Development of radioiodinated pyrimidinopyridone derivatives as targeted imaging probes of activated p38α for single photon emission computed tomography

Article abstract of DOI:10.1007/s12149-021-01669-6

Objective: p38α, a member of the mitogen-activated protein kinase superfamily, is ubiquitously expressed in a variety of mammalian cells. Activated p38α induces inflammatory responses to external stimuli, suggesting that non-invasive detection of activate

Full text of DOI:10.1007/s12149-021-01669-6

Annals of Nuclear Medicine
https://doi.org/10.1007/s12149-021-01669-6
ORIGINAL ARTICLE
Development of radioiodinated pyrimidinopyridone derivatives
as targeted imaging probes of activated p38α for single photon
emission computed tomography
Tomoyuki Hashimoto1 · Naoya Kondo1 · Masahiko Hirata1 · Takashi Temma1
Received: 11 May 2021 / Accepted: 9 August 2021
© The Japanese Society of Nuclear Medicine 2021
Abstract
Objective p38α, a member of the mitogen-activated protein kinase superfamily, is ubiquitously expressed in a variety of
mammalian cells. Activated p38α induces infammatory responses to external stimuli, suggesting that non-invasive detec-
tion of activated p38α would be valuable for diagnosing infammatory diseases. For this purpose, we designed radiolabeled
compounds [123I]2-IR and [123I]4-IR based on a potent p38α selective inhibitor R1487 for use with single photon emission
computed tomography (SPECT). In this study, we used 125I instead of 123I due to its more usable radiochemical properties,
synthesized [125I]2-IR and [125I]4-IR, and evaluated their efectiveness as activated p38α imaging probes.
Methods [123I]2-IR and [123I]4-IR were designed by introduction of a 123I atom at the 2- or 4-ositions of the phenoxy ring,
preserving the pyrimidinopyridone structure of R1487. We synthesized 2-IR and 4-IR via a 7-step process. The inhibitory
potencies of 2-IR, 4-IR, and p38α inhibitors were measured using an ADP-Glo™ kinase assay system. Radioiodination of
2-IR and 4-IR was performed via an organotin-radioiodine exchange reaction using the corresponding tributyltin precur-
sors. Biodistributions were evaluated by determining radioactivity in tissues of interest after intravenous administration of
[
125I]2-IR and [125I]4-IR in normal ddY mice and turpentine oil-induced infammation model mice. In vivo inhibition study
was also performed in infammation model mice after intravenous administration of [125I]4-IR with pretreatment of p38α
inhibitors.
Results We synthesized 2-IR and 4-IR at total yields of 17.5% and 19.2%, respectively. 4-IR had higher p38α inhibitory
potency than 2-IR; both compounds were signifcantly less potent than R1487. [125I]2-IR and [125I]4-IR were successfully
obtained from tributyltin precursors with high radiochemical yield (>65%), purity (>97%), and molar activity (~81 GBq/
µmol). [125I]4-IR showed high radioactivity accumulation in the infamed tissue (7.0 1.2%D/g), rapid delivery throughout
the body, and rapid blood clearance, resulting in a high infammation-to-blood ratio (6.2 0.4) and a high infammation-
to-muscle ratio (5.2 1.3) at 30 min, while [125I]2-IR showed low radioactivity accumulation in infamed tissue over the
experimental period. Further, radioactivity accumulation in infamed tissue after [125I]4-IR administration was signifcantly
decreased by pretreatment with selective inhibitors.
Conclusions [123I]4-IR would be a promising imaging agent for detection of activated p38α.
Keywords p38α · Infammation · Single photon emission computed tomography · Pyrimidinopyridone radioiodine
Introduction
p38 is a Ser/Thr kinase that is a mitogen-activated protein
kinase (MAPK) and a member of the MAPK superfamily.
Several MAPK signaling cascades are activated in response
to environmental stresses such as osmotic shock and infec-
tion [1, 2] and are also important regulators of the biosyn-
thesis of inflammatory cytokines at transcriptional and
translational levels. Among the four subtypes, p38α is ubiq-
uitously expressed in a variety of mammalian cells [3] and
* Takashi Temma
takashi.temma@ompu.ac.jp
1
Department of Biofunctional Analysis, Graduate
School of Pharmaceutical Sciences, Osaka Medical
and Pharmaceutical University, 4-20-1 Nasahara, Takatsuki,
Osaka 569-1094, Japan
Vol.:(0123456789)
1 3

Annals of Nuclear Medicine
contributes to production of infammatory cytokines such
as TNF-α, IL-1, and IL-6 in response to lipopolysaccharide
stimulation [4, 5]. p38α inhibitors have an anti-infammatory
efect and have been clinically tested as candidate thera-
peutic agents for rheumatoid arthritis [6]. Based on these
features, non-invasive detection of aberrant p38α activity
in biological tissues may be useful for precise prediction of
the treatment efects of such p38α inhibitors. Nuclear medi-
cal techniques such as single photon emission computed
tomography (SPECT) can be used for this purpose with tar-
geted tracers labeled with radioisotopes such as 123I, 111In,
and 99mTc that are suitable for use in the gamma ray energy
detection range of a SPECT system [7]. However, radio-
metal isotopes require conjugation of a chelating moiety to
the mother skeleton of a candidate lead compound that can
lead to loss of function, particularly for small compounds.
Here we describe the development of radioiodinated probes
for use in SPECT imaging of p38α activity in infammatory
diseases.
were collected with a JMS-700(2)mass spectrometer (JEOL
Ltd., Tokyo, Japan).
Synthesis (Scheme 1)
R1487, 2-IR, and 4-IR were synthesized following a previ-
ous report [8].
Ethyl 4‑methylamino‑2‑methylthiopyrimi‑
dine‑5‑carboxylate (2)
Ethyl 4-chloro-2-ethylthiopyrimidine-5-carboxy-
late (1) (1.00 g, 4.30 mmol) was dissolved in chloroform
(12.5 mL). Then, methylamine in ethanol (33%, 1.75 mL,
14.1 mmol) solution was slowly added dropwise at 0°C, and
the mixture was stirred at 0°C for 30 min. After addition
of 15 mL water, the solution was extracted with chloro-
form (15 mL × 3), and the organic layer was separated. The
organic layer was dried over magnesium sulfate, fltered, and
then the solution was evaporated under reduced pressure to
obtain 2 as white crystals.
In this study, we adopted R1487, a selective and high-
afnity p38α inhibitor with a pyrimidinopyridone skel-
eton, as the lead compound of an imaging agent [8]. We
designed the low-molecular-weight probes [123I]4-I-R1487
([123I]4-IR) and [123I]2-I-R1487 ([123I]2-IR) in which a
fuorine atom at the 4- or 2-osition of the R1487 phenoxy
group was replaced with an 123I atom, with a focus on the
tolerability of the phenoxy moiety predicted in a previous
report [8]. In whole experiments, we used 125I instead of
123I due to its useful radiochemical properties, such as low
gamma ray energy and long half-life. We synthesized both
Yield: 95.3%. mp: 92.5–92.7°C. 1H−NMR (CDCl3): 1.40
(t, J = 7.2 Hz, 3H, CH3), 2.55 (s, 3H, CH3), 2.62 (s, 3H,
CH3), 4.39 (q, J = 7.1 Hz 2H, OCH2), 8.79 (s, 1H, aromat-
ics). EI−MS: m/z: 227. Measured: 227.
4‑Methylamino‑2‑methylthiopyrimidine‑5‑metha‑
nol (3)
Then, 2 (0.50 g, 2.2 mmol) in dry tetrahydrofuran (4.5 mL)
was slowly added dropwise to lithium aluminum hydride
(0.089 g, 2.4 mmol) in dry tetrahydrofuran (3.5 mL) at 5°C.
The reaction mixture was stirred at 5°C for 15 min, and
0.20 mL water was slowly added dropwise. The mixture was
stirred at 5°C for 30 min before 0.090 mL 15% aqueous
sodium hydroxide solution was added dropwise, followed
by 0.28 mL water. The mixture was stirred for 17 h at room
temperature and subsequently fltered. The flter residue was
washed with tetrahydrofuran (5 mL × 2), and the fltrate and
washed liquid were mixed and evaporated under reduced
pressure. The resulting residue was resuspended in 2:1 ethyl
acetate/hexane (3 mL) solution and fltration was performed
to obtain 3 as yellow crystals.
[
125I]2-IR and [125I]4-IR, and evaluated their efectiveness as
a SPECT imaging probe that targets p38α activity in in vitro
and in vivo examinations using mice treated with turpentine
oil to induce infammation.
Methods
Reagents and instruments
2-Fluoro-4-iodophenol and 4-fuoro-2-iodophenol were pur-
chased from BLD Pharmatech (SHA, China). All other rea-
gents were of reagent grade and were purchased from Sigma-
Aldrich Japan (Tokyo, Japan), Nacalai Tesque (Kyoto,
Japan), Wako Pure Chemical Industry (Osaka, Japan), or
Tokyo Chemical Industry (Tokyo, Japan), and used without
further purifcation. Melting points were measured with a
Yanagimoto micro melting point apparatus (Yanaco, Kyoto,
Yield: 84.0%. mp: 145.1–145.4°C. 1H−NMR (CDCl3):
2.52 (s, 3H, CH3), 3.04 (d, J = 5.6 Hz, 3H, CH3), 4.49 (s,
2H, OCH2), 5.90 (s, 1H, NH), 7.64 (s, 1H, aromatics),
EI−MS: m/z: 185. Measured: 185.
1
Japan). H and 13C-NMR spectra were measured using a
4‑Methylamino‑2‑methylthiopyrimidine‑5‑carboxy‑
aldehyde (4)
Varian NMR system (1H 400 MHz, Agilent Technologies,
CA, USA) and chemical shifts are indicated as δ (ppm) val-
ues using tetramethylsilane as an internal standard. Mass
spectra (MS) and high-resolution mass spectra (HRMS)
3 (0.25 g, 1.4 mmol) was dissolved in chloroform
(12.5 mL), and manganese dioxide (1.2 g, 1.4 mmol) was
1 3

