5-O-(4-[125I]iodobenzyl)-L-ascorbic acid: Electrophilic radioiodination and biodistribution in mice

Article abstract of DOI:10.1248/cpb.60.235

As a part of our efforts to develop potential imaging agents for ascorbate bioactivity, 5-O-(4-[¹²⁵I] iodobenzyl)-L-ascorbic acid ([ ¹²⁵I]1) was prepared through a two-step sequence which involved radioiodo-destannylation of a protec

Full text of DOI:10.1248/cpb.60.235

—
February 2012
Regular Article
Chem. Pharm. Bull. 60(2) 235 240 (2012)
235
5-O-(4-[125I]Iodobenzyl)-L-ascorbic Acid: Electrophilic Radioiodination
and Biodistribution in Mice
Jintaek Kim, Tomohiro Kino, Hiroharu Kato, Fumihiko Yamamoto, Kohei Sano,
†
,
‡
,
Takahiro Mukai,* and Minoru Maeda*
Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582,
Japan. Received October 31, 2011; accepted November 28, 2011; published online December 2, 2011
As a part of our efforts to develop potential imaging agents for ascorbate bioactivity, 5-O-(4-[125I]
iodobenzyl)-L-ascorbic acid ([125I]1) was prepared through a two-step sequence which involved radioiodo-
destannylation of a protected tributylstannyl precursor 6, followed by hydrolysis in acidic methanol of the
protecting groups in 61% overall radiochemical yield, with a radiochemical purity of over 98% and a specific
activity of more than 15.4GBq/μmol. Tissue distribution of [125I]1 in tumor-bearing mice showed signs of
distribution profiles similar to the reported results for 6-deoxy-6-[18F]fluoro-L-ascorbic (6-18FAsA) acid and
6-deoxy-6-[131I]iodo-L-ascorbic acid (6-131IAsA) but with notable differences in the adrenal glands, in which
considerably lower uptake of radioactivity and rapid clearance with time were observed. Pretreatment of
mice with a known inhibitor of ascorbate transport, sulfinpyrazone, did not produce any significant change
in the adrenal uptake of radioactivity after injection of [125I]1 compared to the control, suggesting that
uptake in the adrenal glands is independent of the sodium-dependent vitamin C transporter 2 transport
mechanism. Introduction of a bulky substituent at C-5 on AsA, such as an iodobenzyloxy group, may not be
suitable for the design of analogs that may still be able to maintain characteristic distribution properties in
vivo seen with AsA itself.
Key words ascorbic acid analog; radioiodine; iododestannylation; biodistribution; mouse; imaging agent
L-Ascorbic acid (AsA), the reduced form of vitamin C, is the C-6 position of the AsA core, and their potential imaging
highly concentrated in the neurons in the brain in mammalian characteristics were also evaluated in rodents. Among them,
—
13)
bodies, which likely indicates its essential roles in neuronal 6-deoxy-6-[18F]fluoro-L-ascorbic acid (6-18FAsA)10
and
function and protection against oxidative stress.1,2) Currently, 6-deoxy-6-[131I]iodo-L-ascorbic acid (6-131IAsA)14,15) showed
the numerous functions of AsA in the brain have stimulated the expected distribution of highest uptake in the adrenal
multidisciplinary interest in this molecule.3,4) In recent years, glands in rodents, a neuroendocrine organ that is known to
two transport mechanisms by which AsA enters the central highly express the SVCT-2 transporter,16) thus demonstrating
nervous system (CNS) from plasma have been identified and their suitability as radiotracer analogs of AsA. However, the
characterized.5) One is that AsA enters the cerebrospinal fluid application of these radiotracers as brain-targeted imaging
(CSF) directly through the choroid plexus via the sodium- probes for in vivo studies was found to be limited because of
dependent vitamin C transporter 2, SVCT-2, the likely major their poor delivery from blood circulation into the brain.
