Development of a p38α-selective radioactive probe for qualitative diagnosis of cancer using SPECT

Article abstract of DOI:10.1007/s12149-019-01341-0

Objective: p38 mitogen-activated protein (MAP) kinase (p38α) has drawn attention as a new target molecule for the treatment and diagnosis of cancer, and its overexpression and activation have been reported in various types of cancer. In this study, a single photon emission computed tomography (SPECT) imaging probe of p38α was developed to noninvasively image p38α activity for effective qualitative diagnosis of cancer. Methods: Pyrrolepyridine derivatives, m-YTM and p-YTM, were designed and synthesized based on the structure of the p38α-selective inhibitor. Radioactive iodine-labeled m-YTM, [¹²⁵I]m-YTM, was synthesized because m-YTM greatly inhibited the phosphorylation of p38α upon examining the inhibitory effects of the compounds. After investigating the binding affinity of [¹²⁵I]m-YTM to the recombinant p38α, a saturation binding experiment using activated p38α and inactive p38α was performed to determine the binding site. Uptake of [¹²⁵I]m-YTM into various cancer cell lines was investigated, and the pharmacokinetics was evaluated using tumor-bearing mice. Results: The inhibitory activity of m-YTM was approximately 13 times higher than that of SB203580, a p38α-selective inhibitor. The binding site of [¹²⁵I]m-YTM was estimated to be the p38α activating site, similar to that of SB203580, because the [¹²⁵I]m-YTM bound strongly to both activated p38α and inactive p38α. Various different cancer cells incorporated [¹²⁵I]m-YTM; however, its accumulation was significantly reduced by treatment with SB203580. Pharmacokinetics study of [¹²⁵I]m-YTM in B-16 tumor-bearing mice was examined which revealed high accumulation of radioactivity in tumor tissues. The ratios of radioactivity in the B-16 tumor to that in blood were 3.1 and 50 after 1 and 24?h, respectively. The ratio of radioactivity in the tumor to that in blood in the tumor-bearing mice generated using other cancer cell lines was also ≥ 1 at 1?h after the administration of the probe. Conclusions: This study suggests that [¹²³I]m-YTM has potential as a p38α imaging probe effective for various cancer types.

Full text of DOI:10.1007/s12149-019-01341-0

Annals of Nuclear Medicine
https://doi.org/10.1007/s12149-019-01341-0
ORIGINAL ARTICLE
Development of a p38α‑selective radioactive probe for qualitative
diagnosis of cancer using SPECT
Masahiko Hirata1 · Tatsuma Yao1 · Shigeaki Fujimura1 · Yasukazu Kanai1 · Mitsuyoshi Yoshimoto1,2 · Takaji Sato1 ·
Yoshiro Ohmomo1 · Takashi Temma1
Received: 7 December 2018 / Accepted: 23 January 2019
© The Japanese Society of Nuclear Medicine 2019
Abstract
Objective p38 mitogen-activated protein (MAP) kinase (p38α) has drawn attention as a new target molecule for the treatment
and diagnosis of cancer, and its overexpression and activation have been reported in various types of cancer. In this study, a
single photon emission computed tomography (SPECT) imaging probe of p38α was developed to noninvasively image p38α
activity for efective qualitative diagnosis of cancer.
Methods Pyrrolepyridine derivatives, m-YTM and p-YTM, were designed and synthesized based on the structure of the
p38α-selective inhibitor. Radioactive iodine-labeled m-YTM, [125I]m-YTM, was synthesized because m-YTM greatly inhib-
ited the phosphorylation of p38α upon examining the inhibitory efects of the compounds. After investigating the binding
afnity of [125I]m-YTM to the recombinant p38α, a saturation binding experiment using activated p38α and inactive p38α
was performed to determine the binding site. Uptake of [125I]m-YTM into various cancer cell lines was investigated, and the
pharmacokinetics was evaluated using tumor-bearing mice.
Results The inhibitory activity of m-YTM was approximately 13 times higher than that of SB203580, a p38α-selective
inhibitor. The binding site of [125I]m-YTM was estimated to be the p38α activating site, similar to that of SB203580, because
the [125I]m-YTM bound strongly to both activated p38α and inactive p38α. Various diferent cancer cells incorporated
[
125I]m-YTM; however, its accumulation was signifcantly reduced by treatment with SB203580. Pharmacokinetics study
of [125I]m-YTM in B-16 tumor-bearing mice was examined which revealed high accumulation of radioactivity in tumor tis-
sues. The ratios of radioactivity in the B-16 tumor to that in blood were 3.1 and 50 after 1 and 24 h, respectively. The ratio
of radioactivity in the tumor to that in blood in the tumor-bearing mice generated using other cancer cell lines was also≥1
at 1 h after the administration of the probe.
Conclusions This study suggests that [123I]m-YTM has potential as a p38α imaging probe efective for various cancer types.
Keywords P38α · Tumor imaging · SPECT
Introduction
growth factors, which was discovered in the late 1980s. It is
functionally involved in various biological activities, such as
cellular growth and diferentiation, cell death, cell cycle sup-
pression, early development, and brain diseases [1, 2]. MAP
kinase is considered to be activated in response to external
stimuli, after which it moves to the nucleus to regulate gene
expression through the phosphorylation of transcription fac-
tors, among others.
Mitogen-activated protein (MAP) kinase is an intracellu-
lar signaling kinase found in mammalian culture cells; it is
a serine/threonine kinase commonly activated by various
* Takashi Temma
ttemma@gly.oups.ac.jp
The activation of MAP kinase requires the phos-
phorylation of threonine/tyrosine residues in the TEY
(Thr–Glu–Tyr) sequence between subdomains VII and
VIII of the kinase. Enzymes that phosphorylate both
amino acid residues and activate MAP kinase are serine/
threonine/tyrosine kinases, MAP kinase kinases (MAPKK,
1
Department of Biofunctional Analysis, Osaka University
of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki,
Osaka 569-1094, Japan
2
Division of Functional Imaging, Exploratory Oncology
Research & Clinical Trial Center, National Cancer Center,
Kashiwa, Chiba, Japan
Vol.:(0123456789)
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Annals of Nuclear Medicine
MEK). The activation of MAPKK itself also requires the
phosphorylation of serine/threonine residues, and enzymes
that phosphorylate MAPKK are called MAPKK kinases
(MAPKKK). This cascade composed of three diferent
kinases, namely, MAP kinase, MAPKK, and MAPKKK,
is called an MAP kinase cascade. In addition, a group of
MAP kinases carrying a TPY (Thr-Pro-Tyr) sequence or a
TGY (Thr-Gly-Tyr) sequence instead of the TEY sequence
and requiring the phosphorylation of threonine and tyros-
ine residues for their activation has been discovered; these
are collectively referred to as MAP kinase superfamily
molecules [3].
because m-YTM exhibited high inhibitory activity, its
radioactive iodine-labeled form was synthesized. The
compound obtained was examined in vitro and in vivo to
evaluate its efectiveness as a novel SPECT imaging probe
for detecting p38α activity.
