1-(3′-[125I]iodophenyl)-3-methy-2-pyrazolin-5-one: Preparation, solution stability, and biodistribution in normal mice

Article abstract of DOI:10.1248/cpb.58.1020

3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), known as a potent free radical scavenger, has been developed as a medical drug for the treatment of acute cerebral infarction. With the aim of developing radiotracers for imaging free radicals in vivo, 1-(3′-[¹²⁵I]iodophenyl)-3-methy-2- pyrazolin-5-one (¹²⁵I-2) was synthesized by two methods, via isotopic exchange and interhalogen exchange under solvent-free conditions, in which iodo- and bromo-derivatives were used as labeling precursors, respectively. After HPLC purification, ¹²⁵I-2 was obtained in modest isolated radiochemical yields (ca. 20%) with high radiochemical purities by both methods. The former gave specific activities of 0.2-0.6 kBq/μmol, whereas the latter approach achieved specific activities of more than 0.14 GBq/μmol. On attempting to prepare an injectable formulation for ¹²⁵I-2 with high specific activity, its radiochemical purities dropped to about 60-70%. Unlabeled analog 2 was found to have lipophilic and antioxidant properties similar to edaravone. Intravenous injection of ¹²⁵I-2 with low specific radioactivity into normal mice showed signs of distribution profiles similar to reported results for ¹⁴C-labeled edaravone in normal rats.

Full text of DOI:10.1248/cpb.58.1020

1020
Regular Article
Chem. Pharm. Bull. 58(8) 1020—1025 (2010)
Vol. 58, No. 8
1-(3؅-[125I]Iodophenyl)-3-methy-2-pyrazolin-5-one: Preparation, Solution
Stability, and Biodistribution in Normal Mice
Yuuhei SANO, Tomokazu MOTOMURA, Fumihiko YAMAMOTO, Miki FUKUDA, Takahiro MUKAI,* and
Minoru MAEDA*
Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582,
Japan. Received February 1, 2010; accepted May 15, 2010; published online May 18, 2010
3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), known as a potent free radical scavenger, has been
developed as a medical drug for the treatment of acute cerebral infarction. With the aim of developing radiotrac-
ers for imaging free radicals in vivo, 1-(3؅-[125I]iodophenyl)-3-methy-2-pyrazolin-5-one (125I-2) was synthesized by
two methods, via isotopic exchange and interhalogen exchange under solvent-free conditions, in which iodo- and
bromo-derivatives were used as labeling precursors, respectively. After HPLC purification, 125I-2 was obtained in
modest isolated radiochemical yields (ca. 20%) with high radiochemical purities by both methods. The former
gave specific activities of 0.2—0.6 kBq/mmol, whereas the latter approach achieved specific activities of more than
0.14 GBq/mmol. On attempting to prepare an injectable formulation for 125I-2 with high specific activity, its
radiochemical purities dropped to about 60—70%. Unlabeled analog 2 was found to have lipophilic and antioxi-
dant properties similar to edaravone. Intravenous injection of 125I-2 with low specific radioactivity into normal
mice showed signs of distribution profiles similar to reported results for 14C-labeled edaravone in normal rats.
Key words edaravone; radioiodine; labeling; stability; biodistribution; mouse
Recent studies indicate that reactive oxygen species
(ROS), such as hydroxyl radical and hydrogen peroxide, are
generated in a normal physiological process, and that the im-
balance between the antioxidant defense system and exces-
sive levels of ROS in cellular systems is involved in the initi-
ation and progression of various diseases and disorders.1—5)
Various chemical probes of several methods have been devel-
Fig. 1. Edaravone (1) and Its Radical-Induced Oxidation Product (OPB)
oped to measure ROS in biological systems and the most
common approach is the measurement of the levels of
trapped molecules formed as a result of interception of ROS priate radiotracers for nuclear imaging technology are cur-
by probe molecules.6,7) Potential problems in detection and rently available for detecting free radicals in vivo.
quantification of ROS by methods currently available have
3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1, Fig.
been reviewed,8,9) in which the difficulties of precisely ana- 1), known as a potent free radical scavenger, has been used
lyzing ROS-expression in the whole body are discussed, be- clinically for the treatment of cerebral infarction in humans
cause of the low concentration of ROS formed in tissues and to prevent ischemic reperfusion injury15—17) and has also
interference by the presence of endogenous reductants. Nev- been reported to be effective for myocardial and hepatic
ertheless, in vivo imaging technologies that allow visualiza- ischemia as well.18,19) Biochemical and chemical studies have
tion of localized production and accumulation of ROS in a found that the free radical scavenging reaction of edaravone
living system provide useful information on the pathophysio- (1) with free radical species produces mainly 2-oxo-3-
logical status of the living body, and they have attracted a (phenylhydrazono)butanoic acid (OPB) with a more hy-
great deal of interest.
