown as a MOLCAD
surface. The protein backbone is depicted as a red ribbon, and residues
that constitute the binding region are shown in orange. Cys34 is displayed
as atom-colored sticks.
Molecular Modeling of the Interactions between Compounds
1-5 and HSA
Figure 2. Docking of 2 with HSA. Compound 2, HSA residues
Thr83 and Tyr84, and a bound water molecule (2.71 A from Gd) are
displayed as atom-colored sticks. Hydrogen atoms and remaining
water molecules are omitted for clarity.
We postulated that compounds such as 1-5 may bind to
HSA via one or more of the following: formation of a covalent
disulfide bond between the thiol-terminated linker and the
Cys34-SH group, linker-protein interactions, and interac-
tions between the chelate moiety and a hydrophobic pocket
near the surface of the protein. Our current modeling effort
employs a known X-ray crystal structure of HSA34 to design
molecules that will maximize binding based on these three key
interactions. Figure 1 shows a possible binding site located on
the surface of HSA near Cys34. The site consists of a hydro-
phobic binding pocket and a hydrophobic channel leading
from the pocket to Cys34. The pocket is formed by Gly82,
Thr83, Tyr84 (backbone), Gly85, Glu86, and Met87 on one
side and by Gln104, His105, Lys106, Asp107, Asp108, Glu465,
Lys466, and Thr467 on the other side. These residues create a
bowl-shaped depression where the chelate moiety might nest.
The backbone and side chains of Tyr30, Leu31, Gln32, Gln33,
and Tyr84 create a hydrophobic channel where an appropriate
linker might bind while permitting covalent attachment of a
terminal thiol to the Cys34-SH group.
accommodate small molecule docking was allowed. In the
secondstage, a covalentdisulfidelinkage between theterminal
thiol of the small molecule and the Cys34-SH was established,
and the simulations were repeated. At the end of each docking
simulation, the interaction energy value was calculated.
For the DOTA-containing compounds 1and 2, Gdchelation
by the amide group in the linker provides an extra measure of
structural rigidity. Covalent binding of 2 to HSA results in
additional coordination by the backbone CdO groups of
Thr83 or Tyr84 and one water molecule (see Figure 2). It is
hypothesized that the system is dynamic and that Thr83, Tyr84,
and water exchange in the coordination sphere of the gadoli-
nium atom. Compound 2 exhibited a greater interaction energy
(-510 kcal/mol; see Table 1) than did 1 (-290 kcal/mol),
presumably because of increased van der Waals interactions
with the channel. These calculations were qualitatively consis-
tent with the measured binding constants of 1 and 2 (see
Table 1).
Our molecular modeling protocol was designed to simulate
a possible two-stage binding process.35 In the first stage,
noncovalent interactions between compounds 1-5 and the
putative binding site described above were computa-
tionally examined in the presence of water molecules. During
these simulations, movement of the protein side chains to
In contrast with Gd-DOTA chelates in which only one
water molecule may be coordinated with the Gd (q = 1), Gd-
DO3A chelates allow for the possibility of two coordinated
water molecules (q = 2) and may exhibit very different

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6749
Table 1. Binding of Compounds 1, 2, 3a-5a, and 24 to Human Serum
Albumin
calculated interaction
energy (kcal/mol)
calculated distance of
water from Gd in the (A)
covalent noncovalent measured
complexb KA c (mM-1
presence
) of HSA
absence
of HSA
compd complexa
1d
2d
3a
4a
5a
24
-290
-510
-410
-471
-453
naf
-210
-496
-366
-404
-413
-406
5.0 ( 1.5
64 ( 16
mbe
2.76 A
2.71 A
2.54 A
2.75 A
2.75 A
ncg
2.71
2.75
2.65
2.72
2.67
2.89
2.73
2.74
2.73
2.74
ncg
mbe
22 ( 1.4
∼0
Figure 4. Docking of 5a with HSA. Compound 5a, HSA residues
Thr83 and Tyr84, and a bound water molecule (2.67 A from Gd) are
displayed as atom-colored sticks. Hydrogen atoms and the remain-
ing water molecules are omitted for clarity.
a Includes a disulfide bond between the compound thiol and Cys34-
SH. b Without a disulfide bond between the compound thiol and Cys34-
SH. c Measured at 37 °C. d Results taken from ref 31. e Results suggest this
compound binds to multiple sites on HSA; see text for explanation. f Not
applicable. g Not calculated.
Synthesis
A retrosynthetic analysis for construction of Ln-DO3A
conjugates 3-5 is depicted in Scheme 1. A key step would
be the simultaneous deprotection of the three tert-butyl esters
and the tritylthio group in 7 to afford DO3A derivatives
6, which, upon complexation with the desired lanthanide,
would give the complexes 3-5. N-Alkylation of the known
DO3Aderivative8 with primary bromides 9 would give access
to 7.
The required bromides were synthesized as shown in
Scheme 2. Bromide 1136 was prepared by reacting p-xylene
dibromide 10 with trityl mercaptan and sodium hydride in
anhydrous THF. Bromide 13 was similarly prepared by
reacting 1,9-dibromononane (12) with trityl mercaptan and
K2CO3 in acetonitrile. Bromide 17 was prepared in three
steps. 1,3-Dibromopropane (14) was reacted with trityl
mercaptan and K2CO3 in acetonitrile to give 15. Bromide
15 was converted to 16 by reaction with diethylene glycol,
sodium hydride, and TBAI in dry THF.37 Finally, bromi-
nation of 16 with CBr4 and PPh3 gave 17.38
Ester 8 was prepared from commercially available cyclen 18
by treatment with 3 equiv of tert-butyl bromoacetate and
sodium acetate in N,N-dimethylacetamide using a modifica-
tion of a published procedure (Scheme 3).39 N-Alkylation of 8
with bromide 11 in acetonitrile using K2CO3 as base gave 19a
in 80% yield. Simultaneous removal of tert-butyl and trityl
groups was attempted by treatment of 19a with triethylsilane
and trifluoroacetic acid (1% v/v). Under these conditions,
it appeared that the trityl group came off easily, while the
ester groups were incompletely hydrolyzed. Longer reaction
times and the use of solvents such as dichloromethane and
chloroform led to what appeared by mass spectrometry to be
the monoester derivative.40 We reasoned that this was not a
case of incomplete ester hydrolysis but rather of capture of a
tert-butyl cation by the thiol group after loss of the trityl
group. To eliminate this undesired process, an excess of
butanethiol was added to the reaction medium to scavenge
tert-butyl cations. This led to isolation of the fully depro-
tected tricarboxylic acid 20a in 80% yield. Acid 20a was
metalated with Gd(OAc)3 or Eu(OAc)3 in refluxing methanol
to give the corresponding Gd(III) and Eu(III) complexes 3a
and 3b in 75% and 71% yields, respectively, following reverse
phase silica gel chromatography.