Methods
Reagents and instruments
The radioactive compounds [125I]sodium iodide and [γ-32P]
ATP were purchased from Amersham Pharmacia Biotech
Inc. Fetal calf serum (FCS), penicillin–streptomycin solu-
tion, and MEMα were purchased from Trace Biosciences
Pty. Ltd., Wako Chemicals, and Nacalai Tesque, respectively.
p38α (active, inactive) was obtained from Upstate Bio-
technology; SB203580, SB202190, SB202474, PD98059,
U0126, Go6983, and bisindolylmaleimide I were from Cal-
biochem; and SP600125 was from Tocris. All other reagents
obtained were of special grade and used without purifcation.
Melting point was measured with a Yanagimoto micro-
melting point apparatus. IR spectra were measured with
p38 is a MAP kinase family molecule requiring the
phosphorylation of threonine and tyrosine residues in the
TGY sequence for its activation; currently, four genes
encoding having this sequence have been identifed: p38α,
p38β, p38γ, and p38δ [4]. p38α is known to be activated
in response to stimuli such as infammatory cytokines and
is involved in processes such as apoptosis [5, 6]. Aber-
rant activation of p38α is observed in the early stages of
ischemic diseases, such as myocardial infarction [7–9] and
cerebral infarction [10, 11], and it has been reported that
cell death due to ischemia can be prevented by inhibit-
ing this abnormal activity of p38α using the p38-selective
inhibitor SB203580 [8, 12, 13]. Urokinase plasminogen
activator (uPA) and its receptor uPA receptor, which are
involved in the infltration and metastasis of tumor cells,
are known to show increased levels in many cancers [14],
and continuous activation of p38α has been found to be
necessary for this expression [15, 16]. Recently, p38α is
recognized as a potential therapeutic target in tumors [17,
18].
1
the IR-700 (JASCO Corporation). H-NMR spectra were
measured with the Mercury-300 (Varian) and chemical shifts
were reported in δ (ppm) values using tetramethylsilane as
an internal standard. High-resolution mass spectra (HRMS)
were measured with M-80 or DIP-181 (JASCO Corpora-
tion). A CK-40 microscope (Olympus) was employed.
Absorbance was measured using Model 550 Microplate
Reader (Bio-Rad). HPLC was performed using a device
composed of a 600C type pump, 600F type universal injec-
tor, 2487 type detector (Waters), 170 type I detector (Beck-
man), C-18 reverse-phase column (10 × 25 mm; Nacalai
Tesque), and a mobile phase of methanol/water (80:20, v/v)
at a fow rate of 3 ml/min to measure absorbance (254 nm)
and 125I radioactivities. Radioactivities of 125I were measured
using an ARC-300 or ARC-380 auto-well gamma counter
(Aloka). Radioactivities of 32P were measured with a TRI-
CARB1600CA type liquid scintillation analyzer (Packard).
A-375 cells were obtained from the University of Fukui
Biomedical Imaging Research Center. B-16, C-6, SLC,
ME-180, PC-3, and A-431 cell lines were provided by the
Cell Resource Center for Biomedical Research, Institute of
Development, Aging and Cancer, Tohoku University. Mice
were purchased from Japan SLC and raised in accordance
with Osaka University of Pharmaceutical Sciences animal
experiment guidelines.
Based on the evidence described above, we decided
to develop a novel radiopharmaceutical for p38α activ-
ity diagnosis because, if aberrant activity of p38α could
be revealed from outside the body, it would enable early
detection of the aforementioned diseases, which would
be very useful for their diagnosis and treatment. m-YTM
and p-YTM (Fig. 1) were designed based on the struc-
ture–activity relationship of 6-amino-2-(4-fuorophenyl)-
4-benzyloxy-3-(4-pyridyl)-1H-pyrrolo[2,3-b]pyridine
(RWJ-15) [19] with p38α, and synthesized. Subsequently,
Fig. 1 Drug design of m-YTM
and p-YTM as a novel p38α
imaging probe
X=H, Y=I: m-YTM
X=I, Y=H: p-YTM
X=F, Y=H: RWJ-15
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Annals of Nuclear Medicine
Scheme 1 Synthesis of compounds 1–5
Scheme 3 Synthesis of m-YTM, p-YTM, and RWJ-15
dried over sodium sulfate, and then the solvent was distilled
of under reduced pressure. The residue was separated and
purifed by silica gel column chromatography using chloro-
form as an eluate to obtain 2a as an oily matter.
Yield 57.5%. 1H–NMR (CDCl3): 3.35 (s, 3H, CH3), 3.55
(s, 3H, OCH3), 7.14 (t, J=8.1 Hz, 1H, aromatics), 7.64 (d,
J=8.1 Hz, 1H, aromatics), 7.78 (d, J=8.1 Hz, 1H, aromat-
ics), 8.01 (s, 1H, aromatics). HRMS: m/z 290.9756. Found:
290.9756. Anal. calcd. for C9H10INO2: C, 37.14; H, 3.46; N,
4.81. Found: C, 36.94; H, 3.47; N, 4.89.
p‑Iodo‑N‑methoxy‑N‑methyl‑benzamide (2b)
Using p-iodo benzoic acid (1b), it was synthesized in a man-
ner similar to that used for synthesizing 2a.
Scheme 2 Synthesis of compounds 6–11
Yield 45.7%. 1H–NMR (CDCl3): 3.34 (s, 3H, CH3), 3.53
(s, 3H, OCH3), 7.43 (d, J=8.4 Hz, 2H, aromatics), 7.75 (d,
J=8.4 Hz, 2H, aromatics). HRMS: m/z 290.9756. Found:
290.9756. Anal. calcd. for C9H10INO2: C, 37.14; H, 3.46; N,
4.81. Found: C, 36.93; H, 3.38; N, 4.92.
Synthesis (Schemes 1, 2, 3)
m‑Iodo‑N‑methoxy‑N‑methyl‑benzamide (2a)
p‑Fluoro‑N‑methoxy‑N‑methyl‑benzamide
(2c) Using
p-fuoro benzoic acid (1c), it was synthesized in a manner
m-Iodo benzoic acid (1a) (4.0 g, 0.0162 mol) was dissolved
in thionyl chloride (6.8 mL) and then refuxed for 2 h. After
evaporating the reaction mixture under reduced pressure, the
mixture was added dropwise to a mixed solution of anhy-
drous DMF (100 mL) and N,O-dimethylhydroxylamine
hydrochloride (16.0 g, 0.1640 mol) and refuxed for 12 h.
Next, after evaporating the solvent under reduced pressure,
water (100 mL) was added to the residue and the pH of the
mixture was adjusted to 11 with 2 M NaOH. Subsequently,
after extracting with chloroform (100 mL ×2), the extract
was washed with water (100 mL×2). The organic layer was
similar to that used for synthesizing 2a.
Yield 43.2%. 1H–NMR (CDCl3): 3.36 (s, 3H, CH3), 3.54
(s, 3H, OCH3), 7.08 (d, J=8.4 Hz, 2H, aromatics), 7.74 (d,
J=8.4 Hz, 2H, aromatics).