Annals of Nuclear Medicine
Scheme 1 Synthesis of R1487, 2-IR, and 4-IR
added with stirring. The suspension was stirred for 24 h
and then evaporated to replace benzene (12.5 mL) fol-
lowed by fltration through Celite. The flter residue was
washed with benzene (10 mL × 5), and then the combined
fltrate and wash liquid were evaporated to obtain 4 as
white solids.
Methyl 2‑(4‑fuoro‑2‑iodephenoxy)acetate (6b)
4-Fluoro-2-iodephenol (5b) (0.50 g, 2.1 mmol), methyl
bromoacetate (0.33 g, 2.2 mmol), sodium iodate (2.1 mg)
and potassium carbonate (0.59 g) were added in dry acetone
(20 mL), and then the mixture was refuxed at 76°C for 3 h.
After concentrating the mixture under reduced pressure,
25 mL water was added, followed by recrystallization. Crys-
tals were isolated to obtain 6b as white crystals.
Yield: 73.2%. mp: 101.7–102.3°C. 1H−NMR (CDCl3):
2.57 (s, 3H, CH3), 3.12 (d, J = 4.8 Hz, 3H, CH3), 8.30
(s, 1H, CHO), 9.70 (s, 1H, aromatics). EI−MS: m/z: 183.
Measured: 183.
Yield: 92.1%. mp: 44.1–44.2°C. 1H−NMR (CDCl3):
3.81 (s, 3H, OCH3), 4.76 (s, 2H, OCH2), 6.71 (dd, J = 2.4,
4.4 Hz, 1H, aromatics)), 7.01 (s, 1H, aromatics), 7.52–7.54
(m, 1H, aromatics). 13C−NMR (CDCl3): 52.39, 67.04,
86.28 (d, 2JC−F = 8.3 Hz), 113.18 (d, 1JC−F = 8.4 Hz), 115.83
(d, 1JC−F = 22.8 Hz), 126.61 (d, 2JC−F = 25.0 Hz), 153.45
(d, 3JC−F = 2.3 Hz), 158.63 (d, JC−F = 243.6 Hz), 168.76.
EI−MS: m/z: 310. Measured: 310.
Methyl 2‑(2,4‑difuorophenoxy)acetate (6a)
2,4−Difuorophenol (5a) (0.88 g, 6.8 mmol), methyl bro-
moacetate (1.1 g, 7.2 mmol), sodium iodate (6.8 mg) and
potassium carbonate (1.9 g) were added in dry acetone
(20 mL), and then the mixture was refuxed at 76°C for
3 h. After concentrating the mixture under reduced pres-
sure, 25 mL water was added, the solution was extracted
with chloroform (5 mL × 3), and the organic layer was
separated. The organic layer was dried over sodium sul-
fate, and then the solution was concentrated under reduced
pressure to obtain 6a as yellow oily matter.
Methyl 2‑(2‑fuoro‑4‑iodephenoxy)acetate (6c)
6c was synthesized in a manner similar to that for 6b.
Yield: 88.0%. mp: 49.0–49.3°C. 1H−NMR (CDCl3):
3.80 (s, 3H, OCH3), 4.69 (s, 2H, OCH2), 6.68 (dd, J = 8.4,
8.8 Hz, 1H, aromatics), 7.36 (d, J = 10.0 Hz, 1H, aromatics),
7.42 (d, J = 12.4 Hz, 1H, aromatics). 13C−NMR (CDCl3):
52.41, 66.50, 83.16 (d, 2JC−F = 6.8 Hz), 117.70, 125.86 (d,
Yield: 93.6%. 1H−NMR (CDCl3): 3.80 (s, 3H, OCH3),
4.67 (s, 2H, OCH2), 6.76–6.81 (m, 1H, aromatics),
6.85–6.97 (m, 2H, aromatics). EI−MS: m/z: 202. Meas-
ured: 202.
3
1JC−F = 20.5 Hz), 133.41 (d, JC−F = 4.5 Hz), 146.05 (d,
1 3