Figure 3. Overlay of energy-minimized structures for compounds
3a (magenta), 4a (red), and 5a (blue). Sulfur is colored yellow, and
hydrogen atoms are omitted for clarity.
binding to HSA and different MRI relaxivity characteristics.
In modeling Gd-DO3A chelates, the three linkers shown in
compounds 3-5 were selected from a number of possibilities
that were initially considered. Included are linkers based on
a rigid hydrophobic p-xylyl group, a flexible hydrophobic
nonyl chain, and a flexible hydrophilic 3,6-dioxanonyl chain.
Figure 3 shows an overlay of energy-minimized structures for
compounds 3a-5a.
In the computational docking studies, compound 3a pos-
sessing the xylyl linker exhibited somewhat weaker binding
(-410 kcal/mol; see Table 1) than did compounds 4a and 5a
(-471 and -453 kcal/mol, respectively). The xylyl linker has
neither the reach nor the flexibility of the nonyl and 3,6-
dioxanonyl chains which are able to interact more favor-
ably with the channel connecting Cys34 and the hydro-
phobic binding pocket on the surface of HSA. Still, docking
indicates that all three compounds show similar profiles
with respect to binding of the gadolinium chelate moiety.
Backbone amide CdO groups of Thr83 and Tyr84 and one
water molecule are in the inner coordination sphere of the
gadolinium atom (Table 1, Figure 4). In simulations carried
out in water, two water molecules occupy two inner sphere
coordination sites for each of the DO3A chelates 3a-5a.
Thus, one would expect different relaxivity for protein-
bound and unbound chelates, and so these compounds were
selected for synthesis and evaluation of binding and relaxi-
vity properties.

6750 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Raghunand et al.
Scheme 1. Retrosynthetic Analysis of Ln-DO3A Conjugates 3-5
Scheme 2. Synthesis of Bromides 11, 13, and 17
The structures of all compounds, except 3a, 4a, 5a, and 24,
1
were characterized by H NMR, 13C NMR, and HRMS.
Compounds 3a-5a, 3b-5b, and 24 gave appropriate
masses and isotopic patterns in HRMS analysis (see the
Supporting Information). Further evidence for the struc-
tures of the Gd-DO3A macrocycles was obtained by X-ray
crystallographic analysis of 4a. Crystals were grown ac-
cording to a procedure used previously for growing an In-
DO3A-TPP complex.41 An equimolar mixture of 20b and
Gd(OAc)3 was heated at 100 °C for 30 min in NH4OAc
buffer (0.5 M, pH 6). Slow diffusion of acetone into the
reaction mixture produced colorless, flat plates. Com-
pound 4a crystallized in space group P1 as a dihydrate with
an acetate anion coordinated to the gadolinium center (Figure
5) and a charge-balancing ammonium cation present in the
asymmetric unit. Water is also present in the asymmetric unit,
some of which was removed by SQUEEZE.42 There is some
disorder in the ω-thiononyl chain, which adopts a mostly
extended conformation.43
Binding and Relaxometry
The binding of compounds 1, 2, 3a-5a, and 24 to commer-
cially obtained HSA was measured at 37 °C in buffered saline
(see Table 1). For commercialsamples, some percentage of the
HSA present bears homocysteine or another thiol of low
molecular weight covalently attached at Cys34.44 Thus, cal-
culation of an apparent equilibrium association constant (KA)
was possible from the binding experiments described herein.
Compounds 3a and 4a bound to HSA with a stoichiom-
etry of >3:1 (data not shown), while compounds 1, 2, and
5a bound to HSA with a stoichiometry of ∼1:1. For
compound 5a, KA=22 mM-1, a value between the pub-
lished values for DOTA complexes 1 and 2.31 The binding
of compounds 1, 2, and 5a to HSA could be competitively
inhibited by addition of homocysteine (see Table 2). The
binding of 5a to HSA was inhibited even more effectively
by the structurally related Eu chelate 5b. Binding to HSA
was abolished for 24, a compound similar to 5a but
lacking the terminal thiol group.
Following a similar synthetic pathway, amine 8 was alky-
lated with bromide 13 to give 19b in 80% yield. Deprotection
of 19b produced 20b in 75% yield, and metalation of 20b with
Gd(OAc)3 and Eu(OAc)3 in refluxing methanol gave the
complexes 4a and 4b in 57% and 58% yields, respectively,
following reverse phase silica gel chromatography. Finally,
N-alkylation of 8 with 17 gave 19c in 92% yield. Deprotection
of 19c gave 20c in 75% yield, and metalation of 20c with
Gd(OAc)3 and Eu(OAc)3 produced complexes 5a and 5b in
75% and 82% yields, respectively, following reverse phase
silica gel chromatography.
In addition to the syntheses of 3a-5a and 3b-5b, the
synthesis of Gd-DO3A complex 24 is depicted in Scheme 4.
Bromide 21 was prepared from diethylene glycol monopropyl
ether in 90% yield by use of the Appel reaction.38 Alkylation
of 8 with 21 in acetonitrile using K2CO3 as base gave 22 in
87% yield. Deprotection of the tert-butyl ester groups using
TFA in DCM gave tricarboxylic acid 23 in 77% yield.
Metalation with Gd(OAc)3 hydrate in refluxing methanol
gave complex 24 in 75% yield following reverse phase silica
gel chromatography.
The longitudinal water-proton relaxivities (r1) of com-
pounds 1, 2, and 5a were measured in HEPES-buffered saline.
For compounds 3a and 4a, r1 was measured in acetate-
buffered saline because these complexes were poorly soluble
in the absence of acetate. Results reported include r1(free),
determined in the absence of HSA, and r1(bound), determined

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6751
Figure 5. Molecular structure of 4a, with anisotropic displacement parameters at the 30% probability level. The minor disorder component,
hydrogen atoms, water molecules, and the ammonium cation have been omitted for clarity.
Scheme 3. Synthesis of Gd-DO3A Complexes 3a-5a and and Eu-DO3A Complexes 3b-5b
Scheme 4. Synthesis of Gd-DO3A Complex 24

6752 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Table 2. Competitive Binding Study for Compounds 1, 2, and 5aa
Raghunand et al.
two water molecules in the inner coordination sphere (see
Table 1).46 Displacement of inner sphere water molecules by
acetate is a possible reason why the r1(free) values for 3a and
4a are lower than those of the DOTA complexes 1 and 2. In
addition, it has been reported that inner sphere water mole-
cules can be displaced by carboxylate groups on albumin.47
There are 97 aspartate and glutamate residues in HSA.
Examination of the protein crystal structure suggests that
most of the side chain carboxylate groups are present on
the protein surface. Given the low solubility of 3a and 4a
in the absence of acetate and the presence of a bifurcated
acetate ligand in the crystal structure of 4a, it is concei-
vable that carboxylate groups on the HSA surface con-
stitute the multiple sites to which these compounds bind.