drophilic character as a stable oxidative product.17,20,21) In ad-
Of several biological imaging methods, nuclear imaging dition, metabolite studies have indicated that edaravone, after
technology, such as positron emission tomography or single intravenous administration in rats and humans, is exclusively
photon emission computed tomography using radiolabeled metabolized into its glucuronide and/or sulfate conjugates
molecules, is well known for its ability to assess molecular which are excreted into the urine via the kidney,22,23) except
pathways in vivo in both pre-clinical and clinical studies with for oxidation process according to its radical scavenging
very high sensitivity.10) The first reported work in developing functions. Such in vivo behavior of edaravone thus appeared
radiotracers for free radical imaging in vivo includes 125I- to have favorable properties as a lead molecule for the devel-
labeled analogs of a-p-hydroxy-m-iodophenyl-N-t-butyl- opment of radiotracers of the metabolic trapping type suit-
nitrone11) and a-p-iodophenyl-N-tert-butyl-nitrone,12) based on able for detecting free radicals in vivo. In order to investigate
the chemical characteristic that free radicals can be reacted the possibility of developing new radiotracers for probing
with nitrone-based compounds at the nitrone-double bond to free radical expression in vivo, we were interested in iodi-
form organic radical adducts, but none of these proved to be nated analogs of edaravone where an iodine atom was intro-
useful as in vivo probes because of their limited free radical duced at the meta position relative to the phenyl ring of the
specificity. An 18F-labeled analog of N-t-butylnitrone13) or molecule due to the expected stability against in vivo deiodi-
ethylmethylthiourea compound14) has also been prepared, but nation.24) Here we describe the synthesis and some physico-
with no information on its in vivo behavior. Thus, no appro- chemical properties of 1-(3Ј-iodophenyl)-3-methy-2-pyra-
∗
To whom correspondence should be addressed.
e-mail: maeda@phar.kyushu-u.ac.jp; mukai@phar.kyushu-u.ac.jp © 2010 Pharmaceutical Society of Japan

August 2010
1021
zolin-5-one (2), the radiosynthesis of the 125I-labeled analog 2 is slightly more lipophilic than edaravone itself, as ex-
(
125I-2) with low and high specific radioactivity, and its pre- pected. It is thought that edaravone (1) can be distributed be-
liminary biodistribution in normal mice.
tween the aqueous and lipid phases,29,30) thereby preventing
lipid peroxidation in both phases, and a lipophilic sub-
stituent, such as the iodine atom, may be beneficial to the li-
Results and Discussion
According to a similar literature procedure,25—27) 1-(3Ј- pidic peroxidation inhibitory activity of edaravone, as sug-
iodophenyl)-3-methy-2-pyrazolin-5-one (2) was prepared in gested.27) Next, the hydrogen-donating capacity by DPPH
three steps from 3-iodoaniline, involving diazotization in hy- (2,2-diphenyl-1-picrylhydrazyl) scavenging activity using a
drochloride–acetic acid, followed by reduction with stannous simple UV spectroscopic method,31) and the one-electron ox-
chloride and cyclization with ethyl acetoacetate in EtOH, in idation potential (Epa) by the cyclic voltammetry technique,
49% overall yield of the three-step conversion, as presented which are prerequisites for further investigation, were exam-
in Chart 1. It was important to carry out this under a stream ined for 2 in comparison with that for 1. It can be seen from
of argon gas for the reduction stage. Cyclization with ethyl Table 1 that 2 exhibits almost the same rate constant as edar-
acetoacetate to form a pyrazolone ring proceeded sluggishly avone 1 for the reaction with DPPH in a mixture of EtOH
in our initial experiments, giving low yields of the required and Tris buffer (pHϭ7.4). The oxidation potentials were
product. After many attempts, the addition of two equivalents measured in a basic aqueous solution at a pH range of 10.0—
of ethyl acetoacetate in small portions to a solution of the 10.1 because of the poor solubility of compound (2) in acidic
hydrazine compound in MeOH over 10 min was found to and neutral solutions. Iodoedaravone (2) had an oxidation
give much better results, and the formation of the by-prod- potential of 372 mV (vs. Agϩ/AgCl), which showed about a
ucts became significant when ethyl acetoacetate was added 50-mV lower value compared to edaravone itself as shown in
rapidly. 1-(3Ј-Bromophenyl)-3-methy-2-pyrazolin-5-one (3), Table 1. These results suggest that introduction of an iodine
for use as a precursor of radioiodination described below, atom at meta-position of the phenyl ring of edaravone mole-
was also prepared from 3-bromoaniline as similarily de- cule has a little effect on both lipophilicity and antioxidant
scribed for iodoedaravone (2), but it gave a complex mixture activity.