Annals of Nuclear Medicine
1JC−F = 10.6 Hz), 153.872 (d, JC−F = 251.2 Hz), 168.67.
EI−MS: m/z: 310. Measured: 310.
6‑(2,4‑Difuorophenoxy)‑8‑methyl‑2‑(methylsulfony
l)‑pyrido[2,3‑d]pyrimidin‑7(8H)‑one (8a)
6‑(2,4‑Difuorophenoxy)‑8‑methyl‑2‑(methylthio)‑p
yrido[2,3‑d]pyrimidin‑7(8H)‑one (7a)
After 7a (0.17 g, 0.51 mmol) was dissolved in chloroform
(3 mL), formic acid (0.057 g, 1.24 mmol) was added, and
30% aqueous hydrogen peroxide (0.71 g, 0,63 mmol) was
slowly added dropwise to the solution, which was then
stirred at 38 °C. The completeness of reaction was con-
frmed by TLC, and the yellow solution was cooled to 25
°C before extraction with chloroform (3 mL × 3) and wash-
ing with water (0.15 mL). Additionally, the organic layer
was washed with 10% aqueous NaOH solution (0.18 mL),
and extracted with chloroform (3 mL × 3). The solvent
was distilled of under reduced pressure. Then tert−butyl
methyl ether (0.60 mL) was added to the residue, and the
mixture was fltered and washed with tert−butyl methyl
ether (0.20 mL × 2) at <5°C. The residue was then sepa-
rated to obtain 8a as light-yellow crystals.
4 (0.20 g, 1.1 mmol) was frst dissolved in 1−methyl−2−pyr-
rolidinone (2 mL), and then 6a (0.32 g, 1.6 mmol) and
potassium carbonate (0.26 g) were added. The mixture
was refuxed at 120°C for 12 h. In addition, 6a (0.12 g,
0.59 mmol) and potassium carbonate (0.10 g) were added,
and the mixture was refuxed at 120°C for 6 h, and then the
mixture was cooled to room temperature. After 5 mL water
was added, the mixture was stirred for 45 min and then fl-
tered. The residue was washed with water (3 mL × 3) before
3 mL ethyl acetate was added. The resulting suspension was
stirred for 1 h and then fltered. The residue was washed
with ethyl acetate (2 mL × 3) and the residue was isolated
to obtain 7a as light-yellow crystals.
Yield: 75.4%. mp: 154.2–154.7°C. 1H−NMR (CDCl3):
3.40 (s, 3H, CH3), 3.95 (s, 3H, CH3), 6.78 (s, 1H, aro-
matics), 6.99–7.09 (m, 2H, aromatics), 7.24–7.30 (m, 1H,
aromatics), 8.82 (s, 1H, aromatics). EI−MS: m/z: 367
Measured: 367.
Yield: 55.6%. mp: 176.2–176.8°C. 1H−NMR (CDCl3):
2.63 (s, 3H, CH3), 3.85 (s, 3H, CH3), 6.76 (s, 1H, aromatics),
6.91–6.97 (m, 1H, aromatics), 6.98–7.03 (m, 1H, aromat-
ics), 7.16–7.23 (m, 1H, aromatics), 8.49 (s, 1H, aromatics).
EI−MS: m/z: 335. Measured: 335.
6‑(4‑Fluoro‑2‑iodephenoxy)‑8‑methyl‑2‑(methylthio
)‑pyrido[2,3‑d]pyrimidin‑7(8H)‑one (7b)
6‑(4‑Fluoro‑2‑iodephenoxy)‑8‑methyl‑2‑(methylsulf
onyl)‑pyrido[2,3‑d]pyrimidin‑7(8H)‑one (8b)
7b was synthesized in a manner similar to that for 7a.
Yield: 57.9%. mp: 178.5–178.9°C. 1H−NMR (CDCl3):
2.65 (s, 3H, CH3), 3.86 (s, 3H, CH3), 6.70 (s, 1H, aromat-
ics), 7.05 (dd, J = 4.8, 4.8 Hz, 1H, aromatics), 7.11–7.16 (m,
1H, aromatics), 7.63 (dd, J = 2.8, 2.8 Hz, 1H, aromatics),
8.50 (s, 1H, aromatics). 13C−NMR (CDCl3): 14.42, 28.65,
89.07 (d, 2JC−F = 8.4 Hz), 108.66, 113.14, 117.03 (d, 1JC−F
8b was synthesized in a manner similar to that for 8a.
Yield: 71.8%. mp: 182.1–182.4°C. 1H−NMR (CDCl3):
3.40 (s, 3H, CH3), 3.96 (s, 3H, CH3), 6.68 (s, 1H, aromatics),
7.14–7.24 (m, 2H, aromatics), 7.66 (dd, J = 2.4, 2.8 Hz, 1H,
aromatics), 8.82 (s, 1H, aromatics). 13C−NMR (CDCl3):
2
29.44, 39.32, 89.35 (d, JC−F = 6.2 Hz), 109.75, 115.17,
117.46 (d, 1JC−F = 15.3 Hz), 122.43 (d, 2JC−F = 12.7 Hz),
2
1
1
3
= 22.8 Hz), 121.33 (d, JC−F = 8.4 Hz), 127.04 (d, JC−F
127.38 (d, JC−F = 16.8 Hz), 149.76 (d, JC−F = 4.6 Hz),
149.78, 151.86, 155.41, 159.11 (d, JC−F = 145.6 Hz),
160.79, 162.82. EI−MS: m/z: 475 Measured: 475.
3
= 25.1 Hz), 146.18, 150.73 (d, JC−F = 2.3 Hz), 151.54,
155.24, 158.33 (d, JC−F = 21.3 Hz), 160.60, 171.37. EI−MS:
m/z: 443 Measured: 443.
6‑(2‑Fluoro‑4‑iodephenoxy)‑8‑methyl‑2‑(methylthio
)‑pyrido[2,3‑d]pyrimidin‑7(8H)‑one (7c)
6‑(2‑Fluoro‑4‑iodephenoxy)‑8‑methyl‑2‑(methylsulf
onyl)‑pyrido[2,3‑d]pyrimidin‑7(8H)‑one (8c)
7c was synthesized in a manner similar to that for 7a.
Yield: 56.9%. mp: 181.8–182.2°C. 1H−NMR (CDCl3):
2.63 (s, 3H, CH3), 3.83 (s, 3H, CH3), 6.89 (s, 1H, aromat-
ics), 6.93 (dd, J = 8.8, 8.0 Hz, 1H, aromatics), 7.50 (d, J =
8.0 Hz, 1H, aromatics), 7.58 (d, J = 9.6 Hz, 1H, aromatics),
8.52 (s, 1H, aromatics). 13C−NMR (CDCl3): 14.41, 28.59,
87.95 (d, 2JC−F = 6.9 Hz), 108.54, 113.64, 123.74, 126.54
(d, 1JC−F = 20.5 Hz), 134.29 (d, 3JC−F = 3.8 Hz), 142.05 (d,
8c was synthesized in a manner similar to that for 8a.
Yield: 80.7%. mp: 110.0–110.6°C. 1H−NMR (CDCl3):
3.48 (s, 3H, CH3), 4.01 (s, 3H, CH3), 6.94 (s, 1H, aromat-
ics), 7.09 (dd, J = 8.0, 8.4 Hz, 1H, aromatics), 7.67 (d, J =
8.2 Hz, 1H, aromatics), 7.70 (d, J = 9.6 Hz, 1H, aromatics),
8.92 (s, 1H, aromatics). 13C−NMR (CDCl3): 29.42, 39.31,
89.52 (d, 2JC−F = 6.9 Hz), 109.98, 115.06, 124.56, 126.89
(d, 1JC−F = 19.7 Hz), 134.79 (d, 3JC−F = 4.5 Hz), 140.69 (d,
1JC−F = 11.4 Hz), 145.79, 151.63, 152.29, 154.84 (d, JC−F
=
1JC−F = 11.4 Hz), 149.44, 151.91, 152.26, 154.81 (d, JC−F
=
50.1 Hz), 158.27, 171.60. EI−MS: m/z: 443 Measured: 443.
78.9 Hz), 157.60, 162.88. EI−MS: m/z: 475 Measured: 475.
1 3

