Design, synthesis, and evaluation of coumarin-based molecular probes for imaging of myelination

Article abstract of DOI:10.1021/jm101489w

Myelination represents one of the most fundamental biological processes in the vertebrate nervous system. Abnormalities and changes in myelination in the central nervous system (CNS) are seen in many neurodegenerative disorders, such as multiple sclerosis

Full text of DOI:10.1021/jm101489w

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Evaluation of Coumarin-Based Molecular
Probes for Imaging of Myelination
Changning Wang,† Chunying Wu,† Junqing Zhu,† Robert H. Miller,‡ and Yanming Wang*,†
†Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve
University, Cleveland, Ohio 44106, United States
‡Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106, United States
S Supporting Information
b
ABSTRACT: Myelination represents one of the most funda-
mental biological processes in the vertebrate nervous system.
Abnormalities and changes in myelination in the central ner-
vous system (CNS) are seen in many neurodegenerative
disorders, such as multiple sclerosis (MS). A long-standing goal
has been to directly detect and quantify myelin content in order
to facilitate diagnosis and therapeutic treatments of myelin-
related diseases. In the course of our studies, we have developed
a series of small-molecule probes (SMP) as myelin-imaging
agents. Among them are coumarin derivatives, which exhibit
promising brain permeability and myelin-binding properties.
Herein we report a full account of the design and synthesis of coumarin-based SMPs as myelin-imaging agents. Systematic evaluation
of these SMPs in both the CNS and peripheral nervous system (PNS) allowed us to identify some lead agents for potential use as
fluorescent dyes for intraoperative nerve mapping in surgical operations or as radiotracers for positron emission tomography (PET)
imaging of myelination.
’ INTRODUCTION
bloodꢀbrain barrier and selectively bind to myelin with high
affinity and specificity17ꢀ22 (Figure 1). Compared to conven-
tional myelin stains such as luxol fast blue and fluoromyelin, these
molecular probes are capable of direct imaging of myelination
in vivo. For example, some of these SMPs have been used for
positron emission tomography (PET), which is widely used to
study biochemical and metabolic changes at the molecular
level.17,20 One of the lead myelin-imaging agents has also been
used for myelin imaging in nonhuman primates.17
In the course of our studies, we have focused our efforts on the
design and synthesis of a series of coumarin derivatives as myelin-
imaging agents23 (Table 1). Coumarin is a natural product
occurring in plants and has many important biological
activities.24,25 We have previously reported a coumarin deriva-
tive, 3-(4-aminophenyl)-2H-chromen-2-one, termed CMC,
which exhibits promising myelin-binding properties.23 To fully
examine the potential of this class of compounds as myelin-
imaging agents, we evaluated 36 coumarin derivatives through in
vitro fluorescent tissue staining and in vivo fluorescent imaging.
Herein we report a full account of structureꢀactivity studies of
these coumarin-based agents, which allow us to identify some
lead compounds suitable for myelin imaging in clinically relevant
settings.
Multiple sclerosis (MS) is the most commonly acquired
demyelinating disease in humans, currently affecting 350,000
Americans and 2 million people worldwide.1 MS is characteristic
of myelin loss in the central nervous system, leading to axonal
damage.2ꢀ5 In order to restore new myelin sheaths to demyeli-
nated regions to protect axons, efforts have been made to
promote endogenous repair mechanisms and/or transplant an
exogenous source of myelinating cells to the demyelinated
regions.6ꢀ12 To achieve this goal, significant progress has
been made and several clinical trials are currently being
conducted.13,14
A major challenge in such clinical studies has been the need to
assess and quantify any changes in myelin content in vivo. To
date, magnetic resonance imaging (MRI) has been used as the
primary tool for detection of brain lesions in MS.15 However,
MRI cannot distinguish lesions caused by demyelination or
inflammation, which often coexist in the brain. This is due to
the fact that any changes in signal intensity on a dual echo T2-
weighted sequence are based on tissue water content. As a result,
the use of MRI as a primary outcome measure of MS disease
activity has been shown to be uncorrelated with clinical
outcomes.16 For this reason, we set out to develop small-
molecule probes (SMP) that bind to myelin membranes with
high affinity and specificity. Over the past years, we have
developed a series of SMPs that readily penetrate the
Received: November 19, 2010
Published: March 10, 2011
r
2011 American Chemical Society
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Figure 1. Myelin-imaging agents for in vivo imaging studies. BMB, BDB, and DAS were precursors of PET-imaging radiotracers. DBT was a novel
myelin-specific optical imaging probe. BMB, (E,E)-1,4-bis(p-aminostyryl)-2-methoxybenzene; BDB, (E,E)-1,4-bis(40-aminostyryl)-2,5-dimethoxyben-
zene; DAS, 4,40-diamino-trans-stilbene; CMC, 3-(4-aminophenyl)-2H-chromen-2-one; DBT, 3,30-diethylthiatricarbocyanine iodide.
’ RESULTS
interaction is dependent on the geometry of the aniline moiety;
(3) substituents in the R2 and R3 positions can be switched
without affecting the binding properties; and (4) with the fluor-
alkyl side chains, the substituents on R2 show higher binding
selectivity than on R3.
