125I-labeled 2-O- and 3-O-m-iodobenzyl, and 6-O-m-iodophenyl derivatives of L-ascorbic acid: Synthesis and preliminary tissue distribution

Article abstract of DOI:10.1002/jlcr.714

Three ¹²⁵I-labeled 2-O- and 3-O-m-iodobenzyl, and 6-O-m-iodophenyl derivatives of L-ascorbic acid were prepared by melt exchange procedures in isolated radiochemical yields of 12-60% after HPLC purification. Biodistribution studies in tumor-bearing mice showed very different in vivo tissue uptake properties from previous results obtained with ¹⁴C-labeled ascorbic acid and 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid. None of these seems to be suitable radioiodinated analogs of L-ascorbic acid for imaging study of its in vivo biochemistry. Copyright

Full text of DOI:10.1002/jlcr.714

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 737–750.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.714
Research Article
125I-Labeled 2-O- and 3-O-m-iodobenzyl, and 6-O-
m-iodophenyl derivatives of L-ascorbic acid:
synthesis and preliminary tissue distribution
Fumihiko Yamamoto, Eri Kuwano, Tetsuo Kaneshiro, Shigeki Sasaki
and Minoru Maeda*
Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan
Summary
Three 125I-labeled 2-O- and 3-O-m-iodobenzyl, and 6-O-m-iodophenyl
derivatives of l-ascorbic acid were prepared by melt exchange procedures in
isolated radiochemical yields of 12–60% after HPLC purification. Biodistribu-
tion studies in tumor-bearing mice showed very different in vivo tissue uptake
properties from previous results obtained with 14C-labeled ascorbic acid and 6-
deoxy-6-[18F]fluoro-l-ascorbic acid. None of these seems to be suitable
radioiodinated analogs of l-ascorbic acid for imaging study of its in vivo
biochemistry. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: iodine-125; exchange radiolabeling; ascorbic acid; tissue distribu-
tion
Introduction
l-Ascorbic acid is an essential nutrient required for an array of
biological functions, including antioxidation and enzymatic reactions.
In addition, it has been reported that l-ascorbic acid has anti-tumor
*Correspondence to: M. Maeda, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: maeda@phar.kyushu-u.ac.jp
Contract/grant sponsor: Ministry of Education, Science, Sports and Culture of Japan.
Copyright # 2003 John Wiley & Sons, Ltd.
Received 27 December 2002
Revised 3 March 2003
Accepted 22 March 2003

738
F. YAMAMOTO ET AL.
activity and certain human tumors contain increased concentrations of
l-ascorbic acid compared with normal tissue, although the precise
mechanisms are still unknown.1 Various derivatives of l- ascorbic acid
have been synthesized and studied for their multifold biological and
pharmacological interest.2 We developed 6-deoxy-6-[18F]fluoro-l-ascor-
bic acid (18F-DFA) as a positron emitter-labeled analog of l-ascorbic
acid for studying the in vivo biochemistry of ascorbic acid by positron
emission tomography.3 Previous tissue distribution studies in rats and
tumor-bearing mice3 demonstrated that the uptake and distribution
pattern of 18F-DFA, showing the highest uptake of radioactivity in the
adrenal glands with moderate capability to accumulate in tumors, has a
remarkable resemblance to that reported for 14C-labeled ascorbic
acid.4,5
The present study was undertaken to develop ascorbic acid analogs
labeled with radionuclides of iodine with the intention of potential
application for use in single photon emission tomography. The
iodophenyl group was chosen as a supplementary unit for iodine
radiolabeling in our initial development work, because of the known in
vivo stability of the iodine bound to the phenyl ring.6 This paper
describes the synthesis and radiolabeling with 125I of m-iodobenzyl ether
and m-iodophenyl ether derivatives of l-ascorbic acid, and their
biodistribution in tumor-bearing mice is also reported.
Results and discussion
Synthesis
The synthesis of 2-O-(m-iodobenzyl)-l-ascorbic acid (2-IBA)(4), as
depicted in Scheme 1, started with 5,6-O-isopropylidene-l-ascorbic acid
(1). The hydroxy group at the 3-position of (1) was protected with a
methoxymethyl group by use of chloromethyl methyl ether and m-
iodobenzylation with m-iodobenzyl bromide in the presence of
potassium carbonate, followed by acid hydrolysis, gave the required
2-IBA (4) in good yield. 3-O-(m-iodobenzyl)-l-ascorbic acid (3-IBA)(7)
was obtained from direct m-iodobenzylation of (1) followed by acid
hydrolysis in good yield. Initial attempts to prepare 6-O-iodobenzyl-l-
ascorbic acid failed to give the desired iodobenzylation product.