of products and only a very low yield of 3 as a crystalline
Iodine-125 was selected as the radiolabel for our radio-
compound due to the technical difficulty in chromatographic tracer development work because the long half-life (60 d) and
purification to obtain a product of satisfactory purity and in- low radiation energy of this radioisotope simplify radiosyn-
stability for the hydrazine base. The tributylstannylated pre- thesis. A number of radioiodination methods have been
cursor (4) for use in the electrophilic labeling approach was developed for the radiolabeling of biological and medical
also prepared in 23% yield by refluxing a solution of 2 and interests34) and, among them, a solvent-free procedure for the
bistributyltin in dry toluene in the presence of a palladium radioiodination of aromatic compounds has often been em-
catalyst.
ployed when the compound to be labeled is stable at its melt-
The log partition coefficient (1-octanol/67 mM phosphate ing point. Our initial approach to the synthesis of 125I-2 was
buffer, pHϭ7.4) of 2, determined by the shake-flask tech- via a conventional exchange reaction, under solvent-free con-
nique and HPLC quantification, was found to be 1.35Ϯ0.17 ditions, of nonradioactive 2 with no-carrier-added (NCA)
whereas that of edaravone (1) was 1.05Ϯ0.18, indicating that Na[125I]I which had been previously dried by azeotropic
evaporation with dry CH3CN under a stream of nitrogen at
room temperature. During the labeling reaction, which
required a high temperature of 140 °C in a closed vessel, the
label incorporation began to occur from 10—15 min, fol-
lowed by a gradual incorporation by additional 15—20 min,
but either prolonged heating over 30 min or temperatures
higher than 140 °C led to the decomposition of the labeled
product formed. An average isolated radiochemical yield of
23% with specific radioactivities between 0.2—0.6 kBq/mmol
and high radiochemical purity (Ͼ98%) was obtained after
reversed-phase HPLC purification (Fig. 2).
Specific activity appears to influence the in vivo radical
(i) NaNO2, HCl–AcOH; (ii) SnCl2, HCl; (iii) CH3COCH2COOEt, EtOH; (iv)
Bu6Sn2, Pd(Ph3)4, toluene.
Chart 1
scavenging ability of the radiotracer, since the presence of
the carrier molecule may have a negative effect on the con-
centration of radioactivity in targeted tissues. In order to
achieve high specific radioactivity in the preparation of 125I-
2, the electrophilic demetallation labeling method using an
organotin precursor was attempted.35) The stannylated pre-
cursor (4) was subjected to a radioiodo-destannylation reac-
tion with NCA Na[125I]I under classical reaction conditions,
involving a combination of H2O2–AcOH, chloramine-T or N-
chlorosuccinimide, but no detectable 125I-2 was observed,
even with an excess amount of oxidizing agents or an in-
crease in reaction time. It is most likely that the observed
lack of any response of 4 toward the radioiodine-destannyla-
Radiosynthesis of 1-(3Ј-[125I]iodophenyl)-3-methy-2-pyrazolin-5-one (125I-2) by
exchange of no-carrier-added Na[125I]I for iodinated and brominated edaravone (2,
3) under solvent-free conditions.
Chart 2

1022
Vol. 58, No. 8
Table 1. The Lipophilicity, DPPH Scavenging Activity and Oxidation
Potential
tion reaction was caused by the antioxidant property of its
pyrazolone ring.
Therefore, we examined an alternative method which does
not utilize an electrophilic iodine source. Thus, heating bro-
moedaravone (3) with NCA Na[125I]I at 140 °C for 60 min in
a solvent-free procedure provided 125I-2 in an average iso-
lated radiochemical yield of 18% after HPLC separation
(Fig. 2), reflecting lower reactivity of 3 in regard to the dis-
placement reaction. The obtained 125I-2 had Ͼ98% radio-
chemical purity without contamination from the labeling pre-
cursor and the specific radioactivity was estimated to be in
excess of 0.14 GBq/mmol based on the UV absorbance detec-
tion limit of the HPLC system used. Thus, use of bromoedar-
avone (3) for the interhalogen exchange reaction produced
125I-2 with a much higher specific activity compared to that
obtained by the isotopic exchange method.