Displacement of inner sphere water molecules would
also help explain the surprisingly low value of r1(bound)
observed for 3a. Compound 5a might possess only one
water molecule in the inner coordination sphere, since
the oxygen atom in the PEG side chain proximal to the
DO3A moiety can coordinate to gadolinium.48 Thus, 5a
might exhibit properties more like those of a DOTA com-
plex. However, r1(free) and r1(bound) for 5a were signifi-
cantly lower than values for 1 and 2, possibly because
of suboptimal coordination geometry or decreased basi-
city of the macrocyclic nitrogen that bears the pendent
arm.49
KA (mM-1
at inhibitor concentration of
)
compd
inhibitor
0.0 mM
0.5 mM 1.0 mM 2.0 mM
1
homocysteine 5.0 ( 1.5 2.0 ( 0.12 1.5 ( 0.2 0.87 ( 0.05
2
homocysteine 64 ( 16 17 ( 2.2
homocysteine 22 ( 1.4 11 ( 3.4
6.1 ( 0.54 3.6 ( 0.23
6.6 ( 2.7 3.4 ( 0.84
5a
5a
5b
22 ( 1.4 6.2 ( 1.2 3.0 ( 0.91 ∼ 0
a Measured at 37 °C.
Table 3. Relaxivities of Compounds 1, 2, and 3a-5aa
compd
r1,free (1/(mM s))
r1,bound (1/(mM s))
3
3
1
3.86 ( 0.44
3.23 ( 0.33
2.37 ( 0.18
2.27 ( 0.37
2.18 ( 0.58
4.54 ( 0.14
4.74 ( 0.32
1.54 ( 0.15
3.28 ( 0.17
3.15 ( 0.51
2
3a
4a
5a
a Measured at 37 °C and 7 T.
in the presence of this protein (see Table 3). The DO3A-based
complexes had smaller relaxivities, in both bound and free
states, thanthe DOTA-basedcomplexes. For compounds1, 2,
4a, and 5a, r1(free) was smaller than r1(bound), most likely
because of increased times for rotational reorientation of the
complexes when bound to HSA. In the case of 3a, r1(free) was
comparable to values observed for the other DO3A chelates,
4a and 5a, but r1(bound) was smaller than r1(free), suggesting
that other factors limit the relaxivity of 3a when bound to
HSA.
Conclusion
A general method for the synthesis of DO3A-based lantha-
nide chelates possessing different nitrogen-attached side arms
thatterminatewithredox-activethiolgroupshasbeen demon-
strated. The synthesis is relatively short and efficient. To the
best of our knowledge, the first X-ray crystal structure of a
gadolinium-DO3A complex has been obtained. Taken to-
gether with protein binding and relaxivity data, the crystal
structure supports the suggestion that coordination by surface
carboxylate groups may be important in protein binding and
water relaxivity for DO3A chelates in vivo.47 Direct and
competition binding assays indicate that for DOTA chelates
and DOTA-like DO3A chelates bearing terminal thiol
groups, covalent attachment through a disulfide linkage with
Cys34 is an important mode of HSA binding. Studies to
further examine the redox chemistry of gadolinium DO3A
and DOTA chelates and the associated MRI properties are
underway.
Discussion
Calculated interaction energies for compounds 1, 2, 3a-5a,
and 24 with HSA are given in Table 1. The covalent interac-
tion energies, which include a disulfide linkage, are lower (i.e.,
the covalent complex is more stable) than the corresponding
noncovalent interaction energies. The differences in the cal-
culated covalent and noncovalent interaction energies are 80,
14, 44, 67, and 40 kcal/mol for compounds 1, 2, 3a, 4a, and 5a,
respectively, in reasonable agreement with typical disulfide
bondstrengths (70 kcal/mol).45 In the case of compound 2, the
energy gained through disulfide bond formation is presum-
ably counterbalanced by losses of other stabilizing interac-
tions. Consistent with this interpretation, the calculated
noncovalent interaction energies for 5a and compound 24,
which lacks a terminal thiol group and cannot form a disulfide
bond, are very similar. In addition, the calculated covalent
interaction energies correlate reasonably well with experimen-
tally determined apparent equilibrium binding constants
(KA, Table 1).
Homocysteine is known to bond covalently with Cys34 of
HSA.44 Increasing concentrations of homocysteine resulted in
corresponding decreases inKA forcompounds1, 2, and5a(see
Table 2), suggesting that these compounds also bond with
HSA at Cys34. In contrast, compounds 3a and 4a exhibited
essentially complete and irreversible binding to HSA. Because
of insolubility, binding assays for these compounds were
carried out in acetate-buffered saline. In the centrifugal filtra-
tion assay used to quantify binding, essentially no Gd was
recovered in the filtrates from incubations of up to 2.0 mM 3a
or 4a with 0.66 mM HSA (3:1). These results were unchanged
when up to 2.0 mM homocysteine was included, indicating
that 3a and 4a bind to HSA at sites other than Cys34. In
aqueous solutions 3a and 4a would be expected to possess
Experimental Section
Small Molecule Docking with HSA. An X-ray crystal struc-
ture of HSA (PDB code 1AO6) was refined and used as the basis
for docking simulations that were carried out using Insight II
2005L.35 Compounds to be docked were placed in the vicinity of
the Cys34-SH group, and the protein-small molecule system
was surrounded with a 7.5 A layer of water. Charges were
assigned using extensible systematic force field (ESFF).50 The
gadolinium atom was assigned a þ3.0 formal charge. The
system was then energy minimized using 5000 steps, followed by
a molecular dynamics simulation with a 50 ps equilibration and
a 100 ps simulation at 300 K. Frames were collected after every
100 fs during the 100 ps simulation phase. At the end of the
molecular dynamics run, trajectories were analyzed on the basis
of potential energy. The frame with the lowest potential energy
was further minimized using 5000 steps. During covalent simu-
lations, an S-S linkage was first established between the thiol
groups of the small molecule and Cys34. This was then followed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6753
by energy minimization and molecular dynamics protocols as
described above. Interaction energies were calculated as the
difference between the energy of the small molecule-protein
complex and the sum of the individual energies of the protein
and the small molecule.
(5 mL) was added a solution of diethylene glycol (1.6 g, 15.1
mmol, 1.43 mL) in THF (25 mL) dropwise. After 30 min,
a solution of 15 (2.0 g, 5 mmol) in dry THF (30 mL) was
added dropwise, and the mixture was heated at reflux overnight.
The reaction mixture was cooled, cautiously quenched with
water (50 mL), and the mixture extracted with ether (3 ꢀ
50 mL). The combined organic layers were washed with brine,
dried (MgSO4), filtered, and concentrated in vacuo. The residue
was subjected to flash chromatography on silica gel, eluting with
hexanes/EtOAc (2:1 f 1:1) to give 16 (0.76 g, 1.8 mmol, 36%) as
a white solid, mp 63-64 °C. Rf = 0.29 (1:1 hexanes/EtOAc on
Einteraction ¼ Ecomplex - ðEsmall molecule þ Eprotein
Þ
Simulations in Aqueous Solution. The small molecule of
interest was surrounded with a 7.5 A layer of TIP3P water
molecules. The system was then minimized using 5000 steps.