4‑(tert‑Butyl‑dimethyl‑silanyloxymethyl)‑pyridine [4]
4-Pyridinemethanol [3] (10 g, 0.0916 mol), 4-dimethyl-
aminopyridine (5.8 g, 0.0474 mol), and tert-butyldimeth-
ylchlorosilane (15.2 g, 0.1008 mol) were dissolved in
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Annals of Nuclear Medicine
chloroform (190 mL). After triethylamine was added drop-
wise (15.2 mL, 0.1096 mol) into the solution, it was stirred
at room temperature for 3 h.
the mixture was refuxed overnight. The resulting precipitate
was removed by suction fltration, and the reaction solu-
tion was evaporated under reduced pressure. After extract-
ing with chloroform (30 mL × 2) and washing with water
(30 mL× 2), the organic layer was dried over sodium sul-
fate and evaporated under reduced pressure. The residue was
separated and purifed by silica gel column chromatography
using ether/hexane (1/1, v/v) as an eluate to obtain 8 as white
crystals.
After washing with water (100 mL× 3) and drying the
organic layer over sodium sulfate, the solvent was evaporated
under reduced pressure. The residue was separated and puri-
fed by silica gel column chromatography using chloroform/
methanol (100/1, v/v) as an eluate to obtain 4 as an oily
matter.
Yield 82.5% 1H–NMR (CDCl3): 0.20 (s, 6H, CH3), 1.05
(s, 9H, CH3), 4.82 (s, 2H, OCH2), 7.33 (d, J=6.0 Hz, 2H,
aromatics), 8.62 (d, J = 6.0 Hz, 2H, aromatics). MS: m/z
223. Found: 223.
Yield 54.2%. mp 79–81 °C, lit [20], mp 79–81 °C.
1H–NMR (CDCl3): 1.45 (t, J=7.2 Hz, 6H, CH3), 4.47 (q,
J=7.2 Hz, 4H, CH2), 5.22 (s, 2H, OCH2), 7.43 (m, 5H, aro-
matics), 7.86 (s, 2H, aromatics). MS: m/z 329. Found: 329.
2‑(tert‑Butyl‑dimethyl‑silanyloxy)‑1‑(3,4‑iodo,4‑fuorophe
nyl)‑2‑pyridin‑4‑yl‑ethanone (5a–c) Under conditions with
an argon stream and a temperature of −78 °C, 4 (2.1 g,
0.0094 mol) was dissolved in THF (40 mL) to which LDA
(7.9 mL, 0.0158 mol) was slowly added dropwise over 1 h.
Following stirring for 1 h, a solution in which 2a–c (2.3 g,
0.0079 mol) was dissolved in THF (10 mL) was added drop-
wise over 1 h and stirred for 15 min. After evaporation under
reduced pressure, a small amount of chloroform and water
was added, and the precipitated impurities were fltered.
After extracting with chloroform (100 mL×3), the product
was washed with brine (100 mL×3), dried over sodium sul-
fate, and evaporated under reduced pressure. The fraction
of 5a–c was separated by silica gel column chromatography
using chloroform as an eluate and was used without purif-
cation.
4‑Benzyloxy‑pyridine‑2,6‑dicarboxylic acid dihydrazide [9]
In ethanol (260 mL), 8 (18.3 g, 0.0555 mol) was dissolved,
and after adding hydrazine monohydrate (23 mL, 0.474 mol)
dropwise, the solution was refuxed for 2 h. The reaction
solution was cooled and the precipitate was suction fltered
to obtain 9 as white crystals.
Yield 95.6%. mp 206–208 °C, lit [20], mp 206–208.5 °C.
1H–NMR (DMSO): 4.62 (d, J=4.2 Hz, 4H, NH2), 5.35 (s,
2H, OCH2), 7.40 (m, 5H, aromatics), 7.67 (s, 2H, aromat-
ics), 10.59 (t, J = 4.2 Hz, 2H, NH). MS: m/z 301. Found:
301.
4‑Benzyloxy‑2,6‑dicarboethoxyamino pyridine [10]
In 1 M HCl solution (60 mL), 9 (3 g, 0.01 mol) was added,
and a solution in which NaNO2 (2.8 g, 0.04 mol) was dis-
solved in a mixture of DMF/water (2/1, v/v) (21 mL) was
slowly added dropwise and kept at 10–15 °C. The resulting
gummy product was removed, and benzene and brine were
added. After extraction and washing with water, the organic
layer was dried over sodium sulfate and evaporated under
reduced pressure. The residue was thoroughly dried, then
added to ethanol (110 mL), refuxed for 1 h, and then evap-
orated under reduced pressure. The product was extracted
with chloroform, washed with water, and the organic layer
was dried over sodium sulfate and evaporated under reduced
pressure. The residue was separated and purifed by silica
gel column chromatography using chloroform as an eluate
to obtain 10 as a gummy matter.
4‑Hydroxy‑pyridine‑2,6‑dicarboxylic acid diethyl ester [7]
Chelidamic acid [6] (10.0 g, 0.0546 mol) was added to
ethanol (150 mL), after which sulfuric acid (1.0 mL) was
added dropwise; the solution was then refuxed overnight.
After evaporating under reduced pressure, the reaction was
extracted with chloroform (100 mL×3), washed with water
(50 mL×2), dried over sodium sulfate, and evaporated under
reduced pressure. The residue was recrystallized with etha-
nol to obtain 7 in the form of white crystals.
1
Yield 46.2%. mp 120–121 °C. H–NMR (CDCl3): 1.33
(t, J=7.2 Hz, 6H, CH3), 4.35 (q, J=7.2 Hz, 4H, CH2), 7.57
(s, 2H, aromatics), 11.57 (broad s, 1H, OH). MS: m/z 239.
Found: 239.
Yield 44.01%. mp 118–120 °C, lit [20], mp 118–120 °C.
1H–NMR (CDCl3): 1.31 (t, J = 7.2 Hz, CH3), 4.24 (q,
J = 7.2 Hz, 4H, CH2), 5.14 (s, 2H, OCH2), 7.40 (m, 7H,
aromatics), 8.33 (s, 2H, NH). MS: m/z 359. Found: 359.
4‑Benzyloxy‑pyridine‑2,6‑dicarboxylic acid diethyl ester [8]
4‑Benzyloxy‑2,6‑diaminopyridine [11]
In THF (30 mL), 7 (1.5 g, 0.00627 mol) was dissolved, then
triethylamine (8.25 mL, 0.006 mol) was added, and the
mixture was stirred at room temperature for 30 min. Subse-
quently, benzylchloride (5.5 mL) was added dropwise and
After dissolving 10 (2.53 g, 0.007 mol) in ethanol (100 mL),
an ethanol solution of KOH (2.53 g, 0.045 mol) (40 mL) was
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Annals of Nuclear Medicine
added dropwise, and then the solution was refuxed for 12 h.
