Synthesis of radioiodinated probes to evaluate the biodistribution of a potent TRPC3 inhibitor

Article abstract of DOI:10.1039/c6md00023a

The transient receptor potential canonical 3 (TRPC3) channel is a member of the TRPC family that contributes to the entry of Ca²⁺ through the plasma membrane or modulates the driving force for Ca²⁺ entry channels. The pyrazole compound Pyr3 has recently been reported to be a selective TRPC3 inhibitor and has become an attractive research tool and therapeutic agent for the treatment of heart failure. However, the in vivo characteristics of Pyr3 have not been investigated. To monitor the fate of Pyr3 in vivo, we designed and synthesized a radioiodinated Pyr3 probe ([¹²⁵I]I-Pyr3) by introducing radioiodine at the 2-position of the central phenyl ring of Pyr3. I-Pyr3 was shown to have direct TRPC3 inhibition activity similar to that of Pyr3 in TRPC3-overexpressing HEK293 cells. Using the tributyltin derivative as a radioiodination precursor, [¹²⁵I]I-Pyr3 was successfully prepared with high radiochemical purity. Biodistribution studies of [¹²⁵I]I-Pyr3 and [¹²⁵I]I-Pyr8 (the esterolysis product of [¹²⁵I]I-Pyr3) indicated high uptake of intact [¹²⁵I]I-Pyr3 in the lung and rapid metabolism to [¹²⁵I]I-Pyr8. These findings provide useful information about the in vivo kinetics of the selective TRPC inhibitor Pyr3.

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RESEARCH ARTICLE
Synthesis of radioiodinated probes to evaluate the
biodistribution of a potent TRPC3 inhibitor†‡
Cite this: DOI: 10.1039/c6md00023a
Masayori Hagimori,a Takahiro Murakami,a Kinue Shimizu,b Motohiro Nishida,bc
b
a
*
Takashi Ohshima and Takahiro Mukai
The transient receptor potential canonical 3 (TRPC3) channel is a member of the TRPC family that contrib-
utes to the entry of Ca2+ through the plasma membrane or modulates the driving force for Ca2+ entry
channels. The pyrazole compound Pyr3 has recently been reported to be a selective TRPC3 inhibitor and
has become an attractive research tool and therapeutic agent for the treatment of heart failure. However,
the in vivo characteristics of Pyr3 have not been investigated. To monitor the fate of Pyr3 in vivo, we
designed and synthesized a radioiodinated Pyr3 probe ([125I]I-Pyr3) by introducing radioiodine at the 2-po-
sition of the central phenyl ring of Pyr3. I-Pyr3 was shown to have direct TRPC3 inhibition activity similar to
that of Pyr3 in TRPC3-overexpressing HEK293 cells. Using the tributyltin derivative as a radioiodination pre-
cursor, [125I]I-Pyr3 was successfully prepared with high radiochemical purity. Biodistribution studies of [125I]
I-Pyr3 and [125I]I-Pyr8 (the esterolysis product of [125I]I-Pyr3) indicated high uptake of intact [125I]I-Pyr3 in
the lung and rapid metabolism to [125I]I-Pyr8. These findings provide useful information about the in vivo
kinetics of the selective TRPC inhibitor Pyr3.
Received 12th January 2016,
Accepted 12th March 2016
DOI: 10.1039/c6md00023a
www.rsc.org/medchemcomm
nels.6 It has been reported that TPRC channels are closely re-
lated to heart diseases, including cardiac hypertrophy and
1 Introduction
Changes in the intracellular concentration of the calcium ion
([Ca2+]i) modulate a wide range of cellular processes, includ-
ing secretion, gene expression, proliferation, and cell
death.1–4 Although [Ca2+]i is normally maintained in the 10–
100 nM range, it is increased by the release of Ca2+ from
intracellular stores and the endoplasmic reticulum (ER) on
activation. In addition, [Ca2+]i is increased by the influx of
Ca2+ across the plasma membrane in response to extracellular
stimulation.5 Abnormal changes in [Ca2+]i influence intracel-
lular Ca2+ homeostasis and can cause diseases.5
fibrosis, and cancer.7–10 TRPC channels can be divided into
seven isoforms (TRPC1–TRPC7), and TRPC1 and TRPC3–7
are broadly expressed in mammals.11,12 TRPC channels are
activated in response to membrane receptors linked to phos-
