Reactivity of 6-halopurine analogs with glutathione as a radiotracer for assessing function of multidrug resistance-associated protein 1

Article abstract of DOI:10.1021/jm901332c

6-Bromo-7-[¹¹C]methylpurine is reported to react with glutathione via glutathione S-transferases in the brain and to be converted into a substrate for multidrug resistance-associated protein 1 (MRP1), an efflux pump. The compound with a rapid c

Full text of DOI:10.1021/jm901332c

7
284 J. Med. Chem. 2009, 52, 7284–7288
DOI: 10.1021/jm901332c
Reactivity of 6-Halopurine Analogs with Glutathione as a Radiotracer for Assessing Function of
Multidrug Resistance-Associated Protein 1
Toshimitsu Okamura, Tatsuya Kikuchi,* Kiyoshi Fukushi, and Toshiaki Irie
Probe Research Section, Department of Molecular Probe, Molecular Imaging Center, National Institute of Radiological Sciences,
4
-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
Received June 15, 2009
1
1
-Bromo-7-[ C]methylpurine is reported to react with glutathione via glutathione S-transferases in the
6
brain and to be converted into a substrate for multidrug resistance-associated protein 1 (MRP1), an
efflux pump. The compound with a rapid conversion rate allows quantitative assessment of MRP1
function, but this rate is probably susceptible to interspecies differences. Hence, for application to
different species, including humans, it is necessary to adjust the conversion rate by modifying the
1
4
chemical structure. We therefore designed 6-halo-9-(or 7)-[ C]methylpurine (halogen: F, Cl, Br, or I),
and evaluated them in vitro with respect to enzymatic reactivity with glutathione using brain
homogenates from the mouse, rat, or monkey. There was a marked difference in reactivity between
these species. Changes in the position of the methyl group and halogen on N-methyl-6-halopurine
provided various compounds possessing wide-ranging reactivity with glutathione. In conclusion, the
1
1
adjustment of reactivity of 6-bromo-7-[ C]methylpurine may allow assessment of MRP1 function in
the brain in various species.
Introduction
blood-brain barrier after intravenous injection. It is immedi-
ately converted to a hydrophilic substrate for MRP1, a glu-
tathione (GSH) conjugate, in the brain. This allows assessment
of MRP1 function by an estimation of the efflux rate of the
substrate from the time-radioactivity curve after complete
disappearance of the unconjugated radiotracer. The efflux rate
of the GSH conjugate S-[6-(7-methylpurinyl)]glutathione
a
Multidrug resistance-associated protein 1 (MRP1 ), a
member of the ATP binding cassette superfamily of transpor-
ters, contributes to various physiological functions, including
defense against xenobiotics and toxic metabolites, as well as
1
-4
protection from the toxic effect of oxidative stress.
This
protein has an unusually broad substrate specificity and can
transport numerous hydrophobic compounds, including ther-
(
approximately 90% in Mrp1 knockout mice compared with
PSG) was estimated with high precision and decreased by
5
apeutic drugs and hydrophilic compounds. It has been
reported that adriamycin, a chemotherapeutic agent, affects
8
wild-type mice.
6
MRP1 expression in the mouse brain. A slight increase in
MRP1 expression was observed in hippocampal samples
Small-animal models of human disease, in particular,
mouse and rat models, are valuable for investigating the
underlying mechanisms of human disease. Nonhuman pri-
mate studies provide a crucial link between small-animal and
human studies. It is therefore desirable to assess MRP1
function in various species. In the MEM, the conversion rate
is an extremely important factor because a radiotracer with a
low conversion rate in the brain does not allow quantitative
assessment of MRP1 function in the brain. GSH conjugation
is typically mediated by GSH S-transferases (GSTs), and thus
the conversion rate is probably susceptible to interspecies
7
obtained from patients with Alzheimer’s disease. Therefore,
an in vivo quantitative assessment of MRP1 function in the
brain is useful for examining the alteration of MRP1 expres-
sion caused by brain diseases and for directly evaluating the
effect of drugs, including modulators used to improve drug
delivery to the brain, on MRP1.
6-Bromo-7-[ C]methylpurine has recently been developed as
a radiotracer for quantitative assessment of MRP1 function in
the living brain using positron emission tomography (PET).
This radiotracer was designed based on the metabolite extrusion
11
8
1
1,12
differences.
sion rate by modifying the chemical structure of 6-bromo-
7
It is therefore necessary to adjust the conver-
8
-10
method (MEM), a type of prodrug/drug approach.
shows the rationale of MEM for assessing MRP1 function in the
brain. 6-Bromo-7-[ C]methylpurine in the blood crosses the
Figure 1
1
-[ C]methylpurine for application of the MEM to different
1
1
1
types of species, including humans. This requires examination
of the effect of substituents of this compound on the conver-
sion rate.
