Carbazole as Linker for Dinuclear Gadolinium-Based MRI Contrast Agents

Article abstract of DOI:10.1002/ejic.201700847

Ligands able to complex two gadolinium ions have been synthesized and characterized in view of the ability of the complexes to increase the spin relaxation of water protons. All ligands are based on the heptadentate diethylenetriaminetetraacetic acid (DTTA) chelator and carbazole as a rigid linker. Depending on the derivatization on the nitrogen atom of the five-membered ring, the compounds form small aggregates in aqueous solution, self-assemble to form micelles or bind to human serum albumin. In all cases, this leads to a marked increase in ¹H relaxivity at nuclear Larmor frequencies between 20 and 60 MHz. Water exchange on the gadolinium ions as measured by ¹⁷O NMR relaxation is fast enough not to limit relaxivity. ¹H nuclear magnetic relaxation dispersion profiles were also measured and analyzed using Solomon–Bloembergen–Morgan theory including Lipari–Szabo treatment to include internal motion or anisotropic rotation.

Full text of DOI:10.1002/ejic.201700847

A Journal of
Accepted Article
Title: Carbazole as linker for dinuclear gadolinium based MRI Contrast
Agents
Authors: BibiMaryam Mousavi, Anne-Sophie Chauvin, Loïck Moriggi,
and Lothar Helm
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201700847
Link to VoR: http://dx.doi.org/10.1002/ejic.201700847

10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
Carbazole as linker for dinuclear gadolinium based MRI Contrast
Agents
Bibimaryam Mousavi,[a,b] Anne-Sophie Chauvin,[a] Loïck Moriggi,[a] and Lothar Helm*[a]
Abstract: Ligands able to complex two gadolinium ions have been
synthesized and characterized in view of the ability of the complexes
to increase spin relaxation of water protons. All ligands are based on
the heptadentate DTTA chelator and carbazole as a rigid linker.
Depending on the derivatization on the nitrogen atom of the five-
membered ring, the compounds form small aggregates in aqueous
solution, self-assemble to form micelles or bind to human serum
albumin. In all cases, this leads to a marked increase in 1H relaxivity
at nuclear Larmor frequencies between 20 and 60 MHz. Water
exchange on the gadolinium ions as measured by 17O NMR relaxation
correlation times of 0.5 to 1 ns will give best results at magnetic
fields above 3 T [5]
.
τ
global
-OOC
τ
H
-OOC
N
local
O
COO-
COO-
H
H
O
Gd3+
1
is fast enough not to limit relaxivity. H nuclear magnetic relaxation
linker
N
H-
N
N
OOC
-OOC
dispersion profiles have been measured and analysed using
Solomon-Bloembergen-Morgan theory including Lipari-Szabo
treatment to include internal motion or anisotropic rotation.
N
Gd3+
H
O
N
H
-
O
COO
COO-
H
H
Introduction
In recent years, the number of installed high field MRI scanners
working at magnetic fields of 3 T continuously increased. Even if
1.5 T scanners are still the most widely used, the portion of higher
field instruments is expected to grow in the next years.[1] This
evolution has consequences for the development of contrast
agents administered in more than a third of all MRI examinations.
Most gadolinium based contrast agents used in clinics have been
developed at a time when low field scanners working at 60 MHz
or below were mostly installed.[2,3] There is a need for new
contrast agents which are adapted for higher magnetic fields and
for new application which become feasible with this new
instruments.[4–6] Increasing efficiency of MRI contrast agents at
low magnetic field is, at least in theory, relatively simple: rotational
diffusion and electron spin relaxation have to be slow and
exchange of inner sphere water has to be optimal.[3,7–9] At high
magnetic field the situation is more complex. Both, rotational
diffusion and water exchange have to be optimal; relaxation of the
Gd3+ electron spin is however of no importance. From theoretical
considerations follows that mid-size compounds with rotational
Figure 1. Schematic representation of a dinuclear complex showing global and
local rotational diffusion
To achieve a high local enhancement of relaxation it is therefore
a good choice to construct relatively small compounds that can
accommodate several paramagnetic centres. Examples of such
[10–18]
compounds are dinuclear complexes with two Gd3+ ions
(Figure 1) and trinuclear complexes with three Gd3+ ions
[19–22]
.
These compounds have rotational correlation times below 1 ns
and relaxation enhancement is two to three times higher than on
compounds with only one paramagnetic centre. A closer look to
the parameters governing relaxivity, the relaxation enhancement
per mM of Gd3+, reveals that besides the global rotation of the
compound local internal motion can strongly influence the
interaction between the 1H nuclear spin of bound water molecules
and the spin of the seven unpaired electrons of the lanthanide
ion.[8,23,24]
An essential part of di- or in general multinuclear compounds is
the linker connecting the chelates bearing the paramagnetic
centres (Figure 1). Some linkers used are designed to make the
compound responsive for example by its ability to bind cations like
Ca2+.[11,12,16,25] Other linkers are hydrophobic organic molecules
like benzene or poly-benzene. It has been shown that these
compounds tend to form aggregates in aqueous solution, most
probably due to interaction between the aromatic rings.[15,18,22] The
aggregation leads to longer rotational correlation times and
therefore to higher relaxivities mainly in the frequency range
between 20 and 80 MHz. These aggregates are very dynamic
with fast exchange between monomeric and multimeric
species.[18,22] In this situation, a unique rotational correlation time
[a]
[b]
Institut des Sciences et Ingénieries Chimiques
Ecole Polytechnique Fédérale de Lausanne (EPFL)
1015 Lausanne, Switzerland
E-mail: lothar.helm@epfl.ch
State Key Laboratory of Advanced Technology fort Materials
Synthesis and Processing
Wuhan University of Technology
Wuhan, 430070, PR China
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
2-
cannot describe the rotational diffusion responsible for the
fluctuation of the local magnetic fields.