Therefore, we turned our attention to 6-O-(m-iodophenyl)-l-ascorbic
acid (6-IPA)(11), which was prepared by an alternative route involving
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND BIODISTRIBUTION OF RADIOIODINATED ASCORBIC ACID 739
Scheme 1. (a) MOMCl, K2CO3, rt, 5 h (30%), (b) m-iodobenzyl bromide,
K2CO3, rt, 40 min (57%), (c) 10% HCl, MeOH, rt, 15 h (96%), (d) Na125l,
1608 C, 9 min, (e) m-iodobenzyl bromide, K2CO3, rt, 3 hr (49%), (f) TsOH,
MeOH, rt, 15 h (89%), (g) Na 125l, 1508 C, 13 min.
Scheme 2. (a) potassium m-iodophenoxide CH3CN, reflux, 1 h, (b), HCl/H2O,
reflux, 2 h (76%), (c) methylal, P2O5, CHCl3, rt, 18h (64%), (d) Na 125I, 1508 C,
10 min, (e) HCl/MeOH, 608 C, 30 min.
the nucleophilic ring opening of the cyclic sulfate (9)3 with potassium m-
iodophenoxide, followed by acid hydrolysis (Scheme 2). The structures
1
were determined on the basis of analysis in H- and 13C-NMR spectra,
consistent with those reported for related ascorbic acid derivatives.7,8
The structures of regioisomers 2-IBA and 3-IBA were also confirmed by
13C-NMR chemical shifts depending on pH (Table 1). In the 13C-NMR
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740
F. YAMAMOTO ET AL.
Table 1. 13C-NMR chemical shifts of 4 and 7 depending on pH
13C chemical shiftsa d (ppm)
Compound 4
pH>10b
Compound 7
Carbon position
pH=3.5
Dc
pH=6
pH410b
Dc
1
2
3
4
5
6
172.61
121.52
161.86
76.94
70.63
63.42
179.70
118.06
177.93
80.18
72.05
64.20
+7.09
ꢀ3.46
+16.07
+3.24
+1.42
+0.78
172.72
121.36
151.17
76.72
70.51
63.42
179.58
132.35
145.63
76.89
71.58
64.25
+6.86
+10.99
ꢀ5.54
+0.17
+1.07
+0.83
aSpectra recorded in CD3OD. bTwo drops of 20% NaOH/D2O was added to adjust pH. cD=d(pH
3.5 or 6)ꢀd(pH410).
Table 2. Reducing activity and partition coefficient
Compounds
Reducing activity (%)a
log P (octanol/water)b
2-IBA
3-IBA
6-IPA
Ascorbic acid
92
60
72
96
ꢀ1.16
+0.78
ꢀ0.42
ꢀ1.27
aReducing activity of 1 ꢁ 10ꢀ4 M antioxidants against a,a-diphenyl-b-picrylhydrazyl (DPPH).
bValues were determined by shaking solutions of compounds in 2.5 ml 1-octanol with 2.5 ml
potassium phosphate buffer (pH 7.4) for 20 min and the data are the average of three separate
experiments.
spectrum of 2-IBA, the signal of the carbon at the 3-position moved
most downfield when the pH of the solution was changed from 3.5 to
>10. On the other hand, the 13C-NMR spectrum of 3-IBA revealed an
11 ppm downfield shift of the carbon at the 2-position with a similar
increase in pH. These observations are in accord with those found
earlier.7,8
The reducing abilities of the three iodinated ascorbic acid analogs
were determined by use of a stable radical a,a-diphenyl-b-picrylhydrazyl
(Table 2). 2-IBA exhibited almost the same reducing potency as ascorbic
acid while the activities of 3-IBA and 6-IPA were lower than that of
ascorbic acid. The octanol/water distribution (logP value, Table 2)
showed that, as expected, the attachment of a iodobenzyl or iodophenyl
group to the ascorbic acid molecule decreased the hydrophilicity to
some extent; 3-IBA exhibited a somewhat higher partition coefficient
compared to the two other iodinated derivatives, likely due to the
predominantly uncharged character of the 3-O-substituted derivative at
a physiological pH.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND BIODISTRIBUTION OF RADIOIODINATED ASCORBIC ACID 741
Radiolabeling
A number of different methods of introducing a radioiodine atom into a
phenyl ring have been documented in the literature.9 However,
radioiodination via organometallics or direct electrophilic radioiodina-
tion that required oxidation conditions was found to be unsuitable for
use with ascorbic acid susceptible to oxidation. Thus, a direct exchange
of radioiodine for aromatic iodide was chosen in this work: A high
specific activity was not required because l-ascorbic acid is normally
present at high concentrations in mammalian cells.10 2-125IBA and
3-125IBA were prepared by simple heating with Na125I in the absence of
any solvent in a sealed tube. The exchange required 13 min at 1508C
with 3-IBA and 9 min at 1608C with 2-IBA to give an isolated
radiochemical yield of 27–60 and 12–45%, respectively, after HPLC
purification. Similarly, the preparation of 6-125IPA was achieved by
isotope exchange of the MOM-protected (12) in a melt procedure
(1508C, 10 min) and subsequent removal of the protecting group with
HCl in an overall radiochemical yield of 20–48% after HPLC
purification. The time required for both the radioiodination and
purification was less than 2 h for all preparations and the radiochemical
purity of the labeled materials was excellent. When the exchange
reaction was performed with 1 mg of the iodinated compounds and
2.5–3.3 MBq of Na125I, specific activities in the range of 200–350 MBq/
mmol were produced, which were of the same level as that of
commercially available 14C-labeled ascorbic acid and were considered
to be sufficient to permit preliminary biodistribution studies.