This was followed by a molecular dynamics simulation with a
50 ps equilibration and a 100 ps simulation at 300 K. Frames
were collected after every 100 fs during the 100 ps simulation
phase. At the end of the molecular dynamics run, trajectories
were analyzed on the basis of potential energy. The frame with
the lowest potential energy was further minimized using 5000
steps. This minimized frame was used to study the coordination
of the gadolinium atom by water molecules.
1
silica gel 60 F254). H NMR (500 MHz, CDCl3) δ 1.64 (2H,
quintet, J = 7, 6.5 Hz), 2.23 (2H, t, J = 7 Hz), 2.33 (1H, br s),
3.40 (2H, t, J = 6.5 Hz), 3.50 (2H, m), 3.55-3.60 (4H, m), 3.68
(2H, m), 7.19 (3H, m), 7.26 (6H, m), 7.38-7.42 (6H, m). 13C
NMR (125 MHz, CDCl3) δ 28.6, 61.8, 66.5, 69.9, 70.1, 70.3,
72.4, 126.5, 127.7, 129.5, 144.8. HRMS-ESI calculated for
C26H30O3SNa (M þ Na)þ 445.1808, found 445.1809. Anal.
Calcd for C26H30O3S: C 73.90, H 7.16. Found C 73.52, H 7.19.
1-Bromo-9-tritylthio-3,6-dioxanonane (17). To a solution of
16 (1.0 g, 2.36 mmol) in DCM (20 mL) was added CBr4 (0.86 g,
2.60 mmol) followed by PPh3 (0.68 g, 2.60 mmol), and the
mixture was stirred at room temperature. After 4 h, the pale-
brown solution was concentrated under reduced pressure and
the residue subjected to flash chromatography on silica gel.
Elution with hexanes/EtOAc (85:15) gave 17 (0.88 g, 1.88 mmol,
80%) as a white solid, mp 68-70 °C. Rf = 0.53 (4:1 hexanes/
EtOAc on silica gel 60 F254). 1H NMR (500 MHz, CDCl3) δ 1.64
(2H, quintet, J = 7 Hz, 6.5 Hz), 2.23 (2H, t, J = 7 Hz),
3.38-3.43 (4H, m), 3.49 (2H, m), 3.59 (2H, m), 3.75 (2H, t,
J = 6.5 Hz), 7.19 (3H, m), 7.26 (6H, m), 7.40 (6H, m). 13C NMR
(125 MHz, CDCl3) δ 28.7, 30.2, 66.4, 69.9, 70.0, 70.4, 71.2,
126.5, 127.7, 129.5, 144.8. HRMS-ESI calculated for C26H29O2-
BrSNa (M þ Na)þ 507.0964, found 507.0946. Anal. Calcd for
C26H29O2BrS: C 64.32, H 6.02. Found: C 64.28, H 6.07.
1,4,7-Tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclo-
dodecane (8).39 To a suspension of cyclen 18 (2.00 g, 11.62 mmol)
and sodium acetate (2.85 g, 34.8 mmol) in N,N-dimethylaceta-
mide (DMA, 25 mL) at 0 °C was added a solution of tert-butyl
bromoacetate (6.79 g, 34.82 mmol, 5.14 mL) in DMA (10 mL)
dropwise. The reaction mixture was stirred at room temperature
for 5 d, after which it was poured into water (125 mL) to give a
clear yellow solution. Solid NaHCO3 was added portionwise
until 8 precipitated as a white solid. The precipitate was collected
by filtration and dissolved in CHCl3 (150 mL). The solution was
washed with water (75 mL), dried (MgSO4), filtered, and con-
centrated to about 20 mL. Ether (150mL) was added, after which
8 crystallized as a white fluffy solid, mp 201-202 °C. Rf = 0.37
(10:1 DCM/MeOH on silica gel 60 F254). Yield: 4.7 g (9.1 mmol,
78%). 1H NMR (500 MHz, CDCl3) δ 1.38 (27H, s), 2.78-2.88
(12H, m), 3.01 (4H, m), 3.21 (2H, br s), 3.30 (4H, br s), 10.18 (1H,
br s). 13C NMR (125 MHz, CDCl3) δ 28.0, 28.1, 47.4, 48.5, 49.1,
51.0, 51.2, 58.1, 81.4, 81.6, 169.4, 170.3. HRMS-ESI calcd for
C26H51N4O6 (M þ H)þ 515.3809, found 515.3817.
Synthesis.51 1-Bromomethyl-4-(triphenylmethylthio)methylbenzene
(11)36. To a suspension of NaH (46 mg, 1.89 mmol) in dry THF
(10 mL) was added a solution of trityl mercaptan (523 mg, 1.89
mmol) in dry THF (10 mL) with stirring. After 0.5 h, the solution
was added dropwise to a solution of 10 (1.0 g, 3.7 mmol) in dry
THF (20 mL) and the reaction mixture stirred overnight at room
temperature. Ether (20 mL) and water (25 mL) were added to the
reaction mixture, and the organic layer was separated, washed
with water (25 mL), brine (25 mL), dried (MgSO4), filtered, and
concentrated. Flash chromatography (10:1 f 5:1, hexanes/
DCM) afforded 678 mg (1.4 mmol, 74%) of 11 as a white solid,
mp 117-118 °C. Rf = 0.54 (2:1 hexanes/DCM on silica gel
60 F254). 1H NMR (500 MHz, CDCl3) δ 3.29 (2H, s), 4.41 (2H,
s), 7.07 (2H, d, J = 8 Hz), 7.19-7.25 (5H, m), 7.26-7.3 (6H, m),
7.44 (6H, m). 13C NMR (125 MHz, CDCl3) δ 33.3, 36.6, 67.5,
126.7, 127.9, 129.1, 129.4, 129.5, 136.4, 137.4, 144.5. Anal. Calcd
for C27H23BrS: C 70.58, H 5.05, S 6.98. Found: C 70.64, H 5.56,
S 6.11.
1-Bromo-9-tritylthiononane (13). To a solution of 1,9-dibro-
mononane (12, 5.72 g, 20 mmol, 4 mL) and trityl mercaptan
(2.76 g, 10 mmol) in CH3CN (50 mL) was added anhydrous
K2CO3 (10.35 g, 75 mmol), and the mixture was stirred over-
night at room temperature. The reaction mixture was filtered
and the filtrate evaporated under reduced pressure. The residue
was subjected to flash chromatography on silica gel, eluting with
hexanes to give 13 (2.82 g, 5.86 mmol, 59%) as a white solid, mp
58-60 °C. Rf = 0.48 (3:1 hexanes/DCM on silica gel 60 F254).