H
H
OOC
N
Gd3+
COO
H
O
O
H
H
OOC
COO
Gd3+
N
H
N
O
O
H
H
OOC
OOC
COO
COO
N
N
N
NH
2-
O)4
[Gd2(Lcarb)(H2
]
2-
H
H
OOC
N
COO
N
H
O
O
H
H
O
H
H
OOC
Gd3+
COO
Gd3+
N
O
H
OOC
COO
N
N
N
OOC
COO
N
2-
O)4
[Gd2(Lcarb-biphen)(H2
]
H
H
O
OOC
N
COO
N
O
2-
H
H
H
OOC
COO
Gd3+
H
H
O
O
H
Gd3+
OOC
OOC
COO
COO
N
N
N
N
N
2-
O)4
[Gd2(Lcarb-C18)(H2
]
2-
H
OOC
COO
N
H
O
O
H
OOC
COO
H
N
H
H
O
O
H
Gd3+
Gd3+
H
N
N
N
N
OOC
OOC
COO
COO
N
2-
O)4
[Gd2(Lbipa-C18)(H2
]
Figure 3. Structures of the gadolinium complexes of this study:
[Gd2(Lcarb)(H2O)4]2-,
[Gd2(Lbipa-C18)(H2O)4]2-.
[Gd2(Lcarb-biphen)(H2O)4]2-,
[Gd2(Lcarb-C18)(H2O)4]2- and
IS
Figure 2. Inner sphere relaxivity r1 as a function of the Larmor frequency ν
calculated for different global correlation times τg and different order parameters
S2 (SBM theory, kex = 108 s-1, τv = 10 ps, Δ2 = 10-19 s-2, rGdH = 3.1 Å, τl = 200 ps).
These observations let us to choose a new linker to build
dinuclear compounds in view of application as gadolinium based
MRI contrast agent. Carbazole is a rigid molecule with the
possibility to attach two chelating units to the benzene rings
(Figure 3). By attaching the central nitrogen of an acyclic
polyaminocarboxylate directly to an aromatic carbon, a relatively
high rigidity of the dinuclear compound should be obtained.
Carbazole can be further derivatized by adding substituents to the
nitrogen atom of the five-membered ring. It is interesting to note
that carbazole is also known as chromophore. For example,
lanthanide complexes with β-diketonate functionalized carbazoles
show interesting photophysical properties which could eventually
be exploited using for example Eu3+.[29–32]
A simple theoretical description of rotational motion with two
characteristic correlation times has been developed by Lipari and
Szabo to describe nuclear spin relaxation in macromolecules.[26,27]
This so called model free approach has been successfully
adapted to describe the rotational behaviour of contrast agents.[28]
Adding a local correlation time and an order parameter opens the
parameter space but finally does not resolve the problem of
relatively low relaxivities at high frequencies (Figure 2). At 400
MHz (9.4 T) for example global correlation times around 1 ns are
still optimal. From these simulations, one can see that high order
parameters (S2 > 0.5) lead to highest r1 values. High order
IS
parameters are in general obtained by using rigid linkers or by
forming stable densely packed aggregates like micelles.
Therefore, we have synthetized three ligands in that series:
[Gd2(Lcarb)(H2O)4]2- for which the carbazole moiety was attached
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10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
to two acyclic polyaminocarboxylate moieties. Addition of RESULTS
substituents on the nitrogen atom of the five-membered ring was
also performed. [Gd2(Lcarb-C18)(H2O)4]2- was designed in order to
For the synthesis of ligand H8Lcarb 6, 3,6-diaminocarbazole was
synthesized in two steps, and subsequently reacted with four
form aggregates in soluteon and [Gd2(Lcarb-biphen)(H2O)4]2- in order
to bind macromolecules like Human serum albumin (HSA). The
last ligand contained a biphenylamine core with a long C18 alkyl
chain instead of the carbazole one [Gd2(Lbipa-C18)(H2O)4]2-, and
was synthetized for comparative purposes.
equivalents
of
2-[bis-(tert-butyloxycarbonylmethyl)amino]
ethylbromide (Figure 4). At this step we could have used
protected diethylentriamine; however, using 2-[bis-(tert-
butyloxycarbonyl-methyl)-amino]ethylbromide was preferred to
decrease steric problems.
ROOC
ROOC
COOR
COOR
COOtBu
COOtBu
ROOC
ROOC
N
N
N
Br
COOR
N
X
X
4
N
H
2O
COOR
Cu(NO3)2.2.5
N
N
HAc, AC2O 100°C
Diisopropylethylamine
DMF
N
H
N
H
N
1
2
X=NO2
H
Sn, Conc. HCl,
reflux 30 h
3 X= NH2
5
R= tBu
TFA/DCM
50:50
6 R= H
Lcarb
H8
ROOC
COOR
COOR
ROOC
ROOC
ROOC
N
N
COOR
N
COOR
N
N
N
NaH, THF
5
N
Br
7
R= tBu
TFA/DCM
50:50
8 R= H
H8Lcarb-biphen
ROOC
ROOC
ROOC
ROOC
COOR
COOR
X
O2N
NO2
DMF, K2CO3
N
N
N
4
COOR
COOR
N
N
1-bromooctadecane
X
Diisopropylethylamine,
DMF
N
N
N
C18H38
H
2
9
X=NO2
MeOH
N
H2 Pd/C
,
10 X= NH2
11 R1
NO2
=
tBu
H
TFA/DCM
50:50
H2N
NH2
12 R1
H8
=
Lcarb-C18
+
2
O
DMSO
ROOC
ROOC
COOR
COOR
O2N
NO2
X
X
4
DMF
K2CO3
N
N
N
N
Diisopropylethylamine
DMF
H
C18H37
1-bromooctadecane
N
N
13
ROOC
N
N
COOR
COOR
14 X=NO2
15 X= NH2
N
ROOC
MeOH
H2 Pd/C
,
16 R1 tBu
=
TFA/DCM
50:50
17 R1
L
H8
=
H
bipa-C18
Figure 4. Synthesis of the 4 ligands H8Lcarb , H8Lcarb-biphen , H8Lcarb-C18 and H8Lbipa-C18
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10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
For the synthesis of ligand H8Lcarb-biphen, compound 5 was reacted
with 4-(bromo-methyl) biphenyl using a strong base (NaH) in dry
THF. Due to lots of aromatic groups, the purification of this
compound was not possible by normal phase chromatography or
even using a C18-column in reverse phase chromatography. We
prepared a homemade reverse-phase called C3 column which
was used for purification. After purification, the tert-butyl
protecting groups of compounds 5 and 7 were removed by
TFA/DCM to give both ligands.