Animal studies
The distributions of 2-125IBA, 3-125IBA and 6-125IPA after intravenous
injections of these compounds in mice bearing 3-methylcholanthrene-
induced fibrosarcoma are shown in Tables 3–5. There are marked
differences in the distribution of these compounds. A fast disappearance
of radioactivity from the blood was similar for 2-125IBA and 3-125IBA,
but the level of radioactivity in the blood of 6-125IPA remained much
higher relative to the other two compounds throughout the during of
the study. At an early time following injection, 3-125IBA showed the
highest uptake of radioactivity in the liver, whereas 2-125IBA had the
highest kidney uptake of radioactivity and the highest uptake of
radioactivity of 6-125IPA was found in the lungs. These organs showed
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F. YAMAMOTO ET AL.
Table 3. Biodistribution of 2-125IBA in mice bearing fibrosarcoma
Uptake (% dose/g)a
Tissue
1 min
10 min
30 min
60 min
Blood
Lung
Liver
Adrenals
Kidneys
Tumor
Muscle
Brain
15.66+0.83
7.66 ꢂ 0.05
7.18 ꢂ 0.45
3.18 ꢂ 0.10
46.38 ꢂ 2.02
1.56 ꢂ 0.20
1.61 ꢂ 0.25
0.74 ꢂ 0.07
6.02 ꢂ 1.34
6.15+4.82
2.88 ꢂ 2.20
4.20 ꢂ 2.15
2.03 ꢂ 2.17
17.70 ꢂ 5.09
1.43 ꢂ 0.72
1.14 ꢂ 0.73
0.28 ꢂ 0.34
3.03 ꢂ 1.73
1.30 ꢂ 0.82
0.75 ꢂ 0.55
1.04 ꢂ 0.42
0.67 ꢂ 0.69
4.47 ꢂ 2.73
0.69 ꢂ 0.53
0.37 ꢂ 0.28
0.05 ꢂ 0.05
2.00 ꢂ 0.30
0.53 ꢂ 0.32
0.44 ꢂ 0.32
0.68 ꢂ 0.31
0.45 ꢂ 0.38
1.14 ꢂ 0.67
0.41 ꢂ 0.15
0.17 ꢂ 0.0.8
0.04 ꢂ 0.04
6.28 ꢂ 1.58
Thyroid
ameans ꢂ SD (n=3).
Table 4. Biodistribution of 3-125IBA in mice bearing fibrosarcoma
Uptake (% dose/g)a
Tissue
1 min
10 minb
30 min
60 minb
Blood
Lung
Liver
Adrenals
Kidneys
Tumor
Muscle
Brain
12.03+0.55
10.17 ꢂ 1.26
21.15 ꢂ 1.00
6.65 ꢂ 0.10
16.70 ꢂ 2.34
1.10 ꢂ 0.28
1.92 ꢂ 0.38
0.55 ꢂ 0.04
4.55 ꢂ 0.53
3.39+0.35
2.79 ꢂ 0.45
17.51 ꢂ 5.58
2.33 ꢂ 0.46
18.08 ꢂ 2.71
1.86 ꢂ 0.30
1.21 ꢂ 0.12
0.20 ꢂ 0.03
2.28 ꢂ 0.71
0.65 ꢂ 0.12
0.50 ꢂ 0.08
2.20 ꢂ 0.62
0.63 ꢂ 0.21
3.22 ꢂ 0.21
0.60 ꢂ 0.17
0.24 ꢂ 0.07
0.06 ꢂ 0.01
1.47 ꢂ 0.26
0.43 ꢂ 0.23
0.30 ꢂ 0.13
1.47 ꢂ 0.74
0.36 ꢂ 0.16
1.93 ꢂ 0.99
0.41 ꢂ 0.23
0.17 ꢂ 0.12
0.03 ꢂ 0.01
1.04 ꢂ 0.33
Thyroid
ameans ꢂ SD (n=3). bn=4.