In addition to the lipophilicity (LogPoct), the fluorescent
intensity ratios between the white matter and gray matter of
those myelin-binding compounds were also determined as a
parameter to quantitatively evaluate the binding specificity.
Based on these structureꢀactivity relationship studies, particu-
larly the binding ratios between the white matter and gray matter,
32 stands out with the highest binding specificity for myelin.
Thus, we selected 32 (termed FIC) as the lead compound for
further studies.
32 Binds to Myelin in Both the CNS and PNS. The myelin-
binding properties of 32 were further evaluated in both the CNS
and PNS through in vitro fluorescent staining. Both myelinated
corpus callosum and cerebellar regions in CNS and sciatic nerve
in PNS were then examined by fluorescent microscopy. At 100
μM concentration, 32 selectively labeled corpus callosum
(Figure 3A) and cerebellum (Figure 3C) in the CNS as well as
the sciatic nerves in the PNS (Figure 3E). The 32 staining
patterns were found to be virtually identical to the patterns
observed in immunohistochemical staining for MBP (Figure 3B
and D) or Black-Gold II staining (Figure 3F).
Quantitative analysis indicated that the fluorescent intensity is
proportional to the level of myelination present in wild-type
(Figure 4A and C) and shiverer mouse (Figure 4B and D) brains.
As shown in Figure 4E and F, the MBP antibody staining showed
a fluorescent intensity of shiverer mouse brain which is only 14%
of that detected in the wild-type control brains. Consistently, 32
staining also exhibited similar fluorescent intensity, which is
proportional to the level of myelination. In the shiverer mouse
brains, the fluorescent intensity of 32 was 12% of that detected in
the wild-type mouse brains, which was consistent with MBP
immunostaining.
Chemical Synthesis. Synthesis of some coumarin derivatives
(3, 18, 19, 29ꢀ36) was achieved through reactions as shown in
Scheme 1. Briefly, substituted salicaldehyde (37, 39, and 40) and
arylacetic acid (38 and 41) were reacted with sodium acetate to
yield the corresponding coumarin derivatives 3, 18, 29, and 33.
The acetyl groups in 29 and 33 were removed in acidic solution
to obtain 30 and 34 and then coupled with side chains to obtain
final products 31, 32, 35, and 36. Further reduction of 3 with
SnCl2 in ethanol yielded compound 19.
Compound Screening. All of these compounds are fluores-
cent, which allowed us to examine their myelin-binding proper-
ties by fluorescent microscopy. Thus, 1ꢀ36 were screened for
binding specificity for myelin by histological staining of mouse
brain tissue sections. At a concentration of 100 μM of each
compound, whole mouse brain tissue sections containing the
white matter and gray matter were incubated for 20 min. The
myelin-binding specificity was quantitatively examined by fluor-
escent intensity in different brain regions.
Based on the fluorescent tissue staining and fluorescent
intensity in the predefined region of the corpus callosum,
1ꢀ36 can be divided into three groups: (1) compounds includ-
ing 1ꢀ17, which showed no binding to compact myelin; (2)
18ꢀ26, which showed moderate binding to myelin; and (3)
27ꢀ36, which showed high binding. We then further calculated
the fluorescence intensity ratio between the white matter and
gray matter using predefined regions of interest (ROI) as shown
in Figure 2A.
The structureꢀbinding relationship studies led us to propose
some molecular based generalities on myelin-binding properties:
(1) an aniline moiety in the R6 position is normally necessary for
the compound to bind to myelin. The amino group of the aniline
moiety can bear methyl, ethyl, or other small alkyl groups
(27ꢀ36), but the myelin-binding property is diminished or even
lost once the amino group is removed or substituted by a nitro
group (1ꢀ17), which suggests that binding takes place through
direct interaction of the amino group with myelin membranes;
(2) an aniline moiety in the R2 position, such as 20ꢀ26, shows
only moderate binding to myelin, which indicates that binding
32 Stains Myelinated CNS Regions In Situ. Following in vitro
tissue staining, we then evaluated the brain permeability and
subsequent myelin-binding properties of 32 in mice. A dose of 32
(40 mg/kg) was administered to wild-type mice via tail vein
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Table 1. Coumarin-Based Compound Library for Screening of Myelin-Imaging Probes
injection. One hour post injection, the mouse brains were
perfused with saline, followed by 4% paraformaldehyde (PFA),
and removed. The fresh frozen brains were then sectioned.
Fluorescently stained myelinated regions such as the cerebellum
were then directly examined under a fluorescent microscope. As
shown in Figure 5, 32 readily entered the mouse brain and
selectively labeled myelinated cerebellum in situ.
The nature of 32 binding to myelin was revealed by compar-
ison of 32 staining with immunohistochemical staining for MBP
in the cerebellum, caudate putamen, and the temporal cortex
regions (Figure 5). In the cerebellum, 32 staining was confined to
myelin tracts whereas MBP staining was also observed in the
granule cell layers. In the caudate putamen, 32 selectively stained
myelin fibers while MBP also stained oligodendrocyte cell soma
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Scheme 1. Synthesis of Some Coumarin Derivativesa
a Reagents and conditions: (i) sodium acetate, Ac2O, reflux; (ii) 2 N HCl, MeOH, reflux; (iii) 1-fluoro-2-iodoethane, cerium carbonate, dry acetonitrile,
reflux; (iv) 1-fluoro-3-iodopropane, cerium carbonate, dry acetonitrile, reflux; (v) SnCl2, EtOH, reflux.