Antioxidant activity
log Pa)
DPPH
Epa (mV)c)
scavenging activityb)
Iodoedaravone (2)
Edaravone (1)
1.35Ϯ0.17
1.05Ϯ0.18
0.78d)
0.264Ϯ0.010/s
0.237Ϯ0.007/s
372Ϯ1.5
422Ϯ5.0
483e)
a) Expressed as log P (1-octanol–67 mM phosphate buffer pHϭ7.4). Values repre-
sent the meanϮS.D. of three experiments. b) Reducing activity of 2.0ϫ10Ϫ4
M
antioxidant against 2,2-diphenyl-1-picrylhydrazyl (DPPH) in EtOH–50 mM Tris–buffer
(pHϭ7.4)ϭ3 : 2. Rate constants were obtained from the linear portion of the decrease
in optical density at 517 nm. Values represent the meanϮS.D. of five experiments.
c) Conditions for measurement: 0.025 mmol samples in aqueous solution (pHϭ10.0—
10.1); the oxidation potentials were expressed versus Agϩ/AgCl and the values repre-
sent the meanϮS.D. of three experiments. d) Taken from ref. 28. e) Taken from
refs. 32, 33.
After removal of the solvent (MeOH–H2Oϭ45 : 55 (v/v)
Table 2. Distribution of Radioactivity in Tissues of Normal Mice Following Intravenous Administration of 1-(3Ј-[125I]Iodophenyl)-3-methy-2-pyrazolin-5-
one (125I-2), Obtained by the Isotopic Exchange Method
% Injected dose per gram (nϭ3—5), meanϮS.D.
Organ
5 min
30 min
60 min
6 h
24 h
Blood
Spleen
9.21Ϯ0.58
2.06Ϯ0.14
2.77Ϯ0.52
2.36Ϯ0.39
0.98Ϯ0.10
7.39Ϯ1.81
20.06Ϯ5.30
2.31Ϯ0.44
0.42Ϯ0.21
26.03Ϯ6.65
10.35Ϯ2.43
2.83Ϯ0.25
6.26Ϯ0.42
1.63Ϯ0.49
0.48Ϯ0.04
2.10Ϯ0.31
10.04Ϯ1.15
0.12Ϯ0.03
3.26Ϯ0.40
0.88Ϯ0.15
1.01Ϯ0.32
0.88Ϯ0.16
0.50Ϯ0.34
1.47Ϯ0.11
1.90Ϯ0.68
0.64Ϯ0.11
0.57Ϯ0.41
3.15Ϯ0.30
2.38Ϯ0.24
1.62Ϯ0.49
2.44Ϯ0.26
0.60Ϯ0.28
0.20Ϯ0.04
1.19Ϯ0.75
4.28Ϯ0.87
0.11Ϯ0.07
2.01Ϯ0.35
0.85Ϯ0.08
0.44Ϯ0.16
0.72Ϯ0.12
0.44Ϯ0.13
1.07Ϯ0.27
1.02Ϯ0.44
0.86Ϯ0.24
5.28Ϯ3.79
1.95Ϯ0.53
1.28Ϯ0.20
0.81Ϯ0.09
1.50Ϯ0.22
0.79Ϯ0.50
0.10Ϯ0.03
0.68Ϯ0.20
3.11Ϯ0.10
0.09Ϯ0.03
0.36Ϯ0.07
0.36Ϯ0.09
0.14Ϯ0.03
0.42Ϯ0.22
0.67Ϯ1.15
0.22Ϯ0.08
0.26Ϯ0.15
0.23Ϯ0.07
2.08Ϯ0.67
0.56Ϯ0.09
0.24Ϯ0.02
0.18Ϯ0.04
0.60Ϯ0.10
0.22Ϯ0.08
0.03Ϯ0.01
0.16Ϯ0.03
1.19Ϯ0.26
0.14Ϯ0.02
0.07Ϯ0.02
0.14Ϯ0.04
0.04Ϯ0.01
0.09Ϯ0.01
0.04Ϯ0.02
0.08Ϯ0.04
0.07Ϯ0.02
0.07Ϯ0.03
0.18Ϯ0.10
0.28Ϯ0.04
0.05Ϯ0.02
0.05Ϯ0.01
0.27Ϯ0.05
0.09Ϯ0.06
0.02Ϯ0.01
0.21Ϯ0.15
1.07Ϯ0.48
0.33Ϯ0.11
Pancreas
Stomach
S. contenta)
Small intestine
S. i. contentb)
Large intestine
L. i. contentc)
Kidney
Liver
Heart
Lung
Muscle
Brain
Bone
Artery
Thyroidd)
a) s.contentϭstomach content, b) s.i.contentϭsmall intestine content, c) l.i.contentϭlarge intestine content, d) expressed as % of injected dose/organ.
Fig. 2. Preparative HPLC-Chromatograms for Isolation of 125I-2 from the Reaction Mixtures on a Reversed-Phase Column
(A) Isotopic exchange between iodinated edaravone (2) and NCA Na[125I]I; (B) nonisotopic exchange between brominated edaravone (3) and NCA Na[125I]I.