Table 5. Biodistribution of 6-125IBA in mice bearing fibrosarcoma
Uptake (% dose/g)a
Tissue
1 min
10 min
30 min
60 min
Blood
Lung
Liver
Adrenals
Kidneys
Tumor
Muscle
Brain
54.18 ꢂ 25.45
215.73 ꢂ 136.24
36.72 ꢂ 13.09
25.69 ꢂ 9.12
26.20 ꢂ 11.69b
1.86 ꢂ 0.64
3.84 ꢂ 1.45
6.03 ꢂ 3.05
17.82 ꢂ 5.04
8.83 ꢂ 2.91
17.32 ꢂ 6.69
16.49 ꢂ 3.98
4.72 ꢂ 2.05
22.24 ꢂ 5.34
2.38 ꢂ 0.58
1.58 ꢂ 0.47
0.63 ꢂ 0.17
7.95 ꢂ 2.65
5.28 ꢂ 0.30
5.10 ꢂ 1.56
6.13 ꢂ 0.73
2.40 ꢂ 0.46
7.47 ꢂ 0.58
1.85 ꢂ 0.18
0.92 ꢂ 0.15
0.26 ꢂ 0.01
17.56 ꢂ 4.63
4.54 ꢂ 0.885c
26.07 ꢂ 31.28c
9.71 ꢂ 5.59c
4.58 ꢂ 2.58c
9.21 ꢂ 5.24c
2.21 ꢂ 1.40c
1.20 ꢂ 3.80
0.26 ꢂ 0.07c
21.79 ꢂ 8.32c
Thyroid
c
ameans ꢂ SD (n=4). bn=3. n=5.
subsequent fast washout of radioactivity with time. The thyroid showed
relatively low radioactivity concentrations for 2-125IBA and 3-125IBA,
suggesting the reasonable stability of the label in regard to metabolic
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SYNTHESIS AND BIODISTRIBUTION OF RADIOIODINATED ASCORBIC ACID 743
deiodination. On the other hand, a much higher thyroid accumulation
was observed with 6-125IPA. Very little concentration of radioactivity in
the brain indicated very poor blood-brain permeability of all these
compounds. Biodistribution studies indicated that the propensity for
tumor uptake as well as the preferential uptake in the adrenal glands,
characteristic of tissue distribution of exogenously administrated L-
ascorbic acid,4,5 were not mirrored by any of these radioiodinated
analogs of ascorbic acid. The reasons for these different behaviors
are not clear, but it appears likely that increased lipophilicities of
the 125I-analogs has a significant influence on in vivo distribution
behavior. It is also conceivable that structural modification of
ascorbic acid by substitution with iodobenzyl or iodophenyl
groups may have led to a loss of the affinities for the transporters
of ascorbic acid which play central roles in the absorption and
accumulation of ascorbic acid in tissues.11,12
Conclusion
The three 125I-labelled forms of ascorbic acid possessing a m-iodobenzyl
or m-iodophenyl moiety on the hydroxy group were prepared by a melt
exchange procedure. These radioiodinated analogs of ascorbic acid
markedly differed in their biodistribution profiles from each other, and
from 14C-labeled ascorbic acid or 18F-DFA. Therefore, none of these
seems to be suitable radioiodinated analogs of l-ascorbic acid for
imaging study of its in vivo biochemistry.
Experimental
All melting points were obtained on a Yanaco micro melting point
1
apparatus and are uncorrected. H-NMR spectra were obtained on a
JEOL GX-270 spectrometer (270 MHz), and the chemical shifts are
reported in parts per million downfield from tetramethylsilane. 13C-
NMR spectra were obtained on a Varian Unity 500 spectrometer
(125 MHz), and the chemical shifts are reported in parts per million
downfield from tetramethylsilane. Infrared (IR) spectra were recorded
with a JASCO IR Report-100 spectrometer. Mass spectra and high
resolution-FAB-mass spectra were obtained with a JEOL JMS DX-610
and JEOL SX-102 mass spectrometer, respectively. The elemental
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744
F. YAMAMOTO ET AL.
analyses were performed by the staff of the micro-analytical section of
Kyushu University. Column chromatography was performed on
Kieselgel 60 (70–230 mesh, Merck), and analytical TLC was carried
out on Silica gel 60F 254 (Merck) or FL-60D (Fuji-gel). In the synthetic
procedures, organic extracts were routinely dried over anhydrous
Na2SO4 and evaporated with a rotary evaporator under reduced
pressure. HPLC was done using a Waters Associate Model 440 HPLC
system fitted with a Whatman Partisil 5PAC (10 ꢁ 100 mm) with
monitoring of the radioactivity as well as UV absorption (at 254 nm).