Figure 2. (A) Regions of interest (ROI) used for quantification of fluorescent intensity and calculation of binding ratios between the white matter and gray
matter. (B) Chemical properties and fluorescence intensity analysis. Log Pand molecular weight values were calculated by ChemBioOffice 2010, and fluorescence
intensity ratios were measured by Image J. Statistical analysis was performed with GraphPad Prism 5, La Jolla, CA (average( SD, n = 3). (C) Comparison of the
electron density of nitrogen and fluorescence intensity ratios of 31, 32, 35, and 36; less electron density of the nitrogen is preferred to enhance the white/gray
matter ratio. Therefore, 32 was an ideal probe for further studies. The electron density of nitrogen is calculated using the H€uckel method by ChemBioOffice 2010.
and processes. In the temporal cortex, no chemical staining was
observed whereas MBP staining revealed the presence of
individual myelinated fibers in the subcortical gray matter. These
studies suggest that 32 selectively binds to compact myelin.
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32 Stains PNS Nerves In Vivo. To examine the ability of 32 to
visualize PNS nerves in vivo, 32 (40 mg/kg) was administered to
wild-type mice via tail vein injection. As shown in Figure 6, 10
min post injection, bright fluorescence was observed in the sciatic
nerves exposed under anesthesia. The fluorescence intensities of
the nerves were significantly higher than that detected in the
surrounding bones or muscles. Thus, 32 could be used intrao-
peratively to visualize delicate nerves that are prone to damage
during surgical procedures in the operating room. These studies
indicated that 32 allowed selective visualization of myelinated
nerves in vivo.
’ DISCUSSION
Myelin possesses a unique structure of a highly differentiated
multilamellar assembly. It is primarily present in vertebrate
species, providing an insulative sheath wrapping around axons
to facilitate saltatory conduction in the nervous system.26 Myelin
sheaths are comprised of 70% lipids and 30% proteins, which are
distributed according to charge, lipophilicity, and relative molec-
ular weight.27 Due to its high lipid-to-protein ratio, myelin is
considered lipophilic in nature. Thus, SMPs have long been
believed to serve as myelin-binding agents that interact with
myelin sheaths solely based on lipid-to-lipid interactions with no
structural discrimination at the molecular level. Consequently, all
molecules that are lipophilic are thought to bind to myelin
regardless of their structural differences. Thus, the structural
basis of myelin-binding interactions has not been thoroughly
examined. Through the structureꢀactivity relationship studies
reported herein, we demonstrate that variation of molecular
structures leads to very different, often completely opposite
myelin-binding properties despite the fact that the lipophilicity
of these molecules was found to be very similar. These results
suggest that there are specific molecule-to-molecule interactions
between SMPs and lipids which determine their binding sensi-
tivity and specificity for myelin.
The precise molecular basis for the selective binding of the
coumarin derivatives to myelin remains to be determined but
most likely reflects interactions with the unique myelin structure.
Myelin sheaths consist of 70% lipid and 30% proteins. In the
proteins present in myelin sheaths, the amino acid composition
shows a high proportion of nonpolar amino acids, excluding
glycine.28 The binding interaction between the coumarin deri-
vatives and myelin is likely to take place at the hydrophobic sites,
which are formed by a focal concentration of nonpolar amino
acids, the fatty acids of bound lipids, or some combination of
both. The coumarin derivatives could gain access to these sites by
virtue of specific interactions with the NMe2 group, since
compounds without an NMe2 group showed virtually no affinity
for myelin. Because the NMe2 could be retained in the hydro-
phobic sites, less electron density of the nitrogen is preferred to
enhance the lipophilic interactions. Indeed, according to our
calculations based on the H€uckel method, the electron density of
Figure 3. In vitro 32 staining of the whole mouse brain (A), cerebellum
(C), and sciatic nerve (E). For comparison, the same sections were used
for MBP staining of the whole mouse brain (B) and cerebellum (D) and
for Black-Gold II staining of sciatic nerves (F). Scale bar: A, B = 500 μm,
C, D = 300 μm, E, F = 100 μm.
Figure 4. Comparison of 32 staining of the wild-type mouse brain (A) and myelin-deficient shiverer mouse brain (B). Staining with anti-MBP of the
same brain sections (C, wild-type; D, shiverer) was also conducted for comparison. Fluorescent intensities in the same corpus callosum region following
MBP staining and 32 staining were quantified. The data were analyzed using a GraphPad Prism. (E) P = 0.0005, n = 3, unpaired t test; (F) P < 0.0001,
n = 3, unpaired t test. Scale bar: A, B = 300 μm, C, D = 500 μm.
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the nitrogen in 32 with a fluoropropoxyl group in the 2-position
is much lower than that in 36 with a 3-fluoroproxyl group
(Figure 2C). The same trend was also observed with the
fluoroethoxyl analogues (31 and 35). As a result, both 31 and
32 showed a similar binding profile in terms of white/gray matter
ratio, which is much higher than that of 35 and 36.
derivatives and binding involves, at least in part, hydrogen
bonding between the amino group and the amino acids of
myelin proteins. Previous studies showed that proteins
present in myelin sheaths such as myelin basic protein
(MBP) have an overall positive charge at physiological
pH because of an excess of lysine and arginine residues.30
Such residues can act as the hydrogen-bonding donor,
and nitrogen in the coumarins acts as an acceptor.
