Towards an Improved Design of MRI Contrast Agents: Synthesis and Relaxometric Characterisation of Gd-HPDO3A Analogues

Article abstract of DOI:10.1002/chem.202000479

The properties of Ln^III-HPDO3A complexes as relaxation enhancers and paraCEST agents are essentially related to the hydroxylpropyl moiety. A series of three HPDO3A derivatives, with small modifications to the hydroxyl arm, were herein investigated to understand how heightened control can be gained over the parameters involved in the design of these agents. A full ¹H and ¹⁷O-NMR relaxometric analysis was conducted and demonstrated that increasing the length of the OH group from the lanthanide centre significantly enhanced the water exchange rate of the gadolinium complex, but with a subsequent reduction in kinetic stability. Alternatively, the introduction of an additional methyl group, which increased the steric bulk around the OH moiety, resulted in the formation of almost exclusively the TSAP isomer (95 %) as identified by ¹H-NMR of the europium complex. The gadolinium analogue of this complex also exhibited a very fast water exchange rate, but with no detectable loss of kinetic stability. This complex therefore demonstrates a notable improvement over Gd-HPDO3A.

Full text of DOI:10.1002/chem.202000479

A Journal of
Accepted Article
Title: Towards an improved design of MRI Contrast Agents: Synthesis
and Relaxometric Characterisation of Gd-HPDO3A Analogues
Authors: Louise Rachel Tear, Carla Carrera, Eliana Gianolio, and Silvio
Aime
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To be cited as: Chem. Eur. J. 10.1002/chem.202000479
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Towards an improved design of MRI Contrast Agents: Synthesis
and Relaxometric Characterisation of Gd-HPDO3A Analogues
Louise R. Tear,[a] Carla Carrera,[b] Eliana Gianolio,*[a] and Silvio Aime*[a]
[a]
Dr. L.R. Tear, Dr. E. Gianolio and Prof. S. Aime
Department of Molecular Biotechnology and Health Sciences
Molecular Imaging Centre, University of Torino
Via Nizza 52, 10126 Torino, Italy
E-mail: eliana.gianolio@unito.it
[b]
Institute of Biostructures and Bioimaging, National Research Council, Turin, Italy
Supporting information for this article is given via a link at the end of the document.
Abstract: The properties of Ln(III)-HPDO3A complexes as relaxation
enhancers and paraCEST agents are essentially related to the
hydroxylpropyl moiety. A series of three HPDO3A derivatives, with
small modifications to the hydroxyl arm, were herein investigated to
understand how heightened control can be gained over the
parameters involved in the design of these agents. A full 1H and 17O-
NMR relaxometric analysis was conducted and demonstrated that
increasing the length of the OH group from the lanthanide centre
significantly enhanced the water exchange rate of the gadolinium
complex, but with a subsequent reduction in kinetic stability.
Alternatively, the introduction of an additional methyl group, which
increased the steric bulk around the OH moiety, resulted in formation
of almost exclusively the TSAP isomer (95 %) as identified by 1H-NMR
of the europium complex. The gadolinium analogue of this complex
also exhibited a very fast water exchange rate, but with no detectable
loss of kinetic stability. This complex therefore demonstrates a notable
improvement over Gd-HPDO3A.
ratio of SAP to TSAP isomers in aqueous solution is 70 % SAP to
30 % TSAP. TSAP isomers are known in general to display
shorter water residency times (τm) than SAP isomers, due to the
increased steric hindrance surrounding the coordinated water
molecule.
Therefore,
the
proportion of
these two
diastereoisomers affects the overall observed τM value.[15]
It has also been shown that the slow exchange rate of the
hydroxyl proton allows the use of paramagnetic Ln-HPDO3A
complexes as para-CEST agents.[16] In particular, these
complexes can act as ratiometric pH reporters due to the two
CEST hydroxyl signals, which correspond to the SAP and TSAP
isomers.[17]
The overall properties of Gd-HPDO3A as relaxation enhancers
and of Yb and Eu-HPDO3A as para-CEST agents are therefore
essentially related to the occurrence of the hydroxypropyl moiety.
This group affects the overall neutral charge of the complexes,
their stability, the ratio of TSAP/SAP, the rate of water exchange
etc., when compared to the parent DOTA complexes. On this
basis we deemed it of interest to introduce chemical changes to
the OH bearing arm to assess whether one may acquire
heightened control of the parameters involved in the design of
relaxation enhancers or of para-CEST agents.
Introduction
Contrast agents are widely used in magnetic resonance imaging
(MRI) to enhance the differentiation and characterisation of
pathological tissue.[1] One of the contrast agents most commonly
applied in clinical practice is Gd-HPDO3A (Prohance, Bracco),
which was first reported almost 30 years ago.[2] In recent years
there has been concern regarding the discovery of Gd deposition
in the brain and other tissues of patients, after contrast-enhanced
MRI scans.[3–6] Gd-HPDO3A appears to be one of the agents
which displays a lower level of retention in the body than most
other Gd contrast agents,[7–9] in part due to its high kinetic stability
as a macrocyclic agent.[10,11]
Figure 1. Structure of HPDO3A and derivatives: HEDO3A (10‐(2‐hydroxyethyl)‐
1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic),
HIBDO3A
(10‐(2‐
hydroxyisobutyl)‐1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic
acid),
The relaxometric properties of Gd-HPDO3A have been
investigated extensively.[12] These studies suggest that while the
prototropic exchange of the OH group significantly enhances the
relaxivity of the complex, this effect may only contribute at basic
pH. However, it has been determined that the OH-proton has a
shorter Gd-H distance than Gd-H2O and therefore T1M of the
hydroxyl proton is approximately 50 % shorter than that of the co-
ordinated water protons.[13,14]
HMPDO3A (10‐(3‐hydroxy-2-methylpropyl)‐1,4,7,10‐tetraazacyclododecane‐
1,4,7‐triacetic acid).