For the synthesis of compound 12, two different strategies could
be used. One was direct reaction of 5 with 1-bromooctadecane
using NaH in dry tetrahydrofuran which resulted in a very low 9%
yield. Another route was elaborated from carbazole as a starting
material. Subsequently, the reaction of 2 with 1-bromooctadecane
in DMF using potassium carbonate lead to 3,6-dinitro-9-
octadecyl-9H-carbazole 9. The reduction of the nitro groups of
compound 9 to amine groups was performed using palladium on
activated charcoal in methanol and compound 10 was obtained.
The reduction step followed by reaction of the intermediate 10
Figure 5. 1H NMR relaxivity as a function of [Gd3+] for [Gd2(Lcarb)(H2O)4]2- ()
and [Gd2(Lcarb-biphen)(H2O)4]2- () (B0 = 0.94 T; ν = 40 MHz; 37°C; 1/T1d = 0.37 s-
)
1
In case of [Gd2(Lcarb-biphen)(H2O)4]2- the relaxivity increases by
more than 70% if the concentration of [Gd3+] is raised from 0.033
mM to 1 mM (Figure 5). At low concentration relaxivity is close to
that of [Gd2(Lcarb)(H2O)4]2-. After a marked increase up to [Gd3+]
~0.7 mM r1 reaches a plateau with r1~ 22 mM-1 s-1. A simple
interpretation of that data is that the monomer of biphenyl-
carbazole [Gd2(Lcarb-biphen)(H2O)4]2- has about the same relaxivity
and therefore rotational correlation time as [Gd2(Lcarb)(H2O)4]2-.
Starting at concentrations of 0.05 mM [Gd2(Lcarb-biphen)(H2O)4]2-
forms aggregates in aqueous solution leading to slower rotational
diffusion and therefore higher relaxivities at 40 MHz. The
increased hydrophilicity due to the introduction of the biphenyl
group is clearly responsible for the formation of aggregates. The
increase of r1 by a factor of about two indicates that the
aggregates formed are small comprising two or three monomer
units at most.
with
excess
2-[bis-(t-butyloxycarbonylmethyl)-amino]-ethyl-
bromide led to compound 11. The final product 12 was obtained
by hydrolysis of the ester precursor with TFA in DCM solvent.
Compound 17 was designed and synthesized to have a similar
structure as 12.
Concentration effect on relaxivity
The more or less hydrophobic linkers are suspected to show
aggregation in aqueous solution due to the carbazole itself and
due to the attached biphenyl- or C18 groups. The two complexes
with carbazole [Gd2(Lcarb)(H2O)4]2- and biphenyl-carbazole
[Gd2(Lcarb-biphen)(H2O)4]2- are suspected to form small aggregates
composed of a few monomers.[15,18,22] The compound with C18-
carbazole [Gd2(Lcarb-C18)(H2O)4]2- should be able to form
nanosized micelles due to interactions between the long
hydrophobic chains.[33] To check for formation of aggregates and
micelles in aqueous solution 1H relaxivities have been measured
as a function of concentration at 40 MHz and 37 °C. Formation of
aggregates should lead to a marked increase in relaxivity at that
Larmor frequency due to the increase in rotational correlation
time.
Complexes [Gd2(Lcarb-C18)(H2O)4]2- and [Gd2(Lbipa-C18)(H2O)4]2-
have a long hydrophobic chain attached to the core which will
drive the formation of micelles in aqueous solution. The nanosized
micelles are formed above a critical micellar concentration, CMC.
The formation of micelles leads to slower rotational diffusion of
the gadolinium chelates and therefore a marked increase in
relaxivity at magnetic fields between 0.5 and 1.5 T.
The biphenyl compound [Gd2(Lbipa-C18)(H2O)4]2- shows a marked
The relaxivity of [Gd2(Lcarb)(H2O)4]2- with the two chelates linked to
a simple carbazole is nearly independent on concentration up to
increase in r1 at [Gd3+] ~ 0.1 mM (Figure 6). Using equations
[33]
developed by Nicolle
(see ESI) relaxivities of 12 mM-1 s-1
4 mM in [Gd3+] (Figure 5). The mean value calculated, r1
11.7±0.3 mM-1s-1, corresponds to values expected for
∼
a
(below the CMC) and 43 mM-1 s-1 (above the CMC) are calculated
for [Gd2(Lbipa-C18)(H2O)4]2-. The complex with the carbazole linker
[Gd2(Lcarb-C18)(H2O)4]2- shows high relaxivity already at the lowest
concentration measured ([Gd3+] = 0.04 mM, Figure 6). T small
change in relaxivity can be assigned to a change in size of the
micelles around [Gd3+] = 0.1 mM. The relaxivity plateau with r1 ~
46 mM-1 s-1 is reached at [Gd3+] ~ 0.1 mM. Only an upper limit of
the CMC can therefore be given for this compound: CMC < 0.1
mM.
monomeric dinuclear compound. This relaxivity is similar to that
[19]
of [Gd2bpy(DTTA)2(H2O)4]2-
(r1= 12.4 mM-1s-1 at 20 MHz and
o
37 C) and higher than r1 found for other previously studied
dinuclear complexes such as [pip[GdDO3A(H2O)]2] (r1= 5.6 mM-1
s-1) and [bisoxa[GdDO3A(H2O)]2] (r1= 4.4 mM-1 s-1) at 40 MHz and
37 oC [10]. This increase in relaxivity by a factor of two can be
explained by the presence of two inner-sphere water molecules
in case of the DTTA chelator.