Annals of Nuclear Medicine
6‑(2,4‑Difuorophenoxy)‑8‑methyl‑2‑(tetrahydro‑2H
‑pyran‑4‑ylamino)pyrido[2,3‑d]pyrimidin‑7(8H)‑one
(9a: R1487)
6‑(4‑Fluoro‑2‑iodephenoxy)‑8‑methyl‑2‑(tetrah
ydro‑2H‑pyran‑4‑ylamino)pyrido[2,3‑d]pyrimi‑
din‑7(8H)‑one (9c: 4‑IR)
8a, potassium carbonate and 4-aminotetrahydropyran
were mixed in toluene, and the mixture was refluxed at
80°C overnight. The mixture was diluted with toluene
(5 mL), and the hot suspension was filtered. The filtrate
was concentrated under reduced pressure, and then the
solution was cooled and the solids precipitated. The sus-
pension was filtered and the residue was washed with
water and then with 1:1 water/methanol, after which the
residue was separated. The residue was recrystallized
with ethanol to obtain 9a (R1487) as white crystals.
Yield: 75.2%. mp: 177.1–177.3°C. 1H−NMR (CDCl3)
δ: 1.71 (s, 2H, CH2), 2.08 (d, J = 13.2 Hz, 2H, CH2), 3.57
(t, J = 11.0 Hz, 2H, CH2), 3.75 (s, 3H, CH3) 4.05 (d, J =
12.8 Hz, 2H, CH2), 4.12 (m, 1H, CH), 5.30 (s, 1H, NH),
6.76 (s, 1H, aromatics), 6.96 (d, J = 14.6 Hz, 2H, aro-
matics), 7.17 (s, 1H, aromatics), 8.39 (s, 1H, aromatics).
EI−HRMS: m/z Calcd. for C19H18N4O3F2 [M+]:
388.1347. Measured: 388.1351.
9c (4-IR) was synthesized in a manner similar to that for 9a.
Yield: 50.6%. mp: 181.0–181.4°C. 1H−NMR (CDCl3) δ:
1.66 (s, 2H, CH2), 2.08 (d, J = 12.4 Hz, 2H, CH2), 3.57 (t, J
= 11.6 Hz, 2H, CH2), 3.71 (s, 3H, CH3), 4.02 (d, J = 12.0 Hz,
2H, CH2), 4.11 (s, 1H, CH), 5.37 (s, 1H, NH), 6.83 (dd, J
= 8.4, 8.0 Hz, 1H, aromatics), 6.98 (s, 1H, aromatics), 7.43
(d, J = 8.4 Hz, 1H, aromatics), 7.52 (s, 1H, aromatics), 8.53
(s, 1H, aromatics). 13C−NMR (DMSO−d6): 27.94, 31.72,
31.72, 47.36, 65.75, 65.75, 86.62 (d, 2JC−F = 20.8 Hz), 103.45,
120.13, 121.32, 125.31 (d, 1JC−F = 19.7 Hz), 133.83 (d, 3JC−F
1
= 3.0 Hz), 140.27, 143.47 (d, JC−F = 10.6 Hz), 150.82,
153.32, 153.94 (d, JC−F = 27.3 Hz), 156.62, 158.11.
EI−HRMS: m/z Calcd. for C19H18N4O3FI [M+]:
496.0408. Measured: 496.0408.
Radiosynthesis (Scheme 2)
[
125I]Sodium iodide was purchased from PerkinElmer Japan
(Kanagawa, Japan). Radioactivity levels of 125I were measured
with a 2480 WIZARD2™ instrument (PerkinElmer).
6‑(4‑Fluoro‑2‑iodephenoxy)‑8‑methyl‑2‑(tetrah
ydro‑2H‑pyran‑4‑ylamino)pyrido[2,3‑d]pyrimi‑
din‑7(8H)‑one (9b: 2‑IR)
6‑(4‑Fluoro‑2‑tributylstanylphenoxy)‑8‑me‑
thyl‑2‑(tetrahydro‑2H‑pyran‑4‑ylamino)
pyrido[2,3‑d]pyrimidin‑7(8H)‑one (10b)
9b (2-IR) was synthesized in a manner similar to that
for 9a.
2-IR, bis(tributyltin) and tetrakis(triphenylphosphine)palla-
dium(0) were added to anhydrous toluene, and the mixture was
refuxed at 140°C for 14 h before fltration through Celite. The
fltrate was concentrated under reduced pressure and separated
and purifed by silica gel column chromatography using ethyl
acetate/hexane (1/1, v/v) as an eluate to obtain 10b as white
oily matter.
Yield: 71.8%. mp: 223.7–223.9°C. 1H−NMR (CDCl3)
δ: 1.61 (s, 2H, CH2), 2.08 (d, J = 11.6 Hz, 2H, CH2), 3.57
(m, 2H, CH2), 3.73 (s, 3H, CH3), 4.04 (m, J = 4.0 Hz,
2H, CH2), 4.12 (m, J = 4.5 Hz, 1H, CH), 6.83(s, 1H,
NH), 6.93 (m, J = 4.5 Hz, 1H, aromatics), 7.06 (s, 1H,
aromatics), 7.59 (dd, J = 2.6 Hz, 1H, aromatics), 7.59
(dd, J = 2.6 Hz, 1H, aromatics), 8.33 (s, 1H, aromatics).
13C−NMR (DMSO−d6): 28.25, 32.95, 32.95, 47.64,
Yield: 20.0%. 1H−NMR (CDCl3) δ: 0.80–1.66 (m, 29H,
Bu3, CH2), 2.08 (d, J = 11.2 Hz, 2H, CH2), 3.57 (t, J =
10.6 Hz, 2H, CH2), 3.73 (s, 3H, CH3), 4.03 (d, J = 11.6 Hz,
2H, CH2), 4.12 (m, 1H, CH), 5.26 (s, 1H, NH) 6.75 (s, 1H,
aromatics), 6.84 (dd, J = 4.0, 4.0 Hz, 1H, aromatics), 6.98
(ddd, J = 3.2, 2.8, 2.8 Hz, 1H, aromatics), 7.18 (dd, J = 2.8,
2.8 Hz, 1H, aromatics), 8.28 (s, 1H, aromatics).
2
66.75, 66.75, 88.20 (d, JC−F = 9.1 Hz), 104.21, 116.49
(d, 1JC−F = 22.8 Hz), 117.00, 119.54 (d, 2JC−F = 8.4 Hz),
1
3
126.58 (d, JC−F = 25.0 Hz), 142.66, 151.74 (d, JC−F
=
3.0 Hz), 153.36, 157.50, 157.54, 159.02, 160.05 (d, JC−F
= 6.9 Hz).
EI−HRMS: m/z Calcd. for C19H18N4O3FI [M+]:
496.0408. Measured: 496.0412.
FAB−HRMS: m/z Calcd. for C31H45N4O3FSn [M+H]+:
661.2576. Measured: 661.2582.
Scheme 2 Radiosynthesis of
[
125I]2-IR and [125I]4-IR
1 3

