MRI ParaCEST agents that improve amide based pH measurements by limiting inner sphere water T2 exchange

Article abstract of DOI:10.1039/c3ra43537g

A series of Dy³⁺ and Tm³⁺ tetra-substituted DOTAM paraCEST agents incorporating para-substituted anilines has been synthesized and their paraCEST and relaxation properties evaluated. The response of selected agents (aniline and p-met

Full text of DOI:10.1039/c3ra43537g

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MRI ParaCEST agents that improve amide based pH
measurements by limiting inner sphere water T2
exchange†
Cite this: RSC Adv., 2014, 4, 1666
Mark Milne,a Melissa Lewis,a Nevin McVicar,b Mojmir Suchy,ab Robert Barthab
a
*
and Robert H. E. Hudson
A series of Dy3+ and Tm3+ tetra-substituted DOTAM paraCEST agents incorporating para-substituted
anilines has been synthesized and their paraCEST and relaxation properties evaluated. The response of
selected agents (aniline and p-methoxyaniline) to changes in pH near the physiological range has also
been studied. A crystal structure of Tm3+-p-OMe agent indicates a TSAP geometry with the absence of
an inner sphere water molecule (denoted as TSAP’), which is thought to be due to the bulky nature of
the amide nitrogen substituent. Two distinct amide signals are observed in the CEST spectrum for this
compound, one for each of the isomers that exist in solution: SAP and TSAP with the larger chemical
shift centred at ꢀ83 ppm compared to bulk water. Due to the non-optimal inner sphere water
exchange, these agents produce minimal shortening of T1 and T2 relaxation time constants and therefore
decrease relaxation induced signal losses during imaging.
Received 11th July 2013
Accepted 28th October 2013
DOI: 10.1039/c3ra43537g
www.rsc.org/advances
A promising technique for the in vivo measurement of
tissue environmental conditions is the use of chemical
Introduction
exchange saturation transfer (CEST) or paraCEST, when a
paramagnetic species is used as a hyperne shi agent.9–13
The CEST and paraCEST techniques rely on the exchange of
shied protons to transfer energy and create an overall
decrease in the bulk water magnetization.14–16 The exchange-
Lanthanide based contrast agents (CAs) have been exploited for
a number of years in magnetic resonance imaging (MRI) with
coordinated gadolinium (Gd3+) agents being used primarily in
clinical environments.1–5 Of these agents cyclen-based macro-
cyclic agents have been predominantly utilized as chelators
because of the high stability of the resulting metal complexes
under in vivo conditions.
It has been shown that cyclen-based agents can adopt two
main geometries in solution. The rst geometry is a square
antiprismatic (SAP) while the second is a twisted square anti-
prismatic (TSAP).6,7 In solution, these geometries can be iden-
tied by the chemical shis of the cyclen backbone with their
interconversion measured by EXSY NMR experiments. In solid
state, the two geometries can be distinguished by the angle
between planes of N–Ln–N and O–Ln–O. When the angle
between the aforementioned planes is ꢁ40ꢂ, the structure is
assigned to the SAP form, while angles closer to 30ꢂ are assigned
the TSAP form. Of these two forms, it has been shown that the
TSAP geometry possesses a faster exchange of bound water
compared to the SAP form, which is thought to occur because of
the more open structure TSAP displays.8
able proton sources are usually bound water as seen in Eu3+
-
containing paraCEST agents or protons associated amide,
amine or alcohol functional groups in either diamagnetic
CEST agents or in lanthanide paraCEST complexes.17–20
Because these agents generate contrast following the transfer
of magnetization by the exchangeable protons, the contrast
can be directly related to the environment around the agent if
it modies the exchange rate. For example, temperature and
pH both modulate the proton exchange rate, particularly when
using the highly shied paraCEST agents that utilize Eu3+
,
Dy3+, Tb3+ or Tm3+ ions.21,22 While there have been numerous
agents that are capable of reporting pH and temperature,13,23–27
some of these agents either do not function at physiological
temperatures, or their optimal pH response is above 7, which
is beyond the physiological range of interest. These drawbacks
demonstrate the need to design agents that are responsive to
the physiological parameters of interest, most notably when
the tissue is slightly acidic (pH 6–7) and at temperatures
between 35 ꢂC and 45 ꢂC. To address these issues, we have
synthesized a series of Dy3+ and Tm3+ DOTAM anilides that are
para-substituted, which should allow for the modulation of
the amide’s pKa based on the electron donating/withdrawing
nature of the substituent (Fig. 1).
aDepartment of Chemistry, The University of Western Ontario, London, Ontario N6A
5B7, Canada. E-mail: robert.hudson@uwo.ca; Fax: +1-519-661-3022
bRobarts Research Institute, Department of Medical Biophysics, The University of
Western Ontario, London, Ontario, N6A 5C1, Canada
† Electronic supplementary information (ESI) available: CEST images of Tm3+
DOTAM-Gly-Lys, Tm3+-para-OMe, 1H and 13C NMR spectra of electrophiles and
tetra analide compounds. See DOI: 10.1039/c3ra43537g
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Fig. 1 Lanthanide-based CAs synthesised.
Although paraCEST agents are generally inefficient at
inducing T1 relaxation, such agents are capable of producing
signicant signal loss during in vivo imaging,28,29 due to
increased transverse relaxation mediated by water exchange (T2
exchange) from the inner sphere to the bulk water. The use of
ultra-short echo-time (UTE) pulse sequences can reduce this
effect, as observed with the SWIFT pulse sequence when used
for paraCEST imaging.30 However, such sequences are not
standard on most clinical scanners limiting their applicability.