1
4
We designed and synthesized 6-halo-9-[ C]methylpurines
*To whom correspondence should be addressed. Phone: 81-43-206-
1
(9-isomer) and 6-halo-7-[ C]methylpurines (7-isomer) based
4
3
191. Fax: 81-43-206-3261. E-mail: kiku@nirs.go.jp.
a
11
on 6-bromo-7-[ C]methylpurine (Table 1). We examined
Abbreviations: S
N
Ar, aromatic nucleophilic substitution; GSH,
glutathione; GSTs, glutathione S-transferases; LUMO, lowest unoccu-
pied molecular orbital; MEM, metabolite extrusion method; MRP1,
multidrug resistance-associated protein 1; PET, positron emission to-
mography; PSG, S-[6-(7-methylpurinyl)]glutathione.
nonenzymatic and enzymatic reaction rates with GSH in
brain homogenates from the mouse, rat, or monkey under
tracer dose conditions. The lowest unoccupied molecular
r
pubs.acs.org/jmc
Published on Web 10/27/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7285
Table 2. Nonenzymatic Reactivity with GSH
a
b
-1
cmpd
position
X
k
non (h )
1
1
1
1
a
b
c
9
9
9
9
F
0.41 (0.013)
Cl
Br
I
0.024 (0.0060)
0.046 (0.0036)
0.046 (0.0036)
d
2
2
2
2
a
b
c
d
a
7
7
7
7
F
1.4 (0.036)
Cl
Br
I
0.096 (0.012)
0.28 (0.0060)
0.33 (0.012)
b
Position of the methyl group on the purine ring. Rate constant in
phosphate buffer (0.1 M, pH 7.4) containing 2 mM GSH. Values
are the mean of triplicate determinations with standard deviation in
parentheses.
Figure 1. MEM for assessment of MRP1 function in the brain.
After a radiotracer (open circles) penetrates the blood-brain
barrier (BBB) by diffusion, it is attacked by the nucleophilic thiolate
anion of GSH to form its GSH conjugate (circle-SG), which is
extruded by MRP1 from the brain to the blood. The efflux rate
constant (keff) is influenced by the pumping action of MRP1. Brain
radioactivity is constituted by a mixture of the radiotracer and
conjugate in the early phase after injection but only the conjugate
after complete conversion. The conversion rate is therefore impor-
tant for quantitative assessment of MRP1 function in the living
brain.
1
nonenzymatic reaction rate (knon) of C-labeled 6-halopur-
4
14
ine analogs with GSH (physiological concentration, 2 mM)
in phosphate buffer. The rank order of knon was 1a > 1d ≈ 1c
> 1b for the 9-isomer and 2a > 2d g 2c > 2b for the
7-isomer. This was similar to the rank order for aromatic
nucleophilic substitution (S Ar) reactions of 6-halopurine
N
nucleosides with thiolate nucleophiles (thioacetate and
3
-methylbutane-1-thiol) in organic solvents: F > Br ≈ I >
15
Cl. Most S Ar reactions are thought to proceed via a
N
Table 1. Analogs of 6-Halopurine
16
Meisenheimer complex or a σ-complex intermediate. In
SNAr reactions, the relative reactivity of the halogen is
dependent upon the rate-limiting step, which is formation
or decomposition of the σ-complex intermediate. In a reac-
tion of 1-substituted-2,4-dinitrobenzenes with piperidine,
1
7
the rank order was F . Br ≈ Cl > I. The differences in
rate between Cl, Br, and I were small, and the maximum
variation was only 4-fold. When the 1-substituent was F, the
relative rate was 3300 compared with I. This behavior would
not be expected in a reaction in which decomposition of the
σ-complex is rate limiting. Given these findings, our result
suggested that σ-complex formation was the rate-limiting
step for the nonenzymatic reaction of 6-halopurine analogs
with GSH.
orbital (LUMO) energy values were determined to describe
the electrophilicity of purine analogs.
Comparing the effect of the methyl position (9 or 7), the
non of the 7-isomer was several times higher than that of the
-isomer, despite the fact that the methyl group at the 7-
k
9
position was supposed to sterically hinder the thiolate anion
of GSH from approaching the purine carbon at the
6-position. Introduction of a methyl group into the 7-posi-
tion of the purine appeared to enhance the electrophilicity of
6-halopurine analogs, which was supported by the difference
in LUMO energy between the 9-isomer and the 7-isomer,
although the LUMO energy did not reflect the high reactivity
of fluorine (Table 3).
