Towards the rational design of MRI contrast agents: δ-Substitution of lanthanide(III) NB-DOTA-tetraamide chelates influences but does not control coordination geometry

Article abstract of DOI:10.1002/chem.201101007

LnDOTA-tetraamide chelates (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid) have received considerable recent attention as a result of their potential to act as PARACEST contrast agents for magnetic resonance imaging (MRI). Although PARACE

Full text of DOI:10.1002/chem.201101007

DOI: 10.1002/chem.201101007
Towards the Rational Design of MRI Contrast Agents: d-Substitution of
LanthanideACHTUNGTRENNUNG(III) NB-DOTA-Tetraamide Chelates Influences but Does Not
Control Coordination Geometry**
Christiane E. Carney,[a] Anh D. Tran,[b] Jing Wang,[b] Matthias C. Schabel,[c]
A. Dean Sherry,[b, d] and Mark Woods*[a, c]
Abstract: LnDOTA-tetraamide che-
lates (DOTA=1,4,7,10-tetraazacyclo-
portant goal. In this study we investi-
gate the potential to extend conforma-
tional control of LnDOTA-type ligands
to those applicable to PARACEST.
Furthermore, the question of whether
d- rather than a-substitution of the
pendant arms could be used to control
the chelate coordination geometry is
addressed. Although d-substitution
does influence coordination geometry
it does not afford control. However, it
can play an important role in governing
the conformation of the amide sub-
stituent relative to the chelate in such
as way that suggests a PARACEST
agent could be designed that has detec-
tion limits at least as low as a conven-
tional MRI contrast agent.
dodecane-1,4,7,10-tetraacetic
acid)
have received considerable recent at-
tention as a result of their potential to
act as PARACEST contrast agents for
magnetic resonance imaging (MRI).
Although PARACEST agents afford
several advantages over conventional
contrast agents they suffer from sub-
stantially higher detection limits; thus,
Keywords: conformational control ·
coordination modes · lanthanides ·
PARACEST agents
change
· water ex-
improving
the
effectiveness
of
LnDOTA-tetraamide chelates is an im-
Introduction
formation of the macrocyclic ring or a reorientation of the
pendant arms.[3,4] Substitution can effectively halt these in-
tramolecular processes affording a single coordination ge-
ometry.[1,2] The coordination geometry thus obtained is then
determined by the relative configuration of the substituents
on the macrocyclic ring and the pendant arms; when the
configurations at these two locations are the same, a TSAP
isomer results and when they are opposed, a SAP isomer re-
sults.[1,2] The selection of coordination geometry in this type
of chelate is of interest since it may facilitate the design of
more effective Gd3+-based MRI contrast agents. TSAP iso-
mers have consistently been found to have water exchange
rates that are 50–100-fold faster than those of SAP iso-
mers,[5–7] a critical factor in the behavior of Gd3+-based con-
trast agents. LnDOTA-tetraamide chelates exhibit a similar
coordination chemistry to DOTA chelates but much slower
water proton exchange kinetics as a result of the reduced
electron density on the central metal ion. These slower ex-
change rates have sparked interest in this class of chelate as
potential PARACEST (paramagnetic chemical exchange
saturation transfer) agents.[8,9] Since slow exchange is desira-
ble for PARACEST, SAP isomers are generally preferred. It
has been observed that LnDOTA-tetraamides with more
bulky amide substituents have a general preference for the
SAP coordination geometry, but recent results suggest that
changes in the local environment, and in particular the sol-
vent, may cause changes in the coordination chemistry of
LnDOTA-tetraamide chelates.[10] Furthermore, addition of a
second amide substituent seems to increase the proportion
We have recently reported a method by which the coordina-
tion geometry adopted by Ln3+ chelates of DOTA-type li-
gands (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-
raacetic acid) may be selected by appropriate substitution of
the pendant arms and macrocyclic ring.[1,2] LnDOTA che-
lates can interconvert between mono-capped square anti-
prism (SAP) and mono-capped twisted square antiprism
(TSAP) coordination geometries either by a flip in the con-
[a] C. E. Carney, Dr. M. Woods
Department of Chemistry, Portland State University
1719 SW 10th Ave, Portland, OR 97201 (USA)
E-mail: mark.woods@pdx.edu
[b] A. D. Tran, Dr. J. Wang, Prof. A. D. Sherry
Department of Chemistry, University of Texas at Dallas
800 W. Campbell Road, Richardson, TX 75080 (USA)
[c] Dr. M. C. Schabel, Dr. M. Woods
Advanced Imaging Research Center
Oregon Health & Science University
3181 S.W. Sam Jackson Park Road, Portland, OR 97239 (USA)
E-mail: woodsmar@ohsu.edu
[d] Prof. A. D. Sherry
Advanced Imaging Research Center
University of Texas Southwestern Medical Center
Harry Hines Blvd., Dallas, TX 75235 (USA)
[**] NB-DOTA=2-(S)-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101007.