Therefore, we hypothesized that a paraCEST agent designed to
reduce T2 signal losses would therefore be of benet. To this
end, we have developed agents that either do not contain inner
sphere water or their water exchange is outside the optimal
exchange rate regime and therefore are inefficient modulators
to the bulk water T2 relaxation time constant. It must be noted
that the amides that generate the CEST effect will still induce T2
relaxation, but to a much less extent compared to bound water.
Similar studies have been performed to monitor and adjust
the water exchange rate on a series of Eu3+-based ParaCEST
agents.31 While those results demonstrate the ability to modu-
late the bound water exchange rate based on the electron
donating and withdrawing properties of the substituents, the
effect of para-substitution on amide proton exchange of anilides
was not studied. The use of Eu3+ as the lanthanide limits the
ability to monitor how the amide is affected by the substitutions
because the amides will exhibit a small shi compared to that
induced by other lanthanides such as Dy3+, Tm3+ or Tb3+.
Scheme 1 Conditions for the synthesis of lanthanide-based CAs. (i)
Acetonitrile, K2CO3, room temperature, 18 h. (ii) Acetonitrile, K2CO3,
55–80 ꢂC, 2–3 days. (iii) 1 : 1H2O : dioxane, DyCl3 or TmCl3, 60–80 ꢂC,
4 days.
reactions was monitored by UPLC MS. When the reaction was
deemed complete, the mixtures were cooled and the product
was allowed to precipitate along with the K2CO3. The mixture
was then ltered and the precipitate was washed with cold water
to remove the K2CO3. At this point, attempts to recrystallize
these compounds from methanol were unsuccessful due to
their solubility properties. As such, the crude products were
used without further purication. This procedure worked well
except for products 2d,e which showed the presence of some
minor impurities by 1H NMR (ESI†). It is our belief that the MR
properties described are not affected by the presence of impu-
rities. Crude yields ranged from of 94% to quantitative. The
Results and discussion
Synthesis of agents 3a–e, 4a–e
The electrophiles 1a–e were synthesised by adding chloroacetyl tetra-substituted cyclen products were then metallated using
chloride to the appropriate aniline in acetonitrile while cooled either DyCl3 or TmCl3 in a 1 : 1 water : dioxane mixture at 80 ꢂC
in an ice bath in the presence of potassium carbonate over 4 days. The metallated complexes were subjected to dialysis
(Scheme 1). The reactions were stirred overnight at room against water across a membrane with a 500 Dalton molecular
temperature, then ltered and the crude products were recrys- weight cutoff to remove salts carried through from the previous
tallized from methanol. The average yield of these reactions was step as well as unchelated lanthanide ion. The nal products
good (ꢁ75%). Syntheses of the tetra-substituted cyclens were were isolated and lyophilized to give white powders, with
performed by mixing cyclen with 4.5 eq of the appropriate exception of the p-nitro compound being a yellow powder.
electrophiles in acetonitrile in the presence of potassium Identication of the metallated products was accomplished by
carbonate. It was necessary to heat the reaction at 55–80 ꢂC over high resolution mass spectrometry along with their UPLC trace
1–3 days to ensure complete tetraalkylation as the nal products which showed all of the samples to be homogeneous tetra-
were found to have limited solubility. The progress of these substituted product.
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while the signal less shied is closer to the normally observed
SAP geometry amide.11,32 This feature appears to be limited to
Tm3+ agents and could be due to the smaller lanthanide size
compared to the Dy3+, thus exacerbating steric interactions. The
CEST spectra as a function of pH for 3a and 3b are shown in
Fig. 2, with the CEST response for each of the 4 agents measured
at pH 7.0 reported in Table 1.
Fig. 3 shows how the CEST effect of agents 3a and 3b changes
over the pH ranges measured. The signal for the Tm3+-para-OMe
TSAP amide (ꢀ83 ppm) has a maximum signal at a slightly more
acidic pH (7.0) compared to the SAP (ꢀ43 ppm) amide (pH 7.5).