Results
Chemistry. Radiotracers were synthesized by methylation
1
of 6-halopurine analogs with CH I as a model for CH3I
4
11
3
in acetone. This resulted in the formation of two isomers: the
major product being the 9-isomer (1a-1d) and the minor
product being the 7-isomer (2a-2d). Their radiochemical
purities were >95%. The radiochemical yield, although not
optimized, was 35-50% for the 9-isomer and 8-20% for the
Enzymatic Reactivity. Table 4 shows the enzymatic reac-
tion rate (kenz) of 6-halopurine analogs in brain homogenates
from the mouse, rat, and monkey supplemented with 2 mM
GSH, and Table 5 exhibits the ratio of kenz to knon for each
compound. There was a marked difference in kenz between
species, and the rank order of kenz among compounds was
not identical. As expected, kenz of each 6-halopurine analog
was much higher than knon. Considering that 2b and 2c are
rapidly converted into a GSH conjugate of PSG in the rodent
7
-isomer, which was similar to an earlier study on alkylation
1
3
of 6-chloropurine. The low yield of the 7-isomer was
probably due to the steric hindrance between the halogen
at the 6-position and the methyl group at the 7-position. The
1
4
radiotracers were rapidly labeled with C by a one-step
reaction. They were therefore expected to be well suited for
1
labeling with C nuclide for PET studies after slight mod-
1
1
1
ification. Indeed, 6-bromo-7-[ C]methylpurine was ob-
tained in a total synthesis time of 30 min, including HPLC
purification and formulation.
Nonenzymatic Reactivity. Because a small amount of a
radiotracer is injected into humans or animals in PET
studies, reactivity of 6-halopurine analogs with GSH was
investigated under tracer dose conditions. Table 2 shows the
8
,10
brain in vivo,
these results indicate that GSTs would
8
catalyze the reaction of 6-halopurine analogs with GSH.
GSTs have two substrate-binding sites: the GSH-binding site
and the substrate-binding site for hydrophobic com-
1
8
pounds. The partition coefficient of 6-halopurine analogs
was determined to investigate the relationship between

7
286 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Okamura et al.
Table 3. Comparison of Electrophilic Characteristics between the 9-
Isomer and the 7-Isomer
b
E(LUMO) (eV)
a
cmpd
position
X
AM1
PM3
1
1
1
1
a
b
c
9
9
9
9
F
-0.784
-0.763
-0.838
-0.831
-0.900
-0.830
-0.865
-0.767
Cl
Br
I
d
2a
2b
2c
2d
a
7
7
7
7
F
-0.806
-0.793
-0.859
-0.848
b
-0.960
-0.913
-0.923
-1.09
Cl
Br
I
Position of the methyl group on the purine ring. LUMO energy was
calculated with the AM1 and PM3 Hamiltonian.
Table 4. Enzymatic Reactivity with GSH
b
-1 -1
-1
k
enz (h
g
mL )
Figure 2. Hydrophobic nature of 6-halopurine analogs. Partition
coefficients were determined in a mixture of 1-octanol/phosphate
buffer (0.1 M, pH 7.4). Data are mean ( standard deviation of
triplicate determinations.
a
cmpd
position
X
mouse
rat
monkey
32 (0.19)
1
1
1
1
a
b
c
9
9
9
9
F
8.4 (0.091)
1.5 (0.14)
3.6 (0.26)
2.6 (0.079)
4.2 (0.084)
0.43 (0.0090)
1.5 (0.069)
2.0 (0.035)
c
Cl
Br
I
2.0 (0)
2.0 (0.019)
1.3 (0.070)
6-halopurine analogs with other thiol groups (e.g., proteins
and cysteine). Because 6-halopurine analogs possess high kenz
d
/
knon, they specifically produce an MRP1 substrate of the GSH
2
2
2
2
a
b
c
d
a
7
7
7
7
F
41 (0.9)
21 (0.68)
83 (1.2)
150 (3.4)
47 (3.9)
31 (0.73)
92 (4.6)
260 (14)
b
120 (4.5)
7.8 (0.033)
26 (0.067)
80 (0.51)
conjugate. Similarly to 2c, 2b was efficiently converted to the
although
Cl
Br
I
8,10
GSH conjugate of PSG in the rat brain in vivo,
kenz of 2b was lower than that of 2c. Brain radioactivity was
almost completely constituted by PSG from 15 min after
intravenous injection of 2b or 2c. Radiotracers with greater
Position of the methyl group on the purine ring. Rate constant in
cerebral cortical homogenate from three species supplemented with
mM GSH. Values are the mean (n = 3, rat) and the mean of triplicate
determinations of homogenate from a single monkey or the combined
homogenate from seven mice. Standard deviation is shown in paren-
theses. All values are identical.