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10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
Figure 6. 1H NMR relaxivity as a function of [Gd3+] for [Gd2(Lcarb-C18)(H2O)4]2- (∆)
and [Gd2(Lbipa-C18)(H2O)4]2- (); (B0 = 0.7 T; ν = 30 MHz; 25°C)
17O and 1H NMRD measurements
To obtain detailed information on rotational diffusion behaviour
and on water exchange on the carbazole based dinuclear
compounds we measured 1H nuclear magnetic relaxation
dispersion (NMRD) profiles and 17O transverse relaxation
enhancements, 1/T2r, (Figure 7).
Figure 7 Left: 1H NMRD profiles measured at 25 °C (blue) and 37°C (red); right: 17O transverse relaxation enhancement 1/T2r measured at 9.4 T; A:
[Gd2(Lcarb)(H2O)4]2- [Gd3+] = 4.01 mM; B: [Gd2(Lcarb-biphen)(H2O)4]2- [Gd3+] = 0.66 mM (), [Gd3+] = 0.58 mM (▲); C: [Gd2(Lcarb-C18)(H2O)4]2- [Gd3+] = 2.16 mM (), [Gd3+
]
= 7.09 mM (▲),[Gd2(Lbipa-C18)(H2O)4]2- [Gd3+] = 0.62 mM (),[Gd3+] = 0.04 mM (). The lines correspond to a simultaneous fit of the experimental data points using
SBM theory.
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10.1002/ejic.201700847
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The relaxivities of all compounds show a maximum in the MRI
relevant frequency region between 20 and 60 MHz (Figure 7, left).
The exact position and the highest relaxivity at the maximum
depends on electron spin relaxation and on the rotational
correlation time.[34] Comparison of the relaxivities in Figure 7
shows that the frequency of the maximum shifts to lower values
and the value of r1 increases by replacing the proton on the
carbazole nitrogen by a biphenyl group (B) or by a C18 chain (C).
Qualitatively, an increase of the rotational correlation time can be
deduced from this data.
The relaxivity curves of both micelle forming compounds,
[Gd2(Lcarb-C18)(H2O)4]2- and [Gd2(Lbipa-C18)(H2O)4]2-, both measured
at concentrations above the CMC, are identical (Figure 7 C, left).
This shows that both form micelles of similar size at those
concentrations (see ESI for DLS and zeta potentials). The NMRD
profile of [Gd2(Lbipa-C18)(H2O)4]2- has also been measured for a
concentration below the CMC. The maximum of relaxivity is <10
s-1 mM-1, clearly showing the absence of the formation of
micelles at that concentration.
Surprisingly, the transverse relaxation rates measured by 17O
NMR differ for the three carbazole compounds (Figure 7 right).
1/T2r gives direct access to water exchange rate constants, kex.[35]
The chelators used for all dinuclear compounds are all the same,
DTTA, and therefore one would expect that water exchange does
not differ markedly between the three compounds.
The combined analysis of 1H NMRD and 17O 1/T2r using standard
Solomon-Bloembergen-Morgan (SBM) theory together with the
Lipari-Szabo approach for anisotropic or internal rotation allowed
getting rotational correlation times and water exchange rate
constants. (Table 1, for details see ESI). Only relaxivities at
Larmor frequencies > 5 MHz have been included in the fit to
account for the limited validity of SBM theory.
The water exchange rate constants, kex298, obtained from the fit
(Table 1) confirm the observation made on the experimental data.
At 25°
C
water exchange is three times faster on
[Gd2(Lcarb)(H2O)4]2- compared to [Gd2(Lcarb-biphen)(H2O)4]2- and the
micellar [Gd2(Lcarb-C18)(H2O)4]2-. One should however keep in mind
that the statistical error is relatively big due to strong correlation
of the parameters.
Table 1. Best fit parameters of 1H NMRD and 17O NMR using Solomon-Bloembergen-Morgan theory including Lipari-Szabo approach.
Parameters
[Gd2(Lcarb)(H2O)4]2-
1/T2 17O, NMRD
58 ± 10
[Gd2(Lcarb-biphen)(H2O)4]2-
1/T2 17O, NMRD
35 ± 3
HSA-[Gd2(Lcarb-biphen)(H2O)4]2-
[Gd2(Lcarb-C18)(H2O)4]2-
1/T2 17O, NMRD
52 ± 4
NMRD, fit A NMRD, fit B
∆H‡ (kJ mol-1)
58
-
36
∆S‡ (J K-1 mol-1)
+90 ± 23
+8 ± 7
-
+64 ± 13
298
kex
(106 s-1)
(ps)
24 ± 12
8 ± 2
24
8
8 ± 3
298
725 ± 220
1050 ± 300
360 ± 80
3400 ± 460
71 ± 11
0.28 ± 0.02
3100 ± 470
71 ± 14
0.29 ± 0.02
6000 ± 1600
75± 10
τg
τl298
S2
(ps)
170 ± 25
0.19 ± 0.09
0.35 ± 0.16
0.42 ± 0.02
Concerning the rotational motion as characterized by rotational
correlation times in all cases two correlation times are necessary
to fit the data: a global rotational correlation time τg and a local
rotational correlation time τl (Table 1). The carbazole and the
biphenyl-carbazole compounds are characterized by τg ≤ 1 ns and
τl of 170 – 350 ps. The main difference in relaxivity comes from
the markedly higher order parameter for the aggregate forming
biphenyl-carbazole [Gd2(Lcarb-biphen)(H2O)4]2- (S2 ~0.35). The
micelle forming compound [Gd2(Lcarb-C18)(H2O)4]2- shows a longer
global correlation time τg ~ 6 ns but a shorter local one τl ~ 75 ps.