pathway to brain cells. The other route of entry into the CNS
In an attempt to improve the brain targeting of a radiola-
involves the uptake of dehydroascorbic acid (DHA), the oxi- beled AsA analog, we have become interested in the use of
dized form of AsA, on glucose transporters of the GLUT fam- the oxidized form of AsA, in view of the uptake behavior
ily in the blood-brain barrier endothelium, followed by rapid of the pro-drug type of DHA in vivo, and have designed
intracellular reduction of transported DHA to AsA. However, 5-O-(4-iodobenzyl)-L-ascorbic acid (1) as a potential probe
it is argued that this DHA transport is unlikely to play a major molecule. This 5-O-substituted AsA analog can be expected
role in AsA supply to the brain due to only minimal quanti- to have the hydrated bicyclic hemiketal structure like AsA in
ties of DHA in the plasma and competition with glucose for aqueous solution (Chart 1), which appears to be a molecular
GLUT transport.5,6) Literature reports have also shown that species capable of interacting with the GLUTs.17,18) Recently,
AsA can enter neurons through SVCT-2,1,7) and that there is we developed a synthetic route for the preparation of 5-O-
a regional difference in the concentration of AsA within the (4-iodobenzyl)-L-ascorbic acid through a multi-step sequence
brain, being potentially reflective of the level of SVCT-2 dis- starting from AsA, such that it would be amenable to prepara-
tribution.4,8,9)
tion of the corresponding labeled analog, and this compound
We are interested in exploring radiotracer probes of AsA
analogs for the visualization of biochemical events associ-
ated with the functions and transport of AsA in the brain,
utilizing a nuclear imaging technique. In previous studies,
we reported the synthesis of several AsA analogs, focusing
on the introduction of radioisotopes such as 18F and 131I at
† Present address: Kobe Pharmaceutical University; 4–19–1 Moto-
yamakita-machi, Higashinada-ku, Kobe 658–8558, Japan.
‡ Present address: Daiichi University of Pharmacy; 22–1 Tamagawa-cho,
Minami-ku, Fukuoka 815–8511, Japan.
Chart 1. 5-O-(4-Iodobenzyl)-L-ascorbic Acid (1) and Predicted Con-
version to Its Oxidized Form (2)
© 2012 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: tmukai@kobepharma-u.ac.jp
m-maeda@daiichi-cps.ac.jp

236
Vol. 60, No. 2
Reagents and conditions: (i) 4-trimethylsilylbenzyl bromide, Ag2O, CaSO4,
benzene, r.t. (ii) 4-bromobenzyl bromide, Ag2O, CaSO4, r.t. (iii) Pd(PPh3)4,
Sn2(n-Bu)6, toluene, reflux.
Chart 2. Synthesis of Labeling Precursors
Reagents and conditions: (i) Na[125I]I, N-chlorosuccinimide, AcOH or
AcOH–CF3COOH (ii) Na[125I]I, chloramine-T, AcOH–EtOH, r.t., 2h (iii) ery-
Fig. 1. (A) Analytical HPLC Chromatogram of Deprotection Reaction
Mixture of Intermediate [125I]7 with 1.0M HCl–MeOH at 50 C for
°
thorbic acid, 1.0M HCl–MeOH, 60 C, 30min.
°
Chart 3. Radiosynthesis of 5-O-(4-[125I]Iodobenzyl)-L-ascorbic Acid
([125I]1)
30min, Showing Radioactive Profile, and (B) Under the Same Reaction
Conditions in the Presence of Erythorbic Acid, Showing Radioactive
Profile
HPLC analytical conditions: COSMOSIL 5C18AR-II, 15mM phosphate buffer
(pH=6.0)/MeOH=60/40, flow rate=0.8mL/min.
was found to have almost the same reducing activity as AsA
itself.19) We report here the radiosynthesis of 5-O-(4-[125I]
iodobenzyl)-L-ascorbic acid ([125I]1) via a two-step procedure acid hydrolysis reaction to remove all the protecting groups,
and its biodistribution in tumor-bearing mice, in order to ob- this step yielded complicated reaction mixtures, giving an
tain reference information for the subsequent development of unacceptable low yield of the desired product, as illustrated by
its oxidized form.