-
1
-1
-1
2
than the rat kenz of 2b (31 h
g
mL ) would therefore
satisfy the requirement of an efficient conversion in the MEM
(Table4). The7-isomerappearedtobealmostapplicabletothe
mouse, rat, and mokey, but the 9-isomer (except for 1a)
showed low reactivity with GSH in these species. The low
reactivity of the 9-isomer would delay complete disappearance
of the unchanged form in the brain in vivo. This may make
estimation of the efflux rate of the GSH conjugate inaccurate.
However, if sufficient brain radioactivity persists for a long
time, the extension of duration of PET may allow accurate
estimation of the efflux rate. The 9-isomer is therefore not
always a poor radiotracer for assessment of MRP1 function.
In addition to reactivity with GSH, the MRP1-mediated
transport rate may be susceptible to interspecies dif-
ferences. We examined the kinetics of human MRP1- and rat
Mrp1-mediated transport of PSG, the GSH conjugate of
the promising radiotracer (the 7-isomer). The Km
c
Table 5. Ratio of Enzymatic (kenz) to Nonenzymatic (knon) Reaction
Rates of Each Compound
k
enz/knon
a
cmpd
position
X
mouse
rat
monkey
1
1
1
1
a
b
c
9
9
9
9
F
21
64
78
57
10
18
32
44
79
84
42
29
Cl
Br
I
d
2
2
2
2
a
b
c
d
a
7
7
7
7
F
28
33
85
81
Cl
Br
I
214
295
462
321
325
781
93
241
Position of the methyl group on the purine ring.
(Michaelis-Menten constant) and Vmax (maximum uptake
rate) for human MRP1 were 127 ( 18 μM and 1051 (
55 pmol/min/mg protein, respectively, whereas those for rat
Mrp1 were 78.9 ( 10 μM and 1053 ( 42 pmol/min/mg
protein, respectively. Assuming that the expression level of
MRP1 in human brain is equal to that of Mrp1 in rat brain,
there may not be a critical difference between the efflux rate in
kenz and hydrophobicity (Figure 2). The partition coeffi-
cients of the 7-isomer were approximately half of those of the
corresponding 9-isomer, and the rank order of the partition
coefficient for each isomer was I > Br > Cl > F. Partition
coefficients were approximately correlated with the ratio of
kenz/knon of the 7-isomer for each species and of the 9-isomer
for the rat, suggesting that hydrophobicity in addition to
11
humans and rats, suggesting that the C-labeled 7-isomer
may be applicable to human studies.
nonenzymatic reactivity may contribute to the degree of kenz
.
In conclusion, reactivity of 2c with GSH can be adjusted by
changing the position of the halogen and the methyl group.
This may allow assessment of MRP1 function in the brain in
different species, including humans.
Discussion and Conclusions
For the application of the MEM to different kinds of
species, including humans, we investigated kenz and knon of
-halopurine analogs, which can be used to estimate the in vivo
Experimental Section
6
conversion rate in the MEM. The nonenzymatic reactivity
presumably contributes to the nonspecific reaction of
General. All commercially available starting materials and
solvents were of reagent grade or better and were used without

Article
further purification. [ C]Methyl iodide (specific activity:
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7287
1
4
S-[6-(7-Methylpurinyl)]glutathione. This compound was pre-
pared from 6-chloro-7-methylpurine with GSH in the same
3
.15 GBq/mmol) and [ S]GSH (36.1 TBq/mmol) were pur-
5
2
1
0
chased from Amersham Biosciences Corp. (Piscataway, NJ) and
PerkinElmer Life Sciences, Inc. (Boston, MA), respectively.