1H NMR (500 MHz, CDCl3) δ 1.12-1.28 (8H, m), 1.38 (4H,
quintet, J = 7.5 Hz, 7 Hz), 1.82 (2H, quintet, J = 7.5 Hz), 2.12
(2H, t, J = 7.5 Hz), 3.38 (2H, t, J = 7 Hz), 7.17-7.21 (3H, m),
7.24-7.28 (6H, m), 7.39-7.42 (6H, m). 13C NMR (125 MHz,
CDCl3) δ 28.1, 28.5, 28.6, 29.0, 29.2, 32.0, 32.8, 33.9, 66.3, 126.4,
127.7, 129.5, 145.0. Anal. Calcd for C28H33BrS: C 69.84, H 6.91,
S 6.66. Found: C 70.01, H 6.77, S 6.44.
1-Bromo-3-tritylthiopropane (15). To a solution of 1,3-dibro-
mopropane 14 (8.0 g, 40 mmol, 4 mL) and trityl mercaptan (5.4
g, 20 mmol) in CH3CN (150 mL) was added anhydrous K2CO3
(20.7 g, 150 mmol), and the mixture was stirred overnight at
room temperature. The reaction mixture was filtered and the
filtrate evaporated under reduced pressure. The residue was
subjected to flash chromatography on silica gel, eluting with
hexanes to give 15 (5.1 g, 12.9 mmol, 65%) as a white solid, mp
90-92 °C. Rf = 0.45 (3:1 hexanes/DCM on silica gel 60 F254).
1H NMR (500 MHz, CDCl3) δ 1.80 (2H, quintet, J = 7 Hz, 6.5
Hz), 2.31 (2H, t, J = 7 Hz), 3.31 (2H, t, J = 6.5 Hz), 7.18-7.23
(3H, m), 7.25-7.29 (6H, m), 7.40-7.41 (6H, m). 13C NMR (125
MHz, CDCl3) δ 30.2, 31.6, 32.2, 66.7, 126.6, 127.8, 129.5, 144.6.
Anal. Calcd for C22H21BrS: C 66.50, H 5.33, S 8.07. Found: C
66.76, H 5.26, S 7.42.
1,4,7-Tris(tert-butoxycarbonylmethyl)-10-[4-(triphenylmethyl-
thio)methyl)phenyl]methyl-1,4,7,10-tetraazacyclododecane (19a).
To a solution of 8 (2.0 g, 3.9 mmol) in dry CH3CN (40 mL) were
added 11 (1.9 g, 4.1 mmol) and K2CO3 (4.3 g, 31.1 mmol), and
the mixture was heated at 60 °C. After 2 h, the reaction mixture
was filtered and the filtrate evaporated to dryness with reduced
pressure. The residue was dissolved in DCM (100 mL), and the
resulting solution was washed with 1 N HCl (2 ꢀ 50 mL),
saturated aqueous NaHCO3 (50 mL), and brine. The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo.
The residue was subjected to flash chromatography on silica gel,
eluting with DCM/MeOH (25:1) to give 19a (2.8 g, 3.14 mmol,
80%) as a white foam. Rf = 0.38 (10:1 DCM/MeOH on silica gel
1
60 F254). H NMR (500 MHz, CDCl3) δ 1.43 (9H, br s), 1.46
9-Tritylthio-3,6-dioxanonan-1-ol (16). To a mixture of NaH
(0.25 g, 10.6 mmol) and TBAI (0.19 g, 0.5 mmol) in dry THF
(18H, br s), 2.20 (4H, m), 2.35 (6H, m), 2.50-2.90 (6H, m), 3.02
(4H, m), 3.23 (2H, m), 7.10 (2H, d, J = 7.5 Hz), 7.22 (2H, m),

6754 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Raghunand et al.
7.29 (9H, m), 7.45 (6H, m). 13C NMR (125 MHz, CDCl3) δ 27.8,
27.9, 28.0, 28.1, 36.5, 53.3, 55.6, 55.8, 59.2, 67.2, 82.2, 82.6, 126.6,
127.8, 129.3, 129.4, 130.0, 136.0, 136.3, 144.4, 172.4, 173.3.
HRMS-ESI calcd for C53H73N4O6S (M þ H)þ 893.5251, found
893.5216.
stirred overnight at room temperature. Volatiles were removed in
vacuo, a minimum amount of MeOH was added to effect a
solution, and ether (30 mL) was added. The precipitated white
solid was collected by filtration and purified by flash chromatog-
raphy on silica gel, eluting with CHCl3/MeOH/aq NH4OH (5:3:1)
to give 20b as a waxy solid (0.88 g, 1.74 mmol, 74%). Rf = 0.54
(5:3:1 DCM/MeOH/aq NH4OH on silica gel 60 F254). 1H NMR
(500 MHz, TFA-d) δ 1.43 (12H, m), 1.72 (2H, m), 1.86 (2H, m),
2.67 (1H, m), 3.13-3.22 (4H, m), 3.30-3.45 (6H, m), 3.57 (4H,
br s), 3.67-3.84 (8H, m), 4.34 (2H, br s). 13C NMR (125 MHz,
CDCl3) δ 25.3, 25.8, 28.1, 29.8, 30.4, 30.6, 30.8, 35.0, 50.3, 50.9,
52.0, 54.5, 54.7, 56.5, 57.6, 171.4, 178.2. HRMS-ESI calcd for
C23H45N4O6S (M þ H)þ 505.3059, found 505.3052.
1,4,7-Tris(tert-butoxycarbonylmethyl)-10-[9-(triphenylmethyl-
thio)nonyl]-1,4,7,10-tetraazacyclododecane (19b). To a solution
of 8 (2.0 g, 3.9 mmol) in dry CH3CN (40 mL) was added 13 (2.0 g,
4.1 mmol) and K2CO3 (4.3 g, 31.1 mmol), and the mixture was
heated at 60 °C. After 4 h, the reaction mixture was filtered and
the filtrate evaporated to dryness under reduced pressure. The
residue was dissolved in DCM (100 mL) and the resulting
solution was washed with 1 N HCl (2 ꢀ 50 mL), saturated
aqueous NaHCO3 (50 mL), and brine. The organic layer was
dried (MgSO4), filtered, and concentrated in vacuo. The residue
was subjected to flash chromatography on silica gel, eluting with
DCM/MeOH (25:1) to give 19b (2.84 g, 3.1 mmol, 80%) as a
white foam. Rf = 0.40 (10:1 DCM/MeOH on silica gel 60 F254).
1H NMR (500 MHz, CDCl3) δ 1.11-1.26 (10H, m), 1.34-1.41
(4H, m), 1.42-1.47 (27H, m), 2.12 (2H, t, J = 7 HZ), 2.25-2.50
(10H, m), 2.79 (4H, m), 3.00-3.22 (6H, m), 3.28 (2H, br s), 3.47
(2H, m), 7.19 (3H, m), 7.23-7.29 (6H, m), 7.36-7.41 (6H, m).