a typical HPLC profile in Fig. 1A (reaction conditions: 1.0M
°
HCl–MeOH, 50 C, 30min). It was assumed that the forma-
tion of byproducts, probably resulting from oxidative damage
Results and Discussion
Two organometallic compounds (4, 6) were prepared as of the desired product, would be induced by exposure to both
precursors for electrophilic radioiodination,20) as depicted in the acidic medium and elevated temperatures. We considered,
Chart 2. Trimethylsilylated precursor 4 was synthesized by therefore, incorporation of anti-oxidant stabilizers into the re-
4-trimethylsilylbenzylation of the 2,3,6-tri-O-modified deriva- action mixture. Thus, the presence of erythorbic acid (10μg),
tive of AsA (3) and tributylstannane 6 was prepared by heat- a stereoisomer of AsA and a well-known antioxidant food
ing to reflux a mixture of 5-O-bromobenzylated compound 5 additive,21) in a reaction mixture was found to be effective in
with an excess of hexabutylditin, and a catalytic amount of minimizing the formation of byproducts occurring during the
Pd(PPh3)4 in dry toluene for 5h. The first attempt to prepare reaction, as analyzed by HPLC (Fig. 1B). Based on these pre-
[
125I]7 by radioiodo-desilylation of 4 with a mixture of no-car- liminary observations, the deprotection reactions of intermedi-
rier-added Na[125I]I and N-chlorosuccinimide in AcOH, or ad- ate [125I]7 in the presence of erythorbic acid were carried out
ditionally containing a small amount of trifluoroacetic acid at under some different conditions, giving good conversion to
125
°
[ I]1 on heating with 1.0M HCl–MeOH at 60 C for 30min as
various temperatures proved to be inefficient, and the silicon
precursor showed significant decomposition, leading to no for- shown in Table 1. Thus, the preparation of [125I]1 from inter-
mation of the desired compound (Chart 3). On the other hand, mediate [125I]7 and isolation by HPLC (COSMOSIL 5C18AR-
treatment of tributylstannyl precursor 6 with no-carrier-added II, 15mM phosphate buffer (pH=6.0)/MeOH=60/40) was
Na[125I]I in the presence of chloramine-T in an AcOH–EtOH repeated. However, considerable decomposition of the final
solution at room temperature for 2h yielded a radiochemically product was observed in the HPLC isolation step, demonstrat-
and chemically pure product of [125I]7 after HPLC purifica- ing an inherent instability of the radiotracer. In an additional
tion, with a radiochemical yield of 77.5 8.3%.
attempt to overcome this unwanted outcome and, also, to find
In this study, deprotection of intermediate [125I]7 was found a suitable means for retaining high radiochemical purity for
to be a critical step. During the initial phases of studying the some period of storage, we prepared an HPLC solvent system

February 2012
237
Table 1. Deprotection Reaction of Intermediate [125I]7 by Acid Hydrolysis in an H2O–MeOH Mixture Containing Erythorbic Acid
Yielda) (%)
°
Entry
Temperature ( C)
Solvent
Time (min)
1
2
3
4
r.t.
50
60
60
HCl (0.1M)
HCl (0.1M)
HCl (0.1M)
HCl (1.0M)
30
30
30
30
No reaction
22.4
68.0
78.7
a) Isolated radiochemical yield by HPLC (COSMOSIL 5C18AR-II, 15mM phosphate buffer (pH=6.0)/MeOH=60/40, flow rate=0.8mL/min).
of [125I]1 to reach the brain. Moreover, tumor accumulation
in the fibrosarcoma was not significant, also similar to those
of 6-18FAsA and 6-131IAsA. Recent studies have shown that
high-grade tumor tissue has reduced capacity to accumulate
AsA relative to normal tissue.23) On the other hand, contrary
to our expectations, the maximum adrenal uptake of radio-
activity (12.52 2.52 ID%/g) was seen at 2min postinjection,
which was 3.9-fold lower than that observed for 6-131IAsA in
mice at the same time point,14) and then it rapidly decreased
to 2.20 1.44 ID%/g at 30min after injection with an adrenal-
to-liver ratio of only 0.4 for [125I]1 versus 4.3 for 6-131IAsA
at 10min postinjection. Thus, mouse biodistribution studies
showed very low accessibility of [125I]1 to the adrenal glands,
different from 14C-AsA,24) 6-18FAsA and 6-131IAsA, all of
which showed the preferential uptake of radioactivity in the
adrenal glands. Attempts to identify accumulated radioactivity
in the adrenals and blood of mice failed to show optimal con-
ditions for analysis: TLC analysis was considerably complex,
being accompanied by the formation of many radioactive spe-
cies probably produced in the attempted tissue homogeniza-
tion and/or extraction steps, and chromatographic separation
due to the labile nature of compound.