Here, we report the results obtained by comparing the parent Gd-
HPDO3A complex and three new derivatives (Figure 1). The
ligands have been designed to alter the properties of the hydroxyl
group through its structural environment. These involve either the
addition or removal of a simple methyl group (HIBDO3A and
HEDO3A respectively), or the elongation of the hydroxyl chain by
Studies have investigated extensively the solution structure of Ln-
HPDO3A complexes, which have shown how the SAP to TSAP
ratio changes along the lanthanide series.[12] In Gd-HPDO3A the
1
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one carbon (HMPDO3A). A full relaxometric study of the Gd
complexes has been carried out, including analysis of the
SAP/TSAP ratio with high resolution 1H-NMR of the Eu complexes
and 17O-NMR and NMRD of the Gd complexes. The new
complexes have been thoroughly assessed to gain insight into the
modulation of the properties of the Ln complex through the
modifications introduced at the OH-bearing arm.
Twisted Square Anti-Prismatic (TSAP) isomers, which depend on
the torsion angle (δ or λ) of the macrocyclic ring and the
orientation of the pendant arms (Δ or Λ). This leads to four
possible diastereoisomers: Δ(λλλλ) (SAP1), Λ(δδδδ) (SAP2),
Λ(λλλλ) (TSAP1) and Δ(δδδδ) (TSAP2). In addition to this, Ln-
HMPDO3A (like Ln-HPDO3A) has a chiral carbon which can be
either R or S and creates the possibility of a total of 8 isomers.
Based on crystallography data of Gd-HPDO3A,[13] the dominant
diastereoisomers in solution of Eu-HPDO3A were assumed to be
SAP2-R and TSAP1-R and their enantiomeric pairs SAP1-S and
TSAP2-S.[12]
Results and Discussion
It was early established that the axial ring proton region is highly
diagnostic for assessing the SAP/TSAP isomeric ratio. These
protons are highly shifted downfield in EuDOTA-type complexes
(10 – 50 ppm), with the SAP isomer experiencing a greater
chemical shift than the TSAP isomer protons. Figure 2 shows the
axial region of Eu-HMPDO3A with assignment of the protons for
each isomer. Interestingly, in respect to the parent Eu-HPDO3A
complex and to the Eu-HIBDO3A and Eu-HEDO3A derivatives
(see Supp. Info. Figures S10 and S13), an additional minor set of
peaks in the SAP and TSAP region are present. These are most
likely ascribable to the SAP1 and TSAP2 isomers, which in this
case have a different chemical shift to those of SAP2 and TSAP1,
as has previously been observed for Eu-HPDO3MA.[20] In analogy
with Eu-HPDO3A, the diastereoisomers of Eu-HMPDO3A can be
assigned as SAP2-R (35 %), SAP1-R (15 %) and TSAP1-R
(35 %), TSAP2-R (15 %), with their corresponding S enantiomers
also present.
Synthesis
The HPDO3A derivatives were synthesised through the scheme
shown in Scheme 1. The syntheses of all the ligands involved the
alkylation of the protected triester 1 with the appropriate
substituent to give intermediates 3, 5 and 7. In the case of 3, 2-
bromoethanol was protected with tetrahydropyran (2) prior to
addition to 1. The intermediates of the ligands were then
deprotected by TFA and purified via chromatography on
Amberchrom® to give the final ligands 4, 6 and 8. Triester 1 was
synthesised according to literature procedures and used as the
starting material for all three ligands.[18] HEDO3A was previously
synthesised in the literature with a different procedure, which
involved the direct addition of 2-bromoethanol to DO3A
(deprotected 1).[19] Our strategy allows an easier purification of the
product with satisfactory yields.
Figure 2. Axial region of 1H-NMR of Eu-HMPDO3A in D2O at pD 7.4, 600 MHz.
Peaks labelled as follows: SAP2-R/1-S = blue, SAP1-R/2-S = orange, TSAP1-
R/2-S = green, TSAP2-R/1-S = red. * peaks excluded from TSAP contribution
based on EXSY information.
Scheme 1. Synthesis of HPDO3A derivatives: i) pyridinium p-toluenesulfonate,
DCM, 12 h, RT; ii) K2CO3, ACN, 12 h, RT; iii) DIPEA, ACN, 48 h, 50 °C; iv) DCM,
TFA then TFA with triisopropylsilane, 60 h, RT; v) water, THF, acetic acid, 24 h,
RT then TFA with triisopropylsilane, 72 h, RT.
2D EXSY-NMR were acquired to identify the exchange between
isomers and to aid the assignment of isomer peaks (SI). In Eu-
HMPDO3A, the EXSY NMR (Figure 3) indicates ring inversion of
the major SAP2-R/1-S and TSAP1-R/2-S isomers (box A) and
one arm rotation between the minor SAP1-R/2-S protons and the
major TSAP1-R/2-S protons (box B). With longer mixing time (10
ms instead of 5 ms, Figure S17) exchange via ring inversion of
the minor SAP1-R/2-S isomer is also observable and indicates
exchange to the same protons as the major TSAP1-R/2-S isomer.
The other peaks in the TSAP axial region (red in Figure 2) have
been assigned as the TSAP2-R/1-S minor isomer. These protons
do not demonstrate exchange via arm rotation with that of the
High resolution solution 1H-NMR
As Europium is one of the closest elements to Gadolinium it is
customarily to acquire the 1H-NMR of the europium complexes in
order to determine the ratio of isomers in aqueous solution (SI).