Again, the order parameter is relatively high, S2 ~0.4.
than 95% of [Gd2(Lcarb-biphen)(H2O)4]2- is bound to HSA (for binding
constants see ESI). The NMRD profile looks similar to that of
[38]
HSA-[triphenyl-[Gd(DOTMA)(H2O)]-]
which possesses a
similar HSA binding site. The relaxivity increases if the
temperature is lowered indicating that water exchange is not
limiting relaxivity in this case. No 17O NMR study could be
performed due to the very low concentration of gadolinium in the
sample.
The NMRD profiles have been fitted neglecting the small amount
of unbound complex and using water exchange parameters from
[Gd2(Lcarb)(H2O)4]2- (fit A) or from [Gd2(Lcarb-biphen)(H2O)4]2- (fit B,
see Table 1). The correlation times and the order parameter fitted
change only within less than one standard deviation confirming
that water exchange is not limiting relaxivity. The global rotational
correlation time τg ~3-4 ns is of the order of that of micelles.[39] The
HSA-[Gd2(Lcarb-biphen)(H2O)4]2- construct has however a lower
order parameter compared to that of the micelles.
[Gd2(Lcarb-biphen)(H2O)4]2- bound to HSA
Binding of gadolinium based MRI contrast agents to HSA results
in a remarkable enhancement in relaxivity in the frequency region
of 20 to 80 MHz.[36,37] The 1H NMRD profiles of a HSA-[Gd2(Lcarb-
biphen)(H2O)4]2- sample is shown in Figure 8 and, as expected,
indicates a marked relaxivity hump between 10 and 100 MHz. The
ratio of HSA to [Gd2(Lcarb-biphen)(H2O)4]2- is 3.9 and therefore more
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10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
Table 2. Comparison of water exchange parameters measured on Gd-DTTA
complexes
298
kex
∆H‡
∆S‡
(106 s-1)
(kJ mol-1)
(J K-1 mol-1)
[Gd2(Lcarb)(H2O)4]2-
24
8
58
36
52
+90
+8
This
work
[Gd2(Lcarb-biphen)(H2O)4]2-
This
work
[Gd2(Lcarb-C18)(H2O)4]2-
8
+64
This
work
Figure 8. 1H nuclear magnetic relaxation dispersion profiles of HSA-[Gd2(Lcarb-
biphen)(H2O)4]2- ([Gd3+]=77 ꢀM) at 25 oC (○) and 37 oC (□). The lines represent
the simultaneous least squares fit of points above 6 MHz (full line: fit A, dashed
line: fit B, Table 1).
[Gd2(pX(DTTA)2(H2O)4]2-
[Gd2(mX(DTTA)2(H2O)4]2-
[Gd2(bpy(DTTA)2(H2O)4]2-
{Ph4[Gd(DTTA)(H2O)2]-3}
[Gd(DTTA-Me)(H2O)]-
9.0
8.9
8.1
17
45.4
39.2
43.7
39.9
50
+41
+20
+34
+27
+64
Ref. [13]
Ref. [13]
Ref. [19]
Ref. [22]
Ref. [40]
DISCUSSION
Water exchange rate constants have now been measured on a
series of gadolinium chelates with the heptadentate DTTA
chelator. Most of the kex298 values measured are around 8106 s-
24.6
1
(see Table 2). Three compounds show an up to three times
faster water exchange, namely the monomer [Gd(DTTA-
Me)(H2O)]- [40], the dimer [Gd2(Lcarb)(H2O)4]2- and the tetramer
{Ph4[Gd(DTTA)(H2O)2]-3} [22]. The reason for this change in water
exchange is unclear. All exchange rate constants have been
measured by 17O NMR The charge of the Gd-DTTA units is -1
and the substitution is in all cases at the central nitrogen of the
DTTA. A dissociative activation mode should operate in all cases
as can be seen from the positive activation entropies and the
activation volume ∆V‡ of +8 cm3 mol-1 measured for water
CONCLUSION
Several novel compounds based on [carbazole-DTTA2]8- have
been synthesized all being able to complex two gadolinium(III)
ions. The carbazole unit has been chosen due to its rigid
structure, the possibility to connect the chelator directly to the
aromatic ring and the relatively simple possibility to connect
other groups to the carbazole nitrogen. It has been shown that
depending on the connected groups the dinuclear compounds
can in aqueous solution either build small aggregates, or self-
aggregate to form micelles or bind non-covalently to proteins like
HSA. The slow global rotational diffusion of the micelles formed
(τg ~6 ns) and the HSA bound complexes (τg ~3.2 ns) together
with the presence of two inner-sphere water molecules per Gd3+
leads to relaxivities > 40 s-1 mM-1 (at 30 MHz and 25 °C).
Exchange of the inner-sphere water molecules is fast enough
not to limit relaxivity. By replacing one gadolinium ion by another
lanthanide the dinuclear carbazole based compounds could be
investigated in view of its used as multimodal imaging agent.
[40]
exchange on [Gd(DTTA-Me)(H2O)]-.