pholipase C (PLC), including PLCβ and PLCγ. TRPC3, TRPC6,
and TRPC7 are directly activated by PLC-derived diac-
ylglycerol (DAG).6
To elucidate the functions of a TRPC channel, several
small molecules, such as SKF 96365, LOE908, and 2-APB, have
been evaluated as TRP inhibitors.13,14 However, the roles
played by the TRPC channels have not been adequately char-
acterized because these inhibitors exhibit low selectivity to-
ward TRPC channels. BTP2 has been reported to be a TRPC
channel inhibitor that does not influence mitochondrial Ca2+
signaling, ER Ca2+ release, or K+ channels in T-cells. However,
BTP2 lacks TRPC subtype selectivity.15–17 The pyrazole com-
pound Pyr3 has been recently reported to be a selective TRPC3
inhibitor that directly inhibits the TRPC3 channel by binding
to its extracellular side.18 Pyr3 regulated the TRPC3-mediated
Ca2+ influx in B lymphocytes and eliminated the B cell
receptor-induced Ca2+ oscillation. Pyr3 also inhibited pressure
overload-induced cardiac hypertrophy in mice with transverse
aortic constriction.18 Furthermore, in an in vitro model of
stent-induced arterial injury, Pyr3 prevented arterial injury by
suppressing agonist-induced Ca2+ entry into vascular smooth
muscles.19
The transient receptor potential canonical (TRPC) channel
is a subfamily of TRP channels and contributes to changes in
[Ca2+]i by serving as a Ca2+ entry pathway in the plasma mem-
brane or by modulating the driving force for Ca2+ entry chan-
a Kobe Pharmaceutical University, 4-19-1 Motoyamakita Machi, Higashinada-ku,
Kobe 658-8558, Japan. E-mail: tmukai@kobepharma-u.ac.jp;
Fax: +81 78 4417541; Tel: +81 78 4417539
b Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan
c Okazaki Institute for Integrative Bioscience (National Institute for Physiological
Sciences), National Institutes of Natural Sciences, 5-1 Higashiyama, Okazaki,
Aichi 444-8787, Japan
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6md00023a
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These findings suggest that Pyr3 could be used as a re-
search tool and a therapeutic agent for the treatment of heart
failure. Nevertheless, little is known about the in vivo charac-
teristics of Pyr3, including its distribution, metabolism, and
elimination. In addition, because the ester moiety of Pyr3 is
easily hydrolyzed, Pyr3 is converted to Pyr8, the carboxylic
acid derivative, which shows little TRPC3 inhibition.
Radiolabeled probes are widely used to study the in vivo char-
acteristics of drugs. Iodine radioisotopes (123I, 125I, and 131I)
3
have higher specific activities than β-emitters, such as H or
14C, and are commonly used in biological assay and in vivo
imaging. 125I has a relatively long half-life and emits low en-
ergy photons, thus the pyrazole compounds were labeled with
125I. In this study, we synthesized the radiolabeled probes
([125I]I-Pyr3 and [125I]I-Pyr8) (Fig. 1) and used them to moni-
tor the fate of Pyr3 in vivo.
Scheme 1 Syntheses of I-Pyr3 and I-Pyr8. Reagents and conditions:
(a) NH2NH2·H2O, EtOH, reflux, 2 h, (b) ethyl 2-(ethoxymethylene)-
4,4,4-trifluoro-3-oxobutyrate, H2SO4, EtOH, reflux, 4 h, (c) zinc dust,
aqueous NH4Cl solution, EtOAc, 0 °C, 4 h, (d) Pd(PPh3)4, SnBu3, tolu-
ene, 60 °C, 15 h, (e) I2, CH2Cl2, rt, 2.5 h, (f) 2,3,3-trichloroacryloyl
chloride, CH2Cl2, N,N-diisopropylethylamine, 0 °C, 1 h, and (g) 1 M
KOH (aq.), EtOH, H2O, rt, 8 h.
2 Results and discussion
2.1 Chemistry
Pyr3 comprises two structural motifs: an ethyl-3-
HEK293 cells (Fig. 2). I-Pyr3 showed potent inhibition of the
TRPC3 channel-mediated Ca2+ influx that was only slightly
poorer than that of the mother compound (Pyr3).18 This re-
sult suggested that the introduction of iodine at the 2-posi-
tion of the central phenyl ring of Pyr3 did not affect its ability
to inhibit the TRPC3-mediated Ca2+ influx and that 125I-la-
beled I-Pyr3 could be a promising probe to monitor the fate
of Pyr3 in plasma.