Fig. 2. An HPLC Analysis of 5-O-(4-[125I]Iodobenzyl)-L-ascorbic Acid
([125I]1), Coinjected with Authentic Sample, after Purification by HPLC
15mM phosphate buffer (pH=6.0)–MeOH=60:40 containing 400μM DL-homo-
cysteine, flow rate=0.8mL/min.
DL-Homocysteine, a sulfur amino acid, is an intermediate
metabolite of methionine. An increased homocysteine level in
of 15mM phosphate buffer (pH=6.0)–MeOH=60:40 contain- plasma is a risk factor for several chronic pathologies, includ-
ing 400μM DL-homocysteine, a sulfur–containing amino acid, ing cardiovascular diseases, atherosclerosis and chronic renal
as an antioxidant stabilizer, based on literature reports of vari- failure.25) The amount of DL-homocysteine administered for
ous HPLC analytical techniques for AsA analysis.21,22) A con- the distribution studies was estimated to be 0.04μmol/mouse.
sequence was that a buffer solution of [125I]1, obtained after In separate experiments, mice (n=3) bearing fibrosarcoma
HPLC isolation using this solvent system, had a reproducible were injected with a buffer solution of [125I]1 in the absence
radiochemical purity of above 98% (Fig. 2), and its radio- of DL-homocysteine, although its radiochemical purity was not
chemical purity was maintained at 92% up to 3h after storage determined (probably less than 70% based on our experience),
°
at 4 C, but dropped to about 70% in 12h.
and its biodistribution at 10min postinjection was examined
Thus, by the two-step procedure described above, the target for comparison with that (n=3) using an injectable solution
[
125I]1, free of erythorbic acid contaminants, was successfully containing DL-homocysteine. The accumulation pattern of ra-
isolated in 61% overall radiochemical yield, ready for intrave- dioactivity in the tissues was similar between the two groups
nous injection into mice with specific activity of over 15GBq/ (data not shown), seemingly indicating that homocysteine was
μmol. The total preparation time, including two HPLC purifi- without significant influence on the mouse distribution profile
cations and isolation procedures, was about 4h.
of [125I]1, although under limited experimental conditions.
Sulfinpyrazone is a uricosuric medication used to treat gout
To explore the in vivo tissue distribution characteristics of
[
125I]1, biodistribution studies were performed using C3H/He and has also been used as a blocker of ascorbate transport
mice bearing fibrosarcoma, and the results are expressed as % in some cell types in vitro.26,27) We previously reported that
of injected dose (ID)/g of tissue, as shown in Table 2. There 6-131IAsA uptake by the adrenal glands of rats was signifi-
was a somewhat slower clearance of the radioactivity from cantly inhibited by pretreatment with sulfinpyrazone (1.5μg/g
—
13)
the blood when compared to 6-18FAsA10
and 6-131IAsA14,15) per animal) in vivo.15) In this study a similar experiment was
studied previously. The accumulation of radioactivity in se- carried out to see the response to sulfinpyrazone-treatment
lected organs was highest in the kidneys>liver>lungs>adre for [125I]1. As shown in Fig. 3, predosing of mice with sulfin-
nals>heart at 2min postinjection and then the radioactivity pyrazone (1.5μg/g per animal) did not produce any change of
levels gradually decreased with time, similar to those seen uptake in the adrenal glands compared to the control group.