To fit the NMRD profiles at least two rotational correlation times
are needed for all dinuclear carbazole compounds. In case of
[Gd2(Lcarb)(H2O)4]2- which does not aggregate over the
concentration range used in the study either anisotropic overall
rotation or internal rotation around the carbazole-DTTA bond can
be assigned to the two correlation times. In case of the
aggregate forming [Gd2(Lcarb-biphen)(H2O)4]2- it is difficult to assign
the rotational diffusion processes leading to τg and τl. Aggregates
of different size as well as monomers are present in solution. The
micelle forming [Gd2(Lcarb-C18)(H2O)4]2- shows the longest global
correlation time τR ~ 6 ns. Together with a high order parameter
this leads to the highest relaxivities at 30 MHz. Replacing the
carbazole by the less rigid biphenylamine does not change
relaxivity which allows us to conclude that the rotational
behaviour of [Gd2(Lcarb-C18)(H2O)4]2- and [Gd2(Lbipa-C18)(H2O)4]2-
micelles is identical. Global rotational correlation times of
[Gd2(Lcarb-biphen)(H2O)4]2- bound to HSA are nearly as long as
those of the micelles. Comparison with correlation times
obtained for other HSA bound contrast agents is not trivial
because often high field relaxivities (ν > 100 MHz) are missing
and therefore no analysis is possible using two rotational
correlation times.
Experimental Section
General: All manipulations were carried out under inert nitrogen
atmosphere using standard Schlenk technique unless otherwise
mentioned. Human serum albumin (HSA), product number A-1653
(Fraction V Powder 96-99% albumin), was purchased from Sigma
Chemical Co. Other reagents and HPLC solvents were supplied by
Sigma- Aldrich and Fluka Chemical Co. and were used without further
purification.
Compounds 2 and 3 were synthesized and characterized according to
Maity et al. [41]. 3,6-Dinitrocarbazole 2 gave a yellow solid (4.4g 70%) as
well as 3,6-Diaminocarbazole 3 (0.99 g, 51%.). 1H NMR for 2: (CD3CN,
This article is protected by copyright. All rights reserved.
7

10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
400MHz) δ in ppm: 7.72 (d, 2H), 8.38 (d, 2H), 9.2 (s, 2H), 10.43 (s, 1H).
disappearance of tert-butyl peaks and observation of broad peaks in the
expected aromatic and aliphatic frequency range confirm the complete
deprotection reaction. MS (ESI) m/z (%): 1000.0 (15) [M+H]+.
1
MS (ESI): 258.2 [M+1]+. H NMR for 3: (DMSO-d6, 400MHz) δ in ppm:
4.11 (s, 4H), 6.72 (d, 2H), 7.17 (s, 2H), 7.18 (d, 2H), 10.18 (s, 1H).
2-[bis-(tert-butyloxycarbonylmethyl) amino]-ethyl bromide 4 was
synthesized according to Achilefu
ppm: 1.49 (s, 18H), 3.18 (t, J=8 Hz, 2H), 3.45 (t, J=8 Hz, 2H), 3.5 (s, 4H).
MS (ESI) m/z (%): 352.63 (95) [M+H]+.
3,6-dinitro-9-octadecyl-9H-carbazole 9: 4,4'-dinitrocarbazole 2 (3.28
mmol, m=2.52 g), 1-bromooctadecane (3.6 mmol, m=1.20 g) and K2CO3
(9.8 mmol, m= 1.35g), were dissolved in dimethyformamide and stirred
[42]
.
1HNMR (CDCl3, 400 MHz) δ in
o
for 36 h at 80 C. The mixture was allowed to reach room temperature.
After addition of water the resulting precipitate was filtered, washed
several times with water and dried under reduced pressure. Normal
phase chromatography (CH2Cl2-MeOH 3%) was performed to obtain the
final pure compound as a pale yellow solid (1.13 g, 68 %). 1H NMR
(CDCl3 ppm, 400MHz) δ in ppm, 0.88 (t, 3H), 1.23 (m, 30H), 1.93 (m, 2H),
4.42 (t, 2H), 7.53 (dd, J:8 Hz, 2H), 8.48 (dd, J:8 Hz, 2H), 9.27 (s, 2H). MS
(ESI) m/z (%): 510.5 [M+H]+.
Tert-butyl 2,2',2'',2''',2'''',2''''',2'''''',2'''''''-(2,2',2'',2'''- (9H-carbazole-
3,6-diyl) bis (azanetriyl) tetrakis (ethane-2,1-diyl)) tetrakis
(azanetriyl) octaacetate 5: 2-[bis-(tert-butyloxy carbonylmethyl)amino]-
ethyl-bromide 4 (1.11 g, 3.15 mmol,), compound 3 (154 mg, 0.78 mmol),
and N,N-diisopropylethylamine, (0.83 mg, 6.45 mmol), were refluxed for
36 hours in anhydrous DMF (33 mL) under a nitrogen atmosphere. The
solvent was evaporated under vacuum at 40 oC. The crude product was
extracted in dichloromethane (2x33 mL) and the organic phase was
washed with water (33 mL), then brine (33 mL). The dichloromethane
layer was dried over sodium sulfate. The solvent was evaporated under
vacuum at 40 oC. Purification of the resulting compound was performed
using auto-preparative HPLC model Waters (4.6 × 150 mm Vydac C18)
using the gradient mixture of (H2O 99.89%, HCOOH 0.1%, TFA 0.01%)
and (CH3CN 99.89%, HCOOH 0.1% TFA 0.01%)) (see ESI for more
details regarding the gradient concentration). Compound 5 was obtained
with 96% purity as a pink solid (0.25 g, 25%). 1H NMR (DMSO-d6,
400MHz) δ in ppm: 1.38 (s, 72H), 2.75 (t, 8H), 3.34 (t, 8H + s,16H), 6.89
(d, J = 8 Hz, 2H), 7.17 (d, J=8 Hz, 2H), 7.41 (s, 2H), 10.35 (s, 1H). 13C
NMR (CD2Cl2, 54.00 MHz) δ in ppm: 28.72, 53.20, 57.36, 81.43, 106.75,
112.1, 116.15, 125.19, 135.1, 142.96, 171.38. (ESI): 1283 (33) [M+H]+,
9-octadecyl-9H-carbazole-3,6-diamine 10: A mixture of 9 (200 mg,
0.39 mmol) and 150 mg Pd/C in methanol, was stirred under H2
atmosphere. The reduction of the nitro groups was monitored by ESI-MS.
