Discovery of 1,2,3-Triazole Derivatives for Multimodality PET/CT/Cryoimaging of Myelination in the Central Nervous System

Article abstract of DOI:10.1021/acs.jmedchem.6b01328

Myelin pathology is present in many neurological conditions such as multiple sclerosis (MS) and traumatic spinal cord injury (SCI). To facilitate development of novel therapies aimed at myelin repair, we set out to develop imaging agents that permit direct quantification of myelination in vivo. In this work, we designed and synthesized a series of fluorescent fluorinated myelin imaging agents that can be used for in vivo positron emission tomography (PET) imaging combined with subsequent post-mortem fluorescent cryoimaging. Structure-activity relationship (SAR) studies of the newly developed myelin imaging agents led us to identify a lead compound (TAFDAS, 21) that readily enters the brain and spinal cord and selectively binds to myelin. By conducting sequential PET and 3D cryoimaging in an SCI rat model, we demonstrated for the first time that PET and cryoimaging can be combined as a novel technique to image the spinal cord with high sensitivity and spatial resolution.

Full text of DOI:10.1021/acs.jmedchem.6b01328

Article
pubs.acs.org/jmc
Discovery of 1,2,3-Triazole Derivatives for Multimodality PET/CT/
Cryoimaging of Myelination in the Central Nervous System
Chunying Wu,*,† Brendan Eck,§ Sheng Zhang,† Junqing Zhu,† Anand Dev Tiwari,† Yifan Zhang,§
Yunjie Zhu,†,¶ Jinming Zhang,∥ Bin Wang,⊥ Xizhen Wang,⊥ Xu Wang,⊥ Jingqiang You,‡ Jian Wang,#
Yihui Guan,∇ Xingdang Liu,∇ Xin Yu,§ Bruce D. Trapp,◆ Robert Miller,○ Jerry Silver,‡ David Wilson,§
and Yanming Wang*,†,⊥
‡
§
†Department of Radiology, Department of Neuroscience, Department of Biomedical Engineering, Case Western Reserve
University, 11100 Euclid Avenue, Cleveland, Ohio 44106, United States,
∥Department of Nuclear Medicine, PLA General Hospital, Beijing 100853, China
⊥Department of Radiology, Bingzhou Medical University, Binzhou, Shandong 256603, China
#Department of Neurology, ∇Department of Nuclear Medicine, Huashan Hospital, Shanghai, 200040, China
○Department of Anatomy and Regenerative Biology, George Washington University, Washington, D.C. 20037, United States
◆Department of Neurosciences, Cleveland Clinic, Cleveland, Ohio 44195, United States
S
* Supporting Information
ABSTRACT: Myelin pathology is present in many neurological conditions such as multiple sclerosis (MS) and traumatic spinal
cord injury (SCI). To facilitate development of novel therapies aimed at myelin repair, we set out to develop imaging agents that
permit direct quantification of myelination in vivo. In this work, we designed and synthesized a series of fluorescent fluorinated
myelin imaging agents that can be used for in vivo positron emission tomography (PET) imaging combined with subsequent
post-mortem fluorescent cryoimaging. Structure−activity relationship (SAR) studies of the newly developed myelin imaging
agents led us to identify a lead compound (TAFDAS, 21) that readily enters the brain and spinal cord and selectively binds to
myelin. By conducting sequential PET and 3D cryoimaging in an SCI rat model, we demonstrated for the first time that PET and
cryoimaging can be combined as a novel technique to image the spinal cord with high sensitivity and spatial resolution.
Currently, conventional magnetic resonance imaging (MRI)
is widely used to detect lesions formed in the brain and spinal
cord.6−9 However, the use of MRI as a primary measure of
disease activity is only indirectly related to clinical outcome.
This is due to the fact that MRI relies solely on changes of
diffusivity of water to detect lesions, which is a nonspecific
measure of the overall changes in macroscopic tissue injury that
ranges from edema to inflammation, demyelination, and axonal
loss. Thus, MRI does not provide a direct measure of myelin
content to specifically detect and quantify demyelination or
remyelination,
INTRODUCTION
■
Myelin membranes constitute an insulating layer around axons
which allow electrical impulses to transmit quickly and
efficiently along the nerve cell.1,2 Abnormalities or destruction
of myelin (demyelination) occurs in many neurodegenerative
conditions such as multiple sclerosis (MS)3,4 and traumatic
injury of the spinal cord.5 In the United States alone, an
estimated 350000 people are afflicted by MS and over 10000
spinal cord injuries (SCIs) occur every year. Tremendous
efforts are being made to develop novel therapeutic agents to
prevent demyelination and/or promote remyelination. For
efficacy evaluation of myelin-repair therapies, myelin-specific
imaging tools are needed for longitudinal evaluation and
quantification of myelin in vivo.
Received: September 9, 2016
© XXXX American Chemical Society
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a
Scheme 1. Click Synthesis of Fluorinated MeDAS Analogues (18−23)
a
(i) Boc2O, THF; (ii) R-I, NaH, THF; (iii) NaH, 4-(prop-2-yn-1-yloxy)benzaldehyde or tert-butyl (4-formylphenyl) (prop-2-yn-1-yl)carbamate
(24), DMF; (iv) 1-azido-2-fluoroethane, CuI, Et3N, DMF, MeOH; (v) HCl, H2O, NaOH.
To overcome the limitations of MRI, we have demonstrated
the feasibility to directly detect and quantify myelin changes in
the central nervous system (CNS) based on positron emission
tomography (PET), which is a functional imaging modality
widely used in clinical settings with high sensitivity and
quantitation capability. Over the past decade, we have
developed a wide array of myelin-specific radiotracers based
on different pharmacophores.10−19 Using a C-11 labeled
radiotracer, [11C]MeDAS, we have demonstrated proof-of-
principle for using PET as an imaging end point for monitoring
treatment response for myelin-repair therapies.18 To further
enhance its potential for routine clinical use, we set out to
develop fluorinated analogues of MeDAS, which potentially can
be radiolabeled with fluorine-18 and distributed by a central
radiopharmacy and used in medical facilities that are not
equipped with an on-site cyclotron. Our studies showed that
selected compounds readily entered the brain and selectively
bound to myelin.19 To further optimize the pharmacokinetic
profiles and achieve rapid, reliable, and reproducible F-18
labeling, we designed and synthesized a novel series of
fluorinated and fluorescent compounds using click chemistry
through Cu(I)-catalyzed Huisgen cycloaddition. In this work,
we report the design, synthesis, and imaging studies of a series
of fluorinated triazole analogues of MeDAS. Systematic
structure−activity relationship (SAR) studies led us to identify
a lead compound termed TAFDAS (21) that can be used for
both in vivo PET imaging and subsequent post-mortem 3D
cryoimaging. We hypothesize that, using the same imaging
agent, PET can be coregistered with microscopic 3D
cryoimaging to seamlessly combine the unique features of
quantitative physiologic information provided by PET with
microscopic histologic information in high resolution provided
by cryoimaging.
synthesized and radiolabeled through a click reaction.
Currently, the most common method of F-18 labeling is the
direct nucleophilic substitution of sulfonic acid esters (such as
tosylates, triflates, or mesylates) into the precursor with
fluorine-18, but this reaction condition raises the possibility
of side reactions like E-2 elimination and substitution of other
potential leaving groups. Over the past decade,20−23 a 1,3-
dipolar cycloaddition reaction between alkynes and azides has
been widely applied to radiolabeling with high specificity and
good yields, which thereby allows efficient F-18 labeling of
radiopharmaceuticals.
As shown in Scheme 1, the synthesis starts with Boc
protection of the amino group of compound diethyl (4-
aminobenzyl) phosphonate (1) to generate compound 2,
followed by alkylation with iodoalkanes to obtain various
alkylated tert-butyl (4-((diethoxyphosphoryl)methyl)-phenyl)-
carbamate (3−5). The obtained compounds 3−5 were coupled
with corresponding aldehydes through the Horner−Wads-
worth−Emmons reaction to generate N-alkylated tert-butyl
(E)-(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)carbamate (6−8)
having a propargyloxyl group in 72−74% yield, and N-alkylated
tert-butyl (E)-(4-(4-((tert-butoxycarbonyl)(prop-2-yn-1-yl)-
amino)styryl)phenyl)carbamate (9−11) with a Boc-protected
propargylamino group in 68−72% yield. Click reactions
between compounds 6−11 and 1-azido-2-fluoroethane cata-
lyzed by copper(I) yielded fluorinated triazoles (12−17) in
50−60% yield, which underwent Boc-deprotection to give
compounds 18−23 as the final products in 70−75% yield.
Fluorescence Properties. The excitation and emission
spectra (0.3 mM in dichloromethane (DCM)) of the newly
synthesized compounds were recorded using a Cary Eclipse
fluorescent spectrophotometer. As shown in Figure 1, all
compounds were fluorescent with excitation wavelengths in the
range of 300−415 nm and emission wavelengths in the range of
394−600 nm.