In addition, we conducted further SAR analysis to address the
following important topics:
(1) The necessity of the amino substituent in the active
coumarin analogues. As discussed above, myelin sheets
consist of 70% lipids and 30% proteins made of a high
proportion of nonpolar amino acids. We thus hypothesize
that the coumarin derivatives bind to myelin at hydro-
phobic sites formed by a focal concentration of nonpolar
amino acids of myelin proteins and fatty acids of bound
lipid. Our SAR studies indicated that the presence of an
amino group is critical for active analogues to bind to
myelin. However, compounds with a free amino group
such as 6, 7, 13, and 14 are too hydrophilic to gain access
to the hydrophobic sites. The binding interaction was
significantly enhanced when the amino group was di-
methylated to increase the lipophilicity. Further SAR
analysis shows that the electron density of the nitrogen
is also an important factor in the binding interactions.
(2) The importance of a specific range of logPoct in enhan-
cing activity. Lipophilicity is an important property that
impacts not only the binding interaction between the
coumarin derivatives and myelin but also brain perme-
ability. Previous study has suggested that small molecules
with logPoct values ranging from 1.5 to 3 can passively
and freely penetrate the bloodꢀbrain barrier.29 Our SAR
studies suggest that the logPoct values of active coumarin
analogues should be higher than 2.7 in order to exhibit
appropriate binding to myelin. This is likely due to the fact
that myelin membranes are expected to be more lipophilic
than the membranes present in the bloodꢀbrain barrier.
Thus, we propose that logPoct values between 2.5 and 4.0
are the optimal range for imaging agents to readily enter
the brain and selectively bind to myelin. If the lipophilicity
is too high, significant nonspecific binding is observed.
Thus, with a LogPoct value of 3.82, 32 shows an optimal
binding profile and brain permeability.
The structureꢀactivity relationship results led us to identify
32 as a lead myelin-imaging agent with promising in vitro and
in vivo binding properties. The fluorescent 32 staining in the
CNS was comparable to that of immunohistochemical staining
for MBP. High-magnification views of both types of staining in
different brain regions suggested that 32 staining was confined to
compact myelinated fibers, whereas MBP staining also labeled
individual myelinated fibers. 32 fluoresces with relatively high
resistance to fading in tissue. This allows us to quantify fluores-
cence intensity and to correlate changes with myelin content.
Using part of the corpus callosum in the white matter and a
subcortical region in the gray matter as regions of interest, the
ratios of fluorescent intensity between the white matter and gray
matter were determined for all myelin-binding compounds
(Figure 2B). The ratio of fluorescent intensity can be used as
the parameter to examine the binding specificity for myelin in the
same brain. As shown in Figure 2B, 32 exhibits the highest ratio
of fluorescent intensity between the white matter and gray matter
regions.
The binding specificity of 32 was further evaluated by
comparison of 32 retention in wild-type brains with a my-
elin-deficient shiverer mouse model (Figure 4). Compared to
Figure 6. In vivo visualization of myelinated nerves following injection
of 32 in mice. Image A shows the exposure of sciatic nerves in a mouse
under anesthesia at 10 min post injection of saline. No fluorescence was
observed in sciatic nerves under a dissection fluorescent microscope. At
10 min post injection of 40 mg/kg of 32, strong fluorescence from 32
was observed in the sciatic nerve (B), which also showed better contrast
between the sciatic nerve and surrounding tissues. This remained
unchanged after perfusion (C), suggesting that 32 fluorescence was in
nerves and not blood vessels. Scale bar: A = 1 mm. B, C = 0.1 mm.
(3) The significance of hydrogen bonding in interactions
between coumarins and the myelin environment. Given
the importance of the dimethyl amino group, we pro-
posed that the binding interaction between coumarin
Figure 5. In situ 32 chemical staining of the brain (A), striatum (C), fibers in cortex (E), and cerebellum (G). 32 did not show binding with small fibers
in the cortex. Then the stained sections were double-stained with anti-MBP, and the merged images show the brain (B), striatum (D), fibers in cortex
(F), and cerebellum (H). The lack of staining with anti-MBP on the brain, striatum, and cerebellum indicated that 32 can bind MBP on the big bundle of
myelin sheaths. Scale bar: A, B = 500 μm, C, D = 50 μm, E, F, G, H = 100 μm.
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wild-type controls, 32 retention was significantly reduced by
80%, as confirmed by immunohistochemical staining. These
studies suggest that 32 is a sensitive and specific myelin-imaging
probe that can be selectively retained in the brain proportionally
to the degree of myelination.
’ EXPERIMENTAL SECTION
General Remarks. Compounds 1, 2, 4ꢀ17, and 20ꢀ27 are
commercially available from Sigma-Aldrich, TCI American, or Alfa
Aesar and used without further purification. All NMR spectra were
acquired on an Inova 400 NMR equipped with a 5 mm broadband probe.
