Synthesis and characterization of a novel gadolinium-based contrast agent for magnetic resonance imaging of myelination

Article abstract of DOI:10.1021/jm301435z

Myelin is a membrane system that fosters nervous impulse conduction in the vertebrate nervous system. Myelin sheath disruption is a common characteristic of several neurodegenerative diseases such as multiple sclerosis (MS) and various leukodystrophies. To date, the diagnosis of MS is obtained using a set of criteria in which MRI observations play a central role. However, because of the lack of specificity for myelin integrity, the use of MRI as the primary diagnostic tool has not yet been accepted. In order to improve MR specificity, we began developing MR probes targeted toward myelin. In this work we describe a new myelin-targeted MR contrast agent, Gd-DODAS, based on a stilbene binding moiety and demonstrate its ability to specifically bind to myelin in vitro and in vivo. We also present evidence that Gd-DODAS generates MR contrast in vivo in T₁-weighed images and in T₁ maps that correlates to the myelin content.

Full text of DOI:10.1021/jm301435z

Article
pubs.acs.org/jmc
Synthesis and Characterization of a Novel Gadolinium-Based
Contrast Agent for Magnetic Resonance Imaging of Myelination
Luca Frullano,† Junqing Zhu,† Robert H. Miller,‡ and Yanming Wang*,†
†Departments of Radiology, Chemistry, and Biomedical Engineering, Case Center for Imaging Research, Division of
Radiopharmaceutical Science, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106, United States
‡Department of Neuroscience, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106, United States
S
* Supporting Information
ABSTRACT: Myelin is a membrane system that fosters
nervous impulse conduction in the vertebrate nervous system.
Myelin sheath disruption is a common characteristic of several
neurodegenerative diseases such as multiple sclerosis (MS)
and various leukodystrophies. To date, the diagnosis of MS is
obtained using a set of criteria in which MRI observations play
a central role. However, because of the lack of specificity for
myelin integrity, the use of MRI as the primary diagnostic tool
has not yet been accepted. In order to improve MR specificity,
we began developing MR probes targeted toward myelin. In
this work we describe a new myelin-targeted MR contrast
agent, Gd-DODAS, based on a stilbene binding moiety and
demonstrate its ability to specifically bind to myelin in vitro
and in vivo. We also present evidence that Gd-DODAS generates MR contrast in vivo in T1-weighed images and in T1 maps that
correlates to the myelin content.
is associated with both demyelination and inflammation. The
mechanism of myelin disruption has been extensively studied,
and a wealth of experimental data have been reported.8−11
Accordingly, various pathways underlying the process of
demyelination have been proposed based on mitochondrial
injury caused by oxidative stress or based on autoimmune
response to autoantigens.
INTRODUCTION
■
Myelination is one of the most fundamental biological
processes in the vertebrate nervous system.1 The presence of
myelin sheaths wrapped around axons provides the necessary
electrical insulation for efficient nervous impulse conduction.
Disruption of the compacted myelin sheath of axons is
associated with a number of debilitating diseases such as
multiple sclerosis (MS) and various leukodystrophies.2,3 MS is
the most common demyelinating disease, and it is characterized
by demyelination in the central nervous system (CNS). MS
affects an estimated 400 000 people in the U.S. and 2 million
people worldwide.4,5 The etiology of MS is complex and has
both genetic and environmental factors contributing to the risk
of disease. Risk factors include Epstein−Barr virus, vitamin D
deficiency, and smoking.6 MS is an inflammatory demyelinating
disease that is considered by many to have an autoimmune
origin. In the early phase of the disease, periods of active
demyelination are followed by periodic and deteriorating
remyelination. This phase is known as relapsing-remitting
phase (RRMS). The RRMS phase is followed by a phase of
continuous disease progression known as secondary progressive
MS (SPMS). In some cases, during the transition from RRMS
to SPMS, the continuous clinical deterioration is marked by
active MS attacks (relapsing progressive multiple sclerosis,
RPMS). Finally, about 10−20% of MS sufferers do not
experience RRMS but present a continuous disease progression
from the onset (primary progressive multiple sclerosis,
PPMS).7 In RRMS and in progressive MS, active tissue injury
Over the past decade, magnetic resonance imaging (MRI)
has played an important role in the diagnosis and disease
management of MS.12 Diagnosis of MS is normally guided by
the revised McDonald criteria as recommended by the
International Panel on the Diagnosis of Multiple Sclerosis.13
In 2001, MRI was first incorporated into the criteria as an
imaging tool to facilitate diagnosis and subtyping of MS. Since
then, the McDonald criteria have been frequently revised, but
they always included MRI as an indispensable tool to simplify
diagnosis and provide a more accurate identification of the
disease. Furthermore, tremendous efforts are being made to
promote myelin repair in the brain as a new therapeutic
strategy to restore neurological functions in the treatment of
MS.14 Development of myelin repair therapies must be
accompanied by an imaging tool that can specifically and
longitudinally monitor myelination. While MRI is very effective
in detecting brain lesions, conventional MR techniques do not
provide specific information on myelin pathology in the course
Received: October 3, 2012
Published: January 11, 2013
© 2013 American Chemical Society
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Figure 1. (A) Chemical structure of myelin-targeting probes previously identified by our group (MIC = gadolinium(III) [4,10-bis-carboxymethyl-7-
({3-[3-(4-dimethylaminophenyl)-2-oxo-2H-chromen-7-yloxy]-propylcarbamoyl}methyl)-1,4,7,10-tetraazacyclododec-1-yl]acetate; CMC = 3-(4-
aminophenyl)-2H-chromen-2-one; BMB = 4,4′-((1E,1′E)-(2-methoxy-1,4-phenylene)bis(ethene-2,1-diyl))dianiline; BDB = 4,4′-((1E,1′E)-(2,5-
dimethoxy-1,4-phenylene)bis(ethene-2,1-diyl))dianiline; MeDAS = (E)-4-(4-aminostyryl)-N-methylaniline). In bold is highlighted a shared
structural motif that is involved in binding with myelin. (B) Chemical structure of complexes that are discussed in this paper (Gd-DODAS,
gadolinium (E)-2,2′,2″-(10-(2-((3-(4-((4-(4-(methylamino)styryl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-2-oxoethyl)-1,4,7,10-tet-
raazacyclododecane-1,4,7-triyl)triacetic acetate; compound 11, gadolinium 2,2′,2″-(10-(2-oxo-2-((2-phenoxyethyl)amino)ethyl)-1,4,7,10-tetraazacy-
clododecane-1,4,7-triyl)triacetate).
of disease progression.15,16 For example, lesions detected by T2-
weighed images are not directly related to demyelination or
remyelination: they could also originate from an increased
fraction of mobile water protons in regions where myelin is
replaced with gliosis or from edema where myelin sheaths may
remain intact. MS lesions can also be detected in T1-weighed
images, but they are less sensitive than T2-weighed images and,
likewise, do not correlate with myelin pathology of underlying
tissues. Clinical contrast agents such as Gd-DTPA (DTPA =
diethylenetriamine pentaacetic acid) are widely used for
contrast-enhanced MR studies in MS patients. The use of
these contrast agents is possible because of the fact that the
blood−brain barrier (BBB) is often disrupted in MS patients.