This effect has been observed previously for DOTAM agents
where the signal possessing the greater chemical shi displays a
maximum signal at lower pH compared to the signal with a
smaller chemical shi.33
CEST evaluation
To study the effect of the para-substitution on the amide
exchange rate of the Tm3+ and Dy3+ DOTAM anilides (3a–e, 4a–
e), we began with a pH study of the series at 20 mM. Quickly, we
discovered many of the compounds were not sufficiently soluble
to achieve this concentration, except for the unsubstituted
anilides (3a, 4a) and the para-OMe anilides (3b, 4b). Unfortu-
nately, the rest of the series (3c–e, 4c–e) was not soluble enough
to permit collection of reliable CEST spectra even at 5 mM. It is
commonly reported in literature to use an organic co-solvent,
such as acetonitrile, to increase CA solubility. However, we
choose not to used a mixed-solvent system due to the difficulty
in evaluating the true pH under such conditions. For the soluble
3a,b and 4a,b complexes, CEST experiments were performed by
applying a 15 mT, 5 s saturation pulse at frequencies between
ꢀ100 ppm and 100 pm in 1 ppm steps. Solutions were tested in
the pH range of 6.5 to 9.0 for the unsubstituted anilides and the
range of 5.1 to 8.0 for the para-OMe anilides. These ranges were
chosen because of the observation that the maximum CEST
effect was close to 8.0 for the unsubstituted anilides while the
maximum CEST effect was between 7.0 and 7.5 for the para-
OMe anilides. Of interest, we observed that the Tm3+ para-OMe
(3b) compound displayed two CEST signals due to the slow
conformational equilibrium between the SAP and the TSAP
geometries while the Dy3+ agent (4b) produced only one
measurable signal. We have previously observed this feature
during the study of a series of alkyl amides and designated the
rarely seen higher shied amide signal to a TSAP geometry
An effective MRI CA for pH measurement must show a
monotonic response to pH and must provide a concentration
Table 1 CEST % measured at pH 7, 37 ꢂC, 20 mM with a 15 mT, 5 s
continuous wave saturation pulse
Agent
ppm (d)
CEST %
3a
3b
ꢀ41
ꢀ43
ꢀ83
70
12%
17%
12%
6%
4a
4b
74
3%
Fig. 2 (Top) CEST spectra of 3a over the pH range of 6.5–9.0. Fig. 3 Maximum CEST effect as a function of pH. (Top) 3a shows a pH
(Bottom) CEST spectra of 3b over the pH range of 5.1–7.0. CEST maximum at 8.0. (Bottom) 3b shows a pH maximum at 7.5 for the
spectra were acquired at 37 ꢂC, 15 uT, 5 s, 20 mM.
signal at ꢀ43 ppm and at 7.0 for the signal at ꢀ83 ppm. CAs at 20 mM.
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independent measurement. Both conditions are violated by
these agents, although the rst issue can be minimized by
rationalizing that it would be rare to observe a pH above 8
in vivo. One method to overcome the concentration dependence
is illustrated in Fig. 4. Using a line width analysis, Tm3+ p-H (3a)
can be used as a pH reporter with a monotonic response to pH
over the range of 6.5 to 8.5 by following the procedure described
in McVicar et al.13
Another approach to overcome concentration dependence is
to implement a ratiometric approach as previously described,
where two intramolecular signals are evaluated simulta-
neously.23,34 In particular, when two CEST responses arise from a
single agent a ratio of the CEST effect eliminates the concentra-
tion dependence and enables the agent to be used as a physio-
logical reporter. Since Tm3+-para-OMe possesses two
intramolecular amide signals that behave slightly different based
on pH, we examined the possibility of using it as a ratiometric pH
reporter. However, the small pH responses and limited solubility
of the Tm3+-para-OMe agent frustrated attempts to demonstrate
how this agent could be used as a concentration independent pH
reporter (data not shown). We are currently developing synthetic
avenues to make this agent more pH responsive, as well as
increase its solubility to extend its functionality.
Fig. 5 Molecular representation of the solid state structure of Tm3+
-
para-OMe (3b) determined by single crystal X-ray studies. Hydrogens
have been omitted for clarity. (Left) Side on view of 3b showing
eightfold coordination of the Tm3+ by the nitrogens of cyclen and the
oxygens of the amide pendant groups. 4 denotes the trans O–Ln–O
bond angle. (Right) Top down view of 3b. The aniline rings are omitted
for clarity. a indicates the angle created between the planes of N–Ln–
N and O–Ln–O and is listed in Table 2 along with selected angles and
bond lengths.
coordinate the metal centre. This distinction has been observed
before for lanthanide based CAs and has been denoted by
ascribing a prime (0) to the end of the geometry abbrevia-
tion.7,35–38 It should be noted that a lack of inner sphere water in
the solid state does not reect the solvation environment in
solution where a metal bound water may be present.
For an agent to have space to occupy the ninth site with a
bound water there needs to be an angle at the metal center (4,
trans O–Ln–O) of at least 135ꢂ.38,39 With an average angle of 123ꢂ
(Table 2) it is not surprising that water is not observed coordi-
nated to the metal center in the crystal structure. The steric
crowding of the metal center is exacerbated due to the rigid nature
of the phenyl rings that are enforced into an northern orientation
due to the thermodynamically favoured s-trans amide geometry.
The a angle created between the planes of N–Ln–N and O–
Ln–O is used in determining which geometry is present. Angles
that near 40ꢂ are in a SAP geometry while angles that are closer
X-ray structures
Crystals suitable for X-ray diffraction were grown from slow
evaporation of a concentrated solution of Tm3+-para-OMe (3b)
in water. Fig. 5 illustrates a molecular representation of 3b at
50% probability for the ellipsoids. It has been shown that
macrocyclic CAs based on cyclen can exhibit a 9 coordination
geometry around the lanthanide, where 8 of the coordination
sites are lled with 4 nitrogens of the cyclen and 4 oxygens from
the carbonyls pendant groups. The ninth coordination site is
typically occupied by a water molecule that can be exchanged
with the bulk water system and can be used to enhance relax-
ation or transfer saturation to the bulk system.