RESULTS
■
In Vitro Staining of Intact Myelin. The newly synthesized
compounds were first examined for myelin binding by in vitro
staining of frozen mouse brain tissue sections. Because of the
inherent fluorescence, the myelin-binding properties were
directly examined by fluorescent microscopy after staining
Chemical Synthesis. A series of fluorinated triazole
derivatives of MeDAS with different N-alkyl groups were
designed and synthesized as shown in Scheme 1. Incorporation
of triazole provides a pharmacophore that can be readily
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(Figure 2A,B). Because myelin is rich in white matter and
deficient in gray matter, the difference of staining between the
white matter and gray matter is expected to reflect binding
specificity of the test compounds. We thus calculated the
fluorescent intensity ratio (FIR) of each test compound in the
same region of interest (ROI) between the white matter and
gray matter using ImageJ.12,19 As shown in Figure 2G, ROIs of
same size were drawn on a representative region in the genu of
the corpus callosum (gcc) and in the subcortical gray matter
(cortex), respectively. The newly synthesized compounds fell
into two categories based on the calculated FIR (Figure 2F).
The three O-linked phenyl ring compounds (E)-4-(4-((1-(2-
fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-N-alkane-
aniline (18−20) showed FIRs ∼ 1. The other three N-linked
phenyl ring compounds (E)-N-((1-(2-fluoroethyl)-1H-1,2,3-
triazol-4-yl)methyl)-4-(4-(alkane-amino)styryl)-aniline (21−
23) showed FIRs ∼ 1.5. The relatively higher FIRs indicated
a higher degree of specific binding. This study suggests that an
N-linked phenyl ring is more myelin-specific than an O-linked
phenyl ring in terms of binding to myelin sheaths.
Figure 1. Excitation (Ex) and emission (Em) spectra of compounds
18−23 (0.3 mM in methylene chloride). Excitation spectra scans from
300 to 415 nm and emission spectra scans from 394 to 600 nm.
Bandwidth at 5 nm, scan at 120 nm/min, and integration time of 0.5 s.
Maximal excitation wavelengths of compounds 18−23 were at 375 and
395 nm, while maximal emission wavelengths of compounds 18−23
were at 430 nm.
Lipophilicity. To penetrate the blood−brain barrier (BBB),
lipophilicity is one of the three primary requirements that small
molecules have to meet. Research showed that a lipophilicity
value of 1.0−3.5 is ideal for a small molecule to cross the
BBB.24 On the basis of the structures, we calculated the
with each tested compound at 10 μM concentration. All
compounds labeled intact myelin sheaths present in the white
matter included the corpus callosum, striatum, and cerebellum
Figure 2. Tissue staining of compound 21 in wild-type mouse brain and demyelination LPC rat model brain, and preliminary compound screening
with FIR calculated based on ROIs. Fresh frozen wild-type mouse brain sections were stained with compound 21, and highly myelinated corpus
callosum (A) and cerebellum myelin track (B) were clearly visualized. In vitro tissue staining showed that compound 21 can be used to detect
demyelinated lesion present in a rat brain of LPC model (C), which is further confirmed by LFB and cresyl violet staining (D) on an adjacent tissue
section. In situ staining proved that compound 21 crosses the BBB and stains myelinated areas of the mouse brain (E) and spinal cord (F).
Representative in vitro tissue staining of coronal sections showing ROIs used for calculation of FIRs between white matter and gray matter (G), and
FIRs of compounds 18−23 were calculated by ImageJ (H).
C
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lipophilicity of the newly designed compounds as shown in
Table 1. The ClogP value of N-linked compound 21 is 2.86,
which is very similar to our model compound MeDAS (2.91).
Thus, compound 21 was selected for further evaluation.
be used to detect brain lesions based on specific binding to
myelin.
In Situ Staining of Compound 21. Following the in vitro
studies, we then investigated the ability of compound 21 to
readily enter the brain and bind to myelin in situ. A dose of 1.0
mg of compound 21 (50 mg/kg) was administered via the tail
vein to wild-type (WT) mice. One hour after injection, the
mice were perfused with saline followed by 4% paraformalde-
hyde (PFA) and the brain and spinal cord were removed and
sectioned. The distribution of compound 21 was then directly
examined under a fluorescent microscope. As shown in Figure
2E,F, strong fluorescence was visualized in the myelinated
regions including the corpus callosum, striatum, cerebellar
white matter, and the white matter of spinal cord. These studies
suggest that compound 21 readily entered the brain and spinal
cord and localized in all the myelinated regions in proportion to
the myelin content.
Table 1. Structures and ClogP of the Compounds
Synthesized
Radiosynthesis. Encouraged by the in vitro and in situ
results, we next evaluated the in vivo pharmacokinetic profiles
of compound 21 after labeling with positron-emitting nuclide
fluorine-18. As shown in Scheme 2, the radiosynthesis of [18F]
21 was accomplished through a click reaction between
compound 9 and 2-[18F]fluoroethylazide ([18F]26). [18F]26
was prepared through a nucleophilic substitution of tosylate
compound 25 with [18F]KF in the presence of K2CO3 and
Kryptofix (K222) in MeCN at 95 °C for 10 min. The identity
of the product was confirmed using analytical HPLC by
coinjection with cold standard compound 21. The radio-
chemical purity (RCP) of the final products was >98%,
determined by analytical radio-HPLC. The specific activity at
the end of synthesis was in the range of 1−2.5 Ci/μmol.
Quantitative MicroPET/CT Imaging Studies in WT
Rats. Following radiosynthesis, the pharmacokinetic profile of
[18F]21 was fully characterized by quantitative microPET/CT
imaging in WT rats (n = 3). As shown in Figure 3, [18F]21
exhibited high uptake in the brain. The brain radioactivity
peaked at 3 min postinjection followed by rapid clearance
before reaching a plateau at 40−60 min postinjection. The
time−radioactivity curve fitted well the equation C(SUV)
=
1.1189 e−0.0514t + 0.3041, with a calculated time constant of
clearance of 19.45 min.
In Situ Autoradiography. To validate the PET results and
confirm that the PET signals were indeed from specific binding
to myelin, we conducted ex vivo autoradiography in the mouse
brain following administration of [18F]21 through tail vein
injections. After 1 h, the mouse brain was dissected for coronal
tissue sectioning. As shown in Figure 3E, [18F]21 distinctly
labeled the corpus callosum, which is the region with a high
density of myelin sheaths. The autoradiographic visualization
showed that the distribution of [18F]21 was consistent with the
histological staining of myelinated regions with the correspond-
ing nonlabeled compound 21 (see Figure 2A). Thus,
combination of in situ autoradiography and PET imaging
confirmed that [18F]21 binds specifically to myelin in vivo.
Quantitative MicroPET/CT Imaging Studies in Shiver-
er Mice. To further evaluate the in vivo binding specificity of
[18F]21, we investigated the pharmacokinetic profiles of [18F]
21 in a Shiverer mouse model that is deficient in myelin in the
brain. Thus, the pharmacokinetic profiles of [18F]21 were
quantitatively compared between Shiverer mice (C3Fe.SWV-
Mbpshi/J, n = 2) and age-matched WT mice (CB57J, n = 2).
During PET image acquisition, the Shiverer and WT mice were
imaged side by side on the same bed for direct comparison.
In Vitro Detection of Focal Demyelination in Rats. We
then examined if compound 21 can be used to detect
demyelinated lesions present in a rat model of focal
demyelination. In this model, demyelination was induced by
lysolecithin (LPC), which was stereotaxically injected into the
corpus callosum of the right hemisphere. As shown in Figure
2C, compound 21 was capable of detecting the demyelinated
focal lesion by fluorescent staining. The same foci of
demyelination in the adjacent section were confirmed by
conventional Luxol-Fast-Blue (LFB) and cresyl violet staining
(Figure 2D). This result demonstrates that compound 21 can
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Scheme 2. Radiosynthesis of [18F]21 by Click Reaction Followed by Boc Deprotection
Figure 3. Representative (A) coronal, (B) sagittal, and (C) axial microPET/CT fusion images of the rat brain following iv administration of [18F]21,
showing high uptake in the white matter region of the brain. (D) Quantitative analysis of average radioactivity concentration of target compound
[18F]21 in the whole brain of rats (n = 3) in terms of SUV as a function of time. (E) In situ autoradiography showing [18F]21 binds to myelinated
corpus callosum in mouse brain (coronal) .
Dynamic emission scans were acquired for 60 min in 3D list
mode immediately after [18F]21 (0.3 mCi each mouse,
11.1MBq) was administered. The brain uptake of [18F]21
was lower in the Shiverer brain than in the WT littermates
(1.25 vs 1.64). After the brain concentration of [18F]21 reached
a plateau, the SUVs from 40 to 60 min were summarized and
compared. As shown in Figure 4, [18F]21 uptake in the Shiverer
models. We conducted [18F]21-PET imaging in a rat model of
thoracic contusive SCI. In this model, the contusion was made
at T13 to introduce demyelination. In vivo [18F]21 PET
imaging was performed before (as a baseline scan) and 1 day
after contusion. The uptake of [18F]21 in every spine segment
was then normalized to the average spine uptake. As shown in
Figure 5A,B, the whole intact thoracic region of the spinal cord
could be clearly visualized by [18F]21-PET imaging in the
control rats, but the lesion at vertebrae level T13 was clearly
visualized by [18F]21-PET in the SCI model (Figure 5C,D).
Quantitative analysis of [18F]21 uptake in the whole thoracic
region (T1-T13), and part of the lumbar vertebra showed
distinct patterns of uptake. As shown in Figure 5E, the uptake
of [18F]21 in T13 in the SCI group was 0.81, which was
significantly lower than that in their own baseline scans (1.02).