Thin-layer chromatography (TLC) plates were purchased from
Merck Ltd. (Tokyo, Japan). Membrane vesicles containing
manner as that described previously and was analyzed using
an HPLC system consisting of a model L-2130 pump (Hitachi
High-Technologies Corporation, Tokyo, Japan), a model
L-2420 UV-vis absorbance detector set at 288 nm (Hitachi
High-Technologies Corporation), and a COSMOSIL 5C18-
AR-II column (4.6 I.D. ꢀ 250 mm; Nacalai Tesque Inc.). The
column was eluted with a mobile phase of acetonitrile/water
containing 0.1% trifluoroacetic acid (10:90; v/v) at a flow rate of
1.0 mL/min.
human MRP1 or rat Mrp1 were purchased from BD Biosciences
(
1
Tokyo, Japan). Proton nuclear magnetic resonance ( H NMR)
spectroscopy was carried out on JEOL JNM-ALPHA 500 FT-
NMR (JEOL Ltd., Tokyo, Japan) with chemical shifts reported
in units of parts per million (ppm). Fast atom bombardment
mass spectrometry (FAB-MS) and electron impact mass spec-
trometry (EI-MS) were carried out using a JEOL JMS-HX-
14
Radiochemical Synthesis. 6-Halo-9-[ C]methylpurines (9-isomer,
14
1a-1d) and 6-halo-7-[ C]methylpurines (7-isomer, 2a-2d) were
prepared by N-methylation using [ C]methyl iodide. A solution
14
1
(
10A mass spectrometer and a GC-MATE mass spectrometer
JEOL Ltd.). Radioactivity was measured using a liquid
14
of 9.25 MBq of [ C]methyl iodide in 1 mL of acetone was added
to a mixture of 6-halopurine (60 μmol) and potassium carbonate
(70 μmol). In a sealed vial, the mixture was heated in a water bath
at 55 °C for 30 min. Acetone was removed in a stream of nitrogen.
The residue was dissolved in 200 μL of acetone and applied to
silica gel TLC plates. It was developed with a mixture of chloro-
form/ethanol (9:1) for the 9-isomer or ethyl acetate/ethanol (9:1)
scintillation counter (LS 6000; Beckman Instruments, Fuller-
ton, CA). The relative radioactivity of compounds on TLC
was quantified using an imaging phosphor plate system (BAS
1
800 system; Fuji Photo Film Co., Tokyo, Japan). Purity of the
test compounds was determined by TLC or HPLC to be greater
than 95%.
6-Fluoropurine. This compound was prepared by nucleophilic
f
for the 7-isomer. The radioactive zone with an R value corre-
displacement of the trimethylammonio group with potassium
hydrogen fluoride, after the synthesis of trimethylpurin-6-
ylammonium chloride from 6-chloropurine, as previously des-
sponding to the authentic compound was scratched, collected, and
extracted with acetone. Radiochemical yields of the 9-isomer and
the 7-isomer were 35-50% and 8-20%, respectively. Radio-
chemical purities (>95%) were determined by TLC using chloro-
form/ethanol (9:1; v/v) and ethyl acetate/ethanol (9:1; v/v).
19 1
cribed. H NMR (δ, CD OD) 8.50 (1H, s, H-8), 8.59 (1H, s,
3
þ
H-2); FAB-MS (m/z) calcd, 139 (M þ H) ; found, 139; yield 17%.
3
5
6
-Iodopurine. This compound was prepared by reaction of
[ S]S-[6-(7-methylpurinyl)]glutathione was synthesized by
the conjugation of 6-chloro-7-methylpurine with [ S]GSH. A
2
0
35
6
-chloropurine with hydriodic acid, as previously described.
1
H NMR (δ, DMSO-d
1H, br, NH); FAB-MS (m/z) calcd, 247 (M þ H) ; found, 247;
yield 72%.
6
) 8.58 (1H, s, H-8), 8.63 (1H, s, H-2), 13.8
solution of 6-chloro-7-methylpurine (2.2 mg) dissolved in
200 μL of ethanol was added to a solution of 9.25 MBq of
þ
(
3
5
[ S]GSH in 225 μL of phosphate buffer (0.1 M, pH 7.4). In a
sealed vial, the mixture was heated in a water bath at 55 °C for
60 min, and the solution was concentrated in a stream of
nitrogen. The residue was purified by preparative TLC, as
Nonradiolabeled 6-Halo-9-methylpurine and 6-Halo-7-methyl-
purine. These compounds were prepared by a standard alkyla-
1
3,19
tion procedure as previously reported.
iodide (12 mmol) was added to a solution of 6-halopurine
6 mmol) and potassium carbonate (6 mmol) dissolved in
0 mL of dimethylsulfoxide. After the reaction mixture was
Briefly, methyl
1
0
reported previously, and was analyzed using the HPLC system
as described above. The eluate from the column was collected in
0.5 mL fractions, and radioactivity in the fractions was mea-
sured by a liquid scintillation counter. Radiochemical yield was
(
3
stirred at room temperature for 24 h, the solvent was evaporated
in vacuo. The residue was then purified by column chromatog-
raphy (silica gel C-200) using one of the following eluent
systems: (A) ethyl acetate/ethanol (9:1), (B) chloroform/ethanol
6
0%; the radiochemical purity (98%) was calculated from the
radioactivity in the eluate fraction corresponding to the authen-
tic sample and the total radioactivity injected into HPLC.