13C NMR (125 MHz,CDCl3) δ 27.7, 27.9, 28.0, 28.1, 28.5, 28.9,
29.0, 29.1, 29.2, 29.3, 29.5, 31.9, 47.7, 50.2, 50.3, 50.5, 52.6, 52.9,
54.4, 55.7, 56.3, 56.8, 66.2, 81.5, 81.6, 82.2, 82.5, 126.3, 127.6,
129.4, 144.8, 169.7, 170.2, 172.4. HRMS-ESI calcd for C54H83-
N4O6S (M þ H)þ 915.6028, found 915.6019.
1,4,7-Tris(carboxymethyl)-10-(9-mercapto-3,6-dioxanonyl)-
1,4,7,10-tetraazacyclododecane (20c). To a mixture of 19c
(2.4 g, 2.6 mmol), Et3SiH (0.34 g, 2.9 mmol, 460 μL), and
butanethiol (25 mL) was added TFA (25 mL) dropwise. The
mixture was stirred overnight at room temperature. Volatiles
were removed in vacuo, a minimum amount of MeOH was
added to effect a solution, and ether (30 mL) was added. The
precipitated white solid was collected by filtration and purified by
flash chromatography on silica gel, eluting with CHCl3/MeOH/aq
NH4OH (5:3:1) to afford 20c as a waxy solid (0.98 g, 1.92 mmol,
74%). Rf = 0.52 (5:3:1 DCM/MeOH/aq NH4OH on silica gel 60
F254). 1H NMR (500 MHz, TFA-d) δ 1.89 (2H, m), 2.2 (1H, t, J =
7 Hz), 2.95-3.18 (8H, m), 3.45 (2H, m), 3.54-3.75 (18H, m), 3.85
(3H, m), 4.21 (2H, br s). 13C NMR (125 MHz, CDCl3) δ 21.8, 33.6,
50.1, 50.7, 53.1, 54.4, 54.7, 56.4, 56.6, 66.0, 70.9, 71.6, 71.9, 170.9,
177.4. HRMS-ESI calcd for C21H41N4O6S (M þ H)þ 508.2640,
found 508.2634.
{1,4,7-Tris(carboxymethyl)-10-[4-(thiomethyl)phenyl]methyl-
1,4,7,10-tetraazacyclododecanato}gadolinium (3a). To a solu-
tion of 20a (100 mg, 0.207 mmol) in MeOH (3 mL) was added
Gd(OAc)3 (69.2 mg, 0.207 mmol), and the mixture was heated at
reflux. After 2 h volatiles were removed in vacuo. The residue
was subjected to flash chromatography on reverse-phase silica
gel 60 RP-18 eluted with MeOH/H2O (7:3) to give 3a as a white
solid (0.1 g, 0.15 mmol, 75%). Rf = 0.16 (MeOH/H2O 19:1 on
silica gel 60 RP-18 F254). ESI-MS calcd for C20H32GdN4O6S
(M þ H)þ 638.1287, found 638.1307.
1,4,7-Tris(tert-butoxycarbonylmethyl)-10-[9-(triphenylmethyl-
thio)-3,6-dioxanonyl]-1,4,7,10-tetraazacyclododecane (19c). To a
solution of 8 (1.72 g, 3.37 mmol) in dry CH3CN (40 mL) was
added 17 (1.73 g, 3.54 mmol) and K2CO3 (3.9 g, 28.32 mmol),
and the mixture was heated at 60 °C. After 4 h, the reaction
mixture was filtered and the filtrate evaporated to dryness under
reduced pressure. The residue was dissolved in 1 N HCl (50 mL)
and brine (50 mL), and the resulting solution was extracted with
DCM (3 ꢀ 50 mL). The organic extracts were combined, washed
with saturated aqueous NaHCO3 (50 mL), brine, dried(MgSO4),
filtered, and concentrated in vacuo. The residue was subjected to
flash chromatography on silica gel, eluting with DCM/MeOH
(25:1) to give 19c (2.84 g, 3.09 mmol, 92%) as a white foam. Rf =
1
0.43 (10:1 DCM/MeOH on silica gel 60 F254). H NMR (500
[1,4,7-Tris(carboxymethyl)-10-(9-mercaptononyl)-1,4,7,10-
tetraazacyclododecanato]gadolinium (4a). To a solution of 20b
(100 mg, 0.198 mmol) in MeOH (3 mL) was added Gd(OAc)3
(66.2 mg, 0.198 mmol), and the mixture was heated at reflux.
After 2 h volatiles were removed in vacuo. The residue was
subjected to flash chromatography on reverse-phase silica gel
60 RP-18, eluting with MeOH/H2O (7:3) to give 4a as a white
solid (75 mg, 0.113 mmol, 57%). Rf = 0.17 (MeOH/H2O 19:1
on silica gel 60 RP-18 F254). ESI-MS calcd for C23H42GdN4-
O6S (M þ H)þ 660.2061, found 660.2069. Crystals were grown
for X-ray diffraction analysis according to a procedure used
previously for growing an In-DO3A-TPP complex.41 An equi-
molar mixture of 20b and Gd(OAc)3 was heated at 100 °C for
30 min in NH4OAc buffer (0.5 M, pH 6). Slow diffusion of acetone
into the reaction mixture produced colorless, flat plates.
{1,4,7-Tris(carboxymethyl)-10-[(9-mercapto-3,6-dioxa)nonyl]-
1,4,7,10-tetraazacyclododecanato}gadolinium (5a). To a solution
of 20c (100 mg, 0.196 mmol) in MeOH (3 mL) was added Gd-
(OAc)3 (65.8 mg, 0.196 mmol), and the mixture was heated at
reflux. After 2 h volatileswereremoved in vacuo. Theresidue was
subjected to flash chromatography on reverse-phase silica gel 60
RP-18, eluting with MeOH/H2O (1:4) to give 5a as a white solid
(0.1 g, 0.15 mmol, 75%). Rf = 0.23 (MeOH/H2O 1:4 on silica gel
60 RP-18 F254). ESI-MS calcd for C21H38GdN4O8S (M þ H)þ
664.1646, found 664.1650.
MHz, CDCl3) δ 1.44 (27, m), 1.61 (2H, quintet, J = 7 Hz, 6.5
Hz), 2.20 (2H, t, J = 7 Hz), 2.22-2.50 (8H, cluster of m), 2.82
(8H, m), 3.02 (8H, m), 3.33 (2H, t, J = 6.5 Hz), 3.48 (4H, m), 3.60
(2H, m), 7.20 (3H, m), 7.26 (6H, m), 7.37 (6H, m). 13C NMR (125
MHz, CDCl3) δ 27.9, 28.0, 28.1, 28.2, 28.6, 28.7, 49.7, 50.5, 52.2,
53.4, 55.6, 56.3, 66.5, 67.5, 69.5, 69.9, 70.0, 82.0, 82.1, 126.5,
127.7, 129.4, 144.7, 172.4. HRMS-ESI calcd for C52H79N4O8S
(M þ H)þ 919.5613, found 919.5612.