Thus, Gd-DTPA enhancement increases the reliability and
sensitivity of detecting active lesions. However, none of the
clinical contrast agents exhibit any affinity and specificity for
myelin; instead, lesion enhancement by these agents is mainly
indicative of disruption of the BBB. No information can be
extracted from these images regarding myelin integrity in
detected lesions. Recently gadofluorine, a fluorinated T1 MR
agent, has been reported. Gadofluorine binds to extracellular
matrix proteins with high affinity and was found to detect brain
lesions with high sensitivity in MR images.17,18 Disruption of
the BBB or blood to nerve barrier (BNB) allows the
extravasation of gadofluorine, and binding to extracellular
matrix protein provides lesion enhancement. However,
gadofluorine enhancement does not reflect any changes in
myelin integrity.
In order to improve the specificity of MR enhancement, we
set out to develop myelin-specific contrast agents with optimal
MR properties. Such myelin-targeting MR probes are crucial to
establish the use of MRI for efficacy evaluation of novel myelin
repair therapies currently under development. Over the past
decade, our laboratory has developed a series of myelin-
targeting molecular probes for multimodality imaging based on
positron emission tomography (PET), MRI, and near-infrared
(NIR).19−25 Structure−activity relationship (SAR) studies of a
library of compounds suggest a common structural motif that is
important for binding to myelin (Figure 1A). On the basis of
this knowledge, we explored the incorporation of some of these
probes into the design of gadolinium-based contrast agents for
MR studies of myelin pathology. We first developed
gadolinium(III) [4,10-bis-carboxymethyl-7-({3-[3-(4-dimethy-
laminophenyl)-2-oxo-2H-chromen-7-yloxy]propylcarbamoyl}-
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a
Scheme 1. Synthesis of Compound 10
a
(i) Boc2O, THF, 96%; (ii) MeI, NaH, THF, 94%; (iii) NaH, DMF, 83%; (iv) 3-azidopropan-1-amine, CuI, DIPEA, 54%; (v) HBTU, HOBT,
DIPEA, DMF, 92%; (vi) TFA; (vii) GdCl3, pH 5.5, 50% (over two steps).
methyl)-1,4,7,10-tetraazacyclododec-1-yl]acetate), a myelin-tar-
geting MR agent termed MIC (Figure 1), which is based on a
coumarin binding moiety, and demonstrated that MIC binds to
myelin sheaths in vitro and in vivo and could be used to
characterize myelin distribution based on T1w MR imaging in
murine models.26,27 This demonstrated the validity of this
strategy and that it is possible to integrate previously developed
myelin-imaging agents into the design of MR contrast agents
without negative impact on the myelin-binding properties. In
order to increase the structural diversity of the pool of myelin
targeted agents, we designed and synthesized a second type of
myelin-targeted MR contrast agent bearing an aminostilbene
moiety, Gd-DODAS (compound 10, Figure 1B). The synthesis
of 10, along with biological evaluation of binding properties and
MR relaxometric properties in vivo, is described in comparison
with the previously developed MIC.
RESULTS
■
Synthesis. The myelin-targeting MR contrast agent 10 was
prepared in seven steps from p-aminobenzyldiethyl phospho-
nate 1 (Scheme 1). The amino group was initially protected
with Boc and subsequently methylated with methyl iodide to
generate intermediate 3 in 90% yield over two steps.
Intermediate 5, carrying a terminal alkyne group, was prepared
from Horner−Wadsworth−Emmons reaction of 3 and 4 in
83% yield. Copper(I)-catalyzed azide−alkyne cycloaddition
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Figure 2. Chemical staining of representative frozen sections of wild-type mouse brain showing that 10 selectively stains various myelinated white
matter regions in the brain such as corpus callosum, striatum, anterior commissure, and cerebellum (A−D, respectively). Gd-DODAS staining were
correlated with Black-Gold II staining of adjacent sections (E−H). Atlas figures representing the stained regions are also shown (I−L).30 Scale bar =
500 μm.
Figure 3. (A) Chemical staining with 10 of a brain section of LPC-treated rat. (B) The correspondence of compound 10 fluorescence with myelin
distribution within the brain was confirmed by black Black-Gold II staining. The demyelinated lesion is indicated by the arrow. Scale bar = 500 μm.
(C) Atlas figure shows the represented region.31
between intermediate 5 and 3-azidopropylamine was used to
prepare compound 6, which was subsequently coupled to tris-t-
Bu protected DOTA to give the protected ligand 8 in 50% yield
over two steps. Deprotection of the t-Bu groups in neat TFA
and metalation with GdCl3 afforded 10 in 50% yield over the
last two steps with a 19% overall yield.
In Vitro Chemical Staining. The absorption spectrum of
compound 10 shows a single intense long wavelength band
with a maximum of absorbance at 341 nm. The fluorescent
spectrum of 10 in water is similar to those of other p-
aminostilbenes with a fluorescence maximum at 443 nm and a
Stokes shift of 6848 cm−1.28 The emission spectrum does not
show any vibrational structure.
Because of the fluorescent nature of 10, its myelin binding
properties were first investigated by analyzing the fluorescent
staining pattern on freshly frozen tissue sections of 2-month-old
Swiss−Webster R/J mouse brains. Axial sections of the whole
mouse brain close to the bregma were used to examine the
myelin-binding properties of 10 in both myelin-deficient gray
and myelin-rich white matter regions. Axial sections were used
to examine staining in the cerebellum.
As shown in Figure 2, 10 preferentially stains myelin-rich
white matter regions such as the corpus callosum (Figure 2A),
the external capsule (Figure 2B), striatum (Figure 2B), the
anterior commissure (Figure 2C), and the cerebellum (Figure
2D). Myelin distribution was covalidated by chemical staining
with Black-Gold II, a commonly used myelin stain, on adjacent
sections.29 As displayed in Figure 2E−H, chemical staining with
Black-Gold II and with compound 10 showed an identical
pattern, which suggests that compound 10 binds to myelin with
high specificity.
Compound 10 was also able to detect demyelinated lesions
in vitro in a rat model of focal demyelination. For this purpose,
Sprague−Dawley rats were treated with lysolecithin (LPC), a
neurotoxin that induces demyelination at the site of injection.