August 2010
1023
Fig. 3. Typical HPLC Chromatograms of the Formulated Solution after Removal of the HPLC Solvent of the Fractions Containing Low Specific Activity
125I-2; tϭ0 h (A) and tϭ3 h (B) after Storage at 37 °C
with 0.1% CF3COOH in both methanol and water) of the from these organs washed out with time, approaching the
HPLC fractions containing the isolated radioactive com- levels seen in other tissues. The accumulation of radioactiv-
pound under reduced pressure, the residue was redissolved in ity in the thyroid was low (0.14Ϯ0.02%ID/organ at 6 h) and
1% Tween-80-physiological saline containing 5% EtOH as remained constant at all points of the study, confirming the
an injectable solution. The formulated solution of 125I-2 with expected stability of the iodophenyl group in regard to in
low specific activity remained stable up to 6 h at 0 °C, but the vivo deiodination. The presence of a substantial amount of
radiochemical purity dropped off to 80% in 1 h and 40% in radioactivity in the small and large intestines, and also intes-
3 h after storage at 37 °C. HPLC analysis of the formulated tinal content seem to be an indication of extensive biliary
solution, as shown in Fig. 3, revealed the presence of at least excretions of radioactivity. 125I-2 showed very little uptake in
four radioactive peaks newly produced, in a form more the mouse brain even at 5 min after injection, thus demon-
lipophilic than the parent tracer, as judged from their longer strating limited permeability through the blood–brain barrier
retention times by HPLC. The chemical forms of these in the normal mouse brain, as observed for ‘phenyl ring’-14C-
radioactivity peaks are of unknown identity at present, labeled edaravone (specific activity: 4.5 GBq/mmol) in
although neither HPLC nor TLC analysis gave any indication rats.23) 125I-2 had a relatively higher radioactivity concentra-
of the formation of free 125I-iodide ion. In contrast to the low tion in the aorta tissue relative to other tissues at 30 and
specific activity 125I-2, we found that, on attempting to 60 min after injection. Similar uptake and retention of
remove the HPLC solvent containing 125I-2 with high specific radioactivity in aorta tissue was reported in a biodistribution
activity, the formation of new radioactive substances was study of rats injected with 14C-labeled edaravone by Komatsu
frequently occurring. A consequence was that the radiochem- et al.,36) in which it was proposed as a chemical trap for 14C-
ical purity in the formulated solution decreased to a level of labeled edaravone with free radical species existing in this
60—70%. Additional storage at 37 °C promoted a further tissue, although it is largely speculation in the absence of de-
decrease of 125I-2 and after 6 h no radioactivity remained as tailed studies. Thus, from the tissue biodistribution in normal
the parent 125I-2. HPLC analysis gave the same HPLC peaks mice, a general clearance of the radioactivity over time in all
of radioactive substances as observed after storage at 37 °C organs was found, qualitatively similar to that seen with 14C-
of the formulated solution of of the 125I-2 with low specific labeled edaravone in rats,23) in spite of the large differences
radioactivity. The lability observed in the formulation step of in specific activities of these agents.
125I-2 with high specific radioactivity appears to be related to
both self-absorbed radiation and chemical instability of the Conclusion
compound itself. In the present study we have not been able
In the present study, two radiolabeling methods via iso-
to develop a suitable method for stabilization of the high spe- topic exchange and interhalogen exchange were evaluated
cific activity 125I-2 after preparation, and therefore, in this to synthesize 1-(3Ј-[125I]iodophenyl)-3-methy-2-pyrazolin-5-
work, the in vivo application of this agent was not carried one (125I-2) with regard to radiochemical yield, radiochemi-
out.
cal purity, and specific activity in order to develop a radio-
Tissue biodistribution of 125I-2 with low specific activity tracer for probing free radicals in vivo. The radiosynthesis
was thus evaluated in normal mice by the dissection proce- provided radiochemically pure 125I-2 in reasonable quantities
dure and the results are expressed as % injected dose (ID)/g by both methods, although further optimization is possible.
of tissue or %ID/organ as shown in Table 2. 125I-2 demon- The biodistribution showed significant elimination of radio-
strated low initial blood activity (3.26Ϯ0.40%ID/g at 30 min activity from normal tissues and relatively low levels of cir-
after injection) and relatively fast clearance after 24 h (0.07Ϯ culating radioactivity in the blood pool during the 6—24 h.