After completion of the reaction, the precipitate formed by
cooling was removed by fltration. The fltrate was adjusted
to an acidic state (pH 3) by adding 1 M HCl aqueous solu-
tion, followed by refuxing for 30 min. The solution was
then adjusted to a basic state (pH 11) by slowly adding 1 M
NaOH aqueous solution, followed by evaporation under
reduced pressure. Water (30 mL) was added to the residue,
after which the solution was extracted with ethyl acetate
(100 mL×3), dried over sodium sulfate, evaporated under
reduced pressure, and separated and purifed by silica gel
column chromatography using chloroform/methanol (10/1,
v/v) as an eluate to obtain 11 as white crystals.
6‑Amino‑2‑(4‑iodophenyl)‑4‑benzyloxy‑3‑(4‑pyridyl)‑1H‑
pyrrolo[2,3‑b]pyridine (p‑YTM) p-YTM was synthesized
using 5b by a method similar to that used for synthesizing
m-YTM.
Yield 40.5%. mp 278–280 °C. 1H–NMR (DMSO): 5.08
(s, 2H, OCH2), 5.87 (s, 2H, NH2), 6.02 (s, 1H, NH), 7.09 (d,
J=8.4 Hz, 2H, aromatics), 7.19 (d, J=3.3 Hz, 2H, aromat-
ics), 7.29 (d, J=5.7 Hz, 2H, aromatics), 7.35 (t, J=3.3 Hz,
3H, aromatics), 7.67 (d, J = 8.4 Hz, 2H, aromatics), 8.36
(d, J = 5.7 Hz, 2H, aromatics), 11.58 (s, 1H, aromatics).
HRMS: m/z 518.0604. Found: 518.0609. Anal. calcd. for
C25H19IN4O: C, 57.93; H, 3.69; N, 10.81. Found: C,57.14;
H, 3.65; N, 10.35. IR(KBr): 3470, 3320, 1621, 1586, 1419,
1232 cm−1.
Yield 58.5%. mp 166–169 °C, lit [20], mp 166–168.5 °C.
1H–NMR (CDCl3): 4.28 (broad s, 4H, NH2), 5.01 (s, 2H,
OCH2), 5.55 (s, 2H, aromatics), 7.38 (m, 5H, aromatics).
MS: m/z 215. Found: 215.
6‑Amino‑2‑(4‑fuorophenyl)‑4‑benzyloxy‑3‑(4‑pyridyl)‑1H‑
pyrrolo[2,3‑b]pyridine (RWJ‑15) RWJ-15 was synthesized
using 5c by a method similar to that for synthesizing m-
YTM.
6‑Amino‑2‑(3‑iodophenyl)‑4‑benzyloxy‑3‑(4‑pyridyl)‑1H
‑pyrrolo[2,3‑b]pyridine (m‑YTM) In DME (120 mL), 5a
(1.64 g) and 11 (0.77 g, 0.0036 mol) were dissolved, after
which sulfuric acid (0.96 mL, 0.0181 mol) was added drop-
wise and refuxed for 6 h. After evaporating the solution
under reduced pressure, an appropriate amount of metha-
nol was added, and then the reaction was neutralized with
sodium bicarbonate. After evaporating the product under
reduced pressure, extracting with ethyl acetate, and washing
with water, the organic layer was dried over sodium sulfate
and then evaporated under reduced pressure. The residue
was subjected to separation by silica gel column chromatog-
raphy using chloroform, followed by chloroform/methanol
(100/1, v/v) as eluates, and recrystallized with methanol to
obtain m-YTM as yellow crystals.
Yield 42.3%. mp 275–276 °C, lit [21], mp 275–276 °C.
HRMS: m/z 410.1543. Found: 410.1532.
Radiosynthesis (Scheme 4)
6‑Amino‑2‑(3‑tributylstanyl‑phenyl)‑4‑benzyloxy‑3‑(4‑py
ridyl)‑1H‑pyrrolo[2,3‑b]pyridine [12]
m-YTM (0.07 g, 0.14 mmol), bis(tributyltin) (0.21 mL,
0.42 mmol), and tetrakis (triphenylphosphine) palladium(0)
(0.0085 g, 0.0074 mmol) were refluxed in anhydrous
1,4-dioxane (12 mL) overnight. After cooling, the reaction
was fltered through Celite, and the fltrate was concentrated
under reduced pressure. The residue was separated and puri-
fed by silica gel column chromatography using ethyl ace-
tate/hexane (10/1, v/v) as an eluate to obtain 12 as yellow
crystals.
Yield 44.1%. mp 296–298 °C. 1H–NMR (DMSO): 5.02
(s, 2H, OCH2), 5.84 (s, 2H, NH2), 5.96 (s, 1H, NH), 7.03
(t, J=7.8 Hz, 1H, aromatics), 7.24 (d, J=5.7 Hz, 2H, aro-
matics), 7.27 (m, 6H, aromatics), 7.54 (d, J= 7.8 Hz, 1H,
aromatics), 7.62 (s, 1H, aromatics), 8.33 (d, J=5.7 Hz, 2H,
aromatics), 11.54 (s, 1H, aromatics). HRMS: m/z 518.0604.
Found: 518.0602. Anal. calcd. for C25H19IN4O: C, 57.93; H,
3.69; N, 10.81. Found: C, 57.23; H, 3.73; N, 10.53. IR(KBr):
3470, 3311, 1622, 1588, 1420, 1233 cm−1.
1
Yield 31.4%. mp 160–165 °C. H–NMR (CDCl3):
0.86–1.47 (m, 27H, Bu3), 4.01 (broad s, 2H, NH2), 5.05 (s,
2H, OCH2), 5.84 (s, 1H, NH), 7.29 (m, 11H, aromatics),
8.32 (d, J=6.0 Hz, 2H, aromatics), 10.61 (s, 1H, aromatics).
MS: m/z 682. Found: 682.
Scheme 4 Radiosynthesis of
[
125I]m-YTM
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Annals of Nuclear Medicine
6‑Amino‑2‑(3‑[125I]iodophenyl)‑4‑benzyloxy‑3‑(4‑pyridyl)
‑1H‑pyrrolo[2,3‑b]pyridine ([125I]m‑YTM) In a sealed vial,
99% ethanol (25 μL), 0.1 M HCl solution (25 μL), [125I]
sodium iodide (1 μL, 3.7 MBq), and 30% w/v hydrogen
peroxide solution (10 μL), were sequentially added to pre-
cursor 12 (25 μL, 1 mg/ml methanol solution); the mixture
was then allowed to react at room temperature for 10 min.
A target compound was purifed by HPLC using metha-
nol/0.01 M phosphate bufer (80/20, v/v) as a mobile
phase followed by radiochemical purity analysis.
binding amounts with 30 µL of 0.1% Triton X-100 1%
DMSO solution containing sample compounds (m-YTM,
RWJ-15, SB203580, SB202190, SB202474, PD98059,
U0126, SP600125, Go6985, and bisindolylmaleimide I at a
fnal concentration of 1 μM) were measured and expressed
as a percentage relative to the control.