High Resolution ESI-MS were acquired on an ESI-FTICRMS (4.7 T) at
the University of Cincinnati Mass Spectrometry Facility. All the inter-
mediates and final product were synthesized in g95% purity, as
determined by a high pressure liquid chromatography (HPLC) system
(Agilent 1100 series, Phenomenex C-18 column, 10 μm, 250 mm ꢁ 10
mm, acetonitrile/H2O = 80:20 (v/v), flow rate 2.0 mL/min) equipped
with a UV detector.
6-Fluoro-3-(4-nitrophenyl)-2H-chromen-2-one (3). To a
round-bottom flask were added 40 (1.4 g, 10.0 mmol), 41 (1.8 g, 10.0
mmol), and sodium acetate (1.6 g, 20.0 mmol), which were then
dissolved in Ac2O (30 mL) and refluxed overnight. After the reaction
mixture was cooled to room temperature, the mixture was neutralized
with 1 M aqueous NaOH; the solid then was collected, washed with
EtOAc, and further purified by recrystallization from EtOAc. The
A unique aspect of this work is the ability of 32 to image
myelinated nerves in both the CNS and PNS. To further evaluate
the potential of 32 staining of myelinated nerves in the PNS, we
examined 32 uptake and retention in sciatic nerves following tail-
vein injection. As shown in Figure 6, as early as 10 min after
injection of 32, strong fluorescence appeared in the sciatic nerve,
but no significant distribution of 32 was seen in regions
surrounding sciatic nerves. Following subsequent perfusion,
the sciatic nerves were still readily visualized under the micro-
scope with very good contrast with the surrounding tissues.
These studies thus suggest that 32 selectively stained the
myelinated nerves in the PNS. Thus, 32 can be used as a
fluorescent probe for intraoperative nerve mapping, which is
often needed during surgical procedures in which surgeons need
to identify myelinated nerves prior to dissecting diseased tissues
to preserve functions of normal organs.
A foremost application of 32 may be in PET imaging of
demyelination. In our previous studies, we identified some lead
compounds that readily entered the brain and selectively loca-
lized in the myelinated regions. These findings provide proof-of-
the-concept that small-molecule myelin-imaging probes can be
developed via direct binding to myelin membranes. However, all
of these imaging probes are radiolabeled with C-11. Due to short
half-life (20 min), C-11-labeled probes can only be synthesized
on site using a dedicated cyclotron and, therefore, are not suitable
for remote distribution. Since most clinical facilities do not have
an on site cyclotron, use of C-11-labeled imaging agents is
limited. In order to enhance the potential of their clinical
applications, myelin-imaging agents have to be radiolabeled with
fluorine-18, a positron-emitting radionuclide with a half-life of
110 min. F-18-Labeled agents are suitable for regional distribu-
tion and thus can be used by PET facilities without an on-site
cyclotron. Thus, in future studies, we plan to thoroughly evaluate
F-18-labeled 32 for PET studies in a way similar to widely used
2-fluorodeoxyglucose (FDG). Once developed, it can be com-
mercially distributed across the country for routine clinical
studies, as is currently done with FDG.
In summary, we have demonstrated that fluorescent coumarin
derivatives represented by 32 can be developed as myelin-
imaging agents for imaging of myelination in the CNS and nerve
mapping in the CNS. In this study, we first show that global
lipophilicity is an important factor for brain permeability and
binding affinity for myelin. Compounds with a logPoct value
between 2.5 and 4 tend to bind to myelin with high affinity.
Further SAR analysis indicated that, when lipophilicity is similar,
some other physicochemical properties become the determining
factors. Because the binding interaction is likely to take place in a
hydrophobic environment, the presence of hydrophilic groups
such as hydroxyl diminishes the binding affinity. The lower
electron density of nitrogen enhances the binding affinity.
Through structureꢀactivity studies, we have identified 32, which
readily penetrates the BBB and selectively stained intact myelin
sheaths in the brain. 32 can also be used as a fluorescent probe for
intraoperative nerve mapping in the PNS. In addition, 32 is
capable of radiolabeling with F-18, thus making it a promising
candidate as PET radiotracer for routine clinical studies, which
will be the primary focus for future investigations.
1
product 3 (2.2 g) was obtained as a yellow solid, yield = 78%. H
NMR (400 MHz, CDCl3): δ = 8.32 (d, J = 9.2 Hz, 2H), 7.89ꢀ7.92
(m, 3H), 7.26ꢀ7.42 (m, 3H). HRMS: calcd for C15H8FNO4Na, 308.0335;
found, 308.0333. HPLC purity: 99.95%, retention time: 10.8 min.
3-(4-(Dimethylamino)phenyl)-6-fluoro-2H-chromen-2-one
(18). To a round-bottom flask were added 40 (1.8 g, 12.5 mmol), 38
(2.2 g, 12.5 mmol), and sodium acetate (2.1 g, 25.0 mmol), which were
then dissolved in Ac2O (30 mL) and refluxed overnight. After the
reaction mixture was cooled to room temperature, the mixture was
neutralized with 1 M aqueous NaOH; the solid then was collected,
washed with EtOAc, and further purified by recrystallization from
EtOAc. The product 18 (2.3 g) was obtained as a yellow solid, yield =
65.3%. 1H NMR (400 MHz, CDCl3): δ = 7.64ꢀ7.68 (m, 3H), 7.31 (t,
J1 = J2 = 4.4 Hz, 1H), 7.28 (d, J = 12.4 Hz, 1H), 6.75ꢀ6.79 (m, 2H), 3.02
(s, 6H). HRMS: calcd for C17H14FNO2Na, 306.0906; found, 306.0914.
