Synthesis and biological evaluation of radioiodinated 3-phenylcoumarin derivatives targeting myelin in multiple sclerosis

Article abstract of DOI:10.1016/j.bmcl.2020.127562

Myelin is a lipid multilayer involved in the rate of nerve transmission, and its loss is a pathological feature of multiple sclerosis in brains. Since in vivo imaging of myelin may be useful for drug development, early diagnosis, and monitoring the disease stage, we designed, synthesized, and evaluated eight novel radioiodinated 3-phenylcoumarin derivatives as imaging probes targeting myelin. In the biodistribution study using normal mice, all compounds displayed sufficient brain uptake, ranging from 2.5 to 5.0% ID/g, at 2 min postinjection. On ex vivo autoradiography, [¹²⁵I]18 and [¹²⁵I]21, which have a dimethylamino group, showed high binding affinity for myelin in the normal mouse brain. In addition, the radioactivity accumulation of [¹²⁵I]21 in the white matter of the spinal cord in the experimental autoimmune encephalomyelitis mice was lower than that in naive mice. These results suggest that [¹²³I]21 shows potential as a single photon emission computed tomography probe targeting myelin.

Full text of DOI:10.1016/j.bmcl.2020.127562

Bioorganic&MedicinalChemistryLetters30(2020)127562
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of radioiodinated 3-phenylcoumarin
derivatives targeting myelin in multiple sclerosis
⁎
Hiroyuki Watanabea, , Shiori Sakaia, Shimpei Iikunia, Yoichi Shimizua,b, Hisashi Shirakawac,
⁎
Shuji Kanekoc, Masahiro Onoa,
a Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-
8501, Japan
b Department of Diagnostic Imaging and Nuclear Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
c Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501,
Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Myelin is a lipid multilayer involved in the rate of nerve transmission, and its loss is a pathological feature of
multiple sclerosis in brains. Since in vivo imaging of myelin may be useful for drug development, early diagnosis,
and monitoring the disease stage, we designed, synthesized, and evaluated eight novel radioiodinated 3-phe-
nylcoumarin derivatives as imaging probes targeting myelin. In the biodistribution study using normal mice, all
compounds displayed sufficient brain uptake, ranging from 2.5 to 5.0% ID/g, at 2 min postinjection. On ex vivo
autoradiography, [125I]18 and [125I]21, which have a dimethylamino group, showed high binding affinity for
myelin in the normal mouse brain. In addition, the radioactivity accumulation of [125I]21 in the white matter of
the spinal cord in the experimental autoimmune encephalomyelitis mice was lower than that in naive mice.
These results suggest that [123I]21 shows potential as a single photon emission computed tomography probe
targeting myelin.
Myelin
Radioiodinated phenylcoumarin
Multiple sclerosis
Single photon emission computed tomography
Myelin is a sheath-like multilayer lipid membrane that wraps
around axons and acts to increase nerve conduction velocity.1 Demye-
lination of the central nervous system is involved in the pathological
process of many neurodegenerative diseases such as multiple sclerosis
(MS) and spinal cord injury.2,3 The prevalence of MS has been in-
creasing, and the number of patients was estimated at about 2.3 million
worldwide in 2013.3,4 Since MS is a disease that repeats relapses and