Binding afnity of [125I]m‑YTM
To a 60-µL suspension of p38α (active or inactive form)
(25 ng) in 0.1% Triton X-100 ADB (pH 7.2), 20 µL of
0.1% Triton X-100 and 1% DMSO solution with or with-
out SB203580 (fnal concentration of 10 μM) was added.
Further, a mixture containing 10 µL of m-YTM (fnal con-
centration of 1–100 nM) and 10 µL of 0.1% Triton X-100
containing [125I]m-YTM (1.67 kBq) was added to make a
total volume of 100 µL, and the resulting solution was incu-
bated at 30 °C for 30 min. After the reaction, the solution
was suction fltered using a glass flter and washed six times
with 3 mL of ice-cold 0.2 mM phosphate bufer (pH 7.2).
Subsequently, the radioactivity of the flter was measured
by a gamma counter to determine the levels of total binding
and nonspecifc binding of [125I]m-YTM to p38α (active or
inactive form). The specifc binding amount was determined
by subtracting the nonspecifc binding amount from the total
binding amount at various concentrations. Kd and Bmax
were calculated by Scatchard analysis on the basis of the
obtained specifc binding curves.
Measurement of the inhibition of p38 phosphoryla‑
tion by m, p‑YTM
Assay dilution bufer (ADB: 20 mM MOPS-Na, 25 mM
β-glycerol phosphate, 5 mM EGTA, 1 mM sodium ortho-
vanadate, and 1 mM dithiothreitol; pH 7.2), Mg/ATP cock-
tail (500 μM cold ATP and 75 mM magnesium chloride in
ADB), and [γ-32P]ATP (0.2 μCi/μL in Mg/ATP cocktail)
were prepared.
Myelin basic protein (20 μg) was suspended in the ADB
(10 μL), and 10 µL of 5% DMSO 0.01 M HCl solution
containing a test compound (m-YTM and RWJ-15: fnal
concentration of 0.1 nM–1 μM; SB203580 and p-YTM:
1 nM–10 μM) was added. p38α (25 ng in 10 μL of ADB)
and [γ-32P]ATP (10 μL) were sequentially added and incu-
bated at 30 °C for 30 min. After completion of the reac-
tion, the reaction mixture was immediately spotted on P81
phosphocellulose paper. One minute later, the paper was
washed three times with 0.75% phosphoric acid aqueous
solution, once with acetone, and then dried well. Radio-
activity was measured by a liquid scintillation counter
after adding 10 mL of the Clear-sol I scintillator (Nacalai
Tesque). Ten microliters of 5% DMSO 0.01 M HCl solu-
tion containing no sample was used as a control. The phos-
phorylation inhibition of each compound was expressed as
a percentage relative to the control.
Cellular uptake study
Cancer cell lines were inoculated at 1.0 × 106 cells per
75 cm2 dish and incubated in a CO2 incubator (5% CO2 at
37 °C, humidifed), in accordance with a standard method.
The media used were DMEM for A-375, B-16, C-6, and
A-431; and RPMI 1640 for SLC, ME-180, and PC-3. A-431
was cultured with 5% FCS, and all others were cultured with
medium containing 10% FCS supplemented with penicil-
lin–streptomycin solution.
A-375 cells (1.0 × 106) were suspended in DMEM
medium (5% FCS, 800 μL), and 100 µL of DMEM solu-
tion containing 5% DMSO with or without various sam-
ple compounds (m-YTM, RWJ-15, SB203580, SB202190,
SB202474, PD98059, U0126, SP600125, Go6985, and
bisindolylmaleimide I at a fnal concentration of 1 μM) was
added to the suspension. To the mixed solution, 100 μL
of 5% DMSO and 0.01 M HCl containing [125I]m-YTM
(1.08 kBq) was added to a total volume of 1 mL and incu-
bated at 37 °C for 1 h. After the reaction, the solution was
suction fltered using a glass flter and washed eight times
with 3 mL ice-cold 0.2 mM phosphate bufer (pH 7.2). The
amount of radioactivity accumulated in the cells over time
was examined by measuring the radioactivity of the flter.
Binding of [125I]m‑YTM to the recombinant p38α
Sixty microliters of p38α (25 ng) suspension in ADB (pH
7.2) containing 0.1% Triton X-100 (Nacalai Tesque), 30 µL
of 0.1% Triton X-100 1% DMSO solution, and 10 µL of
[
125I]m-YTM in 0.1% Triton X-100 solution (1.67 kBq)
were sequentially added to a total volume of 100 µL and
incubated at 30 °C for 30 min. After the reaction, the solu-
tion was suction fltered using a glass flter and washed
six times with 3 mL of ice-cold 0.2 mM phosphate bufer
(pH 7.2). Subsequently, the radioactivity of the flter was
measured by a gamma counter to measure the total amount
of [125I]m-YTM bound to p38. In a similar manner, the
1 3

Annals of Nuclear Medicine
The accumulation of radioactivity was expressed as a per-
centage relative to the total binding amount of the control.
Various cancer cells (1.0 × 106) were suspended in
medium (5% FCS, 800 μL), to which 100 µL of DMEM
solution containing 5% DMSO with or without SB203580
(fnal concentration of 10 μM) was added. To this mix-
ture, 100 μL of 5% DMSO and 0.01 M HCl containing
Results and discussion
Synthesis and radiosynthesis (Schemes 1, 2, 3, 4)
As shown in Fig. 1, in this study, we designed m-YTM and
p-YTM based on a previous report of the structure–activity
relationship which indicated possible introduction sites of
a halogen atom in derivatives of RWJ-15 [19, 22]. Since
the introduction of a large halogen atom on the m posi-
tion reportedly contributed to high inhibitory potency
toward p38α while RWJ-15 has a fuorine atom on the
p position, we decided to test the possibilities of both m-
YTM and p-YTM as a p38α imaging probe. In accordance
with Schemes 1, 2, and 3 constructed following the previ-
ous report of RWJ-15 synthesis [19, 21, 23] with slight
modifcations, m-YTM, p-YTM, and RWJ-15 were suc-
cessfully synthesized. On the other hand, o-YTM was not
obtained in our preliminary synthetic trial probably due to
a steric hindrance during a ring formation reaction shown
in Scheme 3. Radioactive iodine labeling of m-YTM was
performed by an organotin-radioactive iodine exchange
reaction (Scheme 4). Based on the analytical HPLC fol-
lowing the labeling reaction, a single radioactive peak
was observed at 19.1 min, consistent with the retention
time of m-YTM; therefore, the peak was confrmed as
[
125I]m-YTM (1.08 kBq) was added to make a total volume
of 1 mL, which was then incubated at 37 °C for 1 h. After the
reaction, the solution was suction fltered using a glass flter
and washed eight times with 3 mL of ice-cold 0.2 mM phos-
phate bufer (pH 7.2). Subsequently, the amount of radioac-
tivity accumulated in the cells was examined by measuring
the radioactivity of the flter using a gamma counter.