trifluoromethylpyrazole-4-carboxylate
group
and
a
trichloroacrylic amide group. The trichloroacrylic amide
group of Pyr3 is critical for the observed TRPC3-selective inhi-
bition.18 The effect of introducing a functional group to the
central phenyl ring of Pyr3 on TRPC3 channel inhibition or
selectivity has not been reported. We designed ethyl
1-(2-[125I]iodo-4-(2,3,3-trichloroacrylamido)phenyl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxylate, [125I]I-Pyr3, to in-
troduce radioiodine at the 2-position of the central phenyl
ring of Pyr3. Nonradioactive I-Pyr3 was synthesized via the
route outlined in Scheme 1. Reaction of 2-bromo-1-fluoro-4-
nitrobenzene with hydrazine hydrate, followed by treatment
with ethyl 2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutyrate,
afforded the pyrazole derivative 3 in 94% yield. Treatment of
3 with zinc dust and aqueous NH4Cl afforded the amine 4 in
95% yield. A bromo to tributyltin exchange reaction using
PdIJPPh3)4 and a subsequent iododestannylation reaction
yielded the iodine derivative 6. I-Pyr3 was prepared from 6 in
60% yield by treatment with 2,3,3-trichloroacryloyl chloride.
Alkaline hydrolysis of the ester moiety of I-Pyr3 afforded the
carboxylic acid derivative, I-Pyr8, in 18% yield.
2.3 Radiosynthesis
The radioiodinated analog [125I]I-Pyr3 was obtained via the
iododestannylation of tributyltin precursor 7, which was
obtained from the reaction of amine
5 with 2,3,3-
2.2 Measurement of intracellular Ca2+ concentration
The ability of Pyr derivatives to inhibit TRPC3-mediated Ca2+
influx in vitro was evaluated using TRPC3-overexpressing
Fig. 2 Concentration-dependent inhibition of TRPC3-mediated Ca2+
influx by I-Pyr3. Upper panels: average changes in intracellular Ca2+
concentration ([Ca2+]i) induced by endogenous purinergic receptor
stimulation in TRPC3-overexpressing HEK293 cells (n = 45–92 cells) in
the presence or absence of extracellular Ca2+ (2 mM). The cells were
treated with Pyr3
2 min prior to ATP stimulation. Lower panels:
concentration-dependent inhibition of TRPC3-mediated Ca2+ influx by
I-Pyr3.
Fig. 1 Structure of TRPC3-targeted compounds.
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ylate analog [125I]I-Pyr8 was also obtained in high yield (84%)
via the alkaline hydrolysis of the ester moiety of [125I]I-Pyr3.
The radiochemical purities and specific activities of these
[
125I]I-labeled compounds were greater than 98% and greater
than 26.3 GBq μmol−1, respectively.
2.4 Stability studies
The in vitro stability of [125I]I-Pyr3 in 20 mM phosphate buffer
(PB; pH 7.4) or mouse plasma at 37 °C was evaluated using
radio-TLC analysis (Fig. 3). The radioactivity of [125I]I-Pyr3
remained >95% intact for 24 h in PB, while the radioactivity
of [125I]I-Pyr3 rapidly decreased and completely disappeared
after 6 h in mouse plasma. The metabolite of [125I]I-Pyr3 was
identified as [125I]I-Pyr8, indicating the rapid hydrolysis of
the ester group of [125I]I-Pyr3.
Scheme 2 Syntheses of [125I]I-Pyr3 and [125I]I-Pyr8. Reagents and
conditions: (a) 2,3,3-trichloroacryloyl chloride, CH2Cl2, DIEPA, 0 °C, 1 h,
(b) chloramine-T, AcOH, MeOH, [125I]NaI, 35 °C, 2 h, and (c) 1 M KOH
(aq.), EtOH, H2O, rt, 8 h.
2.5 In vivo biodistribution in normal mice
To characterize the in vivo kinetics of Pyr3, a biodistribution
study of [125I]I-Pyr3 was carried out in normal ddY mice
(Table 1). The radioactivity of [125I]I-Pyr3 cleared rapidly from
the blood pool and showed considerable accumulation in the
lung, small intestine, and liver 10 min after injection. In ad-
dition, accumulation of radioactivity was broadly observed in
the stomach, heart, kidney, and muscle. The thyroid accumu-
lation was low, indicating high stability against in vivo
deiodination. Because [125I]I-Pyr3 was hydrolysed to [125I]I-
Pyr8, the distribution of radioactivity after administration of
[
125I]I-Pyr3 might reflect the distribution of [125I]I-Pyr8. There-
fore, we performed a biodistribution study of [125I]I-Pyr8
(Table 2). Similar to [125I]I-Pyr3, [125I]I-Pyr8 showed rapid
blood clearance and considerable accumulation of radioactiv-
ity in the small intestine and liver. However, the lung radio-
activity after administration of [125I]I-Pyr8 was dramatically
lower compared to that after [125I]I-Pyr3 administration.