After microPET/CT imaging, we conducted in situ histological
staining of the spinal cord 60 min after injection of nonlabeled
compound 21 through the tail vein. As shown in Figure 6, in
situ histological staining of the SCI (T13) tissue section with
compound 21 showed that a demyelinated lesion was present at
the dorsal column (A), which was confirmed by LFB and cresyl
violet staining of the adjacent sections (B).
Figure 4. Representative coronal microPET images of the shiverer
mouse brain (A) and WT mouse brain (B) following iv administration
of [18F]21, showing higher brain uptake in the control than that in the
shiverer mouse. Quantitative analysis of total uptake of [18F]21 in
terms of SUV at 40−60 min postinjection in shiverer mice [Shi] and
control mouse [WT], showing significant higher uptake in WT mouse
brain (p = 0.00028, two-tailed t test).
Coregistration of in Situ 3D Cryoimaging with
MicroPET/CT Imaging. To confirm the in vivo imaging of
the spinal white matter by PET/CT, we acquired 2D
fluorescence images and generated a 3D reconstructed image
stack. Thus, nonlabeled compound 21 was administered
through tail vein injection into the rat after microPET/CT
imaging of the spinal cord of SCI rats. One hour later, the rat
was perfused with saline followed by 4% paraformaldehyde
(PFA) and the spinal cord (T7-L2) was removed, sequentially
sectioned, and imaged using a Leica fluorescent microscope.
mouse brain was significantly decreased compared to the
control mouse brain. These results suggested that [18F]21
indeed binds to myelin in the brain with high specificity.
Quantitative MicroPET/CT Imaging Studies in the
Spinal Cord. So far, imaging of the spinal cord remains a great
challenge. Our studies showed that compound 21 is capable of
detecting focal demyelinated lesions induced in the rat brain in
vitro. Next, we asked whether [18F]21 PET is capable of in vivo
imaging of myelin deficiency or myelin damage in living animal
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Figure 5. MicroPET/CT imaging. Representative PET/CT fusion images in rats acquired before SCI surgery (baseline scan) with higher
magnification coronal (A) and sagittal images (B) in the T13 spinal cord. Representative PET/CT fusion images in rats acquired 1 day after SCI
surgery with higher magnification coronal (C) and sagittal images (D) in the T13 spinal cord. Note the decrease signal contrast in the SCI animal
compared with the baseline scan. (E) Quantification of the total cumulative [18F]21 uptake in the T13 ROI 30−60 post injection showed
significantly lower uptake in the SCI group compared with baseline scans.
groups. Our previous studies suggested that the two terminal
amino groups of MeDAS are essential moieties for specific
binding to myelin. Alkylation of the amino groups does not
adversely alter the binding properties, which makes it possible
to introduce fluorine to the pharmacophore.
We first examined the lipophilicity of the newly synthesized
compounds, which has a significant impact on brain
permeability. Because introduction of fluorine to one amino
group often renders the compound less lipophilic, we designed
a series of fluorinated compounds by introducing an alkyl group
Figure 6. In situ histological staining of the SCI (T13) tissue section
into the other amino group to compensate for the decreased
with reference compound 21 after microPET/CT imaging showing a
lipophilicity. The fluoro group was introduced through a Cu(I)-
catalyzed Huisgen cycloaddition, known as the “click reaction”,
demyelinated lesion at dorsal portion (A), which is consistent with
LFB and cresyl violet staining using adjacent sections (B).
which can be conducted under mild conditions with high
yield.21,25−29 This striking versatility of Cu(I) catalyzed azide−
alkyne cycloaddition has proven valuable in preparing PET
radiopharmaceuticals.
Slices were aligned sequentially using a semiautomated image
registration algorithm through Matlab, giving a 3D recon-
structed fluorescence image volume. Image volumes from
fluorescence, PET, and CT were visualized in Amira using
volume rendering and aligned based on the shape of the spine
and fiducials from the vertebrae. As shown in Figure 7, 3D
reconstructed fluorescence images (blue) were coregistered
with microPET/CT images (yellow), where the reduced
fluorescence signal was consistent with the reduced microPET
signal in the same spinal cord region T13, thereby confirming
that the reduced PET signal at the injury site was actually
caused by demyelination after the T13 contusion.
After synthesis of compounds 18−23, we conducted in vitro
staining of mouse brain tissue sections. The binding specificity
for myelin of each compound was estimated by calculating the
FIR between the myelin-rich white matter and myelin-deficient
gray matter.17 As shown in Figure 1, these compounds had
similar fluorescence properties (same excitation and emission
wavelength), making it possible for direct comparison of myelin
binding specificity based on the FIR when tissue staining was
conducted using the same thickness of sections and the same
exposure time of image acquisition. Our study suggested that
N-linked analogues (21−23) exhibit higher myelin binding
specificity than O-linked analogues (18−20). This may be in
part due to the fact that introduction of O to the molecules
renders the compounds more lipophilic as is evidenced by the
higher LogP values shown in Table 1, which increase
nonspecific binding.
DISCUSSION AND CONCLUSION
■
In this study, our goal was to developed a novel F-18-labeled
radiotracer [18F]21 that can be used to quantitatively monitor
myelin content in vivo. F-18-labeled radiotracers are often used
to facilitate remote distribution of the tracer from a centralized
radiopharmacy production site. On the basis of our previously
developed C-11 myelin-imaging agent, MeDAS, we designed
and synthesized a series of fluorinated analogues for SAR
studies in order to identify lead candidates capable of in vivo
imaging of myelin. In this work, we designed and synthesized a
series of compounds by alkylating one of the terminal amino
Among those compounds with higher FIR, compound 21
was selected for further study as it has a logPoct value in the
range between 1.5 and 3.5, which is often required for optimal
brain uptake.30 Combination of these studies led us to focus on
compound 21 for further in situ staining, which is usually a
practical way to determine brain permeability and in vivo
F
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Figure 7. Coregistration of in situ 3D fluorescence imaging with microPET/CT imaging. (A) [18F]21 PET/CT imaging of spinal cord in SCI rat
showing reduced uptake after contusive injury at T13 by the arrowhead. (B) 3D in situ fluorescence images of the spinal cord coregistered with CT
image of the spinal cord in SCI rat showing reduced fluorescence intensity at T13 by the arrowhead. (C) Fusion images of PET, CT, and 3D
fluorescence images. (D) Prior to 3D coregistration, a sagittal slice of the 3D fluorescence image stack indicates demyelination corresponding with
microPET/CT imaging.
binding specificity of fluorescent compounds. Our studies
indicate that compound 21 readily enters the brain and
specifically binds to myelin in white matter regions such as the
corpus callosum, striatum, and spinal cord. Furthermore,
compound 21 is capable of detecting demyelinated lesions in
a rat model of focal demyelination induced by LPC. These
studies further confirmed that the characteristic brain uptake
and staining of compound 21 is proportional to myelin content
in the CNS.
Following in vitro and ex vivo studies, we proceeded with
radiosynthesis of [18F]21 for in vivo PET imaging studies. The
radiosynthesis was achieved by coupling 2-[18F]fluoroethyl
azide with its alkyne precursor compound 9 in the presence of
excess Cu2+/ascorbate and bathophenanthrolinedisulfonic acid
disodium salt (BPDS) in aqueous solution, yielding the 1,2,3-
triazoles product with the desired radiochemical yield (80−
90%, n = 5, decay corrected) and radiochemical purity of over
98%. 2-[18F]fluoroethyl azide is the key intermediate. Although
the entire radiosynthesis could be conducted in a “one-pot”
fashion, the radiochemical yield was relatively low. Separate
isolation and purification of 2-[18F]fluoroethyl azide was found
to be critical for successful radiolabeling. Although [18F]-
fluoroethyl azide is highly volatile with a boiling point of 50 °C,
distillation of [18F]fluoroethyl azide from the reaction mixture
was not practical. We thus used solid phase extraction (SPE)
instead with two series of Waters Oasis HLB cartridges to
successfully trap [18F]fluoroethyl azide with high yield and
purity.31
Following radiosynthesis, we conducted quantitative micro-
PET/CT imaging studies in wild-type rats. An ideal radiotracer
for in vivo PET imaging of myelin should meet several criteria
such as high brain uptake and prolonged retention due to high
myelin-binding potential. As expected, quantitative PET data
analysis showed a high and rapid accumulation of [18F]21 in
the brain followed by a rapid nonspecific binding clearance. In
situ autoradiography further confirmed that the radioactivity
signal detected by PET indeed reflects the specific spatial
distribution of [18F]21 after binding to myelinated fibers. The
combination of in situ autoradiography with [18F]21 and with
PET imaging studies confirmed that [18F]21 is a specific
imaging marker of myelin content.
Given the fact that [18F]21 binds to myelin sheaths in vivo,
we further determined whether [18F]21-PET is capable of
imaging myelin deficiency or damage as tested in two animal
models. The Shiverer mouse model was used to represent a
myelin-deficiency in the brain, while the rat model with
traumatic SCI was used to represent myelin damage in the
spinal cord. As shown in Figure 5, quantitative analysis of the
uptake of [18F]21 in the brain showed that retention in the
Shiverer mice was significantly lower than that in the WT
control (p = 0.00028, two-tailed t test). MicroPET/CT imaging
in the SCI rat model further confirmed that [18F]21 is capable
of detecting and quantifying myelin damage in the spinal cord.