Annals of Nuclear Medicine
[
125I]4-IR (47.1 kBq) were added to a mixture of 1−octanol
6‑(2‑Fluoro‑4‑tributylstanylphenoxy)‑8‑me‑
thyl‑2‑(tetrahydro‑2H‑pyran‑4‑ylamino)
pyrido[2,3‑d]pyrimidin‑7(8H)‑one (10c)
and PBS (−) (pH = 7.4), stirred with shaking, and centri-
fuged. Then, the liquid was collected from each liquid layer,
the radioactivity was measured, and the log D value was
calculated.
10c was synthesized in a manner similar to that for 10b.
Yield: 90.3%. 1H−NMR (CDCl3) δ: 0.88–1.65 (m, 29H,
Bu3, CH2), 2.08 (d, J = 11.2 Hz, 2H, CH2), 3.57 (t, J =
10.8 Hz, 2H, CH2), 3.74 (s, 3H, CH3), 4.02 (d, J = 11.2 Hz,
2H, CH2), 4.11 (s, 1H, CH), 5.33 (s, 1H, NH) 6.83 (s, 1H,
aromatics), 7.01 (dd, J = 7.6, 7.6 Hz, 1H, aromatics), 7.18
(d, J = 6.8 Hz 1H, aromatics), 7.25 (d, J = 6.8 Hz 1H, aro-
matics), 8.31 (s, 1H, aromatics).
In vitro kinase inhibition assay
The kinase inhibitory activities of 2-IR, 4-IR, R1487, and
SB203580 (AdooQ BioScience, CA, USA) were measured
using an ADP-Glo™ kinase assay system (Promega, MSN,
USA) according to the method recommended by Promega.
Briefy, the test compound (2-IR, 4-IR, or SB203580, fnal
concentration of 1 nM−100 μM; R1487, fnal concentra-
tion of 0.033 nM−3.3 μM), active p38α, p38 substrate, and
150 mM ATP included in the assay solution were mixed and
incubated at room temperature for 60 min. The ADP-Glo™
reagent was added to the solution and incubated at room
temperature for 40 min. A kinase detection reagent was then
added and incubated at room temperature for another 30 min.
Finally, the luminescence was measured with a Hidex Sense
Beta Photometer (Hidex, Turku, Finland) with an integration
time of 1 s. The inhibition activity of the tested compounds
was evaluated by determining the 50% inhibitory potency
(IC50) from inhibition curves.
EI−HRMS: m/z Calcd. for C31H45N4O3FSn [M+]:
660.2498. Measured: 660.2504.
6‑(4‑Fluoro‑2‑[125I]iodephenoxy)‑8‑methyl‑2‑(tetr
ahydro‑2H‑pyran‑4‑ylamino)‑pyrido[2,3‑d]pyrimi‑
din‑7(8H)‑one ([125I]2‑IR)
10b (10 μL, 1 mg/mL ethanol solution), 0.1 M HCl solution
(100 μL), [125I] sodium iodide (0.2 μL, 7.4 MBq), and 5%
aqueous hydrogen peroxide (10 μL) were mixed in a sealed
vial, and then the mixture was shaken and stirred at room
temperature for 15 min. After the mixture was fltered with
a 4 mm Cosmonice Filter W (Aqueous; Nacalai Tesque),
the target compound was purifed by a reverse-phase HPLC
composed of two pumps (LC-20AT, Shimadzu, Kyoto,
Japan), an absorbance detector (SPD-20A, Shimadzu), a
170 type I radioactivity detector (Beckman Coulter, CA,
USA), and a Cosmosil 5C18-AR-II column (10×250 mm,
Nacalai Tesque). A mobile phase of methanol/water (75/25,
v/v) at a fow rate of 3 mL/min was used. Subsequently, 2-IR
and [125I]2-IR were spotted on TLC Silica gel 60 F254 TLC
plates (Aluminum Sheet 20 × 20 cm; Merck, Darmstadt,
Germany) and simultaneouslydeveloped with chloroform/
methanol (30/1). After the unlabeled 2-IR spot was detected
on the TLC plate under illumination at 254 nm, the plate was
divided into 10 regions, and the radioactivity in each region
was measured. The radiochemical purity was estimated as
the percentage of the radioactivity in a given region of the
plate relative to the total radioactivity.
Preparation of turpentine oil‑induced infammation
model mice
The animal experiments in this study were conducted in
accordance with Osaka University of Pharmaceutical Sci-
ences guidelines for experiments involving animals. The
study protocol was approved by the Experimental Animal
Committee at Osaka University of Pharmaceutical Sciences
(Permission Number: 18–76, 19–76, and 20–76). Male ddY
mice (4 weeks old) were purchased from Japan SLC (Shi-
zuoka, Japan) and allowed access to food and water ad libi-
tum. Animals were given intramuscular injections with
turpentine oil (50 μL) in the left thigh region under isofu-
rane anesthesia and used for experiments 2 days after the
injection. Muscle from contralateral untreated regions of the
same mouse was used as the untreated control.
6‑(2‑Fluoro‑4‑[125I]iodephenoxy)‑8‑methyl‑2‑(tetr
ahydro‑2H‑pyran‑4‑ylamino)‑pyrido[2,3‑d]pyrimi‑
din‑7(8H)‑one ([125I]4‑IR)
In vitro stability analysis
[
125I]2-IR (10 μL, 27.7 kBq), and [125I]4-IR (10 μL,
[
[
125I]4-IR was synthesized in a manner similar to that for
125I]2-IR.
18.3 kBq) were incubated in ddY mice plasma (100 μL) for
0, 1, 2, 6, and 24 h at 37 °C. Methanol was then added and
the mixture was centrifuged. The resulting supernatant was
analyzed in a manner similar to that used for radiochemical
purity analysis. The intact rate (%) was calculated as the
percentage of the radioactivity detected in the intact form
region relative to the total radioactivity.
Measurement of Log D values
Log D values of [125I]2-IR and [125I]4-IR were measured as
previously described [9]. In brief, [125I]2-IR (52.0 kBq) and
1 3

Annals of Nuclear Medicine
Tween 80 and R1487 (30 mg/kg) or ralimetinib (30 mg/kg,
Selleck Chemicals, TX, USA) 60 min prior to the probe
administration.
Western blotting analysis
Infamed and contralateral uninfamed muscle tissues were
excised from infammation model mice (n = 3), homog-
enized in Passive Lysis Bufer (Promega) and centrifuged
to remove debris. After determining the protein concentra-
tion of the samples using the Pierce BCA Protein Assay
Kit (Thermo Scientific, MA, USA) and measuring the
absorbance at 562 nm with a Hidex Sense Beta Photometer
(Hidex), the samples were diluted with sample bufer con-
taining dithiothreitol to a protein concentration of 0.2 mg/
mL. The samples (5μL) were then loaded onto a polyacryla-
mide gel and western blotting was performed using human/
mouse/rat p38 alpha antibody (0.4 μg/mL, AF8691, R&D
Systems, MSP, USA) and phospho-p38 MAPK (Thr180,
Tyr182) polyclonal antibody (1:1000 dilution, 36–8500,
Invitrogen, CA, USA) as the primary antibodies and rabbit
IgG HRP−conjugated antibody (1:3000 dilution, HAF008,
R&D systems) as the secondary antibody. GAPDH levels in
the samples were used as protein loading controls and were
measured with rabbit polyclonal GAPDH antibody (Gene-
Tex, GTX110118, LAX, USA). Immunoreactive bands were
visualized using Chemi−Lumi One Ultra (Nacalai Tesque).
The FastGene Bluestar prestained protein marker (NIPPON
genetics, Tokyo, Japan) was used as a molecular weight
marker to estimate molecular weights. An Amersham Imager
600 (GE Healthcare Japan, Tokyo, Japan) was used to visu-
alize bands on the western blot.
Statistical analysis
Data are shown as means standard deviations (S.D.). Sta-
tistical analysis was performed with GraphPad Prism 8 using
Tukey’s multiple comparison test for in vitro kinase inhibi-
tion assay analysis and in vivo inhibition study analysis, and
paired t test for western blotting analysis. A two-tailed value
of p<0.05 was considered as statistically signifcant.
Results
Synthesis (Scheme 1)
We designed and synthesized two pyrimidinopyridone
derivatives, 2-IR and 4-IR, based on the structure of R1487,
a well−characterized selective p38α inhibitor. In the frst
route, 1 was reacted with methylamine to obtain 2, which
was subsequently reduced with LiAlH4 to obtain 3, and then
oxidized using MnO2 to obtain 4. In another route, 5a−c was
reacted with methyl bromoacetate to obtain 6a−c according
to a previously reported method [10]. Next, 4 and 6a−c were
condensed to obtain 7a−c, which was then oxidized with
performic acid to obtain 8a−c having a sulfone group that
was displaced with 4−aminotetrahydropyran to obtain 9a
(R1487), 9b (2-IR), and 9c (4-IR). R1487, 2-IR, and 4-IR
were synthesized at total yields of 24.2%, 17.5%, and 19.2%,
respectively.
Biodistribution study
[
125I]2-IR (23.9–28.9 kBq/100 μL saline containing 0.2%
Tween 80) and [125I]4-IR (17.1–19.0 kBq/100 μL saline con-
taining 0.2% Tween 80) were intravenously injected into ddY
mice and infammation model mice. The mice were sacri-
fced under isofurane anesthesia at 5, 15, 30, 60, or 120 min
(n = 4 for each time point) after probe administration. The
infamed site was carefully identifed by appearance such as
abscess, color, and hardness. Samples of infamed tissue and
tissues of interest were excised for measurement of weight
and radioactivity. Radioactivity accumulation in the tissues
is expressed in terms of the percentage of the injected dose
per gram of tissue (%ID/g).
In vitro kinase inhibition assay
The IC50 values of the tested compounds were determined
(Table 1). The IC50 values were 139.4 26.5 nM and 88.9
14.5 nM for 2-IR and 4-IR, respectively, indicating a signif-
cantly lower potency of both compounds relative to R1487
(11.8 8.3 nM); however, 4-IR exhibited significantly
Table 1 IC50 values of 4-IR,
Compound
IC50 (nM)
2-IR, and p38α inhibitors
2-IR
4-IR
R1487
SB203580
139.4 26.5*†
88.9 14.5†
11.8 8.3*
In vivo inhibition study
142.2 33.1*†
Radioactivity accumulation in the infamed tissue and nor-
mal muscle 30 min after intravenous injection of [125I]4-IR
(17.2 kBq/100 μL saline containing 0.2% Tween 80) in
infammation model mice (n = 3 each) was measured with
or without intraperitoneal injection of saline containing 0.2%
Data represent mean SD, n =
5
*p<0.05 vs. 4-IR
†p<0.05 vs. R1487
1 3