Octa-coordinated, tetra-aza-cyclododecane based macrocyclic
ligands are known to wrap around Lanthanide (III) ions to yield
four isomers, namely two Square Anti-Prismatic (SAP) and two
2
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SAP2-R/1-S major isomer, but there is evidence of ring inversion
of both isomers, which indicates that arm rotation between these
two isomers is most likely blocked.
assumption that exchange by arm rotation is too fast to observe
on the NMR timescale. However, in these derivatives, arm
rotation of the less hindered Eu-HEDO3A and longer armed Eu-
HMPDO3A, would be anticipated to be faster than that of Eu-
HPDO3A. Therefore, it is possible that arm rotation is blocked in
the parent compound and that only the two dominant
diastereoisomers are populated (SAP2-R/1-S) and TSAP1-R/2-
S).
All three derivatives also have an additional proton resonance in
the TSAP axial region (* in Figure 2, S10 and S13), which has
been excluded from the TSAP axial protons, due to the absence
of corresponding cross peaks that would indicate arm rotation with
the SAP isomer in the EXSY spectrum. In Figure 2, the proton
resonances assigned to the TSAP2-R/1-S minor isomer (red)
most likely also have an additional proton present, as indicated by
the integrals of these regions. Eu-HPDO3A also displays this
additional proton resonance within this region, while Eu-DOTA
does not, therefore indicating it probably corresponds to one of
the protons on the hydroxyl arm. A comparison of these three
complexes with Eu-HPDO3A would suggest that the CH2 group
on the hydroxyl arm is the most likely candidate, as it is the only
group which remains unchanged between all complexes.
The relative populations of each isomer were calculated by
integration of the protons in the axial region and are displayed in
Table 1.
Figure 3. 2D EXSY 1H-NMR of 100 mM EuHMPDO3A, with 5 ms mixing time
in D2O at pD 7.4, 275 K, 600 MHz. Cross peaks show the exchange between
isomers by A) ring inversion and B) arm rotation.
The high resolution 1H-NMR spectrum of Eu-HEDO3A in D2O
(Figure S10) indicates the presence of both SAP and TSAP
isomers in a ratio of SAP 50 % and TSAP 50 %. The pairs of TSAP
and SAP isomers are indistinguishable in the 1H-NMR, as with Eu-
HPDO3A. The 2D EXSY spectrum (Figure S11) indicates proton
exchange between the isomers via ring inversion and also via arm
rotation. Each proton resonance in the axial region (SAP and
TSAP) has three corresponding cross peaks, two which show
exchange to equatorial protons and one to the axial protons of the
alternate isomer (i.e. SAPax to TSAPax). The proton resonances
which are linked by arm rotation (SAPax and TSAPax) exchange
with the same set of equatorial protons through ring inversion.
This indicates that all four isomers are populated, as previously
observed for DOTA,[21] and that the chemical shifts of SAP and
TSAP diastereoisomers are almost equivalent and therefore
appear together in the spectrum.
Table 1. Percentage of SAP and TSAP isomers in europium and gadolinium
complexes.
Ligand
Eu Complex[a]
SAP %
Gd complex[b]
SAP %
TSAP %
TSAP %
HPDO3A
HEDO3A
HIBDO3A
HMPDO3A
60
50
5
40
50
95
70
60
6
30
40
94
40
50
50
60
(35 – 2R/1S
15 – 1R/2S)
(35 – 1R/2S
15 – 2R/1S)
1
In contrast to the other complexes, the H-NMR of Eu-HIBDO3A
[a] From 1H-NMR peak integrals at 275 K, 600 MHz. [b] Empirically extrapolated
from Eu ratios using the equation: χSAP(Gd)= χSAP(Eu)×1.16; χ TSAP(Gd)= 100 -
χSAP(Gd)
(Figure S13) shows almost only the TSAP isomer (95 %). TSAP
most likely dominates due to the isobutyl group increasing the
steric bulk around the bound metal, which limits isomerization and
forces a specific conformation.[22] This has been previously
reported for chiral complexes,[23,24] as well as for DOTMA and
HPDO3MA,[20,25] which display a high proportion of the TSAP
isomer in part due to blocked isomer exchange. The very small
quantity (5%) of SAP isomer present in the EXSY spectrum
(Figure S14) demonstrates some proton exchange via arm
rotation and ring inversion between the SAP and TSAP isomers
in solution. It is therefore possible that both TSAP enantiomers
are populated and there is exchange between these two (either
by direct enantiomeric exchange, or via the SAP isomer which
does not remain significantly populated). The proton resonances
of TSAP1 and TSAP2 isomers would therefore occur with the
same chemical shifts and the contribution of each to the 95 %
observed is unknown.
Relaxometric Analysis
It has been consistently reported that TSAP isomers display faster
water exchange rates than those of SAP isomers. In general, the
overall observed relaxivity is frequently limited by the relatively
slow exchange rates due to a low proportion of the TSAP isomer.
The proportion of TSAP and SAP isomers and their differing water
exchange rate characterise the variable-temperature 17O-NMR
transverse relaxation rate profiles (R2) and influence the
amplitude of the NMRD profiles. Therefore, the higher proportion
of TSAP isomer observed for some of these complexes, may
allow improved relaxometric properties.
The NMRD profiles in water and in human serum at 25 °C and
37 °C (Figure 4A and S20) and the variable-temperature 17O-NMR
R2 profiles (Figure 4B and S22), were measured in order to define
Exchange of isomers via arm rotation was observable in the
EXSY NMR of all three derivatives, but has not been observed in
that of Eu-HPDO3A.[12] This has previously been attributed to the
3
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the determinants of the relaxometric properties of the investigated
complexes.
complexes are not too far from those of the corresponding Eu-
complexes and comparable with what was observed for Eu- and
Gd-HPDO3A (i.e. χSAP(Gd)= χ SAP(Eu)×1.16; χ TSAP(Gd)= 100 -
χSAP(Gd), Table 1). A single isomer model was used for the fitting
of Gd-HIBDO3A 17O-NMR data, as it was found that the low
calculated proportion of SAP isomer in the Gd complex (0.06)
made the two-isomer fitting model unreliable.