As shown in Figure 6, a distinct myelin-imaging defect at T13
contused tissue could be visualized and quantified directly by
PET/CT imaging. Before contusion, [18F]21 uptakes at T12,
T13, and L1 spine segments were respectively 0.99, 1.02, and
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1.05, while the uptakes were 0.99, 0.81, and 1.08 1 day post-
T13 contusion. [18F]21 uptake decreased dramatically at the
site of injury (T13) compared with its own baseline scan.
To date, to expedite the translation of myelin imaging into
clinical practice, some radiotracers such as PIB and florbetaben
that were originally developed for amyloid imaging have been
repurposed for myelin imaging.32,33 These radiotracers have
already been approved by the FDA for clinical use. Such
radiotracers were found to bind to the myelin-rich white matter
in the brain, albeit to a lesser extent than amyloid deposits. It is
thus believed that they could directly be used in MS patients to
image myelin distribution in the white matter region where no
amyloid deposits would be present, even in Alzheimer’s
patients. Thus, the utility of these amyloid-imaging radiotracers
were tested in various subtypes of MS patients for correlation of
tracer uptake with MRI characterization of brain lesions, disease
progression patterns, and functional disability. These studies
provided proof-of-the-concept that PET imaging is a valid
clinical imaging tool to observe white matter changes in terms
of myelin distribution. However, these radiotracers originally
were so optimized for amyloid imaging that any binding
potential in the white matter was minimized. This makes it
difficult to address several important issues concerning imaging
sensitivity and specificity under an inflammatory condition that
is typical of MS. Independent studies often led to controversial
interpretations of imaging results.
fluorescent compound 21 following microPET/CT imaging
using [18F]21. As shown in Figure 6A, a distinct demyelination
at the dorsal portion was observed. This was further confirmed
by standard LFB and cresyl violet staining of myelin sheaths on
the adjacent sections, which suggests there was a significant
demyelinated lesion in the white matter region at the injury
site. In contrast, no tissue loss was observed and myelinated
white matter outside the injured region remained intact (Figure
7B). After tomographic 3D reconstruction, the cryoimages were
coregistered with PET/CT images (Figure 7). We observed a
significant reduction of the fluorescence signal in the lesion
region of the spinal cord, validating the sensitivity and
specificity of PET. These findings suggest that [18F]21-PET
imaging can be used as a diagnostic marker of myelin damage.
In addition, we demonstrated, for the first time, the feasibility of
simultaneous coregistration of PET/CT with and validation by
high-resolution, 3D cryoimaging, which allows for seamless
combination of physiological information with the high
sensitivity and quantification capability provided by PET and
microscopic histology provided by cryoimaging. This newly
developed multimodality imaging technique should greatly
facilitate radiotracer development and efficacy evaluation of
novel therapies.
In summary, a series of fluorinated fluorescent myelin-
binding agents was synthesized for PET and 3D cryoimaging.
Following systematic SAR studies, we identified a novel myelin-
imaging agent (TAFDAS, 21) that readily penetrates the BBB
and binds to myelin membranes in the brain and spinal cord.
Sequential [18F]21-PET imaging and 3D cryoimaging in a
contusion rat model of SCI demonstrate, for the first time, that
combination of PET and cryoimaging is a new imaging tool
with high sensitivity, specificity, and spatial resolution.
To overcome these limitations and enhance the myelin-
imaging sensitivity and specificity, new myelin-imaging radio-
tracers must be developed whereby the binding potential for
myelin can be maximized in the white matter. The parent
compound, MeDAS, was thus optimized to be specific for
myelin and to minimize any effect of inflammation. On the
basis of solid preclinical validation, it will significantly improve
the imaging sensitivity and specificity.
EXPERIMENTAL SECTION
■
An important innovative aspect of this work lies in the
combination of PET/CT with 3D cryoimaging. PET is a
tomographic, functional imaging modality with high quantita-
tive capacity. Yet, PET quantitative analysis must be validated
by correlation with histological characterization. This is often a
very challenging task, particularly in spinal cord imaging due to
its small and mobile structure. Tissue preparation and
sectioning are very time-consuming, and only selected tissue
sections can be used for validation of PET-imaging results,
which is likely biased by the limited scope of sampling.
Tomographic cryoimaging thus can overcome this limitation as
it can provide high-resolution, three-dimensional images of the
entire spinal cord for histological characterization. The whole
process can be fully automated using a CryoViz system
(BioInVision, Inc.).34,35 The imaging results are thus less
subjective as the sampling bias can be essentially eliminated.
More importantly, the 3D cryoimaging of the spinal cord was
carried out in situ using nonlabeled, otherwise identical
compound 21, which was administered via tail vein injection
in a way similar to the PET scan. This promotes accurate image
coregistration between PET and cryoimaging for quantitative
analysis.
Methods and Materials. Chemicals and reagents were used as
received without further purification. Glassware was dried in an oven at
130 °C and purged with a dry atmosphere prior to use. Unless
otherwise mentioned, reactions were performed open to air. Reactions
were monitored by TLC and visualized by a dual short/long wave UV
lamp. Column flash chromatography was performed using 230−400
mesh silica gel (Fisher). Preparative TLC was performed on Analtech
Preparative TLC Uniplates with UV254 fluorescence indicator (500
μm). NMR spectra were recorded on a Varian Inova 400 spectrometer
and 500 MHz Bruker Ascend Avance III HD at room temperature.
Florescence spectra were recorded, and chemical shifts for 1H and 13
C
NMR were reported as δ, part per million (ppm), and referenced to an
internal deuterated solvent central line. The abbreviations s, br s, d, dd,
ddd, br d, t, dt, q, m, and br m stand for their resonance multiplicity
singlet, broad singlet, doublet, doublet of doublets, doublet of doublet
of doublets, broad doublets, triplet, triplets of doublets, quartet,
multiplet, and broad multiplet, respectively, which were calculated
automatically on a MestReNova 10.0. The purity of tested compounds
as determined by analytical HPLC was 95%. HRMS-ESI mass spectra
were acquired on an Agilent Q-TOF. 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 spectropho-
tometer using a 1 cm × 1 cm quartz cuvette.
General Method for Alkylation of tert-Butyl (4-
((Diethoxyphosphoryl)methyl)phenyl)carbamate (3−5). To an
oven-dried 100 mL round-bottom flask purged with argon gas and
fitted with a magnetic stir bar, sodium hydride (2 equiv 95%) and tert-
butyl (4-((diethoxyphosphoryl) methyl)phenyl)carbamate (1 equiv)
were added together with dry THF (25 mL) at 0 °C and purged with
argon gas. Iodo-alkane (3 equiv) was added slowly dropwise after 30
min at 0 °C under argon gas. The reaction was stirred under argon and
allowed to reach room temperature overnight. After completion, the
This new multimodality PET/cryoimaging technique was
first applied in the studies of SCI. In vivo imaging of myelin
changes after SCI is of particular importance as demyelination
and remyelination have direct effects on disability and
functional recovery. To confirm that the contusive injury at
T13 was associated directly with demyelination rather than
tissue loss, we conducted in situ 3D cryoimaging with
H
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Journal of Medicinal Chemistry
Article
reaction was quenched with water and THF was removed in vacuum.
The residue was dissolved in DCM and water and the aqueous layer
was extracted three times with DCM (30 mL). The organic layers were
combined and washed twice with water (50 mL) and once with brine
(50 mL). The organic layer was dried over Na2SO4 and concentrated
to give the desired product as a sticky oil, which was used for the next
step without further purification.
General Method for Horner−Wadsworth−Emmons Coupling
Reaction (6−11). To an oven-dried 100 mL round-bottom flask with a
magnetic stir bar was added sodium hydride (2 equiv, 95%). The flask
was purged with argon, and 2 mL of dry DMF was added. This
solution was cooled to 0 °C, and the phosphonate in DMF (2 mL)
was added. The mixture was allowed to stir for 1 h at 0 °C. Then the
aldehyde in 2 mL of DMF was added slowly. The reaction was
continued for another 2 h at 0 °C under argon followed by quenching
with water (50 mL) and then extracted with ethyl acetate (50 mL)
three times. The organic layer was washed with saturated NaHCO3
(20 mL) and brine (50 mL), dried with Na2SO4, and concentrated to
give the crude product, which was purified with flash chromatography
using hexanes and ethyl acetate as eluents.
added. The reaction mixture was stirred overnight at room
temperature, and saturated NaHCO3 (20 mL) was then added
followed by extraction with ethyl acetate (20 mL) three times. The
combined organic layers were washed with water (50 mL) and brine
(50 mL), dried with Na2SO4, and concentrated to give the crude
product, which was purified by column chromatography eluted with
hexanes and ethyl acetate.