X-ray crystallography determined an 8 coordination system
around the Tm3+ ion, with the 8 sites lled by either nitrogen
atoms or oxygen atoms. The ninth potential coordination site
was unoccupied – no bound water was found to directly
Table 2 Summary of torsion angle a (ꢂ) and selected bond lengths (A˚)
for Tm3+ para-OMe (3b)
Average a angle
4 angle O1–Tm–O3
4 angle O2–Tm–O4
N1–C–C–O1
N2–C–C–O2
N3–C–C–O3
N4–C–C–O4
N1–Tm
N2–Tm
N3–Tm
N4–Tm
O1–Tm
O2–Tm
O3–Tm
O4–Tm
Centeroid O4–Tm
Centeroid N4–Tm
Centeroid O4–centeroid N4
26.95
123.64(7)
122.43(8)
12.4(3)
15.1(4)
14.7(4)
2.9(3)
2.508(2)
2.492(2)
2.469(3)
2.500(3)
2.296(2)
2.245(2)
2.313(2)
2.265(2)
1.087
Fig. 4 Line width analysis vs. pH of a 20 mM solution of Tm3+ p-H (3a)
at 37 ꢂC, using 15 mT, 5 s saturation.
1.382
2.470
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to 30ꢂ are in the TSAP geometry.40 In the case of the Tm3+ para-
OMe, the observed solid-state geometry is TSAP’ system with an
a angle of 27ꢂ and lacking inner sphere water. The distance
between the centroids of the O4 and N4 planes is also indicative
Table 3 Crystallographic information for 3b
Formula
C
44H67Cl3N8O13Tm
Formula weight (g molꢀ1
)
1191.34
Crystal dimensions (mm)
Crystal colour and habit
Crystal system
0.27 ꢃ 0.18 ꢃ 0.08
Colourless prism
Triclinic
ꢀ
P1
110
10.948(3)
14.438(3)
17.436(5)
77.345(10)
78.428(9)
85.152(10)
2632.1(12)
˚
of the geometry of the crystal. With a distance of 2.47 A, this
crystal is comparable to other TSAP structures whereas the
38,41
Space group
˚
typical distance in SAP structures is smaller (ꢁ2.35 A).
The
Temperature, K
distance between the centeroid of the 4 nitrogen atoms of cyclen
˚
a, A
˚
and the metal center is 1.38 A, which is shorter then a series of
˚
b, A
previously reported SAP DOTAM complexs (Nd3+ ¼ 1.66 A,
˚
˚
c, A
3+
3+
Dy3+ ¼ 1.59 A, Tb ¼ 1.59 A, Yb ¼ 1.56 A).42 By chelating the
metal lower into the cyclen ring the 4 carbonyls tend to wrap
overtop the metal effectively limiting water access giving rise to
the low 4 angle of 123ꢂ previously discussed.37,38 It is possible the
compacting of the overall structure results in a highly shied
amide signal as the chemical shi observed for the exchangeable
amide proton is distance dependent.43 The average distance of
a, ꢂ
b, ꢂ
g, ꢂ
˚
˚
˚
3
˚
V, A
Number of reections to determine nal unit cell 9059
Min and max 2q for cell determination
4.88, 64.1
2
1222
1.503
0.71073
1.904
0.989
87264
22979
0.0335
Z, ꢂ
F(000)
r (g cmꢀ1
)
˚
the amide nitrogen to the metal center is 4.28 A while the average
˚
l, A, (MoKa)
m, (cmꢀ1
)
distance for a series of previously reported SAP DOTAM
3+
complexes was signicantly longer (Nd3+ ¼ 4.56 A, Dy ¼ 4.48 A,
˚
˚
3+
Measured fraction of data
Number of reections measured
Unique reections measured
Rmerge
Tb3+ ¼ 4.50, Yb3+ ¼ 4.45 A)42 We have observed a signal for a Tm
˚
agent above 100 ppm previously; however, we were unable to
grow a crystal to help determine if there was an inner sphere
water which can contribute to tighter packing and increased
signal dispersion. In solution, the other geometry that is present
that gives rise to a CEST peak at ꢀ43 ppm is thought to be in a
SAP geometry, which exhibits a similar limited water exchange
due to the bulkiness of the ligands, however we are unable to
determine the exact number of inner sphere water molecules.
Selected angles and distances are listed in Table 2 and more
detailed crystallographic parameters in Table 3.
Table 4 r1 and r2 values of four Tm3+ based agents at 37 ꢂC, pH 7
Agent
r1 (sꢀ1 mMꢀ1
)
r2 (sꢀ1 mMꢀ1
)
Tm3+ para-H (3a)
Tm3+ para-OMe (3b)
Tm3+ DOTAM-Gly
Tm3+ DOTAM-Gly-
Lys
0.06
0.05
0.06
0.09
0.66
0.40
2.00
5.11
Relaxation evaluation
With the ninth site of Tm3+ para-OMe (3b) either vacant of inner roughly 500 ns to 1 ms for Dy3+ complexes and could generate r2
3+
sphere water or possessing a water which exchanges outside the values of 18 mMꢀ1 sꢀ1
.
Because Eu based agents typically
45,46
optimal rate regime for T2 relaxation, which was not deter- utilize the exchange of bound water from the inner sphere to
mined, theory predicts that there should be sizable differences generate the saturation used in paraCEST imaging such agents
in the ability of this agent to induce bulk water relaxation may also produce T2 related signal loss when images are
compared to similar agents that contain bound water or are acquired with non-zero echo-time.