10372
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10372 – 10378

FULL PAPER
of TSAP isomer present.[10] In light of these observations,
we were interested to know whether it was possible to selec-
tively control the coordination geometry of DOTA-tetraa-
mide chelates through substitution in a manner analogous
that used for DOTA chelates. Of particular interest was the
effect of introducing chirality into the d-position of an
amide substituent of the pendant arm rather than in the a-
position as employed previously with DOTA chelates. The
motivation for this approach is that a-substitution necessari-
ly involves a synthetic transformation at a chiral center, with
all the attendant problems. Control from the d-position
would eliminate these synthetic complications. Chiral amide
substituents have been reported to influence both the orien-
tation of pendant arms and configuration at phosphorus in
triphosphinate-monoamide chelates.[11] The question we
sought to address in this work is whether, in DOTA-tetraa-
mide systems, this approach would not merely influence the
orientation of the pendant arms but define it.
Scheme 1. The preparation of the two diastereoisomeric ligands 1. Re-
agents and conditions: S-RRRR-1, R=Et: i. BrCH2COBr/K2CO3/CH2Cl2/
08C; ii. S-NB-cyclen/K2CO3/MeCN/608C; iii. NaOH/H2O/THF; iv. HCl:
S-SSSS-1, R=Bn: i. BrCH2COBr/K2CO3/CH2Cl2/08C; ii. S-NB-cyclen/
K2CO3/MeCN/608C; iii. BCl3/CH2Cl2/THF; iv. H2O.
Results and Discussion
age—saponification for the ethyl esters and BCl3 in CH2Cl2
in the case of benzyl esters[17]—afforded S-RRRR-1 and S-
SSSS-1. The Ln3+ chelates were then prepared from the cor-
responding Ln3+ triflate in water at pH 6 and purified by
preparative RP-HPLC (C18) to afford each chelate in its
protonated form.
EuDOTAM-Gly has been widely studied as a potential
PARACEST agent[12–14] and, given its potential suitability for
use in vivo,[15,16] was therefore an ideal starting point for this
investigation. The conformation of the macrocyclic ring was
The coordination geometry of DOTA and DOTA-tetraa-
mide type chelates are known to be strongly affected by the
ionic radius of the Ln3+ ion. A preference for the TSAP ge-
ometry is observed for earlier (larger) Ln3+ ions, switching
to the SAP geometry as the ionic radius decreases across
the series.[18,19] To account for the effect of ionic radius the
Pr3+, Eu3+, and Yb3+ chelates of each ligand were prepared
1
and examined by H NMR spectroscopy. In the case of Pr3+
,
only one coordination geometry could be detected for each
chelate (Figure 1), indicating that, as expected, the S-RRRR-
isomer adopts the L
ACHTUNGTRENNUNG
the S-SSSS-isomer is in the DAHCTNUGTRENNNUG
exclusively. Reducing the ionic radius of the Ln3+ ion (Pr3+
!Eu3+!Yb3+) was found to have no effect on the chelates
of S-RRRR-1, which were found to exist in only one coordi-
nation geometry (the LACHTUNRGTNE(NUG dddd) conformation) across the lan-
thanide series (Figure 1, 2, and 3). In contrast, the chelates
of S-SSSS-1 were found to be profoundly affected by chang-
ing the ionic radius of the Ln3+ ion with increasing amounts
of chelate in a SAP coordination geometry being discerna-
ble in the NMR spectrum of the Eu3+ and Yb3+ chelates
(Figure 2 and Figure 3). For EuS-SSSS-1 the amount of SAP
isomer observed is quite small, approximately 5%, but for
YbS-SSSS-1 the amount increases to almost 50%. It is also
notable that considerable line broadening can be observed
in the spectrum of the TSAP isomer of EuS-SSSS-1, but not
the SAP isomer. The line broadening is also not observed in
the corresponding Yb3+ chelate. The origin of this effect re-
mains obscure but perhaps relates to an increase in internal
vibrational or torsional motions in the ligand, shortening T2,
controlled with the same nitrobenzyl substituent employed
in our previous systems[1,2]—an S- configuration conferring a
(dddd) conformation upon the macrocyclic ring. Alanine
was substituted for glycine in the synthesis of the pendant
arms. (R)-alanine ethyl ester and (S)-alanine benzyl ester
were used to prepare the two diastereoisomeric ligands S-
RRRR-1 and S-SSSS-1 (Scheme 1). In each case the alanine
ester was condensed with bromoacetyl bromide in CH2Cl2
with K2CO3 as base. The resulting bromoacetamides were
then used to alkylate the S-2-nitrobenzyl cyclen. Ester cleav-
Chem. Eur. J. 2011, 17, 10372 – 10378
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10373

M. Woods et al.
Figure 2. The 1H NMR spectra of EuS-RRRR-1 (top), EuS-SSSS-1
(bottom) recorded at 400 MHz and 300 K in D2O at pD 4. The four shift-
ed peaks between 22 and 27 ppm (both spectra) and 7 and 10 ppm
(bottom) are typical of those observed for the SAP and TSAP isomers of
EuDOTA-tetraamide chelates, respectively.