remissions and the course varies from patient to patient, the monitoring
of disease activity is important. Currently, magnetic resonance imaging
(MRI) is mainly used for the diagnosis and monitoring of MS. MRI can
measure changes in diffusivity of water in tissue, but cannot specifically
detect de- and remyelination.5–8 Therefore, it is necessary to develop a
novel method to specifically detect changes in myelin for the accurate
evaluation of the disease stage and early definitive diagnosis of MS.
Nuclear medicine techniques can be used for in vivo imaging of bio-
chemical and physiological processes. Therefore, nuclear medicine
techniques with radiolabeled probes targeting myelin may be attractive
for diagnostic imaging and monitoring of MS.7–9 Over the past decade,
some positron emission tomography (PET) probes for in vivo imaging of
myelin, such as [11C]N-methyl-4,4′-diaminostilbene ([11C]MeDAS), [11C]
1,4-bis (p-aminostyryl)-2-methoxybenzene ([11C]BMB), [18F](E)-4-(4-
aminostyryl)-N-(5-fluoropentyl)aniline ([18F]PENDAS), and [18F] (E)-N
-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-(methylamino)
styryl)aniline ([18F]TAFDAS) have been developed and evaluated re-
garding their utility.10–13 Among them, [11C]MeDAS has been well-
evaluated, but the results of these studies suggest that further optimi-
zation of the pharmacokinetics, stability, and physical half-life is
needed.8,13,14 Conversely, few myelin imaging probes for single photon
emission computed tomography (SPECT) have been reported.15 In ad-
dition, it is generally known that SPECT is more routinely used than PET.
This situation encouraged us to develop novel myelin imaging probes for
SPECT.
Almost all PET probes targeting myelin reported previously were
based on a stilbene structure,10–13,15–17 but this backbone has some
problems including high lipophilicity and photoisomerization. Ac-
cordingly, there is a need to develop novel radiolabeled myelin imaging
probes with other backbones. Previous reports stated that 3-phe-
nylcoumarin derivatives, including 3-(4-(dimethylamino)phenyl)-7-(3-
fluoropropoxy)-2H-chromen-2-one (FCP) and 3-(4-aminophenyl)-2H-
chromen-2-one (CMC), bound to myelin in vitro.18,19 In addition, it has
⁎ Corresponding authors.
E-mail addresses: hwatanabe@pharm.kyoto-u.ac.jp (H. Watanabe), ono@pharm.kyoto-u.ac.jp (M. Ono).
https://doi.org/10.1016/j.bmcl.2020.127562
Received 18 June 2020; Received in revised form 11 September 2020; Accepted 16 September 2020
Availableonline22September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127562
O
H
NH2
N
N
O
125I
125I
125I
125I
O
O
O
O
O
O
O
O
Fig. 1. Chemical structures of 3-phenylcoumarin derivatives developed in this study.
Scheme 1. Synthetic route for 3-phenylcoumarin derivatives. Reagents: (a) Ac2O, Et3N; (b) SnCl2, EtOH; (c) (SnBu3)2, (PPh3)4Pd, Et3N, 1,4-dioxane; (d) I2, CHCl3; (e)
NaOMe, paraformaldehyde, NaBH4, MeOH; (f) paraformaldehyde, NaCNBH3, AcOH.
been reported that the type and position of substituted groups in the 3-
phenylcoumarin scaffold affect the affinity for myelin.19 However,
there have been no reports on the development of radiolabeled 3-phe-
nylcoumarin derivatives targeting myelin in the brain. Therefore, in
this study, we newly designed and synthesized eight radioiodinated 3-
phenylcoumarin derivatives and evaluated their utility as SPECT ima-
ging probes for myelin (Fig. 1).
We synthesized target 3-phenylcoumarin derivatives (6, 9, 12, 15,
2