HPLC purity: 99.88%, retention time: 8.7 min.
3-(4-Aminophenyl)-6-fluoro-2H-chromen-2-one (19). To a
round-bottom flask were added 3 (2.0 g, 7.0 mmol) and SnCl2 (8.0 g,
42.0 mmol); ethanol then was added, and the mixture was refluxed
overnight. and the ethanol was removed, the residue was neutralized
with aqueous 1 M NaOH, and the precipitate was collected. It was then
dissolved in a large volume of CHCl3 and filtered, and the filtrate was
concentrated to obtain the product 19 (0.63 g) as a yellow solid, yield =
35%. 1H NMR (400 MHz, CDCl3): δ = 7.66 (s, 1H), 7.56 (d, J = 8.8 Hz,
2H), 7.33 (d, J = 4.4 Hz, 1H), 7.30 (d, J = 4.4 Hz, 2H), 6.74 (d, J = 8.4 Hz,
2H), 3.88 (s, 2H). HRMS: calcd for C15H10FNO2Na, 278.0593; found,
278.0593. HPLC purity: 99.95%, retention time: 14.3 min.
3-(4-(Dimethylamino)phenyl)-2-oxo-2H-chromen-7-yl Acet-
ate(29). To a round-bottom flask were added 37 (2.0 g, 14.5 mmol), 38
(2.6 g, 14.5 mmol), and sodium acetate (2.4 g, 29.0 mmol), which were
then dissolved in Ac2O (30 mL) and refluxed overnight. After the
reaction mixture was cooled to room temperature, the mixture was
neutralized with 1 M aqueous NaOH; the solid then was collected,
washed with EtOAc, and further purified by recrystallization from
EtOAc. The product 29 (1.7 g) was collected as a yellow solid, yield =
36.2%. 1H NMR (400 MHz, CDCl3): δ = 7.70 (s, 1H), 7.64 (d, J = 9.2
Hz, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.02ꢀ7.05
(m, 1H), 6.76 (d, J = 8.8 Hz, 2H), 3.01 (s, 6H), 2.34 (s, 3H). HRMS:
calcd for C19H17NO4Na, 346.1055; found, 346.1047. HPLC purity:
97.21%, retention time: 12.0 min.
3-(4-(Dimethylamino)phenyl)-7-hydroxy-2H-chromen-2-
one (30). To a round-bottom flask was charged 29 (3.0 g, 9.3 mmol),
and then 20 mL of MeOH and 10 mL of 2 N HCl were added and
refluxed for 4 h. After the reaction mixture was cooled to room
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Journal of Medicinal Chemistry
ARTICLE
temperature, the mixture was neutralized with 1 M aqueous NaOH; the
solid then was collected, washed with EtOAc, and further purified by
recrystallization from EtOAc. The product 30 (1.96 g) was collected as a
yellow solid, yield = 75%.1H NMR (400 MHz, DMSO-d6): δ = 7.97
(s, 1H), 7.50ꢀ7.55 (m, 3H), 6.68ꢀ6.73 (m, 3H), 2.90 (s, 6H). HRMS:
calcd for C17H15NO3Na, 304.0950; found, 304.0958. HPLC purity:
99.92%, retention time: 9.0 min.
suspended in water, filtered, washed with EtOAc, and further purified by
recrystallization from EtOAc. The product 35 (0.38 g) was obtained as a
yellow solid, yield = 58.4%. 1H NMR (400 MHz, CDCl3): δ = 7.67ꢀ7.67
(m, 3H), 7.27 (d, J = 9.2 Hz, 1H), 7.06ꢀ7.09 (m, 1H), 6.97 (d, J = 2.8 Hz,
1H), 6.76 (d, J = 8.8 Hz, 2H), 4.72ꢀ4.85 (m, 2H), 4.21ꢀ4.30 (m, 2H),
3.01 (s, 6H). HRMS: calcd for C19H18NO4 (M þ Hþ), 328.1349; found,
328.1357. HPLC purity: 98.93%, retention time: 12.1 min.
3-(4-(Dimethylamino)phenyl)-6-(3-fluoropropoxy)-2H-chro-
men-2-one (36). To a round-bottom flask were charged 34 (0.42 g,
1.5 mmol), 1-fluoro-3-iodopropane (0.34 g, 1.8 mmol), and cerium
carbonate (0.98 g, 3.0 mmol), which were then dissolved in dry
acetonitrile (30 mL) and refluxed for 3 h under argon. After the reaction
mixture was cooled to room temperature, the solvent was removed and
then suspended in water, filtered, washed with EtOAc, and further
purified by recrystallization from EtOAc. The product 36 (0.30 g) was
obtained as a yellow solid, yield = 59.1%. 1H NMR (400 MHz, CDCl3):
δ = 7.64ꢀ7.87 (m, 3H), 7.24ꢀ7.27 (m, 1H), 6.96ꢀ7.05 (m, 2H), 6.97
(d, J = 2.8 Hz, 1H), 4.60ꢀ4.74 (m, 2H), 4.13 (t, J1 = J2 = 6.0 Hz, 2H),
3.01 (s, 6H), 2.15ꢀ2.25 (m, 2H). HRMS: calcd for C20H21FNO3 (M þ
Hþ), 342.1505; found, 342.1514. HPLC purity: 98.88%, retention time:
14.9 min.