The radioactivity was also quantified with a Capintec radioisotope
calibrator (CRC-5). The identity of radiolabeled compounds was
supported by HPLC co-injection studies and specific activities
were determined by HPLC using cold mass calibration lines. Radio-
chemical yields were expressed at the end-of-synthesis, relative to the
amount of the Na125I as total activity present in the reaction vessel. All
chemicals were purchased from Nacalai Tesque, Japan and Na125I was
obtained from Amersham Pharmacia Biotech. All animal experiments
were carried out in accordance with the regulations on animal experiments
of the Faculty of Pharmaceutical Sciences, Kyushu University.
5,6-O-Isopropylidene-3-O-methoxymethyl-L-ascorbic acid (2). Under
argon atmosphere, to a solution of 5,6-O-isopropylidene-l-ascorbic
acid (1) (216 mg, 1.0 mmol) in DMF (2 ml) was added K2CO3 (276.4 mg,
2.0 mmol) and chloromethyl methyl ether (95.4 mg, 1.2 mmol). The
mixture was stirred at room temperature for 5 h. The reaction mixture
was diluted with water (5 ml) and then extracted with ethyl acetate
(30 ml). The organic layer was washed with a saturated NaCl aqueous
solution, dried and evaporated to dryness. The residue was purified by
chromatography on silica gel (chloroform and then chloroform:metha-
nol=10:1) to give compound (2) (77.2 mg, 30%) as a colorless powder,
1
mp 66–698C. H-NMR (CDCl3) d(ppm): 5.32(2 H, s), 4.62 (1 H, d,
J ꢀ 2:97 Hz), 4.33 (1 H, dt, J ¼ 2:97, 6.77 Hz), 4.16 (1 H, dd, J ¼ 6:76,
6.77 Hz), 4.06 (1 H, dd, J ¼ 6:60, 8.58 Hz), 3.61 (3 H, s), 1.39 (3 H, s),
1.36 (3 H, s). IR(neat): 3350, 1740, 1680 cmꢀ1. FAB-MS m/z:
261(MH+).
5,6-O-Isopropylidene-2-O-(m-iodobenzyl)-3-O-methoxymethyl-L-ascor-
bic acid (3). Under argon atmosphere, to a solution of (2) (680 mg,
2.61 mmol) in DMF (7 ml) was added K2CO3 (433 mg, 3.12 mmol) and
m-iodobenzyl bromide (775 mg, 2.61 mmol). The mixture was stirred at
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SYNTHESIS AND BIODISTRIBUTION OF RADIOIODINATED ASCORBIC ACID 745
room temperature for 40 min.The reaction mixture was diluted with
water (30 ml) and extracted with ethyl acetate (150 ml). The organic
layer was washed with saturated NaCl aqueous solution, dried and
evaporated to remove the solvent. The residue was purified by
chromatography on silica gel (hexane: ethyl acetate=4:1) to give
1
compound (3) (706 mg, 57%) as a colorless powder, mp 68–718C. H-
NMR (CDCl3) d (ppm): 7.80 (1 H, t, J ¼ 1:49 Hz), 7.67 (1 H, dt,
J ¼ 7:91, 1.32 Hz), 7.39 (1 H, dt, J ¼ 8:24, 1.32 Hz), 7.10 (1 H, t,
J ¼ 7:75 Hz), 5.36 (1 H, d, J ¼ 5:61 Hz), 5.32 (1 H, d, J ¼ 5:28 Hz), 5.12
(1 H, d, J ¼ 10:89 Hz), 5.05 (1 H, d, J ¼ 11:22 Hz), 4.58 (1 H, d,
J ¼ 2:97 Hz), 4.32 (1 H, dt, J ¼ 2:97, 6.7 Hz), 4.15 (1 H, dd, J ¼ 6:77,
8.42 Hz), 4.06 (1 H, dd, J ¼ 6:76, 8.41 Hz), 3.50 (3 H, s), 1.39 (3 H, s),
1.37 (3 H,s). IR(neat): 1760, 1690 cmꢀ1. FAB-MS m=z: 477 (MH+).