exchanging within the optimal regime.2,44 While Tm3+ lantha-
Development of agents that do not contain inner sphere
nide agents are not generally efficient T1 relaxation agents they water or agents that have water exchange rates outside the
have recently been shown to produce sizable T2 relaxation optimal exchange regime, such as Tm3+ para-OMe (3b), should
effects (Table 4). Soesbe et al.29 compared several Eu3+ based have an advantage in –NH based paraCEST imaging as the
agents that contained inner sphere water with one that did not, major T2 shortening mechanism from water exchange has been
and showed a darkening during in vivo paraCEST imaging of removed. It should be noted that the exchange of –NH protons
kidneys for those agents that had intermediate inner sphere of the anilides still contributes to the T2 relaxation of bulk
water exchange rates. The darkening was linked to T2 short- water, but to a lesser extent compared to highly shied bound
ening which is largely caused by the frequency shi of bound water.29 Fig. 6 shows the relaxation rate (R2) as a function of
water which is in exchange with a paramagnetic metal. This concentration for Tm3+ based agents that are thought to either
shortening of the T2 time constant lead to a reduction in the have bound water (Tm3+ DOTAM-Gly, Tm3+ DOTAM-Gly-Lys)
image signal intensity and reduced CEST contrast. Another Eu3+ and those that are thought to have no bound water or are
agent that lacked an inner sphere water (Eu3+ TETA) showed exchanging outside the optimal rate (Tm3+ para-H (3a), Tm3+
very little T2 shortening and limited darkening. Since this para-OMe (3b)). The associated longitudinal and transverse
seminal publication, Soesbe et al. have prepared and studied a relaxivity (r1, r2) is provided in Table 4.
series of Dy3+ agents to ne tune the optimal water exchange to
generate T2 contrast.45 The optimal lifetime was found to be relaxation rate (Fig. 6) because they do not possess an inner
The two anilide agents (3a, 3b) produced a signicantly lower
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Lys as an example of a short T2 agent (large r2) and the second
containing Tm3+ para-OMe (3b) as an example of a long T2 agent
(small r2). T1 and T2 weighted images were acquired and
demonstrate that Tm3+ DOTAM Gly-Lys (Fig. 7c) produces signal
loss due to the T2 exchange effect, while Tm3+ para-OMe
(Fig. 7d) does not have a large T2 exchange effect. The two
phantoms were imaged at a concentration of 20 mM and
compared to aqueous solutions containing no agents (outer
circles in Fig. 7). Both the signal to noise ratio (SNR) and CEST
effect are important properties that can be used to characterize
CEST images. In medical imaging practice however it is
important to calculate the contrast to noise ratio (CNR) of CEST
images to quantify the likelihood of distinguishing between
diseased and surrounding normal tissue.47 In Table 5, several
CEST image properties including the SNR, CEST effect and CNR
are provided for Tm3+-para-OMe and Tm3+ DOTAM-Gly-Lys.
While Tm3+ DOTAM-Gly-Lys produces the largest CEST effect,
the agent with the longest T2, Tm3+-para-OMe, provides a
signicantly greater CNR.
To visualize the signicance of SNR and CNR, Fig. 7 provides
a direct comparison of T1- and T2-weighted images of 20 mM
Tm3+-para-OMe and Tm3+ DOTAM-Gly-Lys phantoms sur-
rounded by an outer cylinder of distilled water. A 3.5% decrease
in SNR was calculated for the long T2 agent (Tm3+-para-OMe)
while a 85% decrease was measured for the short T2 agent (Tm3+
DOTAM-Gly-Lys). It is apparent that even with a large CEST
effect, the overall detectability of Tm3+ Gly-Lys is substantially
lower than Tm3+-para-OMe due to the short bulk water T2
induced by Tm3+ DOTAM-Gly-Lys.
Fig. 6 Transverse relaxation rate (R2) for agents Tm3+ para-H (3a),
Tm3+ para-OMe (3b), Tm3+ DOTAM-Gly and Tm3+ DOTAM-Gly-Lys.
The relaxivity (r2) is given by the slope of the linear fit and reported in
Table 4. Error bars are from the standard deviation using n ¼ 3 values.
Experimental
General experimental procedures
CEST fast spin-echo (FSE) images were acquired on a 9.4T MRI
scanner (Agilent, Palo Alto, CA) with the following parameters,
FOV (eld of view): 25.6 ꢃ 25.6 mm2, matrix: 64 ꢃ 64, TR:
5000 ms, TE: 10 ms, ETL ¼ 32, 1 average, 2 prescans, 1 slice
3 mm thick. CEST spectra (Tm3+ and Dy3+) were acquired using
a 15 mT, 5 s continuous wave presaturation pulse at offset
Fig. 7 T1 and T2 weighted images collected at 37 ꢂC and pH 7.0. Inner
circle contains 20 mM CAs. Outer circle contains water with no agents.
T1 weighted images of (a) Tm3+ DOTAM Gly-Lys (b) Tm3+ para-OMe (3b).
T2 weighted images of (c) Tm3+ DOTAM Gly-Lys (d) Tm3+ para-OMe (3b).
sphere water or the metal-bound water exchanges outside the frequencies ranging from ꢀ100 to 100 ppm in steps of 1 ppm,
optimal exchange regime (which was not determined). None of averaged n ¼ 3. CEST signals were tted to a Gaussian curve and
the agents tested produced strong T1 relaxation of the bulk line widths were taken from the ttings. T1 weighted images
water, which is not surprising as Tm3+ is not known to act as a were acquired using a gradient echo pulse sequence with TR ¼
T1 relaxation enhancer.28 The benet of having CEST agents 200 ms, TE ¼ 2.62 ms, ip angle ¼ 20ꢂ, 10 averages, matrix ¼
with minimal effect on T2 relaxation is demonstrated in Fig. 7. 128 ꢃ 128, FOV ¼ 19.2 ꢃ 19.2 mm2, 1 slick 4 mm thick. T2
Two phantoms are shown, one containing Tm3+ DOTAM-Gly- weighted images were acquired using the FSE pulse sequence
Table 5 In vitro MR image contrast parameters for Tm3+ para-OMe and Tm3+ DOTAM-Gly-Lys
msignal
SNRa
w/o saturation
msignal
Agent (mobile pool frequency)
w/o saturation
w/o saturation
CEST (%)
CNRa
Bulk water
58 030
56 050
3627
3503
—
—
—
Tm3+ para-OMe (ꢀ43 ppm)
Tm3+ para-OMe (ꢀ83 ppm)
Tm3+ DOTAM-Gly-Lys (ꢀ46 ppm)
46 540
49 190
5039
16.9
12.2
43.3
59.0
42.6
23.9
8891
556
a
SNR and CNR were calculated as dened in ref. 41. snoise used in the calculations was 161.