These compounds were prepared without adding carriers,
thus, each specific activity may be considered to be equal to that
(
9:1), (C) ethyl acetate/ethanol (95:5), and (D) ethyl acetate.
1
6-Fluoro-9-methylpurine. H NMR (δ, CDCl
3
) 3.92 (3H, s,
1
4
35
NCH ), 8.06 (1H, s, H-8), 8.59 (1H, s, H-2); EI-MS (m/z) calcd,
3
3
of the original radioactive compound, CH I or [ S]GSH.
þ
1
1
52 (M ); found, 152; eluent system (A); yield 36%; mp
Reaction Rates of Purine Analogs with GSH in Phosphate
Buffer. A solution (2 mM GSH in 0.1 M phosphate buffer, pH
21
30-132 °C (lit., 135-136 °C).
-Fluoro-7-methylpurine. H NMR (δ, CDCl
1
6
3
) 4.07 (3H, s,
7
1
1
.4; 150 μL) was placed in tubes and preincubated at 37 °C for
14
5 min. Addition of each [ C]-purine solution (18.5 kBq in
NCH ), 8.16 (1H, s, H-8), 8.72 (1H, s, H-2); EI-MS (m/z) calcd, 152
3
þ
(
M ); found, 152; eluent system (B); yield 13%; mp 140-142 °C.
-Chloro-9-methylpurine and 6-Chloro-7-methylpurine. These
0 μL buffer) to each tube initiated the reaction. At designated
6
intervals, 20 μL of the solution was immediately added to 40 μL
of ethanol containing excess diethyl maleate (50 mM), the latter
acting as a stop solution. The resultant solution (5 μL) was
applied to silica gel TLC plates, and developed with ethyl
acetate/ethanol (9:1; v/v). The air-dried TLC plate was covered
with a thin plastic film (lumilar film) and placed in a cassette in
contact with an imaging phosphor plate for several hours.
1
0
compounds were characterized as previously reported.
-Bromo-9-methylpurine. This compound was recrystallized
from benzene after separation by column chromatography. H
6
1
NMR (δ, CDCl
(
3
) 3.92 (3H, s, NCH
3
), 8.10 (1H, s, H-8), 8.71
1H, s, H-2); EI-MS (m/z) calcd, 212 and 214 (M ); found, 212
þ
and 214; eluent system (C); yield 28%; mp 157-159 °C.
-Bromo-7-methylpurine. This compound was characterized
as reported.
6
Radioactivity corresponding to the R value of unchanged
f
8
purines was quantified using the BAS 1800 system. The pseudo
first-order rate constants (knon) were calculated by plotting
6-Iodo-9-methylpurine. This compound was recrystallized
from chloroform after separation by column chromatography.
1
4
changes in the log of the radioactivity of [ C]-purine analogs
against time. Determinations were made in triplicate.
Reaction Rates of Purine Analogs with GSH in Tissue Homo-
genates. Animals were treated and handled according to the
“Recommendations for Handling of Laboratory Animals for
Biomedical Research”, compiled by the Committee on Safety
and Ethical Handling Regulations for Laboratory Animal
Experiment, NIRS.
1
H NMR (δ, DMSO-d ) 3.83 (3H, s, NCH ), 8.61 (1H, s, H-8),
6
3
þ
8
2
2
.63 (1H, s, H-2); FAB-MS (m/z) calcd, 261 (M þ H) ; found,
2
2
61; eluent system (D); yield 20%; mp >211 °C dec (lit.,
12-215 °C dec.)
6
from chloroform after separation by column chromatography.
-Iodo-7-methylpurine. This compound was recrystallized
1
H NMR (δ, DMSO-d
6
) 4.07 (3H, s, NCH
3
), 8.59 (1H, s, H-8),
þ
8
2
.72 (1H, s, H-2); FAB-MS (m/z) calcd, 261 (M þ H) ; found,
Male ddY mice (35-38 g; 8 weeks old) and Wistar rats
(185-205 g; 8 weeks old) were purchased from Japan SLC
61; eluent system (D); yield 1.0%; mp >190 °C dec.

7
288 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Okamura et al.