As shown in Figure 3, the LPC-induced lesions in the corpus
callosum can be readily detected by fluorescent staining with 10
as characterized by decreased fluorescence intensity. The same
demyelinated lesion was confirmed by Black-Gold II staining of
myelin in an adjacent brain tissue section.
The binding specificity and sensitivity of 10 for myelin was
then further evaluated using a hypermyelinated Plp-Akt-DD
transgenic mouse model.32,33 Chemical staining with com-
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Figure 4. (A) Chemical staining with compound 10 of a brain section of a hypermyelinated Plp-Akt-DD D transgenic mouse. (B) The
correspondence of compound 10 fluorescence with myelin distribution within the brain was confirmed by Black-Gold II staining. Scale bar = 250
μm. (C) Atlas figure shows the represented region.30
Figure 5. Chemical staining with compound 10 of a brain section of a cuprizone mouse model showing demyelinated lesions (arrows) in the corpus
callosum (A) and external capsule (B). Demyelination was confirmed by Black-Gold II staining (D, E). Atlas figures show the represented regions
(C, F).30
Figure 6. Chemical staining with compound 10 of an axial spinal cord section of a wild type mouse (A) and Black-Gold II staining of an adjacent
section (B). A large demyelinated lesion (arrow) can be detected in a spinal cord section of an LPC-induced mouse model of demyelination (D).
The demyelinated lesion was confirmed by Black-Gold II staining of an adjacent section (E). Spinal cord diagrams show the represented regions (C,
F).
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Figure 7. (A) In situ fluorescent microscopy of a brain tissue section following in vivo contrast enhanced MR imaging. (B) Bright field microscopy
Black-Gold II myelin staining of an adjacent section. Scale bar = 500 μm. (C) Atlas figure shows the represented region.31
pound 10 of hypermyelinated Plp-Akt-DD transgenic mice
frozen brain sections reveals the hypertrophic corpus callosum
characteristic of this model (Figure 4). The same staining
pattern is also observed by Black-Gold II staining using
adjacent brain tissue sections.
In order to prove that compound 10 is able to identify
demyelinated lesions in an animal model of the MS disease,
frozen brain sections of cuprizone mouse brains were
chemically stained with compound 10. In this model of toxic
demyelination, mice are fed with the copper chelator cuprizone,
which induces oligodendrocyte cell death and consequently
demyelination. Sections stained with compound 10 revealed
large demyelinated lesions in the corpus callosum and the
Figure 8. Dependence of the relaxation rate of PBS buffered solutions
(pH 7.4) of 10 upon Gd concentration at 0.47 T (40 °C, squares),
1.41 T (40 °C, triangles), and 9.4 T (21 °C, diamonds).
external capsule. The demyelinated nature of these lesions was
confirmed by Black-Gold II staining (Figure 5).
We investigated the capability of compound 10 to bind
myelin other than in the brain white matter. For this purpose
spinal cord sections of wild type mice were chemically stained
with compound 10. The staining pattern observed correctly
reflects the distribution of white matter in the spinal cord as
covalidated by Black-Gold II staining of adjacent sections
(Figure 6A,B). Furthermore, we demonstrated that compound
10, similar to what was observed in the brain, is able to detect
demyelinated lesions induced by LPC injection in the spinal
cord (Figure 6D,E).
Table 1. Longitudinal Relaxivity of Compound 10 Compared
to MIC and the Clinically Approved MR Contrast Agents
GdDTPA and GdDOTA
r1 (s−1 mM−1
)
magnetic
field (T)
10
MIC
GdDTPA
GdDOTA
a
a
b
a
a
b
a
c
a
c
0.47
1.41
5.8 0.2
5.1 0.2
5.8 0.2
5.1 0.1
3.4 0.234
3.4 0.234
,
,
3.3 0.234
2.9 0.234
,
,
d
,
d
,
9.4
5.5 0.3
5.2 0.1
3.935
4.135
In Situ Chemical Staining. In situ staining of compound
10 was studied following intraventricular infusion in wild-type
Sprague−Dawley rats. The rats were euthanized and perfused
with 4% paraformaldehyde to remove the blood. The brains
were extracted and dissected to prepare frozen axial sections.
Fluorescent microscopy shows that 10 is selectively localized in
myelinated regions such as corpus callosum and striatum
(Figure 7). Adjacent sections were then subjected to Black-
Gold II staining for myelin. As shown in Figure 6, the pattern of
compound 10 staining (blue) is consistent with the pattern of
Black-Gold II staining. These data suggest that 10 can
selectively label myelin fibers in situ and potentially be used
to monitor myelin changes in vivo.
Relaxometric Properties. The relaxometric properties of
10 were measured at 0.47 T (40 °C), 1.41 T (40 °C), and 9.4 T
(21 °C), three magnetic field strengths commonly used in
clinical and preclinical studies. Compound 10 relaxivity is
dependent upon concentration (Figure 8). At high concen-
trations a sharp increase in relaxivity is observed at the two
lower magnetic fields. At 9.4 T, the relaxivity at higher
concentration is almost identical to the low concentration
relaxivity. The high dilution relaxivities of 10 at the measured
fields in comparison with previously reported MIC and other
clinical contrast agents are reported in Table 1.
a
b
c
d
40 °C. 21 °C. 1.5 T, 37 °C. 25 °C.
In Vivo MR Imaging. Similar to other Gd-based contrast
agents, 10 is a hydrophilic compound that is not permeable
across the BBB. For this reason, we evaluated compound 10
brain biodistribution after intracerebroventricular infusion to
bypass the BBB. Sprague−Dawley rats were anesthetized, and
10 (10 μL, 70 nmol) was administered via stereotaxic injection
to the lateral ventricles (LV). The animals were imaged twice
using a spin−echo multiple TR saturation recovery method 5
and 24 h after injection. As shown in Figure 9A, compound 10
induced a dramatic shortening of T1 localized in correspond-
ence of the corpus callosum. T1 shortening was clearly visible at
a dose of 70 nmol (∼3.2 mg/kg) and persisted at least for 24 h
after infusion (data not shown). In contrast, brain T1 maps of
untreated rats show little contrast between highly myelinated
white matter and less myelinated gray matter regions (Figure
9B). T1 maps of the rat brain infused with compound 11 (10
μL, 70 nmol), acquired 5 h after infusion, also lacked contrast
between the white matter and gray matter, suggesting that
compound 11, which contains only part of the pharmacophore
responsible for myelin binding, is not retained in highly
myelinated regions of the brain and produces a uniform
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Figure 9. MR T1 maps (spin−echo multiple TR saturation recovery, TE = 6.99 ms, TR = 385−12500 ms, spatial resolution = 0.195 × 0.195 mm/
pixel, matrix size = 256 × 256, 30 slices, slice thickness = 0.5 mm, 1 average) of a (A) Sprague−Dawley rat brain 5 h after compound 10 injection,
(B) Sprague−Dawley rat brain without infusion of contrast agent, (C) Sprague−Dawley rat brain after injection of compound 11 and corresponding
anatomical images (D−F).
shortening of the longitudinal relaxation time throughout the
brain (Figure 9C).
solvent. Relaxivity is not a constant but depends on a number
of external parameters, such as the applied magnetic field
strength and temperature, as well as molecular parameters
including the number of coordinated water molecule(s) (q),
the mean residence time (τM) of the coordinated water
molecule(s), and the reorientational correlation time (τR). The
relaxivity of small Gd complexes such as compound 10 is
strongly influenced by the reorientational correlation time
especially at magnetic fields lower than 1.5 T.