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used for CEST imaging with TR ¼ 3000 ms, echo spacing ¼ 10
ms, echo train length ¼ 4, effective TE ¼ 40 ms, 4 averages,
matrix ¼ 128 ꢃ 128, FOV ¼ 19.2 ꢃ 19.2 mm2, 1 slick 4 mm
thick. All samples were studied at 20 mM concentration, 37 ꢂC.
All solvents were HPLC grade and used as such, except for
dioxane (dried by passing through columns of activated Al2O3)
and water (18.2 MU cmꢀ1 deionized). Solvents were removed
under reduced pressure in a rotary evaporator. NMR spectra
2-Chloro-N-(4-chlorophenyl)acetamide (1e). White solid
(1.08 g, 5.3 mmol), yield 68%. 1H NMR (400 MHz, DMSO-d6): d
10.43 (1H, s); 7.62 (2H, d, J ¼ 8.6 Hz); 7.38 (2H, d, J ¼ 8.6 Hz);
4.26 (2H, s). 13C NMR (100 MHz, DMSO-d6): d 165.2, 137.8,
129.2, 127.9, 121.3, 43.9. ESI-TOF. m/z calcd for C8H8Cl2NO (M +
H)+, 203.9983, found 203.9983.
1
were recorded on a 400 MHz spectrometer; for H (400 MHz),
chemical shi values (d) are reported relative to TMS and
General procedures for tetra substituted cyclen 2a–e
were referenced to the residual proton in the deuterated Electrophiles 1a–e (2.25 mmol) in 3 mL acetonitrile were added
solvents as follows: CDCl3 (7.26 ppm); DMSO-d6 (2.49 ppm); to a stirring suspension of cyclen (0.5 mmol) and K2CO3
D2O (4.75 ppm). Mass spectra (MS) were obtained using elec- (2.25 mmol) in acetonitrile (12 mL). The reaction was then
ꢂ
trospray ionization (ESI). T1 relaxation time constant measure- stirred for 1–3 days at 55 –80 C and monitored by UPLC MS.
ments were made for 4 different concentrations (1, 2, 4, 8 mM, Upon completion the reaction was ltered and the precipitate
averaged n ¼ 3) of CA using an inversion recovery sequence washed with water, then collected and used without further
(10 inversion times in the range of 10 ms to 10 s) with a 20 purication. This procedure worked well except for compounds
seconds repetition time to ensure full recovery, pH 7 and 37 ꢂC. 2d and 2e which possessed some minor impurities (see ESI†).
T2 relaxation time constant measurements were made for 4
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
different concentrations (1, 2, 4, 8 mM, averaged n ¼ 3) of CA tetrakis(N-phenylacetamide) (2a). The title compound was
1
using a CPMG pulse sequence (10 train echo times in the range obtained as a white powder (0.348 g) yield quantitative (q). H
of 10 ms to 10 s) with a 20 seconds repetition time to ensure full NMR (400 MHz, DMSO-d6): d 10.32 (4H, broad s); 7.61 (8H, d, J ¼
recovery, pH 7 and 37 ꢂC. Tm3+ DOTAM-Gly was prepared 7.8 Hz); 7.22 (8H, t, J ¼ 7.8 Hz); 7.01 (4H, t, J ¼ 7.8 Hz); 3.38
previously.12 Tm3+ DOTAM-Gly-Lys was prepared previously.48
(16H, s); 2.81 (8H, broad s). 13C NMR (100 MHz, DMSO-d6): d
169.3, 138.5, 128.3, 123.0, 119.3, 58.1, 51.9. ESI-TOF. m/z calcd
for C40H49N8O4 (M + H)+, 705.3877, found 705.399.
General procedure for electrophiles 1a–e
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
Chloroacetyl chloride (15.7 mmol) was added slowly on ice to a tetrakis(N-(4-methoxyphenyl)acetamide)
(2b).
The
title
stirring suspension of aniline (a–e) (7.84 mmol) and K2CO3 compound was obtained as a white powder (0.389 g) yield 94%.
(15.7 mmol) in acetonitrile (50 mL). The solution was allowed to 1H NMR (400 MHz, DMSO-d6): d 10.04 (4H, broad s); 7.51 (8H,
stir overnight at room temperature followed by ltration. The s); 6.80 (8H, s); 3.68 (16H, s); 3.25 (12H, s); 2.84 (8H, broad s).
ltrate was concentrated and the product was crystallized from 13C NMR (100 MHz, DMSO-d6): d 168.8, 155.1, 131.7, 120.8,
methanol.