Inc. (Shizuoka, Japan). Nonanesthetized mice and rats anesthe-
tized with diethyl ether were decapitated. Their brains were
removed and the cerebral cortices dissected. A rhesus monkey
(2) Minich, T.; Riemer, J.; Schulz, J. B.; Wielinga, P.; Wijnholds, J.;
Dringen, R. The multidrug resistance protein 1 (Mrp1), but not
Mrp5, mediates export of glutathione and glutathione disulfide
from brain astrocytes. J. Neurochem. 2006, 97, 373–384.
(
23 years old) was anesthetized with pentobarbital and killed by
saline perfusion. The monkey brain was removed and stored at
80 °C before use; the tissue of the mouse and rat was used
(
3) Dallas, S.; Miller, D. S.; Bendayan, R. Multidrug resistance-
associated proteins: expression and function in the central nervous
system. Pharmacol. Rev. 2006, 58, 140–161.
-
immediately after dissection. The cerebral cortex was weighed
and homogenized in phosphate buffer (0.1 M, pH 7.4). The
homogenate (150 μL) supplemented with 2 mM GSH was
placed in tubes and preincubated at 37 °C for 15 min. Each
(4) Renes, J.; de Vries, E. E.; Hooiveld, G. J.; Krikken, I.; Jansen, P. L.;
Muller, M., Multidrug resistance protein MRP1 protects against
the toxicity of the major lipid peroxidation product 4-hydroxyno-
nenal. Biochem. J. 2000, 350 Pt 2, 555-561.
(
5) Loscher, W.; Potschka, H. Role of drug efflux transporters in the
brain for drug disposition and treatment of brain diseases. Prog.
Neurobiol. 2005, 76, 22–76.
1
4
[
C]-purine solution (18.5 kBq in 10 μL buffer) was added to
each tube to initiate the reaction. At designated intervals, 20 μL
of the homogenate was immediately added to 40 μL of the stop
solution described above and then centrifuged. The supernatant
(6) Joshi, G.; Sultana, R.; Tangpong, J.; Cole, M. P.; St Clair, D. K.;
Vore, M.; Estus, S.; Butterfield, D. A. Free radical mediated
oxidative stress and toxic side effects in brain induced by the anti
cancer drug adriamycin: insight into chemobrain. Free Radical Res.
(
5 μL) was applied to silica gel TLC plates, and developed with
ethyl acetate/ethanol (9:1; v/v). First-order rate constants were
calculated as described above and corrected for homogenate
concentration after subtracting knon to yield the reaction rate
2
005, 39, 1147–1154.
(
7) Sultana, R.; Butterfield, D. A. Oxidatively modified GST and
MRP1 in Alzheimer’s disease brain: implications for accumulation
of reactive lipid peroxidation products. Neurochem. Res. 2004, 29,
-
1
-1
-1
k
homogenate (n = 3) and in triplicate with homogenate from a
enz (h
g
mL ). Experiments were carried out with rat
2
215–2220.
(
8) Okamura, T.; Kikuchi, T.; Okada, M.; Toramatsu, C.; Fukushi,
K.; Takei, M.; Irie, T. Noninvasive and quantitative assessment
of the function of multidrug resistance-associated protein 1 in the
living brain. J. Cereb. Blood Flow Metab. 2009, 29, 504–511.
single monkey or the combined homogenate from seven mice.
Partition Coefficient. Each C-labeled purine analog was
1
4
added to a 1:1 mixture of 1-octanol and 0.1 M phosphate buffer
(
pH 7.4), shaken vigorously, and allowed to equilibrate for 1 h at
(9) Okamura, T.; Igarashi, J.; Kikuchi, T.; Fukushi, K.; Arano, Y.;
Irie, T. A radiotracer method to study efflux transport of iodide
liberated from thyroid hormones via deiodination metabolism in
the brain. Life Sci. 2009, 84, 791–795.
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A novel noninvasive method for assessing glutathione-conjugate
efflux systems in the brain. Bioorg. Med. Chem. 2007, 15, 3127–
3133.
11) Polidoro, G.; Di Ilio, C.; Sacchetta, P.; Del Boccio, G.; Federici, G.
Isoelectric focusing of brain cortex GSH S-transferase activity in
mammals: evidence that polymorphism is absent in man. Int. J.
Biochem. 1984, 16, 741–746.
(12) Das, M.; Dixit, R.; Seth, P. K.; Mukhtar, H. Glutathione-
S-transferase activity in the brain: species, sex, regional, and age
differences. J. Neurochem. 1981, 36, 1439–1442.
13) Montgomery, J. A.; Temple, C., Jr. Synthesis of potential
anticancer agents. XXVI. The alkylation of 6-chloropurine.