Figure 1. The 1H NMR spectra of PrS-RRRR-1 (top) PrS-SSSS-1
(bottom) and recorded at 400 MHz and 300 K in D2O at pD 4. The four
highly shifted peaks between d=ꢀ28 and ꢀ35 ppm (top) and d=ꢀ10
and ꢀ16 ppm (bottom), are characteristic of the shifts of the axS protons
in SAP and TSAP isomers of PrDOTA-tetraamide chelates, respectively.
site orientation, then the bulky substituent is placed in a
pseudo-axial position that is presumably higher in energy
(Figure 4). Examples of this situation in the crystallographic
literature suggest that this occurs primarily when a second
factor forces the arms into the orientation that opposes the
configuration of the chiral substituent.[21] It seems likely that
the decrease in ionic radius, and perhaps the associated in-
crease in charge density, contributes to an increase in this
second factor for LnS-SSSS-1 chelates as one passes along
the Ln3+ series. All LnDOTAM-Gly chelates exhibit an ex-
clusive preference for the SAP geometry and although the
reasons for this are unclear, it does explain why no TSAP
isomer was observed for any of the LnS-RRRR-1 chelates,
even for Pr3+ which often exhibits a preference for this co-
ordination geometry. This phenomenon, which drives
LnDOTAM-Gly chelates into exclusively SAP geometries,
is presumably the same as that which results in SAP isomers
being observed for the LnS-SSSS-1 chelates. The SAP
as it attempts, but fails, to overcome the energy barrier asso-
ciated with isomeric exchange.
For a chelate of S-SSSS-1 to adopt a SAP coordination
geometry, either the macrocyclic ring or the pendant arms
must adopt a conformation that opposes the configuration
of the chiral center located on that group. A flip in the con-
formation of the macrocyclic ring would place the nitroben-
zyl substituent in an axial position, substantially increasing
the torsional strain in the system and may be considered
highly unlikely. Rather it seems that the SAP form arises
from the pendant arms rotating into the unexpected confor-
mation (L rather than D for the S-SSSS-isomer).[20] In an a-
substituted system, torsional strain is minimized by placing
ꢀ
the substituent as far from the metal (trans- across the N Ca
bond) as possible (Figure 4). This determines the orientation
of the pendant arms. The origins of control observed in
some d-substituted systems remain ambiguous; however, a
survey of crystallographic data on related systems suggests
that a preferred conformation exists.[11,21–27] It appears that
the lowest energy conformation points the hydrogen of the
chiral center towards the metal ion, the bulkiest substituent
is then placed in a pseudo-equatorial position relative to the
chelate (Figure 4). This can occur only if the pendant arms
are orientated appropriately: L for an R-configuration, D
for S-configuration. If the arms rotate and adopt the oppo-
isomer that is observed would be the same LACTHUNRTGNEUNG(dddd) confor-
mation observed for the LnS-RRRR-1 chelates but with one
significant difference: the carboxylate substituent must
occupy a pseudo-axial rather than a pseudo-equatorial posi-
tion.
Although the substitution of the d-position does not
afford control over the coordination geometry in LnS-SSSS-
1 chelates, preliminary results suggest that the intramolecu-
10374
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10372 – 10378

d-Substitution of LanthanideACHTNUTRGNE(NUG III) NB-DOTA-Tetraamide Chelates
FULL PAPER
From the point of view of PARACEST agent design, che-
lates that adopt a SAP coordination geometry are preferred
because, in general, they possess slower water proton ex-
change kinetics. The unexpected appearance of a second
conformer of the SAP coordination geometry, that adopted
by EuS-SSSS-1, afforded a unique opportunity to examine
how the orientation of the carboxylate group affects CEST.
NMR samples were prepared to compare the CEST of EuS-
RRRR-1 with that of EuS-SSSS-1 at 1) the same overall con-
centration of chelate (274 mm) and 2) the same concentra-
tion of SAP isomer (13.7 mm) (Figure 5). CEST peaks at ap-
proximately +50 ppm were observed for both chelates:
52 ppm for EuS-RRRR-1 and 49 ppm for EuS-SSSS-1, con-
sistent with CEST arising from a SAP coordination geome-
try in each case. When the overall chelate concentration
([Eu3+]) is the same, the magnitude of CEST arising from
EuS-RRRR-1 is approximately threefold greater than that of
EuS-SSSS-1 (Figure 5, open symbols), but the SAP isomer
constitutes only 1/20 of the EuS-SSSS-1 chelate. When the
concentrations of the SAP isomer in each sample were nor-
malized ([SAP]=13.7 mm), no CEST was observed from Eu-
S-RRRR-1, whereas EuS-SSSS-1 produces a change in bulk
water signal of approximately 10% (Figure 5, circles). Clear-
ly the SAP isomer of EuS-SSSS-1 is a substantially more ef-
fective PARACEST agent than that of EuS-RRRR-1.
To better understand the difference between these two
SAP isomers these CEST data were quantitatively analyzed.
In general CEST spectra may be fitted to the Bloch equa-
tion modified for exchange,[28] however, a reasonable fit be-
tween this model and the data for EuS-SSSS-1 could not be
obtained when the concentration of water shifted to
+49 ppm was the same as that of the SAP isomer (see Fig-
ure S2 in the Supporting Information). The water proton
residence lifetime (tMH) for the SAP isomer of each chelate
was therefore determined independently by the so-called ꢁw-
method’,[29] affording values of tMH =116 ms (EuS-RRRR-1)
and 65 ms (EuS-SSSS-1) (Figure 6). These values could then
Figure 3. The 1H NMR spectra of YbS-RRRR-1 (top), YbS-SSSS-1
(bottom) recorded at 400 MHz and 275 K in D2O at pD 4. The four shift-
ed peaks between 82 and 103 ppm (both spectra) and 20 and 35 ppm
(bottom) are typical of those observed for the SAP and TSAP isomers of
YbDOTA-tetraamide chelates, respectively.