In vivo biodistribution study
A suspension of B-16 cells (4×106 cells in 200 μL) was sub-
cutaneously injected into the thigh of 4-week-old BALB/c-
nu male nude mice (20–25 g) and raised for 2 weeks.
[
125I]m-YTM (100 μL, 37 kBq in saline) was injected into
the B-16 tumor-bearing mice via the tail vein. The mice
were sacrifced over time and blood was collected; next, the
tumor and each organ were excised to measure their weight
and radioactivity. Radioactivity accumulation in each tissue
was expressed as a percentage of the total dose per gram of
tissue (% dose/g tissue).
[
125I]m-YTM. The radiochemical yield was 95.8%, the
radiochemical purity was 99% or higher, and the molar
activity was approximately 74.3 TBq/mmol.
Various tumor-bearing mice were generated to compare
the accumulation dynamics of the compound in cancer tis-
sues. Various cancer cell lines were suspended in a medium
used for culture, and the numbers of cells per 200 µL of cell
suspension were adjusted as follows: 5.0× 106 for A-375,
B-16, and SLC; and 1.0 × 107 for C-6, ME-180, A-431,
and PC-3. A cell suspension of 200 µL was subcutaneously
injected into the thigh of 4-week-old BALB/c-nu male
nude mice (20–25 g) and raised for 2 weeks for the A-375,
B-16, and SLC cell lines, 3 weeks for C-6 and A-431 cell
lines, or 4 weeks for ME-180 and PC-3 cell lines, and mice
with a tumor of approximately 1×1 cm were used for the
experiment.
Measurement of the inhibition of p38 phosphoryla‑
tion by m, p‑YTM
The calculated IC50 value was 4.8 nM for m-YTM, which
corresponds to inhibitory activity comparable to that of
the base compound RWJ-15 (2.6 nM). It was confrmed
m-YTM
RWJ-15
Statistical analysis
SB203580
SB202190
SB202474
PD98059
Data are presented as the mean standard deviation (S.D.).
Statistical analysis was performed using the unpaired t test.
A two-tailed value of p<0.05 was considered statistically
signifcant.
U0126
SP600125
Go6983
Bisindolylmaleimide I
0
20
40
60
80
100
120
[
125I]m-YTM bound (% of control)
Fig. 2 Binding of [125I]m-YTM to recombinant p38α with treatment
of several inhibitors
1 3

Annals of Nuclear Medicine
that the inhibitory potency of m-YTM was about 13
times higher than a representative p38-selective inhibi-
tor, SB203580 (63 nM). Meanwhile, the IC50 of p-YTM
was 690 nM, which indicates lower potency than those of
RWJ-15 and m-YTM; therefore, the results are consistent
with that of a previous report [19, 22], which states that
the inhibition of a derivative having a large halogen intro-
duced at the m position is greater than that with a large
halogen at the p position. Thus, it was proved that the
method of drug design in this study was valid.
upon pretreatment with p38-selective inhibitors such as
RWJ-15, SB203580, and SB202190. Alternatively, no
change was noted in the binding amount of [125I]m-YTM
when pretreated with a compound having no inhibitory
activity toward p38, such as SB202474, PD98059, U0126,
SP600125, Go6983, and bisindolylmaleimide I. Conse-
quently, it was suggested that [125I]m-YTM selectively
binds to the recombinant p38α.
The binding afnity of [125I]m‑YTM (Fig. 3)
Binding of [125I]m‑YTM to the recombinant p38α
The cascade by which p38α transmits the signal from
upstream into the nucleus comprises two stages, and p38α
has two ATP binding pockets [24, 25]. Hence, two possi-
bilities of p38α, activation site and autophosphorylation
site, can be considered as candidates for the binding site of
m-YTM that exhibits inhibitory activity toward p38α. We
therefore performed a binding saturation assay using the
active or inactive form of p38α to elucidate the binding site
for m-YTM. The active form of p38α is a phosphorylated
p38α which has the two active ATP binding pockets, activa-
tion site and autophosphorylation site, while the inactive
form of p38α is a naïve p38α which has only one active
ATP binding pocket, that is, activation site. The two forms
of p38α protein could not be interconverted in the reaction
solutions due to lack of a corresponding kinase. The specifc
binding amount of [125I]m-YTM was calculated by using the
(Fig. 2)
Because m-YTM exhibited excellent potential to inhibit
p38 phosphorylation, the binding of the compound to
p38α was examined. Human recombinant p38α was
used as an enzyme sample. The amount of binding of
[
125I]m-YTM upon pretreatment with various inhibi-
tors [p38-selective inhibitors: RWJ-15, SB203580, and
SB202190; p38 inactive analog: SB202474; MEK inhibi-
tors: PD98059 and U0126; c-Jun N-terminal kinase (JNK)
inhibitor: SP600125; Protein kinase C (PKC) inhibitors:
Go6983 and bisindolylmaleimide I] was determined and
expressed as a percentage relative to that of the control.
The results indicated that the amount of [125I]m-YTM
binding to p38 was significantly lower than for the control
Fig. 3 Binding saturation assay
of [125I]m-YTM using active (A)
and inactive (B) forms of p38α
6
5
4
3
2
1
0
0.20
0.15
0.10
0.05
0
(A)
0
20
40
60
80
100
0
1
2
3
4
5
Drug concentration (nM)
Bound (nmol/mg protein)
6
0.20
(B)
5
4
3
2
1
0
0.15
0.10
0.05
0
0
20
40
60
80
100
0
1
2
3
4
5
Drug concentration (nM)
Bound(nmol/mg protein)
1 3

Annals of Nuclear Medicine
Table 1 Radioactivity biodistribution after intravenous administration
of [125I]m-YTM in B-16 cell-implanted model mice
m-YTM
RWJ-15
SB203580
SB202190
SB202474
PD98059
Time after administration (hours)
1
6
12
24
Blood
Pancreas
Spleen
Stomach
Liver
Kidney
Heart
Lung
Brain
Muscle
Tumor
0.31 0.07
0.06 0.02
0.03 0.01
0.01 0.01
0.03 0.02
0.12 0.14
0.30 0.07
0.10 0.02
0.00 0.00
0.07 0.03
0.00 0.00
0.01 0.02
0.98 0.15
0.02 0.00
0.00 0.00
0.08 0.08
0.07 0.06
0.32 0.08
0.13 0.02
0.01 0.01
0.10 0.04
0.00 0.00
0.01 0.01
1.00 0.08
U0126
0.86 0.13
1.24 0.13
2.82 1.57
2.95 0.36
3.42 0.43
0.43 0.06
0.84 0.10
0.05 0.00
0.59 0.21
1.79 0.20
0.07 0.06
0.07 0.07
0.32 0.14
0.98 0.33
0.21 0.11
0.02 0.02
0.16 0.04
0.00 0.00
0.02 0.01
1.04 0.15
SP600125
Go6983
Bisindolylmaleimide I
0
20
40
60
80
100
120
140
Radioactivity accumulation (% of control)
Fig. 4 Cellular uptake study of [125I]m-YTM in A-375 cells with
treatment of several inhibitors
8
Data are presented as % injected dose per gram. Each value repre-
sents the mean S.D. for four animals at each interval
Control
SB203580 treatment
6
*p<0.05 vs. Control
Table 2 Tumor/blood and tumor/muscle ratios obtained from bio-
distribution studies of [125I]m-YTM in a variety of tumor-implanted
model mice
4
*
2
Time after administration (hours)
*
C-6
*
*
*
*
*
Tumor/muscle ratio
Tumor/blood ratio
0
A-375
B-16
SLC ME-180 PC-3
A-431
1
24
1
24
Fig. 5 Cellular uptake study of [125I]m-YTM in several cancer cells
A-375
B-16
C-6
1.52 0.25
3.03 0.28
1.42 0.81
1.22 0.24
1.06 0.24
1.17 0.09
1.04 0.16
1.47 0.38
47.6 7.29
0.92 0.06
5.88 0.83
1.66 0.24
2.43 1.28
7.11 1.12
3.49 0.41
6.56 1.32
3.54 1.84
3.15 0.59
1.38 0.47
1.98 0.35
2.53 0.15
0.91 0.38
213 27.6
1.11 0.27
1.87 0.43
1.40 1.41
0.84 0.79
3.24 0.85
with treatment of SB203580
SLC
active or inactive form of human recombinant p38α; next,
the dissociation constant Kd and maximum binding number
Bmax were calculated using the Scatchard analysis method.