After completion of the reaction (30 h) the Pd/C was filtered and the
solvent was removed under reduced pressure. The resulting products
1
(125 mg) was used in the next step without further purification. H NMR
(MeOD, 400MHz) δ in ppm, 0.91 (t, 3H), 1.27 (m, 30), 1.84 (m, 2H), 4.39
(t, 3H), 7.31 (dd, J:8 Hz, 2H), 7.56 (dd, J:8 Hz, 2H), 7.89 (s, 2H). MS (ESI)
m/z (%): 450.5 [M+H]+.
Octa-tert-butyl2,2',2'',2''',2'''',2''''',2'''''',2'''''''((((9-octadecyl-
9Hcarbazole
-3,6-diyl)
bis(
azanetriyl))tetrakis(ethane-2,1-
diyl))tetrakis(azanetriyl)) octaacetate 11: The procedure was similar to
that of 5. Compound 4 (2.50 mmol m=0.88g), diisopropylethylamine (4.0
mmol, m= 0.52 g) and 10 (0.24 g, 0.54 mmol) in 15 mL of anhydrous
dimethylforamide were refluxed for 16 hours under N2 atmosphere. The
work up was identical. The crude product was purified by normal phase
chromatography on silica gel with hexane/ ethyl acetate 2:1 (Yield: 0.31
g, 36%). 1HNMR (CD2Cl2=4.85 ppm, 400MHz) δ in ppm 0.85 (t, 3H), 1.27
(m, 30H), 1.47 (s, 72), 1.72 (m, 2H), 2.92 (t, 8H), 3.49 (s,16), 3.72 (t, 8H),
4.25 (t, 2H), 7-7.6 (m, 6H). MS (ESI) m/z (%): 1535.17 (12) [M+H]+, 768.5
642.25 (100) [M+2H]2+
.
2,2',2'',2''',2'''',2''''',2'''''',2'''''''
-((((9H-carbazole-3,6-diyl)bis
(azanetriyl))tetrakis(ethane-2,1-diyl))tetrakis(azanetriyl))octaacetic
acid 6: The compound 5 (0.12 mmol) was stirred in a mixture of
TFA/DCM (50:50 mL), over 24 hours and the reaction monitored by MS
(ESI) and/or NMR (tert-butyl peaks disappearance) until completion. The
solvents were removed by evaporation, the remaining residue was
dissolved in water (10 mL) and the solution was re-evaporated to
dryness. This procedure was repeated five times to ensure total loss of
acids. H8Lcarb was obtained as a pale yellow solid (91 mg, 92 %). 1H-NMR
(D2O= 4.75, 400 MHz) δ in ppm: 3.43 (t, J=8 Hz, 8 H), 3.73 (t, J=8 Hz, 8
H), 3.89 (s, 16 H), 7.42 (d, J=8 Hz, 2 H), 7.60 (d, J=8 Hz, 2 H), 8.03 (s,
(100) [M+2H]2+
.
2,2',2'',2''',2'''',2''''',2'''''',2'''''''-((((9-octadecyl-9H-carbazole-3,6-
diyl)bis(azanetriyl)) tetrakis (ethane-2,1-diyl))tetrakis(azanetriyl))
octaacetic acid 12: Same procedure than for compound 6, starting from
(0.31 g, 0.20 mmol) of 11. Occasionally ethanol was added to the solution
to minimize the formation of foam. (Yield: 0.18g 85%). 1H NMR (MeOD,
d4 = 4.87 ppm, 400MHz) δ in ppm 0.86 (t, 3H), 1.25 (m, 30H), 1.88 (m,
2H), 2.92 (m, 8H), 3.57 (s, 16H), 3.82 (m, 8H), 4.47 (t, 2H), 7.78 (m, 4H),
8.46 (m, 2H). MS (ESI) m/z (%): 1086.83 (11) [M+H]+.
2H). MS (ESI-TOF) m/z (%): 417.66 (100) [M+2H]+2
.
Octa-tert-butyl 2,2',2'',2''',2'''',2''''',2'''''',2'''''''-((((9-([1,1'-biphenyl]-4-
ylmethyl)-9H-carbazole-3,6-diyl) bis(azanetriyl))tetrakis(ethane-2,1-
diyl)) tetrakis(azanetriyl))octaacetate 7: Compound 5 (154 mg, 0.12
mmol) and dry sodium hydride (5.76 mg, 0.24 mmol) were dissolved in
dry THF (10 mL) and heated to 70 oC under a nitrogen atmosphere for 2
hours. 4-(bromo-methyl) 1,1'-biphenyl was added to the solution and the
mixture was refluxed for 36 hours. Completion of the reaction was
monitored by ESI-MS. The solvent was evaporated under vacuum, and
the residue was extracted by dichloromethane (CH2Cl2/H2O 30:30 mL).
The organic phase was separated and washed with water (30 mL), and
then with brine (30 mL). Subsequently the dichloromethane layer was
dried over sodium sulfate. The compound 7 was further purified by
reverse phase chromatography on a homemade C3 column (see ESI),
using a gradient of ACN: H2O from 60:40 to 90:10%. 1H NMR (CDCl3,
400MHz) δ in ppm: 1.43 (s, 72H), 2.91 (t, 8H), 3.5 (t, 8H), 3.5 (s, 16H),
5.39 (s, 2H), 6.9 - 7.7, (15 ArH). MS (ESI) m/z (%): 724.91 (100)
[M+2H]+2, 1448.89 (3) [M+H]+.