Annals of Nuclear Medicine
higher potency than the p38α inhibitor SB203580 (142.2
33.1 nM).
values were 2.50 0.05 and 2.10 0.02 for [125I]2-IR and
[
125I]4-IR, respectively.
Radiosynthesis (Scheme 2)
In vitro stability analysis
Radioiodination of 2-IR and 4-IR was performed via an
organotin−radioiodine exchange reaction using the cor-
responding tributyltin precursors. After an initial HPLC
purifcation, on analytical HPLC, [125I]2-IR and [125I]4-IR
radioactivity had retention times of 11.1 min and 14.3 min,
respectively, which coincided with the retention times of
nonradioactive 2-IR and 4-IR as detected by UV absorb-
ance. The radiochemical yield and radiochemical purity
for [125I]2-IR were 65.6% and 99.7%, respectively, whereas
those for [125I]4-IR were 84.2% and 97.5%. The molar activi-
ties for both were estimated to be 81 GBq/µmol. The log D
The intact percentages after incubation of [125I]2-IR and
[
[
125I]4-IR in mouse plasma were shown in Fig. 1. Both
125I]2-IR and [125I]4-IR were highly stable in mouse plasma
for 24 h.
Preparation of turpentine oil‑induced infammation
model mice
Infammation model mice were prepared by intramuscular
injection of turpentine oil in male ddY mice. Expression
levels of p-p38α and phosphorylated p38α (p-p38α) proteins
in infamed muscle from the treated area and muscle from
an unafected area from the same mouse were evaluated by
western blotting (Fig. 2, Supplementary Fig. 1). The p38α/
GAPDH ratios were comparable between the infamed and
untreated muscle (Fig. 2b), whereas the p-p38α/GAPDH
ratio was signifcantly higher in infamed than uninfamed
muscle (Fig. 2c).
Biodistribution study
The biodistribution of [125I]2-IR and [125I]4-IR was frst
evaluated in normal ddY mice (Supplementary Tables 1
and 2). Both probes showed a similar biodistribution profle
characterized by fast radioactivity delivery and clearance
throughout the body, except for the small intestine and the
colon, which are recognized as major excretion routes for
Fig. 1 In vitro stability of [125I]2-IR and [125I]4-IR in plasma. Data
represent mean SD, n = 4
Fig. 2 Protein expression levels
of p38α in muscle tissue. a Rep-
resentative western blot bands
for p38α, p-p38α, and GAPDH
in homogenates of infamed
and uninfamed muscle tissue
from diferent gels. Expres-
sion of b p38α and c p-p38α
in the infammation compared
with muscle expression cor-
rected to 1 after normalization
to GAPDH. Data represent
mean SD, n = 3 *p<0.05
1 3

Annals of Nuclear Medicine
Fig. 3 Biodistribution of probes
in infammation model mice.
In vivo biodistribution of a
[
125I]2-IR and b [125I]4-IR in
infammation model mice. Data
represent mean %ID/g SD,
n = 4
Table 2 In vivo biodistribution of [125I]2-IR in infammation model
Table 3 In vivo biodistribution of [125I]4-IR in infammation model
mice
mice
Time after injection (min)
Time after injection (min)
5
15
30
60
120
5
15
30
60
120
Infamed
2.5 0.4 2.8 0.6 1.8 0.5 1.1 0.2 0.3 0.0
Infamed
2.8 0.3 5.8 1.1 7.0 1.2 4.0 1.3 2.2 0.7
tissue
tissue
Blood
Pancreas
Spleen
1.4 0.1 1.2 0.1 0.7 0.1 0.4 0.1 0.2 0.0
5.2 0.4 3.3 0.6 1.2 0.2 0.6 0.1 0.3 0.0
2.9 0.3 2.0 0.3 0.7 0.1 0.4 0.1 0.2 0.1
Blood
Pancreas
Spleen
2.0 0.2 1.9 0.7 1.0 0.1 0.8 0.1 0.5 0.1
4.7 0.7 3.5 0.3 2.1 0.3 1.3 0.2 0.8 0.1
2.4 0.3 1.9 0.1 1.3 0.1 0.8 0.1 0.4 0.1
1.3 0.1 1.9 0.2 3.5 0.4 3.8 1.0 3.9 1.7
4.1 0.4 7.4 0.3 14.5 2.7 23.1 1.5 24.8 2.1
Stomach* 1.3 0.6 5.3 3.0 2.1 0.6 3.0 1.1 2.3 0.4
Stomach*
Small
5.2 0.8 8.0 2.6 15.7 4.7 27.1 2.5 28.3 8.3
Small
intestine
intestine
Colon
Liver
Kidney
Heart
1.8 0.3 2.0 0.4 2.0 0.3 4.0 2.1 18.8 13.1
11.6 0.8 8.2 0.7 4.7 0.5 4.6 1.1 2.3 1.2
6.3 0.3 4.5 0.5 3.3 0.4 2.4 0.2 1.0 0.2
4.4 0.6 2.6 0.5 1.1 0.1 0.6 0.1 0.2 0.1
4.1 0.8 2.8 0.3 1.1 0.2 0.7 0.3 0.3 0.1
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
2.7 0.4 1.8 0.2 0.7 0.1 0.4 0.1 0.1 0.0
1.7 0.6 1.0 0.2 0.3 0.0 0.3 0.2 0.1 0.0
2.7 0.3 1.4 0.2 0.5 0.0 0.2 0.0 0.1 0.0
Colon
Liver
Kidney
Heart
1.6 0.2 2.0 0.1 2.3 0.1 3.6 0.5 16.5 4.4
10.7 1.0 8.1 0.9 5.3 0.5 3.7 0.6 2.9 0.9
6.0 1.1 4.6 0.5 3.5 0.3 2.3 0.2 1.4 0.3
3.9 0.5 3.0 0.4 1.7 0.2 1.2 0.1 0.5 0.1
3.7 0.3 3.1 0.4 1.8 0.1 1.2 0.1 0.7 0.1
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
2.4 0.4 1.8 0.2 1.4 0.1 0.8 0.1 0.4 0.1
1.2 0.1 1.2 0.2 0.7 0.1 0.6 0.1 0.2 0.1
2.8 0.4 1.7 0.2 1.0 0.2 0.6 0.0 0.3 0.0
Lung
Lung
Thyroid*
Muscle
Bone
Thyroid*
Muscle
Bone
Brain
Brain
Data represent mean %ID/g SD, n=4
*Data represent mean %ID SD, n=4
Data represent mean %ID/g SD, n=4
*Data represent mean %ID SD, n=4
such lipophilic compounds. In particular, the low and neg-
ligible radioactivity levels detected in the stomach and the
thyroid, respectively, indicated high stability against deio-
dination reactions in vivo.
levels in the blood and muscle were low and rapidly cleared,
which resulted in a high infammation-to-blood ratio (6.2
0.4) and a high infammation-to-muscle ratio (5.2 1.3)
observed 30 min post-injection. In contrast, radioactivity
accumulation in infamed tissue after [125I]2-IR administra-
tion peaked at 15 min post−injection (2.8 0.6%ID/g) and
then gradually decreased to 0.3 0.0%ID/g at 2 h post-
injection. For this probe, the infammation-to-blood ratio
and infammation-to-muscle ratio at 15 min post−injection
were 2.4 0.5 and 1.6 0.4, respectively.
Biodistribution of [125I]2-IR and [125I]4-IR were next
investigated in the infammation model mice (Fig. 3, Tables 2
and 3). The radioactivity accumulation in the infamed tissue
after [125I]4-IR administration peaked at 30 min post-injec-
tion (7.0 1.2%ID/g) and then gradually decreased to 2.2
0.7%ID/g at 2 h post-injection. In addition, radioactivity
1 3