1,4,7-Tris(carboxymethyl)-10-[4-(thiomethyl)phenyl]methyl-
1,4,7,10-tetraazacyclododecane (20a). To a mixture of 19a (2.0
g, 2.2 mmol), Et3SiH (0.28 g, 2.4 mmol, 386 μL), and buta-
nethiol (20 mL) was added TFA (20 mL) dropwise. The
mixture was stirred overnight at room temperature. Volatiles
were removed in vacuo, a minimum amount of MeOH was
added to effect a solution, and ether (30 mL) was added. The
precipitated white solid was collected by filtration and purified
by flash chromatography on silica gel, eluting with CHCl3/
MeOH/aq NH4OH (5:3:1) to give 20a as a waxy solid (0.85 g,
1.76 mmol, 80%). Rf = 0.41 (5:3:1 DCM/MeOH/aq NH4OH
on silica gel 60 F254). 1H NMR (600 MHz, TFA-d) δ 3.21 (4H, m),
3.33 (3H, m), 3.42 (3H, m), 3.58 (4H, m), 3.78 (2H, m), 3.72 (2H,
m), 3.77(4H, m), 3.87 (2H, brs), 4.44(2H, br s), 4.66(2H, br s). 13
C
NMR (150 MHz, TFA-d) δ 29.1, 49.9, 50.3, 51.7, 54.1, 54.5, 56.4,
60.7, 127.4, 131.5, 133.1, 146.9, 171.0, 177.4. HRMS-ESI calcd for
C20H35N4O6S (M þ H)þ 483.2277, found 483.2273.
{1,4,7-Tris(carboxymethyl)-10-[4-(thiomethyl)phenyl]methyl-
1,4,7,10-tetraazacyclododecanato}europium (3b). To a solution
of 20a (100 mg, 0.207 mmol) in MeOH (3 mL) was added
Eu(OAc)3 (68.3 mg, 0.207 mmol), and the mixture was heated at
reflux. After 2 h volatiles were removed in vacuo. The residue
[1,4,7-Tris(carboxymethyl)-10-(9-mercaptononyl)-1,4,7,10-
tetraazacyclododecane (20b). To a mixture of 19b (2.16 g, 2.36
mmol), Et3SiH (0.30 g, 2.6 mmol, 415 μL), and butanethiol
(20 mL) was added TFA (20 mL) dropwise. The mixture was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6755
was subjected to flash chromatography on reverse-phase silica
gel 60 RP-18, eluting with MeOH/H2O (7:3) to give 3b as a white
solid (93 mg, 0.147 mmol, 71%). Rf = 0.13 (MeOH/H2O 19:1
on silica gel 60 RP-18 F254). ESI-MS calcd for C20H32EuN4O6S
(M þ H)þ 633.1259, found 633.1243.
55.2, 57.1, 57.3, 67.0, 71.2, 72.5, 76.2, 171.9, 178.3. HRMS calcd
for (M þ H)þ C21H41N4O8 477.2918, found 477.2926.
[1,4,7-Tris(carboxymethyl)-10-(3,6-dioxa)nonyl-1,4,7,10-tetra-
azacyclododecanato]gadolinium (24). To a solution of 23 (100 mg,
0.21 mmol) in MeOH (3 mL) was added Gd(OAc)3 (70.2 mg, 0.21
mmol), and the mixture was heated at reflux. After 2 h volatiles
were removed in vacuo. The residue was subjected to flash chro-
matography on reverse-phase silica gel 60 RP-18, eluting with
MeOH/H2O (1:4) to give 24 as a white solid (120 mg, 0.19 mmol,
75%). Rf = 0.25 (MeOH/H2O 3:7 on silica gel 60 RP-18 F254).
ESI-MS calcd for C21H39GdN4O6 (M þ H)þ 632.1930, found
632.1932.
[1,4,7-Tris(carboxymethyl)-10-(9-mercaptononyl)-1,4,7,10-
tetraazacyclododecanato]europium (4b). To a solution of 20b
(100 mg, 0.198 mmol) in MeOH (3 mL) was added Eu(OAc)3
(65.3 mg, 0.198 mmol), and the mixture was heated at reflux.
After 2 h volatiles were removed in vacuo. The residue was
subjected to flash chromatography on reverse-phase silica gel
60 RP-18, eluting with MeOH/H2O (7:3) to give 4b as a white
solid (75 mg, 0.114 mmol, 58%). Rf = 0.10 (MeOH/H2O 19:1
on silica gel 60 RP-18 F254). ESI-MS calcd for C23H42Eu-
N4O6S (M þ H)þ 655.2032, found 655.2035.
Binding of Gd Thiols to Human Serum Albumin. Solutions of
Gd complexes were made in phosphate-buffered saline (PBS),
acetate-buffered saline, or HEPES-buffered saline and in corre-
sponding solutions that contained 0.66 mM human serum
albumin (HSA, Sigma), as well as 0-2.0 mM homocysteine
(Sigma), to final gadolinium concentrations of 0-1.0 mM. All
solutions also contained 10 mM sodium azide, and the final pH of
all solutions was 7.40 ( 0.05 at room temperature. Solutions were
allowed to equilibrate overnight at 37 °C prior to measurements.
Aliquots (500 μL) of each solution containing HSA were placed in
prewarmed ultrafiltration units (Amicon UItra-4 centrifugal filter
units, 30000 MW cutoff, Millipore Corporation) and immediately
centrifuged at 7451g for 10.7 min, inclusive of braking time. It was
assumed that gadolinium bound to HSA (Gdbound) would not pass
{1,4,7-Tris(carboxymethyl)-10-[(9-mercapto-3,6-dioxa)nonyl]-
1,4,7,10-tetraazacyclododecanato}europium (5b). To a solution
of 20c (100 mg, 0.196 mmol) in MeOH (3 mL) was added Eu-
(OAc)3 (64.8 mg, 0.196 mmol), and the mixture was heated at
reflux. After 2 h volatiles were removed in vacuo. The residue
was subjected to flash chromatography on reverse-phase silica
gel 60 RP-18, eluting with MeOH/H2O (1:4) to give 5b as a white
solid (106 mg, 0.161 mmol, 82%). Rf = 0.20 (MeOH/H2O 1:4 on
silica gel 60 RP-18 F254). ESI-MS calcd for C21H38EuN4O6S
(M þ H)þ 659.1617, found 659.1617.
1-Bromo-3,6-dioxanonane (21). To a solution of diethylene
glycol monopropyl ether (5.0 g, 30 mmol) in DCM (150 mL) was
added CBr4 (12.3 g, 37 mmol) followed by PPh3 (9.7 g, 37 mmol).
The mixture was stirred at room temperature. After 12 h, the
pale-brown solution was concentrated in vacuo, and the residue
was subjected to flash chromatography on silica gel. Elution
with hexanes/EtOAc (80:20) gave 21 (5.7 g, 27 mmol, 90%) as a
colorless oil. Rf = 0.54 (4:1 hexanes/EtOAc on silica gel 60 F254).