At 0.47 and 1.41 T (40 °C), the low concentration relaxivity
of 10 is nearly identical to the relaxivity of MIC and about 1.5−
1.7 times higher than that of commercial MR contrast agents
such as GdDOTA or GdDTPA. Unlike MIC, compound 10
tends to aggregate in solution at concentrations above ∼0.4
mM, which is apparent from the increase in relaxivity observed
at high concentration. The higher relaxivity observed under
these conditions is a reflection of the increased rotational
correlation time of the aggregated complex. At 9.4 T, the
increase in relaxivity is less pronounced, since the influence of
the rotational correlation time upon the relaxivity becomes less
important.36 An increase in relaxivity can also be expected upon
binding of compound 10 to myelin, provided that the metal
center retains access to bulk water and that the bound water
exchange rate is not significantly reduced.
Compound 10 is fluorescent, and therefore, it is possible to
investigate its myelin-binding properties using fluorescent
microscopy. For this purpose the ability of 10 to selectively
bind myelin was investigated by chemical staining of wild type
mouse brain sections. As shown in Figure 2, the fluorescence
originating from 10 is more intense in highly myelinated white
matter brain regions such as the corpus callosum, the external
capsule, the caudate putamen, and the anterior commissure and
in the cerebellar arbor vitae. The colocalization of compound
10 with the myelin distribution in the brain was confirmed by
chemical staining with Black-Gold II, a commonly used myelin
stain. The ability of 10 to detect and identify areas of decreased
or increased myelination was tested by in vitro histochemistry
of murine animal models of abnormal myelination. Chemical
DISCUSSION
■
The inability of conventional MR to probe myelination status
in the CNS and PNS prompted us to prepare MR contrast
agents that could selectively interact with myelin sheaths. The
design of the proposed agent is based on the structure of
myelin probes for PET imaging that were previously identified
in our group. SAR studies on a library of compounds revealed a
common structural motif that is involved in binding with
myelin (Figure 1A). These previously identified myelin probes,
while sharing a common structural motif, can be categorized
into coumarin and stilbene derivatives. We have recently
reported the preparation and the myelin binding and MR
properties of MIC, a myelin targeted MR agent derived from
coumarin. In order to establish if myelin-targeted probes based
on a stilbene core are also amenable to be functionalized with
an MR reporter, we synthesized and characterized a novel
myelin-targeting agent derived from an aminostilbene moiety
conjugated with a DOTA monoamide gadolinium(III)
complex. Compound 10 was synthesized in seven steps with
an overall yield of ∼19%.
Gd(III) ions, because of their high magnetic moment and
long electron relaxation time, are able to efficiently shorten the
longitudinal or transversal relaxation rate (R1 and R2) of the
solvent water protons. The effectiveness with which a
paramagnetic agent is able to shorten the relaxation rate of
solvent water protons is given by a parameter called relaxivity
(ri; i = 1, 2) which represents the increase in relaxation rate of a
water solution occurring after the addition of the agent,
normalized to a concentration of 1 mM (eq 1).
1
Ri =
= r[Gd] + Risolv
;
i = 1, 2
i
T
(1)
i
Here Ti is the longitudinal or transversal relaxation time of the
solution, ri is the relaxivity, and Risolv is the relaxation rate of the
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staining with compound 10 of brain sections of an LPC rat
model of demyelination (Figure 3) demonstrates that
compound 10 can be used to identify focal demyelinated
lesions. Decreased myelination was also easily detected in the
corpus callosum and external capsule of a cuprizone mouse
model of demyelinating disease (Figure 5). Furthermore,
histochemical staining of AKT mouse brain with compound
10 showed an increased fluorescence in highly myelinated
white matter that correlates well to the enhanced myelination
that is characteristic of this kind of animal model (Figure 4).
Myelin is a complex blend of proteins and lipids, and its
composition changes not only between the CNS and PNS but
also within the CNS.1 In order to test if compound 10 retains
its binding ability to myelin fibers outside the brain, we stained
axial sections of a wild type spinal cord with compound 10
(Figure 6). The staining pattern observed for compound 10
reflects the myelin distribution within the spinal cord.
Moreover, compound 10 could detect LPC-induced demyeli-
nated lesion in the spinal cord of a wild type mouse similar to
what was observed for brain lesions.
Once established that compound 10 is able to selectively
bind myelin sheaths in vitro, its ability to bind myelin sheaths in
vivo was tested by measuring its distribution in the brain of live
animals by magnetic resonance imaging relaxometry. Com-
pound 10 is a hydrophilic, highly water-soluble gadolinium
complex with a molecular weight (904 Da) that exceeds 500
Da, a value that is usually recognized as the upper limit for BBB
permeability by passive diffusion. Thus, like other clinical
contrast agents, compound 10 is not permeable across the BBB.
It is important to note that in MS the BBB is normally
disrupted in correspondence of new or reactivated lesions.
Therefore, compound 10 can be expected to cross the
compromised BBB similar to GdDTPA. In the present study,
we examined the distribution of 10 in the brain of wild type rats
with an intact BBB. In order to bypass the BBB, compound 10
was administered by intracerebroventricular infusion. T1 maps
generated 5 h postinfusion show that compound 10
accumulates preferentially in the highly myelinated genus of
the corpus callosum. The diffusion from the site of injection is
slow, and the contrast pattern in T1 maps acquired 24 h
postinjection remains unchanged (data not shown). The
contrast pattern generated by compound 10 was compared
with the pattern generated by compound 11. Compound 11 is
characterized by the same coordination cage as compound 10
modified with a hydrophobic moiety. T1 maps obtained 5 h
postinfusion show that compound 11 does not accumulate in
highly myelinated region and produces a uniform decrease of
the longitudinal relaxation time throughout the entire brain
(Figure 9C).
can provide deep tissue penetration, high resolution, and an
excellent soft tissue contrast. For this reason, we decided to
modify our previously developed probes for MR imaging. We
recently reported MIC, a Gd complex containing a coumarin
binding moiety, as the first myelin-targeting MR contrast agent.