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127562
Scheme 2. Radiosynthesis of 3-phenylcoumarin derivatives. Reagents: (a) [125I]NaI, H2O2, 1 N HCl.
18, 21, 24, and 27) according to Scheme 1. The 3-phenylcoumarin
scaffold was synthesized by the reaction of substituted salicylaldehyde,
aryl acetic acid, and Et3N by following the method reported pre-
viously.20 The free amino derivatives (4, 5, and 6) were prepared from
nitro derivatives (1, 2, and 3) by reduction with SnCl2 in yields of 54,
74, and 89%, respectively. The amino derivatives (4, 5, and 6) were
converted to monomethylamino derivatives (10, 11, and 12) or di-
methylamino derivatives (16, 17, and 18) by the method reported
previously.21,22 The bromo derivatives (4, 5, 10, 11, 16, 17, 22, and
23) were converted to the tributyltin derivatives (7, 8, 13, 14, 19, 20,
25, and 26) by the exchange reaction of bromine to tributyltin in yields
of 11–53%. These tributyltin compounds were reacted with iodine at
room temperature to give the iodo derivatives (9, 15, 21, and 27) in
yields of 70–99%.
Table 1
Brain uptake of radioactivity after intravenous injection of [125I]6, [125I]9,
[
125I]12, [125I]15, [125I]18, [125I]21, [125I]24, and [125I]27 in normal micea
and the ratio of their radioactivity accumulation (2 min/60 min).
Compd
%ID/g in the brain
Ratio
2 min
10 min
30 min
60 min
2 min/
60 min
[
[
[
[
[
[
[
[
125I]6
3.67
4.79
0.88 2.77
0.75 5.60
0.33 3.64
0.24 2.06
0.20 2.91
0.29 3.12
0.24 2.56
0.61 1.90
0.25 1.27
0.17 0.65
0.06
0.16
0.11
0.04
0.10
0.18
5.6
4.3
6.2
4.5
6.1
2.8
125I]9
0.44 2.63
1.23 1.34
0.16 1.07
0.17 1.44
0.27 1.74
0.21 0.74
0.32 0.44
0.43 1.11
0.21 0.61
0.12 0.55
0.23 0.56
0.16 1.00
0.07 0.27
0.05 0.13
125I]12 3.76
125I]15 2.48
125I]18 3.40
125I]21 2.84
125I]24 5.02
125I]27 2.87
0.03 18.6
0.01 22.1
We used the tributyltin derivatives as the precursor materials for
radioiodination. 125I-labeled 3-phenylcoumarin derivatives ([125I]6,
a
Each value represents the mean
SD of five animals.
[
125I]9, [125I]12, [125I]15, [125I]18, [125I]21, [125I]24, and [125I]27)
were prepared by an iododestannylation reaction using [125I]NaI and
hydrogen peroxide as the oxidant (Scheme 2). The radioiodinated
compounds were purified by reverse phase high-performance liquid
chromatography (RP-HPLC) after the radioiodination reaction. We
identified the radioiodinated ligands from their HPLC profiles by co-
injection with non-radioiodinated compounds. [125I]6, [125I]9, [125I]
12, [125I]15, [125I]18, [125I]21, [125I]24, and [125I]27 were obtained in
a radiochemical yield of 63.7, 78.8, 69.9, 75.5, 80.3, 88.1, 86.5, and
71.8%, respectively, with a radiochemical purity of over 95%.
normal mouse brain. Since all compounds except [125I]9 and [125I]15
showed low radioactivity in the thyroid until 60 min postinjection
(0.02–0.54% ID) (Table S1), they are stable against deiodination in vivo.
In order to evaluate selective binding affinity to myelin in vivo, we
performed ex vivo autoradiography (ARG) using brain sections prepared
from normal mice at 60 min after injection of 125I-labeled 3-phe-
nylcoumarin derivatives (Fig. 2). The location of myelin was confirmed
by histochemical staining with Luxole Fast Blue (LFB) (Fig. 2I). [125I]6,
[
125I]9, [125I]12, [125I]15, [125I]18, and [125I]21 showed radioactivity
First, we performed biodistribution experiments to evaluate uptake
into and retention in the brain after the injection of 125I-labeled 3-
phenylcoumarin derivatives into wild-type mice (Table 1). All com-
pounds displayed sufficient brain uptake, ranging from 2.5 to 5.0%
injected dose (ID)/g, at 2 min postinjection. This suggested that all 125I-
labeled 3-phenylcoumarin derivatives have the potential of binding to
myelin in the brain. Among these compounds, [125I]24 and [125I]27,
which have a 1,3-dioxolan group, showed a high 2 min/60 min ratio.
On the other hand, [125I]21 exhibited the lowest 2 min/60 min ratio
among these derivatives, indicating that [125I]21 was retained in the
accumulation in the white matter, which contains myelin. On the other
hand, [125I]24 and [125I]27 did not show marked radioactivity accu-
mulation in the brain due to low accumulation in the brain at 60 min
postinjection. To compare the selective binding affinity for myelin
among 3-phenylcoumarin derivatives, the region of interest (ROI) was
set to white and gray matter of brain sections (Fig. S2), and the ratio of
radioactivity accumulation in the white matter against the gray matter
was calculated (Table 2). Due to the extremely low radioactivity on the
brain sections, it was difficult to calculate the ratios of [125I]24 and
3