Figure 4. The orientation of chiral pendant arms and how they are affect-
ed by a- (top) and d-substitution (bottom), in each case the configuration
at carbon is R.
lar exchange processes between the two coordination geo-
metries have been “frozen out”. No evidence of exchange
could be detected by EXSY or by saturation transfer experi-
ments on the highly shifted acetate peaks of YbS-SSSS-1,
suggesting that the two isomers (SAP and TSAP) are either
not in exchange or exchange so slowly that the process
cannot be detected by these techniques.
Figure 5. CEST spectra of solutions of EuS-RRRR-1 ([Eu3+]=[SAP]=
274 mm open diamonds; [Eu3+]=[SAP]=13.7 mm closed circles) and EuS-
SSSS-1 ([Eu3+]=274 mm, [SAP]=13.7 mm open circles) in CD3CN (10%
H2O) recorded at 400 MHz; 298 K; irr. time=10 s; B1 =30 mT.
Chem. Eur. J. 2011, 17, 10372 – 10378
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10375

M. Woods et al.
linear relationship between CEST and concentration (which
is approximately true at very low concentrations),[30] 137 mm
could generate 10% CEST. A change in water signal inten-
sity of only about 3% is required to observe contrast
changes in an MR image, so just 46 mm of the SAP isomer of
EuS-SSSS-1 should afford an MRI detectable level of CEST
in pure water.
Although these measurements were performed by using
admittedly high B1 powers (upwards of 1 kHz) there is
scope for B1 to be substantially reduced before the detection
limit of the active form of this agent exceeds 0.1 mm, the
typical dose of
a
clinical MRI contrast agent
(0.1 mmolkgꢀ1). Clearly further experimentation is required
to better understand the potential of the type of system for
PARACEST design. Nonetheless, these preliminary data do
suggest that attending to the coordination chemistry of
LnDOTA-tetraamide chelates may provide a means by
which the detection limit problem in CEST may be over-
come.
Figure 6. The w-plots of EuS-RRRR-1 (filled circles) and EuS-SSSS-1
(open diamonds), data taken from the CEST spectra in Figure S1 in the
Supporting Information. The difference in slope of the two lines can be
attributed to the difference in SAP isomer concentration between the
two samples. The water proton residence lifetimes are afforded by the in-
tercept of the x axis.
be applied to modeling the data to the modified Bloch equa-
tions. The spectrum of Eu-S-RRRR-1 fitted to theory satis-
factorily (Figure 5), however, the spectrum of EuS-SSSS-1
continued to prove difficult to fit. A satisfactory fit for these
data could only be obtained when the concentration of pro-
tons in the highly shifted pool (+49 ppm) was allowed to in-
crease beyond the concentration of the SAP isomer. When
the concentration of water molecule in this pool was 3.7
times that of the SAP isomer a good fit was obtained sug-
gesting that between two and three additional water mole-
cules may be present in the highly shifted pool of EuS-
SSSS-1 (Figure 5). These additional water molecules must
be closely held and highly shifted by Eu3+, although perhaps
not so highly shifted as the coordinated water, which may
account for the slightly smaller shift of the CEST peak in
this chelate. These additional water protons would certainly
explain why the SAP isomer of EuS-SSSS-1 is a much more
effective PARACEST agent than EuS-RRRR-1.
Experimental Section
General remarks: All solvents and reagents were purchased from com-
mercial sources and used as received except where noted. ꢁWaterꢂ refers
to deionized water with a resistivity of <18.2 MWcm. 1H and 13C NMR
spectra were acquired on a JOEL Eclipse 270 spectrometer operating at
270.17 and 67.94 MHz, respectively. CEST spectra were acquired on a
Bruker Avance IIa 400 spectrometer operating at 400.13 MHz. Infrared
spectra were recorded on a Nicolet Avatar 360 FTIR spectrophotometer.
Melting points were recorded on a Fisher–Johns melting point apparatus
and are uncorrected. RP-HPLC purification was performed on a Waters
d-prep HPLC system using a 30ꢃ250 mm Phenomenex Luna C18(2)
column. In all cases ligands were purified by eluting for 5 min of 100%
water (0.037% HCl) followed by a linear gradient over 35 min to 40%
acetonitrile and 60% water (0.037% HCl) at a flow rate of 30 mLminꢀ1
.
Chelates were purified by eluting for 5 min with 100% water (0.037%
HCl) followed by a linear gradient over 35 min to 10% acetonitrile and
90% water (0.037% HCl) at a flow rate of 30 mLminꢀ1. Elution of prod-
ucts was monitored by UV absorbance at 210 and 270 nm.