In this work we describe the synthesis, relaxometric character-
ization, and myelin binding properties of compound 10, the
first MR myelin-targeting agent based on a stilbene binding
moiety. Compound 10 binding to myelin was demonstrated
both in vitro and in situ by fluorescent microscopy and in vivo
by T1w MR imaging. The fact that both myelin-targeted
coumarin and stilbene probes can be successfully modified and
functionalized with MR reporter while retaining their targeting
specificity suggests that it is possible to create structurally
diverse libraries of myelin-targeted MR probes by modifying
the lanthanide-chelating unit. Like all the other hydrophilic MR
probes, BBB permeability of 10 remains a challenge. In this
work we exploited intracerebroventricular infusion to bypass
the BBB. A number of methodologies have been reported in
the literature to shuttle hydrophilic compounds across the BBB,
and we are currently exploring the application of some of these
techniques to deliver these agents. The utility of these probes,
however, is not entirely dependent on their ability to cross the
intact BBB. In neurodegenerative diseases such as MS, the BBB
is transiently open in active lesions, and this is already exploited
clinically for GdDTPA-enhanced MR imaging. For this reason,
we believe that the development of myelin-targeting MR probes
can prove to be an extremely useful tool for neuroimaging.
EXPERIMENTAL SECTION
■
Materials and Methods. All chemicals, unless otherwise stated,
were purchased from commercial sources and used without further
purification. L-α-Lysophosphatidylcholine from egg yolk was pur-
chased from Sigma-Aldrich. All NMR spectra were acquired on an
Inova 400 NMR system (Varian) equipped with a 5 mm broadband
probe. Analytical HPLC was performed on an Agilent 1100 series
system equipped with a dual channel UV/vis detector using a
Phenomenex 5 μm C18(2) 100A (250 mm × 4.56 mm, 5 μm) column
(4.6 mm × 250 mm); eluent A, H2O/0.1% TFA; B, MeOH/0.1%
TFA; elution, 10% B 3 min, 10% B to 100% B in 15 min; flow rate 1
mL/min. The purity of tested compounds as determined by analytical
HPLC was >95%. ESI mass spectra were acquired on a Finnigan LCQ
Deca. pH was measured using a PHM210 standard pH meter
(Radiometer Analytical) connected to a sympHony pH glass electrode
(VWR). UV absorption was measured on a Cary 50 Bio
spectrophotometer using a standard 1 cm × 1 cm quartz cuvette.
Fluorescence was measured with a Cary Eclipse spectrophotofluorim-
eter using a standard 1 cm × 1 cm quartz cuvette.
tert-Butyl (4-((Diethoxyphosphoryl)methyl)phenyl)-
carbamate (2). To a 100 mL round-bottom flask fitted with a
magnetic stir bar were added diethyl 4-aminobenzylphosphonate (1,
2.500 g, 10.28 mmol), di-tert-butyl dicarbonate (2.240 g, 10.27 mmol),
THF (15 mL), and water (6.0 mL). The mixture was stirred at room
temperature overnight. THF was removed in vacuo, and the resulting
residue was diluted with water and extracted with EtOAC three times.
The organic layers were combined and washed twice with water and
once with brine. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuum. The resulting white solid (3.393 g, 96%) was
used without further purification. 1H NMR (CDCl3, 400 MHz) δ: 1.22
(6H, td, 3JH,H = 6.8 Hz, 4JH,P = 0.4 Hz, CH2CH3), 1.49 (9H, s, CCH3),
3.07 (2H, d, 2JH,P = 21.2 Hz, PCH2), 3.91−4.04 (4H, m, OCH2), 6.87
(1H, br s, NH), 7.17 (2H, m, ArH), 7.30 (2H, br d, J = 8.4 Hz, ArH).
13C NMR (CDCl3, 100 MHz) δ: 16.3 (CH2CH3), 28.8 (CCH3), 32.9
CONCLUSION
■
There is an urgent and unmet need to improve the sensitivity of
MR techniques toward myelin changes in the nervous system.
Such improvements will facilitate the early diagnosis of
neurodegenerative diseases such as MS, provide a powerful
tool to monitor response to current and new therapies, and
assist in drug development.
A number of myelin-targeting probes for PET and optical
imaging have been previously developed in our group. Optical
imaging is an effective tool for preclinical drug development;
however, it lacks deep tissue penetration, while PET, despite
having a superb sensitivity, is characterized by a low resolution
and lack of anatomical information. MRI, on the other hand,
1
(d, JC,P = 138 Hz, PCH2), 62.0 (OCH2), 80.3 (CMe3), 118.4
(Caromatic), 125.5 (Caromatic), 130.1 (Caromatic), 137.4 (Caromatic), 152.8-
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(Caromatic). ESI-MS (m/z): measured 366.03 [M + Na]+; expected
366.14
MHz) δ: 28.28 (OCH3), 33.30 (CH2CH2CH2), 37.15 (NCH3), 38.48
(H2NCH2), 47.73 (NCH2), 62.03 (OCH2), 80.32 (CMe3), 114.92
(CHaromatic), 122.77 (Caromatic triazol), 125.39 (CHaromatic), 126.11
(Caromatic), 126.26 (CHaromatic), 127.62 (CHaromatic), 127.71(CHalkene),
130.55 (CHalkene), 134.48 (Caromatic), 142.74 (Caromatic), 143.97
(Caromatic triazol), 154.61 (Caromatic), 157.78 (CO). ESI-MS (m/z):
measured 464.07 [M + H]+; expected 464.27
tert-Butyl (4-((Diethoxyphosphoryl)methyl)phenyl)(methyl)-
carbamate (3). To an oven-dried 100 mL round-bottom flask purged
with argon and fitted with a magnetic stir bar was added sodium
hydride (0.35 g, 8.74 mmol, 60% dispersion in mineral oil). The
sodium hydride was washed three times with hexanes (6 mL), and the
solvent was discarded. Compound 2 (2.0g, 5.82 mmol) was added
under argon, and the mixture was suspended in dry THF (18 mL).