Annals of Nuclear Medicine
known to be activated in response to a variety of external
stimuli such as infammatory cytokines [2], lipopolysaccha-
ride [5], and osmotic shock [1]. In this study, we used mice
treated with turpentine oil as a model of infammation. We
confrmed high expression levels of the active form of p38α,
p-p38α, in infamed lesions that arose following treatment
compared to normal, untreated muscle, which verifes that
this animal model of infammation is relevant for testing
these probes. p-p38α is p38α phosphorylated at residues
Thr180/Tyr182 by MAP kinase kinases. Since R1487 and
ralimetinib both bind to an ATP-binding site in p-p38α [8,
11] that is covered by a crossover connection in a naive p38α
prior to phosphorylation of these two residues [12], it is
suggested that [125I]2-IR and [125I]4-IR can bind to the ATP-
binding site of p-p38α and thus would be valuable probes
to detect sites with disease activity as evidenced by high
p-p38α expression levels.
In vivo inhibition study
Mice were frst treated or not with R1487 or ralimetinib
delivered by intraperitoneal injection. At 60 min after
administration, [125I]4-IR was delivered and 30 min later
samples of infamed and uninfamed muscle tissue were
taken. The accumulation of radioactivity in the infamed
tissue was signifcantly decreased by systemic pretreatment
with both p38α inhibitors, while levels in the uninfamed
muscle were not afected (Fig. 4).
Discussion
In this study, we designed the probes [123I]2-IR and
[
123I]4-IR based on the pyrimidinopyridone structure of
a potent p38α-selective inhibitor, R1487, as an activated
p38α imaging probe for SPECT, synthesized radiolabeled
compounds using an easy-to-use radioiodine-125 ([125I]2-IR
and [125I]4-IR) instead of radioiodine-123, and evaluated the
usefulness of each compound in in vitro and in vivo experi-
ments. 4-IR showed signifcantly higher inhibitory activity
toward p38α relative to 2-IR and SB203580. In particular,
R1487 was selected as the parent compound for the radi-
otracer in this study based on its high afnity and selectivity
toward activated p38α [8], as well as the strong interaction
between the R1487 pyrimidinopyridone moiety and the
ATP-binding site (Thr106-Gly110) of p-p38α [8]. We pre-
dicted that the residual phenoxy group could be used as an
imaging moiety after the introduction of radioiodine-123.
Moreover, the phenoxy group also contributes to the pref-
erential recognition of p38α compared to p38β [8]. The two
R1487 derivatives having phenoxy group fuorination at
positions 2- or 4- showed similar selectivity [8]. As such,
we tested the performance of both 2- or 4-radioiodinated
R1487 tracers.
[
125I]4-IR exhibited high radioactivity accumulation in
infamed tissue, depending on the levels of p-p38α expres-
sion, as well as favorable pharmacokinetic characteristics,
such as rapid delivery and clearance and high stability. These
results indicated that [123I]4-IR, in particular, is a promising
imaging probe for use with SPECT to identify regions hav-
ing high p-p38α expression that would occur in a variety of
infammatory diseases.
We observed a signifcant decrease in inhibitory potency
for both 2-IR and 4-IR relative to unmodified R1487,
and 4-IR had reduced specificity between p38α/p38β
p38α is an intracellular signaling kinase that is ubiq-
uitously expressed in mammalian cells [3]. This kinase is
Fig. 4 In vivo inhibition of
[
125I]4-IR radioactivity accumu-
lation in a infamed tissue and
b muscle using p38α selective
inhibitors. Data represent mean
%ID/g SD, n = 3 *p<0.05
1 3

Annals of Nuclear Medicine
(Supplementary Fig. 2). These results indicate that the phe-
noxy moiety makes a clear contribution to the p38α interac-
tion and that the introduction of a large atom like iodine in
the phenoxy group could produce steric hinderance at amino
acid residues that comprise the ATP-binding pocket that in
turn afects interactions with and recognition of p-p38α [8].
The in vivo biodistribution study could clearly diferen-
tiate radioactivity accumulation profles for [125I]2-IR and
contrast, [123/125I]4-IR designed using R1487, which specif-
cally binds to activated p38α, exhibits superior potential as
an imaging probe targeting biological functions of the p38α
pathway activated in disease settings, compared to [125I]
m-YTM.
Activation of p38α contributes not only to infamma-
tory responses, but also to regulation of several pathologi-
cal functions, such as angiogenesis [16–18] and apoptosis
[19, 20]. Therefore, activated p38α imaging with [123I]4-IR
could be versatile enough to be applied to cancer and athero-
sclerosis, and of worth to be evaluated using other various
disease model animals in the future, aside from the turpen-
tine oil-induced infammation model mice used in this study.
Considering the sensitivity of [123I]4-IR for infammatory
disease imaging, comparable radioactivity accumulation
in the infamed tissue with that observed for [18F]FDG in
the model mice suggests the potential use of [123I]4-IR for
this purpose. We used model mice recognized as an acute
infammation animal model mainly due to the utility and
high degree of expression of activated p38α. Because several
other cascades relevant to NF-κB [21] and JNK [22] are also
involved in the infammatory response, in addition to the
p38α pathway, careful evaluation is required in future stud-
ies to determine the utility of [123I]4-IR in other pathological
disease model animals.
[
125I]4-IR in infamed tissue. [125I]4-IR showed cumula-
tive radioactivity accumulation in the infamed tissue over
the frst 30 min, accompanied by a monotonic decrease in
blood radioactivity that suggests strong active targeting of
the probe toward p-p38α in the infamed tissue. This result is
likely associated with the signifcant diference in the inhibi-
tion potency of the two probes, given that the physicochemi-
cal characteristics are similar, as is the biodistribution profle
in normal mice. Based on a previously reported docking
simulation, we hypothesized that the fuorine at positions
2- and 4- of the R1487 phenoxy ring can both be replaced
by a large atom such as iodine without having a signifcant
efect on the inhibitory potency [8]. However, the results of
this study indicate that replacement of fuorine with iodine at
the 2-position produces more steric hindrance compared to
the 4-position. The in vivo biodistribution study of [125I]4-IR
showed not only high radioactivity accumulation in the
infamed tissue, but also high infammation-to-blood and
infammation-to-muscle ratios (indices for in vivo imaging
with SPECT) within 1 h post-injection of the tracer. Accu-
mulation levels were similar to those observed for 2-[18F]
fuoro-2-deoxy-D-glucose ([18F]FDG), a well-known infam-
mation imaging tracer [13], obtained in the same infamma-
tion model mice (Supplementary Fig. 3). While [18F]FDG
has been widely used as an infammation imaging agent,
[18F]FDG also accumulates in normal brain, heart, and
cramped and post-exercise muscles, leading to difculty in
detecting infamed tissues in images [14]. These fndings,
along with the results of the present study, may support the
development of other imaging agents based on diferent
accumulation mechanisms, such as the p38 MAPK path-
way investigated in this study. Together the in vivo results
suggest that [123I]4-IR would be a valuable probe for in vivo
SPECT imaging of p-p38α in a variety of disease settings.
As already mentioned above, naive p38α is ubiquitously
expressed in mammalian cells [3] and phosphorylated to
become active p38α in response to external stimuli. There-
fore, in vivo imaging targeting the p38α pathway in disease
pathology may be achieved by the development of an imag-
ing probe that is specifc for the activated form of p38α.
The radioiodinated probe [125I]m-YTM previously reported
by our group, which is the only p38α probe that has been
developed to our knowledge, targeted total p38α, including
both naive and activated forms [15], indicating limited utility
as an imaging probe for p38α activation in disease states. In
This study does have some limitations. The inhibitory
activity of 4-IR was about 10 times signifcantly lower than
that of R1487, although it was still signifcantly higher than
that of the p38α inhibitor SB203580. The diminished selec-
tivity of 4-IR for p38α over p38β compared to R1487 should
also be addressed. As mentioned above, the introduction of
a large atom like iodine at the 4-position of the R1487 phe-
noxy moiety could afect the binding afnity due to steric
hindrance. Introduction of radiofuorine-18 instead of radi-
oiodine-123/125 at the 4-position could address this issue,
considering recent reports concerning direct 18F labelling
of a benzene ring from corresponding boronic acid pinacol
ester [23–25] or tributylstannyl [26, 27] precursors. [18F]
R1487 obtained using the direct radiofuorination reaction
could preserve the binding profle of the mother compound
R1487 and would still allow for positron emission tomogra-
phy (PET) imaging applications. We are currently pursuing
development of [18F]R1487 for p-p38α PET imaging.
In summary, [125I]4-IR newly designed as an activated
p38α targeting probe could be synthesized with high radio-
chemical yield, purity, and molar activity. [125I]4-IR showed
high inhibitory activity toward p38α, high stability, and
favorable pharmacokinetic characteristics, such as high radi-
oactivity accumulation in infamed tissue and rapid blood
clearance in a mouse model of infammation. Together,
[
123I]4-IR can target areas with high p-p38α expression and
thus could be a valuable probe for use with SPECT imaging
of various infammatory diseases.
1 3