The Kd value and the Bmax of m-YTM for activated p38α
were 26.1 nM and 4.3 nmol/mg protein, respectively; those
for inactivated p38α were 29.6 nM and 4.5 nmol/mg protein,
respectively. m-YTM showed similar levels of binding afni-
ties for active and inactive forms. Because the Hill coef-
cient was one for both, it was suggested that m-YTM has one
binding site and there is no interaction with other binding
sites. If m-YTM binds only to the active form, it would be
considered to bind to an autophosphorylation site; if it binds
to both active and inactive forms, it would be considered to
bind to the p38α activation site. Therefore, it was suggested
that the binding site of m-YTM is the p38α activation site,
as in the case using the p38α-selective inhibitor SB203580
[26, 27]. Accordingly, the amount of m-YTM accumulated
in cancer cells was considered to represent the amount of
p38α present in the cells.
ME-180
PC-3
A-431
Each value represents the mean S.D. for four animals at each inter-
val
Cellular uptake study (Figs. 4–5)
The cellular uptake of [125I]m-YTM was examined using
A-375 cells. The efects of various inhibitors were inves-
tigated by pretreating the cells and measuring the uptake
of [125I]m-YTM, which was determined as a percentage
relative to that of the control. It was shown that the uptake
of [125I]m-YTM in A-375 cells was signifcantly decreased
only with the p38α-selective inhibitors relative to that of
the control, whereas the pretreatment with other inhibi-
tors had no efect on the uptake amount. Accordingly, it
was suggested that [125I]m-YTM incorporated in the cancer
cells selectively binds to p38α and is retained in the cells.
1 3

Annals of Nuclear Medicine
The tumor to muscle ratio of radioactivity accumulation
was 3.1 at 1 h post-administration, then increased over time,
and reached a high value of more than 100 at 12 h post-
administration. Similarly, the tumor to blood ratio was 5.8 at
1 h post-administration, and then increased further, reaching
50 at 24 h post-administration. The tumor to liver and tumor
to kidney ratios were 3.2 and 9.5 at 12 h post-administration,
respectively. The tumor to lung ratio was 14.2 at 12 h post-
administration, and the tumor to stomach ratio was as high
as 15.1 at 24 h post-administration.
2.5
2.0
1.5
1.0
0.5
0
1 hr
24 hr
The accumulation of [125I]m-YTM in the tumor was
examined using various tumor-bearing mice (Table 2,
Fig. 6). At 1 h post-administration, the accumulation ranged
from 0.55 to 1.84% ID/g. In B-16-bearing mice, the accumu-
lation was 1.00% ID/g at 24 h post-administration; however,
it was 0.01%–0.05% ID/g in other cancer tissues. The reason
for the diference in the radioactivity accumulations between
B-16 and other tumors remained unsolved, but somehow
might be attributed to binding with an indeterminate factor
diferent from those evaluated in Fig. 4, and not to non-
specifc retention. This is because the contribution of p38α
specifc accumulation on B-16 radioactivity was estimated
approximately a half from the cellular uptake study in Fig. 5.
Therefore, the elucidation of this issue and further quanti-
tative assessment of the radioactivity accumulation profle
corresponding to the expression and activity levels of p38α
would be demanded in future. Nevertheless, the ratios of
accumulation in the tumor to that in blood and in tumor
to that in muscle were≥1 at 1 h post-administration in all
tumors tested; therefore, [125I]m-YTM seems to be applica-
ble to various types of cancer. Cumulatively, these fndings
suggest that [125I]m-YTM is an efective p38α imaging probe
that can be employed for diferent types of tumor.
A-375
B-16
C-6
SLC ME-180 PC-3 A-431
Fig. 6 Radioactivity accumulation in the tumor after intravenous
administration of [125I]m-YTM in a variety of tumor-implanted model
mice
Then, the applicability of [125I]m-YTM to various can-
cers was investigated. The cancer cell lines used were
A-375 human malignant melanoma, B-16 mouse malignant
melanoma, C6 rat brain tumor, SLC human lung tumor,
ME-180 human cervical carcinoma, PC-3 human prostate
cancer, and A-431 human squamous cell carcinoma. Using
these cell lines, the uptake of [125I]m-YTM was examined.
The results revealed the uptake of [125I]m-YTM in vari-
ous cancer cells, which was signifcantly reduced by the
p38α-selective inhibitor SB203580. Accordingly, it was
suggested that [125I]m-YTM is retained inside various can-
cer cells through the binding to p38α.
In vivo biodistribution study (Tables 1–2, Fig. 6)
p38α is a ubiquitous protein kinase activated in response
to external stimuli and is closely implicated in the modula-
tion of multiple cellular processes such as apoptosis [5, 6].
Also, p38α is recognized as a potential therapeutic target
because of contribution on the progression of tumors [17,
18]. Therefore, [123I]m-YTM can be an efective imaging
probe for usefulness evaluation of such p38α specifc tar-
geting agents as well as therapeutic agents targeting other
kinases or receptors locating in the upstream of the p38α
pathway in view of the fnal therapeutic efects. In addition,
In a study of pharmacokinetics of [125I]m-YTM over time
in B-16 tumor-bearing mice (Table 1), the radioactive accu-
mulation in the tumor was 1.79% ID/g 1 h after adminis-
tration, and was retained in the tumor for a long time; for
example, even after 6, 12, and 24 h, it showed high accu-
mulation of 1.04, 0.98, and 1.00% ID/g, respectively. Con-
versely, the liver and kidney showed high accumulation of
2.95 and 3.42% ID/g 1 h after the administration; however,
the amount was decreased to lower than that in the tumor
6 h post-administration, and then continued to decrease over
time. The accumulation in other organs was lower than that
in the tumor starting from 1 h post-administration and it con-
tinued to decrease further thereafter. In particular, the accu-
mulation was decreased to the background level 12 h post-
administration and later, which confrms that [125I]m-YTM
has excellent excretion. In addition, it was suggested that
[
123I]m-YTM would have a great potential for the diagnosis
of a broad range of diseases such as cancers and ischemic
diseases that activate the p38α pathway.