113.6, 58.1, 55.0, 52.0. ESI-TOF. m/z calcd for C44H57N8O8 (M +
2-Chloro-N-phenylacetamide (1a). White solid (1.06 g, 6.2 H)+, 825.4299, found 825.4284.
mmol) yield 80%. 1H NMR (400 MHz, DMSO-d6): d 10.36 (1H, s);
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
7.60 (2H, d, J ¼ 7.4 Hz); 7.33 (2H, t, J ¼ 7.4 Hz); 7.08 (1H, t, J ¼ tetrakis(N-(p-tolyl)acetamide) (2c). The title compound was
7.4 Hz); 4.26 (2H, s). 13C NMR (100 MHz, DMSO-d6): d 165.0, obtained as a white powder (0.375 g) yield (q). 1H NMR
138.9, 129.2, 124.2, 119.8, 44.0. ESI-TOF. m/z calcd for (400 MHz, DMSO-d6): d 10.05 (4H, broad s); 7.47 (8H, d, J ¼
C8H9ClNO (M + H)+, 170.0373, found 170.0366.
6.6 Hz); 7.03 (8H, d, J ¼ 6.6 Hz); 3.26 (16H, s); 2.82 (8H, broad s);
2-Chloro-N-(4-methoxyphenyl)acetamide (1b). Silver solid 2.22 (12H, s). 13C NMR (100 MHz, DMSO-d6): d 169.0, 135.9,
(1.29 g, 6.5 mmol), yield 82%. 1H NMR (400 MHz, DMSO-d6): d 131.9, 128.7, 119.3, 58.1, 52.3, 20.1 ESI-TOF. m/z calcd for
10.17 (1H, s); 7.51 (2H, d, J ¼ 8.6 Hz); 6.90 (2H, d, J ¼ 8.6 Hz);
C
44H57N8O4 (M + H)+, 761.4503, found 761.4529.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
4.22 (2H, s); 3.72 (3H, s). 13C NMR (100 MHz, DMSO-d6): d 164.5,
156.0, 132.0, 121.4, 114.3, 55.6. 43.9. ESI-TOF. m/z calcd for tetrakis(N-(4-nitrophenyl)acetamide) (2d). The title compound
C9H11ClNO2 (M + H)+, 200.0478, found 200.0477. was obtained as a yellow powder (0.437 g) yield (q). 1H NMR (400
2-Chloro-N-(p-tolyl)acetamide (1c). White solid (1.08 g, MHz, DMSO-d6): d 10.69 (4H, broad s); 8.10 (8H, s); 7.81 (8H, s);
1
5.9 mmol), yield 75%. H NMR (400 MHz, DMSO-d6): d 10.21 3.39 (16H, s); 2.87 (8H, broad s). 13C NMR (100 MHz, DMSO-d6):
(1H, s); 7.50 (2H, d, J ¼ 7.4 Hz); 7.13 (2H, d, J ¼ 7.4 Hz); 4.24 (2H, d 170.2, 144.5, 142.0124.3, 118.7, 57.7, 52.0. ESI-TOF. m/z calcd
s); 2.26 (3H, s). 13C NMR (100 MHz, DMSO-d6): d 164.8, 136.4, for C40H45N12O12 (M + H)+, 885.3280, found, 885.3254.
133.2, 129.6, 119.8, 44.0, 20.8. ESI-TOF. m/z calcd for C9H11ClNO
2,20,20,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
(M + H)+, 184.0529, found 184.0524.
tetrakis(N-(4-chlorophenyl)acetamide) (2e). The title compound
1
2-Chloro-N-(4-nitrophenyl)acetamide (1d). Yellow solid was obtained as a white powder (0.394 g) yield 94%. H NMR
(1.80 g, 7.9 mmol), yield 79%. 1H NMR (400 MHz, DMSO-d6): d (400 MHz, DMSO-d6): d 10.6 (4H, broad s); 7.64 (8H, d, J ¼
10.97 (1H, s); 8.22 (2H, d, J ¼ 9.0 Hz); 7.84 (2H, d, J ¼ 9.0 Hz); 8.8 Hz); 7.26 (8H, d, J ¼ 8.8 Hz); 3.37 (16H, s); 2.78 (8H, broad s).
4.35 (2H, s). 13C NMR (100 MHz, DMSO-d6): d 166.0, 145.0, 13C NMR (100 MHz, DMSO-d6): d 169.5, 137.4, 128.1, 126.6,
143.0, 125.4, 119.5, 44.0. ESI-TOF. m/z calcd for C8H8ClN2O3 120.8, 57.8, 51.7. ESI-TOF. m/z calcd for C40H45Cl4N8O4 (M + H)+,
(M + H)+, 215.0223, found 215.0220.
841.2318, found, 841.2325.
1672 | RSC Adv., 2014, 4, 1666–1674
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62%). ESI-TOF. m/z calcd for C40H42N12O12Dy (M ꢀ 2H)+,
1046.2337, found 1046.2336.
General procedure for metallation of tetra substituted cyclen
products 3a–e, 4a–e
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)
tetrakis(N-(4-chlorophenyl)acetamide), dysprosium(III) salt (4e).
The title compound was obtained as a white powder (49 mg,
70%). ESI-TOF. m/z calcd for C40H42Cl4N8O4Dy (M ꢀ 2H)+,
1002.1375, found, 1002.1343.