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127562
[
125I]27 accurately. The ratios of other compounds were over 1, in-
dicating that [125I]6, [125I]9, [125I]12, [125I]15, [125I]18, and [125I]21
are selectively accumulated in the white matter of the living mouse
brain. In addition, these values were higher than or comparable with
FCP (1.54), PENDAS (> 1.2), and TAFDAS (< 1.5).12,13,19 Since [18F]
PENDAS and [18F]TAFDAS imaged myelin in the brain and spinal cord
with PET, [125I]6, [125I]9, [125I]12, [125I]15, [125I]18, and [125I]21
show sufficient binding specificity for myelin imaging. Among them,
[
125I]18 and [125I]21 showed higher ratios (2.01 and 2.07, respec-
tively), suggesting that these compounds have a high binding affinity
for myelin. Since the blood-to-brain ratio of [125I]12 and [125I]18 was
over 1 at 30 min after injection, we also performed ex vivo ARG studies
at this time-point (Fig. S1). The ARG images showed radioactivity ac-
cumulation in the white matter, but the ratio ([125I]12: 1.45, [125I]18:
1.51) was lower than that at 60 min after injection ([125I]12: 1.56,
[
125I]18: 2.01). One of the possible reasons for this result may be high
radioactivity accumulation in the brain other than white matter at
30 min after the injection of these compounds.
The results of biodistribution and in vitro ARG using normal mice
suggest that [125I]21 is a promising as a myelin imaging probe.
Therefore, we performed a further study with MS model mice. EAE
model rodents are well-known animal models of MS that show axon
damage resulting from local immune attacks and accompanying de-
myelination. The axon damage often occurs at multiple sites in white
matter of the central nervous system.23 Therefore, many studies on MS
including myelin imaging have been performed using EAE model ro-
dents.24,25 Since spinal cords are generally used for these studies, we
performed ex vivo ARG using a spinal cord section of an EAE mouse. The
experiment with a naive mouse was also performed as a control. ARG
images showed a decrease in radioactivity accumulation in the white
matter of the spinal cord of an EAE mouse (Fig. 3A and B). Compared
with the spinal cord section obtained from naive mice, a decrease in
myelin was also observed in EAE mice by histochemical staining
(Fig. 3C and D). These results suggest that [123I]21 has the potential to
visualize demyelination in living mice.
We newly designed and synthesized eight novel radioiodinated 3-
phenylcoumarin derivatives with various substituted groups. Among
these compounds, [125I]21, which has a dimethylamino group, showed
sufficient initial uptake and high retention in the brain. In addition,
selective binding affinity for myelin in the normal mouse brain was
Fig. 2. Comparison of ex vivo autoradiograms with [125I]6 (A), [125I]9 (B),
[
125I]12 (C), [125I]15 (D), [125I]18 (E), [125I]21 (F), [125I]24 (G), and [125I]27
(H) in normal mouse brain slices. The presence and localization of myelin were
confirmed with LFB staining (I).
Table 2
Ratio of radioactivity accumulation in the white matter against the gray
matter.
Compounds
Ratio* (white matter/gray matter)
[
[
[
[
[
[
[
[
125I]6
1.79
0.35
125I]9
1.77
0.28
125I]12
125I]15
125I]18
125I]21
125I]24
125I]27
1.56
0.32**
0.42***
0.38
1.69
2.01
2.07
0.17
n.d.****
n.d.****
* Each value represents the mean
SD for 3 animals.
** Indicates p < 0.01 compared to [125I]21.
*** Indicates p < 0.05 compared to [125I]21.
**** n.d.: not determined.
Fig. 3. Comparison of ex vivo autoradiograms with [125I]21 (A and B) and LFB
staining (C and D) between EAE (A and C) and naive (B and D) mouse spinal
cords.
4

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127562
observed. Furthermore, [125I]21 detected demyelination in the spinal
cord of the EAE mouse, suggesting that [123I]21 might be useful as a
SPECT probe targeting myelin.
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Declaration of Competing Interest
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sion tomography. Proc Natl Acad Sci U S A. 2006;103(24):9304–9309.
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myelination. Bioorg Med Chem. 2010;18(24):8592–8599.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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This study was supported by JSPS KAKENHI Grant Number
17H04260 and 17H05092. We thank Ms. Sakie Miyamura for preparing
EAE model mice.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127562.
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