for 6-18FAsA and 6-131IAsA. As expected, the brain uptake This is a strong indication that the distribution of [125I]1 to the
was limited, ranging from 0.62 0.08 ID%/g at 2min postin- adrenal glands is not SVCT-mediated (the SVCT-2 subtype
jection to 0.22 09 ID%/g at 60min, suggesting the inability that is the main isoform in adrenal glands), although we do

238
Vol. 60, No. 2
Table 2. Biodistribution of Radioactivity in C3H/He Mice Bearing Fibrosarcoma at Various Time Points after i.v. Injection of 5-O-(4-[125I]
Iodobenzyl)-L-ascorbic Acid ([125I]1)
Tissue concentrations of radioactivity (% injected dose/g tissue)
Tissue
2min
10min
30min
60min
Blood
Adrenals
15.05 0.41
12.52 2.52
4.51 0.43
4.02 0.29
3.04 0.13
0.07 0.05
4.13 0.65
2.18 0.83
3.38 0.53
0.57 0.26
61.30 20.21
28.97 3.84
8.17 0.57
15.56 4.31
1.99 0.21
3.07 0.51
5.33 0.74
0.62 0.08
2.92 0.51
4.23 0.74
1.69 0.26
8.15 1.00
7.49 2.25
2.16 0.14
1.89 0.21
2.24 0.54
0.07 0.02
4.72 1.13
8.50 2.56
3.25 2.72
0.38 0.12
19.69 4.66
12.70 3.14
3.76 0.34
5.40 0.51
1.34 0.24
1.87 0.34
2.40 0.76
0.52 0.11
1.43 0.24
2.32 1.06
1.63 0.20
4.30 0.30
2.20 1.44
0.81 0.19
0.82 0.24
0.82 0.24
0.14 0.09
3.93 1.64
8.53 3.61
1.59 0.56
7.53 8.11
9.38 6.29
3.34 1.55
1.32 0.36
2.33 1.04
0.52 0.11
1.09 0.24
1.00 0.33
0.34 0.02
0.54 0.29
1.90 0.41
0.91 0.06
3.30 0.72
1.83 0.73
0.70 0.15
0.85 0.49
0.91 0.38
0.87 1.46
6.82 3.60
13.93 3.32
2.10 0.68
2.66 2.89
5.28 4.88
2.38 0.79
1.16 0.25
2.29 0.49
0.53 0.38
1.13 0.96
2.41 1.12
0.22 0.09
0.71 0.38
1.30 0.57
0.59 0.13
Spleen
Pancreas
Stomach
Stomach content
Small intestine
Small intestine content
Large intestine
Large intestine content
Kidneys
Liver
Heart
Lungs
Muscle
Skin
Thyroid
Brain
Bone
Ovaries
Tumor
Results expressed as mean %ID/g tissue S.D., n=3 per data point.
hydrophobic iodobenzyl substituent at the C-5 position of AsA
may not be able to interact with the transporter, probably due
to its steric hindrance within the site.
Conclusion
We successfully prepared 5-O-(4-[125I]iodobenzyl)-L-ascor-
bic acid ([125I]1) with reasonable radiochemical yields, high
radiochemical purity and specific activity. Biodistribution
studies in mice showed low uptake of radioactivity for [125I]1
in the adrenal glands, followed by rapid elimination of radio-
activity with time. The insensitivity of a blocking effect by
pretreatment with sulfinpyrazone is indicative of no or only
poor affinity of this radiotracer for the SVCT-2 transporter,
although further experiments are needed to confirm this point.
Such biological properties of [125I]1 might be problematic for
in vivo application of its oxidized form as a prodrug-type
radiotracer of AsA, because, unlike AsA itself, this analog
may be not able to enter neurons in the brain through SVCT2
Fig. 3. Radioactivity Levels in Selected Mice Tissues in Control and
Sulfinpyrazone Pre-treatment Conditions, 2min Following i.v. Injection
of 5-O-(4-[125I]Iodobenzyl)-L-ascorbic Acid ([125I]1)
—
Results expressed as mean %ID/g tissue S.D., n=3 4 for each group.