2-O-(m-Iodobenzyl)-L-ascorbic acid (4). To a solution of (3) (706 mg,
1.48 mmol) in methanol (50 ml) was added 10% HCl aqueous solution
(9.9 ml). The reaction mixture was stirred for 15.5 hrs at room
temperature followed by the evaporation of the solvent. The residue
was dissolved in water (10 ml) and extracted with ethyl acetate (30 ml).
The organic layer was washed with saturated NaCl aqueous solution,
dried and evaporated to dryness. The residue was recrystallized from
ethyl acetate to give compound (4)(558 mg, 96%) as a colorless powder,
mp 148–1508C. 1H-NMR (acetone-d6) d (ppm): 7.84 (1 H, d,
J ¼ 1:65 Hz), 7.67 (1 H, d, J ¼ 7:92 Hz), 7.46 (1 H, dd, J ¼ 7:59,
1.65 Hz), 7.16 (1 H, t, J ¼ 7:76 Hz), 4.99 (2 H, s), 4.83 (1 H, d,
J ¼ 1:98 Hz), 3.94 (1 H, ddd, J ¼ 1:98, 6.27, 9.57 Hz), 3.69 (1 H, dd,
J ¼ 6:27, 10.56 Hz), 3.64 (1 H, dd, J ¼ 7:42, 10.72 Hz). 13C-NMR
(CD3OD) d (ppm): 172.61, 161.86, 140.94, 138.44, 138.28, 131.19,
128.68, 121.52, 94.64, 76.94, 73.34, 70.63, 63.42. IR(neat): 3300, 1730,
1640 cmꢀ1. FAB-MS m=z: 393 (MH+). Analytically Calculated for
C13H13IO6: C, 39.82; H, 3.34. Found: C, 40.02; H, 3.32.
2-O-(m-[125I]Iodobenzyl)-L-ascorbic acid (5). To a glass vessel contain-
ing a solution of 2-O-(m-iodobenzyl)-l-ascorbic acid (4) (1.3 mg,
0.0033 mmol) in acetonitrile (130 ml) was added an aqueous NaOH
solution of Na125I (carrier free, 4–5 ml, 0.3–5.9 MBq). The mixture was
evaporated to dryness. The vessel was closed tightly and heated at
1608C in an oil bath for 9 min, and then cooled to room temperature.
The labeled (5) was purified by HPLC (Whatman Partisil 5PAC, 0.003 M
potassium phosphate buffer (pH=6): methanol=1:1, 3 ml/min). The
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F. YAMAMOTO ET AL.
radioactive fraction eluting at tR ¼ 16 min, corresponding to authentic 2-
IBA, was collected. The radiochemical yield was 27–60% and the specific
activity was 200–360 MBq/mmol. Chemical purity and radiochemical
purity were higher than 98%, respectively, as determined by HPLC.
5,6-O-Isopropylidene-3-O-(m-iodobenzyl)-L-ascorbic acid (6). Under
argon atmosphere, to a solution of 5,6-O-isopropylidene-l-ascorbic
acid (1) (1.0 g, 4.63 mmol) in DMF (15 ml) was added K2CO3 (830 mg,
6.00 mmol) and m-iodobenzyl bromide (1.79 g, 6.00 mmol). The reaction
mixture was stirred for 3.5 h at room temperature, then diluted with
water (50 ml) and extracted with ethyl acetate (180 ml). The organic
layer was washed with a saturated NaCl aqueous solution, dried and
evaporated to dryness. The residue was purified by chromatography on
silica gel (hexane:ethyl acetate=8:2 ! 6:4) to give compound (6)
1
(984 mg, 49%) as pale yellow oil. H-NMR (CDCl3) d(ppm): 7.76
(1 H, d, J ¼ 1:65 Hz), 7.70 (1 H, dd, J ¼ 7:92, 1.65 Hz), 7.36 (1 H, m),
7.13 (1 H, m), 5.43 (2 H, s), 4.58 (1 H, d, J ¼ 2:97 Hz), 4.30 (1 H, dt,
J ¼ 3:30, 6.60 Hz), 4.13 (1 H, dd, J ¼ 6:93, 8.25 Hz), 4.04 (1 H, dd,
J ¼ 6:60, 8.57 Hz), 1.39 (3 H, s), 1.37 (3 H,s). IR(neat): 3300, 1760,
1680 cmꢀ1. FAB-MS m=z: 433 (MH+).