3-(4-(Dimethylamino)phenyl)-7-(2-fluoroethoxy)-2H-chro-
men-2-one (31). To a round-bottom flask were charged 30 (0.5 g, 1.8
mmol), 1-fluoro-2-iodoethane (0.37 g, 2.2 mmol), and cerium carbonate
(1.8 g, 3.6 mmol), which were then dissolved in dry acetonitrile (30 mL)
and refluxed for 3 h under argon. After the reaction mixture was cooled
to room temperature, the solvent was removed and the solid then was
suspended in water, filtered, washed with EtOAc, and further purified by
recrystallization from EtOAc. The product 31 (0.38 g) was collected as a
yellow solid, yield = 64.2%. 1H NMR (400 MHz, CDCl3): δ = 7.62ꢀ
7.67 (m, 3H), 7.42 (d, J = 8.4 Hz, 1H), 6.84ꢀ6.90 (m, 2H), 6.77 (d, J =
8.8 Hz, 2H), 4.73ꢀ4.88 (m, 2H), 4.23ꢀ4.32 (m, 2H), 3.00 (s, 6H).
HRMS: calcd for C19H18FNO3Na, 350.1169; found, 350.1169. HPLC
purity: 99.66%, retention time: 12.6 min.
3-(4-(Dimethylamino)phenyl)-7-(3-fluoropropoxy)-2H-chro-
men-2-one (32). To a round-bottom flask were charged 30 (0.4 g, 1.4
mmol), 1-fluoro-3-iodopropane (0.32 g, 1.7 mmol), and cerium carbo-
nate (0.91 g, 2.8 mmol), which were then dissolved in dry acetonitrile
(30 mL) and refluxed for 3 h under argon. After the reaction mixture was
cooled to room temperature, the solvent was removed and then
suspended in water, filtered, washed with EtOAc, and further purified
by recrystallization from EtOAc. The product 32 (0.32 g) was collected
as a yellow solid, yield = 68%. 1H NMR (400 MHz, CDCl3): δ = 8.04
(s, 1H), 7.56ꢀ7.64 (m, 3H), 6.93ꢀ7.00 (m, 2H), 6.73 (d, J = 8.8 Hz,
2H), 4.52ꢀ4.67 (m, 2H), 4.14ꢀ4.17 (m, 2H), 2.92 (s, 6H), 2.06ꢀ2.16
(m, 2H). HRMS: calcd for C20H20FNO3Na, 364.1325; found, 364.1316.
HPLC purity: 95.70%, retention time: 15.8 min.
’ ANIMAL PREPARATION AND STUDIES
Animals. All animal experiments were performed in accor-
dance with the guidelines approved by the Institutional Animal
Care and Use Committee of Case Western Reserve University
(Protocol 2010-0007). The animals were subjected to minimal
stress during tail vein injections. The 8-week old C57BL/6 mice
and C3Fe.SWV-Mbpshi/J shiverer mice were obtained from The
Jackson Laboratory, Bar Harbor, MN.
Euthanasia. Mice were deeply anesthetized with isoflurane
and perfused via the ascending aorta with 1xPBS followed by 4%
paraformaldehyde in 1xPBS. Brains and sciatic nerves were
removed and incubated for 24 h in 4% paraformaldehyde at 4 °C.
Tissue Processing. Brain and sciatic nerve tissue were rinsed
in 1xPBS. After treatment with 4% PFA overnight, the tissues
were incubated in 30% sucrose until submerged. 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.
3-(4-(Dimethylamino)phenyl)-2-oxo-2H-chromen-6-yl
acetate (33). To a round-bottom flask were added 39 (2.5 g, 18.0
mmol), 38 (3.2 g, 18.0 mmol), and sodium acetate (3.0 g, 36.0 mmol),
which were then dissolved in Ac2O (40 mL) and refluxed overnight.
After the reaction mixture was cooled to room temperature, the mixture
was neutralized with aqueous 1 M NaOH; the solid then was collected,
washed with EtOAc, and further purified by recrystallization from
EtOAc. The product 33 (4.3 g) was obtained as a yellow solid, yield =
1
73.1%. H NMR (400 MHz, CDCl3): δ = 7.64ꢀ7.66 (m, 3H), 7.33
(d, J = 8.8 Hz, 1H), 7.26ꢀ7.27 (m, 1H), 7.16ꢀ7.19 (m, 1H), 6.76
(d, J = 8.8 Hz, 2H), 3.01 (s, 6H), 2.33 (s, 3H). HRMS: calcd for
C17H15NO3Na, 346.1055; found, 346.1053. HPLC purity: 99.56%,
retention time: 11.7 min.
Chemical staining. Fresh frozen sections with 20 μm in
thickness were incubated in 0.1% Triton-100 in 1xPBS for 10
min and then incubated in a solution of compounds (100 μM) in
10%DMSO/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
Leica DMI5000 inverted microscope (BP470/40 nm filter).