not have information on the levels of the SVCT transporters in from cerebrospinal fluid. Nevertheless, our proposed strategy
our tissue sample. is now a starting point for the development of radiolabeled
There are some literature reports on the substrate specificity AsA analogs targeting the central nervous system, and further
—
32)
of the AsA transporter using biological tissues or cells.28
research is going to prepare the oxidized form of [125I]1 to
As most AsA exists as a monovalent anion at physiological evaluate its biodistribution.
pH and SVCT has a high affinity for the ionic form, it has
been suggested that the ionic interactions between SVCT and
its substrates are the predominant driving forces for the bind-
Experimental
Chemical reagents and solvents were of commercial quality
ing of SVCT.31,32) Studies by Rumsey et al.33) demonstrated and were used without further purification unless otherwise
that one of the structural requisites of AsA and its analogs noted. Benzene and toluene were purified by drying over CaH2
1
for transport in cells is an S-absolute configuration at the C-4 and distillation. H-NMR spectra were obtained on a Varian
position in a five-membered reduced ring with no substitution Inova 400 (400MHz) and were referenced to tetramethylsilane
on the C-2 and C-3 positions, and that 6-deoxy-6-halogeno-L- (δ=0ppm). IR spectra were recorded with a Shimadzu FTIR-
ascorbic acid is an effective compound for inhibiting AsA 8400 spectrometer. Mass spectra were obtained with a JEOL
uptake. For the case of [125I]1, it is conceivable that the bulky JMS DX-610 (FAB-MS) or an Applied Biosystems Mariner

February 2012
239
System 5299 spectrometer (electrospray ionization (ESI)-MS)). J=5.5Hz), 5.57 (d, 1H, J=5.5Hz), 7.14 (d, 2H, J=8.5Hz), 7.44
Column chromatography was performed on Silica gel 60N (dd, 2H, J=1.8, 6.5Hz); IR (neat) cm−1: 1770, 1681; FAB-MS
(63 210 mesh, Kanto Chemical Co., Inc., Japan), the progress (m/z): 635.2 [M]+.
—
of the reaction was monitored by TLC on Silica gel 60F 254
6 - O -tert-Butyldimethylsilyl-2, 3 - O -dimethoxy-
plates (Merck, Germany), and the spots were visualized with ethoxymethyl-5-O-(4′-tri-n-butylstynylbenzyl)-L-ascorbic
UV light or by spraying with 5% alcoholic molybdophos- Acid (6) A solution of 5 (202mg, 318μmol), Pd(PPh3)4
phoric acid. In the synthetic procedures, the organic extracts (24.5mg, 21.2μmol) and hexabutylditin (369μL, 636μmol) in
were routinely dried over anhydrous Na2SO4 and evaporated dry toluene (15mL) was heated to reflux for 5h. The reaction
with a rotary evaporator under reduced pressure. HPLC was mixture was diluted with CHCl3 and filtered through a short
done by using a Liquid Chromatograph system (GL-7410/GL- pad of Celite®. The combined filtrate was concentrated in
7450, GL Science, Japan) coupled in series with an NaI(Tl) vacuo. The residue was purified with silica flash chromatog-
detector (B-FC-3200, Bioscan Inc., Washington, DC, U.S.A.) raphy (hexane–EtOAc, 1:1 v/v) to give 6 (127mg, 47%) as a
1
by monitoring the radioactivity as well as the UV absorp- pale yellow oil. H-NMR (CDCl3) δ: 0.04 (s, 3H), 0.05 (s, 3H),
—
—
tion (at 254nm). All solvents used as mobile phases in HPLC 0.87 0.94 (m, 18H), 1.04 (t, 6H, J=8.1Hz), 1.28 1.42 (m,
—
—
procedures were bubbled with nitrogen gas before use. The 6H), 1.49 1.63 (m, 6H), 3.34 (s, 3H), 3.35 (s, 3H), 3.49 3.53
—
—
radioactivity was quantified with an auto-well gamma counter (m, 4H), 3.76 (s, 3H), 3.78 3.82 (m, 2H), 3.85 3.89 (m, 2H),
(ARC-370, Aloka, Japan). Identity of the labeled compound 4.55 (s, 2H), 4.90 (s, 1H), 5.21 (d,1H, J=5.8Hz), 5.22 (d, 1H,
was confirmed from co-injection with authentic samples by J=5.8Hz), 5.42 (d, 1H, J=5.5Hz), 5.60 (d, 1H, J=5.5Hz),
HPLC under the same conditions. Specific radioactivity and 7.21 (d, 2H, J=7.6Hz), 7.41 (d, 2H, J=7.6Hz) ; IR (neat)
radiochemical purity were determined by the same HPLC cm−1: 2954, 1770; ESI(+)-MS (m/z) Calcd for C39H71O10SiSn
system. No-carrier-added sodium [125I]iodide (0.01N NaOH so- 847.3842 [M+H]+, Found 847.3813 [M+H]+.