The mixture was cooled to 0 °C, and methyl iodide (0.75 mL, 11.7
mmol, 2.28 g/mL) was added dropwise. The mixture was allowed to
warm to room temperature and stirred under argon overnight. The
reaction was quenched with water, and the THF was removed under
vacuum. The residue was dissolved in EtOAc and water, and the
aqueous layer was extracted three times with EtOAc. The organic
layers were combined and washed twice with water and once with
brine. The organic layer was dried over MgSO4, filtered, and
(E)-Tri-tert-butyl 2,2′,2″-(10-(2-((3-(4-((4-(4-((tert-
Butoxycarbonyl)(methyl)amino)styryl)phenoxy)methyl)-1H-
1,2,3-triazol-1-yl)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraaza-
cyclododecane-1,4,7-triyl)triacetate (8). A solution of 7 (0.48 g,
0.83 mmol), 6 (0.35 g, 0.76 mmol), HBTU (0.30 g, 0.79 mmol),
HOBT (0.12 g, 0.79 mmol), and DIPEA (0.15 g, 1.13 mmol) in
dimethylformamide (7 mL) was stirred at room temperature for 12 h.
The solution was diluted with EtOAc (25 mL) and extracted with
water (3 × 25 mL). The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel, eluting with a gradient of
methanol in dichloromethane from 0% to 5%. The fractions containing
pure product 8 were evaporated. The fractions containing the product
contaminated with aromatic byproducts were combined and
evaporated. The residue was dissolved in dichloromethane (30 mL)
and extracted with CH3COONaaq 0.1 M (6 × 50 mL) and brine (1 ×
50 mL). The organic phase was dried over Na2SO4, filtered, and
evaporated under reduced pressure to give compound 8 as an amber
1
concentrated to give pure 3 as a yellow oil (1.96 g, 94%). H NMR
(CDCl3, 400 MHz) δ: 1.23 (6H, t, J = 7.2 Hz, CH2CH3), 1.42 (9H, s,
3
CCH3), 3.11 (2H, d, JH,P = 21.6 Hz, PCH2), 3.22 (3H, s, NCH3),
3.94−4.07 (4H, m, OCH2), 7.16 (2H, br d, J = 8.0 Hz, ArH), 7.24
(2H, m, ArH). 13C NMR (CDCl3, 100 MHz) δ: 16.3 (CH2CH3), 28.1
(CCH3), 33.0 (d, 2JC,P = 138 Hz, PCH2), 37.1 (NCH3), 61.9 (OCH2),
80.1 (CMe3), 125.4 (Caromatic), 128.4 (Caromatic), 129.7 (Caromatic), 142.4
(Caromatic), 154.5 (Caromatic). ESI-MS (m/z): measured 380.06 [M +
Na]+; expected 380.16
1
glassy solid. The combined yield of product 8 was 92% (0.71 g). H
NMR (400 MHz, CDCl3) δ: 1.28−1.51 (36H, m, br, CCH3), 1.67−
3.64 (28H, br, CH2 macrocycle, NCH2), 3.23 (3H, s, NCH3), 4.38 (2H,
m, CH2N), 5.17 (2H, s, OCH2), 6.87−7.02 (4H, m, ArH and
CHalkene), 7.17 (2H, d, J = 8.4 Hz, ArH), 7.40 (4H, d, J = 8.6 Hz, ArH),
7.88 (1H, s, CHtriazol). 13C NMR (CDCl3, 100 MHz) δ: 27.72
(OC(CH3)3, br), 28.18 (OC(CH3)3), 29.89 (CH2CH2CH2), 36.02
(H2NCH2), 37.06 (NCH3), 47.61 (NCH2), 47.1−53.5 (NCH2 ring, br),
55.2−55.7 (NCH2 acetic, br), 61.42 (OCH2), 80.20 (CMe3), 81.5−82.05
(CMe3 ester, br), 114.90 (CHaromatic), 123.91 (CHaromatic), 125.27
(CHaromatic), 125.74 (Caromatic), 126.14 (CHaromatic), 127.49 (CHaromatic),
127.76 (CHalkene), 130.16 (CHalkene), 134.45 (Caromatic), 142.55
(Caromatic), 143.14 (Caromatic triazol), 154.53 (Caromatic), 157.87 (NCO),
171.9−173.9 (COmacrocycle).
(E)-2,2′,2″-(10-(2-((3-(4-((4-(4-(Methylamino)styryl)-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-2-ox-
oethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
Acid (9). A solution of 8 (0.16 g, 0.16 mmol) in trifluoroacetic acid (2
mL) was stirred at room temperature for 24 h. Trifluoroacetic acid
(TFA) was evaporated under reduced pressure. The residue was
dissolved in a minimum amount of water and loaded on a Hypersep
C18 SPE cartridge (1 g bed). The cartridge was eluted with a gradient
of water/TFA (99.9:0.1, eluant A) and acetonitrile/TFA (99.9:0.1,
eluant B). The cartridge was eluted extensively with solvent A and
then with solvent A/solvent B 9/1 (15 mL). The product was
recovered eluting with solvent A/solvent B 8/2. The fraction
containing the product was evaporated to give 9·xTFA (0.085 g) as
an amber glass solid. 1H NMR (400 MHz, pH ≈ 3, D2O, external ref t-
BuOH) δ: 1.96 (2H, q, J = 6.6 Hz CH2CH2CH2), 2.70−4.44 (26H, br
m, CH2 macrocycle, NCH2), 2.91 (3H, s, NCH3), 4.32 (2H, t, J = 6.5 Hz,
CH2N), 4.95 (2H, s, OCH2), 6.57 (1H, d, J = 16.1 Hz, CHalkene),
6.68−6.78 (3H, m, ArH, CHalkene), 7.15 (2H, d, J = 8.7 Hz, ArH), 7.23
(4H, s, ArH) 7.94 (1H, s, CHtriazol); 13C NMR (600 MHz, 50 C, pH ≈
3, D2O, external ref t-BuOH) δ: 30.68 (CH2CH2CH2), 38.34 (NCH2),
38.78 (NCH2), 40.93 (NCH2), 50.03 (NCH2 ring, br), 51.04
(NCH2 ring, br), 52.5 (NCH2 ring, br), 55.34 (NCH2 acetic, br), 56.05
(NCH2 acetic, br), 56.66 (NCH2 acetic, br), 63.03 (OCH2), 117.40
(CHaromatic), 118.24 (q, J = 292 Hz, CF3) 124.00 (CHaromatic), 126.94
(Caromatic), 127.01 (CHaromatic), 129.57 (CHalkene), 130.02 (CHalkene),
131.63 (CHaromatic), 132.19 (CHaromatic), 136.78 (Caromatic), 140.6
(Caromatic), 144.98 (Caromatic triazol), 159.4 (Caromatic), 164.29 (q, J =
35.3 Hz, CF3COOH), 173.84 (very br, CO). ESI-MS (m/z): measured
750.32 [M + H]+ ; expected 750.39
(E)-tert-Butyl Methyl(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)-
carbamate (5). A mixture of compound 3 (3, 1.12 g, 3.14 mmol) and
sodium hydride (0.25 g, 6.29 mmol) in dry DMF (5 mL) was stirred
under Ar for 1 h. A solution of 4-(prop-2-yn-1-yloxy)benzaldehyde (4,
0.40 g, 2.51 mmol) in dry DMF (2 mL) was added and the mixture
stirred for an additional 12 h. The reaction mixture was diluted with
EtOAc (50 mL) and extracted with K2CO3(aq) 10% (2 × 30 mL). The
organic layer was dried over sodium sulfate, filtered, and evaporated.