0.02%ID/g). Early renal and hepatic uptake was higher than Solution stability studies demonstrated that 125I-2 has the dis-
that in other tissues except the arteries, but the radioactivity advantage of inherent instability in an aqueous solution. Fur-

1024
Vol. 58, No. 8
was obtained from 3-bromoaniline (688 mg), by diazotization in hydrochlo-
ride–acetic acid, followed by reduction with stannous chloride dihydrate
(2.7 g) and cyclization with ethyl acetoacetate (182 mg) in EtOH, according
to the procedure described for 2. Removal of the solvent under vacuum gave
a thick oily residue, which was purified by repeated column chromatography
on silica gel(chloroform–hexaneϭ1 : 3) to give 3 as an orange solid (38 mg,
4%), mp 132—134 °C. 1H-NMR (CDCl3) d: 2.32 (s, 3H), 3.58 (s, 2H), 7.33
(m, 1H), 7.43 (d, 1H, Jϭ8.0 Hz), 7.54 (d, 1H, Jϭ8.0 Hz), 7.65 (s, 1H); IR
ther study will focus on the development of formulation pro-
cedure, capable of maintaining radiochemical purity of 125I-2
with high specific radioactivity, and it is also necessary to
address the labeled products resulting from the exposure of
this radiotracer to tissues in vitro and in vivo.
Experimental
Edaravone was supplied by Mitsubishi Pharma Co. (Tokyo, Japan) and (KBr) cmϪ1: 1684, 1626, 1578, 1481; ESI-HR-MS (m/z): Calcd for
had a purity of >95%. Chemical reagents and solvents were of commercial C10H10N2OBr (MϩH) 252.9967, Found 252.9971; Calcd for C10H11N2OBr
quality and were used without further purification unless otherwise noted.
(Mϩ3H) 254.9952, Found 254.9951.
Lipophilicity The partition coefficients of 2 and edaravone (1) were
1H-NMR spectra were obtained on a Varian Unity 400 (400 MHz) and are
referenced to tetramethylsilane (TMS) (dϭ0 ppm). Infrared (IR) spectra determined at room temperature from the distribution of the compounds
were recorded with a Shimadzu FTIR-8400 spectrometer and mass spectra
between a mixture of 1-octanol (2.5 ml) and a 67 mM phosphate buffer (pHϭ
were obtained with a JEOL JMS DX-610 (FAB Mass) or an Applied Biosys- 7.4, 2.5 ml). The mixture was vortexed for 3 min and was left to stand for
tems Mariner System 5299 spectrometer electrospray ionization-mass spec- 10 min. This procedure was repeated three times, followed by centrifugation
tra (ESI-MS). All melting points were determined on a Yanaco melting point at 3000ϫg for 5 min. The concentration of the compound in both phases
apparatus (Yanagimoto Ind. Co., Japan) and are uncorrected. Column chro-
before and after the shaking were analyzed by HPLC (column; Nacalai
matography was performed on Silica gel 60N (63—210 mesh, Kanto Chem-
Tesque COSMOSIL 5C18-AR-II, 4.6ϫ250 mm, mobile phase: MeOH–0.1%
ical Co., Inc., Japan) and the progress of the reaction was monitored by TLC aqueous CF3COOH 72 : 28, v/v, flow rate: 0.5 ml/min, UV: 254 nm, retention
on Silica gel 60F 254 plates (Merck, Germany), and the spots were visual-
time: 13.5 min for 2 and 7.5 min for edaravone). The log P value represents
ized with UV light or by spraying with 5% alcoholic molybdophosphoric the meanϮS.D. of three independent experiments.
acid. In the synthetic procedures, the organic extracts were routinely dried
DPPH Radical-Scavenging Assay The radical scavenging activity
over anhydrous Na2SO4 and evaporated with a rotary evaporator under re- against the DPPH radical was determined according to the method described
duced pressure. HPLC was done using a Shimadzu Liquid Chromatograph
in the literature, with slight modifications.31) Briefly, changes in the ab-
System (SPD-10A) (COSMOSIL 5C18-PAQ column, 250ϫ4.6 mm, Nacalai sorbance at 517 nm due to the scavenging of the DPPH radical was continu-
Tesque, Japan) and by monitoring the radioactivity (NaI(Tl) detector) as well ously monitored with a spectrophotometer (Hitachi U-2810 spectrometer)
as UV absorption (at 254 nm). The radioactivity was quantified with an auto- immediately after mixing (1.5 ml) a freshly prepared 2.0ϫ10-4 M solution of
well gamma counter (ARC-605 II, Aloka, Japan). Analysis of the radioactiv-
ity of the TLC plates was performed by cutting into 0.5 cm-wide strips and
DPPH in a mixture of a 50 mM Tris–HCl buffer (pH 7.4) and EtOH (2 : 3,
v/v), and a 2.0ϫ10Ϫ4 M solution of 2 or edaravone (1) in the same solvent
counting their radioactivity with a gamma-well counter. Identity of the la- system at 25 °C. The first-order of rate constant for the disappearance of the
beled compound, specific radioactivity and radiochemical purity were deter- DPPH color at 517 nm was calculated from the slope of the linear portion of
mined by the same HPLC system (COSMOSIL 5C18-AR-II, 250ϫ4.6 mm,
the disappearance curves, and the result was expressed as the meanϮS.D. of
Nacalai Tesque, Japan) or radio TLC. No-carrier-added Na[125I]iodide five determinations.