These results indicated that [125I]I-Pyr3 is broadly distributed,
especially to the lung, as an intact form at early times post-in-
jection, and that this is followed by rapid metabolism to
Fig. 3 Stability studies of (a) [125I]I-Pyr3 in 20 mM phosphate buffer
(pH 7.4) and (b) [125I]I-Pyr3 in mouse plasma.
trichloroacryloyl chloride. However, its radiochemical yield
was only 15% when N-chlorosuccinimide was used as an oxi-
dant. After the optimization of reaction conditions, the radio-
chemical yield of
[
125I]I-Pyr3 improved to 39% when
chloramine-T was used as an oxidant (Scheme 2). The carbox-
[
125I]I-Pyr8.
Table 1 Biodistribution in normal mice after iv injection of [125I]I-Pyr3a
Organ
10 min
30 min
1 h
3 h
0.40 0.10
24 h
0.26 0.09
Blood
1.74 0.31
1.52 0.21
1.20 0.12
3.14 2.97
18.20 2.25
3.10 0.45
14.59 2.91
2.68 0.67
20.58 2.64
2.24 0.46
1.11 0.12
0.13 0.03
0.84 0.48
1.02 0.17
0.98 0.14
3.42 2.64
21.09 2.28
1.23 0.40
6.16 2.57
1.14 0.15
5.65 1.12
2.66 0.78
0.94 0.09
0.14 0.03
0.73 0.10
0.93 0.08
1.10 0.16
3.27 2.96
26.51 3.75
0.93 0.17
7.80 2.64
0.98 0.03
4.68 0.78
1.92 0.24
1.04 0.25
0.16 0.05
Spleen
Pancreas
Stomach
Intestine
Kidney
Liver
0.98 0.14
0.88 0.21
1.47 1.04
26.91 3.72
0.60 0.09
4.29 1.51
0.82 0.05
1.61 0.23
2.02 0.81
0.57 0.11
0.19 0.08
0.98 0.19
0.62 0.08
0.07 0.02
0.05 0.01
0.27 0.03
0.21 0.03
0.62 0.06
0.66 0.10
1.29 0.34
0.23 0.04
0.29 0.11
3.77 1.45
73.81 10.52
Heart
Lung
Muscle
Brain
Thyroidb
Urineb
Fecesb
a
Each value represents the mean s.d. (n = 5). Expressed as % injection dose per gram of organ. b Expressed as % injection dose.
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Table 2 Biodistribution in normal mice after iv injection of [125I]I-Pyr8a
Organ
10 min
30 min
1 h
0.36 0.04
3 h
0.31 0.04
24 h
Blood
0.64 0.04
1.40 0.71
1.75 1.70
1.59 0.46
18.06 7.65
2.34 0.68
12.22 2.60
1.00 0.36
1.31 1.02
2.34 0.78
0.31 0.07
0.10 0.02
0.46 0.14
0.97 0.18
0.64 0.13
0.47 0.25
22.83 6.08
1.03 0.43
9.37 1.87
0.84 0.32
0.76 0.40
1.90 0.85
0.36 0.06
0.10 0.01
0.28 0.05
0.69 0.17
0.60 0.16
0.12 0.04
0.10 0.04
0.23 0.03
0.12 0.02
0.63 0.16
0.45 0.07
1.03 0.35
0.23 0.05
0.13 0.03
2.50 0.65
77.08 8.38
Spleen
Pancreas
Stomach
Intestine
Kidney
Liver
0.92 0.23
0.54 0.20
0.91 0.13
27.53 3.09
0.83 0.17
8.94 6.27
0.75 0.07
0.59 0.03
1.93 0.83
0.55 0.49
0.10 0.01
0.89 0.11
0.88 0.30
1.15 0.41
21.44 1.48
0.85 0.58
8.08 2.75
0.64 0.04
0.57 0.10
1.78 0.99
0.31 0.04
0.13 0.02
Heart
Lung
Muscle
Brain
Thyroidb
Urineb
Fecesb
a
Each value represents the mean s.d. (n = 5). Expressed as % injection dose per gram of organ. b Expressed as % injection dose.
3 Conclusions
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We successfully designed and synthesized [125I]I-Pyr3 and
[
125I]I-Pyr8 and used these compounds for the in vivo charac-
terization of the selective TRPC3 inhibitor Pyr3. The ability of
I-Pyr3 to inhibit TRPC3-mediated Ca2+ influx was comparable
to Pyr3. Biodistribution studies of [125I]I-Pyr3 and [125I]I-Pyr8
in normal mice showed that [125I]I-Pyr3 is highly distributed
to the lung as an intact form at early times post-injection and
that this is followed by rapid metabolism to [125I]I-Pyr8.
These findings provide useful information about the in vivo
kinetics of the selective TRPC inhibitor Pyr3.
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