Funding This work was partly supported by a Grant-in-Aid for Sci-
entifc Research from the Japan Society for the Promotion of Science
(No. 12770512). There are no conficts of interest.
[
125I]m-YTM is stably present in the body because low radio-
activity accumulation in the stomach was observed, which
is an index of deiodination.
1 3

Annals of Nuclear Medicine
by phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA.
1996;93(18):9641–4.
References
15. Han Q, Leng J, Bian D, Mahanivong C, Carpenter KA, Pan ZK,
et al. Rac1-MKK3-p38-MAPKAPK2 pathway promotes urokinase
plasminogen activator mRNA stability in invasive breast cancer
cells. J Biol Chem. 2002;277(50):48379–85.
1. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase and
MAP kinase. Curr Opin Genet Dev. 1994;4(1):82–9.
2. Nishida E, Gotoh Y. The MAP kinase cascade is essential for
diverse signal transduction pathways. Trends Biochem Sci.
1993;18(4):128–31.
16. Huang S, New L, Pan Z, Han J, Nemerow GR. Urokinase plas-
minogen activator/urokinase-specifc surface receptor expression
and matrix invasion by breast cancer cells requires constitutive
p38alpha mitogen-activated protein kinase activity. J Biol Chem.
2000;275(16):12266–72.
3. Kyriakis JM, Avruch J. Protein kinase cascades activated by stress
and infammatory cytokines. BioEssays. 1996;18(7):567–77.
4. New L, Han J. The p38 MAP kinase pathway and its biological
function. Trends Cardiovasc Med. 1998;8(5):220–8.
17. Igea A, Nebreda AR. The stress kinase p38alpha as a target for
cancer therapy. Cancer Res. 2015;75(19):3997–4002.
18. Koul HK, Pal M, Koul S. Role of p38 MAP kinase signal trans-
duction in solid tumors. Genes Cancer. 2013;4(9–10):342–59.
19. Henry JR, Rupert KC, Dodd JH, Turchi IJ, Wadsworth SA, Cav-
ender DE, et al. Potent inhibitors of the MAP kinase p38. Bioorg
Med Chem Lett. 1998;8(23):3335–40.
5. Lo U, Selvaraj V, Plane JM, Chechneva OV, Otsu K, Deng W.
p38alpha (MAPK14) critically regulates the immunological
response and the production of specifc cytokines and chemokines
in astrocytes. Sci Rep. 2014;4:7405.
6. Sui X, Kong N, Ye L, Han W, Zhou J, Zhang Q, et al. p38 and
JNK MAPK pathways control the balance of apoptosis and
autophagy in response to chemotherapeutic agents. Cancer Lett.
2014;344(2):174–9.
20. Markees DG, Dewey VC, Kidder GW. The synthesis and bio-
logical activity of substituted 2,6-diaminopyridines. J Med Chem.
1968;11(1):126–9.
7. Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, Fuller SJ,
Ben-Levy R, Ashworth A, et al. Stimulation of the stress-acti-
vated mitogen-activated protein kinase subfamilies in perfused
heart. p38/RK mitogen-activated protein kinases and c-Jun N-ter-
minal kinases are activated by ischemia/reperfusion. Circ Res.
1996;79(2):162–73.
21. Henry JR, Rupert KC, Dodd JH, Turchi IJ, Wadsworth SA,
Cavender DE, et al. 6-Amino-2-(4-fuorophenyl)-4-methoxy-3-
(4-pyridyl)-1H-pyrrolo[2, 3-b]pyridine (RWJ 68354): a potent and
selective p38 kinase inhibitor. J Med Chem. 1998;41(22):4196–8.
22. Wilson KP, McCafrey PG, Hsiao K, Pazhanisamy S, Galullo
V, Bemis GW, et al. The structural basis for the specifcity of
pyridinylimidazole inhibitors of p38 MAP kinase. Chem Biol.
1997;4(6):423–31.
8. Ma XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher
TA, et al. Inhibition of p38 mitogen-activated protein kinase
decreases cardiomyocyte apoptosis and improves cardiac func-
tion after myocardial ischemia and reperfusion. Circulation.
1999;99(13):1685–91.
23. Henry JR, Dodd JH. Synthesis of RWJ 68354: a potent inhibitor
of the MAP kinase p38. Tetrahedron Lett. 1998;39(48):8763–4.
24. Cohen P. The search for physiological substrates of MAP and SAP
kinases in mammalian cells. Trends Cell Biol. 1997;7(9):353–61.
25. Moriguchi T, Gotoh Y, Nishida E. Roles of the MAP kinase cas-
cade in vertebrates. Adv Pharmacol. 1996;36:121–37.
26. Han H, Wang H, Long H, Nattel S, Wang Z. Oxidative pre-
conditioning and apoptosis in L-cells. Roles of protein
kinase B and mitogen-activated protein kinases. J Biol Chem.
2001;276(28):26357–64.
9. Yue TL, Wang C, Gu JL, Ma XL, Kumar S, Lee JC, et al. Inhibi-
tion of extracellular signal-regulated kinase enhances Ischemia/
Reoxygenation-induced apoptosis in cultured cardiac myocytes
and exaggerates reperfusion injury in isolated perfused heart. Circ
Res. 2000;86(6):692–9.
10. Sugino T, Nozaki K, Takagi Y, Hattori I, Hashimoto N, Morigu-
chi T, et al. Activation of mitogen-activated protein kinases after
transient forebrain ischemia in gerbil hippocampus. J Neurosci.
2000;20(12):4506–14.
27. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Oppos-
ing efects of ERK and JNK-p38 MAP kinases on apoptosis. Sci-
ence. 1995;270(5240):1326–31.
11. Walton KM, DiRocco R, Bartlett BA, Koury E, Marcy VR, Jarvis
B, et al. Activation of p38MAPK in microglia after ischemia. J
Neurochem. 1998;70(4):1764–7.
12. Barancik M, Htun P, Strohm C, Kilian S, Schaper W. Inhibition
of the cardiac p38-MAPK pathway by SB203580 delays ischemic
cell death. J Cardiovasc Pharmacol. 2000;35(3):474–83.
13. Mackay K, Mochly-Rosen D. An inhibitor of p38 mitogen-acti-
vated protein kinase protects neonatal cardiac myocytes from
ischemia. J Biol Chem. 1999;274(10):6272–9.
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14. Symes K, Mercola M. Embryonic mesoderm cells spread
in response to platelet-derived growth factor and signaling
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