J. Am. Chem. Soc. 1961, 83, 630–635.
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and cysteine depletion in rats and mice following acute
intoxication with diethylmaleate. Biochem. Pharmacol. 1992, 43,
1
4
room temperature. C concentrations in the organic and aqu-
eous phases were measured with a liquid scintillation counter.
Each partition coefficient was calculated as the ratio of con-
centration in the organic phase to that in the aqueous phase.
Determinations were made in triplicate.
(
(
Molecular Orbital Calculations. Molecular orbital calcula-
tions were carried out on computers based on Intel Pentium 4
running on Windows XP. The semiempirical molecular orbital
method was used, applying the AMI or PM3 Hamiltonian from
the MOPAC program. Calculations were carried out using
PRECISE criteria. Molecular structures were optimized using
eigenvector Following (a geometry optimization procedure
within MOPAC2002).
(
Vesicular Transport Studies. Transport studies were carried
with some modifica-
1
0,23
out using the rapid filtration technique
tion. Briefly, membrane vesicles (50 μg of protein) were incu-
bated in the presence of 4 mM ATP or AMP in 50 μL of transport
(
4
51–456.
medium (40 mM MOPS-Tris, 50 mM KCl, 6 mM MgCl
2
, 5.2
kBq of [ S]PSG with 5, 15, 50, 150, or 500 μM of unlabeled PSG,
mM GSH) at 37 °C. The transport reaction was stopped at a
given time by the addition of 200 μL of ice-cold buffer containing
0 mM MOPS-Tris and 70 mM KCl. The stopped reaction
35
(15) Liu, J.; Robins, M. J. S(N)Ar displacements with 6-(fluoro,
chloro, bromo, iodo, and alkylsulfonyl)purine nucleosides: synth-
esis, kinetics, and mechanism. J. Am. Chem. Soc. 2007, 129, 5962–
2
5
968.
4
(16) Miller, J. Variation of Leaving Groups. In Reaction Mechanisms in
Organic Chemistry; Eaborn, C., Chapman, N. B., Eds.; Elsevier: New
York, 1968; pp 137-179.
17) Bunnett, J. F.; Garbisch, E. W., Jr.; Pruitt, K. M. The “Element
Effect” as a criterion of mechanism in activated aromatic
nucleophilic substitution reactions. J. Am. Chem. Soc. 1957, 79,
385–391.
(18) Danielson, U. H.; Mannervik, B. Kinetic independence of the
subunits of cytosolic glutathione transferase from the rat. Biochem.
J. 1985, 231, 263–267.
19) Kiburis, J.; Lister, J. H. Nucleophilic displacement of the trimethy-
lammonio-group as a new route to fluoropurines. J. Chem. Soc.,
Perkin Trans. 1 1971, 23, 3942–3947.
mixture was filtered through a GF/F glass fiber filter (Whatman)
and then washed five times with 200 μL of the ice-cold buffer.
Radioactivity retained on the filters was determined using a
liquid scintillation counter. ATP-dependent transport was cal-
culated by subtracting the activity values obtained with AMP
from those in the presence of ATP. The uptake was measured at
three time points (0.5, 1, and 2 min) for substrate concentrations
of 5, 15, and 50 μM, and at two time points (1 and 2 min) for
(
(
(
(
m
substrate concentrations of 150 and 500 μM. K and Vmax were
estimated by the nonlinear least-squares method.
20) Elion, G. B.; Hitchings, G. H. Studies on condensed pyrimidine
systems. XVII. Some halogenopurines. J. Am. Chem. Soc. 1956, 78,
Acknowledgment. This research was partially supported by
the Ministry of Education, Culture, Sports, Science and Tech-
nology, Grant-in-Aid for Young Scientists (B), 20790920,
3
508–3510.
21) Beaman, A. G.; Robins, R. K. The synthesis of 6-fluoro-9-methyl-
purine. J. Med. Pharm. Chem. 1962, 91, 1067–1074.
(22) Dyer, E.; Reitz, J. M.; Farris, R. E., Jr. Carbamates derived from
2009-2010.
aminopurines. J. Med. Chem. 1963, 6, 289–291.
(
23) Loe, D. W.; Almquist, K. C.; Deeley, R. G.; Cole, S. P. Multidrug
resistance protein (MRP)-mediated transport of leukotriene C4
and chemotherapeutic agents in membrane vesicles. Demonstra-
tion of glutathione-dependent vincristine transport. J. Biol. Chem.
1996, 271, 9675–9682.
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