The residue was purified by column chromatography over silica gel,
eluting with DCM. The fractions containing the product were joined
and evaporated under reduced pressure to give 5 (0.76 g, 83%) as a
white solid. 1H NMR (CDCl3, 400 MHz) δ: 1.46 (9H, s, OCH3), 2.54
(1H, t, J = 2.4 Hz, CCH), 3.27 (3H, s, NCH3), 4.70 (2H, d, J = 2.4 Hz,
OCH2), 6.93−7.05 (4H, m, CHalkene and ArH), 7.22 (2H, d, J = 8.4
Hz, ArH), 7.42−7.47 (4H, m, ArH). 13C NMR (CDCl3, 100 MHz) δ:
28.29 (OCH3), 37.15 (NCH3), 55.76 (OCH2), 75.60 (CHalkyne), 78.38
(Calkyne), 80.32 (OCMe3), 115.03 (CHaromatic), 125.40 (CHaromatic),
126.29 (CHaromatic + Caromatic), 127.56 (CHaromatic), 127.70 (CHalkene),
130.90 (CHalkene), 134.47 (Caromatic), 142.77 (Caromatic), 154.61
(Caromatic), 157.06 (CO). ESI-MS (m/z): measured 364.33 [M +
H]+; expected 364.19
(E)-tert-Butyl (4-(4-((1-(3-aminopropyl)-1H-1,2,3-triazol-4-
yl)methoxy)styryl)phenyl)methylcarbamate (6). A suspension
of 5 (0.62 g, 1.71 mmol), 3-azidopropan-1-amine (0.26 g, 2.57 mmol),
Hunig’s base (0.55 g, 4.28 mmol), and copper(I) iodide (0.049 g, 0.26
mmol) in dichloromethane (25 mL) was stirred under argon. After 3 h
a fresh aliquot of 3-azidopropan-1-amine (0.21 g, 2.12 mmol) was
added in a dichloromethane solution (2 mL). The mixture was stirred
under Ar at room temperature for 12 h. The solvent was removed
under reduced pressure, and the residue was dissolved in ethyl acetate
(25 mL) and extracted with NH4OHaq 0.1 M (2 × 25 mL). The
aqueous extracts were combined and washed with dichloromethane (2
× 25 mL). The dichloromethane solutions were combined and
extracted with NH4OHaq 0.1 M (4 × 50 mL) and brine (1 × 50 mL).
The organic layer was dried over sodium sulfate and filtered. The
solvent was removed under reduced pressure, and the residue was
purified by column chromatography over silica gel, eluting with a
gradient of methanol in dichloromethane from 0% to 5%. The
fractions containing the product were combined and evaporated to
give 6 (0.43 g, 54%) as a white solid. 1H NMR (400 MHz, CDCl3) δ:
1.45 (9H, s, OCH3), 2.02 (2H, q, J = 6.8 Hz, CH2CH2CH2), 2.72 (2H,
br, NH2), 3.25 (3H, s, NCH3), 4.47 (2H, t, J = 6.9 Hz, CH2N), 5.21
(2H, s, OCH2), 6.90−7.05 (4H, m, ArH and CHalkene), 7.20 (2H, m,
ArH), 7.42 (4H, m, ArH), 7.63 (1H, s, NCH). 13C NMR (CDCl3, 100
Gadolinium (E)-2,2′,2″-(10-(2-((3-(4-((4-(4-(Methylamino)-
styryl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-2-
oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
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Acetate (10). The pH of a solution of 9·xTFA (0.019 g) in deionized
water (10 mL) was adjusted to 5 with NaOHaq 1 M. A solution of
GdCl3 (6.09 mg, 0.023 mmol) in water (4.0 mL) was added in four
aliquots, maintaining the pH between 5.5 and 6 with NaOHaq 1 M.
The solution was stirred at room temperature for 5 h, and
subsequently the temperature was raised to 60 C and the reaction
continued for 12 h. The solution was allowed to cool to room
temperature, and the pH was adjusted to 9.5 with NaOHaq 1 M. The
solution was then filtered through a 0.2 μm syringe filter and
immediately loaded onto a Hypersep C18 SPE cartridge (1 g bed).
The cartridge was eluted with a gradient of water and acetonitrile from
water/acetonitrile 100/0 to water/acetonitrile 60/40. The fractions
containing the product were concentrated under reduced pressure to
remove the organic solvent and then lyophilized to give 10 (0.017g,
50% over two steps) as a yellow solid. ESI-MS (m/z): measured 905.2
[M + H]+; expected 905.29. A correct isotopic pattern was observed.
Elemental analysis calcd (%) for C37H48N9O8Gd·3H2O: C 46.68, H
5.64, N 13.24. Found: C 46.76, H 5.54, N 12.93.
Animals. All animal experiments were performed in accordance
with guidelines approved by the Institutional Animal Care and Use
Committee of Case Western Reserve University (Protocols 2010-
0006, 2010-0007). Two-month-old Swiss-Webster R/J mice were
purchased from Harlan Laboratories, Indianapolis, IN. Two-month-old
C57BL/6 mice were obtained from The Jackson Laboratory, Bar
Harbor, ME. Sprague−Dawley rats were purchased from Harlan
Laboratories, Haslett, MI. The Plp-Akt-DD transgenic mice were
generated as described previously and used at 2 months of age.32,33
Briefly, the transgenic mice expressing constitutively active Akt
(HAAkt308D473D, Akt-DD) driven by the Plp promoter were
generated and used as a hypermyelinated animal model. The Akt
cDNA was inserted into the AscI/PacI sites of the modified Plp
promoter cassette, and hypermyelination was induced after the Plp
promoter/Akt-DD insert was injected into SJL/SWR F1 mice. Positive
founders were identified by PCR amplification of tail DNA using
IntronSV40F (5′-GCAGTGGACCACGGTCAT-3′) and Akt lower
(5′-CTGGCAACTAGAAGGCACAG-3′) primer pairs. Analyses were
done from wild-type littermate mice in all developmental experiments
and, when possible, with older animals.