Immunohistochemistry. Fresh frozen sections were rinsed in
3% normal goat serum in 0.1% TritonX-100/1xPBS three times
for 5 min each and washed with 1xPBS three times for 5 min each.
The sections then were immunostained overnight at 4 °C with
purified mouse antibody monoclonal (Covance, Inc., Princeton,
NJ) 1:500 dilution in 3% normal goat serum in 1xPBS. Sections
were incubated for 1 h at room temperature in Cy3 AffiniPure
Goat Anti-Mouse IgG (HþL) (Jackson ImmunoResearch La-
boratories, Inc. West Grove, PA) and rinsed in PBS three times
for 5 min each. Images of the stained mouse brain sections were
3-(4-(Dimethylamino)phenyl)-6-hydroxy-2H-chromen-2-
one (34). To a round-bottom flask was charged 33 (2.9 g, 9.0 mmol),
and then 20 mL of MeOH and 10 mL of 2 N HCl were added and
refluxed for 4 h. After the reaction mixture was cooled to room
temperature, the mixture was neutralized with aqueous 1 M NaOH;
the solid then was collected, washed with EtOAc, and further purified by
recrystallization from EtOAc. The product 34 (1.8 g) was obtained as a
yellow solid, yield = 70.6%. 1H NMR (400 MHz, CDCl3):
δ = 7.63ꢀ7.67 (m, 3H), 7.20ꢀ7.26 (m, 1H), 6.93ꢀ6.97 (m, 2H),
6.77 (d, J = 9.2 Hz, 2H), 4.80 (s, 1H), 3.01 (s, 6H). HRMS: calcd for
C17H15NO3Na, 304.0950; found, 304.0958. HPLC purity: 97.70%,
retention time: 8.7 min.
3-(4-(Dimethylamino)phenyl)-6-(2-fluoroethoxy)-2H-chro-
men-2-one (35). To a round-bottom flask were charged 34 (0.56 g, 2.0
mmol), 1-fluoro-2-iodoethane (0.40 g, 2.4 mmol), and cerium carbonate
(2.0 g, 4.0 mmol), which were then dissolved in dry acetonitrile (30 mL)
and refluxed for 3 h under argon. After the reaction mixture was cooled to
room temperature, the solvent was removed and the solid then was
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ARTICLE
acquired on a Leica DMI5000 inverted microscope (BP 645/75
filter).
’ ABBREVIATIONS USED
CNS, central nervous system; PNS, peripheral nervous system; MS,
multiple sclerosis; MRI, magnetic resonance imaging; SMP, small-
molecule probes; PET, positron emission tomography; BMB,
(E,E)-1,4-bis(p-aminostyryl)-2-methoxybenzene; BDB, (E,E)-1,4-bis-
(40-aminostyryl)-2,5-dimethoxybenzene; DAS, 4,40-diamino-trans-stil-
bene; CMC, 3-(4-aminophenyl)-2H-chromen-2-one; DBT, 3,30-die-
thylthiatricarbocyanine iodide; FIC, fluorinated imaging compound;
ROI, regions of interest; FDG, 2-fluorodeoxyglucose
Black-Gold II Staining. A 0.3% solution of Black-Gold II was
made by adding 150 mg of Black-Gold II to 50 mL of 0.9% NaCl.
The solution was fully equilibrated to 60 °C in a water bath. The
slide mounted tissue sections were transferred to this warm Black
Gold II impregnating solution in the water bath, and the staining
was visually monitored. Images of the stained mouse brain
sections were acquired on a Leica DMI5000 inverted microscope
(bright field).
Quantification and Statistical Analysis. Image J software
(http://rsb.info.nih.gov/ij/) was used to quantify pixel intensi-
ties values. The corpus callosum between the midline and below
the apex of the cingulum was defined as ROI. The density of
myelin in the corpus callosum of wild-type mice was given the
arbitrary value of 100, and the density of myelin in shiverer mice
was determined as a percentage of wild-type mice. The data were
analyzed using the GraphPad Prism, GraphPad Software, La
Jolla, CA, with a nonpaired Student’s t test.
In Situ Tissue Staining of Normal Control Mice Brain
Section. Under anesthesia, wild-type mice were injected with
32 (40 mg/kg) via the tail vein, and the mice were then perfused
transcardially with saline followed by 4% PFA in 1xPBS. Brain
tissues were then removed, postfixed by immersion in 4% PFA
overnight, dehydrated in 30% sucrose solution, cryostat sec-
tioned at 20 μm on a microtome, and mounted on superfrost
slides (Fisher Scientific). For the direct detection, the sections
were coverslipped with fluoromounce mounting media (Vector
Laboratories, Burlingame, CA). Images of the stained mouse
brain sections were acquired on a Leica DMI5000 inverted
microscope (BP470/40 nm filter). For the immunohistochem-
ical costaining, the sections were stained with anti-MBP antibody
following the procedure described above.
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Corresponding Author
*Telephone: 216 844 3288. Fax: 216 844 8062. E-mail: yanming.
wang@case.edu.
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We gratefully acknowledge the financial support of this project
by grants from the NIH/NINDS (R01 NS061837), the Depart-
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for his assistance with preparation of the manuscript.
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