In human serum, the NMRD profiles of Gd-HIBDO3A and Gd-
HMPDO3A both indicate a small ‘hump’ in the high field region
(10 – 100 MHz, Figure 4A.i.), which is not observable for Gd-
HPDO3A and Gd-HEDO3A. An increase in relaxivity in the high-
field region is indicative of binding interactions with
macromolecules and therefore a slow tumbling time (τR). To gain
more insight into the observed behaviour, the NMRD profile of Gd-
HMPDO3A with human serum albumin (HSA, 35 mg/ml) was also
measured. However, it did not display any increase in relaxivity in
the high-field region (Figure S21). This has previously been
observed for the complex Gd-PhenDO3A, that has a phenyl
substituent instead of a methyl group, which demonstrated a
larger ‘hump’ than the complexes described here but had no such
interaction with HSA.[26] It is possible that hydrophobic interactions
with another undetermined protein in serum result in a reduction
in molecular tumbling time yielding the small increase in relaxivity
in this region. Though small, this effect indicates how minor
structural modifications may control specific interactions with
blood serum components and outlines the high detection
sensitivity of the relaxometric measurements through the complex
interplay between τR, τm and the other parameters.
The parameters determined from fitting the 17O-NMR and NMRD
data are displayed in Table 2. The values of the exchange lifetime
(τm) and activation energy for water exchange (ΔHM) were derived
from the fitting of the 17O-NMR data. The derived weighted
average of τm for the two isomers was then fixed in the fitting of
the NMRD data and the rotational correlation time (τR), squared
mean transient zero-field splitting energy (Δ2) and electronic
relaxation time (τV) determined. These values are comparable
across the complexes to those of Gd-HPDO3A, while the τm
values differ markedly. Whereas Gd-HEDO3A and Gd-HPDO3A
display very similar τm values, Gd-HMPDO3A and Gd-HIBDO3A
have significantly shorter average water exchange lifetimes. For
Gd-HMPDO3A, the τm value of the TSAP isomer is comparable to
that of Gd-HPDO3A, while that of the SAP isomer is significantly
shorter (around 26 times shorter). As the TSAP isomer is almost
exclusively present in Gd-HIBDO3A, the complex displays a short
water exchange lifetime (average τm ca. 16 ns). The average
water exchange lifetimes of both Gd-HIBDO3A and Gd-
HMPDO3A are close to what has been determined the optimal
range for proton relaxation enhancers at clinical imaging fields (24
ns).
Table 2. Fitting parameters from analysis of 17O-NMR and 1H-NMRD[a] profiles
for Gd-HPDO3A derivatives.
Parameters
Gd-
HPDO3A[b]
Gd-
HEDO3A[c]
Gd-
HIBDO3A[d]
Gd-
HMPDO3A[c]
Mol.Fracn
TSAP
0.3
0.4
-
0.4
298
r
water[e]
4.2
5.4
640
8.9
451
4.2
4.3
5.7
4.3
1P
1P
298
r
serum[e]
5.3
5.9
τm (ns) SAP
546.3 ± 25.7
6.6 ± 2.7
330
24.4 ± 3.9
7.0 ± 2.9
17.4
16.1 ± 2.8
16.1
TSAP
Weighted avg.
τm (ns)
[f]
ΔHM SAP
53
54.8 ± 2.5
43.3 ± 13.2
54.2 ± 1.0
2.7 ± 0.2
61.6 ± 9.9
27.8 ± 8.3
56.0 ± 0.1
3.7 ± 0.1
19.5 ± 3.2
Figure 4. A) NMRD profiles in i) water and ii) human serum measured at 298 K.
B) 17O-NMR profiles measured at variable temperature and 600 MHz, of 20 mM
Gd complex in H2O. Gd complexes measured in A and B are: Gd-HMPDO3A
(blue), Gd-HEDO3A (green) and Gd-HIBDO3A (red), compared to Gd-HPDO3A
(black), at pH 7.
TSAP
τR (ps)
15
65
62.1 ± 2.7
5.4 ± 1.0
12.9 ± 1.8
Δ2 (1019 s-2)
τv (ps)
7.4
14.6
21.3 ± 1.6
20.7 ± 0.1
The NMRD and 17O-NMR data were analysed independently
using the Swift-Connick theory for 17O relaxation, and the
Solomon-Bloembergen-Morgan (SBM) and Freed’s model for
inner- and outer-sphere proton relaxation.[27] A two-isomer model
was used in the fitting of the 17O-NMR data for Gd-HMPDO3A and
Gd-HEDO3A, where the ratio of isomers in the Gd complexes was
extrapolated from the corresponding Eu complexes. In analogy
with what has been previously reported,[20,28] we considered that
it was reasonable to assume the SAP/TSAP ratios for Gd-
[a] aNMRD profiles used for fitting were acquired at 298 K in water, with the
following parameters fixed during the fitting procedure: q = 1, rGdH = 3.1 Å, aGdH
= 3.8 Å, DGdH = 2.24 x 10-5 cm2s-1. [b] From ref.[12] [c] Two-isomer model was
used for fitting of the 17O-NMR data with fixed parameters: rGdO = 2.5 Å, Er = 10
KJmol-1, Ev = 10 KJmol-1, A/ћ = -3.5 x 106 rad s-1, τR0 = 90 ps. [d] Single isomer
model used for fitting of Gd-HIBDO3A17O-NMR data with the same fixed
parameters as the two isomer model except Er = 20 KJmol-1 and Ev was left
unfixed. [e] mM-1s-1. [f] KJmol-1.