To a stirring solution of appropriate tetra substituted cyclen (2a–
e) (0.07 mmol) in 4 mL 1 : 1H2O : dioxane, DyCl3$6H2O or
TmCl3$H2O (0.07 mmol) was added and the reaction was stirred
over a period of 1–4 days at 60 –80 ꢂC. The metalation was
monitored by UPLC MS and upon completion, the reaction
mixture was concentrated and re-dissolved in 1 mL H2O followed
by dialysis (for 3 days) against water to remove any unchelated
lanthanide ions. Aer dialysis, the absence of free lanthanide ion
was conrmed by the xylene orange test. The homogeneity of
dialyzed chelates was analyzed by UPLC and their identity were
conrmed by ESI-HRMS. UV traces of the chromatograms for
compounds 3a,b and 4a,b are given in the Supplemental Infor-
mation. The chelates were then used without further purication.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-phenylacetamide), thulium(III) salt (3a). The title
compound was obtained as a white powder (41 mg, 67%). ESI-
TOF. m/z calcd for C40H46N8O4Tm (M ꢀ 2H)+, 871.2984, found
871.2974.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-(4-methoxyphenyl)acetamide), thulium(III) salt (3b).
The title compound was obtained as a white powder (58 mg,
83%). ESI-TOF. m/z calcd for C44H54N8O8Tm (M ꢀ 2H)+,
991.3407, found 991.3387.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-(p-tolyl)acetamide), thulium(III) salt (3c). The title
compound was obtained as a white powder (30 mg, 46%). ESI-
TOF. m/z calcd for C44H54N8O4Tm (M ꢀ 2H)+, 927.3610, found
927.3647.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-(4-nitrophenyl)acetamide), thulium(III) salt (3d). The
title compound was obtained as a yellow powder (61 mg, 82%).
ESI-TOF. m/z calcd for C40H42N12O12Tm (M ꢀ 2H)+, 1051.2387,
found 1051.2408.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-(4-chlorophenyl)acetamide), thulium(III) salt (3e).
The title compound was obtained as a white powder (40 mg,
56%). ESI-TOF. m/z calcd for C40H42Cl4N8O4Tm (M ꢀ 2H)+,
1007.1425, found 1007.1412.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)tet-
rakis(N-phenylacetamide), dysprosium(III) salt (4a). The title
compound was obtained as a white powder (45 mg, 75%). ESI-TOF.
m/z calcd for C40H46N8O4Dy (M ꢀ 2H)+, 866.934, found 866.2903.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-(4-methoxyphenyl)acetamide), dysprosium(III) salt
(4b). The title compound was obtained as a white powder
(53 mg, 77%). ESI-TOF. m/z calcd for C44H54N8O8Dy (M ꢀ 2H)+,
986.3356, found, 986.3383.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)tet-
rakis(N-(p-tolyl)acetamide), dysprosium(III) salt (4c). The title
compound was obtained as a white powder (43 mg, 69%) ESI-TOF.
m/z calcd for C44H54N8O4Dy (M ꢀ 2H)+, 922.3560, found 922.3586.
2,20,200,2000-(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)-
tetrakis(N-(4-nitrophenyl)acetamide), dysprosium(III) salt (4d).
The title compound was obtained as a yellow powder (45 mg,
Conclusions
A series of Dy3+ and Tm3+ para-substituted anilide CAs were
synthesised and tested for their ability to respond to pH
changes in a range of interest for in vivo reporting. Poor solu-
bility of many of the compounds limited the possible
measurements and prevented us from making a complete
comparison of structure and effects. Even with the limited
solubility, there were a number of interesting observations in
the agents that were soluble. It was observed that the unsub-
stituted anilines had a maximum CEST effect at a pH near 8,
while the maximum CEST effect for the para-OMe agents was
between pH 7–7.5. The Tm3+ para-OMe CAS possesses a highly
shied signal at ꢀ83 ppm. A pH and concentration study was
attempted to determine if Tm3+ para-OMe could be used as a
ratiometric agent to measure pH in vivo, independent of
concentration, but due to solubility limitations, no suitable
measurements could be performed. Modications of this ligand
are currently underway to overcome the poor solubility.
Despite the limited data set collected, a comparison of
agents that create large T2 relaxation with those that do not was
possible. Examination of the molecular structure of Tm3+
DOTAM p-methoxyanilide revealed that the large amide sub-
situtents impinge on the ability of the metal center to possess
water in the solid state and is designated as TSAP’. The space
normally occupied by a ninth ligand (e.g. bound water) is
diminished by the metal ion being held deeply in the chelate at
a relatively acute trans O–Ln–O angle (123ꢂ). Within this
chelation geometry, the amide nitrogens are positioned rela-
tively closer to the metal, as compared to SAP geometry, thus
resulting in highly shied exchangeable protons. The solution
behaviour of this chelate also mirrors the lack of bound water in
an exchange regime that causes T2 relaxation. These features
produce an increase in CEST sensitivity and also add the benet
of moving the CEST signal outside the MT background of in vivo
imaging.11 Due to these promising properties, modication of
these agents are underway to allow for in vivo testing of par-
aCEST agents with high shiing CEST signals and limited T2
relaxation for improved CNR during imaging.
Acknowledgements
The authors gratefully acknowledge funding from the Natural
Sciences and Engineering Research Council of Canada (RHEH)
and Canadian Institutes for Health Research (RB). The authors
also thank Dr Todd Soesbe (University of Texas Southwestern
Medical Centre, Dallas TX) for his helpful discussion of T2
exchange and Dr Paul Boyle (Western) for performing the
single-crystal X-ray diffraction study.
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