4,4'-Dinitrodiphenylamine 13: The compound was synthesized
according to a literature procedure [43]. Yield: 2.8 g, 54%. 1HNMR (DMSO
= 2.5 ppm, 400MHz) δ in ppm 7.32 (d, J = 8 Hz, 4H), 8.23 (d, J = 8 Hz,
4H), 10.0 (s, 1H).
4-nitro-N-(4-nitrophenyl)-N-octadecylaniline 14: The same procedure
than that for 9 was used, starting from 4,4'-dinitrodiphenylamine (1.9
mmol m=0.51 g), 1-bromooctadecane (0.5 mmol m=0.17 g) and K2CO3
(2.0 mmol) to give a pale yellow solid 14 (0.08 g, 31%). 1H NMR (CD2Cl2
= 5.32 ppm, 400MHz) δ in ppm: 0.88 (t, 3H), 1.25 (m, 30H), 1.70 (m, 2H),
3.85 (t, 2H), 7.14 (d, 4H), 8.18 (d, 4H).
N1-(4-aminophenyl)-N1-octadecylbenzene-1,4-diamine 15: The
same procedure than that for 10 was used, starting from 14 (55 mg, 0.11
mmol) and 50mg Pd/C in methanol. Compound 15 (29 mg, 58%) was
used in the next step without further purification. 1HNMR (CDCl3, 400
MHz) δ in ppm: 0.86 (m, 3H), 1.25 (s, 30H), 3.5 (t, 2H), 3.33-3.52 (broad
band, 4H), 6.61 (dd, 4H), 6.74 (dd, 4H). MS (ESI) m/z (%): 452.6 (100)
[M+H]+.
2,2',2'',2''',2'''',2''''',2'''''',2'''''''-((((9-([1,1'-biphenyl]-4-ylmethyl)-9H-
carbazole-3,6-diyl)bis(azanetriyl))tetrakis(ethane-2,1-diyl))tetrakis
(azanetriyl))octaacetic acid 8: Same procedure than for compound 6,
starting from 0.23mmol (0.33 g) of 7. H8Lcarb-phen was obtained as a pale
yellow solid (0.20 g, 90%). It was not possible to obtain a good 1H NMR
for ligand 8 in aqueous solution, possibly due to aggregation. However,
This article is protected by copyright. All rights reserved.
8

10.1002/ejic.201700847
European Journal of Inorganic Chemistry
FULL PAPER
[48]
Octa-tert-butyl 2,2',2'',2''',2'''',2''''',2'''''',2'''''''-(((((octadecylazanediyl)
bis (4,1-phenylene))bis(azanetriyl))tetrakis(ethane-2,1-diyl))tetrakis
(azanetriyl))octaacetate 16: The procedure was similar to that of 11.
Compound 16 was obtained as a pale yellow solid (0.14 g, 18.2 %). H
NMR (CD2Cl2, 400MHz) δ in ppm 0.83 (t, 3H), 1.29 (s, 30), 1.49 (s,72 H),
measured using the inversion recovery
and the Carr-Purcell-
Meiboom-Gill pulse sequences [49], respectively.
1
Data Analysis: The simultaneous least squares fits of the 1H NMRD, 17
O
NMR and the determination of the HSA-ligand binding affinity constant
were carried out using the programs Visualizeur/Optimiseur running on a
Matlab platform, version 7.6.0 (R2008).[50]
2.80 (t, 8H), 3.42 (t, 8H), 3.48 (s, 16H), 6.68 (dd, 4H), 6.79 (dd, 4H). MS
(ESI) m/z (%): 1537.0 (26) [M+H]+, 769.42 (100) [M+2H]2+
.
2,2',2'',2''',2'''',2''''',2'''''',2'''''''-(((((octadecylazanediyl)
bis
(4,1-
phenylene))
bis(azanetriyl))tetrakis(ethane-2,1-diyl))tetrakis
Acknowledgments
(azanetriyl))octaacetate 17: The procedure was similar to that of 12.
Compound 17 was obtained as a pale yellow solid (89mg, 90 %). 1H NMR
spectrum of the compound 17 shows broad peaks due to aggregation in
solution. MS (ESI) m/z (%): [M] (15 %) 1087.
MM and LH acknowledge financial support from the Swiss
National Science Foundation.
Preparation of gadolinium complexes: A solution of GdCl3 in water
was prepared and the exact concentration of Gd3+ was measured by bulk
magnetic susceptibility (BMS) [44] at 21.2 oC on a Bruker DRX-400 (9.4 T,
400 MHz). For preparation of [Gd2(Lcarb)(H2O)4]2-, H8Lcarb (6) was
dissolved in water at room temperature and it's concentration was
determined by back titration of a Gd3+ excess with Na2H2EDTA (0.022
mM) using a Metrohm 665 Dosimat for titration (indicator: xylenol
orange). The final complexation with Gd3+ was performed with a small
excess of ligand (3%). The pH was adjusted to 5.9 with NaOH (0.05 M).
Absence of free Gd3+ was checked by the xylenol orange test [45]. After
complexation, the exact concentration of Gd3+ was measured by BMS,
[Gd3+] = 4.01 mM. Preparation of the other complexes was performed by
the same way. Gadolinium concentrations as measured by BMS were
Gd3+ = 0.66 mM for [Gd2(Lcarb-biphen)(H2O)4]2-, 2.16 mM for [Gd2(Lcarb-
C18)(H2O)4]2- and 1.15 mM for [Gd2(Lbipa-C18)(H2O)4]2-.
Keywords: Gadolinium • Imaging agents • Chelates •
Dinuclear complexes • Aggregation
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