4
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The remarkably short exchange lifetime of the coordinated water
molecule in Gd-HMPDO3A parallels well with previously reported
results obtained on macrocyclic complexes containing an
elongated carboxo-amide arm. In fact, Toth and co- workers
showed that the increased steric crowding at the water
coordination site, obtained by replacing an ethylene bridge of
DOTA4− by a propylene bridge, resulted in a dramatic increase of
the water exchange rate.[29]
Stability and Anion Binding
The Gd complexes were tested for their kinetic stability by
measuring the rate of transmetallation with zinc chloride in
phosphate buffer.[32] Gd-HEDO3A and Gd-HIBDO3A maintain the
same kinetic stability as Gd-HPDO3A as they display no evidence
of transmetallation with zinc over the time course of the
experiment (Figure 6). However, Gd-HMPDO3A displays a
reduction in kinetic stability as zinc is able to displace Gd from the
complex with approximately a 25 % displacement over 3 days.
This is due to the elongation of the hydroxypropyl arm, which
reduces the stability of the coordinating hydroxyl group as it is
located further from the gadolinium centre than in the prior Gd-
HPDO3A complexes. Gd-HPDO3A appears to show a small
increase in the relaxivity after 4 days, which was not observed in
the data recorded by Laurent et al. as they did not record beyond
this time point.[32] The reason for this increase is unclear, but as it
is known that phosphate can enhance the second sphere
relaxation contribution, perhaps there is some slow coordination
of zinc and phosphate to the ligand sphere of Gd-HPDO3A that
increases the observed relaxation rate over time.[33]
The rate of transmetallation of Gd-HMPDO3A with zinc is slower
than that reported for any of the linear chelates. After 3 days, R1P(t
= 4320)/R1P(t=0) is 0.76 for Gd-HMPDO3A while for all linear
chelates it was reported to be below 0.7. In addition to this, the
linear chelates show a quicker initial decrease over the first couple
of days, as the data shows it takes Gd-HMPDO3A 3650 min to
reach R1P/R1P(t=0) = 0.8, while for the linear chelates it takes
between 1500 min (Gd-EOB-DTPA) and 70 min (Gd-DTPA-
BMA).[32] Thus, these results suggest that the stability of Gd-
HMPDO3A is not low enough to prevent in vivo studies, given the
improvement over linear agents.
Prototropic exchange
The relaxivity of Gd-HPDO3A has been previously shown to be
markedly pH dependent, with a significant increase in relaxivity at
high pH values (> 8 – 10.4) as a result of base catalysed
exchange of the hydroxyl proton.[30] The pKa of the hydroxyl proton
[31]
in Gd-HPDO3A is 11.36,
and therefore the decrease in
relaxivity above pH 10.4 was attributed to the formation of the
deprotonated anionic complex.[30] Similar behaviour is observed
for the Gd-HPDO3A derivatives (Figure 5). Gd-HEDO3A
remained the most similar to Gd-HPDO3A, as it exhibited an
increase of 1.3 mM-1s-1 at pH 10.6 compared to 1.4 mM-1s-1 at pH
10.4 for Gd-HPDO3A. Gd-HIBDO3A demonstrates a maximum
increase of 1.6 mM-1s-1 at pH 11 which is a notable enhancement
in the maximum relaxivity as well as a shift towards a higher pH.
The higher pH value of the maximum relaxivity enhancement is
consistent with a higher pKa of tertiary alcohols. However, the
cause of the higher relaxivity enhancement is uncertain, perhaps
the OH group is located closer to the Gd in this structure than the
other HPDO3A derivatives, or the exchange of the hydroxyl
proton is faster.
Gd-HMPDO3A shows a distinct lower enhancement of the
relaxivity with increasing pH. The complex displays a maximum
enhancement of 0.8 mM-1s-1 at pH 9.8. The shift in pH is again
consistent with the lower pKa of primary alcohols, while the lower
enhancement is most likely the result of the increased Gd-H
distance in Gd-HMPDO3A compared to Gd-HPDO3A. An
increase of 0.8 mM-1s-1 corresponds to the exchange of one
proton at a Gd-H distance of 3.3 Å, in comparison to 3 Å in Gd-
HPDO3A.[30]
Figure 6. Zinc transmetallation as modelled by the change in R1P(t)/ R1P(t=0)
over time for the different Gd-HPDO3A derivatives at 310 K.
The reduced strength of the coordinating hydroxyl group in Gd-
HMPDO3A allows some displacement by strongly coordinating
bidentate anions such as lactate. The complexes were tested for
their interaction with phosphate (Na2HPO4), Carbonate (NaHCO3),
citrate and lactate in 150 mM NaCl,10 mM HEPES buffer. In the
presence of 50 mM lactate the relaxivity of Gd-HMPDO3A is
Figure 5. Relaxivity of Gd-HPDO3A derivatives versus pH measured at 298 K
and 0.47 T. Symbols indicate experimental data, solid lines are drawn freehand
to aid visualisation of the data.
reduced from 4.45 mM-1s-1 (r1P in NaCl/HEPES) to 3.6 mM-1s-1
–
a 19 % decrease, while with carbonate, citrate and phosphate the
relaxivity remains comparable. (Figure S23) At more basic pH
values (pH 8) it was found that some binding of carbonate does
5
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10.1002/chem.202000479
Chemistry - A European Journal
FULL PAPER
occur. The other complexes, including Gd-HPDO3A, do not show
significant changes in relaxivity even with high concentrations (50
mM) of these anions.
bromoethoxy)tetrahydro-2H-pyran boils at 85°C and 147 mbar
(29.2 g).
1,4,7,10-tetraazacyclododecane-10-(2-((tetrahydro-2H-pyran-
2-yl)oxy)ethyl)-1,4,7-triacetic acid 1,4,7 tri-tert-butyl ester (3).