tert-Butyl (E)-(4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)-
methoxy)styryl)phenyl)(methyl)carbamate (12). 1H NMR (400
MHz, chloroform-d) δ 7.70 (d, J = 1.0 Hz, 1H), 7.40 (dd, J = 8.7,
3.0 Hz, 4H), 7.17 (d, J = 8.5 Hz, 2H), 6.95 (s, 3H), 6.93 (d, J = 22.8
Hz, 4H), 6.90 (d, J = 16.5 Hz, 2H), 4.81 (dd, J = 5.2, 4.1 Hz, 1H), 4.67
(ddd, J = 14.2, 5.5, 4.3 Hz, 2H), 4.59 (dd, J = 5.2, 3.9 Hz, 1H), 3.22 (s,
3H), 1.42 (s, 9H). 13C NMR (100 MHz, chloroform-d) δ 158.0, 154.9,
144.6, 143.0, 134.7, 130.8, 128.0, 127.9, 126.5, 126.4, 125.7, 124.0,
115.2, 114.8, 82.5, 80.8, 80.6, 62.1, 50.9, 50.7, 37.4, 28.5.
tert-Butyl (E)-(4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4yl)-
1
methoxy)styryl)phenyl)(ethyl)carbamate (13). H NMR (400 MHz,
chloroform-d) δ 7.72−7.70 (m, 1H), 7.40 (dd, J = 8.6, 1.8 Hz, 4H),
7.12 (d, J = 8.1 Hz, 2H), 6.98 (d, J = 16.3 Hz, 1H), 6.96−6.93 (m,
2H), 6.91 (d, J = 15.9 Hz, 1H), 5.19 (s, 2H), 4.81 (dd, J = 5.2, 4.1 Hz,
1H), 4.75−4.65 (m, 2H), 4.59 (dd, J = 5.2, 4.0 Hz, 1H), 3.64 (q, J =
7.1 Hz, 2H), 1.40 (s, 9H), 1.12 (t, J = 7.1 Hz, 3H). 13C NMR (100
MHz, chloroform-d) δ 158.0, 154.7, 144.6, 141.7, 135.3, 130.8, 128.1,
127.9, 127.2, 126.7, 126.4, 124.0, 115.2, 114.8, 82.5, 80.8, 80.2, 62.1,
50.9, 50.7, 45.1, 28.6, 14.1.
tert-Butyl (E)-(4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)-
methoxy)styryl)phenyl)(propyl)carbamate (14). 1H NMR (400
MHz, chloroform-d) δ 7.70 (s, 1H), 7.40 (dd, J = 8.7, 1.9 Hz, 4H),
7.12 (d, J = 8.1 Hz, 2H), 6.98 (d, J = 16.2 Hz, 1H), 6.96−6.93 (m,
2H), 6.91 (d, J = 16.2 Hz, 1H), 5.18 (s, 2H), 4.80 (dd, J = 5.2, 4.1 Hz,
1H), 4.67 (ddd, J = 14.2, 5.4, 4.3 Hz, 2H), 4.58 (dd, J = 5.2, 4.0 Hz,
1H), 3.59−3.52 (m, 2H), 1.57−1.47 (m, 2H), 1.40 (s, 9H), 0.84 (t, J =
7.4 Hz, 3H). 13C NMR (100 MHz, chloroform-d) δ 158.1, 154.9,
144.7, 141.8, 135.3, 130.9, 128.1, 127.9, 127.3, 126.7, 126.4, 124.0,
115.2, 114.8, 82.5, 80.8, 80.2, 62.2, 51.7, 50.9, 50.7, 28.5, 21.9, 11.4.
tert-Butyl (E)-(4-(4-((tert-Butoxycarbonyl)((1-(2-fluoroethyl)-1H-
1,2,3-triazol-4-yl)methyl)amino)-styryl)phenyl)(methyl)carbamate
(15). 1H NMR (400 MHz, chloroform-d) δ 7.68 (s, 1H), 7.44 (dd, J =
8.7, 6.9 Hz, 4H), 7.28 (d, J = 7.8 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H),
7.02 (s, 2H), 4.92 (s, 2H), 4.84 (dd, J = 5.2, 4.1 Hz, 1H), 4.71 (ddd, J
= 13.2, 5.3, 4.3 Hz, 2H), 4.62 (dd, J = 5.1, 4.1 Hz, 1H), 3.27 (s, 3H),
1.46 (s, 9H), 1.45 (s, 9H). 13C NMR (100 MHz, chloroform-d) δ
154.6, 154.5, 145.9, 143.3, 142.1, 135.0, 134.5, 128.1, 127.9, 126.8,
126.7, 126.4, 125.6, 123.9, 82.6, 81.2, 80.9, 80.6, 50.8, 50.6, 46.0, 37.4,
28.5, 28.5.
tert-Butyl (E)-Ethyl(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)-
carbamate (7). 1H NMR (400 MHz, chloroform-d) δ 7.43−7.36
(m, 4H), 7.13 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 16.3 Hz, 1H), 6.93 (d, J
= 1.9 Hz, 2H), 6.90 (d, J = 7.4 Hz, 1H), 4.63 (dd, J = 2.5, 1.1 Hz, 2H),
3.65 (q, J = 7.1 Hz, 2H), 2.50 (t, J = 2.4 Hz, 1H), 1.42 (s, 9H), 1.13 (t,
J = 7.1 Hz, 3H). 13C NMR (100 MHz, chloroform-d) δ 157.4, 154.6,
141.7, 135.3, 131.1, 128.1, 127.9, 127.2, 126.7, 126.5, 115.3, 80.2, 78.7,
76.0, 56.0, 45.1, 28.6, 14.2.
tert-Butyl (E)-Propyl(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)-
1
carbamate (8). H NMR (400 MHz, chloroform-d) δ 7.40 (dd, J =
8.7, 2.1 Hz, 4H), 7.14 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 16.5 Hz, 1H),
6.93 (s, 2H), 6.90 (d, J = 7.1 Hz, 1H), 4.63 (d, J = 2.4 Hz, 2H), 3.62−
3.54 (m, 2H), 2.50 (s, 1H), 1.61−1.49 (m, 2H), 1.43 (s, 9H), 0.87 (t, J
= 7.4 Hz, 3H). 13C NMR (100 MHz, chloroform-d) δ 157.4, 154.9,
141.8, 135.3, 131.1, 128.2, 127.9, 127.3, 126.7, 126.5, 115.3, 80.2, 78.7,
76.0, 55.99, 51.8, 28.6, 22.0, 11.4.
tert-Butyl (E)-(4-(4-((tert-Butoxycarbonyl)(prop-2-yn-1-yl)amino)-
1
styryl)phenyl)(methyl)carbamate (9). H NMR (400 MHz, chloro-
form-d) δ 7.46 (dd, J = 8.7, 6.7 Hz, 4H), 7.31 (d, J = 8.2 Hz, 2H), 7.22
(d, J = 8.6 Hz, 2H), 7.03 (s, 2H), 4.37 (d, J = 2.5 Hz, 2H), 3.26 (s,
3H), 2.27−2.24 (t, J = 4.8 1H), 1.46 (s, 18H). 13C NMR (100 MHz,
chloroform-d) δ 154.8, 154.1, 143.3, 141.5, 135.4, 134.4, 128.2, 127.9,
127.8, 126.9, 126.8, 126.5, 125.6, 81.4, 80.6, 80.2, 72.1, 39.9, 37.4, 28.5,
28.5.
tert-Butyl (E)-(4-(4-((tert-Butoxycarbonyl)(prop-2-yn-1-yl)amino)-
1
styryl)phenyl)(ethyl)carbamate (10). H NMR (400 MHz, chloro-
tert-Butyl (E)-(4-(4-((tert-Butoxycarbonyl)((1-(2-fluoroethyl)-1H-
form-d) δ 7.45−7.42 (m, 4H), 7.28 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.5
Hz, 2H), 7.01 (s, 2H), 4.32 (d, J = 2.4 Hz, 2H), 3.65 (q, J = 7.0 Hz,
2H), 2.23 (t, J = 2.4 Hz, 1H), 1.44 (s, 9H), 1.44 (s, 9H), 1.12 (t, J =
7.1 Hz, 3H). 13C NMR (100 MHz, chloroform-d) δ 154.6, 154.1,
142.0, 141.6, 135.4, 135.0, 128.3, 128.0, 127.2, 126.9, 126.9, 126.5,
81.4, 80.3, 80.2, 72.1, 45.0, 39.9, 28.6, 28.5, 14.1.
1,2,3-triazol-4-yl)methyl)amino)-styryl)phenyl)(ethyl)carbamate
1
(16). H NMR (400 MHz, chloroform-d) δ 7.73−7.62 (m, 1H), 7.45
(t, J = 8.5 Hz, 4H), 7.31−7.26 (m, 2H), 7.17 (d, J = 8.5 Hz, 2H), 7.03
(s, 2H), 4.92 (s, 2H), 4.82 (dd, J = 5.2, 4.1 Hz, 1H), 4.69 (ddd, J =
12.4, 5.5, 4.3 Hz, 2H), 4.61 (dd, J = 5.2, 4.0 Hz, 1H), 3.68 (q, J = 7.1
Hz, 2H), 1.45 (s, 9H), 1.44 (s, 9H), 1.16 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, chloroform-d) δ 154.6, 154.45, 145.9, 142.1, 141.95, 135.0,
128.1, 128.0, 127.2, 126.9, 126.8, 126.4, 123.9, 82.6, 81.2, 80.9, 80.3,
50.8, 50.6, 46.0, 45.0, 28.5, 28.5, 14.1.
tert-Butyl (E)-(4-(4-((tert-Butoxycarbonyl)(prop-2-yn-1-yl)amino)-
1
styryl)phenyl)(propyl)carbamate (11). H NMR (400 MHz, chloro-
form-d) δ 7.48−7.45 (m, 4H), 7.31 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.3
Hz, 2H), 7.05 (s, 2H), 4.37 (d, J = 2.5 Hz, 2H), 3.62−3.58 (m, 2H),
2.26 (t, J = 2.4 Hz, 1H), 1.63−1.52 (m, 2H), 1.47 (s, 9H), 1.44 (s,
9H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, chloroform-d) δ
154.9, 154.1, 142.2, 141.6, 135.4, 135.0, 128.3, 128.0, 127.3, 126.9,
126.5, 81.4, 80.2, 80.2, 72.0, 51.7, 39.9, 28.5, 28.4, 21.9, 11.4.