Annals of Nuclear Medicine
3-cyano/ethoxycarbonyl-2-methyl-benzo[b]furans. Tetrahedron.
2004;60(52):12231–7.
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s12149-021-01669-6.
11. Campbell RM, Anderson BD, Brooks NA, Brooks HB, Chan EM,
De Dios A, et al. Characterization of LY2228820 dimesylate, a
potent and selective inhibitor of p38 MAPK with antitumor activ-
ity. Mol Cancer Ther. 2014;13(2):364–74.
Acknowledgements We are grateful for the technical assistance of Ms.
Atsuko Takeguchi, Ms. Kaede Hanazono, Mr. Ryuji Kakisaka, Mr.
Naofumi Yoshida, and Ms. Nami Nakai.
12. Zhang YY, Wu JW, Wang ZX. Mitogen-activated protein kinase
(MAPK) phosphatase 3-mediated cross-talk between MAPKs
ERK2 and p38alpha. J Biol Chem. 2011;286(18):16150–62.
13. Pellegrino D, Bonab AA, Dragotakes SC, Pitman JT, Mariani
G, Carter EA. Infammation and infection: imaging properties of
18F-FDG-labeled white blood cells versus 18F-FDG. J Nucl Med.
2005;46(9):1522–30.
Author contributions TH, MH, and TT conceptualized and designed
the study. TH, NK, and MH performed the experiments and collected
the data. TH, NK, MH, and TT interpreted the data. TH drafted the
initial manuscript, which was critically reviewed by NK and TT. All
authors approved the fnal manuscript and are accountable for all of
the work.
14. Parida GK, Roy SG, Kumar R. FDG-PET/CT in skeletal muscle:
pitfalls and pathologies. Semin Nucl Med. 2017;47(4):362–72.
15. Hirata M, Yao T, Fujimura S, Kanai Y, Yoshimoto M, Sato T,
et al. Development of a p38α-selective radioactive probe for
qualitative diagnosis of cancer using SPECT. Ann Nucl Med.
2019;33(5):333–43.
Funding This work was supported in part by JSPS KAKENHI
(19H03606).
Declarations
16. Patnaik A, Haluska P, Tolcher AW, Erlichman C, Papadopou-
los KP, Lensing JL, et al. A first-in-human phase I study of
the oral p38 MAPK inhibitor, Ralimetinib (LY2228820 Dime-
sylate), in patients with advanced cancer. Clin Cancer Res.
2016;22(5):1095–102.
Conflict of interest All authors declare no conficts of interests. The
funding bodies had no role in study design, data collection and analy-
sis, decision to publish, or manuscript preparation.
17. Leelahavanichkul K, Amornphimoltham P, Molinolo AA, Basile
JR, Koontongkaew S, Gutkind JS. A role for p38 MAPK in head
and neck cancer cell growth and tumor-induced angiogenesis and
lymphangiogenesis. Mol Oncol. 2014;8(1):105–18.
References
18. Reustle A, Torzewski M. Role of p38 MAPK in atherosclerosis
and aortic valve sclerosis. Int J Mol Sci. 2018;19(12):3761.
19. Miura H, Kondo Y, Matsuda M, Aoki K. Cell-to-cell heterogene-
ity in p38-mediated cross-inhibition of JNK causes stochastic cell
death. Cell Rep. 2018;24(10):2658–68.
1. Cohen DM. Mitogen-activated protein kinase cascades and the
signaling of hyperosmotic stress to immediate early genes. Comp
Biochem Physiol A Physiol. 1997;117(3):291–9.
2. Johnson GL, Lapadat R. Mitogen-activated protein kinase path-
ways mediated by ERK, JNK, and p38 protein kinases. Science.
2002;298(5600):1911–2.
20. Lin CL, Lee CH, Chen CM, Cheng CW, Chen PN, Ying TH,
et al. Protodioscin induces apoptosis through ROS-mediated
endoplasmic reticulum stress via the JNK/p38 activation path-
ways in human cervical cancer cells. Cell Physiol Biochem.
2018;46(1):322–34.
3. Ono K, Han J. The p38 signal transduction pathway: activation
and function. Cell Signal. 2000;12(1):1–13.
4. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green
D, et al. A protein kinase involved in the regulation of infamma-
tory cytokine biosynthesis. Nature. 1994;372(6508):739–46.
5. Beyaert R, Cuenda A, Vanden Berghe W, Plaisance S, Lee JC,
Haegeman G, et al. The p38/RK mitogen-activated protein kinase
pathway regulates interleukin-6 synthesis response to tumor
necrosis factor. Embo j. 1996;15(8):1914–23.
21. Lawrence T. The nuclear factor NF-kappaB pathway in infamma-
tion. Cold Spring Harb Perspect Biol. 2009;1(6):a001651.
22. Grynberg K, Ma FY, Nikolic-Paterson DJ. The JNK signaling
pathway in renal Fibrosis. Front Physiol. 2017;8:829.
23. Tredwell M, Preshlock SM, Taylor NJ, Gruber S, Huiban M, Pass-
chier J, et al. A general copper-mediated nucleophilic 18F fuorina-
tion of arenes. Angew Chem Int Ed Engl. 2014;53(30):7751–5.
24. Lim SSD, Jeon S, Kim Y, Kim H, Lee S, Cho H, Lee BC, Kim
SE, Kim K, Lee E. Cobalt-catalyzed C-F bond borylation of aryl
fuorides. Org Lett. 2018;20(22):7249–52.
6. Cohen SB, Cheng TT, Chindalore V, Damjanov N, Burgos-Vargas
R, Delora P, et al. Evaluation of the efcacy and safety of pamapi-
mod, a p38 MAP kinase inhibitor, in a double-blind, methotrex-
ate-controlled study of patients with active rheumatoid arthritis.
Arthritis Rheum. 2009;60(2):335–44.
25. Niwa TOH, Watanabe Y, Hosoya T. Ni/Cu-catalyzed defuorobo-
rylation of fuoroarenes for diverse C-F Bond functionalizations.
J Am Chem Soc. 2015;137(45):14313–8.
7. Khalil MM, Tremoleda JL, Bayomy TB, Gsell W. Molec-
ular SPECT imaging: an overview. Int J Mol Imaging.
2011;2011:796025.
26. Airaksinen AJ. The radiopharmaceutical chemistry of Fluo-
rine-18: next-generation fluorinations. Radiopharmaceutical
Chemistry. 2019:297–310.
8. Goldstein DM, Soth M, Gabriel T, Dewdney N, Kuglstat-
ter A, Arzeno H, et al. Discovery of 6-(2,4-difuorophenoxy)-
2-[3-hydroxy-1-(2-hydroxyethyl)propylamino]-8-me-
thyl-8H-pyrido[2,3-d]pyrimidin-7-one (Pamapimod) and
6-(2,4-difluorophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-
ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (R1487) as orally
bioavailable and highly selective inhibitors of p38α mitogen-
activated protein kinase. J Med Chem. 2011;54(7):2255–65.
9. OECD. Test No. 107: Partition Coefcient (n-octanol/water):
Shake Flask Method. 1995.
27. Makaravage KJBA, Mossine AV, Sanford MS, Scott PJH. Copper-
mediated radiofuorination of arylstannanes with [18F]KF. Org
Lett. 2016;18(20):5440–3.
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
10. RamaRao VV, Reddy GV, Maitraie D, Ravikanth S, Yadla R,
Narsaiah B, Rao PS. One-pot synthesis of fuorine containing
1 3