To a solution of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid 1,4,7-tris(1,1-dimethylethyl) ester hydrobromide (1) (15 g; 25
mmol) in 150 ml of ACN, K2CO3 (−325 mesh, 10.4 g; 75 mmol)
and 2-(2-bromoethoxy)tetrahydropyran (2) (5.75 g; 342 µl; 27.5
mmol) in 50 ml of ACN were added. After one night the mixture
was fitered and evaporated. The residue was dissolved in ethyl
acetate (100 ml) and washed with water (2 x 25 ml) and brine (15
x 20 ml) to obtain the substitution of bromide with chloride, then
evaporated (15 g).
Conclusion
Three ligands have been synthesised and investigated for their
relaxometric properties relating to the hydroxylpropyl moiety. In
particular, it was found that increasing the distance of the hydroxyl
group from the lanthanide centre by one carbon group
(HMPDO3A) enhanced the water exchange dynamics of the
complex, but at the expense of decreasing kinetic stability.
Removal of the methyl group in HPDO3A to give HEDO3A was
shown to have little effect on the properties of the Gd complex,
both in the water exchange dynamics and also in the overall
observed relaxivity. Meanwhile, Gd-HIBDO3A demonstrated
significant differences to Gd-HPDO3A in terms of water exchange
and the ratio of isomers that results from the addition of one
methyl group. The observed relaxivity profiles remain relatively
unchanged since the molecular tumbling time limits the
achievable enhancement of relaxivity beyond water exchange
rates. With a larger molecular weight and slower rotational
correlation time it is expected that higher relaxivity values could
be achieved. Overall, Gd-HIBDO3A maintains all the benefits of
Gd-HPDO3A while increasing the rate of water exchange to give
a τm value within the optimal range.[34] These results demonstrate
how small modifications to the hydroxyl group in HPDO3A allow
significant alterations to the observed properties of the lanthanide
complexes. Understanding how this clinical probe can be adapted
to further enhance or modify its application in MRI and CEST
provides a useful insight for the future development of high
relaxation enhancers or reporter probes.
1,4,7,10-tetraazacyclododecane-10-(2-hydroxy-ethyl)-1,4,7-
triacetic
acid
(4,
HEDO3A)
The
1,4,7,10-
tetraazacyclododecane-10-(2-((tetrahydro-2H-pyran-2-
yl)oxy)ethyl)-1,4,7-triacetic acid 1,4,7 tri-tert-butyl ester (3) was
dissolved in water (30 ml), THF (60 ml) and Acetic Acid (120 ml)
and stirred at room temperature; after 24 hours the mixture was
evaporated to give a residue. Cooling with an ice bath, the product
was dissolved in TFA (40 ml) with 2 drops of Triisopropylsylane
and stirred at room temperature for 3 days. The product was
precipitated with diethyl ether, filtered and dried under vacuum.
The crude product was purified by chromatography on an
Amberchrom CG161 resin (eluent: 0.2% TFA in water). The
fraction containing the pure product was freeze dried. (8.3 g). HCl
1N (60 ml) was added and the solution was stirred for 1 hour, then
freeze dried (7.3 g of 1,4,7,10-tetraazacyclododecane-10-(2-
hydroxy-ethyl)-1,4,7-triacetic acid tetrahydrochloride). 1H-NMR
(600 MHz, D2O): δH = 4.14 (s, 2 H, -CH2COO), 3.99 (t, 2 H, -
CH2OH), 3.63-3.09 (m, 22 H, -NCH2). 13C-NMR (150 MHz, D2O):
δC = 173.4 (-COO), 168.3 (-COO), 54.7 (-CH2OH), 54.3(-
CH2COO), 54.2 (-CH2CH2OH) 52.2 (-CH2COO), 51.0, 49.8, 47.5,
47.2 (CH2 cyclen ring) ppm. MS ESI: m/z cald 391.22 [M+H+];
found 391.33.
Experimental Section
1,4,7,10-tetraazacyclododecane-10-(2-hydroxy-2-
methylpropyl)-1,4,7-triacetic acid 1,4,7 tri-tert-butyl ester (5).
To a solution of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid 1,4,7-tris(1,1-dimethylethyl) ester (1) (20 g; 38.8 mmol) and
DIPEA (15 g; 20.3 ml; 116.5 mmol) in 100 ml of ACN, 1,2-Epoxy-
2-methylpropane (4.2 g; 5.2 ml; 58.3 mmol) was added. The
reaction was monitored by TLC (eluent DCM/MeOH 1:1, stain
KMnO4): after 2 days at 50°C the mixture was evaporated an
redissolved in ethyl acetate (200 ml), washed with water (5 x 30
ml), dried over Na2SO4 and evaporated to residue (19.7 g).
1,4,7,10-tetraazacyclododecane-10-(2-hydroxy-2-
methylpropyl)-1,4,7-triacetic acid (6, HIBDO3A). The crude
1,4,7,10-tetraazacyclododecane-10-(2-hydroxy-2-methylpropyl)-
1,4,7-triacetic acid 1,4,7 tri-tert-butyl ester (5) (19,7 g; 33,6 mmol)
was dissolved in DCM (50 ml), acidified with TFA (10 ml),
evaporated and dissolved in TFA (50 ml) with triisopropylsilane (2
drops). After one night the TFA was evaporated and replaced:
after 2 days the deprotection was complete and the product was
precipitated with diethyl ether, filtered and dried under vacuum.