(R)-N-(Ethyl-2-propionate) bromoacetamide (2): To a solution of bro-
moacetyl bromide (2.40 mL, 26.3 mmol) in dichloromethane (100 mL)
was added potassium carbonate (6.04 g, 43.8 mmol) and the resulting sus-
pension was cooled to 08C. A solution of (R)-alanine ethyl ester (2.57 g,
21.9 mmol) in dichloromethane (50 mL) was added dropwise over a
period of 20 min. The mixture was stirred for a further 30 min at 08C and
18 more hours at room temperature. Water (30 mL) was added cautiously
and the two layers separated. The aqueous phase was extracted with di-
chloromethane (50 mL), the organic phases were combined, dried
(Na2SO4), and the solvents removed under reduced pressure. The residue
was taken up into a minimum of dichloromethane and recrystallized by
addition ethyl ether and hexanes at ꢀ208C to afford a colorless solid
(1.98 g 38%). M.p. 66–678C; 1H NMR (CDCl3, 270 MHz): d=7.13 (s br,
1H, NH), 4.52 (dq, 3JHH =7 Hz, 3JHH =7 Hz, 1H, NHCHCH3), 4.26 (q,
3JHH =8 Hz, 2H, OCH2CH3), 3.82 (s, 2H, BrCH2), 1.45 (d, 3JHH =7 Hz,
Although two conformational isomers (pseudo-axial/equa-
torial) have been postulated previously[11] the two diastereo-
isomeric chelates of Eu1 have for the first time afforded an
opportunity to examine the CEST properties of each. The
S-SSSS (pseudo-axial) isomer is a significantly more effec-
tive PARACEST agent possibly because it closely holds
more than one water molecule in a highly shifted pool. In
H
such a situation the value of tM reflects all protons in this
pool, it is therefore difficult to understand exactly how the
exchange kinetics of these chelates modulate CEST.
These results demonstrate, for the first time, that it may
be possible to design PARACEST agents that have detec-
tion limits for MRI comparable to current MRI contrast
agents. In Figure 4 EuS-SSSS-1 generates about 10% CEST
at an overall chelate concentration of 0.274 mm. However,
only about 5% of the chelate is responsible for this CEST
so the active concentration is just 13.7 mm, but these meas-
urements are in only 10% water in acetonitrile. Assuming a
3
3H, CHCH3), 1.27 ppm (t, JHH =8 Hz, 3H, CH2CH3); 13C NMR (CDCl3,
68 MHz): d=171.6 (C=O), 164.5 (NHC=O), 60.1 (OCH2), 47.8 (BrCH2),
27.6 (CH), 17.3 (OCH2CH3), 13.4 ppm (CHCH3); nmax (KBr disc)=3743
(NH), 1738 (CO2), 1647 cmꢀ1 (NHC=O); m/z (ESMS EIꢀ): 236 (100%,
[MꢀH+]ꢀ), appropriate isotope patterns were observed.
(S)-N-(Benzyl-2-propionate) bromoacetamide (3): l-Alanine benzyl ester
hydrochloride (4.0 g, 18.5 mmol) was dissolved in water (20 mL) and
sodium carbonate added until the solution pH exceeded 12. The solution
was then extracted with dichloromethane (2ꢃ200 mL). The organic ex-
10376
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10372 – 10378

d-Substitution of LanthanideACHTNUTRGNE(NUG III) NB-DOTA-Tetraamide Chelates
FULL PAPER
tracts were combined, dried (Na2SO4), and the solvents were removed
under reduced pressure. The residue was taken up into dichloromethane
(50 mL) and the resulting solution added dropwise to a solution of bro-
moacetyl bromide (2.0 mL, 22.2 mmol) in dichloromethane (100 mL)
containing potassium carbonate (5.11 g, 37.0 mmol) and cooled to 08C in
an ice-bath. Addition was carried out over a period of 20 min and the re-
action stirred for a further 30 min at 08C. The reaction mixture was al-
lowed to warm to room temperature and stirred for 18 h at ambient tem-
perature. Water (30 mL) was added cautiously and the two layers sepa-
rated. The aqueous phase was extracted with dichloromethane (50 mL),
the organic phases were combined, dried (Na2SO4), and the solvents re-
moved under reduced pressure. The residue was taken up into a mini-
mum of dichloromethane and recrystallized by addition ethyl ether and
hexanes at ꢀ208C to afford a colorless solid (3.44 g, 62%). M.p. 59–
618C; 1H NMR (CDCl3, 270 MHz): d=7.34 (m, 5H, Ar), 7.06 (s br, 1H,
NH), 5.12 (d, JHH =10 Hz, 1H, aaꢂ system, ArCH2) 5.11 (d, JHH =10 Hz,
1H, aaꢂ system, ArCH2), 4.6 (dq, 3JHH =7 Hz, 3JHH =7 Hz, 1H,
NHCHCH3), 3.82 (s, 2H, BrCH2), 1.47 ppm (d, 3JHH =7 Hz, 3H, CH3);
13C NMR (CDCl3, 68 MHz): d=172.4 (CO2), 165.6 (NHC=O), 135.2
(Ar), 129.9 (Ar), 129.1 (Ar), 128.4 (Ar), 67.7 (ArCH2), 49.2 (BrCH2),
29.4 (CH), 18.7 ppm (CH3); nmax (KBr disc): 1734 (CO2), 1717 (CO2),
1653 cmꢀ1 (NHC=O); m/z (ESMS EIꢀ): 298 (100%, [MꢀH+]ꢀ) an ap-
propriate isotope pattern was observed.