3-O-(m-Iodobenzyl)-L-ascorbic acid (7). To a solution of 5,6-O-
isopropylidene-3-O-(m-iodobenzyl)-l-ascorbic acid (6) (150 mg,
0.35 mmol) in methanol–water (10:1, 4.4 ml) was added p-toluenesulfo-
nic acid monohydrate (66 mg, 0.35 mmol). The mixture was stirred for
12 h at room temperature and evaporated to dryness. The residue was
dissolved in water (10 ml) and extracted with ethyl acetate (30 ml). The
organic layer was washed with a saturated NaCl aqueous solution, dried
and evaporated to dryness. Recrystallization from ethyl acetate gave
compound (7) as a colorless powder (112 mg, 89%), mp 137–1408C. 1H-
NMR (acetone-d6) d(ppm): 7.87 (1 H, m), 7.72 (1 H, m), 7.52 (1 H, m),
7.20 (1 H, t, J ¼ 7:76 Hz), 5.54 (1 H, d, J ¼ 12:2 Hz), 5.43 (1 H, d,
J ¼ 12:2 Hz), 4.85 (1 H, d, J ¼ 1:65 Hz), 4.20 (1 H, bs), 3.92 (1 H, t,
J ¼ 6:43 Hz), 3.70 (2 H,m). 13C-NMR (CD3OD) d(ppm): 172.72,
151.17, 140.30, 138.50, 137.86, 131.35, 128.19, 121.36, 94.73, 76.72,
72.85, 70.51, 63.42. IR(neat): 3350, 1740, 1680 cmꢀ1. FAB-MS m=z: 393
(MH+). Analytically Calculated for C13H13IO6: C, 39.82; H, 3.34.
Found: C, 39.72; H, 3.33.
3-O-(m-[125I]Iodobenzyl)-L-ascorbic acid (8). To a glass vessel contain-
ing a solution of 3-O-(m-iodobenzyl)-l-ascorbic acid (7) (1.0 mg,
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND BIODISTRIBUTION OF RADIOIODINATED ASCORBIC ACID 747
0.0026 mmol) in acetonitrile (100 ml) was added an aqueous NaOH
solution of Na125I (carrier free, 10 ml, 2.9–4.7 MBq). The mixture was
evaporated to dryness under a stream of argon. The vessel was closed
tightly and heated at 1508C in an oil bath for 13 min, and then cooled to
room temperature. The labeled (8) was purified by HPLC (Whatman
Partisil 5PAC, H2O:methanol=7:3, 3 ml/min). The radioactive fraction
eluting at tR ¼ 8 min, corresponding to authentic 3-IBA, was collected.
The radiochemical yield was 12–45% and the specific activity was 200–
340 MBq/mmol. Chemical purity and radiochemical purity were higher
than 98%, respectively, as determined by HPLC.
6-O-(m-Iodophenyl)-L-ascorbic acid (11). Under argon atmosphere, to a
solution of potassium m-iodophenoxide (1.173 g, 4.55 mmol) in
acetonitrile (10 ml) was added methyl 2,3-O-isopropylidene-2-keto-L-
glonate 4,6-cyclic sulfate3 (9) (403 mg, 1.3 mmol). The mixture
was refluxed for 1 h and evaporated to dryness. To the residue was
added 35% HCl (6.5 ml, 5 ml/mmol), and the mixture was
refluxed for 2 hrs. After cooling to room temperature, the
reaction mixture was extracted with ethyl acetate (60 ml). The
organic layer was washed with a saturated NaCl aqueous
solution, dried and evaporated to dryness. The residue was
purified by chromatography on silica gel (ethyl acetate and then
ethyl acetate: methanol=9:1 ! 8:2 ! methanol) to give com-
pound (11) (370.8 mg, 76%) as a yellow powder. Further
purification by HPLC (nacalai tesque COSMOSIL 5C18MS-
AR300, H2O:methanol=45:55, flow rate=3 ml/min) afforded an
1
analytical sample, mp 155–1578C. H-NMR (CD3OD) d(ppm):
7.31–7.26 (2 H, m), 7.05–6.94 (2 H, m), 4.53 (1 H, s), 4.25 (1 H, bt,
J ¼ 5:2 Hz), 4.14 (1 H, dd, J ¼ 5:5, 9.57 Hz), 4.07 (1 H, dd,
J ¼ 7:3, 9.3 Hz). IR(neat): 3300, 1710, 1590 cmꢀ1. FAB-MS m=z:
401 (M+Na+), 379 (MH+). HR FAB-MS m=z calculated for
C12H12INaO6: 401.9576, found: 401.9577.
6-O-(m-Iodophenyl)-2,3,5-tri-O-methoxymethyl-L-ascorbic acid (12).