The crude product was purified by chromatography on an
Amberchrom CG161 resin (eluent: water/MeOH gradient). The
fraction containing the pure product was evaporated and freeze
dried. (4.8 g). 1H-NMR (600 MHz, D2O): δH = 4.06 (s, 2 H,
CH2COO), 3.93 (d, 2 H, CH2COO), 3.63-3.09 (m, 18 H, CH2), 3.39
(s, 2H, -CH2C(CH3)2OH), 1.36 (s, 6 H, CH3) ppm. 13C-NMR (150
General: All chemicals were purchased from Sigma-Aldrich Co.
and
were
used
without
purification.
acid
1,4,7,10-
1,4,7-tris(1,1-
tetraazacyclododecane-1,4,7-triacetic
dimethylethyl) ester (1) was synthesized according to literature
procedures.[18] Mass spectra were recorded on a Waters Acquity
UPLC H class coupled with QDa detector mass spectrometer
(direct infusion with H2O 0.1% COOH (or TFA)/ACN 0.1% COOH
(or TFA) 50:50). pH measurements were made using an AS
instruments pH meter equipped with
a glass electrode.
Chromatographic purification was performed using an AKTA
Purifier equipped with a UV-900 system, P-900 pump, frac-920
fraction collector and an Amberchrom® CG161 resin column. 1H-
NMR spectra were acquired at 14.1 T on a Bruker Avance 600
spectrometer equipped with a 5 mm probe and standard
temperature control unit.
Synthesis: 2-(2-bromoethoxy)tetrahydro-2H-pyran (2).
A
solution of 2-bromoethanol (40.5 g; 23 ml; 0.33 mol) and
pyridinium p-toluenesulfonate (8.3 g; 33 mmol) in DCM (150 ml)
was cooled with an ice bath and 3,4-dihydropyran (41.6 g; 45 ml;
0.5 mol) in DCM (50 ml) was slowly added. After one night at room
temperature, the mixture was washed with water (3 x 100ml) and
brine (100 ml), dried over Na2SO4 and distilled: the 2-(2-
6
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10.1002/chem.202000479
Chemistry - A European Journal
FULL PAPER
MHz, D2O): δC = 173.2 (-COO), 168.6 (-COO), 67.7 (-C(CH3)2OH), Gd-HMPDO3A MS ESI: m/z cald 574.15 [M+H+]; found 574.24.
62.7 (-CH2C(CH3)2OH), 54.7 (-CH2COO), 52.6 (CH2COO), 52.3,
50.6, 47.5, 46.6 (CH2 cyclen ring), 26.6 (-CH3) ppm. MS ESI m/z
cald 419.25 [M+H+]; found 418.95.
Eu-HMPDO3A 1H-NMR (600 MHz, D2O): δH = 38.95, 33.92, 33.69,
32.87, 32.44, 31.53, 27.39, 22.44, 15.86, 13.93, 13.37, 12.99,
11.34, 10.39, 9.03, 5.64, 5.42, 3.39, 1.77, 1.07, 0.94, 0.58, -1.11,
-1.31, -2.03, -4.08, -4.71, -5.49, -6.17, -6.62, -7.16, -7.34, -7.60, -
7.71, -8.31, -8.79, -9.29, -9.58, -9.70, -11.07, -11.45, -11.84, -
12.07, -12.44, -13.50, -14-44, -14.67, -18.46, 19.43, -20.48, -
20.74, -21.05, -21.24, -22.12, -24.32 ppm. MS ESI: m/z cald
569.15 [M+H+]; found 569.27.
1,4,7,10-tetraazacyclododecane-10-(3-hydroxy-2-
methylpropyl)-1,4,7-triacetic acid 1,4,7 tri-tert-butyl ester (7).
To a solution of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid 1,4,7-tris(1,1-dimethylethyl) ester hydrobromide (1) (1.5 g;
2,5 mmol) in 25 ml of ACN, K2CO3 (−325 mesh, 1.4 g; 10 mmol)
and 3-bromo-2-methyl-1-propanol (0.5 g; 342 µl; 3.2 mmol)
dissolved in 5 ml of ACN were added. The reaction was monitored
by MS ESI+. The mixture was fitered and evaporated to residue
(1 g).
High Resolution 1H-NMR: For high resolution 1H-NMR of
europium complexes, samples were dissolved in D2O at ca. 100
mM concentration and the pH adjusted with NaOD or DCl to 7 (pD
= 7.4). The temperature was varied from 275 to 350 K according
to the experiments. EXSY experiments were acquired using the
standard NOESY pulse sequence (90°-t1-90°-τM-90°) with the
mixing time (τM) set to 5 ms unless otherwise stated.
1,4,7,10-tetraazacyclododecane-10-(3-hydroxy-2-
methylpropyl)-1,4,7-triacetic acid. (8, HMPDO3A) The crude
10-[2-methyl-3-hydroxypropyl]-1,4,7,10-tetraazacyclododecan-
1,4,7-triacetic acid 1,4,7-tris(1,1-dimethylethyl) ester (7) was
dissolved in DCM (15 ml), acidified with TFA (3 ml), then
evaporated and dissolved in TFA (15 ml) with triisopropylsilane (2
drops). After one night the TFA was evaporated and replaced:
after 2 days the deprotection was complete and the product was
precipitated with diethyl ether, filtered and dried under vacuum.
The crude product was purified by chromatography on an
Amberchrom CG161 resin (eluent: water). The fraction containing
the pure product was freeze dried. (450 mg). 1H-NMR (600 MHz,
Relaxometric Measurements: R1obs values were determined by
inversion recovery at 21.5 MHz and 25 °C using a Stelar
SpinMaster spectrometer (Stelar Snc, Meade (PV), Italy).
Temperature was controlled with a Stelar VTC-91 airflow heater
and the temperature inside the probe checked with a calibrated
RS PRO RS55-11 digital thermometer. Data was acquired using
a recovery time ≥ 5 x T1 and with 2 scans per data point. The
absolute error in R1 measurements was less than 1 %.