CH2C=O, CH2Ar, CHCH3), 1.28 ppm (m, 12H, CHCH3); m/z (ESMS
EI+): 824 (100%, [M+H+]+); nmax (iTR): 3169 (NH), 1727 (CO2), 1617
(C=ONH), 1455, 1346, 1253, 1155, 957, 821, 751 cmꢀ1
.
General procedure for the preparation of Ln3+ chelates: Europium tri-
flate (15 mg, 24.6 mmol) and 1 (20 mg, 22.3 mmol) were dissolved in water
and the pH adjusted to 6 by addition of NaOH (1m aqueous solution).
The resulting solution was heated at 60 C for 24 h and then allowed to
cool to room temperature. After filtration through a 20 mm syringe filter
the reaction mixed was injected directly onto a preparative HPLC system
for purification. Chelates were obtained by lyophilization of the fraction
containing the major reaction product.
Praseodymium
ACHTUNGTREN(NUNG III) (1R, 4R, 7R, 10R)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-
(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetate-
A
(100%, [MꢀH+]ꢀ) an appropriate isotope pattern was observed.
2
2
EuropiumACTHUNRTGNEUNG(III) (1R, 4R, 7R, 10R)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-ni-
trobenzyl)-1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetateacetamide
(HEuS-RRRR-1): RT =24.96 min; m/z (ESMS EIꢀ): 975 (100%,
[MꢀH+]ꢀ) an appropriate isotope pattern was observed.
YtterbiumACTHNUTRGNEU(GN III) (1R, 4R, 7R, 10R)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-ni-
trobenzyl)-1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetateacetamide
(HYbS-RRRR-1): RT =31.31 min; m/z (ESMS EIꢀ): 996 (100%,
[MꢀH+]ꢀ) an appropriate isotope pattern was observed.
(1R, 4R, 7R, 10R)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-nitrobenzyl)-
Praseodymium
nitrobenzyl)-1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetateacet-
amide (HPrS-SSSS-1): RT =25.33 min; m/z (ESMS EIꢀ): 963 (100%,
[MꢀH+]ꢀ) an appropriate isotope pattern was observed.
Europium(III) (1S, 4S, 7S, 10S)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-nitro-
ACHTUNGERTN(NUNG III) (1S, 4S, 7S, 10S)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-
1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetateacetamide
RRRR-1): 2-S-(p-Nitrobenzyl) cyclen (150 mg, 0.49 mmol) and the bro-
moacetamide (581 mg, 2.44 mmol) were dissolved in acetonitrile
(H4S-
AHCTUNGTRENNUNG
2
AHCTUNGTRENNUNG
(30 mL), and potassium carbonate (337 mg, 2.44 mmol) was added. The
resulting suspension was heated under reflux with stirring for seven days.
The solvents were then removed under reduced pressure and the residue
taken up into water (20 mL) and dichloromethane (100 mL). The two
layers were separated and the aqueous phase extracted with dichlorome-
thane (2ꢃ50 mL). The organic phases were combined, dried (Na2SO4),
and the solvents removed under reduced pressure. The residue was taken
up into tetrahydrofuran (5 mL) and water (10 mL) and a 1m sodium hy-
droxide solution (2.4 mL) added. The resulting monophasic solution was
heated to 608C for 48 h. The solvents were removed under reduced pres-
sure and the residue taken up into water and the pH adjusted to 7 with
HCl prior to RP-HPLC purification. After lyophilization of the solvents
the dihydrochloride salt of title compound was obtained as a colorless
solid (147 mg, 30%). RT =27.79 min; m.p.>1708C; 1H NMR (D2O,
270 MHz): d=8.11 (d, 3JHH =8 Hz, 2H, Ar), 7.40 (d, 3JHH =8 Hz, 2H,
Ar), 2.66–4.43 (m br, 29H, ring NCH2, CH2C=O, CH2Ar, CHCH3),
1.31 ppm (m, 12H, CHCH3); m/z (ESMS EI+): 824 (100%, [M+H+]);
nmax (iTR): 3023 (NH), 1733 (CO2), 1656 (C=ONH), 1546, 1519, 1454,
AHCTUNGTRENNUNG
benzyl)-1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetateacetamide
(HEuS-SSSS-1): RT =30.24 min; m/z (ESMS EIꢀ): 975 (100%,
A
YtterbiumACTHUNRTGNEUNG(III) (1S, 4S, 7S, 10S)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-nitro-
benzyl)-1,4,7,10-tetraazacyclododecane]-1,4,7,10-tetraacetateacetamide
(HYbS-SSSS-1): RT =20.45 min; m/z (ESMS EIꢀ): 996 (100%,
A
CEST fitting: CEST spectra were recorded at six different B1 powers for
each chelate: 42.7 mT, 36.3 mT, 34.3 mT, 30.1 mT, 27.4 mT, and 24.3 mT (see
Figure S1 in the Supporting Information). The water proton exchange ki-
netics for each SAP isomer were determined by plotting the magnetiza-
tion at the CEST peak minimum as a function of the initial magnetiza-
tion (M /
N
1346, 1210, 1157, 943, 735 cmꢀ1
.