Induction of LPC Lesions. One Sprague−Dawley rat was
anesthetized by ip injection of 225 μL of rodent cocktail [9 parts
ketamine (100 mg/mL) + 9 parts xylazine (20 mg/mL) + 3 parts
acepromazine (10 mg/mL) + 79 parts sterile saline]. The skull was
shaved, and a 3 cm longitudinal incision was made on the scalp. A burr
hole was created using a spherical dental burr over the site of the
intended injection. Then 6 μL of a 1% LPC solution was injected at a
rate 0.25 μL/min. The injection coordinates were AP = 0.0 mm, ML =
2.0 mm, DV = 3.2 mm, corresponding to the corpus callosum. After
the injection the incision was sutured and the animal allowed to
recover while being warmed with a heating pad. The lesions were
allowed to develop for 7 days and subsequently the animal brain was
used to prepare frozen sections as described below.
Preparation of Frozen Sections. Mice were deeply anesthetized
with isoflurane and perfused via the ascending aorta with PBS followed
by 4% paraformaldehyde in PBS. Brains were removed and incubated
for 24 h in 4% paraformaldehyde at 4 °C, and then the tissues were
incubated in 30% sucrose at 4 °C until submerged. Frozen sections
were used for fluorescence microscopy. For preparation of fresh frozen
sections, the cryoprotected tissues were first frozen in OCT on dry ice
before axial sectioning (20 μm) with a cryostat at −20 °C. Tissue
sections from the midline of the brain containing the whole corpus
callosum were selected for staining. Stained sections were covered with
fluorescence mounting medium (Vectashield, Vector Laboratories)
and stored at 4 °C for future analysis.
340−380; DM, 400; BA, 435−485) or with a Leica DM5000B
microscope equipped with an HCX PL FLUOTAR 1.25×/0.04
objective and using the A4 filter (360/40 nm band-pass excitation, 400
nm dichromatic mirror, 470/40 nm band-pass suppression).
Intracerebroventricular Injection of Gd Complexes.
Sprague−Dawley rats were anesthetized by ip injection of 200−250
μL of rodent cocktail (9 parts ketamine (100 mg/mL) + 9 parts
xylazine (20 mg/mL) + 3 parts acepromazine (10 mg/mL) + 79 parts
sterile saline) and were fixed to the rat head-restraining stereotaxic
surgical table. The skull was shaved and a 3 cm longitudinal incision
was made on the scalp. A burr hole was created using a spherical dental
burr over the site of the intended injection. A water solution of 10 or
compound 11 (7 mM, 10 μL, 70 nmol) was injected into the brain
through a 10 μL syringe equipped with a 28 gauge needle. After
injection the incision was sutured and the animals allowed to recover
consciousness while warmed with a heating pad. The injections were
performed on both sides of the brain in the lateral ventricles with half
of the dose injected on each side. Typical coordinates for the injection
sites relative to the bregma are AP = −0.5 0.1 mm, ML = 2.3 0.1
mm, DV = 3.5 0.1 mm.
In Situ Staining of Myelin. The rats that were used for MR
imaging were euthanized less than 48 h after compound 10
intracerebroventricular infusion and perfused to remove the blood,
and the brains were extracted and sectioned to prepare frozen axial
sections. Images of the stained mouse brain sections were acquired on
a Nikon TE2000 inverted microscope (UV-2E/C Ex, 340−380; DM,
400; BA, 435−485).
Relaxometry. The relaxivity of 10 was measured at 1.41 T (40 °C)
and 0.47 T (40 °C) on a Bruker minispec and at 9.4 T (21 °C) on a
Varian Inova 400 NMR system equipped with a 5 mm broadband
probe. The longitudinal relaxation rates of five water solutions, with
concentrations of compound 10 ranging from 0 to 0.7 mM, were
measured using an inversion recovery pulse sequence with at least 10
inversion times. Attention was paid to ensure that the relaxation delay
between pulses was at least 5T1. The absence of free Gd(III) was
confirmed using a standard colorimetric test with xylenol orange.
MR Imaging. All MR experiments were performed at 9.4 T
(Bruker “BIOSPEC”, Bruker, Germany) using a 60 mm diameter
volume coil. Anesthesia was maintained by mask inhalation of
isoflurane vaporized at concentrations of up to 4% in the induction
phase, at 1.5% during the imaging experiments. Prior to measurement
of the longitudinal relaxation time, axial images of the rat brain were
acquired, using a multislice RARE pulse sequence (TE = 11.3 ms, TR
= 5000 ms, spatial resolution = 0.195 × 0.195 mm/pixel, matrix size
256 × 256, 30 slices, slice thickness = 0.5 mm, 1 average), for the
identification of the region of interest (ROI). T1 measurements were
carried out using a spin−echo multiple TR saturation recovery method
with at least 10 TRs (TE = 6.99 ms, TR = 385−12500 ms, spatial
resolution = 0.195 × 0.195 mm/pixel, matrix size = 256 × 256, 30
slices, slice thickness = 0.5 mm, 1 average). T1 maps were generated
using the QuickVol II plug-in in ImageJ. The animals were imaged
twice using a spin−echo multiple TR saturation recovery method 5−7
h after injection and then again 20−24 h after injection.
ASSOCIATED CONTENT
■
S
* Supporting Information
Fluorimetric characterization of compound 10 and synthesis
and characterization of compound 11. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Chemical Staining. Freshly frozen sections 20 μm in thickness
were incubated in 0.1% Triton-100 in 1× PBS for 10 min and then
incubated in a solution of 10 (100 μM) in H2O for 30 min at room
temperature. The fresh frozen sections were then washed three times
for 5 min each with PBS before coverslipping with fluorescence
mounting medium. Images of the stained mouse brain sections were
acquired on a Nikon TE2000 inverted microscope (UV-2E/C Ex,
Corresponding Author
*Telephone: (216) 844-3288. Fax: (216) 844-8062. E-mail:
yanming.wang@case.edu.
Notes
The authors declare no competing financial interest.
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Article
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ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support through grants
from the Department of Defense (Grant W81XWH-10-1-
0842), National Multiple Sclerosis Society (Grant RG 4339-A-
2), and NIH/NINDS (Grant R01 NS061837).
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DIPEA, N,N-diisopropylethylamine; DMF, dimethylforma-
mide; DOTA, tetraazacyclododecanetetraacetic acid; DTPA,
diethylenetriamine pentaacetic acid; Gd-DODAS, gadolinium
(E)-2,2′,2″-(10-(2-((3-(4-((4-(4-(methylamino)styryl)-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-2-ox-
oethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic ace-
tate; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate; HOBT, 1-hydroxybenzotriazole;
MBP, myelin basic protein; MIC, gadolinium(III) [4,10-bis-
carboxymethyl-7-({3-[3-(4-dimethylaminophenyl)-2-oxo-2H-
chromen-7-yloxy]propylcarbamoyl}methyl)-1,4,7,10-tetraazacy-
clododec-1-yl]acetate; PET, positron emission tomography;
TFA, trifluoroacetic acid; THF, tetrahydrofuran
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