(0.04 M NaOH solution; 185 MBq, Ͼ600 GBq/mg) was purchased from
Cyclic Voltammetry Cyclic voltammograms were measured using BAS
American Radiolabeled Chemicals Inc. Animal experiments were carried 100B/W (CV-50W) version 2 potentiometer consisting of platinum elec-
out in accordance with our institutional guidelines and were approved by the trodes as working and counter electrodes. Potentials were measured relative
Animal Care and Use Committee, Kyushu University.
to the Agϩ/AgCl reference electrode. An internal reference (potassium ferri-
cyanide) was used as well. Edaravone (4.35 mg, 0.025 mmol) or 2 (7.5 mg,
0.025 mmol) was dissolved in 2 ml of 1 M NaOH and then the solutions were
adjusted to pH 10.0—10.1 with 1 M HCl (final concentrations: 6.4 mM for
edaravone and 6.6 mM for 2). Oxygen was removed by purging the solution
Synthesis. 1-(3؅-Iodophenyl)-3-methy-2-pyrazolin-5-one (2) A solu-
tion of 3-iodoaniline (438 mg, 2 mmol) in acetic acid (1.64 ml) and concen-
trated HCl (4.78 ml) was treated with an ice-cold solution of NaNO2 (207
mg, 3 mmol) in H2O (776 ml). The mixture was stirred for 1 h at this temper-
ature. Stannous chloride dihydrate (1.354 g, 6 mmol) was dissolved in con- with argon gas and a continuous stream of this gas was passed over the solu-
centrated HCl (925.4 ml) and the solution was cooled to 0 °C under an argon tions during the measurements. Cyclic voltammograms were obtained at a
atomosphere. To the diazonium mixture produced was slowly added a solu- scan rate of 50 mV/s. The values of the oxidation potential represent the
tion of the stannous chloride over a period of 2 h under an argon atmosphere, meanϮS.D. of three independent measurements.
while the temperature was always kept at 0 °C. The mixture was made basic
by the addition of an excess of 10% NaOH and extracted several times with
Radiochemistry. 1-(3؅-[125I]Iodophenyl)-3-methy-2-pyrazolin-5-one
(
125I-2) (A) Iodine-Radioiodine Exchange: No-carrier-added Na[125I]I
ether. The combined organic extracts were dried and evaporated to dryness. (0.37—7.2 MBq) was transferred to a reaction vial and to this was added dry
The reddish brown residue was dissolved in EtOH (5 ml) and to the mixture CH3CN (30 ml), followed by evaporation to dryness under a stream of nitro-
was slowly added ethyl acetoacetate (234 mg, 1.8 mmol) over a period of 10 gen at room temperature. To the residue was then added a solution of 2
min at 50 °C under stirring. The mixture was further heated under reflux for
3 h. After removal of the solvent under vacuum, the residue was chro- vial was flushed with a stream of nitrogen for a few seconds, tightly sealed
(50—500 mg) in dry CH3CN (500 ml) and evaporation was repeated. The
matographed on silica gel (chloroform–hexaneϭ1 : 3) to give 2 as an orange
and heated at 140 °C for 30 min. The entire content of the reaction vessel
was dissolved in methanol (100 ml) and was injected onto the HPLC column
1
solid (296 mg, 49%), mp 128—132 °C. H-NMR (CDCl3) d: 2.21 (s, 3H),
3.47 (s, 2H), 7.10 (t, 1H, Jϭ16.1 Hz), 7.51 (d, 1H, Jϭ8.1 Hz), 7.90 (d, 1H, (Nacalai Tesque COSMOSIL 5C18-PAQ 4.5ϫ250 mm, MeOH/H2Oϭ45/55
Jϭ10.3 Hz), 8.24 (t, 1H, Jϭ3.6 Hz); IR (KBr) cmϪ1: 1716, 1582, 1474; (v/v) with 0.1% CF3COOH in both methanol and water which were bubbled
FAB-MS (m/z): 301.08 (MϩH)ϩ; ESI-HR-MS (m/z): Calcd for C10H10N2OI
(MϩH) 300.9832, Found 300.9847.