General Method for Click Chemistry (12−17). A portion of 1-
(4-methylbenzenesulfonate)-2-fluoroethanol (1.67 equiv) in DMF (4
mL) was stirred with a suspension of sodium azide (5.6 equiv) at room
temperature. After 48 h, the solution was filtered through Celite and
the crude 1-azido-2-fluoroethane was used immediately in the next
step without further purification. In a separate flask, copper(I) iodide
(5.4 equiv) was suspended in methanol (2 mL) under an argon
atmosphere with vigorous stirring. In rapid succession, the alkyne
precursor (1 equiv) dissolved in methanol (1 mL), the crude 1-azido-
2-fluoroethane dissolved in DMF, and triethylamine (5.4 equiv) were
tert-Butyl (E)-(4-(4-((tert-Butoxycarbonyl)((1-(2-fluoroethyl)-1H-
1,2,3-triazol-4-yl)methyl)amino)-styryl)phenyl)(propyl)carbamate
(17). 1H NMR (400 MHz, chloroform-d) δ 7.68 (s, 1H), 7.45 (dd, J =
9.0, 7.2 Hz, 4H), 7.28 (d, J = 9.0 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H),
7.03 (s, 2H), 4.92 (s, 2H), 4.84 (dd, J = 5.2, 4.1 Hz, 1H), 4.71 (ddd, J
= 13.2, 5.5, 4.3 Hz, 2H), 4.63 (dd, J = 5.2, 4.0 Hz, 1H), 1.62−1.53 (m,
2H), 1.45 (s, 9H), 1.44 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H), 0.89 (t, J =
12.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 154.9, 154.5, 146.0,
142.1, 135.0, 128.1, 128.0, 127.3, 126.9, 126.4, 123.9, 82.6, 81.2, 80.9,
80.26, 51.7, 50.8, 50.6, 46.0, 28.5, 28.5, 21.9, 11.4.
General Method for Boc Deprotection (18−23). The click
product was dissolved in 2 mL of methanol. To this mixture was added
2 mL of HCl (1.2 M), and the mixture was stirred for 1.5 h at 60 °C
and then sufficient NaOH (1 M) was added to bring the pH to 10.
Water was added to the mixture and extracted with ethyl acetate (10
I
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Journal of Medicinal Chemistry
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mL) three times, washed with brine (30 mL), dried with Na2SO4, and
concentrated to give the crude product. Prep TLC or flash
chromatography (hexanes/acetone) yielded the deprotected product.
(E)-4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-
4.49 (s, 2H), 3.10 (t, J = 7.1 Hz, 2H), 1.65 (q, J = 7.2 Hz, 3H), 1.00 (t,
J = 7.4 Hz, 3H). 13C NMR (125 MHz, chloroform-d) δ 147.6, 146.6,
146.5, 128.5, 127.3, 127.2, 127.2, 125.4, 124.4, 122.5, 113.3, 112.8,
82.2, 80.8, 50.6, 50.5, 45.8, 39.9, 31.9, 22.7, 11.6. HR-MS (ESI) m/z
calculated for (C22H27FN5) [M + H]+ 380.2245, found 380.2248.
HPLC purity: 99.93%, retention time 8.13 min. C-18 reversed-phase
HPLC (Phenomenex, 10 mm × 250 mm); eluent, acetonitrile:H2O =
30:70; flow rate of 1.0 mL/min.
tert-Butyl (4-Formylphenyl)(prop-2-yn-1-yl)carbamate (24). To
an oven-dried 100 mL round-bottom flask purged with argon gas and
fitted with a magnetic stir bar was added sodium hydride (140 mg,
95%) and tert-butyl (4-formylphenyl)carbamate (1.02 g), which was
purged with argon gas under dry THF (25 mL) at 0 °C. 3-Bromoprop-
1-yne (1.55 mL, 80% in toluene) was added slowly dropwise after 30
min at 0 °C under argon gas. The reaction was stirred under argon and
allowed to reach room temperature. After 3 h, the reaction was
quenched with water and extracted with ethyl acetate (30 mL × 3).
The organic layers were combined and washed twice with water (50
mL) and once with brine (50 mL). The organic layer was dried over
Na2SO4 and concentrated to give the crude product. Flash
chromatography (Hex:EA/8:1) yielded 24 (1.06 g, 89%) as an oil.
1H NMR (400 MHz, chloroform-d) δ 9.92 (s, 1H), 7.87−7.78 (m,
2H), 7.55−7.47 (m, 2H), 4.39 (dd, J = 2.5, 0.9 Hz, 2H), 2.28 (t, J = 2.4
Hz, 1H), 1.46 (s, 9H). 13C NMR (100 MHz, chloroform-d) δ 191.2,
153.1, 147.6, 133.4, 130.2, 125.4, 82.1, 79.4, 72.4, 39.4, 28.2.
1
N-methylaniline (18). H NMR (400 MHz, DMSO-d6) δ 8.27 (s,
1H), 7.44 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.6 Hz, 2H), 7.07−6.98 (m,
2H), 6.94 (d, J = 16.4 Hz, 1H), 6.85 (d, J = 16.4 Hz, 1H), 6.52 (d, J =
8.7 Hz, 2H), 5.82 (q, J = 5.0 Hz, 1H), 5.16 (s, 2H), 4.89 (dd, J = 5.3,
4.1 Hz, 1H), 4.81−4.75 (m, 2H), 4.73−4.67 (m, 1H), 2.69 (d, J = 5.1
Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 157.4, 149.9, 143.4,
131.5, 127.8, 127.5, 127.4, 125.4, 125.3, 122.9, 115.3, 112.1, 83.0, 81.7,
61.5, 50.6, 50.4, 30.1. HR-MS (ESI) m/z calculated for (C20H22FN4O)
[M + H]+ 353.1772, found 353.1773. HPLC purity: 96.94%, retention
time 4.00 min. C-18 reversed-phase HPLC (Phenomenex, 10 mm ×
250 mm); eluent, acetonitrile:H2O = 40:60; flow rate of 1.0 mL/min.
(E)-4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-
1
N-ethylaniline (19). H NMR (500 MHz, chloroform-d) δ 7.74 (s,
1H), 7.40 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.1
Hz, 2H), 6.90 (d, J = 16.3 Hz, 1H), 6.84 (d, J = 16.3 Hz, 1H), 6.59 (d,
J = 8.0 Hz, 2H), 5.24 (s, 2H), 4.85 (t, J = 4.8 Hz, 1H), 4.76 (t, J = 4.8
Hz, 1H), 4.71 (t, J = 4.8 Hz, 1H), 4.65 (t, J = 4.8 Hz, 1H), 3.18 (q, J =
7.1 Hz, 2H), 1.27 (t, J = 6.7 Hz, 3H). 13C NMR (125 MHz,
chloroform-d) δ 157.2, 147.8, 144.7, 131.6, 127.5, 127.3, 127.2, 126.9,
123.8, 123.7, 115.0, 112.8, 82.2, 80.8, 62.0, 50.7, 50.5, 38.5, 14.8. HR-
MS (ESI) m/z calculated for (C21H24FN4O) [M + H]+ 367.1929,
found 367.1932. HPLC purity: 96.22%, retention time 4.02 min. C-18
reversed-phase HPLC (Phenomenex, 10 mm × 250 mm); eluent,
acetonitrile:H2O = 40:60; flow rate of 1.0 mL/min.
2-Azidoethyl 4-Methylbenzenesulfonate (25). To an oven-dried
100 mL round-bottom flask purged with argon gas and fitted with a
magnetic stir bar was added DCM (15 mL), 2-azidoethan-1-ol (2.3
mL, 0.5 M in methyl tert-butyl ether), 4-toluenesulfonyl chloride (0.33
g), and Et3N (0.32 mL). The reaction mixture was allowed to stir
overnight at room temperature, and water was then added followed by
two extractions with DCM (30 mL). The organic layers were
combined and washed with water (50 mL) and brine (50 mL). Flash
(E)-4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-
1
N-propylaniline (20). H NMR (400 MHz, chloroform-d) δ 7.68 (s,
1H), 7.33 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.7
Hz, 2H), 6.83 (d, J = 16.2 Hz, 1H), 6.77 (d, J = 16.3 Hz, 1H), 6.51 (d,
J = 8.5 Hz, 2H), 5.17 (s, 2H), 4.80 (dd, J = 5.2, 4.1 Hz, 1H), 4.67 (dt, J
= 13.4, 4.6 Hz, 2H), 4.61−4.56 (m, 1H), 3.04 (t, J = 7.1 Hz, 2H), 1.57
(p, J = 7.3 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz,
chloroform-d) δ 157.4, 148.2, 144.9, 131.8, 127.7, 127.4, 127.3, 126.9,
123.9, 115.1, 112.9, 82.5, 80.8, 62.2, 50.9, 50.7, 45.9, 22.9, 11.8. HR-
MS (ESI) m/z calculated for (C22H26FN4O) [M + H]+ 381.2085,
found 381.2090. HPLC purity: 96.28%, retention time 6.50 min. C-18
reversed-phase HPLC (Phenomenex, 10 mm × 250 mm); eluent,
acetonitrile:H2O = 40:60; flow rate of 1.0 mL/min.