The CEST spectra were then fitted to the Bloch equations modified for
exchange as previously described.[28] A 2-pool model was used to fit the
data for EuS-RRRR-1 representing the bulk water pool, the bound or
shifted water pool. The inclusion of a pool for the amide protons was
found not to affect the fitting and therefore excluded from the all fitting
models. A 3-pool model was used to fit the data for EuS-RRRR-1 repre-
senting the bulk water, the bound water or shifted water associated with
the SAP isomer and the bound or shifted water associated with the
TSAP isomer.
(1S, 4S, 7S, 10S)-d, d’, d’’, d’’’-Tetramethyl-[2-(S)-(p-nitrobenzyl)-1,4,7,10-
tetraazacyclododecane]-1,4,7,10-tetraacetateacetamide (H4S-SSSS-1): 2-
S-(p-Nitrobenzyl) cyclen (250 mg, 0.81 mmol) and the bromoacetamide 3
(1.22 g, 4.07 mmol) were dissolved in acetonitrile (30 mL), and potassium
carbonate (562 mg, 4.07 mmol) was added. The resulting suspension was
heated under reflux with stirring for seven days. The solvents were then
removed under reduced pressure and the residue taken up into water
(20 mL) and dichloromethane (100 mL). The two layers were separated
and the aqueous phase extracted with dichloromethane (2ꢃ50 mL). The
organic phases were combined, dried (Na2SO4), and the solvents removed
under reduced pressure. The residue was taken up into dry chloroform
(10 mL); 5 mL of the resulting solution was transferred to a reaction
flask which was purged with argon. The reaction mixture was cooled to
ꢀ108C in a salt/ice-bath, and a 1m solution of BCl3 in dichloromethane
(1.6 mL) was added. After addition the reaction mixture was allowed to
warm to ambient temperature and was then stirred for 14 h. Water
(2 mL) was added dropwise to quench the reaction, followed by 1m HCl
(8 mL) and the ligand was extracted into the aqueous phase. The ligand
solution was used directly to purify the title compound by RP-HPLC.
After lyophilization of the solvents the dihydrochloride salt of title com-
pound was obtained as a colorless solid (78.4 mg, 20%). RT =25.94 min;
m.p.>1708C; 1H NMR (D2O, 270 MHz): d=8.21 (d, 3JHH =8 Hz, 2H,
Ar), 7.42 (d, 3JHH =8 Hz, 2H, Ar), 2.72–4.31 (m br, 29H, ring NCH2,
The pre-saturation power (B1) was held fixed during fitting. The water
proton exchange lifetime (tMH) was constrained to lie within 10% of the
value determined by the w-fitting. In initial fittings the concentration of
each pool was deconstrained to reflect the confidence level of the con-
centration determination. T1 values for the bulk water pool were deter-
mined experimentally using an inversion recovery pulse sequence and
held fixed during fitting. T1 values for the other pools could not be deter-
mined by direct measurement owing to the low chelate concentrations of
each sample. The T1s of these pools and all T2 values were allowed to
float over the range of values previously found in CEST fitting routines
H
in EuDOTA-tetramide chelates.[31] The value of tM for the TSAP
isomer, which does appear to influence the shape of the direct saturation
peak at 0 ppm (witness the downfield edge of the peak), was constrained
H
to have a ratio of between 1/40 and 1/60 of the tM value of the SAP
Chem. Eur. J. 2011, 17, 10372 – 10378
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10377

M. Woods et al.
isomer. This ratio is consistent with previously reported behavioral pat-
terns in this type of chelate.
[14] S. Zhang, C. R. Malloy, A. D. Sherry, J. Am. Chem. Soc. 2005, 127,
17572.
[15] M. Woods, P. Caravan, C. F. G. C. Geraldes, M. T. Greenfield, G. E.
Kiefer, M. Lin, K. McMillan, M. I. M. Prata, A. C. Santos, X. Sun, J.
Wang, S. Zhang, P. Zhao, A. D. Sherry, Invest. Radiol. 2008, 43, 861.
[16] A. D. Sherry, P. Caravan, R. E. Lenkinski, J. Magn. Reson. Imaging
2009, 30, 1240.
[17] U. Schmidt, M. Kroner, H. Griesser, Synthesis 1991, 294.
[18] S. Aime, M. Botta, M. Fasano, M. P. M. Marques, C. F. G. C. Ger-
aldes, D. Pubanz, A. E. Merbach, Inorg. Chem. 1997, 36, 2059.
[19] J. Vipond, M. Woods, P. Zhao, G. Tircso, J. M. Ren, S. G. Bott, D.
Ogrin, G. E. Kiefer, Z. Kovacs, A. D. Sherry, Inorg. Chem. 2007, 46,
2584.
[20] T. Mani, G. Tircso, P. Zhao, A. D. Sherry, M. Woods, Inorg. Chem.
2009, 48, 10338.
[21] R. S. Dickins, A. S. Batsanov, J. A. K. Howard, D. Parker, H. Pusch-
mann, S. Salamano, Dalton Trans. 2004, 70.
[22] R. S. Dickins, J. A. K. Howard, J. M. Moloney, D. Parker, R. D. Pea-
cock, G. Siligardi, Chem. Commun. 1997, 1747.