with nitrogen gas beforehand, flow rate 0.8 ml/min). The product fraction
was collected after a retention time of 30.1 min. The isolated radiochemical
1-(3؅-Tri-n-butylstannylphenyl)-3-methy-2-pyrazolin-5-one (4) 1-(3Ј- yield of 125I-2 ranged from 6.6 to 41.3% (average of 23%) in a total prepara-
Iodophenyl)-3-methy-2-pyrazolin-5-one (2) (30 mg, 0.1 mmol) was dis- tion time of 60 min. The specific activities of the obtained 125I-2 varied from
solved in dry toluene (30 ml) which had been degassed by bubbling argon 0.2 to 0.6 kBq/mmol, as determined by direct measurement of the HPLC elu-
gas through it for 5 min. Tetrakis(triphenylphosphine)palladium(0) (11.55
mg, 0.01 mmol) and bis(tri-n-butyl)tin (145 mg, 0.25 mmol) were succes-
ate using a UV detector. Quality control HPLC showed a single radioactive
peak with the corresponding carrier, suggesting that the preparations were
sively added. The reaction mixture was heated under reflux for 3 h. The more than 98% chemically and radiochemically pure. The product was for-
black reaction mixture was diluted with dichloromethane and filtered, and mulated by evaporating the fraction collected from HPLC under reduced
the solvent was removed under vacuum. Purification was carried out using pressure, and dissolving in 1% Tween-80-physiological saline containing 5%
column chromatography on silica gel (chloroform–hexaneϭ1 : 1) to give EtOH.
1
compound (4) as a yellow-brown oil (10.8 mg, 23%). H-NMR (CDCl3) d:
0.89 (t, 9H, Jϭ7.3 Hz), 1.07 (t, 6H, Jϭ8.2 Hz), 1.33 (m, 6H), 1.53 (m, 6H),
2.19 (s, 3H), 3.41 (s, 2H), 7.34 (t, 2H, Jϭ7.7 Hz), 7.77 (d, 1H, Jϭ8.1 Hz),
(B) Bromine-Radioiodine Exchange: To no-carrier-added Na[125I]I
(0.37—3.7 MBq), which had been dried as described above, was added a
solution of 4 (100—500 mg) in dry CH3CN (500 ml) and evaporation was
7.92 (d, 1H, Jϭ2.2 Hz); IR (neat) cmϪ1: 2957, 2927,1725, 1580; ESI-MS carried out under a stream of nitrogen at room temperature. The vial was
(m/z): 465.1 (MϩH).
flushed with a stream of nitrogen for a few seconds, then tightly sealed and
1-(3؅-Bromophenyl)-3-methy-2-pyrazolin-5-one (3) Compound (3) heated at 140 °C for 60 min. The entire content of the reaction vessel was

August 2010
1025
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dissolved in methanol (100 ml) and was injected onto the HPLC column
(Nacalai Tesque COSMOSIL 5C18-PAQ 4.5ϫ250 mm, MeOH/H2Oϭ40 : 60
(v/v) with 0.1% CF3COOH in both methanol and water which were bubbled
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Solution Stability The formulated solution of 125I-2 was allowed to
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were withdrawn and analyzed for stability by analytical HPLC and radio-
TLC. HPLC analysis was performed using a COSMOSIL 5C18-AR-II col-
umn (250ϫ4.6 mm, Nacalai Tesque). The column was eluted with a gradient
of 0.1% CF3COOH in MeOH (A) and 0.1% CF3COOH in H2O (B) at
0.7 ml/min. The percentage of (A) was increased linearly to 100% over
45 min and maintained at that value for another 15 min. Under these condi-
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a stream of nitrogen gas using Merck TLC aluminium backed sheets Sil-
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Tissue Distribution Studies in Normal Rats Normal male ICR mice
(6 weeks old, weighing 30—35 g), deprived of food 18 h prior to and during
the course of experiment, with free access to water, were used in this investi-
gation. The animals were intravenously injected through the tail vein with a
solution of 125I-2 (9.0—11.1 kBq, 100 ml, specific activity 0.2—0.6 kBq/
mmol) in 1% Tween-80-physiological saline containing 5% EtOH, immedi-
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Acknowledgements Support for this study from the Asahi Glass Foun-
dation and the program for the Creation of Innovation Centers for Advanced
Interdisciplinary Research Areas from Special Coordination Funds for Pro-
moting Science and Technology (SCF) commissioned by the Ministry of Ed-
ucation, Culture, Sports, Science and Technology (MEXT) of Japan, is
gratefully acknowledged. The authors also thank Mr. Jintaek Kim for his
support in preparing this manuscript.
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