lution; >14.8GBq/mL) was purchased from MP Biomedicals
Radiochemistry. 2,3-O-Dimethoxyethoxymethyl)-5-O-
(U.S.A.). Animal experiments were carried out in accor- 4-[125I]iodobenzyl-6-O-tert-butyldimethylsilyl-L-ascorbic
dance with our institutional guidelines and were approved Acid ([125I]7) No-carrier-added Na125
I
(3.7 18.5MBq),
—
by the Animal Care and Use Committee, Kyushu University. chloramine-T (10μg), and acetic acid (3.3μL) were dissolved
Statistical analysis: Quantitative data were expressed as sequentially in a solution of 6 (100μg) in EtOH (50μL). The
’
mean S.D. Means were compared using Student s t-test and p mixture was allowed to stand for 2h at room temperature and
values <0.05 were considered statistically significant. the entire reaction mixture was injected into an HPLC column
6 - O -tert-Butyldimethylsilyl-2, 3 - O -dimethoxy- (Nacalai Tesque COSMOSIL 5C18 AR-II, 4.6×250mm, H2O/
ethoxymethyl-5-O-(4′-trimethylsilylbenzyl)-L-ascorbic Acid MeOH=13/87, flow rate 0.8mL/min). The product fraction
(4) To a solution of 6-O-tert-butyldimethylsilyl-2,3-O-di- corresponding to 7 was collected after a retention time of
methoxyethoxymethyl-L-ascorbic acid 3 (511mg, 1.10mmol)19) 13min. The isolated radiochemical yield was 77.5 8.3% with
in dry benzene (15mL), in a flask covered with aluminum foil, radiochemical purity of above 98%, as determined by HPLC.
was added CaSO4 (530mg), Ag2O (570mg, 1.37mmol) and
5-O-(4′-[125I]Iodobenzyl)-L-ascorbic Acid ([125I]1) A so-
4-trimethylsilylbenzyl bromide (332mg, 1.37mmol) sequen- lution of intermediate [125I]7 (3.7 11.1MBq) in an HPLC
tially. The mixture was stirred for 24h. Additional aliquots eluate (a mixture of H2O and MeOH) obtained by electrophilic
of Ag2O, CaSO4 and 4-trimethylsilylbenzyl bromide were radioiodination was added to a reaction vial and the solvent
then added in the same quantities as before and the reaction was evaporated at room temperature. To find out the appropri-
mixture was stirred for an additional 4d. The mixture was ate reaction conditions for the deprotection step, to this resi-
diluted with EtOAc and filtered through a short pad of Celite®. due erythorbic acid (10μg) and methanol containing 0.1M–HCl
The combined filtrate was evaporated to dryness. The residue or 1.0M HCl (100μL) were added, and the vial was sealed. The
was chromatographed on silica gel (EtOAc:hexane=1:3) to mixture was then allowed to proceed at room temperature,
—
°
°
give the required product 4 (325mg, 47.2%) as a viscous oil. or heated at 50 C or 60 C for 30min, as shown in Table 1.