D2O): δH = 4.04-2.93 (m, 26 H, CH2 cyclen ring and pendant arms), The gadolinium concentration of the complexes was determined
2.43 (br, 1 H, -CHCH3CH2OH), 0.94 (d, 3 H, CH3). 13C-NMR (150
MHz, D2O): δC = 173.5 (-COO), 173.1 (-COO), 169.4 (-COO), 66.2
(-CH2OH), 60.0 (-NCH2CH-), 55.9 (-CH2COO), 52.1 (-CH2COO),
52.0 (-CH2COO), 51.6, 50.8, 49.8, 49.1, 48.5, 48.0, 46.0, 44.5
(CH2 cyclen ring), 28.5 (-CHCH3), 12.7 (-CH3) ppm. MS ESI: m/z
cald 419.25 [M+H+]; found 419.32.
using the previously described relaxometric technique.[36] Briefly,
gadolinium complex solutions were mixed in equal volumes with
37 % HCl and heated in sealed vials at 120 °C overnight to
solubilize the free Gd3+ aqua ion. The R1 of the solution was
measured at 25 °C and 21.5 MHz and the concentration
determined using the equation: R1obs = R1d + r1PGd [Gd]. Where R1d
Gd
is the diamagnetic contribution (0.5 s-1) and r1P is the relaxivity
Complexation with Ln(III): A similar method to the following
procedure was used for all lanthanide complexations.
Characterisation data for all complexes are listed. The isotope
patterns in the mass spec data were consistent with Eu(III) and
Gd(III). Only fully resolved peaks are reported for 1H-NMR
recorded at 275 K.
(13.5 mM-1s-1) under the same experimental conditions.
Transmetallation experiments were performed as follows: Gd
complexes were dissolved in phosphate buffer (26 mM KH2PO4
and 41 mM Na2HPO4) at pH 7. ZnCl2 solution (250 mM ZnCl2 in
H2O) was added to give an equimolar Gd : Zn solution. Two
samples were measured for each complex and the average of
R1obs taken. The relaxation rate of the solution at 298 K, 21 MHz
was measured (t=0) the tubes were kept at 310 K and subsequent
measurements were taken over the course of a week.
Gd-HEDO3A
The
1,4,7,10-tetraazacyclododecane-10-(2-
hydroxy-ethyl)-1,4,7-triacetic acid tetrahydrochloride (4) (150
mg;0.3 mmol) was dissolved in water (10 ml) and basified to pH 7
with NaOH 0.1N. A solution of GdCl3 in water was gradually added, Anion interaction was determined by measurement of the
while maintaining pH 7 with NaOH 0.1N, until the presence of
residual free Gd3+ in the solution was confirmed by the orange
xylenol UV method.[35] The product was purified by
chromatography on an Amberchrom CG161 resin (eluent:
water/ACN gradient). The fraction containing the pure product
was evaporated and freeze dried. (97 mg). MS ESI: m/z cald
546.12 [M+H+]; found 546.05.
relaxation rate at 298 K and 21 MHz in 10 mM HEPES with 150
mM NaCl at pH 7.4, with addition of a known concentration of
anion (0-50 mM).
NMRD and 17O-NMR: NMRD profiles were obtained using a
Stelar SpinMaster FFC NMR relaxometer from 0.01 – 20 MHz and
a Bruker WP80 NMR electromagnet for variable higher-field
measurements (21.5 – 70 MHz), both equipped with a Stelar VTC-
Eu-HEDO3A 1H-NMR (600 MHz, D2O): δH = 35.66, 34.90, 29.86,
24.71, 13.33, 11.37, 10.57, 7.55, 5.49, 3.98, 3.34, 2.05, 1.34, 0.39, 91 for temperature control. Aqueous and human serum solutions
-0.83, -1.96, -2.44, -3.57, -4.52, -6.06, -7.49, -8.19, -9.07, -9.79, -
10.58, -11.31, -11.97, -14.54, -15.37, -16.06, -17.35, -19.02, -
21.30 ppm. MS ESI: m/z cald 541.12 [M+H+]; found 541.21.
Gd-HIBDO3A MS ESI: m/z cald 574.15 [M+H+]; found 574.24.
Eu-HIBDO3A 1H-NMR (600 MHz, D2O): δH = 15.36, 12.59, 12.17,
of the Gd complexes ([Gd] = 0.5 - 1.5 mM) were measured at 298
K and 310 K.
For 17O-NMR measurements, 10 mM Gd solutions of the
complexes in H2O with 1% H217O isotope (Cambridge Isotope)
were made with capillary inserts of D2O and measured on a
10.87, 6.60, 3.99, 3.52, 2.33, 0.19, -1.51. -4.17, -6.16, -6.72, -7.16, Bruker Avance 600 MHz spectrometer with temperature control.
-8.77, -11.18, -12.21. -13.05, -16.05 ppm. MS ESI: m/z cald
569.15 [M+H+]; found 569.21.
The full width at half maximum (Δω) was used to calculate the
7
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10.1002/chem.202000479
Chemistry - A European Journal
FULL PAPER
observed transverse relaxation rate using the equation: R2
π[Δωobs – ΔωH2O].
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8
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FULL PAPER
Entry for the Table of Contents
O
N
R
O
R =
OH
N
Gd
OH
N
O
N
O
OH
O
O
500
400
300
200
100
0
OH
280
300
320
340
T (K)
The hydroxyl arm of Gd-HPDO3A has been modified to form three derivatives, which have been fully analysed for their relaxometric
properties. It was found that these small modifications could significantly alter the proportion of isomers and increase the water
exchange rate through minor adjustments to the hydroxyl group environment. In particular, increasing the steric bulk around the OH
group allowed the properties of Gd-HPDO3A to be optimised.
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