1
chromatography (Hex:EA/4:1) yielded 25 (0.22 g, 79%). H NMR
(400 MHz, chloroform-d) δ 7.85−7.76 (m, 2H), 7.42−7.34 (m, 2H),
4.15 (ddd, J = 5.2, 4.5, 0.9 Hz, 2H), 3.53−3.42 (m, 2H), 2.45 (s, 3H).
Animal Preparation and Studies. All animal experiments were
performed in accordance with guidance protocol approved by the
Institutional Animal Care and Use Committee (IACUC) of Case
Western Reserve University (protocols 2016-0028, 2016-0023). The
8-week old WT C57BL/6 mice were used for all of the in vitro and ex
vivo tissue staining. and SD rats (Harlan Laboratory, Indianapolis, IN)
and Shiverer mice (Jackson Laboratory, Bar Harbor, ME) were used
for microPET/CT imaging studies. The animals were fasted overnight
prior to imaging but had access to water. Their diet was then
replenished after microPET/CT imaging.
Brain Focal Demyelination Rat Model. Sprague−Dawley female
rats (220−250 g, 8 weeks old) were anesthetized and positioned in a
stereotaxic frame (Stoelting). A small incision was made in the scalp,
and the corpus callosum was targeted using the following stereotaxic
coordinates, relative to bregma: anterior−posterior, 0.0 mm; medial−
lateral, 2.0 mm; and dorsal−ventral, 3.4 mm. A small hole was drilled
in the skull, and a 26S-gauge needle attached to a 10 μL Hamilton
Syringe was lowered into the corpus callosum according to the dorsal−
ventral coordinate. A microinjector pump (Stoelting) controlled the
infusion of 6 μL of lysolecithin (LPC, 0.1% in saline) at a rate of 0.25
μL/min, after which the needle was left in place for 2 min in order to
prevent liquid reflux out of the brain parenchyma. The incision was
then closed using 5−0 Ethicon sutures, and the animals were allowed
to recover on a heating pad. After 5−7 days, the animals were ready for
study.
(E)-N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-
1
(methylamino)styryl)aniline (21). H NMR (400 MHz, chloroform-
d) δ 7.55 (s, 1H), 7.32 (dd, J = 8.6, 1.8 Hz, 4H), 6.83 (d, J = 1.7 Hz,
2H), 6.64 (d, J = 8.6 Hz, 2H), 6.58 (d, J = 8.6 Hz, 2H), 4.82 (dd, J =
5.1, 4.2 Hz, 1H), 4.71 (dd, J = 5.2, 4.1 Hz, 1H), 4.65 (dd, J = 5.2, 4.2
Hz, 1H), 4.59 (dd, J = 5.2, 4.1 Hz, 1H), 2.85 (s, 3H). 13C NMR (100
MHz, chloroform-d) δ 148.7, 146.7, 146.7, 128.6, 127.6, 127.4, 127.4,
125.5, 124.7, 122.7, 113.5, 112.7, 82.6, 80.8, 50.8, 50.6, 40.1, 30.9. HR-
MS (ESI) m/z calculated for (C20H23FN5) [M + H]+ 352.1932, found
352.1933. HPLC purity: 95.64%, retention time 4.47 min. C-18
reversed-phase HPLC (Phenomenex, 10 mm × 250 mm); eluent,
acetonitrile:H2O = 50:50; flow rate of 1.0 mL/min.
(E)-N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-
(ethylamino)styryl)aniline (22). 1H NMR (400 MHz, chloroform-d) δ
7.57 (s, 1H), 7.31 (dd, J = 8.5, 3.6 Hz, 4H), 6.82 (d, J = 1.5 Hz, 2H),
6.64 (d, J = 8.5 Hz, 2H), 6.58 (d, J = 8.5 Hz, 2H), 4.89−4.82 (m, 1H),
4.72 (dd, J = 5.2, 4.0 Hz, 1H), 4.69−4.63 (m, 1H), 4.63−4.56 (m,
1H), 4.49 (s, 2H), 3.18 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
13C NMR (100 MHz, chloroform-d) δ 147.8, 146.8, 146.65, 128.6,
127.5, 127.4, 127.4, 125.5, 124.6, 122.7, 113.5, 113.0, 82.6, 80.7, 50.9,
50.6, 40.1, 38.7, 15.1. HR-MS (ESI) m/z calculated for (C21H25FN5)
[M + H]+ 366.2089, found 366.2089. HPLC purity: 98.41%, retention
time 5.05 min. C-18 reversed-phase HPLC (Phenomenex, 10 mm ×
250 mm); eluent, acetonitrile:H2O = 30:70; flow rate of 1.0 mL/min.
(E)-N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-
(propylamino)styryl)aniline (23). 1H NMR (400 MHz, chloroform-d)
δ 7.57 (s, 1H), 7.31 (dd, J = 8.5, 5.1 Hz, 4H), 6.82 (d, J = 1.8 Hz, 2H),
6.65 (d, J = 8.4 Hz, 2H), 6.58 (d, J = 8.5 Hz, 2H), 4.84 (t, J = 4.6 Hz,
1H), 4.76−4.70 (m, 1H), 4.70−4.64 (m, 1H), 4.64−4.58 (m, 1H),
Rat SCI Model. Sprague−Dawley female rats (220−250 g, 8 weeks
old) were anesthetized and a restricted laminectomy was conducted to
expose the dorsal surface of T13. The vertebral column between T12
and L1 was then stabilized with clamps and forceps fixed to the base of
an Infinite Horizon impact device. The midpoint of T13 was impacted
with a force of 250 kDyn using a 2.5 mm stainless steel impactor tip,
which was used to induce a moderately severe contusive injury to the
spinal cord. The musculature was then sutured over the laminectomy
site and the skin closed with wound clips followed by subsequent
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treatment with Marcaine at the incision site. The force/displacement
graph was used to monitor impact consistency. After surgery, the
animals were carefully monitored daily for pain and body weight with
manual bladder expression 2−3 times daily to stimulate reflex voiding
until the animals could urinate independently.
QMA cartridge (Waters, preconditioned with 5 mL of water followed
by 10 mL of air in a syringe) and was eluted by K2CO3 solution (6 mg
in 0.6 mL of water) followed by K222 solution (12 mg in 1 mL
ofacetonitrile). The solvent was evaporated under a steam of helium at
85 °C for 5 min, and the residue was vacuumed at 55 °C for another 3
min to get the anhydrous K222/[18F] complex. A solution of the
tosylated precursors (25, 3−5 mg, in 0.5 mL of acetonitrile) was added
to the above dried complex, and the mixture was heated at 95 °C for
10 min. Cooling water (10 mL) was then added to the reaction vessel,
and the mixture was passed through a C18 plus cartridge and an Oasis
HLB plus cartridge in series (Waters, preconditioned with acetonitrile
(10 mL), water (10 mL), and followed by air (∼10 mL)). An
additional 20 mL of water was used to rinse the reaction vial and the
cartridges, followed by air (∼20 mL) to remove the residual water
from the HLB cartridge. DMF (400 μL + 500 μL) was used to elute
[18F]26 from the Waters HLB cartridge into a reaction vial prefilled
with a mixture of the click reaction precursor 9 (5 mg), 1.25 mg of
CuSO4·5H2O, 4 mg of sodium ascorbate, and 3 mg of BPDS in 50 μL
(water/DMF = 4/1). The mixture was then stirred for 10 min at 90
°C. After cooling, 0.5 mL of HCl (1 M) was added and the resulting
mixture was heated at 90 °C for 10−20 min. A NaOH solution (0.5
mL, 1 M) and water (15 mL) were then added, and the resulting
mixture was passed through a preconditioned Sep-Pak C-18 cartridge.
The cartridge was then washed with another 20 mL of water, and the
crude products were eluted with 1 mL of acetonitrile which was further
purified by semipreparative HPLC (Phenomenex C-18, 10 mm × 250
mm; acetonitrile:H2O = 65:35; flow rate of 3 mL/min). The
radioactive fraction containing the desired products was collected,
diluted with water, loaded onto a Sep-Pak C-18 cartridge, and eluted
with 1 mL of ethanol. After evaporation, the residue was redissolved in
5% ethanol in saline solution and filtered (0.22 μm) into a sterile
injection bottle for animal use. RCP and specific activity (SA) were
determined by analytical HPLC (Phenomenex C-18; 4.6 mm × 250
mm; acetonitrile:H2O = 65:35; flow rate of 1 mL/min). SA was
calculated from the area of the UV peak of purified F-18 compound
and titrated with the standard curve of the nonradioactive reference
compound of known concentration.