1H-NMR (CDCl3) δ: 0.04 (s, 3H Hz), 0.05 (s, 3H), 0.25 (s, In preparative runs, the deprotection reaction of intermediate
125
—
°
[ I]7 was carried out by heating with 1.0M HCl at 60 C for
9H), 0.88 (s, 9H), 3.34 (s, 3H), 3.35 (s, 3H), 3.52 3.49 (q,
—
4H, J=4.3Hz), 3.77 3.89 (m, 7H), 4.55 (d, 1H, J=12.0Hz), 30min in the presence of erythorbic acid. This acidic solution
4.59 (d, 1H, J=12.0Hz), 4.89 (s, 1H), 5.20 (d, 1H, J=5.8Hz), was then neutralized with saturated aqueous NaHCO3 and
5.26 (d, 1H, J=5.8Hz), 5.39 (d, 1H, J=5.5Hz), 5.58 (d, 1H, the resulting entire mixture was injected into an HPLC col-
J=5.5Hz), 7.23 (d, 2H, J=9.1Hz), 7.47 (d, 2H, J=7.6Hz); IR umn (Nacalai Tesque COSMOSIL 5C18 AR-II, 4.6×250mm,
(neat) cm−1: 1770, 1687; FAB-MS (m/z): 629.3 [M+H]+.
15mM phosphate buffer containing 400μM DL-homocysteine
6 - O -tert-Butyldimethylsilyl-2, 3 - O -dimethoxy- (pH= 6.0)/MeOH=60/40, flow rate 0.8mL/min). The radioac-
ethoxymethyl-5-O-(4′-bromobenzyl)-L-ascorbic Acid (5) tive fraction containing the required product (retention time:
Compound 5 was prepared from 3 using 4-bromobenzyl 13min) was collected in a flask and the MeOH from the eluent
°
bromide following a procedure similar to that for 4. After was evaporated at 30 C. By the two-step procedure described
the reaction mixture was stirred for 3d, the crude product above, the target [125I]1, free of erythorbic acid contaminants,
was chromatographed on silica gel (EtOAc:hexane=1:3) to was isolated in 61% overall radiochemical yield. Total prepa-
1
give the required product 5 (37%) as a viscous oil. H-NMR ration time starting from the radoiodination step was 4h. The
(CDCl3) δ: 0.06 (s, 3H), 0.07 (s, 3H), 0.89 (s, 9H), 3.35 (s, specific activity of the obtained [125I]1 was above 15.4GBq/
—
6H), 3.51 (q, 4H, J=4.9Hz), 3.76 3.87 (m, 7H), 4.49 (d, 1H, μmol, as determined by direct measurement of the HPLC
J=12.0Hz), 4.58 (d, 1H, J=12.0Hz), 4.87 (d, 1H, J=1.2Hz), eluate using a UV detector. The HPLC-collected fraction
5.20 (d, 1H, J=5.8Hz), 5.25 (d, 1H, J=5.8Hz), 5.42 (d, 1H, of [125I]1 was allowed to stand at 4 C and at several inter-
°

240
Vol. 60, No. 2
—
70 75 (1999).
vals from zero to 6h, and the samples were withdrawn and
analyzed for stability by HPLC using the above conditions.
At 12h, two radioactive species eluted before intact [125I]1,
appeared with a radioactive ratio of about 30%, without any
indication of the formation of the free 125I-iodide ion.
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C3H/He mice (5 weeks old, 15 18g). These mice bearing
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tumors which developed with a diameter of about 1cm at
—
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14d after inoculation were used for the in vivo biodistri-
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bution studies. The tumor-bearing mice were intravenously
injected through the tail vein with a solution of [125I]1 (37kBq,
100μL) in an HPLC eluent containing DL-homocysteine
(0.04μmol/mouse). The mice were sacrificed by ether an-
esthesia at a predetermined time after injection. A sample
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radioactivity as described above.
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Acknowledgments Support for this study from the
Asahi Glass Foundation and the program for the Creation of
Innovation Centers for Advanced Interdisciplinary Research
Areas from Special Coordination Funds for Promoting
Science and Technology (SCF) commissioned by the Ministry
of Education, Culture, Sports, Science and Technology
(MEXT) of Japan is gratefully acknowledged. We also thank
Prof. Takashi Ohshima of Graduate School of Pharmaceutical
Sciences, Kyushu University for valuable discussion.
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