1H NMR (500 MHz, CDCl3) δ 0.92 (2H, t, J=6.5 Hz), 1.61
(2H, m), 3.43 (2H, m), 3.47 (2H, m), 3.59 (2H, m), 3.67 (2H, m),
3.82 (2H, t, J = 6.5 Hz). 13C NMR (125 MHz, CDCl3) δ 10.4,
22.8, 30.2, 70.0, 70.5, 71.2.
through the membrane and that gadolinium in the filtrate (Gdfree
)
accurately represented the unbound gadolinium in each sample.
Gadolinium concentrations in the filtrates were determined
by MRI relaxometry. The apparent equilibrium binding con-
stant (KA) for each complex was calculated from the law of
mass action.52
Relaxivity of Gd Complexes in the Absence of HSA. Measure-
ments of the longitudinal water-proton relaxivities of Gd com-
plexes in buffered saline were made at 37 °C on a 7 T Bruker
Biospec MR instrument (Bruker Biospin, Billerica, MA). 96-
Well tissue culture plates (Falcon) were cut down to 6 ꢀ 8 wells,
creating a sample tray that fit inside a 72 mm i.d. birdcage radio
frequency transmitter-receiver coil (Bruker). Aliquots (250 μL)
of the gadolinium solutions were loaded into these wells. Spaces
between the wells were filled with water in order to reduce
air-water susceptibility artifacts in the images. The sample tray
was maintained at 37 °C during imaging by flowing heated air
over the sample tray. Sample temperature was continuously
monitored using a fluoroptic temperature probe (Luxtron Cor-
poration, Santa Clara, CA). Spin-echo MR images of cross
sections of the wells were acquired with recycle times (TR)
ranging between 35 and 8000 ms and an echo time (TE) of
4 ms. Signal intensity S in each well was fitted to eq 1 to extract
the T1 at each solution condition. The factor c was between 0.98
and 1.0 in all regressions.
1,4,7-Tris(tert-butoxycarbonylmethyl)-10-(3,6-dioxanonyl)-
1,4,7,10-tetraazacyclododecane (22). To a solution of 8 (2.0 g,
4 mmol) in dry CH3CN (40 mL) were added 21 (0.88 g, 4.2
mmol) and K2CO3 (4.4 g, 32 mmol), and the mixture was
heated at 60 °C. After 12 h, the reaction mixture was filtered
and the filtrate evaporated to dryness in vacuo. The residue
was dissolved in 1 N HCl (50 mL) and brine (50 mL), and the
resulting solution was extracted with DCM (3 ꢀ 50 mL). The
organic extracts were combined, washed with saturated aqu-
eous NaHCO3 (50 mL), brine, dried (MgSO4), filtered, and
concentrated in vacuo. The residue was subjected to flash
chromatography on silica gel, eluting with DCM/MeOH
(25:1) to give 22 (2.47 g, 3.8 mmol, 96%) as a white foam.
Rf = 0.52 (10:1 DCM/MeOH on silica gel 60 F254). 1H NMR
(500 MHz, CDCl3) δ 0.89 (3H, t, J = 7.5 Hz), 1.45 (27, m), 1.55
(2H, m), 2.22-2.60 (8H, cluster of m), 2.70-2.90 (10H, m),
3.05 (4H, m), 3.33 (2H, m), 3.50 (4H, m), 3.70 (4H, m). 13C
NMR (125 MHz, CDCl3) δ 10.5, 22.7, 27.9, 28.0, 28.1, 28.2,
49.8, 50.4, 52.2, 55.7, 56.4, 67.5, 69.3, 69.9, 70.1, 82.0, 82.1,
172.4, 172.7. HRMS-ESI calcd for (M þ H)þ C33H65N4O8
645.4796, found 645.4799.
1,4,7-Tris(carboxymethyl)-10-(3,6-dioxanonyl)-1,4,7,10-tetra-
azacyclododecane (23). To a solution of 22 (0.60 g, 0.93 mmol) in
DCM (4 mL) was added TFA (4 mL) dropwise. The mixture was
stirred overnight at room temperature. Volatiles were removed
in vacuo and the residue was subjected to flash chromatography
on silica gel, eluting with CHCl3/MeOH/aq NH4OH (4:2:1) to
afford 23 as a waxy solid (0.34 g, 0.72 mmol, 77%). Rf = 0.54
(4:2:1 DCM/MeOH/aq NH4OH on silica gel 60 F254). 1H NMR
(500 MHz, TFA-d) δ 1.03 (3H, t, J = 7 Hz), 1.79 (2H, m),
3.15-3.29 (4H, m), 3.30-3.42 (4H, m), 3.68 (2H, m), 3.7-3.8
(8H, m), 3.88 (2H, m), 3.93 (2H, m), 4.08 (2H, m), 4.45 (2H, br s).
13C NMR (125 MHz, CDCl3) δ 10.8, 23.9, 50.7, 51.3, 53.9, 55.0,
!
ꢀ
ꢁ
- TR
T1
S ¼ S0 1 - cexp
ð1Þ
The T1 relaxation times of the solutions of gadolinium in saline
containing 0-2.0 mM homocysteine thus calculated were then
fit to eq 2, and a longitudinal relaxivity r1 was obtained for each
solution condition.
1
1
¼
þ r1½Gdꢁ
ð2Þ
T1
T10
Here 1/T10 is the relaxation rate in the absence of contrast
agent, and [Gd] is the concentration of gadolinium in the
solution.
Relaxivity of Gd Complexes in the Presence of HSA. The
relaxivity of gadolinium in HSA-containing solutions was ap-
proximated to have only two components: “free” (monomer,
homodimer, heterodimer) and “bound” (to HSA). The T1 times
of the solutions of both gadolinium complexes in saline þ HSA

6756 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Raghunand et al.
were inserted into eq 3 and fitted for the relaxivity of bound
gadolinium, r1(bound).
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1
1
¼
þ r1ðfreeÞ½GdðfreeÞꢁ þ r1ðboundÞ½GdðtotalÞ - GdðfreeÞꢁ
ð3Þ
T1
T10
where [Gd(free)] was obtained as described previously31 with
KA constrained to equal the binding constant calculated for
the respective HSA-containing solution. In these regressions,
r1(free) was constrained to equal the relaxivity measured in the
corresponding HSA-free solution.
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Acknowledgment. This work was supported by Grants
R01-CA118359 and P30-CA023074 from the National Insti-
tutes of Health. The single crystal X-ray diffractometer was
purchased with funds from Grant CHE-9610347 from the
National Science Foundation.
Supporting Information Available: General experimental
procedures, proton and carbon NMR spectra for compounds
3b-5b, 8, 11, 13, 17, 19a-c, 20a-c, and 21-23, mass spectra for
compounds 3a-5a, 3b-5b, and 24, and an X-ray crystallo-
graphic information file (CIF format) for compound 4a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(23) Yoh, K.; Hirayama, A.; Ishizaki, K.; Yamada, A.; Takeuchi, M.;
Yamagichi, S.; Morito, N.; Nakano, T.; Ojima, M.; Shimohata, H.;
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Oxidative and Nitrosative Stress and Increases Renal Functional
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