In Vivo MicroPET/CT Imaging Studies. MicroPET/CT imaging
was performed using a Siemens Inveon microPET/CT scanner in the
Case Center for Imaging Research. For better anatomic localization,
CT coregistration was applied. Before microPET imaging, CT scout
views were taken to ensure the brain tissues were placed in the co-scan
field of view (FOV) where the highest image resolution and sensitivity
are achieved. Under anesthesia, radiotracer was administered via tail
vein injection and immediately followed by a dynamic PET acquisition
up to 60 min. After the microPET acquisition was done, the rat was
moved into the CT field and a two-bed CT scan was performed. A
two-dimensional ordered subset expectation maximization (OSEM)
algorithm was used for image reconstruction using CT as attenuation
correction. For quantitative analysis, the resultant PET images were
registered to the CT images, which enabled us to accurately define the
ROI and quantify the radioactivity concentrations. In this study, the
whole brains of rats were used as ROI and the radioactivity
concentrations were determined in terms of SUVs.
In Vitro Tissue Staining of Brain. Wild-type mice (20−22 g, 8
weeks old) were deeply anesthetized and perfused with precooled
saline (4 °C, 10 mL/min for 1 min followed by 7 mL/min for 6 min),
which was followed by fixation with precooled 4% PFA in PBS (4 °C,
10 mL/min for 1 min followed by 7 mL/min for 6 min). Brain tissues
were then removed, postfixed by immersion in 4% PFA overnight,
dehydrated in 10%, 20%, and 30% sucrose solution, embedded in a
freezing compound (OCT, Fisher Scientific, Suwanee, GA), and
sectioned at 20 μm increments with a cryostat (Thermo HM525,
Thermo Fisher Scientific Inc., Chicago, IL, USA). To provide staining
sections for preliminary FIR measurement, brain sections were
collected from AP (1.0) to AP (−0.1), and the first 12 sections
were mounted in order on the bottom of 12 superfrost slides (Fisher
Scientific) with one section on each slide. Sections 13−24 were
mounted in order on the middle of each slide, and sections 25−36
were mounted in order on the top of each slide. Sections were then
incubated with tested compounds (1 mM, 5% DMSO in 1× PBS (pH
7.0), 6 sections per compound) for 25 min at room temperature in the
dark. Excess compounds were washed by briefly rinsing the slides in
PBS (1×) and coverslipped with fluoromount-G mounting media
(Vector Laboratories, Burlingame, CA). Sections were then examined
under a microscope (Leica DM4000B, Leica Microsystem Inc., Buffalo
Grove, IL, USA) equipped for fluorescence (DFC7000T), and images
of the stained mouse whole brain sections were acquired with the same
exposure time.
FIR Measurement. ImageJ software was used to quantify
fluorescent intensity on six sections of each tested compound. A
ROI was selected on the genu of the corpus callosum (gcc, white
matter), and the same size of ROI was applied on the midline between
gcc and the edge of the section (see Figure 2A), which is considered to
be gray matter. Images were analyzed by two experienced individuals.
The FIR values of white matter to gray matter were then calculated.
Ex Vivo Tissue Staining. Wild-type mice were administered the
newly synthesized compounds (40 mg/kg) via tail vein injection, and
30−60 min later the mice were perfused transcardially with saline
followed by 4% PFA in PBS. Brains and spinal cords were then
removed, postfixed by immersion in 4% PFA overnight, dehydrated in
30% sucrose solution, cryostat sectioned at 100 μm, mounted on
superfrost slides, and images were acquired directly using a Leica
fluorescent microscope.
In Vitro Staining of Rat Brain Treated with LPC. Five days after
surgery, the rats were anesthetized and perfused with saline followed
by 4% PFA. Brain tissues were then removed, postfixed in 4% PFA
overnight, dehydrated in 10%, 20%, and 30% sucrose solution,
embedded in OCT, and sectioned at 20 μm with a cryostat. To
determine if the selected compound can differentiate demyelinated
regions from normal myelinated sheaths, we conducted in vitro tissue
staining using spinal cord sections taken from LPC-treated rats. LPC-
treated spinal cord sections were then incubated with tested
compounds (1 mM, 5% DMSO in 1× PBS (pH 7.0)) for 25 min at
room temperature in the dark. Excess compounds were washed by
briefly rinsing the slides in PBS (1×) and coverslipped with
fluoromount-G mounting media (Vector Laboratories, Burlingame,
CA). Sections were then examined under a microscope (Leica
DM4000B, Leica Microsystem Inc., Buffalo Grove, IL, USA) equipped
for fluorescence (DFC7000T), and images of the stained mouse whole
brain sections were acquired with the same exposure time. In the
meantime, standard Luxol-Fast-Blue (LFB) staining was performed on
the adjacent LPC-treated spinal cord section for comparison.
Radiosynthesis. No carrier-added (nca) [18F] fluoride was
produced by a cyclotron (Eclipse High Production, Siemens) via the
nuclear reaction 18O (p,n) 18F. At the end of bombardment (EOB),
the activity of aqueous [18F]fluoride (50−100 mCi) was transferred to
the GE Tracerlab FXn synthesizer by high helium pressure. After
delivery, the radioactive solution was passed through a Sep-Pak light
Coregistration of in Situ 3D Cryoimaging with PET/CT
Imaging. After microPET imaging studies, the rats were administered
compound 21 (3−5 mg) via tail vein injection, and 60 min later the
rats were perfused transcardially with saline followed by 4% PFA in
PBS. Spinal cords were then removed, postfixed by immersion in 4%
PFA overnight, dehydrated in 30% sucrose solution, cryostat sectioned
at 100 μm, mounted consecutively on superfrost slides, and images
were sequentially acquired directly using a Leica fluorescent
microscope to give a series of 2D microscopic images along the
length of the spinal cord. Slices were aligned sequentially using a
semiautomated image registration algorithm. Starting from the first
slice image, the current slice was used as the reference image for
alignment and the next slice was the floating image. The floating image
was aligned via control point pairs between corresponding anatomic
features in the two images. A 2D rigid-body transformation
(translation, rotation) was used to transform the floating image to
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Journal of Medicinal Chemistry
Article
align with the reference and a minimum of three control point pairs
were used for the transformation. Once two slices were aligned, the
algorithm continued with the newly aligned image serving as the
reference image and the next slice in the sequence serving as the
floating image. All slices in the sequence were aligned by this
technique, giving a 3D reconstructed fluorescence image volume.
Image volumes from fluorescence, PET, and CT were visualized in
Amira using volume rendering and aligned based on the shape of the
spine and fiducials from the vertebrae.
ethyl acetate; MeCN, acetonitrile; EtOH, ethanol; SUV,
standardized uptake value; CT, computerized tomography;
PFA, paraformaldehyde; FOV, field of view; SAR, structure−
activity relationship; TLC, thin-layer chromography; OSEM,
ordered subset expectation maximization; RPC, radiochemical
purity; SA, specific activity; DMF, dimethylformamide;
NaHCO3, sodium bicarbonate; Na2SO4, sodium sulfate
In Situ Autoradiography. Wild-type mice were euthanized at 30
min post iv injection of [18F]21 (111 MBq, 3.0 mCi). The brains were
rapidly removed, placed in OCT embedding medium, and frozen at
−20 °C. After reaching equilibrium at this temperature, the brains
were coronally sectioned at 60 μm on a cryostat and mounted on
superfrost slides. After drying by air at room temperature, the slides
were put in a cassette and exposed for 20 min to film to obtain images.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01328.
■
S
Additional 1H NMR and 13C NMR spectra for all
intermediate and final compounds synthesized; HPLC
and HRMS spectra for all of the final compounds (18−
23) (PDF)
PET-CT cryo fusion imaging(AVI)
Molecular formula strings (CSV)
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AUTHOR INFORMATION
Corresponding Authors
*For C.W.: phone, 216-983-4235; fax, 216-844-8062; E-mail,
cxw130@case.edu.
*For Y.W.: phone, 216-844-3288; fax, 216-844-8062; E-mail,
yxw91@case.edu.
ORCID
■
Chunying Wu: 0000-0002-6782-2610
Present Address
¶For Yunjie Zhu: Department of Chemistry and Biochemistry,
Oberlin College, Oberlin, Ohio 44074, United States
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported in part by the National Multiple
Sclerosis Society (RG5174-A-4), Genzyme Corporation, and
the Bryon Riesch Paralysis Foundation.
ABBREVIATIONS USED
■
s, singlet; br s, broad singlet; dd, doublet of doublets; ddd,
doublet of doublet of doublets; br d, broad doublets; t, triplet;
dt, triplets of doublets; q, quartet; m, multiplet; br m, broad
multiplet; MS, multiple sclerosis; PET, positron emission
tomography; SCI, spinal cord injury; MRI, magnetic resonance
imaging; MeDAS, methyl diamino stilbene; TAFDAS, triazole
fluorinated diamino stilbene; CNS, central nervous system;
DCM, dichloromethane; FIR, fluorescent intensity ratio; ROI,
region of interest; gcc, genu of the corpus callosum; BBB,
blood−brain barrier; LPC, lysolecithin; LFB, Luxol-Fast-Blue;
K
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DOI: 10.1021/acs.jmedchem.6b01328
J. Med. Chem. XXXX, XXX, XXX−XXX