Fitting the CEST data acquired at B1 =1282.8 Hz afforded a good fit to
the data for EuS-RRRR-1. However, a suitable fit to the S-SSSS-isomer
could not be obtained when the concentration was constrained as de-
scribed (see Figure S2 in the Supporting Information). Only when the
concentration of the highly shifted pool is allowed to float and fit to
values 3.7 times higher than the 13.7 mm chelate concentration is an ade-
quate fit achieved.
Acknowledgements
The authors acknowledge grant support from the National Institutes of
Health (EB-11687 (M.W.); RR-02584 and CA-115531 (A.D.S.)); ONAMI
(N00014–11–1–0193), the M. J. Murdock Charitable Trust and the Robert
A Welch Foundation (AT-584).
[23] R. S. Dickins, J. A. K. Howard, C. W. Lehmann, J. Moloney, D.
Parker, R. D. Peacock, Angew. Chem. 1997, 109, 541; Angew. Chem.
Int. Ed. Engl. 1997, 36, 521.
[24] R. S. Dickins, J. A. K. Howard, C. L. Maupin, J. M. Moloney, D.
Parker, J. P. Riehl, G. Siligardi, J. A. G. Williams, Chem. Eur. J. 1999,
5, 1095.
[25] R. S. Dickins, S. Aime, A. S. Batsanov, A. Beeby, M. Botta, J. I.
Bruce, J. A. K. Howard, C. S. Love, D. Parker, R. D. Peacock, H.
Puschmann, J. Am. Chem. Soc. 2002, 124, 12697.
[26] T. Gunnlaugsson, R. J. H. Davies, M. Nieuwenhuyzen, J. E. OꢂBrien,
C. S. Stevenson, S. Mulready, Polyhedron 2003, 22, 711.
[27] A. L. Thompson, D. Parker, D. A. Fulton, J. A. K. Howard, S. U.
Pandya, H. Puschmann, K. Senanayake, P. A. Stenson, A. Badari,
M. Botta, S. Avedano, S. Aime, Dalton Trans. 2006, 5605.
[28] D. E. Woessner, S. Zhang, M. E. Merritt, A. D. Sherry, Magn. Reson.
Med. 2005, 53, 790.
[29] W. T. Dixon, J. Ren, A. J. M. Lubag, J. Ratnakar, E. Vinogradov, I.
Hancu, R. E. Lenkinski, A. D. Sherry, Magn. Reson. Med. 2010, 63,
625.
[30] Y. Wu, Y. Zhou, O. Ouari, M. Woods, P. Zhao, T. C. Soesbe, G. E.
Kiefer, A. D. Sherry, J. Am. Chem. Soc. 2008, 130, 13854.
[31] M. Woods, D. E. Woessner, P. Zhao, A. Pasha, M.-Y. Yang, C.-H.
Huang, O. Vasalitiy, J. R. Morrow, A. D. Sherry, J. Am. Chem. Soc.
2006, 128, 10155.
[1] M. Woods, Z. Kovacs, S. Zhang, A. D. Sherry, Angew. Chem. 2003,
115, 6069; Angew. Chem. Int. Ed. 2003, 42, 5889.
[2] M. Woods, M. Botta, S. Avedano, J. Wang, A. D. Sherry, Dalton
Trans. 2005, 3829.
[3] S. Aime, M. Botta, G. Ermondi, Inorg. Chem. 1992, 31, 4291.
[4] S. Hoeft, K. Roth, Chem. Ber. 1993, 126, 869.
[5] S. Aime, A. Barge, J. I. Bruce, M. Botta, J. A. K. Howard, J. M. Mo-
loney, D. Parker, A. S. de Sousa, M. Woods, J. Am. Chem. Soc. 1999,
121, 5762.
[6] F. A. Dunand, S. Aime, A. E. Merbach, J. Am. Chem. Soc. 2000,
122, 1506.
[7] M. Woods, S. Aime, M. Botta, J. A. K. Howard, J. M. Moloney, M.
Navet, D. Parker, M. Port, O. Rousseaux, J. Am. Chem. Soc. 2000,
122, 9781.
[8] M. Woods, D. E. Woessner, A. D. Sherry, Chem. Soc. Rev. 2006, 35,
500.
[9] S. Zhang, P. Winter, K. Wu, A. D. Sherry, J. Am. Chem. Soc. 2001,
123, 1517.
[10] K. J. Miller, A. A. Saherwala, B. C. Webber, Y. Wu, A. D. Sherry, M.
Woods, Inorg. Chem. 2010, 49, 8662.
[11] S. Aime, M. Botta, R. S. Dickins, C. L. Maupin, D. Parker, J. P.
Riehl, J. A. G. Williams, J. Chem. Soc. Dalton Trans. 1998, 881.
[12] Z. Baranyai, E. Brꢄcher, T. Ivanyi, R. Kiraly, I. Lazar, L. Zekany,
Helv. Chim. Acta 2005, 88, 604.
[13] E. Terreno, D. Castelli Daniela, G. Cravotto, L. Milone, S. Aime,
Invest. Radiol. 2004, 39, 235.
Received: April 1, 2011
Published online: August 11, 2011
10378
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10372 – 10378