Under argon atmosphere, to an ice-cooled solution of dimethoxy-
methane (1 ml, 11.5 mmol) and P2O5 (ca.100 mg) in CHCl3 (1 ml) was
added a solution of 6-O-(m-iodophenyl)-l-ascorbic acid (11) (35.0 mg,
0.0926 mmol) in CHCl3 (1 ml). The mixture was stirred for 18 h at room
temperature. The reaction mixture was cooled at 08C and a saturated
NaHCO3 aqueous solution (10 ml) was added. The mixture was
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748
F. YAMAMOTO ET AL.
extracted with ethyl acetate (50 ml). The organic layer was dried and
evaporated to dryness. The residue was purified by chromatography on
silica gel with ethyl acetate and then ethyl acetate: methanol=2:1 to give
1
compound (12) (30 mg, 64%) as yellow oil. H-NMR (CDCl3) d(ppm):
7.34–7.26 (2 H, m), 7.04–6.86 (2 H,m), 5.56 (1 H, d, J ¼ 5:27 Hz), 5.42
(1 H, d, J ¼ 5:61 Hz), 5.20 (2 H, dd, J ¼ 14:52, 5.94 Hz), 4.96 (1 H, d,
J ¼ 1:65 Hz), 4.68 (2 H, dd, J ¼ 10:23, 6.93 Hz), 4.19 (1 H, ddd,
J ¼ 5:60, 5.60, 1.65 Hz), 4.18 (2 H, s), 3.55 (3 H, s), 3.54 (3 H, s), 3.38
(3 H, s). IR(KBr): 2500, 1760, 1670, 1580 cmꢀ1. FAB-MS m=z: 549
(M+K+), 533 (M+Na+), 511 (MH+).
6-O-(m-[125I]Iodophenyl)-L-ascorbic acid (13). To a glass vessel
containing 6-O-(m-iodophenyl)-2,3,5-tri-O-methoxymethyl-l-ascorbic
acid (12) (1.0 mg, 0.0020 mmol) was added an aqueous NaOH solution
of Na125I (carrier free, 5 ml, 1.9–2.7 MBq). The mixture was evaporated
to dryness under a stream of argon. The vessel was closed tightly and
heated at 1508C for 10 min in an oil bath, and then cooled to room
temperature. To the reaction mixture was added 35% HCl (1 gtt) and
the mixture was heated at 608C for 30 min.The labeled compound (13)
was purified by HPLC (Whatman Partisil 5PAC, 0.003M potassium
phosphate buffer (pH=6):methanol=1:2, 2.5 ml/min). The radioactive
fraction eluting at tR ¼ 9:3 min, corresponding to authentic 6-IPA, was
collected. The radiochemical yield was 20–48%, and the specific activity
was 200–350 MBq/mmol. Chemical purity and radiochemical purity
were higher than 98%, respectively, as determined by HPLC.
Partition coefficients. The log P values were measured using a
standard shake flask method. The non-radioactive samples were well
shaken with a mixture of 1-octanol (2.5 ml) and 0.05M phosphate buffer
(2.5 ml, pH7.4) for 20 min at 258C, after which aliquots of both phases
were taken for analysis by HPLC quantitation.
Reducing activity. According to the literature procedure3, 2-IBA, 3-
IBA, 6-IPA or l-ascorbic acid was added to a solution of a,a-diphenyl-
b-picrylhydrazyl (DPPH) (10ꢀ4 M) in ethanol. After 20 min, the
absorbance at 517 nm was measured. The percentage of the absorbance
change to that of the control was taken as the reducing activity.
Biodistribution in tumor-bearing mice. 3-Methylcholanthrene-induced
fibrosarcoma (FSa) was inoculated s.c. into the right hind leg muscle of
female C3H/He mice (5 weeks old, 16–18 g). Tumors which developed
with a diameter of about 1 cm at 9–14 days after inoculation were used.3
The animals were allowed free access to water and food at all times.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND BIODISTRIBUTION OF RADIOIODINATED ASCORBIC ACID 749
Aliquots of 2-125IBA, 3-125IBA or 6-125IPA in about 500 ml of saline
solution, with activities ranging from 40–460 kBq, were injected through
the tail vein of unanesthetized mice. At 1-, 10-, 30- and 60-min
postinjection intervals, the animals were killed by cervical dislocation
while under ether anesthesia. Samples of blood and the tissues of
interest, blotted free of blood, were taken, weighed, and assayed for
radioactivity in an Aloka Auto Well Gamma System ARC-300. The
results were expressed as percent injected dose per gram of tissue weight
(%dose/g).
Acknowledgements
The support of this work by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports and Culture of Japan is
gratefully acknowledged.
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